**A Review of Radiation Therapy's Role in Early-Stage Breast Cancer and an Introduction to Electronic Brachytherapy**

Brent Herron, Alex Herron, Kathryn Howell, Daniel Chin and Luann Roads

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55621

### **1. Introduction**

With the exception of skin cancer, breast cancer is the most commonly diagnosed cancer in women in the United States and the most developed European countries [1]. Although breast cancer has been known to be a major cause of mortality in women living in affluent countries, this disease does not discriminate crossing racial, gender, geograph‐ ic, and economic lines. Encouraging reports indicate there may be a trend toward decreasing breast cancer incidence in countries where there is a decline in hormone replacement therapy [31,6]. Furthermore, it has been reported that breast cancer mortality has fallen in industrialized countries in the last decade [7,36,6]. Reasons for declining mortality may include early detection and better treatment.

Treatment of breast cancer requires a multidisciplinary approach. The surgeon, medical oncologist, radiation oncologist, and pathologist play a role in developing treatment options for the patient. Radiation therapy has a significant part in the treatment of breast cancer, both for noninvasive and invasive cancers.

Breast conserving surgery includes partial mastectomy, lumpectomy, tylectomy, wide local excision, and quadrantectomy. These techniques followed by 5-7 weeks of radia‐ tion therapy have been known for over two decades as breast conservation therapy. Initially accepted as a form of breast cancer treatment in Europe, breast conservation therapy is now accepted throughout the world and has gained popularity in the United States since the early 1980s.

© 2013 Herron et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

With the advancements in computed tomography imaging, simulation, treatment planning and delivery systems, more accurate and homogenous radiation treatment can be delivered. Radiation therapy options following breast conserving surgery include whole breast radiation, accelerated partial breast radiation with external beam treatment or brachytherapy, and hypofractionated whole breast radiation treatment. This chapter includes a review of these techniques and also introduces a relatively new option: electronic brachytherapy as the role of radiation therapy in early breast cancer management has continued to evolve.

systems. Today, for patients with node negative disease, local recurrence for patients undergoing breast conserving surgery with radiation therapy and systemic chemothera‐

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225

The American College of Radiology Practice Guidelines and the National Comprehensive Network Practice Guidelines serve as tools for selecting patients as candidates for breast conserving therapy. Most women diagnosed with localized breast cancer are candidates. However, there are contraindications to breast conserving therapy including large tumor size to size of the breast, multicentric breast cancer, diffuse malignant appearing, pregnancy, prior radiation to the chest, persistent positive margins after several re-excisions, pacemaker in radiation portal, and morbid obesity exceeding the radiation therapy table limit. Having a collagen vascular disease, such as active lupus, is a relative contraindication to breast con‐

At least two-thirds of patients are eligible for breast conserving surgery at diagnosis [6]. Several factors influence local regional recurrence. Obtaining gross negative margins at the time of surgery is no longer considered acceptable. Margins should be microscopically negative and as wide as possible. Most surgeons consider a 2-3mm clear margin as acceptable. The median rates of ipsilateral breast recurrence has been shown to be 2%, 3%, and 6% when margins of

The presence or absence of extensive intraductal component (EIC) has traditionally been felt to affect local recurrence. Holland et al. [26] showed that the presence of EIC is associated with breast recurrence. In a series of 214 patients who underwent a mastectomy, 71% of patients with EIC had residual intraductal tumor, whereas only 28% of patients without EIC had

In certain studies, young age, usually 40, 35 or 30 years or less, has been associated with an increase of ipsilateral tumor recurrence following breast conserving surgery. [22,50,34]. However, many of these studies also show young age to correlate with other high risk features such as high grade and the presence of EIC. A boost dose delivered to the surgical cavity following whole breast radiation therapy is particularly significant for younger patients since

External beam radiation therapy typically begins 3 to 6 weeks following surgery unless systemic chemotherapy is given. Treatment planning starts with the simulation process. Breast boards, wing boards, or customized cradles or molds are created or fitted to the individual patient. This allows the patient to be in a reproducible position with e ach treatment. Patients

clearance were determined clear, 1mm clear, and 2mm clear respectively [41].

higher doses tend to correlate with lower recurrences.

**4. External beam radiation therapy**

py has dropped to 0.5% annually [12, 13, 14].

serving treatment.

residual disease.

**3. Patient selection and factors affecting local recurrence**

### **2. Support of breast conservation treatment**

Multiple international trials have demonstrated the efficacy of breast-conserving surgery followed by radiation therapy [16,47,20,21].Early detection promotions has resulted in patients presenting with even smaller and more favorable tumors than years ago [15].The improvement of mammographic imaging and screening has led to the increase incidence of patients presenting with noninvasive breast cancer from 5% to 30% [44].

Randomized trials worldwide comparing mastectomy to breast conserving surgery followed by radiation therapy have clearly shown equivalent long-term survival in both groups [20,21, 47,37,38, 9]. The Milan trial was one of the first landmark trials on this subject. From 1973 to 1980, 701 women with Stage 1 breast cancer were randomized to radical mastectomy versus breast conserving surgery with adjuvant whole breast radiation therapy (50 Gy plus a 10 Gy boost). Patients with positive lymph node metastases also received adjuvant chemotherapy. There were no significant differences between the two groups in the development of contrala‐ teral breast cancers, distant metastases, or secondary primary cancers. At a median follow-up of 20 years, survival was shown to be equivalent between the two groups.

Other landmark European trials comparing mastectomy and breast conserving surgery included the Institut Gustave-Roussy and the European Organization for Research and Treatment of Cancer (EORTC). The Institut trial randomized women with 2cm or smaller tumors to mastectomy or local excision followed by radiation therapy. 15 year survival rates and local recurrences were statistically similar in this trial as well as the EORTC trial.

In the United States, the National Surgical Adjuvant Breast and Bowel Project (NSABP) initiated the B-04 study in 1971. A total of 2163 women with 4 cm or less breast cancers were randomized to one of three treatment arms: total mastectomy, lumpectomy alone, and lumpectomy plus radiation therapy. Twenty-year follow-up analysis showed no differences in overall survival in the three arms. However, patients who underwent lumpectomy alone had a 39.2% risk of local recurrence versus 14.3% risk of recurrence in the lumpectomy plus radiation arm. Radiation therapy also showed a marginally significant decrease in breast cancer related deaths when compared to the lumpectomy alone arm [20,21].

Fortunately, the outcome for patients treated with breast conserving surgery continues to improve. There have been tremendous advancements in the last decade in surgical techniques, systemic treatment, diagnostic imaging, and radiation therapy delivery systems. Today, for patients with node negative disease, local recurrence for patients undergoing breast conserving surgery with radiation therapy and systemic chemothera‐ py has dropped to 0.5% annually [12, 13, 14].

### **3. Patient selection and factors affecting local recurrence**

With the advancements in computed tomography imaging, simulation, treatment planning and delivery systems, more accurate and homogenous radiation treatment can be delivered. Radiation therapy options following breast conserving surgery include whole breast radiation, accelerated partial breast radiation with external beam treatment or brachytherapy, and hypofractionated whole breast radiation treatment. This chapter includes a review of these techniques and also introduces a relatively new option: electronic brachytherapy as the role

Multiple international trials have demonstrated the efficacy of breast-conserving surgery followed by radiation therapy [16,47,20,21].Early detection promotions has resulted in patients presenting with even smaller and more favorable tumors than years ago [15].The improvement of mammographic imaging and screening has led to the increase incidence of patients

Randomized trials worldwide comparing mastectomy to breast conserving surgery followed by radiation therapy have clearly shown equivalent long-term survival in both groups [20,21, 47,37,38, 9]. The Milan trial was one of the first landmark trials on this subject. From 1973 to 1980, 701 women with Stage 1 breast cancer were randomized to radical mastectomy versus breast conserving surgery with adjuvant whole breast radiation therapy (50 Gy plus a 10 Gy boost). Patients with positive lymph node metastases also received adjuvant chemotherapy. There were no significant differences between the two groups in the development of contrala‐ teral breast cancers, distant metastases, or secondary primary cancers. At a median follow-up

Other landmark European trials comparing mastectomy and breast conserving surgery included the Institut Gustave-Roussy and the European Organization for Research and Treatment of Cancer (EORTC). The Institut trial randomized women with 2cm or smaller tumors to mastectomy or local excision followed by radiation therapy. 15 year survival rates

In the United States, the National Surgical Adjuvant Breast and Bowel Project (NSABP) initiated the B-04 study in 1971. A total of 2163 women with 4 cm or less breast cancers were randomized to one of three treatment arms: total mastectomy, lumpectomy alone, and lumpectomy plus radiation therapy. Twenty-year follow-up analysis showed no differences in overall survival in the three arms. However, patients who underwent lumpectomy alone had a 39.2% risk of local recurrence versus 14.3% risk of recurrence in the lumpectomy plus radiation arm. Radiation therapy also showed a marginally significant decrease in breast

Fortunately, the outcome for patients treated with breast conserving surgery continues to improve. There have been tremendous advancements in the last decade in surgical techniques, systemic treatment, diagnostic imaging, and radiation therapy delivery

and local recurrences were statistically similar in this trial as well as the EORTC trial.

of radiation therapy in early breast cancer management has continued to evolve.

**2. Support of breast conservation treatment**

224 Cancer Treatment - Conventional and Innovative Approaches

presenting with noninvasive breast cancer from 5% to 30% [44].

of 20 years, survival was shown to be equivalent between the two groups.

cancer related deaths when compared to the lumpectomy alone arm [20,21].

The American College of Radiology Practice Guidelines and the National Comprehensive Network Practice Guidelines serve as tools for selecting patients as candidates for breast conserving therapy. Most women diagnosed with localized breast cancer are candidates. However, there are contraindications to breast conserving therapy including large tumor size to size of the breast, multicentric breast cancer, diffuse malignant appearing, pregnancy, prior radiation to the chest, persistent positive margins after several re-excisions, pacemaker in radiation portal, and morbid obesity exceeding the radiation therapy table limit. Having a collagen vascular disease, such as active lupus, is a relative contraindication to breast con‐ serving treatment.

At least two-thirds of patients are eligible for breast conserving surgery at diagnosis [6]. Several factors influence local regional recurrence. Obtaining gross negative margins at the time of surgery is no longer considered acceptable. Margins should be microscopically negative and as wide as possible. Most surgeons consider a 2-3mm clear margin as acceptable. The median rates of ipsilateral breast recurrence has been shown to be 2%, 3%, and 6% when margins of clearance were determined clear, 1mm clear, and 2mm clear respectively [41].

The presence or absence of extensive intraductal component (EIC) has traditionally been felt to affect local recurrence. Holland et al. [26] showed that the presence of EIC is associated with breast recurrence. In a series of 214 patients who underwent a mastectomy, 71% of patients with EIC had residual intraductal tumor, whereas only 28% of patients without EIC had residual disease.

In certain studies, young age, usually 40, 35 or 30 years or less, has been associated with an increase of ipsilateral tumor recurrence following breast conserving surgery. [22,50,34]. However, many of these studies also show young age to correlate with other high risk features such as high grade and the presence of EIC. A boost dose delivered to the surgical cavity following whole breast radiation therapy is particularly significant for younger patients since higher doses tend to correlate with lower recurrences.

### **4. External beam radiation therapy**

External beam radiation therapy typically begins 3 to 6 weeks following surgery unless systemic chemotherapy is given. Treatment planning starts with the simulation process. Breast boards, wing boards, or customized cradles or molds are created or fitted to the individual patient. This allows the patient to be in a reproducible position with e ach treatment. Patients are typically placed in the supine position with their torso angled 10-15 degrees. The ipsilateral arm is abducted usually between 100-120 degrees and the shoulder is externally rotated. At this time, radio opaque wires are placed and secured along the surgical scars. The radiation oncologist then defines the treatment field borders to encompass the breast target and regional lymph nodes, if needed. CT simulation is performed. The isocenter can be selected and the daily set-up marks are placed on the patient's skin. 3–dimensional treatment planning is performed. Treatment volumes and critical structures are identified and outlined. Optimal beam arrangements are chosen. The goal is to deliver the prescribed dose to the target with a homogenous distribution, minimizing hot and cold spots, to minimize doses delivered to critical structures, such as lungs and heart, and minimize the volumes of the critical structures within the treatment fields.

ing approximately 50 Gy in 25 to 28 fractions. For the boost treatment, electrons typically are used. The lumpectomy cavity is boosted for another 10-16 Gy at 1.8 to 2 Gy per fraction.

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227

New advances in radiation treatment planning and delivery have led to the development of intensity modulated radiation therapy (IMRT) or forward planning IMRT to treat the breast. The dose to the contralateral breast is reduced with IMRT [10]. By conforming doses along the breast and blocking normal structures with multi-leaf collimators, the normal structures like the lungs or heart for left sided breast cancer treatment also receive reduced doses. The dose to the breast could be more homogenous with concave isodose curves, conforming to the target. Studies have shown that forward planning IMRT when compared to standard radiotherapy, can produce homogenous plans with fewer hot spots [4,25]. This could particularly benefit large-breasted women or those with large breast separation. Whether this translates to better

In some elderly patients, particularly those over 70 years of age with early disease who receive adjuvant hormonal therapy, breast conserving surgery alone may be an option. There could be biological differences in the tumors in some elderly women. Additionally, some elderly patients tend to have more transportation, social, and other health-related issues that may affect their ability to receive daily radiation therapy. The Canadian trial [23] and the Cancer and Leukemia Group B (CALGB/Radiation Therapy Oncology Group (RTOG)/Eastern Cooperative Oncology Group (ECOG) trial [28,42,23] both randomized older women with estrogen-receptor-positive early breast cancer following breast-conserving surgery to tamox‐ ifen with or without radiation therapy. Although both trials showed absolute benefits to

The many trials supporting breast-conserving surgery followed by adjuvant radiation therapy have also shown that the risk of recurrence outside the tumor cavity is similar whether or not whole breast radiation was given [20,21,47,27]. This suggests that additional radiation given

Breast brachytherapy was historically used to treat the lumpectomy cavity as a "boost" following external whole breast radiation therapy. Many centers have now adapted the use of accelerated partial breast irradiation (APBI), either with interstitial needle implants, various applicators (i.e. Mammosite balloon, Contoura multilumen balloon, Savi), or even through the use of 3D conformal external radiation therapy as the sole radiation treatment modality following breast-conserving surgery. By irradiating less volume, higher radiation doses can be given per fraction to the tumor bed. This shortens treatment times significantly, decreasing the patient's travel time when compared to daily whole breast external beam radiation therapy. Patients are potential candidates for APBI if they have Stage 0, 1, or II tumors, with a single tumor less than 3 cm in maximum dimension. Minimal nodal involvement and clear surgical margins are also required. Typically, partial breast radiation is delivered twice a day, with

cosmetic outcomes is unknown until these trials mature.

women receiving radiation therapy, the benefits overall were small.

**5. Breast brachytherapy for partial breast irradiation**

outside the tumor cavity may not be of additional benefit to patients.

each treatment separated at least 6 hours apart, for a total of ten fractions.

For early stage breast cancer, tangential fields that include the most anterior thorax are typically used (Fig 1). These fields can include level I and II lymph node chains. Attention to tangent field borders, especially the cranial and posterior chest wall interface, is important if most of levels I and II axillary nodes are to be included [40]. Radiation therapy to the supra‐ clavicular fossa plus or minus a posterior axillary boost is sometimes offered to patients with undissected nodes, four or more lymph node metastases or to patients with one to three positive nodes. A typical supraclavicular field is a half beam block field matched to the tangents with the beam angled 10-15 degrees away from the cord. A table kick is utilized for the tangential fields to account for the divergence of the beam into the supraclavicular field. The posterior axillary beam supplements dose to the midaxillary plane. Pierce et al. discusses several techniques to treat the internal mammary nodes [36].

**Figure 1.** Depiction of tangential fields used for treatment in external beam radiation therapy.

Four to six MV photon energy is most commonly selected for treating the breast and lymph nodes. Whole breast radiation treatments are administered Monday through Friday, deliver‐ ing approximately 50 Gy in 25 to 28 fractions. For the boost treatment, electrons typically are used. The lumpectomy cavity is boosted for another 10-16 Gy at 1.8 to 2 Gy per fraction.

are typically placed in the supine position with their torso angled 10-15 degrees. The ipsilateral arm is abducted usually between 100-120 degrees and the shoulder is externally rotated. At this time, radio opaque wires are placed and secured along the surgical scars. The radiation oncologist then defines the treatment field borders to encompass the breast target and regional lymph nodes, if needed. CT simulation is performed. The isocenter can be selected and the daily set-up marks are placed on the patient's skin. 3–dimensional treatment planning is performed. Treatment volumes and critical structures are identified and outlined. Optimal beam arrangements are chosen. The goal is to deliver the prescribed dose to the target with a homogenous distribution, minimizing hot and cold spots, to minimize doses delivered to critical structures, such as lungs and heart, and minimize the volumes of the critical structures

For early stage breast cancer, tangential fields that include the most anterior thorax are typically used (Fig 1). These fields can include level I and II lymph node chains. Attention to tangent field borders, especially the cranial and posterior chest wall interface, is important if most of levels I and II axillary nodes are to be included [40]. Radiation therapy to the supra‐ clavicular fossa plus or minus a posterior axillary boost is sometimes offered to patients with undissected nodes, four or more lymph node metastases or to patients with one to three positive nodes. A typical supraclavicular field is a half beam block field matched to the tangents with the beam angled 10-15 degrees away from the cord. A table kick is utilized for the tangential fields to account for the divergence of the beam into the supraclavicular field. The posterior axillary beam supplements dose to the midaxillary plane. Pierce et al. discusses

several techniques to treat the internal mammary nodes [36].

**Figure 1.** Depiction of tangential fields used for treatment in external beam radiation therapy.

Four to six MV photon energy is most commonly selected for treating the breast and lymph nodes. Whole breast radiation treatments are administered Monday through Friday, deliver‐

within the treatment fields.

226 Cancer Treatment - Conventional and Innovative Approaches

New advances in radiation treatment planning and delivery have led to the development of intensity modulated radiation therapy (IMRT) or forward planning IMRT to treat the breast. The dose to the contralateral breast is reduced with IMRT [10]. By conforming doses along the breast and blocking normal structures with multi-leaf collimators, the normal structures like the lungs or heart for left sided breast cancer treatment also receive reduced doses. The dose to the breast could be more homogenous with concave isodose curves, conforming to the target. Studies have shown that forward planning IMRT when compared to standard radiotherapy, can produce homogenous plans with fewer hot spots [4,25]. This could particularly benefit large-breasted women or those with large breast separation. Whether this translates to better cosmetic outcomes is unknown until these trials mature.

In some elderly patients, particularly those over 70 years of age with early disease who receive adjuvant hormonal therapy, breast conserving surgery alone may be an option. There could be biological differences in the tumors in some elderly women. Additionally, some elderly patients tend to have more transportation, social, and other health-related issues that may affect their ability to receive daily radiation therapy. The Canadian trial [23] and the Cancer and Leukemia Group B (CALGB/Radiation Therapy Oncology Group (RTOG)/Eastern Cooperative Oncology Group (ECOG) trial [28,42,23] both randomized older women with estrogen-receptor-positive early breast cancer following breast-conserving surgery to tamox‐ ifen with or without radiation therapy. Although both trials showed absolute benefits to women receiving radiation therapy, the benefits overall were small.

### **5. Breast brachytherapy for partial breast irradiation**

The many trials supporting breast-conserving surgery followed by adjuvant radiation therapy have also shown that the risk of recurrence outside the tumor cavity is similar whether or not whole breast radiation was given [20,21,47,27]. This suggests that additional radiation given outside the tumor cavity may not be of additional benefit to patients.

Breast brachytherapy was historically used to treat the lumpectomy cavity as a "boost" following external whole breast radiation therapy. Many centers have now adapted the use of accelerated partial breast irradiation (APBI), either with interstitial needle implants, various applicators (i.e. Mammosite balloon, Contoura multilumen balloon, Savi), or even through the use of 3D conformal external radiation therapy as the sole radiation treatment modality following breast-conserving surgery. By irradiating less volume, higher radiation doses can be given per fraction to the tumor bed. This shortens treatment times significantly, decreasing the patient's travel time when compared to daily whole breast external beam radiation therapy.

Patients are potential candidates for APBI if they have Stage 0, 1, or II tumors, with a single tumor less than 3 cm in maximum dimension. Minimal nodal involvement and clear surgical margins are also required. Typically, partial breast radiation is delivered twice a day, with each treatment separated at least 6 hours apart, for a total of ten fractions.

Interstitial breast brachytherapy alone has been successfully used for over 10 years following breast conserving surgery (Fig 2). A trial was started by Vinci et al. in 1993 using brachytherapy as the only radiation treatment modality for patients following breast-conserving surgery [48]. By 2001, 120 patients were enrolled in this trial. Four patients developed local recurrence at a median follow-up of 82 months. During 1997-2000, 100 patients were enrolled in a Radiation Therapy Oncology Group (RTOG), prospective Phase I/II study of breast brachytherapy. Patients were either high-dose or low-dose-rate brachytherapy. For the high-dose-rate group at a median follow-up of 6.14 years; 5-year estimates of ipsilateral breast, regional, and contralateral breast failures were 3%, 5%, and 2% respectively. For patients receiving low-doserate brachytherapy at a median follow-up of 6.22 years; 5-year estimates of ipsilateral breast, regional, and contra-lateral breast failures were 6%, 0%, and 6%, respectively. Both groups experienced good cosmesis and local control [3]. Several institutions have shown low recur‐ rences with brachytherapy at 5 and 10 years [2,5]

Subsequent evaluation of 43 patients eligible for the therapy revealed only mild to moderate self-limited side effects [30], Most recently, the American Society of Breast Surgeons reported results from their registry trial involving 1,440 women treated with the MammoSite® catheter following breast-conserving surgery. The 3-year actuarial rates of ipsilateral breast cancer and axillary recurrences were 2.15% and 0.36%, respectively. Cosmetic outcomes were reported to be acceptable and similar to patients treated with other forms of partial breast irradiation [33]. The advantages of the balloon catheter are that it is easier to place in the cavity, placement is more reproducible, and patient comfort is improved. It has become the most widely used device and has the longest track record [43]. The single catheter needs to be temporarily placed in the lumpectomy cavity, as opposed to 10-20 catheters with traditional interstitial implants. However, the balloon needs to "conform" properly to the tumor cavity and optimal dosimetry could be problematic if a large air pocket develops along the periphery of the cavity. The dose distribution is spherical or elliptical depending on the balloon chosen. Balloon-skin spacing should be at least 7 mm. The American Society of Breast Surgeons showed that skin spacing in addition to the use of chemotherapy and breast wound infection were the most important factors of cosmesis at 36 months in their MammoSite® Breast Brachytherapy Registry Trial [24].

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229

**Figure 3.** Hologic Mammosite balloon used for APBI treatments.

**Figure 2.** Tube placement for interstitial brachytherapy.

In 2002, the FDA approved Proxima Therapeutics MammoSite\* balloon catheter for intracavitary high dose rate breast brachytherapy (Fig 3). Seventy patients were initially enrolled in a prospective multi-center trial evaluating the safety of the MammoSite® balloon catheter. Subsequent evaluation of 43 patients eligible for the therapy revealed only mild to moderate self-limited side effects [30], Most recently, the American Society of Breast Surgeons reported results from their registry trial involving 1,440 women treated with the MammoSite® catheter following breast-conserving surgery. The 3-year actuarial rates of ipsilateral breast cancer and axillary recurrences were 2.15% and 0.36%, respectively. Cosmetic outcomes were reported to be acceptable and similar to patients treated with other forms of partial breast irradiation [33]. The advantages of the balloon catheter are that it is easier to place in the cavity, placement is more reproducible, and patient comfort is improved. It has become the most widely used device and has the longest track record [43]. The single catheter needs to be temporarily placed in the lumpectomy cavity, as opposed to 10-20 catheters with traditional interstitial implants. However, the balloon needs to "conform" properly to the tumor cavity and optimal dosimetry could be problematic if a large air pocket develops along the periphery of the cavity. The dose distribution is spherical or elliptical depending on the balloon chosen. Balloon-skin spacing should be at least 7 mm. The American Society of Breast Surgeons showed that skin spacing in addition to the use of chemotherapy and breast wound infection were the most important factors of cosmesis at 36 months in their MammoSite® Breast Brachytherapy Registry Trial [24].

Interstitial breast brachytherapy alone has been successfully used for over 10 years following breast conserving surgery (Fig 2). A trial was started by Vinci et al. in 1993 using brachytherapy as the only radiation treatment modality for patients following breast-conserving surgery [48]. By 2001, 120 patients were enrolled in this trial. Four patients developed local recurrence at a median follow-up of 82 months. During 1997-2000, 100 patients were enrolled in a Radiation Therapy Oncology Group (RTOG), prospective Phase I/II study of breast brachytherapy. Patients were either high-dose or low-dose-rate brachytherapy. For the high-dose-rate group at a median follow-up of 6.14 years; 5-year estimates of ipsilateral breast, regional, and contralateral breast failures were 3%, 5%, and 2% respectively. For patients receiving low-doserate brachytherapy at a median follow-up of 6.22 years; 5-year estimates of ipsilateral breast, regional, and contra-lateral breast failures were 6%, 0%, and 6%, respectively. Both groups experienced good cosmesis and local control [3]. Several institutions have shown low recur‐

In 2002, the FDA approved Proxima Therapeutics MammoSite\* balloon catheter for intracavitary high dose rate breast brachytherapy (Fig 3). Seventy patients were initially enrolled in a prospective multi-center trial evaluating the safety of the MammoSite® balloon catheter.

rences with brachytherapy at 5 and 10 years [2,5]

228 Cancer Treatment - Conventional and Innovative Approaches

**Figure 2.** Tube placement for interstitial brachytherapy.

**Figure 3.** Hologic Mammosite balloon used for APBI treatments.

Other applicator devices have come onto the market recently, with the advantages of having the potential for improved dosimetry in select patients when compared to the MammoSite applicator. The Contura™ Multi-Lumen Balloon catheter, depicted in Figure 4, allows multiple offset lumens to provide dose shaping opportunities to reduce skin and rib doses [13]. This product may have the advantages of using a balloon type applicator, in which many surgeons and radiation oncologists are familiar and comfortable. Additionally, air and blood around the cavity could be removed with the Contura™ catheter before treatment, potentially reducing air pockets and seroma formation. However, dosimetry is still limited to the confines of a "balloon catheter."

Another recent addition to the brachytherapy options is the SAVI device (Fig 5), a single-entry multicatheter applicator which allows a radiation oncologist to selectively direct radiation through up to eleven catheter channels, allowing more tailored manipulation of the isodose lines. The device is a bundle of expandable catheters around a central lumen. This applicator tries to blend in the advantages of interstitial brachytherapy with a single-entry device. Dose feathering could be done along the skin and chest. Studies have shown the device to give good tumor bed conformance with minimal normal tissue exposure [39]. Patient positioning as well asmaintaining a consistentinter-fractionposition is important.Apotentialdisadvantage is that removalofthedevicemaybemoredifficultwhencomparedtothesmallerballoontypecatheters.

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Three-dimension (3D) conformal radiation technology has been developed in recent years. This technique of APBI has the advantage of being noninvasive, eliminating an additional procedure, allowing many medical groups that do not perform brachytherapy to offer partial breast radiation therapy. No adverse side effects were seen in 28 patients treated with 3D conformal radiation in a 1999 pilot study [49]. A potential disadvantage is that the breast is not a stationary target and there is the potential for a geographical miss with external radiation

**Figure 5.** Cianna Medical Savi applicators.

therapy to a small target.

**Figure 4.** Bard Medical Systems Contoura Multi-lumen balloon.

The ClearPath™ multicatheter device is one of the newest brachytherapy devices available. The catheter is placed through a single entry point but without the constraints of having a single radiation source. The use of a multicatheter hybrid can reduce doses to the skin and normal tissues in the breast when compared to a single catheter system [18,19,8]. Both highdose-rate as well as low-dose continuous release brachytherapy can be delivered. Therefore, facilities without high-rate-rate equipment can now offer brachytherapy. Additionally, patients can get continuous release treatments at home without having to make twice-daily trips to the treatment facility. Strands of I-125 seeds are inserted in the outer catheters. Patients must wear a fully shielded bra if low-dose continuous release treatment is given.

Another recent addition to the brachytherapy options is the SAVI device (Fig 5), a single-entry multicatheter applicator which allows a radiation oncologist to selectively direct radiation through up to eleven catheter channels, allowing more tailored manipulation of the isodose lines. The device is a bundle of expandable catheters around a central lumen. This applicator tries to blend in the advantages of interstitial brachytherapy with a single-entry device. Dose feathering could be done along the skin and chest. Studies have shown the device to give good tumor bed conformance with minimal normal tissue exposure [39]. Patient positioning as well asmaintaining a consistentinter-fractionposition is important.Apotentialdisadvantage is that removalofthedevicemaybemoredifficultwhencomparedtothesmallerballoontypecatheters.

Other applicator devices have come onto the market recently, with the advantages of having the potential for improved dosimetry in select patients when compared to the MammoSite applicator. The Contura™ Multi-Lumen Balloon catheter, depicted in Figure 4, allows multiple offset lumens to provide dose shaping opportunities to reduce skin and rib doses [13]. This product may have the advantages of using a balloon type applicator, in which many surgeons and radiation oncologists are familiar and comfortable. Additionally, air and blood around the cavity could be removed with the Contura™ catheter before treatment, potentially reducing air pockets and seroma formation. However, dosimetry is still limited to the confines

The ClearPath™ multicatheter device is one of the newest brachytherapy devices available. The catheter is placed through a single entry point but without the constraints of having a single radiation source. The use of a multicatheter hybrid can reduce doses to the skin and normal tissues in the breast when compared to a single catheter system [18,19,8]. Both highdose-rate as well as low-dose continuous release brachytherapy can be delivered. Therefore, facilities without high-rate-rate equipment can now offer brachytherapy. Additionally, patients can get continuous release treatments at home without having to make twice-daily trips to the treatment facility. Strands of I-125 seeds are inserted in the outer catheters. Patients

must wear a fully shielded bra if low-dose continuous release treatment is given.

of a "balloon catheter."

230 Cancer Treatment - Conventional and Innovative Approaches

**Figure 4.** Bard Medical Systems Contoura Multi-lumen balloon.

Three-dimension (3D) conformal radiation technology has been developed in recent years. This technique of APBI has the advantage of being noninvasive, eliminating an additional procedure, allowing many medical groups that do not perform brachytherapy to offer partial breast radiation therapy. No adverse side effects were seen in 28 patients treated with 3D conformal radiation in a 1999 pilot study [49]. A potential disadvantage is that the breast is not a stationary target and there is the potential for a geographical miss with external radiation therapy to a small target.

### **6. Introduction to electronic brachytherapy**

Alternative methods of balloon-based APBI have been explored. A modified form of balloonbased brachytherapy called Xoft Axxent Electronic Brachytherapy™ (EBX) received FDA clearance for the treatment of breast cancer in January, 2006. This device uses a unique miniaturized x-ray source (Fig 6) and a mobile controller, which generates kilovoltage (kV) xrays. This approach to APBI requires minimal shielding and thus has the potential to increase the number of settings in which radiation treatments can be offered. In addition, EBX is not limited by rigorous radiation source regulations associated with high dose rate afterloaders used for other methods of APBI, which utilize radioisotope sources. The early results of a clinical trial to evaluate the performance and safety of EBX in the outpatient treatment of earlystage breast cancer patients were presented at the American Society of Clinical Oncology (ASCO) Breast Cancer Symposium. Treatment with EBX was found to be feasible and associ‐ ated with minimal acute side effects [32].

external beam radiation therapy and as a primary treatment in APBI have shown favorable

A Review of Radiation Therapy's Role in Early-Stage Breast Cancer and an Introduction to Electronic Brachytherapy

http://dx.doi.org/10.5772/55621

233

The use of EBX in the operating room is still in its infant stages, but the acceptance of this technique is growing rapidly. The following outlines the steps of an intraoperative procedure:

**2.** Send tumor mass and excision margins for permanent section analysis. Identified sentinel

**3.** During surgery, the pathology department reviews the lymph node specimen and informs

**4.** Remove additional breast tissue posterior to the lumpectomy cavity down to the depth of

**5.** Place a pliable piece of lead over the chest wall to shield the ribs, lung, and heart, from

**6.** Insert a cavity evaluation device into the lumpectomy cavity through a small incision in

**7.** Evaluate the conformity of the cavity evaluation device to the surrounding breast tissue

**8.** Deflate and remove the cavity evaluation device. Based on the fill volume, choose appropriate balloon catheter kit and insert and secure with retention sutures.

**9.** Verify x-ray source calibration and adjust atlas plan treatment dwell times accordingly.

**10.** During machine calibration, perform an intraoperative ultrasound to evaluate that the

**11.** Prepare for treatment by placing a sterile drape over the operative field. Add a flexible lead equivalent shield on top of the drape to decrease transmission to the patient and

**12.** The radiation oncologist connects the x-ray source into the balloon. Provide the radiation

The treatment prescription is the delivery of 20 Gy to the balloon surface. The treatment is accomplished in 10-25 minutes based on the balloon size, fill volume, and the x-ray source calibration. The duration of the entire procedure including lumpectomy, sentinel lymph node biopsy, balloon catheter placement, radiation therapy, and closing the incisions is approxi‐

Long-term data regarding the safety and efficacy of IORT are not available. The TARGIT trial is a phase III prospective, randomized trial comparing single fraction IORT delivered via EBX to conventional whole breast external beam radiation therapy. Sixteen international institu‐

under direct visualization. Determine balloon fill volume to nearest 5cc.

the treating physicians that the node was uninvolved by cancer.

the superficial pectoralis fascia to accommodate a chest wall shield.

outcomes [45,46,29].

scatter radiation.

hospital staff.

treatment.

mately two hours.

**1.** Perform lumpectomy and sentinel node biopsy.

node sent for frozen section evaluation.

the lateral breast and inflate with saline.

Transfer dwell times to a control USB drive.

minimum balloon-to-skin distance is>.6 cm.

**Figure 6.** Xoft Accent miniature x-ray source.

Investigators have also explored delivering APBI in the operating room immediately after lumpectomy. Intraoperative radiation therapy (IORT) allows the patient to receive all her radiation in a single fraction before she awakens from surgery. Additional potential advan‐ tages include delivering the radiation before tumor cells have a chance to proliferate, per‐ forming the radiation under direct visualization at the time of surgery, and decreasing healthcare costs. Published results using IORT both as a tumor bed boost in conjunction with external beam radiation therapy and as a primary treatment in APBI have shown favorable outcomes [45,46,29].

The use of EBX in the operating room is still in its infant stages, but the acceptance of this technique is growing rapidly. The following outlines the steps of an intraoperative procedure:

**1.** Perform lumpectomy and sentinel node biopsy.

**6. Introduction to electronic brachytherapy**

232 Cancer Treatment - Conventional and Innovative Approaches

ated with minimal acute side effects [32].

**Figure 6.** Xoft Accent miniature x-ray source.

Alternative methods of balloon-based APBI have been explored. A modified form of balloonbased brachytherapy called Xoft Axxent Electronic Brachytherapy™ (EBX) received FDA clearance for the treatment of breast cancer in January, 2006. This device uses a unique miniaturized x-ray source (Fig 6) and a mobile controller, which generates kilovoltage (kV) xrays. This approach to APBI requires minimal shielding and thus has the potential to increase the number of settings in which radiation treatments can be offered. In addition, EBX is not limited by rigorous radiation source regulations associated with high dose rate afterloaders used for other methods of APBI, which utilize radioisotope sources. The early results of a clinical trial to evaluate the performance and safety of EBX in the outpatient treatment of earlystage breast cancer patients were presented at the American Society of Clinical Oncology (ASCO) Breast Cancer Symposium. Treatment with EBX was found to be feasible and associ‐

Investigators have also explored delivering APBI in the operating room immediately after lumpectomy. Intraoperative radiation therapy (IORT) allows the patient to receive all her radiation in a single fraction before she awakens from surgery. Additional potential advan‐ tages include delivering the radiation before tumor cells have a chance to proliferate, per‐ forming the radiation under direct visualization at the time of surgery, and decreasing healthcare costs. Published results using IORT both as a tumor bed boost in conjunction with


The treatment prescription is the delivery of 20 Gy to the balloon surface. The treatment is accomplished in 10-25 minutes based on the balloon size, fill volume, and the x-ray source calibration. The duration of the entire procedure including lumpectomy, sentinel lymph node biopsy, balloon catheter placement, radiation therapy, and closing the incisions is approxi‐ mately two hours.

Long-term data regarding the safety and efficacy of IORT are not available. The TARGIT trial is a phase III prospective, randomized trial comparing single fraction IORT delivered via EBX to conventional whole breast external beam radiation therapy. Sixteen international institu‐ tions are enrolling patients in the trial. Eligible patients include patients >35 years of age with T1-T3, N0 tumors eligible for breast conserving therapy. Patients with multi-focal or multicentric lesions, clinically positive lymph nodes, extensive intraductal component, or invasive lobular cancers are not eligible for enrollment [46].

[2] Antonucci, J, Wallace, M, Goldstein, N, et al. (2009). Differences in patterns of failure in patients treated with accelerated partial breast irradiation versus whole-breast ir‐ radiation: a matched-pair analysis with 10-year follow-up. Int J Radiat Oncol Biol

A Review of Radiation Therapy's Role in Early-Stage Breast Cancer and an Introduction to Electronic Brachytherapy

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235

[3] Arthur, D, Winter, K, Kuske, R, et al. (2008). A phase II trial of brachytherapy alone after lumpectomy for select breast cancer: tumor control and survival outcomes of

[4] Bamett, G. C, Wilkinson, J, Moody, A. M, et al. (2009). A randomized controlled trial of forward-planned radiotherapy (IMRT) for early breast cancer: baseline characteris‐

[5] Benitez, P, Keisch, M, Vicini, F, et al. (2007). Five year results; the initial clinical trial of MammosSite balloon brachytherapy for partial breast irradiation in early-stage

[6] Benson, J. R, Jotoi, I, Keisch, M, et al. (2009). Early breast cancer. Lancet , 373,

[7] Beral, V, Hermon, C, Reeves, G, et al. (1995). Sudden fall in breast cancer death rates

[8] Beriwal, S, Coon, D, Kim, H, et al. (2008). Multicatheter hybrid breast brachytherapy: a potential alternative for patients with inadequate skin distance. Brachytherapy ,

[9] Blichert-toft, M, Nielsen, M, During, M, et al. (2008). Long-term results of breast con‐ serving surgery vs. mastectomy for early stage breast cancer: 20-year follow-up of

[10] Borghero, Y. O, Salehpuor, M, Mcneese, M. D, et al. (2007). Multileaf field-in-field forward-planned intensity-modulated dose compensation for whole-breast irradia‐ tion is associated with reduced contralateral breast dose: a phantom model compari‐

[11] Brown, S, Mclaughlin, M, Pope, K, et al. (2009). Initial radiation experience evaluat‐ ing early tolerance and toxicities in patients undergoing accelerated partial breast ir‐ radiationusing the Contura Multi-Lumen Balloon breast brachytherapy catheter.

[12] Buchholz, T. A. (2009). Radiation therapy for early-stage breast cancer after breast-

[13] Buchholz, T. A, Tucker, S. L, Erwin, J, et al. (2001). Impact of systemic treatment on local control for patients with lymph node-negative breast cancer treated with breats-

the Danish randomized DBCG-82TM protocol. Acta Oncol , 47(4), 672-681.

tics and dosimetry results. Radiother Oncol Epub (ahead of publication)

RTOG 95-17. Int J Radiat Oncol Biol Phys , 72(2), 463-473.

breast cancer. Am J Surg , 194(4), 456-462.

son. Radiother Oncol , 82(3), 324-328.

conserving surgery. N Engl J Med , 360(1), 63-70.

conservation therapy. J ClinOncol , 19(8), 2240-2246.

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in England and Wales. Lancet , 356(8965), 1670-1674.

Phys , 74(2), 447-452.

1463-1479.

7(4), 301-304.

The results of the TARGIT trial will help determine whether IORT is an equivalent alternative to standard whole breast external beam radiation therapy. If IORT methods, including EBX, are established as a standard treatment option, this may allow increased access to breast conserving therapy, as well as, improved quality of life and decreased medical costs for patients with a diagnosis of early-stage breast cancer.

### **7. Conclusion**

Early detection and treatment of breast cancer has significantly improved in recent years. Diagnostic imaging advancements have led to finer and tighter target definition for radiation therapy planning. Treatment delivery systems have changed and continue to change with a movement to shorter and less aggressive therapy. Several treatment options are available for some patients after breast conserving surgery. A 5-7 week course of whole breast external beam therapy now competes with one week of partial breast radiotherapy or a single fraction intraoperative treatment highlighted in this report. As these techniques evolve, focus remains on control, recurrence, and normal tissue response. However, the focus on treatment options also now includes patient schedules, lifestyles as well as the economic impact of the therapy. The multiple risk factors associated with the disease and the variable presentation amongst patients calls out for the need of molecular profiling to assist the oncologist possibly with information on not just who to treat, but how.

### **Author details**

Brent Herron, Alex Herron\* , Kathryn Howell, Daniel Chin and Luann Roads

\*Address all correspondence to: medical.physics@hotmail.com

Radiation Oncology, PSL Medical Center, Denver, CO, USA

### **References**

[1] American Cancer Society ((2008). Cancer facts and figures. American Cancer Society, Atlanta.

[2] Antonucci, J, Wallace, M, Goldstein, N, et al. (2009). Differences in patterns of failure in patients treated with accelerated partial breast irradiation versus whole-breast ir‐ radiation: a matched-pair analysis with 10-year follow-up. Int J Radiat Oncol Biol Phys , 74(2), 447-452.

tions are enrolling patients in the trial. Eligible patients include patients >35 years of age with T1-T3, N0 tumors eligible for breast conserving therapy. Patients with multi-focal or multicentric lesions, clinically positive lymph nodes, extensive intraductal component, or invasive

The results of the TARGIT trial will help determine whether IORT is an equivalent alternative to standard whole breast external beam radiation therapy. If IORT methods, including EBX, are established as a standard treatment option, this may allow increased access to breast conserving therapy, as well as, improved quality of life and decreased medical costs for

Early detection and treatment of breast cancer has significantly improved in recent years. Diagnostic imaging advancements have led to finer and tighter target definition for radiation therapy planning. Treatment delivery systems have changed and continue to change with a movement to shorter and less aggressive therapy. Several treatment options are available for some patients after breast conserving surgery. A 5-7 week course of whole breast external beam therapy now competes with one week of partial breast radiotherapy or a single fraction intraoperative treatment highlighted in this report. As these techniques evolve, focus remains on control, recurrence, and normal tissue response. However, the focus on treatment options also now includes patient schedules, lifestyles as well as the economic impact of the therapy. The multiple risk factors associated with the disease and the variable presentation amongst patients calls out for the need of molecular profiling to assist the oncologist possibly with

, Kathryn Howell, Daniel Chin and Luann Roads

[1] American Cancer Society ((2008). Cancer facts and figures. American Cancer Society,

lobular cancers are not eligible for enrollment [46].

234 Cancer Treatment - Conventional and Innovative Approaches

patients with a diagnosis of early-stage breast cancer.

information on not just who to treat, but how.

\*Address all correspondence to: medical.physics@hotmail.com

Radiation Oncology, PSL Medical Center, Denver, CO, USA

**7. Conclusion**

**Author details**

**References**

Atlanta.

Brent Herron, Alex Herron\*


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[27] Holli, K, Saaristo, R, Isalo, J, et al. (2001). Lumpectomy with or without postopertive radiotherapy for breast cancer with favorable prognostic features: results of a

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[35] Peto, R, Boreham, J, Clarke, M, et al. (2000). UK and USA breast cancer deaths down

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**Chapter 11**

**Radiosurgery and Hypofractionated Stereotactic**

Dante Amelio, Marco Cianchetti, Barbara Rombi,

Sabina Vennarini, Francesco Dionisi,

http://dx.doi.org/10.5772/55692

**1. Introduction**

Maurizio Amichetti and Giuseppe Minniti

Additional information is available at the end of the chapter

desirable in order to avoid acute and late side effects.

**Skull Base**

**Irradiation with Photons or Protons for Tumours of the**

The fundamental goal of improving radiation therapy (RT) is to maximize dose to the tumour while limiting dose to normal tissues. Higher radiation dose to the tumour can result in better disease control, and possibly also in improving survival. Decreasing dose to normal tissues is

Recent technological advances in photon-RT have allowed an improvement in targeting accuracy, dose escalation, delivery of multiple large fractions (hypofractionated stereotactic radiation therapy - HSRT) or single fraction stereotactic ablative radiation therapy (radiosur‐ gery - SRS). Radiosurgery delivers a single large dose with very steep dose fall-off outside the lesion to very small volumes in order to be tumouricidal through DNA damage or ablative causing necrosis via a vascular endothelial damage [1]. Furthermore, it has been shown that molecular responses to radiation differ based on dose per fraction. More recently, HSRT in two to five sessions has been employed to deliver extremely hypofractionated regimens. Prelimi‐ nary data suggest that HSRT may represent an effective treatment associated with lower risk of radiation-related adverse effects in patients with perioptic or large benign tumours as compared with single fraction SRS, although potential benefits remain to be demonstrated.

Different techniques are used to deliver both HSRT and SRS, enabling patient immobilization, set-up uncertainty reduction, targeting accuracy, delivery of high doses, and heterogeneous dose distribution with a steep dose gradient. Despite these enhancements in delivery with

and reproduction in any medium, provided the original work is properly cited.

© 2013 Amelio et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.


## **Radiosurgery and Hypofractionated Stereotactic Irradiation with Photons or Protons for Tumours of the Skull Base**

Dante Amelio, Marco Cianchetti, Barbara Rombi, Sabina Vennarini, Francesco Dionisi, Maurizio Amichetti and Giuseppe Minniti

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55692

**1. Introduction**

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[45] Vaidya, J. S, Walton, L, & Dewar, J. (2006). Single dose targeted intraoperative radio‐ therapy (TARGIT) for breast cancer can be delivered as a second procedure under lo‐

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Radiat Oncol Biol Phys , 51(3), 671-678.

238 Cancer Treatment - Conventional and Innovative Approaches

cancer. Surg Clin North Am , 79(5), 1091-1115.

early breast cancer. N Engl J Med, 347(16), 1227-1232.

cal anaesthetic. World]Surg Oncol 4:2.

Breast , 12(6), 491-496.

Biol Phys , 66, 1335-8.

Monogr (11): 33-39.

Clin Oncol , 19(7), 1993-2001.

Radiat Oncol Biol Phys , 57(5), 1247-1253.

The fundamental goal of improving radiation therapy (RT) is to maximize dose to the tumour while limiting dose to normal tissues. Higher radiation dose to the tumour can result in better disease control, and possibly also in improving survival. Decreasing dose to normal tissues is desirable in order to avoid acute and late side effects.

Recent technological advances in photon-RT have allowed an improvement in targeting accuracy, dose escalation, delivery of multiple large fractions (hypofractionated stereotactic radiation therapy - HSRT) or single fraction stereotactic ablative radiation therapy (radiosur‐ gery - SRS). Radiosurgery delivers a single large dose with very steep dose fall-off outside the lesion to very small volumes in order to be tumouricidal through DNA damage or ablative causing necrosis via a vascular endothelial damage [1]. Furthermore, it has been shown that molecular responses to radiation differ based on dose per fraction. More recently, HSRT in two to five sessions has been employed to deliver extremely hypofractionated regimens. Prelimi‐ nary data suggest that HSRT may represent an effective treatment associated with lower risk of radiation-related adverse effects in patients with perioptic or large benign tumours as compared with single fraction SRS, although potential benefits remain to be demonstrated.

Different techniques are used to deliver both HSRT and SRS, enabling patient immobilization, set-up uncertainty reduction, targeting accuracy, delivery of high doses, and heterogeneous dose distribution with a steep dose gradient. Despite these enhancements in delivery with

© 2013 Amelio et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

better conformality indices, photons still have a relatively high exit dose (beyond the tumour target), which can produce significant normal-tissue exposure.

This section provides a brief overview on the radiobiological principles underlying radiation therapy and their applications to single or (hypo)fractionated radiotherapy. For a compre‐ hensive analysis of the topic, the reader is referred to specific textbooks and articles [4-6].

Radiosurgery and Hypofractionated Stereotactic Irradiation with Photons or Protons for Tumours of the Skull Base

http://dx.doi.org/10.5772/55692

241

The first principle points out that hypoxic cells are highly resistant to the radiation-related

Secondly, the dose-response relationship differs according to the type of tissues. Those containing mainly non-cycling cells (classified as "late reacting tissues" [8]), are more sensitive to large doses per fraction than tissues containing mainly cycling cells (classified as "early

Finally, experimental data show that cells have different radiation sensitivities in different

Taking into account such knowledge, the employment of a fractionated regimen allows hypoxic cells to reestablish their oxygenation state [6] so that they will be more sensitive to a second, and subsequent, dose fraction. Dose fractionation also spares late reacting (healthy) tissues more than early reacting (malignant tumor) tissues [5]. Finally, it allows part of the cells to leave the resistant phase while entering in a more sensitive phase. As an overall result, a

Conversely, SRS exploits a different pattern of dose distribution, rather than radiobiological differences between normal and tumour tissue, to achieve effective tumour destruction. Theoretically, the use of large single dose does not allow reoxygenation [4] and may be more damaging if the target is in close proximity of or embedded within late responding organs at risk [10]. Provided that SRS dose falloff is steep enough to spare surrounding structures, the delivery of high single dose should translate into a greater rate of local tumour control, while still offering a low rate of complications. Moreover, the delivery of a single large dose does not allow redistribution of cells into a more radiosensitive phase of the cell cycle. However, the argument for the use of SRS is the relevant radiobiological effect of single-session radiation

Protons and photons differ in terms of physical properties and interaction with matter, which ultimately translate into different dose distribution as well as biological effectiveness [2]. To date, such a difference has been quantified in a 1.1 relative biological effectiveness (RBE) of protons over photons [11]. Despite the fact that this generic RBE may not be true, its variations do not show sufficient degree to be clinically relevant [11]. As a consequence, all of the above stated radiobiological principles as well as the corresponding clinical applications keep their validity regardless the employed type of radiation so that there is no difference between

However, experimental data [11] have shown that proton RBE values increase over the last few millimeters of the range, ultimately leading to an increased linear energy transfer. The corresponding effect may be equivalent to the expansion of 2 mm or more of the distal penumbra [11], which may be clinically relevant for the surrounding healthy structures. From the radiobiological standpoint, this issue probably represents the most relevant difference

cell kill or cell division capability arrest, regardless of the mitotic phase [10].

killing [7] when radiations with low linear energy transfer are employed.

reacting tissues" [8]).

parts of the cell cycle [9].

more effective cell killing takes place.

photon- and proton-based stereotactic irradiation.

Protons are positively-charged elementary particles, with similar biological effectiveness to conventional photon radiation. They have a defined range exhibited by the Bragg peak resulting in an energy deposition with no exit dose beyond the target volume. Thanks to these fundamental physics characteristics, proton radiation therapy offers superior dose distribution and reduced low-dose integral irradiated volume [2] enabling more radiation dose to be delivered to the tumour while significantly lowering the dose to the surrounding normal tissues.

The skull base (SB) is a very complex anatomical region that includes portions of the anterior cranial fossa, clivus, petrous bone, middle cranial fossa, cavernous sinus and infratemporal fossa encompassing several critical neurovascular structures. Tumours of the SB are challeng‐ ing lesions because of their anatomical location and close proximity to several critical neuro‐ vascular structures. Surgical treatment is considered the first managing step and it has the aim to remove (completely or partially) the tumour. The deep location of SB tumour requires extensive experience in surgical procedures; in fact, surgical damage could severely affect vision, hearing, speech, swallowing, and could even be life-threatening.

New RT techniques allow targeting SB tumours when surgery is not feasible, macroscopic residual is left after surgical intervention or even as an alternative, definitive treatment. Most patients with lesions of the SB have a benign tumour and a long-life expectancy. They need to be treated with techniques allowing target irradiation with a conformal isodose configuration and a steep fall-off into other surrounding structures in order to provide long-term tumour control with a low morbidity profile.

Several machines have been developed or implemented to deliver stereotactic treatments and are currently in use: Gamma Knife, linear accelerators, Cyberknife, and dedicated proton equipments are the most used and have been compared in several plan-comparison studies. At the same time, in the treatment of SB tumours, many studies have shown the effectiveness of SRS and HSRT with photons and, though less frequently, with protons. Considering the continuous advancement of technology in delivering SRS and HSRT with photons and the increased use of protons, we deemed it useful to review this topic by evaluating differences with photons and possible advantages of the use of protons. An analysis of the fundamental principles and differences underlying photon- and proton-based SRS/HSRT as well as clinical outcomes in SB tumours is provided and discussed.

### **2. Radiobiological background**

Experimental and clinical data suggest that the radiobiological principles may differ when irradiation is delivered with different fractionation regimens (fractionated versus large single doses) [3] or when a different type of radiation, such as protons, is employed [4].

This section provides a brief overview on the radiobiological principles underlying radiation therapy and their applications to single or (hypo)fractionated radiotherapy. For a compre‐ hensive analysis of the topic, the reader is referred to specific textbooks and articles [4-6].

better conformality indices, photons still have a relatively high exit dose (beyond the tumour

Protons are positively-charged elementary particles, with similar biological effectiveness to conventional photon radiation. They have a defined range exhibited by the Bragg peak resulting in an energy deposition with no exit dose beyond the target volume. Thanks to these fundamental physics characteristics, proton radiation therapy offers superior dose distribution and reduced low-dose integral irradiated volume [2] enabling more radiation dose to be delivered to the tumour while significantly lowering the dose to the surrounding normal

The skull base (SB) is a very complex anatomical region that includes portions of the anterior cranial fossa, clivus, petrous bone, middle cranial fossa, cavernous sinus and infratemporal fossa encompassing several critical neurovascular structures. Tumours of the SB are challeng‐ ing lesions because of their anatomical location and close proximity to several critical neuro‐ vascular structures. Surgical treatment is considered the first managing step and it has the aim to remove (completely or partially) the tumour. The deep location of SB tumour requires extensive experience in surgical procedures; in fact, surgical damage could severely affect

New RT techniques allow targeting SB tumours when surgery is not feasible, macroscopic residual is left after surgical intervention or even as an alternative, definitive treatment. Most patients with lesions of the SB have a benign tumour and a long-life expectancy. They need to be treated with techniques allowing target irradiation with a conformal isodose configuration and a steep fall-off into other surrounding structures in order to provide long-term tumour

Several machines have been developed or implemented to deliver stereotactic treatments and are currently in use: Gamma Knife, linear accelerators, Cyberknife, and dedicated proton equipments are the most used and have been compared in several plan-comparison studies. At the same time, in the treatment of SB tumours, many studies have shown the effectiveness of SRS and HSRT with photons and, though less frequently, with protons. Considering the continuous advancement of technology in delivering SRS and HSRT with photons and the increased use of protons, we deemed it useful to review this topic by evaluating differences with photons and possible advantages of the use of protons. An analysis of the fundamental principles and differences underlying photon- and proton-based SRS/HSRT as well as clinical

Experimental and clinical data suggest that the radiobiological principles may differ when irradiation is delivered with different fractionation regimens (fractionated versus large single

doses) [3] or when a different type of radiation, such as protons, is employed [4].

target), which can produce significant normal-tissue exposure.

240 Cancer Treatment - Conventional and Innovative Approaches

vision, hearing, speech, swallowing, and could even be life-threatening.

control with a low morbidity profile.

outcomes in SB tumours is provided and discussed.

**2. Radiobiological background**

tissues.

The first principle points out that hypoxic cells are highly resistant to the radiation-related killing [7] when radiations with low linear energy transfer are employed.

Secondly, the dose-response relationship differs according to the type of tissues. Those containing mainly non-cycling cells (classified as "late reacting tissues" [8]), are more sensitive to large doses per fraction than tissues containing mainly cycling cells (classified as "early reacting tissues" [8]).

Finally, experimental data show that cells have different radiation sensitivities in different parts of the cell cycle [9].

Taking into account such knowledge, the employment of a fractionated regimen allows hypoxic cells to reestablish their oxygenation state [6] so that they will be more sensitive to a second, and subsequent, dose fraction. Dose fractionation also spares late reacting (healthy) tissues more than early reacting (malignant tumor) tissues [5]. Finally, it allows part of the cells to leave the resistant phase while entering in a more sensitive phase. As an overall result, a more effective cell killing takes place.

Conversely, SRS exploits a different pattern of dose distribution, rather than radiobiological differences between normal and tumour tissue, to achieve effective tumour destruction. Theoretically, the use of large single dose does not allow reoxygenation [4] and may be more damaging if the target is in close proximity of or embedded within late responding organs at risk [10]. Provided that SRS dose falloff is steep enough to spare surrounding structures, the delivery of high single dose should translate into a greater rate of local tumour control, while still offering a low rate of complications. Moreover, the delivery of a single large dose does not allow redistribution of cells into a more radiosensitive phase of the cell cycle. However, the argument for the use of SRS is the relevant radiobiological effect of single-session radiation cell kill or cell division capability arrest, regardless of the mitotic phase [10].

Protons and photons differ in terms of physical properties and interaction with matter, which ultimately translate into different dose distribution as well as biological effectiveness [2]. To date, such a difference has been quantified in a 1.1 relative biological effectiveness (RBE) of protons over photons [11]. Despite the fact that this generic RBE may not be true, its variations do not show sufficient degree to be clinically relevant [11]. As a consequence, all of the above stated radiobiological principles as well as the corresponding clinical applications keep their validity regardless the employed type of radiation so that there is no difference between photon- and proton-based stereotactic irradiation.

However, experimental data [11] have shown that proton RBE values increase over the last few millimeters of the range, ultimately leading to an increased linear energy transfer. The corresponding effect may be equivalent to the expansion of 2 mm or more of the distal penumbra [11], which may be clinically relevant for the surrounding healthy structures. From the radiobiological standpoint, this issue probably represents the most relevant difference between photon- and proton-based stereotactic radiotherapy. Therefore, it is wise to take into account the biological effects of this high-RBE component during the planning.

**4. Clinical results of photon and proton SRS/HSRT for skull base tumours**

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http://dx.doi.org/10.5772/55692

243

Data for this review were obtained searching MEDLINE databases for publications dated

The search terms were: "skull base" and "stereotactic radiosurgery". Further research was conducted by adding the definitions of different SB tumours ("meningioma", "schwannoma / acoustic neuroma", "pituitary adenoma", "chordoma", "chondrosarcoma", "craniopharyng‐ ioma", "olfactory neuroblastoma / esthesioneuroblastoma", "glomus jugulare / chemodecto‐

This search was limited to articles written in English. Editorials, case reports, letters of opinion, and congress abstracts were excluded, even if they added valuable information. In case of repeated publications by the same institution, only the most updated was used for the analysis. Papers were reviewed and prioritized according to content relevancy. Reference lists from these sources were searched for additional publications. A systematic review was beyond the aim of the paper; the following results are reported in the form of a narrative

Photon-based SRS and HSRT have been increasingly employed as primary or post-operative treatments with more than 10.000 patients reported in published studies over the last two decades. Less data are available on the treatment with proton-based SRS and HSRT even though several types of tumour in benign and malignant settings, and also non-tumoural lesions as arteriovenous malformations, have been treated since its early use showing that it

To date, no randomized or non-randomized study has compared photon SRS/HSRT with proton SRS/HSRT, and almost all the studies available in literature are retrospective. Clinical outcomes are presented in the following, according to histopathological classification.

**Tumor type Delivery technique Doses Five-year tumor control**

HSRT 14 – 25 Gy / two-five

HSRT 17 – 25 Gy / three-five

SRS 15 – 22 Gy (non functioning)

SRS 12 – 18 Gy > 92%

fractions

18 – 26 Gy (secreting)

93.5%

94%

fractions 98% (three-years)

**4.1. Search strategy and selection criteria**

between January 1980 and December 2011.

summary.

**4.2. Clinical outcomes**

Benign meningioma

Pituitary adenoma

as a viable option for larger volumes.

A brief summary is provided in Table 1.

ma", "proton") to the previously-searched keywords.

### **3. Dosimetrical features**

The stereotactic delivering modalities and techniques have been compared in several plan comparison studies [12-15]. The corresponding efficacy has been investigated also on the basis of the normal tissue complication probability (NTCP) and tumour control probability (TCP) models in the attempt to set the results also on a biological basis [14,16].

In general, the differences among photon-based techniques (GK, multi- non-coplanar arcs or shaped beams linac treatment) are negligible [12-15]. Conversely, the modality (photons or protons) can be what is more important. Target features such as size, shape and location within the brain can influence the choice for the best stereotactic modality. In fact, all modalities are equally good if the target is small and regular [14,15].

Based on the normal brain dose, the dosimetrical advantage of charged particles relative to photons is evident in all types of targets [12,14,15]. Such a difference is more relevant under the 60% dose level regardless of the target features [14]. Moreover, the larger the target volume the greater the difference [12,14,15], which peaks for regular shaped targets larger than 24-26 cc, even though it can be relevant even for smaller and irregular targets (about 6 cc) [14,15].

All of the above-stated considerations also apply with respect to the lesion's shape and location: charged particles perform better than photon techniques.

All of the above-mentioned quantitative differences have been confirmed when the analysis was approached on a biological basis, being the NTCP different according to the treatment modality, size, shape and location of the target [14,16]. Again, protons demonstrated the lowest NTCP for medium-large regular and irregular shaped lesions [14,16]. In this scenario, charged particles scored NTCP values 4-6% smaller than photon techniques.

In this context, it is noteworthy that radiation-induced tumours have been reported after photon SRS [17,18]. It is well-known that protons feature a low integral dose to healthy structures providing the potential to reduce this risk. However, the tissue volume that can benefit from this feature may be very small in SRS and the corresponding clinical gain may be difficult to detect.

In conclusion, in the attempt to customize the treatment according to the clinical scenario it is possible to state that small to medium regularly-shaped lesions can be effectively managed by all photon-based techniques although at the expense of some target dose inhomogeneity. The charged particle capability to simultaneously provide high-target conformity and dose homogeneity maximizes for regularly and irregularly-shaped, medium to large lesions.

Finally, it is noteworthy that despite such comparisons included several planning and treatment strategies, further improvements, such as intensity-modulated photon and proton RT, have been introduced. These certainly deserve further investigation.

### **4. Clinical results of photon and proton SRS/HSRT for skull base tumours**

### **4.1. Search strategy and selection criteria**

between photon- and proton-based stereotactic radiotherapy. Therefore, it is wise to take into

The stereotactic delivering modalities and techniques have been compared in several plan comparison studies [12-15]. The corresponding efficacy has been investigated also on the basis of the normal tissue complication probability (NTCP) and tumour control probability (TCP)

In general, the differences among photon-based techniques (GK, multi- non-coplanar arcs or shaped beams linac treatment) are negligible [12-15]. Conversely, the modality (photons or protons) can be what is more important. Target features such as size, shape and location within the brain can influence the choice for the best stereotactic modality. In fact, all modalities are

Based on the normal brain dose, the dosimetrical advantage of charged particles relative to photons is evident in all types of targets [12,14,15]. Such a difference is more relevant under the 60% dose level regardless of the target features [14]. Moreover, the larger the target volume the greater the difference [12,14,15], which peaks for regular shaped targets larger than 24-26 cc, even though it can be relevant even for smaller and irregular targets (about 6 cc) [14,15]. All of the above-stated considerations also apply with respect to the lesion's shape and location:

All of the above-mentioned quantitative differences have been confirmed when the analysis was approached on a biological basis, being the NTCP different according to the treatment modality, size, shape and location of the target [14,16]. Again, protons demonstrated the lowest NTCP for medium-large regular and irregular shaped lesions [14,16]. In this scenario, charged

In this context, it is noteworthy that radiation-induced tumours have been reported after photon SRS [17,18]. It is well-known that protons feature a low integral dose to healthy structures providing the potential to reduce this risk. However, the tissue volume that can benefit from this feature may be very small in SRS and the corresponding clinical gain may be

In conclusion, in the attempt to customize the treatment according to the clinical scenario it is possible to state that small to medium regularly-shaped lesions can be effectively managed by all photon-based techniques although at the expense of some target dose inhomogeneity. The charged particle capability to simultaneously provide high-target conformity and dose homogeneity maximizes for regularly and irregularly-shaped, medium to large lesions.

Finally, it is noteworthy that despite such comparisons included several planning and treatment strategies, further improvements, such as intensity-modulated photon and proton

account the biological effects of this high-RBE component during the planning.

models in the attempt to set the results also on a biological basis [14,16].

equally good if the target is small and regular [14,15].

charged particles perform better than photon techniques.

particles scored NTCP values 4-6% smaller than photon techniques.

RT, have been introduced. These certainly deserve further investigation.

**3. Dosimetrical features**

242 Cancer Treatment - Conventional and Innovative Approaches

difficult to detect.

Data for this review were obtained searching MEDLINE databases for publications dated between January 1980 and December 2011.

The search terms were: "skull base" and "stereotactic radiosurgery". Further research was conducted by adding the definitions of different SB tumours ("meningioma", "schwannoma / acoustic neuroma", "pituitary adenoma", "chordoma", "chondrosarcoma", "craniopharyng‐ ioma", "olfactory neuroblastoma / esthesioneuroblastoma", "glomus jugulare / chemodecto‐ ma", "proton") to the previously-searched keywords.

This search was limited to articles written in English. Editorials, case reports, letters of opinion, and congress abstracts were excluded, even if they added valuable information. In case of repeated publications by the same institution, only the most updated was used for the analysis. Papers were reviewed and prioritized according to content relevancy. Reference lists from these sources were searched for additional publications. A systematic review was beyond the aim of the paper; the following results are reported in the form of a narrative summary.

#### **4.2. Clinical outcomes**

Photon-based SRS and HSRT have been increasingly employed as primary or post-operative treatments with more than 10.000 patients reported in published studies over the last two decades. Less data are available on the treatment with proton-based SRS and HSRT even though several types of tumour in benign and malignant settings, and also non-tumoural lesions as arteriovenous malformations, have been treated since its early use showing that it as a viable option for larger volumes.

To date, no randomized or non-randomized study has compared photon SRS/HSRT with proton SRS/HSRT, and almost all the studies available in literature are retrospective. Clinical outcomes are presented in the following, according to histopathological classification.


A brief summary is provided in Table 1.


Protons have been used in this context usually with conventional fractionation and in associ‐ ation with photons for benign [28-32] or atypical lesions [33,34], but also with HSRT or single session SRS [35-37]. In reference [35], 23 patients were treated, 18 with three fractions HSRT, and five with HSRT in 16 or more fractions: the mean reference dose was 20.3 Cobalt Gray equivalent (CGyE). In the HSRT group, clinical control was 89% (16/18) and radiological local control was 88%. Two patients (11%) developed transient new cranial nerve neuropathy after radiosurgery, which gradually recovered. Two more patients (11%) developed late side effects. The results of 51 cases of benign meningioma treated, between 1996 and 2007, with proton SRS as primary treatment (n = 32) or for residual tumour following surgery (n = 8), or recurrent tumour following surgery (n = 10) were recently published [37]. The median dose delivered was 13 CGyE (range, 10 -15.5 CGyE) prescribed to the 90% isodose line. After a median followup of 32 months (range, 6-133 months), MRI revealed 33 meningiomas with stable, 13 with decreased, and five with increased size. The 3-year actuarial tumour control rate was 94%. Symptoms were improved in 47% (16/34) of patients. Potentially permanent adverse effects after SRS were recorded in 3/51 (5.9%) patients. The main limitation of these studies is that

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245

There are two general categories of pituitary tumours: non-secreting and secreting lesions. Functioning tumours cause an excess secretion of one or more pituitary hormones. Although pituitary adenomas are histologically benign, successful management of these tumours can be challenging. Treatment options include microresection, medical therapy, fractionated RT, and SRS. The role of RT in pituitary adenomas is well-established [38], particularly when medical and surgical options have been exhausted. Therapeutic goals when performing RT for pituitary tumours are: stopping the tumour growth by preventing problems from mass effect, and

All main published results on the long-term effectiveness of SRS in patients with nonfunctioning and secreting pituitary adenomas have been recently reviewed [39,40]. In 15 studies reporting 684 patients with non-functioning adenomas treated with SRS at doses of 15-22 Gy, the reported 5-year actuarial tumour control rate was 94%. A similar local control was observed in patients with secreting pituitary adenomas, although higher doses in the range of 18-26 Gy are usually employed with the aim to achieve normalization of hormone hypersecretion. SRS data for 1215 patients with acromegaly have been reported in 29 studies [39]. At a median follow-up of 50 months, the 5-year and 10-year biochemical remission rates were 44% (range, 15-60%) and 74% (range, 46-86%), respectively. Time to response ranged from 12 to 66 months. Results of SRS were reported for 280 patients with Cushing's disease in 12 studies [40]. At a corrected median follow-up of 45 months, 48% of patients had biochemical remission of disease, with a reported time to hormonal response ranging from 3 months to 3 years. SRS is rarely used in the treatment of prolactinomas since medical treatment with dopamine agonists can achieve tumour shrinkage and normalize prolactin (PRL) levels in more than 80% of patients. When employed in patients who fail surgery and medical therapy, at a median follow-up of 29 months normalization of elevated PRL levels has been observed in 33% of 353 patients included in 18 studies, with a reported time to hormonal response ranging

longer follow-up is needed to assess the durability of tumour control.

normalization of excessive hormone secretion.

**•** Pituitary adenoma

**Table 1.** Main clinical outcomes regarding the most frequent tumours of the skull base treated with stereotactic radiosurgery (SRS) and hypofractionated stereotactic radiotherapy (HSRT).

#### **•** Meningioma

Meningiomas represent approximately 25% of all intracranial tumours, the majority of which are benign (grade I according to the World Health Organization classification).

These lesions can be observed or treated with surgery or RT. Surgical resection is the preferred treatment for accessible tumours that can be safely removed. RT is used when surgery is not possible for the location of the lesion or when the patient is not a suitable surgical candidate. Other indications are the risk of progression after partial excision or the salvage after a relapse. Atypical or malignant meningioma are usually irradiated adjuvantly after complete surgical excision [19]. Recently-published multicenter series [20] and review [21] on benign lesions show a 5-year control rate ≥ 92%. Radiosurgical doses between 12 and 18 Gy have been used in the control of skull base meningiomas. A similar 5-year actuarial tumour control rate in the range of 90-95% has been observed with doses of 15-16 Gy or 12-14 Gy. Large meningiomas are associated with worse long-term local control [22,23].

Radiation-induced toxicity has been shown in up to 40% after SRS, being represented by either transient or permanent neurological complications; however, the reported rate of significant complications at doses of 12-15 Gy, as currently used in most centres, is relatively low. Reference [22] reported permanent neurological deficits of 6.3% for cavernous sinus menin‐ giomas treated with GK SRS. Reference [24] showed late transient or permanent complications in 4.5% of patients, and similar complication rates have been reported in the majority of published series [21]. Other complications, such as epilepsy, internal carotid occlusion, and hypopituitarism have been rarely reported (less than 1-2%).

Only few studies report on the use of HSRT for skull base meningiomas [25-27]. In a series of 157 patients treated with Cyberknife [27], 5-year control was 93.5%. Interestingly, local control in tumours bigger than 8 ml and/or situated close to critical structures and treated with two to five daily fractions was similar to that obtained in smaller meningiomas treated with single fraction SRS.

Protons have been used in this context usually with conventional fractionation and in associ‐ ation with photons for benign [28-32] or atypical lesions [33,34], but also with HSRT or single session SRS [35-37]. In reference [35], 23 patients were treated, 18 with three fractions HSRT, and five with HSRT in 16 or more fractions: the mean reference dose was 20.3 Cobalt Gray equivalent (CGyE). In the HSRT group, clinical control was 89% (16/18) and radiological local control was 88%. Two patients (11%) developed transient new cranial nerve neuropathy after radiosurgery, which gradually recovered. Two more patients (11%) developed late side effects. The results of 51 cases of benign meningioma treated, between 1996 and 2007, with proton SRS as primary treatment (n = 32) or for residual tumour following surgery (n = 8), or recurrent tumour following surgery (n = 10) were recently published [37]. The median dose delivered was 13 CGyE (range, 10 -15.5 CGyE) prescribed to the 90% isodose line. After a median followup of 32 months (range, 6-133 months), MRI revealed 33 meningiomas with stable, 13 with decreased, and five with increased size. The 3-year actuarial tumour control rate was 94%. Symptoms were improved in 47% (16/34) of patients. Potentially permanent adverse effects after SRS were recorded in 3/51 (5.9%) patients. The main limitation of these studies is that longer follow-up is needed to assess the durability of tumour control.

**•** Pituitary adenoma

**Tumor type Delivery technique Doses Five-year tumor control**

HSRT 30 – 36 Gy /six fractions

HSRT 20 – 43.6 Gy / two-five

**Table 1.** Main clinical outcomes regarding the most frequent tumours of the skull base treated with stereotactic

Meningiomas represent approximately 25% of all intracranial tumours, the majority of which

These lesions can be observed or treated with surgery or RT. Surgical resection is the preferred treatment for accessible tumours that can be safely removed. RT is used when surgery is not possible for the location of the lesion or when the patient is not a suitable surgical candidate. Other indications are the risk of progression after partial excision or the salvage after a relapse. Atypical or malignant meningioma are usually irradiated adjuvantly after complete surgical excision [19]. Recently-published multicenter series [20] and review [21] on benign lesions show a 5-year control rate ≥ 92%. Radiosurgical doses between 12 and 18 Gy have been used in the control of skull base meningiomas. A similar 5-year actuarial tumour control rate in the range of 90-95% has been observed with doses of 15-16 Gy or 12-14 Gy. Large meningiomas

Radiation-induced toxicity has been shown in up to 40% after SRS, being represented by either transient or permanent neurological complications; however, the reported rate of significant complications at doses of 12-15 Gy, as currently used in most centres, is relatively low. Reference [22] reported permanent neurological deficits of 6.3% for cavernous sinus menin‐ giomas treated with GK SRS. Reference [24] showed late transient or permanent complications in 4.5% of patients, and similar complication rates have been reported in the majority of published series [21]. Other complications, such as epilepsy, internal carotid occlusion, and

Only few studies report on the use of HSRT for skull base meningiomas [25-27]. In a series of 157 patients treated with Cyberknife [27], 5-year control was 93.5%. Interestingly, local control in tumours bigger than 8 ml and/or situated close to critical structures and treated with two to five daily fractions was similar to that obtained in smaller meningiomas treated with single

SRS 15 – 20 Gy

are benign (grade I according to the World Health Organization classification).

radiosurgery (SRS) and hypofractionated stereotactic radiotherapy (HSRT).

are associated with worse long-term local control [22,23].

hypopituitarism have been rarely reported (less than 1-2%).

SRS 12 – 13 Gy 92%-100%

SRS 3 – 25 Gy 36%-91.6%

fractions

HSRT NA NA

20 – 25 Gy /five fractions

94%-100%

years)

Chordoma: 32-72% Chondrosarcoma: 63-100%

Chordoma: 100% (two-

Acoustic neuroma

244 Cancer Treatment - Conventional and Innovative Approaches

Craniopharyngioma

Chordoma and chondrosarcoma

**•** Meningioma

fraction SRS.

There are two general categories of pituitary tumours: non-secreting and secreting lesions. Functioning tumours cause an excess secretion of one or more pituitary hormones. Although pituitary adenomas are histologically benign, successful management of these tumours can be challenging. Treatment options include microresection, medical therapy, fractionated RT, and SRS. The role of RT in pituitary adenomas is well-established [38], particularly when medical and surgical options have been exhausted. Therapeutic goals when performing RT for pituitary tumours are: stopping the tumour growth by preventing problems from mass effect, and normalization of excessive hormone secretion.

All main published results on the long-term effectiveness of SRS in patients with nonfunctioning and secreting pituitary adenomas have been recently reviewed [39,40]. In 15 studies reporting 684 patients with non-functioning adenomas treated with SRS at doses of 15-22 Gy, the reported 5-year actuarial tumour control rate was 94%. A similar local control was observed in patients with secreting pituitary adenomas, although higher doses in the range of 18-26 Gy are usually employed with the aim to achieve normalization of hormone hypersecretion. SRS data for 1215 patients with acromegaly have been reported in 29 studies [39]. At a median follow-up of 50 months, the 5-year and 10-year biochemical remission rates were 44% (range, 15-60%) and 74% (range, 46-86%), respectively. Time to response ranged from 12 to 66 months. Results of SRS were reported for 280 patients with Cushing's disease in 12 studies [40]. At a corrected median follow-up of 45 months, 48% of patients had biochemical remission of disease, with a reported time to hormonal response ranging from 3 months to 3 years. SRS is rarely used in the treatment of prolactinomas since medical treatment with dopamine agonists can achieve tumour shrinkage and normalize prolactin (PRL) levels in more than 80% of patients. When employed in patients who fail surgery and medical therapy, at a median follow-up of 29 months normalization of elevated PRL levels has been observed in 33% of 353 patients included in 18 studies, with a reported time to hormonal response ranging from 5 to 40 months [40]. The reported overall rate of serious complications after SRS is low. The main complication is hypopituitarism, which is reported in up to 47% of patients, with higher rates in those series with a longer median follow-up.

ance at presentation [52]. Neurological toxicity, including facial and trigeminal neuropathies, and balance disturbances may occur in 0-3% of patients. Hydrocephalus has been observed in 1-2% of patients, whereas radiation-induced tumors or malignant transformation of acoustic

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There are only few reports regarding the use of HSRT in patients with vestibular schwannomas [53,54]. The reference [53] employed a regimen of 30 or 36 Gy in six fractions. At a median follow-up of 4.5 years, absolute tumor control, as well as hearing, facial and trigeminal

In reference [54], 20 or 25 Gy were delivered in five fractions. Five-year tumor control, trigeminal, facial, and hearing preservation were 94%, 98%, 97%, and 61%, respectively. It is noteworthy that in the latter reference HSRT provided equivalent tumor control, facial and hearing preservation with respect to SRS. Conversely, trigeminal preservation was signifi‐

Also in this site, proton beam has been used with conventional fractionation [55] or with SRS with a satisfactory level of hearing, facial nerve, trigeminal nerve preservation, and with tumor-control rates of 84-100% [56-58]. Reference [56] reported on 88 patients with vestibular schwannomas treated at the Massachusetts General Hospital between 1992 and 2000 with proton SRS. At a median follow-up period of 38.7 months (range, 12-102), the actuarial 2- and 5-year tumor control was 95.3% and 93.6%, respectively. Hearing was preserved in 33% of 21 (24% of the total) patients with functional hearing before treatment. Actuarial 5-year normal facial and trigeminal nerve function preservation rates were 91% and 89%, respectively. Three

Figure 1 shows a representative case of vestibular schwannoma treated with proton SRS.

Courtesy of Francis H. Burr Proton Therapy Center – Massachusetts General Hospital, Boston (USA).

**Figure 1.** Dose distribution in axial (A), sagittal (B), and coronal (C) views of a right vestibular schwannoma treated by proton radiosurgery. Tumor volume (in red) was 1 cubic centimeter. A dose of 12 Cobalt Gray Equivalent was prescri‐ bed to 90% isodose. Three equally weighted passive scattering beams were employed. The dose-volume histogram graph (D) shows the doses to organs at risk (right cochlea in blue and brainstem in green) and tumor volume (in red).

neuroma have been reported rarely [49].

cantly improved in patients treated with HSRT.

patients (3.4%) underwent shunting for hydrocephalus.

preservation were 100%.

HSRT has been employed in patients with tumours involving the optic apparatus and patients who are not considered suitable for SRS. Initial experiences with Cyberknife in treating patients with pituitary adenomas are promising [25,41,42]. Reference [25] reported high rates of tumour control and preservation of visual function in a small group of patients with pituitary adeno‐ mas within two mm of the optic apparatus treated to doses of 18-24 Gy delivered in two to five sessions. Although hypofractionated treatment schedules may offer a reduced risk of radiation-related adverse effects as compared to single fraction SRS, its efficacy needs to be evaluated in large prospective studies.

Data on proton treatment in pituitary adenomas are available both with the option of conven‐ tional fractionation [43] or with SRS [44-47]. In a small series of 22 patients treated with proton SRS for persistent acromegaly at a median follow-up of 6.3 years, the biochemical remission of disease was observed in 13 patients (59%) [44]. Time to response was 42 (range, 6-62) months. In a retrospective series of 33 patients with Cushing's disease at a median follow-up of 62 months, normalization of plasma and urinary free cortisol was achieved in 17 (52%) patients, with a time to remission of 18 (range, 5-49) months [45]. In both series, the only reported toxicity was represented by new pituitary deficits, which occurred in up to 52% of patients, whereas no visual complications, seizures, or secondary tumours were noted. The small number of cases treated and limited follow-up precludes drawing firm conclusions, even though it supports the hypothesis that proton SRS may offer better dosimetric coverage of the pituitary gland than photon-based treatments.

**•** Acoustic neuroma/vestibular schwannoma

Acoustic neuroma is a benign primary intracranial tumor of the vestibulocochlear nerve that can be treated with surgery or with several stereotactic irradiation techniques. Studies in the literaturearepoorlycomparablebecauseofthelackofuniform-reportingevaluationcriteria[48].

SRS as an effective treatment for acoustic neuroma has evolved over the last decades, leading to an improvement of local control and reduction of long-term toxicity. At doses of 12-13 Gy, as used in most recent studies, SRS results in an actuarial 5-year tumour control between 92 and 100% with a low incidence of radiation-induced complications [49]. The reported local control is similar to that reported with higher doses in the range of 15-18 Gy as used in early experiences of SRS, however with a lower incidence of radiation-induced complications. A recent review of more than 2000 patients included in 23 studies has shown an overall facial nerve preservation rate of 96% after GK, with a significant better facial nerve preservation rate in patients receiving ≤ 13 Gy of radiation at the marginal dose and with a tumor volume ≤ 1.5 cm3 [50]. Using similar doses, an overall hearing preservation, as defined by the maintenance of Gardner-Robertson Grade I or II after SRS, has been reported in 51% (range, 32-71%) of 4234 patients included in 45 publications [51]. Equivalent tumor control and hearing preservation rates have been reported for larger acoustic neuromas compressing the brainstem, with a reported balance improvement or stabilization in more than 85% of patients who had imbal‐ ance at presentation [52]. Neurological toxicity, including facial and trigeminal neuropathies, and balance disturbances may occur in 0-3% of patients. Hydrocephalus has been observed in 1-2% of patients, whereas radiation-induced tumors or malignant transformation of acoustic neuroma have been reported rarely [49].

from 5 to 40 months [40]. The reported overall rate of serious complications after SRS is low. The main complication is hypopituitarism, which is reported in up to 47% of patients, with

HSRT has been employed in patients with tumours involving the optic apparatus and patients who are not considered suitable for SRS. Initial experiences with Cyberknife in treating patients with pituitary adenomas are promising [25,41,42]. Reference [25] reported high rates of tumour control and preservation of visual function in a small group of patients with pituitary adeno‐ mas within two mm of the optic apparatus treated to doses of 18-24 Gy delivered in two to five sessions. Although hypofractionated treatment schedules may offer a reduced risk of radiation-related adverse effects as compared to single fraction SRS, its efficacy needs to be

Data on proton treatment in pituitary adenomas are available both with the option of conven‐ tional fractionation [43] or with SRS [44-47]. In a small series of 22 patients treated with proton SRS for persistent acromegaly at a median follow-up of 6.3 years, the biochemical remission of disease was observed in 13 patients (59%) [44]. Time to response was 42 (range, 6-62) months. In a retrospective series of 33 patients with Cushing's disease at a median follow-up of 62 months, normalization of plasma and urinary free cortisol was achieved in 17 (52%) patients, with a time to remission of 18 (range, 5-49) months [45]. In both series, the only reported toxicity was represented by new pituitary deficits, which occurred in up to 52% of patients, whereas no visual complications, seizures, or secondary tumours were noted. The small number of cases treated and limited follow-up precludes drawing firm conclusions, even though it supports the hypothesis that proton SRS may offer better dosimetric coverage of the pituitary

Acoustic neuroma is a benign primary intracranial tumor of the vestibulocochlear nerve that can be treated with surgery or with several stereotactic irradiation techniques. Studies in the literaturearepoorlycomparablebecauseofthelackofuniform-reportingevaluationcriteria[48].

SRS as an effective treatment for acoustic neuroma has evolved over the last decades, leading to an improvement of local control and reduction of long-term toxicity. At doses of 12-13 Gy, as used in most recent studies, SRS results in an actuarial 5-year tumour control between 92 and 100% with a low incidence of radiation-induced complications [49]. The reported local control is similar to that reported with higher doses in the range of 15-18 Gy as used in early experiences of SRS, however with a lower incidence of radiation-induced complications. A recent review of more than 2000 patients included in 23 studies has shown an overall facial nerve preservation rate of 96% after GK, with a significant better facial nerve preservation rate in patients receiving ≤ 13 Gy of radiation at the marginal dose and with a tumor volume ≤ 1.5

 [50]. Using similar doses, an overall hearing preservation, as defined by the maintenance of Gardner-Robertson Grade I or II after SRS, has been reported in 51% (range, 32-71%) of 4234 patients included in 45 publications [51]. Equivalent tumor control and hearing preservation rates have been reported for larger acoustic neuromas compressing the brainstem, with a reported balance improvement or stabilization in more than 85% of patients who had imbal‐

higher rates in those series with a longer median follow-up.

evaluated in large prospective studies.

246 Cancer Treatment - Conventional and Innovative Approaches

gland than photon-based treatments.

cm3

**•** Acoustic neuroma/vestibular schwannoma

There are only few reports regarding the use of HSRT in patients with vestibular schwannomas [53,54]. The reference [53] employed a regimen of 30 or 36 Gy in six fractions. At a median follow-up of 4.5 years, absolute tumor control, as well as hearing, facial and trigeminal preservation were 100%.

In reference [54], 20 or 25 Gy were delivered in five fractions. Five-year tumor control, trigeminal, facial, and hearing preservation were 94%, 98%, 97%, and 61%, respectively. It is noteworthy that in the latter reference HSRT provided equivalent tumor control, facial and hearing preservation with respect to SRS. Conversely, trigeminal preservation was signifi‐ cantly improved in patients treated with HSRT.

Also in this site, proton beam has been used with conventional fractionation [55] or with SRS with a satisfactory level of hearing, facial nerve, trigeminal nerve preservation, and with tumor-control rates of 84-100% [56-58]. Reference [56] reported on 88 patients with vestibular schwannomas treated at the Massachusetts General Hospital between 1992 and 2000 with proton SRS. At a median follow-up period of 38.7 months (range, 12-102), the actuarial 2- and 5-year tumor control was 95.3% and 93.6%, respectively. Hearing was preserved in 33% of 21 (24% of the total) patients with functional hearing before treatment. Actuarial 5-year normal facial and trigeminal nerve function preservation rates were 91% and 89%, respectively. Three patients (3.4%) underwent shunting for hydrocephalus.

Figure 1 shows a representative case of vestibular schwannoma treated with proton SRS.

Courtesy of Francis H. Burr Proton Therapy Center – Massachusetts General Hospital, Boston (USA).

**Figure 1.** Dose distribution in axial (A), sagittal (B), and coronal (C) views of a right vestibular schwannoma treated by proton radiosurgery. Tumor volume (in red) was 1 cubic centimeter. A dose of 12 Cobalt Gray Equivalent was prescri‐ bed to 90% isodose. Three equally weighted passive scattering beams were employed. The dose-volume histogram graph (D) shows the doses to organs at risk (right cochlea in blue and brainstem in green) and tumor volume (in red).

Reference [58] reported on 51 patients treated with proton HSRT with a dose of 26 CGyE in three fractions. At a median follow-up of 72 months, the 5-year local control was 98%. Hearing, facial nerve, and trigeminal nerve preservation rates were 42%, 90.5% and 93% at 5 years, respectively.

Even though such data need to be confirmed at a longer follow-up, it seems that SRS and HSRT could represent viable treatment options for small sized chordomas and chondrosarcomas

Radiosurgery and Hypofractionated Stereotactic Irradiation with Photons or Protons for Tumours of the Skull Base

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249

Radiation has been found to be helpful in controlling glomus jugulare tumour growth by inducing fibrosis around the supplying vessels. A recent comprehensive review identified 109 studies for a total of 869 patients described outcomes for patients with glomus jugulare tumours [72]. Patients undergoing SRS had the lowest rates of recurrence and the most favourable rates of tumour control. In particular, those treated with subtotal resection plus SRS had a control rate of 71% at 96 months of follow-up. At a median follow-up of 71 months, patients undergoing SRS alone had a tumour control rate of 95%. A recent meta-analysis [73] found 19 eligible studies. SRS marginal dose ranged between 12 and 20.4 Gy. Ninety-seven percent of patients achieved tumour control, and 95% of patients achieved clinical control suggesting the useful utilization of SRS for the primary management of glomus jugulare tumours in particular for patients with preserved glossopharyngeal and vagus nerve function, after surgical recurrence, in the elderly, and in patients with serious pre-existing medical

So far, data dealing with the use of HSRT in patients with chemodectoma or tumors of glomus jugulare are very limited. In reference [75], part of the patient sample received a slight hypofractionated regimen: 2.67 Gy per fraction (median dose 45 Gy). Overall, the 10-year tumour control rate was 92%. In reference [76] 49 patients received a median dose of 45 Gy in 15 or 16 fractions. At both 5 and 10 years, 92% of cases were recurrence-free. More recently, 18 patients were treated with a median dose of 20 Gy in 3 fractions [77]. At a median follow-up

Olfactory neuroblastoma or esthesioneuroblastoma is a rare tumour of the frontal SB. It is characterized by high rates of tumour recurrence and mortality. A recent meta-analysis [78] demonstrated that the most effective management of these lesions is usually based on surgery followed by post-operative irradiation. For early stage tumours, where the risk of cervical nodes involvement is very low, the combination of SRS (15-34 Gy marginal dose) with

To date, there are no data dealing with the delivery of HSRT in patients with olfactory

In the only report concerning the use of protons in this field, 14 patients received irradiation as definitive treatment [81]. Total dose was 65 CGyE, with 2.5 CGyE per fraction. Actuarial 5-

No data are available at this moment concerning the use of proton SRS or HSRT.

residual after surgery or relapsing.

conditions [74].

of 22 months, local control was 100%.

neuroblastoma or esthesioneuroblastoma.

year local progression-free survival rate was 84%.

No data are available for the use of protons in this field.

endoscopic sinus surgery seems a promising treatment option [79,80].

**•** Olfactory neuroblastoma/Esthesioneuroblastoma

**•** Chemodectoma/glomus jugulare tumours

**•** Craniopharyngioma

Craniopharyngioma is a rare and mostly benign epithelial paediatric brain tumour of the sellar and suprasellar region. The treatment is based on a surgical approach with transcranial approaches or endoscopic endonasal surgery followed by RT, mainly in form of fractionated regimens with a local control of 80-90% at 5-10 years [59]. The proximity of craniopharyngio‐ mas to the optic pathways provides a major limitation to the use of SRS, although in selected series of relatively small residual tumours, a local control of 34-88% has been reported [60-62]. Tumor control was achieved with a median dose of 22-24 Gy (marginal dose 11-12 Gy), whereas the use of lower radiation results in an unsatisfactory tumor control [63]. The reported late toxicity after SRS ranges from 0 to 38%, mainly represented by visual and endocrinological deficits [59].

To date, there are no data dealing with the delivery of HSRT in patients with craniopharyng‐ ioma.

Protons have been recently used for the treatment of this tumour but only with conventional fractionated regimens [64-66], and to date no experience with proton SRS has been reported.

**•** Chordoma and chondrosarcoma

Chordomas and chondrosarcomas of the SB are rare bone tumours with locally aggressive behaviour. Safe, maximal resection is the mainstay of treatment, usually followed by adjuvant RT. Protons are used with conventional fractionation schemes utilizing doses of 70 Gy in chondrosarcoma and, 74-78 in chordoma with valuable results [67,68]. The improvements in surgical techniques allow more radical tumour resection, while frequently providing small residual lesions suitable also for SRS or HSRT. Few clinical data was published on this issue, showing, however, promising preliminary results. Both for chordomas and chondrosarcomas, SRS has been employed to treat small tumour volumes: with few exceptions, corresponding median or mean values were less than 20 cm3 . Median delivered dose was 15-20 Gy in most series, depending on the proximity with organs at risk. Such dose levels translated into actuarial local control rates of 32-72% at 5 years for chordomas [67] and 63-100% for chondro‐ sarcomas [68]. It is noteworthy that most series have mean or median follow-up of less than 5 years.

Concerning the delivery of HSRT, literature shows very limited data [69-71]. None of them reports outcomes regarding chondrosarcomas. Total dose was 20-43.6 Gy delivered in 2 to 5 fractions. When reported, tumour volumes were again limited in size (less than 20 cm3 ). Only one series reports specific outcomes for patients treated with HSRT: at a median follow-up of just 24 months, absolute local control was 100% [70].

Both for SRS and HSRT severe radiation-related side effects were rare [67].

Even though such data need to be confirmed at a longer follow-up, it seems that SRS and HSRT could represent viable treatment options for small sized chordomas and chondrosarcomas residual after surgery or relapsing.

No data are available at this moment concerning the use of proton SRS or HSRT.

**•** Chemodectoma/glomus jugulare tumours

Reference [58] reported on 51 patients treated with proton HSRT with a dose of 26 CGyE in three fractions. At a median follow-up of 72 months, the 5-year local control was 98%. Hearing, facial nerve, and trigeminal nerve preservation rates were 42%, 90.5% and 93% at 5 years,

Craniopharyngioma is a rare and mostly benign epithelial paediatric brain tumour of the sellar and suprasellar region. The treatment is based on a surgical approach with transcranial approaches or endoscopic endonasal surgery followed by RT, mainly in form of fractionated regimens with a local control of 80-90% at 5-10 years [59]. The proximity of craniopharyngio‐ mas to the optic pathways provides a major limitation to the use of SRS, although in selected series of relatively small residual tumours, a local control of 34-88% has been reported [60-62]. Tumor control was achieved with a median dose of 22-24 Gy (marginal dose 11-12 Gy), whereas the use of lower radiation results in an unsatisfactory tumor control [63]. The reported late toxicity after SRS ranges from 0 to 38%, mainly represented by visual and endocrinological

To date, there are no data dealing with the delivery of HSRT in patients with craniopharyng‐

Protons have been recently used for the treatment of this tumour but only with conventional fractionated regimens [64-66], and to date no experience with proton SRS has been reported.

Chordomas and chondrosarcomas of the SB are rare bone tumours with locally aggressive behaviour. Safe, maximal resection is the mainstay of treatment, usually followed by adjuvant RT. Protons are used with conventional fractionation schemes utilizing doses of 70 Gy in chondrosarcoma and, 74-78 in chordoma with valuable results [67,68]. The improvements in surgical techniques allow more radical tumour resection, while frequently providing small residual lesions suitable also for SRS or HSRT. Few clinical data was published on this issue, showing, however, promising preliminary results. Both for chordomas and chondrosarcomas, SRS has been employed to treat small tumour volumes: with few exceptions, corresponding

series, depending on the proximity with organs at risk. Such dose levels translated into actuarial local control rates of 32-72% at 5 years for chordomas [67] and 63-100% for chondro‐ sarcomas [68]. It is noteworthy that most series have mean or median follow-up of less than 5

Concerning the delivery of HSRT, literature shows very limited data [69-71]. None of them reports outcomes regarding chondrosarcomas. Total dose was 20-43.6 Gy delivered in 2 to 5 fractions. When reported, tumour volumes were again limited in size (less than 20 cm3

one series reports specific outcomes for patients treated with HSRT: at a median follow-up of

Both for SRS and HSRT severe radiation-related side effects were rare [67].

. Median delivered dose was 15-20 Gy in most

). Only

respectively.

deficits [59].

**•** Chordoma and chondrosarcoma

median or mean values were less than 20 cm3

just 24 months, absolute local control was 100% [70].

ioma.

years.

**•** Craniopharyngioma

248 Cancer Treatment - Conventional and Innovative Approaches

Radiation has been found to be helpful in controlling glomus jugulare tumour growth by inducing fibrosis around the supplying vessels. A recent comprehensive review identified 109 studies for a total of 869 patients described outcomes for patients with glomus jugulare tumours [72]. Patients undergoing SRS had the lowest rates of recurrence and the most favourable rates of tumour control. In particular, those treated with subtotal resection plus SRS had a control rate of 71% at 96 months of follow-up. At a median follow-up of 71 months, patients undergoing SRS alone had a tumour control rate of 95%. A recent meta-analysis [73] found 19 eligible studies. SRS marginal dose ranged between 12 and 20.4 Gy. Ninety-seven percent of patients achieved tumour control, and 95% of patients achieved clinical control suggesting the useful utilization of SRS for the primary management of glomus jugulare tumours in particular for patients with preserved glossopharyngeal and vagus nerve function, after surgical recurrence, in the elderly, and in patients with serious pre-existing medical conditions [74].

So far, data dealing with the use of HSRT in patients with chemodectoma or tumors of glomus jugulare are very limited. In reference [75], part of the patient sample received a slight hypofractionated regimen: 2.67 Gy per fraction (median dose 45 Gy). Overall, the 10-year tumour control rate was 92%. In reference [76] 49 patients received a median dose of 45 Gy in 15 or 16 fractions. At both 5 and 10 years, 92% of cases were recurrence-free. More recently, 18 patients were treated with a median dose of 20 Gy in 3 fractions [77]. At a median follow-up of 22 months, local control was 100%.

No data are available for the use of protons in this field.

**•** Olfactory neuroblastoma/Esthesioneuroblastoma

Olfactory neuroblastoma or esthesioneuroblastoma is a rare tumour of the frontal SB. It is characterized by high rates of tumour recurrence and mortality. A recent meta-analysis [78] demonstrated that the most effective management of these lesions is usually based on surgery followed by post-operative irradiation. For early stage tumours, where the risk of cervical nodes involvement is very low, the combination of SRS (15-34 Gy marginal dose) with endoscopic sinus surgery seems a promising treatment option [79,80].

To date, there are no data dealing with the delivery of HSRT in patients with olfactory neuroblastoma or esthesioneuroblastoma.

In the only report concerning the use of protons in this field, 14 patients received irradiation as definitive treatment [81]. Total dose was 65 CGyE, with 2.5 CGyE per fraction. Actuarial 5 year local progression-free survival rate was 84%.

### **5. Conclusions**

Stereotactic irradiation is highly effective in the management of SB benign tumours and longterm data clearly indicate a tumour control in more than 90% of cases after 5 and 10 years, with an acceptable incidence of complications. In most series, radiosurgical dose has been delivered using GK, although outcome is similar for patients with SB tumours treated with Linac SRS.

The authors have no conflicts of interest. All authors have contributed to the review.

[1] Balagamwala EH. Principles of Radiobiology of Stereotactic Radiosurgery and Clini‐ cal Applications in the Central Nervous System. Technol Cancer Res Treat 2012;11(1)

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http://dx.doi.org/10.5772/55692

251

[2] Suit H. Proton beams to replace photon beams in radical dose treatments. Acta Oncol

[3] Kirkpatrick JP. The linear-quadratic model is inappropriate to model high dose per

[4] Breuer H, Smit BJ. Proton therapy and radiosurgery. Heidelberg: Springer-Verlag;

[5] Chin LS, Regine WF. Principles and practice of stereotactic radiosurgery. New York:

[6] Hall EJ. The radiobiology of radiosurgery: rationale for different treatment regimens for AVMs and malignancies. Int J Radiat Oncol Biol Phys 1993;25(2) 381-385.

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Even though protons are increasingly used in the clinical community, only few studies have been performed to assess the efficacy and toxicity of proton SRS and HSRT in skull base tumors. The number of Institutions that are currently using protons is small, particularly those performing proton-based stereotactic techniques. Proton beam, while utilizing a different type of radiation, also represents a similar highly focused and targeted radiation tool. The physical properties of protons offer superior conformality in dose distribution with respect to photons. This advantage becomes more apparent as the lesion volume increases. However, current results do not clearly indicate that proton SRS/HSRT is superior to photon SRS/HSRT. Clinical results are probably confounded by a bias toward reserving proton beams for the treatment of larger and more complex lesions; but conclusions about the presumed superiority of protons in comparison to other photons-based techniques are difficult to draw.

With respect to the small number of treated patients and short follow-up, toxicity was similar with the use of the different techniques; however, the evaluation of complications is often completely subjective and unsatisfactory. Proton SRS may represent a treatment alternative to photon SRS especially for larger and/or irregularly shaped tumors close to sensitive structures. The difference between techniques may be small and large numbers of patients followed for long periods would be required to demonstrate any clinically significant advantage. The more widespread use of protons could allow comparative multi-institutional trials to select the appropriate modality for each tumour type.

### **Acknowledgements**

We thank Valentina Piffer (ATreP, Trento – Italy) for her language editing of the manuscript.

### **Author details**

Dante Amelio1 , Marco Cianchetti1 , Barbara Rombi1 , Sabina Vennarini1 , Francesco Dionisi1 , Maurizio Amichetti1 and Giuseppe Minniti2

1 ATreP – Provincial Agency for Proton Therapy, Trento, Italy

2 Department of Radiation Oncology, University La Sapienza, S. Andrea Hospital, Rome, Italy

The authors have no conflicts of interest. All authors have contributed to the review.

### **References**

**5. Conclusions**

250 Cancer Treatment - Conventional and Innovative Approaches

Stereotactic irradiation is highly effective in the management of SB benign tumours and longterm data clearly indicate a tumour control in more than 90% of cases after 5 and 10 years, with an acceptable incidence of complications. In most series, radiosurgical dose has been delivered using GK, although outcome is similar for patients with SB tumours treated with Linac SRS.

Even though protons are increasingly used in the clinical community, only few studies have been performed to assess the efficacy and toxicity of proton SRS and HSRT in skull base tumors. The number of Institutions that are currently using protons is small, particularly those performing proton-based stereotactic techniques. Proton beam, while utilizing a different type of radiation, also represents a similar highly focused and targeted radiation tool. The physical properties of protons offer superior conformality in dose distribution with respect to photons. This advantage becomes more apparent as the lesion volume increases. However, current results do not clearly indicate that proton SRS/HSRT is superior to photon SRS/HSRT. Clinical results are probably confounded by a bias toward reserving proton beams for the treatment of larger and more complex lesions; but conclusions about the presumed superiority of protons

With respect to the small number of treated patients and short follow-up, toxicity was similar with the use of the different techniques; however, the evaluation of complications is often completely subjective and unsatisfactory. Proton SRS may represent a treatment alternative to photon SRS especially for larger and/or irregularly shaped tumors close to sensitive structures. The difference between techniques may be small and large numbers of patients followed for long periods would be required to demonstrate any clinically significant advantage. The more widespread use of protons could allow comparative multi-institutional trials to select the

We thank Valentina Piffer (ATreP, Trento – Italy) for her language editing of the manuscript.

, Barbara Rombi1

2 Department of Radiation Oncology, University La Sapienza, S. Andrea Hospital, Rome,

, Sabina Vennarini1

, Francesco Dionisi1

,

in comparison to other photons-based techniques are difficult to draw.

appropriate modality for each tumour type.

, Marco Cianchetti1

and Giuseppe Minniti2

1 ATreP – Provincial Agency for Proton Therapy, Trento, Italy

**Acknowledgements**

**Author details**

Maurizio Amichetti1

Dante Amelio1

Italy


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199 patients. Neurosurgery 2009;64(Suppl 2) A7-13.

Strahlenther Onkol 2002;178(9) 480-485.

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865-871.

diosurgery in a series of 4565 patients. Neurosurgery 2012;70(1) 32-39.

and control probabilities. Int J Radiat Oncol Biol Phys 1998;40(2):507-513.

road? Neurosurgery 2003;52(6) 1436-40; discussion 1440-1442.

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252 Cancer Treatment - Conventional and Innovative Approaches

at Oncol 2009;4 42.

2004;60(5) 1515–1519.

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ment. J Neurooncol 2010;99(3) 433-443.


[47] Aghi MK. Management of Recurrent and Refractory Cushing's Disease with Reoper‐ ation and/or Proton Beam Radiosurgery. Clin Neurosurg 2008;55 141-144.

[63] Ulfarsson E. Gamma knife radiosurgery for craniopharyngiomas: long-term results

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[64] Beltran C. On the Benefits and Risks of Proton Therapy in Pediatric Craniopharyng‐

[65] Luu QT. Fractionated proton radiation treatment for pediatric craniopharyngioma:

[66] Fitzek MM. Combined proton and photon irradiation for craniopharyngioma: longterm results of the early cohort of patients treated at Harvard Cyclotron Laboratory and Massachusetts General Hospital. Int J Radiat Oncol Biol Phys 2006;64(5)

[67] Amichetti M. Proton therapy in chordoma of the base of the skull: a systematic re‐

[68] Amichetti M. A systematic review of proton therapy in the treatment of chondrosar‐

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[70] Gwak HS. Hypofractionated stereotactic radiation therapy for skull base and upper cervical chordoma and chondrosarcoma: preliminary results. Stereotact Funct Neu‐

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[72] Ivan ME. A meta-analysis of tumor control rates and treatment-related morbidity for patients with glomus jugulare tumors. J Neurosurg 2011;114(5) 1299-1305.

[73] Guss ZD. Radiosurgery of glomus jugulare tumors: a meta-analysis. Int J Radiat On‐

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[75] Krych AJ. Long-term results of irradiation for paraganglioma. Int J Radiat Oncol Biol

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[48] Bassim MK. Radiation therapy for the treatment of vestibular schwannoma: a critical

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[50] Yang I. A comprehensive analysis of hearing preservation after radiosurgery for ves‐

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[54] Meijer OW. Single-fraction vs. fractionated LINAC-based stereotactic radiosurgery for vestibular schwannoma: a single institution study. Int J Radiat Oncol Biol Phys

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[79] Walch C. The minimally invasive approach to olfactory neuroblastoma: combined endoscopic and stereotactic treatment. Laryngoscope 2000;110(4) 635-640.

**Chapter 12**

**Hyperthermia: Cancer Treatment and Beyond**

The three mainstays for cancer treatment include surgical removal of tumors, radiation therapy and chemotherapy, which have led to improved patient survival for certain types of cancer, but there is still much room for improvement. Cancer is one of the leading causes of death worldwide and accounted for 7.6 million deaths (13% of all deaths) in 2008 (World Health Organization, 2012). The 2012 Report to the Nation on the Status of Cancer indicated that there was a decrease in overall cancer mortality and incidence in the U.S.A. from 1999 to 2008, particularly for the four major cancer sites: lung, colorectum, breast and prostate [1]. However, there were increases in the incidence of other types of cancer, including those of the pancreas, kidney, thyroid and liver, as well as melanoma and adenocarcinoma of the esophagus, from

Over the past decades, the struggle against cancer has led to the discovery of new strategies to fight this disease and to bring hope to patients. These new strategies include hyperthermia (also commonly known as thermal therapy or thermotherapy), biological therapies (e.g. immunotherapy), photodynamic therapy, laser treatment, gene therapy, and inhibitors of angiogenesis. Most of these strategies still need optimization, and in some cases (e.g. hyper‐ thermia, photodynamic therapy), improved equipment is required. Moreover, a better understanding of the biological mechanisms involved in their anticancer action would certainly be beneficial. Hyperthermia is one of the few strategies to be adopted as a promising

Hyperthermia is defined as moderate elevation in temperature. Hyperthermia can either have a pathological origin, resulting from the fever response of the organism to viral or bacterial infections, or may occur during exposure to high temperatures as during heat stroke. It is relatively recent as a clinical procedure, in which body tissues are exposed to elevated

and reproduction in any medium, provided the original work is properly cited.

© 2013 Bettaieb et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Ahmed Bettaieb, Paulina K. Wrzal and

Additional information is available at the end of the chapter

therapy among the alternative methods to treat cancer.

Diana A. Averill-Bates

http://dx.doi.org/10.5772/55795

**1. Introduction**

1999 to 2008.


### **Hyperthermia: Cancer Treatment and Beyond**

Ahmed Bettaieb, Paulina K. Wrzal and Diana A. Averill-Bates

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55795

### **1. Introduction**

[79] Walch C. The minimally invasive approach to olfactory neuroblastoma: combined endoscopic and stereotactic treatment. Laryngoscope 2000;110(4) 635-640.

[80] Unger F. Combined endoscopic surgery and radiosurgery as treatment modality for olfactory neuroblastoma (esthesioneuroblastoma). Acta Neurochir (Wien) 2005;147(6)

[81] Nishimura H. Proton-beam therapy for olfactory neuroblastoma. Int J Radiat Oncol

595-601.

Biol Phys 2007;68(3) 758-762.

256 Cancer Treatment - Conventional and Innovative Approaches

The three mainstays for cancer treatment include surgical removal of tumors, radiation therapy and chemotherapy, which have led to improved patient survival for certain types of cancer, but there is still much room for improvement. Cancer is one of the leading causes of death worldwide and accounted for 7.6 million deaths (13% of all deaths) in 2008 (World Health Organization, 2012). The 2012 Report to the Nation on the Status of Cancer indicated that there was a decrease in overall cancer mortality and incidence in the U.S.A. from 1999 to 2008, particularly for the four major cancer sites: lung, colorectum, breast and prostate [1]. However, there were increases in the incidence of other types of cancer, including those of the pancreas, kidney, thyroid and liver, as well as melanoma and adenocarcinoma of the esophagus, from 1999 to 2008.

Over the past decades, the struggle against cancer has led to the discovery of new strategies to fight this disease and to bring hope to patients. These new strategies include hyperthermia (also commonly known as thermal therapy or thermotherapy), biological therapies (e.g. immunotherapy), photodynamic therapy, laser treatment, gene therapy, and inhibitors of angiogenesis. Most of these strategies still need optimization, and in some cases (e.g. hyper‐ thermia, photodynamic therapy), improved equipment is required. Moreover, a better understanding of the biological mechanisms involved in their anticancer action would certainly be beneficial. Hyperthermia is one of the few strategies to be adopted as a promising therapy among the alternative methods to treat cancer.

Hyperthermia is defined as moderate elevation in temperature. Hyperthermia can either have a pathological origin, resulting from the fever response of the organism to viral or bacterial infections, or may occur during exposure to high temperatures as during heat stroke. It is relatively recent as a clinical procedure, in which body tissues are exposed to elevated

© 2013 Bettaieb et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

temperatures in the range of 39°C to 45°C. These high temperatures can damage and kill cancer cells with minimal injury to normal tissues [2]. During the last two decades, hyperthermia has been used as an efficient complement to standard cancer treatments such as radiation therapy and chemotherapy [2,3] (Figure 1). A further advantage is that hyperthermia can eliminate drug-resistant and radio-resistant tumour cells. Another form of hyperthermia involves very high temperatures (> 60°C), which can destroy or «cook» tumours by a technique known as thermal ablation (see review, [4]). The present review will address the therapeutic potential of moderate hyperthermia (39°C to 45°C).

vations during several decades, these cancer treatments were difficult to administer in a controlled manner, and responses were unpredictable [10]. Using a different approach, Westermark reported the use of localized, non-fever producing heat treatments (42-44°C) by means of water-circulating cisterns that resulted in the long-term remission of inoperable cancer of the cervix [11]. As different techniques were developed, such as surgery, radiation therapy and chemotherapy, further development of hyperthermia for cancer treatment was put on the back burner. There was a resurgence of interest in the use of hyperthermia in cancer treatment based on scientific studies initiated in the 1960s and 1970s. A turning point was a study conducted in transplanted mouse tumors that illustrated novel biological phenomena: cytotoxicity of hyperthermia was dependent on time and temperature; increased sensitivity of large versus small tumors to hyperthermia (later attributed to vascular events); heat-induced thermotolerance of normal and tumor tissue; and hyperthermia-induced sensitization to radiation [12]. These promising observations led to quantitative experimental studies and a rapid increase in our understanding of the biological effects of hyperthermia. Furthermore, they frame the rationale for the clinical use of hyperthermia, and the development of more effective technologies for the precise application of heat to tumors and for the measurement

Temperatures in the range of moderate hyperthermia can be non-lethal (39 to 42°C) or lethal (>42°C). Temperatures above 42°C were shown to kill cancer cells in a time- and temperaturedependent manner that was measured by the clonogenic cell survival assay [13]. However, despite numerous studies during at least three decades, which have improved our under‐ standing of hyperthermia biology, the mechanisms involved in heat-induced cytotoxicity are still ill-defined [14]. Hyperthermia causes many changes in cells and leads to a loss of cellular homeostasis [15-17]. A key event appears to be protein denaturation and aggregation, which results in cell cycle arrest, inactivation of protein synthesis, and inhibition of DNA repair processes [18]. Other cellular effects of hyperthermia include: (1) the inhibition of DNA synthesis, transcription, RNA processing and translation; (2) increased degradation of aggregated/misfolded proteins through the proteasomal and lysosomal pathways; (3) disrup‐ tion of the membrane cytoskeleton; (4) metabolic changes (e.g. uncoupling of oxidative phosphorylation) that lead to decreased levels of ATP; and (5) alterations in membrane

Hyperthermia can cause changes in lipids but these appear to be reversible [21]. The viscosity of the plasma membrane decreases with increasing temperature [22], and this may be associ‐ ated with altered transport functions of the membrane. Changes in membrane viscosity were linked to an elevation in the activity of the ATP-dependent sodium-potassium pump [22],

gradient. During hyperthermia, membrane permeability towards several compounds is

Despite the large number of documented cellular changes, the nature of the critical lesions that lead to cell death following heat treatment remains unknown. Proteins appear to be the first

levels across the plasma membrane against a concentration

, H+ and Ca2+ (see reviews, [19,20].

Hyperthermia: Cancer Treatment and Beyond

http://dx.doi.org/10.5772/55795

259

of heat distribution in tumors by thermometry.

permeability that cause increases in intracellular levels of Na+

altered, including polyamines, glucose, and anticancer drugs [23-25].

and K+

**2.2. Cellular changes**

which maintains Na+

**Figure 1.** Hyperthermia complements standard cancer treatments such as chemotherapy and radiation therapy in de‐ stroying tumour cells.

### **2. Hyperthermia**

### **2.1. Scientific history**

The use of heat to treat disease, including cancer, is a concept that dates back to early Egyptian times, over 5000 years ago (see review, [5]). Indeed, the Egyptian medical papyrus recounts an attempt to treat breast cancer with a "heated stick" [6]. Likewise, many Greek doctors, among them Hippocrates, suggested cauterizing superficial tumours by using heated metal. Many ancient cultures, including the Roman, Chinese, Indian and Japanese cultures have used this concept for the treatment of a variety of diseases. During the late 1800s, there were numerous observations by astute clinicians of spontaneous remissions of cancer in patients suffering from a variety of infections [7]. Dr. William B. Coley found 47 case reports in which simultaneous infection seemed to have caused the remission of an incurable neoplastic malignancy (see review, [8]). In the late 1800s, he used "Coley's Mixed Toxins" (bacterial pyrogenic toxins) as a deliberate fever-inducing treatment to control tumor growth [9]. Despite promising obser‐ vations during several decades, these cancer treatments were difficult to administer in a controlled manner, and responses were unpredictable [10]. Using a different approach, Westermark reported the use of localized, non-fever producing heat treatments (42-44°C) by means of water-circulating cisterns that resulted in the long-term remission of inoperable cancer of the cervix [11]. As different techniques were developed, such as surgery, radiation therapy and chemotherapy, further development of hyperthermia for cancer treatment was put on the back burner. There was a resurgence of interest in the use of hyperthermia in cancer treatment based on scientific studies initiated in the 1960s and 1970s. A turning point was a study conducted in transplanted mouse tumors that illustrated novel biological phenomena: cytotoxicity of hyperthermia was dependent on time and temperature; increased sensitivity of large versus small tumors to hyperthermia (later attributed to vascular events); heat-induced thermotolerance of normal and tumor tissue; and hyperthermia-induced sensitization to radiation [12]. These promising observations led to quantitative experimental studies and a rapid increase in our understanding of the biological effects of hyperthermia. Furthermore, they frame the rationale for the clinical use of hyperthermia, and the development of more effective technologies for the precise application of heat to tumors and for the measurement of heat distribution in tumors by thermometry.

#### **2.2. Cellular changes**

temperatures in the range of 39°C to 45°C. These high temperatures can damage and kill cancer cells with minimal injury to normal tissues [2]. During the last two decades, hyperthermia has been used as an efficient complement to standard cancer treatments such as radiation therapy and chemotherapy [2,3] (Figure 1). A further advantage is that hyperthermia can eliminate drug-resistant and radio-resistant tumour cells. Another form of hyperthermia involves very high temperatures (> 60°C), which can destroy or «cook» tumours by a technique known as thermal ablation (see review, [4]). The present review will address the therapeutic potential of

**Hyperthermia**

Drug resistant cells: Radiosensitization • Primary resistance

**Figure 1.** Hyperthermia complements standard cancer treatments such as chemotherapy and radiation therapy in de‐

The use of heat to treat disease, including cancer, is a concept that dates back to early Egyptian times, over 5000 years ago (see review, [5]). Indeed, the Egyptian medical papyrus recounts an attempt to treat breast cancer with a "heated stick" [6]. Likewise, many Greek doctors, among them Hippocrates, suggested cauterizing superficial tumours by using heated metal. Many ancient cultures, including the Roman, Chinese, Indian and Japanese cultures have used this concept for the treatment of a variety of diseases. During the late 1800s, there were numerous observations by astute clinicians of spontaneous remissions of cancer in patients suffering from a variety of infections [7]. Dr. William B. Coley found 47 case reports in which simultaneous infection seemed to have caused the remission of an incurable neoplastic malignancy (see review, [8]). In the late 1800s, he used "Coley's Mixed Toxins" (bacterial pyrogenic toxins) as a deliberate fever-inducing treatment to control tumor growth [9]. Despite promising obser‐

**Radiation Therapy**

**Tumor cell death**

moderate hyperthermia (39°C to 45°C).

258 Cancer Treatment - Conventional and Innovative Approaches

• Aquired multidrug resistance

(Pgp, MRP‐1)

stroying tumour cells.

**2. Hyperthermia**

**2.1. Scientific history**

**Chemotherapy**

Temperatures in the range of moderate hyperthermia can be non-lethal (39 to 42°C) or lethal (>42°C). Temperatures above 42°C were shown to kill cancer cells in a time- and temperaturedependent manner that was measured by the clonogenic cell survival assay [13]. However, despite numerous studies during at least three decades, which have improved our under‐ standing of hyperthermia biology, the mechanisms involved in heat-induced cytotoxicity are still ill-defined [14]. Hyperthermia causes many changes in cells and leads to a loss of cellular homeostasis [15-17]. A key event appears to be protein denaturation and aggregation, which results in cell cycle arrest, inactivation of protein synthesis, and inhibition of DNA repair processes [18]. Other cellular effects of hyperthermia include: (1) the inhibition of DNA synthesis, transcription, RNA processing and translation; (2) increased degradation of aggregated/misfolded proteins through the proteasomal and lysosomal pathways; (3) disrup‐ tion of the membrane cytoskeleton; (4) metabolic changes (e.g. uncoupling of oxidative phosphorylation) that lead to decreased levels of ATP; and (5) alterations in membrane permeability that cause increases in intracellular levels of Na+ , H+ and Ca2+ (see reviews, [19,20].

Hyperthermia can cause changes in lipids but these appear to be reversible [21]. The viscosity of the plasma membrane decreases with increasing temperature [22], and this may be associ‐ ated with altered transport functions of the membrane. Changes in membrane viscosity were linked to an elevation in the activity of the ATP-dependent sodium-potassium pump [22], which maintains Na+ and K+ levels across the plasma membrane against a concentration gradient. During hyperthermia, membrane permeability towards several compounds is altered, including polyamines, glucose, and anticancer drugs [23-25].

Despite the large number of documented cellular changes, the nature of the critical lesions that lead to cell death following heat treatment remains unknown. Proteins appear to be the first target of hyperthermia in the clinically-relevant temperature range of 39 to 45°C (Figure 2). The alteration of cellular homeostasis after exposure to hyperthermia entails a certain number of post-translational modifications such as glycosylation, acylation, phosphorylation, farne‐ lysation and ubiquitination [18,26]. Several studies reported that hyperthermia can cause DNA fragmentation and the formation of double strand breaks (DBSs) [27,28], which could arise from the inhibition of DNA repair mechanisms [21]. However, it appears that nuclear protein damage may be the key factor rather than direct DNA damage itself. Nuclear proteins, in particular, appear to be very sensitive to hyperthermia and undergo aggregation [21]. Nuclear protein aggregation has been linked to the inhibition of transcription and DNA replication.

phosphate pathway [35], which maintains the intracellular antioxidant glutathione in its reduced form, GSH [36]. An increase in the generation of ROS can cause oxidative damage to proteins, lipids and nucleic acids. A hyperthermia-induced decrease in tumor growth was accompanied by an increase in lipid peroxidation in rabbits [37]. Another consequence of increased ROS generation by hyperthermia is that molecules such as H2O2 can perturb mitochondrial membrane potential [38]. A temperature-induced increase in cell metabolism

Hyperthermia: Cancer Treatment and Beyond

http://dx.doi.org/10.5772/55795

261

As a consequence of different cellular changes, hyperthermia causes mitotic catastrophe, permanent G1 arrest and a loss of clonogenic or reproductive cell capacity [21] (Figure 2). Cells can die by processes such as apoptosis and/or necrosis, which are dependent on the cell type as well as the temperature and duration of heat exposure [32,41]. Another consequence is that cells can become sensitized to other cytotoxic modalities such as radiation [16]. Hyperthermia was reported to cause centrosomal dysfunction and mitotic catastrophe [42], which have been implicated in thermal radio-sensitization [43]. Hyperthermia (42 to 44°C) has been reported to cause chromatin condensation and apoptotic DNA fragmentation (formation of DNA ladders) leading to apoptosis in many different cell types including HeLa cells [44], T lymphocytes [45,46], HL-60 leukemic cells [47], and mice embryonic fibroblasts [48]. In rats treated with whole body hyperthermia (41.5°C for 2 h), both the extent and kinetics of hyperthermiainduced apoptosis differed between two different tumor types (fibrosarcoma and colon carcinoma) [49]. Additionally, the same study revealed another important advantage; the induction of apoptosis was higher in tumor tissues in comparison to normal tissues. Most of the studies that have investigated the mechanisms of heat shock-induced cytotoxicity con‐ cluded that apoptosis is the main form of cell death and proposed the pro-apoptotic effects of

hyperthermia as the potential desired outcome of hyperthermia in cancer therapy.

Several physiological factors including oxygenation, pH and blood flow were shown to play a role in the sensitivity of cells/tissues to moderate hyperthermia. The intrinsic sensitivity to heat varies significantly among different cell types. Several studies indicate that cancer cells are more susceptible to heat injury than normal cells [21,50]. This could be caused, at least in part, by the differential expression of heat shock proteins (Hsps) and other proteins involved in the cellular defense system against different stressors, including heat shock. However, there is no consistency in findings about heat sensitivity between tumor and normal cells [21]. The sensitivity of cells to heat also varies with phase of the cell cycle, where cells in S phase and

Another reason for the use of hyperthermia in cancer treatment is the fact that tumor tissues are poorly vascularized in comparison to normal tissues. This may lead to a differential heating, with higher temperatures being achieved in tumors compared with normal tissue, where heat may be dissipated by circulating blood. Hyperthermia also appears to be complementary to other forms of treatment by being able to destroy tumor

could also cause acidosis of the tumor tissue [39,40].

**2.4. Hyperthermia and physiological changes**

mitosis were reported to be most sensitive [51].

**2.3. Cytotoxicity of hyperthermia**

**Figure 2.** Hyperthermia-induced cellular changes that could lead to tumour cell death.

Elevated temperatures can increase the rates of biochemical reactions and this would increase cell metabolism, which should cause increased oxidative stress (Figure 2). Levels of reactive oxygen species (ROS) were shown to increase after exposure to both lethal (≥42°C) [29-31] and non-lethal (40°C) temperatures [32,33]. This would arise principally from the increased generation of ROS such as superoxide and hydrogen peroxide (H2O2), likely as a result of dysfunction of the mitochondrial respiratory chain. Other potential sources of increased ROS generation would be increased activity of superoxide-producing enzymes such as NADPH oxidase and xanthine oxidase at elevated temperatures. Hyperthermia could also increase the reactivity of these ROS; indeed, the cytotoxicity of hydrogen peroxide was increased at elevated temperatures (41 to 43°C) compared to the physiological temperature (37°C) [34]. Hyperthermia also inactivated cellular antioxidant defenses against H2O2 such as the pentose phosphate pathway [35], which maintains the intracellular antioxidant glutathione in its reduced form, GSH [36]. An increase in the generation of ROS can cause oxidative damage to proteins, lipids and nucleic acids. A hyperthermia-induced decrease in tumor growth was accompanied by an increase in lipid peroxidation in rabbits [37]. Another consequence of increased ROS generation by hyperthermia is that molecules such as H2O2 can perturb mitochondrial membrane potential [38]. A temperature-induced increase in cell metabolism could also cause acidosis of the tumor tissue [39,40].

### **2.3. Cytotoxicity of hyperthermia**

target of hyperthermia in the clinically-relevant temperature range of 39 to 45°C (Figure 2). The alteration of cellular homeostasis after exposure to hyperthermia entails a certain number of post-translational modifications such as glycosylation, acylation, phosphorylation, farne‐ lysation and ubiquitination [18,26]. Several studies reported that hyperthermia can cause DNA fragmentation and the formation of double strand breaks (DBSs) [27,28], which could arise from the inhibition of DNA repair mechanisms [21]. However, it appears that nuclear protein damage may be the key factor rather than direct DNA damage itself. Nuclear proteins, in particular, appear to be very sensitive to hyperthermia and undergo aggregation [21]. Nuclear protein aggregation has been linked to the inhibition of transcription and DNA replication.

Hyperthermia

Increased metabolic rate

Increased generation of reactive oxygen species

**Membrane damage** • Altered transport functions • Altered cell signaling mechanisms • Altered receptor functions

**Protein damage**

**Tumor cell death**

Elevated temperatures can increase the rates of biochemical reactions and this would increase cell metabolism, which should cause increased oxidative stress (Figure 2). Levels of reactive oxygen species (ROS) were shown to increase after exposure to both lethal (≥42°C) [29-31] and non-lethal (40°C) temperatures [32,33]. This would arise principally from the increased generation of ROS such as superoxide and hydrogen peroxide (H2O2), likely as a result of dysfunction of the mitochondrial respiratory chain. Other potential sources of increased ROS generation would be increased activity of superoxide-producing enzymes such as NADPH oxidase and xanthine oxidase at elevated temperatures. Hyperthermia could also increase the reactivity of these ROS; indeed, the cytotoxicity of hydrogen peroxide was increased at elevated temperatures (41 to 43°C) compared to the physiological temperature (37°C) [34]. Hyperthermia also inactivated cellular antioxidant defenses against H2O2 such as the pentose

**Inhibition of cell proliferation** • G1 cell cycle arrest • Mitotic catastrophe

• Apoptosis (low heat dose) • Necrosis (high heat dose)

**Figure 2.** Hyperthermia-induced cellular changes that could lead to tumour cell death.

**Nuclear protein damage** • Inhibition of DNA repair mechanisms

260 Cancer Treatment - Conventional and Innovative Approaches

• Oxidation • Aggregation • Denaturation As a consequence of different cellular changes, hyperthermia causes mitotic catastrophe, permanent G1 arrest and a loss of clonogenic or reproductive cell capacity [21] (Figure 2). Cells can die by processes such as apoptosis and/or necrosis, which are dependent on the cell type as well as the temperature and duration of heat exposure [32,41]. Another consequence is that cells can become sensitized to other cytotoxic modalities such as radiation [16]. Hyperthermia was reported to cause centrosomal dysfunction and mitotic catastrophe [42], which have been implicated in thermal radio-sensitization [43]. Hyperthermia (42 to 44°C) has been reported to cause chromatin condensation and apoptotic DNA fragmentation (formation of DNA ladders) leading to apoptosis in many different cell types including HeLa cells [44], T lymphocytes [45,46], HL-60 leukemic cells [47], and mice embryonic fibroblasts [48]. In rats treated with whole body hyperthermia (41.5°C for 2 h), both the extent and kinetics of hyperthermiainduced apoptosis differed between two different tumor types (fibrosarcoma and colon carcinoma) [49]. Additionally, the same study revealed another important advantage; the induction of apoptosis was higher in tumor tissues in comparison to normal tissues. Most of the studies that have investigated the mechanisms of heat shock-induced cytotoxicity con‐ cluded that apoptosis is the main form of cell death and proposed the pro-apoptotic effects of hyperthermia as the potential desired outcome of hyperthermia in cancer therapy.

### **2.4. Hyperthermia and physiological changes**

Several physiological factors including oxygenation, pH and blood flow were shown to play a role in the sensitivity of cells/tissues to moderate hyperthermia. The intrinsic sensitivity to heat varies significantly among different cell types. Several studies indicate that cancer cells are more susceptible to heat injury than normal cells [21,50]. This could be caused, at least in part, by the differential expression of heat shock proteins (Hsps) and other proteins involved in the cellular defense system against different stressors, including heat shock. However, there is no consistency in findings about heat sensitivity between tumor and normal cells [21]. The sensitivity of cells to heat also varies with phase of the cell cycle, where cells in S phase and mitosis were reported to be most sensitive [51].

Another reason for the use of hyperthermia in cancer treatment is the fact that tumor tissues are poorly vascularized in comparison to normal tissues. This may lead to a differential heating, with higher temperatures being achieved in tumors compared with normal tissue, where heat may be dissipated by circulating blood. Hyperthermia also appears to be complementary to other forms of treatment by being able to destroy tumor

cells that are relatively resistant to radiation therapy or chemotherapy. Tumor cells located in the hypoxic centers of tumors are relatively resistant to chemotherapy due to poor drug delivery. Several chemotherapeutic drugs also require oxygen to generate free radicals in order to cause tumor cytotoxicity. Further, most chemotherapeutic drugs are more effective against proliferating cells. However, hypoxia has been shown to cause decreased proliferation, which may partially explain the reason for resistance of tumor cells to chemotherapy [52-54]. Cells located in hypoxic areas of tumors are also resist‐ ant to radiation therapy.

**2.5. Thermotolerance: The other side of hyperthermia**

necrosis.

or structure.

ethanol, toxins and bacterial infections.

The exposure of cells to lethal temperatures such as 43 to 45°C during short periods of time, ranging from 10 to 30 minutes, allows the development of tolerance towards subsequent exposure to multiple stresses; this phenomenon is termed "thermotolerance" [66,67]. Ther‐ motolerance is an adaptive survival response induced by heat preconditioning whereby cells become resistant to a subsequent lethal insult such as that triggered by heat shock, reactive oxygen species (ROS), and environmental stressors including heavy metals [68,69]. If the level of stress is very low, cells attempt to survive by activating stress responses that protect essential biochemical processes such as DNA repair, protein folding, and the elimination of damaged proteins [70]. Once the stress stimulus is removed, cells can recover their normal cellular function. If the stress continues or is too severe, then the cell will likely die by apoptosis or

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The acquisition of thermotolerance is characterized by numerous biochemical and molecular changes. Thermotolerance is generally associated with the accumulation of Hsps [19,63,68,71-73]. Hsp expression is regulated by a stress-responsive transcription factor known as heat shock factor 1 (HSF-1), through its interaction with the heat shock element (HSE) [74]. In addition, changes in the expression of about 50 to 200 other genes, not traditionally considered Hsps, have been found during or after heat stress (see review, [20]). These include genes for transcription factors, protein degradation, DNA repair enzymes, metabolic enzymes, cell cycle arrest, transport and detoxification, and signal transduction. The reason for the induction of these other cell-protective pathways by heat shock is probably to protect nascent chain synthesis and folding, prevent protein misfolding and aggregation, and to promote recovery from stress-induced damage [75]. Proteomic analyses showed a change in the phosphorylation of 93 proteins between control RIF-1 and their thermotolerant derivatives, TR-RIF-1 cells [76]. These phosphorylated proteins are responsible for a range of cellular functions, which include chaperones, ion channels, signal transduction, transcription and translation, biosynthesis of amino acids, oxidoreduction, energy metabolism, and cell motility

The heat shock response is highly conserved in all organisms from yeast to humans, which suggests that it is important for survival in a stressful environment [74]. In addition to heat, the heat shock response can be induced by other insults such as oxidative stress, heavy metals,

The major classes of Hsps induced by the heat shock response are Hsp90, Hsp70, Hsp60, and Hsp27. Hsps appear to play an important role in thermotolerance. Many studies suggest a correlation between the accumulation of Hsp70 and the acquisition of a thermotolerant state in mammals, amphibians, insects [77-79] and fish [80]. Under conditions of stress, Hsp70s can prevent the formation of protein aggregates and assist the refolding of aggregated proteins into their native structures [19]. Other studies have shown that the state of thermotolerance correlated with an increase in the expression of Hsp110 [81]. Hsp110 is as effective as Hsp70 in preventing protein aggregation, and contributes, along with Hsp70 and Hsp40, to the refolding of denatured proteins. In addition to their protective role against a subsequent lethal

Heating of human tumours is heterogeneous. Some areas of the tumour reach cytotoxic temperatures such as 43 to 45°C, whereas other areas only reach 39 to 42°C. It is more difficult to heat larger or deep-seated tumours to cytotoxic temperatures that are adequate to cause cell death or vascular damage.

Tumors are unable to adapt their blood circulation to the effects of high temperatures (≥42°C), which enables hyperthermia to cut off the supply of nutrients and oxygen, leading to lower interstitial pH and a collapse in tumor vasculature [55]. These conditions render cells more susceptible to heat treatment. Indeed, cells at lower (acidic) pH and decreased oxygen tension, as in the center of tumors, are more sensitive to heat treatment [56,57]. Cells in a nutrientdeprived environment are also more sensitive to elevated temperatures. This effect appears to correlate with changes in cellular ATP levels [58]. Cells that were deprived of glucose exhibited increased sensitivity to the cytotoxicity of hyperthermia [35]. This effect could be linked to a decrease in antioxidant defenses involving the glutathione redox cycle, since glucose metab‐ olism, through the pentose phosphate pathway, is required for maintaining intracellular levels of GSH. On the other hand, heating at milder temperatures (e.g. 39° to 42°C) can increase tumor blood flow, which leads to improved tumor oxygenation [59,60]. This could render tumors more sensitive to radiation and certain anticancer drugs.

Hyperthermia (≥42°C) has been shown to cause vascular damage in rodent tumours, which leads to decreased oxygenation and necrosis [61]. Although, the vasculature of human tumours appears to be more resistant to hyperthermia than that of rodent tissues, hyperthermia has been shown to cause disturbances in the microcirculation of cancer tissue in human osteosar‐ coma [62].

Milder temperatures in the range of 40 to 41°C appear to be able to stimulate various elements of the immune system, thus increasing immune surveillance and protecting against tumor growth (see reviews, [63-65]). The exposure of immune effector cells (e.g. macrophages, T cells, and natural killer (NK) cells) to mild temperatures has been shown to: (1) enhance the migration of immune cells to target sites, which could allow better control of infection and tumor burden; (2) increase the expression of cell surface molecules (e.g. involved in antigen presentation); (3) increase the release of soluble factors involved in immune effector cell activity (e.g. pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), IL-6, IL-10, and IL-12; (4) regulate immune cell proliferation; and (5) increase the cytotoxicity of immune cells against target (tumor) cells.

### **2.5. Thermotolerance: The other side of hyperthermia**

cells that are relatively resistant to radiation therapy or chemotherapy. Tumor cells located in the hypoxic centers of tumors are relatively resistant to chemotherapy due to poor drug delivery. Several chemotherapeutic drugs also require oxygen to generate free radicals in order to cause tumor cytotoxicity. Further, most chemotherapeutic drugs are more effective against proliferating cells. However, hypoxia has been shown to cause decreased proliferation, which may partially explain the reason for resistance of tumor cells to chemotherapy [52-54]. Cells located in hypoxic areas of tumors are also resist‐

Heating of human tumours is heterogeneous. Some areas of the tumour reach cytotoxic temperatures such as 43 to 45°C, whereas other areas only reach 39 to 42°C. It is more difficult to heat larger or deep-seated tumours to cytotoxic temperatures that are adequate to cause cell

Tumors are unable to adapt their blood circulation to the effects of high temperatures (≥42°C), which enables hyperthermia to cut off the supply of nutrients and oxygen, leading to lower interstitial pH and a collapse in tumor vasculature [55]. These conditions render cells more susceptible to heat treatment. Indeed, cells at lower (acidic) pH and decreased oxygen tension, as in the center of tumors, are more sensitive to heat treatment [56,57]. Cells in a nutrientdeprived environment are also more sensitive to elevated temperatures. This effect appears to correlate with changes in cellular ATP levels [58]. Cells that were deprived of glucose exhibited increased sensitivity to the cytotoxicity of hyperthermia [35]. This effect could be linked to a decrease in antioxidant defenses involving the glutathione redox cycle, since glucose metab‐ olism, through the pentose phosphate pathway, is required for maintaining intracellular levels of GSH. On the other hand, heating at milder temperatures (e.g. 39° to 42°C) can increase tumor blood flow, which leads to improved tumor oxygenation [59,60]. This could render tumors

Hyperthermia (≥42°C) has been shown to cause vascular damage in rodent tumours, which leads to decreased oxygenation and necrosis [61]. Although, the vasculature of human tumours appears to be more resistant to hyperthermia than that of rodent tissues, hyperthermia has been shown to cause disturbances in the microcirculation of cancer tissue in human osteosar‐

Milder temperatures in the range of 40 to 41°C appear to be able to stimulate various elements of the immune system, thus increasing immune surveillance and protecting against tumor growth (see reviews, [63-65]). The exposure of immune effector cells (e.g. macrophages, T cells, and natural killer (NK) cells) to mild temperatures has been shown to: (1) enhance the migration of immune cells to target sites, which could allow better control of infection and tumor burden; (2) increase the expression of cell surface molecules (e.g. involved in antigen presentation); (3) increase the release of soluble factors involved in immune effector cell activity (e.g. pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), IL-6, IL-10, and IL-12; (4) regulate immune cell proliferation; and (5)

ant to radiation therapy.

262 Cancer Treatment - Conventional and Innovative Approaches

death or vascular damage.

coma [62].

more sensitive to radiation and certain anticancer drugs.

increase the cytotoxicity of immune cells against target (tumor) cells.

The exposure of cells to lethal temperatures such as 43 to 45°C during short periods of time, ranging from 10 to 30 minutes, allows the development of tolerance towards subsequent exposure to multiple stresses; this phenomenon is termed "thermotolerance" [66,67]. Ther‐ motolerance is an adaptive survival response induced by heat preconditioning whereby cells become resistant to a subsequent lethal insult such as that triggered by heat shock, reactive oxygen species (ROS), and environmental stressors including heavy metals [68,69]. If the level of stress is very low, cells attempt to survive by activating stress responses that protect essential biochemical processes such as DNA repair, protein folding, and the elimination of damaged proteins [70]. Once the stress stimulus is removed, cells can recover their normal cellular function. If the stress continues or is too severe, then the cell will likely die by apoptosis or necrosis.

The acquisition of thermotolerance is characterized by numerous biochemical and molecular changes. Thermotolerance is generally associated with the accumulation of Hsps [19,63,68,71-73]. Hsp expression is regulated by a stress-responsive transcription factor known as heat shock factor 1 (HSF-1), through its interaction with the heat shock element (HSE) [74]. In addition, changes in the expression of about 50 to 200 other genes, not traditionally considered Hsps, have been found during or after heat stress (see review, [20]). These include genes for transcription factors, protein degradation, DNA repair enzymes, metabolic enzymes, cell cycle arrest, transport and detoxification, and signal transduction. The reason for the induction of these other cell-protective pathways by heat shock is probably to protect nascent chain synthesis and folding, prevent protein misfolding and aggregation, and to promote recovery from stress-induced damage [75]. Proteomic analyses showed a change in the phosphorylation of 93 proteins between control RIF-1 and their thermotolerant derivatives, TR-RIF-1 cells [76]. These phosphorylated proteins are responsible for a range of cellular functions, which include chaperones, ion channels, signal transduction, transcription and translation, biosynthesis of amino acids, oxidoreduction, energy metabolism, and cell motility or structure.

The heat shock response is highly conserved in all organisms from yeast to humans, which suggests that it is important for survival in a stressful environment [74]. In addition to heat, the heat shock response can be induced by other insults such as oxidative stress, heavy metals, ethanol, toxins and bacterial infections.

The major classes of Hsps induced by the heat shock response are Hsp90, Hsp70, Hsp60, and Hsp27. Hsps appear to play an important role in thermotolerance. Many studies suggest a correlation between the accumulation of Hsp70 and the acquisition of a thermotolerant state in mammals, amphibians, insects [77-79] and fish [80]. Under conditions of stress, Hsp70s can prevent the formation of protein aggregates and assist the refolding of aggregated proteins into their native structures [19]. Other studies have shown that the state of thermotolerance correlated with an increase in the expression of Hsp110 [81]. Hsp110 is as effective as Hsp70 in preventing protein aggregation, and contributes, along with Hsp70 and Hsp40, to the refolding of denatured proteins. In addition to their protective role against a subsequent lethal heat shock, Hsps are known to protect cells against other forms of stress, such as oxidative stress and radiation [82].

have reported that Hsp27 may be involved in the development of resistance to chemothera‐

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265

The biological rational for the use of hyperthermia in cancer treatment is very strong. Tem‐ peratures of 42.5°C and above are able to kill cancer cells. Findings from *in vitro* studies generally indicate that there is no intrinsic difference in heat sensitivity between normal and tumour cells [105]. However, a tumour selective effect of hyperthermia could occur at higher temperatures *in vivo*. In solid tumours, the vascular system is chaotic, which results in regions with hypoxia and low pH levels, compared to normal tissues. These conditions render cells more sensitive to the cytotoxic effects of hyperthermia. Therefore, hyperthermia can be beneficial by causing direct cytotoxicity to tumour cells, in addition to selective destruction of tumour cells in hypoxic and low pH environments within solid tumours. A further benefit is that mild hyperthermia can activate certain responses of the immune system, which could also provide protection against tumour growth [64,106]. In the clinic, hyperthermia has been shown to be most beneficial when used in combination with radiation therapy and/or chemotherapy.

One of the most promising aspects of hyperthermia in cancer treatment is the ability to eliminate radiation-resistant tumour cells [see review, 5]. Indeed, this renders hyperthermia as one of the most effective radiation sensitizers known. The basis for this effect is that hyperthermia has the ability to kill cells that are under conditions of hypoxia, low pH and that are in the S-phase of cell division, which are all conditions that render cells resistant to radiation. The mechanisms responsible for heat-induced radio-sensitization are not entirely understood, particularly for milder temperatures [21]. For temperatures of 43°C and above, nuclear protein damage is considered to be a critical event [107]. It was suggested that hyperthermia interferes with the repair of radiation-induced DNA damage. In support of this idea, hyperthermia increased the amount of radiation-induced chromosomal aberrations [13, 108]. It was suggested that heat-induced enhancement of chromosomal aberrations could arise from the inhibition of repair of radiation-induced DNA damage. Hyperthermia could exert its major effect on radio-sensitization by specifically inhibiting base excision repair of DNA

The combined use of regional hyperthermia with systemic chemotherapy has considerable potential in cancer treatment mainly because localized heat delivery could enhance cytotoxic activity of anticancer drugs within a defined target region. This may lead to an improved therapeutic ratio by allowing targeting of chemotherapy, as can be achieved with radiation therapy. At present, targeted treatment with anticancer drugs can only be accomplished when they are administered either topically or intra-arterially. There is also evidence to suggest that the cytotoxic effects of hyperthermia and anticancer drugs may prove to be complementary. Tumour cells that are located in less well-vascularized regions of a tumour, such as the tumour

peutic agents such as doxorubicin and cisplatin [100-104].

*2.6.1. Hyperthermia in combination with radiotherapy*

*2.6.2. Hyperthermia in combination with chemotherapy*

damage [109,110].

**2.6. Hyperthermia in cancer therapy**

Hsps play an important and yet complex role in the regulation of apoptosis. The specific roles of different Hsps such as Hsp27, Hsp60, Hsp70 and Hsp90 in the regulation of the mitochon‐ drial and death receptor pathways of apoptosis have been reviewed [82-85]. The induction of apoptosis through the Fas death receptor can be regulated by Hsp70 and Hsp27 [86,87]. Hsp27 and Hsp70 can regulate the death receptor pathway of apoptosis by preventing t-Bid translo‐ cation to mitochondria, which in turn inhibits cytochrome c release [88, 89]. Hsp90 was shown to be a negative regulator of caspase-2 activation [90]. Hsp27, Hsp70, and Hsp90 can attenuate apoptosis upstream of mitochondria [91], as well as interfering with apoptosome formation, post-mitochondrial events, and caspase activation [92]. Furthermore, Hsp70 and phosphory‐ lated Hsp27 can protect cells against oxidative stress, a potent activator of apoptosis [93,94].

The development of thermotolerance by lethal hyperthermia has been the subject of intensive studies during the past three decades, whereas thermotolerance induced at mild, fever-range temperatures has received relatively little attention. Thermotolerance can be developed following exposure for shorter times (e.g. 30 min) to lethal temperatures (42 to 45°C) [68,71], or during continuous heating (e.g. 3 to 24 h) at non-lethal temperatures (39 to 41.5°C) [95,96]. The development of thermotolerance by exposure of cells to mild hyperthermia (40°C) for 3 to 24 h led to the accumulation of Hsps 27, 32, 60, 70, 90 and 110 [32,95]. This phenomenon is of notable importance for fundamental research given that it is a physiological fever-range temperature and suggests that thermotolerance could protect healthy tissue against stressors during clinical therapies. The treatment of BALB/c mice *in vivo* with fever-range whole body hyperthermia (39.5 to 40°C) for 6h led to increased expression of Hsp70 and Hsp110 in several mouse tissues [97].

Mild thermotolerance developed at 40°C created an apoptosis-resistant phenotype. The activation of the mitochondrial pathway of apoptosis by moderate hyperthermia (42 to 43°C) was attenuated in these thermotolerant cells [44]. Similarly, activation of the death receptor signaling pathway through the Fas receptor by lethal heat shock (42 to 43°C) was inhibited in thermotolerant cells [32]. Furthermore, thermotolerance developed at 40°C protected cells against the induction of apoptosis by oxidative stress (H2O2), mediated through the mitochon‐ drial and death receptor pathways [33,38]. This apoptosis-resistant phenotype could be conferred by increased levels of both Hsps (Hsps 27, 32, 60, 70, 90, and 110 kDa) and antioxi‐ dants (catalase, manganese superoxide dismutase, glutathione) [32,33]. Mild thermotolerance also inhibited hyperthermia-induced ROS generation [32], and this could be explained by the ROS-inhibitory effect of Hsps such as Hsp27 and Hsp70 [93,94].

Hsps play overlapping roles in tumour development and growth by promoting cell prolifer‐ ation and by inhibiting cell death pathways [98]. Hsp70 is a survival protein that is overex‐ pressed in various malignant tumors and its expression correlates with increased cell proliferation, poor differentiation and poor therapeutic outcome in human breast cancer [99]. The increased expression of Hsp70 in tumors can prevent the activation of caspases and proteases, and thus abolish apoptotic cell death [98]. Moreover, the increased expression of Hsps appears to be involved in the acquisition of drug-resistant phenotypes. Several studies have reported that Hsp27 may be involved in the development of resistance to chemothera‐ peutic agents such as doxorubicin and cisplatin [100-104].

