**Part 1**

**Surgical Therapy of Glioma** 

**1** 

*USA* 

**Innovative Surgical Management of Glioma** 

Glioma is one of the most common primary brain tumors accounting for 30 to 40% of all intracranial tumors. Gliomas can be divided into two types based upon histopathalogic diagnosis according to the World Health Organization (WHO) classification, low grade (WHO I and II) and high grade (WHO III and IV). High grade malignant glioblastoma accounts for the majority of diagnoses and carries the worst prognosis. Prognosis with glioma depends on the patient's age and Karnofsky performance score (KPS) as well as the histological grade of the tumor. For the United States in 2010 there were 6.8 new cases of glioblastoma for every 100,000 people1. The current standard of care for patients with newly diagnosed high-grade glioma is surgical resection followed by fractionated external beam radiation therapy and systemic chemotherapeutic treatment mostly with temozolomide2.

The single best therapeutic option in the treatment of glioma is extensive surgical resection, and the extent of resection directly correlates with the greatest survival benefit. Despite our best efforts however, the outcomes for malignant glioblastoma are still very poor with less than 5% of patients surviving five years post diagnosis even with the best current treatment3. In light of these poor results there is significant room for innovation and improvement in the area of surgical management particularly with an objective to increase the extent of surgical resection. Throughout this chapter we will discuss the importance of surgical resection in the treatment of glioma as well as emerging innovative surgical methods and technologies for increasing the extent of resection and improving upon the associated survival benefit. We will also explore adjuvant therapies in glioma management and the important role that surgery plays in maximizing the potential benefit of these

Three options are available for the surgical management of gliomas. The first option is to refrain from surgery for as long as possible, sometimes referred to as the "wait and see" approach. The second option involves biopsy of the lesion or subtotal resection of surgically accessible areas within the tumor in order to obtain histopathology diagnosis. Biopsy and subtotal resection are often followed by adjuvant therapies (radiation or chemotherapy). The

**1. Introduction** 

adjuvant therapies.

**2. Current surgical management of glioma** 

Dave Seecharan1, Faris Farassati1 and Ania Pollack2

*2Department of Medicine, Molecular Medicine Laboratory, The University of Kansas Medical Centre, Kansas City, KS,* 

*1Department of Neurosurgery,* 

## **Innovative Surgical Management of Glioma**

Dave Seecharan1, Faris Farassati1 and Ania Pollack2

*1Department of Neurosurgery, 2Department of Medicine, Molecular Medicine Laboratory, The University of Kansas Medical Centre, Kansas City, KS, USA* 

#### **1. Introduction**

Glioma is one of the most common primary brain tumors accounting for 30 to 40% of all intracranial tumors. Gliomas can be divided into two types based upon histopathalogic diagnosis according to the World Health Organization (WHO) classification, low grade (WHO I and II) and high grade (WHO III and IV). High grade malignant glioblastoma accounts for the majority of diagnoses and carries the worst prognosis. Prognosis with glioma depends on the patient's age and Karnofsky performance score (KPS) as well as the histological grade of the tumor. For the United States in 2010 there were 6.8 new cases of glioblastoma for every 100,000 people1. The current standard of care for patients with newly diagnosed high-grade glioma is surgical resection followed by fractionated external beam radiation therapy and systemic chemotherapeutic treatment mostly with temozolomide2.

The single best therapeutic option in the treatment of glioma is extensive surgical resection, and the extent of resection directly correlates with the greatest survival benefit. Despite our best efforts however, the outcomes for malignant glioblastoma are still very poor with less than 5% of patients surviving five years post diagnosis even with the best current treatment3. In light of these poor results there is significant room for innovation and improvement in the area of surgical management particularly with an objective to increase the extent of surgical resection. Throughout this chapter we will discuss the importance of surgical resection in the treatment of glioma as well as emerging innovative surgical methods and technologies for increasing the extent of resection and improving upon the associated survival benefit. We will also explore adjuvant therapies in glioma management and the important role that surgery plays in maximizing the potential benefit of these adjuvant therapies.

#### **2. Current surgical management of glioma**

Three options are available for the surgical management of gliomas. The first option is to refrain from surgery for as long as possible, sometimes referred to as the "wait and see" approach. The second option involves biopsy of the lesion or subtotal resection of surgically accessible areas within the tumor in order to obtain histopathology diagnosis. Biopsy and subtotal resection are often followed by adjuvant therapies (radiation or chemotherapy). The

Innovative Surgical Management of Glioma 5

prognosis than the low grade lesions. The first line therapy for high-grade glioma is cytoreductive surgery with the goal of removing as much abnormal tissue as possible without causing further damage to normal parenchyma. The reduction in tumor volume results in improved survival and quality of life by delaying recurrence and malignant

There are many studies over the past two decades that examine the extent of cytoreductive surgery; gross total resection verses biopsy and subtotal resection in regards to increasing patient survival. Tumor location as well as extent of neurologic deficits plays a large role in the decision making process for surgical management. One retrospective study in 1996 showed significant increased mean survival time of 292 days vs 184 days between a group undergoing cytoreductive surgery and a group undergoing stereotactic biopsy respectively8. However quality of life measured by KPS was not significantly different between the two

A prospective study utilizing 60 patients, examined the extent of resection compared to survival. These authors found median survival of 64 weeks for the group who underwent gross total resection compared to 36 weeks for the group undergoing subtotal resection9. They concluded that patients undergoing subtotal resection have 6.6 times higher risk of death. Another large prospective of study 645 patients showed that patients undergoing total resection had a median survival of 11.3 months compared to 10.4 months for subtotal resection, both of which were significant increases in survival compared to 6.6 months for

Increased survival with resection holds true even for the elderly. A study by Vuorienen, focusing specifically on elderly patients over the age of 65 showed median survival of 5.7 months with open craniotomy and surgical resection compared to 2.8 months with stereotactic biopsy alone for an increased estimated survival time of 2.7 times longer in the resection group11. However, this increased survival time is modest in this patient population providing only a 2-3 month survival benefit. The role of aggressive surgical management in the elderly population is still a controversial subject. Chronological age however, is not necessarily the most important factor to consider in deciding to pursue an aggressive surgical course. Rather biological age which takes into account the patients general health

A recent review article from 2008 examined over thirty published articles from the neurosurgical and neuro-oncologic literature regarding extent of resection and the effects on survival for malignant glioma. Only one study failed to support the idea that extent of surgical resection correlates with an increased survival advantage12. These authors recommend that based on the current prospective and retrospective data that for newly diagnosed malignant glioma in adults, maximal cytoreductive surgery should be

Studies have also been performed in order to quantify exactly what percent of tumor resection is necessary to provide maximal survival benefit. The original landmark study was published in 2001 by Lacroix and colleagues in which they performed a retrospective analysis of 416 patients with glioblastoma treated with surgical resection13. Patients who received resection of greater than 98% had a mean survival of 13 months compared to

undertaken provided that postoperative neurological deficits are minimized.

patients receiving resection of less than 98% with mean survival of 8.8 months.

progression.

groups.

patients with biopsy only10.

and functional level should be used as a guideline.

best surgical option is gross total resection (GTR), usually defined as removal of all areas of contrast enhancement on T1 weighted MRI obtained postoperatively. GTR provides the best surgical treatment and is associated with the best survival rates; however, it also carries the greatest risks of postoperative neurologic deficits and disability.

Current treatment for glioma, particularly high grade lesions, is not curative and the majority of patients experience reoccurrence following initial resection. Reoccurrence is due to the infiltrative nature of these lesions and the inability of current treatment modalities to fully remove and destroy all tumor cells. The rationale behind surgical management of these aggressive lesions is based on the Gompertzian phenomenon. In 1825, the English mathematician Benjamin Gompertz postulated that the biological growth of normal organs and malignancies follows a characteristic curve and that cell number increases with time, but the relative rate of increase falls exponentially as the mass reaches a "plateau phase" of a very slow actual growth4. Therapy can induce regression in tumor volume, however, there is always regrowth between cycles of treatment and this "regrowth" will follow the same Gompertzian growth curve. The only escape from Gompertzian phenomenon is complete tumor cell eradication. Therefore, the ultimate goal of surgical therapy in these tumors should be the complete eradication of all abnormal neoplastic cells. With the currently available surgical techniques we are still unable to fully resect most of these tumors. Therefore we need new techniques to improve surgical resection as well as adjuvant therapies to eradicate remaining tumor cells and thereby maximize the survival benefit of surgery.

Low grade gliomas (WHO I and II) are a broad group of tumors that are clinically, histologically and molecularly diverse. WHO grade I tumors comprise the group of pilocytic astrocytoma and subependymal giant cell astrocytoma the more common being pilocytic. Management of WHO grade I glioma consists of gross total resection as the treatment of choice and carries an excellent prognosis. Following resection, 25-year survival rates of 50- 94% have been reported5. Grade I lesions are the only glioma subtype where gross total resection is considered curative.

WHO grade II gliomas consist of diffuse fibrillary astrocytoma, oligodendrogliomas, and oligoastrocytomas, all of which have similar invasive and malignant potential. Grade II gliomas that are symptomatic and surgically accessible should undergo maximal cytoreductive surgery as the treatment of choice. Predictors of incomplete tumor resection in low grade lesions include tumor involvement of the cortico-spinal tract, large tumor volume, and oligodendroglioma histopathologic type. 5 year survival of up to 95-97% has been reported following gross total resection in these lesions6. In 2008, Smith and colleagues reported a large series of 216 patients with biologically aggressive grade II lesions and examined the extent of resection and the effect on overall survival7. Patients with at least 90% resection showed 5 and 8 year survivals of 97% and 91% respectively compared to those with less than 90% resection who showed survival rates of 76% and 60% at 5 and 8 years.

For both grade I and grade II tumors, maximal surgical resection is the single best treatment for obtaining increased survival. In cases where there is progressive tumor growth following resection or progressive neurological symptoms in unresectable tumors, adjuvant chemotherapy or radiation treatment may be used.

High grade glioma's (WHO III and IV) consist of anaplastic astrocytoma (Grade III), and glioblastoma (Grade IV). These tumors are malignant and carry a significantly poorer

best surgical option is gross total resection (GTR), usually defined as removal of all areas of contrast enhancement on T1 weighted MRI obtained postoperatively. GTR provides the best surgical treatment and is associated with the best survival rates; however, it also carries the

Current treatment for glioma, particularly high grade lesions, is not curative and the majority of patients experience reoccurrence following initial resection. Reoccurrence is due to the infiltrative nature of these lesions and the inability of current treatment modalities to fully remove and destroy all tumor cells. The rationale behind surgical management of these aggressive lesions is based on the Gompertzian phenomenon. In 1825, the English mathematician Benjamin Gompertz postulated that the biological growth of normal organs and malignancies follows a characteristic curve and that cell number increases with time, but the relative rate of increase falls exponentially as the mass reaches a "plateau phase" of a very slow actual growth4. Therapy can induce regression in tumor volume, however, there is always regrowth between cycles of treatment and this "regrowth" will follow the same Gompertzian growth curve. The only escape from Gompertzian phenomenon is complete tumor cell eradication. Therefore, the ultimate goal of surgical therapy in these tumors should be the complete eradication of all abnormal neoplastic cells. With the currently available surgical techniques we are still unable to fully resect most of these tumors. Therefore we need new techniques to improve surgical resection as well as adjuvant therapies to eradicate

Low grade gliomas (WHO I and II) are a broad group of tumors that are clinically, histologically and molecularly diverse. WHO grade I tumors comprise the group of pilocytic astrocytoma and subependymal giant cell astrocytoma the more common being pilocytic. Management of WHO grade I glioma consists of gross total resection as the treatment of choice and carries an excellent prognosis. Following resection, 25-year survival rates of 50- 94% have been reported5. Grade I lesions are the only glioma subtype where gross total

WHO grade II gliomas consist of diffuse fibrillary astrocytoma, oligodendrogliomas, and oligoastrocytomas, all of which have similar invasive and malignant potential. Grade II gliomas that are symptomatic and surgically accessible should undergo maximal cytoreductive surgery as the treatment of choice. Predictors of incomplete tumor resection in low grade lesions include tumor involvement of the cortico-spinal tract, large tumor volume, and oligodendroglioma histopathologic type. 5 year survival of up to 95-97% has been reported following gross total resection in these lesions6. In 2008, Smith and colleagues reported a large series of 216 patients with biologically aggressive grade II lesions and examined the extent of resection and the effect on overall survival7. Patients with at least 90% resection showed 5 and 8 year survivals of 97% and 91% respectively compared to those with

less than 90% resection who showed survival rates of 76% and 60% at 5 and 8 years.

chemotherapy or radiation treatment may be used.

For both grade I and grade II tumors, maximal surgical resection is the single best treatment for obtaining increased survival. In cases where there is progressive tumor growth following resection or progressive neurological symptoms in unresectable tumors, adjuvant

High grade glioma's (WHO III and IV) consist of anaplastic astrocytoma (Grade III), and glioblastoma (Grade IV). These tumors are malignant and carry a significantly poorer

greatest risks of postoperative neurologic deficits and disability.

remaining tumor cells and thereby maximize the survival benefit of surgery.

resection is considered curative.

prognosis than the low grade lesions. The first line therapy for high-grade glioma is cytoreductive surgery with the goal of removing as much abnormal tissue as possible without causing further damage to normal parenchyma. The reduction in tumor volume results in improved survival and quality of life by delaying recurrence and malignant progression.

There are many studies over the past two decades that examine the extent of cytoreductive surgery; gross total resection verses biopsy and subtotal resection in regards to increasing patient survival. Tumor location as well as extent of neurologic deficits plays a large role in the decision making process for surgical management. One retrospective study in 1996 showed significant increased mean survival time of 292 days vs 184 days between a group undergoing cytoreductive surgery and a group undergoing stereotactic biopsy respectively8. However quality of life measured by KPS was not significantly different between the two groups.

A prospective study utilizing 60 patients, examined the extent of resection compared to survival. These authors found median survival of 64 weeks for the group who underwent gross total resection compared to 36 weeks for the group undergoing subtotal resection9. They concluded that patients undergoing subtotal resection have 6.6 times higher risk of death. Another large prospective of study 645 patients showed that patients undergoing total resection had a median survival of 11.3 months compared to 10.4 months for subtotal resection, both of which were significant increases in survival compared to 6.6 months for patients with biopsy only10.

Increased survival with resection holds true even for the elderly. A study by Vuorienen, focusing specifically on elderly patients over the age of 65 showed median survival of 5.7 months with open craniotomy and surgical resection compared to 2.8 months with stereotactic biopsy alone for an increased estimated survival time of 2.7 times longer in the resection group11. However, this increased survival time is modest in this patient population providing only a 2-3 month survival benefit. The role of aggressive surgical management in the elderly population is still a controversial subject. Chronological age however, is not necessarily the most important factor to consider in deciding to pursue an aggressive surgical course. Rather biological age which takes into account the patients general health and functional level should be used as a guideline.

A recent review article from 2008 examined over thirty published articles from the neurosurgical and neuro-oncologic literature regarding extent of resection and the effects on survival for malignant glioma. Only one study failed to support the idea that extent of surgical resection correlates with an increased survival advantage12. These authors recommend that based on the current prospective and retrospective data that for newly diagnosed malignant glioma in adults, maximal cytoreductive surgery should be undertaken provided that postoperative neurological deficits are minimized.

Studies have also been performed in order to quantify exactly what percent of tumor resection is necessary to provide maximal survival benefit. The original landmark study was published in 2001 by Lacroix and colleagues in which they performed a retrospective analysis of 416 patients with glioblastoma treated with surgical resection13. Patients who received resection of greater than 98% had a mean survival of 13 months compared to patients receiving resection of less than 98% with mean survival of 8.8 months.

Innovative Surgical Management of Glioma 7

Fig. 1. Neuronavigation. Operating room setup with patient positioned and

guided biopsy showing a temporal lobe glioma in three planes.

neuronavigational stereotactic equipment (A). Neuronavigational MRI for stereotactic

A more recent 2011 retrospective study by Sanai and colleagues also quantified the percent of tumor resection required for maximal survival benefit14. 500 patients with gliobastoma were treated with surgical resection followed by standard radiation and chemotherapy. They found a survival benefit with extent of surgical resection of as low as 78%. However, for maximal survival benefit, resection of greater than 95% was observed

Overall, these studies demonstrate that prolonged survival time correlates with the extent of surgical resection. The data also suggests that for high grade lesions, resection of greater than 95% of the tumor volume should be performed in order to provide the maximal survival benefit. In some cases, however, such a high level of resection is unable to be obtained. This is usually due to tumor involvement in areas of eloquent brain tissue (areas involved with speech production, motor function and sensory perception), and is associated with a high risk of postoperative deficits. Maximizing the extent of tumor resection while preserving normal brain function and optimizing quality of life postoperatively represents a major challenge in neurosurgery. Several strategies and innovative techniques have been developed to assist the surgeon in safely resecting tumors located in these eloquent areas, particularly in the areas of neuroimaging, neuronavigation, functional mapping, and photodynamics. Theses new developments provide for greater resection volumes and better survival rates.

#### **3. Neuronavigation**

Image guided neuronavigation utilizes the principle of stereotaxis. The brain is considered as a geometric volume which can be divided by three imaginary intersecting spatial planes based on a Cartesian coordinate system. Any point within the brain can be specified by measuring its distance along these three planes. This provides a precise surgical guidance by referencing this coordinate system of the brain with a parallel coordinate system of the three-dimensional image data of the patient that is displayed on a computer-workstation so that the medical images become point-to-point maps of the corresponding actual locations within the brain15. Neuronavigation provides intraoperative orientation to the surgeon, helps in planning a precise surgical approach to the targeted lesion and defines the surrounding neurovascular structures. Conventional neuronavigation typically utilizes a preoperative MRI which is registered to the patients skull at the beginning of the procedure and is used throughout the case without any update in imaging or reregistration of the imaging to the corresponding brain tissue. Conventional neuronavigation is readily available at most centers providing neurosurgical care and is not particularly cost prohibitive. (Figure 1)

Intraoperative MRI (iMRI) guided intracranial surgery improves upon the benefits of conventional stereotactic guided neurosurgery by providing a real time updated view of the anatomic relationship between tumor and normal brain structures. During surgical resection utilizing traditional cranial neuronavigation, the brain parenchyma becomes distorted due to changes in tumor volume, edema and volume of cerebrospinal fluid resulting in brain shift, which is not reflected in the preoperatively obtained MRI. This results in less reliability of the stereotactic guidance as the surgery progresses. Intraoperatively obtained MRI allows updating of the images used for neuronavigation as well as updated visualization of the contrast enhancing tissue that remains.

A more recent 2011 retrospective study by Sanai and colleagues also quantified the percent of tumor resection required for maximal survival benefit14. 500 patients with gliobastoma were treated with surgical resection followed by standard radiation and chemotherapy. They found a survival benefit with extent of surgical resection of as low as 78%. However,

Overall, these studies demonstrate that prolonged survival time correlates with the extent of surgical resection. The data also suggests that for high grade lesions, resection of greater than 95% of the tumor volume should be performed in order to provide the maximal survival benefit. In some cases, however, such a high level of resection is unable to be obtained. This is usually due to tumor involvement in areas of eloquent brain tissue (areas involved with speech production, motor function and sensory perception), and is associated with a high risk of postoperative deficits. Maximizing the extent of tumor resection while preserving normal brain function and optimizing quality of life postoperatively represents a major challenge in neurosurgery. Several strategies and innovative techniques have been developed to assist the surgeon in safely resecting tumors located in these eloquent areas, particularly in the areas of neuroimaging, neuronavigation, functional mapping, and photodynamics. Theses new developments provide for greater resection volumes and better

Image guided neuronavigation utilizes the principle of stereotaxis. The brain is considered as a geometric volume which can be divided by three imaginary intersecting spatial planes based on a Cartesian coordinate system. Any point within the brain can be specified by measuring its distance along these three planes. This provides a precise surgical guidance by referencing this coordinate system of the brain with a parallel coordinate system of the three-dimensional image data of the patient that is displayed on a computer-workstation so that the medical images become point-to-point maps of the corresponding actual locations within the brain15. Neuronavigation provides intraoperative orientation to the surgeon, helps in planning a precise surgical approach to the targeted lesion and defines the surrounding neurovascular structures. Conventional neuronavigation typically utilizes a preoperative MRI which is registered to the patients skull at the beginning of the procedure and is used throughout the case without any update in imaging or reregistration of the imaging to the corresponding brain tissue. Conventional neuronavigation is readily available at most centers providing neurosurgical care and is not particularly cost

Intraoperative MRI (iMRI) guided intracranial surgery improves upon the benefits of conventional stereotactic guided neurosurgery by providing a real time updated view of the anatomic relationship between tumor and normal brain structures. During surgical resection utilizing traditional cranial neuronavigation, the brain parenchyma becomes distorted due to changes in tumor volume, edema and volume of cerebrospinal fluid resulting in brain shift, which is not reflected in the preoperatively obtained MRI. This results in less reliability of the stereotactic guidance as the surgery progresses. Intraoperatively obtained MRI allows updating of the images used for neuronavigation as well as updated visualization of the

for maximal survival benefit, resection of greater than 95% was observed

survival rates.

**3. Neuronavigation** 

prohibitive. (Figure 1)

contrast enhancing tissue that remains.

Fig. 1. Neuronavigation. Operating room setup with patient positioned and neuronavigational stereotactic equipment (A). Neuronavigational MRI for stereotactic guided biopsy showing a temporal lobe glioma in three planes.

