of brain mets

Analysis; SD: Stable disease; SIR: Score Index for Radiosurgery

**Table 7.** Risk Stratification of Patients with Brain Metastases from Melanoma

MM-GKR Age, KPS, Adverse

BS-BM KPS, control of primary tumor, presence of ECM

DS-GPA RPA factors (KPS, age, extra-cranial disease status, control of primary disease site) and number of brain mets

CNS lesion locations (brainstem, posterior fossa, nuclei, cerebellum)

**(all tumor types)**

Factor 0 1 1.5 2 NR Score Intended for

Factor 0 1 Score NR Not validated in

1 2 3

1.5-2.5 3.0 3.5-4.0

CNS: Central Nervous System; CR: Complete response; DS-GPA: Diagnosis-Specific Graded Prognostic Assessment; ECM: Extracranial metastases; KPS: Karnofsky performance status; LM: Leptomeningeal metastasis; MM-GKR: Malignant Melanoma-Gamma Knife Radiosurgery score; ND: Not defined; NED: No evidence of disease; NR: Not reported; OS: Overall survival; PD: Progressive disease; PI: Prognostic Index; PR: Partial Response; QOL: Quality of life; RPA: Recursive Partitioning

An important caveat in discussing outcomes estimates derived using these risk stratification systems is that they all were first developed prior to 2011. Prior to that time, reliably effective and proven treatments for advanced melanoma were not available for general clinical use. Development of drugs with proven activity, such as ipilimumab and vemurafenib discussed above, are changing the outlook for melanoma patients. This includes patients with brain metastases. With these drugs, and more being developed with potentially even greater activity, the risk estimates of these systems will certainly change for the better. This is especially likely

3.4m 6.4m 11.6m 14.8m

KPS <70 70-80 90-100 2010 [127]

0.0-1.0 1.5-2.5 3.0 3.5-4.0 3.4m 4.7m 8.8m 13.2m In melanoma, only 2 significant prognostic factors: KPS (*p*<0.0001) and number of brain mets (*p*<0.0001)

KPS <80 - - ≤80 0 *et al.,* 2006 [23] 1-2 ≥2.5

KPS 50-70 "/>70 0 2004 [128]

1.9 m 3.3 m 13.1 m ND

**Median OS (melanoma)** **Comments References**

http://dx.doi.org/10.5772/55175

Gaudy-Marqueste

309

Lorenzoni *et al.,*

Sperduto *et al.,*

assessment of outcome after SRS.

Management of Brain Metastasis in Melanoma Patients

melanoma, although study included 19 melanoma patients out of 110 total (17%). Score of 3 had OS not reaching median with 30 m of follow-up. Intended for assessment of outcome after SRS.

7.1 m 5 m 2.2 m

Median survival times predicted by studies of brain metastasis patients generally are similar to those reported for melanoma patients with CNS involvement. With minor differences, the systems described utilize similar and easily available data to arrive at their risk estimations. RPA has probably been examined in the widest array of clinical trial settings. It also does not seem to be specific to a given treatment modality. Its components are fairly simple to derive from clinical parameters. It will therefore be used for further discussion.



CNS: Central Nervous System; CR: Complete response; DS-GPA: Diagnosis-Specific Graded Prognostic Assessment; ECM: Extracranial metastases; KPS: Karnofsky performance status; LM: Leptomeningeal metastasis; MM-GKR: Malignant Melanoma-Gamma Knife Radiosurgery score; ND: Not defined; NED: No evidence of disease; NR: Not reported; OS: Overall survival; PD: Progressive disease; PI: Prognostic Index; PR: Partial Response; QOL: Quality of life; RPA: Recursive Partitioning Analysis; SD: Stable disease; SIR: Score Index for Radiosurgery

**Table 7.** Risk Stratification of Patients with Brain Metastases from Melanoma

CNS disease progression prior to WBRT, and the presence of meningeal disease. This system is focused on those with extensive disease, not amenable to local therapy with SRS or surgery.

Median survival times predicted by studies of brain metastasis patients generally are similar to those reported for melanoma patients with CNS involvement. With minor differences, the systems described utilize similar and easily available data to arrive at their risk estimations. RPA has probably been examined in the widest array of clinical trial settings. It also does not seem to be specific to a given treatment modality. Its components are fairly simple to derive

> **(all tumor types)**

Class I: 7.1 m Class II: 4.2 m Class III: 2.3 m **Median OS (melanoma)**

Class I: 6.5-10.5 m Class II: 3.5-5.9 m Class III: 1.8-2.5 m

NR Score To determine

138 d 80 d 42 d 18 d 15 d

4 m

5-6 7-8 9-10 11+

≤6 "/>6

Factor 0 1 2 Score Score Point values for

2.9 m 7 m 31 m

4-7 8-10

KPS ≤50 60-70 ≥70 7 m

or NED **Comments References**

Gaspar *et al.,* 1997

Gaspar *et al.,* 2000

[122]

[123] Buchsbaum *et al.,* 2002 [10] Lutterbach *et al.,* 2002 [124] Harrison *et al.,* 2003 [42] Morris *et al.,* 2004

[43]

[35]

[19]

outcome following palliative WBRT.

individual factors are added to derive score. Intended for assessment of outcome after SRS.

2-4 [43]

Radbill *et al.,* 2004

Brown *et al.,* 2002

Morris et al., 2004

Weltman *et al.,* 2000 [129] Selek *et al.,* 2004

[38]

Validated for radiation therapy and surgery.

from clinical parameters. It will therefore be used for further discussion.

**System Prognostic Factors Prognostic Classification Median OS**

Class II: Not Class I or III Class III: KPS<70

disease

308 Melanoma - From Early Detection to Treatment

Class I: KPS≥70, age<65 y., controlled primary disease site, no extra-cranial

Index= Number of ECM sites + (2 x RPA class) + (2 if PD on pre-WBRT imaging) +

Age ≥60 51-59 ≤50 0-3

PD PR/SD CR

"/>13 5-13 <5

(4 if LM present)

Systemic disease status

Largest Lesion Volume (cm<sup>3</sup>)

RPA KPS, age, extracranial disease status, control of primary disease site

PI Number of ECM, RPA class (see above), PD on imaging prior to WBRT, presence of

LM

SIR Age, KPS, extracranial disease status, volume of largest CNS lesion, number of CNS lesions

An important caveat in discussing outcomes estimates derived using these risk stratification systems is that they all were first developed prior to 2011. Prior to that time, reliably effective and proven treatments for advanced melanoma were not available for general clinical use. Development of drugs with proven activity, such as ipilimumab and vemurafenib discussed above, are changing the outlook for melanoma patients. This includes patients with brain metastases. With these drugs, and more being developed with potentially even greater activity, the risk estimates of these systems will certainly change for the better. This is especially likely to be the case in patients with very high risk/poor prognosis disease. Treatment recommen‐ dations for the brain metastasis problem in melanoma will therefore likely be very fluid over the next several years, as new treatment paradigms for melanoma evolve.

For someone with a poor performance status unlikely to live that long, ipilimumab is unlikely to provide benefit, despite preliminary evidence of CNS activity. For these patients, further developments in melanoma therapy are awaited. Pallitative WBRT likely remains the standard

Management of Brain Metastasis in Melanoma Patients

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311

One peculiar circumstance remains: some patients present with RPA class III advanced disease, including brain metastases and poor performance status, but their BRAF mutational status is unknown. Given their overall condition and location of disease, obtaining a tumor specimen to determine BRAF mutation status may not be possible. A wait of 1-2 weeks for results of mutational testing may consume a significant portion of their remaining lifespan. In such patients, standard care would be supportive, potentially with the addition of WBRT. Given a frequency of BRAF mutations targeted by presently available drugs of about 50% and the lack of other proven options, a therapeutic trial of BRAF inhibition is unlikely to cause significant harm, and might lead to dramatic benefit if the patient possesses an appropriate mutation. Again, before embarking on such a treatment course, the patient must be aware of the

Patients of RPA class I have a relatively good prognosis and warrant an aggressive treatment approach. Such patients are young, have a good performance status, and no active extracranial disease. However, among patients with metastatic melanoma, true RPA class I patients are infrequently encountered, especially those completely lacking detectable extracranial disease. The major treatment decision for these patients relates to local therapy of existing brain lesions (Figure 1). The goal would be to treat all evident CNS disease by some form of definitive therapy (surgery or SRS). Traditionally, surgery was favored in cases involving one surgically accessible lesion, and benefit was reported in surgeries targeting up to 3 lesions [14, 133]. Surgery is also especially useful in specific situations where SRS is less favorable, such as large lesion size (> 3cm) or symptomatology (for example, bleeding). Surgery also yields a specimen to confirm the diagnosis and analyze for targetable alterations in the tumor, such as BRAF mutations status. In patients lacking any other evident disease, these data can be very impor‐ tant and only obtainable from a surgically resected CNS specimen. Otherwise, SRS is emerging as the preferred local therapy, both for its simpler administration and possibly for better local control [44]. SRS may also be able to provide definitive treatment at sites inaccessible to surgery. Surgery and SRS are not mutually exclusive: both may be necessary to provide

SRS has been a remarkable addition to our armamentarium for treatment of brain metastases. Previously, surgery was the only approach to definitive treatment. If more than 3 lesions were present, or they were located in surgically inaccessible locations, surgery could not be used with the intention of long-term control. SRS allows treatment of multiple lesions, in sites inaccessible to surgery. It also offers the possibility of re-treatment. At some point, presumably, the number of lesions exceeds the ability of SRS to control the disease. The exact number is not defined, but some have advocated SRS to control up to five CNS lesions [37]. Beyond this, it may be unreasonable to expect a local treatment modality like SRS to control what is clinically

definitive treatment of all lesions in multi-focal metastatic CNS disease.

therapy for these patients.

limitations of our current dataset.

**4.2. Favorable/good risk**

## **4. Therapy of CNS disease**

## **4.1. Unfavorable/poor risk**

By definition, RPA class III patients have a KPS less than 70%. Often, they have multi-focal brain metastases, active extracranial disease, or both. Historically, their life expectancy was very limited. The PI prognostic system, intended to assess prognosis in this group as described above, uses days rather than months as the unit of time for its estimates [43].

Conventionally, surgery or SRS would only be used judiciously with palliative intent and welldefined goals. WBRT may be undertaken for symptom palliation and a very modest survival benefit [8, 12, 86, 130-132]. The anticipated duration of survival played an important part in designing any treatment approach, as even therapy of several weeks duration could consume a significant proportion of a patient's remaining lifespan. The burden of coming to repeated treatments (as might be the case with palliative WBRT, frequently administered as 10 treat‐ ments over 2 weeks) may lead to a significant QOL decrement in patients with poor perform‐ ance status. By definition, RPA class III patients have such a poor performance status.

Prior to the approval in the United States of ipilimumab and vemurafenib in 2011, systemic therapy played a minimal role in this group. Exceptions included steroid therapy for tumorassociated edema and anti-convulsants for seizures. The low performance status and CNS disease in these patients excluded them from virtually all clinical trials. Activity of systemic agents with CNS penetration, such as temozolmide and fotemustine, was limited, with an onset of action too slow to benefit most patients with melanoma who were in this category.

As of 2011, BRAF mutational status serves as an important factor in making treatment decisions. This may be especially important in patients with RPA class III melanoma with CNS involvement. The BRAF inhibitors vemurafenib and dabrafenib, discussed above, have rapid onset of action, high response rates, preliminary evidence of CNS activity, oral administration and manageable toxicity profiles. As of November 2012, vemurafenib is approved in the United States and Europe, and debrafanib's approval is pending. For patients possessing an appro‐ priate BRAF mutation, treatment with one of these agents would be reasonable to consider, even with RPA class III. Of course, the patient must be aware that information about this drug in the brain metastasis population is presently very limited. Data regarding combinations with radiotherapy is also very limited at this time. While a clinical trial would be the preferred setting to treat these patients, use of BRAF inhibition therapy would be reasonable to offer to BRAF-mutant melanoma patients with brain metastases and RPA class III.

For patients in whom a targetable BRAF mutation is not present, fewer options are available. Ipilimumab, discussed above, has a relatively slow onset of action, taking 3-4 months in phase 3 trials to confer a survival benefit versus controls [90, 91], with an overall low response rate. For someone with a poor performance status unlikely to live that long, ipilimumab is unlikely to provide benefit, despite preliminary evidence of CNS activity. For these patients, further developments in melanoma therapy are awaited. Pallitative WBRT likely remains the standard therapy for these patients.

One peculiar circumstance remains: some patients present with RPA class III advanced disease, including brain metastases and poor performance status, but their BRAF mutational status is unknown. Given their overall condition and location of disease, obtaining a tumor specimen to determine BRAF mutation status may not be possible. A wait of 1-2 weeks for results of mutational testing may consume a significant portion of their remaining lifespan. In such patients, standard care would be supportive, potentially with the addition of WBRT. Given a frequency of BRAF mutations targeted by presently available drugs of about 50% and the lack of other proven options, a therapeutic trial of BRAF inhibition is unlikely to cause significant harm, and might lead to dramatic benefit if the patient possesses an appropriate mutation. Again, before embarking on such a treatment course, the patient must be aware of the limitations of our current dataset.

## **4.2. Favorable/good risk**

to be the case in patients with very high risk/poor prognosis disease. Treatment recommen‐ dations for the brain metastasis problem in melanoma will therefore likely be very fluid over

By definition, RPA class III patients have a KPS less than 70%. Often, they have multi-focal brain metastases, active extracranial disease, or both. Historically, their life expectancy was very limited. The PI prognostic system, intended to assess prognosis in this group as described

Conventionally, surgery or SRS would only be used judiciously with palliative intent and welldefined goals. WBRT may be undertaken for symptom palliation and a very modest survival benefit [8, 12, 86, 130-132]. The anticipated duration of survival played an important part in designing any treatment approach, as even therapy of several weeks duration could consume a significant proportion of a patient's remaining lifespan. The burden of coming to repeated treatments (as might be the case with palliative WBRT, frequently administered as 10 treat‐ ments over 2 weeks) may lead to a significant QOL decrement in patients with poor perform‐ ance status. By definition, RPA class III patients have such a poor performance status.

Prior to the approval in the United States of ipilimumab and vemurafenib in 2011, systemic therapy played a minimal role in this group. Exceptions included steroid therapy for tumorassociated edema and anti-convulsants for seizures. The low performance status and CNS disease in these patients excluded them from virtually all clinical trials. Activity of systemic agents with CNS penetration, such as temozolmide and fotemustine, was limited, with an onset of action too slow to benefit most patients with melanoma who were in this category. As of 2011, BRAF mutational status serves as an important factor in making treatment decisions. This may be especially important in patients with RPA class III melanoma with CNS involvement. The BRAF inhibitors vemurafenib and dabrafenib, discussed above, have rapid onset of action, high response rates, preliminary evidence of CNS activity, oral administration and manageable toxicity profiles. As of November 2012, vemurafenib is approved in the United States and Europe, and debrafanib's approval is pending. For patients possessing an appro‐ priate BRAF mutation, treatment with one of these agents would be reasonable to consider, even with RPA class III. Of course, the patient must be aware that information about this drug in the brain metastasis population is presently very limited. Data regarding combinations with radiotherapy is also very limited at this time. While a clinical trial would be the preferred setting to treat these patients, use of BRAF inhibition therapy would be reasonable to offer to

the next several years, as new treatment paradigms for melanoma evolve.

above, uses days rather than months as the unit of time for its estimates [43].

BRAF-mutant melanoma patients with brain metastases and RPA class III.

For patients in whom a targetable BRAF mutation is not present, fewer options are available. Ipilimumab, discussed above, has a relatively slow onset of action, taking 3-4 months in phase 3 trials to confer a survival benefit versus controls [90, 91], with an overall low response rate.

**4. Therapy of CNS disease**

310 Melanoma - From Early Detection to Treatment

**4.1. Unfavorable/poor risk**

Patients of RPA class I have a relatively good prognosis and warrant an aggressive treatment approach. Such patients are young, have a good performance status, and no active extracranial disease. However, among patients with metastatic melanoma, true RPA class I patients are infrequently encountered, especially those completely lacking detectable extracranial disease.

The major treatment decision for these patients relates to local therapy of existing brain lesions (Figure 1). The goal would be to treat all evident CNS disease by some form of definitive therapy (surgery or SRS). Traditionally, surgery was favored in cases involving one surgically accessible lesion, and benefit was reported in surgeries targeting up to 3 lesions [14, 133]. Surgery is also especially useful in specific situations where SRS is less favorable, such as large lesion size (> 3cm) or symptomatology (for example, bleeding). Surgery also yields a specimen to confirm the diagnosis and analyze for targetable alterations in the tumor, such as BRAF mutations status. In patients lacking any other evident disease, these data can be very impor‐ tant and only obtainable from a surgically resected CNS specimen. Otherwise, SRS is emerging as the preferred local therapy, both for its simpler administration and possibly for better local control [44]. SRS may also be able to provide definitive treatment at sites inaccessible to surgery. Surgery and SRS are not mutually exclusive: both may be necessary to provide definitive treatment of all lesions in multi-focal metastatic CNS disease.

SRS has been a remarkable addition to our armamentarium for treatment of brain metastases. Previously, surgery was the only approach to definitive treatment. If more than 3 lesions were present, or they were located in surgically inaccessible locations, surgery could not be used with the intention of long-term control. SRS allows treatment of multiple lesions, in sites inaccessible to surgery. It also offers the possibility of re-treatment. At some point, presumably, the number of lesions exceeds the ability of SRS to control the disease. The exact number is not defined, but some have advocated SRS to control up to five CNS lesions [37]. Beyond this, it may be unreasonable to expect a local treatment modality like SRS to control what is clinically widespread involvement in an organ system, even if limited to the CNS. Surgery and SRS may be able to control specific lesions that are symptomatic in such patients, but the overall treatment approach relies primarily on therapeutic WBRT and systemic therapy, discussed below under "Intermediate Risk."

to treat extracranial disease, an important prognostic factor once CNS disease was controlled.

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313

Newly developed agents, such as vemurafenib and ipilimumab, may have a role defined in the future for adjuvant therapy in patients with RPA class I CNS disease from melanoma. They may be able to affect both CNS recurrence rates and progression of extracranial disease. However, data supporting such use is not available at present. Their use as adjuvant therapies

**Figure 1.** Treatment of melanoma patients with brain metastases with favorable risk profile, equivalent to RPA class I. In patients with more than one brain lesion who are to receive local therapy, it may be necessary to use both surgery and radiosurgery to treat all lesions. By definition, RPA class I patients have no active extracranial disease. Thus, sys‐ temic therapy is not indicated except in an experimental trial. CNS: central nervous system; KPS: Karnofsky perform‐ ance status; RPA: Recursive Partitioning Analysis; SRS: stereotactic radiosurgery; WBRT: whole brain radiotherapy.

Treatment decisions in intermediate risk patients (equivalent to RPA class II) are probably most difficult of all (Figure 2). This relates to their variable clinical presentation. They have better performance status than those with unfavorable RPA class III. They are also of advanced age (according to RPA, anyone older than 65 years), have active extracranial disease, or both, conveying a negative prognosis relative to RPA class I. A logical way to divide this population is into those with CNS disease amenable to local definitive therapy and those with CNS disease

too extensive for complete, definitive local therapy of all lesions.

**4.3. Intermediate risk**

By definition, true RPA class I patients have no active extracranial disease.

in this population is not warranted, outside the setting of a clinical trial.

In patients with RPA class I disease from melanoma, risk of failure in the CNS is high if treatment focuses solely on radiologically evident disease. This likely reflects the underlying biology, in which specific neurotropic sub-clones of melanoma develop that colonize the CNS, leading to brain metastases. Limiting treatment to surgery and/or SRS of only radiologically evident lesions ignores this biological reality. This observation is confirmed in the multiple randomized trials of adjuvant WBRT enrolling patients with multiple tumor types, including melanoma. Adjuvant WBRT decreases intracranial recurrence rates when combined with definitive local therapy. This effect is evident at both definitively treated macroscopic sites (treating residual contamination) and at distant sites within the CNS. At distant sites, adjuvant WBRT must accomplish this by either treating pre-existing radiologically undetectable micrometastatic disease or making the CNS less receptive to colonization from extracranial sites. The former is the more plausible biological explanation.

Approaches to address the problem of distant CNS recurrence have been discussed in detail earlier. Basically, these come down to either administering adjuvant WBRT up-front, or using an expectant management strategy, with regular imaging and re-treatment (primarily with SRS), at the time of CNS progression. Arguments against adjuvant WBRT include concern regarding its cognitive toxicity, its lack of clear survival benefit and inability to undertake retreatment. As described earlier, cognitive effects of adjuvant WBRT, while not absent, are not unreasonable in the setting of CNS metastases, especially when balanced against the cognitive effects of tumor progression and those of re-treatment (as, for example, by SRS). Adjuvant WBRT is unlikely to be associated, in general, with an overall survival benefit overall due to extracranial disease as a competing cause of death. In the setting of RPA class I patients, who lack active extracranial disease, adjuvant WBRT may very well have a survival benefit [54, 134].

As noted above, the use of a salvage strategy, relying on SRS in the event of tumor progression, is associated with high rates of intracranial failure. The cognitive effects of such a strategy have not been assessed in detail, but the data regarding cognitive effects of allowing tumor pro‐ gression have been reviewed and are clearly unfavorable. Whether the effects are better or worse than those due to adjuvant use of WBRT can only be answered by a randomized comparison of the two strategies.

We concede that the decision regarding use of adjuvant WBRT is not simple and clear-cut. To determine whether to recommend its use, the benefit of decreased intracranial progression rates must be balanced against its adverse effects. Overall, we believe that the published evidence generally supports the use of adjuvant WBRT in melanoma patients.

Systemic adjuvant therapy might be an alternative to adjuvant WBRT. Traditional cytotoxic therapy agents with known CNS activity, such as fotemustine or temozolomide, have not been shown clearly to impact the subsequent development of CNS disease in melanoma patients [113, 135]. In the setting of melanoma patients with CNS disease, their primary purpose was to treat extracranial disease, an important prognostic factor once CNS disease was controlled. By definition, true RPA class I patients have no active extracranial disease.

Newly developed agents, such as vemurafenib and ipilimumab, may have a role defined in the future for adjuvant therapy in patients with RPA class I CNS disease from melanoma. They may be able to affect both CNS recurrence rates and progression of extracranial disease. However, data supporting such use is not available at present. Their use as adjuvant therapies in this population is not warranted, outside the setting of a clinical trial.

**Figure 1.** Treatment of melanoma patients with brain metastases with favorable risk profile, equivalent to RPA class I. In patients with more than one brain lesion who are to receive local therapy, it may be necessary to use both surgery and radiosurgery to treat all lesions. By definition, RPA class I patients have no active extracranial disease. Thus, sys‐ temic therapy is not indicated except in an experimental trial. CNS: central nervous system; KPS: Karnofsky perform‐ ance status; RPA: Recursive Partitioning Analysis; SRS: stereotactic radiosurgery; WBRT: whole brain radiotherapy.

#### **4.3. Intermediate risk**

widespread involvement in an organ system, even if limited to the CNS. Surgery and SRS may be able to control specific lesions that are symptomatic in such patients, but the overall treatment approach relies primarily on therapeutic WBRT and systemic therapy, discussed

In patients with RPA class I disease from melanoma, risk of failure in the CNS is high if treatment focuses solely on radiologically evident disease. This likely reflects the underlying biology, in which specific neurotropic sub-clones of melanoma develop that colonize the CNS, leading to brain metastases. Limiting treatment to surgery and/or SRS of only radiologically evident lesions ignores this biological reality. This observation is confirmed in the multiple randomized trials of adjuvant WBRT enrolling patients with multiple tumor types, including melanoma. Adjuvant WBRT decreases intracranial recurrence rates when combined with definitive local therapy. This effect is evident at both definitively treated macroscopic sites (treating residual contamination) and at distant sites within the CNS. At distant sites, adjuvant WBRT must accomplish this by either treating pre-existing radiologically undetectable micrometastatic disease or making the CNS less receptive to colonization from extracranial

Approaches to address the problem of distant CNS recurrence have been discussed in detail earlier. Basically, these come down to either administering adjuvant WBRT up-front, or using an expectant management strategy, with regular imaging and re-treatment (primarily with SRS), at the time of CNS progression. Arguments against adjuvant WBRT include concern regarding its cognitive toxicity, its lack of clear survival benefit and inability to undertake retreatment. As described earlier, cognitive effects of adjuvant WBRT, while not absent, are not unreasonable in the setting of CNS metastases, especially when balanced against the cognitive effects of tumor progression and those of re-treatment (as, for example, by SRS). Adjuvant WBRT is unlikely to be associated, in general, with an overall survival benefit overall due to extracranial disease as a competing cause of death. In the setting of RPA class I patients, who lack active extracranial disease, adjuvant WBRT may very well have a survival benefit [54, 134].

As noted above, the use of a salvage strategy, relying on SRS in the event of tumor progression, is associated with high rates of intracranial failure. The cognitive effects of such a strategy have not been assessed in detail, but the data regarding cognitive effects of allowing tumor pro‐ gression have been reviewed and are clearly unfavorable. Whether the effects are better or worse than those due to adjuvant use of WBRT can only be answered by a randomized

We concede that the decision regarding use of adjuvant WBRT is not simple and clear-cut. To determine whether to recommend its use, the benefit of decreased intracranial progression rates must be balanced against its adverse effects. Overall, we believe that the published

Systemic adjuvant therapy might be an alternative to adjuvant WBRT. Traditional cytotoxic therapy agents with known CNS activity, such as fotemustine or temozolomide, have not been shown clearly to impact the subsequent development of CNS disease in melanoma patients [113, 135]. In the setting of melanoma patients with CNS disease, their primary purpose was

evidence generally supports the use of adjuvant WBRT in melanoma patients.

below under "Intermediate Risk."

312 Melanoma - From Early Detection to Treatment

comparison of the two strategies.

sites. The former is the more plausible biological explanation.

Treatment decisions in intermediate risk patients (equivalent to RPA class II) are probably most difficult of all (Figure 2). This relates to their variable clinical presentation. They have better performance status than those with unfavorable RPA class III. They are also of advanced age (according to RPA, anyone older than 65 years), have active extracranial disease, or both, conveying a negative prognosis relative to RPA class I. A logical way to divide this population is into those with CNS disease amenable to local definitive therapy and those with CNS disease too extensive for complete, definitive local therapy of all lesions.

Considerations regarding the use of adjuvant WBRT and the desirability for treatment in the context of a clinical trial are essentially the same as for favorable prognosis patients. The key differentiating question is whether local therapy of CNS lesions with surgery or SRS should be attempted at all. Several clinical situations argue against their use. Lesions may be inacces‐ sible for surgery or too large for SRS or they may simply be too numerous. If definitive treatment of all CNS disease sites is possible, then it should be attempted. If CNS disease is not amenable to local therapy of all lesions, then treatment must rely on therapeutic WBRT and systemic therapy, with surgery or SRS reserved for large or symptomatic lesions.

Europe). Although these drugs treated extracranial disease as well, they were not highly ac‐ tive in either the CNS or extracranial compartments, and did not demonstrate clear or dra‐

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315

Systemic therapy of melanoma in RPA class II patients is appearing brighter than it has in the past. Clinical trials are opening which permit these patients to enroll, and highly active agents, with CNS activity moreover, such as vemurafenib are available non-experimentally for patients with BRAF mutations. For patients lacking BRAF mutations, treatment with ipilimu‐ mab is a reasonable consideration, due to its survival benefit in a phase 3 trial, which included patients with pre-existing, treated brain metastases [90]. Most of these patients will live long enough to derive benefit from ipilimumab. As new agents are developed, their use in RPA class II melanoma patients is justified, even without demonstrable CNS activity, due to the active extracranial disease present in most of this population as a competing cause of death.

Brain metastasis is part of the natural history of metastatic melanoma. It is a common problem and has a major adverse impact on treatment outcomes and QOL. The bulk of melanomaspecific research consists of retrospective analyses and single-center studies. Prospectively validated, comprehensive treatment paradigms do not yet exist. The preceding discussion

Optimal use of SRS technology remains undefined. One question relates to the treatment of multiplelesions.TheonlymajorrandomizedtrialofSRSinbrainmetastasistherapydemonstrat‐ ed a survival benefit in the presence of only a single brain metastasis [15]. No prospective data supportasurvivalbenefitfromSRSwhenmorethanonelesionispresent;retrospectivedatafrom several sources indicate that multiple CNS lesions are associated with worse survival [22, 26, 35].

At some point, the absolute number of CNS lesions poses a barrier to effective SRS therapy. Some argue that the presence of multiple lesions (up to about 5) should not preclude therapy, based on results indicating that the number of CNS melanoma lesions did not predict subse‐ quent survival [39, 136]. Whether some threshold number of lesions exists is an unanswered

SRS itself is a generic term for a rapidly evolving technology. The relevance of even recently published results to current treatment technologies may be questioned. What is unlikely to change is the local nature of SRS therapy: SRS treats the radiated region, but not that which is unradiated. As discussed extensively, concurrent micrometastatic disease is not addressed by SRS, as it is also not by surgery. The use of adjuvant therapy after local treatment with surgery or SRS lacks melanoma-specific prospective data. Five randomized trials, described above, indicate that adjuvant WBRT can decrease intracranial recurrence rates, both at sites treated with surgery/SRS and at untreated sites. The adverse neurocognitive effects of WBRT and the efficacy of this modality in the metastatic melanoma population are valid questions. As noted

matic survival benefits.

**5. Directions for the future**

question appropriate for investigation.

above, such a study is in progress (NCT01503827) [55].

suggests some important research questions for the future.

**Figure 2.** Treatment of melanoma patients with brain metastases with intermediate prognosis, equivalent to RPA class II. In patients with more than one brain lesion who are to receive local therapy, it may be necessary to use both surgery and radio‐ surgery to treat all lesions. Patients with active extracranial disease should be considered for systemic therapy. While in the past, few active systemic agents were available, newer therapies might have relevance early in the treatment of intermedi‐ ate prognosis patients. For example, BRAF inhibition with vemurafenib has rapid onset of control, high response rates, and even preliminary evidence of CNS activity. Thus, early use of systemic therapy might be able to impact both CNS disease and extracranial disease. Extracranial disease activity is a consistent adverse prognostic factor in intermediate patients, once CNS disease is controlled. CNS: central nervous system; KPS: Karnofsky performance status; RPA: Recursive Partitioning Analysis; SRS: stereotactic radiosurgery; WBRT: whole brain radiotherapy.

In RPA class II patients with CNS melanoma, active extracranial disease status is a key prog‐ nostic factor. Disease outside the CNS represents a competing cause of death. Before the ap‐ proval in 2011-2012 of agents with proven anti-melanoma activity (such as vemurafenib and ipilimumab), systemic therapy of the CNS was limited to temozolomide or fotemustine (in Europe). Although these drugs treated extracranial disease as well, they were not highly ac‐ tive in either the CNS or extracranial compartments, and did not demonstrate clear or dra‐ matic survival benefits.

Systemic therapy of melanoma in RPA class II patients is appearing brighter than it has in the past. Clinical trials are opening which permit these patients to enroll, and highly active agents, with CNS activity moreover, such as vemurafenib are available non-experimentally for patients with BRAF mutations. For patients lacking BRAF mutations, treatment with ipilimu‐ mab is a reasonable consideration, due to its survival benefit in a phase 3 trial, which included patients with pre-existing, treated brain metastases [90]. Most of these patients will live long enough to derive benefit from ipilimumab. As new agents are developed, their use in RPA class II melanoma patients is justified, even without demonstrable CNS activity, due to the active extracranial disease present in most of this population as a competing cause of death.

## **5. Directions for the future**

Considerations regarding the use of adjuvant WBRT and the desirability for treatment in the context of a clinical trial are essentially the same as for favorable prognosis patients. The key differentiating question is whether local therapy of CNS lesions with surgery or SRS should be attempted at all. Several clinical situations argue against their use. Lesions may be inacces‐ sible for surgery or too large for SRS or they may simply be too numerous. If definitive treatment of all CNS disease sites is possible, then it should be attempted. If CNS disease is not amenable to local therapy of all lesions, then treatment must rely on therapeutic WBRT

314 Melanoma - From Early Detection to Treatment

and systemic therapy, with surgery or SRS reserved for large or symptomatic lesions.

**Figure 2.** Treatment of melanoma patients with brain metastases with intermediate prognosis, equivalent to RPA class II. In patients with more than one brain lesion who are to receive local therapy, it may be necessary to use both surgery and radio‐ surgery to treat all lesions. Patients with active extracranial disease should be considered for systemic therapy. While in the past, few active systemic agents were available, newer therapies might have relevance early in the treatment of intermedi‐ ate prognosis patients. For example, BRAF inhibition with vemurafenib has rapid onset of control, high response rates, and even preliminary evidence of CNS activity. Thus, early use of systemic therapy might be able to impact both CNS disease and extracranial disease. Extracranial disease activity is a consistent adverse prognostic factor in intermediate patients, once CNS disease is controlled. CNS: central nervous system; KPS: Karnofsky performance status; RPA: Recursive Partitioning

In RPA class II patients with CNS melanoma, active extracranial disease status is a key prog‐ nostic factor. Disease outside the CNS represents a competing cause of death. Before the ap‐ proval in 2011-2012 of agents with proven anti-melanoma activity (such as vemurafenib and ipilimumab), systemic therapy of the CNS was limited to temozolomide or fotemustine (in

Analysis; SRS: stereotactic radiosurgery; WBRT: whole brain radiotherapy.

Brain metastasis is part of the natural history of metastatic melanoma. It is a common problem and has a major adverse impact on treatment outcomes and QOL. The bulk of melanomaspecific research consists of retrospective analyses and single-center studies. Prospectively validated, comprehensive treatment paradigms do not yet exist. The preceding discussion suggests some important research questions for the future.

Optimal use of SRS technology remains undefined. One question relates to the treatment of multiplelesions.TheonlymajorrandomizedtrialofSRSinbrainmetastasistherapydemonstrat‐ ed a survival benefit in the presence of only a single brain metastasis [15]. No prospective data supportasurvivalbenefitfromSRSwhenmorethanonelesionispresent;retrospectivedatafrom several sources indicate that multiple CNS lesions are associated with worse survival [22, 26, 35].

At some point, the absolute number of CNS lesions poses a barrier to effective SRS therapy. Some argue that the presence of multiple lesions (up to about 5) should not preclude therapy, based on results indicating that the number of CNS melanoma lesions did not predict subse‐ quent survival [39, 136]. Whether some threshold number of lesions exists is an unanswered question appropriate for investigation.

SRS itself is a generic term for a rapidly evolving technology. The relevance of even recently published results to current treatment technologies may be questioned. What is unlikely to change is the local nature of SRS therapy: SRS treats the radiated region, but not that which is unradiated. As discussed extensively, concurrent micrometastatic disease is not addressed by SRS, as it is also not by surgery. The use of adjuvant therapy after local treatment with surgery or SRS lacks melanoma-specific prospective data. Five randomized trials, described above, indicate that adjuvant WBRT can decrease intracranial recurrence rates, both at sites treated with surgery/SRS and at untreated sites. The adverse neurocognitive effects of WBRT and the efficacy of this modality in the metastatic melanoma population are valid questions. As noted above, such a study is in progress (NCT01503827) [55].

An alternative to the use of adjuvant WBRT is a planned radiosurgical salvage strategy. This presumably minimizes exposure of the CNS to WBRT and its adverse effects. Little data is available regarding this treatment approach. A randomized clinical trial would be most helpful, in which patients are randomized to receive immediate adjuvant WBRT after SRS therapy or undergo planned SRS salvage treatments, with WBRT only when SRS is not possible. This study would provide data to balance the neurocognitive consequences of immediate WBRT with those due to an increased rate of later macroscopic CNS progression. Further, some estimate of the neurocognitive cost of SRS re-treatment would be obtained.

from other conditions or is based on retrospective analyses from individual centers. Data from well-designed, prospective trials is lacking in many regards. This deficiency has been noted at least eight years previously by others [139]. At present, many of the same questions posed by those workers remain unanswered. Fortunately, melanoma treatment itself has not remained static, with new agents generating new questions regarding optimal treatment of the condition.

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317

Well-designed, rigorous trials will allow our patients to receive the best and most cost-effective treatments available. Melanoma patients with brain metastases can look forward to a brighter future. We must, however, demand rigorous investigations to allow the best use possible of

BS-BM: Basic Score for Brain Metastases; CNS: Central Nervous System; CR: Complete Response; CT: Computed Tomography; DS-GPA: Diagnosis-Specific Graded Prognostic Assessment; Gy: Grey, unit of radiation dose; HVLT: Hopkin's Verbal Learning Test; KPS: Karnofsky Performance Status; MM-GKR: Malignant Melanoma-Gamma Knife Radiosurgery; MMSE: Mini-Mental Status Examination; MRI: Magnetic Resonance Imaging; NCF: Neuro‐ cognitive Function; NSCLC: Non Small Cell Lung Cancer; OIRR: Overall Intracranial Response Rate; ORR: Objective Response Rate; OS: Overall Survival; QOL: Quality-of-Life; PCI: Pro‐ phylactic Cranial Irradiation; PFS: Progression-Free Survival; PI: Prognostic Index; PR: Partial Response; RPA: Recursive Partitioning Analysis; RTOG: Radiation Therapy Oncology Group; SCLC: Small Cell Lung Cancer; SD: Stable Disease; SIR: Score Index for Radiosurgery; SRS: Stereotactic Radiosurgery; TCI: Therapeutic Cranial Irradiation; WBRT: Whole Brain Radia‐

, Evan M. Hersh1

1 Section of Hematology and Oncology, Melanoma/Sarcoma Program, University of Arizo‐

2 Department of Radiation Oncology, College of Medicine, University of Arizona, USA

**Conflict of Interest:** The authors would like to disclose the following conflicts of interest:

Evan Hersh: GlaxoSmithKline, Bristol-Meyers Squibb, Pfizer, Genentech/Roche, Celgene.

, Sun K. Yi2

and Lee D. Cranmer\*1\*

the arsenal being placed at our disposal to treat this challenging problem.

**Abbreviations**

tion Therapy

**Author details**

Sherif S. Morgan\*1

, Joanne M. Jeter1

na Cancer Center, University of Arizona, USA

\*Both of these authors contributed equally.

Sherif Morgan: None to disclose.

\*Address all correspondence to: lcranmer@azcc.arizona.edu

SRS itself is used for adjuvant purposes after surgical metastectomy to treat residual disease at the resection site. The efficacy of this has not been defined. Additionally, such therapy does not treat occult disease at other sites within the CNS. A randomized trial comparing the efficacy of adjuvant SRS to either no adjuvant therapy or to adjuvant WBRT would be appropriate.

Finally, and perhaps most significantly, systemic therapy of melanoma is evolving rapidly, and those advances will have a major impact on treatment of CNS disease. Even now, convincing preliminary evidence of CNS activity of these several new agents has been presented. Previous‐ ly, melanoma patients with CNS disease were excluded from clinical trials in the belief that the blood-brain barrier posed to great a hurdle to clinical efficacy. This no longer appears to be a valid assumption. As new agents emerge, their activity in the CNS should either be addressed in CNSspecific trials, or patients with CNS melanoma should be considered similar to any other melanoma patient, so long as their CNS disease is minimally or asymptomatic.

Much of this review has focused on the controversy of adjuvant therapy in the CNS. Adjuvant WBRT is not an optimal solution to this problem. It does not prevent CNS re-seeding from extracranial sites and cannot be used repeatedly. Adverse cognitive effects of WBRT are clearly demonstrable, even if their clinical impact is arguable. Critically, adjuvant WBRT also does not address the problem of extracranial disease, a major prognostic factor. Optimal adjuvant therapy to address these limitations is likely to be systemic. The development of highly active agents with CNS penetration opens the possibility of their use in melanoma patients after definitive treatment of brain metastases.

Several prior studies can provide necessary baseline data regarding rates of CNS progression for sample size calculations [113, 135]. Neurocognitive effects must be a secondary endpoint in any study, as it cannot be assumed that systemic agents are devoid of adverse neurocognitive effects. For example, case reports of melanoma patients treated with fotemustine reported toxic leukoencephalopathy with progressive dementia in several patients, [137, 138].

## **6. Conclusions**

Brain metastasis is a frequent and serious problem for melanoma patients. New technologies, such as SRS and agents, such as vemurafenib and ipilimumab, are expanding our ability to treat this condition. Melanoma-specific studies guiding optimal employment of new technol‐ ogies are limited. Most information regarding CNS treatment in melanoma is extrapolated from other conditions or is based on retrospective analyses from individual centers. Data from well-designed, prospective trials is lacking in many regards. This deficiency has been noted at least eight years previously by others [139]. At present, many of the same questions posed by those workers remain unanswered. Fortunately, melanoma treatment itself has not remained static, with new agents generating new questions regarding optimal treatment of the condition.

Well-designed, rigorous trials will allow our patients to receive the best and most cost-effective treatments available. Melanoma patients with brain metastases can look forward to a brighter future. We must, however, demand rigorous investigations to allow the best use possible of the arsenal being placed at our disposal to treat this challenging problem.

## **Abbreviations**

An alternative to the use of adjuvant WBRT is a planned radiosurgical salvage strategy. This presumably minimizes exposure of the CNS to WBRT and its adverse effects. Little data is available regarding this treatment approach. A randomized clinical trial would be most helpful, in which patients are randomized to receive immediate adjuvant WBRT after SRS therapy or undergo planned SRS salvage treatments, with WBRT only when SRS is not possible. This study would provide data to balance the neurocognitive consequences of immediate WBRT with those due to an increased rate of later macroscopic CNS progression. Further, some estimate of the neurocognitive cost of SRS re-treatment would be obtained.

SRS itself is used for adjuvant purposes after surgical metastectomy to treat residual disease at the resection site. The efficacy of this has not been defined. Additionally, such therapy does not treat occult disease at other sites within the CNS. A randomized trial comparing the efficacy of adjuvant SRS to either no adjuvant therapy or to adjuvant WBRT would be appropriate.

Finally, and perhaps most significantly, systemic therapy of melanoma is evolving rapidly, and those advances will have a major impact on treatment of CNS disease. Even now, convincing preliminary evidence of CNS activity of these several new agents has been presented. Previous‐ ly, melanoma patients with CNS disease were excluded from clinical trials in the belief that the blood-brain barrier posed to great a hurdle to clinical efficacy. This no longer appears to be a valid assumption. As new agents emerge, their activity in the CNS should either be addressed in CNSspecific trials, or patients with CNS melanoma should be considered similar to any other

Much of this review has focused on the controversy of adjuvant therapy in the CNS. Adjuvant WBRT is not an optimal solution to this problem. It does not prevent CNS re-seeding from extracranial sites and cannot be used repeatedly. Adverse cognitive effects of WBRT are clearly demonstrable, even if their clinical impact is arguable. Critically, adjuvant WBRT also does not address the problem of extracranial disease, a major prognostic factor. Optimal adjuvant therapy to address these limitations is likely to be systemic. The development of highly active agents with CNS penetration opens the possibility of their use in melanoma patients after

Several prior studies can provide necessary baseline data regarding rates of CNS progression for sample size calculations [113, 135]. Neurocognitive effects must be a secondary endpoint in any study, as it cannot be assumed that systemic agents are devoid of adverse neurocognitive effects. For example, case reports of melanoma patients treated with fotemustine reported toxic

Brain metastasis is a frequent and serious problem for melanoma patients. New technologies, such as SRS and agents, such as vemurafenib and ipilimumab, are expanding our ability to treat this condition. Melanoma-specific studies guiding optimal employment of new technol‐ ogies are limited. Most information regarding CNS treatment in melanoma is extrapolated

leukoencephalopathy with progressive dementia in several patients, [137, 138].

melanoma patient, so long as their CNS disease is minimally or asymptomatic.

definitive treatment of brain metastases.

316 Melanoma - From Early Detection to Treatment

**6. Conclusions**

BS-BM: Basic Score for Brain Metastases; CNS: Central Nervous System; CR: Complete Response; CT: Computed Tomography; DS-GPA: Diagnosis-Specific Graded Prognostic Assessment; Gy: Grey, unit of radiation dose; HVLT: Hopkin's Verbal Learning Test; KPS: Karnofsky Performance Status; MM-GKR: Malignant Melanoma-Gamma Knife Radiosurgery; MMSE: Mini-Mental Status Examination; MRI: Magnetic Resonance Imaging; NCF: Neuro‐ cognitive Function; NSCLC: Non Small Cell Lung Cancer; OIRR: Overall Intracranial Response Rate; ORR: Objective Response Rate; OS: Overall Survival; QOL: Quality-of-Life; PCI: Pro‐ phylactic Cranial Irradiation; PFS: Progression-Free Survival; PI: Prognostic Index; PR: Partial Response; RPA: Recursive Partitioning Analysis; RTOG: Radiation Therapy Oncology Group; SCLC: Small Cell Lung Cancer; SD: Stable Disease; SIR: Score Index for Radiosurgery; SRS: Stereotactic Radiosurgery; TCI: Therapeutic Cranial Irradiation; WBRT: Whole Brain Radia‐ tion Therapy

## **Author details**

Sherif S. Morgan\*1 , Joanne M. Jeter1 , Evan M. Hersh1 , Sun K. Yi2 and Lee D. Cranmer\*1\*

\*Address all correspondence to: lcranmer@azcc.arizona.edu

1 Section of Hematology and Oncology, Melanoma/Sarcoma Program, University of Arizo‐ na Cancer Center, University of Arizona, USA

2 Department of Radiation Oncology, College of Medicine, University of Arizona, USA

\*Both of these authors contributed equally.

**Conflict of Interest:** The authors would like to disclose the following conflicts of interest: Sherif Morgan: None to disclose.

Evan Hersh: GlaxoSmithKline, Bristol-Meyers Squibb, Pfizer, Genentech/Roche, Celgene.

Joanne Jeter: None to disclose.

Sun K Yi: None to disclose.

Lee Cranmer: Bristol-Meyers Squibb, Merck, Genentech/Roche, Celgene, Prometheus Labo‐ ratories.

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319

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**Chapter 12**

**Surgical Treatment of Nevi and Melanoma in the**

Surgical care of children affected by melanocytic lesions is a complex area of pediatric surgery, where psychosocial aspects, involving parents and children of different ages, overlap with oncologic implications. In this challenging field results may be frustrating despite knowledge

Due to its rarity, the occurrence of malignant melanoma in children may be underestimated by involved professionals. Melanoma in children is rare, but it does exist and every effort should be done to assure proper treatment, which should be guaranteed in a pediatric tertiary

The role of dermatologists is essential to achieve proper selection of indications to surgery, by clinical follow up and dermatoscopy techniques. On the other hand surgeon is expected to share a profound knowledge of indications and techniques of treatment. In fact, the awareness that simple excision of a nevus may represent the first and most important therapeutic intervention of a MM, before knowing the definitive diagnosis, is sometimes lacking even among pediatric care professionals and the occasional occurrence of a MM diagnosis may be

Decisions about surgical treatment of congenital giant nevi in some cases may need psycho‐ logical assessment because of complex relationships between patient's and parents' awareness

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

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

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

**Pediatric Age**

Martino Ascanio

**1. Introduction**

and experience.

and willingness.

http://dx.doi.org/10.5772/54632

Andrea Zangari, Federico Zangari,

care center by a multi-specialist approach.

confounding and cause of an incomplete care strategy.

Mercedes Romano, Elisabetta Cerigioni,

Maria Giovanna Grella, Anna Chiara Contini and

Additional information is available at the end of the chapter

## **Surgical Treatment of Nevi and Melanoma in the Pediatric Age**

Andrea Zangari, Federico Zangari, Mercedes Romano, Elisabetta Cerigioni, Maria Giovanna Grella, Anna Chiara Contini and Martino Ascanio

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54632

**1. Introduction**

Surgical care of children affected by melanocytic lesions is a complex area of pediatric surgery, where psychosocial aspects, involving parents and children of different ages, overlap with oncologic implications. In this challenging field results may be frustrating despite knowledge and experience.

Due to its rarity, the occurrence of malignant melanoma in children may be underestimated by involved professionals. Melanoma in children is rare, but it does exist and every effort should be done to assure proper treatment, which should be guaranteed in a pediatric tertiary care center by a multi-specialist approach.

The role of dermatologists is essential to achieve proper selection of indications to surgery, by clinical follow up and dermatoscopy techniques. On the other hand surgeon is expected to share a profound knowledge of indications and techniques of treatment. In fact, the awareness that simple excision of a nevus may represent the first and most important therapeutic intervention of a MM, before knowing the definitive diagnosis, is sometimes lacking even among pediatric care professionals and the occasional occurrence of a MM diagnosis may be confounding and cause of an incomplete care strategy.

Decisions about surgical treatment of congenital giant nevi in some cases may need psycho‐ logical assessment because of complex relationships between patient's and parents' awareness and willingness.

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

Pediatric anesthesia offers a variety of techniques that can be personalized to suite patients from the newborn to the adolescent.

increased intracranial pressure due to hydrocephalus or a mass lesion. The prognosis of patients with symptomatic neurocutaneous melanosis is very poor, even in the absence of malignancy [8]. Significant association is between giant congenital nevi and neurofibromato‐ sis, with development of neurofibromas [9]. The histology is characterized by the presence of melanocytes in the epidermis ordered in theques and/or malanocytes in the dermis as sheets,

Surgical Treatment of Nevi and Melanoma in the Pediatric Age

http://dx.doi.org/10.5772/54632

331

The histology of large congenital nevus may be delivered into nevus cell, neuroid, epithelioid

In the *nevus cell type* histology may appear identical to acquired nevi, but in the congenital nevus melanocytes are more in the lower two-thirds of the reticular dermis or deeper and more associated with neurovascular structures in the reticular dermis. In the *neuroid type* of giant congenital nevi, the dermis imelanocyte cells appear to be arranged in palisaded around a cellular mass of homogeneous material (Varocay Body) and sheating of nerves by neuroid tissue (neuroid tubes). The neuroid type of giant congenital nevus may be associated with congenital anomalies of bone (club foot, spina bifida, atrophy). In the *Spindle cell and/or epithelioid cell type* of giant congenital nevus, the dermis is infiltrated in whole or in part by nests or sheets of epithelioid and/or spindle cells, but unlike acquired variety is involved deeper the reticular dermis, with neuroid elements. Sometime, in giant congenital nevi, architectural and cellular features may be so atypical making differentiation with melanoma very difficult. In the *dermal melanocytic type* of giant congenital nevi, appearance may be that

In large congenital nevi are occasionally present, within melanocytes, trace of other tissue like muscle, bone, placenta. Other tissues occasionally present intermixed with melanocytic elements are hemangiomas, increased numbers of mast cells, cartilage, calcificacion. Associ‐ ated tumors include schwannoma, neuroid tumors, lipoma, rhabdomyosarcoma, neurofibro‐ ma, sebaceous nevus, blue nevus, hemangioma, lymphangioma and mastocytoma, nevi of Ota and Ito, Spitz nevus [12]. The etiology of congenital melanocytic nevi has not been elucidated. One possible cause is a mutation. An association between infantile hemangiomas and con‐ genital melanocytic nevi has been suggested [13]. Future investigation may yield more definitive causative factors. A review of dermoscopy patterns in congenital nevi found that most nevi demonstrate a reticular, globular, or reticuloglobular pattern. The findings varied with age and the anatomic location of the nevus, with the globular pattern found more often in younger children and the reticular pattern found in patients aged 12 years or older [14]. The

The risk of melanoma development is proportional to to the size of congenital nevus., with a clear evidence of increased risk in patients with congenital nevi involving over 5% of the body surface. For giant congenital melanocytic nevi, the risk of developing melanoma has been reported to be as high as 5-7% [15]. Risk for the development of melanoma in smaller nevi has not been well quantified and the matter is still controversial (Fig.1). Also suggested is that melanoma developing within smaller congenital nevi usually occurs at puberty or later and

nests, cords and/or single cells [10].

of a giant blue nevus.

cell and/or spindle cell, dermal melanocytic and mixed [11].

role of dermoscopy in congenital nevi is currently recruiting.

**2.2. Association between congenital nevi and melanoma**

The intent of this chapter is to contribute to knowledge of this multifaceted field of pediatric surgery.

## **2. Nevomelanocytic lesions in the pediatric age**

Current strategies for the treatment of nevomelanocytic lesions in children mostly derived from the more extensive experience in adults. Nevertheless, further knowledge and experience in this field have shown some peculiarities that require special considerations relevant to the pediatric age. In fact, due to the multifaceted field of congenital nevi, to the rarity of melanoma in children and to peculiar features of some nevic lesions in this age range, important impli‐ cations related to treatment emerge.

## **2.1. Congenital nevi**

Congenital nevi are present at birth and occur approximately in 1% of newborn infants. They result from a proliferation of benign melanocytes in the dermis, epidermis, or both. Occasion‐ ally, nevi that are histologically identical to congenital nevi may develop approximately during the first 2 years of life. These are referred to be considered tardive congenital nevi [1].

The etiology of congenital melanocytic nevi remains unclear. The melanocytes of the skin originate in the neuroectoderm, although the specific cell type from which they derive remains controversial [3, 4, 5].

One hypothesis is that pluripotential nerve sheath precursor cells migrate from the neural crest to the skin along paraspinal ganglia and peripheral nerve sheaths and differentiate into melanocytes upon reaching the skin [6]. There are many reports of familial aggregation of congenital nevi.

One study found that the MC1R (melanocortin-1-receptor) genotype, which corresponds to a red-haired genotype and a tendency to increased birthweight, was overrepresented in a cohort of congenital melanocytic nevi affected Northern European patients. How MC1R variants promote growth of congenital melanocytic nevi and the fetus itself is unknown as is the application of this finding to non-european and more darkly pigmented races [7].

Congenital nevi have been stratified into 3 groups according to size. Small nevi are less than 1.5 cm in greatest diameter, medium nevi are 1.5-19.9 cm in greatest diameter, and large or giant nevi are greater than 20 cm. Giant nevi are often surrounded by several smaller satellite nevi.

Large congenital nevi of the head or posterior midline may also be seen as a component of neurocutaneous disorder, with cranial and/or leptomeningeal melanosis. Neurocutaneous melanosis may result from an error in the morphogenesis of the neuroectoderm, which gives rise to the melanotic cells of both the skin and meninges. Clinically, patients may present with increased intracranial pressure due to hydrocephalus or a mass lesion. The prognosis of patients with symptomatic neurocutaneous melanosis is very poor, even in the absence of malignancy [8]. Significant association is between giant congenital nevi and neurofibromato‐ sis, with development of neurofibromas [9]. The histology is characterized by the presence of melanocytes in the epidermis ordered in theques and/or malanocytes in the dermis as sheets, nests, cords and/or single cells [10].

Pediatric anesthesia offers a variety of techniques that can be personalized to suite patients

The intent of this chapter is to contribute to knowledge of this multifaceted field of pediatric

Current strategies for the treatment of nevomelanocytic lesions in children mostly derived from the more extensive experience in adults. Nevertheless, further knowledge and experience in this field have shown some peculiarities that require special considerations relevant to the pediatric age. In fact, due to the multifaceted field of congenital nevi, to the rarity of melanoma in children and to peculiar features of some nevic lesions in this age range, important impli‐

Congenital nevi are present at birth and occur approximately in 1% of newborn infants. They result from a proliferation of benign melanocytes in the dermis, epidermis, or both. Occasion‐ ally, nevi that are histologically identical to congenital nevi may develop approximately during

The etiology of congenital melanocytic nevi remains unclear. The melanocytes of the skin originate in the neuroectoderm, although the specific cell type from which they derive remains

One hypothesis is that pluripotential nerve sheath precursor cells migrate from the neural crest to the skin along paraspinal ganglia and peripheral nerve sheaths and differentiate into melanocytes upon reaching the skin [6]. There are many reports of familial aggregation of

One study found that the MC1R (melanocortin-1-receptor) genotype, which corresponds to a red-haired genotype and a tendency to increased birthweight, was overrepresented in a cohort of congenital melanocytic nevi affected Northern European patients. How MC1R variants promote growth of congenital melanocytic nevi and the fetus itself is unknown as is the

Congenital nevi have been stratified into 3 groups according to size. Small nevi are less than 1.5 cm in greatest diameter, medium nevi are 1.5-19.9 cm in greatest diameter, and large or giant nevi are greater than 20 cm. Giant nevi are often surrounded by several smaller satellite

Large congenital nevi of the head or posterior midline may also be seen as a component of neurocutaneous disorder, with cranial and/or leptomeningeal melanosis. Neurocutaneous melanosis may result from an error in the morphogenesis of the neuroectoderm, which gives rise to the melanotic cells of both the skin and meninges. Clinically, patients may present with

application of this finding to non-european and more darkly pigmented races [7].

the first 2 years of life. These are referred to be considered tardive congenital nevi [1].

from the newborn to the adolescent.

330 Melanoma - From Early Detection to Treatment

cations related to treatment emerge.

**2.1. Congenital nevi**

controversial [3, 4, 5].

congenital nevi.

nevi.

**2. Nevomelanocytic lesions in the pediatric age**

surgery.

The histology of large congenital nevus may be delivered into nevus cell, neuroid, epithelioid cell and/or spindle cell, dermal melanocytic and mixed [11].

In the *nevus cell type* histology may appear identical to acquired nevi, but in the congenital nevus melanocytes are more in the lower two-thirds of the reticular dermis or deeper and more associated with neurovascular structures in the reticular dermis. In the *neuroid type* of giant congenital nevi, the dermis imelanocyte cells appear to be arranged in palisaded around a cellular mass of homogeneous material (Varocay Body) and sheating of nerves by neuroid tissue (neuroid tubes). The neuroid type of giant congenital nevus may be associated with congenital anomalies of bone (club foot, spina bifida, atrophy). In the *Spindle cell and/or epithelioid cell type* of giant congenital nevus, the dermis is infiltrated in whole or in part by nests or sheets of epithelioid and/or spindle cells, but unlike acquired variety is involved deeper the reticular dermis, with neuroid elements. Sometime, in giant congenital nevi, architectural and cellular features may be so atypical making differentiation with melanoma very difficult. In the *dermal melanocytic type* of giant congenital nevi, appearance may be that of a giant blue nevus.

In large congenital nevi are occasionally present, within melanocytes, trace of other tissue like muscle, bone, placenta. Other tissues occasionally present intermixed with melanocytic elements are hemangiomas, increased numbers of mast cells, cartilage, calcificacion. Associ‐ ated tumors include schwannoma, neuroid tumors, lipoma, rhabdomyosarcoma, neurofibro‐ ma, sebaceous nevus, blue nevus, hemangioma, lymphangioma and mastocytoma, nevi of Ota and Ito, Spitz nevus [12]. The etiology of congenital melanocytic nevi has not been elucidated. One possible cause is a mutation. An association between infantile hemangiomas and con‐ genital melanocytic nevi has been suggested [13]. Future investigation may yield more definitive causative factors. A review of dermoscopy patterns in congenital nevi found that most nevi demonstrate a reticular, globular, or reticuloglobular pattern. The findings varied with age and the anatomic location of the nevus, with the globular pattern found more often in younger children and the reticular pattern found in patients aged 12 years or older [14]. The role of dermoscopy in congenital nevi is currently recruiting.

#### **2.2. Association between congenital nevi and melanoma**

The risk of melanoma development is proportional to to the size of congenital nevus., with a clear evidence of increased risk in patients with congenital nevi involving over 5% of the body surface. For giant congenital melanocytic nevi, the risk of developing melanoma has been reported to be as high as 5-7% [15]. Risk for the development of melanoma in smaller nevi has not been well quantified and the matter is still controversial (Fig.1). Also suggested is that melanoma developing within smaller congenital nevi usually occurs at puberty or later and develops more superficially in the skin, where it is easier to detect clinically. The lifetime risk of melanoma for patients with very large congenital nevi has benn estimated, approximately and considering variations in several countries and studies, at least 6%.

oral, ocular, genital mucosae. They first can appear after 6-12 months of life. Histology classifies acquired melanocytic nevi as a collection of melanocytic cells in the epidermis (Junctional), dermis (Intradermal), or both (Compound), disposed in isolated elements (epidermal variety, lentiginous pattern) or aggregated (junctional, intradermal and compound variety). There is evidence that number and size of common acquired nevi is associated with familiarity. Studies documented an increased number of nevi for pale skin, blond hair, blue or green eyes, tendency to sunburn. Typical acquired nevi usually have a round or oval, symmetric shape and relatively well-demarcated, smooth borders. The surface of nevi may be flat-topped, dome-shaped, papillomatous or peduncolated. More elevated acquired nevi tend to be more lightly pig‐ mented, and flatter acquired nevi tend to be more darkly pigmented. More elevated and less pigmented lesions tend to have a prominent intradermal melanocytic component, whereas flatter and darker lesions have a more prominent junctional melanocytic component and a less prominent dermal component. Changes in acquired melanocytic nevi can be physiologic in puberty, pregnancy, corticosteroid administration and sun exposure; also changes may occur slowly during the years as normal evolution of nevi. Though most of times changes are benign, in presence of alterations of symmetry, color, borders, extension or regression, especially in a short time (months), a periodic monitoring of all nevi on the skin and mucosa is necessary, preferably with dermoscopy. When melanoma occurs on a melanocytic acquired nevi, changes may be global or, most of times, partial, that is the reason why asymmetry is a predominant

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parameter to be evaluated [17].

**Figure 2.** Spitz nevus

**Figure 1.** Malignant melanoma diagnosed in a small congenital lesion of the dorsal foot

### **2.3. Spitz nevus**

It is also defined by other terms, including epithelioid cell and/or Spindle cell nevus, juvenile melanoma, benign juvenile melanoma. It occurs normally in children, but may appear in 15% of adolescents and adults. Spitz nevus is a unique, acquired, usually benign melanocytic tumor, so alarming in its clinical presentation and sometimes histologically confused with melanoma. It is possible that some lesions regress spontaneously. It can appear pink or tan, as a papule, often with teleangiectasies on the surface (Fig.2). A variety called Reed nevus, more frequent In adults, may also be confused with melanoma, but histologically is an acquired, predomi‐ nantly spindle cells variety, darkly pigmented [16].

#### **2.4. Common acquired melanocytic nevus**

Acquired melanocytic nevus is a common disorder of melanocytes, occurring as a pigmented benign lesion, possibly localized in every part of the skin (palmoplantar areas included) and

**Figure 2.** Spitz nevus

develops more superficially in the skin, where it is easier to detect clinically. The lifetime risk of melanoma for patients with very large congenital nevi has benn estimated, approximately

and considering variations in several countries and studies, at least 6%.

332 Melanoma - From Early Detection to Treatment

**Figure 1.** Malignant melanoma diagnosed in a small congenital lesion of the dorsal foot

nantly spindle cells variety, darkly pigmented [16].

**2.4. Common acquired melanocytic nevus**

It is also defined by other terms, including epithelioid cell and/or Spindle cell nevus, juvenile melanoma, benign juvenile melanoma. It occurs normally in children, but may appear in 15% of adolescents and adults. Spitz nevus is a unique, acquired, usually benign melanocytic tumor, so alarming in its clinical presentation and sometimes histologically confused with melanoma. It is possible that some lesions regress spontaneously. It can appear pink or tan, as a papule, often with teleangiectasies on the surface (Fig.2). A variety called Reed nevus, more frequent In adults, may also be confused with melanoma, but histologically is an acquired, predomi‐

Acquired melanocytic nevus is a common disorder of melanocytes, occurring as a pigmented benign lesion, possibly localized in every part of the skin (palmoplantar areas included) and

**2.3. Spitz nevus**

oral, ocular, genital mucosae. They first can appear after 6-12 months of life. Histology classifies acquired melanocytic nevi as a collection of melanocytic cells in the epidermis (Junctional), dermis (Intradermal), or both (Compound), disposed in isolated elements (epidermal variety, lentiginous pattern) or aggregated (junctional, intradermal and compound variety). There is evidence that number and size of common acquired nevi is associated with familiarity. Studies documented an increased number of nevi for pale skin, blond hair, blue or green eyes, tendency to sunburn. Typical acquired nevi usually have a round or oval, symmetric shape and relatively well-demarcated, smooth borders. The surface of nevi may be flat-topped, dome-shaped, papillomatous or peduncolated. More elevated acquired nevi tend to be more lightly pig‐ mented, and flatter acquired nevi tend to be more darkly pigmented. More elevated and less pigmented lesions tend to have a prominent intradermal melanocytic component, whereas flatter and darker lesions have a more prominent junctional melanocytic component and a less prominent dermal component. Changes in acquired melanocytic nevi can be physiologic in puberty, pregnancy, corticosteroid administration and sun exposure; also changes may occur slowly during the years as normal evolution of nevi. Though most of times changes are benign, in presence of alterations of symmetry, color, borders, extension or regression, especially in a short time (months), a periodic monitoring of all nevi on the skin and mucosa is necessary, preferably with dermoscopy. When melanoma occurs on a melanocytic acquired nevi, changes may be global or, most of times, partial, that is the reason why asymmetry is a predominant parameter to be evaluated [17].

#### **2.5. Blue nevus**

The blue nevus consists of an acquired or congenital blue, blue-gray or blue-black papule, plaque or nodule, histologically composed by dermal dendritic, fibroblast-like cells containing melanin. Most of times it is localized on dorsa of hands and feet, usually singular. Common blue nevi remain unchanged or possibly regress. Particular types of blue nevi are:

**2.8. Nevus of Ota and Ito**

**2.9. Melanoma**

**3.1. Lentigo maligna**

Ota first described this nevus and called it "nevus fuscocaeruleus ophtalmomaxillaris. Nevus of Ota is usually congenital but may appear in early childhood or in puberty. It is usually characterized by unilateral, flat, blue-black macules in the skin innervated by the first and second branches of the trigeminal nerve. Oral, nasal and pharingeal mucosae, conjunctivae and tympanic membranes may be involved. More rarely pigmentation may extend to cornea, optic nerve, fundus oculi, retrobulbar fat and periosteum. Enlargement and darkening may be observed over time. Histology shows stellate melanocytes widely scattered in the reticular dermis. Overlying melanocytes may be reduced in size and contain increased melanin. Nevus of Ota does not improve with time. 66 cases of melanoma development in nevus of Ota have been reported. Effective treatment is photothermolysis with Q-Switched LASER needing multiple sessions, with good results. Nevus of Ito is analogous to nevus of Ota and may coexist in the same patient. The difference between the two types of nevi is that nevus of Ito involves

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Melanoma is a malignant tumor resulting from the trasformation of melanocytes of the skin and less frequently of mucosae. During embryonic life, melanoblasts migrate from neural crest to the basal-cell layer of epidermis and a minus part to skin appendages and dermis. Melanoma

Risk for the development of melanoma remains low in pre-pubertal age, with an annual incidence of 0.7 cases per million children aged 0-9 years. Reaching adolescence the incidence of melanoma increases, with a rate of 13.2 cases per million children aged 15-19 years [2]. Prevention and early recognition of melanoma is mainly applied to adults through periodic clinical and dermoscopic controls. Only in recent years data documented alarm about increas‐ ing incidence of melanoma in adolescents. This increase, combined with other data about congenital nevi, allow physicians in the last years to play a crucial role in the identification of children at risk for melanoma, with particular regard to detection of risk factors in children

Lentigo maligna is a precursor lesion that may progress into invasive melanoma. It appears as a macular, freckled-like lesion of irregular shape, occurring most often in *elderly patients* (age over60years)insunexposedandsun-damaged,atrophic skin.Lentigomalignanormallygrows slowlyforlongperiods(years)withaprolongedradialgrowthphase,beforeevolutioninLentigo Maligna Melanoma. Hstopathology reveals atrophic epidermis and increased numbers of atypical basilar melanocytes that may extend down the hair follicles and skin appendages.

and adolescents, and education about sun and artificial ultraviolet exposure.

the distribution of the lateral supraclavicular and brachial nerves.

can arise from melanocytes located in these sites.

**3. Types of primary melanoma of the skin**


Malignant blue nevus, may develop in contiguity with cellular blue nevus, nevus of Ota, or de novo [18].

## **2.6. Dysplastic melanocytic nevi**

Dysplastic nevus is an acquired, usually atypical-appearing melanocytic tumor, characterized histologically by epidermic and/or dermal melanocytic dysplasia. Dysplasia refers to abnormal tissue development. When applied to melanocytic tumors, dysplasia is referred to a disordered melanocytic proliferation in association with discontinuous and variable cellular atypia (mild, moderate and severe). About this spectrum of atypia, from slight to marked may be said that intraepidermal melanocytes in dysplastic melanocytic nevi occupy an intermediate position between typical and malignant, basing on nuclear and cytoplasmic features. Not all atypicalappearing melanocytic lesions have an atypical histology. It is generally believed that a melanocytic nevus appearing asymmetric, irregular in borders and pigmentation and with a diameter equal or more than 6 mm is considered dysplastic, but these characteristics are referred to "Atypical acquired Nevus", also called "Clark Nevus". Although the diagnosis of dysplastic nevus is suspected because of the atypical appearing, histological confirmation is required to establish the presence or not of dysplasia. It is important to define if atypical nevus is dysplastic, because it is a potential histogenic precursor of melanoma and marker of increased melanoma risk.

#### **2.7. Halo nevi**

Halo nevus, also referred as "Sutton's nevus" or "leukoderma acquisitum centrifugum", is a nevus surrounded by a macule of leukoderma (hypopigmented or apigmented area). It occurs in up to 1% of general population, with a peak of incidence in the second decade. It is commonly composed of a central pigmented nevus and an acquired surrounding depigmented halo. From 25 to 50% of patients have more than one halo nevi. Nevus regression can be complete and is caused by a lymphocytic aggression against nevus melanocytes with involvement of sur‐ rounding epidermal melanocytes. Association with vitiligo needs clinical and anamnestic analysis as a history for melanoma.

#### **2.8. Nevus of Ota and Ito**

**2.5. Blue nevus**

melanoma.

de novo [18].

the buttock or sacrum.

334 Melanoma - From Early Detection to Treatment

**2.6. Dysplastic melanocytic nevi**

increased melanoma risk.

analysis as a history for melanoma.

**2.7. Halo nevi**

The blue nevus consists of an acquired or congenital blue, blue-gray or blue-black papule, plaque or nodule, histologically composed by dermal dendritic, fibroblast-like cells containing melanin. Most of times it is localized on dorsa of hands and feet, usually singular. Common

**•** Cellular blue nevus is a blue-gray nodule or plaque 1 to 3 cm diameter mostly located on

**•** Combined blue nevus-melanocytic nevus, sometimes confused with atypical nevi or

Malignant blue nevus, may develop in contiguity with cellular blue nevus, nevus of Ota, or

Dysplastic nevus is an acquired, usually atypical-appearing melanocytic tumor, characterized histologically by epidermic and/or dermal melanocytic dysplasia. Dysplasia refers to abnormal tissue development. When applied to melanocytic tumors, dysplasia is referred to a disordered melanocytic proliferation in association with discontinuous and variable cellular atypia (mild, moderate and severe). About this spectrum of atypia, from slight to marked may be said that intraepidermal melanocytes in dysplastic melanocytic nevi occupy an intermediate position between typical and malignant, basing on nuclear and cytoplasmic features. Not all atypicalappearing melanocytic lesions have an atypical histology. It is generally believed that a melanocytic nevus appearing asymmetric, irregular in borders and pigmentation and with a diameter equal or more than 6 mm is considered dysplastic, but these characteristics are referred to "Atypical acquired Nevus", also called "Clark Nevus". Although the diagnosis of dysplastic nevus is suspected because of the atypical appearing, histological confirmation is required to establish the presence or not of dysplasia. It is important to define if atypical nevus is dysplastic, because it is a potential histogenic precursor of melanoma and marker of

Halo nevus, also referred as "Sutton's nevus" or "leukoderma acquisitum centrifugum", is a nevus surrounded by a macule of leukoderma (hypopigmented or apigmented area). It occurs in up to 1% of general population, with a peak of incidence in the second decade. It is commonly composed of a central pigmented nevus and an acquired surrounding depigmented halo. From 25 to 50% of patients have more than one halo nevi. Nevus regression can be complete and is caused by a lymphocytic aggression against nevus melanocytes with involvement of sur‐ rounding epidermal melanocytes. Association with vitiligo needs clinical and anamnestic

blue nevi remain unchanged or possibly regress. Particular types of blue nevi are:

Ota first described this nevus and called it "nevus fuscocaeruleus ophtalmomaxillaris. Nevus of Ota is usually congenital but may appear in early childhood or in puberty. It is usually characterized by unilateral, flat, blue-black macules in the skin innervated by the first and second branches of the trigeminal nerve. Oral, nasal and pharingeal mucosae, conjunctivae and tympanic membranes may be involved. More rarely pigmentation may extend to cornea, optic nerve, fundus oculi, retrobulbar fat and periosteum. Enlargement and darkening may be observed over time. Histology shows stellate melanocytes widely scattered in the reticular dermis. Overlying melanocytes may be reduced in size and contain increased melanin. Nevus of Ota does not improve with time. 66 cases of melanoma development in nevus of Ota have been reported. Effective treatment is photothermolysis with Q-Switched LASER needing multiple sessions, with good results. Nevus of Ito is analogous to nevus of Ota and may coexist in the same patient. The difference between the two types of nevi is that nevus of Ito involves the distribution of the lateral supraclavicular and brachial nerves.

### **2.9. Melanoma**

Melanoma is a malignant tumor resulting from the trasformation of melanocytes of the skin and less frequently of mucosae. During embryonic life, melanoblasts migrate from neural crest to the basal-cell layer of epidermis and a minus part to skin appendages and dermis. Melanoma can arise from melanocytes located in these sites.

Risk for the development of melanoma remains low in pre-pubertal age, with an annual incidence of 0.7 cases per million children aged 0-9 years. Reaching adolescence the incidence of melanoma increases, with a rate of 13.2 cases per million children aged 15-19 years [2]. Prevention and early recognition of melanoma is mainly applied to adults through periodic clinical and dermoscopic controls. Only in recent years data documented alarm about increas‐ ing incidence of melanoma in adolescents. This increase, combined with other data about congenital nevi, allow physicians in the last years to play a crucial role in the identification of children at risk for melanoma, with particular regard to detection of risk factors in children and adolescents, and education about sun and artificial ultraviolet exposure.

## **3. Types of primary melanoma of the skin**

#### **3.1. Lentigo maligna**

Lentigo maligna is a precursor lesion that may progress into invasive melanoma. It appears as a macular, freckled-like lesion of irregular shape, occurring most often in *elderly patients* (age over60years)insunexposedandsun-damaged,atrophic skin.Lentigomalignanormallygrows slowlyforlongperiods(years)withaprolongedradialgrowthphase,beforeevolutioninLentigo Maligna Melanoma. Hstopathology reveals atrophic epidermis and increased numbers of atypical basilar melanocytes that may extend down the hair follicles and skin appendages.

## **3.2. Lentigo maligna melanoma**

It is a melanoma in situ slow growing progressing to invasive melanoma with nests of malignant melanocytes invading the dermis. Lentigo Maligna Melanoma represents 4 to 15 % of all melanoma.

**3.6. Melanoma of the mucosa**

**3.7. Desmoplastic melanoma**

of malignancy [24, 25, 26].

a radial growth phase.

nerves.

It involves oral, nasal, vulva, anorectal, conjunctival mucosae and it may occur with or without

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It is a rare subtype of melanoma, locally aggressive and with high rates of local recurrence. It may arise in association with LM, ALM and mucosal melanoma or "de novo". DM may appear as a pigmented macule, papule, nodule or reddish. Histologically it is caracterized by fibrous tissue and atypical spindle-shaped melanocytes that show a propensity to infiltrate cutaneous

The incidence of malignant melanoma is rapidly increasing in the last decades. The surveil‐ lance, epidemiology and results program (SEER) has documented a 32,7% increased in mortality rates over the period 1973 to 1995. on the other hand, the overall survival rate has been improving for melanoma in the last decades. Currently, cutaneous melanoma accounts

More recent studies indicate that the rate of increase in all age groups was 2.8% per year from 1981 to 2001 and in children (age < 20 years) was 1.1% per year from 1975 to 2001.1 The diagnosis is often delayed in children since melanoma is rare (300 to 420 new cases per year), and benign lesions, especially Spitz nevus, may mimic melanoma. The prognosis is good when there is prompt identification and wide local excision of early disease, but poor for those with advanced disease at presentation [23]. Case-control studies in adults have identified multiple host and environmental factors associated with increased risk of malignant melanoma. Host factors include fair skin, white race, blond or red hair, light eye color, tendency to burn with UV radiation exposure, increased number of benign nevi, dysplastic nevi, family history of melanoma, and xeroderma pigmentosum.6,7 Environmental factors include sunburns, often as a child, and increased exposure to UV radiation. Proposed risk factors for paediatric melanoma include congenital, dysplastic, or increased number of nevi; inability to tan; blue eyes; facial freckling; family history of melanoma; disorders of DNA excision repair like xeroderma pigmentosum; acquired or congenital immunosuppression and a previous history

Analysis regarding children and young adults with melanoma between 1973 and 2001 show how older age, more recent year of diagnosis, female sex, white race, and increased environ‐ mental UV radiation were all associated with a significant increase in the risk of melanoma. In the first year of life, the incidence of melanoma is similar by race, but it diverges by age 5

The increase in incidence of melanoma in children, especially in adolescents, is similar to that seen in young adults. This may reflect increased cumulative UV exposure during childhood or adolescence, greater awareness and more frequent diagnosis of melanoma (eg, versus atypical Spitz nevus), differences in genetic predisposition, and/or other environmental factors. The increased risk of melanoma in girls, particularly on the lower extremities, may be a result of increased UV exposure. In adults, sun-related behaviours differ between men and

to 9 years and is more than 40-fold higher in white individuals by age 20 to 24 years.

for approximatively 1% of all cancer deaths [19, 20, 21, 22].

## **3.3. Superficial spreading melanoma**

SSM is the most frequent type of melanocytic malignancy representing 70% of all melanomas.

Most commonly it occurs on the upper back of men and on the legs of women, although it can develop at any site, mucosa included. The usual history is that of slow change (months to 1-5 years) of a preexisting melanocytic lesion or can arise "de novo".

SSM most frequently presents as a macule asymmetric, variegated pigmentation (from brown to black, with variable presence of blue-gray, gray-white or pink areas as sign of regression along the borders or inside the lesion. Borders may present intact initially in presence of a precursor melanocytic nevus, but mostly are irregular. Dermoscopy can reveal better all these alterations, and more other parameters typical of SSM (salt-peppering areas, star-bust aspect, pseudopodes at borders, blue-white veil and so on). Diagnosis of SSM may be done from a macroscopic view, but dermoscopy helps much for *confirmation diagnosis* and for *early diagno‐ sis*, when it is difficult to find macroscopically some alterations typical of melanoma.

Histopathology reveals a "pagetoid" distribution of atypical large melanocytes thruoughout the epidermis. The large cells may occur singularly or in nests and have a monomorphous appearance. In the dermis areas of invasion of atypical melanocytes are present.

## **3.4. Nodular melanoma**

The second most common subtype of melanoma is nodular melanoma with a frequency of 15 to 30 percent of all types. NM is remarkable for its rapid evolution and may arise from melanocytic nevi or normal skin (de novo), but in lack of an apparent radial growth phase. NM is more common to arise "de novo" than from a preexisting nevus.

NM appears typically as a blue-black, blue-red or amelanotic nodule or papule. In certain cases it may be difficult to diagnose especially when it appears as an amelanotic reddish lesion.

Histopathology may demonstrates a little tendency for intraepidermal growth, but it typically arises at dermal-epidermal junction and from its onset with extention to the dermis, composed of large epithelioid cells, spindle cells, small cells, or a mix of these different cells.

#### **3.5. Acral lentiginous melanoma**

ALM is more common in darker skin individuals (60 to 72 percent in blacks, 29 to 46 percent in asians). In white skin people it represents only 2 to 8 percent of melanomas. ALM occurs on palms and more often on soles, or beneath the nail plate.

The biologic behavior of ALM is traditionally considered more aggressive with a poorer prognosis and this may be due to late diagnosis and/or to the different biologic origin of it.

### **3.6. Melanoma of the mucosa**

**3.2. Lentigo maligna melanoma**

336 Melanoma - From Early Detection to Treatment

**3.3. Superficial spreading melanoma**

of all melanoma.

**3.4. Nodular melanoma**

**3.5. Acral lentiginous melanoma**

palms and more often on soles, or beneath the nail plate.

It is a melanoma in situ slow growing progressing to invasive melanoma with nests of malignant melanocytes invading the dermis. Lentigo Maligna Melanoma represents 4 to 15 %

SSM is the most frequent type of melanocytic malignancy representing 70% of all melanomas. Most commonly it occurs on the upper back of men and on the legs of women, although it can develop at any site, mucosa included. The usual history is that of slow change (months to 1-5

SSM most frequently presents as a macule asymmetric, variegated pigmentation (from brown to black, with variable presence of blue-gray, gray-white or pink areas as sign of regression along the borders or inside the lesion. Borders may present intact initially in presence of a precursor melanocytic nevus, but mostly are irregular. Dermoscopy can reveal better all these alterations, and more other parameters typical of SSM (salt-peppering areas, star-bust aspect, pseudopodes at borders, blue-white veil and so on). Diagnosis of SSM may be done from a macroscopic view, but dermoscopy helps much for *confirmation diagnosis* and for *early diagno‐*

*sis*, when it is difficult to find macroscopically some alterations typical of melanoma.

appearance. In the dermis areas of invasion of atypical melanocytes are present.

of large epithelioid cells, spindle cells, small cells, or a mix of these different cells.

NM is more common to arise "de novo" than from a preexisting nevus.

Histopathology reveals a "pagetoid" distribution of atypical large melanocytes thruoughout the epidermis. The large cells may occur singularly or in nests and have a monomorphous

The second most common subtype of melanoma is nodular melanoma with a frequency of 15 to 30 percent of all types. NM is remarkable for its rapid evolution and may arise from melanocytic nevi or normal skin (de novo), but in lack of an apparent radial growth phase.

NM appears typically as a blue-black, blue-red or amelanotic nodule or papule. In certain cases it may be difficult to diagnose especially when it appears as an amelanotic reddish lesion. Histopathology may demonstrates a little tendency for intraepidermal growth, but it typically arises at dermal-epidermal junction and from its onset with extention to the dermis, composed

ALM is more common in darker skin individuals (60 to 72 percent in blacks, 29 to 46 percent in asians). In white skin people it represents only 2 to 8 percent of melanomas. ALM occurs on

The biologic behavior of ALM is traditionally considered more aggressive with a poorer prognosis and this may be due to late diagnosis and/or to the different biologic origin of it.

years) of a preexisting melanocytic lesion or can arise "de novo".

It involves oral, nasal, vulva, anorectal, conjunctival mucosae and it may occur with or without a radial growth phase.

### **3.7. Desmoplastic melanoma**

It is a rare subtype of melanoma, locally aggressive and with high rates of local recurrence. It may arise in association with LM, ALM and mucosal melanoma or "de novo". DM may appear as a pigmented macule, papule, nodule or reddish. Histologically it is caracterized by fibrous tissue and atypical spindle-shaped melanocytes that show a propensity to infiltrate cutaneous nerves.

The incidence of malignant melanoma is rapidly increasing in the last decades. The surveil‐ lance, epidemiology and results program (SEER) has documented a 32,7% increased in mortality rates over the period 1973 to 1995. on the other hand, the overall survival rate has been improving for melanoma in the last decades. Currently, cutaneous melanoma accounts for approximatively 1% of all cancer deaths [19, 20, 21, 22].

More recent studies indicate that the rate of increase in all age groups was 2.8% per year from 1981 to 2001 and in children (age < 20 years) was 1.1% per year from 1975 to 2001.1 The diagnosis is often delayed in children since melanoma is rare (300 to 420 new cases per year), and benign lesions, especially Spitz nevus, may mimic melanoma. The prognosis is good when there is prompt identification and wide local excision of early disease, but poor for those with advanced disease at presentation [23]. Case-control studies in adults have identified multiple host and environmental factors associated with increased risk of malignant melanoma. Host factors include fair skin, white race, blond or red hair, light eye color, tendency to burn with UV radiation exposure, increased number of benign nevi, dysplastic nevi, family history of melanoma, and xeroderma pigmentosum.6,7 Environmental factors include sunburns, often as a child, and increased exposure to UV radiation. Proposed risk factors for paediatric melanoma include congenital, dysplastic, or increased number of nevi; inability to tan; blue eyes; facial freckling; family history of melanoma; disorders of DNA excision repair like xeroderma pigmentosum; acquired or congenital immunosuppression and a previous history of malignancy [24, 25, 26].

Analysis regarding children and young adults with melanoma between 1973 and 2001 show how older age, more recent year of diagnosis, female sex, white race, and increased environ‐ mental UV radiation were all associated with a significant increase in the risk of melanoma. In the first year of life, the incidence of melanoma is similar by race, but it diverges by age 5 to 9 years and is more than 40-fold higher in white individuals by age 20 to 24 years.

The increase in incidence of melanoma in children, especially in adolescents, is similar to that seen in young adults. This may reflect increased cumulative UV exposure during childhood or adolescence, greater awareness and more frequent diagnosis of melanoma (eg, versus atypical Spitz nevus), differences in genetic predisposition, and/or other environmental factors. The increased risk of melanoma in girls, particularly on the lower extremities, may be a result of increased UV exposure. In adults, sun-related behaviours differ between men and women. [22] The increased rates of melanoma in adolescent and young women may reflect sunbathing or the widespread (> 25%) practice of indoor tanning [28].

matter of controversy. In the lack of consensus about systematic removal of small congenital nevi, careful dermatologic monitoring and prompt excision after clinical changing is recom‐ mended [33] In the Literature there is a large agreement on the excision of large congenital nevi. In fact they show an increased risk for development of malignant melanoma, varying from 1% to 31%. Nonetheless malignancy is reported to occur despite complete excision and

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Indications for surgical excision [36] of acquired nevi are mainly related to lesions resembling malignant melanoma, such as atypical nevi, Spitz nevi, and lesions presenting clinical signs and symptoms as diameter of more than 5 mm or increasing diameter, irregular margins, border notching, irregular pigmentation, asymmetry, rapid onset or increase in diameter, ulceration, bleeding, pain and itching. These last signs seem to represent the presentation symptoms of malignant melanoma in 85% of cases [37] Although the risk of malignant melanoma is higher in familial atypical nevi rather than in nonfamilial, features of atypical

Some special consideration is given to Spitz nevus for possible misdiagnosis with MM. Most dermatologists and physicians recommend biopsy. In an interview with dermatologists, most responding specialists (93%) recommended the biopsy of suspected Spitz nevi. Sixty-nine percent of physicians would completely excise a lesion that was histologically diagnosed as an incompletely removed Spitz nevus. Seventy percent of general dermatologists and 80% of pediatric dermatologists would recommend excision with a 1- to 2-mm margin of normalappearing skin around a Spitz nevus [39]. The lack of consensus about the nature and the ideal management of Spitz nevus reflects the uncertainty in histopathologic distinction between Spitz nevus and melanoma, and such a concern influences management. By some authors 2 mm margins excisional biopsy of clinically appearing Spitz nevus, as of any nevic lesion, is

In some cases nevi located in sites of clinically difficult monitoring or placed in sites of exposure

Acquired melanocytic nevi begin to appear after the first 6 months of life and increase in number during childhood and adolescence, typically reaching a peak count in the third decade and then slowly regressing with age. They are classified as junctional nevi if the nests of melanocytes are in the dermal – epidermal junction, intradermal nevi if the nests are in dermis

They usually have a diameter of less than 6 - 8 mm, a homogeneous surface, pigmentation, round or oval shape, regular outline, and demarcated border, sometimes with pigmentary

These are rarely complicated by evolution in malignant melanoma so conservative treatment,

nevi have been reported to be the most frequent indication to surgery [38]

to frequent trauma are an indication for excision [38, 40]

and compound nevi if the nests are located in both sites.

stippling or perifollicular hypopigmentation.

that is clinical periodic control, is usually enough.