#### **2.6. Hyperthermia in cancer therapy**

heat shock, Hsps are known to protect cells against other forms of stress, such as oxidative

Hsps play an important and yet complex role in the regulation of apoptosis. The specific roles of different Hsps such as Hsp27, Hsp60, Hsp70 and Hsp90 in the regulation of the mitochon‐ drial and death receptor pathways of apoptosis have been reviewed [82-85]. The induction of apoptosis through the Fas death receptor can be regulated by Hsp70 and Hsp27 [86,87]. Hsp27 and Hsp70 can regulate the death receptor pathway of apoptosis by preventing t-Bid translo‐ cation to mitochondria, which in turn inhibits cytochrome c release [88, 89]. Hsp90 was shown to be a negative regulator of caspase-2 activation [90]. Hsp27, Hsp70, and Hsp90 can attenuate apoptosis upstream of mitochondria [91], as well as interfering with apoptosome formation, post-mitochondrial events, and caspase activation [92]. Furthermore, Hsp70 and phosphory‐ lated Hsp27 can protect cells against oxidative stress, a potent activator of apoptosis [93,94]. The development of thermotolerance by lethal hyperthermia has been the subject of intensive studies during the past three decades, whereas thermotolerance induced at mild, fever-range temperatures has received relatively little attention. Thermotolerance can be developed following exposure for shorter times (e.g. 30 min) to lethal temperatures (42 to 45°C) [68,71], or during continuous heating (e.g. 3 to 24 h) at non-lethal temperatures (39 to 41.5°C) [95,96]. The development of thermotolerance by exposure of cells to mild hyperthermia (40°C) for 3 to 24 h led to the accumulation of Hsps 27, 32, 60, 70, 90 and 110 [32,95]. This phenomenon is of notable importance for fundamental research given that it is a physiological fever-range temperature and suggests that thermotolerance could protect healthy tissue against stressors during clinical therapies. The treatment of BALB/c mice *in vivo* with fever-range whole body hyperthermia (39.5 to 40°C) for 6h led to increased expression of Hsp70 and Hsp110 in several

Mild thermotolerance developed at 40°C created an apoptosis-resistant phenotype. The activation of the mitochondrial pathway of apoptosis by moderate hyperthermia (42 to 43°C) was attenuated in these thermotolerant cells [44]. Similarly, activation of the death receptor signaling pathway through the Fas receptor by lethal heat shock (42 to 43°C) was inhibited in thermotolerant cells [32]. Furthermore, thermotolerance developed at 40°C protected cells against the induction of apoptosis by oxidative stress (H2O2), mediated through the mitochon‐ drial and death receptor pathways [33,38]. This apoptosis-resistant phenotype could be conferred by increased levels of both Hsps (Hsps 27, 32, 60, 70, 90, and 110 kDa) and antioxi‐ dants (catalase, manganese superoxide dismutase, glutathione) [32,33]. Mild thermotolerance also inhibited hyperthermia-induced ROS generation [32], and this could be explained by the

Hsps play overlapping roles in tumour development and growth by promoting cell prolifer‐ ation and by inhibiting cell death pathways [98]. Hsp70 is a survival protein that is overex‐ pressed in various malignant tumors and its expression correlates with increased cell proliferation, poor differentiation and poor therapeutic outcome in human breast cancer [99]. The increased expression of Hsp70 in tumors can prevent the activation of caspases and proteases, and thus abolish apoptotic cell death [98]. Moreover, the increased expression of Hsps appears to be involved in the acquisition of drug-resistant phenotypes. Several studies

ROS-inhibitory effect of Hsps such as Hsp27 and Hsp70 [93,94].

stress and radiation [82].

264 Cancer Treatment - Conventional and Innovative Approaches

mouse tissues [97].

The biological rational for the use of hyperthermia in cancer treatment is very strong. Tem‐ peratures of 42.5°C and above are able to kill cancer cells. Findings from *in vitro* studies generally indicate that there is no intrinsic difference in heat sensitivity between normal and tumour cells [105]. However, a tumour selective effect of hyperthermia could occur at higher temperatures *in vivo*. In solid tumours, the vascular system is chaotic, which results in regions with hypoxia and low pH levels, compared to normal tissues. These conditions render cells more sensitive to the cytotoxic effects of hyperthermia. Therefore, hyperthermia can be beneficial by causing direct cytotoxicity to tumour cells, in addition to selective destruction of tumour cells in hypoxic and low pH environments within solid tumours. A further benefit is that mild hyperthermia can activate certain responses of the immune system, which could also provide protection against tumour growth [64,106]. In the clinic, hyperthermia has been shown to be most beneficial when used in combination with radiation therapy and/or chemotherapy.

### *2.6.1. Hyperthermia in combination with radiotherapy*

One of the most promising aspects of hyperthermia in cancer treatment is the ability to eliminate radiation-resistant tumour cells [see review, 5]. Indeed, this renders hyperthermia as one of the most effective radiation sensitizers known. The basis for this effect is that hyperthermia has the ability to kill cells that are under conditions of hypoxia, low pH and that are in the S-phase of cell division, which are all conditions that render cells resistant to radiation. The mechanisms responsible for heat-induced radio-sensitization are not entirely understood, particularly for milder temperatures [21]. For temperatures of 43°C and above, nuclear protein damage is considered to be a critical event [107]. It was suggested that hyperthermia interferes with the repair of radiation-induced DNA damage. In support of this idea, hyperthermia increased the amount of radiation-induced chromosomal aberrations [13, 108]. It was suggested that heat-induced enhancement of chromosomal aberrations could arise from the inhibition of repair of radiation-induced DNA damage. Hyperthermia could exert its major effect on radio-sensitization by specifically inhibiting base excision repair of DNA damage [109,110].

#### *2.6.2. Hyperthermia in combination with chemotherapy*

The combined use of regional hyperthermia with systemic chemotherapy has considerable potential in cancer treatment mainly because localized heat delivery could enhance cytotoxic activity of anticancer drugs within a defined target region. This may lead to an improved therapeutic ratio by allowing targeting of chemotherapy, as can be achieved with radiation therapy. At present, targeted treatment with anticancer drugs can only be accomplished when they are administered either topically or intra-arterially. There is also evidence to suggest that the cytotoxic effects of hyperthermia and anticancer drugs may prove to be complementary. Tumour cells that are located in less well-vascularized regions of a tumour, such as the tumour center, may be relatively resistant to systemic chemotherapy because they are exposed to lower concentrations of drug. The benefit of hyperthermia is that it kills cells most efficiently in the low pH and hypoxic environment of the tumour core. Furthermore, the temperature achieved in poorly vascularized regions of the tumour may be higher because of less efficient cooling by circulating blood. Another potential benefit is that regional hyperthermia at 40–43°C causes an increase in tumour blood supply [111]. Blood flow and vascular permeability, which are increased by hyperthermia, are critical factors for drug uptake [112].

glucuronate, or sulfate [124], as well as cysteinyl leukotriene (LTC4), prostaglandins, and the

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Eventually, other distinct mechanisms were also implicated in the MDR phenotype [126]. These mechanisms engage other proteins involved in cellular defenses such as glutathione Stransferase (GST), an enzyme involved in the cellular detoxification of xenobiotics, which include certain anticancer drugs, toxins and environmental pollutants that undergo conjuga‐ tion with the antioxidant GSH [127]. Other cellular defenses utilized by the MDR phenotype include metallothionein, thioredoxin, thymidylate synthase, dihydrofolate reductase, Hsps

Clinical drug resistance appears to be a very complex and multifactorial problem [128] with multiple mechanisms involved. There is often overlapping substrate specificity between different drug transporters, and they are commonly co-expressed in many normal tissues and tumours. Overcoming MDR in cancer treatment presents a formidable challenge [129].

To date, three generations of inhibitors have been used to increase the efficacy of chemotherapy by inhibiting transporter-mediated drug efflux. However, the development of clinical inhibi‐ tors of ABC transporters as targets for clinical intervention in oncology has been difficult and new approaches are clearly needed. Clinical drug resistance is a major barrier which, if

A beneficial effect of hyperthermia is its ability to reverse resistance to certain chemothera‐ peutic drugs [130]. Hyperthermia increased the cytotoxicity of anticancer drugs such as methotrexate [131], cisplatin [132], and mitomycin c [133] in cells exhibiting primary drug resistance. In addition, hyperthermia enhanced the cytotoxicity of melphalan in MDR Chinese hamster ovary CHRC5 cells that overexpress Pgp [117]. CHRC5 cells are resistant to anticancer

Among the earlier strategies to overcome MDR, Pgp-modulating agents such as cyclosporin A and verapamil were developed. These chemosensitizers appear to act by decreasing Pgpmediated efflux of anticancer drugs from cells, which allows increased accumulation of drugs to more cytotoxic levels inside cells. However, clinical studies showed that these chemosen‐ sitizers were effective only at toxic doses [128]. Therefore, chemosensitizers with improved MDR-reversing ability and lower toxicity need to be developed, as well as novel approaches. Hyperthermia (42 to 43°C) showed beneficial effects by reversing MDR involving Pgp when melphalan or Adriamycin was combined with Pgp modulators such as cyclosporin A [135,136]) or verapamil [137,138]. When combined with hyperthermia (43°C), the Pgp modu‐ lator PSC 833 reduced resistance to vinblastine in MDR K562 leukaemia cells and MESSA leiomyosarcoma cells [139]. Moreover, ultrasound-induced hyperthermia (USHT) increased Adriamycin cytotoxicity in the MDR human lung adenocarcinoma cell line MV522 [140]. The alkylating agent melphalan is mainly detoxified through conjugation with GSH, which can be catalyzed by GST [141]. In addition to overexpression of Pgp, CHRC5 cells also overexpress the alpha and pi forms of GST, compared to the drug-sensitive AuxB1 cells [142]. Hyperthermia

overcome, should lead to a significant improvement in patient survival.

drugs such as colchicine, Vinca alkaloids, Adriamycin, and melphalan [134].

*2.6.2.2. Hyperthermia and reversal of resistance to chemotherapeutic agents*

anticancer drug methotrexate [125].

and topoisomerase II [126].

Laboratory and *in vivo* studies have shown that the combined use of hyperthermia and chemotherapy leads to increased cytotoxic effects of several anticancer drugs such as cisplatin, anthracyclines, cyclophosphamide, ifosfamide, nitrosoureas, bleomycin, mitomycin, and nitrogen mustards such as melphalan [16,25,105,113-118]. Optimal heat enhancement of drug cytotoxicity generally occurs between 40.5°C and 43°C. For drugs such as cisplatin, alkylating agents, and nitrosoureas, interactions between heat and drug are more than additive (or synergistic), whereas in other cases, interactions are simply additive [119]. For bleomycin and Adriamycin, there is a threshold temperature of about 42.5°C to 43°C for enhancement of drug cytotoxicity. The antimetabolites (e.g. 5-fluorodeoxyuridin and methotrexate) and Vinca alkaloids or taxanes have independent interactions with hyperthermia. In general, the most effective heat-drug sequence is drug treatment immediately before heat delivery. The mech‐ anisms of heat-induced enhancement of drug cytotoxicity are not well understood. Possible mechanisms include improved drug delivery to the tumour due to increased blood perfusion, increased intracellular uptake of drugs, and increased rates of reaction of drugs with cellular targets (e.g. increased drug alkylation, increased DNA damage).

#### *2.6.2.1. Resistance to chemotherapeutic agents*

One of the major limitations to the successful use of chemotherapy in cancer treatment is the development of resistance to multiple anticancer drugs. Cross-resistance occurs between different anticancer agents that have distinct structures and mechanisms of cytotoxicity. Multidrug resistance (MDR) is characterized by cross-resistance to four classes of commonly used anticancer drugs such as Vinca alkaloids, anthracyclines, taxanes, and epipodophyllo‐ toxins. Classical MDR was discovered about 35 years ago and was initially related to the overexpression of the cellular 170-kDa protein P-glycoprotein (Pgp) [120], a member of the ATP-binding cassette (ABC) transporters. Pgp acts as an ATP-dependent transmembrane pump. Once anticancer drugs enter cells, they are immediately expulsed out of cells by Pgp. This results in decreased levels of drugs inside cells, rendering the drugs less effective against the tumour cells. In addition to Pgp, several other transporter proteins have been implicated in MDR in human cancer: multidrug resistance-associated protein 1 (MRP1), lung resistance protein (LRP) and breast cancer resistance protein (BCRP) [121]. MRP1 is a 190-kDa member of the ABC transporter family of proteins [122]. MRP1-mediated transport requires GSH, as well as ATP binding and hydrolysis. The overexpression of the protein MRP1 can cause cellular resistance to several anticancer drugs, including Adriamycin (doxorubicin), epipodophyllo‐ toxins, and Vinca alkaloids such as vincristine, [123]. The substrate spectrum of MRP proteins also comprises amphiphilic anion conjugates of lipophilic compounds with glutathione (GSH), glucuronate, or sulfate [124], as well as cysteinyl leukotriene (LTC4), prostaglandins, and the anticancer drug methotrexate [125].

center, may be relatively resistant to systemic chemotherapy because they are exposed to lower concentrations of drug. The benefit of hyperthermia is that it kills cells most efficiently in the low pH and hypoxic environment of the tumour core. Furthermore, the temperature achieved in poorly vascularized regions of the tumour may be higher because of less efficient cooling by circulating blood. Another potential benefit is that regional hyperthermia at 40–43°C causes an increase in tumour blood supply [111]. Blood flow and vascular permeability, which are

Laboratory and *in vivo* studies have shown that the combined use of hyperthermia and chemotherapy leads to increased cytotoxic effects of several anticancer drugs such as cisplatin, anthracyclines, cyclophosphamide, ifosfamide, nitrosoureas, bleomycin, mitomycin, and nitrogen mustards such as melphalan [16,25,105,113-118]. Optimal heat enhancement of drug cytotoxicity generally occurs between 40.5°C and 43°C. For drugs such as cisplatin, alkylating agents, and nitrosoureas, interactions between heat and drug are more than additive (or synergistic), whereas in other cases, interactions are simply additive [119]. For bleomycin and Adriamycin, there is a threshold temperature of about 42.5°C to 43°C for enhancement of drug cytotoxicity. The antimetabolites (e.g. 5-fluorodeoxyuridin and methotrexate) and Vinca alkaloids or taxanes have independent interactions with hyperthermia. In general, the most effective heat-drug sequence is drug treatment immediately before heat delivery. The mech‐ anisms of heat-induced enhancement of drug cytotoxicity are not well understood. Possible mechanisms include improved drug delivery to the tumour due to increased blood perfusion, increased intracellular uptake of drugs, and increased rates of reaction of drugs with cellular

One of the major limitations to the successful use of chemotherapy in cancer treatment is the development of resistance to multiple anticancer drugs. Cross-resistance occurs between different anticancer agents that have distinct structures and mechanisms of cytotoxicity. Multidrug resistance (MDR) is characterized by cross-resistance to four classes of commonly used anticancer drugs such as Vinca alkaloids, anthracyclines, taxanes, and epipodophyllo‐ toxins. Classical MDR was discovered about 35 years ago and was initially related to the overexpression of the cellular 170-kDa protein P-glycoprotein (Pgp) [120], a member of the ATP-binding cassette (ABC) transporters. Pgp acts as an ATP-dependent transmembrane pump. Once anticancer drugs enter cells, they are immediately expulsed out of cells by Pgp. This results in decreased levels of drugs inside cells, rendering the drugs less effective against the tumour cells. In addition to Pgp, several other transporter proteins have been implicated in MDR in human cancer: multidrug resistance-associated protein 1 (MRP1), lung resistance protein (LRP) and breast cancer resistance protein (BCRP) [121]. MRP1 is a 190-kDa member of the ABC transporter family of proteins [122]. MRP1-mediated transport requires GSH, as well as ATP binding and hydrolysis. The overexpression of the protein MRP1 can cause cellular resistance to several anticancer drugs, including Adriamycin (doxorubicin), epipodophyllo‐ toxins, and Vinca alkaloids such as vincristine, [123]. The substrate spectrum of MRP proteins also comprises amphiphilic anion conjugates of lipophilic compounds with glutathione (GSH),

increased by hyperthermia, are critical factors for drug uptake [112].

targets (e.g. increased drug alkylation, increased DNA damage).

*2.6.2.1. Resistance to chemotherapeutic agents*

266 Cancer Treatment - Conventional and Innovative Approaches

Eventually, other distinct mechanisms were also implicated in the MDR phenotype [126]. These mechanisms engage other proteins involved in cellular defenses such as glutathione Stransferase (GST), an enzyme involved in the cellular detoxification of xenobiotics, which include certain anticancer drugs, toxins and environmental pollutants that undergo conjuga‐ tion with the antioxidant GSH [127]. Other cellular defenses utilized by the MDR phenotype include metallothionein, thioredoxin, thymidylate synthase, dihydrofolate reductase, Hsps and topoisomerase II [126].

Clinical drug resistance appears to be a very complex and multifactorial problem [128] with multiple mechanisms involved. There is often overlapping substrate specificity between different drug transporters, and they are commonly co-expressed in many normal tissues and tumours. Overcoming MDR in cancer treatment presents a formidable challenge [129].

To date, three generations of inhibitors have been used to increase the efficacy of chemotherapy by inhibiting transporter-mediated drug efflux. However, the development of clinical inhibi‐ tors of ABC transporters as targets for clinical intervention in oncology has been difficult and new approaches are clearly needed. Clinical drug resistance is a major barrier which, if overcome, should lead to a significant improvement in patient survival.

### *2.6.2.2. Hyperthermia and reversal of resistance to chemotherapeutic agents*

A beneficial effect of hyperthermia is its ability to reverse resistance to certain chemothera‐ peutic drugs [130]. Hyperthermia increased the cytotoxicity of anticancer drugs such as methotrexate [131], cisplatin [132], and mitomycin c [133] in cells exhibiting primary drug resistance. In addition, hyperthermia enhanced the cytotoxicity of melphalan in MDR Chinese hamster ovary CHRC5 cells that overexpress Pgp [117]. CHRC5 cells are resistant to anticancer drugs such as colchicine, Vinca alkaloids, Adriamycin, and melphalan [134].

Among the earlier strategies to overcome MDR, Pgp-modulating agents such as cyclosporin A and verapamil were developed. These chemosensitizers appear to act by decreasing Pgpmediated efflux of anticancer drugs from cells, which allows increased accumulation of drugs to more cytotoxic levels inside cells. However, clinical studies showed that these chemosen‐ sitizers were effective only at toxic doses [128]. Therefore, chemosensitizers with improved MDR-reversing ability and lower toxicity need to be developed, as well as novel approaches. Hyperthermia (42 to 43°C) showed beneficial effects by reversing MDR involving Pgp when melphalan or Adriamycin was combined with Pgp modulators such as cyclosporin A [135,136]) or verapamil [137,138]. When combined with hyperthermia (43°C), the Pgp modu‐ lator PSC 833 reduced resistance to vinblastine in MDR K562 leukaemia cells and MESSA leiomyosarcoma cells [139]. Moreover, ultrasound-induced hyperthermia (USHT) increased Adriamycin cytotoxicity in the MDR human lung adenocarcinoma cell line MV522 [140]. The alkylating agent melphalan is mainly detoxified through conjugation with GSH, which can be catalyzed by GST [141]. In addition to overexpression of Pgp, CHRC5 cells also overexpress the alpha and pi forms of GST, compared to the drug-sensitive AuxB1 cells [142]. Hyperthermia was beneficial by enhancing melphalan cytotoxicity in MDR cells when GST was inhibited using ethacrynic acid [142].

**2.7. Hyperthermia in the cancer clinic**

*2.7.1.1. Local hyperthermia*

cancer [158].

*2.7.1.2. Perfusion hyperthermia*

avoid drug toxicity.

*2.7.1.3. Whole body hyperthermia*

*2.7.1. Techniques to increase tumour temperatures*

In the cancer clinic, hyperthermia is administered by exposing tumour tissues to conductive heat sources, or non-ionizing radiation (e.g. electromagnetic or ultrasonic fields). Hyperther‐ mia can be applied by either invasive or noninvasive techniques, using externally applied power. To increase tumour temperatures, hyperthermia can be applied by several different techniques: local hyperthermia by external or internal energy sources, perfusion hyperthermia

Hyperthermia: Cancer Treatment and Beyond

http://dx.doi.org/10.5772/55795

269

Local hyperthermia entails elevating the temperature of superficial or deep-seated subcuta‐ neous tumours while sparing the surrounding normal tissue, using external, intraluminal or interstitial heating modalities. The area can be heated externally with high-frequency waves (e.g. electromagnetic or ultrasound energy) aimed at the tumour from a device outside the body. To achieve internal heating, one of several types of sterile probes may be used, including thin heated wires, hollow tubes filled with warm water, implanted microwave antennae, radiofrequency electrodes and ultrasound. Local hyperthermia has allowed the use of hyperthermia in conjunction with other modalities of antineoplastic therapy. Local hyperthermia is more appropriate for the treatment of solid tumours, rather than blood diseases such as leukaemia. Despite advances in the technology of heating, the non-homogeneous character of the treatment region (i.e. tissue characteristics and blood flow) can often affect the uniformity of the heat dispersion in the treated area. This means that it can be difficult to obtain a uniform regional rise in the temperature that is reproducible [151-155]. Deep regional hyperthermia combined with chemotherapy, also known as hyperthermic intraperitoneal chemotherapy, is one of the promising methods for the treatment of prostate carcinoma [156,157] and bladder

This technique involves regional heating through the perfusion of a limb, organ (liver, pelvis, stomach), or body cavity using heated fluids [159-161]. In perfusion, the patient's blood can be removed, heated, and then pumped into the region that is to be heated internally. Perfusion hyperthermia can be applied with or without a cytotoxic drug. When applied to limbs without a cytotoxic agent, a temperature of about 43°C can be used for about two hours. Lower temperatures are used when perfusion is performed in combination with cytotoxic agents, to

Externally-induced whole body hyperthermia can be used to treat metastatic cancers that have spread throughout the body. Whole body hyperthermia can be applied using different methods and involves heating the patient to a maximum temperature of 41.8 to 42°C. A newer

of organs, limbs, or body cavities, and whole body hyperthermia [150].

#### *2.6.2.3. Sensitivity of multidrug resistant cells to hyperthermia*

Another important advantage for the clinical use of hyperthermia is that MDR cells overex‐ pressing Pgp or MRP1 do not display cross-resistance to heat [25,143]. Indeed, these MDR cells exhibit equivalent sensitivity to the cytotoxic and apoptosis-inducing effects of hyperthermia (41-45°C) as their drug-sensitive counterparts. Moreover, drug-resistant sub-clones of human T-lineage acute lymphoblastic leukaemia (ALL) and acute myeloblastic leukaemia (AML) cells were as sensitive to hyperthermia as were the drug-sensitive sub-clones [144]. Results from these studies indicate that, in addition to enhancing drug cytotoxicity in resistant cells, hyperthermia alone can successfully eliminate MDR cells. Together, these findings clearly show that hyperthermia could be useful by destroying subpopulations of drug-resistant tumour cells, which have survived chemotherapy treatments, where the overexpression of Pgp and MRP1 is involved.

Apoptosis is considered to be a physiological mechanism for the elimination of damaged and abnormal cells, such as tumour cells. One of the hallmark characteristics of tumour cells is their ability to evade destruction by apoptosis [145]. The up-regulation of differ‐ ent anti-apoptotic proteins, to provide a survival advantage, has been a frequent explana‐ tion for the resistance of cancer cells to elimination by apoptosis [146]. The induction of death receptor and mitochondria-mediated signaling pathways of apoptosis by hyperther‐ mia (41 to 43°C) in MDR CHRC5 cells was compared to drug-sensitive CHO cells [147]. Differences were found between MDR and drug-sensitive cells in terms of induction of apoptosis by hyperthermia. For death receptor-mediated apoptosis, MDR cells contained higher levels of the anti-apoptosis protein c-FLIP and they had a lower level of activa‐ tion of initiator caspase-8 and caspase-10 in response to hyperthermia. In the mitochondriamediated pathway of heat-induced apoptosis, MDR cells showed higher mitochondrial levels of the pro-apoptosis proteins Bax and tBid, more pronounced mitochondrial membrane depolarization, and increased levels of the apoptosome protein Apaf-1 (apopto‐ sis protease activating factor 1). The MDR cells appeared to show some resistance to death receptor-mediated apoptosis [147], in agreement with other studies in leukaemia cells [148, 149], but this resistance appeared to be compensated for by the pro-apoptosis changes in mitochondrial apoptosis. For the execution stage of apoptosis, the MDR and drug-sensi‐ tive cells showed similar levels of hyperthermia-induced caspase-3 activation, as well cleavage of caspase-3 substrates poly (ADP-ribose) polymerase (PARP) and inhibitor of caspase-activated DNase (ICAD) [147]. Similar levels of nuclear chromatin condensation were induced by hyperthermia, showing that overall, MDR cells are not resistant to hyperthermia-induced apoptosis compared to the drug-sensitive cells. In summary, MDR and drug-sensitive cells showed similar responses to heat in terms of clonogenic cell survival and apoptosis, which indicates that hyperthermia could be a promising strategy for eradicating MDR tumour cells in the cancer clinic.

### **2.7. Hyperthermia in the cancer clinic**

#### *2.7.1. Techniques to increase tumour temperatures*

In the cancer clinic, hyperthermia is administered by exposing tumour tissues to conductive heat sources, or non-ionizing radiation (e.g. electromagnetic or ultrasonic fields). Hyperther‐ mia can be applied by either invasive or noninvasive techniques, using externally applied power. To increase tumour temperatures, hyperthermia can be applied by several different techniques: local hyperthermia by external or internal energy sources, perfusion hyperthermia of organs, limbs, or body cavities, and whole body hyperthermia [150].

#### *2.7.1.1. Local hyperthermia*

was beneficial by enhancing melphalan cytotoxicity in MDR cells when GST was inhibited

Another important advantage for the clinical use of hyperthermia is that MDR cells overex‐ pressing Pgp or MRP1 do not display cross-resistance to heat [25,143]. Indeed, these MDR cells exhibit equivalent sensitivity to the cytotoxic and apoptosis-inducing effects of hyperthermia (41-45°C) as their drug-sensitive counterparts. Moreover, drug-resistant sub-clones of human T-lineage acute lymphoblastic leukaemia (ALL) and acute myeloblastic leukaemia (AML) cells were as sensitive to hyperthermia as were the drug-sensitive sub-clones [144]. Results from these studies indicate that, in addition to enhancing drug cytotoxicity in resistant cells, hyperthermia alone can successfully eliminate MDR cells. Together, these findings clearly show that hyperthermia could be useful by destroying subpopulations of drug-resistant tumour cells, which have survived chemotherapy treatments, where the overexpression of Pgp

Apoptosis is considered to be a physiological mechanism for the elimination of damaged and abnormal cells, such as tumour cells. One of the hallmark characteristics of tumour cells is their ability to evade destruction by apoptosis [145]. The up-regulation of differ‐ ent anti-apoptotic proteins, to provide a survival advantage, has been a frequent explana‐ tion for the resistance of cancer cells to elimination by apoptosis [146]. The induction of death receptor and mitochondria-mediated signaling pathways of apoptosis by hyperther‐ mia (41 to 43°C) in MDR CHRC5 cells was compared to drug-sensitive CHO cells [147]. Differences were found between MDR and drug-sensitive cells in terms of induction of apoptosis by hyperthermia. For death receptor-mediated apoptosis, MDR cells contained higher levels of the anti-apoptosis protein c-FLIP and they had a lower level of activa‐ tion of initiator caspase-8 and caspase-10 in response to hyperthermia. In the mitochondriamediated pathway of heat-induced apoptosis, MDR cells showed higher mitochondrial levels of the pro-apoptosis proteins Bax and tBid, more pronounced mitochondrial membrane depolarization, and increased levels of the apoptosome protein Apaf-1 (apopto‐ sis protease activating factor 1). The MDR cells appeared to show some resistance to death receptor-mediated apoptosis [147], in agreement with other studies in leukaemia cells [148, 149], but this resistance appeared to be compensated for by the pro-apoptosis changes in mitochondrial apoptosis. For the execution stage of apoptosis, the MDR and drug-sensi‐ tive cells showed similar levels of hyperthermia-induced caspase-3 activation, as well cleavage of caspase-3 substrates poly (ADP-ribose) polymerase (PARP) and inhibitor of caspase-activated DNase (ICAD) [147]. Similar levels of nuclear chromatin condensation were induced by hyperthermia, showing that overall, MDR cells are not resistant to hyperthermia-induced apoptosis compared to the drug-sensitive cells. In summary, MDR and drug-sensitive cells showed similar responses to heat in terms of clonogenic cell survival and apoptosis, which indicates that hyperthermia could be a promising strategy

using ethacrynic acid [142].

268 Cancer Treatment - Conventional and Innovative Approaches

and MRP1 is involved.

*2.6.2.3. Sensitivity of multidrug resistant cells to hyperthermia*

for eradicating MDR tumour cells in the cancer clinic.

Local hyperthermia entails elevating the temperature of superficial or deep-seated subcuta‐ neous tumours while sparing the surrounding normal tissue, using external, intraluminal or interstitial heating modalities. The area can be heated externally with high-frequency waves (e.g. electromagnetic or ultrasound energy) aimed at the tumour from a device outside the body. To achieve internal heating, one of several types of sterile probes may be used, including thin heated wires, hollow tubes filled with warm water, implanted microwave antennae, radiofrequency electrodes and ultrasound. Local hyperthermia has allowed the use of hyperthermia in conjunction with other modalities of antineoplastic therapy. Local hyperthermia is more appropriate for the treatment of solid tumours, rather than blood diseases such as leukaemia. Despite advances in the technology of heating, the non-homogeneous character of the treatment region (i.e. tissue characteristics and blood flow) can often affect the uniformity of the heat dispersion in the treated area. This means that it can be difficult to obtain a uniform regional rise in the temperature that is reproducible [151-155]. Deep regional hyperthermia combined with chemotherapy, also known as hyperthermic intraperitoneal chemotherapy, is one of the promising methods for the treatment of prostate carcinoma [156,157] and bladder cancer [158].

#### *2.7.1.2. Perfusion hyperthermia*

This technique involves regional heating through the perfusion of a limb, organ (liver, pelvis, stomach), or body cavity using heated fluids [159-161]. In perfusion, the patient's blood can be removed, heated, and then pumped into the region that is to be heated internally. Perfusion hyperthermia can be applied with or without a cytotoxic drug. When applied to limbs without a cytotoxic agent, a temperature of about 43°C can be used for about two hours. Lower temperatures are used when perfusion is performed in combination with cytotoxic agents, to avoid drug toxicity.

#### *2.7.1.3. Whole body hyperthermia*

Externally-induced whole body hyperthermia can be used to treat metastatic cancers that have spread throughout the body. Whole body hyperthermia can be applied using different methods and involves heating the patient to a maximum temperature of 41.8 to 42°C. A newer approach is to increase the temperature to about 40°C for a longer duration, and use a combination of mild hyperthermia with cytokines and/or cytotoxic drugs [118].

**3. Conclusion**

Throughout the past two decades, hyperthermia has been used as a particularly efficient complement to standard cancer treatments such as radiation therapy and chemotherapy. Furthermore, considerable progress has been made in our understanding of the biology, physics and bioengineering involved in hyperthermia. Significant improvement in clinical outcome has been demonstrated for many different types of tumours, including head and neck, breast, brain, bladder, cervix, rectum, lung, esophagus, liver, prostate, melanoma and sarcoma [150]. In Europe, hyperthermia is a standard for the treatment of cervical cancer and some sarcomas. It is a successful alternative for the treatment of other types of cancer such as brain, bladder, rectal and esophageal cancer. Moreover, transurethral microwave thermotherapy (TUMT) has been found to be safe and effective as an alternative to surgery and drug treatment for chronic urogenital pathologies such as benign prostatic hyperplasia [176]. TUMT is a

Hyperthermia: Cancer Treatment and Beyond

http://dx.doi.org/10.5772/55795

271

In spite of good clinical results, hyperthermia has received little attention [150]. Several problems associated with the acceptance of this promising treatment modality concern the limited availability of equipment for heating tumours, the lack of awareness concerning clinical results, and the lack of financial resources. Hyperthermia is currently under study in many clinical trials, particularly in Europe, Japan and the US, to improve and better understand this promising technique. Future areas of challenge and opportunity for hyperthermia include: improved understanding of thermal biology; improved technologies for delivery and moni‐ toring of heat treatments in patients; successful high-quality clinical trials; and combination of

Financial support is gratefully acknowledged from the Natural Sciences and Engineering

and Diana A. Averill-Bates3

2 Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec,

3 Département des sciences biologiques, Université du Québec à Montréal, Succursale Cen‐

minimally invasive therapy that aims to maintain a good quality of life.

hyperthermia with emerging cancer therapies [170].

Research Council of Canada (NSERC) (DAB).

, Paulina K. Wrzal2

tre-Ville, Montréal, Québec, Canada

1 Department of Nutrition, University of California, Davis, California, USA

**Acknowledgements**

**Author details**

Ahmed Bettaieb1

Canada

Many studies are focusing on improving the heating techniques. This is one of the main challenges that currently limit the clinical use of hyperthermia. Furthermore, improvements are required to heat effectively the deep-seated tumours that are localized in internal organs. The use of nanoparticles and the induction heating of magnetic materials that are implanted into tumors are among the new approaches that are currently being investigated for the improved application of hyperthermia.

### *2.7.2. Progress in the cancer clinic*

In the cancer clinic, hyperthermia (40 to 44°C) is mainly used as an adjuvant to radiation and chemotherapy[2,5,16,150].Themajorlimitationsoftheseconventionalcancertreatmentsarelack of specificity and normal tissue toxicity. An important advantage of hyperthermia is that the cytotoxicity of radiotherapy and chemotherapy can be targeted to the tumour volume, thereby decreasing toxic side effects. The effectiveness of hyperthermia depends on the temperature rise and the duration of treatment at the elevated temperature. At least 19 randomized studies using a combination of hyperthermia with radiotherapy, chemotherapy or both, have shown signifi‐ cant improvement in clinical outcome in oncology patients, without a significant increase in side effects[150].Inallofthesestudies,thedifferenceswereverylarge.Thecombinationofhyperther‐ mia with radiation resulted in higher (complete) response rates, accompanied by improved local tumour control rates, better palliative effects, and/or better overall survival rates in many Phase II clinical trials [162-171]. These studies focused on many types of cancer including tumors of the head and neck, cervix, rectum, breast, brain, bladder, lung, esophagus, liver, appendix, pros‐ tate, peritoneal lining (mesothelioma), soft-tissue sarcoma and melanoma [2,3,105]. Based on results from a randomized study [171], radiation combined with hyperthermia was included in the2007BreastCancerGuidelinesforrecurrentbreastcancerandotherlocalizedcancerrecurrenc‐ es by the National Comprehensive Cancer Network (NCCN, U.S.A.).

Despite positive phase III trials, the clinical application of hyperthermia remains limited. This could be partly due to inadequate monitoring of tumour temperatures or thermal dose, during heat treatments. The temperature distribution throughout a tumour during clinical treatment is not homogeneous due to variable tissue properties and changes in blood flow [172] To ensure high quality of treatments, precise tumour temperature measurements and rigorous thermal dosimetric data are essential. Most hyperthermia centers obtain a sparse number of tempera‐ ture measurements within intraluminal or interstitial catheters [173]. Thermal dose parameters are dependent on the number of measurement sites and on characteristics such as blood flow and tumor size [174]. It is eventually hoped that temperature measurements during hyper‐ thermia treatment can be improved by measuring 3D thermal distribution in tumours by magnetic resonance imaging (MRI) techniques.

Ingeneral,hyperthermiatreatmentsarewelltoleratedbypatients.Hyperthermiacancausesome toxicity, including skin burns, but this is usually of limited clinical relevance [166]. Normal tissue damageandtoxicitydonotgenerallyoccurduring1houroftreatmentwithtemperaturesthatare below 44°C [175]. Nervous and gastrointestinal tissues appear to be most sensitive.

### **3. Conclusion**

approach is to increase the temperature to about 40°C for a longer duration, and use a

Many studies are focusing on improving the heating techniques. This is one of the main challenges that currently limit the clinical use of hyperthermia. Furthermore, improvements are required to heat effectively the deep-seated tumours that are localized in internal organs. The use of nanoparticles and the induction heating of magnetic materials that are implanted into tumors are among the new approaches that are currently being investigated for the

In the cancer clinic, hyperthermia (40 to 44°C) is mainly used as an adjuvant to radiation and chemotherapy[2,5,16,150].Themajorlimitationsoftheseconventionalcancertreatmentsarelack of specificity and normal tissue toxicity. An important advantage of hyperthermia is that the cytotoxicity of radiotherapy and chemotherapy can be targeted to the tumour volume, thereby decreasing toxic side effects. The effectiveness of hyperthermia depends on the temperature rise and the duration of treatment at the elevated temperature. At least 19 randomized studies using a combination of hyperthermia with radiotherapy, chemotherapy or both, have shown signifi‐ cant improvement in clinical outcome in oncology patients, without a significant increase in side effects[150].Inallofthesestudies,thedifferenceswereverylarge.Thecombinationofhyperther‐ mia with radiation resulted in higher (complete) response rates, accompanied by improved local tumour control rates, better palliative effects, and/or better overall survival rates in many Phase II clinical trials [162-171]. These studies focused on many types of cancer including tumors of the head and neck, cervix, rectum, breast, brain, bladder, lung, esophagus, liver, appendix, pros‐ tate, peritoneal lining (mesothelioma), soft-tissue sarcoma and melanoma [2,3,105]. Based on results from a randomized study [171], radiation combined with hyperthermia was included in the2007BreastCancerGuidelinesforrecurrentbreastcancerandotherlocalizedcancerrecurrenc‐

Despite positive phase III trials, the clinical application of hyperthermia remains limited. This could be partly due to inadequate monitoring of tumour temperatures or thermal dose, during heat treatments. The temperature distribution throughout a tumour during clinical treatment is not homogeneous due to variable tissue properties and changes in blood flow [172] To ensure high quality of treatments, precise tumour temperature measurements and rigorous thermal dosimetric data are essential. Most hyperthermia centers obtain a sparse number of tempera‐ ture measurements within intraluminal or interstitial catheters [173]. Thermal dose parameters are dependent on the number of measurement sites and on characteristics such as blood flow and tumor size [174]. It is eventually hoped that temperature measurements during hyper‐ thermia treatment can be improved by measuring 3D thermal distribution in tumours by

Ingeneral,hyperthermiatreatmentsarewelltoleratedbypatients.Hyperthermiacancausesome toxicity, including skin burns, but this is usually of limited clinical relevance [166]. Normal tissue damageandtoxicitydonotgenerallyoccurduring1houroftreatmentwithtemperaturesthatare

below 44°C [175]. Nervous and gastrointestinal tissues appear to be most sensitive.

combination of mild hyperthermia with cytokines and/or cytotoxic drugs [118].

es by the National Comprehensive Cancer Network (NCCN, U.S.A.).

magnetic resonance imaging (MRI) techniques.

improved application of hyperthermia.

270 Cancer Treatment - Conventional and Innovative Approaches

*2.7.2. Progress in the cancer clinic*

Throughout the past two decades, hyperthermia has been used as a particularly efficient complement to standard cancer treatments such as radiation therapy and chemotherapy. Furthermore, considerable progress has been made in our understanding of the biology, physics and bioengineering involved in hyperthermia. Significant improvement in clinical outcome has been demonstrated for many different types of tumours, including head and neck, breast, brain, bladder, cervix, rectum, lung, esophagus, liver, prostate, melanoma and sarcoma [150]. In Europe, hyperthermia is a standard for the treatment of cervical cancer and some sarcomas. It is a successful alternative for the treatment of other types of cancer such as brain, bladder, rectal and esophageal cancer. Moreover, transurethral microwave thermotherapy (TUMT) has been found to be safe and effective as an alternative to surgery and drug treatment for chronic urogenital pathologies such as benign prostatic hyperplasia [176]. TUMT is a minimally invasive therapy that aims to maintain a good quality of life.

In spite of good clinical results, hyperthermia has received little attention [150]. Several problems associated with the acceptance of this promising treatment modality concern the limited availability of equipment for heating tumours, the lack of awareness concerning clinical results, and the lack of financial resources. Hyperthermia is currently under study in many clinical trials, particularly in Europe, Japan and the US, to improve and better understand this promising technique. Future areas of challenge and opportunity for hyperthermia include: improved understanding of thermal biology; improved technologies for delivery and moni‐ toring of heat treatments in patients; successful high-quality clinical trials; and combination of hyperthermia with emerging cancer therapies [170].

### **Acknowledgements**

Financial support is gratefully acknowledged from the Natural Sciences and Engineering Research Council of Canada (NSERC) (DAB).

### **Author details**

Ahmed Bettaieb1 , Paulina K. Wrzal2 and Diana A. Averill-Bates3

1 Department of Nutrition, University of California, Davis, California, USA

2 Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec, Canada

3 Département des sciences biologiques, Université du Québec à Montréal, Succursale Cen‐ tre-Ville, Montréal, Québec, Canada

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[165] Datta NR et al. Head and neck cancers: results of thermoradiotherapy versus radio‐

[166] van der Zee J et al. Point-counterpoint: what is the optimal trial design to test hyper‐ thermia for carcinoma of the cervix? Point: addition of hyperthermia or cisplatin to

der cancer. Oncology (Williston Park), 2010. 24(12): 1149-55.

sociation of oxyhemoglobin]. Biofizika, 1972. 17(3): 446-52.

tients. Am J Clin Oncol, 1989. 12(5): 378-83.

Oncology. Lancet, 1995. 345(8949): 540-3.

therapy. Int J Hyperthermia, 1990. 6(3): 479-86.

indications]. Bull Cancer, 2008. 95(1): 141-5.

282 Cancer Treatment - Conventional and Innovative Approaches

113(3): 435-41.

165(10): 721-3.

35(4): 731-44.

28(1): 163-9.

gen, 1976. 73(33): 691.

kol, 2005. 181(1): 35-41.


**Chapter 13**

**Glioblastomas, Astrocytomas: Survival Amelioration**

**Temozolomide — Use of Pegylated Doxorubicin and**

**Hyperthermia in the Treatment of a Recurrent Case**

Gianfranco Baronzio, Gurdev Parmar,

http://dx.doi.org/10.5772/55966

Growth Factor (EGFR) [4,6].

**1. Introduction**

Michela De Santis and Alberto Gramaglia

Additional information is available at the end of the chapter

**Adding Hyperthermia to Conformal Radiotherapy and**

Fifty per cent of primary brain tumours are Glioblastoma (GBM), the rest is constituted by astrocytomas and less aggressive tumours [1,2]. GBM is biologically an aggressive tumor quickly developing genetic hetereoneity and therapeutic resistance [2,3]. No specific markers exist for GBM at the moment as reported by Kesari [4], but new imaging techniques including diffusion weighted imaging (DWI), perfusion weighted imaging (PWI or MR), are aiding in better defining disease development and progression. According to the World Health Organ‐ ization (WHO), astrocytomas are classified into four prognostic grades [4,5]. Grade I and two are classified as low grade gliomas whereas grade III and IV are classified as high grade gliomas, despite the fact that these grades indicate tumor aggressiveness they have not been reliable in predicting the response to therapy. Kesari has outlined that other factors such as overexpression of some growth factors and their receptors have prognostic relevance [4]. Overexpression of Platelet derived growth factor (PDGF) and its receptors indicates a tumor is less aggressive and has a better prognosis compared to GBM expressing the Epidermal

Almost 45% of GBM have molecular alterations of the enzyme 06-methylguanine-DNA methyltransferase(MGMT) and mutations of isocitrate dehydrogenase 1 (IDH1) [7]. MGMT, affects signalling pathways and has an important role with alkylating cytostatic drugs such as Temozolomide (TMZ). Methylation of MGMT is associated to a better prognosis and response

and reproduction in any medium, provided the original work is properly cited.

© 2013 Baronzio et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, **Glioblastomas, Astrocytomas: Survival Amelioration Adding Hyperthermia to Conformal Radiotherapy and Temozolomide — Use of Pegylated Doxorubicin and Hyperthermia in the Treatment of a Recurrent Case**

Gianfranco Baronzio, Gurdev Parmar, Michela De Santis and Alberto Gramaglia

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55966

**1. Introduction**

Fifty per cent of primary brain tumours are Glioblastoma (GBM), the rest is constituted by astrocytomas and less aggressive tumours [1,2]. GBM is biologically an aggressive tumor quickly developing genetic hetereoneity and therapeutic resistance [2,3]. No specific markers exist for GBM at the moment as reported by Kesari [4], but new imaging techniques including diffusion weighted imaging (DWI), perfusion weighted imaging (PWI or MR), are aiding in better defining disease development and progression. According to the World Health Organ‐ ization (WHO), astrocytomas are classified into four prognostic grades [4,5]. Grade I and two are classified as low grade gliomas whereas grade III and IV are classified as high grade gliomas, despite the fact that these grades indicate tumor aggressiveness they have not been reliable in predicting the response to therapy. Kesari has outlined that other factors such as overexpression of some growth factors and their receptors have prognostic relevance [4]. Overexpression of Platelet derived growth factor (PDGF) and its receptors indicates a tumor is less aggressive and has a better prognosis compared to GBM expressing the Epidermal Growth Factor (EGFR) [4,6].

Almost 45% of GBM have molecular alterations of the enzyme 06-methylguanine-DNA methyltransferase(MGMT) and mutations of isocitrate dehydrogenase 1 (IDH1) [7]. MGMT, affects signalling pathways and has an important role with alkylating cytostatic drugs such as Temozolomide (TMZ). Methylation of MGMT is associated to a better prognosis and response

© 2013 Baronzio et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

to TMZ [8]. More recently, a specific and target therapy has been suggested by authors such as Reardon and Wen [3] who have provided a complete list of these inhibitors for this disease including: Gefitinib (Iressa®), Ernolitib (Tarceva®),Thalidomide (Thalidomid®), Bezacizu‐ mab (Avastin®), and proteasome inhibitors (bortezomid).

at a mean interval of 65 days, with a large range [40-90] days for those patients with a disease

Glioblastomas, Astrocytomas: Survival Amelioration Adding Hyperthermia to Conformal Radiotherapy…

 36 F GBM X 45 65.6 2 D 25 52 M GBM X 42 101 1 D 12 54 F GBM X 60 1 A 75 57 M GBM X 14 42 3 A 51 51 M GBM X 20 57 2 A 20 45 F GBM X 65 43.4 1 A 20 70 M GBM X 28 28.4 5 A 96 31 M GBM X 14 64 1 D 7 41 F GBM 3 41 64 1 D 12 33 M GBM 28 28.4 2 A 83 60 F GBM 1 65 64 1 D 12 40 F Astro IV X X 10 25 72.5 1 D 9 43 M Astro IV X X 2 47.6 33.6 2 D 7 59 M Astro IV X X 4 45 32.9 1 A 19 37 M Astro IV X 1 42 101 1 D 12 45 F Astro IV 1 44 113 1 A 98 44 M Astro IV 10 39.6 3 A 82 44 M AstroII 10 45 65.6 4 A 111 34 M AstroII X 39.6 44 1 D 23 44 F AstroII 10 43 112 6 D 17 34 M AstroII 45 1 A 76 40 F Astro II X 4 D 12 30 M Astro II X 6 A 60 46 M Astro II X 6 A 30 46 M Astro II X 9 A 17

> 39.9±14. 39

Pts: Patients; GBM: Glioblastomas, Astro: Astrocytomas; RTV: radiotherapeutic volume; Gy: Gray; D: dead, A: Alive; Ms:

Computer tomographic (CT) scans using a spiral CT scanner and magnetic resonance scanner (MR) were performed on each patients. These scans are elaborated for determin‐

62.91±27. 95

**Cycle**

**A/D Survival**

http://dx.doi.org/10.5772/55966

13A-12D 39.44±34.2

5

**Ms**

287

**PTS Year Gender Histology TMZ CCNU ECOG Gy RTV Ner HT**

stabilization.

25> 9F- 16M 11GBM, 6

**1.1. Conformal radiotherapy**

months

Astro IV, 8 Astro II

**Table 1.** Characteristics of patients with GBM – ASTRO- II- IV treated with CFRT+ HT

Another approach at the moment not completely in use is immunotherapy [9, 10]. EGFR is expressedin nearthe 50% ofpatients withGBM[11].Inhibitors ofEGFR, like Iressa andTarceva have beenused with limitedsuccess [12].Aphase II study withGefitinib has been conductedby Rich et al. in which fifty-seven patients have been treated orally once daily with 500 mg of oral Iressa®. No objective responses were seen, possibly because only 21% of patients had measura‐ ble disease at treatment initiation. Iressa® was well tolerated and a dose increase was thus suggested [13]. Studies with Tarceva® has given similar results with a minimum benefit not superior to standard treatment of care with radiotherapy and TMZ [14]. Another modest response has been obtained inhibiting the PDGF receptors using imatinib (Gleevec®) [10, 16].

Lasty, as angiogenesis due tumor hypoxia is a common processes in GBM [17,18], the inhibitor of Vascular Endothelial Growth Factor (VEGF) bevacizumab (Avastin) has been studied extensively in treating resistant GBM [17, 19]. Avastin has recently been used as single agent or in combination with several drugs, such as carboplatin [20], Ectoposide [21], Lomustine and TMZ [17]. Avastin, is a recombinant monoclonal immunoglobulin able to inhibit VEGF and to normalize tumor vasculature [21,22], but, the promises on GBM have not been confirmed and 98.8% of patients experienced adverse effects such as fatigue, headache, hypertension, thromboembolism, cerebral haemorrhage, convulsion and infarction [22]. Another antiangio‐ genetic factor Thalidomide has been used in GBL [23]. Thalidomide exert its anti tumoral activity through several mechanisms, such as: inhibition of angiogenesis, cytokine-mediated pathways and adhesion molecules modulation, inhibition of cyclooxygenase-2 and stimula‐ tion of immuno response [24]. Another approach is immunotherapy. As known GBL is an immunogenic tumor exerting an immunosuppressive effects on cell mediated immunity partially by regulatory T cells [25,26,27]. This kind of therapy is however experimental and dependent on laboratory skilled personal.

However, none of these association are really superior to TMZ + radiotherapy, regarding survival, and are also becoming cost - prohibitive [28]. Understanding this issue we have started to add hyperthermia (HT) to TMZ and Conformal Radiotherapy (CRT). The basis for adding HT together radiotherapy have been described since 2006 [29]. Hypoxia, the increased apoptosis induced by heat and the additive response to CRT are the most important reasons for adding HT in the treatment of GBM [29]. Animal and human studies have also indicated that there are significant chemosensitizing effects of adding HT to chemotherapy such as nitrosureas derivates [29, 30]. Previous studies not yet published by our group have shown that HT with TMZ is additive. We have thus be (TMZ) (5F,11 M median age 44.64 ± 9.95 yr), and were eligible to be compared with 15 patients with (GBM) treated with CFRT plus TMZ (see table 2)( 15 GBM, 7F,8M; 52.13±16.17 yr). Four of them (Astrocytoma IV ) were previous treated with CCNU, with disease progression (see. Table. 1). All patients had a histological proven malignant GBM or anaplastic astrocytomas. Since 2001, we have begun to use capac‐ itive HT in association to TMZ and conformal radiotherapy. Follow up of patients was collected at a mean interval of 65 days, with a large range [40-90] days for those patients with a disease stabilization.


Pts: Patients; GBM: Glioblastomas, Astro: Astrocytomas; RTV: radiotherapeutic volume; Gy: Gray; D: dead, A: Alive; Ms: months

**Table 1.** Characteristics of patients with GBM – ASTRO- II- IV treated with CFRT+ HT

#### **1.1. Conformal radiotherapy**

to TMZ [8]. More recently, a specific and target therapy has been suggested by authors such as Reardon and Wen [3] who have provided a complete list of these inhibitors for this disease including: Gefitinib (Iressa®), Ernolitib (Tarceva®),Thalidomide (Thalidomid®), Bezacizu‐

Another approach at the moment not completely in use is immunotherapy [9, 10]. EGFR is expressedin nearthe 50% ofpatients withGBM[11].Inhibitors ofEGFR, like Iressa andTarceva have beenused with limitedsuccess [12].Aphase II study withGefitinib has been conductedby Rich et al. in which fifty-seven patients have been treated orally once daily with 500 mg of oral Iressa®. No objective responses were seen, possibly because only 21% of patients had measura‐ ble disease at treatment initiation. Iressa® was well tolerated and a dose increase was thus suggested [13]. Studies with Tarceva® has given similar results with a minimum benefit not superior to standard treatment of care with radiotherapy and TMZ [14]. Another modest response has been obtained inhibiting the PDGF receptors using imatinib (Gleevec®) [10, 16].

Lasty, as angiogenesis due tumor hypoxia is a common processes in GBM [17,18], the inhibitor of Vascular Endothelial Growth Factor (VEGF) bevacizumab (Avastin) has been studied extensively in treating resistant GBM [17, 19]. Avastin has recently been used as single agent or in combination with several drugs, such as carboplatin [20], Ectoposide [21], Lomustine and TMZ [17]. Avastin, is a recombinant monoclonal immunoglobulin able to inhibit VEGF and to normalize tumor vasculature [21,22], but, the promises on GBM have not been confirmed and 98.8% of patients experienced adverse effects such as fatigue, headache, hypertension, thromboembolism, cerebral haemorrhage, convulsion and infarction [22]. Another antiangio‐ genetic factor Thalidomide has been used in GBL [23]. Thalidomide exert its anti tumoral activity through several mechanisms, such as: inhibition of angiogenesis, cytokine-mediated pathways and adhesion molecules modulation, inhibition of cyclooxygenase-2 and stimula‐ tion of immuno response [24]. Another approach is immunotherapy. As known GBL is an immunogenic tumor exerting an immunosuppressive effects on cell mediated immunity partially by regulatory T cells [25,26,27]. This kind of therapy is however experimental and

However, none of these association are really superior to TMZ + radiotherapy, regarding survival, and are also becoming cost - prohibitive [28]. Understanding this issue we have started to add hyperthermia (HT) to TMZ and Conformal Radiotherapy (CRT). The basis for adding HT together radiotherapy have been described since 2006 [29]. Hypoxia, the increased apoptosis induced by heat and the additive response to CRT are the most important reasons for adding HT in the treatment of GBM [29]. Animal and human studies have also indicated that there are significant chemosensitizing effects of adding HT to chemotherapy such as nitrosureas derivates [29, 30]. Previous studies not yet published by our group have shown that HT with TMZ is additive. We have thus be (TMZ) (5F,11 M median age 44.64 ± 9.95 yr), and were eligible to be compared with 15 patients with (GBM) treated with CFRT plus TMZ (see table 2)( 15 GBM, 7F,8M; 52.13±16.17 yr). Four of them (Astrocytoma IV ) were previous treated with CCNU, with disease progression (see. Table. 1). All patients had a histological proven malignant GBM or anaplastic astrocytomas. Since 2001, we have begun to use capac‐ itive HT in association to TMZ and conformal radiotherapy. Follow up of patients was collected

mab (Avastin®), and proteasome inhibitors (bortezomid).

286 Cancer Treatment - Conventional and Innovative Approaches

dependent on laboratory skilled personal.

Computer tomographic (CT) scans using a spiral CT scanner and magnetic resonance scanner (MR) were performed on each patients. These scans are elaborated for determin‐ ing the cancer size and shape in 3 dimensions (3D). In this way precisely focused, high dose, radiation beams can be delivered to cancer mass in multiple treatment sessions. Before radiation, patients are fitted with a head frame, meantime CT and MR scans are performed to determine treatment planning. After the acquisition of these informations, patients are positioned on treatment couch and the linear accelerator directs arcs of radioactive photon beams to tumor. The pattern of the arc is computer - matched to the tumor shape using specific multileaf collimators. The CT/MR data of patients are sent to a computerized treatment platform. Through, this platform CT/MR images are fused using an image fusion software and the CTV is defined and contoured manually by clinicians and physicists. In our department, an integration of images with metabolic information such as single photon emission computed tomography (SPECT) has also been used. This permits sometimes to obtain a more accurate tumor visualization. The fusion of images is obtained by a commercial software package (Ergo- 3Dline®). Obtained the CTV, normal‐ ly, a margin over these countered borders must be defined to take into account the possible microscopic extension of the tumour not evidenced on the CT/MR scans. These margins are generally countered 10-30 mm around the CTV obtaining the planned target volume (PTV). After PTV determination a new contour is done ensuring PTV coverage by 95% isodose line with the aim for obtaining a uniform dose homogeneity. Treatment is performed using a Varian Clinac 2100, 6mv. The median dose used for GBM and anaplas‐ tic astrocytomas treated with HT were of 36.87±17.75 Gy versus 39.2 ± 15.57 Gy for patients treated with CFRT+TMZ alone. The radiotherapeutic volume were 54.04 ± 22.53 mm3 versus 85.12 ± 50.37 mm3 respectively.

**2. TMZ administration**

fatty acids and silymarin for preventing radionecrosis.

**3. Rationale for using liposomal doxorubicin**

TMZ was administered orally 200mg/m2 /day on day one to five, after written informed consent. A full blood examination was performed before each new cycle. The treatment cycles were repeated every 28 days. The patients fasted 4 hours before TMZ use, and were submitted to conformal radiotherapy (CFRT), following our standards supplementation with omega-3

Glioblastomas, Astrocytomas: Survival Amelioration Adding Hyperthermia to Conformal Radiotherapy…

http://dx.doi.org/10.5772/55966

289

Temozolomide is an imido-tetrazine readily absorbed orally and able to cross the Blood brain barrier. TMZ has demonstrated activity against Glioblastoma, astrocytoma of various degree and brain metastases [3, 15]. Although TMZ has become the drug of choice in association with radiotherapy for Glioblastoma, many Glioblastoma develop resistance to the drug and become incurable. The reasons for this resistance is at the moment not completely understood and seems linked to the presence of certain subpopulations of cancer- stem cells inside the tumor mass [32]. This possible resistance has forced our group to look for other drugs active on GBM. We have chosed liposomal doxorubicin for various reasons that we will be describe here.

Liposomal doxorubicin (Caelyx®), is a formulation of hydrochloride doxorubicin wrap‐ ped by a film composed by phospholipids and polymers of methoxypolyethylene (mPEG) embedded in the lipid surface [33]. This association determines favorable pharmacokinet‐ ic profile characterised by an extended circulation time, a reduced volume of distribu‐ tion, thereby promoting an increased tumour uptake [34, 35]. Tumor abnormal microcirculation and permeability is responsible for the increased uptake and retention of liposomal drugs, see Maeda [36]. This phenomenon is greatly increased by Hyperther‐ mia, as demonstrated by Ponce and Dvorak [37, 38]. Dvorak was one of the first to use the combination of Caelyx® and hyperthermia on hepatocarcinoma and after his study reported that the combination of hyperthermia and doxorubicin itself may be supraadditive, resulting in enhanced antitumor efficacy in the heated region and in decrease of toxicity [38]. Caelyx® has been investigated by Koukoukaris in glioblastoma and in metastatic brain tumours [39]. These authors in agreement with Chua and Lesniak group concluded that liposomal doxorubicin selectively overcome the blood brain barrier and accumulate 13-19 times higher in the Glioblastoma [40, 41]. Furthermore, Chua [40] has demonstated the possibility of using Caelyx® in association with temozolomide in recurrent Glioblastoma. Liposomal doxorubicin was associated with disease stabilization and a modest haematologic toxicity. These studies have convinced our group to use pegylated doxorubicin in a recurrent case of GBM. The case is here briefly described.

#### **1.2. Capacitive hyperthermia**

For treating our patients we used a device (Synchrotherm) developed by Duer ®, Vigeva‐ no, Italy. This device consists of the following components: 1) a Radiofrequency genera‐ tor (13.56 Mhz) 2) a pair of mobile plates or electrodes with independent superficial cooling system, 3) a heat exchanger, 4) a computerized control console. The thermal profiles to obtain a probable deposition of the energy were obtained by heating patterns produced in a static phantoms. A cylindrical phantom made of 4% agar gel plus 02% NaCl was made and the various isotherms were monitored and reconstructed through computerizations of images obtained by a special film sensible to temperature. A flexible vinyl sheet, forming a space filled with 0.4% NaCl solution, covered the surfaces of the electrodes. The saline solution circulated between the electrode and the heat exchangers. Differently to other cooling system, the two electrodes were independently controlled and were simply adaptable to the contour of the brain patients, thanks to their flexibility These plates are coupled to opposite side of the patient 's brain and kept in place thanks to a girdle permitting a better contact over the irregularity of the skull contour. Capacitive hyperther‐ mia treatment were given to the patients 30 ' after the CFRT; during the HT session 500cc of mannitol at 19%, plus 4-8 mg of desamethazone were infused. HT treatments lasted for all patients 1 hour, and for five days consecutively. Some of these patients received more than one HT treatment/month (median 3 maximum 5 HT treatments).

### **2. TMZ administration**

ing the cancer size and shape in 3 dimensions (3D). In this way precisely focused, high dose, radiation beams can be delivered to cancer mass in multiple treatment sessions. Before radiation, patients are fitted with a head frame, meantime CT and MR scans are performed to determine treatment planning. After the acquisition of these informations, patients are positioned on treatment couch and the linear accelerator directs arcs of radioactive photon beams to tumor. The pattern of the arc is computer - matched to the tumor shape using specific multileaf collimators. The CT/MR data of patients are sent to a computerized treatment platform. Through, this platform CT/MR images are fused using an image fusion software and the CTV is defined and contoured manually by clinicians and physicists. In our department, an integration of images with metabolic information such as single photon emission computed tomography (SPECT) has also been used. This permits sometimes to obtain a more accurate tumor visualization. The fusion of images is obtained by a commercial software package (Ergo- 3Dline®). Obtained the CTV, normal‐ ly, a margin over these countered borders must be defined to take into account the possible microscopic extension of the tumour not evidenced on the CT/MR scans. These margins are generally countered 10-30 mm around the CTV obtaining the planned target volume (PTV). After PTV determination a new contour is done ensuring PTV coverage by 95% isodose line with the aim for obtaining a uniform dose homogeneity. Treatment is performed using a Varian Clinac 2100, 6mv. The median dose used for GBM and anaplas‐ tic astrocytomas treated with HT were of 36.87±17.75 Gy versus 39.2 ± 15.57 Gy for patients treated with CFRT+TMZ alone. The radiotherapeutic volume were 54.04 ± 22.53 mm3

For treating our patients we used a device (Synchrotherm) developed by Duer ®, Vigeva‐ no, Italy. This device consists of the following components: 1) a Radiofrequency genera‐ tor (13.56 Mhz) 2) a pair of mobile plates or electrodes with independent superficial cooling system, 3) a heat exchanger, 4) a computerized control console. The thermal profiles to obtain a probable deposition of the energy were obtained by heating patterns produced in a static phantoms. A cylindrical phantom made of 4% agar gel plus 02% NaCl was made and the various isotherms were monitored and reconstructed through computerizations of images obtained by a special film sensible to temperature. A flexible vinyl sheet, forming a space filled with 0.4% NaCl solution, covered the surfaces of the electrodes. The saline solution circulated between the electrode and the heat exchangers. Differently to other cooling system, the two electrodes were independently controlled and were simply adaptable to the contour of the brain patients, thanks to their flexibility These plates are coupled to opposite side of the patient 's brain and kept in place thanks to a girdle permitting a better contact over the irregularity of the skull contour. Capacitive hyperther‐ mia treatment were given to the patients 30 ' after the CFRT; during the HT session 500cc of mannitol at 19%, plus 4-8 mg of desamethazone were infused. HT treatments lasted for all patients 1 hour, and for five days consecutively. Some of these patients received more

than one HT treatment/month (median 3 maximum 5 HT treatments).

versus 85.12 ± 50.37 mm3 respectively.

288 Cancer Treatment - Conventional and Innovative Approaches

**1.2. Capacitive hyperthermia**

TMZ was administered orally 200mg/m2 /day on day one to five, after written informed consent. A full blood examination was performed before each new cycle. The treatment cycles were repeated every 28 days. The patients fasted 4 hours before TMZ use, and were submitted to conformal radiotherapy (CFRT), following our standards supplementation with omega-3 fatty acids and silymarin for preventing radionecrosis.

### **3. Rationale for using liposomal doxorubicin**

Temozolomide is an imido-tetrazine readily absorbed orally and able to cross the Blood brain barrier. TMZ has demonstrated activity against Glioblastoma, astrocytoma of various degree and brain metastases [3, 15]. Although TMZ has become the drug of choice in association with radiotherapy for Glioblastoma, many Glioblastoma develop resistance to the drug and become incurable. The reasons for this resistance is at the moment not completely understood and seems linked to the presence of certain subpopulations of cancer- stem cells inside the tumor mass [32]. This possible resistance has forced our group to look for other drugs active on GBM. We have chosed liposomal doxorubicin for various reasons that we will be describe here.

Liposomal doxorubicin (Caelyx®), is a formulation of hydrochloride doxorubicin wrap‐ ped by a film composed by phospholipids and polymers of methoxypolyethylene (mPEG) embedded in the lipid surface [33]. This association determines favorable pharmacokinet‐ ic profile characterised by an extended circulation time, a reduced volume of distribu‐ tion, thereby promoting an increased tumour uptake [34, 35]. Tumor abnormal microcirculation and permeability is responsible for the increased uptake and retention of liposomal drugs, see Maeda [36]. This phenomenon is greatly increased by Hyperther‐ mia, as demonstrated by Ponce and Dvorak [37, 38]. Dvorak was one of the first to use the combination of Caelyx® and hyperthermia on hepatocarcinoma and after his study reported that the combination of hyperthermia and doxorubicin itself may be supraadditive, resulting in enhanced antitumor efficacy in the heated region and in decrease of toxicity [38]. Caelyx® has been investigated by Koukoukaris in glioblastoma and in metastatic brain tumours [39]. These authors in agreement with Chua and Lesniak group concluded that liposomal doxorubicin selectively overcome the blood brain barrier and accumulate 13-19 times higher in the Glioblastoma [40, 41]. Furthermore, Chua [40] has demonstated the possibility of using Caelyx® in association with temozolomide in recurrent Glioblastoma. Liposomal doxorubicin was associated with disease stabilization and a modest haematologic toxicity. These studies have convinced our group to use pegylated doxorubicin in a recurrent case of GBM. The case is here briefly described.

### **4. Case of patient treated with Caelyx**

The patient (right handed man) was first surgically treated (Dec 2005) for left posterior parietal Glioblastoma then the patient underwent RT (45 Gy CFRT in 18 fractions followed by a boost CFRT to reduced target of 20 Gy in 4 fractions) and started and continued Temodar (10 cycles) until progression (Jan 07) followed by ACNU (2 Cycles) until progression (March 2007) and then we started LD and Radiofrequency Hyperthermia (HT).

**Pts Gender Age RTV(cm3) Gy D / A Ms Survival**

Glioblastomas, Astrocytomas: Survival Amelioration Adding Hyperthermia to Conformal Radiotherapy…

http://dx.doi.org/10.5772/55966

291

15 7F- 8M 52.13±16.177 85.12±50.37 39.2±15.57 15 D 14.87±10.7

**Figure 1.** Survival curve of patients treated with HT + CFRT + Temodal, compared with CFRT + Temodal

Pts: Patients; RTV: radiotherapeutic volume; Gy: Gray; D: dead, A: Alive; Ms: months

**Table 2.** Characteristics of Patients with GBM treated with CFRT + TMZ

1 F 30 31.6 36.4 D 30 2 M 52 65.4 20 D 13 3 F 64 108 40.5 D 37 4 M 22 56.8 25 D 9 5 M 69 76.9 45.6 D 15 6 M 57 64.1 70 D 1 7 F 61 175 18 D 2 8 F 65 64.5 35 D 10 9 F 47 19.8 54 D 25 10 M 66 136.9 45.5 D 4 11 M 50 226 42 D 13 12 F 45 19.9 54 D 19 13 M 67 67.9 20.4 D 26 14 M 65 119 20 D 14 15 F 22 45 45 D 5

During the first period the cycles were done at 45 days interval then, after a initial good response and stabilization, the treatment was done at larger interval up to 9 CT+HT (the last treatment was done in Nov 2007).

The treatment was as follows: 12 mgm2 IV + steroids in glucose solution and assumption of 200 mg of Quercetin in day 1 and one hour of HT at least four hour later. From day 2 to 5 the patient underwent 4 further consecutive days of HT and quercetin (100 mg before and after the completion of treatment).

HT was delivered by means of a 13.56 MHz radiofrequency capacitive device (Synchrotherm Duer) via two opposite plates at the maximum tolerated power for at least one hour for five consecutive days.

### **4.1. Statistical analysis**

Survival curves were calculated according to Kaplan-Meier method, Starting on the first day of HT. Survival was compared using the log-rank test and the K-test. Significance was posed as p ≤ 0.05 [11].

### **4.2. Results**

As it is possible to see comparing table 1 and table 2 there is no statistical difference between the two groups regarding age, gy administered and volume of tumour treated. The difference on survival curves (see Fig. 1) using the log-rank test and the K-test are important and more than the 50% of patients treated with CFRT+HT+TMZ are alive at 26 months. We can object than the comparison of the two groups is not completely homogeneous. In fact, the group in table 2 treated with TMZ + CFRT are only GBM, on the contrary in table 1 there is the presence of Astrocytoma of II degree. At a second look at table 1, an important observation emerges however, the GBM group has a long survival that is sometimes 3 times that of table 2, see pts 1, 3 table 2 and pts 3,7,10 of table1. It seems clear that HT adds something regarding survival and no side effects have been reported by patients. The patient treated with Caelyx® after surgery Dec 2005, had progression of the disease. He received CFRT in February 2006 and 10 cycles of Temodal. The patient on March 2009 shows progression and possible resistance to Temodal, we decided to treat the patient with 11 cycles of Caelyx + HT. The patient situation is illustrated in Fig 1( progression of disease after 10 cycle of Temodal, 2006) and Fig 2 after 11 cycles of Caelyx + HT (2009). The decrease of volume is evident. The patient is still alive and in treatment with a dose of 80 mg of Talidomide taken orally at evening, as a single dose.

Glioblastomas, Astrocytomas: Survival Amelioration Adding Hyperthermia to Conformal Radiotherapy… http://dx.doi.org/10.5772/55966 291


Pts: Patients; RTV: radiotherapeutic volume; Gy: Gray; D: dead, A: Alive; Ms: months

**Table 2.** Characteristics of Patients with GBM treated with CFRT + TMZ

**4. Case of patient treated with Caelyx**

290 Cancer Treatment - Conventional and Innovative Approaches

treatment was done in Nov 2007).

the completion of treatment).

consecutive days.

as p ≤ 0.05 [11].

**4.2. Results**

**4.1. Statistical analysis**

The treatment was as follows: 12 mgm2

then we started LD and Radiofrequency Hyperthermia (HT).

The patient (right handed man) was first surgically treated (Dec 2005) for left posterior parietal Glioblastoma then the patient underwent RT (45 Gy CFRT in 18 fractions followed by a boost CFRT to reduced target of 20 Gy in 4 fractions) and started and continued Temodar (10 cycles) until progression (Jan 07) followed by ACNU (2 Cycles) until progression (March 2007) and

During the first period the cycles were done at 45 days interval then, after a initial good response and stabilization, the treatment was done at larger interval up to 9 CT+HT (the last

200 mg of Quercetin in day 1 and one hour of HT at least four hour later. From day 2 to 5 the patient underwent 4 further consecutive days of HT and quercetin (100 mg before and after

HT was delivered by means of a 13.56 MHz radiofrequency capacitive device (Synchrotherm Duer) via two opposite plates at the maximum tolerated power for at least one hour for five

Survival curves were calculated according to Kaplan-Meier method, Starting on the first day of HT. Survival was compared using the log-rank test and the K-test. Significance was posed

As it is possible to see comparing table 1 and table 2 there is no statistical difference between the two groups regarding age, gy administered and volume of tumour treated. The difference on survival curves (see Fig. 1) using the log-rank test and the K-test are important and more than the 50% of patients treated with CFRT+HT+TMZ are alive at 26 months. We can object than the comparison of the two groups is not completely homogeneous. In fact, the group in table 2 treated with TMZ + CFRT are only GBM, on the contrary in table 1 there is the presence of Astrocytoma of II degree. At a second look at table 1, an important observation emerges however, the GBM group has a long survival that is sometimes 3 times that of table 2, see pts 1, 3 table 2 and pts 3,7,10 of table1. It seems clear that HT adds something regarding survival and no side effects have been reported by patients. The patient treated with Caelyx® after surgery Dec 2005, had progression of the disease. He received CFRT in February 2006 and 10 cycles of Temodal. The patient on March 2009 shows progression and possible resistance to Temodal, we decided to treat the patient with 11 cycles of Caelyx + HT. The patient situation is illustrated in Fig 1( progression of disease after 10 cycle of Temodal, 2006) and Fig 2 after 11 cycles of Caelyx + HT (2009). The decrease of volume is evident. The patient is still alive and in treatment with a dose of 80 mg of Talidomide taken orally at evening, as a single dose.

IV + steroids in glucose solution and assumption of

**Figure 1.** Survival curve of patients treated with HT + CFRT + Temodal, compared with CFRT + Temodal

**5. Conclusions**

**Author details**

**References**

533-43.

Gianfranco Baronzio1\*, Gurdev Parmar2

\*Address all correspondence to: barongf@intercom.it

2 Integrated Health Clinic, Fort Langley, B.C., Canada

Opin Cell Biol. (2009). Apr;, 21(2), 311-6.

1 Centro Medico Kines, Castano primo, (Mi), Italy

Our study, is in agreement with the previous studies on HT [29], and show that the association with themozolomide is feasible and that this triple treatment (Chemotherapy, Conformal Radiotherapy and hyperthermia) represents a promising new approach to the treatment of glioblastomas. In this initial work we have not analyzed patients treated with hyperthermia without TMZ, so we cannot understand if the increase in life survival must be ascribed simply to HT or to the combination with temozolamide. The quantity of patients is limited but we are encouraged to use external capacitive HT in association with the standard therapy of our institution. In a near future, we hope to be able to distinguish the effects of the two therapies. We hope also to analyze the compliance of the patients to hyperthermia and to be able to study the side effects of HT on brain. We want to point out that most of works done on brain and hyperthermia regards studies with interstitial HT [29]. Interstitial HT represents to our opinion a limit to a larger use of this technique in association with CFRT and chemotherapy, whereas

Glioblastomas, Astrocytomas: Survival Amelioration Adding Hyperthermia to Conformal Radiotherapy…

, Michela De Santis3

[1] Parkin, D. M. Global cancer statistics in the year 2000. Lancet Oncol. (2001). Sep;, 2(9),

[2] Lino, M, & Merlo, A. Translating biology into clinic: the case of Glioblastoma. Curr

[3] Reardon, D. A, & Wen, P. Y. Therapeutic advances in the treatment of Glioblastoma: rationale and potential role of targeted agents. Oncologist. (2006). Feb;, 11(2), 152-64.

[4] Kesari, S. Understanding glioblastoma tumor biology: the potential to improve cur‐ rent diagnosis and treatments. Semin Oncol. (2011). Dec;38 Suppl 4:S, 2-10.

and Alberto Gramaglia3

http://dx.doi.org/10.5772/55966

293

capacitive HT show a simpler use and seems to be well tolerated by patients.

3 Radiotherapy and Hyperthermia Department, Policlinico di Monza, Monza, Italy

**Figure 2.** a. Patient with recurrent and drug resistance before Hyperthermia and Caelyx treatmen. b. Decrease of GBM volume after Hyperthermia and Caelyx treatment

### **5. Conclusions**

Our study, is in agreement with the previous studies on HT [29], and show that the association with themozolomide is feasible and that this triple treatment (Chemotherapy, Conformal Radiotherapy and hyperthermia) represents a promising new approach to the treatment of glioblastomas. In this initial work we have not analyzed patients treated with hyperthermia without TMZ, so we cannot understand if the increase in life survival must be ascribed simply to HT or to the combination with temozolamide. The quantity of patients is limited but we are encouraged to use external capacitive HT in association with the standard therapy of our institution. In a near future, we hope to be able to distinguish the effects of the two therapies. We hope also to analyze the compliance of the patients to hyperthermia and to be able to study the side effects of HT on brain. We want to point out that most of works done on brain and hyperthermia regards studies with interstitial HT [29]. Interstitial HT represents to our opinion a limit to a larger use of this technique in association with CFRT and chemotherapy, whereas capacitive HT show a simpler use and seems to be well tolerated by patients.

### **Author details**

(a)

292 Cancer Treatment - Conventional and Innovative Approaches

(b)

volume after Hyperthermia and Caelyx treatment

**Figure 2.** a. Patient with recurrent and drug resistance before Hyperthermia and Caelyx treatmen. b. Decrease of GBM

Gianfranco Baronzio1\*, Gurdev Parmar2 , Michela De Santis3 and Alberto Gramaglia3


### **References**


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[6] Ekstrand, A. J, Sugawa, N, James, C. D, & Collins, V. P. Amplified and rearranged epidermal growth factor receptor genes in human glioblastomas reveal deletions of sequences encoding portions of the N- and/or C-terminal tails. Proc Natl Acad Sci U

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[13] Rich, J. N, Reardon, D. A, Peery, T, Dowell, J. M, Quinn, J. A, Penne, K. L, Wikstrand, C. J, Van Duyn, L. B, Dancey, J. E, Mclendon, R. E, Kao, J. C, & Stenzel, T. T. Ahmed Rasheed BK,Tourt-Uhlig SE, Herndon JE 2nd, Vredenburgh JJ, Sampson JH, Fried‐ man AH, Bigner DD, Friedman HS. Phase II trial of gefitinib in recurrent glioblasto‐

[14] Raizer, J. J, Abrey, L. E, Lassman, A. B, Chang, S. M, Lamborn, K. R, Kuhn, J. G, Yung, W. K, Gilbert, M. R, Aldape, K. A, Wen, P. Y, Fine, H. A, Mehta, M, Deangelis, L. M, Lieberman, F, Cloughesy, T. F, Robins, H. I, & Dancey, J. Prados MD; North American Brain TumorConsortium. A phase II trial of erlotinib in patients with re‐ current malignant gliomas and nonprogressive glioblastoma multiforme postradia‐

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ing a chimera? Expert Opin Investig Drugs. (2011). Jul;, 20(7), 881-95.

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**Section 3**

**The Immunotherapy of Cancer**


## **The Immunotherapy of Cancer**

on malignat Brain tumors. In Hyperthermia in Cancer treatment. A primer. Baronzio

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[35] Holloway, R. W, Grendys, E. C, Lefebvre, P, Vekeman, F, & Mcmeekin, S. Tolerabili‐ ty,efficacy, and safety of pegylated liposomal Doxorubicin in combination with Car‐ boplatin versus gemcitabine-Carboplatin for the treatment of platinum-sensitive recurrent ovarian cancer: a systematic review. Oncologist.(2010). , 15(10), 1073-82.

[36] Maeda, H. Macromolecular therapeutics in cancer treatment: The EPR effect and be‐

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[38] Dvorak, J, Zoul, Z, Melichar, B, Petera, J, Vesely, P, Vosmik, M, & Dolezel, M. Liposo‐ mal doxorubicin combined with regional hyperthermia: reducing systemic toxicity and improving locoregional efficacy in the treatment of solid tumors. J Chemother.

[39] Koukourakis, M. I, Koukouraki, S, Fezoulidis, I, Kelekis, N, Kyrias, G, Archimandri‐ tis, S, & Karkavitsas, N. High intratumoural accumulation of stealth liposomal dox‐ orubicin (Caelyx) in glioblastomas and in metastatic brain tumours. Br J Cancer.

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**Chapter 14**

**Targeted Cancer Therapy by Dendritic Cell Vaccine**

The unpredictability of efficacy and toxicity of treatment are limitations of current standard cancer treatments. The clinical results obtained by standard therapies suggest a need for a paradigm change in cancer treatment. In recent years, immune cell therapy has been in the spotlight with the expectation of opening the door to a new area of cancer therapy. Targeted cancer therapy, which selectively takes action against targets expressed in the tumor surface,

The immune system can control various types of tumors. Antigen-non-specific innate immun‐

The identification of tumor antigens recognized by T cells has facilitated the development of immune cell therapy in clinical oncology. The dendritic cell based cancer vaccine aims to induce tumor specific effector T cells (cytotoxic T lymphocyte, CTL) that can reduce tumor

In this text, immune cell target therapy in clinical oncology will be discussed and hopefully

Natural killer (NK) cells are present in the peripheral blood and number approximately 10-15% in the lymphocyte fraction. NK cells are the most important innate immune cell because of their ability to directly kill target cells as well as produce immunoregulatory cytokines. NK cells are defined by the surface expression of CD56, a neural cell adhesion molecule and lacks

and reproduction in any medium, provided the original work is properly cited.

© 2013 Abe et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

mass as well as tumor specific memory T cells that can control tumor relapse.

Hiroyuki Abe, Touko Shimamoto, Shinichiro Akiyama and Minako Abe

http://dx.doi.org/10.5772/55450

**1. Introduction**

seems to be promising.

Additional information is available at the end of the chapter

ity and antigen-specific adaptive immunity can reject tumors.

this will be helpful in daily clinical practice.

**2. Role of natural killer cells**

### **Targeted Cancer Therapy by Dendritic Cell Vaccine**

Hiroyuki Abe, Touko Shimamoto, Shinichiro Akiyama and Minako Abe

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55450

**1. Introduction**

The unpredictability of efficacy and toxicity of treatment are limitations of current standard cancer treatments. The clinical results obtained by standard therapies suggest a need for a paradigm change in cancer treatment. In recent years, immune cell therapy has been in the spotlight with the expectation of opening the door to a new area of cancer therapy. Targeted cancer therapy, which selectively takes action against targets expressed in the tumor surface, seems to be promising.

The immune system can control various types of tumors. Antigen-non-specific innate immun‐ ity and antigen-specific adaptive immunity can reject tumors.

The identification of tumor antigens recognized by T cells has facilitated the development of immune cell therapy in clinical oncology. The dendritic cell based cancer vaccine aims to induce tumor specific effector T cells (cytotoxic T lymphocyte, CTL) that can reduce tumor mass as well as tumor specific memory T cells that can control tumor relapse.

In this text, immune cell target therapy in clinical oncology will be discussed and hopefully this will be helpful in daily clinical practice.

### **2. Role of natural killer cells**

Natural killer (NK) cells are present in the peripheral blood and number approximately 10-15% in the lymphocyte fraction. NK cells are the most important innate immune cell because of their ability to directly kill target cells as well as produce immunoregulatory cytokines. NK cells are defined by the surface expression of CD56, a neural cell adhesion molecule and lacks

the T cell antigen CD3 [1]. The function of NK cells are direct cytotoxic activity against virusinfected cells and tumor cells [2]. There are two distinct subsets of human NK cells based on the density of surface CD56 expression [3]. Approximately 90% of human NK cells are CD 56dim and have high density expression of CD16, others are CD56bright and CD16dim/neg. The CD56bright and CD56dim NK cell subsets show an important difference in cytotoxic potential, capacity for cytokine production and response to cytokine activation (Table 1) [4].

type lectin family. In this text, these receptors are not discussed in order to simplify the clinical application of NK cell therapy. Although the activating KIR and CD95/NKG2 receptors are important in mediating NK cytotoxicity, Natural Cytotoxicity Receptors (NCR) and the homodimeric NKG 2D receptors may be important in mediating cytotoxicity against abnormal, MHC class I-deficient, or class I-negative targets. This biological information will result in the clinical application of NK cell-based therapies for cancer. Table 2 shows the incidence of MHC

Targeted Cancer Therapy by Dendritic Cell Vaccine

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301

**Tumor cell Incidence (%)**

Primary 52 Metastatic 88

Primary 32 Metastatic 72

Primary 16 Metastatic 58

Uterine cervical cancer 90

Breast cancer 81 Pancreatic cancer 76 Prostate cancer 74

Sarcoma 62

Head and neck cancer 49 Hepatoma 42 Renal cell carcinoma 38 Lung cancer 38 Ovarian cancer 37 Pharyngeal cancer 56 Urinary bladder cancer 25

class I deficient or negative cancer cells.

Osteosarcoma

Colon cancer

Melanoma

**Table 2.** Incidence of MHC class I deficient or negative cancer cells [11]


\*KIR indicated killer immunoglobulin-like receptor; IL, interleukin; ADCC, antibody-dependent cellular cytotoxicity.

**Table 1.** Functional Differences in Natural Killer (NK) cell Subsets\* [2]

NK cells can mediate antibody-dependent cellular cytotoxicity (ADCC) through membrane FcγRIII (CD16) expressed on the majority of NK cells. CD56dim NK cells are more cytotoxic against NK-sensitive targets than CD56bright NK cells and respond to IL-2 with increased cytotoxicity. It is of clinical importance to know that CD56 bright cells, after activation with IL-2, can exhibit similar or enhanced cytotoxicity against NK targets compared with CD56dim cells [5-7]. In addition, more than 95% of all CD56dim NK cells express CD16 (FcγRIII) and are capable of ADCC. On the other hand, 50% to 70% of CD56bright NK cells lack expression of CD16 or have only low-density expression of CD16 and therefore function minimally in ADCC.

It is well known that major histocompatibility complex (MHC) class I molecules are critical for the inhibition of NK cell-mediated lysis of normal autologous cells [8, 9]. NK cells selectively lyse autologous cell that have lost MHC class I self-expression [10].

In humans, two families of paired inhibitory and activating NK receptors have been identified, killer immunoglobulin (Ig)-like receptor (KIR) family and the heterodimeric CD94/NKG2 C- type lectin family. In this text, these receptors are not discussed in order to simplify the clinical application of NK cell therapy. Although the activating KIR and CD95/NKG2 receptors are important in mediating NK cytotoxicity, Natural Cytotoxicity Receptors (NCR) and the homodimeric NKG 2D receptors may be important in mediating cytotoxicity against abnormal, MHC class I-deficient, or class I-negative targets. This biological information will result in the clinical application of NK cell-based therapies for cancer. Table 2 shows the incidence of MHC class I deficient or negative cancer cells.

the T cell antigen CD3 [1]. The function of NK cells are direct cytotoxic activity against virusinfected cells and tumor cells [2]. There are two distinct subsets of human NK cells based on the density of surface CD56 expression [3]. Approximately 90% of human NK cells are CD 56dim and have high density expression of CD16, others are CD56bright and CD16dim/neg. The CD56bright and CD56dim NK cell subsets show an important difference in cytotoxic potential,

**CD56bright CD56dim**

capacity for cytokine production and response to cytokine activation (Table 1) [4].

FcγRIII (CD16) -/+ + + + KIR -/+ + + + CD94/NKG2 + -/+

IL-2Rαβγ + + - IL-2Rβγ + + + + CCR7 + + - Adhesion molecules + + -/+

ADCC -/+ + + + Natural cytotoxicity -/+ + + + Cytokine production + + + -/+

**Table 1.** Functional Differences in Natural Killer (NK) cell Subsets\* [2]

\*KIR indicated killer immunoglobulin-like receptor; IL, interleukin; ADCC, antibody-dependent cellular cytotoxicity.

NK cells can mediate antibody-dependent cellular cytotoxicity (ADCC) through membrane FcγRIII (CD16) expressed on the majority of NK cells. CD56dim NK cells are more cytotoxic against NK-sensitive targets than CD56bright NK cells and respond to IL-2 with increased cytotoxicity. It is of clinical importance to know that CD56 bright cells, after activation with IL-2, can exhibit similar or enhanced cytotoxicity against NK targets compared with CD56dim cells [5-7]. In addition, more than 95% of all CD56dim NK cells express CD16 (FcγRIII) and are capable of ADCC. On the other hand, 50% to 70% of CD56bright NK cells lack expression of CD16 or have only low-density expression of CD16 and therefore function minimally in ADCC.

It is well known that major histocompatibility complex (MHC) class I molecules are critical for the inhibition of NK cell-mediated lysis of normal autologous cells [8, 9]. NK cells selectively

In humans, two families of paired inhibitory and activating NK receptors have been identified, killer immunoglobulin (Ig)-like receptor (KIR) family and the heterodimeric CD94/NKG2 C-

lyse autologous cell that have lost MHC class I self-expression [10].

NK receptors

300 Cancer Treatment - Conventional and Innovative Approaches

Cytokine receptors

Effector functions


**Table 2.** Incidence of MHC class I deficient or negative cancer cells [11]

It is presumed that the difference of MHC class I expression may be the difference in the clinical efficacy of NK cell based immune therapy. Because of the variety of cancer cells, it is not enough to kill all cancer cells by NK cell-based therapy. It is necessary to target a cancer specific antigen by cytotoxic T cells, which are activated by a Dendritic Cell (DC) based cancer vaccine (Figure 1).

**Figure 1.** Cytotoxicity of NK cells [11]

### **3. Strategies using dendritic cell vaccine**

Dendritic cells (DCs) are key regulators of both T- and B-cell immunity, because of their superior ability to take up, process and present antigens compared with other antigen presenting cells (APCs) [12]. DCs can also activate NK cells and natural killer T (NKT) cells [13]. Because of these functions, DCs can conduct all of the elements of the immune orchestra and they are therefore a fundamental target and tool for vaccines [14]. Cancer related antigens are a key factor implicated in the design of DC vaccine strategies. If a patient's own cancer cells are available for lysate, this will be used for the production of an individual DC-based vaccine, which is utilized for the optimally matched tumor surface antigen. In most instances, however, if a patient's own cancer cells are not available, then artificial cancer antigens are utilized for the production of a DC-based cancer vaccine.

A pilot project by the National Cancer Institute reported on the prioritization of cancer antigens to develop a well-vetted, priority-ranked list of cancer vaccine target antigens based on predefined and preweighted objective criteria [15]. Antigen prioritization involves developing a list of ideal cancer antigen criteria (Figure 2).

**Figure 2.** Cancer antigen pilot prioritization: representation of ranking based on predefined and preweighted criteria and subcriteria. Inset, the color used to designate each criterion and its relative weight. Number at the end of each

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303

Among these, frequently used artificial tumor antigens are listed below. Some of these are restricted by HLA-A1 or HLA-A24, so that an HLA study is needed to select and match the

bar, relative rank of that antigen. [15]

It is presumed that the difference of MHC class I expression may be the difference in the clinical efficacy of NK cell based immune therapy. Because of the variety of cancer cells, it is not enough to kill all cancer cells by NK cell-based therapy. It is necessary to target a cancer specific antigen by cytotoxic T cells, which are activated by a Dendritic Cell (DC)-

Dendritic cells (DCs) are key regulators of both T- and B-cell immunity, because of their superior ability to take up, process and present antigens compared with other antigen presenting cells (APCs) [12]. DCs can also activate NK cells and natural killer T (NKT) cells [13]. Because of these functions, DCs can conduct all of the elements of the immune orchestra and they are therefore a fundamental target and tool for vaccines [14]. Cancer related antigens are a key factor implicated in the design of DC vaccine strategies. If a patient's own cancer cells are available for lysate, this will be used for the production of an individual DC-based vaccine, which is utilized for the optimally matched tumor surface antigen. In most instances, however, if a patient's own cancer cells are not available, then artificial cancer antigens are utilized for

A pilot project by the National Cancer Institute reported on the prioritization of cancer antigens to develop a well-vetted, priority-ranked list of cancer vaccine target antigens based on predefined and preweighted objective criteria [15]. Antigen prioritization involves developing

based cancer vaccine (Figure 1).

302 Cancer Treatment - Conventional and Innovative Approaches

**Figure 1.** Cytotoxicity of NK cells [11]

**3. Strategies using dendritic cell vaccine**

the production of a DC-based cancer vaccine.

a list of ideal cancer antigen criteria (Figure 2).

**Figure 2.** Cancer antigen pilot prioritization: representation of ranking based on predefined and preweighted criteria and subcriteria. Inset, the color used to designate each criterion and its relative weight. Number at the end of each bar, relative rank of that antigen. [15]

Among these, frequently used artificial tumor antigens are listed below. Some of these are restricted by HLA-A1 or HLA-A24, so that an HLA study is needed to select and match the tumor antigens to the DCs (Table 3). Certain artificial tumor antigens cannot apply depending on HLA types on DCs and tumor cells.

Wild sequence: RMFPNAPYL (126-135), HLA-A2 restricted

lymphoid leukemia, salivary gland and prostate, etc.

lung (adenocarcinoma), prostate and so on.

**Figure 4.** Sequence of MUC1 peptide.

for cancer vaccines [16, 17].

Modified sequence: CYTWNOMNL (235-243), HLA-A24 restricted

The sequence RMFPNAPYL is called wild type and is HLA-A2 restricted. Another sequence CYTWNQMNL is a modified type and is HLA-A24 restricted. WT1 is expressed on the cell surface of carcinomas such as esophageal, gastric, colorectal, pancreas, biliary tract, liver, breast, uterus, brain, lung (non-small cell), malignant melanoma, sarcoma, acute myeloid and

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Another important tumor antigen to be targeted is MUC1 which is the cell membrane associ‐ ated protein. The sequence of this peptide is TRPAPGSTAPPAHGVTSAPDTRPAPGSTAP and is HLA-non restricted (Figure 4). MUC1 is presented over the surface of cancer cells of the esophagus, stomach, colorectal, pancreas, biliary tract, breast, uterus, ovary, salivary gland,

Recently, new therapy strategies that focus on tumor associated antigens (TAAs) have been suggested as additional options to currently available treatments, due to their fewer adverse events and better tolerability. The establishment and maintenance of immune cell therapy for cancer relies on special TAAs, such as WT1 and MUC1 which have become primary targets

The high risk of metastatic recurrence suggests that cancer cell dissemination may occur early in most carcinomas, and therefore it seems that active immunotherapy may have a place among treatment modalities [18]. Among TAAs, above mentioned WT1 and MUC1 have received


**Table 3.** Typical artificial tumor antigens.

WT1 peptide is a part of long chain of WT1 protein (Figure 3).


**Figure 3.** Amino acid sequence of WT1 protein (Total length 449, mass (Da) 49,188)

Wild sequence: RMFPNAPYL (126-135), HLA-A2 restricted

tumor antigens to the DCs (Table 3). Certain artificial tumor antigens cannot apply depending

MUC1 CA125 PSA NY-ESO-2

10 20 30 40 50

60 70 80 90 100

110 120 130 140 150

160 170 180 190 200

210 220 230 240 250

310 320 330 340 350

360 370 380 390 400

410 420 430 440 449

MGSDVRDLNA LLPAVPSLGG GGGCALPVSG AAQWAPVLDF APPGASAYGS

LGGPAPPPAP PPPPPPPPHS FIKQEPSWGG AEPHEEQCLS AFTVHFSGQF

TGTAGACRYG PFGPPPPSQA SSGQARMFPN APYLPSCLES QPAIRNQGYS

TVTFDGTPSY GHTPSHHAAQ FPNHSFKHED PMGQQGSLGE QQYSVPPPVY

GCHTPTDSCT GSQALLLRTP YSSDNLYQMT SQLECYTWNQ MNLGATLKGV

RRVPGVAPTL VRSASETSEK RPFMCAYPGC NKRYFKLSHL QMHSRKHTGE

KPYQCDFKDC ERRFSRSDQL KRHQRRHTGV KPFQCKTCQR KFSRSDHLKT

HTRTHTGKTS EKPFSCRWPS CQKKFARSDE LVRHHNMHQR NMTKLQLAL

**Figure 3.** Amino acid sequence of WT1 protein (Total length 449, mass (Da) 49,188)

260 270 280 290 AAGSSSSVKW TEGQSNHSTG YESDNHTTPI LCGAQYRIHT HGVFRGIQDV

on HLA types on DCs and tumor cells.

304 Cancer Treatment - Conventional and Innovative Approaches

WT1 CEA

HER 2 gp100 NY-ESO-1 PSMA

MAGE-1, MAGE-3

SART-1, SART-3 MART-1 Melan-A HPV16-E7

**Table 3.** Typical artificial tumor antigens.

**HLA-A2 or HLA-A24 restricted Non-restricted**

WT1 peptide is a part of long chain of WT1 protein (Figure 3).

Modified sequence: CYTWNOMNL (235-243), HLA-A24 restricted

The sequence RMFPNAPYL is called wild type and is HLA-A2 restricted. Another sequence CYTWNQMNL is a modified type and is HLA-A24 restricted. WT1 is expressed on the cell surface of carcinomas such as esophageal, gastric, colorectal, pancreas, biliary tract, liver, breast, uterus, brain, lung (non-small cell), malignant melanoma, sarcoma, acute myeloid and lymphoid leukemia, salivary gland and prostate, etc.

Another important tumor antigen to be targeted is MUC1 which is the cell membrane associ‐ ated protein. The sequence of this peptide is TRPAPGSTAPPAHGVTSAPDTRPAPGSTAP and is HLA-non restricted (Figure 4). MUC1 is presented over the surface of cancer cells of the esophagus, stomach, colorectal, pancreas, biliary tract, breast, uterus, ovary, salivary gland, lung (adenocarcinoma), prostate and so on.

Recently, new therapy strategies that focus on tumor associated antigens (TAAs) have been suggested as additional options to currently available treatments, due to their fewer adverse events and better tolerability. The establishment and maintenance of immune cell therapy for cancer relies on special TAAs, such as WT1 and MUC1 which have become primary targets for cancer vaccines [16, 17].

The high risk of metastatic recurrence suggests that cancer cell dissemination may occur early in most carcinomas, and therefore it seems that active immunotherapy may have a place among treatment modalities [18]. Among TAAs, above mentioned WT1 and MUC1 have received particular attention as potential targets for vaccine-based immunotherapy, because with the exception of very few tissues such as the splenic capsule and stroma, they are not expressed in normal human tissues and become activated in a number of cancers [19-21]. Therefore vaccination is an effective medical procedure in clinical oncology, based on the induction of a long-lasting immunologic memory and characterized by mechanisms endowed with high destructive potential and specificity. These functions will elicit a persistent immune memory that can eliminate residual cancer cells and protect against relapses.

WT1 20μg/mL MUC1, long peptide (30-mer) 20μg/mL CEA peptides 20μg/mL CA125 protein 500μg/mL HER2 20μg/mL Autologous tumor lysates 50μg/mL

determined using flow cytometry. The cells defined as mature DCs are CD14-

and CCR7+

To prepare the autologous tumor lysates, tumor masses were obtained by surgical resection exclusion and are homogenized. Aliquots of isolated tumor cells were then lysed by 10 cycles of repeated freeze in liquid nitrogen and thaw in a 37deg C water bath. The lysed cells were centrifuged at 14,000G for 5 min, and supernatants are passed through a 0.22µm filter (Millipore Corporation, Bedford, MA). Protein concentrations in the resultant cell-free lysates are determined using DC protein assay kits (Bio-Rad Laboratories, Hercules, CA). Aliquots (500µg/tube) are then cryopreserved at -135deg C until use [30]. Surface molecules are

follow; All vaccines are subjected to quality control evaluation, which involves assessing the total number of live DCs, monocyte-derived DC characteristics and percentage of viable cells. For vaccine to be deemed "adequate" 4x107 viable DCs are required. The frozen DC cells are allowed to thaw quickly in a 37deg C water bath and are retrieved from the cryopreservation tube by rinsing with 0.02% albumin-containing FACS buffer cell WashTM (Bioscience, San Hose, CA). The FACS analysis is performed for cell surface antigen detection. FITC-labeled antihuman CD14, CD40, CD80, HLA-A, B, C, PE-labeled anti-human CD11C, CD83, CD197

), HLA-DR and the FACS Calibur flow cytometer were used from DC Biosciences

Hyperthermia is widely used to enhance the efficiency of chemotherapy or radiation in patients with inoperable cancer [31]. It has been given much attention for the cellular response to heat stress with respect to the immune system in cancer. The anti-tumor immune response can be markedly enhanced by treatment with hyperthermia particularly in the fever range [32]. Immunological effects of mild hyperthermia are twofold. One is the effect on dendritic and other immune cells [33]. The other is the effect is on tumor cells. Protein or peptides derived from cancer which are chaperoned by heat-shock protein (HSP) are possible sources of

tumor-derived HSP70 peptide complexes (HSP70-PC) have the immunogenic potential to instruct DCs and cross-present endogenously expressed, nonmutated, tumor antigenic peptides. The cross-presentation of a shared human tumor Ag together with its exquisite

, HLA-DR+

T cell responses [34]. Human

. Vaccine quality control and FACS analysis are as

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, HLA-

**Table 4.** Concentrations of peptide using DC vaccine

ABC+

(CCR7+

, CD83+

(Franklin Lakes, NJ).

, CD86+

, CD40+

**4. Role of hyperthermia in DC vaccine therapy**

antigens, transferred to antigen presenting cells for priming CD8+

On this basis, vaccination strategies employing DCs have been regarded as a promising therapeutic approach, even for advanced cancer. DCs internalize the cancer antigen, process their protein and then displays them, as short peptides on their extra cellular surface in conjunction with major histocompatibility complex (MHC) class I and II molecules. DCs then migrate into corresponding lymph nodes, where they mature and present the antigen to naïve T lymphocytes. Helper T cells (CD4+ ) recognize their cognate antigens (MHC class II molecules) on DCs, where CD8+ cytotoxic T lymphocytes (CTLs) recognize foreign or cancer cells that display the complementary peptide-MHC class I molecule on their cell surface. Adapting single peptides for the development of vaccines is not an optimal approach. It has been shown that after a complete objective response to the NY-ESO-1 peptide vaccine, a NY-SEO-1 negative tumor later recurred, showing that single-target immunization approaches can result in the development of immune escape tumor variants [22]. Since MHC expression levels vary with tumor types and stages, it is difficult to eradicate cancer by administration of NK cells alone. So, it is rational to use NK cell together with cancer vaccine, which we call hybrid immune therapy. CTLs activated by DC-based vaccine target MHC expressing cancer cells, where NK cells attack cancers that do not express MHC. We have proposed WT1, MUC1, CEA, CA125 and HER2/neu as potential cancer antigens for DC-based cancer vaccine, according to the patient's primary lesions and the tumor markers [23-26]. It has been reported that WT1 and MUC1 are antigens with high immunogenicity and their targeted immunotherapy has confirmed their safety and clinical efficiency. However, there are few studies regarding cancer vaccines that simultaneously use WT1 and MUC1 as antigens [27].

Preparations of DCs are as follows; PBMC-rich fraction is obtained usually by leukapheresis using COM. TEC (Fresenius Kabi, Hamburg, Germany). PBMCs were isolated from the heparinized leukapheresis products by Fi-coll-Hypaque gradient density centrifugation [28]. These PBMCs are placed into 100mm plastic tissue culture plates (Becton Dickinson Labware, Franklin Lakes, NJ) in AIM-V medium (Gibco, Gaithersburg, MD). Following 30 min incuba‐ tion at 37deg C, non-adherent cells are removed and adherent cells were cultured in AIM-V medium containing granulocyte-macrophage colony stimulating factor (GM-CSF, 500ng/ml, Primmune Inc., Kobe, Japan) and IL-4 (250ng/ml, R&D Systems Inc., Minneapolis, MN), to generate immature DCs [29]. The population of adherent cells remaining in the wells is composed of 95.6 +/- 3.3% CD14+ cells. After 5 days of cultures, the immature DCs are stimu‐ lated with OK-432 (10µ/mL) and prostaglandin E2 (50ng/mL, Daiichi Fine Chemical Co., Ltd., Toyama, Japan) for 24 hours to induce differentiation. Then, WT1, MUC1 and other antigens or proteins are pulsed onto the DCs in the same culture media are incubated for 24 hours. The concentrations usually used are shown (Table 4).


**Table 4.** Concentrations of peptide using DC vaccine

particular attention as potential targets for vaccine-based immunotherapy, because with the exception of very few tissues such as the splenic capsule and stroma, they are not expressed in normal human tissues and become activated in a number of cancers [19-21]. Therefore vaccination is an effective medical procedure in clinical oncology, based on the induction of a long-lasting immunologic memory and characterized by mechanisms endowed with high destructive potential and specificity. These functions will elicit a persistent immune memory

On this basis, vaccination strategies employing DCs have been regarded as a promising therapeutic approach, even for advanced cancer. DCs internalize the cancer antigen, process their protein and then displays them, as short peptides on their extra cellular surface in conjunction with major histocompatibility complex (MHC) class I and II molecules. DCs then migrate into corresponding lymph nodes, where they mature and present the antigen to naïve

display the complementary peptide-MHC class I molecule on their cell surface. Adapting single peptides for the development of vaccines is not an optimal approach. It has been shown that after a complete objective response to the NY-ESO-1 peptide vaccine, a NY-SEO-1 negative tumor later recurred, showing that single-target immunization approaches can result in the development of immune escape tumor variants [22]. Since MHC expression levels vary with tumor types and stages, it is difficult to eradicate cancer by administration of NK cells alone. So, it is rational to use NK cell together with cancer vaccine, which we call hybrid immune therapy. CTLs activated by DC-based vaccine target MHC expressing cancer cells, where NK cells attack cancers that do not express MHC. We have proposed WT1, MUC1, CEA, CA125 and HER2/neu as potential cancer antigens for DC-based cancer vaccine, according to the patient's primary lesions and the tumor markers [23-26]. It has been reported that WT1 and MUC1 are antigens with high immunogenicity and their targeted immunotherapy has confirmed their safety and clinical efficiency. However, there are few studies regarding cancer

Preparations of DCs are as follows; PBMC-rich fraction is obtained usually by leukapheresis using COM. TEC (Fresenius Kabi, Hamburg, Germany). PBMCs were isolated from the heparinized leukapheresis products by Fi-coll-Hypaque gradient density centrifugation [28]. These PBMCs are placed into 100mm plastic tissue culture plates (Becton Dickinson Labware, Franklin Lakes, NJ) in AIM-V medium (Gibco, Gaithersburg, MD). Following 30 min incuba‐ tion at 37deg C, non-adherent cells are removed and adherent cells were cultured in AIM-V medium containing granulocyte-macrophage colony stimulating factor (GM-CSF, 500ng/ml, Primmune Inc., Kobe, Japan) and IL-4 (250ng/ml, R&D Systems Inc., Minneapolis, MN), to generate immature DCs [29]. The population of adherent cells remaining in the wells is composed of 95.6 +/- 3.3% CD14+ cells. After 5 days of cultures, the immature DCs are stimu‐ lated with OK-432 (10µ/mL) and prostaglandin E2 (50ng/mL, Daiichi Fine Chemical Co., Ltd., Toyama, Japan) for 24 hours to induce differentiation. Then, WT1, MUC1 and other antigens or proteins are pulsed onto the DCs in the same culture media are incubated for 24 hours. The

) recognize their cognate antigens (MHC class II molecules)

cytotoxic T lymphocytes (CTLs) recognize foreign or cancer cells that

that can eliminate residual cancer cells and protect against relapses.

vaccines that simultaneously use WT1 and MUC1 as antigens [27].

concentrations usually used are shown (Table 4).

T lymphocytes. Helper T cells (CD4+

306 Cancer Treatment - Conventional and Innovative Approaches

on DCs, where CD8+

To prepare the autologous tumor lysates, tumor masses were obtained by surgical resection exclusion and are homogenized. Aliquots of isolated tumor cells were then lysed by 10 cycles of repeated freeze in liquid nitrogen and thaw in a 37deg C water bath. The lysed cells were centrifuged at 14,000G for 5 min, and supernatants are passed through a 0.22µm filter (Millipore Corporation, Bedford, MA). Protein concentrations in the resultant cell-free lysates are determined using DC protein assay kits (Bio-Rad Laboratories, Hercules, CA). Aliquots (500µg/tube) are then cryopreserved at -135deg C until use [30]. Surface molecules are determined using flow cytometry. The cells defined as mature DCs are CD14- , HLA-DR+ , HLA-ABC+ , CD83+ , CD86+ , CD40+ and CCR7+ . Vaccine quality control and FACS analysis are as follow; All vaccines are subjected to quality control evaluation, which involves assessing the total number of live DCs, monocyte-derived DC characteristics and percentage of viable cells. For vaccine to be deemed "adequate" 4x107 viable DCs are required. The frozen DC cells are allowed to thaw quickly in a 37deg C water bath and are retrieved from the cryopreservation tube by rinsing with 0.02% albumin-containing FACS buffer cell WashTM (Bioscience, San Hose, CA). The FACS analysis is performed for cell surface antigen detection. FITC-labeled antihuman CD14, CD40, CD80, HLA-A, B, C, PE-labeled anti-human CD11C, CD83, CD197 (CCR7+ ), HLA-DR and the FACS Calibur flow cytometer were used from DC Biosciences (Franklin Lakes, NJ).

### **4. Role of hyperthermia in DC vaccine therapy**

Hyperthermia is widely used to enhance the efficiency of chemotherapy or radiation in patients with inoperable cancer [31]. It has been given much attention for the cellular response to heat stress with respect to the immune system in cancer. The anti-tumor immune response can be markedly enhanced by treatment with hyperthermia particularly in the fever range [32]. Immunological effects of mild hyperthermia are twofold. One is the effect on dendritic and other immune cells [33]. The other is the effect is on tumor cells. Protein or peptides derived from cancer which are chaperoned by heat-shock protein (HSP) are possible sources of antigens, transferred to antigen presenting cells for priming CD8+ T cell responses [34]. Human tumor-derived HSP70 peptide complexes (HSP70-PC) have the immunogenic potential to instruct DCs and cross-present endogenously expressed, nonmutated, tumor antigenic peptides. The cross-presentation of a shared human tumor Ag together with its exquisite efficacy is an important new aspect for HSP70-based immunotherapy in clinical anticancer vaccination strategies, and suggest a potential extension of HSP70-based vaccination protocols from a patient-individual treatment modality to its use in an allogeneic setting [35]. The other studies support various clinical use of hyperthermia as part of an immunotherapeutic strategy in treating cancer [32, 36].

Autologous DCs (1x107 cells) were administered intradermally at 14-day intervals, for a total of 6-8 times. Tolerable 1 to 5 KE doses of OK-432 (Chugai Pharmaceutical Co., Ltd., Tokyo, a streptococcus immunological adjuvant) was administered together with the DC vaccine. NK cells were simultaneously injected intravenously in some patients at 14-day intervals. Clinical response was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.0 as follows: complete remission (CR), partial remission (PR), stable disease (SD) and progressive disease (PD). Adverse events were evaluated by grading the toxicity according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. The most common advanced cancers that were refractory to standard treatments that were treated by our DC-based cancer vaccine were as follows: breast cancer, lung cancer, pancreatic cancer, and colorectal cancer (Figures 5-8). The most important factor for prolonged survival was good PS before entry into DC vaccine treatment. Patients in the better PS group had a significantly longer survival time compared to those in the poorer group. The overall survival (OS) based on our risk score was significantly better for patients with clinical response of CR, PR and SD compared to those with a response of PD group. Therapy was well tolerated during treatment and for 3 months after the final treatment. None of the patients experienced adverse events of grade 3 or higher during the treatment period. Grade 1 to 2 fevers and grade 1 injection site reactions, consisting of erythema, induration and tenderness lasting 1-5 days after injection occurred in most patients and did not result in any dosage modifications or delayed treatments. No signs of autoimmune disease (arthritis, rash,

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colitis, etc.) were observed either during or after therapy.

**Figure 5.** Clinical response of DC vaccine in advanced breast cancer refractory to standard treatment.

The mechanisms by which a tumor cell can escape CTL is critical for the design and modifi‐ cation of effective vaccine strategies against cancer. These mechanisms fall into four broad categories: (i) inadequate antigen presentation by tumor cells resulting in their poor sensitivity to lysis by CTL; (ii) inhibitory signals provided by the tumor microenvironment; (iii) inability of TAA-specific CTL to localize at a tumor site; and (iv) inability of the tumor microenviron‐ ment to sustain T cell function in vivo [37]. Therefore, adequate antigen expression by tumor cells is of crucial importance. Many research papers show the possible augmentation of MHC class I antigen presentation via heat shock protein expression by hyperthermia. It has been demonstrated that the cell surface presentation of MHC class I antigen is increased in tandem with increased heat shock protein 70 (HSP 70) [38]. It is clear that mild hyperthermia enhances both the expression of TAA on the surface of tumors and also increased presentation of TAA chaperoned by HSP on the dendritic cell. These findings are encouraging for usage of hyper‐ thermia at the time of DC vaccination.

### **5. Clinical results in miscellaneous cancers**

Indications for the DC-based cancer vaccine is summarized as follows:


Prevention of relapse or metastasis after surgery or other standard treatments.

In this communication, the results of the retrospective study of the DC-based vaccine are presented in cancers common in Japan. Most of these patients are called "cancer refugees" who are told that no further effective treatments are available. Patient evaluations included a medical history and physical examination which include measurement of performance status (PS), total protein, albumin, hemoglobin, WBC count, platelet count, blood urea nitrogen (BUN), creatinine, alkaline phosphatase, LDH, AST, ALT, bilirubin, HbA1c, tumor marker level and HLA. As an image marker, computed tomography (CT) scans or magnetic resonance image (MRI) as well as ultrasound studies were included. To be eligible, patients were required to have an ECOG PS of <3. They were also required to have adequate hematologic and hepatorenal functions as determined by the flowing parameters:

WBC counts of≧2,500/µL, platelet counts of≥80,000/µL, hemoglobin value of≧9.0g/dL, BUN<50mg/dL, serum bilirubin level <5.0mg/dL and AST level<50DIU/L.

Autologous DCs (1x107 cells) were administered intradermally at 14-day intervals, for a total of 6-8 times. Tolerable 1 to 5 KE doses of OK-432 (Chugai Pharmaceutical Co., Ltd., Tokyo, a streptococcus immunological adjuvant) was administered together with the DC vaccine. NK cells were simultaneously injected intravenously in some patients at 14-day intervals. Clinical response was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.0 as follows: complete remission (CR), partial remission (PR), stable disease (SD) and progressive disease (PD). Adverse events were evaluated by grading the toxicity according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. The most common advanced cancers that were refractory to standard treatments that were treated by our DC-based cancer vaccine were as follows: breast cancer, lung cancer, pancreatic cancer, and colorectal cancer (Figures 5-8). The most important factor for prolonged survival was good PS before entry into DC vaccine treatment. Patients in the better PS group had a significantly longer survival time compared to those in the poorer group. The overall survival (OS) based on our risk score was significantly better for patients with clinical response of CR, PR and SD compared to those with a response of PD group. Therapy was well tolerated during treatment and for 3 months after the final treatment. None of the patients experienced adverse events of grade 3 or higher during the treatment period. Grade 1 to 2 fevers and grade 1 injection site reactions, consisting of erythema, induration and tenderness lasting 1-5 days after injection occurred in most patients and did not result in any dosage modifications or delayed treatments. No signs of autoimmune disease (arthritis, rash, colitis, etc.) were observed either during or after therapy.

efficacy is an important new aspect for HSP70-based immunotherapy in clinical anticancer vaccination strategies, and suggest a potential extension of HSP70-based vaccination protocols from a patient-individual treatment modality to its use in an allogeneic setting [35]. The other studies support various clinical use of hyperthermia as part of an immunotherapeutic strategy

The mechanisms by which a tumor cell can escape CTL is critical for the design and modifi‐ cation of effective vaccine strategies against cancer. These mechanisms fall into four broad categories: (i) inadequate antigen presentation by tumor cells resulting in their poor sensitivity to lysis by CTL; (ii) inhibitory signals provided by the tumor microenvironment; (iii) inability of TAA-specific CTL to localize at a tumor site; and (iv) inability of the tumor microenviron‐ ment to sustain T cell function in vivo [37]. Therefore, adequate antigen expression by tumor cells is of crucial importance. Many research papers show the possible augmentation of MHC class I antigen presentation via heat shock protein expression by hyperthermia. It has been demonstrated that the cell surface presentation of MHC class I antigen is increased in tandem with increased heat shock protein 70 (HSP 70) [38]. It is clear that mild hyperthermia enhances both the expression of TAA on the surface of tumors and also increased presentation of TAA chaperoned by HSP on the dendritic cell. These findings are encouraging for usage of hyper‐

in treating cancer [32, 36].

308 Cancer Treatment - Conventional and Innovative Approaches

thermia at the time of DC vaccination.

**5. Clinical results in miscellaneous cancers**

Indications for the DC-based cancer vaccine is summarized as follows: **1.** Patients with advanced cancer refractory to standard treatments.

**2.** Cancer patients treated with standard treatments without satisfactory results.

Prevention of relapse or metastasis after surgery or other standard treatments.

hepatorenal functions as determined by the flowing parameters:

BUN<50mg/dL, serum bilirubin level <5.0mg/dL and AST level<50DIU/L.

**3.** Efficiency of current standard therapy is not expected, but there are some possibilities to improve quality of life and prolong survival time by use of DC-based vaccine.

In this communication, the results of the retrospective study of the DC-based vaccine are presented in cancers common in Japan. Most of these patients are called "cancer refugees" who are told that no further effective treatments are available. Patient evaluations included a medical history and physical examination which include measurement of performance status (PS), total protein, albumin, hemoglobin, WBC count, platelet count, blood urea nitrogen (BUN), creatinine, alkaline phosphatase, LDH, AST, ALT, bilirubin, HbA1c, tumor marker level and HLA. As an image marker, computed tomography (CT) scans or magnetic resonance image (MRI) as well as ultrasound studies were included. To be eligible, patients were required to have an ECOG PS of <3. They were also required to have adequate hematologic and

WBC counts of≧2,500/µL, platelet counts of≥80,000/µL, hemoglobin value of≧9.0g/dL,

**Figure 5.** Clinical response of DC vaccine in advanced breast cancer refractory to standard treatment.

**Figure 6.** Clinical response of DC vaccine in advanced lung cancer refractory to standard treatment.

**Figure 8.** Clinical response of DC vaccine in advanced pancreatic cancer refractory to standard treatment.

of positive cells, and cancer stem cell expression.

The current results show that the DC-based vaccine is clinically applicable to any patient with a good PS at the initiation of DC-based therapy and can clinically benefit from continuing therapy beyond disease progression. Of the professional APCs, DCs are the most potent stimulators of T cell responses and play a crucial role in the initiation of primary immune responses [12]. Despite several immunotherapeutic approaches tested in colon cancer patients, only one has reported clinical results in a prospective randomized trial [39]. Preclinical data suggest that DC-based vaccines exert cytotoxic actions and that prolonged vaccine exposure is necessary for continued cancer suppression [40]. However, precisely when the full efficacy of antitumor vaccines will be realized, and when this approach will become routine therapy is difficult to predict. To date, most of peptide-based vaccines have targeted HLA class Irestricted peptides. However, there is increasing evidence that tumor-specific CD4+ T cells may also be important in inducing effective antitumor immunity. An ideal TAA is a protein that is essential for sustaining the malignant phenotype but which is not removed or down-regulated by the immune reaction. TAAs have been categorized according to their characteristics, such as therapeutic function, immunogenicity, oncogenicity, specificity, expression level, number

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It is important to understand the immunological mechanisms underlying the significant increase in cancer control ratios. Our results indicate that WT1 and /or MUC1-pulsed DC-based vaccination can have significant clinical benefits, even for advanced cancer patients that are refractory to standard therapies. These encouraging preliminary results suggest that WT1 and/or MUC1-pulsed DC-based vaccination strategies warrant further study as novel thera‐

**Figure 7.** Clinical response of DC vaccine in advanced colorectal cancer refractory to standard treatment.

**Figure 8.** Clinical response of DC vaccine in advanced pancreatic cancer refractory to standard treatment.

**Figure 6.** Clinical response of DC vaccine in advanced lung cancer refractory to standard treatment.

310 Cancer Treatment - Conventional and Innovative Approaches

**Figure 7.** Clinical response of DC vaccine in advanced colorectal cancer refractory to standard treatment.

The current results show that the DC-based vaccine is clinically applicable to any patient with a good PS at the initiation of DC-based therapy and can clinically benefit from continuing therapy beyond disease progression. Of the professional APCs, DCs are the most potent stimulators of T cell responses and play a crucial role in the initiation of primary immune responses [12]. Despite several immunotherapeutic approaches tested in colon cancer patients, only one has reported clinical results in a prospective randomized trial [39]. Preclinical data suggest that DC-based vaccines exert cytotoxic actions and that prolonged vaccine exposure is necessary for continued cancer suppression [40]. However, precisely when the full efficacy of antitumor vaccines will be realized, and when this approach will become routine therapy is difficult to predict. To date, most of peptide-based vaccines have targeted HLA class Irestricted peptides. However, there is increasing evidence that tumor-specific CD4+ T cells may also be important in inducing effective antitumor immunity. An ideal TAA is a protein that is essential for sustaining the malignant phenotype but which is not removed or down-regulated by the immune reaction. TAAs have been categorized according to their characteristics, such as therapeutic function, immunogenicity, oncogenicity, specificity, expression level, number of positive cells, and cancer stem cell expression.

It is important to understand the immunological mechanisms underlying the significant increase in cancer control ratios. Our results indicate that WT1 and /or MUC1-pulsed DC-based vaccination can have significant clinical benefits, even for advanced cancer patients that are refractory to standard therapies. These encouraging preliminary results suggest that WT1 and/or MUC1-pulsed DC-based vaccination strategies warrant further study as novel thera‐ peutic approaches to patients with advanced carcinomas. The combination of cytotoxic therapy with immune stimulation against cancer has been studied preclinically for a variety of common tumor types and could be directly translated to clinical use [41-43]. The current result clearly supports the idea that quality specifications are of the highest priority and must be important considerations in any future vaccine-based study.

the DC vaccine and or combination with natural killer cells are discussed. The response rate and cancer control rate of advanced breast cancer, lung cancer, colorectal cancer and pancreatic cancer are 12% and 38%, 22.7% and 68.2%, 21.9% and 59.4%, 16.9% and 42.9%, respectively. Overall survival rates were more than that of expected in advanced cancer refractory to standard therapy. These findings of DC based vaccines suggest the usefulness for treating cancer patients. Given the wide interest for targeted vaccine intervention in treating miscella‐ neous cancers, our findings may help in guiding and designing future trials and the develop‐

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, Touko Shimamoto, Shinichiro Akiyama and Minako Abe

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effector cells. Cell Immunol. 1990;131: 352-365.

ment of novel cancer treatment strategies.

\*Address all correspondence to: drabeqqq@yahoo.co.jp

**Author details**

Abe Cancer Clinic, Japan

1195-1203.

Hiroyuki Abe\*

**References**

Moreover, the induction of memory T and B cells underlies immunological memory induced by vaccination [44]. The ability of memory T cells to confer protective immunity depends on the number and quality of the cells produced [45, 46]. In the current study patients with an outcome of SD lived for a relatively long time, which is unusual for other therapeutic modal‐ ities. This tendency may due to CTL proliferation and differentiation into effector memory CD8 cells.

Given the wide interest for vaccine intervention in treating miscellaneous cancers, our findings may help in guiding and designing future trials. Additional studies are necessary to identify appropriate targets for vaccine development in this new era of molecular-targeted agents for cancer treatment.

### **6. Discussion and conclusion**

A better understanding of cancer molecular biology would enhance the design of novel therapies for cancer. Currently the scope of cancer immunotherapy is limited because most targeted antigens are restricted to a subset of patients. Molecular target DC vaccines evoke the power of each patient's immune system to help prevent recurrence and increase the long-term survival rate. If the patient's resected tumor is available, lysate is used as molecular antigens. Using this lysate, the vaccine induces an immune response against cancerous cells and creates immunologic memory. Because it is derived from the individual patient's tumor cells, this vaccine is a true targeted and personalized cancer therapy. When patient's own tumor cells are not available, integrating several candidates of peptides such as WT1, MUC1, CEA, CA125, Her2, PSA etc. can be used for the design of an anti-tumor vaccine which are restricted to the patient's HLA typing. Among these antigens, it is known that WT1 and MUC1 are the most important antigens expressed in cancer stem cells. Cancer stem cells form new tumors and may not be eliminated by chemotherapy or radiation. This has changed the perspective with regard to new approaches for treating cancer. Cancer stem cells are slow-dividing and inherently chemotherapy resistant. Eradication of these cancer stem cells may be necessary for the long-term success in cancer treatment. Using this strategy, a DC vaccine pulsed with WT1 and MUC1 and other tumor specific antigens would be used to eliminate cancer stem cells in individual patients. Hyperthermia is often used to activate immune system. There is evidence that when DCs take up HSPs together with the peptide they chaperone, the accompanying peptides are delivered into the antigen-processing pathways, leading to peptide presentation by MHC molecules. When DCs travel to the lymph nodes, T cells recognize the antigenic peptides and are specifically activated against cancer cells bearing these peptides [47]. Finally, the clinical results of molecular target cell therapy for cancers involving different organs, using the DC vaccine and or combination with natural killer cells are discussed. The response rate and cancer control rate of advanced breast cancer, lung cancer, colorectal cancer and pancreatic cancer are 12% and 38%, 22.7% and 68.2%, 21.9% and 59.4%, 16.9% and 42.9%, respectively. Overall survival rates were more than that of expected in advanced cancer refractory to standard therapy. These findings of DC based vaccines suggest the usefulness for treating cancer patients. Given the wide interest for targeted vaccine intervention in treating miscella‐ neous cancers, our findings may help in guiding and designing future trials and the develop‐ ment of novel cancer treatment strategies.

### **Author details**

peutic approaches to patients with advanced carcinomas. The combination of cytotoxic therapy with immune stimulation against cancer has been studied preclinically for a variety of common tumor types and could be directly translated to clinical use [41-43]. The current result clearly supports the idea that quality specifications are of the highest priority and must

Moreover, the induction of memory T and B cells underlies immunological memory induced by vaccination [44]. The ability of memory T cells to confer protective immunity depends on the number and quality of the cells produced [45, 46]. In the current study patients with an outcome of SD lived for a relatively long time, which is unusual for other therapeutic modal‐ ities. This tendency may due to CTL proliferation and differentiation into effector memory

Given the wide interest for vaccine intervention in treating miscellaneous cancers, our findings may help in guiding and designing future trials. Additional studies are necessary to identify appropriate targets for vaccine development in this new era of molecular-targeted agents for

A better understanding of cancer molecular biology would enhance the design of novel therapies for cancer. Currently the scope of cancer immunotherapy is limited because most targeted antigens are restricted to a subset of patients. Molecular target DC vaccines evoke the power of each patient's immune system to help prevent recurrence and increase the long-term survival rate. If the patient's resected tumor is available, lysate is used as molecular antigens. Using this lysate, the vaccine induces an immune response against cancerous cells and creates immunologic memory. Because it is derived from the individual patient's tumor cells, this vaccine is a true targeted and personalized cancer therapy. When patient's own tumor cells are not available, integrating several candidates of peptides such as WT1, MUC1, CEA, CA125, Her2, PSA etc. can be used for the design of an anti-tumor vaccine which are restricted to the patient's HLA typing. Among these antigens, it is known that WT1 and MUC1 are the most important antigens expressed in cancer stem cells. Cancer stem cells form new tumors and may not be eliminated by chemotherapy or radiation. This has changed the perspective with regard to new approaches for treating cancer. Cancer stem cells are slow-dividing and inherently chemotherapy resistant. Eradication of these cancer stem cells may be necessary for the long-term success in cancer treatment. Using this strategy, a DC vaccine pulsed with WT1 and MUC1 and other tumor specific antigens would be used to eliminate cancer stem cells in individual patients. Hyperthermia is often used to activate immune system. There is evidence that when DCs take up HSPs together with the peptide they chaperone, the accompanying peptides are delivered into the antigen-processing pathways, leading to peptide presentation by MHC molecules. When DCs travel to the lymph nodes, T cells recognize the antigenic peptides and are specifically activated against cancer cells bearing these peptides [47]. Finally, the clinical results of molecular target cell therapy for cancers involving different organs, using

be important considerations in any future vaccine-based study.

312 Cancer Treatment - Conventional and Innovative Approaches

CD8 cells.

cancer treatment.

**6. Discussion and conclusion**

Hiroyuki Abe\* , Touko Shimamoto, Shinichiro Akiyama and Minako Abe

\*Address all correspondence to: drabeqqq@yahoo.co.jp

Abe Cancer Clinic, Japan

### **References**


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**Chapter 15**

**Immunotherapy of**

http://dx.doi.org/10.5772/55283

for Tis that is not feasible for TURBT.

**1. Introduction**

Yi Luo, Eric J. Askeland, Mark R. Newton,

Jonathan R. Henning and Michael A. O'Donnell

Additional information is available at the end of the chapter

**Urinary Bladder Carcinoma: BCG and Beyond**

Urothelial carcinoma of the bladder is the second most common urologic neoplasm after prostate carcinoma in the United States, with an estimated 70,510 new cases and 14,880 deaths in 2012 [1]. Global prevalence of bladder cancer is estimated at >1 million and is steadily increasing. This disease places enormous economic burden on the U.S. health care system due to its requirements of surgical resection, repeated intravesical therapies, and lifelong medical follow-up. Urothelial carcinoma accounts for 90% of bladder tumors. At the time of diagnosis, 20-25% of cases are muscle invasive (stage T2 or higher) and are typically treated with surgical resection (radical cystectomy) [2]. The remainders are confined to layers above the muscularis propria – so-called nonmuscle invasive bladder cancer (NMIBC). These cancers (also termed "superficial bladder cancer") include tumors confined to the urothelium (Ta), tumors invading the lamina propria (T1), and carcinoma *in situ* (Tis, a flat erythematous lesion), occurring in 70%, 20% and 10% of NMIBC cases, respectively [2]. Transurethral resection of bladder tumor (TURBT) is the standard primary treatment for Ta and T1 lesions; however, recurrence rates for TURBT alone can be as high as 70% with up to 30% progressing to muscle invasive disease requiring cystectomy [3]. The high rates of recurrence and significant risk of progression in higher grade tumors mandate additional therapy with intravesical agents. While limiting the systemic exposure, intravesical therapy allows the destruction of residual microscopic tumor and circulating tumor cells after TURBT by exposure to therapeutic agents, thereby preventing reimplantation. To date, intravesical therapy has been used as an adjuvant treatment after TURBT to prevent recurrence and progression of the disese and is also the treatment of choice

> © 2013 Luo et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

## **Immunotherapy of Urinary Bladder Carcinoma: BCG and Beyond**

Yi Luo, Eric J. Askeland, Mark R. Newton, Jonathan R. Henning and Michael A. O'Donnell

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55283

### **1. Introduction**

Urothelial carcinoma of the bladder is the second most common urologic neoplasm after prostate carcinoma in the United States, with an estimated 70,510 new cases and 14,880 deaths in 2012 [1]. Global prevalence of bladder cancer is estimated at >1 million and is steadily increasing. This disease places enormous economic burden on the U.S. health care system due to its requirements of surgical resection, repeated intravesical therapies, and lifelong medical follow-up. Urothelial carcinoma accounts for 90% of bladder tumors. At the time of diagnosis, 20-25% of cases are muscle invasive (stage T2 or higher) and are typically treated with surgical resection (radical cystectomy) [2]. The remainders are confined to layers above the muscularis propria – so-called nonmuscle invasive bladder cancer (NMIBC). These cancers (also termed "superficial bladder cancer") include tumors confined to the urothelium (Ta), tumors invading the lamina propria (T1), and carcinoma *in situ* (Tis, a flat erythematous lesion), occurring in 70%, 20% and 10% of NMIBC cases, respectively [2]. Transurethral resection of bladder tumor (TURBT) is the standard primary treatment for Ta and T1 lesions; however, recurrence rates for TURBT alone can be as high as 70% with up to 30% progressing to muscle invasive disease requiring cystectomy [3]. The high rates of recurrence and significant risk of progression in higher grade tumors mandate additional therapy with intravesical agents. While limiting the systemic exposure, intravesical therapy allows the destruction of residual microscopic tumor and circulating tumor cells after TURBT by exposure to therapeutic agents, thereby preventing reimplantation. To date, intravesical therapy has been used as an adjuvant treatment after TURBT to prevent recurrence and progression of the disese and is also the treatment of choice for Tis that is not feasible for TURBT.

© 2013 Luo et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Chemotherapeutic agents such as mitomycin C, doxorubicin and epirubicin have long been used as intravesical therapies for NMIBC [3,4]. Recently, intravesical use of gemcitabine, valrubicin, and apaziquone have also been evaluated [5-7]. With respect to immunotherapy, BCG, a live attenuated strain of *Mycobacterium bovis* widely used as a vaccine against tuber‐ culosis, was first introduced as an intravesical therapy for bladder cancer in 1976 by Morales and associates [8]. Since then, BCG has been extensively evaluated and demonstrated to be superior to any other single chemotherapeutic agent for reducing recurrence and preventing progression of the disease [3,9]. To date, BCG has become the mainstay of therapy for NMIBC and remains the most effective treatment [3,9]. However, despite its favorable effects, a significant proportion of patients do not respond to BCG or tolerate treatment. In addition, recurrence and side effects are common. Therefore, research has been pursued and efforts made to improve BCG therapy. During the past decades, cytokine-based therapies have been developed. To date, multiple cytokines with Th1 stimulating properties, such as IFN-α, IL-2 and IL-12, have been evaluated, alone or in combination with BCG for the treatment of bladder cancer. In addition, pre-clinical research continues, aiming to identify new BCG therapeutic modalities. This chapter reviews the progress of bladder cancer immunotherapy, focusing on the clinical use of BCG and cytokines. In addition, we describe our own experience with BCG and cytokine therapies as well as research on BCG combination therapy and genetic engineer‐ ing of BCG to secrete Th1 cytokines. Finally, we describe the future directions for research with regard to BCG immunotherapy.

healing of the urothelium and reduce the risk of side effects. The induction course consists of six weekly intravesical instillations. The recommended dose varies in weight from strain to

mycobacteria. Lyophilized powder BCG is reconstituted in 50 ml of saline and administered via urethral catheter into an empty bladder with a dwell time of 2 hours. Maintenance is given as three weekly intravesical instillations at 3 and 6 months and then every 6 months for up to 3 years. Maintenance BCG is more effective in decreasing recurrence as compared to induction therapy alone. Multiple meta-analyses support BCG maintenance and it is now firmly established in clinical practice. The European Association of Urology (EAU) and the AUA recommend at least one year of maintenance for high risk patients [10,15]. The optimum schedule and duration of therapy have yet to be determined; however, most who use main‐ tenance follow some permutation of the Southwest Oncology Group (SWOG) program, a 3 week "mini" series given at intervals of 3, 6, 12, 18, 24, 30 and 36 months for a total of 27 instillations over 3 years [3,9,16]. Other schedules, such as single maintenance instillations of BCG at 3, 6, 9 and 12 months after induction therapy, have also produced promising results [17]. Recently, the EAU updated its guidelines on NMIBC and recommended a minimum of one year of intravesical BCG therapy for intermediate or high risk disease [18]. The Interna‐ tional Bladder Cancer Group (IBCG) also reviewed the current guidelines and recommended the use of intravesical BCG with maintenance for intermediate or high risk disease [19]. Intravesical BCG is contraindicated under the following situations: TURBT within the past 2 weeks, traumatic catheterization, macroscopic hematuria, urethral stenosis, active tuberculo‐

colony-forming units (CFU) of viable

http://dx.doi.org/10.5772/55283

321

Immunotherapy of Urinary Bladder Carcinoma: BCG and Beyond

strain, but each provides approximately 1-5 X 108

sis, prior BCG sepsis, immunosuppression, and urinary tract infection.

which time it is extended to every 6 months for 2 years, and then annually.

**2.2. Mechanism of BCG action**

At our own institution, a BCG induction course is typically initiated at 2-3 weeks post-TURBT with six weekly installations and a 1-2 hour dwell time. For patients with Tis, severe dysplasia, Grade 3/high grade or poorly differentiated pathology, and/or stage T1 disease, formal restaging under anesthesia is performed 6 weeks later and includes bilateral upper tract cytology, retrograde pyelograms, 4-5 random bladder biopsies, and prostatic urethral biopsies. If this pathology and restaging is negative, maintenance cycles may be initiated in 6 weeks. We classify three maintenance cycles A, B and C. Maintenance A consists of 3 weekly instil‐ lations followed by cystoscopy 6 weeks later. Cytology and fluorescence *in situ* hybridization (FISH) in urine specimens may be obtained at this time. If cystoscopy/cytology is negative, maintenance B may be initiated 6 months after the conclusion of cycle A, again for three weekly treatments. Maintenance C is initiated 6 months after the conclusion of cycle B. Following cycle C, cystoscopy/cytology is repeated every 3 months for 2 years from the original diagnosis at

Understanding of the mechanisms of BCG action is critical to improving the efficacy of BCG therapy. Although the exact mechanisms of BCG action currently remain elusive, many details have been discovered during the past decades. It has become clear that a functional host immune system is a necessary prerequisite for successful BCG therapy. It has also been known that the effects of intravesical BCG depend on the induction of a complex inflammatory cascade

### **2. BCG immunotherapy of bladder cancer**

#### **2.1. Clinical use of BCG in bladder cancer treatment**

Intravesical administration of BCG is currently the most common therapy employed for NMIBC. Since its advent in 1976, BCG has been extensively used to reduce recurrence and progression of NMIBC in an attempt to preserve the bladder. Although various BCG strains (e.g. Pasteur, Tice, Connaught, Frappier, RIVM and Tokyo) have been used, there is no evidence of a difference in efficacy or toxicity profile among these strains [9]. Many prospective randomized studies and meta-analyses have demonstrated the effectiveness of intravesical BCG therapy. Typical complete response rates are 55-65% for papillary tumors and 70-75% for Tis, which inversely indicates that 30-45% of patients will fail BCG treatment [3,9-12]. Adjuvant intravesical therapy was noted by the 2007 American Urological Association (AUA) panel to reduce recurrences by 24% and treatment with BCG was recommended by the panel [10]. Unfortunately, of complete responders, up to 50% will develop recurrent tumors within the first 5 years [13]. Furthermore, up to 90% of patients experience side effects ranging from cystitis and irritative voiding symptoms to much more uncommon life-threatening BCG sepsis. Up to 20% of patients are BCG intolerant due to these side effects [14].

The optimum dosing, schedule and duration for BCG treatment of NMIBC are unknown. Both induction and maintenance courses are largely empirical. According to the AUA's 2007 clinical practice guidelines [10], BCG therapy should be initiated 2-3 weeks following TURBT to allow healing of the urothelium and reduce the risk of side effects. The induction course consists of six weekly intravesical instillations. The recommended dose varies in weight from strain to strain, but each provides approximately 1-5 X 108 colony-forming units (CFU) of viable mycobacteria. Lyophilized powder BCG is reconstituted in 50 ml of saline and administered via urethral catheter into an empty bladder with a dwell time of 2 hours. Maintenance is given as three weekly intravesical instillations at 3 and 6 months and then every 6 months for up to 3 years. Maintenance BCG is more effective in decreasing recurrence as compared to induction therapy alone. Multiple meta-analyses support BCG maintenance and it is now firmly established in clinical practice. The European Association of Urology (EAU) and the AUA recommend at least one year of maintenance for high risk patients [10,15]. The optimum schedule and duration of therapy have yet to be determined; however, most who use main‐ tenance follow some permutation of the Southwest Oncology Group (SWOG) program, a 3 week "mini" series given at intervals of 3, 6, 12, 18, 24, 30 and 36 months for a total of 27 instillations over 3 years [3,9,16]. Other schedules, such as single maintenance instillations of BCG at 3, 6, 9 and 12 months after induction therapy, have also produced promising results [17]. Recently, the EAU updated its guidelines on NMIBC and recommended a minimum of one year of intravesical BCG therapy for intermediate or high risk disease [18]. The Interna‐ tional Bladder Cancer Group (IBCG) also reviewed the current guidelines and recommended the use of intravesical BCG with maintenance for intermediate or high risk disease [19]. Intravesical BCG is contraindicated under the following situations: TURBT within the past 2 weeks, traumatic catheterization, macroscopic hematuria, urethral stenosis, active tuberculo‐ sis, prior BCG sepsis, immunosuppression, and urinary tract infection.

At our own institution, a BCG induction course is typically initiated at 2-3 weeks post-TURBT with six weekly installations and a 1-2 hour dwell time. For patients with Tis, severe dysplasia, Grade 3/high grade or poorly differentiated pathology, and/or stage T1 disease, formal restaging under anesthesia is performed 6 weeks later and includes bilateral upper tract cytology, retrograde pyelograms, 4-5 random bladder biopsies, and prostatic urethral biopsies. If this pathology and restaging is negative, maintenance cycles may be initiated in 6 weeks. We classify three maintenance cycles A, B and C. Maintenance A consists of 3 weekly instil‐ lations followed by cystoscopy 6 weeks later. Cytology and fluorescence *in situ* hybridization (FISH) in urine specimens may be obtained at this time. If cystoscopy/cytology is negative, maintenance B may be initiated 6 months after the conclusion of cycle A, again for three weekly treatments. Maintenance C is initiated 6 months after the conclusion of cycle B. Following cycle C, cystoscopy/cytology is repeated every 3 months for 2 years from the original diagnosis at which time it is extended to every 6 months for 2 years, and then annually.

#### **2.2. Mechanism of BCG action**

Chemotherapeutic agents such as mitomycin C, doxorubicin and epirubicin have long been used as intravesical therapies for NMIBC [3,4]. Recently, intravesical use of gemcitabine, valrubicin, and apaziquone have also been evaluated [5-7]. With respect to immunotherapy, BCG, a live attenuated strain of *Mycobacterium bovis* widely used as a vaccine against tuber‐ culosis, was first introduced as an intravesical therapy for bladder cancer in 1976 by Morales and associates [8]. Since then, BCG has been extensively evaluated and demonstrated to be superior to any other single chemotherapeutic agent for reducing recurrence and preventing progression of the disease [3,9]. To date, BCG has become the mainstay of therapy for NMIBC and remains the most effective treatment [3,9]. However, despite its favorable effects, a significant proportion of patients do not respond to BCG or tolerate treatment. In addition, recurrence and side effects are common. Therefore, research has been pursued and efforts made to improve BCG therapy. During the past decades, cytokine-based therapies have been developed. To date, multiple cytokines with Th1 stimulating properties, such as IFN-α, IL-2 and IL-12, have been evaluated, alone or in combination with BCG for the treatment of bladder cancer. In addition, pre-clinical research continues, aiming to identify new BCG therapeutic modalities. This chapter reviews the progress of bladder cancer immunotherapy, focusing on the clinical use of BCG and cytokines. In addition, we describe our own experience with BCG and cytokine therapies as well as research on BCG combination therapy and genetic engineer‐ ing of BCG to secrete Th1 cytokines. Finally, we describe the future directions for research with

Intravesical administration of BCG is currently the most common therapy employed for NMIBC. Since its advent in 1976, BCG has been extensively used to reduce recurrence and progression of NMIBC in an attempt to preserve the bladder. Although various BCG strains (e.g. Pasteur, Tice, Connaught, Frappier, RIVM and Tokyo) have been used, there is no evidence of a difference in efficacy or toxicity profile among these strains [9]. Many prospective randomized studies and meta-analyses have demonstrated the effectiveness of intravesical BCG therapy. Typical complete response rates are 55-65% for papillary tumors and 70-75% for Tis, which inversely indicates that 30-45% of patients will fail BCG treatment [3,9-12]. Adjuvant intravesical therapy was noted by the 2007 American Urological Association (AUA) panel to reduce recurrences by 24% and treatment with BCG was recommended by the panel [10]. Unfortunately, of complete responders, up to 50% will develop recurrent tumors within the first 5 years [13]. Furthermore, up to 90% of patients experience side effects ranging from cystitis and irritative voiding symptoms to much more uncommon life-threatening BCG sepsis.

The optimum dosing, schedule and duration for BCG treatment of NMIBC are unknown. Both induction and maintenance courses are largely empirical. According to the AUA's 2007 clinical practice guidelines [10], BCG therapy should be initiated 2-3 weeks following TURBT to allow

regard to BCG immunotherapy.

320 Cancer Treatment - Conventional and Innovative Approaches

**2. BCG immunotherapy of bladder cancer**

**2.1. Clinical use of BCG in bladder cancer treatment**

Up to 20% of patients are BCG intolerant due to these side effects [14].

Understanding of the mechanisms of BCG action is critical to improving the efficacy of BCG therapy. Although the exact mechanisms of BCG action currently remain elusive, many details have been discovered during the past decades. It has become clear that a functional host immune system is a necessary prerequisite for successful BCG therapy. It has also been known that the effects of intravesical BCG depend on the induction of a complex inflammatory cascade event in the bladder mucosa reflecting activation of multiple types of immune cells and bladder tissue cells [20,21] (Figure 1). The initial step after BCG instillation is binding of BCG to fibronectin expressed on the urothelial lining through fibronectin attachment protein (FAP) [22]. Attached BCG is then internalized and processed by both normal and malignant cells, resulting in secretion of an array of proinflammatory cytokines and chemokines such as IL-1, IL-6, IL-8, tumor necrosis factor (TNF)-α, and granulocyte-macrophage colony stimulating factor (GM-CSF) [23,24]. Following urothelial cell activation, an influx of various leukocyte types into the bladder wall occurs including neutrophils, monocytes/macrophages, lympho‐ cytes, natural killer (NK) cells, and dendritic cells (DC) [25-27]. These infiltrating leukocytes are activated and produce a variety of additional proinflammatory cytokines and chemokines and also form BCG-induced granuloma structures in the bladder wall [25,27]. Subsequently, a large number of leukocyte types such as neutrophils, T cells and macrophages are expelled into the bladder lumen and appear in patients' voided urine [28-31]. In addition, transient massive cytokines and chemokines can be detected in voided urine including IL-1β, IL-2, IL-6, IL-10, IL-12, IL-18, IFN-γ, TNF-α, GM-CSF, macrophage colony-stimulating factor (M-CSF), macrophage-derived chemokine (MDC), monocyte chemoattractant protein (MCP)-1, macro‐ phage inflammatory protein (MIP)-1α, interferon-inducible protein (IP)-10, monokine induced by γ-interferon (MIG), and eosinophil chemoattractant activity (Eotaxin) [30,32-37]. The urine of animals treated with intravesical BCG also showed increased levels of numerous cytokines and chemokines [27]. It has been noted that the development of a predominant Th1 cytokine profile (e.g. IFN-γ, IL-2 and IL-12) is associated with the therapeutic effects of BCG, whereas the presence of a high level of Th2 cytokines (e.g. IL-10) is associated with BCG failure [33,35, 36]. Thus, a shift of the cytokines produced towards a Th1 milieu is necessary for succesful BCG immunotherapy of bladder cancer. To support this, it has been observed that both IFNγ and IL-12 but not IL-10 are required for local tumor surveillance in an animal model of bladder cancer [38]. Mice deficint in IL-10 genetically (IL-10-/-) or functionally via antibody neutralization or receptor blockage can also develop enhanced anti-bladder cancer immunity in response to intravesical BCG [36,39].

Neutrophils also compose the early responding cells to BCG instillation of the bladder and can be observed in the bladder wall and urine shortly after BCG instillation [27,28,30]. Neutrophils are central mediators of the innate immunity in BCG infection and are activated by signalling through TLR2 and TLR4 in conjunction with the adaptor protein myeloid differentiation factor 88 (MyD88) [47]. In addition to secretion of proinflammatory cytokines and chemokines (e.g. IL-1α, IL-1β, IL-8, MIP-1α, MIP-1β, MCP-1, transforming growth factor (TGF)-β, and growthrelated oncogene (GRO)-α) that lead to the recruitment of other immune cells [48], recent studies revealed that neutrophils are the primary source of TNF-related apoptosis-inducing ligand (TRAIL) found in the urine after BCG instillation [49,50]. TRAIL is a member of the TNF family that induces apoptosis in malignant cells but not in normal cells. Studies have indicated that the neutrophil TRAIL response is specific to BCG stimulation rather than nonspecific immune activation. Studies have also revealed a positive correlation between urinary TRAIL level and a favorable response to BCG treatment [49]. These observations suggest an important

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**Figure 1.** Suggested cascade of immune responses in bladder mucosa induced by intravesical BCG instillation. Attach‐ ment of BCG to urothelial cells including carcinoma cells triggers release of cytokines and chemokines from these cells, resulting in recruitment of various types of immune cells into the bladder wall. Activation of phagocytes and the new cytokine environment lead to the differentiation of naïve CD4+ T cells into TH1 and/or TH2 cells that direct immune responses toward cellular or humoral immunity, respectively. The therapeutic effect of BCG depends on a proper in‐ duction of TH1 immune responses. IL-10 inhibits TH1 immune responses whereas IFN-γ inhibits TH2 immune respons‐ es. Blocking IL-10 or inducing IFN-γ can lead to a TH1 dominated immunity that is essential for BCG-mediated bladder

cancer destruction.

Multiple immune cell types participate in the inflammatory response induced by BCG in the bladder. It is well accepted that macrophages, an indispensable cellular component of the innate immune system, serve as the first line of defense in mycobacterial infection. Activation, maturation and cytokine production of macrophages are primarily induced by Toll-like receptor (TLR) 2 ligation [40]. Following BCG instillation, an increased number of macrophag‐ es can be observed in bladder cancer infiltrates and the peritumoral bladder wall. Voided urine after BCG instillation also contains an increased number of macrophages and the cytokines and chemokines predominantly produced by macrophages such as TNF-α, IL-6, IL-10, IL-12 and IL-18 [28,30,32,35-37]. In addition to presenting BCG antigens, macrophages are capable of functioning as tumoricidal cells toward bladder cancer cells upon activation by BCG [41-45]. The killing of bladder cancer cells by macrophages relies on direct cell-to-cell contact and release of various soluble effector factors such as cytotoxic cytokines TNF-α and IFN-γ and apoptotic mediators such as nitric oxide (NO) [43-45,46]. Th1 cytokines (e.g. IFN-γ) enhance the induction of macrophage cytotoxicity whereas Th2 cytokines (e.g. IL-10) inhibit the induction of macrophage cytotoxicity [44,45].