Innovative Surgical Management of Glioma 9

anesthesia is the best technique to locate eloquent domains as well as to distinguish functional area from nonfunctional area. In 2008, Duffau and colleagues, reported the largest experience with cortical and subcortical mapping of gliomas affecting the language area19. They performed resection using awake craniotomy and direct electrical stimulation in 115 patients. 98% of patients improved or returned to their preoperative baseline following resection guided by direct electrostimulation. Awake craniotomy however presents several challenges in regards to anesthesia, patient comfort and anxiety as well as

Functional magnetic resonance imaging (fMRI) is another improving technology that shows promise for increasing the extent of surgical resection while minimizing neurological deficits by providing functional mapping with the use of newly developed imaging modalities. The use of functional magnetic resonance imaging (fMRI) allows information regarding the location of specific brain functions such as speech, motor function, and sensory perception to be mapped to three-dimensional reconstructed MRI images. Blood oxygen level dependent (BOLD) fMRI is one of the most commonly used forms of fMRI. BOLD fMRI provides functional information based on cerebral hemodynamic responses by measuring changes in the ratio of blood oxyhemoglobin and deoxyhemoglobin during the presentation of a various stimuli to the patient. This data can then be used to map taskdriven regional cortical activity in patients to noninvasively locate the brains essential

In 2010, Talacchi and colleges retrospectively examined the use of preoperative fMRI and neuronavigation compared to traditional intraoperative neurostimulation in 171 patients20. They found that preoperative fMRI provided equivalent rate of GTR when compared to the invasive neurostimulation 71% vs 73%c compared to 40% in resections not utilizing either modality. Similar findings have been demonstrated by additional authors21,22. Together these studies support fMRI as a viable alternative to awake craniotomy and functional neurostimulation with the benefit of reduced operative time as well as elimination of the

Diffusion tensor imaging (DTI) is an additional form of functional magnetic resonance imaging used to delineate white matter anatomy. DTI is based upon the principle that water preferentially diffuses along the long axis of white matter tracts and the degree and direction of water diffusion can be measured. Tractography uses algorithms to process this data and to reconstruct three-dimensional maps representing subcortical fiber tracts23. Tractography can be used in surgical planning to show the relationship between white matter tracts and tumor. It can reveal whether tracts are displaced, disrupted, or infiltrated

In 2007, Wu and colleges reported a large prospective randomized controlled trial of 238 patients with gliomas<sup>24</sup>. A randomized study group of 118 patients underwent resection with DTI tractography and neuronavigation while a control group of 120 patients underwent resection with neuronavigation alone. They found a significant increase in the ability to achieve GTR with the use of DTI tractography with 74.4 % of patients in the study group achieving GTR compared to 33.3% of patients in the control group. This translated into an increased mean survival time 21.2 months in the study group compared to 14.0 months mean survival in the control group. They also found better outcomes with DTI

prolongation of operative time.

eloquent areas and guide surgical planning. (Figure 2)

challenges associated with awake craniotomy.

by tumor. (Figure 3)

Over the past decade several studies have looked at iMRI guided resection vs conventional neuronavigation in patients with glioma. In 2000, Wirtz and colleagues examined 68 cases of high grade glioma resected with iMRI16. Of the 68 cases, 27% of showed GTR on the first iMRI scan and 66% percent underwent continued resection. Median survival was 13.3 months for GTR vs 9.2 months for subtotal resection.

In 2005, Hisrschberg and colleagues examined the use of iMRI in 32 patients with glioblastoma compared to a matched control group of 32 patients using conventional neuronavigation. They found a mean survival time of 14.5 months in the iMRI group vs 12.1 months in the control group. They also saw a significant increase in length of surgical time with iMRI 5.1 hours vs 3.4 with conventional navigation. They also reported postoperative functional performance results, which were not significantly different between the two groups. Neurologic improvement was seen in 16% of patients, 55% showed no change, and 19% showed some worsening of symptoms.

In 2010, Senft and colleagues reported on 41 patients with glioblastoma, 10 of whom underwent resection with iMRI and 31 who received resection by conventional means17. GTR was seen in all 10 iMRI cases and 19 of the conventional group. Median survival was 88 weeks for the iMRI group and 68 weeks for the conventional group. Median survival in regards to the extent of resection was 74 weeks for the 29 patients who obtained GTR vs 46 weeks for the 12 patients who obtained subtotal resection.

A recent 2011 review article of 12 studies from the current literature examined the benefits of iMRI vs conventional stereotactic surgery for glioblastoma. The authors concluded that iMRI guided surgery is more effective that conventional neuronavigational surgery in increasing the extent of resection and prolonging survival in patients with glioblastoma18. However there are currently no randomized trials with validated endpoints that demonstrate the additional value of iMRI guided surgery. Intraoperative MRI is currently of limited availability, adds significant expense and prolongs surgical time. Therefore, the decision to use this modality should be made judiciously on a case by case basis.

These systems are not perfect and continued improvements are needed. Advances in real time surgical imaging are important to reduce the neuronavigational inaccuracies due to brain shift as well as to provide a clearer more accurate representation of the tumor margins throughout the case. Currently these systems still rely heavily on a rigid fixation of the patient's head and a registration landmark. Movement of the patient's head, pin slippage, and loss of registration can drastically limit the utility of these surgical tools and more robust technologies are needed. Despite these limitations, the use of improved neuroimaging and newer methods of neuronavigation can significantly improve the extent of resection and thereby increase the survival rates for patients with glioma.

#### **4. Functional mapping**

Functional mapping is another tool that is changing the surgical management of glioma therapy. These new techniques allow the protection of eloquent areas of the brain while permitting the extent of surgical resection to be maximized. With this technology, lesions that were previously thought to be inoperable due to location are now often resectable. One such procedure is awake craniotomy. Intraoperative direct electrostimulation under awake

Over the past decade several studies have looked at iMRI guided resection vs conventional neuronavigation in patients with glioma. In 2000, Wirtz and colleagues examined 68 cases of high grade glioma resected with iMRI16. Of the 68 cases, 27% of showed GTR on the first iMRI scan and 66% percent underwent continued resection. Median survival was 13.3

In 2005, Hisrschberg and colleagues examined the use of iMRI in 32 patients with glioblastoma compared to a matched control group of 32 patients using conventional neuronavigation. They found a mean survival time of 14.5 months in the iMRI group vs 12.1 months in the control group. They also saw a significant increase in length of surgical time with iMRI 5.1 hours vs 3.4 with conventional navigation. They also reported postoperative functional performance results, which were not significantly different between the two groups. Neurologic improvement was seen in 16% of patients, 55% showed no change, and

In 2010, Senft and colleagues reported on 41 patients with glioblastoma, 10 of whom underwent resection with iMRI and 31 who received resection by conventional means17. GTR was seen in all 10 iMRI cases and 19 of the conventional group. Median survival was 88 weeks for the iMRI group and 68 weeks for the conventional group. Median survival in regards to the extent of resection was 74 weeks for the 29 patients who obtained GTR vs 46

A recent 2011 review article of 12 studies from the current literature examined the benefits of iMRI vs conventional stereotactic surgery for glioblastoma. The authors concluded that iMRI guided surgery is more effective that conventional neuronavigational surgery in increasing the extent of resection and prolonging survival in patients with glioblastoma18. However there are currently no randomized trials with validated endpoints that demonstrate the additional value of iMRI guided surgery. Intraoperative MRI is currently of limited availability, adds significant expense and prolongs surgical time. Therefore, the

These systems are not perfect and continued improvements are needed. Advances in real time surgical imaging are important to reduce the neuronavigational inaccuracies due to brain shift as well as to provide a clearer more accurate representation of the tumor margins throughout the case. Currently these systems still rely heavily on a rigid fixation of the patient's head and a registration landmark. Movement of the patient's head, pin slippage, and loss of registration can drastically limit the utility of these surgical tools and more robust technologies are needed. Despite these limitations, the use of improved neuroimaging and newer methods of neuronavigation can significantly improve the extent

Functional mapping is another tool that is changing the surgical management of glioma therapy. These new techniques allow the protection of eloquent areas of the brain while permitting the extent of surgical resection to be maximized. With this technology, lesions that were previously thought to be inoperable due to location are now often resectable. One such procedure is awake craniotomy. Intraoperative direct electrostimulation under awake

decision to use this modality should be made judiciously on a case by case basis.

of resection and thereby increase the survival rates for patients with glioma.

months for GTR vs 9.2 months for subtotal resection.

19% showed some worsening of symptoms.

**4. Functional mapping** 

weeks for the 12 patients who obtained subtotal resection.

anesthesia is the best technique to locate eloquent domains as well as to distinguish functional area from nonfunctional area. In 2008, Duffau and colleagues, reported the largest experience with cortical and subcortical mapping of gliomas affecting the language area19. They performed resection using awake craniotomy and direct electrical stimulation in 115 patients. 98% of patients improved or returned to their preoperative baseline following resection guided by direct electrostimulation. Awake craniotomy however presents several challenges in regards to anesthesia, patient comfort and anxiety as well as prolongation of operative time.

Functional magnetic resonance imaging (fMRI) is another improving technology that shows promise for increasing the extent of surgical resection while minimizing neurological deficits by providing functional mapping with the use of newly developed imaging modalities. The use of functional magnetic resonance imaging (fMRI) allows information regarding the location of specific brain functions such as speech, motor function, and sensory perception to be mapped to three-dimensional reconstructed MRI images. Blood oxygen level dependent (BOLD) fMRI is one of the most commonly used forms of fMRI. BOLD fMRI provides functional information based on cerebral hemodynamic responses by measuring changes in the ratio of blood oxyhemoglobin and deoxyhemoglobin during the presentation of a various stimuli to the patient. This data can then be used to map taskdriven regional cortical activity in patients to noninvasively locate the brains essential eloquent areas and guide surgical planning. (Figure 2)

In 2010, Talacchi and colleges retrospectively examined the use of preoperative fMRI and neuronavigation compared to traditional intraoperative neurostimulation in 171 patients20. They found that preoperative fMRI provided equivalent rate of GTR when compared to the invasive neurostimulation 71% vs 73%c compared to 40% in resections not utilizing either modality. Similar findings have been demonstrated by additional authors21,22. Together these studies support fMRI as a viable alternative to awake craniotomy and functional neurostimulation with the benefit of reduced operative time as well as elimination of the challenges associated with awake craniotomy.

Diffusion tensor imaging (DTI) is an additional form of functional magnetic resonance imaging used to delineate white matter anatomy. DTI is based upon the principle that water preferentially diffuses along the long axis of white matter tracts and the degree and direction of water diffusion can be measured. Tractography uses algorithms to process this data and to reconstruct three-dimensional maps representing subcortical fiber tracts23. Tractography can be used in surgical planning to show the relationship between white matter tracts and tumor. It can reveal whether tracts are displaced, disrupted, or infiltrated by tumor. (Figure 3)

In 2007, Wu and colleges reported a large prospective randomized controlled trial of 238 patients with gliomas<sup>24</sup>. A randomized study group of 118 patients underwent resection with DTI tractography and neuronavigation while a control group of 120 patients underwent resection with neuronavigation alone. They found a significant increase in the ability to achieve GTR with the use of DTI tractography with 74.4 % of patients in the study group achieving GTR compared to 33.3% of patients in the control group. This translated into an increased mean survival time 21.2 months in the study group compared to 14.0 months mean survival in the control group. They also found better outcomes with DTI

Innovative Surgical Management of Glioma 11

Fig. 3. Tractography. 3-Dimensional reconstruction showing fiber tracts generated from DTI

Another problem arises in surgery when dealing with the infiltrating malignant cells present at the tumor margins. These cells often lie outside the area of enhancement on neuroimaging and intraoperatively appear grossly and microscopically indistinguishable from normal brain tissue. These areas of infiltrating cells contribute to reoccurrence and negatively effect

Fluorescence image guided surgical resection (FIGS) is another innovative surgical technique which uses fluorescence intraoperatively to enhance the visualization of abnormal tumor cells intraoperatively and allow for maximal extent of resection. 5-Aminolevulinic acid (ALA) is injected systemically prior to surgery. High grade gliomas and other metabolically active tumors take up ALA at a rapid rate. After gross resection of the visible tumor, a specially filtered blue light can illuminate the areas of high uptake within the

In 2000, Stummer and colleagues reported an initial study of the efficacy of FIGS which demonstrated FIGS to be quite specific (only 0.4 % of fluorescent biopsy sites did not contain tumor cells) and quite sensitive (81.6% of fluorescent biopsy sites contained tumor cells)26. In 2006 this same author reported a multicenter phase III trial comparing FIGS to placebo and found 64% complete surgical excision with fluorescence guided resection compared to 38%

MRI sequence in a patient with large glioblastoma multiforme.

cavity allowing selective resection of the residual areas25.

**5. Florescence guided resection** 

long-term tumor control if not resected.

tractography with motor strength deterioration occurring in 15.3% of patients in the study group compared to 32.8% in the control group. Improved outcome was also demonstrated in 6 month Karnofsky Performace Scale scores with a mean score of 77 for study group patients and 53 for the control group. When combined, fMRI, neuronavigation, and DTI allows precise surgical resection of the maximal tumor volume while sparing intact fiber tracts as well as eloquent areas of the brain. This results increased patient survival and improved functional outcomes.

Fig. 2. Functional MRI. MRI generated from BOLD data in a patient with large frontal lobe glioma showing areas of activation during a sentence completion task.

tractography with motor strength deterioration occurring in 15.3% of patients in the study group compared to 32.8% in the control group. Improved outcome was also demonstrated in 6 month Karnofsky Performace Scale scores with a mean score of 77 for study group patients and 53 for the control group. When combined, fMRI, neuronavigation, and DTI allows precise surgical resection of the maximal tumor volume while sparing intact fiber tracts as well as eloquent areas of the brain. This results increased patient survival and

Fig. 2. Functional MRI. MRI generated from BOLD data in a patient with large frontal lobe

glioma showing areas of activation during a sentence completion task.

improved functional outcomes.

Fig. 3. Tractography. 3-Dimensional reconstruction showing fiber tracts generated from DTI MRI sequence in a patient with large glioblastoma multiforme.

#### **5. Florescence guided resection**

Another problem arises in surgery when dealing with the infiltrating malignant cells present at the tumor margins. These cells often lie outside the area of enhancement on neuroimaging and intraoperatively appear grossly and microscopically indistinguishable from normal brain tissue. These areas of infiltrating cells contribute to reoccurrence and negatively effect long-term tumor control if not resected.

Fluorescence image guided surgical resection (FIGS) is another innovative surgical technique which uses fluorescence intraoperatively to enhance the visualization of abnormal tumor cells intraoperatively and allow for maximal extent of resection. 5-Aminolevulinic acid (ALA) is injected systemically prior to surgery. High grade gliomas and other metabolically active tumors take up ALA at a rapid rate. After gross resection of the visible tumor, a specially filtered blue light can illuminate the areas of high uptake within the cavity allowing selective resection of the residual areas25.

In 2000, Stummer and colleagues reported an initial study of the efficacy of FIGS which demonstrated FIGS to be quite specific (only 0.4 % of fluorescent biopsy sites did not contain tumor cells) and quite sensitive (81.6% of fluorescent biopsy sites contained tumor cells)26. In 2006 this same author reported a multicenter phase III trial comparing FIGS to placebo and found 64% complete surgical excision with fluorescence guided resection compared to 38%

Innovative Surgical Management of Glioma 13

Novel methods of administering radiation therapy are also being developed. Following optimal surgical resection, image guided stereotactic radiosurgery (SRS) is now being evaluated for treatment these lesions. This new modality utilizes a single high doses of radiation specifically targeted to a well-defined lesions using detailed neuroimaging. This allows delivery of focused radiation to the tumor with a much lower dose to adjacent nontargeted tissue which results in reduced side effects compared with traditional methods.

For low grade gliomas, adjuvant RT following resection has failed to show a survival benefit. In cases of disease progression or inoperable lesions with neurologic symptoms, delayed RT appears to provide the same survival advantage as postoperative RT34. For newly diagnosed high grade gliomas studies have demonstrated that SRS does not provide a significant survival advantage over conventional radiotherapy35,36. The use of SRS has also been explored as a treatment for recurrent high grade lesions. A recent article by Romanelli and colleagues reviewed 17 retrospective studies examining the role of SRS in the treatment of recurrent high grade glioma. This review demonstrated that SRS is associated with prolonged survival in patients with recurrent GBM with median survival times ranging from 7.5 to 30 months37. SRS has been shown to have no significant advantage as a first line radiation therapy for glioma and the results for recurrent glioma are inconclusive, therefore

In addition to externally administered sources of radiation therapy, interstitial brachytherapy is another form of radiation therapy used to treat glioma, which refers to surgical placement of the radioactive source a short distance from or within the tumor being treated. Brachytherapy was developed due to the observation that 80% of malignant gliomas reoccur within 2cm of the initial tumor site following resection. By placing the radioactive source directly in the tumor or tumor bed, continuous high dose radiation increases damage to nearby proliferating tumor cells located at the margin with a rapid fall off in the dose

There are multiple surgical methods for delivering the radiation source to the tumor. During surgery, temporary implants can be placed which provide a source of radiation for a specified duration and are then removed at a later time, however this usually requires multiple procedures. To avoid multiple procedures, small radioactive seed have been developed which are surgically implanted and are left permanently to gradually decay over a period of weeks to months to a state of zero radiation emission. Another novel approach uses a surgically implanted catheter system with an expandable balloon reservoir implanted at the site of resection and a catheter connecting to a subcutaneous access port. A radioactive solution can then be injected percutaneously into the implanted reservoir and retrieved at a later time. The most commonly radiation sources used for glioma brachytherapy are iodine-

Brachytherapy is usually reserved for cases of high grade gliomas which have shown reoccurrence since several studies have shown no significant survival benefit for brachytherapy in newly diagnosed glioma38,39. One large randomized study by Selker and colleagues with 270 patients examined the use of interstitial brachytherapy in newly diagnosed high grade glioma40. Patients were randomized to two groups, one receiving resection, external beam radiation, and chemotherapy with the other group receiving resection, external beam radiation, chemotherapy, and 125I permanent interstitial

further study on the role of SRS in glioma treatment is needed.

delivered to normal cells which are located farther from the source.

125 and iridium-192.

complete excision in the placebo group27. In 2007 Stepp and colleagues reported similar findings with GTR in 65% of patients undergoing FIGS compared to 36% in placebo group28. They also demonstrated improved 6 month progression free survival in 41% of patients in the study group vs 21% in the control group.

Despite the positive initial results with FIGS, there are several limitations of this modality. The blue light used to illuminate 5-ALA has a depth of penetration of only a few millimeters and is easily obscured by blood products. Also 5-ALA has minimal uptake in tumors that have minimal contrast enhancement or that do not enhance at all, such as low grade glioma29,30. Advances in florescence guidance, particularly in its use for resection of these low grade, non-enhancing lesions are needed. As this technology continues to progress it will allow for greater extent of resection and increased survival in patients with glioma.

#### **6. Photodynamic treatment**

Photodynamic treatment (PDT) is another novel surgical method for the treatment of malignant glioblastoma. It utilizes the selective uptake of a photosensitizer by the individual tumor cells followed by irradiation of the tumor with light of a specific wavelength during surgery, which activates the photosensitizer to destroy the tumor cells selectively via oxidative reactions. Many different photosensitisers have been studied with the most promising being haematoporphyrin derivative (HPD). One of the greatest benefits with PDT is that it is a localized treatment, which lacks the systemic side effects associated with chemotherapy and radiation. The major side effect associated with PTD is cerebral edema in the irradiated area which can usually be managed with steroids.

In 2005, Stylli and colleagues reported one of the largest series of PDT for high grade glioma in 136 patients31. They utilized HPD administered IV preoperatively followed by irradiation with laser light during surgery. Median survival time following treatment for the 78 patients with glioblastoma was 14.3 months and median survival for the patients with anaplastic astrocytoma was 76.5 months. The same authors have also reported a review of the literature examining 10 studies, which show similar results although with fewer numbers of patients32. They conclude that PDT shows potential as a novel adjuvant therapy for glioma treatment along with chemotherapy and radiation therapy. However, further controlled clinical trials are needed to standardize HPD dosage as well as type and dose of light irradiation.

#### **7. Radiation therapy**

Following surgical resection, radiation therapy (RT) is considered the next step in the treatment of glioma. The principal goal of RT is to destroy residual tumor cells that were not removed with surgery, therefore preventing or postponing tumor reoccurrence. RT is most affective on smaller lesions and is therefore an adjuvant therapy in addition to surgical resection. Resection of maximal tumor bulk results in smaller residual volumes which are more responsive to RT and thereby increase the efficacy of RT. Historically, conventional radiation therapy using external beam radiation has been the main modality of radiotherapy used for glioma. Due to the risk of radiation induced injury to normal brain tissue, conventional radiation therapy is fractionated and the total dose is delivered over several treatments. Standard therapy usually consists of a total radiation dose in the range of 50–60 Gy administered over 20-30 fractions each with a dose of 1.8–2.0 Gy 33.

complete excision in the placebo group27. In 2007 Stepp and colleagues reported similar findings with GTR in 65% of patients undergoing FIGS compared to 36% in placebo group28. They also demonstrated improved 6 month progression free survival in 41% of patients in

Despite the positive initial results with FIGS, there are several limitations of this modality. The blue light used to illuminate 5-ALA has a depth of penetration of only a few millimeters and is easily obscured by blood products. Also 5-ALA has minimal uptake in tumors that have minimal contrast enhancement or that do not enhance at all, such as low grade glioma29,30. Advances in florescence guidance, particularly in its use for resection of these low grade, non-enhancing lesions are needed. As this technology continues to progress it will allow for greater extent of resection and increased survival in patients with glioma.

Photodynamic treatment (PDT) is another novel surgical method for the treatment of malignant glioblastoma. It utilizes the selective uptake of a photosensitizer by the individual tumor cells followed by irradiation of the tumor with light of a specific wavelength during surgery, which activates the photosensitizer to destroy the tumor cells selectively via oxidative reactions. Many different photosensitisers have been studied with the most promising being haematoporphyrin derivative (HPD). One of the greatest benefits with PDT is that it is a localized treatment, which lacks the systemic side effects associated with chemotherapy and radiation. The major side effect associated with PTD is cerebral edema in

In 2005, Stylli and colleagues reported one of the largest series of PDT for high grade glioma in 136 patients31. They utilized HPD administered IV preoperatively followed by irradiation with laser light during surgery. Median survival time following treatment for the 78 patients with glioblastoma was 14.3 months and median survival for the patients with anaplastic astrocytoma was 76.5 months. The same authors have also reported a review of the literature examining 10 studies, which show similar results although with fewer numbers of patients32. They conclude that PDT shows potential as a novel adjuvant therapy for glioma treatment along with chemotherapy and radiation therapy. However, further controlled clinical trials are needed to standardize HPD dosage as well as type and dose of light

Following surgical resection, radiation therapy (RT) is considered the next step in the treatment of glioma. The principal goal of RT is to destroy residual tumor cells that were not removed with surgery, therefore preventing or postponing tumor reoccurrence. RT is most affective on smaller lesions and is therefore an adjuvant therapy in addition to surgical resection. Resection of maximal tumor bulk results in smaller residual volumes which are more responsive to RT and thereby increase the efficacy of RT. Historically, conventional radiation therapy using external beam radiation has been the main modality of radiotherapy used for glioma. Due to the risk of radiation induced injury to normal brain tissue, conventional radiation therapy is fractionated and the total dose is delivered over several treatments. Standard therapy usually consists of a total radiation dose in the range of 50–60

the irradiated area which can usually be managed with steroids.

Gy administered over 20-30 fractions each with a dose of 1.8–2.0 Gy 33.

the study group vs 21% in the control group.

**6. Photodynamic treatment** 

irradiation.

**7. Radiation therapy** 

Novel methods of administering radiation therapy are also being developed. Following optimal surgical resection, image guided stereotactic radiosurgery (SRS) is now being evaluated for treatment these lesions. This new modality utilizes a single high doses of radiation specifically targeted to a well-defined lesions using detailed neuroimaging. This allows delivery of focused radiation to the tumor with a much lower dose to adjacent nontargeted tissue which results in reduced side effects compared with traditional methods.