**5. Treatment of acquired nevi**

may be not preventable [34, 35]

recommended

Prognosis for young children, adolescents, and young adults with melanoma appears to be similar. Also increased is the risk of death in male children, those with regional or distant metastasis, primary sites other than the extremities or torso, increasing thickness of the primary lesion, earlier year of diagnosis, and previous cancer.

Melanoma-specific survival in children has improved by approximately 4% per year during the last 3 decades. It is difficult to explain this dramatic improvement. Although earlier diagnosis could be associated with improved survival, there has been no decrease in lesion thickness over the last decade. Furthermore, survival has improved for all stages of paediatric melanoma. The most notable improvement has been in the "unstaged" group, likely due to more complete staging. There are important differences in young children (age < 10 years) with melanoma compared with adolescents and young adults that may reflect distinct tumor biology and/or host characteristics.

Published large series of paediatric melanoma report 5-year survival rates of 74% to 80%. This is significantly worse than the 91% 5-year overall survival seen in recent analysis, after the exclusion of cases of melanoma in situ [29, 30]. Accepted prognostic factors in adult melanoma include primary lesion thickness, ulceration, and non-extremity site; increased age; regional lymph node involvement; satellite or in-transit metastases; elevated serum lactate dehydro‐ genase level; visceral or brain metastases.[4,9 However, prognosis and prognostic factors in children are less defined. In a recent review of more than 300 cases, the outcome for paediatric patients (5-year survival of 74%) was slightly worse than that of young adults, but these survival estimates have limitations. In a large European registry study of children, male sex, unfavorable site (lesions on the trunk), and/or second primary or regional or distant metastasis. [10] Advanced stage has been associated with poor prognosis in other paediatric studies [27].

In summary, paediatric melanoma is an important and increasing problem. Factors conferring risk of adult melanoma, including older age, white race (blue eyes, blond or red hair, freckling tendency, liability to tan and tendence to sunburn), family history of melanoma, elevate number of acquired melanocytic nevi (double risk in 50 to 99 of acquired melanocytic nevi), dysplastic nevi, environmental exposure to UV radiation, congenital or acquired immuno‐ suppression are also important in paediatric melanoma.

## **4. Indications for excision of nevi**

Indication for excision are usually assessed by dermatologists, paediatric surgeons, paedia‐ trician and physicians.

Nevi can be divided into congenital and acquired. In turn congenital nevi can be small, intermediate and large (>20cm). Melanoma risk in small congenital nevi is debated, although in some reports it seems to be of importance [32] Some studies demonstrated an increased risk of malignant melanoma in small lesions as well as in intermediate lesions, but this is still a matter of controversy. In the lack of consensus about systematic removal of small congenital nevi, careful dermatologic monitoring and prompt excision after clinical changing is recom‐ mended [33] In the Literature there is a large agreement on the excision of large congenital nevi. In fact they show an increased risk for development of malignant melanoma, varying from 1% to 31%. Nonetheless malignancy is reported to occur despite complete excision and may be not preventable [34, 35]

Indications for surgical excision [36] of acquired nevi are mainly related to lesions resembling malignant melanoma, such as atypical nevi, Spitz nevi, and lesions presenting clinical signs and symptoms as diameter of more than 5 mm or increasing diameter, irregular margins, border notching, irregular pigmentation, asymmetry, rapid onset or increase in diameter, ulceration, bleeding, pain and itching. These last signs seem to represent the presentation symptoms of malignant melanoma in 85% of cases [37] Although the risk of malignant melanoma is higher in familial atypical nevi rather than in nonfamilial, features of atypical nevi have been reported to be the most frequent indication to surgery [38]

Some special consideration is given to Spitz nevus for possible misdiagnosis with MM. Most dermatologists and physicians recommend biopsy. In an interview with dermatologists, most responding specialists (93%) recommended the biopsy of suspected Spitz nevi. Sixty-nine percent of physicians would completely excise a lesion that was histologically diagnosed as an incompletely removed Spitz nevus. Seventy percent of general dermatologists and 80% of pediatric dermatologists would recommend excision with a 1- to 2-mm margin of normalappearing skin around a Spitz nevus [39]. The lack of consensus about the nature and the ideal management of Spitz nevus reflects the uncertainty in histopathologic distinction between Spitz nevus and melanoma, and such a concern influences management. By some authors 2 mm margins excisional biopsy of clinically appearing Spitz nevus, as of any nevic lesion, is recommended

In some cases nevi located in sites of clinically difficult monitoring or placed in sites of exposure to frequent trauma are an indication for excision [38, 40]

## **5. Treatment of acquired nevi**

women. [22] The increased rates of melanoma in adolescent and young women may reflect

Prognosis for young children, adolescents, and young adults with melanoma appears to be similar. Also increased is the risk of death in male children, those with regional or distant metastasis, primary sites other than the extremities or torso, increasing thickness of the primary

Melanoma-specific survival in children has improved by approximately 4% per year during the last 3 decades. It is difficult to explain this dramatic improvement. Although earlier diagnosis could be associated with improved survival, there has been no decrease in lesion thickness over the last decade. Furthermore, survival has improved for all stages of paediatric melanoma. The most notable improvement has been in the "unstaged" group, likely due to more complete staging. There are important differences in young children (age < 10 years) with melanoma compared with adolescents and young adults that may reflect distinct tumor

Published large series of paediatric melanoma report 5-year survival rates of 74% to 80%. This is significantly worse than the 91% 5-year overall survival seen in recent analysis, after the exclusion of cases of melanoma in situ [29, 30]. Accepted prognostic factors in adult melanoma include primary lesion thickness, ulceration, and non-extremity site; increased age; regional lymph node involvement; satellite or in-transit metastases; elevated serum lactate dehydro‐ genase level; visceral or brain metastases.[4,9 However, prognosis and prognostic factors in children are less defined. In a recent review of more than 300 cases, the outcome for paediatric patients (5-year survival of 74%) was slightly worse than that of young adults, but these survival estimates have limitations. In a large European registry study of children, male sex, unfavorable site (lesions on the trunk), and/or second primary or regional or distant metastasis. [10] Advanced stage has been associated with poor prognosis in other paediatric studies [27]. In summary, paediatric melanoma is an important and increasing problem. Factors conferring risk of adult melanoma, including older age, white race (blue eyes, blond or red hair, freckling tendency, liability to tan and tendence to sunburn), family history of melanoma, elevate number of acquired melanocytic nevi (double risk in 50 to 99 of acquired melanocytic nevi), dysplastic nevi, environmental exposure to UV radiation, congenital or acquired immuno‐

Indication for excision are usually assessed by dermatologists, paediatric surgeons, paedia‐

Nevi can be divided into congenital and acquired. In turn congenital nevi can be small, intermediate and large (>20cm). Melanoma risk in small congenital nevi is debated, although in some reports it seems to be of importance [32] Some studies demonstrated an increased risk of malignant melanoma in small lesions as well as in intermediate lesions, but this is still a

sunbathing or the widespread (> 25%) practice of indoor tanning [28].

lesion, earlier year of diagnosis, and previous cancer.

suppression are also important in paediatric melanoma.

**4. Indications for excision of nevi**

trician and physicians.

biology and/or host characteristics.

338 Melanoma - From Early Detection to Treatment

Acquired melanocytic nevi begin to appear after the first 6 months of life and increase in number during childhood and adolescence, typically reaching a peak count in the third decade and then slowly regressing with age. They are classified as junctional nevi if the nests of melanocytes are in the dermal – epidermal junction, intradermal nevi if the nests are in dermis and compound nevi if the nests are located in both sites.

They usually have a diameter of less than 6 - 8 mm, a homogeneous surface, pigmentation, round or oval shape, regular outline, and demarcated border, sometimes with pigmentary stippling or perifollicular hypopigmentation.

These are rarely complicated by evolution in malignant melanoma so conservative treatment, that is clinical periodic control, is usually enough.

However, when removal of a naevus is necessary we have to do some considerations especially in order to the characteristics of the nevus and its position.

The elliptical excisional biopsy, as the other type of excisional biopsy, must include a portion of healthy tissue of 0,2 cm wideness from the perimeter of the lesion and the subcutaneous tissue. The possible exuberant tissue, called "dog - ear" can be corrected by extending the

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The wedge excisional biopsy is usually utilized for the lesions located on or close to the free margin of some particular structures as eyelid, lip, nose and ear. In the eyelids it is possible to make an incision along the edge and remove only the skin, although some lesions require excision of full thickness eyelid. 1/4 of eyelid or 1/3 of the lower lip can be completely excised

The circular excisionalbiopsyisusedwhena skinlimitedincisionisneededas inthe caseofnose or in the anterior region of the auricle. The defect can be closed with a skin graft or a skin flap.

When repairing the loss of substance by combination of skin edges is impossible, other

A flap is a portion of one or more tissues transferred from a donor site to a receiver maintaining

The use of flap has many advantages including that of allow the repair of the defect by means of tissue similar or equal to those of the receiving site. It is very important for specialized tissues

The skin flap, composed by skin only or including subcutaneous tissue, is transferred from one part of the body to another with its neurovascular pedicle or attached by just a margin to

There are two types of skin flap: those which rotate around a pivot point (rotation, transposi‐ tion and interpolation flaps) and advancement flaps (single pedicle, V – Y, Y – V and bi –

The rotation flap is a semicircular flap that rotates from its pivot point to the receiving site (Fig.3). If tension is too high, the incision can be extended by a reverse incision from pivot point

The transposition flap is composed by a rectangle or square of skin and subcutaneous tissue

This can have only one pedicle, so feeding is maintained by exploiting the elasticity of the skin.

The advancement V – Y flap more than a flap is a V incision whose sides are closed in such a

The advancement flap is brought forward on the lesion without rotation.

and the defect closed with a simple suture without elaborated reconstructions.

ellipse or removing the excess skin with a L or Y incision.

Grafts, rarely used for the excision of acquired nevi

a neurovascular connection ("pedicle").

techniques are necessary:

*5.2.2. Flaps*

as lip or eyelid.

pedunculated.

preserve vascular support.

along the base of the flap (backcut).

way that the final suture gives a Y.

that rotate around a pivot point close to lesion.

## **5.1. Anesthesia**

Indications and surgical techniques of the excision of a skin lesion are similar to the adult and so the actual difference with children is the anesthetic management.

Analgesics are commonly administered prior to surgical procedure in children. The concept of preemptive analgesia is still controversial and its effectiveness may depend on the type of surgery. Nowadays the most commonly used medications are acetaminophen alone or with codeine, but also non – steroidal anti – inflammatory medications (ibuprofen) are effective analgesics for perioperative pain, but less used for the impact on bleeding.

In association with analgesics and anesthetic medicament the use of sedative is very common and useful also in children as in the adult. Midazolam is very convenient in children because not only alleviates the anxiety of surgery, but also induces an anterograde amnesia, useful for treatments requiring multiple visits. Furthermore, it can be administrated in various vehicles via the nasal, rectal, sublingual that are less traumatic than intramuscular injection in children.

Fentanyl is 100 times more potent than morphine and has less influence on GI motility than morphine, so reducing the effect of emesis and oxygen desaturation becoming more tolerable in children.

General anaesthesia is used in the excisional surgery of large lesions and occasionally in non – cooperative children. It is a very safe procedure, but the rate of complication increases in case of surgery in the first year of life and when the anesthesiologist has no pediatric experience.

In recent years the use of topical anesthetics, especially EMLA cream (eutectic mixture of local anesthetics), knows an important development in office procedures in children, although the incorrect use of EMLA causes failure in reducing pain of dermatologic procedure. In particular for an effective absorption of the anesthetic into the skin an occlusive dressing is useful.

#### **5.2. Surgery**

Any skin lesion presenting features of malignant melanoma are biopsied. Although melanoma in children is rare, the most frequent indication for excision of an acquired nevus is early diagnostic evaluation because of melanoma concern.

Excisional biopsy is the treatment of choice. It may be elliptical, wedge or circular, but the first is the most commonly used.

#### *5.2.1. Simple excision*

Skin incisions should be planned along or parallel to the relaxed skin tension lines (RSTLs). This allows, on one hand, a better wound healing, an easier matching of edge of the wound and lower tension of the sutures and, on the other hand, to hide the wound in a skin fold.

The elliptical excisional biopsy, as the other type of excisional biopsy, must include a portion of healthy tissue of 0,2 cm wideness from the perimeter of the lesion and the subcutaneous tissue. The possible exuberant tissue, called "dog - ear" can be corrected by extending the ellipse or removing the excess skin with a L or Y incision.

The wedge excisional biopsy is usually utilized for the lesions located on or close to the free margin of some particular structures as eyelid, lip, nose and ear. In the eyelids it is possible to make an incision along the edge and remove only the skin, although some lesions require excision of full thickness eyelid. 1/4 of eyelid or 1/3 of the lower lip can be completely excised and the defect closed with a simple suture without elaborated reconstructions.

The circular excisionalbiopsyisusedwhena skinlimitedincisionisneededas inthe caseofnose or in the anterior region of the auricle. The defect can be closed with a skin graft or a skin flap.

When repairing the loss of substance by combination of skin edges is impossible, other techniques are necessary:

Grafts, rarely used for the excision of acquired nevi

## *5.2.2. Flaps*

However, when removal of a naevus is necessary we have to do some considerations especially

Indications and surgical techniques of the excision of a skin lesion are similar to the adult and

Analgesics are commonly administered prior to surgical procedure in children. The concept of preemptive analgesia is still controversial and its effectiveness may depend on the type of surgery. Nowadays the most commonly used medications are acetaminophen alone or with codeine, but also non – steroidal anti – inflammatory medications (ibuprofen) are effective

In association with analgesics and anesthetic medicament the use of sedative is very common and useful also in children as in the adult. Midazolam is very convenient in children because not only alleviates the anxiety of surgery, but also induces an anterograde amnesia, useful for treatments requiring multiple visits. Furthermore, it can be administrated in various vehicles via the nasal, rectal, sublingual that are less traumatic than intramuscular injection in children.

Fentanyl is 100 times more potent than morphine and has less influence on GI motility than morphine, so reducing the effect of emesis and oxygen desaturation becoming more tolerable

General anaesthesia is used in the excisional surgery of large lesions and occasionally in non – cooperative children. It is a very safe procedure, but the rate of complication increases in case of surgery in the first year of life and when the anesthesiologist has no pediatric experience.

In recent years the use of topical anesthetics, especially EMLA cream (eutectic mixture of local anesthetics), knows an important development in office procedures in children, although the incorrect use of EMLA causes failure in reducing pain of dermatologic procedure. In particular for an effective absorption of the anesthetic into the skin an occlusive dressing is useful.

Any skin lesion presenting features of malignant melanoma are biopsied. Although melanoma in children is rare, the most frequent indication for excision of an acquired nevus is early

Excisional biopsy is the treatment of choice. It may be elliptical, wedge or circular, but the first

Skin incisions should be planned along or parallel to the relaxed skin tension lines (RSTLs). This allows, on one hand, a better wound healing, an easier matching of edge of the wound and lower tension of the sutures and, on the other hand, to hide the wound in a skin fold.

diagnostic evaluation because of melanoma concern.

in order to the characteristics of the nevus and its position.

so the actual difference with children is the anesthetic management.

analgesics for perioperative pain, but less used for the impact on bleeding.

**5.1. Anesthesia**

340 Melanoma - From Early Detection to Treatment

in children.

**5.2. Surgery**

is the most commonly used.

*5.2.1. Simple excision*

A flap is a portion of one or more tissues transferred from a donor site to a receiver maintaining a neurovascular connection ("pedicle").

The use of flap has many advantages including that of allow the repair of the defect by means of tissue similar or equal to those of the receiving site. It is very important for specialized tissues as lip or eyelid.

The skin flap, composed by skin only or including subcutaneous tissue, is transferred from one part of the body to another with its neurovascular pedicle or attached by just a margin to preserve vascular support.

There are two types of skin flap: those which rotate around a pivot point (rotation, transposi‐ tion and interpolation flaps) and advancement flaps (single pedicle, V – Y, Y – V and bi – pedunculated.

The rotation flap is a semicircular flap that rotates from its pivot point to the receiving site (Fig.3). If tension is too high, the incision can be extended by a reverse incision from pivot point along the base of the flap (backcut).

The transposition flap is composed by a rectangle or square of skin and subcutaneous tissue that rotate around a pivot point close to lesion.

The advancement flap is brought forward on the lesion without rotation.

This can have only one pedicle, so feeding is maintained by exploiting the elasticity of the skin.

The advancement V – Y flap more than a flap is a V incision whose sides are closed in such a way that the final suture gives a Y.

often experience appearance-related teasing and bullying during the course of their school career [46]. A link between appearance-related teasing, body dissatisfaction and general psychological disturbance has been discussed by Gilbert and Thompson [47]. The physical and psychological changes associated with adolescence increase the importance of physical appearance, and having a disfigurement during this period may present particular challenges. Image counts in the dating game, and joining an acceptable social grouping can be difficult if social confidence has been in some way weakened. Harter [48] reported that teenagers who believed their appearance determined their self-worth had lower self-esteem and greater depression than adolescents who believed their self-worth determined their feelings about

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Nonetheless, indications to surgical treatment should be carefully evaluated when the motivation of patient is doubtful. Treatment decisions made during childhood, adolescence and adulthood can be stressful. Deciding whether or not to undergo appearance-altering surgery may not be easily accepted, and those affected can question any motivation for putting themselves through the associated stress [49] Furthermore, expectations about outcomes may be unrealistic, and can generate disappointment when the aesthetic result becomes apparent. Given the multifaceted nature of surgical adjustment, the prevailing model of care needs to be expanded to offer psychosocial support and intervention as routine adjuncts or alternatives to surgical treatment. Interventions need to be carefully planned to take account of individual physical (e.g. growth) and social issues and the child with a visible difference or disfiguring lesion should receive continuing psychological support, preoperative assessment and follow

Surgical excision with reconstruction, is the mainstay of treatment. If direct closure after complete excision is not possible, reconstruction may include excision with skin grafts, skin flaps, tissue expansion with subsequent flap rotation or full thickness skin grafting, autologous cultured human epithelium, artificial skin replacement, and free tissue transfer after tissue expansion [50, 51, 52] Chemical peels, dermabrasion, and laser treatments are adjunctive treatment choices, that have not been demonstrated to decrease the malignant potential, because of incomplete removal of cells in the treated area. If surgical excision is not feasible, management consists of examination and high-quality photographic documentation for life.

Serial excision of large congenital nevi by skin expansion should preferably start in early months of life, for their malignant potential and for their size, requiring many surgical stages. It is usually addressed at age 6 months, to decrease anesthetic and surgical risks [52, 53, 54]. Attempts to complete the treatment of particularly disfiguring lesions is preferably carried out before age 5, when possible, or at least in the pre-adolescent, to prevent important psychosocial implications linked to the different developmental stages of the child and to parental behavior [55]. The goals of treatment are to remove all or as much as feasible of the CNN and reconstruct the defect, preserving function and maintaining the aesthetic appearance. Each case requires tailoring of the operations to fit the anatomic defect and to respect anatomic units and relaxed skin tension lines when possible. Excision begins in the 6-9 month range, placing procedures

their appearance.

3-6 months apart.

up during the course of treatment.

**Figure 3.** Rotational flap to excise a peliungueal nevus

## **6. Treatment of congenital nevi in the pediatric age**

#### **6.1. Indications and timing**

Surgery of congenital nevi is predominantly indicated for preventive reasons, related to the risk of developing a malignant melanoma within the lesion during life. This indication has been discussed more extensively elsewhere in the chapter. Evaluation of all small and medium CNN for prophylactic excision should take place before the patient is aged 12 years. After this age, malignant potential rises sharply. Some authors advocate prophylactic excision of all CNN [35, 41, 42],

whereas others advocate clinical monitoring of small [38] or both small and medium nevi [43] The incidence of malignant melanoma appears higher in large congenital nevi, in the scalp, back, and buttocks and requires removal first. This increase in incidence is likely secondary to the total body surface area. The presence of an enlarging nodular mass indicates malignant change and requires immediate treatment. This mass may represent a rare neuroectodermal sarcoma.

The other important indication to treatment is the possible discomfort due to the presence of a visible difference or even disfigurement. Psychological implications of this condition can involve both parents and children, in a different manner for different ages. Parents feelings about their child's appearance are likely to influence the child's perception of his or her disfigurement, the developing body image and feelings of self-worth [44]. Parental strategies to deal with a physical difference vary considerably. Some discuss it openly, others may act as if it does not exist, parents may be over-protective or children may avoid issues related to their appearance for fear of upsetting their parents 45. Most children with a visible difference often experience appearance-related teasing and bullying during the course of their school career [46]. A link between appearance-related teasing, body dissatisfaction and general psychological disturbance has been discussed by Gilbert and Thompson [47]. The physical and psychological changes associated with adolescence increase the importance of physical appearance, and having a disfigurement during this period may present particular challenges. Image counts in the dating game, and joining an acceptable social grouping can be difficult if social confidence has been in some way weakened. Harter [48] reported that teenagers who believed their appearance determined their self-worth had lower self-esteem and greater depression than adolescents who believed their self-worth determined their feelings about their appearance.

Nonetheless, indications to surgical treatment should be carefully evaluated when the motivation of patient is doubtful. Treatment decisions made during childhood, adolescence and adulthood can be stressful. Deciding whether or not to undergo appearance-altering surgery may not be easily accepted, and those affected can question any motivation for putting themselves through the associated stress [49] Furthermore, expectations about outcomes may be unrealistic, and can generate disappointment when the aesthetic result becomes apparent. Given the multifaceted nature of surgical adjustment, the prevailing model of care needs to be expanded to offer psychosocial support and intervention as routine adjuncts or alternatives to surgical treatment. Interventions need to be carefully planned to take account of individual physical (e.g. growth) and social issues and the child with a visible difference or disfiguring lesion should receive continuing psychological support, preoperative assessment and follow up during the course of treatment.

**6. Treatment of congenital nevi in the pediatric age**

Surgery of congenital nevi is predominantly indicated for preventive reasons, related to the risk of developing a malignant melanoma within the lesion during life. This indication has been discussed more extensively elsewhere in the chapter. Evaluation of all small and medium CNN for prophylactic excision should take place before the patient is aged 12 years. After this age, malignant potential rises sharply. Some authors advocate prophylactic excision of all CNN

whereas others advocate clinical monitoring of small [38] or both small and medium nevi [43] The incidence of malignant melanoma appears higher in large congenital nevi, in the scalp, back, and buttocks and requires removal first. This increase in incidence is likely secondary to the total body surface area. The presence of an enlarging nodular mass indicates malignant change and requires immediate treatment. This mass may represent a rare neuroectodermal

The other important indication to treatment is the possible discomfort due to the presence of a visible difference or even disfigurement. Psychological implications of this condition can involve both parents and children, in a different manner for different ages. Parents feelings about their child's appearance are likely to influence the child's perception of his or her disfigurement, the developing body image and feelings of self-worth [44]. Parental strategies to deal with a physical difference vary considerably. Some discuss it openly, others may act as if it does not exist, parents may be over-protective or children may avoid issues related to their appearance for fear of upsetting their parents 45. Most children with a visible difference

**6.1. Indications and timing**

342 Melanoma - From Early Detection to Treatment

**Figure 3.** Rotational flap to excise a peliungueal nevus

[35, 41, 42],

sarcoma.

Surgical excision with reconstruction, is the mainstay of treatment. If direct closure after complete excision is not possible, reconstruction may include excision with skin grafts, skin flaps, tissue expansion with subsequent flap rotation or full thickness skin grafting, autologous cultured human epithelium, artificial skin replacement, and free tissue transfer after tissue expansion [50, 51, 52] Chemical peels, dermabrasion, and laser treatments are adjunctive treatment choices, that have not been demonstrated to decrease the malignant potential, because of incomplete removal of cells in the treated area. If surgical excision is not feasible, management consists of examination and high-quality photographic documentation for life.

Serial excision of large congenital nevi by skin expansion should preferably start in early months of life, for their malignant potential and for their size, requiring many surgical stages. It is usually addressed at age 6 months, to decrease anesthetic and surgical risks [52, 53, 54]. Attempts to complete the treatment of particularly disfiguring lesions is preferably carried out before age 5, when possible, or at least in the pre-adolescent, to prevent important psychosocial implications linked to the different developmental stages of the child and to parental behavior [55]. The goals of treatment are to remove all or as much as feasible of the CNN and reconstruct the defect, preserving function and maintaining the aesthetic appearance. Each case requires tailoring of the operations to fit the anatomic defect and to respect anatomic units and relaxed skin tension lines when possible. Excision begins in the 6-9 month range, placing procedures 3-6 months apart.

## **6.2. Surgical techniques**

Technical choices vary depending on nevus size and anatomical site and may be challenging in some cases of giant nevi.

General considerations, including anatomical and surgical principles, should be remembered.

Incisions are planned according to the orientation of the relaxed skin tension lines (RSTL) when possible, with attention to the most favorable and less visible site of the resulting scar, especially when the use of skin expanders is required.

A variable amount of subcutaneous tissue should be included in the excision, for its diagnostic value in the rare occasional finding of melanoma in the specimen. This amount is thinner in facial areas, to avoid nerve injuries, but enough to include hair follicles.

#### *6.2.1. Small congenital nevi*

Excision of small congenital nevi (diameter ≤1.5 cm) is usually performed with 2 mm margins of normally appearing skin, by simple excision. In special areas, as some parts of the nose, lip, eyelid or ear, serial excision or rotation, advancement or transposition flaps are often necessary.

### *6.2.2. Medium size congenital nevi*

Excision of medium size nevi (diameter >1.5 <19 cm) can be achieved by serial excision or tissue expansion.

#### **Serial excision**

The efficacy of serial excision for the treatment of medium size congenital nevi has been reported by different authors [56] and it is the indication of choice when the procedure can be easily planned in 2 stages [57].

In children this indication can be extended to larger lesions, requiring more than 2 stages, when considering the possibility of avoiding morbidity related to tissue expansion, longer operating time of every stage, multiple expanding percutaneous injections and poor compliance by the patient. When surgical planning suggests too many operations to complete the removal of the lesion, tissue expansion should be seriously considered as an alternative.

When the resulting scar is desired to fall in a crease or for the treatment of particular anatomical sites, as nasal ala, oral commissure, lateral canthus, some modifications may be required. The fusiform excision may be planned to be eccentric and the skin undermined more on one side, to move the tissue in one direction rather than the opposite one. The direction of the prevalent movement can be towards a natural crease, the border of an aesthetic unit or an anatomical

**Figure 5.** Serial excision of medium size congenital nevus, with final positioning of the scar in the nasogenien crease

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In difficult anatomical sites and for wider intermediate sized lesions flap surgery is also indicated to obtain excision without tissue loss or distortion. An advancement flap may be used when its incision lines can be drawn along the borders of different aesthetic units. For example, in the case of a round nevus on the lateral aspect of nasal pyramid, incisions of an advancement flap could be outlined on the infraorbital and nasogenien folds to hide scars in

area not to be distorted, as nasal ala or oral commissure (Fig.5)

**Figure 4.** First stage of serial excision with the long axis of the lesion parallel tothe RSTL

**Flap surgery**

#### **Techniques of serial excision:**

A symmetric, fusiform ellipse is drawn within the lesion, parallel to the RSTLs and the margins are undermined enough to obtain a tension free suture (Fig. 4)

In the subsequent period the surrounding skin is going to stretch and and adapt, releasing tension on the scar. After a minimum of 3 months a second excision is performed to complete nevus removal.

**Figure 4.** First stage of serial excision with the long axis of the lesion parallel tothe RSTL

**Figure 5.** Serial excision of medium size congenital nevus, with final positioning of the scar in the nasogenien crease

When the resulting scar is desired to fall in a crease or for the treatment of particular anatomical sites, as nasal ala, oral commissure, lateral canthus, some modifications may be required. The fusiform excision may be planned to be eccentric and the skin undermined more on one side, to move the tissue in one direction rather than the opposite one. The direction of the prevalent movement can be towards a natural crease, the border of an aesthetic unit or an anatomical area not to be distorted, as nasal ala or oral commissure (Fig.5)

## **Flap surgery**

**6.2. Surgical techniques**

344 Melanoma - From Early Detection to Treatment

in some cases of giant nevi.

*6.2.1. Small congenital nevi*

*6.2.2. Medium size congenital nevi*

easily planned in 2 stages [57].

**Techniques of serial excision:**

expansion.

**Serial excision**

nevus removal.

especially when the use of skin expanders is required.

facial areas, to avoid nerve injuries, but enough to include hair follicles.

Technical choices vary depending on nevus size and anatomical site and may be challenging

General considerations, including anatomical and surgical principles, should be remembered.

Incisions are planned according to the orientation of the relaxed skin tension lines (RSTL) when possible, with attention to the most favorable and less visible site of the resulting scar,

A variable amount of subcutaneous tissue should be included in the excision, for its diagnostic value in the rare occasional finding of melanoma in the specimen. This amount is thinner in

Excision of small congenital nevi (diameter ≤1.5 cm) is usually performed with 2 mm margins of normally appearing skin, by simple excision. In special areas, as some parts of the nose, lip, eyelid or ear, serial excision or rotation, advancement or transposition flaps are often necessary.

Excision of medium size nevi (diameter >1.5 <19 cm) can be achieved by serial excision or tissue

The efficacy of serial excision for the treatment of medium size congenital nevi has been reported by different authors [56] and it is the indication of choice when the procedure can be

In children this indication can be extended to larger lesions, requiring more than 2 stages, when considering the possibility of avoiding morbidity related to tissue expansion, longer operating time of every stage, multiple expanding percutaneous injections and poor compliance by the patient. When surgical planning suggests too many operations to complete the removal of the

A symmetric, fusiform ellipse is drawn within the lesion, parallel to the RSTLs and the margins

In the subsequent period the surrounding skin is going to stretch and and adapt, releasing tension on the scar. After a minimum of 3 months a second excision is performed to complete

lesion, tissue expansion should be seriously considered as an alternative.

are undermined enough to obtain a tension free suture (Fig. 4)

In difficult anatomical sites and for wider intermediate sized lesions flap surgery is also indicated to obtain excision without tissue loss or distortion. An advancement flap may be used when its incision lines can be drawn along the borders of different aesthetic units. For example, in the case of a round nevus on the lateral aspect of nasal pyramid, incisions of an advancement flap could be outlined on the infraorbital and nasogenien folds to hide scars in

*6.2.4. Tissue expansion technique*

excision of giant congenital nevi (Fig.8).

their pre-expanded size and number [58, 59].

avoided whenever possible.

Although multistaged direct excision, described elsewhere about medium size nevi, is sometimes feasible for the treatment of large lesions, skin expansion is the treatment of choice and will be discussed in more detail. In general terms, expansion of tissue is used to improve rotation, transposition or advancement of local or regional flaps, or to increase the harvest of full-thickness skin grafts. In adults, aside from their use in breast reconstruction, tissue expanders are used primarily for secondary burn and trauma reconstruction in the head and neck region. In the pediatric population, expanders have been used in a variety of reconstruc‐ tive procedures. The most common indication in children is to reconstruct defects left by

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**Figure 8.** Skin expansion to excise a large congenital nevus of the gluteal region

Tissue expansion is contraindicated in infected skin. Although expansion is possible in radiated or scarred tissue, it is associated with a much higher complication rate and should be

Surgical technique of skin expansion relies on the ability of skin and soft tissues to grow by generation of new tissue in response to tension. Tensive impulse is generated by implanting a subcutaneous device (expander) that is inflated over a period of weeks; new tissue is generated in response to the constant stretch caused by the progressive inflation. An increase in skin surface area after expansion is due to generation of new tissue rather than the stretching of existing skin, as supported by numerous studies. Fibroblast and epidermal hyperplasia induced by mechanical stress have been observed in culture. Histological response to expan‐ sion is similar in adult and pediatric skin. Within 1-3 weeks of expansion, the epidermis begins to thicken and the dermis thins while skin appendages do not change. The subcutaneous fat atrophy. Cellular proliferation reduces the resting tension of the skin over time, enabling further expansion to take place. Once the process is complete, the expanded skin eventually returns to its baseline thickness. The vessels of the skin and subcutaneous tissue also resume

Expanders are available in a variety of shapes and sizes, and there is no absolute ideal expander for a given site or condition. Expanders have different types of filling ports. These can be internal to the expander or remote and connected by a tube of various length, that is usually adjustable by the surgeon. Most experienced surgeons recommend using remote ports. These should be placed away from the expander. Internal ports have both a higher failure rate and

**Figure 6.** An advancement flap is drawn in the infraorbital area along infraorbital and nasogenien creases.

**Figure 7.** A bilobed flap is transposed from the retroauricular crease.

these creases (Fig.6). On the auricle, due to the adherence of local skin, a sufficient amount of tissue can be obtained from the retroauricular fold by a transposition flap (Fig.7).

#### *6.2.3. Large or giant congenital nevi*

Treatment of large or giant congenital melanocytic nevi (CGMN) (diameter>19 cm) always requires multiple surgical stages and complex strategies.

#### *6.2.4. Tissue expansion technique*

these creases (Fig.6). On the auricle, due to the adherence of local skin, a sufficient amount of

Treatment of large or giant congenital melanocytic nevi (CGMN) (diameter>19 cm) always

tissue can be obtained from the retroauricular fold by a transposition flap (Fig.7).

**Figure 6.** An advancement flap is drawn in the infraorbital area along infraorbital and nasogenien creases.

*6.2.3. Large or giant congenital nevi*

346 Melanoma - From Early Detection to Treatment

requires multiple surgical stages and complex strategies.

**Figure 7.** A bilobed flap is transposed from the retroauricular crease.

Although multistaged direct excision, described elsewhere about medium size nevi, is sometimes feasible for the treatment of large lesions, skin expansion is the treatment of choice and will be discussed in more detail. In general terms, expansion of tissue is used to improve rotation, transposition or advancement of local or regional flaps, or to increase the harvest of full-thickness skin grafts. In adults, aside from their use in breast reconstruction, tissue expanders are used primarily for secondary burn and trauma reconstruction in the head and neck region. In the pediatric population, expanders have been used in a variety of reconstruc‐ tive procedures. The most common indication in children is to reconstruct defects left by excision of giant congenital nevi (Fig.8).

Tissue expansion is contraindicated in infected skin. Although expansion is possible in radiated or scarred tissue, it is associated with a much higher complication rate and should be avoided whenever possible.

Surgical technique of skin expansion relies on the ability of skin and soft tissues to grow by generation of new tissue in response to tension. Tensive impulse is generated by implanting a subcutaneous device (expander) that is inflated over a period of weeks; new tissue is generated in response to the constant stretch caused by the progressive inflation. An increase in skin surface area after expansion is due to generation of new tissue rather than the stretching of existing skin, as supported by numerous studies. Fibroblast and epidermal hyperplasia induced by mechanical stress have been observed in culture. Histological response to expan‐ sion is similar in adult and pediatric skin. Within 1-3 weeks of expansion, the epidermis begins to thicken and the dermis thins while skin appendages do not change. The subcutaneous fat atrophy. Cellular proliferation reduces the resting tension of the skin over time, enabling further expansion to take place. Once the process is complete, the expanded skin eventually returns to its baseline thickness. The vessels of the skin and subcutaneous tissue also resume their pre-expanded size and number [58, 59].

Expanders are available in a variety of shapes and sizes, and there is no absolute ideal expander for a given site or condition. Expanders have different types of filling ports. These can be internal to the expander or remote and connected by a tube of various length, that is usually adjustable by the surgeon. Most experienced surgeons recommend using remote ports. These should be placed away from the expander. Internal ports have both a higher failure rate and a greater incidence of accidental expander rupture. In children, the use of internal ports is associated with a higher rate of exposure of the expander due to the pressure exerted on the skin by the port. As a rule, expansion proceeds best when the expander rests on a firm base like the ribs or skull. When placed within the abdominal wall, for example, expansion tends to be tess predictable. The incisions for expander placement and the remote port should be placed where they will not interfere with later advancement or compromise the blood supply to the expanded tissue. If possible the incisions for expander placement are placed in the proposed area to be excised. Incisions are never placed parallel to the edges of the expander. This creates a situation that increases implant exposure, additional scar tissue outside the lesion, possible stretching of the scar and a delay in inflation of expander. Incisions should be radial or almost perpendicular to the expander or in the form of a V or W

in order to gain extra length. The donor site should be closed in layers after the implant capsule

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**Scalp**. Although tissue expansion does not increase the number of hair follicles, the size of the hair-bearing region can be doubled without a noticeable decrease in hair density. As such, tissue expansion may be used to reconstruct the scalp when removal of a medium or large nevus is needed. Expanders are most commonly placed in the occipital or posterior parietal regions. They are placed under the galea, superficial to the periosteum. It usually requires up to 12 weeks to complete the expansion in children. Radial scoring of the galea at the time of surgery can facilitate the process. Once the expansion is complete, flaps are advanced or transposed, based on named arteries of the scalp. It is important to orient flaps so that the correct direction of hair growth is maintained. Although galeal scoring or capuslotomy

**Forehead**. The brow position is the most important structure to preserve during forehead expansion. When possible, two or more expanders are used with incisions hidden within the

Midforehead nevi are best treated using an expansion of bilateral normal forehead segments and medial advancement of the flaps, placing scars along the brow and at or posterior to the hairline. Hemiforehead nevi often require serial expansion of the uninvolved area of the forehead to reduce the need for a back-cut. Nevi of the supraorbital and temporal forehead can be treated with a transposition of the expanded normal skin medial to the nevus. When the temporal scalp is minimally involved with nevus, the parietal scalp can be expanded and advanced to create the new hairline. When the temporoparietal scalp is also involved with nevus, a combined advancement and transposition flap provides the proper hair direction for the temporal hairline and allows significantly greater movement of the expanded flap. Once the brow is significantly elevated on either the ipsilateral or contralateral side from the reconstruction, it can only be returned to the preoperative position with the interposition of additional, non–hair-bearing forehead skin. The largest expander possible beneath the uninvolved forehead skin should always be used, occasionally even carrying the expander

**Face and Neck.** The skin of the neck and face is relatively thin. Therefore, multiple expanders with smaller volumes are preferable to a single large expander. In general, however, a single larger expander is preferable to several smaller expanders. Careful planning is essential in determining where to place the expanders, and where incisions should be located in order to preserve aesthetic units, facial symmetry and matching skin color and to avoid distortion of the eyelids and oral commissure. The expander is usually placed above the platysma muscle to avoid risk of facial nerve injury and to keep the flap from being excessively bulky. The expanded flaps are positioned by advancement, rotation, or transposition. Incisions should be placed in skin creases such as the nasolabial fold or along the margins of aesthetic units. Expanding the hairless skin adjacent to the mastoid region can increase the available tissue for reconstructive procedures of the ear. The skin above the clavicle can be expanded to provide

incisions can be useful, wide undermining is a safer method of recruiting tissue.

is excised.

hairline.

under the lesion [62].

full-thickness skin grafts to the face.

The broad base of the V or W should be directed toward the expander, thus facilitating implant insertion and inflation because the lines of tension are perpendicular to the wound. The open end of the V should be at least 2 to 3 cm from the pocket to accommodate expansion. In addition, a sigma (lazy S) incision can also be beneficial in instances where partial excision of some of the lesion might be helpful during the insertion phase. By this approach, partial excision can be done while an expander is placed. Once the wounds heal, the expander can then be inflated without worry because the end of the incision is almost radial to the expansion process [60]. The expander should be placed on top of the deep fascia (or subgaleal in the scalp), unless the plan is to incorporate muscle into the expanded flap. The pocket should always be larger than the base diameter of the expander. Blunt dissection in a single fascial plane is safest for preserving blood supply. Filling the expanders intraoperatively with sufficient saline to eliminate dead space can prevent postoperative bleeding and hematoma. An alternative to traditional prolonged expansion is immediate intraoperative expansion combined with broad undermining of the defect. In rapid expansion, the skin initially expands due to its elasticity and the displacement of interstitial fluid. Within minutes, the alignment of the collagen fibers changes due to the stretch. This process yields up to 20% more tissue for flap coverage. Intraoperative expansion is indicated for relatively small defects, such as in coverage of defects of the ear.

The rate of inflation is variable and largely based on surgeon preference. Patient comfort and signs of tissue perfusion, such as tension, color, and capillary refill, guide the filling rate. Filling is usually initiated 7-10 days postoperatively and performed once or twice a week, based on the above mentioned criteria and patient tolerance. The rate of expansion depends both on the body site as well as patient factors. Some skin is more amenable to expansion, and some patients can tolerate the discomfort better than others [61]. Tissue expansion should continue until the expanded area is larger than the defect, usually up to 2 months. As a general rule, the diameter of the expanded flap should be 2-3 times the diameter of the skin that is to be excised

Most surgeons overinflate tissue expanders beyond the manufacturer's recommended maximum capacity. Studies have demonstrated that significant overinflation is possible before weakening or rupturing.

The use of rotation and transposition flaps enables the transfer of tension from the tip of the flap more proximally to its base. A single or double back-cut can be performed prior to inset in order to gain extra length. The donor site should be closed in layers after the implant capsule is excised.

a greater incidence of accidental expander rupture. In children, the use of internal ports is associated with a higher rate of exposure of the expander due to the pressure exerted on the skin by the port. As a rule, expansion proceeds best when the expander rests on a firm base like the ribs or skull. When placed within the abdominal wall, for example, expansion tends to be tess predictable. The incisions for expander placement and the remote port should be placed where they will not interfere with later advancement or compromise the blood supply to the expanded tissue. If possible the incisions for expander placement are placed in the proposed area to be excised. Incisions are never placed parallel to the edges of the expander. This creates a situation that increases implant exposure, additional scar tissue outside the lesion, possible stretching of the scar and a delay in inflation of expander. Incisions should be

The broad base of the V or W should be directed toward the expander, thus facilitating implant insertion and inflation because the lines of tension are perpendicular to the wound. The open end of the V should be at least 2 to 3 cm from the pocket to accommodate expansion. In addition, a sigma (lazy S) incision can also be beneficial in instances where partial excision of some of the lesion might be helpful during the insertion phase. By this approach, partial excision can be done while an expander is placed. Once the wounds heal, the expander can then be inflated without worry because the end of the incision is almost radial to the expansion process [60]. The expander should be placed on top of the deep fascia (or subgaleal in the scalp), unless the plan is to incorporate muscle into the expanded flap. The pocket should always be larger than the base diameter of the expander. Blunt dissection in a single fascial plane is safest for preserving blood supply. Filling the expanders intraoperatively with sufficient saline to eliminate dead space can prevent postoperative bleeding and hematoma. An alternative to traditional prolonged expansion is immediate intraoperative expansion combined with broad undermining of the defect. In rapid expansion, the skin initially expands due to its elasticity and the displacement of interstitial fluid. Within minutes, the alignment of the collagen fibers changes due to the stretch. This process yields up to 20% more tissue for flap coverage. Intraoperative expansion is indicated for relatively small defects, such as in coverage of defects

The rate of inflation is variable and largely based on surgeon preference. Patient comfort and signs of tissue perfusion, such as tension, color, and capillary refill, guide the filling rate. Filling is usually initiated 7-10 days postoperatively and performed once or twice a week, based on the above mentioned criteria and patient tolerance. The rate of expansion depends both on the body site as well as patient factors. Some skin is more amenable to expansion, and some patients can tolerate the discomfort better than others [61]. Tissue expansion should continue until the expanded area is larger than the defect, usually up to 2 months. As a general rule, the diameter of the expanded flap should be 2-3 times the diameter of the skin that is to be excised Most surgeons overinflate tissue expanders beyond the manufacturer's recommended maximum capacity. Studies have demonstrated that significant overinflation is possible before

The use of rotation and transposition flaps enables the transfer of tension from the tip of the flap more proximally to its base. A single or double back-cut can be performed prior to inset

radial or almost perpendicular to the expander or in the form of a V or W

348 Melanoma - From Early Detection to Treatment

of the ear.

weakening or rupturing.

**Scalp**. Although tissue expansion does not increase the number of hair follicles, the size of the hair-bearing region can be doubled without a noticeable decrease in hair density. As such, tissue expansion may be used to reconstruct the scalp when removal of a medium or large nevus is needed. Expanders are most commonly placed in the occipital or posterior parietal regions. They are placed under the galea, superficial to the periosteum. It usually requires up to 12 weeks to complete the expansion in children. Radial scoring of the galea at the time of surgery can facilitate the process. Once the expansion is complete, flaps are advanced or transposed, based on named arteries of the scalp. It is important to orient flaps so that the correct direction of hair growth is maintained. Although galeal scoring or capuslotomy incisions can be useful, wide undermining is a safer method of recruiting tissue.

**Forehead**. The brow position is the most important structure to preserve during forehead expansion. When possible, two or more expanders are used with incisions hidden within the hairline.

Midforehead nevi are best treated using an expansion of bilateral normal forehead segments and medial advancement of the flaps, placing scars along the brow and at or posterior to the hairline. Hemiforehead nevi often require serial expansion of the uninvolved area of the forehead to reduce the need for a back-cut. Nevi of the supraorbital and temporal forehead can be treated with a transposition of the expanded normal skin medial to the nevus. When the temporal scalp is minimally involved with nevus, the parietal scalp can be expanded and advanced to create the new hairline. When the temporoparietal scalp is also involved with nevus, a combined advancement and transposition flap provides the proper hair direction for the temporal hairline and allows significantly greater movement of the expanded flap. Once the brow is significantly elevated on either the ipsilateral or contralateral side from the reconstruction, it can only be returned to the preoperative position with the interposition of additional, non–hair-bearing forehead skin. The largest expander possible beneath the uninvolved forehead skin should always be used, occasionally even carrying the expander under the lesion [62].

**Face and Neck.** The skin of the neck and face is relatively thin. Therefore, multiple expanders with smaller volumes are preferable to a single large expander. In general, however, a single larger expander is preferable to several smaller expanders. Careful planning is essential in determining where to place the expanders, and where incisions should be located in order to preserve aesthetic units, facial symmetry and matching skin color and to avoid distortion of the eyelids and oral commissure. The expander is usually placed above the platysma muscle to avoid risk of facial nerve injury and to keep the flap from being excessively bulky. The expanded flaps are positioned by advancement, rotation, or transposition. Incisions should be placed in skin creases such as the nasolabial fold or along the margins of aesthetic units. Expanding the hairless skin adjacent to the mastoid region can increase the available tissue for reconstructive procedures of the ear. The skin above the clavicle can be expanded to provide full-thickness skin grafts to the face.

**Trunk**. Unlike the head and neck, there are very few critical landmarks on the trunk that must be preserved. Aside from the breast and nipple-areola complex, distortion of the skin and soft tissues of the trunk is well-tolerated. For defects requiring excision, multiple expanders surrounding the defect are often employed. Expanders can also be used to expand the skin of the abdomen for use as a donor site of full-thickness skin grafts.

Once decided to remove a suspicious lesion, it is recommended to perform a 1- to 2-mm circumferential margin. There are no prospective data to provide an evidence-based approach

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It has long been suggested that malignant cells may be shed into the bloodstream during any given surgical procedure for cancer. While there is no evidence to suggest that an incisional

There may be times that the incision is to big that the tissue are not able to cover the skin defect, in this case is possible to use a skin graft, or in order to avoid a graft, the surgical defect may

The orientation of the incision should follow the relaxed skin tension lines (RSTLs, also known as lines of Langer), however, at the level of the limbs incisions parallel to the major axis of the

In the setting of dysplastic changes or once the diagnosis of melanoma is established, in children, surgical excision should be performed with the same excision margins recommended for adults by the National Comprehensive Cancer Network (NCCN) in 2007, and depends on the Breslow depth of the primary lesion, Clark's level of tumor invasion may provide addi‐

The basic oncologic criteria of surgery are: the resection margins and the depth of the skin

When there is an in situ melanoma, excision should include 0,5 centimeter of normal skin surronding the tumor and takes off the skin layers down to the fat; in removing an invasive melanoma that is 2 mm thick the margins are extended to 1 cm and the excision goes through all skin layers and down to the fascia; margins are 2 cm for lesions greater than 2 mm in

The depth of excision can reach muscolaris fascia, whose removal has no oncological meaning,

An exception is the localization to the face, in these cases also with melanomas more than 4

In recent years, great importance was served to sentinel lymph node biopsy (SLNB) for the detection of lymph node metastases, in fact in adult melanoma therapy, it has become a mandatory procedure in the current AJCC staging system; however its use in the pediatric

Lymph node are the most common site of initial metastases [70], the lack of disease in the

SLNB will select, with a minimally invasive technique, patients who should undergo regional lymph node dissection for clinically occult loco-regional metastases, so as to avoid completion

furthermore the preservation of the fascia allows a better aesthetic result.

sentinel lymph node should indicate the lack of dissemination.

lymph node dissection if the sentinel node is negative.

biopsy does cause local spread of melanoma, it is generally not advocated. [67]

be closed using a rotational or advancement flap

tional prognostic value for thin melanomas [68].

limb are used, not to alter the paths of lymphatic drainage.

in this setting.

excision.

thickness. [69]

mm thick margins of 1 cm are used.

population has been limited.

**Extremities**. Tissue expansion in the extremities has been reported to have a higher compli‐ cation rate, in comparison to other regions and therefore, especially in children, should not be a first choice. Blood supply and drainage of the extremities is inferior to that of the trunk and head. This predisposes limbs, especially below the knee, to an increased rate of wound complications such as infection, dehiscence and prosthesis extrusion. Multiple expanders are usually required in the extremites.

**Complications**. Among all patients, the major complication rate is about 10% and includes implant exposure, deflation, and wound dehiscence. Minor complications also occur in about 10% of patients. These include filling port problems, seroma, hematoma, infection and delayed healing.

Patients under the age of 7 have the highest risk of complications. One explanation for this is that young children are more prone to expander rupture due to external pressure on the expanded skin. Expansion in the extremities caries twice the risk of complication compared to other regions. The use of tissue expansion in congenital nevi has a 5-7% complication rate. Tissue that has undergone serial expansion (two or more prior expansions) is at a higher risk for a major complication.

## **7. Surgery of primary melanoma**

Pediatric melanoma is rare but increasing in incidence [63] limited options are possible for treatment. Early diagnosis and surgical management are the cornerstone of therapy and must adhere to the guidelines estabilished by the American Joint Committee on Cancer (AJCC) [64] Diagnosis of melanoma in children is more difficult than in adults, it relates to a number of variables, so many criteria used in adults are of limited value, for example the natural evolution of congenital and acquired nevi during childhood and adolescence [65] Historically, a wide excision with 5-cm margins with regional lymph node dissection was recommended for all melanomas. This indication, dating back 1907, was based on evaluations following a single necropsy, on a patient with advanced melanoma, assuming that in this way, all possible neoplastic foci would have been eliminated.

Furthermore, the indications suggested to extend the excision below the fascia, so as to also remove the superficial vascular and lymphatic structures.

This attitude has remained unchanged, until Breslow and Match described the treatment of melanoma with narrow margins [66]

Once decided to remove a suspicious lesion, it is recommended to perform a 1- to 2-mm circumferential margin. There are no prospective data to provide an evidence-based approach in this setting.

**Trunk**. Unlike the head and neck, there are very few critical landmarks on the trunk that must be preserved. Aside from the breast and nipple-areola complex, distortion of the skin and soft tissues of the trunk is well-tolerated. For defects requiring excision, multiple expanders surrounding the defect are often employed. Expanders can also be used to expand the skin of

**Extremities**. Tissue expansion in the extremities has been reported to have a higher compli‐ cation rate, in comparison to other regions and therefore, especially in children, should not be a first choice. Blood supply and drainage of the extremities is inferior to that of the trunk and head. This predisposes limbs, especially below the knee, to an increased rate of wound complications such as infection, dehiscence and prosthesis extrusion. Multiple expanders are

**Complications**. Among all patients, the major complication rate is about 10% and includes implant exposure, deflation, and wound dehiscence. Minor complications also occur in about 10% of patients. These include filling port problems, seroma, hematoma, infection and delayed

Patients under the age of 7 have the highest risk of complications. One explanation for this is that young children are more prone to expander rupture due to external pressure on the expanded skin. Expansion in the extremities caries twice the risk of complication compared to other regions. The use of tissue expansion in congenital nevi has a 5-7% complication rate. Tissue that has undergone serial expansion (two or more prior expansions) is at a higher risk

Pediatric melanoma is rare but increasing in incidence [63] limited options are possible for treatment. Early diagnosis and surgical management are the cornerstone of therapy and must adhere to the guidelines estabilished by the American Joint Committee on Cancer (AJCC) [64] Diagnosis of melanoma in children is more difficult than in adults, it relates to a number of variables, so many criteria used in adults are of limited value, for example the natural evolution of congenital and acquired nevi during childhood and adolescence [65] Historically, a wide excision with 5-cm margins with regional lymph node dissection was recommended for all melanomas. This indication, dating back 1907, was based on evaluations following a single necropsy, on a patient with advanced melanoma, assuming that in this way, all possible

Furthermore, the indications suggested to extend the excision below the fascia, so as to also

This attitude has remained unchanged, until Breslow and Match described the treatment of

the abdomen for use as a donor site of full-thickness skin grafts.

usually required in the extremites.

350 Melanoma - From Early Detection to Treatment

for a major complication.

**7. Surgery of primary melanoma**

neoplastic foci would have been eliminated.

melanoma with narrow margins [66]

remove the superficial vascular and lymphatic structures.

healing.

It has long been suggested that malignant cells may be shed into the bloodstream during any given surgical procedure for cancer. While there is no evidence to suggest that an incisional biopsy does cause local spread of melanoma, it is generally not advocated. [67]

There may be times that the incision is to big that the tissue are not able to cover the skin defect, in this case is possible to use a skin graft, or in order to avoid a graft, the surgical defect may be closed using a rotational or advancement flap

The orientation of the incision should follow the relaxed skin tension lines (RSTLs, also known as lines of Langer), however, at the level of the limbs incisions parallel to the major axis of the limb are used, not to alter the paths of lymphatic drainage.

In the setting of dysplastic changes or once the diagnosis of melanoma is established, in children, surgical excision should be performed with the same excision margins recommended for adults by the National Comprehensive Cancer Network (NCCN) in 2007, and depends on the Breslow depth of the primary lesion, Clark's level of tumor invasion may provide addi‐ tional prognostic value for thin melanomas [68].

The basic oncologic criteria of surgery are: the resection margins and the depth of the skin excision.

When there is an in situ melanoma, excision should include 0,5 centimeter of normal skin surronding the tumor and takes off the skin layers down to the fat; in removing an invasive melanoma that is 2 mm thick the margins are extended to 1 cm and the excision goes through all skin layers and down to the fascia; margins are 2 cm for lesions greater than 2 mm in thickness. [69]

The depth of excision can reach muscolaris fascia, whose removal has no oncological meaning, furthermore the preservation of the fascia allows a better aesthetic result.

An exception is the localization to the face, in these cases also with melanomas more than 4 mm thick margins of 1 cm are used.

In recent years, great importance was served to sentinel lymph node biopsy (SLNB) for the detection of lymph node metastases, in fact in adult melanoma therapy, it has become a mandatory procedure in the current AJCC staging system; however its use in the pediatric population has been limited.

Lymph node are the most common site of initial metastases [70], the lack of disease in the sentinel lymph node should indicate the lack of dissemination.

SLNB will select, with a minimally invasive technique, patients who should undergo regional lymph node dissection for clinically occult loco-regional metastases, so as to avoid completion lymph node dissection if the sentinel node is negative.

SLNB was first described by Morton et al. in 1992 [71] The procedure is usually performed concurrent with re-excision of the primary lesion, and is advised for lesions thicker than 1 mm or for those between 0,76 mm and 1 mm with ulceration or reticular dermal invasion.

localised disease, ulceration has been recognized as an important predictor of outcome and growing consideration is given to the significance of melanoma thickness. New importance has been recognized to the number of lymph nodes involved, the significance of in-transit or satellite metastases, the description of the sites of metastases and the prognostic value of serum

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Future trials including paediatric and adolescent melanoma patients should incorporate this new staging system to achieve a wider interpretation of results from institutions and patient populations. In addition, the routine use of sentinel node biopsy for the staging of paediatric and adolescent melanoma is mandatory, in order to determine the prognostic and therapeutic value of this procedure in young patients and to compare these results with those reported in

Although in adult patients the routine use of chest and abdomen computed tomography is not recommended in literature, in paediatric patients it has been found useful in about 25% of cases to identify clinically undetectable metastases from thick localised melanomas or patients with

The routine use of magnetic resonance imaging (MRI) to detect brain metastases is not advocated. For localised lesions under 1.5mm thick, investigations include a complete blood

Positron emission tomography (PET) is a very useful tool in adults, but its use in paediatric

Early primary excision of melanoma is the mainstay of definitive treatment of the tumour. With the introduction of sentinel lymph node biopsy (SLNB) the treatment of patients with

The adoption of SLNB has led to selection of patients who do not need elective lymph node dissection (ELND) and in which the morbidity linked to this procedure can be avoided. The techniques of preoperative lymphoscintigraphy and sentinel lymph node (SLN) biopsy have become the standard of care for staging adult patients after detection of a primary melanoma. SLNB is particularly important in intermediate-thickness (1.2-3.5 mm) primary melanomas in order to indicate elective lymphadenectomy and has also a prognostic value [83]. SLNB is a very promising technique also in paediatric patients [84, 85, 86]. However, due to paucity of available data, the role of SLNB in paediatric patients is still debated, as concerns both its

SLN biopsy should be included in the surgical management of children. The indications for SLN biopsy in paediatric and adolescent patients are based on the adult literature and include the presence of lesions thicker than 1 mm, the presence of ulceration or a Clark's level of invasion of IV or V in patients with lesion thickness of less than 1 mm. The technique is the same as in adults. Excision with 2 mm margins of normal skin is performed. After diagnosis of melanoma, the patient undergoes SLNB for tumour thickness ≥ 1 mm followed by wide

count, serum chemistries including liver function tests, and a chest radiograph.

lactic dehydrogenase [23].

the adult literature [80]

patients has not been validated [23]

melanoma has been revolutioned.

prognostic [87] and therapeutic implications.

*8.2.2. Sentinel lymph nodes*

melanoma arising at an unknown primary site [81, 82]

The procedure involves injection of the primary cutaneous lesion site with technectium-99m sulfur colloid followed by lymphoscintigraphy in the nuclear medicine suite. This is done on the morning of scheduled re-excision, and the patient is brought to the operating room in the afternoon. The lesion is injected with approximately 1 ml of 1% isosulfan blue dye. The dye is allowed to travel through lymphatics for several minutes, and a hand held gamma counter is used to determine the area of maximal radiolabeled tracer intensity for lymph node sampling. An incision is made over the area identified to have the most active uptake of radiolabeled tracer as determined by the handled gamma probe and the preoperative lymphoscintigraphy. Upon examination of the draining lymph node basin, all nodes that are blue, palpable, or show significant activity with the gamma probe are excised and sent fresh to pathology [72].

The incision must be oriented so as to allow an eventual loco-regional lymphadenectomy, the lymph node is identified with the gamma camera and visually with the blue dye; the lymph node is removed after ligation of the afferent and efferent lymphatic vessels, after the removal it is necessary to evaluate "ex vivo" the radioactivity of the lymph node and the possible presence of other involved nodes.

A small lymphocele may result in postoperative period, usually with the possibility of spontaneous regression.

Elective regional lymph node dissection is subsequently performed if the result of the SLNB is positive for metastases [38].

## **8. Treatment of metastatic disease**

#### **8.1. Congenital melanoma and transplacental metastases**

Congenital melanoma as a result of placental transmission from a mother with metastatic melanoma is extremely rare, with only a few cases described in literature [73, 74, 75, 76, 77]

To date, metastatic disease transmission from fetus to mother has never been reported. [78, 79]

#### **8.2. Lymph node metastases**

#### *8.2.1. Staging*

After primary surgery and diagnosis of melanoma, staging of the disease is completed by pathologic detection of lymphatic involvement. Comprehensive staging guidelines for paediatric and adolescent melanoma have not been clearly established.

The American Joint Committee on Cancer (AJCC) provides a reproducible model on the natural history of melanoma and a detailed description of important prognostic variables. For localised disease, ulceration has been recognized as an important predictor of outcome and growing consideration is given to the significance of melanoma thickness. New importance has been recognized to the number of lymph nodes involved, the significance of in-transit or satellite metastases, the description of the sites of metastases and the prognostic value of serum lactic dehydrogenase [23].

Future trials including paediatric and adolescent melanoma patients should incorporate this new staging system to achieve a wider interpretation of results from institutions and patient populations. In addition, the routine use of sentinel node biopsy for the staging of paediatric and adolescent melanoma is mandatory, in order to determine the prognostic and therapeutic value of this procedure in young patients and to compare these results with those reported in the adult literature [80]

Although in adult patients the routine use of chest and abdomen computed tomography is not recommended in literature, in paediatric patients it has been found useful in about 25% of cases to identify clinically undetectable metastases from thick localised melanomas or patients with melanoma arising at an unknown primary site [81, 82]

The routine use of magnetic resonance imaging (MRI) to detect brain metastases is not advocated. For localised lesions under 1.5mm thick, investigations include a complete blood count, serum chemistries including liver function tests, and a chest radiograph.

Positron emission tomography (PET) is a very useful tool in adults, but its use in paediatric patients has not been validated [23]

## *8.2.2. Sentinel lymph nodes*

SLNB was first described by Morton et al. in 1992 [71] The procedure is usually performed concurrent with re-excision of the primary lesion, and is advised for lesions thicker than 1 mm

The procedure involves injection of the primary cutaneous lesion site with technectium-99m sulfur colloid followed by lymphoscintigraphy in the nuclear medicine suite. This is done on the morning of scheduled re-excision, and the patient is brought to the operating room in the afternoon. The lesion is injected with approximately 1 ml of 1% isosulfan blue dye. The dye is allowed to travel through lymphatics for several minutes, and a hand held gamma counter is used to determine the area of maximal radiolabeled tracer intensity for lymph node sampling. An incision is made over the area identified to have the most active uptake of radiolabeled tracer as determined by the handled gamma probe and the preoperative lymphoscintigraphy. Upon examination of the draining lymph node basin, all nodes that are blue, palpable, or show significant activity with the gamma probe are excised and sent fresh to pathology [72].

The incision must be oriented so as to allow an eventual loco-regional lymphadenectomy, the lymph node is identified with the gamma camera and visually with the blue dye; the lymph node is removed after ligation of the afferent and efferent lymphatic vessels, after the removal it is necessary to evaluate "ex vivo" the radioactivity of the lymph node and the possible

A small lymphocele may result in postoperative period, usually with the possibility of

Elective regional lymph node dissection is subsequently performed if the result of the SLNB

Congenital melanoma as a result of placental transmission from a mother with metastatic melanoma is extremely rare, with only a few cases described in literature [73, 74, 75, 76, 77]

To date, metastatic disease transmission from fetus to mother has never been reported. [78, 79]

After primary surgery and diagnosis of melanoma, staging of the disease is completed by pathologic detection of lymphatic involvement. Comprehensive staging guidelines for

The American Joint Committee on Cancer (AJCC) provides a reproducible model on the natural history of melanoma and a detailed description of important prognostic variables. For

paediatric and adolescent melanoma have not been clearly established.

presence of other involved nodes.

352 Melanoma - From Early Detection to Treatment

spontaneous regression.

is positive for metastases [38].

**8.2. Lymph node metastases**

*8.2.1. Staging*

**8. Treatment of metastatic disease**

**8.1. Congenital melanoma and transplacental metastases**

or for those between 0,76 mm and 1 mm with ulceration or reticular dermal invasion.

Early primary excision of melanoma is the mainstay of definitive treatment of the tumour. With the introduction of sentinel lymph node biopsy (SLNB) the treatment of patients with melanoma has been revolutioned.

The adoption of SLNB has led to selection of patients who do not need elective lymph node dissection (ELND) and in which the morbidity linked to this procedure can be avoided. The techniques of preoperative lymphoscintigraphy and sentinel lymph node (SLN) biopsy have become the standard of care for staging adult patients after detection of a primary melanoma. SLNB is particularly important in intermediate-thickness (1.2-3.5 mm) primary melanomas in order to indicate elective lymphadenectomy and has also a prognostic value [83]. SLNB is a very promising technique also in paediatric patients [84, 85, 86]. However, due to paucity of available data, the role of SLNB in paediatric patients is still debated, as concerns both its prognostic [87] and therapeutic implications.

SLN biopsy should be included in the surgical management of children. The indications for SLN biopsy in paediatric and adolescent patients are based on the adult literature and include the presence of lesions thicker than 1 mm, the presence of ulceration or a Clark's level of invasion of IV or V in patients with lesion thickness of less than 1 mm. The technique is the same as in adults. Excision with 2 mm margins of normal skin is performed. After diagnosis of melanoma, the patient undergoes SLNB for tumour thickness ≥ 1 mm followed by wide excision of the tumour site with 2 cm margins and primary closure or skin graft. SLNB is performed using preoperative lymphoscintigraphy, intraoperative blue dye injection around the site of excision and hand-held gamma probe for radio-localization [38,88]. One day before the operation, between 18.5 and 40 MBq of Tc-99m microcolloid is injected intradermally around the scar. The drainage of the colloid is localized by detecting radiation, and the location of the SLN is marked on the skin. The position of the SLN is confirmed with a handheld gamma probe before starting the operation. At the author's center the procedure is performed under epidural anaesthesia and sedation or general anaesthesia. As reported by some authors, subcutaneous infusion anaesthesia (SIA) can be useful [89]. Patent blue is additionally injected intradermally around the scar as standard procedure. Sentinel lymph node biopsy is then accomplished with the help of repeated measurements with the handheld gamma probe. The SLN(s) is (are) removed, and the wound is closed.

**8.3. Distant metastases**

*8.3.2. Radiotherapy*

cure for cutaneous melanoma [97].

**8.4. Prognosis**

people with metastatic melanoma.

*8.3.1. Treatment of disseminated disease*

The incidence of metastatic melanoma has increased over the last three decades, and the death rate continues to climb faster than that of most other cancers. According to the American Cancer Society, there were approximately 68,000 new cases of melanoma in the United States in 2009, and 8,700 melanoma-related deaths. Melanoma is difficult to treat once it has spread beyond the skin to other parts of the body (metastasized). Very few treatment options exist for

Surgical Treatment of Nevi and Melanoma in the Pediatric Age

http://dx.doi.org/10.5772/54632

355

Most reports describing the treatment of paediatric melanoma are from single institutions in which diagnostic criteria, staging and pathological evaluation of the primary tumour have varied significantly. Dacarbazine, which is the most active agent in adult melanoma, showed encouraging activity in four children with melanoma treated between 1975 and 1984 [96] Other traditional chemotherapeutic regimens have shown some efficacy in metastatic melanoma [23]. The availability of investigational therapies, such as interleukin-2, interferon alfa-2b and vaccines, has been generally restricted to patients who are older than 18 years of age and no prospective trials in adolescents have been performed. Collaborative efforts, now under discussion between paediatric and adult cooperative groups, should help facilitate the

Radiotherapy is rarely indicated in the management of primary paediatric melanoma. However, it should be considered in patients with head and neck melanomas at high risk for parotid or cervical metastases and in those who develop brain metastases. Brain metastases have been reported to occur during the course of the disease in up to 18% of children with melanoma [23]. Ultimately, as in adults, there is no effective therapy for metastatic melanoma in children. Therefore, the main focus of the parent, the paediatrician, and the dermatologist should be risk reduction and early detection of melanoma. The former consists primarily of avoiding intense sunlight exposure, using protective clothing and broad-spectrum sunblock, and educating children. Early detection requires a high index of clinical suspicion, especially by the paediatrician, who sees children with much more regularity than a dermatologist, of any rapidly growing or otherwise atypical pigmented lesion. In addition, the physician should recognize the elevated risk of any child with a family history of melanoma, GCMN, or dysplastic nevi. Again, prevention and early clinical diagnosis are the only current effective

The outcome for children and adolescents with melanoma also appears to be similar to that reported for adults and is dependent on the initial stage of the tumour. Patients with localised disease have an excellent outcome, whereas those with nodal and distant metastases have estimated 10-year survivals of only 60 and 25%, respectively. Outcome is also stage-dependent

enrollment of younger patients onto trials that use experimental therapies.

A comparison between adults and patients younger than 21 years who underwent either lymph node dissection or SLNB showed a higher rate of lymph node metastasis in the paediatric age (44%) as compared to the adult (23.9%). However this finding had no statistical significance. In this series, paediatric patients either with Stage I or Stage II disease showed a 94.4% 10-year survival, while patients with Stage III melanoma had a 60.1% 10-year survival [90]

Recent data show that although the SLNB positivity rate is higher in paediatric and adolescent melanoma patients than in adults, non SLNB positivity and melanoma specific death rate are low [91]

### *8.2.3. Regional lymph nodes*

In case of positive SLNB many surgeons would proceed to a completion lymph node dissection (CLND),

however survival advantage of this procedure is unclear, and is currently being investigated [92, 93, 94]

In a large series of paediatric melanoma cases 18 patients underwent SNLB, and 7 proceeded to undergo CLND because of findings of metastatic disease to the SLN; two of these had tumour-positive lymph nodes on pathologic analysis of the CLND specimen.

Similarly, the presence of metastases in regional lymph nodes after CLND has been diagnosed in 1 of 3 patients by some authors and in 1 of 4 patients by others [38, 92]

#### *8.2.4. Adjuvant therapy*

Consideration of systemic therapy after regional lymph nodes involvement by melanoma cells is under investigation. Treatment plans for children must be extrapolated from adult studies.

Interferon alfa-2b is currently used for adjuvant therapy in high-risk melanoma after surgery in adult patients and can also be used in paediatric melanoma patients with acceptable toxicity [95]

#### **8.3. Distant metastases**

excision of the tumour site with 2 cm margins and primary closure or skin graft. SLNB is performed using preoperative lymphoscintigraphy, intraoperative blue dye injection around the site of excision and hand-held gamma probe for radio-localization [38,88]. One day before the operation, between 18.5 and 40 MBq of Tc-99m microcolloid is injected intradermally around the scar. The drainage of the colloid is localized by detecting radiation, and the location of the SLN is marked on the skin. The position of the SLN is confirmed with a handheld gamma probe before starting the operation. At the author's center the procedure is performed under epidural anaesthesia and sedation or general anaesthesia. As reported by some authors, subcutaneous infusion anaesthesia (SIA) can be useful [89]. Patent blue is additionally injected intradermally around the scar as standard procedure. Sentinel lymph node biopsy is then accomplished with the help of repeated measurements with the handheld gamma probe. The

A comparison between adults and patients younger than 21 years who underwent either lymph node dissection or SLNB showed a higher rate of lymph node metastasis in the paediatric age (44%) as compared to the adult (23.9%). However this finding had no statistical significance. In this series, paediatric patients either with Stage I or Stage II disease showed a 94.4% 10-year survival, while patients with Stage III melanoma had a 60.1% 10-year

Recent data show that although the SLNB positivity rate is higher in paediatric and adolescent melanoma patients than in adults, non SLNB positivity and melanoma specific death rate are

In case of positive SLNB many surgeons would proceed to a completion lymph node dissection

however survival advantage of this procedure is unclear, and is currently being investigated

In a large series of paediatric melanoma cases 18 patients underwent SNLB, and 7 proceeded to undergo CLND because of findings of metastatic disease to the SLN; two of these had

Similarly, the presence of metastases in regional lymph nodes after CLND has been diagnosed

Consideration of systemic therapy after regional lymph nodes involvement by melanoma cells is under investigation. Treatment plans for children must be extrapolated from adult studies.

Interferon alfa-2b is currently used for adjuvant therapy in high-risk melanoma after surgery in adult patients and can also be used in paediatric melanoma patients with acceptable

tumour-positive lymph nodes on pathologic analysis of the CLND specimen.

in 1 of 3 patients by some authors and in 1 of 4 patients by others [38, 92]

SLN(s) is (are) removed, and the wound is closed.

354 Melanoma - From Early Detection to Treatment

survival [90]

*8.2.3. Regional lymph nodes*

*8.2.4. Adjuvant therapy*

toxicity [95]

low [91]

(CLND),

[92, 93, 94]

The incidence of metastatic melanoma has increased over the last three decades, and the death rate continues to climb faster than that of most other cancers. According to the American Cancer Society, there were approximately 68,000 new cases of melanoma in the United States in 2009, and 8,700 melanoma-related deaths. Melanoma is difficult to treat once it has spread beyond the skin to other parts of the body (metastasized). Very few treatment options exist for people with metastatic melanoma.

#### *8.3.1. Treatment of disseminated disease*

Most reports describing the treatment of paediatric melanoma are from single institutions in which diagnostic criteria, staging and pathological evaluation of the primary tumour have varied significantly. Dacarbazine, which is the most active agent in adult melanoma, showed encouraging activity in four children with melanoma treated between 1975 and 1984 [96] Other traditional chemotherapeutic regimens have shown some efficacy in metastatic melanoma [23]. The availability of investigational therapies, such as interleukin-2, interferon alfa-2b and vaccines, has been generally restricted to patients who are older than 18 years of age and no prospective trials in adolescents have been performed. Collaborative efforts, now under discussion between paediatric and adult cooperative groups, should help facilitate the enrollment of younger patients onto trials that use experimental therapies.

## *8.3.2. Radiotherapy*

Radiotherapy is rarely indicated in the management of primary paediatric melanoma. However, it should be considered in patients with head and neck melanomas at high risk for parotid or cervical metastases and in those who develop brain metastases. Brain metastases have been reported to occur during the course of the disease in up to 18% of children with melanoma [23]. Ultimately, as in adults, there is no effective therapy for metastatic melanoma in children. Therefore, the main focus of the parent, the paediatrician, and the dermatologist should be risk reduction and early detection of melanoma. The former consists primarily of avoiding intense sunlight exposure, using protective clothing and broad-spectrum sunblock, and educating children. Early detection requires a high index of clinical suspicion, especially by the paediatrician, who sees children with much more regularity than a dermatologist, of any rapidly growing or otherwise atypical pigmented lesion. In addition, the physician should recognize the elevated risk of any child with a family history of melanoma, GCMN, or dysplastic nevi. Again, prevention and early clinical diagnosis are the only current effective cure for cutaneous melanoma [97].

#### **8.4. Prognosis**

The outcome for children and adolescents with melanoma also appears to be similar to that reported for adults and is dependent on the initial stage of the tumour. Patients with localised disease have an excellent outcome, whereas those with nodal and distant metastases have estimated 10-year survivals of only 60 and 25%, respectively. Outcome is also stage-dependent and the thickness of the primary lesion correlates with the risk of nodal involvement and subsequent disease recurrence [23]

[2] Krengel, S, Hauschild, A, & Schafer, T. Melanoma risk in congenital melanocytic nae‐

Surgical Treatment of Nevi and Melanoma in the Pediatric Age

http://dx.doi.org/10.5772/54632

357

[3] Ansarin, H, Soltani-arabshahi, R, Mehregan, D, Shayanfar, N, & Soltanzadeh, P. Giant congenital melanocytic nevus with neurofibroma-like changes and spina bifida

[4] Cruz, M. A, Cho, E. S, Schwartz, R. A, & Janniger, C. K. Congenital neurocutaneous

[5] Silfen, R, Skoll, P. J, & Hudson, D. A. Congenital giant hairy nevi and neurofibroma‐ tosis: the significance of their common origin. *Plast Reconstr Surg*. Oct (2002). , 110(5),

[6] Cramer, S. F. The melanocytic differentiation pathway in congenital melanocytic ne‐

[7] Kinsler, V. A, Abu-amero, S, Budd, P, Jackson, I. J, Ring, S. M, Northstone, K, et al. Germline Melanocortin-Receptor Genotype Is Associated with Severity of Cutaneous Phenotype in Congenital Melanocytic Nevi: A Role for MC1R in Human Fetal Devel‐

[8] Kadonaga, J. N, & Frieden, I. J. Neurocutaneous melanosis: definition and review of

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[10] Everett, M. A. Histopathology of congenital pigmented nevi. *Am J Dermatopathol*. Feb

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Melanomas arising on congenital nevi seem to have a better prognosis if they arise during early infancy than in childhood; moreover, metastatic melanoma associated with giant nevi have a worse prognosis than those associated with other skin lesions [32]

Melanoma has also reported to be more frequently metastatic in young children than in adolescents.This can be due to several causative factors but can also reflect a true biologic difference [98, 99]. There were significant differences in baseline characteristics of young children (age < 10 years) compared with adolescents and young adults: the former were more likely to be non-white, to have metastases, to have nodular or other histology, head, face, or neck primaries, thicker lesions and history of cancer.

Multivariate analysis for melanoma survival in children showed significantly worse survival for males, patients with regional or unstaged disease, nodular histology, increasing thickness of the primary tumor, primary disease in the head, face, neck, eye, orbit, central nervous system, genitals, or overlapping sites, earlier year of diagnosis and previous cancer. Five-year melanoma-specific survival for pediatric cases (age < 20 years) was 100% for in situ disease, 96.1% for localized disease, 77.2% for regional disease and 57.3% for distant disease. Five-year overall survival was 88.9% for young children (age < 10 years), 91.5% for adolescents (age 10 to 19 years) and 90.9% for young adults, but the latter data had not statistical significance [100]. Recent data confirm that paediatric melanoma patients in younger ages have an increased risk of lymph node metastasis and thicker tumors. This suggests that the younger paediatric patients may have a disease that differs biologically from that of the older ones [101].

## **Author details**

Andrea Zangari1 , Federico Zangari2 , Mercedes Romano2 , Elisabetta Cerigioni2 , Maria Giovanna Grella3 , Anna Chiara Contini3 and Martino Ascanio2


## **References**

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and the thickness of the primary lesion correlates with the risk of nodal involvement and

Melanomas arising on congenital nevi seem to have a better prognosis if they arise during early infancy than in childhood; moreover, metastatic melanoma associated with giant nevi

Melanoma has also reported to be more frequently metastatic in young children than in adolescents.This can be due to several causative factors but can also reflect a true biologic difference [98, 99]. There were significant differences in baseline characteristics of young children (age < 10 years) compared with adolescents and young adults: the former were more likely to be non-white, to have metastases, to have nodular or other histology, head, face, or

Multivariate analysis for melanoma survival in children showed significantly worse survival for males, patients with regional or unstaged disease, nodular histology, increasing thickness of the primary tumor, primary disease in the head, face, neck, eye, orbit, central nervous system, genitals, or overlapping sites, earlier year of diagnosis and previous cancer. Five-year melanoma-specific survival for pediatric cases (age < 20 years) was 100% for in situ disease, 96.1% for localized disease, 77.2% for regional disease and 57.3% for distant disease. Five-year overall survival was 88.9% for young children (age < 10 years), 91.5% for adolescents (age 10 to 19 years) and 90.9% for young adults, but the latter data had not statistical significance [100]. Recent data confirm that paediatric melanoma patients in younger ages have an increased risk of lymph node metastasis and thicker tumors. This suggests that the younger paediatric

patients may have a disease that differs biologically from that of the older ones [101].

, Mercedes Romano2

[1] Clemmensen, O. J, & Kroon, S. The histology of "congenital features" in early ac‐

quired melanocytic nevi. *J Am Acad Dermatol*. Oct (1988). , 19(4), 742-6.

, Elisabetta Cerigioni2

and Martino Ascanio2

,

have a worse prognosis than those associated with other skin lesions [32]

neck primaries, thicker lesions and history of cancer.

, Federico Zangari2

3 Catholic University of the Sacred Heart, Roma, Italy

, Anna Chiara Contini3

1 Pediatric Surgery Department San Camillo Hospital, Roma, Italy

2 Pediatric Surgery Department, University Hospital of Ancona, Italy

subsequent disease recurrence [23]

356 Melanoma - From Early Detection to Treatment

**Author details**

Andrea Zangari1

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Maria Giovanna Grella3


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**Chapter 13**

**Adoptive Cell Therapy of Melanoma:**

Jennifer Makalowski and Hinrich Abken

http://dx.doi.org/10.5772/53619

**1. Introduction**

heart" of melanoma.

Additional information is available at the end of the chapter

**The Challenges of Targeting the Beating Heart**

The identification of melanoma-associated antigens, the isolation of tumor infiltrating T cells from melanoma lesions, and the significant progress in engineering redirected T cells has favored the development of various strategies in the adoptive immunotherapy of mel‐ anoma. Recent trials in adoptive cell therapy (ACT) have achieved spectacular results in inducing remission in advanced stages of the disease, although produced on-target off-tu‐ mor toxicities, emphasizing the tremendous potential benefit of harnessing the immune system for fighting the disease. Moreover, the identification of so-called melanoma stem cells along with strategies for selectively eliminating subsets of melanoma cells implies that there is a need for redefining therapeutic targets in melanoma. This review discusses current challenges in the rational design of adoptive cell therapy to target "the beating

Surgical resection of tumor lesions in early stages of the disease is the curative option for combating melanoma; a 10-year-survival rate of 75 - 85% can be achieved for melanoma in stage I or II. However, melanoma in stage III or IV is still associated with low survival rates of less than 1 year upon diagnosis [1]. Despite the development of novel drugs and major improvements in therapeutic regimens, significant responses were only achieved in prede‐ fined groups and of short duration. Treatment with the chemotherapeutic drug dacarbazine (DTIC) and vemurafenib, an inhibitor of mutated BRAF, produced a median progressionfree survival of 64% with dacarbazine, respectively 84% with vemurafenib of approximately 6 months [2-4]. The biology of melanoma and the heterogeneity of malignant cells are thought to be responsible for this unsatisfactory situation. First, melanoma cells can persist

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

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

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

**1.1. Advanced stages of melanoma resist conventional therapeutic regimens**

## **Adoptive Cell Therapy of Melanoma: The Challenges of Targeting the Beating Heart**

Jennifer Makalowski and Hinrich Abken

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53619

## **1. Introduction**

The identification of melanoma-associated antigens, the isolation of tumor infiltrating T cells from melanoma lesions, and the significant progress in engineering redirected T cells has favored the development of various strategies in the adoptive immunotherapy of mel‐ anoma. Recent trials in adoptive cell therapy (ACT) have achieved spectacular results in inducing remission in advanced stages of the disease, although produced on-target off-tu‐ mor toxicities, emphasizing the tremendous potential benefit of harnessing the immune system for fighting the disease. Moreover, the identification of so-called melanoma stem cells along with strategies for selectively eliminating subsets of melanoma cells implies that there is a need for redefining therapeutic targets in melanoma. This review discusses current challenges in the rational design of adoptive cell therapy to target "the beating heart" of melanoma.

#### **1.1. Advanced stages of melanoma resist conventional therapeutic regimens**

Surgical resection of tumor lesions in early stages of the disease is the curative option for combating melanoma; a 10-year-survival rate of 75 - 85% can be achieved for melanoma in stage I or II. However, melanoma in stage III or IV is still associated with low survival rates of less than 1 year upon diagnosis [1]. Despite the development of novel drugs and major improvements in therapeutic regimens, significant responses were only achieved in prede‐ fined groups and of short duration. Treatment with the chemotherapeutic drug dacarbazine (DTIC) and vemurafenib, an inhibitor of mutated BRAF, produced a median progressionfree survival of 64% with dacarbazine, respectively 84% with vemurafenib of approximately 6 months [2-4]. The biology of melanoma and the heterogeneity of malignant cells are thought to be responsible for this unsatisfactory situation. First, melanoma cells can persist

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

for long periods of time in a "dormant" stage without any progression in tumor formation [5]. Second, melanoma cells can disseminate early into distant organs including the brain forming micro-metastases, which are small in cell numbers and frequently beyond the de‐ tection limit of current imaging procedures [6, 7]. Third, many melanoma cells are notori‐ ously resistant to chemo- and radiation therapy [8-10], making alternative strategies in tumor cell elimination necessary.

the prevalence of TIL's in primary melanoma lesions and metastases is not a prognos‐

Adoptive Cell Therapy of Melanoma: The Challenges of Targeting the Beating Heart

http://dx.doi.org/10.5772/53619

367

Protocols according to GMP standards have been established in several centers to isolate and amplify TIL's to numbers appropriate for adoptive therapy. Melanoma reactive T cells are expanded in the presence of IL-2 by culture on feeder cells expressing melanoma anti‐ gens [23]. Subsequent to TIL re-infusions, metastases regressed in the majority of patients and a stable disease phase followed. However, only few patients remained in complete re‐ mission [21]. The disappointing therapeutic efficacy, despite high numbers of infused TIL's is thought to be due to low responsiveness of highly expanded T cells which are unable to execute a productive anti-melanoma attack after administration to the patient. Current TIL protocols therefore attempt to administer so-called "young TIL's" (Figure 1), i.e. melanoma infiltrating T cells which underwent short-term culture expansions and therefore passed through fewer cell division cycles prior to re-infusion and thereby exhibit a less differentiat‐ ed phenotype [24]. Another change in protocols is that TIL's are not selected for melanoma reactivity; the rationale behind this is that re-infusion of *ex vivo* IFN-γ secreting TIL's exhibit‐ ed no major benefit compared to non-responding TIL's [16]. Early phase I trials showed im‐ proved persistence of young TIL's [25] and 50% response rates in a cohort of 20 patients [26], which is just as effective as traditionally grown TIL's [27]. Different non-randomized phase II trials at the NCI and at Sheba Medical Center confirmed these early observations (Table 1) [28, 29]. A roadmap describing critical steps for comparative testing the TIL strategy in a randomized multi-center setting was recently published in a White Paper on adoptive cell

**Figure 1.** Adoptive cell therapy for metastatic melanoma. Adoptive cell therapy with tumor infiltrating lympho‐ cytes (TIL´s) makes use of melanoma-specific TIL´s which are isolated from a melanoma biopsy, amplified *ex vivo* by stimulation with melanoma biopsy cells and propagated to high numbers in the presence of IL-2. In more re‐ cent trials, TIL´s are propagated short-term ex vivo without stimulation by melanoma cells and administered as

tic factor itself.

therapy [30].

"young" TIL´s.

Therefore, in more progressed stages of the disease the recruitment of the cellular im‐ mune defense to eliminate cancer cells is thought to be an alternative. Administration of high dose interleukin-2 (IL-2) [11] and anti-cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) antibody [12] as well as interferon (IFN) α-2b prolongs the disease-free survival although at a relatively low response rate and without being curative over time [13, 14]. However, these and other observations imply that activation or modulation of the pa‐ tient's immune response may be effective in the treatment of melanoma. A number of ap‐ proaches for enhancing the immune cell response against melanoma are currently explored with some success. In particular, the adoptive transfer of autologous T cells iso‐ lated from melanoma lesions and expanded to large numbers ex vivo has produced en‐ couraging phase II results [15, 16]. The administration of patient's blood T cells engineered with defined specificity for melanoma-associated antigens are additionally be‐ ing explored in a number of trials. In this review, we summarize evidence for the potency of adoptive T cell therapy in the treatment of melanoma and discuss current challenges in achieving long-term remission. Upcoming strategies in selective targeting cancer stem cells are also discussed.

## **2. Adoptive cell therapy can successfully fight melanoma**

Melanoma can trigger a curative immune response; this conclusion is drawn from the clinical observation of spontaneous and complete melanoma regressions and of the higher frequency of melanomas among immune compromised patients [17, 18]. More direct evi‐ dence for the immune cell control of melanoma growth was obtained by the treatment with high dose IL-2, which produces an objective response rate of 16%. Indeed, some of the patients receiving thus treatment exhibit a long-term complete response for years [11, 19]. These observations are remarkable in light of the low and short-lived response rates after chemotherapy and currently drive the development of adoptive T cell therapy for treatment of late stage melanoma.