Neutrophils also compose the early responding cells to BCG instillation of the bladder and can be observed in the bladder wall and urine shortly after BCG instillation [27,28,30]. Neutrophils are central mediators of the innate immunity in BCG infection and are activated by signalling through TLR2 and TLR4 in conjunction with the adaptor protein myeloid differentiation factor 88 (MyD88) [47]. In addition to secretion of proinflammatory cytokines and chemokines (e.g. IL-1α, IL-1β, IL-8, MIP-1α, MIP-1β, MCP-1, transforming growth factor (TGF)-β, and growthrelated oncogene (GRO)-α) that lead to the recruitment of other immune cells [48], recent studies revealed that neutrophils are the primary source of TNF-related apoptosis-inducing ligand (TRAIL) found in the urine after BCG instillation [49,50]. TRAIL is a member of the TNF family that induces apoptosis in malignant cells but not in normal cells. Studies have indicated that the neutrophil TRAIL response is specific to BCG stimulation rather than nonspecific immune activation. Studies have also revealed a positive correlation between urinary TRAIL level and a favorable response to BCG treatment [49]. These observations suggest an important

event in the bladder mucosa reflecting activation of multiple types of immune cells and bladder tissue cells [20,21] (Figure 1). The initial step after BCG instillation is binding of BCG to fibronectin expressed on the urothelial lining through fibronectin attachment protein (FAP) [22]. Attached BCG is then internalized and processed by both normal and malignant cells, resulting in secretion of an array of proinflammatory cytokines and chemokines such as IL-1, IL-6, IL-8, tumor necrosis factor (TNF)-α, and granulocyte-macrophage colony stimulating factor (GM-CSF) [23,24]. Following urothelial cell activation, an influx of various leukocyte types into the bladder wall occurs including neutrophils, monocytes/macrophages, lympho‐ cytes, natural killer (NK) cells, and dendritic cells (DC) [25-27]. These infiltrating leukocytes are activated and produce a variety of additional proinflammatory cytokines and chemokines and also form BCG-induced granuloma structures in the bladder wall [25,27]. Subsequently, a large number of leukocyte types such as neutrophils, T cells and macrophages are expelled into the bladder lumen and appear in patients' voided urine [28-31]. In addition, transient massive cytokines and chemokines can be detected in voided urine including IL-1β, IL-2, IL-6, IL-10, IL-12, IL-18, IFN-γ, TNF-α, GM-CSF, macrophage colony-stimulating factor (M-CSF), macrophage-derived chemokine (MDC), monocyte chemoattractant protein (MCP)-1, macro‐ phage inflammatory protein (MIP)-1α, interferon-inducible protein (IP)-10, monokine induced by γ-interferon (MIG), and eosinophil chemoattractant activity (Eotaxin) [30,32-37]. The urine of animals treated with intravesical BCG also showed increased levels of numerous cytokines and chemokines [27]. It has been noted that the development of a predominant Th1 cytokine profile (e.g. IFN-γ, IL-2 and IL-12) is associated with the therapeutic effects of BCG, whereas the presence of a high level of Th2 cytokines (e.g. IL-10) is associated with BCG failure [33,35, 36]. Thus, a shift of the cytokines produced towards a Th1 milieu is necessary for succesful BCG immunotherapy of bladder cancer. To support this, it has been observed that both IFNγ and IL-12 but not IL-10 are required for local tumor surveillance in an animal model of bladder cancer [38]. Mice deficint in IL-10 genetically (IL-10-/-) or functionally via antibody neutralization or receptor blockage can also develop enhanced anti-bladder cancer immunity

Multiple immune cell types participate in the inflammatory response induced by BCG in the bladder. It is well accepted that macrophages, an indispensable cellular component of the innate immune system, serve as the first line of defense in mycobacterial infection. Activation, maturation and cytokine production of macrophages are primarily induced by Toll-like receptor (TLR) 2 ligation [40]. Following BCG instillation, an increased number of macrophag‐ es can be observed in bladder cancer infiltrates and the peritumoral bladder wall. Voided urine after BCG instillation also contains an increased number of macrophages and the cytokines and chemokines predominantly produced by macrophages such as TNF-α, IL-6, IL-10, IL-12 and IL-18 [28,30,32,35-37]. In addition to presenting BCG antigens, macrophages are capable of functioning as tumoricidal cells toward bladder cancer cells upon activation by BCG [41-45]. The killing of bladder cancer cells by macrophages relies on direct cell-to-cell contact and release of various soluble effector factors such as cytotoxic cytokines TNF-α and IFN-γ and apoptotic mediators such as nitric oxide (NO) [43-45,46]. Th1 cytokines (e.g. IFN-γ) enhance the induction of macrophage cytotoxicity whereas Th2 cytokines (e.g. IL-10) inhibit the

in response to intravesical BCG [36,39].

322 Cancer Treatment - Conventional and Innovative Approaches

induction of macrophage cytotoxicity [44,45].

**Figure 1.** Suggested cascade of immune responses in bladder mucosa induced by intravesical BCG instillation. Attach‐ ment of BCG to urothelial cells including carcinoma cells triggers release of cytokines and chemokines from these cells, resulting in recruitment of various types of immune cells into the bladder wall. Activation of phagocytes and the new cytokine environment lead to the differentiation of naïve CD4+ T cells into TH1 and/or TH2 cells that direct immune responses toward cellular or humoral immunity, respectively. The therapeutic effect of BCG depends on a proper in‐ duction of TH1 immune responses. IL-10 inhibits TH1 immune responses whereas IFN-γ inhibits TH2 immune respons‐ es. Blocking IL-10 or inducing IFN-γ can lead to a TH1 dominated immunity that is essential for BCG-mediated bladder cancer destruction.

role of neutrophils in BCG-induced anti-bladder cancer immunity. Indeed, it has been observed that depletion of neutrophils resulted in a reduced BCG-induced anti-bladder cancer response in a mouse model of bladder cancer [48].

of intravesical BCG depends on its proper induction of a localized Th1 immune response. However, a systemic immune response appears also to be involved in intravesical BCG therapy. It has been documented that purified protein derivative (PPD) skin test often converts from negative to positive after BCG instillation and the effective treatment is associated with the development of delayed-type hypersensitivity (DTH) reaction to PPD [68]. Animal studies have also demonstrated the importance of DTH in the antitumor activity of intravesical BCG therapy [36]. Moreover, studies have shown increased levels of cytokines and chemokines in the serum (e.g. IL-2, IFN-γ, MCP-1 and RANTES), along with production of these cytokines and chemokines in the urine and/or bladder, during the course of BCG instillation [34,69]. Furthermore, studies have also shown an increase in PBMC cytotoxicity against urothelial

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In addition to the ability of BCG to elicit host immune responses, evidence supports a direct effect of BCG on the biology of UCC. *In vitro* studies have shown that BCG is anti-proliferative and even cytotoxic to UCC [41,70], and induces UCC expression of cytokines and chemokines (e.g. IL-1β, IL-6, IL-8, TNF-α and GM-CSF) [24], antigen-presenting molecules (e.g. MHC class II, CD1 and B7-1) [71], and intercellular adhesion molecules (e.g. ICAM-1) [71]. Analysis of tumor biopsy specimens from bladder cancer patients who underwent intravesical BCG therapy further supported the ability of BCG to induce UCC expression of these molecules *in vivo* [26]. Moreover, the bladder urothelium of animals treated with intravesical BCG shows upregulation of HLA antigens (e.g. MHC class I and II) and changes of many other molecules [72]. Recent studies have revealed that by cross-linking α5β1 integrin receptors, BCG exerts its direct biological effects on UCC, including activation of the signal transduction pathways involving activator protein (AP) 1, NFkB and CCAAT-enhancer-binding protein (C/EBP) [73], upregulation of gene expressions such as IL-6 and cyclin dependant kinase inhibitor p21 [73,74], and cell cycle arrest at the G1/S transition [75]. Although some studies showed the ability of BCG to induce apoptosis in UCC [76], other studies showed no such an ability or even induction of apoptotic resistance in UCC [77]. Further studies revealed that BCG induced UCC death in a caspase-independent manner [77] and that p21 played an important role in

carcinoma cells (UCC) after BCG instilation [34].

modulating the direct effects of BCG on UCC [78].

preserving or even enhancing BCG efficacy.

**3. Recombinant cytokines for bladder cancer treatment**

Prompted by the burden of patients either with BCG refractory disease or intolerance to BCG treatment, the search goes on for therapeutic improvements. Given that the effect of BCG depends on a proper induction of Th1 immune responses, decades of research have focused on enhancing the BCG induction of Th1 immune responses. Th1 stimulating cytokines, such as IFN-α, IL-2, IL-12, IFN-γ, TNF-α and GM-CSF, have been used alone or in combination with BCG and demonstrated to be favorable in the treatment of bladder cancer. Particularly, combination therapies potentially allow the use of a lower and safer dose of BCG while

Following the activation of macrophages and neutrophils in the bladder wall, driven by chemoattractants, recruitment of other immune cell types including CD4+ T cells, CD8+ T cells, NK cells, and DC takes place [25,26]. As for neutrophils and macrophages, these cell types can be found in the voided urine of patients after BCG instillation [28-30]. These effector cells produce various cytokines and chemokines to further promote BCG-induced anti-bladder cancer immune responses in the local milieu. In addition, DC, together with macrophages, trigger an anti-BCG specific immune response via antigen presentation to T cells that also amplifies the BCG-induced antitumor immunity. Like neutrophils and macrophages, both T cells and NK cells are cytotoxic toward bladder cancer cells upon activation. They kill target cells via the major histocompatibility complex (MHC) restricted (e.g. for cytotoxic T lympho‐ cytes (CTL)) and/or MHC non-restricted pathways (e.g. for NK cells) [41,51,52]. Perforinmediated lysis and apoptosis-associated killing (e.g. via Fas ligand and TRAIL) have been implicated as the major molecular effector mechanisms underlying the eradication of bladder cancer cells. These effector cell types are crucial for BCG immunotherapy of bladder cancer, as depletion of these cell types failed to develop effective anti-bladder cancer responses *in vivo* and kill bladder cancer cells *in vitro* [53,54].

Ithasbeenshownthatstimulationofhumanperipheralbloodmononuclearcells(PBMC)byviable BCG *in vitro* leads to the generation of a specialized cell population called BCG-activated killer (BAK) cells [55,56]. BAK cells are a CD3- CD8+ CD56+ cell population whose cytotoxicity is MHC non-restricted [56,57]. BAK cells kill bladder cancer cells through the perforin-mediated lysis pathwayandeffectivelylyseNKcell-resistantbladdercancercells[55-57].MacrophagesandCD4+ T cells have been found to be indispensable for the induction of BAK cell killing activity but have no such activity by themselves [56]. Th1 cytokines IFN-γ and IL-2 have been found to be re‐ quired for the induction of BAK cell cytotoxicity, as neutralizing antibodies specific to these cytokines could inhibit BCG-induced cytotoxicity [56]. BAK cells, together with lymphokineactivated killer (LAK) cells, a diverse population with NK or T cell phenotypes that are generat‐ ed by IL-2 [58-60], have been suggested to be the major effector cells during intravesical BCG therapy of bladder cancer. Other potential cytotoxic effector cells include CD1 restricted CD8+ T cells [61], γδ T cells [62-64], and natural killer T (NKT) cells [63-65].

Activation of the innate immune system is a prerequisite for BCG-induced inflammatory responses and the subsequent eradication of bladder cancer by intravesical BCG. In BCG instillation, TLRs participate in neutrophil, macrophage and DC recognition, maturation and activation. Both TLR2 and TLR4 appear to serve important but distinct roles in the induction of host immune responses to BCG or BCG cell-wall skeleton [40]. TLR9 also contributes to DC recognition of BCG [66]. Like other microbes, BCG has surface components called pathogenassociated molecular patterns (PAMPs) that are recognized by cells of the innate immune system through TLRs during infection [67]. It is this interaction between TLRs and PAMPs that activates the cells of the innate immune system, leading to BCG-induced inflammatory responses and subsequent eradication of bladder cancer. It is known that the antitumor effect of intravesical BCG depends on its proper induction of a localized Th1 immune response. However, a systemic immune response appears also to be involved in intravesical BCG therapy. It has been documented that purified protein derivative (PPD) skin test often converts from negative to positive after BCG instillation and the effective treatment is associated with the development of delayed-type hypersensitivity (DTH) reaction to PPD [68]. Animal studies have also demonstrated the importance of DTH in the antitumor activity of intravesical BCG therapy [36]. Moreover, studies have shown increased levels of cytokines and chemokines in the serum (e.g. IL-2, IFN-γ, MCP-1 and RANTES), along with production of these cytokines and chemokines in the urine and/or bladder, during the course of BCG instillation [34,69]. Furthermore, studies have also shown an increase in PBMC cytotoxicity against urothelial carcinoma cells (UCC) after BCG instilation [34].

role of neutrophils in BCG-induced anti-bladder cancer immunity. Indeed, it has been observed that depletion of neutrophils resulted in a reduced BCG-induced anti-bladder cancer

Following the activation of macrophages and neutrophils in the bladder wall, driven by

NK cells, and DC takes place [25,26]. As for neutrophils and macrophages, these cell types can be found in the voided urine of patients after BCG instillation [28-30]. These effector cells produce various cytokines and chemokines to further promote BCG-induced anti-bladder cancer immune responses in the local milieu. In addition, DC, together with macrophages, trigger an anti-BCG specific immune response via antigen presentation to T cells that also amplifies the BCG-induced antitumor immunity. Like neutrophils and macrophages, both T cells and NK cells are cytotoxic toward bladder cancer cells upon activation. They kill target cells via the major histocompatibility complex (MHC) restricted (e.g. for cytotoxic T lympho‐ cytes (CTL)) and/or MHC non-restricted pathways (e.g. for NK cells) [41,51,52]. Perforinmediated lysis and apoptosis-associated killing (e.g. via Fas ligand and TRAIL) have been implicated as the major molecular effector mechanisms underlying the eradication of bladder cancer cells. These effector cell types are crucial for BCG immunotherapy of bladder cancer, as depletion of these cell types failed to develop effective anti-bladder cancer responses *in*

Ithasbeenshownthatstimulationofhumanperipheralbloodmononuclearcells(PBMC)byviable BCG *in vitro* leads to the generation of a specialized cell population called BCG-activated killer

CD56+

non-restricted [56,57]. BAK cells kill bladder cancer cells through the perforin-mediated lysis pathwayandeffectivelylyseNKcell-resistantbladdercancercells[55-57].MacrophagesandCD4+ T cells have been found to be indispensable for the induction of BAK cell killing activity but have no such activity by themselves [56]. Th1 cytokines IFN-γ and IL-2 have been found to be re‐ quired for the induction of BAK cell cytotoxicity, as neutralizing antibodies specific to these cytokines could inhibit BCG-induced cytotoxicity [56]. BAK cells, together with lymphokineactivated killer (LAK) cells, a diverse population with NK or T cell phenotypes that are generat‐ ed by IL-2 [58-60], have been suggested to be the major effector cells during intravesical BCG therapy of bladder cancer. Other potential cytotoxic effector cells include CD1 restricted CD8+

Activation of the innate immune system is a prerequisite for BCG-induced inflammatory responses and the subsequent eradication of bladder cancer by intravesical BCG. In BCG instillation, TLRs participate in neutrophil, macrophage and DC recognition, maturation and activation. Both TLR2 and TLR4 appear to serve important but distinct roles in the induction of host immune responses to BCG or BCG cell-wall skeleton [40]. TLR9 also contributes to DC recognition of BCG [66]. Like other microbes, BCG has surface components called pathogenassociated molecular patterns (PAMPs) that are recognized by cells of the innate immune system through TLRs during infection [67]. It is this interaction between TLRs and PAMPs that activates the cells of the innate immune system, leading to BCG-induced inflammatory responses and subsequent eradication of bladder cancer. It is known that the antitumor effect

CD8+

cells [61], γδ T cells [62-64], and natural killer T (NKT) cells [63-65].

T cells, CD8+

cell population whose cytotoxicity is MHC

T cells,

T

chemoattractants, recruitment of other immune cell types including CD4+

response in a mouse model of bladder cancer [48].

324 Cancer Treatment - Conventional and Innovative Approaches

*vivo* and kill bladder cancer cells *in vitro* [53,54].

(BAK) cells [55,56]. BAK cells are a CD3-

In addition to the ability of BCG to elicit host immune responses, evidence supports a direct effect of BCG on the biology of UCC. *In vitro* studies have shown that BCG is anti-proliferative and even cytotoxic to UCC [41,70], and induces UCC expression of cytokines and chemokines (e.g. IL-1β, IL-6, IL-8, TNF-α and GM-CSF) [24], antigen-presenting molecules (e.g. MHC class II, CD1 and B7-1) [71], and intercellular adhesion molecules (e.g. ICAM-1) [71]. Analysis of tumor biopsy specimens from bladder cancer patients who underwent intravesical BCG therapy further supported the ability of BCG to induce UCC expression of these molecules *in vivo* [26]. Moreover, the bladder urothelium of animals treated with intravesical BCG shows upregulation of HLA antigens (e.g. MHC class I and II) and changes of many other molecules [72]. Recent studies have revealed that by cross-linking α5β1 integrin receptors, BCG exerts its direct biological effects on UCC, including activation of the signal transduction pathways involving activator protein (AP) 1, NFkB and CCAAT-enhancer-binding protein (C/EBP) [73], upregulation of gene expressions such as IL-6 and cyclin dependant kinase inhibitor p21 [73,74], and cell cycle arrest at the G1/S transition [75]. Although some studies showed the ability of BCG to induce apoptosis in UCC [76], other studies showed no such an ability or even induction of apoptotic resistance in UCC [77]. Further studies revealed that BCG induced UCC death in a caspase-independent manner [77] and that p21 played an important role in modulating the direct effects of BCG on UCC [78].

### **3. Recombinant cytokines for bladder cancer treatment**

Prompted by the burden of patients either with BCG refractory disease or intolerance to BCG treatment, the search goes on for therapeutic improvements. Given that the effect of BCG depends on a proper induction of Th1 immune responses, decades of research have focused on enhancing the BCG induction of Th1 immune responses. Th1 stimulating cytokines, such as IFN-α, IL-2, IL-12, IFN-γ, TNF-α and GM-CSF, have been used alone or in combination with BCG and demonstrated to be favorable in the treatment of bladder cancer. Particularly, combination therapies potentially allow the use of a lower and safer dose of BCG while preserving or even enhancing BCG efficacy.

#### **3.1. Recombinant IFN-α**

IFNs are glycoproteins initially isolated in the 1950s and valued for their anti-viral properties. Three types have been isolated, IFN-α (which is actually a family of interferons), IFN-β, and IFN-γ. IFN-α and IFN-β are grouped as "Type I" interferons whereas IFN-γ is a "Type II" interferon. The Type I interferon receptor has 2 components, IFNAR-1 and IFNAR-2, which subsequently bind and phosphorylate Jak molecules initiating a cascade resulting in gene transcription [79]. The IFN-α family is well known to stimulate NK cells, induce MHC class I response, and increase antibody recognition [80]. They have antineoplastic properties by direct antiproliferative effects and complex immunomodulatory effects [79], both of which could be advantageous for bladder cancer treatment. Clinically available preparations include IFN-α2a (Roferon-A, recombinant, Roche Laboratories, Nutley, NJ) and IFN-α2b (Intron-A, recombi‐ nant, Schering Plough, Kenilworth, NJ), though to date most research involves IFN-α2b. There has been interest in IFN-α2b both alone and combination with BCG, where a synergistic response has been described. Conceptually, combining BCG and IFN makes sense. BCG efficacy depends on the induction of a robust Th1 cytokine profile and IFN-α2b has been shown to potentiate the Th1 immune response [81]. However, despite theoretical promise, data after translation to clinical practice has been mixed.

*In vivo* monotherapy with IFN-α2b for bladder cancer has been explored by multiple groups. In 1990, Glashan published data from a randomized controlled trial evaluating high dose (100 million unit) and low dose (10 million unit) IFN-α2b regimens in patients with Tis [90]. Patients were treated weekly for 12 weeks and monthly thereafter for 1 year. The high and low dose groups had complete response rates of 43% and 5%, respectively. Of the high dose patients achieving a complete response, 90% remained disease-free at a notably short 6 months of follow-up. The primary side effects of treatment were flu-like symptoms (8% low dose, 17% high dose) but without the irritative symptoms seen so often in BCG therapy. When IFN-α2b was investigated alone to treat BCG failures, eight of twelve patients had recurrence at initial three-month evaluation and only one of twelve was disease-free at 24 months [91]. Another trial conducted by Portillo and associates randomized 90 pT1 bladder cancer patients to either intravesical treatment or placebo groups as primary prophylaxis after complete resection [92]. They utilized a similar dosing schedule but used 60 million units IFN-α2b. At 12 months of follow-up, recurrence rates were significantly lower for IFN-α2b group than placebo, 28.2% vs 35.8%, respectively. However, after 43 months rates were similar - 53.8% and 51.2%

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respectively, indicating that treatment benefit of IFN-α2b alone may not be durable.

and 20-30%, respectively [94].

Given the described antiproliferative and immunomodulatory effects of IFN-α, combination therapy with BCG has held tantalizing promise. Gan and associates found significantly greater antitumor activity with combination therapy than BCG alone: 14/15 mice receiving BCG/IFNα2b versus 8/15 mice receiving only BCG became tumor-free after 5 weekly intralesional treatments [93]. In an *in vitro* study comparing BCG plus IFN-α2b to BCG alone, our group demonstrated a 66-fold increase in IFN-γ production in peripheral blood mononuclear cell (PBMC) cultures [81]. Since IFN-γ is a major Th1 restricted cytokine found in patients re‐ sponding to BCG therapy, it has been used routinely as a surrogate marker for Th1 immune response in studies examining effect of IFN-α [81]. It appears that IFN-α2b by itself generates a negligible Th1 response, as no significant levels of IFN-γ were detected after IFN-α2b was incubated alone with the PBMCs. We have also demonstrated that the augmented IFN-γ production persisted even with reduced doses of BCG. These findings give credence to the idea that adding Th1 stimulating cytokines may allow for a decrease in BCG doses, thereby decreasing side effects thought to be directly related to BCG. Further augmenting Th1 differentiation, IFN-α was found to increase levels of several Th1 cytokines, including IL-12 and TNF-α as well as decreasing known Th1 inhibitory cytokines IL-10 and IL-6 by 80-90%

Clinical investigations with the combination of IFN-α2b and BCG began initially in BCG refractory patients but were subsequently expanded to BCG naïve patients. Stricker and associates found the combination to be safe, with a similar side effect profile to BCG alone [95]. In 2001, O'Donnell and associates reported on combination therapy administered to 40 patients who had failed at least 1 course of BCG alone [96]. At 24 months, 53% of patients were diseasefree. Patients with two or more prior BCG failures faired similarly to patients with only one. Lam and associates in 2003 reported on the treatment of 32 patients, of which 20 (63%) were BCG failures. At 22 months' median follow-up, 12 of the 20 BCG failure patients (60%) remained disease-free [97]. In a smaller trial, Punnen and associates documented a 50%

For many years, IFN-α was thought to exert antitumor activity primarily through direct antiproliferative properties [82]. At least part of this effect has been shown to be mediated by directly inducing tumor cell death. IFN-α has been documented to independently induce tumor necrosis factor related apoptosis inducing ligand (TRAIL) expression in UM-UC-12 bladder cancer cells [83], which subsequently triggers apoptosis in cells expressing the appropriate cell death receptor. Cell death occurs ultimately by Fas-associated protein with death domain (FADD) dependent activation of the death inducing signaling complex (DISC) followed by activation of caspase-8. Furthermore, Tecchio and associates have demonstrated that IFN-α can stimulate TRAIL mRNA as well as the release of a bioactive soluble TRAIL protein from neutrophils and monocytes, which induces apoptotic activity on TRAIL sensitive leukemic cell lines [84]. It also appears that IFN-α apoptotic effects may not be limited to TRAIL; rather it may trigger caspase-8 via both cell death receptor dependent and independent pathways [85]. Much like IFN-α, BCG has also been shown to induce TRAIL [49,50], which has correlated with patient response to BCG therapy and has been a source of overlapping research interest. Other direct IFN-α effects include enhancing cytotoxicity of CD4<sup>+</sup> T cells, increasing antigen detection by up-regulating MHC class I expression [82,86,87]. Direct suppression of proliferation by induction of tumor suppressor genes or inhibition of tumor oncogenes has also been described [82]. Also contributing to antiproliferative properties, IFNα has been documented to decrease angiogenesis and basic fibroblast growth factor. Addi‐ tionally, it down-regulates matrix metalloprotease-9 (MMP-9) mRNA as well as the MMP-9 translational protein in murine bladder tumors [88]. Interestingly, it has also been demon‐ strated that an optimal biologic dose with higher frequency, rather than maximal tolerated dose, produced the most significant decreases in angiogenesis. Significantly decreased angiogenesis has also been documented in human urothelium during and after IFN-α2b treatment following TURBT [89].

*In vivo* monotherapy with IFN-α2b for bladder cancer has been explored by multiple groups. In 1990, Glashan published data from a randomized controlled trial evaluating high dose (100 million unit) and low dose (10 million unit) IFN-α2b regimens in patients with Tis [90]. Patients were treated weekly for 12 weeks and monthly thereafter for 1 year. The high and low dose groups had complete response rates of 43% and 5%, respectively. Of the high dose patients achieving a complete response, 90% remained disease-free at a notably short 6 months of follow-up. The primary side effects of treatment were flu-like symptoms (8% low dose, 17% high dose) but without the irritative symptoms seen so often in BCG therapy. When IFN-α2b was investigated alone to treat BCG failures, eight of twelve patients had recurrence at initial three-month evaluation and only one of twelve was disease-free at 24 months [91]. Another trial conducted by Portillo and associates randomized 90 pT1 bladder cancer patients to either intravesical treatment or placebo groups as primary prophylaxis after complete resection [92]. They utilized a similar dosing schedule but used 60 million units IFN-α2b. At 12 months of follow-up, recurrence rates were significantly lower for IFN-α2b group than placebo, 28.2% vs 35.8%, respectively. However, after 43 months rates were similar - 53.8% and 51.2% respectively, indicating that treatment benefit of IFN-α2b alone may not be durable.

**3.1. Recombinant IFN-α**

326 Cancer Treatment - Conventional and Innovative Approaches

translation to clinical practice has been mixed.

treatment following TURBT [89].

IFNs are glycoproteins initially isolated in the 1950s and valued for their anti-viral properties. Three types have been isolated, IFN-α (which is actually a family of interferons), IFN-β, and IFN-γ. IFN-α and IFN-β are grouped as "Type I" interferons whereas IFN-γ is a "Type II" interferon. The Type I interferon receptor has 2 components, IFNAR-1 and IFNAR-2, which subsequently bind and phosphorylate Jak molecules initiating a cascade resulting in gene transcription [79]. The IFN-α family is well known to stimulate NK cells, induce MHC class I response, and increase antibody recognition [80]. They have antineoplastic properties by direct antiproliferative effects and complex immunomodulatory effects [79], both of which could be advantageous for bladder cancer treatment. Clinically available preparations include IFN-α2a (Roferon-A, recombinant, Roche Laboratories, Nutley, NJ) and IFN-α2b (Intron-A, recombi‐ nant, Schering Plough, Kenilworth, NJ), though to date most research involves IFN-α2b. There has been interest in IFN-α2b both alone and combination with BCG, where a synergistic response has been described. Conceptually, combining BCG and IFN makes sense. BCG efficacy depends on the induction of a robust Th1 cytokine profile and IFN-α2b has been shown to potentiate the Th1 immune response [81]. However, despite theoretical promise, data after

For many years, IFN-α was thought to exert antitumor activity primarily through direct antiproliferative properties [82]. At least part of this effect has been shown to be mediated by directly inducing tumor cell death. IFN-α has been documented to independently induce tumor necrosis factor related apoptosis inducing ligand (TRAIL) expression in UM-UC-12 bladder cancer cells [83], which subsequently triggers apoptosis in cells expressing the appropriate cell death receptor. Cell death occurs ultimately by Fas-associated protein with death domain (FADD) dependent activation of the death inducing signaling complex (DISC) followed by activation of caspase-8. Furthermore, Tecchio and associates have demonstrated that IFN-α can stimulate TRAIL mRNA as well as the release of a bioactive soluble TRAIL protein from neutrophils and monocytes, which induces apoptotic activity on TRAIL sensitive leukemic cell lines [84]. It also appears that IFN-α apoptotic effects may not be limited to TRAIL; rather it may trigger caspase-8 via both cell death receptor dependent and independent pathways [85]. Much like IFN-α, BCG has also been shown to induce TRAIL [49,50], which has correlated with patient response to BCG therapy and has been a source of overlapping research interest. Other direct IFN-α effects include enhancing cytotoxicity of CD4<sup>+</sup> T cells, increasing antigen detection by up-regulating MHC class I expression [82,86,87]. Direct suppression of proliferation by induction of tumor suppressor genes or inhibition of tumor oncogenes has also been described [82]. Also contributing to antiproliferative properties, IFNα has been documented to decrease angiogenesis and basic fibroblast growth factor. Addi‐ tionally, it down-regulates matrix metalloprotease-9 (MMP-9) mRNA as well as the MMP-9 translational protein in murine bladder tumors [88]. Interestingly, it has also been demon‐ strated that an optimal biologic dose with higher frequency, rather than maximal tolerated dose, produced the most significant decreases in angiogenesis. Significantly decreased angiogenesis has also been documented in human urothelium during and after IFN-α2b

Given the described antiproliferative and immunomodulatory effects of IFN-α, combination therapy with BCG has held tantalizing promise. Gan and associates found significantly greater antitumor activity with combination therapy than BCG alone: 14/15 mice receiving BCG/IFNα2b versus 8/15 mice receiving only BCG became tumor-free after 5 weekly intralesional treatments [93]. In an *in vitro* study comparing BCG plus IFN-α2b to BCG alone, our group demonstrated a 66-fold increase in IFN-γ production in peripheral blood mononuclear cell (PBMC) cultures [81]. Since IFN-γ is a major Th1 restricted cytokine found in patients re‐ sponding to BCG therapy, it has been used routinely as a surrogate marker for Th1 immune response in studies examining effect of IFN-α [81]. It appears that IFN-α2b by itself generates a negligible Th1 response, as no significant levels of IFN-γ were detected after IFN-α2b was incubated alone with the PBMCs. We have also demonstrated that the augmented IFN-γ production persisted even with reduced doses of BCG. These findings give credence to the idea that adding Th1 stimulating cytokines may allow for a decrease in BCG doses, thereby decreasing side effects thought to be directly related to BCG. Further augmenting Th1 differentiation, IFN-α was found to increase levels of several Th1 cytokines, including IL-12 and TNF-α as well as decreasing known Th1 inhibitory cytokines IL-10 and IL-6 by 80-90% and 20-30%, respectively [94].

Clinical investigations with the combination of IFN-α2b and BCG began initially in BCG refractory patients but were subsequently expanded to BCG naïve patients. Stricker and associates found the combination to be safe, with a similar side effect profile to BCG alone [95]. In 2001, O'Donnell and associates reported on combination therapy administered to 40 patients who had failed at least 1 course of BCG alone [96]. At 24 months, 53% of patients were diseasefree. Patients with two or more prior BCG failures faired similarly to patients with only one. Lam and associates in 2003 reported on the treatment of 32 patients, of which 20 (63%) were BCG failures. At 22 months' median follow-up, 12 of the 20 BCG failure patients (60%) remained disease-free [97]. In a smaller trial, Punnen and associates documented a 50% disease-free rate after combination therapy at 12 months' follow-up in 12 patients with BCG refractory disease [98]. A subsequent large community based phase II clinical trial examined 1106 patients from 125 sites with NMIBC, which were split into BCG naïve and BCG refractory groups [99]. At median 24 months' follow-up, tumor-free rates were 59% and 45%, respec‐ tively. In this larger trial, patients who had two or more courses of prior BCG therapy had a worse outcome when compared to patients who had 1 or less, likely indicating more resistant disease. A recent study limited to BCG naïve patients demonstrated similar disease-free rate of 62% but with much longer median follow-up of 55.8 months [100]. Furthermore, after evaluating failure patterns and response rates to BCG plus IFN-α, Gallagher and associates found that patients who recurred more than 12 months after initial BCG treatments had similar tumor-free rates at 24 months when compared to BCG naïve patients [101]. However, patients who recurred within a year of receiving their initial BCG treatments did significantly worse, with disease-free rates of 34-43% at 24 months, indicating that additional immunotherapy may not be appropriate. Overall, while promising, these data are unable to define any treatment benefit of combination therapy over BCG alone in previously BCG untreated patients.

enhanced antiproliferative effects of IFN-α2b, with >95% growth reduction in T24 bladder cancer cells [107]. Cell cycle analysis also revealed G1 cell cycle arrest, with Western blots confirming expression of G1 cell cycle regulators. Lastly, several groups have investigated gene therapy with a recombinant adenovirus delivery system (rAd-IFN/Syn3), which could potentially result in sustained therapeutic IFN-α2b levels for long periods of time. Nagab‐ hushnan and associates were able to demonstrate delivery and expression of IFN in the bladder as well as significant tumor regression in mice. Phase I trials with rAd-IFN/Syn3 were ongoing

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329

The discovery and characterization of IL-2 was one of the most important breakthroughs in the field of immunology. Prior to its discovery, lymphocytes were thought to be terminally differentiated and incapable of proliferation [109,110]. In 1975 it was discovered that the supernatant of murine splenic cell cultures could stimulate thymocytes, suggesting a native effector protein was responsible for this mitogenic activity [110,111]. When initially examined independently by different investigators, this "effector protein" was given multiple working names including thymocyte stimulating factor (TSF), thymocyte mitogenic factor (TMF), T cell growth factor (TCGF), co-stimulator, killer cell helper factor (KHF), and secondary cytotoxic T cell-inducing factor (SCIF) [112]. In 1979 it was recognized that these factors likely repre‐ sented the same entity and the nomenclature was standarized with the term "interleukin" (between leukocytes). Thus, the "effector protein" was named IL-2, differentiating it from the only other interleukin known at that time, IL-1 [112]. Regardless of the nomenclature, this protein was recognized to promote proliferation of primary T cells *in vitro* which revolution‐

ized the experimental armamentarium in the field of immunology [109,111,113].

is important for the activation of B cells [119]. As the CD4+

Since the discovery of IL-2 mediated control of T cell growth in culture, there has been much progress in elucidating its mechanisms. It was discovered relatively early that IL-2 enhances the production of cytotoxic lymphocytes which are capable of lysing tumor cells while leaving normal cells unharmed [113-116]. These IL-2 activated lymphocytes became known as "lymphokine-activated killer" (LAK) cells and were thought to play a large role in antitumor immune function [113-116]. Additionally, it was noted that IL-2 functions to augment the cytotoxic activity of NK cells and monocytes [117,118]. It has even been discovered that IL-2

were defined, it became clear that IL-2 is predominantly a Th1 secreted cytokine [120].

The cytotoxic antitumor capabilities induced in lymphocytes by IL-2 make it a potential cancer immunotherapeutic agent. To date, multiple studies have demonstrated regression of meta‐ static disease following systemic IL-2 treatment in some cancers [121]. Rosenberg and asso‐ ciates reported on 157 patients with a heterogenous mix of metastatic cancers refractory to other treatments including renal cell, colon cancer, breast cancer and lymphoma. Patients were treated with either IL-2 and LAK cells or IL-2 alone. Between the two groups, 9 complete and 20 partial responses were obtained. Significant morbidity has been reported with systemic IL-2 much of which is secondary to increased capillary permeability [121,122] and includes weight gain, hypotension, oliguria, elevated creatinine and bilirubin. These tend to resolve with

Th1 and Th2 cell cytokine profiles

at the time of their publication in 2007 [108].

**3.2. Recombinant IL-2**

To date, the only randomized trial comparing BCG alone to BCG plus IFN was a multi-center study of 670 BCG naïve patients with Tis, Ta, or T1 urothelial carcinoma [102]. This was a fourarm trial evaluating efficacy of megadose vitamins as well as BCG and IFN. Patients were randomized to 1 of 4 groups: BCG plus recommended daily vitamins, BCG plus megadose daily vitamins, BCG plus IFN-α2b plus recommended daily vitamins, and BCG plus IFN-α2b plus megadose daily vitamins. At 24 month follow up, median recurrence-free survival was similar across all groups, though the two IFN-α2b groups experienced higher incidence of constitutional symptoms and fever (p<0.05).

In general, a BCG/IFN-α2b combination thearpy is appropriate for patients with previous BCG failures, those with Tis, and the elderly [103]. Optimal dose and schedule have yet to be defined in controlled trials and debate continues on the subject. At our institution, we use 1/3 the standard dose of BCG plus 50 MU of IFN-α2b. The dose may be lowered for those patients experiencing lower urinary tract symptoms or low grade fever. For maintenance cycle A, we adjust the BCG dose for week 1 consisting of 1/3 the standard dose of BCG plus 50 MU of IFNα2b. For weeks 2 and 3, the BCG dose is lowered to 1/10 the standard dose plus 50 MU of rIFNα2b. Maintenance cycles B and C utilize similar dosing.

There are multiple areas where additional research is warranted. A recent evolution in combination therapy has been the development of an IFN-α2b expressing strain of recombi‐ nant BCG (rBCG-IFN-α) from the Pasteur strain of BCG. An initial *in vitro* study documented enhanced IFN-γ expression in PBMCs after incubation with rBCG-IFN-α as compared to standard BCG [104]. A subsequent study reported that rBCG-IFN-α increased cytotoxicity up to 2-fold over standard BCG in PBMC cultures. Both CD56+ CD8- NK cells and CD8+ T cells were identified as primary contributors to the increased cytotoxicity [105]. Combining IFNα2b with other antiproliferative agents has shown *in vitro* promise. Louie and associates reported that a combination of IFN-α2b and maitake mushroom D-fraction (PDF) could reduce T24 bladder cancer cell proliferation by 75%, accompanied by G1 cell cycle arrest [106]. A recently reported study indicated that adding grape seed proanthocyanin significantly enhanced antiproliferative effects of IFN-α2b, with >95% growth reduction in T24 bladder cancer cells [107]. Cell cycle analysis also revealed G1 cell cycle arrest, with Western blots confirming expression of G1 cell cycle regulators. Lastly, several groups have investigated gene therapy with a recombinant adenovirus delivery system (rAd-IFN/Syn3), which could potentially result in sustained therapeutic IFN-α2b levels for long periods of time. Nagab‐ hushnan and associates were able to demonstrate delivery and expression of IFN in the bladder as well as significant tumor regression in mice. Phase I trials with rAd-IFN/Syn3 were ongoing at the time of their publication in 2007 [108].

### **3.2. Recombinant IL-2**

disease-free rate after combination therapy at 12 months' follow-up in 12 patients with BCG refractory disease [98]. A subsequent large community based phase II clinical trial examined 1106 patients from 125 sites with NMIBC, which were split into BCG naïve and BCG refractory groups [99]. At median 24 months' follow-up, tumor-free rates were 59% and 45%, respec‐ tively. In this larger trial, patients who had two or more courses of prior BCG therapy had a worse outcome when compared to patients who had 1 or less, likely indicating more resistant disease. A recent study limited to BCG naïve patients demonstrated similar disease-free rate of 62% but with much longer median follow-up of 55.8 months [100]. Furthermore, after evaluating failure patterns and response rates to BCG plus IFN-α, Gallagher and associates found that patients who recurred more than 12 months after initial BCG treatments had similar tumor-free rates at 24 months when compared to BCG naïve patients [101]. However, patients who recurred within a year of receiving their initial BCG treatments did significantly worse, with disease-free rates of 34-43% at 24 months, indicating that additional immunotherapy may not be appropriate. Overall, while promising, these data are unable to define any treatment benefit of combination therapy over BCG alone in previously BCG untreated patients.

To date, the only randomized trial comparing BCG alone to BCG plus IFN was a multi-center study of 670 BCG naïve patients with Tis, Ta, or T1 urothelial carcinoma [102]. This was a fourarm trial evaluating efficacy of megadose vitamins as well as BCG and IFN. Patients were randomized to 1 of 4 groups: BCG plus recommended daily vitamins, BCG plus megadose daily vitamins, BCG plus IFN-α2b plus recommended daily vitamins, and BCG plus IFN-α2b plus megadose daily vitamins. At 24 month follow up, median recurrence-free survival was similar across all groups, though the two IFN-α2b groups experienced higher incidence of

In general, a BCG/IFN-α2b combination thearpy is appropriate for patients with previous BCG failures, those with Tis, and the elderly [103]. Optimal dose and schedule have yet to be defined in controlled trials and debate continues on the subject. At our institution, we use 1/3 the standard dose of BCG plus 50 MU of IFN-α2b. The dose may be lowered for those patients experiencing lower urinary tract symptoms or low grade fever. For maintenance cycle A, we adjust the BCG dose for week 1 consisting of 1/3 the standard dose of BCG plus 50 MU of IFNα2b. For weeks 2 and 3, the BCG dose is lowered to 1/10 the standard dose plus 50 MU of rIFN-

There are multiple areas where additional research is warranted. A recent evolution in combination therapy has been the development of an IFN-α2b expressing strain of recombi‐ nant BCG (rBCG-IFN-α) from the Pasteur strain of BCG. An initial *in vitro* study documented enhanced IFN-γ expression in PBMCs after incubation with rBCG-IFN-α as compared to standard BCG [104]. A subsequent study reported that rBCG-IFN-α increased cytotoxicity up

were identified as primary contributors to the increased cytotoxicity [105]. Combining IFNα2b with other antiproliferative agents has shown *in vitro* promise. Louie and associates reported that a combination of IFN-α2b and maitake mushroom D-fraction (PDF) could reduce T24 bladder cancer cell proliferation by 75%, accompanied by G1 cell cycle arrest [106]. A recently reported study indicated that adding grape seed proanthocyanin significantly

CD8-

NK cells and CD8+

T cells

constitutional symptoms and fever (p<0.05).

328 Cancer Treatment - Conventional and Innovative Approaches

α2b. Maintenance cycles B and C utilize similar dosing.

to 2-fold over standard BCG in PBMC cultures. Both CD56+

The discovery and characterization of IL-2 was one of the most important breakthroughs in the field of immunology. Prior to its discovery, lymphocytes were thought to be terminally differentiated and incapable of proliferation [109,110]. In 1975 it was discovered that the supernatant of murine splenic cell cultures could stimulate thymocytes, suggesting a native effector protein was responsible for this mitogenic activity [110,111]. When initially examined independently by different investigators, this "effector protein" was given multiple working names including thymocyte stimulating factor (TSF), thymocyte mitogenic factor (TMF), T cell growth factor (TCGF), co-stimulator, killer cell helper factor (KHF), and secondary cytotoxic T cell-inducing factor (SCIF) [112]. In 1979 it was recognized that these factors likely repre‐ sented the same entity and the nomenclature was standarized with the term "interleukin" (between leukocytes). Thus, the "effector protein" was named IL-2, differentiating it from the only other interleukin known at that time, IL-1 [112]. Regardless of the nomenclature, this protein was recognized to promote proliferation of primary T cells *in vitro* which revolution‐ ized the experimental armamentarium in the field of immunology [109,111,113].

Since the discovery of IL-2 mediated control of T cell growth in culture, there has been much progress in elucidating its mechanisms. It was discovered relatively early that IL-2 enhances the production of cytotoxic lymphocytes which are capable of lysing tumor cells while leaving normal cells unharmed [113-116]. These IL-2 activated lymphocytes became known as "lymphokine-activated killer" (LAK) cells and were thought to play a large role in antitumor immune function [113-116]. Additionally, it was noted that IL-2 functions to augment the cytotoxic activity of NK cells and monocytes [117,118]. It has even been discovered that IL-2 is important for the activation of B cells [119]. As the CD4+ Th1 and Th2 cell cytokine profiles were defined, it became clear that IL-2 is predominantly a Th1 secreted cytokine [120].

The cytotoxic antitumor capabilities induced in lymphocytes by IL-2 make it a potential cancer immunotherapeutic agent. To date, multiple studies have demonstrated regression of meta‐ static disease following systemic IL-2 treatment in some cancers [121]. Rosenberg and asso‐ ciates reported on 157 patients with a heterogenous mix of metastatic cancers refractory to other treatments including renal cell, colon cancer, breast cancer and lymphoma. Patients were treated with either IL-2 and LAK cells or IL-2 alone. Between the two groups, 9 complete and 20 partial responses were obtained. Significant morbidity has been reported with systemic IL-2 much of which is secondary to increased capillary permeability [121,122] and includes weight gain, hypotension, oliguria, elevated creatinine and bilirubin. These tend to resolve with cessation of IL-2 therapy [121]; however, Rosenberg reported 4 treatment related deaths among their 157 patients. Despite the reports of morbidity, IL-2 seemed to offer hope to patients with few treatment options.

It didn't take long to discover that these factors represented the same entity, thus the nomen‐

Although initially discovered in a B cell lymphoma, it was subsequently found that IL-12 is primarilyinvolvedwiththeregulationofTcells,causingproliferationofbothactivatedCD4+ and

cific LAK cells and facilitate the generation of an allogeneic CTL response [160]. IL-12 has even been found to play a role in the activation of neutrophils [161,162]. Multiple studies have shown that IL-12 strongly inhibits neovascularization, thought to be mediated through its induction of IFN-γ [163,166]. Furthermore, the mechanism by which IL-12 enhances the cytolytic effect of NK

Multiple animal studies have shown tumor responsiveness to immunomodulation with IL-12. Using systemic or peri-tumoral injections, IL-12 showed antitumor properties in murine sarcoma, melanoma, renal cell carcinoma, lung cancer, colon cancer, breast cancer, and bladder cancer models [164,169-173]. Increases in serum IFN-γ were observed in mice treated with IL-12 [170]. Antitumor efficacy was lost in CD8+ depleted mice, but not CD4+ depleted mice or NK deficient mice, suggesting that the primary mediators of the antitumor IL-12 effect are

 T cells [169,170]. Some of these studies saw effectiveness even with metastatic disease, including bladder cancer [169,170,173]. Multiple murine studies have also revealed added effectiveness with IL-12 administered in combination with chemotherapeutic agents [171,174-176]. Additionally, IL-12 therapy has shown synergistic activity when combined with radiation therapy in mice [172,177]. Various delivery systems for IL-12 therapy have been tested in mice using viral and retroviral vectors to elicit an IL-12 response [178-182]. These constructs have shown some effectiveness as antitumor therapeutics [178-181]. IL-12 as intravesical therapy for bladder cancer has shown great success in mouse models. BCG was found to be a potent stimulus for IL-12 expression, and neutralization of IL-12 significantly dampened the induction of IFN-γ by BCG [183]. BCG therapy for murine bladder cancer was essentially found to be ineffective in IL-12 knock-out mice, suggesting a crucial role for IL-12 in the BCG response [184]. When IL-12 is used as a therapy with BCG it causes a synergistic induction of IFN-γ [183]. Intravesical IL-12 treatment alone was found to be effective for the treatment of orthotopically placed bladder tumors in mice, and urinary IFN-γ was subse‐ quently found to be significantly elevated [173,185,186]. These observations further support to importance of IFN-γ induction for effective immunotherapy of bladder cancer. More recently, multiple attempts have been made to improve the delivery of intravesical IL-12 to the bladder mucosa to improve efficacy. One method utilized cationic liposome-mediated IL-12 gene therapy which showed improved survival and tumor-specific immunologic memory in mice [187]. Another method utilized chitosan, a mucoadhesive biopolymer, to increase IL-12 delivery to urothelial surfaces [188]. This method showed improved efficacy

 T cell subsets while causing minimal proliferation of resting PBMCs [152,154]. This concept is supported by studies demonstrating that the IL-12 receptor is upregulated in activated T and NK cells, but not in activated B cells [157]. IL-12 potentiates a Th1 specific immune response, and it was later discovered that DCs produce IL-12 and thus direct the development of Th1 cells from

T cells [158,159]. Additionally, IL-12 can, by itself, stimulate the activation of nonspe‐

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331

clature converged and this protein was termed IL-12 [153-157].

cells has been found to be via the perforin pathway [167,168].

over IL-12 alone in a mouse model [188].

CD8+

CD8+

naïve CD4+

With regard to bladder cancer, interest was stimulated after multiple investigators identified elevated IL-2 levels (as well as other cytokines) in urine of patients following BCG, suggesting an immunomodulatory effect of BCG [30,32,33,123-129]. Additionally, an elevation in IL-2 receptor expression has been documented on T cells in voided urine after BCG therapy [30,128]. Increased levels of urinary IL-2 have also been found to correlate with BCG response, which supports the concept that a Th1 cytokine profile confers a favorable response to BCG [35]. Furthermore, elevated IL-2 has been reported in the serum of patients following BCG instillation, which suggests both a local and systemic immune response to therapy [34,130]. These findings led to the conclusion that IL-2 may have a therapeutic use in bladder cancer.

One of the first clinical trials reported evidence of bladder tumor regression following intralesional injections of IL-2, with no adverse events recorded [131]. Multiple murine studies have demonstrated that systemic administration of IL-2, with or without BCG, can significantly decrease tumor size, suppress tumor growth and improve mean survival [132-134]. A small clinical study investigating systemic IL-2 administration effects on low stage bladder cancer found a complete and partial response rate in 5 of 12 patients, though 2 patients discontinued therapy due to toxicity [135]. The poor side effect profile of systemic IL-2 administration subsequently prompted a shift to utilize IL-2 as an intravesical therapy. Reports of intravesical use revealed a much improved side effect profile as well as some efficacy alone or when combined with BCG [136-141]. Den Otter and associates administered intravesical IL-2 alone after incomplete transurethral resection of grade 1-2, T1 papillary urothelial carcinoma, and documented "marker lesion" regression in 8 of 10 patients [142]. Additional experiments have focused on developing recombinant-IL-2 secreting strains of BCG [42,143-147]. Animal models using this approach have shown that compared to native BCG, IL-2 secreting BCG strains have increased IFN-γ production, induced a more favorable IFN-γ to IL-4 ratio, improved antigenspecific proliferation, enhanced antitumor cytotoxicity, and mounted a Th1 cytokine profile even in immunosuppressed or IL-4 transgenic mice (two conditions which favor a Th2 response) [42,143-147]. More recent animal and *in vitro* studies have investigated IL-2 trans‐ fecting dendritic cells (DCs), immobilized streptavidin-tagged bioactive IL-2 on the biotiny‐ lated surface of murine bladder mucosa, and development of a murine IL-2 surface modified bladder cancer vaccine [148-151]. Since IL-2 plays a crucial role in the Th1 response, it will continue to be a source of interest for immunotherapy of bladder cancer.

### **3.3. Recombinant IL-12**

IL-12 has been the focus of significant cancer research among cytokines as well. In 1987, it was discovered through *in vitro* experiments that there existed a factor which synergized with IL-2 in promoting a CTL response [151]. This factor was given the name cytotoxic lymphocyte maturation factor (CLMF) [151]. Shortly thereafter a factor was discovered that induced IFNγ production, enhanced T cell responses to mitogens, and augmented NK cell cytotoxicity [152]. This factor was provisionally called natural killer cell stimulatory factor (NKSF) [152]. It didn't take long to discover that these factors represented the same entity, thus the nomen‐ clature converged and this protein was termed IL-12 [153-157].

cessation of IL-2 therapy [121]; however, Rosenberg reported 4 treatment related deaths among their 157 patients. Despite the reports of morbidity, IL-2 seemed to offer hope to patients with

With regard to bladder cancer, interest was stimulated after multiple investigators identified elevated IL-2 levels (as well as other cytokines) in urine of patients following BCG, suggesting an immunomodulatory effect of BCG [30,32,33,123-129]. Additionally, an elevation in IL-2 receptor expression has been documented on T cells in voided urine after BCG therapy [30,128]. Increased levels of urinary IL-2 have also been found to correlate with BCG response, which supports the concept that a Th1 cytokine profile confers a favorable response to BCG [35]. Furthermore, elevated IL-2 has been reported in the serum of patients following BCG instillation, which suggests both a local and systemic immune response to therapy [34,130]. These findings led to the conclusion that IL-2 may have a therapeutic use in bladder cancer.

One of the first clinical trials reported evidence of bladder tumor regression following intralesional injections of IL-2, with no adverse events recorded [131]. Multiple murine studies have demonstrated that systemic administration of IL-2, with or without BCG, can significantly decrease tumor size, suppress tumor growth and improve mean survival [132-134]. A small clinical study investigating systemic IL-2 administration effects on low stage bladder cancer found a complete and partial response rate in 5 of 12 patients, though 2 patients discontinued therapy due to toxicity [135]. The poor side effect profile of systemic IL-2 administration subsequently prompted a shift to utilize IL-2 as an intravesical therapy. Reports of intravesical use revealed a much improved side effect profile as well as some efficacy alone or when combined with BCG [136-141]. Den Otter and associates administered intravesical IL-2 alone after incomplete transurethral resection of grade 1-2, T1 papillary urothelial carcinoma, and documented "marker lesion" regression in 8 of 10 patients [142]. Additional experiments have focused on developing recombinant-IL-2 secreting strains of BCG [42,143-147]. Animal models using this approach have shown that compared to native BCG, IL-2 secreting BCG strains have increased IFN-γ production, induced a more favorable IFN-γ to IL-4 ratio, improved antigenspecific proliferation, enhanced antitumor cytotoxicity, and mounted a Th1 cytokine profile even in immunosuppressed or IL-4 transgenic mice (two conditions which favor a Th2 response) [42,143-147]. More recent animal and *in vitro* studies have investigated IL-2 trans‐ fecting dendritic cells (DCs), immobilized streptavidin-tagged bioactive IL-2 on the biotiny‐ lated surface of murine bladder mucosa, and development of a murine IL-2 surface modified bladder cancer vaccine [148-151]. Since IL-2 plays a crucial role in the Th1 response, it will

continue to be a source of interest for immunotherapy of bladder cancer.

IL-12 has been the focus of significant cancer research among cytokines as well. In 1987, it was discovered through *in vitro* experiments that there existed a factor which synergized with IL-2 in promoting a CTL response [151]. This factor was given the name cytotoxic lymphocyte maturation factor (CLMF) [151]. Shortly thereafter a factor was discovered that induced IFNγ production, enhanced T cell responses to mitogens, and augmented NK cell cytotoxicity [152]. This factor was provisionally called natural killer cell stimulatory factor (NKSF) [152].

few treatment options.

330 Cancer Treatment - Conventional and Innovative Approaches

**3.3. Recombinant IL-12**

Although initially discovered in a B cell lymphoma, it was subsequently found that IL-12 is primarilyinvolvedwiththeregulationofTcells,causingproliferationofbothactivatedCD4+ and CD8+ T cell subsets while causing minimal proliferation of resting PBMCs [152,154]. This concept is supported by studies demonstrating that the IL-12 receptor is upregulated in activated T and NK cells, but not in activated B cells [157]. IL-12 potentiates a Th1 specific immune response, and it was later discovered that DCs produce IL-12 and thus direct the development of Th1 cells from naïve CD4+ T cells [158,159]. Additionally, IL-12 can, by itself, stimulate the activation of nonspe‐ cific LAK cells and facilitate the generation of an allogeneic CTL response [160]. IL-12 has even been found to play a role in the activation of neutrophils [161,162]. Multiple studies have shown that IL-12 strongly inhibits neovascularization, thought to be mediated through its induction of IFN-γ [163,166]. Furthermore, the mechanism by which IL-12 enhances the cytolytic effect of NK cells has been found to be via the perforin pathway [167,168].

Multiple animal studies have shown tumor responsiveness to immunomodulation with IL-12. Using systemic or peri-tumoral injections, IL-12 showed antitumor properties in murine sarcoma, melanoma, renal cell carcinoma, lung cancer, colon cancer, breast cancer, and bladder cancer models [164,169-173]. Increases in serum IFN-γ were observed in mice treated with IL-12 [170]. Antitumor efficacy was lost in CD8+ depleted mice, but not CD4+ depleted mice or NK deficient mice, suggesting that the primary mediators of the antitumor IL-12 effect are CD8+ T cells [169,170]. Some of these studies saw effectiveness even with metastatic disease, including bladder cancer [169,170,173]. Multiple murine studies have also revealed added effectiveness with IL-12 administered in combination with chemotherapeutic agents [171,174-176]. Additionally, IL-12 therapy has shown synergistic activity when combined with radiation therapy in mice [172,177]. Various delivery systems for IL-12 therapy have been tested in mice using viral and retroviral vectors to elicit an IL-12 response [178-182]. These constructs have shown some effectiveness as antitumor therapeutics [178-181]. IL-12 as intravesical therapy for bladder cancer has shown great success in mouse models. BCG was found to be a potent stimulus for IL-12 expression, and neutralization of IL-12 significantly dampened the induction of IFN-γ by BCG [183]. BCG therapy for murine bladder cancer was essentially found to be ineffective in IL-12 knock-out mice, suggesting a crucial role for IL-12 in the BCG response [184]. When IL-12 is used as a therapy with BCG it causes a synergistic induction of IFN-γ [183]. Intravesical IL-12 treatment alone was found to be effective for the treatment of orthotopically placed bladder tumors in mice, and urinary IFN-γ was subse‐ quently found to be significantly elevated [173,185,186]. These observations further support to importance of IFN-γ induction for effective immunotherapy of bladder cancer. More recently, multiple attempts have been made to improve the delivery of intravesical IL-12 to the bladder mucosa to improve efficacy. One method utilized cationic liposome-mediated IL-12 gene therapy which showed improved survival and tumor-specific immunologic memory in mice [187]. Another method utilized chitosan, a mucoadhesive biopolymer, to increase IL-12 delivery to urothelial surfaces [188]. This method showed improved efficacy over IL-12 alone in a mouse model [188].

The great success of IL-12 in treating various murine cancers subsequently led to experiments testing its use on human cancers, though with mixed success. Initial trials focused on systemic IL-12 treatment for metastatic cancer, though progress was initially halted when several patients suffered severe toxic effects from the treatment and two patients died from the therapy [189]. A phase I trial of systemically administered IL-12 in 40 patients with advanced malig‐ nancy found a dose-dependent increase in circulating IFN-γ with administration [190]. Experiments on the peripheral blood of these patients showed augmented NK cell cytolytic activity and enhanced T cell proliferation [191]. Unfortunately, of these 40 patients there was only one partial response and one transient complete response [190]. Further studies looking at chronic administration of twice weekly IL-12 in patients with metastatic cancer found that it is well tolerated and induces co-stimulatory cytokines (including IFN-γ) [192]. However, in a cohort of 28 patients there was only one patient with a partial response and two with prolonged disease stabilization, with one of these patients eventually exhibiting tumor regression [192]. Similar low response rates have been seen with systemic IL-12 in other studies of advanced malignancies [193-197]. Various combinations of immunotherapy have been tested with systemic IL-12 in humans. A phase I study examined systemic IL-12 with low dose IL-2 and showed it was well tolerated, and the addition of IL-2 significantly augmented IFNγ production as well as the NK response [198]. Of 28 patients there was one partial response and two pathologic responses [198]. Another phase I study using systemic IL-12 with IFN-α2b showed acceptable toxicity, but with no response in 41 patients [199]. As discussed previously, intravesical IL-12 showed great promise for the topical treatment of bladder cancer in a mouse model, however this success has not translated clinically. A phase I study of intravesical IL-12 therapy in patients with superficial bladder cancer showed minimal toxicity, but disappointing efficacy [200]. A total of 15 patients were enrolled in this study, of which 12 had no visible pretreatment lesions [200]. Of these 12 patients, 7 remained disease-free and 5 had recurrence within 4 weeks. The remaining 3 patients with pretreatment lesions had persistent disease at follow-up [200]. Perhaps the most disparaging results were that there was negligible IFN-γ induced in the urine and serum of these patients post-treatment, suggesting minimal immu‐ nologic effect from intravesical IL-12 therapy [200]. Despite the disappointing results from human studies, IL-12 remains an important target for the treatment of bladder cancer.

TURBT during a mean follow-up time of 12.1 months [202]. Serretta and associates demon‐ strated in two studies that intravesical TNF-α therapy was well tolerated and resulted in approximately a 24.5% complete response rate in 42 patients with superficial bladder cancer [203,204]. Two separate studies also supported the excellent tolerability and some antitumor effects of intravesical TNF therapy in patients with superficial bladder cancer [205,206]. Studies demonstrated that intravesical administration of GM-CSF for patients with stage Ta/T1 tumors after TURBT induced immunomodulatory effects on macrophage activities [207]. In correla‐ tion with regression of marker lesions, migration of macrophages to the surface layer was observed. Macrophages showed an extensive lysosomal system and pseudopodia. In addition, intravesical GM-CSF therapy was also observed to enhance lymphocyte recruitment into the bladder wall and activation in the bladder mucosa [208]. These clinical trials suggest that intravesical use of recombinant cytokines are favorable for the treatment of bladder cancer and

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Unlike Th1 stimulating cytokines discussed above, IL-10 is distinct in that its primary effect is to promote a Th2 response and thus dampen the immunotherapeutic effects of BCG for the treatment of bladder cancer [36,45]. As a result, it may have therapeutic value not by its native function, but by abrogation of its native function. IL-10 was first characterized in 1989. It was initially termed cytokine synthesis inhibitory factor (CSIF), a rather fitting name, because it was found to inhibit the production of several cytokines produced by Th1 clones [209]. The most important of these cytokines was IFN-γ, which was recognized as an important player in the Th1 response. As discussed previously, it is a key contributor in the immunotherapeutic effectiveness of BCG [209,210]. Further studies showed that IL-10 prevented DTH response to BCG and the neutralization or abrogation of IL-10 prolonged a response, thus further sup‐ porting its role in the Th1/2 response [36,211]. Several human tumors, including melanoma, non-small cell lung carcinoma, renal cell carcinoma and bladder cancer, have been found to have elevated expression of IL-10 [212-216]. It is speculated that production of IL-10 by tumor cells may represent an "escape mechanism" whereby tumor cells avoid Th1 immune mediated

There has been significant progress in determining the regulation and mechanism of IL-10 function since its discovery, particularly with regard to its role in tumor immunology. It is secreted by multiple cell types including Th2 cells, B cells and monocytes/macrophages [209,217-219]. Like many other cytokines, IL-10 is known to auto-regulate itself by downregulating its own mRNA synthesis [219]. It has been shown to block the accumulation of macrophages and DCs at tumor sites, which are important players in the cellular immune response [220,221]. Additionally, it compromises DCs ability to stimulate T cells causing induction of antigen-specific anergy of T cells [222]. Furthermore, it down-regulates the expression of MHC class II and co-stimulatory molecules, thus preventing a cellular immune

further investigations are warranted.

tumoricidal effects [212].

**4. Advances in BCG immunotherapy research**

**4.1. BCG therapy in conjunction with IL-10 blockage**

#### **3.4. Other recombinant cytokines**

In addition to the above-mentioned cytokines, several phase I and II trials have shown that other Th1 stimulating cytokines such as IFN-γ, TNF-α and GM-CSF, when intravesically administrated, are well tolerated and effective in the treatment of bladder cancer. Giannopou‐ los and associates conducted a study of 123 patients with stage Ta/T1, grade 2 tumors who were followed for a median of 26.5 months. They demonstrated that intravesical IFN-γ therapy prevented tumor recurrence after TURBT and was more effective than intravesical mitomycin C therapy [201]. The effect of IFN-γ was associated with significant increases of leukocytes in the bladder wall including CD4+ T cells, CD8+ T cells, NK cells and B cells, suggesting the involvement of a primary cellular immune response in the mechanism of IFN-γ action. A separate study consisting of 54 patients with stage Ta/T1 tumors also supported the safety and anti-bladder cancer activity of intravesical IFN-γ therapy in preventing tumor recurrence after TURBT during a mean follow-up time of 12.1 months [202]. Serretta and associates demon‐ strated in two studies that intravesical TNF-α therapy was well tolerated and resulted in approximately a 24.5% complete response rate in 42 patients with superficial bladder cancer [203,204]. Two separate studies also supported the excellent tolerability and some antitumor effects of intravesical TNF therapy in patients with superficial bladder cancer [205,206]. Studies demonstrated that intravesical administration of GM-CSF for patients with stage Ta/T1 tumors after TURBT induced immunomodulatory effects on macrophage activities [207]. In correla‐ tion with regression of marker lesions, migration of macrophages to the surface layer was observed. Macrophages showed an extensive lysosomal system and pseudopodia. In addition, intravesical GM-CSF therapy was also observed to enhance lymphocyte recruitment into the bladder wall and activation in the bladder mucosa [208]. These clinical trials suggest that intravesical use of recombinant cytokines are favorable for the treatment of bladder cancer and further investigations are warranted.

### **4. Advances in BCG immunotherapy research**

### **4.1. BCG therapy in conjunction with IL-10 blockage**

The great success of IL-12 in treating various murine cancers subsequently led to experiments testing its use on human cancers, though with mixed success. Initial trials focused on systemic IL-12 treatment for metastatic cancer, though progress was initially halted when several patients suffered severe toxic effects from the treatment and two patients died from the therapy [189]. A phase I trial of systemically administered IL-12 in 40 patients with advanced malig‐ nancy found a dose-dependent increase in circulating IFN-γ with administration [190]. Experiments on the peripheral blood of these patients showed augmented NK cell cytolytic activity and enhanced T cell proliferation [191]. Unfortunately, of these 40 patients there was only one partial response and one transient complete response [190]. Further studies looking at chronic administration of twice weekly IL-12 in patients with metastatic cancer found that it is well tolerated and induces co-stimulatory cytokines (including IFN-γ) [192]. However, in a cohort of 28 patients there was only one patient with a partial response and two with prolonged disease stabilization, with one of these patients eventually exhibiting tumor regression [192]. Similar low response rates have been seen with systemic IL-12 in other studies of advanced malignancies [193-197]. Various combinations of immunotherapy have been tested with systemic IL-12 in humans. A phase I study examined systemic IL-12 with low dose IL-2 and showed it was well tolerated, and the addition of IL-2 significantly augmented IFNγ production as well as the NK response [198]. Of 28 patients there was one partial response and two pathologic responses [198]. Another phase I study using systemic IL-12 with IFN-α2b showed acceptable toxicity, but with no response in 41 patients [199]. As discussed previously, intravesical IL-12 showed great promise for the topical treatment of bladder cancer in a mouse model, however this success has not translated clinically. A phase I study of intravesical IL-12 therapy in patients with superficial bladder cancer showed minimal toxicity, but disappointing efficacy [200]. A total of 15 patients were enrolled in this study, of which 12 had no visible pretreatment lesions [200]. Of these 12 patients, 7 remained disease-free and 5 had recurrence within 4 weeks. The remaining 3 patients with pretreatment lesions had persistent disease at follow-up [200]. Perhaps the most disparaging results were that there was negligible IFN-γ induced in the urine and serum of these patients post-treatment, suggesting minimal immu‐ nologic effect from intravesical IL-12 therapy [200]. Despite the disappointing results from human studies, IL-12 remains an important target for the treatment of bladder cancer.

In addition to the above-mentioned cytokines, several phase I and II trials have shown that other Th1 stimulating cytokines such as IFN-γ, TNF-α and GM-CSF, when intravesically administrated, are well tolerated and effective in the treatment of bladder cancer. Giannopou‐ los and associates conducted a study of 123 patients with stage Ta/T1, grade 2 tumors who were followed for a median of 26.5 months. They demonstrated that intravesical IFN-γ therapy prevented tumor recurrence after TURBT and was more effective than intravesical mitomycin C therapy [201]. The effect of IFN-γ was associated with significant increases of leukocytes in

involvement of a primary cellular immune response in the mechanism of IFN-γ action. A separate study consisting of 54 patients with stage Ta/T1 tumors also supported the safety and anti-bladder cancer activity of intravesical IFN-γ therapy in preventing tumor recurrence after

T cells, NK cells and B cells, suggesting the

T cells, CD8+

**3.4. Other recombinant cytokines**

332 Cancer Treatment - Conventional and Innovative Approaches

the bladder wall including CD4+

Unlike Th1 stimulating cytokines discussed above, IL-10 is distinct in that its primary effect is to promote a Th2 response and thus dampen the immunotherapeutic effects of BCG for the treatment of bladder cancer [36,45]. As a result, it may have therapeutic value not by its native function, but by abrogation of its native function. IL-10 was first characterized in 1989. It was initially termed cytokine synthesis inhibitory factor (CSIF), a rather fitting name, because it was found to inhibit the production of several cytokines produced by Th1 clones [209]. The most important of these cytokines was IFN-γ, which was recognized as an important player in the Th1 response. As discussed previously, it is a key contributor in the immunotherapeutic effectiveness of BCG [209,210]. Further studies showed that IL-10 prevented DTH response to BCG and the neutralization or abrogation of IL-10 prolonged a response, thus further sup‐ porting its role in the Th1/2 response [36,211]. Several human tumors, including melanoma, non-small cell lung carcinoma, renal cell carcinoma and bladder cancer, have been found to have elevated expression of IL-10 [212-216]. It is speculated that production of IL-10 by tumor cells may represent an "escape mechanism" whereby tumor cells avoid Th1 immune mediated tumoricidal effects [212].

There has been significant progress in determining the regulation and mechanism of IL-10 function since its discovery, particularly with regard to its role in tumor immunology. It is secreted by multiple cell types including Th2 cells, B cells and monocytes/macrophages [209,217-219]. Like many other cytokines, IL-10 is known to auto-regulate itself by downregulating its own mRNA synthesis [219]. It has been shown to block the accumulation of macrophages and DCs at tumor sites, which are important players in the cellular immune response [220,221]. Additionally, it compromises DCs ability to stimulate T cells causing induction of antigen-specific anergy of T cells [222]. Furthermore, it down-regulates the expression of MHC class II and co-stimulatory molecules, thus preventing a cellular immune response to tumor cells [223-225]. During activation of CD4+ T cells, the presence of IL-10 can cause them to differentiate into T regulatory cells 1 (Tr1), leading to peripheral tolerance [226]. IL-10 further reduces cellular tumoricidal activity by preventing release of reactive nitrogen/ oxygen intermediates by macrophages and NK cells, a key step in their efficacy during cellular immune defense [45,227].

[229-250] (Table 1). Most of these rBCG strains have demonstrated to be superior to BCG in the

Anti-BCG: anti-BCG infection; Anti-*M.tb*: anti-Mycobacterium tuberculosis infection; CI: cellular immunity; DC act: dendritic cell activation; h: human; HI: humoral immunity; m: mouse; r: rat; Th1 cyt prod: T helper type 1 cytokine

BCG is a potent immunoadjuvant and induces a primary Th1 immune response that is required for effective treatment of most cancer types. Genetic manipulation of BCG to secrete Th1

production; Th2 cyt prod: T helper type 2 cytokine production.

**Table 1.** Cytokine- and chemokine-expressing rBCG strains

**Strain Cytokine Species Immunological Effect Reference**

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induction of Th1 immune responses and antitumor immunity in pre-clinical settings.

IL-2 BCG (RBD) IL-2 m Th1 cyt prod, Antitumor, Cytotoxicity [143] IL-2 BCG (MAO) IL-2 r Th1 cyt prod [143] BCG-CI IL-2 h Anti-BCG [229] BCG-CII IL-2 h Anti-BCG [229] BCG-IL-2 IL-2 m CI, Th1 & Th2 cyt prod [144] BCG-GM-CSF GM-CSF m CI, Th1 & Th2 cyt prod, DC act, Anti-*M.tb* [144, 230] BCG-IFN-γ IFN-γ m CI, Th1 & Th2 cyt prod, Anti-BCG [144, 231] rBCG/IL-2 IL-2 m CI, Th1 cyt prod, Anti-BCG [145, 147, 232] rBCG-IL-2/GFP IL-2 m CI, Th1 cyt prod, Anti-BCG [146] rBCG(α-Ag-IL-2) IL-2 m Th1 cyt prod, Cytotoxicity [42] BCG-IFN-γ IFN-γ m Th1 cyt prod, Anti-BCG [233] rBCG-IFN-α IFN-α2b h Th1 cyt prod, Cytotoxicity [104] rBCG/IL-18 IL-18 m no clear effect [232] BCG IL-18 IL-18 m Th1 & Th2 cyt prod [234, 235] BCG-hIL2MUC1 IL-2 h CI, Th1 cyt prod, Antitumor [236, 237] rBCG-IFN-γ IFN-γ m CI, Th1 cyt prod, Antitumor [238] rBCG-IL-18 IL-18 m Th1 cyt prod, Anti-BCG, Cytotoxicity [43] rBCG-huIL-2-ESAT6 IL-2 h CI, Th1 cyt prod, Cytotoxicity, HI [239] rBCG-IL-2 IL-2 h Th1 cyt prod [240] BCGMCP-3 MCP-3 m CI, Anti-BCG [241] rBCG-AEI IFN-γ m CI, HI, Anti-*M.tb* [242] rBCG-Ag85B-IL15 IL-15 m CI, Th1 cyt prod, Anti-*M.tb* [243] rBCG-MVNTR4-CSF GM-CSF h CI, Th1 cyt prod, Antitumor [244, 245] rBCG-MVNTR8-CSF GM-CSF h CI, Th1 cyt prod, Antitumor [244, 245] rBCG-Ag85B-Esat6-TNF-α TNF-α m CI, HI [246] rBCG-IE IL-12 h CI, Th1 cyt prod [247] rBCG:GE GM-CSF h CI, Th1 cyt prod, HI [248] rBCG::Ag85B-CFP10-IL-12 IL-12 h CI, HI, Anti-*M.tb* [249] rBCG-IFN-α−2b IFN-α2b h CI, Cytotoxicity [250]

Successful treatment of bladder cancer with BCG, as discussed previously, requires a Th1 cytokine profile. IL-10 antagonizes the production of a Th1 milieu, thus its neutralization has been targeted as a potential means to augment the BCG response. Several murine studies have demonstrated that after IL-10 knock-out mice are inoculated with bladder cancer, they have improved BCG response with amplified local immune response, increased bladder mononu‐ clear infiltrate, enhanced DTH responses, greater antitumor activity, and prolonged survival [36,212]. Although murine IL-10 knock-out studies are conceptually important, studies focused on IL-10 neutralization hold more promise as clinically useful therapeutics. Murine bladder cancer studies utilizing anti-IL-10 neutralizing monoclonal antibody (mAb) have shown similar results, with BCG treatment inducing an enhanced DTH response and increased bladder mononuclear infiltrate [36,211]. More recent efforts have been placed at targeting the IL-10 receptor. The IL-10 receptor is composed of two known subunits (IL-10R1 and IL-10R2) and the IL-10R1 subunit plays the predominant role in signal transduction [228]. In *in vitro* studies we have recently shown that splenocytes incubated with BCG and anti-IL-10R1 mAb produced significantly higher IFN-γ than those incubated with BCG plus anti-IL-10 neutral‐ izing mAb [39], suggesting that interference with IL-10 signal transduction may be more effective than neutralizing IL-10 protein. In *in vivo* studies mice treated with BCG and anti-IL-10R1 mAb showed increased urinary IFN-γ production compared to BCG controls [39]. In a similar murine experiment, there was improved overall and tumor-free state in mice treated with BCG plus anti-IL-10R1 mAb compared to BCG treatment controls, though this difference did not reach statistical significance [39]. Most recently, in an experiment designed to follow murine survival after inoculation with a luciferase-expressing MB49 bladder cancer cells, we discovered that control mice and BCG only treated mice developed histologically confirmed lung metastasis, whereas mice treated with BCG and anti-IL-10R1 mAb developed no metastasis [unpublished data]. This difference was statistically significant and raises questions as to anti-IL-10R1 mAb's role as more than just an augmentation to BCG for local bladder cancer control. Confirmatory experiments and mechanistic studies are necessary, but anti-IL-10R1 mAb shows great potential in not only local bladder cancer control, but also possibly systemic immunomodulation for the prevention of metastatic bladder cancer.

#### **4.2. Development of recombinant BCG strains**

BCG in combination with Th1 stimulating cytokines (e.g. IFN-α2b) has demonstrated to im‐ proveBCGefficacyinthetreatmentofbladdercancer.However,thesestrategiesrequiremultiple applications and a large quantity of recombinant cytokines. Genetic manipulation of BCG to secrete Th1 stimulating cytokines provides an opportunity to overcome the drawbacks. To date, numerousrecombinantBCG(rBCG)strainscapableofsecretingcytokinesorchemokines,mainly Th1 stimulating cytokines such as IL-2, IL-12, IL-18, IFN-γ and IFN-α, have been developed


[229-250] (Table 1). Most of these rBCG strains have demonstrated to be superior to BCG in the induction of Th1 immune responses and antitumor immunity in pre-clinical settings.

response to tumor cells [223-225]. During activation of CD4+

334 Cancer Treatment - Conventional and Innovative Approaches

immune defense [45,227].

cause them to differentiate into T regulatory cells 1 (Tr1), leading to peripheral tolerance [226]. IL-10 further reduces cellular tumoricidal activity by preventing release of reactive nitrogen/ oxygen intermediates by macrophages and NK cells, a key step in their efficacy during cellular

Successful treatment of bladder cancer with BCG, as discussed previously, requires a Th1 cytokine profile. IL-10 antagonizes the production of a Th1 milieu, thus its neutralization has been targeted as a potential means to augment the BCG response. Several murine studies have demonstrated that after IL-10 knock-out mice are inoculated with bladder cancer, they have improved BCG response with amplified local immune response, increased bladder mononu‐ clear infiltrate, enhanced DTH responses, greater antitumor activity, and prolonged survival [36,212]. Although murine IL-10 knock-out studies are conceptually important, studies focused on IL-10 neutralization hold more promise as clinically useful therapeutics. Murine bladder cancer studies utilizing anti-IL-10 neutralizing monoclonal antibody (mAb) have shown similar results, with BCG treatment inducing an enhanced DTH response and increased bladder mononuclear infiltrate [36,211]. More recent efforts have been placed at targeting the IL-10 receptor. The IL-10 receptor is composed of two known subunits (IL-10R1 and IL-10R2) and the IL-10R1 subunit plays the predominant role in signal transduction [228]. In *in vitro* studies we have recently shown that splenocytes incubated with BCG and anti-IL-10R1 mAb produced significantly higher IFN-γ than those incubated with BCG plus anti-IL-10 neutral‐ izing mAb [39], suggesting that interference with IL-10 signal transduction may be more effective than neutralizing IL-10 protein. In *in vivo* studies mice treated with BCG and anti-IL-10R1 mAb showed increased urinary IFN-γ production compared to BCG controls [39]. In a similar murine experiment, there was improved overall and tumor-free state in mice treated with BCG plus anti-IL-10R1 mAb compared to BCG treatment controls, though this difference did not reach statistical significance [39]. Most recently, in an experiment designed to follow murine survival after inoculation with a luciferase-expressing MB49 bladder cancer cells, we discovered that control mice and BCG only treated mice developed histologically confirmed lung metastasis, whereas mice treated with BCG and anti-IL-10R1 mAb developed no metastasis [unpublished data]. This difference was statistically significant and raises questions as to anti-IL-10R1 mAb's role as more than just an augmentation to BCG for local bladder cancer control. Confirmatory experiments and mechanistic studies are necessary, but anti-IL-10R1 mAb shows great potential in not only local bladder cancer control, but also possibly

systemic immunomodulation for the prevention of metastatic bladder cancer.

BCG in combination with Th1 stimulating cytokines (e.g. IFN-α2b) has demonstrated to im‐ proveBCGefficacyinthetreatmentofbladdercancer.However,thesestrategiesrequiremultiple applications and a large quantity of recombinant cytokines. Genetic manipulation of BCG to secrete Th1 stimulating cytokines provides an opportunity to overcome the drawbacks. To date, numerousrecombinantBCG(rBCG)strainscapableofsecretingcytokinesorchemokines,mainly Th1 stimulating cytokines such as IL-2, IL-12, IL-18, IFN-γ and IFN-α, have been developed

**4.2. Development of recombinant BCG strains**

T cells, the presence of IL-10 can

Anti-BCG: anti-BCG infection; Anti-*M.tb*: anti-Mycobacterium tuberculosis infection; CI: cellular immunity; DC act: dendritic cell activation; h: human; HI: humoral immunity; m: mouse; r: rat; Th1 cyt prod: T helper type 1 cytokine production; Th2 cyt prod: T helper type 2 cytokine production.

**Table 1.** Cytokine- and chemokine-expressing rBCG strains

BCG is a potent immunoadjuvant and induces a primary Th1 immune response that is required for effective treatment of most cancer types. Genetic manipulation of BCG to secrete Th1 stimulating cytokines with simultaneous coexpression of tumor-associated antigens may therefore potentiate the induction of specific antitumor immune responses. Early studies demonstrated that IL-2 secreting rBCG was at least equally effective to wild-type BCG when used as an intratumoral injection or a vaccine therapy in conjunction with irradiated tumor cells in a murine melanoma model [251]. However, it was not until recently that the potential of rBCG for treating cancer has gained further appreciation. We and others have developed rBCG strains that deliver the breast cancer-associated antigen mucin-1 (MUC1) in a form of multiple tandem repeats with coexpression of human IL-2 or human GM-CSF [236,237,244,245]. Severe combined immunodeficient (SCID) mice reconstituted with human peripheral blood lymphocytes (PBL) followed by immunization with the rBCG strains developed MUC1-specific cellular immune respnses and enhanced protection against MUC1 positive human breast cancer xenografts, compared to control mice reconstituted with human PBL and immunized with non-cytokine secreting BCG. Studies have also demonstrated that the antitumor effects of the rBCG strains were correlated with the number of MUC1 tandem repeats delivered by BCG [244,245]. These results suggest that these MUC1 rBCG strains coexpressing Th1 stimulating cytokines are promising candidates as breast cancer vaccines and thus warrant further investigation.

played an important role in BCG-induced macrophage cytotoxicity, as blockage of these cytokines during BCG stimulation led to substantially reduced macrophage cytotoxicity toward bladder cancer cells [44]. In contrast, supplementation of BCG with Th1 cytokines (e.g. IL-2, IL-12 or IL-18) increased macrophage cytotoxicity by approximately 2-fold. Consistent with these observations, rBCG strains secreting murine IL-2 or IL-18 showed enhanced macrophage-mediated killing on bladder cancer MBT-2 cells, which was correlated with increased expression of IFN-γ, TNF-α and IL-6 by rBCG stimulated macrophages [44]. The effect of murine IL-2 secreting rBCG strain on the induction of macrophage cytotoxicity toward

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Although the *in vitro* studies have suggested the potential usefulness of Th1 cytokine-secreting rBCG strains for the treatment of bladder cancer, unfortunately, the effect of rBCG on treating bladder cancer *in vivo* has not well been studied. Up to date, only an rBCG strain secreting IFN-γ (rBCG-IFN-γ) has been studied in a murine MB49 syngeneic orthotopic tumor model [238]. This study showed that, with a low-dose treatment regimen, intravesical administration of rBCG-IFN-γ significantly prolonged animal survival compared to medium-treated controls, whereas BCG carrying an empty vector only slightly increased survival. In a similar experi‐ ment using the MB49 syngeneic orthotopic tumor model in IFN-γ knockout mice, intravesical treatment with rBCG-IFN-γ failed to prolong survival of mice, indicating that rBCG-derived IFN-γ had no measurable antitumor effect in the absence of endogenous IFN-γ. Studies also provided the mechanisms underlying the effect of rBCG-IFN-γ on treating bladder cancer. As demonstrated, this rBCG-IFN-γ strain could specifically upregulate the expression of MHC class I molecules on MB49 cells *in vitro* compared to control BCG, as the MHC class I upregu‐ lation could be blocked by an inhibitory antibody to IFN-γ. This rBCG strain also enhanced recruitment of CD4+ T cells into the bladder and further induced the local expression of IL-2 and IL-4 mRNA compared to control BCG. In addition, we have also evaluated the effects of rBCG strains secreting murine IL-2 or IP-10 (a Th1 chemokine) on treating bladder cancer in the MB49 syngeneic orthotopic tumor model and observed survival benefits of these rBCG strains [unpublished data]. All these observations suggest that rBCG strains secreting Th1 cytokines or chemokines possess improved antitumor properties and may offer new oppor‐

Supporting Th1 cytokine-secreting rBCG, *Mycobacterium smegmatis* (*M. smegmatis*), a closely related non-pathogenic mycobacterial organism, has been engineered to secrete murine TNFα (*M. smegmatis*/TNF-α) and tested in a transplantable MB49 tumor model [252]. Studies demonstrated that lymphocytes from tumor-bearing mice vaccinated with *M. smegmatis*/TNFα produced elevated and prolonged IFN-γ but no IL-10 in response to mycobacterial antigen or tumor lysate stimulation *in vitro*. Histopathology revealed significantly increased infiltrat‐

compared to those of mice receiving wild-type bacteria. These observations indicated that *M. smegmatis*/TNF-α induced cell-mediated immunity. Importantly, mice implanted subcutane‐ ously with MB49 tumor and treated at an adjacent site with the recombinant vaccine exhibited significantly reduced tumor growth with a 70% durable tumor-free survival compared to those treated with wild-type bacteria or BCG (a 10-20% long-term survival). Interestingly, treatment

lymphocytes in the tumor nodules of mice receiving the recombinant vaccine

bladder cancer MBT-2 cells was also demonstrated by a separate study [42].

tunities for the treatment of bladder cancer.

ing CD3+

It has been known that BCG stimulation of human PBMC leads to the generation of effector cells cytotoxic to bladder cancer cells *in vitro* [55,56]. We recently demonstrated that stimulation of human PBMC with rBCG-IFN-α, a rBCG strain secreting human IFN-α2b [104], *in vitro* for 7 days induced enhanced PBMC cytotoxicity toward human bladder cancer cell lines T24, J82, 5637, TCCSUP and UMUC-3 by up to 2-fold compared to control BCG carrying an empty vector [105]. This induction of enhanced PBMC cytotoxicity was correlated with increased production of IFN-γ and IL-2 by rBCG stimulated PBMC. Studies further revealed that this enhancement in PBMC cytotoxicity was dependent on BCG secreted IFN-α as well as endogenously expressed IFN-γ and IL-2, as blockage of IFN-α, IFN-γ or IL-2 by neutralizing antibodies during BCG stimulation reduced or abolished the induction of this enhanced PBMC cytotox‐ icity. Studies using NK and CD8+ T cells isolated from human PBMC revealed that both cell types were responsible for the enhanced PBMC cytotoxicity induced by rBCG-IFN-α with the former cell type being more predominant [105]. A similar rBCG strain secreting human IFNα2b has also been recently demonstrated to stimulate PBMC proliferation and cytotoxicity toward bladder cancer cell lines T24 and 5637 [250].

An early study demonstrated that human peripheral monocytes/macrophages were capable of functioning as tumoricidal cells toward bladder cancer UCRU-BL-17 cells upon activation by BCG *in vitro* [41]. It was observed that the cytotoxic activity of human monocytes/macro‐ phages was significantly enhanced after BCG stimulation, while the naïve cells exhibited only minimum cytotoxicity. Later, more studies including ours further demonstrated that murine macrophages could also function as tumoricidal cells toward bladder cancer cells upon activation by BCG *in vitro* [42-45]. Stimulation of thioglycollate-elicited peritoneal macrophag‐ es by BCG for 24 hour resulted in macrophage-mediated killing of bladder cancer MBT-2 (C3H background) and MB49 (C57BL/6 background) cells in a dose-dependent manner [44,45]. Studies also revealed that endogenous Th1 cytokines (e.g. IL-12, IL-18, IFN-γ and TNF-α) played an important role in BCG-induced macrophage cytotoxicity, as blockage of these cytokines during BCG stimulation led to substantially reduced macrophage cytotoxicity toward bladder cancer cells [44]. In contrast, supplementation of BCG with Th1 cytokines (e.g. IL-2, IL-12 or IL-18) increased macrophage cytotoxicity by approximately 2-fold. Consistent with these observations, rBCG strains secreting murine IL-2 or IL-18 showed enhanced macrophage-mediated killing on bladder cancer MBT-2 cells, which was correlated with increased expression of IFN-γ, TNF-α and IL-6 by rBCG stimulated macrophages [44]. The effect of murine IL-2 secreting rBCG strain on the induction of macrophage cytotoxicity toward bladder cancer MBT-2 cells was also demonstrated by a separate study [42].

stimulating cytokines with simultaneous coexpression of tumor-associated antigens may therefore potentiate the induction of specific antitumor immune responses. Early studies demonstrated that IL-2 secreting rBCG was at least equally effective to wild-type BCG when used as an intratumoral injection or a vaccine therapy in conjunction with irradiated tumor cells in a murine melanoma model [251]. However, it was not until recently that the potential of rBCG for treating cancer has gained further appreciation. We and others have developed rBCG strains that deliver the breast cancer-associated antigen mucin-1 (MUC1) in a form of multiple tandem repeats with coexpression of human IL-2 or human GM-CSF [236,237,244,245]. Severe combined immunodeficient (SCID) mice reconstituted with human peripheral blood lymphocytes (PBL) followed by immunization with the rBCG strains developed MUC1-specific cellular immune respnses and enhanced protection against MUC1 positive human breast cancer xenografts, compared to control mice reconstituted with human PBL and immunized with non-cytokine secreting BCG. Studies have also demonstrated that the antitumor effects of the rBCG strains were correlated with the number of MUC1 tandem repeats delivered by BCG [244,245]. These results suggest that these MUC1 rBCG strains coexpressing Th1 stimulating cytokines are promising candidates as breast cancer vaccines

It has been known that BCG stimulation of human PBMC leads to the generation of effector cells cytotoxic to bladder cancer cells *in vitro* [55,56]. We recently demonstrated that stimulation of human PBMC with rBCG-IFN-α, a rBCG strain secreting human IFN-α2b [104], *in vitro* for 7 days induced enhanced PBMC cytotoxicity toward human bladder cancer cell lines T24, J82, 5637, TCCSUP and UMUC-3 by up to 2-fold compared to control BCG carrying an empty vector [105]. This induction of enhanced PBMC cytotoxicity was correlated with increased production of IFN-γ and IL-2 by rBCG stimulated PBMC. Studies further revealed that this enhancement in PBMC cytotoxicity was dependent on BCG secreted IFN-α as well as endogenously expressed IFN-γ and IL-2, as blockage of IFN-α, IFN-γ or IL-2 by neutralizing antibodies during BCG stimulation reduced or abolished the induction of this enhanced PBMC cytotox‐

types were responsible for the enhanced PBMC cytotoxicity induced by rBCG-IFN-α with the former cell type being more predominant [105]. A similar rBCG strain secreting human IFNα2b has also been recently demonstrated to stimulate PBMC proliferation and cytotoxicity

An early study demonstrated that human peripheral monocytes/macrophages were capable of functioning as tumoricidal cells toward bladder cancer UCRU-BL-17 cells upon activation by BCG *in vitro* [41]. It was observed that the cytotoxic activity of human monocytes/macro‐ phages was significantly enhanced after BCG stimulation, while the naïve cells exhibited only minimum cytotoxicity. Later, more studies including ours further demonstrated that murine macrophages could also function as tumoricidal cells toward bladder cancer cells upon activation by BCG *in vitro* [42-45]. Stimulation of thioglycollate-elicited peritoneal macrophag‐ es by BCG for 24 hour resulted in macrophage-mediated killing of bladder cancer MBT-2 (C3H background) and MB49 (C57BL/6 background) cells in a dose-dependent manner [44,45]. Studies also revealed that endogenous Th1 cytokines (e.g. IL-12, IL-18, IFN-γ and TNF-α)

T cells isolated from human PBMC revealed that both cell

and thus warrant further investigation.

336 Cancer Treatment - Conventional and Innovative Approaches

icity. Studies using NK and CD8+

toward bladder cancer cell lines T24 and 5637 [250].

Although the *in vitro* studies have suggested the potential usefulness of Th1 cytokine-secreting rBCG strains for the treatment of bladder cancer, unfortunately, the effect of rBCG on treating bladder cancer *in vivo* has not well been studied. Up to date, only an rBCG strain secreting IFN-γ (rBCG-IFN-γ) has been studied in a murine MB49 syngeneic orthotopic tumor model [238]. This study showed that, with a low-dose treatment regimen, intravesical administration of rBCG-IFN-γ significantly prolonged animal survival compared to medium-treated controls, whereas BCG carrying an empty vector only slightly increased survival. In a similar experi‐ ment using the MB49 syngeneic orthotopic tumor model in IFN-γ knockout mice, intravesical treatment with rBCG-IFN-γ failed to prolong survival of mice, indicating that rBCG-derived IFN-γ had no measurable antitumor effect in the absence of endogenous IFN-γ. Studies also provided the mechanisms underlying the effect of rBCG-IFN-γ on treating bladder cancer. As demonstrated, this rBCG-IFN-γ strain could specifically upregulate the expression of MHC class I molecules on MB49 cells *in vitro* compared to control BCG, as the MHC class I upregu‐ lation could be blocked by an inhibitory antibody to IFN-γ. This rBCG strain also enhanced recruitment of CD4+ T cells into the bladder and further induced the local expression of IL-2 and IL-4 mRNA compared to control BCG. In addition, we have also evaluated the effects of rBCG strains secreting murine IL-2 or IP-10 (a Th1 chemokine) on treating bladder cancer in the MB49 syngeneic orthotopic tumor model and observed survival benefits of these rBCG strains [unpublished data]. All these observations suggest that rBCG strains secreting Th1 cytokines or chemokines possess improved antitumor properties and may offer new oppor‐ tunities for the treatment of bladder cancer.

Supporting Th1 cytokine-secreting rBCG, *Mycobacterium smegmatis* (*M. smegmatis*), a closely related non-pathogenic mycobacterial organism, has been engineered to secrete murine TNFα (*M. smegmatis*/TNF-α) and tested in a transplantable MB49 tumor model [252]. Studies demonstrated that lymphocytes from tumor-bearing mice vaccinated with *M. smegmatis*/TNFα produced elevated and prolonged IFN-γ but no IL-10 in response to mycobacterial antigen or tumor lysate stimulation *in vitro*. Histopathology revealed significantly increased infiltrat‐ ing CD3+ lymphocytes in the tumor nodules of mice receiving the recombinant vaccine compared to those of mice receiving wild-type bacteria. These observations indicated that *M. smegmatis*/TNF-α induced cell-mediated immunity. Importantly, mice implanted subcutane‐ ously with MB49 tumor and treated at an adjacent site with the recombinant vaccine exhibited significantly reduced tumor growth with a 70% durable tumor-free survival compared to those treated with wild-type bacteria or BCG (a 10-20% long-term survival). Interestingly, treatment with *M. smegmatis*/TNF-α also resulted in similar tumor growth inhibition in T cell-deficient athymic nude mice and reduced but not abolished tumor growth inhibition in NK cell-deficient Beige mice. These observations indicated that NK cells contribute to the antitumor effect of *M. smegmatis*/TNF-α but are not solely responsible for the eradication of tumor. Like immu‐ nocompetent mice, Beige mice also developed tumor specific memory after treatment with *M. smegmatis*/TNF-α. A study also demonstrated enhanced immunotherapeutic potential of a human TNF-α secreting recombinant *M. smegmatis* for treating bladder cancer [253]. The ability to deliver immunomodulatory cytokines with no pathogenic effects makes *M. smegmatis* attractive as an alternative intravesical mycobacterial agent for bladder cancer treatment.

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### **5. Conclusion and future perspectives**

Intravesical administration of BCG for NMIBC represents one of the most successful immuno‐ therapies for solid malignancy. However, BCG therapy is associated with a considerable sideeffect profile and is ineffective in a significant proportion of patients. Therefore, multiple Th1 stimulating cytokines (e.g. IFN-α, IL-2 and IL-12) have been investigated either as adjuncts with BCGorasasoloreplacementtherapyinbothclinicalandpre-clinicalstudies.CombinationofBCG with IL-10 blocking mAb and genetic engineering of BCG to secrete Th1 cytokines have also been conducted in pre-clinical studies. These treatment strategies potentially allow the use of a lower and safer dose of BCG while preserving or even enhancing BCG efficacy. Despite a multitude of encouraging *in vitro* and murine studies, no clinical data has yet been reported which is compel‐ ling enough to change the current standard of care, yet many practitioners continue to use adjunctive immunotherapy based on basic science data and theoretical benefit. Further studies are needed and should focus on the optimization of combination therapies including dosing, schedule and duration. The mechanisms through which supplemental agents enhance BCGinducedTh1immuneresponsesandantitumorimmunityneedtobeexploredinbotheffectorand memory phases. In addition to classical effector cells, influence of combination therapy on Th17 and regulatory T (Treg) cells should be evaluated, as the importance of these cell types in bladder cancer has emerged. Today, research continues and efforts have been made to increase our understanding of tumor biology, human immunology, and the treatment of urothelial carcino‐ ma. The pace of research must be maintained if we are to improve this gold standard therapy for bladder cancer. BCG combination therapy merits further appraisal as an improved modality for the treatment of bladder cancer.

### **Author details**

Yi Luo, Eric J. Askeland, Mark R. Newton, Jonathan R. Henning and Michael A. O'Donnell University of Iowa, Department of Urology Iowa City, Iowa, USA

### **References**

with *M. smegmatis*/TNF-α also resulted in similar tumor growth inhibition in T cell-deficient athymic nude mice and reduced but not abolished tumor growth inhibition in NK cell-deficient Beige mice. These observations indicated that NK cells contribute to the antitumor effect of *M. smegmatis*/TNF-α but are not solely responsible for the eradication of tumor. Like immu‐ nocompetent mice, Beige mice also developed tumor specific memory after treatment with *M. smegmatis*/TNF-α. A study also demonstrated enhanced immunotherapeutic potential of a human TNF-α secreting recombinant *M. smegmatis* for treating bladder cancer [253]. The ability to deliver immunomodulatory cytokines with no pathogenic effects makes *M. smegmatis* attractive as an alternative intravesical mycobacterial agent for bladder cancer treatment.

Intravesical administration of BCG for NMIBC represents one of the most successful immuno‐ therapies for solid malignancy. However, BCG therapy is associated with a considerable sideeffect profile and is ineffective in a significant proportion of patients. Therefore, multiple Th1 stimulating cytokines (e.g. IFN-α, IL-2 and IL-12) have been investigated either as adjuncts with BCGorasasoloreplacementtherapyinbothclinicalandpre-clinicalstudies.CombinationofBCG with IL-10 blocking mAb and genetic engineering of BCG to secrete Th1 cytokines have also been conducted in pre-clinical studies. These treatment strategies potentially allow the use of a lower and safer dose of BCG while preserving or even enhancing BCG efficacy. Despite a multitude of encouraging *in vitro* and murine studies, no clinical data has yet been reported which is compel‐ ling enough to change the current standard of care, yet many practitioners continue to use adjunctive immunotherapy based on basic science data and theoretical benefit. Further studies are needed and should focus on the optimization of combination therapies including dosing, schedule and duration. The mechanisms through which supplemental agents enhance BCGinducedTh1immuneresponsesandantitumorimmunityneedtobeexploredinbotheffectorand memory phases. In addition to classical effector cells, influence of combination therapy on Th17 and regulatory T (Treg) cells should be evaluated, as the importance of these cell types in bladder cancer has emerged. Today, research continues and efforts have been made to increase our understanding of tumor biology, human immunology, and the treatment of urothelial carcino‐ ma. The pace of research must be maintained if we are to improve this gold standard therapy for bladder cancer. BCG combination therapy merits further appraisal as an improved modality for

Yi Luo, Eric J. Askeland, Mark R. Newton, Jonathan R. Henning and Michael A. O'Donnell

University of Iowa, Department of Urology Iowa City, Iowa, USA

**5. Conclusion and future perspectives**

338 Cancer Treatment - Conventional and Innovative Approaches

the treatment of bladder cancer.

**Author details**


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[240] Chen X, O'Donnell MA, Luo Y. Dose-dependent synergy of Th1-stimulating cyto‐ kines on bacille Calmette-Guérin-induced interferon-gamma production by human mononuclear cells. Clinical and Experimental Immunology 2007;149(1):178-185. [241] Ryan AA, Spratt JM, Britton WJ, Triccas JA. Secretion of functional monocyte chemo‐ tactic protein 3 by recombinant Mycobacterium bovis BCG attenuates vaccine viru‐ lence and maintains protective efficacy against M. tuberculosis infection. Infection

[242] Xu Y, Zhu B, Wang Q, Chen J, Qie Y, Wang J, Wang H, Wang B, Wang H. Recombi‐ nant BCG coexpressing Ag85B, ESAT-6 and mouse-IFN-gamma confers effective protection against Mycobacterium tuberculosis in C57BL/6 mice. FEMS Immunology

[243] Tang C, Yamada H, Shibata K, Maeda N, Yoshida S, Wajjwalku W, Ohara N, Yamada T, Kinoshita T, Yoshikai Y. Efficacy of recombinant bacille Calmette-Guérin vaccine secreting interleukin-15/antigen 85B fusion protein in providing protection against Mycobacterium tuberculosis. Journal of Infectious Diseases 2008;197(9):1263-1274.

novel breast cancer vaccine. Cancer Research 2003;63(6):1280-1287.

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mice. Infection and Immunity 2002;70(12):6549-6557.

Journal of Oncology 2002;20(6):1305-1311.

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and Medcal Microbiology 2007;51(3):480-487.

175-185.

360 Cancer Treatment - Conventional and Innovative Approaches

1065-1072.


**Chapter 16**

**Anti-Angiogenic Active Immunotherapy for Cancers:**

The formation of blood vessels occurs by two mechanisms [1]: vasculogenesis and angiogen‐ esis. Vasculogenesis is the process during which blood vessels are formed *de novo* by *in situ* differentiation of the primitive progenitor (i.e. angioblasts) into mature endothelial cells, which was thought to take place only during embryonic development. However, angiogenesis occurs both during embryonic development and postnatal life. Angiogenesis is defined as a process which gives rise to new blood vessels by proliferation and migration of preexisting differen‐ tiated endothelial cells. In embryonic life, angiogenesis is a critical process that leads to formation of stable vasculature comprised of endothelial cells, mural cells (pericytes) and basement membrane in the adult [2]. Vasculature in healthy adult is very stable with the exception of rare events such as cyclical growth of vessels in the ovarian corpus luteum or during pregnancy, angiogenesis activities are rare in adult individuals [2]. In addition to normal development, angiogenesis is known to be an important event in pathological condi‐

tions such as tissue repair during wound healing and in the growth of tumors [3].

Tumor angiogenesis, the formation of new blood vessels supplying the tumor mass, plays a critical role in tumor growth, progression, persistence and metastasis, because the proliferation and metastasis of malignant tumors are dependent on the sufficient nutrition supplied by the new vessels [4-6]. Many molecules have been demonstrated as positive regulators of angio‐ genesis, including vascular endothelial growth factor (VEGF), acidic or basic fibroblast growth factor (aFGF, bFGF), epidermal growth factor (EGF), transforming growth factor-α/β (TGF-α, TGF-β), placental growth factor (PlGF), angiopoietin, angiogenin, endoglin (CD105), prostatespecific membrane antigen (PSMA), the anthrax-toxin-receptor (ATR, TEM8), connective

and reproduction in any medium, provided the original work is properly cited.

© 2013 Pan and Zhang; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Dawn of a New Era?**

http://dx.doi.org/10.5772/55323

**1. Introduction**

Jianping Pan and Lihuang Zhang

**1.1. Angiogenesis in health and disease**

Additional information is available at the end of the chapter

## **Anti-Angiogenic Active Immunotherapy for Cancers: Dawn of a New Era?**

Jianping Pan and Lihuang Zhang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55323

### **1. Introduction**

### **1.1. Angiogenesis in health and disease**

The formation of blood vessels occurs by two mechanisms [1]: vasculogenesis and angiogen‐ esis. Vasculogenesis is the process during which blood vessels are formed *de novo* by *in situ* differentiation of the primitive progenitor (i.e. angioblasts) into mature endothelial cells, which was thought to take place only during embryonic development. However, angiogenesis occurs both during embryonic development and postnatal life. Angiogenesis is defined as a process which gives rise to new blood vessels by proliferation and migration of preexisting differen‐ tiated endothelial cells. In embryonic life, angiogenesis is a critical process that leads to formation of stable vasculature comprised of endothelial cells, mural cells (pericytes) and basement membrane in the adult [2]. Vasculature in healthy adult is very stable with the exception of rare events such as cyclical growth of vessels in the ovarian corpus luteum or during pregnancy, angiogenesis activities are rare in adult individuals [2]. In addition to normal development, angiogenesis is known to be an important event in pathological condi‐ tions such as tissue repair during wound healing and in the growth of tumors [3].

Tumor angiogenesis, the formation of new blood vessels supplying the tumor mass, plays a critical role in tumor growth, progression, persistence and metastasis, because the proliferation and metastasis of malignant tumors are dependent on the sufficient nutrition supplied by the new vessels [4-6]. Many molecules have been demonstrated as positive regulators of angio‐ genesis, including vascular endothelial growth factor (VEGF), acidic or basic fibroblast growth factor (aFGF, bFGF), epidermal growth factor (EGF), transforming growth factor-α/β (TGF-α, TGF-β), placental growth factor (PlGF), angiopoietin, angiogenin, endoglin (CD105), prostatespecific membrane antigen (PSMA), the anthrax-toxin-receptor (ATR, TEM8), connective

© 2013 Pan and Zhang; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

tissue growth factor (CTGF, CCN2), urokinase plasminogen activator (uPA), and several others [7-10]. However, VEGF-mediated signaling through its receptor VEGFR-2 is the key rate-limiting step in tumor angiogenesis, and plays the most important role in neovasculari‐ zation, development, and progression of various tumors including hematopoietic malignan‐ cies [11, 12], breast cancer [13], bladder cancer [14], and renal cell cancer [15]. Importantly, it has been found that tumor growth can be attenuated *via* the suppression of angiogenesis [7].

ing VEGF and other signaling pathways have been developed. Some of the most

**Compound Company Targets Indications** Sunitinib (SU11248) Pfizer VEGFRs, PDGFRB, CSF1R, c-Kit RCC, GIST Pazopanib (Votrient) GSK VEGFRs, PDGFRs and c-kit RCC

Cabozantinib (XL184) Exelixis VEGFRs Met Prostate Cancer

Axitinib (AG-013736) Pfizer VEGFRs mRCC Linifanib (ABT-869) Abbott VEGFRs, PDGFRB, CSF1R RCC, NSCLC

**3.** Therapy with angiogenesis negative regulators: Many negative regulators, such as angiostatin, endostatin, interferon-γ etc., are involved in the angiogenesis [25]. Therefore the use of these agents to negatively regulate angiogenesis is another strategy for cancer treatment. For example, recombinant human endostatin has been approved in September 2005 by the State Food and Drug Administration (SFDA), P. R. China. The phase III clinical trial of endostatin in China showed promising effects. Combination of endostatin and NP regimen (vinorelbine and cisplatin) significantly improved the therapeutic efficacy in patients with advanced nonsmall-cell lung cancer and safely extended the median time

**4.** Therapy with vascular disrupting agents: A strategy directly induce vascular collapse. ASA404, a flavonoid compound, is one of the vascular disrupting agents that induce apoptosis of tumor associated endothelial cells, resulting in the inhibition of blood flow, causing hypoxia and necrosis in tumor mass. ASA404 is currently in advanced stage of clinical trial in combination with standard of care in non-small-cell lung cancer [27]. **5.** Active immunotherapy: Active immunotherapy targeting tumor angiogenesis is a novel modality for treatment of cancers which is based on several assumptions: a) Tumorderived endothelial cells (ECs) possess characteristics distinct from those of normal tissue [18]. b) Specific immune responses against self-antigen can be elicited. c) Tumor growth can be attenuated via suppression of angiogenesis [7]. The main aim of the active immu‐ notherapy targeting tumor vessels is to break self immunological tolerance to the positive regulators of angiogenesis, hereby inhibiting tumor angiogenesis and thus leading to the inhibition of tumor growth and metastasis. Anti-angiogenic active immunotherapies can

VEGFR-2 and -3, PDGFR-b, Flt-3, c-kit, Raf

VEGFRs and EGFRs, RET- tyrosine kinases

kinases RCC, Inoperable HCC

Anti-Angiogenic Active Immunotherapy for Cancers: Dawn of a New Era?

VEGFRs RCC

Late-stage Medullary Thyroid Cancer

http://dx.doi.org/10.5772/55323

365

clinically relevant RTKIs are summarized in Table 1.

AVEO Pharmaceutical

**Table 1.** VEGF RTKIs and their indications in caner patients (adopted from [2])

) Bayer

Vendatanib (Caprelsa) AstraZeneca

of tumor progression [26].

Sorafenib (Bay 43- 9006; Nexavar®

> Tivozanib (AV-951, KRN-951)

#### **1.2. Anti-angiogenic strategies for cancer treatment**

Given the role of angiogenesis in tumor growth and progression, therapeutic strategies targeting tumor vascular endothelia rather than tumor cells have several merits in comparison to conventional anti-tumor therapies [16, 17]: (a) Vascular endothelial cells have genetically stable MHC expression on the surface, which will not be down-regulated, in contrast to the surface of tumor cells [18]; (b) Effector cells or antibodies can reach targeted endothelial cells more readily than they can reach tumor cells [17]; (c) Treatment by targeting endothelial cells is not restricted to specific tumor entities [16, 17, 19]; (d) As each tumor vessel supplies hundreds of tumor cells, the inhibition or diminishment of a large amount of tumor cells could be achieved merely by the comparatively limited impairment of neovascularized endothelial cells; as a consequence, the efficiency of targeting tumor blood vessel endothelium should be higher than that of targeting tumor cells themselves [15]. (e) Several specific anti-angiogenic agents, such as IFN-γ, have very low toxicity in some cases of drug combination-therapy regimens in both patients and animal models [16]. In recent years, the field of anti-angiogenic therapy for cancers has attracted much attention. In general, anti-angiogenic strategies can be divided into the following five categories:



ing VEGF and other signaling pathways have been developed. Some of the most clinically relevant RTKIs are summarized in Table 1.

**Table 1.** VEGF RTKIs and their indications in caner patients (adopted from [2])

tissue growth factor (CTGF, CCN2), urokinase plasminogen activator (uPA), and several others [7-10]. However, VEGF-mediated signaling through its receptor VEGFR-2 is the key rate-limiting step in tumor angiogenesis, and plays the most important role in neovasculari‐ zation, development, and progression of various tumors including hematopoietic malignan‐ cies [11, 12], breast cancer [13], bladder cancer [14], and renal cell cancer [15]. Importantly, it has been found that tumor growth can be attenuated *via* the suppression of angiogenesis [7].

Given the role of angiogenesis in tumor growth and progression, therapeutic strategies targeting tumor vascular endothelia rather than tumor cells have several merits in comparison to conventional anti-tumor therapies [16, 17]: (a) Vascular endothelial cells have genetically stable MHC expression on the surface, which will not be down-regulated, in contrast to the surface of tumor cells [18]; (b) Effector cells or antibodies can reach targeted endothelial cells more readily than they can reach tumor cells [17]; (c) Treatment by targeting endothelial cells is not restricted to specific tumor entities [16, 17, 19]; (d) As each tumor vessel supplies hundreds of tumor cells, the inhibition or diminishment of a large amount of tumor cells could be achieved merely by the comparatively limited impairment of neovascularized endothelial cells; as a consequence, the efficiency of targeting tumor blood vessel endothelium should be higher than that of targeting tumor cells themselves [15]. (e) Several specific anti-angiogenic agents, such as IFN-γ, have very low toxicity in some cases of drug combination-therapy regimens in both patients and animal models [16]. In recent years, the field of anti-angiogenic therapy for cancers has attracted much attention. In general, anti-angiogenic strategies can be

**1.** Passive immunotherapy: The use of antibody to neutralize angiogenesis positive regula‐ tors such as VEGF. In 2003, the Food & Drug Administration (FDA) of the United States approved Bevacizumab (Avastin®; Genentech Inc.), a humanized variant of VEGF neutralizing monoclonal antibody, as the first anti-angiogenic agent for combinatorial treatment with standard of care in metastatic colorectal cancer [20] and subsequently for treatment of patients with non-small-cell lung cancer [21] or metastatic breast cancer [22]. The combinatorial treatment of Bevacizumab with conventional chemotherapy showed increased therapeutic efficacy, for example in patients with metastatic colorectal cancer, the median survival time was extended by 4.7 months [20]. Besides combining with conventional chemotherapy, bevacizumab could combine with radiation therapy safely

**2.** Therapy with VEGF inhibitors: Since the approval of Bevacizumab in the clinical use by FDA, several VEGF inhibitors including small molecules targeting VEGF or its receptors came into different stages of clinical development. For example VEGF-TrapR1- R2 (Aflibercept; Regeneron Inc.), a chimeric soluble receptor containing structural elements from VEGFR1 and VEGFR2, has the ability to bind to and neutralize the circulating VEGF [24]. VEGF-TrapR1-R2 has shown potent anti-tumor activity in preclinical animal models and is currently in clinical trials [24]. In addition to VEGF inhibitors, several small molecule receptor tyrosine kinase inhibitors (RTKIs) target‐

**1.2. Anti-angiogenic strategies for cancer treatment**

364 Cancer Treatment - Conventional and Innovative Approaches

divided into the following five categories:

and effectively [23].


be divided into two categories: one is based on the immunological cross-reactions mediated by vaccination with xenogeneic homologous molecules associated with angiogenesis, and the other targets non-xenogeneic homologous molecules. Therapeutic targets, vaccines and tumor models used in anti-angiogenic active immunotherapy for cancers are summarized in Table 2.

**Strategies Therapeutic targets Vaccines Tumor models References**

Murine sVEGFR-2 and IFN-γ fusion gene-transfected DC

Recombinant plasmid encoding flk-1

H-2Db-restricted KDR2 or KDR3 peptide

HLA-A\*0201-restricted VEGFR-2 epitope peptide

VEGFR2-169

domain peptide

ectodomain pulsed DC

murine MHC class I antigen epitopes of Legumain

endoglin

Listeria-based vaccines encoding murine endoglin

Notes: B16: B16 melanoma; B16BL6: B16BL6 tumor cells; D121: D121 lung carcinoma; D2F2: breast carcinoma cells; D2F2/E2: mammary tumor cells derived from BALB/c mouse and were transfected with the cDNA encoding ERBB2; EL-4: EL-4 lymphoma; FM3A: mammary cancer; H22: H22 hepatoma; LL/2: LL/2 Lewis lung carcinoma; LLC-LM: LLC-LM tumor cells; MA782/5S: MA782/5S mammary cancer; Meth A: Meth A fibrosarcoma; MBT-2: MBT-2 bladder tu‐ mor; MC38: MC38 murine colon cancer; MOPC-315: MOPC-315 plasmacytoma; TC-1: TC-1 carcinoma; 3LL: 3LL Lewis

Endothelium Endothelium lysates pulsed DC Colon-26 colon cancer 89

Glutaraldehyde-fixed HUVECs Patients with recurrent

Human VEGFR-2 HLA-A\*2402-restricted

Murine FGF-2 FGF-2 heparin-binding structural

Murine EGFR Recombinant mouse EGFR

Legumain Minigene plasmid DNA encoding

Endoglin Plasmid DNA encoding murine

Murine VEGFR-2 flk-1 protein pulsed DC B16, 3LL 62

flk1 mRNA-transfected DC B16, MBT-2 64

Anti-Angiogenic Active Immunotherapy for Cancers: Dawn of a New Era?

*L. monocytogene*-based Flk-1 4T1-Luc 71, 72 pSG.SS.Flk-1ECD.C3d3 BTT739 73

B16, 3LL 68

http://dx.doi.org/10.5772/55323

367

MC38 69

MC38, B16 70

B16BL6, LLC-LM 39

D2F2 85

D2F2 86

74

80

87

90

Patients with advanced pancreatic cancer

Lewis lung carcinoma and mammary cancer

4T1-Luc and NT-2 breast cancer

malignant brain tumour or metastatic colorectal cancer

B16, MC38, D121 63

Vaccination based on non-xenogeneic homologous molecules

> VEGFR-2 in transgenic mice expressing HLA-A\*0201

lung carcinoma; HUVECs: human umbilical vein endothelial cells.

**Table 2.** Anti-angiogenic active immunotherapy for cancers



Notes: B16: B16 melanoma; B16BL6: B16BL6 tumor cells; D121: D121 lung carcinoma; D2F2: breast carcinoma cells; D2F2/E2: mammary tumor cells derived from BALB/c mouse and were transfected with the cDNA encoding ERBB2; EL-4: EL-4 lymphoma; FM3A: mammary cancer; H22: H22 hepatoma; LL/2: LL/2 Lewis lung carcinoma; LLC-LM: LLC-LM tumor cells; MA782/5S: MA782/5S mammary cancer; Meth A: Meth A fibrosarcoma; MBT-2: MBT-2 bladder tu‐ mor; MC38: MC38 murine colon cancer; MOPC-315: MOPC-315 plasmacytoma; TC-1: TC-1 carcinoma; 3LL: 3LL Lewis lung carcinoma; HUVECs: human umbilical vein endothelial cells.

**Table 2.** Anti-angiogenic active immunotherapy for cancers

be divided into two categories: one is based on the immunological cross-reactions mediated by vaccination with xenogeneic homologous molecules associated with angiogenesis, and the other targets non-xenogeneic homologous molecules. Therapeutic targets, vaccines and tumor models used in anti-angiogenic active immunotherapy for

**Strategies Therapeutic targets Vaccines Tumor models References**

Human umbilical vein endothelial cells, human dermal microvascular endothelial cells, and bovine glomerular endothelial cells

VEGF of xenopus laevis

pMAE5△5 vectors harboring human VEGF 121 gene and mutated human VEGF 121 gene

VEGFR-2

Plasmid DNA encoding the C terminal 37 amino acids of hCGβ, 5 different CTL epitopes from human surviving and the 3rd and 4th extracellular domains of VEGFR-2

*Bifidobacterium infantis* expressing human sKDR

Murine FGFR-1 cDNA encoding Xenopus FGFR-1 Meth A, H22, and

binding domain of chicken integrin β3

MMP-2

RHAMM

angiomotin

Murine DLL4 Plasmid encoding human DLL4 D2F2/E2 55

Murine αvβ3 Plasmid DNA encoding the ligand-

Murine MMP-2 Plasmid DNA encoding chicken

Murine RHAMM Plasmid encoding *Xenopus*

Murine Angiomotin Plasmid encoding human

Canine VEGF Liposome-DNA adjuvant Soft tissue sarcoma 34

Murine VEGF Recombinant plasmid encoding

Murine VEGFR-2 Plasmid DNA encoding quail

Meth A, H22, MA782/5S, FM3A, and Lewis Lung carcinoma

> Meth A, H22 and MA782/5S

> Meth A, EL-4 and MOPC-315,

EL-4, B16, and TC-1 33

LL/2 lung carcinoma 37

Lewis lung cancer 38

LL/2, Meth A, H22 48

TUBO breast cancer 57, 61

B16 49

MA782/5S

Meth A, H22, and MA782/5S

29

32

36

41

43

cancers are summarized in Table 2.

366 Cancer Treatment - Conventional and Innovative Approaches

Murine vascular endothelial cells

Vaccination based on xenogeneic homologous molecules

### **2. Anti-angiogenic active immunotherapy**

#### **2.1. Anti-angiogenic active immunotherapy based on xenogeneic homologous molecules**

(such as VEGER2, von Willebrand factor, CD105) [30]. EO-EPCs also incorporated DiLDL and bound UEA-I, which are endothelial features, and additionally, they formed vascular-like structures on Matrigel [30]. Thus, it might be a promising strategy toward anti-angiogenic cancer treatment to use EO-EPCs as cell vaccine to inhibit tumor angiogenesis, since such cells might function both as dendritic-like cells to augment anti-tumor immunity and as xenogeneic proliferative endothelial cells to break self-tolerance, thereby inducing profound anti-angio‐

Anti-Angiogenic Active Immunotherapy for Cancers: Dawn of a New Era?

http://dx.doi.org/10.5772/55323

369

VEGF is a potent and crucial vasculogenic and angiogenic factor, which can induce endothelial cell proliferation, promote cell migration, and inhibit endothelial cell apoptosis [5, 6]. In most types of cancers, VEGF is often present at elevated levels, and strategies aimed at blocking its activity usually lead to suppression of tumor angiogenesis and consequently tumor growth inhibition [31]. The amino acid sequence of VEGF in *Xenopus laevis* shares 75 % and 73 % homology with that of VEGF164 in mice and that of VEGF165 in humans, respectively [32]. Recombinant eukaryotic expression plasmids harboring VEGF-encoding gene of mice and *Xenopus laevis*, respectively, designated as MVEGF-P and XVEGF-P, have been constructed. Immunization of mice with XVEGF-P provoked protective and therapeutic anti-tumor immunological effects in mouse tumor models with Meth A fibrosarcoma, MA782/5S mam‐ mary cancer and H22 hepatoma [32]. Anti-VEGF specific autoantibody was detected in serum of mice vaccinated with XVEGF-P by Western blot and ELISA [32]. The VEGF levels in the tumor-bearing mice immunized with XVEGF-P was lower than that in the control groups [32]. Furthermore, the frequency of anti-VEGF antibody-producing B cells in the spleen of mice immunized with XVEGF-P was remarkably higher than that in the spleen of control groups where such B cells were undetectable [32]. VEGF-mediated proliferation of ECs could be inhibited *in vitro* by purified immunoglobulins from XVEGF-P-immunized mice. Adoptive transfer of the purified immunoglobulins into non-immunized tumor-bearing mice could also inhibit tumor angiogenesis *in vivo* and generate anti-tumor effects [32]. Anti-CD4+ monoclonal antibody could obstruct the escalation of concentration of immunoglobulin IgG1 and IgG2 in serum and also block the anti-tumor effects of XVEGF-P DNA vaccines, indicating that CD4+ T lymphocytes were responsible for XVEGF-P-induced anti-tumor effects [32]. The possibility that the anti-tumor activity may result from nonspecifically augmented immune response could be ruled out by the findings that no increase in NK activity of spleen cells or in the level of cytokines such as IFN-α, IFN-β, TNF-α, or β-chemokine in sera was found in immunized mice [32]. Recently, it was reported that when immunized with human VEGF isoform 121 gene (hVEGF121) inserted into pMAE5△5 vector (pM-VEGF) and later challenged with melanoma or lung carcinoma tumor cells, a reduction of tumor growth and an increased survival of tumor-bearing C57BL/6 mice were observed because the hVEGF121 gene is highly homolo‐ gous to its murine counterpart [33]. A decrease in tumor cell density around vessels and in mitotic figures, as well as an increase in apoptotic tumor cells were manifested by histopatho‐ logical analyses of tumors from C57BL/6 mice immunized with hVEGF121 [33]. Spleen cells

genic effects *in vivo*.

*2.1.2. Non-cell vaccines*

*2.1.2.1. VEGF/VEGFR2*

Homologous molecules in different species are formed as the result of evolution. Molecules with essential functions keep the stability of their molecular sequences, although some moderate degree of evolution is essential for adaptation to different environments and physiological requirements in different species. Many genes in the human and mouse genome are similar (but not identical) to the corresponding genome sequences of the fruit fly Droso‐ phila melanogaster and other non-vertebrates such as *Xenopus laevis* [28]. In consequence, effective immune response to self antigens associated with angiogenesis can thus be induced by vaccination with xenogeneic homologous molecules.

#### *2.1.1. Cell vaccine*

Neovascular endothelial cells in tumor tissues express proteins not present or not detectable in normal vascular endothelial cells, such as αvβ3 integrin and receptors for certain angiogenic growth factors [18]. These proteins in murine vascular endothelial cells share homology to varying degree with counterparts of other species including human [18]. Vaccination of mice with paraformaldehyde-fixed xenogeneic human and bovine proliferative vascular endothe‐ lial cells, such as human umbilical vein endothelial cells, human dermal microvascular endothelial cells, and bovine glomerular endothelial cells, resulted in successful breaking of the immunological tolerance to autogeneic vascular endothelial cells in several murine tumor models, such as Meth A fibrosarcoma, MA782/5S and FM3A mammary cancer, H22 hepatoma, and Lewis lung carcinoma, generating a protective and therapeutic anti-tumor immunological reaction [29]. Antibodies against the receptors associated with tumor angiogenesis generated in mice immunized with the xenogeneic homologous proliferative vascular endothelial cell vaccines might inhibit the proliferation of endothelial cells in vivo, leading to the regression of established tumor, and the prolonged survival of tumor-bearing mice [29]. Tumor angio‐ genesis could be suppressed by the adoptive transfer of autoreactive immunoglobulins purified from the immunized mouse, resulting in inhibition of tumor growth in mice [29]. Autoantibody sediments were detected on ECs within tumor tissues in the immunized mice by immunohistochemical analysis [29]. Furthermore, Western blot analysis showed that reactions between the extract from murine ECs and the serum from the immunized mice resulted in several positive bands, at least two of which, with the molecular weight of 220 kDa and 130 kDa, had similar molecular sizes to those of ligand-binding sites of known VEGFR2 and αv integrin, respectively [29], although the authors did not provide direct evidence to demonstrate that the two positive bands aforementioned contained VEGFR2 and αv integrin respectively. Immune cell subset depletion experiments showed that the production of autoantibodies against tumor vascular ECs and the anti-tumor effect were dependent on CD4+ T lymphocytes [29].

In 2006, early-outgrowth progenitor endothelial cells (EO-EPCs) have been characterized on the basis of their dendritic-like phenotypes (such as expression of HLA-DR, CD40, CD54, CD80, and CD86), phagocytotic and antigen-presenting functions, and endothelial markers (such as VEGER2, von Willebrand factor, CD105) [30]. EO-EPCs also incorporated DiLDL and bound UEA-I, which are endothelial features, and additionally, they formed vascular-like structures on Matrigel [30]. Thus, it might be a promising strategy toward anti-angiogenic cancer treatment to use EO-EPCs as cell vaccine to inhibit tumor angiogenesis, since such cells might function both as dendritic-like cells to augment anti-tumor immunity and as xenogeneic proliferative endothelial cells to break self-tolerance, thereby inducing profound anti-angio‐ genic effects *in vivo*.

#### *2.1.2. Non-cell vaccines*

**2. Anti-angiogenic active immunotherapy**

368 Cancer Treatment - Conventional and Innovative Approaches

by vaccination with xenogeneic homologous molecules.

*2.1.1. Cell vaccine*

CD4+

T lymphocytes [29].

**2.1. Anti-angiogenic active immunotherapy based on xenogeneic homologous molecules** Homologous molecules in different species are formed as the result of evolution. Molecules with essential functions keep the stability of their molecular sequences, although some moderate degree of evolution is essential for adaptation to different environments and physiological requirements in different species. Many genes in the human and mouse genome are similar (but not identical) to the corresponding genome sequences of the fruit fly Droso‐ phila melanogaster and other non-vertebrates such as *Xenopus laevis* [28]. In consequence, effective immune response to self antigens associated with angiogenesis can thus be induced

Neovascular endothelial cells in tumor tissues express proteins not present or not detectable in normal vascular endothelial cells, such as αvβ3 integrin and receptors for certain angiogenic growth factors [18]. These proteins in murine vascular endothelial cells share homology to varying degree with counterparts of other species including human [18]. Vaccination of mice with paraformaldehyde-fixed xenogeneic human and bovine proliferative vascular endothe‐ lial cells, such as human umbilical vein endothelial cells, human dermal microvascular endothelial cells, and bovine glomerular endothelial cells, resulted in successful breaking of the immunological tolerance to autogeneic vascular endothelial cells in several murine tumor models, such as Meth A fibrosarcoma, MA782/5S and FM3A mammary cancer, H22 hepatoma, and Lewis lung carcinoma, generating a protective and therapeutic anti-tumor immunological reaction [29]. Antibodies against the receptors associated with tumor angiogenesis generated in mice immunized with the xenogeneic homologous proliferative vascular endothelial cell vaccines might inhibit the proliferation of endothelial cells in vivo, leading to the regression of established tumor, and the prolonged survival of tumor-bearing mice [29]. Tumor angio‐ genesis could be suppressed by the adoptive transfer of autoreactive immunoglobulins purified from the immunized mouse, resulting in inhibition of tumor growth in mice [29]. Autoantibody sediments were detected on ECs within tumor tissues in the immunized mice by immunohistochemical analysis [29]. Furthermore, Western blot analysis showed that reactions between the extract from murine ECs and the serum from the immunized mice resulted in several positive bands, at least two of which, with the molecular weight of 220 kDa and 130 kDa, had similar molecular sizes to those of ligand-binding sites of known VEGFR2 and αv integrin, respectively [29], although the authors did not provide direct evidence to demonstrate that the two positive bands aforementioned contained VEGFR2 and αv integrin respectively. Immune cell subset depletion experiments showed that the production of autoantibodies against tumor vascular ECs and the anti-tumor effect were dependent on

In 2006, early-outgrowth progenitor endothelial cells (EO-EPCs) have been characterized on the basis of their dendritic-like phenotypes (such as expression of HLA-DR, CD40, CD54, CD80, and CD86), phagocytotic and antigen-presenting functions, and endothelial markers

#### *2.1.2.1. VEGF/VEGFR2*

VEGF is a potent and crucial vasculogenic and angiogenic factor, which can induce endothelial cell proliferation, promote cell migration, and inhibit endothelial cell apoptosis [5, 6]. In most types of cancers, VEGF is often present at elevated levels, and strategies aimed at blocking its activity usually lead to suppression of tumor angiogenesis and consequently tumor growth inhibition [31]. The amino acid sequence of VEGF in *Xenopus laevis* shares 75 % and 73 % homology with that of VEGF164 in mice and that of VEGF165 in humans, respectively [32]. Recombinant eukaryotic expression plasmids harboring VEGF-encoding gene of mice and *Xenopus laevis*, respectively, designated as MVEGF-P and XVEGF-P, have been constructed. Immunization of mice with XVEGF-P provoked protective and therapeutic anti-tumor immunological effects in mouse tumor models with Meth A fibrosarcoma, MA782/5S mam‐ mary cancer and H22 hepatoma [32]. Anti-VEGF specific autoantibody was detected in serum of mice vaccinated with XVEGF-P by Western blot and ELISA [32]. The VEGF levels in the tumor-bearing mice immunized with XVEGF-P was lower than that in the control groups [32]. Furthermore, the frequency of anti-VEGF antibody-producing B cells in the spleen of mice immunized with XVEGF-P was remarkably higher than that in the spleen of control groups where such B cells were undetectable [32]. VEGF-mediated proliferation of ECs could be inhibited *in vitro* by purified immunoglobulins from XVEGF-P-immunized mice. Adoptive transfer of the purified immunoglobulins into non-immunized tumor-bearing mice could also inhibit tumor angiogenesis *in vivo* and generate anti-tumor effects [32]. Anti-CD4+ monoclonal antibody could obstruct the escalation of concentration of immunoglobulin IgG1 and IgG2 in serum and also block the anti-tumor effects of XVEGF-P DNA vaccines, indicating that CD4+ T lymphocytes were responsible for XVEGF-P-induced anti-tumor effects [32]. The possibility that the anti-tumor activity may result from nonspecifically augmented immune response could be ruled out by the findings that no increase in NK activity of spleen cells or in the level of cytokines such as IFN-α, IFN-β, TNF-α, or β-chemokine in sera was found in immunized mice [32]. Recently, it was reported that when immunized with human VEGF isoform 121 gene (hVEGF121) inserted into pMAE5△5 vector (pM-VEGF) and later challenged with melanoma or lung carcinoma tumor cells, a reduction of tumor growth and an increased survival of tumor-bearing C57BL/6 mice were observed because the hVEGF121 gene is highly homolo‐ gous to its murine counterpart [33]. A decrease in tumor cell density around vessels and in mitotic figures, as well as an increase in apoptotic tumor cells were manifested by histopatho‐ logical analyses of tumors from C57BL/6 mice immunized with hVEGF121 [33]. Spleen cells from mice immunized with pM-VEGF showed a significant enhanced cytotoxic activity against VEGF-secreting tumor cells, including EL-4 lymphoma, B16-F10 melanoma, and TC-1 carcinoma, as compared with those obtained from the mice immunized with the pMAE5△5 ''empty'' vector [33]. IFN-γ ELISPOT assay revealed a significant increase in the number of spots in spleen cells from mice immunized with pM-VEGF [33]. Vaccination with a mutated hVEGF121 gene inserted into the pMAE5△5 vector (pM-VEGFmut) produced similar *in vitro* and *in vivo* results, and remarkably reduced the number of spontaneous metastases in a murine model with Lewis lung carcinoma [33]. Serum VEGF levels decreased 8-fold in mice vaccinated with pM-VEGF or pM-VEGFmut as compared with those in pMAE5△5 treated mice [33]. A significant correlation was also found between the elevation of serum VEGF level and the increase of the tumor dimensions [33]. However, antibody responses against the GSThVEGF121 fusion protein or GST alone used as capture antigens in ELISA were undetect‐ able in animals vaccinated with pM-VEGF or pM-VEGFmut [33]. These findings indicate that human VEGF-harboring DNA vaccine can be employed for anti-angiogenic active immuno‐ therapy for cancers in mice and direct cell cytotoxicity contributes to the overall anti-tumor effects observed in immunized mice [33].

immunized mice recognized not only recombinant qVEGFR-2, but also recombinant mouse VEGFR-2 (mVEGFR) in Western blot analysis [36]. In contrast, the sera isolated from controls showed negative staining [36]. Sera from mice immunized with qVEGFR-2 recognized a single band in flk-1-positive mouse SVEC4-10 endothelial cells and KDRpositive human umbilical vein endothelial cells, with the same size as recognized by commercially available flk-1 or KDR antibodies [36]. Sera from qVEGFR-2-immunized mice also recognized recombinant protein qVEGFR-2 and mVEGFR-2 in ELISA [36]. Detectable IgG1 and IgG2b with significantly elevated concentration in sera were found to be responsible for the immunoglobulin response to VEGFR-2 [36]. Anti-VEGFR-2 specific antibody-producing B cells were detected by ELISPOT. The number of anti-VEGFR-2 antibody-producing B cells was elevated in the spleens of mice immunized with qVEGFR-2, compared with that in controls [36]. Deposition of immunoglobulins on endothelial cells was found within tumors from qVEGFR-2-immunized mice, but not from controls [36]. Adoptive transfer of the purified immunoglobulins from qVEGFR-2-immunized mice resulted in inhibition of VEGF-mediated endothelial cell proliferation and effective protection against tumor growth [36]. Angiogenesis was markedly suppressed within the tumors, and the vascularization of alginate beads was also diminished [36]. Depletion of CD4+ T lymphocyte could abrogate the anti-tumor activity and the production of autoanti‐

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Very recently, a DNA vaccine designed by synergizing different tumor antigens with VEGFR2 was constructed. A DNA fragment (HSV) encoding the C terminal 37 amino acids of human chorionic gonadotropin β chain (hCGβ), 5 different HLA-restricted cytotoxic T lymphocyte epitopes from human survivin and the third and fourth extracellular domains of VEGFR2 was inserted into the sequence between the luminal and transmembrane domain of human lysosome-associated membrane protein-1 cDNA for the construction of a novel DNA vaccine (p-L/HSV) [37]. Vaccination of the mice with p-L/HSV elicited potent and long-lasting cellular and humoral immune responses to the specific antigens and showed a prominent anti-tumor effect on the LL/2 lung carcinoma model in syngeneic C57BL/6 mice. In addition, the tumor vasculature was abrogated as observed by immunohistochemistry in p-L/HSV immunized mice [37]. These data indicates that the strategies of combining anti-tumor with anti-angio‐ genesis cooperate well. Such a study may shed new light on the designing of vaccine for cancer

Again in 2012, a *Bifidobacterium infantis*-based vaccine that express human extracellular domain of VEGFR2 (sKDR) was established [38]. Immunization of the mice with the *Bifidobacterium infantis*-based vaccine through caudal vein could significantly suppress the tumor growth and prolong the survival of the tumor-bearing mice. On the other hand, this immunization strategy could significantly increase the tumor necrosis, and obviously decrease microvessel density

Fibroblast growth factor receptor-1 (FGFR-1) is expressed on endothelial cells and many types of tumors [39, 40]. The Xenopus homologue of FGFR-1 is 80% and 74% identical at the amino

bodies against flk-1 [36].

in the future.

*2.1.2.2. FGFR-1*

and the blood flow signals in tumor [38].

Previous studies in rodent tumor models have indicated that immunization against xenogeneic growth factors is more likely to induce effective anti-tumor responses than immunization against the syngeneic growth factor [34]. In 2007, an investigation was conducted to assess the safety and anti-tumor and anti-angiogenic effects of a xenogeneic VEGF vaccine in pet dogs with spontaneous cancer. Nine dogs with soft tissue sarcoma were immunized with a recombinant human VEGF vaccine over a 16-week period [34]. The xenogeneic VEGF vaccine was well-tolerated by all dogs and resulted in induction of humoral responses against both human and canine VEGF in animals that remained in the study long enough to receive multiple immunizations [34]. Three of five multiply immunized dogs also experienced sustained decreases in circulating plasma VEGF concentrations and two dogs had a significant decrease in tumor microvessel density [34]. The overall tumor response (>50% decrease in tumor volume) rate was 30% for all treated dogs in the study. Thus, it was concluded that a xenogeneic VEGF vaccine may be a safe and effective alternative means of controlling tumor growth and angiogenesis [34].

VEGF receptor-2 (VEGFR-2, also known as fetal liver kinse-1 (flk-1) in mouse and kinasecontaining domain receptor (KDR) in human) is the main receptor responsible for the VEGF-mediated angiogenic activity [6]. The impairment of vasculogenesis and death of embryo at day 8.5 were observed as the result of the targeted inactivation of flk-1 gene in mice [35]. Overexpression of KDR was found on activated endothelial cells of newly formed vessels [6]. It was discovered that the primary sequence of quail VEGFR-2 (qVEGFR-2) was 67% and 70% identical at the amino acid level with mouse and human homologues (flk-1 and KDR), respectively [36]. Immunotherapy with a vaccine based on quail homologous VEGFR-2 elicited protective and therapeutic anti-tumor immunity in both solid and hematopoietic tumor models in mice, such as LL/2 Lewis lung carcinoma, CT26 colon carcinoma, Meth A fibrosarcoma, MOPC-315 plasmacytoma, and EL-4 lymphoma [36]. Autoantibodies against flk-1 in the immunized mice were identified. Sera from qVEGFR-2immunized mice recognized not only recombinant qVEGFR-2, but also recombinant mouse VEGFR-2 (mVEGFR) in Western blot analysis [36]. In contrast, the sera isolated from controls showed negative staining [36]. Sera from mice immunized with qVEGFR-2 recognized a single band in flk-1-positive mouse SVEC4-10 endothelial cells and KDRpositive human umbilical vein endothelial cells, with the same size as recognized by commercially available flk-1 or KDR antibodies [36]. Sera from qVEGFR-2-immunized mice also recognized recombinant protein qVEGFR-2 and mVEGFR-2 in ELISA [36]. Detectable IgG1 and IgG2b with significantly elevated concentration in sera were found to be responsible for the immunoglobulin response to VEGFR-2 [36]. Anti-VEGFR-2 specific antibody-producing B cells were detected by ELISPOT. The number of anti-VEGFR-2 antibody-producing B cells was elevated in the spleens of mice immunized with qVEGFR-2, compared with that in controls [36]. Deposition of immunoglobulins on endothelial cells was found within tumors from qVEGFR-2-immunized mice, but not from controls [36]. Adoptive transfer of the purified immunoglobulins from qVEGFR-2-immunized mice resulted in inhibition of VEGF-mediated endothelial cell proliferation and effective protection against tumor growth [36]. Angiogenesis was markedly suppressed within the tumors, and the vascularization of alginate beads was also diminished [36]. Depletion of CD4+ T lymphocyte could abrogate the anti-tumor activity and the production of autoanti‐ bodies against flk-1 [36].

Very recently, a DNA vaccine designed by synergizing different tumor antigens with VEGFR2 was constructed. A DNA fragment (HSV) encoding the C terminal 37 amino acids of human chorionic gonadotropin β chain (hCGβ), 5 different HLA-restricted cytotoxic T lymphocyte epitopes from human survivin and the third and fourth extracellular domains of VEGFR2 was inserted into the sequence between the luminal and transmembrane domain of human lysosome-associated membrane protein-1 cDNA for the construction of a novel DNA vaccine (p-L/HSV) [37]. Vaccination of the mice with p-L/HSV elicited potent and long-lasting cellular and humoral immune responses to the specific antigens and showed a prominent anti-tumor effect on the LL/2 lung carcinoma model in syngeneic C57BL/6 mice. In addition, the tumor vasculature was abrogated as observed by immunohistochemistry in p-L/HSV immunized mice [37]. These data indicates that the strategies of combining anti-tumor with anti-angio‐ genesis cooperate well. Such a study may shed new light on the designing of vaccine for cancer in the future.

Again in 2012, a *Bifidobacterium infantis*-based vaccine that express human extracellular domain of VEGFR2 (sKDR) was established [38]. Immunization of the mice with the *Bifidobacterium infantis*-based vaccine through caudal vein could significantly suppress the tumor growth and prolong the survival of the tumor-bearing mice. On the other hand, this immunization strategy could significantly increase the tumor necrosis, and obviously decrease microvessel density and the blood flow signals in tumor [38].

#### *2.1.2.2. FGFR-1*

from mice immunized with pM-VEGF showed a significant enhanced cytotoxic activity against VEGF-secreting tumor cells, including EL-4 lymphoma, B16-F10 melanoma, and TC-1 carcinoma, as compared with those obtained from the mice immunized with the pMAE5△5 ''empty'' vector [33]. IFN-γ ELISPOT assay revealed a significant increase in the number of spots in spleen cells from mice immunized with pM-VEGF [33]. Vaccination with a mutated hVEGF121 gene inserted into the pMAE5△5 vector (pM-VEGFmut) produced similar *in vitro* and *in vivo* results, and remarkably reduced the number of spontaneous metastases in a murine model with Lewis lung carcinoma [33]. Serum VEGF levels decreased 8-fold in mice vaccinated with pM-VEGF or pM-VEGFmut as compared with those in pMAE5△5 treated mice [33]. A significant correlation was also found between the elevation of serum VEGF level and the increase of the tumor dimensions [33]. However, antibody responses against the GSThVEGF121 fusion protein or GST alone used as capture antigens in ELISA were undetect‐ able in animals vaccinated with pM-VEGF or pM-VEGFmut [33]. These findings indicate that human VEGF-harboring DNA vaccine can be employed for anti-angiogenic active immuno‐ therapy for cancers in mice and direct cell cytotoxicity contributes to the overall anti-tumor

Previous studies in rodent tumor models have indicated that immunization against xenogeneic growth factors is more likely to induce effective anti-tumor responses than immunization against the syngeneic growth factor [34]. In 2007, an investigation was conducted to assess the safety and anti-tumor and anti-angiogenic effects of a xenogeneic VEGF vaccine in pet dogs with spontaneous cancer. Nine dogs with soft tissue sarcoma were immunized with a recombinant human VEGF vaccine over a 16-week period [34]. The xenogeneic VEGF vaccine was well-tolerated by all dogs and resulted in induction of humoral responses against both human and canine VEGF in animals that remained in the study long enough to receive multiple immunizations [34]. Three of five multiply immunized dogs also experienced sustained decreases in circulating plasma VEGF concentrations and two dogs had a significant decrease in tumor microvessel density [34]. The overall tumor response (>50% decrease in tumor volume) rate was 30% for all treated dogs in the study. Thus, it was concluded that a xenogeneic VEGF vaccine may be a safe and effective alternative means of controlling tumor growth and

VEGF receptor-2 (VEGFR-2, also known as fetal liver kinse-1 (flk-1) in mouse and kinasecontaining domain receptor (KDR) in human) is the main receptor responsible for the VEGF-mediated angiogenic activity [6]. The impairment of vasculogenesis and death of embryo at day 8.5 were observed as the result of the targeted inactivation of flk-1 gene in mice [35]. Overexpression of KDR was found on activated endothelial cells of newly formed vessels [6]. It was discovered that the primary sequence of quail VEGFR-2 (qVEGFR-2) was 67% and 70% identical at the amino acid level with mouse and human homologues (flk-1 and KDR), respectively [36]. Immunotherapy with a vaccine based on quail homologous VEGFR-2 elicited protective and therapeutic anti-tumor immunity in both solid and hematopoietic tumor models in mice, such as LL/2 Lewis lung carcinoma, CT26 colon carcinoma, Meth A fibrosarcoma, MOPC-315 plasmacytoma, and EL-4 lymphoma [36]. Autoantibodies against flk-1 in the immunized mice were identified. Sera from qVEGFR-2-

effects observed in immunized mice [33].

370 Cancer Treatment - Conventional and Innovative Approaches

angiogenesis [34].

Fibroblast growth factor receptor-1 (FGFR-1) is expressed on endothelial cells and many types of tumors [39, 40]. The Xenopus homologue of FGFR-1 is 80% and 74% identical at the amino acid level with mouse FGFR-1 and human FGFR-1, respectively [41]. Therefore, FGFR-1 may be used as another ideal target for anti-angiogenesis therapy. Vaccination with Xenopus FGFR-1 (pxFR1) provoked protective and therapeutic effects in three murine tumor models, including Meth A fibrosarcoma cells, H22 hepatoma cells, and MA782/5S mammary carcinoma [41]. FGFR-1-specific autoantibodies were detected in sera of pxFR1-immunized mice by Western blot analysis, and the purified immunoglobulins effectively inhibited endothelial cell proliferation *in vitro* [41]. However, the immunoglobulins had no direct inhibitory effect on the proliferation of above three tumor cell lines [41]. Adoptive transfer of sera or purified immunoglobulin isolated from pxFR1-immunized mice into unimmunized mice provided effective protection against tumor growth, while adsorption of sera or immunoglobulin with FGFR-1-positive endothelial cells before adoptive transfer could abrogate its anti-tumor activity [41]. Autoantibodies deposited on the endothelial cells within tumor tissues and significantly suppressed intratumoral angiogenesis were found in pxFR1-immunized mice by histological examination [41]. Furthermore, this anti-tumor activity and production of FGFR-1 specific autoantibodies were abrogated by depletion of CD4+ T lymphocytes, again pointing to their essential helper function for antibody production [41].

dases, can selectively degrade components of the extracellular matrix [44]. *In vivo*, elevated stromal MMP-2 and MMP-9 activity is highly correlated with increased metastatic potential in most malignant tumors [45]. Increased activity of MMPs appears to permit the tumor to remodel its surrounding microenvironment, to grow in a permissive space, and to promote the development of supporting stroma, including angiogenesis [46]. Moreover, numerous pathological and clinical studies demonstrated that the MMPs were frequently overexpressed in various solid tumor cells and peritumoral stromal cells [46]. It was reported that the abrogation of MMP-2 alone resulted in the inhibition of the transition from the prevascular to the vascular stage during tumor development and then of tumor growth [47]. Furthermore, the suppression of tumor-induced angiogenesis and of invasion and metastasis of tumor cells could be observed in MMP-2-deficient mice [47]. These findings indicated that MMP-2 alone played an important role in angiogenesis and tumor growth. Sequence comparison analysis showed that the primary sequence of mouse MMP-2 at the amino acid level was 82% and 91% identical with chicken and human homologues, respectively [48]. It was reported that the plasmid DNA vaccination with chicken homologous MMP-2 (c-MMP-2)-based model antigen could induce both protective and therapeutic anti-tumor immunity in murine tumor models with LL/2 Lewis lung carcinoma, Meth A fibrosarcoma, and H22 hepatoma [48]. The elevation of MMP-2 in the sera of tumor-bearing mice was abrogated with the vaccination of c-MMP-2 [48]. The autoimmune response against MMP-2 may be provoked in a cross-reaction by the immunization with c-MMP-2, and the autoantibody targeting to MMP-2 was elevated and probably responsible for the anti-tumor activity [48]. Moreover, gelatinase activity of MMP-2, including both latent MMP-2 and active MMP-2, derived from the above mentioned three murine tumor models was apparently inhibited by the vaccination with c-MMP-2 [48]. However, the vaccination did not inhibit the gelatinase activity of MMP-9 [48]. These findings indicate that the activity of MMP-2 is impaired by immunization with c-MMP-2 in mice. Angiogenesis was apparently inhibited within tumors in immunized mice. The anti-tumor activity and production of auto-antibodies against MMP-2 were abrogated by depletion of

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In 2010, Yang et al. [49] used a cross-reactive serological expression cloning (SEREX) strategy (CR-SEREX) to identify novel xenogenic angiogenesis- and tumor-associated antigens in oocytes of *Xenopus laevis* and found that *Xenopus* receptor for hyaluronic-acid-mediated motility (xRHAMM) was the most frequently clone among 78 CR-SEREX positive clones, suggesting that xRHAMM has the strongest immunogenic potential for xenogenic immuno‐ therapy. It was demonstrated that expression of RHAMM is restricted to the testis, thymus, placenta, vascular endothelial cells, and various cancer cells, and RHAMM functions in vascular endothelial cell migration, angiogenesis, and in hyaluronic-acid-induced cell mobility [50]. In order to examine the anti-angiogenic effects, a DNA vaccine based on xRHAMM (pcDNA3.1-xRHAMM) was constructed [49]. Intramuscular vaccination of the cationic liposome encapsulated pcDNA3.1-xRHAMM DNA effectively induced a protective antitumor immunity against local tumor and lung metastasis in B16 melanoma mouse models. Angiogenesis was inhibited and cell apoptosis was increased within tumors. Anti-tumor

CD4+

*2.1.2.5. xRHAMM*

T lymphocytes [48].

#### *2.1.2.3. Integrins*

Integrins are heterodimeric transmembrane proteins consisting of α and β subunits with large extracellular domain and short cytoplasmic tail. They play very crucial roles in angiogenesis as the migration of endothelial cells is dependent on their adhesion to extracellular matrix proteins such as vitronectin [42]. αvβ3 is not generally found on blood vessels in normal tissues, but its expression is enhanced on newly developing blood vessels in human wound tissue, tumors, diabetic retinopathy, macular degeneration and rheumatoid arthritis, which implies that this integrin may play an important role in angiogenesis and development of neovascu‐ larization [42]. This distributive characteristic also makes αvβ3 an attractive target for tumor therapy [42]. A plasmid DNA encoding the ligand-binding domain of chicken integrin β3 was constructed to test this assumption. Immunization with chicken homologous integrin β3-based vaccine could elicit both protective and therapeutic anti-tumor immunity in murine tumor models with Meth A fibrosarcoma, H22 hepatoma, or MA782/5S mammary carcinoma [43]. Autoantibodies against integrin β3 in sera of the immunized mice were found by Western blot analysis and ELISA [43]. The purified immunoglobulins could effectively inhibit endothelial cell proliferation *in vitro*, and adoptive transfer of the purified immunoglobulins into nonimmunized mice could provide effective protection against tumor growth and markedly inhibit tumor angiogenesis [43]. The anti-tumor activity and the production of integrin β3 specific autoantibodies were CD4+ T lymphocyte-dependent [43].