For low grade gliomas, adjuvant RT following resection has failed to show a survival benefit. In cases of disease progression or inoperable lesions with neurologic symptoms, delayed RT appears to provide the same survival advantage as postoperative RT34. For newly diagnosed high grade gliomas studies have demonstrated that SRS does not provide a significant survival advantage over conventional radiotherapy35,36. The use of SRS has also been explored as a treatment for recurrent high grade lesions. A recent article by Romanelli and colleagues reviewed 17 retrospective studies examining the role of SRS in the treatment of recurrent high grade glioma. This review demonstrated that SRS is associated with prolonged survival in patients with recurrent GBM with median survival times ranging from 7.5 to 30 months37. SRS has been shown to have no significant advantage as a first line radiation therapy for glioma and the results for recurrent glioma are inconclusive, therefore further study on the role of SRS in glioma treatment is needed.

In addition to externally administered sources of radiation therapy, interstitial brachytherapy is another form of radiation therapy used to treat glioma, which refers to surgical placement of the radioactive source a short distance from or within the tumor being treated. Brachytherapy was developed due to the observation that 80% of malignant gliomas reoccur within 2cm of the initial tumor site following resection. By placing the radioactive source directly in the tumor or tumor bed, continuous high dose radiation increases damage to nearby proliferating tumor cells located at the margin with a rapid fall off in the dose delivered to normal cells which are located farther from the source.

There are multiple surgical methods for delivering the radiation source to the tumor. During surgery, temporary implants can be placed which provide a source of radiation for a specified duration and are then removed at a later time, however this usually requires multiple procedures. To avoid multiple procedures, small radioactive seed have been developed which are surgically implanted and are left permanently to gradually decay over a period of weeks to months to a state of zero radiation emission. Another novel approach uses a surgically implanted catheter system with an expandable balloon reservoir implanted at the site of resection and a catheter connecting to a subcutaneous access port. A radioactive solution can then be injected percutaneously into the implanted reservoir and retrieved at a later time. The most commonly radiation sources used for glioma brachytherapy are iodine-125 and iridium-192.

Brachytherapy is usually reserved for cases of high grade gliomas which have shown reoccurrence since several studies have shown no significant survival benefit for brachytherapy in newly diagnosed glioma38,39. One large randomized study by Selker and colleagues with 270 patients examined the use of interstitial brachytherapy in newly diagnosed high grade glioma40. Patients were randomized to two groups, one receiving resection, external beam radiation, and chemotherapy with the other group receiving resection, external beam radiation, chemotherapy, and 125I permanent interstitial

Innovative Surgical Management of Glioma 15

One advancement has come in the area of surgically implantable polymers, which are infused with chemotherapeutic agents. These polymers are then placed into the surgical cavity following resection to provide direct application of the chemotherapeutic agent to the tumor bed over a prolonged period of time as the polymer degrades and releases the agent. The most widely studied therapy of this nature utilizes a polyanhydride wafer embedded with carmustine (gliadel wafers). Gliadel is currently the only interstitial chemotherapy treatment approved for use with malignant glioma. Wafers can be implanted at the time of initial surgery or reserved for episodes of reoccurrence. Several trials have demonstrated increased survival following the use of implantable wafers. For initial tumor treatment, increased survival has been shown of 13.9 months compared to 11.6 months in nontreatment group. Wafer usage for treatment of tumor reoccurrence provided increased

Overall, these studies show a 35% risk reduction of death with the use of gliadel, with a median survival of 14 months, which is a 2.5 month improvement over placebo. 1 year survival is approximately 10% better with use of gliadel48. Care must be taken with the use of this therapy as side effects of necrosis, cerebral edema, and seizure are common but can

Use of gliadel can also exclude from further clinical trials and newer treatments. It can cause confounding effects on another trial because all of these are so new. Improvement of

Another novel approach still in the early phases of development is the use of surgically implantable nanoparticles to deliver chemotherapeutic agents. Small spherical particles 7 to 10 nanometers in size comprised of polymers or liposomes are non-covalently attached to slow sustained release formulations of chemotherapeutic drugs for delivery. These nanoparticles are small enough to cross the blood brain tumor barrier (BBTB) and transmit

Nanoparticles can be administered intravascularly or directly into the brain via a surgically implanted catheter. Delivery of nanoparticles via surgically implanted catheter has the benefit of greater volume of distribution directly to the brain tissue compared with diffusion alone50. Development of these nanoparticle systems for treatment of malignant brain tumors is currently in the animal model phase and no human studies are currently available. Rat models of glioblastoma have shown increased survival using doxorubicin bonded to cyanoacrylate nanoparticles for delivery to tumor cells. The drug transport across the BBB by nanoparticles appears to be due to a receptor-mediated interaction with the brain capillary endothelial cells, which is facilitated by certain plasma apolipoproteins adsorbed by nanoparticles in the blood. Nanoparticle uptake appears selective to tumor cells in these

In addition to drug delivery, nanoparticles are being investigated for use in neuroimaging. Current imaging techniques have a maximum resolution of 1 mm. Nanoparticles could

survival of 31 weeks compared to 23 weeks in non-treated patients45–47.

be controlled with steroid and antiepliptic therapy.

surgical resection can eliminate these problems.

drug directly into individual tumor cells49.

models as the animals did not manifest signs of neurotoicity51.

**11. Nanoparticles** 

**10. Implantable polymers** 

brachytherapy. They found no statistical difference in the median survival time between the two groups for newly diagnosed high grade glioma.

The results for brachytherapy in the case of recurrent high grade glioma appear to show improved survival41–43. One large study with 95 patients by Gabayan and colleagues examined the use of the GliaSite Radiation Therapy System in the treatment of recurrent high grade glioma. This system utilizes 125I administered via a surgically implanted balloon catheter system. All patient were initially treated with resection followed by external beam radiation. Following reoccurrence, patients underwent maximal surgical debulking followed by implantation of an expandable balloon catheter system. Radioactive solution was then administered between 2-6 weeks following the debulking procedure. Patient undergoing this treatment showed median survival of 36.3 weeks from the time of the debulking surgery with a 1 year survival of 31.1%. Although no control group was used in this study, survival times were compared to matched patients from another published study matched for age, KPS, and surgical management. These control patients showed a median survival of 23 weeks following resection for reoccurrence. The results from the brachytherapy group compare favorably with the control group. Further studies including randomized trials are still needed but brachytherapy appears to show promise as adjuvant therapy in recurrent glioma.

#### **8. Chemotherapy**

In addition to surgical resection and radiation therapy the other mainstay of treatment for malignant glioma is chemotherapeutics. Chemotherapy is another adjuvant therapy to be utilized along with surgical resection. As with radiation therapy, chemotherapy is more effective in treating smaller tumor volumes and therefore maximizing the extent of surgical resection is important in order to provide a more favorable response to chemotherapy44. Two main classes of drugs are currently used, alkylating agents (carmusitine, temozolomide) and antiangiogenic agents (bevacizumab).

#### **9. Blood brain barrier**

The blood brain barrier (BBB) presents difficulty in the chemotherapuetic treatment of gliomas as many agents that are effective for the treatment of systemic disease are unable to cross the BBB. There have been several developments designed to open the BBB and provide direct treatment of the central nervous system. Initial therapies to open the BBB made use of small lipophilic molecule drugs administered systemically for increased permeation across the BBB. This approach was limited by drug binding to plasma protein as well as extravasation of the drug back across the BBB into the systemic circulation. Other early treatments used osmotic modification of the BBB with agents such as mannitol given intraarterially to disrupt the BBB followed by the chemotherapeutic agent of choice. The effectiveness of these early methods were limited due to the transient elevation of drug concentration within the brain tissues and short drug half–life which did not allow for accumulation of the drug at a therapeutic concentration. Over the past decade, several innovative surgical methods of opening the BBB have been developed to provide longer acting and direct treatment to the tumor cells.

#### **10. Implantable polymers**

14 Novel Therapeutic Concepts in Targeting Glioma

brachytherapy. They found no statistical difference in the median survival time between the

The results for brachytherapy in the case of recurrent high grade glioma appear to show improved survival41–43. One large study with 95 patients by Gabayan and colleagues examined the use of the GliaSite Radiation Therapy System in the treatment of recurrent high grade glioma. This system utilizes 125I administered via a surgically implanted balloon catheter system. All patient were initially treated with resection followed by external beam radiation. Following reoccurrence, patients underwent maximal surgical debulking followed by implantation of an expandable balloon catheter system. Radioactive solution was then administered between 2-6 weeks following the debulking procedure. Patient undergoing this treatment showed median survival of 36.3 weeks from the time of the debulking surgery with a 1 year survival of 31.1%. Although no control group was used in this study, survival times were compared to matched patients from another published study matched for age, KPS, and surgical management. These control patients showed a median survival of 23 weeks following resection for reoccurrence. The results from the brachytherapy group compare favorably with the control group. Further studies including randomized trials are still needed but brachytherapy appears to show promise as adjuvant

In addition to surgical resection and radiation therapy the other mainstay of treatment for malignant glioma is chemotherapeutics. Chemotherapy is another adjuvant therapy to be utilized along with surgical resection. As with radiation therapy, chemotherapy is more effective in treating smaller tumor volumes and therefore maximizing the extent of surgical resection is important in order to provide a more favorable response to chemotherapy44. Two main classes of drugs are currently used, alkylating agents (carmusitine,

The blood brain barrier (BBB) presents difficulty in the chemotherapuetic treatment of gliomas as many agents that are effective for the treatment of systemic disease are unable to cross the BBB. There have been several developments designed to open the BBB and provide direct treatment of the central nervous system. Initial therapies to open the BBB made use of small lipophilic molecule drugs administered systemically for increased permeation across the BBB. This approach was limited by drug binding to plasma protein as well as extravasation of the drug back across the BBB into the systemic circulation. Other early treatments used osmotic modification of the BBB with agents such as mannitol given intraarterially to disrupt the BBB followed by the chemotherapeutic agent of choice. The effectiveness of these early methods were limited due to the transient elevation of drug concentration within the brain tissues and short drug half–life which did not allow for accumulation of the drug at a therapeutic concentration. Over the past decade, several innovative surgical methods of opening the BBB have been developed to provide longer

two groups for newly diagnosed high grade glioma.

temozolomide) and antiangiogenic agents (bevacizumab).

acting and direct treatment to the tumor cells.

therapy in recurrent glioma.

**8. Chemotherapy** 

**9. Blood brain barrier** 

One advancement has come in the area of surgically implantable polymers, which are infused with chemotherapeutic agents. These polymers are then placed into the surgical cavity following resection to provide direct application of the chemotherapeutic agent to the tumor bed over a prolonged period of time as the polymer degrades and releases the agent.

The most widely studied therapy of this nature utilizes a polyanhydride wafer embedded with carmustine (gliadel wafers). Gliadel is currently the only interstitial chemotherapy treatment approved for use with malignant glioma. Wafers can be implanted at the time of initial surgery or reserved for episodes of reoccurrence. Several trials have demonstrated increased survival following the use of implantable wafers. For initial tumor treatment, increased survival has been shown of 13.9 months compared to 11.6 months in nontreatment group. Wafer usage for treatment of tumor reoccurrence provided increased survival of 31 weeks compared to 23 weeks in non-treated patients45–47.

Overall, these studies show a 35% risk reduction of death with the use of gliadel, with a median survival of 14 months, which is a 2.5 month improvement over placebo. 1 year survival is approximately 10% better with use of gliadel48. Care must be taken with the use of this therapy as side effects of necrosis, cerebral edema, and seizure are common but can be controlled with steroid and antiepliptic therapy.

Use of gliadel can also exclude from further clinical trials and newer treatments. It can cause confounding effects on another trial because all of these are so new. Improvement of surgical resection can eliminate these problems.

#### **11. Nanoparticles**

Another novel approach still in the early phases of development is the use of surgically implantable nanoparticles to deliver chemotherapeutic agents. Small spherical particles 7 to 10 nanometers in size comprised of polymers or liposomes are non-covalently attached to slow sustained release formulations of chemotherapeutic drugs for delivery. These nanoparticles are small enough to cross the blood brain tumor barrier (BBTB) and transmit drug directly into individual tumor cells49.

Nanoparticles can be administered intravascularly or directly into the brain via a surgically implanted catheter. Delivery of nanoparticles via surgically implanted catheter has the benefit of greater volume of distribution directly to the brain tissue compared with diffusion alone50. Development of these nanoparticle systems for treatment of malignant brain tumors is currently in the animal model phase and no human studies are currently available. Rat models of glioblastoma have shown increased survival using doxorubicin bonded to cyanoacrylate nanoparticles for delivery to tumor cells. The drug transport across the BBB by nanoparticles appears to be due to a receptor-mediated interaction with the brain capillary endothelial cells, which is facilitated by certain plasma apolipoproteins adsorbed by nanoparticles in the blood. Nanoparticle uptake appears selective to tumor cells in these models as the animals did not manifest signs of neurotoicity51.

In addition to drug delivery, nanoparticles are being investigated for use in neuroimaging. Current imaging techniques have a maximum resolution of 1 mm. Nanoparticles could

Innovative Surgical Management of Glioma 17

Treatments using active immunotherapy via cell-based and peptide vaccines are also under study. Cells from glioma tumors are thought to be poor antigen-presenting cells because they often secrete immunosuppressive cytokines as well as growth factors such as transforming growth factor and vascular endothelial growth factor which can have a negative effect on T cell and natural killer cell activity. Tumor vaccines are designed to augment tumor-specific cellular immunity and enhance low-level immunity by stimulating

Chang and colleagues published results from one vaccine based phase II clinical trial in 2011. 16 patients with glioblastoma (8 newly diagnosed, 8 recurrent) underwent craniotomy for maximal cytoreduction followed by standard external beam radiation therapy in the newly diagnosed patients. Tumor cells obtained from the surgical resection were cultured in the laboratory and combined with autologous dendritic cells to produce vaccine. The vaccine was administered to patients via subcutaneous injection over lymph nodes for a total of 10 treatments over a 6 month period. Median survival and 5 year survival was 381 days and 12.5% for the newly diagnosed group, 966 days and 25% for the recurrent group compared to 380 days mean survival and 0% five years survival for a 16 patient age and sex

Although most published results are from preliminary studies with small numbers of patients, immunotherapy for the treatment of glioma is a promising area currently undergoing multiple phase II and phase III trials for FDA approval54. Once the basic efficacy of these initial studies have been verified as a plausible modality for the treatment of glioma, randomized and controlled clinical trials can be undertaken to further explore the

Gene therapy is another novel modality used in the treatment of glioma, which focuses on the delivery of apoptotic genes at the time of surgical intervention and implantation in the surgical cavity. One of the most effective methods of in vivo gene delivery is the use of viral vectors as gene carriers. Retroviruses, adenovirus, and herpes simplex virus-1 (HSV-1) are all currently undergoing trials as vectors for viral brain tumor therapy Replication competent retroviruses have been shown to have infection rates of 97% with specificity for tumor cells without significant effects on non-tumor cells56. Adenovirus and HSV-1 are used in both replication competent and replication defective forms and have shown high

In 2003, Germano and colleagues reported a series of 11 patients with high grade glioma treated with gene therapy using adenovirus as a viral vector57. Adenovirus was used to transfer the herpes simplex-thymidine kinase gene into malignant glioma cells. This gene then phosphorylates ganciclovir, a non-cytotoxic nucleotide analog, into a compound that halts the transcription of DNA in dividing cells. Since normal brain cells are not rapidly dividing they are not affected. At the time of surgery, following gross total resection, the viral solution was injected directly into the tumor bed. This was followed by administration of ganciclovir systemically over 7 days. Of the 11 patients 10 had a survival of > 52 weeks following treatment. This survival time was associated with maintaining quality of life. 8 patients maintained a KPS of greater than 70 after 3 months and 5 patients 6 months after

the production of higher-avidity T cells specific to a tumor55.

matched historical control group.

full potential of this therapy.

transgenic capacity and persistent gene expression57.

**13. Gene therapy** 

treatment.

improve the resolution by a factor of ten or more, allowing detection of smaller tumors and more precise surgical resection. Several nanoparticle-based contrast materials have been used to enhance MRI imaging. One iron oxide nanoparticle currently under study has shown an innocuous toxicity profile as well as sustained retention in mouse tumors52. These fluorescent nanoparticles improved the contrast between the tumor tissue and the normal tissue in both MRI and optical imaging, which can be used during surgery to see the tumor boundary more precisely.

Another promising role of nanoparticle in the treatment of glioma involves hyperthermia treatment. The heating of cancerous tissues between 41 and 45°C, has been shown to improve the efficacy of cancer therapy when used in conjunction with chemotherapy and radiation52. Magnetic nanocomposites based on iron oxide can be used as implantable biomaterials for thermal cancer therapy applications at the time of surgery. These implanted particles can then be remotely heated by exposure to an external alternating magnetic field.

In 2011, Maier-Hauff and colleagues reported a trial of magnetic nanoparticle thermotherapy in conjunction with radiation treatments in 59 patients with recurrent glioblastoma53. Magnetic fluid was instilled within the tumor site using a neuronavigational procedure comparable to a brain needle biopsy and an external magnetic field was then applied for multiple treatments. Patients undergoing this procedure showed a mean survival time of 13.4 months after the first re-occurrence. Thermotherapy using magnetic nanoparticles in conjunction with a reduced radiation dose is safe and effective and leads to longer overall survival compared with conventional therapies in the treatment of recurrent GBM.

#### **12. Immunotherapies**

Using the body's own immune system to fight glioma, immunotherapy, is a new field which has seen significant advancements over the past decade. There are two categories of immunotherapy for glioma that are currently undergoing clinical research. Passive immunotherapy involves the activation of cytotoxic effector cells ex vivo and thee transfer of these activated cells back into the patient's body. Active immunotherapy, on the other hand, uses an exogenous trigger which causes activation of endogenous effector cells within the patients own body to target tumor cells. Active immunotherapy is generally employed in the tumor vaccine model. Surgery plays a large role in these therapies, as sizeable volumes of tumor tissue must be surgically obtained in order to construct the immunotherapeutic agents. Also several of these therapies utilize direct delivery of these agents into the brain during surgery.

Current clinical trials utilizing passive immunotherapy focus on the activation of effector cytotoxic T lymphocytes, natural killer cells, or lymphokine activated killer cells sensitized to glioma-associated antigens. Once such trial for glioblastoma treatment uses donor cytotoxic T lymphoyctes which are sensitized ex vivo to recognize patient human leukocyte antigen (HLA) groups expressed on the surface of glioma cells but not on normal neurons or glia. After surgical resection, the sensitized CTL cells are placed in the resection cavity as well as surgical placement of an intraparanchemal catheter and reservoir system to allow future delivery of CTL cells. An initial pilot study with this model showed significant survival benefit in 3 of 6 patients with 2 patients surviving >15 years since beginning immunotherapy54.

improve the resolution by a factor of ten or more, allowing detection of smaller tumors and more precise surgical resection. Several nanoparticle-based contrast materials have been used to enhance MRI imaging. One iron oxide nanoparticle currently under study has shown an innocuous toxicity profile as well as sustained retention in mouse tumors52. These fluorescent nanoparticles improved the contrast between the tumor tissue and the normal tissue in both MRI and optical imaging, which can be used during surgery to see the tumor

Another promising role of nanoparticle in the treatment of glioma involves hyperthermia treatment. The heating of cancerous tissues between 41 and 45°C, has been shown to improve the efficacy of cancer therapy when used in conjunction with chemotherapy and radiation52. Magnetic nanocomposites based on iron oxide can be used as implantable biomaterials for thermal cancer therapy applications at the time of surgery. These implanted particles can then be remotely heated by exposure to an external alternating magnetic field. In 2011, Maier-Hauff and colleagues reported a trial of magnetic nanoparticle thermotherapy in conjunction with radiation treatments in 59 patients with recurrent glioblastoma53. Magnetic fluid was instilled within the tumor site using a neuronavigational procedure comparable to a brain needle biopsy and an external magnetic field was then applied for multiple treatments. Patients undergoing this procedure showed a mean survival time of 13.4 months after the first re-occurrence. Thermotherapy using magnetic nanoparticles in conjunction with a reduced radiation dose is safe and effective and leads to longer overall survival compared with

Using the body's own immune system to fight glioma, immunotherapy, is a new field which has seen significant advancements over the past decade. There are two categories of immunotherapy for glioma that are currently undergoing clinical research. Passive immunotherapy involves the activation of cytotoxic effector cells ex vivo and thee transfer of these activated cells back into the patient's body. Active immunotherapy, on the other hand, uses an exogenous trigger which causes activation of endogenous effector cells within the patients own body to target tumor cells. Active immunotherapy is generally employed in the tumor vaccine model. Surgery plays a large role in these therapies, as sizeable volumes of tumor tissue must be surgically obtained in order to construct the immunotherapeutic agents. Also several of these therapies utilize direct delivery of these agents into the brain

Current clinical trials utilizing passive immunotherapy focus on the activation of effector cytotoxic T lymphocytes, natural killer cells, or lymphokine activated killer cells sensitized to glioma-associated antigens. Once such trial for glioblastoma treatment uses donor cytotoxic T lymphoyctes which are sensitized ex vivo to recognize patient human leukocyte antigen (HLA) groups expressed on the surface of glioma cells but not on normal neurons or glia. After surgical resection, the sensitized CTL cells are placed in the resection cavity as well as surgical placement of an intraparanchemal catheter and reservoir system to allow future delivery of CTL cells. An initial pilot study with this model showed significant survival benefit in 3 of 6 patients with 2 patients surviving >15 years since beginning

boundary more precisely.

**12. Immunotherapies** 

during surgery.

immunotherapy54.

conventional therapies in the treatment of recurrent GBM.

Treatments using active immunotherapy via cell-based and peptide vaccines are also under study. Cells from glioma tumors are thought to be poor antigen-presenting cells because they often secrete immunosuppressive cytokines as well as growth factors such as transforming growth factor and vascular endothelial growth factor which can have a negative effect on T cell and natural killer cell activity. Tumor vaccines are designed to augment tumor-specific cellular immunity and enhance low-level immunity by stimulating the production of higher-avidity T cells specific to a tumor55.

Chang and colleagues published results from one vaccine based phase II clinical trial in 2011. 16 patients with glioblastoma (8 newly diagnosed, 8 recurrent) underwent craniotomy for maximal cytoreduction followed by standard external beam radiation therapy in the newly diagnosed patients. Tumor cells obtained from the surgical resection were cultured in the laboratory and combined with autologous dendritic cells to produce vaccine. The vaccine was administered to patients via subcutaneous injection over lymph nodes for a total of 10 treatments over a 6 month period. Median survival and 5 year survival was 381 days and 12.5% for the newly diagnosed group, 966 days and 25% for the recurrent group compared to 380 days mean survival and 0% five years survival for a 16 patient age and sex matched historical control group.