The development of adoptive cell therapy (ACT) was further strengthened by upcom‐ ing technologies in isolating tumor infiltrating lymphocytes (TIL's) from melanoma bi‐ opsies (Figure 1). First described in 1969 [20], TIL's from melanoma lesions consisted of both effector and helper T cell subsets and can be expanded *ex vivo* in the presence of IL-2. The expanded cells are then selected for melanoma reactivity. A strong ration‐ ale for using these T cells in adoptive therapy is provided by the observation that the infusion of high TIL numbers correlates with better clinical outcome [21, 22] although the prevalence of TIL's in primary melanoma lesions and metastases is not a prognos‐ tic factor itself.

for long periods of time in a "dormant" stage without any progression in tumor formation [5]. Second, melanoma cells can disseminate early into distant organs including the brain forming micro-metastases, which are small in cell numbers and frequently beyond the de‐ tection limit of current imaging procedures [6, 7]. Third, many melanoma cells are notori‐ ously resistant to chemo- and radiation therapy [8-10], making alternative strategies in

Therefore, in more progressed stages of the disease the recruitment of the cellular im‐ mune defense to eliminate cancer cells is thought to be an alternative. Administration of high dose interleukin-2 (IL-2) [11] and anti-cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) antibody [12] as well as interferon (IFN) α-2b prolongs the disease-free survival although at a relatively low response rate and without being curative over time [13, 14]. However, these and other observations imply that activation or modulation of the pa‐ tient's immune response may be effective in the treatment of melanoma. A number of ap‐ proaches for enhancing the immune cell response against melanoma are currently explored with some success. In particular, the adoptive transfer of autologous T cells iso‐ lated from melanoma lesions and expanded to large numbers ex vivo has produced en‐ couraging phase II results [15, 16]. The administration of patient's blood T cells engineered with defined specificity for melanoma-associated antigens are additionally be‐ ing explored in a number of trials. In this review, we summarize evidence for the potency of adoptive T cell therapy in the treatment of melanoma and discuss current challenges in achieving long-term remission. Upcoming strategies in selective targeting cancer stem

**2. Adoptive cell therapy can successfully fight melanoma**

Melanoma can trigger a curative immune response; this conclusion is drawn from the clinical observation of spontaneous and complete melanoma regressions and of the higher frequency of melanomas among immune compromised patients [17, 18]. More direct evi‐ dence for the immune cell control of melanoma growth was obtained by the treatment with high dose IL-2, which produces an objective response rate of 16%. Indeed, some of the patients receiving thus treatment exhibit a long-term complete response for years [11, 19]. These observations are remarkable in light of the low and short-lived response rates after chemotherapy and currently drive the development of adoptive T cell therapy for

The development of adoptive cell therapy (ACT) was further strengthened by upcom‐ ing technologies in isolating tumor infiltrating lymphocytes (TIL's) from melanoma bi‐ opsies (Figure 1). First described in 1969 [20], TIL's from melanoma lesions consisted of both effector and helper T cell subsets and can be expanded *ex vivo* in the presence of IL-2. The expanded cells are then selected for melanoma reactivity. A strong ration‐ ale for using these T cells in adoptive therapy is provided by the observation that the infusion of high TIL numbers correlates with better clinical outcome [21, 22] although

tumor cell elimination necessary.

366 Melanoma - From Early Detection to Treatment

cells are also discussed.

treatment of late stage melanoma.

Protocols according to GMP standards have been established in several centers to isolate and amplify TIL's to numbers appropriate for adoptive therapy. Melanoma reactive T cells are expanded in the presence of IL-2 by culture on feeder cells expressing melanoma anti‐ gens [23]. Subsequent to TIL re-infusions, metastases regressed in the majority of patients and a stable disease phase followed. However, only few patients remained in complete re‐ mission [21]. The disappointing therapeutic efficacy, despite high numbers of infused TIL's is thought to be due to low responsiveness of highly expanded T cells which are unable to execute a productive anti-melanoma attack after administration to the patient. Current TIL protocols therefore attempt to administer so-called "young TIL's" (Figure 1), i.e. melanoma infiltrating T cells which underwent short-term culture expansions and therefore passed through fewer cell division cycles prior to re-infusion and thereby exhibit a less differentiat‐ ed phenotype [24]. Another change in protocols is that TIL's are not selected for melanoma reactivity; the rationale behind this is that re-infusion of *ex vivo* IFN-γ secreting TIL's exhibit‐ ed no major benefit compared to non-responding TIL's [16]. Early phase I trials showed im‐ proved persistence of young TIL's [25] and 50% response rates in a cohort of 20 patients [26], which is just as effective as traditionally grown TIL's [27]. Different non-randomized phase II trials at the NCI and at Sheba Medical Center confirmed these early observations (Table 1) [28, 29]. A roadmap describing critical steps for comparative testing the TIL strategy in a randomized multi-center setting was recently published in a White Paper on adoptive cell therapy [30].

**Figure 1.** Adoptive cell therapy for metastatic melanoma. Adoptive cell therapy with tumor infiltrating lympho‐ cytes (TIL´s) makes use of melanoma-specific TIL´s which are isolated from a melanoma biopsy, amplified *ex vivo* by stimulation with melanoma biopsy cells and propagated to high numbers in the presence of IL-2. In more re‐ cent trials, TIL´s are propagated short-term ex vivo without stimulation by melanoma cells and administered as "young" TIL´s.


**3. Adoptive cell therapy with antigen-specific T cells**

counterparts, TCR engineered T cells responded to gp100+

The rationale for using melanoma antigen-specific T cells is based on the observation that the success of TIL therapy in some patients correlates with the presence of melanoma-reac‐ tive T cells, in particular with those cells specific for Melan-A, MART-1 or gp100 [23, 31]. The median survival of patients treated with Melan-A specific TIL's was 53.5 months com‐ pared to 3.5 months for patients who received TIL's without Melan-A specificity [32]. These observations together with a number of technical obstacles in obtaining TIL's from biopsies strengthened efforts to derive melanoma-specific T cell clones from peripheral blood lym‐ phocytes for the use in adoptive cell therapy. The strategy was corroborated by a 50% re‐ sponse rate obtained after transfer of MART-1 or gp100 specific T cell clones isolated and propagated *ex vivo* from peripheral blood lymphocytes (Table 1) [33]. Melanoma reactive T cell clones in peripheral blood are rare, TIL therapy increases the otherwise low magnitude of the tumor-reactive T cell compartment *in vivo*, which matches the reactivity in the TIL product [34]. Interestingly, individual TIL products from different patients contain unique patterns of reactivity against shared melanoma-associated antigens [34]. TIL isolation and expansion *in vitro*, however, is extremely laborious. This limit leads to attempts to engineer patient's blood T cells with pre-defined specificity for more specifically redirecting the cyto‐ toxic response toward melanoma. It is therefore assumed that the clinical efficacy of TIL therapy can be improved by application of T cells with more defined tumor-reactivity.

Adoptive Cell Therapy of Melanoma: The Challenges of Targeting the Beating Heart

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369

To engineer specificity for melanoma, T cell receptors (TCR's) were cloned from TIL's of re‐ sponding melanoma patients and transferred to peripheral blood T cells of the same patient (Figure 2) [35-38]. The gp100 specific TCR was one of the first TCR's, cloned from melanoma TIL's and introduced *ex vivo* by retrovirus-mediated gene transfer into blood T cells, which thus obtained redirected specificity for gp100 positive cells. In contrast to their non-modified

inflammatory cytokines including IFN-γ and by lysing the target cells [45, 46]. Similarly, blood T cells were engineered with recombinant TCR's with specificity for MART-1 or MAGE-A1. The functional avidity of cloned TCR's was improved and engineered T cells were successfully used in subsequent trials [47, 48]. About 30% of patients receiving ACT with MART-1 specific T cells responded with melanoma regression; 19% of patients treated with gp100 specific TCR T cells exhibited objective response, most responses were persistent [38]. TCR engineered T cells also showed efficacy towards brain metastases, which indicates that patients with otherwise incurable metastatic sites may benefit from ACT (Table 1) [115]. In patients with prolonged clinical remission, engineered T cells were present in the circula‐ tion for more than a year after initiation of treatment; this indicates that therapeutic efficacy and long-term anti-melanoma immunity may correlate with T cell persistence [49, 50].

However, the enthusiasm for adoptive cell therapy with TCR modified T cells has been dampened by several limitations. Tumor cells including those of the melanoma undergo clo‐ nal evolution, and some of these evolved cells evade T cell recognition, for instance, as a re‐ sult of repression of their MHC complex [51], of mutations in their β2 microglobulin chain [52], and of deficiencies in their antigen processing machinery [51, 53]. Each of these altera‐

melanoma cells by secreting pro-

**CHUV**, Centre Hospitalier Universitaire Vaudois; **DFCI**, Dana-Farber Cancer Institute; **FHCRC**, Fred Hutchinson Cancer Research Center; **HMO**, Hadassah Medical Organization; **LUMC**, Leiden University Medical Center; **MDACC**, M.D. Anderson Cancer Center; **MOFFITT**, H. Lee Moffitt Cancer Center and Research Institute; **NIH**, National Institutes of Health; **NUH**, Nantes University Hospital; **PI**, principal investigator; **SMC**, Sheba Medical Center; **UC**, University of California; **UR**, University of Regensburg

**Table 1.** Adoptive cell therapy trials in patients with metastatic melanoma

## **3. Adoptive cell therapy with antigen-specific T cells**

**Target antigen Adoptively transferred T cells NCT ID / Reference Center**

368 Melanoma - From Early Detection to Treatment

MART-1 MART-1 specific CD8+ T cells [113] DFCI MART-1 MART-1 specific CD8+ T cells NCT00512889 DFCI MART-1 MART-1 specific CD8+ T cells [87] UR MART-1 MART-1 specific CD8+ T cells [33] UNH MART-1 MART-1 specific CD8+ T cells NCT00324623 CHUV MART-1 MART-1 specific CD8+ T cells NCT01106235 FHCRC NY-ESO-1 NY-ESO-1 specific CD8+ T cells and anti-CTLA-4 antibody NCT00871481 FHCRC

MART-1 MART-1 specific TILs NCT00720031 NUH MART-1 MART-1 specific TILs (DMF5) NCT00924001 CC

NY-ESO-1 anti-NY-ESO-1 TCR [121] NIH NY-ESO-1 anti-NY-ESO-1 TCR NCT00670748 NIH MART-1 anti-MART-1 TCR (low-affinity) [49] NIH MART-1 anti-MART-1 TCR NCT00910650 UC MART-1 anti-MART-1 TCR (high-affinity) [38] NIH gp-100 anti-gp-100 TCR [38] NIH MART-1 anti-MART-1 TCR [114] NIH gp-100 anti-gp-100 TCR [114] NIH MART-1 anti-MART-1 TCR NCT00612222 NIH gp-100 anti-gp-100 TCR NCT00610311 NIH MART-1 anti-MART-1 TCR plus MART-1 vaccination NCT00923195 NIH gp-100 anti-gp-100 TCR plus gp-100 vaccination NCT00923195 NIH p53 anti-p53 TCR NCT00393029 NIH VEGFR2 anti-VEGFR2 CAR engineered CD8+ T cells NCT01218867 NIH Ganglioside GD-3 anti-GD-3 CAR PI: M. Davies MDACC

**CHUV**, Centre Hospitalier Universitaire Vaudois; **DFCI**, Dana-Farber Cancer Institute; **FHCRC**, Fred Hutchinson Cancer Research Center; **HMO**, Hadassah Medical Organization; **LUMC**, Leiden University Medical Center; **MDACC**, M.D. Anderson Cancer Center; **MOFFITT**, H. Lee Moffitt Cancer Center and Research Institute; **NIH**, National Institutes of Health; **NUH**, Nantes University Hospital; **PI**, principal investigator;

**SMC**, Sheba Medical Center; **UC**, University of California; **UR**, University of Regensburg

**Table 1.** Adoptive cell therapy trials in patients with metastatic melanoma

IL-2 engineered TILs [117] NIH IL-2 engineered TIL NCT00062036 NIH IL-12 engineered TIL NCT01236573 NIH CXCR2 engineered TIL [86] MDACC

melanoma specific CD8+ T cells [118] FHCRC melanoma specific T cells [119] LUMC

TILs [114] NIH TIL [120] NIH TILs [27] NIH TILs [29] NIH TILs [115] NIH TILs NCT00287131 SMC TILs NCT000604136 HMO TILs NCT01005745 MOFFITT TILs and IFN-γ NCT01082887 NUH "young" TILs [116] NIH "young" TILs [28] SMC "young" TILs NCT01118091 NIH "young" TILs NCT01319565 NIH "young" TILs NCT01369888 MIH "young" TILs NCT01468818 NIH "young" TILs NCT00513604 NIH

The rationale for using melanoma antigen-specific T cells is based on the observation that the success of TIL therapy in some patients correlates with the presence of melanoma-reac‐ tive T cells, in particular with those cells specific for Melan-A, MART-1 or gp100 [23, 31]. The median survival of patients treated with Melan-A specific TIL's was 53.5 months com‐ pared to 3.5 months for patients who received TIL's without Melan-A specificity [32]. These observations together with a number of technical obstacles in obtaining TIL's from biopsies strengthened efforts to derive melanoma-specific T cell clones from peripheral blood lym‐ phocytes for the use in adoptive cell therapy. The strategy was corroborated by a 50% re‐ sponse rate obtained after transfer of MART-1 or gp100 specific T cell clones isolated and propagated *ex vivo* from peripheral blood lymphocytes (Table 1) [33]. Melanoma reactive T cell clones in peripheral blood are rare, TIL therapy increases the otherwise low magnitude of the tumor-reactive T cell compartment *in vivo*, which matches the reactivity in the TIL product [34]. Interestingly, individual TIL products from different patients contain unique patterns of reactivity against shared melanoma-associated antigens [34]. TIL isolation and expansion *in vitro*, however, is extremely laborious. This limit leads to attempts to engineer patient's blood T cells with pre-defined specificity for more specifically redirecting the cyto‐ toxic response toward melanoma. It is therefore assumed that the clinical efficacy of TIL therapy can be improved by application of T cells with more defined tumor-reactivity.

To engineer specificity for melanoma, T cell receptors (TCR's) were cloned from TIL's of re‐ sponding melanoma patients and transferred to peripheral blood T cells of the same patient (Figure 2) [35-38]. The gp100 specific TCR was one of the first TCR's, cloned from melanoma TIL's and introduced *ex vivo* by retrovirus-mediated gene transfer into blood T cells, which thus obtained redirected specificity for gp100 positive cells. In contrast to their non-modified counterparts, TCR engineered T cells responded to gp100+ melanoma cells by secreting proinflammatory cytokines including IFN-γ and by lysing the target cells [45, 46]. Similarly, blood T cells were engineered with recombinant TCR's with specificity for MART-1 or MAGE-A1. The functional avidity of cloned TCR's was improved and engineered T cells were successfully used in subsequent trials [47, 48]. About 30% of patients receiving ACT with MART-1 specific T cells responded with melanoma regression; 19% of patients treated with gp100 specific TCR T cells exhibited objective response, most responses were persistent [38]. TCR engineered T cells also showed efficacy towards brain metastases, which indicates that patients with otherwise incurable metastatic sites may benefit from ACT (Table 1) [115]. In patients with prolonged clinical remission, engineered T cells were present in the circula‐ tion for more than a year after initiation of treatment; this indicates that therapeutic efficacy and long-term anti-melanoma immunity may correlate with T cell persistence [49, 50].

However, the enthusiasm for adoptive cell therapy with TCR modified T cells has been dampened by several limitations. Tumor cells including those of the melanoma undergo clo‐ nal evolution, and some of these evolved cells evade T cell recognition, for instance, as a re‐ sult of repression of their MHC complex [51], of mutations in their β2 microglobulin chain [52], and of deficiencies in their antigen processing machinery [51, 53]. Each of these altera‐

progression. An additional advantage over transgenic TCR's is that CAR's can be used inde‐ pendently of the individual HLA subtype. However, the T-body strategy is restricted to an‐ tigens expressed on the surface of the target cell; intracellular antigens are not visible to CAR's. Due to the broad variety of antibodies available, a nearly unlimited panel of antigens can be targeted with high affinity and specificity, including those which are not classical T cell antigens, e.g. carbohydrates. High affinity CAR's activate engineered T cells even after binding to low amounts of target antigen; this not only makes the approach highly sensitive,

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**Figure 3. Recombinant receptors to redirect T cells for use in antigen-specific cell therapy.** The physiologic T cell receptor (TCR)/CD3 complex consists of the α and β TCR chains, which recognize major histocompatibility complex (MHC)-presented antigen by binding through both variable regions Vα Vβ, and of the CD3 chains. Antigen engage‐ ment induces clustering of the TCR complex and the primary signal for T cell activation is generated by the intracellu‐ lar CD3ζ chain. Recombinant TCR α and β chains can be engineered to T cells in order to provide a new specificity. Alternatively, the V regions of the TCR chains can be combined and fused to the intracellular CD3ζ chain to produce a T cell activation signal upon binding to antigen. The chimeric antigen receptor (CAR) makes use of an antibody bind‐ ing domain for antigen recognition which is enigneered by fusing the variable (V) regions of the immunoglobulin heavy (H) and light (L) chain. The VH-VL single chain antibody is linked via a spacer to the intracellular CD3ζ chain to produce the primary T cell activation signal upon antigen binding. Intracellular signaling domains of costimulatory molecules like CD28 can be added to provide appropriate costimulation in addition to the primary CD3ζ signal.

T cells require two signals for full and lasting activation, one provided by the TCR and the other by costimulatory co-receptors; the prototype of which is CD28. The corresponding li‐ gands are usually not present in the tumor micro-environment. Some effector functions in‐ cluding IL-2 secretion require CD28 costimulation along with the primary TCR/CD3ζ signal; this provides a rationale for combining the intracellular CD3ζ with the CD28 signaling do‐ main in one polypeptide chain (Figure 3) [59]. Other costimulatory domains, such as 4-1BB (CD137) and OX40 (CD134), were also linked to CD3ζ; each domain has a different impact on T cell effector functions [60]. Costimulatory domains were furthermore combined in socalled 3rd generation CAR's, and a number of additional modifications have been intro‐

but also makes the choice of the appropriate melanoma-selective antigen difficult.

**Figure 2. Adoptive cell therapy with redirected T cells.** T cells from the peripheral blood of the patient are engi‐ neered *ex vivo* by retro- or lentiviral gene transfer with cDNA coding for a T cell receptor (TCR) with specificity for a melanoma-associated antigen. Alternatively, T cells are engineered with a chimeric antigen receptor (CAR) which rec‐ ognizes a melanoma-associated antigen by an antibody-derived binding domain. Engineered T cells are expanded *ex vivo* prior to administration to the patient.

tions renders the melanoma cell invisible to a TCR-mediated T cell attack. A possible safety hazard moreover became apparent when analyzing in more detail the transgenic TCR, which is co-expressed with the physiological TCR in the same T cell. The transgenic TCR turned out to create new but unpredictable specificities by forming hetero-dimers of the re‐ combinant α and β TCR chains with the respective chains of the physiological TCR. Unde‐ sirable mispairing of TCR chains may result in loss of specificity and may induce severe auto-reactivity [54, 55]. Tremendous efforts were subsequently made to solve the problem including replacement of TCR constant moieties by the homologous murine domains [56] and creation of additional cysteine bridges [57] to enforce preferential pairing of the re‐ combinant αβ TCR chains in the presence of the physiological TCR.

These and other technical difficulties promoted the development of an artificial "one-chainreceptor" molecule to redirect T cells in an antigen-restricted manner (Figure 3). In a seminal paper Zelig Eshhar of the Weizmann Institute of Science described a chimeric antigen recep‐ tor (CAR), also named immunoreceptor, which is composed in the extracellular part of a sin‐ gle chain antibody for antigen binding and in the intracellular part of the TCR/CD3ζ endodomain for provision of T cell activation [58]. The CAR modified T cell, also known as "T-body", becomes activated by binding to antigen, and secretes pro-inflammatory cyto‐ kines, amplifies and lyses target cells expressing the respective antigen. By using an anti‐ body for binding, the CAR recognizes the target in a MHC-independent fashion and is therefore not affected by loss of HLA molecules, which frequently occurs during neoplastic progression. An additional advantage over transgenic TCR's is that CAR's can be used inde‐ pendently of the individual HLA subtype. However, the T-body strategy is restricted to an‐ tigens expressed on the surface of the target cell; intracellular antigens are not visible to CAR's. Due to the broad variety of antibodies available, a nearly unlimited panel of antigens can be targeted with high affinity and specificity, including those which are not classical T cell antigens, e.g. carbohydrates. High affinity CAR's activate engineered T cells even after binding to low amounts of target antigen; this not only makes the approach highly sensitive, but also makes the choice of the appropriate melanoma-selective antigen difficult.

**Figure 3. Recombinant receptors to redirect T cells for use in antigen-specific cell therapy.** The physiologic T cell receptor (TCR)/CD3 complex consists of the α and β TCR chains, which recognize major histocompatibility complex (MHC)-presented antigen by binding through both variable regions Vα Vβ, and of the CD3 chains. Antigen engage‐ ment induces clustering of the TCR complex and the primary signal for T cell activation is generated by the intracellu‐ lar CD3ζ chain. Recombinant TCR α and β chains can be engineered to T cells in order to provide a new specificity. Alternatively, the V regions of the TCR chains can be combined and fused to the intracellular CD3ζ chain to produce a T cell activation signal upon binding to antigen. The chimeric antigen receptor (CAR) makes use of an antibody bind‐ ing domain for antigen recognition which is enigneered by fusing the variable (V) regions of the immunoglobulin heavy (H) and light (L) chain. The VH-VL single chain antibody is linked via a spacer to the intracellular CD3ζ chain to produce the primary T cell activation signal upon antigen binding. Intracellular signaling domains of costimulatory molecules like CD28 can be added to provide appropriate costimulation in addition to the primary CD3ζ signal.

tions renders the melanoma cell invisible to a TCR-mediated T cell attack. A possible safety hazard moreover became apparent when analyzing in more detail the transgenic TCR, which is co-expressed with the physiological TCR in the same T cell. The transgenic TCR turned out to create new but unpredictable specificities by forming hetero-dimers of the re‐ combinant α and β TCR chains with the respective chains of the physiological TCR. Unde‐ sirable mispairing of TCR chains may result in loss of specificity and may induce severe auto-reactivity [54, 55]. Tremendous efforts were subsequently made to solve the problem including replacement of TCR constant moieties by the homologous murine domains [56] and creation of additional cysteine bridges [57] to enforce preferential pairing of the re‐

**Figure 2. Adoptive cell therapy with redirected T cells.** T cells from the peripheral blood of the patient are engi‐ neered *ex vivo* by retro- or lentiviral gene transfer with cDNA coding for a T cell receptor (TCR) with specificity for a melanoma-associated antigen. Alternatively, T cells are engineered with a chimeric antigen receptor (CAR) which rec‐ ognizes a melanoma-associated antigen by an antibody-derived binding domain. Engineered T cells are expanded *ex*

These and other technical difficulties promoted the development of an artificial "one-chainreceptor" molecule to redirect T cells in an antigen-restricted manner (Figure 3). In a seminal paper Zelig Eshhar of the Weizmann Institute of Science described a chimeric antigen recep‐ tor (CAR), also named immunoreceptor, which is composed in the extracellular part of a sin‐ gle chain antibody for antigen binding and in the intracellular part of the TCR/CD3ζ endodomain for provision of T cell activation [58]. The CAR modified T cell, also known as "T-body", becomes activated by binding to antigen, and secretes pro-inflammatory cyto‐ kines, amplifies and lyses target cells expressing the respective antigen. By using an anti‐ body for binding, the CAR recognizes the target in a MHC-independent fashion and is therefore not affected by loss of HLA molecules, which frequently occurs during neoplastic

combinant αβ TCR chains in the presence of the physiological TCR.

*vivo* prior to administration to the patient.

370 Melanoma - From Early Detection to Treatment

T cells require two signals for full and lasting activation, one provided by the TCR and the other by costimulatory co-receptors; the prototype of which is CD28. The corresponding li‐ gands are usually not present in the tumor micro-environment. Some effector functions in‐ cluding IL-2 secretion require CD28 costimulation along with the primary TCR/CD3ζ signal; this provides a rationale for combining the intracellular CD3ζ with the CD28 signaling do‐ main in one polypeptide chain (Figure 3) [59]. Other costimulatory domains, such as 4-1BB (CD137) and OX40 (CD134), were also linked to CD3ζ; each domain has a different impact on T cell effector functions [60]. Costimulatory domains were furthermore combined in socalled 3rd generation CAR's, and a number of additional modifications have been intro‐ duced in the last years to improve T cell persistence and activation [61, 62]. CAR's with a costimulatory domain clearly demonstrated clinical benefit and improved T cell persistence compared to CAR's targeting the same antigen but with only the CD3ζ domain [63-65].

To avoid mispairing of the recombinant TCR with the physiological TCR chains and the re‐ sulting unpredictable auto-immunity, TCR-like single chain antibodies were used as target‐ ing domain in a CAR. Thus combining the MHC-restricted recognition of antigen with the T-body strategy. T cells with TCR-like CAR were redirected towards NY-ESO-1 and MAGE-A1, respectively [41, 42]. The possible advantages of these MHC restricted CAR's compared

Adoptive Cell Therapy of Melanoma: The Challenges of Targeting the Beating Heart

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The antibody-derived binding domain of a CAR displays extraordinary high affinity com‐ pared to a TCR. However, an increase in affinity, for instance, by affinity maturation, does not necessarily improve CAR redirected T cell activation above threshold [41, 43], which is not additionally modulated by CD28 costimulation [44]. A similar effect is also assumed for TCR mediated T cell activation. The TCR or CAR binding avidity probably affects the persis‐ tence of engineered T cells at the targeted tumor site. Strong binding to a target antigen may cause the T cells to be trapped and to become fully activated for a cytolytic attack, whereas low avidity interactions may not provide sufficiently long T cell – melanoma cell contacts. In addition to the binding avidity, the amount of target antigen on the cell surface also impacts on the selectivity of redirected T cell activation. In essence, low affinity binding directs the activity of engineered T cells preferentially toward target cells with abundant antigen levels; high affinity binding is likewise effective against low antigen levels on target cells. The opti‐ mized affinity to sustain a more selective T cell trafficking to the tumor and activation while avoiding targeting healthy cells that are expressing low quantities of the same antigen, how‐

A beneficial T cell-to-target cell ratio at the tumor site seems to be required for efficient tu‐ mor elimination. Higher numbers of engineered T cells applied per dose will probably in‐ crease clinical efficacy; the majority of recent trials have applied up to 1010 cells per dose [27]. These and higher numbers of engineered T cells can be generated by extended expan‐ sion protocols; however, cells with a "young" phenotype may not be generated for adoptive transfer under these conditions. Short-term amplification protocols are therefore envisioned for both TIL's and engineered blood T cells. However, the majority of recent trials targeting

leukemia provided evidence for therapeutic efficacy at numbers less than or equal to

engineered T cells [73]. This once again raises the question of whether high T cell doses

The clinical outcome of adoptive cell therapy correlates with the persistence of adoptively transferred T cells [81]. As long as T cells engage their cognate antigen, T cells will expand and persist in detectable numbers; but when the antigen is no longer present, the T cell pop‐ ulation will contract to potentially undetectable levels and disappear from circulation. To improve survival of CAR T cells, Epstein-Barr virus (EBV)-specific T cells were engineered with a tumor-specific CAR based on the rationale that T cells recognizing the low amounts of EBV antigens by their physiological TCR will be maintained in a sizable population in cir‐ culation and in the process providing enough CAR T cells to recognize and kill melanoma cells in the surrounding tissues. A clinical trial with EBV-specific T cells engineered with an anti-GD2 CAR thus showed benefit over non-virus-specific, CAR engineered T cells in the

to the use of recombinant TCR's still has to be determined in trials.

ever, still has to be determined.

are required for a therapeutic effect.

treatment of neuroblastoma [81].

CD19+

105

Various CARs were engineered for targeting melanoma-associated antigens, including HMW-MAA, also known as MCSP [67, 68], melanotransferrin [69], the ganglioside GD2 [70] and GD3 [71]. A clinical trial targeting melanoma cells with CAR engineered T cells is cur‐ rently recruiting participants [66]. Recent phase I trials using CAR redirected T cells in the treatment of lymphoma/leukemia exhibited spectacular efficacy [72, 73]. However, the en‐ thusiasm was dampened by reports on serious adverse events and even fatalities after CAR T cell therapy [74, 75]. Targeting ErbB2 produced a cytokine storm and respiratory failure in one case [76] which is thought to be due to low levels of antigen on a number of healthy cells which can trigger CAR T cell activation. On the one hand, this event points out that ACT with CAR modified T cells may be a powerful therapy; but, on the other hand, empha‐ sizes the necessity for careful T cell dose escalation studies to balance anti-tumor efficacy and auto-immunity[61, 77, 78].

## **4. Challenges and premises in the adoptive cell therapy of melanoma**

To date, approximately half of the melanoma patients treated with TIL ACT benefit from this therapy; genetic modification of T cells may further improve clinical response to mela‐ noma, but this will have to be proven in upcoming trials. However, the strategy has poten‐ tial challenges which need to be addressed.

A major challenge of redirected T cells is the tumor selectivity for the target antigen itself, which in most cases is not exclusively expressed on tumor cells but also on healthy cells [79], although almost always at lower levels: for instance MART-1, which is also expressed by melanocytes. When targeting these antigens, vitiligo and inner ear toxicity resulting in a cer‐ tain degree of deafness are frequently observed side effects [38]. From this perspective it is reasonable to assume that off-target toxicities may be adverse reactions for clinical efficacy in an anti-melanoma response [80]. Since nearly all tumor-associated antigens are self-anti‐ gens, strategies will have to be developed to ensure that off-target toxicities are kept to a minimum. Whether T cells with low-avidity TCR or CAR are less prone to induce such un‐ desirable side effects is currently under investigation.

Melanoma cells, like other cancer cells, down-regulate components of the MHC and become increasingly deficient in antigen processing. As a consequence, TCR engineered T cells can no longer bind to and destroy those melanoma cells. However, they may be visible to a CAR recognizing surface antigens in a MHC independent manner, because of the antibody-de‐ rived binding domain (Figure 3). TCR redirected T cells, on the one hand, may also recog‐ nize cross-presented targeted antigen, for instance by stroma cells, but this is not the case for CAR engineered T cells. Cross-presented antigen, on the other hand, may help to destroy stroma, which is required to eliminate large tumor lesions [39, 40].

To avoid mispairing of the recombinant TCR with the physiological TCR chains and the re‐ sulting unpredictable auto-immunity, TCR-like single chain antibodies were used as target‐ ing domain in a CAR. Thus combining the MHC-restricted recognition of antigen with the T-body strategy. T cells with TCR-like CAR were redirected towards NY-ESO-1 and MAGE-A1, respectively [41, 42]. The possible advantages of these MHC restricted CAR's compared to the use of recombinant TCR's still has to be determined in trials.

duced in the last years to improve T cell persistence and activation [61, 62]. CAR's with a costimulatory domain clearly demonstrated clinical benefit and improved T cell persistence compared to CAR's targeting the same antigen but with only the CD3ζ domain [63-65].

Various CARs were engineered for targeting melanoma-associated antigens, including HMW-MAA, also known as MCSP [67, 68], melanotransferrin [69], the ganglioside GD2 [70] and GD3 [71]. A clinical trial targeting melanoma cells with CAR engineered T cells is cur‐ rently recruiting participants [66]. Recent phase I trials using CAR redirected T cells in the treatment of lymphoma/leukemia exhibited spectacular efficacy [72, 73]. However, the en‐ thusiasm was dampened by reports on serious adverse events and even fatalities after CAR T cell therapy [74, 75]. Targeting ErbB2 produced a cytokine storm and respiratory failure in one case [76] which is thought to be due to low levels of antigen on a number of healthy cells which can trigger CAR T cell activation. On the one hand, this event points out that ACT with CAR modified T cells may be a powerful therapy; but, on the other hand, empha‐ sizes the necessity for careful T cell dose escalation studies to balance anti-tumor efficacy

**4. Challenges and premises in the adoptive cell therapy of melanoma**

To date, approximately half of the melanoma patients treated with TIL ACT benefit from this therapy; genetic modification of T cells may further improve clinical response to mela‐ noma, but this will have to be proven in upcoming trials. However, the strategy has poten‐

A major challenge of redirected T cells is the tumor selectivity for the target antigen itself, which in most cases is not exclusively expressed on tumor cells but also on healthy cells [79], although almost always at lower levels: for instance MART-1, which is also expressed by melanocytes. When targeting these antigens, vitiligo and inner ear toxicity resulting in a cer‐ tain degree of deafness are frequently observed side effects [38]. From this perspective it is reasonable to assume that off-target toxicities may be adverse reactions for clinical efficacy in an anti-melanoma response [80]. Since nearly all tumor-associated antigens are self-anti‐ gens, strategies will have to be developed to ensure that off-target toxicities are kept to a minimum. Whether T cells with low-avidity TCR or CAR are less prone to induce such un‐

Melanoma cells, like other cancer cells, down-regulate components of the MHC and become increasingly deficient in antigen processing. As a consequence, TCR engineered T cells can no longer bind to and destroy those melanoma cells. However, they may be visible to a CAR recognizing surface antigens in a MHC independent manner, because of the antibody-de‐ rived binding domain (Figure 3). TCR redirected T cells, on the one hand, may also recog‐ nize cross-presented targeted antigen, for instance by stroma cells, but this is not the case for CAR engineered T cells. Cross-presented antigen, on the other hand, may help to destroy

and auto-immunity[61, 77, 78].

372 Melanoma - From Early Detection to Treatment

tial challenges which need to be addressed.

desirable side effects is currently under investigation.

stroma, which is required to eliminate large tumor lesions [39, 40].

The antibody-derived binding domain of a CAR displays extraordinary high affinity com‐ pared to a TCR. However, an increase in affinity, for instance, by affinity maturation, does not necessarily improve CAR redirected T cell activation above threshold [41, 43], which is not additionally modulated by CD28 costimulation [44]. A similar effect is also assumed for TCR mediated T cell activation. The TCR or CAR binding avidity probably affects the persis‐ tence of engineered T cells at the targeted tumor site. Strong binding to a target antigen may cause the T cells to be trapped and to become fully activated for a cytolytic attack, whereas low avidity interactions may not provide sufficiently long T cell – melanoma cell contacts. In addition to the binding avidity, the amount of target antigen on the cell surface also impacts on the selectivity of redirected T cell activation. In essence, low affinity binding directs the activity of engineered T cells preferentially toward target cells with abundant antigen levels; high affinity binding is likewise effective against low antigen levels on target cells. The opti‐ mized affinity to sustain a more selective T cell trafficking to the tumor and activation while avoiding targeting healthy cells that are expressing low quantities of the same antigen, how‐ ever, still has to be determined.

A beneficial T cell-to-target cell ratio at the tumor site seems to be required for efficient tu‐ mor elimination. Higher numbers of engineered T cells applied per dose will probably in‐ crease clinical efficacy; the majority of recent trials have applied up to 1010 cells per dose [27]. These and higher numbers of engineered T cells can be generated by extended expan‐ sion protocols; however, cells with a "young" phenotype may not be generated for adoptive transfer under these conditions. Short-term amplification protocols are therefore envisioned for both TIL's and engineered blood T cells. However, the majority of recent trials targeting CD19+ leukemia provided evidence for therapeutic efficacy at numbers less than or equal to 105 engineered T cells [73]. This once again raises the question of whether high T cell doses are required for a therapeutic effect.

The clinical outcome of adoptive cell therapy correlates with the persistence of adoptively transferred T cells [81]. As long as T cells engage their cognate antigen, T cells will expand and persist in detectable numbers; but when the antigen is no longer present, the T cell pop‐ ulation will contract to potentially undetectable levels and disappear from circulation. To improve survival of CAR T cells, Epstein-Barr virus (EBV)-specific T cells were engineered with a tumor-specific CAR based on the rationale that T cells recognizing the low amounts of EBV antigens by their physiological TCR will be maintained in a sizable population in cir‐ culation and in the process providing enough CAR T cells to recognize and kill melanoma cells in the surrounding tissues. A clinical trial with EBV-specific T cells engineered with an anti-GD2 CAR thus showed benefit over non-virus-specific, CAR engineered T cells in the treatment of neuroblastoma [81].

Adoptively transferred CD8+ T cell clones may be less persistent than CD4+ T cell clones due to T cell exhaustion after extensive *ex vivo* amplification and multiple rounds of activation. In addition, CD4+ T cell help is essential for CD8+ T cell persistence *in vivo*; adoptively trans‐ ferred pure CD8+ T cell clones may fail to persist [82]. T cell therapy may be combined with antibody therapy to prolong the initiated immune response. For instance, CTLA-4 is upre‐ gulated on the surface of activated T cells, where it acts as negative regulator to return the T cell to a resting stage. Co-application of the anti-CTLA-4 blocking antibody, ipilimumab, may prolong the anti-tumor activation of transferred T cells, although it would also affect all the other T cells.

This may be counteracted by expansion in the presence of IL-15 and IL-21 and/or by co-stim‐

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Metastatic melanoma patients with the B-raf activating mutation V600E transiently benefit from a small molecule drug, PLX4032 or vemurafenib, which inhibits the mitogen-activated protein kinase (MAPK) pathway. Treatment with vemurafenib is accompanied by increased T cell infiltrations in the melanoma lesions [94, 95]. Combination of B-raf inhibition with

Although the TCR downstream signaling machinery is used by the prototype CAR, mono‐ cytes, macrophages as well as NK cells can also be redirected by CAR's in an antigen-spe‐ cific fashion [96, 97]. Whether redirected non-T cells are advantageous in tumor elimination to cancer patients in general and to melanoma patients in particular has to be

melanoma-specific ACT may provide an option to prolong the clinical response.

**5. Does targeting "melanoma stem cells" provide hope for long-term**

Observations that a number of malignant lesions display a tremendous cellular and pheno‐ typic heterogeneity and contain pluripotent stem cells led to the hypothesis that cancer is initiated and maintained by so-called cancer stem cells (CSC's). Low abundance, induction of tumors upon transplantation under limiting conditions, radiation and chemo-resistance, self-renewal and a-symmetric differentiation into a variety of cell types are properties postu‐ lated for CSC's. The concept was sustained by deciphering the hierarchical organization in hematological malignancies [98], and subsequently in solid cancers including mammary, prostate, pancreatic, colon carcinoma and glioma [99-103]. Transplantation of melanoma cell subsets under limiting dilution conditions showed that a subset of cancer cells can induce tumors of the same histological phenotype as the parental tumor [99, 104, 105]. A first study using the limiting dilution transplantation assay identified a melanoma cell subset which ex‐ hibits stem-like capacities and expresses CD20 [106]. A conclusion drawn from these and other experiments was that melanoma is organized in a hierarchical manner originating from an initiator cell. In this context, several phenomena in melanoma biology which have been clinically observed but not well understood are described by the CSC model, for in‐ stance, metastatic relapse more than a decade after surgical treatment of the primary lesion. Residual CSC's are thought to drive cancer relapse even after years of "dormancy" [107]. Moreover, melanoma initiating cells were identified as expressing either the transporter pro‐ tein ABCB5 [104] or the nerve growth factor receptor CD271; the latter occurs in melanoma

However, transplantation under more rigorous conditions, i.e., ideally of one isolated mela‐ noma cell, revealed that nearly every fourth randomly taken melanoma cell (1/2 - 1/15) can induce tumors and raising the question of the validity the stem cell paradigm for melanoma [109, 110]. From these and subsequent studies, it has been concluded that the potential of melanoma induction is not closely associated with a particular phenotype and that the num‐

ulation via 4-1BB by an agonistic antibody [93].

explored in clinical trials.

**remission from melanoma?**

in a frequency of approximately 1/2000 cells [108].

Besides maintaining a high number of T cells in circulation, another challenge is to accumu‐ late significant numbers of effector T cells in the tumor lesion. A tightly controlled network of chemokines controls the migration of cells in the body; adoptively transferred T cells use these networks to accumulate at the tumor site. The expression of specific chemokine recep‐ tors controls how cells will migrate against the chemokine gradient into the targeted lesion. Melanoma cells secrete a number of chemokines including CXCL1. However, early imaging studies revealed that melanoma-specific T cells massively infiltrate the lungs, spleen and liv‐ er with some accumulation at the tumor site, which clearly represents a minority of the transferred cells, before the cells decline to undetectable levels in circulation [83-85]. Since those T cells do not express CXCR2, the receptor for melanoma secreted CXCL1, TIL's were engineered with CXCR2 which generated improved melanoma accumulation and anti-tu‐ mor activity in a mouse model [86]. The strategy is currently being explored in an early phase I trial (Table 1) [86].

One of the major hurdles of redirected immunotherapy of cancer in general is the tremen‐ dous heterogeneity of cancer cells with respect to the expression of the targeted antigen. Low or lack of antigen expression within the malignant lesions will negatively affect the long-term therapeutic efficacy of the approach. Several reports document relapse of anti‐ gen-loss tumor metastases after adoptive therapy with melanoma-reactive T cell clones [87-89] and argue for the use of polyclonal T cells with various melanoma specificities. Melanoma cells expressing the target antigen may successfully be eliminated by redirect‐ ed T cells, whereas antigen-negative tumor cells will not be recognized. T cell populations modified with different CAR's recognizing different antigens expressed by the same tu‐ mor may be able to overcome these limitations. However, pro-inflammatory cytokines se‐ creted by redirected T cells into the tumor micro-environment upon activation may attract a second wave of non-antigen restricted effector cells, which in turn may eradiate antigennegative tumor cells. At least in an animal model, antigen-negative melanoma cells are in‐ deed eliminated when co-inoculated with antibody-targeted cytokines [90]. Moreover, T cells engineered with induced expression of transgenic IL-12 attract innate immune cells including macrophages into the tumor tissue; they eliminate antigen-negative tumor cells in the same lesion [91].

Highly expanded T cells, such as TIL's, become hypo-responsive to CD28 costimulation and rapidly enter activation induced cell death, in particular upon IL-2 driven expansion [92]. This may be counteracted by expansion in the presence of IL-15 and IL-21 and/or by co-stim‐ ulation via 4-1BB by an agonistic antibody [93].

Adoptively transferred CD8+ T cell clones may be less persistent than CD4+ T cell clones due to T cell exhaustion after extensive *ex vivo* amplification and multiple rounds of activation. In addition, CD4+ T cell help is essential for CD8+ T cell persistence *in vivo*; adoptively trans‐

antibody therapy to prolong the initiated immune response. For instance, CTLA-4 is upre‐ gulated on the surface of activated T cells, where it acts as negative regulator to return the T cell to a resting stage. Co-application of the anti-CTLA-4 blocking antibody, ipilimumab, may prolong the anti-tumor activation of transferred T cells, although it would also affect all

Besides maintaining a high number of T cells in circulation, another challenge is to accumu‐ late significant numbers of effector T cells in the tumor lesion. A tightly controlled network of chemokines controls the migration of cells in the body; adoptively transferred T cells use these networks to accumulate at the tumor site. The expression of specific chemokine recep‐ tors controls how cells will migrate against the chemokine gradient into the targeted lesion. Melanoma cells secrete a number of chemokines including CXCL1. However, early imaging studies revealed that melanoma-specific T cells massively infiltrate the lungs, spleen and liv‐ er with some accumulation at the tumor site, which clearly represents a minority of the transferred cells, before the cells decline to undetectable levels in circulation [83-85]. Since those T cells do not express CXCR2, the receptor for melanoma secreted CXCL1, TIL's were engineered with CXCR2 which generated improved melanoma accumulation and anti-tu‐ mor activity in a mouse model [86]. The strategy is currently being explored in an early

One of the major hurdles of redirected immunotherapy of cancer in general is the tremen‐ dous heterogeneity of cancer cells with respect to the expression of the targeted antigen. Low or lack of antigen expression within the malignant lesions will negatively affect the long-term therapeutic efficacy of the approach. Several reports document relapse of anti‐ gen-loss tumor metastases after adoptive therapy with melanoma-reactive T cell clones [87-89] and argue for the use of polyclonal T cells with various melanoma specificities. Melanoma cells expressing the target antigen may successfully be eliminated by redirect‐ ed T cells, whereas antigen-negative tumor cells will not be recognized. T cell populations modified with different CAR's recognizing different antigens expressed by the same tu‐ mor may be able to overcome these limitations. However, pro-inflammatory cytokines se‐ creted by redirected T cells into the tumor micro-environment upon activation may attract a second wave of non-antigen restricted effector cells, which in turn may eradiate antigennegative tumor cells. At least in an animal model, antigen-negative melanoma cells are in‐ deed eliminated when co-inoculated with antibody-targeted cytokines [90]. Moreover, T cells engineered with induced expression of transgenic IL-12 attract innate immune cells including macrophages into the tumor tissue; they eliminate antigen-negative tumor cells

Highly expanded T cells, such as TIL's, become hypo-responsive to CD28 costimulation and rapidly enter activation induced cell death, in particular upon IL-2 driven expansion [92].

T cell clones may fail to persist [82]. T cell therapy may be combined with

ferred pure CD8+

374 Melanoma - From Early Detection to Treatment

the other T cells.

phase I trial (Table 1) [86].

in the same lesion [91].

Metastatic melanoma patients with the B-raf activating mutation V600E transiently benefit from a small molecule drug, PLX4032 or vemurafenib, which inhibits the mitogen-activated protein kinase (MAPK) pathway. Treatment with vemurafenib is accompanied by increased T cell infiltrations in the melanoma lesions [94, 95]. Combination of B-raf inhibition with melanoma-specific ACT may provide an option to prolong the clinical response.

Although the TCR downstream signaling machinery is used by the prototype CAR, mono‐ cytes, macrophages as well as NK cells can also be redirected by CAR's in an antigen-spe‐ cific fashion [96, 97]. Whether redirected non-T cells are advantageous in tumor elimination to cancer patients in general and to melanoma patients in particular has to be explored in clinical trials.

## **5. Does targeting "melanoma stem cells" provide hope for long-term remission from melanoma?**

Observations that a number of malignant lesions display a tremendous cellular and pheno‐ typic heterogeneity and contain pluripotent stem cells led to the hypothesis that cancer is initiated and maintained by so-called cancer stem cells (CSC's). Low abundance, induction of tumors upon transplantation under limiting conditions, radiation and chemo-resistance, self-renewal and a-symmetric differentiation into a variety of cell types are properties postu‐ lated for CSC's. The concept was sustained by deciphering the hierarchical organization in hematological malignancies [98], and subsequently in solid cancers including mammary, prostate, pancreatic, colon carcinoma and glioma [99-103]. Transplantation of melanoma cell subsets under limiting dilution conditions showed that a subset of cancer cells can induce tumors of the same histological phenotype as the parental tumor [99, 104, 105]. A first study using the limiting dilution transplantation assay identified a melanoma cell subset which ex‐ hibits stem-like capacities and expresses CD20 [106]. A conclusion drawn from these and other experiments was that melanoma is organized in a hierarchical manner originating from an initiator cell. In this context, several phenomena in melanoma biology which have been clinically observed but not well understood are described by the CSC model, for in‐ stance, metastatic relapse more than a decade after surgical treatment of the primary lesion. Residual CSC's are thought to drive cancer relapse even after years of "dormancy" [107]. Moreover, melanoma initiating cells were identified as expressing either the transporter pro‐ tein ABCB5 [104] or the nerve growth factor receptor CD271; the latter occurs in melanoma in a frequency of approximately 1/2000 cells [108].

However, transplantation under more rigorous conditions, i.e., ideally of one isolated mela‐ noma cell, revealed that nearly every fourth randomly taken melanoma cell (1/2 - 1/15) can induce tumors and raising the question of the validity the stem cell paradigm for melanoma [109, 110]. From these and subsequent studies, it has been concluded that the potential of melanoma induction is not closely associated with a particular phenotype and that the num‐ ber of potential CSC's in melanoma may not necessarily be low. This resulted in a further conclusion that nearly every melanoma cell is capable to re-program to a tumor initiating cell under certain experimental conditions of xeno-transplantation irrespectively which par‐ ticular marker phenotype the cell expressed at the time of isolation from a melanoma lesion.

the remaining melanoma sustaining cells, which are extraordinary resistant to chemothera‐ peutics. This resistance is probably due to transporter molecules like ABCB5, which are highly expressed by a number of CSC's including melanoma [104] and therefore efficiently counteract chemotherapy. Melanoma maintaining cells like other CSC's are merely in a "dormant" state and replicate less frequently than the majority of cancer cells in the same le‐ sion, which reduces the efficacy of anti-proliferative drugs. Low proliferative capacities to‐ gether with the efficient export of chemotherapeutics contribute to CSC resistance toward a variety of therapeutic drugs. As a consequence, alternative strategies that specifically induce cell death of those cells are required. Moreover, the situation is exacerbated by the fact that the melanoma maintaining cells in the lesion are rare and unlikely to be eliminated by the random targeting provided by most therapeutic agents. Specific targeting by cytotoxic T cells redirected towards CD20 or by CD20-specific therapeutic antibodies like Rituxan™ (rit‐ uximab) or Arzerra™ (ofatumumab), probably as adjunct to a tumor de-bulking strategy,

Adoptive Cell Therapy of Melanoma: The Challenges of Targeting the Beating Heart

http://dx.doi.org/10.5772/53619

377

Second, whether the prevalence of CD20+ melanoma maintaining cells in a tumor lesion may correlate with clinical progression or relapse has to be addressed. If so, the frequency of CD20+ melanoma cells may serve as a surrogate marker for therapeutic efficacy and/or prog‐ nosis. Chemotherapy and/or radiation may induce amplification of these cells thus contribu‐

Third, melanoma maintaining cells may exhibit an extraordinary functional and phenotyp‐ ic plasticity. As a consequence, continuous presence of targeting therapeutic agents will be required to eliminate those cells, which exhibit newly acquired melanoma initiating and/or maintaining capacities. In their pre-clinical model, Schmidt and colleagues [69] used CAR engineered T cells which penetrate tissues, scan for targets and persist for longterm acting as an antigen-specific guardian. These T cells are present in the targeted le‐ sion as long as cells expressing the target antigen appear. Repetitive restimulation of these T cells, for instance by engaging their TCR with EBV-specific antigens [63, 81], may sus‐ tain persistence of CAR T cells in sufficient numbers over long periods of time. In this constellation, cellular therapy has a major advantage compared to pharmaceutical drugs, which are present in therapeutic levels for short periods; in the case of melanoma the re‐ quired period for screening for re-appearance of such melanoma initiating cells may be many years. The development of an antigen-specific memory by adoptively transferred CAR T cells, as recently shown in a pre-clinical model [112], may be of benefit to patients

Work in the author's laboratory was supported by the Deutsche Krebshilfe, Bonn and Ziel 2. NRW Programm of the Ministerium für Innovation, Wissenschaft, Forschung und Technolo‐

gie des Landes Nordrhein-Westfalen and of the European Union.

ting to their accumulation during tumor progression and metastasis.

may improve the situation.

in preventing a melanoma relapse.

**Acknowledgements**

Once the tumor is established, a minor subset seems to take over control of melanoma progres‐ sion. Evidence is provided by recent observations from a pre-clinical model [69], which ad‐ dressed the question of whether specific elimination of defined melanoma cells from an established xeno-transplanted lesion causes tumor regression by adoptive transfer of antigenspecific cytotoxic T cell. The rationale is that, if there is a clearly defined hierarchy of cancer cells in an established tumor, specific ablation of the melanoma sustaining cells from the estab‐ lished tumor tissue must inevitably lead to a decay of the tumor lesion independently of target‐ ing the cancer cell mass. However, the melanoma sustaining cell may, but must not, be identical to CSC's identified by the transplantation assay. Targeted elimination of a minor sub‐ set of CD20+ melanoma cells completely eradicated transplanted melanoma lesions, whereas targeted elimination of any random melanoma cell population in the same lesion did not. CD20+ melanoma cells are rare, i.e. approximately 1-2%, in melanoma, independently of the histological type and the transplanted tumor tissue. A caveat is that in approximately 20% of melanoma samples, no CD20+ melanoma cells could be detected by histological screening. When these tumors were transplanted, adoptive transfer of CD20-specific CAR T cells did not induce tumor regression. Interestingly, CD20 re-expression in a random subpopulation of those tumor cells did not render the tumor lesion sensitive for complete eradication with CD20 specific T cells. This indicates that CD20 expression *per se* is not dominant in maintaining mela‐ noma progression. However, the phenotype of CD20+ melanoma cells may be flexible and associated with additional capabilities which mediate the dominant effect.

The first clinical evidence confirming this concept was recently provided by a case report [111]. A patient with stage III/IV metastatic melanoma, which harbored CD20+ melanoma cells at a frequency of 2%, received intra-lesional injections of the anti-CD20 therapeutic an‐ tibody rituximab and concomitant dacarbazine treatment. Dacarbazine as mono-therapy had already proved to be ineffective. This treatment produced lasting complete and partial remission accompanied by a decline of the melanoma serum marker S-100 to physiological levels, a switch of a T helper-2 to a more pro-inflammatory T helper-1 response, all without treatment related grade 3/4 toxicity. Although anecdotic, this data provides the first clinical evidence that targeting the subset of CD20+ melanoma sustaining cells can produce regres‐ sion of chemotherapy-refractory melanoma. Moreover, the report highlights the potency of selective cancer cell targeting in the treatment of melanoma.

These observations although so far based on a pre-clinical model and a clinical observation which will have to be reproduced in larger cohorts have major impact on the future devel‐ opment of melanoma therapy.

First, the melanoma maintaining cells may be more resistant to current therapy regimens than the bulk of melanoma cells. Standard therapy strategies attempt to eliminated all can‐ cer cells in a tumor lesion; elimination of any other cancer cells than the tumor progressing cells will rapidly de-bulk the tumor lesion. The melanoma will inevitably relapse, driven by the remaining melanoma sustaining cells, which are extraordinary resistant to chemothera‐ peutics. This resistance is probably due to transporter molecules like ABCB5, which are highly expressed by a number of CSC's including melanoma [104] and therefore efficiently counteract chemotherapy. Melanoma maintaining cells like other CSC's are merely in a "dormant" state and replicate less frequently than the majority of cancer cells in the same le‐ sion, which reduces the efficacy of anti-proliferative drugs. Low proliferative capacities to‐ gether with the efficient export of chemotherapeutics contribute to CSC resistance toward a variety of therapeutic drugs. As a consequence, alternative strategies that specifically induce cell death of those cells are required. Moreover, the situation is exacerbated by the fact that the melanoma maintaining cells in the lesion are rare and unlikely to be eliminated by the random targeting provided by most therapeutic agents. Specific targeting by cytotoxic T cells redirected towards CD20 or by CD20-specific therapeutic antibodies like Rituxan™ (rit‐ uximab) or Arzerra™ (ofatumumab), probably as adjunct to a tumor de-bulking strategy, may improve the situation.

Second, whether the prevalence of CD20+ melanoma maintaining cells in a tumor lesion may correlate with clinical progression or relapse has to be addressed. If so, the frequency of CD20+ melanoma cells may serve as a surrogate marker for therapeutic efficacy and/or prog‐ nosis. Chemotherapy and/or radiation may induce amplification of these cells thus contribu‐ ting to their accumulation during tumor progression and metastasis.

Third, melanoma maintaining cells may exhibit an extraordinary functional and phenotyp‐ ic plasticity. As a consequence, continuous presence of targeting therapeutic agents will be required to eliminate those cells, which exhibit newly acquired melanoma initiating and/or maintaining capacities. In their pre-clinical model, Schmidt and colleagues [69] used CAR engineered T cells which penetrate tissues, scan for targets and persist for longterm acting as an antigen-specific guardian. These T cells are present in the targeted le‐ sion as long as cells expressing the target antigen appear. Repetitive restimulation of these T cells, for instance by engaging their TCR with EBV-specific antigens [63, 81], may sus‐ tain persistence of CAR T cells in sufficient numbers over long periods of time. In this constellation, cellular therapy has a major advantage compared to pharmaceutical drugs, which are present in therapeutic levels for short periods; in the case of melanoma the re‐ quired period for screening for re-appearance of such melanoma initiating cells may be many years. The development of an antigen-specific memory by adoptively transferred CAR T cells, as recently shown in a pre-clinical model [112], may be of benefit to patients in preventing a melanoma relapse.

## **Acknowledgements**

ber of potential CSC's in melanoma may not necessarily be low. This resulted in a further conclusion that nearly every melanoma cell is capable to re-program to a tumor initiating cell under certain experimental conditions of xeno-transplantation irrespectively which par‐ ticular marker phenotype the cell expressed at the time of isolation from a melanoma lesion. Once the tumor is established, a minor subset seems to take over control of melanoma progres‐ sion. Evidence is provided by recent observations from a pre-clinical model [69], which ad‐ dressed the question of whether specific elimination of defined melanoma cells from an established xeno-transplanted lesion causes tumor regression by adoptive transfer of antigenspecific cytotoxic T cell. The rationale is that, if there is a clearly defined hierarchy of cancer cells in an established tumor, specific ablation of the melanoma sustaining cells from the estab‐ lished tumor tissue must inevitably lead to a decay of the tumor lesion independently of target‐ ing the cancer cell mass. However, the melanoma sustaining cell may, but must not, be identical to CSC's identified by the transplantation assay. Targeted elimination of a minor sub‐

melanoma cells completely eradicated transplanted melanoma lesions, whereas

targeted elimination of any random melanoma cell population in the same lesion did not.

The first clinical evidence confirming this concept was recently provided by a case report [111]. A patient with stage III/IV metastatic melanoma, which harbored CD20+ melanoma cells at a frequency of 2%, received intra-lesional injections of the anti-CD20 therapeutic an‐ tibody rituximab and concomitant dacarbazine treatment. Dacarbazine as mono-therapy had already proved to be ineffective. This treatment produced lasting complete and partial remission accompanied by a decline of the melanoma serum marker S-100 to physiological levels, a switch of a T helper-2 to a more pro-inflammatory T helper-1 response, all without treatment related grade 3/4 toxicity. Although anecdotic, this data provides the first clinical

sion of chemotherapy-refractory melanoma. Moreover, the report highlights the potency of

These observations although so far based on a pre-clinical model and a clinical observation which will have to be reproduced in larger cohorts have major impact on the future devel‐

First, the melanoma maintaining cells may be more resistant to current therapy regimens than the bulk of melanoma cells. Standard therapy strategies attempt to eliminated all can‐ cer cells in a tumor lesion; elimination of any other cancer cells than the tumor progressing cells will rapidly de-bulk the tumor lesion. The melanoma will inevitably relapse, driven by

melanoma sustaining cells can produce regres‐

associated with additional capabilities which mediate the dominant effect.

evidence that targeting the subset of CD20+

opment of melanoma therapy.

selective cancer cell targeting in the treatment of melanoma.

 melanoma cells are rare, i.e. approximately 1-2%, in melanoma, independently of the histological type and the transplanted tumor tissue. A caveat is that in approximately 20% of melanoma samples, no CD20+ melanoma cells could be detected by histological screening. When these tumors were transplanted, adoptive transfer of CD20-specific CAR T cells did not induce tumor regression. Interestingly, CD20 re-expression in a random subpopulation of those tumor cells did not render the tumor lesion sensitive for complete eradication with CD20 specific T cells. This indicates that CD20 expression *per se* is not dominant in maintaining mela‐ noma progression. However, the phenotype of CD20+ melanoma cells may be flexible and

set of CD20+

376 Melanoma - From Early Detection to Treatment

CD20+

Work in the author's laboratory was supported by the Deutsche Krebshilfe, Bonn and Ziel 2. NRW Programm of the Ministerium für Innovation, Wissenschaft, Forschung und Technolo‐ gie des Landes Nordrhein-Westfalen and of the European Union.

## **Abbreviations**

**ACT**, adoptive cell therapy; **CAR**, chimeric antigen receptor; **CTLA-4**, anti-cytotoxic T-lym‐ phocyte-associated antigen-4; **CSC**, cancer stem cell; **EBV**, Epstein-Barr virus; **GMP**, Good Manufacturing Practice; **IFN**, interferon; **IL**, interleukin; **TCR**, T cell receptor; **TIL**, tumor in‐ filtrating lymphocyte

[6] Denninghoff VC, Kahn AG, Falco J, Curutchet HP, Elsner B. Sentinel lymph node: detection of micrometastases of melanoma in a molecular study. Molecular Diagno‐

Adoptive Cell Therapy of Melanoma: The Challenges of Targeting the Beating Heart

http://dx.doi.org/10.5772/53619

379

[7] Bedikian AY, Wei C, Detry M, Kim KB, Papadopoulos NE, Hwu WJ, Homsi J, Davies M, McIntyre S, Hwu P. Predictive Factors for the Development of Brain Metastasis in Advanced Unresectable Metastatic Melanoma. American Journal of Clinical Oncolo‐

[8] Bradbury PA, Middleton MR. DNA repair pathways in drug resistance in melanoma.

[9] Pak BJ, Chu W, Lu SJ, Kerbel RS, Ben-David Y. Lineage-specific mechanism of drug and radiation resistance in melanoma mediated by tyrosinase-related protein 2. Can‐

[10] Pak BJ, Lee J, Thai BL, Fuchs SY, Shaked Y, Ronai Z, Kerbel RS, Ben-David Y. Radia‐ tion resistance of human melanoma analysed by retroviral insertional mutagenesis reveals a possible role for dopachrome tautomerase. Oncogene 2004;23(1) 30-38. [11] Atkins MB, Lotze MT, Dutcher JP, Fisher RI, Weiss G, Margolin K, Abrams J, Sznol M, Parkinson D, Hawkins M, Paradise C, Kunkel L, Rosenberg SA. High-dose re‐ combinant interleukin 2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and 1993. Journal of Clinical Oncology 1999;17( 7)

[12] Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, Gonzalez R, Robert C, Schadendorf D, Hassel JC, Akerley W, van den Eertwegh AJ, Lutzky J, Lor‐ igan P, Vaubel JM, Linette GP, Hogg D, Ottensmeier CH, Lebbé C, Peschel C, Quirt I, Clark JI, Wolchok JD, Weber JS, Tian J, Yellin MJ, Nichol GM, Hoos A, Urba WJ. Im‐ proved survival with ipilimumab in patients with metastatic melanoma. The New

[13] Kirkwood JM, Ibrahim JG, Sondak VK, Richards J, Flaherty LE, Ernstoff MS, Smith TJ, Rao U, Steele M, Blum RH. High- and low-dose interferon alfa-2b in high-risk melanoma: first analysis of intergroup trial E1690/S9111/C9190. Journal of Clinical

[14] Kirkwood JM, Strawderman MH, Ernstoff MS, Smith TJ, Borden EC, Blum RH. Inter‐ feron alfa-2b adjuvant therapy of high-risk resected cutaneous melanoma: the East‐ ern Cooperative Oncology Group Trial EST 1684. Journal of Clinical Oncology

[15] Galluzzi L, Vacchelli E, Eggermont A, Fridman WH, Galon J, Sautès-Fridman C, Tar‐ tour E, Zitvogel L, Kroemer G. Trial Watch: Adoptive cell transfer immunotherapy.

[16] Bernatchez C, Radvanyi LG, Hwu P. Advances in the treatment of metastatic mela‐

noma: adoptive T-cell therapy. Seminars in Oncology 2012;39(2) 215-226.

sis 2004;8(4) 253-258.

gy 2010;34(6) 603-610.

2105-2116.

Anti-cancer Drugs 2004;15(5) 421-426.

cer Metastasis Reviews 2001;20(1-2) 27-32.

England Journal of Medicine 2010;363(8) 711-723.

Oncology 2000;18(12) 2444-2458.

Oncoimmunology 2012;1(3) 306-315.

1996;14(1) 7-17.

## **Author details**

Jennifer Makalowski1,2 and Hinrich Abken1,2\*

\*Address all correspondence to: hinrich.abken@uk-koeln.de

1 Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany

2 Dept. I Internal Medicine, University Hospital Cologne, Cologne, Germany

## **References**


[6] Denninghoff VC, Kahn AG, Falco J, Curutchet HP, Elsner B. Sentinel lymph node: detection of micrometastases of melanoma in a molecular study. Molecular Diagno‐ sis 2004;8(4) 253-258.

**Abbreviations**

378 Melanoma - From Early Detection to Treatment

filtrating lymphocyte

**Author details**

**References**

(26) 2507-2516.

Jennifer Makalowski1,2 and Hinrich Abken1,2\*

\*Address all correspondence to: hinrich.abken@uk-koeln.de

Journal of Cancer 2010;46(2) 270-283.

**ACT**, adoptive cell therapy; **CAR**, chimeric antigen receptor; **CTLA-4**, anti-cytotoxic T-lym‐ phocyte-associated antigen-4; **CSC**, cancer stem cell; **EBV**, Epstein-Barr virus; **GMP**, Good Manufacturing Practice; **IFN**, interferon; **IL**, interleukin; **TCR**, T cell receptor; **TIL**, tumor in‐

1 Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany

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2 Dept. I Internal Medicine, University Hospital Cologne, Cologne, Germany


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[121] Robbins PF, Morgan RA, Feldman SA, Yang JC, Sherry RM, Dudley ME, Wunderlich JR, Nahvi AV, Helman LJ, Mackall CL, Kammula US, Hughes MS, Restifo NP, Raf‐

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**Chapter 14**

**Cellular and Molecular Mechanisms of Methotrexate**

Melanoma is a cancer that develops in melanocytes, the pigment cells present in the skin. It can be more serious than the other forms of skin cancer because it may spread to other parts of the body (metastasize) and cause serious illness and death. For malignant melanomas standard treatment options have remained remarkably static over the past 30 years [1,2]. At present, the incidence of melanoma continues to increase despite public health initiatives that have promoted protection against the sun. Thus, during the past ten years, the incidence and annual mortality of melanoma has increased more rapidly than any other cancer and according to the American Cancer Society estimate, there will have been approximately 76,250 new cases of invasive melanoma diagnosed in 2012 in the United States, which resulted in approximately

Unfortunately, the increase in incidence has not been paralleled by the development of new therapeutic agents with a significant impact on survival. Although many patients with mela‐ noma localized to the skin are cured by surgical excision, increased time to diagnosis is asso‐ ciated with higher stage of disease, and those with regional lymphatic or metastatic disease respond poorly to conventional radiation and chemotherapy with 5-year survival rates rang‐ ing from 10 to 50% [4]. Currently, limited therapeutic options exist for patients with metastatic melanomas, and all standard combinations currently used in metastasis therapy have low efficacy and poor response rates. For instance, the only approved chemotherapy for metastatic melanoma, dacarbacine, has a response rate of about 10% and a median survival of 8-9 months.

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

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

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

**Resistance in Melanoma**

Juan Cabezas-Herrera and

http://dx.doi.org/10.5772/52414

**1. Introduction**

9,180 deaths [3].

Jose Neptuno Rodriguez-Lopez

Luis Sanchez del-Campo, Maria F. Montenegro, Magali Saez-Ayala, María Piedad Fernández-Pérez,

Additional information is available at the end of the chapter

## **Cellular and Molecular Mechanisms of Methotrexate Resistance in Melanoma**

Luis Sanchez del-Campo, Maria F. Montenegro, Magali Saez-Ayala, María Piedad Fernández-Pérez, Juan Cabezas-Herrera and Jose Neptuno Rodriguez-Lopez

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52414

**1. Introduction**

feld M, Lee CC, Levy CL, Li YF, El-Gamil M, Schwarz SL, Laurencot C, Rosenberg SA. Tumor regression in patients with metastatic synovial cell sarcoma and melano‐ ma using genetically engineered lymphocytes reactive with NY-ESO-1. Journal of

Clinical Oncology 2011;29(7) 917-924.

390 Melanoma - From Early Detection to Treatment

Melanoma is a cancer that develops in melanocytes, the pigment cells present in the skin. It can be more serious than the other forms of skin cancer because it may spread to other parts of the body (metastasize) and cause serious illness and death. For malignant melanomas standard treatment options have remained remarkably static over the past 30 years [1,2]. At present, the incidence of melanoma continues to increase despite public health initiatives that have promoted protection against the sun. Thus, during the past ten years, the incidence and annual mortality of melanoma has increased more rapidly than any other cancer and according to the American Cancer Society estimate, there will have been approximately 76,250 new cases of invasive melanoma diagnosed in 2012 in the United States, which resulted in approximately 9,180 deaths [3].

Unfortunately, the increase in incidence has not been paralleled by the development of new therapeutic agents with a significant impact on survival. Although many patients with mela‐ noma localized to the skin are cured by surgical excision, increased time to diagnosis is asso‐ ciated with higher stage of disease, and those with regional lymphatic or metastatic disease respond poorly to conventional radiation and chemotherapy with 5-year survival rates rang‐ ing from 10 to 50% [4]. Currently, limited therapeutic options exist for patients with metastatic melanomas, and all standard combinations currently used in metastasis therapy have low efficacy and poor response rates. For instance, the only approved chemotherapy for metastatic melanoma, dacarbacine, has a response rate of about 10% and a median survival of 8-9 months.

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

The other approved agent for advanced melanoma is high dose interleukin-2, which can induce dramatic complete and durable responses [2]. However, only one patient in twenty derives lasting benefit. These data indicate the needed for alternative therapies for this disease and recent results indicated that combined therapies could became an attractive strategy to fight melanoma [2].

as a mechanism by which antifolate drugs promote apoptosis in cancer cells [15,16]. Although the mechanism of dTTP depletion-induced apoptosis is yet to be determined, Pardee's group recently postulated that dTTP controls E2F1, which regulates both DNA synthesis and apop‐ tosis. This hypothesis was based on the observation that MTX increased E2F1 levels in sensitive

Cellular and Molecular Mechanisms of Methotrexate Resistance in Melanoma

http://dx.doi.org/10.5772/52414

393

Eukaryotic cells have developed complex checkpoint pathways that monitor DNA for damage or incomplete replication. Checkpoint pathways are amplified upon detection of aberrant DNA structures and lead to a delay in cell cycle progression during which damage can be repaired or replication be completed. Alternatively, in case of heavily damaged or seriously deregulated cells, checkpoint activation can result in apoptosis. As such, checkpoint mecha‐ nisms are essential for the maintenance of genomic integrity [17]. When vertebrate cells expe‐ rience replication arrest or undergo DNA damage by UV irradiation, the ATR kinase [ataxia telangiectasia mutated (ATM)- and Rad3-related kinase] phosphorylates and activates the Chk1 protein kinase. Activated Chk1 inhibits Cdc25 phosphatases, which control inhibitory phosphorylation sites on cyclin-dependent kinases, the latter being critical regulators of cell cycle transitions [18,19]. Because the ability of cells to delay cell cycle progression and halt DNA synthesis represents a defensive mechanism that spares potential toxicity [20], the acti‐ vation of Chk1 by MTX could constitute a key event in the resistance of melanoma to MTX. In addition to these cellular mechanisms of resistance to MTX in melanoma, other mechanism that includes liver transformation of the drug has also been reported. A paradoxical response of malignant melanoma to MTX *in vivo* and *in vitro* has been described [21]. The authors ob‐ served that MTX showed consistent cytotoxicity for melanoma cells *in vitro* but was ineffective at equivalent concentrations *in vivo*. MTX undergoes oxidation to its primary metabolite 7 hydroxy-MTX (7-OH-MTX) in the liver by the enzyme aldehyde oxidase [11] and therefore, this transformation has been proposed as a novel mechanism of resistance to explain this par‐ adox [11,21]. In contrast to the large body of literature available on the multiple modalities of MTX resistance, very little is known regarding the ability of 7-OH-MTX to provoke antifolateresistance phenomena that may disrupt MTX activity. Recent studies seem to indicate that 7- OH-MTX which exceeds by far MTX in the plasma of MTX-treated patients can provoke distinct modalities of antifolate-resistance that severely compromise the efficacy of the parent

cancer cells, resulting in an increase in the E2F1-mediated apoptotic cascade.

**3. Melanoma-specific mechanisms of resistance to MTX**

**3.1. The critical role of alpha-folate receptor in the resistance of melanoma to MTX**

Experiments from our laboratory and others provide evidence that melanosomes contribute to the refractory properties of melanoma cells by sequestering cytotoxic drugs and increasing melanosome-mediated drug export [6,12,13]. Concretely, we have described that folate recep‐ tor α (FRα)-endocytotic transport of MTX facilitates drug melanosomal sequestration and cel‐ lular exportation in melanoma cells, which ensures reduced accumulation of MTX in intracellular compartments [6]. An important observation in this study was that MTX was a

drug MTX [22].

Other example of the complications involved in melanoma chemotherapy is the limited effec‐ tiveness of antifolates. Although methotrexate (MTX), the most frequently used antifolate, is an efficient drug for several types of cancer, it is not active against melanoma [5-7]. Undoubt‐ edly, unravelling the mechanisms of melanoma resistance to MTX could yield important in‐ formation on how to circumvent this resistance and could have important pharmacological implications for the design of novel combined therapies. Thus, although an old drug, MTX could become a valuable tool with which to improve melanoma therapy.

## **2. General mechanisms of resistance to classical antifolates**

The antifolate methotrexate was rationally-designed nearly 70 years ago to potently block the folate-dependent enzyme dihydrofolate reductase (DHFR). DHFR (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) catalyses the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) in the presence of coenzyme NADPH as follows: DHF + NADPH + H+ → THF + NADP+. This enzyme is necessary for maintaining intracellular pools of THF and its derivatives which are essential cofactors in one-carbon metabolism. Coupled with thymidylate synthase (TS) [8], it is directly involved in thymidylate (dTMP) production through a *de novo* pathway. DHFR is therefore pivotal in providing purines and pyrimidine precursors for the biosynthesis of DNA, RNA and amino acids. In addition, it is the target enzyme [9] for antifolate drugs such as the antineoplastic drug MTX and the antibacterial drug trimethoprim (TMP). The mechanisms of resistance to MTX have been extensively studied, mainly in experimental tumours propagated *in vitro* and *in vivo* [5,10,11]; however, the specific basis for the resistance of melanoma cells to MTX is unclear. During decades the mechanism of resistance of melanoma to MTX was associated with general mechanisms of resistance de‐ tected in other epithelial cancer cell including reduced cellular uptake of this drug, high in‐ tracellular levels of DHFR and/or insufficient rate of MTX polyglutamylation, which diminishes long-chain MTX polyglutamates from being preferentially retained intracellularly [11]. However, recently, a melanoma-specific mechanism of resistance to cytotoxic drugs, in‐ cluding MTX, has been described [6,12,13].

Antifolate resistance in cancer cells is believed to be a multifactorial process in which dysre‐ gulation of apoptosis, insufficient rates of MTX polyglutamylation, and enhanced DNA repair play important roles [11,14]. In melanoma, another classical mechanism of resistance to MTX, the upregulation of endogenous dihydrofolate reductase (DHFR) activity, has been described [5]; however, the contribution of this mechanism to the overall resistance of melanoma to MTX as well as its possible impact on DNA damage response pathways in cells is unknown. 'Thy‐ mineless' death, which occurs upon the depletion of cellular dTTP pools, has been proposed as a mechanism by which antifolate drugs promote apoptosis in cancer cells [15,16]. Although the mechanism of dTTP depletion-induced apoptosis is yet to be determined, Pardee's group recently postulated that dTTP controls E2F1, which regulates both DNA synthesis and apop‐ tosis. This hypothesis was based on the observation that MTX increased E2F1 levels in sensitive cancer cells, resulting in an increase in the E2F1-mediated apoptotic cascade.

The other approved agent for advanced melanoma is high dose interleukin-2, which can induce dramatic complete and durable responses [2]. However, only one patient in twenty derives lasting benefit. These data indicate the needed for alternative therapies for this disease and recent results indicated that combined therapies could became an attractive strategy to fight

Other example of the complications involved in melanoma chemotherapy is the limited effec‐ tiveness of antifolates. Although methotrexate (MTX), the most frequently used antifolate, is an efficient drug for several types of cancer, it is not active against melanoma [5-7]. Undoubt‐ edly, unravelling the mechanisms of melanoma resistance to MTX could yield important in‐ formation on how to circumvent this resistance and could have important pharmacological implications for the design of novel combined therapies. Thus, although an old drug, MTX

The antifolate methotrexate was rationally-designed nearly 70 years ago to potently block the folate-dependent enzyme dihydrofolate reductase (DHFR). DHFR (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) catalyses the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) in the presence of coenzyme NADPH as follows: DHF + NADPH + H+ → THF + NADP+. This enzyme is necessary for maintaining intracellular pools of THF and its derivatives which are essential cofactors in one-carbon metabolism. Coupled with thymidylate synthase (TS) [8], it is directly involved in thymidylate (dTMP) production through a *de novo* pathway. DHFR is therefore pivotal in providing purines and pyrimidine precursors for the biosynthesis of DNA, RNA and amino acids. In addition, it is the target enzyme [9] for antifolate drugs such as the antineoplastic drug MTX and the antibacterial drug trimethoprim (TMP). The mechanisms of resistance to MTX have been extensively studied, mainly in experimental tumours propagated *in vitro* and *in vivo* [5,10,11]; however, the specific basis for the resistance of melanoma cells to MTX is unclear. During decades the mechanism of resistance of melanoma to MTX was associated with general mechanisms of resistance de‐ tected in other epithelial cancer cell including reduced cellular uptake of this drug, high in‐ tracellular levels of DHFR and/or insufficient rate of MTX polyglutamylation, which diminishes long-chain MTX polyglutamates from being preferentially retained intracellularly [11]. However, recently, a melanoma-specific mechanism of resistance to cytotoxic drugs, in‐

Antifolate resistance in cancer cells is believed to be a multifactorial process in which dysre‐ gulation of apoptosis, insufficient rates of MTX polyglutamylation, and enhanced DNA repair play important roles [11,14]. In melanoma, another classical mechanism of resistance to MTX, the upregulation of endogenous dihydrofolate reductase (DHFR) activity, has been described [5]; however, the contribution of this mechanism to the overall resistance of melanoma to MTX as well as its possible impact on DNA damage response pathways in cells is unknown. 'Thy‐ mineless' death, which occurs upon the depletion of cellular dTTP pools, has been proposed

could become a valuable tool with which to improve melanoma therapy.

**2. General mechanisms of resistance to classical antifolates**

cluding MTX, has been described [6,12,13].

melanoma [2].

392 Melanoma - From Early Detection to Treatment

Eukaryotic cells have developed complex checkpoint pathways that monitor DNA for damage or incomplete replication. Checkpoint pathways are amplified upon detection of aberrant DNA structures and lead to a delay in cell cycle progression during which damage can be repaired or replication be completed. Alternatively, in case of heavily damaged or seriously deregulated cells, checkpoint activation can result in apoptosis. As such, checkpoint mecha‐ nisms are essential for the maintenance of genomic integrity [17]. When vertebrate cells expe‐ rience replication arrest or undergo DNA damage by UV irradiation, the ATR kinase [ataxia telangiectasia mutated (ATM)- and Rad3-related kinase] phosphorylates and activates the Chk1 protein kinase. Activated Chk1 inhibits Cdc25 phosphatases, which control inhibitory phosphorylation sites on cyclin-dependent kinases, the latter being critical regulators of cell cycle transitions [18,19]. Because the ability of cells to delay cell cycle progression and halt DNA synthesis represents a defensive mechanism that spares potential toxicity [20], the acti‐ vation of Chk1 by MTX could constitute a key event in the resistance of melanoma to MTX.

In addition to these cellular mechanisms of resistance to MTX in melanoma, other mechanism that includes liver transformation of the drug has also been reported. A paradoxical response of malignant melanoma to MTX *in vivo* and *in vitro* has been described [21]. The authors ob‐ served that MTX showed consistent cytotoxicity for melanoma cells *in vitro* but was ineffective at equivalent concentrations *in vivo*. MTX undergoes oxidation to its primary metabolite 7 hydroxy-MTX (7-OH-MTX) in the liver by the enzyme aldehyde oxidase [11] and therefore, this transformation has been proposed as a novel mechanism of resistance to explain this par‐ adox [11,21]. In contrast to the large body of literature available on the multiple modalities of MTX resistance, very little is known regarding the ability of 7-OH-MTX to provoke antifolateresistance phenomena that may disrupt MTX activity. Recent studies seem to indicate that 7- OH-MTX which exceeds by far MTX in the plasma of MTX-treated patients can provoke distinct modalities of antifolate-resistance that severely compromise the efficacy of the parent drug MTX [22].

## **3. Melanoma-specific mechanisms of resistance to MTX**

#### **3.1. The critical role of alpha-folate receptor in the resistance of melanoma to MTX**

Experiments from our laboratory and others provide evidence that melanosomes contribute to the refractory properties of melanoma cells by sequestering cytotoxic drugs and increasing melanosome-mediated drug export [6,12,13]. Concretely, we have described that folate recep‐ tor α (FRα)-endocytotic transport of MTX facilitates drug melanosomal sequestration and cel‐ lular exportation in melanoma cells, which ensures reduced accumulation of MTX in intracellular compartments [6]. An important observation in this study was that MTX was a cytostatic agent on melanoma cells. These cells were resistant to MTX-induced apoptosis but responded to the drug by arresting their growth. A similar response was observed when the murine B16/F10 melanoma cell line was grown in low folate. After 3 days in folate-deficient medium the cells had restricted proliferative activity and also increased their metastatic po‐ tential [23]. Taking this into consideration, the results indicate that MTX might also induce depletion of intracellular reduced folate coenzymes by reducing their transport though the FRα and/or competing with them for the reduced folate carrier (RFC). Melanoma cells may be highly sensitive to intracellular depletion of folate coenzymes, and in this situation may enter into a "latent" state. This form of melanoma should indeed be highly resistant to MTX, since antifolate drugs are more effective on fast-dividing cells, which require continuous DNA syn‐ thesis. Most likely, the high increases of DHFR expression in cells treated with MTX [5] would represent an adaptation mechanism that allows cells to survive with low intracellular concen‐ trations of folate coenzymes. Increasing the recycling of folate molecules the cells would main‐ tain other cellular functions that are dependent on folate coenzymes, such as the synthesis of purines, pyrimidines, amino acids and methylation reactions. The presence of this "latent" form of melanoma should be critical for the resistance to MTX during *in vivo* therapies. Al‐ though MTX chemotherapy could initially halt the development of the tumor, after clearance of the drug from the body the melanoma cells may reinitiate their progression, possibly with an increased metastatic potential [23].

A defect in intracellular folate retention is another recognized mechanism of drug resist‐ ance [5,10,11,21]. In addition to a decrease in antifolate polyglutamylation, melanoma cells may also export cytotoxic drugs by melanosome sequestration [12]. The results pre‐ sented in this study indicated that drug exportation was an operative mechanism of re‐ sistance to MTX in melanoma cells. Although the mechanism by which cytotoxic drugs are sequestered into melanosomes remains unclear, we demonstrated that MTX-melano‐ some trapping may be a consequence of its FRα-endosomal transport [6]. To test the im‐ portance of this process on the resistance of melanoma to antifolates, we silenced the expression of the melanosomal structural protein gp100/Pmel17, which is known to play a critical role in melanosome biogenesis [24]. Recently, Xie and collaborators [13] provid‐ ed the first direct evidence that disruption of the process of normal melanosome biogen‐ esis, by mutation of gp100/Pmel17, increased sensitivity to cisplatin. We also observed that effective silencing of gp100/Pmel17 significantly increased the sensitivity of melano‐ ma cells to MTX, favouring MTX-induced apoptosis. This observation strongly supports the hypothesis which indicates that melanosome biogenesis is a specialization of the en‐ docytic pathway [25,26]; however, the exact mechanism by which MTX induces abnor‐ mal trafficking of early endosomes in melanoma cells, favoring the exportation of melanosomes, is still unclear. Whether MTX blocks the formation of carrier vesicles op‐ erating between early and late endosomes, inhibits the delivery of endocytosed material from endosomes to lysosomes, promoting, thus, the generation of exosomes [26] and/or induces a failure of lysosomal acidification, which is essential for normal endocytosis [27], remains to be determined.

**Figure 1.** A) Possible mechanisms for transport and trafficking of folates in melanoma cells. (B) Mechanisms to explain the MTX-induced depletion of DHF in melanoma cells. (C) Folate deficiency induces DHF depletion and enhances the transactivational potential of E2F1. (D) Excess of dTTP inhibits E2F1-mediated apoptosis and activates Chk1 in melano‐ ma cells. High levels of DHFR and TS could reactivate *de novo* dTMP biosynthesis impeding depletion of dTTP. Excess of dTTP would prevent apoptosis by several mechanisms. First, dTTP is an allosteric inhibitor of ribonucleotide reductase

Cellular and Molecular Mechanisms of Methotrexate Resistance in Melanoma

http://dx.doi.org/10.5772/52414

395

To explore the relationship between MTX exportation and melanosome trafficking, we studied the possible interaction of MTX with melanin [6]. Such interaction was confirmed by incubating this drug with synthetic 3,4-dihydroxyphenylalanine (DOPA)-melanin. Importantly, folic acid and 5-methyl-THF (5-MTHF), the natural source of cellular folates, did not appear to interact with synthetic DOPA-melanin. A comparison of the interaction of several folates (folic acid and 5-MTHF) and antifolates (MTX and aminopterin) with synthetic DOPA-melanin indicated that the double amino group of the pterin ring is an important molecular requirement for the drug-melanin interaction. Therefore, the physiological importance of the high affinity of mel‐ anin for antifolates, such as MTX and aminopterin, for drug melanosomal sequestration is also another important issue that remains to be addressed. Endocytic transport of molecules in‐ volves several processes, including the fusion of early and late endosomes and the dissociation

(RR), the enzyme which reduces cytidine diphosphate (CDP) and uridine diphosphate to dCDP and dUDP.

cytostatic agent on melanoma cells. These cells were resistant to MTX-induced apoptosis but responded to the drug by arresting their growth. A similar response was observed when the murine B16/F10 melanoma cell line was grown in low folate. After 3 days in folate-deficient medium the cells had restricted proliferative activity and also increased their metastatic po‐ tential [23]. Taking this into consideration, the results indicate that MTX might also induce depletion of intracellular reduced folate coenzymes by reducing their transport though the FRα and/or competing with them for the reduced folate carrier (RFC). Melanoma cells may be highly sensitive to intracellular depletion of folate coenzymes, and in this situation may enter into a "latent" state. This form of melanoma should indeed be highly resistant to MTX, since antifolate drugs are more effective on fast-dividing cells, which require continuous DNA syn‐ thesis. Most likely, the high increases of DHFR expression in cells treated with MTX [5] would represent an adaptation mechanism that allows cells to survive with low intracellular concen‐ trations of folate coenzymes. Increasing the recycling of folate molecules the cells would main‐ tain other cellular functions that are dependent on folate coenzymes, such as the synthesis of purines, pyrimidines, amino acids and methylation reactions. The presence of this "latent" form of melanoma should be critical for the resistance to MTX during *in vivo* therapies. Al‐ though MTX chemotherapy could initially halt the development of the tumor, after clearance of the drug from the body the melanoma cells may reinitiate their progression, possibly with

A defect in intracellular folate retention is another recognized mechanism of drug resist‐ ance [5,10,11,21]. In addition to a decrease in antifolate polyglutamylation, melanoma cells may also export cytotoxic drugs by melanosome sequestration [12]. The results pre‐ sented in this study indicated that drug exportation was an operative mechanism of re‐ sistance to MTX in melanoma cells. Although the mechanism by which cytotoxic drugs are sequestered into melanosomes remains unclear, we demonstrated that MTX-melano‐ some trapping may be a consequence of its FRα-endosomal transport [6]. To test the im‐ portance of this process on the resistance of melanoma to antifolates, we silenced the expression of the melanosomal structural protein gp100/Pmel17, which is known to play a critical role in melanosome biogenesis [24]. Recently, Xie and collaborators [13] provid‐ ed the first direct evidence that disruption of the process of normal melanosome biogen‐ esis, by mutation of gp100/Pmel17, increased sensitivity to cisplatin. We also observed that effective silencing of gp100/Pmel17 significantly increased the sensitivity of melano‐ ma cells to MTX, favouring MTX-induced apoptosis. This observation strongly supports the hypothesis which indicates that melanosome biogenesis is a specialization of the en‐ docytic pathway [25,26]; however, the exact mechanism by which MTX induces abnor‐ mal trafficking of early endosomes in melanoma cells, favoring the exportation of melanosomes, is still unclear. Whether MTX blocks the formation of carrier vesicles op‐ erating between early and late endosomes, inhibits the delivery of endocytosed material from endosomes to lysosomes, promoting, thus, the generation of exosomes [26] and/or induces a failure of lysosomal acidification, which is essential for normal endocytosis

an increased metastatic potential [23].