#### *2.1.2.4. MMP*

Angiogenesis is an invasive process, requiring proteolysis of the extracellular matrix [44]. Inappropriate destruction of extracellular matrix components is involved in certain patholog‐ ical conditions, including arteriosclerosis, rheumatoid arthritis, and tumor aggression and metastasis [44]. The matrix metalloproteinases (MMPs), a family of extracellular endopepti‐ dases, can selectively degrade components of the extracellular matrix [44]. *In vivo*, elevated stromal MMP-2 and MMP-9 activity is highly correlated with increased metastatic potential in most malignant tumors [45]. Increased activity of MMPs appears to permit the tumor to remodel its surrounding microenvironment, to grow in a permissive space, and to promote the development of supporting stroma, including angiogenesis [46]. Moreover, numerous pathological and clinical studies demonstrated that the MMPs were frequently overexpressed in various solid tumor cells and peritumoral stromal cells [46]. It was reported that the abrogation of MMP-2 alone resulted in the inhibition of the transition from the prevascular to the vascular stage during tumor development and then of tumor growth [47]. Furthermore, the suppression of tumor-induced angiogenesis and of invasion and metastasis of tumor cells could be observed in MMP-2-deficient mice [47]. These findings indicated that MMP-2 alone played an important role in angiogenesis and tumor growth. Sequence comparison analysis showed that the primary sequence of mouse MMP-2 at the amino acid level was 82% and 91% identical with chicken and human homologues, respectively [48]. It was reported that the plasmid DNA vaccination with chicken homologous MMP-2 (c-MMP-2)-based model antigen could induce both protective and therapeutic anti-tumor immunity in murine tumor models with LL/2 Lewis lung carcinoma, Meth A fibrosarcoma, and H22 hepatoma [48]. The elevation of MMP-2 in the sera of tumor-bearing mice was abrogated with the vaccination of c-MMP-2 [48]. The autoimmune response against MMP-2 may be provoked in a cross-reaction by the immunization with c-MMP-2, and the autoantibody targeting to MMP-2 was elevated and probably responsible for the anti-tumor activity [48]. Moreover, gelatinase activity of MMP-2, including both latent MMP-2 and active MMP-2, derived from the above mentioned three murine tumor models was apparently inhibited by the vaccination with c-MMP-2 [48]. However, the vaccination did not inhibit the gelatinase activity of MMP-9 [48]. These findings indicate that the activity of MMP-2 is impaired by immunization with c-MMP-2 in mice. Angiogenesis was apparently inhibited within tumors in immunized mice. The anti-tumor activity and production of auto-antibodies against MMP-2 were abrogated by depletion of CD4+ T lymphocytes [48].

#### *2.1.2.5. xRHAMM*

acid level with mouse FGFR-1 and human FGFR-1, respectively [41]. Therefore, FGFR-1 may be used as another ideal target for anti-angiogenesis therapy. Vaccination with Xenopus FGFR-1 (pxFR1) provoked protective and therapeutic effects in three murine tumor models, including Meth A fibrosarcoma cells, H22 hepatoma cells, and MA782/5S mammary carcinoma [41]. FGFR-1-specific autoantibodies were detected in sera of pxFR1-immunized mice by Western blot analysis, and the purified immunoglobulins effectively inhibited endothelial cell proliferation *in vitro* [41]. However, the immunoglobulins had no direct inhibitory effect on the proliferation of above three tumor cell lines [41]. Adoptive transfer of sera or purified immunoglobulin isolated from pxFR1-immunized mice into unimmunized mice provided effective protection against tumor growth, while adsorption of sera or immunoglobulin with FGFR-1-positive endothelial cells before adoptive transfer could abrogate its anti-tumor activity [41]. Autoantibodies deposited on the endothelial cells within tumor tissues and significantly suppressed intratumoral angiogenesis were found in pxFR1-immunized mice by histological examination [41]. Furthermore, this anti-tumor activity and production of FGFR-1 specific autoantibodies were abrogated by depletion of CD4+ T lymphocytes, again pointing

Integrins are heterodimeric transmembrane proteins consisting of α and β subunits with large extracellular domain and short cytoplasmic tail. They play very crucial roles in angiogenesis as the migration of endothelial cells is dependent on their adhesion to extracellular matrix proteins such as vitronectin [42]. αvβ3 is not generally found on blood vessels in normal tissues, but its expression is enhanced on newly developing blood vessels in human wound tissue, tumors, diabetic retinopathy, macular degeneration and rheumatoid arthritis, which implies that this integrin may play an important role in angiogenesis and development of neovascu‐ larization [42]. This distributive characteristic also makes αvβ3 an attractive target for tumor therapy [42]. A plasmid DNA encoding the ligand-binding domain of chicken integrin β3 was constructed to test this assumption. Immunization with chicken homologous integrin β3-based vaccine could elicit both protective and therapeutic anti-tumor immunity in murine tumor models with Meth A fibrosarcoma, H22 hepatoma, or MA782/5S mammary carcinoma [43]. Autoantibodies against integrin β3 in sera of the immunized mice were found by Western blot analysis and ELISA [43]. The purified immunoglobulins could effectively inhibit endothelial cell proliferation *in vitro*, and adoptive transfer of the purified immunoglobulins into nonimmunized mice could provide effective protection against tumor growth and markedly inhibit tumor angiogenesis [43]. The anti-tumor activity and the production of integrin β3-

T lymphocyte-dependent [43].

Angiogenesis is an invasive process, requiring proteolysis of the extracellular matrix [44]. Inappropriate destruction of extracellular matrix components is involved in certain patholog‐ ical conditions, including arteriosclerosis, rheumatoid arthritis, and tumor aggression and metastasis [44]. The matrix metalloproteinases (MMPs), a family of extracellular endopepti‐

to their essential helper function for antibody production [41].

372 Cancer Treatment - Conventional and Innovative Approaches

*2.1.2.3. Integrins*

specific autoantibodies were CD4+

*2.1.2.4. MMP*

In 2010, Yang et al. [49] used a cross-reactive serological expression cloning (SEREX) strategy (CR-SEREX) to identify novel xenogenic angiogenesis- and tumor-associated antigens in oocytes of *Xenopus laevis* and found that *Xenopus* receptor for hyaluronic-acid-mediated motility (xRHAMM) was the most frequently clone among 78 CR-SEREX positive clones, suggesting that xRHAMM has the strongest immunogenic potential for xenogenic immuno‐ therapy. It was demonstrated that expression of RHAMM is restricted to the testis, thymus, placenta, vascular endothelial cells, and various cancer cells, and RHAMM functions in vascular endothelial cell migration, angiogenesis, and in hyaluronic-acid-induced cell mobility [50]. In order to examine the anti-angiogenic effects, a DNA vaccine based on xRHAMM (pcDNA3.1-xRHAMM) was constructed [49]. Intramuscular vaccination of the cationic liposome encapsulated pcDNA3.1-xRHAMM DNA effectively induced a protective antitumor immunity against local tumor and lung metastasis in B16 melanoma mouse models. Angiogenesis was inhibited and cell apoptosis was increased within tumors. Anti-tumor activity of xRHAMM was mediated by both the antigen-specific cellular and humoral responses against RHAMM, as confirmed by the depletion of immune cell subsets *in vivo*. Furthermore, the anti-angiogenic and anti-tumor effects induced by vaccination of pcDNA3.1 xRHAMM were significantly stronger than that induced by vaccination of the corresponding autologous counterpart pcDNA3.1-mRHAMM [49].

effect of angiostatin. These antibodies inhibit endothelial cell migration, block tumor cell- and bFGF-induced angiogenesis in the matrigel plug assay and prevent growth of transplanted tumors without impairing normal stromal or retina vessels [57]. Very recently, Arigoni et al. further showed that the pAmot-induced antibodies alter tumor vessel permeability and structure. These combined effects of vaccine-induced anti-Amot antibodies lead to inhibition of established clinically evident mammary tumors, massive tumor perivascular necrosis, and an effective tumor antigen presentation in a form of epitope spreading that induces an immune response against other oncoantigens overexpressed by tumor cells [61]. Greater tumor vessel permeability also markedly boosts the local accumulation of doxorubicin and enhances the anti-tumor effect of the drugs [61]. These data provide a rationale for the development of fresh anticancer treatments based on anti-Amot vaccination in conjunction with chemotherapy

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Taken together, it is obvious that vaccination with xenogeneic homologous molecules associ‐ ated with angiogenesis, such as pro-angiogenic factors, integrins, MMP, could induce antitumor immunity and thus might be a feasible strategy for cancer therapy with potential clinical

Given that vaccination with xenogeneic homologous molecules associated with tumor angiogenesis could effectively induce anti-tumor immunity, it can be assumed that vaccines based on non-xenogeneic homologous molecules, such as allogeneic homologues of some proangiogenic factors or other important molecules associated with angiogenesis, could also successfully induce specific and potent anti-tumor immunity. To date, several vaccines based on non-xenogeneic homologous molecules were used in anti-angiogenic active immunother‐

As has been discussed above, VEGF-mediated signaling pathway through VEGFR-2 is a ratelimiting step during tumor angiogenesis. Thus, VEGF/VEGFR-2 is still an ideal target in the non-xenogeneic homologous molecules-based anti-angiogenic strategy. Immunization of mice with VEGF receptor-2 (flk-1)-pulsed dendritic cells (DC) can break self-tolerance to VEGFR-2, induce CTL and antibody responses to VEGFR-2 [62]. Significant inhibition of tumor growth and metastasis was observed in both melanoma and Lewis lung carcinoma metastasis murine models [62]. Oral administration of mice with DNA vaccines encoding murine VEGFR-2 carried by attenuated *Salmonella typhimurium* could break the immune tolerance to VEGFR-2, induce CTL response to VEGFR-2, inhibit tumor cell-induced neoangiogenesis, and suppress the formation of spontaneous and experimental pulmonary metastases, with slight impact on wounds healing and no influence on hematopoiesis and pregnancy [63]. Immunization of mice with flk1-encoding mRNA-transfected DC could induce specific CTL response to VEGFR-2, partially inhibit the tumor cell-induced neoangiogenesis, and suppress tumor growth and metastasis in murine B16/F10.9 melanoma and MBT-2 bladder tumor models [64]. We studied

**2.2. Anti-angiogenic active immunotherapies based on non-xenogeneic homologous**

regimens.

applications.

**molecules**

apy for tumors.

*2.2.1. VEGFR-2*

### *2.1.2.6. DLL4*

Notch signaling has recently emerged as a critical regulator of developmental and tumor angiogenesis. Notch signaling in both endothelial and smooth muscle cells appears to provide critical regulatory information to these cells downstream of the initiating signal induced by VEGF [51, 52]. Studies in humans and mice have demonstrated that Notch ligand delta-like 4 (DLL4) is strongly expressed by the tumor vasculature and generally not by the tumor cells themselves. In various mouse models, strong DLL4 expression was observed in the majority of tumor vessels, contrasting with significantly lower vascular expression in adjacent normal tissues [51]. In humans, DLL4 expression was analyzed in tumors from kidney, bladder, colon, brain and breast [53, 54]. Robust DLL4 expression was observed specifically in the tumor vasculature in all of these tumor types, whereas DLL4 expression was low to undetectable in the vasculature of adjacent normal tissue. Furthermore, at least in the case of breast cancer, the degree of DLL4 expression correlated with outcome: tumors with high DLL4 in the vasculature progressed more rapidly [54]. These findings suggest that DLL4 is an attractive new thera‐ peutic anti-angiogenesis target. To generate the DLL4 plasmid vaccine, the cDNA encoding human DLL4 was cloned into the pVAX1 expression vector (DLL4 vaccine), which is specifically designed for the development of DNA vaccines and approved for use in humans. Immunization of Balb/c mice with DLL4 vaccine could bring about a break in tolerance against the self-antigen, DLL4. Readily detectable titers of serum antibodies against DLL4 were induced. Moreover, immunization with DLL4-encoding plasmid DNA severely retarded the growth of orthotopically implanted D2F2/E2 mammary carcinomas in mice by induction of a non-productive angiogenic response. In addition to the promising therapeutic effects, no evidence for a delayed wound healing response, or for toxicity associated with pharmacolog‐ ical blockade of DLL4 signaling, was observed in mice immunized with the DLL4 vaccine [55].

### *2.1.2.7. Angiomotin*

Angiomotin (Amot), one of angiostatin receptors [56], is a membrane-associated protein present on the endothelial cell surface of angiogenic tissues [57] characterized by conserved coiledcoil and carboxy termini-PDZ domains [58]. A shorter Amot isoform (p80) confers a hyper-migratory and invasive phenotype in transfected cells [59] and induces endothelial cell migration during angiogenesis [60]. The longer (p130) isoform localizes to tight junctions, regulates cell shape and appears to play a role in the later phase of angiogenesis [60]. It was demonstrated that increased Amot expression on tumor endothelia concomitant with the progression from pre-neoplastic lesions to full-fledged carcinoma, therefore, plasmid vaccine encoding human p80 Amot (pAmot) was constructed [57]. Immunization of mice with pAmot can overcome immune tolerance and induce a significant antibody response that mimic the effect of angiostatin. These antibodies inhibit endothelial cell migration, block tumor cell- and bFGF-induced angiogenesis in the matrigel plug assay and prevent growth of transplanted tumors without impairing normal stromal or retina vessels [57]. Very recently, Arigoni et al. further showed that the pAmot-induced antibodies alter tumor vessel permeability and structure. These combined effects of vaccine-induced anti-Amot antibodies lead to inhibition of established clinically evident mammary tumors, massive tumor perivascular necrosis, and an effective tumor antigen presentation in a form of epitope spreading that induces an immune response against other oncoantigens overexpressed by tumor cells [61]. Greater tumor vessel permeability also markedly boosts the local accumulation of doxorubicin and enhances the anti-tumor effect of the drugs [61]. These data provide a rationale for the development of fresh anticancer treatments based on anti-Amot vaccination in conjunction with chemotherapy regimens.

Taken together, it is obvious that vaccination with xenogeneic homologous molecules associ‐ ated with angiogenesis, such as pro-angiogenic factors, integrins, MMP, could induce antitumor immunity and thus might be a feasible strategy for cancer therapy with potential clinical applications.

#### **2.2. Anti-angiogenic active immunotherapies based on non-xenogeneic homologous molecules**

Given that vaccination with xenogeneic homologous molecules associated with tumor angiogenesis could effectively induce anti-tumor immunity, it can be assumed that vaccines based on non-xenogeneic homologous molecules, such as allogeneic homologues of some proangiogenic factors or other important molecules associated with angiogenesis, could also successfully induce specific and potent anti-tumor immunity. To date, several vaccines based on non-xenogeneic homologous molecules were used in anti-angiogenic active immunother‐ apy for tumors.

### *2.2.1. VEGFR-2*

activity of xRHAMM was mediated by both the antigen-specific cellular and humoral responses against RHAMM, as confirmed by the depletion of immune cell subsets *in vivo*. Furthermore, the anti-angiogenic and anti-tumor effects induced by vaccination of pcDNA3.1 xRHAMM were significantly stronger than that induced by vaccination of the corresponding

Notch signaling has recently emerged as a critical regulator of developmental and tumor angiogenesis. Notch signaling in both endothelial and smooth muscle cells appears to provide critical regulatory information to these cells downstream of the initiating signal induced by VEGF [51, 52]. Studies in humans and mice have demonstrated that Notch ligand delta-like 4 (DLL4) is strongly expressed by the tumor vasculature and generally not by the tumor cells themselves. In various mouse models, strong DLL4 expression was observed in the majority of tumor vessels, contrasting with significantly lower vascular expression in adjacent normal tissues [51]. In humans, DLL4 expression was analyzed in tumors from kidney, bladder, colon, brain and breast [53, 54]. Robust DLL4 expression was observed specifically in the tumor vasculature in all of these tumor types, whereas DLL4 expression was low to undetectable in the vasculature of adjacent normal tissue. Furthermore, at least in the case of breast cancer, the degree of DLL4 expression correlated with outcome: tumors with high DLL4 in the vasculature progressed more rapidly [54]. These findings suggest that DLL4 is an attractive new thera‐ peutic anti-angiogenesis target. To generate the DLL4 plasmid vaccine, the cDNA encoding human DLL4 was cloned into the pVAX1 expression vector (DLL4 vaccine), which is specifically designed for the development of DNA vaccines and approved for use in humans. Immunization of Balb/c mice with DLL4 vaccine could bring about a break in tolerance against the self-antigen, DLL4. Readily detectable titers of serum antibodies against DLL4 were induced. Moreover, immunization with DLL4-encoding plasmid DNA severely retarded the growth of orthotopically implanted D2F2/E2 mammary carcinomas in mice by induction of a non-productive angiogenic response. In addition to the promising therapeutic effects, no evidence for a delayed wound healing response, or for toxicity associated with pharmacolog‐ ical blockade of DLL4 signaling, was observed in mice immunized with the DLL4 vaccine [55].

Angiomotin (Amot), one of angiostatin receptors [56], is a membrane-associated protein present on the endothelial cell surface of angiogenic tissues [57] characterized by conserved coiledcoil and carboxy termini-PDZ domains [58]. A shorter Amot isoform (p80) confers a hyper-migratory and invasive phenotype in transfected cells [59] and induces endothelial cell migration during angiogenesis [60]. The longer (p130) isoform localizes to tight junctions, regulates cell shape and appears to play a role in the later phase of angiogenesis [60]. It was demonstrated that increased Amot expression on tumor endothelia concomitant with the progression from pre-neoplastic lesions to full-fledged carcinoma, therefore, plasmid vaccine encoding human p80 Amot (pAmot) was constructed [57]. Immunization of mice with pAmot can overcome immune tolerance and induce a significant antibody response that mimic the

autologous counterpart pcDNA3.1-mRHAMM [49].

374 Cancer Treatment - Conventional and Innovative Approaches

*2.1.2.6. DLL4*

*2.1.2.7. Angiomotin*

As has been discussed above, VEGF-mediated signaling pathway through VEGFR-2 is a ratelimiting step during tumor angiogenesis. Thus, VEGF/VEGFR-2 is still an ideal target in the non-xenogeneic homologous molecules-based anti-angiogenic strategy. Immunization of mice with VEGF receptor-2 (flk-1)-pulsed dendritic cells (DC) can break self-tolerance to VEGFR-2, induce CTL and antibody responses to VEGFR-2 [62]. Significant inhibition of tumor growth and metastasis was observed in both melanoma and Lewis lung carcinoma metastasis murine models [62]. Oral administration of mice with DNA vaccines encoding murine VEGFR-2 carried by attenuated *Salmonella typhimurium* could break the immune tolerance to VEGFR-2, induce CTL response to VEGFR-2, inhibit tumor cell-induced neoangiogenesis, and suppress the formation of spontaneous and experimental pulmonary metastases, with slight impact on wounds healing and no influence on hematopoiesis and pregnancy [63]. Immunization of mice with flk1-encoding mRNA-transfected DC could induce specific CTL response to VEGFR-2, partially inhibit the tumor cell-induced neoangiogenesis, and suppress tumor growth and metastasis in murine B16/F10.9 melanoma and MBT-2 bladder tumor models [64]. We studied the regulatory effects of IFN-γ on the differentiation and development of DC and found that IFN-γ is an autocrine mediator for DC maturation [65]. IFN-γ gene transfection could promote differentiation, development, and functional maturation of DC [66]. IFN-γ gene-modified DC had increased capacity to induce Th1 type immune response, and intratumoral injection of IFN-γ gene-modified DC in a murine model with pre-established B16 melanoma resulted in the potentiation of the anti-tumor effect of DC [66]. On the other hand, it was demonstrated that IFN-γ itself is also a negative regulator of neoangiogenesis [67]. In order to combine the anti-angiogenic immunotherapy with the cytokine immunotherapy, we constructed recombi‐ nant plasmid expressing murine VEGFR-2 extracellular domain (sVEGFR-2) and IFN-γ fusion protein, pcDNA3.1/sVEGFR-2-IFN-γ, and found that the fusion protein expressed by recombi‐ nant plasmid shared biological activities of both sVEGFR-2 and IFN-γ [68]. Immunization of mice with murine sVEGFR-2-IFN-γ fusion gene-transfected DC could significantly augment the CTL response to murine VEGFR-2 and pronouncedly inhibit tumor cell-induced angioge‐ nensis and tumor metastasis in comparison with murine sVEGFR2 gene-transfected DC [68].

In 2009, Seavey, et al developed *Listeria monocytogens* based VEGFR-2 vaccines that encode the peptide of VEGFR-2 extracellular domain fused to the first 441 residues of the microbial adjuvant listeriolysin O (*Lm*-LLO-Flk-E1 and *Lm*-LLO-Flk-E2) and the peptide of VEGFR-2 intracellular domain that also fused to LLO (*Lm*-LLO-Flk-I1), respectively [71, 72]. Immuni‐ zation of the mice with the *Listeria*-based Flk1 vaccines elicited potent antitumor CTL respons‐ es. Lm-LLO-Flk-1 was able to eradicate some established Her-2/neu+ breast tumors, reduce microvascular density in the remaining tumors, protect against tumor rechallenge and experimental metastases, and induce epitope spreading to various regions of the tumorassociated antigen Her-2/neu. Tumor eradication was found to be dependent on epitope spreading to Her-2/neu and was not solely due to the reduction of tumor vasculature [71]. In an autochthonous model for Her-2/neu+ breast cancer, theses vaccines could significantly delay tumor onset, while tumors that grew out overtime accumulated mutations in the Her-2/ neu molecule near or within CTL epitopes [72]. Moreover, vaccine efficacy did not affect normal wound healing nor have toxic side effects on pregnancy [71]. These data suggest that an anti-angiogenesis vaccine can overcome tolerance to the host vasculature driving epitope

Anti-Angiogenic Active Immunotherapy for Cancers: Dawn of a New Era?

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377

spreading to an endogenous tumor protein and drive active tumor regression.

the optimal dose for further clinical trials might be 2 mg/body or higher.

*2.2.2. bFGF*

Recently, a DNA vaccine (pSG.SS.Flk-1ECD.C3d3) encoding Flk-1 extracellular domain and the complement fragment C3d fusion protein was constructed [73]. Vaccination of mice with pSG.SS.Flk-1ECD.C3d3 could also elicit Flk-1 specific antibody response, leading to suppression of angiogenesis and tumor growth in bladder translational cell carcinoma mouse model, suggesting that C3d can be used as an adjuvant to enhance the immune response [73].

In 2010, Miyazawa, et al. [74] reported the results of phase I clinical trial combining of epitope peptide for VEGFR-2 (VEGFR2-169) with gemcitabine for patients with advanced pancreatic cancer. 18 HLA-A\*2402-positive patients with metastatic and unresectable pancreatic cancer were enrolled in the trial. Gemcitabine was administered at a dose of 1000 mg/m2 on days 1, 8, and 15 in a 28-day cycle. The VEGFR2-169 peptide was subcutaneously injected weekly in a dose-escalation manner (doses of 0.5, 1, and 2 mg/body, six patients/one cohort). Safety and immunological parameters were assessed. No severe adverse effect of grade 4 or higher was observed. Of the 18 patients who completed at least one course of the treatment, 15 (83%) developed immunological reactions at the injection sites. VEGFR2-169 specific CTLs were induced in 11 (61%) of the 18 patients. The disease control rate was 67%, and the median overall survival time was 8.7 months. This combination therapy for pancreatic cancer patients was tolerable at all doses. Peptide-specific CTL could be induced by the VEGFR2-169 peptide vaccine at a high rate, even in combination with gemcitabine. Therefore, they suggested that

Basic fibroblast growth factor (bFGF/FGF2) is an important proangiogenic factor, which is secreted by tumor cells and macrophages or released by extracelluar matrix, and functions in the autocrine or paracrine manner. FGF2 can upregulate the expression of several dominant pro-angiogenic factors, such as VEGF [75], and activator of plasminogen [76], and inhibit apoptosis of endothelial cells by bcl-2 pathway [77]. bFGF exerts its biological activities

In 2006, three CTL epitope candidates, designated as KDR1, KDR2 and KDR3, respectively, from VEGFR-2 with high binding affinity to the H-2Db molecule were predicted by two computer programs: Bimas and SYFPEITH [69]. Two of them, KDR2 and KDR3, were from the extracellular domain; KDR1 was from the intracellular part of the receptor [69]. Immuni‐ zation of mice with KDR2 or KDR3 peptide in combination with murine GM-CSF and agonist anti-mouse CD40 antibodies as adjuvant could break self-tolerance and induce specific immune responses in C57BL/6 mice [69]. Furthermore, immunization of mice with these two peptide epitopes elicited pronounced specific CTL responses to murine VEGFR-2, effectively inhibited VEGF-induced angiogenesis, and suppressed tumor growth in MC38 murine colon cancer model [69]. Similarly, the epitope peptides of human VEGFR-2 restricted by HLA-A\*0201 and HLA-A\*2402 were also identified by analyzing the binding affinities to the corresponding HLA molecules [70]. Antigen based on the epitope peptide with high binding affinity to human HLA-A\*0201 could successfully induce specific CTL response *in vitro* [70]. Furthermore, transgenic mice expressing HLA-A\*0201, A2/Kb, were generated, and the vascular endothelial cells in that mice could not only express human VEGFR-2 (KDR), but also express human MHC class Ι molecules [70]. After inoculation of A2/Kb with HLA-A\*0201 restricted VEGFR-2 epitope peptide, specific IFN-γ-expressed CTL was induced [70]. Immu‐ nization of tumor-bearing A2/Kb transgenic mice with VEGFR-2 epitope peptide could markedly inhibit tumor-induced angiogenesis, hereby inhibiting tumor growth in MC38 colon cancer and B16 melanoma models, and prolong survival of the tumor-bearing animals without fatal adverse effects [70]. To further study whether specific CTL response to KDR can be elicited in human or not, KDR epitope peptide vaccines were used to stimulate peripheral blood mononuclear cells derived from 6 cancer patients *in vitro*, and CTLs specific for the peptide epitope were successfully induced in all patients [70].

In comparison with the full-length protein, peptide vaccines like the aforementioned KDR epitope peptides can be easily synthesized in high purity and are less expensive. Moreover, immunization with such vaccines could avoid the potential dangers involving induction of an infection by recombinant viruses or exposure to a latently allergenic exogenous protein.

In 2009, Seavey, et al developed *Listeria monocytogens* based VEGFR-2 vaccines that encode the peptide of VEGFR-2 extracellular domain fused to the first 441 residues of the microbial adjuvant listeriolysin O (*Lm*-LLO-Flk-E1 and *Lm*-LLO-Flk-E2) and the peptide of VEGFR-2 intracellular domain that also fused to LLO (*Lm*-LLO-Flk-I1), respectively [71, 72]. Immuni‐ zation of the mice with the *Listeria*-based Flk1 vaccines elicited potent antitumor CTL respons‐ es. Lm-LLO-Flk-1 was able to eradicate some established Her-2/neu+ breast tumors, reduce microvascular density in the remaining tumors, protect against tumor rechallenge and experimental metastases, and induce epitope spreading to various regions of the tumorassociated antigen Her-2/neu. Tumor eradication was found to be dependent on epitope spreading to Her-2/neu and was not solely due to the reduction of tumor vasculature [71]. In an autochthonous model for Her-2/neu+ breast cancer, theses vaccines could significantly delay tumor onset, while tumors that grew out overtime accumulated mutations in the Her-2/ neu molecule near or within CTL epitopes [72]. Moreover, vaccine efficacy did not affect normal wound healing nor have toxic side effects on pregnancy [71]. These data suggest that an anti-angiogenesis vaccine can overcome tolerance to the host vasculature driving epitope spreading to an endogenous tumor protein and drive active tumor regression.

Recently, a DNA vaccine (pSG.SS.Flk-1ECD.C3d3) encoding Flk-1 extracellular domain and the complement fragment C3d fusion protein was constructed [73]. Vaccination of mice with pSG.SS.Flk-1ECD.C3d3 could also elicit Flk-1 specific antibody response, leading to suppression of angiogenesis and tumor growth in bladder translational cell carcinoma mouse model, suggesting that C3d can be used as an adjuvant to enhance the immune response [73].

In 2010, Miyazawa, et al. [74] reported the results of phase I clinical trial combining of epitope peptide for VEGFR-2 (VEGFR2-169) with gemcitabine for patients with advanced pancreatic cancer. 18 HLA-A\*2402-positive patients with metastatic and unresectable pancreatic cancer were enrolled in the trial. Gemcitabine was administered at a dose of 1000 mg/m2 on days 1, 8, and 15 in a 28-day cycle. The VEGFR2-169 peptide was subcutaneously injected weekly in a dose-escalation manner (doses of 0.5, 1, and 2 mg/body, six patients/one cohort). Safety and immunological parameters were assessed. No severe adverse effect of grade 4 or higher was observed. Of the 18 patients who completed at least one course of the treatment, 15 (83%) developed immunological reactions at the injection sites. VEGFR2-169 specific CTLs were induced in 11 (61%) of the 18 patients. The disease control rate was 67%, and the median overall survival time was 8.7 months. This combination therapy for pancreatic cancer patients was tolerable at all doses. Peptide-specific CTL could be induced by the VEGFR2-169 peptide vaccine at a high rate, even in combination with gemcitabine. Therefore, they suggested that the optimal dose for further clinical trials might be 2 mg/body or higher.

#### *2.2.2. bFGF*

the regulatory effects of IFN-γ on the differentiation and development of DC and found that IFN-γ is an autocrine mediator for DC maturation [65]. IFN-γ gene transfection could promote differentiation, development, and functional maturation of DC [66]. IFN-γ gene-modified DC had increased capacity to induce Th1 type immune response, and intratumoral injection of IFN-γ gene-modified DC in a murine model with pre-established B16 melanoma resulted in the potentiation of the anti-tumor effect of DC [66]. On the other hand, it was demonstrated that IFN-γ itself is also a negative regulator of neoangiogenesis [67]. In order to combine the anti-angiogenic immunotherapy with the cytokine immunotherapy, we constructed recombi‐ nant plasmid expressing murine VEGFR-2 extracellular domain (sVEGFR-2) and IFN-γ fusion protein, pcDNA3.1/sVEGFR-2-IFN-γ, and found that the fusion protein expressed by recombi‐ nant plasmid shared biological activities of both sVEGFR-2 and IFN-γ [68]. Immunization of mice with murine sVEGFR-2-IFN-γ fusion gene-transfected DC could significantly augment the CTL response to murine VEGFR-2 and pronouncedly inhibit tumor cell-induced angioge‐ nensis and tumor metastasis in comparison with murine sVEGFR2 gene-transfected DC [68].

In 2006, three CTL epitope candidates, designated as KDR1, KDR2 and KDR3, respectively,

computer programs: Bimas and SYFPEITH [69]. Two of them, KDR2 and KDR3, were from the extracellular domain; KDR1 was from the intracellular part of the receptor [69]. Immuni‐ zation of mice with KDR2 or KDR3 peptide in combination with murine GM-CSF and agonist anti-mouse CD40 antibodies as adjuvant could break self-tolerance and induce specific immune responses in C57BL/6 mice [69]. Furthermore, immunization of mice with these two peptide epitopes elicited pronounced specific CTL responses to murine VEGFR-2, effectively inhibited VEGF-induced angiogenesis, and suppressed tumor growth in MC38 murine colon cancer model [69]. Similarly, the epitope peptides of human VEGFR-2 restricted by HLA-A\*0201 and HLA-A\*2402 were also identified by analyzing the binding affinities to the corresponding HLA molecules [70]. Antigen based on the epitope peptide with high binding affinity to human HLA-A\*0201 could successfully induce specific CTL response *in vitro* [70]. Furthermore, transgenic mice expressing HLA-A\*0201, A2/Kb, were generated, and the vascular endothelial cells in that mice could not only express human VEGFR-2 (KDR), but also express human MHC class Ι molecules [70]. After inoculation of A2/Kb with HLA-A\*0201 restricted VEGFR-2 epitope peptide, specific IFN-γ-expressed CTL was induced [70]. Immu‐ nization of tumor-bearing A2/Kb transgenic mice with VEGFR-2 epitope peptide could markedly inhibit tumor-induced angiogenesis, hereby inhibiting tumor growth in MC38 colon cancer and B16 melanoma models, and prolong survival of the tumor-bearing animals without fatal adverse effects [70]. To further study whether specific CTL response to KDR can be elicited in human or not, KDR epitope peptide vaccines were used to stimulate peripheral blood mononuclear cells derived from 6 cancer patients *in vitro*, and CTLs specific for the peptide

In comparison with the full-length protein, peptide vaccines like the aforementioned KDR epitope peptides can be easily synthesized in high purity and are less expensive. Moreover, immunization with such vaccines could avoid the potential dangers involving induction of an infection by recombinant viruses or exposure to a latently allergenic exogenous protein.

molecule were predicted by two

from VEGFR-2 with high binding affinity to the H-2Db

376 Cancer Treatment - Conventional and Innovative Approaches

epitope were successfully induced in all patients [70].

Basic fibroblast growth factor (bFGF/FGF2) is an important proangiogenic factor, which is secreted by tumor cells and macrophages or released by extracelluar matrix, and functions in the autocrine or paracrine manner. FGF2 can upregulate the expression of several dominant pro-angiogenic factors, such as VEGF [75], and activator of plasminogen [76], and inhibit apoptosis of endothelial cells by bcl-2 pathway [77]. bFGF exerts its biological activities through its binding to high affinity receptor, fibroblast growth factor receptor-1 (FGFR1). It was found that both peptide segments of synthetic human FGF2 heparin-binding structural domain and receptor-binding structural domain could inhibit the *in vitro* proliferation of human umbilical vein endothelial cells [39]. Immunization of mice with vaccine based on heparin-binding structural domain peptide could induce production of anti-FGF2 specific antibody, which could hamper the binding of FGF2 to heparin sulphate, and inhibit tumorinduced angiogenesis in a gelatin sponge model and tumor growth in a tumor metastatic model [39]. Surprisingly, despite an immune response toward FGF2, this modality of treatment did not affect wound healing as shown by the fact that the treatment did not alter the mean time of wound healing [78]. It also did not affect fertility, because the vaccinated females were not impaired in their ability to become pregnant, to support the growth and development of their embryos, and to deliver viable offspring when compared with control animals [78]. Furthermore, histological analyses did not reveal any alterations in organogenesis in these offsprings [78]. Therefore, the authors concluded that although vaccination against FGF2 induced a specific FGF2 antibody response and inhibited angiogenesis and tumor develop‐ ment in a pathological setting, it did not adversely alter normal physiological events dependent on FGF2.

*2.2.4. Legumain*

a prophylactic setting [85].

*2.2.5. Endoglin (CD105)*

, AroA–

(dam–

Tumor associated macrophages (TAMs) are well known to play a very important role in tumor angiogenesis and metastasis, as the abrogation of TAMs in tumor tissues effectively reduced several pro-tumor growth and angiogenesis factors, such as VEGF, TGF-β, TNF-α and MMP-9 [81]. Thus, the suppression of TAMs in the tumor-microenvironment provides a novel strategy to inhibit tumor growth and dissemination by remodeling the tumor's stroma. Legumain is an asparaginyl endopeptidase and a member of the C13 family of cystine proteases which was found to be highly upregulated in many murine and human tumor tissues and, furthermore, also overexpressed on TAMs in the murine tumor stroma, but absent or present at only very low levels in all normal tissues from which such tumors arose [81-84]. Recently, several oral minigene vaccines against murine MHC class I antigen epitopes of Legumain were constructed based on the binding predictions for these MHC class I molecules by the HLA peptide binding predictions program [85]. Expression vectors encoding these epitopes were designated as pLegu-H-2Dd and pLegu-H-2Kd respectively [85]. Oral administration of those vaccines by

Anti-Angiogenic Active Immunotherapy for Cancers: Dawn of a New Era?

significant suppression of angiogenesis in tumor tissues of D2F2 breast carcinoma in syngeneic BALB/c mice [85]. The possible mechanism of angiogenic inhibition involved the induction of a specific CTL response capable of killing Legumain positive cells, especially TAMs, which is likely to be responsible for anti-tumor angiogenesis [85]. Generally, the anti-angiogenic effect aided in the protection of BALB/c mice from lethal challenges with D2F2 breast tumor cells in

Endoglin, a 95 kDa cell surface protein expressed as a homodimer, functions as an accessory protein for kinase receptor complexes of the TGF-β superfamily and modulates TGF-β signaling [8]. Expression of CD105 is correlated with vascular density and poor prognosis [8]. Endoglin is over-expressed on proliferating endothelial cells in the breast tumor neovascula‐ ture and thus offers a target for anti-angiogenic therapy [8]. It was reported that an oral murine endoglin-encoding DNA vaccine carried by double attenuated Salmonella typhimurium

, AroA–

http://dx.doi.org/10.5772/55323

379

s patches, resulted in activation of

) resulted in

T cells

transforming them into attenuated Salmonella typhimurium (Dam–

) to a secondary lymphoid organ, i.e., Peyer'

antigen-presenting dendritic cells, induction of immune responses mediated by CD8+

against endoglin-positive target cells, and suppression of angiogenesis and dissemination of pulmonary metastases of D2F2 breast carcinoma cells presumably by eliminating proliferating endothelial cells in the tumor vasculature, thus providing an promising strategy to therapies for breast cancer [86]. More recently, Wood et al. [87] developed *Listeria*-based vaccines directed against CD105, Lm-LLO-CD105A and Lm-LLO-CD105B. The region of CD105 in Lm-LLO-CD105A vaccine contains at least three predicted H-2Kd epitopes, while the region of CD105 in Lm-LLO-CD105B contains at least two predicted H-2Kd epitopes. Immunization of the *Listeria*-based vaccines led to therapeutic responses against primary and metastatic tumors in the 4T1-Luc and NT-2 mouse models of breast cancer. In a mouse model for autochthonous Her-2/neu-driven breast cancer, Lm-LLO-CD105A vaccination prevented tumor incidence in 20% of mice by week 58 after birth while all control mice developed tumors by week 40. In

#### *2.2.3. EGFR*

Epidermal growth factor receptor (EGFR), a membrane surface sensor with tyrosine kinase activity, is widely distributed on the membrane of mammalian cells [79]. In the physiological condition, EGFR exerts, through binding to ligands (epidermal growth factor, EGF), its physiological activities in regulation of cell division, proliferation and differentiation [79]. Results from clinical studies show that high expression level of EGFR is frequently observed in non-small cell lung cancer, and has been implicated in aggressive biological behavior of tumor cells and poor prognosis of tumor patients [79]. Therefore, immunotherapy targeting EGFR should be another attractive approach to the treatment of EGFR-positive tumors. In murine tumor models with Lewis lung carcinoma and mammary cancer, immunization of mice with DC pulsed with recombinant ectodomain of mouse EGFR (DC-edMER) inhibited tumor angiogenesis, reduced tumor growth, and prolonged the survival of tumor-bearing mice [80]. Spleen cells isolated from DC-edMER-immunized mice showed a high frequency of EGFR-specific antibody-producing cells [80]. Anti-EGFR specific antibody was markedly elevated in sera of immunized mice and was shown to be effective against tumor growth by adoptive transfer [80]. Immunization with DC-edMER vaccine also elicited CTL responses [80]. Depletion of CD4+ T lymphocytes could completely abrogate the anti-tumor activity and generation of EGFR-specific antibody responses, whereas depletion of CD8+ T lymphocytes showed partial abrogation of the anti-tumor activity but antibody was still detected [80]. Furthermore, tumor-induced angiogenesis was suppressed in DC-edMER-immunized mice or mice treated with antibody adoptive transfer [80]. These findings indicate that vaccination with DC-edMER can induce both humoral and cellular anti-tumor immunity, and may suggest novel strategies for the treatment of EGFR-positive tumors through the induction of active immunity against EGFR [80].

### *2.2.4. Legumain*

through its binding to high affinity receptor, fibroblast growth factor receptor-1 (FGFR1). It was found that both peptide segments of synthetic human FGF2 heparin-binding structural domain and receptor-binding structural domain could inhibit the *in vitro* proliferation of human umbilical vein endothelial cells [39]. Immunization of mice with vaccine based on heparin-binding structural domain peptide could induce production of anti-FGF2 specific antibody, which could hamper the binding of FGF2 to heparin sulphate, and inhibit tumorinduced angiogenesis in a gelatin sponge model and tumor growth in a tumor metastatic model [39]. Surprisingly, despite an immune response toward FGF2, this modality of treatment did not affect wound healing as shown by the fact that the treatment did not alter the mean time of wound healing [78]. It also did not affect fertility, because the vaccinated females were not impaired in their ability to become pregnant, to support the growth and development of their embryos, and to deliver viable offspring when compared with control animals [78]. Furthermore, histological analyses did not reveal any alterations in organogenesis in these offsprings [78]. Therefore, the authors concluded that although vaccination against FGF2 induced a specific FGF2 antibody response and inhibited angiogenesis and tumor develop‐ ment in a pathological setting, it did not adversely alter normal physiological events dependent

378 Cancer Treatment - Conventional and Innovative Approaches

Epidermal growth factor receptor (EGFR), a membrane surface sensor with tyrosine kinase activity, is widely distributed on the membrane of mammalian cells [79]. In the physiological condition, EGFR exerts, through binding to ligands (epidermal growth factor, EGF), its physiological activities in regulation of cell division, proliferation and differentiation [79]. Results from clinical studies show that high expression level of EGFR is frequently observed in non-small cell lung cancer, and has been implicated in aggressive biological behavior of tumor cells and poor prognosis of tumor patients [79]. Therefore, immunotherapy targeting EGFR should be another attractive approach to the treatment of EGFR-positive tumors. In murine tumor models with Lewis lung carcinoma and mammary cancer, immunization of mice with DC pulsed with recombinant ectodomain of mouse EGFR (DC-edMER) inhibited tumor angiogenesis, reduced tumor growth, and prolonged the survival of tumor-bearing mice [80]. Spleen cells isolated from DC-edMER-immunized mice showed a high frequency of EGFR-specific antibody-producing cells [80]. Anti-EGFR specific antibody was markedly elevated in sera of immunized mice and was shown to be effective against tumor growth by adoptive transfer [80]. Immunization with DC-edMER vaccine also elicited CTL responses [80].

generation of EGFR-specific antibody responses, whereas depletion of CD8+

showed partial abrogation of the anti-tumor activity but antibody was still detected [80]. Furthermore, tumor-induced angiogenesis was suppressed in DC-edMER-immunized mice or mice treated with antibody adoptive transfer [80]. These findings indicate that vaccination with DC-edMER can induce both humoral and cellular anti-tumor immunity, and may suggest novel strategies for the treatment of EGFR-positive tumors through the induction of active

T lymphocytes could completely abrogate the anti-tumor activity and

T lymphocytes

on FGF2.

*2.2.3. EGFR*

Depletion of CD4+

immunity against EGFR [80].

Tumor associated macrophages (TAMs) are well known to play a very important role in tumor angiogenesis and metastasis, as the abrogation of TAMs in tumor tissues effectively reduced several pro-tumor growth and angiogenesis factors, such as VEGF, TGF-β, TNF-α and MMP-9 [81]. Thus, the suppression of TAMs in the tumor-microenvironment provides a novel strategy to inhibit tumor growth and dissemination by remodeling the tumor's stroma. Legumain is an asparaginyl endopeptidase and a member of the C13 family of cystine proteases which was found to be highly upregulated in many murine and human tumor tissues and, furthermore, also overexpressed on TAMs in the murine tumor stroma, but absent or present at only very low levels in all normal tissues from which such tumors arose [81-84]. Recently, several oral minigene vaccines against murine MHC class I antigen epitopes of Legumain were constructed based on the binding predictions for these MHC class I molecules by the HLA peptide binding predictions program [85]. Expression vectors encoding these epitopes were designated as pLegu-H-2Dd and pLegu-H-2Kd respectively [85]. Oral administration of those vaccines by transforming them into attenuated Salmonella typhimurium (Dam– , AroA– ) resulted in significant suppression of angiogenesis in tumor tissues of D2F2 breast carcinoma in syngeneic BALB/c mice [85]. The possible mechanism of angiogenic inhibition involved the induction of a specific CTL response capable of killing Legumain positive cells, especially TAMs, which is likely to be responsible for anti-tumor angiogenesis [85]. Generally, the anti-angiogenic effect aided in the protection of BALB/c mice from lethal challenges with D2F2 breast tumor cells in a prophylactic setting [85].

### *2.2.5. Endoglin (CD105)*

Endoglin, a 95 kDa cell surface protein expressed as a homodimer, functions as an accessory protein for kinase receptor complexes of the TGF-β superfamily and modulates TGF-β signaling [8]. Expression of CD105 is correlated with vascular density and poor prognosis [8]. Endoglin is over-expressed on proliferating endothelial cells in the breast tumor neovascula‐ ture and thus offers a target for anti-angiogenic therapy [8]. It was reported that an oral murine endoglin-encoding DNA vaccine carried by double attenuated Salmonella typhimurium (dam– , AroA– ) to a secondary lymphoid organ, i.e., Peyer' s patches, resulted in activation of antigen-presenting dendritic cells, induction of immune responses mediated by CD8+ T cells against endoglin-positive target cells, and suppression of angiogenesis and dissemination of pulmonary metastases of D2F2 breast carcinoma cells presumably by eliminating proliferating endothelial cells in the tumor vasculature, thus providing an promising strategy to therapies for breast cancer [86]. More recently, Wood et al. [87] developed *Listeria*-based vaccines directed against CD105, Lm-LLO-CD105A and Lm-LLO-CD105B. The region of CD105 in Lm-LLO-CD105A vaccine contains at least three predicted H-2Kd epitopes, while the region of CD105 in Lm-LLO-CD105B contains at least two predicted H-2Kd epitopes. Immunization of the *Listeria*-based vaccines led to therapeutic responses against primary and metastatic tumors in the 4T1-Luc and NT-2 mouse models of breast cancer. In a mouse model for autochthonous Her-2/neu-driven breast cancer, Lm-LLO-CD105A vaccination prevented tumor incidence in 20% of mice by week 58 after birth while all control mice developed tumors by week 40. In comparison with previous *Listeria*-based vaccines (Lm-LLO-HMWMAA-C [88] and Lm-LLO-FLK-I1 and Lm-LLO-FLK-E2 [71] ) targeting tumor vasculature, Lm-LLO-CD105A and Lm-LLO-CD105B demonstrated equivalent or superior efficacy against two transplantable mouse models of breast cancer. Mechanism analysis revealed that the anti-tumor therapeutic efficacy of *Listeria*-based CD105 vaccines was mediated by epitope spreading to endogenous tumor antigens and reduction in tumor vascularity [87]. These data suggest that CD105 therapeutic vaccines are highly effective in stimulating anti-angiogenesis and anti-tumor immune responses leading to therapeutic efficacy against primary and metastatic breast cancer.

inhibitors and angiogenic antibodies, anti-angiogenic active immunotherapy has its obvious merits. Provided that a break of immunological tolerance to positive regulators of angiogenesis is successfully induced, the long-lasting immune response to angiogenesis-related molecule will be present in the body, hereby providing long-lasting inhibitory effects on angiogenesis. Therefore, it is expected to be the more cost-effective strategy than angiogenic inhibitor or anti-

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Here we divided anti-angiogenic active immunotherapy into two categories: therapies based on vaccination with xenogeneic homologous molecules and with non-xenogeneic homologous molecules related to angiogenesis. Presently, it is difficult to point out which one is better for clinical application because most of the outcomes reported to date were based on pre-clinical animal experiments. As VEGF-mediated signaling through its receptor VEGFR-2 is the key rate-limiting step in tumor angiogenesis, and plays the most impor‐ tant role in neovascularization, development, and progression of various tumors [6], as well as human VEGFR2-169 peptide vaccination could effectively break peripheral self toler‐ ance against VEGFR-2 in patients with metastatic and unresectable pancreatic cancer [74], anti-angiogenic active immunotherapy targeting VEGF or VEGFR-2 might be the most effective strategy among all these therapies. Moreover, considering the potential clinical application of anti-angiogenic immunotherapy based on the specific antibodies raised against a variety of angiogenesis-associated molecules in different tumor entities like glioma, renal cell cancer, and breast cancer, etc, a promising clinical application of antiangiogenic active immunotherapy alone or in combination with other anti-tumor strat‐ egies could be expected. However, there exist as well *caveats* and deficiencies in this strategy. Firstly, in the early phase of tumor growth when the tumor diameter is less than 2-3 mm, tumor cells simply depend on passive diffusion rather than blood perfusion to acquire enough oxygen and nutrition indispensable for growth. Therefore, anti-angiogen‐ ic therapy against tumor in this early stage might be ineffective when applied alone. Secondly, although current anti-angiogenic active immunotherapy is focused on specific targets, potential adverse effects might include impairment of wound healing and menstru‐ al cycle. Furthermore, this approach has also limited application perspectives in children with cancers. Therefore, along with recent developments in molecular biology and immunology, future studies will focus on multiple approaches, such as series analysis of gene expression to analyze the gene expression in normal endothelial cells and in prolifer‐ ative endothelial cells, phage display technology to search for new endothelial cell receptors, and proteomics to discover peptide segments or proteins regulating endothelial cell growth. These approaches are expected to discover more tumor-specific endothelial cell markers for the purpose of selecting specific targets for anti-angiogenic active immunother‐ apy. In addition, further studies are also required to optimize protocols how to construct vaccines to effectively break self-tolerance and to induce efficient immune response. With these issues being solved continuously, anti-angiogenic active immunotherapy for cancers

angiogenic antibody therapy where continuous use of the drugs is needed.

will become more applicable and effective.

#### *2.2.6. Endothelial cell lysates-pulsed dendritic cells*

Dendritic cells (DCs) are the most potent professional antigen-presenting cells, they play crucial roles in the initiation of an immune response. DCs prepared from BALB/c mouse were pulsed with lysates of autologous or xenogeneic endothelium, and their anti-tumor effects were tested in two syngeneic models of colon cancer [89]. Immunization of endothelium lysates pulsed DCs could induce a break in self tolerance against endothelial cells and mount both the endothelium-specific CTL response and antibody response, leading to significant inhibition of tumor angiogenesis and the growth of subcutaneous tumors as well as pulmonary meta‐ stases in mice. Furthermore, the decrease in the mean vascular density of tumors correlates well with the extent of tumor inhibition [89]. Therefore, immunization of endothelium lysates pulsed DCs is also an effective modality of anti-angiogenic active immunotherapy for cancers, and should have important clinical implications for adjuvant cancer therapy.

### *2.2.7. Endothelial cell vaccine*

In 2008, Okaji Y, et al. [90] reported a pilot phase I clinical study in which glutaraldehydefixed human umbilical vein endothelial cells (HUVECs) were used as the vaccine. Six patients with recurrent malignant brain tumour and three patients with metastatic colorectal cancer were given intradermal injections of 5x107 HUVECs/dose, first month weekly, and then every 2 weeks (in total 230 vaccinations). ELISA and flow cytometry revealed immunoglobulin response against HUVECs' membrane antigens. ELISPOT and 51Cr-release cytotoxicity assay revealed a specific cellular immune response against HUVECs, which were lysed in an effectors:targets ratio-dependent manner. Gadoliniumcontrasted MRI showed partial or complete tumour responses in three malignant brain tumour patients. Except for a DTH-like skin reaction at the injection site, no adverse effect of vaccination was observed. These results suggested that the endothelial vaccine can overcome peripheral tolerance of self-angiogenic antigens in clinical settings, and there‐ fore could be useful for adjuvant immunotherapy of cancer.

### **3. Concluding remarks**

Recent research achievements have disclosed inspiring pragmatic perspectives of antiangiogenic active immunotherapy for cancers. In comparison with application of angiogenic inhibitors and angiogenic antibodies, anti-angiogenic active immunotherapy has its obvious merits. Provided that a break of immunological tolerance to positive regulators of angiogenesis is successfully induced, the long-lasting immune response to angiogenesis-related molecule will be present in the body, hereby providing long-lasting inhibitory effects on angiogenesis. Therefore, it is expected to be the more cost-effective strategy than angiogenic inhibitor or antiangiogenic antibody therapy where continuous use of the drugs is needed.

comparison with previous *Listeria*-based vaccines (Lm-LLO-HMWMAA-C [88] and Lm-LLO-FLK-I1 and Lm-LLO-FLK-E2 [71] ) targeting tumor vasculature, Lm-LLO-CD105A and Lm-LLO-CD105B demonstrated equivalent or superior efficacy against two transplantable mouse models of breast cancer. Mechanism analysis revealed that the anti-tumor therapeutic efficacy of *Listeria*-based CD105 vaccines was mediated by epitope spreading to endogenous tumor antigens and reduction in tumor vascularity [87]. These data suggest that CD105 therapeutic vaccines are highly effective in stimulating anti-angiogenesis and anti-tumor immune responses leading to therapeutic efficacy against primary and metastatic breast cancer.

Dendritic cells (DCs) are the most potent professional antigen-presenting cells, they play crucial roles in the initiation of an immune response. DCs prepared from BALB/c mouse were pulsed with lysates of autologous or xenogeneic endothelium, and their anti-tumor effects were tested in two syngeneic models of colon cancer [89]. Immunization of endothelium lysates pulsed DCs could induce a break in self tolerance against endothelial cells and mount both the endothelium-specific CTL response and antibody response, leading to significant inhibition of tumor angiogenesis and the growth of subcutaneous tumors as well as pulmonary meta‐ stases in mice. Furthermore, the decrease in the mean vascular density of tumors correlates well with the extent of tumor inhibition [89]. Therefore, immunization of endothelium lysates pulsed DCs is also an effective modality of anti-angiogenic active immunotherapy for cancers,

In 2008, Okaji Y, et al. [90] reported a pilot phase I clinical study in which glutaraldehydefixed human umbilical vein endothelial cells (HUVECs) were used as the vaccine. Six patients with recurrent malignant brain tumour and three patients with metastatic colorectal cancer were given intradermal injections of 5x107 HUVECs/dose, first month weekly, and then every 2 weeks (in total 230 vaccinations). ELISA and flow cytometry revealed immunoglobulin response against HUVECs' membrane antigens. ELISPOT and 51Cr-release cytotoxicity assay revealed a specific cellular immune response against HUVECs, which were lysed in an effectors:targets ratio-dependent manner. Gadoliniumcontrasted MRI showed partial or complete tumour responses in three malignant brain tumour patients. Except for a DTH-like skin reaction at the injection site, no adverse effect of vaccination was observed. These results suggested that the endothelial vaccine can overcome peripheral tolerance of self-angiogenic antigens in clinical settings, and there‐

Recent research achievements have disclosed inspiring pragmatic perspectives of antiangiogenic active immunotherapy for cancers. In comparison with application of angiogenic

and should have important clinical implications for adjuvant cancer therapy.

fore could be useful for adjuvant immunotherapy of cancer.

*2.2.6. Endothelial cell lysates-pulsed dendritic cells*

380 Cancer Treatment - Conventional and Innovative Approaches

*2.2.7. Endothelial cell vaccine*

**3. Concluding remarks**

Here we divided anti-angiogenic active immunotherapy into two categories: therapies based on vaccination with xenogeneic homologous molecules and with non-xenogeneic homologous molecules related to angiogenesis. Presently, it is difficult to point out which one is better for clinical application because most of the outcomes reported to date were based on pre-clinical animal experiments. As VEGF-mediated signaling through its receptor VEGFR-2 is the key rate-limiting step in tumor angiogenesis, and plays the most impor‐ tant role in neovascularization, development, and progression of various tumors [6], as well as human VEGFR2-169 peptide vaccination could effectively break peripheral self toler‐ ance against VEGFR-2 in patients with metastatic and unresectable pancreatic cancer [74], anti-angiogenic active immunotherapy targeting VEGF or VEGFR-2 might be the most effective strategy among all these therapies. Moreover, considering the potential clinical application of anti-angiogenic immunotherapy based on the specific antibodies raised against a variety of angiogenesis-associated molecules in different tumor entities like glioma, renal cell cancer, and breast cancer, etc, a promising clinical application of antiangiogenic active immunotherapy alone or in combination with other anti-tumor strat‐ egies could be expected. However, there exist as well *caveats* and deficiencies in this strategy. Firstly, in the early phase of tumor growth when the tumor diameter is less than 2-3 mm, tumor cells simply depend on passive diffusion rather than blood perfusion to acquire enough oxygen and nutrition indispensable for growth. Therefore, anti-angiogen‐ ic therapy against tumor in this early stage might be ineffective when applied alone. Secondly, although current anti-angiogenic active immunotherapy is focused on specific targets, potential adverse effects might include impairment of wound healing and menstru‐ al cycle. Furthermore, this approach has also limited application perspectives in children with cancers. Therefore, along with recent developments in molecular biology and immunology, future studies will focus on multiple approaches, such as series analysis of gene expression to analyze the gene expression in normal endothelial cells and in prolifer‐ ative endothelial cells, phage display technology to search for new endothelial cell receptors, and proteomics to discover peptide segments or proteins regulating endothelial cell growth. These approaches are expected to discover more tumor-specific endothelial cell markers for the purpose of selecting specific targets for anti-angiogenic active immunother‐ apy. In addition, further studies are also required to optimize protocols how to construct vaccines to effectively break self-tolerance and to induce efficient immune response. With these issues being solved continuously, anti-angiogenic active immunotherapy for cancers will become more applicable and effective.

### **Acknowledgements**

This work was supported by a grant from the Science and Technology Bureau of Hangzhou, Zhejiang Province, P.R. China (No. 20120633B30).

[11] Caldini R, Barletta E, Del Rosso M, et al. FGF2-mediated upregulation of urokinasetype plasminogen activator expression requires a MAP-kinase dependent activation

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### **Author details**

Jianping Pan\* and Lihuang Zhang

\*Address all correspondence to: jppan@zucc.edu.cn

Department of Clinical Medicine, Zhejiang University City College School of Medicine, Hangzhou, P.R. China

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poietic cells. Immunity 2000; 12:677-686.

dritic cell maturation. Immunol Lett 2004; 94:141-151.

gene 2010; 29:4276-4286.

386 Cancer Treatment - Conventional and Innovative Approaches

Acta 2008; 1783: 429-437.

2002; 195: 1575-1584.

8:1369-1375.

152: 1247-1254.

9208-9213.


[80] Hu B, Wei YQ, Tian L, et al. Active antitumor immunity elicited by vaccine based on recombinant form of epidermal growth factor receptor. J Immunother 2005; 28:236-244.

**Section 4**

**Multidisciplinarity in Cancer Therapy: Nutrition**

**and Beyond**


**Multidisciplinarity in Cancer Therapy: Nutrition and Beyond**

[80] Hu B, Wei YQ, Tian L, et al. Active antitumor immunity elicited by vaccine based on recombinant form of epidermal growth factor receptor. J Immunother 2005;

[81] Luo Y, Zhou H, Krueger J, et al. Targeting tumor-associated macrophages as a novel

[82] Liu C, Sun C, Huang H, et al. Overexpression of legumain in tumors is significant for invasion / metastasis and a candidate enzymatic target for prodrug therapy. Cancer

[83] Murthy RV, Arbman G, Gao J, et al. Legumain expression in relation to clinicopatho‐ logic and biological variables in colorectal cancer. Clin Cancer Res 2005; 11:2293-2299.

[84] Oosterling SJ, van der Bij GJ, Meijer GA, et al. Macrophages direct tumor histology

[85] Lewen S, Zhou H, Hu HD, et al. A Legumain-based minigene vaccine targets the tu‐ mor stroma and suppresses breast cancer growth and angiogenesis. Cancer Immunol

[86] Lee SH, Mizutani N, Mizutani M, et al. Endoglin (CD105) is a target for an oral DNA vaccine against breast cancer. Cancer Immunol Immunother 2006; 55:1565-1574. [87] Wood LM, Pan ZK, Guirnalda P, et al. Targeting tumor vasculature with novel Liste‐ ria-based vaccines directed against CD105. Cancer Immunol Immunother 2011;

[88] Maciag PC, Seavey MM, Pan ZK, et al. Cancer immunotherapy targeting the high molecular weight melanoma-associated antigen protein results in a broad antitumor response and reduction of pericytes in the tumor vasculature. Cancer Res 2008; 68:

[89] Yoneyama S, Okaji Y, Tsuno NH, et al. A study of dendritic and endothelial cell in‐ teractions in colon cancer in a cell line and small mammal model. Eur J Surg Oncol

[90] Okaji Y, Tsuno NH, Tanaka M, et al. Pilot study of anti-angiogenic vaccine using fixed whole endothelium in patients with progressive malignancy after failure of

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388 Cancer Treatment - Conventional and Innovative Approaches

Immunother 2008; 57:507-515.

**Chapter 17**

**Nutrigenomics and Cancer Prevention**

Cancer is fundamentally a genetic disease. At the beginning of the process is an alteration in the DNA of a single cell. This change in DNA can be caused by several factors, chemical, physical or biological phenomena. The stage of promotion is the second stage of carcinogenesis. The genetically altered cells, ie, "initiated," suffer the effects of carcinogens classified as oncopromo‐ tores. The initiated cell is transformed into a malignant cell, a slow and gradual process. For this transformation to occur, you need a long and continuous contact with the carcinogen promot‐ er. The stage of progression is the third and final stage and is characterized by uncontrolled proliferationof cells andirreversiblychanged.Atthis stage canceris alreadyinstalled,progress‐ ing to the emergence of the first clinical manifestations of the disease [1]. In this sense, the diet plays a key role in various stages of cancer development. The process of carcinogenesis may be affected by nutritional factors through mechanisms that promote or inhibit its development. Some foods can contain not only carcinogens, but also other substances that act to reduce the damage to the cell's genetic material caused by environmental mutagens. The observation of cancer in an individual does not identify the causative agent(s). However, epidemiological data on populations do indicate that a large fraction of human cancers are associated with lifestyle/ diet. Such studies may also help identify the etiologic agents but unless there are good doseresponse data for humans and/or animal models, the probability of identifying the agent is not high. Cancers may result from endogenous reactions, such as oxidations or from exogenous agents, such as tobacco smoke (lung cancer), sunlight exposure (skin cancer), aflatoxin (liver

cancer), and relatively high doses of ionizing radiations (many types of cancers) [2].

The importance of nutrition in health is not a new idea. More than two thousand years ago, Hippocrates, the father of Western medicine, wrote: "Let food be thy medicine and medicine be thy food." What has changed since the time of Hippocrates is our understanding of the details of how nutrition affects our health. Researchers are getting more knowledge as to what foods or bioactive food compounds and how they can interact with our bodies promoting

and reproduction in any medium, provided the original work is properly cited.

© 2013 Nepomuceno; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Additional information is available at the end of the chapter

Júlio César Nepomuceno

http://dx.doi.org/10.5772/55429

**1. Introduction**

### **Chapter 17**

### **Nutrigenomics and Cancer Prevention**

### Júlio César Nepomuceno

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55429

### **1. Introduction**

Cancer is fundamentally a genetic disease. At the beginning of the process is an alteration in the DNA of a single cell. This change in DNA can be caused by several factors, chemical, physical or biological phenomena. The stage of promotion is the second stage of carcinogenesis. The genetically altered cells, ie, "initiated," suffer the effects of carcinogens classified as oncopromo‐ tores. The initiated cell is transformed into a malignant cell, a slow and gradual process. For this transformation to occur, you need a long and continuous contact with the carcinogen promot‐ er. The stage of progression is the third and final stage and is characterized by uncontrolled proliferationof cells andirreversiblychanged.Atthis stage canceris alreadyinstalled,progress‐ ing to the emergence of the first clinical manifestations of the disease [1]. In this sense, the diet plays a key role in various stages of cancer development. The process of carcinogenesis may be affected by nutritional factors through mechanisms that promote or inhibit its development. Some foods can contain not only carcinogens, but also other substances that act to reduce the damage to the cell's genetic material caused by environmental mutagens. The observation of cancer in an individual does not identify the causative agent(s). However, epidemiological data on populations do indicate that a large fraction of human cancers are associated with lifestyle/ diet. Such studies may also help identify the etiologic agents but unless there are good doseresponse data for humans and/or animal models, the probability of identifying the agent is not high. Cancers may result from endogenous reactions, such as oxidations or from exogenous agents, such as tobacco smoke (lung cancer), sunlight exposure (skin cancer), aflatoxin (liver cancer), and relatively high doses of ionizing radiations (many types of cancers) [2].

The importance of nutrition in health is not a new idea. More than two thousand years ago, Hippocrates, the father of Western medicine, wrote: "Let food be thy medicine and medicine be thy food." What has changed since the time of Hippocrates is our understanding of the details of how nutrition affects our health. Researchers are getting more knowledge as to what foods or bioactive food compounds and how they can interact with our bodies promoting

© 2013 Nepomuceno; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

health. The Human Genome Project was one of the key factors that enable the study of genefood interactions and promotion of health. Discoveries in genetics make it possible to under‐ stand the effects of nutrients in processes at the molecular level in the body and also the variable effects of dietary components on each individual. Research has shown that the nutrients affect gene expression and formation of several proteins that are important in the formation and maintenance of tissues. So, faced with this interaction genomics and nutrition, emerges Nutrigenomics aiming to understand the functions of all genes and their interactions with food, in order to promote health and reduce the risk of developing diseases [3].

nucleotide, resulting in two methylated cytosine residues sitting diagonally to each other on opposing DNA strands [6]. DNA methylation, which modifies a cytosine base at the CpG dinucleotide residues with methyl groups, is catalyzed by DNA methyltransferases (Dnmt) and regulates gene expression patterns by altering chromatin structures. Currently, 5 different Dnmt are known: Dnmt1, Dnmt2, Dnmt 3a, Dnmt3b and DnmtL [7]. The Polycomb group protein EZH2 directly controls DNA methylation (Figure 1). EZH2 serves as a recruitment platform for DNA methyltransferases, thus highlighting a previously unrecognized direct

Nutrigenomics and Cancer Prevention http://dx.doi.org/10.5772/55429 393

**Figure 1.** Polycomb Group (PcG) protein EZH2 serves as a recruitment platform for DNA methyltransferases (http://

DNA methylation is essential for cell differentiation and embryonic development. Moreover, in some cases, methylation has observed to play a role in mediating gene expression. In mammals, methylation is found sparsely but globally, distributed in definite CpG sequences throughout the entire genome, with the exception of CpG islands, or certain stretches (ap‐ proximately 1 kilobase in length) where high CpG contents are found. The methylation of these sequences can lead to inappropriate gene silencing, such as the silencing of tumor suppressor genes in cancer cells [6]. A large amount of research on DNA methylation and disease has focused on cancer and tumor suppressor genes. Tumor suppressor genes are often silenced in cancer cells due to hypermethylation. In contrast, the genomes of cancer cells have been shown to be hypomethylated overall when compared to normal cells, with the exception of hyper‐ methylation events at genes involved in cell cycle regulation, tumor cell invasion, DNA repair, and other events in which silencing propagates metastasis. In fact, in certain cancers, such as that of the colon, hypermethylation is detectable early and might serve as a biomarker for the

In the nutritional field, epigenetics is exceptionally important, because nutrients and bioactive food components can modify epigenetic phenomena and alter the expression of genes at the transcriptional level. Nutrients can reverse or change epigenetic phenomena such as DNA

connection between two key epigenetic repression systems [8].

www.ulb.ac.be/medecine/fukslab/research.htm).

disease [6] (See Figure 2).

Nutrigenomics studies the modulating effect of the chemical compounds in foods and on the stability of DNA synthesis and gene expression. The nutrients are able to affect the genome and its expression through the synthesis of nucleotides, prevention and repair of DNA damage, or through epigenetic mechanisms including methylation of histones, proteins responsible for chromatin structure that play an important role in regulating gene expression. Those meth‐ odological approaches are based on nutrition, molecular biology, and genomics. Integration of these disciplines is leading to identification and understanding of individual and population differences and similarities in gene expression, or phenotype, in response to diet. We can consider nutrigenomics as a multidisciplinary science that applies the genomic techniques besides the biochemical and epidemiological aspects, with the aim to understand the etiologic aspects of chronic diseases such as cardiovascular diseases, diabetes, obesity and cancer [4].

An understanding of scientific information about the composition and functions of genomes, has created unprecedented opportunities for increasing our understanding of how nutrients modulate gene and protein expression and ultimately influence cellular and organismal metabolism. On that basis, the purpose of this chapter is to make a broad review study to evaluate the modulation between compounds found in nutrients and their interactions with on the genomic stability and control of gene expression.

### **2. Nutrition and epigenetics**

All the cells in the body have identical genomes. However, each cell has one of many "epige‐ nomes", unique sets of epigenetic instructions for establishing and maintaining lineagespecific expression profiles. The genome is programmed to express appropriate sets of genes, in particular tissues, at specific time points during the individual's life. Epigenetic events create a memory of cell identity, maintaining genomic functions such as the maintenance of cell identity after differentiation, the propagation of essential features of chromosomal architecture and dosage compensation [5]. Epigenetic mechanisms are capable of modulating gene expression through changes in the chromosomes structure. Chromosomes are formed from the condensation of the chromatin, which is formed by a complex of DNA, and unique proteins called histone. Examples of epigenetic mechanisms may be mentioned as DNA methylation and histone acetylation [3].

DNA methylation occurs at the cytosine bases of eukaryotic DNA, which are converted to 5 methylcytosine. The altered cytosine residues are usually immediately adjacent to a guanine nucleotide, resulting in two methylated cytosine residues sitting diagonally to each other on opposing DNA strands [6]. DNA methylation, which modifies a cytosine base at the CpG dinucleotide residues with methyl groups, is catalyzed by DNA methyltransferases (Dnmt) and regulates gene expression patterns by altering chromatin structures. Currently, 5 different Dnmt are known: Dnmt1, Dnmt2, Dnmt 3a, Dnmt3b and DnmtL [7]. The Polycomb group protein EZH2 directly controls DNA methylation (Figure 1). EZH2 serves as a recruitment platform for DNA methyltransferases, thus highlighting a previously unrecognized direct connection between two key epigenetic repression systems [8].

health. The Human Genome Project was one of the key factors that enable the study of genefood interactions and promotion of health. Discoveries in genetics make it possible to under‐ stand the effects of nutrients in processes at the molecular level in the body and also the variable effects of dietary components on each individual. Research has shown that the nutrients affect gene expression and formation of several proteins that are important in the formation and maintenance of tissues. So, faced with this interaction genomics and nutrition, emerges Nutrigenomics aiming to understand the functions of all genes and their interactions with

Nutrigenomics studies the modulating effect of the chemical compounds in foods and on the stability of DNA synthesis and gene expression. The nutrients are able to affect the genome and its expression through the synthesis of nucleotides, prevention and repair of DNA damage, or through epigenetic mechanisms including methylation of histones, proteins responsible for chromatin structure that play an important role in regulating gene expression. Those meth‐ odological approaches are based on nutrition, molecular biology, and genomics. Integration of these disciplines is leading to identification and understanding of individual and population differences and similarities in gene expression, or phenotype, in response to diet. We can consider nutrigenomics as a multidisciplinary science that applies the genomic techniques besides the biochemical and epidemiological aspects, with the aim to understand the etiologic aspects of chronic diseases such as cardiovascular diseases, diabetes, obesity and cancer [4]. An understanding of scientific information about the composition and functions of genomes, has created unprecedented opportunities for increasing our understanding of how nutrients modulate gene and protein expression and ultimately influence cellular and organismal metabolism. On that basis, the purpose of this chapter is to make a broad review study to evaluate the modulation between compounds found in nutrients and their interactions with

All the cells in the body have identical genomes. However, each cell has one of many "epige‐ nomes", unique sets of epigenetic instructions for establishing and maintaining lineagespecific expression profiles. The genome is programmed to express appropriate sets of genes, in particular tissues, at specific time points during the individual's life. Epigenetic events create a memory of cell identity, maintaining genomic functions such as the maintenance of cell identity after differentiation, the propagation of essential features of chromosomal architecture and dosage compensation [5]. Epigenetic mechanisms are capable of modulating gene expression through changes in the chromosomes structure. Chromosomes are formed from the condensation of the chromatin, which is formed by a complex of DNA, and unique proteins called histone. Examples of epigenetic mechanisms may be mentioned as DNA methylation

DNA methylation occurs at the cytosine bases of eukaryotic DNA, which are converted to 5 methylcytosine. The altered cytosine residues are usually immediately adjacent to a guanine

food, in order to promote health and reduce the risk of developing diseases [3].

on the genomic stability and control of gene expression.

**2. Nutrition and epigenetics**

392 Cancer Treatment - Conventional and Innovative Approaches

and histone acetylation [3].

**Figure 1.** Polycomb Group (PcG) protein EZH2 serves as a recruitment platform for DNA methyltransferases (http:// www.ulb.ac.be/medecine/fukslab/research.htm).

DNA methylation is essential for cell differentiation and embryonic development. Moreover, in some cases, methylation has observed to play a role in mediating gene expression. In mammals, methylation is found sparsely but globally, distributed in definite CpG sequences throughout the entire genome, with the exception of CpG islands, or certain stretches (ap‐ proximately 1 kilobase in length) where high CpG contents are found. The methylation of these sequences can lead to inappropriate gene silencing, such as the silencing of tumor suppressor genes in cancer cells [6]. A large amount of research on DNA methylation and disease has focused on cancer and tumor suppressor genes. Tumor suppressor genes are often silenced in cancer cells due to hypermethylation. In contrast, the genomes of cancer cells have been shown to be hypomethylated overall when compared to normal cells, with the exception of hyper‐ methylation events at genes involved in cell cycle regulation, tumor cell invasion, DNA repair, and other events in which silencing propagates metastasis. In fact, in certain cancers, such as that of the colon, hypermethylation is detectable early and might serve as a biomarker for the disease [6] (See Figure 2).

In the nutritional field, epigenetics is exceptionally important, because nutrients and bioactive food components can modify epigenetic phenomena and alter the expression of genes at the transcriptional level. Nutrients can reverse or change epigenetic phenomena such as DNA

methylation in *Apis* is used for storing epigenetic information, that the use of that information can be differentially altered by nutritional input, and that the flexibility of epigenetic modifi‐ cations underpins, profound shifts in developmental fates, with massive implications for

During our lifetime, nutrients can modify physiologic and pathologic processes through epigenetic mechanisms that are critical for gene expression (summarized in Table 1). Modu‐ lation of these processes through diet or specific nutrients may prevent diseases and maintain health. However, it is very hard to delineate the precise effect of nutrients or bioactive food components on each epigenetic modulation and their associations with physiologic and pathologic processes in our body, because the nutrients also interact with genes, other nutrients, and other lifestyle factors. Furthermore, each epigenetic phenomenon also interacts

**Nutrient or diet Epigenetic mechanism**

modifications

Nutrigenomics and Cancer Prevention http://dx.doi.org/10.5772/55429 395

methylation

methylation

Choline DNA methylation Protein restriction DNA methylation, histone

Alcohol DNA methylation

Calorie restriction Histone acetylation

Genistein DNA methylation, microRNA (-)-Epigallocatechin-3-gallate DNA methylation, PRC

Embryonic Folate DNA methylation, imprinting

Stem cell Butyrate Histone acetylation, DNA

Aging Folate DNA methylation

Immune function Folate DNA methylation

Retinoic acid PRC

Cancer Methyl-deficient diet Histone modification, microRNA

Obesity, insulin High-fat diet DNA methylation, microRNA

Inflammation Resveratrol Histone acetylation

**Table 1.** Epigenetic roles of nutrition in physiologic and pathologic processes

Neurocognition Choline DNA methylation, histone

Curcumin microRNA

Methyl-deficient diet microRNA

Methyl-deficient diet DNA methylation Curcumin Histone acetylation

AdoMet Histone methylation

reproductive and behavioral status.

development

resistance

Data in reference [7].

with the others, adding to the complexity of the system [7].

**Figure 2.** DNA methylation and cancer. This diagram shows a representative region of genomic DNA in a normal cell. The region contains repeat-rich, hypermethylated pericentromeric heterochromatin and an actively transcribed tumor suppressor gene (TSG) associated with a hypomethylated CpG island (indicated in red). In tumor cells, repeat-rich het‐ erochromatin becomes hypomethylated, and this contributes to genomic instability (a hallmark of tumor cells) through increased mitotic recombination events. *De novo* methylation of CpG islands also occurs in cancer cells, and it can result in the transcriptional silencing of growth-regulatory genes. These changes in methylation are early events in tumorigenesis. (See reference [9].)

methylation and histone modifications, thereby modifying the expression of critical genes associated with physiologic and pathologic processes, including embryonic development, aging, and carcinogenesis [7].

The most interesting study linking diet and epigenetics was made by Kucharski et al. [10], about nutritional control of reproductive status in honeybees via DNA methylation. Fertile queens and sterile workers are alternative forms of the adult female honeybee that develop from genetically identical larvae following differential feeding with royal jelly. Royal jelly is a complex, protein-rich substance secreted from glands on the heads of worker bees. A larva destined to become a queen is fed large quantities of royal jelly inside a specially constructed compartment called a queen cup. The authors observed that larvae fed with royal jelly developed functional ovaries and a larger abdomen for egg laying, while worker bees remain sterile. She'll also develop the necessary behaviors to act as queen, such as killing rival queens, making communication sounds known as "piping," and going on "mating flights." The queen is fed royal honey exclusively for the rest of her life. They showed that royal jelly silences a key gene (Dnmt3), which codes for an enzyme involved in genome-wide gene silencing. When Dnmt3 is active in bee larvae, the queen genes are epigenetically silenced and the larvae develop into the default "worker" variety. But when royal jelly turns Dnmt3 off, certain genes jump into action that turn the lucky larvae into queens. The authors suggested that DNA methylation in *Apis* is used for storing epigenetic information, that the use of that information can be differentially altered by nutritional input, and that the flexibility of epigenetic modifi‐ cations underpins, profound shifts in developmental fates, with massive implications for reproductive and behavioral status.

During our lifetime, nutrients can modify physiologic and pathologic processes through epigenetic mechanisms that are critical for gene expression (summarized in Table 1). Modu‐ lation of these processes through diet or specific nutrients may prevent diseases and maintain health. However, it is very hard to delineate the precise effect of nutrients or bioactive food components on each epigenetic modulation and their associations with physiologic and pathologic processes in our body, because the nutrients also interact with genes, other nutrients, and other lifestyle factors. Furthermore, each epigenetic phenomenon also interacts with the others, adding to the complexity of the system [7].


**Table 1.** Epigenetic roles of nutrition in physiologic and pathologic processes

methylation and histone modifications, thereby modifying the expression of critical genes associated with physiologic and pathologic processes, including embryonic development,

**Figure 2.** DNA methylation and cancer. This diagram shows a representative region of genomic DNA in a normal cell. The region contains repeat-rich, hypermethylated pericentromeric heterochromatin and an actively transcribed tumor suppressor gene (TSG) associated with a hypomethylated CpG island (indicated in red). In tumor cells, repeat-rich het‐ erochromatin becomes hypomethylated, and this contributes to genomic instability (a hallmark of tumor cells) through increased mitotic recombination events. *De novo* methylation of CpG islands also occurs in cancer cells, and it can result in the transcriptional silencing of growth-regulatory genes. These changes in methylation are early events in

The most interesting study linking diet and epigenetics was made by Kucharski et al. [10], about nutritional control of reproductive status in honeybees via DNA methylation. Fertile queens and sterile workers are alternative forms of the adult female honeybee that develop from genetically identical larvae following differential feeding with royal jelly. Royal jelly is a complex, protein-rich substance secreted from glands on the heads of worker bees. A larva destined to become a queen is fed large quantities of royal jelly inside a specially constructed compartment called a queen cup. The authors observed that larvae fed with royal jelly developed functional ovaries and a larger abdomen for egg laying, while worker bees remain sterile. She'll also develop the necessary behaviors to act as queen, such as killing rival queens, making communication sounds known as "piping," and going on "mating flights." The queen is fed royal honey exclusively for the rest of her life. They showed that royal jelly silences a key gene (Dnmt3), which codes for an enzyme involved in genome-wide gene silencing. When Dnmt3 is active in bee larvae, the queen genes are epigenetically silenced and the larvae develop into the default "worker" variety. But when royal jelly turns Dnmt3 off, certain genes jump into action that turn the lucky larvae into queens. The authors suggested that DNA

aging, and carcinogenesis [7].

tumorigenesis. (See reference [9].)

394 Cancer Treatment - Conventional and Innovative Approaches

### **2.1. Diet and genomic stability**

Eukaryotic DNA replication starts at multiple sites throughout the genome and is necessarily coordinated with transcription, sister chromatid cohesion, nucleosome assembly and cell cycle progression. In addition to the complexity of the replication reaction it, during replication cells need to deal with DNA damage and stalled forks, originated inevitably by the action of exogenous and endogenous agents. The success of this process is crucial to preserve genome stability, and the inability to deal with DNA lesions during replication or to protect or restart stalled forks leads to DNA breaks, chromosomal rearrangements, and mutations that can cause the loss of cell viability, but in addition errors in DNA replication result in a large number of human syndromes, including premature aging, various cancer predispositions and genetic abnormalities. To solve or reduce these problems, cells use repair and detoxification pathways as well as surveillance mechanisms, called checkpoints, which serve to detect the problem and coordinate repair with chromosome segregation and progression through the cell cycle (see Figure 3. www.genomic-instability.org/).

Maintaining genomic stability in the face of replication and recombination requires a huge variety of different damage response proteins. A cell's ability to decide when and where to deploy this DNA repair kit is critical to prevent tumor development [11].

There is evidence that inappropriate nutrient supply can cause sizeable levels of genome mutation and alter expression of genes required for genome maintenance. Deficiencies in several micronutrients have been shown to cause DNA damage and are thought to be associated with a number of serious human diseases: folic acid, niacin, vitamin B6 and B12 deficiency may increase the risk of colon cancer, heart disease and neurological dysfunction due to chromosome breaks and disabled DNA repair [12]. On the other hand, as seen in reference [13], the authors believe that caloric restriction (CR) is an 'intervention' that alters the activation of specific 'stress response genes', key enzymes in DNA repair pathways, which then results in upregulation of 'DNA repair' capacity. Enhanced DNA repair reduces the levels of DNA damage, consequently reducing mutation frequency, which would result in mainte‐ nance of genomic stability.

**3. Diet and cancer prevention**

ty.org/).

Current cancer models comprise those that are inherited through the germline and represent only ∼5% of total cases of human cancers. These tumors originate because of mutational events. The remaining ∼95% originate as sporadic events and evolve as a result of exposure to the environment, which includes exposure to both environmental contaminants and dietary agents. The multistage model of carcinogenesis identifies various phases, initiation, promo‐ tion, and progression, appears to be influenced by tissue microenvironment and organization. Significant opportunities in nutrition and cancer prevention exist in the early stages of initiation and promotion prior to clonal expansion of heterogeneous populations. Nutrige‐ nomics represents a strategy that can be applied to the study and prevention of many diseases including cancer. DNA methylation and histone modifications are epigenetic events that mediate heritable changes in gene expression and chromatin organization in the absence of changes in the DNA sequence. The age-increased susceptibility to cancer may derive from accumulation of epigenetic changes and represents a potential target for therapies with bioactive compounds. Factors that mediate the response to dietary factors include nuclear receptors and transcription factors, which function as sensors to dietary components and determine changes in the profile of transcripts [15]. Milner and Romagnolo [15] affirm that the opportunity of targeting nutrients–gene interactions to influence the cancer process is modu‐ lated by genetic variations in human populations, epigenetic modifications that selectively and permanently alter gene expression, by complex interactions/associations among dietary components, and heterogeneity of cells within a certain tumor. Therefore, integration of information about gene polymorphisms, identification of gene targets that regulate cell and

**Figure 3.** A general view of DNA insults and consequences on cell cycle and DNA repair (www.genomic-instabili‐

Nutrigenomics and Cancer Prevention http://dx.doi.org/10.5772/55429 397

Recommended dietary allowances (RDAs) of micronutrients have been traditionally defined as those levels necessary to prevent symptoms of deficiency diseases. There is increasing evidence that higher levels of many such micronutrients may be necessary for various DNA maintenance reactions, and that the current RDAs for some micronutrients may be inadequate to protect against genomic instability. Dietary imbalance may increase gene mutation and chromosome aberrations in human populations, similar to exposure to radiation, mutagens and carcinogens. Diet may well be a key factor in determining genomic stability since it impacts on all relevant pathways, i.e. exposure to dietary carcinogens, activation/detoxification of carcinogens, DNA repair, DNA synthesis and apoptosis, as mentioned previously. Many micronutrient minerals and vitamins act as substrates and/or co-factors in key DNA mainte‐ nance reactions, and the exact concentration of these in the cell may be critical. Sub-optimal levels of key micronutrients required for DNA maintenance will reduce genomic stability, producing similar effects to inherited genetic disorders or exposure to carcinogens [14].

**Figure 3.** A general view of DNA insults and consequences on cell cycle and DNA repair (www.genomic-instabili‐ ty.org/).

### **3. Diet and cancer prevention**

**2.1. Diet and genomic stability**

396 Cancer Treatment - Conventional and Innovative Approaches

Figure 3. www.genomic-instability.org/).

nance of genomic stability.

Eukaryotic DNA replication starts at multiple sites throughout the genome and is necessarily coordinated with transcription, sister chromatid cohesion, nucleosome assembly and cell cycle progression. In addition to the complexity of the replication reaction it, during replication cells need to deal with DNA damage and stalled forks, originated inevitably by the action of exogenous and endogenous agents. The success of this process is crucial to preserve genome stability, and the inability to deal with DNA lesions during replication or to protect or restart stalled forks leads to DNA breaks, chromosomal rearrangements, and mutations that can cause the loss of cell viability, but in addition errors in DNA replication result in a large number of human syndromes, including premature aging, various cancer predispositions and genetic abnormalities. To solve or reduce these problems, cells use repair and detoxification pathways as well as surveillance mechanisms, called checkpoints, which serve to detect the problem and coordinate repair with chromosome segregation and progression through the cell cycle (see

Maintaining genomic stability in the face of replication and recombination requires a huge variety of different damage response proteins. A cell's ability to decide when and where to

There is evidence that inappropriate nutrient supply can cause sizeable levels of genome mutation and alter expression of genes required for genome maintenance. Deficiencies in several micronutrients have been shown to cause DNA damage and are thought to be associated with a number of serious human diseases: folic acid, niacin, vitamin B6 and B12 deficiency may increase the risk of colon cancer, heart disease and neurological dysfunction due to chromosome breaks and disabled DNA repair [12]. On the other hand, as seen in reference [13], the authors believe that caloric restriction (CR) is an 'intervention' that alters the activation of specific 'stress response genes', key enzymes in DNA repair pathways, which then results in upregulation of 'DNA repair' capacity. Enhanced DNA repair reduces the levels of DNA damage, consequently reducing mutation frequency, which would result in mainte‐

Recommended dietary allowances (RDAs) of micronutrients have been traditionally defined as those levels necessary to prevent symptoms of deficiency diseases. There is increasing evidence that higher levels of many such micronutrients may be necessary for various DNA maintenance reactions, and that the current RDAs for some micronutrients may be inadequate to protect against genomic instability. Dietary imbalance may increase gene mutation and chromosome aberrations in human populations, similar to exposure to radiation, mutagens and carcinogens. Diet may well be a key factor in determining genomic stability since it impacts on all relevant pathways, i.e. exposure to dietary carcinogens, activation/detoxification of carcinogens, DNA repair, DNA synthesis and apoptosis, as mentioned previously. Many micronutrient minerals and vitamins act as substrates and/or co-factors in key DNA mainte‐ nance reactions, and the exact concentration of these in the cell may be critical. Sub-optimal levels of key micronutrients required for DNA maintenance will reduce genomic stability, producing similar effects to inherited genetic disorders or exposure to carcinogens [14].

deploy this DNA repair kit is critical to prevent tumor development [11].

Current cancer models comprise those that are inherited through the germline and represent only ∼5% of total cases of human cancers. These tumors originate because of mutational events. The remaining ∼95% originate as sporadic events and evolve as a result of exposure to the environment, which includes exposure to both environmental contaminants and dietary agents. The multistage model of carcinogenesis identifies various phases, initiation, promo‐ tion, and progression, appears to be influenced by tissue microenvironment and organization. Significant opportunities in nutrition and cancer prevention exist in the early stages of initiation and promotion prior to clonal expansion of heterogeneous populations. Nutrige‐ nomics represents a strategy that can be applied to the study and prevention of many diseases including cancer. DNA methylation and histone modifications are epigenetic events that mediate heritable changes in gene expression and chromatin organization in the absence of changes in the DNA sequence. The age-increased susceptibility to cancer may derive from accumulation of epigenetic changes and represents a potential target for therapies with bioactive compounds. Factors that mediate the response to dietary factors include nuclear receptors and transcription factors, which function as sensors to dietary components and determine changes in the profile of transcripts [15]. Milner and Romagnolo [15] affirm that the opportunity of targeting nutrients–gene interactions to influence the cancer process is modu‐ lated by genetic variations in human populations, epigenetic modifications that selectively and permanently alter gene expression, by complex interactions/associations among dietary components, and heterogeneity of cells within a certain tumor. Therefore, integration of information about gene polymorphisms, identification of gene targets that regulate cell and tissue specific pathways, and development of diagnostic strategies to control for clinical heterogeneity are important to understand how nutrigenomics may be used in cancer prevention.

**Study and year of**

Linxian, China, 1993

Linxian, China, 1993

Adapted from reference [16].

relative risk

**publication Agent Primary end point Relative risk**

ATBC, 1994 Alpha tocopherol Lung cancer 0.99 0.64\*\* for prostate

minerals\*\*\* Esophagus/cardias 0.98 PHS, 1996 Beta-carotene All cancers 0.98 0.95 for lung cancer

APPP, Australian Polyp Prevention Project; ATBC, Alpha Tocopherol Beta Carotene study; CARET, Carotene and Retinol Efficacy Trial; ECPOS, European Cancer Prevention Organisation Study Group; EUROSCAN, European Organization for Research and Treatment of Head and Neck Cancer and Lung Cancer Cooperative Group; NPCS, Nutritional Prevention of Skin Cancer; PHS, Physicians Health Study; PPS, Polyp Prevention Study Group; PPT, Polyp Prevention Trial; SCPS, Skin Cancer Prevention Study Group; SWCPS, Sothwest Skin Cancer Prevention Study; TPPT, Toronto Polyp Prevention Trial.

**Table 2.** Randomized controlled trials of dietary supplements to prevent cancer or colorectal adenomas, ordered by

American Cancer Society Guidelines on Nutrition and Physical Activity for Cancer Prevention [17] says that many epidemiologic studies have reported a modest but significant association

All cancers 0.93

Linxian, China, 1993 Retinol + Zn All cancers 1.00

EUROSCAN, 2000 Retinilpalmitate Lung cancer 1.00 Alberts et al., 2000 Cereal fiber Colon adenoma 0.99

14 vitamins + 12

Se + Vit E + betacarotene

Linxian, China, 1993 Riboflavin+niacin All cancers 0.95

Baron et al., 1999 Calcium Colon adenoma 0.83 SWCPS, 1997 Retinol Skin, squamous cell 0.74\*\* ECPOS, 2000 Calcium Colon adenoma 0.66

\*and low fat diet; \*\*P < 0.05; \*\*\*including selenium, vitamin E and beta-carotene.

**Relative risk for secondary end points** 399

0.96 for stomach

1.66\*\* for colon adenoma

0.83 for colorectal

1.26 for stomach

1.18\*\* for stomach

1.04 for stomach

0.79\*\* for stomach

0.91\*\* for total mortality

cancer

Nutrigenomics and Cancer Prevention http://dx.doi.org/10.5772/55429

cancer

cancer

cancer

cancer

cancer

cancer

Berrino, Krogh and Riboli [16] were made a review which showed an epidemiology studies on diet and cancer (see Table 2 that summarizes the results of the randomized studies pub‐ lished). The authors summarized (Table 3) the results of the World Cancer Research Fund (WCRF) evaluation on major foods and nutrients and major cancer sites. The 'probable' and 'possible' judgements provide a frame of hypotheses to be addressed in further studies. The overall pattern indicates that vegetarian food, except sugar and alcoholic beverages, is usually associated with cancer prevention, whereas animal food is frequently associated with cancer risk. The first WCRF dietary recommendation to reduce cancer, indeed, is: "Choose predom‐ inantly plant-based diets rich in a variety of vegetables and fruits, pulses (legumes) and minimally processed starchy staple foods". This seems to open a new perspective in nutrition and cancer research: from chemoprevention studies based on a single or a few micronutrients to an experimental strategy requiring a comprehensive modification of dietary habits.



\*and low fat diet; \*\*P < 0.05; \*\*\*including selenium, vitamin E and beta-carotene.

tissue specific pathways, and development of diagnostic strategies to control for clinical heterogeneity are important to understand how nutrigenomics may be used in cancer

Berrino, Krogh and Riboli [16] were made a review which showed an epidemiology studies on diet and cancer (see Table 2 that summarizes the results of the randomized studies pub‐ lished). The authors summarized (Table 3) the results of the World Cancer Research Fund (WCRF) evaluation on major foods and nutrients and major cancer sites. The 'probable' and 'possible' judgements provide a frame of hypotheses to be addressed in further studies. The overall pattern indicates that vegetarian food, except sugar and alcoholic beverages, is usually associated with cancer prevention, whereas animal food is frequently associated with cancer risk. The first WCRF dietary recommendation to reduce cancer, indeed, is: "Choose predom‐ inantly plant-based diets rich in a variety of vegetables and fruits, pulses (legumes) and minimally processed starchy staple foods". This seems to open a new perspective in nutrition and cancer research: from chemoprevention studies based on a single or a few micronutrients

to an experimental strategy requiring a comprehensive modification of dietary habits.

NPCS, 1996 Selenium Skin, squamous cell 1.14 0.50\*\* for all cancers

**Relative risk for secondary end points**

0.98 for colon adenoma

cancer

cancer

cancer

cancer

1.05 for colorectal

1.26 for stomach

1.23 for prostate

1.10 for stomach

**publication Agent Primary end point Relative risk**

ECPOS, 2000 Ispaghula fiber Colon adenoma 1.67\*\* APPP, 1995 Beta-carotene Colon adenoma 1.50\*\* CARET, 1996 Beta-carotene Lung cancer 1.28\*\* APPP, 1995 Cereal fibre Colon adenoma 1.20 TPPT, 1994 Cereal fibre\* Colon adenoma 1.20

ATBC, 1994 Beta-carotene Lung cancer 1.18\*\*

NPCS, 1996 Selenium Skin, basal cell 1.10 PPS, 1994 Vit C + Vit E Colon adenoma 1.08 SWCPS, 1997 Retinol Skin, basal cell 1.06

Linxian, China, 1993 Vit C + Mb All cancers 1.06

SCPS, 1990 Beta-carotene Skin 1.05 PPS, 1994 Beta-carotene Colon adenoma 1.01

prevention.

398 Cancer Treatment - Conventional and Innovative Approaches

**Study and year of**

APPP, Australian Polyp Prevention Project; ATBC, Alpha Tocopherol Beta Carotene study; CARET, Carotene and Retinol Efficacy Trial; ECPOS, European Cancer Prevention Organisation Study Group; EUROSCAN, European Organization for Research and Treatment of Head and Neck Cancer and Lung Cancer Cooperative Group; NPCS, Nutritional Prevention of Skin Cancer; PHS, Physicians Health Study; PPS, Polyp Prevention Study Group; PPT, Polyp Prevention Trial; SCPS, Skin Cancer Prevention Study Group; SWCPS, Sothwest Skin Cancer Prevention Study; TPPT, Toronto Polyp Prevention Trial. Adapted from reference [16].

**Table 2.** Randomized controlled trials of dietary supplements to prevent cancer or colorectal adenomas, ordered by relative risk

American Cancer Society Guidelines on Nutrition and Physical Activity for Cancer Prevention [17] says that many epidemiologic studies have reported a modest but significant association


possible that the fat content in meat contributes to risk through increasing the concentration of secondary bile acids and other compounds in the stool that could be carcinogens or

Nutrigenomics and Cancer Prevention http://dx.doi.org/10.5772/55429 401

According to Davis [18] epidemiologic evidence suggests that regular consumption of fruits, vegetables, and whole grains may reduce cancer risk in some individuals. This association has been attributed to these foods being rich sources of numerous bioactive compounds. Plant foods contain a variety of components, including, but not limited to, essential nutrients, polyunsaturated fatty acids, and phytochemicals such as glucosinolates and flavonoids, many of which can inhibit cell proliferation and induce apoptosis, and which may act additively or

Polyphenols are common constituents of foods of plant origin and major antioxidants of our diet. The main dietary sources of polyphenols are fruits and beverages. Fruits like apple, grape, pear, cherry, and various berries contain up to 200–300 mg polyphenols per 100 g fresh weight. Typically, a glass of red wine or a cup of tea or coffee contains about 100 mg polyphenols. Cereals, chocolate, and dry legumes also contribute to the polyphenol intake [19]. Red wine polyphenols, which consisted of various powerful antioxidants such as flavonoids and stilbenes, have been implicated in cancer prevention and that promote human health without recognizable side effects. Experimental studies have shown that polyphenols from red wine, like resveratrol, quercetin, (+)-catechin and gallic acid, were potential cancer chemopreventive agents. However, red wine contains a wide range of different polyphenols and protective effects have not been assigned to a specific fraction or compound, so it is not yet clear which compounds present in red wine are endowed with protective activity [20]. Among the most highly cited class of polyphenols are the flavonoids, which comprise a large and diverse family of compounds synthesized by plants. Flavonoid subclasses include anthocyanidins in berries and grapes, flavanols in tea, flavanones in citrus fruits, flavonols in onions, flavones in herbs

Zhou et al. [22] evaluated combined effects of soy phytochemical concentrate (SPC) and tea (green tea and black tea) components on the growth and metastasis of androgen-sensitive LNCaP human prostate cancer. The authors find that both black tea and green tea inhibited tumorigenicity rates of LNCaP tumors. For them the combination of soy phytochemicals and tea synergistically inhibited tumorigenicity, final tumor weight and metastasis to lymph nodes in vivo. This study supports further investigations using soy and tea combinations as effective nutritional regimens for prevention of prostate cancer. According to authors, studies of tea polyphenols suggest that epigallocatechin gallate (EGCG) is the major bioactive component in green tea and less is present in black tea. Black tea also contains other tea polyphenols such as theaflavins and thearubigins. They also affirm that chemopreventive properties of the soy

Lambert and Yang [23] affirm that although numerous health benefits have been proposed for the consumption of tea, the effectiveness of tea as a cancer preventive agent in humans remains unclear. Animal models of carcinogenesis may be different from the human situation (e.g., the

isoflavone genistein have been the subject of extensive in vitro and in vivo.

promoters of carcinogenesis [17].

**3.1. Polyphenols**

synergistically when combined in the human diet.

and peppers, and isoflavones in soy [21].

Increased risk: +++, convincing; ++, probable; +, possible; decreased risk: – – –, convincing; – –, probable; –, possible. Data adapted from reference [16].

**Table 3.** Matrix summary of the WCRF/AICR judgments on the role of various foods in the risk of cancer

between high intakes of processed meats (such as bacon, sausage, luncheon meats) and red meats (defined as beef, pork, or lamb) and increases in cancer incidence and mortality as well as death from other causes. The American Cancer Society says that current evidence supports approximately a 15% to 20% increased risk of cancers of the colon and/or rectum per 100 grams (g) of red meat or 50 g of processed meat consumed per day, while the evidence for some other cancers (those of the esophagus, stomach, lung, pancreas, breast, prostate, stomach, endome‐ trium, renal, and ovarian) is considered limited and suggestive. According to American Cancer Society meat contains several constituents that could increase the risk of cancer. Mutagens and carcinogens (heterocyclic amines and polycyclic aromatic hydrocarbons) are produced by cooking meat at high temperatures and/or by charcoal grilling. Nitrates/nitrites and salt used to process meat contribute to the formation of nitrosamines, which are known mutagens and carcinogens in animals. Iron from the heme group of myoglobin in red meat may act as a catalyst to nitrosamine formation, and generate free radicals that may damage DNA. It is also possible that the fat content in meat contributes to risk through increasing the concentration of secondary bile acids and other compounds in the stool that could be carcinogens or promoters of carcinogenesis [17].

According to Davis [18] epidemiologic evidence suggests that regular consumption of fruits, vegetables, and whole grains may reduce cancer risk in some individuals. This association has been attributed to these foods being rich sources of numerous bioactive compounds. Plant foods contain a variety of components, including, but not limited to, essential nutrients, polyunsaturated fatty acids, and phytochemicals such as glucosinolates and flavonoids, many of which can inhibit cell proliferation and induce apoptosis, and which may act additively or synergistically when combined in the human diet.

### **3.1. Polyphenols**

between high intakes of processed meats (such as bacon, sausage, luncheon meats) and red meats (defined as beef, pork, or lamb) and increases in cancer incidence and mortality as well as death from other causes. The American Cancer Society says that current evidence supports approximately a 15% to 20% increased risk of cancers of the colon and/or rectum per 100 grams (g) of red meat or 50 g of processed meat consumed per day, while the evidence for some other cancers (those of the esophagus, stomach, lung, pancreas, breast, prostate, stomach, endome‐ trium, renal, and ovarian) is considered limited and suggestive. According to American Cancer Society meat contains several constituents that could increase the risk of cancer. Mutagens and carcinogens (heterocyclic amines and polycyclic aromatic hydrocarbons) are produced by cooking meat at high temperatures and/or by charcoal grilling. Nitrates/nitrites and salt used to process meat contribute to the formation of nitrosamines, which are known mutagens and carcinogens in animals. Iron from the heme group of myoglobin in red meat may act as a catalyst to nitrosamine formation, and generate free radicals that may damage DNA. It is also

**Table 3.** Matrix summary of the WCRF/AICR judgments on the role of various foods in the risk of cancer

Increased risk: +++, convincing; ++, probable; +, possible; decreased risk: – – –, convincing; – –, probable; –, possible. Data

Prostate – + +

Kidney – + +

**Vegetables Fruits**

400 Cancer Treatment - Conventional and Innovative Approaches

Gallbladder

Cervix – – Endometrium – – Ovary – –

Bladder – – – –

Thyroid –

adapted from reference [16].

Mouth, pharynx – – – – – – +++

Esophagus – – – – – – + +++

Liver – +++

Larynx – – – – +++ Lung – – – – – – +

**Grains, fibers**

Nasopharynx +++

Stomach – – – – – – – – ++

Pancreas – – – – – +

Colon, rectum – – – – ++ +

Breast – – – – – ++ +

**Tea Sugar Alcohol**

**Salt & salting**

**Meat Eggs**

**Milk & dairy**

> Polyphenols are common constituents of foods of plant origin and major antioxidants of our diet. The main dietary sources of polyphenols are fruits and beverages. Fruits like apple, grape, pear, cherry, and various berries contain up to 200–300 mg polyphenols per 100 g fresh weight. Typically, a glass of red wine or a cup of tea or coffee contains about 100 mg polyphenols. Cereals, chocolate, and dry legumes also contribute to the polyphenol intake [19]. Red wine polyphenols, which consisted of various powerful antioxidants such as flavonoids and stilbenes, have been implicated in cancer prevention and that promote human health without recognizable side effects. Experimental studies have shown that polyphenols from red wine, like resveratrol, quercetin, (+)-catechin and gallic acid, were potential cancer chemopreventive agents. However, red wine contains a wide range of different polyphenols and protective effects have not been assigned to a specific fraction or compound, so it is not yet clear which compounds present in red wine are endowed with protective activity [20]. Among the most highly cited class of polyphenols are the flavonoids, which comprise a large and diverse family of compounds synthesized by plants. Flavonoid subclasses include anthocyanidins in berries and grapes, flavanols in tea, flavanones in citrus fruits, flavonols in onions, flavones in herbs and peppers, and isoflavones in soy [21].

> Zhou et al. [22] evaluated combined effects of soy phytochemical concentrate (SPC) and tea (green tea and black tea) components on the growth and metastasis of androgen-sensitive LNCaP human prostate cancer. The authors find that both black tea and green tea inhibited tumorigenicity rates of LNCaP tumors. For them the combination of soy phytochemicals and tea synergistically inhibited tumorigenicity, final tumor weight and metastasis to lymph nodes in vivo. This study supports further investigations using soy and tea combinations as effective nutritional regimens for prevention of prostate cancer. According to authors, studies of tea polyphenols suggest that epigallocatechin gallate (EGCG) is the major bioactive component in green tea and less is present in black tea. Black tea also contains other tea polyphenols such as theaflavins and thearubigins. They also affirm that chemopreventive properties of the soy isoflavone genistein have been the subject of extensive in vitro and in vivo.

> Lambert and Yang [23] affirm that although numerous health benefits have been proposed for the consumption of tea, the effectiveness of tea as a cancer preventive agent in humans remains unclear. Animal models of carcinogenesis may be different from the human situation (e.g., the

doses of tea and tea components used in animal studies are often much higher than those consumed by humans), and many confounding factors are involved in epidemiological studies. Interindividual variation in biotransformation and bioavailability may also affect the efficacy of tea as a cancer preventive agent. For them further studies on definitive mechanisms of cancer preventive activities of tea in animal models are needed. Although many possible mechanisms have been proposed, their relevance in vivo needs to be demonstrated. With some exceptions, the concentrations of catechins or theaflavins used in cell culture systems exceed the plasma concentrations obtained in animal studies by 10- to 100-fold. Mechanisms based on the use of such high concentrations may be relevant for cancers of the gastrointestinal tract but not for sites such as the lung, prostate and breast, which depend on systemic bioavailability. In spite of many in vitro and in vivo studies, the molecular mechanisms for the cancer preventive actions of these compounds are not clearly known. The relationship between tea consumption and cancer risk has not been conclusively demonstrated, and the relationship may become clearer if we consider the effects of specific types of tea, at defined doses, in populations with certain dietary patterns or genetic polymorphisms. Human intervention trials and large prospective studies are needed to further assess cancer preventive activities of tea constituents [24]. For the National Cancer Institute [25] more than 50 epidemiologic studies of the association between tea consumption and cancer risk have been published since 2006. The results of these studies have often been inconsistent, but some have linked tea consump‐ tion to reduced risks of cancers of the colon, breast, ovary, prostate, and lung. They also believe that the inconsistent results may be due to variables such as differences in tea preparation and consumption, the types of tea studied (green, black, or both), the methods of tea production, the bioavailability of tea compounds, genetic variation in how people respond to tea con‐ sumption, the concomitant use of tobacco and alcohol, and other lifestyle factors that may influence a person's risk of developing cancer, such as physical activity or weight status.

lipophilic antioxidants whereas vitamin C is hydrophilic antioxidant. Vitamin E function as a free radical chain breaker particularly it interferes with the propagation step of lipid peroxi‐ dation. Vitamin A and Beta-carotene have actions by quenching both singlet oxygen and other

Nutrigenomics and Cancer Prevention http://dx.doi.org/10.5772/55429 403

The changes in the DNA by a deficiency of some micronutrients (folic acid, vitamin B12, vitamin B6, niacin, vitamin C, vitamin E, iron and zinc) are considered as the most likely cause

Studies investigating the interactions between dietary exposure and genetic polymor‐ phisms have the potential to clarify mechanisms and identify susceptible subgroups so that preventative strategies can be focused on the subgroups for maximum benefit. Red meat or meat cooking methods such as frying and doneness levels have been associated with the increased risk of colorectal and other cancers [30]. It is not clear whether it is red meat intake or the way meat is cooked that is involved in the etiology of colorectal cancer, as stated above. Both cooking methods and doneness level of red meat are thought to be surrogates for heterocyclic amines (HCA) consumption [31]. Sinha and Caporaso [31] affirm that genetics polymorphisms may interact with various dietary components and thus define subgroups of individuals who may be at a higher risk of getting cancer. For them there are also other polymorphic enzymes that may interact with various dietary components and play a role in human carcinogenesis. The authors describe categories of susceptibility genes, potential dietary carcinogens and anticarcinogens, and cancer sites in which they may be involved (see Table 4). Many studies are currently investigating the role of circulating vitamin D metabolites and dietary calcium. Because the vitamin D receptor is involved in vitamin D and calcium metabolism, the vitamin D receptor polymorphisms may also be important for colorectal cancers. Martinez et al. [32] investigated the associations between the intake of calcium and vitamin D and the occurrence of colorectal cancer. They found that vitamin D is suggestive of an inverse association, particularly for total vitamin D in relation to rectal cancer. However, since most of the support for this protective effect was seen for total vitamin D. They not rule out the possibility that something other than vitamin D in multivitamin supplements contributes to this apparent effect. The relation between vitamin D and colorectal cancer may be better elucidated with additional dietary measure‐ ments and further follow-up. They conclude that available evidence does not warrant an increase in calcium intake to prevent colon cancer, but longer-term studies of both calcium

and especially vitamin D in relation to colorectal cancer risk are needed.

Carotenoids are the pigments that give fruits and vegetables such as carrots, cantaloupe, sweet potato, and kale their vibrant orange, yellow, and green colors. Beta-carotene, lycopene, and lutein are all different varieties of carotenoids. They all act as antioxidants with strong cancerfighting properties. Preclinical studies have shown that some carotenoids have potent antitumor effects both in vitro and in vivo, suggesting potential preventive and/or therapeutic roles for the compounds. Since chemoprevention is one of the most important strategies in the control of cancer development, molecular mechanism-based cancer chemoprevention using carotenoids seems to be an attractive approach [33]. Epidemiologic studies have shown an

free radicals generated by photochemical reactions [28].

of some types of cancer [29].

A double-blind intervention trial conducted in patients with oral mucosa leukoplakia using a mixed tea showed some direct evidence on the protective effects of tea on oral cancer. In this study developed by Li et al. [26] fifty-nine oral mucosa leukoplakia patients, diagnosed by established clinical and pathological criteria, were randomly divided into a treated group (3 g mixed tea oral administration and topical treatment) and a control group (placebo and glycerin treatment). After the 6-month trial, the size of oral lesion was decreased in 37.9% of the 29 treated patients and increased in 3.4%; whereas the oral lesion was decreased in 10.0% of the 30 control patients and increased in 6.7%.

#### **3.2. Vitamins and micronutrients**

Natural inhibitors of oxidizing agents that are found in the diet are important in preventing cancer and typically do not have the undesirable side effects of many xenobiotic compounds. Some vitamins, such as the antioxidant Vitamins A, E, and C, demonstrate these protective effects. The daily ingestion of antioxidants has the potential of not only protecting against cancer, but also cardiovascular disorders and neurological degenerative diseases [28]. Anti‐ oxidants nutrients such as vitamin E, vitamin C, vitamin A, and Beta-carotene are involved in detoxification of the Reactive oxygen species (ROS). Vitamin E, A, and Beta-carotene are lipophilic antioxidants whereas vitamin C is hydrophilic antioxidant. Vitamin E function as a free radical chain breaker particularly it interferes with the propagation step of lipid peroxi‐ dation. Vitamin A and Beta-carotene have actions by quenching both singlet oxygen and other free radicals generated by photochemical reactions [28].

doses of tea and tea components used in animal studies are often much higher than those consumed by humans), and many confounding factors are involved in epidemiological studies. Interindividual variation in biotransformation and bioavailability may also affect the efficacy of tea as a cancer preventive agent. For them further studies on definitive mechanisms of cancer preventive activities of tea in animal models are needed. Although many possible mechanisms have been proposed, their relevance in vivo needs to be demonstrated. With some exceptions, the concentrations of catechins or theaflavins used in cell culture systems exceed the plasma concentrations obtained in animal studies by 10- to 100-fold. Mechanisms based on the use of such high concentrations may be relevant for cancers of the gastrointestinal tract but not for sites such as the lung, prostate and breast, which depend on systemic bioavailability. In spite of many in vitro and in vivo studies, the molecular mechanisms for the cancer preventive actions of these compounds are not clearly known. The relationship between tea consumption and cancer risk has not been conclusively demonstrated, and the relationship may become clearer if we consider the effects of specific types of tea, at defined doses, in populations with certain dietary patterns or genetic polymorphisms. Human intervention trials and large prospective studies are needed to further assess cancer preventive activities of tea constituents [24]. For the National Cancer Institute [25] more than 50 epidemiologic studies of the association between tea consumption and cancer risk have been published since 2006. The results of these studies have often been inconsistent, but some have linked tea consump‐ tion to reduced risks of cancers of the colon, breast, ovary, prostate, and lung. They also believe that the inconsistent results may be due to variables such as differences in tea preparation and consumption, the types of tea studied (green, black, or both), the methods of tea production, the bioavailability of tea compounds, genetic variation in how people respond to tea con‐ sumption, the concomitant use of tobacco and alcohol, and other lifestyle factors that may influence a person's risk of developing cancer, such as physical activity or weight status.

A double-blind intervention trial conducted in patients with oral mucosa leukoplakia using a mixed tea showed some direct evidence on the protective effects of tea on oral cancer. In this study developed by Li et al. [26] fifty-nine oral mucosa leukoplakia patients, diagnosed by established clinical and pathological criteria, were randomly divided into a treated group (3 g mixed tea oral administration and topical treatment) and a control group (placebo and glycerin treatment). After the 6-month trial, the size of oral lesion was decreased in 37.9% of the 29 treated patients and increased in 3.4%; whereas the oral lesion was decreased in 10.0% of the

Natural inhibitors of oxidizing agents that are found in the diet are important in preventing cancer and typically do not have the undesirable side effects of many xenobiotic compounds. Some vitamins, such as the antioxidant Vitamins A, E, and C, demonstrate these protective effects. The daily ingestion of antioxidants has the potential of not only protecting against cancer, but also cardiovascular disorders and neurological degenerative diseases [28]. Anti‐ oxidants nutrients such as vitamin E, vitamin C, vitamin A, and Beta-carotene are involved in detoxification of the Reactive oxygen species (ROS). Vitamin E, A, and Beta-carotene are

30 control patients and increased in 6.7%.

402 Cancer Treatment - Conventional and Innovative Approaches

**3.2. Vitamins and micronutrients**

The changes in the DNA by a deficiency of some micronutrients (folic acid, vitamin B12, vitamin B6, niacin, vitamin C, vitamin E, iron and zinc) are considered as the most likely cause of some types of cancer [29].

Studies investigating the interactions between dietary exposure and genetic polymor‐ phisms have the potential to clarify mechanisms and identify susceptible subgroups so that preventative strategies can be focused on the subgroups for maximum benefit. Red meat or meat cooking methods such as frying and doneness levels have been associated with the increased risk of colorectal and other cancers [30]. It is not clear whether it is red meat intake or the way meat is cooked that is involved in the etiology of colorectal cancer, as stated above. Both cooking methods and doneness level of red meat are thought to be surrogates for heterocyclic amines (HCA) consumption [31]. Sinha and Caporaso [31] affirm that genetics polymorphisms may interact with various dietary components and thus define subgroups of individuals who may be at a higher risk of getting cancer. For them there are also other polymorphic enzymes that may interact with various dietary components and play a role in human carcinogenesis. The authors describe categories of susceptibility genes, potential dietary carcinogens and anticarcinogens, and cancer sites in which they may be involved (see Table 4). Many studies are currently investigating the role of circulating vitamin D metabolites and dietary calcium. Because the vitamin D receptor is involved in vitamin D and calcium metabolism, the vitamin D receptor polymorphisms may also be important for colorectal cancers. Martinez et al. [32] investigated the associations between the intake of calcium and vitamin D and the occurrence of colorectal cancer. They found that vitamin D is suggestive of an inverse association, particularly for total vitamin D in relation to rectal cancer. However, since most of the support for this protective effect was seen for total vitamin D. They not rule out the possibility that something other than vitamin D in multivitamin supplements contributes to this apparent effect. The relation between vitamin D and colorectal cancer may be better elucidated with additional dietary measure‐ ments and further follow-up. They conclude that available evidence does not warrant an increase in calcium intake to prevent colon cancer, but longer-term studies of both calcium and especially vitamin D in relation to colorectal cancer risk are needed.

Carotenoids are the pigments that give fruits and vegetables such as carrots, cantaloupe, sweet potato, and kale their vibrant orange, yellow, and green colors. Beta-carotene, lycopene, and lutein are all different varieties of carotenoids. They all act as antioxidants with strong cancerfighting properties. Preclinical studies have shown that some carotenoids have potent antitumor effects both in vitro and in vivo, suggesting potential preventive and/or therapeutic roles for the compounds. Since chemoprevention is one of the most important strategies in the control of cancer development, molecular mechanism-based cancer chemoprevention using carotenoids seems to be an attractive approach [33]. Epidemiologic studies have shown an


1 Abbreviations used: NAT, *N*-acetyltminsferase; CYP, cytochrome p450; GST, glutathione-S-transferase; EPHX, epoxide hydrolase; ADH, alcohol dehydrogenase; MTHFR, metheylenetetrahydrofolate reductase. Adapted from reference [31].

#### **Table 4.** Polymorphic genes, dietary components and cancer: possible candidates

inverse relationship between the presence of various cancers and dietary or blood carotenoid levels. According to Tanaka, Shnimizu and Moriwaki [33] the epidemiologic observations of the possible protective effects of high dietary (not supplemental) β-carotene intakes against cancer, along with what is known about carotenoid biochemical functions, has led to further study of the effect of β-carotene on cancer risk. Long-term large randomized intervention trials were designed to test the efficacy of high doses of β-carotene (20–30 mg/day) in the prevention of cancer. These results are summarized in Table 5.

**Figure 4.** Proposed mechanisms by which certain carotenoids suppress carcinogenesis. Adapted from reference [33].

**Study Designs**

18% increase in lung cancer; 8% increase in

Nutrigenomics and Cancer Prevention http://dx.doi.org/10.5772/55429 405

28% increase in lung cancer; 17% increase in

incidence of cancer

13% decrease in total cancers; 9% decrease in

overall deaths

No effect of supplementation in incidence of cancer

No effect of supplementation

mortality

deaths

in

**Studies Population Intervention Duration Cancer outcome**

vitamin E, 50 mg/day 5–8 years

vitamin A, 25,000 IU <4 years

alternate days 12 years

5 years

4.1 years (2.1 years' treatment and 2.0 years' follow-up)

β-carotene, 20 mg/day;

β-carotene, 30 mg/day;

β-carotene, 50 mg on

β-carotene, 15 mg/day; selenium, 50 mg/day; α-tocopherol, 30 mg/day

β-carotene, 50 mg on alternate days

CARET, Beta-Carotene and Retinol Efficacy Trial; ATBC, Alpha Tocopherol and Beta-Carotene Cancer Prevention; PHS,

ATBC

CARET

Linxian

Women's Health Study

29,133 Finish male smokers (50–69 years

18,314 men and women and asbestoss workers (45–74 years of age)

(40–84 years of age)

39,876 female health professionals (over 45 years of age)

29,584 men and women, vitamin and mineral deficient (40–69 years of age)

of age)

PHS 22,071 male physicians

Data adapted from reference [33].

**Table 5.** β-Carotene supplementation trials.

Physicians' Health Study.


Data adapted from reference [33].

inverse relationship between the presence of various cancers and dietary or blood carotenoid levels. According to Tanaka, Shnimizu and Moriwaki [33] the epidemiologic observations of the possible protective effects of high dietary (not supplemental) β-carotene intakes against cancer, along with what is known about carotenoid biochemical functions, has led to further study of the effect of β-carotene on cancer risk. Long-term large randomized intervention trials were designed to test the efficacy of high doses of β-carotene (20–30 mg/day) in the prevention

1 Abbreviations used: NAT, *N*-acetyltminsferase; CYP, cytochrome p450; GST, glutathione-S-transferase; EPHX, epoxide hydrolase; ADH, alcohol dehydrogenase; MTHFR, metheylenetetrahydrofolate reductase. Adapted from reference [31].

gene/phenotype1 **Cancer site**

NAT2, (NAT1), CYP1A2 Colorectal, breast,

Retinoic acid receptor Head and neck,

stomach

Colorectal, other sites

Acute promylocytic Leukemia, skin,

Heterocyclic amines (CYP1A1) other sites

Aflatoxins GSTM1, EPHX Liver

Alcohol ADH (ALDH, CYP2E1) Colorectal, oral

Fruits and vegetables CYP1A2, GST Many sites

Retinoids Variant breast MTHFR, Methionine Folate, methionine Synthase Colorectal, cervix

Calcium/vitamin D Vitamin D receptor Colorectal, prostate

Polycyclic hydrocarbons CYP1A1, GSTM1 Gastrointestinal tract Nasophyrangeal,

of cancer. These results are summarized in Table 5.

**Table 4.** Polymorphic genes, dietary components and cancer: possible candidates

**Dietary component** Polymorphic

404 Cancer Treatment - Conventional and Innovative Approaches

Nitrosamines CYP2E1

vegetables CYP1A2, GST

Carcinogens

Anticarcinogens

Cruciferous

CARET, Beta-Carotene and Retinol Efficacy Trial; ATBC, Alpha Tocopherol and Beta-Carotene Cancer Prevention; PHS, Physicians' Health Study.

**Table 5.** β-Carotene supplementation trials.

**Figure 4.** Proposed mechanisms by which certain carotenoids suppress carcinogenesis. Adapted from reference [33].

The authors [33] wrote an important review which showed a table (Table 6) about cancer prevention by means of carotenoids with dietary sources, function e effects. According to these authors the mechanisms underlying the anticancer and/or cancer chemopreventive activities of carotenoids may involve changes in pathways leading to cell growth or cell death. These include immune modulation, hormone and growth factor signaling, regulatory mechanisms of cell cycle progression, cell differentiation and apoptosis. In this sense the authors also showed an interesting figure proposing possible mechanisms by which certain carotenoids suppress carcinogenesis (see Figure 4 on the left).

**Carotenoids Dietary Sources Function Effects**

Provitamin A activity; Anti-oxidant

Provitamin A activity; Antioxidant

Anti-oxidant

Provitamin A activity; Anti-oxidant

Anti-photosensitizing

Antioxidant; Coloration

agent and photosynthetic pigment; Acts as antioxidants and blue

light filters

Canthaxanthin Salmon, crustacean Antioxidant; Coloration Immune enhancement; Decreases

Immune- enhancement; Stimulate cell to cell communication; Decreases risk of some cancers

Nutrigenomics and Cancer Prevention http://dx.doi.org/10.5772/55429 407

Immune-enhancement; Decreases risk of some cancers and some cardiovascular events; high-dose supplementation may increase the risk of lung cancer among smokers

Decreases risk of some cancers and some cardiovascular events, diabetes, and osteoporosis

Anti-inflammatory effects; Inhibits

risks of some cancer and cardiovascular events; Immune

Decrease age-related macular degeneration, cataract, and risk of cardiovascular disease and certain

Prevent certain cancers, cataract, diabetes, and inflammatory neurodegenerative and cardiovascular diseases

Anti-cancer, anti-allergic, anti-obese,

risk of some cancers

anti-inflammatory, and anti-osteoporotic activities

enhancemen

cancers

Yellow-orange vegetables (carrots, sweet totatoes, pumpkin) and Dark-green vegetables (broccoli, green

Green leafy vegetables and orange and yellow fruits and vegetables (carrots, apricots, spinach, sweet potetoes, pumpkin, pepper, kale,

Tomatoes, water melon, apricot, peaches

Orange fruits (mandarin orange and papaya, etc.), corn, peas, and egg yolks

Dark green leafy vegetables (spinach, kale), red peppers, maize, tomatoes, corn, and

egg yolks

Focoxanthin Brown algae, heterokonts Antioxidant

**Table 6.** Sources, function, and effects of different carotenoids.

Astaxanthin Green algae, salmon, trout, Crustacean

beans, spinach)

cantaloupe)

α-Carotene

β-Carotene

Lycopene

β-Cyptoxanthin

Lutein/Zeaxanthin

Adapted from reference [33].

Studies involving the use of vitamin C in cancer prevention are the most contradictory. Vitamin C is an essential vitamin the human body needs to function well. It is a water-soluble vitamin that cannot be made by the body, and must be obtained from foods or other sources. Vitamin C is found in abundance in citrus fruits such as oranges, grapefruit, and lemons, and in green leafy vegetables, potatoes, strawberries, bell peppers, and cantaloupe. American Cancer Society [34] wrote that many studies have shown a connection between eating foods rich in vitamin C, such as fruits and vegetables, and a reduced risk of cancer. On the other hand, evidence indicates that vitamin C supplements do not reduce cancer risk. This suggests that the activity of fruits and vegetables in preventing cancer is due to a combination of many vitamins and other phytochemicals and not to vitamin C alone. Clinical trials of high doses vitamin C as a treatment for cancer have not shown any benefit. High doses of vitamin C can cause a number of side effects.

According to Block [35] epidemiologic evidence of a protective effect of vitamin C for nonhormone-dependent cancers is strong. Of the 46 such studies in which a dietary vitamin C index was calculated, 33 found statistically significant protection, with high intake conferring approximately a twofold protective effect compared with low intake. 0f 29 additional studies that assessed fruit intake, 2 1 found significant protection. For cancers of the esophagus, larynx, oral cavity, and pancreas, evidence for a protective effect of vitamin C or some component in fruit is strong and consistent. For cancers of the stomach, rectum, breast, and cervix there is also strong evidence. Several recent lung cancer studies found significant protective effects of vitamin C or of foods that are better sources of vitamin C than of /3-carotene. It is likely that ascorbic acid, carotenoids, and other factors in fruits and vegetables act jointly.

Several lines of evidence suggest that vitamin C is a powerful antioxidant in biological systems in vitro. However, its antioxidant role in humans has not been supported by currently available clinical studies. Diets high in fruits and vegetables protect against cardiovascular disease and cancer, but such a protective effect cannot as yet be ascribed to vitamin C. In vivo markers of oxidative damage are being developed, and these have yet not shown major changes with vitamin C intake in humans [36]. The most important problem about vitamin C is that it can exert a pro-oxidant activity under certain conditions, particularly in the presence of transition metal ions or alkali. Thus, vitamin C *in vitro* reduces free ferric iron that generates hydrogen peroxide in the Fenton reaction and results in the production of hydroxyl radicals. The reactive hydroxyl radical quickly reacts with critical cellular macromolecules, including DNA, which may lead to mutagenesis and the initiation of cancer [37]. According to authors, the high


**Table 6.** Sources, function, and effects of different carotenoids.

The authors [33] wrote an important review which showed a table (Table 6) about cancer prevention by means of carotenoids with dietary sources, function e effects. According to these authors the mechanisms underlying the anticancer and/or cancer chemopreventive activities of carotenoids may involve changes in pathways leading to cell growth or cell death. These include immune modulation, hormone and growth factor signaling, regulatory mechanisms of cell cycle progression, cell differentiation and apoptosis. In this sense the authors also showed an interesting figure proposing possible mechanisms by which certain carotenoids

Studies involving the use of vitamin C in cancer prevention are the most contradictory. Vitamin C is an essential vitamin the human body needs to function well. It is a water-soluble vitamin that cannot be made by the body, and must be obtained from foods or other sources. Vitamin C is found in abundance in citrus fruits such as oranges, grapefruit, and lemons, and in green leafy vegetables, potatoes, strawberries, bell peppers, and cantaloupe. American Cancer Society [34] wrote that many studies have shown a connection between eating foods rich in vitamin C, such as fruits and vegetables, and a reduced risk of cancer. On the other hand, evidence indicates that vitamin C supplements do not reduce cancer risk. This suggests that the activity of fruits and vegetables in preventing cancer is due to a combination of many vitamins and other phytochemicals and not to vitamin C alone. Clinical trials of high doses vitamin C as a treatment for cancer have not shown any benefit. High doses of vitamin C can

According to Block [35] epidemiologic evidence of a protective effect of vitamin C for nonhormone-dependent cancers is strong. Of the 46 such studies in which a dietary vitamin C index was calculated, 33 found statistically significant protection, with high intake conferring approximately a twofold protective effect compared with low intake. 0f 29 additional studies that assessed fruit intake, 2 1 found significant protection. For cancers of the esophagus, larynx, oral cavity, and pancreas, evidence for a protective effect of vitamin C or some component in fruit is strong and consistent. For cancers of the stomach, rectum, breast, and cervix there is also strong evidence. Several recent lung cancer studies found significant protective effects of vitamin C or of foods that are better sources of vitamin C than of /3-carotene. It is likely that

Several lines of evidence suggest that vitamin C is a powerful antioxidant in biological systems in vitro. However, its antioxidant role in humans has not been supported by currently available clinical studies. Diets high in fruits and vegetables protect against cardiovascular disease and cancer, but such a protective effect cannot as yet be ascribed to vitamin C. In vivo markers of oxidative damage are being developed, and these have yet not shown major changes with vitamin C intake in humans [36]. The most important problem about vitamin C is that it can exert a pro-oxidant activity under certain conditions, particularly in the presence of transition metal ions or alkali. Thus, vitamin C *in vitro* reduces free ferric iron that generates hydrogen peroxide in the Fenton reaction and results in the production of hydroxyl radicals. The reactive hydroxyl radical quickly reacts with critical cellular macromolecules, including DNA, which may lead to mutagenesis and the initiation of cancer [37]. According to authors, the high

ascorbic acid, carotenoids, and other factors in fruits and vegetables act jointly.

suppress carcinogenesis (see Figure 4 on the left).

406 Cancer Treatment - Conventional and Innovative Approaches

cause a number of side effects.

consumption of vitamin C–rich fruit and vegetables is not likely to be harmful. In general, data from in vitro and in vivo experiments and population-based studies do not indicate that high doses of vitamin C are linked to increased oxidative DNA damage or an elevated risk of cancer.

vitamin C therapy is not effective against advanced malignant disease regardless of whether

Nutrigenomics and Cancer Prevention http://dx.doi.org/10.5772/55429 409

The terms folic acid and folate are often used interchangeably for this water-soluble B-complex vitamin. Folic acid, the more stable form, occurs rarely in foods or the human body but is the form most often used in vitamin supplements and fortified foods. Folic acid is essential to numerous bodily functions ranging from nucleotide biosynthesis to the remethylation of homocysteine. The human body needs folate to synthesize DNA, repair DNA, and methylate

Considerable epidemiological evidence suggests that a low-folate diet is associated with an increased risk of colorectal neoplasia. Much animal data support an antineoplastic effect of folate. However, in some animal studies, folate deficiency protects against, and supplemen‐ tation increases, experimental carcinogenesis. Cole et al. [39] developed a double-blind, placebo-controlled, 2-factor, phase 3, randomized clinical trial conducted at 9 clinical centers between July 6, 1994, and October 1, 2004. Participants included 1021 men and women with a recent history of colorectal adenomas and no previous invasive large intestine carcinoma. Participants were randomly assigned in a 1:1 ratio to receive 1 mg/d of folic acid (n=516) or placebo (n=505), and were separately randomized to receive aspirin (81 or 325 mg/d) or placebo. Follow-up consisted of 2 colonoscopic surveillance cycles (the first interval was at 3 years and the second at 3 or 5 years later). In this double-blind, placebo-controlled, randomized clinical trial, was found that folic acid supplementation did not decrease the risk of adenoma occurrence among participants with a recent history of adenomas. The authors concluded that folate, when administered as folic acid for up to 6 years, does not decrease the risk of adenoma formation in the large intestine among individuals with previously removed adenomas. For them, the evidence for an increased risk of adenomas is equivocal and requires further research. In March of 1996, the U.S. Food and Drug Administration mandated that all enriched flour and uncooked cereal grains sold in the United States should be fortified with 140 µg folic acid/ 100 g of flour no later than January of 1998. Following the institution of fortification populationbased studies showed the effectiveness of this measure: plasma levels of folate in the adult population increased ~2-fold as a result and the incidence of births complicated by neural tube defects was variously reported to decline by 20% to 50%. However, analyses of several cereal grains that were purchased after the institution of fortification showed that in many instances the actual amount of folate was 150% to 300% greater than the mandate, suggesting that in this early era of fortification, manufacturers often included ''overage'' to ensure that they were meeting the minimal level of mandated fortification [40]. Thus, the authors hypothesize, by means of an epidemiological study, that the institution of folic acid fortification may have been wholly or partly responsible for the observed increase in colorectal cancer rates in the mid-1990s. The authors affirm that wish to highlight the potential complexity of the response to this nutrient and emphasize prior observations that have been made in both preclinical and clinical studies that indicate that administering high doses of folic acid to susceptible individ‐ uals or in an inappropriate time frame may accelerate the growth of existing neoplasms.

Vitamin C, also known as ascorbic acid, is a water-soluble vitamin. Unlike most mammals and other animals, humans do not have the ability to make their own vitamin C. Therefore, we

DNA as well as to act as a cofactor in biological reactions involving folate.

the patient has had any prior chemotherapy [38].

Lee et al. [37] believe that the cancer preventive effects of vegetables and fruit may result from multiple combined effects of various phenolic phytochemicals, vitamins, dietary fibers, indoles, allium compounds, and selenium rather than from the effect of a single active ingredient. For them, many dietary phenolic phytochemicals may have stronger antioxidant and antitumor promotion effects than do antioxidant vitamins, which may contribute to the chemopreventive effects of the phytochemicals in carcinogenesis. However, these authors suggest that the chemopreventive effects of vitamin C in carcinogenesis may be linked to the protective effects of vitamin C against epigenetic mechanisms, such as the inflammation and inhibition of gap junction intercellular communication (GJIC), as well as to antioxidant activities (see Figure 5).

**Figure 5.** Possible chemopreventive mechanisms of vitamin C in carcinogenesis. ROIs, reactive oxygen intermediates; GPx, glutathione peroxidase; GST, glutathione *S*-transferase; QR, quinone oxidoreductase; SOD, superoxide dismutase; CAT, catalase. Adapted from reference [37].

Regarding the use of vitamin C in cancer patient the results were not promising. In a doubleblind study 100 patients with advanced colorectal cancer were randomly assigned to treatment with either high-dose vitamin C (10 g daily) or placebo. Overall, these patients were in very good general condition, with minimal symptoms. None had received any previous treatment with cytotoxic drugs. Vitamin C therapy showed no advantage over placebo therapy with regard to either the interval between the beginning of treatment and disease progression or patient survival. Among patients with measurable disease, none had objective improvement. On the basis of this and our previous randomized study, it can be concluded that high-dose vitamin C therapy is not effective against advanced malignant disease regardless of whether the patient has had any prior chemotherapy [38].

consumption of vitamin C–rich fruit and vegetables is not likely to be harmful. In general, data from in vitro and in vivo experiments and population-based studies do not indicate that high doses of vitamin C are linked to increased oxidative DNA damage or an elevated risk of cancer.

Lee et al. [37] believe that the cancer preventive effects of vegetables and fruit may result from multiple combined effects of various phenolic phytochemicals, vitamins, dietary fibers, indoles, allium compounds, and selenium rather than from the effect of a single active ingredient. For them, many dietary phenolic phytochemicals may have stronger antioxidant and antitumor promotion effects than do antioxidant vitamins, which may contribute to the chemopreventive effects of the phytochemicals in carcinogenesis. However, these authors suggest that the chemopreventive effects of vitamin C in carcinogenesis may be linked to the protective effects of vitamin C against epigenetic mechanisms, such as the inflammation and inhibition of gap junction intercellular communication (GJIC), as well as to antioxidant

**Figure 5.** Possible chemopreventive mechanisms of vitamin C in carcinogenesis. ROIs, reactive oxygen intermediates; GPx, glutathione peroxidase; GST, glutathione *S*-transferase; QR, quinone oxidoreductase; SOD, superoxide dismutase;

Regarding the use of vitamin C in cancer patient the results were not promising. In a doubleblind study 100 patients with advanced colorectal cancer were randomly assigned to treatment with either high-dose vitamin C (10 g daily) or placebo. Overall, these patients were in very good general condition, with minimal symptoms. None had received any previous treatment with cytotoxic drugs. Vitamin C therapy showed no advantage over placebo therapy with regard to either the interval between the beginning of treatment and disease progression or patient survival. Among patients with measurable disease, none had objective improvement. On the basis of this and our previous randomized study, it can be concluded that high-dose

activities (see Figure 5).

408 Cancer Treatment - Conventional and Innovative Approaches

CAT, catalase. Adapted from reference [37].

The terms folic acid and folate are often used interchangeably for this water-soluble B-complex vitamin. Folic acid, the more stable form, occurs rarely in foods or the human body but is the form most often used in vitamin supplements and fortified foods. Folic acid is essential to numerous bodily functions ranging from nucleotide biosynthesis to the remethylation of homocysteine. The human body needs folate to synthesize DNA, repair DNA, and methylate DNA as well as to act as a cofactor in biological reactions involving folate.

Considerable epidemiological evidence suggests that a low-folate diet is associated with an increased risk of colorectal neoplasia. Much animal data support an antineoplastic effect of folate. However, in some animal studies, folate deficiency protects against, and supplemen‐ tation increases, experimental carcinogenesis. Cole et al. [39] developed a double-blind, placebo-controlled, 2-factor, phase 3, randomized clinical trial conducted at 9 clinical centers between July 6, 1994, and October 1, 2004. Participants included 1021 men and women with a recent history of colorectal adenomas and no previous invasive large intestine carcinoma. Participants were randomly assigned in a 1:1 ratio to receive 1 mg/d of folic acid (n=516) or placebo (n=505), and were separately randomized to receive aspirin (81 or 325 mg/d) or placebo. Follow-up consisted of 2 colonoscopic surveillance cycles (the first interval was at 3 years and the second at 3 or 5 years later). In this double-blind, placebo-controlled, randomized clinical trial, was found that folic acid supplementation did not decrease the risk of adenoma occurrence among participants with a recent history of adenomas. The authors concluded that folate, when administered as folic acid for up to 6 years, does not decrease the risk of adenoma formation in the large intestine among individuals with previously removed adenomas. For them, the evidence for an increased risk of adenomas is equivocal and requires further research.

In March of 1996, the U.S. Food and Drug Administration mandated that all enriched flour and uncooked cereal grains sold in the United States should be fortified with 140 µg folic acid/ 100 g of flour no later than January of 1998. Following the institution of fortification populationbased studies showed the effectiveness of this measure: plasma levels of folate in the adult population increased ~2-fold as a result and the incidence of births complicated by neural tube defects was variously reported to decline by 20% to 50%. However, analyses of several cereal grains that were purchased after the institution of fortification showed that in many instances the actual amount of folate was 150% to 300% greater than the mandate, suggesting that in this early era of fortification, manufacturers often included ''overage'' to ensure that they were meeting the minimal level of mandated fortification [40]. Thus, the authors hypothesize, by means of an epidemiological study, that the institution of folic acid fortification may have been wholly or partly responsible for the observed increase in colorectal cancer rates in the mid-1990s. The authors affirm that wish to highlight the potential complexity of the response to this nutrient and emphasize prior observations that have been made in both preclinical and clinical studies that indicate that administering high doses of folic acid to susceptible individ‐ uals or in an inappropriate time frame may accelerate the growth of existing neoplasms.

Vitamin C, also known as ascorbic acid, is a water-soluble vitamin. Unlike most mammals and other animals, humans do not have the ability to make their own vitamin C. Therefore, we must obtain vitamin C through our diet. Vitamin C is required for the synthesis of collagen, an important structural component of blood vessels, tendons, ligaments, and bone. Vitamin C also plays an important role in the synthesis of the neurotransmitter, norepinephrine. Neuro‐ transmitters are critical to brain function and are known to affect mood. Vitamin C is also a highly effective antioxidant. Even in small amounts vitamin C can protect indispensable molecules in the body, such as proteins, lipids (fats), carbohydrates, and nucleic acids (DNA and RNA), from damage by free radicals and reactive oxygen species that can be generated during normal metabolism as well as through exposure to toxins and pollutants (e.g., cigarette smoke). In the U.S., the recommended dietary allowance (RDA) for vitamin C was revised in 2000 upward from the previous recommendation of 60 mg daily for men and women. The RDA continues to be based primarily on the prevention of deficiency disease, rather than the prevention of chronic disease and the promotion of optimum health. The recommended intake for smokers is 35 mg/day higher than for non-smokers, because smokers are under increased oxidative stress from the toxins in cigarette smoke and generally have lower blood levels of vitamin C (see Table 7 – reference [41]).

prevention. Due to the role of vitamin C in the formation of N-nitrosocompounds, this may also be of importance for stomach cancer prevention. Another hand, 8 prospective studies does not suggest that intakes of vitamins A, C and E and folate reduce the risk of lung cancer. The results were similar with different analytic approaches and across studies, sex, smoking status

Nutrigenomics and Cancer Prevention http://dx.doi.org/10.5772/55429 411

Data on intake of specific carotenoids and breast cancer risk are limited. Furthermore, studies of vitamins A, C, and E in relation to breast cancer risk are inconclusive. Zhang et al. [44] were made studies, using multivariate analysis, demonstrated associations between intakes of specific carotenoids, vitamins A, C, and E, consumption of fruits and vegetables, and breast cancer risk in a cohort of 83,234 women (aged 33-60 years in 1980). Through 1994, they identified 2,697 incident cases of invasive breast cancer (784 premenopausal and 1913 post‐ menopausal). The results demonstrated that intakes of beta-carotene from food and supple‐ ments, lutein/zeaxanthin, and vitamin A from foods were weakly inversely associated with breast cancer risk in premenopausal women. Strong inverse associations were found for increasing quintiles of alpha-carotene, beta-carotene, lutein/zeaxanthin, total vitamin C from foods, and total vitamin A among premenopausal women with a positive family history of breast cancer. An inverse association was also found for increasing quintiles of beta-carotene among premenopausal women who consumed 15 g or more of alcohol per day. Premenopausal women who consumed five or more servings per day of fruits and vegetables had modestly lower risk of breast cancer than those who had less than two servings per day (relative risk [RR] = 0.77; 95% confidence interval [CI] = 0.58-1.02); this association was stronger among premenopausal women who had a positive family history of breast cancer (RR = 0.29; 95% CI = 0.13-0.62) or those who consumed 15 g or more of alcohol per day (RR = 0.53; 95% CI = 0.27-1.04). The author concluded that consumption of fruits and vegetables high in specific

carotenoids and vitamins may reduce premenopausal breast cancer risk [44].

In recent years, the intake of vitamins, minerals and herbs as a dietary supplement has increased dramatically. The supplementation with vitamins and minerals are used more often than the herbs. The most common supplements among users in the U.S. are multivitamins (75%), followed by vitamin C (38%), and iron (38%) [45]. Food supplementation with vitamins is a polemic question and it differs among authors. There are evidences that dietary supple‐ mentation with vitamin C may reduce the incidence of gastric cancer in certain populations, but it is unclear whether it was the antioxidant, vitamin or other property, responsible for this action [46]. However, the author states that it does not justify, in terms of cancer prevention to make a diet supplemented with vitamin C if the person has a good diet. Claycombe and Meydani [47] were made a review reporting the protective effect of vitamin E against chro‐ mosomal alterations induced by oxidation of DNA. However, the authors call attention to the careful supplementation, simultaneous with C vitamin E, considering a possible genotoxicity in the association of the two vitamins. Although most animal studies have shown cancerpreventive effects, a few recent studies suggest that soy phytoestrogens may stimulate breast cancer cell growth under certain circumstances. Before recommendations regarding phytoes‐ trogen supplements can be safely made, we must have more information on the effects of the extracts on bone, heart and breast health. Until safety with respect to breast cancer is estab‐ lished, phytoestrogen supplements should not be recommended, particularly for women at

and lung cancer cell type [43].

high risk of breast cancer [48].


**Table 7.** Recommended Dietary Allowance (RDA) for Vitamin C

The relations between the intake of beta-carotene, vitamin C, selenium, and 25-yr mortality from lung cancer and total cancer were analyzed within the Zutphen Study, a cohort study on diet and chronic diseases [42]. The Zutphen Study is a prospective study on the relations between diet, other risk factors, and the incidence of chronic diseases. The results of this study suggest that vitamin C intake may be more important for prevention of lung cancer than betacarotene. It can, however, not be ruled out that substances present in fruit other than vitamin C (eg, phenols, flavones, and terpenes) may also be of importance in lung cancer prevention. The results suggest that a vitamin C intake of ≥ 70 mg/d may be of importance in lung cancer prevention. Due to the role of vitamin C in the formation of N-nitrosocompounds, this may also be of importance for stomach cancer prevention. Another hand, 8 prospective studies does not suggest that intakes of vitamins A, C and E and folate reduce the risk of lung cancer. The results were similar with different analytic approaches and across studies, sex, smoking status and lung cancer cell type [43].

must obtain vitamin C through our diet. Vitamin C is required for the synthesis of collagen, an important structural component of blood vessels, tendons, ligaments, and bone. Vitamin C also plays an important role in the synthesis of the neurotransmitter, norepinephrine. Neuro‐ transmitters are critical to brain function and are known to affect mood. Vitamin C is also a highly effective antioxidant. Even in small amounts vitamin C can protect indispensable molecules in the body, such as proteins, lipids (fats), carbohydrates, and nucleic acids (DNA and RNA), from damage by free radicals and reactive oxygen species that can be generated during normal metabolism as well as through exposure to toxins and pollutants (e.g., cigarette smoke). In the U.S., the recommended dietary allowance (RDA) for vitamin C was revised in 2000 upward from the previous recommendation of 60 mg daily for men and women. The RDA continues to be based primarily on the prevention of deficiency disease, rather than the prevention of chronic disease and the promotion of optimum health. The recommended intake for smokers is 35 mg/day higher than for non-smokers, because smokers are under increased oxidative stress from the toxins in cigarette smoke and generally have lower blood levels of

**Life Stage Age Males (mg/day) Females (mg/day)** Infants 0-6 months 40 (AI) 40 (AI) Infants 7-12 months 50 (AI) 50 (AI) Children 1-3 years 15 15 Children 4-8 years 25 25 Children 9-13 years 45 45 Adolescents 14-18 years 75 65 Adults 19 years and older 90 75 Smokers 19 years and older 125 110 Pregnancy 18 years and younger - 80 Pregnancy 19 years and older - 85 Breast-feeding 18 years and younger - 115 Breast-feeding 19 years and older - 120

The relations between the intake of beta-carotene, vitamin C, selenium, and 25-yr mortality from lung cancer and total cancer were analyzed within the Zutphen Study, a cohort study on diet and chronic diseases [42]. The Zutphen Study is a prospective study on the relations between diet, other risk factors, and the incidence of chronic diseases. The results of this study suggest that vitamin C intake may be more important for prevention of lung cancer than betacarotene. It can, however, not be ruled out that substances present in fruit other than vitamin C (eg, phenols, flavones, and terpenes) may also be of importance in lung cancer prevention. The results suggest that a vitamin C intake of ≥ 70 mg/d may be of importance in lung cancer

vitamin C (see Table 7 – reference [41]).

410 Cancer Treatment - Conventional and Innovative Approaches

Data from reference [41].

**Table 7.** Recommended Dietary Allowance (RDA) for Vitamin C

Data on intake of specific carotenoids and breast cancer risk are limited. Furthermore, studies of vitamins A, C, and E in relation to breast cancer risk are inconclusive. Zhang et al. [44] were made studies, using multivariate analysis, demonstrated associations between intakes of specific carotenoids, vitamins A, C, and E, consumption of fruits and vegetables, and breast cancer risk in a cohort of 83,234 women (aged 33-60 years in 1980). Through 1994, they identified 2,697 incident cases of invasive breast cancer (784 premenopausal and 1913 post‐ menopausal). The results demonstrated that intakes of beta-carotene from food and supple‐ ments, lutein/zeaxanthin, and vitamin A from foods were weakly inversely associated with breast cancer risk in premenopausal women. Strong inverse associations were found for increasing quintiles of alpha-carotene, beta-carotene, lutein/zeaxanthin, total vitamin C from foods, and total vitamin A among premenopausal women with a positive family history of breast cancer. An inverse association was also found for increasing quintiles of beta-carotene among premenopausal women who consumed 15 g or more of alcohol per day. Premenopausal women who consumed five or more servings per day of fruits and vegetables had modestly lower risk of breast cancer than those who had less than two servings per day (relative risk [RR] = 0.77; 95% confidence interval [CI] = 0.58-1.02); this association was stronger among premenopausal women who had a positive family history of breast cancer (RR = 0.29; 95% CI = 0.13-0.62) or those who consumed 15 g or more of alcohol per day (RR = 0.53; 95% CI = 0.27-1.04). The author concluded that consumption of fruits and vegetables high in specific carotenoids and vitamins may reduce premenopausal breast cancer risk [44].

In recent years, the intake of vitamins, minerals and herbs as a dietary supplement has increased dramatically. The supplementation with vitamins and minerals are used more often than the herbs. The most common supplements among users in the U.S. are multivitamins (75%), followed by vitamin C (38%), and iron (38%) [45]. Food supplementation with vitamins is a polemic question and it differs among authors. There are evidences that dietary supple‐ mentation with vitamin C may reduce the incidence of gastric cancer in certain populations, but it is unclear whether it was the antioxidant, vitamin or other property, responsible for this action [46]. However, the author states that it does not justify, in terms of cancer prevention to make a diet supplemented with vitamin C if the person has a good diet. Claycombe and Meydani [47] were made a review reporting the protective effect of vitamin E against chro‐ mosomal alterations induced by oxidation of DNA. However, the authors call attention to the careful supplementation, simultaneous with C vitamin E, considering a possible genotoxicity in the association of the two vitamins. Although most animal studies have shown cancerpreventive effects, a few recent studies suggest that soy phytoestrogens may stimulate breast cancer cell growth under certain circumstances. Before recommendations regarding phytoes‐ trogen supplements can be safely made, we must have more information on the effects of the extracts on bone, heart and breast health. Until safety with respect to breast cancer is estab‐ lished, phytoestrogen supplements should not be recommended, particularly for women at high risk of breast cancer [48].