Although most published results are from preliminary studies with small numbers of patients, immunotherapy for the treatment of glioma is a promising area currently undergoing multiple phase II and phase III trials for FDA approval54. Once the basic efficacy of these initial studies have been verified as a plausible modality for the treatment of glioma, randomized and controlled clinical trials can be undertaken to further explore the full potential of this therapy.

#### **13. Gene therapy**

Gene therapy is another novel modality used in the treatment of glioma, which focuses on the delivery of apoptotic genes at the time of surgical intervention and implantation in the surgical cavity. One of the most effective methods of in vivo gene delivery is the use of viral vectors as gene carriers. Retroviruses, adenovirus, and herpes simplex virus-1 (HSV-1) are all currently undergoing trials as vectors for viral brain tumor therapy Replication competent retroviruses have been shown to have infection rates of 97% with specificity for tumor cells without significant effects on non-tumor cells56. Adenovirus and HSV-1 are used in both replication competent and replication defective forms and have shown high transgenic capacity and persistent gene expression57.

In 2003, Germano and colleagues reported a series of 11 patients with high grade glioma treated with gene therapy using adenovirus as a viral vector57. Adenovirus was used to transfer the herpes simplex-thymidine kinase gene into malignant glioma cells. This gene then phosphorylates ganciclovir, a non-cytotoxic nucleotide analog, into a compound that halts the transcription of DNA in dividing cells. Since normal brain cells are not rapidly dividing they are not affected. At the time of surgery, following gross total resection, the viral solution was injected directly into the tumor bed. This was followed by administration of ganciclovir systemically over 7 days. Of the 11 patients 10 had a survival of > 52 weeks following treatment. This survival time was associated with maintaining quality of life. 8 patients maintained a KPS of greater than 70 after 3 months and 5 patients 6 months after treatment.

Innovative Surgical Management of Glioma 19

drug 5-flourouracil62. These stem cells were then implanted in rats with induced glioblastoma and the animals were subsequently treated with the nontoxic compound. After 10 days, the animals treated with the modified NSCs showed significant 50% decrease in tumoral mass compared to a control group. After histopathological examination of the treated tissue, they also found high levels of the toxic 5-flourouracil drug in adjacent tissue demonstrating in vivo conversion of the nontoxic compound to the toxic drug with

Neural stem cells are also being used as vehicles for the tracking and suppression of glioblastoma. These methods exploit the tendency of NSCs to preferentially migrate towards brain tumors. This allows NSCs to be labeled and used as diagnostic imaging tools to identify extent of tumor invasion. One such animal model used NSCs modified to express the firefly luciferase gene63. These cells were then implanted into the contralateral brain parenchyma as well as injected into the ventricles of mice with intracranial gliomas. Over a period of 3 weeks, serial bioluminescence imaging was performed, which showed migration of the implanted cell across the corpus callosum with a maximal density at the site of the tumors. A subsequent study using the same model but with the addition of an apoptosispromoting gene to the NSCs was performed to evaluate the therapeutic possibilities of this model. NSCs were modified not only to express the luciferase gene but also the tumor necrosis factor related apoptosis inducing ligand S-TRAIL. The transformed NSCs were then stereotactically implanted into the left frontal lobe of mice and glioma cells were stereotactically injected into the right frontal lobe of the same animals. Serial imaging was again performed which initially showed increased tumor volumes at the site of glioma injection as well as migration of the NSCs towards the tumor areas. After 16 days however there was a considerable decrease in tumor growth and a significant reduction in tumor cells on quantitative analysis compared to control animals. They also found expression of the S-TRAIL gene product at the tumor site on histopathological examination. These studies

demonstrate the promising role that stem cells can play in the treatment of glioma.

Surgical resection remains the single most important primary treatment in the management of patients with glioma and extent of surgical resection directly correlates with increased patient survival. In this chapter we reviewed several innovative technologies and surgical methods for increasing the extent of resection as well as several adjuvant therapies and the important role that surgery plays in maximizing the potential benefit of these therapies in the treatment of glioma. As our ability to increase the extent of resection improves and new innovative technologies are perfected, we will continue to see improvements in long term

[1] Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. *CA Cancer J Clin*. 2010;60(5):277-

[2] Schneider T, Mawrin C, Scherlach C, Skalej M, Firsching R. Gliomas in adults. *Deutsches* 

therapeutic benefit.

**14. Conclusion** 

**15. References** 

300.

survival in patients affected with glioma.

*\Ärzteblatt International*. 2010;107(45):799.

Oncolytic viruses are also currently being used for clinical trials. These virus replicate selectively within tumor cells and can lead to increased intratumoral viral titers and cell death58. In 2004, Harrow and colleges used a modified herpes simplex virus with an affinity for glioblastoma cells which replicates only in rapidly diving cells causing cell lysis while sparing normal terminally differentiated cells. Twelve patients, 6 with newly diagnosed glioblastoma and 6 with recurrent glioblastoma, underwent craniotomy and surgical resection. Following resection during the same surgical procedure they were injected with the modified herpes virus at 8-10 sights adjacent to the tumor bed. Four patients, 2 newly diagnosed and 2 with recurrent disease showed survival of greater than 15 months following treatment.

Viral vectors, although promising, do have several limitations. Viral vectors suffer from low levels of gene incorporation due to limited diffusion into the brain parenchyma as well as low transfection rates of some cell types. Formation of antigenicity to vectors and introduced gene products causes additional difficulties. Also the use of retroviruses that incorporate genes into the host chromosome can result in insertional mutagenesis and propensity to form new tumors. The use of genetically modified cells to deliver gene therapy to the CNS may avoid some of these limitations. The use of stem cells is one area of research being used to avoid these limitations seen with traditional gene therapy.

Neural stem cells (NSC) are self-renewing multipotent cells found in the fetal brain within the ventricular zone, midbrain and spinal cord. These cells have the ability to repopulate a degenerated CNS region and can migrate toward pathologically altered tissues, including stroke, trauma and tumors. Genetic changes in NSCs may result in tumorigenesis by activation of oncogenes and/or inactivation of tumor suppressor genes.

Within brain tumors, a population of cells known brain tumor stem cells (BTSC) have been found. They are resistant to current treatments and capable of maintaining and propagating these tumors59. BTSCs are thought to arise from aberrant NSCs or from mature cells that have undergone mutation and dedifferentiation. BTSCs are similar to normal stem cells with regards to self-renewal, capacity, multi-potentiality, tumorigenicity as well as migratory capability. Transformation of these neural stem cells and their progenitor cells may therefore lead to the formation of BTSCs and eventually malignancy60. Gene therapies focusing on preventing the malignant transformation of

NSCs into BTSCs as well as the use of normal stem cells as a method of targeted delivery of therapeutic agents to glioma cells are currently under investigation.

Genetic modification of NSCs to secrete anti-tumor agents allows targeted delivery as well as provides a high level of active compounds at the local site of neoplasm. These types of therapies are still in the early stages and have not been evaluated in human glioma patients, however there are studies which have shown good results using animal models. One model using neural stem cells modified to produce high quantities of interleukin-4 in vivo was examined in Spraque-Dawley rats61. They implanted the modified NSCs into the brain tissue of rats affected with malignant gliobastoma and found a long term survival of 50% compared to control animals.

Another rat study used immortalized neural progenitor stem cells to express a eukaryotic catalytic enzyme that converts the nontoxic compound 5-flourocytosine into the highly toxic

Oncolytic viruses are also currently being used for clinical trials. These virus replicate selectively within tumor cells and can lead to increased intratumoral viral titers and cell death58. In 2004, Harrow and colleges used a modified herpes simplex virus with an affinity for glioblastoma cells which replicates only in rapidly diving cells causing cell lysis while sparing normal terminally differentiated cells. Twelve patients, 6 with newly diagnosed glioblastoma and 6 with recurrent glioblastoma, underwent craniotomy and surgical resection. Following resection during the same surgical procedure they were injected with the modified herpes virus at 8-10 sights adjacent to the tumor bed. Four patients, 2 newly diagnosed and 2 with recurrent disease showed survival of greater than 15 months

Viral vectors, although promising, do have several limitations. Viral vectors suffer from low levels of gene incorporation due to limited diffusion into the brain parenchyma as well as low transfection rates of some cell types. Formation of antigenicity to vectors and introduced gene products causes additional difficulties. Also the use of retroviruses that incorporate genes into the host chromosome can result in insertional mutagenesis and propensity to form new tumors. The use of genetically modified cells to deliver gene therapy to the CNS may avoid some of these limitations. The use of stem cells is one area of

Neural stem cells (NSC) are self-renewing multipotent cells found in the fetal brain within the ventricular zone, midbrain and spinal cord. These cells have the ability to repopulate a degenerated CNS region and can migrate toward pathologically altered tissues, including stroke, trauma and tumors. Genetic changes in NSCs may result in tumorigenesis by

Within brain tumors, a population of cells known brain tumor stem cells (BTSC) have been found. They are resistant to current treatments and capable of maintaining and propagating these tumors59. BTSCs are thought to arise from aberrant NSCs or from mature cells that have undergone mutation and dedifferentiation. BTSCs are similar to normal stem cells with regards to self-renewal, capacity, multi-potentiality, tumorigenicity as well as migratory capability. Transformation of these neural stem cells and their progenitor cells may therefore lead to the formation of BTSCs and eventually malignancy60. Gene therapies focusing on

NSCs into BTSCs as well as the use of normal stem cells as a method of targeted delivery of

Genetic modification of NSCs to secrete anti-tumor agents allows targeted delivery as well as provides a high level of active compounds at the local site of neoplasm. These types of therapies are still in the early stages and have not been evaluated in human glioma patients, however there are studies which have shown good results using animal models. One model using neural stem cells modified to produce high quantities of interleukin-4 in vivo was examined in Spraque-Dawley rats61. They implanted the modified NSCs into the brain tissue of rats affected with malignant gliobastoma and found a long term survival of 50%

Another rat study used immortalized neural progenitor stem cells to express a eukaryotic catalytic enzyme that converts the nontoxic compound 5-flourocytosine into the highly toxic

research being used to avoid these limitations seen with traditional gene therapy.

activation of oncogenes and/or inactivation of tumor suppressor genes.

therapeutic agents to glioma cells are currently under investigation.

preventing the malignant transformation of

compared to control animals.

following treatment.

drug 5-flourouracil62. These stem cells were then implanted in rats with induced glioblastoma and the animals were subsequently treated with the nontoxic compound. After 10 days, the animals treated with the modified NSCs showed significant 50% decrease in tumoral mass compared to a control group. After histopathological examination of the treated tissue, they also found high levels of the toxic 5-flourouracil drug in adjacent tissue demonstrating in vivo conversion of the nontoxic compound to the toxic drug with therapeutic benefit.

Neural stem cells are also being used as vehicles for the tracking and suppression of glioblastoma. These methods exploit the tendency of NSCs to preferentially migrate towards brain tumors. This allows NSCs to be labeled and used as diagnostic imaging tools to identify extent of tumor invasion. One such animal model used NSCs modified to express the firefly luciferase gene63. These cells were then implanted into the contralateral brain parenchyma as well as injected into the ventricles of mice with intracranial gliomas. Over a period of 3 weeks, serial bioluminescence imaging was performed, which showed migration of the implanted cell across the corpus callosum with a maximal density at the site of the tumors. A subsequent study using the same model but with the addition of an apoptosispromoting gene to the NSCs was performed to evaluate the therapeutic possibilities of this model. NSCs were modified not only to express the luciferase gene but also the tumor necrosis factor related apoptosis inducing ligand S-TRAIL. The transformed NSCs were then stereotactically implanted into the left frontal lobe of mice and glioma cells were stereotactically injected into the right frontal lobe of the same animals. Serial imaging was again performed which initially showed increased tumor volumes at the site of glioma injection as well as migration of the NSCs towards the tumor areas. After 16 days however there was a considerable decrease in tumor growth and a significant reduction in tumor cells on quantitative analysis compared to control animals. They also found expression of the S-TRAIL gene product at the tumor site on histopathological examination. These studies demonstrate the promising role that stem cells can play in the treatment of glioma.

#### **14. Conclusion**

Surgical resection remains the single most important primary treatment in the management of patients with glioma and extent of surgical resection directly correlates with increased patient survival. In this chapter we reviewed several innovative technologies and surgical methods for increasing the extent of resection as well as several adjuvant therapies and the important role that surgery plays in maximizing the potential benefit of these therapies in the treatment of glioma. As our ability to increase the extent of resection improves and new innovative technologies are perfected, we will continue to see improvements in long term survival in patients affected with glioma.

#### **15. References**


Innovative Surgical Management of Glioma 21

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**EGFR and Glioma Therapy** 

[63] Tang Y, Shah K, Messerli SM, et al. In vivo tracking of neural progenitor cell migration to glioblastomas. *Hum. Gene Ther.* 2003;14(13):1247-1254. **Part 2**  to glioblastomas. *Hum. Gene Ther.* 2003;14(13):1247-1254. **Part 2** 

**EGFR and Glioma Therapy** 

24 Novel Therapeutic Concepts in Targeting Glioma

[63] Tang Y, Shah K, Messerli SM, et al. In vivo tracking of neural progenitor cell migration

**2** 

Terrance Johns *Monash University,* 

*Australia* 

**Advances in the Development of EGFR Targeted** 

The epidermal growth factor receptor (EGFR) is receptor tyrosine kinase (RTK) dysregulated in glioblastoma (GBM) through overexpression, mutation or inappropriate expression of ligand. Activation of the EGFR by these mechanisms contributes to the development and progression of GBM by engaging downstream targets, such as the PI3K pathway. The de2-7 EGFR (or EGFRvIII), a naturally occurring mutation of the EGFR frequently expressed in GBM, preferentially activates this pathway. Clinical trials with EGFR-specific tyrosine kinase inhibitors (TKIs) have been disappointing with very little antitumor activity observed. The outcome of controlled clinical trials with EGFR-specific antibodies is yet to be reported. Encouraging preclinical and preliminary clinical data suggests that the combination of EGFR therapeutics and compounds that target molecules downstream of EGFR might have increased efficacy. Finally, the identification of biomarkers that predict those patients most likely to respond to EGFR inhibition is

The EGFR is frequently expressed in GBM (Jungbluth et al., 2003), the most common and deadly form of malignant brain cancer (DeAngelis, 2001). Extensive co-expression of EGFR ligands such as EGF and TGF-α has also been reported (Ekstrand et al., 1991), suggesting the existence of a robust autocrine loop in many cases of GBM. Furthermore, overexpression of the EGFR has been reported in up to 60% of GBM cases depending on the technique used (Libermann et al., 1985; Schlegel et al., 1994; Jungbluth et al., 2003), with overexpression leading to ligand-independent activation of the receptor (Thomas et al., 2003). The activation and subsequent phosphorylation of EGFR stimulates several downstream pathways including Ras/MAPK, PI3K/Akt, PLC-gamma and STAT3 (Halatsch et al., 2006; Nakamura, 2007). All four pathways contribute to the tumorigenicity of GBM, but the PI3K/Akt pathway appears to have a central role in the development and maintenance of this cancer (Chakravarti et al., 2004). Indeed, inactivation/deletion/mutation of PTEN, an endogenous inhibitor of the PI3K pathway, is also a common event in GBM (Rasheed et al., 1997). Of note, there is an emerging role for EGFR-mediated activation of STAT3 in the development

of GBM (Weissenberger et al., 2004; Mizoguchi et al., 2006; Sherry et al., 2009).

**1. Introduction** 

desperately needed.

**2. Expression of EGFR and its ligands in GBM** 

**Therapies for the Treatment of Glioblastoma** 

## **Advances in the Development of EGFR Targeted Therapies for the Treatment of Glioblastoma**

Terrance Johns *Monash University, Australia* 

#### **1. Introduction**

The epidermal growth factor receptor (EGFR) is receptor tyrosine kinase (RTK) dysregulated in glioblastoma (GBM) through overexpression, mutation or inappropriate expression of ligand. Activation of the EGFR by these mechanisms contributes to the development and progression of GBM by engaging downstream targets, such as the PI3K pathway. The de2-7 EGFR (or EGFRvIII), a naturally occurring mutation of the EGFR frequently expressed in GBM, preferentially activates this pathway. Clinical trials with EGFR-specific tyrosine kinase inhibitors (TKIs) have been disappointing with very little antitumor activity observed. The outcome of controlled clinical trials with EGFR-specific antibodies is yet to be reported. Encouraging preclinical and preliminary clinical data suggests that the combination of EGFR therapeutics and compounds that target molecules downstream of EGFR might have increased efficacy. Finally, the identification of biomarkers that predict those patients most likely to respond to EGFR inhibition is desperately needed.

#### **2. Expression of EGFR and its ligands in GBM**

The EGFR is frequently expressed in GBM (Jungbluth et al., 2003), the most common and deadly form of malignant brain cancer (DeAngelis, 2001). Extensive co-expression of EGFR ligands such as EGF and TGF-α has also been reported (Ekstrand et al., 1991), suggesting the existence of a robust autocrine loop in many cases of GBM. Furthermore, overexpression of the EGFR has been reported in up to 60% of GBM cases depending on the technique used (Libermann et al., 1985; Schlegel et al., 1994; Jungbluth et al., 2003), with overexpression leading to ligand-independent activation of the receptor (Thomas et al., 2003). The activation and subsequent phosphorylation of EGFR stimulates several downstream pathways including Ras/MAPK, PI3K/Akt, PLC-gamma and STAT3 (Halatsch et al., 2006; Nakamura, 2007). All four pathways contribute to the tumorigenicity of GBM, but the PI3K/Akt pathway appears to have a central role in the development and maintenance of this cancer (Chakravarti et al., 2004). Indeed, inactivation/deletion/mutation of PTEN, an endogenous inhibitor of the PI3K pathway, is also a common event in GBM (Rasheed et al., 1997). Of note, there is an emerging role for EGFR-mediated activation of STAT3 in the development of GBM (Weissenberger et al., 2004; Mizoguchi et al., 2006; Sherry et al., 2009).

Advances in the Development of EGFR Targeted Therapies for the Treatment of Glioblastoma 29

(Nishikawa et al., 1994). Furthermore, expression of the de2-7 EGFR is consistently lost when GBM cell lines are established *in vitro* using serum*,* yet is retained if GBM samples are implanted and subsequently passaged directly in nude mice (Sarkaria et al., 2007). Taken together, this indicates that the de2-7 EGFR contributes primarily to aspects of *in vivo* growth. While the increased tumorigenicity mediated by the de2-7 EGFR is primarily due to the receptor's constitutive tyrosine kinase activity (Huang et al., 1997), attempts to identify intracellular molecules and signaling pathways associated with its growth advantage remain ongoing. Transfection of the de2-7 EGFR into U87MG human GBM cells results in an increase in PI3K activity that is important to the growth advantage mediated by the mutant receptor, an observation confirmed by several papers (Moscatello et al., 1998; Li et al., 2004; Luwor et al., 2004). U87MG cells transfected with the de2-7 EGFR also co-express wild-type (wt) EGFR, a scenario that probably mimics the situation in GBM patients. The significance of a possible interaction between the de2-7 EGFR and wtEGFR is not fully known, but it has been shown that the de2-7 EGFR can directly activate PI3K in the absence of the wtEGFR in non-GBM cell lines (Moscatello et al., 1998). We reported that the de2-7 EGFR and the wt EGFR can heterodimerize leading to increased PI3K signaling (Luwor et al., 2004), suggesting that an interaction between the mutant and wt receptor could enhance de2-7 EGFR signaling. One clear consequence of PI3K activation by de2-7 EGFR is the increased production of VEGF, both under normoxic and hypoxic conditions (Feldkamp et al., 1999),

ascribing a pro-angiogenic function to this receptor.

**Mutation Frequency (%) Biological Effect** 

**Δ521–603 <10 Unknown** 

Table 1. Selected mutations of the EGFR expressed in GBM

**Δ6–273 30 Ligand-independent, failure to** 

**Δ959–1186 <10 Increased ligand-dependant kinase** 

**Δ959–1030 and Δ959-1043 <10 Increased basal activity especially** 

**R84K <5 Increased basal activity but** 

**T239P <5 Increased basal activity but** 

**A265V/D/T <5 Increased basal activity but** 

**G574V <1 Increased basal activity but** 

**Kinase mutation (L861Q) <1 Increased basal activity, failure to** 

Very recently de2-7 EGFR has been shown to stimulate the production of cytokines, including IL-6 and LIF, which signal through the gp130 complex (Inda et al., 2010). Importantly these cytokines were shown to activate the wtEGFR when it is overexpressed in neighboring GBM cells, through a mechanism involving cross-talk between gp130 and

**down-regulate** 

**responds to ligand** 

**responds to ligand** 

**responds to ligand** 

**responds to ligand** 

**downregulate** 

**Δ959–1030 but responds to ligand** 

**activity** 

#### **3. Amplification of the EGFR gene in GBM**

Amplification of the *EGFR* gene was the first reported genetic alteration in GBM (Libermann et al., 1985). Subsequent studies have confirmed that approximately 40% of GBMs display amplification of the *EGFR* gene (Wong et al., 1987). Gene amplification invariably leads to overexpression of the EGFR at the cell surface (Wong et al., 1987), although given that overexpression of the receptor occurs in 60% of GBMs, gene amplification is not the only route to increased expression. The majority of GBMs develop rapidly, without evidence of pre-existing malignant lesion, and are known as primary (or *de novo*) GBMs, while secondary GBMs arise from low-grade diffuse astrocytomas or anaplastic astrocytomas (Furnari et al., 2007; Ohgaki & Kleihues, 2007). *EGFR* gene amplification is more commonly associated with primary GBMs than secondary GBMs, where it occurs at a frequency of less than 10% (Watanabe et al., 1996; Ohgaki & Kleihues, 2007). Overexpressed EGFR not only activates in a ligand-independent manner, but shows enhanced signaling through the STATs, including STAT3 (Thomas et al., 2003; Pedersen et al., 2005), which in turn can induce expression of IL-6. Since IL-6 autocrine loops and amplification of the *IL6* gene have been reported at high frequency in GBM (Weissenberger et al., 2004; Tchirkov et al., 2007), this could be an important, but largely overlooked, consequence of EGFR overexpression.

Recent studies have shown that GBM can be classified into at least 4 distinct molecular subtypes; classical, pro-neural, neural and mesenchymal (Brennan et al., 2009; Verhaak et al., 2010). Pro-neural GBMs largely constitutes the secondary GBMs and therefore does not display EGFR amplification and/or overexpression. In contrast nearly all GBM in the classical sub-type overexpress EGFR (Verhaak et al., 2010). Furthermore, the GBM specific mutation, de2-7 EGFR, is found almost exclusively in the classical sub-type. Neural and mesenchymal GBMs have variable levels of EGFR with some showing increased expression and others decreased expression.