394 Melanoma - From Early Detection to Treatment

[27], remains to be determined.

**Figure 1.** A) Possible mechanisms for transport and trafficking of folates in melanoma cells. (B) Mechanisms to explain the MTX-induced depletion of DHF in melanoma cells. (C) Folate deficiency induces DHF depletion and enhances the transactivational potential of E2F1. (D) Excess of dTTP inhibits E2F1-mediated apoptosis and activates Chk1 in melano‐ ma cells. High levels of DHFR and TS could reactivate *de novo* dTMP biosynthesis impeding depletion of dTTP. Excess of dTTP would prevent apoptosis by several mechanisms. First, dTTP is an allosteric inhibitor of ribonucleotide reductase (RR), the enzyme which reduces cytidine diphosphate (CDP) and uridine diphosphate to dCDP and dUDP.

To explore the relationship between MTX exportation and melanosome trafficking, we studied the possible interaction of MTX with melanin [6]. Such interaction was confirmed by incubating this drug with synthetic 3,4-dihydroxyphenylalanine (DOPA)-melanin. Importantly, folic acid and 5-methyl-THF (5-MTHF), the natural source of cellular folates, did not appear to interact with synthetic DOPA-melanin. A comparison of the interaction of several folates (folic acid and 5-MTHF) and antifolates (MTX and aminopterin) with synthetic DOPA-melanin indicated that the double amino group of the pterin ring is an important molecular requirement for the drug-melanin interaction. Therefore, the physiological importance of the high affinity of mel‐ anin for antifolates, such as MTX and aminopterin, for drug melanosomal sequestration is also another important issue that remains to be addressed. Endocytic transport of molecules in‐ volves several processes, including the fusion of early and late endosomes and the dissociation of receptor-ligand complexes through the acidic pH of preformed vesicles [28]. After melano‐ some biogenesis from MTX-loaded endosomes, dissociated MTX could be trapped in the mel‐ anosomes by its interaction with melanins. In contrast, folate substrates would not be sequestered in melanosomes due to their low affinities for melanin; facilitated by the acidic pH of this organelle, uncharged reduced folates would leave the melanosome by passive dif‐ fusion and reach the cytosol, where they would become available for cellular functions. There‐ fore, elucidation of the molecular basis for the (anti)folate interaction with melanins could have important therapeutic implications, and this study might be used as a guide for the synthesis of new antifolates or for using existing antifolates in ways that escape melanin trapping.

melanoma cells [6] would suggest that MTX might also induce depletion of reduced folate coenzymes associated to endocytic compartments (Figure 1A and 1B). Therefore, a possible explanation for the depletion of DHF during MTX exposure could be that this drug diminishes the required supply of folates to the nucleus for the maintenance of both dTMP and DHF synthesis; however, how melanoma cells can control endocytic pathways to supply their own nucleus with folates is unknown. Recent studies have indicated that some endocytic proteins are also involved in direct signaling pathways from membranes to the nucleus, and mecha‐ nisms for the nuclear translocation of intact or fragmented endosome-localized proteins have been identified [33]. Another possibility is the existence of a late endosome-lysosome transport mechanism for folate [34]. The proximity of lysosomes to the nucleus suggests that folates could be released into the perinuclear region of the cytoplasm, perhaps facilitating their nuclear

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entry during cell division following the disassembly of the nuclear membrane [29].

the unknown function of overexpressed FRα in cancer cells [38].

**MTX resistance**

Although the uptake of 5-MTHF into mammalian cells is mainly mediated by the RFC, the expression of FRα in several epithelial tissues and especially its overexpression in cancerous cells indicate that this receptor may confer a growth advantage to these cells [35]. The high affinity of FRα for 5-MTHF suggest that this GPI-anchored receptor may play an important role in maintaining nuclear folates even at low extracellular concentrations of this vitamin. This hypothesis is supported by the finding that induction of FRα expression in cells that normally do not express this receptor allows the cells to grow in low nanomolar folate con‐ centrations [36]. On the other hand, the observation that methionine synthase was localized in the nucleus of melanoma cells could explain many of the unanswered questions on the role and regulation of the folate metabolism in the nucleus of these cancer cells. The methionine synthase -mediated catalysis of 5-MTHF would first supply THF and methionine to maintain both dTTP synthesis and the methylation reactions in the nucleus of the cells (Figure 1C) and second would prevent the nuclear accumulation of 5-MTHF, a potent inhibitor of SHMT [29]. Therefore, in melanoma, the existence of a specific folate transport pathway from the plasma membrane to the nucleus, mediated by FRα, is possible [37] and could shed light on

**4. Melanoma coordinates general and cell-specific mechanisms to promote**

MTX acts as a cytostatic agent in melanoma cells [6]. To discriminate between the mechanisms by which MTX could induce cell growth arrest without inducing apoptosis, the effect of this drug on the cell cycle of several melanoma cell lines was analysed [7]. The results indicated that, in all the tested melanoma cell lines, MTX conferred an arrest in early S phase; the G1 peak shifted toward the G1/S border, and cells were arrested with a minimal increase in their DNA content. Because S phase arrest has been recognized as a major mechanism of resistance in response to non-toxic concentrations of drugs that induce DNA replication stress, these pre‐

**4.1. MTX induces E2F1 demethylation and prevents dTTP depletion in melanoma**

### **3.2. MTX disrupts folate trafficking in melanoma cells**

Although MTX is exported within a few hours in contact with cells, in this short time, MTX is capable of inducing important changes in folate metabolism by depleting dihydrofolate (DHF) early on and by inducing the expression of folate-dependent enzymes later on [7]. The in‐ creased expression of DHFR is a common occurrence in melanoma and other cancer cells in response to MTX treatment; however, the observed depletion of DHF was completely unex‐ pected. The pathways that comprise folate-mediated one-carbon metabolism have been sug‐ gested to function in a metabolic network that interconnects the three biosynthetic pathways, namely *de novo* purine biosynthesis, *de novo* dTMP biosynthesis, and homocysteine remethy‐ lation. Recent studies provide direct evidence for cell cycle–dependent nuclear dTMP biosyn‐ thesis in the nucleus [29]. However, there are many unanswered questions regarding the role and regulation of nuclear *de novo* dTMP biosynthesis. Nothing is known about the transport, processing, and accumulation of folates into the nucleus, the one-carbon forms of folate present in the nucleus, and the relationship between cell cycle dependency of *de novo* dTMP biosyn‐ thesis and cell cycle-dependent accumulation of nuclear folate [29]. Although there is no data of how the homocysteine remethylation cycle is compartmentalized, the observation that MTX affected both DHF synthesis and E2F1 methylation (see below) seem to indicate that both the *de novo* dTMP biosynthesis and the homocysteine remethylation cycles might operate simul‐ taneously in the nucleus.

Using HeLa and MCF-7 cells, Stover and coworkers observed that cytoplasmic serine hydrox‐ ymethyltransferase (SMTH), TS, and DHFR are all translocated into the nucleus during S and G2/M phases following their modification by the small ubiquitin-like modifier (SUMO) [30,31]. This finding indicated that the folate cycle may be compartmentalized and that dTMP and DHF synthesis may occur in the nucleus during DNA synthesis. In a recent study, Wollack et al. [32] characterized 5-MTHF uptake and metabolism by primary rat choroid plexus epithelial cells *in vitro*. They distinguish two different processes for 5-MTHF transport, one that was FRα dependent and the other that was independent of this receptor and mediated by the proton couple folate transporter or reduced folate carrier (RFC). This investigation revealed that cel‐ lular metabolism of 5-MTHF depends on the route of folate entry into the cell. Thus, 5-MTHF taken up via a non-FRα–mediated process was rapidly metabolized to folylpolyglutamates, whereas 5-MTHF that accumulates via FRα remained non-metabolized and associated to en‐ docytic compartments. The observation that MTX induces the overall depletion of FRα in melanoma cells [6] would suggest that MTX might also induce depletion of reduced folate coenzymes associated to endocytic compartments (Figure 1A and 1B). Therefore, a possible explanation for the depletion of DHF during MTX exposure could be that this drug diminishes the required supply of folates to the nucleus for the maintenance of both dTMP and DHF synthesis; however, how melanoma cells can control endocytic pathways to supply their own nucleus with folates is unknown. Recent studies have indicated that some endocytic proteins are also involved in direct signaling pathways from membranes to the nucleus, and mecha‐ nisms for the nuclear translocation of intact or fragmented endosome-localized proteins have been identified [33]. Another possibility is the existence of a late endosome-lysosome transport mechanism for folate [34]. The proximity of lysosomes to the nucleus suggests that folates could be released into the perinuclear region of the cytoplasm, perhaps facilitating their nuclear entry during cell division following the disassembly of the nuclear membrane [29].

of receptor-ligand complexes through the acidic pH of preformed vesicles [28]. After melano‐ some biogenesis from MTX-loaded endosomes, dissociated MTX could be trapped in the mel‐ anosomes by its interaction with melanins. In contrast, folate substrates would not be sequestered in melanosomes due to their low affinities for melanin; facilitated by the acidic pH of this organelle, uncharged reduced folates would leave the melanosome by passive dif‐ fusion and reach the cytosol, where they would become available for cellular functions. There‐ fore, elucidation of the molecular basis for the (anti)folate interaction with melanins could have important therapeutic implications, and this study might be used as a guide for the synthesis of new antifolates or for using existing antifolates in ways that escape melanin trapping.

Although MTX is exported within a few hours in contact with cells, in this short time, MTX is capable of inducing important changes in folate metabolism by depleting dihydrofolate (DHF) early on and by inducing the expression of folate-dependent enzymes later on [7]. The in‐ creased expression of DHFR is a common occurrence in melanoma and other cancer cells in response to MTX treatment; however, the observed depletion of DHF was completely unex‐ pected. The pathways that comprise folate-mediated one-carbon metabolism have been sug‐ gested to function in a metabolic network that interconnects the three biosynthetic pathways, namely *de novo* purine biosynthesis, *de novo* dTMP biosynthesis, and homocysteine remethy‐ lation. Recent studies provide direct evidence for cell cycle–dependent nuclear dTMP biosyn‐ thesis in the nucleus [29]. However, there are many unanswered questions regarding the role and regulation of nuclear *de novo* dTMP biosynthesis. Nothing is known about the transport, processing, and accumulation of folates into the nucleus, the one-carbon forms of folate present in the nucleus, and the relationship between cell cycle dependency of *de novo* dTMP biosyn‐ thesis and cell cycle-dependent accumulation of nuclear folate [29]. Although there is no data of how the homocysteine remethylation cycle is compartmentalized, the observation that MTX affected both DHF synthesis and E2F1 methylation (see below) seem to indicate that both the *de novo* dTMP biosynthesis and the homocysteine remethylation cycles might operate simul‐

Using HeLa and MCF-7 cells, Stover and coworkers observed that cytoplasmic serine hydrox‐ ymethyltransferase (SMTH), TS, and DHFR are all translocated into the nucleus during S and G2/M phases following their modification by the small ubiquitin-like modifier (SUMO) [30,31]. This finding indicated that the folate cycle may be compartmentalized and that dTMP and DHF synthesis may occur in the nucleus during DNA synthesis. In a recent study, Wollack et al. [32] characterized 5-MTHF uptake and metabolism by primary rat choroid plexus epithelial cells *in vitro*. They distinguish two different processes for 5-MTHF transport, one that was FRα dependent and the other that was independent of this receptor and mediated by the proton couple folate transporter or reduced folate carrier (RFC). This investigation revealed that cel‐ lular metabolism of 5-MTHF depends on the route of folate entry into the cell. Thus, 5-MTHF taken up via a non-FRα–mediated process was rapidly metabolized to folylpolyglutamates, whereas 5-MTHF that accumulates via FRα remained non-metabolized and associated to en‐ docytic compartments. The observation that MTX induces the overall depletion of FRα in

**3.2. MTX disrupts folate trafficking in melanoma cells**

396 Melanoma - From Early Detection to Treatment

taneously in the nucleus.

Although the uptake of 5-MTHF into mammalian cells is mainly mediated by the RFC, the expression of FRα in several epithelial tissues and especially its overexpression in cancerous cells indicate that this receptor may confer a growth advantage to these cells [35]. The high affinity of FRα for 5-MTHF suggest that this GPI-anchored receptor may play an important role in maintaining nuclear folates even at low extracellular concentrations of this vitamin. This hypothesis is supported by the finding that induction of FRα expression in cells that normally do not express this receptor allows the cells to grow in low nanomolar folate con‐ centrations [36]. On the other hand, the observation that methionine synthase was localized in the nucleus of melanoma cells could explain many of the unanswered questions on the role and regulation of the folate metabolism in the nucleus of these cancer cells. The methionine synthase -mediated catalysis of 5-MTHF would first supply THF and methionine to maintain both dTTP synthesis and the methylation reactions in the nucleus of the cells (Figure 1C) and second would prevent the nuclear accumulation of 5-MTHF, a potent inhibitor of SHMT [29]. Therefore, in melanoma, the existence of a specific folate transport pathway from the plasma membrane to the nucleus, mediated by FRα, is possible [37] and could shed light on the unknown function of overexpressed FRα in cancer cells [38].

## **4. Melanoma coordinates general and cell-specific mechanisms to promote MTX resistance**

#### **4.1. MTX induces E2F1 demethylation and prevents dTTP depletion in melanoma**

MTX acts as a cytostatic agent in melanoma cells [6]. To discriminate between the mechanisms by which MTX could induce cell growth arrest without inducing apoptosis, the effect of this drug on the cell cycle of several melanoma cell lines was analysed [7]. The results indicated that, in all the tested melanoma cell lines, MTX conferred an arrest in early S phase; the G1 peak shifted toward the G1/S border, and cells were arrested with a minimal increase in their DNA content. Because S phase arrest has been recognized as a major mechanism of resistance in response to non-toxic concentrations of drugs that induce DNA replication stress, these pre‐ liminary results suggest that moderate DNA damage could be responsible for the cytostatic effect of MTX on melanoma cells.

peptide analysis of immunoprecipitated E2F1, after trypsin digestion (Figure 3), indicated that MTX promoted the demethylation of E2F1 at Lys185 (Figures 3B and 3D). A negative crosstalk between methylation and other posttranslational modifications of E2F1, such as acetylation and phosphorylation, has been recently described [39]. We observed that MTX induced the transient co-immunoprecipitation of E2F1 with p300/CBP-associated factor (P/CAF) (Figure 3B), an interaction that has been associated with the transcriptionally active hyperacetylated form of this transcription factor [40]. The hyperacetylated status of E2F1 after MTX treatment was also confirmed by MALDI-TOF mass spectrometry (Figures 3B and 3D). In response to severe DNA damage, the E2F1 protein is stabilized through distinct mechanisms, including direct phosphorylation by Chk2 at Ser364 [41] or ATM kinase at Ser31 [42]. As we did not observe phosphorylation of E2F1 after MTX treatment (Figures 3C and 3D), these data further suggest that MTX induced moderate DNA damage without inducing double strand breaks (DSBs) [43].

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MTX increased E2F1 levels in sensitive cancer cells [16]. However, we did not observe an MTXmediated increase in E2F1 levels in melanoma cells (Figure 2A) [7], a result that could be explained, at least in part, by the results obtained after determination of dNTP pools in mel‐ anoma cells (Figure 4). Contrary to the effects of MTX in most cancer cells [16], this drug increased the levels of dTTP in melanoma. Increased levels of dTTP were accompanied by a decrease in dCTP levels, which resulted in a nucleotide imbalance that favored thymidine excess. The MTX-induced expression of DHFR and TS (Figure 2A) and the low levels of MTX accumulated in melanoma cells [6] could explain this paradoxical response of melanoma cells

The data obtained in our study indicate that melanoma cells respond to the lack of folate coenzymes by enhancing the transactivational potential of E2F1. We observed that treatment of melanoma cells with MTX transiently affected the stability of Rb and the posttranslational state of E2F1 [7]. A crosstalk between the methylated and acetylated forms of E2F1 has been suggested [39]. Methylated E2F1 is prone to ubiquitination and degradation, whereas the de‐ methylation of E2F1 favors its P/CAF-dependent acetylation. Together, the results suggest a model whereby the MTX-induced degradation of Rb and the demethylation of E2F1 would result in the accumulation of E2F1 in its 'free' state, and in the absence of DNA damage, free E2F1 would be acetylated, leading to the transcription of genes required for S phase (Figure 1C). The activation of E2F1 by MTX would allow S phase transition in melanoma cells, and importantly for melanoma survival, cells would recover an operative folate cycle, thereby re‐ storing the original status of the Rb/E2F1 system. In the absence of exported MTX, high levels of TS and DHFR would impede the lethal depletion of dTTP and in turn, would produce a nucleotide imbalance that would favor a dTTP excess. Contrary to thymidine depletion, excess thymidine stops cells in S phase by blocking synthesis of DNA, an effect known as 'thymidine block' (Figure 1D) [15]. Recently, a mechanism by which dTTP allosterically feedback controls E2F1 has been proposed [15,16]. According to this mechanism, excess of dTTP inhibits E2F1 accumulation acting either upon production of E2F1 or its degradation. Because control of E2F1 is essential for cell survival, this mechanism would prevent E2F1 accumulation, which would result in activation of apoptosis through a process that involves p53 or p73, cytochrome

to a cytotoxic drug that typically depletes dTTP levels.

c, and caspases (Figure 1D) [44].

**Figure 2.** MTX enhances the transactivation potential of E2F1 in melanoma cells. (A) The time-dependent effect of MTX treatment (1 µM) on the expression of E2F1, DHFR, and TS proteins as assayed by western blot (WB). (B) ChIP experiments showing the occupancy of E2F1 and Rb on the DHFR promoter of B16/F10 melanoma cells (*\*P < 0.05*). (C) The upper panels represent the time-dependent effects of MTX (1 μM) treatment on the expression and phosphoryla‐ tion state of the Rb protein as assayed by WB. The lower panel depicts the Rb mRNA expression as assayed by qRT-PCR. The changes observed after MTX treatment were not statistically significant. (D) Co-immunoprecipitation assays were performed to test the interaction between Rb and E2F1.

To understand the mechanisms involved in G1 cell cycle progression in MTX-treated melano‐ ma cells, the effect of this drug on several G1 cell cycle components was analysed. Although the protein levels of E2F1 were not affected by MTX (Figure 2A), this drug significantly in‐ creased the protein levels of DHFR and thymidylate synthase (TS), two E2F1-target genes involved in folate metabolism and required for G1 progression and DNA synthesis (Figure 2A). Chromatin immunoprecipitation (ChIP) experiments that were designed to analyze the occupancy of E2F1 on the DHFR promoter of B16/F10 melanoma cells indicated that MTX stimulated the transcriptional activity of E2F1 (Figure 2B). First, we observed that MTX in‐ duced a transient decrease in the hypophosphorylated Rb protein in melanoma cells (Figure 2C) as evidenced by a noticeable lack of Rb co-immunoprecipitation with E2F1 in 10 h MTXtreated SK-MEL-28 cells when compared to untreated controls (Figure 2D). In addition, mass peptide analysis of immunoprecipitated E2F1, after trypsin digestion (Figure 3), indicated that MTX promoted the demethylation of E2F1 at Lys185 (Figures 3B and 3D). A negative crosstalk between methylation and other posttranslational modifications of E2F1, such as acetylation and phosphorylation, has been recently described [39]. We observed that MTX induced the transient co-immunoprecipitation of E2F1 with p300/CBP-associated factor (P/CAF) (Figure 3B), an interaction that has been associated with the transcriptionally active hyperacetylated form of this transcription factor [40]. The hyperacetylated status of E2F1 after MTX treatment was also confirmed by MALDI-TOF mass spectrometry (Figures 3B and 3D). In response to severe DNA damage, the E2F1 protein is stabilized through distinct mechanisms, including direct phosphorylation by Chk2 at Ser364 [41] or ATM kinase at Ser31 [42]. As we did not observe phosphorylation of E2F1 after MTX treatment (Figures 3C and 3D), these data further suggest that MTX induced moderate DNA damage without inducing double strand breaks (DSBs) [43].

liminary results suggest that moderate DNA damage could be responsible for the cytostatic

**Figure 2.** MTX enhances the transactivation potential of E2F1 in melanoma cells. (A) The time-dependent effect of MTX treatment (1 µM) on the expression of E2F1, DHFR, and TS proteins as assayed by western blot (WB). (B) ChIP experiments showing the occupancy of E2F1 and Rb on the DHFR promoter of B16/F10 melanoma cells (*\*P < 0.05*). (C) The upper panels represent the time-dependent effects of MTX (1 μM) treatment on the expression and phosphoryla‐ tion state of the Rb protein as assayed by WB. The lower panel depicts the Rb mRNA expression as assayed by qRT-PCR. The changes observed after MTX treatment were not statistically significant. (D) Co-immunoprecipitation assays

To understand the mechanisms involved in G1 cell cycle progression in MTX-treated melano‐ ma cells, the effect of this drug on several G1 cell cycle components was analysed. Although the protein levels of E2F1 were not affected by MTX (Figure 2A), this drug significantly in‐ creased the protein levels of DHFR and thymidylate synthase (TS), two E2F1-target genes involved in folate metabolism and required for G1 progression and DNA synthesis (Figure 2A). Chromatin immunoprecipitation (ChIP) experiments that were designed to analyze the occupancy of E2F1 on the DHFR promoter of B16/F10 melanoma cells indicated that MTX stimulated the transcriptional activity of E2F1 (Figure 2B). First, we observed that MTX in‐ duced a transient decrease in the hypophosphorylated Rb protein in melanoma cells (Figure 2C) as evidenced by a noticeable lack of Rb co-immunoprecipitation with E2F1 in 10 h MTXtreated SK-MEL-28 cells when compared to untreated controls (Figure 2D). In addition, mass

were performed to test the interaction between Rb and E2F1.

effect of MTX on melanoma cells.

398 Melanoma - From Early Detection to Treatment

MTX increased E2F1 levels in sensitive cancer cells [16]. However, we did not observe an MTXmediated increase in E2F1 levels in melanoma cells (Figure 2A) [7], a result that could be explained, at least in part, by the results obtained after determination of dNTP pools in mel‐ anoma cells (Figure 4). Contrary to the effects of MTX in most cancer cells [16], this drug increased the levels of dTTP in melanoma. Increased levels of dTTP were accompanied by a decrease in dCTP levels, which resulted in a nucleotide imbalance that favored thymidine excess. The MTX-induced expression of DHFR and TS (Figure 2A) and the low levels of MTX accumulated in melanoma cells [6] could explain this paradoxical response of melanoma cells to a cytotoxic drug that typically depletes dTTP levels.

The data obtained in our study indicate that melanoma cells respond to the lack of folate coenzymes by enhancing the transactivational potential of E2F1. We observed that treatment of melanoma cells with MTX transiently affected the stability of Rb and the posttranslational state of E2F1 [7]. A crosstalk between the methylated and acetylated forms of E2F1 has been suggested [39]. Methylated E2F1 is prone to ubiquitination and degradation, whereas the de‐ methylation of E2F1 favors its P/CAF-dependent acetylation. Together, the results suggest a model whereby the MTX-induced degradation of Rb and the demethylation of E2F1 would result in the accumulation of E2F1 in its 'free' state, and in the absence of DNA damage, free E2F1 would be acetylated, leading to the transcription of genes required for S phase (Figure 1C). The activation of E2F1 by MTX would allow S phase transition in melanoma cells, and importantly for melanoma survival, cells would recover an operative folate cycle, thereby re‐ storing the original status of the Rb/E2F1 system. In the absence of exported MTX, high levels of TS and DHFR would impede the lethal depletion of dTTP and in turn, would produce a nucleotide imbalance that would favor a dTTP excess. Contrary to thymidine depletion, excess thymidine stops cells in S phase by blocking synthesis of DNA, an effect known as 'thymidine block' (Figure 1D) [15]. Recently, a mechanism by which dTTP allosterically feedback controls E2F1 has been proposed [15,16]. According to this mechanism, excess of dTTP inhibits E2F1 accumulation acting either upon production of E2F1 or its degradation. Because control of E2F1 is essential for cell survival, this mechanism would prevent E2F1 accumulation, which would result in activation of apoptosis through a process that involves p53 or p73, cytochrome c, and caspases (Figure 1D) [44].

**Figure 4.** MTX does not deplete dTTP levels in melanoma cells. dNTP quantification in SK-MEL-28 control cells and cells subjected to MTX (1 μM) treatment (\**P* < 0.05). Data collected from the left panel was used to determine the total amounts of each dNTP at each time point. The percent contribution of each dNTP to the total pool after 24 h of

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Excess thymidine induces little detectable DNA damage in the form of DSBs. The ATR-medi‐ ated response appears to play a more prominent role under these cellular conditions [45]. As it is known that the central mechanism responsible for Chk1 activation upon DNA damage is the distribution of ATR into nuclear foci [46], the effects of MTX on the localization of ATR and the phosphorylation of Chk1 at Ser345 were analyzed by confocal microscopy and western blot, respectively (Figures 5A and 5B). Time- and dose-dependent experiments clearly indi‐ cated that MTX induced Chk1 phosphorylation in melanoma cells. Because Chk1 phosphor‐ ylation may not directly correspond to Chk1 activation, we next analyzed the dose-dependent effects of MTX on the stability of Cdc25A (Figure 5B). We found that Chk1 phosphorylation led to a corresponding decrease in Cdc25A abundance, indicating that MTX not only conferred Chk1 phosphorylation, but it also activated Chk1. Conversely, phosphorylation of Chk2 was not observed in melanoma cells that had been treated with MTX for as long as 48 h (Figure 5B), indicating that this drug specifically induced Chk1 activation in response to DNA single strand breaks (SSBs). To determine the extent to which Chk1 activation affected the resistance of melanoma to MTX, we took two independent experimental approaches. First, we silenced the expression of Chk1 in SK-MEL-28 (p53 mutant) cells and studied the sensitivity of the cells to MTX (Figure 5C). The results indicated that the downregulation of Chk1 increased the sen‐ sitivity of SK-MEL-28 cells to MTX and led to apoptosis. As a second approach, we evaluated the ability of Chk1 to protect B16/F10 murine cells (p53 wild-type) from MTX-induced apop‐ tosis by first inducing an S phase arrest with MTX and then treating the S-arrested cells with a combination of MTX and 7-hydroxystaurosporine (UCN-01). We observed that B16/F10 S phase-arrested cells were sensitive to MTX treatment after the effective inhibition of Chk1

**4.2. Excess of dTTP favours Chk1 activation in melanoma after MTX treatment**

treatment is represented.

(Figure 5C).

**Figure 3.** MTX induces demethylation and hyperacetylation of E2F1 in melanoma cells. (A) Schematic representation of the E2F1 protein. Residues susceptible to methylation (K185), acetylation (K117, K120, and K125), and phosphory‐ lation (S31 and S364) are shown. (B) Relative intensity of unmethylated [(K)NHIQWLGSHTTVGVGGR(L); m/z 1820.0229] and hyperacetylated [(R)HPGKAcGVKAcSPGEKAcSR(Y); m/z 1589.8399] peptides in E2F1-trypsin digested samples. Peptides were analyzed in untreated SK-MEL-28 cells (CN) or treated for 10 h with 1 μM MTX (\**P* < 0.05). Intensities were normalized with respect to an internal matrix control. (C) Cell lysates from SK-MEL-28 cells that had been treated with 1 μM MTX were used for IP assays with E2F1 to test the co-immunoprecipitation of E2F1 with P/CAF and the phosphorylated state of E2F1. (D) MALDI-TOF mass spectra of tryptic digests of immunoprecipitated E2F1. The characteristics peptides involving posttranslational modifications of E2F1 (methylation = Me, acetylation = Ac, and phosphorylation = P), as well as their measured and theoretical m/z are shown.

**Figure 4.** MTX does not deplete dTTP levels in melanoma cells. dNTP quantification in SK-MEL-28 control cells and cells subjected to MTX (1 μM) treatment (\**P* < 0.05). Data collected from the left panel was used to determine the total amounts of each dNTP at each time point. The percent contribution of each dNTP to the total pool after 24 h of treatment is represented.

#### **4.2. Excess of dTTP favours Chk1 activation in melanoma after MTX treatment**

Excess thymidine induces little detectable DNA damage in the form of DSBs. The ATR-medi‐ ated response appears to play a more prominent role under these cellular conditions [45]. As it is known that the central mechanism responsible for Chk1 activation upon DNA damage is the distribution of ATR into nuclear foci [46], the effects of MTX on the localization of ATR and the phosphorylation of Chk1 at Ser345 were analyzed by confocal microscopy and western blot, respectively (Figures 5A and 5B). Time- and dose-dependent experiments clearly indi‐ cated that MTX induced Chk1 phosphorylation in melanoma cells. Because Chk1 phosphor‐ ylation may not directly correspond to Chk1 activation, we next analyzed the dose-dependent effects of MTX on the stability of Cdc25A (Figure 5B). We found that Chk1 phosphorylation led to a corresponding decrease in Cdc25A abundance, indicating that MTX not only conferred Chk1 phosphorylation, but it also activated Chk1. Conversely, phosphorylation of Chk2 was not observed in melanoma cells that had been treated with MTX for as long as 48 h (Figure 5B), indicating that this drug specifically induced Chk1 activation in response to DNA single strand breaks (SSBs). To determine the extent to which Chk1 activation affected the resistance of melanoma to MTX, we took two independent experimental approaches. First, we silenced the expression of Chk1 in SK-MEL-28 (p53 mutant) cells and studied the sensitivity of the cells to MTX (Figure 5C). The results indicated that the downregulation of Chk1 increased the sen‐ sitivity of SK-MEL-28 cells to MTX and led to apoptosis. As a second approach, we evaluated the ability of Chk1 to protect B16/F10 murine cells (p53 wild-type) from MTX-induced apop‐ tosis by first inducing an S phase arrest with MTX and then treating the S-arrested cells with a combination of MTX and 7-hydroxystaurosporine (UCN-01). We observed that B16/F10 S phase-arrested cells were sensitive to MTX treatment after the effective inhibition of Chk1 (Figure 5C).

**Figure 3.** MTX induces demethylation and hyperacetylation of E2F1 in melanoma cells. (A) Schematic representation of the E2F1 protein. Residues susceptible to methylation (K185), acetylation (K117, K120, and K125), and phosphory‐ lation (S31 and S364) are shown. (B) Relative intensity of unmethylated [(K)NHIQWLGSHTTVGVGGR(L); m/z 1820.0229] and hyperacetylated [(R)HPGKAcGVKAcSPGEKAcSR(Y); m/z 1589.8399] peptides in E2F1-trypsin digested samples. Peptides were analyzed in untreated SK-MEL-28 cells (CN) or treated for 10 h with 1 μM MTX (\**P* < 0.05). Intensities were normalized with respect to an internal matrix control. (C) Cell lysates from SK-MEL-28 cells that had been treated with 1 μM MTX were used for IP assays with E2F1 to test the co-immunoprecipitation of E2F1 with P/CAF and the phosphorylated state of E2F1. (D) MALDI-TOF mass spectra of tryptic digests of immunoprecipitated E2F1. The characteristics peptides involving posttranslational modifications of E2F1 (methylation = Me, acetylation =

Ac, and phosphorylation = P), as well as their measured and theoretical m/z are shown.

400 Melanoma - From Early Detection to Treatment

Inhibitors of DNA synthesis, such as excess thymidine, hydroxyurea, and camptothecin, are normally poor inducers of apoptosis; however, these agents become potent inducers of death in S phase cells upon the small interfering RNA-mediated depletion of Chk1 [45]. Here, we observed that MTX activated Chk1 and induced an early S phase arrest in melanoma cells lines that were harboring either wild-type or mutant p53. The impact of MTX on the survival of Chk1-silenced melanoma cells and cells co-treated with UCN-01 indicates that MTX provokes a 'thymidine block'-like effect and that S phase arrest, as a result of Chk1 activation, might constitute a major and general p53-independent mechanism that is responsible for the resist‐ ance of melanomas to MTX. However, it would be difficult to understand this extreme resist‐ ance without taking into account the melanosome-mediated exportation of MTX. The activation of the DNA damage response pathway reflects the magnitude and extent of DNA damage that occurs in response to a specific genotoxic agent, and a dual role of Chk1, de‐ pending on the extent of DNA damage, has been proposed [45]. Thus, Chk1 may play an antiapoptotic role in response to weaker replication fork stresses, whereas more catastrophic damage, such as the accumulation of DNA strand breaks, may result in the activation of apop‐ tosis by Chk1. Together, the results indicate that low intracellular levels of MTX in melanoma induce moderate DNA damage that favors the anti-apoptotic role of Chk1 (Figure 1D).

Cellular and Molecular Mechanisms of Methotrexate Resistance in Melanoma

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403

Although melanoma resistance to MTX was initially thought to be due to the classical mechanisms of resistance that have been observed in other epithelial cells, recent discov‐ eries indicate that the resistance of melanoma to MTX might be due to the idiosyncra‐ sies of these cancer cells [6,12] where drug melanosomal sequestration and its subsequent cellular exportation may have a marked protagonist. Unravelling the mecha‐ nisms of melanoma resistance to MTX could, therefore, yield important information on how to circumvent this resistance and could have important pharmacological implica‐ tions for the design of novel combined therapies. Taking into account these observa‐ tions, uses of combined treatments with MTX, to prevent melanosomal drug sequestration [6,12] or to avoid MTX-induced S phase arrest [19], are rational therapeuti‐ cal approaches. The observation that MTX induces cellular depletion of DHF in melano‐ ma [7] could generate novel combined therapies to efficiently inhibit DHFR with antifolates transported into the cells by FRα-independent processes. Also, of great inter‐ est is the observed effect of MTX on the posttranslational status of E2F1 in melanoma (Figure 3). Various studies have suggested that E2F1 plays dual roles in cell survival/ apoptosis [47-50]. Therefore, the MTX-induced demethylation and acetylation of E2F1 could favour melanoma cell death when combined with E2F1-stabilizing drugs. In addi‐ tion to E2F1 phosphorylation, acetylation has also been recognized to play a role in the activation and stabilization of the E2F1 protein during DNA damage and apoptosis [40]. A possible strategy to favour E2F1 apoptosis in melanoma by the combination of MTX

**5. Therapeutical implications**

with E2F1-stabilizing drugs is depicted in Figure 6.

**Figure 5.** MTX activates Chk1 in melanoma cells. (A) SK-MEL-28 cells were treated with 1 μM MTX for 24 h and then examined for ATR nuclear foci. Nuclei were stained with DAPI. (B) The dose-dependent effects of MTX on Chk1 phos‐ phorylation and Cdc25A degradation in SK-MEL-28 after 24 h of drug exposure (\**P* < 0.05). MTX (1 μM) induced the time-dependent phosphorylation of Chk1, but not Chk2, in different melanoma cell lines. (C) Chk1 siRNA sensitizes SK-MEL-28 cells to MTX-induced toxicity (left panel). siControl (siCN)- and siChk1-transfected cells were treated with in‐ creasing doses of MTX for 48 h (\**P* < 0.05). The effective silencing of Chk1 was tested by WB. The induction of the phosphorylated form of Chk1 was analyzed after 24 h of MTX treatment (1 μM). The induction of apoptosis by UCN-01 in MTX-arrested B16/F10 cells is shown in the right-side panel. Cells were incubated with 1 μM MTX continu‐ ously for 32 h, and 50 nM UCN-01 was added at 24 h to one group of cells following splitting of the culture. As a control experiment, SK-MEL-28 cells were treated with 50 nM UCN-01 only for 32 h.

Inhibitors of DNA synthesis, such as excess thymidine, hydroxyurea, and camptothecin, are normally poor inducers of apoptosis; however, these agents become potent inducers of death in S phase cells upon the small interfering RNA-mediated depletion of Chk1 [45]. Here, we observed that MTX activated Chk1 and induced an early S phase arrest in melanoma cells lines that were harboring either wild-type or mutant p53. The impact of MTX on the survival of Chk1-silenced melanoma cells and cells co-treated with UCN-01 indicates that MTX provokes a 'thymidine block'-like effect and that S phase arrest, as a result of Chk1 activation, might constitute a major and general p53-independent mechanism that is responsible for the resist‐ ance of melanomas to MTX. However, it would be difficult to understand this extreme resist‐ ance without taking into account the melanosome-mediated exportation of MTX. The activation of the DNA damage response pathway reflects the magnitude and extent of DNA damage that occurs in response to a specific genotoxic agent, and a dual role of Chk1, de‐ pending on the extent of DNA damage, has been proposed [45]. Thus, Chk1 may play an antiapoptotic role in response to weaker replication fork stresses, whereas more catastrophic damage, such as the accumulation of DNA strand breaks, may result in the activation of apop‐ tosis by Chk1. Together, the results indicate that low intracellular levels of MTX in melanoma induce moderate DNA damage that favors the anti-apoptotic role of Chk1 (Figure 1D).

## **5. Therapeutical implications**

**Figure 5.** MTX activates Chk1 in melanoma cells. (A) SK-MEL-28 cells were treated with 1 μM MTX for 24 h and then examined for ATR nuclear foci. Nuclei were stained with DAPI. (B) The dose-dependent effects of MTX on Chk1 phos‐ phorylation and Cdc25A degradation in SK-MEL-28 after 24 h of drug exposure (\**P* < 0.05). MTX (1 μM) induced the time-dependent phosphorylation of Chk1, but not Chk2, in different melanoma cell lines. (C) Chk1 siRNA sensitizes SK-MEL-28 cells to MTX-induced toxicity (left panel). siControl (siCN)- and siChk1-transfected cells were treated with in‐ creasing doses of MTX for 48 h (\**P* < 0.05). The effective silencing of Chk1 was tested by WB. The induction of the phosphorylated form of Chk1 was analyzed after 24 h of MTX treatment (1 μM). The induction of apoptosis by UCN-01 in MTX-arrested B16/F10 cells is shown in the right-side panel. Cells were incubated with 1 μM MTX continu‐ ously for 32 h, and 50 nM UCN-01 was added at 24 h to one group of cells following splitting of the culture. As a

control experiment, SK-MEL-28 cells were treated with 50 nM UCN-01 only for 32 h.

402 Melanoma - From Early Detection to Treatment

Although melanoma resistance to MTX was initially thought to be due to the classical mechanisms of resistance that have been observed in other epithelial cells, recent discov‐ eries indicate that the resistance of melanoma to MTX might be due to the idiosyncra‐ sies of these cancer cells [6,12] where drug melanosomal sequestration and its subsequent cellular exportation may have a marked protagonist. Unravelling the mecha‐ nisms of melanoma resistance to MTX could, therefore, yield important information on how to circumvent this resistance and could have important pharmacological implica‐ tions for the design of novel combined therapies. Taking into account these observa‐ tions, uses of combined treatments with MTX, to prevent melanosomal drug sequestration [6,12] or to avoid MTX-induced S phase arrest [19], are rational therapeuti‐ cal approaches. The observation that MTX induces cellular depletion of DHF in melano‐ ma [7] could generate novel combined therapies to efficiently inhibit DHFR with antifolates transported into the cells by FRα-independent processes. Also, of great inter‐ est is the observed effect of MTX on the posttranslational status of E2F1 in melanoma (Figure 3). Various studies have suggested that E2F1 plays dual roles in cell survival/ apoptosis [47-50]. Therefore, the MTX-induced demethylation and acetylation of E2F1 could favour melanoma cell death when combined with E2F1-stabilizing drugs. In addi‐ tion to E2F1 phosphorylation, acetylation has also been recognized to play a role in the activation and stabilization of the E2F1 protein during DNA damage and apoptosis [40]. A possible strategy to favour E2F1 apoptosis in melanoma by the combination of MTX with E2F1-stabilizing drugs is depicted in Figure 6.

**6. Conclusions**

**Acknowledgements**

ington, Oxford, UK.

**Author details**

Spain

Luis Sanchez del-Campo1

María Piedad Fernández-Pérez1

Jose Neptuno Rodriguez-Lopez1

Melanoma, the most aggressive form of skin cancer, is notoriously resistant to all current mo‐ dalities of cancer therapy, including to the drug MTX. Melanosomal sequestration and cellular exportation of methotrexate have been proposed to be important melanoma-specific mecha‐ nisms that contribute to the resistance of melanoma to methotrexate. In addition, other mecha‐ nisms of resistance that are present in most epithelial cancer cells are also operative in melanoma. This chapter reviews how melanoma orchestrates these mechanisms to become ex‐ tremely resistant to methotrexate, where both E2F1 and Chk1, two molecules with dual roles in survival/apoptosis, play prominent roles. The results indicated that MTX induced the depletion of DHF in melanoma cells, which stimulated the transcriptional activity of E2F1. The elevate ex‐ pression of DHFR and TS, two E2F1-target genes involved in folate metabolism and required for G1 progression, favoured dTTP accumulation, which promoted DNA single strand breaks and the subsequent activation of Chk1. Under these conditions, melanoma cells are protected from apoptosis by arresting their cell cycle in S phase. Excess of dTTP could also inhibit E2F1 mediated apoptosis in melanoma cells. In addition, these discoveries could open the way for the development of new combined and directed therapies against this elusive skin pathology.

Cellular and Molecular Mechanisms of Methotrexate Resistance in Melanoma

http://dx.doi.org/10.5772/52414

405

Research described was supported in part by a grant from Ministerio de Ciencia e Innovación (MICINN) (Project SAF2009-12043-C02-01), Fundación Séneca, Región de Murcia (FS-RM) (15230/PI/10) and EU ERA293514. J.C-H is contracted by the Translational Cancer Research Group (Fundación para la Formación e Investigación Sanitarias). MPF-P has a fellowship from Ministerio de Educación, Cultura y Deporte. M.F.M is contracted by an agreement with the Fundación de la Asociación Española contra el Cáncer (FAECC). L.S-d-C has a postdoctoral fellowship from Fundación Séneca (Región de Murcia) for application in the Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Head‐

, Magali Saez-Ayala1

and

,

, Maria F. Montenegro1

, Juan Cabezas-Herrera2

2 Research Unit of Clinical Analusis Service, University Hospital Virgen de la Arrixaca,

1 Department of Biochemistry & Molecular Biology A, University of Murcia, Spain

**Figure 6.** Proposed mechanism for the regulation of E2F1 by MTX. E2F1 is regulated by its interaction with Rb and by several posttranslational modifications, including methylation (Me), acetylation (Ac) and phosphorylation (P) [39]. The effects of MTX (red dashed line) on E2F1 status and that result in melanoma resistance are shown. A possible strategy to stabilize E2F1 (green dashed lines) to induce apoptosis in melanoma cells is also displayed.

## **6. Conclusions**

Melanoma, the most aggressive form of skin cancer, is notoriously resistant to all current mo‐ dalities of cancer therapy, including to the drug MTX. Melanosomal sequestration and cellular exportation of methotrexate have been proposed to be important melanoma-specific mecha‐ nisms that contribute to the resistance of melanoma to methotrexate. In addition, other mecha‐ nisms of resistance that are present in most epithelial cancer cells are also operative in melanoma. This chapter reviews how melanoma orchestrates these mechanisms to become ex‐ tremely resistant to methotrexate, where both E2F1 and Chk1, two molecules with dual roles in survival/apoptosis, play prominent roles. The results indicated that MTX induced the depletion of DHF in melanoma cells, which stimulated the transcriptional activity of E2F1. The elevate ex‐ pression of DHFR and TS, two E2F1-target genes involved in folate metabolism and required for G1 progression, favoured dTTP accumulation, which promoted DNA single strand breaks and the subsequent activation of Chk1. Under these conditions, melanoma cells are protected from apoptosis by arresting their cell cycle in S phase. Excess of dTTP could also inhibit E2F1 mediated apoptosis in melanoma cells. In addition, these discoveries could open the way for the development of new combined and directed therapies against this elusive skin pathology.

## **Acknowledgements**

Research described was supported in part by a grant from Ministerio de Ciencia e Innovación (MICINN) (Project SAF2009-12043-C02-01), Fundación Séneca, Región de Murcia (FS-RM) (15230/PI/10) and EU ERA293514. J.C-H is contracted by the Translational Cancer Research Group (Fundación para la Formación e Investigación Sanitarias). MPF-P has a fellowship from Ministerio de Educación, Cultura y Deporte. M.F.M is contracted by an agreement with the Fundación de la Asociación Española contra el Cáncer (FAECC). L.S-d-C has a postdoctoral fellowship from Fundación Séneca (Región de Murcia) for application in the Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Head‐ ington, Oxford, UK.

## **Author details**

**Figure 6.** Proposed mechanism for the regulation of E2F1 by MTX. E2F1 is regulated by its interaction with Rb and by several posttranslational modifications, including methylation (Me), acetylation (Ac) and phosphorylation (P) [39]. The effects of MTX (red dashed line) on E2F1 status and that result in melanoma resistance are shown. A possible strategy

to stabilize E2F1 (green dashed lines) to induce apoptosis in melanoma cells is also displayed.

404 Melanoma - From Early Detection to Treatment

Luis Sanchez del-Campo1 , Maria F. Montenegro1 , Magali Saez-Ayala1 , María Piedad Fernández-Pérez1 , Juan Cabezas-Herrera2 and Jose Neptuno Rodriguez-Lopez1

1 Department of Biochemistry & Molecular Biology A, University of Murcia, Spain

2 Research Unit of Clinical Analusis Service, University Hospital Virgen de la Arrixaca, Spain

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**Chapter 15**

**Surgery and the Staging of Melanoma**

An estimated 166,900 patients were diagnosed with malignant melanoma in developed countries last year [1]. The reported incidence of malignant melanoma continues to rise de‐ spite increasing understanding of its aetiology. In the United States 76,250 new cases are ex‐ pected in 2012 with melanoma far outstripping other skin cancers in terms of mortality [2]. Similarly, in the UK, 12,818 new cases of malignant melanoma were diagnosed in 2010 [3]. Approximately, 85% percent of patients with cutaneous melanoma are diagnosed at a local‐ ized stage, while 10% have associated regional lymph node involvement and 5% of patients will have distant metastatic disease at presentation. The corresponding 5-year overall sur‐ vival rates are 98.2% for localized disease, 62.4% for regional lymph node involvement and

Advances in the understanding of the molecular mechanisms and immunology of mela‐ noma have lead to the development of promising novel therapeutic agents. Surgery, however, remains the mainstay of treatment and changes in the surgical approach have been guided by the greater understanding of melanoma pathogenesis. The management of the primary tumour has become more conservative, with acceptance of narrower exci‐ sion margins. In addition, there has been a move away from the routine performance of elective regional lymph node dissection towards sentinel lymph node biopsy which is as‐

The new American Joint Committee on Cancer (AJCC) guidelines for the staging of melano‐ ma were introduced into clinical practice in 2010 [7]. The two most important distinctions with previous guidelines are the incorporation of the mitotic rate of the primary tumor and the key role of the sentinel lymph node, including methods of analysis, in accurately staging

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

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

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

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

Z. Al-Hilli, D. Evoy, J.G. Geraghty, E.W. McDermott and R.S. Prichard

http://dx.doi.org/10.5772/53626

15.1% for distant melanomas [4, 5].

sociated with less morbidity [6].

clinically occult nodal disease [8].

**1. Introduction**

Additional information is available at the end of the chapter

## **Chapter 15**

## **Surgery and the Staging of Melanoma**

Z. Al-Hilli, D. Evoy, J.G. Geraghty, E.W. McDermott and R.S. Prichard

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53626

## **1. Introduction**

An estimated 166,900 patients were diagnosed with malignant melanoma in developed countries last year [1]. The reported incidence of malignant melanoma continues to rise de‐ spite increasing understanding of its aetiology. In the United States 76,250 new cases are ex‐ pected in 2012 with melanoma far outstripping other skin cancers in terms of mortality [2]. Similarly, in the UK, 12,818 new cases of malignant melanoma were diagnosed in 2010 [3]. Approximately, 85% percent of patients with cutaneous melanoma are diagnosed at a local‐ ized stage, while 10% have associated regional lymph node involvement and 5% of patients will have distant metastatic disease at presentation. The corresponding 5-year overall sur‐ vival rates are 98.2% for localized disease, 62.4% for regional lymph node involvement and 15.1% for distant melanomas [4, 5].

Advances in the understanding of the molecular mechanisms and immunology of mela‐ noma have lead to the development of promising novel therapeutic agents. Surgery, however, remains the mainstay of treatment and changes in the surgical approach have been guided by the greater understanding of melanoma pathogenesis. The management of the primary tumour has become more conservative, with acceptance of narrower exci‐ sion margins. In addition, there has been a move away from the routine performance of elective regional lymph node dissection towards sentinel lymph node biopsy which is as‐ sociated with less morbidity [6].

The new American Joint Committee on Cancer (AJCC) guidelines for the staging of melano‐ ma were introduced into clinical practice in 2010 [7]. The two most important distinctions with previous guidelines are the incorporation of the mitotic rate of the primary tumor and the key role of the sentinel lymph node, including methods of analysis, in accurately staging clinically occult nodal disease [8].

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

The purpose of this chapter is twofold. Firstly, this chapter describes the appropriate surgi‐ cal management of the primary tumour, the associated regional lymph node basin and dis‐ tant metastatic disease. Secondly, the updated and revised AJCC staging system will be discussed and current controversies addressed.

**Seven point checklist The ABCDE lesion system**

Irregular shape B Irregular Border

An excision biopsy is indicated for lesions suspected of being a melanoma. An excision bi‐ opsy is the recommended method for suspected malignant melanoma as it enables diagno‐ sis and staging of the tumour and may determine future treatment and prognosis [20, 21]. The whole lesion should be excised with a 1-3 mm margin of normal skin including sub-der‐ mal fat. It is crucial to plan this excision carefully with a view towards definitive treatment. Knowledge of lymphatic drainage and subsequent need for sentinel node biopsy should lead to narrow margin excision potentially avoiding interference with subsequent lymphatic mapping. In addition, a longitudinal orientation is preferred in the extremities and incision orientation should be along Langer's lines on the trunk. This allows for subsequent closure of a wide local excision and reduces the need for skin grafting if primary closure is to be

In certain areas (such as the face, palm of hand, sole of foot, ears, digits and subungal le‐ sions) an excision biopsy may not be appropriate. In these cases, an incisional or punch bi‐ opsy of the thickest portion of the lesion may be performed [21]. Shave biopsy is avoided as it makes characterising the lesion difficult by underestimating tumour thickness, which is important in determining further treatment [21]. It also risks leaving residual tumour at the

Obtaining an adequate biopsy specimen is crucial for histopathological diagnosis and tu‐ mour staging. The tumour thickness, which remains the most powerful prognostic parame‐ ter, provides a guide to the margin clearance required for delayed wide excision and need for adjuvant therapy [20, 22]. Pathological examination should evaluate macroscopic fea‐

Largest diameter 7mm or more E Elevation

**Table 1.** Seven point checklist and ABCDE system for assessment of pigmented lesions [19]

Change in size A Geometrical Asymmetry in 2 axes

Surgery and the Staging of Melanoma http://dx.doi.org/10.5772/53626 413

Irregular colour C At least 2 different Colours in lesion

*Minor features* D Maximum Diameter >6mm

*Major features*

Inflammation

Oozing

Itch/ change in sensation

achieved.

radial and deep margins.

## **2. Risk factors**

The worldwide incidence of melanoma doubles every ten to fifteen years [9]. Risk factors associated with the development of malignant melanoma are varied and include genetic susceptibility, exposure to ultraviolet radiation, and immunologic factors. The most im‐ portant of these is ultraviolet exposure where intermittent, unaccustomed sun exposure and sunburn were found to have considerable roles as risk factors for melanoma. How‐ ever, despite the increase in public awareness, the practice of ultraviolet radiation protec‐ tion behaviour is low. Also worryingly a survey performed in the US in 2005 documented that up to 14% of adults, primarily women and young adults used an in‐ door tanning device on at least one occasion [10].

Epidemiological studies have found that blue, green or grey eyes, blonde or red hair, light complexion, freckles, sun sensitivity, and an inability to tan, are risk factors for the develop‐ ment of melanoma [11, 12]. Countries with close proximity to the equator with predomi‐ nantly fair-skinned populations have shown a higher preponderance to developing melanoma. Risk factors for melanoma also include a positive family history or personal his‐ tory of melanoma/non-melanoma cancer or in-situ skin carcinomas, large numbers of mela‐ nocitic naevi in childhood, and xeroderma pigmentosum [13].

It is suggested that minimising radiation, and the adoption of photo-protective measures, can significantly reduce the risk of developing melanoma [13-15].

## **3. Surgery**

#### **3.1. Initial surgical biopsy**

Melanoma can develop either in a pre-existing pigmented lesion or de novo. Features raising suspicion of melanoma in a pre-existing pigmented lesion include a change in size, irregular shape, irregular colour, diameter 7 mm or more, inflammation, oozing or a change in sensation [5,16]. The ABCD system of diagnosis (Asymmetry, Border irregulari‐ ty, Colour change, and a Diameter greater than 6 mm) has also been advocated to assist early clinical diagnosis, to which 'E' (Evolving or Elevation) has been added [5,17,18]. Ta‐ ble 1 illustrates the seven point checklist and ABCDE system for the assessment of pig‐ mented lesions.


**Table 1.** Seven point checklist and ABCDE system for assessment of pigmented lesions [19]

The purpose of this chapter is twofold. Firstly, this chapter describes the appropriate surgi‐ cal management of the primary tumour, the associated regional lymph node basin and dis‐ tant metastatic disease. Secondly, the updated and revised AJCC staging system will be

The worldwide incidence of melanoma doubles every ten to fifteen years [9]. Risk factors associated with the development of malignant melanoma are varied and include genetic susceptibility, exposure to ultraviolet radiation, and immunologic factors. The most im‐ portant of these is ultraviolet exposure where intermittent, unaccustomed sun exposure and sunburn were found to have considerable roles as risk factors for melanoma. How‐ ever, despite the increase in public awareness, the practice of ultraviolet radiation protec‐ tion behaviour is low. Also worryingly a survey performed in the US in 2005 documented that up to 14% of adults, primarily women and young adults used an in‐

Epidemiological studies have found that blue, green or grey eyes, blonde or red hair, light complexion, freckles, sun sensitivity, and an inability to tan, are risk factors for the develop‐ ment of melanoma [11, 12]. Countries with close proximity to the equator with predomi‐ nantly fair-skinned populations have shown a higher preponderance to developing melanoma. Risk factors for melanoma also include a positive family history or personal his‐ tory of melanoma/non-melanoma cancer or in-situ skin carcinomas, large numbers of mela‐

It is suggested that minimising radiation, and the adoption of photo-protective measures,

Melanoma can develop either in a pre-existing pigmented lesion or de novo. Features raising suspicion of melanoma in a pre-existing pigmented lesion include a change in size, irregular shape, irregular colour, diameter 7 mm or more, inflammation, oozing or a change in sensation [5,16]. The ABCD system of diagnosis (Asymmetry, Border irregulari‐ ty, Colour change, and a Diameter greater than 6 mm) has also been advocated to assist early clinical diagnosis, to which 'E' (Evolving or Elevation) has been added [5,17,18]. Ta‐ ble 1 illustrates the seven point checklist and ABCDE system for the assessment of pig‐

discussed and current controversies addressed.

412 Melanoma - From Early Detection to Treatment

door tanning device on at least one occasion [10].

nocitic naevi in childhood, and xeroderma pigmentosum [13].

can significantly reduce the risk of developing melanoma [13-15].

**2. Risk factors**

**3. Surgery**

mented lesions.

**3.1. Initial surgical biopsy**

An excision biopsy is indicated for lesions suspected of being a melanoma. An excision bi‐ opsy is the recommended method for suspected malignant melanoma as it enables diagno‐ sis and staging of the tumour and may determine future treatment and prognosis [20, 21]. The whole lesion should be excised with a 1-3 mm margin of normal skin including sub-der‐ mal fat. It is crucial to plan this excision carefully with a view towards definitive treatment. Knowledge of lymphatic drainage and subsequent need for sentinel node biopsy should lead to narrow margin excision potentially avoiding interference with subsequent lymphatic mapping. In addition, a longitudinal orientation is preferred in the extremities and incision orientation should be along Langer's lines on the trunk. This allows for subsequent closure of a wide local excision and reduces the need for skin grafting if primary closure is to be achieved.

In certain areas (such as the face, palm of hand, sole of foot, ears, digits and subungal le‐ sions) an excision biopsy may not be appropriate. In these cases, an incisional or punch bi‐ opsy of the thickest portion of the lesion may be performed [21]. Shave biopsy is avoided as it makes characterising the lesion difficult by underestimating tumour thickness, which is important in determining further treatment [21]. It also risks leaving residual tumour at the radial and deep margins.

Obtaining an adequate biopsy specimen is crucial for histopathological diagnosis and tu‐ mour staging. The tumour thickness, which remains the most powerful prognostic parame‐ ter, provides a guide to the margin clearance required for delayed wide excision and need for adjuvant therapy [20, 22]. Pathological examination should evaluate macroscopic fea‐ tures of the tumour such as width, symmetry, and circumscription, and microscopic features such as ulceration, microsatellitosis, angiolymphatic invasion and mitotic rate [22].

domly assigned to either a 2cm or 5cm resection margin. At a median follow-up of 11 years the local recurrence rate for all groups was less than 1%. Again there was no significant dif‐ ference noted in the overall or disease-free survival between the two groups. A third trial, the French Cooperative Group, included 362 patients with melanomas ≤2mm in thickness. Patients were randomly assigned to a wide local excision with either a 2cm or 5cm resection margin. No difference was noted between the groups in terms of local recurrence or overall survival. Therefore at present, a resection margin of 1cm is recommended for melanomas

Surgery and the Staging of Melanoma http://dx.doi.org/10.5772/53626 415

Melanomas between 2 - 4mm are considered intermediate thickness melanomas. Once again, there are a number of trials looking specifically at this cohort of patients which failed to show a benefit of greater than a 2cm excision margin. The Melanoma Intergroup Trial in‐ cluded 468 patients with melanomas of 1 to 4mm thickness. Patients were randomly as‐ signed to an excision margin of either 2cm or 4cm. Forty two percent of patients in the group undergoing 2cm excision had a melanoma thickness >2.0mm, while 46% of patients in the 4cm resection group had melanomas >2.0mm. At mean follow up, a 2cm margin was shown to be as effective as a 4cm margin in both the local control and overall survival for patients with intermediate thickness melanomas. Local recurrence however, was primarily determined by the thickness of the primary lesion and the presence or absence of ulceration [29, 30]. A multi-centre European trial was also performed to tease out the need for wider margins in deeper, intermediate thickness melanomas. In total, 936 patients were included who were assigned randomly to have either a 2cm or 4cm resection margin. At a follow-up of almost 7 years there was no statistically significant difference noted for recurrence or sur‐ vival between the two groups [31]. Finally a British trial was performed which recruited 900 patients with lesions greater than 2mm to a wide local excision with either a 1cm or 3 cm margin Interestingly, this study demonstrated a higher local recurrence rate when a 1cm margin was used. However, there was no statistically significant difference noted in overall survival. The authors therefore concluded that a margin of 1cm should be restricted to pa‐ tients with a melanoma thickness of less than 2mm [32]. Therefore, at present a 2cm excision

margin is recommended for intermediate (2 – 4mm) thickness melanomas.

There is unfortunately limited evidence or published data on the optimal resection margin for melanomas with a thickness of 4mm or greater. The British Trial included 243 patients with melanomas of > 4mm thickness and the results showed a higher local recurrence rate associated with a margin of 1cm [32]. However, the local recurrence rates with a 3cm margin appeared similar to other trials with only a 2cm margin of excision. In a retrospective review from MD Anderson which assessed patients with melanomas of greater than 6mm thick‐ ness, excision margins greater than 2 cm were not found to effect overall survival when compared to margins of 2cm or less. The 5-year overall and disease free survival rates were 55% and 30% in node negative compared to node positive patients which were included in the study. Nodal status, thickness, and ulceration were significantly associated with overall survival by multivariate analysis. However, the neither the disease free nor overall survival was effected by the presence of a local recurrence or the original excision margin in this study [36]. The study authors therefore concluded that a 2 cm margin of excision is adequate

<1mm and 2cm for melanomas 1 - 2mm thick [26-28].

#### **3.2. Management of the primary tumour**

The surgical management of the primary tumour has shifted from extensive surgical resec‐ tion, which was not only debilitating but also disfiguring, to a more conservative approach. A multidisciplinary team in a tertiary referral centre should ideally manage patients with malignant melanoma. This team should include: a surgeon, dermatologist,,medical oncolo‐ gist, pathologist, radiologist, counsellor, specialist nurse and palliative care specialist [23].

Pathological assessment of the surgically excised biopsy specimen allows for staging of the tumour while the thickness of the melanoma at initial biopsy serves as a guide to the subse‐ quent resection. The Breslow thickness, which is the most important prognostic indicator of localised disease, is defined as the distance of invasion and is measured from the granular layer of the epidermis to the point of deepest invasion by tumour cells [5, 24, 25].

Large randomised controlled trials have been performed in an attempt to elucidate the opti‐ mal resection margin in melanoma of various thickness (thin, intermediate, and thick mela‐ nomas) [26-31]. The trials reported data with not only differing lengths of follow-up but also differing margin excision widths. Therefore interpretation of the results is largely restricted to survival outcomes as a result of this heterogeneity.

The management of lentigo maligna and in situ melanoma present unique problems because of the characteristic, yet unpredictable, subclinical extension of atypical junctional melano‐ cytic hyperplasia, which may extend several centimeters beyond the visible margins [33]. There are no randomized trials looking at the optimal resection margin in these lesions. Guidelines from the American Academy of Dermatology in 2011 recommend a resection margin of 0.5 to 1.0 cm for melanoma in situ [34]. The NCCN recommends a margin of 0.5 cm around the visible lesion. For large in-situ lentigo maligna melanoma, it is felt that surgi‐ cal margins greater than 0.5 cm may be necessary to achieve a histologically negative mar‐ gin [33]. More recently, topical imiquimod has been used in lentigo melanoma treatment prior to definitive surgical resection. In a study that included 40 patients, 33 of these were found to have a complete clinical response after the use of imiquimod 5% cream. On histo‐ logical review, 30 of the patients had no evidence of melanoma. While studies have shown a limited role for this treatment, it does not replace surgery [35].

Three main trials (The World Health Organisation Trial, Swedish Melanoma Study and the French Cooperative Group) looked at the optimal resection margin for T1 and T2 melano‐ mas. The World Health Organisation (WHO) trial included 612 patients with melanomas less than 2.0mm with patients being randomly assigned to a wide local excision with a either a 3cm margin or 1cm margin. At 12 years of follow up, similar survival rates between the groups were noted (87% and 85% respectively) with no statistically significant difference in recurrence dependent upon margin width. As a consequence of this trial recommendations were made that a 1cm margin be used for melanomas ≤1mm. Similarly, the Swedish Mela‐ noma Study Group studied 989 patients with melanomas 0.8 to 2mm thick who were ran‐ domly assigned to either a 2cm or 5cm resection margin. At a median follow-up of 11 years the local recurrence rate for all groups was less than 1%. Again there was no significant dif‐ ference noted in the overall or disease-free survival between the two groups. A third trial, the French Cooperative Group, included 362 patients with melanomas ≤2mm in thickness. Patients were randomly assigned to a wide local excision with either a 2cm or 5cm resection margin. No difference was noted between the groups in terms of local recurrence or overall survival. Therefore at present, a resection margin of 1cm is recommended for melanomas <1mm and 2cm for melanomas 1 - 2mm thick [26-28].

tures of the tumour such as width, symmetry, and circumscription, and microscopic features

The surgical management of the primary tumour has shifted from extensive surgical resec‐ tion, which was not only debilitating but also disfiguring, to a more conservative approach. A multidisciplinary team in a tertiary referral centre should ideally manage patients with malignant melanoma. This team should include: a surgeon, dermatologist,,medical oncolo‐ gist, pathologist, radiologist, counsellor, specialist nurse and palliative care specialist [23].

Pathological assessment of the surgically excised biopsy specimen allows for staging of the tumour while the thickness of the melanoma at initial biopsy serves as a guide to the subse‐ quent resection. The Breslow thickness, which is the most important prognostic indicator of localised disease, is defined as the distance of invasion and is measured from the granular

Large randomised controlled trials have been performed in an attempt to elucidate the opti‐ mal resection margin in melanoma of various thickness (thin, intermediate, and thick mela‐ nomas) [26-31]. The trials reported data with not only differing lengths of follow-up but also differing margin excision widths. Therefore interpretation of the results is largely restricted

The management of lentigo maligna and in situ melanoma present unique problems because of the characteristic, yet unpredictable, subclinical extension of atypical junctional melano‐ cytic hyperplasia, which may extend several centimeters beyond the visible margins [33]. There are no randomized trials looking at the optimal resection margin in these lesions. Guidelines from the American Academy of Dermatology in 2011 recommend a resection margin of 0.5 to 1.0 cm for melanoma in situ [34]. The NCCN recommends a margin of 0.5 cm around the visible lesion. For large in-situ lentigo maligna melanoma, it is felt that surgi‐ cal margins greater than 0.5 cm may be necessary to achieve a histologically negative mar‐ gin [33]. More recently, topical imiquimod has been used in lentigo melanoma treatment prior to definitive surgical resection. In a study that included 40 patients, 33 of these were found to have a complete clinical response after the use of imiquimod 5% cream. On histo‐ logical review, 30 of the patients had no evidence of melanoma. While studies have shown a

Three main trials (The World Health Organisation Trial, Swedish Melanoma Study and the French Cooperative Group) looked at the optimal resection margin for T1 and T2 melano‐ mas. The World Health Organisation (WHO) trial included 612 patients with melanomas less than 2.0mm with patients being randomly assigned to a wide local excision with a either a 3cm margin or 1cm margin. At 12 years of follow up, similar survival rates between the groups were noted (87% and 85% respectively) with no statistically significant difference in recurrence dependent upon margin width. As a consequence of this trial recommendations were made that a 1cm margin be used for melanomas ≤1mm. Similarly, the Swedish Mela‐ noma Study Group studied 989 patients with melanomas 0.8 to 2mm thick who were ran‐

layer of the epidermis to the point of deepest invasion by tumour cells [5, 24, 25].

such as ulceration, microsatellitosis, angiolymphatic invasion and mitotic rate [22].

**3.2. Management of the primary tumour**

414 Melanoma - From Early Detection to Treatment

to survival outcomes as a result of this heterogeneity.

limited role for this treatment, it does not replace surgery [35].

Melanomas between 2 - 4mm are considered intermediate thickness melanomas. Once again, there are a number of trials looking specifically at this cohort of patients which failed to show a benefit of greater than a 2cm excision margin. The Melanoma Intergroup Trial in‐ cluded 468 patients with melanomas of 1 to 4mm thickness. Patients were randomly as‐ signed to an excision margin of either 2cm or 4cm. Forty two percent of patients in the group undergoing 2cm excision had a melanoma thickness >2.0mm, while 46% of patients in the 4cm resection group had melanomas >2.0mm. At mean follow up, a 2cm margin was shown to be as effective as a 4cm margin in both the local control and overall survival for patients with intermediate thickness melanomas. Local recurrence however, was primarily determined by the thickness of the primary lesion and the presence or absence of ulceration [29, 30]. A multi-centre European trial was also performed to tease out the need for wider margins in deeper, intermediate thickness melanomas. In total, 936 patients were included who were assigned randomly to have either a 2cm or 4cm resection margin. At a follow-up of almost 7 years there was no statistically significant difference noted for recurrence or sur‐ vival between the two groups [31]. Finally a British trial was performed which recruited 900 patients with lesions greater than 2mm to a wide local excision with either a 1cm or 3 cm margin Interestingly, this study demonstrated a higher local recurrence rate when a 1cm margin was used. However, there was no statistically significant difference noted in overall survival. The authors therefore concluded that a margin of 1cm should be restricted to pa‐ tients with a melanoma thickness of less than 2mm [32]. Therefore, at present a 2cm excision margin is recommended for intermediate (2 – 4mm) thickness melanomas.

There is unfortunately limited evidence or published data on the optimal resection margin for melanomas with a thickness of 4mm or greater. The British Trial included 243 patients with melanomas of > 4mm thickness and the results showed a higher local recurrence rate associated with a margin of 1cm [32]. However, the local recurrence rates with a 3cm margin appeared similar to other trials with only a 2cm margin of excision. In a retrospective review from MD Anderson which assessed patients with melanomas of greater than 6mm thick‐ ness, excision margins greater than 2 cm were not found to effect overall survival when compared to margins of 2cm or less. The 5-year overall and disease free survival rates were 55% and 30% in node negative compared to node positive patients which were included in the study. Nodal status, thickness, and ulceration were significantly associated with overall survival by multivariate analysis. However, the neither the disease free nor overall survival was effected by the presence of a local recurrence or the original excision margin in this study [36]. The study authors therefore concluded that a 2 cm margin of excision is adequate for patients with thick melanoma [36].However, overall there is insufficient data to support the preferred use of either a 2cm or 3cm margin, and consequently, it may be reasonable to allow the patient to decide, following an informed discussion of surgical options. The use of the larger 3cm margin may be recommended in patients with deep tumours (> 4mm depth), due to the higher risk of loco-regional recurrence [32]. In selected cases, however, margin size may be modified to accommodate individual anatomic or cosmetic considerations [23].

found localized around the primary tumour or may be widespread throughout the affected limb or on the head and neck or trunk, depending on the primary site [40] (Figure 1). It is thought that these metastases arise from dissemination of melanoma cells via the lymphatics to tissues located between the primary tumor and the regional lymph node basin. Other the‐ ories include that of drift metastases within tissue fluid of the limb or the local implantation

Surgery and the Staging of Melanoma http://dx.doi.org/10.5772/53626 417

The presence of small in-transit metastatic melanoma presents specific surgical problems. Unlike nodal disease, which can be managed by regional lymph node dissection, in-transit disease is often widespread and may necessitate multiple surgeries as the disease progresses and new deposits become apparent. In its most severe form, in-transit metastasis may be‐ come severely disabling and may be refractory to treatment. Treatment is therefore, pallia‐ tive, even if staging investigations fail to show evidence of distant metastatic disease [40]. Recent studies have recommended that treatment should be tailored to the extent of the dis‐ ease, with treatments associated with significant morbidity being reserved for bulky ad‐

Several therapies have been proposed for the management of in-transit metastasis including surgery, radiotherapy, and intra-lesional therapy. In-transit metastasis are sharply circum‐ scribed with a clear line of demarcation from normal dermis and epidermis. This line does not contain any in-situ component. Therefore, wide excision margins are not recommended for these lesions and a complete macroscopic excision and primary closure is sufficient. If

lesions are grouped closely together, an en bloc excision is acceptable [40].

of circulating haematogenous melanoma cells [41, 42].

vanced metastases [40].

**Figure 1.** In-transit metastases on the left lower limb

Although radial excision margins remain somewhat controversial, the depth of excision in clinical practice is defined as an excision down to but not including the deep fascia [37]. This definition has been internationally accepted and forms the basis of the current gold-stand‐ ard management of melanoma. Unfortunately in facial areas where the 'deep fascia' is less clearly defined (for example, on the ear, nose, or eyelid), or other anatomic sites such as over the breast, existing studies provide no clear guidelines for optimal depth of excision [5].


**Table 2.** Recommended excision margins based on tumor size [23]

Despite all the evidence discussed above, controversy still remains regarding the optimal width of the surgical excision margins in malignant melanoma and current evidence is not sufficient to address the optimal surgical management for all melanomas. Indeed a Co‐ chrane review which has been recently published attempted to address this complex ques‐ tion [5]. Overall, there was no statistically significant difference in overall survival between either a narrow or wide excision, but this meta-analysis was confounded by the fact that ex‐ cision margins were not standardized between studies within the overall analysis. Therefore the dilemma regarding surgical margin remains. However, guidelines regarding margin width have been published and should be adhered to where feasible. Further studies are re‐ quired to determine the appropriate local treatment for thick melanoma which has not been comprehensively addressed in trials thus far.

#### **3.3. In-transit metastasis**

The treatment of advanced or recurrent melanoma remains controversial. Around 10% of patients develop in-transit or multiple cutaneous metastases but at least half will survive for two years without developing distant disease [38, 39]. Unfortunately, the 5-year survival has been reported as 12% with a median survival of 19 months [39].

In-transit metastases are defined as cutaneous or subcutaneous deposits of melanoma be‐ tween the site of the primary disease and regional lymph nodes [40]. These deposits may be found localized around the primary tumour or may be widespread throughout the affected limb or on the head and neck or trunk, depending on the primary site [40] (Figure 1). It is thought that these metastases arise from dissemination of melanoma cells via the lymphatics to tissues located between the primary tumor and the regional lymph node basin. Other the‐ ories include that of drift metastases within tissue fluid of the limb or the local implantation of circulating haematogenous melanoma cells [41, 42].

The presence of small in-transit metastatic melanoma presents specific surgical problems. Unlike nodal disease, which can be managed by regional lymph node dissection, in-transit disease is often widespread and may necessitate multiple surgeries as the disease progresses and new deposits become apparent. In its most severe form, in-transit metastasis may be‐ come severely disabling and may be refractory to treatment. Treatment is therefore, pallia‐ tive, even if staging investigations fail to show evidence of distant metastatic disease [40]. Recent studies have recommended that treatment should be tailored to the extent of the dis‐ ease, with treatments associated with significant morbidity being reserved for bulky ad‐ vanced metastases [40].

Several therapies have been proposed for the management of in-transit metastasis including surgery, radiotherapy, and intra-lesional therapy. In-transit metastasis are sharply circum‐ scribed with a clear line of demarcation from normal dermis and epidermis. This line does not contain any in-situ component. Therefore, wide excision margins are not recommended for these lesions and a complete macroscopic excision and primary closure is sufficient. If lesions are grouped closely together, an en bloc excision is acceptable [40].

**Figure 1.** In-transit metastases on the left lower limb

for patients with thick melanoma [36].However, overall there is insufficient data to support the preferred use of either a 2cm or 3cm margin, and consequently, it may be reasonable to allow the patient to decide, following an informed discussion of surgical options. The use of the larger 3cm margin may be recommended in patients with deep tumours (> 4mm depth), due to the higher risk of loco-regional recurrence [32]. In selected cases, however, margin size may be modified to accommodate individual anatomic or cosmetic considerations [23].

Although radial excision margins remain somewhat controversial, the depth of excision in clinical practice is defined as an excision down to but not including the deep fascia [37]. This definition has been internationally accepted and forms the basis of the current gold-stand‐ ard management of melanoma. Unfortunately in facial areas where the 'deep fascia' is less clearly defined (for example, on the ear, nose, or eyelid), or other anatomic sites such as over the breast, existing studies provide no clear guidelines for optimal depth of excision [5].

Tis Histologically clear margins are adequate

Despite all the evidence discussed above, controversy still remains regarding the optimal width of the surgical excision margins in malignant melanoma and current evidence is not sufficient to address the optimal surgical management for all melanomas. Indeed a Co‐ chrane review which has been recently published attempted to address this complex ques‐ tion [5]. Overall, there was no statistically significant difference in overall survival between either a narrow or wide excision, but this meta-analysis was confounded by the fact that ex‐ cision margins were not standardized between studies within the overall analysis. Therefore the dilemma regarding surgical margin remains. However, guidelines regarding margin width have been published and should be adhered to where feasible. Further studies are re‐ quired to determine the appropriate local treatment for thick melanoma which has not been

The treatment of advanced or recurrent melanoma remains controversial. Around 10% of patients develop in-transit or multiple cutaneous metastases but at least half will survive for two years without developing distant disease [38, 39]. Unfortunately, the 5-year survival has

In-transit metastases are defined as cutaneous or subcutaneous deposits of melanoma be‐ tween the site of the primary disease and regional lymph nodes [40]. These deposits may be

T1 1cm margin is recommended T2 1-2cm margin recommended T3 2-3cm margin recommended

**Margins**

416 Melanoma - From Early Detection to Treatment

**Table 2.** Recommended excision margins based on tumor size [23]

comprehensively addressed in trials thus far.

been reported as 12% with a median survival of 19 months [39].

**3.3. In-transit metastasis**

There are numerous treatments available for the management of in-transit metastases that are not suitable for surgical treatment. Carbon dioxide laser therapy has been used in the management of small in-transit metastasis that are not amenable for surgical excision. This is performed as a day case under local anesthetic. Small lesions may be vaporized complete‐ ly, while larger lesions are first circumscribed with the laser prior to excision of the central core. This well tolerated procedure is more suitable for smaller lesions.

may result in significant deformity. Local rotation flaps, such as rhomboid flaps, have been found to be safe, versatile, and more aesthetically pleasing when used in these areas [15, 48].

Surgery and the Staging of Melanoma http://dx.doi.org/10.5772/53626 419

The presence of regional lymph node metastatic disease is a significant predictor of out‐ come in melanoma and is associated with a 50% reduction in overall survival compared to that of patients without nodal involvement [23]. Indeed the regional lymph node sta‐ tus is thought to be the most powerful prognostic indicator in clinically localised melano‐ ma. The risk of patients developing lymph node metastases increases exponentially with the increasing thickness of the primary melanoma. Melanomas less than 1mm rarely met‐ astasise (less than 10%), while at least 25% of melanomas 1.5- 4.0mm and over 60% of melanomas greater than 4.0mm thick will have lymph node metastasis at presenta‐ tion[49]. These data form the basis for the current guidelines on which patients should

Patients with melanoma can present with either a clinically normal regional lymph node ba‐ sin or palpable regional lymphadenopathy. Patients with stage III disease commonly have clinically negative lymph nodes but are found to have micro-metastatic disease on their sen‐ tinel lymph node biopsy. Such patients have been found to have a more favourable outcome than patients with clinically involved nodes at presentation [8]. The outcome of patients with stage III disease is determined by the number of metastatic nodes and the presence of either microscopic or macroscopic disease. The 5-year survival rate for patients with stage IIIA disease is 67%, and the 10-year survival is 60%. Patients with stage IIIB disease have survival rates estimated at 53%, while stage IIIC disease patients have the worst prognosis with a 5-year survival of approximately 26% [49]. The surgical management of the associat‐

Metastasis to regional lymph nodes is an important prognostic factor in patients with mela‐ noma, upstaging patients to stage III disease and has been shown to occur in about 20% of patients with intermediate thickness melanoma [50]. A sentinel lymph node biopsy (SLNB) is a minimally invasive procedure that aims to identify patients with microscopic lymph node metastasis who would benefit from further lymph node dissection and adjuvant treat‐ ment. The sentinel node is defined as any lymph node that receives lymphatic drainage di‐

The technical details of sentinel lymph node biopsy can be broken down into a number of steps. First, the patient undergoes preoperative lymphoscintigraphy which identifies the re‐ gional nodal basin and estimates the location of the sentinel node. Four intra-dermal injec‐ tions of 0.1–0.2 ml of 10 MBq radio-colloid are performed around the melanoma or melanoma scar: the injection should raise a small wheal on the skin. The most commonly used radiotracers are 99mTc-labeled albumin (Europe), 99mTc-labeled sulphur colloid and

ed lymph node basin depends on the initial presentation of the patient.

**4. Management of the regional lymph node basin**

be offered a sentinel lymph node biopsy.

**4.1. The sentinel lymph node biopsy**

rectly from a primary tumour site [51] (Figure 2).

In more advanced diseased, isolated limb perfusion has traditionally been the main method of treatment. This invasive procedure has been replaced by isolated limb infusion, which is simpler, minimally invasive, and a more economical alternative with comparable results [38, 39]. Isolated limb perfusion with chemotherapeutic agents was developed in New Orleans in the mid 1950s by Creech *et al* [38, 39, 43]. It is based on the principle of vascular isolation of the affected limb using a cardiopulmonary bypass circuit through open surgical cannula‐ tion of the major limb vessels. This procedure is technically difficult, expensive, and compli‐ cations are common. Repeated limb perfusions are difficult to perform and morbidity rates increase from 28% to 51% [38]. A simpler alternative, isolated limb infusion was developed by Dr John Thompson in the Sydney Melanoma Unit [44]. It is a less invasive procedure, which involves percutaneous placement of venous and arterial catheters and the infusion of chemotherapeutic agents. This negates the need for a bypass circuit. As opposed to isolated limb perfusion, autologous blood or autologous transfusion of allogenic units is not re‐ quired. The operating time is reduced from four hours to one hour, and the complication rates are documented to be lower, at only 1% [38, 43].

The presence of in-transit metastases indicates a poor prognosis. The development of intransit disease may be rapidly followed by distant metastases [40]. The American Commit‐ tee on Cancer Staging (AJCC) classify it as stage IIIB or IIIC disease, along with regional lymph node metastases. Five year survival rates in patients with stage III disease ranges from 18% to 60%. However, patients with in-transit metastasis have the worst prognosis, with 5 year survival of approximately 25%.

#### **3.4. Reconstruction**

The optimal treatment of patients undergoing melanoma excision is primary closure of the wound. Unfortunately, this is not always possible especially in patients with thick melano‐ mas requiring wider excision margins. Therefore, in these cases reconstructive surgery must be considered and where feasible offered to the patient. This will usually depend on the site and extent of the excision to be performed. Skin grafting is the commonest technique em‐ ployed to ensure skin cover of the anatomical defect. Traditionally, the graft is harvested from the contralateral limb, as melanoma was thought to metastasize primarily via lymphat‐ ic routes [15, 45, 46]. However, a recent study looking at the recurrence rates within skin graft donor sites, reported no difference in local recurrence rates when either the ipsilateral or contralateral limbs were used as graft sites. The authors of this study recommended that to improve patient recovery, harvesting the graft from the same limb as the primary tumor is both oncologically safe and technically superior to contralateral skin graft harvest [47]. In certain sites, such as the head and neck, the use of skin grafts may not always be ideal and may result in significant deformity. Local rotation flaps, such as rhomboid flaps, have been found to be safe, versatile, and more aesthetically pleasing when used in these areas [15, 48].

## **4. Management of the regional lymph node basin**

There are numerous treatments available for the management of in-transit metastases that are not suitable for surgical treatment. Carbon dioxide laser therapy has been used in the management of small in-transit metastasis that are not amenable for surgical excision. This is performed as a day case under local anesthetic. Small lesions may be vaporized complete‐ ly, while larger lesions are first circumscribed with the laser prior to excision of the central

In more advanced diseased, isolated limb perfusion has traditionally been the main method of treatment. This invasive procedure has been replaced by isolated limb infusion, which is simpler, minimally invasive, and a more economical alternative with comparable results [38, 39]. Isolated limb perfusion with chemotherapeutic agents was developed in New Orleans in the mid 1950s by Creech *et al* [38, 39, 43]. It is based on the principle of vascular isolation of the affected limb using a cardiopulmonary bypass circuit through open surgical cannula‐ tion of the major limb vessels. This procedure is technically difficult, expensive, and compli‐ cations are common. Repeated limb perfusions are difficult to perform and morbidity rates increase from 28% to 51% [38]. A simpler alternative, isolated limb infusion was developed by Dr John Thompson in the Sydney Melanoma Unit [44]. It is a less invasive procedure, which involves percutaneous placement of venous and arterial catheters and the infusion of chemotherapeutic agents. This negates the need for a bypass circuit. As opposed to isolated limb perfusion, autologous blood or autologous transfusion of allogenic units is not re‐ quired. The operating time is reduced from four hours to one hour, and the complication

The presence of in-transit metastases indicates a poor prognosis. The development of intransit disease may be rapidly followed by distant metastases [40]. The American Commit‐ tee on Cancer Staging (AJCC) classify it as stage IIIB or IIIC disease, along with regional lymph node metastases. Five year survival rates in patients with stage III disease ranges from 18% to 60%. However, patients with in-transit metastasis have the worst prognosis,

The optimal treatment of patients undergoing melanoma excision is primary closure of the wound. Unfortunately, this is not always possible especially in patients with thick melano‐ mas requiring wider excision margins. Therefore, in these cases reconstructive surgery must be considered and where feasible offered to the patient. This will usually depend on the site and extent of the excision to be performed. Skin grafting is the commonest technique em‐ ployed to ensure skin cover of the anatomical defect. Traditionally, the graft is harvested from the contralateral limb, as melanoma was thought to metastasize primarily via lymphat‐ ic routes [15, 45, 46]. However, a recent study looking at the recurrence rates within skin graft donor sites, reported no difference in local recurrence rates when either the ipsilateral or contralateral limbs were used as graft sites. The authors of this study recommended that to improve patient recovery, harvesting the graft from the same limb as the primary tumor is both oncologically safe and technically superior to contralateral skin graft harvest [47]. In certain sites, such as the head and neck, the use of skin grafts may not always be ideal and

core. This well tolerated procedure is more suitable for smaller lesions.

rates are documented to be lower, at only 1% [38, 43].

with 5 year survival of approximately 25%.

418 Melanoma - From Early Detection to Treatment

**3.4. Reconstruction**

The presence of regional lymph node metastatic disease is a significant predictor of out‐ come in melanoma and is associated with a 50% reduction in overall survival compared to that of patients without nodal involvement [23]. Indeed the regional lymph node sta‐ tus is thought to be the most powerful prognostic indicator in clinically localised melano‐ ma. The risk of patients developing lymph node metastases increases exponentially with the increasing thickness of the primary melanoma. Melanomas less than 1mm rarely met‐ astasise (less than 10%), while at least 25% of melanomas 1.5- 4.0mm and over 60% of melanomas greater than 4.0mm thick will have lymph node metastasis at presenta‐ tion[49]. These data form the basis for the current guidelines on which patients should be offered a sentinel lymph node biopsy.

Patients with melanoma can present with either a clinically normal regional lymph node ba‐ sin or palpable regional lymphadenopathy. Patients with stage III disease commonly have clinically negative lymph nodes but are found to have micro-metastatic disease on their sen‐ tinel lymph node biopsy. Such patients have been found to have a more favourable outcome than patients with clinically involved nodes at presentation [8]. The outcome of patients with stage III disease is determined by the number of metastatic nodes and the presence of either microscopic or macroscopic disease. The 5-year survival rate for patients with stage IIIA disease is 67%, and the 10-year survival is 60%. Patients with stage IIIB disease have survival rates estimated at 53%, while stage IIIC disease patients have the worst prognosis with a 5-year survival of approximately 26% [49]. The surgical management of the associat‐ ed lymph node basin depends on the initial presentation of the patient.

### **4.1. The sentinel lymph node biopsy**

Metastasis to regional lymph nodes is an important prognostic factor in patients with mela‐ noma, upstaging patients to stage III disease and has been shown to occur in about 20% of patients with intermediate thickness melanoma [50]. A sentinel lymph node biopsy (SLNB) is a minimally invasive procedure that aims to identify patients with microscopic lymph node metastasis who would benefit from further lymph node dissection and adjuvant treat‐ ment. The sentinel node is defined as any lymph node that receives lymphatic drainage di‐ rectly from a primary tumour site [51] (Figure 2).