Cancer prevention can be done with a diet rich in vegetables, fruits, and low in red meat, saturated fats, salt and sugar. Carbohydrates should be consumed in the form of cereals - wheat bread and brown rice. The addition of fats should be in the form of fats dehydrogenated [49]. The types of vegetables or fruit that most often appear to be protective against cancer are allium vegetables, carrots, green vegetables, cruciferous vegetables, and tomatoes. Substances present in some vegetable and fruit may help cancer prevention and they include dithiolthiones, isothiocyanates, indole-3-carbinol, allium compounds, isoflavones, protease inhibitors, saponins, phytosterols, inositol hexaphosphate, vitamin C, D-limonene, lutein, folic acid, beta carotene, lycopene, selenium, vitamin E, flavonoids, and dietary fiber. Current US vegetable and fruit intake, which averages about 3.4 servings per day, is discussed, as are possible noncancer-related effects of increased vegetable and fruit consumption, including benefits against cardiovascular disease, diabetes, stroke, obesity, diverticulosis, and cataracts [50].

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animal models. Mutat Res 2001;477(1-2): 1-6.

Foodtechnology 2003;57(4): 60-67.

(accessed 215 August 2012).

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597-610.

2001;475:1-6

2003;89: 581-585.

Humana Press. 824p., 2010.

### **4. Conclusion**

Cancer incidence is projected to increase in the future and an effectual preventive strategy is required to face this challenge. Alteration of dietary habits is potentially an effective approach for reducing cancer risk. Assessment of biological effects of a specific food or bioactive component that is linked to cancer and prediction of individual susceptibility as a function of nutrient-nutrient interactions and genetics is an essential element to evaluate the beneficiaries of dietary interventions [51]. We know that diet is an important factor both to minimize, as to increase the risk of cancer development. But diet is not the only factor. There are several risk factors that can trigger a process of tumor formation. Sedentary life, environmental issues, viruses, smoking, alcohol in excess, are factors that contribute to and are also strategic points that should be worked in cancer prevention.

### **Author details**

Júlio César Nepomuceno

Universidade Federal de Uberlândia/ Instituto de Genética e Bioquímica; Centro Universi‐ tário de Patos de Minas /Laboratório de Citogenética e Mutagênese, Brazil

### **References**

[1] Pitot HC, Goldsworthy T., Moran S. The natural history of carcinogenesis: Implica‐ tions of experimental carcinogenesis in the genesis of human cancer. Journal of Su‐ pramolecular Structure and Cellular Biochemistry. 2004;17: 133-146.


Cancer prevention can be done with a diet rich in vegetables, fruits, and low in red meat, saturated fats, salt and sugar. Carbohydrates should be consumed in the form of cereals - wheat bread and brown rice. The addition of fats should be in the form of fats dehydrogenated [49]. The types of vegetables or fruit that most often appear to be protective against cancer are allium vegetables, carrots, green vegetables, cruciferous vegetables, and tomatoes. Substances present in some vegetable and fruit may help cancer prevention and they include dithiolthiones, isothiocyanates, indole-3-carbinol, allium compounds, isoflavones, protease inhibitors, saponins, phytosterols, inositol hexaphosphate, vitamin C, D-limonene, lutein, folic acid, beta carotene, lycopene, selenium, vitamin E, flavonoids, and dietary fiber. Current US vegetable and fruit intake, which averages about 3.4 servings per day, is discussed, as are possible noncancer-related effects of increased vegetable and fruit consumption, including benefits against

cardiovascular disease, diabetes, stroke, obesity, diverticulosis, and cataracts [50].

Cancer incidence is projected to increase in the future and an effectual preventive strategy is required to face this challenge. Alteration of dietary habits is potentially an effective approach for reducing cancer risk. Assessment of biological effects of a specific food or bioactive component that is linked to cancer and prediction of individual susceptibility as a function of nutrient-nutrient interactions and genetics is an essential element to evaluate the beneficiaries of dietary interventions [51]. We know that diet is an important factor both to minimize, as to increase the risk of cancer development. But diet is not the only factor. There are several risk factors that can trigger a process of tumor formation. Sedentary life, environmental issues, viruses, smoking, alcohol in excess, are factors that contribute to and are also strategic points

Universidade Federal de Uberlândia/ Instituto de Genética e Bioquímica; Centro Universi‐

[1] Pitot HC, Goldsworthy T., Moran S. The natural history of carcinogenesis: Implica‐ tions of experimental carcinogenesis in the genesis of human cancer. Journal of Su‐

pramolecular Structure and Cellular Biochemistry. 2004;17: 133-146.

tário de Patos de Minas /Laboratório de Citogenética e Mutagênese, Brazil

**4. Conclusion**

**Author details**

**References**

Júlio César Nepomuceno

that should be worked in cancer prevention.

412 Cancer Treatment - Conventional and Innovative Approaches


Committee. American Cancer Society Guidelines on Nutrition and Physical Activity for Cancer Prevention Reducing the Risk of Cancer With Healthy Food Choices and Physical Activity. CA Cancer J Clin 2012;62:30–67.

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[32] Martinez M, Giovannucci EL, Colditz GA, Stampfer M, Hunter DJ, Speizer FE, Wing A and Willet WC. Calcium, Vitamin D, and the Occurrence of Colorectal Cancer

[33] Tanaka T, Shnimizu M and Moriwaki H. Cancer Chemoprevention by Carotenoids,

[34] American Cancer Society. Vitamin C. http://www.cancer.org/Treatment/Treatment‐ sandSideEffects/ComplementaryandAlternativeMedicine/HerbsVitaminsandMiner‐

[35] Block G. Vitamin C and cancer prevention: the epidemiologic evidence. Am J Clin

[36] Padayatty, S.J., Katz, A., Wang, Y., Eck, P., Kwon, O., Lee, J.H., Chen, S., Corpe, C., Dutta, A., Dutta, S.K. & Levine, M. (2003). Vitamin C as an Antioxidant: Evaluation

[37] Lee KW, Lee HJ, Surh YJ and Lee CY. Vitamin C and cancer chemoprevention: reap‐

[38] Moertel CG, Fleming TR, Creagan ET, Rubin J, O'Connell MJ and Ames MM. High-Dose Vitamin C versus Placebo in the Treatment of Patients with Advanced Cancer Who Have Had No Prior Chemotherapy — A Randomized Double-Blind Compari‐

[39] Cole BF, Baron JA, Sandler RS, Haile RW, Ahnen DJ, Bresalier RS, McKeown-Eyssen G, Summers RW, Rothstein RI, Burke CA, Snover DC, Church TR, Allen JI, Robert‐ son DJ, Beck GJ, Bond JH, Byers T, Mandel JS, Mott LA, Pearson LH, Barry EL, Rees JR, Marcon N, Saibil F, Ueland PM and Greenberg ER; Polyp Prevention Study Group. Folic Acid for the Prevention of Colorectal Adenomas - A Randomized Clini‐

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[41] Linus Pauling Institute. Micronutrient Information Center; Vitamin C. http:// lpi.oregonstate.edu/infocenter/vitamins/vitaminC/ (accessed 12 September 2012). [42] Kromhout D. Essential micronutrients in relation to carcinogenesis. Am J Clin Nutr

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[19] Scalbert A, Manach C, Morand C, Rémésy C, Jiménez L. Dietary Polyphenols and the

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[28] Nepomuceno, JC. Antioxidants in Cancer Treatment, Current Cancer Treatment: In‐ tech, 2011. http://www.intechopen.com/books/current-cancer-treatment-novel-be‐ yond-conventional-approaches/antioxidants-in-cancer-treatment (accessed 20

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**Chapter 18**

**The Impact of Vitamin D in Cancer**

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55324

**1. Introduction**

Khanh vinh quoc Luong and Lan Thi Hoang Nguyen

The relationship between vitamin D and cancer has previously been reported in the literature. A systemic review and meta-analysis of prospective cohort studies revealed that a 20 nmol/L increase in the 25-hydroxyvitamin D3 (25OHD) levels was associated with an 8% lower mortality in the elderly population (Schöttker et al., 2012). Oncology patients had significantly lower mean serum vitamin D levels than non-cancer primary care patients from the same geographic region (Churilla et al., 2011). In a community oncology experience, vitamin D deficiency is widespread in cancer patients and correlates with advanced stage disease (Churilla et al., 2012). A high prevalent of vitamin D deficiency has been associated with head and neck cancer (Orell-Kotikangas et al., 2011), breast cancer (Crew et al., 2009; Peppone et al., 2012), vulvar cancer (Salehin et al., 2012), prostate cancer (Varsavsky et al., 2011), pancreatic cancer (Wolpin et al., 2011), gastric cancer (Ren et al., 2012), colon and rectal cancer (Tangrea et al., 1997), ovarian cancer (Lefkowitz et al., 1994), oral cavity and esophagus cancers (Lipworth et al., 2009), myelo-proliferative neoplasms and myelo-dysplastic syndromes (Pardanani et al., 2011), multiple myeloma (Ng et at., 2009), non-Hodgkin's lymphoma (Drake et al., 2010), and chronic lymphocytic leukemia (Shamafelt et al., 2011). On the other hand, a serum 25OHD concentration of 25 nmol/L was associated with a 17% reduction in incidence of cancer, a 29% reduction in total cancer mortality, and a 45% reduction in digestive system cancer mortality (Giovannucci et al., 2006). Improving vitamin D status may also help lower the risk of colorectal cancer (Wu et al., 2011a). In a case-control study, a higher vitamin D intake is associated with a lower risk of esophageal squamous cell carcinoma (Launoy et al., 1998). A meta-analysis revealed that an increase of serum 25OHD by 50 nmol/L was associated with a risk reduction of 59% for rectal cancer and 22% for colon cancer (Yin et al., 2009). High 25OHD levels were associated with better prognosis in breast, colon, prostate cancer, and lung cancer relative to patients with lower 25OHD levels (Robsahm et al., 2004; Zhou et al., 2007). In a murine model, dietary vitamin D may play an important role as a preventive agent in andro‐

> © 2013 Luong and Nguyen; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

> © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

and reproduction in any medium, provided the original work is properly cited.


### **The Impact of Vitamin D in Cancer**

Khanh vinh quoc Luong and Lan Thi Hoang Nguyen

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55324

### **1. Introduction**

bohm RA, Graham S, Miller AB, Rohan TE, Sellers TA, Virtamo J, Willett WC, Smith-Warner SA. Intakes of vitamins A, C and E and folate and multivitamins and lung cancer: a pooled analysis of 8 prospective studies. Int J Cancer 2006;118(4):970-8. [44] Zhang S, Hunter DJ, Forman MR, Rosner BA, Speizer FE, Colditz GA, Manson JE, Hankinson SE, Willett WC. Dietary carotenoids and vitamins A, C, and E and risk of

[45] Yu C. Contribution of Dietary Supplements to the Nutritional Status of College Stu‐ dents. (2011). Honors Scholar Theses. Paper 180. http://digitalcommons.uconn.edu/

[47] Claycombe KJ, Meydani SN. Vitamin E and genome stability. Mutat Res

[48] Kurzer MS. Phytoestrogen Supplement Use by Women. J Nutr 2003;133(6):

[50] Steinmetz KA, Potter JD. Vegetables, fruit, and cancer prevention: a review. J Am Di‐

[51] Ardekani AM and Jabbari S. Nutrigenomics and Cancer. Avicenna J Med Biotech

[49] Willett WC. Diet and Cancer An Evolving Picture. JAMA 2005;293(2): 233-234.

[46] Halliwell B. Vitamin C and genomic stability. Mutation Res 2001;475: 29-35.

breast cancer. J Natl Cancer Inst 1999;91(6):547-56.

srhonors\_theses/180

416 Cancer Treatment - Conventional and Innovative Approaches

2001;475(1-2):37-44.

et Assoc. 1996;96(10):1027-39.

1983S-1986S.

2009;1(1): 9-17.

The relationship between vitamin D and cancer has previously been reported in the literature. A systemic review and meta-analysis of prospective cohort studies revealed that a 20 nmol/L increase in the 25-hydroxyvitamin D3 (25OHD) levels was associated with an 8% lower mortality in the elderly population (Schöttker et al., 2012). Oncology patients had significantly lower mean serum vitamin D levels than non-cancer primary care patients from the same geographic region (Churilla et al., 2011). In a community oncology experience, vitamin D deficiency is widespread in cancer patients and correlates with advanced stage disease (Churilla et al., 2012). A high prevalent of vitamin D deficiency has been associated with head and neck cancer (Orell-Kotikangas et al., 2011), breast cancer (Crew et al., 2009; Peppone et al., 2012), vulvar cancer (Salehin et al., 2012), prostate cancer (Varsavsky et al., 2011), pancreatic cancer (Wolpin et al., 2011), gastric cancer (Ren et al., 2012), colon and rectal cancer (Tangrea et al., 1997), ovarian cancer (Lefkowitz et al., 1994), oral cavity and esophagus cancers (Lipworth et al., 2009), myelo-proliferative neoplasms and myelo-dysplastic syndromes (Pardanani et al., 2011), multiple myeloma (Ng et at., 2009), non-Hodgkin's lymphoma (Drake et al., 2010), and chronic lymphocytic leukemia (Shamafelt et al., 2011). On the other hand, a serum 25OHD concentration of 25 nmol/L was associated with a 17% reduction in incidence of cancer, a 29% reduction in total cancer mortality, and a 45% reduction in digestive system cancer mortality (Giovannucci et al., 2006). Improving vitamin D status may also help lower the risk of colorectal cancer (Wu et al., 2011a). In a case-control study, a higher vitamin D intake is associated with a lower risk of esophageal squamous cell carcinoma (Launoy et al., 1998). A meta-analysis revealed that an increase of serum 25OHD by 50 nmol/L was associated with a risk reduction of 59% for rectal cancer and 22% for colon cancer (Yin et al., 2009). High 25OHD levels were associated with better prognosis in breast, colon, prostate cancer, and lung cancer relative to patients with lower 25OHD levels (Robsahm et al., 2004; Zhou et al., 2007). In a murine model, dietary vitamin D may play an important role as a preventive agent in andro‐

© 2013 Luong and Nguyen; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

gen-insensitive human prostate tumor growth (Ray et al., 2012). The season in which patients were operated on seemed to have an effect on survival of patients undergoing resection of nonsmall cell lung cancer (Turna et al., 2012). The survival of patients who had surgery in winter was statistically significantly shorter than that of patients who underwent surgery in the summer. In Australia, prostate cancer mortality rates are inversely correlated with solar radiation exposure (Loke et al., 2011). Dietary vitamin D3 and calcitriol have been shown to demonstrate equivalent anticancer activity in mouse xenograft models of breast and prostate cancers (Swani et al., 2012). The combination of calcitriol and dietary soy resulted in substan‐ tially greater inhibition of tumor growth than the inhibition achieved with either agent alone in a mouse xenograft model of prostate cancer (Wang et al., 2012a). Soy diets alone caused a modest elevation in serum calcitriol. Vitamin D3 treatment significantly suppressed the viability of gastric cancer and cholangiocarcinoma cells and also had a synergistic effect with other anti-cancer drugs, such as paclitaxel, adriamycin, and vinblastine (Baek et al., 2011). The vitamin D analog, 19-Nor-2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D3, is a potent cell growth regulator with enhanced chemotherapeutic potency in liver cancer cells (Chiang et al., 2011). Alphacalcidol, a vitamin D analogue, has been demonstrated significant antitumor activity in patients with low-grade non-Hodgkin's lymphoma of the follicular, small-cleaved cell type (Raina et al., 1991). In patient with parathyroid cancer, vitamin D has been shown to prevent or delay the progression of recurrence (Palmieri-Sevier et al., 1993). In locally advanced or cutaneous metastatic breast cancer, topical calcipotriol treatment reduced the diameter of treated lesions that contained vitamin D receptor (*VDR*) (Bower et al., 1991). In a clinical trial, high-dose calcitriol decreased prostatic-specific antigen (PSA) levels by 50% and reduced thrombosis in prostate cancer patients (Beer et al., 2003 & 2006). In hepatocellular carcinoma, calcitriol and its analogs have been reported to reduce tumor volume, increase hepatocarci‐ noma cell apoptosis by 21.4%, and transient stabilize serum alpha-fetoprotein levels (Dalhoff et al., 2003; Luo et al., 2004; Morris et al., 2002). These findings suggested a relationship between vitamin D and cancer. In this chapter, we will discuss the role of vitamin D in cancer.

strongly associated with thyroid carcinoma (Panza et al., 1982). The HLA-DR was also increased in poorly differentiated thyroid carcinoma, especially in the anaplastic type (Lindhorst et al., 2002). The DQA1\*0102 and DPB1\*0501 alleles were significantly more common in Chinese patients with hepatocellular carcinoma (HCC) (Donaldson et al., 2001). The frequency of DRB1\*0404 allele was significantly higher in the gastric cancer group compared with the gastritis group in Koreans (Lee et al., 2009). However, the frequencies of the DRB1\*0405 and DQB1\*0401 alleles were increased in the Japanese patients with intestinaltype gastric cancer compared with controls (Ando et al., 2009). Somatic mutations affecting HLA class II genes may lead to loss of HLA class II expression due to the formation of microsatellites in unstable colorectal carcinomas (Michel et al., 2010). The DRB1\*15 allele and the haplotype DRB1\*15 DQB1\*0602 were associated with human papillomavirus (HPV)-16 positive invasive cervical cancer in Mexican women (Hernández-Hernández et al., 2009). The DRB1\*0410 allele was the susceptibility allele in Japanese patients with testicular germ cell carcinoma (Ozdemir et al., 1997). Furthermore, the frequencies of the DRB1\*09 and DQB1\*03 alleles were increased in patients with non-Hodgkin's lymphoma and diffuse large B cell lymphoma compared with normal controls (Choi et al., 2008). The frequencies of the DRB1\*04 and DRB1\*15 alleles were significantly higher in Turkish children with acute leukemia compared with controls (Ozdilli et al., 2010). The DRB1\*16 allele was a marker for a signigni‐ ficant risk of chronic myelogenous leukemia in Eastern Canada (Naugler and Liwski, 2009). The DRB1\*04 and DRB5 alleles are associated with disease progression in Iranian patients with chronic lymphocytic leukemia (Hojattat-Farsangi et al., 2008). On the other hand, calcitriol is known to stimulate phagocytosis and suppress MHC class II antigen expression in human mononuclear phagocytes (Tokuda et al., 1992 & 1996), thereby preventing antigen-specific T cell proliferation. In addition, calcitriol exerts effects that opposes the effect of IL-4 on MHC class II antigen expression in human monocytes (Xu et al., 1993) and specifically modulates human monocyte phenotype and function by altering HLA-DR antigen expression and antigen presentation, while leaving lytic function intact (Rigby et al., 1990). Calcitriol also decreases interferon-γ-induced HLA-DR antigen expression in normal and transformed human kerati‐ nocytes (Tamaki et al., 1990-1991 & Tone et al., 1991) and reduces the levels of HLA-DR mRNA in cultured epithelial tumor cell lines (Tone et al., 1993). In addition, 1α-calcidol significantly modulates the expression of HLA-DR in human peripheral blood monocytes (Scherberich et al., 2005). These findings suggest that calcitriol may have an effect on cancer by suppressing

The Impact of Vitamin D in Cancer http://dx.doi.org/10.5772/55324 419

The expression of VDR in a variety of cell lines, coupled with increased evidence of *VDR* involvement in cell differentiation, inhibition of cellular proliferation and angiogenesis in many tumor types, suggest that vitamin D plays a role in cancer (Luong and Nguyen, 2010 & Lương and Nguyễn, 2012). VDR ablation is associated with ductal ectasia of the primary mammary ducts, loss of secondary and tertiary branches and atrophy of the mammary fad pad (Welsh et al., 2011). Breast cancer patients with high VDR expression showed significant better in progression-free survival and overall survival than patients with moderate/negative VDR expression scores (Ditch et al., 2012). Certain allelic variations in the *VDR* may also be

the expression of MHC class II antigens.

**2.2. Vitamin D Receptor (VDR)**

### **2. Genetic factors related to vitamin D and cancer**

#### **2.1. The Major Histocompatibility Complex (MHC) class II molecules**

The major histocompatibility complex (MHC) class II molecules play an important role in the immune system and are essential in the defense against infection. The human MHC class II molecules are encoded by three different human leukocytic antigen (HLA) isotypes: HLA-DR, -DQ, and -DP. Studies have suggested that several genes within MHC region promote cancer susceptibility. A chimeric DR4 homozygous transgenic mouse line is reported to spontane‐ ously develop diverse hematological malignancies at a high frequency (Raffegerst et al., 2009). Most of these neoplasms were highly similar to those found in human diseases. HLA-DR antigen expression was correlated with the histopathological type and to the degree of cell differentiation in cutaneous squamous cell carcinomas (Garcia-Plata et al., 1993). The DRB1\*03 and DR-B1\*13 alleles were significantly more frequent in patients with nasopharyngeal carcinoma compared with controls in southern Tunisia (Makni et al., 2010). The DR1 gene is strongly associated with thyroid carcinoma (Panza et al., 1982). The HLA-DR was also increased in poorly differentiated thyroid carcinoma, especially in the anaplastic type (Lindhorst et al., 2002). The DQA1\*0102 and DPB1\*0501 alleles were significantly more common in Chinese patients with hepatocellular carcinoma (HCC) (Donaldson et al., 2001). The frequency of DRB1\*0404 allele was significantly higher in the gastric cancer group compared with the gastritis group in Koreans (Lee et al., 2009). However, the frequencies of the DRB1\*0405 and DQB1\*0401 alleles were increased in the Japanese patients with intestinaltype gastric cancer compared with controls (Ando et al., 2009). Somatic mutations affecting HLA class II genes may lead to loss of HLA class II expression due to the formation of microsatellites in unstable colorectal carcinomas (Michel et al., 2010). The DRB1\*15 allele and the haplotype DRB1\*15 DQB1\*0602 were associated with human papillomavirus (HPV)-16 positive invasive cervical cancer in Mexican women (Hernández-Hernández et al., 2009). The DRB1\*0410 allele was the susceptibility allele in Japanese patients with testicular germ cell carcinoma (Ozdemir et al., 1997). Furthermore, the frequencies of the DRB1\*09 and DQB1\*03 alleles were increased in patients with non-Hodgkin's lymphoma and diffuse large B cell lymphoma compared with normal controls (Choi et al., 2008). The frequencies of the DRB1\*04 and DRB1\*15 alleles were significantly higher in Turkish children with acute leukemia compared with controls (Ozdilli et al., 2010). The DRB1\*16 allele was a marker for a signigni‐ ficant risk of chronic myelogenous leukemia in Eastern Canada (Naugler and Liwski, 2009). The DRB1\*04 and DRB5 alleles are associated with disease progression in Iranian patients with chronic lymphocytic leukemia (Hojattat-Farsangi et al., 2008). On the other hand, calcitriol is known to stimulate phagocytosis and suppress MHC class II antigen expression in human mononuclear phagocytes (Tokuda et al., 1992 & 1996), thereby preventing antigen-specific T cell proliferation. In addition, calcitriol exerts effects that opposes the effect of IL-4 on MHC class II antigen expression in human monocytes (Xu et al., 1993) and specifically modulates human monocyte phenotype and function by altering HLA-DR antigen expression and antigen presentation, while leaving lytic function intact (Rigby et al., 1990). Calcitriol also decreases interferon-γ-induced HLA-DR antigen expression in normal and transformed human kerati‐ nocytes (Tamaki et al., 1990-1991 & Tone et al., 1991) and reduces the levels of HLA-DR mRNA in cultured epithelial tumor cell lines (Tone et al., 1993). In addition, 1α-calcidol significantly modulates the expression of HLA-DR in human peripheral blood monocytes (Scherberich et al., 2005). These findings suggest that calcitriol may have an effect on cancer by suppressing the expression of MHC class II antigens.

#### **2.2. Vitamin D Receptor (VDR)**

gen-insensitive human prostate tumor growth (Ray et al., 2012). The season in which patients were operated on seemed to have an effect on survival of patients undergoing resection of nonsmall cell lung cancer (Turna et al., 2012). The survival of patients who had surgery in winter was statistically significantly shorter than that of patients who underwent surgery in the summer. In Australia, prostate cancer mortality rates are inversely correlated with solar radiation exposure (Loke et al., 2011). Dietary vitamin D3 and calcitriol have been shown to demonstrate equivalent anticancer activity in mouse xenograft models of breast and prostate cancers (Swani et al., 2012). The combination of calcitriol and dietary soy resulted in substan‐ tially greater inhibition of tumor growth than the inhibition achieved with either agent alone in a mouse xenograft model of prostate cancer (Wang et al., 2012a). Soy diets alone caused a modest elevation in serum calcitriol. Vitamin D3 treatment significantly suppressed the viability of gastric cancer and cholangiocarcinoma cells and also had a synergistic effect with other anti-cancer drugs, such as paclitaxel, adriamycin, and vinblastine (Baek et al., 2011). The vitamin D analog, 19-Nor-2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D3, is a potent cell growth regulator with enhanced chemotherapeutic potency in liver cancer cells (Chiang et al., 2011). Alphacalcidol, a vitamin D analogue, has been demonstrated significant antitumor activity in patients with low-grade non-Hodgkin's lymphoma of the follicular, small-cleaved cell type (Raina et al., 1991). In patient with parathyroid cancer, vitamin D has been shown to prevent or delay the progression of recurrence (Palmieri-Sevier et al., 1993). In locally advanced or cutaneous metastatic breast cancer, topical calcipotriol treatment reduced the diameter of treated lesions that contained vitamin D receptor (*VDR*) (Bower et al., 1991). In a clinical trial, high-dose calcitriol decreased prostatic-specific antigen (PSA) levels by 50% and reduced thrombosis in prostate cancer patients (Beer et al., 2003 & 2006). In hepatocellular carcinoma, calcitriol and its analogs have been reported to reduce tumor volume, increase hepatocarci‐ noma cell apoptosis by 21.4%, and transient stabilize serum alpha-fetoprotein levels (Dalhoff et al., 2003; Luo et al., 2004; Morris et al., 2002). These findings suggested a relationship between

418 Cancer Treatment - Conventional and Innovative Approaches

vitamin D and cancer. In this chapter, we will discuss the role of vitamin D in cancer.

The major histocompatibility complex (MHC) class II molecules play an important role in the immune system and are essential in the defense against infection. The human MHC class II molecules are encoded by three different human leukocytic antigen (HLA) isotypes: HLA-DR, -DQ, and -DP. Studies have suggested that several genes within MHC region promote cancer susceptibility. A chimeric DR4 homozygous transgenic mouse line is reported to spontane‐ ously develop diverse hematological malignancies at a high frequency (Raffegerst et al., 2009). Most of these neoplasms were highly similar to those found in human diseases. HLA-DR antigen expression was correlated with the histopathological type and to the degree of cell differentiation in cutaneous squamous cell carcinomas (Garcia-Plata et al., 1993). The DRB1\*03 and DR-B1\*13 alleles were significantly more frequent in patients with nasopharyngeal carcinoma compared with controls in southern Tunisia (Makni et al., 2010). The DR1 gene is

**2. Genetic factors related to vitamin D and cancer**

**2.1. The Major Histocompatibility Complex (MHC) class II molecules**

The expression of VDR in a variety of cell lines, coupled with increased evidence of *VDR* involvement in cell differentiation, inhibition of cellular proliferation and angiogenesis in many tumor types, suggest that vitamin D plays a role in cancer (Luong and Nguyen, 2010 & Lương and Nguyễn, 2012). VDR ablation is associated with ductal ectasia of the primary mammary ducts, loss of secondary and tertiary branches and atrophy of the mammary fad pad (Welsh et al., 2011). Breast cancer patients with high VDR expression showed significant better in progression-free survival and overall survival than patients with moderate/negative VDR expression scores (Ditch et al., 2012). Certain allelic variations in the *VDR* may also be genetic risk factors for developing tumors. There are five important common polymorphisms within the *VDR* gene region that are likely to exert functional effects on *VDR* expression. The anti-carcinogenic potential of vitamin D might be mediated by VDR expression. The associa‐ tion between plasma 25OHD levels and colorectal adenoma was modified by the *Taq*I polymorphism of the VDR gene (Yamaji et al., 2011). There is a significant association between single nucleotide polymorphisms (SNPs) in the VDR gene and vitamin D intake in African Americans with colorectal cancer (Kupfer et al., 2011). The *Bsm*I polymorphism of the VDR gene also modified the association between dietary vitamin D intake and breast cancer (Rollison et al., 2012). The *AA* genotype of VDR is reported to be associated with colorectal cancer, with a stronger association in female patients (Mahmoudi et al., 2012). The *Fok*I and *Bsm*I genotypes of VDR gene are implicated in the pathogenesis of renal cell carcinoma (RCC) in a North Indian population (Arjumand et al., 2012). Altered VDR expression was associated with RCC carcinogenesis via the expression of epithelial Ca2+ channel transient receptor potential vanilloid subfamily 5 and 6 (TRPV5/6) (Wu et al., 2011b). There is a significant association between shorter progression-free survival time in patients with head and neck squamous cell carcinoma and the *Fok*I *TT* genotype, as well as the *Cdx2*-*Fox*I-*Apa*I haplotype (Hama et al., 2011). In Spanish children, osteosarcoma patients showed a significantly higher frequency of the *Ff* genotype of the *Fok*I *VDR* gene than the control group (Ruza et al., 2003). In a German population, the *AaTtBb* genotype of the VDR gene is associated with basal cell carcinoma risk, whereas the *aaTTbb* genotype is found at a high frequency in both basal cell carcinomas and cutaneous squamous cell carcinomas compared with controls (Köststner et al., 2012). In a systematic review, *Taq*I, *Bsm*I and *Fok*I polymorphisms of the VDR gene were found to be associated with malignant melanoma (Denzer et al., 2011). Furthermore, the presence of specific VDR *Bsm*I and *Taq*I alleles was associated with a higher C-reactive protein (CRP) level in cancer patients with cachectic syndrome (Punzi et al., 2012). In another pro‐ spective study, plasma 25OHD levels and common variation among several vitamin D-related genes (*CYP27A1*, *CYP2R1*, *CYP27B1*, *CYP24A1*, *GC*, *RXRA*, and *VDR*) were associated with lethal prostate cancer risk (Shui et al., 2012). Slattery et al. (2009) examined genetic variants that are linked to the pathway that contribute to colon cancer. They revealed that *Fox*I *VDR* polymorphism was associated with CpG Island methylator phenotype (CIMP) positive/Ki-ras mutated tumors, whereas the Poly *A* and *Cdx2 VDR* polymorphisms were associated only with Ki-ras mutated tumors.

tissue and causes high CYP24 protein expression, which catalyzes the inactivation of calcitriol (Komagata et al., 2009). Stress induced by serum starvation caused significant alteration in the expression of multiple miRNAs including miRNA-182, but calcitriol effectively reversed this alterationinbreastepithelial cells (Pengetal.,2010).VitaminD3up-regulatedprotein1(VDUP1) is regulated by miRNA-17-5p at the post-transcriptional levels in senescent fibroblasts (Zhuo et al., 2010). VDUP1 expression is increased in cancer cells (Takahashi et al., 2002; Dutta et al., 2005). In melanoma cell lines, the endogenous VDR mRNA level is inversely associated with expression of miRNA-125b (Essa et al., 2010), and calcitriol also reduced the miRNA-27b expression in these cell lines. In human colon cells, calcitriol induced miRNA-22 and may contribute to its antitumor action against this neoplasm (Alvarez-Diaz et al., 2012). Fifteen miRNAs are also differentially regulated by calcitriol in prostate cancer cells (LNCaP) (Wang etal2011a).Furthermore, calcitriolregulatedmiRNA-32 andmiRNA-181expressions inhuman myeloid leukemia cells (Gocek et al., 2011; Zimmerman et al., 2011; Wang et al., 2009a).

The Impact of Vitamin D in Cancer http://dx.doi.org/10.5772/55324 421

The primary function of the renin-angiotensin system (RAS) is to maintain fluid homeostasis and regulate blood pressure. The angiotensin converting enzyme (ACE) is a key enzyme in the RAS and converts angiotensin (AT) I to the potent vasoconstrictor AT II (Johnston, 1994). The local RAS may influence tissue angiogenesis, cellular proliferation, apoptosis, and inflammation (Deshayes and Nahmias, 2005). Epidemiological and experimental studies suggested that the RAS may contribute to the paracrine regulation of tumor growth. The renin levels are elevated in patients with liver cirrhosis and HCC and positively correlated with αfetoprotein (Lotfy et al., 2010). The over-expression of ACE is reported in extrahepatic cholangiocarcinoma (Beyazit et al., 2011), leukemic myeloid blast cells (Aksu et al., 2006), and macrophages in the lymph nodes of Hodgkin's disease patients (Koca et al., 2007). The AT II receptors were also expressed in all human gastric cancer lines (Huang et al., 2008), premalignant and malignant prostate cells (Louis et al., 2007), human lung cancer xenografts (Feng et al., 2011a), and ovarian cancer (Ino et al., 2006). The RAS mutation in codon 61 was the most common genetic alteration in poorly differentiated thyroid carcinomas (Volante et al., 2009). The ACE *I/D* polymorphism is a possible target for developing genetic markers for breast cancer in Brazilian women (Alves Corrêa et al., 2009). The ACE *I/D* polymorphisms play an important role in breast cancer risk and disease-free survival in Caucasian postmenopausal women (González-Zuloeta Ladd et al., 2012). Carriers of the high-activity *DD* genotype had an increased risk of breast cancer compared with low activity *II/ID* genotype carriers (van der Knaap et al., 2008). The *DD* genotype was associated with patients with an aggressive stage of prostate cancer (Wang et al., 2011b). ACE2 expression was decreased in non-small-cell lung cancer and pancreatic ductal adenocarcinoma in which AT II levels were higher than those in controls (Feng et al., 2010; Zhou et al., 2009). ACE2 has been suggested as a potential molecular target for pancreatic cancer therapy (Zhou et al., 2011). The AT II concentration in gastric cancer region was significantly higher than those of normal region (Kinoshita et al., 2009). Further‐ more, AT II receptor blockers (ARB) suppress the cell proliferation effects of AT II in breast cancer cells (Du et al., 2012). The addition of ACE inhibitor or ARB to platinum-based first line chemotherapy contributed to prolong survival in patients with advanced lung cancer (Wilop

**2.4. Renin-Angiotensin System (RAS)**

#### **2.3. MicroRNA (miRNA)**

MiRNAs are endogenous noncoding RNAs that regulate gene expression through the transla‐ tional repression or degradation of target mRNA (Bartel, 2004). Aberrant miRNA expression has been well characterized in cancer (Lu et al., 2005). Circulating miRNAs are suggested to be diagnostic and prognostic markers in breast cancer (Cortez et al., 2012). Circulating miR‐ NA-125b expression is associated with chemotherapeutic resistance of breast cancer (Wang et al., 2012b). Several miRNAs are found to share 125b complementarity with a sequence in the 3' unstranslated region of human VDR mRNA. The overexpression miRNA-125b significantly decreased the endogenous VDR protein level in human breast adenocarcinoma cells lines (MCF-7) to 40% of the control (Mohri et al., 2009). This miRNA is down-regulated in cancer tissue and causes high CYP24 protein expression, which catalyzes the inactivation of calcitriol (Komagata et al., 2009). Stress induced by serum starvation caused significant alteration in the expression of multiple miRNAs including miRNA-182, but calcitriol effectively reversed this alterationinbreastepithelial cells (Pengetal.,2010).VitaminD3up-regulatedprotein1(VDUP1) is regulated by miRNA-17-5p at the post-transcriptional levels in senescent fibroblasts (Zhuo et al., 2010). VDUP1 expression is increased in cancer cells (Takahashi et al., 2002; Dutta et al., 2005). In melanoma cell lines, the endogenous VDR mRNA level is inversely associated with expression of miRNA-125b (Essa et al., 2010), and calcitriol also reduced the miRNA-27b expression in these cell lines. In human colon cells, calcitriol induced miRNA-22 and may contribute to its antitumor action against this neoplasm (Alvarez-Diaz et al., 2012). Fifteen miRNAs are also differentially regulated by calcitriol in prostate cancer cells (LNCaP) (Wang etal2011a).Furthermore, calcitriolregulatedmiRNA-32 andmiRNA-181expressions inhuman myeloid leukemia cells (Gocek et al., 2011; Zimmerman et al., 2011; Wang et al., 2009a).

#### **2.4. Renin-Angiotensin System (RAS)**

genetic risk factors for developing tumors. There are five important common polymorphisms within the *VDR* gene region that are likely to exert functional effects on *VDR* expression. The anti-carcinogenic potential of vitamin D might be mediated by VDR expression. The associa‐ tion between plasma 25OHD levels and colorectal adenoma was modified by the *Taq*I polymorphism of the VDR gene (Yamaji et al., 2011). There is a significant association between single nucleotide polymorphisms (SNPs) in the VDR gene and vitamin D intake in African Americans with colorectal cancer (Kupfer et al., 2011). The *Bsm*I polymorphism of the VDR gene also modified the association between dietary vitamin D intake and breast cancer (Rollison et al., 2012). The *AA* genotype of VDR is reported to be associated with colorectal cancer, with a stronger association in female patients (Mahmoudi et al., 2012). The *Fok*I and *Bsm*I genotypes of VDR gene are implicated in the pathogenesis of renal cell carcinoma (RCC) in a North Indian population (Arjumand et al., 2012). Altered VDR expression was associated with RCC carcinogenesis via the expression of epithelial Ca2+ channel transient receptor potential vanilloid subfamily 5 and 6 (TRPV5/6) (Wu et al., 2011b). There is a significant association between shorter progression-free survival time in patients with head and neck squamous cell carcinoma and the *Fok*I *TT* genotype, as well as the *Cdx2*-*Fox*I-*Apa*I haplotype (Hama et al., 2011). In Spanish children, osteosarcoma patients showed a significantly higher frequency of the *Ff* genotype of the *Fok*I *VDR* gene than the control group (Ruza et al., 2003). In a German population, the *AaTtBb* genotype of the VDR gene is associated with basal cell carcinoma risk, whereas the *aaTTbb* genotype is found at a high frequency in both basal cell carcinomas and cutaneous squamous cell carcinomas compared with controls (Köststner et al., 2012). In a systematic review, *Taq*I, *Bsm*I and *Fok*I polymorphisms of the VDR gene were found to be associated with malignant melanoma (Denzer et al., 2011). Furthermore, the presence of specific VDR *Bsm*I and *Taq*I alleles was associated with a higher C-reactive protein (CRP) level in cancer patients with cachectic syndrome (Punzi et al., 2012). In another pro‐ spective study, plasma 25OHD levels and common variation among several vitamin D-related genes (*CYP27A1*, *CYP2R1*, *CYP27B1*, *CYP24A1*, *GC*, *RXRA*, and *VDR*) were associated with lethal prostate cancer risk (Shui et al., 2012). Slattery et al. (2009) examined genetic variants that are linked to the pathway that contribute to colon cancer. They revealed that *Fox*I *VDR* polymorphism was associated with CpG Island methylator phenotype (CIMP) positive/Ki-ras mutated tumors, whereas the Poly *A* and *Cdx2 VDR* polymorphisms were associated only with

420 Cancer Treatment - Conventional and Innovative Approaches

MiRNAs are endogenous noncoding RNAs that regulate gene expression through the transla‐ tional repression or degradation of target mRNA (Bartel, 2004). Aberrant miRNA expression has been well characterized in cancer (Lu et al., 2005). Circulating miRNAs are suggested to be diagnostic and prognostic markers in breast cancer (Cortez et al., 2012). Circulating miR‐ NA-125b expression is associated with chemotherapeutic resistance of breast cancer (Wang et al., 2012b). Several miRNAs are found to share 125b complementarity with a sequence in the 3' unstranslated region of human VDR mRNA. The overexpression miRNA-125b significantly decreased the endogenous VDR protein level in human breast adenocarcinoma cells lines (MCF-7) to 40% of the control (Mohri et al., 2009). This miRNA is down-regulated in cancer

Ki-ras mutated tumors.

**2.3. MicroRNA (miRNA)**

The primary function of the renin-angiotensin system (RAS) is to maintain fluid homeostasis and regulate blood pressure. The angiotensin converting enzyme (ACE) is a key enzyme in the RAS and converts angiotensin (AT) I to the potent vasoconstrictor AT II (Johnston, 1994). The local RAS may influence tissue angiogenesis, cellular proliferation, apoptosis, and inflammation (Deshayes and Nahmias, 2005). Epidemiological and experimental studies suggested that the RAS may contribute to the paracrine regulation of tumor growth. The renin levels are elevated in patients with liver cirrhosis and HCC and positively correlated with αfetoprotein (Lotfy et al., 2010). The over-expression of ACE is reported in extrahepatic cholangiocarcinoma (Beyazit et al., 2011), leukemic myeloid blast cells (Aksu et al., 2006), and macrophages in the lymph nodes of Hodgkin's disease patients (Koca et al., 2007). The AT II receptors were also expressed in all human gastric cancer lines (Huang et al., 2008), premalignant and malignant prostate cells (Louis et al., 2007), human lung cancer xenografts (Feng et al., 2011a), and ovarian cancer (Ino et al., 2006). The RAS mutation in codon 61 was the most common genetic alteration in poorly differentiated thyroid carcinomas (Volante et al., 2009). The ACE *I/D* polymorphism is a possible target for developing genetic markers for breast cancer in Brazilian women (Alves Corrêa et al., 2009). The ACE *I/D* polymorphisms play an important role in breast cancer risk and disease-free survival in Caucasian postmenopausal women (González-Zuloeta Ladd et al., 2012). Carriers of the high-activity *DD* genotype had an increased risk of breast cancer compared with low activity *II/ID* genotype carriers (van der Knaap et al., 2008). The *DD* genotype was associated with patients with an aggressive stage of prostate cancer (Wang et al., 2011b). ACE2 expression was decreased in non-small-cell lung cancer and pancreatic ductal adenocarcinoma in which AT II levels were higher than those in controls (Feng et al., 2010; Zhou et al., 2009). ACE2 has been suggested as a potential molecular target for pancreatic cancer therapy (Zhou et al., 2011). The AT II concentration in gastric cancer region was significantly higher than those of normal region (Kinoshita et al., 2009). Further‐ more, AT II receptor blockers (ARB) suppress the cell proliferation effects of AT II in breast cancer cells (Du et al., 2012). The addition of ACE inhibitor or ARB to platinum-based first line chemotherapy contributed to prolong survival in patients with advanced lung cancer (Wilop et al., 2009) and affected the prognosis of advanced pancreatic cancer patients receiving gemcitabine (Nakai et al., 2010). The RAS inhibitors also improved the outcome of sunitinib treatment in metastatic renal cell carcinoma (Keizman et al., 2011). On the other hand, the administration of ACE inhibitors in patients with the ACE *DD* genotype has been shown to decrease the level of calcitriol required (Pérez-Castrillón et al., 2006). In a hypertensive Turkish population, the presence of the ACE *D* allele, which correlates negatively with serum 25OHD levels, is linked to a higher left ventricular mass index value and elevated ambulatory blood pressure measurements (Kulah et al., 2007). In addition, genetic disruption of the *VDR* gene resulted in overstimulation of the RAS with increased renin and angiotensin II production, which lead to high blood pressure and cardiac hypertrophy. However, treatment with captopril reduced cardiac hypertrophy in VDR-knockout mice (Xiang et al., 2005), suggesting that calcitriol may function as an endocrine suppressor of renin biosynthesis. Moreover, calcitriol suppresses renin gene transcription by blocking the activity of the cyclic AMP response element in the renin core promoter (Yuan et al., 2007) and decreases ACE activity in bovine endothelial cells (Higiwara et al., 1988).

been shown to inhibit the induction of cathelicidin mRNA by more than 80%, thereby reducing the protein expression of this antimicrobial agent by approximately 70% (Yim et al., 2007). Cathelicidinwasabundantintumor-infiltratingNK1.1+ cellsinmice.Cathelicidinknockoutmice (*Camp*-/-) permitted faster tumor growth than wild type controls; NK cells derived from *Camp*-/ mice showed impaired cytotoxic activity toward tumor targets compared with wild-type mice (Büchau et al., 2010). The human cathelicidin LL-37, which inhibits gastric cancer cell prolifera‐ tion,isdown-regulatedingastricadenocarcinomas(Wuetal.,2010).Gastrointestinalcancercells lacked LL-37 expression; Cathelicidin expression is modulated by histone-deacetylase (HDAC) inhibitors in various gastrointestinal cells, including gastric and hepatocellular cells (Schauer et al., 2004). HDAC inhibitors enhance the acetylation of core proteins, which is linked to the formation of transcriptionally active chromatin in various cells. The expression of the LL-37/ hCAP-18 gene was also reduced in some leukemia cells (Yang et al., 2003). In patients with acute myeloid leukemia, there was a marked reduction of LL-37/hCAP-18 expression in the peripher‐ al blood compared with the level in healthy donors (An et al., 2005). In myeloid cells, cathelici‐ din gene is a direct target of the VDR and is strongly up-regulated by calcitriol (Gombart et al., 2005). The combination of TLR ligands (CpG oligodeoxynucleotides, CpG-ODN) LL-37 generat‐ edsignificantlybettertherapeutictumoreffectsandenhancedsurvivalinmurineovariantumor-

The Impact of Vitamin D in Cancer http://dx.doi.org/10.5772/55324 423

bearing mice compared with CpG-ODN or LL-37 alone (Chuang et al., 2009).

The BCG vaccine was developed to provide protection against tuberculosis and has also been demonstrated to offer protection against cancer. The combination of BCG and ionizing radiation resulted in the induction of autophagy in colon cancer cells (Yuk et al., 2010). Intravesical BCG therapy has been demonstrated to reduce the recurrence rate and the risk of progression to muscle-invasive disease in patients with superficial bladder tumors (Herr et al., 1988). The BCG vaccination significantly prolongs the survival of patients with a malignant melanoma after initial surgical removed (Kölmel et al., 2005) and improved survival rates in patients with resected lung cancer (Repin, 1992). BCG inoculation delayed the tumor growth and prolonged the survival time in nude mice with leukemia (Wang et al., 2011c). BCG vaccination reduced the risk of lymphomas in a Danish population (Villumsen et al., 2009) and demonstrated to reduce the mortality, morbidity, and frequency of myeloic and chronic leukemia in children (Ambrosch et al., 1981). On the other hand, BCG-vaccinated infants are almost 6 times more likely to have sufficient vitamin D concentrations than unvaccinated infants 3 months after BCG vaccination, and this association remains strong even after adjusting for season, ethnic group and sex (Lalor et al., 2011). Among the vaccinated group, there was also a strong inverse correlation between the IFN-γ response to *M. tuberculosis* PPD and vitamin D concentration; infants with higher vitamin D concentrations had lower IFN-γ responses. Similarly, tuberculosis in cattle usually presents with a rapid transient increase in serum calcitriol within the first two weeks following infection (Rhodes et al., 2003). 1,25OHDpositive mononuclear cells were later identified in all of the tuberculous granulomas. During

**3. Role of vitamin D and its analog in cancer**

**3.1. The bacillus Calmette-Guerin (BCG) vaccination**

### **2.5. Toll-Like Receptor (TLR)**

Toll-like receptors (TLRs) are a group of glycoproteins that functions as surface trans-mem‐ brane receptors and are involved in the innate immune responses to exogenous pathogenic microorganisms. Substantial evidence exists for an important role of TLRs in the pathogenesis andoutcomesofcancer.TLR2expressionwassignificantlyhigherinsporadiccolorectalcancerous tissue than in non-cancerous tissue (Nihon-Yanagi et al., 2012). The TLR5 play an important role in tumor progression of gastric cancer (Song et al., 2011). The TLR7 and TLR9 showed high expression in laryngeal carcinoma cells (Shikora et al., 2010). The over-expression of TLR9 was reported oral squamous cell carcinoma (Min et al., 2011), esophageal squamous cell carcinoma (Takala et al., 2011), and breast cancer cells (Qiu et al., 2011; Sandholm et al., 2012). The expres‐ sion levels of TLR1, TLR2, TLR4, TLR5, TLR6, TLR8, and TLR10 are significantly higher in the humanrenalcarcinomacellline(780-6)thanthoseinnormalrenalcell(HK-2)line(Yuetal.,2011). Chronic lymphocytic leukemia cells express all TLRs expressed by normal activated B cells, with a high expression of TLR9 and CD180 and an intermediate expression of TLR1, TLR6, and TLR10 (Arvaniti et al., 2011). The TLR4 polymorphisms are reported in patients with the risk of pros‐ tate cancer (Kim et al., 2012), head and neck squamous cell carcinomas (Bergmann et al., 2011), HCC (Minmin et al., 2011), and colon cancer (Eyking et al., 2011). Furthermore, multiple SNPs in TLR2 and TLR4 were associated with colon cancer survival (Slattery et al., 2012). On the other hand, vitamin D deficiency increases the expression of hepatic mRNA levels of TLR2, TLR4, and TLR9 in obese rats (Roth et al., 2011). However, calcitriol suppresses the expression of TLR2 and TLR4 protein and mRNA in human monocytes and triggers hypo-responsiveness to pathogenassociated molecular patterns (Sadeghi et al., 2006). Calcitriol has also been shown to downregulateintracellularTLR2,TLR4andTLR9expressioninhumanmonocytes(Dickieetal.,2010). TLR activation results in the expression of the VDR and 1α-vitamin D hydroxylase in human monocytes (Liu et al., 2006). Additionally, calcitriol can cause the vitamin D-induced expres‐ sion of cathelicidin in bronchial epithelial cells (Yim et al., 2007) and may enhance the produc‐ tion of cathelicidin LL-37 (Rivas-Santiago et al., 2008). The addition of a VDR antagonist has also

been shown to inhibit the induction of cathelicidin mRNA by more than 80%, thereby reducing the protein expression of this antimicrobial agent by approximately 70% (Yim et al., 2007). Cathelicidinwasabundantintumor-infiltratingNK1.1+ cellsinmice.Cathelicidinknockoutmice (*Camp*-/-) permitted faster tumor growth than wild type controls; NK cells derived from *Camp*-/ mice showed impaired cytotoxic activity toward tumor targets compared with wild-type mice (Büchau et al., 2010). The human cathelicidin LL-37, which inhibits gastric cancer cell prolifera‐ tion,isdown-regulatedingastricadenocarcinomas(Wuetal.,2010).Gastrointestinalcancercells lacked LL-37 expression; Cathelicidin expression is modulated by histone-deacetylase (HDAC) inhibitors in various gastrointestinal cells, including gastric and hepatocellular cells (Schauer et al., 2004). HDAC inhibitors enhance the acetylation of core proteins, which is linked to the formation of transcriptionally active chromatin in various cells. The expression of the LL-37/ hCAP-18 gene was also reduced in some leukemia cells (Yang et al., 2003). In patients with acute myeloid leukemia, there was a marked reduction of LL-37/hCAP-18 expression in the peripher‐ al blood compared with the level in healthy donors (An et al., 2005). In myeloid cells, cathelici‐ din gene is a direct target of the VDR and is strongly up-regulated by calcitriol (Gombart et al., 2005). The combination of TLR ligands (CpG oligodeoxynucleotides, CpG-ODN) LL-37 generat‐ edsignificantlybettertherapeutictumoreffectsandenhancedsurvivalinmurineovariantumorbearing mice compared with CpG-ODN or LL-37 alone (Chuang et al., 2009).

### **3. Role of vitamin D and its analog in cancer**

et al., 2009) and affected the prognosis of advanced pancreatic cancer patients receiving gemcitabine (Nakai et al., 2010). The RAS inhibitors also improved the outcome of sunitinib treatment in metastatic renal cell carcinoma (Keizman et al., 2011). On the other hand, the administration of ACE inhibitors in patients with the ACE *DD* genotype has been shown to decrease the level of calcitriol required (Pérez-Castrillón et al., 2006). In a hypertensive Turkish population, the presence of the ACE *D* allele, which correlates negatively with serum 25OHD levels, is linked to a higher left ventricular mass index value and elevated ambulatory blood pressure measurements (Kulah et al., 2007). In addition, genetic disruption of the *VDR* gene resulted in overstimulation of the RAS with increased renin and angiotensin II production, which lead to high blood pressure and cardiac hypertrophy. However, treatment with captopril reduced cardiac hypertrophy in VDR-knockout mice (Xiang et al., 2005), suggesting that calcitriol may function as an endocrine suppressor of renin biosynthesis. Moreover, calcitriol suppresses renin gene transcription by blocking the activity of the cyclic AMP response element in the renin core promoter (Yuan et al., 2007) and decreases ACE activity in

Toll-like receptors (TLRs) are a group of glycoproteins that functions as surface trans-mem‐ brane receptors and are involved in the innate immune responses to exogenous pathogenic microorganisms. Substantial evidence exists for an important role of TLRs in the pathogenesis andoutcomesofcancer.TLR2expressionwassignificantlyhigherinsporadiccolorectalcancerous tissue than in non-cancerous tissue (Nihon-Yanagi et al., 2012). The TLR5 play an important role in tumor progression of gastric cancer (Song et al., 2011). The TLR7 and TLR9 showed high expression in laryngeal carcinoma cells (Shikora et al., 2010). The over-expression of TLR9 was reported oral squamous cell carcinoma (Min et al., 2011), esophageal squamous cell carcinoma (Takala et al., 2011), and breast cancer cells (Qiu et al., 2011; Sandholm et al., 2012). The expres‐ sion levels of TLR1, TLR2, TLR4, TLR5, TLR6, TLR8, and TLR10 are significantly higher in the humanrenalcarcinomacellline(780-6)thanthoseinnormalrenalcell(HK-2)line(Yuetal.,2011). Chronic lymphocytic leukemia cells express all TLRs expressed by normal activated B cells, with a high expression of TLR9 and CD180 and an intermediate expression of TLR1, TLR6, and TLR10 (Arvaniti et al., 2011). The TLR4 polymorphisms are reported in patients with the risk of pros‐ tate cancer (Kim et al., 2012), head and neck squamous cell carcinomas (Bergmann et al., 2011), HCC (Minmin et al., 2011), and colon cancer (Eyking et al., 2011). Furthermore, multiple SNPs in TLR2 and TLR4 were associated with colon cancer survival (Slattery et al., 2012). On the other hand, vitamin D deficiency increases the expression of hepatic mRNA levels of TLR2, TLR4, and TLR9 in obese rats (Roth et al., 2011). However, calcitriol suppresses the expression of TLR2 and TLR4 protein and mRNA in human monocytes and triggers hypo-responsiveness to pathogenassociated molecular patterns (Sadeghi et al., 2006). Calcitriol has also been shown to downregulateintracellularTLR2,TLR4andTLR9expressioninhumanmonocytes(Dickieetal.,2010). TLR activation results in the expression of the VDR and 1α-vitamin D hydroxylase in human monocytes (Liu et al., 2006). Additionally, calcitriol can cause the vitamin D-induced expres‐ sion of cathelicidin in bronchial epithelial cells (Yim et al., 2007) and may enhance the produc‐ tion of cathelicidin LL-37 (Rivas-Santiago et al., 2008). The addition of a VDR antagonist has also

bovine endothelial cells (Higiwara et al., 1988).

422 Cancer Treatment - Conventional and Innovative Approaches

**2.5. Toll-Like Receptor (TLR)**

### **3.1. The bacillus Calmette-Guerin (BCG) vaccination**

The BCG vaccine was developed to provide protection against tuberculosis and has also been demonstrated to offer protection against cancer. The combination of BCG and ionizing radiation resulted in the induction of autophagy in colon cancer cells (Yuk et al., 2010). Intravesical BCG therapy has been demonstrated to reduce the recurrence rate and the risk of progression to muscle-invasive disease in patients with superficial bladder tumors (Herr et al., 1988). The BCG vaccination significantly prolongs the survival of patients with a malignant melanoma after initial surgical removed (Kölmel et al., 2005) and improved survival rates in patients with resected lung cancer (Repin, 1992). BCG inoculation delayed the tumor growth and prolonged the survival time in nude mice with leukemia (Wang et al., 2011c). BCG vaccination reduced the risk of lymphomas in a Danish population (Villumsen et al., 2009) and demonstrated to reduce the mortality, morbidity, and frequency of myeloic and chronic leukemia in children (Ambrosch et al., 1981). On the other hand, BCG-vaccinated infants are almost 6 times more likely to have sufficient vitamin D concentrations than unvaccinated infants 3 months after BCG vaccination, and this association remains strong even after adjusting for season, ethnic group and sex (Lalor et al., 2011). Among the vaccinated group, there was also a strong inverse correlation between the IFN-γ response to *M. tuberculosis* PPD and vitamin D concentration; infants with higher vitamin D concentrations had lower IFN-γ responses. Similarly, tuberculosis in cattle usually presents with a rapid transient increase in serum calcitriol within the first two weeks following infection (Rhodes et al., 2003). 1,25OHDpositive mononuclear cells were later identified in all of the tuberculous granulomas. During tuberculosis infection, alveolar macrophage-produced calcitriol plays a beneficial role by limiting inflammation-mediated tissue injury, potentiating NO production by stimulated monocytes/macrophages, inhibiting INF-γ production by stimulated CD4+ cells, and sup‐ pressing the growth of *M. tuberculosis* (Ametaj et al., 1996; Rockett et al., 1998).

(Nguyen et al., 2011). Moreover, calciferol, calcitriol, and vitamin D analogs decreased MMP-2 and MMP-9 activities and inhibited prostate cancer cell invasion (Tokar and Webber, 2005; Schartz et al., 1997; Iglesias-Gato et al., 2011; Stio et al., 2011). A vitamin D analog has also been reported to reduce the expression of MMP-2, MMP-9, vascular endothelial growth factor (VEGF) and PTH-related peptide in Lewis lung carcinoma cells (Nakagawa et al., 2005). Taken together, these studies suggest that calcitriol may play an important role in the pathological processes in cancer by down-regulating the level of MMPs and regulating the level of TIMPs.

The Impact of Vitamin D in Cancer http://dx.doi.org/10.5772/55324 425

The Wnt/β-catenin signaling pathway plays a pivotal role in the regulation of cell growth, cell development and the differentiation of normal stem cells. Wnt/β-catenin signaling is implicat‐ ed in many human cancers, including gastrointestinal cancer, gastric cancer, colon cancer, melanoma, HCC, endometrial carcinoma, ovarian carcinoma, cervical cancer, papillary thyroid carcinoma, renal cell carcinoma, prostate cancer, parathyroid carcinoma, and hematological malignancies (White et al., 2012; Nuñez et al., 2011; Polakis, 2000; Li et al., 2012; Yoshioka et al., 2012; Guturi et al., 2012; Bulut et al., 2011; Gilber-Sirieix et al., 2011; Ueno et al., 2011; Svedlund et al., 2010; Ge and Wang, 2010). Calcitriol inhibits β-catenin transcriptional activity by promoting VDR binding to β-catenin and the induction of E-cadherin expression (Palmer et al., 2001). Paricalcitol, a vitamin D analog, suppressed β-catenin-mediated gene transcription and amelio‐ rated proteinuria and kidney injury in adriamycin nephropathy (He et al., 2011). Most VDR variants fail to activate the vitamin D-responsive promoter and also fail to bind β-catenin or regulateitsactivity(ByersandShah,2007).VDRdepletionenhancesWnt/β-cateninsignalingand the tumor burden in colon cancer (Larriba et al., 2011). The action of cacitriol on colon carcinoma cells depends on the dual action of VDR as a transcription factor and a nongenomic activator of RhoA-ROCK and p38MAPK-MSK1, which are required for the inhibition of the Wnt/β-catenin signaling pathway and cell proliferation (Ordóñez-Morán et al., 2008). The *DICKKOFF-*4 gene induces a malignant phenotype, promotes tumor cell invasion, and angiogenesis in colon cancer cells and is repressed by calcitriol (Pendás-Franco et al., 2008a); whereas *DICKKOFF-*1 gene acts as a tumor suppressor in human colon cells and is up-regulated by calcitriol (Aguilera et al., 2007; Pendás-Franco et al., 2008b). The transcription factor TCF-4 acts as transcriptional repressor in breast and colorectal cancer cell growth. The TCF-4 and β-catenin binding partner are indirect targets of the VDR pathway. In the VDR knockout mouse, TCF-4 is decreased in the mammary gland when compared with a wild-type mouse. In addition, calcitriol increases TCF-4 RNA and proteinlevelsinseveralhumancolorectalcancercelllines(Beildecketal.,2009).Furthermore,the Snail1 gene is associated with gastric cancer, melanoma, breast cancer, HCC, and colon carcino‐ ma. Calcitriol inhibits the Wnt/β-catenin signaling pathway and is abrogated by Snail1 in human

**3.3. Wnt/β-catenin**

colon cancer cells (Larriba et al., 2007).

**3.4. The Mitogen-Activated Protein Kinase (MAPK) pathways**

The MAPK pathways provide a key link between the membrane bound receptors that receive these cues and changes in the pattern of gene expression, including the extracellular signalregulated kinase (ERK) cascade, the stress activated protein kinases/c-jun N-terminal kinase

#### **3.2. Matrix Metalloproteinase (MMPs)**

MMPs are proteolytic enzymes responsible for extracellular matrix remodeling and the regulation of leukocyte migration through the extracellular matrix, which is an important step in inflammatory and infectious pathophysiology. MMPs are produced by many cell types including lymphocytes, granulocytes, astrocytes and activated macrophages. The MMP-1 expression is linked to sarcoma cell invasion (Garamszegi et al., 2011). MMP-2 expression is increased in gastric cancer cells (Partyka et al., 2012) and colorectal cancer (Dong et al., 2011). MMP-9 is expressed in many cancer cells, such as those associated with non-small-cell lung cancer (Peng et al., 2012), ovarian cancer invasion and metastasis (Zhang et al., 2011a), glioblastoma multiforme (Yan et al., 2011), and adamantinous craniopharyndioma (Xia et al., 2011). The MMP-2 and MMP-9 secreted by leukemic cells increase the permeability of blood brain barrier of the CNS by disrupting tight junction proteins (Feng et al., 2011b). In gastric cancer, MMP-2 and MMP-9 play an important role in tumor invasion and metastasis (Parsons et al., 1998). The risks for the development of hypophyseal adenoma and cervical neoplasia are greater in patients with MMP-1 polymorphisms (Altaş et al., 2010; Tee et al., 2012) than those with the wild-type allele. The MMP-2 polymorphism contributed to prostate cancer susceptibility in North India (Srivastava et al., 2012) and to the clinical outcome of Chinese patients with non-small cell lung cancer treated with first-line, platinum-based chemotherapy (Zhao et al., 2011). The MMP-7 polymorphisms are associated with esophageal squamous cell carcinoma and colorectal cancers (Manzoor et al., 2011; Dziki et al., 2011). The SPNs in the MMP-2 and MMP-9 region are associated with susceptibility to head and neck squamous cell carcinoma in an Indian population (Chaudhary et al., 2011). The SNPs of genes encoding MMPs (-1, -2, -3, -7, -8, -9, -12, -13, and -21) are related to breast cancer risk, progression, and survival (Wieczorek et al., 2012). Based on meta-analysis studies, the MMP-2 allele (-1306T) is a protective factor for digestive cancer risk (Zhang and Ren, 2011), the MMP-9 polymorphism is associated with a lower risk of colorectal cancer (Zhang et al., 2012a), and polymorphisms in the promoter regions of MMP-1, -3, -7, and -9 are associated with metastasis in some cancers (Liu et al., 2012). On the other hand, VDR-knock-out mice were shown to have an influx of inflammatory cells, phospho-acetylation of NF-κв, and up-regulated expression of MMP-2, MMP-9, and MMP-12 in the lung (Sundar et al., 2011). The *VDR TaqI* polymorphism is associated with decreased production of TIMP-1, a natural inhibitor of MMP-9 (Timms et al., 2002). In addition, calcitriol modulates tissue MMP expression under experimental conditions (Dean et al., 1996), down-regulates MMP-9 levels in keratinocytes, and may attenuate the deleterious effects of excessive TNF-α-induced proteolytic activity associated with cutaneous inflammation (Bahar-Shang et al., 2010). Calcitriol decreased the invasive properties of breast carcinoma cells and decreased MMP-9 levels in association with the increased levels of the tissue inhibitor of MMP-1 activity (Koli and Keshi-Oja, 2000). Calcitriol also inhibits endome‐ trial cancer cell growth and is associated with decreased MMP-2 and MMP-9 expression (Nguyen et al., 2011). Moreover, calciferol, calcitriol, and vitamin D analogs decreased MMP-2 and MMP-9 activities and inhibited prostate cancer cell invasion (Tokar and Webber, 2005; Schartz et al., 1997; Iglesias-Gato et al., 2011; Stio et al., 2011). A vitamin D analog has also been reported to reduce the expression of MMP-2, MMP-9, vascular endothelial growth factor (VEGF) and PTH-related peptide in Lewis lung carcinoma cells (Nakagawa et al., 2005). Taken together, these studies suggest that calcitriol may play an important role in the pathological processes in cancer by down-regulating the level of MMPs and regulating the level of TIMPs.

### **3.3. Wnt/β-catenin**

tuberculosis infection, alveolar macrophage-produced calcitriol plays a beneficial role by limiting inflammation-mediated tissue injury, potentiating NO production by stimulated monocytes/macrophages, inhibiting INF-γ production by stimulated CD4+ cells, and sup‐

MMPs are proteolytic enzymes responsible for extracellular matrix remodeling and the regulation of leukocyte migration through the extracellular matrix, which is an important step in inflammatory and infectious pathophysiology. MMPs are produced by many cell types including lymphocytes, granulocytes, astrocytes and activated macrophages. The MMP-1 expression is linked to sarcoma cell invasion (Garamszegi et al., 2011). MMP-2 expression is increased in gastric cancer cells (Partyka et al., 2012) and colorectal cancer (Dong et al., 2011). MMP-9 is expressed in many cancer cells, such as those associated with non-small-cell lung cancer (Peng et al., 2012), ovarian cancer invasion and metastasis (Zhang et al., 2011a), glioblastoma multiforme (Yan et al., 2011), and adamantinous craniopharyndioma (Xia et al., 2011). The MMP-2 and MMP-9 secreted by leukemic cells increase the permeability of blood brain barrier of the CNS by disrupting tight junction proteins (Feng et al., 2011b). In gastric cancer, MMP-2 and MMP-9 play an important role in tumor invasion and metastasis (Parsons et al., 1998). The risks for the development of hypophyseal adenoma and cervical neoplasia are greater in patients with MMP-1 polymorphisms (Altaş et al., 2010; Tee et al., 2012) than those with the wild-type allele. The MMP-2 polymorphism contributed to prostate cancer susceptibility in North India (Srivastava et al., 2012) and to the clinical outcome of Chinese patients with non-small cell lung cancer treated with first-line, platinum-based chemotherapy (Zhao et al., 2011). The MMP-7 polymorphisms are associated with esophageal squamous cell carcinoma and colorectal cancers (Manzoor et al., 2011; Dziki et al., 2011). The SPNs in the MMP-2 and MMP-9 region are associated with susceptibility to head and neck squamous cell carcinoma in an Indian population (Chaudhary et al., 2011). The SNPs of genes encoding MMPs (-1, -2, -3, -7, -8, -9, -12, -13, and -21) are related to breast cancer risk, progression, and survival (Wieczorek et al., 2012). Based on meta-analysis studies, the MMP-2 allele (-1306T) is a protective factor for digestive cancer risk (Zhang and Ren, 2011), the MMP-9 polymorphism is associated with a lower risk of colorectal cancer (Zhang et al., 2012a), and polymorphisms in the promoter regions of MMP-1, -3, -7, and -9 are associated with metastasis in some cancers (Liu et al., 2012). On the other hand, VDR-knock-out mice were shown to have an influx of inflammatory cells, phospho-acetylation of NF-κв, and up-regulated expression of MMP-2, MMP-9, and MMP-12 in the lung (Sundar et al., 2011). The *VDR TaqI* polymorphism is associated with decreased production of TIMP-1, a natural inhibitor of MMP-9 (Timms et al., 2002). In addition, calcitriol modulates tissue MMP expression under experimental conditions (Dean et al., 1996), down-regulates MMP-9 levels in keratinocytes, and may attenuate the deleterious effects of excessive TNF-α-induced proteolytic activity associated with cutaneous inflammation (Bahar-Shang et al., 2010). Calcitriol decreased the invasive properties of breast carcinoma cells and decreased MMP-9 levels in association with the increased levels of the tissue inhibitor of MMP-1 activity (Koli and Keshi-Oja, 2000). Calcitriol also inhibits endome‐ trial cancer cell growth and is associated with decreased MMP-2 and MMP-9 expression

pressing the growth of *M. tuberculosis* (Ametaj et al., 1996; Rockett et al., 1998).

**3.2. Matrix Metalloproteinase (MMPs)**

424 Cancer Treatment - Conventional and Innovative Approaches

The Wnt/β-catenin signaling pathway plays a pivotal role in the regulation of cell growth, cell development and the differentiation of normal stem cells. Wnt/β-catenin signaling is implicat‐ ed in many human cancers, including gastrointestinal cancer, gastric cancer, colon cancer, melanoma, HCC, endometrial carcinoma, ovarian carcinoma, cervical cancer, papillary thyroid carcinoma, renal cell carcinoma, prostate cancer, parathyroid carcinoma, and hematological malignancies (White et al., 2012; Nuñez et al., 2011; Polakis, 2000; Li et al., 2012; Yoshioka et al., 2012; Guturi et al., 2012; Bulut et al., 2011; Gilber-Sirieix et al., 2011; Ueno et al., 2011; Svedlund et al., 2010; Ge and Wang, 2010). Calcitriol inhibits β-catenin transcriptional activity by promoting VDR binding to β-catenin and the induction of E-cadherin expression (Palmer et al., 2001). Paricalcitol, a vitamin D analog, suppressed β-catenin-mediated gene transcription and amelio‐ rated proteinuria and kidney injury in adriamycin nephropathy (He et al., 2011). Most VDR variants fail to activate the vitamin D-responsive promoter and also fail to bind β-catenin or regulateitsactivity(ByersandShah,2007).VDRdepletionenhancesWnt/β-cateninsignalingand the tumor burden in colon cancer (Larriba et al., 2011). The action of cacitriol on colon carcinoma cells depends on the dual action of VDR as a transcription factor and a nongenomic activator of RhoA-ROCK and p38MAPK-MSK1, which are required for the inhibition of the Wnt/β-catenin signaling pathway and cell proliferation (Ordóñez-Morán et al., 2008). The *DICKKOFF-*4 gene induces a malignant phenotype, promotes tumor cell invasion, and angiogenesis in colon cancer cells and is repressed by calcitriol (Pendás-Franco et al., 2008a); whereas *DICKKOFF-*1 gene acts as a tumor suppressor in human colon cells and is up-regulated by calcitriol (Aguilera et al., 2007; Pendás-Franco et al., 2008b). The transcription factor TCF-4 acts as transcriptional repressor in breast and colorectal cancer cell growth. The TCF-4 and β-catenin binding partner are indirect targets of the VDR pathway. In the VDR knockout mouse, TCF-4 is decreased in the mammary gland when compared with a wild-type mouse. In addition, calcitriol increases TCF-4 RNA and proteinlevelsinseveralhumancolorectalcancercelllines(Beildecketal.,2009).Furthermore,the Snail1 gene is associated with gastric cancer, melanoma, breast cancer, HCC, and colon carcino‐ ma. Calcitriol inhibits the Wnt/β-catenin signaling pathway and is abrogated by Snail1 in human colon cancer cells (Larriba et al., 2007).

### **3.4. The Mitogen-Activated Protein Kinase (MAPK) pathways**

The MAPK pathways provide a key link between the membrane bound receptors that receive these cues and changes in the pattern of gene expression, including the extracellular signalregulated kinase (ERK) cascade, the stress activated protein kinases/c-jun N-terminal kinase (SAPK/JNK)cascade,andthep38MAPK/RK/HOGcascade(HipskindandBilbe,1998).Inhuman coloncancercells,calcitriolincreasescytosolicCa2+concentrationandtransientlyactivatesRhoA-ROCK, and then activates the p38MAPK-MSK signaling pathway (Ordóñez-Morán et al., 2008). In breast cancer cells, the MARK (JNK and p38) signaling pathway involved in calcitriol-in‐ ducedbreastcelldeath(Brosseauetal.,2010)andpotentiatedthecytotoxicactionofcalcitrioland TNF-α (Weitsman, et al., 2004). In murine squamous cell carcinoma cells, vitamin D induced apoptosis and selective induction of caspase-dependent MEK cleavage (McGuire et al., 2001). In an ovarian cancer animal model, vitamin D induced cell death and is mediated by the p38MAPK signalingpathway(Langeetal.,2010).Inhumanpromyeloblasticleukemiacells(HL60),vitamin D derivatives had anti-proliferative activity and activated MAPK signaling pathways (Ji et al., 2002). In human acute myeloid leukemia cells, calcitriol-induced differentiation is enhanced by the activation of MAPK signaling pathways (Zhang et al., 2011b).

Eleraetal.,2012).Singlenucleotidepolymorphismsofantioxidantdefensegenesmaysignificant‐ ly modify the functional activity of the encoded proteins. Women with genetic variability in the iron-relatedoxidativestresspathwaysmaybeatincreasedriskofpost-menopausalbreastcancer (Hong et al., 2007). The *ala* variant of superoxide dismutase (SOD) is associated with a moderate‐ ly increased risk of prostate cancer (Woodson et al., 2003). Based on meta-analysis studies, manganese SOD (MnSOD) polymorphisms may contribute to cancer development (Val-9Ala) (Wang et al., 2009b), prostate cancer susceptibility (Val-16Ala) (Mao et al., 2010), but not to breast cancer susceptibility (Val-16Ala) (Ma et al., 2010a). Calcitriol can also protect nonmalignant prostate cells from oxidative stress-induced cell death through the prevention of reactive oxygen species (ROS)-induced cellular injuries (Bao et al., 2008). Vitamin D metabolites and vitamin D analogshavebeenreportedtoinducelipoxygenasemRNAexpression,lipoxygenaseactivityand ROS in a human bone cell line (Somjen et al., 2011). Vitamin D can also reduce the extent of lipid peroxidationandinduceSODactivityinthehepaticanti-oxidantsystemofrats(Sardaretal.,1996). Moreover,theactivationofmacrophage1α-hydroxylaseresultsinanincreasein1,25OHD,which inhibits iNOS expression and reduces nitric oxide (NO) production by LPS-stimulated macro‐ phages (Chang et al., 2004). This calcitriol production by macrophages may provide protection against the oxidative injuries caused by the NO burst. Calcitriol is known to inhibit LPS-in‐ duced immune activation in human endothelial cells (Equil et al., 2005), and calcitriol has also been shown to enhance intracellular glutathione pools and significantly reduce the nitrite productioninducedbytheLPS(Garcionetal.,1999).Furthermore,overproductionofROSinduces DNA damage and leads to carcinogenesis. In the mouse colon, there was an inverse relationship betweenVDRlevelsandcolonichyperproliferation;theexpressionof8-hydroxy-2'-deoxyguano‐ sine (8-OHdG), a maker of oxidative DNA damage, significantly increased with complete loss of VDR (Kállay et al., 2002). Vitamin D decrease 8-OHdG by 22% in the normal human colorectal mucosa (Fedirko et al., 2012). Calcitriol contributes to a reduction of the DNA intensify replica‐ tion stress in lymphocytes (Halicka et al., 2012). In addition, vitamin D3 up-regulated protein 1(VDUP1)isaregularforredoxsignalingandstress-mediateddiseases(Chungetal.,2006).Taken

The Impact of Vitamin D in Cancer http://dx.doi.org/10.5772/55324 427

together, these findings suggest that vitamin D modulates oxidative stress in cancer.

A number of clinical trials have used vitamin D3 and calcitriol alone or in combination with anti-tumor agents. Most preclinical suggest that that the optimal anti-tumor effect of calcitriol and other analogs is seen with the administration of high dose calcitriol on intermittent schedule. A small number of single agent trials utilizing vitamin D3 and calcitriol hace been

Fifteen patients were given 2,000 IU (50 microg) of cholecalciferol daily and monitored prospectively every 2-3 mo. There was a statistically significant decrease in the rate of PSA rise after administration of cholecalciferol compared with that before cholecalciferol. The median PSA doubling time increased from 14.3 months prior to commencing cholecalciferol to 25

**4. The use of vitamin D in cancer treatment**

conducted with limited success.

**4.1. Vitamin D3 trials**

### **3.5. The Prostaglandins (PGs)**

Prostaglandins (PGs) play a role in inflammatory processes, and cyclooxygenase (COX) participates in the conversion of arachidonic acid in PGs. A variety studies have shown that prostaglandin signaling stimulates cancer cell growth and cancer progression. The regulation of PG metabolism and biological actions contribute to its anti-proliferation effects in prostate cells and calcitriol has been reported to regulate the expression of several key genes involved in the PG pathway, resulting in decreased PG synthesis (Moreno et al., 2005). The expression of the COX-2 gene is significantly increased in human gastric adenocarcinoma tissues com‐ pared with adjuvant normal gastric mucosal specimens (Ristimäki et al., 1997). There is inversely association between elevated COX-2 levels and decreased VDR expression in patients with breast and ovarian cancers compared with healthy women (Cordes et al., 2012). Calcitriol differentiated the human leukemic cell line (HL-60) and metabolized exogenous arachidonic acid to both COX products (predominantly thromboxane B2 and PG E2) and lipoxygenase products, including leukotriene B<sup>4</sup> (Stenson et al., 1988). In a mouse xenograft model of prostate cancer, the combination of cacitriol and dietary soy enhanced cacitriol activity in regulating target gene expression and increased the suppression of PG synthesis and signaling, such as COX-2, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), and PG receptors. (Wang et al., 2012a). Calcitriol and its analogs have also been shown to selectively inhibit the activity of COX-2 (Aparna et al., 2008), and an inverse correlation exists between the expression of PG-metabolizing enzymes and reduced VDR expression in malignant breast cell lines (Thill et al., 2012). Taken together, these findings suggest that vitamin D may play a role in modulating the inflammatory process in cancer.

#### **3.6. Oxidative stress**

Reactiveoxygenspecies(ROS)playamajorroleinvariouscell-signalingpathways.ROSactivates various transcription factors and increases in the expression of proteins that control cellular transformation, tumor cell survival, tumor cell proliferation and invasion, angiogenesis, and metastasis. ROS has an important role in the initiation and progression of many cancers (Gupta et al., 2012; Marra et al., 2011; Zhang et al., 2011c; Wang et al., 2011c; Rogalska et al., 2011; GuptaEleraetal.,2012).Singlenucleotidepolymorphismsofantioxidantdefensegenesmaysignificant‐ ly modify the functional activity of the encoded proteins. Women with genetic variability in the iron-relatedoxidativestresspathwaysmaybeatincreasedriskofpost-menopausalbreastcancer (Hong et al., 2007). The *ala* variant of superoxide dismutase (SOD) is associated with a moderate‐ ly increased risk of prostate cancer (Woodson et al., 2003). Based on meta-analysis studies, manganese SOD (MnSOD) polymorphisms may contribute to cancer development (Val-9Ala) (Wang et al., 2009b), prostate cancer susceptibility (Val-16Ala) (Mao et al., 2010), but not to breast cancer susceptibility (Val-16Ala) (Ma et al., 2010a). Calcitriol can also protect nonmalignant prostate cells from oxidative stress-induced cell death through the prevention of reactive oxygen species (ROS)-induced cellular injuries (Bao et al., 2008). Vitamin D metabolites and vitamin D analogshavebeenreportedtoinducelipoxygenasemRNAexpression,lipoxygenaseactivityand ROS in a human bone cell line (Somjen et al., 2011). Vitamin D can also reduce the extent of lipid peroxidationandinduceSODactivityinthehepaticanti-oxidantsystemofrats(Sardaretal.,1996). Moreover,theactivationofmacrophage1α-hydroxylaseresultsinanincreasein1,25OHD,which inhibits iNOS expression and reduces nitric oxide (NO) production by LPS-stimulated macro‐ phages (Chang et al., 2004). This calcitriol production by macrophages may provide protection against the oxidative injuries caused by the NO burst. Calcitriol is known to inhibit LPS-in‐ duced immune activation in human endothelial cells (Equil et al., 2005), and calcitriol has also been shown to enhance intracellular glutathione pools and significantly reduce the nitrite productioninducedbytheLPS(Garcionetal.,1999).Furthermore,overproductionofROSinduces DNA damage and leads to carcinogenesis. In the mouse colon, there was an inverse relationship betweenVDRlevelsandcolonichyperproliferation;theexpressionof8-hydroxy-2'-deoxyguano‐ sine (8-OHdG), a maker of oxidative DNA damage, significantly increased with complete loss of VDR (Kállay et al., 2002). Vitamin D decrease 8-OHdG by 22% in the normal human colorectal mucosa (Fedirko et al., 2012). Calcitriol contributes to a reduction of the DNA intensify replica‐ tion stress in lymphocytes (Halicka et al., 2012). In addition, vitamin D3 up-regulated protein 1(VDUP1)isaregularforredoxsignalingandstress-mediateddiseases(Chungetal.,2006).Taken together, these findings suggest that vitamin D modulates oxidative stress in cancer.

### **4. The use of vitamin D in cancer treatment**

A number of clinical trials have used vitamin D3 and calcitriol alone or in combination with anti-tumor agents. Most preclinical suggest that that the optimal anti-tumor effect of calcitriol and other analogs is seen with the administration of high dose calcitriol on intermittent schedule. A small number of single agent trials utilizing vitamin D3 and calcitriol hace been conducted with limited success.

### **4.1. Vitamin D3 trials**

(SAPK/JNK)cascade,andthep38MAPK/RK/HOGcascade(HipskindandBilbe,1998).Inhuman coloncancercells,calcitriolincreasescytosolicCa2+concentrationandtransientlyactivatesRhoA-ROCK, and then activates the p38MAPK-MSK signaling pathway (Ordóñez-Morán et al., 2008). In breast cancer cells, the MARK (JNK and p38) signaling pathway involved in calcitriol-in‐ ducedbreastcelldeath(Brosseauetal.,2010)andpotentiatedthecytotoxicactionofcalcitrioland TNF-α (Weitsman, et al., 2004). In murine squamous cell carcinoma cells, vitamin D induced apoptosis and selective induction of caspase-dependent MEK cleavage (McGuire et al., 2001). In an ovarian cancer animal model, vitamin D induced cell death and is mediated by the p38MAPK signalingpathway(Langeetal.,2010).Inhumanpromyeloblasticleukemiacells(HL60),vitamin D derivatives had anti-proliferative activity and activated MAPK signaling pathways (Ji et al., 2002). In human acute myeloid leukemia cells, calcitriol-induced differentiation is enhanced by

Prostaglandins (PGs) play a role in inflammatory processes, and cyclooxygenase (COX) participates in the conversion of arachidonic acid in PGs. A variety studies have shown that prostaglandin signaling stimulates cancer cell growth and cancer progression. The regulation of PG metabolism and biological actions contribute to its anti-proliferation effects in prostate cells and calcitriol has been reported to regulate the expression of several key genes involved in the PG pathway, resulting in decreased PG synthesis (Moreno et al., 2005). The expression of the COX-2 gene is significantly increased in human gastric adenocarcinoma tissues com‐ pared with adjuvant normal gastric mucosal specimens (Ristimäki et al., 1997). There is inversely association between elevated COX-2 levels and decreased VDR expression in patients with breast and ovarian cancers compared with healthy women (Cordes et al., 2012). Calcitriol differentiated the human leukemic cell line (HL-60) and metabolized exogenous arachidonic acid to both COX products (predominantly thromboxane B2 and PG E2) and lipoxygenase products, including leukotriene B<sup>4</sup> (Stenson et al., 1988). In a mouse xenograft model of prostate cancer, the combination of cacitriol and dietary soy enhanced cacitriol activity in regulating target gene expression and increased the suppression of PG synthesis and signaling, such as COX-2, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), and PG receptors. (Wang et al., 2012a). Calcitriol and its analogs have also been shown to selectively inhibit the activity of COX-2 (Aparna et al., 2008), and an inverse correlation exists between the expression of PG-metabolizing enzymes and reduced VDR expression in malignant breast cell lines (Thill et al., 2012). Taken together, these findings suggest that vitamin D may play a

Reactiveoxygenspecies(ROS)playamajorroleinvariouscell-signalingpathways.ROSactivates various transcription factors and increases in the expression of proteins that control cellular transformation, tumor cell survival, tumor cell proliferation and invasion, angiogenesis, and metastasis. ROS has an important role in the initiation and progression of many cancers (Gupta et al., 2012; Marra et al., 2011; Zhang et al., 2011c; Wang et al., 2011c; Rogalska et al., 2011; Gupta-

the activation of MAPK signaling pathways (Zhang et al., 2011b).

role in modulating the inflammatory process in cancer.

**3.6. Oxidative stress**

**3.5. The Prostaglandins (PGs)**

426 Cancer Treatment - Conventional and Innovative Approaches

Fifteen patients were given 2,000 IU (50 microg) of cholecalciferol daily and monitored prospectively every 2-3 mo. There was a statistically significant decrease in the rate of PSA rise after administration of cholecalciferol compared with that before cholecalciferol. The median PSA doubling time increased from 14.3 months prior to commencing cholecalciferol to 25 months after commencing cholecalciferol. Fourteen of 15 patients had a prolongation of PSA doubling time after commencing cholecalciferol (Woo et al., 2005). Breast cancer patients with bone metastases received 10,000 IU of vitamin D3 daily for 4 months. There was a significant reduction in the number of sites of pain (Amir et al., 2010). Arlet al. (2012) reported on an unexpected observation of a spectacular 13-month remission of chronic lymphocytic leukemia after the administration of cholecalciferol in an elderly patient. Dietary vitamin D3 and calcitriol have been shown to demonstrate equivalent anticancer activity in mouse xenograft models of breast and prostate cancers (Swani et al., 2012).

advanced or cutaneous metastatic breast cancer, topical calcipotriol treatment reduced the diameter of treated lesions that contained vitamin D receptor (*VDR*) (Bower et al., 1991). Calci‐ triolpotentiatesbothcarboplatinandcisplatin-mediatedgrowthinhibitioninbreastandprostate cancer cell lines (Cho et al., 1991; Moffatt et al., 1999). Tamoxifen and calcitriol or its analog used together to enhance growth inhibition in breast cancer cells than either agent alone (Vink-van Wijngaarden et al., 1994). Cacitriol sensitizes breast cancer cells to doxorubicin through the inhibition of the expression and activity of cytoplasmic antioxidant enzyme (Ravid et al., 1999). Calcitriolmayincreasecisplatinsensitivityinchemotherapy-resistanttesticulargermcellcancerderived cell lines (Jørgensen et al., 2012). Combination of retinoic acid and vitamin D analog exert synergistic growth inhibition and apoptosis induction on hepatocellular cancers cells (Zhang et al.,2012b).Thecombinationofcalcitriolanddietarysoyresultedinsubstantiallygreaterinhibition of tumor growth than the inhibition achieved with either agent alone in a mouse xenograft model

The Impact of Vitamin D in Cancer http://dx.doi.org/10.5772/55324 429

VitaminDhasaroleinthepreventionandtreatmentofcancer.Geneticstudieshaveprovidedthe opportunitytodeterminewhatproteinslinkvitaminDtothepathologyofcancer.VitaminDalso exerts its effect on cancer via non-genomic mechanisms. As a result, it is imperative that vitamin D levels in patients with cancer be followed. Many studies use the relationship between serum PTHand25OHDtodefinethenormalrangeofserum25OHD.AccordingtothereportonDietary Reference Intakes for vitamin D and calcium by the Institute of Medicine (IOM), persons are at risk of deficiency at serum 25OHD levels less than 30 nmol/L. Saliba et al. (2011) suggested that a 25OHD threshold of 50 nmol/L is sufficient for PTH suppression and prevention of secondary hyperparathyroidism in persons with normal renal function. It is necessary to check serum 25OHD3andparathyroidhormone(PTH)statusincancerpatients.SerumlevelsofPTHhavebeen reportedtocorrelatewithPSAlevelsandcolorectalcancer(Skinner&Schwartz,2009;Charalam‐ popoulos et al., 2010). Some authors proposed that, in patients with normal calcium levels, the serum 25OHD3 levels should be stored to > 55ng/ml in cancer patients (colon, breast, and ovary) (Garland et al., 2007). Calcitriol, 1,25OHD3, is best used for cancer treatment, because of its active form of vitamin D3 metabolite, suppression of PTH levels (acted as cellular growth factor), and theirreceptorspresentedinmostofhumancells.However,monitorofserum25OHD3aftertaking calcitriol is not necessary because calcitriol inhibits the production of serum 25OHD3 by the liver (Bell et al., 1984; Luong & Nguyen, 1996). The main limitation to the clinical widespread evolu‐

of prostate cancer (Wang et al., 2012a).

tion of 1,25OHD3 is its hypercalcemic side-effects.

Khanh vinh quoc Luong and Lan Thi Hoang Nguyen

Vietnamese American Medical Research Foundation, Westminster, California, USA

**5. Conclusion**

**Author details**

### **4.2. Calcitriol trials — Single agent**

In a clinical trial, high-dose calcitriol decreased prostatic-specific antigen (PSA) levels by 50% and reduced thrombosis in prostate cancer patients (Beer et al., 2003 & 2006). In hepatocellular carcinoma, calcitriol and its analogs have been reported to reduce tumor volume, increase hepatocarcinoma cell apoptosis by 21.4%, and transient stabilize serum alpha-fetoprotein levels (Dalhoff et al., 2003; Luo et al., 2004; Morris et al., 2002). The vitamin D analog, 19- Nor-2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D3, is a potent cell growth regulator with enhanced chemotherapeutic potency in liver cancer cells (Chiang et al., 2011). Alphacalcidol, a vitamin D analogue, has been demonstrated significant antitumor activity in patients with low-grade non-Hodgkin's lymphoma of the follicular, small-cleaved cell type (Raina et al., 1991). In patient with parathyroid cancer, vitamin D has been shown to prevent or delay the progression of recurrence (Palmieri-Sevier et al., 1993). Treatment with paricalcitol inhibited gastric cancer cell growth and peritoneal metastatic gastric cancer volume was significantly lower in paricalcitol treated mice (Park et al., 2012). Calcitriol treatment of breast cancer cell lines led to significantly fewer inflammatory breast cancer experimental metastases as compared to control (Hillyer et al., 2012).

### **4.3. Calcitriol trials — In combination**

Calcitrioladditivelyorsynergisticallypotentiatestheantitumorofothertypesofchemotherapeu‐ tic agents. Calcitriol enhances cellular sensitivity of human colon cancer cells to 5-fluorouracil (Liuetal.,2010).Combinationofcalcitriolandcytarabineprolongedremissioninelderlypatients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) (Slapak et al., 1992; Ferrero,etal.,2004).Arenalcellcarcinomapatientwithmultiplebonemetastasesthatwerealmost completely resolved after treatment with vitamin D and interferon-α (Fujioka et al., 1988). In a prospective study, a combination of active vitamin D and α- interferon has shown to be effective in patients with metastatic renal cell carcinoma (Obara et al., 2008). Calcitriol promotes the antiproliferative effects of gemcitabine and cisplatin in human bladder cancer models (Ma et al., 2010b), and also potentiates antitumor activity of paclitaxel and docetaxel (Hershberger et al., 2001; Ting et al. 2007). A phase II study showed that high-dose calcitriol with docetaxel may increase time to progression in patients with incurable pancreatic cancer when compared with docetaxel monotherapy (Blanke, 2009).Vitamin D3 treatment significantly suppressed the viabilityofgastriccancerandcholangiocarcinomacellsandalsohadasynergisticeffectwithother anti-cancer drugs, such as paclitaxel, adriamycin, and vinblastine (Baek et al., 2011). In locally advanced or cutaneous metastatic breast cancer, topical calcipotriol treatment reduced the diameter of treated lesions that contained vitamin D receptor (*VDR*) (Bower et al., 1991). Calci‐ triolpotentiatesbothcarboplatinandcisplatin-mediatedgrowthinhibitioninbreastandprostate cancer cell lines (Cho et al., 1991; Moffatt et al., 1999). Tamoxifen and calcitriol or its analog used together to enhance growth inhibition in breast cancer cells than either agent alone (Vink-van Wijngaarden et al., 1994). Cacitriol sensitizes breast cancer cells to doxorubicin through the inhibition of the expression and activity of cytoplasmic antioxidant enzyme (Ravid et al., 1999). Calcitriolmayincreasecisplatinsensitivityinchemotherapy-resistanttesticulargermcellcancerderived cell lines (Jørgensen et al., 2012). Combination of retinoic acid and vitamin D analog exert synergistic growth inhibition and apoptosis induction on hepatocellular cancers cells (Zhang et al.,2012b).Thecombinationofcalcitriolanddietarysoyresultedinsubstantiallygreaterinhibition of tumor growth than the inhibition achieved with either agent alone in a mouse xenograft model of prostate cancer (Wang et al., 2012a).

### **5. Conclusion**

months after commencing cholecalciferol. Fourteen of 15 patients had a prolongation of PSA doubling time after commencing cholecalciferol (Woo et al., 2005). Breast cancer patients with bone metastases received 10,000 IU of vitamin D3 daily for 4 months. There was a significant reduction in the number of sites of pain (Amir et al., 2010). Arlet al. (2012) reported on an unexpected observation of a spectacular 13-month remission of chronic lymphocytic leukemia after the administration of cholecalciferol in an elderly patient. Dietary vitamin D3 and calcitriol have been shown to demonstrate equivalent anticancer activity in mouse xenograft models of

In a clinical trial, high-dose calcitriol decreased prostatic-specific antigen (PSA) levels by 50% and reduced thrombosis in prostate cancer patients (Beer et al., 2003 & 2006). In hepatocellular carcinoma, calcitriol and its analogs have been reported to reduce tumor volume, increase hepatocarcinoma cell apoptosis by 21.4%, and transient stabilize serum alpha-fetoprotein levels (Dalhoff et al., 2003; Luo et al., 2004; Morris et al., 2002). The vitamin D analog, 19- Nor-2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D3, is a potent cell growth regulator with enhanced chemotherapeutic potency in liver cancer cells (Chiang et al., 2011). Alphacalcidol, a vitamin D analogue, has been demonstrated significant antitumor activity in patients with low-grade non-Hodgkin's lymphoma of the follicular, small-cleaved cell type (Raina et al., 1991). In patient with parathyroid cancer, vitamin D has been shown to prevent or delay the progression of recurrence (Palmieri-Sevier et al., 1993). Treatment with paricalcitol inhibited gastric cancer cell growth and peritoneal metastatic gastric cancer volume was significantly lower in paricalcitol treated mice (Park et al., 2012). Calcitriol treatment of breast cancer cell lines led to significantly fewer inflammatory breast cancer experimental metastases as

Calcitrioladditivelyorsynergisticallypotentiatestheantitumorofothertypesofchemotherapeu‐ tic agents. Calcitriol enhances cellular sensitivity of human colon cancer cells to 5-fluorouracil (Liuetal.,2010).Combinationofcalcitriolandcytarabineprolongedremissioninelderlypatients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) (Slapak et al., 1992; Ferrero,etal.,2004).Arenalcellcarcinomapatientwithmultiplebonemetastasesthatwerealmost completely resolved after treatment with vitamin D and interferon-α (Fujioka et al., 1988). In a prospective study, a combination of active vitamin D and α- interferon has shown to be effective in patients with metastatic renal cell carcinoma (Obara et al., 2008). Calcitriol promotes the antiproliferative effects of gemcitabine and cisplatin in human bladder cancer models (Ma et al., 2010b), and also potentiates antitumor activity of paclitaxel and docetaxel (Hershberger et al., 2001; Ting et al. 2007). A phase II study showed that high-dose calcitriol with docetaxel may increase time to progression in patients with incurable pancreatic cancer when compared with docetaxel monotherapy (Blanke, 2009).Vitamin D3 treatment significantly suppressed the viabilityofgastriccancerandcholangiocarcinomacellsandalsohadasynergisticeffectwithother anti-cancer drugs, such as paclitaxel, adriamycin, and vinblastine (Baek et al., 2011). In locally

breast and prostate cancers (Swani et al., 2012).

428 Cancer Treatment - Conventional and Innovative Approaches

**4.2. Calcitriol trials — Single agent**

compared to control (Hillyer et al., 2012).

**4.3. Calcitriol trials — In combination**

VitaminDhasaroleinthepreventionandtreatmentofcancer.Geneticstudieshaveprovidedthe opportunitytodeterminewhatproteinslinkvitaminDtothepathologyofcancer.VitaminDalso exerts its effect on cancer via non-genomic mechanisms. As a result, it is imperative that vitamin D levels in patients with cancer be followed. Many studies use the relationship between serum PTHand25OHDtodefinethenormalrangeofserum25OHD.AccordingtothereportonDietary Reference Intakes for vitamin D and calcium by the Institute of Medicine (IOM), persons are at risk of deficiency at serum 25OHD levels less than 30 nmol/L. Saliba et al. (2011) suggested that a 25OHD threshold of 50 nmol/L is sufficient for PTH suppression and prevention of secondary hyperparathyroidism in persons with normal renal function. It is necessary to check serum 25OHD3andparathyroidhormone(PTH)statusincancerpatients.SerumlevelsofPTHhavebeen reportedtocorrelatewithPSAlevelsandcolorectalcancer(Skinner&Schwartz,2009;Charalam‐ popoulos et al., 2010). Some authors proposed that, in patients with normal calcium levels, the serum 25OHD3 levels should be stored to > 55ng/ml in cancer patients (colon, breast, and ovary) (Garland et al., 2007). Calcitriol, 1,25OHD3, is best used for cancer treatment, because of its active form of vitamin D3 metabolite, suppression of PTH levels (acted as cellular growth factor), and theirreceptorspresentedinmostofhumancells.However,monitorofserum25OHD3aftertaking calcitriol is not necessary because calcitriol inhibits the production of serum 25OHD3 by the liver (Bell et al., 1984; Luong & Nguyen, 1996). The main limitation to the clinical widespread evolu‐ tion of 1,25OHD3 is its hypercalcemic side-effects.

### **Author details**

Khanh vinh quoc Luong and Lan Thi Hoang Nguyen

Vietnamese American Medical Research Foundation, Westminster, California, USA

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452 Cancer Treatment - Conventional and Innovative Approaches

46-60.


**Chapter 19**

**The Treatment of Cancer: A Comprehensive Therapeutic**

Although an overall rise in cancer incidence has been observed over the past 300 years concomitantly with the industrial revolution, a more prominent increase has been recorded

Genetic factors are thought to account for 5-10% of all malignant neoplasms, even though hereditary susceptibility will be variably relevant depending on histotype, anatomic site, and epidemiologic context; additionally, a key role is played by environmental factors. Socioeco‐ nomic improvements have resulted in an increase in food availability as well as significant changes in lifestyle habits; with new technologies allowing for automation of manual work, an overall physical activity reduction has been observed leading to unbalances between caloric

Cancer is no longer a rapidly lethal disease for an increasing number of patients. Knowledge of the main risk factors for cancer development is essential for establishing a comprehensive

Cancer patients receiving treatment combinations of surgery, radiation therapy and chemo‐

Pain, heightened risk of infection, neural deficits, lymphedema, fatigue, nausea and vomiting, loss of flexibility, myopathies, muscle weakness, cachexia, dehydration, emotional distress, shortness of breath are common side-effects capable of negatively affecting patients' lifestyle and physical activities. Any combination of surgical treatments, chemotherapy, and radio‐ therapy must be integrated within a global therapeutic plan aimed to reduce the above-

and reproduction in any medium, provided the original work is properly cited.

© 2013 Saggini and Calvani; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Model Entailing a Complex of Interaction Modalities**

R. Saggini and M. Calvani

http://dx.doi.org/10.5772/55696

intake and energy expenditure.

and integrated treatment plan (tab 1).

**1. Introduction**

Additional information is available at the end of the chapter

since the '30s, with a further acceleration during the last 2 decades.

therapy are prone to developing several treatment-related diseases.

**Chapter 19**

## **The Treatment of Cancer: A Comprehensive Therapeutic Model Entailing a Complex of Interaction Modalities**

R. Saggini and M. Calvani

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55696

### **1. Introduction**

Although an overall rise in cancer incidence has been observed over the past 300 years concomitantly with the industrial revolution, a more prominent increase has been recorded since the '30s, with a further acceleration during the last 2 decades.

Genetic factors are thought to account for 5-10% of all malignant neoplasms, even though hereditary susceptibility will be variably relevant depending on histotype, anatomic site, and epidemiologic context; additionally, a key role is played by environmental factors. Socioeco‐ nomic improvements have resulted in an increase in food availability as well as significant changes in lifestyle habits; with new technologies allowing for automation of manual work, an overall physical activity reduction has been observed leading to unbalances between caloric intake and energy expenditure.

Cancer is no longer a rapidly lethal disease for an increasing number of patients. Knowledge of the main risk factors for cancer development is essential for establishing a comprehensive and integrated treatment plan (tab 1).

Cancer patients receiving treatment combinations of surgery, radiation therapy and chemo‐ therapy are prone to developing several treatment-related diseases.

Pain, heightened risk of infection, neural deficits, lymphedema, fatigue, nausea and vomiting, loss of flexibility, myopathies, muscle weakness, cachexia, dehydration, emotional distress, shortness of breath are common side-effects capable of negatively affecting patients' lifestyle and physical activities. Any combination of surgical treatments, chemotherapy, and radio‐ therapy must be integrated within a global therapeutic plan aimed to reduce the above-


**Table 1.** The 9 modifiable risk factors responsible for a third of all cancer deaths in the world

mentioned negative effects that may become apparent immediately as well as after several months or years.

Calories intake directly correlates with risk of developing obesity as well as cancer.

Cancer is responsible of approximately 25% of all deaths in the US.

ment in obesity physiopatology.

million per year, respectively.

obesity range (> 30).

and squared height (centimeters2

just to name a few examples.

to height.

intakes.

been increased by 42% compared to now.

key role in up to 70% of colorectal cancer-related deaths.

writing, or accomplishing ordinary housework actions).

Obesity *per se* is considered to be to blame for up to 14% and 20% of all men and women deaths. Approximately 50% of all primary malignant cancers arise in tissues with a primary involve‐

The Treatment of Cancer: A Comprehensive Therapeutic Model Entailing a Complex of Interaction Modalities

http://dx.doi.org/10.5772/55696

457

According to recent predictions, by 2020 the global world population will have reached 7,5 billion, with a cancer incidence and disease-specific mortality of 15 million per year and 12

At present the total US cancer survivors population is made of 5-y cancer survivors for up to 66%, and by 2020 it has been estimated that cancer survivors aged at least 65 years will have

The diet is responsible for approximately 30-35 % of total mortality in the US, with its impact on cancer development depending on histotype and anatomic location; nutrition may play a

Nowadays, men and women in Occidental countries are progressively increasing in body size, with average body-mass indexes (BMI, i.e. the ratio between weight and squared height) relentlessly soaring beyond the normal range (18.5-24.9); conversely, an increas‐ ing number of individuals is falling into the overweight range (25-29.9) as well as the overt

Obesity is easily diagnosed by assessing the increase in horizontal body dimensions compared

One method for measuring such imbalance is the BMI, i.e. the ratio between weight (kilograms)

BMI, however, being frequently used in epidemiological studies to assess the effect of diet as a risk factor, may become a confounding factor; indeed, BMI is less reliable in elderly patients, with height being gradually reduced due to spinal degenerative processes. Likewise, children BMI measurements may be biased by different growth rates in different body areas. Addi‐ tionally, BMI fail to provide any definite information regarding body composition, i.e. the percentage of lean body mass versus fat mass, bone mineralization status, and total body water,

The value of lean body mass is critical because it is the body component consuming higher energy values per weight unit, being therefore critical for any estimations of appropriate caloric

Any diet based on caloric restriction alone would be ineffective as well as potentially danger‐ ous if no caloric intake assessment were to be calculated according to body composition and estimated energy requirements for performing daily physical activity (including walking,

overweight and different obesity degrees (mild and severe) have been defined.

). BMI ranges identifying malnutrition, normal weight,

Mullan (1985) classified the life of cancer survivors into three stages: 1) Acute Stage, spanning from diagnosis to the first year after primary treatment; 2) Extended Stage, until the 5th year after primary treatment; 3) Permanent Stage, from the 5th year after primary treatment onwards.

The first year after primary treatment should be considered just as the "tip of the iceberg", and it is crucial that any approach to cancer treatment is holistic and comprehensive, based on the assumption that cancer is a chronic illness rather than an acute condition.

The aim of this chapter is not to describe the specifics of early management of patients diagnosed with cancer; however, the authors' view is that such approach should be as integrative and comprehensive as possible.

It is essential that physicians in the process of planning specific therapeutic interventions (either actions specifically aimed to the primary disease or supportive therapies) extensively profile patients according to their physical status in order to establish an individual patienttailored strategy.

The integrative management approach relies on a number of basic interventions, including:


### **2. Therapeutic changes of lifestyle habits and daily diet**

#### **2.1. What do you know?**

Up to 30-40% of all malignant cancers could be prevented by interventions on diet, physical activities, and daily lifestyle.

Calories intake directly correlates with risk of developing obesity as well as cancer.

Obesity *per se* is considered to be to blame for up to 14% and 20% of all men and women deaths.

Approximately 50% of all primary malignant cancers arise in tissues with a primary involve‐ ment in obesity physiopatology.

Cancer is responsible of approximately 25% of all deaths in the US.

mentioned negative effects that may become apparent immediately as well as after several

Mullan (1985) classified the life of cancer survivors into three stages: 1) Acute Stage, spanning from diagnosis to the first year after primary treatment; 2) Extended Stage, until the 5th year after primary treatment; 3) Permanent Stage, from the 5th year after primary treatment

The first year after primary treatment should be considered just as the "tip of the iceberg", and it is crucial that any approach to cancer treatment is holistic and comprehensive, based on the

The aim of this chapter is not to describe the specifics of early management of patients diagnosed with cancer; however, the authors' view is that such approach should be as

It is essential that physicians in the process of planning specific therapeutic interventions (either actions specifically aimed to the primary disease or supportive therapies) extensively profile patients according to their physical status in order to establish an individual patient-

The integrative management approach relies on a number of basic interventions, including:

Up to 30-40% of all malignant cancers could be prevented by interventions on diet, physical

assumption that cancer is a chronic illness rather than an acute condition.

integrative and comprehensive as possible.

8. Indoor air pollution due to household use of solid fuels

456 Cancer Treatment - Conventional and Innovative Approaches

9. Spread of bacterial and viral infections through unsafe health care procedures

**Table 1.** The 9 modifiable risk factors responsible for a third of all cancer deaths in the world

**1.** Therapeutic changes of lifestyle habits and daily diet;

**2.** Specific physical exercises and walking prescriptions;

**3.** Physical therapies coupled with psychophysical techniques.

**2. Therapeutic changes of lifestyle habits and daily diet**

months or years.

1. Obesity and overweight 2. Low fruit and vegetable intake

3. Physical inactivity 4. Smoking

5. Alcohol consumption 6. Unprotected sex 7. Urban air pollution

tailored strategy.

**2.1. What do you know?**

activities, and daily lifestyle.

onwards.

According to recent predictions, by 2020 the global world population will have reached 7,5 billion, with a cancer incidence and disease-specific mortality of 15 million per year and 12 million per year, respectively.

At present the total US cancer survivors population is made of 5-y cancer survivors for up to 66%, and by 2020 it has been estimated that cancer survivors aged at least 65 years will have been increased by 42% compared to now.

The diet is responsible for approximately 30-35 % of total mortality in the US, with its impact on cancer development depending on histotype and anatomic location; nutrition may play a key role in up to 70% of colorectal cancer-related deaths.

Nowadays, men and women in Occidental countries are progressively increasing in body size, with average body-mass indexes (BMI, i.e. the ratio between weight and squared height) relentlessly soaring beyond the normal range (18.5-24.9); conversely, an increas‐ ing number of individuals is falling into the overweight range (25-29.9) as well as the overt obesity range (> 30).

Obesity is easily diagnosed by assessing the increase in horizontal body dimensions compared to height.

One method for measuring such imbalance is the BMI, i.e. the ratio between weight (kilograms) and squared height (centimeters2 ). BMI ranges identifying malnutrition, normal weight, overweight and different obesity degrees (mild and severe) have been defined.

BMI, however, being frequently used in epidemiological studies to assess the effect of diet as a risk factor, may become a confounding factor; indeed, BMI is less reliable in elderly patients, with height being gradually reduced due to spinal degenerative processes. Likewise, children BMI measurements may be biased by different growth rates in different body areas. Addi‐ tionally, BMI fail to provide any definite information regarding body composition, i.e. the percentage of lean body mass versus fat mass, bone mineralization status, and total body water, just to name a few examples.

The value of lean body mass is critical because it is the body component consuming higher energy values per weight unit, being therefore critical for any estimations of appropriate caloric intakes.

Any diet based on caloric restriction alone would be ineffective as well as potentially danger‐ ous if no caloric intake assessment were to be calculated according to body composition and estimated energy requirements for performing daily physical activity (including walking, writing, or accomplishing ordinary housework actions).

Obesity plays a critical role in cancer promotion, progression, and therapy resistance; obesity oncogenic actions are thought to be mediated by dysregulation of hormonal networks (i.e., circulating insuline, IGF-1, testosterone, and estrogens levels) as well as through pro-inflam‐ matory effects due to adipose tissues cytokines.

Diets rich in saturated fatty acids closely correlate with metabolic syndrome and inflammation, especially inflammation of the white adipose tissue, which is not only a storage organ for lipids

The Treatment of Cancer: A Comprehensive Therapeutic Model Entailing a Complex of Interaction Modalities

http://dx.doi.org/10.5772/55696

459

It has been known since 1885 that hyperglycemia is more frequent among cancer patients than

Warburg in 1930 highlighted the abnormal glucidic metabolism occurring in cancer cells, i.e. the so-called aerobic glycolysis, defined as the tendency of the cancer tissues to produce lactic acid even in the presence of sufficient oxygen to sustain Krebs cycle and mitochondrial

Glucose intolerance is an established risk factor for several cancers (including colorectal, breast, prostatic, pancreatic, and gastric cancer). Obesity and glucose intolerance are part of the metabolic syndrome, a condition characterized by increased insulin levels both during fasting and after glucose load. Metabolic syndrome, first described by Reaven in 1988, is defined by the presence of at least three of the following components: intra-abdominal or visceral obesity, glucose intolerance, hypertension, low HDL blood levels, and high triglyceride levels. In 2001, the National Cholesterol Education Program developed an alternative definition, which required the presence of at least 3 of the following 5 factors: increased waist circumference, hypertriglyceridemia, low HDL cholesterol, hypertension, and high levels of fasting glycemic levels. At the roots of metabolic syndrome there are increase in visceral fat, excessive caloric

The prevalence of metabolic syndrome is steadily increasing all over the world together with

In subjects with glucose intolerance (IGT), both the levels of glycemia and fasting insulin are increased. The latter are coupled until glycemia reaches the concentration of 7-8 mM, a level beyond which insulin does not show further increases and may even begin to decline as a result of functional failure of pancreatic β-cells (De Fronzo 1992). This is paralleled by the

Many people with newly diagnosed cancer are obese, with further changes in body structure

Chemotherapy often changes, even a year later, body composition, increasing fat mass and reducing muscle mass, creating a phenotype that could be defined as post-cancer sarcopenic

Modifications in body composition in cancer patients imply that many studies conducted through questionnaires, perhaps using only one scale, were affected by significant biases. The reduction in caloric intake as a strategy to reduce obesity should be assessed on a case by case basis, followed over time, and maintained proportional with nutritional needs of the whole

The caloric intake, however, should be calibrated according to the composition of energy sources (carbohydrates, lipids, proteins); the latter, in a typical Mediterranean diet, should be

being induced by chemotherapy, surgery, and therapy-related physical inactivity.

obesity; the latter appears to correlate with a high risk of cancer recurrence.

gradual increase in glycemia, starting with postprandial glycemia.

body in order to prevent secondary nutritional deficiencies.

in the ratio of 60%, 25%, 15%, respectively.

but also an endocrine organ.

in the healthy population.

membrane oxidation processes.

intake, and low physical activity.

the increase in several types of cancer.

Increased BMI values correlate with circulating inflammatory cytokines levels, that appear to be related to insulin resistance.

A positive correlation between high BMI values (>30) and cancer risk is being observed in different areas worldwide, with significant increases in cancer risk being recorded for every 5 Kg/m2 -gain in BMI.

Obesity directly promotes tissue inflammation. Lipids intake should be proportional to that of other nutrients in order to reach an adequate energy balance; in this regard, it should be remembered that 1g of fat provides approximately 9 Kcal of energy, while 1g of carbohydrates or proteins only provides 4.5 Kcal. However, specific lipids significantly differ in their chemical structure and will result in different metabolic responses when given at equal calories levels. Increased amounts of fat per portion, a phenomenon commonly occurring in restaurant and cafèteria, leads to significant inflammatory response spikes, that can be quantified by assessing increases of circulating inflammatory factors; the latter are capable of inducing insulin resistance and free radicals production, resulting in oxidation of cell structures such as nucleic acids, proteins, and membrane lipids. Other lipids possess an anti-inflammatory activity. There is plenty of literature addressing the beneficial administration of omega-3 unsaturated lipids for lessening the inflammatory consequences of several chronic diseases. Omega-3 unsaturated lipids are available either as dedicated over-the-counter preparations or through several common foods, more prominently fish and dried fruit. Omega-3 lipids are unsaturated lipids, i.e. they are in liquid form at room temperature (oils); they can easily undergo oxidation if not protected by intrinsic animals antioxidant systems or by vitamin E addition in commer‐ cially available preparations. Their content in fish meat changes according to the species, the fishing site, temperature, type of feeding (algae or other kinds of food for livestock); these features make difficult to calculate the omega-3 unsaturated lipids daily dose. Many public health authorities have been encouraging increases in diet fish intake, but it is important to know diet fish origins because of the risk related to heavy metals; it is therefore necessary to avoid eating exceedingly large amounts fish. Of course, such details are hardly specified, if ever, in epidemiological studies assessing the effects of fish-based diets. Obesity results in a status of enduring subclinical inflammation within fat tissues. In obese individuals both visceral and subcutaneous adipose tissues are infiltrated by macrophages surrounding necrotic adipocytes forming the so-called crown-like structures (CLS). The infiltrating macro‐ phages release inflammatory cytokines whose plasma levels in post-menopausal breast cancer patients were shown to correlate with cancer progression and disease-specific mortality. In both experimental animals and humans the CLS number is directly related to BMI values.

Diets with high concentration in saturated fatty acids (cafeteria food, sausages, dairy products, red meat) are becoming more and more frequent worldwide, leading to a global escalation in overnutrition-related diseases.

Diets rich in saturated fatty acids closely correlate with metabolic syndrome and inflammation, especially inflammation of the white adipose tissue, which is not only a storage organ for lipids but also an endocrine organ.

Obesity plays a critical role in cancer promotion, progression, and therapy resistance; obesity oncogenic actions are thought to be mediated by dysregulation of hormonal networks (i.e., circulating insuline, IGF-1, testosterone, and estrogens levels) as well as through pro-inflam‐

Increased BMI values correlate with circulating inflammatory cytokines levels, that appear to

A positive correlation between high BMI values (>30) and cancer risk is being observed in different areas worldwide, with significant increases in cancer risk being recorded for every 5

Obesity directly promotes tissue inflammation. Lipids intake should be proportional to that of other nutrients in order to reach an adequate energy balance; in this regard, it should be remembered that 1g of fat provides approximately 9 Kcal of energy, while 1g of carbohydrates or proteins only provides 4.5 Kcal. However, specific lipids significantly differ in their chemical structure and will result in different metabolic responses when given at equal calories levels. Increased amounts of fat per portion, a phenomenon commonly occurring in restaurant and cafèteria, leads to significant inflammatory response spikes, that can be quantified by assessing increases of circulating inflammatory factors; the latter are capable of inducing insulin resistance and free radicals production, resulting in oxidation of cell structures such as nucleic acids, proteins, and membrane lipids. Other lipids possess an anti-inflammatory activity. There is plenty of literature addressing the beneficial administration of omega-3 unsaturated lipids for lessening the inflammatory consequences of several chronic diseases. Omega-3 unsaturated lipids are available either as dedicated over-the-counter preparations or through several common foods, more prominently fish and dried fruit. Omega-3 lipids are unsaturated lipids, i.e. they are in liquid form at room temperature (oils); they can easily undergo oxidation if not protected by intrinsic animals antioxidant systems or by vitamin E addition in commer‐ cially available preparations. Their content in fish meat changes according to the species, the fishing site, temperature, type of feeding (algae or other kinds of food for livestock); these features make difficult to calculate the omega-3 unsaturated lipids daily dose. Many public health authorities have been encouraging increases in diet fish intake, but it is important to know diet fish origins because of the risk related to heavy metals; it is therefore necessary to avoid eating exceedingly large amounts fish. Of course, such details are hardly specified, if ever, in epidemiological studies assessing the effects of fish-based diets. Obesity results in a status of enduring subclinical inflammation within fat tissues. In obese individuals both visceral and subcutaneous adipose tissues are infiltrated by macrophages surrounding necrotic adipocytes forming the so-called crown-like structures (CLS). The infiltrating macro‐ phages release inflammatory cytokines whose plasma levels in post-menopausal breast cancer patients were shown to correlate with cancer progression and disease-specific mortality. In both experimental animals and humans the CLS number is directly related to BMI values.

Diets with high concentration in saturated fatty acids (cafeteria food, sausages, dairy products, red meat) are becoming more and more frequent worldwide, leading to a global escalation in

matory effects due to adipose tissues cytokines.

458 Cancer Treatment - Conventional and Innovative Approaches

be related to insulin resistance.


overnutrition-related diseases.

Kg/m2

It has been known since 1885 that hyperglycemia is more frequent among cancer patients than in the healthy population.

Warburg in 1930 highlighted the abnormal glucidic metabolism occurring in cancer cells, i.e. the so-called aerobic glycolysis, defined as the tendency of the cancer tissues to produce lactic acid even in the presence of sufficient oxygen to sustain Krebs cycle and mitochondrial membrane oxidation processes.

Glucose intolerance is an established risk factor for several cancers (including colorectal, breast, prostatic, pancreatic, and gastric cancer). Obesity and glucose intolerance are part of the metabolic syndrome, a condition characterized by increased insulin levels both during fasting and after glucose load. Metabolic syndrome, first described by Reaven in 1988, is defined by the presence of at least three of the following components: intra-abdominal or visceral obesity, glucose intolerance, hypertension, low HDL blood levels, and high triglyceride levels. In 2001, the National Cholesterol Education Program developed an alternative definition, which required the presence of at least 3 of the following 5 factors: increased waist circumference, hypertriglyceridemia, low HDL cholesterol, hypertension, and high levels of fasting glycemic levels. At the roots of metabolic syndrome there are increase in visceral fat, excessive caloric intake, and low physical activity.

The prevalence of metabolic syndrome is steadily increasing all over the world together with the increase in several types of cancer.

In subjects with glucose intolerance (IGT), both the levels of glycemia and fasting insulin are increased. The latter are coupled until glycemia reaches the concentration of 7-8 mM, a level beyond which insulin does not show further increases and may even begin to decline as a result of functional failure of pancreatic β-cells (De Fronzo 1992). This is paralleled by the gradual increase in glycemia, starting with postprandial glycemia.

Many people with newly diagnosed cancer are obese, with further changes in body structure being induced by chemotherapy, surgery, and therapy-related physical inactivity.

Chemotherapy often changes, even a year later, body composition, increasing fat mass and reducing muscle mass, creating a phenotype that could be defined as post-cancer sarcopenic obesity; the latter appears to correlate with a high risk of cancer recurrence.

Modifications in body composition in cancer patients imply that many studies conducted through questionnaires, perhaps using only one scale, were affected by significant biases. The reduction in caloric intake as a strategy to reduce obesity should be assessed on a case by case basis, followed over time, and maintained proportional with nutritional needs of the whole body in order to prevent secondary nutritional deficiencies.

The caloric intake, however, should be calibrated according to the composition of energy sources (carbohydrates, lipids, proteins); the latter, in a typical Mediterranean diet, should be in the ratio of 60%, 25%, 15%, respectively.

The American Cancer Society guidelines suggests that carbohydrates should be in the ratio of 40-65% of the energy pool, the same as for healthy population, lipids in the ratio of 20-35%, of which <10% saturated fats, and proteins should be 10-35%.

**3. Diet, caloric restriction and cooking: A therapeutic way**

of their content in terms of micronutrients.

increase the excessive energy introduction.

stress.

tables.

The nutritional sources of food themselves are different from those used by our ancestors. The production doesn't respect the proximity criteria (0 km), seasonality criteria, or crop rotation criteria, resulting in a loss of micro-elements in soil. Fruits and vegetables generally meet more the preservation criteria instead of those of maturation with the result of the unpredictability

The Treatment of Cancer: A Comprehensive Therapeutic Model Entailing a Complex of Interaction Modalities

http://dx.doi.org/10.5772/55696

461

The taste for food has been gradually changing giving priority to a rapid food intake (fast food), high levels of fat, flour and refined sugar. The large use of sweetened drinks contributes to

As for oxygen free radicals (ROS) production, it is related to inflammation during oxidative

In obese patients and in those with cancer the ROS problem has a special role; supplements or diets with high content of vegetables with antioxidant activity have been given. The use of fruits and vegetables showed positive results in reducing the risk for cancer and recurrences. Data, however, are not univocal. Each vegetable contains many different compounds, their availability is not always in relation with their content (it is a typical example for Beta carotene of carrot), the contents of a type of antioxidant may differ for the production site, stage of maturation to collection, preservation, and preparation methods (tomato sauce contains more available lycopene than raw tomato). The availability of a substance may change in different individuals according to the integrity of the intestinal mucosa (often damaged by chemother‐ apy) or to the kind of intestinal flora (1-1,5 kg of bacteria). This condition can also modify the food chemical structure, producing harmful or healthy substances for our health as in the case of soy isoflavones transformed into the much more active Equol only in subjects with suitable bacteria. In our blood and urine there's a large amount of products of bacterial metabolism which may influence our health; it may differ depending on the breed, gender, functional states (pregnancy) and dietary habits: there's much more complexity in epidemiological studies with

the use of the food or nutritional supplements than expected in the research protocol.

The real availability (absorption) of substances in food or in supplements has a good chance to be different from that hypothesized and calculated with questionnaires or bromatological

Diet should not cause any further increase in insulin levels, either basal or food-induced.

rehabilitation system to reduce overweight and improve muscular function).

The daily intake of carbohydrates (i.e., glycemic load) should be in proportional with the body composition, the energy percentage (calculated in relation with other energy sources), and the degree of physical activity (including daily activities as well as activities planned by the

Carbohydrates intake should be progressively reduced throughout the day according to the circadian increase in insulin resistance, more prominently observed during the last day hours. Last but not least, it is necessary to avoid foods with high glycemic index (GI). The GI is determined by comparing the postprandial glycemic response of a food with the postprandial

Daily protein intake should not be less than 0.8-1 grams per Kg of body weight.

Nutrition does not mean only caloric intake, but also replenishment of the very primary elements that the body uses to live. Nutritionists from different countries define the optimal daily replenishment levels of micronutrients depending on gender, age, and functional status (i.e., pregnancy, sporting activities, etc.). However, patients suffering from cancer will be almost always exhibiting to nutritional deficiencies.

Obesity itself is a malnutrition disease characterized by several deficiencies, including vitamin D deficiency. Many other deficits can be induced by specific therapies (i.e., those impairing renal tubular reabsorption through tubular damage, or intestinal absorption through muco‐ sitis, anorexia, and vomiting) and by treatments for related comorbidities (cholesterollowering agents, diuretics, anti-hypertensive drugs, etc...) resulting in minerals and antioxidants loss. These events may worsen the peroxidation phenomena of several biological structures, that will have been already compromised by metabolic syndrome and administra‐ tion of chemotherapy.

Obesity is also associated with insulin resistance, i.e. the insulin inability, despite being available in physiological concentrations, of exerting its metabolic tasks in different body districts.

Insulin resistance assessment is performed in specialized centers, at times requiring expensive and complex methods. Such assessment could be easier by evaluation of blood glucose levels and fasting insulin levels according to the HOMA-IR algorithm, with values above 2.5 being indicative of insulin resistance.

Diet should not cause any further increase in insulin levels, either basal or food-induced.

The daily intake of carbohydrates (i.e., glycemic load) should be proportional with the body composition, the energy percentage (calculated in relation with other energy sources), and the degree of physical activity (including daily activities as well as activities planned by the rehabilitation system to reduce overweight and improve muscular function).

Carbohydrates intake should be progressively reduced throughout the day in light of the circadian increase in insulin resistance, more prominently observed during the last day hours.

Last but not least, it is necessary to avoid foods with high glycemic index (GI). The GI is determined by comparing the post prandial glycemic response of a food with the postprandial glycemic response to the same amount of available carbohydrate from a standard food in the same individual.

Baseline plasma levels of cytokines in obese people return to normal values after weight loss.

### **3. Diet, caloric restriction and cooking: A therapeutic way**

The American Cancer Society guidelines suggests that carbohydrates should be in the ratio of 40-65% of the energy pool, the same as for healthy population, lipids in the ratio of 20-35%, of

Nutrition does not mean only caloric intake, but also replenishment of the very primary elements that the body uses to live. Nutritionists from different countries define the optimal daily replenishment levels of micronutrients depending on gender, age, and functional status (i.e., pregnancy, sporting activities, etc.). However, patients suffering from cancer will be

Obesity itself is a malnutrition disease characterized by several deficiencies, including vitamin D deficiency. Many other deficits can be induced by specific therapies (i.e., those impairing renal tubular reabsorption through tubular damage, or intestinal absorption through muco‐ sitis, anorexia, and vomiting) and by treatments for related comorbidities (cholesterollowering agents, diuretics, anti-hypertensive drugs, etc...) resulting in minerals and antioxidants loss. These events may worsen the peroxidation phenomena of several biological structures, that will have been already compromised by metabolic syndrome and administra‐

Obesity is also associated with insulin resistance, i.e. the insulin inability, despite being available in physiological concentrations, of exerting its metabolic tasks in different body

Insulin resistance assessment is performed in specialized centers, at times requiring expensive and complex methods. Such assessment could be easier by evaluation of blood glucose levels and fasting insulin levels according to the HOMA-IR algorithm, with values above 2.5 being

Diet should not cause any further increase in insulin levels, either basal or food-induced.

rehabilitation system to reduce overweight and improve muscular function).

The daily intake of carbohydrates (i.e., glycemic load) should be proportional with the body composition, the energy percentage (calculated in relation with other energy sources), and the degree of physical activity (including daily activities as well as activities planned by the

Carbohydrates intake should be progressively reduced throughout the day in light of the circadian increase in insulin resistance, more prominently observed during the last day

Last but not least, it is necessary to avoid foods with high glycemic index (GI). The GI is determined by comparing the post prandial glycemic response of a food with the postprandial glycemic response to the same amount of available carbohydrate from a standard food in the

Baseline plasma levels of cytokines in obese people return to normal values after weight

Daily protein intake should not be less than 0.8-1 grams per Kg of body weight.

which <10% saturated fats, and proteins should be 10-35%.

460 Cancer Treatment - Conventional and Innovative Approaches

almost always exhibiting to nutritional deficiencies.

tion of chemotherapy.

indicative of insulin resistance.

districts.

hours.

loss.

same individual.

The nutritional sources of food themselves are different from those used by our ancestors. The production doesn't respect the proximity criteria (0 km), seasonality criteria, or crop rotation criteria, resulting in a loss of micro-elements in soil. Fruits and vegetables generally meet more the preservation criteria instead of those of maturation with the result of the unpredictability of their content in terms of micronutrients.

The taste for food has been gradually changing giving priority to a rapid food intake (fast food), high levels of fat, flour and refined sugar. The large use of sweetened drinks contributes to increase the excessive energy introduction.

As for oxygen free radicals (ROS) production, it is related to inflammation during oxidative stress.

In obese patients and in those with cancer the ROS problem has a special role; supplements or diets with high content of vegetables with antioxidant activity have been given. The use of fruits and vegetables showed positive results in reducing the risk for cancer and recurrences.

Data, however, are not univocal. Each vegetable contains many different compounds, their availability is not always in relation with their content (it is a typical example for Beta carotene of carrot), the contents of a type of antioxidant may differ for the production site, stage of maturation to collection, preservation, and preparation methods (tomato sauce contains more available lycopene than raw tomato). The availability of a substance may change in different individuals according to the integrity of the intestinal mucosa (often damaged by chemother‐ apy) or to the kind of intestinal flora (1-1,5 kg of bacteria). This condition can also modify the food chemical structure, producing harmful or healthy substances for our health as in the case of soy isoflavones transformed into the much more active Equol only in subjects with suitable bacteria. In our blood and urine there's a large amount of products of bacterial metabolism which may influence our health; it may differ depending on the breed, gender, functional states (pregnancy) and dietary habits: there's much more complexity in epidemiological studies with the use of the food or nutritional supplements than expected in the research protocol.

The real availability (absorption) of substances in food or in supplements has a good chance to be different from that hypothesized and calculated with questionnaires or bromatological tables.

Diet should not cause any further increase in insulin levels, either basal or food-induced.

The daily intake of carbohydrates (i.e., glycemic load) should be in proportional with the body composition, the energy percentage (calculated in relation with other energy sources), and the degree of physical activity (including daily activities as well as activities planned by the rehabilitation system to reduce overweight and improve muscular function).

Carbohydrates intake should be progressively reduced throughout the day according to the circadian increase in insulin resistance, more prominently observed during the last day hours.

Last but not least, it is necessary to avoid foods with high glycemic index (GI). The GI is determined by comparing the postprandial glycemic response of a food with the postprandial glycemic response to the same amount of available carbohydrate from a standard food in the same individual.

During the cooking process an improper use of heat can turn food into a non-profit element, even dangerous for health. The use of high temperatures for long periods can produce carcinogenic substances. The use of cooking helps the extraction of carotenoids from tomatoes and carrots, but degrades the antioxidants in cruciferous vegetables, often investigated for their anticancer properties. The problem regarding the cooking should be extended to the used

The Treatment of Cancer: A Comprehensive Therapeutic Model Entailing a Complex of Interaction Modalities

http://dx.doi.org/10.5772/55696

463

All food should be cooked with adequate methods, tools and cooking times. A typical example may be that of the french fries, for which the interest in compositional characteristics of nutritional caused a controversy about their potential toxicity, related to frying due to the

Caloric restriction is an integral part of religion requirements in several countries (Islamic Ramadan, Orthodox Church abstinence during Christmas, Easter, Assumption, the Jewish

Over the past 30 years there have been more and more studies addressing health benefits

Data seem to show that maximum benefits may be achieved by applying the highest possible calory reduction without resulting in overt malnutrition, and by prolonging this status as long

In animal models, caloric reduction of not more than 10-40% of the normal calories intake exerts

Caloric restriction induces changes in metabolic and hormonal status in a similar way among

Caloric restriction can increase life expectancy in animals; however, the restriction of carbo‐ hydrates or lipids alone does not seem to influence this result, which instead appears to be related to the reduction in methionine intake by lowering consumption of animal proteins. One year-long caloric restriction alone, even without physical activity, can reduce several markers of inflammation in obese postmenopausal women, including C-reactive protein,

Accordingly, the excess of caloric intake induces obesity and represents a risk factor for cancer.

From rodents to primates, including humans, caloric restriction has been shown to be one of

Caloric restriction improves sensitivity to insulin and improves glucose metabolism.

related to caloric intake reduction in animal models and in humans.

an anticancer effect which is directly related to its duration.

the most powerful tools in the prevention of carcinogenesis.

Caloric restriction can reduce oxidative stress.

instruments types (oven, microwave, fry, steam, etc.).

formation of acrylamide.

**4. Caloric restriction**

as possible.

animals and humans.

serum amyloid, and IL-6.

tradition of Daniel's fasting, etc.).

Often using fruit we take more attention to the amount (5 servings a day) and to the concen‐ tration in antioxidants rather than the sugar content, which brings us back to the problem of calories and metabolic syndrome (fructose plugged to lead to a lower insulin response, is indeed much more dangerous than glucose for the pathogenesis of metabolic syndrome).

Diet is often unbalanced, not respecting the right proportions between carbohydrates (60%), lipids (25%) and proteins (15%).

The use of processed foods induces a higher salt intake, with effects on blood pressure and 10 on the integrity of structures such as the gastric mucosa with possible susceptibility to cancer.

The use of sweetened drinks and refined flour, without fibers, which are characteristics of white bread and pasta, causes a rapid absorption of carbohydrates and a rapid elevation of blood glucose, followed by a massive insulin response. Insulin is a hormone with multiple activities involved in the regulation of blood glucose, the transport of amino acids, the mobilization of fat from their deposits, the monitoring of urine output and of cell proliferation.

Persistent high levels of insulin indicate a loss of activity of the hormone (insulin resistance) that goes together with obesity, dyslipidemia (low HDL cholesterol, high triglycerides), high blood pressure and, according to data, even the cancer.

Fast food diets, also known with the term "Cafeteria Diet", are often characterized by an excessive fat content, often saturated, (those who melt at higher temperatures) contained in marbled meat, so defined because at a thin shear it shows impregnation of lipids within the muscle structure, typical of those animals kept under movement restriction.

A high-fatty acids diet an altered ratio between saturated and unsaturated fats, an alteration in the ratio of unsaturated omega-6 (those that have a double bond in position 6 from terminal COOH) and omega-3 (those that have the double bond in position 3, typical of fish, nuts, etc.) causes increase in blood inflammatory markers. In a state of inflammation it leads to resistance to insulin receptors, which is the first step for obesity and metabolic syndrome.

Foods with sugar and refined flour should be reduced or abolished. Bread and pasta should be made with whole grain flours, that give them a distinctive dark color, rice should be strictly integral.

As for pasta it should be investigated whether the product is integral outset or if fibers have been added to starch in a second time. The difference is huge because the slow release of the starch in an originally integral flour can give an IG <40% than the refined flour = 75%. Rice and pasta should never be overcooked.

It is absolutely necessary to avoid using fructose as an alternative to sucrose.

Salt is an important part in the preparation and storage of food. It is blamed for stomach cancer, but may be also critical for its action on blood pressure and, indirectly, on the metabolic and inflammatory situation. Very often it is not calculated in nutritional epidemiological studies in oncology.

During the cooking process an improper use of heat can turn food into a non-profit element, even dangerous for health. The use of high temperatures for long periods can produce carcinogenic substances. The use of cooking helps the extraction of carotenoids from tomatoes and carrots, but degrades the antioxidants in cruciferous vegetables, often investigated for their anticancer properties. The problem regarding the cooking should be extended to the used instruments types (oven, microwave, fry, steam, etc.).

All food should be cooked with adequate methods, tools and cooking times. A typical example may be that of the french fries, for which the interest in compositional characteristics of nutritional caused a controversy about their potential toxicity, related to frying due to the formation of acrylamide.

### **4. Caloric restriction**

glycemic response to the same amount of available carbohydrate from a standard food in the

Often using fruit we take more attention to the amount (5 servings a day) and to the concen‐ tration in antioxidants rather than the sugar content, which brings us back to the problem of calories and metabolic syndrome (fructose plugged to lead to a lower insulin response, is indeed much more dangerous than glucose for the pathogenesis of metabolic syndrome).

Diet is often unbalanced, not respecting the right proportions between carbohydrates (60%),

The use of processed foods induces a higher salt intake, with effects on blood pressure and 10 on the integrity of structures such as the gastric mucosa with possible susceptibility to cancer. The use of sweetened drinks and refined flour, without fibers, which are characteristics of white bread and pasta, causes a rapid absorption of carbohydrates and a rapid elevation of blood glucose, followed by a massive insulin response. Insulin is a hormone with multiple activities involved in the regulation of blood glucose, the transport of amino acids, the mobilization of fat from their deposits, the monitoring of urine output and of cell proliferation. Persistent high levels of insulin indicate a loss of activity of the hormone (insulin resistance) that goes together with obesity, dyslipidemia (low HDL cholesterol, high triglycerides), high

Fast food diets, also known with the term "Cafeteria Diet", are often characterized by an excessive fat content, often saturated, (those who melt at higher temperatures) contained in marbled meat, so defined because at a thin shear it shows impregnation of lipids within the

A high-fatty acids diet an altered ratio between saturated and unsaturated fats, an alteration in the ratio of unsaturated omega-6 (those that have a double bond in position 6 from terminal COOH) and omega-3 (those that have the double bond in position 3, typical of fish, nuts, etc.) causes increase in blood inflammatory markers. In a state of inflammation it leads to resistance

Foods with sugar and refined flour should be reduced or abolished. Bread and pasta should be made with whole grain flours, that give them a distinctive dark color, rice should be strictly

As for pasta it should be investigated whether the product is integral outset or if fibers have been added to starch in a second time. The difference is huge because the slow release of the starch in an originally integral flour can give an IG <40% than the refined flour = 75%. Rice and

Salt is an important part in the preparation and storage of food. It is blamed for stomach cancer, but may be also critical for its action on blood pressure and, indirectly, on the metabolic and inflammatory situation. Very often it is not calculated in nutritional epidemiological studies

muscle structure, typical of those animals kept under movement restriction.

to insulin receptors, which is the first step for obesity and metabolic syndrome.

It is absolutely necessary to avoid using fructose as an alternative to sucrose.

same individual.

integral.

in oncology.

pasta should never be overcooked.

lipids (25%) and proteins (15%).

462 Cancer Treatment - Conventional and Innovative Approaches

blood pressure and, according to data, even the cancer.

Caloric restriction is an integral part of religion requirements in several countries (Islamic Ramadan, Orthodox Church abstinence during Christmas, Easter, Assumption, the Jewish tradition of Daniel's fasting, etc.).

Over the past 30 years there have been more and more studies addressing health benefits related to caloric intake reduction in animal models and in humans.

Data seem to show that maximum benefits may be achieved by applying the highest possible calory reduction without resulting in overt malnutrition, and by prolonging this status as long as possible.

In animal models, caloric reduction of not more than 10-40% of the normal calories intake exerts an anticancer effect which is directly related to its duration.

Caloric restriction induces changes in metabolic and hormonal status in a similar way among animals and humans.

Caloric restriction improves sensitivity to insulin and improves glucose metabolism.

Caloric restriction can reduce oxidative stress.

Caloric restriction can increase life expectancy in animals; however, the restriction of carbo‐ hydrates or lipids alone does not seem to influence this result, which instead appears to be related to the reduction in methionine intake by lowering consumption of animal proteins. One year-long caloric restriction alone, even without physical activity, can reduce several markers of inflammation in obese postmenopausal women, including C-reactive protein, serum amyloid, and IL-6.

Accordingly, the excess of caloric intake induces obesity and represents a risk factor for cancer.

From rodents to primates, including humans, caloric restriction has been shown to be one of the most powerful tools in the prevention of carcinogenesis.

However, epidemiological data deriving from forced restrictions during the events of II World War showed conflicting results.

The recovery of residual capacity is designed to recovery joint mobility and to increase the

The Treatment of Cancer: A Comprehensive Therapeutic Model Entailing a Complex of Interaction Modalities

http://dx.doi.org/10.5772/55696

465

Flexibility is one of the physiological parameters involved in almost all forms of the human movement and is similar to aerobic capacity, strength, and neuromuscular endurance in being

Flexibility has been defined as mobility compliance and, alternatively, as the reciprocal counterpart of stiffness. Most of the authors define flexibility either as range of motion at or about a joint. Another definition represents flexibility like the ability of a joint to move throughout its potential range of motion. Those definitions confuse the property of flexibility with the criterion able to measure the range of motion and using hardly synonymous; since potential range of motion is a variable factor among others in deterring flexibility, flexibility

We define flexibility like the disposition of body tissues to allow, without injury, excursions at a joint or set of joints. This property is measured by, but not equivalent to, range of motion. Both joint tissues and the surrounding soft tissues contribute to flexibility, although only the

To increase this capability is possible to use yoga, slow / static and dynamic stretching techniques, Pilates method; in our experience we prefer anyway Elispheric Imoove method (fig. 5) and exercises deriving from proprioceptive neuromuscular facilitation (PNF). This last technique is designed as a manual, partner-assisted stretching; a partner is needed to provide the fixed resistance against which the lengthened agonist isometrical contracted at or near

Some factors that affect flexibility are modifiable, subject to voluntary control to some or large

Flexibility decreases with age. In cancer patients, it suggests that regular activities, in order to

Gender is another factor that influences flexibility. Females are generally more flexible than males especially during the same stretching program; probably women have a larger percent‐

Flexibility varies during the course of the day. There is greater flexibility of cervical spine during the late afternoon and evening hours and about the lower lumbar spine data show an

About the anatomical constraints, the excessive fatty tissue limits range of motion related to the tightness of soft tissue structures. This problem is connected with some conditions of diseases like arthritis, diabetes mellitus, hemophilia and finally the cancer but also is correlated

maintain elasticity, or to do specific stretching programs, are important for aging.

In the cancer patient there is usually a marked reduction of the flexibility.

uninjured muscle tone after reprogram of flexibility.

cannot be understood simple as relative to it.

maximum (to use spindle facilitation).

extent, others are not modifiable.

age of elastin in their miofascia.

improvement during daytime later hours.

to bad posture in orthostasis or with seated flexed posture.

latter should be modified in order to enhance flexibility.

a trainable fitness parameter.

Conversely, Norwegians with a mean caloric intake reduction of about 50%, maintaining a balanced diet, showed a reduction in the incidence of breast cancer compared to controls.

In the Netherlands, a caloric intake reduction (70% in adults, 50% children) was paralleled by an increase in breast cancer but not in other forms of cancer.

The survivors of German and Russian concentration camps showed a sharp increase in all forms of cancer.

This apparent inconsistency of results can be due, in our opinion, to the distinction between caloric restriction and forced malnutrition characterized by the presence of other factors such as emotional stress, infections, etc.

### **5. Physical exercise and walking prescriptions**

### **5.1. What do you know?**

About the component of physical exercise, the American Cancer Society recommends the exercise like part of a continuum of cancer survival care.

The physical exercise is able to reduce the risk to develop the breast cancer and colon on 25% and pulmonary cancer on 30%, uterine cancer and ovary cancer about on 20% and on 9% about the prostate cancer.

After the diagnosis and the treatment there is a reduction from 26 to 40% of recruitment of Brest cancer and of colon cancer with daily physical exercise and also good quality of life.

Also during the prostate cancer the aerobic and endurance physical activity can reduce the fatigue and improve the life's quality.

During the hematological cancer especially in non-Hodgkin lymphoma and multiple myelo‐ ma, the physical exercise can improve the quality of life with reduction of fatigue and also the aerobic capability in bone marrow transplantation.

The general benefits of physical exercise in cancer treatment are numerous and include: improved cardiac output, increased ventilation, improved flexibility and range of motion; increased muscular strength and endurance; decreased resting heart rate; improved stroke volume, vasodilatation, perfusion; improved metabolic efficiency; improved blood counts; improved psychological attitude to resist to the cancer disease. The cancer-specific benefits are related to cancer treatment toxicity especially to muscular degeneration with 1) fatigue and weakness, 2) neurotoxicity, 3) cardiotoxicity, 4) pulmonary toxicity.

Our therapeutic approach using the physical exercise and walking prescriptions is divided in 3 phases to: 1) recovery of residual capacity; 2) sensory-motor and functional recovery capacity; 3) the quality of life improvement.

The recovery of residual capacity is designed to recovery joint mobility and to increase the uninjured muscle tone after reprogram of flexibility.

In the cancer patient there is usually a marked reduction of the flexibility.

However, epidemiological data deriving from forced restrictions during the events of II World

Conversely, Norwegians with a mean caloric intake reduction of about 50%, maintaining a balanced diet, showed a reduction in the incidence of breast cancer compared to controls.

In the Netherlands, a caloric intake reduction (70% in adults, 50% children) was paralleled by

The survivors of German and Russian concentration camps showed a sharp increase in all

This apparent inconsistency of results can be due, in our opinion, to the distinction between caloric restriction and forced malnutrition characterized by the presence of other factors such

About the component of physical exercise, the American Cancer Society recommends the

The physical exercise is able to reduce the risk to develop the breast cancer and colon on 25% and pulmonary cancer on 30%, uterine cancer and ovary cancer about on 20% and on 9% about

After the diagnosis and the treatment there is a reduction from 26 to 40% of recruitment of Brest cancer and of colon cancer with daily physical exercise and also good quality of life.

Also during the prostate cancer the aerobic and endurance physical activity can reduce the

During the hematological cancer especially in non-Hodgkin lymphoma and multiple myelo‐ ma, the physical exercise can improve the quality of life with reduction of fatigue and also the

The general benefits of physical exercise in cancer treatment are numerous and include: improved cardiac output, increased ventilation, improved flexibility and range of motion; increased muscular strength and endurance; decreased resting heart rate; improved stroke volume, vasodilatation, perfusion; improved metabolic efficiency; improved blood counts; improved psychological attitude to resist to the cancer disease. The cancer-specific benefits are related to cancer treatment toxicity especially to muscular degeneration with 1) fatigue and

Our therapeutic approach using the physical exercise and walking prescriptions is divided in 3 phases to: 1) recovery of residual capacity; 2) sensory-motor and functional recovery capacity;

an increase in breast cancer but not in other forms of cancer.

**5. Physical exercise and walking prescriptions**

exercise like part of a continuum of cancer survival care.

War showed conflicting results.

464 Cancer Treatment - Conventional and Innovative Approaches

as emotional stress, infections, etc.

fatigue and improve the life's quality.

3) the quality of life improvement.

aerobic capability in bone marrow transplantation.

weakness, 2) neurotoxicity, 3) cardiotoxicity, 4) pulmonary toxicity.

**5.1. What do you know?**

the prostate cancer.

forms of cancer.

Flexibility is one of the physiological parameters involved in almost all forms of the human movement and is similar to aerobic capacity, strength, and neuromuscular endurance in being a trainable fitness parameter.

Flexibility has been defined as mobility compliance and, alternatively, as the reciprocal counterpart of stiffness. Most of the authors define flexibility either as range of motion at or about a joint. Another definition represents flexibility like the ability of a joint to move throughout its potential range of motion. Those definitions confuse the property of flexibility with the criterion able to measure the range of motion and using hardly synonymous; since potential range of motion is a variable factor among others in deterring flexibility, flexibility cannot be understood simple as relative to it.

We define flexibility like the disposition of body tissues to allow, without injury, excursions at a joint or set of joints. This property is measured by, but not equivalent to, range of motion. Both joint tissues and the surrounding soft tissues contribute to flexibility, although only the latter should be modified in order to enhance flexibility.

To increase this capability is possible to use yoga, slow / static and dynamic stretching techniques, Pilates method; in our experience we prefer anyway Elispheric Imoove method (fig. 5) and exercises deriving from proprioceptive neuromuscular facilitation (PNF). This last technique is designed as a manual, partner-assisted stretching; a partner is needed to provide the fixed resistance against which the lengthened agonist isometrical contracted at or near maximum (to use spindle facilitation).

Some factors that affect flexibility are modifiable, subject to voluntary control to some or large extent, others are not modifiable.

Flexibility decreases with age. In cancer patients, it suggests that regular activities, in order to maintain elasticity, or to do specific stretching programs, are important for aging.

Gender is another factor that influences flexibility. Females are generally more flexible than males especially during the same stretching program; probably women have a larger percent‐ age of elastin in their miofascia.

Flexibility varies during the course of the day. There is greater flexibility of cervical spine during the late afternoon and evening hours and about the lower lumbar spine data show an improvement during daytime later hours.

About the anatomical constraints, the excessive fatty tissue limits range of motion related to the tightness of soft tissue structures. This problem is connected with some conditions of diseases like arthritis, diabetes mellitus, hemophilia and finally the cancer but also is correlated to bad posture in orthostasis or with seated flexed posture.

Other ways to improve flexibility: massage, warm-up and stretching are three basic techniques used to increase flexibility but neither massage or warm-up is as efficient as a proper stretching regimen in increasing flexibility.

stimulation at high intensity with Vissone ( fig. 4 Vissman, Italy ) and after anaerobic work with TRX system. Vibrations are able to induce muscular adaptions to the recovery of muscle tone at the 300 Hz, of frequency and to stimulate the upper motors centers in order to obtain a better performance of controls, responsible for the muscle recruitment. Is noted that so is possible to 1) activate the aerobic metabolism; 2) determine an analgesic effect; 3) increase local circulation and bone density; 4) finally increase the contractile capacity and elasticity of the

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To elicit the sensory-motor and functional recovery we need to get acceptable walking.

Human movement usually is defined by the walk and is not limited to bipedal locomotion; however, such locomotion is a fundamental part of daily life and is a prominent focus of public

The human gait is more complex; going one step forward, although it can start from the hip flexors of the Deep Frontal Line, especially the psoas and iliacus, afterwards, it involves the hip flexion, the knee extension, and the ankle dorsiflexion necessary to step forward, thanks to the myofascia of the Superficial Frontal Line. As the leg travels forward, the entire myofascia

Once the heel places on the ground and the step begins, the Superficial Back Line takes over as the back of leg engages into hip extension and plantar flexion. The abductors of Lateral Line, Ischio-Tibial-Tract, and the lateral compartment of the lower leg provide stability that prevents the hip adduction, while the adductor group and the other tissues of the Deep Frontal Line assist the flexion- extension motions and provide stability to the inner arch of the foot and up the inside of the leg. In the upper body, the common contralateral walking pattern involves the Functional Lines bringing the right shoulder forward to counterbalance the left leg when it swings forward and vice versa. Therefore the gist of walking capability is to improve the

The walking objective monitoring evolution, using pedometer and accelerometer technology, offers an opportunity to perform guidelines, including recommendations for cancer patients.