#### **4. Mutations of EGFR described in GBM**

Amplification of the *EGFR* gene in GBM is associated with gene rearrangements. The first rearrangement to be described in detail was an extracellular domain (ECD) deletion producing a mutant known as the de2-7 EGFR (or EGFRvIII) (Sugawa et al., 1990; Yamazaki et al., 1990; Ekstrand et al., 1992; Wong et al., 1992). Several other deletion mutants have since been described and categorized (Table 1) (Ekstrand et al., 1992; Frederick et al., 2000). Numerous subsequent studies have shown that the most common mutation is the de2-7 EGFR, occurring in about 50% of cases where the *EGFR* gene is amplified (Wikstrand et al., 1998; Frederick et al., 2000; Pedersen et al., 2001). This cancer-specific EGFR mutant has a specific deletion between exons 2 and 7 of *EGFR* (Sugawa et al., 1990). The truncation of exons 2–7 leads to the elimination of 267 amino acids from the ECD and the insertion of a novel glycine at the fusion junction. This renders the mutant EGFR unable to bind any known ligand (Sugawa et al., 1990; Wikstrand et al., 1998; Pedersen et al., 2001). Despite this, the de2-7 EGFR is capable of low-level constitutive signaling, which is augmented by the mutant receptor's impaired internalization and downregulation (Nishikawa et al., 1994; Schmidt et al., 2003).

GBM cell lines transfected with the de2-7 EGFR display enhanced tumorigenicity when grown as xenografts in nude mice, but only a marginal effect on growth is observed *in vitro* 

Amplification of the *EGFR* gene was the first reported genetic alteration in GBM (Libermann et al., 1985). Subsequent studies have confirmed that approximately 40% of GBMs display amplification of the *EGFR* gene (Wong et al., 1987). Gene amplification invariably leads to overexpression of the EGFR at the cell surface (Wong et al., 1987), although given that overexpression of the receptor occurs in 60% of GBMs, gene amplification is not the only route to increased expression. The majority of GBMs develop rapidly, without evidence of pre-existing malignant lesion, and are known as primary (or *de novo*) GBMs, while secondary GBMs arise from low-grade diffuse astrocytomas or anaplastic astrocytomas (Furnari et al., 2007; Ohgaki & Kleihues, 2007). *EGFR* gene amplification is more commonly associated with primary GBMs than secondary GBMs, where it occurs at a frequency of less than 10% (Watanabe et al., 1996; Ohgaki & Kleihues, 2007). Overexpressed EGFR not only activates in a ligand-independent manner, but shows enhanced signaling through the STATs, including STAT3 (Thomas et al., 2003; Pedersen et al., 2005), which in turn can induce expression of IL-6. Since IL-6 autocrine loops and amplification of the *IL6* gene have been reported at high frequency in GBM (Weissenberger et al., 2004; Tchirkov et al., 2007), this could be an important, but largely overlooked, consequence of EGFR overexpression. Recent studies have shown that GBM can be classified into at least 4 distinct molecular subtypes; classical, pro-neural, neural and mesenchymal (Brennan et al., 2009; Verhaak et al., 2010). Pro-neural GBMs largely constitutes the secondary GBMs and therefore does not display EGFR amplification and/or overexpression. In contrast nearly all GBM in the classical sub-type overexpress EGFR (Verhaak et al., 2010). Furthermore, the GBM specific mutation, de2-7 EGFR, is found almost exclusively in the classical sub-type. Neural and mesenchymal GBMs have variable levels of EGFR with some showing increased expression

Amplification of the *EGFR* gene in GBM is associated with gene rearrangements. The first rearrangement to be described in detail was an extracellular domain (ECD) deletion producing a mutant known as the de2-7 EGFR (or EGFRvIII) (Sugawa et al., 1990; Yamazaki et al., 1990; Ekstrand et al., 1992; Wong et al., 1992). Several other deletion mutants have since been described and categorized (Table 1) (Ekstrand et al., 1992; Frederick et al., 2000). Numerous subsequent studies have shown that the most common mutation is the de2-7 EGFR, occurring in about 50% of cases where the *EGFR* gene is amplified (Wikstrand et al., 1998; Frederick et al., 2000; Pedersen et al., 2001). This cancer-specific EGFR mutant has a specific deletion between exons 2 and 7 of *EGFR* (Sugawa et al., 1990). The truncation of exons 2–7 leads to the elimination of 267 amino acids from the ECD and the insertion of a novel glycine at the fusion junction. This renders the mutant EGFR unable to bind any known ligand (Sugawa et al., 1990; Wikstrand et al., 1998; Pedersen et al., 2001). Despite this, the de2-7 EGFR is capable of low-level constitutive signaling, which is augmented by the mutant receptor's impaired internalization and downregulation (Nishikawa et al., 1994;

GBM cell lines transfected with the de2-7 EGFR display enhanced tumorigenicity when grown as xenografts in nude mice, but only a marginal effect on growth is observed *in vitro* 

**3. Amplification of the EGFR gene in GBM** 

and others decreased expression.

Schmidt et al., 2003).

**4. Mutations of EGFR described in GBM** 

(Nishikawa et al., 1994). Furthermore, expression of the de2-7 EGFR is consistently lost when GBM cell lines are established *in vitro* using serum*,* yet is retained if GBM samples are implanted and subsequently passaged directly in nude mice (Sarkaria et al., 2007). Taken together, this indicates that the de2-7 EGFR contributes primarily to aspects of *in vivo* growth. While the increased tumorigenicity mediated by the de2-7 EGFR is primarily due to the receptor's constitutive tyrosine kinase activity (Huang et al., 1997), attempts to identify intracellular molecules and signaling pathways associated with its growth advantage remain ongoing. Transfection of the de2-7 EGFR into U87MG human GBM cells results in an increase in PI3K activity that is important to the growth advantage mediated by the mutant receptor, an observation confirmed by several papers (Moscatello et al., 1998; Li et al., 2004; Luwor et al., 2004). U87MG cells transfected with the de2-7 EGFR also co-express wild-type (wt) EGFR, a scenario that probably mimics the situation in GBM patients. The significance of a possible interaction between the de2-7 EGFR and wtEGFR is not fully known, but it has been shown that the de2-7 EGFR can directly activate PI3K in the absence of the wtEGFR in non-GBM cell lines (Moscatello et al., 1998). We reported that the de2-7 EGFR and the wt EGFR can heterodimerize leading to increased PI3K signaling (Luwor et al., 2004), suggesting that an interaction between the mutant and wt receptor could enhance de2-7 EGFR signaling. One clear consequence of PI3K activation by de2-7 EGFR is the increased production of VEGF, both under normoxic and hypoxic conditions (Feldkamp et al., 1999), ascribing a pro-angiogenic function to this receptor.


Table 1. Selected mutations of the EGFR expressed in GBM

Very recently de2-7 EGFR has been shown to stimulate the production of cytokines, including IL-6 and LIF, which signal through the gp130 complex (Inda et al., 2010). Importantly these cytokines were shown to activate the wtEGFR when it is overexpressed in neighboring GBM cells, through a mechanism involving cross-talk between gp130 and

Advances in the Development of EGFR Targeted Therapies for the Treatment of Glioblastoma 31

strategy. In fact, antibodies specific for the de2-7 EGFR have been generated; the monoclonal antibodies (mAbs) DH8.3 (Hills et al., 1995), L8A4 and Y10 (Wikstrand et al., 1995) all preferentially recognize the unique junctional peptide. DH8.3 and Y10 have been shown to specifically target cells expressing the de2-7 EGFR (i.e. they do not bind the wtEGFR) (Hills et al., 1995; Wikstrand et al., 1995). Although DH8.3 has been shown to target de2-7 EGFRexpressing xenografts *in vivo* (Johns et al., 2002), its efficacy has not been reported. The Y10 antibody has been shown to have *in vivo* antitumor activity against murine B16 melanoma cells transfected with a murine homolog of the human de2-7 EGFR (Sampson et al., 2000), an unusual system for its evaluation since expression of the de2-7 EGFR has not been reported in melanoma. Furthermore, the antitumor activity seen was completely dependent on the Fc function of the Y10 antibody and not a direct inhibitory effect on de2-7 EGFR signaling (Sampson et al., 2000). There have been no reports to date of clinical trials using de2-7 EGFR-specific antibodies. These antibodies are internalized and could be used for delivery of radiotherapy or cytotoxics given their specificity (Foulon et al., 2000), but this approach

Numerous therapeutic antibodies to the wtEGFR have been described and several have been used in the context of GBM (Quang & Brady, 2004; Belda-Iniesta et al., 2006). These antibodies all function in a similar manner by interacting with the L2 domain of the EGFR and inhibiting the binding of ligand. Structural studies with Cetuximab suggest that these antibodies also prevent EGFR dimerization, a crucial step in its activation (Li et al., 2005). Antibodies directed to the wtEGFR do show antitumor activity in GBM xenograft models even when the tumors are grown intracranially (Eller et al., 2002; Perera et al., 2005). Furthermore, these antibodies can bind the de2-7 EGFR and can inhibit GBM cells coexpressing the de2-7 and wtEGFR (Perera et al., 2005). However, the large intratumoral pressure found in GBM, the 'remnants' of a blood brain barrier (BBB) and the inefficient nature of GBM vascularization have all raised concerns about the effective targeting of antibodies to GBM following systemic administration. Despite these concerns, three antibodies targeting wtEGFR are have been tested in GBM using systemic administration, including mAb 425. 131I-mAb 425 has been used in several clinical trials and clearly demonstrates targeting to GBM following systemic administration (Quang & Brady, 2004). Unfortunately, this antibody is of murine origin and can only be administered on a few

Cetuximab is a chimeric antibody directed to the EGFR that has been approved in several human cancers including that of the colon (Moosmann & Heinemann, 2007). There are anecdotal reports of this antibody being used for the treatment of GBM, but no rigorously controlled studies have been implemented to date (Belda-Iniesta et al., 2006). Preclinical *in vitro* and *in vivo* studies suggest that cetuximab and related antibodies have significant antitumor activity in GBM, encouraging the establishment of a Phase I/II clinical trial (Combs et al., 2006). In this trial, cetuximab will be co-administered with a combination of temozolomide and radiotherapy, which is the standard of care following initial resection of a GBM (Trial Number: NCT00311857). Outcomes from this trial should be reported shortly. Recently cetuximab was trialed with a combination with bevacizumab (i.e. avastin) and irinotecan but did not increase the efficacy of this combination in GBM patients (Hasselbalch et al., 2010). There are several other EGFR-directed antibodies either approved or in development including Panitumumab (Rivera et al., 2008); however, there have been no

has not been examined clinically.

occasions before immune responses render it ineffective.

reports of their systematic evaluation in GBM.

EGFR. Activation of the wtEGFR in this manner leads to enhanced proliferation. Thus, de2-7 EGFR contributes to the growth of surrounding GBM cells through this field effect. This work also shows the functional link between EGFR and IL-6. More generally this indicates that the de2-7 EGFR actively contributes to the heterogeneity of GBM by acting indirectly with neighboring de2-7 EGFR negative cells (Inda et al., 2010). This hypothesis is entirely consistent with the observation that wtEGFR amplification and de2-7 EGFR expression are usually seen together. It may also explain why the pronounced growth advantage mediated by the de2-7 EGFR does not lead in patients to a homogenous population of cells in patients all expressing the receptor.

Lee *et al* sequenced the entire *EGFR* gene in a panel of eight GBM cell lines and 132 GBM samples (Table 1) (Lee et al., 2006). Interestingly, they identified a series of missense mutations in the ECD of the EGFR expressed in 14% of the GBM samples and 12% of the cell lines (Lee et al., 2006). In general, the missense mutations were found to be independent of the de2-7 EGFR but were associated with *EGFR* gene amplification; approximately 60% of samples with missense mutations also had *EGFR* gene amplification. Subsequent studies showed that these single amino acid mutations led to ligand-independent activation of the EGFR, and unlike the wtEGFR, were transforming in NR6 cells; a variant of mouse 3T3 cells lacking the EGFR (Lee et al., 2006). However, unlike the de2-7 EGFR, these mutants could also respond to ligand stimulation. Recently, we extended these studies and showed that some of these mutations also provide a significant advantage to *in vivo* growth (Ymer et al., 2011).

The presence of activating kinase mutations, such as those commonly found in lung cancer (Sharma et al., 2007), is extremely rare in GBM, with only one sample displaying this type of mutation (Lee et al., 2006). Interestingly, a subsequent analysis of 119 lung cancer samples failed to find a single missense mutation in the ECD, although 13% of the samples contained kinase domain mutations, as expected (Lee et al., 2006). Thus, mutations of the EGFR in GBM appear to cluster in the ECD and lead to ligand independence. Therefore, the lessons learned with respect to EGFR therapeutics for the treatment of lung cancer are probably of minimal value in the context of GBM. Finally, these mutations further emphasize just how frequently the EGFR is perturbed in GBM; in fact, taking into account EGFR autocrine loops, activation of the EGFR probably occurs in over 70% of GBMs.

### **5. EGFR as a therapeutic target in GBM**

Given that the EGFR is activated or dysregulated in a large percentage of GBM cases it is a rational target for therapeutic intervention in this disease. There are two major classes of EGFR inhibitors either currently approved or being evaluated for the treatment of various cancers; antibodies that target the ECD and small molecule TKIs that target the intracellular kinase domain (Marshall, 2006). No specific agent from either class has been approved for the treatment of GBM, but as described below, a number of clinical trials have been reported or are ongoing.

#### **5.1 Antibodies directed to the EGFR**

Overexpression of the de2-7 EGFR on the cell surface, the unique junctional peptide created by the deletion and the aggressive phenotype associated with this receptor, suggests that targeting the de2-7 EGFR with antibodies that are cancer-specific is an attractive therapeutic

EGFR. Activation of the wtEGFR in this manner leads to enhanced proliferation. Thus, de2-7 EGFR contributes to the growth of surrounding GBM cells through this field effect. This work also shows the functional link between EGFR and IL-6. More generally this indicates that the de2-7 EGFR actively contributes to the heterogeneity of GBM by acting indirectly with neighboring de2-7 EGFR negative cells (Inda et al., 2010). This hypothesis is entirely consistent with the observation that wtEGFR amplification and de2-7 EGFR expression are usually seen together. It may also explain why the pronounced growth advantage mediated by the de2-7 EGFR does not lead in patients to a homogenous population of cells in patients

Lee *et al* sequenced the entire *EGFR* gene in a panel of eight GBM cell lines and 132 GBM samples (Table 1) (Lee et al., 2006). Interestingly, they identified a series of missense mutations in the ECD of the EGFR expressed in 14% of the GBM samples and 12% of the cell lines (Lee et al., 2006). In general, the missense mutations were found to be independent of the de2-7 EGFR but were associated with *EGFR* gene amplification; approximately 60% of samples with missense mutations also had *EGFR* gene amplification. Subsequent studies showed that these single amino acid mutations led to ligand-independent activation of the EGFR, and unlike the wtEGFR, were transforming in NR6 cells; a variant of mouse 3T3 cells lacking the EGFR (Lee et al., 2006). However, unlike the de2-7 EGFR, these mutants could also respond to ligand stimulation. Recently, we extended these studies and showed that some of these

mutations also provide a significant advantage to *in vivo* growth (Ymer et al., 2011).

activation of the EGFR probably occurs in over 70% of GBMs.

**5. EGFR as a therapeutic target in GBM** 

**5.1 Antibodies directed to the EGFR** 

or are ongoing.

The presence of activating kinase mutations, such as those commonly found in lung cancer (Sharma et al., 2007), is extremely rare in GBM, with only one sample displaying this type of mutation (Lee et al., 2006). Interestingly, a subsequent analysis of 119 lung cancer samples failed to find a single missense mutation in the ECD, although 13% of the samples contained kinase domain mutations, as expected (Lee et al., 2006). Thus, mutations of the EGFR in GBM appear to cluster in the ECD and lead to ligand independence. Therefore, the lessons learned with respect to EGFR therapeutics for the treatment of lung cancer are probably of minimal value in the context of GBM. Finally, these mutations further emphasize just how frequently the EGFR is perturbed in GBM; in fact, taking into account EGFR autocrine loops,

Given that the EGFR is activated or dysregulated in a large percentage of GBM cases it is a rational target for therapeutic intervention in this disease. There are two major classes of EGFR inhibitors either currently approved or being evaluated for the treatment of various cancers; antibodies that target the ECD and small molecule TKIs that target the intracellular kinase domain (Marshall, 2006). No specific agent from either class has been approved for the treatment of GBM, but as described below, a number of clinical trials have been reported

Overexpression of the de2-7 EGFR on the cell surface, the unique junctional peptide created by the deletion and the aggressive phenotype associated with this receptor, suggests that targeting the de2-7 EGFR with antibodies that are cancer-specific is an attractive therapeutic

all expressing the receptor.

strategy. In fact, antibodies specific for the de2-7 EGFR have been generated; the monoclonal antibodies (mAbs) DH8.3 (Hills et al., 1995), L8A4 and Y10 (Wikstrand et al., 1995) all preferentially recognize the unique junctional peptide. DH8.3 and Y10 have been shown to specifically target cells expressing the de2-7 EGFR (i.e. they do not bind the wtEGFR) (Hills et al., 1995; Wikstrand et al., 1995). Although DH8.3 has been shown to target de2-7 EGFRexpressing xenografts *in vivo* (Johns et al., 2002), its efficacy has not been reported. The Y10 antibody has been shown to have *in vivo* antitumor activity against murine B16 melanoma cells transfected with a murine homolog of the human de2-7 EGFR (Sampson et al., 2000), an unusual system for its evaluation since expression of the de2-7 EGFR has not been reported in melanoma. Furthermore, the antitumor activity seen was completely dependent on the Fc function of the Y10 antibody and not a direct inhibitory effect on de2-7 EGFR signaling (Sampson et al., 2000). There have been no reports to date of clinical trials using de2-7 EGFR-specific antibodies. These antibodies are internalized and could be used for delivery of radiotherapy or cytotoxics given their specificity (Foulon et al., 2000), but this approach has not been examined clinically.

Numerous therapeutic antibodies to the wtEGFR have been described and several have been used in the context of GBM (Quang & Brady, 2004; Belda-Iniesta et al., 2006). These antibodies all function in a similar manner by interacting with the L2 domain of the EGFR and inhibiting the binding of ligand. Structural studies with Cetuximab suggest that these antibodies also prevent EGFR dimerization, a crucial step in its activation (Li et al., 2005). Antibodies directed to the wtEGFR do show antitumor activity in GBM xenograft models even when the tumors are grown intracranially (Eller et al., 2002; Perera et al., 2005). Furthermore, these antibodies can bind the de2-7 EGFR and can inhibit GBM cells coexpressing the de2-7 and wtEGFR (Perera et al., 2005). However, the large intratumoral pressure found in GBM, the 'remnants' of a blood brain barrier (BBB) and the inefficient nature of GBM vascularization have all raised concerns about the effective targeting of antibodies to GBM following systemic administration. Despite these concerns, three antibodies targeting wtEGFR are have been tested in GBM using systemic administration, including mAb 425. 131I-mAb 425 has been used in several clinical trials and clearly demonstrates targeting to GBM following systemic administration (Quang & Brady, 2004). Unfortunately, this antibody is of murine origin and can only be administered on a few occasions before immune responses render it ineffective.

Cetuximab is a chimeric antibody directed to the EGFR that has been approved in several human cancers including that of the colon (Moosmann & Heinemann, 2007). There are anecdotal reports of this antibody being used for the treatment of GBM, but no rigorously controlled studies have been implemented to date (Belda-Iniesta et al., 2006). Preclinical *in vitro* and *in vivo* studies suggest that cetuximab and related antibodies have significant antitumor activity in GBM, encouraging the establishment of a Phase I/II clinical trial (Combs et al., 2006). In this trial, cetuximab will be co-administered with a combination of temozolomide and radiotherapy, which is the standard of care following initial resection of a GBM (Trial Number: NCT00311857). Outcomes from this trial should be reported shortly. Recently cetuximab was trialed with a combination with bevacizumab (i.e. avastin) and irinotecan but did not increase the efficacy of this combination in GBM patients (Hasselbalch et al., 2010). There are several other EGFR-directed antibodies either approved or in development including Panitumumab (Rivera et al., 2008); however, there have been no reports of their systematic evaluation in GBM.

Advances in the Development of EGFR Targeted Therapies for the Treatment of Glioblastoma 33

Why these trials have produced conflicting outcomes is not clear, but a comprehensive Phase III study is probably required to finally determine if erlotinib improves standard therapy. Overall though the data suggests that erlotinib's benefits are relatively small at

**Objective** 

**16 0 12% (6** 

**5 patients remain on study** 

**Response# PFS Median OS** 

**months) <sup>10</sup>**

**months) <sup>6</sup>**

**months) <sup>12</sup>**

**months) <sup>6</sup>**

**(median) 9<sup>Ϯ</sup>**

**(median) <sup>19</sup>**

**(median) 15<sup>Ϯ</sup>**

**3 months** 

**(months)** 

**(***n***)** 

**Rich** *et al* **(Rich et al., 2004) 53 0 13% (6** 

**Uhm** *et al* **(Uhm et al., 2011) 98 N/A 17% (12** 

**Raizer** *et al (Raizer et al., 2010)* **38 0 3% (6** 

**Prados** *et al* **(Prados et al., 2009) 65 N/A 8 months** 

**Brown** *et al* **(Brown et al., 2008) 97 N/A 7 months** 

**PFS, progression-free survival; OS, overall survival. \*Abstract form only. #Excludes stable disease. <sup>Ϯ</sup> Significant toxicity** 

Table 2. Selected Phase II clinical trials of EGFR TKI's in GBM

**Abbreviations: CR, complete response; PR, partial response; N/A not available;** 

A group of GBM patients treated with erlotinib or gefitinib who had responded or failed to respond to therapy was analyzed retrospectively to determine factors that might explain the different responses (Mellinghoff et al., 2005). The majority of responders expressed the de2-7 EGFR. Furthermore, loss of PTEN, an endogenous inhibitor of PI3K that leads to reduced phosphorylated AKT (Akt), was highly predictive of treatment failure. Not surprisingly, the co-expression of de2-7 EGFR and PTEN was predictive of response to EGFR-specific TKI therapy. A panel of human GBMs directly established as xenografts in nude mice retains expression of de2-7 EGFR in some cases (Sarkaria et al., 2006). This model system also confirmed that the presence of de2-7 EGFR and PTEN in a xenograft was predictive of response (Sarkaria et al., 2007). The mechanistic reasons for this observation remain unknown, but it provides a potential method for screening for patients likely to respond to TKI therapy. Finally, none of the current trials have shown a correlation between response to EGFR TKIs and *EGFR* gene amplification (Brown et al., 2008), although one immunohistochemical study reported a correlation between response to erlotinib and high

**Trial Patients** 

*Gefitinib (single agent, recurrent)*

*Erlotinib (single agent, recurrent)*

*primary)*

**(Peereboom et al., 2010) <sup>27</sup>**

**Franceschi** *et al* **(Franceschi et al., 2007)**  *Gefitinib (single agent, primary)*

*Erlotinib (plus standard care,* 

**Peereboom** *et al* 

best.