The technical details of sentinel lymph node biopsy can be broken down into a number of steps. First, the patient undergoes preoperative lymphoscintigraphy which identifies the re‐ gional nodal basin and estimates the location of the sentinel node. Four intra-dermal injec‐ tions of 0.1–0.2 ml of 10 MBq radio-colloid are performed around the melanoma or melanoma scar: the injection should raise a small wheal on the skin. The most commonly used radiotracers are 99mTc-labeled albumin (Europe), 99mTc-labeled sulphur colloid and 99mTc-antimony trisulphide colloid. Scintillation cameras are used to obtain dynamic im‐ ages. These images allow identification of sentinel nodes within the regional nodal basin. They also allow discrimination of second-tier nodes, which may be falsely interpreted as sentinel nodes on delayed imaging. The surface location of the sentinel node may be marked on the skin preoperatively or, alternatively, a gamma probe can be use to locate the node intra-operatively. Intra-operative lymphatic mapping involves injection of vital blue dye (Isosulfan blue (Lymphazurin), Methylene Blue or Patent Blue V are used). A combination of radiotracers and blue dye has been shown to allow sentinel node identification in 99% of cases. The blue dye is injected intra-dermally in 2-4 locations at the site of the primary le‐ sion, 10-15 minutes before skin incision. The dye is used to visualize the sentinel node intraoperatively. A gamma probe (covered in a sterile plastic sheath), which detects radiation, may be used to locate the sentinel node (Figure 3). Counts should be obtained over the skin before incision, to confirm the location of the sentinel node. A short skin incision is made, bearing in mind the potential need for complete lymph node dissection. The sentinel nodes are then identified using the blue dye and gamma probe as a guide, and they are removed with minimal dissection. An ex-vivo count should be obtained, by measuring the radioactiv‐ ity of the sentinel node(s) after removal. A bed count is then also obtained following remov‐ al of the sentinel node(s), to ensure that no sentinel nodes remain [15, 52].

survival for patients with positive SLN status was 72.3%, compared to 90.2% in those with

Surgery and the Staging of Melanoma http://dx.doi.org/10.5772/53626 421

The AJCC Melanoma Staging Committee recommends that a sentinel lymph node biopsy be performed as a staging procedure in patients for whom the information will be useful in planning subsequent treatments and follow-up regimens. Significant controversy surrounds the use of sentinel lymph node biopsy in thin, early melanomas. There are a number of rea‐ sons for this. Firstly, patients with a low-risk of nodal metastases are exposed to the morbid‐ ity of a potentially unnecessary procedure. Secondly, the routine use of sentinel lymph node biopsy is expensive: global application of sentinel lymph node biopsy in all patients is esti‐ mated to cost between \$700,000 and \$1,000,000 for every sentinel node metastasis detected [15, 54]. Therefore, for thin melanomas, the routine use of SLNB has not been advocated as the risk of positive nodes is around 5.1% [55]. Indeed, a rate of only 2.7% has been docu‐ mented with melanomas thinner than 0.75mm [55]. SLNB may be considered, however, in patients with high risk features such as ulceration, a mitotic rate of greater than or equal to

 especially in patients with melanomas of ≥ 0.76 mm as they are associated with an approximately 10% risk of occult metastases in their sentinel lymph nodes [8]. SLNB is also recommended for patients with intermediate thickness melanoma (2 – 4mm). With regards to thick melanomas, it is expected that around 30% of patients will have evidence of lymph node involvement and the role for SLNB is less clear. It is, however, recommended that SLNB be performed in patients with no clinically evidence positive nodes as it allows for

Recent editions of the AJCC melanoma guidelines have altered the criteria for the presence of regional lymph node disease. Originally, the 6th Edition of the AJCC melanoma guidelines

negative SLN status [53].

**Figure 3.** Gamma probe used to locate sentinel lymph node

better chances at local disease control [50].

1/mm2

**Figure 2.** The Sentinel Lymph Node Biopsy

The Multicenter Selective Lymphadenectomy Trial-1 (MSLT-1) is the largest trial to address the role of lymphatic mapping with SLNB in determining prognosis and its impact on sur‐ vival [53]. Patients with a primary cutaneous melanoma were randomly assigned to wide excision and postoperative observation of the regional lymph nodes with lymphadenectomy being performed only if nodal relapse was confirmed or to wide excision and sentinel-node biopsy with immediate lymphadenectomy if nodal micro-metastases were detected on biop‐ sy [53]. The MSLT-1 trial confirmed the prognostic importance of SLN status, demonstrating that SLN status is the most statistically significant predictor of survival for clinically local‐ ized (stage I/II) intermediate thickness melanoma (1.2 to 3.5 mm). The 5-year disease-free survival for patients with positive SLN status was 72.3%, compared to 90.2% in those with negative SLN status [53].

**Figure 3.** Gamma probe used to locate sentinel lymph node

99mTc-antimony trisulphide colloid. Scintillation cameras are used to obtain dynamic im‐ ages. These images allow identification of sentinel nodes within the regional nodal basin. They also allow discrimination of second-tier nodes, which may be falsely interpreted as sentinel nodes on delayed imaging. The surface location of the sentinel node may be marked on the skin preoperatively or, alternatively, a gamma probe can be use to locate the node intra-operatively. Intra-operative lymphatic mapping involves injection of vital blue dye (Isosulfan blue (Lymphazurin), Methylene Blue or Patent Blue V are used). A combination of radiotracers and blue dye has been shown to allow sentinel node identification in 99% of cases. The blue dye is injected intra-dermally in 2-4 locations at the site of the primary le‐ sion, 10-15 minutes before skin incision. The dye is used to visualize the sentinel node intraoperatively. A gamma probe (covered in a sterile plastic sheath), which detects radiation, may be used to locate the sentinel node (Figure 3). Counts should be obtained over the skin before incision, to confirm the location of the sentinel node. A short skin incision is made, bearing in mind the potential need for complete lymph node dissection. The sentinel nodes are then identified using the blue dye and gamma probe as a guide, and they are removed with minimal dissection. An ex-vivo count should be obtained, by measuring the radioactiv‐ ity of the sentinel node(s) after removal. A bed count is then also obtained following remov‐

al of the sentinel node(s), to ensure that no sentinel nodes remain [15, 52].

The Multicenter Selective Lymphadenectomy Trial-1 (MSLT-1) is the largest trial to address the role of lymphatic mapping with SLNB in determining prognosis and its impact on sur‐ vival [53]. Patients with a primary cutaneous melanoma were randomly assigned to wide excision and postoperative observation of the regional lymph nodes with lymphadenectomy being performed only if nodal relapse was confirmed or to wide excision and sentinel-node biopsy with immediate lymphadenectomy if nodal micro-metastases were detected on biop‐ sy [53]. The MSLT-1 trial confirmed the prognostic importance of SLN status, demonstrating that SLN status is the most statistically significant predictor of survival for clinically local‐ ized (stage I/II) intermediate thickness melanoma (1.2 to 3.5 mm). The 5-year disease-free

**Figure 2.** The Sentinel Lymph Node Biopsy

420 Melanoma - From Early Detection to Treatment

The AJCC Melanoma Staging Committee recommends that a sentinel lymph node biopsy be performed as a staging procedure in patients for whom the information will be useful in planning subsequent treatments and follow-up regimens. Significant controversy surrounds the use of sentinel lymph node biopsy in thin, early melanomas. There are a number of rea‐ sons for this. Firstly, patients with a low-risk of nodal metastases are exposed to the morbid‐ ity of a potentially unnecessary procedure. Secondly, the routine use of sentinel lymph node biopsy is expensive: global application of sentinel lymph node biopsy in all patients is esti‐ mated to cost between \$700,000 and \$1,000,000 for every sentinel node metastasis detected [15, 54]. Therefore, for thin melanomas, the routine use of SLNB has not been advocated as the risk of positive nodes is around 5.1% [55]. Indeed, a rate of only 2.7% has been docu‐ mented with melanomas thinner than 0.75mm [55]. SLNB may be considered, however, in patients with high risk features such as ulceration, a mitotic rate of greater than or equal to 1/mm2 especially in patients with melanomas of ≥ 0.76 mm as they are associated with an approximately 10% risk of occult metastases in their sentinel lymph nodes [8]. SLNB is also recommended for patients with intermediate thickness melanoma (2 – 4mm). With regards to thick melanomas, it is expected that around 30% of patients will have evidence of lymph node involvement and the role for SLNB is less clear. It is, however, recommended that SLNB be performed in patients with no clinically evidence positive nodes as it allows for better chances at local disease control [50].

Recent editions of the AJCC melanoma guidelines have altered the criteria for the presence of regional lymph node disease. Originally, the 6th Edition of the AJCC melanoma guidelines recommended histological confirmation of all immunohistochemically (IHC) detected meta‐ stasis by routine H&E staining and only after this confirmation could metastatic disease be documented [56]. However, the more recently published guidelines state that positive nodes may be confirmed by either H&E staining or IHC staining with melanoma associated mark‐ ers [7]. The three most commonly used IHC markers for melanoma are S-100, HMB-45, and Melan A/MART 1. Currently, S-100 remains the most sensitive marker for detection of mela‐ noma, while HMB-45 and Melan A/MART 1 are used for their specificity [57].

pared with those who had a delayed lymphadenectomy only when they presented with

Surgery and the Staging of Melanoma http://dx.doi.org/10.5772/53626 423

However, a significant survival benefit has been noted in patients with a positive senti‐ nel lymph node biopsy, who undergo a complete lymph node dissection, when com‐ pared with patients undergoing complete lymph node dissection after nodal metastases become apparent [68]. In a study conducted by Morton et al, a 5-year survival rate of 72% was seen in patients with positive sentinel lymph nodes, followed by immediate lymph node dissection, whereas patients undergoing a delayed lymph node dissection had a 5-year survival rate of only 52% [53]. Further positive non-sentinel lymph nodes are found in a relatively small proportion of patients: previously quoted figures ranged from 17%-24% [15, 69-71]. Interestingly a recent study has shown rates of further positive

Researchers have sought to identify factors which may increase a patient's likelihood of non-sentinel node metastases. Increasing Breslow depth has been associated with increased risk of non-sentinel node metastases, while a depth of less than 1mm has no association with any further positive nodes on completion lymph node dissection [15,65]. Studies have failed to show an association between specific tumour and patient characteristics with an in‐ creased rate of non-sentinel nodal metastasis [15, 71], However, a number of histopathologi‐ cal features have been shown to be associated with positive complete lymph node dissections. These include: nodular melanoma, ulceration, melanoma regression, and nae‐ vus association [15, 65]. Using a size/ulceration score, Reeves et al. showed ulceration to be

Recent studies have examined the association between the size of the sentinel lymph node deposits and the rate of positive complete lymph node dissection. Kunte et al. did not report any patients with micro-metastatic deposits on sentinel lymph node biopsy to have positive findings on complete lymph node dissection [15, 65, 73]. Another study showed a 3-year survival rate in patients with 1mm sentinel lymph node metastasis to be 100%, while 3-year survival in patients with deposits greater than 1mm was 80% [15, 74]. Ollila et al., however, found a significantly higher rate of recurrence in patients with submicrometastatic disease (ie. sentinel lymph node deposits less than 0.1mm), compared

A significant number of these questions will be address by the publication of the results of The Multicenter Selective Lymphadenectomy Trial-II (MSTL-II) which are currently awaited [76, 77]. This trial aims to address the importance of SLN metastases, the relevance of molec‐ ular assessment of the SLN and to evaluate the therapeutic benefit of CLNB after SLNB. Within the trial, all patients with primary melanoma ≥1.2 mm or ≤1.2 mm with Clark level IV / V or ulceration undergo a SLNB. This will be analyzed by both H&E and IHC techni‐ ques. Patients with a negative SLNB by H&E and IHC will undergo RT-PCR. All SLN-posi‐ tive patients identified by H&E/IHC or RT-PCR are randomized to one of two groups: observation of lymph node basin with clinical examination and repeated follow-up ultra‐ sound scanning or to immediate CLND. Patients with negative SLN as determined by RT-PCR are assigned to routine follow-up. The primary endpoint of this study is to determine if

clinically evident nodal metastasis [15, 53, 67].

findings to be as low as 14.8% [15, 53].

with node-negative patients [15, 75].

an independent predictor of non-sentinel node deposits [72].

More recently, reverse transcriptase polymerase chain reaction (RT-PCR) has been shown to be a promising staging tool used to identify patients with histologically unidentified micrometastatic disease. This technique relies on detection of distinct mRNA expressed by mela‐ noma cells, such as tyrosinase, MAGE-3, MART-1, gp100 and other markers [58, 59]. There has been evidence suggesting the correlation between RT-PCR positive results in blood with stage of melanoma, tumor thickness and known prognostic indicators. The value of RT-PCR in regional lymph nodes is less clear. The number of false positives due to the presence of melanocytic naevi and Schwann cells has limited its use. However, there are results that show that positive results correlate with melanoma thickness [60]. Initial results from 30 month follow-up of the Sunbelt Melanoma Trial did not show any difference in disease-free or overall survival in RT-PCR positive and negative patients [61]. The results were subse‐ quently included in meta-analysis where it has been suggested that RT-PCR may have val‐ uable prognostic use in the prediction of overall and disease free survival [62]. The clinical relevance of the ability to detect micro-metastases by RT-PCR is still under investigation.

#### **4.2. Elective regional lymph node dissection**

Completion lymph node dissection (CLND) is recommended for patients with a positive SLN biopsy. It is performed with the intention of halting metastatic spread of melanoma in the early stages of the disease [15, 62, 64]. The five-year survival rate in patients with nega‐ tive complete lymph node dissection stands at 62.5%, compared with 20.3% in patients with positive non-sentinel nodes [65]. However, the exact role of this and its reflection on overall survival in the setting of positive sentinel nodes has yet to be fully elucidated.

Currently, a complete lymph node dissection is carried out for all patients with a posi‐ tive sentinel lymph node, irrespective of the type of metastases (micro or macro-metasta‐ sis) identified. The value of a complete lymph node dissection in this group of patients has not been extensively investigated and it must constantly be borne in mind that com‐ pletion lymph node dissection is associated with significant patient morbidity [66]. In‐ deed, in the MSLT-1, no improvement in OS was seen in the total group randomized to receive SLNB followed by completion lymph node dissection (CLND) if the SLN was positive compared to those randomized to WLE and observation, with nearly identical 5 year melanoma specific-survival of 87.1% versus 86.6% (P = 0.58) [53]. Studies that looked at this difference did not show any statistical significant between the two groups. In ad‐ dition, it is felt that micro-metastases will become evident if left untreated. Patients with nodal metastases were shown to have a survival advantage with early intervention com‐ pared with those who had a delayed lymphadenectomy only when they presented with clinically evident nodal metastasis [15, 53, 67].

recommended histological confirmation of all immunohistochemically (IHC) detected meta‐ stasis by routine H&E staining and only after this confirmation could metastatic disease be documented [56]. However, the more recently published guidelines state that positive nodes may be confirmed by either H&E staining or IHC staining with melanoma associated mark‐ ers [7]. The three most commonly used IHC markers for melanoma are S-100, HMB-45, and Melan A/MART 1. Currently, S-100 remains the most sensitive marker for detection of mela‐

More recently, reverse transcriptase polymerase chain reaction (RT-PCR) has been shown to be a promising staging tool used to identify patients with histologically unidentified micrometastatic disease. This technique relies on detection of distinct mRNA expressed by mela‐ noma cells, such as tyrosinase, MAGE-3, MART-1, gp100 and other markers [58, 59]. There has been evidence suggesting the correlation between RT-PCR positive results in blood with stage of melanoma, tumor thickness and known prognostic indicators. The value of RT-PCR in regional lymph nodes is less clear. The number of false positives due to the presence of melanocytic naevi and Schwann cells has limited its use. However, there are results that show that positive results correlate with melanoma thickness [60]. Initial results from 30 month follow-up of the Sunbelt Melanoma Trial did not show any difference in disease-free or overall survival in RT-PCR positive and negative patients [61]. The results were subse‐ quently included in meta-analysis where it has been suggested that RT-PCR may have val‐ uable prognostic use in the prediction of overall and disease free survival [62]. The clinical relevance of the ability to detect micro-metastases by RT-PCR is still under investigation.

Completion lymph node dissection (CLND) is recommended for patients with a positive SLN biopsy. It is performed with the intention of halting metastatic spread of melanoma in the early stages of the disease [15, 62, 64]. The five-year survival rate in patients with nega‐ tive complete lymph node dissection stands at 62.5%, compared with 20.3% in patients with positive non-sentinel nodes [65]. However, the exact role of this and its reflection on overall

Currently, a complete lymph node dissection is carried out for all patients with a posi‐ tive sentinel lymph node, irrespective of the type of metastases (micro or macro-metasta‐ sis) identified. The value of a complete lymph node dissection in this group of patients has not been extensively investigated and it must constantly be borne in mind that com‐ pletion lymph node dissection is associated with significant patient morbidity [66]. In‐ deed, in the MSLT-1, no improvement in OS was seen in the total group randomized to receive SLNB followed by completion lymph node dissection (CLND) if the SLN was positive compared to those randomized to WLE and observation, with nearly identical 5 year melanoma specific-survival of 87.1% versus 86.6% (P = 0.58) [53]. Studies that looked at this difference did not show any statistical significant between the two groups. In ad‐ dition, it is felt that micro-metastases will become evident if left untreated. Patients with nodal metastases were shown to have a survival advantage with early intervention com‐

survival in the setting of positive sentinel nodes has yet to be fully elucidated.

noma, while HMB-45 and Melan A/MART 1 are used for their specificity [57].

**4.2. Elective regional lymph node dissection**

422 Melanoma - From Early Detection to Treatment

However, a significant survival benefit has been noted in patients with a positive senti‐ nel lymph node biopsy, who undergo a complete lymph node dissection, when com‐ pared with patients undergoing complete lymph node dissection after nodal metastases become apparent [68]. In a study conducted by Morton et al, a 5-year survival rate of 72% was seen in patients with positive sentinel lymph nodes, followed by immediate lymph node dissection, whereas patients undergoing a delayed lymph node dissection had a 5-year survival rate of only 52% [53]. Further positive non-sentinel lymph nodes are found in a relatively small proportion of patients: previously quoted figures ranged from 17%-24% [15, 69-71]. Interestingly a recent study has shown rates of further positive findings to be as low as 14.8% [15, 53].

Researchers have sought to identify factors which may increase a patient's likelihood of non-sentinel node metastases. Increasing Breslow depth has been associated with increased risk of non-sentinel node metastases, while a depth of less than 1mm has no association with any further positive nodes on completion lymph node dissection [15,65]. Studies have failed to show an association between specific tumour and patient characteristics with an in‐ creased rate of non-sentinel nodal metastasis [15, 71], However, a number of histopathologi‐ cal features have been shown to be associated with positive complete lymph node dissections. These include: nodular melanoma, ulceration, melanoma regression, and nae‐ vus association [15, 65]. Using a size/ulceration score, Reeves et al. showed ulceration to be an independent predictor of non-sentinel node deposits [72].

Recent studies have examined the association between the size of the sentinel lymph node deposits and the rate of positive complete lymph node dissection. Kunte et al. did not report any patients with micro-metastatic deposits on sentinel lymph node biopsy to have positive findings on complete lymph node dissection [15, 65, 73]. Another study showed a 3-year survival rate in patients with 1mm sentinel lymph node metastasis to be 100%, while 3-year survival in patients with deposits greater than 1mm was 80% [15, 74]. Ollila et al., however, found a significantly higher rate of recurrence in patients with submicrometastatic disease (ie. sentinel lymph node deposits less than 0.1mm), compared with node-negative patients [15, 75].

A significant number of these questions will be address by the publication of the results of The Multicenter Selective Lymphadenectomy Trial-II (MSTL-II) which are currently awaited [76, 77]. This trial aims to address the importance of SLN metastases, the relevance of molec‐ ular assessment of the SLN and to evaluate the therapeutic benefit of CLNB after SLNB. Within the trial, all patients with primary melanoma ≥1.2 mm or ≤1.2 mm with Clark level IV / V or ulceration undergo a SLNB. This will be analyzed by both H&E and IHC techni‐ ques. Patients with a negative SLNB by H&E and IHC will undergo RT-PCR. All SLN-posi‐ tive patients identified by H&E/IHC or RT-PCR are randomized to one of two groups: observation of lymph node basin with clinical examination and repeated follow-up ultra‐ sound scanning or to immediate CLND. Patients with negative SLN as determined by RT-PCR are assigned to routine follow-up. The primary endpoint of this study is to determine if CLND will improve melanoma specific survival in patients with a positive SLNB. Secondary endpoints include assessing the predictive value of immune responses to melanoma- associ‐ ated antigens, to analyze blood samples from patients for molecular markers of melanoma, both before and after surgery and to assess the quality of life of patients undergoing either CLND or observation after SLNB. Finally the study analyses the predictive value of certain DNA markers of the primary tumor in relation to disease outcome [76, 77].

newer highly selective inhibitor with promising results. The main limitation of this novel agent is its limited response with an approximately 40% to 50% response rate in patients with a V600-mutated BRAF gene. Unfortunately, the median duration of response is only 5 to 6 months [33]. GSK2118436 is a newer highly selective inhibitor of BRAF that is still

Melanoma is an immunogenic tumor. Ipilimumab is a monoclonal antibody directed to the cytotoxic T lymphocyte antigen-4 (CTLA-4). Results of two randomized phase III trial of pa‐ tients with unresectable metastatic disease that progressed during systemic therapy showed an overall improvement in survival in patients randomized to the ipilimumab arm [33,81,82]). In another phase III study looking at the role of ipilumumab and dacarbazine in patients with previously untreated metastatic melanoma, ipilumumab and dacarbazine was shown to have improved patient survival in comparison to the group receiving dacarbazine alone [83[. The limitation of ipilimumab is its association with autoimmune toxicity. In addi‐ tion, clinical responses may take months to become apparent, and the overall response rate is less than 20% [33]. Research is ongoing in this area. The EORTC18071 trial is ongoing and compares adjuvant treatment with ipilimumab with observation in patients with high risk

The role of biochemotherapy has also been studied. This involves using a combination of chemotherapy and biologic agents [33]. The results, however, show no additional surviv‐ al benefit with this treatment. Finally, palliative radiotherapy may have a role in the set‐ ting of metastatic melanoma and has been shown to have good palliation of symptomatic

An updated Cancer Staging Manual was recently published by the AJCC [7]. Modifications of the melanoma staging guidelines, which have been used since 2002, were based on a mul‐ tivariate analysis on 38,918 patients [8]. In the revised guidelines melanoma patients have been categorised into 3 groups; those with localised disease with no evidence of metastases (stage I - II), patients with regional disease (stage III), and those with distant metastatic dis‐ ease (stage IV). Primary tumour thickness remains the factor most associated with progno‐ sis. Tumour thickness is defined in even integers (1.0, 2.0 and 4.0mm) with increasing thickness corresponding with worsening survival. Within each tumour thickness category,

Mitotic rate is an indicator of tumour proliferation and is measured as the number of mito‐

factor in patients with melanoma [88-91]. The AJCC guidelines now recommend the "hot spot" technique for calculating the mitotic rate, where the pathologist begins the mitotic count with the most active tumour focus. This is calculated as mitosis/mm2 [8]. Multiple thresholds of mitotic rate were examined statistically, and the most significant correlation

. Several studies have shown the mitotic rate to be an independent prognostic

, where a mitotic rate greater

Surgery and the Staging of Melanoma http://dx.doi.org/10.5772/53626 425

the presence of ulceration further upgrades the classification (Table 3).

with survival was identified at a threshold of at least 1/mm2

in pre-clinical trials [80].

lymph node positive disease [84].

disease [85-87].

**6. Staging**

ses per mm2

In conclusion, in the setting of a negative sentinel lymph node biopsy, a completion lymph node dissection is clearly not indicated. The presence of positive nodes warrants considera‐ tion of complete lymph node dissection of the involved lymph node basin. Results of the MSLT-II trial are awaited and will give answers to the option of nodal observation.

## **5. Management of distant metastatic disease**

The management of patients with metastatic melanoma remains challenging. Despite im‐ proved therapeutic options the prognosis remains poor. A complete surgical resection of metastatic disease in distant sites offers the best chance to improve survival. Patients with in-transit metastasis may be offered further surgical resection of the lesions. Favourable prognostic factors in patients with metastatic disease include a longer disease free survival, single site disease, complete resection and non-visceral metastases [78]. Patients that under‐ go resection of their non-visceral metastasis have been shown to have a medium survival of between 17 - 50 months, and a 5 - year survival of 9 - 35%. Patients with pulmonary metasta‐ sis, who have a complete resection, have a median survival of 8 - 20 months and a 5 year survival of 10 -25%. Brain and gastrointestinal tract metastasis confers a median survival of only 7-10 months [78]. Surgical resection in cases of advanced melanoma has been shown to give good palliation, if all the disease is completely removed. More recently, new systemic biological therapies have been developed, and when combined with surgery may be shown to aid in improved survival. These combinations, however, are still under review [79].

Chemotherapeutic agents have little role to play in the management of metastatic melano‐ ma. Regimens that have previously been utilised include dacarbazine, temozolomide, high dose interleukin-2, paclitaxel and cisplatin or carboplatin. These show a response rate of less than 20% [33]. There is little evidence of its value in metastatic melanoma, however with combination treatments their role is yet to be fully examined.

In 2011, the FDA approved two newer therapies for metastasis melanoma. These include the highly selective BRAF inhibitor, vemurafenib, and ipilimumab, a fully human IgG1 monoclonal antibody. Around 40% to 60% of melanomas are shown to harbor a mutation in the gene encoding for the serine / threonine kinase protein kinase B-raf (BRAF) with 90% of the mutations resulting in a substitution of valine for glutamate at amino acid 600 (V600E) [80]. Mutated BRAF leads to constitutive activation of the mitogen-activated pro‐ tein kinase pathway (MAPK) that in turn increases cellular proliferation and drives onco‐ genic activity. Sorafenib, the initial BRAF inhibitor failed to demonstrate significant response rates in melanoma and its use has been largely discontinued. Vemurafenib is a newer highly selective inhibitor with promising results. The main limitation of this novel agent is its limited response with an approximately 40% to 50% response rate in patients with a V600-mutated BRAF gene. Unfortunately, the median duration of response is only 5 to 6 months [33]. GSK2118436 is a newer highly selective inhibitor of BRAF that is still in pre-clinical trials [80].

Melanoma is an immunogenic tumor. Ipilimumab is a monoclonal antibody directed to the cytotoxic T lymphocyte antigen-4 (CTLA-4). Results of two randomized phase III trial of pa‐ tients with unresectable metastatic disease that progressed during systemic therapy showed an overall improvement in survival in patients randomized to the ipilimumab arm [33,81,82]). In another phase III study looking at the role of ipilumumab and dacarbazine in patients with previously untreated metastatic melanoma, ipilumumab and dacarbazine was shown to have improved patient survival in comparison to the group receiving dacarbazine alone [83[. The limitation of ipilimumab is its association with autoimmune toxicity. In addi‐ tion, clinical responses may take months to become apparent, and the overall response rate is less than 20% [33]. Research is ongoing in this area. The EORTC18071 trial is ongoing and compares adjuvant treatment with ipilimumab with observation in patients with high risk lymph node positive disease [84].

The role of biochemotherapy has also been studied. This involves using a combination of chemotherapy and biologic agents [33]. The results, however, show no additional surviv‐ al benefit with this treatment. Finally, palliative radiotherapy may have a role in the set‐ ting of metastatic melanoma and has been shown to have good palliation of symptomatic disease [85-87].

## **6. Staging**

CLND will improve melanoma specific survival in patients with a positive SLNB. Secondary endpoints include assessing the predictive value of immune responses to melanoma- associ‐ ated antigens, to analyze blood samples from patients for molecular markers of melanoma, both before and after surgery and to assess the quality of life of patients undergoing either CLND or observation after SLNB. Finally the study analyses the predictive value of certain

In conclusion, in the setting of a negative sentinel lymph node biopsy, a completion lymph node dissection is clearly not indicated. The presence of positive nodes warrants considera‐ tion of complete lymph node dissection of the involved lymph node basin. Results of the

The management of patients with metastatic melanoma remains challenging. Despite im‐ proved therapeutic options the prognosis remains poor. A complete surgical resection of metastatic disease in distant sites offers the best chance to improve survival. Patients with in-transit metastasis may be offered further surgical resection of the lesions. Favourable prognostic factors in patients with metastatic disease include a longer disease free survival, single site disease, complete resection and non-visceral metastases [78]. Patients that under‐ go resection of their non-visceral metastasis have been shown to have a medium survival of between 17 - 50 months, and a 5 - year survival of 9 - 35%. Patients with pulmonary metasta‐ sis, who have a complete resection, have a median survival of 8 - 20 months and a 5 year survival of 10 -25%. Brain and gastrointestinal tract metastasis confers a median survival of only 7-10 months [78]. Surgical resection in cases of advanced melanoma has been shown to give good palliation, if all the disease is completely removed. More recently, new systemic biological therapies have been developed, and when combined with surgery may be shown to aid in improved survival. These combinations, however, are still under review [79].

Chemotherapeutic agents have little role to play in the management of metastatic melano‐ ma. Regimens that have previously been utilised include dacarbazine, temozolomide, high dose interleukin-2, paclitaxel and cisplatin or carboplatin. These show a response rate of less than 20% [33]. There is little evidence of its value in metastatic melanoma, however with

In 2011, the FDA approved two newer therapies for metastasis melanoma. These include the highly selective BRAF inhibitor, vemurafenib, and ipilimumab, a fully human IgG1 monoclonal antibody. Around 40% to 60% of melanomas are shown to harbor a mutation in the gene encoding for the serine / threonine kinase protein kinase B-raf (BRAF) with 90% of the mutations resulting in a substitution of valine for glutamate at amino acid 600 (V600E) [80]. Mutated BRAF leads to constitutive activation of the mitogen-activated pro‐ tein kinase pathway (MAPK) that in turn increases cellular proliferation and drives onco‐ genic activity. Sorafenib, the initial BRAF inhibitor failed to demonstrate significant response rates in melanoma and its use has been largely discontinued. Vemurafenib is a

MSLT-II trial are awaited and will give answers to the option of nodal observation.

DNA markers of the primary tumor in relation to disease outcome [76, 77].

**5. Management of distant metastatic disease**

424 Melanoma - From Early Detection to Treatment

combination treatments their role is yet to be fully examined.

An updated Cancer Staging Manual was recently published by the AJCC [7]. Modifications of the melanoma staging guidelines, which have been used since 2002, were based on a mul‐ tivariate analysis on 38,918 patients [8]. In the revised guidelines melanoma patients have been categorised into 3 groups; those with localised disease with no evidence of metastases (stage I - II), patients with regional disease (stage III), and those with distant metastatic dis‐ ease (stage IV). Primary tumour thickness remains the factor most associated with progno‐ sis. Tumour thickness is defined in even integers (1.0, 2.0 and 4.0mm) with increasing thickness corresponding with worsening survival. Within each tumour thickness category, the presence of ulceration further upgrades the classification (Table 3).

Mitotic rate is an indicator of tumour proliferation and is measured as the number of mito‐ ses per mm2 . Several studies have shown the mitotic rate to be an independent prognostic factor in patients with melanoma [88-91]. The AJCC guidelines now recommend the "hot spot" technique for calculating the mitotic rate, where the pathologist begins the mitotic count with the most active tumour focus. This is calculated as mitosis/mm2 [8]. Multiple thresholds of mitotic rate were examined statistically, and the most significant correlation with survival was identified at a threshold of at least 1/mm2 , where a mitotic rate greater than or equal to 1/mm2 was found to be independently associated with a poorer disease-spe‐ cific survival in patients with T1 disease. For non-ulcerated, thin melanomas the 10-year sur‐ vival was 95% if there were fewer than 1 mitosis per mm2 , compared with 88% 10-year survival if at least one mitosis per mm2 was present. In addition, the level of invasion, as defined by Wallace Clark, was found to have no statistical significance in staging with the mitotic rate replacing it as an upstaging criterion from stages 1a to 1b [92].

**N Classification Nodes involved Nodal metastatic mass**

a: micro-metastasis b: macro-metastasis

Surgery and the Staging of Melanoma http://dx.doi.org/10.5772/53626 427

a: micro-metastasis b: macro-metastasis c: in transit mets(s)/ satellite(s) without metastatic nodes

Normal

Normal- visceral met(s) Elevated- Distant met(s)

previously removed for another reason)

4 or more metastatic nodes, or matted nodes, or in transit met(s)/ satellite(s) with metastatic node(s)

Finally, the database for stage IV patients was expanded to include 7972 patients and the new guidelines now incorporate the serum lactate dehydrogenase as a prognostic marker included in staging (Table 5). An elevated serum LDH was found to be an independent and a highly significant predictor of survival outcome. In a study that looked at the correlation between survival in advanced melanoma from two large trials (Oblimersen GM301 and EORTC 189510), the authors reported an elevated LDH in melanoma patients compared to the normal population. A relationship was found between LD and survival [95]. Patients with elevated serum LDH at diagnosis of melanoma are staged as M1c according to the

**M Classification Site Serum LDH**

nodes

Metastases to all other visceral sites or distant metastases to any site combined with an elevated serum LDH

M1b Metastases to lung Normal

M0 No detectable evidence of distant metastases

M1a Metastases to skin, subcutaneous, or distant lymph

Nx Regional nodes cannot be assessed (for example,

N0 No regional metastasis noted

N1 1 node

N2 2-3 nodes

**Table 4.** N Classification as recommended by the AJCC [7]

N3

AJCC guidelines.

M1c

**Table 5.** M Classification as recommended by the AJCC [7]


**Table 3.** T Classification as recommended by the AJCC [7]

Stage III patients have documented lymph node metastasis (microscopic of macroscopic) (Table 4). S-100 is the most sensitive marker for melanocytic lesions while others such as HMB-45, MART-1/Melan-A, tyrosinase, and MITF are very specific but less sensitive [93]. In terms of documenting micro-metastasis, the AJCC accepts immunohistochemical staining of at least one melanoma specific marker to make the diagnosis. Around 5% to 40% of patients will be upstaged to stage III based on the presence of micro-metastatic disease. These pa‐ tients have a better prognosis than those presenting with macro-metastatic disease as shown in several studies [8, 95]. The new AJCC guidelines reviewed the results of 3307 patients and make a clear distinction between each group. Staging of this group includes defining the number of nodes involved, the presence of microscopic versus macroscopic disease, as well as intra-lymphatic (in-transit or satellite) metastasis, the presence or absence of primary tu‐ mour ulceration, and the thickness of the primary melanoma. These factors were found to be predictive of survival on multivariate analysis. In the absence of nodal metastases, patients with intra-lymphatic metastases (N2c) have 5-year and 10-year survival rates of 69% and 52%, respectively while those with combined intra-lymphatic metastases and nodal metasta‐ ses (N3) have survival rates of 46% and 33%, respectively [8].


**Table 4.** N Classification as recommended by the AJCC [7]

than or equal to 1/mm2 was found to be independently associated with a poorer disease-spe‐ cific survival in patients with T1 disease. For non-ulcerated, thin melanomas the 10-year sur‐

defined by Wallace Clark, was found to have no statistical significance in staging with the

**T Classification Thickness Ulceration status/mitosis**

Stage III patients have documented lymph node metastasis (microscopic of macroscopic) (Table 4). S-100 is the most sensitive marker for melanocytic lesions while others such as HMB-45, MART-1/Melan-A, tyrosinase, and MITF are very specific but less sensitive [93]. In terms of documenting micro-metastasis, the AJCC accepts immunohistochemical staining of at least one melanoma specific marker to make the diagnosis. Around 5% to 40% of patients will be upstaged to stage III based on the presence of micro-metastatic disease. These pa‐ tients have a better prognosis than those presenting with macro-metastatic disease as shown in several studies [8, 95]. The new AJCC guidelines reviewed the results of 3307 patients and make a clear distinction between each group. Staging of this group includes defining the number of nodes involved, the presence of microscopic versus macroscopic disease, as well as intra-lymphatic (in-transit or satellite) metastasis, the presence or absence of primary tu‐ mour ulceration, and the thickness of the primary melanoma. These factors were found to be predictive of survival on multivariate analysis. In the absence of nodal metastases, patients with intra-lymphatic metastases (N2c) have 5-year and 10-year survival rates of 69% and 52%, respectively while those with combined intra-lymphatic metastases and nodal metasta‐

, compared with 88% 10-year

a: without ulceration and mitosis <1/mm2 b: with ulceration or mitoses ≥1/mm2

> a: without ulceration b: with ulceration

> a: without ulceration b: with ulceration

> a: without ulceration b: with ulceration

was present. In addition, the level of invasion, as

vival was 95% if there were fewer than 1 mitosis per mm2

T0 No evidence of primary tumor Tis Melanoma in situ

T1 Melanoma is 1.0mm or less in thickness

T2 Melanoma 1.01-2.0mm

T3 Melanoma 2.01- 4.0mm

T4 Melanoma more than 4.0mm

ses (N3) have survival rates of 46% and 33%, respectively [8].

**Table 3.** T Classification as recommended by the AJCC [7]

mitotic rate replacing it as an upstaging criterion from stages 1a to 1b [92].

Primary tumour cannot be assessed (for example, curettaged or severely regressed melanoma)

survival if at least one mitosis per mm2

426 Melanoma - From Early Detection to Treatment

Tx

Finally, the database for stage IV patients was expanded to include 7972 patients and the new guidelines now incorporate the serum lactate dehydrogenase as a prognostic marker included in staging (Table 5). An elevated serum LDH was found to be an independent and a highly significant predictor of survival outcome. In a study that looked at the correlation between survival in advanced melanoma from two large trials (Oblimersen GM301 and EORTC 189510), the authors reported an elevated LDH in melanoma patients compared to the normal population. A relationship was found between LD and survival [95]. Patients with elevated serum LDH at diagnosis of melanoma are staged as M1c according to the AJCC guidelines.


**Table 5.** M Classification as recommended by the AJCC [7]

## **7. Follow-up**

All patients with invasive melanoma should be followed up post-operatively, except for pa‐ tients with melanoma in-situ. The aim of follow-up is to detect evidence of recurrent disease or a new primary melanoma early [97,98]. The primary site and adjacent skin should be ex‐ amined for recurrence of new suspicious lesions, as well as the draining lymph node basins [23]. It is estimated that the lifetime risk of developing a second melanoma is around 4 - 6%. Furthermore, around 60 - 80% of recurrences are found at local and/or regional nodal sites. Around two thirds of these will occur within the first three years, 16% after the first five years. Recurrence after more than ten years is also recognised [23].

[2] American Cancer Society: Cancer Facts and Figures 2012. Atlanta, Ga: American

Surgery and the Staging of Melanoma http://dx.doi.org/10.5772/53626 429

[3] Cancer Research UK. http://info.cancerresearchuk.org/cancerstats/types/skin/ (Ac‐

[4] National Cancer Institute. http://seer.cancer.gov/statfacts/html/melan.html (Accessed

[5] Sladden MJ, Balch C, Barzilai DA, Berg D, Freiman A, Handiside T, Hollis S, Lens MB, Thompson JF. Surgical excision margins for primary cutaneous melanoma. Co‐ chrane Database of Systematic Reviews 2009, Issue 4. Art. No.: CD004835. DOI:

[6] Stebbins WG, Garibyan, Sober AJ. Sentinel lymph node biopsy and melanoma: 2010 Part I. Journal of the American Academy of Dermatology. 2010; 62(5) 723-734

[7] Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A (Eds.): AJCC Cancer

[8] Balch CM, Gershenwald JE, Soong SJ, Thompson JF, Atkins MB, Byrd DR, Buzaid AC, Cochran AJ, Coit DG, Ding S, Eggermont AM, Flaherty KT, Gimotty PA, Kirk‐ wood JM, McMasters KM, Mihm Jr MC, Morton DL, Ross MI, Sober AJ, Sondak VK. Final Version of 2009 AJCC Melanoma Staging and Classification. Journal of Clinical

[9] Cascinelli N, Marchesini R. Increasing incidence of cutaneous melanoma, ultraviolet radiation and the clinician. Photochemistry and Photobiology. 1989; 50 497-505

[10] The American Cancer Society. (2010). What are the key statistics about Melanoma?, In : The American Cancer Society, Available from http://www.cancer.org/Cancer/ SkinCancerMelanoma/DetailedGuide/melanoma-skin-cancer-key-statistics (Accessed

[11] Evans RD, Kopf, Lew RA, Rigel DS, Bart RS, Friedman RJ, Rivers JK. Risk factors for the development of malignant melanoma: I. Review of case-control studies. The jour‐

[12] Gellin GA, Kopf AW, Garfinkel L. Malignant melanoma: A controlled study of possi‐

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There is little evidence for the optimum protocol for follow-up. It appears reasonable that all patients with invasive melanoma should be followed up 6-monthly for 2 years. Thereafter, those with melanomas less than 1.0 mm in depth may be discharged from routine follow-up; other patients should be followed up for a further 3 years at 6-monthly intervals. Patients with stage III or IV disease require lifelong follow up [23].

## **8. Conclusion**

The incidence of melanoma continues to rise steadily in the Western World. Despite increased awareness of the disease this does not appear to have a significant impact on its overall poor prognosis. Surgery remains the mainstay of treatment as there is little in the way of adjuvant systemic treatment. Adequate surgical margins with or without local reconstruction can im‐ prove local recurrence rates. The utilisation of the sentinel lymph node biopsy has allowed for accurate staging of the disease. The finding of positive sentinel lymph nodes requires patients to undergo further regional lymph node dissection to reduce the risk of loco-regional disease. The impact of this on overall survival has not yet been clearly elucidated. Increased understanding of the melanoma pathogenesis and molecular biology may lead to the development of novel promising therapeutic agents and individualised treatment plans for these patients..

## **Author details**


## **References**

[1] Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA: A Cancer Journal for Clinicians 2011; 61(2) 69–90

[2] American Cancer Society: Cancer Facts and Figures 2012. Atlanta, Ga: American Cancer Society, 2012. Last accessed Aug 3rd 2012

**7. Follow-up**

428 Melanoma - From Early Detection to Treatment

**8. Conclusion**

**Author details**

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All patients with invasive melanoma should be followed up post-operatively, except for pa‐ tients with melanoma in-situ. The aim of follow-up is to detect evidence of recurrent disease or a new primary melanoma early [97,98]. The primary site and adjacent skin should be ex‐ amined for recurrence of new suspicious lesions, as well as the draining lymph node basins [23]. It is estimated that the lifetime risk of developing a second melanoma is around 4 - 6%. Furthermore, around 60 - 80% of recurrences are found at local and/or regional nodal sites. Around two thirds of these will occur within the first three years, 16% after the first five

There is little evidence for the optimum protocol for follow-up. It appears reasonable that all patients with invasive melanoma should be followed up 6-monthly for 2 years. Thereafter, those with melanomas less than 1.0 mm in depth may be discharged from routine follow-up; other patients should be followed up for a further 3 years at 6-monthly intervals. Patients

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**Chapter 16**

**Melanoma: Treatments and Resistance**

In the past two decades, it has been observed an increased incidence of skin cancer around the world [1-4]. This increase is particularly important in melanoma [5]. Latin-American da‐ ta have shown both an increase in incidence rates of skin cancer [6] and in mortality from malignant melanoma [7]. The number of melanoma cases worldwide is increasing faster than any other cancer. Although early detection, appropriate surgery, and adjuvant therapy have improved outcomes, the prognosis of metastatic melanoma remains very poor. Ad‐ vanced melanoma is still associated with an extremely poor median survival, ranging from 2 to 8 months, with only 5% surviving more than 5 years and remains one of the most treat‐

The only way to cure a malignant melanoma is early detection and appropriate surgical treatment, because once it reaches an advanced stage, is highly resistant to conventional ra‐ diotherapy and chemotherapy [9]. The median survival for patients with metastatic disease is approximately 8 months [10], and chemotherapy has so far failed to improve survival. Treatment options include radiation therapy, chemotherapy, immunotherapy and bioche‐

The use of adjuvant radiotherapy (RT) in melanomas has been controversial. *In vitro* studies have shown that melanoma cells possess a broad shoulder on the cell survival curve and

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

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

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

Additional information is available at the end of the chapter

Jonathan Castillo Arias and Miriam Galvonas Jasiulionis

http://dx.doi.org/10.5772/54202

ment-refractory malignancy [8]

motherapy which are summarized below.

**2. Treatments**

**2.1. Radiotherapy**

**1. Introduction**

## **Melanoma: Treatments and Resistance**

Jonathan Castillo Arias and Miriam Galvonas Jasiulionis

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54202

## **1. Introduction**

In the past two decades, it has been observed an increased incidence of skin cancer around the world [1-4]. This increase is particularly important in melanoma [5]. Latin-American da‐ ta have shown both an increase in incidence rates of skin cancer [6] and in mortality from malignant melanoma [7]. The number of melanoma cases worldwide is increasing faster than any other cancer. Although early detection, appropriate surgery, and adjuvant therapy have improved outcomes, the prognosis of metastatic melanoma remains very poor. Ad‐ vanced melanoma is still associated with an extremely poor median survival, ranging from 2 to 8 months, with only 5% surviving more than 5 years and remains one of the most treat‐ ment-refractory malignancy [8]

## **2. Treatments**

The only way to cure a malignant melanoma is early detection and appropriate surgical treatment, because once it reaches an advanced stage, is highly resistant to conventional ra‐ diotherapy and chemotherapy [9]. The median survival for patients with metastatic disease is approximately 8 months [10], and chemotherapy has so far failed to improve survival. Treatment options include radiation therapy, chemotherapy, immunotherapy and bioche‐ motherapy which are summarized below.

#### **2.1. Radiotherapy**

The use of adjuvant radiotherapy (RT) in melanomas has been controversial. *In vitro* studies have shown that melanoma cells possess a broad shoulder on the cell survival curve and

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

thus have a large capacity for DNA repair. As a result, hypofractionated RT schedules have been developed to counteract this perceived radioresistance, producing excellent locoregion‐ al control rates of 85% and higher [11,12]. Radiation Therapy Oncology Group (RTOG) Trial 83-05 was a prospective randomized study comparing hypofractionation to conventional fractionation. The results showed no difference in partial or complete response rates be‐ tween the two schedules, and the overall response rates were approximately 70% [13]. The role of adjuvant radiation therapy (RT) following nodal surgery in malignant melanoma re‐ mains controversial. Despite the high incidence of distant metastases, loco-regional control remains an important goal in the management of melanoma. Surgery and adjuvant RT pro‐ vides excellent loco-regional control, although distant metastases remain the major cause of mortality.[14]

months. Doses up to 150 mg/m2

in combination with amifostine produced tumor responses

Melanoma: Treatments and Resistance http://dx.doi.org/10.5772/54202 441

in 53% of patients. However, all of those responses were partial, and the median response duration was only 4 months [26]. Regarding carboplatin, in a study on 26 chemotherapy-na‐ ive metastatic melanoma patients, a response rate of 19% with 5 partial responses was re‐

The vinca alkaloids, especially vindesine and vinblastine, have induced responses in ap‐ proximately 14% of melanoma patients and they are usually used in combination with other drugs [28]. Docetaxel or paclitaxel, do not have a significant activity in melanoma [29-32]. The role of tamoxifen (TAM) as single agent at standard or high-doses in the treatment of melanoma is negligible with a response rate ranging between 0% and 10%. Currently all of

In a phase II study, Lattanzi et al. [33] reported their experience with the addition of TAM to the three-drug combination regimen of cisplatin, carmustine and dacarbazine (the Dart‐ mouth regimen) and showed high response rates (55%) with a 20% complete response. Since then several randomized clinical trials have been conducted to confirm the therapeutic bene‐

Cocconi et al. [34] published a small phase III trial demonstrating an improvement of re‐ sponse and survival with the addition of tamoxifen to dacarbazine compared to dacarbazine alone. However, two large randomized trials with low and high-dose tamoxifen in combina‐ tion with either dacarbazine alone or the Dartmouth regimen failed to demonstrate an ad‐

The efficacy of the combination of paclitaxel and carboplatin in the treatment of metastatic melanoma was reported some years ago. Although originally tested in two small phase II clinical trials and deemed not sufficiently clinically active, this evidence suggests that the

Immunotherapy in melanoma consists of various approaches leading to specific or non-spe‐ cific immunomodulation. Immunotherapies are being used for melanoma patients in stage II–III patients in the adjuvant setting, where only a fraction of patients have widespread (mi‐ croscopic) disease with the aim to prevent relapse of disease, prolong relapse-free survival and, ideally, prolong overall survival (OS). In patients with stage IV disease, there is a need for adequate systemic therapies as median OS for this patient group is only 6–9 months [38]. However, for the first time in >30 years, prospective randomized trials in patients with dis‐

Some agents used in the treatment against the melanoma are ipilimumab and tremelimu‐ mab, fully human IgG1 and IgG2 monoclonal antibodies, respectively. They block cytotoxic T-lymphocyte- associated antigen 4 (CTLA-4), a negative regulator of T cells, and thus aug‐

combination of paclitaxel and carboplatin may be worth further consideration [37].

ported and thrombocytopenia was the dose-limiting toxicity [27].

*2.2.2. Chemotheraphy with combined drugs in melanoma*

fit of TAM in combination with chemotherapy.

vantage to the addition of tamoxifen [35,36].

tant metastatic melanoma demonstrated an OS benefit [39].

**2.3. Immunotherapy**

these drugs are rarely used as single agent therapy in metastatic melanoma.

## **2.2. Chemotherapy**

Chemotherapeutic agents are cytotoxic anticancer drugs which aim is impair the cell division, resulting in the death of rapidly dividing cells. They are widely used in the treatment of malig‐ nancies; however, melanomas are resistant to many forms of traditional chemotherapy.

### *2.2.1. Chemotherapy with single drugs in melanoma*

Several antitumoral drugs have been used to treat the melanoma. One of the most known is dacarbazine. In 1975, dacarbazine (DTIC) became the first US Food and Drug Administra‐ tion (FDA) approved chemotherapeutic agent for the treatment of metastatic melanoma. The response rates with dacarbazine were 15–25%, with median response ranging from 5 to 6 months, but with less than 5% of complete responses [17-19]. Long-term follow-up of pa‐ tients treated with DTIC alone shows that less than 2% of the patients could survive for 6 years [15,16]. In a meta-analysis comparing two or three-drugs combination regimens with DTIC alone, Huncharek et al. [20] concluded that there was no advantage for the combina‐ tion in terms of response or survival. Since survival was not improved by the use of single or combination chemotherapy for metastatic melanoma, treatment decisions remain contro‐ versial, and quality of life and toxicity issues from treatment assume greater importance.

An orally analogue of DTIC is temozolomide whose activity has been tested in several clini‐ cal studies as single agent in metastatic malignant melanoma [18,21,22]. A randomized phase III trial comparing TMZ to DTIC on patients with advanced melanoma demonstrated a statistically significant increase in progression-free survival (1.9 months vs 1.5 months) when TMZ was administered [18].

Fotemustine (FTMU) is the most active nitrosourea used against the metastatic melanoma. It has been widely tested in Europe and has shown overall response of 20–25% including 5–8% of complete response rates and it was the first drug to show significant efficacy in brain metastases [23,24]. However, at conventional doses, little or no activity was observed against melanoma brain metastases [25].

Platinum-based drugs are widely used in the treatment of cancer. In patients with melano‐ ma, cisplatin was shown to induce a 15% response rate with a short median duration of 3 months. Doses up to 150 mg/m2 in combination with amifostine produced tumor responses in 53% of patients. However, all of those responses were partial, and the median response duration was only 4 months [26]. Regarding carboplatin, in a study on 26 chemotherapy-na‐ ive metastatic melanoma patients, a response rate of 19% with 5 partial responses was re‐ ported and thrombocytopenia was the dose-limiting toxicity [27].

The vinca alkaloids, especially vindesine and vinblastine, have induced responses in ap‐ proximately 14% of melanoma patients and they are usually used in combination with other drugs [28]. Docetaxel or paclitaxel, do not have a significant activity in melanoma [29-32]. The role of tamoxifen (TAM) as single agent at standard or high-doses in the treatment of melanoma is negligible with a response rate ranging between 0% and 10%. Currently all of these drugs are rarely used as single agent therapy in metastatic melanoma.

## *2.2.2. Chemotheraphy with combined drugs in melanoma*

In a phase II study, Lattanzi et al. [33] reported their experience with the addition of TAM to the three-drug combination regimen of cisplatin, carmustine and dacarbazine (the Dart‐ mouth regimen) and showed high response rates (55%) with a 20% complete response. Since then several randomized clinical trials have been conducted to confirm the therapeutic bene‐ fit of TAM in combination with chemotherapy.

Cocconi et al. [34] published a small phase III trial demonstrating an improvement of re‐ sponse and survival with the addition of tamoxifen to dacarbazine compared to dacarbazine alone. However, two large randomized trials with low and high-dose tamoxifen in combina‐ tion with either dacarbazine alone or the Dartmouth regimen failed to demonstrate an ad‐ vantage to the addition of tamoxifen [35,36].

The efficacy of the combination of paclitaxel and carboplatin in the treatment of metastatic melanoma was reported some years ago. Although originally tested in two small phase II clinical trials and deemed not sufficiently clinically active, this evidence suggests that the combination of paclitaxel and carboplatin may be worth further consideration [37].

### **2.3. Immunotherapy**

thus have a large capacity for DNA repair. As a result, hypofractionated RT schedules have been developed to counteract this perceived radioresistance, producing excellent locoregion‐ al control rates of 85% and higher [11,12]. Radiation Therapy Oncology Group (RTOG) Trial 83-05 was a prospective randomized study comparing hypofractionation to conventional fractionation. The results showed no difference in partial or complete response rates be‐ tween the two schedules, and the overall response rates were approximately 70% [13]. The role of adjuvant radiation therapy (RT) following nodal surgery in malignant melanoma re‐ mains controversial. Despite the high incidence of distant metastases, loco-regional control remains an important goal in the management of melanoma. Surgery and adjuvant RT pro‐ vides excellent loco-regional control, although distant metastases remain the major cause of

Chemotherapeutic agents are cytotoxic anticancer drugs which aim is impair the cell division, resulting in the death of rapidly dividing cells. They are widely used in the treatment of malig‐

Several antitumoral drugs have been used to treat the melanoma. One of the most known is dacarbazine. In 1975, dacarbazine (DTIC) became the first US Food and Drug Administra‐ tion (FDA) approved chemotherapeutic agent for the treatment of metastatic melanoma. The response rates with dacarbazine were 15–25%, with median response ranging from 5 to 6 months, but with less than 5% of complete responses [17-19]. Long-term follow-up of pa‐ tients treated with DTIC alone shows that less than 2% of the patients could survive for 6 years [15,16]. In a meta-analysis comparing two or three-drugs combination regimens with DTIC alone, Huncharek et al. [20] concluded that there was no advantage for the combina‐ tion in terms of response or survival. Since survival was not improved by the use of single or combination chemotherapy for metastatic melanoma, treatment decisions remain contro‐ versial, and quality of life and toxicity issues from treatment assume greater importance.

An orally analogue of DTIC is temozolomide whose activity has been tested in several clini‐ cal studies as single agent in metastatic malignant melanoma [18,21,22]. A randomized phase III trial comparing TMZ to DTIC on patients with advanced melanoma demonstrated a statistically significant increase in progression-free survival (1.9 months vs 1.5 months)

Fotemustine (FTMU) is the most active nitrosourea used against the metastatic melanoma. It has been widely tested in Europe and has shown overall response of 20–25% including 5–8% of complete response rates and it was the first drug to show significant efficacy in brain metastases [23,24]. However, at conventional doses, little or no activity was observed

Platinum-based drugs are widely used in the treatment of cancer. In patients with melano‐ ma, cisplatin was shown to induce a 15% response rate with a short median duration of 3

nancies; however, melanomas are resistant to many forms of traditional chemotherapy.

mortality.[14]

**2.2. Chemotherapy**

440 Melanoma - From Early Detection to Treatment

*2.2.1. Chemotherapy with single drugs in melanoma*

when TMZ was administered [18].

against melanoma brain metastases [25].

Immunotherapy in melanoma consists of various approaches leading to specific or non-spe‐ cific immunomodulation. Immunotherapies are being used for melanoma patients in stage II–III patients in the adjuvant setting, where only a fraction of patients have widespread (mi‐ croscopic) disease with the aim to prevent relapse of disease, prolong relapse-free survival and, ideally, prolong overall survival (OS). In patients with stage IV disease, there is a need for adequate systemic therapies as median OS for this patient group is only 6–9 months [38]. However, for the first time in >30 years, prospective randomized trials in patients with dis‐ tant metastatic melanoma demonstrated an OS benefit [39].

Some agents used in the treatment against the melanoma are ipilimumab and tremelimu‐ mab, fully human IgG1 and IgG2 monoclonal antibodies, respectively. They block cytotoxic T-lymphocyte- associated antigen 4 (CTLA-4), a negative regulator of T cells, and thus aug‐ ment T-cell activation and proliferation [40,41]. A phase-III trial was completed first and its results were reported in 2010 [39]. This trial compared ipilimumab alone or in combination with a gp100-peptide vaccine, compared to the vaccine alone in patients who had failed pri‐ or therapy or therapies. Melanoma patients receiving ipilimumab and ipilimumab + vacci‐ nation had a significantly better survival outcome than those receiving the vaccine alone. Ipilimumab was combined with high-dose IL-2 in 36 patients in the surgery branch of the NCI, with some remarkable observations. There were six patients (17%) with long-lasting complete response, all over 5 years, and none of the patients relapsed. Moreover, there was no increased toxicity as compared to high-dose IL-2 alone [42]. Other study showing a com‐ bination of tremelimumab with high-dose interferon yielded a high overall response rate of 30% in 33 melanoma patients, with three complete responses and seven partial responses, all long-lasting responses. Again, there was no increased toxicity compared to high-dose IFN therapy alone [43].

sion of 9 MIU/m2 of IL-2 + IFN-α) because of concern of toxicity when drugs were given simul‐ taneously or concurrent with chemo-immunotherapy. Both approaches have produced promising results with overall response rates between 40% and 60% and a long-term remission rate of about 9%. The sequential approach was compared to chemotherapy alone in a random‐ ized trial conducted at the MD Anderson Cancer Center. Although both response rate and time to progression were improved in the sequential biochemotherapy group, the survival differ‐ ence was at borderline significance and the toxicity was very high [53]. The results of the largest phase III trial (ECOG/Intergroup E3695 trial) and most definitive test for biochemotherapy comparing concurrent CVD-Bio to CVD alone showed that biochemotherapy produced slight‐ ly higher response rates and significantly longer median progression-free survival than CVD alone, but once again failed to show any improvement in either overall survival or durable re‐ sponses. Considering the extra toxicity and complexity, this concurrent biochemotherapy regi‐

Melanoma: Treatments and Resistance http://dx.doi.org/10.5772/54202 443

In the past decades, no significant impact on survival has been made in spite of increased re‐ sponse rates achieved with combinations of chemotherapeutics or with the combination of che‐ motherapy and cytokines such as interferon (IFN) or interleukin-2 (IL-2). However, great advances have been made in a very short time, both in terms of targeted drugs that kill melano‐

Sorafenib was designed to inhibit tyrosine kinase activity of CRAF, but this drug inhibits both the wild-type RAF protein as the V600E mutant protein. Subsequently, it was shown that sora‐ fenib is actually a multikinase inhibitor, can inhibit many other molecules such as VEGFR2 and 3, PDGFR, p38 MAPK, FLT3, c-Kit and RET [55]. Although preclinical experiments, both *in vi‐ tro* and in animal models, seemed to be encouraging, the results of clinical trials have not con‐

After the failure of sorafenib in melanoma, was synthesized a more specific BRAF inhibitors, in particular against the protein with the V600E mutation: PLX4032, a low molecular weight drug, for oral administration. In the first clinical trial published in 2010 [57], the objective response was observed in 81% of the BRAFV600E melanoma patients with 2 complete respons‐ es and 24 partial responses. Responses occurred in patients with visceral metastases in loca‐ tions usually resistant to treatment such as liver, intestine and bone. However, despite having achieved a good response, relapses occur early, usually in a period of 8-12 months

The possibility that c-Kit was a therapeutic target in melanoma has long since shuffled. In fact, c-Kit is a protein that acts as a receptor for a growth factor essential for epidermal mela‐ nocytes and has a role in the differentiation and migration of melanocytic cells during em‐ bryonic development [59]. In 2011, a phase-II study from China reported 20–30% response

From 15 to 30% of melanomas have mutations of NRAS. RAS activation mutations stimulate MAP kinase pathway, but also the route of PI3K/AKT among others. A phase II trial using

rates and prolongation of progression-free survival with imatinib treatment [60].

men should not be recommended for patients with metastatic melanoma [54].

firmed the efficacy of sorafenib for the treatment of disseminated melanoma [56]

**2.5. Signal transduction inhibitors**

ma cells.

after treatment [58].

Interferon-α (IFN-α) has been approved in the adjuvant setting for the treatment of high-risk melanoma based on clinical trials in the early 1990s [44,45]. In a metastatic situation, melanoma patients treated with the single agent IFN-α showed approximately 15% of responses, with less than 5% of complete response rates and median response duration between 6 and 9 months with a maximum of 12 months for the best studies [46]. These response rates, while encourag‐ ing, were not significant enough to lead to its widespread use in the treatment of metastatic melanoma. However, observations that patients with non-visceral disease were more likely to respond suggested that the use of IFN-α may demonstrated a grater impact in patients with mi‐ crometastasis [46, 47]. Other combination studied was IL-2 with IFN-α. This association did not seem to achieve better results (median response rate of 18% with three complete responses) than if these agents were given alone [48-50]. By contrast, in a small randomized phase III trial comparing continuous infusion IL- 2 plus interferon vs. continuous infusion decreasing IL-2 plus interferon, Keilholtz and colleagues [51], demonstrated improved response rates and re‐ duced toxicity with decreasing doses of IL-2.

#### **2.4. Biochemotherapy**

Because chemotherapy and cytokines have different and synergistic mechanisms of action and in order to improve response rates and durable remissions, several groups developed in the early 1990s the concept of biochemotherapy, a combination of chemotherapy and biolog‐ ic response modifiers.

Dacarbazine/IFN-α is one of the most evaluated combinations in metastatic malignant mela‐ noma. In a randomized phase II trial, Falkson et al. [52] reported that the association of IFNα with dacarbazine resulted in an encouraging response rate (53% vs. 20% for dacarbazine alone) and a higher duration of response (8.9 months vs. 2.5 months) but IFN-α significantly increased the toxicity. However, a follow up of a large randomized trial demonstrated no benefit for the addition of IFN-α to dacarbazine and significantly more severe toxic events occurred with treatments containing IFN-α [36].

The other approaches of biochemotherapy have involved sequential chemotherapy (cisplatin, vinblastine, and dacarbazine, CVD) followed by biologic response modifiers (continuous infu‐ sion of 9 MIU/m2 of IL-2 + IFN-α) because of concern of toxicity when drugs were given simul‐ taneously or concurrent with chemo-immunotherapy. Both approaches have produced promising results with overall response rates between 40% and 60% and a long-term remission rate of about 9%. The sequential approach was compared to chemotherapy alone in a random‐ ized trial conducted at the MD Anderson Cancer Center. Although both response rate and time to progression were improved in the sequential biochemotherapy group, the survival differ‐ ence was at borderline significance and the toxicity was very high [53]. The results of the largest phase III trial (ECOG/Intergroup E3695 trial) and most definitive test for biochemotherapy comparing concurrent CVD-Bio to CVD alone showed that biochemotherapy produced slight‐ ly higher response rates and significantly longer median progression-free survival than CVD alone, but once again failed to show any improvement in either overall survival or durable re‐ sponses. Considering the extra toxicity and complexity, this concurrent biochemotherapy regi‐ men should not be recommended for patients with metastatic melanoma [54].

## **2.5. Signal transduction inhibitors**

ment T-cell activation and proliferation [40,41]. A phase-III trial was completed first and its results were reported in 2010 [39]. This trial compared ipilimumab alone or in combination with a gp100-peptide vaccine, compared to the vaccine alone in patients who had failed pri‐ or therapy or therapies. Melanoma patients receiving ipilimumab and ipilimumab + vacci‐ nation had a significantly better survival outcome than those receiving the vaccine alone. Ipilimumab was combined with high-dose IL-2 in 36 patients in the surgery branch of the NCI, with some remarkable observations. There were six patients (17%) with long-lasting complete response, all over 5 years, and none of the patients relapsed. Moreover, there was no increased toxicity as compared to high-dose IL-2 alone [42]. Other study showing a com‐ bination of tremelimumab with high-dose interferon yielded a high overall response rate of 30% in 33 melanoma patients, with three complete responses and seven partial responses, all long-lasting responses. Again, there was no increased toxicity compared to high-dose IFN

Interferon-α (IFN-α) has been approved in the adjuvant setting for the treatment of high-risk melanoma based on clinical trials in the early 1990s [44,45]. In a metastatic situation, melanoma patients treated with the single agent IFN-α showed approximately 15% of responses, with less than 5% of complete response rates and median response duration between 6 and 9 months with a maximum of 12 months for the best studies [46]. These response rates, while encourag‐ ing, were not significant enough to lead to its widespread use in the treatment of metastatic melanoma. However, observations that patients with non-visceral disease were more likely to respond suggested that the use of IFN-α may demonstrated a grater impact in patients with mi‐ crometastasis [46, 47]. Other combination studied was IL-2 with IFN-α. This association did not seem to achieve better results (median response rate of 18% with three complete responses) than if these agents were given alone [48-50]. By contrast, in a small randomized phase III trial comparing continuous infusion IL- 2 plus interferon vs. continuous infusion decreasing IL-2 plus interferon, Keilholtz and colleagues [51], demonstrated improved response rates and re‐

Because chemotherapy and cytokines have different and synergistic mechanisms of action and in order to improve response rates and durable remissions, several groups developed in the early 1990s the concept of biochemotherapy, a combination of chemotherapy and biolog‐

Dacarbazine/IFN-α is one of the most evaluated combinations in metastatic malignant mela‐ noma. In a randomized phase II trial, Falkson et al. [52] reported that the association of IFNα with dacarbazine resulted in an encouraging response rate (53% vs. 20% for dacarbazine alone) and a higher duration of response (8.9 months vs. 2.5 months) but IFN-α significantly increased the toxicity. However, a follow up of a large randomized trial demonstrated no benefit for the addition of IFN-α to dacarbazine and significantly more severe toxic events

The other approaches of biochemotherapy have involved sequential chemotherapy (cisplatin, vinblastine, and dacarbazine, CVD) followed by biologic response modifiers (continuous infu‐

therapy alone [43].

442 Melanoma - From Early Detection to Treatment

**2.4. Biochemotherapy**

ic response modifiers.

duced toxicity with decreasing doses of IL-2.

occurred with treatments containing IFN-α [36].

In the past decades, no significant impact on survival has been made in spite of increased re‐ sponse rates achieved with combinations of chemotherapeutics or with the combination of che‐ motherapy and cytokines such as interferon (IFN) or interleukin-2 (IL-2). However, great advances have been made in a very short time, both in terms of targeted drugs that kill melano‐ ma cells.

Sorafenib was designed to inhibit tyrosine kinase activity of CRAF, but this drug inhibits both the wild-type RAF protein as the V600E mutant protein. Subsequently, it was shown that sora‐ fenib is actually a multikinase inhibitor, can inhibit many other molecules such as VEGFR2 and 3, PDGFR, p38 MAPK, FLT3, c-Kit and RET [55]. Although preclinical experiments, both *in vi‐ tro* and in animal models, seemed to be encouraging, the results of clinical trials have not con‐ firmed the efficacy of sorafenib for the treatment of disseminated melanoma [56]

After the failure of sorafenib in melanoma, was synthesized a more specific BRAF inhibitors, in particular against the protein with the V600E mutation: PLX4032, a low molecular weight drug, for oral administration. In the first clinical trial published in 2010 [57], the objective response was observed in 81% of the BRAFV600E melanoma patients with 2 complete respons‐ es and 24 partial responses. Responses occurred in patients with visceral metastases in loca‐ tions usually resistant to treatment such as liver, intestine and bone. However, despite having achieved a good response, relapses occur early, usually in a period of 8-12 months after treatment [58].

The possibility that c-Kit was a therapeutic target in melanoma has long since shuffled. In fact, c-Kit is a protein that acts as a receptor for a growth factor essential for epidermal mela‐ nocytes and has a role in the differentiation and migration of melanocytic cells during em‐ bryonic development [59]. In 2011, a phase-II study from China reported 20–30% response rates and prolongation of progression-free survival with imatinib treatment [60].

From 15 to 30% of melanomas have mutations of NRAS. RAS activation mutations stimulate MAP kinase pathway, but also the route of PI3K/AKT among others. A phase II trial using the RAS inhbitor Tipifarnib was performed; however, it was closed for lack of response. None of the patients was selected based on the presence of mutations of NRAS [61].

changes during exposure to chemotherapy and changes in the levels of ceramides [75] or changes in the cell cycle machinery, which triggers checkpoints and prevent initiation of apoptosis. Below we present several mechanisms of resistance to the treatments that have

Melanoma: Treatments and Resistance http://dx.doi.org/10.5772/54202 445

Melanocytes and their stem cell precursors are activated to secrete melanin and protect neighboring keratinocytes and other epidermal cells from further damage [76]. Thus, melanocytes should be programmed to survive. Keratinocytes promote melanocyte ex‐ pression of Bcl-2 by secreting neuronal growth factor (NGF) and stem cell grow factor (SCF). NGF binds to its receptors in the melanocyte membrane and increases the levels of Bcl-2 [77]. SCF interacts with its receptor c-KIT on the membrane and leads to the ac‐ tivation of transcription factor Mitf, which induces proliferation and differentiation of melanocyte precursors [78]. Tumorigenic melanoma cells may take advantage of high en‐ dogenous Bcl-2 levels to survive under adverse environmental conditions that they may encounter during metastatic progression and, given the connection between apoptosis and drug sensitivity, bypass the effects of chemotherapeutic drugs. Similarly, BclxL and Mcl-1, other anti-apoptotic members of the Bcl-2 family, are strongly expressed in nor‐ mal melanocytes, benign nevi, primary melanoma and melanoma metastases, and may

In melanoma, two members of the IAP family, survivin and ML-IAP, have been associated with tumor progression, as they become detectable in melanocytic nevi and further overex‐ pressed in invasive and metastatic melanomas [81,82]. Survivin is abundantly expressed, and its subcellular localization varies depending upon tumor thickness and invasiveness. Survivin overexpression has been shown in squamous cell carcinoma (SCC), and it is in‐ volved in UVB-induced carcinogenesis. The presence of survivin both in the nucleus and in the cytoplasm throughout the epidermal layers of psoriatic lesions suggests the involvement of this protein in the keratinocyte alterations typical of this disease [81]. Similarly, suppres‐ sion of survivin can increase the sensitivity of melanoma cells to chemotherapeutic agents [83,84]. ML-IAP is also upregulated in melanoma cell lines and absent in normal melano‐ cytes [85]. ML-IAP's effects on the mitochondrial pathways are considered to be related to a direct inhibition of the pro-apoptotic factor Smac/Diablo, and the caspases 9 and 3 [86]. The role of ML-IAP on melanoma chemoresistance has not been proven yet, but the overexpres‐ sion of ML-IAP in breast cancer cell lines (MCF-7) or in HeLa cells protects against the drug

Adriamycin and other apoptotic inducers, including TNF-α, FADD or BAX [86,87].

p53 suppresses tumor development through multiple activities including induction of growth arrest, apoptosis, senescence, and autophagy [88,89]. Environmental agents such as UV that induce cellular damage activate the p53 tumor suppressor and p53 activation re‐ sults in p53-dependent programmed cell death (apoptosis) in many cell types. Melanocytes are resistant to UV-induced apoptosis suggesting that p53 activity is somehow blocked

been described in melanoma.

**3.2. p53 pathway**

**3.1. Antipoptotic characteristics in melanoma**

contribute to melanoma resistance to therapy [79,80]

MEK is a protein of the MAP kinase pathway, located downstream BRAF. Several MEK in‐ hibitors (PD0325901, AZD6244, GSK1120212, and E6201) have been synthesized. Bases on some results, it appears that these pharmacological agents may be effective as single agents in the treatment of melanoma. However, there are many preclinical studies suggesting that it would be a good alternative to the combined treatments, both to avoid resistance in the use of drugs directed against BRAF/V600E mutation, as for the treatment of BRAF muta‐ tions other than V600E or mutations of NRAS, especially if associated with inhibitors of PI3K/AKT pathway [62-65]

Different derivatives of rapamycin (CCI-779 or temsirolimus) have been used as inhibitors of the PI3K/AKT pathway. These inhibitors act on mTOR molecule downstream AKT/PKB. There are also dual inhibitors of PI3K and mTOR, PI3K and AKT [66]. Although clinical out‐ comes of these drugs in phase II trials have not been good, there are several authors propos‐ ing their use in combined therapies especially with drugs that inhibit the MAP kinase pathway [62, 63, 65, 67] or even, simultaneous inhibition via PI3K/AKT [68].

## **3. Resistance to the treatments in melanoma**

Simultaneous resistance to several structurally unrelated drugs that do not necessarily have a common mechanism of action is called multidrug resistance phenomena. An important principle in multidrug resistance is that cancer cells are genetically heterogeneous. Although the process results in uncontrolled cell growth for clonal expansion of cancer, tumor cells ex‐ posed to chemotherapeutic agents will be selected by their ability to survive and grow in the presence of cytotoxic drugs. Therefore, in any population of cancer cells that are exposed to chemotherapy, more than one mechanism of multidrug resistance may be present [69]. Dif‐ ferent types of multidrug resistance mechanisms have been described in cancer cells. Natu‐ ral resistance to hydrophobic drugs sometimes known as classical multidrug resistance, usually results in the expression of efflux pumps with an ATP-dependent drug broad specif‐ icity. These pumps belong to a family of conveyors called ABC transporters (ATP-binding cassette) that show sequence and structural homology [70]. The resistance is caused by in‐ creased output by lowering the intracellular concentration of the drug. Resistance may also occur due to reduced entry of the drug. Water-soluble drugs, which are returned by carriers that are used to carry nutrients into the cell, or agents that enter through endocytosis, could fail without evidencing of increased output. Examples of this kind of drugs include the anti‐ folate methotrexate, nucleotide analogues such as 5-fluorouracil and 8-azaguanine, and al‐ kylating agents such as cisplatin [71,72]. Multidrug resistance can also result from the activation of coordinated systems of detoxification, such as DNA repair systems and cyto‐ chrome P-450 [73]. In another hand, resistance can also result from a defective apoptotic pathway. This can occur because of malignant transformation, such as in cancer, or as a re‐ sult of non-functional mutant p53 [74]. Alternatively, cells may acquire apoptotic pathways changes during exposure to chemotherapy and changes in the levels of ceramides [75] or changes in the cell cycle machinery, which triggers checkpoints and prevent initiation of apoptosis. Below we present several mechanisms of resistance to the treatments that have been described in melanoma.

## **3.1. Antipoptotic characteristics in melanoma**

the RAS inhbitor Tipifarnib was performed; however, it was closed for lack of response.

MEK is a protein of the MAP kinase pathway, located downstream BRAF. Several MEK in‐ hibitors (PD0325901, AZD6244, GSK1120212, and E6201) have been synthesized. Bases on some results, it appears that these pharmacological agents may be effective as single agents in the treatment of melanoma. However, there are many preclinical studies suggesting that it would be a good alternative to the combined treatments, both to avoid resistance in the use of drugs directed against BRAF/V600E mutation, as for the treatment of BRAF muta‐ tions other than V600E or mutations of NRAS, especially if associated with inhibitors of

Different derivatives of rapamycin (CCI-779 or temsirolimus) have been used as inhibitors of the PI3K/AKT pathway. These inhibitors act on mTOR molecule downstream AKT/PKB. There are also dual inhibitors of PI3K and mTOR, PI3K and AKT [66]. Although clinical out‐ comes of these drugs in phase II trials have not been good, there are several authors propos‐ ing their use in combined therapies especially with drugs that inhibit the MAP kinase

Simultaneous resistance to several structurally unrelated drugs that do not necessarily have a common mechanism of action is called multidrug resistance phenomena. An important principle in multidrug resistance is that cancer cells are genetically heterogeneous. Although the process results in uncontrolled cell growth for clonal expansion of cancer, tumor cells ex‐ posed to chemotherapeutic agents will be selected by their ability to survive and grow in the presence of cytotoxic drugs. Therefore, in any population of cancer cells that are exposed to chemotherapy, more than one mechanism of multidrug resistance may be present [69]. Dif‐ ferent types of multidrug resistance mechanisms have been described in cancer cells. Natu‐ ral resistance to hydrophobic drugs sometimes known as classical multidrug resistance, usually results in the expression of efflux pumps with an ATP-dependent drug broad specif‐ icity. These pumps belong to a family of conveyors called ABC transporters (ATP-binding cassette) that show sequence and structural homology [70]. The resistance is caused by in‐ creased output by lowering the intracellular concentration of the drug. Resistance may also occur due to reduced entry of the drug. Water-soluble drugs, which are returned by carriers that are used to carry nutrients into the cell, or agents that enter through endocytosis, could fail without evidencing of increased output. Examples of this kind of drugs include the anti‐ folate methotrexate, nucleotide analogues such as 5-fluorouracil and 8-azaguanine, and al‐ kylating agents such as cisplatin [71,72]. Multidrug resistance can also result from the activation of coordinated systems of detoxification, such as DNA repair systems and cyto‐ chrome P-450 [73]. In another hand, resistance can also result from a defective apoptotic pathway. This can occur because of malignant transformation, such as in cancer, or as a re‐ sult of non-functional mutant p53 [74]. Alternatively, cells may acquire apoptotic pathways

pathway [62, 63, 65, 67] or even, simultaneous inhibition via PI3K/AKT [68].

**3. Resistance to the treatments in melanoma**

None of the patients was selected based on the presence of mutations of NRAS [61].

PI3K/AKT pathway [62-65]

444 Melanoma - From Early Detection to Treatment

Melanocytes and their stem cell precursors are activated to secrete melanin and protect neighboring keratinocytes and other epidermal cells from further damage [76]. Thus, melanocytes should be programmed to survive. Keratinocytes promote melanocyte ex‐ pression of Bcl-2 by secreting neuronal growth factor (NGF) and stem cell grow factor (SCF). NGF binds to its receptors in the melanocyte membrane and increases the levels of Bcl-2 [77]. SCF interacts with its receptor c-KIT on the membrane and leads to the ac‐ tivation of transcription factor Mitf, which induces proliferation and differentiation of melanocyte precursors [78]. Tumorigenic melanoma cells may take advantage of high en‐ dogenous Bcl-2 levels to survive under adverse environmental conditions that they may encounter during metastatic progression and, given the connection between apoptosis and drug sensitivity, bypass the effects of chemotherapeutic drugs. Similarly, BclxL and Mcl-1, other anti-apoptotic members of the Bcl-2 family, are strongly expressed in nor‐ mal melanocytes, benign nevi, primary melanoma and melanoma metastases, and may contribute to melanoma resistance to therapy [79,80]

In melanoma, two members of the IAP family, survivin and ML-IAP, have been associated with tumor progression, as they become detectable in melanocytic nevi and further overex‐ pressed in invasive and metastatic melanomas [81,82]. Survivin is abundantly expressed, and its subcellular localization varies depending upon tumor thickness and invasiveness. Survivin overexpression has been shown in squamous cell carcinoma (SCC), and it is in‐ volved in UVB-induced carcinogenesis. The presence of survivin both in the nucleus and in the cytoplasm throughout the epidermal layers of psoriatic lesions suggests the involvement of this protein in the keratinocyte alterations typical of this disease [81]. Similarly, suppres‐ sion of survivin can increase the sensitivity of melanoma cells to chemotherapeutic agents [83,84]. ML-IAP is also upregulated in melanoma cell lines and absent in normal melano‐ cytes [85]. ML-IAP's effects on the mitochondrial pathways are considered to be related to a direct inhibition of the pro-apoptotic factor Smac/Diablo, and the caspases 9 and 3 [86]. The role of ML-IAP on melanoma chemoresistance has not been proven yet, but the overexpres‐ sion of ML-IAP in breast cancer cell lines (MCF-7) or in HeLa cells protects against the drug Adriamycin and other apoptotic inducers, including TNF-α, FADD or BAX [86,87].

#### **3.2. p53 pathway**

p53 suppresses tumor development through multiple activities including induction of growth arrest, apoptosis, senescence, and autophagy [88,89]. Environmental agents such as UV that induce cellular damage activate the p53 tumor suppressor and p53 activation re‐ sults in p53-dependent programmed cell death (apoptosis) in many cell types. Melanocytes are resistant to UV-induced apoptosis suggesting that p53 activity is somehow blocked (non-functional p53), a state shared with melanoma cells [90], which are resistant to conven‐ tional modes of chemotherapy that aim to stimulate p53-dependent apoptosis.

nositide 3-kinase (PI3K)/AKT, either as consequences of genetic alterations or resulting from environmental stimulations, is known to play a central role in the resistance of melanoma to

Melanoma: Treatments and Resistance http://dx.doi.org/10.5772/54202 447

One-third of primary melanomas and about 50% of metastatic melanoma cell lines showed reduced expression of PTEN as a result of allelic deletion, mutation or transcriptional silenc‐ ing [109,110], suggesting that inactivation of PTEN is a late, but frequent, event on melano‐ magenesis [111,112]. Multiple lines of evidence point to the PI3K/AKT/PTEN pathway as a putative candidate for therapeutic intervention in melanoma because PTEN overexpression can revert the invasive phenotype of human and mouse melanoma cell lines [113,114] and

Recent progress in the identification of genes relevant for melanomagenesis was made, revealing the importance of several signaling pathways. Sinnberg et al. [116] suggest that the oncogenic transcription factor Y-box binding protein-1 (YB-1) play a pivotal role in melanoma cells. YB-1 could be a key player, activated by the signalling pathways MAPK and PI3K⁄AKT. Indeed, was demostrated that both signaling pathways are able to in‐ crease S102-phosphorylation and nuclear translocation of YB-1. It is known that S102 phosphorylated YB-1 can induce the expression of the catalytic subunit of PI3K and by

In melanoma cells, the NF-kB pathway can be altered by upregulation of the NF-kB sub‐ units p50 and RelA [118,119] and downregulation of the NF-kB inhibitor IkB [120,121]. Consequently, downstream NF-kB targets like c-myc, cyclin D1, the anti-apoptotic factor TRAF2, the invasion-associated proteins Mel-CAM or the pro-angiogenic chemokine GRO are also frequently upregulated in melanoma [122]. Recent studies have highlighted that some components of NF- kB family, such as p50 and p65/ RelA proteins, are overex‐ pressed in the nuclei of dysplastic nevi and melanoma cells compared to those of normal nevi and healthy melanocytes, respectively [123]. Other data show that a hyperactivation of NF-kB can be also caused by an increased expression of other factors involved indi‐ rectly in NF-kB pathway. Recent studies on the gene expression profile of melanoma cells have shown an increased expression of Osteopontin (OPN) [124], a secreted glyco‐ phosphoprotein that induces NF-kB activation through enhancement of the IKK activity based on phosphorylation and degradation of IkBa [125]. Indeed, OPN induces AKT phosphorylation and, in turn, phosphorylated AKT binds to IKKa/b and activates IKK complex [125]. Mutational activation of BRAF, common in human melanomas, has been also associated with an enhanced IKK activity and a concomitant increase in the rate of IkBa ubiquitination and its subsequent degradation. This process overall entails a constit‐ utive induction of NF-kB activity and an increased survival of melanoma cells [126]. Combination of these data with others reported in literature strongly suggests that the enhanced activation of NF-kB may be due to deregulations occurring in upstream signal‐

Oncogenic mutations on Ras-family members, RAS and B-RAF, have been shown to im‐ pinge at multiple levels on AKT/NF-kB, RAF/MAPK and RAL/Rho signaling pathways [127] producing survival signals to disengage cell cycle checkpoint controls, favor metastasis and

elevated PTEN activity may sensitize cells to chemotherapeutic drugs [115].

ing pathways such as RAS/RAF, PI3K/AKT and NIK [121].

apoptosis [107,108].

this increases PI3K activity [117].

Melanoma is one of a number of tumor types where p53 is still wild type, indicating that other events are contributing to p53 inactivation, in fact p53 function could be disabled by lesions that disrupt other components of the pathway. Studies using mouse models of mela‐ noma have shown that disruption of the upstream p53 regulator p14 ARF can functionally re‐ place p53 loss during melanomagenesis [91]. Analogous to the human situation, tumors arising in these mouse models present wild type p53 [91]. Moreover, the abnormal phos‐ phorylation of p53 by Chk2 kinase may contribute to the resistance of melanoma cells to ra‐ diotherapy [92]. Disruption of apoptosis downstream of p53 may alleviate pressure to mutate p53 and simultaneously decrease drug sensitivity [93]. For example, Apaf-1 and cas‐ pase 9 can be essential downstream effectors of p53-induced apoptosis and their disruption can facilitate oncogenic transformation of cultured fibroblasts [94]. In melanomas, Apaf-1 protein and mRNA expression are frequently downregulated in metastatic cell lines and tu‐ mor specimens [95]. Interestingly, Apaf-1 protein levels can be restored by addition of the methylation inhibitor 5-aza-2´-deoxycytidine (5azaCdR), suggesting that DNA methylation contributes to suppression of Apaf-1 levels. Whether methylation blocks Apaf-1 mRNA ex‐ pression directly by interfering with the recruitment of transcription factors at the Apaf-1 promoter or by affecting a regulator of Apaf-1 expression remains an open question. In any case, Apaf-1 downregulation compromises the apoptotic response of melanoma cells in re‐ sponse to p53 activation [95] or E2F-1 [96]. Restoring physiological levels of Apaf-1 through gene transfer or 5aza2dC treatment enhances chemosensitivity, alleviating cell death defects associated with reduced Apaf-1 expression [95].

In tumor cells, the selective pressure to delete or inactivate p53 is very high. This primarily occurs through mutations in p53, amplification/overexpression of its inhibitors like Mdm2, Mdm4 (Mdm2 family member) [97]. The key molecule in the p53 regulatory network is Mdm2, an E3 ubiquitin ligase with potentially oncogenic activity. Dynamic fine-tuning of the Mdm2-centered network dictates the proper rapidity, intensity, and duration of a p53 re‐ sponse, resulting in the appropriate biological outcomes [98]. Although p53 is one of the most frequently mutated tumor suppressor genes in cancer, it is mutated in only about 13% of uncultured melanoma specimens [99-101]. The absence of p53 mutations in melanoma has been attributed to the epistatic loss of ARF [101] or amplification of HDM2 [102], both of which lead to a functionally debilitating interaction between HDM2 and p53. Ji et al. have provided important data that HDM2 antagonism can effectively restore p53 function, sup‐ press melanoma growth, and synergize with MEK inhibition [103].

#### **3.3. Signaling pathways in melanoma**

In malignant melanoma, the PI3K⁄AKT signaling pathway is frequently constitutively acti‐ vated [104]. Several studies indicate that only a combinatorial inhibition of PI3K⁄AKT and MAPK signalling induces apoptosis in melanoma cells efficiently [105,106]. On the other hand, inappropriate activation of survival signaling pathways such as those mediated by mitogen-activated protein kinase (MEK)/extracellular-regulated kinase (ERK) and phosphoi‐ nositide 3-kinase (PI3K)/AKT, either as consequences of genetic alterations or resulting from environmental stimulations, is known to play a central role in the resistance of melanoma to apoptosis [107,108].