All the studies in literature have used a variety of objective parameters using instruments that have been previously validated. The Yamax pedometer is considered a criterion research quality pedometer (Schneider et al., 2004), the Lifecorder's validity is well documented (Crouter et al., 2003; Schneider et al., 2004), and the ActiGraph has been adopted by national surveillance strategies (Troiano et al., 2008) and is probably the most utilized accelerometer in

Therefore is possible to define with the pedometer the sedentary level into < 2,500 steps/ day (basally active) and into < 2,500 to 4,999 steps/day (limited activity); but using an established step-defined physical activity scale is possible to establish a level one for sedentary < 5,000

prepares to receive the weight of the body and the ground reaction.

muscle treated.

**6. Walking prescriptions**

health physical activity guidelines.

miofascial flexibility.

research today.

The best method to realize stretching involves a series of less than maximal isometric contrac‐ tions of the agonist muscles in a pre-lengthened state (to set up the stretch), followed by concentric contractions of the antagonist muscle group (to lengthen the agonist) in conjunction with light pressure from a partner when needed and with an instrumentation like sensorized postural bench system (TecnoBody, Italy).Though this mode the objectives are to alleviate muscle tension, to facilitate healing by increasing blood flow, to decrease muscle pain by reducing vasoconstriction. This work is to applied day by day using at the cancer patients home a specific personalized postural bench like Fleximat postural bench (fig. 1 DeltaDue, Italy).

#### **Figure 1.** Fleximat

When it is not possible to get a flexibility increase in cancer treatment: there are specific contraindications, due to time and circumstances, where stretching should not be performed to get flexibility improvement. Especially when there are reduced joint receptor and pain sensation, when mobilization of tissue is not possible, for example in post-acute cancer surgical treatment or when stretching or tension in tissue elicits pain.

After the recovery the joint mobility with the flexibility replanning, the improvement of the uninjured muscle tone and strength should be possible using before focused vibratory acoustic stimulation at high intensity with Vissone ( fig. 4 Vissman, Italy ) and after anaerobic work with TRX system. Vibrations are able to induce muscular adaptions to the recovery of muscle tone at the 300 Hz, of frequency and to stimulate the upper motors centers in order to obtain a better performance of controls, responsible for the muscle recruitment. Is noted that so is possible to 1) activate the aerobic metabolism; 2) determine an analgesic effect; 3) increase local circulation and bone density; 4) finally increase the contractile capacity and elasticity of the muscle treated.

### **6. Walking prescriptions**

Other ways to improve flexibility: massage, warm-up and stretching are three basic techniques used to increase flexibility but neither massage or warm-up is as efficient as a proper stretching

The best method to realize stretching involves a series of less than maximal isometric contrac‐ tions of the agonist muscles in a pre-lengthened state (to set up the stretch), followed by concentric contractions of the antagonist muscle group (to lengthen the agonist) in conjunction with light pressure from a partner when needed and with an instrumentation like sensorized postural bench system (TecnoBody, Italy).Though this mode the objectives are to alleviate muscle tension, to facilitate healing by increasing blood flow, to decrease muscle pain by reducing vasoconstriction. This work is to applied day by day using at the cancer patients home a specific personalized postural bench like Fleximat postural bench (fig. 1 DeltaDue,

When it is not possible to get a flexibility increase in cancer treatment: there are specific contraindications, due to time and circumstances, where stretching should not be performed to get flexibility improvement. Especially when there are reduced joint receptor and pain sensation, when mobilization of tissue is not possible, for example in post-acute cancer surgical

After the recovery the joint mobility with the flexibility replanning, the improvement of the uninjured muscle tone and strength should be possible using before focused vibratory acoustic

treatment or when stretching or tension in tissue elicits pain.

regimen in increasing flexibility.

466 Cancer Treatment - Conventional and Innovative Approaches

Italy).

**Figure 1.** Fleximat

To elicit the sensory-motor and functional recovery we need to get acceptable walking.

Human movement usually is defined by the walk and is not limited to bipedal locomotion; however, such locomotion is a fundamental part of daily life and is a prominent focus of public health physical activity guidelines.

The human gait is more complex; going one step forward, although it can start from the hip flexors of the Deep Frontal Line, especially the psoas and iliacus, afterwards, it involves the hip flexion, the knee extension, and the ankle dorsiflexion necessary to step forward, thanks to the myofascia of the Superficial Frontal Line. As the leg travels forward, the entire myofascia prepares to receive the weight of the body and the ground reaction.

Once the heel places on the ground and the step begins, the Superficial Back Line takes over as the back of leg engages into hip extension and plantar flexion. The abductors of Lateral Line, Ischio-Tibial-Tract, and the lateral compartment of the lower leg provide stability that prevents the hip adduction, while the adductor group and the other tissues of the Deep Frontal Line assist the flexion- extension motions and provide stability to the inner arch of the foot and up the inside of the leg. In the upper body, the common contralateral walking pattern involves the Functional Lines bringing the right shoulder forward to counterbalance the left leg when it swings forward and vice versa. Therefore the gist of walking capability is to improve the miofascial flexibility.

The walking objective monitoring evolution, using pedometer and accelerometer technology, offers an opportunity to perform guidelines, including recommendations for cancer patients.

All the studies in literature have used a variety of objective parameters using instruments that have been previously validated. The Yamax pedometer is considered a criterion research quality pedometer (Schneider et al., 2004), the Lifecorder's validity is well documented (Crouter et al., 2003; Schneider et al., 2004), and the ActiGraph has been adopted by national surveillance strategies (Troiano et al., 2008) and is probably the most utilized accelerometer in research today.

Therefore is possible to define with the pedometer the sedentary level into < 2,500 steps/ day (basally active) and into < 2,500 to 4,999 steps/day (limited activity); but using an established step-defined physical activity scale is possible to establish a level one for sedentary < 5,000 steps/day ; a level two >5,000 <7,499 steps/day for low active; a level three >7,500 <9,999 steps/ day for somewhat active ; a level four >10,000 <12,499 steps/day for active; and a level five ≥ 12,500 steps/day for highly active.

**7. Physical therapies connected with psychophysical techniques**

changes in patient with cancer especially in some specific T-cell populations.

better the efficiency of the cell itself to achieve its correct metabolism.

elements fluctuate in phase in the those regions called coherence domains.

the ions and of the fundamental elements to the cellular economy.

ion from its orbit of rotation in order to escape.

theory (G. Preparata, E. Del Giudice, G. Talpo 1999).

consequence of the manifestations of the diseases.

adjustment, but always deeply medical care.

be "read and decoded".

During the first year after the cancer treatment the immune system shows some specific

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There is no scientific evidence that physical therapies, like magnetic fields, are effective in the treatment of cancer itself. Global physics community perfectly knows what the extreme low frequencies and intensity of magnetic fields are. They also know how they provoke the resonance of ions (Ion Cyclotron Resonance), with the exact frequency in order to remove an

Only in the last decades the studies in biophysics have shown that with the ion cyclotron resonance is possible to stimulate the passage of ions through the membranes of the cells of the living beings changing their permeability and therefore improving the ion exchange on both sides of the membrane itself. The increase of the bioavailability of the essential ions, makes

The role of electromagnetic fields for control of cancer pain and chemotherapy nausea-induced symptoms remains controversial but this theory is to be correlate to water coherence domains'

The activities and the exchanges of the molecules in the body doesn't happen by chance, but they follow an "order" dictated by the magnetic field produced by the water, where all the

Only the molecules which react to the frequency of this magnetic field, interact with each other, starting in ordered way the correct chemical reactions necessary for life of the cell and the organism. An imbalance of this 'order' jeopardizes the functioning of the cell, with the

The 40% of the water is coherent and it can receive and deliver electromagnetic information, while the remaining 60% is not coherent, equally essential for life; it represents the solvent of

Also Montagnier L. in 2009 has recognized the validity of the coherence domains, stating how the water is not an inert substance, but may take special configurations emitting electromag‐ netic waves that can become an not pharmacological instrument of the therapy and the

The cells' DNA emits extremely low frequency waves, from zero to a few hundred of Hertz. The studies were published on the unbalance of this "range" that disturbs the harmony of the cell, with the onset of the manifestations of diseases. Some chronic diseases such as Alzheimer's, Parkinson's, multiple sclerosis, rheumatoid arthritis, and the viral diseases such as HIV -AIDS, influenza A and hepatitis C, "inform" the water of our body (biological water) of their presence issuing a special electromagnetic signals that can then

We also noticed that healthy adults can perform between approximately 4,000 and 18,000 steps/ day, and, in our opinion, also 7,500-9,9990 steps / day, resulting in between 50/ 85 steps /minute. That would be a reasonable target for the cancer patients in the first Mullan phase.

In order to get a better walking performance in the first phase of Mullan, and also in the second phase, we adopt two integrate procedures: 1) normalization of the foot-ground reaction forces using a personalized viscoelastic insoles to control vertical and shear forces on the foot during the stance phase without the obligatory use of athletic shoes; 2) use of the microgravitary system S.P.A.D (fig. 2) that determine the sensory-motor and functional recovery of the posture during the walking in combination to the development of proprioceptive information from the periphery to the cortical central system.

**PHYSICAL THERAPIES COUPLED WITH PSYCHOPHYSICAL TECHNIQUES** 

**During the first year after the cancer treatment the immune system show a some characteristic changes in patient with cancer especially in some specific T-cell populations. There is no scientific evidence that physical therapies like magnetic fields is effective in the treatment of cancer itself. In the world of the physics it is perfectly known what are the extremely low frequencies and intensity of magnetic fields that put in resonance the ions (Ion Cyclotron Resonance), that is the exact frequency with which an ion is removed from its orbit** 

**Only in the last decades the studies in biophysics have shown that with the ion cyclotron resonance is possible to stimulate the passage of ions through the membranes of the cells of the living beings changing its permeability and therefore improving the ion exchange on both sides of the membrane itself. The increase of the bioavailability of the essential ions,** 

**makes better the efficiency of the cell itself to achieve its correct metabolism.** 

**Figure 2.** SPAD

**Fig.2 SPAD** 

**of rotation to take the street of escape.**

### **7. Physical therapies connected with psychophysical techniques**

steps/day ; a level two >5,000 <7,499 steps/day for low active; a level three >7,500 <9,999 steps/ day for somewhat active ; a level four >10,000 <12,499 steps/day for active; and a level five ≥

We also noticed that healthy adults can perform between approximately 4,000 and 18,000 steps/ day, and, in our opinion, also 7,500-9,9990 steps / day, resulting in between 50/ 85 steps /minute.

In order to get a better walking performance in the first phase of Mullan, and also in the second phase, we adopt two integrate procedures: 1) normalization of the foot-ground reaction forces using a personalized viscoelastic insoles to control vertical and shear forces on the foot during the stance phase without the obligatory use of athletic shoes; 2) use of the microgravitary system S.P.A.D (fig. 2) that determine the sensory-motor and functional recovery of the posture during the walking in combination to the development of proprioceptive information from

**PHYSICAL THERAPIES COUPLED WITH PSYCHOPHYSICAL TECHNIQUES** 

**During the first year after the cancer treatment the immune system show a some characteristic changes in patient with cancer especially in some specific T-cell populations. There is no scientific evidence that physical therapies like magnetic fields is effective in the treatment of cancer itself. In the world of the physics it is perfectly known what are the extremely low frequencies and intensity of magnetic fields that put in resonance the ions (Ion Cyclotron Resonance), that is the exact frequency with which an ion is removed from its orbit** 

**Only in the last decades the studies in biophysics have shown that with the ion cyclotron resonance is possible to stimulate the passage of ions through the membranes of the cells of the living beings changing its permeability and therefore improving the ion exchange on both sides of the membrane itself. The increase of the bioavailability of the essential ions,** 

**makes better the efficiency of the cell itself to achieve its correct metabolism.** 

That would be a reasonable target for the cancer patients in the first Mullan phase.

12,500 steps/day for highly active.

468 Cancer Treatment - Conventional and Innovative Approaches

the periphery to the cortical central system.

**Fig.2 SPAD** 

**Figure 2.** SPAD

**of rotation to take the street of escape.**

During the first year after the cancer treatment the immune system shows some specific changes in patient with cancer especially in some specific T-cell populations.

There is no scientific evidence that physical therapies, like magnetic fields, are effective in the treatment of cancer itself. Global physics community perfectly knows what the extreme low frequencies and intensity of magnetic fields are. They also know how they provoke the resonance of ions (Ion Cyclotron Resonance), with the exact frequency in order to remove an ion from its orbit of rotation in order to escape.

Only in the last decades the studies in biophysics have shown that with the ion cyclotron resonance is possible to stimulate the passage of ions through the membranes of the cells of the living beings changing their permeability and therefore improving the ion exchange on both sides of the membrane itself. The increase of the bioavailability of the essential ions, makes better the efficiency of the cell itself to achieve its correct metabolism.

The role of electromagnetic fields for control of cancer pain and chemotherapy nausea-induced symptoms remains controversial but this theory is to be correlate to water coherence domains' theory (G. Preparata, E. Del Giudice, G. Talpo 1999).

The activities and the exchanges of the molecules in the body doesn't happen by chance, but they follow an "order" dictated by the magnetic field produced by the water, where all the elements fluctuate in phase in the those regions called coherence domains.

Only the molecules which react to the frequency of this magnetic field, interact with each other, starting in ordered way the correct chemical reactions necessary for life of the cell and the organism. An imbalance of this 'order' jeopardizes the functioning of the cell, with the consequence of the manifestations of the diseases.

The 40% of the water is coherent and it can receive and deliver electromagnetic information, while the remaining 60% is not coherent, equally essential for life; it represents the solvent of the ions and of the fundamental elements to the cellular economy.

Also Montagnier L. in 2009 has recognized the validity of the coherence domains, stating how the water is not an inert substance, but may take special configurations emitting electromag‐ netic waves that can become an not pharmacological instrument of the therapy and the adjustment, but always deeply medical care.

The cells' DNA emits extremely low frequency waves, from zero to a few hundred of Hertz. The studies were published on the unbalance of this "range" that disturbs the harmony of the cell, with the onset of the manifestations of diseases. Some chronic diseases such as Alzheimer's, Parkinson's, multiple sclerosis, rheumatoid arthritis, and the viral diseases such as HIV -AIDS, influenza A and hepatitis C, "inform" the water of our body (biological water) of their presence issuing a special electromagnetic signals that can then be "read and decoded".

With Ion Cyclotron Resonance we have the possibility to intervene in a not invasive, natural and precise adjustment mechanisms of the body's homeostasis, where the only pharmacolog‐ ical support can be not complete.

Therefore you get the possibility:


**Figure 3.** Qps 1

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**Figure 4.** Viss


In a preliminary observational study of 43 cancer patient group, they were divided into 3 groups of 14 patients, using also the Ion Cyclotron Resonance with QUEC PHISIS QPS1 (fig. 3) we observed the initial and final values of d-ROMs Test.

The first group only used the QUEC PHISIS QPS1

The second group used the QUEC PHISIS QPS1 and the antioxidants.

The third group only used the antioxidants.

The study shows a significant improvement after 90 minutes before the beginning of the first treatment. The values are improved and consolidated in the time after a month about the end of the cycle of treatments with the values well below average.

The Treatment of Cancer: A Comprehensive Therapeutic Model Entailing a Complex of Interaction Modalities http://dx.doi.org/10.5772/55696 471

**Figure 3.** Qps 1

With Ion Cyclotron Resonance we have the possibility to intervene in a not invasive, natural and precise adjustment mechanisms of the body's homeostasis, where the only pharmacolog‐

**2.** To adjust the enzyme functions, the ion channels and the body pH

**6.** To stimulate healing in all kinds of wounds, even after surgery.

**4.** To encourage the bioavailability and absorption of nutrients for cell metabolism

**9.** To detoxify and to allow antioxidant function against free radicals, metabolites, toxins

**12.** To improve the homeostasis recovery under stress (physiological micro trauma and

In a preliminary observational study of 43 cancer patient group, they were divided into 3 groups of 14 patients, using also the Ion Cyclotron Resonance with QUEC PHISIS QPS1 (fig.

The study shows a significant improvement after 90 minutes before the beginning of the first treatment. The values are improved and consolidated in the time after a month about the end

ical support can be not complete.

Therefore you get the possibility:

**1.** To rebalance subjective metabolism

470 Cancer Treatment - Conventional and Innovative Approaches

**3.** To strengthen the immune system

**7.** To balance the water retention

muscle protein catabolism)

**5.** To treat neuralgia, headaches and migraines

**8.** To enhance the effect of drugs and supplements

**10.** To stimulate a pain-killer function (acute and chronic)

**11.** To get muscle relaxation, from anxiety and stress

**13.** To improve the quality of life for cancer patients.

The first group only used the QUEC PHISIS QPS1

The third group only used the antioxidants.

3) we observed the initial and final values of d-ROMs Test.

of the cycle of treatments with the values well below average.

The second group used the QUEC PHISIS QPS1 and the antioxidants.

**Figure 4.** Viss

**Author details**

Chieti, Italy

**References**

R. Saggini1,2 and M. Calvani1,2

*Diabetes* 53: 1966–1971, 2004.

Diabete Metab 1993;19:458–62.

88:1721-1726.

2000

ISSN: 0394-6320

Nutr 2002;132:3451S–5S)

1 Dept. of Neuroscience and Imaging, "G. d'Annunzio" University, Chieti, Italy

the American College of Nutrition, Vol. 28, No. 4, 439S–445S (2009).

the American College of Nutrition, Vol. 28, No. 4, 439S–445S (2009).

2 Specialitation school of Physical Medicine and Rehabilitation, "G. d'Annunzio" University,

The Treatment of Cancer: A Comprehensive Therapeutic Model Entailing a Complex of Interaction Modalities

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473

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[4] Bachelot T, Ray-Coquard I, Menetrier-Caux C, et al. Prognostic value of serum levels of interleukin 6 and of serum and plasmalevels of vascular endothelial growth factor in hormonerefractory metastatic breast cancer patients. *Br J Cancer*. 2003;

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**Figure 5.** Imoove

### **8. Conclusion**

The integration between the pharmacology, the biochemistry, the biophysics and the lifestyle with energetic modulation using therapeutic diet through the use of the information and the signals, probably will be able to restore a robust immune response in the tumor-bearing host or to promote by adoptive transfer of activated effector cells or tumor-specific antibodies into the tumor-bearing host.

### **Author details**

R. Saggini1,2 and M. Calvani1,2

1 Dept. of Neuroscience and Imaging, "G. d'Annunzio" University, Chieti, Italy

2 Specialitation school of Physical Medicine and Rehabilitation, "G. d'Annunzio" University, Chieti, Italy

### **References**

**Figure 5.** Imoove

472 Cancer Treatment - Conventional and Innovative Approaches

**8. Conclusion**

the tumor-bearing host.

The integration between the pharmacology, the biochemistry, the biophysics and the lifestyle with energetic modulation using therapeutic diet through the use of the information and the signals, probably will be able to restore a robust immune response in the tumor-bearing host or to promote by adoptive transfer of activated effector cells or tumor-specific antibodies into


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[21] Colman,R.J. et al. (2009) Caloric restriction delays disease onset and mortality in rhe‐

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**Section 5**

**Supportive Care for Cancer Patients**

**Supportive Care for Cancer Patients**

**Chapter 20**

**Supportive and Palliative Care in Solid Cancer Patients**

During this century, cancer has become one of the major problem and diseases which has caused predominant death and it will even surpass heart diseases. Many of the researchers begin to use the term lifetime risk for cancer patients which refer to the time that cancer will progress and developed or the time that the patient will die because of cancer. There are many problems (i.e., side effects) associated with cancer diseases either solid type or hematological type such as nausea, vomiting, diarrhea, constipation, hypercalcemia, pain, lost of appetite, anemia, fatigue, cachexia, leucopenia, neutropenia and thrombocytopenia. However the major problems are nausea and vomiting, neutropenia, anemia, thrombocytopenia and hypercalce‐ mia. Hence due to these reasons cancer is consider as one of the major diseases that will effect

Chemotherapy was developed and used since the Word War I from the chemical weapon program of the United State of America (USA). Since then chemotherapy has became as one of the most important and significant treatment of cancer. Its main mechanism of action is by destroying the cancer cells which are characterized by their high multiplication and growth speed. However when comparing chemotherapy with other types of treatments, it still remain potentially high risk with many side effects which are difficult to manage. Chemotherapy used required the involvement of various clinical professionals during its various stages of admin‐ istration and enormous patient health care is needed to overcome its side effects [7-8].

> © 2013 Hassan et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Bassam Abdul Rasool Hassan, Zuraidah Binti Mohd Yusoff,

http://dx.doi.org/10.5772/55358

**1. Introduction**

**1.1. Cancer background**

on the quality of life for human [1-6].

**1.2. Chemotherapy background**

Mohamed Azmi Hassali and Saad Bin Othman

Additional information is available at the end of the chapter

### **Supportive and Palliative Care in Solid Cancer Patients**

Bassam Abdul Rasool Hassan, Zuraidah Binti Mohd Yusoff, Mohamed Azmi Hassali and Saad Bin Othman

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55358

### **1. Introduction**

#### **1.1. Cancer background**

During this century, cancer has become one of the major problem and diseases which has caused predominant death and it will even surpass heart diseases. Many of the researchers begin to use the term lifetime risk for cancer patients which refer to the time that cancer will progress and developed or the time that the patient will die because of cancer. There are many problems (i.e., side effects) associated with cancer diseases either solid type or hematological type such as nausea, vomiting, diarrhea, constipation, hypercalcemia, pain, lost of appetite, anemia, fatigue, cachexia, leucopenia, neutropenia and thrombocytopenia. However the major problems are nausea and vomiting, neutropenia, anemia, thrombocytopenia and hypercalce‐ mia. Hence due to these reasons cancer is consider as one of the major diseases that will effect on the quality of life for human [1-6].

### **1.2. Chemotherapy background**

Chemotherapy was developed and used since the Word War I from the chemical weapon program of the United State of America (USA). Since then chemotherapy has became as one of the most important and significant treatment of cancer. Its main mechanism of action is by destroying the cancer cells which are characterized by their high multiplication and growth speed. However when comparing chemotherapy with other types of treatments, it still remain potentially high risk with many side effects which are difficult to manage. Chemotherapy used required the involvement of various clinical professionals during its various stages of admin‐ istration and enormous patient health care is needed to overcome its side effects [7-8].

© 2013 Hassan et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1.3. Chemotherapy side effects**

The goal of chemotherapy is to be as effective as possible with tolerable side effects, since the dose of chemotherapy will be toxic to the cancer cells as well as to the normal cells. A proportion of the cancer patients suffer from only mild side effects whereas others may suffer from serious side effects. These side effects are classified as:

*2.1.1. Nausea and vomiting in solid cancer patients*

intracranial pressure and tumor burden [21].

impulses to the VC [22].

of both nausea and vomiting which are:

associated with chemotherapy and cancer diseases [11, 18-20].

*2.1.2. Understanding nausea and vomiting in advanced solid cancer*

*2.1.3. Pathophysiology of chemotherapy-induced nausea and vomiting*

*2.1.4. Major patients risk factors related with nausea and vomiting*

History of drinking alcohol, 5-Patient anxiety [11, 16, 18, 23-25]

*2.1.5. Major chemotherapy factors responsible for incidence of nausea and vomiting*

Nausea and vomiting are two of the major problems that are associated with cancer patients and 50%-55% of cancer patients suffer from both nausea and vomiting even with the use of antiemetic drugs. The main causes for this are either due to the chemotherapy or because of the cancer progression. Some of the cancer patients who were treated with chemotherapy did not suffer from nausea or vomiting because the chemotherapy used were not significantly emetogenic. Nausea and vomiting still remain the major side effects that occur and are

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Both nausea and vomiting are very common problems especially with advanced stages of solid cancer diseases like breast cancer and stomach cancer where 50 to 60% of the patients are mainly female under 65 years of age [21]. In this situation, nausea and vomiting occur because of the advanced stages of solid cancer diseases characterized by more severe complications than that caused by chemoradiotherapy or other treatments. The main causes for those problems are gastric stasis, obstruction of the intestine, opioid use, constipation caused by morphine uses, hypercalcemia, brain metastasis, renal failure, hyponatremia, increases in the

Chemotherapy cause nausea by stimulating the autonomic nervous system (ANS), while vomiting is triggered when afferent impulses from chemoreceptor trigger zone (CTZ), pharynx, cerebral cortex and vagal afferent fiber stimulate the vomiting center (VC) located in the medulla. The stimulation of the VC leads to contraction of muscles of abdomen, chest wall and diaphragm, so this will lead to an expulsion of stomach and intestine contents [11-20]. The main mechanism of chemotherapy induced vomiting is the stimulation of the entrochromaffin cells lining the wall of the gastrointestinal tract (GIT) hence causes the release of the serotonin. The serotonin will then bind to the vagal afferent 5-HT3 recep‐ tors in the GIT which will send impulses to the CTZ and VC. Vomiting will be triggered when afferent impulses from CTZ, pharynx, cerebral cortex and vagal afferent fiber transfer

1- Gender, 2-Age, 3- History of motion sickness and history of vomiting during pregnancy, 4-

There are several chemotherapeutics factors that play a major role in the incidence and severity


Occurrence of specific side effects will vary according to the chemotherapy or medications used. The most common side effects experienced are nausea and vomiting, anemia, hair lost, bleeding (thrombocytopenia), hyperuricemia, neurotoxicity, cardiotoxicity, bone marrow depression, alopecia, nephrotoxicity, pulmonary toxicity, dehydration, cystitis and mucositis. So different parameters must be taken into consideration to prevent, reduce and overcome these side effects [8-10]. This chapter will focus on the main side effects caused by cancer disease and/ or chemotherapy.

### **2. Main problems caused by cancer disease itself and/ or chemotherapy treatment**

### **2.1. Nausea and vomiting**

Both nausea and vomiting are recognized as two separate and distinct conditions. Nausea is an unpleasant sensation of being vomit or urge to vomit which may or may not result in vomiting. While, vomiting or emesis is the process of expelling of undigested food through the mouth. Nausea and vomiting can arises from different or wide spectrum of etiologies which are either directly associated to cancer disease itself or to its treatment. According to the new ranking of chemotherapy side effects, nausea is the number one or the most disturbing side effect while vomiting is the third and sometimes the fifth disturbing chemotherapy side effects. Even so, not all cancer patients suffer from nausea and/ or vomiting because not all of them were treated with emetogenic chemotherapy [11-17].

### *2.1.1. Nausea and vomiting in solid cancer patients*

**1.3. Chemotherapy side effects**

treatments.

side effects. These side effects are classified as:

488 Cancer Treatment - Conventional and Innovative Approaches

The goal of chemotherapy is to be as effective as possible with tolerable side effects, since the dose of chemotherapy will be toxic to the cancer cells as well as to the normal cells. A proportion of the cancer patients suffer from only mild side effects whereas others may suffer from serious

**2.** Delayed, which developed after 24 hours and up to 6 to 8 weeks after chemotherapy

**4.** Late/ long term, which developed after months or years of chemotherapy treatment.

Occurrence of specific side effects will vary according to the chemotherapy or medications used. The most common side effects experienced are nausea and vomiting, anemia, hair lost, bleeding (thrombocytopenia), hyperuricemia, neurotoxicity, cardiotoxicity, bone marrow depression, alopecia, nephrotoxicity, pulmonary toxicity, dehydration, cystitis and mucositis. So different parameters must be taken into consideration to prevent, reduce and overcome these side effects [8-10]. This chapter will focus on the main side effects caused by cancer

**2. Main problems caused by cancer disease itself and/ or chemotherapy**

Both nausea and vomiting are recognized as two separate and distinct conditions. Nausea is an unpleasant sensation of being vomit or urge to vomit which may or may not result in vomiting. While, vomiting or emesis is the process of expelling of undigested food through the mouth. Nausea and vomiting can arises from different or wide spectrum of etiologies which are either directly associated to cancer disease itself or to its treatment. According to the new ranking of chemotherapy side effects, nausea is the number one or the most disturbing side effect while vomiting is the third and sometimes the fifth disturbing chemotherapy side effects. Even so, not all cancer patients suffer from nausea and/ or vomiting because not all of them

**1.** Acute, which develop within 24 hours after chemotherapy administration.

**3.** Short term, combination of both acute and delayed effect.

**5.** Expected, which developed among 75% of the patients.

**7.** Uncommon, happened is less than 15% of the patients.

**6.** Common, occurred in 25%-75% of the patients.

**9.** Very rare, occur with less than 1% of the patients.

were treated with emetogenic chemotherapy [11-17].

**8.** Rare, occur in only 5% of the patients.

disease and/ or chemotherapy.

**2.1. Nausea and vomiting**

**treatment**

Nausea and vomiting are two of the major problems that are associated with cancer patients and 50%-55% of cancer patients suffer from both nausea and vomiting even with the use of antiemetic drugs. The main causes for this are either due to the chemotherapy or because of the cancer progression. Some of the cancer patients who were treated with chemotherapy did not suffer from nausea or vomiting because the chemotherapy used were not significantly emetogenic. Nausea and vomiting still remain the major side effects that occur and are associated with chemotherapy and cancer diseases [11, 18-20].

### *2.1.2. Understanding nausea and vomiting in advanced solid cancer*

Both nausea and vomiting are very common problems especially with advanced stages of solid cancer diseases like breast cancer and stomach cancer where 50 to 60% of the patients are mainly female under 65 years of age [21]. In this situation, nausea and vomiting occur because of the advanced stages of solid cancer diseases characterized by more severe complications than that caused by chemoradiotherapy or other treatments. The main causes for those problems are gastric stasis, obstruction of the intestine, opioid use, constipation caused by morphine uses, hypercalcemia, brain metastasis, renal failure, hyponatremia, increases in the intracranial pressure and tumor burden [21].

### *2.1.3. Pathophysiology of chemotherapy-induced nausea and vomiting*

Chemotherapy cause nausea by stimulating the autonomic nervous system (ANS), while vomiting is triggered when afferent impulses from chemoreceptor trigger zone (CTZ), pharynx, cerebral cortex and vagal afferent fiber stimulate the vomiting center (VC) located in the medulla. The stimulation of the VC leads to contraction of muscles of abdomen, chest wall and diaphragm, so this will lead to an expulsion of stomach and intestine contents [11-20]. The main mechanism of chemotherapy induced vomiting is the stimulation of the entrochromaffin cells lining the wall of the gastrointestinal tract (GIT) hence causes the release of the serotonin. The serotonin will then bind to the vagal afferent 5-HT3 recep‐ tors in the GIT which will send impulses to the CTZ and VC. Vomiting will be triggered when afferent impulses from CTZ, pharynx, cerebral cortex and vagal afferent fiber transfer impulses to the VC [22].

### *2.1.4. Major patients risk factors related with nausea and vomiting*

1- Gender, 2-Age, 3- History of motion sickness and history of vomiting during pregnancy, 4- History of drinking alcohol, 5-Patient anxiety [11, 16, 18, 23-25]

### *2.1.5. Major chemotherapy factors responsible for incidence of nausea and vomiting*

There are several chemotherapeutics factors that play a major role in the incidence and severity of both nausea and vomiting which are:

1- Emetogenic potential of the drug, 2- Dosage level, 3- Schedule of administration, 4- Route of administration, 5- History of previous chemotherapy, 6- Rate of I.V infusion [11, 16, 18, 23, 26].

ing 70%-80% of nausea and vomiting problems. Combination antiemetic treatment becomes the standard regimen used for the control of nausea and vomiting caused by chemothera‐ py [30]. The different types of treatments are as follows: Serotonin-receptor antagonists (*5- HT3*), Dopamine-2-receptor antagonists, Corticosteroids, Neurokinin-1-recptor antagonists,

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Interindividual diversity in drug metabolism is caused by many factors including environ‐ mental factors, cultural factors related with type of diet, concomitant drug therapy as well as genetic factors i.e., ethnic variation. All of these variations play an important role in changing pharmacokinetic and pharmacodynamic properties, volume of distribution, elimination, disposition and clinical effect for many drugs [31, 32]. Much of this distinction has shown to be caused by genetic polymorphisms of the human cytochrome P450 enzymes (CYP) [32]. CYP is the most vital enzymatic system concerned with drug metabolism. Approximately 65% of common drugs used are metabolized by cytochrome P450 enzymes and half of them are

This is a condition characterized by lack of blood or in other word a reduction of total quantity of erythrocyte (red blood cells, RBC) or hemoglobin in the circulation which are necessary for normal function. This is caused by the inability of the bone marrow to replace the erythrocyte lost. The normal level of RBC for the male is 5.4×106 cell/ µl and for female is 4.8×106 cell/ µl [11, 33-35]. It is considered as one of the most frequent hematological demonstration of malignant diseases, which will lead to momentous impairment in every tissues and organs of cancer patients and put them under serious stress. This major problem may arise because of the underlining diseases (i.e., cancer diseases) or radiother‐

The large proportion of body iron (20 mg per day) is used in the synthesis of erythrocyte cells. The body absorbed about 1 mg of iron per day from the gut to compensate the amount of daily iron lost. After the transition from erythroblast to reticulocyte, it will then remain for 3 to 4 days in the bone marrow after which being released into the blood circulation and circulate for about 100-120 days. Red blood cell (RBC) has no mitochondria so are totally dependent on ATP generated during glycolysis process. In the circulation RBC loss about 20% of its hemo‐ globin and shows physiological steps of aging. They will be phagocytes by the macrophage leading to destruction of the erythrocyte and the removal of the iron from the hemoglobin (Hb)

Cannabinoids & Benzodiazepines [29].

mediated by the CYP3A subfamily [32].

apy or chemotherapy treatment received [36, 37].

There are different types of anemia as follows:

which will be released into the plasma and redistributed again [38].

*2.2.1. Red blood cell (RBC) and iron*

*2.2.2. Types of anemia*

**2.2. Anemia**

*2.1.9. Genetic polymorphism and incidence of nausea and vomiting*

### *2.1.6. Classification of chemotherapy induced nausea and vomiting*

This classification is based on the emetogenic potential of the chemotherapeutic drug.


#### *2.1.7. Classification and incidence of chemotherapy induced nausea and vomiting*

CINV are clinically classified as:

1- Acute chemotherapy related nausea and vomiting, 2- Delayed emesis, 3- Anticipatory emesis [11, 16, 18, 28].

#### *2.1.8. Nausea and vomiting treatment options*

The main goal of the antiemetic treatment is to abolish nausea and vomiting which in the last twenty years consider as an inevitable chemotherapy side effect. This prevention is focused on the entire period of emetic risk which is 4 days for patients who received highly or moderately emetogenic chemotherapy [22, 29]. This could be perfectly achieved by understanding the mechanisms of these antiemetic drugs either alone or in combination so as to get their maximum benefit [30]. Modern antiemetic treatments help in prevent‐ ing 70%-80% of nausea and vomiting problems. Combination antiemetic treatment becomes the standard regimen used for the control of nausea and vomiting caused by chemothera‐ py [30]. The different types of treatments are as follows: Serotonin-receptor antagonists (*5- HT3*), Dopamine-2-receptor antagonists, Corticosteroids, Neurokinin-1-recptor antagonists, Cannabinoids & Benzodiazepines [29].

### *2.1.9. Genetic polymorphism and incidence of nausea and vomiting*

Interindividual diversity in drug metabolism is caused by many factors including environ‐ mental factors, cultural factors related with type of diet, concomitant drug therapy as well as genetic factors i.e., ethnic variation. All of these variations play an important role in changing pharmacokinetic and pharmacodynamic properties, volume of distribution, elimination, disposition and clinical effect for many drugs [31, 32]. Much of this distinction has shown to be caused by genetic polymorphisms of the human cytochrome P450 enzymes (CYP) [32]. CYP is the most vital enzymatic system concerned with drug metabolism. Approximately 65% of common drugs used are metabolized by cytochrome P450 enzymes and half of them are mediated by the CYP3A subfamily [32].

### **2.2. Anemia**

1- Emetogenic potential of the drug, 2- Dosage level, 3- Schedule of administration, 4- Route of administration, 5- History of previous chemotherapy, 6- Rate of I.V infusion [11, 16, 18,

**1.** Severe (90% of the patients will experience nausea and vomiting) Example: Carmustine

**3.** Moderate (30%-60%) Example: Altretamine I.V PO dose, Asparginase, Cyclophospha‐

**4.** Low (10%-30%) Example: Aldesleukin, Amsacrine, Bortezomib, Capecitabine, PO dose, Docetaxel, Doxorubicin liposomal, Etoposide all dose I.V or PO, Erlotrinib PO dose, Fluorouracil, Gefitinib PO dose, Gemcitabine, Methotrexate (I.V) 50-250 mg/ m2

**5.** Very low (less than 10%) Example: Bleomycin, Busulfan PO dose, Chlorambucil PO dose, Cladribine, Fludaradine, Hydroxyurea PO dose, Interferon, Levamisole, Melphalan PO

1- Acute chemotherapy related nausea and vomiting, 2- Delayed emesis, 3- Anticipatory

The main goal of the antiemetic treatment is to abolish nausea and vomiting which in the last twenty years consider as an inevitable chemotherapy side effect. This prevention is focused on the entire period of emetic risk which is 4 days for patients who received highly or moderately emetogenic chemotherapy [22, 29]. This could be perfectly achieved by understanding the mechanisms of these antiemetic drugs either alone or in combination so as to get their maximum benefit [30]. Modern antiemetic treatments help in prevent‐

Epirubicin I.V, Idarubicin, Ifosfamide, Lomustine PO dose, Methotrexate (I.V) 250-1000

to 1500 mg/ m2

, Cyclophosphamide PO dose, Doxorubicin (I.V) 20 to 60 mg/ m2

, Cyclophosphamide I.V > 1500 mg/ m2

, Pemetrexed, Raltitrexed, Temozalamide and

, Rituximab, Thalidomide, Thioguanine, Thiotepa, Vin‐

,

,

,

, Mito‐

, Cisplatin I.V < 50

, Cytarabine I.V > 1gm/ m2

, Irinotecan, Methotrexate I.V

This classification is based on the emetogenic potential of the chemotherapeutic drug.

, Cisplatin I.V ≥ 50 mg/ m2

**2.** High (60%-90%) Example: Carboplatin, Carmustine I.V ≤ 250 mg/ m2

Dacarbazine, Mechlorethamine, Nitrogen mustard and Streptozocin.

*2.1.6. Classification of chemotherapy induced nausea and vomiting*

490 Cancer Treatment - Conventional and Innovative Approaches

, Cyclophosphamide I.V 750 mg/ m2

and Procarbazine PO dose.

, Mitoxantrone (I.V) < 15 mg/ m2

Dactinomycin, Daunorubicin, Doxorubicin I.V > 60 mg/ m2

mycin, Paclitaxel, Porfimer, Teniposide and Trastuzumab.

blastine, Vincristine, Vinorelbine and Vindesine [25, 27].

*2.1.7. Classification and incidence of chemotherapy induced nausea and vomiting*

23, 26].

I.V ≥ 250 mg/ m2

mide (I.V) ≤ 750 mg/ m<sup>2</sup>

dose, Methotrexate < 50mg/ m2

*2.1.8. Nausea and vomiting treatment options*

CINV are clinically classified as:

emesis [11, 16, 18, 28].

mg/ m2

mg/ m2

Topotecan.

> 1 gm/ m2

This is a condition characterized by lack of blood or in other word a reduction of total quantity of erythrocyte (red blood cells, RBC) or hemoglobin in the circulation which are necessary for normal function. This is caused by the inability of the bone marrow to replace the erythrocyte lost. The normal level of RBC for the male is 5.4×106 cell/ µl and for female is 4.8×106 cell/ µl [11, 33-35]. It is considered as one of the most frequent hematological demonstration of malignant diseases, which will lead to momentous impairment in every tissues and organs of cancer patients and put them under serious stress. This major problem may arise because of the underlining diseases (i.e., cancer diseases) or radiother‐ apy or chemotherapy treatment received [36, 37].

### *2.2.1. Red blood cell (RBC) and iron*

The large proportion of body iron (20 mg per day) is used in the synthesis of erythrocyte cells. The body absorbed about 1 mg of iron per day from the gut to compensate the amount of daily iron lost. After the transition from erythroblast to reticulocyte, it will then remain for 3 to 4 days in the bone marrow after which being released into the blood circulation and circulate for about 100-120 days. Red blood cell (RBC) has no mitochondria so are totally dependent on ATP generated during glycolysis process. In the circulation RBC loss about 20% of its hemo‐ globin and shows physiological steps of aging. They will be phagocytes by the macrophage leading to destruction of the erythrocyte and the removal of the iron from the hemoglobin (Hb) which will be released into the plasma and redistributed again [38].

#### *2.2.2. Types of anemia*

There are different types of anemia as follows:

1- Iron deficiency anemia, 2- Folic acid deficiency anemia, 3- Vitamin (Vit) B12 deficiency anemia, 4- Vit C deficiency anemia, 5- Hemolytic anemia. It is an acquired type of anemia, 6- Thalassemias, 7- Sickle cell anemia, 8- Anemia of chronic diseases (ACD) [36, 37].

Mild Anemia Hb= 10g/ dL

Moderate Anemia Hb= 8.0 g/ dL- 10.0 g/ dL

Life Threatening Anemia Hb= < 6.5 g/ dL [33, 36, 43].

ment of several body organs and activities [36, 46].

*2.2.8. Role of cancer patients ages*

*2.2.9. Cancer patients gender and anemia*

female as a gender more than male [45].

*2.2.10. Cancer patients race*

*2.2.7. Clinical signs and symptoms of anemia in cancer patients*

The severity of signs and symptoms of anemia depend on several factors like Hb level, age, extent of the underlining malignant, comorbidity, rate of anemia onset, biological activity of patients vital organs and intensity of treatment used for anemia. Generally in elderly patients the clinical signs and symptoms appear with Hb level higher than that in younger patients. These symptoms usually appear gradually, starting with fatigue which is considered as one of the major signs happing in 60% to more than 90% of the anemic patients. Lethargy and lost of concentration will also take place as the Hb becomes lower than 12 g/ dL. When anemia becomes severe and chronic this will lead to decompensation of cadiorespiratory and impair‐

Supportive and Palliative Care in Solid Cancer Patients

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It has been found that the incidence of anemia and cancer increases as the age of the patient increases too. Anemia is much more related and significantly present as the age became higher than 60 years old and with steeper increases after age 80 years. Many studies showed that the hemoglobin levels remain stable between age 60 to 98 years old but there are several causes for the high incidence of anemia in the old age since there were high comorbidity, hemato‐ poietic stress and reduce in function of many vital organs. For this reason there will be great

As mention above anemia highly occur in patients older than 60 years old, but it has been found that among women, anemia happen at a younger age. The main difference between men and women are the presence of menstrual cycle i.e., blood loss and childbearing iron loss which make incidence and association of anemia higher in younger women as compared to men [44]. Besides that men and women who do not have menstruation, the amount of iron lost in one day is 1 mg. While, in women still with menstrual, the loss is about 0.6 to 2.5 times more than previous mentioned amount. The amount of iron lost during each menstruation cycle depends on the severity of bleeding. The standard iron lost per menstrual cycle for woman weighting about 60 kg is about 10 mg. So all of these evidences showed that anemia is associated with

Race also play an important role in incidence of anemia since it is consider as one of the risk factor which play role in its occurrence. The prevalence of anemia in USA among white women

association and increases in occurrence of anemia in elderly patients [44].

Severe Anemia Hb= 6.5 g/ dL- 7.9 g/ dL

### *2.2.3. Erythropoietin (EPO) description and action*

EPO is a glycoprotein hormone consists of 165 amino acid with a peptide mass of 18.2 kDa. It is mainly produce by the liver during fetal stage but after birth the kidneys become the primary production sites. It has been realized that most of EPO in the circulation comes and produce from the cortex of the kidney [39]. EPO production is mainly controlled by the feedback system between kidney and bone marrow. The kidneys mainly depend on the renal oxygen sensor for EPO production. Kidney cells response greatly towards hypoxia by increasing the EPO production. Serum level of EPO ranges between 10 to 20 mU/ mL and for normal situation the observed EPO concentration/ predictive EPO concentration (O/P) ratio must range between 0.8-1.2 [41]. EPO maintain erythropoiesis is by preventing the colony forming unit-erythroid (CFU-E) from death by apoptosis process. By this way these progenitor cells will keep proliferating and differentiating to produce erythrocyte [39].

### *2.2.4. Causes of anemia of chronic diseases (ACD)*

Anemia remain as one of the serious and frequent problem of cancer mainly cancer of the gastrointestinal, liver, head and neck, ovarian and cervix. This is mainly caused by cytokines including interlukine-1, interlukine-6, interferon-γ and tumor necrosis factor-α produced by these cancer diseases. These cytokines caused impairment of erythropoietin (EPO) synthesis, reduce erythrocytes life span and prevent normal iron utilization. Other direct effect of tumor that cause anemia is bone marrow replacement which is associated with inhibition of the body ability for the production of RBC. This condition of bone marrow suppression is associated with specific types of cancers like breast, prostate, myeloma, lymphoma and acute leukemia. Also bone marrow suppression is mainly caused by chemotherapy and radiotherapy which are the main treatment for cancer. Mainly in cancer patients the major risk factors responsible for incidence and severity of anemia are the form of cancer as well as type and dose of chemotherapy administered to the cancer patients. [11, 33-36, 40, 41].

#### *2.2.5. Diagnosis of anemia*

Several parameters need to be checked for anemia diagnosis since each one is considered important and they are as follow: Family history, laboratory tests, X-ray, biopsy and bone examination [33, 35, 42].

#### *2.2.6. Grades of anemia (levels)*

The grades or severity of anemia will depend on several factors like hemoglobin level, velocity of onset of anemia, age, co-morbidities, extent of the underlining malignancy, intensity of treatment and the biological function of the patients organ. Anemia grades as follows:

Normal level (women Hb= 12.0 g/ dL-16.0 g/ dL, men Hb= 14.0 g/ dL- 18.0 g/ dL)

Mild Anemia Hb= 10g/ dL Moderate Anemia Hb= 8.0 g/ dL- 10.0 g/ dL Severe Anemia Hb= 6.5 g/ dL- 7.9 g/ dL Life Threatening Anemia Hb= < 6.5 g/ dL [33, 36, 43].

### *2.2.7. Clinical signs and symptoms of anemia in cancer patients*

The severity of signs and symptoms of anemia depend on several factors like Hb level, age, extent of the underlining malignant, comorbidity, rate of anemia onset, biological activity of patients vital organs and intensity of treatment used for anemia. Generally in elderly patients the clinical signs and symptoms appear with Hb level higher than that in younger patients. These symptoms usually appear gradually, starting with fatigue which is considered as one of the major signs happing in 60% to more than 90% of the anemic patients. Lethargy and lost of concentration will also take place as the Hb becomes lower than 12 g/ dL. When anemia becomes severe and chronic this will lead to decompensation of cadiorespiratory and impair‐ ment of several body organs and activities [36, 46].

#### *2.2.8. Role of cancer patients ages*

1- Iron deficiency anemia, 2- Folic acid deficiency anemia, 3- Vitamin (Vit) B12 deficiency anemia, 4- Vit C deficiency anemia, 5- Hemolytic anemia. It is an acquired type of anemia, 6-

EPO is a glycoprotein hormone consists of 165 amino acid with a peptide mass of 18.2 kDa. It is mainly produce by the liver during fetal stage but after birth the kidneys become the primary production sites. It has been realized that most of EPO in the circulation comes and produce from the cortex of the kidney [39]. EPO production is mainly controlled by the feedback system between kidney and bone marrow. The kidneys mainly depend on the renal oxygen sensor for EPO production. Kidney cells response greatly towards hypoxia by increasing the EPO production. Serum level of EPO ranges between 10 to 20 mU/ mL and for normal situation the observed EPO concentration/ predictive EPO concentration (O/P) ratio must range between 0.8-1.2 [41]. EPO maintain erythropoiesis is by preventing the colony forming unit-erythroid (CFU-E) from death by apoptosis process. By this way these progenitor cells will keep

Anemia remain as one of the serious and frequent problem of cancer mainly cancer of the gastrointestinal, liver, head and neck, ovarian and cervix. This is mainly caused by cytokines including interlukine-1, interlukine-6, interferon-γ and tumor necrosis factor-α produced by these cancer diseases. These cytokines caused impairment of erythropoietin (EPO) synthesis, reduce erythrocytes life span and prevent normal iron utilization. Other direct effect of tumor that cause anemia is bone marrow replacement which is associated with inhibition of the body ability for the production of RBC. This condition of bone marrow suppression is associated with specific types of cancers like breast, prostate, myeloma, lymphoma and acute leukemia. Also bone marrow suppression is mainly caused by chemotherapy and radiotherapy which are the main treatment for cancer. Mainly in cancer patients the major risk factors responsible for incidence and severity of anemia are the form of cancer as well as type and dose of

Several parameters need to be checked for anemia diagnosis since each one is considered important and they are as follow: Family history, laboratory tests, X-ray, biopsy and bone

The grades or severity of anemia will depend on several factors like hemoglobin level, velocity of onset of anemia, age, co-morbidities, extent of the underlining malignancy, intensity of treatment and the biological function of the patients organ. Anemia grades as follows:

Normal level (women Hb= 12.0 g/ dL-16.0 g/ dL, men Hb= 14.0 g/ dL- 18.0 g/ dL)

Thalassemias, 7- Sickle cell anemia, 8- Anemia of chronic diseases (ACD) [36, 37].

*2.2.3. Erythropoietin (EPO) description and action*

492 Cancer Treatment - Conventional and Innovative Approaches

proliferating and differentiating to produce erythrocyte [39].

chemotherapy administered to the cancer patients. [11, 33-36, 40, 41].

*2.2.4. Causes of anemia of chronic diseases (ACD)*

*2.2.5. Diagnosis of anemia*

examination [33, 35, 42].

*2.2.6. Grades of anemia (levels)*

It has been found that the incidence of anemia and cancer increases as the age of the patient increases too. Anemia is much more related and significantly present as the age became higher than 60 years old and with steeper increases after age 80 years. Many studies showed that the hemoglobin levels remain stable between age 60 to 98 years old but there are several causes for the high incidence of anemia in the old age since there were high comorbidity, hemato‐ poietic stress and reduce in function of many vital organs. For this reason there will be great association and increases in occurrence of anemia in elderly patients [44].

#### *2.2.9. Cancer patients gender and anemia*

As mention above anemia highly occur in patients older than 60 years old, but it has been found that among women, anemia happen at a younger age. The main difference between men and women are the presence of menstrual cycle i.e., blood loss and childbearing iron loss which make incidence and association of anemia higher in younger women as compared to men [44]. Besides that men and women who do not have menstruation, the amount of iron lost in one day is 1 mg. While, in women still with menstrual, the loss is about 0.6 to 2.5 times more than previous mentioned amount. The amount of iron lost during each menstruation cycle depends on the severity of bleeding. The standard iron lost per menstrual cycle for woman weighting about 60 kg is about 10 mg. So all of these evidences showed that anemia is associated with female as a gender more than male [45].

### *2.2.10. Cancer patients race*

Race also play an important role in incidence of anemia since it is consider as one of the risk factor which play role in its occurrence. The prevalence of anemia in USA among white women is 7.1% and 25.1% among black women even after adjustment of iron level. Besides that black women are characterized by lower mean hemoglobin level compared to white women. Also black woman has a wide standard deviation in mean of hemoglobin than the white one has [45]. *2.2.12. Indications and options for anemia treatments*

incidence is high among acute leukemia patients [56-63].

macrophages cells predominantly in the spleen and liver [66].

*2.3.1. Platelets morphology and structure*

anemic patients (QOL) [36].

**2.3. Thrombocytopenia**

Anemia and its related symptoms have serious negative effects on patients quality of life (QOL) and anticancer treatment since it will leads to treatment delay. These effects may be tolerated in young patients even with very low hemoglobin levels. While in patients with multimor‐ bidity would not be able to tolerate this and as a result of that many severe clinical signs and symptoms will developed even with minor reduction in the Hb levels [36, 54, 55]. The treatment strategies of anemia mainly based on the clinical situation, clinical signs and symptoms and on the underlining cause of anemia. These treatments will include red blood cell transfusion, corticosteroids, VitB12 and Epoetin alfa (recombinant human erythropoietin, rHuEPO). All these treatments were used to overcome anemia related signs, symptoms and to improve the

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Thrombocytopenia is a term used to denote abnormal decrease or drop in platelets numbers. The main function of these platelets is clot formation during bleeding in order to prevent blood lost. Thus a decrease in platelet number will leads to bleeding condition which ranges from mild bleeding from small blood vessels to severe bleeding from large blood vessels. Severe bleeding in the presence of severe thrombocytopenia or when is coupled with other clotting disorders can leads to serious morbidity or death. Thrombocytopenia is a com‐ mon problem experience by cancer patients, which usually resulted from the use of conventional chemotherapy and at times is a dose limiting factor for chemotherapy administration. The incidence of thrombocytopenia among solid cancer patients is rather low i.e., ranging between 10%-25% among breast cancer, ovarian and germ cell cancer patients who were treated with intensive chemotherapy. However thrombocytopenia

Platelets or thrombocytes are irregular, disc shaped cells which are considered as the smallest cells in the blood (0.5 to 3.0 µm diameter). They are usually produce from the megakaryocytes which are large cells (80 to 150 µm diameter) found specifically in the spongy center of long bones by the stimulation of thrombopoietin (TPO) by process called endomitosis whereby each megakaryocyte cell produce about 2000 platelets. These platelets shared a characteristic of having a very short life span (five to nine days only) so the bone marrow of healthy individual continuously keep producing new platelets cells to replace the old dead ones. The thrombo‐ poietin hormone is mainly synthesis and produce by the liver and plays a major role in stimulation of proliferation and maturation of platelets. The circulating platelets have no nucleus but they have alpha granules and dense granules [57, 64, 65]. Physiologically the platelets are removed from the blood circulation by two mechanisms. The first is being used at common sites of vascular injury like in the microcirculation, secondly to be phagocyte by

#### *2.2.11. Mechanisms of anemia in cancer patients*

#### *2.2.11.1. Role of cancer disease*

Occurrence and association of anemia with cancer depends on several factors including patients age, stage of cancer, presence or absence of infection and other comorbidities. Anemia prevalence is highly associated especially with lymphomas, genitourinary tumor, lung and multiply myeloma. The incidence of mild to moderate anemia with solid tumor is higher than incidence of severe anemia which occurs highly with hematological can‐ cers than solid one [36, 46, 47]. The main mechanism whereby cancer causes anemia is by producing cytokines which are mainly tumor necrosis factor (TNF-α) and interleukin-1 and they have the ability to hamper EPO production and action, reduce the life span of RBC and preclude ordinary utilization of iron. Other mechanisms which will cause association of anemia with cancer will be separated from the cancer itself like Vit B12, folic acid and iron deficiency. Renal, endocrinal disorders splenomegaly, clonogenic and cachexia occurrence with cancer also play a major role in occurrence of anemia [36].

#### *2.2.11.2. Role of chemotherapy*

Anemia is one of the common side effect of chemotherapy especially with the myelosuppres‐ sive type. Incidence and severity of anemia depend on several different factors which are the chemotherapy type, schedule and intensity as well as type of cancer. Chemotherapy cycles also play an important role in increasing the severity of anemia since multiply cycles will cumulatively inhibit or reduce erythropoiesis. It has been found by the European Cancer Anaemia Survey (ECAS) that the incidence of anemia after the first cycle is 19.5% and after second cycle is 34.3% while after the third the incidence was more than 40%. Also single or combination chemotherapy play a serious and major role in anemia incidence and severity since the use of combination chemotherapy regimen will leads to severe anemia more than the use of single chemotherapy drug [48-50]. Besides chemotherapy myelosuppression, anemia can take place as a result of direct destruction of the RBC (i.e., direct effect on the erythropoiesis in the bone marrow) or reduced erythropoietin production (i.e., impact on EPO production). When this chemotherapy drug or other drugs used repetitively this may lead to prolong production of anemia. Also the results that obtained from clinical trials showed that the probability of mild anemia incidence after the use of chemotherapy is 100%, while the probability of severe anemia incidence after chemotherapy is 80%. From these results and data it has been proven that chemotherapy is the major impact factor for anemia onset and severity in cancer patients [41, 48, 51-53].

### *2.2.12. Indications and options for anemia treatments*

Anemia and its related symptoms have serious negative effects on patients quality of life (QOL) and anticancer treatment since it will leads to treatment delay. These effects may be tolerated in young patients even with very low hemoglobin levels. While in patients with multimor‐ bidity would not be able to tolerate this and as a result of that many severe clinical signs and symptoms will developed even with minor reduction in the Hb levels [36, 54, 55]. The treatment strategies of anemia mainly based on the clinical situation, clinical signs and symptoms and on the underlining cause of anemia. These treatments will include red blood cell transfusion, corticosteroids, VitB12 and Epoetin alfa (recombinant human erythropoietin, rHuEPO). All these treatments were used to overcome anemia related signs, symptoms and to improve the anemic patients (QOL) [36].

### **2.3. Thrombocytopenia**

is 7.1% and 25.1% among black women even after adjustment of iron level. Besides that black women are characterized by lower mean hemoglobin level compared to white women. Also black woman has a wide standard deviation in mean of hemoglobin than the white one has [45].

Occurrence and association of anemia with cancer depends on several factors including patients age, stage of cancer, presence or absence of infection and other comorbidities. Anemia prevalence is highly associated especially with lymphomas, genitourinary tumor, lung and multiply myeloma. The incidence of mild to moderate anemia with solid tumor is higher than incidence of severe anemia which occurs highly with hematological can‐ cers than solid one [36, 46, 47]. The main mechanism whereby cancer causes anemia is by producing cytokines which are mainly tumor necrosis factor (TNF-α) and interleukin-1 and they have the ability to hamper EPO production and action, reduce the life span of RBC and preclude ordinary utilization of iron. Other mechanisms which will cause association of anemia with cancer will be separated from the cancer itself like Vit B12, folic acid and iron deficiency. Renal, endocrinal disorders splenomegaly, clonogenic and cachexia

Anemia is one of the common side effect of chemotherapy especially with the myelosuppres‐ sive type. Incidence and severity of anemia depend on several different factors which are the chemotherapy type, schedule and intensity as well as type of cancer. Chemotherapy cycles also play an important role in increasing the severity of anemia since multiply cycles will cumulatively inhibit or reduce erythropoiesis. It has been found by the European Cancer Anaemia Survey (ECAS) that the incidence of anemia after the first cycle is 19.5% and after second cycle is 34.3% while after the third the incidence was more than 40%. Also single or combination chemotherapy play a serious and major role in anemia incidence and severity since the use of combination chemotherapy regimen will leads to severe anemia more than the use of single chemotherapy drug [48-50]. Besides chemotherapy myelosuppression, anemia can take place as a result of direct destruction of the RBC (i.e., direct effect on the erythropoiesis in the bone marrow) or reduced erythropoietin production (i.e., impact on EPO production). When this chemotherapy drug or other drugs used repetitively this may lead to prolong production of anemia. Also the results that obtained from clinical trials showed that the probability of mild anemia incidence after the use of chemotherapy is 100%, while the probability of severe anemia incidence after chemotherapy is 80%. From these results and data it has been proven that chemotherapy is the major impact factor for anemia onset and severity

occurrence with cancer also play a major role in occurrence of anemia [36].

*2.2.11. Mechanisms of anemia in cancer patients*

494 Cancer Treatment - Conventional and Innovative Approaches

*2.2.11.1. Role of cancer disease*

*2.2.11.2. Role of chemotherapy*

in cancer patients [41, 48, 51-53].

Thrombocytopenia is a term used to denote abnormal decrease or drop in platelets numbers. The main function of these platelets is clot formation during bleeding in order to prevent blood lost. Thus a decrease in platelet number will leads to bleeding condition which ranges from mild bleeding from small blood vessels to severe bleeding from large blood vessels. Severe bleeding in the presence of severe thrombocytopenia or when is coupled with other clotting disorders can leads to serious morbidity or death. Thrombocytopenia is a com‐ mon problem experience by cancer patients, which usually resulted from the use of conventional chemotherapy and at times is a dose limiting factor for chemotherapy administration. The incidence of thrombocytopenia among solid cancer patients is rather low i.e., ranging between 10%-25% among breast cancer, ovarian and germ cell cancer patients who were treated with intensive chemotherapy. However thrombocytopenia incidence is high among acute leukemia patients [56-63].

#### *2.3.1. Platelets morphology and structure*

Platelets or thrombocytes are irregular, disc shaped cells which are considered as the smallest cells in the blood (0.5 to 3.0 µm diameter). They are usually produce from the megakaryocytes which are large cells (80 to 150 µm diameter) found specifically in the spongy center of long bones by the stimulation of thrombopoietin (TPO) by process called endomitosis whereby each megakaryocyte cell produce about 2000 platelets. These platelets shared a characteristic of having a very short life span (five to nine days only) so the bone marrow of healthy individual continuously keep producing new platelets cells to replace the old dead ones. The thrombo‐ poietin hormone is mainly synthesis and produce by the liver and plays a major role in stimulation of proliferation and maturation of platelets. The circulating platelets have no nucleus but they have alpha granules and dense granules [57, 64, 65]. Physiologically the platelets are removed from the blood circulation by two mechanisms. The first is being used at common sites of vascular injury like in the microcirculation, secondly to be phagocyte by macrophages cells predominantly in the spleen and liver [66].

### *2.3.2. Platelets function*

Platelets have vital functions in immunity, wound repair and homeostasis. These functions mainly depend on platelets concentration in blood circulation. Platelets prevent bleeding by either sealing the hole in the blood vessel wall or by forming haemostatic plug or by liberating several chemicals that will activate more clotting formation by breaking down more of the platelets. The main steps for platelets action to form clot are the following:

**2.** Solid cancer.

**4.** Spleen cancer.

**5.** Anemia.

**3.** Blood cancer (Leukemia).

production [57, 59, 65].

*2.3.6. Chemotherapy and thrombocytopenia*

*2.3.7. Mechanism of thrombocytopenia in solid cancer*

malignant unless all the above characteristic are all present [62].

*2.3.5. Role of age and gender*

**6.** Hemorrhage which will lead to increases loss of platelets.

**7.** When the rate of platelets destruction is higher than the rate of bone marrow platelets

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Repetto (2003) mentioned that anemia is highly prevalent and happened in the elderly cancer patients who receive chemotherapy. This is specifically because their senescent cells have low ability to repair DNA and their low mass of the hematopoietic stem cell causing slowing of their recovery ability. Repetto also mentioned in his study that the occurrence of grade 3 thrombocytopenia is highly associated with older female suffering from breast cancer and found that there is an association between age and gender with thrombocytopenia. While others retrospective studies of solid tumor patients found that there is no association between age and myelosuppression i.e., neutropenia, anemia and thrombocytopenia occurrence [68].

Thrombocytopenia is a detrimental side effect of chemotherapy since it will lead to hemorrhage from vital organ particularly the brain specifically within solid cancer patients who were treated with chemotherapy. These chemotherapies caused thrombocytopenia by different mechanisms either by suppressing megakaryopoiesis leading to prevention of platelets production or by direct damaging of the platelets. Chemotherapies like antimetabolites and alkylating agents induced severe thrombocytopenia due to their ability in causing bone marrow suppression and specifically after the first cycle of chemotherapy [62, 69, 70, 71].

The association between bleeding and thrombocytopenia in patient suffering from leuke‐ mia was first described in 1962. Later in 1878 and 1984 this was reported happening among patient suffering from solid cancer [72]. Thrombocytopenia as a serious side effect is usually associated with solid cancer as a result of its metastasis to bone marrow. Theoretically most of solid tumors can metastasis to bone marrow but the most frequent are breast, lung and prostate cancers. These cancers when metastasized to bone marrow will lead to bone marrow suppression resulting in neutropenia and thrombocytopenia with serious morbid‐ ity and mortality (Kilickap *et al*., 2007). Besides that Elting and his colleagues mentioned that solid cancer patients are characterized by several things which are poor performance status, low baseline for platelets count and bone marrow metastasis. Despite that the bleeding situation among solid cancer patients remain poor compare with hematological


From this it is clear that thrombocytopenia which is associated with decrease in platelets count in the blood of cancer patients such as leukemic patients is considered as a very serious problem. Thrombocytopenia prevalence in hematological patients is very high. While in case of solid tumor, thrombocytopenia happens because of chemotherapy uses and thus the incidence is rather low. However in some subgroups the incidence is higher than 20% and it still remain as a serious and dangerous problem [62].

### *2.3.3. Thrombopoietin hormone (TPO)*

It is a single 353- amino acid protein, synthesized primarily in the liver. Its level will increase during thrombocytopenia and keep increasing in response to the decline in platelet mass. For this reason most of the studies found that when platelets is transfused to the thrombocytopenic patients the TPO level will decreased. TPO mainly act by increasing the numbers of megakar‐ yocyte colony forming cells (Meg-CFC), increases their ploidy, size and growth to produce more of the platelets. Moreover, it will stimulate the hematopoietic stem cell of the bone marrow and it has been found that high doses of TPO will lead to reactivation of the mature platelets to some aggregation stimuli [61].

### *2.3.4. Main causes of thrombocytopenia*

The main causes leading to occurrence of thrombocytopenia are:

**1.** Chemotherapy drugs.

### **2.** Solid cancer.

*2.3.2. Platelets function*

496 Cancer Treatment - Conventional and Innovative Approaches

the chemical changes.

formation [64, 67].

*2.3.3. Thrombopoietin hormone (TPO)*

platelets to some aggregation stimuli [61].

The main causes leading to occurrence of thrombocytopenia are:

*2.3.4. Main causes of thrombocytopenia*

**1.** Chemotherapy drugs.

of thromboxane A2 released by the platelets.

still remain as a serious and dangerous problem [62].

Platelets have vital functions in immunity, wound repair and homeostasis. These functions mainly depend on platelets concentration in blood circulation. Platelets prevent bleeding by either sealing the hole in the blood vessel wall or by forming haemostatic plug or by liberating several chemicals that will activate more clotting formation by breaking down more of the

**1.** Adhesion (Step 1): This reaction is mediated by release of granules and characterized by shape change of the platelets from disc shape to spiny spheres after their adhesion to

**2.** Aggregation (Step 2): In this step more of platelets adhere to each other and there will be an obvious shape change of these platelets. The main factors that stimulate this step are

**3.** Release (Step 3): Here the aggregation caused by the dense granules released by the platelets themselves is irreversible. In addition vasoconstriction will take place as a result

**4.** Stabilization of the clot (Step 4): This is the main reaction which is responsible for the thrombus formation, whereby the aggregate platelets will release factor V that will accelerate the aggregation of other platelets and this will lead to stabilized clot

From this it is clear that thrombocytopenia which is associated with decrease in platelets count in the blood of cancer patients such as leukemic patients is considered as a very serious problem. Thrombocytopenia prevalence in hematological patients is very high. While in case of solid tumor, thrombocytopenia happens because of chemotherapy uses and thus the incidence is rather low. However in some subgroups the incidence is higher than 20% and it

It is a single 353- amino acid protein, synthesized primarily in the liver. Its level will increase during thrombocytopenia and keep increasing in response to the decline in platelet mass. For this reason most of the studies found that when platelets is transfused to the thrombocytopenic patients the TPO level will decreased. TPO mainly act by increasing the numbers of megakar‐ yocyte colony forming cells (Meg-CFC), increases their ploidy, size and growth to produce more of the platelets. Moreover, it will stimulate the hematopoietic stem cell of the bone marrow and it has been found that high doses of TPO will lead to reactivation of the mature

platelets. The main steps for platelets action to form clot are the following:

collagen. The aggregation of the platelets in this face is reversible.


#### *2.3.5. Role of age and gender*

Repetto (2003) mentioned that anemia is highly prevalent and happened in the elderly cancer patients who receive chemotherapy. This is specifically because their senescent cells have low ability to repair DNA and their low mass of the hematopoietic stem cell causing slowing of their recovery ability. Repetto also mentioned in his study that the occurrence of grade 3 thrombocytopenia is highly associated with older female suffering from breast cancer and found that there is an association between age and gender with thrombocytopenia. While others retrospective studies of solid tumor patients found that there is no association between age and myelosuppression i.e., neutropenia, anemia and thrombocytopenia occurrence [68].

### *2.3.6. Chemotherapy and thrombocytopenia*

Thrombocytopenia is a detrimental side effect of chemotherapy since it will lead to hemorrhage from vital organ particularly the brain specifically within solid cancer patients who were treated with chemotherapy. These chemotherapies caused thrombocytopenia by different mechanisms either by suppressing megakaryopoiesis leading to prevention of platelets production or by direct damaging of the platelets. Chemotherapies like antimetabolites and alkylating agents induced severe thrombocytopenia due to their ability in causing bone marrow suppression and specifically after the first cycle of chemotherapy [62, 69, 70, 71].

### *2.3.7. Mechanism of thrombocytopenia in solid cancer*

The association between bleeding and thrombocytopenia in patient suffering from leuke‐ mia was first described in 1962. Later in 1878 and 1984 this was reported happening among patient suffering from solid cancer [72]. Thrombocytopenia as a serious side effect is usually associated with solid cancer as a result of its metastasis to bone marrow. Theoretically most of solid tumors can metastasis to bone marrow but the most frequent are breast, lung and prostate cancers. These cancers when metastasized to bone marrow will lead to bone marrow suppression resulting in neutropenia and thrombocytopenia with serious morbid‐ ity and mortality (Kilickap *et al*., 2007). Besides that Elting and his colleagues mentioned that solid cancer patients are characterized by several things which are poor performance status, low baseline for platelets count and bone marrow metastasis. Despite that the bleeding situation among solid cancer patients remain poor compare with hematological malignant unless all the above characteristic are all present [62].

### *2.3.8. Diagnosis of thrombocytopenia*

Different parameters are taken into consideration in order to diagnose thrombocytopenia such as medical history and laboratory test. Platelets count which is considered as part of the complete blood count (CBC) is the main key for the diagnosis of thrombocytopenia. It measures the exact numbers of platelets in a measured volume of blood. If the test shows low number of platelets then a careful examination for spleen and bone marrow biopsy must be done since both have a direct association with thrombocytopenia occurrence. Usually in adults when the platelets count is less than 100,000 cell/ microliter it is consid‐ ered low but sometimes this happen without any symptoms. Other important tests which are used to diagnose thrombocytopenia are the prothrombin time (PT), activated partial thromboplastin time (aPTT) and thrombin time (TT). The results of these tests play a critical role in the diagnosis and certification of the presence of thrombocytopenia. In addition to liver enzymes test, renal function test, erythrocyte sedimentation rate (ESR), Vit B12 and folic acid levels are also carried out [57, 59, 64, 65, 73, 74].

alternative drugs or use of platelets growth factors (i.e., thrombopoietic growth factor) (Oprelvekin, Neumega®) should be made. Recombinant human interleukin-11 (rhIL-11) will stimulate megakaryocyte maturation and proliferation and maintain platelets production. It has been proven by the Food and Drug Administration (FDA) that rhIL-11 is very effective in reducing and preventing of severe thrombocytopenia as well as it will decrease the need for platelet transfusions especially after myelosuppressive chemotherapy which could be contin‐ ue with the same doses. In the case of severe thrombocytopenia (i.e., platelet level ≤ 20,000/ µL) which is due to intensive chemotherapeutics drugs treatment of hematological and solid cancer patients, in this case platelet transfusion is needed. At this point, patient will suffer from severe bleeding and the laboratory tests signify that platelets transfusion is very important and a required treatment. Platelets transfusion is one of the most important treatments for acute and severe thrombocytopenia, but there are some limitations to its use which are: the availability of the blood products since it must be freshly taken and used within 5 days, cost,

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refractoriness, transfusion reaction and diseases transmission [57-59, 63, 76, 77].

with thrombocytopenia together has not been defined yet [78].

Acute thrombocytopenia has been described in patients given Hematopoietic Growth Factors. The main factor which play role in this incidence is neutropenia treatments which are either GM-CSF and/ or G-CSF. This rapid incidence for thrombocytopenia in association with these treatments is mainly because these treatments sometimes target the platelets or caused their destruction. But the main mechanism which is responsible for the incidence of neutropenia

Hypercalcemia is a life threatening situation in which serum calcium level is elevated greater than 10.5 mg/ dl, while albumin concentration is lower than 4 g/ dl. It is a serious problem that occurs in about 10%-20% of all cancer patients especially lung, breast, head and neck cancer patients. While, in hematological cancer hypercalcemia also takes place specifically in the advanced phases of both myeloma and lymphoma. Besides that a very important point is that hypercalcemia is mainly caused by cancer without any effect or role from anticancer treat‐ ments. So many references consider hypercalcemia as a very serious and dangerous compli‐ cation that caused a significant morbidity and mortality frequently in breast cancer patients. It can occur in patients with and without bone metastasis and the main cause of hypercalcemia is the pathological bone resorption. Bone resorption is caused by the secretion of cytokine like parathyroid hormone-related protein (PTHrP) leading to activation and differentiation of osteoclast cell. In normal condition normal breast cells also secreted PTHrP during lactation so as to stimulate bone resorption and skeletal calcium release which will be used in milk synthesis. In this situation hypercalcemia is asymptomatic since the elevation of calcium level is mild, but when serum calcium elevation became very high it will leads to significant morbidity and mortality. Hypercalcemia is highly associated with breast cancer more than

*2.3.12. Thrombocytopenia and neutropenia*

**2.4. Hypercalcemia**

other types of cancers [78-83].

### *2.3.9. Grades of thrombocytopenia*

The normal range of the platelets is between 150,000 and 450,000 cells per microliter of blood (i.e., 150-450×109 / L) while thrombocytopenia could be classified into three levels as follows:


### *2.3.10. Clinical signs and symptoms of thrombocytopenia*

There are several signs and symptoms which occasionally happen with thrombocytopenia. These are bloody stool, dizziness, headache, hemorrhage, oral bleeding, nose bleeding, vaginal bleeding, black stool and petechiae (reddish purple spots in the skin) [57, 58, 74].

#### *2.3.11. Options for thrombocytopenia treatments*

There are different options for thrombocytopenia treatment but the selection will mainly depends on the etiology and severity of thrombocytopenia. Sometimes with asymptomatic thrombocytopenia treatment is not required like that in children with viral infection. But if thrombocytopenia incidence is because of spleen enlargement then splenectomy will be beneficial and effective in increasing the platelets counts. While for thrombotic thrombocyto‐ penic purpura (TTP) treatment is needed since it can leads to renal failure. In case of idiopathic thrombocytopenic purpura (ITP) the treatment depends on severity of the case and the platelets counts. In the case of heparin induce thrombocytopenia and thrombosis (HITT) the treatment is by stopping heparin administration. If the cause of thrombocytopenia is due to patient's immune system causing destruction to the platelets then the use of corticosteroids is very effective so as to suppress immune response. While if the cause is due to chemotherapy then the decision to either continue the treatment with low chemotherapy doses or use of alternative drugs or use of platelets growth factors (i.e., thrombopoietic growth factor) (Oprelvekin, Neumega®) should be made. Recombinant human interleukin-11 (rhIL-11) will stimulate megakaryocyte maturation and proliferation and maintain platelets production. It has been proven by the Food and Drug Administration (FDA) that rhIL-11 is very effective in reducing and preventing of severe thrombocytopenia as well as it will decrease the need for platelet transfusions especially after myelosuppressive chemotherapy which could be contin‐ ue with the same doses. In the case of severe thrombocytopenia (i.e., platelet level ≤ 20,000/ µL) which is due to intensive chemotherapeutics drugs treatment of hematological and solid cancer patients, in this case platelet transfusion is needed. At this point, patient will suffer from severe bleeding and the laboratory tests signify that platelets transfusion is very important and a required treatment. Platelets transfusion is one of the most important treatments for acute and severe thrombocytopenia, but there are some limitations to its use which are: the availability of the blood products since it must be freshly taken and used within 5 days, cost, refractoriness, transfusion reaction and diseases transmission [57-59, 63, 76, 77].

### *2.3.12. Thrombocytopenia and neutropenia*

Acute thrombocytopenia has been described in patients given Hematopoietic Growth Factors. The main factor which play role in this incidence is neutropenia treatments which are either GM-CSF and/ or G-CSF. This rapid incidence for thrombocytopenia in association with these treatments is mainly because these treatments sometimes target the platelets or caused their destruction. But the main mechanism which is responsible for the incidence of neutropenia with thrombocytopenia together has not been defined yet [78].

### **2.4. Hypercalcemia**

*2.3.8. Diagnosis of thrombocytopenia*

498 Cancer Treatment - Conventional and Innovative Approaches

*2.3.9. Grades of thrombocytopenia*

(i.e., 150-450×109

folic acid levels are also carried out [57, 59, 64, 65, 73, 74].

**1.** Mild thrombocytopenia if platelets count < 150 and ≥ 100 × 10<sup>9</sup>

**3.** Severe thrombocytopenia if platelets count < 20 × 109

*2.3.10. Clinical signs and symptoms of thrombocytopenia*

*2.3.11. Options for thrombocytopenia treatments*

**2.** Moderate thrombocytopenia if platelets count < 100 and ≥ 50 × 10<sup>9</sup>

Different parameters are taken into consideration in order to diagnose thrombocytopenia such as medical history and laboratory test. Platelets count which is considered as part of the complete blood count (CBC) is the main key for the diagnosis of thrombocytopenia. It measures the exact numbers of platelets in a measured volume of blood. If the test shows low number of platelets then a careful examination for spleen and bone marrow biopsy must be done since both have a direct association with thrombocytopenia occurrence. Usually in adults when the platelets count is less than 100,000 cell/ microliter it is consid‐ ered low but sometimes this happen without any symptoms. Other important tests which are used to diagnose thrombocytopenia are the prothrombin time (PT), activated partial thromboplastin time (aPTT) and thrombin time (TT). The results of these tests play a critical role in the diagnosis and certification of the presence of thrombocytopenia. In addition to liver enzymes test, renal function test, erythrocyte sedimentation rate (ESR), Vit B12 and

The normal range of the platelets is between 150,000 and 450,000 cells per microliter of blood

There are several signs and symptoms which occasionally happen with thrombocytopenia. These are bloody stool, dizziness, headache, hemorrhage, oral bleeding, nose bleeding, vaginal

There are different options for thrombocytopenia treatment but the selection will mainly depends on the etiology and severity of thrombocytopenia. Sometimes with asymptomatic thrombocytopenia treatment is not required like that in children with viral infection. But if thrombocytopenia incidence is because of spleen enlargement then splenectomy will be beneficial and effective in increasing the platelets counts. While for thrombotic thrombocyto‐ penic purpura (TTP) treatment is needed since it can leads to renal failure. In case of idiopathic thrombocytopenic purpura (ITP) the treatment depends on severity of the case and the platelets counts. In the case of heparin induce thrombocytopenia and thrombosis (HITT) the treatment is by stopping heparin administration. If the cause of thrombocytopenia is due to patient's immune system causing destruction to the platelets then the use of corticosteroids is very effective so as to suppress immune response. While if the cause is due to chemotherapy then the decision to either continue the treatment with low chemotherapy doses or use of

bleeding, black stool and petechiae (reddish purple spots in the skin) [57, 58, 74].

/ L) while thrombocytopenia could be classified into three levels as follows:

/ L.

/ L [75, 76].

/ L.

Hypercalcemia is a life threatening situation in which serum calcium level is elevated greater than 10.5 mg/ dl, while albumin concentration is lower than 4 g/ dl. It is a serious problem that occurs in about 10%-20% of all cancer patients especially lung, breast, head and neck cancer patients. While, in hematological cancer hypercalcemia also takes place specifically in the advanced phases of both myeloma and lymphoma. Besides that a very important point is that hypercalcemia is mainly caused by cancer without any effect or role from anticancer treat‐ ments. So many references consider hypercalcemia as a very serious and dangerous compli‐ cation that caused a significant morbidity and mortality frequently in breast cancer patients. It can occur in patients with and without bone metastasis and the main cause of hypercalcemia is the pathological bone resorption. Bone resorption is caused by the secretion of cytokine like parathyroid hormone-related protein (PTHrP) leading to activation and differentiation of osteoclast cell. In normal condition normal breast cells also secreted PTHrP during lactation so as to stimulate bone resorption and skeletal calcium release which will be used in milk synthesis. In this situation hypercalcemia is asymptomatic since the elevation of calcium level is mild, but when serum calcium elevation became very high it will leads to significant morbidity and mortality. Hypercalcemia is highly associated with breast cancer more than other types of cancers [78-83].

### *2.4.1. Calcium homeostasis*

Calcium in human body has multiply functions, it is one of the major components and mineral of the body skeleton and its concentration is maintained by influx and efflux to the extracellular fluid from kidney, bone and gut. This vital process is regulated by two hormones which are parathyroid hormone (PTH) and 1, 25 dihydroxyvitamin D. Serum calcium consist of calcium bounded to albumin 37%, bounded with globulin (10%), biologically active calcium (47%) i.e., ionized form and calcium complex with anion (10%) (like: phosphate, citrate, bicarbonate). The ionized serum calcium is the only form metabolically active and is regulated by homeo‐ static mechanisms [87, 90, 98]. Calcium also has other important role in regulation of the cellular metabolism function, since it is a co-factor for many of body enzymes reactions. Also, calcium is needed for cell adhesion, cell death, an important component of cellular electrical current and has a very important function in muscular contraction process [84, 85].

is usually consider as a catastrophic situation. Metastasis to bone marrow happens in 30% of breast cancer patients and causing disturbances in the plasma calcium concentration. Thus it is clear that bone play a critical role in the maintenance of serum calcium level [86, 88].

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The extra cellular calcium concentration is maintained in a narrow range of 8.5-10.2 mg/ dL

Both act on the three main organs which are kidney, gut and skeleton but PTH is more important since it regulate calcium level from minute to minute (i.e., very rapid effect), PTH it is consists of 84 amino acid single chain polypeptides and is mainly secreted by the chief cells of the four parathyroid glands besides the thyroid gland in the neck. PTH secretion is regulated by the serum calcium level of the extracellular fluid. When calcium concentration increases, the PTH secretion will be suppressed and when the calcium concentration decreases, PTH secretion increases. PTH mainly regulates the calcium transportation between extracel‐ lular fluids and kidney, bone and gut. PTH has a direct effect on bone and plays a critical role in increasing the rate of bone formation and turnover. Its effect on bone came from its stimulation and activation for the osteoclast cells which will lead to increase in bone turnover and it also increases the rate for bone formation. This effect has been found to be dependent on the presence of other hormones like 1, 25-dihydroxyvitamin D. PTH effect on kidney will leads to an increase in distal tubules reabsorption of calcium. Here its effect is enhanced by 1, 25-dihydroxyvitamin D, but it has no direct effect on the gut [78, 85, 88]. 1, 25 dihydroxyvitamin D is the major biological active metabolite of vitamin D. This steroid-like metabolite is derived either from skin during its exposure to ultraviolet light (i.e., sun light) or from plant ergosterol after its ingestion in the gut. It increases the absorption of calcium and phosphorus from the gut by active transport as well as it increases the bone resorption. 1, 25 dihydroxyvitamin D is characterized by its slower action than PTH but it is more effective than PTH in long term control of the serum calcium level [83, 86, 89]. Besides these two hormones, calcitonin which is 32 amino acid peptide also is involved in calcium content. It is synthesis and secreted by parafollicular cells of the thyroid gland. Its main action is by inhibition of the osteoclast cell

from resorption of the bone by causing their dissolution to mononuclear cells [85].

The main causes of hypercalcemia during solid or hematological malignancy are as follows: **1.** The direct effect of cancer diseases on the bone by causing bone destruction such as with breast cancer, lung cancer, multiply myeloma and leukemia. Hypercalcemia occurs in about 10%-20% of all cancer patients during specific stages of their malignant diseases. Lung and breast cancers are highly associated with hypercalcemia incidence beside head

*2.4.5. Main hormones responsible for calcium control*

**2.** 1,25-dihydroxy-vitamin D [1,25 (OH)2 D]

**1.** Parathyroid hormone (PTH)

*2.4.6. Causes of hypercalcemia*

(2.1-2.55 mmol/ L) by two main hormones which are:

### *2.4.2. Kidney role in calcium homeostasis*

Kidney plays a very important role in regulation of calcium concentration in the extracel‐ lular fluid and its capacity to clear the calcium is about 600 mg per day (15 mmol/ day). In adult approximately 98% of calcium is resorbed by kidney. This process of calcium absorption is mainly controlled by PTH and 65% of total reabsorbed calcium happens at the proximal tubules, 20-25% in the ascending loop of Henle, while only 10% in the distal convoluted tubules [86].

### *2.4.3. Gut role in calcium homeostasis*

The daily amount of calcium absorbed ranges between 150-200 mg/ day and this is send to the extracellular fluid. This process of absorption is mainly regulated by 1, 25 dihydroxyvitamin D hormone and calcium concentration in the blood circulation. Besides that the amounts of calcium absorbed from the daily diet is affected by the amount of calcium in the diet and presence of other dietary components which may serve to increase (lactose, fatty acid) or decreases (oxalate, phosphate and phytate) calcium absorption. The absorption of calcium from the daily diet varies even in healthy adult from 20% to 70%. The main parts responsible for absorption are the ileum (65%) and jejunum (17%). This is because these parts are the longest parts and hence the longest time calcium will be absorbed [87].

#### *2.4.4. Bone role in calcium homeostasis*

Bone consider as the main storehouse of calcium which store 99% of the body calcium. The role of bone in calcium homeostasis is important in normal conditions since the process of bone formation is tightly coupled with processes of bone resorption i.e., the velocity of calcium influx and efflux between the extracellular fluid and bone. The extracellular calcium concen‐ tration will be disturbed when the rates of bone resorption increase more than the rate of bone formation. This is seen in cases of advanced cancer diseases which caused activation of the osteoclast cell of the bone marrow leading to increase in bone marrow destruction and increase in calcium efflux. This mainly happy when the cancer disease metastasis to bone marrow and is usually consider as a catastrophic situation. Metastasis to bone marrow happens in 30% of breast cancer patients and causing disturbances in the plasma calcium concentration. Thus it is clear that bone play a critical role in the maintenance of serum calcium level [86, 88].

### *2.4.5. Main hormones responsible for calcium control*

The extra cellular calcium concentration is maintained in a narrow range of 8.5-10.2 mg/ dL (2.1-2.55 mmol/ L) by two main hormones which are:

**1.** Parathyroid hormone (PTH)

*2.4.1. Calcium homeostasis*

500 Cancer Treatment - Conventional and Innovative Approaches

*2.4.2. Kidney role in calcium homeostasis*

convoluted tubules [86].

*2.4.3. Gut role in calcium homeostasis*

*2.4.4. Bone role in calcium homeostasis*

Calcium in human body has multiply functions, it is one of the major components and mineral of the body skeleton and its concentration is maintained by influx and efflux to the extracellular fluid from kidney, bone and gut. This vital process is regulated by two hormones which are parathyroid hormone (PTH) and 1, 25 dihydroxyvitamin D. Serum calcium consist of calcium bounded to albumin 37%, bounded with globulin (10%), biologically active calcium (47%) i.e., ionized form and calcium complex with anion (10%) (like: phosphate, citrate, bicarbonate). The ionized serum calcium is the only form metabolically active and is regulated by homeo‐ static mechanisms [87, 90, 98]. Calcium also has other important role in regulation of the cellular metabolism function, since it is a co-factor for many of body enzymes reactions. Also, calcium is needed for cell adhesion, cell death, an important component of cellular electrical

current and has a very important function in muscular contraction process [84, 85].

Kidney plays a very important role in regulation of calcium concentration in the extracel‐ lular fluid and its capacity to clear the calcium is about 600 mg per day (15 mmol/ day). In adult approximately 98% of calcium is resorbed by kidney. This process of calcium absorption is mainly controlled by PTH and 65% of total reabsorbed calcium happens at the proximal tubules, 20-25% in the ascending loop of Henle, while only 10% in the distal

The daily amount of calcium absorbed ranges between 150-200 mg/ day and this is send to the extracellular fluid. This process of absorption is mainly regulated by 1, 25 dihydroxyvitamin D hormone and calcium concentration in the blood circulation. Besides that the amounts of calcium absorbed from the daily diet is affected by the amount of calcium in the diet and presence of other dietary components which may serve to increase (lactose, fatty acid) or decreases (oxalate, phosphate and phytate) calcium absorption. The absorption of calcium from the daily diet varies even in healthy adult from 20% to 70%. The main parts responsible for absorption are the ileum (65%) and jejunum (17%). This is because these parts are the longest

Bone consider as the main storehouse of calcium which store 99% of the body calcium. The role of bone in calcium homeostasis is important in normal conditions since the process of bone formation is tightly coupled with processes of bone resorption i.e., the velocity of calcium influx and efflux between the extracellular fluid and bone. The extracellular calcium concen‐ tration will be disturbed when the rates of bone resorption increase more than the rate of bone formation. This is seen in cases of advanced cancer diseases which caused activation of the osteoclast cell of the bone marrow leading to increase in bone marrow destruction and increase in calcium efflux. This mainly happy when the cancer disease metastasis to bone marrow and

parts and hence the longest time calcium will be absorbed [87].

**2.** 1,25-dihydroxy-vitamin D [1,25 (OH)2 D]

Both act on the three main organs which are kidney, gut and skeleton but PTH is more important since it regulate calcium level from minute to minute (i.e., very rapid effect), PTH it is consists of 84 amino acid single chain polypeptides and is mainly secreted by the chief cells of the four parathyroid glands besides the thyroid gland in the neck. PTH secretion is regulated by the serum calcium level of the extracellular fluid. When calcium concentration increases, the PTH secretion will be suppressed and when the calcium concentration decreases, PTH secretion increases. PTH mainly regulates the calcium transportation between extracel‐ lular fluids and kidney, bone and gut. PTH has a direct effect on bone and plays a critical role in increasing the rate of bone formation and turnover. Its effect on bone came from its stimulation and activation for the osteoclast cells which will lead to increase in bone turnover and it also increases the rate for bone formation. This effect has been found to be dependent on the presence of other hormones like 1, 25-dihydroxyvitamin D. PTH effect on kidney will leads to an increase in distal tubules reabsorption of calcium. Here its effect is enhanced by 1, 25-dihydroxyvitamin D, but it has no direct effect on the gut [78, 85, 88]. 1, 25 dihydroxyvitamin D is the major biological active metabolite of vitamin D. This steroid-like metabolite is derived either from skin during its exposure to ultraviolet light (i.e., sun light) or from plant ergosterol after its ingestion in the gut. It increases the absorption of calcium and phosphorus from the gut by active transport as well as it increases the bone resorption. 1, 25 dihydroxyvitamin D is characterized by its slower action than PTH but it is more effective than PTH in long term control of the serum calcium level [83, 86, 89]. Besides these two hormones, calcitonin which is 32 amino acid peptide also is involved in calcium content. It is synthesis and secreted by parafollicular cells of the thyroid gland. Its main action is by inhibition of the osteoclast cell from resorption of the bone by causing their dissolution to mononuclear cells [85].

#### *2.4.6. Causes of hypercalcemia*

The main causes of hypercalcemia during solid or hematological malignancy are as follows:

**1.** The direct effect of cancer diseases on the bone by causing bone destruction such as with breast cancer, lung cancer, multiply myeloma and leukemia. Hypercalcemia occurs in about 10%-20% of all cancer patients during specific stages of their malignant diseases. Lung and breast cancers are highly associated with hypercalcemia incidence beside head and neck cancer. While myeloma and lymphoma are the most common hematological types of cancers associated with hypercalcemia.

hyperparathyroidism is usually mild or moderate and the patient will be asymptomatic or only suffer from minor clinical signs mentioned above. While hypercalcemia occurs as a result of breast cancer is usually acute or subacute and the calcium level will be highly elevated and many of the clinical signs mentioned above will be manifested. While mild hypercalcemic patients will be asymptomatic and hypercalcemia will be detected fortui‐

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There are different types of treatments used for hypercalcemic patients whereby some are often

While for emergency cases with calcium level exceeding 13 mg/ dl the following treatments

Hypercalcemia is usually seen in aged female patients more than male where the main characteristic is the presence of hypercalcemia without any symptoms. The main cause is either

Mechanism of hypercalcemia incidence in solid cancer patients can be divided into two groups. In the first group, hypercalcemia may or may not be associated with bone metastasis and the main factor is the solid cancer itself since it will produce systematic circulating humoral factors which will ultimately cause loss of calcium from the bone i.e., bone resorption. Moreover these factors will lead to increase in calcium reabsorption from renal tubules. So this group is named as humoral hypercalcemia of malignancy (HHM) which include lung, ovarian, head and neck, pancreas and kidney cancer but the most frequent are the lung and head and neck cancers. The main factors produced by the cancer cells responsible for this situation are PTH, PTH-like factors, transforming growth factors, colony stimulating factors and leukocyte cytokines. In the second group, hypercalcemia is mainly caused or produced by extensive bone metastasis (i.e., extensive localized bone destruction) which include breast cancer. Breast cancer is considered as the highest and the most frequent solid cancer associated with hypercalcemia

use for daily cases and some others used for emergency cases of hypercalcemia:

**1.** Bisphosphonates (Etidronate, Clodronate and Pamidronate):

**2.** Furosemide (Lasix®) 200-400 ml/ hour [83, 86, 87, 97-99].

*2.4.12. Mechanisms of hypercalcemia occurrence with malignancy*

malignant disease or hyperparathyroidism [83, 86].

tously during routine laboratory screening [83, 97-99].

*2.4.10. Hypercalcemia treatments and options*

**2.** Plicamycin (Mithramycin)

**4.** Zoledronic acid (Zometa®) **5.** Glucocorticoids (Prednisone)

**1.** Normal saline 200-400 ml/ hour I.V.

**3.** Calcitonin (Calcimar®)

*2.4.11. Role of age and gender*

are preferred:


#### *2.4.7. Hypercalcemia diagnosis*

Diagnosis of hypercalcemia is made based on serum calcium level and also on levels of phosphate, chloride, PTH and alkaline phosphates. Other tests for kidney function especially urea level, creatinine level and albumin level tests also performed because in hypercalcemia these are elevated. Bone scan, prospective computed tomography (CT) scan for neck, chest and magnetic resonance imaging (MRI) may help to determined whether the tumor has metasta‐ sized to the bone [95].

### *2.4.8. Hypercalcemia levels*

Normal level of calcium in the blood ranges between 8.7 – 10.4 mg/ dl. Correct calcium level in the blood could be determined by using the following equation:

Corrected calcium (mg/ dl) = measured calcium + ([4- albumin (g/ dl)] × 0.8).

Serum calcium ranging between 10.5 – 12.0 mg/ dl indicates mild hypercalcemia.

Moderate hypercalcemia is being diagnosed when serum calcium is between 12.0 – ≤ 14.0 mg/ dl.

Severe hypercalcemia (hypercalcemia crisis) occurs when serum calcium is higher than 14.0 mg/ dl and is associated with acute signs and symptoms [87, 90-96].

### *2.4.9. Signs and symptoms of hypercalcemia*

Since calcium has a wide range of physiological actions so it has a myriad of clinical effects on multi organs. On central nervous system (CNS), hypercalcemia will cause fatigue, depression, confusion, headache, difficulty in thinking and stupor. Cardiovascular system effects manifestation will range from abnormal electrocardiogram to arrhythmias. Gastroin‐ testinal system signs will involve constipation, nausea and vomiting. Hypercalcemia will cause impaired kidney function and as a consequence will lead to decrease in the renal excretion of calcium and thus increase in the severity of hypercalcemia. Dehydration, bone pain and lost of appetite has also been observed. The hypercalcemia due to primary hyperparathyroidism is usually mild or moderate and the patient will be asymptomatic or only suffer from minor clinical signs mentioned above. While hypercalcemia occurs as a result of breast cancer is usually acute or subacute and the calcium level will be highly elevated and many of the clinical signs mentioned above will be manifested. While mild hypercalcemic patients will be asymptomatic and hypercalcemia will be detected fortui‐ tously during routine laboratory screening [83, 97-99].

### *2.4.10. Hypercalcemia treatments and options*

There are different types of treatments used for hypercalcemic patients whereby some are often use for daily cases and some others used for emergency cases of hypercalcemia:


and neck cancer. While myeloma and lymphoma are the most common hematological

**2.** Some cancers diseases lead to production of parathyroid hormone-related protein (PTHrP) which is mainly associated with solid cancer but not with malignant cancer. **3.** Some cancer diseases decrease the ability of the kidneys to remove excess calcium also

**4.** Dehydration due to nausea and vomiting which will lead to difficulties of the kidneys to

**5.** Decreases in the movement and activity of cancer patients which will lead to breakdown of the bone and hence increase in the release of the calcium into the blood [90-94].

Diagnosis of hypercalcemia is made based on serum calcium level and also on levels of phosphate, chloride, PTH and alkaline phosphates. Other tests for kidney function especially urea level, creatinine level and albumin level tests also performed because in hypercalcemia these are elevated. Bone scan, prospective computed tomography (CT) scan for neck, chest and magnetic resonance imaging (MRI) may help to determined whether the tumor has metasta‐

Normal level of calcium in the blood ranges between 8.7 – 10.4 mg/ dl. Correct calcium level

Moderate hypercalcemia is being diagnosed when serum calcium is between 12.0 – ≤ 14.0

Severe hypercalcemia (hypercalcemia crisis) occurs when serum calcium is higher than 14.0

Since calcium has a wide range of physiological actions so it has a myriad of clinical effects on multi organs. On central nervous system (CNS), hypercalcemia will cause fatigue, depression, confusion, headache, difficulty in thinking and stupor. Cardiovascular system effects manifestation will range from abnormal electrocardiogram to arrhythmias. Gastroin‐ testinal system signs will involve constipation, nausea and vomiting. Hypercalcemia will cause impaired kidney function and as a consequence will lead to decrease in the renal excretion of calcium and thus increase in the severity of hypercalcemia. Dehydration, bone pain and lost of appetite has also been observed. The hypercalcemia due to primary

in the blood could be determined by using the following equation:

mg/ dl and is associated with acute signs and symptoms [87, 90-96].

*2.4.9. Signs and symptoms of hypercalcemia*

Corrected calcium (mg/ dl) = measured calcium + ([4- albumin (g/ dl)] × 0.8).

Serum calcium ranging between 10.5 – 12.0 mg/ dl indicates mild hypercalcemia.

types of cancers associated with hypercalcemia.

leading to decreases in the urination.

502 Cancer Treatment - Conventional and Innovative Approaches

remove excess calcium from the blood.

*2.4.7. Hypercalcemia diagnosis*

sized to the bone [95].

mg/ dl.

*2.4.8. Hypercalcemia levels*


While for emergency cases with calcium level exceeding 13 mg/ dl the following treatments are preferred:


### *2.4.11. Role of age and gender*

Hypercalcemia is usually seen in aged female patients more than male where the main characteristic is the presence of hypercalcemia without any symptoms. The main cause is either malignant disease or hyperparathyroidism [83, 86].

### *2.4.12. Mechanisms of hypercalcemia occurrence with malignancy*

Mechanism of hypercalcemia incidence in solid cancer patients can be divided into two groups. In the first group, hypercalcemia may or may not be associated with bone metastasis and the main factor is the solid cancer itself since it will produce systematic circulating humoral factors which will ultimately cause loss of calcium from the bone i.e., bone resorption. Moreover these factors will lead to increase in calcium reabsorption from renal tubules. So this group is named as humoral hypercalcemia of malignancy (HHM) which include lung, ovarian, head and neck, pancreas and kidney cancer but the most frequent are the lung and head and neck cancers. The main factors produced by the cancer cells responsible for this situation are PTH, PTH-like factors, transforming growth factors, colony stimulating factors and leukocyte cytokines. In the second group, hypercalcemia is mainly caused or produced by extensive bone metastasis (i.e., extensive localized bone destruction) which include breast cancer. Breast cancer is considered as the highest and the most frequent solid cancer associated with hypercalcemia caused by bone metastasis. This hypercalcemia is called local osteolytic hypercalcemia (LOH). The main difference is that in LOH, hypercalcemia is caused by localized bone destruction resulting from bone metastasis by the solid cancer, while in HHM the systematic humoral factor is the sole responsible factor and that hypercalcemia is unrelated to the extent of bone metastasis. In LOH, hypercalcemia is produced by direct effect of the solid cancer cells on the bone i.e., by acting like osteoclast cell producing acid protease (lysosomal enzymes) and collagens responsible for removing of mineral from bone and mainly lead to resorption of bone matrix and causing an increase in cAMP and inhibition of microtubule assembly by agents like colchicine. Resorption could also happened or take place independently of osteoclast cell activity. While for hematological cancers i.e., myeloma the main causes for hypercalcemia are increase bone resorption and glomerular filtration impairment. The main cause of hypercal‐ cemia during lymphoma is bone resorption associated with increase in absorption of calcium from the gut [82, 86, 100].

*2.5.2. Blood-brain barrier and it's role in protecting CNS*

ability to cross the blood-brain barrier [106].

1- Platinum compounds, 2- Taxanes, 3- Vinca alkaloid [104].

*2.5.4. Factors associated with the incidence of neurotoxicity*

**2.** Route of chemotherapy administration [107].

*2.5.3. Neurotoxicity and chemotherapy*

are the following::

*2.5.5. Other factors*

factors are:

**1.** Chemotherapy doses.

**3.** Neurological deficits [108].

*2.5.6. Neurotoxicity evaluation and management*

Blood-brain barrier consider as a very efficient part of the nervous system that determine whether a chemotherapy agent is able to reach the nervous system or not. This barrier has the ability to block certain chemotherapy agents from entering nervous system at the cellular level [105]. Blood-brain barrier which surrounding the CNS varies from the one which surrounding the peripheral nervous system, as a result of this variation some chemotherapy agents such as vincristine significantly affect the peripheral nervous system but not the CNS. Chemotherapy agent will produce neurotoxic effects only if it has the

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Chemotherapy agents that significantly associated with neurotoxicity include the following:

There are many factors play role in the incidence of neurotoxicity but the most critical factors

The incidence of neurotoxicity can be related to factors other than chemotherapy, these

Treatment used for neurotoxicity that caused by chemotherapy agents is limited. The focus of care should be on early recognition of neurotoxicity and careful monitoring of patients at high risk of toxicity [109]. There are various agents that either block the development and/ or incidence of neurotoxicity that caused by chemotherapy agents. Even so the mechanisms of action for these agents still mysterious [110]. Example for agent used as antidote for encephal‐ opathy cause by ifosfamide is the methylene blue [111], besides that amifostine and adreno‐ corticotropic hormone analogues have also been found to be an effective neuroprotective agents. But farther investigation still required to clarifying the role of these agents in over‐ coming and/ or preventing neurotoxicity problem which leads to either delay in chemotherapy schedule, reduction in chemotherapy doses or substitution with an alternative agent [104].

**1.** Primary or secondary tumor deposits, which may involve the nervous system.

**2.** Metabolic or electrolyte imbalance which will leads to neurological disturbance.

### *2.4.13. Relation of hypercalcemia with nausea and vomiting*

The main mechanism of hypercalcemia incidence in solid cancer is the metastasis of the cancer to the bone. Breast cancer which is the highest type of the LOH has shown to cause bone marrow destruction leading to hypercalcemia. Hypercalcemia will lead to many side effects mainly nausea and vomiting and there are studies indicating that hypercalcemia is one of the main risk factor for nausea and vomiting [16, 101].

#### **2.5. Neurotoxicity**

Neurotoxicity which induced by chemotherapy can occurs because of the direct or indirect effect and/ or damage that chemotherapy will cause to the central nervous system (CNS) or peripheral nervous system or any combination of these [102]. It is a critical matter to distinct between the two components of the nervous system. The CNS consists from the brain and the spinal cord. CNS mainly responsible for controlling neurological function of mental status, level of consciousness, motor power, sensory function, cerebral function and cranial nerve function. While for the peripheral nervous system it consists of peripheral nerves, this system mainly responsible for sensing pain, temperature and sensation [103].

This side effect i.e., neurological toxicity remain as one of the major critical side effect of chemotherapy treatment. Its clinical presentation varies significantly as a result of that it became very difficult to confirm the diagnosis [104].

### *2.5.1. General signs and symptoms of neurotoxicity*

Symptoms associated with neurotoxicity may include cerebellar effects i.e., (tremor, loss of balance and fine motor movements), confusion, visual impairment, peripheral neuropathy, somnolence and auditory [102].

It has been found that neurotoxicity problems usually temporary i.e., resolving once treatment is completed, even so sometimes permanent neurological deficits may happened [102].

### *2.5.2. Blood-brain barrier and it's role in protecting CNS*

Blood-brain barrier consider as a very efficient part of the nervous system that determine whether a chemotherapy agent is able to reach the nervous system or not. This barrier has the ability to block certain chemotherapy agents from entering nervous system at the cellular level [105]. Blood-brain barrier which surrounding the CNS varies from the one which surrounding the peripheral nervous system, as a result of this variation some chemotherapy agents such as vincristine significantly affect the peripheral nervous system but not the CNS. Chemotherapy agent will produce neurotoxic effects only if it has the ability to cross the blood-brain barrier [106].

### *2.5.3. Neurotoxicity and chemotherapy*

Chemotherapy agents that significantly associated with neurotoxicity include the following: 1- Platinum compounds, 2- Taxanes, 3- Vinca alkaloid [104].

#### *2.5.4. Factors associated with the incidence of neurotoxicity*

There are many factors play role in the incidence of neurotoxicity but the most critical factors are the following::


#### *2.5.5. Other factors*

caused by bone metastasis. This hypercalcemia is called local osteolytic hypercalcemia (LOH). The main difference is that in LOH, hypercalcemia is caused by localized bone destruction resulting from bone metastasis by the solid cancer, while in HHM the systematic humoral factor is the sole responsible factor and that hypercalcemia is unrelated to the extent of bone metastasis. In LOH, hypercalcemia is produced by direct effect of the solid cancer cells on the bone i.e., by acting like osteoclast cell producing acid protease (lysosomal enzymes) and collagens responsible for removing of mineral from bone and mainly lead to resorption of bone matrix and causing an increase in cAMP and inhibition of microtubule assembly by agents like colchicine. Resorption could also happened or take place independently of osteoclast cell activity. While for hematological cancers i.e., myeloma the main causes for hypercalcemia are increase bone resorption and glomerular filtration impairment. The main cause of hypercal‐ cemia during lymphoma is bone resorption associated with increase in absorption of calcium

The main mechanism of hypercalcemia incidence in solid cancer is the metastasis of the cancer to the bone. Breast cancer which is the highest type of the LOH has shown to cause bone marrow destruction leading to hypercalcemia. Hypercalcemia will lead to many side effects mainly nausea and vomiting and there are studies indicating that hypercalcemia is one of the

Neurotoxicity which induced by chemotherapy can occurs because of the direct or indirect effect and/ or damage that chemotherapy will cause to the central nervous system (CNS) or peripheral nervous system or any combination of these [102]. It is a critical matter to distinct between the two components of the nervous system. The CNS consists from the brain and the spinal cord. CNS mainly responsible for controlling neurological function of mental status, level of consciousness, motor power, sensory function, cerebral function and cranial nerve function. While for the peripheral nervous system it consists of peripheral nerves, this system

This side effect i.e., neurological toxicity remain as one of the major critical side effect of chemotherapy treatment. Its clinical presentation varies significantly as a result of that it

Symptoms associated with neurotoxicity may include cerebellar effects i.e., (tremor, loss of balance and fine motor movements), confusion, visual impairment, peripheral neuropathy,

It has been found that neurotoxicity problems usually temporary i.e., resolving once treatment is completed, even so sometimes permanent neurological deficits may happened [102].

mainly responsible for sensing pain, temperature and sensation [103].

from the gut [82, 86, 100].

**2.5. Neurotoxicity**

*2.4.13. Relation of hypercalcemia with nausea and vomiting*

504 Cancer Treatment - Conventional and Innovative Approaches

main risk factor for nausea and vomiting [16, 101].

became very difficult to confirm the diagnosis [104].

*2.5.1. General signs and symptoms of neurotoxicity*

somnolence and auditory [102].

The incidence of neurotoxicity can be related to factors other than chemotherapy, these factors are:


#### *2.5.6. Neurotoxicity evaluation and management*

Treatment used for neurotoxicity that caused by chemotherapy agents is limited. The focus of care should be on early recognition of neurotoxicity and careful monitoring of patients at high risk of toxicity [109]. There are various agents that either block the development and/ or incidence of neurotoxicity that caused by chemotherapy agents. Even so the mechanisms of action for these agents still mysterious [110]. Example for agent used as antidote for encephal‐ opathy cause by ifosfamide is the methylene blue [111], besides that amifostine and adreno‐ corticotropic hormone analogues have also been found to be an effective neuroprotective agents. But farther investigation still required to clarifying the role of these agents in over‐ coming and/ or preventing neurotoxicity problem which leads to either delay in chemotherapy schedule, reduction in chemotherapy doses or substitution with an alternative agent [104].

### **2.6. Cardiotoxicity**

The major function of the heart is to pump the blood to the whole body to supply body organs with adequate oxygen and nutrition they need. This process will happened by contracting muscular walls of the left ventricle [112]. There are various factors which can leads to cardiac injury in the cancer patients. This may happened as a result of either infiltration of metastases to infections and/ or because of chemotherapy toxicity [112].

*2.7.1. Chemotherapy and pulmonary toxicity*

effects on pulmonary function:

*2.7.2. Assessment of pulmonary function*

*2.7.3. Treatments used for pulmonary toxicity cases*

7- Aminophylline and theophylline [128-129].

**3. Conclusion**

Chemotherapeutic agents will be divided into three groups, this will mainly based on their

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**1.** Hypersensitive pulmonary reaction: Bleomycin, 6-mercaptopurine, methotrexate, mitomycin and procarbazine. This condition take place as a result of either desquamative

**2.** Non-cardiogenic pulmonary oedema: Cyclophosphamide, cytarabine and methotrexate. This condition will take place after few days of strating using of chemotherapy treatment.

**3.** Chronic pulmonary fibrosis: Bleomycin, busulfan, carmustine, cyclophosphamide, fludarabine, ifosfamide, methotrexate and mitomycin [122, 123]. This clinical condition

Besides that it has been found that when mitomycin used in combination with vinca alkaloids and/ or gemcitabine with docetaxel or when the later two agents i.e., gemcitabine and docetaxel

It is very important to assess patients pulmonary function before start administration of chemotherapy, the assessment will include the following:1- Chest X-ray 2- Lung biopsy

Managements used for pulmonary problems i.e., toxicity will include the following: 1- Bronchodilator, 2- Corticosteroid 3- Expectorant 4- Oxygen 5- Antibiotics 6- Nebulised saline

Cancer has become a major killer in the world which almost surpasses the cardiovascular diseases and will become the main lethal cause in this century. Although the global war against cancer leads to remarkable gain in understanding the main molecular mechanism for the cancer cell, this progress is still consider as slow and not enough especially in case of treatment of common solid tumor in adults. Besides that there are so many types of serious side effects

Therefore it is an obligate for all the clinicians and physicians to focus on these main side effects that emerged as a result of cancer itself or its treatment and working to built and develop

interstitial pneumonitis or an eosinophilic pneumonitis [122, 123].

will take place within months of using chemotherapy treatment.

used alone they can cause pulmonary toxicity [124, 125, 126, 127].

required to differentiate chronic fibrosis from lung metastasis [121].

caused by the tumor itself or because of its chemotherapy treatment.

treatment guidelines to overcome or palliate these major side effects.

### *2.6.1. Major factors which cause cardiac damage in cancer patients*

a-Cardiac tumors, b-Bacterial infections, c-Chemotherapy induce toxicity, e-Radiation induce toxicity, f-Fungal and/ or viral infection [113, 114].

Chemotherapy effects will be classified into two types: acute and chronic effects.

### *2.6.2. Acute toxic effect*

Acute cardiotoxicity caused by doxorubicin came from combination of factors which are: mitochondrial changes, cellular degeneration and a loss of myocardial fibrils. The incidence of cardiotoxicity will be either during or after doxorubicin administration, this cardiotoxicity will leads to cardiac abnormalities which include: ST and T wave changes, sinus tachycardia, atrial and ventricular ectopics, complete heart block, supraventricular tachycardia and ventricular tachycardia [116, 117].

Although doxorubicin cause cardiotoxicity there is no specific treatment for this condition, but there is only a supportive treatments. Researchers and clinicians keep on using of cardiopro‐ tective agents that allow chemotherapy agents specifically anthracycline to be used at a higher dose without causing cardiotoxicity [114]. Example for these cardioprotective agents are dexrazoxane and amifostine [118].

### *2.6.3. Chronic toxicity*

This type of toxicity is one of the most common toxicity caused by doxorubicin it is character‐ ized by chronic dilated cardiomyopathy. This condition i.e., cardiomyopathy usually hap‐ pened either at late of chemotherapy cycle or shortly after the end of it [119]. Cardiomyopathy is significantly attenuated by the chelation of iron. Moreover, cardiomyopathy has been diagnosed among the survivors of cancer patients who have been treated with doxorubicin during their childhood [120].

### **2.7. Pulmonary toxicity**

It is one of the main side effects of chemotherapy, which become clinically obvious after weeks, months or even years of termination of chemotherapy. It usually associated with several clinical symptoms which are: dry cough, dyspnoea and progressive worsening of symptoms with a poor prognosis for recovery [121].

### *2.7.1. Chemotherapy and pulmonary toxicity*

**2.6. Cardiotoxicity**

*2.6.2. Acute toxic effect*

ventricular tachycardia [116, 117].

dexrazoxane and amifostine [118].

during their childhood [120].

with a poor prognosis for recovery [121].

**2.7. Pulmonary toxicity**

*2.6.3. Chronic toxicity*

The major function of the heart is to pump the blood to the whole body to supply body organs with adequate oxygen and nutrition they need. This process will happened by contracting muscular walls of the left ventricle [112]. There are various factors which can leads to cardiac injury in the cancer patients. This may happened as a result of either infiltration of metastases

a-Cardiac tumors, b-Bacterial infections, c-Chemotherapy induce toxicity, e-Radiation induce

Acute cardiotoxicity caused by doxorubicin came from combination of factors which are: mitochondrial changes, cellular degeneration and a loss of myocardial fibrils. The incidence of cardiotoxicity will be either during or after doxorubicin administration, this cardiotoxicity will leads to cardiac abnormalities which include: ST and T wave changes, sinus tachycardia, atrial and ventricular ectopics, complete heart block, supraventricular tachycardia and

Although doxorubicin cause cardiotoxicity there is no specific treatment for this condition, but there is only a supportive treatments. Researchers and clinicians keep on using of cardiopro‐ tective agents that allow chemotherapy agents specifically anthracycline to be used at a higher dose without causing cardiotoxicity [114]. Example for these cardioprotective agents are

This type of toxicity is one of the most common toxicity caused by doxorubicin it is character‐ ized by chronic dilated cardiomyopathy. This condition i.e., cardiomyopathy usually hap‐ pened either at late of chemotherapy cycle or shortly after the end of it [119]. Cardiomyopathy is significantly attenuated by the chelation of iron. Moreover, cardiomyopathy has been diagnosed among the survivors of cancer patients who have been treated with doxorubicin

It is one of the main side effects of chemotherapy, which become clinically obvious after weeks, months or even years of termination of chemotherapy. It usually associated with several clinical symptoms which are: dry cough, dyspnoea and progressive worsening of symptoms

Chemotherapy effects will be classified into two types: acute and chronic effects.

to infections and/ or because of chemotherapy toxicity [112].

*2.6.1. Major factors which cause cardiac damage in cancer patients*

toxicity, f-Fungal and/ or viral infection [113, 114].

506 Cancer Treatment - Conventional and Innovative Approaches

Chemotherapeutic agents will be divided into three groups, this will mainly based on their effects on pulmonary function:


Besides that it has been found that when mitomycin used in combination with vinca alkaloids and/ or gemcitabine with docetaxel or when the later two agents i.e., gemcitabine and docetaxel used alone they can cause pulmonary toxicity [124, 125, 126, 127].

#### *2.7.2. Assessment of pulmonary function*

It is very important to assess patients pulmonary function before start administration of chemotherapy, the assessment will include the following:1- Chest X-ray 2- Lung biopsy required to differentiate chronic fibrosis from lung metastasis [121].

#### *2.7.3. Treatments used for pulmonary toxicity cases*

Managements used for pulmonary problems i.e., toxicity will include the following: 1- Bronchodilator, 2- Corticosteroid 3- Expectorant 4- Oxygen 5- Antibiotics 6- Nebulised saline 7- Aminophylline and theophylline [128-129].

### **3. Conclusion**

Cancer has become a major killer in the world which almost surpasses the cardiovascular diseases and will become the main lethal cause in this century. Although the global war against cancer leads to remarkable gain in understanding the main molecular mechanism for the cancer cell, this progress is still consider as slow and not enough especially in case of treatment of common solid tumor in adults. Besides that there are so many types of serious side effects caused by the tumor itself or because of its chemotherapy treatment.

Therefore it is an obligate for all the clinicians and physicians to focus on these main side effects that emerged as a result of cancer itself or its treatment and working to built and develop treatment guidelines to overcome or palliate these major side effects.

### **Acknowledgements**

I would like to show and express my great appreciation and heartfelt thanks to my main supervisor Associate Prof. Dr. Zuraidah Mohd Yusoff, for her great support and guidance. Moreover, I'd like to express my great appreciation for my co-supervisors Associate Prof. Saad Bin Othman and Associate Prof. Dr Mohamed Azmi Hassali for their creative advice and guidance.

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Also I'd like to express my grateful appreciation to Universiti Sains Malaysia and a special thanks to the School of Pharmaceutical Sciences. I'd like to thank those who represent the greatest support in my whole life, those who fill my life with all of colorful beauties of hope and nature, who always by their skillful advice made the correct scope for my life, my family specifically my great and marvelous father (Abdul Rasool), mother (Basma) and my daughter (Shams).

### **Author details**

Bassam Abdul Rasool Hassan1\*, Zuraidah Binti Mohd Yusoff1 , Mohamed Azmi Hassali2 and Saad Bin Othman1

\*Address all correspondence to: bassamsunny@yahoo.com

1 Clinical Pharmacy Discipline, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden, Penang, Malaysia

2 Discipline of Social and Administrative Pharmacy, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden Penang, Malaysia

### **References**


[4] Henry, L. Malnutrition. In: Brighton D., Wood M. (ed.) The Royal Marsden Hospital Handbook of Cancer Chemotherapy. Churchill Livingstone: Elsevier; (2005). , 177-184.

**Acknowledgements**

508 Cancer Treatment - Conventional and Innovative Approaches

guidance.

(Shams).

**Author details**

Saad Bin Othman1

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Minden, Penang, Malaysia

I would like to show and express my great appreciation and heartfelt thanks to my main supervisor Associate Prof. Dr. Zuraidah Mohd Yusoff, for her great support and guidance. Moreover, I'd like to express my great appreciation for my co-supervisors Associate Prof. Saad Bin Othman and Associate Prof. Dr Mohamed Azmi Hassali for their creative advice and

Also I'd like to express my grateful appreciation to Universiti Sains Malaysia and a special thanks to the School of Pharmaceutical Sciences. I'd like to thank those who represent the greatest support in my whole life, those who fill my life with all of colorful beauties of hope and nature, who always by their skillful advice made the correct scope for my life, my family specifically my great and marvelous father (Abdul Rasool), mother (Basma) and my daughter

1 Clinical Pharmacy Discipline, School of Pharmaceutical Sciences, Universiti Sains Malaysia,

2 Discipline of Social and Administrative Pharmacy, School of Pharmaceutical Sciences,

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**Chapter 21**

**Impact of Cancer Treatment on Reproductive Health**

Cancer is in general regarded as a disease of elderly people. Nevertheless, although age is the most significant risk factor for cancer in both sexes, young adults and children may also develop cancer diseases. Overall, men have a 45% risk of developing cancer at some time during their lives. For women, this risk is a bit lower, approximately 37% [1] and thus, in many cases, male and female cancer patients would be young and may not have been started building their families at the time of diagnosis. In people younger than 39 years, the risk of cancer is of about 1/72 for men and 1/51 for women. This risk increases with aging and between 40-59

The majority of children, adolescents, and young adults diagnosed with cancer today will become long-term survivors. One primary concern of cancer survivors will be the ability to reproduce and have children. Detrimental effects on the reproductive system following cancer treatment have shown to negatively affect quality of life[2], [3]. Large studies of women and men with cancer have reported that the risk of infertility related to their treatments may be an important issue for those who have not yet started or completed their family size [2], [4].

**2. Cancer treatment modalities with impact in reproductive health**

Cancer surgery may have impact in fertility by removing reproductive organs or damaging structures needed for reproduction. Chemotherapy and radiotherapy have toxic effects on the gonads and may in certain cases induce ovarian and testicular failure, affecting thus all aspects

and reproduction in any medium, provided the original work is properly cited.

© 2013 Rodriguez-Wallberg; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**and Options for Fertility Preservation**

Additional information is available at the end of the chapter

Kenny A. Rodriguez-Wallberg

**1.1. Cancer in patients of reproductive age**

years, 1/12 men and 1/11 women will develop cancer [1].

http://dx.doi.org/10.5772/55980

**1. Introduction**

## **Impact of Cancer Treatment on Reproductive Health and Options for Fertility Preservation**

Kenny A. Rodriguez-Wallberg

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55980

**1. Introduction**

[129] Filshi, J, Penn, K, & Ashley, S. Acupuncture For The Relief of Cancer-Related Breath‐

lessness. Palliative Medicine (1996). , 10, 145-150.

518 Cancer Treatment - Conventional and Innovative Approaches

### **1.1. Cancer in patients of reproductive age**

Cancer is in general regarded as a disease of elderly people. Nevertheless, although age is the most significant risk factor for cancer in both sexes, young adults and children may also develop cancer diseases. Overall, men have a 45% risk of developing cancer at some time during their lives. For women, this risk is a bit lower, approximately 37% [1] and thus, in many cases, male and female cancer patients would be young and may not have been started building their families at the time of diagnosis. In people younger than 39 years, the risk of cancer is of about 1/72 for men and 1/51 for women. This risk increases with aging and between 40-59 years, 1/12 men and 1/11 women will develop cancer [1].

The majority of children, adolescents, and young adults diagnosed with cancer today will become long-term survivors. One primary concern of cancer survivors will be the ability to reproduce and have children. Detrimental effects on the reproductive system following cancer treatment have shown to negatively affect quality of life[2], [3]. Large studies of women and men with cancer have reported that the risk of infertility related to their treatments may be an important issue for those who have not yet started or completed their family size [2], [4].

### **2. Cancer treatment modalities with impact in reproductive health**

Cancer surgery may have impact in fertility by removing reproductive organs or damaging structures needed for reproduction. Chemotherapy and radiotherapy have toxic effects on the gonads and may in certain cases induce ovarian and testicular failure, affecting thus all aspects

© 2013 Rodriguez-Wallberg; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of reproductive health, including pubertal development, impairment of hormone production and sexual function in adults. Effects of cancer biological therapy on gametes and reproductive organs are not yet established.

young female patients may be offered a single oophorectomy aiming at preserving the uterus

**Diagnosis Type of surgery Description Obstetric outcome Oncologic outcome**

Spontaneous pregnancies described in up to 70%. Risk of second trimester pregnancy loss and preterm delivery

Impact of Cancer Treatment on Reproductive Health and Options for Fertility Preservation

Pregnancies have been reported and favorable obstetric

Pregnancies have been reported and favorable obstetric

Pregnancies have been reported and favorable obstetric

Pregnancies have been reported

outcome

outcome

outcome

Rates of recurrence and

Oncologic outcome is comparable with the more radical approach of removing both ovaries and the uterus. Recurrence 0-20% vs 12-58% when only cystectomy was performed

7% recurrence of the ovarian malignancy and

Risk of recurrence similar to historical

Recurrence rate 30-40%. Five percent recurrence during progesterone treatment

5% deaths

controls

mortality are comparable to those described for similar cases treated by means of radical hysterectomy or radiation therapy

http://dx.doi.org/10.5772/55980

521

Laparoscopic pelvic lymphadenectomy. Vaginal resection of the cervix and surrounding parametria keeping the corpus of the uterus and the ovaries intact

Removal of the affected ovary only, keeping in place the unaffected one and the

Removal of the affected ovary only, keeping in place the unaffected one and the

Removal of the affected

Follow-up with endometrial biopsies every 3 months

**Table 1.** Fertility-sparing interventions in female patients. Reprinted, with permission from Rodriguez-Macias

The most established surgical procedure for fertility preservation of women is the radical trachelectomy described first by Dargent in 1994 [7]. It is currently offered in cases of invasive cervical cancer in early stages to patients interested in preserving fertility. About 500 cases have been reported worldwide, most of them in European countries, Japan, U.S.A, Canada

uterus

uterus

ovary only

and the contralateral ovary[6].

Radical vaginal trachelectomy

Unilateral oophorectomy

Unilateral oophorectomy

Unilateral oophorectomy

Hormonal treatment with progestational agents for 6 months

Wallberg et al, *J Pediatric Blood & Cancer, 2009*, Ref [6].

Cervical cancer stage IA1,1A2,1B1

Borderline ovarian tumors FIGO stage I

Ovarian epithelial cancer stage I, grade 1

Malignant ovarian germ cell tumors/sex cord stromal tumors

Endometrial adenocarcinoma Grade 1, stage 1A (without myometrial or cervical invasion)

and China [8-12].

### **3. Cancer surgery may cause infertility**

Surgery is currently the most effective treatment for cancer and eventually up to 100% of patients may be cured when complete removal of the tumor is achieved. Surgery may also be indicated for cancer prophylaxis, such as the case of premalignant disease of the cervix in female patients. In those very early stages of cervix cancer, the conization of a significant part of the cervix, may offer to patients a complete disease-free survival. However, in case of loop excisions, even if they are small, surgery of the cervix may induce subfertility by affecting the normal functioning of the cervix and its glandular secretion. Infertility induced by those interventions may be overcomed by using assisted reproductive technologies, such as treating the patient with intrauterine insemination or performing In Vitro Fertilization, IVF.

Surgery may also affect future fertility if there is removal or damage of the reproductive organs. In male patients, surgery for pelvic cancer such as for prostate, bladder or colon cancer may damage nerves and affect potency or ejaculation. Further on, surgical adjuvant treatment by removing the gonads may be indicated in female and male patients with hormone sensitive tumors.

In case of large tumors, neo-adjuvant chemotherapy and radiation may be indicated as first line treatment aiming to a reduction of tumor size and control of subclinical metastatic disease before surgical treatment. Neo-adjuvant therapy is usually planned before surgery in female patients with stage III breast cancer and young male cancer patients with bulky testicular cancer.

### **3.1. Cancer surgery aiming at preserving fertility**

Fertility-sparing surgery may be an option for selected patients who wish to retain the ability to achieve a pregnancy. In many cases, pregnancies will occur spontaneously, nevertheless, causes of subfertility may be present in some patients, and a number of those may further require assisted reproduction treatments [5]. In gynecologic and urologic oncologic surgery, there has been a gradual development of fertility-sparing surgery aiming at preserving reproductive organs without compromising survival. Indications for fertility-sparing surgery include, in general, a well-differentiated low-grade tumor in its early stages or with low malignant potential.

#### **3.2. Fertility sparing surgery in female patients**

Table 1 presents a compilation of current data on fertility sparing surgery for young female patients with gynaecological cancer. In cases of selected ovarian tumors i.e. borderline tumors, young female patients may be offered a single oophorectomy aiming at preserving the uterus and the contralateral ovary[6].

of reproductive health, including pubertal development, impairment of hormone production and sexual function in adults. Effects of cancer biological therapy on gametes and reproductive

Surgery is currently the most effective treatment for cancer and eventually up to 100% of patients may be cured when complete removal of the tumor is achieved. Surgery may also be indicated for cancer prophylaxis, such as the case of premalignant disease of the cervix in female patients. In those very early stages of cervix cancer, the conization of a significant part of the cervix, may offer to patients a complete disease-free survival. However, in case of loop excisions, even if they are small, surgery of the cervix may induce subfertility by affecting the normal functioning of the cervix and its glandular secretion. Infertility induced by those interventions may be overcomed by using assisted reproductive technologies, such as treating

the patient with intrauterine insemination or performing In Vitro Fertilization, IVF.

Surgery may also affect future fertility if there is removal or damage of the reproductive organs. In male patients, surgery for pelvic cancer such as for prostate, bladder or colon cancer may damage nerves and affect potency or ejaculation. Further on, surgical adjuvant treatment by removing the gonads may be indicated in female and male patients with hormone sensitive

In case of large tumors, neo-adjuvant chemotherapy and radiation may be indicated as first line treatment aiming to a reduction of tumor size and control of subclinical metastatic disease before surgical treatment. Neo-adjuvant therapy is usually planned before surgery in female patients with stage III breast cancer and young male cancer patients with bulky testicular

Fertility-sparing surgery may be an option for selected patients who wish to retain the ability to achieve a pregnancy. In many cases, pregnancies will occur spontaneously, nevertheless, causes of subfertility may be present in some patients, and a number of those may further require assisted reproduction treatments [5]. In gynecologic and urologic oncologic surgery, there has been a gradual development of fertility-sparing surgery aiming at preserving reproductive organs without compromising survival. Indications for fertility-sparing surgery include, in general, a well-differentiated low-grade tumor in its early stages or with low

Table 1 presents a compilation of current data on fertility sparing surgery for young female patients with gynaecological cancer. In cases of selected ovarian tumors i.e. borderline tumors,

organs are not yet established.

tumors.

cancer.

malignant potential.

**3. Cancer surgery may cause infertility**

520 Cancer Treatment - Conventional and Innovative Approaches

**3.1. Cancer surgery aiming at preserving fertility**

**3.2. Fertility sparing surgery in female patients**


**Table 1.** Fertility-sparing interventions in female patients. Reprinted, with permission from Rodriguez-Macias Wallberg et al, *J Pediatric Blood & Cancer, 2009*, Ref [6].

The most established surgical procedure for fertility preservation of women is the radical trachelectomy described first by Dargent in 1994 [7]. It is currently offered in cases of invasive cervical cancer in early stages to patients interested in preserving fertility. About 500 cases have been reported worldwide, most of them in European countries, Japan, U.S.A, Canada and China [8-12].

The global utilization of fertility-sparing surgery in female patients is currently unknown. A recent European study collecting data from several countries demonstrated a low incidence of those procedures and it raised concerns on the need to centralize fertility sparing treatments of gynaecological cancer at accredited units, to ensure a sufficient number of patients at each center aiming at maintaining thus healthcare quality [13].

Except for the bone marrow, the most sensitive organs to radiation therapy in the body are the gonads, both the male testis and the female ovary. The extent of damage in the female and male gonads depends on the dose, fractionation schedule and irradiation field [19] [20]. Radiation therapy can be administered as teletherapy, which aims at treating a large volume of tissue. For small volumes of tissue, such as in the case of cervix cancer in the female, radiation therapy can be administered in encapsulated sources of radiation that can be implanted

Impact of Cancer Treatment on Reproductive Health and Options for Fertility Preservation

http://dx.doi.org/10.5772/55980

523

Whenever female reproductive organs are involved in the irradiated field, i.e., the ovaries, the uterus and the vagina may be compromised and damaged by direct irradiation. Scattered radiation may also damage reproductive organs. In the female, radiation therapy results in dose-related damage of the gonads by the destruction of primordial follicles, which constitute the nonrenewable follicle pool. In women, the degree and persistence of the damage is also influenced by age at the time of exposure to radiotherapy and due to a reduced reserve of primordial follicles in older women, the number of follicles remaining may be also be reduced at older ages [21]. Table 2 presents a compilation of current knowledge on the impact of radiation doses and age at radiotherapy in male and female gonadal function [22]. In general, a dose of about 2 Gy applied to the gonadal area destroys up to 50 % of the ovarian follicle reserve. In pediatric patients, failure in pubertal development may be the first sign of gonadal failure in both sexes. Total body irradiation (TBI) given in conjunction with myeloablative conditioning prior to bone marrow transplantation is one of the most toxic treatments for the

gonads and it is highly related to gonadal failure in both sexes [23] [24].

Total Body Irradiation (TBI) for bone marrow transplant/stem cell transplant (9,15,16)

Pelvic or whole abdominal radiation dose ≥ 10 Gy in post-pubertal girls (21,22,23,24) Pelvic radiation or whole abdominal dose ≥ 15 Gy in pre-pubertal girls (21,22,23,24)

Testicular radiation dose 1-6 Gy from scattered pelvic or abdominal radiation (13,16) Pelvic or whole abdominal radiation dose 5-10 Gy in post-pubertal girls (21,24) Pelvic or whole abdominal radiation dose 10-15 Gy in pre-pubertal girls (21,22,24)

permission from Rodriguez-Wallberg and Oktay, *J Ped Hematol Oncol*, 2010, Ref [22].

**Table 2.** Radiotherapy protocols with high or intermediate impact on ovarian and testicular function. Reprinted, with

In men, the gonadal stem cells responsible for the continual differentiation and production of mature spermatozoa, the spermatogoniae, are extremely sensitive to radiation. The Leydig cells, which are responsible for the hormonal production of testosterone, are on the contrary more resistant to radiotherapy and adult patients may thus preserve hormonal production

**High risk of prolonged azoospermia in men or amenorrhea in women**

Pelvic or whole abdominal radiation dose ≥ 6 Gy in adult women (20,21,22)

Testicular radiation dose "/>2.5 Gy in adult men (9,17) Testicular radiation dose ≥ 6 Gy in pre-pubertal boys (18,19)

Craniospinal radiotherapy dose ≥ 25 Gy (14)

**Intermediate risk**

directly into or adjacent to tumor tissue.

### **3.3. Cervical cancer and fertility sparing surgery during pregnancy**

In pregnant women, the gynaecological cancer most commonly diagnosed is also the cancer of the cervix, usually detected at an early stage in those patients. The treatment of pregnant women should be established in the same way as in non-pregnant patients, based on the stage of the disease according to the International Federation of Gynecology (FIGO). Nevertheless, individualization of the treatment should be considered based on the desire to continue the pregnancy, the gestational age and the risks of modifying or delaying cancer treatment during the pregnancy. Clinical practice guidelines by the European Society for Medical Oncology ESMO are available on this respect [14]. Both abdominal radical trachelectomy [15] and vaginal trachelectomy [16] with lymphadenectomies have been reported during pregnancy to preserve an ongoing pregnancy and female fertility.

### **3.4. Fertility sparing surgery in males**

In men, a partial orchidectomy has become an established method to preserve hormonal and sperm production in carefully selected patients. This method, originally developed for treatment of benign teratomas in prepubertal patients, has shown good results when adopted for treatment of testicular malignancies in adults [17]. Data from The German Testicular Cancer Study Group reported a 98.6% disease-free survival rate at 7 years follow-up after conservative surgery of tumors <2 cm [18].

### **4. Radiotherapy treatments and their impact in reproductive health**

Radiation therapy is a component of curative therapy for a number of diseases, including those presenting frequently in young patients such as breast cancer, Hodgkin's disease, head and neck cancer and gynecologic cancers. It is often indicated for the treatment of prostate cancer as well.

It is known that cancer cells present with defects in their ability to repair sub-lethal DNA whereas normal cells have the ability to recover. Although radiation therapy is aimed to a locoregional application and although cancer cells are the target, radiation may also induce damage to normal cells in the tissues.

The response to radiation therapy depends on various factors such as the phase of cell cycle the cells are (cells in late G1 and S are more resistant), the degree of cell ability to repair the DNA damage and other factors such as hypoxia (hypoxic cells are more resistant), tumor mass and growth fraction. Non-dividing cells are more resistant than dividing cells.

Except for the bone marrow, the most sensitive organs to radiation therapy in the body are the gonads, both the male testis and the female ovary. The extent of damage in the female and male gonads depends on the dose, fractionation schedule and irradiation field [19] [20]. Radiation therapy can be administered as teletherapy, which aims at treating a large volume of tissue. For small volumes of tissue, such as in the case of cervix cancer in the female, radiation therapy can be administered in encapsulated sources of radiation that can be implanted directly into or adjacent to tumor tissue.

The global utilization of fertility-sparing surgery in female patients is currently unknown. A recent European study collecting data from several countries demonstrated a low incidence of those procedures and it raised concerns on the need to centralize fertility sparing treatments of gynaecological cancer at accredited units, to ensure a sufficient number of patients at each

In pregnant women, the gynaecological cancer most commonly diagnosed is also the cancer of the cervix, usually detected at an early stage in those patients. The treatment of pregnant women should be established in the same way as in non-pregnant patients, based on the stage of the disease according to the International Federation of Gynecology (FIGO). Nevertheless, individualization of the treatment should be considered based on the desire to continue the pregnancy, the gestational age and the risks of modifying or delaying cancer treatment during the pregnancy. Clinical practice guidelines by the European Society for Medical Oncology ESMO are available on this respect [14]. Both abdominal radical trachelectomy [15] and vaginal trachelectomy [16] with lymphadenectomies have been reported during pregnancy to preserve

In men, a partial orchidectomy has become an established method to preserve hormonal and sperm production in carefully selected patients. This method, originally developed for treatment of benign teratomas in prepubertal patients, has shown good results when adopted for treatment of testicular malignancies in adults [17]. Data from The German Testicular Cancer Study Group reported a 98.6% disease-free survival rate at 7 years follow-up after conservative

**4. Radiotherapy treatments and their impact in reproductive health**

Radiation therapy is a component of curative therapy for a number of diseases, including those presenting frequently in young patients such as breast cancer, Hodgkin's disease, head and neck cancer and gynecologic cancers. It is often indicated for the treatment of prostate cancer

It is known that cancer cells present with defects in their ability to repair sub-lethal DNA whereas normal cells have the ability to recover. Although radiation therapy is aimed to a locoregional application and although cancer cells are the target, radiation may also induce damage

The response to radiation therapy depends on various factors such as the phase of cell cycle the cells are (cells in late G1 and S are more resistant), the degree of cell ability to repair the DNA damage and other factors such as hypoxia (hypoxic cells are more resistant), tumor mass

and growth fraction. Non-dividing cells are more resistant than dividing cells.

center aiming at maintaining thus healthcare quality [13].

522 Cancer Treatment - Conventional and Innovative Approaches

an ongoing pregnancy and female fertility.

**3.4. Fertility sparing surgery in males**

surgery of tumors <2 cm [18].

to normal cells in the tissues.

as well.

**3.3. Cervical cancer and fertility sparing surgery during pregnancy**

Whenever female reproductive organs are involved in the irradiated field, i.e., the ovaries, the uterus and the vagina may be compromised and damaged by direct irradiation. Scattered radiation may also damage reproductive organs. In the female, radiation therapy results in dose-related damage of the gonads by the destruction of primordial follicles, which constitute the nonrenewable follicle pool. In women, the degree and persistence of the damage is also influenced by age at the time of exposure to radiotherapy and due to a reduced reserve of primordial follicles in older women, the number of follicles remaining may be also be reduced at older ages [21]. Table 2 presents a compilation of current knowledge on the impact of radiation doses and age at radiotherapy in male and female gonadal function [22]. In general, a dose of about 2 Gy applied to the gonadal area destroys up to 50 % of the ovarian follicle reserve. In pediatric patients, failure in pubertal development may be the first sign of gonadal failure in both sexes. Total body irradiation (TBI) given in conjunction with myeloablative conditioning prior to bone marrow transplantation is one of the most toxic treatments for the gonads and it is highly related to gonadal failure in both sexes [23] [24].


**Table 2.** Radiotherapy protocols with high or intermediate impact on ovarian and testicular function. Reprinted, with permission from Rodriguez-Wallberg and Oktay, *J Ped Hematol Oncol*, 2010, Ref [22].

In men, the gonadal stem cells responsible for the continual differentiation and production of mature spermatozoa, the spermatogoniae, are extremely sensitive to radiation. The Leydig cells, which are responsible for the hormonal production of testosterone, are on the contrary more resistant to radiotherapy and adult patients may thus preserve hormonal production although becoming infertile. In prepubertal boys, the sensitivity to radiation therapy of Leydig cells is greater than that of older males at very high doses [25]. Prepubertal patients may retain Leydig cell function after radiation therapy during childhood and in those cases they will present with normal pubertal development and well-preserved sexual function later in life. Nevertheless, most of those patients present at adulthood with reduced testicular size, impaired spermatogenesis and infertility.

cols combine several agents and there is a possibility of a synergistic gonadotoxic ef‐ fect[29]. In the female, primordial ovarian follicles including their oocytes and granulosa cells are particularly sensitive to alkylating agents, which induce apoptosis, as demonstrat‐ ed in vitro [26], and in vivo using human ovarian tissue xenotransplanted in SCID mouse

Impact of Cancer Treatment on Reproductive Health and Options for Fertility Preservation

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525

Because of a high ovarian reserve with high numbers of follicles in young women, the risk of developing ovarian failure and permanent infertility after a cancer treatment is lower in younger than in older women [21]. Younger patients at the time of cancer treatment have thus a higher chance of recovering ovarian function following chemotherapy, nevertheless their fertility window might be reduced, and they should be recommended not to delay childbearing

The development of amenorrhea should be considered unfavorable as it may be due to permanent gonadal failure. On the other hand, the presence of cycles should not be interpreted as proof of fertility. In the clinical setting, a gynecological examination includ‐ ing ultrasonography and estimation of antral follicle counts together with the determina‐ tion of hormones such as follicle-stimulating hormone (FSH) and estradiol, inhibin and antimullerian hormone (AMH), may help the clinician in evaluating patient's remaining ovarian reserve after a cancer treatment and providing counseling on her chances to obtain a

Due to toxicity of cancer treatments on growing oocytes, patients should be advised to avoid conception in the 6 -12 month period immediately following completion of chemother‐ apy treatment [32]. There is a high risk of teratogenesis during or immediately following chemotherapy, nevertheless DNA integrity has shown to return over time after a cancer treatment and thus no increase in childhood malignancies or genetic malformations have been shown in a large follow-up of more than 4000 children of cancer survivors [33].

In male patients, prepubertal status does not provide protection from gonadal damage and alkylating agents at high doses induce germ cell injury although Leydig cell function is commonly preserved [29]. Because most chemotherapy agents are given as part of a combination regimen, it has been difficult to quantify the gonadotoxicty of individual drugs.

Table 3 summarizes the gonadotoxic impact of chemotherapy agents on the female ovary and

[30]. Ovarian failure is thus common after alkylating treatment [22].

for too long [31].

pregnancy.

male testis.

**5.1. Clinical evaluation of ovarian reserve**

**5.2. Conception following chemotherapy**

**5.3. Chemotherapy in males**

### **4.1. Gonadal shielding and ovarian transposition**

The standard medical procedure currently offered to reduce scatter radiation to reproductive organs and preserve fertility in male and female patients, both adult and prepubertal, is the use of shielding. When shielding of the gonadal area is not possible, the surgical fixation of the ovaries in females far from the radiation field known as oophoropexy (ovarian transposi‐ tion) may be considered. It is estimated that this procedure significantly reduces the risk of ovarian failure by about 50% and those patients may retain some menstrual function and fertility [26]. Scattered radiation and damage of the blood vessels that supply the ovaries are related to the failure of this procedure [26].

#### **4.2. Radiotherapy of the uterus**

Radiotherapy of the uterus in young women and girls has shown to induce tissue fibrosis, restricted uterine capacity, restricted blood flow and impaired uterine growth during preg‐ nancy, as shown by follow-up of cancer survivors [27] [28]. The uterine damage seems to be more pronounced in the youngest patients at the time of radiotherapy. As a consequence, radiotherapy-treated female patients present with a high risk of unfavorable pregnancy outcomes such as spontaneous abortion, premature labor and low birth weight offspring [27] [28]. Irradiation of the vagina is related to fertility and sexual issues due to loss of lubrication, anatomical impairments and in some cases vaginal stenosis.

### **4.3. Cranial irradiation and hormonal dysfunction**

Cranial irradiation may induce disruption of the hypothalamic-pituitary-gonadal axis, which is a recognized potential complication that can lead to infertility in both female and male patients. Follow-up of female patients treated for brain tumors with cranial irradiation postand pre-pubertally has evidenced a high incidence of primary hypothalamic and pituitary dysfunction with consecuent disturbance in gonadotropin secretion. In some cases, precocious puberty may also be induced by cranial irradiation in childhood, which has been attributed to cortical disruption and loss of inhibition by the hypothalamus.

### **5. Impact of chemotherapy in reproductive health**

Chemotherapy given as only treatment may be curative for a series of cancer presenting in young adults and children. In a vast majority of cancer treatments, chemotherapy proto‐ cols combine several agents and there is a possibility of a synergistic gonadotoxic ef‐ fect[29]. In the female, primordial ovarian follicles including their oocytes and granulosa cells are particularly sensitive to alkylating agents, which induce apoptosis, as demonstrat‐ ed in vitro [26], and in vivo using human ovarian tissue xenotransplanted in SCID mouse [30]. Ovarian failure is thus common after alkylating treatment [22].

Because of a high ovarian reserve with high numbers of follicles in young women, the risk of developing ovarian failure and permanent infertility after a cancer treatment is lower in younger than in older women [21]. Younger patients at the time of cancer treatment have thus a higher chance of recovering ovarian function following chemotherapy, nevertheless their fertility window might be reduced, and they should be recommended not to delay childbearing for too long [31].

### **5.1. Clinical evaluation of ovarian reserve**

although becoming infertile. In prepubertal boys, the sensitivity to radiation therapy of Leydig cells is greater than that of older males at very high doses [25]. Prepubertal patients may retain Leydig cell function after radiation therapy during childhood and in those cases they will present with normal pubertal development and well-preserved sexual function later in life. Nevertheless, most of those patients present at adulthood with reduced testicular size,

The standard medical procedure currently offered to reduce scatter radiation to reproductive organs and preserve fertility in male and female patients, both adult and prepubertal, is the use of shielding. When shielding of the gonadal area is not possible, the surgical fixation of the ovaries in females far from the radiation field known as oophoropexy (ovarian transposi‐ tion) may be considered. It is estimated that this procedure significantly reduces the risk of ovarian failure by about 50% and those patients may retain some menstrual function and fertility [26]. Scattered radiation and damage of the blood vessels that supply the ovaries are

Radiotherapy of the uterus in young women and girls has shown to induce tissue fibrosis, restricted uterine capacity, restricted blood flow and impaired uterine growth during preg‐ nancy, as shown by follow-up of cancer survivors [27] [28]. The uterine damage seems to be more pronounced in the youngest patients at the time of radiotherapy. As a consequence, radiotherapy-treated female patients present with a high risk of unfavorable pregnancy outcomes such as spontaneous abortion, premature labor and low birth weight offspring [27] [28]. Irradiation of the vagina is related to fertility and sexual issues due to loss of lubrication,

Cranial irradiation may induce disruption of the hypothalamic-pituitary-gonadal axis, which is a recognized potential complication that can lead to infertility in both female and male patients. Follow-up of female patients treated for brain tumors with cranial irradiation postand pre-pubertally has evidenced a high incidence of primary hypothalamic and pituitary dysfunction with consecuent disturbance in gonadotropin secretion. In some cases, precocious puberty may also be induced by cranial irradiation in childhood, which has been attributed to

Chemotherapy given as only treatment may be curative for a series of cancer presenting in young adults and children. In a vast majority of cancer treatments, chemotherapy proto‐

impaired spermatogenesis and infertility.

524 Cancer Treatment - Conventional and Innovative Approaches

related to the failure of this procedure [26].

anatomical impairments and in some cases vaginal stenosis.

cortical disruption and loss of inhibition by the hypothalamus.

**5. Impact of chemotherapy in reproductive health**

**4.3. Cranial irradiation and hormonal dysfunction**

**4.2. Radiotherapy of the uterus**

**4.1. Gonadal shielding and ovarian transposition**

The development of amenorrhea should be considered unfavorable as it may be due to permanent gonadal failure. On the other hand, the presence of cycles should not be interpreted as proof of fertility. In the clinical setting, a gynecological examination includ‐ ing ultrasonography and estimation of antral follicle counts together with the determina‐ tion of hormones such as follicle-stimulating hormone (FSH) and estradiol, inhibin and antimullerian hormone (AMH), may help the clinician in evaluating patient's remaining ovarian reserve after a cancer treatment and providing counseling on her chances to obtain a pregnancy.

### **5.2. Conception following chemotherapy**

Due to toxicity of cancer treatments on growing oocytes, patients should be advised to avoid conception in the 6 -12 month period immediately following completion of chemother‐ apy treatment [32]. There is a high risk of teratogenesis during or immediately following chemotherapy, nevertheless DNA integrity has shown to return over time after a cancer treatment and thus no increase in childhood malignancies or genetic malformations have been shown in a large follow-up of more than 4000 children of cancer survivors [33].

### **5.3. Chemotherapy in males**

In male patients, prepubertal status does not provide protection from gonadal damage and alkylating agents at high doses induce germ cell injury although Leydig cell function is commonly preserved [29]. Because most chemotherapy agents are given as part of a combination regimen, it has been difficult to quantify the gonadotoxicty of individual drugs.

Table 3 summarizes the gonadotoxic impact of chemotherapy agents on the female ovary and male testis.


**6.1. Sperm banking for male patients**

**Figure 1.** Strategies for fertility preservation in males and females

and electro ejaculation.