A novel EGFR antibody (mAb 806) was generated against cells expressing the de2-7 EGFR but, unexpectedly, was also found to bind to a small proportion of the wtEGFR in GBM samples that overexpress the receptor (Jungbluth et al., 2003). mAb 806 does not bind the unique junctional peptide found in the de2-7 EGFR; rather, it binds to a short cysteine loop on the ECD that is only transiently exposed as the wtEGFR moves from its inactive to active conformation (Johns et al., 2004). The loop is constitutively exposed in the de2-7 EGFR, consistent with our original desire to generate a de2-7 EGFR-specific antibody. Thus, mAb 806 reactivity is found only in cells with favorable conditions for receptor activation, such as the presence of mutations (e.g. de2-7 EGFR), overexpression of the wt receptor or increased presence of EGFR ligands. In the case of EGFR overexpression, there is increased activation as a result of ligand-independent EGFR activation and changes in glycosylation (Johns et al., 2005). These conditions are common in malignant cells but are rare in normal tissues, thereby allowing mAb 806 to preferentially target malignancy such as GBM but not normal organs such as the liver. Our recent Phase I clinical trial confirmed that a chimeric version of mAb 806 did not bind normal tissue and could target GBM following systemic administration (Scott et al., 2007). Since mAb 806, unlike Cetuximab, does not target organs such as the liver it is easier to deliver a therapeutically relevant dose to the site of the GBM. Furthermore, the lack of normal tissue uptake will allow the labeling of mAb 806 with cytotoxic compounds or radioisotopes to enhance its already substantial antitumor efficacy (Johns et al., 2007). This antibody has been licensed and has re-entered clinical trial.

#### **5.2 Tyrosine kinase inhibitors that target the EGFR**

TKIs are small molecules that specifically target the kinase domain of tyrosine kinases, preventing binding of ATP and subsequent activation (Marshall, 2006). Two EGFR-specific TKIs have been approved for the treatment of certain cancers (Mendelsohn & Baselga, 2006), while several others are in clinical trials. Similar to EGFR-specific antibodies, these compounds have shown antitumor activity in *in vitro* and *in vivo* preclinical models (Mendelsohn & Baselga, 2006). They are active against both the wtEGFR and the de2-7 EGFR (Stea et al., 2003; Halatsch et al., 2006; Sarkaria et al., 2007), although they might be less effective against the latter, especially if the de2-7 EGFR is expressed in the absence of the wtEGFR (Learn et al., 2004). Given that there are fewer concerns with regard to delivery of TKIs to the site of GBM when compared with antibodies, the development of these reagents for the treatment of GBM is more advanced. In fact, excellent targeting of these agents to the site of GBM has been well demonstrated (Hofer et al., 2006; Hegi et al., 2011).

The two clinically approved EGFR TKIs, erlotinib and gefitinib, have been used as monotherapy or in combination with temozolomide and/or radiotherapy in clinical trials of GBM patients (Table 2 lists selected Phase II trials). No significant clinical activity has been observed for either erlotinib or gefitinib in either primary or recurrent GBM when used as monotherapies (Table 2). Surgery, followed by temozolomide/radiotherapy is standard of care in primary GBM. The addition or erlotinib to this standard of care has been assessed in 3 different Phase II trials (Table 2). Two of these trials failed to show any benefit when erlotinib was added to standard of care and was associated with significant toxicity (Brown et al., 2008; Peereboom et al., 2010). In a third trial however, the authors reported encouraging PFS and median survival when compared to their previous studies using other agents (Prados et al., 2009). All three studies were conducted in newly diagnosed patients. Why these trials have produced conflicting outcomes is not clear, but a comprehensive Phase III study is probably required to finally determine if erlotinib improves standard therapy. Overall though the data suggests that erlotinib's benefits are relatively small at best.


**PFS, progression-free survival; OS, overall survival. \*Abstract form only. #Excludes stable disease. <sup>Ϯ</sup>**

**Significant toxicity** 

32 Novel Therapeutic Concepts in Targeting Glioma

A novel EGFR antibody (mAb 806) was generated against cells expressing the de2-7 EGFR but, unexpectedly, was also found to bind to a small proportion of the wtEGFR in GBM samples that overexpress the receptor (Jungbluth et al., 2003). mAb 806 does not bind the unique junctional peptide found in the de2-7 EGFR; rather, it binds to a short cysteine loop on the ECD that is only transiently exposed as the wtEGFR moves from its inactive to active conformation (Johns et al., 2004). The loop is constitutively exposed in the de2-7 EGFR, consistent with our original desire to generate a de2-7 EGFR-specific antibody. Thus, mAb 806 reactivity is found only in cells with favorable conditions for receptor activation, such as the presence of mutations (e.g. de2-7 EGFR), overexpression of the wt receptor or increased presence of EGFR ligands. In the case of EGFR overexpression, there is increased activation as a result of ligand-independent EGFR activation and changes in glycosylation (Johns et al., 2005). These conditions are common in malignant cells but are rare in normal tissues, thereby allowing mAb 806 to preferentially target malignancy such as GBM but not normal organs such as the liver. Our recent Phase I clinical trial confirmed that a chimeric version of mAb 806 did not bind normal tissue and could target GBM following systemic administration (Scott et al., 2007). Since mAb 806, unlike Cetuximab, does not target organs such as the liver it is easier to deliver a therapeutically relevant dose to the site of the GBM. Furthermore, the lack of normal tissue uptake will allow the labeling of mAb 806 with cytotoxic compounds or radioisotopes to enhance its already substantial antitumor efficacy

(Johns et al., 2007). This antibody has been licensed and has re-entered clinical trial.

TKIs are small molecules that specifically target the kinase domain of tyrosine kinases, preventing binding of ATP and subsequent activation (Marshall, 2006). Two EGFR-specific TKIs have been approved for the treatment of certain cancers (Mendelsohn & Baselga, 2006), while several others are in clinical trials. Similar to EGFR-specific antibodies, these compounds have shown antitumor activity in *in vitro* and *in vivo* preclinical models (Mendelsohn & Baselga, 2006). They are active against both the wtEGFR and the de2-7 EGFR (Stea et al., 2003; Halatsch et al., 2006; Sarkaria et al., 2007), although they might be less effective against the latter, especially if the de2-7 EGFR is expressed in the absence of the wtEGFR (Learn et al., 2004). Given that there are fewer concerns with regard to delivery of TKIs to the site of GBM when compared with antibodies, the development of these reagents for the treatment of GBM is more advanced. In fact, excellent targeting of these agents to the site of GBM has been well demonstrated (Hofer et al., 2006; Hegi et al., 2011). The two clinically approved EGFR TKIs, erlotinib and gefitinib, have been used as monotherapy or in combination with temozolomide and/or radiotherapy in clinical trials of GBM patients (Table 2 lists selected Phase II trials). No significant clinical activity has been observed for either erlotinib or gefitinib in either primary or recurrent GBM when used as monotherapies (Table 2). Surgery, followed by temozolomide/radiotherapy is standard of care in primary GBM. The addition or erlotinib to this standard of care has been assessed in 3 different Phase II trials (Table 2). Two of these trials failed to show any benefit when erlotinib was added to standard of care and was associated with significant toxicity (Brown et al., 2008; Peereboom et al., 2010). In a third trial however, the authors reported encouraging PFS and median survival when compared to their previous studies using other agents (Prados et al., 2009). All three studies were conducted in newly diagnosed patients.

**5.2 Tyrosine kinase inhibitors that target the EGFR** 

Table 2. Selected Phase II clinical trials of EGFR TKI's in GBM

A group of GBM patients treated with erlotinib or gefitinib who had responded or failed to respond to therapy was analyzed retrospectively to determine factors that might explain the different responses (Mellinghoff et al., 2005). The majority of responders expressed the de2-7 EGFR. Furthermore, loss of PTEN, an endogenous inhibitor of PI3K that leads to reduced phosphorylated AKT (Akt), was highly predictive of treatment failure. Not surprisingly, the co-expression of de2-7 EGFR and PTEN was predictive of response to EGFR-specific TKI therapy. A panel of human GBMs directly established as xenografts in nude mice retains expression of de2-7 EGFR in some cases (Sarkaria et al., 2006). This model system also confirmed that the presence of de2-7 EGFR and PTEN in a xenograft was predictive of response (Sarkaria et al., 2007). The mechanistic reasons for this observation remain unknown, but it provides a potential method for screening for patients likely to respond to TKI therapy. Finally, none of the current trials have shown a correlation between response to EGFR TKIs and *EGFR* gene amplification (Brown et al., 2008), although one immunohistochemical study reported a correlation between response to erlotinib and high

Advances in the Development of EGFR Targeted Therapies for the Treatment of Glioblastoma 35

a non-critical manner. Indeed, we have very recently shown that de2-7 EGFR has a role in

EGFR antibodies do not have any efficacy in colon cancer in patients containing mutations in Ras or Raf (Laurent-Puig et al., 2009). While mutations in these molecules are comparatively rare in GBM (McLendon et al., 2008), and thus cannot be used to stratify patients, the pathways associated with Ras/Raf can be activated by other RTKs on the cell surface. The activation of Ras/Raf by these RTKs might cause *de novo* resistance to EGFR therapeutics. Raf can be targeted directly by TKIs already in the clinic, however Ras cannot be inhibited by this approach. However, the signalling of both kinases can be targeted by blocking downstream targets such as ERK and MEK (Pratilas & Solit, 2010). If the toxicity issues can be managed, targeting these kinases in combination with EGFR inhibitors is a logical next step in GBM. This is not a problem in NSCLC as EGFR and ras mutations are

Recent data suggests that mutations in PI3K may also cause resistance to EGFR antibodies in colon cancer (Weickhardt et al., 2010). This is highly relevant to GBM as the PI3K pathway appears to be dysregulated in most GBMs through direct mutation of PI3K, the deletion or mutation of the negative PI3K regulator PTEN or activation through other RTKs (McLendon et al., 2008). Indeed, as discussed above, the absence of PTEN was associated with clinical resistance to EGFR TKIs in GBM patients expressing the de2-7 EGFR while responsiveness was associated with co-expression of PTEN and de2-7 EGFR. The recent translation of PI3K inhibitors into the clinic provides a strategy for overcoming PI3K-mediated resistance to EGFR inhibitors. All the evidence suggests that this combinational approach might finally unleash the potential of EGFR inhibitors for the treatment of GBM (Fan & Weiss, 2010); once

Since most RTKs can activate Ras/Raf and/or PI3K, the dual inhibition of EGFR and additional activated RTKs is an obvious therapeutic approach. Stommel *et al* clearly showed that multiple RTKs can be activated in GBM and that these RTKs activate overlapping downstream pathways causing resistance to TKIs that only targeting a single RTK (Stommel et al., 2007). The RTK c-Met is also commonly activated in GBM through a number of mechanisms and also has an important role in angiogenesis (Abounader & Laterra, 2005). We recently showed that co-expression of de2-7 EGFR and c-Met in GBM xenografts causes therapeutic resistant to single agents directed to either of these RTK. However, the combination of EGFR and c-Met inhibitors produced synergistic anti-tumor activity (Pillay et al., 2009), confirming that dual inhibition of RTKs is a valid approach in GBM. A number of other RTKs have been shown to be activated in GBM including the FGFR family, Axl, ErbB2/3/4, EphA2/7, VEGFR2 and PDGFRα/β (Ren et al., 2007; Pillay et al., 2009). Given the range of potential RTKs activated in a given patient, the most effective therapeutic strategy may have to be determined by screening patient tissues for RTK mutation and/or

We recently showed that EGFR and Src-family kinases (SFK) are frequently coactivated in GBM (Lu et al., 2009). Furthermore, the de2-7 EGFR physically associated with SFKs and

regulating GBM response to low glucose (Cvrljevic et al, *in press*).

mutually exclusive in this cancer type.

again management of toxicity remains as a concern.

**6.2 Combination of EGFR inhibitors with other targeted therapies** 

activation (i.e. phosphorylation) before commencing treatment.

expression of EGFR in combination with low levels of pAkt (Haas-Kogan et al., 2005). Once again this supports the notion that activated EGFR in the presence of low pAkt may be indicative of increased levels of response to EGFR inhibition.

Recently bevacizumab (i.e. avastin) was approved for the treatment of recurrent GBM. A Phase II trial of erlotinib and bevacizumab showed that this combination was adequately tolerated, but provided no additional benefit to that seen in other bevacizumab containing regimens (Sathornsumetee et al., 2010). mTOR is downstream of the PI3K/Akt pathway and therefore represents a therapeutic target in GBM. The combination of sirolimus (a mTor inhibitor), and erlotinib has been reported in recurrent GBM patients (Reardon et al., 2010). While the combination was well tolerated, it displayed negligible anti-tumor activity.

#### **6. Future developments**

Both Cetuximab and Panitumumab have been approved for the treatment of colon cancer and erlotinib and gefitinib are approved for non-small cell lung cancer (NSCLC), validating the EGFR as a genuine therapeutic target (Van den Eynde et al., 2011). Given the dysregulation of the EGFR in GBM through the range of mechanisms described above, the failure of these agents to show significant anti-tumor activity in this cancer is somewhat surprising. The possibility that the BBB is responsible for this lack of activity has been conclusively discounted for EGFR TKI's and appears not to be a factor for anti-EGFR antibodies, although formal demonstration of this is still required for antibodies. Future research should focus the identification of patients more likely to respond to EGFR inhibition and identifying other targeted therapies that might work synergistically with EGFR inhibitors. Further classification of GBM into unique molecular sub-types beyond the four already described (Verhaak et al., 2010) will hopefully help identify EGFR levels of tumor subtypes more likely to respond to EGFR inhibition.

#### **6.1 Lessons from other cancers**

In NSCLC the sub-set of patients most likely to respond to EGFR TKI's are those containing activating mutations in the kinase domain of *EGFR (da Cunha Santos et al., 2011)*. Significant clinical and laboratory evidence suggests that these mutations lead to "oncogenic addiction"; the phenomenon whereby a tumor becomes largely dependent on a single activated kinase. Interestingly, *EGFR* amplification is not strongly associated with response to TKI's in NSCLC (De Luca & Normanno, 2010). In contrast, EGFR activation in GBM is most often driven by gene amplification and/or mutations found in the ECD domain of the receptor (Lee et al., 2006), although a recent report described several c-terminal deleted EGFR molecules that are constitutively active (Pines et al., 2010). The clinical data in GBM implies that mutations of this nature do not lead to EGFR addiction. The reason for this remains speculative but suggests that the primary role of EGFR in GBM may not be tumor proliferation or survival but some other important biological function that supports, but is not essential to, GBM growth. A recent paper showing that inactive EGFR has a critical role in upregulating glucose transport highlights other unanticipated roles for this receptor (Weihua et al., 2008). Importantly inhibition of EGFR does not block this activity, rather EGFR needed to be removed from the cell surface. This result suggests at least one mechanism by which the presence of inactive EGFR may contribute to GBM development in

expression of EGFR in combination with low levels of pAkt (Haas-Kogan et al., 2005). Once again this supports the notion that activated EGFR in the presence of low pAkt may be

Recently bevacizumab (i.e. avastin) was approved for the treatment of recurrent GBM. A Phase II trial of erlotinib and bevacizumab showed that this combination was adequately tolerated, but provided no additional benefit to that seen in other bevacizumab containing regimens (Sathornsumetee et al., 2010). mTOR is downstream of the PI3K/Akt pathway and therefore represents a therapeutic target in GBM. The combination of sirolimus (a mTor inhibitor), and erlotinib has been reported in recurrent GBM patients (Reardon et al., 2010). While the combination was well tolerated, it displayed negligible anti-tumor activity.

Both Cetuximab and Panitumumab have been approved for the treatment of colon cancer and erlotinib and gefitinib are approved for non-small cell lung cancer (NSCLC), validating the EGFR as a genuine therapeutic target (Van den Eynde et al., 2011). Given the dysregulation of the EGFR in GBM through the range of mechanisms described above, the failure of these agents to show significant anti-tumor activity in this cancer is somewhat surprising. The possibility that the BBB is responsible for this lack of activity has been conclusively discounted for EGFR TKI's and appears not to be a factor for anti-EGFR antibodies, although formal demonstration of this is still required for antibodies. Future research should focus the identification of patients more likely to respond to EGFR inhibition and identifying other targeted therapies that might work synergistically with EGFR inhibitors. Further classification of GBM into unique molecular sub-types beyond the four already described (Verhaak et al., 2010) will hopefully help identify EGFR levels of

In NSCLC the sub-set of patients most likely to respond to EGFR TKI's are those containing activating mutations in the kinase domain of *EGFR (da Cunha Santos et al., 2011)*. Significant clinical and laboratory evidence suggests that these mutations lead to "oncogenic addiction"; the phenomenon whereby a tumor becomes largely dependent on a single activated kinase. Interestingly, *EGFR* amplification is not strongly associated with response to TKI's in NSCLC (De Luca & Normanno, 2010). In contrast, EGFR activation in GBM is most often driven by gene amplification and/or mutations found in the ECD domain of the receptor (Lee et al., 2006), although a recent report described several c-terminal deleted EGFR molecules that are constitutively active (Pines et al., 2010). The clinical data in GBM implies that mutations of this nature do not lead to EGFR addiction. The reason for this remains speculative but suggests that the primary role of EGFR in GBM may not be tumor proliferation or survival but some other important biological function that supports, but is not essential to, GBM growth. A recent paper showing that inactive EGFR has a critical role in upregulating glucose transport highlights other unanticipated roles for this receptor (Weihua et al., 2008). Importantly inhibition of EGFR does not block this activity, rather EGFR needed to be removed from the cell surface. This result suggests at least one mechanism by which the presence of inactive EGFR may contribute to GBM development in

indicative of increased levels of response to EGFR inhibition.

tumor subtypes more likely to respond to EGFR inhibition.

**6. Future developments** 

**6.1 Lessons from other cancers** 

a non-critical manner. Indeed, we have very recently shown that de2-7 EGFR has a role in regulating GBM response to low glucose (Cvrljevic et al, *in press*).

EGFR antibodies do not have any efficacy in colon cancer in patients containing mutations in Ras or Raf (Laurent-Puig et al., 2009). While mutations in these molecules are comparatively rare in GBM (McLendon et al., 2008), and thus cannot be used to stratify patients, the pathways associated with Ras/Raf can be activated by other RTKs on the cell surface. The activation of Ras/Raf by these RTKs might cause *de novo* resistance to EGFR therapeutics. Raf can be targeted directly by TKIs already in the clinic, however Ras cannot be inhibited by this approach. However, the signalling of both kinases can be targeted by blocking downstream targets such as ERK and MEK (Pratilas & Solit, 2010). If the toxicity issues can be managed, targeting these kinases in combination with EGFR inhibitors is a logical next step in GBM. This is not a problem in NSCLC as EGFR and ras mutations are mutually exclusive in this cancer type.

Recent data suggests that mutations in PI3K may also cause resistance to EGFR antibodies in colon cancer (Weickhardt et al., 2010). This is highly relevant to GBM as the PI3K pathway appears to be dysregulated in most GBMs through direct mutation of PI3K, the deletion or mutation of the negative PI3K regulator PTEN or activation through other RTKs (McLendon et al., 2008). Indeed, as discussed above, the absence of PTEN was associated with clinical resistance to EGFR TKIs in GBM patients expressing the de2-7 EGFR while responsiveness was associated with co-expression of PTEN and de2-7 EGFR. The recent translation of PI3K inhibitors into the clinic provides a strategy for overcoming PI3K-mediated resistance to EGFR inhibitors. All the evidence suggests that this combinational approach might finally unleash the potential of EGFR inhibitors for the treatment of GBM (Fan & Weiss, 2010); once again management of toxicity remains as a concern.

#### **6.2 Combination of EGFR inhibitors with other targeted therapies**

Since most RTKs can activate Ras/Raf and/or PI3K, the dual inhibition of EGFR and additional activated RTKs is an obvious therapeutic approach. Stommel *et al* clearly showed that multiple RTKs can be activated in GBM and that these RTKs activate overlapping downstream pathways causing resistance to TKIs that only targeting a single RTK (Stommel et al., 2007). The RTK c-Met is also commonly activated in GBM through a number of mechanisms and also has an important role in angiogenesis (Abounader & Laterra, 2005). We recently showed that co-expression of de2-7 EGFR and c-Met in GBM xenografts causes therapeutic resistant to single agents directed to either of these RTK. However, the combination of EGFR and c-Met inhibitors produced synergistic anti-tumor activity (Pillay et al., 2009), confirming that dual inhibition of RTKs is a valid approach in GBM. A number of other RTKs have been shown to be activated in GBM including the FGFR family, Axl, ErbB2/3/4, EphA2/7, VEGFR2 and PDGFRα/β (Ren et al., 2007; Pillay et al., 2009). Given the range of potential RTKs activated in a given patient, the most effective therapeutic strategy may have to be determined by screening patient tissues for RTK mutation and/or activation (i.e. phosphorylation) before commencing treatment.

We recently showed that EGFR and Src-family kinases (SFK) are frequently coactivated in GBM (Lu et al., 2009). Furthermore, the de2-7 EGFR physically associated with SFKs and

Advances in the Development of EGFR Targeted Therapies for the Treatment of Glioblastoma 37

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#### **7. Conclusion**

The seemingly central role of EGFR in GBM biology would suggest that it should be an excellent therapeutic target in GBM; clinical trials clearly show that this is not the case when EGFR is targeted alone or in combination with standard GBM therapy. The further development of EGFR inhibitors in GBM must be underpinned by additional studies into the biology of EGFR in this cancer and the identification of signalling events associated with resistance to EGFR therapeutics. On-going rational trials using EGFR inhibitors in combination with other TKIs are justified, possibly underpinned by some basic stratification of patients based on mutations present in tumors. Finally, a prospective study formally proving that therapeutic antibodies do actually enter the GBMs and bind target cells would be informative. In conclusion, inhibition of the EGFR pathway in GBM is ineffective as a therapeutic strategy even when used in combination with current therapies. However, their effectiveness in combination with other targeted therapeutics should form the next stage of their development as a therapeutic target.