(non-functional p53), a state shared with melanoma cells [90], which are resistant to conven‐

Melanoma is one of a number of tumor types where p53 is still wild type, indicating that other events are contributing to p53 inactivation, in fact p53 function could be disabled by lesions that disrupt other components of the pathway. Studies using mouse models of mela‐ noma have shown that disruption of the upstream p53 regulator p14 ARF can functionally re‐ place p53 loss during melanomagenesis [91]. Analogous to the human situation, tumors arising in these mouse models present wild type p53 [91]. Moreover, the abnormal phos‐ phorylation of p53 by Chk2 kinase may contribute to the resistance of melanoma cells to ra‐ diotherapy [92]. Disruption of apoptosis downstream of p53 may alleviate pressure to mutate p53 and simultaneously decrease drug sensitivity [93]. For example, Apaf-1 and cas‐ pase 9 can be essential downstream effectors of p53-induced apoptosis and their disruption can facilitate oncogenic transformation of cultured fibroblasts [94]. In melanomas, Apaf-1 protein and mRNA expression are frequently downregulated in metastatic cell lines and tu‐ mor specimens [95]. Interestingly, Apaf-1 protein levels can be restored by addition of the methylation inhibitor 5-aza-2´-deoxycytidine (5azaCdR), suggesting that DNA methylation contributes to suppression of Apaf-1 levels. Whether methylation blocks Apaf-1 mRNA ex‐ pression directly by interfering with the recruitment of transcription factors at the Apaf-1 promoter or by affecting a regulator of Apaf-1 expression remains an open question. In any case, Apaf-1 downregulation compromises the apoptotic response of melanoma cells in re‐ sponse to p53 activation [95] or E2F-1 [96]. Restoring physiological levels of Apaf-1 through gene transfer or 5aza2dC treatment enhances chemosensitivity, alleviating cell death defects

In tumor cells, the selective pressure to delete or inactivate p53 is very high. This primarily occurs through mutations in p53, amplification/overexpression of its inhibitors like Mdm2, Mdm4 (Mdm2 family member) [97]. The key molecule in the p53 regulatory network is Mdm2, an E3 ubiquitin ligase with potentially oncogenic activity. Dynamic fine-tuning of the Mdm2-centered network dictates the proper rapidity, intensity, and duration of a p53 re‐ sponse, resulting in the appropriate biological outcomes [98]. Although p53 is one of the most frequently mutated tumor suppressor genes in cancer, it is mutated in only about 13% of uncultured melanoma specimens [99-101]. The absence of p53 mutations in melanoma has been attributed to the epistatic loss of ARF [101] or amplification of HDM2 [102], both of which lead to a functionally debilitating interaction between HDM2 and p53. Ji et al. have provided important data that HDM2 antagonism can effectively restore p53 function, sup‐

In malignant melanoma, the PI3K⁄AKT signaling pathway is frequently constitutively acti‐ vated [104]. Several studies indicate that only a combinatorial inhibition of PI3K⁄AKT and MAPK signalling induces apoptosis in melanoma cells efficiently [105,106]. On the other hand, inappropriate activation of survival signaling pathways such as those mediated by mitogen-activated protein kinase (MEK)/extracellular-regulated kinase (ERK) and phosphoi‐

tional modes of chemotherapy that aim to stimulate p53-dependent apoptosis.

associated with reduced Apaf-1 expression [95].

446 Melanoma - From Early Detection to Treatment

**3.3. Signaling pathways in melanoma**

press melanoma growth, and synergize with MEK inhibition [103].

One-third of primary melanomas and about 50% of metastatic melanoma cell lines showed reduced expression of PTEN as a result of allelic deletion, mutation or transcriptional silenc‐ ing [109,110], suggesting that inactivation of PTEN is a late, but frequent, event on melano‐ magenesis [111,112]. Multiple lines of evidence point to the PI3K/AKT/PTEN pathway as a putative candidate for therapeutic intervention in melanoma because PTEN overexpression can revert the invasive phenotype of human and mouse melanoma cell lines [113,114] and elevated PTEN activity may sensitize cells to chemotherapeutic drugs [115].

Recent progress in the identification of genes relevant for melanomagenesis was made, revealing the importance of several signaling pathways. Sinnberg et al. [116] suggest that the oncogenic transcription factor Y-box binding protein-1 (YB-1) play a pivotal role in melanoma cells. YB-1 could be a key player, activated by the signalling pathways MAPK and PI3K⁄AKT. Indeed, was demostrated that both signaling pathways are able to in‐ crease S102-phosphorylation and nuclear translocation of YB-1. It is known that S102 phosphorylated YB-1 can induce the expression of the catalytic subunit of PI3K and by this increases PI3K activity [117].

In melanoma cells, the NF-kB pathway can be altered by upregulation of the NF-kB sub‐ units p50 and RelA [118,119] and downregulation of the NF-kB inhibitor IkB [120,121]. Consequently, downstream NF-kB targets like c-myc, cyclin D1, the anti-apoptotic factor TRAF2, the invasion-associated proteins Mel-CAM or the pro-angiogenic chemokine GRO are also frequently upregulated in melanoma [122]. Recent studies have highlighted that some components of NF- kB family, such as p50 and p65/ RelA proteins, are overex‐ pressed in the nuclei of dysplastic nevi and melanoma cells compared to those of normal nevi and healthy melanocytes, respectively [123]. Other data show that a hyperactivation of NF-kB can be also caused by an increased expression of other factors involved indi‐ rectly in NF-kB pathway. Recent studies on the gene expression profile of melanoma cells have shown an increased expression of Osteopontin (OPN) [124], a secreted glyco‐ phosphoprotein that induces NF-kB activation through enhancement of the IKK activity based on phosphorylation and degradation of IkBa [125]. Indeed, OPN induces AKT phosphorylation and, in turn, phosphorylated AKT binds to IKKa/b and activates IKK complex [125]. Mutational activation of BRAF, common in human melanomas, has been also associated with an enhanced IKK activity and a concomitant increase in the rate of IkBa ubiquitination and its subsequent degradation. This process overall entails a constit‐ utive induction of NF-kB activity and an increased survival of melanoma cells [126]. Combination of these data with others reported in literature strongly suggests that the enhanced activation of NF-kB may be due to deregulations occurring in upstream signal‐ ing pathways such as RAS/RAF, PI3K/AKT and NIK [121].

Oncogenic mutations on Ras-family members, RAS and B-RAF, have been shown to im‐ pinge at multiple levels on AKT/NF-kB, RAF/MAPK and RAL/Rho signaling pathways [127] producing survival signals to disengage cell cycle checkpoint controls, favor metastasis and block pro-apoptotic stimuli. In support of this hypothesis, overexpression of N-RAS in hu‐ man melanoma cells enhances Bcl-2 expression and contributes to a higher tumorigenicity and drug resistance in mouse xenotransplant models (i.e. subcutaneous injections) [128]. Chin and collaborators have generated melanomas in the context of a specific genetic back‐ ground (INK4a/ARF deficiency) by conditional overexpression of H-RAS in melanocytes. Once the tumors were formed, downregulation of H-RAS expression led to a marked tumor regression by enhanced apoptosis of the tumor cells and also on the host-derived endothe‐ lial cells [129]. High-throughput analyses of genetic alterations in human cancers demon‐ strate that specifically, B-RAF, a RAS effector, was found to be mutated in 66% of human melanomas. Mutations are restricted to a few single amino-acid changes (primarily on V599) that render a constitutive active kinase with transforming properties in NIH3T3 cells [130]. Interestingly, previous studies indicate that wild-type B-RAF may inhibit programmed cell death downstream of cytocrome C release [131].

resistance factors is still unclear. In a retrospective study, the expression levels of the MMR proteins, hMSH2, hMSH6 and hMLH1, Ma et al. [137] analysed by immunohisto‐ chemistry in melanoma metastases from 64 patients, who had received dacarbazine (DTIC) based chemotherapy. All tumours showed positive nuclear staining for hMLH1. The response rates were similar in patients with hMSH2 and/or hMSH6 positive tu‐ mours to these in patients with negative tumours. In other retrospective study, Ma et al. [138] analysed the levels of the DNA repair protein *O*(6)-methylguanine-DNA methyl‐ transferase (MGMT) in melanoma metastases from patients receiving dacarbazine (DTIC) either as a single drug or as part of combination chemotherapy regimens, and related the expression levels to the clinical response to treatment. DTIC as single agent was given to 44 patients, while 21 received combination chemotherapy. Objective responses to chemo‐ therapy were seen in 12 patients, while 53 patients failed to respond to treatment. The expression of MGMT was determined according to the proportion of antibody-stained tu‐ mor cells, using a cut-off level of 50%. In 12 of the patients, more than one metastasis was analyzed, and in seven of these cases, the MGMT expression differed between tu‐ mours in the same individual. Among the responders a larger proportion (six out of 12, 50%) had tumors containing less than 50% MGMT-positive tumor cells than among the non-responders (12 out of 53, 23%). These data are consistent with the hypothesis that MGMT contributes to resistance to DTIC-based treatment. The conclusion that can be drawn from the fact that the development of drug resistance in melanoma cells is accom‐ panied by down modulation of certain components of the MMR system and by an in‐ crease in MGMT activity when *O*6-alkylating agents are applied has several far-reaching implications regarding primary and acquired clinical resistance to these drugs. Further‐ more, reduction or deficiency in MMR may increase the mutation rate in affected cells leading subsequently to an increased rate of development of resistance to other drugs having different targets. In addition, an enhanced mutation rate may contribute to in‐ creased phenotypic variation and therefore the clinical aggressiveness of melanomas and

Melanoma: Treatments and Resistance http://dx.doi.org/10.5772/54202 449

Recently, Li et al. [139] demonstrated the expression of DNA repair genes ERCC1 and XPF is induced by cisplatin in melanoma cells and that this induction is regulated by the MAPK pathway, with the role of DUSP6 phosphatase being particularly important. This induction contributes to increased drug resistance, which is one of the major obstacles to melanoma treatment, suggesting that ERCC1 or XPF inhibitors could be used to enhance the effective‐

The intrinsic multidrug resistance and sensitivity in melanomas and in pigment-producing cells involves multiple ABC transporters and melanosome biogenesis [140]. Melanoma cells express a group of ABC transporters, including ABCA9, ABCB1, ABCB5, ABCB8, ABCC1,

ABCC1 was shown to cooperate with glutathione S-transferase M1 to help melanoma cells escape the cytotoxicity of vincristine [141]. Have been described too that B16 melanoma

their metastases.

ness of cisplatin treatment.

ABCC2, and ABCD1 [140,141].

**3.5. Multidrug Resistance Proteins (MRP)**

Although >50 mutations in BRAF have now been described, the most common BRAF muta‐ tion in melanoma, accounting for 80% of all of the BRAF mutations, is a valine to glutamic acid (V600E) substitution [130,132]. Acquisition of a V600E mutation in BRAF destabilizes the inactive kinase conformation switching the equilibrium towards the active form, leading to constitutive activity [132]. Mechanistically, mutated BRAF exerts most of its oncogenic ef‐ fects through the activation of the MAPK pathway [133]. MAPK activity drives the uncon‐ trolled growth of melanoma cells by upregulating the expression of cyclin D1 and through the suppression of the cyclin dependent kinase inhibitor p27KIP1. Pre-clinical studies have shown that introduction of mutated BRAF into immortalized melanocytes leads to anchor‐ age independent growth and tumor formation in immunocompromised mice [133]. Con‐ versely, downregulation of mutated BRAF using RNAi causes cell cycle arrest and apoptosis in both *in vitro* and *in vivo* BRAFV600E mutant melanoma models [133]. Although it has been suggested that the acquisition of the BRAFV600E mutation is an early event in melanoma de‐ velopment, with 80% of all benign nevi showing to be BRAF mutant, the available evidence indicates that mutant BRAF alone cannot initiate melanoma [134,135].

#### **3.4. DNA Mismatch Repair (MMR) proteins**

Late et al. [136] determined that melanoma cells exhibiting resistance to cisplatin, etoposide and vindesine present a reduction of 30 to 70% in the nuclear content of each of the DNA mismatch repair (MMR) proteins hMLH1, hMSH2 and hMSH6. A decreased expression lev‐ el of up to 80% of mRNAs encoding hMLH1 and hMSH2 was observed in drug-resistant melanoma cells selected for cisplatin, etoposide and fotemustine. In melanoma cells that ac‐ quired resistance to fotemustine, the activity of *O*6-methylguanine-DNA methyltransferase (MGMT) was considerably enhanced. The data of this group indicate that modulation of both MMR components and MGMT expression level may contribute to the drug-resistant phenotype of melanoma cells.

DNA mismatch repair (MMR) deficiency and increased *O*6-methylguanine-DNA methyl‐ transferase (MGMT) activity have been related to resistance to *O*6-guanine methylating agents in tumour cell lines. However, the clinical relevance of MMR and MGMT as drug resistance factors is still unclear. In a retrospective study, the expression levels of the MMR proteins, hMSH2, hMSH6 and hMLH1, Ma et al. [137] analysed by immunohisto‐ chemistry in melanoma metastases from 64 patients, who had received dacarbazine (DTIC) based chemotherapy. All tumours showed positive nuclear staining for hMLH1. The response rates were similar in patients with hMSH2 and/or hMSH6 positive tu‐ mours to these in patients with negative tumours. In other retrospective study, Ma et al. [138] analysed the levels of the DNA repair protein *O*(6)-methylguanine-DNA methyl‐ transferase (MGMT) in melanoma metastases from patients receiving dacarbazine (DTIC) either as a single drug or as part of combination chemotherapy regimens, and related the expression levels to the clinical response to treatment. DTIC as single agent was given to 44 patients, while 21 received combination chemotherapy. Objective responses to chemo‐ therapy were seen in 12 patients, while 53 patients failed to respond to treatment. The expression of MGMT was determined according to the proportion of antibody-stained tu‐ mor cells, using a cut-off level of 50%. In 12 of the patients, more than one metastasis was analyzed, and in seven of these cases, the MGMT expression differed between tu‐ mours in the same individual. Among the responders a larger proportion (six out of 12, 50%) had tumors containing less than 50% MGMT-positive tumor cells than among the non-responders (12 out of 53, 23%). These data are consistent with the hypothesis that MGMT contributes to resistance to DTIC-based treatment. The conclusion that can be drawn from the fact that the development of drug resistance in melanoma cells is accom‐ panied by down modulation of certain components of the MMR system and by an in‐ crease in MGMT activity when *O*6-alkylating agents are applied has several far-reaching implications regarding primary and acquired clinical resistance to these drugs. Further‐ more, reduction or deficiency in MMR may increase the mutation rate in affected cells leading subsequently to an increased rate of development of resistance to other drugs having different targets. In addition, an enhanced mutation rate may contribute to in‐ creased phenotypic variation and therefore the clinical aggressiveness of melanomas and their metastases.

Recently, Li et al. [139] demonstrated the expression of DNA repair genes ERCC1 and XPF is induced by cisplatin in melanoma cells and that this induction is regulated by the MAPK pathway, with the role of DUSP6 phosphatase being particularly important. This induction contributes to increased drug resistance, which is one of the major obstacles to melanoma treatment, suggesting that ERCC1 or XPF inhibitors could be used to enhance the effective‐ ness of cisplatin treatment.

#### **3.5. Multidrug Resistance Proteins (MRP)**

block pro-apoptotic stimuli. In support of this hypothesis, overexpression of N-RAS in hu‐ man melanoma cells enhances Bcl-2 expression and contributes to a higher tumorigenicity and drug resistance in mouse xenotransplant models (i.e. subcutaneous injections) [128]. Chin and collaborators have generated melanomas in the context of a specific genetic back‐ ground (INK4a/ARF deficiency) by conditional overexpression of H-RAS in melanocytes. Once the tumors were formed, downregulation of H-RAS expression led to a marked tumor regression by enhanced apoptosis of the tumor cells and also on the host-derived endothe‐ lial cells [129]. High-throughput analyses of genetic alterations in human cancers demon‐ strate that specifically, B-RAF, a RAS effector, was found to be mutated in 66% of human melanomas. Mutations are restricted to a few single amino-acid changes (primarily on V599) that render a constitutive active kinase with transforming properties in NIH3T3 cells [130]. Interestingly, previous studies indicate that wild-type B-RAF may inhibit programmed cell

Although >50 mutations in BRAF have now been described, the most common BRAF muta‐ tion in melanoma, accounting for 80% of all of the BRAF mutations, is a valine to glutamic acid (V600E) substitution [130,132]. Acquisition of a V600E mutation in BRAF destabilizes the inactive kinase conformation switching the equilibrium towards the active form, leading to constitutive activity [132]. Mechanistically, mutated BRAF exerts most of its oncogenic ef‐ fects through the activation of the MAPK pathway [133]. MAPK activity drives the uncon‐ trolled growth of melanoma cells by upregulating the expression of cyclin D1 and through the suppression of the cyclin dependent kinase inhibitor p27KIP1. Pre-clinical studies have shown that introduction of mutated BRAF into immortalized melanocytes leads to anchor‐ age independent growth and tumor formation in immunocompromised mice [133]. Con‐ versely, downregulation of mutated BRAF using RNAi causes cell cycle arrest and apoptosis in both *in vitro* and *in vivo* BRAFV600E mutant melanoma models [133]. Although it has been suggested that the acquisition of the BRAFV600E mutation is an early event in melanoma de‐ velopment, with 80% of all benign nevi showing to be BRAF mutant, the available evidence

Late et al. [136] determined that melanoma cells exhibiting resistance to cisplatin, etoposide and vindesine present a reduction of 30 to 70% in the nuclear content of each of the DNA mismatch repair (MMR) proteins hMLH1, hMSH2 and hMSH6. A decreased expression lev‐ el of up to 80% of mRNAs encoding hMLH1 and hMSH2 was observed in drug-resistant melanoma cells selected for cisplatin, etoposide and fotemustine. In melanoma cells that ac‐ quired resistance to fotemustine, the activity of *O*6-methylguanine-DNA methyltransferase (MGMT) was considerably enhanced. The data of this group indicate that modulation of both MMR components and MGMT expression level may contribute to the drug-resistant

DNA mismatch repair (MMR) deficiency and increased *O*6-methylguanine-DNA methyl‐ transferase (MGMT) activity have been related to resistance to *O*6-guanine methylating agents in tumour cell lines. However, the clinical relevance of MMR and MGMT as drug

indicates that mutant BRAF alone cannot initiate melanoma [134,135].

death downstream of cytocrome C release [131].

448 Melanoma - From Early Detection to Treatment

**3.4. DNA Mismatch Repair (MMR) proteins**

phenotype of melanoma cells.

The intrinsic multidrug resistance and sensitivity in melanomas and in pigment-producing cells involves multiple ABC transporters and melanosome biogenesis [140]. Melanoma cells express a group of ABC transporters, including ABCA9, ABCB1, ABCB5, ABCB8, ABCC1, ABCC2, and ABCD1 [140,141].

ABCC1 was shown to cooperate with glutathione S-transferase M1 to help melanoma cells escape the cytotoxicity of vincristine [141]. Have been described too that B16 melanoma (B16M) cells presenting high ABCC1 and GSH content show high metastatic activity and high multidrug and radiation resistance [142]. Elevated expression of ABCC2 was shown to cause cisplatin resistance by reducing nuclear DNA damage, decreasing cell cycle G2-arrest, and increasing reentry into the cell cycle [4].

tion patterns [162, 163]. In particular, hydroxyl radicals that produce DNA lesions, such as 8-hydroxyl-2-deoxyguanosine, 8-hydroxyguanine, 8 -OHdG [164-166], and damage to the single strand of DNA [167] have been shown to decrease DNA methylation by means of interfering with the ability of DNA to function as a substrate for the DNA

Melanoma: Treatments and Resistance http://dx.doi.org/10.5772/54202 451

Oxidative stress may play different roles in the pathogenesis of melanoma and non-melano‐ ma skin cancer. It is likely that in non-melanoma skin cancers, a diminished antioxidant de‐ fense caused by chronic UV exposure contributes to the occurrence of mutations and carcinogenesis, whereas melanoma cells are equipped with a high antioxidant capacity and might use their ability to generate ROS for damaging surrounding tissue and thus support‐ ing tumour progression and metastasis [169]. Gidanian et al. showed that melanosomes de‐ rived from melanoma cells in comparison to melanocytes actively produce excessive amounts of ROS [170]. Higher intracellular levels of ROS in melanoma cells were also de‐ tected by the studies by Meyskens et al. [171]. They furthermore showed that due to these elevated levels of ROS, melanin itself becomes progressively more oxidized and starts to function as a pro‐oxidant [172]. They also showed that oxidation of melanin can be further increased by binding of metals, such as iron. These melanin‐metal complexes can be con‐ verted by the Fenton reaction thereby producing even more ROS [173]. There is supportive evidence that sustained oxidative stress is related to oxidative DNA damage [174]. Atypical melanocytes have increased levels of oxidative stress and oxidative DNA damage [175, 176]. In line with these observations, Leikam et al. found that ROS production was accompanied

The cytotoxicity of some antitumoral drugs like actinomycin-D (AMD), adriamycin (ADR), cisplatin (Cis-Pt), vincristine (VCR), cytosine arabinoside (Ara-C) and dacarbazine (DTIC) are, to a greater or lesser extent, linked to the generation of free radicals and/or to the antiox‐ idant defense of the cells. AMD and ADR are xenobiotics, which, in the cell, enter to cycles of oxidation and reduction, generating ROS [178,179]. Cis-Pt does not produce ROS; howev‐ er, during its detoxification the level of glutathione (GSH) decreases [180]. In the case of DTIC, it has been shown that the resistance of melanoma cells to that drug is also partly linked to changes in the level of GSH [17,181]. ROS generated by mitochondria intensify the

Radiotherapy is a cornerstone in the treatment of several cancers. Ionic irradiation exposes all cells to high levels of oxidative stress, thus resulting in the formation of ROS, increasing DNA damage and ultimately leading to cell death. Another mechanism of the action of radi‐ otherapy is to alter cellular homeostasis, thus modifying the signal transduction pathways and predisposing to apoptosis [183]. However, there are conflicting reports on the effect of radiotherapy on oxidative stress. Some studies have reported increased oxidative stress after radiotherapy [184], while others have reported decreased oxidative stress after radiotherapy

methyltransferases (DNMTs) and thus resulting in global hypomethylation [168].

by enhanced DNA damage [177].

in cancer patients [185, 186].

**4.1. Oxidative stress by antitumoral treatments**

apoptosis induced by cytosine arabinoside [182].

Has been reported that ABCB5 and ABCB8 mediate doxorubicin resistance in melanoma cells [143, 144]. ABCB5 shares 73% of sequence homology with the classic and the most stud‐ ied multidrug resistance protein ABCB1 (P-gp, MDR1) [145,146] and was firstly detected in tissues derived from the neuroectodermal lineage including melanocyte progenitors [145], melanoma cell lines and patient specimens [143,146-148]. In melanoma, ABCB5-expressing cells are endowed with self-renewal, differentiation and tumorigenicity abilities [149,150]. Their abundance in clinical melanoma specimens correlates positively with the neoplasic progression suggesting that ABCB5 expression is associated with tumor aggressiveness. Moreover, the growth of melanoma xenografts in mice was delayed when the animals were treated with a monoclonal anti-ABCB5 antibody [149]. As a member of the ABC transporter family, ABCB5 is thought to play a role in drug efflux. This was supported by experiments measuring the intracellular accumulation of Rhodamine 123 [145]. These data suggest that ABC proteins may be important molecular targets for the reversal of multidrug resistance in melanoma cells.

## **4. Does oxidative stress contribute to the resistance in melanoma?**

Free radicals are implicated in the pathogenesis of a multistage process of carcinogenesis. They can cause DNA base alterations, strand breaks, damage to tumor suppressor genes and enhanced expression of proto-oncogenes. The burst of reactive oxygen species (ROS) and the reactive nitrogen species (RNS) has been implicated in the development of can‐ cer [151,152]. Excessive production of ROS can be harmful to both normal and cancer cells. High levels of ROS cause damage to lipids, DNA and cellular proteins, disrupting their normal function. However, some cancer cells can develop mechanisms that use ROS for purposes such as mitogenic upregulation of the expression of antioxidant en‐ zymes [153-155]. Several studies have investigated the role of antioxidant enzymes in cancer and it has been shown that these enzymes play a significant role in regulating cancer growth and survival [156,157]. The carcinogenic effect of oxidative stress is attrib‐ uted primarily to the genotoxicity of ROS in various cellular processes [158]. For exam‐ ple, hydroxyl radicals can react with purines andor pyrimidines as well as chromatin proteins, resulting in base modifications and genomic instability which can cause altera‐ tions in gene expression [159]. These data have suggested the accumulation of ROS as a common phenomenon in many cancer cells. Such accumulations can cause direct damage to DNA by increasing the cellular mutation and/or promoting and maintaining the tu‐ morigenic phenotype by activating a second messenger in intracellular signaling cascades [160]. In addition, ROS have been determined to cause epigenetic alterations that affect the genome and play a major role in the development of carcinogenesis in humans [161]. More specifically, the production of ROS is associated with alterations in DNA methyla‐ tion patterns [162, 163]. In particular, hydroxyl radicals that produce DNA lesions, such as 8-hydroxyl-2-deoxyguanosine, 8-hydroxyguanine, 8 -OHdG [164-166], and damage to the single strand of DNA [167] have been shown to decrease DNA methylation by means of interfering with the ability of DNA to function as a substrate for the DNA methyltransferases (DNMTs) and thus resulting in global hypomethylation [168].

Oxidative stress may play different roles in the pathogenesis of melanoma and non-melano‐ ma skin cancer. It is likely that in non-melanoma skin cancers, a diminished antioxidant de‐ fense caused by chronic UV exposure contributes to the occurrence of mutations and carcinogenesis, whereas melanoma cells are equipped with a high antioxidant capacity and might use their ability to generate ROS for damaging surrounding tissue and thus support‐ ing tumour progression and metastasis [169]. Gidanian et al. showed that melanosomes de‐ rived from melanoma cells in comparison to melanocytes actively produce excessive amounts of ROS [170]. Higher intracellular levels of ROS in melanoma cells were also de‐ tected by the studies by Meyskens et al. [171]. They furthermore showed that due to these elevated levels of ROS, melanin itself becomes progressively more oxidized and starts to function as a pro‐oxidant [172]. They also showed that oxidation of melanin can be further increased by binding of metals, such as iron. These melanin‐metal complexes can be con‐ verted by the Fenton reaction thereby producing even more ROS [173]. There is supportive evidence that sustained oxidative stress is related to oxidative DNA damage [174]. Atypical melanocytes have increased levels of oxidative stress and oxidative DNA damage [175, 176]. In line with these observations, Leikam et al. found that ROS production was accompanied by enhanced DNA damage [177].

### **4.1. Oxidative stress by antitumoral treatments**

(B16M) cells presenting high ABCC1 and GSH content show high metastatic activity and high multidrug and radiation resistance [142]. Elevated expression of ABCC2 was shown to cause cisplatin resistance by reducing nuclear DNA damage, decreasing cell cycle G2-arrest,

Has been reported that ABCB5 and ABCB8 mediate doxorubicin resistance in melanoma cells [143, 144]. ABCB5 shares 73% of sequence homology with the classic and the most stud‐ ied multidrug resistance protein ABCB1 (P-gp, MDR1) [145,146] and was firstly detected in tissues derived from the neuroectodermal lineage including melanocyte progenitors [145], melanoma cell lines and patient specimens [143,146-148]. In melanoma, ABCB5-expressing cells are endowed with self-renewal, differentiation and tumorigenicity abilities [149,150]. Their abundance in clinical melanoma specimens correlates positively with the neoplasic progression suggesting that ABCB5 expression is associated with tumor aggressiveness. Moreover, the growth of melanoma xenografts in mice was delayed when the animals were treated with a monoclonal anti-ABCB5 antibody [149]. As a member of the ABC transporter family, ABCB5 is thought to play a role in drug efflux. This was supported by experiments measuring the intracellular accumulation of Rhodamine 123 [145]. These data suggest that ABC proteins may be important molecular targets for the reversal of multidrug resistance in

**4. Does oxidative stress contribute to the resistance in melanoma?**

Free radicals are implicated in the pathogenesis of a multistage process of carcinogenesis. They can cause DNA base alterations, strand breaks, damage to tumor suppressor genes and enhanced expression of proto-oncogenes. The burst of reactive oxygen species (ROS) and the reactive nitrogen species (RNS) has been implicated in the development of can‐ cer [151,152]. Excessive production of ROS can be harmful to both normal and cancer cells. High levels of ROS cause damage to lipids, DNA and cellular proteins, disrupting their normal function. However, some cancer cells can develop mechanisms that use ROS for purposes such as mitogenic upregulation of the expression of antioxidant en‐ zymes [153-155]. Several studies have investigated the role of antioxidant enzymes in cancer and it has been shown that these enzymes play a significant role in regulating cancer growth and survival [156,157]. The carcinogenic effect of oxidative stress is attrib‐ uted primarily to the genotoxicity of ROS in various cellular processes [158]. For exam‐ ple, hydroxyl radicals can react with purines andor pyrimidines as well as chromatin proteins, resulting in base modifications and genomic instability which can cause altera‐ tions in gene expression [159]. These data have suggested the accumulation of ROS as a common phenomenon in many cancer cells. Such accumulations can cause direct damage to DNA by increasing the cellular mutation and/or promoting and maintaining the tu‐ morigenic phenotype by activating a second messenger in intracellular signaling cascades [160]. In addition, ROS have been determined to cause epigenetic alterations that affect the genome and play a major role in the development of carcinogenesis in humans [161]. More specifically, the production of ROS is associated with alterations in DNA methyla‐

and increasing reentry into the cell cycle [4].

450 Melanoma - From Early Detection to Treatment

melanoma cells.

The cytotoxicity of some antitumoral drugs like actinomycin-D (AMD), adriamycin (ADR), cisplatin (Cis-Pt), vincristine (VCR), cytosine arabinoside (Ara-C) and dacarbazine (DTIC) are, to a greater or lesser extent, linked to the generation of free radicals and/or to the antiox‐ idant defense of the cells. AMD and ADR are xenobiotics, which, in the cell, enter to cycles of oxidation and reduction, generating ROS [178,179]. Cis-Pt does not produce ROS; howev‐ er, during its detoxification the level of glutathione (GSH) decreases [180]. In the case of DTIC, it has been shown that the resistance of melanoma cells to that drug is also partly linked to changes in the level of GSH [17,181]. ROS generated by mitochondria intensify the apoptosis induced by cytosine arabinoside [182].

Radiotherapy is a cornerstone in the treatment of several cancers. Ionic irradiation exposes all cells to high levels of oxidative stress, thus resulting in the formation of ROS, increasing DNA damage and ultimately leading to cell death. Another mechanism of the action of radi‐ otherapy is to alter cellular homeostasis, thus modifying the signal transduction pathways and predisposing to apoptosis [183]. However, there are conflicting reports on the effect of radiotherapy on oxidative stress. Some studies have reported increased oxidative stress after radiotherapy [184], while others have reported decreased oxidative stress after radiotherapy in cancer patients [185, 186].

## **4.2. Transcription factors Nrf1 and Nrf2 are regulators of oxidative stress signaling**

These results suggest that Nrf2 may be involved in the positive regulation of the *ABCB1*

Maher and collaborators examined the possibility that Nrf2 is also involved in the expres‐ sion levels of ABCC1 in mouse embryo fibroblasts. The constitutive expression levels of Mrp1 mRNA and protein were significantly lower in Nrf2 (-/-) cells compared with those in wild type cells. In addition, significant induction by diethyl maleate was observed in wild type, but not in Nrf2 (-/-) cells, suggesting the involvement of Nrf2 in both the constitutive and inducible mRNA and protein expression of ABCC1. In addition, the uptake of [3

dinitrophenyl-S-glutathione, a typical substrate of ABCC1, into isolated membrane vesicles also demonstrated that Nrf2 regulates the transport activity of glutathione conjugates in mouse fibroblasts [206]. In another hand, Maher evaluated whether oxidative conditions (that is, the disruption of hepatic GSH synthesis) or the administration of nuclear factor-E2 related factor-2 (Nrf2) activators (oltipraz and butylated hydroxyanisole) can induce hepatic ABC transporters and whether that induction is through the NRF2 transcriptional pathway. Livers from hepatocyte-specific glutamate-cysteine ligase catalytic subunit-null mice had in‐ creased nuclear NRF2 levels, marked gene and protein induction of the Nrf2 target gene NAD(P)H: quinone oxidoreductase 1, as well as ABCC2, ABCC3, and ABCC4 expression. The treatment of wild type and Nrf2-null mice with oltipraz and butylated hydroxyanisole demonstrated that the induction of ABCC2, ABCC3, and ABCC4 is NRF2-dependent. In Hepa1c1c7 cells treated with the Nrf2 activator tert-butyl hydroquinone, chromatin immu‐ noprecipitation with Nrf2 antibodies revealed the binding of NRF2 to antioxidant response elements in the promoter regions of mouse ABCC2 [-185 base pairs (bp)], ABCC3 (-9919 bp), and ABCC4 (-3767 bp). In this way, the activation of the Nrf2 regulatory pathway was

You et al. [207] confirmed that Nrf2 is directly involved in the basal expression of Mdm2 through the antioxidant response element, which is located in the first intron of this gene. This linkage between Nrf2 and Mdm2 appears to cause the accumulation of p53 protein in Nrf2-deficent MEFs. They also showed that ovarian carcinoma A2780 cells silenced for Nrf2 by shRNA displayed higher levels of p53 activation in response to hydrogen peroxide treat‐ ment, leading to increased cell death. Collectively, those results suggest novel evidence that the inhibition of Nrf2 can suppress Mdm2 expression, which may result in p53 signaling modulation. Thus, forced inhibition of Nrf2 expression in cancer cells may be lead to activa‐

The functional interaction between the Keap1-Nrf2 pathway and PTEN-PI3K-AKT pathway has been reported in several studies using cell lines. The pharmacological inhibition of the PI3K-AKT pathway represses the nuclear translocation of Nrf2 [208, 209]. In another hand, Beyer et al. showed that AKT phosphorylation was robustly augmented in the P/K-Alb mice in Nrf2-dependent manner, which is consistent with the previous report that Nrf2 positively

shown to stimulate the coordinated induction of hepatic ABCs [190].

tion of apoptosis response through the activation of p53 signaling.

**4.4. NRF2 represses the p53 pathway**

**4.5. Nrf2 and signalling pathways**

H]2,4-

453

Melanoma: Treatments and Resistance http://dx.doi.org/10.5772/54202

gene transcription [205].

Nrf1 (NF-E2 related factor-1) and Nrf2 (NF-E2 related factor-2) nowadays are known as two oxidative stress sensitive transcription factors that belong to the CNC/bZIP family of tran‐ scription factors consisting of NF-E2, Nrf1, Nrf2, Nrf3, BACH1, and BACH2 [45–48]. Both Nrf1 and Nrf2 are responsible for regulating the expression of many antioxidant genes in‐ cluding peroxiredoxin-1 (Prx-1), thioredoxin-1 (Txn-1), GCLC (Glutamate cysteine ligase cat‐ alytic subunit - an enzyme responsible for catalyzing the formation of glutathione), glutathione peroxidase (GPX-1), drug metabolizing enzymes (cytochrome P-450s), and sev‐ eral ATP Binding Cassette (ABC) transporters that are responsible for drug efflux [187-190]. All of these genes are essential for the maintenance of oxidative homeostasis and contain an Electrophile Response Element (EpRE) to which Nrf1 and Nrf2 bind (also known as the An‐ tioxidant Response Element). Both Nrf1 and Nrf2 are essential to the cellular response to ox‐ idative stress and several studies have shown that knockdown of Nrf1 and/or Nrf2 expression sensitizes cells to oxidative stress [191-193]. It has also been suggested that Nrf2 responds to inducible oxidative stimuli and that Nrf1 regulates oxidative stress [194]. In‐ creased oxidative stress has been shown to promote tumor proliferation and survival through deregulation of redox-sensitive pathways [153,195,196]. Nrf2 resides predominantly in the cytoplasm where it interacts with the actin-associated cytosolic protein INrf2, which is also known as Keap1 (Kelch-like ECH-associated protein 1). INrf2 functions as a substrate adaptor protein for a Cul3/Rbx1-dependent E3 ubiquitin ligase complex to ubiquitinate and degrade Nrf2, thus maintaining a steady-state level of Nrf2 [197].

Data from tumor cell lines isolated and profiled from human patients have indicated that many tumors have adapted to exploit the cytoprotective actions of Nrf2 both *in vivo* and *in vitro* through mutations of Keap1 and Nrf2, which lead to the constitutive upregulation and permanent activation of Nrf2-signaling to enhance the tolerance of the cancer cells to toxins and thereby limit the efficacy of chemotherapeutic agents. The loss of INrf2 (Keap1) function is shown to lead to nuclear accumulation of Nrf2, activation of metabolizing enzymes and drug resistance [198]. Studies have reported mutations resulting in dysfunctional Nrf2 in lung, breast and bladder cancers [199-203].

In a study carried out by Matundan et al. [204], they demonstrated the basal Nrf2 expression pattern in human melanoma was increased in 7 of 8 human melanoma cell lines. Immuno‐ blots of Nrf2 showed over-expression in 6 of 8 metastatic melanoma cell lines and they de‐ termined that Nrf2's contribution was protective against redox stress in melanoma, and that decreased Nrf2 activation sensitizes melanoma cell lines to existing chemotherapeutics [204].

#### **4.3. NRF2 are related with the expression of multidrug resistance proteins**

Ogura and colleagues reported previously that Nrf2 binds within the *ABCB1* promoter's -126 and -102 regions, which contain the ATTCAGTCA motif. They have purified Nrf2 from the nuclear extract of K562/ADM cells, a multidrug-resistant cell line derived from human myelogenous leukemia K562 cells. This group determined that ATTCAGTCA motif is a pos‐ itive regulatory element of MDR1 gene and that the motif is important for Nrf2 binding. These results suggest that Nrf2 may be involved in the positive regulation of the *ABCB1* gene transcription [205].

Maher and collaborators examined the possibility that Nrf2 is also involved in the expres‐ sion levels of ABCC1 in mouse embryo fibroblasts. The constitutive expression levels of Mrp1 mRNA and protein were significantly lower in Nrf2 (-/-) cells compared with those in wild type cells. In addition, significant induction by diethyl maleate was observed in wild type, but not in Nrf2 (-/-) cells, suggesting the involvement of Nrf2 in both the constitutive and inducible mRNA and protein expression of ABCC1. In addition, the uptake of [3 H]2,4 dinitrophenyl-S-glutathione, a typical substrate of ABCC1, into isolated membrane vesicles also demonstrated that Nrf2 regulates the transport activity of glutathione conjugates in mouse fibroblasts [206]. In another hand, Maher evaluated whether oxidative conditions (that is, the disruption of hepatic GSH synthesis) or the administration of nuclear factor-E2 related factor-2 (Nrf2) activators (oltipraz and butylated hydroxyanisole) can induce hepatic ABC transporters and whether that induction is through the NRF2 transcriptional pathway. Livers from hepatocyte-specific glutamate-cysteine ligase catalytic subunit-null mice had in‐ creased nuclear NRF2 levels, marked gene and protein induction of the Nrf2 target gene NAD(P)H: quinone oxidoreductase 1, as well as ABCC2, ABCC3, and ABCC4 expression. The treatment of wild type and Nrf2-null mice with oltipraz and butylated hydroxyanisole demonstrated that the induction of ABCC2, ABCC3, and ABCC4 is NRF2-dependent. In Hepa1c1c7 cells treated with the Nrf2 activator tert-butyl hydroquinone, chromatin immu‐ noprecipitation with Nrf2 antibodies revealed the binding of NRF2 to antioxidant response elements in the promoter regions of mouse ABCC2 [-185 base pairs (bp)], ABCC3 (-9919 bp), and ABCC4 (-3767 bp). In this way, the activation of the Nrf2 regulatory pathway was shown to stimulate the coordinated induction of hepatic ABCs [190].

#### **4.4. NRF2 represses the p53 pathway**

**4.2. Transcription factors Nrf1 and Nrf2 are regulators of oxidative stress signaling**

degrade Nrf2, thus maintaining a steady-state level of Nrf2 [197].

lung, breast and bladder cancers [199-203].

452 Melanoma - From Early Detection to Treatment

Nrf1 (NF-E2 related factor-1) and Nrf2 (NF-E2 related factor-2) nowadays are known as two oxidative stress sensitive transcription factors that belong to the CNC/bZIP family of tran‐ scription factors consisting of NF-E2, Nrf1, Nrf2, Nrf3, BACH1, and BACH2 [45–48]. Both Nrf1 and Nrf2 are responsible for regulating the expression of many antioxidant genes in‐ cluding peroxiredoxin-1 (Prx-1), thioredoxin-1 (Txn-1), GCLC (Glutamate cysteine ligase cat‐ alytic subunit - an enzyme responsible for catalyzing the formation of glutathione), glutathione peroxidase (GPX-1), drug metabolizing enzymes (cytochrome P-450s), and sev‐ eral ATP Binding Cassette (ABC) transporters that are responsible for drug efflux [187-190]. All of these genes are essential for the maintenance of oxidative homeostasis and contain an Electrophile Response Element (EpRE) to which Nrf1 and Nrf2 bind (also known as the An‐ tioxidant Response Element). Both Nrf1 and Nrf2 are essential to the cellular response to ox‐ idative stress and several studies have shown that knockdown of Nrf1 and/or Nrf2 expression sensitizes cells to oxidative stress [191-193]. It has also been suggested that Nrf2 responds to inducible oxidative stimuli and that Nrf1 regulates oxidative stress [194]. In‐ creased oxidative stress has been shown to promote tumor proliferation and survival through deregulation of redox-sensitive pathways [153,195,196]. Nrf2 resides predominantly in the cytoplasm where it interacts with the actin-associated cytosolic protein INrf2, which is also known as Keap1 (Kelch-like ECH-associated protein 1). INrf2 functions as a substrate adaptor protein for a Cul3/Rbx1-dependent E3 ubiquitin ligase complex to ubiquitinate and

Data from tumor cell lines isolated and profiled from human patients have indicated that many tumors have adapted to exploit the cytoprotective actions of Nrf2 both *in vivo* and *in vitro* through mutations of Keap1 and Nrf2, which lead to the constitutive upregulation and permanent activation of Nrf2-signaling to enhance the tolerance of the cancer cells to toxins and thereby limit the efficacy of chemotherapeutic agents. The loss of INrf2 (Keap1) function is shown to lead to nuclear accumulation of Nrf2, activation of metabolizing enzymes and drug resistance [198]. Studies have reported mutations resulting in dysfunctional Nrf2 in

In a study carried out by Matundan et al. [204], they demonstrated the basal Nrf2 expression pattern in human melanoma was increased in 7 of 8 human melanoma cell lines. Immuno‐ blots of Nrf2 showed over-expression in 6 of 8 metastatic melanoma cell lines and they de‐ termined that Nrf2's contribution was protective against redox stress in melanoma, and that decreased Nrf2 activation sensitizes melanoma cell lines to existing chemotherapeutics [204].

Ogura and colleagues reported previously that Nrf2 binds within the *ABCB1* promoter's -126 and -102 regions, which contain the ATTCAGTCA motif. They have purified Nrf2 from the nuclear extract of K562/ADM cells, a multidrug-resistant cell line derived from human myelogenous leukemia K562 cells. This group determined that ATTCAGTCA motif is a pos‐ itive regulatory element of MDR1 gene and that the motif is important for Nrf2 binding.

**4.3. NRF2 are related with the expression of multidrug resistance proteins**

You et al. [207] confirmed that Nrf2 is directly involved in the basal expression of Mdm2 through the antioxidant response element, which is located in the first intron of this gene. This linkage between Nrf2 and Mdm2 appears to cause the accumulation of p53 protein in Nrf2-deficent MEFs. They also showed that ovarian carcinoma A2780 cells silenced for Nrf2 by shRNA displayed higher levels of p53 activation in response to hydrogen peroxide treat‐ ment, leading to increased cell death. Collectively, those results suggest novel evidence that the inhibition of Nrf2 can suppress Mdm2 expression, which may result in p53 signaling modulation. Thus, forced inhibition of Nrf2 expression in cancer cells may be lead to activa‐ tion of apoptosis response through the activation of p53 signaling.

#### **4.5. Nrf2 and signalling pathways**

The functional interaction between the Keap1-Nrf2 pathway and PTEN-PI3K-AKT pathway has been reported in several studies using cell lines. The pharmacological inhibition of the PI3K-AKT pathway represses the nuclear translocation of Nrf2 [208, 209]. In another hand, Beyer et al. showed that AKT phosphorylation was robustly augmented in the P/K-Alb mice in Nrf2-dependent manner, which is consistent with the previous report that Nrf2 positively regulates the activation of AKT [210]. Recently, Mitsuishi et al. [211] demonstrated a contri‐ bution of Nrf2 to cellular metabolic activities in proliferating cells, and the positive feedback loop between the PTEN-PI3K-AKT and Keap1-Nrf2 pathways, which appears to be one of the most substantial mechanisms for promoting the malignant evolution of cancers. It should be noted that Nrf2 accumulation, which is achieved by the functional impairment of Keap1 combined with the sustained activation of PI3K-AKT pathway, allows Nrf2 to get in‐ volved in the modulation of metabolism under pathological conditions. In contrast, tempo‐ rary accumulation of Nrf2 at a low level is sufficient for Nrf2 to exert the cytoprotective function under physiological conditions [211].

ered a double-edged sword because it participates in the regulation of oxidative stress, however has been shown that overexpression of Nrf2 is a common phenomenon in several cancer types, participating in chemoresistance and tumor survival. We assume that this phe‐ nomenon also overlaps in melanoma, thus the intrinsic or extrinsic resistance produced in melanoma cells is partly due to overexpression of Nrf2, which can promote cell survival through mechanisms already reviewed in this chapter. Although these mechanisms present‐ ed in the last part of this chapter were not studied in melanoma, we believe that future stud‐ ies endorse our theory. The knowledge about melanoma treatment has been widespread in recent years, but still is not enough, hence we must deepen in this area in order to improve the existing treatments and create effective targeted therapeutic target against this disease.

Melanoma: Treatments and Resistance http://dx.doi.org/10.5772/54202 455

**Acknowledgements**

**Author details**

**References**

Jonathan Castillo Arias1

This work was supported by FAPESP (2011/12306-1).

\*Address all correspondence to: mjasiulionis@gmail.com

tional du cancer. 1999;81(4):555-9. Epub 1999/05/04.

1996;22(3):217-26. Epub 1996/03/01.

Universidad Austral de Chile, Valdivia, Chile

and Miriam Galvonas Jasiulionis2

2 Pharmacology Department, Universidade Federal de São Paulo, São Paulo, Brazil

1 Pharmacological Biochemistry Laboratory, Institute of Biochemistry, Faculty of Sciences,

[1] Karagas MR, Greenberg ER, Spencer SK, Stukel TA, Mott LA. Increase in incidence rates of basal cell and squamous cell skin cancer in New Hampshire, USA. New Hampshire Skin Cancer Study Group. International journal of cancer Journal interna‐

[2] Gloster HM, Jr., Brodland DG. The epidemiology of skin cancer. Dermatologic sur‐ gery : official publication for American Society for Dermatologic Surgery [et al].

[3] Plesko I, Severi G, Obsitnikova A, Boyle P. Trends in the incidence of non-melanoma skin cancer in Slovakia, 1978-1995. Neoplasma. 2000;47(3):137-42. Epub 2000/10/24. [4] Liedert B, Materna V, Schadendorf D, Thomale J, Lage H. Overexpression of cMOAT (MRP2/ABCC2) is associated with decreased formation of platinum-DNA adducts

Su et al. [212] reported the first evidence that Nrf2 is phosphorylated by MAPKs *in vivo,* however the nuclear accumulation of Nrf2 was slightly enhanced by its phosphorylation. This group concluded that direct phosphorylation of Nrf2 by MAPKs has a limited contribu‐ tion in regulating the Nrf2-dependent antioxidant responses.

#### **4.6. Nrf2 and anti-apoptotic features**

Nrf2 resides predominantly in the cytoplasm where it interacts with the actin-associated cytosolic protein INrf2, which is also known as Keap1 (Kelch-like ECH-associated pro‐ tein 1). INrf2 functions as a substrate adaptor protein for a Cul3/Rbx1-dependent E3 ubiquitin ligase complex to ubiquitinate and degrade Nrf2, thus maintaining a steadystate level of Nrf2 [197]. A study conducted by Niture et al. demonstrated that INrf2, in association with Cul3/Rbx1, ubiquitinates and degrades Bcl-2 [213]. However they recent‐ ly demonstrated that Nrf2 binds to Bcl-2 ARE and regulates expression and induction of the Bcl-2 gene. Nrf2 mediated the up-regulation of Bcl-2, down regulated the activity of pro-apoptotic Bax protein and caspases 3/7, and protected cells from etoposide/radiationmediated apoptosis that leads to drug resistance. Thus, they demonstrate that Nrf2-medi‐ ated up-regulation of Bcl-2 plays a significant role in preventing apoptosis, increasing cell survival, and drug resistance [214].

## **5. Conclusion**

Melanoma continues to increase in incidence in many parts of the world, but there is cur‐ rently no curative treatment once the disease has spread beyond the primary site because of the absence of effective therapies. This is believed to be largely due to the resistance of mela‐ noma cells to induction of apoptosis by available chemotherapeutic drugs and biological re‐ agents. Drug resistance is likely not only a primary consequence of acquired genetic alterations selected during or after therapy, but rather inherent to the malignant behavior of melanoma cells at diagnosis. Data support the existing hypothesis that talks about melano‐ ma cells are "born to survive". Their aggressive behavior stems from intrinsic survival fea‐ tures of their paternal melanocytes nourished by additional alterations acquired during tumor progression. These inherent survival mechanisms may be partly caused by the oxida‐ tive stress to which melanoma cells are exposed. Nrf2 is a transcription factor that is consid‐ ered a double-edged sword because it participates in the regulation of oxidative stress, however has been shown that overexpression of Nrf2 is a common phenomenon in several cancer types, participating in chemoresistance and tumor survival. We assume that this phe‐ nomenon also overlaps in melanoma, thus the intrinsic or extrinsic resistance produced in melanoma cells is partly due to overexpression of Nrf2, which can promote cell survival through mechanisms already reviewed in this chapter. Although these mechanisms present‐ ed in the last part of this chapter were not studied in melanoma, we believe that future stud‐ ies endorse our theory. The knowledge about melanoma treatment has been widespread in recent years, but still is not enough, hence we must deepen in this area in order to improve the existing treatments and create effective targeted therapeutic target against this disease.

## **Acknowledgements**

This work was supported by FAPESP (2011/12306-1).

## **Author details**

regulates the activation of AKT [210]. Recently, Mitsuishi et al. [211] demonstrated a contri‐ bution of Nrf2 to cellular metabolic activities in proliferating cells, and the positive feedback loop between the PTEN-PI3K-AKT and Keap1-Nrf2 pathways, which appears to be one of the most substantial mechanisms for promoting the malignant evolution of cancers. It should be noted that Nrf2 accumulation, which is achieved by the functional impairment of Keap1 combined with the sustained activation of PI3K-AKT pathway, allows Nrf2 to get in‐ volved in the modulation of metabolism under pathological conditions. In contrast, tempo‐ rary accumulation of Nrf2 at a low level is sufficient for Nrf2 to exert the cytoprotective

Su et al. [212] reported the first evidence that Nrf2 is phosphorylated by MAPKs *in vivo,* however the nuclear accumulation of Nrf2 was slightly enhanced by its phosphorylation. This group concluded that direct phosphorylation of Nrf2 by MAPKs has a limited contribu‐

Nrf2 resides predominantly in the cytoplasm where it interacts with the actin-associated cytosolic protein INrf2, which is also known as Keap1 (Kelch-like ECH-associated pro‐ tein 1). INrf2 functions as a substrate adaptor protein for a Cul3/Rbx1-dependent E3 ubiquitin ligase complex to ubiquitinate and degrade Nrf2, thus maintaining a steadystate level of Nrf2 [197]. A study conducted by Niture et al. demonstrated that INrf2, in association with Cul3/Rbx1, ubiquitinates and degrades Bcl-2 [213]. However they recent‐ ly demonstrated that Nrf2 binds to Bcl-2 ARE and regulates expression and induction of the Bcl-2 gene. Nrf2 mediated the up-regulation of Bcl-2, down regulated the activity of pro-apoptotic Bax protein and caspases 3/7, and protected cells from etoposide/radiationmediated apoptosis that leads to drug resistance. Thus, they demonstrate that Nrf2-medi‐ ated up-regulation of Bcl-2 plays a significant role in preventing apoptosis, increasing

Melanoma continues to increase in incidence in many parts of the world, but there is cur‐ rently no curative treatment once the disease has spread beyond the primary site because of the absence of effective therapies. This is believed to be largely due to the resistance of mela‐ noma cells to induction of apoptosis by available chemotherapeutic drugs and biological re‐ agents. Drug resistance is likely not only a primary consequence of acquired genetic alterations selected during or after therapy, but rather inherent to the malignant behavior of melanoma cells at diagnosis. Data support the existing hypothesis that talks about melano‐ ma cells are "born to survive". Their aggressive behavior stems from intrinsic survival fea‐ tures of their paternal melanocytes nourished by additional alterations acquired during tumor progression. These inherent survival mechanisms may be partly caused by the oxida‐ tive stress to which melanoma cells are exposed. Nrf2 is a transcription factor that is consid‐

function under physiological conditions [211].

**4.6. Nrf2 and anti-apoptotic features**

454 Melanoma - From Early Detection to Treatment

cell survival, and drug resistance [214].

**5. Conclusion**

tion in regulating the Nrf2-dependent antioxidant responses.

Jonathan Castillo Arias1 and Miriam Galvonas Jasiulionis2

\*Address all correspondence to: mjasiulionis@gmail.com

1 Pharmacological Biochemistry Laboratory, Institute of Biochemistry, Faculty of Sciences, Universidad Austral de Chile, Valdivia, Chile

2 Pharmacology Department, Universidade Federal de São Paulo, São Paulo, Brazil

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**Chapter 17**

**Management of Acral Lentiginous Melanoma**

Cutaneous malignant melanoma is the most common cause of mortality from skin cancers in Caucasian populations. The incidence rates of malignant melanoma show considerable varia‐ tion worldwide. Annual incidence rates per 100,000 people vary between about 40 in Australia and New Zealand to about 20 in the United States [1,2]. In contrast, a significantly lower inci‐ dence rate has been reported in Asian populations with rates of 0.65 to 1/100,000 [3-5]. In addi‐ tion, the most common sites of melanoma occurrence in Asians are the extremities at a rate of

In 1976, RJ Reed first described the fourth variant of melanoma as "pigmented lesions on the extremities, particularly on palmoplantar regions, that are characterized by a lentiginous (ra‐ dial) growth phase evolving over months or years to a dermal (vertical) invasive stage" [9]. He named this anatomical subgroup of melanoma as "plantar lentiginous melanoma (PLM) ", which had a characteristic lentiginous, radial component of melanocytic prolifera‐ tion and mentioned for the first time that this subgroup was the most common in Blacks and

In 1986, malignant melanoma was classified into four subtypes by Clark et al. according to histological features; nodular melanoma (NM), superficial spreading melanoma (SSM), len‐ tigo maligna melanoma (LMM) and acral lentiginous melanoma (ALM) [11]. In the United States, the incidence rates of SSM, NM, LMM and ALM are approximately 70%, 15%, 13% and 2-3% respectively [12,13]. Although, ALM is the most common expression of malignant melanoma in Asian and Black populations, the rate of ALM is 41% in Japan [14], 65% in Ko‐

The prognosis of each subtype differs due to delayed diagnosis rather than an actual differen‐ ces in the biological nature of tumour and the prognosis of ALM is generally poorer than other subtypes [17]. The lesion, especially on soles and nail beds, is likely to be overlooked by pa‐

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

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

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

about 50% of all cases [6,7], compared to only 2-3% in Caucasian populations [8].

Yoshitaka Kai and Sakuhei Fujiwara

http://dx.doi.org/10.5772/54266

the very poor prognosis group [10].

rea [15] and 62% in the American Blacks [16].

**1. Introduction**

Additional information is available at the end of the chapter

## **Management of Acral Lentiginous Melanoma**

Yoshitaka Kai and Sakuhei Fujiwara

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54266

## **1. Introduction**

Cutaneous malignant melanoma is the most common cause of mortality from skin cancers in Caucasian populations. The incidence rates of malignant melanoma show considerable varia‐ tion worldwide. Annual incidence rates per 100,000 people vary between about 40 in Australia and New Zealand to about 20 in the United States [1,2]. In contrast, a significantly lower inci‐ dence rate has been reported in Asian populations with rates of 0.65 to 1/100,000 [3-5]. In addi‐ tion, the most common sites of melanoma occurrence in Asians are the extremities at a rate of about 50% of all cases [6,7], compared to only 2-3% in Caucasian populations [8].

In 1976, RJ Reed first described the fourth variant of melanoma as "pigmented lesions on the extremities, particularly on palmoplantar regions, that are characterized by a lentiginous (ra‐ dial) growth phase evolving over months or years to a dermal (vertical) invasive stage" [9]. He named this anatomical subgroup of melanoma as "plantar lentiginous melanoma (PLM) ", which had a characteristic lentiginous, radial component of melanocytic prolifera‐ tion and mentioned for the first time that this subgroup was the most common in Blacks and the very poor prognosis group [10].

In 1986, malignant melanoma was classified into four subtypes by Clark et al. according to histological features; nodular melanoma (NM), superficial spreading melanoma (SSM), len‐ tigo maligna melanoma (LMM) and acral lentiginous melanoma (ALM) [11]. In the United States, the incidence rates of SSM, NM, LMM and ALM are approximately 70%, 15%, 13% and 2-3% respectively [12,13]. Although, ALM is the most common expression of malignant melanoma in Asian and Black populations, the rate of ALM is 41% in Japan [14], 65% in Ko‐ rea [15] and 62% in the American Blacks [16].

The prognosis of each subtype differs due to delayed diagnosis rather than an actual differen‐ ces in the biological nature of tumour and the prognosis of ALM is generally poorer than other subtypes [17]. The lesion, especially on soles and nail beds, is likely to be overlooked by pa‐

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

tients. Moreover, Metzger et al. found that ALM had a high likelihood of being clinically mis‐ diagnosed as a benign melanocytic lesion, which leads to a delay in the initiation of treatment [18]. However, this report was made in the pre-dermoscopic era and now it has become much easier with dermoscopy to distinguish the early stage of ALM from a benign lesion.

tral plaque-like thickening of malignant cells in the papillary dermis, with (3) extension of the spindle cells into the deeper levels, accompanied by prominent dysplasia; (4) there is ep‐ idermal hyperplasia with elongation of the rete ridges and acanthosis and central ulceration;

> Thigh 1 (1.6) Lower leg 6 (9.8) Dorsum of foot 1 (1.6) Sole 23 (37.7) Toe 8 (13.1) Toenail 2 (3.3) Total 41

Upper arm 0 (0) Forearm 1 (1.6) Dorsum of Hand 1 (1.6) Palm 4 (6.6) Finger 4 (6.6) Fingernail 9 (14.8) Total 19

Dermoscopic observations help the diagnosis in the early stage of ALM. In 66 cases of volar skin melanomas, irregular diffuse pigmentation (60%) with variable shades from tan to black without parallel disposition of pigment (figure 1a) and the parallel ridge pattern (53%) with pigmentation along the ridges (figure 1b) were the two most prevalent patterns [23]. According to Saida et al., the sensitivity and specificity of the parallel ridge pattern in diag‐ nosing all melanoma on volar skins were 86% and 99% respectively and those of irregular diffuse pigmentation were 85% and 97% respectively. Only in diagnosing melanoma in situ on volar skin, the sensitivity of parallel ridge pattern (86%) was significantly higher than that of irregular diffuse pigmentation (69%) [24]. A parallel furrow pattern with pigmenta‐ tion along the furrows (figure 1c) and a lattice-like pattern with longitudinal and transversal thicker lines surrounding the eccrine pores (figure 1d) are more common in melanocytic ne‐ vi. The sensitivity and specificity of a parallel furrow pattern or lattice-like pattern in diag‐

Unknown 1 (1.6) Total 61 (100)

nosing melanocytic nevi were 67% and 93% respectively [24].

**Table 1.** Primary sites of cutaneous melanoma experienced in our institute from 2004 to 2011

**Location Case Number (%)**

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and (5) host immune response is active, with areas of tumor regression" [22].

Lower extremities

Upper extremities

Human extremities, especially palms and soles, are not exposed to ultraviolet light and there is no evidence of overexposure to UV light as a risk factor of ALM [19]. In contrast, UV light plays an important role in the pathogenesis of LMM.

In 2005, Bastian et al. proposed new classification of melanoma according to genetic altera‐ tions at different sites. They classified melanoma into four distinct groups, each of which has a different degree of exposure to UV light: chronic sun-damaged melanoma (CSD) which nearly corresponds to LMM, non-CSD melanoma which also corresponds to SSM, acral mel‐ anoma (AM) which also corresponds to ALM, and mucosal melanoma [20]. They found that 81% of non-CSD melanoma had mutations in BRAF or N-RAS and the other groups had no mutations in either gene. Otherwise, melanoma with wild-type BRAF or N-RAS frequently had an increase in the number of copies of the genes for cyclin-dependent kinase 4 (CDK4) and cyclin D1 (CCND1). Furthermore, a recent study showed that AM and mucosal melano‐ ma had frequent mutation or amplification of the KIT gene [21]. Although these findings have led to molecular targeted therapy today, this new therapeutic approach has just begun and therefore we will only touch upon these new directions.

Today, there are some difficulties and controversies in the treatment of ALM caused by the anatomical and biological specificity of ALM. The standardized treatment of ALM is not easy to establish due to the unique characteristics. This chapter includes our experiences and a review of the literature focusing on the surgical treatment of ALM, and in particular, dis‐ cusses the controversies surrounding the treatment of ALM.

### **Clinical presentation and dermoscopic findings of ALM**

ALM occurs more frequently on lower extremities than on upper extremities. In our insti‐ tute, 41 cases of all 61 ALM cases occurred on lower extremities. The soles of the feet are the most frequent sites of ALM, where 56% of ALM on lower extremities occurred. In contrast, most frequent sites on upper extremities are fingernails, where 45% of ALM on upper ex‐ tremities appeared.

Clinically, ALMs begin with pale brown macules, enlarge slowly and form irregularly pig‐ mented, asymmetric macular lesions with notching at the periphery over the years. After that, nodules appear on the pigmented lesion and form ulceration. In the past ALM was considered to occur from benign malanocytic lesions, however, de novo synthesis in major cases of ALM has been confirmed by dermoscopic findings (see below). Due to the very slow progress, it tends to be overlooked and even when the tumour becomes larger, it is easily underestimated.

Histologically, "(1) the radial growth phase consists of lentiginous dysplastic melanocytes, extending along the basal cell layer, with extension of single atypical melanocytes up into the thickened epidermis; (2) the vertical growth phase usually consists of a progressive cen‐ tral plaque-like thickening of malignant cells in the papillary dermis, with (3) extension of the spindle cells into the deeper levels, accompanied by prominent dysplasia; (4) there is ep‐ idermal hyperplasia with elongation of the rete ridges and acanthosis and central ulceration; and (5) host immune response is active, with areas of tumor regression" [22].

tients. Moreover, Metzger et al. found that ALM had a high likelihood of being clinically mis‐ diagnosed as a benign melanocytic lesion, which leads to a delay in the initiation of treatment [18]. However, this report was made in the pre-dermoscopic era and now it has become much

Human extremities, especially palms and soles, are not exposed to ultraviolet light and there is no evidence of overexposure to UV light as a risk factor of ALM [19]. In contrast, UV light

In 2005, Bastian et al. proposed new classification of melanoma according to genetic altera‐ tions at different sites. They classified melanoma into four distinct groups, each of which has a different degree of exposure to UV light: chronic sun-damaged melanoma (CSD) which nearly corresponds to LMM, non-CSD melanoma which also corresponds to SSM, acral mel‐ anoma (AM) which also corresponds to ALM, and mucosal melanoma [20]. They found that 81% of non-CSD melanoma had mutations in BRAF or N-RAS and the other groups had no mutations in either gene. Otherwise, melanoma with wild-type BRAF or N-RAS frequently had an increase in the number of copies of the genes for cyclin-dependent kinase 4 (CDK4) and cyclin D1 (CCND1). Furthermore, a recent study showed that AM and mucosal melano‐ ma had frequent mutation or amplification of the KIT gene [21]. Although these findings have led to molecular targeted therapy today, this new therapeutic approach has just begun

Today, there are some difficulties and controversies in the treatment of ALM caused by the anatomical and biological specificity of ALM. The standardized treatment of ALM is not easy to establish due to the unique characteristics. This chapter includes our experiences and a review of the literature focusing on the surgical treatment of ALM, and in particular, dis‐

ALM occurs more frequently on lower extremities than on upper extremities. In our insti‐ tute, 41 cases of all 61 ALM cases occurred on lower extremities. The soles of the feet are the most frequent sites of ALM, where 56% of ALM on lower extremities occurred. In contrast, most frequent sites on upper extremities are fingernails, where 45% of ALM on upper ex‐

Clinically, ALMs begin with pale brown macules, enlarge slowly and form irregularly pig‐ mented, asymmetric macular lesions with notching at the periphery over the years. After that, nodules appear on the pigmented lesion and form ulceration. In the past ALM was considered to occur from benign malanocytic lesions, however, de novo synthesis in major cases of ALM has been confirmed by dermoscopic findings (see below). Due to the very slow progress, it tends to be overlooked and even when the tumour becomes larger, it is

Histologically, "(1) the radial growth phase consists of lentiginous dysplastic melanocytes, extending along the basal cell layer, with extension of single atypical melanocytes up into the thickened epidermis; (2) the vertical growth phase usually consists of a progressive cen‐

easier with dermoscopy to distinguish the early stage of ALM from a benign lesion.

plays an important role in the pathogenesis of LMM.

476 Melanoma - From Early Detection to Treatment

and therefore we will only touch upon these new directions.

cusses the controversies surrounding the treatment of ALM.

**Clinical presentation and dermoscopic findings of ALM**

tremities appeared.

easily underestimated.


**Table 1.** Primary sites of cutaneous melanoma experienced in our institute from 2004 to 2011

Dermoscopic observations help the diagnosis in the early stage of ALM. In 66 cases of volar skin melanomas, irregular diffuse pigmentation (60%) with variable shades from tan to black without parallel disposition of pigment (figure 1a) and the parallel ridge pattern (53%) with pigmentation along the ridges (figure 1b) were the two most prevalent patterns [23]. According to Saida et al., the sensitivity and specificity of the parallel ridge pattern in diag‐ nosing all melanoma on volar skins were 86% and 99% respectively and those of irregular diffuse pigmentation were 85% and 97% respectively. Only in diagnosing melanoma in situ on volar skin, the sensitivity of parallel ridge pattern (86%) was significantly higher than that of irregular diffuse pigmentation (69%) [24]. A parallel furrow pattern with pigmenta‐ tion along the furrows (figure 1c) and a lattice-like pattern with longitudinal and transversal thicker lines surrounding the eccrine pores (figure 1d) are more common in melanocytic ne‐ vi. The sensitivity and specificity of a parallel furrow pattern or lattice-like pattern in diag‐ nosing melanocytic nevi were 67% and 93% respectively [24].

es occurred on the thumbnail, 27% on the nail of the great toe, 2-4% on the nail of the index,

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It is known that subungual melanoma has a very poor prognosis among all subtypes of cu‐ taneous melanomas. The reason for this is because the majority of subungual melanomas are already been quite deep when diagnosed [28]. Delayed diagnosis of subungual melanoma is common because it is very difficult to distinguish the early stage of subungual melanoma from longitudinal melanonychia. According to Cohen et al., 38 of 43 patients (88%) had de‐

Subungual melanomas begin with fine pigmented striata which could not be clinically dis‐ tinguished from benign longitudinal melanonychia at an early stage and grow wider with colour variegation and the presence of nail plate fissuring or splitting eventually forming a triangular shape which has a broader proximal lesion rather than a distal lesion, blurred lat‐ eral borders and Hutchinson's sign - indicating the peripheral pigmentation beyond the nail

Baran et al. mentioned the clinical clues to the diagnosis of subungual melanoma in detail. Hutchinson's sign is the most important sign of subungual melanoma. Other clues are when longitudinal melanonychia (a) begins in a single digit of a person over six decades or more, (b) develops abruptly in a previous normal nail plate, (c) becomes suddenly darker or wider, (d) occurs in either the thumb, index finger or giant toe, (e) is accompanied by nail destruc‐

In addition to these clinical clues, dermoscopy provides useful information for the diagnosis of subungual melanoma. The prominent dermoscopic features of subungual melanoma are brown pigmentation of the background with longitudinal brown to black lines which are ir‐ regular in their colouration, spacing, thickness and parallelism [32]. This irregularity was significantly associated with melanoma when compared with all other benign diseases. The micro-Hutchinson's sign is the suspicious dermoscopic feature, which consists of the irregu‐

Since the early stage of subungual melanoma has minimal histopathological change, it may be difficult to distinguish subungual melanoma from benign lesion with only histopatholog‐ ical findings. Thus, both clinical features, including present history and histopathological

We propose a diagnostic algorithm for the early stage of subungual melanoma (figure 2). When a case falls under any of the clinical features mentioned above (a-g), dermoscopic ex‐ amination is recommended. If Hutchinson's sign and colour change in overall nail to dark black are present, excisional biopsy is recommended. When those characteristic appearances are absent, but a nail streak has irregularity, biopsy is also recommended. On the contrary, when nail streaks are monotonous pale brown, subungual melanoma is not suspicious. Even if a streak is dark brown or black, no irregularity of lines on dermoscopy allows careful fol‐ low-up without biopsies. If the streak increases in width or has colour variegation during a period of follow-up, the necessity of excision or biopsy should be discussed according to

tion or disappearance, (f) has colour variegation, (g) has a wide band and so on [31].

lar lines in the cuticle area and can be observed only on dermoscopy [32,33].

findings, are necessary for diagnosis.

further dermoscopic examination.

layed diagnosis and the median delay time was 24 months (range 4 to 132) [29].

middle and ring finger, 1.6% on the nail of the second toe [27].

apparatus [30].

**Figure 1.** Dermoscopy of early stage ALM (a,b) and melanocytic nevi (c,d). (a) Irregular diffuse pigmentation, (b) paral‐ lel ridge pattern, (c) parallel furrow pattern, (d) lattice-like pattern with longitudinal and transversal thicker lines sur‐ rounding the eccrine pores (arrows).

Ulceration on nodules following pigmented macules reveals a polymorphous vascular pat‐ tern with a combination of milky-red areas (95%) which are larger areas of fuzzy or unfo‐ cused milky-red colour corresponding to an elevated part of the lesion, linear irregular vessels (49%), dotted vessels (43%) and hairpin vessels (41%) [25].

## **2. Subungual melanoma**

The incidence of subungual melanoma also has a racial difference. It is more frequent in Asian and Blacks than in Caucasians. Its frequency has been reported to be approximately 2-4% in all cutaneous melanomas in Caucasians and 10% in Japanese [26]. Of the 108 subun‐ gual melanomas in Japan, the cases involving fingers and toes were 76% and 24% respec‐ tively. On both fingers and toes, the thumb and the great toe were the most common sites [26]. Among the subungual melanoma, the occurrence rate of ALM on the fingernail is high‐ er than on toenails. According to the literature, of 64 cases of subungual melanoma, 55% cas‐ es occurred on the thumbnail, 27% on the nail of the great toe, 2-4% on the nail of the index, middle and ring finger, 1.6% on the nail of the second toe [27].

It is known that subungual melanoma has a very poor prognosis among all subtypes of cu‐ taneous melanomas. The reason for this is because the majority of subungual melanomas are already been quite deep when diagnosed [28]. Delayed diagnosis of subungual melanoma is common because it is very difficult to distinguish the early stage of subungual melanoma from longitudinal melanonychia. According to Cohen et al., 38 of 43 patients (88%) had de‐ layed diagnosis and the median delay time was 24 months (range 4 to 132) [29].

Subungual melanomas begin with fine pigmented striata which could not be clinically dis‐ tinguished from benign longitudinal melanonychia at an early stage and grow wider with colour variegation and the presence of nail plate fissuring or splitting eventually forming a triangular shape which has a broader proximal lesion rather than a distal lesion, blurred lat‐ eral borders and Hutchinson's sign - indicating the peripheral pigmentation beyond the nail apparatus [30].

Baran et al. mentioned the clinical clues to the diagnosis of subungual melanoma in detail. Hutchinson's sign is the most important sign of subungual melanoma. Other clues are when longitudinal melanonychia (a) begins in a single digit of a person over six decades or more, (b) develops abruptly in a previous normal nail plate, (c) becomes suddenly darker or wider, (d) occurs in either the thumb, index finger or giant toe, (e) is accompanied by nail destruc‐ tion or disappearance, (f) has colour variegation, (g) has a wide band and so on [31].

In addition to these clinical clues, dermoscopy provides useful information for the diagnosis of subungual melanoma. The prominent dermoscopic features of subungual melanoma are brown pigmentation of the background with longitudinal brown to black lines which are ir‐ regular in their colouration, spacing, thickness and parallelism [32]. This irregularity was significantly associated with melanoma when compared with all other benign diseases. The micro-Hutchinson's sign is the suspicious dermoscopic feature, which consists of the irregu‐ lar lines in the cuticle area and can be observed only on dermoscopy [32,33].

**Figure 1.** Dermoscopy of early stage ALM (a,b) and melanocytic nevi (c,d). (a) Irregular diffuse pigmentation, (b) paral‐ lel ridge pattern, (c) parallel furrow pattern, (d) lattice-like pattern with longitudinal and transversal thicker lines sur‐

Ulceration on nodules following pigmented macules reveals a polymorphous vascular pat‐ tern with a combination of milky-red areas (95%) which are larger areas of fuzzy or unfo‐ cused milky-red colour corresponding to an elevated part of the lesion, linear irregular

The incidence of subungual melanoma also has a racial difference. It is more frequent in Asian and Blacks than in Caucasians. Its frequency has been reported to be approximately 2-4% in all cutaneous melanomas in Caucasians and 10% in Japanese [26]. Of the 108 subun‐ gual melanomas in Japan, the cases involving fingers and toes were 76% and 24% respec‐ tively. On both fingers and toes, the thumb and the great toe were the most common sites [26]. Among the subungual melanoma, the occurrence rate of ALM on the fingernail is high‐ er than on toenails. According to the literature, of 64 cases of subungual melanoma, 55% cas‐

vessels (49%), dotted vessels (43%) and hairpin vessels (41%) [25].

rounding the eccrine pores (arrows).

478 Melanoma - From Early Detection to Treatment

**2. Subungual melanoma**

Since the early stage of subungual melanoma has minimal histopathological change, it may be difficult to distinguish subungual melanoma from benign lesion with only histopatholog‐ ical findings. Thus, both clinical features, including present history and histopathological findings, are necessary for diagnosis.

We propose a diagnostic algorithm for the early stage of subungual melanoma (figure 2). When a case falls under any of the clinical features mentioned above (a-g), dermoscopic ex‐ amination is recommended. If Hutchinson's sign and colour change in overall nail to dark black are present, excisional biopsy is recommended. When those characteristic appearances are absent, but a nail streak has irregularity, biopsy is also recommended. On the contrary, when nail streaks are monotonous pale brown, subungual melanoma is not suspicious. Even if a streak is dark brown or black, no irregularity of lines on dermoscopy allows careful fol‐ low-up without biopsies. If the streak increases in width or has colour variegation during a period of follow-up, the necessity of excision or biopsy should be discussed according to further dermoscopic examination.

bone. However, not all cases of nail destruction are accompanied with invasion. Some cases of nail destruction may be melanomas in situ. Since the distance between nail bed and bone is wider at the distal side than at the proximal side, the possibility of avoiding amputation is

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Moehrle et al. proposed 'functional' surgery for subungual melanoma, by which the ampu‐ tation of the distal phalanx could be avoided and the more digital function could be pre‐ served [35]. The tumour was surgically removed with measurable excision margins and a partial resection of the distal part of the distal phalanx was performed with a Luer instru‐ ment. After the resection, three-dimensional histology was performed as described in the lit‐ erature [36]. Two of 31 patients had local recurrence after this operation. This method did not lead to shorter survival when compared to amputation, thus, it is worth considering.

According to a review of the literature on the margins of radical excision for melanomas thinner than 2mm, the French Cooperative Group Trial [37] and the Scandinavian Melano‐ ma Group Study [38] compared 2cm with 5cm margins and the World Health Organization (WHO) Melanoma Program Trial 10 [39] compared 1cm with 3cm margins. All three trials

Although a 5mm margin for melanoma in situ is frequently recommended in some national guidelines, Kunishige et al. demonstrated that 86% of 1120 melanomas in situ were success‐ fully excised with a 6mm margin and 98.9% with a 9mm margin. They concluded that a 6mm margin for melanomas in situ was inadequate and a 9mm margin was necessary [40]. A 1cm margin of excision has been proposed for melanomas less than 1mm thick and a wid‐ er margin for more than 1mm thick [41,42]. Although many national guidelines recommend that a 1 cm margin is appropriate for 1-2mm thick invasive melanoma, this is less clear be‐ cause there has been very little data indicating that a 1cm margin for 1-2mm thick melano‐ ma is safer than 2cm margin [43]. For more than 2mm thick melanomas, a 2cm margin is considered to be sufficient in almost all cases. Depth of excision has been recommended to be at least the level of muscle fascia and deeper excision under it has not been shown to im‐

For melanomas on the extremities, especially on fingers, amputation impairs the function. Thus, even if finger amputation is necessary, it is desirable that the defect is smaller so that functional impairment can be minimal. Detailed histopathological examination of resected specimens may allow surgeons to excise a smaller part of the fingers. We show the patho‐ logical specimen as illustrated on Figure 3. Because the resected margins are usually intri‐ cately curved, the specimen is divided into several parts so that a marginal side of each part becomes planar and paraffin sections can be made so that the whole surface of the marginal side can be examined. This technique provides highly accurate detection of continuous le‐ sions with a small possibility of missing skip lesions. If the margin is negative, additional

excision is not necessary and that provides preservation of more digital functions.

higher when the nail destruction is modest and located at the distal side of the nail.

**3. Wide local excision**

prove outcome [43-45].

demonstrated no benefits for wider margins.

**Figure 2.** Diagnostic algorithm for early stage subungual melanoma.

Since nail biopsies cause cosmetic problems, biopsy methods should be selected as follows. If streaks are more likely to be melanoma, complete excisional biopsies are desirable. If it is less likely, punch biopsies around the origin of the longitudinal melanonychia (which is fre‐ quently located on the nail matrix) can be chosen. When excisional biopsies are performed, we should keep in mind that an insufficient margin at the proximal side of the nail may cause incision in between the lesional nail matrix without including the whole lesion. Ultra‐ sound echography provides useful information on the location of the nail matrix [34], so that a sufficient margin can be ensured. Furthermore, it is desirable that extent of the excisional biopsy includes the periosteum of the distal phalanx. Since the distance between the nail matrix and bone is extremely close at the proximal side, excisional biopsies excluding peri‐ osteum may incise the nail matrix and leave some lesion on the body. Excisional biopsies including the periosteum causes very little disadvantage compared with those excluding the periosteum and afterwards good granulation tissue will be formed on the bone when the ar‐ tificial dermis is used. If it is histopathologically diagnosed as subungual melanoma, a local wide excision is selected, excluding the case when a sufficient margin is ensured at the pre‐ vious excisional biopsy.

Excisional biopsies in a good manner allow us to determine correct tumour thickness, which provides important information on the choice of SLNB, local wide excision and chemothera‐ py. However, biopsy specimens easily break down if the biopsy procedure for histological examination is not performed well, which may cause incorrect choices for treatment.

If nail destruction is present under diagnosis of subungual melanoma, amputation of the distal phalanx will be applied on the assumption that the lesion invades the periosteum or bone. However, not all cases of nail destruction are accompanied with invasion. Some cases of nail destruction may be melanomas in situ. Since the distance between nail bed and bone is wider at the distal side than at the proximal side, the possibility of avoiding amputation is higher when the nail destruction is modest and located at the distal side of the nail.

Moehrle et al. proposed 'functional' surgery for subungual melanoma, by which the ampu‐ tation of the distal phalanx could be avoided and the more digital function could be pre‐ served [35]. The tumour was surgically removed with measurable excision margins and a partial resection of the distal part of the distal phalanx was performed with a Luer instru‐ ment. After the resection, three-dimensional histology was performed as described in the lit‐ erature [36]. Two of 31 patients had local recurrence after this operation. This method did not lead to shorter survival when compared to amputation, thus, it is worth considering.

## **3. Wide local excision**

**Figure 2.** Diagnostic algorithm for early stage subungual melanoma.

480 Melanoma - From Early Detection to Treatment

vious excisional biopsy.

Since nail biopsies cause cosmetic problems, biopsy methods should be selected as follows. If streaks are more likely to be melanoma, complete excisional biopsies are desirable. If it is less likely, punch biopsies around the origin of the longitudinal melanonychia (which is fre‐ quently located on the nail matrix) can be chosen. When excisional biopsies are performed, we should keep in mind that an insufficient margin at the proximal side of the nail may cause incision in between the lesional nail matrix without including the whole lesion. Ultra‐ sound echography provides useful information on the location of the nail matrix [34], so that a sufficient margin can be ensured. Furthermore, it is desirable that extent of the excisional biopsy includes the periosteum of the distal phalanx. Since the distance between the nail matrix and bone is extremely close at the proximal side, excisional biopsies excluding peri‐ osteum may incise the nail matrix and leave some lesion on the body. Excisional biopsies including the periosteum causes very little disadvantage compared with those excluding the periosteum and afterwards good granulation tissue will be formed on the bone when the ar‐ tificial dermis is used. If it is histopathologically diagnosed as subungual melanoma, a local wide excision is selected, excluding the case when a sufficient margin is ensured at the pre‐

Excisional biopsies in a good manner allow us to determine correct tumour thickness, which provides important information on the choice of SLNB, local wide excision and chemothera‐ py. However, biopsy specimens easily break down if the biopsy procedure for histological

If nail destruction is present under diagnosis of subungual melanoma, amputation of the distal phalanx will be applied on the assumption that the lesion invades the periosteum or

examination is not performed well, which may cause incorrect choices for treatment.

According to a review of the literature on the margins of radical excision for melanomas thinner than 2mm, the French Cooperative Group Trial [37] and the Scandinavian Melano‐ ma Group Study [38] compared 2cm with 5cm margins and the World Health Organization (WHO) Melanoma Program Trial 10 [39] compared 1cm with 3cm margins. All three trials demonstrated no benefits for wider margins.

Although a 5mm margin for melanoma in situ is frequently recommended in some national guidelines, Kunishige et al. demonstrated that 86% of 1120 melanomas in situ were success‐ fully excised with a 6mm margin and 98.9% with a 9mm margin. They concluded that a 6mm margin for melanomas in situ was inadequate and a 9mm margin was necessary [40].

A 1cm margin of excision has been proposed for melanomas less than 1mm thick and a wid‐ er margin for more than 1mm thick [41,42]. Although many national guidelines recommend that a 1 cm margin is appropriate for 1-2mm thick invasive melanoma, this is less clear be‐ cause there has been very little data indicating that a 1cm margin for 1-2mm thick melano‐ ma is safer than 2cm margin [43]. For more than 2mm thick melanomas, a 2cm margin is considered to be sufficient in almost all cases. Depth of excision has been recommended to be at least the level of muscle fascia and deeper excision under it has not been shown to im‐ prove outcome [43-45].

For melanomas on the extremities, especially on fingers, amputation impairs the function. Thus, even if finger amputation is necessary, it is desirable that the defect is smaller so that functional impairment can be minimal. Detailed histopathological examination of resected specimens may allow surgeons to excise a smaller part of the fingers. We show the patho‐ logical specimen as illustrated on Figure 3. Because the resected margins are usually intri‐ cately curved, the specimen is divided into several parts so that a marginal side of each part becomes planar and paraffin sections can be made so that the whole surface of the marginal side can be examined. This technique provides highly accurate detection of continuous le‐ sions with a small possibility of missing skip lesions. If the margin is negative, additional excision is not necessary and that provides preservation of more digital functions.

institution study in Japan, SLNs were identified in 253 nodal basins from 117 patients and interval SLNs were found in six patients. They recognized 41 (17%) SLN metastases in 246

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Tumours on upper extremities almost always drain to the axillary region. The axillary re‐ gion is divided into three parts based on the pectoralis minor. Level 1, 2 and 3 are located lateral, deep and medial to the pectoralis minor respectively. Outside this conventional re‐ gion, SLNs are recognized in the cubital region and other areas. Figure 4 is the local sites of

SLNs were identified in all 10 cases (100%) for the axillary region, three cases (30%) for the cubital region, three cases (30%) for the upper arm, one case (10%) for the forearm, one case (10%) for the subclavicular region (level 3) and one case (10%) for the supraclavicular re‐ gion. In all 10 cases, SLNs were present in anatomic level 1 of the axillary region. Although it has been considered that there is very little chance of finding SLNs in level 3, one case

SLNs between the primary lesion and the axillary region are regarded as interval nodes on the upper extremities. Manganomi et al. reported that the interval SLN identification rate on upper extremities was 0.4% (two out of 480 cases) [53] and Kelly et al. reported 3.8% (16 out of 423 cases) [54]. Cubital region is the most common site of interval SLN on upper extremi‐ ties. In our 3 cases of interval SLNs identified in cubital region, those were present on cubital

conventional nodal basins and one (14%) in seven interval SLNs [52].

**5. Sentinel lymph node biopsy on upper extremities**

**Figure 4.** The sites of SLNs in primary melanomas on upper extremities.

with SLN in level 3 was present in our data.

SLNs in our experience of 10 cases.

**Figure 3.** How to make pathological specimen of an amputated finger tip was illustrated. In order to examine the tumor cells at the marginal side accurately and continuously, curved rim of the excised margin was cut as linear as possible, and the outside of the margin is examined. (arrow)

## **4. Sentinel lymph node biopsy**

Sentinel lymph node biopsy (SLNB) has become the standard procedure used to determine whether a tumour has metastasized to lymph nodes and more accurate staging of the mela‐ noma. It is a less invasive technique than lymph node dissection allowing patients with node negative (N0) melanoma to avoid unnecessary lymph node dissection. In the case of SLN positive melanoma, additional surgery of lymph node dissection is necessary.

The false-negative rate in SLN mapping for melanoma has been reported to be very low with a rate of 0 to 2 % [46-49]. The multicenter selective lymphadenectomy trial-1 (MSLT-1) demonstrated immediate lymph node dissection following microscopic positive node at SLNB could bring about better prognosis than the lymph node dissection after clinical nodal observation [50].

For more correct mapping of SLNs, a combination of blue dye and radioisotope 99mTc label‐ led phytate is generally used. SLNs are identified by the presence of blue stained lymph ves‐ sels and lymph nodes, and the radioactivity measured by gamma probe. Furthermore, distinction between SLNs and secondary non-SLNs is achieved by using pre-operative dy‐ namic cutaneous lymphoscintigraphy [51].

Although most melanomas drain to conventional regional nodes, unexpected drainage out‐ side of these basins is observed in some cases. Pre-operative lymphscintigraphy and a hand‐ held gamma probe are required for detection of these interval SLNs. According to a singleinstitution study in Japan, SLNs were identified in 253 nodal basins from 117 patients and interval SLNs were found in six patients. They recognized 41 (17%) SLN metastases in 246 conventional nodal basins and one (14%) in seven interval SLNs [52].

## **5. Sentinel lymph node biopsy on upper extremities**

Tumours on upper extremities almost always drain to the axillary region. The axillary re‐ gion is divided into three parts based on the pectoralis minor. Level 1, 2 and 3 are located lateral, deep and medial to the pectoralis minor respectively. Outside this conventional re‐ gion, SLNs are recognized in the cubital region and other areas. Figure 4 is the local sites of SLNs in our experience of 10 cases.

**Figure 4.** The sites of SLNs in primary melanomas on upper extremities.

**Figure 3.** How to make pathological specimen of an amputated finger tip was illustrated. In order to examine the tumor cells at the marginal side accurately and continuously, curved rim of the excised margin was cut as linear as

Sentinel lymph node biopsy (SLNB) has become the standard procedure used to determine whether a tumour has metastasized to lymph nodes and more accurate staging of the mela‐ noma. It is a less invasive technique than lymph node dissection allowing patients with node negative (N0) melanoma to avoid unnecessary lymph node dissection. In the case of

The false-negative rate in SLN mapping for melanoma has been reported to be very low with a rate of 0 to 2 % [46-49]. The multicenter selective lymphadenectomy trial-1 (MSLT-1) demonstrated immediate lymph node dissection following microscopic positive node at SLNB could bring about better prognosis than the lymph node dissection after clinical nodal

For more correct mapping of SLNs, a combination of blue dye and radioisotope 99mTc label‐ led phytate is generally used. SLNs are identified by the presence of blue stained lymph ves‐ sels and lymph nodes, and the radioactivity measured by gamma probe. Furthermore, distinction between SLNs and secondary non-SLNs is achieved by using pre-operative dy‐

Although most melanomas drain to conventional regional nodes, unexpected drainage out‐ side of these basins is observed in some cases. Pre-operative lymphscintigraphy and a hand‐ held gamma probe are required for detection of these interval SLNs. According to a single-

SLN positive melanoma, additional surgery of lymph node dissection is necessary.

possible, and the outside of the margin is examined. (arrow)

**4. Sentinel lymph node biopsy**

482 Melanoma - From Early Detection to Treatment

namic cutaneous lymphoscintigraphy [51].

observation [50].