As many children are born after fertility treatments using frozen-thawed sperm, the cryopreser‐ vation of ejaculated semen is regarded as an established fertility preservation method in adult patients and pubertal boys. Although spermatogenesis starts in the pre-pubertal period and mature spermatozoa can be found at a Tanner III stage with a testis volume above 5 ml, sperma‐ tozoa production is generally effective only at the age of 13-14 years [33]. Sperm cryopreserva‐ tion has been reported in adolescent patients from the age of 13 years with a high prevalence of normal sperm counts and semen volumes [34] [35]. Traditionally, sperm banking by cryopreser‐ vation of at least three semen samples with an abstinence period of at least 48 hours in between the samples has been recommended for adult males desiring to preserve their fertility [36].

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In the situation of ejaculation failure, the search for spermatozoa in a urine sample could be proposed. When failure in obtaining a semen sample in young men and adolescents, a testicular sperm extraction TESE can be performed to retrieve spermatozoa [35]. Other methods described to retrieve spermatozoa in adolescents include penile vibratory stimulation

**6.2. Cryopreservation of embryos or oocytes after controlled ovarian stimulation in females**

Adult women wishing to preserve fertility may undergo controlled ovulation stimulation with gonadotropins, for retrieval of matured oocytes and egg freezing, or, if the woman wishes, for in vitro fertilization (IVF) of the retrieved eggs and freezing of embryos. In general, controlled

**Table 3.** Chemotherapy agents with high or intermediate gonadotoxic impact in women and men

### **6. Options to preserve fertility by using cryopreservation methods**

In 2006, an expert panel commissioned by the American Society of Clinical Oncology ASCO published guidelines for fertility preservation for male and female patients [26]. Established cryopreservation methods for fertility preservation available for adult female and male cancer patients before starting cancer treatments included sperm freezing for male patients and embryo cryopreservation following ovarian stimulation with gonadotropins and In Vitro Fertilization, IVF for females. All remaining options were still considered experimental at that time and they included the freezing of unfertilized oocytes for adult women and the cryopre‐ servation of gonadal tissue, ovarian or testicular, both methods still under development which constitute the only options that can be offered to pre-pubertal children (Figure 1).

Recently, by the end of 2012, the methods for cryopreservation of oocytes by vitrification techniques have markedly improved and thus freezing of unfertilized eggs is currently becoming an established clinical option for female patients.

**Figure 1.** Strategies for fertility preservation in males and females

#### **6.1. Sperm banking for male patients**

**6. Options to preserve fertility by using cryopreservation methods**

**Table 3.** Chemotherapy agents with high or intermediate gonadotoxic impact in women and men

constitute the only options that can be offered to pre-pubertal children (Figure 1).

becoming an established clinical option for female patients.

**High risk of prolonged azoospermia in men or amenorrhea in women**

526 Cancer Treatment - Conventional and Innovative Approaches

Treatment protocols for Hodgkin lymphoma without alkylating agents

Cyclophosphamide Ifosphamide Melphalan Busulfan

Nitrogen mustard Procarbazine Chlorambucil **Intermediate risk**

Adriamycin Low risk

Bleomycin Actinomycin D Vincristine Methotrexate 5 fluorouracil

Cisplatin with low cumulative dose Carboplatin with low cumulative dose

Recently, by the end of 2012, the methods for cryopreservation of oocytes by vitrification techniques have markedly improved and thus freezing of unfertilized eggs is currently

In 2006, an expert panel commissioned by the American Society of Clinical Oncology ASCO published guidelines for fertility preservation for male and female patients [26]. Established cryopreservation methods for fertility preservation available for adult female and male cancer patients before starting cancer treatments included sperm freezing for male patients and embryo cryopreservation following ovarian stimulation with gonadotropins and In Vitro Fertilization, IVF for females. All remaining options were still considered experimental at that time and they included the freezing of unfertilized oocytes for adult women and the cryopre‐ servation of gonadal tissue, ovarian or testicular, both methods still under development which

As many children are born after fertility treatments using frozen-thawed sperm, the cryopreser‐ vation of ejaculated semen is regarded as an established fertility preservation method in adult patients and pubertal boys. Although spermatogenesis starts in the pre-pubertal period and mature spermatozoa can be found at a Tanner III stage with a testis volume above 5 ml, sperma‐ tozoa production is generally effective only at the age of 13-14 years [33]. Sperm cryopreserva‐ tion has been reported in adolescent patients from the age of 13 years with a high prevalence of normal sperm counts and semen volumes [34] [35]. Traditionally, sperm banking by cryopreser‐ vation of at least three semen samples with an abstinence period of at least 48 hours in between the samples has been recommended for adult males desiring to preserve their fertility [36].

In the situation of ejaculation failure, the search for spermatozoa in a urine sample could be proposed. When failure in obtaining a semen sample in young men and adolescents, a testicular sperm extraction TESE can be performed to retrieve spermatozoa [35]. Other methods described to retrieve spermatozoa in adolescents include penile vibratory stimulation and electro ejaculation.

#### **6.2. Cryopreservation of embryos or oocytes after controlled ovarian stimulation in females**

Adult women wishing to preserve fertility may undergo controlled ovulation stimulation with gonadotropins, for retrieval of matured oocytes and egg freezing, or, if the woman wishes, for in vitro fertilization (IVF) of the retrieved eggs and freezing of embryos. In general, controlled ovarian stimulation with gonadotropins for IVF may require only two weeks to achieve, as it has been shown that a random-start in the stimulation cycle, independently of cycle day, does not have a negative impact on the number and quality of oocytes retrieved.

**6.4. Cryopreservation of immature oocytes obtained without hormonal stimulation**

few fertility centers worldwide offer treatments using this technique.

first courses of chemotherapy, if the procedure was not possible before [6].

heterotopically, i.e. at other places including extrapelvic sites [52, 53].

percentage of these have returned for ovarian transplantation.

to preserve fertility by this method.

**6.6. Ovarian tissue transplantation**

**6.5. Ovarian tissue freezing for prepubertal and adult patients**

Freezing immature oocytes is also an option for female fertility preservation in case of patients having a contraindication for hormonal stimulation or when there is not time available for stimulation. The oocytes are retrieved in the natural cycle and frozen at an immature stage or after maturation *in vitro* (IVM) [46]. Immature oocytes survive cryopreservation better than mature metaphase II oocytes [47]. After thawing they can be matured in vitro and fertilized. In vitro maturation of oocytes is at an experimental stage and needs further development. Only

Impact of Cancer Treatment on Reproductive Health and Options for Fertility Preservation

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529

As the vast majority of eggs making up the ovarian reserve are within primordial follicles in the ovarian cortex, small cortical ovarian biopsies may provide a high number of eggs to be preserved. This procedure is usually performed by laparoscopy, can be planned immediately after the diagnosis of malignant disease and does not require hormonal stimulation. In cases when the patient needs to undergo abdominal surgery for the treatment of cancer, the ovarian tissue can be harvested during the same surgical procedure. Although it is preferable to carry out cryopreservation of ovarian tissue before a gonadotoxic treatment, young women, adolescents and girls have normally an abundant number of primordial follicles in their ovaries and attempts to harvest ovarian tissue for cryopreservation may still be worthwhile after the

Cryopreservation of ovarian tissue is the only option in prepubertal girls, as sexual maturity is not required. As this procedure does not cause any significant delay to initiation of cancer treatment and it does not require ovarian stimulation, some adult female patients also prefer

Transplantation of frozen-thawn ovarian cortex has shown to be a new promising method for recovery of ovarian function [48] and in some cases sufficient to restore fertility [49-51]. Ovarian tissue can be transplanted orthotopically, i.e. at the anatomical intrapelvic ovarian site or

There have been hundreds of patients undergoing ovarian tissue freezing but only a small

Autotransplantation is only possible if absence of malignant cells in the graft is confirmed. Methods for detection of cancer cells in the ovarian tissue of patients having suffered from hematological malignancies are under development including immunohistochemistry or the polymerase chain reaction applied to the tissue [54]. The investigation of residual malignant cells in the ovarian tissue may also be performed by xeno-transplantation to immunodeficient SCID mouse. Autotransplantation of ovarian tissue in patients having suffered from systemic hematological malignancies is not recommended due to the high risk of retransmission of malignancy and only patients with cancer diagnosis associated with a negligible or no risk of

Oocyte retrieval is undertaken usually by vaginal ultrasound assistance under sedation or general anaesthesia. Fertilization of the oocytes for embryo cryopreservation has traditionally been offered to woman having a partner. Transfer of frozen/thawed embryos today is a clinical routine in fertility clinics worldwide and it has been used for over 25 years. Intact embryos after thawing have similar implantation potential as fresh embryos and this treatment can lead to a 59% pregnancy rate and a 26% live birth rate [37].

Freezing unfertilized oocytes aiming at later thawing and fertilizing them by IVF is also a promising option to preserve fertility today. As the methods for cryopreservation of eggs have notably developed in recent years with the development of vitrification techniques, improving success in oocyte survival and fertilization rates has been achieved, approaching that of fresh oocytes. Worldwide, an increasing number of pregnancies and children born after fertilization offrozen-thawnoocyteshasbeenreportedandalthoughoverallpregnancyratesarestillrelatively lower than those with embryo freezing [38-40], pregnancy rates and livebirths after thawing and fertilizingfrozeneggsare currentlyreachingthoseobtainedafter embryocryopreservation[41].

### **6.3. Ovarian stimulation using aromatase inhibitors to maintain low systemic estradiol levels in case of breast cancer**

Ovarian stimulation with gonadotropins before egg retrieval aims at obtaining more than one oocyte per cycle and it is a key component of the success of IVF.

In women with an estrogen-sensitive tumor, the elevation of circulating estradiol levels during ovulation stimulation is undesirable and it has been regarded as potentially harmful. There‐ fore, hormone positive breast cancer patients have been largely excluded of the option to preserve fertility aiming at freezing eggs or embryos [42].

Alternative protocols, including natural cycle IVF (without hormone stimulation) or inducing ovulation by using Selective Estrogen Receptor Modulators (SERMs) and aromatase inhibitors alone or in combination with gonadotropins have been proposed, as they might be potentially safer. Natural cycle IVF gives only one oocyte or embryo per cycle and this treatment protocol has a high rate of cycle cancellation.

Both tamoxifen and letrozole can be administered alone or alongside with gonadotropins to increase the number of oocytes yielded for cryopreservation. Stimulation protocols using letrozole alongside with gonadotropins have shown to be most effective resulting in higher number of oocytes obtained and fertilized when compared to tamoxifen protocols [43]. The short-term follow-up of breast cancer patients having undergone ovarian stimulation with letrozole for fertility preservation has not shown any detrimental effects on survival [44].

Although aromatase inhibitors are contraindicated during pregnancy, data indicate that fertility treatments with letrozole are safe and the use of letrozole before conception does not induce any increased risks for the fetus [45]. Letrozole is currently used in the treatment of anovulatory infertility in many countries.

### **6.4. Cryopreservation of immature oocytes obtained without hormonal stimulation**

Freezing immature oocytes is also an option for female fertility preservation in case of patients having a contraindication for hormonal stimulation or when there is not time available for stimulation. The oocytes are retrieved in the natural cycle and frozen at an immature stage or after maturation *in vitro* (IVM) [46]. Immature oocytes survive cryopreservation better than mature metaphase II oocytes [47]. After thawing they can be matured in vitro and fertilized. In vitro maturation of oocytes is at an experimental stage and needs further development. Only few fertility centers worldwide offer treatments using this technique.

### **6.5. Ovarian tissue freezing for prepubertal and adult patients**

As the vast majority of eggs making up the ovarian reserve are within primordial follicles in the ovarian cortex, small cortical ovarian biopsies may provide a high number of eggs to be preserved. This procedure is usually performed by laparoscopy, can be planned immediately after the diagnosis of malignant disease and does not require hormonal stimulation. In cases when the patient needs to undergo abdominal surgery for the treatment of cancer, the ovarian tissue can be harvested during the same surgical procedure. Although it is preferable to carry out cryopreservation of ovarian tissue before a gonadotoxic treatment, young women, adolescents and girls have normally an abundant number of primordial follicles in their ovaries and attempts to harvest ovarian tissue for cryopreservation may still be worthwhile after the first courses of chemotherapy, if the procedure was not possible before [6].

Cryopreservation of ovarian tissue is the only option in prepubertal girls, as sexual maturity is not required. As this procedure does not cause any significant delay to initiation of cancer treatment and it does not require ovarian stimulation, some adult female patients also prefer to preserve fertility by this method.

#### **6.6. Ovarian tissue transplantation**

ovarian stimulation with gonadotropins for IVF may require only two weeks to achieve, as it has been shown that a random-start in the stimulation cycle, independently of cycle day, does

Oocyte retrieval is undertaken usually by vaginal ultrasound assistance under sedation or general anaesthesia. Fertilization of the oocytes for embryo cryopreservation has traditionally been offered to woman having a partner. Transfer of frozen/thawed embryos today is a clinical routine in fertility clinics worldwide and it has been used for over 25 years. Intact embryos after thawing have similar implantation potential as fresh embryos and this treatment can lead

Freezing unfertilized oocytes aiming at later thawing and fertilizing them by IVF is also a promising option to preserve fertility today. As the methods for cryopreservation of eggs have notably developed in recent years with the development of vitrification techniques, improving success in oocyte survival and fertilization rates has been achieved, approaching that of fresh oocytes. Worldwide, an increasing number of pregnancies and children born after fertilization offrozen-thawnoocyteshasbeenreportedandalthoughoverallpregnancyratesarestillrelatively lower than those with embryo freezing [38-40], pregnancy rates and livebirths after thawing and fertilizingfrozeneggsare currentlyreachingthoseobtainedafter embryocryopreservation[41].

**6.3. Ovarian stimulation using aromatase inhibitors to maintain low systemic estradiol**

Ovarian stimulation with gonadotropins before egg retrieval aims at obtaining more than one

In women with an estrogen-sensitive tumor, the elevation of circulating estradiol levels during ovulation stimulation is undesirable and it has been regarded as potentially harmful. There‐ fore, hormone positive breast cancer patients have been largely excluded of the option to

Alternative protocols, including natural cycle IVF (without hormone stimulation) or inducing ovulation by using Selective Estrogen Receptor Modulators (SERMs) and aromatase inhibitors alone or in combination with gonadotropins have been proposed, as they might be potentially safer. Natural cycle IVF gives only one oocyte or embryo per cycle and this treatment protocol

Both tamoxifen and letrozole can be administered alone or alongside with gonadotropins to increase the number of oocytes yielded for cryopreservation. Stimulation protocols using letrozole alongside with gonadotropins have shown to be most effective resulting in higher number of oocytes obtained and fertilized when compared to tamoxifen protocols [43]. The short-term follow-up of breast cancer patients having undergone ovarian stimulation with letrozole for fertility preservation has not shown any detrimental effects on survival [44]. Although aromatase inhibitors are contraindicated during pregnancy, data indicate that fertility treatments with letrozole are safe and the use of letrozole before conception does not induce any increased risks for the fetus [45]. Letrozole is currently used in the treatment of

not have a negative impact on the number and quality of oocytes retrieved.

to a 59% pregnancy rate and a 26% live birth rate [37].

528 Cancer Treatment - Conventional and Innovative Approaches

oocyte per cycle and it is a key component of the success of IVF.

preserve fertility aiming at freezing eggs or embryos [42].

**levels in case of breast cancer**

has a high rate of cycle cancellation.

anovulatory infertility in many countries.

Transplantation of frozen-thawn ovarian cortex has shown to be a new promising method for recovery of ovarian function [48] and in some cases sufficient to restore fertility [49-51]. Ovarian tissue can be transplanted orthotopically, i.e. at the anatomical intrapelvic ovarian site or heterotopically, i.e. at other places including extrapelvic sites [52, 53].

There have been hundreds of patients undergoing ovarian tissue freezing but only a small percentage of these have returned for ovarian transplantation.

Autotransplantation is only possible if absence of malignant cells in the graft is confirmed. Methods for detection of cancer cells in the ovarian tissue of patients having suffered from hematological malignancies are under development including immunohistochemistry or the polymerase chain reaction applied to the tissue [54]. The investigation of residual malignant cells in the ovarian tissue may also be performed by xeno-transplantation to immunodeficient SCID mouse. Autotransplantation of ovarian tissue in patients having suffered from systemic hematological malignancies is not recommended due to the high risk of retransmission of malignancy and only patients with cancer diagnosis associated with a negligible or no risk of ovarian compromise should be considered for future autotransplantation [55]. Ovarian tissue cryopreservation and transplantation has shown not to interfere with proper genomic imprinting in mice pups [56] but additional studies in other animal models are needed.

women having received GnRHa but none has demonstrated a beneficial effect regarding fertility recovery. Although data indicate that infertility is increased after a chemotherapy treatment, even if menstrual cycles are resumed [65], studies suggesting a beneficial effect of GnRHa co-treatment on preserving menstruation have had a great impact in the medical community and the empirical use of GnRHa for ovarian protection during chemotherapy is

Impact of Cancer Treatment on Reproductive Health and Options for Fertility Preservation

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531

**7. Counseling and prompt referal increase the chances to preserve fertility**

Despite the fact that fertility issues are recognized in young people with cancer, health care professionals still report never referring cancer patients of reproductive age to a reproductive specialists for fertility preservation, indicating that many patients still do not receive adequate and timely information [66] [67]. This contrasts to data indicating that approximately three out of four cancer patients younger than 35 years and childless at the time of cancer treatment may be interested in having children in the future [2]. Because incidence of most cancers increases with age, the trend of delaying childbearing in Western societies will naturally result in more

In despite of this, recent data indicate that female cancer patients are still poorly informed on fertility threats of cancer treatment and options to preserve fertility in comparison with their gender counterparts. A recent Swedish survey found that less than half of female patients recalled having received appropriate information on reproductive threats of cancer therapy whereas 80% of male patients recalled having had an appropriate discussion [67]. Only a small number of female patients used fertility preservation methods compared to a rate >70% of sperm freezing in male patients in that study. Urgency to start cancer treatment and lack of appropriate time, lack of knowledge on fertility preservation and awareness of the costs of assisted reproduction methods are recognized barriers to counseling and referring patients to

Infertility due to gonadal failure is one of the major consequences of cancer therapy, particu‐ larly in patients who receive aggressive chemotherapy and/or radiotherapy treatment. Many surveys of cancer survivors have found that those patients are at increased risk of emotional distress if they become infertile as a result of their treatment. Evidence suggests that long-term survival after treatment for cancer during childhood is associated with increased risk of impaired quality of life and higher frequency of psychosocial problems often related to infertility issues. Adolescent cancer survivors have increased anxieties about body image and dating, and pediatric cancer survivors are less likely to marry than matched controls. Although cancer survivors can become parents by adoption or gamete donation, most would prefer to

currently widely spread [26].

fertility preservation [68].

**8. Conclusion**

female cancer patients interested in fertility preservation.

have biological parenthood and biologically related children.

### **6.7. In vitro culture of ovarian follicles**

Although many improvements have been reported on the in vitro culture of follicles at early stages aiming at developing them into competent mature follicles, those methods are still on development [57-59]. Follicles cultured isolated or within a piece of thawed tissue will be the option for patients with hematological and ovarian malignancies. The normality of imprinted genesof cryobankedoocytes culturedandmaturedinvitrohasyettobeverifiedexperimentally.

### **6.8. Testicular tissue cryopreservation**

This technique involves removal of testicular tissue from the male patient before cytotoxic therapy is initiated. In prepubertal boys, as there is absence of spermatozoa and spermatids, studies have been going on to cryopreserve the testicular totipotent precursors, i.e. the spermatogonial stem cells. Success has been reported in cryopreservation methods of testicular tissue [60] but more research is still needed in how to use the frozen-thawed tissue and obtain mature spermatozoa in vitro. Research suggests that in vitro spermatogenesis is likely to be the safest option for boys suffering from haematological malignancies, which might be retransmitted by retransplantation, but this technique is still to be fully developed [61]. Although there are promising results in experimental animal studies of autologous retransplantation of spermatogonial stem cells showing re-colonization of seminiferous tubules generating complete spermatogenesis and mature germ cells and thus restoring natural fertility, the technique is still experimental in humans [61].

Cryopreservation of gonadal tissue offers hope to childhood cancer survivors, however it also raises several medical and ethical questions. Experimental methods for fertility preservation should only be offered to patients at specialized centers working with ethics board-approved research protocols and only in case when the recognized risks associated to the procedure are minimal.

#### **6.9. Ovarian suppression to prevent gonadal damage**

It has been hypothesized that suppressing the gonadal function transiently during chemo‐ therapy could prevent ovarian follicle destruction in female patients by maintaining the follicles dormant. However, the pool of primordial follicles is normally non-proliferating. Those follicles lack FSH receptors [62] and their initial recruitment is not controlled by gonadotropins [63], therefore hormonal manipulation by suppressing gonadotropin release is not likely to affect them [64]. The vast majority of available studies having investigating gonadal protection by gonadotropin-releasing hormone analogues (GnRHa) agonists or antagonists during chemotherapy in females have been small, retrospective and uncontrolled. A significant number of those studies had used resumption of menses as a surrogate marker for fertility and many of them had reported higher frequency of resumption of menses in women having received GnRHa but none has demonstrated a beneficial effect regarding fertility recovery. Although data indicate that infertility is increased after a chemotherapy treatment, even if menstrual cycles are resumed [65], studies suggesting a beneficial effect of GnRHa co-treatment on preserving menstruation have had a great impact in the medical community and the empirical use of GnRHa for ovarian protection during chemotherapy is currently widely spread [26].

### **7. Counseling and prompt referal increase the chances to preserve fertility**

Despite the fact that fertility issues are recognized in young people with cancer, health care professionals still report never referring cancer patients of reproductive age to a reproductive specialists for fertility preservation, indicating that many patients still do not receive adequate and timely information [66] [67]. This contrasts to data indicating that approximately three out of four cancer patients younger than 35 years and childless at the time of cancer treatment may be interested in having children in the future [2]. Because incidence of most cancers increases with age, the trend of delaying childbearing in Western societies will naturally result in more female cancer patients interested in fertility preservation.

In despite of this, recent data indicate that female cancer patients are still poorly informed on fertility threats of cancer treatment and options to preserve fertility in comparison with their gender counterparts. A recent Swedish survey found that less than half of female patients recalled having received appropriate information on reproductive threats of cancer therapy whereas 80% of male patients recalled having had an appropriate discussion [67]. Only a small number of female patients used fertility preservation methods compared to a rate >70% of sperm freezing in male patients in that study. Urgency to start cancer treatment and lack of appropriate time, lack of knowledge on fertility preservation and awareness of the costs of assisted reproduction methods are recognized barriers to counseling and referring patients to fertility preservation [68].

### **8. Conclusion**

ovarian compromise should be considered for future autotransplantation [55]. Ovarian tissue cryopreservation and transplantation has shown not to interfere with proper genomic imprinting in mice pups [56] but additional studies in other animal models are needed.

Although many improvements have been reported on the in vitro culture of follicles at early stages aiming at developing them into competent mature follicles, those methods are still on development [57-59]. Follicles cultured isolated or within a piece of thawed tissue will be the option for patients with hematological and ovarian malignancies. The normality of imprinted genesof cryobankedoocytes culturedandmaturedinvitrohasyettobeverifiedexperimentally.

This technique involves removal of testicular tissue from the male patient before cytotoxic therapy is initiated. In prepubertal boys, as there is absence of spermatozoa and spermatids, studies have been going on to cryopreserve the testicular totipotent precursors, i.e. the spermatogonial stem cells. Success has been reported in cryopreservation methods of testicular tissue [60] but more research is still needed in how to use the frozen-thawed tissue and obtain mature spermatozoa in vitro. Research suggests that in vitro spermatogenesis is likely to be the safest option for boys suffering from haematological malignancies, which might be retransmitted by retransplantation, but this technique is still to be fully developed [61]. Although there are promising results in experimental animal studies of autologous retransplantation of spermatogonial stem cells showing re-colonization of seminiferous tubules generating complete spermatogenesis and mature germ cells and thus restoring natural fertility, the

Cryopreservation of gonadal tissue offers hope to childhood cancer survivors, however it also raises several medical and ethical questions. Experimental methods for fertility preservation should only be offered to patients at specialized centers working with ethics board-approved research protocols and only in case when the recognized risks associated to the procedure are

It has been hypothesized that suppressing the gonadal function transiently during chemo‐ therapy could prevent ovarian follicle destruction in female patients by maintaining the follicles dormant. However, the pool of primordial follicles is normally non-proliferating. Those follicles lack FSH receptors [62] and their initial recruitment is not controlled by gonadotropins [63], therefore hormonal manipulation by suppressing gonadotropin release is not likely to affect them [64]. The vast majority of available studies having investigating gonadal protection by gonadotropin-releasing hormone analogues (GnRHa) agonists or antagonists during chemotherapy in females have been small, retrospective and uncontrolled. A significant number of those studies had used resumption of menses as a surrogate marker for fertility and many of them had reported higher frequency of resumption of menses in

**6.7. In vitro culture of ovarian follicles**

530 Cancer Treatment - Conventional and Innovative Approaches

**6.8. Testicular tissue cryopreservation**

technique is still experimental in humans [61].

**6.9. Ovarian suppression to prevent gonadal damage**

minimal.

Infertility due to gonadal failure is one of the major consequences of cancer therapy, particu‐ larly in patients who receive aggressive chemotherapy and/or radiotherapy treatment. Many surveys of cancer survivors have found that those patients are at increased risk of emotional distress if they become infertile as a result of their treatment. Evidence suggests that long-term survival after treatment for cancer during childhood is associated with increased risk of impaired quality of life and higher frequency of psychosocial problems often related to infertility issues. Adolescent cancer survivors have increased anxieties about body image and dating, and pediatric cancer survivors are less likely to marry than matched controls. Although cancer survivors can become parents by adoption or gamete donation, most would prefer to have biological parenthood and biologically related children.

Oncologists should thus be prepared to discuss the negative impact of cancer therapy on reproductive potential with their patients in the same way as any other risks of cancer treatment are discussed. Furthermore, patients interested in fertility preservation should be promptly referred to a reproductive medicine expert to offer timely and appropriate counsel‐ ing and improve success of fertility preservation. Close collaboration between the oncology team and the reproductive endocrinologists should be encouraged.

[8] Morice, P., et al., *[Effects of radiotherapy (external and/or internal) and chemotherapy on female fertility].* Bull Acad Natl Med, 2010. 194(3): p. 481-92; discussion 492-4, 529-30.

Impact of Cancer Treatment on Reproductive Health and Options for Fertility Preservation

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533

[9] Liou, W.S., et al., *Innovations in fertility preservation for patients with gynecologic cancers.*

[10] Abu-Rustum, N.R. and Y. Sonoda, *Fertility-sparing surgery in early-stage cervical cancer: indications and applications.* J Natl Compr Canc Netw, 2010. 8(12): p. 1435-8.

[11] Plante, M., et al., *The vaginal radical trachelectomy: An update of a series of 125 cases and*

[12] Li, J., et al., *Radical abdominal trachelectomy for cervical malignancies: Surgical, oncological and fertility outcomes in 62 patients.* Gynecologic oncology, 2011. 121(3): p. 565-70.

[13] Kesic, V., et al., *Fertility Preserving Management in Gynecologic Cancer Patients: The Need for Centralization.* International Journal of Gynecological Cancer, 2010. 20(9): p.

[14] Pentheroudakis, G., et al., *Cancer, fertility and pregnancy: ESMO Clinical Practice Guide‐ lines for diagnosis, treatment and follow-up.* Ann Oncol, 2010. 21 Suppl 5: p. v266-73.

[15] Ungar, L., et al., *Abdominal radical trachelectomy during pregnancy to preserve pregnancy*

[16] Sioutas, A., et al., *Three cases of vaginal radical trachelectomy during pregnancy.* Gyneco‐

[17] Sabanegh, E.S. and A.M. Ragheb, *Male fertility after cancer.* Urology, 2009. 73(2): p.

[18] Heidenreich, A., et al., *Organ sparing surgery for malignant germ cell tumor of the testis.*

[19] Gosden, R.G., et al., *Impact of congenital or experimental hypogonadotrophism on the radia‐ tion sensitivity of the mouse ovary.* Human reproduction (Oxford, England), 1997.

[20] Speiser, B., P. Rubin, and G. Casarett, *Aspermia following lower truncal irradiation in*

[21] Wallace, W.H.B., R.A. Anderson, and D.S. Irvine, *Fertility preservation for young pa‐ tients with cancer: who is at risk and what can be offered?* The lancet oncology, 2005. 6(4):

[22] Rodriguez-Wallberg, K.A. and K. Oktay, *Fertility preservation medicine: options for young adults and children with cancer.* Journal of pediatric hematology/oncology : offi‐ cial journal of the American Society of Pediatric Hematology/Oncology, 2010. 32(5):

*106 pregnancies.* Gynecologic oncology, 2011. 121(2): p. 290-7.

*and fertility.* Obstet Gynecol, 2006. 108(3 Pt 2): p. 811-4.

logic oncology, 2011. 121(2): p. 420-1.

The Journal of urology, 2001. 166(6): p. 2161-5.

*Hodgkin's disease.* Cancer, 1973. 32(3): p. 692-8.

Fertil Steril, 2005. 84(6): p. 1561-73.

1613-9.

225-31.

12(11): p. 2483-8.

p. 209-18.

p. 390-6.

### **Author details**

Kenny A. Rodriguez-Wallberg1,2,3

Address all correspondence to: kenny.rodriguez-wallberg@karolinska.se

1 Clinical Responsible of Fertility Preservation Programme, Karolinska University Hospital, Stockholm, Sweden

2 Karolinska Institutet, Department of Clinical Science, Intervention and Technology, Divi‐ sion of Obstetrics and Gynecology, Sweden

3 Karolinska University Hospital Huddinge, Fertility Unit, Stockholm, Sweden

### **References**


[8] Morice, P., et al., *[Effects of radiotherapy (external and/or internal) and chemotherapy on female fertility].* Bull Acad Natl Med, 2010. 194(3): p. 481-92; discussion 492-4, 529-30.

Oncologists should thus be prepared to discuss the negative impact of cancer therapy on reproductive potential with their patients in the same way as any other risks of cancer treatment are discussed. Furthermore, patients interested in fertility preservation should be promptly referred to a reproductive medicine expert to offer timely and appropriate counsel‐ ing and improve success of fertility preservation. Close collaboration between the oncology

1 Clinical Responsible of Fertility Preservation Programme, Karolinska University Hospital,

2 Karolinska Institutet, Department of Clinical Science, Intervention and Technology, Divi‐

[1] SEER Cancer Statistics Review 1975-2007, N.C.I., updated January 7, 2011 2011.

[2] Schover, L.R., et al., *Having children after cancer. A pilot survey of survivors' attitudes and*

[3] Rosen, A., K.A. Rodriguez-Wallberg, and L. Rosenzweig, *Psychosocial distress in young*

[4] Schover, L.R., et al., *Knowledge and experience regarding cancer, infertility, and sperm banking in younger male survivors.* Journal of clinical oncology : official journal of the

[5] Wong, I., et al., *Assisted conception following radical trachelectomy.* Human reproduction

[6] Wallberg, K.A., V. Keros, and O. Hovatta, *Clinical aspects of fertility preservation in fe‐*

[7] Dargent, D., et al., *La trachelectomie elargie(te), une alternative a l'hysterectomie radicale dans le traitement des cancer infiltrants developpes sur la face externe du col uterin.* 1994. 2:

team and the reproductive endocrinologists should be encouraged.

Address all correspondence to: kenny.rodriguez-wallberg@karolinska.se

3 Karolinska University Hospital Huddinge, Fertility Unit, Stockholm, Sweden

**Author details**

Stockholm, Sweden

**References**

p. 285-292.

Kenny A. Rodriguez-Wallberg1,2,3

sion of Obstetrics and Gynecology, Sweden

532 Cancer Treatment - Conventional and Innovative Approaches

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[38] Oktay, K., A.P. Cil, and H. Bang, *Efficiency of oocyte cryopreservation: a meta-analysis.*

[39] Practice Committee of the American Society for Reproductive Medicine, P.C.o.t.S.f.A.R.T., *Ovarian tissue and oocyte cryopreservation.* Fertil Steril, 2008. 90: p.

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[42] Rodriguez-Wallberg, K.A. and K. Oktay, *Fertility preservation in women with breast can‐*

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[44] Azim, A.A., M. Costantini-Ferrando, and K. Oktay, *Safety of fertility preservation by ovarian stimulation with letrozole and gonadotropins in patients with breast cancer: a pro‐ spective controlled study.* Journal of clinical oncology : official journal of the American

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[46] Chian, R.C., et al., *Live birth after vitrification of in vitro matured human oocytes.* Fertil

[47] Boiso, I., et al., *A confocal microscopy analysis of the spindle and chromosome configura‐ tions of human oocytes cryopreserved at the germinal vesicle and metaphase II stage.* Hum

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[49] Andersen, C.Y., et al., *Two successful pregnancies following autotransplantation of frozen/*

[50] von Wolff, M., et al., *Cryopreservation and autotransplantation of human ovarian tissue prior to cytotoxic therapy--a technique in its infancy but already successful in fertility preser‐ vation.* European journal of cancer (Oxford, England : 1990), 2009. 45(9): p. 1547-53.

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Fertility and sterility, 2006. 86(1): p. 70-80.

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134-5.


[37] Marrs, R.P., J. Greene, and B.A. Stone, *Potential factors affecting embryo survival and clinical outcome with cryopreserved pronuclear human embryos.* Am J Obstet Gynecol, 2004. 190(6): p. 1766-71; discussion 1771-2.

[23] Sklar, C., *Growth and endocrine disturbances after bone marrow transplantation in child‐ hood.* Acta paediatrica (Oslo, Norway : 1992) Supplement, 1995. 411: p. 57-61; discus‐

[24] Thibaud, E., et al., *Ovarian function after bone marrow transplantation during childhood.*

[25] Shalet, S.M., et al., *Vulnerability of the human Leydig cell to radiation damage is dependent*

[26] Lee, S.J., et al., *American Society of Clinical Oncology recommendations on fertility preser‐ vation in cancer patients.* Journal of clinical oncology : official journal of the American

[27] Wo, J.Y. and A.N. Viswanathan, *Impact of radiotherapy on fertility, pregnancy, and neo‐ natal outcomes in female cancer patients.* International journal of radiation oncology, bi‐

[28] Green, D.M., et al., *Ovarian failure and reproductive outcomes after childhood cancer treat‐ ment: results from the Childhood Cancer Survivor Study.* Journal of clinical oncology : of‐ ficial journal of the American Society of Clinical Oncology, 2009. 27(14): p. 2374-81.

[29] Hudson, M.M., *Reproductive outcomes for survivors of childhood cancer.* Obstetrics and

[30] Oktem, O. and K. Oktay, *A novel ovarian xenografting model to characterize the impact of chemotherapy agents on human primordial follicle reserve.* Cancer research, 2007. 67(21):

[31] Wallberg, K.A.R.-M., V. Keros, and O. Hovatta, *Clinical aspects of fertility preservation*

[32] Meirow, D., et al., *Administration of cyclophosphamide at different stages of follicular ma‐ turation in mice: effects on reproductive performance and fetal malformations.* Human re‐

[33] Guerin, J.F., *[Testicular tissue cryoconservation for prepubertal boy: indications and feasibil‐*

[34] Bahadur, G., et al., *Semen quality and cryopreservation in adolescent cancer patients.* Hum

[35] Menon, S., et al., *Fertility preservation in adolescent males: experience over 22 years at*

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534 Cancer Treatment - Conventional and Innovative Approaches


[51] Oktay, K., I. Turkcuoglu, and K.A. Rodriguez-Wallberg, *Four spontaneous pregnancies and three live births following subcutaneous transplantation of frozen banked ovarian tissue: what is the explanation?* Fertility and sterility, 2011. 95(2): p. 804 e7-10.

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2009. 54(4): p. 203-7.


[65] Letourneau, J.M., et al., *The prevalence of self-reported reproductive impairment in young female cancer survivors throuhgout California.* Fertil Steril, 2010. 94(4): p. 510 (Abstract).

[51] Oktay, K., I. Turkcuoglu, and K.A. Rodriguez-Wallberg, *Four spontaneous pregnancies and three live births following subcutaneous transplantation of frozen banked ovarian tissue:*

[52] Oktay, K., et al., *Embryo development after heterotopic transplantation of cryopreserved*

[53] Sonmezer, M. and K. Oktay, *Orthotopic and heterotopic ovarian tissue transplantation.*

[54] Meirow, D., et al., *Searching for evidence of disease and malignant cell contamination in ovarian tissue stored from hematologic cancer patients.* Hum Reprod, 2008. 23(5): p.

[55] Ajala, T., et al., *Fertility preservation for cancer patients: a review.* Obstet Gynecol Int,

[56] al, S.e., *Immature cryopreserved ovary restores puberty and fertility in mice without altera‐*

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[58] Smitz, J., et al., *Current achievements and future research directions in ovarian tissue cul‐ ture, in vitro follicle development and transplantation: implications for fertility preservation.*

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**Section 6**

**Perspectives in Cancer Biology and Modeling**

**Perspectives in Cancer Biology and Modeling**

**Chapter 22**

**Sialyl Salivary–Type Amylase Associated with Ovarian**

Studies of tumor-producing amylase originated with Weiss et al.'s 1951 report illustrating a case of bronchogenic carcinoma associated with elevated serum amylase levels [1]. Since then, many reports have focused on lung cancer-producing amylase [2-11], and there have been similar reports in pancreatic [12], stomach [13], uterine [14], and ovarian cancers [15, 16]. Moreover, there have been reports of non-epithelial amylase-producing osteosarcoma [17] and multiple myeloma [18-20]. In these cases, the amylase had the salivary phenotype. However, pancreatic-type amylase has been reported in one case of uterine cancer [21] and two cases of breast cancer [22, 23]. Among those reported cases, determine of the total amylase activity in the sera and amylase isoenzyme electrophoretic analysis have been contributed much to the

In contrast, Sudo and Kanno [24] reported so-called sialic-acid-containing amylase in the sera of patients with lung and pancreatic cancer. It was similarly detected in the sera of patients with IgA-type [25] and IgD-type multiple myeloma [26], and identified to be sialyl salivarytype amylase by electrophoretic study with neuraminidase treatment and immunological characterization [26]. In 2004, Shigemura et al. [27] demonstrated, using cell culture and immunohistochemical techniques, that sialyl salivary-type amylase, together with normal salivary amylase (defined by electrophoretic characteristics), was produced by myeloma cells. In 2006, Yokouchi et al. [28] also detected the same type of amylase in culture medium from the amylase-producing lung adenocarcinoma cell line IMEC-2. In 2008, the author reported that the characterization of sialyl salivary-type amylase associated with ovarian cancer using conserved sera that were obtained from a retrospective study of amylase zymograms [29]. That

> © 2013 Moriyama; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Cancer**

Takanori Moriyama

**1. Introduction**

http://dx.doi.org/10.5772/55452

**1.1. Review of related literature**

diagnosis and treatment.

Additional information is available at the end of the chapter

## **Sialyl Salivary–Type Amylase Associated with Ovarian Cancer**

Takanori Moriyama

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55452

### **1. Introduction**

### **1.1. Review of related literature**

Studies of tumor-producing amylase originated with Weiss et al.'s 1951 report illustrating a case of bronchogenic carcinoma associated with elevated serum amylase levels [1]. Since then, many reports have focused on lung cancer-producing amylase [2-11], and there have been similar reports in pancreatic [12], stomach [13], uterine [14], and ovarian cancers [15, 16]. Moreover, there have been reports of non-epithelial amylase-producing osteosarcoma [17] and multiple myeloma [18-20]. In these cases, the amylase had the salivary phenotype. However, pancreatic-type amylase has been reported in one case of uterine cancer [21] and two cases of breast cancer [22, 23]. Among those reported cases, determine of the total amylase activity in the sera and amylase isoenzyme electrophoretic analysis have been contributed much to the diagnosis and treatment.

In contrast, Sudo and Kanno [24] reported so-called sialic-acid-containing amylase in the sera of patients with lung and pancreatic cancer. It was similarly detected in the sera of patients with IgA-type [25] and IgD-type multiple myeloma [26], and identified to be sialyl salivarytype amylase by electrophoretic study with neuraminidase treatment and immunological characterization [26]. In 2004, Shigemura et al. [27] demonstrated, using cell culture and immunohistochemical techniques, that sialyl salivary-type amylase, together with normal salivary amylase (defined by electrophoretic characteristics), was produced by myeloma cells. In 2006, Yokouchi et al. [28] also detected the same type of amylase in culture medium from the amylase-producing lung adenocarcinoma cell line IMEC-2. In 2008, the author reported that the characterization of sialyl salivary-type amylase associated with ovarian cancer using conserved sera that were obtained from a retrospective study of amylase zymograms [29]. That

© 2013 Moriyama; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

by this paper, universally seen sialyl salivary-type amylase has been revealed in the patients' sera with those malignancies.

**2.3. Amylase isoenzyme electrophoresis**

**2.4. Treatment with neuraminidase**

**2.5. Reaction with anti-human salivary monoclonal antibody**

amylase isoenzyme electrophoresis of the mixture was performed.

**2.6. High performance liquid chromatography (HPLC)**

kit purchased from Iatoron Labs, Tokyo, Japan.

**clinical diagnoses of selected sera**

**3. Results**

a high resolution.

The electrophoresis was performed for 60 min at 300 V using a cellulose acetate membrane (Titan III lipo, Helena Labs, Beaumont, TX, USA) with discontinuous buffer system [47]. Amylase activity was detected by blue starch staining, according to the technique described by Leclerc and Forest [48]. This electrophoretic technique is the most convenient way to have

Sialyl Salivary–Type Amylase Associated with Ovarian Cancer

http://dx.doi.org/10.5772/55452

543

Neuraminidase from *Arthrobacter ureafaciens* (specificities: α-2→3, α-2→6 and α-2→8) and *Clostridium perfringens* (specificities: α-2→3 and α-2→6) were purchased from Nakalai Tesque (Kyoto, Japan) and Sigma (St. Louis, MO, USA), respectively. Neuraminidase treatment was performed at 37ºC for 1 h and the treated sample was analyzed by amylase isoenzyme electrophoresis. It was confirmed, using samples from previous reports that the results of both treatments did not differ between whole serum and a purified amylase fraction sample obtained by size-exclusion chromatography [25, 26]. Whole sera were used for this treatment and the next reaction with monoclonal antibody, because the sample volumes were very low.

Inhibitory monoclonal antibody against human salivary amylase was obtained from an amylase isoenzyme PNP kit (Roche Diagnostics, Tokyo, Japan) based on the method of Gerber et al. [49]. The monoclonal antibody solution was concentrated 5-fold with Minicon B15 clinical sample concentrators (Millipore, Billerica, MA, USA). The monoclonal antibody binds specifically to salivary amylase and inhibits *ca*. 90% of total activity. The whole serum was mixed with this antibody, and incubated at 37˚C for 1 h then at 4˚C for 18 h. After the reaction,

Size-exclusion HPLC analysis was carried out on a Pharmacia (Uppsala, Sweden) fast-protein liquid chromatography (FPLC) apparatus with a Superose 12 HR column (30 cm 1.0 cm) [50]. The serum (100 µL) was eluted with a phosphate buffer (50 mmol/L, pH 7.2) containing NaCl (150 mmol/L). The volume of each fraction was 0.8 mL. Protein was monitored by absorbance at 280 nm, and amylase activity (absorbance at 600 nm) was monitored with an amylase test

**3.1. Ages, serum total amylase activities, amylase isoenzyme electrophoretic analyses, and**

Three specimens came from female patients, and afterward it was found out that they had been diagnosed with ovarian cancer based on medical histories. The patients' ages, total serum

#### **1.2. Ovarian cancer–producing amylase**

When you focus on ovarian cancer-producing amylase, many studies have been published so far [30-46]. In these reports, the important thing is the following three points.


In those reports [30-46], in 1988, Henriksen and Brock had been already reported about "fastmigrated amylase isoenzymes" in the patient's serum, cyst fluid, and tumor tissue. They reported that electrophoretic separation of the amylase revealed fast-migration forms in serum 10 of 47 (21.3%) patients with malignant ovarian neoplasms. Unfortunately, they did not characterize the fast-migrating amylase isoenzyme forms [45], however, it is considered in the perspective of today think and as "sialyl salivary-type amylase" similar to our reports [25, 28, 29]. In the following, describe the research results that led to the identification of the amylase found in the sera with ovarian cancer.

### **2. Materials and methods**

#### **2.1. Subjects**

Three patients' sera were chosen from strictly retrospective observation of routine amylase isoenzyme electrophoresis data, 2,850 specimens, which were analyzed from April 1988 to March 1999 in the Clinical Laboratory, Asahikawa Medical College Hospital, Hokkaido, Japan. The criteria were: a S3 to S2 ratio of over 1.0 and/or acidic fast-migrated sub-bands from S4 to S6. The sera were stored at -80ºC until required.

A sample with a normal amylase isoenzyme electrophoretic pattern was used as control in the analyses of neuraminidase treatment, reaction with anti-salivary monoclonal antibody, and size-exclusion HPLC.

#### **2.2. Measurement of total amylase activity**

Total serum amylase activity was measured on a Hitachi 7170 automated analyzer with G4- CNP as substrate (Toyobo, Osaka, Japan) at 37ºC. The reference interval of total serum amylase activity was from 40 to 160 U/L.

### **2.3. Amylase isoenzyme electrophoresis**

by this paper, universally seen sialyl salivary-type amylase has been revealed in the patients'

When you focus on ovarian cancer-producing amylase, many studies have been published so

**1.** Amylase is directly produced from tumor cell and it can be thought of as of one of the

In those reports [30-46], in 1988, Henriksen and Brock had been already reported about "fastmigrated amylase isoenzymes" in the patient's serum, cyst fluid, and tumor tissue. They reported that electrophoretic separation of the amylase revealed fast-migration forms in serum 10 of 47 (21.3%) patients with malignant ovarian neoplasms. Unfortunately, they did not characterize the fast-migrating amylase isoenzyme forms [45], however, it is considered in the perspective of today think and as "sialyl salivary-type amylase" similar to our reports [25, 28, 29]. In the following, describe the research results that led to the identification of the amylase

Three patients' sera were chosen from strictly retrospective observation of routine amylase isoenzyme electrophoresis data, 2,850 specimens, which were analyzed from April 1988 to March 1999 in the Clinical Laboratory, Asahikawa Medical College Hospital, Hokkaido, Japan. The criteria were: a S3 to S2 ratio of over 1.0 and/or acidic fast-migrated sub-bands from S4 to

A sample with a normal amylase isoenzyme electrophoretic pattern was used as control in the analyses of neuraminidase treatment, reaction with anti-salivary monoclonal antibody, and

Total serum amylase activity was measured on a Hitachi 7170 automated analyzer with G4- CNP as substrate (Toyobo, Osaka, Japan) at 37ºC. The reference interval of total serum amylase

**2.** Serum levels of amylase is decreased after removal the tumor and/or treatments.

far [30-46]. In these reports, the important thing is the following three points.

**3.** The phenotype has been deflected to the salivary-type.

sera with those malignancies.

**1.2. Ovarian cancer–producing amylase**

542 Cancer Treatment - Conventional and Innovative Approaches

important tumor marker.

found in the sera with ovarian cancer.

S6. The sera were stored at -80ºC until required.

**2.2. Measurement of total amylase activity**

activity was from 40 to 160 U/L.

**2. Materials and methods**

**2.1. Subjects**

size-exclusion HPLC.

The electrophoresis was performed for 60 min at 300 V using a cellulose acetate membrane (Titan III lipo, Helena Labs, Beaumont, TX, USA) with discontinuous buffer system [47]. Amylase activity was detected by blue starch staining, according to the technique described by Leclerc and Forest [48]. This electrophoretic technique is the most convenient way to have a high resolution.

### **2.4. Treatment with neuraminidase**

Neuraminidase from *Arthrobacter ureafaciens* (specificities: α-2→3, α-2→6 and α-2→8) and *Clostridium perfringens* (specificities: α-2→3 and α-2→6) were purchased from Nakalai Tesque (Kyoto, Japan) and Sigma (St. Louis, MO, USA), respectively. Neuraminidase treatment was performed at 37ºC for 1 h and the treated sample was analyzed by amylase isoenzyme electrophoresis. It was confirmed, using samples from previous reports that the results of both treatments did not differ between whole serum and a purified amylase fraction sample obtained by size-exclusion chromatography [25, 26]. Whole sera were used for this treatment and the next reaction with monoclonal antibody, because the sample volumes were very low.

### **2.5. Reaction with anti-human salivary monoclonal antibody**

Inhibitory monoclonal antibody against human salivary amylase was obtained from an amylase isoenzyme PNP kit (Roche Diagnostics, Tokyo, Japan) based on the method of Gerber et al. [49]. The monoclonal antibody solution was concentrated 5-fold with Minicon B15 clinical sample concentrators (Millipore, Billerica, MA, USA). The monoclonal antibody binds specifically to salivary amylase and inhibits *ca*. 90% of total activity. The whole serum was mixed with this antibody, and incubated at 37˚C for 1 h then at 4˚C for 18 h. After the reaction, amylase isoenzyme electrophoresis of the mixture was performed.

### **2.6. High performance liquid chromatography (HPLC)**

Size-exclusion HPLC analysis was carried out on a Pharmacia (Uppsala, Sweden) fast-protein liquid chromatography (FPLC) apparatus with a Superose 12 HR column (30 cm 1.0 cm) [50]. The serum (100 µL) was eluted with a phosphate buffer (50 mmol/L, pH 7.2) containing NaCl (150 mmol/L). The volume of each fraction was 0.8 mL. Protein was monitored by absorbance at 280 nm, and amylase activity (absorbance at 600 nm) was monitored with an amylase test kit purchased from Iatoron Labs, Tokyo, Japan.

### **3. Results**

### **3.1. Ages, serum total amylase activities, amylase isoenzyme electrophoretic analyses, and clinical diagnoses of selected sera**

Three specimens came from female patients, and afterward it was found out that they had been diagnosed with ovarian cancer based on medical histories. The patients' ages, total serum amylase activities, and amylase isoenzyme electrophoretic data at the time of amylase electrophoretic analysis are summarized in Table 1. Their total amylase activities were 300, 772, and 798 U/L and the ratios of activity to the upper reference interval were 1.88, 4.20, and 4.99, respectively. Amylase isoenzymes with abnormal anodic migration were detected in all three patients' sera and are shown in Fig. 1A (lane 3), B (lane 3), and C (lane 3). Table 1 gives the ratios of total fast-migrated isoenzymes to S1 and S2 isoenzymes ((S3+S4+S5+S6)/(S1+S2)) and of S3 to S2 (S3/S2). These ratios approximately indicate the proportion of sialyl salivarytype amylase in the total salivary amylase fraction. In cases 2 and 3, the S3 sub-bands were slightly more dominant than the S2 sub-bands, but the ratio of S3 to S2 was below 1.00 only in case 1. The ratio of abnormal anodic bands (from S3 to S6) to normal salivary sub-bands (S1 and S2) was highest for case 3. Unfortunately, these cases were not recognized as having an abnormal amylase pattern in the routine electrophoretic analyses. It was considered that the S3 sub-bands were obviously dominant over the S2 sub-band in previous cases of multiple myeloma [25, 26]; this was less pronounced in the cases here.

**3.3 Reaction with an anti-salivary amylase monoclonal antibody**

following similar treatment [25].

**3.4. HPLC analysis**

The fast-migrating bands found in the three cases disappeared from the electrophoretic patterns, together with residual normal salivary bands, on reaction with the anti-salivary amylase monoclonal antibody, and a faint broad band of amylase activity was observed on the original patterns. Formation of the faint broad band is evidence that the fast-migrating amylase reacted completely with the monoclonal antibody [25]. These results are shown in Fig. 1A (lane 1) 1B (lane 1), and 1C (lane 1), respectively. It was confirmed previously that the salivary amylase bands in normal serum disappeared from the electrophoretic pattern

A, Elution profile of normal serum amylase. Protein concentration and amylase activity were monitored at 280 nm (blue line) and 600 nm (red line), respectively. Peak 1, IgM; peak 2, IgG; peak 3, alubumin; peak 4, normal amylase. B, Elution pro‐ file of amylase in the serum of case 1; C, that of case 2; D, that of case 3. Amylase activity was monitored at 600 nm. Amy‐ lase activities of normal and patient were indicated by the broken line and solid line, respectively. In case 1 and 3, amylase activity was eluted in a broad peak. In case 2, amylase activity was eluted in two peaks. Fraction numbers 7 and 9 corre‐

**Figure 2.** Elution profiles of amylase from normal serum and three patients' sera by HPLC on a Superose 12 HR column.

Normal serum and the patients' sera were subjected to HPLC using a Superose 12 column, and the elution patterns are shown in Fig. 2. Typically, normal serum amylase is eluted as a single peak far from sharp in the low-molecular-weight albumin, indicated in Fig. 2A. From Fig. 2B to 2D, elution patterns of the three cases are shown in comparison with normal amylase peak. Normal serum amylase eluted in fraction number 9, illustrated with a broken line. In contrast, two amylase activity peaks were noted in case 2, in fractions 7 and 9 (Fig. 2C). In cases 1 and 3, the amylase activity eluted in a large peak, fraction number 8 (Fig. 2B and D). It has been confirmed using isoamylase electrophoretic characterization that fraction 9 corresponds

spond to the peak of sialyl salivary-type amylase and the normal serum amylase, respectively.

(a) (b)

Sialyl Salivary–Type Amylase Associated with Ovarian Cancer

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545

(c) (d)

#### **3.2. Neuraminidase treatment**

The serum samples of the three cases were treated with neuraminidase and submitted to electrophoretic analyses. The results using neuraminidase from *Arthrobacter ureafaciens* are shown in Fig. 1A (lane 2), 1B (lane 2), and 1C (lane 2). The abnormal anodic bands (from S3 to S6) showed a reduction of electrophoretic mobility compared with those in untreated sera, and shifted to the cathodic side corresponding to normal salivary isoenzymes in all cases. Both S1 and S2 bands were resultantly stained more strongly, respectively. These densitometric data are shown in Table 1 together with original amylase isoenzyme data. Neuraminidase from *Clostridium perfringens* had similar effects (data not shown). Therefore, it was considered that the binding pattern of the sialic acid residue was α-2→3 or α-2→6 in those cases. It has been previously confirmed that normal serum shows no change in electrophoresis under the same neuraminidase treatment conditions [25].

A, Case 1; B, Case 2; C, Case 3. 1, After reaction with anti-salivary amylase monoclonal antibody; 2, after neuramini‐ dase treatment; 3, patient's original serum; 4, normal serum. The fast-migrated amylase isoenzymes were indicated by the bracket.

**Figure 1.** Amylase isoenzyme electrophoretic analysis of three patients' sera treated with anti-salivary monoclonal an‐ tibody and neuraminidase.

### **3.3 Reaction with an anti-salivary amylase monoclonal antibody**

The fast-migrating bands found in the three cases disappeared from the electrophoretic patterns, together with residual normal salivary bands, on reaction with the anti-salivary amylase monoclonal antibody, and a faint broad band of amylase activity was observed on the original patterns. Formation of the faint broad band is evidence that the fast-migrating amylase reacted completely with the monoclonal antibody [25]. These results are shown in Fig. 1A (lane 1) 1B (lane 1), and 1C (lane 1), respectively. It was confirmed previously that the salivary amylase bands in normal serum disappeared from the electrophoretic pattern following similar treatment [25].

A, Elution profile of normal serum amylase. Protein concentration and amylase activity were monitored at 280 nm (blue line) and 600 nm (red line), respectively. Peak 1, IgM; peak 2, IgG; peak 3, alubumin; peak 4, normal amylase. B, Elution pro‐ file of amylase in the serum of case 1; C, that of case 2; D, that of case 3. Amylase activity was monitored at 600 nm. Amy‐ lase activities of normal and patient were indicated by the broken line and solid line, respectively. In case 1 and 3, amylase activity was eluted in a broad peak. In case 2, amylase activity was eluted in two peaks. Fraction numbers 7 and 9 corre‐ spond to the peak of sialyl salivary-type amylase and the normal serum amylase, respectively.

**Figure 2.** Elution profiles of amylase from normal serum and three patients' sera by HPLC on a Superose 12 HR column.

#### **3.4. HPLC analysis**

amylase activities, and amylase isoenzyme electrophoretic data at the time of amylase electrophoretic analysis are summarized in Table 1. Their total amylase activities were 300, 772, and 798 U/L and the ratios of activity to the upper reference interval were 1.88, 4.20, and 4.99, respectively. Amylase isoenzymes with abnormal anodic migration were detected in all three patients' sera and are shown in Fig. 1A (lane 3), B (lane 3), and C (lane 3). Table 1 gives the ratios of total fast-migrated isoenzymes to S1 and S2 isoenzymes ((S3+S4+S5+S6)/(S1+S2)) and of S3 to S2 (S3/S2). These ratios approximately indicate the proportion of sialyl salivarytype amylase in the total salivary amylase fraction. In cases 2 and 3, the S3 sub-bands were slightly more dominant than the S2 sub-bands, but the ratio of S3 to S2 was below 1.00 only in case 1. The ratio of abnormal anodic bands (from S3 to S6) to normal salivary sub-bands (S1 and S2) was highest for case 3. Unfortunately, these cases were not recognized as having an abnormal amylase pattern in the routine electrophoretic analyses. It was considered that the S3 sub-bands were obviously dominant over the S2 sub-band in previous cases of multiple

The serum samples of the three cases were treated with neuraminidase and submitted to electrophoretic analyses. The results using neuraminidase from *Arthrobacter ureafaciens* are shown in Fig. 1A (lane 2), 1B (lane 2), and 1C (lane 2). The abnormal anodic bands (from S3 to S6) showed a reduction of electrophoretic mobility compared with those in untreated sera, and shifted to the cathodic side corresponding to normal salivary isoenzymes in all cases. Both S1 and S2 bands were resultantly stained more strongly, respectively. These densitometric data are shown in Table 1 together with original amylase isoenzyme data. Neuraminidase from *Clostridium perfringens* had similar effects (data not shown). Therefore, it was considered that the binding pattern of the sialic acid residue was α-2→3 or α-2→6 in those cases. It has been previously confirmed that normal serum shows no change in electrophoresis under the same

(a) (b) (c) A, Case 1; B, Case 2; C, Case 3. 1, After reaction with anti-salivary amylase monoclonal antibody; 2, after neuramini‐ dase treatment; 3, patient's original serum; 4, normal serum. The fast-migrated amylase isoenzymes were indicated by

**Figure 1.** Amylase isoenzyme electrophoretic analysis of three patients' sera treated with anti-salivary monoclonal an‐

myeloma [25, 26]; this was less pronounced in the cases here.

**3.2. Neuraminidase treatment**

544 Cancer Treatment - Conventional and Innovative Approaches

neuraminidase treatment conditions [25].

the bracket.

tibody and neuraminidase.

Normal serum and the patients' sera were subjected to HPLC using a Superose 12 column, and the elution patterns are shown in Fig. 2. Typically, normal serum amylase is eluted as a single peak far from sharp in the low-molecular-weight albumin, indicated in Fig. 2A. From Fig. 2B to 2D, elution patterns of the three cases are shown in comparison with normal amylase peak. Normal serum amylase eluted in fraction number 9, illustrated with a broken line. In contrast, two amylase activity peaks were noted in case 2, in fractions 7 and 9 (Fig. 2C). In cases 1 and 3, the amylase activity eluted in a large peak, fraction number 8 (Fig. 2B and D). It has been confirmed using isoamylase electrophoretic characterization that fraction 9 corresponds to normal salivary and pancreatic amylase, fraction 8 corresponds to a mixture of fastmigrating abnormal amylases with normal amylases, and fraction 7 corresponds to the fastmigrating abnormal amylase [25].

type amylase from myeloma was determined by immunoblotting to be approximately 60,000 Da, the same as normal salivary amylase [25]. Unfortunately, the equivalent experiment could not be repeated in this study, owing to the small serum sample sizes, but as the elution profiles in this report were the same as in previous reports [25-28], the molecular mass is assumed to be the same. However, the sialyl salivary-type amylases were well separated by Superose 12 HPLC analyses. Moreover, the peak of the amylases did not change following neuraminidase treatment; such elution behavior can probably be explained by the unusual protein confor‐

Sialyl Salivary–Type Amylase Associated with Ovarian Cancer

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547

Recently, Shigemura et al.[55] reported that sialyl salivary-type amylase was detected in serum from 7 out of 11 (63.6%) subjects with multiple myeloma. It was emphasized that sialyl salivarytype amylase is a useful marker of disease activity in multiple myeloma, and that sialylation of the amylase molecule might be concerned with oncogenic transformation or chromosomal abnormalities. Moreover, it was disclosed that sialyl salivary-type amylases could be detected in the serum of patients with a normal serum amylase level and apparently normal electropho‐ retic patterns. In our cases, although even cases 2 and 3 were not recognized at the time of the samples were taken, the electrophoretic pattern of case 1 was close to normal. However, there certainly were some (small) abnormal fast-migrating sub-bands; those observations were extremely significant. Accordingly, if amylase isoenzyme electrophoresis is more widely and carefully applied to hyperamylasemia with ovarian cancer, it seems likely that more cases will be detected. Serum sialyl salivary-type amylase will no doubt prove a useful marker of ovari‐

In this study, a case of salivary amylase genetic variant [56] might be experienced by unex‐ pectedly. In amylase zymogram of case 1, S2 sub-band was equal or dominant to S1 sub-band. However, further studies could not be performed in this study and there are no evidences. Isoamylase analysis of saliva and/or tumor extract should been carried out to characterize the

Sialyl salivary-type amylase has been found in the sera of patients with lung cancer [24, 28], pancreatic cancer [24], multiple myeloma [25], and ovarian cancer [29]. Therefore, it is expected that the sialyl salivary-type amylase will be found generally in patients with amylaseproducing tumors. The author especially recommends that amylase isoenzyme electrophoresis should be applied to hyperamylasemia with malignancies, in place of immunological amylase isoenzyme analysis [49] or that a rapid immunological technique for sialyl salivary-type

Zakrzewska and Pietrynczak [58] had already elucidated that the total serum and urinary amylase activity and salivary isoenzyme were significantly decreased after surgical removal of the tumor with different types of ovarian cancer. Moreover, they demonstrated that those activities in the serum of the patients with ovarian carcinoma with various types were significantly decreased after radiotherapy [59]. Although the frequency of salivary amylase and/or sialyl salivary-type amylase in ovarian cancer has not been revealed those amylase could be definitely considered as a nonspecific tumor maker [60]. Therefore, the author would like to propose that this old and new amylase should be added with standard tumor marker

CA125 [61, 62] in the routine treatment and surgery in ovarian cancer

mation of this abnormal salivary-type amylase [25].

an cancer, as for multiple myeloma [55].

amylase should be developed in future.

variant [57].

### **4. Discussion**

The fast-migrating amylase isoenzyme bands found in the three cases of ovarian cancer were determined to be a sialyl salivary-type amylase from the following results:


These characteristics of sialyl salivary-type amylase were also demonstrated in the author's first report of myeloma [25]. The three characteristics above were considered simultaneously as strict criteria to detect sialyl salivary-type amylase. The author would like to recommend that, in the future, at least neuraminidase treatment and size-exclusion HPLC analysis should be used for identification.

Many investigators have reported amylase-producing ovarian cancer and reported that serum amylase is an important tumor marker [15, 30-44]. In particular, amylase isoenzyme electro‐ phoresis has been helpful [6, 32] in making an early diagnosis and distinguishing from pancreatitis; the amylase phenotype was generally salivary. In contrast, there have been a few unique reports [30, 40, 44, 45] describing acidic amylase and/or fast-migrating amylase found in the sera or ascites associated with ovarian cancer. Unfortunately, neuramidase treatment and characterization of other properties were not performed in these studies. The author considers those amylases, from the findings of this and previous reports [25-28], to be most likely sialyl salivary-type. It seems likely that sialylated salivary-type amylase is directly produced together with normal salivary amylase by ovarian cancer cells, as in multiple mylelona cells [27] and lung cancer cells [28].

In contrast, acidic amylase from ovarian cystic fluids [51, 52] can be distinguished clearly from sialyl salivary-type amylase because the cystic amylases are unaffected by treatment with neuraminidase. These amylases are thought to result from aging transformation of cystic amylase, as reported by Warshaw and Lee [53], and Weaver et al.[54]. Therefore, neuramini‐ dase treatment is expected to provide a very important and useful means of distinguishing between aging salivary amylase and sialyl salivary-type amylase. For contrast, we previously published the electrophoretic pattern of aging sialyl salivary-type amylase in pleural effusion with IgD-type multiple myeloma [26].

Size-exclusion HPLC characteristic is another important means of distinguishing sialyl salivary-type amylase from normal salivary amylase. The molecular mass of sialyl salivarytype amylase from myeloma was determined by immunoblotting to be approximately 60,000 Da, the same as normal salivary amylase [25]. Unfortunately, the equivalent experiment could not be repeated in this study, owing to the small serum sample sizes, but as the elution profiles in this report were the same as in previous reports [25-28], the molecular mass is assumed to be the same. However, the sialyl salivary-type amylases were well separated by Superose 12 HPLC analyses. Moreover, the peak of the amylases did not change following neuraminidase treatment; such elution behavior can probably be explained by the unusual protein confor‐ mation of this abnormal salivary-type amylase [25].

to normal salivary and pancreatic amylase, fraction 8 corresponds to a mixture of fastmigrating abnormal amylases with normal amylases, and fraction 7 corresponds to the fast-

The fast-migrating amylase isoenzyme bands found in the three cases of ovarian cancer were

**1.** The isoenzyme bands showed reduced electrophoretic mobility to the cathodic side

**2.** The isoenzyme bands disappeared from the amylase zymograms, and faint broad bands were formed, on reaction with anti-human salivary amylase monoclonal antibody. **3.** The isoenzyme bands could be separated by Superose 12 HR size-exclusion HPLC. Thus, an apparent extra high-molecular-mass peak was observed on the chromatogram. These characteristics of sialyl salivary-type amylase were also demonstrated in the author's first report of myeloma [25]. The three characteristics above were considered simultaneously as strict criteria to detect sialyl salivary-type amylase. The author would like to recommend that, in the future, at least neuraminidase treatment and size-exclusion HPLC analysis should

Many investigators have reported amylase-producing ovarian cancer and reported that serum amylase is an important tumor marker [15, 30-44]. In particular, amylase isoenzyme electro‐ phoresis has been helpful [6, 32] in making an early diagnosis and distinguishing from pancreatitis; the amylase phenotype was generally salivary. In contrast, there have been a few unique reports [30, 40, 44, 45] describing acidic amylase and/or fast-migrating amylase found in the sera or ascites associated with ovarian cancer. Unfortunately, neuramidase treatment and characterization of other properties were not performed in these studies. The author considers those amylases, from the findings of this and previous reports [25-28], to be most likely sialyl salivary-type. It seems likely that sialylated salivary-type amylase is directly produced together with normal salivary amylase by ovarian cancer cells, as in multiple

In contrast, acidic amylase from ovarian cystic fluids [51, 52] can be distinguished clearly from sialyl salivary-type amylase because the cystic amylases are unaffected by treatment with neuraminidase. These amylases are thought to result from aging transformation of cystic amylase, as reported by Warshaw and Lee [53], and Weaver et al.[54]. Therefore, neuramini‐ dase treatment is expected to provide a very important and useful means of distinguishing between aging salivary amylase and sialyl salivary-type amylase. For contrast, we previously published the electrophoretic pattern of aging sialyl salivary-type amylase in pleural effusion

Size-exclusion HPLC characteristic is another important means of distinguishing sialyl salivary-type amylase from normal salivary amylase. The molecular mass of sialyl salivary-

determined to be a sialyl salivary-type amylase from the following results:

migrating abnormal amylase [25].

546 Cancer Treatment - Conventional and Innovative Approaches

following neuraminidase treatment.

mylelona cells [27] and lung cancer cells [28].

with IgD-type multiple myeloma [26].

**4. Discussion**

be used for identification.

Recently, Shigemura et al.[55] reported that sialyl salivary-type amylase was detected in serum from 7 out of 11 (63.6%) subjects with multiple myeloma. It was emphasized that sialyl salivarytype amylase is a useful marker of disease activity in multiple myeloma, and that sialylation of the amylase molecule might be concerned with oncogenic transformation or chromosomal abnormalities. Moreover, it was disclosed that sialyl salivary-type amylases could be detected in the serum of patients with a normal serum amylase level and apparently normal electropho‐ retic patterns. In our cases, although even cases 2 and 3 were not recognized at the time of the samples were taken, the electrophoretic pattern of case 1 was close to normal. However, there certainly were some (small) abnormal fast-migrating sub-bands; those observations were extremely significant. Accordingly, if amylase isoenzyme electrophoresis is more widely and carefully applied to hyperamylasemia with ovarian cancer, it seems likely that more cases will be detected. Serum sialyl salivary-type amylase will no doubt prove a useful marker of ovari‐ an cancer, as for multiple myeloma [55].

In this study, a case of salivary amylase genetic variant [56] might be experienced by unex‐ pectedly. In amylase zymogram of case 1, S2 sub-band was equal or dominant to S1 sub-band. However, further studies could not be performed in this study and there are no evidences. Isoamylase analysis of saliva and/or tumor extract should been carried out to characterize the variant [57].

Sialyl salivary-type amylase has been found in the sera of patients with lung cancer [24, 28], pancreatic cancer [24], multiple myeloma [25], and ovarian cancer [29]. Therefore, it is expected that the sialyl salivary-type amylase will be found generally in patients with amylaseproducing tumors. The author especially recommends that amylase isoenzyme electrophoresis should be applied to hyperamylasemia with malignancies, in place of immunological amylase isoenzyme analysis [49] or that a rapid immunological technique for sialyl salivary-type amylase should be developed in future.

Zakrzewska and Pietrynczak [58] had already elucidated that the total serum and urinary amylase activity and salivary isoenzyme were significantly decreased after surgical removal of the tumor with different types of ovarian cancer. Moreover, they demonstrated that those activities in the serum of the patients with ovarian carcinoma with various types were significantly decreased after radiotherapy [59]. Although the frequency of salivary amylase and/or sialyl salivary-type amylase in ovarian cancer has not been revealed those amylase could be definitely considered as a nonspecific tumor maker [60]. Therefore, the author would like to propose that this old and new amylase should be added with standard tumor marker CA125 [61, 62] in the routine treatment and surgery in ovarian cancer

### **5. Conclusion**

Sialyl salivary-type amylase was detected in the sera of the patients with ovarian cancer. The amylase was considered to have been directly produced together with salivary-type amylase from tumor cells. These studies have contributed to the research into amylase-producing tumors, particularly into amylase-producing ovarian cancer.

[6] Maeda M, Otsuki M, Yuu H, Saeki S, Yamasaki T, Baba S. Salivary-type hyperamyla‐ semia in primary lung cancer: observation of a possible precursor of the salivary-

Sialyl Salivary–Type Amylase Associated with Ovarian Cancer

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549

[7] Yoshida Y, Mori M, Sonoda T, Sakauchi F, Sugawara H, Suzuki A. Ultrastructural, immunohistochemical and biochemical studies on amylase and ACTH producing

[8] Tomita N, Matsuura N, Horii A, Emi M, Nishide T, Ogawa M, et al. Expression of alpha-amylase in human lung cancers. Cancer Res. 1988 Jun 1;48(11):3292-6.

[9] Tsukawaki M, Izawa M, Yoshida M, Araki N, Hashiba Y, Nakagawa H, et al. A case

[10] Grove A. Amylase in lung carcinomas. An ultrastructural and immunohistochemical study of two adenocarcinomas, and a review of the literature. APMIS. 1994 Feb;

[11] Lenler-Petersen P, Grove A, Brock A, Jelnes R. alpha-Amylase in resectable lung can‐

[12] Shimamura J, Fridhandler L, Berk JE. Nonpancreatic-type hyperamylasemia associat‐

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### **Acknowledgements**

The author extremely grateful to the deceased Professor Tatsuo Tozawa, Department of Laboratory Medicine, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan, for encour‐ agement and helpful discussions.

### **Author details**

Takanori Moriyama

Address all correspondence to: moriyama@hs.hokudai.ac.jp

Medical Laboratory Science, Faculty of Health Sciences, Hokkaido University, Kitaku, Sap‐ poro, Japan

### **References**


[6] Maeda M, Otsuki M, Yuu H, Saeki S, Yamasaki T, Baba S. Salivary-type hyperamyla‐ semia in primary lung cancer: observation of a possible precursor of the salivarytype isoamylase. Eur J Cancer Clin Oncol. 1982 Feb;18(2):123-8.

**5. Conclusion**

**Acknowledgements**

**Author details**

Takanori Moriyama

poro, Japan

**References**

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1980;387(2):125-32.

agement and helpful discussions.

Sialyl salivary-type amylase was detected in the sera of the patients with ovarian cancer. The amylase was considered to have been directly produced together with salivary-type amylase from tumor cells. These studies have contributed to the research into amylase-producing

The author extremely grateful to the deceased Professor Tatsuo Tozawa, Department of Laboratory Medicine, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan, for encour‐

Medical Laboratory Science, Faculty of Health Sciences, Hokkaido University, Kitaku, Sap‐

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[3] Gomi K, Kameya T, Tsumuraya M, Shimosato Y, Zeze F, Abe K, et al. Ultrastructural, histochemical, and biochemical studies of two cases with amylase, ACTH, and beta-

[4] Yokoyama M, Natsuizaka T, Ishii Y, Ohshima S, Kasagi A, Tateno S. Amylase-pro‐ ducing lung cancer: ultrastructural and biochemical studies. Cancer. 1977 Aug;40(2):

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tumors, particularly into amylase-producing ovarian cancer.

548 Cancer Treatment - Conventional and Innovative Approaches

Address all correspondence to: moriyama@hs.hokudai.ac.jp


[21] Matsuyama M, Inoue T, Ariyoshi Y, Doi M, Suchi T, Sato T, et al. Argyrophil cell car‐ cinoma of the uterine cervix with ectopic production of ACTH, beta-MSH, serotonin, histamine, and amylase. Cancer. 1979 Nov;44(5):1813-23.

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[24] Sudo K, Kanno T. Sialic acid containing abnormal amylases in human sera. Clin

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[33] Takeuchi T, Fujiki H, Kameya T. Characterization of amylases produced by tumors.

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**Chapter 23**

**Role of CREB Protein Family Members in Human**

Cyclic AMP Response Element Binding (CREB) protein is a member of the CREB/ATF (Activating Transcription Factor) family of transcription factors playing an important role in the nuclear responses to a variety of external signals that lead to proliferation, differentiation, apoptosis and survival. Other authors' evidences have highlighted a critical role of CREB in the regulation of normal haematopoiesis and leukemogenesis due to the interaction with target genes crucially involved in the cell cycle machinery. Recent findings of our research group have demonstrated that CREB and ATF-1 phosphorylation levels are related to a different sensitivity of T leukaemia cell clones to the cytotoxic action of TNF-related apoptosis inducing ligand (TRAIL) and that low dose radiation treatment of erythroleukaemia cells (K562) can trigger CREB activation and deliver a survival signal. Since one fundamental problem of most malignancies, including those of haematological origin, is the development of multiple mechanisms of resistance, which progressively reduce or suppress the therapeutic efficacy of anticancer treatment, the early identification of biological markers of responsiveness/unre‐ sponsiveness and the follow-up of individual response are highly desirable to adjust thera‐ peutic treatments. In light of all these considerations and of the complex molecular interactions involving CREB/ATF family members, the present chapter is aimed at revising literature focusing, in particular, on the involvement of CREB/ATF family members in leukemogenesis and lymphomagenesis, in order to gain more insight into this matter that could result useful

The CREB or CREB/ATF multigenic family is composed by several nuclear transcription factors. The prototype of this family is CREB, a 43 kDa – basic-region leucine zipper (bZIP)

and reproduction in any medium, provided the original work is properly cited.

© 2013 D'Auria and Di Pietro; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Haematological Malignancies**

Francesca D'Auria and Roberta Di Pietro

http://dx.doi.org/10.5772/55368

**1. Introduction**

Additional information is available at the end of the chapter

to the treatment of leukaemia and lymphoma diseases.

**2. CREB family members**

## **Role of CREB Protein Family Members in Human Haematological Malignancies**

Francesca D'Auria and Roberta Di Pietro

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55368

### **1. Introduction**

Cyclic AMP Response Element Binding (CREB) protein is a member of the CREB/ATF (Activating Transcription Factor) family of transcription factors playing an important role in the nuclear responses to a variety of external signals that lead to proliferation, differentiation, apoptosis and survival. Other authors' evidences have highlighted a critical role of CREB in the regulation of normal haematopoiesis and leukemogenesis due to the interaction with target genes crucially involved in the cell cycle machinery. Recent findings of our research group have demonstrated that CREB and ATF-1 phosphorylation levels are related to a different sensitivity of T leukaemia cell clones to the cytotoxic action of TNF-related apoptosis inducing ligand (TRAIL) and that low dose radiation treatment of erythroleukaemia cells (K562) can trigger CREB activation and deliver a survival signal. Since one fundamental problem of most malignancies, including those of haematological origin, is the development of multiple mechanisms of resistance, which progressively reduce or suppress the therapeutic efficacy of anticancer treatment, the early identification of biological markers of responsiveness/unre‐ sponsiveness and the follow-up of individual response are highly desirable to adjust thera‐ peutic treatments. In light of all these considerations and of the complex molecular interactions involving CREB/ATF family members, the present chapter is aimed at revising literature focusing, in particular, on the involvement of CREB/ATF family members in leukemogenesis and lymphomagenesis, in order to gain more insight into this matter that could result useful to the treatment of leukaemia and lymphoma diseases.

### **2. CREB family members**

The CREB or CREB/ATF multigenic family is composed by several nuclear transcription factors. The prototype of this family is CREB, a 43 kDa – basic-region leucine zipper (bZIP)

transcription factor that elicits responses to a variety of extracellular signals, including stress and growth factors, and that is involved in several cellular processes such as glucose homeo‐ stasis, proliferation, ageing and differentiation, survival and apoptosis, memory and learning [1]. The CREB/ATF family of transcription factors includes three homologous genes: cAMP response element binding (*CREB)*, cAMP response element modulator (*CREM*), and activating transcription factor-1 (*ATF-1*), whose structure domains are illustrated in a recent review [2]. These genes generate a group of highly homologous proteins that have been named after their prototypes: CREB, CREM, and ATF-1, respectively [3].

expression of a diverse set of target genes, depending on the cell type and/or the nature of the

Role of CREB Protein Family Members in Human Haematological Malignancies

http://dx.doi.org/10.5772/55368

557

While both CREBs and ATF-1 are ubiquitously expressed, CREMs are mainly present in spermatids [6] and in the neuroendocrine system [18]. Interestingly, a recently published paper on the effects of traumatic brain injury demonstrated the nuclear co-localization of CREM-1 and active caspase-3 in the ispilateral cortex of adult rats, suggesting a possible role for CREM-1 in neuronal apoptosis [19]. In a recent report of our research group on Jurkat leukaemia cells [20], we observed a different cell compartmentalization of CREB protein in dependence of the TRAIL dose employed and induced cytotoxicity. Indeed, both under normal or low serum culture conditions an evident nuclear translocation of phospho-CREB was detected after 1 h treatment only with the lower dose of TRAIL (100 ng/mL) and prevented in the presence of PI3K/Akt and p38 mitogen-activated protein kinase (MAPK) specific inhibitors [20]. In another model under investigation in our laboratories and represented by K562 erythroleukaemia cells induced to differentiation [21], the nuclear localization of the active form of CREB was clearly evident after only 1 h treatment with haemin. Interestingly, CREB positive nuclei resembled the features of apoptotic nuclei, suggesting that CREB phosphorylation is possibly required to determine the nuclear structural changes occurring during erythroblast maturation [21, 22]. Concerning other family members, it has been recently shown that ATF-2 is a nucleocy‐ toplasmic shuttling protein and that its subcellular localization is regulated by AP-1 dimeri‐ zation [23]. ATF-3 is ubiquitously expressed and localized in the nucleus but maintained at low levels in the absence of cellular stresses. Instead, it is rapidly transcriptionally induced under different conditions, among which hypoxia, DNA damage (induced by UV radiation, ionizing radiation, etoposide), heat or cold shock, serum starvation or stimulation [13, 15]. ATF-4 is of particular interest since it has been demonstrated to translocate from the cytoplas‐ mic membrane to the nucleus in neuronal cells upon γ aminobutyric acid (GABA) receptor activation, to be likely involved in neuronal plasticity by coupling receptor activity to gene expression [24]. Finally, a number of immunofluorescent and cell fractionation experiments indicate that ATF-6 is linked to the endoplasmic reticulum (ER) chaperone Bip/Crp78 and localizes in the precursor form on the ER membrane [25]. Upon ER stress induced by prolonged nutrient deprivation, it translocates to the Golgi where it is cleaved by resident proteases to liberate its active N-terminal domain. In this active form it translocates to the nucleus where

it up-regulates a number of target genes involved in energy homeostasis [25].

The human CREB-binding protein (CBP) and its paralogue, p300, are highly related proteins that are well conserved amongst mammals. Due to their high degree of sequence similarity, these two proteins are most often functionally interchangeable although they also possess unique functions [26, 27]. CBP was initially recognized as an interaction partner for CREB nuclear transcription factor [28], whereas p300 cDNA was cloned encoding the 300 kDa protein known to be associated with the adenoviral protein E1A [29]. Though encoded by different genes, CBP/p300 share several conserved regions that constitute most of their known func‐ tional domains [for details see 27]. Both CBP and p300 have originally been described as transcriptional co-activators that bridge DNA-binding transcription factors to components of

**3. CREB binding proteins**

stimuli [16, 17].

CREB/ATF proteins were initially identified for their binding to the cyclic AMP response element (CRE) in various gene promoters that contain the octanucleotide consensus sequence TGACGTCA [4]. Over the years, cDNA clones encoding identical or homologous proteins have been isolated. Up to now, at least 20 different mammalian proteins with the prefix CREB or ATF have been characterized and grouped into subgroups on the basis of their amino acid similarity [5, 6]. CREB/ATF family members include CREB-1 (also known as CREB), CREB-2 (recently named ATF-4), CREB-3, CREB-5, CREM, ATF-1 (also known as TREB36), ATF-2 (also known as CRE-BP1), ATF-3, ATF-4 (previously named CREB-2), ATF-5 (also known as ATFX), ATF-6, ATF-7 and B-ATF subgroups [7, 8]. Proteins belonging to this class represent a large group of bZIP transcription factors containing highly divergent N-terminal domains, but sharing a C-terminal leucine zipper domain. The basic region in the bZIP domain is rich in basic amino acids and is responsible for specific DNA binding, while the leucine zipper region contains leucine residues and is responsible for dimerization of the proteins by resembling a zipper. Based on the sequence of each bZip domain, these proteins form homodimers or heterodimers both with other members of the family and with other bZIP containing proteins like the activator protein-1 (AP-1), C/EBP, Fos, Jun or Maf family proteins [8]. That implies the expansion of the repertoire and different opportunities of target gene regulation that are further increased by the alternative splice products of *CREB* and *CREM* genes that show repressor or activator properties [5, 7]. Whereas CREB, CREM, and ATF-1 are relatively well characterized and known to regulate gene transcription via binding to CRE sites, ATF-2, ATF-3, and ATF-4 are structurally more distant and their functional properties remain poorly understood. Rather than being activated by the cAMP cascade, ATF-2 is activated by c-Jun Nterminal kinase (JNK) and can dimerize with members of the AP-1 family such as c-Jun to bind to CRE or AP-1 sites [9, 10]. Additionally, ATF-2 homodimers and ATF-2/c-Jun heterodimers can bind to certain CRE-like sites that are insensitive to CREB [11]. ATF-3 and ATF-4 also dimerize with various Jun species and can shift c-Jun DNA binding site preferences from AP-1 to CRE, thereby promoting crosstalk among AP-1 and CREB protein families [9]. In addition, ATF-4 is able to dimerize with Nrf1 (NF-E2 related factor 1) and Nrf2 (NF-E2 related factor 2) and then interact with the antioxidant responsive element (ARE) present in the promoters of many antioxidant genes [12]. ATF-2, ATF-3, and ATF-4 have been considered as cellular stress response proteins [5, 13, 14] but recently they have been also involved in non-stress adapta‐ tions. In fact, extensive studies have demonstrated that ATF-3 is an adaptive response gene that is activated by a wide variety of signals including those initiated by cytokines, genotoxic agents or physiological stresses [15]. Interestingly, unlike other ATF family members, emerg‐ ing evidences have implicated ATF-3 in the host defence against invading pathogens and cancer. These processes are controlled by the efficient coordination of cell responses and genetic regulatory networks which allow this key transcription factor to modulate the expression of a diverse set of target genes, depending on the cell type and/or the nature of the stimuli [16, 17].

While both CREBs and ATF-1 are ubiquitously expressed, CREMs are mainly present in spermatids [6] and in the neuroendocrine system [18]. Interestingly, a recently published paper on the effects of traumatic brain injury demonstrated the nuclear co-localization of CREM-1 and active caspase-3 in the ispilateral cortex of adult rats, suggesting a possible role for CREM-1 in neuronal apoptosis [19]. In a recent report of our research group on Jurkat leukaemia cells [20], we observed a different cell compartmentalization of CREB protein in dependence of the TRAIL dose employed and induced cytotoxicity. Indeed, both under normal or low serum culture conditions an evident nuclear translocation of phospho-CREB was detected after 1 h treatment only with the lower dose of TRAIL (100 ng/mL) and prevented in the presence of PI3K/Akt and p38 mitogen-activated protein kinase (MAPK) specific inhibitors [20]. In another model under investigation in our laboratories and represented by K562 erythroleukaemia cells induced to differentiation [21], the nuclear localization of the active form of CREB was clearly evident after only 1 h treatment with haemin. Interestingly, CREB positive nuclei resembled the features of apoptotic nuclei, suggesting that CREB phosphorylation is possibly required to determine the nuclear structural changes occurring during erythroblast maturation [21, 22]. Concerning other family members, it has been recently shown that ATF-2 is a nucleocy‐ toplasmic shuttling protein and that its subcellular localization is regulated by AP-1 dimeri‐ zation [23]. ATF-3 is ubiquitously expressed and localized in the nucleus but maintained at low levels in the absence of cellular stresses. Instead, it is rapidly transcriptionally induced under different conditions, among which hypoxia, DNA damage (induced by UV radiation, ionizing radiation, etoposide), heat or cold shock, serum starvation or stimulation [13, 15]. ATF-4 is of particular interest since it has been demonstrated to translocate from the cytoplas‐ mic membrane to the nucleus in neuronal cells upon γ aminobutyric acid (GABA) receptor activation, to be likely involved in neuronal plasticity by coupling receptor activity to gene expression [24]. Finally, a number of immunofluorescent and cell fractionation experiments indicate that ATF-6 is linked to the endoplasmic reticulum (ER) chaperone Bip/Crp78 and localizes in the precursor form on the ER membrane [25]. Upon ER stress induced by prolonged nutrient deprivation, it translocates to the Golgi where it is cleaved by resident proteases to liberate its active N-terminal domain. In this active form it translocates to the nucleus where it up-regulates a number of target genes involved in energy homeostasis [25].

### **3. CREB binding proteins**

transcription factor that elicits responses to a variety of extracellular signals, including stress and growth factors, and that is involved in several cellular processes such as glucose homeo‐ stasis, proliferation, ageing and differentiation, survival and apoptosis, memory and learning [1]. The CREB/ATF family of transcription factors includes three homologous genes: cAMP response element binding (*CREB)*, cAMP response element modulator (*CREM*), and activating transcription factor-1 (*ATF-1*), whose structure domains are illustrated in a recent review [2]. These genes generate a group of highly homologous proteins that have been named after their

CREB/ATF proteins were initially identified for their binding to the cyclic AMP response element (CRE) in various gene promoters that contain the octanucleotide consensus sequence TGACGTCA [4]. Over the years, cDNA clones encoding identical or homologous proteins have been isolated. Up to now, at least 20 different mammalian proteins with the prefix CREB or ATF have been characterized and grouped into subgroups on the basis of their amino acid similarity [5, 6]. CREB/ATF family members include CREB-1 (also known as CREB), CREB-2 (recently named ATF-4), CREB-3, CREB-5, CREM, ATF-1 (also known as TREB36), ATF-2 (also known as CRE-BP1), ATF-3, ATF-4 (previously named CREB-2), ATF-5 (also known as ATFX), ATF-6, ATF-7 and B-ATF subgroups [7, 8]. Proteins belonging to this class represent a large group of bZIP transcription factors containing highly divergent N-terminal domains, but sharing a C-terminal leucine zipper domain. The basic region in the bZIP domain is rich in basic amino acids and is responsible for specific DNA binding, while the leucine zipper region contains leucine residues and is responsible for dimerization of the proteins by resembling a zipper. Based on the sequence of each bZip domain, these proteins form homodimers or heterodimers both with other members of the family and with other bZIP containing proteins like the activator protein-1 (AP-1), C/EBP, Fos, Jun or Maf family proteins [8]. That implies the expansion of the repertoire and different opportunities of target gene regulation that are further increased by the alternative splice products of *CREB* and *CREM* genes that show repressor or activator properties [5, 7]. Whereas CREB, CREM, and ATF-1 are relatively well characterized and known to regulate gene transcription via binding to CRE sites, ATF-2, ATF-3, and ATF-4 are structurally more distant and their functional properties remain poorly understood. Rather than being activated by the cAMP cascade, ATF-2 is activated by c-Jun Nterminal kinase (JNK) and can dimerize with members of the AP-1 family such as c-Jun to bind to CRE or AP-1 sites [9, 10]. Additionally, ATF-2 homodimers and ATF-2/c-Jun heterodimers can bind to certain CRE-like sites that are insensitive to CREB [11]. ATF-3 and ATF-4 also dimerize with various Jun species and can shift c-Jun DNA binding site preferences from AP-1 to CRE, thereby promoting crosstalk among AP-1 and CREB protein families [9]. In addition, ATF-4 is able to dimerize with Nrf1 (NF-E2 related factor 1) and Nrf2 (NF-E2 related factor 2) and then interact with the antioxidant responsive element (ARE) present in the promoters of many antioxidant genes [12]. ATF-2, ATF-3, and ATF-4 have been considered as cellular stress response proteins [5, 13, 14] but recently they have been also involved in non-stress adapta‐ tions. In fact, extensive studies have demonstrated that ATF-3 is an adaptive response gene that is activated by a wide variety of signals including those initiated by cytokines, genotoxic agents or physiological stresses [15]. Interestingly, unlike other ATF family members, emerg‐ ing evidences have implicated ATF-3 in the host defence against invading pathogens and cancer. These processes are controlled by the efficient coordination of cell responses and genetic regulatory networks which allow this key transcription factor to modulate the

prototypes: CREB, CREM, and ATF-1, respectively [3].

556 Cancer Treatment - Conventional and Innovative Approaches

The human CREB-binding protein (CBP) and its paralogue, p300, are highly related proteins that are well conserved amongst mammals. Due to their high degree of sequence similarity, these two proteins are most often functionally interchangeable although they also possess unique functions [26, 27]. CBP was initially recognized as an interaction partner for CREB nuclear transcription factor [28], whereas p300 cDNA was cloned encoding the 300 kDa protein known to be associated with the adenoviral protein E1A [29]. Though encoded by different genes, CBP/p300 share several conserved regions that constitute most of their known func‐ tional domains [for details see 27]. Both CBP and p300 have originally been described as transcriptional co-activators that bridge DNA-binding transcription factors to components of the basal transcriptional machinery, including the TATA-box-binding protein (TBP) [30], TFIIB [31] and, via RNA helicase A, also RNA polymerase II [32]. Due to the huge size of over 2400 amino acids, CBP/p300 can also behave as a scaffold, bridging together a variety of cofactor proteins at the same time and leading to the assembly of multi-competent co-activator complexes [26, 27]. In addition, CBP/p300 interact with protein kinases such as the MAPKs and the cyclin E-Cdk2 complex, thus mediating the phosphorylation of CBP/p300-interacting transcription factors such as ER81 and E2F family members. Both CBP and p300 have been found originally to possess histone acetyltransferase (HAT) activity [33]. This acetyltransferase function has profound consequences for nucleosomal structure and the activity of transcription factors, and thereby affects gene activity in multiple ways. In fact, it is well known that acetylation of multiple sites in the histone tails has been directly associated with transcriptional up-regulation, whereas de-acetylation correlates with transcriptional repression. Mechanisti‐ cally, histone acetylation promotes the accessibility of DNA to transcription protein complexes, by facilitating the "unwiring" of the chromatin structure. During the last years, both CBP and p300 have been regarded as protein acetyltransferases rather than only HAT since they have shown the capacity to acetylate a number of non-histone nuclear proteins, including the tumour suppressor protein p53, dTCF, EKLF (erythroid Kruppel-like factor), GATA-1, NF-Y and other basal transcription factors [34, 35]. Thus, in light of the number of proteins interacting with CBP/p300, it is not surprising to find that many physiological processes, including cell growth, cell division, cell differentiation, cell transformation, embryogenesis and apoptosis, are dependent on CBP/p300 function [27, 28, 34]. Moreover, the importance of CBP/p300 is underscored by the fact that genetic alterations as well as their functional dysregulation are strongly linked to human diseases [36, 37].

propensity to develop cancer, including leukaemia [36]. Similarly, *CREBBP*(+/-) mice show abnormalities in bone, haematopoietic tissues and neural tissues and an increased tendency to develop haematological malignancies with age [41]. In earlier studies, in *CREBBP*(+/-) HSCs (haematopoietic stem cells) a number of cell-intrinsic defects have been described, including diminished HSC self-renewal and excessive myeloid differentiation [42]. The combination of skeletal and haematopoietic defects in *CREBBP*(+/-) mice suggests the involvement of the bone marrow (BM) microenvironment in the haematopoietic phenotype of these mice. One of the genes whose transcription is directly regulated by CBP is matrix metalloproteinase 9 (MMP9) that was reported to be a microenvironmental regulator of haematopoiesis [43]. Interestingly, *CREBBP* heterozygosity in the BM microenvironment results in reduced levels of MMP9 and soluble kit ligand (KITL) and increased expression of endothelial cell adhesion molecule 1 (ESAM1) and cadherin 5 (CDH5) on a subset of endothelial cells. In addition, it has been reported that the loss of a single *CREBBP* allele is deleterious for the BM microenvironment, leading to defective haematopoiesis. In fact, the *CREBBP*(+/-) microenvironment poorly supports HSCs, promotes excessive myelopoiesis and reduces lymphopoiesis. Furthermore, it has been reported that *CREBBP*(+/-) mice have reduced bone volume due to increased osteoclastogenesis. A concomitant reduction in CFU-fibroblasts (CFU-Fs) and osteoblasts per tissue area was also identified and likely contributes to fewer HSC niches [41]. Thus, all these findings reveal the importance of CBP in the development and function of the BM microen‐ vironment and underscore the multiple levels at which this protein acts to regulate haemato‐ poiesis. Indeed, half of the normal complement of *CREBBP*, but not of *EP300*, in the BM microenvironment has a deleterious effect on haematopoiesis via multiple mechanisms, leading to the development of excessive myelopoiesis, disrupting the proper architecture of

Role of CREB Protein Family Members in Human Haematological Malignancies

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559

the BM and resulting in poor maintenance of HSC number and quality.

CREB is a multi-functional transcriptional activator that is involved in many signalling pathways under normal and pathologic conditions. CREB mediates its transcriptional responses following phosphorylation at Ser133 [7] and the consequent association with the 256 kDa co-activator CBP [28] or related family members like p300 [29]. Both Ser133 phosphory‐ lation and CBP association play an essential role for gene transactivation mediated by an octanucleotide CRE consensus sequence placed in the promoters of many cellular genes [29]. In more detail, CREB transactivation domain, that is the site able to interact with other nuclear factors, contains a constitutive glutamine rich domain termed Q2 and an inducible domain, termed the kinase-inducible domain (KID), regulated by cellular kinases [2]. The Q2 domain interacts with a TATA binding protein-associated factor and is constitutively active; instead, the KID region promotes isomerization by recruiting the co-activator factors CBP and p300 to the gene promoters and is active only when it is phosphorylated at Ser133 by a variety of cellular kinases. Recent studies using a genome-wide analysis showed that the number of putative target genes for CREB is about 5000, among which immediate-early genes, including *c-FOS*, *AP-1/JunB* and early growth response protein 1 (*EGR-1*) [44], as well as genes crucially

**4. CREB physiological roles and signalling pathways**

Previous studies have shown that CBP and p300 play distinct roles in haematopoiesis and act non-redundantly in microenvironment-mediated haematopoietic regulation in spite of their high homology [38-40]. It has been widely documented that both proteins interact with crucial transcriptional regulators in virtually all haematopoietic lineages. Intriguing‐ ly, CBP/p300 can promote, on one hand, normal differentiation and cell cycle arrest (by cooperating with GATA-1) and, on the other hand, cell cycle progression and transforma‐ tion by cooperating with c-Myb and PU.1, an Ets family transcription factor. It is conceiv‐ able that an overexpressed oncoprotein might compete with differentiation-inducing factors for CBP/p300 function. Furthermore, during normal development, CBP/p300 could differentially partition among transcriptional regulators with opposing functions, thus controlling the balance between proliferation and differentiation. As an example, the downmodulation of the proto-oncoproteins PU.1 and c-Myb during the erythroleukaemia MEL cell line maturation might increase availability of CBP/p300 for differentiation-associated factors such as GATA-1, NF-E2 and EKLF. Moreover, besides the involvement in eryth‐ roid cell lineage differentiation, CBP and, very likely, p300 target a broad range of myeloid and lymphoid expressed transcription factors [38-40].

Because of its central role in transcription, it is not surprising that aberrations in *CREBBP* can affect many tissues [17]. In humans, chromosomal translocations involving the *CREBBP* gene have been observed in leukaemia and myelodysplastic syndrome [38]. Mutations of *CREBBP* in the germline have been associated to the Rubinstein-Taybi syndrome (RTS), an autosomal dominant disease characterized by mental retardation, skeletal abnormalities and a high propensity to develop cancer, including leukaemia [36]. Similarly, *CREBBP*(+/-) mice show abnormalities in bone, haematopoietic tissues and neural tissues and an increased tendency to develop haematological malignancies with age [41]. In earlier studies, in *CREBBP*(+/-) HSCs (haematopoietic stem cells) a number of cell-intrinsic defects have been described, including diminished HSC self-renewal and excessive myeloid differentiation [42]. The combination of skeletal and haematopoietic defects in *CREBBP*(+/-) mice suggests the involvement of the bone marrow (BM) microenvironment in the haematopoietic phenotype of these mice. One of the genes whose transcription is directly regulated by CBP is matrix metalloproteinase 9 (MMP9) that was reported to be a microenvironmental regulator of haematopoiesis [43]. Interestingly, *CREBBP* heterozygosity in the BM microenvironment results in reduced levels of MMP9 and soluble kit ligand (KITL) and increased expression of endothelial cell adhesion molecule 1 (ESAM1) and cadherin 5 (CDH5) on a subset of endothelial cells. In addition, it has been reported that the loss of a single *CREBBP* allele is deleterious for the BM microenvironment, leading to defective haematopoiesis. In fact, the *CREBBP*(+/-) microenvironment poorly supports HSCs, promotes excessive myelopoiesis and reduces lymphopoiesis. Furthermore, it has been reported that *CREBBP*(+/-) mice have reduced bone volume due to increased osteoclastogenesis. A concomitant reduction in CFU-fibroblasts (CFU-Fs) and osteoblasts per tissue area was also identified and likely contributes to fewer HSC niches [41]. Thus, all these findings reveal the importance of CBP in the development and function of the BM microen‐ vironment and underscore the multiple levels at which this protein acts to regulate haemato‐ poiesis. Indeed, half of the normal complement of *CREBBP*, but not of *EP300*, in the BM microenvironment has a deleterious effect on haematopoiesis via multiple mechanisms, leading to the development of excessive myelopoiesis, disrupting the proper architecture of the BM and resulting in poor maintenance of HSC number and quality.

### **4. CREB physiological roles and signalling pathways**

the basal transcriptional machinery, including the TATA-box-binding protein (TBP) [30], TFIIB [31] and, via RNA helicase A, also RNA polymerase II [32]. Due to the huge size of over 2400 amino acids, CBP/p300 can also behave as a scaffold, bridging together a variety of cofactor proteins at the same time and leading to the assembly of multi-competent co-activator complexes [26, 27]. In addition, CBP/p300 interact with protein kinases such as the MAPKs and the cyclin E-Cdk2 complex, thus mediating the phosphorylation of CBP/p300-interacting transcription factors such as ER81 and E2F family members. Both CBP and p300 have been found originally to possess histone acetyltransferase (HAT) activity [33]. This acetyltransferase function has profound consequences for nucleosomal structure and the activity of transcription factors, and thereby affects gene activity in multiple ways. In fact, it is well known that acetylation of multiple sites in the histone tails has been directly associated with transcriptional up-regulation, whereas de-acetylation correlates with transcriptional repression. Mechanisti‐ cally, histone acetylation promotes the accessibility of DNA to transcription protein complexes, by facilitating the "unwiring" of the chromatin structure. During the last years, both CBP and p300 have been regarded as protein acetyltransferases rather than only HAT since they have shown the capacity to acetylate a number of non-histone nuclear proteins, including the tumour suppressor protein p53, dTCF, EKLF (erythroid Kruppel-like factor), GATA-1, NF-Y and other basal transcription factors [34, 35]. Thus, in light of the number of proteins interacting with CBP/p300, it is not surprising to find that many physiological processes, including cell growth, cell division, cell differentiation, cell transformation, embryogenesis and apoptosis, are dependent on CBP/p300 function [27, 28, 34]. Moreover, the importance of CBP/p300 is underscored by the fact that genetic alterations as well as their functional dysregulation are

Previous studies have shown that CBP and p300 play distinct roles in haematopoiesis and act non-redundantly in microenvironment-mediated haematopoietic regulation in spite of their high homology [38-40]. It has been widely documented that both proteins interact with crucial transcriptional regulators in virtually all haematopoietic lineages. Intriguing‐ ly, CBP/p300 can promote, on one hand, normal differentiation and cell cycle arrest (by cooperating with GATA-1) and, on the other hand, cell cycle progression and transforma‐ tion by cooperating with c-Myb and PU.1, an Ets family transcription factor. It is conceiv‐ able that an overexpressed oncoprotein might compete with differentiation-inducing factors for CBP/p300 function. Furthermore, during normal development, CBP/p300 could differentially partition among transcriptional regulators with opposing functions, thus controlling the balance between proliferation and differentiation. As an example, the downmodulation of the proto-oncoproteins PU.1 and c-Myb during the erythroleukaemia MEL cell line maturation might increase availability of CBP/p300 for differentiation-associated factors such as GATA-1, NF-E2 and EKLF. Moreover, besides the involvement in eryth‐ roid cell lineage differentiation, CBP and, very likely, p300 target a broad range of myeloid

Because of its central role in transcription, it is not surprising that aberrations in *CREBBP* can affect many tissues [17]. In humans, chromosomal translocations involving the *CREBBP* gene have been observed in leukaemia and myelodysplastic syndrome [38]. Mutations of *CREBBP* in the germline have been associated to the Rubinstein-Taybi syndrome (RTS), an autosomal dominant disease characterized by mental retardation, skeletal abnormalities and a high

strongly linked to human diseases [36, 37].

558 Cancer Treatment - Conventional and Innovative Approaches

and lymphoid expressed transcription factors [38-40].

CREB is a multi-functional transcriptional activator that is involved in many signalling pathways under normal and pathologic conditions. CREB mediates its transcriptional responses following phosphorylation at Ser133 [7] and the consequent association with the 256 kDa co-activator CBP [28] or related family members like p300 [29]. Both Ser133 phosphory‐ lation and CBP association play an essential role for gene transactivation mediated by an octanucleotide CRE consensus sequence placed in the promoters of many cellular genes [29]. In more detail, CREB transactivation domain, that is the site able to interact with other nuclear factors, contains a constitutive glutamine rich domain termed Q2 and an inducible domain, termed the kinase-inducible domain (KID), regulated by cellular kinases [2]. The Q2 domain interacts with a TATA binding protein-associated factor and is constitutively active; instead, the KID region promotes isomerization by recruiting the co-activator factors CBP and p300 to the gene promoters and is active only when it is phosphorylated at Ser133 by a variety of cellular kinases. Recent studies using a genome-wide analysis showed that the number of putative target genes for CREB is about 5000, among which immediate-early genes, including *c-FOS*, *AP-1/JunB* and early growth response protein 1 (*EGR-1*) [44], as well as genes crucially involved in the cell cycle machinery, namely *Cyclin A1* and *D1* [7]. In this respect, it has been found that Cyclin A is up-regulated in cell lines, transgenic mice and patient bone marrow that show increased CREB levels [44]. It is still to unravel whether this occurs through a direct or indirect mechanism. To address this issue or, in other words, to determine whether CREB overexpression results in target gene activation through increased occupancy of binding sites or by altering levels of Ser133 phosphorylation, several authors proposed to use chromatin immunoprecipitation assays. Moreover, microarray analysis of potential CREB target genes will help in understanding the downstream pathways through which CREB contributes to normal and aberrant haematopoiesis. By interacting with its huge number of target genes CREB plays a critical role in the regulation of various biological processes including haema‐ topoiesis, liver gluconeogenesis, pituitary gland physiology, circadian rhythm, spermatogen‐ esis, learning and memory [1, 45, 46]. Concerning haematopoiesis, CREB is a downstream target of haematopoietic growth factor signalling activated by granulocyte-macrophage– colony stimulating factor (GM-CSF) and interleukin-3 (IL-3), thus resulting a crucial factor for normal myelopoiesis [44]. In addition, it appears to play a role in primary erythroblast differentiation [47] as well as in megacaryocyte differentiation where it is activated in a MAPKdependent manner [48]. More recently, it has also been involved in HSC and uncommitted progenitor proliferation and survival through its effects on cell cycle control [45, 46]. A growing body of evidences is unravelling the role of CREB in the regulation of the immune system [49]. Indeed, several immune-related genes contain a cAMP responsive element, as in the case of IL-2, IL-6, IL-10 and TNF-α. In monocytes and macrophages CREB exerts anti-apoptotic survival effects. Moreover, CREB promotes normal B and T cell survival and proliferation when it is phosphorylated in response to signalling by the B-cell receptor or different kinases [49]. Particularly well characterized is the regulatory role that CREB plays in the nervous system. Actually, numerous papers have demonstrated CREB involvement in promoting neuronal survival, precursor proliferation, neurite outgrowth and neuronal differentiation in certain neuronal populations [50], highlighting the importance of CREB signalling in learning and memory processes in several organisms [2, 51].

kinase that targets Ser133 in many processes [1, 3]. Other signalling molecules responsible for CREB Ser133 phosphorylation include mitogen- and stress-activated kinase 1 (MSK-1), extracellular signal-regulated kinase (ERK), calcium/calmodulin-dependent kinases (CaMKs), p90 ribosomal S6 kinase (RSK), MAPKs and Akt/protein kinase B (PKB) [1, 3, 7, 55, 56]. Both MAPK and Akt have been shown to enhance the survival of cultured cells by stimulating CREB-dependent target gene expression [56]. CREB activity is also regulated by a family of cytoplasmic co-activators known as transducers of regulated CREB activity (TORCs) and including TORC1, TORC2 and TORC3. TORCs are activated by extracellular stimuli repre‐ sented by nutrients (glucose) and hormones. Once activated, they translocate into the nucleus where they bind to the bZIP domain of CREB exerting its activation through a phospho-Ser133 independent mechanism. All TORCs are regarded as strong activators of CREB-dependent

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In Fig. 1 the main factors and signalling molecules leading to CREB activation in haemato‐

**Figure 1.** Schematic representation of the main factors and signalling molecules leading to CREB activation in haema‐ topoietic cells. A various array of extracellular stimuli promote CREB activation through phosphorylation or through interaction with CREB co-activators to enhance the expression of CREB responsive genes. CREB target genes, including Cyclin A1, are able to mediate effects on cellular proliferation, apoptosis, survival and differentiation. PLC: phospholi‐

transcription [57].

poietic cells are schematically represented.

pase-C; DAG: 1,2-diacylglycerol; PKC-ε: protein kinase C-ε.

In the late 1980s, it was discovered that cAMP mediates the hormonal stimulation of several cellular processes by regulating the phosphorylation of critical proteins among which CREB transcription factor [52]. Although it was initially identified as a target of the cAMP signalling pathway, studies on activation of immediate-early genes revealed that CREB is a substrate for kinases other than cAMP-dependent protein kinase A (PKA) and that various signalling routes converge on CREB and CREM, controlling their function by modulating their phosphorylation states [52, 53]. As above mentioned, almost all the signalling pathways that activate CREB lead to the phosphorylation of Ser133, which is required for CREB-induced gene transcription, but additional sites on CREB or on linked proteins can be phosphorylated exerting a modulation of CREB activity [35]. For example, Ser133 phosphorylation primes CREB for phosphorylation by Glycogen synthase kinase 3 (GSK-3) at Ser129. However, unlike Ser133 phosphorylation, the physiologic consequences of Ser129 phosphorylation are not well defined, although evidence suggests that it is also linked to CREB activation [54]. In different systems a number of different kinases have been shown to stimulate CREB phosphorylation and several CREB kinase candidates have been identified so far. PKA, which is activated by cAMP, is the major kinase that targets Ser133 in many processes [1, 3]. Other signalling molecules responsible for CREB Ser133 phosphorylation include mitogen- and stress-activated kinase 1 (MSK-1), extracellular signal-regulated kinase (ERK), calcium/calmodulin-dependent kinases (CaMKs), p90 ribosomal S6 kinase (RSK), MAPKs and Akt/protein kinase B (PKB) [1, 3, 7, 55, 56]. Both MAPK and Akt have been shown to enhance the survival of cultured cells by stimulating CREB-dependent target gene expression [56]. CREB activity is also regulated by a family of cytoplasmic co-activators known as transducers of regulated CREB activity (TORCs) and including TORC1, TORC2 and TORC3. TORCs are activated by extracellular stimuli repre‐ sented by nutrients (glucose) and hormones. Once activated, they translocate into the nucleus where they bind to the bZIP domain of CREB exerting its activation through a phospho-Ser133 independent mechanism. All TORCs are regarded as strong activators of CREB-dependent transcription [57].

involved in the cell cycle machinery, namely *Cyclin A1* and *D1* [7]. In this respect, it has been found that Cyclin A is up-regulated in cell lines, transgenic mice and patient bone marrow that show increased CREB levels [44]. It is still to unravel whether this occurs through a direct or indirect mechanism. To address this issue or, in other words, to determine whether CREB overexpression results in target gene activation through increased occupancy of binding sites or by altering levels of Ser133 phosphorylation, several authors proposed to use chromatin immunoprecipitation assays. Moreover, microarray analysis of potential CREB target genes will help in understanding the downstream pathways through which CREB contributes to normal and aberrant haematopoiesis. By interacting with its huge number of target genes CREB plays a critical role in the regulation of various biological processes including haema‐ topoiesis, liver gluconeogenesis, pituitary gland physiology, circadian rhythm, spermatogen‐ esis, learning and memory [1, 45, 46]. Concerning haematopoiesis, CREB is a downstream target of haematopoietic growth factor signalling activated by granulocyte-macrophage– colony stimulating factor (GM-CSF) and interleukin-3 (IL-3), thus resulting a crucial factor for normal myelopoiesis [44]. In addition, it appears to play a role in primary erythroblast differentiation [47] as well as in megacaryocyte differentiation where it is activated in a MAPKdependent manner [48]. More recently, it has also been involved in HSC and uncommitted progenitor proliferation and survival through its effects on cell cycle control [45, 46]. A growing body of evidences is unravelling the role of CREB in the regulation of the immune system [49]. Indeed, several immune-related genes contain a cAMP responsive element, as in the case of IL-2, IL-6, IL-10 and TNF-α. In monocytes and macrophages CREB exerts anti-apoptotic survival effects. Moreover, CREB promotes normal B and T cell survival and proliferation when it is phosphorylated in response to signalling by the B-cell receptor or different kinases [49]. Particularly well characterized is the regulatory role that CREB plays in the nervous system. Actually, numerous papers have demonstrated CREB involvement in promoting neuronal survival, precursor proliferation, neurite outgrowth and neuronal differentiation in certain neuronal populations [50], highlighting the importance of CREB signalling in learning

In the late 1980s, it was discovered that cAMP mediates the hormonal stimulation of several cellular processes by regulating the phosphorylation of critical proteins among which CREB transcription factor [52]. Although it was initially identified as a target of the cAMP signalling pathway, studies on activation of immediate-early genes revealed that CREB is a substrate for kinases other than cAMP-dependent protein kinase A (PKA) and that various signalling routes converge on CREB and CREM, controlling their function by modulating their phosphorylation states [52, 53]. As above mentioned, almost all the signalling pathways that activate CREB lead to the phosphorylation of Ser133, which is required for CREB-induced gene transcription, but additional sites on CREB or on linked proteins can be phosphorylated exerting a modulation of CREB activity [35]. For example, Ser133 phosphorylation primes CREB for phosphorylation by Glycogen synthase kinase 3 (GSK-3) at Ser129. However, unlike Ser133 phosphorylation, the physiologic consequences of Ser129 phosphorylation are not well defined, although evidence suggests that it is also linked to CREB activation [54]. In different systems a number of different kinases have been shown to stimulate CREB phosphorylation and several CREB kinase candidates have been identified so far. PKA, which is activated by cAMP, is the major

and memory processes in several organisms [2, 51].

560 Cancer Treatment - Conventional and Innovative Approaches

In Fig. 1 the main factors and signalling molecules leading to CREB activation in haemato‐ poietic cells are schematically represented.

**Figure 1.** Schematic representation of the main factors and signalling molecules leading to CREB activation in haema‐ topoietic cells. A various array of extracellular stimuli promote CREB activation through phosphorylation or through interaction with CREB co-activators to enhance the expression of CREB responsive genes. CREB target genes, including Cyclin A1, are able to mediate effects on cellular proliferation, apoptosis, survival and differentiation. PLC: phospholi‐ pase-C; DAG: 1,2-diacylglycerol; PKC-ε: protein kinase C-ε.

### **5. CREB family members and leukemogenesis**

Recent data suggest that CREB acts as a proto-oncogene in haematopoietic cells and contributes to the leukaemia phenotype [37, 38, 45, 46]. It has been shown anyway that CREB is able to promote tumour formation only when other oncogenes are also activated. In fact, its overex‐ pression is not sufficient to induce acute leukaemia *in vivo*. This is consistent with similar observations obtained with translocations, such as AML1-ETO (Acute Myeloid Leukaemia Eight-Twenty-One), a chimeric protein that requires additional mutations to develop leukae‐ mia in mouse models [58]. In previous works different strategies have been delineated to identify the oncogenes cooperating with CREB to drive leukemogenesis: one way is repre‐ sented by crossing different transgenic mice of known oncogenes such as *K-RAS*, *MEIS 1*, *PML/ RAR*α etc. to *CREB* transgenic mice; another approach consists in infecting *CREB* transgenics with a retrovirus such as the Molony murine leukaemia virus to insertionally activate coop‐ erating oncogenes. The latter approach has also the potential to identify novel collaborators of CREB besides the already known CBP and p300. Identifying novel oncogenic alterations that cause leukaemia and discovering the signalling pathways involved will be of great value to gain a better knowledge of the disease and to lead to novel and more efficient therapeutic measures.

expressing CREB at elevated levels show an increased growth/proliferation rate in nor‐ mal conditions and an increased survival when exposed to stress like serum starvation [61]. On the contrary, down-regulation of endogenous CREB in leukaemia cell lines by siRNA resulted in reduced cell viability [20, 45], indicating that CREB is a critical regulator of growth and survival in both myeloid and lymphoid leukaemia cells. Unfortunately, chromosomal translocations have also been involved in drug-induced leukaemia. For instance, the involvement of 11q23-balanced translocations in acute leukaemia after treatment with drugs that inhibit the function of DNA topoisomerase II (topo II) is being recognized with increasing frequency. It has been shown that the gene at 11q23, involved in all of these treatment-related leukaemias, is *MLL* (also called ALL1, Htrx, and HRX). In general, the translocations occurring in these leukaemias are the same as those occurring in *de novo* leukaemia [eg. t(9;11), t(11;19), and t(4;11)]. Interestingly, the t(11;16)(q23;p13.3) has been cloned and has been shown to involve both *MLL* and *CREBBP* [62]. Besides chromosomal translocations, another way for CREB to contribute to tumorigenesis is through the suppression of cellular genes either by competing with or binding to sites occupied by other transcription factors or by confiscating the transcriptional machinery [63].

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Acute leukaemia derives from the clonal expansion of haematopoietic stem/progenitor cells that have lost their ability to undergo terminal differentiation. Since transcription factors control HSC production and differentiation, it is conceivable that disorders of the haemato‐ poietic system often involve alterations of the regulatory network of transcription factors. In haematological malignancies transcription factors can be overexpressed, involved in chromo‐ somal translocations or become targets of somatic mutations that disrupt their normal function [37, 60-63]. Previous studies have demonstrated that *CREB* is a proto-oncogene whose overexpression promotes cellular proliferation in haematopoietic cells [1, 3]. The abnormal proliferation and survival of myeloid cells *in vitro* and *in vivo* appears to be due to the upregulation of CREB target genes such as *Cyclin A1* [60, 63]. Transgenic mice that overexpress CREB in myeloid cells develop a myeloproliferative disease with splenomegaly and aberrant myelopoiesis. However, CREB overexpressing mice do not spontaneously develop acute myeloid leukaemia (AML) [61]. To identify genes that accelerate leukaemia in CREB transgenic mice retroviral insertional mutagenesis has been used. The mutagenesis screen identified several integration sites, including oncogenes *Gfi1*, *Myb*, and *Ras*. Among transcription factors, *Sox4* was identified with the screen as a gene that cooperates with *CREB* in myeloid leukemo‐ genesis by contributing to increased proliferation of haematopoietic progenitor cells [64]. Moreover, chromatin immunoprecipitation assays have demonstrated that *CREB* is a direct target of *Sox4*. In fact, it has been shown that the transduction of *CREB* transgenic mouse bone marrow cells with a Sox4 retrovirus increases survival and self-renewal of cells *in vitro* and results in increased expression of CREB target genes. Consistently, leukaemia blasts from the majority of AML patients have higher levels of CREB, phospho-CREB, and Sox4 protein expression in the bone marrow [64]. The increase in both CREB protein and mRNA levels in primary AML cells is possibly due to *CREB* gene amplification in the blast cells. Furthermore, a higher level of CREB has been found to correlate with a less favourable prognosis and an

**5.1. Acute myeloid leukaemia**

Several CREB family members have been implicated in different malignant conditions. The first malignancy to be discovered was the clear cell sarcoma of the soft tissue (CSST). In this solid tumour, the cells are induced to proliferation by an Ewing's Sarcoma (EWS)- ATF-1 fusion oncoprotein derived by a chromosomal translocation that fuses the DNAbinding and bZip domain of ATF-1 to the EWS gene. In haematological malignancies, CREB has been implicated in the pathogenesis of human T lymphotropic virus I (HTLV-I) related T-cell leukaemia [59] and also associated with the genesis of follicular lymphoma, where CREB binds to the CRE site in the promoter of translocated Bcl-2 [46]. Other leukaemiaassociated chromosomal translocations involving the CBP and p300 genes were also linked to haematological malignancies. These translocations generally result in fusion products that preserve most of the CBP and p300 molecules, suggesting that the disease mecha‐ nism does not simply involve loss of function of CBP, as is the case in Rubinstein-Taybi syndrome, but often implies an altered cofactor function (dominant positive or dominant negative) through fusion to another molecule. The most frequent chromosomal transloca‐ tions targeting *CREBBP* and *EP300* have been described in specific subtypes of myeloid leukaemia and are represented by Mixed-Lineage Leukaemia 1 (*MLL)-EP300*, *MLL-CREBBP*, *MOZ-CREBBP* and *MOZ-EP300* [37, 60]. Interestingly, most translocations involving CREB-related genes result in leukaemia of the myeloid/monocytic lineage, highlighting the importance of CREB and CREB-interacting proteins in the regulation of haematopoietic cell differentiation and proliferation [45, 46]. Actually, previous work demonstrated that bone marrow cells from patients with acute myeloid or lymphoid leukaemia expressed higher levels of CREB compared to patients not affected by leukae‐ mia or with normal bone marrow [60]. Moreover, it appears that an elevated CREB expression is associated with an increased risk of relapse or persistent disease and decreased event-free survival [45]. This is consistent with the observation that leukaemia cell lines expressing CREB at elevated levels show an increased growth/proliferation rate in nor‐ mal conditions and an increased survival when exposed to stress like serum starvation [61]. On the contrary, down-regulation of endogenous CREB in leukaemia cell lines by siRNA resulted in reduced cell viability [20, 45], indicating that CREB is a critical regulator of growth and survival in both myeloid and lymphoid leukaemia cells. Unfortunately, chromosomal translocations have also been involved in drug-induced leukaemia. For instance, the involvement of 11q23-balanced translocations in acute leukaemia after treatment with drugs that inhibit the function of DNA topoisomerase II (topo II) is being recognized with increasing frequency. It has been shown that the gene at 11q23, involved in all of these treatment-related leukaemias, is *MLL* (also called ALL1, Htrx, and HRX). In general, the translocations occurring in these leukaemias are the same as those occurring in *de novo* leukaemia [eg. t(9;11), t(11;19), and t(4;11)]. Interestingly, the t(11;16)(q23;p13.3) has been cloned and has been shown to involve both *MLL* and *CREBBP* [62]. Besides chromosomal translocations, another way for CREB to contribute to tumorigenesis is through the suppression of cellular genes either by competing with or binding to sites occupied by other transcription factors or by confiscating the transcriptional machinery [63].

#### **5.1. Acute myeloid leukaemia**

**5. CREB family members and leukemogenesis**

562 Cancer Treatment - Conventional and Innovative Approaches

measures.

Recent data suggest that CREB acts as a proto-oncogene in haematopoietic cells and contributes to the leukaemia phenotype [37, 38, 45, 46]. It has been shown anyway that CREB is able to promote tumour formation only when other oncogenes are also activated. In fact, its overex‐ pression is not sufficient to induce acute leukaemia *in vivo*. This is consistent with similar observations obtained with translocations, such as AML1-ETO (Acute Myeloid Leukaemia Eight-Twenty-One), a chimeric protein that requires additional mutations to develop leukae‐ mia in mouse models [58]. In previous works different strategies have been delineated to identify the oncogenes cooperating with CREB to drive leukemogenesis: one way is repre‐ sented by crossing different transgenic mice of known oncogenes such as *K-RAS*, *MEIS 1*, *PML/ RAR*α etc. to *CREB* transgenic mice; another approach consists in infecting *CREB* transgenics with a retrovirus such as the Molony murine leukaemia virus to insertionally activate coop‐ erating oncogenes. The latter approach has also the potential to identify novel collaborators of CREB besides the already known CBP and p300. Identifying novel oncogenic alterations that cause leukaemia and discovering the signalling pathways involved will be of great value to gain a better knowledge of the disease and to lead to novel and more efficient therapeutic

Several CREB family members have been implicated in different malignant conditions. The first malignancy to be discovered was the clear cell sarcoma of the soft tissue (CSST). In this solid tumour, the cells are induced to proliferation by an Ewing's Sarcoma (EWS)- ATF-1 fusion oncoprotein derived by a chromosomal translocation that fuses the DNAbinding and bZip domain of ATF-1 to the EWS gene. In haematological malignancies, CREB has been implicated in the pathogenesis of human T lymphotropic virus I (HTLV-I) related T-cell leukaemia [59] and also associated with the genesis of follicular lymphoma, where CREB binds to the CRE site in the promoter of translocated Bcl-2 [46]. Other leukaemiaassociated chromosomal translocations involving the CBP and p300 genes were also linked to haematological malignancies. These translocations generally result in fusion products that preserve most of the CBP and p300 molecules, suggesting that the disease mecha‐ nism does not simply involve loss of function of CBP, as is the case in Rubinstein-Taybi syndrome, but often implies an altered cofactor function (dominant positive or dominant negative) through fusion to another molecule. The most frequent chromosomal transloca‐ tions targeting *CREBBP* and *EP300* have been described in specific subtypes of myeloid leukaemia and are represented by Mixed-Lineage Leukaemia 1 (*MLL)-EP300*, *MLL-CREBBP*, *MOZ-CREBBP* and *MOZ-EP300* [37, 60]. Interestingly, most translocations involving CREB-related genes result in leukaemia of the myeloid/monocytic lineage, highlighting the importance of CREB and CREB-interacting proteins in the regulation of haematopoietic cell differentiation and proliferation [45, 46]. Actually, previous work demonstrated that bone marrow cells from patients with acute myeloid or lymphoid leukaemia expressed higher levels of CREB compared to patients not affected by leukae‐ mia or with normal bone marrow [60]. Moreover, it appears that an elevated CREB expression is associated with an increased risk of relapse or persistent disease and decreased event-free survival [45]. This is consistent with the observation that leukaemia cell lines Acute leukaemia derives from the clonal expansion of haematopoietic stem/progenitor cells that have lost their ability to undergo terminal differentiation. Since transcription factors control HSC production and differentiation, it is conceivable that disorders of the haemato‐ poietic system often involve alterations of the regulatory network of transcription factors. In haematological malignancies transcription factors can be overexpressed, involved in chromo‐ somal translocations or become targets of somatic mutations that disrupt their normal function [37, 60-63]. Previous studies have demonstrated that *CREB* is a proto-oncogene whose overexpression promotes cellular proliferation in haematopoietic cells [1, 3]. The abnormal proliferation and survival of myeloid cells *in vitro* and *in vivo* appears to be due to the upregulation of CREB target genes such as *Cyclin A1* [60, 63]. Transgenic mice that overexpress CREB in myeloid cells develop a myeloproliferative disease with splenomegaly and aberrant myelopoiesis. However, CREB overexpressing mice do not spontaneously develop acute myeloid leukaemia (AML) [61]. To identify genes that accelerate leukaemia in CREB transgenic mice retroviral insertional mutagenesis has been used. The mutagenesis screen identified several integration sites, including oncogenes *Gfi1*, *Myb*, and *Ras*. Among transcription factors, *Sox4* was identified with the screen as a gene that cooperates with *CREB* in myeloid leukemo‐ genesis by contributing to increased proliferation of haematopoietic progenitor cells [64]. Moreover, chromatin immunoprecipitation assays have demonstrated that *CREB* is a direct target of *Sox4*. In fact, it has been shown that the transduction of *CREB* transgenic mouse bone marrow cells with a Sox4 retrovirus increases survival and self-renewal of cells *in vitro* and results in increased expression of CREB target genes. Consistently, leukaemia blasts from the majority of AML patients have higher levels of CREB, phospho-CREB, and Sox4 protein expression in the bone marrow [64]. The increase in both CREB protein and mRNA levels in primary AML cells is possibly due to *CREB* gene amplification in the blast cells. Furthermore, a higher level of CREB has been found to correlate with a less favourable prognosis and an increased risk of relapse and decreased event-free survival in a small cohort of AML patients [45, 61]. Generally, AML in adults has a 20% five-year disease free survival despite treatment with aggressive cytotoxic chemotherapy and two thirds of AML patients do not experience significant periods of remission. Therefore, in light of its important role in the pathogenesis of leukaemia, CREB has been indicated as a potential prognostic marker of disease progression in AML and a molecular target for future treatment of leukaemia.

ferative neoplasm within 9-12 months of age. They are also hypersensitive to ionizing radiation and show a marked decrease in poly(ADP-ribose) polymerase-1 activity after irradiation. In addition, protein levels of XRCC1 (X-ray repair complementing defective repair in Chinese hamster cells 1) and APEX1 (APEX nuclease), key components of base excision repair machi‐ nery, are reduced in un-irradiated *CREBBP*(+/-) cells or upon targeted knockdown of *CREBBP* levels. These results provide validation of a new myelodysplastic/myeloproliferative neo‐ plasm mouse model and, more importantly, point at a defective repair of DNA damage as a

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Acute lymphoblastic leukaemia (ALL) is a heterogeneous disease characterized by the predominance of immature haematopoietic cells, in which malignant cells express phenotypes of either T-cell or B-cell lineages [61]. ALLs account for the 25-30% of all cancer diagnoses in children. CREB involvement in the molecular events related to *in vitro* and *in vivo* lympho‐ blastic proliferation is still little known, whereas a lot of evidences disclose a role of *CREB* as a proto-oncogene in haematopoiesis and in AML. *CREB* can be overexpressed in the 84% of ALL patients (73/86) at diagnosis but neither in remission nor in non-leukaemia samples [70]. By contrast, the parallel expression of the cAMP early inducible repressor (*ICER*), which represses CREB activity by competing for the CRE consensus site, appears down-regulated at diagnosis but neither in remission nor in control samples [70]. Thus, it is presumable that *CREB* overexpression leads to target gene up-regulation and increase in cell proliferation and survival that are not counteracted by the insufficient level of *ICER* expression. Besides this hypothesis, Pigazzi et al. [71] have also demonstrated the co-expression of miR34b in *CREB* overexpressing myeloid leukaemia cells providing new information about myeloid transfor‐ mation and therapeutic strategies. Despite the apparently good prognosis, the 15% of high hyper-diploid (HD) childhood ALL cases relapse [72, 73]. Relapsed ALL is a leading cause of death due to disease in young people, but the molecular mechanisms of treatment failure are still to be elucidated. Genome-wide profiling of structural DNA alterations in ALL identified multiple sub-microscopic somatic mutations targeting key cellular pathways and demonstrat‐ ed evolution in genetic alterations from diagnosis to relapse [74]. Many of the mutations that have been identified concern the transcriptional co-activators *CREBBP* and *NCOR1*, the transcription factors *ERG*, *SPI1*, *TCF4* and *TCF7L2*, components of the Ras signalling pathway, histone genes, genes involved in histone modification (*CREBBP* and *CTCF*) and genes target of DNA copy number alterations [74]. The parallel analysis of an extended cohort of diagnosisrelapsed cases and acute leukaemia cases that did not relapse showed that the 18.3% of relapsed cases had sequence or deletion mutations of *CREBBP* [72, 74]. *CREBBP* is expressed in leukaemia cells and normal B-cell progenitors, and the mutant *CREBBP* alleles are expressed in ALL cell lines harbouring mutations. Mutations at diagnosis or acquired at relapse consist in truncated alleles or deleterious substitutions in conserved residues of the histone acetyl‐ transferase domain, impairing histone acetylation and transcriptional regulation of *CREBBP* targets, including glucocorticoid responsive genes. In mice the homozygous deletion of *CREBBP* or *EP300* is lethal due to developmental abnormalities whereas *CREBBP*(+/−) mice show defects in B lymphoid development and an increased incidence of haematopoietic

contributing factor to the pathogenesis of this currently incurable disease [46].

**5.2. Acute lymphoblastic leukaemia**

Clinical and experimental findings underline that AML is induced by numerous functionally cooperating genetic alterations, including chromosomal translocations that lead to the expression of fusion proteins often behaving as aberrant transcription factors. Several AMLassociated lesions target chromatin regulators like histone methyltransferases or histone acetyltransferases, including MLL1 or CBP/p300 [65]. As already mentioned, CBP is an adapter protein that is involved in regulating transcription and histone acetylation, through which it is thought to contribute to an increased level of gene expression. The *CBP* gene was recently identified as a partner gene in the t(8;16) that occurs in *de novo* acute myelomonocytic leukae‐ mia (AML-M4) and rarely in treatment-related AML [66]. The fusion gene could alter the CBP protein so that it becomes constitutively active or, alternatively, it could modify the chromatinassociation functions of *MLL* gene [38, 40]. *MLL* and *HOXB4*, a member of the homeobox domain transcription factors, have been identified as regulators of HSC maturation during early haematopoiesis [67]. *HOXB4* belongs to the *HOX* genes, a family of oncogenes implicated in the pathogenesis of various human cancers and highly expressed in the majority of AML. In a recent report Wang et al. [54] demonstrated the association of CREB and its co-activators TORC and CBP with homeodomain protein MEIS1, a HOX DNA-binding cofactor and critical downstream mediator of the *MLL* oncogenic program. This MEIS-CREB nexus is regulated by GSK-3, a multi-functional serine/threonine kinase that impairs the proliferation and induces the differentiation of a variety of cancers, including leukaemias, induced by *MLL* oncogenes. This kinase mediates CREB activation through phosphorylation at Ser129. In fact, CREB Ser129 mutation antagonizes *HOX/MEIS* activity and decreases colony-forming abilities of *HOX/ MEIS* or *MLL* transformed cells. These and other similar observations provide a molecular rationale for targeting *HOX*-associated transcription through GSK-3 inhibition in a subset of leukaemias.

Myelodysplastic syndromes (MDS) include a heterogeneous group of clonal haematopoietic stem cell malignancies with significant morbidity and high mortality. The incidence of MDS increases markedly with age and the disease is most prevalent in individuals who are white and male. Because of an ageing population and an improving awareness of the disease, the documented disease burden is expected to worsen in the near future. Due to the poor survival of individuals with MDS, it is important to identify prognostic factors to better risk-stratify patients for more effective treatments [68]. Genomic instability is associated with progression of the disease so that a part of patients develops AML. It has been reported that an increased incidence of haematological malignancies occurs in *CREBBP* heterozygous mice and other authors have shown that *CREBBP* is one of the genes altered by chromosomal translocations in patients suffering from therapy-related myelodysplastic syndrome [69]. Moreover, it has been demonstrated that *CREBBP*(+/-) mice invariably develop myelodysplastic/myeloproli‐ ferative neoplasm within 9-12 months of age. They are also hypersensitive to ionizing radiation and show a marked decrease in poly(ADP-ribose) polymerase-1 activity after irradiation. In addition, protein levels of XRCC1 (X-ray repair complementing defective repair in Chinese hamster cells 1) and APEX1 (APEX nuclease), key components of base excision repair machi‐ nery, are reduced in un-irradiated *CREBBP*(+/-) cells or upon targeted knockdown of *CREBBP* levels. These results provide validation of a new myelodysplastic/myeloproliferative neo‐ plasm mouse model and, more importantly, point at a defective repair of DNA damage as a contributing factor to the pathogenesis of this currently incurable disease [46].

#### **5.2. Acute lymphoblastic leukaemia**

increased risk of relapse and decreased event-free survival in a small cohort of AML patients [45, 61]. Generally, AML in adults has a 20% five-year disease free survival despite treatment with aggressive cytotoxic chemotherapy and two thirds of AML patients do not experience significant periods of remission. Therefore, in light of its important role in the pathogenesis of leukaemia, CREB has been indicated as a potential prognostic marker of disease progression

Clinical and experimental findings underline that AML is induced by numerous functionally cooperating genetic alterations, including chromosomal translocations that lead to the expression of fusion proteins often behaving as aberrant transcription factors. Several AMLassociated lesions target chromatin regulators like histone methyltransferases or histone acetyltransferases, including MLL1 or CBP/p300 [65]. As already mentioned, CBP is an adapter protein that is involved in regulating transcription and histone acetylation, through which it is thought to contribute to an increased level of gene expression. The *CBP* gene was recently identified as a partner gene in the t(8;16) that occurs in *de novo* acute myelomonocytic leukae‐ mia (AML-M4) and rarely in treatment-related AML [66]. The fusion gene could alter the CBP protein so that it becomes constitutively active or, alternatively, it could modify the chromatinassociation functions of *MLL* gene [38, 40]. *MLL* and *HOXB4*, a member of the homeobox domain transcription factors, have been identified as regulators of HSC maturation during early haematopoiesis [67]. *HOXB4* belongs to the *HOX* genes, a family of oncogenes implicated in the pathogenesis of various human cancers and highly expressed in the majority of AML. In a recent report Wang et al. [54] demonstrated the association of CREB and its co-activators TORC and CBP with homeodomain protein MEIS1, a HOX DNA-binding cofactor and critical downstream mediator of the *MLL* oncogenic program. This MEIS-CREB nexus is regulated by GSK-3, a multi-functional serine/threonine kinase that impairs the proliferation and induces the differentiation of a variety of cancers, including leukaemias, induced by *MLL* oncogenes. This kinase mediates CREB activation through phosphorylation at Ser129. In fact, CREB Ser129 mutation antagonizes *HOX/MEIS* activity and decreases colony-forming abilities of *HOX/ MEIS* or *MLL* transformed cells. These and other similar observations provide a molecular rationale for targeting *HOX*-associated transcription through GSK-3 inhibition in a subset of

Myelodysplastic syndromes (MDS) include a heterogeneous group of clonal haematopoietic stem cell malignancies with significant morbidity and high mortality. The incidence of MDS increases markedly with age and the disease is most prevalent in individuals who are white and male. Because of an ageing population and an improving awareness of the disease, the documented disease burden is expected to worsen in the near future. Due to the poor survival of individuals with MDS, it is important to identify prognostic factors to better risk-stratify patients for more effective treatments [68]. Genomic instability is associated with progression of the disease so that a part of patients develops AML. It has been reported that an increased incidence of haematological malignancies occurs in *CREBBP* heterozygous mice and other authors have shown that *CREBBP* is one of the genes altered by chromosomal translocations in patients suffering from therapy-related myelodysplastic syndrome [69]. Moreover, it has been demonstrated that *CREBBP*(+/-) mice invariably develop myelodysplastic/myeloproli‐

in AML and a molecular target for future treatment of leukaemia.

564 Cancer Treatment - Conventional and Innovative Approaches

leukaemias.

Acute lymphoblastic leukaemia (ALL) is a heterogeneous disease characterized by the predominance of immature haematopoietic cells, in which malignant cells express phenotypes of either T-cell or B-cell lineages [61]. ALLs account for the 25-30% of all cancer diagnoses in children. CREB involvement in the molecular events related to *in vitro* and *in vivo* lympho‐ blastic proliferation is still little known, whereas a lot of evidences disclose a role of *CREB* as a proto-oncogene in haematopoiesis and in AML. *CREB* can be overexpressed in the 84% of ALL patients (73/86) at diagnosis but neither in remission nor in non-leukaemia samples [70]. By contrast, the parallel expression of the cAMP early inducible repressor (*ICER*), which represses CREB activity by competing for the CRE consensus site, appears down-regulated at diagnosis but neither in remission nor in control samples [70]. Thus, it is presumable that *CREB* overexpression leads to target gene up-regulation and increase in cell proliferation and survival that are not counteracted by the insufficient level of *ICER* expression. Besides this hypothesis, Pigazzi et al. [71] have also demonstrated the co-expression of miR34b in *CREB* overexpressing myeloid leukaemia cells providing new information about myeloid transfor‐ mation and therapeutic strategies. Despite the apparently good prognosis, the 15% of high hyper-diploid (HD) childhood ALL cases relapse [72, 73]. Relapsed ALL is a leading cause of death due to disease in young people, but the molecular mechanisms of treatment failure are still to be elucidated. Genome-wide profiling of structural DNA alterations in ALL identified multiple sub-microscopic somatic mutations targeting key cellular pathways and demonstrat‐ ed evolution in genetic alterations from diagnosis to relapse [74]. Many of the mutations that have been identified concern the transcriptional co-activators *CREBBP* and *NCOR1*, the transcription factors *ERG*, *SPI1*, *TCF4* and *TCF7L2*, components of the Ras signalling pathway, histone genes, genes involved in histone modification (*CREBBP* and *CTCF*) and genes target of DNA copy number alterations [74]. The parallel analysis of an extended cohort of diagnosisrelapsed cases and acute leukaemia cases that did not relapse showed that the 18.3% of relapsed cases had sequence or deletion mutations of *CREBBP* [72, 74]. *CREBBP* is expressed in leukaemia cells and normal B-cell progenitors, and the mutant *CREBBP* alleles are expressed in ALL cell lines harbouring mutations. Mutations at diagnosis or acquired at relapse consist in truncated alleles or deleterious substitutions in conserved residues of the histone acetyl‐ transferase domain, impairing histone acetylation and transcriptional regulation of *CREBBP* targets, including glucocorticoid responsive genes. In mice the homozygous deletion of *CREBBP* or *EP300* is lethal due to developmental abnormalities whereas *CREBBP*(+/−) mice show defects in B lymphoid development and an increased incidence of haematopoietic tumours [75]. Both *CREBBP* and *EP300* sequence mutations have been reported in solid tumours and, more recently, also in haematological malignancies, whereas rare *EP300* mutations have been detected in an ALL cell lines and myelodysplasia [74, 76]. A lot of detected mutations at relapse, the same identified at diagnosis in other clones, prove that mutations confer resistance to therapy. Many identified mutations are target in transcriptional and epigenetic regulation as a mechanism of resistance in ALL. It is worth outlining that the high incidence of *CREBBP* mutations found in relapse-prone HD ALL cases discloses the possibility of a targeted customized treatment in this genetic subgroup [73].

[82]. The same authors previously showed that inhibition of CREB by using RNA interfer‐ ence (RNAi) technology resulted in decreased proliferation and survival of bcr-abl-express‐ ing K562 cells [45, 83], whereas other authors reported that CREB antisense oligonucleotides were able to induce death of human leukaemia cells and bone marrow cells from patients affected with both AML and CML [84]. A critical factor for the genesis of acute leukaemia or acute transformation of CML appears to be the formation of fusion genes between *NUP98* and members of the *HOX* gene family [85]. Interestingly, all the NUP98-HOX-involved fu‐ sion products exhibit dual binding ability to both CREB binding protein, a co-activator, and histone deacetylase 1, a co-repressor, acting as both trans-activators and trans-repressors and contributing to the genesis of acute leukaemia or acute transformation of CML [86].

Role of CREB Protein Family Members in Human Haematological Malignancies

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**Figure 2. a, b: Phospho-CREB localization in Jurkat T cells upon TRAIL treatment.** An evident nuclear translocation of phospho-CREB (green fluorescence) was detected upon 1 h treatment only with the lower dose of TRAIL (panel a), whereas the labelling was located at cytoplasm level upon the higher more cytotoxic dose (panel b). Nuclei were counterstained with 6-diamino-2-phenylindole (DAPI) (blue fluorescence). Green and blue fluorescence single emis‐ sions are overlapped in the merge panels. The insets show green fluorescence single emission. Original magnification:

40X. The figure has been adapted from [20].

In our laboratory we have investigated the role of PI3K/Akt pathway and CREB family members in a number of lymphoid and erythroleukaemia cell lines treated with chemical and physical agents inducing cell death by apoptosis or necrosis [20, 21, 47, 77-80]. We first detected with Western Blotting a high constitutive level of CREB phosphorylation at Ser133 in Jurkat T cells under normal serum culture conditions [20]. Under low serum culture conditions, an early (within 1 h) and transient increase in CREB phosphorylation was observed in response to TRAIL treatment and reduced upon pre-treatment with LY294002 or SB253580, demonstrating the PI3K/Akt- and p38 MAPK-dependency of this effect. Interestingly, both phospho-CREB and phospho-ATF-1 were down-regulated in response to TRAIL treatment of normal primary cells derived from haematopoietic precursors (HUVEC, HEMA), whereas both of them were up-regulated in the neoplastic counterparts (K562 cell line) [20, 21]. The PI3K/Akt pathway dependency of CREB/ATF-1 phosphorylation induced by TRAIL treatment was demonstrated both in primary cells and in leukaemia cell lines of different origin and TRAIL sensitivity, showing that the observed phenomenon is a general feature of TRAIL action in leukaemia [77, 80]. In addition, the observation of CREB cleavage products upon TRAIL/LY294002 combined treatment of sensitive leukaemia cells was consistent with previous reports on other neoplastic cell lines [81] and compatible with the TRAIL-mediated activation of the caspase cascade and cleavage of anti-apoptotic molecules. The parallel analysis with immune fluorescence dem‐ onstrated the nuclear translocation of the phosphorylated form of CREB upon treatment with 100 ng/mL TRAIL, whereas the immune labelling was mainly detectable in the cytoplasm compartment upon the higher more cytotoxic dose (1000 ng/mL) as shown in Fig. 2. A further enhancement of apoptotic cell death was obtained with the use of CREB1 siRNA technology leading us to hypothesize that CREB activation can have an important role in the complex crosstalk among pro- and anti-apoptotic pathways in Jurkat T cells [20, 80].

#### **5.3. Chronic myelogenous leukaemia**

Chronic myelogenous leukaemia (CML) is characterized in the 85-90% of the cases by the presence of the Philadelphia (Ph) chromosome and the *BCR-ABL* fusion gene. A further 5-10% of the cases display other translocations, most commonly complex variants, that in‐ volve one or more chromosomal regions in addition to bands 9q34 and 22q11, but also sim‐ ple variants that typically involve 22q11 and a chromosome other than 9q34. However, genes that cooperate with *BCR-ABL* leading to acute leukaemia are not well understood nei‐ ther the role played by CREB in CML has been clarified. Preliminary observations of the group of Kathleen Sakamoto indicate that CREB is highly expressed in blood and bone mar‐ row cells from patients with CML in chronic phase, but not in normal bone marrow cells [82]. The same authors previously showed that inhibition of CREB by using RNA interfer‐ ence (RNAi) technology resulted in decreased proliferation and survival of bcr-abl-express‐ ing K562 cells [45, 83], whereas other authors reported that CREB antisense oligonucleotides were able to induce death of human leukaemia cells and bone marrow cells from patients affected with both AML and CML [84]. A critical factor for the genesis of acute leukaemia or acute transformation of CML appears to be the formation of fusion genes between *NUP98* and members of the *HOX* gene family [85]. Interestingly, all the NUP98-HOX-involved fu‐ sion products exhibit dual binding ability to both CREB binding protein, a co-activator, and histone deacetylase 1, a co-repressor, acting as both trans-activators and trans-repressors and contributing to the genesis of acute leukaemia or acute transformation of CML [86].

tumours [75]. Both *CREBBP* and *EP300* sequence mutations have been reported in solid tumours and, more recently, also in haematological malignancies, whereas rare *EP300* mutations have been detected in an ALL cell lines and myelodysplasia [74, 76]. A lot of detected mutations at relapse, the same identified at diagnosis in other clones, prove that mutations confer resistance to therapy. Many identified mutations are target in transcriptional and epigenetic regulation as a mechanism of resistance in ALL. It is worth outlining that the high incidence of *CREBBP* mutations found in relapse-prone HD ALL cases discloses the possibility

In our laboratory we have investigated the role of PI3K/Akt pathway and CREB family members in a number of lymphoid and erythroleukaemia cell lines treated with chemical and physical agents inducing cell death by apoptosis or necrosis [20, 21, 47, 77-80]. We first detected with Western Blotting a high constitutive level of CREB phosphorylation at Ser133 in Jurkat T cells under normal serum culture conditions [20]. Under low serum culture conditions, an early (within 1 h) and transient increase in CREB phosphorylation was observed in response to TRAIL treatment and reduced upon pre-treatment with LY294002 or SB253580, demonstrating the PI3K/Akt- and p38 MAPK-dependency of this effect. Interestingly, both phospho-CREB and phospho-ATF-1 were down-regulated in response to TRAIL treatment of normal primary cells derived from haematopoietic precursors (HUVEC, HEMA), whereas both of them were up-regulated in the neoplastic counterparts (K562 cell line) [20, 21]. The PI3K/Akt pathway dependency of CREB/ATF-1 phosphorylation induced by TRAIL treatment was demonstrated both in primary cells and in leukaemia cell lines of different origin and TRAIL sensitivity, showing that the observed phenomenon is a general feature of TRAIL action in leukaemia [77, 80]. In addition, the observation of CREB cleavage products upon TRAIL/LY294002 combined treatment of sensitive leukaemia cells was consistent with previous reports on other neoplastic cell lines [81] and compatible with the TRAIL-mediated activation of the caspase cascade and cleavage of anti-apoptotic molecules. The parallel analysis with immune fluorescence dem‐ onstrated the nuclear translocation of the phosphorylated form of CREB upon treatment with 100 ng/mL TRAIL, whereas the immune labelling was mainly detectable in the cytoplasm compartment upon the higher more cytotoxic dose (1000 ng/mL) as shown in Fig. 2. A further enhancement of apoptotic cell death was obtained with the use of CREB1 siRNA technology leading us to hypothesize that CREB activation can have an important role in the complex

of a targeted customized treatment in this genetic subgroup [73].

566 Cancer Treatment - Conventional and Innovative Approaches

crosstalk among pro- and anti-apoptotic pathways in Jurkat T cells [20, 80].

Chronic myelogenous leukaemia (CML) is characterized in the 85-90% of the cases by the presence of the Philadelphia (Ph) chromosome and the *BCR-ABL* fusion gene. A further 5-10% of the cases display other translocations, most commonly complex variants, that in‐ volve one or more chromosomal regions in addition to bands 9q34 and 22q11, but also sim‐ ple variants that typically involve 22q11 and a chromosome other than 9q34. However, genes that cooperate with *BCR-ABL* leading to acute leukaemia are not well understood nei‐ ther the role played by CREB in CML has been clarified. Preliminary observations of the group of Kathleen Sakamoto indicate that CREB is highly expressed in blood and bone mar‐ row cells from patients with CML in chronic phase, but not in normal bone marrow cells

**5.3. Chronic myelogenous leukaemia**

**Figure 2. a, b: Phospho-CREB localization in Jurkat T cells upon TRAIL treatment.** An evident nuclear translocation of phospho-CREB (green fluorescence) was detected upon 1 h treatment only with the lower dose of TRAIL (panel a), whereas the labelling was located at cytoplasm level upon the higher more cytotoxic dose (panel b). Nuclei were counterstained with 6-diamino-2-phenylindole (DAPI) (blue fluorescence). Green and blue fluorescence single emis‐ sions are overlapped in the merge panels. The insets show green fluorescence single emission. Original magnification: 40X. The figure has been adapted from [20].