#### **8. References**


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**3** 

*Germany* 

**Future Perspectives of Enhancing the** 

Georg Karpel-Massler and Marc-Eric Halatsch

*University of Ulm School of Medicine, Ulm,* 

**Therapeutic Efficacy of Epidermal Growth** 

**Factor Receptor Inhibition in Malignant Gliomas** 

In adults, glioblastoma multiforme (GBM) represents the most common malignant brain tumor (Karpel-Massler et al., 2009). Unfortunately, even with the best available standard of care, patients with this disease still face a poor clinical outcome (Stupp et al., 2005). Based on the discovery of molecular targets that are involved in tumorigenesis and maintenance of the malignant cellular phenotype, new therapeutic strategies were developed. In about half of all glioblastomas, the epidermal growth factor receptor (HER1/EGFR) was shown to be amplified and overexpressed, rendering it an outstanding target in this disease (Libermann et al., 1985; Ekstrand et al., 1991). Thus, great interest was generated in the creation of HER1/EGFR-targeted agents. The clinically most advanced compounds that were developed to target HER1/EGFR for the treatment of GBM are small-molecule tyrosine kinase (TK) inhibitors such as erlotinib (Tarceva®, Genentech Inc., San Francisco, CA, U.S.A.). TK inhibitors reversibly bind to the intracellular catalytic TK domain of HER1/EGFR followed by the inhibition of autophosphorylation of the receptor as well as further downstream signaling involving phosphatidylinositol 3-kinase/murine thymoma viral oncogene homolog (PI3-K/AKT) and mitogen-activated protein kinase (MAPK) pathways ( Arteaga, 2001; Busse et al., 2000; Scagliotti et al., 2004). Erlotinib does not only inhibit HER1/EGFR but also EGFRvIII, the most frequent mutant form of HER1/EGFR which is characterized by ligand-independent activation (Chu et al., 1997). In experimental studies, erlotinib was shown to inhibit the expression of genes encoding pro-invasive proteins and to significantly diminish EGFRvIII expression in transfected glioblastoma cells (Lal et al., 2002). Moreover, the extent of erlotinib-mediated inhibition of anchorageindependent growth of glioblastoma-derived cell lines was shown to correlate inversely with the cellular capability to induce *HER1/EGFR* mRNA (Halatsch et al., 2004). However, clinical studies examining the therapeutic efficacy of erlotinib in the setting of GBM have so far failed to prove a therapeutic benefit (Raizer et al., 2010; van den Bent et al., 2009). In a randomized, controlled phase II trial, only 11.4% of the patients with recurrent glioblastoma treated with erlotinib were free of progression after 6 months compared to 24.1% of the patients treated with temozolomide or carmustine (van den Bent et al., 2009). In addition, overall survival of the two treatment groups was found to be similar (7.7 months for the

erlotinib group versus 7.3 months for the temozolomide/carmustine group).

**1. Introduction**

Ymer, S. I., et al. (2011). Glioma Specific Extracellular Missense Mutations in the First Cysteine Rich Region of Epidermal Growth Factor Receptor (EGFR) Initiate Ligand Independent Activation. *Cancers*, Vol. 3, No. 2, pp. 2032-2049.

## **Future Perspectives of Enhancing the Therapeutic Efficacy of Epidermal Growth Factor Receptor Inhibition in Malignant Gliomas**

Georg Karpel-Massler and Marc-Eric Halatsch *University of Ulm School of Medicine, Ulm, Germany* 

#### **1. Introduction**

42 Novel Therapeutic Concepts in Targeting Glioma

Ymer, S. I., et al. (2011). Glioma Specific Extracellular Missense Mutations in the First

Independent Activation. *Cancers*, Vol. 3, No. 2, pp. 2032-2049.

Cysteine Rich Region of Epidermal Growth Factor Receptor (EGFR) Initiate Ligand

In adults, glioblastoma multiforme (GBM) represents the most common malignant brain tumor (Karpel-Massler et al., 2009). Unfortunately, even with the best available standard of care, patients with this disease still face a poor clinical outcome (Stupp et al., 2005). Based on the discovery of molecular targets that are involved in tumorigenesis and maintenance of the malignant cellular phenotype, new therapeutic strategies were developed. In about half of all glioblastomas, the epidermal growth factor receptor (HER1/EGFR) was shown to be amplified and overexpressed, rendering it an outstanding target in this disease (Libermann et al., 1985; Ekstrand et al., 1991). Thus, great interest was generated in the creation of HER1/EGFR-targeted agents. The clinically most advanced compounds that were developed to target HER1/EGFR for the treatment of GBM are small-molecule tyrosine kinase (TK) inhibitors such as erlotinib (Tarceva®, Genentech Inc., San Francisco, CA, U.S.A.). TK inhibitors reversibly bind to the intracellular catalytic TK domain of HER1/EGFR followed by the inhibition of autophosphorylation of the receptor as well as further downstream signaling involving phosphatidylinositol 3-kinase/murine thymoma viral oncogene homolog (PI3-K/AKT) and mitogen-activated protein kinase (MAPK) pathways ( Arteaga, 2001; Busse et al., 2000; Scagliotti et al., 2004). Erlotinib does not only inhibit HER1/EGFR but also EGFRvIII, the most frequent mutant form of HER1/EGFR which is characterized by ligand-independent activation (Chu et al., 1997). In experimental studies, erlotinib was shown to inhibit the expression of genes encoding pro-invasive proteins and to significantly diminish EGFRvIII expression in transfected glioblastoma cells (Lal et al., 2002). Moreover, the extent of erlotinib-mediated inhibition of anchorageindependent growth of glioblastoma-derived cell lines was shown to correlate inversely with the cellular capability to induce *HER1/EGFR* mRNA (Halatsch et al., 2004). However, clinical studies examining the therapeutic efficacy of erlotinib in the setting of GBM have so far failed to prove a therapeutic benefit (Raizer et al., 2010; van den Bent et al., 2009). In a randomized, controlled phase II trial, only 11.4% of the patients with recurrent glioblastoma treated with erlotinib were free of progression after 6 months compared to 24.1% of the patients treated with temozolomide or carmustine (van den Bent et al., 2009). In addition, overall survival of the two treatment groups was found to be similar (7.7 months for the erlotinib group versus 7.3 months for the temozolomide/carmustine group).

Future Perspectives of Enhancing the Therapeutic

malignant transformation of gliomas.

Duensing, 2010; Heinrich et al., 2002; Woodman & Davies, 2010).

Efficacy of Epidermal Growth Factor Receptor Inhibition in Malignant Gliomas 45

pathways such as MAPK, JAK/STAT and PI3K/AKT pathways ( Duensing et al., 2004; Mol et al., 2003). Kit was found to be expressed by a variety of cell types including the interstitial cells of Cajal, mast cells, haemopoietic progenitor cells or melanocytes (Natali et al., 1992; Nocka et al., 1989; Turner et al., 1992; Ishikawa et al., 1997), and its dysregulation has been associated with the pathogenesis of various different human malignancies (Duensing &

In glioma, about 75% of the tumors were reported to express Kit (Cetin et al., 2005). Interestingly, amplification and expression of Kit were shown to be significantly higher in high-grade gliomas when compared to low-grade gliomas (Joensuu et al., 2005; Puputti et al., 2006). These findings suggest that Kit may be involved in the tumorigenesis and

Different mutational changes of Kit have been described, such as the D816V mutation conferring an enhanced catalytic activity and an increased affinity for adenosine triphosphate or small in-frame deletions or insertions in the inhibitory juxtamembrane region causing ligand-independent activation of the receptor (Heinrich et al., 2002). Such genetic alterations of Kit have not been reported for gliomas yet. In other human malignancies including gastrointestinal stromal tumors (GIST) or mast cell leukemia, these mutations are quite frequently encountered (Duensing & Duensing, 2010). As a consequence, Kit-targeted agents such as imatinib mesylate (Gleevec®, Novartis, East Hanover, NJ, U.S.A.), a small molecule tyrosine kinase inhibitor, were developed. Imatinib was shown to significantly increase median overall survival of patients with GIST from 19

Imatinib was shown to inhibit the proliferation of certain glioblastoma cell lines *in vitro* (Hagerstrand et al., 2006). In another experimental study, imatinib significantly inhibited the proliferation of human U87 glioblastoma cells and significantly increased the radiosensitivity of this glioma cell line *in vitro* and *in vivo* (Oertel et al., 2006). However, in clinical phase I and II trials, imatinib was shown to exert only moderate antitumor activity (Razis et al., 2009; Wen et al., 2006). In a phase I/II study, 34 patients with glioblastoma were treated with imatinib monotherapy at a dose of 800 mg/d (Wen et al., 2006). Progressionfree survival at 6 months was only 3%, no patient achieved complete response and only 6 patients reached stable disease while 2 patients showed partial response. In a different phase II study, 20 patients with glioblastoma were diagnosed by tumor biopsy and treated with 400 mg imatinib administered twice a day for a period of 7 days prior to re-biopsy or tumor resection. Molecular examination of the tumor specimens showed that treatment with imatinib did not significantly change Ki67 expression, suggesting that treatment with

The fact that inhibition of Kit and co-targeted tyrosine kinases such as the platelet-derived growth factor (PDGFR), alone, does not sufficiently suppress tumor growth in glioblastoma might be explained by co-activation of other growth factor receptors such as HER1/EGFR. Cellular signaling derived from activated HER1/EGFR might interfere with the inhibitory effects of imatinib on Kit and preserve the cancerous cellular phenotype. In this regard, additional inhibition of HER1/EGFR by erlotinib might prove beneficial in terms of a more pronounced therapeutic efficacy. To date, no experimental or clinical data exist with respect to a combined therapeutic approach with erlotinib and an inhibitor of Kit in this disease.

months to more than 50 months (Blanke et al., 2008a, 2008b; Gold et al., 2007).

imatinib did not affect tumor proliferation (Razis et al., 2009).

In addition, several studies examined the therapeutic efficacy of erlotinib when combined with standard radiochemotherapy (Brown et al., 2008; Peereboom et al., 2010; Prados et al., 2009). Overall, the results of these studies appear unfavorable and discourage the use of erlotinib in combination with temozolomide and radiotherapy.

Combined inhibition of HER1/EGFR and downstream key regulators such as mammalian target of rapamycin (mTOR) and PI3-K represents another approach that has been evaluated. In an experimental study, combined treatment with erlotinib and rapamycin, an mTOR inhibitor, resulted in significantly increased anti-proliferative effects on phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-deficient U87 and SF295 glioblastoma cells when compared to cells receiving erlotinib alone (Wang et al., 2006). Moreover, additional inhibition of PI3-K using a dual mTOR/PI3-K inhibitor (PI-103) was shown to result in even more pronounced antineoplastic effects when combined with erlotinib in comparison to erlotinib combined with either mTOR or PI3-K inhibition (Fan et al., 2007). In the clinical setting, in a pilot study, a 6-month progression-free survival of 25% was reported for 22 recurrent glioblastoma patients who were treated with erlotinib or gefitinib in combination with sirolimus (rapamycine, Rapamune®, Wyeth Pharmaceuticals Inc., Ayerst, PA, U.S.A.) (Doherty et al., 2006). In a phase II clinical trial, no complete or partial responses were observed in 32 patients with recurrent glioblastoma treated with erlotinib and sirolimus in combination (Reardon et al., 2010). Median progression-free survival and median overall survival were shown to be 6.9 weeks and 33.8 weeks, respectively.

The therapeutic efficacy of a combined treatment with erlotinib and bevacizumab, a humanized anti-vascular endothelial growth factor (VEGF) monoclonal antibody, on patients with recurrent high-grade glioma was recently evaluated by a phase II clinical trial (Sathornsumetee et al., 2010). For glioblastoma patients, median 6-month progression-free survival and overall survival were reported as 28% and 42 weeks, respectively. In addition, for 48% of the glioblastoma patients radiographic response was reported. However, progression-free survival and radiographic response were similar to historical data of patients treated with bevacizumab alone.

In conclusion, current data suggest that the targeted therapeutic approach against HER1/EGFR may require a synergistic drug combination strategy involving other targeted agents in addition to HER1/EGFR-targeted TK inhibitors. This chapter focuses on innovative therapeutic strategies combining HER1/EGFR-targeted TK inhibitors with novel agents aiming to enhance the antineoplastic effect exerted by erlotinib. Most of the agents discussed in this chapter have not been evaluated for the treatment of GBM yet but constitute worthy candidates for further evaluation in this setting.

#### **2. Promising candidates for enhancing the antineoplastic activity of erlotinib**

#### **2.1 Inhibitors of Kit**

Kit (CD117) is a receptor tyrosine kinase which is related to the macrophage colonystimulating factor receptor (c-fms) and to the platelet-derived growth factor receptor (PDGFR) ( Heinrich et al., 2002; Yarden et al., 1987). Its physiologic ligand is stem cell factor, also known as mast cell factor or steel factor (Nocka et al., 1990). Ligand-binding is followed by receptor dimerization, autophosphorylation and activation of downward signaling

In addition, several studies examined the therapeutic efficacy of erlotinib when combined with standard radiochemotherapy (Brown et al., 2008; Peereboom et al., 2010; Prados et al., 2009). Overall, the results of these studies appear unfavorable and discourage the use of

Combined inhibition of HER1/EGFR and downstream key regulators such as mammalian target of rapamycin (mTOR) and PI3-K represents another approach that has been evaluated. In an experimental study, combined treatment with erlotinib and rapamycin, an mTOR inhibitor, resulted in significantly increased anti-proliferative effects on phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-deficient U87 and SF295 glioblastoma cells when compared to cells receiving erlotinib alone (Wang et al., 2006). Moreover, additional inhibition of PI3-K using a dual mTOR/PI3-K inhibitor (PI-103) was shown to result in even more pronounced antineoplastic effects when combined with erlotinib in comparison to erlotinib combined with either mTOR or PI3-K inhibition (Fan et al., 2007). In the clinical setting, in a pilot study, a 6-month progression-free survival of 25% was reported for 22 recurrent glioblastoma patients who were treated with erlotinib or gefitinib in combination with sirolimus (rapamycine, Rapamune®, Wyeth Pharmaceuticals Inc., Ayerst, PA, U.S.A.) (Doherty et al., 2006). In a phase II clinical trial, no complete or partial responses were observed in 32 patients with recurrent glioblastoma treated with erlotinib and sirolimus in combination (Reardon et al., 2010). Median progression-free survival and median overall survival were shown to be 6.9 weeks and 33.8 weeks,

The therapeutic efficacy of a combined treatment with erlotinib and bevacizumab, a humanized anti-vascular endothelial growth factor (VEGF) monoclonal antibody, on patients with recurrent high-grade glioma was recently evaluated by a phase II clinical trial (Sathornsumetee et al., 2010). For glioblastoma patients, median 6-month progression-free survival and overall survival were reported as 28% and 42 weeks, respectively. In addition, for 48% of the glioblastoma patients radiographic response was reported. However, progression-free survival and radiographic response were similar to historical data of

In conclusion, current data suggest that the targeted therapeutic approach against HER1/EGFR may require a synergistic drug combination strategy involving other targeted agents in addition to HER1/EGFR-targeted TK inhibitors. This chapter focuses on innovative therapeutic strategies combining HER1/EGFR-targeted TK inhibitors with novel agents aiming to enhance the antineoplastic effect exerted by erlotinib. Most of the agents discussed in this chapter have not been evaluated for the treatment of GBM yet but

**2. Promising candidates for enhancing the antineoplastic activity of erlotinib** 

Kit (CD117) is a receptor tyrosine kinase which is related to the macrophage colonystimulating factor receptor (c-fms) and to the platelet-derived growth factor receptor (PDGFR) ( Heinrich et al., 2002; Yarden et al., 1987). Its physiologic ligand is stem cell factor, also known as mast cell factor or steel factor (Nocka et al., 1990). Ligand-binding is followed by receptor dimerization, autophosphorylation and activation of downward signaling

erlotinib in combination with temozolomide and radiotherapy.

respectively.

**2.1 Inhibitors of Kit** 

patients treated with bevacizumab alone.

constitute worthy candidates for further evaluation in this setting.

pathways such as MAPK, JAK/STAT and PI3K/AKT pathways ( Duensing et al., 2004; Mol et al., 2003). Kit was found to be expressed by a variety of cell types including the interstitial cells of Cajal, mast cells, haemopoietic progenitor cells or melanocytes (Natali et al., 1992; Nocka et al., 1989; Turner et al., 1992; Ishikawa et al., 1997), and its dysregulation has been associated with the pathogenesis of various different human malignancies (Duensing & Duensing, 2010; Heinrich et al., 2002; Woodman & Davies, 2010).

In glioma, about 75% of the tumors were reported to express Kit (Cetin et al., 2005). Interestingly, amplification and expression of Kit were shown to be significantly higher in high-grade gliomas when compared to low-grade gliomas (Joensuu et al., 2005; Puputti et al., 2006). These findings suggest that Kit may be involved in the tumorigenesis and malignant transformation of gliomas.

Different mutational changes of Kit have been described, such as the D816V mutation conferring an enhanced catalytic activity and an increased affinity for adenosine triphosphate or small in-frame deletions or insertions in the inhibitory juxtamembrane region causing ligand-independent activation of the receptor (Heinrich et al., 2002). Such genetic alterations of Kit have not been reported for gliomas yet. In other human malignancies including gastrointestinal stromal tumors (GIST) or mast cell leukemia, these mutations are quite frequently encountered (Duensing & Duensing, 2010). As a consequence, Kit-targeted agents such as imatinib mesylate (Gleevec®, Novartis, East Hanover, NJ, U.S.A.), a small molecule tyrosine kinase inhibitor, were developed. Imatinib was shown to significantly increase median overall survival of patients with GIST from 19 months to more than 50 months (Blanke et al., 2008a, 2008b; Gold et al., 2007).

Imatinib was shown to inhibit the proliferation of certain glioblastoma cell lines *in vitro* (Hagerstrand et al., 2006). In another experimental study, imatinib significantly inhibited the proliferation of human U87 glioblastoma cells and significantly increased the radiosensitivity of this glioma cell line *in vitro* and *in vivo* (Oertel et al., 2006). However, in clinical phase I and II trials, imatinib was shown to exert only moderate antitumor activity (Razis et al., 2009; Wen et al., 2006). In a phase I/II study, 34 patients with glioblastoma were treated with imatinib monotherapy at a dose of 800 mg/d (Wen et al., 2006). Progressionfree survival at 6 months was only 3%, no patient achieved complete response and only 6 patients reached stable disease while 2 patients showed partial response. In a different phase II study, 20 patients with glioblastoma were diagnosed by tumor biopsy and treated with 400 mg imatinib administered twice a day for a period of 7 days prior to re-biopsy or tumor resection. Molecular examination of the tumor specimens showed that treatment with imatinib did not significantly change Ki67 expression, suggesting that treatment with imatinib did not affect tumor proliferation (Razis et al., 2009).

The fact that inhibition of Kit and co-targeted tyrosine kinases such as the platelet-derived growth factor (PDGFR), alone, does not sufficiently suppress tumor growth in glioblastoma might be explained by co-activation of other growth factor receptors such as HER1/EGFR. Cellular signaling derived from activated HER1/EGFR might interfere with the inhibitory effects of imatinib on Kit and preserve the cancerous cellular phenotype. In this regard, additional inhibition of HER1/EGFR by erlotinib might prove beneficial in terms of a more pronounced therapeutic efficacy. To date, no experimental or clinical data exist with respect to a combined therapeutic approach with erlotinib and an inhibitor of Kit in this disease.

Future Perspectives of Enhancing the Therapeutic

al., 2009; Prince et al., 2009; Whittaker et al., 2010).

lines.

glioblastoma cells.

**2.3 Vascular disrupting agents** 

Efficacy of Epidermal Growth Factor Receptor Inhibition in Malignant Gliomas 47

intravenous infusion on days 1, 8, and 15 of a 28-day cycle, an overall response rate of 34% was found (Piekarz et al., 2009). Partial response, complete response and stable disease were reported as 26%, 7% and 38%, respectively. Similar findings were reported by a different group (Whittaker et al., 2010). Overall, the safety profile of romidepsin has been favorable, and serious adverse events were shown to be rare (Byrd et al., 2005; Odenike et al., 2008; Piekarz et

There is no clinical data on romidepsin in glioblastoma and only little data on other HDAC inhibitors in this setting. However, in experimental studies, a radiosensitizing effect was observed in glioblastoma cells treated with HDAC inhibitors. The fraction of surviving SF539 and U251 glioblastoma cells that were treated with valproic acid (VA), an anticonvulsive drug known to also inhibit HDACs, and radiation was significantly lower in comparison to cells that were treated with radiation only (Camphausen et al., 2005). Moreover, in a murine heterotopic U251 xenograft model, treatment with VA and irradiation was shown to result in a significantly greater delay of tumor growth when compared to animals treated with either VA or irradiation alone. These findings were confirmed by other groups using different HDAC inhibitors (Entin-Meer et al., 2007; Lucio-Eterovic et al., 2008). In another experimental study, treatment with the HDAC inhibitor trichostatin A or 4-phenyl-butyrate was shown to induce cellular differentiation of different human glioblastoma cell lines (Svechnikova et al., 2008). In addition, both HDAC inhibitors were shown to inhibit cellular proliferation and to promote apoptosis in glioblastoma cell

In the setting of glioblastoma, so far only one experimental study was published examining the effects of romidepsin. In that study, treatment with romidepsin at a concentration of 1 ng/ml was shown to significantly reduce proliferation of T98G, U251MG and U87MG glioblastoma cells (Sawa et al., 2004). In addition, U251MG cells treated with romidepsin were shown to be significantly less invasive when compared to controls. Moreover, in a heterotopic xenograft model, mice treated with romidepsin were shown to have significantly reduced tumor growth of subcutaneously inoculated EGFRvIII-bearing U87MG

Both erlotinib and romidepsin are promising anticancer agents fitting a reasonable safety profile. However, further studies are needed to elucidate if combining the antineoplastic effects of erlotinib and HDAC inhibitors such as romidepsin may result in a significant

Tumor angiogenesis stands for cancers' development of their own blood supply. This process was found to be crucial for the growth and metastasis of solid tumors and can be achieved by different mechanisms such as sprouting angiogenesis, recruitment of bone marrow-derived endothelial progenitor cells or the longitudinal splitting of existing blood

Different anti-angiogenic agents were developed for the treatment of human malignancies including high-grade glioma. One such agent is bevacizumab (Avastin®, Genentech Inc., San Francisco, CA, U.S.A.), a humanized monoclonal antibody targeted to VEGF. Numerous clinical studies were conducted evaluating the therapeutic efficacy of bevacizumab in

improvement of the current clinical course of glioblastoma.

vessels called intussusception (reviewed in Heath & Bicknell, 2009).

However, in the setting of recurrent glioblastoma, encouraging results were reported by a phase II study evaluating the therapeutic efficacy of a combination therapy with imatinib and hydroxyurea, a ribonucleotide reductase inhibitor (Reardon et al., 2005). Median overall survival, progression-free survival at 6 months and median progression-free survival were 48.9 weeks, 27% and 14.4 weeks, respectively. Nine percent of the patients achieved radiographic response and 42% had stable disease within a median follow-up of 58 weeks.