SLNs were identified in all 10 cases (100%) for the axillary region, three cases (30%) for the cubital region, three cases (30%) for the upper arm, one case (10%) for the forearm, one case (10%) for the subclavicular region (level 3) and one case (10%) for the supraclavicular re‐ gion. In all 10 cases, SLNs were present in anatomic level 1 of the axillary region. Although it has been considered that there is very little chance of finding SLNs in level 3, one case with SLN in level 3 was present in our data.

SLNs between the primary lesion and the axillary region are regarded as interval nodes on the upper extremities. Manganomi et al. reported that the interval SLN identification rate on upper extremities was 0.4% (two out of 480 cases) [53] and Kelly et al. reported 3.8% (16 out of 423 cases) [54]. Cubital region is the most common site of interval SLN on upper extremi‐ ties. In our 3 cases of interval SLNs identified in cubital region, those were present on cubital fossa and on ulnar side of cubital region. We have experienced five cases of other interval region rather than cubital region.

cases for external iliac lymph nodes and one case for the obturator region). Three of the 23 cases with SLN on the inguinal region had positive nodes and there were no positive nodes

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One of the problems with SLNB of lower extremities is the presence of pelvic SLNs. Kaout‐ zanis et al. showed 11 of 82 cases with tumour on lower extremities had SLNs on the pelvic region and underwent SLNB [60]. They also showed that 19 of 82 cases (24%) had positive SLNs and all the positive SLNs were located in the inguinal region. No positive SLNs were

Even in SLNB, removing the lymph nodes in the external iliac and obturator region is a rela‐ tively invasive technique. It is still controversial whether or not SLNs in the pelvic region should be harvested because SLNs in the pelvic region may be considered as secondary or third lymphatic basin, even when radioisotope is accumulated in pelvic lymph nodes.

Even now there is no consensus on the clinical definition of an SLN [61]. The SLN has been described as the hottest node, the blue node, first node visualized on lymphoscintigraphy and a node with radioactivity greater than twice or three times background radioactivity [61-63]. McMaster et al. recommended that all blue nodes and all nodes that measure 10% or higher of the 'ex vivo' radioactive count of hottest SLNs should be removed in order to de‐ crease false-negative cases [64]. In our institute, SLN is defined as the node which showed higher than one tenth of the radioactivity of the hottest node. Because radioactivity depends on the distance from the surface of skin to the nodes, the measured radioactivity of the pel‐ vic lymph nodes from the surface of skin is much less than superficial lymph nodes and tends to be underestimated [65]. Therefore, Bagaria et al. provided an answer that the back‐

on the popliteal and pelvic region.

**Figure 6.** The sites of SLNs in primary melanomas on lower extremities.

present in the pelvic region as our cases.

Tumours on upper extremities rarely drain to the subclavicular region (level 3) rather than to level 1 or 2. Our case with SLNs on the subclavicular region had positive with non-posi‐ tive SLNs in level 11 and no SLNs in level 2 (Figure 5).

**Figure 5.** The case with positive SLN in level 3 and non-positive SLNs in level 1 and the upper arm. This metastatic pattern is extremely rare.

There may be cases where it is uncertain whether or not SLNB should be applied when tu‐ mour thickness is unknown. Some literature demonstrated that the patients with primary le‐ sions on their extremities have a lower risk of misidentification of SLNs, even after wide local excision, than patients with axial primary lesions [55-59]. Tumors on central trunk may drain to both bilateral, or both axillary and inguinal regions. By contrast, tumours on ex‐ tremities tend to drain more simply to the expected region. Although it is preferable that wide local excision and SLNB are performed at the same time, SLNB after wide local exci‐ sion is less disruptive to lymphatic drainage in the case of primary lesions on the extremities than on axial sites.

## **6. Sentinel lymph node biopsy on lower extremities**

In almost all cases, tumours on lower extremities drain to the inguinal region. Figure 6 dem‐ onstrates lymphatic drainage for 23 cases with tumours on lower extremities in our institute. Of all 23 cases, the lymph node identification rate was 23 cases (100%) for the inguinal re‐ gion, five cases (21%) for the popliteal region and 10 cases (43%) for the pelvic region (nine cases for external iliac lymph nodes and one case for the obturator region). Three of the 23 cases with SLN on the inguinal region had positive nodes and there were no positive nodes on the popliteal and pelvic region.

**Figure 6.** The sites of SLNs in primary melanomas on lower extremities.

fossa and on ulnar side of cubital region. We have experienced five cases of other interval

Tumours on upper extremities rarely drain to the subclavicular region (level 3) rather than to level 1 or 2. Our case with SLNs on the subclavicular region had positive with non-posi‐

**Figure 5.** The case with positive SLN in level 3 and non-positive SLNs in level 1 and the upper arm. This metastatic

There may be cases where it is uncertain whether or not SLNB should be applied when tu‐ mour thickness is unknown. Some literature demonstrated that the patients with primary le‐ sions on their extremities have a lower risk of misidentification of SLNs, even after wide local excision, than patients with axial primary lesions [55-59]. Tumors on central trunk may drain to both bilateral, or both axillary and inguinal regions. By contrast, tumours on ex‐ tremities tend to drain more simply to the expected region. Although it is preferable that wide local excision and SLNB are performed at the same time, SLNB after wide local exci‐ sion is less disruptive to lymphatic drainage in the case of primary lesions on the extremities

In almost all cases, tumours on lower extremities drain to the inguinal region. Figure 6 dem‐ onstrates lymphatic drainage for 23 cases with tumours on lower extremities in our institute. Of all 23 cases, the lymph node identification rate was 23 cases (100%) for the inguinal re‐ gion, five cases (21%) for the popliteal region and 10 cases (43%) for the pelvic region (nine

**6. Sentinel lymph node biopsy on lower extremities**

region rather than cubital region.

484 Melanoma - From Early Detection to Treatment

pattern is extremely rare.

than on axial sites.

tive SLNs in level 11 and no SLNs in level 2 (Figure 5).

One of the problems with SLNB of lower extremities is the presence of pelvic SLNs. Kaout‐ zanis et al. showed 11 of 82 cases with tumour on lower extremities had SLNs on the pelvic region and underwent SLNB [60]. They also showed that 19 of 82 cases (24%) had positive SLNs and all the positive SLNs were located in the inguinal region. No positive SLNs were present in the pelvic region as our cases.

Even in SLNB, removing the lymph nodes in the external iliac and obturator region is a rela‐ tively invasive technique. It is still controversial whether or not SLNs in the pelvic region should be harvested because SLNs in the pelvic region may be considered as secondary or third lymphatic basin, even when radioisotope is accumulated in pelvic lymph nodes.

Even now there is no consensus on the clinical definition of an SLN [61]. The SLN has been described as the hottest node, the blue node, first node visualized on lymphoscintigraphy and a node with radioactivity greater than twice or three times background radioactivity [61-63]. McMaster et al. recommended that all blue nodes and all nodes that measure 10% or higher of the 'ex vivo' radioactive count of hottest SLNs should be removed in order to de‐ crease false-negative cases [64]. In our institute, SLN is defined as the node which showed higher than one tenth of the radioactivity of the hottest node. Because radioactivity depends on the distance from the surface of skin to the nodes, the measured radioactivity of the pel‐ vic lymph nodes from the surface of skin is much less than superficial lymph nodes and tends to be underestimated [65]. Therefore, Bagaria et al. provided an answer that the back‐ ground radioactivity of the regional nodal basin was measured before incision and all blue nodes and all hot nodes that have radioactivity greater than the background were harvested [61]. According to Soteldo et al., the rate of the cases that had metastasized lymph nodes in the pelvic region after SLNB that had indicated non-positive SLNs in the inguinal region was 2.4% [65]. This indicated that there might be cases with positive lymph nodes in the pel‐ vic region with non-positive lymph nodes in the inguinal region. This rate should not be un‐ derestimated and gives a reason for removing SLNs in the pelvic region.

dissection including level 1 and 2was 5 % (14 cases out of 270 cases) [67]. Guggenheim et al. also reported a rate of 4.5% after axillary dissection which mainly included level 1 and 2 [68]. On the other hand, according to Kretschmer et al., the local recurrence rate was 9.5% (six out of 63 cases) after dissection including level 1,2 and 3. There is no direct evidence that

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The complication rate of dissection for level 1and 2 is less severe than that of dissection for level 1,2 and 3. The rates of infection and seroma after the former operation were 8% and 2%

There are very few reported cases in which positive SLNs in level 3 were harvested except for our case. In addition, the lymph node ratio (LNR: the ratio of involved lymph nodes to total retrieved nodes in lymph node dissection) provides prognostic information [71-73]. There is very little possibility that positive lymph nodes are harvested only in level 3 with‐ out positive SLNs in level 1 or 2. Although there is data not on upper extremities but on lower extremities, a larger number of cases involved lymph nodes in the inguinal region with a higher rate of pelvic lymph node metastases [74]. This indicates that the number of superficial involved lymph nodes is related to the possibility of metastasis in the deep re‐ gion. It has been reported that the size of SLN metastases predicts other nodal disease and survival in malignant melanoma [75,76]. Due to these findings, axillary dissection including level 3 is not always necessary when there are one or two micrometastatic lymph nodes in

dissection including level 3 is superior to dissection without level 3 [69].

respectively, whereas those for the latter operation were 20% and 18% [67,70].

level 1, but it is necessary when a case falls under any of the following conditions:

**•** There is a relatively large clinically involved lymph node in level 1 or 2.

**•** There is negative SLNs in level 3 with positive lymph nodes in level 1 or 2.

**•** There are involved lymph nodes in level 3 evaluated with radiological examination such

There is also controversy surrounding inguinal and pelvic lymph node dissection. The most controversial question is whether routine dissection with the primary tumour on lower ex‐ tremities includes only a superficial inguinal lymph node dissection (SLND) or includes ad‐ ditional iliac and obturator lymph node dissection (deep pelvic/inguinal lymph node dissection : DLND) [77]. Like the axillary dissection, decision on the area to be dissected is

Hughes et al. reported that in cases of palpable inguinal lymph node metastases, pelvic lymph node recurrence occurred in one of 72 patients who had DLND and seven of 60 pa‐ tients who had SLND (p=0.01) [74]. In this study, patients with one positive superficial node

difficult from the viewpoint of local control, overall survival and complications.

**•** There are many metastatic lymph nodes in level 1 or 2.

**9. Inguinal and pelvic lymph node dissection**

**•** There are positive SLNs in level 3.

as computed tomography (CT).

On the other hand, there have been no published results of positive pelvic lymph nodes with negative inguinal SLNs for melanoma located below the knee. Pelvic lymph nodes for melanoma located below the knee were considered as secondary lymphatic basin because they were not stained blue and the radioactivity of the pelvic lymph nodes was significantly less than that of the inguinal nodes removed from the same patients [66]. By contrast, the pelvic lymph nodes for melanoma located on the trunk and thigh are possibly SLNs. In the case of melanoma below the knee in which there is a risk or difficulty in removing the pelvic lymph nodes, for example the case of pelvic adhesion after surgery in the pelvis, SLNB could be clinically avoided in terms of the cost-benefit relationship.

There are two approaches to removing the pelvic lymph nodes. One is the technique with incision above the inguinal ligament and the other is the technique with median incision in the lower abdomen. Each technique has an advantage. With median incision, it is easier to approach a deep site in the pelvic region such as the obturator area or the external iliac re‐ gion near to the common iliac region. By contrast, it is easier and less invasive to approach the external iliac region near to the inguinal ligament with an incision above the inguinal ligament.

## **7. Lymph node dissection**

Lymph node dissection is the primary management for regional lymph node metastasis. It is applied in cases of clinical metastasis, positive SLNs after SLNB and histological lymphatic invasion for a resected or biopsied primary lesion. Surgical technique, extent of dissection, morbidity and complication vary widely in the published literature. Although lymph node dissection has been a standard treatment and the technique has not drastically changed for many years, even now there is much controversy surrounding lymph node dissection. Some of the controversies will be mentioned in the following section.

## **8. Axillary lymph node dissection**

Since the tumours on upper extremities drain to the axillary region, axillary dissection is necessary and performed by way of cure or local control of the metastatic melanoma in the upper extremities. On the area of axillary dissection, it is controversial whether a level 3 dis‐ section should be included. Namm et al. reported that the local recurrence rate of axillary dissection including level 1 and 2was 5 % (14 cases out of 270 cases) [67]. Guggenheim et al. also reported a rate of 4.5% after axillary dissection which mainly included level 1 and 2 [68]. On the other hand, according to Kretschmer et al., the local recurrence rate was 9.5% (six out of 63 cases) after dissection including level 1,2 and 3. There is no direct evidence that dissection including level 3 is superior to dissection without level 3 [69].

The complication rate of dissection for level 1and 2 is less severe than that of dissection for level 1,2 and 3. The rates of infection and seroma after the former operation were 8% and 2% respectively, whereas those for the latter operation were 20% and 18% [67,70].

There are very few reported cases in which positive SLNs in level 3 were harvested except for our case. In addition, the lymph node ratio (LNR: the ratio of involved lymph nodes to total retrieved nodes in lymph node dissection) provides prognostic information [71-73]. There is very little possibility that positive lymph nodes are harvested only in level 3 with‐ out positive SLNs in level 1 or 2. Although there is data not on upper extremities but on lower extremities, a larger number of cases involved lymph nodes in the inguinal region with a higher rate of pelvic lymph node metastases [74]. This indicates that the number of superficial involved lymph nodes is related to the possibility of metastasis in the deep re‐ gion. It has been reported that the size of SLN metastases predicts other nodal disease and survival in malignant melanoma [75,76]. Due to these findings, axillary dissection including level 3 is not always necessary when there are one or two micrometastatic lymph nodes in level 1, but it is necessary when a case falls under any of the following conditions:


ground radioactivity of the regional nodal basin was measured before incision and all blue nodes and all hot nodes that have radioactivity greater than the background were harvested [61]. According to Soteldo et al., the rate of the cases that had metastasized lymph nodes in the pelvic region after SLNB that had indicated non-positive SLNs in the inguinal region was 2.4% [65]. This indicated that there might be cases with positive lymph nodes in the pel‐ vic region with non-positive lymph nodes in the inguinal region. This rate should not be un‐

On the other hand, there have been no published results of positive pelvic lymph nodes with negative inguinal SLNs for melanoma located below the knee. Pelvic lymph nodes for melanoma located below the knee were considered as secondary lymphatic basin because they were not stained blue and the radioactivity of the pelvic lymph nodes was significantly less than that of the inguinal nodes removed from the same patients [66]. By contrast, the pelvic lymph nodes for melanoma located on the trunk and thigh are possibly SLNs. In the case of melanoma below the knee in which there is a risk or difficulty in removing the pelvic lymph nodes, for example the case of pelvic adhesion after surgery in the pelvis, SLNB

There are two approaches to removing the pelvic lymph nodes. One is the technique with incision above the inguinal ligament and the other is the technique with median incision in the lower abdomen. Each technique has an advantage. With median incision, it is easier to approach a deep site in the pelvic region such as the obturator area or the external iliac re‐ gion near to the common iliac region. By contrast, it is easier and less invasive to approach the external iliac region near to the inguinal ligament with an incision above the inguinal

Lymph node dissection is the primary management for regional lymph node metastasis. It is applied in cases of clinical metastasis, positive SLNs after SLNB and histological lymphatic invasion for a resected or biopsied primary lesion. Surgical technique, extent of dissection, morbidity and complication vary widely in the published literature. Although lymph node dissection has been a standard treatment and the technique has not drastically changed for many years, even now there is much controversy surrounding lymph node dissection. Some

Since the tumours on upper extremities drain to the axillary region, axillary dissection is necessary and performed by way of cure or local control of the metastatic melanoma in the upper extremities. On the area of axillary dissection, it is controversial whether a level 3 dis‐ section should be included. Namm et al. reported that the local recurrence rate of axillary

derestimated and gives a reason for removing SLNs in the pelvic region.

could be clinically avoided in terms of the cost-benefit relationship.

of the controversies will be mentioned in the following section.

ligament.

**7. Lymph node dissection**

486 Melanoma - From Early Detection to Treatment

**8. Axillary lymph node dissection**

**•** There are involved lymph nodes in level 3 evaluated with radiological examination such as computed tomography (CT).

## **9. Inguinal and pelvic lymph node dissection**

There is also controversy surrounding inguinal and pelvic lymph node dissection. The most controversial question is whether routine dissection with the primary tumour on lower ex‐ tremities includes only a superficial inguinal lymph node dissection (SLND) or includes ad‐ ditional iliac and obturator lymph node dissection (deep pelvic/inguinal lymph node dissection : DLND) [77]. Like the axillary dissection, decision on the area to be dissected is difficult from the viewpoint of local control, overall survival and complications.

Hughes et al. reported that in cases of palpable inguinal lymph node metastases, pelvic lymph node recurrence occurred in one of 72 patients who had DLND and seven of 60 pa‐ tients who had SLND (p=0.01) [74]. In this study, patients with one positive superficial node and those with more than one positive superficial node were 17% and 51% of 72 patients with DLND respectively. The number of positive superficial lymph nodes and the presence of extracapsular spread were significant prognostic factors for overall survival [77].

**•** There is a palpable inguinal metastasis.

**10. Molecular targeted therapy**

sary to confirm the effect of this agent.

ternational guidelines are yet to be established.

metastasis.

**11. Conclusion**

scopic findings.

tion and follow-up are necessary.

**•** CT indicates metastatic pelvic lymph nodes. **•** There is a Cloquet's lymph node metastasis.

**•** There is more than one superficial lymph node metastasis.

**•** Lymphoschintigraphy indicates SLNs in the pelvic region with a superficial lymph node

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Recent discoveries in cell signalling have provided greater understanding of the biology that underlines melanoma and these advances are being exploited to provide targeted drugs and new therapeutic approaches [81]. In some cases of ALM and mucosal melanoma, the muta‐ tions of KIT, a transmenbrane receptor tyrosine kinase, are reported and these mutations lead to marked expression of KIT in tumour cells. These cases have marked a tendency to respond to imatinib mesylate which inhibits tyrosine kinase. Although case reports are accu‐ mulating [82,83], more data, including long-term control and prognostic data, will be neces‐

Controversies remain regarding the surgical treatment of ALM as described above, thus, in‐

It is not still known whether the thickness of the nail tumour is the same as that on other sites because the distance between the nail bed and bone is very narrow and a mild degree of invasion can reach the bone easily. Considering the poor prognosis of cases with subun‐ gual melanoma, the tumour thickness of the subungual melanoma needs to be evaluated. Because biopsies of the nail bed may cause cosmetic and occasionally functional problems, surgeons may hesitate to do biopsies and lose a vital chance of early diagnosis. Suspicious lesions should be actively biopsied with fully informed consent. When taking a wait-and-see approach, careful observation is necessary so as not to overlook any minor change of dermo‐

On the excision margins around the primary lesion, 2cm is regarded as sufficient for inva‐ sive melanomas. Although some guidelines suggest a 0.5 cm margin for melanomas in situ, some data indicated that resection with 0.5 cm margin caused significant high rates of local recurrence. As mentioned here according to the report, a 1cm surgical margin is a better an‐ swer for melanomas in situ, except for tumours on cosmetic or functional sites such as the face or fingers. When an excision margin is less than 1cm, more careful histological examina‐

In addition, the patients who had DLND with the presence of pelvic lymph node metastases had significantly poorer five year survival than the patients without the pelvic lymph node metastases. However, there was no difference in postoperative morbidity between SLND and DLND [74]. Van der Ploeg et al. also reported that survival and local control did not differ for patients with palpable inguinal metastases treated by DLND or SLND and pelvic lymph node metastases was a significant prognostic factor [78]. In their series of 169 patients with palpable nodes in the inguinal region, five year estimated overall survival rates were 33% for DLND and 29% for SLND.

However, there is no evidence on how the recurrence affects quality of life when DLND is not performed and how the recurrence occurs in the pelvic region for melanomas on lower extremities. It is likely that enlargement of a tumour in pelvic region causes lymphedema, congestion of lower extremities, ileus and so on. Although these symptoms may be due to DLND, also it is possible that DLND increases patients' quality of life during the remaining life time by decreasing the risk of recurrence in the pelvic region. However, there is no evi‐ dence indicating this.

Cloquet's node is an indicator of pelvic lymph node metastases. According to Shen et al.'s study, positive pelvic lymph nodes were identified in the DLND specimen from 20 of 30 (67%) patients with a positive Cloquet's node and negative pelvic lymph nodes were identi‐ fied from 27 of 35 (77%) patients with a negative Cloquet's node (p=0.0019) [79].

Pre-operative computed tomography (CT) is also a good tool to use in predicting the meta‐ stasis. Out of 44 patients with negative pelvic lymph nodes evaluated with pre-operative CT, 40 patients had in fact histologically negative pelvic lymph nodes (negative predictive value = 90.9%). On the other hand, the positive predictive value of pre-operative CT for pel‐ vic metastases, was 59% [78].

A recent study shows pre-operative lymphoscintigraphy can be used to guide the extent of inguinal lymph node dissection [66]. Chu et al. reported on 42 cases of DLND with positive inguinal SLNs. The frequency of synchronous pelvic disease was five of 42 (11.9%) [80]. All five cases with pelvic disease had primary melanomas on extremities. Upon review on the lymphoscintigraphic findings, pelvic drainage was present in four of five cases with pelvic disease (80%) and in 18 of the 32 cases (56%) without pelvic disease, though neither was statistically significant. This strategy is based on the idea that when lymphoscintigraphy shows secondary nodes to be located in the next drainage basin, this basin should be includ‐ ed as the dissecting area [66]. More data is necessary to prove that treatment based on the idea improves the mortality and local control.

For now, there is no guideline on choosing between SLND and DLND. However, many findings provide useful information and surgeons should actively select DLND when a case falls under any of the following conditions:

**•** There is a palpable inguinal metastasis.

and those with more than one positive superficial node were 17% and 51% of 72 patients with DLND respectively. The number of positive superficial lymph nodes and the presence

In addition, the patients who had DLND with the presence of pelvic lymph node metastases had significantly poorer five year survival than the patients without the pelvic lymph node metastases. However, there was no difference in postoperative morbidity between SLND and DLND [74]. Van der Ploeg et al. also reported that survival and local control did not differ for patients with palpable inguinal metastases treated by DLND or SLND and pelvic lymph node metastases was a significant prognostic factor [78]. In their series of 169 patients with palpable nodes in the inguinal region, five year estimated overall survival rates were

However, there is no evidence on how the recurrence affects quality of life when DLND is not performed and how the recurrence occurs in the pelvic region for melanomas on lower extremities. It is likely that enlargement of a tumour in pelvic region causes lymphedema, congestion of lower extremities, ileus and so on. Although these symptoms may be due to DLND, also it is possible that DLND increases patients' quality of life during the remaining life time by decreasing the risk of recurrence in the pelvic region. However, there is no evi‐

Cloquet's node is an indicator of pelvic lymph node metastases. According to Shen et al.'s study, positive pelvic lymph nodes were identified in the DLND specimen from 20 of 30 (67%) patients with a positive Cloquet's node and negative pelvic lymph nodes were identi‐

Pre-operative computed tomography (CT) is also a good tool to use in predicting the meta‐ stasis. Out of 44 patients with negative pelvic lymph nodes evaluated with pre-operative CT, 40 patients had in fact histologically negative pelvic lymph nodes (negative predictive value = 90.9%). On the other hand, the positive predictive value of pre-operative CT for pel‐

A recent study shows pre-operative lymphoscintigraphy can be used to guide the extent of inguinal lymph node dissection [66]. Chu et al. reported on 42 cases of DLND with positive inguinal SLNs. The frequency of synchronous pelvic disease was five of 42 (11.9%) [80]. All five cases with pelvic disease had primary melanomas on extremities. Upon review on the lymphoscintigraphic findings, pelvic drainage was present in four of five cases with pelvic disease (80%) and in 18 of the 32 cases (56%) without pelvic disease, though neither was statistically significant. This strategy is based on the idea that when lymphoscintigraphy shows secondary nodes to be located in the next drainage basin, this basin should be includ‐ ed as the dissecting area [66]. More data is necessary to prove that treatment based on the

For now, there is no guideline on choosing between SLND and DLND. However, many findings provide useful information and surgeons should actively select DLND when a case

fied from 27 of 35 (77%) patients with a negative Cloquet's node (p=0.0019) [79].

of extracapsular spread were significant prognostic factors for overall survival [77].

33% for DLND and 29% for SLND.

488 Melanoma - From Early Detection to Treatment

dence indicating this.

vic metastases, was 59% [78].

idea improves the mortality and local control.

falls under any of the following conditions:


## **10. Molecular targeted therapy**

Recent discoveries in cell signalling have provided greater understanding of the biology that underlines melanoma and these advances are being exploited to provide targeted drugs and new therapeutic approaches [81]. In some cases of ALM and mucosal melanoma, the muta‐ tions of KIT, a transmenbrane receptor tyrosine kinase, are reported and these mutations lead to marked expression of KIT in tumour cells. These cases have marked a tendency to respond to imatinib mesylate which inhibits tyrosine kinase. Although case reports are accu‐ mulating [82,83], more data, including long-term control and prognostic data, will be neces‐ sary to confirm the effect of this agent.

## **11. Conclusion**

Controversies remain regarding the surgical treatment of ALM as described above, thus, in‐ ternational guidelines are yet to be established.

It is not still known whether the thickness of the nail tumour is the same as that on other sites because the distance between the nail bed and bone is very narrow and a mild degree of invasion can reach the bone easily. Considering the poor prognosis of cases with subun‐ gual melanoma, the tumour thickness of the subungual melanoma needs to be evaluated. Because biopsies of the nail bed may cause cosmetic and occasionally functional problems, surgeons may hesitate to do biopsies and lose a vital chance of early diagnosis. Suspicious lesions should be actively biopsied with fully informed consent. When taking a wait-and-see approach, careful observation is necessary so as not to overlook any minor change of dermo‐ scopic findings.

On the excision margins around the primary lesion, 2cm is regarded as sufficient for inva‐ sive melanomas. Although some guidelines suggest a 0.5 cm margin for melanomas in situ, some data indicated that resection with 0.5 cm margin caused significant high rates of local recurrence. As mentioned here according to the report, a 1cm surgical margin is a better an‐ swer for melanomas in situ, except for tumours on cosmetic or functional sites such as the face or fingers. When an excision margin is less than 1cm, more careful histological examina‐ tion and follow-up are necessary.

Although SLNB is the standard technique for the management of malignant melanoma, the definition of SLN itself has not been established. This creates differences in the extent of SLNB between each institute. SLNs in patients with melanomas on upper extremities are very rarely located in level 3. There are very few cases with positive SLNs only in level 3 without positive SLNs in level 1or 2, thus, the lymph nodes in level 3 can be regarded as secondary nodes for melanomas on upper extremities in almost all cases. Surgeons should also pay additional attention to SLNs in other sites such as supraclavicular, the cubital re‐ gion and interval nodes.

[3] Ishihara K, Saida T, Otsuka F, Yamazaki N; Prognosis and Statistical Investigation Committee of the Japanese Skin Cancer Society. Statistical profiles of malignant mel‐ anoma and other skin cancers in Japan: 2007 update. International Journal of Clinical

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491

[4] Makredes M, Hui SK, Kimball AB. Melanoma in Hong Kong between 1983 and 2002: a decreasing trend in incidence observed in a complex socio-political and economic

[5] Chang JW. Cutaneous melanoma: Taiwan experience and literature review. Chang

[6] Luk NM, Ho CL, Choi CL, Wong KH, Yu KH, Yeung WK. Clinicopathological fea‐ tures and prognostic factors of cutaneous melanoma among Hong Kong Chinese.

[7] Chen YJ, Wu CY, Chen JT, Shen JL, Chen CC, Wang HC. Clinicopathologic analysis of malignant melanoma in Taiwan. Journal of American Academy of Dermatology

[8] Bradford PT, Goldstein AM, McMaster ML, Tucker MA. Acral lentiginous melano‐ ma. Incidence and survival patterns in the United States 1986-2005. Archives of Der‐

[9] Reed, RJ. New Concepts in Surgical Pathology of the Skin. New York: John Wiley &

[10] Reed RJ, Ichinose H, Krementz ET. Plantar lentiginous melanoma: a distinctive var‐ iant of human cutaneous malignant melanoma. American Journal of Surgical Pathol‐

[11] Clark, W. H., Jr., Elder, D. E. & Van Horn, M. The biologic forms of malignant mela‐

[12] Surveillance, Epidemiology and End Results (SEER) Program. National Cancer Insti‐ tute, DCCPS,Surveillance Research Program, Cancer Statistics Branch. 2008

[13] Markovic SN, Erickson LA, Rao RD, et al. Malignant Melanoma in the 21st Century, Part 1:Epidemiology, Risk Factors, Screening, Prevention, and Diagnosis. Mayo Clin‐

[14] Ishihara, K., Saida, T., Otsuka, F. & Yamazaki, N. Statistical profiles of malignant melanoma and other skin cancers in Japan. International Journal of Clinical Oncolo‐

[15] Mi Ryung Roh, Jihyun Kim, and Kee Yang Chung. Treatment and Outcomes of Mel‐ anoma in Acral Location in Korean Patients. Yonsei Medical Journal 2010;51(4)

Oncology 2008; 13(1) 33-41.

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There is controversy around the management of lymph nodes in the pelvic region. Accord‐ ing to the literature, there were patients without positive SLNs in the inguinal region, who had metastatic lymph nodes in the pelvic region during follow-up. Thus, at the moment, it is better not to regard all pelvic lymph nodes as secondary nodes and not to exclude all pelvic lymph nodes from SLNs. There are no reported cases with primary melanomas below the knee in which only positive pelvic lymph nodes are present without positive inguinal lymph nodes. Thus, surgeons should decide whether to harvest pelvic lymph nodes taking into consideration the sites of primary lesion, Brethlow thickness and the possibility of com‐ plication on a case by case basis.

Whether or not dissection in cases with primary lesions on upper extremities should include the extent of level 3 is controversial. When a case falls under any of the lists described above, the dissection including level 3 should be actively performed. However, not all cases with positive lymph nodes in level 1 or 2 need to undergo dissection including level 3.

Similarly, it is difficult to choose between SLND and DLND in the case of primary melano‐ mas on lower extremities. The cases which are likely to have metastatic lymph nodes in the pelvic region were mentioned above. It is better that the indication of DLND is determined by referring to the list.

## **Author details**

Yoshitaka Kai and Sakuhei Fujiwara\*

\*Address all correspondence to: fujiwara@oita-u.ac.jp

Department of Dermatology, Faculty of Medicine, Oita University, Oita, Japan

## **References**


[3] Ishihara K, Saida T, Otsuka F, Yamazaki N; Prognosis and Statistical Investigation Committee of the Japanese Skin Cancer Society. Statistical profiles of malignant mel‐ anoma and other skin cancers in Japan: 2007 update. International Journal of Clinical Oncology 2008; 13(1) 33-41.

Although SLNB is the standard technique for the management of malignant melanoma, the definition of SLN itself has not been established. This creates differences in the extent of SLNB between each institute. SLNs in patients with melanomas on upper extremities are very rarely located in level 3. There are very few cases with positive SLNs only in level 3 without positive SLNs in level 1or 2, thus, the lymph nodes in level 3 can be regarded as secondary nodes for melanomas on upper extremities in almost all cases. Surgeons should also pay additional attention to SLNs in other sites such as supraclavicular, the cubital re‐

There is controversy around the management of lymph nodes in the pelvic region. Accord‐ ing to the literature, there were patients without positive SLNs in the inguinal region, who had metastatic lymph nodes in the pelvic region during follow-up. Thus, at the moment, it is better not to regard all pelvic lymph nodes as secondary nodes and not to exclude all pelvic lymph nodes from SLNs. There are no reported cases with primary melanomas below the knee in which only positive pelvic lymph nodes are present without positive inguinal lymph nodes. Thus, surgeons should decide whether to harvest pelvic lymph nodes taking into consideration the sites of primary lesion, Brethlow thickness and the possibility of com‐

Whether or not dissection in cases with primary lesions on upper extremities should include the extent of level 3 is controversial. When a case falls under any of the lists described above, the dissection including level 3 should be actively performed. However, not all cases with

Similarly, it is difficult to choose between SLND and DLND in the case of primary melano‐ mas on lower extremities. The cases which are likely to have metastatic lymph nodes in the pelvic region were mentioned above. It is better that the indication of DLND is determined

positive lymph nodes in level 1 or 2 need to undergo dissection including level 3.

Department of Dermatology, Faculty of Medicine, Oita University, Oita, Japan

[1] Sneyd M, Cox B. The control of melanoma in New Zealand. New Zealand Medical

[2] Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA: A Cancer

gion and interval nodes.

490 Melanoma - From Early Detection to Treatment

plication on a case by case basis.

by referring to the list.

Yoshitaka Kai and Sakuhei Fujiwara\*

Journal 2006;119 U2169.

\*Address all correspondence to: fujiwara@oita-u.ac.jp

Journal for Clinicians 2005; 55(2) 74-108

**Author details**

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496 Melanoma - From Early Detection to Treatment

18(1) 16-21.


**Chapter 18**

**Sentinel Lymph Node Biopsy for Melanoma and**

The surgical approach to cutaneous melanoma patients with clinically uninvolved regional lymph nodes has been controversial. Although most patients with melanoma have no clini‐ cally palpable nodal disease at the time of presentation, some patients whose primary tumor increases in thickness, has ulceration, and shows a high mitotic rate histologically harbor

While some authors have advocated wide excision of the primary tumor with elective lymph node dissection (ELND), others had recommended excision of the primary site alone and therapeutic lymph node dissection (TLND) only when clinical nodal disease is present. ELND is based on the concept that metastasis arises by passage of the tumor from the pri‐ mary to the regional lymph nodes and distant sites, in which case early LND will prevent this metastatic progression. In contrast, TLND, which is a "watch and wait" approach, sug‐ gests that regional lymph node metastases are markers for disease progression and that hematogenous distant metastases could occur without lymph node metastasis. Four randomized prospective studies comparing ELND with TLND were reported[2-5]. The earli‐ er 2 studies conducted in the 1970s demonstrated no overall survival advantage for ELND[2, 3]. Accordingly, ELND was once contested and largely abandoned. Thereafter, the latter 2 studies conducted in the 1990s suggested the tendency, albeit statistically insignificant, that patients with early regional metastases may benefit from ELND[4, 5]. However, in most melanoma patients with no clinical nodal disease, microscopic nodal disease is absent at presentation. These patients cannot benefit from ELND; if ELND were to be performed, they

would suffer from the cost, time, and morbidity of an unnecessary operation.

With respect to this controversy surrounding ELND, the technique of lymphatic mapping and sentinel lymph node biopsy (SLNB) was introduced as a minimally invasive method for

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

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

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

**Surgical Approach to Lymph Node Metastasis**

Yasuhiro Nakamura and Fujio Otsuka

http://dx.doi.org/10.5772/53625

**1. Introduction**

Additional information is available at the end of the chapter

clinically undetectable regional lymph node metastasis[1].

## **Sentinel Lymph Node Biopsy for Melanoma and Surgical Approach to Lymph Node Metastasis**

Yasuhiro Nakamura and Fujio Otsuka

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53625

## **1. Introduction**

The surgical approach to cutaneous melanoma patients with clinically uninvolved regional lymph nodes has been controversial. Although most patients with melanoma have no clini‐ cally palpable nodal disease at the time of presentation, some patients whose primary tumor increases in thickness, has ulceration, and shows a high mitotic rate histologically harbor clinically undetectable regional lymph node metastasis[1].

While some authors have advocated wide excision of the primary tumor with elective lymph node dissection (ELND), others had recommended excision of the primary site alone and therapeutic lymph node dissection (TLND) only when clinical nodal disease is present. ELND is based on the concept that metastasis arises by passage of the tumor from the pri‐ mary to the regional lymph nodes and distant sites, in which case early LND will prevent this metastatic progression. In contrast, TLND, which is a "watch and wait" approach, sug‐ gests that regional lymph node metastases are markers for disease progression and that hematogenous distant metastases could occur without lymph node metastasis. Four randomized prospective studies comparing ELND with TLND were reported[2-5]. The earli‐ er 2 studies conducted in the 1970s demonstrated no overall survival advantage for ELND[2, 3]. Accordingly, ELND was once contested and largely abandoned. Thereafter, the latter 2 studies conducted in the 1990s suggested the tendency, albeit statistically insignificant, that patients with early regional metastases may benefit from ELND[4, 5]. However, in most melanoma patients with no clinical nodal disease, microscopic nodal disease is absent at presentation. These patients cannot benefit from ELND; if ELND were to be performed, they would suffer from the cost, time, and morbidity of an unnecessary operation.

With respect to this controversy surrounding ELND, the technique of lymphatic mapping and sentinel lymph node biopsy (SLNB) was introduced as a minimally invasive method for

detection of microscopic regional lymph node metastases in the early 1990s[6]. Lymphatic mapping is based on the concept that the lymphatic drainage from the skin to the regional lymph node basins runs in an orderly, stepwise fashion. These lymphatic drainage patterns would be the same as the dissemination of melanoma through the lymphatic system and therefore predict the routes of metastatic spread of melanoma cells to the regional lymph nodes (Fig. 1). Morton et al. first reported the details of the SLN technique using intradermal blue dye injection around the primary site and reported that the SLN identification rate was 82% among 237 patients[6], which was considered a high identification rate at that time. In the early 1990s, several authors evaluated this concept by performing synchronous ELND at the time of SLNB[7-9]. A "false-negative" SLN was defined as microscopic metastasis in a non-SLN despite the SLN showing no metastasis. These studies indicated that 5.8% of pa‐ tients had a false-negative SLN. In addition, Gershenwald et al. reported that only 4.1% (10/243) of patients with a histologically negative SLN developed a nodal recurrence in the previously mapped basin during a follow-up period of over 3 years[10]. This low false-nega‐ tive rate supported the SLN concept described above.

**2. Technical advances in SLNB**

as that in the SLN in the regional nodal basins[14].

axillary regions (96.6%)[18].

Although the initial SLN identification rate using blue dye injections alone was approxi‐ mately 82%[6], the advent of lymphoscintigraphy and the intraoperative hand-held gamma probe drastically improved the SLN identification rate. Studies comparing blue dye injection alone with combined techniques using blue dye, lymphoscintigraphy, and an intraoperative hand-held gamma probe showed a significant increase in SLN identification of up to 99% with the combined techniques[11, 12], which has come to be recognized as the standard technique of SLNB (Fig. 2). This combined technique also enables the surgeon to identify the interval (unusual, in-transit, ectopic) nodes located outside the named regional nodal basins (Fig. 3)[13-17]. The rate of interval SLN identification is reported to be approximately 5% to 10%, and the rate of microscopic metastasis in the interval nodes is approximately the same

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However, SLNB in the head and neck has particular problems because the lymphatic drain‐ age in the head and neck is much more complex than those in the axillary and inguinal re‐ gions. Furthermore, the cervical and parotid lymph nodes are smaller and located in sites that are not easily accessible, for example in the parotid gland, through which the facial nerve passes [18, 19]. In addition, it is sometimes difficult to detect the lymphatic drainage and SLN with lymphoscintigraphy because the SLN is often close to the highly radioactive site where the tracer was injected, the so-called shine-through phenomenon[18, 19]. In addi‐ tion, in some cases the naked eye cannot confirm that an SLN has been dyed blue even after injection of the blue dye because of the short staining period for blue dye in cervical SLNs resulting from the rapid and complex cervical lymphatic flow[19]. In our experience too, over half of the SLNs did not show any blue staining. Furthermore, some authors reported a high false-negative rate of up to 44%, which leads to increased morbidity[20-22]. This high rate may be caused by partially obstructed lymphatic vessels that do not allow for smooth flow of nanocolloids with a size of 6 to 12 nm[23]. Although several authors have reported a high identification rate in SLNB for head and neck melanoma[24-26], the identification rate of SLNs for the standard technique in the cervical region is generally less than that in the inguinal or axillary regions. In the MSLT-I trial reported by Morton et al., the SLN identifica‐ tion rate in the cervical region (84.5%) was clearly lower than that in the inguinal (99.3%) or

Several studies on the SLNB technique using indocyanine green (ICG) injection in skin can‐ cer patients have demonstrated high SLN detection and identification rates, although these studies involved mainly axillary and inguinal SLNBs and only a small number of cervical SLNBs[23, 27-29]. ICG is a diagnostic reagent used in various examinations such as exami‐ nation for cardiac output or hepatic function and retinal angiography. It has a size of only 2.1 nm, binds with albumin, and generates a peak wavelength of 840 nm near-infrared fluo‐ rescence when excited with 765-nm light[30]. Using a near-infrared camera intraoperatively, it is possible to observe the ICG as a subcutaneous lymphatic flow as well as SLNs in the fluorescence images after intradermal injection of ICG around the primary tumor. (Fig. 4) In our experience, the mean and median numbers of SLNs per basin were higher in the ICG

**Figure 1. Lymphatic drainage from a primary tumor to sentinel lymph nodes.** A sentinel node is sometimes locat‐ ed between the primary tumor and the regional nodal basins, in which case it is called an interval (unusual, in-transit, ectopic) node. If the SLN has microscopic nodal metastasis, some of the second-tier nodes may also have metastasis.

## **2. Technical advances in SLNB**

detection of microscopic regional lymph node metastases in the early 1990s[6]. Lymphatic mapping is based on the concept that the lymphatic drainage from the skin to the regional lymph node basins runs in an orderly, stepwise fashion. These lymphatic drainage patterns would be the same as the dissemination of melanoma through the lymphatic system and therefore predict the routes of metastatic spread of melanoma cells to the regional lymph nodes (Fig. 1). Morton et al. first reported the details of the SLN technique using intradermal blue dye injection around the primary site and reported that the SLN identification rate was 82% among 237 patients[6], which was considered a high identification rate at that time. In the early 1990s, several authors evaluated this concept by performing synchronous ELND at the time of SLNB[7-9]. A "false-negative" SLN was defined as microscopic metastasis in a non-SLN despite the SLN showing no metastasis. These studies indicated that 5.8% of pa‐ tients had a false-negative SLN. In addition, Gershenwald et al. reported that only 4.1% (10/243) of patients with a histologically negative SLN developed a nodal recurrence in the previously mapped basin during a follow-up period of over 3 years[10]. This low false-nega‐

**Figure 1. Lymphatic drainage from a primary tumor to sentinel lymph nodes.** A sentinel node is sometimes locat‐ ed between the primary tumor and the regional nodal basins, in which case it is called an interval (unusual, in-transit, ectopic) node. If the SLN has microscopic nodal metastasis, some of the second-tier nodes may also have metastasis.

tive rate supported the SLN concept described above.

500 Melanoma - From Early Detection to Treatment

Although the initial SLN identification rate using blue dye injections alone was approxi‐ mately 82%[6], the advent of lymphoscintigraphy and the intraoperative hand-held gamma probe drastically improved the SLN identification rate. Studies comparing blue dye injection alone with combined techniques using blue dye, lymphoscintigraphy, and an intraoperative hand-held gamma probe showed a significant increase in SLN identification of up to 99% with the combined techniques[11, 12], which has come to be recognized as the standard technique of SLNB (Fig. 2). This combined technique also enables the surgeon to identify the interval (unusual, in-transit, ectopic) nodes located outside the named regional nodal basins (Fig. 3)[13-17]. The rate of interval SLN identification is reported to be approximately 5% to 10%, and the rate of microscopic metastasis in the interval nodes is approximately the same as that in the SLN in the regional nodal basins[14].

However, SLNB in the head and neck has particular problems because the lymphatic drain‐ age in the head and neck is much more complex than those in the axillary and inguinal re‐ gions. Furthermore, the cervical and parotid lymph nodes are smaller and located in sites that are not easily accessible, for example in the parotid gland, through which the facial nerve passes [18, 19]. In addition, it is sometimes difficult to detect the lymphatic drainage and SLN with lymphoscintigraphy because the SLN is often close to the highly radioactive site where the tracer was injected, the so-called shine-through phenomenon[18, 19]. In addi‐ tion, in some cases the naked eye cannot confirm that an SLN has been dyed blue even after injection of the blue dye because of the short staining period for blue dye in cervical SLNs resulting from the rapid and complex cervical lymphatic flow[19]. In our experience too, over half of the SLNs did not show any blue staining. Furthermore, some authors reported a high false-negative rate of up to 44%, which leads to increased morbidity[20-22]. This high rate may be caused by partially obstructed lymphatic vessels that do not allow for smooth flow of nanocolloids with a size of 6 to 12 nm[23]. Although several authors have reported a high identification rate in SLNB for head and neck melanoma[24-26], the identification rate of SLNs for the standard technique in the cervical region is generally less than that in the inguinal or axillary regions. In the MSLT-I trial reported by Morton et al., the SLN identifica‐ tion rate in the cervical region (84.5%) was clearly lower than that in the inguinal (99.3%) or axillary regions (96.6%)[18].

Several studies on the SLNB technique using indocyanine green (ICG) injection in skin can‐ cer patients have demonstrated high SLN detection and identification rates, although these studies involved mainly axillary and inguinal SLNBs and only a small number of cervical SLNBs[23, 27-29]. ICG is a diagnostic reagent used in various examinations such as exami‐ nation for cardiac output or hepatic function and retinal angiography. It has a size of only 2.1 nm, binds with albumin, and generates a peak wavelength of 840 nm near-infrared fluo‐ rescence when excited with 765-nm light[30]. Using a near-infrared camera intraoperatively, it is possible to observe the ICG as a subcutaneous lymphatic flow as well as SLNs in the fluorescence images after intradermal injection of ICG around the primary tumor. (Fig. 4) In our experience, the mean and median numbers of SLNs per basin were higher in the ICG group than in the standard-technique group. The small size of ICG allows a smooth flow along the lymphatic vessels. It may lead to detection of SLNs not detectable by lymphoscin‐ tigraphy (Fig. 4C, D) owing to poor flow of the radioactive tracer and may reduce the falsenegative rate. Indeed, Stoffels et al. reported that 2 of 11 additional SLNs that were only identified by the ICG technique showed microscopic metastasis[23].

In addition, the recently introduced hybrid single-photon emission computed tomography with computed tomography (SPECT/CT) can visualize the exact anatomic location of the SLN and second-tier nodes, which would be of great help in identifying the SLN, especially those in the head and neck region[31, 32], as well as the interval nodes.

**Figure 3. Detection of interval SLN.** (A) Primary melanoma on the right heel. (B) Lymphoscintigraphy revealed accu‐ mulation in the right popliteal fossa. (C) Radioactive and blue-stained popliteal node, which had microscopic metasta‐

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**Figure 4. SLNB using ICG.** (A) SLNB for melanoma of the nose. The X mark on the left mandible indicates accumulation of radioisotope (arrow). (B) A fluorescent submandibular SLN is visible through the incision using the near-infrared camera (arrow). (C) SLNB for melanoma of the left temporal region. The X marks indicate accumulation of radioisotope. (D) An ad‐ ditional fluorescent SLN (arrow), which was not detected by lymphoscintigraphy, is observed through the overlying skin.

sis. (D) Popliteal lymph node dissection was performed.

**Figure 2. The technique of lymphatic mapping and sentinel lymph node biopsy (SLNB).** (A) Primary melanoma on the left chest. (B) Lymphoscintigraphy shows accumulation of 99Tc-tin colloid which was intradermally injected around the primary tumor in the left axilla (arrow). (C) Intradermal injection of 2% isosulfan blue injection around the primary site. (D) The exploration of the location of SLN using a hand-held gamma-probe and identification of a bluestained SLN. (E) Histopathologic detection of microscopic nodal metastasis.

group than in the standard-technique group. The small size of ICG allows a smooth flow along the lymphatic vessels. It may lead to detection of SLNs not detectable by lymphoscin‐ tigraphy (Fig. 4C, D) owing to poor flow of the radioactive tracer and may reduce the falsenegative rate. Indeed, Stoffels et al. reported that 2 of 11 additional SLNs that were only

In addition, the recently introduced hybrid single-photon emission computed tomography with computed tomography (SPECT/CT) can visualize the exact anatomic location of the SLN and second-tier nodes, which would be of great help in identifying the SLN, especially

**Figure 2. The technique of lymphatic mapping and sentinel lymph node biopsy (SLNB).** (A) Primary melanoma on the left chest. (B) Lymphoscintigraphy shows accumulation of 99Tc-tin colloid which was intradermally injected around the primary tumor in the left axilla (arrow). (C) Intradermal injection of 2% isosulfan blue injection around the primary site. (D) The exploration of the location of SLN using a hand-held gamma-probe and identification of a blue-

stained SLN. (E) Histopathologic detection of microscopic nodal metastasis.

identified by the ICG technique showed microscopic metastasis[23].

502 Melanoma - From Early Detection to Treatment

those in the head and neck region[31, 32], as well as the interval nodes.

**Figure 3. Detection of interval SLN.** (A) Primary melanoma on the right heel. (B) Lymphoscintigraphy revealed accu‐ mulation in the right popliteal fossa. (C) Radioactive and blue-stained popliteal node, which had microscopic metasta‐ sis. (D) Popliteal lymph node dissection was performed.

**Figure 4. SLNB using ICG.** (A) SLNB for melanoma of the nose. The X mark on the left mandible indicates accumulation of radioisotope (arrow). (B) A fluorescent submandibular SLN is visible through the incision using the near-infrared camera (arrow). (C) SLNB for melanoma of the left temporal region. The X marks indicate accumulation of radioisotope. (D) An ad‐ ditional fluorescent SLN (arrow), which was not detected by lymphoscintigraphy, is observed through the overlying skin.

## **3. Does SLNB-guided early lymph node dissection improve survival rate?**

question because only 10% to 25% of patients with positive SLNs will have additional micro‐ scopic metastasis in non-SLNs[40-42], which means that approximately 80% of patients with positive SLNs may be spared CLND. Several authors categorized the SLN as several varia‐ bles and tried to find a reliable indicator of non-SLN status[43, 44]. However, it remains un‐ clear what size of microscopic metastasis of the SLN or which histopathologic location of metastasis in the SLN, such as subcapsular, parenchymal, multifocal, and extensive, would

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The choice of the extent of CLND is ultimately decided by the individual surgeon. Few spe‐ cific recommendations are available in the published guidelines, with the common descrip‐ tion being ''a thorough dissection'' and reports of low levels of evidence supporting the appropriate surgical extent of CLND of the cervical, axillary, and inguinal regions[45-47].

The purpose of neck dissection is to control regional disease; it has little impact on overall sur‐ vival. However, the extent of neck dissection is still controversial and various extents of neck dissection have been advocated by several authors. Radical neck dissection (RND) including removal of level I-V (Fig. 5A) and nonlymphatic tissue such as the sternocleidomastoid muscle, the internal jugular vein, and the spinal accessary nerve has been the gold standard for neck dissection for melanoma[48]. Despite extensive areas of dissection, O'Brien et al. reported that regional control with RND was unsatisfactory, with regional recurrence of 28% in patients with

Generally, RND is associated with significant morbidity. Therefore, some authors have con‐ sidered modified RND (MRND) or functional neck dissection including preservation of any or all of the sternocleidomastoid muscle, the internal jugular vein, and the spinal accessory nerve[49, 50]. In studies of patients with clinical nodal disease, several authors demonstrat‐ ed that regional recurrence rates were 14-32% after RND, 0% after MRND, and 23% to 29% after selective neck dissection (SND), which is not statistically significant among the groups[51-53]. Byers also reported a 16% recurrence rate after MRND[54]. From these stud‐

In addition, as an even more selective approach, the lymphatic drainage patterns of head and neck melanoma have been described by O'Brien et al. based on a consecutive series of over 270 neck dissections and parotidectomies (Fig. 5B)[52]. As described above, although several authors reported relatively high regional recurrence rates of 23% to 29% after SND, these studies include clinical N2-N3 (multiple involved nodes) disease, which will have a higher risk of recurrence than N1 disease[51, 52]. In a study of 37 consecutive patients with clinically N1 neck disease reported by White et al., 6 patients underwent RND, 24, MRND, and 7, SND. None of the 3 groups had any cases of local recurrence during a mean followup of 46 months[55], indicating that SND may be an alternative to RND or MRND for the

be a reliable indicator of non-SLN status[44].

**5.1. Extent of dissection and regional recurrence rate**

all nodal disease and of 34% in patients with clinical nodal disease[48].

ies, MRND has been advocated even in the setting of clinical nodal disease.

**5. Neck dissection**

clinically N1 neck in melanoma[55].

Whether patients who undergo complete lymph node dissection (CLND) after confirmation of a positive SLN have a better prognosis than patients who undergo TLND after occurrence of clinical nodal disease is controversial. The results of retrospective studies that compared survival after CLND for a positive SLN with survival after TLND for clinical nodal disease remain controversial. Several retrospective studies, including a multicentric study and a matched control study, demonstrated a significant survival benefit for patients who under‐ went CLND for a positive SLN[33, 34]. In addition, a survival benefit was also demonstrated for patients whose primary tumor thickness was between 1 mm and 4 mm and who under‐ went CLND for a positive SLN[35]. In contrast, other retrospective studies demonstrated no significant difference in overall survival between patients who underwent CLND for a posi‐ tive SLN and those who underwent TLND for clinical nodal disease[36, 37].

The third interim analysis of the Multicenter Selective Lymphadenectomy Trial 1 (MLST-1), the only randomized control trial with available results, failed to demonstrate a 5-year sur‐ vival advantage for the SLNB group when compared with the observation group and only a disease-free survival benefit for the SLNB group[38]. In a subgroup analysis, patients who underwent CLND for a positive SLN showed an improvement in 5-year survival of about 20% when compared with patients who underwent TLND after nodal observation and sub‐ sequently occurring clinical nodal disease (72.3% vs 52.4%; P=.004). The nodal recurrence was lower in patients who had a negative SLN (4.0%) than in those who had a positive SLN but were observed without early CLND (15.6%). From these results, the authors concluded that microscopic metastasis would develop within the lymph nodes and that early LND may lead to accurate staging and survival improvement.

However, whether SLNB and/or CLND would be a therapeutic procedure remains unclear, and several authors have questioned this conclusion from the results of the MLST-1. First, they claim that it was inappropriate to conclude that early CLND would improve survival because this result was based on a postrandomization subgroup analysis[39]. Second, they question whether all microscopic metastases will develop into clinical nodal disease. That is, some microscopic metastases may show indolent behavior and not develop into clinical no‐ dal disease for a long time. In that case, comparison of the nodal recurrence rate between the 2 arms described above is an inappropriate analysis[37]. As a result, all that is currently clear is that SLNB can provide staging information that predicts prognosis and may impact clinical management.

## **4. Complete lymph node dissection**

#### **4.1. The role of complete lymph node dissection**

The therapeutic value of CLND and appropriate selection of patients for CLND remain questionable. The role of CLND in patients with positive SLNs is also a clinically important question because only 10% to 25% of patients with positive SLNs will have additional micro‐ scopic metastasis in non-SLNs[40-42], which means that approximately 80% of patients with positive SLNs may be spared CLND. Several authors categorized the SLN as several varia‐ bles and tried to find a reliable indicator of non-SLN status[43, 44]. However, it remains un‐ clear what size of microscopic metastasis of the SLN or which histopathologic location of metastasis in the SLN, such as subcapsular, parenchymal, multifocal, and extensive, would be a reliable indicator of non-SLN status[44].

The choice of the extent of CLND is ultimately decided by the individual surgeon. Few spe‐ cific recommendations are available in the published guidelines, with the common descrip‐ tion being ''a thorough dissection'' and reports of low levels of evidence supporting the appropriate surgical extent of CLND of the cervical, axillary, and inguinal regions[45-47].

## **5. Neck dissection**

**3. Does SLNB-guided early lymph node dissection improve survival rate?**

Whether patients who undergo complete lymph node dissection (CLND) after confirmation of a positive SLN have a better prognosis than patients who undergo TLND after occurrence of clinical nodal disease is controversial. The results of retrospective studies that compared survival after CLND for a positive SLN with survival after TLND for clinical nodal disease remain controversial. Several retrospective studies, including a multicentric study and a matched control study, demonstrated a significant survival benefit for patients who under‐ went CLND for a positive SLN[33, 34]. In addition, a survival benefit was also demonstrated for patients whose primary tumor thickness was between 1 mm and 4 mm and who under‐ went CLND for a positive SLN[35]. In contrast, other retrospective studies demonstrated no significant difference in overall survival between patients who underwent CLND for a posi‐

The third interim analysis of the Multicenter Selective Lymphadenectomy Trial 1 (MLST-1), the only randomized control trial with available results, failed to demonstrate a 5-year sur‐ vival advantage for the SLNB group when compared with the observation group and only a disease-free survival benefit for the SLNB group[38]. In a subgroup analysis, patients who underwent CLND for a positive SLN showed an improvement in 5-year survival of about 20% when compared with patients who underwent TLND after nodal observation and sub‐ sequently occurring clinical nodal disease (72.3% vs 52.4%; P=.004). The nodal recurrence was lower in patients who had a negative SLN (4.0%) than in those who had a positive SLN but were observed without early CLND (15.6%). From these results, the authors concluded that microscopic metastasis would develop within the lymph nodes and that early LND

However, whether SLNB and/or CLND would be a therapeutic procedure remains unclear, and several authors have questioned this conclusion from the results of the MLST-1. First, they claim that it was inappropriate to conclude that early CLND would improve survival because this result was based on a postrandomization subgroup analysis[39]. Second, they question whether all microscopic metastases will develop into clinical nodal disease. That is, some microscopic metastases may show indolent behavior and not develop into clinical no‐ dal disease for a long time. In that case, comparison of the nodal recurrence rate between the 2 arms described above is an inappropriate analysis[37]. As a result, all that is currently clear is that SLNB can provide staging information that predicts prognosis and may impact

The therapeutic value of CLND and appropriate selection of patients for CLND remain questionable. The role of CLND in patients with positive SLNs is also a clinically important

tive SLN and those who underwent TLND for clinical nodal disease[36, 37].

may lead to accurate staging and survival improvement.

clinical management.

504 Melanoma - From Early Detection to Treatment

**4. Complete lymph node dissection**

**4.1. The role of complete lymph node dissection**

## **5.1. Extent of dissection and regional recurrence rate**

The purpose of neck dissection is to control regional disease; it has little impact on overall sur‐ vival. However, the extent of neck dissection is still controversial and various extents of neck dissection have been advocated by several authors. Radical neck dissection (RND) including removal of level I-V (Fig. 5A) and nonlymphatic tissue such as the sternocleidomastoid muscle, the internal jugular vein, and the spinal accessary nerve has been the gold standard for neck dissection for melanoma[48]. Despite extensive areas of dissection, O'Brien et al. reported that regional control with RND was unsatisfactory, with regional recurrence of 28% in patients with all nodal disease and of 34% in patients with clinical nodal disease[48].

Generally, RND is associated with significant morbidity. Therefore, some authors have con‐ sidered modified RND (MRND) or functional neck dissection including preservation of any or all of the sternocleidomastoid muscle, the internal jugular vein, and the spinal accessory nerve[49, 50]. In studies of patients with clinical nodal disease, several authors demonstrat‐ ed that regional recurrence rates were 14-32% after RND, 0% after MRND, and 23% to 29% after selective neck dissection (SND), which is not statistically significant among the groups[51-53]. Byers also reported a 16% recurrence rate after MRND[54]. From these stud‐ ies, MRND has been advocated even in the setting of clinical nodal disease.

In addition, as an even more selective approach, the lymphatic drainage patterns of head and neck melanoma have been described by O'Brien et al. based on a consecutive series of over 270 neck dissections and parotidectomies (Fig. 5B)[52]. As described above, although several authors reported relatively high regional recurrence rates of 23% to 29% after SND, these studies include clinical N2-N3 (multiple involved nodes) disease, which will have a higher risk of recurrence than N1 disease[51, 52]. In a study of 37 consecutive patients with clinically N1 neck disease reported by White et al., 6 patients underwent RND, 24, MRND, and 7, SND. None of the 3 groups had any cases of local recurrence during a mean followup of 46 months[55], indicating that SND may be an alternative to RND or MRND for the clinically N1 neck in melanoma[55].

Furthermore, the appropriate extent of dissection is also unclear in patients with positive SLNs. Pu et al. reported 23 consecutive patients with positive SLNs who underwent MRND or super‐ ficial parotidectomy. Of those patients, 21 (91.3%) had no additional positive non-SLNs and only 2 (8.7 %) had 1 additional positive non-SLN[56]. No patient developed a regional local re‐ currence during a mean follow-up period of 23.7 months. The low prevalence of additional positive non-SLNs in MRND specimens suggests that when microscopic SLN metastasis ex‐ ists, nodal disease is confined to the SLN alone in most patients [56] and SND may be selected.

**5.2. Complication rate and technical variables**

extent of the neck dissection.

**6. Axillary lymph node dissection**

tients with microscopic metastasis[76].

**6.1. Extent of dissection and regional recurrence**

Significant complications associated with radical neck dissection may include injury to the facial and spinal accessory nerves, chylous fistula, and skin flap necrosis[65]. Although it is generally accepted that the rate of morbidity is reduced by MRND and further reduced by SND, detailed complication rates in the treatment of melanoma have not been reported. Ac‐ cording to the literature, neck dissection and parotidectomy is usually safe when appropri‐

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Technical variables mainly include skin incisions. Commonly used incisions are single Y, T, or double Y-type incisions, which provide optimal exposure of the entire neck. However, the edge of the flap sometimes has a poor blood supply and breakdown can result in the exposure of the major vessels. The three-point suture line gives a high incidence of postoper‐ ative scar contracture[66, 67]. The Mcfee incision was designed to eliminate the three-point exposure line, giving a good cosmetic result. However, the exposure is difficult, particularly in a short fat neck, and excessive traction of the skin flaps can result in damaging of the skin edges[67]. Large, single incisions such as the curtain flap, apron flap, U-flap, and Hockey stick incision offer a good blood supply and most of the scar lies within the relaxed skin ten‐ sion lines of the neck[68]. Each incision should be selected appropriately according to the

Axillary LND for patients with melanoma is performed for local control and staging[69]; the therapeutic value is still unclear. The axillary nodes are divided into level I, II, and III nodes. Level I nodes are lateral to the lateral edge of the pectoralis minor muscle. Level II nodes are between the medial and lateral edges of the pectoralis minor muscle. Level III nodes are me‐ dial to the medial edge of the pectorarlis minor muscle, in the apex of the axilla. The gener‐ ally recommended extent of dissection is from level I to III nodes because of the various drainage patterns in the second-tier nodes as well as the potentially increased risk of recur‐ rence with a lesser dissection[70, 71]. Several authors recommended a more extensive dissec‐ tion including the supraaxillary fat pad because approximately 14% of patients will have metastatic nodes in this area[69, 72]. In contrast, several authors have questioned whether a level III dissection is necessary in all melanoma patients with a positive SLN and advocated that level III dissection should be included only when suspicious nodes are present in this level [73-75]. Namm et al. also advocated that level I and II dissection should be performed for positive-SLN patients because of the low regional recurrence rate and low postoperative morbidity and concluded that level III dissection is not necessary for regional control in pa‐

As for the regional recurrence rate, unfortunately, most studies grouped together all of the dissected levels. Several authors reported a 10% to 19% regional recurrence rate during

ately planned preoperatively and when performed by well-experienced surgeons.

As for parotid gland nodes, patients with clinically palpable parotid nodes have a 28% to 58% risk of microscopic metastasis in the cervical nodes[57-59]. Although neck dissection should be included when clinical parotid disease is present, the need to treat the parotid no‐ des when clinical nodal disease of the neck is present is controversial. In such cases, many surgeons selectively perform superficial parotidectomy combined with a neck dissection based on O'Brien's lymphatic map (Fig. 5B) or the protocol of the individual institute[60].

However, the lymphatic drainage in the head and neck is generally complex and 8% to 43% of patients have unexpected drainage patterns in the occipital, postauricular, and contrala‐ teral nodes (Fig. 5A).[26, 61-64] Therefore, SND should be tailored to the individual patient according to the location of the SLN and second-tier nodes.


**Figure 5.** A)Lymphatic anatomy of the head and neck showing the 5 major lymph node levels and superficial nodes (B) Predicted lymphatic drainage and extent of neck dissection recommended by O'Brien et al.

## **5.2. Complication rate and technical variables**

Furthermore, the appropriate extent of dissection is also unclear in patients with positive SLNs. Pu et al. reported 23 consecutive patients with positive SLNs who underwent MRND or super‐ ficial parotidectomy. Of those patients, 21 (91.3%) had no additional positive non-SLNs and only 2 (8.7 %) had 1 additional positive non-SLN[56]. No patient developed a regional local re‐ currence during a mean follow-up period of 23.7 months. The low prevalence of additional positive non-SLNs in MRND specimens suggests that when microscopic SLN metastasis ex‐ ists, nodal disease is confined to the SLN alone in most patients [56] and SND may be selected.

As for parotid gland nodes, patients with clinically palpable parotid nodes have a 28% to 58% risk of microscopic metastasis in the cervical nodes[57-59]. Although neck dissection should be included when clinical parotid disease is present, the need to treat the parotid no‐ des when clinical nodal disease of the neck is present is controversial. In such cases, many surgeons selectively perform superficial parotidectomy combined with a neck dissection based on O'Brien's lymphatic map (Fig. 5B) or the protocol of the individual institute[60].

However, the lymphatic drainage in the head and neck is generally complex and 8% to 43% of patients have unexpected drainage patterns in the occipital, postauricular, and contrala‐ teral nodes (Fig. 5A).[26, 61-64] Therefore, SND should be tailored to the individual patient

**Figure 5.** A)Lymphatic anatomy of the head and neck showing the 5 major lymph node levels and superficial nodes

(B) Predicted lymphatic drainage and extent of neck dissection recommended by O'Brien et al.

according to the location of the SLN and second-tier nodes.

506 Melanoma - From Early Detection to Treatment

Significant complications associated with radical neck dissection may include injury to the facial and spinal accessory nerves, chylous fistula, and skin flap necrosis[65]. Although it is generally accepted that the rate of morbidity is reduced by MRND and further reduced by SND, detailed complication rates in the treatment of melanoma have not been reported. Ac‐ cording to the literature, neck dissection and parotidectomy is usually safe when appropri‐ ately planned preoperatively and when performed by well-experienced surgeons.

Technical variables mainly include skin incisions. Commonly used incisions are single Y, T, or double Y-type incisions, which provide optimal exposure of the entire neck. However, the edge of the flap sometimes has a poor blood supply and breakdown can result in the exposure of the major vessels. The three-point suture line gives a high incidence of postoper‐ ative scar contracture[66, 67]. The Mcfee incision was designed to eliminate the three-point exposure line, giving a good cosmetic result. However, the exposure is difficult, particularly in a short fat neck, and excessive traction of the skin flaps can result in damaging of the skin edges[67]. Large, single incisions such as the curtain flap, apron flap, U-flap, and Hockey stick incision offer a good blood supply and most of the scar lies within the relaxed skin ten‐ sion lines of the neck[68]. Each incision should be selected appropriately according to the extent of the neck dissection.

## **6. Axillary lymph node dissection**

### **6.1. Extent of dissection and regional recurrence**

Axillary LND for patients with melanoma is performed for local control and staging[69]; the therapeutic value is still unclear. The axillary nodes are divided into level I, II, and III nodes. Level I nodes are lateral to the lateral edge of the pectoralis minor muscle. Level II nodes are between the medial and lateral edges of the pectoralis minor muscle. Level III nodes are me‐ dial to the medial edge of the pectorarlis minor muscle, in the apex of the axilla. The gener‐ ally recommended extent of dissection is from level I to III nodes because of the various drainage patterns in the second-tier nodes as well as the potentially increased risk of recur‐ rence with a lesser dissection[70, 71]. Several authors recommended a more extensive dissec‐ tion including the supraaxillary fat pad because approximately 14% of patients will have metastatic nodes in this area[69, 72]. In contrast, several authors have questioned whether a level III dissection is necessary in all melanoma patients with a positive SLN and advocated that level III dissection should be included only when suspicious nodes are present in this level [73-75]. Namm et al. also advocated that level I and II dissection should be performed for positive-SLN patients because of the low regional recurrence rate and low postoperative morbidity and concluded that level III dissection is not necessary for regional control in pa‐ tients with microscopic metastasis[76].

As for the regional recurrence rate, unfortunately, most studies grouped together all of the dissected levels. Several authors reported a 10% to 19% regional recurrence rate during about a 30-month median follow-up[77-79]; however, in all 3 of those studies, the extent of dissection was not documented. Veenstra et al. reported a 4% regional recurrence rate and documented which levels were included when axillary LND was performed; however, they did not tease out the axillary recurrence rate specifically[80]. In the case of level I and II dis‐ section for patients with a positive SLN, a low recurrence rate of 4% during a median fol‐ low-up of approximately 39-month was reported[76].

recommended[74, 85-87]. For patients with clinically palpable nodal disease in the ingui‐ nal region alone, additional pelvic LND has not been widely accepted because of the lack of overall survival advantage[88, 89]. However, some authors advocated ilioinguinal LND because the rate of pelvic lymph node involvement in patients with palpable ingui‐ nal disease is 27% to 52%[87-92]. In a study of predictive factors for pelvic nodal status, Strobbe et al. reported that the Cloquet node has a limited sensitivity of 65% to predict involvement of the pelvic nodes and that the negative predictive value is 78%. In pa‐ tients with clinical inguinal nodal disease, a tumor-positive Cloquet node had a 69% risk (positive predictive value) of additional positive nodes[91]. They also showed that the number of positive nodes in the inguinal region is not a reliable predictive factor for the pelvic nodal status, with a sensitivity of 41% and a negative predictive value of 78%[91].

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Furthermore, the extent of dissection is more controversial in positive inguinal SLN patients. Van der Ploeg et al. reported that there is no lymphatic drainage to the inferior lateral zone, which is just lateral to the femoral artery and inferior to the level of saphenofemoral junction in the inguinal area, in patients with a positive SLN and advocated that this area need not be included in LND for such patients[93]. Pelvic nodes also seem unlikely to be involved when an inguinal SLN shows only microscopic metastasis[94, 95]. Several authors reported that 9% to 17 % of patients with a positive inguinal SLN also have positive pelvic nodes when ilioinguinal LND is performed[96-98]. In addition, a study evaluating lymphatic flow using lymphoscintigraphy and/or SPECT/CT demonstrated that over 50% of patients with a posi‐ tive SLN showed second-tier nodal drainage to the pelvic nodes[93]. This study suggests that a selective pelvic LND based on the location of the second-tier nodes may be appropri‐

As for the regional recurrence rate, published recurrence rates after inguinal or ilioinguinal LND for patients with clinical nodal disease is 0% to 33.6% (inguinal LND: 11.7%-13%; ilioinguinal LND: 0%-17.9%)[74, 85-89]. Sterne et al. reported that patients with palpable no‐ dal disease who underwent inguinal LND alone had a regional recurrence rate of 12.5% (2 of 16 patients), whereas for those who underwent ilioinguinal LND, it was 0% (0 of 25 patients) [85]. Pearlman et al. reported a modification of inguinal LND that does not violate the femo‐

In the field of urology, classical inguinal LND has traditionally been associated with an 80% to 100% risk of surgical morbidity[101]. In the treatment of melanoma, several authors re‐ ported that 20% to 77% of patients who underwent inguinal LND had postoperative mor‐ bidity such as skin necrosis and wound dehiscence (7%-55%), wound infection (5%-15%), lymphocele/seroma (2%-46%), and lymphedema (5%-64%).[102] Although concerns have been raised about the potential for increased morbidity in patients undergoing an additional pelvic LND[87, 103], the addition of pelvic LND to inguinal LND did not significantly in‐ crease the risk for postoperative wound complication[87, 101, 104, 105]. However, lymphe‐ dema was more common after inguinal LND alone in some studies, although 1 study

ral sheath. However, a 16% rate of regional recurrence was reported[100].

ate in positive SLN patients[93, 99].

**7.2. Complication rate and technical variables**

#### **6.2. Complication rate and technical variables**

Wrightson et al. reported a 19.9% complication rate among 262 patients undergoing axillary LND, most of which was thought to be level I-III dissection, for a positive SLN[81]. Several authors reported a complication rate of 14% to 21% for wound infection and of 19% to 36% for lymphocele when performing level I–III dissections[82, 83]. In contrast, Numm et al. re‐ ported that postoperative complications occurred in 11% of patients, with infectious compli‐ cations in 8% when performing level I and II dissection. However, comparative studies of level I-II dissection with and level I-III dissection have not been published. Although the definition of lymphedema varies among studies, a long-term lymphedema rate was report‐ ed to be 1% to 12%[72, 75, 81].

Evidence of an optimal surgical technique for axillary LND has not been shown. As tech‐ nical modifications, 2 incisions are mainly used. One is a transverse incision from the lat‐ eral edge of the pectoralis major muscle to the border of the latissimus dorsi muscle, and the other is an extended incision following the contour of the pectoralis major into the ax‐ illary apex and then down the medial arm[72, 84]. However, these incision variables would not affect the complication rate. Lawton et al. advocated preservation of the pector‐ alis major, the interpectoral, and the latissimus dorsi fascia during axillary LND to try to reduce lymphedema[84]. Over 110 elective and therapeutic fascia-preserving axillary LNDs developed a 5% incidence of long-term lymphedema, which is the same as or slightly lower than the incidence rates reported by the studies [72, 75, 81] described above. Optimal surgical exposure for level III dissection sometimes requires transection of the pectoralis minor muscle, and several authors suggested routine en bloc dissection of the pectoralis minor for TLND[16, 72, 75]. The long thoracic and thoracodorsal nerves are routinely preserved, although the intercostobrachial nerves are often sacrificed in TLND[73, 75]. As a result, no modifications clearly improve the complication rate, and on‐ ly the extent of dissection impacts the complication rate.

## **7. Ilioinguinnal lymph node dissection**

#### **7.1. Extent of dissection and regional recurrence rate**

The dissection areas subject to most controversy are inguinal LND alone or ilioinguinal LND (inguinal LND + iliac/obturator (pelvic) LND). When iliac or obturator node in‐ volvement is suspected clinically or radiologically, additional pelvic LND is generally recommended[74, 85-87]. For patients with clinically palpable nodal disease in the ingui‐ nal region alone, additional pelvic LND has not been widely accepted because of the lack of overall survival advantage[88, 89]. However, some authors advocated ilioinguinal LND because the rate of pelvic lymph node involvement in patients with palpable ingui‐ nal disease is 27% to 52%[87-92]. In a study of predictive factors for pelvic nodal status, Strobbe et al. reported that the Cloquet node has a limited sensitivity of 65% to predict involvement of the pelvic nodes and that the negative predictive value is 78%. In pa‐ tients with clinical inguinal nodal disease, a tumor-positive Cloquet node had a 69% risk (positive predictive value) of additional positive nodes[91]. They also showed that the number of positive nodes in the inguinal region is not a reliable predictive factor for the pelvic nodal status, with a sensitivity of 41% and a negative predictive value of 78%[91].

Furthermore, the extent of dissection is more controversial in positive inguinal SLN patients. Van der Ploeg et al. reported that there is no lymphatic drainage to the inferior lateral zone, which is just lateral to the femoral artery and inferior to the level of saphenofemoral junction in the inguinal area, in patients with a positive SLN and advocated that this area need not be included in LND for such patients[93]. Pelvic nodes also seem unlikely to be involved when an inguinal SLN shows only microscopic metastasis[94, 95]. Several authors reported that 9% to 17 % of patients with a positive inguinal SLN also have positive pelvic nodes when ilioinguinal LND is performed[96-98]. In addition, a study evaluating lymphatic flow using lymphoscintigraphy and/or SPECT/CT demonstrated that over 50% of patients with a posi‐ tive SLN showed second-tier nodal drainage to the pelvic nodes[93]. This study suggests that a selective pelvic LND based on the location of the second-tier nodes may be appropri‐ ate in positive SLN patients[93, 99].

As for the regional recurrence rate, published recurrence rates after inguinal or ilioinguinal LND for patients with clinical nodal disease is 0% to 33.6% (inguinal LND: 11.7%-13%; ilioinguinal LND: 0%-17.9%)[74, 85-89]. Sterne et al. reported that patients with palpable no‐ dal disease who underwent inguinal LND alone had a regional recurrence rate of 12.5% (2 of 16 patients), whereas for those who underwent ilioinguinal LND, it was 0% (0 of 25 patients) [85]. Pearlman et al. reported a modification of inguinal LND that does not violate the femo‐ ral sheath. However, a 16% rate of regional recurrence was reported[100].

#### **7.2. Complication rate and technical variables**

about a 30-month median follow-up[77-79]; however, in all 3 of those studies, the extent of dissection was not documented. Veenstra et al. reported a 4% regional recurrence rate and documented which levels were included when axillary LND was performed; however, they did not tease out the axillary recurrence rate specifically[80]. In the case of level I and II dis‐ section for patients with a positive SLN, a low recurrence rate of 4% during a median fol‐

Wrightson et al. reported a 19.9% complication rate among 262 patients undergoing axillary LND, most of which was thought to be level I-III dissection, for a positive SLN[81]. Several authors reported a complication rate of 14% to 21% for wound infection and of 19% to 36% for lymphocele when performing level I–III dissections[82, 83]. In contrast, Numm et al. re‐ ported that postoperative complications occurred in 11% of patients, with infectious compli‐ cations in 8% when performing level I and II dissection. However, comparative studies of level I-II dissection with and level I-III dissection have not been published. Although the definition of lymphedema varies among studies, a long-term lymphedema rate was report‐

Evidence of an optimal surgical technique for axillary LND has not been shown. As tech‐ nical modifications, 2 incisions are mainly used. One is a transverse incision from the lat‐ eral edge of the pectoralis major muscle to the border of the latissimus dorsi muscle, and the other is an extended incision following the contour of the pectoralis major into the ax‐ illary apex and then down the medial arm[72, 84]. However, these incision variables would not affect the complication rate. Lawton et al. advocated preservation of the pector‐ alis major, the interpectoral, and the latissimus dorsi fascia during axillary LND to try to reduce lymphedema[84]. Over 110 elective and therapeutic fascia-preserving axillary LNDs developed a 5% incidence of long-term lymphedema, which is the same as or slightly lower than the incidence rates reported by the studies [72, 75, 81] described above. Optimal surgical exposure for level III dissection sometimes requires transection of the pectoralis minor muscle, and several authors suggested routine en bloc dissection of the pectoralis minor for TLND[16, 72, 75]. The long thoracic and thoracodorsal nerves are routinely preserved, although the intercostobrachial nerves are often sacrificed in TLND[73, 75]. As a result, no modifications clearly improve the complication rate, and on‐

The dissection areas subject to most controversy are inguinal LND alone or ilioinguinal LND (inguinal LND + iliac/obturator (pelvic) LND). When iliac or obturator node in‐ volvement is suspected clinically or radiologically, additional pelvic LND is generally

low-up of approximately 39-month was reported[76].

ly the extent of dissection impacts the complication rate.

**7. Ilioinguinnal lymph node dissection**

**7.1. Extent of dissection and regional recurrence rate**

**6.2. Complication rate and technical variables**

ed to be 1% to 12%[72, 75, 81].

508 Melanoma - From Early Detection to Treatment

In the field of urology, classical inguinal LND has traditionally been associated with an 80% to 100% risk of surgical morbidity[101]. In the treatment of melanoma, several authors re‐ ported that 20% to 77% of patients who underwent inguinal LND had postoperative mor‐ bidity such as skin necrosis and wound dehiscence (7%-55%), wound infection (5%-15%), lymphocele/seroma (2%-46%), and lymphedema (5%-64%).[102] Although concerns have been raised about the potential for increased morbidity in patients undergoing an additional pelvic LND[87, 103], the addition of pelvic LND to inguinal LND did not significantly in‐ crease the risk for postoperative wound complication[87, 101, 104, 105]. However, lymphe‐ dema was more common after inguinal LND alone in some studies, although 1 study specifically evaluating the incidence of lymphedema found no difference between the 2 pro‐ cedures[87, 106, 107]. The lack of consensus about the complications of additional pelvic LND may suggest that when clinically indicated, concern about increased morbidity should not be a reason to avoid ilioinguinal LND, although patients may suffer from the operating time and cost.

The commonly described technical variables of ilioinguinal LND include different type of skin incision, thick skin flap, preservation of the large saphenous vein, transposition of the sartorius muscle over the femoral vessels, continuity dissection with division of the inguinal ligament, and trimming of the skin edges at the time of closure[108].

> **Figure 6. Ilioinguinal LND using paired incisions.** (A) Incision lines. The incision below the inguinal crease is fusiform to include the skin overlying the metastatic node. (B) Operating field after dissection. The abdominal wall was incised

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As another procedure in an attempt to decrease lymphocele, Nakamura et al. reported a simple method using intraoperative injection of isosulfan blue during inguinal LND without modifications to identify leakage from an injured lymphatic vessels for the prevention of lymphocele (Fig. 7)[112]. There was no incidence of lymphocele in the isosulfan blue injec‐ tion group and the lymphatic drainage output from the inguinal region was clearly less,

Despite many technical variables, it is difficult to evaluate each technique because of the dif‐ ferent study designs, variable definitions of complications, and different patient popula‐ tions. Multicenter, randomized prospective trials with a standardized definition of

**Figure 7.** Intraoperative injection of blue dye during inguinal LND for detection of injured lymphatic vessels. (A) Intra‐ cutaneous injection of isosulfan blue around the right inguinal region just after inguinal LND. (B) Blue-staining lym‐

parallel to the inguinal ligament, which was preserved under the bipedicle flap.

leading to early removal of the suction catheter.

complications are required in the future.

phatic leak (arrow) in the surgical field, which was ligated.

Several skin incisions are used: a Lazy-S incision from just medial to the anterior superi‐ or iliac spine to the inferior margin of the femoral triangle, paired oblique incisions (Fig. 6A), or an oblique/transverse incision above the inguinal crease with a longitudinal inci‐ sion below and a skin bridge between[73, 84, 100]. Lazy-S incision provides optimal ex‐ posure and less subcutaneous lymphatic disruption[108]. In contrast, paired oblique incisions or an oblique/transverse incision can avoid an incision in the inguinal crease to reduce skin necrosis and wound dehiscence[84]. Recently, however, Spillane et al. report‐ ed minimal-access 3- to 6-cm-long paired incisions above and below the inguinal liga‐ ment, which showed no significant difference in wound and lymphedema complications[109]. A thick skin flap elevated at the level of the Scarpa fascia may im‐ prove skin necrosis and wound dehiscence rates; however, a 26% to 34% rate of skin ne‐ crosis and wound infection was reported[84, 100]. The preservation of the saphenous vein and the sartorius transposition flap for vessel coverage were designed to improve lymphedema rates, with no incidence of lymphedema[100]. When performing ilioingui‐ nal LND, technical variables include a continuity dissection by dividing the inguinal liga‐ ment or an abdominal wall incision above and parallel to the inguinal ligament (Fig. 6B) to expose the retroperitoneal space[73, 84, 86]. Although advantages of inguinal ligament division include optimal exposure and possible continuity dissection, the main disadvant‐ age is possible long-term abdominal wall weakness that may lead to abdominal incision‐ al hernia. As another modification, Lawton et al. advocated fascia-preserving ilioinguinal LND, which is similar to the modified axillary dissection described above in the section on axillary LND, and the long-term lymphedema rate was 14%. Video-assisted endoscop‐ ic inguinal LND is currently investigated as a minimally invasive and less morbid ap‐ proach but is not widely used[110, 111].

Despite such modifications, a comparative study reported by Sabel et al. demonstrated no significant difference in wound and lymphedema complications between modified inguinal LND (incision avoiding the inguinal crease, saphenous vein preservation, or sartorius trans‐ position) and conventional inguinal LND[107]. However, although insignificant, saphenous vein preservation decreased the lymphedema rate from 30% to 13% and the wound compli‐ cation rate from 20% to 7%. An incision avoiding the inguinal crease also decreased the wound complication rate from 21% to 9%, which is also statistically insignificant. Thus, these modifications seem to offer promise in decreasing morbidity.

Sentinel Lymph Node Biopsy for Melanoma and Surgical Approach to Lymph Node Metastasis http://dx.doi.org/10.5772/53625 511

specifically evaluating the incidence of lymphedema found no difference between the 2 pro‐ cedures[87, 106, 107]. The lack of consensus about the complications of additional pelvic LND may suggest that when clinically indicated, concern about increased morbidity should not be a reason to avoid ilioinguinal LND, although patients may suffer from the operating

The commonly described technical variables of ilioinguinal LND include different type of skin incision, thick skin flap, preservation of the large saphenous vein, transposition of the sartorius muscle over the femoral vessels, continuity dissection with division of the inguinal

Several skin incisions are used: a Lazy-S incision from just medial to the anterior superi‐ or iliac spine to the inferior margin of the femoral triangle, paired oblique incisions (Fig. 6A), or an oblique/transverse incision above the inguinal crease with a longitudinal inci‐ sion below and a skin bridge between[73, 84, 100]. Lazy-S incision provides optimal ex‐ posure and less subcutaneous lymphatic disruption[108]. In contrast, paired oblique incisions or an oblique/transverse incision can avoid an incision in the inguinal crease to reduce skin necrosis and wound dehiscence[84]. Recently, however, Spillane et al. report‐ ed minimal-access 3- to 6-cm-long paired incisions above and below the inguinal liga‐ ment, which showed no significant difference in wound and lymphedema complications[109]. A thick skin flap elevated at the level of the Scarpa fascia may im‐ prove skin necrosis and wound dehiscence rates; however, a 26% to 34% rate of skin ne‐ crosis and wound infection was reported[84, 100]. The preservation of the saphenous vein and the sartorius transposition flap for vessel coverage were designed to improve lymphedema rates, with no incidence of lymphedema[100]. When performing ilioingui‐ nal LND, technical variables include a continuity dissection by dividing the inguinal liga‐ ment or an abdominal wall incision above and parallel to the inguinal ligament (Fig. 6B) to expose the retroperitoneal space[73, 84, 86]. Although advantages of inguinal ligament division include optimal exposure and possible continuity dissection, the main disadvant‐ age is possible long-term abdominal wall weakness that may lead to abdominal incision‐ al hernia. As another modification, Lawton et al. advocated fascia-preserving ilioinguinal LND, which is similar to the modified axillary dissection described above in the section on axillary LND, and the long-term lymphedema rate was 14%. Video-assisted endoscop‐ ic inguinal LND is currently investigated as a minimally invasive and less morbid ap‐

Despite such modifications, a comparative study reported by Sabel et al. demonstrated no significant difference in wound and lymphedema complications between modified inguinal LND (incision avoiding the inguinal crease, saphenous vein preservation, or sartorius trans‐ position) and conventional inguinal LND[107]. However, although insignificant, saphenous vein preservation decreased the lymphedema rate from 30% to 13% and the wound compli‐ cation rate from 20% to 7%. An incision avoiding the inguinal crease also decreased the wound complication rate from 21% to 9%, which is also statistically insignificant. Thus,

these modifications seem to offer promise in decreasing morbidity.

ligament, and trimming of the skin edges at the time of closure[108].

proach but is not widely used[110, 111].

time and cost.

510 Melanoma - From Early Detection to Treatment

**Figure 6. Ilioinguinal LND using paired incisions.** (A) Incision lines. The incision below the inguinal crease is fusiform to include the skin overlying the metastatic node. (B) Operating field after dissection. The abdominal wall was incised parallel to the inguinal ligament, which was preserved under the bipedicle flap.