#### **5.4. Chronic lymphocytic leukaemia**

Chronic lymphocytic leukaemia (CLL) originates from the abnormal accumulation of antigenstimulated B cells that escape normal cell death mechanisms and/or undergo increased proliferation [87]. CLL is the most prevalent adult leukaemia in the Western world, yet no curative treatment exists. Many studies have explored the use of family-specific cyclic nucleotide phosphodiesterase (PDE) inhibitors in light of the potent effects of cAMP signalling on immune system function [88, 89]. Among the 11 currently known families of cyclic nucleotide PDEs, all but three are capable of catabolizing cAMP and at least 5 PDE families (PDE1-4, PDE7 and PDE8) are expressed in lymphoid cells and regulated by either mitogens or agents that induce cAMP-mediated signalling. Previous work has established that inhibition of PDE4 is sufficient to selectively induce apoptosis in CLL cells by increasing the concentration of cAMP [88]. In a recent paper Meyers et al. [89] examined how CLL cells differ from normal haematopoietic cells with regard to their sensitivity to PDE4 inhibitor-mediated cAMP accumulation, CREB phosphorylation and gene expression. Interestingly, it was discovered that upon exposure to rolipram, a prototypical PDE4 inhibitor, cAMP intracellular levels rapidly rose in both CLL and normal B cells, whereas no such increase was detected in T cells. Likewise, ATF-1/CREB Ser63/133 phosphorylation was induced by rolipram in nearly all CLL and B cells, whereas normal T cells displayed a lower response. Based on these findings and on previous observations of a reduced basal cAMP signalling in CLL cells, the authors suggested the involvement of specific PDE or splice isoforms in the reduced basal apoptotic index of CLL cells [89]. Looking for etiological agents, other authors have identified a stromal cell–derived factor-1 (SDF-1)-dependent mechanism as a microenvironmental regulatory mechanism of CLL cell survival [90]. It is known that SDF-1 is a chemokine that plays an important role in B-cell development. In fact, high levels of SDF-1 are produced by stromal cells within the marrow to retain B-cell precursors in close contact with them, within the supportive haematopoietic microenvironment [91], and to prevent their premature release into the circulation. Upon *in vitro* treatment of CLL cells with synthetic SDF-1α, a rapid and transient activation of p44/42 MAPK (ERK1/2) signalling pathway was observed and related to CLL cell survival. Downward MAPK activation transcription-dependent and -independent mechanisms were involved. In fact, MAPK was able to promote cell survival directly by inactivating the pro-apoptotic BAD protein and indirectly by activating CREB, which, in turn, is important for the transcriptional up-regulation of the anti-apoptotic *BCL-2* gene [92]. Thus, SDF-1 engages B lineage CLL cells through the stromal cell receptor CXCR4 and affects components of the cell death machinery, leading to the noted resistance of CLL cells to apoptosis.

cells. Although the mechanism of HTLV-1 pathogenicity is not fully understood yet, it is widely believed that a virally encoded trans-activator protein, called Tax, is centrally involved in this mechanism. In a recent review Azran et al. [94] provide valuable insights into the molecular mechanisms of HTLV-1 leukemogenesis. In particular, the authors detail the signalling pathways recruited by Tax to set infected T cells into continuous uncontrolled replication and to destabilize their genome, enabling, thereby, accumulation of mutations that can contribute to the leukemogenic process. Tax is able to modulate the expression of many viral genes via the viral long terminal repeat (LTR) and cellular genes through the CREB/ATF-, AP-1-, serum responsive factor (SRF)- and NF-κB-associated pathways, employing the CBP/p300 and p/CAF (p300/CBP-associated factor) co-activators for achieving the full transcriptional activation competence of each of these pathways. It is worth noting that Tax responsive elements (TxRE) contain a centered sequence TGACG(T/A)(C/G)(T/A) that is imperfectly homologous to the consensus cAMP responsive element (CRE; TGACGTCA). Thus, the presence of Tax is necessary for CREB to form a stable complex with the viral CRE. In fact, by interacting with the bZIP region of CREB, Tax enhances CREB dimerization and increases, thereby, its affinity to CRE. In particular, it has been recently shown that CREB is the most prominent factor that cooperates with Tax in activating HTLV-1 LTR region expression [95]. Moreover, it has been demonstrated that while, in the absence of Tax, CREB can activate HTLV-1 LTR expression only if phosphorylated by PKA, another member of the family, namely CREB2, can markedly activate the viral LTR without phosphorylation and can mediate a much stronger activation of the viral LTR by Tax than CREB does [94, 96]. Interestingly, mutant models disrupting Tax activation of the CREB protein resulted in the preferential immortalization of CD8+ lympho‐ cytes, rather than CD4+ lymphocytes, whereas the disruption of Tax interaction with CBP did

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Lymphomas are haematological malignancies of the lymphoid system. Deregulated gene expression is a hallmark of cancer and is well documented in B-cell lymphomas [98]. B cells are particularly susceptible to malignant transformation since the mechanisms involved in antibody diversification can cause chromosomal translocations and oncogenic mutations. Bcell lymphomas include Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (B-NHL). B-NHL consists of a heterogeneous group of diseases whose pathogenesis is associated with multiple genetic lesions affecting oncogenes and tumour-suppressor genes and whose treatment is related to the different grade of malignancy. The most common type of B-NHL is represented by the diffuse large B-cell lymphoma (DLBCL), which generally arises as a clinical evolution of the follicular lymphoma (FL). A number of papers have demonstrated the involvement of CREB family members in the pathogenesis of lymphoma. It has been previ‐ ously found that CREB acts as a positive regulator of the translocated *BCL-2* allele in FLs with the t(14;18) translocation [60] and that the high constitutive expression of *ATF-3* is linked to the viability of Hodgkin/Reed-Sternberg cells and, thus, considered as a molecular hallmark of classical HL [99]. More recently, a number of studies have disclosed the implication of the HAT proteins CBP and p300 as tumour suppressors in B-cell neoplasms [100-102]. Neverthe‐ less, the various mechanisms through which each of these cofactors specifically contributes to

not affect lymphocyte immortalization [97].

**5.6. Lymphoma**

#### **5.5. Human T Lymphotropic Virus 1 (HTLV-1) related T cell leukaemia**

Human T-cell leukaemia virus type-I (HTLV-1) is the first discovered human retrovirus [93]. It has been recognized as the etiological agent of an aggressive malignancy known as adult Tcell leukaemia (ATL) as well as of the neurological syndrome TSP/HAM and of other clinical disorders. *In vitro* HTLV-1 is able to infect a number of different cell types, whereas in natural human infections it generally targets mature CD4+ helper T cells or, less frequently, CD8+ T cells. Although the mechanism of HTLV-1 pathogenicity is not fully understood yet, it is widely believed that a virally encoded trans-activator protein, called Tax, is centrally involved in this mechanism. In a recent review Azran et al. [94] provide valuable insights into the molecular mechanisms of HTLV-1 leukemogenesis. In particular, the authors detail the signalling pathways recruited by Tax to set infected T cells into continuous uncontrolled replication and to destabilize their genome, enabling, thereby, accumulation of mutations that can contribute to the leukemogenic process. Tax is able to modulate the expression of many viral genes via the viral long terminal repeat (LTR) and cellular genes through the CREB/ATF-, AP-1-, serum responsive factor (SRF)- and NF-κB-associated pathways, employing the CBP/p300 and p/CAF (p300/CBP-associated factor) co-activators for achieving the full transcriptional activation competence of each of these pathways. It is worth noting that Tax responsive elements (TxRE) contain a centered sequence TGACG(T/A)(C/G)(T/A) that is imperfectly homologous to the consensus cAMP responsive element (CRE; TGACGTCA). Thus, the presence of Tax is necessary for CREB to form a stable complex with the viral CRE. In fact, by interacting with the bZIP region of CREB, Tax enhances CREB dimerization and increases, thereby, its affinity to CRE. In particular, it has been recently shown that CREB is the most prominent factor that cooperates with Tax in activating HTLV-1 LTR region expression [95]. Moreover, it has been demonstrated that while, in the absence of Tax, CREB can activate HTLV-1 LTR expression only if phosphorylated by PKA, another member of the family, namely CREB2, can markedly activate the viral LTR without phosphorylation and can mediate a much stronger activation of the viral LTR by Tax than CREB does [94, 96]. Interestingly, mutant models disrupting Tax activation of the CREB protein resulted in the preferential immortalization of CD8+ lympho‐ cytes, rather than CD4+ lymphocytes, whereas the disruption of Tax interaction with CBP did not affect lymphocyte immortalization [97].

#### **5.6. Lymphoma**

**5.4. Chronic lymphocytic leukaemia**

568 Cancer Treatment - Conventional and Innovative Approaches

apoptosis.

Chronic lymphocytic leukaemia (CLL) originates from the abnormal accumulation of antigenstimulated B cells that escape normal cell death mechanisms and/or undergo increased proliferation [87]. CLL is the most prevalent adult leukaemia in the Western world, yet no curative treatment exists. Many studies have explored the use of family-specific cyclic nucleotide phosphodiesterase (PDE) inhibitors in light of the potent effects of cAMP signalling on immune system function [88, 89]. Among the 11 currently known families of cyclic nucleotide PDEs, all but three are capable of catabolizing cAMP and at least 5 PDE families (PDE1-4, PDE7 and PDE8) are expressed in lymphoid cells and regulated by either mitogens or agents that induce cAMP-mediated signalling. Previous work has established that inhibition of PDE4 is sufficient to selectively induce apoptosis in CLL cells by increasing the concentration of cAMP [88]. In a recent paper Meyers et al. [89] examined how CLL cells differ from normal haematopoietic cells with regard to their sensitivity to PDE4 inhibitor-mediated cAMP accumulation, CREB phosphorylation and gene expression. Interestingly, it was discovered that upon exposure to rolipram, a prototypical PDE4 inhibitor, cAMP intracellular levels rapidly rose in both CLL and normal B cells, whereas no such increase was detected in T cells. Likewise, ATF-1/CREB Ser63/133 phosphorylation was induced by rolipram in nearly all CLL and B cells, whereas normal T cells displayed a lower response. Based on these findings and on previous observations of a reduced basal cAMP signalling in CLL cells, the authors suggested the involvement of specific PDE or splice isoforms in the reduced basal apoptotic index of CLL cells [89]. Looking for etiological agents, other authors have identified a stromal cell–derived factor-1 (SDF-1)-dependent mechanism as a microenvironmental regulatory mechanism of CLL cell survival [90]. It is known that SDF-1 is a chemokine that plays an important role in B-cell development. In fact, high levels of SDF-1 are produced by stromal cells within the marrow to retain B-cell precursors in close contact with them, within the supportive haematopoietic microenvironment [91], and to prevent their premature release into the circulation. Upon *in vitro* treatment of CLL cells with synthetic SDF-1α, a rapid and transient activation of p44/42 MAPK (ERK1/2) signalling pathway was observed and related to CLL cell survival. Downward MAPK activation transcription-dependent and -independent mechanisms were involved. In fact, MAPK was able to promote cell survival directly by inactivating the pro-apoptotic BAD protein and indirectly by activating CREB, which, in turn, is important for the transcriptional up-regulation of the anti-apoptotic *BCL-2* gene [92]. Thus, SDF-1 engages B lineage CLL cells through the stromal cell receptor CXCR4 and affects components of the cell death machinery, leading to the noted resistance of CLL cells to

**5.5. Human T Lymphotropic Virus 1 (HTLV-1) related T cell leukaemia**

Human T-cell leukaemia virus type-I (HTLV-1) is the first discovered human retrovirus [93]. It has been recognized as the etiological agent of an aggressive malignancy known as adult Tcell leukaemia (ATL) as well as of the neurological syndrome TSP/HAM and of other clinical disorders. *In vitro* HTLV-1 is able to infect a number of different cell types, whereas in natural human infections it generally targets mature CD4+ helper T cells or, less frequently, CD8+ T

Lymphomas are haematological malignancies of the lymphoid system. Deregulated gene expression is a hallmark of cancer and is well documented in B-cell lymphomas [98]. B cells are particularly susceptible to malignant transformation since the mechanisms involved in antibody diversification can cause chromosomal translocations and oncogenic mutations. Bcell lymphomas include Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (B-NHL). B-NHL consists of a heterogeneous group of diseases whose pathogenesis is associated with multiple genetic lesions affecting oncogenes and tumour-suppressor genes and whose treatment is related to the different grade of malignancy. The most common type of B-NHL is represented by the diffuse large B-cell lymphoma (DLBCL), which generally arises as a clinical evolution of the follicular lymphoma (FL). A number of papers have demonstrated the involvement of CREB family members in the pathogenesis of lymphoma. It has been previ‐ ously found that CREB acts as a positive regulator of the translocated *BCL-2* allele in FLs with the t(14;18) translocation [60] and that the high constitutive expression of *ATF-3* is linked to the viability of Hodgkin/Reed-Sternberg cells and, thus, considered as a molecular hallmark of classical HL [99]. More recently, a number of studies have disclosed the implication of the HAT proteins CBP and p300 as tumour suppressors in B-cell neoplasms [100-102]. Neverthe‐ less, the various mechanisms through which each of these cofactors specifically contributes to

lymphomagenesis are still to be elucidated. As before mentioned, CBP and p300 function as co-activators of transcription factors and acetylate proteins relevant to lymphomagenesis such as p53, NF-κB, Bcl-6 and Hsp90 [100, 103, 104]. In particular, p300 acts as a co-activator of NFκB, activates p53 but attenuates Hsp90 chaperone functions and, moreover, transcriptional repressor *BCL-6* is frequently translocated and hyper-mutated in DLBCL where it results inversely correlated with p300 [100]. Importantly, de-acetylated Hsp90 represses p53 but maintains *BCL-6* expression, which suppresses p300 and its essential cofactor BAT3, which is necessary for p53 acetylation and activation. Somatic heterozygous mutations or deletions of the *CREBBP* locus occur in more than the 50% of DLBCL and the 32% of FL cases, whereas *EP300* mutations occur in the 10% of DLBCLs. All cases seem to have in common the disruption of the HAT catalytic domain, and the resulting truncated or mutant proteins may have dominant negative or gain of function properties, or may simply result in a reduced dosage of histone acetyltransferases. Structural alterations inactivating *CREBBP* and, less often, *EP300* have been recently documented and linked to the pathogenesis of both most common types of B-NHL [102]. According to Pasqualucci et al. [102] point mutations at the HAT coding domain of *CREBBP* and *EP300* result in specific defects in acetylation-mediated inactivation of the Bcl-6 oncoprotein and activation of the p53 tumour-suppressor, representing major pathogenetic mechanisms shared by the most common forms of B-NHL. Suppression of p300 either through Bcl-6 or inactivating mutations plays a key role in DLBCL. In fact, treatment of DLBCL cells with Bcl-6 inhibitors leads to p300 protein expression and acetyltransferase activity with subsequent acetylation of p53 (which induces p53 transcriptional functions) and Hsp90 (which suppresses Hsp90 chaperone activity) [100]. Moreover, the combination of Bcl-6 and histone deacetylase inhibitors (HDACI) leads to even higher p300 activity and synergistic killing of lymphoma cells *in vitro* and *in vivo* [100]. Interestingly, the direct effect of HDACI on non-histone proteins as DNA binding transcriptional factors (NF-κB, p53, CREB, GATA, cmyc, Bcl-6, E2F, IRF) can also affect cell growth and differentiation [101]. Furthermore, in light of HDACI effects on cell cycle regulatory molecules (Cyclin D1, p21 and p27) there is enough evidence that indicates these novel pleiotropic drugs as promising compounds for the treatment of B- and even T-cell malignancies in addition to conventional chemotherapy [105].

complex containing CREB [109]. Recent reports have demonstrated that Mcl-1 specific downregulation or repression is able to initiate apoptosis in MM [110]. To this end, proteasome inhibitors like bortezomib have been used though with contrasting results. In fact, it has been shown that accumulated and cleaved Mcl-1 products by proteasome inhibition have either a pro- or an anti-apoptotic function. In particular, Hu et al. [111] have investigated the role of endoplasmic reticulum unfolded protein response (UPR) in order to unravel the mechanisms underlying Mcl-1 accumulation following treatment with proteasome inhibitors, discovering the enhanced translation of ATF-4, an important effector of UPR, upon proteasome inhibition, and indicating ATF-4 as responsible for bortezomib resistance of MM [111]. Besides Mcl-1, novel factors are being identified as important players in the pathogenesis of MM. Recent studies have suggested that X-box–binding protein 1 (XBP1), a bZIP transcription factor of the CREB/ATF family, has an important role in the survival of MM cells [112]. XBP1 is required for B lymphocyte terminal differentiation to plasma cells and is essential for immunoglobulin secretion. Abundant or deregulated expression of *XBP1* has been detected in MM cells [113, 114] and in hepatocellular carcinomas [115]. Due to the production of abundant immunoglo‐ bulins and cytokines, MM cells must be able to survive under conditions of chronic ER stress involving UPR and including constitutive activation of the ER-located transmembrane kinase/ endoribonuclease (RNase) protein IRE1α-XBP1 pathway. This pathway, implicated in the proliferation and survival of MM cells, has been considered as a prognostic factor [116] and, moreover, as a possible target of chemo/immunotherapy [114, 117]. A growing body of evidence attributes a pathogenetic role to several microRNAs (miRNA) resulting up-regulated in MM and targeting p/CAF, a positive regulator of p53 [118]. Other authors have indicated a possible role of CREB family members in IL-6-mediated effects on myeloma cell growth and

Role of CREB Protein Family Members in Human Haematological Malignancies

http://dx.doi.org/10.5772/55368

571

CREB/ATF family is a growing family of transcription factors involved in a number of physiological and pathological processes. Day by day, new family members are being identified for their primary role in normal or aberrant haematopoiesis and proposed as therapeutic targets of anticancer drugs [112]. In fact, by regulating gene expression, transcrip‐ tion factors are often the final mediators of such central processes as proliferation, survival, self-renewal and invasion. Based on these effects, it is conceivable that inhibition of transcrip‐ tion factors can revert the malignant behaviour of many tumour types and can potentially achieve a very high therapeutic index [86]. Actually, in light of its important role in the pathogenesis of leukaemia, CREB has been indicated as a potential prognostic marker of disease progression in AML and a molecular target for future treatment of leukaemia. In addition, CREB has also been implicated in many solid tumours including hepatocellular carcinoma, osteosarcoma, lung adenocarcinoma, melanoma and lymphoma [46]. Indeed, since *CREB* overexpression results in a poor prognosis for the patient, the regulation of CREB activity might represent a useful strategy to treat solid tumours like prostate, breast and lung cancer, as well as haematological malignancies like AML and lymphoma. However, a key question

survival [119].

**6. Concluding remarks**

#### **5.7. Multiple myeloma**

Multiple myeloma (MM), also known as plasma cell myeloma or Kahler's disease, is a B-cell malignancy characterized by the accumulation in the bone marrow of plasma cells with a low proliferation index and an extended life span. Most cases of myeloma also feature the pro‐ duction of a paraprotein, an abnormal antibody that can cause kidney problems. MM cell lines as well as *de novo* MM cells express multiple anti-apoptotic proteins, often do not encode functional p53 and frequently contain a dysregulated Akt pathway [104-107]. A number of factors related to MM cell growth and survival and linked to CREB family members have been identified [108]. Among these factors, the myeloid cell leukaemia-1 (Mcl-1) protein, an antiapoptotic member of the Bcl-2 family, has been considered as a critical regulator of MM cell survival and proposed as an attractive therapeutic target [108]. *Mcl-1* is an immediate early gene activated in response to GM-CSF and IL-3. It has been previously reported that *Mcl-1* activation can occur in dependence of the PI3K/Akt pathway through a transcription factor complex containing CREB [109]. Recent reports have demonstrated that Mcl-1 specific downregulation or repression is able to initiate apoptosis in MM [110]. To this end, proteasome inhibitors like bortezomib have been used though with contrasting results. In fact, it has been shown that accumulated and cleaved Mcl-1 products by proteasome inhibition have either a pro- or an anti-apoptotic function. In particular, Hu et al. [111] have investigated the role of endoplasmic reticulum unfolded protein response (UPR) in order to unravel the mechanisms underlying Mcl-1 accumulation following treatment with proteasome inhibitors, discovering the enhanced translation of ATF-4, an important effector of UPR, upon proteasome inhibition, and indicating ATF-4 as responsible for bortezomib resistance of MM [111]. Besides Mcl-1, novel factors are being identified as important players in the pathogenesis of MM. Recent studies have suggested that X-box–binding protein 1 (XBP1), a bZIP transcription factor of the CREB/ATF family, has an important role in the survival of MM cells [112]. XBP1 is required for B lymphocyte terminal differentiation to plasma cells and is essential for immunoglobulin secretion. Abundant or deregulated expression of *XBP1* has been detected in MM cells [113, 114] and in hepatocellular carcinomas [115]. Due to the production of abundant immunoglo‐ bulins and cytokines, MM cells must be able to survive under conditions of chronic ER stress involving UPR and including constitutive activation of the ER-located transmembrane kinase/ endoribonuclease (RNase) protein IRE1α-XBP1 pathway. This pathway, implicated in the proliferation and survival of MM cells, has been considered as a prognostic factor [116] and, moreover, as a possible target of chemo/immunotherapy [114, 117]. A growing body of evidence attributes a pathogenetic role to several microRNAs (miRNA) resulting up-regulated in MM and targeting p/CAF, a positive regulator of p53 [118]. Other authors have indicated a possible role of CREB family members in IL-6-mediated effects on myeloma cell growth and survival [119].

### **6. Concluding remarks**

lymphomagenesis are still to be elucidated. As before mentioned, CBP and p300 function as co-activators of transcription factors and acetylate proteins relevant to lymphomagenesis such as p53, NF-κB, Bcl-6 and Hsp90 [100, 103, 104]. In particular, p300 acts as a co-activator of NFκB, activates p53 but attenuates Hsp90 chaperone functions and, moreover, transcriptional repressor *BCL-6* is frequently translocated and hyper-mutated in DLBCL where it results inversely correlated with p300 [100]. Importantly, de-acetylated Hsp90 represses p53 but maintains *BCL-6* expression, which suppresses p300 and its essential cofactor BAT3, which is necessary for p53 acetylation and activation. Somatic heterozygous mutations or deletions of the *CREBBP* locus occur in more than the 50% of DLBCL and the 32% of FL cases, whereas *EP300* mutations occur in the 10% of DLBCLs. All cases seem to have in common the disruption of the HAT catalytic domain, and the resulting truncated or mutant proteins may have dominant negative or gain of function properties, or may simply result in a reduced dosage of histone acetyltransferases. Structural alterations inactivating *CREBBP* and, less often, *EP300* have been recently documented and linked to the pathogenesis of both most common types of B-NHL [102]. According to Pasqualucci et al. [102] point mutations at the HAT coding domain of *CREBBP* and *EP300* result in specific defects in acetylation-mediated inactivation of the Bcl-6 oncoprotein and activation of the p53 tumour-suppressor, representing major pathogenetic mechanisms shared by the most common forms of B-NHL. Suppression of p300 either through Bcl-6 or inactivating mutations plays a key role in DLBCL. In fact, treatment of DLBCL cells with Bcl-6 inhibitors leads to p300 protein expression and acetyltransferase activity with subsequent acetylation of p53 (which induces p53 transcriptional functions) and Hsp90 (which suppresses Hsp90 chaperone activity) [100]. Moreover, the combination of Bcl-6 and histone deacetylase inhibitors (HDACI) leads to even higher p300 activity and synergistic killing of lymphoma cells *in vitro* and *in vivo* [100]. Interestingly, the direct effect of HDACI on non-histone proteins as DNA binding transcriptional factors (NF-κB, p53, CREB, GATA, cmyc, Bcl-6, E2F, IRF) can also affect cell growth and differentiation [101]. Furthermore, in light of HDACI effects on cell cycle regulatory molecules (Cyclin D1, p21 and p27) there is enough evidence that indicates these novel pleiotropic drugs as promising compounds for the treatment of B- and even T-cell malignancies in addition to conventional chemotherapy [105].

570 Cancer Treatment - Conventional and Innovative Approaches

Multiple myeloma (MM), also known as plasma cell myeloma or Kahler's disease, is a B-cell malignancy characterized by the accumulation in the bone marrow of plasma cells with a low proliferation index and an extended life span. Most cases of myeloma also feature the pro‐ duction of a paraprotein, an abnormal antibody that can cause kidney problems. MM cell lines as well as *de novo* MM cells express multiple anti-apoptotic proteins, often do not encode functional p53 and frequently contain a dysregulated Akt pathway [104-107]. A number of factors related to MM cell growth and survival and linked to CREB family members have been identified [108]. Among these factors, the myeloid cell leukaemia-1 (Mcl-1) protein, an antiapoptotic member of the Bcl-2 family, has been considered as a critical regulator of MM cell survival and proposed as an attractive therapeutic target [108]. *Mcl-1* is an immediate early gene activated in response to GM-CSF and IL-3. It has been previously reported that *Mcl-1* activation can occur in dependence of the PI3K/Akt pathway through a transcription factor

**5.7. Multiple myeloma**

CREB/ATF family is a growing family of transcription factors involved in a number of physiological and pathological processes. Day by day, new family members are being identified for their primary role in normal or aberrant haematopoiesis and proposed as therapeutic targets of anticancer drugs [112]. In fact, by regulating gene expression, transcrip‐ tion factors are often the final mediators of such central processes as proliferation, survival, self-renewal and invasion. Based on these effects, it is conceivable that inhibition of transcrip‐ tion factors can revert the malignant behaviour of many tumour types and can potentially achieve a very high therapeutic index [86]. Actually, in light of its important role in the pathogenesis of leukaemia, CREB has been indicated as a potential prognostic marker of disease progression in AML and a molecular target for future treatment of leukaemia. In addition, CREB has also been implicated in many solid tumours including hepatocellular carcinoma, osteosarcoma, lung adenocarcinoma, melanoma and lymphoma [46]. Indeed, since *CREB* overexpression results in a poor prognosis for the patient, the regulation of CREB activity might represent a useful strategy to treat solid tumours like prostate, breast and lung cancer, as well as haematological malignancies like AML and lymphoma. However, a key question concerns whether the activation of CREB (or other transcription factors) seen in cancer cells is directly driving the cell malignant phenotype, or whether it is merely a by-product of activation of one of the upstream pathways or only a partner in a more complex scenario. This is a crucial point, since CREB would represent a good molecular target only if it were a main player in the specific tumour biology. Unfortunately, clinical and experimental evidences suggest that several functionally cooperating genetic alterations, including chromosomal translocations, lead to the expression of fusion proteins that play a key role in the pathogenesis of the leukaemia phenotype. CREB itself can promote cellular transformation as a fusion protein or by cooperating with other oncogenes or transcription factors. Furthermore, due to the recruitment of chromatin modulating mechanisms in the transforming activity of leukemo‐ genic factors, transcriptional therapies aimed at inhibiting DNA methyltransferases, histone deacetylases or acetyltransferases, like CBP and p300, are emerging as new frontiers for cancer treatment. Unlike HDACI, which have been used in several phase I/II clinical trials, HAT inhibitors have been less extensively investigated for their potential use in cancer therapy. Indeed, interesting results obtained with clinical treatment of solid tumours [120] suggest that p300 inhibition may be a promising anticancer approach. To overcome the numerous side effects and the mostly transient clinical responses exerted by epigenetic compounds used as a single treatment [121], combinatorial therapy involving epigenetic agents together with conventional or targeted agents is increasingly seen as a more attractive opportunity. There‐ fore, further preclinical investigations aimed at better dissecting epigenetic mechanisms driving induction, maintenance and potential reversibility of the leukaemia state are welcome and functional to select the most potent drugs and combinations and to develop more efficient and long-lasting targeted therapeutic strategies. We hope to have contributed with this chapter to make the state of the art on the role of CREB in leukaemia and lymphoma neoplasms in order to allow further steps moving ahead from bench to bedside.

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### **Acknowledgements**

This book chapter was supported by funds of the Italian Ministry of University and Research (MIUR) granted in 2011 to Prof. Roberta Di Pietro.

### **Author details**

Francesca D'Auria1 and Roberta Di Pietro2\*

\*Address all correspondence to: r.dipietro@unich.it

1 Department of Cardiac and Vascular Surgery, Campus Bio-Medico University of Rome, Italy

2 Department of Medicine and Ageing Sciences, G. d'Annunzio University of Chieti-Pes‐ cara, Italy

### **References**

concerns whether the activation of CREB (or other transcription factors) seen in cancer cells is directly driving the cell malignant phenotype, or whether it is merely a by-product of activation of one of the upstream pathways or only a partner in a more complex scenario. This is a crucial point, since CREB would represent a good molecular target only if it were a main player in the specific tumour biology. Unfortunately, clinical and experimental evidences suggest that several functionally cooperating genetic alterations, including chromosomal translocations, lead to the expression of fusion proteins that play a key role in the pathogenesis of the leukaemia phenotype. CREB itself can promote cellular transformation as a fusion protein or by cooperating with other oncogenes or transcription factors. Furthermore, due to the recruitment of chromatin modulating mechanisms in the transforming activity of leukemo‐ genic factors, transcriptional therapies aimed at inhibiting DNA methyltransferases, histone deacetylases or acetyltransferases, like CBP and p300, are emerging as new frontiers for cancer treatment. Unlike HDACI, which have been used in several phase I/II clinical trials, HAT inhibitors have been less extensively investigated for their potential use in cancer therapy. Indeed, interesting results obtained with clinical treatment of solid tumours [120] suggest that p300 inhibition may be a promising anticancer approach. To overcome the numerous side effects and the mostly transient clinical responses exerted by epigenetic compounds used as a single treatment [121], combinatorial therapy involving epigenetic agents together with conventional or targeted agents is increasingly seen as a more attractive opportunity. There‐ fore, further preclinical investigations aimed at better dissecting epigenetic mechanisms driving induction, maintenance and potential reversibility of the leukaemia state are welcome and functional to select the most potent drugs and combinations and to develop more efficient and long-lasting targeted therapeutic strategies. We hope to have contributed with this chapter to make the state of the art on the role of CREB in leukaemia and lymphoma neoplasms in

order to allow further steps moving ahead from bench to bedside.

and Roberta Di Pietro2\*

(MIUR) granted in 2011 to Prof. Roberta Di Pietro.

572 Cancer Treatment - Conventional and Innovative Approaches

\*Address all correspondence to: r.dipietro@unich.it

This book chapter was supported by funds of the Italian Ministry of University and Research

1 Department of Cardiac and Vascular Surgery, Campus Bio-Medico University of Rome,

2 Department of Medicine and Ageing Sciences, G. d'Annunzio University of Chieti-Pes‐

**Acknowledgements**

**Author details**

Francesca D'Auria1

Italy

cara, Italy


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580 Cancer Treatment - Conventional and Innovative Approaches


**Chapter 24**

**Life-Cycling of Cancer: New Concept**

Marina Shaduri and Marc Bouchoucha

http://dx.doi.org/10.5772/55385

**1. Introduction**

and triggers.

neoplasm?

malignant neoplasm?

something that is not carcinogenic".

Additional information is available at the end of the chapter

**•** What is the main cause of cancer diversity and individualism?

**•** Why do some cancers give metastasis and some do not?

The best way to deal with a tricky and unpredictable disease is to understand its essence, causes

**•** Why and how become some normal cells "rebellious" and aggressive? Are there any common processes and rules that govern the transformation of normal cells into malignant

**•** There are overlaps between benign and malignant lesions. Can we define cancer accurately? Is there a clear margin or a criterion that differentiates benign and slowly progressing

The basic questions about cancer must be answered to demystify this scary disease and solve the "Oncogenic Paradox" described by the Nobel Prize laureate Albert Szent-Gyorgyi [1]: "The malignant transformation of tissues … is a very specific process which must involve very specific changes in a very specific chemical machinery. Accordingly, one would expect that such transformation can be brought about only by a very specific process, as locks can be opened only by their own keys. Contrary to this, a malignant transformation can be brought about by an infinite number of unspecific influences, such as pieces of asbestos, high-energy radiation, irritation, chemicals, viruses, etc. It is getting more and more difficult to find

A new promising way of understanding malignant neoplasia and its paradoxes rests upon integrating biomedical and physical knowledge. Several years ago the US National Cancer Institute funded a major research program to bring insights into the cancer problem from the

and reproduction in any medium, provided the original work is properly cited.

© 2013 Shaduri and Bouchoucha; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Chapter 24**

### **Life-Cycling of Cancer: New Concept**

Marina Shaduri and Marc Bouchoucha

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55385

**1. Introduction**

The best way to deal with a tricky and unpredictable disease is to understand its essence, causes and triggers.


The basic questions about cancer must be answered to demystify this scary disease and solve the "Oncogenic Paradox" described by the Nobel Prize laureate Albert Szent-Gyorgyi [1]: "The malignant transformation of tissues … is a very specific process which must involve very specific changes in a very specific chemical machinery. Accordingly, one would expect that such transformation can be brought about only by a very specific process, as locks can be opened only by their own keys. Contrary to this, a malignant transformation can be brought about by an infinite number of unspecific influences, such as pieces of asbestos, high-energy radiation, irritation, chemicals, viruses, etc. It is getting more and more difficult to find something that is not carcinogenic".

A new promising way of understanding malignant neoplasia and its paradoxes rests upon integrating biomedical and physical knowledge. Several years ago the US National Cancer Institute funded a major research program to bring insights into the cancer problem from the

standpoint of physical science; the hope was that physicists could introduce some radical new ideas to the table. In the manuscript we focus mainly on the physical aspects of cancer origin pushing aside biochemical, immunological and gene-associated findings that do not presently add much to the conceptual framework for cancer theory. Our model of carcinogenesis incorporates certain recently discovered physical phenomena [2-4] that elucidate many peculiarities of malignant processes.

hallmarks of cancers include: 1) Self sufficiency in growth signals, 2) Insensitivity to antigrowth signals, 3) Evading apoptosis, 4) Limitless replicative potential 5) Sustained angiogenesis, 6) Tissue invasion and metastasis, and 7) Genome instability. It is also widely accepted that cancers express aerobic glycolysis regardless of their tissue or cellular origin [9]. Abnormal segregation of chromosomes during mitosis (aneuploidy) and genome instability are found almost in all cancers [10], though the reason(s) of these abnormalities are not

Life-Cycling of Cancer: New Concept http://dx.doi.org/10.5772/55385 585

**The somatic mutation theory** of carcinogenesis has been dominant since the beginning of the 20th century. It is known that cancer cell genomes carry somatic mutations in DNA that may include base substitutions, small insertions and deletions, rearrangements, and copy number alterations. As the tumor progresses, mutations accumulate and the cell eventually becomes cancerous. Apart of successive alterations in genetic material (somatic events), some germ-line mutations can also predispose a person to heritable or familial cancer. Certain defects in DNA are known to be responsible for a variety of hereditary cancer predisposition syndromes including non-polyposis colorectal carcinoma, Bloom syndrome, ataxia-telangiectasia, Fanconi anaemia, etc. [11,12]. Molecular genetics has identified some oncogenes that, along with tumor suppressor genes, can reproduce many aspects of cancer progression. In fact, each tumor is unique in its genetic makeup [13] and, correspondingly, has a unique phenotype akin to an individual organism. Many researchers consider the above theory unsatisfactory because no strict correlation exists between gene mutations and malignancy; besides, it is unclear which factors trigger the gen-associated events that lead to neoplasia. Evidently, the genomic instability per se is not sufficient to initiate a malignant tumor. The somatic mutation theory can explain neither genetic variability within individual tumors, nor many other observable

The **cancer-stem-cell (CSC) concept** is becoming increasingly popular, since nondifferentiated, relatively primitive and pluri- or totipotent cells have the ability to self-renew and to give rise to distinct types of malignant cells. It is now generally accepted that the CSC sub-population of cancer cells plays significant role in initiation, progression and recurrence of cancer. The CSC concept was first demonstrated in the study of leukemia, which was found to be associated with the "stem-cells" having specific surface antigen profiles [14, 15]. Italian researchers who spotted CSCs in human primary bone sarcomas highlighted CD133 as a pivotal marker for their identification [16]. In recent years similar cells were found in human cancers of brain, breast, colon, pancreas and other tissues [17]. Kornelia Polyak from Dana-Farber Cancer Institute (Boston, US) demonstrated that the frequency of tumor cells positive for stem cell–like and more differentiated cell markers varies according to tumor subtype and histological stage [18]; the question whether malignancy arises from normal stem cells due to

maturation arrest or due to transformation of mature cells into CSC is still open.

**The Viral/Microbial Theory of Cancer** that regards viruses/microbes as potential triggers of a neoplastic process has long history. First finding concerned the avian leucosis virus as a cause of leukemia in chickens [19]; Two years later after this discovery P. Rous presented his theory about ultramicroscopic organisms capable to induce cancer in humans and animals [20]. Since then many viral infections have been linked to malignant processes. Recent studies have

clarified.

phenomena in cancer biology.

New concept of cancer origin is a particular example of the more general model of systemgenesis published last year [5]. According to this hypothesis, a malignant neoplasm originates within a small isolated area of a larger organism as a new functional unit with its individual mechanisms of self-control and self-regulation; the cells that are deprived of nutrients and oxygen do pass through several stages of dramatic transformations that lead to the formation of toti- or pluripotent cells with altered genetic makeup. The future fate of this "potential neoplasm" depends on a combination of some physical factors and on the proper timing of successive events that include the unification of enclosed cells and their preparation for aggressive expansion through the physical effect of "Random Lasing" [2]. Hence, contrary to a widely spread opinion of cancer being a chaotic and poorly controlled pull of rebellious cells that are "driven mad" by some mutations, we consider malignant neoplasm to be a strictly controlled and adaptive system of cooperatively acting primitive cells. Some researchers share this point of view regarding cancer as a self-organizing adaptive system or a parasite-like organism [6, 7].

Our model of carcinogenesis is the result of 12-year-long experimental and clinical work in the emerging scientific field of Biological Holography. All illustrations presented in the manuscript are obtained with the computer-assessed device (CID-system) developed specially for cancer detection and visualization [5]. This hardware-software system is the ever first cancer-detecting and monitoring tool convenient for mass-screening purposes; it is capable of detecting and monitoring of any malignant process disregarding its type and location in the body. The non-invasive and automatable method of any cancer detection through a single and short-term procedure is already implemented in diagnostic practice: the patients with and without malignancies are distinguished by spectral information emitted from their body surfaces.

### **2. Cancer origin theories: State-of-the-art**

Malignant neoplasia of normal cells remains a source of misunderstanding and controversy. There is a vast literature on cancer theories. In this section we briefly describe only some of the most acknowledged and interesting ideas. Although none of the debatable hypothesis of carcinogenesis elucidates the general scenario applicable to all cases of cancer, they are nevertheless helpful in generalization of the state-of-the-art knowledge.

A central feature of today's view of cancer is that it does not develop all at once but evolves as a result of complex succession of events over time. According to Hanahan and Weinberg [8] there are several essential alterations in cell physiology typical for malignant cell growth. These hallmarks of cancers include: 1) Self sufficiency in growth signals, 2) Insensitivity to antigrowth signals, 3) Evading apoptosis, 4) Limitless replicative potential 5) Sustained angiogenesis, 6) Tissue invasion and metastasis, and 7) Genome instability. It is also widely accepted that cancers express aerobic glycolysis regardless of their tissue or cellular origin [9]. Abnormal segregation of chromosomes during mitosis (aneuploidy) and genome instability are found almost in all cancers [10], though the reason(s) of these abnormalities are not clarified.

standpoint of physical science; the hope was that physicists could introduce some radical new ideas to the table. In the manuscript we focus mainly on the physical aspects of cancer origin pushing aside biochemical, immunological and gene-associated findings that do not presently add much to the conceptual framework for cancer theory. Our model of carcinogenesis incorporates certain recently discovered physical phenomena [2-4] that elucidate many

New concept of cancer origin is a particular example of the more general model of systemgenesis published last year [5]. According to this hypothesis, a malignant neoplasm originates within a small isolated area of a larger organism as a new functional unit with its individual mechanisms of self-control and self-regulation; the cells that are deprived of nutrients and oxygen do pass through several stages of dramatic transformations that lead to the formation of toti- or pluripotent cells with altered genetic makeup. The future fate of this "potential neoplasm" depends on a combination of some physical factors and on the proper timing of successive events that include the unification of enclosed cells and their preparation for aggressive expansion through the physical effect of "Random Lasing" [2]. Hence, contrary to a widely spread opinion of cancer being a chaotic and poorly controlled pull of rebellious cells that are "driven mad" by some mutations, we consider malignant neoplasm to be a strictly controlled and adaptive system of cooperatively acting primitive cells. Some researchers share this point of view regarding cancer as a self-organizing adaptive system or a parasite-like

Our model of carcinogenesis is the result of 12-year-long experimental and clinical work in the emerging scientific field of Biological Holography. All illustrations presented in the manuscript are obtained with the computer-assessed device (CID-system) developed specially for cancer detection and visualization [5]. This hardware-software system is the ever first cancer-detecting and monitoring tool convenient for mass-screening purposes; it is capable of detecting and monitoring of any malignant process disregarding its type and location in the body. The non-invasive and automatable method of any cancer detection through a single and short-term procedure is already implemented in diagnostic practice: the patients with and without malignancies are distinguished by spectral information emitted from their body

Malignant neoplasia of normal cells remains a source of misunderstanding and controversy. There is a vast literature on cancer theories. In this section we briefly describe only some of the most acknowledged and interesting ideas. Although none of the debatable hypothesis of carcinogenesis elucidates the general scenario applicable to all cases of cancer, they are

A central feature of today's view of cancer is that it does not develop all at once but evolves as a result of complex succession of events over time. According to Hanahan and Weinberg [8] there are several essential alterations in cell physiology typical for malignant cell growth. These

nevertheless helpful in generalization of the state-of-the-art knowledge.

peculiarities of malignant processes.

584 Cancer Treatment - Conventional and Innovative Approaches

organism [6, 7].

surfaces.

**2. Cancer origin theories: State-of-the-art**

**The somatic mutation theory** of carcinogenesis has been dominant since the beginning of the 20th century. It is known that cancer cell genomes carry somatic mutations in DNA that may include base substitutions, small insertions and deletions, rearrangements, and copy number alterations. As the tumor progresses, mutations accumulate and the cell eventually becomes cancerous. Apart of successive alterations in genetic material (somatic events), some germ-line mutations can also predispose a person to heritable or familial cancer. Certain defects in DNA are known to be responsible for a variety of hereditary cancer predisposition syndromes including non-polyposis colorectal carcinoma, Bloom syndrome, ataxia-telangiectasia, Fanconi anaemia, etc. [11,12]. Molecular genetics has identified some oncogenes that, along with tumor suppressor genes, can reproduce many aspects of cancer progression. In fact, each tumor is unique in its genetic makeup [13] and, correspondingly, has a unique phenotype akin to an individual organism. Many researchers consider the above theory unsatisfactory because no strict correlation exists between gene mutations and malignancy; besides, it is unclear which factors trigger the gen-associated events that lead to neoplasia. Evidently, the genomic instability per se is not sufficient to initiate a malignant tumor. The somatic mutation theory can explain neither genetic variability within individual tumors, nor many other observable phenomena in cancer biology.

The **cancer-stem-cell (CSC) concept** is becoming increasingly popular, since nondifferentiated, relatively primitive and pluri- or totipotent cells have the ability to self-renew and to give rise to distinct types of malignant cells. It is now generally accepted that the CSC sub-population of cancer cells plays significant role in initiation, progression and recurrence of cancer. The CSC concept was first demonstrated in the study of leukemia, which was found to be associated with the "stem-cells" having specific surface antigen profiles [14, 15]. Italian researchers who spotted CSCs in human primary bone sarcomas highlighted CD133 as a pivotal marker for their identification [16]. In recent years similar cells were found in human cancers of brain, breast, colon, pancreas and other tissues [17]. Kornelia Polyak from Dana-Farber Cancer Institute (Boston, US) demonstrated that the frequency of tumor cells positive for stem cell–like and more differentiated cell markers varies according to tumor subtype and histological stage [18]; the question whether malignancy arises from normal stem cells due to maturation arrest or due to transformation of mature cells into CSC is still open.

**The Viral/Microbial Theory of Cancer** that regards viruses/microbes as potential triggers of a neoplastic process has long history. First finding concerned the avian leucosis virus as a cause of leukemia in chickens [19]; Two years later after this discovery P. Rous presented his theory about ultramicroscopic organisms capable to induce cancer in humans and animals [20]. Since then many viral infections have been linked to malignant processes. Recent studies have provided cogent evidence that some "oncoviruses", e.g., human papillomavirus, hepatitis B and hepatitis C virus, Epstein-Barr virus, etc. are indeed associated with increased incidence of human cancers [21, 22]. Over the years, scientists have proposed a number of mechanisms to explain this link. However, numerous cases of cancer can originate and develop independently of any viruses, fungi or bacteria.

actively participate in carcinogenesis [31]. Sonnenschein and Soto from Tufts University in Boston [32] put forward the tissue organization field theory arguing that dynamic breakdown of cellular communication and signal transduction prompts disoriented cells to mistakenly

Life-Cycling of Cancer: New Concept http://dx.doi.org/10.5772/55385 587

The theoretical considerations listed above are substantiated by empiric evidence, but they deal with particular events and manifestations of carcinogenesis. These hypotheses are essentially complementary to each other rather than contradictory; they describe various contributing factors and peculiarities of a neoplasm but no data are available concerning the general scenario and common physical processes that take place at early stages of any cancer genesis. No doubt that there is an urgent need for such a theory capable to reconcile existing hypotheses and empiric findings by establishing the reasons and physical laws that drive

Our model of cancer origin has much in common with the embryonal and speciation hypotheses mentioned above; however, it brings new insights into physical mechanisms of cancer emergence and elucidates some details of its "prenatal" life. In this section we will discuss the general peculiarities of complex adaptive systems and show that malignant neoplasm being a system of cooperatively acting cells, behaves as an autonomous organism with its own mechanisms of self-control and self-regulation. Evidently, the whole spectrum of distinct cells, tissues and organs in human body comes out from a bunch of initially identical cells produced by a single zygote - the same processes would be expected in cancers. Lloyd J. Old has found common genetic programs at work in tumor cells and gametes that led him to describe cancer as a "somatic cell pregnancy" [33]. In sections 6 and 7 of the manuscript we will search an answer to the question: how a normal and well-differentiated (somatic) cell

One can suggest that a cluster of young cancer-cells would not survive in the heavily populated competitive environment unless their development is driven by powerful autonomous mechanisms of self-regulation and adaptation. Such self-organizing entities belong to the class of complex adaptive systems (CAS) which are capable to learn from their experience while functioning in variable ambience. Adaptive evolution (evolvability) and the emergence phenomenon are their yet unexplained characteristics. Emergence implies appearance of certain unpredictable and qualitatively new functions that pop up out of the multiplicity of

It is widely accepted, that all CASs share the following common characteristics: 1) robustness – the ability to maintain a basic level of dynamic equilibrium; 2) resilience – all CASs are capable to restore the quasi-equilibrium state after various perturbations; 3) multi-level organization in terms of complex structural and functional hierarchy; 4) self-organization that implies creation of more complex internal structures without external resources or information and, of course, 5) adaptability in the sense that any CAS can vary its strategy and tactics according

revert to pro-growth patterns of behavior.

normal cells towards malignant neoplasia.

**3. Malignant neoplasm as a new organism**

becomes "pregnant" in the absence of fertilizing agents?

relatively simple interactions.

A major cohort of scientists supports the **embryonal theory of cancer.** A type of similarity between embryogenesis and carcinogenesis was first mentioned by John Beard, who put forward The Unitarian Trophoblastic Theory of cancer [23]. The main idea behind his theory is that certain fetal cells or atavistic genes give rise to a neoplasm. Prominent physicist Paul Davis argues that ancient genetic toolkit active in the earliest stages of embryogenesis gets switched back on, re-activating the Proterozoic developmental plan for building cell colonies [7]. Rippert [24] suggested that cells expressing embryonic potential arise due to the process of dedifferentiation. According to the proponents of the embryonal theory, some immature cells such as the remnants of fetal tissues, become eventually malignant due to altered blood supply, e.g., after tissue traumas or mechanical isolation of a small area from nutrients and oxygen. Remarkably, the development of the zygote up to the blastula stage is more or less the same in all mammals, so one can assume that the early phases of cancer "prenatal life" would be of the same nature. Whether we should blame the atavistic genes or there are some other factors that eventually "fertilize" the host-cells producing neoplasm remains an open question.

The embryonal theory is closely related to the hypothesis dubbed **the "speciation theory"** that regards cancers as new species. Duesberg and his UC Berkeley colleagues, who studied aneuploid nature of a cell karyotype across numerous cell cultures, came to a conclusion that some cell-destructive events cause chromosomal mutations and result in cells with totally new phenotypes [25]. The authors argue that carcinogenesis is initiated by a disruption of chromosomes that alters the balance of tens of thousands of genes. The result of these processes is a cell with new traits – that is, a new phenotype or a new organism. According to these researchers, "cancer is comparable to a bacterial level of complexity, but still autonomous; … it doesn't follow orders like other cells in the body, and it can grow where, when and how it likes" [ibid]. M. Vincent [26] also considers cancer as a programmed and evolutionarily conserved formation rather than just a random series of disease-causing mutations.

Malignant neoplasm develops within host tissues, so the state of entire body and traits of **the micro-environment** of a "cancer-nursery" must be taken in account while searching cancer initiation factors. Gene mutations are only part of the process that leads to cancer, which involves an interaction between neoplasm and surrounding tissue. The importance of changes in the micro-environment during tumor progression has been recognized thanks to pertinent enthusiastic scientists, who were moving against the mainstream science to prove their hypothesis [27-29]. The existence of histologically abnormal tissue beyond a neoplastic area that predisposes to tumor formation is a characteristic feature of many cancers. Interesting data were published by a team of American researchers who established that in the course of tumor development the normal cells in tumor stroma may lose more regions of DNA than do the cancer cells [30]. Another team of American scientists demonstrated that stromal cells actively participate in carcinogenesis [31]. Sonnenschein and Soto from Tufts University in Boston [32] put forward the tissue organization field theory arguing that dynamic breakdown of cellular communication and signal transduction prompts disoriented cells to mistakenly revert to pro-growth patterns of behavior.

The theoretical considerations listed above are substantiated by empiric evidence, but they deal with particular events and manifestations of carcinogenesis. These hypotheses are essentially complementary to each other rather than contradictory; they describe various contributing factors and peculiarities of a neoplasm but no data are available concerning the general scenario and common physical processes that take place at early stages of any cancer genesis. No doubt that there is an urgent need for such a theory capable to reconcile existing hypotheses and empiric findings by establishing the reasons and physical laws that drive normal cells towards malignant neoplasia.

### **3. Malignant neoplasm as a new organism**

provided cogent evidence that some "oncoviruses", e.g., human papillomavirus, hepatitis B and hepatitis C virus, Epstein-Barr virus, etc. are indeed associated with increased incidence of human cancers [21, 22]. Over the years, scientists have proposed a number of mechanisms to explain this link. However, numerous cases of cancer can originate and develop

A major cohort of scientists supports the **embryonal theory of cancer.** A type of similarity between embryogenesis and carcinogenesis was first mentioned by John Beard, who put forward The Unitarian Trophoblastic Theory of cancer [23]. The main idea behind his theory is that certain fetal cells or atavistic genes give rise to a neoplasm. Prominent physicist Paul Davis argues that ancient genetic toolkit active in the earliest stages of embryogenesis gets switched back on, re-activating the Proterozoic developmental plan for building cell colonies [7]. Rippert [24] suggested that cells expressing embryonic potential arise due to the process of dedifferentiation. According to the proponents of the embryonal theory, some immature cells such as the remnants of fetal tissues, become eventually malignant due to altered blood supply, e.g., after tissue traumas or mechanical isolation of a small area from nutrients and oxygen. Remarkably, the development of the zygote up to the blastula stage is more or less the same in all mammals, so one can assume that the early phases of cancer "prenatal life" would be of the same nature. Whether we should blame the atavistic genes or there are some other factors that eventually "fertilize" the host-cells producing neoplasm remains an open

The embryonal theory is closely related to the hypothesis dubbed **the "speciation theory"** that regards cancers as new species. Duesberg and his UC Berkeley colleagues, who studied aneuploid nature of a cell karyotype across numerous cell cultures, came to a conclusion that some cell-destructive events cause chromosomal mutations and result in cells with totally new phenotypes [25]. The authors argue that carcinogenesis is initiated by a disruption of chromosomes that alters the balance of tens of thousands of genes. The result of these processes is a cell with new traits – that is, a new phenotype or a new organism. According to these researchers, "cancer is comparable to a bacterial level of complexity, but still autonomous; … it doesn't follow orders like other cells in the body, and it can grow where, when and how it likes" [ibid]. M. Vincent [26] also considers cancer as a programmed and evolutionarily

conserved formation rather than just a random series of disease-causing mutations.

Malignant neoplasm develops within host tissues, so the state of entire body and traits of **the micro-environment** of a "cancer-nursery" must be taken in account while searching cancer initiation factors. Gene mutations are only part of the process that leads to cancer, which involves an interaction between neoplasm and surrounding tissue. The importance of changes in the micro-environment during tumor progression has been recognized thanks to pertinent enthusiastic scientists, who were moving against the mainstream science to prove their hypothesis [27-29]. The existence of histologically abnormal tissue beyond a neoplastic area that predisposes to tumor formation is a characteristic feature of many cancers. Interesting data were published by a team of American researchers who established that in the course of tumor development the normal cells in tumor stroma may lose more regions of DNA than do the cancer cells [30]. Another team of American scientists demonstrated that stromal cells

independently of any viruses, fungi or bacteria.

586 Cancer Treatment - Conventional and Innovative Approaches

question.

Our model of cancer origin has much in common with the embryonal and speciation hypotheses mentioned above; however, it brings new insights into physical mechanisms of cancer emergence and elucidates some details of its "prenatal" life. In this section we will discuss the general peculiarities of complex adaptive systems and show that malignant neoplasm being a system of cooperatively acting cells, behaves as an autonomous organism with its own mechanisms of self-control and self-regulation. Evidently, the whole spectrum of distinct cells, tissues and organs in human body comes out from a bunch of initially identical cells produced by a single zygote - the same processes would be expected in cancers. Lloyd J. Old has found common genetic programs at work in tumor cells and gametes that led him to describe cancer as a "somatic cell pregnancy" [33]. In sections 6 and 7 of the manuscript we will search an answer to the question: how a normal and well-differentiated (somatic) cell becomes "pregnant" in the absence of fertilizing agents?

One can suggest that a cluster of young cancer-cells would not survive in the heavily populated competitive environment unless their development is driven by powerful autonomous mechanisms of self-regulation and adaptation. Such self-organizing entities belong to the class of complex adaptive systems (CAS) which are capable to learn from their experience while functioning in variable ambience. Adaptive evolution (evolvability) and the emergence phenomenon are their yet unexplained characteristics. Emergence implies appearance of certain unpredictable and qualitatively new functions that pop up out of the multiplicity of relatively simple interactions.

It is widely accepted, that all CASs share the following common characteristics: 1) robustness – the ability to maintain a basic level of dynamic equilibrium; 2) resilience – all CASs are capable to restore the quasi-equilibrium state after various perturbations; 3) multi-level organization in terms of complex structural and functional hierarchy; 4) self-organization that implies creation of more complex internal structures without external resources or information and, of course, 5) adaptability in the sense that any CAS can vary its strategy and tactics according to a new or previously experienced situation. The listed hallmarks of autonomously functioning systems are unimaginable without synergy, which implies an orchestrated, synchronized and interdependent behavior of all system-components.

experience) is possible without data storage. No doubt that some mechanisms of data memorizing should exist in all, even in simplest entities capable to adapt and develop: ambient information has to be perceived, processed and stored in a readily accessible (usable) form. We argue that a kind of associative memory must be an embedded feature of all adaptive systems, among them, of cancers, since autonomous functioning, adaptation and development are unimaginable without the available information on previously experienced states [5]. The physical basis of a system memory is closely related to real-time holographic mechanisms that are basic for any CAS. These poorly understood mechanisms that imply the wave-wave and wave-matter interactions ensure the unification and integration of many separate elements

Life-Cycling of Cancer: New Concept http://dx.doi.org/10.5772/55385 589

Cells and other elements of complex biological systems are functionally interdependent – they exhibit evident signs of collective behavior being organized as a hierarchy [37-39]. If cancers are integral and adaptive organisms, the action of malignant cells should be strictly coordinated. Indeed, nontrivial spatial correlations between malignant cells have been found by various researchers. The cooperative behavior, namely, collective migration of malignant cells during their invasion into healthy tissues seems to follow essentially the same pathways as healthy cells that participate in embryological development and damaged tissue reparation [40]. Cells performing collective migration share many biological characteristics with independently migrating cells but, by affecting one another mechanically and via signaling,

Experimental and clinical observations support the suggestion that cancer cells form a complex and integrated system. G. Lambert studied the collective response of breast cancer tissues to drug-induced stress and found a similarity between the rapid evolution of drug resistance in cancers and the behavior of bacterial colonies under starvation conditions [43]. Professor P. Davies, principal investigator of a major research program funded by the National Cancer Institute, argues that cancer is not a random bunch of selfish rogue cells behaving badly, but a highly-efficientpre-programmedresponse tostress,honedbya longperiodof evolution.[7, 44]. Hence, one can regard cancer as a life-tenacious organism created by and incorporated into relatively mature tissues of the host-body. This complex adaptive system, which is doomed to conduct a life-long battle with its superior ancestor - parental body, has its own powerful selfregulation mechanisms, a flexible primitive structure and enough power to hunt the preys –

Cancer remains an elusive, unpredictable and scary disease mostly because the malignant processes are difficult to detect and monitor. The most efficient methods of cancer conventional diagnostics are either harmful or too costly to be used in vivo as often as necessary. Oncologists lack a non-invasive, reliable, user-friendly, automatable and non-expensive test for tracking

into an autonomously functioning system of interdependent agents (see below).

these cell groups are subject to additional regulation and constraints [41, 42].

**4. New approach to the cancer non-invasive study**

**3.2. Collective behavior of malignant cells**

host cells.

We argue that cancer has all the traits typical for any CAS: cancer cells are hard to destroy even by chemical toxins and radiation, since they coordinate their action in order to survive as an entity. Only united and self-organizing system of cooperating cells would be able to start the vital struggle against the powerful host-organism. The cancer-system shares the phase-space with the host-CAS which is its rival and breadwinner at the same time. New organism should either defeat its host, or, alternatively, obey its rules and commands adapting to the variable ambience.

### **3.1. Adaptive behavior and diversity of cancers**

There are about 200 types of cancers each type comprising multiple "families" and sub-types of cells. The scientists from the Wellcome Trust Sanger Institute in Hinxton, England, recently announced 73 different combinations of disease-causing mutations in the breast tumors each involving up to six different genes from a set of 40 driver genes [34]. Canadian researchers have shown that the cells taken from patients with acute lymphoblastic leukemia are actually composed of multiple families of genetically distinct leukemia cells [35]. No doubt, that the treatment of such a diverse pathology would not be efficient without understanding of the most general regulatory mechanisms common for all cancers.

What is the reason of cancer diversity? Are its cells the clones of distinct "cancer-stems" that originate simultaneously, or they emerge as new cells due to clashes with surrounding cells that produce odds and ends of damaged cellular components?

We assume that an interaction of poorly differentiated cells with the bystander elements of stroma can yield various karyo- and phenotypes through the same mechanisms that take place in the first "nursery" of emerging cancer. The tumor micro-environment is a complex system of many cell types, including endothelial cells and their precursors, smooth-muscle cells, fibroblasts, granulocytes, lymphocytes, macrophages, etc. Taking into consideration the features of CAS, one can suggest that adaptation of young, meta-stable and extremely motile cancer cells to variable and heterogeneous micro-environment plays crucial role in the process of cell diversification; however, there is another possibility to provide diverse "stems" and their clones. This "fresh" idea about recurrent (iterative) cycles of carcinogenesis that imply successive production of less complex generations of malignant cells is described in section 7.

Many cancers adapt to chemo- and radiation therapy: according to some researchers, the clonal selection leads to the resistance of recurrent tumors [36]. If "cancer-embryos" are nurtured in various conditions before they proceed to active life-cycling, they might give birth to distinct "clones". This process cannot be considered as selection, but as the emergence of new organisms by the same scenario as in the first act of carcinogenesis.

It should be noted that the adaptation itself is not a well understood phenomenon. Elusive non-molecular processes of information exchange between the cells/tissues are difficult to study. As a result, we often ignore an obvious fact that no process of learning (gaining experience) is possible without data storage. No doubt that some mechanisms of data memorizing should exist in all, even in simplest entities capable to adapt and develop: ambient information has to be perceived, processed and stored in a readily accessible (usable) form. We argue that a kind of associative memory must be an embedded feature of all adaptive systems, among them, of cancers, since autonomous functioning, adaptation and development are unimaginable without the available information on previously experienced states [5]. The physical basis of a system memory is closely related to real-time holographic mechanisms that are basic for any CAS. These poorly understood mechanisms that imply the wave-wave and wave-matter interactions ensure the unification and integration of many separate elements into an autonomously functioning system of interdependent agents (see below).

### **3.2. Collective behavior of malignant cells**

to a new or previously experienced situation. The listed hallmarks of autonomously functioning systems are unimaginable without synergy, which implies an orchestrated,

We argue that cancer has all the traits typical for any CAS: cancer cells are hard to destroy even by chemical toxins and radiation, since they coordinate their action in order to survive as an entity. Only united and self-organizing system of cooperating cells would be able to start the vital struggle against the powerful host-organism. The cancer-system shares the phase-space with the host-CAS which is its rival and breadwinner at the same time. New organism should either defeat its host, or, alternatively, obey its rules and commands adapting to the variable

There are about 200 types of cancers each type comprising multiple "families" and sub-types of cells. The scientists from the Wellcome Trust Sanger Institute in Hinxton, England, recently announced 73 different combinations of disease-causing mutations in the breast tumors each involving up to six different genes from a set of 40 driver genes [34]. Canadian researchers have shown that the cells taken from patients with acute lymphoblastic leukemia are actually composed of multiple families of genetically distinct leukemia cells [35]. No doubt, that the treatment of such a diverse pathology would not be efficient without understanding of the

What is the reason of cancer diversity? Are its cells the clones of distinct "cancer-stems" that originate simultaneously, or they emerge as new cells due to clashes with surrounding cells

We assume that an interaction of poorly differentiated cells with the bystander elements of stroma can yield various karyo- and phenotypes through the same mechanisms that take place in the first "nursery" of emerging cancer. The tumor micro-environment is a complex system of many cell types, including endothelial cells and their precursors, smooth-muscle cells, fibroblasts, granulocytes, lymphocytes, macrophages, etc. Taking into consideration the features of CAS, one can suggest that adaptation of young, meta-stable and extremely motile cancer cells to variable and heterogeneous micro-environment plays crucial role in the process of cell diversification; however, there is another possibility to provide diverse "stems" and their clones. This "fresh" idea about recurrent (iterative) cycles of carcinogenesis that imply successive production of less complex generations of malignant cells is described in section 7. Many cancers adapt to chemo- and radiation therapy: according to some researchers, the clonal selection leads to the resistance of recurrent tumors [36]. If "cancer-embryos" are nurtured in various conditions before they proceed to active life-cycling, they might give birth to distinct "clones". This process cannot be considered as selection, but as the emergence of new

It should be noted that the adaptation itself is not a well understood phenomenon. Elusive non-molecular processes of information exchange between the cells/tissues are difficult to study. As a result, we often ignore an obvious fact that no process of learning (gaining

synchronized and interdependent behavior of all system-components.

**3.1. Adaptive behavior and diversity of cancers**

588 Cancer Treatment - Conventional and Innovative Approaches

most general regulatory mechanisms common for all cancers.

that produce odds and ends of damaged cellular components?

organisms by the same scenario as in the first act of carcinogenesis.

ambience.

Cells and other elements of complex biological systems are functionally interdependent – they exhibit evident signs of collective behavior being organized as a hierarchy [37-39]. If cancers are integral and adaptive organisms, the action of malignant cells should be strictly coordinated. Indeed, nontrivial spatial correlations between malignant cells have been found by various researchers. The cooperative behavior, namely, collective migration of malignant cells during their invasion into healthy tissues seems to follow essentially the same pathways as healthy cells that participate in embryological development and damaged tissue reparation [40]. Cells performing collective migration share many biological characteristics with independently migrating cells but, by affecting one another mechanically and via signaling, these cell groups are subject to additional regulation and constraints [41, 42].

Experimental and clinical observations support the suggestion that cancer cells form a complex and integrated system. G. Lambert studied the collective response of breast cancer tissues to drug-induced stress and found a similarity between the rapid evolution of drug resistance in cancers and the behavior of bacterial colonies under starvation conditions [43]. Professor P. Davies, principal investigator of a major research program funded by the National Cancer Institute, argues that cancer is not a random bunch of selfish rogue cells behaving badly, but a highly-efficientpre-programmedresponse tostress,honedbya longperiodof evolution.[7, 44].

Hence, one can regard cancer as a life-tenacious organism created by and incorporated into relatively mature tissues of the host-body. This complex adaptive system, which is doomed to conduct a life-long battle with its superior ancestor - parental body, has its own powerful selfregulation mechanisms, a flexible primitive structure and enough power to hunt the preys – host cells.

### **4. New approach to the cancer non-invasive study**

Cancer remains an elusive, unpredictable and scary disease mostly because the malignant processes are difficult to detect and monitor. The most efficient methods of cancer conventional diagnostics are either harmful or too costly to be used in vivo as often as necessary. Oncologists lack a non-invasive, reliable, user-friendly, automatable and non-expensive test for tracking the malignant processes on the organism-level. We were lucky to find the solution to this problem thanks to an unexpected discovery of a previously unknown physical effect - "the holographic diffraction" which turned out to be characteristic of all biological objects [4-5, 45]. Detailed description of the innovative technology developed and tested by the authors was published earlier [46, 47]; in this manuscript we present concise information about this principally new approach to the detection and monitoring of malignant processes for a better understanding of our empiric data.

In observational research, results can be changed or biased by the act of measurement itself. The distal areas of human body are used as a source of information because they provide less distorted spectral information about entire body-state avoiding "an observer/measurement effect". A set of images-tomograms of 10 fingertips (each showing a 2D momentary "slice" of the 3D system) is recorded for the detection and monitoring of malignant processes in human body. The fingertips are exposed to electric impulses of distinct frequencies, so that one gets comparable information on the character of resonant responses to electric impulses of particular frequencies in 10 distant areas of human body (necessary for mapping of pathological process). The harmless and short-term procedure of a person examination can be

Life-Cycling of Cancer: New Concept http://dx.doi.org/10.5772/55385 591

In clinical practice various modalities are used for imaging of body parts, including radiography, computed tomography, magnetic resonance imaging (MRI), and positron emission tomography-computed tomography (PET-CT). All these modalities focus on particular areas of human body in order to get the images of organs/tissues that physicians need to examine. In BHT there is no necessity to screen entire body part by part, since the holography-based mechanisms spread the scaled information about the deviations from normal functioning of cells and tissues throughout the whole body acting akin to a wireless system of bio-communication. Experimental and clinical study of various patients (with and without diseases) enabled us to reveal some cancer-specific spectral signatures in fingertip BHT-grams [5, 48] that prompted subsequent research in oncology. The CID is a portable, easyto-use and non-costly tool of the whole body examination; it allows the determination of the spatiotemporal distribution of malignant processes throughout an intact organism. The CID-

Pre-existing devices of the same class [49, 50] failed to provide reliable and reproducible information on cancer-specific emission and on the dynamics of systems. In order to stabilize the air discharge plasma and obtain the informative optical data it became indispensable to modify the device and alter the examination procedure. We have filtered out the most variable and non-informative spectral components thus getting reproducible and comparable recordings of fingertip emission. Stabilization of the discharge plasma and improvement of data quality have been achieved through the limitation of the gas transit-time across the

It became necessary to conduct a plethora of probes on hundreds of patients with distinct types and stages of cancer before we understood the principles of the holographic imaging and developed the system of data interpretation. Close collaboration with clinicians made it possible to define the matrix of correlations between clinical diagnoses and spectral information obtained in various conditions of data acquisition. Experimental and clinical work conducted during several years led us to the conclusion that interference patterns emitted from body surfaces in response to high frequency electric impulses carry encoded information on the shapes, densities, complexity and dynamic features of the most problematic areas/

system is already implemented in routine diagnostic practice.

discharge zone, the restriction of particle upward scattering/dissipation, etc.

processes disregarding their type, size and location in human body (fig. 2).

conducted as often as necessary.

The computer-assessed diagnostic system "CID" provides reliable and comprehensive spectral information valuable for non-invasive detection and monitoring of malignant processes of any location and type [5]. The CID-system belongs to the class of the imaging technology dubbed BHT (Bio-holographic tomography) which is both – a diagnostic and research tool. The device is not cumbersome or difficult to operate: examinations can be conducted right at a patient's bed and the interface is so simple that even novice users can collect data in the form of BHT-grams. The whole procedure of the BHT-examination lasts several minutes: distal body-parts (usually 10 fingertips) are exposed to the pulsed electric fields that are strong enough to initiate the discharge of air; the relaxation of excited atoms and molecules in ionized gas produces optical radiation, which is captured for further processing and analysis; cancer-specific optical signatures are determined by analyzing effects of electric impulses on the body distal "terminals". A computer operates the device and performs analyses of recordings (fig. 1).

**Figure 1.** BHT-examination implies the recording of ten fingertips' emission that takes only 2-3 minutes.

In observational research, results can be changed or biased by the act of measurement itself. The distal areas of human body are used as a source of information because they provide less distorted spectral information about entire body-state avoiding "an observer/measurement effect". A set of images-tomograms of 10 fingertips (each showing a 2D momentary "slice" of the 3D system) is recorded for the detection and monitoring of malignant processes in human body. The fingertips are exposed to electric impulses of distinct frequencies, so that one gets comparable information on the character of resonant responses to electric impulses of particular frequencies in 10 distant areas of human body (necessary for mapping of pathological process). The harmless and short-term procedure of a person examination can be conducted as often as necessary.

the malignant processes on the organism-level. We were lucky to find the solution to this problem thanks to an unexpected discovery of a previously unknown physical effect - "the holographic diffraction" which turned out to be characteristic of all biological objects [4-5, 45]. Detailed description of the innovative technology developed and tested by the authors was published earlier [46, 47]; in this manuscript we present concise information about this principally new approach to the detection and monitoring of malignant processes for a better

The computer-assessed diagnostic system "CID" provides reliable and comprehensive spectral information valuable for non-invasive detection and monitoring of malignant processes of any location and type [5]. The CID-system belongs to the class of the imaging technology dubbed BHT (Bio-holographic tomography) which is both – a diagnostic and research tool. The device is not cumbersome or difficult to operate: examinations can be conducted right at a patient's bed and the interface is so simple that even novice users can collect data in the form of BHT-grams. The whole procedure of the BHT-examination lasts several minutes: distal body-parts (usually 10 fingertips) are exposed to the pulsed electric fields that are strong enough to initiate the discharge of air; the relaxation of excited atoms and molecules in ionized gas produces optical radiation, which is captured for further processing and analysis; cancer-specific optical signatures are determined by analyzing effects of electric impulses on the body distal "terminals". A computer operates the device and performs

**Figure 1.** BHT-examination implies the recording of ten fingertips' emission that takes only 2-3 minutes.

understanding of our empiric data.

590 Cancer Treatment - Conventional and Innovative Approaches

analyses of recordings (fig. 1).

In clinical practice various modalities are used for imaging of body parts, including radiography, computed tomography, magnetic resonance imaging (MRI), and positron emission tomography-computed tomography (PET-CT). All these modalities focus on particular areas of human body in order to get the images of organs/tissues that physicians need to examine. In BHT there is no necessity to screen entire body part by part, since the holography-based mechanisms spread the scaled information about the deviations from normal functioning of cells and tissues throughout the whole body acting akin to a wireless system of bio-communication. Experimental and clinical study of various patients (with and without diseases) enabled us to reveal some cancer-specific spectral signatures in fingertip BHT-grams [5, 48] that prompted subsequent research in oncology. The CID is a portable, easyto-use and non-costly tool of the whole body examination; it allows the determination of the spatiotemporal distribution of malignant processes throughout an intact organism. The CIDsystem is already implemented in routine diagnostic practice.

Pre-existing devices of the same class [49, 50] failed to provide reliable and reproducible information on cancer-specific emission and on the dynamics of systems. In order to stabilize the air discharge plasma and obtain the informative optical data it became indispensable to modify the device and alter the examination procedure. We have filtered out the most variable and non-informative spectral components thus getting reproducible and comparable recordings of fingertip emission. Stabilization of the discharge plasma and improvement of data quality have been achieved through the limitation of the gas transit-time across the discharge zone, the restriction of particle upward scattering/dissipation, etc.

It became necessary to conduct a plethora of probes on hundreds of patients with distinct types and stages of cancer before we understood the principles of the holographic imaging and developed the system of data interpretation. Close collaboration with clinicians made it possible to define the matrix of correlations between clinical diagnoses and spectral information obtained in various conditions of data acquisition. Experimental and clinical work conducted during several years led us to the conclusion that interference patterns emitted from body surfaces in response to high frequency electric impulses carry encoded information on the shapes, densities, complexity and dynamic features of the most problematic areas/ processes disregarding their type, size and location in human body (fig. 2).

**Figure 2.** The geometry and texture of fingertip BHT-grams is altered according to certain characteristics of the most affected tissues and organs: A - a healthy person's uniform emission; B – a case of prostate cancer (a chestnut–like flattened shape); C – cancer of the left kidney (the shape of a bean); D – the lactation state; E – gastrointestinal cancer; F – the shoulder malfunctioning (complex elongated shape); G – lung cancer, advanced stage; H – colorectal cancer (terminal stage).

A system in a quasi-balanced state radiates evenly thanks to intrinsic processes of the destructive interference (similar waves propagating in opposite directions cancel one other and do not affect neighboring waves), whereas any perturbation caused by pathological processes results in constructive interference and phase-shifts that upsets the whole system of interdependent waves. Actually, all non-uniformities on fingertip BHT-grams represent the interference patterns, namely the replicas-holograms of the most malfunctioning tissues and cells – the source of wave-imbalance.

**Figure 3.** Holograms of the small body-structures are displayed on BHT-grams like in a microscope (see section 5 for explanations of the scaling effects). Two fingertip "coronas" and their enlarged parts are shown next to photographs of similar pathologies. Red arrows (upper images) point to a very bright inclusion embedded into a dark zone. Such a contrast between bright "spot" and dark background is typical for invasive cancer: the degrading host-cells around invasive tumors emit weakly. In the case of benign diverticula (bottom image, white arrows) no signs of degradation

Life-Cycling of Cancer: New Concept http://dx.doi.org/10.5772/55385 593

**5. Spectral information is distinct in benign and malignant cases of**

Waves play enormous role in integration and self-organization of complex adaptive systems, though their contribution to the functioning of human body is still grossly underestimated. Some oscillatory and cyclic bio-activities are studied in the process of functional diagnostics in conventional medicine (e.g., ECG, EEG, etc.); however, organization of nonlinear waves within intact body (especially on micro- and nano-scales) was never explored experimentally. All physical objects radiate in response to incident electromagnetic waves that are always present in the environment. The spectrum emitted by simple objects such as particles, atoms, molecules and chemical substances, can be recorded and studied much easier than that of complex dynamical systems where internal waves interact with each other. Natural radiation

are present next to rounded inclusions and the center of relatively intensive emission.

**pathology**

This extraordinary capability of system-waves to scale the information on any abnormal process and to deliver it to all body-elements enables the BHT-analysts to observe many structural nuances of pathological areas like in a microscope (fig.3).

The discovery of the astonishing peculiarity of biological systems that act like "bio-micro‐ scopes" became a great stimulus for subsequent theoretical and experimental research. This natural phenomenon enabled us to get and analyze the interference patterns/holograms of real anatomic structures using human fingertips as a source of the otherwise invisible and non-measurable information. New approach to the evaluation of the body problematic areas can be referred to as the "Holographic Imaging". Owing to non-locality of holographic in‐ formation and because of spectral differences between immature cancer-cells and differenti‐ ated host-cells, it has become possible to detect malignant pathology with high accuracy [45]. It should be noted that contrary to the spectral analysis of BHT-grams, the visual inter‐ pretation of the holographic replicas is not an automatable task.

**Figure 2.** The geometry and texture of fingertip BHT-grams is altered according to certain characteristics of the most affected tissues and organs: A - a healthy person's uniform emission; B – a case of prostate cancer (a chestnut–like flattened shape); C – cancer of the left kidney (the shape of a bean); D – the lactation state; E – gastrointestinal cancer; F – the shoulder malfunctioning (complex elongated shape); G – lung cancer, advanced stage; H – colorectal cancer

A system in a quasi-balanced state radiates evenly thanks to intrinsic processes of the destructive interference (similar waves propagating in opposite directions cancel one other and do not affect neighboring waves), whereas any perturbation caused by pathological processes results in constructive interference and phase-shifts that upsets the whole system of interdependent waves. Actually, all non-uniformities on fingertip BHT-grams represent the interference patterns, namely the replicas-holograms of the most malfunctioning tissues and

This extraordinary capability of system-waves to scale the information on any abnormal process and to deliver it to all body-elements enables the BHT-analysts to observe many

The discovery of the astonishing peculiarity of biological systems that act like "bio-micro‐ scopes" became a great stimulus for subsequent theoretical and experimental research. This natural phenomenon enabled us to get and analyze the interference patterns/holograms of real anatomic structures using human fingertips as a source of the otherwise invisible and non-measurable information. New approach to the evaluation of the body problematic areas can be referred to as the "Holographic Imaging". Owing to non-locality of holographic in‐ formation and because of spectral differences between immature cancer-cells and differenti‐ ated host-cells, it has become possible to detect malignant pathology with high accuracy [45]. It should be noted that contrary to the spectral analysis of BHT-grams, the visual inter‐

structural nuances of pathological areas like in a microscope (fig.3).

pretation of the holographic replicas is not an automatable task.

(terminal stage).

cells – the source of wave-imbalance.

592 Cancer Treatment - Conventional and Innovative Approaches

**Figure 3.** Holograms of the small body-structures are displayed on BHT-grams like in a microscope (see section 5 for explanations of the scaling effects). Two fingertip "coronas" and their enlarged parts are shown next to photographs of similar pathologies. Red arrows (upper images) point to a very bright inclusion embedded into a dark zone. Such a contrast between bright "spot" and dark background is typical for invasive cancer: the degrading host-cells around invasive tumors emit weakly. In the case of benign diverticula (bottom image, white arrows) no signs of degradation are present next to rounded inclusions and the center of relatively intensive emission.

### **5. Spectral information is distinct in benign and malignant cases of pathology**

Waves play enormous role in integration and self-organization of complex adaptive systems, though their contribution to the functioning of human body is still grossly underestimated. Some oscillatory and cyclic bio-activities are studied in the process of functional diagnostics in conventional medicine (e.g., ECG, EEG, etc.); however, organization of nonlinear waves within intact body (especially on micro- and nano-scales) was never explored experimentally.

All physical objects radiate in response to incident electromagnetic waves that are always present in the environment. The spectrum emitted by simple objects such as particles, atoms, molecules and chemical substances, can be recorded and studied much easier than that of complex dynamical systems where internal waves interact with each other. Natural radiation of human body is extremely variable and weak; besides, any perturbation of observable waves causes their instant change, so direct measurements cannot provide reliable spectral information. Only resonant enhancement of body emission and its distant probing can bring the relevant data on the dynamics and character of intrinsic processes. In our model of carcinogenesis we focus mainly on dynamic processes and interactions rather than on various participants (the solid components) in biological processes.

than 99% confidence interval in a preclinical rat breast cancer model and define cancer

Life-Cycling of Cancer: New Concept http://dx.doi.org/10.5772/55385 595

**•** Extremely detailed study of cells in their natural state without the need of fixatives has been

**•** Surface-enhanced Raman spectroscopy in conjunction with imaging was found to be

**•** Fluorescence emission spectrum of blood components was found to be efficient in distinguishing normal from early-stage and advanced-stage breast cancer. The sensitivity and specificity of the method are 80.4% and 100%, respectively, in distinguishing subjects

**•** Fourier Transform Infrared (FTIR) spectroscopic studies and Fluorescence Emission Spectroscopy (FES) have been effectively employed in the qualitative and quantitative analyses of rat tissues. The study showed that the spectral profiles are different when the

**•** Near-infrared light (NIR) is used to differentiate oxygenated vs. deoxygenated forms of hemoglobin and myoglobin. Illumination of intact tissue with NIR allows qualitative

**•** Over the last few years infrared microspectroscopy has been used to study cells and tissues. Research work is now aimed at characterizing spectral biomarkers for cancer diagnosis [61]. Dynamic IR imaging with image-processing-guided frequency analysis is a promising modality for breast cancer detection and may not have the tissue-dependent limitations of mammography. The IR imaging process recognizes the cancer area independently of tissue

**•** Photoacoustic tomography (PAT) is an automatable emerging technique for spectroscopic analysis and imaging live tissue at depths up to 10 cm for detecting tumors and cancer research. The method enables in vivo study of melanomas with both exquisite sensitivity and high specificity [63, 64]. Besides, it can provide anatomical, functional, metabolic, molecular, and genetic contrasts of vasculature, hemodynamics, oxygen metabolism,

**•** Angle-resolved low coherence interferometry (a/LCI) during endoscopic examination has been found to be convenient for esophageal cancer diagnosis [65]. Physicians shine short bursts of light at locations of suspected disease and sensors capture and analyze the light

It is evident that spectral characteristics of actively developing immature cells differ from the emission of normal cells due to increased metabolic rate and proliferative activity of cancercells. Much more difficult is to explain how a small cluster of malignant cells alters the emission

**The "Holographic Imaging" of abnormally functioning internal structures** through nontransparent body is, in fact, a mind-boggling effect. Nobody could ever imagine that it was

informative in the studies of the chemical composition of the live cells [57].

assessment of changes in the tissue concentration of these molecules [60].

density, cancer size, and cancer appearance on mammography [62].

boundaries in fresh unstained tissues.

performed through Raman spectroscopic analysis [56].

tissue of a particular organ is affected with tumor [59].

with breast cancer from normal controls [58].

biomarkers, and gene expression.

of distant body-parts, e.g., human fingertips.

as it is reflected back.

The hardware-software system "CID" records a resonant response of a body to applied electric impulses of high frequencies. This spectral information is valuable for investigation and understanding of yet unknown functional mechanisms in human organism. An elusive and extremely fragile system of organized and interdependent physical waves of various types ensures not only the self-control and self-regulation of the system, but also its interaction with ambient waves and fields [5].

Waves are carriers of energy and information, so their internal "life" and exchange with environment is worth to study. One can ask whether various non-molecular signals, waves and their interference patterns emitted by nontransparent and dynamic organisms carry nondistorted (and interpretable) information on the state of various tissues, cells, intercellular communications and other peculiarities of internal processes. Experimental findings of many researchers prove that the answer to the posed question is positive: G. Hyland has shown that some biological objects emit highly focused coherent electromagnetic waves of ultra-low intensity. The author assumes that such an emission is an outward sign of an orderly functioning metabolism [51]. Japanese researchers caught sound waves generated by bacteria and showed that bacterial cells can enhance the proliferation of neighboring cells through acoustic waves. It is suggested that sounds can function as growth-regulatory signals for entire colony of cells [52]. The alteration of bacterial growth and the synchronization of light emission of adjacent cultures were observed by M. Trushin [53]. An ability of placental mammalian cells to generate pulsating light signals in response to near-ultraviolet light irradiation was discovered by G. Albrecht-Buehler [54]. Such a reversible enhancement of autofluorescence can be used by cells for the "quorum sensing" and coordinated action. And finally, our own experimental and clinical data provide arguments on behalf of the well organized system of interacting waves whose rules and mechanisms are already disclosed (at least partially). The coordinated vibrations and waves of a system medium turned out to be crucial for system integrity and self-regulation via real-time holographic mechanisms (see below).

Although the study of weak radiation of complex biological objects is still in its infancy, spectral analysis of cells, tissues and entire organisms offers great potential being a source of readily automatable biomedical information. The development of spectral methods for Biomedicine was prompted by recent advances in computer sciences, since enormous amount of spectral data requires specific tools and appropriate concepts for data interpretation. There are many approaches to spectroscopic studies of biological samples. Here are some examples that demonstrate the usefulness of spectra for medical studies:

**•** American researchers developed a novel microscopy technique, called nonlinear interferometric vibrational imaging (NIVI) intended for quantitative analysis of tissue specimens [55]. The NIVI can differentiate cancer versus normal tissue sections with greater than 99% confidence interval in a preclinical rat breast cancer model and define cancer boundaries in fresh unstained tissues.

of human body is extremely variable and weak; besides, any perturbation of observable waves causes their instant change, so direct measurements cannot provide reliable spectral information. Only resonant enhancement of body emission and its distant probing can bring the relevant data on the dynamics and character of intrinsic processes. In our model of carcinogenesis we focus mainly on dynamic processes and interactions rather than on various

The hardware-software system "CID" records a resonant response of a body to applied electric impulses of high frequencies. This spectral information is valuable for investigation and understanding of yet unknown functional mechanisms in human organism. An elusive and extremely fragile system of organized and interdependent physical waves of various types ensures not only the self-control and self-regulation of the system, but also its interaction with

Waves are carriers of energy and information, so their internal "life" and exchange with environment is worth to study. One can ask whether various non-molecular signals, waves and their interference patterns emitted by nontransparent and dynamic organisms carry nondistorted (and interpretable) information on the state of various tissues, cells, intercellular communications and other peculiarities of internal processes. Experimental findings of many researchers prove that the answer to the posed question is positive: G. Hyland has shown that some biological objects emit highly focused coherent electromagnetic waves of ultra-low intensity. The author assumes that such an emission is an outward sign of an orderly functioning metabolism [51]. Japanese researchers caught sound waves generated by bacteria and showed that bacterial cells can enhance the proliferation of neighboring cells through acoustic waves. It is suggested that sounds can function as growth-regulatory signals for entire colony of cells [52]. The alteration of bacterial growth and the synchronization of light emission of adjacent cultures were observed by M. Trushin [53]. An ability of placental mammalian cells to generate pulsating light signals in response to near-ultraviolet light irradiation was discovered by G. Albrecht-Buehler [54]. Such a reversible enhancement of autofluorescence can be used by cells for the "quorum sensing" and coordinated action. And finally, our own experimental and clinical data provide arguments on behalf of the well organized system of interacting waves whose rules and mechanisms are already disclosed (at least partially). The coordinated vibrations and waves of a system medium turned out to be crucial for system

integrity and self-regulation via real-time holographic mechanisms (see below).

demonstrate the usefulness of spectra for medical studies:

Although the study of weak radiation of complex biological objects is still in its infancy, spectral analysis of cells, tissues and entire organisms offers great potential being a source of readily automatable biomedical information. The development of spectral methods for Biomedicine was prompted by recent advances in computer sciences, since enormous amount of spectral data requires specific tools and appropriate concepts for data interpretation. There are many approaches to spectroscopic studies of biological samples. Here are some examples that

**•** American researchers developed a novel microscopy technique, called nonlinear interferometric vibrational imaging (NIVI) intended for quantitative analysis of tissue specimens [55]. The NIVI can differentiate cancer versus normal tissue sections with greater

participants (the solid components) in biological processes.

594 Cancer Treatment - Conventional and Innovative Approaches

ambient waves and fields [5].


It is evident that spectral characteristics of actively developing immature cells differ from the emission of normal cells due to increased metabolic rate and proliferative activity of cancercells. Much more difficult is to explain how a small cluster of malignant cells alters the emission of distant body-parts, e.g., human fingertips.

**The "Holographic Imaging" of abnormally functioning internal structures** through nontransparent body is, in fact, a mind-boggling effect. Nobody could ever imagine that it was possible to observe the structural and functional nuances of internal cells, tissues and microscopic areas via assessment of fingertip emission; neither could it be suspected that our organism is able to scale the holograms of real anatomic structures and to expose on a huge scale only those cells and tissues that do not obey the general rules of entire system. This "holographic imaging" is a physical phenomenon and it has been explained as a manifestation of background activity of the system nonlinear medium (phase-space) that acts akin to an organizing holographic grating of a body [5].

**The background order within the medium/space of a system** can explain many peculiarities of CASs. This unifying and organizing realm of a system must be preserved during the whole life-cycle; obviously, the ordered motion of a system-medium and the wave interactions set at initial stage of the system-genesis become more and more complex in parallel with its growth and development. The invisible activity of waves in the phase-space occupied by a CAS can be considered as a "wireless" system of communication between all system-components.

Life-Cycling of Cancer: New Concept http://dx.doi.org/10.5772/55385 597

Information propagates in the form of a signal or a message that cannot alter behavior of the solid matter directly but can instead be sent simultaneously to all system-waves. Obviously, diverse "recipients" of information would not react to one and the same message in a similar way; however, weak interactions ensure delivering of a message to a large "audience", actually to the whole system, so that the instructions and commands would not miss their targets.

The question arises whether there are any specific mechanisms that a biological system utilizes for the reinforcement/amplification of the most urgent and/or essential information; it is also very important to understand how a system controls its "misbehaving agents" and which mechanisms are able to transform weak signals into an effective force? We have reasoned that the system-mechanisms of self-control and self-organization require the interaction between weak (information-associated) and strong (energy-associated) waves; powerful or focused waves can play the role of mediators between the information-associated processes and the processes that affect distinct particles, molecules and cells. The reinforcement of information without actual participation of the solid matter in the process of signal amplification is possible

**The holographic principle and real-time holography** are the only concepts that can explain the imaging of scaled internal structures on the surfaces of autonomous systems. A characteristic feature of any static and dynamic hologram is that any part of a holographic record can be used for the reconstruction of the whole recorded scene. In physics the principle of holography implies that information about a 3D space-volume is encoded in 2D form on its boundary [67-69]. We argue that permanent encoding and decoding of information is a natural phenomenon specific for all autonomously functioning systems; it should not be confused with

The real-time holography enables a rapid successive recording and read-out of the information (interference patterns); in the case of a CAS the amount of the processed information can be very high (terabits/s), since the operation is performed in parallel within the entire volume. When creating a hologram, the ordered reference waves (aka the ordered medium-waves of the body) interfere with disordered waves generated by perturbed waves/particles. This information can be reconstructed if the reference waves are subtracted, e.g., by conjugated waves that propagate in the opposite direction. The original object's field/image is reconstructed when the waves deflect in the hologram structure. The refresh rate (update) of information correlates with the periods of phase-conjugated waves, so the reaction of the entire system to any disorder in a high frequency range would be much more "acute" than in the

via the holography-based mechanisms.

the conventional process of technical holography.

case of a mismatch in slower processes.

In complex adaptive systems (CAS), where all components are well-controlled and there exists a strong subordination between the levels of a system hierarchy, a permanent interaction of "each" and "all" (non-locality) is of utmost importance. In order to achieve interdependence of all agents of a CAS, the periodic grating and synchronicity of vibrations within whole medium must be set from the very first moments of a new system emergence. In the next section we will discuss the role of focused coherent waves in the processes of a systemunification that makes it possible to create an integral system of cooperatively acting agents out of separate elements.

Can the waves generated within human body affect the motion of small neutral particles, molecules and cells? Physicists know that certain waves (e.g., light) can serve to bind neutral matter in new organized forms. It has been established that high frequency oscillations of intense fields interact with micron-size dielectric objects trapping and bounding small particles. The artificial holographic/diffractive setups allow the simultaneous production of very high numbers of such traps generated by superimposing coherent beams either through the wave-interference or through the interaction of several beams previously fanned-out by diffractive optical elements [66]. Hence, a kind of feedback interactions really exists between the waves and solid particles of a CAS. If the suggestion about interdependent action of all system-waves and solid "particles" is correct, the medium waves of a biological system would mirror the state of corresponding solid elements (atoms, molecules, cells, etc.) as all these "agents" are enclosed in the partially bounded space. Any alteration in one of these two complementary realms would affect another - either directly or via some intermediate mechanisms; so, one can evaluate the system-wave behavior/patterns (interference) in order to get information on both - the features of background waves and the state of their complementary (solid) structures.

The permanent wave-wave and wave-matter interactions within a bounded space can explain the effect of the "holographic imaging" discovered by our team 12 years ago; it was an exciting day when we were all huddled round the computer puzzled by the similarity between some BHT-grams and real anatomic structures (see section 8).

As mentioned earlier, an integral system of interfering waves is too sensitive to be studied directly: the wave functions collapse as soon as an observer tries to probe this fragile "structure". That is why we take only the most distant minor areas of human body for BHTexamination – the minor "terminals" of a system provide us with less perturbed systeminformation.

**The background order within the medium/space of a system** can explain many peculiarities of CASs. This unifying and organizing realm of a system must be preserved during the whole life-cycle; obviously, the ordered motion of a system-medium and the wave interactions set at initial stage of the system-genesis become more and more complex in parallel with its growth and development. The invisible activity of waves in the phase-space occupied by a CAS can be considered as a "wireless" system of communication between all system-components.

possible to observe the structural and functional nuances of internal cells, tissues and microscopic areas via assessment of fingertip emission; neither could it be suspected that our organism is able to scale the holograms of real anatomic structures and to expose on a huge scale only those cells and tissues that do not obey the general rules of entire system. This "holographic imaging" is a physical phenomenon and it has been explained as a manifestation of background activity of the system nonlinear medium (phase-space) that acts akin to an

In complex adaptive systems (CAS), where all components are well-controlled and there exists a strong subordination between the levels of a system hierarchy, a permanent interaction of "each" and "all" (non-locality) is of utmost importance. In order to achieve interdependence of all agents of a CAS, the periodic grating and synchronicity of vibrations within whole medium must be set from the very first moments of a new system emergence. In the next section we will discuss the role of focused coherent waves in the processes of a systemunification that makes it possible to create an integral system of cooperatively acting agents

Can the waves generated within human body affect the motion of small neutral particles, molecules and cells? Physicists know that certain waves (e.g., light) can serve to bind neutral matter in new organized forms. It has been established that high frequency oscillations of intense fields interact with micron-size dielectric objects trapping and bounding small particles. The artificial holographic/diffractive setups allow the simultaneous production of very high numbers of such traps generated by superimposing coherent beams either through the wave-interference or through the interaction of several beams previously fanned-out by diffractive optical elements [66]. Hence, a kind of feedback interactions really exists between the waves and solid particles of a CAS. If the suggestion about interdependent action of all system-waves and solid "particles" is correct, the medium waves of a biological system would mirror the state of corresponding solid elements (atoms, molecules, cells, etc.) as all these "agents" are enclosed in the partially bounded space. Any alteration in one of these two complementary realms would affect another - either directly or via some intermediate mechanisms; so, one can evaluate the system-wave behavior/patterns (interference) in order to get information on both - the features of background waves and the state of their

The permanent wave-wave and wave-matter interactions within a bounded space can explain the effect of the "holographic imaging" discovered by our team 12 years ago; it was an exciting day when we were all huddled round the computer puzzled by the similarity between some

As mentioned earlier, an integral system of interfering waves is too sensitive to be studied directly: the wave functions collapse as soon as an observer tries to probe this fragile "structure". That is why we take only the most distant minor areas of human body for BHTexamination – the minor "terminals" of a system provide us with less perturbed system-

organizing holographic grating of a body [5].

596 Cancer Treatment - Conventional and Innovative Approaches

out of separate elements.

complementary (solid) structures.

information.

BHT-grams and real anatomic structures (see section 8).

Information propagates in the form of a signal or a message that cannot alter behavior of the solid matter directly but can instead be sent simultaneously to all system-waves. Obviously, diverse "recipients" of information would not react to one and the same message in a similar way; however, weak interactions ensure delivering of a message to a large "audience", actually to the whole system, so that the instructions and commands would not miss their targets.

The question arises whether there are any specific mechanisms that a biological system utilizes for the reinforcement/amplification of the most urgent and/or essential information; it is also very important to understand how a system controls its "misbehaving agents" and which mechanisms are able to transform weak signals into an effective force? We have reasoned that the system-mechanisms of self-control and self-organization require the interaction between weak (information-associated) and strong (energy-associated) waves; powerful or focused waves can play the role of mediators between the information-associated processes and the processes that affect distinct particles, molecules and cells. The reinforcement of information without actual participation of the solid matter in the process of signal amplification is possible via the holography-based mechanisms.

**The holographic principle and real-time holography** are the only concepts that can explain the imaging of scaled internal structures on the surfaces of autonomous systems. A characteristic feature of any static and dynamic hologram is that any part of a holographic record can be used for the reconstruction of the whole recorded scene. In physics the principle of holography implies that information about a 3D space-volume is encoded in 2D form on its boundary [67-69]. We argue that permanent encoding and decoding of information is a natural phenomenon specific for all autonomously functioning systems; it should not be confused with the conventional process of technical holography.

The real-time holography enables a rapid successive recording and read-out of the information (interference patterns); in the case of a CAS the amount of the processed information can be very high (terabits/s), since the operation is performed in parallel within the entire volume. When creating a hologram, the ordered reference waves (aka the ordered medium-waves of the body) interfere with disordered waves generated by perturbed waves/particles. This information can be reconstructed if the reference waves are subtracted, e.g., by conjugated waves that propagate in the opposite direction. The original object's field/image is reconstructed when the waves deflect in the hologram structure. The refresh rate (update) of information correlates with the periods of phase-conjugated waves, so the reaction of the entire system to any disorder in a high frequency range would be much more "acute" than in the case of a mismatch in slower processes.

We argue that the holographic mechanisms play crucial role in the self-organization of any CAS. These mechanisms imply existence of a hidden order in the background medium where all waves comprise a harmonious structure of vibrations and standing waves; the same mechanisms are critical for the adaptation (decentralized memory) and the resilience of biological systems. Any perturbation, disregarding its actual cause and culprit, would result in constructive interference and phase-shifts of corresponding waves thus altering the entire (scale-invariant) system of background harmonics. Besides, the principle of holography makes it possible to observe the most disordered tissues and organs via assessment of their holographic replicas on distant surfaces of a system (e.g., fingertip BHT-grams), since the "whole" and its "part" can equally reconstruct the entire "holo-image".

In the cases of cancer, two autonomously functioning entities occupy a shared phase-space and compete for available resources. The conflicting organisms that are "trapped" within a shared body are not able to synchronize their individual rhythms and achieve a state of a quasi-balance. Hence, the BHT-grams of the patients with cancer would display the replicas (interference patterns) of aggressive neoplasm with huge resolution. In certain cases of ma‐ lignant pathology the fingertip "coronas" demonstrate dark areas around brighter inclu‐ sions that present the "remnants" of cells destroyed by cancer-cells (fig.3 and fig. 4,a); The effect of illumination by diffuse light is a BHT-characteristic of actively proliferating non-dif‐

Life-Cycling of Cancer: New Concept http://dx.doi.org/10.5772/55385 599

Waves – the carriers of energy and information - are the sole candidates to perform the task of information reinforcement in living systems. In a bounded system of interdependent mechanical and electromagnetic waves of various intensities, wavelengths and frequencies, any perturbation propagates throughout the entire system. The higher coherence and intensity of waves the greater their influence on the solid matter. The interaction of the informationand energy-associated events ensures synergy and coordination of all system-components. The recently discovered physical effect of "Random Lasing" [2, 3] which implies the focusing and amplification of light in a non-uniform and disordered medium, e.g., in biological tissues, casts new light on the interaction between information and energy-related biological mechanisms. The reinforcement of information through the real-time holographic mechanisms differs from the principles that focus and amplify waves in random lasing; the random lasing implies a complex process of wave-trapping and releasing by disordered excitable material. Emitted waves become much more focused and coherent than those that have been initially

Conventional lasers that amplify light through the stimulation of photonic emission, require an excitable medium (gain medium) and some feedback mechanisms that temporarily trap the light before emitting a narrower spectrum beams. Usually the gain medium in lasers is excited by pumped energy supplied as an electrical current, or as light of different wavelength, while

Back in 2000, several teams of researchers announced the creation of microlasers exploiting a disordered dielectric material as gain medium [70, 71]. A disordered material that comprises the scattering elements in random positions was found capable to exhibit a laser-like behavior [72]. Electromagnetic waves bounce from one scattering center/cavity to another and such a recurrent scattering on a microscopic length scale temporarily traps light. Hence, the random lasers do not possess large cavity or mirrors typical for conventional lasers; they contain only multiple non-uniformities that scatter light (or other waves). Small irregularities in the material act just like artificial mirrors in laser resonators preventing the light from escaping too quickly. These non-uniformities can be presented by particles, bubbles, droplets of dye, density fluctuations in fluids, surface roughness, cells in organisms, textile fibers in clothing, etc. In

"arrested", which explains the term "lasing" (light amplification by lasers).

the photons are confined between mirrors in optical resonator.

ferentiated cells (fig. 4,b).

**6. Emergence of cancer via random lasing**

The scaling of information in a system of natural origin depends greatly on the frequency/ wavelengths of the most perturbed intrinsic waves. Thanks to the fractal nature of body wavestructure, its self-similarity and scale-invariance, the high frequency signals from excited cells (short waves correspond to small structures) can reach the body surface only after their scaling through the waves of lower frequency (longer waves correspond to larger structures of a system): the fingertips BHT-grams display the interference patterns with the resolution that is proportional to the frequency of constructively interfering waves.

On the way towards the body surface, the upward propagating waves of high frequency (complementary to cells and other microscopic structures) are scaled through the doubling of their amplitudes and periods at each successive level of the hierarchy; that is why the interference patterns/holograms of cells and their constellations are emitted with higher resolution compared to holograms of larger parts of the body. This peculiarity of the multilevel and self-similar structure of interacting waves enables us to observe and analyze the most active processes and also malignant cells/tissues via assessment of fingertip BHT-grams (see section 8 for examples of the cell-holograms).

**Figure 4.** Examples of cancer-signatures on fingertip BHT-grams. In the case of colorectal cancer (A) a part of growing neoplasm paves its way through degrading surrounding tissues (dark zone around to bright inclusion). In the case of malignant melanoma (B) the rapidly proliferating cancer-cells produce an effect of diffuse illumination. The multidir‐ ectional radiation that illuminates major parts of BHT-grams is a hallmark of the high frequency coherent emission generated by large conglomerates of poorly differentiated cells (see section 6 for more explanations).

In the cases of cancer, two autonomously functioning entities occupy a shared phase-space and compete for available resources. The conflicting organisms that are "trapped" within a shared body are not able to synchronize their individual rhythms and achieve a state of a quasi-balance. Hence, the BHT-grams of the patients with cancer would display the replicas (interference patterns) of aggressive neoplasm with huge resolution. In certain cases of ma‐ lignant pathology the fingertip "coronas" demonstrate dark areas around brighter inclu‐ sions that present the "remnants" of cells destroyed by cancer-cells (fig.3 and fig. 4,a); The effect of illumination by diffuse light is a BHT-characteristic of actively proliferating non-dif‐ ferentiated cells (fig. 4,b).

### **6. Emergence of cancer via random lasing**

We argue that the holographic mechanisms play crucial role in the self-organization of any CAS. These mechanisms imply existence of a hidden order in the background medium where all waves comprise a harmonious structure of vibrations and standing waves; the same mechanisms are critical for the adaptation (decentralized memory) and the resilience of biological systems. Any perturbation, disregarding its actual cause and culprit, would result in constructive interference and phase-shifts of corresponding waves thus altering the entire (scale-invariant) system of background harmonics. Besides, the principle of holography makes it possible to observe the most disordered tissues and organs via assessment of their holographic replicas on distant surfaces of a system (e.g., fingertip BHT-grams), since the

The scaling of information in a system of natural origin depends greatly on the frequency/ wavelengths of the most perturbed intrinsic waves. Thanks to the fractal nature of body wavestructure, its self-similarity and scale-invariance, the high frequency signals from excited cells (short waves correspond to small structures) can reach the body surface only after their scaling through the waves of lower frequency (longer waves correspond to larger structures of a system): the fingertips BHT-grams display the interference patterns with the resolution that is

On the way towards the body surface, the upward propagating waves of high frequency (complementary to cells and other microscopic structures) are scaled through the doubling of their amplitudes and periods at each successive level of the hierarchy; that is why the interference patterns/holograms of cells and their constellations are emitted with higher resolution compared to holograms of larger parts of the body. This peculiarity of the multilevel and self-similar structure of interacting waves enables us to observe and analyze the most active processes and also malignant cells/tissues via assessment of fingertip BHT-grams (see

**Figure 4.** Examples of cancer-signatures on fingertip BHT-grams. In the case of colorectal cancer (A) a part of growing neoplasm paves its way through degrading surrounding tissues (dark zone around to bright inclusion). In the case of malignant melanoma (B) the rapidly proliferating cancer-cells produce an effect of diffuse illumination. The multidir‐ ectional radiation that illuminates major parts of BHT-grams is a hallmark of the high frequency coherent emission

generated by large conglomerates of poorly differentiated cells (see section 6 for more explanations).

"whole" and its "part" can equally reconstruct the entire "holo-image".

proportional to the frequency of constructively interfering waves.

section 8 for examples of the cell-holograms).

598 Cancer Treatment - Conventional and Innovative Approaches

Waves – the carriers of energy and information - are the sole candidates to perform the task of information reinforcement in living systems. In a bounded system of interdependent mechanical and electromagnetic waves of various intensities, wavelengths and frequencies, any perturbation propagates throughout the entire system. The higher coherence and intensity of waves the greater their influence on the solid matter. The interaction of the informationand energy-associated events ensures synergy and coordination of all system-components.

The recently discovered physical effect of "Random Lasing" [2, 3] which implies the focusing and amplification of light in a non-uniform and disordered medium, e.g., in biological tissues, casts new light on the interaction between information and energy-related biological mechanisms. The reinforcement of information through the real-time holographic mechanisms differs from the principles that focus and amplify waves in random lasing; the random lasing implies a complex process of wave-trapping and releasing by disordered excitable material. Emitted waves become much more focused and coherent than those that have been initially "arrested", which explains the term "lasing" (light amplification by lasers).

Conventional lasers that amplify light through the stimulation of photonic emission, require an excitable medium (gain medium) and some feedback mechanisms that temporarily trap the light before emitting a narrower spectrum beams. Usually the gain medium in lasers is excited by pumped energy supplied as an electrical current, or as light of different wavelength, while the photons are confined between mirrors in optical resonator.

Back in 2000, several teams of researchers announced the creation of microlasers exploiting a disordered dielectric material as gain medium [70, 71]. A disordered material that comprises the scattering elements in random positions was found capable to exhibit a laser-like behavior [72]. Electromagnetic waves bounce from one scattering center/cavity to another and such a recurrent scattering on a microscopic length scale temporarily traps light. Hence, the random lasers do not possess large cavity or mirrors typical for conventional lasers; they contain only multiple non-uniformities that scatter light (or other waves). Small irregularities in the material act just like artificial mirrors in laser resonators preventing the light from escaping too quickly. These non-uniformities can be presented by particles, bubbles, droplets of dye, density fluctuations in fluids, surface roughness, cells in organisms, textile fibers in clothing, etc. In polymer films and biological tissues the lasing effects take place because of naturally formed cavities and non-uniformities that temporarily trap energy of waves through internal resonances.

Sugiura were the first to demonstrate that the field can be coupled to the particles in proximity on the order of 100 nanometers [80]. Optical interaction forces are able to organize microscopic objects with sub wavelength accuracy; they can be very long range and oscillate in sign at the optical wavelength [66, 81]. Continuous evanescent field that originates in conditions of multiple internal reflections within a small bounded area can guide a large number of particles

Life-Cycling of Cancer: New Concept http://dx.doi.org/10.5772/55385 601

The field-wave-matter interactions discussed above can be considered the key mechanisms of the self-organization in live cells, since a complex system of organized waves is able to direct

Random lasing creates a perfect order out of extreme disorder. This effect takes place in a chaotic excited medium and it might facilitate creation of a new ordered system out of "ashes" of the host-body degrading cells. Such "Phoenix Paradigm" was proposed by researchers of the Pittsburgh Cancer Institute: their experiments with the Kaposi Sarcoma-associated Herpesvirus resulted in the conclusion that excessive cell death, rather than its absence, may be a defining force that drives the cancer emergence [82]. In a stressful situation, e.g., when deprived of energy and oxygen, living cells can act as a gain medium for wave reinforcement. The increase in internal pool of energy that results in excitement of cellular matrix can be caused by many "cancer-promoting" factors: the degradation of intracellular substances, intrusion of some toxic substances or viruses/microbes into cells, increased temperature during inflammatory reactions, etc. can contribute to random lasing within a small bounded area; however, all these factors should be evaluated from the standpoint of their energy-associated

Coherent radiation of any cellular constellation can reach the body surface if cellular emission is strong and distinct from less intensive radiation of surrounding tissues. The signatures of random lasing are especially prominent on BHT-grams of the patients with aggressive

**Figure 5.** Examples of coherent beams produced by cancer cells. Enlarged and slightly contrasted parts of fingertip coro‐ nas are shown next to the raw BHT-grams. A – gastrointestinal cancer, ongoing radiation therapy; B – non-treated renal cancer with spreading metastasis. White arrows point to the scaled holograms of straight tiny lines (focused light).

into a preferred direction.

effects upon an emerging system.

malignant processes (fig. 5).

and unify diverse elements into an indivisible "whole".

Coherent amplified emission and dramatic spectral narrowing take place only if excitable medium gains energy above the threshold of its excitation [73, 74]. The random micro-laser characteristics can be tuned by varying the geometry of the scatterers' clusters, since each cluster operates at its own specific wavelength, depending on its shape and size.

In some cases constructive interference of backscattered waves brings transport of light to a complete halt (Anderson localization). Philip Anderson was awarded the Nobel Prize in physics for the theory of light localization in disordered medium [75]. In principle, not only electrons and photons, but actually any wave can be localized in a similar way: successful experiments aimed at the sound-wave localization in the strongly disordered 3D samples (composed of aluminum beads) have been described in 2008 [76].

The effects of light amplification and lasing have been found in various vegetable and animal tissues as well as in human tissues from various organs [77]. Even individual cells are capable to produce narrowband laser emission remaining alive after prolonged lasing action: these data were published by researchers from Harvard Medical School, who created biological cell "lasers" based on green fluorescent protein [78]. The team engineered human embryonic kidney cells to produce this protein; when they placed such a cell in the optical micro-resonator and exposed it to pulsed blue light, the cell started to emit a directional laser beam visible with the naked eye.

We have described the random lasing effect and wave interactions in detail because these findings elucidate the energy-mediated mechanism by which information in the form of weak waves affects inert material and creates an "order out of chaos" within the whole system (essential for the system-resilience); besides, the random lasing can account for the appearance of anaplastic cells – the process referred to as the "dedifferentiation" [79]. The spectra narrowing and light amplification are equally important for the understanding of cancer aggressive behavior as the focused light can readily destroy surrounding tissues and facilitate the neoplasm progression.

Indeed, intensity and character of lasing in malignant neoplasm were found to be distinct from benign tissues of the same origin. The Utah University researchers have demonstrated that the malignant colon tissues, when soaked in the laser dye Rhodamine 6G and excited by laser light, emit many more coherent lines than benign tissues in the same colon [2]. The disorder in cancerous tissue was much more chaotic than that in a benign tissue due to a mixture of distinct cells and processes of degradation; however, the increased intensity of coherent radiation in cancerous tissues is indicative of the aggressive behavior and active signaling between elements of neoplasm. The Utah University scientists have experimented with various healthy and cancerous colon tissues taken from different patients, as well as from other parts of the human body such as kidney, with very similar results.

It is acknowledged that the radiation pressure from the focused laser beams is able to trap and physically move small dielectric particles acting like a kind of tweezers. S. Kawata and T. Sugiura were the first to demonstrate that the field can be coupled to the particles in proximity on the order of 100 nanometers [80]. Optical interaction forces are able to organize microscopic objects with sub wavelength accuracy; they can be very long range and oscillate in sign at the optical wavelength [66, 81]. Continuous evanescent field that originates in conditions of multiple internal reflections within a small bounded area can guide a large number of particles into a preferred direction.

polymer films and biological tissues the lasing effects take place because of naturally formed cavities and non-uniformities that temporarily trap energy of waves through internal

Coherent amplified emission and dramatic spectral narrowing take place only if excitable medium gains energy above the threshold of its excitation [73, 74]. The random micro-laser characteristics can be tuned by varying the geometry of the scatterers' clusters, since each

In some cases constructive interference of backscattered waves brings transport of light to a complete halt (Anderson localization). Philip Anderson was awarded the Nobel Prize in physics for the theory of light localization in disordered medium [75]. In principle, not only electrons and photons, but actually any wave can be localized in a similar way: successful experiments aimed at the sound-wave localization in the strongly disordered 3D samples

The effects of light amplification and lasing have been found in various vegetable and animal tissues as well as in human tissues from various organs [77]. Even individual cells are capable to produce narrowband laser emission remaining alive after prolonged lasing action: these data were published by researchers from Harvard Medical School, who created biological cell "lasers" based on green fluorescent protein [78]. The team engineered human embryonic kidney cells to produce this protein; when they placed such a cell in the optical micro-resonator and exposed it to pulsed blue light, the cell started to emit a directional laser beam visible with

We have described the random lasing effect and wave interactions in detail because these findings elucidate the energy-mediated mechanism by which information in the form of weak waves affects inert material and creates an "order out of chaos" within the whole system (essential for the system-resilience); besides, the random lasing can account for the appearance of anaplastic cells – the process referred to as the "dedifferentiation" [79]. The spectra narrowing and light amplification are equally important for the understanding of cancer aggressive behavior as the focused light can readily destroy surrounding tissues and facilitate

Indeed, intensity and character of lasing in malignant neoplasm were found to be distinct from benign tissues of the same origin. The Utah University researchers have demonstrated that the malignant colon tissues, when soaked in the laser dye Rhodamine 6G and excited by laser light, emit many more coherent lines than benign tissues in the same colon [2]. The disorder in cancerous tissue was much more chaotic than that in a benign tissue due to a mixture of distinct cells and processes of degradation; however, the increased intensity of coherent radiation in cancerous tissues is indicative of the aggressive behavior and active signaling between elements of neoplasm. The Utah University scientists have experimented with various healthy and cancerous colon tissues taken from different patients, as well as from other parts of the

It is acknowledged that the radiation pressure from the focused laser beams is able to trap and physically move small dielectric particles acting like a kind of tweezers. S. Kawata and T.

cluster operates at its own specific wavelength, depending on its shape and size.

(composed of aluminum beads) have been described in 2008 [76].

resonances.

600 Cancer Treatment - Conventional and Innovative Approaches

the naked eye.

the neoplasm progression.

human body such as kidney, with very similar results.

The field-wave-matter interactions discussed above can be considered the key mechanisms of the self-organization in live cells, since a complex system of organized waves is able to direct and unify diverse elements into an indivisible "whole".

Random lasing creates a perfect order out of extreme disorder. This effect takes place in a chaotic excited medium and it might facilitate creation of a new ordered system out of "ashes" of the host-body degrading cells. Such "Phoenix Paradigm" was proposed by researchers of the Pittsburgh Cancer Institute: their experiments with the Kaposi Sarcoma-associated Herpesvirus resulted in the conclusion that excessive cell death, rather than its absence, may be a defining force that drives the cancer emergence [82]. In a stressful situation, e.g., when deprived of energy and oxygen, living cells can act as a gain medium for wave reinforcement. The increase in internal pool of energy that results in excitement of cellular matrix can be caused by many "cancer-promoting" factors: the degradation of intracellular substances, intrusion of some toxic substances or viruses/microbes into cells, increased temperature during inflammatory reactions, etc. can contribute to random lasing within a small bounded area; however, all these factors should be evaluated from the standpoint of their energy-associated effects upon an emerging system.

Coherent radiation of any cellular constellation can reach the body surface if cellular emission is strong and distinct from less intensive radiation of surrounding tissues. The signatures of random lasing are especially prominent on BHT-grams of the patients with aggressive malignant processes (fig. 5).

**Figure 5.** Examples of coherent beams produced by cancer cells. Enlarged and slightly contrasted parts of fingertip coro‐ nas are shown next to the raw BHT-grams. A – gastrointestinal cancer, ongoing radiation therapy; B – non-treated renal cancer with spreading metastasis. White arrows point to the scaled holograms of straight tiny lines (focused light).

### **7. New concept of cancer origin: Dramatic events within a "cancer-nursery"**

digesting" mechanism plays an essential role at all stages of the disease, since it helps to prevent tumor cell necrosis by mitigating metabolic stress while acting in concert with apoptosis [93]; the autophagy provides an alternate energy source by degrading damaged proteins and organelles that allow some tumor cells to survive during extended periods of starvation [94]. In the absence of phagocytes, apoptosis would be less efficient as the debris cannot be eliminated from the isolated "nursery" (the disposal of debris is necessary in apoptosis). So, the autophagy seems to dominate over apoptosis in early carcinogenesis though cooperation or alternated action of both mechanisms is not excluded especially just after cessation of the blood supply. Increasing evidence points to the selectivity of autophagy: it helps to "sort" vacuolar enzymes, to remove the aggregate-prone proteins and to destruct only excessive

Life-Cycling of Cancer: New Concept http://dx.doi.org/10.5772/55385 603

There is a kind of similarity between neoplasm and budding primitive organisms (see fig.11 in section 8 – holograms of proliferating cells). A key role of recycling of cellular organelles via autophagy and *de novo* purine biosynthesis was found while studying caloric restriction effects on the longevity of budding yeast (Saccharomyces cerevisiae). This yeast is an effective model for the analysis of genes and cellular pathways. Researchers have shown that additional genes appear to contribute to the restriction of either amino acids or sugar, and that defects in autophagy prevent lifespan extension induced by limitation of nutrients in the growth media [96]. An international team of researchers found that the autophagy helps some starving cells to recover, whereas the cells with a disrupted mitochondrial transmembrane potential

Taking all the above findings into account, one can speculate that a complex action of deathprograms maintains viability of some cells at the expense of others and that debris of sacrificed cells serve as the sources of energy and nutrients for a cluster of rescued cells. The most viable cells with primitive organization, increased pool of free energy, altered genetic material and the capability to proliferate without additional resources, start to colonize the "nursery" and

New genetic makeup of surviving cells might have many reasons, such as partial degradation of cellular DNA, abnormal mitosis due to metabolic stress [98], fusion of cells or their "remnants", functional impairment of DNA repair pathways, the shattering and rebuilding of chromosomes named chromothripsis [99, 100], etc. In chromothripsis the chromosomes exhibit a Humpty Dumpty-like behavior: multiple fragments of chromosomes stuck back together after almost complete "pulverization". Such a massive genomic rearrangement acquired in a single catastrophic event can lead stressed cells towards neoplasia [ibid]; however, the effect of coherent waves on the genetic material of cells should not be ignored, since extreme disorder in overexcited biological tissues would initiate the random lasing processes and the laser-like coherent beams would be able to cut/weld distinct macromolecules and other cellular

Thus, dramatic events within a bounded area are accompanied by the release of free energy that excites the trapped mass of degrading cells. Random lasing takes place in the extremely disordered overexcited medium full of debris where the clusters of nanoparticles, macromolecules and the remnants of cells have their own unique sets of lasing frequencies

inexorably die even under optimal culture conditions [97].

prepare themselves for the cooperative functioning.

organelles [95].

structures.

Cancer, as a new system with altered karyo- and phenotype, originates within a larger and more mature host-system only if a cluster of genetically altered cells builds up its real-time holographic mechanisms of control and regulation. The physical processes within a bounded area of the host-organism play critical role in the cancer-emergence.

We argue that the early carcinogenesis is a multistep process and it starts in a small "nursery", isolated from the matter, energy and information. Such a segregated "nursery" is deprived of oxygen and nutrients having no access to blood supply for this or that reason (a trauma, fibrosis, etc.). A number of starving ischemic cells undergo a chain of metabolic and structural alterations that include the shift of metabolism from aerobic to anaerobic glycolysis, significant increase of Hypoxia-Inducible Factors [83], activation of cell-death programs, disruption of cellular membranes, release of energy from complex substances through their degradation, and other dramatic events typical for metabolic and hypoxic stress in "cut out tissues".

A growing body of evidence supports the view that hypoxia can contribute to the development of cancer. Some researchers established that hypoxia drives cancer progression by promoting genomic instability and that inactivation of apoptosis is essential for tumor-cell survival during this process [84, 85]. Chinese researchers demonstrated that hypoxia inhibits serum withdrawal-induced apoptosis in endothelial progenitor cells [86], while Australian scientists determined that certain monocyte/macrophage populations survive better under conditions of low oxygen [87].

Low oxygen levels characterize the micro-environment of both stem cells and rapidly growing tumors. Moreover, hypoxia is associated with the maintenance of stem-cell–like phenotypes and increased invasion, angiogenesis and metastasis in cancer patients [88]. Recent observations demonstrate the parallelism existing in hypoxia responses of embryonic, adult and cancer stem cells: the mechanisms involved in hypoxia-dependent processes related to stem cell features and tumor progression include the maintenance of the undifferentiated state, cell proliferation, tumor neovascularization, extra-cellular matrix degradation and motility factor up-regulation [89]. Hypoxia often leads to increased aggressiveness and tumor resistance to chemotherapy and radiation [90]. All the findings about the effects of hypoxia and starvation on the state of bounded cellular constellations were taken into account while working on the new concept of cancer emergence.

According to our hypothesis, not only hypoxia, but also isolation from other environmental processes should be considered as the key factors that initiate carcinogenesis. The degradation of starving cells should be tightly regulated in order to rescue at least some of confined cells. It is well known that autophagy is a highly conserved self-digestion process to promote cell survival in response to nutrient starvation and other metabolic stresses [91-92]; however, the role of autophagy that may lead either to cell survival or to cell death is poorly understood in the context of early carcinogenesis.

The autophagy is the chief machinery for bulk elimination and reutilization of aberrant cell components - constituents of cytoplasm and organelles. In the cases of cancer this "selfdigesting" mechanism plays an essential role at all stages of the disease, since it helps to prevent tumor cell necrosis by mitigating metabolic stress while acting in concert with apoptosis [93]; the autophagy provides an alternate energy source by degrading damaged proteins and organelles that allow some tumor cells to survive during extended periods of starvation [94]. In the absence of phagocytes, apoptosis would be less efficient as the debris cannot be eliminated from the isolated "nursery" (the disposal of debris is necessary in apoptosis). So, the autophagy seems to dominate over apoptosis in early carcinogenesis though cooperation or alternated action of both mechanisms is not excluded especially just after cessation of the blood supply. Increasing evidence points to the selectivity of autophagy: it helps to "sort" vacuolar enzymes, to remove the aggregate-prone proteins and to destruct only excessive organelles [95].

**7. New concept of cancer origin: Dramatic events within a "cancer-nursery"**

Cancer, as a new system with altered karyo- and phenotype, originates within a larger and more mature host-system only if a cluster of genetically altered cells builds up its real-time holographic mechanisms of control and regulation. The physical processes within a bounded

We argue that the early carcinogenesis is a multistep process and it starts in a small "nursery", isolated from the matter, energy and information. Such a segregated "nursery" is deprived of oxygen and nutrients having no access to blood supply for this or that reason (a trauma, fibrosis, etc.). A number of starving ischemic cells undergo a chain of metabolic and structural alterations that include the shift of metabolism from aerobic to anaerobic glycolysis, significant increase of Hypoxia-Inducible Factors [83], activation of cell-death programs, disruption of cellular membranes, release of energy from complex substances through their degradation, and other dramatic events typical for metabolic and hypoxic stress in "cut out tissues".

A growing body of evidence supports the view that hypoxia can contribute to the development of cancer. Some researchers established that hypoxia drives cancer progression by promoting genomic instability and that inactivation of apoptosis is essential for tumor-cell survival during this process [84, 85]. Chinese researchers demonstrated that hypoxia inhibits serum withdrawal-induced apoptosis in endothelial progenitor cells [86], while Australian scientists determined that certain monocyte/macrophage populations survive better under conditions

Low oxygen levels characterize the micro-environment of both stem cells and rapidly growing tumors. Moreover, hypoxia is associated with the maintenance of stem-cell–like phenotypes and increased invasion, angiogenesis and metastasis in cancer patients [88]. Recent observations demonstrate the parallelism existing in hypoxia responses of embryonic, adult and cancer stem cells: the mechanisms involved in hypoxia-dependent processes related to stem cell features and tumor progression include the maintenance of the undifferentiated state, cell proliferation, tumor neovascularization, extra-cellular matrix degradation and motility factor up-regulation [89]. Hypoxia often leads to increased aggressiveness and tumor resistance to chemotherapy and radiation [90]. All the findings about the effects of hypoxia and starvation on the state of bounded cellular constellations were taken into account while

According to our hypothesis, not only hypoxia, but also isolation from other environmental processes should be considered as the key factors that initiate carcinogenesis. The degradation of starving cells should be tightly regulated in order to rescue at least some of confined cells. It is well known that autophagy is a highly conserved self-digestion process to promote cell survival in response to nutrient starvation and other metabolic stresses [91-92]; however, the role of autophagy that may lead either to cell survival or to cell death is poorly understood in

The autophagy is the chief machinery for bulk elimination and reutilization of aberrant cell components - constituents of cytoplasm and organelles. In the cases of cancer this "self-

area of the host-organism play critical role in the cancer-emergence.

602 Cancer Treatment - Conventional and Innovative Approaches

of low oxygen [87].

working on the new concept of cancer emergence.

the context of early carcinogenesis.

There is a kind of similarity between neoplasm and budding primitive organisms (see fig.11 in section 8 – holograms of proliferating cells). A key role of recycling of cellular organelles via autophagy and *de novo* purine biosynthesis was found while studying caloric restriction effects on the longevity of budding yeast (Saccharomyces cerevisiae). This yeast is an effective model for the analysis of genes and cellular pathways. Researchers have shown that additional genes appear to contribute to the restriction of either amino acids or sugar, and that defects in autophagy prevent lifespan extension induced by limitation of nutrients in the growth media [96]. An international team of researchers found that the autophagy helps some starving cells to recover, whereas the cells with a disrupted mitochondrial transmembrane potential inexorably die even under optimal culture conditions [97].

Taking all the above findings into account, one can speculate that a complex action of deathprograms maintains viability of some cells at the expense of others and that debris of sacrificed cells serve as the sources of energy and nutrients for a cluster of rescued cells. The most viable cells with primitive organization, increased pool of free energy, altered genetic material and the capability to proliferate without additional resources, start to colonize the "nursery" and prepare themselves for the cooperative functioning.

New genetic makeup of surviving cells might have many reasons, such as partial degradation of cellular DNA, abnormal mitosis due to metabolic stress [98], fusion of cells or their "remnants", functional impairment of DNA repair pathways, the shattering and rebuilding of chromosomes named chromothripsis [99, 100], etc. In chromothripsis the chromosomes exhibit a Humpty Dumpty-like behavior: multiple fragments of chromosomes stuck back together after almost complete "pulverization". Such a massive genomic rearrangement acquired in a single catastrophic event can lead stressed cells towards neoplasia [ibid]; however, the effect of coherent waves on the genetic material of cells should not be ignored, since extreme disorder in overexcited biological tissues would initiate the random lasing processes and the laser-like coherent beams would be able to cut/weld distinct macromolecules and other cellular structures.

Thus, dramatic events within a bounded area are accompanied by the release of free energy that excites the trapped mass of degrading cells. Random lasing takes place in the extremely disordered overexcited medium full of debris where the clusters of nanoparticles, macromolecules and the remnants of cells have their own unique sets of lasing frequencies [73,74]. Intensive motion of the enclosed mass becomes ordered thanks to the organizing effects of powerful waves in the medium [66, 80, 81].

The wave interactions and the motion of solid matter within an isolated area inevitably reach a state when all dynamic processes become synchronous and coordinated. Increasing laserlike radiation of excited cells can help them to break through the isolation and invade hosttissues. "Cancer embryos" do not and cannot manage their logistic problems at the stages of division, compaction and unification that take place in isolation (prenatal phase); such a neoplasm needs to gain power and become "armed" with laser-weapons before it proceeds to the stages of expansion and growth. At the stage of unification via ordered vibrations and organized motion, the entire cluster of new cells acquires its individual rhythm of functioning and becomes a self-organizing entity ready to grow and struggle for resources.

Duration and timing of all "prenatal" stages are the factors of great importance in any systemgenesis. For example, one can deduce that if a "cancer-embryo" is ready for independent functioning but the barrier around its nursery cannot be breached yet, the cells would continue to "chop" internal structures and eventually die. Without supply of nutrients, oxygen and some "building blocks" from surroundings, the trapped energy would be spent on the selfdestructive work yielding a cyst filled with fluids/semi-solid material; if, on the contrary, the passage of nutrients through the isolating barrier is open earlier than enclosed cells become integrated and "armed", the neoplasm would grow and develop like a benign tumor.

In the manuscript we do not discuss the "postnatal" behavior of the neoplasm in detail; however, once adaptive malignant organism left its nursery and started the life-long battle with its host-rival, it would repeat the same (formerly experienced) scenario whenever possible by blocking blood supply to minor areas and creating the nurseries for new "generations" within the host or its own tissues (the latter is a source of metastasis). Cancer easily adapts to variable situation thanks to the holographic mechanisms of data storage [5] and its first "experience" determines the behavior of its clones. This proclivity of the neoplasm to execute the learned schema of action multiple times in the same or in a slightly changed form can explain the exponential progression, diversity, resistance to the stress posed by "aggressive treatment" and other yet unsolved peculiarities of cancer. The arrangement of new nurseries in various host tissues can be regarded as a kind of adaptive de-evolution: malignant cells produce new generations of "stems" whose structure becomes more and more primitive in each successive cycle.

**Figure 6.** The host-cells should pass through "prenatal" stages before the malignant system leaves its "nursery". Note, that some malignant tissues can start the whole process anew. Each successive cycle would generate less differentiat‐

Life-Cycling of Cancer: New Concept http://dx.doi.org/10.5772/55385 605

To summarize, we argue that malignant neoplasia presents an iterative process of the recurrent system-genesis: one and the same scenario is repeated multiple times within various tissues, in various conditions and with accelerated "prenatal" periods. Such course of the disease can explain the capability of many types of neoplasm to give metastasis through successive rejuvenation of its "daughter-spores". Multiple execution of described tactics of the selfreproduction and the creation of new (younger and less differentiated) generations enables the primary clone of malignant neoplasm to progress exponentially, conquer more and more space at the expense of diverse tissues, resist new stresses and ultimately destroy its breadwinner host. From this point of view, certain stresses posed by standard chemo- and radiation therapy should be considered as the factors that in some cases facilitate the genesis

of extremely aggressive and resistant clones of new primitive "organisms".

ed cells.

The schema presented on figure 6 describes the main stages of such an iterative carcinogenesis.

It is established that non-differentiated stem-cell-like sub-populations of cancer (CSCs) are resistant to chemo- and radiation therapy [101]. Since a "cancer-stem" that yields CSCs orig‐ inates from the remnants of partially degraded progenitor cells, a kind of genetic kinship ex‐ ists between the host and malignant tissues. The same can be said about a relatively mature neoplasm and its metastasis: the features of metastasis though distinct from primary cancer cells are usually distinguishable from the metastasis of other types of cancer.

[73,74]. Intensive motion of the enclosed mass becomes ordered thanks to the organizing effects

The wave interactions and the motion of solid matter within an isolated area inevitably reach a state when all dynamic processes become synchronous and coordinated. Increasing laserlike radiation of excited cells can help them to break through the isolation and invade hosttissues. "Cancer embryos" do not and cannot manage their logistic problems at the stages of division, compaction and unification that take place in isolation (prenatal phase); such a neoplasm needs to gain power and become "armed" with laser-weapons before it proceeds to the stages of expansion and growth. At the stage of unification via ordered vibrations and organized motion, the entire cluster of new cells acquires its individual rhythm of functioning

Duration and timing of all "prenatal" stages are the factors of great importance in any systemgenesis. For example, one can deduce that if a "cancer-embryo" is ready for independent functioning but the barrier around its nursery cannot be breached yet, the cells would continue to "chop" internal structures and eventually die. Without supply of nutrients, oxygen and some "building blocks" from surroundings, the trapped energy would be spent on the selfdestructive work yielding a cyst filled with fluids/semi-solid material; if, on the contrary, the passage of nutrients through the isolating barrier is open earlier than enclosed cells become

integrated and "armed", the neoplasm would grow and develop like a benign tumor.

In the manuscript we do not discuss the "postnatal" behavior of the neoplasm in detail; however, once adaptive malignant organism left its nursery and started the life-long battle with its host-rival, it would repeat the same (formerly experienced) scenario whenever possible by blocking blood supply to minor areas and creating the nurseries for new "generations" within the host or its own tissues (the latter is a source of metastasis). Cancer easily adapts to variable situation thanks to the holographic mechanisms of data storage [5] and its first "experience" determines the behavior of its clones. This proclivity of the neoplasm to execute the learned schema of action multiple times in the same or in a slightly changed form can explain the exponential progression, diversity, resistance to the stress posed by "aggressive treatment" and other yet unsolved peculiarities of cancer. The arrangement of new nurseries in various host tissues can be regarded as a kind of adaptive de-evolution: malignant cells produce new generations of "stems" whose structure becomes more and more primitive in

The schema presented on figure 6 describes the main stages of such an iterative carcinogenesis.

It is established that non-differentiated stem-cell-like sub-populations of cancer (CSCs) are resistant to chemo- and radiation therapy [101]. Since a "cancer-stem" that yields CSCs orig‐ inates from the remnants of partially degraded progenitor cells, a kind of genetic kinship ex‐ ists between the host and malignant tissues. The same can be said about a relatively mature neoplasm and its metastasis: the features of metastasis though distinct from primary cancer

cells are usually distinguishable from the metastasis of other types of cancer.

and becomes a self-organizing entity ready to grow and struggle for resources.

of powerful waves in the medium [66, 80, 81].

604 Cancer Treatment - Conventional and Innovative Approaches

each successive cycle.

**Figure 6.** The host-cells should pass through "prenatal" stages before the malignant system leaves its "nursery". Note, that some malignant tissues can start the whole process anew. Each successive cycle would generate less differentiat‐ ed cells.

To summarize, we argue that malignant neoplasia presents an iterative process of the recurrent system-genesis: one and the same scenario is repeated multiple times within various tissues, in various conditions and with accelerated "prenatal" periods. Such course of the disease can explain the capability of many types of neoplasm to give metastasis through successive rejuvenation of its "daughter-spores". Multiple execution of described tactics of the selfreproduction and the creation of new (younger and less differentiated) generations enables the primary clone of malignant neoplasm to progress exponentially, conquer more and more space at the expense of diverse tissues, resist new stresses and ultimately destroy its breadwinner host. From this point of view, certain stresses posed by standard chemo- and radiation therapy should be considered as the factors that in some cases facilitate the genesis of extremely aggressive and resistant clones of new primitive "organisms".

An unpredictable nature of cancer and dubious efficiency of the methods of its treatment often raise the question whether intervention into the disease course is better than the watchful waiting. For instance, the breast ductal carcinoma in situ, which is a low grade (well differentiated) malignant tumor, can become invasive after more than 30 years since its first manifestation [102]; many patients with low-risk prostate cancer lead a normal life for about 10 years without any treatment: "some prostate cancers might never have developed into serious disease… surgery or radiation therapy may not outweigh the substantial side effects of these treatments" [103].

No doubt, it is urgent and critical to understand the most common rules and principles of malignant neoplasia. We hope that an interdisciplinary approach to the problem and fresh ideas would help everyone involved in healthcare and medical decision-making to plot a clear course through the cancer-paradoxes.

> **Figure 8.** Shapes of presented corona's parts are distorted resembling a shoulder joint (blue arrows); a replica of the most affected bone – clavicle - is displayed with higher quality (red arrows). This is a case of the shoulder malfunction‐

Life-Cycling of Cancer: New Concept http://dx.doi.org/10.5772/55385 607

**Figure 9.** Small areas provide their holographic replicas with higher resolution than the large ones. A case of malig‐

nant polyp in the urinary bladder (left) is shown next to the photograph of the bladder cancer.

ing (former trauma).

### **8. Holo-imaging: Some examples**

**Figure 7.** BHT-gram of the patient with treated lung carcinoma. Prominent functional and/or structural disorder in large areas of the body alters major parts of coronas displaying characteristic features of affected tissues in a slightly distorted form. The holographic replica of the most affected lobe of the lung occupies 2/3 of the index finger BHTgram. Red arrow points to the replica of a growing metastasis.

An unpredictable nature of cancer and dubious efficiency of the methods of its treatment often raise the question whether intervention into the disease course is better than the watchful waiting. For instance, the breast ductal carcinoma in situ, which is a low grade (well differentiated) malignant tumor, can become invasive after more than 30 years since its first manifestation [102]; many patients with low-risk prostate cancer lead a normal life for about 10 years without any treatment: "some prostate cancers might never have developed into serious disease… surgery or radiation therapy may not outweigh the substantial side effects

No doubt, it is urgent and critical to understand the most common rules and principles of malignant neoplasia. We hope that an interdisciplinary approach to the problem and fresh ideas would help everyone involved in healthcare and medical decision-making to plot a clear

**Figure 7.** BHT-gram of the patient with treated lung carcinoma. Prominent functional and/or structural disorder in large areas of the body alters major parts of coronas displaying characteristic features of affected tissues in a slightly distorted form. The holographic replica of the most affected lobe of the lung occupies 2/3 of the index finger BHT-

of these treatments" [103].

course through the cancer-paradoxes.

606 Cancer Treatment - Conventional and Innovative Approaches

**8. Holo-imaging: Some examples**

gram. Red arrow points to the replica of a growing metastasis.

**Figure 8.** Shapes of presented corona's parts are distorted resembling a shoulder joint (blue arrows); a replica of the most affected bone – clavicle - is displayed with higher quality (red arrows). This is a case of the shoulder malfunction‐ ing (former trauma).

**Figure 9.** Small areas provide their holographic replicas with higher resolution than the large ones. A case of malig‐ nant polyp in the urinary bladder (left) is shown next to the photograph of the bladder cancer.

**Figure 10.** Spreading metastases are displayed on BHT-grams as bright balls on a dark background (indicated): A – Colorectal carcinoma with liver metastasis; B – Colorectal carcinoma with regional metastasis; C – Renal carcinoma with metastasis in regional lymph-nodes.

**Figure 12.** The case of the breast cancer (relapse). BHT revealed the tumor 7 months prior to its detection by conven‐ tional imaging methods. Pay attention to powerful diffuse light that consists of multidirectional coherent beams. Such a "fireball" is typical for the neoplasm that just came out of its "nursery" (dark surroundings). Several months later the neoplasm became less uniform and poorly outlined; it grows, multiplies and creates the nurseries for new generations

Life-Cycling of Cancer: New Concept http://dx.doi.org/10.5772/55385 609

**Figure 13.** A case of the breast cancer (treated). Almost all BHT-grams display similar replicas of unusually shaped structures. Such a similarity of disordered coronas is typical for an aggravating (sub-acute, transitory) state. A chain of enlarged lymph-nodes in the upper thorax-neck was found to be responsible for the similarity of interference pat‐

(red arrowheads).

terns.

**Figure 11.** A case of refractory dysmyelopoietic anemia transformed to acute leukemia and liver metastasis. BHT-signs of the disease aggravation have been revealed 4 months before the clinical manifestation of acute leukemia (B). Labanalyses were not informative a month prior to urgent hospitalization of the patient (C). Abnormally proliferating cells are displayed with huge resolution (B, enlarged part). Compare these holograms of "budding" cancer-cells to the bone marrow smear of a patient with acute leukemia (color-image from http://www.washington.edu/news/ 2011/09/06/gene-defect-that-predisposes-people-to-leukemia-discovered/).

**Figure 12.** The case of the breast cancer (relapse). BHT revealed the tumor 7 months prior to its detection by conven‐ tional imaging methods. Pay attention to powerful diffuse light that consists of multidirectional coherent beams. Such a "fireball" is typical for the neoplasm that just came out of its "nursery" (dark surroundings). Several months later the neoplasm became less uniform and poorly outlined; it grows, multiplies and creates the nurseries for new generations (red arrowheads).

**Figure 10.** Spreading metastases are displayed on BHT-grams as bright balls on a dark background (indicated): A – Colorectal carcinoma with liver metastasis; B – Colorectal carcinoma with regional metastasis; C – Renal carcinoma

**Figure 11.** A case of refractory dysmyelopoietic anemia transformed to acute leukemia and liver metastasis. BHT-signs of the disease aggravation have been revealed 4 months before the clinical manifestation of acute leukemia (B). Labanalyses were not informative a month prior to urgent hospitalization of the patient (C). Abnormally proliferating cells are displayed with huge resolution (B, enlarged part). Compare these holograms of "budding" cancer-cells to the bone marrow smear of a patient with acute leukemia (color-image from http://www.washington.edu/news/

2011/09/06/gene-defect-that-predisposes-people-to-leukemia-discovered/).

with metastasis in regional lymph-nodes.

608 Cancer Treatment - Conventional and Innovative Approaches

**Figure 13.** A case of the breast cancer (treated). Almost all BHT-grams display similar replicas of unusually shaped structures. Such a similarity of disordered coronas is typical for an aggravating (sub-acute, transitory) state. A chain of enlarged lymph-nodes in the upper thorax-neck was found to be responsible for the similarity of interference pat‐ terns.

### **9. Conclusion**

Presented work was inspired by the discovery of previously unknown physical effects specific to complex adaptive systems of natural origin [5]. The model of carcinogenesis discussed above is a particular example of the more general scenario of system-genesis [5]. According to our data, the "prenatal" life of malignant cells starts in isolation from normally functioning hosttissues. This segregated "nursery" of cancer cells can be compared to a closed box with the famous "Schrödinger's cat" whose fate is totally unpredictable: no direct observation is possible unless the box is transparent to the observer's eye. Conventional biomedical approaches to *in vivo* diagnostics and monitoring are not efficient in such cases. However, we managed to look into the "cancer-nursery" with the help of the waves that in autonomously functioning systems of natural origin act as "wireless" means of the communication between all system-components; back in 2000 we found that cancer provides specific spectral signatures and that aggressive behavior of its cells can be detected via non-invasive analysis of the bodysurface radiation. Thanks to complex nonlinear mechanisms of information scaling and transfer across the system, it became possible to conduct a non-perturbing observation of some physical processes that take place in early carcinogenesis. Presented hypothesis focuses on very early stages of cancer emergence; however, we suggest that the described scenario of the system-genesis within more mature organism is equally responsible for the generation of distinct clones and metastasis.

physicists and friends contributed to this work. We would like to extend our sincere thanks to all of them. We express our gratitude to Dr. Raymond Samak – the oncologist from Nice, France and to Dr. Marina Abuladze from the Institute of Physics (Tbilisi, Georgia), who kindly assisted us in the course of the manuscript preparation. We are also much indebted to Professor Andrea Peracino and Dr. Emanuela Folco from the Lorenzini Foundation (Milan, Italy) for their help, valuable advices and encouragement. Deepest gratitude is due to the staff of many collaborating hospitals, clinics and private medical cabinets in France and Italy; without their

Life-Cycling of Cancer: New Concept http://dx.doi.org/10.5772/55385 611

[1] Szent-Gyorgyi A. The Living State and Cancer. Proc. Natl. Acad. Sci. Biophysics

[2] Polson R, Vardeny Z. Random lasing in human tissues. Appl. Phys. Lett 2004; 85:

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holographic-principle-and-emergence-phenomenon

assistance our work would not have been successful.

Marina Shaduri1,2 and Marc Bouchoucha1

1 Center of Bioholography, Tbilisi, Georgia

1977; 74(7) 2844-2847.

Prolif 2001; 34 115-134

Ancestors, Phys. Biol 2011; 8 015001, p. 7

1289-1292

2 Advanced BioResearch & Technology, Luxemburg

**Author details**

**References**

The general model of iterative carcinogenesis reconciles many existing hypotheses and also significantly reduces the number of possible causes and triggers of malignant neoplasia; besides, it opens wide horizons for new experiments and theoretical considerations that can result in the development of more targeted methods of cancer treatment. Apart of the widely known facts about biochemical and genetic features of cancer cells, our model takes into account some physical aspects of malignant neoplasia.

Fragile non-molecular processes within biological systems were largely ignored by official science due to their elusive and non-measurable nature. The findings of the physicists who demonstrated the ability of live cells to manipulate and focus intra- and intercellular waves (e.g., light) should be acknowledged as a giant leap forward, towards the official recognition of the critical role that background nonlinear processes play in the system life-cycling.

We hope that empiric generalization of the biomedical and physical information together with the new possibility to study the "secret" life of the neoplasm would cast light on many puzzles and paradoxes of malignant processes and also help to build foundation for more efficient diagnostic and cancer-treatment strategies.

### **Acknowledgements**

This research would not have been possible without the support of many enthusiastic and open-minded people. Medical professionals, engineers, software developers, biologists, physicists and friends contributed to this work. We would like to extend our sincere thanks to all of them. We express our gratitude to Dr. Raymond Samak – the oncologist from Nice, France and to Dr. Marina Abuladze from the Institute of Physics (Tbilisi, Georgia), who kindly assisted us in the course of the manuscript preparation. We are also much indebted to Professor Andrea Peracino and Dr. Emanuela Folco from the Lorenzini Foundation (Milan, Italy) for their help, valuable advices and encouragement. Deepest gratitude is due to the staff of many collaborating hospitals, clinics and private medical cabinets in France and Italy; without their assistance our work would not have been successful.

### **Author details**

**9. Conclusion**

610 Cancer Treatment - Conventional and Innovative Approaches

distinct clones and metastasis.

account some physical aspects of malignant neoplasia.

diagnostic and cancer-treatment strategies.

**Acknowledgements**

Presented work was inspired by the discovery of previously unknown physical effects specific to complex adaptive systems of natural origin [5]. The model of carcinogenesis discussed above is a particular example of the more general scenario of system-genesis [5]. According to our data, the "prenatal" life of malignant cells starts in isolation from normally functioning hosttissues. This segregated "nursery" of cancer cells can be compared to a closed box with the famous "Schrödinger's cat" whose fate is totally unpredictable: no direct observation is possible unless the box is transparent to the observer's eye. Conventional biomedical approaches to *in vivo* diagnostics and monitoring are not efficient in such cases. However, we managed to look into the "cancer-nursery" with the help of the waves that in autonomously functioning systems of natural origin act as "wireless" means of the communication between all system-components; back in 2000 we found that cancer provides specific spectral signatures and that aggressive behavior of its cells can be detected via non-invasive analysis of the bodysurface radiation. Thanks to complex nonlinear mechanisms of information scaling and transfer across the system, it became possible to conduct a non-perturbing observation of some physical processes that take place in early carcinogenesis. Presented hypothesis focuses on very early stages of cancer emergence; however, we suggest that the described scenario of the system-genesis within more mature organism is equally responsible for the generation of

The general model of iterative carcinogenesis reconciles many existing hypotheses and also significantly reduces the number of possible causes and triggers of malignant neoplasia; besides, it opens wide horizons for new experiments and theoretical considerations that can result in the development of more targeted methods of cancer treatment. Apart of the widely known facts about biochemical and genetic features of cancer cells, our model takes into

Fragile non-molecular processes within biological systems were largely ignored by official science due to their elusive and non-measurable nature. The findings of the physicists who demonstrated the ability of live cells to manipulate and focus intra- and intercellular waves (e.g., light) should be acknowledged as a giant leap forward, towards the official recognition of the critical role that background nonlinear processes play in the system life-cycling.

We hope that empiric generalization of the biomedical and physical information together with the new possibility to study the "secret" life of the neoplasm would cast light on many puzzles and paradoxes of malignant processes and also help to build foundation for more efficient

This research would not have been possible without the support of many enthusiastic and open-minded people. Medical professionals, engineers, software developers, biologists,

Marina Shaduri1,2 and Marc Bouchoucha1


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618 Cancer Treatment - Conventional and Innovative Approaches

### *Edited by Letícia Rangel*

Cancer Treatment: Conventional and Innovative Approaches is an attempt to integrate into a book volume the various aspects of cancer treatment, compiling comprehensive reviews written by an international team of experts in the field. The volume is presented in six sections: i) Section 1: Cancer treatment: Conventional and innovative pharmacological approaches; ii) Section 2: Combinatorial strategies to fight cancer: Surgery, radiotherapy, backytherapy, chemotherapy, and hyperthermia; iii) Section 3: The immunotherapy of cancer; iv) Section 4: Multidisciplinarity in cancer therapy: nutrition and beyond; v) Section 5: Supportive care for cancer patients; vi) Section 6: Perspectives in cancer biology and modeling. Ultimately, we hope this book can enlighten important issues involved in the management of cancer, summarizing the state-of-the-art knowledge regarding the disease control and treatment; thus, providing means to improve the overall care of patients that daily battle against this potentially lethal condition.

Cancer Treatment - Conventional and Innovative Approaches

Cancer Treatment

Conventional and Innovative Approaches

*Edited by Letícia Rangel*

Photo by Ugreen / iStock