In conclusion, despite rather discouraging results of Kit inhibitors used as single agent therapies in clinical trials, Kit inhibitors may prove as valuable partners for the treatment of glioblastoma when combined with other agents such as erlotinib.

#### **2.2 Histone deacetylase (HDAC) inhibitors**

In humans, 18 HDACs with different tissue distributions and functions have been identified. Class I, IIa and IV HDACs are found in the brain (Marsoni et al., 2008). HDACs induce an increased packaging of chromatin and subsequent suppression of transcription (Lane & Chabner, 2009; Svechnikova et al., 2008). Modulation of the chromatin state through enzymatic histone modification may alter the transcriptional activity of genes involved in cell cycle control which is considered to be an important factor in tumorigenesis (Yoo & Jones, 2006). HDACs were shown to be overexpressed in a variety of human cancers including breast cancer, hematologic malignancies, colorectal cancer or pancreatic carcinoma (Lane & Chabner, 2009; Nakagawa et al., 2007). Moreover, inhibition of HDAC was shown to induce apoptosis by different mechanisms (Insinga et al., 2005; Nebbioso et al., 2005; Zhang et al., 2006; Zhao et al., 2005). In addition, inhibition of HDAC was shown to disrupt the function of the heat shock protein 90 which promotes the degradation of oncogenic proteins such as HER1/EGFR, AKT or BCR-ABL ( Bolden et al., 2006; Kovacs et al., 2005; Whitesell & Lindquist, 2005). Thus, HDAC inhibition may constitute a promising approach in cancer therapy.

Romidepsin is a bicyclic peptide that was shown to have anti-microbial, immunosuppressive and antineoplastic activities ( Ritchie et al., 2009; Ueda et al., 1994). It was shown to selectively inhibit deacetylases such as HDAC or tubulin deacetylase and represents one of the best studied HDAC inhibitors in the clinical setting (Yoo & Jones, 2006). The clinical experience with HDAC inhibitors is most advanced for the treatment of cutaneous T-cell lymphoma (CTCL) and hematologic malignancies (Lane & Chabner, 2009; Prince et al., 2009). In an early phase I trial, 10 patients with chronic lymphocytic leukemia (CLL) and 10 patients with acute myeloid leukemia (AML) were treated with romidepsin at a dose of 13 mg/m2 on day 1, 8, and 15 of a 4-week cycle (Byrd et al., 2005). Despite absence of formal complete or partial responses, all seven CLL patients who had elevated leukocyte counts at the beginning of the therapy showed an improvement in peripheral leukocyte counts, while in the AML group one patient developed a tumor lysis syndrome. Moreover, in a phase II clinical trial, treatment with romidepsin resulted in a decrease of bone marrow blasts in 5 of 7 patients with AML (Odenike et al., 2008). However, within a month after achieving their best response towards romidepsin, these 5 patients developed disease progression. In the clinical setting of refractory CTCL, two phase II clinical trials examining the therapeutic efficacy of romidepsin were recently published (Piekarz et al., 2009; Whittaker et al., 2010). In 71 patients with treatment-refractory or advanced CTCL treated with a starting dose of 14 mg/m2 romidepsin administered as a 4-h

However, in the setting of recurrent glioblastoma, encouraging results were reported by a phase II study evaluating the therapeutic efficacy of a combination therapy with imatinib and hydroxyurea, a ribonucleotide reductase inhibitor (Reardon et al., 2005). Median overall survival, progression-free survival at 6 months and median progression-free survival were 48.9 weeks, 27% and 14.4 weeks, respectively. Nine percent of the patients achieved radiographic response and 42% had stable disease within a median follow-up of 58 weeks. In conclusion, despite rather discouraging results of Kit inhibitors used as single agent therapies in clinical trials, Kit inhibitors may prove as valuable partners for the treatment of

In humans, 18 HDACs with different tissue distributions and functions have been identified. Class I, IIa and IV HDACs are found in the brain (Marsoni et al., 2008). HDACs induce an increased packaging of chromatin and subsequent suppression of transcription (Lane & Chabner, 2009; Svechnikova et al., 2008). Modulation of the chromatin state through enzymatic histone modification may alter the transcriptional activity of genes involved in cell cycle control which is considered to be an important factor in tumorigenesis (Yoo & Jones, 2006). HDACs were shown to be overexpressed in a variety of human cancers including breast cancer, hematologic malignancies, colorectal cancer or pancreatic carcinoma (Lane & Chabner, 2009; Nakagawa et al., 2007). Moreover, inhibition of HDAC was shown to induce apoptosis by different mechanisms (Insinga et al., 2005; Nebbioso et al., 2005; Zhang et al., 2006; Zhao et al., 2005). In addition, inhibition of HDAC was shown to disrupt the function of the heat shock protein 90 which promotes the degradation of oncogenic proteins such as HER1/EGFR, AKT or BCR-ABL ( Bolden et al., 2006; Kovacs et al., 2005; Whitesell & Lindquist, 2005). Thus, HDAC inhibition may constitute a promising

Romidepsin is a bicyclic peptide that was shown to have anti-microbial, immunosuppressive and antineoplastic activities ( Ritchie et al., 2009; Ueda et al., 1994). It was shown to selectively inhibit deacetylases such as HDAC or tubulin deacetylase and represents one of the best studied HDAC inhibitors in the clinical setting (Yoo & Jones, 2006). The clinical experience with HDAC inhibitors is most advanced for the treatment of cutaneous T-cell lymphoma (CTCL) and hematologic malignancies (Lane & Chabner, 2009; Prince et al., 2009). In an early phase I trial, 10 patients with chronic lymphocytic leukemia (CLL) and 10 patients with acute myeloid leukemia (AML) were treated with romidepsin at a dose of 13 mg/m2 on day 1, 8, and 15 of a 4-week cycle (Byrd et al., 2005). Despite absence of formal complete or partial responses, all seven CLL patients who had elevated leukocyte counts at the beginning of the therapy showed an improvement in peripheral leukocyte counts, while in the AML group one patient developed a tumor lysis syndrome. Moreover, in a phase II clinical trial, treatment with romidepsin resulted in a decrease of bone marrow blasts in 5 of 7 patients with AML (Odenike et al., 2008). However, within a month after achieving their best response towards romidepsin, these 5 patients developed disease progression. In the clinical setting of refractory CTCL, two phase II clinical trials examining the therapeutic efficacy of romidepsin were recently published (Piekarz et al., 2009; Whittaker et al., 2010). In 71 patients with treatment-refractory or advanced CTCL treated with a starting dose of 14 mg/m2 romidepsin administered as a 4-h

glioblastoma when combined with other agents such as erlotinib.

**2.2 Histone deacetylase (HDAC) inhibitors** 

approach in cancer therapy.

intravenous infusion on days 1, 8, and 15 of a 28-day cycle, an overall response rate of 34% was found (Piekarz et al., 2009). Partial response, complete response and stable disease were reported as 26%, 7% and 38%, respectively. Similar findings were reported by a different group (Whittaker et al., 2010). Overall, the safety profile of romidepsin has been favorable, and serious adverse events were shown to be rare (Byrd et al., 2005; Odenike et al., 2008; Piekarz et al., 2009; Prince et al., 2009; Whittaker et al., 2010).

There is no clinical data on romidepsin in glioblastoma and only little data on other HDAC inhibitors in this setting. However, in experimental studies, a radiosensitizing effect was observed in glioblastoma cells treated with HDAC inhibitors. The fraction of surviving SF539 and U251 glioblastoma cells that were treated with valproic acid (VA), an anticonvulsive drug known to also inhibit HDACs, and radiation was significantly lower in comparison to cells that were treated with radiation only (Camphausen et al., 2005). Moreover, in a murine heterotopic U251 xenograft model, treatment with VA and irradiation was shown to result in a significantly greater delay of tumor growth when compared to animals treated with either VA or irradiation alone. These findings were confirmed by other groups using different HDAC inhibitors (Entin-Meer et al., 2007; Lucio-Eterovic et al., 2008). In another experimental study, treatment with the HDAC inhibitor trichostatin A or 4-phenyl-butyrate was shown to induce cellular differentiation of different human glioblastoma cell lines (Svechnikova et al., 2008). In addition, both HDAC inhibitors were shown to inhibit cellular proliferation and to promote apoptosis in glioblastoma cell lines.

In the setting of glioblastoma, so far only one experimental study was published examining the effects of romidepsin. In that study, treatment with romidepsin at a concentration of 1 ng/ml was shown to significantly reduce proliferation of T98G, U251MG and U87MG glioblastoma cells (Sawa et al., 2004). In addition, U251MG cells treated with romidepsin were shown to be significantly less invasive when compared to controls. Moreover, in a heterotopic xenograft model, mice treated with romidepsin were shown to have significantly reduced tumor growth of subcutaneously inoculated EGFRvIII-bearing U87MG glioblastoma cells.

Both erlotinib and romidepsin are promising anticancer agents fitting a reasonable safety profile. However, further studies are needed to elucidate if combining the antineoplastic effects of erlotinib and HDAC inhibitors such as romidepsin may result in a significant improvement of the current clinical course of glioblastoma.

#### **2.3 Vascular disrupting agents**

Tumor angiogenesis stands for cancers' development of their own blood supply. This process was found to be crucial for the growth and metastasis of solid tumors and can be achieved by different mechanisms such as sprouting angiogenesis, recruitment of bone marrow-derived endothelial progenitor cells or the longitudinal splitting of existing blood vessels called intussusception (reviewed in Heath & Bicknell, 2009).

Different anti-angiogenic agents were developed for the treatment of human malignancies including high-grade glioma. One such agent is bevacizumab (Avastin®, Genentech Inc., San Francisco, CA, U.S.A.), a humanized monoclonal antibody targeted to VEGF. Numerous clinical studies were conducted evaluating the therapeutic efficacy of bevacizumab in

Future Perspectives of Enhancing the Therapeutic

with erlotinib and a VDA in glioblastoma.

**3. Conclusion** 

frustrating situation.

Efficacy of Epidermal Growth Factor Receptor Inhibition in Malignant Gliomas 49

volume was shown to be reduced by 22.9% at 4 hrs after application of CA4P and by 29.4% after 72 hrs. Moreover, the decrease in blood volume was shown to be more pronounced at the outer rim of the tumor than at its center (51.4% vs 22.8%). These findings suggest that the antivascular effect exerted by CA4P can be enhanced by radiotherapy in the setting of NSCLC. In another study, CA4P was applied for the treatment of patients with different advanced cancers refractory to standard therapy 18-22 hrs prior to a single-agent treatment with paclitaxel or carboplatin or combination therapy with paclitaxel and carboplatin in sequential order (Rustin et al., 2010). A formal response was noted in 7 of 18 patients with ovarian cancer, primary peritoneal carcinoma, or cancer of the fallopian tube. Partial remission was achieved in another 3 out of 30 patients with non-ovarian cancer. Thus, this combinatorial regimen displays antitumor activity in patients with difficult-to-treat cancers. Overall, VDAs are promising anticancer agents and might provide an additional benefit when combined with other antineoplastic drugs. Other therapeutics administered in addition to VDAs might be trapped in the tumor tissue due to the shut-down of tumor blood flow. Thereby, tumor cells might not only die secondary to ischemia, but surviving cells in the outer rim of the tumor may also be eliminated. This way, tumor regrowth might be retarded or prevented. At this point, there is no data on the therapeutic efficacy of a combined treatment with erlotinib and VDAs for the treatment of glioblastoma. Further studies are warranted to examine the overall antineoplastic effect of a combined treatment

Unfortunately, in glioblastoma, HER1/EGFR-targeted small-molecule TK inhibitors such as erlotinib did not fulfill the enthusiastic expectations derived from the promising results obtained by preclinical studies (Brown et al., 2008; van den Bent et al., 2009). Thus, the fate of patients diagnosed with glioblastoma remains dismal despite employing the currently best standard of care. New therapeutic strategies are undoubtedly needed to overcome this

One such new therapeutic approach which aims at enhancing the therapeutic efficacy against glioblastoma involves the combination of erlotinib with other targeted agents in order to inhibit key regulators that are located further downstream of the signaling cascade or with agents inhibiting other signaling pathways. Several clinical studies are ongoing to evaluate this therapeutic option. In patients with recurrent glioblastoma or gliosarcoma, a phase I/II clinical trial currently evaluates the therapeutic effects of a combined treatment with erlotinib, sorafenib (BAY 54-9085, Bayer HealthCare Pharmaceuticals, Montville, NJ, U.S.A.), an inhibitor of murine leukemia viral oncogene homolog (RAF)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and VEGFR-2/PDGFR-β signaling pathways, and temsirolimus (CCI-779, Wyeth Pharmaceuticals, Madison, NJ, U.S.A.), an inhibitor of mTOR. The results are awaited. A different clinical trial investigated the effects of dual therapy with erlotinib and sorafenib in patients with progressive or recurrent

In this chapter, we emphasize the need for a continous search for new agents replenishing our armory for the fight against glioblastoma. Some of the novel agents presented herein may allow to enhance overall antitumor activity when applied together with other

glioblastoma. This study has been completed, and the results are pending.

glioblastoma. In a phase II study, 20 of 35 patients (57%) with recurrent glioblastoma who were treated with bevacizumab in combination with irinotecan showed at least partial response. The 6-month progression-free survival and 6-month overall survival rates were 46% and 77%, respectively (Vredenburgh et al., 2007). Similar findings were reported for patients with recurrent World Health Organization (WHO) grade III gliomas (Desjardins et al., 2008). More recently, Friedman et al. reported the results of a phase II multicenter clinical trial (BRAIN) studying a larger patient population (Friedman et al., 2009). In this study, 167 patients with recurrent glioblastoma were randomly assigned to either treatment with bevacizumab alone (n=85) or in combination with irinotecan (n=82). Median overall survival was 9.2 months and 8.7 months, respectively, 6-month progression-free survival rates were 42.6% and 50.3%, and objective response rates were 28.2% and 37.8%, respectively.

The tumor blood supply may not only be therapeutically attacked by anti-angiogenic means inhibiting the formation of new tumor-supplying blood vessels, but also by destroying already existing tumor blood vessels. The combretastatins are small molecule microtubuledepolymerising agents which cause selective disruption of the tumor-supplying vasculature. The best studied member of this group of agents is represented by CA4P (ZybrestatTM, Oxigene Inc., Lund, Sweden).

The blood supply of spontaneous and ortho- and heterotopically transplanted rodent tumors as well as human xenografted tumors was shown to be significantly reduced within 10-20 min after application of CA4P, an effect lasting for up to 24 hrs in some tumors ( Kanthou & Tozer, 2007; Tozer et al., 2001). However, despite the fact that a single-dose application of CA4P was shown to induce abundant tumor necrosis within a short period of time, cells in the outer rim of the tumor survived (Dark et al., 1997; Tozer et al., 2001). The cells in this niche may continue or restart to grow causing tumor recurrence. In a heterotopic rat glioma model, blood flow in subcutaneous tumors dropped to about half of the initial tumor blood flow during the first 110 min after administration of CA4P (Eikesdal et al., 2000). However, treatment with CA4P at a dose of 50 mg/kg did not significantly affect tumor growth in comparison to controls. Remarkably, when the treatment with CA4P preceded a hyperthermic treatment by 3 hrs, tumor growth was significantly more delayed when compared to animals receiving CA4P immediately before hyperthermia or animals subjected to hyperthermic treatment alone. In conclusion, if applied at the right time, treatment with CA4P may increase thermally induced antitumor activity.

To date, there are no clinical studies examining the effects of CA4P in glioblastoma. However, CA4P was shown to diminish perfusion and blood flow in different advanced solid tumors (Dowlati et al., 2002; Rustin et al., 2003; Stevenson et al., 2003). In addition, some patients were reported to have experienced a notable clinical benefit from the treatment with CA4P. Complete response was reported for a patient with anaplastic thyroid cancer. This patient was free of disease for more than 5 years. Another patient suffering from fibrosarcoma achieved partial response.

Aiming at the elimination of viable tumor cells remaining at the periphery of the tumor despite treatment with VDAs, a therapeutic approach was attempted combining VDAs with radiotherapy or conventional chemotherapy. Eight patients with advanced non-small cell lung cancer (NSCLC) were treated with radiotherapy (27 Gy) and CA4P at a dose of 50 mg/m2 starting after the second fraction of radiotherapy (Ng et al., 2007). The tumor blood volume was shown to be reduced by 22.9% at 4 hrs after application of CA4P and by 29.4% after 72 hrs. Moreover, the decrease in blood volume was shown to be more pronounced at the outer rim of the tumor than at its center (51.4% vs 22.8%). These findings suggest that the antivascular effect exerted by CA4P can be enhanced by radiotherapy in the setting of NSCLC. In another study, CA4P was applied for the treatment of patients with different advanced cancers refractory to standard therapy 18-22 hrs prior to a single-agent treatment with paclitaxel or carboplatin or combination therapy with paclitaxel and carboplatin in sequential order (Rustin et al., 2010). A formal response was noted in 7 of 18 patients with ovarian cancer, primary peritoneal carcinoma, or cancer of the fallopian tube. Partial remission was achieved in another 3 out of 30 patients with non-ovarian cancer. Thus, this combinatorial regimen displays antitumor activity in patients with difficult-to-treat cancers.

Overall, VDAs are promising anticancer agents and might provide an additional benefit when combined with other antineoplastic drugs. Other therapeutics administered in addition to VDAs might be trapped in the tumor tissue due to the shut-down of tumor blood flow. Thereby, tumor cells might not only die secondary to ischemia, but surviving cells in the outer rim of the tumor may also be eliminated. This way, tumor regrowth might be retarded or prevented. At this point, there is no data on the therapeutic efficacy of a combined treatment with erlotinib and VDAs for the treatment of glioblastoma. Further studies are warranted to examine the overall antineoplastic effect of a combined treatment with erlotinib and a VDA in glioblastoma.

#### **3. Conclusion**

48 Novel Therapeutic Concepts in Targeting Glioma

glioblastoma. In a phase II study, 20 of 35 patients (57%) with recurrent glioblastoma who were treated with bevacizumab in combination with irinotecan showed at least partial response. The 6-month progression-free survival and 6-month overall survival rates were 46% and 77%, respectively (Vredenburgh et al., 2007). Similar findings were reported for patients with recurrent World Health Organization (WHO) grade III gliomas (Desjardins et al., 2008). More recently, Friedman et al. reported the results of a phase II multicenter clinical trial (BRAIN) studying a larger patient population (Friedman et al., 2009). In this study, 167 patients with recurrent glioblastoma were randomly assigned to either treatment with bevacizumab alone (n=85) or in combination with irinotecan (n=82). Median overall survival was 9.2 months and 8.7 months, respectively, 6-month progression-free survival rates were 42.6% and 50.3%, and objective response rates were 28.2% and 37.8%,

The tumor blood supply may not only be therapeutically attacked by anti-angiogenic means inhibiting the formation of new tumor-supplying blood vessels, but also by destroying already existing tumor blood vessels. The combretastatins are small molecule microtubuledepolymerising agents which cause selective disruption of the tumor-supplying vasculature. The best studied member of this group of agents is represented by CA4P

The blood supply of spontaneous and ortho- and heterotopically transplanted rodent tumors as well as human xenografted tumors was shown to be significantly reduced within 10-20 min after application of CA4P, an effect lasting for up to 24 hrs in some tumors ( Kanthou & Tozer, 2007; Tozer et al., 2001). However, despite the fact that a single-dose application of CA4P was shown to induce abundant tumor necrosis within a short period of time, cells in the outer rim of the tumor survived (Dark et al., 1997; Tozer et al., 2001). The cells in this niche may continue or restart to grow causing tumor recurrence. In a heterotopic rat glioma model, blood flow in subcutaneous tumors dropped to about half of the initial tumor blood flow during the first 110 min after administration of CA4P (Eikesdal et al., 2000). However, treatment with CA4P at a dose of 50 mg/kg did not significantly affect tumor growth in comparison to controls. Remarkably, when the treatment with CA4P preceded a hyperthermic treatment by 3 hrs, tumor growth was significantly more delayed when compared to animals receiving CA4P immediately before hyperthermia or animals subjected to hyperthermic treatment alone. In conclusion, if applied at the right time,

To date, there are no clinical studies examining the effects of CA4P in glioblastoma. However, CA4P was shown to diminish perfusion and blood flow in different advanced solid tumors (Dowlati et al., 2002; Rustin et al., 2003; Stevenson et al., 2003). In addition, some patients were reported to have experienced a notable clinical benefit from the treatment with CA4P. Complete response was reported for a patient with anaplastic thyroid cancer. This patient was free of disease for more than 5 years. Another patient suffering

Aiming at the elimination of viable tumor cells remaining at the periphery of the tumor despite treatment with VDAs, a therapeutic approach was attempted combining VDAs with radiotherapy or conventional chemotherapy. Eight patients with advanced non-small cell lung cancer (NSCLC) were treated with radiotherapy (27 Gy) and CA4P at a dose of 50 mg/m2 starting after the second fraction of radiotherapy (Ng et al., 2007). The tumor blood

treatment with CA4P may increase thermally induced antitumor activity.

respectively.

(ZybrestatTM, Oxigene Inc., Lund, Sweden).

from fibrosarcoma achieved partial response.

Unfortunately, in glioblastoma, HER1/EGFR-targeted small-molecule TK inhibitors such as erlotinib did not fulfill the enthusiastic expectations derived from the promising results obtained by preclinical studies (Brown et al., 2008; van den Bent et al., 2009). Thus, the fate of patients diagnosed with glioblastoma remains dismal despite employing the currently best standard of care. New therapeutic strategies are undoubtedly needed to overcome this frustrating situation.

One such new therapeutic approach which aims at enhancing the therapeutic efficacy against glioblastoma involves the combination of erlotinib with other targeted agents in order to inhibit key regulators that are located further downstream of the signaling cascade or with agents inhibiting other signaling pathways. Several clinical studies are ongoing to evaluate this therapeutic option. In patients with recurrent glioblastoma or gliosarcoma, a phase I/II clinical trial currently evaluates the therapeutic effects of a combined treatment with erlotinib, sorafenib (BAY 54-9085, Bayer HealthCare Pharmaceuticals, Montville, NJ, U.S.A.), an inhibitor of murine leukemia viral oncogene homolog (RAF)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and VEGFR-2/PDGFR-β signaling pathways, and temsirolimus (CCI-779, Wyeth Pharmaceuticals, Madison, NJ, U.S.A.), an inhibitor of mTOR. The results are awaited. A different clinical trial investigated the effects of dual therapy with erlotinib and sorafenib in patients with progressive or recurrent glioblastoma. This study has been completed, and the results are pending.

In this chapter, we emphasize the need for a continous search for new agents replenishing our armory for the fight against glioblastoma. Some of the novel agents presented herein may allow to enhance overall antitumor activity when applied together with other

Future Perspectives of Enhancing the Therapeutic

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**Part 3** 

**MicroRNAs in Treatment of Glioma** 

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