As another procedure in an attempt to decrease lymphocele, Nakamura et al. reported a simple method using intraoperative injection of isosulfan blue during inguinal LND without modifications to identify leakage from an injured lymphatic vessels for the prevention of lymphocele (Fig. 7)[112]. There was no incidence of lymphocele in the isosulfan blue injec‐ tion group and the lymphatic drainage output from the inguinal region was clearly less, leading to early removal of the suction catheter.

Despite many technical variables, it is difficult to evaluate each technique because of the dif‐ ferent study designs, variable definitions of complications, and different patient popula‐ tions. Multicenter, randomized prospective trials with a standardized definition of complications are required in the future.

**Figure 7.** Intraoperative injection of blue dye during inguinal LND for detection of injured lymphatic vessels. (A) Intra‐ cutaneous injection of isosulfan blue around the right inguinal region just after inguinal LND. (B) Blue-staining lym‐ phatic leak (arrow) in the surgical field, which was ligated.

## **8. Adjuvant radiation therapy**

Regional recurrence occurs in 20% to 50% of patients after TLND. High-risk factors associat‐ ed with regional recurrence include a cervical lymph node basin, large lymph nodes, multi‐ ple positive lymph nodes, and extracapsular extension[113]. Patients with such risk factors are appropriate candidates for adjuvant radiation therapy, and several nonrandomized studies have demonstrated that adjuvant radiation therapy after CLND for patients with re‐ gional nodal disease can reduce the risk of regional recurrence to between 5% and 20% [114-118]. In a prospective phase II study by the Trans Tasman Radiation Oncology Group (TROG Study 96.06) of adjuvant radiation therapy after CLND for patients with regional no‐ dal disease, the regional control rate was 91%[118].

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Although adjuvant radiation therapy can be effective in achieving regional control after TLND, it increases chronic lymphedema, particularly in the inguinal region, which is the major morbidity associated with TLND[119].

## **9. Conclusions**

The surgical approach to regional lymph node metastasis of cutaneous melanoma is chal‐ lenging. SLNB allows accurate staging of nodal status and prediction of prognosis. A posi‐ tive SLN should be treated with CLND for regional control. However, the impact on SLNB on overall survival remains unclear, and the appropriate surgical extent of CLND in the cer‐ vical, axillary, and inguinal regions is also debated. More research is required to provide evidence-based guidelines for surgeons about the extent of LND and to investigate the fac‐ tors that may lead to a more patient-tailored approach.

## **Acknowledgements**

We thank Ms F. Miyamasu, associate professor of the Medical English Communications Center, University of Tsukuba, for expert English revision.

This work was partly supported by the National Cancer Center Research and Development Fund (23-A-22), and the Japanese Association of Dermatologic Surgery.

## **Author details**

Yasuhiro Nakamura\* and Fujio Otsuka

\*Address all correspondence to: ynakamurta3@yahoo.co.jp

Department of Dermatology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan

## **References**

**8. Adjuvant radiation therapy**

512 Melanoma - From Early Detection to Treatment

dal disease, the regional control rate was 91%[118].

tors that may lead to a more patient-tailored approach.

Center, University of Tsukuba, for expert English revision.

and Fujio Otsuka

\*Address all correspondence to: ynakamurta3@yahoo.co.jp

Fund (23-A-22), and the Japanese Association of Dermatologic Surgery.

major morbidity associated with TLND[119].

**9. Conclusions**

**Acknowledgements**

**Author details**

Yasuhiro Nakamura\*

Regional recurrence occurs in 20% to 50% of patients after TLND. High-risk factors associat‐ ed with regional recurrence include a cervical lymph node basin, large lymph nodes, multi‐ ple positive lymph nodes, and extracapsular extension[113]. Patients with such risk factors are appropriate candidates for adjuvant radiation therapy, and several nonrandomized studies have demonstrated that adjuvant radiation therapy after CLND for patients with re‐ gional nodal disease can reduce the risk of regional recurrence to between 5% and 20% [114-118]. In a prospective phase II study by the Trans Tasman Radiation Oncology Group (TROG Study 96.06) of adjuvant radiation therapy after CLND for patients with regional no‐

Although adjuvant radiation therapy can be effective in achieving regional control after TLND, it increases chronic lymphedema, particularly in the inguinal region, which is the

The surgical approach to regional lymph node metastasis of cutaneous melanoma is chal‐ lenging. SLNB allows accurate staging of nodal status and prediction of prognosis. A posi‐ tive SLN should be treated with CLND for regional control. However, the impact on SLNB on overall survival remains unclear, and the appropriate surgical extent of CLND in the cer‐ vical, axillary, and inguinal regions is also debated. More research is required to provide evidence-based guidelines for surgeons about the extent of LND and to investigate the fac‐

We thank Ms F. Miyamasu, associate professor of the Medical English Communications

This work was partly supported by the National Cancer Center Research and Development

Department of Dermatology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan


[14] Sumner WE, 3rd, Ross MI, Mansfield PF et al. Implications of lymphatic drainage to unusual sentinel lymph node sites in patients with primary cutaneous melanoma. Cancer 2002; 95: 354-60.

[28] Fujisawa Y, Nakamura Y, Kawachi Y, Otsuka F. A custom-made, low-cost intraoper‐ ative fluorescence navigation system with indocyanine green for sentinel lymph

Sentinel Lymph Node Biopsy for Melanoma and Surgical Approach to Lymph Node Metastasis

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**Chapter 19**

**Cutaneous Melanoma − Surgical Treatment**

Although surgical excision of the primary melanoma is internationally accepted as the treat‐ ment of choice, several questions concerning the follow-up schedule are still debated contro‐ versially. Incision biopsies should be avoided, except in selected cases (wide lesions or critical anatomic locations). Excision biopsy is preferred to give the dermatopathologist an optimal specimen and to allow evaluation of the excision margins for residual tumor. Since the beginning of the last century, the recommendation has been to excise a primary melano‐ ma with safety margins. In 1907 Handley [1] analyzed the pattern of satellite metastases in melanoma and recommended excision of the primary tumor with a margin of 1 inch (2.54 cm) from the edge of the tumor. In the 1970s and 1980s, safety margins of 5 cm, independent of tumor thickness, were the surgical standard.[2] The World Health Organization Melano‐ ma Group performed the first surgical trial to compare lower safety margins of 1 and 3 cm in primary melanomas with less than 2 mm of tumor thickness.[3] The group found no dif‐ ferences in survival and only slightly increased local recurrence rates in the patients with narrower excision margins. These results led to the recommendation of 1-cm margins in pa‐ tients with primary melanomas with less than 1 mm tumor thickness. Later comparisons of 5- and 2-cm safety margins in thick primary melanomas revealed no significant advantages for the 5-cm margins.[4] A recent trial, however, comparing 1- and 3-cm safety margins in thick primary melanoma with 2 mm and more tumor thickness showed an increased rate of local recurrence in those with the small safety margins and a simultaneous trend towards decreased survival rates. These findings indicate that the safety margin cannot be reduced to

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

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

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

Mario Santinami, Roberto Patuzzo, Roberta Ruggeri, Gianpiero Castelli,

**1.1. Excision margins for primary tumor**

http://dx.doi.org/10.5772/54105

**1. Introduction**

Andrea Maurichi, Giulia Baffa and Carlotta Tinti

Additional information is available at the end of the chapter

## **Cutaneous Melanoma − Surgical Treatment**

Mario Santinami, Roberto Patuzzo, Roberta Ruggeri, Gianpiero Castelli, Andrea Maurichi, Giulia Baffa and Carlotta Tinti

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54105

## **1. Introduction**

## **1.1. Excision margins for primary tumor**

Although surgical excision of the primary melanoma is internationally accepted as the treat‐ ment of choice, several questions concerning the follow-up schedule are still debated contro‐ versially. Incision biopsies should be avoided, except in selected cases (wide lesions or critical anatomic locations). Excision biopsy is preferred to give the dermatopathologist an optimal specimen and to allow evaluation of the excision margins for residual tumor. Since the beginning of the last century, the recommendation has been to excise a primary melano‐ ma with safety margins. In 1907 Handley [1] analyzed the pattern of satellite metastases in melanoma and recommended excision of the primary tumor with a margin of 1 inch (2.54 cm) from the edge of the tumor. In the 1970s and 1980s, safety margins of 5 cm, independent of tumor thickness, were the surgical standard.[2] The World Health Organization Melano‐ ma Group performed the first surgical trial to compare lower safety margins of 1 and 3 cm in primary melanomas with less than 2 mm of tumor thickness.[3] The group found no dif‐ ferences in survival and only slightly increased local recurrence rates in the patients with narrower excision margins. These results led to the recommendation of 1-cm margins in pa‐ tients with primary melanomas with less than 1 mm tumor thickness. Later comparisons of 5- and 2-cm safety margins in thick primary melanomas revealed no significant advantages for the 5-cm margins.[4] A recent trial, however, comparing 1- and 3-cm safety margins in thick primary melanoma with 2 mm and more tumor thickness showed an increased rate of local recurrence in those with the small safety margins and a simultaneous trend towards decreased survival rates. These findings indicate that the safety margin cannot be reduced to

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

zero in melanoma.[5] Different national guidelines now give uniform recommendations for the excision of primary melanoma.[6-9]

SLN biopsy was 7.5%, despite CLND in nearly all patients.[15] Overall, the SLN biopsy pro‐ cedure is well tolerated and associated with low complication rates.[16] Although clinical variables such as older age have been variably reported as lower risk factors,[17-19] there are no specific variables that can reliably identify patients with intermediate-thickness mela‐ nomas at low risk for metastases. The definition of intermediate-thickness melanoma varied by study. Nevertheless, it is clinically consistent with contemporary staging systems to de‐ fine intermediate-thickness melanomas as those measuring 1 to 4 mm.[20] Clinical judgment must be used when considering SLN biopsy in patients with comorbid medical conditions. The individual risks and benefits of the procedure should be weighed against the operative and anesthetic risks as well as potential competing causes of mortality. Complications after SLN biopsy are uncommon. The overall complication rate reported in the Multicenter Selec‐ tive Lymphadenectomy Trial I (MSLT I) was 10.1% after SLN biopsy compared with 32.7% after CLND.[21] The most common complications after SLN removal documented in MSLT I included seroma (5.5%), infection (4.6%), and wound separation (1.2%). The Sunbelt Mela‐ noma Trial similarly showed a low overall rate of complications from SLN biopsy (4.6%) compared with CLND (23.2%).[16,17] Most complications were noted to be short-term is‐ sues that resolved over time with wound care and selective use of antibiotics. Accurate iden‐ tification of patients with node-negative (stage I or II) or node-positive (stage III) disease improves staging and may facilitate regional disease control and decision making for treat‐ ment with adjuvant therapy.[14,22] With substantive changes in the melanoma staging guidelines in 2002, the AJCC staging system effectively linked disease stage and prognosis. [23,24] At that time, the number of nodal metastases and whether nodal disease was occult or clinically apparent (ie, how the N category was defined with regard to burden of disease) were noted to be the most significant independent predictors of survival in patients with stage III melanomas. With later iterations of the last AJCC staging system,[10] additional re‐ finements were made in the N category based on the prognostic value of distinguishing mi‐ crometastases (as would be diagnosed after SLN biopsy) from macrometastases.[25,26] A melanoma macrometastasis is detected by clinical examination (not by size criteria) and con‐ firmed pathologically, whereas a melanoma micrometastasis is a clinically occult nodal metastasis that is detected by a pathologist on microscopic examination of lymph nodes, with or without immunohistochemistry, and is not limited by any minimum or maximum size threshold. Recognizing the value of examining SLNs to detect low volumes of metastat‐ ic disease (aggregates of only a few cells), the current staging system[10,27] incorporates the use of immunohistochemistry and eliminates any minimum size threshold for defining no‐ dal metastases. Molecular diagnostics, such as reverse transcriptase-polymerase chain reac‐ tion, have unproven prognostic significance, and these results are not used to define positive nodes. As a result, more refined definitions of the N category are now used for classification. Distinct differences in classifications have validated prognostic significance. For example, 5 year survival ranges from 70% for patients with one SLN positive with micrometastatic dis‐ ease to 39% for patients with > four involved nodes or with nodes that are extensively involved (eg, matted nodes).1 Although SLN biopsy has been widely accepted for the patho‐ logic staging of patients with intermediate-thickness melanomas, somewhat more contro‐ versy exists regarding the value of this procedure for patients with thick primary tumors

Cutaneous Melanoma − Surgical Treatment http://dx.doi.org/10.5772/54105 525

### **2. Sentinel lymph node biopsy and lymph node dissection**

Metastasis to regional nodes is the most important prognostic factor in patients with earlystage melanoma and has been shown to occur in approximately 20% of patients with inter‐ mediate-thickness tumors.[10,11] As such, it is critically important to identify those patients for whom the expected benefits of resecting regional lymph nodes outweigh the risks of sur‐ gical morbidity. The technique of lymphatic mapping and sentinel lymph node (SLN) biop‐ sy for melanoma has emerged during the last 2 decades as a minimally invasive approach to evaluate regional lymph node basins in patients with intermediate- and high-risk primary cutaneous melanoma. Goals of SLN biopsy include accurate nodal staging, identification of patients with clinically occult, microscopic lymph node disease who may benefit from fur‐ ther treatment, regional nodal control, and a possible survival benefit.[12,13] Moreover, this approach may also identify a subset of patients for whom further treatment is not indicated, sparing them from unnecessary surgical procedures or systemic therapies.[12,13] In this re‐ view, we examine the evolution of SLN biopsy as a technique, the preoperative assessment and operative strategy, the pathologic evaluation of the SLN, the current practice guidelines, the prognostic significance of SLN biopsy findings, and the potential complications of the procedure and address some of the current areas of controversies in the field. Sentinel lymph node (SLN) biopsy is commonly used in melanoma and has been endorsed by the American Joint Committee on Cancer (AJCC) as a valuable staging procedure for patients with melanoma who are at risk of clinically occult nodal metastases. This highly accurate and low-morbidity staging procedure should be used to guide treatment decisions (ie, com‐ pletion lymph node dissection [CLND] and adjuvant therapy) as well as entry into clinical trials.[14] To develop and formalize guideline recommendations for the use of SLN biopsy in oncology practice, the American Society of Clinical Oncology (ASCO) and Society of Sur‐ gical Oncology (SSO) convened a joint Expert Panel in order to better define what are the indications for SLN biopsy as well as what is the role of CLND. SLN biopsy is recommend‐ ed for patients with intermediate-thickness cutaneous melanomas (Breslow thickness, 1 to 4 mm) of any anatomic site. Routine use of SLN biopsy in this population provides accurate staging. Although there are few studies focusing specifically on patients with thick melano‐ mas (T4; Breslow thickness, > 4 mm), use of SLN biopsy in this population may be recom‐ mended for staging purposes and to facilitate regional disease control. There is insufficient evidence to support routine SLN biopsy for patients with thin melanomas (T1; Breslow thickness, < 1 mm), although it may be considered in selected patients with high-risk fea‐ tures when the benefits of pathologic staging may outweigh the potential risks of the proce‐ dure. Such risk factors may include ulceration or mitotic rate ≥ 1/mm2 , especially in the subgroup of patients with melanomas 0.75 to 0.99 mm in Breslow thickness. After a positive SLN biopsy, 97.5% of patients underwent CLND, and 20.1% were found to have additional positive lymph nodes. Overall, the recurrence rate in the same nodal basin after a positive SLN biopsy was 7.5%, despite CLND in nearly all patients.[15] Overall, the SLN biopsy pro‐ cedure is well tolerated and associated with low complication rates.[16] Although clinical variables such as older age have been variably reported as lower risk factors,[17-19] there are no specific variables that can reliably identify patients with intermediate-thickness mela‐ nomas at low risk for metastases. The definition of intermediate-thickness melanoma varied by study. Nevertheless, it is clinically consistent with contemporary staging systems to de‐ fine intermediate-thickness melanomas as those measuring 1 to 4 mm.[20] Clinical judgment must be used when considering SLN biopsy in patients with comorbid medical conditions. The individual risks and benefits of the procedure should be weighed against the operative and anesthetic risks as well as potential competing causes of mortality. Complications after SLN biopsy are uncommon. The overall complication rate reported in the Multicenter Selec‐ tive Lymphadenectomy Trial I (MSLT I) was 10.1% after SLN biopsy compared with 32.7% after CLND.[21] The most common complications after SLN removal documented in MSLT I included seroma (5.5%), infection (4.6%), and wound separation (1.2%). The Sunbelt Mela‐ noma Trial similarly showed a low overall rate of complications from SLN biopsy (4.6%) compared with CLND (23.2%).[16,17] Most complications were noted to be short-term is‐ sues that resolved over time with wound care and selective use of antibiotics. Accurate iden‐ tification of patients with node-negative (stage I or II) or node-positive (stage III) disease improves staging and may facilitate regional disease control and decision making for treat‐ ment with adjuvant therapy.[14,22] With substantive changes in the melanoma staging guidelines in 2002, the AJCC staging system effectively linked disease stage and prognosis. [23,24] At that time, the number of nodal metastases and whether nodal disease was occult or clinically apparent (ie, how the N category was defined with regard to burden of disease) were noted to be the most significant independent predictors of survival in patients with stage III melanomas. With later iterations of the last AJCC staging system,[10] additional re‐ finements were made in the N category based on the prognostic value of distinguishing mi‐ crometastases (as would be diagnosed after SLN biopsy) from macrometastases.[25,26] A melanoma macrometastasis is detected by clinical examination (not by size criteria) and con‐ firmed pathologically, whereas a melanoma micrometastasis is a clinically occult nodal metastasis that is detected by a pathologist on microscopic examination of lymph nodes, with or without immunohistochemistry, and is not limited by any minimum or maximum size threshold. Recognizing the value of examining SLNs to detect low volumes of metastat‐ ic disease (aggregates of only a few cells), the current staging system[10,27] incorporates the use of immunohistochemistry and eliminates any minimum size threshold for defining no‐ dal metastases. Molecular diagnostics, such as reverse transcriptase-polymerase chain reac‐ tion, have unproven prognostic significance, and these results are not used to define positive nodes. As a result, more refined definitions of the N category are now used for classification. Distinct differences in classifications have validated prognostic significance. For example, 5 year survival ranges from 70% for patients with one SLN positive with micrometastatic dis‐ ease to 39% for patients with > four involved nodes or with nodes that are extensively involved (eg, matted nodes).1 Although SLN biopsy has been widely accepted for the patho‐ logic staging of patients with intermediate-thickness melanomas, somewhat more contro‐ versy exists regarding the value of this procedure for patients with thick primary tumors

zero in melanoma.[5] Different national guidelines now give uniform recommendations for

Metastasis to regional nodes is the most important prognostic factor in patients with earlystage melanoma and has been shown to occur in approximately 20% of patients with inter‐ mediate-thickness tumors.[10,11] As such, it is critically important to identify those patients for whom the expected benefits of resecting regional lymph nodes outweigh the risks of sur‐ gical morbidity. The technique of lymphatic mapping and sentinel lymph node (SLN) biop‐ sy for melanoma has emerged during the last 2 decades as a minimally invasive approach to evaluate regional lymph node basins in patients with intermediate- and high-risk primary cutaneous melanoma. Goals of SLN biopsy include accurate nodal staging, identification of patients with clinically occult, microscopic lymph node disease who may benefit from fur‐ ther treatment, regional nodal control, and a possible survival benefit.[12,13] Moreover, this approach may also identify a subset of patients for whom further treatment is not indicated, sparing them from unnecessary surgical procedures or systemic therapies.[12,13] In this re‐ view, we examine the evolution of SLN biopsy as a technique, the preoperative assessment and operative strategy, the pathologic evaluation of the SLN, the current practice guidelines, the prognostic significance of SLN biopsy findings, and the potential complications of the procedure and address some of the current areas of controversies in the field. Sentinel lymph node (SLN) biopsy is commonly used in melanoma and has been endorsed by the American Joint Committee on Cancer (AJCC) as a valuable staging procedure for patients with melanoma who are at risk of clinically occult nodal metastases. This highly accurate and low-morbidity staging procedure should be used to guide treatment decisions (ie, com‐ pletion lymph node dissection [CLND] and adjuvant therapy) as well as entry into clinical trials.[14] To develop and formalize guideline recommendations for the use of SLN biopsy in oncology practice, the American Society of Clinical Oncology (ASCO) and Society of Sur‐ gical Oncology (SSO) convened a joint Expert Panel in order to better define what are the indications for SLN biopsy as well as what is the role of CLND. SLN biopsy is recommend‐ ed for patients with intermediate-thickness cutaneous melanomas (Breslow thickness, 1 to 4 mm) of any anatomic site. Routine use of SLN biopsy in this population provides accurate staging. Although there are few studies focusing specifically on patients with thick melano‐ mas (T4; Breslow thickness, > 4 mm), use of SLN biopsy in this population may be recom‐ mended for staging purposes and to facilitate regional disease control. There is insufficient evidence to support routine SLN biopsy for patients with thin melanomas (T1; Breslow thickness, < 1 mm), although it may be considered in selected patients with high-risk fea‐ tures when the benefits of pathologic staging may outweigh the potential risks of the proce‐

**2. Sentinel lymph node biopsy and lymph node dissection**

dure. Such risk factors may include ulceration or mitotic rate ≥ 1/mm2

subgroup of patients with melanomas 0.75 to 0.99 mm in Breslow thickness. After a positive SLN biopsy, 97.5% of patients underwent CLND, and 20.1% were found to have additional positive lymph nodes. Overall, the recurrence rate in the same nodal basin after a positive

, especially in the

the excision of primary melanoma.[6-9]

524 Melanoma - From Early Detection to Treatment

(T4; Breslow thickness, > 4 mm). Conventional wisdom asserts that patients with thick mela‐ nomas have a high risk of systemic disease at the time of diagnosis and that no survival ben‐ efit can be derived from removal of regional lymph nodes. However, among patients without distant disease, it can be argued that those with thick melanomas have indications for SLN biopsy similar to those of patients with intermediate-thickness melanomas and de‐ rive the same benefits from SLN biopsy as a pathologic staging procedure. One of the main advantages of SLN biopsy in patients with thick melanomas is better regional disease con‐ trol, which is especially important in a population with > 30% chance of lymph node in‐ volvement.[25,28] Evidence from multiple retrospective studies has demonstrated that SLN biopsy provides important staging and prognostic information for patients with thick mela‐ nomas. Seven of eight published studies-each evaluating SLN biopsy in > 100 patients with T4 melanomas-have shown that SLN biopsy is a significant predictor of overall survival. [11,25,26,28-33] The one study that did not show a significant difference in overall survival demonstrated a significant difference in disease-free survival.[29] A majority (70%) of mela‐ nomas diagnosed are thin melanomas (T1; Breslow thickness, < 1 mm).[34] In general, the routine use of SLN biopsy in patients with thin melanomas has not been advocated, because the overall risk of nodal involvement is estimated to be only approximately 5.1%,[35] al‐ though there are reports of positive SLNs in up to 20% of patients in subsets with thin mela‐ nomas (especially those that are 0.75 to 0.99 mm in thickness with ulceration and/or mitotic rate ≥ 1/mm<sup>2</sup> ).[27] An individualized approach to SLN biopsy for patients with thin melano‐ mas has been advocated in many treatment centers based on risk factors that have been shown to be associated with SLN metastasis. Further investigation is also needed to better identify the subgroups of patients with thin melanomas with a greater risk of nodal metasta‐ sis. CLND is recommended for all patients with a positive SLN biopsy. CLND achieves re‐ gional disease control, although whether CLND after a positive SLN biopsy improves survival is the subject of the ongoing Multicenter Selective Lymphadenectomy Trial II (MSLT II). Currently, CLND is the standard recommendation for patients with tumor-posi‐ tive SLNs. The goals of CLND are to improve survival rates, maximize regional disease con‐ trol, and minimize operative morbidity. Whether CLND improves survival is the subject of the ongoing prospective randomized MSLT II study.[36] The main objective of MSLT II is to determine if there is a therapeutic benefit to removing any non-SLNs in patients who have already had their tumor-positive SLN removed. In MSLT I, patients with demonstrated no‐ dal metastases had a survival advantage with early intervention compared with those who had a delayed lymphadenectomy when they presented with clinically evident nodal meta‐ stases.[5] Hence, although two goals of CLND are regional disease control and cure, there is currently insufficient evidence to determine whether omission of CLND is safe. In the two large prospective randomized trials (ie, the Sunbelt Melanoma Trial and MSLT I), the rate of positive non-SLNs among patients who underwent CLND for a tumor-positive SLN was 16%.[17,37] In a retrospective multi-institutional study by Wong et al,[38] which included 134 highly selected patients with positive SLNs who did not undergo CLND, regional nodal metastasis was a component of first recurrence in 15% of these patients. Therefore, it is rea‐ sonable to conclude from these data that the risk of developing regional nodal metastasis as a first site of recurrence, if no CLND is performed, is at least 15% to 20%.[39,40] In MSLT I,

the rate of regional nodal recurrence after CLND was 4.2%5

dures in staging and follow-up.

**3. Treatment of** *in transit* **metastases**

it was 4.9% (unpublished data). These rates are much lower than the 15% rate of regional nodal recurrence as a site of first metastasis and the 41% overall regional nodal recurrence rate when CLND was not performed, reported in the study by Wong et al.[37] Until final results of MSLT II are available, we will not be able to determine, with higher-level evi‐ dence, the impact of CLND on regional disease control. Until that time, the best available evidence suggests that CLND is effective at achieving regional disease control in the majori‐ ty of patients with positive SLNs. MSLT I showed no benefit of CLND with regard to overall survival, likely because only a minority of patients (16%) had tumor-positive SLNs, and the majority of the patients in the study would not have been helped by removal of regional lymph nodes.[37] However, the 5-year survival rate for patients with tumor-positive SLNs who underwent CLND was 72.3% compared with 52.4% for patients who did not undergo SLN biopsy and developed palpable nodal disease (hazard ratio, 0.51; 95% CI, 0.32 to 0.81; P =.004). CLND should be performed until there is convincing evidence that it does not im‐ prove regional disease control or survival. CLND is associated with risks of long-term mor‐ bidity, especially lymphedema. However, morbidity with CLND may be considerably worse when it is delayed until there is clinically evident disease. The observed increases in morbidity for patients who have undergone therapeutic lymphadenectomy for palpable dis‐ ease and the increased morbidity associated with radiation therapy support the continued use of CLND for patients with a positive SLN biopsy rather than delayed CLND for palpa‐ ble disease. There is a need for future clinical trials to address many unresolved research questions related to the use of SLN biopsy in patients with melanoma. These include: deter‐ mining precise criteria for selecting which patients should undergo SLN biopsy, determin‐ ing whether early identification of metastases in the SLN truly improves survival or merely represents lead-time bias, identifying which criteria for individualized risks best inform ap‐ propriate risk stratification for patients at high risk for relapse and those for whom CLND and/or adjuvant therapy are suitable, and establishing the role of prognostic markers from the primary melanoma and SLN to help assign appropriate risk stratification. Results from MSLT II, in which patients were randomly assigned to CLND or observation, will help de‐ termine whether there is any benefit to CLND after a positive sentinel node in patients with melanoma. Answers to these questions will assist clinicians and patients with making deci‐ sions and ultimately help to identify patients who may avoid expensive and intrusive proce‐

In 5–8% of cases, melanoma patients will develop in-transit metastasis (IT-mets). Standard regional treatment options include surgical resection, isolated limb perfusion (ILP), isolated limb infusion (ILI) and Electrochemotherapy. As regional recurrence often precedes system‐ ic disease, amputative surgery is in general no longer practiced, although old series of radi‐ cal surgery have demonstrated that some patients with IT-mets confined to the limb can be cured.[42,43] Simple surgical resection may suffice for incidental and low numbers of IT-

; in the Sunbelt Melanoma Trial,

Cutaneous Melanoma − Surgical Treatment http://dx.doi.org/10.5772/54105 527

the rate of regional nodal recurrence after CLND was 4.2%5 ; in the Sunbelt Melanoma Trial, it was 4.9% (unpublished data). These rates are much lower than the 15% rate of regional nodal recurrence as a site of first metastasis and the 41% overall regional nodal recurrence rate when CLND was not performed, reported in the study by Wong et al.[37] Until final results of MSLT II are available, we will not be able to determine, with higher-level evi‐ dence, the impact of CLND on regional disease control. Until that time, the best available evidence suggests that CLND is effective at achieving regional disease control in the majori‐ ty of patients with positive SLNs. MSLT I showed no benefit of CLND with regard to overall survival, likely because only a minority of patients (16%) had tumor-positive SLNs, and the majority of the patients in the study would not have been helped by removal of regional lymph nodes.[37] However, the 5-year survival rate for patients with tumor-positive SLNs who underwent CLND was 72.3% compared with 52.4% for patients who did not undergo SLN biopsy and developed palpable nodal disease (hazard ratio, 0.51; 95% CI, 0.32 to 0.81; P =.004). CLND should be performed until there is convincing evidence that it does not im‐ prove regional disease control or survival. CLND is associated with risks of long-term mor‐ bidity, especially lymphedema. However, morbidity with CLND may be considerably worse when it is delayed until there is clinically evident disease. The observed increases in morbidity for patients who have undergone therapeutic lymphadenectomy for palpable dis‐ ease and the increased morbidity associated with radiation therapy support the continued use of CLND for patients with a positive SLN biopsy rather than delayed CLND for palpa‐ ble disease. There is a need for future clinical trials to address many unresolved research questions related to the use of SLN biopsy in patients with melanoma. These include: deter‐ mining precise criteria for selecting which patients should undergo SLN biopsy, determin‐ ing whether early identification of metastases in the SLN truly improves survival or merely represents lead-time bias, identifying which criteria for individualized risks best inform ap‐ propriate risk stratification for patients at high risk for relapse and those for whom CLND and/or adjuvant therapy are suitable, and establishing the role of prognostic markers from the primary melanoma and SLN to help assign appropriate risk stratification. Results from MSLT II, in which patients were randomly assigned to CLND or observation, will help de‐ termine whether there is any benefit to CLND after a positive sentinel node in patients with melanoma. Answers to these questions will assist clinicians and patients with making deci‐ sions and ultimately help to identify patients who may avoid expensive and intrusive proce‐ dures in staging and follow-up.

## **3. Treatment of** *in transit* **metastases**

(T4; Breslow thickness, > 4 mm). Conventional wisdom asserts that patients with thick mela‐ nomas have a high risk of systemic disease at the time of diagnosis and that no survival ben‐ efit can be derived from removal of regional lymph nodes. However, among patients without distant disease, it can be argued that those with thick melanomas have indications for SLN biopsy similar to those of patients with intermediate-thickness melanomas and de‐ rive the same benefits from SLN biopsy as a pathologic staging procedure. One of the main advantages of SLN biopsy in patients with thick melanomas is better regional disease con‐ trol, which is especially important in a population with > 30% chance of lymph node in‐ volvement.[25,28] Evidence from multiple retrospective studies has demonstrated that SLN biopsy provides important staging and prognostic information for patients with thick mela‐ nomas. Seven of eight published studies-each evaluating SLN biopsy in > 100 patients with T4 melanomas-have shown that SLN biopsy is a significant predictor of overall survival. [11,25,26,28-33] The one study that did not show a significant difference in overall survival demonstrated a significant difference in disease-free survival.[29] A majority (70%) of mela‐ nomas diagnosed are thin melanomas (T1; Breslow thickness, < 1 mm).[34] In general, the routine use of SLN biopsy in patients with thin melanomas has not been advocated, because the overall risk of nodal involvement is estimated to be only approximately 5.1%,[35] al‐ though there are reports of positive SLNs in up to 20% of patients in subsets with thin mela‐ nomas (especially those that are 0.75 to 0.99 mm in thickness with ulceration and/or mitotic

).[27] An individualized approach to SLN biopsy for patients with thin melano‐

mas has been advocated in many treatment centers based on risk factors that have been shown to be associated with SLN metastasis. Further investigation is also needed to better identify the subgroups of patients with thin melanomas with a greater risk of nodal metasta‐ sis. CLND is recommended for all patients with a positive SLN biopsy. CLND achieves re‐ gional disease control, although whether CLND after a positive SLN biopsy improves survival is the subject of the ongoing Multicenter Selective Lymphadenectomy Trial II (MSLT II). Currently, CLND is the standard recommendation for patients with tumor-posi‐ tive SLNs. The goals of CLND are to improve survival rates, maximize regional disease con‐ trol, and minimize operative morbidity. Whether CLND improves survival is the subject of the ongoing prospective randomized MSLT II study.[36] The main objective of MSLT II is to determine if there is a therapeutic benefit to removing any non-SLNs in patients who have already had their tumor-positive SLN removed. In MSLT I, patients with demonstrated no‐ dal metastases had a survival advantage with early intervention compared with those who had a delayed lymphadenectomy when they presented with clinically evident nodal meta‐ stases.[5] Hence, although two goals of CLND are regional disease control and cure, there is currently insufficient evidence to determine whether omission of CLND is safe. In the two large prospective randomized trials (ie, the Sunbelt Melanoma Trial and MSLT I), the rate of positive non-SLNs among patients who underwent CLND for a tumor-positive SLN was 16%.[17,37] In a retrospective multi-institutional study by Wong et al,[38] which included 134 highly selected patients with positive SLNs who did not undergo CLND, regional nodal metastasis was a component of first recurrence in 15% of these patients. Therefore, it is rea‐ sonable to conclude from these data that the risk of developing regional nodal metastasis as a first site of recurrence, if no CLND is performed, is at least 15% to 20%.[39,40] In MSLT I,

rate ≥ 1/mm<sup>2</sup>

526 Melanoma - From Early Detection to Treatment

In 5–8% of cases, melanoma patients will develop in-transit metastasis (IT-mets). Standard regional treatment options include surgical resection, isolated limb perfusion (ILP), isolated limb infusion (ILI) and Electrochemotherapy. As regional recurrence often precedes system‐ ic disease, amputative surgery is in general no longer practiced, although old series of radi‐ cal surgery have demonstrated that some patients with IT-mets confined to the limb can be cured.[42,43] Simple surgical resection may suffice for incidental and low numbers of IT- mets. In cases of rapid recurrences and multiple IT-mets, other techniques must provide an attractive treatment option that can improve local control markedly and thereby quality of life. ILP, developed by Creech et al., achieves a 20-fold higher concentration of chemothera‐ peutic drugs when compared with systemic therapy.[44,45] Melphalan-based ILP (M-ILP) has been the standard treatment and has been reported to achieve overall complete response (CR) rates in the range of about 50%.[46]In general large IT-mets showed a poor response and inhomogeneous uptake comparable with locally advanced soft tissue sarcomas (STS). The introduction of tumor necrosis factor-α (TNF) changed this situation dramatically. Large tumors now reacted very well to ILP.[47] This led to a successful multicenter trial in Europe and the approval of TNF-based ILP (TM-ILP) for unresectable extremity soft tissue sarcomas (STS).[48] Similar encouraging results were reported for the use of TNF in ILP for melanoma patients.[49] Preclinical and clinical studies suggested that a reduction of the dose of TNF to 1 mg for the arm and 2 mg for the leg might be as effective as the higher doses.[50-53]Isolated limb infusion (ILI) is a minimally invasive technique for delivering high-dose regional chemotherapy in locally advanced melanoma. It was first described by Thompson et al. in 1994 from the Sydney Melanoma Unit as a simplified alternative to ILP [54,55]. Percutaneous arterial and venous catheters are placed in the affected extremity by interventional radiologists and a tourniquet is placed proximal to the catheter tips to allow isolation of the limb from the systemic circulation. High-dose chemotherapy (e.g. melphalan and actinomycin-D) is infused into a hyperthermic, hypoxic limb via the arterial catheter and blood is withdrawn from the venous catheter to be re-infused into the arterial side. Therefore, it is a quicker, safer, and cheaper procedure with reported response rates compa‐ rable to ILP.[56,57] Although the primary indication for this technique is melanoma, it has been successfully applied to other tumors such as soft-tissue sarcomas,[58] Merkel cell tu‐ mor,[59] and cutaneous T-cell lymphoma.[60]

The electrodes were connected to a pulse generator (Cliniporator™; Igea, Modena, Italy). This generator produces high voltages (up to 1000 V), but delivered as a compressed train of eight pulses at a frequency of 5000 Hz and 100 μs duration, and therefore well tolerated by the patient. The software controls and stores the applied voltage and the actual current de‐ livered to each tumor. ECT could be repeated every 8–12 weeks according to local response,

Cutaneous Melanoma − Surgical Treatment http://dx.doi.org/10.5772/54105 529

Conventional teaching maintains that resection is not indicated in patients with distant metastases, except for palliation. This dogma stems from the concept that patients with mul‐ tiple metastases usually also have occult micrometastases and circulating tumor cells. How‐ ever the results of surgical treatment of stage IV melanoma patients have improved considerably over the past two decades. Recent studies [75] provide further evidence of the beneficial role of surgery for distant metastases of melanoma. Our findings indicate a sur‐ vival advantage for a surgical approach, even in patients with high-risk visceral metastases or multiple metastases that may require multiple operations for complete resection. At least 55 % of stage IV patients may be eligible to undergo surgery as part of their treatment plan and the surgeon should play an integral role in evaluation and treatment planning for all patients with stage IV recurrence of melanoma. One potential therapeutic advantage of re‐ section is that it may delay disease progression by interrupting the metastatic cascade asso‐ ciated with hematogenous seeding of cells to other sites.[76]In addition, it immediately reduces tumor burden and thereby decreases tumor-induced immune suppression.[77] Fi‐ nally, metastasectomy may enhance the patient's endogenous immune defences or response to adjuvant immunotherapy and thus maintain a complete clinical remission. Surgery for distant metastases has been improved by development of more advanced imaging techni‐ ques that can detect lesions as small as 5–10 mm.[78] These techniques can differentiate pa‐ tients with multiple versus limited metastases, allowing surgeons to better judge the extent of disease and plan the operative procedure necessary for complete resection. In addition, modern advances in anaesthesia, surgical techniques and supportive care have reduced op‐ erative mortality from multiple metastasectomy with a corresponding reduction in morbidi‐ ty and finally, shorter postsurgical hospitalizations have decreased the total costs of cancer surgery. Surgical therapy for stage IV disease remains controversial. The development of metastases is a complex process and the rationale for surgical resection of metastatic mela‐ noma is multifactorial. First, reduction of tumor burden through surgical resection limits disease progression by interrupting the metastatic cascade associated with haematogenous seeding of cells to other sites. Unlike chemotherapy, surgery can easily eradicate tumor masses 2 cm or larger. Second, surgery may reverse tumor-induced immunosuppression, re‐ storing immune function and inhibiting metastatic progression. Third, most patients tolerate surgical resection to a much greater extent than they can tolerate adverse effects of systemic therapy and recurrences after initial metastasectomy can also be treated through a secon‐ dary resection of metastases. Last, metastasectomy does not preclude systemic therapy;

the appearance of new lesions and the patient's tolerance of the treatment.

**4. Surgical approach for distant metastases**

Electrochemotherapy (ECT) represents an effective therapeutic option for skin tumors that has received experimental and clinical support in recent years.[61-71] The European stand‐ ard operating procedures for ECT emphasize the technical aspects of the procedure and have established this treatment in clinical practice.[72,73] In recent years, the effectiveness of ECT treatment has been confirmed in several small series of patients with melanoma.[71] At present, ECT is employed routinely with encouraging results not only for superficial tumor control but also to preserve quality of life.[70]Patients with regional or distant skin or subcu‐ taneous metastases, with or without visceral disease, could undergo this technique. Eligibili‐ ty criteria were the following: melanoma stage IIIC–IV (American Joint Committee on Cancer, 6th edition)[74] lesions no deeper than 3 cm suitable for electrode insertion; no anti‐ cancer treatments 4 weeks before and 8 weeks after ECT; age more than 18 years; and an Eastern Cooperative Oncology Group performance status equal to or less than 2. Exclusion criteria included: allergy to Bleomycin; pulmonary, cardiac or liver impairment; epilepsy; life expectancy less than 3 months; active infection; brain metastases; and cardiac pacemaker in patients with chest wall metastases. Bleomycin is administered intravenously (15 000 units/m2 in a bolus administered over 60 s) and was followed, within 8 min after intrave‐ nous injection, by the application of brief electric pulses to each tumor nodule. Electric cur‐ rents were delivered by means of a 2–3-cm long needle electrode according to lesion size. The electrodes were connected to a pulse generator (Cliniporator™; Igea, Modena, Italy). This generator produces high voltages (up to 1000 V), but delivered as a compressed train of eight pulses at a frequency of 5000 Hz and 100 μs duration, and therefore well tolerated by the patient. The software controls and stores the applied voltage and the actual current de‐ livered to each tumor. ECT could be repeated every 8–12 weeks according to local response, the appearance of new lesions and the patient's tolerance of the treatment.

## **4. Surgical approach for distant metastases**

mets. In cases of rapid recurrences and multiple IT-mets, other techniques must provide an attractive treatment option that can improve local control markedly and thereby quality of life. ILP, developed by Creech et al., achieves a 20-fold higher concentration of chemothera‐ peutic drugs when compared with systemic therapy.[44,45] Melphalan-based ILP (M-ILP) has been the standard treatment and has been reported to achieve overall complete response (CR) rates in the range of about 50%.[46]In general large IT-mets showed a poor response and inhomogeneous uptake comparable with locally advanced soft tissue sarcomas (STS). The introduction of tumor necrosis factor-α (TNF) changed this situation dramatically. Large tumors now reacted very well to ILP.[47] This led to a successful multicenter trial in Europe and the approval of TNF-based ILP (TM-ILP) for unresectable extremity soft tissue sarcomas (STS).[48] Similar encouraging results were reported for the use of TNF in ILP for melanoma patients.[49] Preclinical and clinical studies suggested that a reduction of the dose of TNF to 1 mg for the arm and 2 mg for the leg might be as effective as the higher doses.[50-53]Isolated limb infusion (ILI) is a minimally invasive technique for delivering high-dose regional chemotherapy in locally advanced melanoma. It was first described by Thompson et al. in 1994 from the Sydney Melanoma Unit as a simplified alternative to ILP [54,55]. Percutaneous arterial and venous catheters are placed in the affected extremity by interventional radiologists and a tourniquet is placed proximal to the catheter tips to allow isolation of the limb from the systemic circulation. High-dose chemotherapy (e.g. melphalan and actinomycin-D) is infused into a hyperthermic, hypoxic limb via the arterial catheter and blood is withdrawn from the venous catheter to be re-infused into the arterial side. Therefore, it is a quicker, safer, and cheaper procedure with reported response rates compa‐ rable to ILP.[56,57] Although the primary indication for this technique is melanoma, it has been successfully applied to other tumors such as soft-tissue sarcomas,[58] Merkel cell tu‐

Electrochemotherapy (ECT) represents an effective therapeutic option for skin tumors that has received experimental and clinical support in recent years.[61-71] The European stand‐ ard operating procedures for ECT emphasize the technical aspects of the procedure and have established this treatment in clinical practice.[72,73] In recent years, the effectiveness of ECT treatment has been confirmed in several small series of patients with melanoma.[71] At present, ECT is employed routinely with encouraging results not only for superficial tumor control but also to preserve quality of life.[70]Patients with regional or distant skin or subcu‐ taneous metastases, with or without visceral disease, could undergo this technique. Eligibili‐ ty criteria were the following: melanoma stage IIIC–IV (American Joint Committee on Cancer, 6th edition)[74] lesions no deeper than 3 cm suitable for electrode insertion; no anti‐ cancer treatments 4 weeks before and 8 weeks after ECT; age more than 18 years; and an Eastern Cooperative Oncology Group performance status equal to or less than 2. Exclusion criteria included: allergy to Bleomycin; pulmonary, cardiac or liver impairment; epilepsy; life expectancy less than 3 months; active infection; brain metastases; and cardiac pacemaker in patients with chest wall metastases. Bleomycin is administered intravenously (15 000

in a bolus administered over 60 s) and was followed, within 8 min after intrave‐

nous injection, by the application of brief electric pulses to each tumor nodule. Electric cur‐ rents were delivered by means of a 2–3-cm long needle electrode according to lesion size.

mor,[59] and cutaneous T-cell lymphoma.[60]

528 Melanoma - From Early Detection to Treatment

units/m2

Conventional teaching maintains that resection is not indicated in patients with distant metastases, except for palliation. This dogma stems from the concept that patients with mul‐ tiple metastases usually also have occult micrometastases and circulating tumor cells. How‐ ever the results of surgical treatment of stage IV melanoma patients have improved considerably over the past two decades. Recent studies [75] provide further evidence of the beneficial role of surgery for distant metastases of melanoma. Our findings indicate a sur‐ vival advantage for a surgical approach, even in patients with high-risk visceral metastases or multiple metastases that may require multiple operations for complete resection. At least 55 % of stage IV patients may be eligible to undergo surgery as part of their treatment plan and the surgeon should play an integral role in evaluation and treatment planning for all patients with stage IV recurrence of melanoma. One potential therapeutic advantage of re‐ section is that it may delay disease progression by interrupting the metastatic cascade asso‐ ciated with hematogenous seeding of cells to other sites.[76]In addition, it immediately reduces tumor burden and thereby decreases tumor-induced immune suppression.[77] Fi‐ nally, metastasectomy may enhance the patient's endogenous immune defences or response to adjuvant immunotherapy and thus maintain a complete clinical remission. Surgery for distant metastases has been improved by development of more advanced imaging techni‐ ques that can detect lesions as small as 5–10 mm.[78] These techniques can differentiate pa‐ tients with multiple versus limited metastases, allowing surgeons to better judge the extent of disease and plan the operative procedure necessary for complete resection. In addition, modern advances in anaesthesia, surgical techniques and supportive care have reduced op‐ erative mortality from multiple metastasectomy with a corresponding reduction in morbidi‐ ty and finally, shorter postsurgical hospitalizations have decreased the total costs of cancer surgery. Surgical therapy for stage IV disease remains controversial. The development of metastases is a complex process and the rationale for surgical resection of metastatic mela‐ noma is multifactorial. First, reduction of tumor burden through surgical resection limits disease progression by interrupting the metastatic cascade associated with haematogenous seeding of cells to other sites. Unlike chemotherapy, surgery can easily eradicate tumor masses 2 cm or larger. Second, surgery may reverse tumor-induced immunosuppression, re‐ storing immune function and inhibiting metastatic progression. Third, most patients tolerate surgical resection to a much greater extent than they can tolerate adverse effects of systemic therapy and recurrences after initial metastasectomy can also be treated through a secon‐ dary resection of metastases. Last, metastasectomy does not preclude systemic therapy; however, if metastasectomy is delayed, increasing tumor burden may make disease unre‐ sectable. In addition the advent of newer and better systemic therapies makes the role of surgical resection more relevant today than ever before. Timing of surgery versus systemic treatment is another important end point. The development of new and effective drugs in the systemic treatment of stage IV melanoma patients have been reported recently, with the BRAF inhibitor Vemurafinib and the monoclonal antibody Ipilimumab; other targeted drugs are being developed, and some are currently being tested in the clinical setting. Thus a ther‐ apeutic strategy combining new drugs with aggressive surgery in selected cases of melano‐ ma metastatic disease could be designed in the following years.

[8] C. Garbe, A. Hauschild, M. Volkenandt et al.Evidence and interdisciplinary consen‐ sus-based German guidelines: surgical treatment and radiotherapy of melanoma.

Cutaneous Melanoma − Surgical Treatment http://dx.doi.org/10.5772/54105 531

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## **Author details**

Mario Santinami1 , Roberto Patuzzo1 , Roberta Ruggeri1 , Gianpiero Castelli1 , Andrea Maurichi1 , Giulia Baffa1 and Carlotta Tinti1

1 Melanoma Sarcoma Unit Fondazione Istituto Tumori Milano, Italy

Dermatology Unit Ospedale Umberto I Siracusa, Italy

## **References**


[8] C. Garbe, A. Hauschild, M. Volkenandt et al.Evidence and interdisciplinary consen‐ sus-based German guidelines: surgical treatment and radiotherapy of melanoma. Melanoma Res, 18: 61–67, 2008.

however, if metastasectomy is delayed, increasing tumor burden may make disease unre‐ sectable. In addition the advent of newer and better systemic therapies makes the role of surgical resection more relevant today than ever before. Timing of surgery versus systemic treatment is another important end point. The development of new and effective drugs in the systemic treatment of stage IV melanoma patients have been reported recently, with the BRAF inhibitor Vemurafinib and the monoclonal antibody Ipilimumab; other targeted drugs are being developed, and some are currently being tested in the clinical setting. Thus a ther‐ apeutic strategy combining new drugs with aggressive surgery in selected cases of melano‐

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, Giulia Baffa1

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Andrea Maurichi1

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**Chapter 20**

**Therapeutic Agents for Advanced Melanoma**

Melanoma is an extremely complicated disease, with many gene mutations and alternations in signaling pathways. Because effective treatment for melanoma is lacking, the prognosis for metastatic melanoma patients remains very poor. Over the past 30 years, significant ef‐ forts have been made to search for better agents or strategies to fight this deadly disease. Numerous clinical trials at different stages have been carried out. Although most of them have failed, some did show very promising results. New treatment strategies have resulted in paradigm shift in our approach to melanoma therapy. These shining examples may mark‐

The year 2011 marked a fruitful year for melanoma research. The FDA approved three drugs for advanced melanoma treatment: ipilimumab (an anti-cytotoxic T lymphocyte anti‐ gen-4 monoclonal antibody), vemurafenib (a selective BRAF inhibitor), and pegylated inter‐ feron-α2b for adjuvant setting usage [1]. However, there are still significant limitations for melanoma treatment. Ipilimumab only prolonged the survival time for metastatic melano‐ ma patient from an average of 6.5 months to an average of 10 months. This treatment also has been associated with strong immunological adverse effects; severe to fatal autoimmune reactions were seen in 12.9% of patients treated with ipilimumab in a clinical trial that enrol‐ led 676 melanoma patients [2]. Vemurafenib is not effective for melanoma patients with wide type BRAF, and confirmation of BRAFV600E mutation-positive melanoma using an FDA-approved test is required before treatment with vemurafenib. This treatment also only prolonged the median survival time for advanced melanoma patients 2~3 months [1]. More importantly, despite the high initial response rate for patients with BRAFV600E mutation to vemurafenib, virtually all the patients developed primary or acquired resistance to this drug in the end [3]. With the rapidly rising incidents of this disease and the high resistance to cur‐ rent therapeutic agents, developing more effective drugs for melanoma is very important.

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

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

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

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

Zhao Wang, Wei Li and Duane D. Miller

http://dx.doi.org/10.5772/53632

**1. Introduction**

Additional information is available at the end of the chapter

edly change our philosophy about melanoma treatment.

## **Therapeutic Agents for Advanced Melanoma**

Zhao Wang, Wei Li and Duane D. Miller

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53632

## **1. Introduction**

Melanoma is an extremely complicated disease, with many gene mutations and alternations in signaling pathways. Because effective treatment for melanoma is lacking, the prognosis for metastatic melanoma patients remains very poor. Over the past 30 years, significant ef‐ forts have been made to search for better agents or strategies to fight this deadly disease. Numerous clinical trials at different stages have been carried out. Although most of them have failed, some did show very promising results. New treatment strategies have resulted in paradigm shift in our approach to melanoma therapy. These shining examples may mark‐ edly change our philosophy about melanoma treatment.

The year 2011 marked a fruitful year for melanoma research. The FDA approved three drugs for advanced melanoma treatment: ipilimumab (an anti-cytotoxic T lymphocyte anti‐ gen-4 monoclonal antibody), vemurafenib (a selective BRAF inhibitor), and pegylated inter‐ feron-α2b for adjuvant setting usage [1]. However, there are still significant limitations for melanoma treatment. Ipilimumab only prolonged the survival time for metastatic melano‐ ma patient from an average of 6.5 months to an average of 10 months. This treatment also has been associated with strong immunological adverse effects; severe to fatal autoimmune reactions were seen in 12.9% of patients treated with ipilimumab in a clinical trial that enrol‐ led 676 melanoma patients [2]. Vemurafenib is not effective for melanoma patients with wide type BRAF, and confirmation of BRAFV600E mutation-positive melanoma using an FDA-approved test is required before treatment with vemurafenib. This treatment also only prolonged the median survival time for advanced melanoma patients 2~3 months [1]. More importantly, despite the high initial response rate for patients with BRAFV600E mutation to vemurafenib, virtually all the patients developed primary or acquired resistance to this drug in the end [3]. With the rapidly rising incidents of this disease and the high resistance to cur‐ rent therapeutic agents, developing more effective drugs for melanoma is very important.

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

In this chapter, we will review available therapeutic agents for advanced melanoma as well as agents that are still under clinical development. We will focus on their mechanism of ac‐ tion, development history, and therapeutic effects. In learning from our efforts in the past, we must continue to challenge the current paradigms of treatment as we forge new paths to more effective treatment options. This will likely involve a multimodal approach to therapy utilizing all of the available tools in our arsenal.

As for the dose, it has been demonstrated that 850~1000 mg/m2

**2.2. Temozolomide**

ated. This single dose administration appears to deliver clinical improvements similar to those observed with multiple doses that provide the same total dose per cycle. This should be the reference standard for randomized trials comparing new therapies with DTIC [7].

Temozolomide (TMZ) is an orally active alkylating agent. It's a prodrug of MTIC and conge‐ ner of DTIC. It has been available in the US since August 1999 and in other countries since the early 2000s. The therapeutic benefit of temozolomide depends on its ability to alkylate/ methylate DNA, which most often occurs at the N-7 or O-6 positions of guanine residues. This methylation damages the DNA and triggers the death of tumor cells. However, some tumor cells are able to repair this type of DNA damage, and therefore diminish the thera‐ peutic efficacy of temozolomide, by expressing an enzyme called O-6-methylguanine-DNA

The single agent activity of TMZ in metastatic melanoma has been established in several phase 1 and 2 studies [9]. In a randomized trial of 305 patients with advanced melanoma, TMZ showed efficacy at least equivalent to that of DTIC in terms of objective response rate, time to progression, and overall disease-free survival [10]. TMZ was tolerated very well and showed an advantage in terms of improvement in the quality of life. More patients showed improvement or maintenance of physical functioning at Week 12. That trial excluded pa‐ tients who had brain metastases. Because the trial design was intended to demonstrate the superiority of TMZ over DTIC, rather than equivalence, the FDA did not accept the results of that trial as grounds for approving a melanoma indication for TMZ. But in clinical prac‐

TMZ has demonstrated efficacy in the treatment of variety of solid tumors, especially in brain malignancies, which is a manifestation of its far greater ability to penetrate the central nervous system (CNS). Taking into account the high rate of CNS recurrence as a site of fail‐ ure after cytotoxic chemotherapy, TMZ may represent a viable alternative to DTIC, which is

Sorafenib (BAY43-9006, developed by Bayer Pharmaceuticals, West Haven CT, trade name Nexavar) is an orally administered tyrosine kinase inhibitor. It is a potent inhibitor of the BRAF kinase that is frequently mutated in melanoma, as well as an inhibitor of the Vascular Endothelial Growth Factor (VEGF) receptor and other kinases. It targets the adenosine tri‐ phosphate-binding site of the BRAF kinase and inhibits both wild-type and mutant BRAF *in vitro*. Sorafenib was approved by the FDA in December 2005 for use in the treatment of ad‐ vanced renal cancer. Preclinical studies demonstrated a significant retardation in the growth of human melanoma tumor xenografts with Sorafenib. In a phase 1 study, the maximum tol‐ erated dose of Sorafenib as a single agent was established at 400 mg twice daily, and the

methyltransferase (MGMT) or O-6-alkylguanine-DNA alkyltransferase [8].

tice, patients with metastatic melanoma often are treated off-label with TMZ.

ineffective against melanoma CNS metastases.

**2.3. Sorafenib**

single dose of DTIC is toler‐

http://dx.doi.org/10.5772/53632

539

Therapeutic Agents for Advanced Melanoma

## **2. Chemotherapy agents**

According to the current 7th edition American Joint Committee of Cancer (AJCC) staging system, melanoma can be pathologically classified in the following stages: 0, IA, IB, IIA, IIB, IIC, IIIA, IIIB, IIIC, and IV. Stage 0 is in situ melanoma. Stage I and II are growth phase of localized cutaneous melanoma with increasing thickness. Stage III has regional involvement of lymph node. Stage IV means distant metastasis. Normally stage III and IV melanoma are called metastatic melanoma. Localized melanoma is curable with complete surgical excision in most patients. But currently treatment for metastatic melanoma is still very challenging. Only two chemotherapy drugs in current use have been approved by the Food and Drug Administration (FDA) for metastatic melanoma: dacarbazine (DTIC) and vemurafenib. Oth‐ er agents that have been tried on melanoma patients are also discussed below.

#### **2.1. Dacarbazine (DTIC)**

DTIC is the first FDA approved chemotherapy drug for metastatic melanoma. This drug gained FDA approval in May 1975 as DTIC-Dome for the treatment of metastatic melanoma. It was initially marketed by Bayer. The therapeutic effect of DTIC is believed to be produced through alkylation of DNA. While its anticancer mechanism of action is still not fully under‐ stood, DTIC is believed to be first metabolically bioactivated through a series of reactions involving CYP450. Initial demethylation to MTIC [3-methyl-(triazen-1-yl)imidazole-4-car‐ boxamide] is followed by formation of diazomethane, the active moiety of DTIC and a po‐ tent methylating agent [4].

DTIC has produced response rates of from 15% to 25% in single-institution trials. But the overall response rate has fallen over the years from 15% to 7% and less than 5% of responses are complete in phase 3 trials. The median response durations to DTIC are 5 to 6 months. Long-term follow-up of patients treated with DTIC alone shows that only <2% can be antici‐ pated to survive for more than 6 years. In recent phase 3 trials that used strict response as‐ sessment criteria, the response rates with single-agent DTIC did not exceed 12% [5].

Over the past 30 years after its approval by the FDA, DTIC remains the only currently used cytotoxic drug for the treatment of metastatic melanoma. Despite its low single-agent activi‐ ty, DTIC has remained the mainstay of many combination chemotherapy regimens and evaluations of resistance-reversing agents. After more than 20 years of research, DTIC is still the standard against which most new chemotherapy agents are compared [6].

As for the dose, it has been demonstrated that 850~1000 mg/m2 single dose of DTIC is toler‐ ated. This single dose administration appears to deliver clinical improvements similar to those observed with multiple doses that provide the same total dose per cycle. This should be the reference standard for randomized trials comparing new therapies with DTIC [7].

## **2.2. Temozolomide**

In this chapter, we will review available therapeutic agents for advanced melanoma as well as agents that are still under clinical development. We will focus on their mechanism of ac‐ tion, development history, and therapeutic effects. In learning from our efforts in the past, we must continue to challenge the current paradigms of treatment as we forge new paths to more effective treatment options. This will likely involve a multimodal approach to therapy

According to the current 7th edition American Joint Committee of Cancer (AJCC) staging system, melanoma can be pathologically classified in the following stages: 0, IA, IB, IIA, IIB, IIC, IIIA, IIIB, IIIC, and IV. Stage 0 is in situ melanoma. Stage I and II are growth phase of localized cutaneous melanoma with increasing thickness. Stage III has regional involvement of lymph node. Stage IV means distant metastasis. Normally stage III and IV melanoma are called metastatic melanoma. Localized melanoma is curable with complete surgical excision in most patients. But currently treatment for metastatic melanoma is still very challenging. Only two chemotherapy drugs in current use have been approved by the Food and Drug Administration (FDA) for metastatic melanoma: dacarbazine (DTIC) and vemurafenib. Oth‐

DTIC is the first FDA approved chemotherapy drug for metastatic melanoma. This drug gained FDA approval in May 1975 as DTIC-Dome for the treatment of metastatic melanoma. It was initially marketed by Bayer. The therapeutic effect of DTIC is believed to be produced through alkylation of DNA. While its anticancer mechanism of action is still not fully under‐ stood, DTIC is believed to be first metabolically bioactivated through a series of reactions involving CYP450. Initial demethylation to MTIC [3-methyl-(triazen-1-yl)imidazole-4-car‐ boxamide] is followed by formation of diazomethane, the active moiety of DTIC and a po‐

DTIC has produced response rates of from 15% to 25% in single-institution trials. But the overall response rate has fallen over the years from 15% to 7% and less than 5% of responses are complete in phase 3 trials. The median response durations to DTIC are 5 to 6 months. Long-term follow-up of patients treated with DTIC alone shows that only <2% can be antici‐ pated to survive for more than 6 years. In recent phase 3 trials that used strict response as‐

Over the past 30 years after its approval by the FDA, DTIC remains the only currently used cytotoxic drug for the treatment of metastatic melanoma. Despite its low single-agent activi‐ ty, DTIC has remained the mainstay of many combination chemotherapy regimens and evaluations of resistance-reversing agents. After more than 20 years of research, DTIC is still

sessment criteria, the response rates with single-agent DTIC did not exceed 12% [5].

the standard against which most new chemotherapy agents are compared [6].

er agents that have been tried on melanoma patients are also discussed below.

utilizing all of the available tools in our arsenal.

**2. Chemotherapy agents**

538 Melanoma - From Early Detection to Treatment

**2.1. Dacarbazine (DTIC)**

tent methylating agent [4].

Temozolomide (TMZ) is an orally active alkylating agent. It's a prodrug of MTIC and conge‐ ner of DTIC. It has been available in the US since August 1999 and in other countries since the early 2000s. The therapeutic benefit of temozolomide depends on its ability to alkylate/ methylate DNA, which most often occurs at the N-7 or O-6 positions of guanine residues. This methylation damages the DNA and triggers the death of tumor cells. However, some tumor cells are able to repair this type of DNA damage, and therefore diminish the thera‐ peutic efficacy of temozolomide, by expressing an enzyme called O-6-methylguanine-DNA methyltransferase (MGMT) or O-6-alkylguanine-DNA alkyltransferase [8].

The single agent activity of TMZ in metastatic melanoma has been established in several phase 1 and 2 studies [9]. In a randomized trial of 305 patients with advanced melanoma, TMZ showed efficacy at least equivalent to that of DTIC in terms of objective response rate, time to progression, and overall disease-free survival [10]. TMZ was tolerated very well and showed an advantage in terms of improvement in the quality of life. More patients showed improvement or maintenance of physical functioning at Week 12. That trial excluded pa‐ tients who had brain metastases. Because the trial design was intended to demonstrate the superiority of TMZ over DTIC, rather than equivalence, the FDA did not accept the results of that trial as grounds for approving a melanoma indication for TMZ. But in clinical prac‐ tice, patients with metastatic melanoma often are treated off-label with TMZ.

TMZ has demonstrated efficacy in the treatment of variety of solid tumors, especially in brain malignancies, which is a manifestation of its far greater ability to penetrate the central nervous system (CNS). Taking into account the high rate of CNS recurrence as a site of fail‐ ure after cytotoxic chemotherapy, TMZ may represent a viable alternative to DTIC, which is ineffective against melanoma CNS metastases.

## **2.3. Sorafenib**

Sorafenib (BAY43-9006, developed by Bayer Pharmaceuticals, West Haven CT, trade name Nexavar) is an orally administered tyrosine kinase inhibitor. It is a potent inhibitor of the BRAF kinase that is frequently mutated in melanoma, as well as an inhibitor of the Vascular Endothelial Growth Factor (VEGF) receptor and other kinases. It targets the adenosine tri‐ phosphate-binding site of the BRAF kinase and inhibits both wild-type and mutant BRAF *in vitro*. Sorafenib was approved by the FDA in December 2005 for use in the treatment of ad‐ vanced renal cancer. Preclinical studies demonstrated a significant retardation in the growth of human melanoma tumor xenografts with Sorafenib. In a phase 1 study, the maximum tol‐ erated dose of Sorafenib as a single agent was established at 400 mg twice daily, and the most common toxicities were gastrointestinal (mainly diarrhea), dermatologic (skin rash, hand-foot syndrome), and fatigue [11].

Vemurafenib's efficacy was measured by overall survival (OS), investigator-assessed pro‐ gression-free survival (PFS) and confirmed investigator-assessed best overall response rate. Overall survival was significantly improved in patients receiving vemurafenib compared with those receiving dacarbazine. The median survival of patients receiving vemurafenib had not been reached and was 7.9 months for those receiving dacarbazine. Progression-free survival (PFS) was also significantly improved in patients receiving vemurafenib. The me‐ dian PFS was 5.3 months for patients receiving vemurafenib and 1.6 months for patients re‐ ceiving dacarbazine. 48.4% for patients who received vemurafenib showed complete or partial response while only 5.5% patients who received dacarbazine showed complete or

Therapeutic Agents for Advanced Melanoma

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541

Arthralgia, rash, photosensitivity, fatigue, alopecia, pruritis, and skin papilloma were ob‐ served in at least 30% of patients treated with vemurafenib. Cutaneous squamous cell carcinomas were detected in approximately 24% of patients. Other adverse reactions re‐ ported in patients treated with vemurafenib included hypersensitivity, Stevens-Johnson syndrome, toxic epidermal necrolysis, uveitis, QT prolongation, and liver enzyme labora‐

Cisplatin and carboplatin have shown modest activity as single agents in patients with metastatic melanoma. Cisplatin as single-agent therapy induced a 15% response rate with a short median duration of 3 months [19]. A response rate of 19% has been reported in 26 che‐ motherapy-naive patients with metastatic melanoma who received carboplatin. In those pa‐ tients, there were 5 partial responses, and thrombocytopenia was the dose-limiting toxicity [20]. *In vitro* studies suggested that oxaliplatin may be more active than cisplatin or carbo‐ platin. But a small phase 2 trial in 10 patients who had received and failed prior chemothera‐

The nitrosoureas (carmustine, lomustine, and semustine) induce objective responses in 13~18% patients. They can cross the blood-brain barrier. But at conventional doses, little or no activity was observed against melanoma brain metastases [22]. Another drawback of the nitrosureas is they induce prolonged myelosuppression. Despite these, they have been in‐ cluded frequently in multi-agent chemotherapy combinations, presumably for their ability

The vinca alkaloids (vindesine and vinblastine) have produced responses in approximately 14% of patients [23]. The taxanes have produced responses in 16~17% patients [24]. All of these response rate data were obtained from phase 2 trials. None of those drugs have been evaluated as single agents in phase 3 trials. Based on the experience with DTIC, it is likely that the phase 3 trial objective response rates would be less than the rates reported from phase 2 trials. All of these drugs are rarely used currently as single-agent therapy in meta‐ static melanoma, but they frequently have been incorporated into combination chemothera‐

to penetrate into the CNS and lack of viable alternatives for metastatic melanoma.

partial response [17].

tory abnormalities [18].

**2.5. Other single chemotherapy agents**

py produced no objective responses [21].

py and biochemotherapy regimens.

But in further phase 2 clinical trials, Sorafenib had shown relatively little activity in metastatic melanoma when using alone. In a phase 2 trial that was conducted in 20 pa‐ tients with refractory metastatic melanoma, Sorafenib showed modest activity with 1 par‐ tial response and 3 patients who achieved stable disease [12]. In another phase 2, randomized, discontinuation trial, no objective responses were achieved, and 19% of pa‐ tients achieved stable disease [13].

Sorafenib combined with other chemotherapy drugs were also tested clinically. In a phase 1 and 2 study that combined carboplatin and paclitaxel with escalating doses of Sorafenib in 35 patients, a promising response rate of 31% was observed, and another 54% of patients ex‐ perienced stable disease that lasted longer than 3 months. That study recently was updated to include 105 patients, and the current response rate is 27% [14]. On this basis, 2 phase 3 trials have been launched to assess the efficacy of carboplatin and paclitaxel plus Sorafenib versus placebo in chemotherapy-naive patients and in previously treated patients. In De‐ cember 2006, Bayer reported the combinations failed to show significant improvement of progression-free survival in melanoma patients [15].

## **2.4. Vemurafenib**

Vemurafenib established a successful model for extracellular chemotherapeutic targeted therapy based on deep understanding cancer biology. It's a paradigm of structured-based drug development. It was first discovered in 2002 that the protein kinase BRAF is mutated in about 70% of malignant melanomas and a significant number of colorectal, ovarian and papillary thyroid cancers, implicating mutated BRAF as a critical promoter of malignancy. Then scientists determined the structure of the BRAF catalytic domain and identified a class of BRAF inhibitors that bind to the active conformation of the protein. Further lead series were developed and crystal structures of complexes combined with molecular modeling studies have resulted in potent selective inhibitors. Vemurafenib is the first one that went into clinical trials and gained FDA approval in August 2011.

Vemurafenib (PLX4032/RG7204) is developed by Plexxikon (now part of the Daiichi Sankyo group and Hoffmann–La Roche) for the treatment of late-stage melanoma. Vemurafenib can induce programmed cell death in melanoma cell lines. It interrupts the BRAF/MEK step on the BRAF/MEK/ERK pathway − if the BRAF has the common V600E mutation [16].

Vemurafenib has very impressive single-agent clinical activity, with unprecedented re‐ sponse rates of about 80% and a clear impact on progression-free survival longer than 6 months. An international randomized open-label trial in patients with previously untreated metastatic or unresectable melanoma with the BRAFV600E mutation led to the FDA approval of Vemurafenib for melanoma. This clinical trial enrolled 675 patients. 337 patients were randomly assigned to vemurafenib with 960 mg orally twice daily. 338 patients were ran‐ domly assigned to dacarbazine with 1000 mg/m2 intravenously every three weeks. Treat‐ ment end-points are disease progression, unacceptable toxicity, and/or consent withdrawal. Vemurafenib's efficacy was measured by overall survival (OS), investigator-assessed pro‐ gression-free survival (PFS) and confirmed investigator-assessed best overall response rate. Overall survival was significantly improved in patients receiving vemurafenib compared with those receiving dacarbazine. The median survival of patients receiving vemurafenib had not been reached and was 7.9 months for those receiving dacarbazine. Progression-free survival (PFS) was also significantly improved in patients receiving vemurafenib. The me‐ dian PFS was 5.3 months for patients receiving vemurafenib and 1.6 months for patients re‐ ceiving dacarbazine. 48.4% for patients who received vemurafenib showed complete or partial response while only 5.5% patients who received dacarbazine showed complete or partial response [17].

Arthralgia, rash, photosensitivity, fatigue, alopecia, pruritis, and skin papilloma were ob‐ served in at least 30% of patients treated with vemurafenib. Cutaneous squamous cell carcinomas were detected in approximately 24% of patients. Other adverse reactions re‐ ported in patients treated with vemurafenib included hypersensitivity, Stevens-Johnson syndrome, toxic epidermal necrolysis, uveitis, QT prolongation, and liver enzyme labora‐ tory abnormalities [18].

#### **2.5. Other single chemotherapy agents**

most common toxicities were gastrointestinal (mainly diarrhea), dermatologic (skin rash,

But in further phase 2 clinical trials, Sorafenib had shown relatively little activity in metastatic melanoma when using alone. In a phase 2 trial that was conducted in 20 pa‐ tients with refractory metastatic melanoma, Sorafenib showed modest activity with 1 par‐ tial response and 3 patients who achieved stable disease [12]. In another phase 2, randomized, discontinuation trial, no objective responses were achieved, and 19% of pa‐

Sorafenib combined with other chemotherapy drugs were also tested clinically. In a phase 1 and 2 study that combined carboplatin and paclitaxel with escalating doses of Sorafenib in 35 patients, a promising response rate of 31% was observed, and another 54% of patients ex‐ perienced stable disease that lasted longer than 3 months. That study recently was updated to include 105 patients, and the current response rate is 27% [14]. On this basis, 2 phase 3 trials have been launched to assess the efficacy of carboplatin and paclitaxel plus Sorafenib versus placebo in chemotherapy-naive patients and in previously treated patients. In De‐ cember 2006, Bayer reported the combinations failed to show significant improvement of

Vemurafenib established a successful model for extracellular chemotherapeutic targeted therapy based on deep understanding cancer biology. It's a paradigm of structured-based drug development. It was first discovered in 2002 that the protein kinase BRAF is mutated in about 70% of malignant melanomas and a significant number of colorectal, ovarian and papillary thyroid cancers, implicating mutated BRAF as a critical promoter of malignancy. Then scientists determined the structure of the BRAF catalytic domain and identified a class of BRAF inhibitors that bind to the active conformation of the protein. Further lead series were developed and crystal structures of complexes combined with molecular modeling studies have resulted in potent selective inhibitors. Vemurafenib is the first one that went

Vemurafenib (PLX4032/RG7204) is developed by Plexxikon (now part of the Daiichi Sankyo group and Hoffmann–La Roche) for the treatment of late-stage melanoma. Vemurafenib can induce programmed cell death in melanoma cell lines. It interrupts the BRAF/MEK step on

Vemurafenib has very impressive single-agent clinical activity, with unprecedented re‐ sponse rates of about 80% and a clear impact on progression-free survival longer than 6 months. An international randomized open-label trial in patients with previously untreated metastatic or unresectable melanoma with the BRAFV600E mutation led to the FDA approval of Vemurafenib for melanoma. This clinical trial enrolled 675 patients. 337 patients were randomly assigned to vemurafenib with 960 mg orally twice daily. 338 patients were ran‐

ment end-points are disease progression, unacceptable toxicity, and/or consent withdrawal.

intravenously every three weeks. Treat‐

the BRAF/MEK/ERK pathway − if the BRAF has the common V600E mutation [16].

hand-foot syndrome), and fatigue [11].

540 Melanoma - From Early Detection to Treatment

tients achieved stable disease [13].

**2.4. Vemurafenib**

progression-free survival in melanoma patients [15].

into clinical trials and gained FDA approval in August 2011.

domly assigned to dacarbazine with 1000 mg/m2

Cisplatin and carboplatin have shown modest activity as single agents in patients with metastatic melanoma. Cisplatin as single-agent therapy induced a 15% response rate with a short median duration of 3 months [19]. A response rate of 19% has been reported in 26 che‐ motherapy-naive patients with metastatic melanoma who received carboplatin. In those pa‐ tients, there were 5 partial responses, and thrombocytopenia was the dose-limiting toxicity [20]. *In vitro* studies suggested that oxaliplatin may be more active than cisplatin or carbo‐ platin. But a small phase 2 trial in 10 patients who had received and failed prior chemothera‐ py produced no objective responses [21].

The nitrosoureas (carmustine, lomustine, and semustine) induce objective responses in 13~18% patients. They can cross the blood-brain barrier. But at conventional doses, little or no activity was observed against melanoma brain metastases [22]. Another drawback of the nitrosureas is they induce prolonged myelosuppression. Despite these, they have been in‐ cluded frequently in multi-agent chemotherapy combinations, presumably for their ability to penetrate into the CNS and lack of viable alternatives for metastatic melanoma.

The vinca alkaloids (vindesine and vinblastine) have produced responses in approximately 14% of patients [23]. The taxanes have produced responses in 16~17% patients [24]. All of these response rate data were obtained from phase 2 trials. None of those drugs have been evaluated as single agents in phase 3 trials. Based on the experience with DTIC, it is likely that the phase 3 trial objective response rates would be less than the rates reported from phase 2 trials. All of these drugs are rarely used currently as single-agent therapy in meta‐ static melanoma, but they frequently have been incorporated into combination chemothera‐ py and biochemotherapy regimens.

## **2.6. Chemotherapy drug combinations**

Theoretically drug combination should be based on laboratory or clinical evidence of syner‐ gistic effect. But since single-agent chemotherapy regimens only provided modest activity against metastatic melanoma and lack of viable alternatives, many combination regimens have been evaluated in clinical trials. Initially two-agent combinations were tested in which DTIC was combined with a nitrosourea, vinca alkaloid, or platinum compound. In most of these trials, only 10~20% response rates were observed. There was little evidence to suggest superiority of these combinations compared with DTIC treatment alone [25-27].

tal in the body's natural response to microbial infection and in discriminating between for‐ eign (non-self) and self. It's a glycosylated 15,500 dalton single protein molecule. IL-2 mediates its effects by binding to IL-2 receptors, which are expressed by lymphocytes, the cells that are responsible for immunity. It is one of the only two FDA approved agents for the treatment of metastatic melanoma. Although the overall response rate is only about 15% and less than 5% of patients achieve complete remission with IL-2, its performance on mela‐

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543

One of the major immunologic effects of IL-2 upon the immune system is to expand the total number of T-lymphocytes (CD4+ and CD8+) and to prevent lymphocyte apoptosis. Another key role of IL-2 is to provide the appropriate cytokine milieu necessary to over‐ come tumor-induced immune tolerance. But the exact molecular and genetic mechanisms involved in this complex interaction between the tumor and the host immune response

There is currently a wide spectrum of dosing schedules and regimens for IL-2 therapy, with the current standard used by most oncologists being 600,000 to 720,000 IU/kg/dose, given at 8 h intervals. Although the optimal dosing schedule resulting in the best clinical response is currently unknown, previous data would suggest that the higher dose regi‐ mens as well as the number of total doses received correlates best with clinical response. Thus, several groups have begun to look at alternative dosing strategies to achieve an in‐ creased drug tolerance and tolerability profile, such as the continuous infusion of IL-2

many IL-2 regimens limits its use. In addition, the tumor-killing cytotoxic T cells and natural killer cells, which are the presumed target cells for IL-2, are frequently inefficient in the tumor environment, partly due to suppressive and apoptosis-inducing signals

Interferons (IFNs) are proteins made and released by the cells of most vertebrates in re‐ sponse to the presence of pathogens or tumor cells. They allow communication between cells to trigger the protective defenses of the immune system that eradicate pathogens or tu‐ mors. IFNs belong to the large class of glycoproteins known as cytokines. They are named after their ability to "interfere" with viral replication within host cells. IFNs have other func‐ tions: they activate immune cells, such as natural killer cells and macrophages; they increase recognition of infection or tumor cells by up-regulating antigen presentation to T lympho‐ cytes; and they increase the ability of uninfected host cells to resist new infection by virus. Based on the type of receptors through which they signal, human interferons have been clas‐ sified into three major types. The type I interferons present in human are IFN-α, IFN-β and IFN-ω [35]. High-dose IFN therapy using IFN-α was the first form of medical therapy to be approved by the FDA for use in high-risk melanoma in the adjuvant setting. Adjuvant nor‐ mally means using IFN- α weeks after the surgical excision of the melanoma tumor. Com‐

/day) over an extended period of 72 h [33]. But the multiorgan toxicity of

/day intravenous injection 5

/day subcutaneous injection 3 days a week for the

noma is better than DTIC.

is still largely unknown [32].

from tumor-infiltrating mononuclear phagocytes [34].

mon treatment scheme is IFN-α2b at 20 million units (MU)/m2

days a week for 4 weeks, then 10 MU/m2

(18 mIU/m2

**3.2. Interferon α**

In order to improve response rates, more aggressive multi-drug combinations using 3 or 4 different drugs were also tested clinically. Two most widely studied combinations are cis‐ platin, vinblastine, and DTIC (CVD) and the Dartmouth regimen. The latter is a 4-drug com‐ bination consisting of cisplatin, DTIC, carmustine, and tamoxifen (also called CDBT). Both combinations showed improved response rates that ranged from 30% to 50% in single-insti‐ tution phase 2 studies [28, 29]. But in further randomized phase 3 trials which involved more patients, they all showed much lower efficiency: In a randomized trial comparing CVD with single-agent DTIC that involved approximately 150 patients, the CVD arm pro‐ duced a 19% response rate compared with 14% for the DTIC arm, and there was no differen‐ ces in either response duration or survival. In another randomized phase 3 trial, the CDBT combination was compared with single-agent DTIC. That cooperative group trial involved 240 patients, and the response rate was 10% for the DTIC regimen compared with 19% for the CDBT regimen (P=0.09). The median survival was 7 months, with no significant differ‐ ence between the 2 treatment arms [6].

The main reason for such discrepancies between the results from single-institution studies and those from large, multicenter, cooperative trials probably is selection bias. Differences in performance status, percentages of patients with visceral involvement, and number of meta‐ static sites easily could account for some of the observed differences. In fact, all of those fac‐ tors are known to have an impact on both response rate and survival [30].

Overall, controlled trials have produced no compelling evidence to support the value of combination chemotherapy, with or without tamoxifen, in patients with metastatic melano‐ ma. Toxicity was substantially greater for the combination regimen, with bone marrow sup‐ pression, nausea, emesis, and fatigue significantly more frequent with CDBT than with DTIC [6]. So it is difficult to justify the use of either CVD or CDBT instead of single-agent DTIC or TMZ for the treatment of most patients with metastatic melanoma.

## **3. Immunotherapy agents**

#### **3.1. Interleukin-s (IL-2)**

In 1998, the FDA approved intermittent high-dose bolus IL-2 based on its ability to mediate durable complete response in metastatic melanoma patients [31]. IL-2 is a type of cytokine immune system signaling molecule, which is a leukocytotrophic hormone that is instrumen‐ tal in the body's natural response to microbial infection and in discriminating between for‐ eign (non-self) and self. It's a glycosylated 15,500 dalton single protein molecule. IL-2 mediates its effects by binding to IL-2 receptors, which are expressed by lymphocytes, the cells that are responsible for immunity. It is one of the only two FDA approved agents for the treatment of metastatic melanoma. Although the overall response rate is only about 15% and less than 5% of patients achieve complete remission with IL-2, its performance on mela‐ noma is better than DTIC.

One of the major immunologic effects of IL-2 upon the immune system is to expand the total number of T-lymphocytes (CD4+ and CD8+) and to prevent lymphocyte apoptosis. Another key role of IL-2 is to provide the appropriate cytokine milieu necessary to over‐ come tumor-induced immune tolerance. But the exact molecular and genetic mechanisms involved in this complex interaction between the tumor and the host immune response is still largely unknown [32].

There is currently a wide spectrum of dosing schedules and regimens for IL-2 therapy, with the current standard used by most oncologists being 600,000 to 720,000 IU/kg/dose, given at 8 h intervals. Although the optimal dosing schedule resulting in the best clinical response is currently unknown, previous data would suggest that the higher dose regi‐ mens as well as the number of total doses received correlates best with clinical response. Thus, several groups have begun to look at alternative dosing strategies to achieve an in‐ creased drug tolerance and tolerability profile, such as the continuous infusion of IL-2 (18 mIU/m2 /day) over an extended period of 72 h [33]. But the multiorgan toxicity of many IL-2 regimens limits its use. In addition, the tumor-killing cytotoxic T cells and natural killer cells, which are the presumed target cells for IL-2, are frequently inefficient in the tumor environment, partly due to suppressive and apoptosis-inducing signals from tumor-infiltrating mononuclear phagocytes [34].

## **3.2. Interferon α**

**2.6. Chemotherapy drug combinations**

542 Melanoma - From Early Detection to Treatment

ence between the 2 treatment arms [6].

**3. Immunotherapy agents**

**3.1. Interleukin-s (IL-2)**

Theoretically drug combination should be based on laboratory or clinical evidence of syner‐ gistic effect. But since single-agent chemotherapy regimens only provided modest activity against metastatic melanoma and lack of viable alternatives, many combination regimens have been evaluated in clinical trials. Initially two-agent combinations were tested in which DTIC was combined with a nitrosourea, vinca alkaloid, or platinum compound. In most of these trials, only 10~20% response rates were observed. There was little evidence to suggest

In order to improve response rates, more aggressive multi-drug combinations using 3 or 4 different drugs were also tested clinically. Two most widely studied combinations are cis‐ platin, vinblastine, and DTIC (CVD) and the Dartmouth regimen. The latter is a 4-drug com‐ bination consisting of cisplatin, DTIC, carmustine, and tamoxifen (also called CDBT). Both combinations showed improved response rates that ranged from 30% to 50% in single-insti‐ tution phase 2 studies [28, 29]. But in further randomized phase 3 trials which involved more patients, they all showed much lower efficiency: In a randomized trial comparing CVD with single-agent DTIC that involved approximately 150 patients, the CVD arm pro‐ duced a 19% response rate compared with 14% for the DTIC arm, and there was no differen‐ ces in either response duration or survival. In another randomized phase 3 trial, the CDBT combination was compared with single-agent DTIC. That cooperative group trial involved 240 patients, and the response rate was 10% for the DTIC regimen compared with 19% for the CDBT regimen (P=0.09). The median survival was 7 months, with no significant differ‐

The main reason for such discrepancies between the results from single-institution studies and those from large, multicenter, cooperative trials probably is selection bias. Differences in performance status, percentages of patients with visceral involvement, and number of meta‐ static sites easily could account for some of the observed differences. In fact, all of those fac‐

Overall, controlled trials have produced no compelling evidence to support the value of combination chemotherapy, with or without tamoxifen, in patients with metastatic melano‐ ma. Toxicity was substantially greater for the combination regimen, with bone marrow sup‐ pression, nausea, emesis, and fatigue significantly more frequent with CDBT than with DTIC [6]. So it is difficult to justify the use of either CVD or CDBT instead of single-agent

In 1998, the FDA approved intermittent high-dose bolus IL-2 based on its ability to mediate durable complete response in metastatic melanoma patients [31]. IL-2 is a type of cytokine immune system signaling molecule, which is a leukocytotrophic hormone that is instrumen‐

tors are known to have an impact on both response rate and survival [30].

DTIC or TMZ for the treatment of most patients with metastatic melanoma.

superiority of these combinations compared with DTIC treatment alone [25-27].

Interferons (IFNs) are proteins made and released by the cells of most vertebrates in re‐ sponse to the presence of pathogens or tumor cells. They allow communication between cells to trigger the protective defenses of the immune system that eradicate pathogens or tu‐ mors. IFNs belong to the large class of glycoproteins known as cytokines. They are named after their ability to "interfere" with viral replication within host cells. IFNs have other func‐ tions: they activate immune cells, such as natural killer cells and macrophages; they increase recognition of infection or tumor cells by up-regulating antigen presentation to T lympho‐ cytes; and they increase the ability of uninfected host cells to resist new infection by virus.

Based on the type of receptors through which they signal, human interferons have been clas‐ sified into three major types. The type I interferons present in human are IFN-α, IFN-β and IFN-ω [35]. High-dose IFN therapy using IFN-α was the first form of medical therapy to be approved by the FDA for use in high-risk melanoma in the adjuvant setting. Adjuvant nor‐ mally means using IFN- α weeks after the surgical excision of the melanoma tumor. Com‐ mon treatment scheme is IFN-α2b at 20 million units (MU)/m2 /day intravenous injection 5 days a week for 4 weeks, then 10 MU/m2 /day subcutaneous injection 3 days a week for the next 48 weeks for a full year's. But IFN-α2b can also be used one month before definitive surgical lymphadenectomy. This is called 'neoadjuvant' treatment [36].

block the negative signal sending by CTLA4 by using anti-CTLA4 antibodies, thus reduce the sensitivity of activated T cells to negative regulatory signals and enhance the immune

Therapeutic Agents for Advanced Melanoma

http://dx.doi.org/10.5772/53632

545

As of October 2007 there are two fully human monoclonal anti-CTLA4 antibodies in ad‐ vanced clinical trials, one from Medarex, Inc. (Princeton, NJ) and Bristol-Myers Squibb (New York), called ipilimumab (MDX-010), and one from Pfizer (New York), called tremelimumab (formerly ticilimumab, CP-675,206) [42]. These antibodies were produced using different types of mice with engineered immune systems, and are thus fully human, with long half-

Ipilimumab (MDX-010) is an IgG1 monoclonal antibody. Preclinical and early clinical stud‐ ies of patients with metastatic melanoma show that ipilimumab promotes antitumor activity as monotherapy and in combination with treatments such as chemotherapy, vaccines, or cy‐ tokines. The initial success with these antibodies has encouraged the rapid development of new agonistic and antagonistic antibodies that alter immune regulation, such as anti-PD-1, anti-4-1BB, anti-CD40, and anti-OX-40. On December 10, 2007, Bristol-Myers Squibb and Medarex released the results of three studies on ipilimumab [42]. The three studies tested 487 patients with metastatic melanoma. Short-term tumor progression prior to delayed re‐ gression has been observed in ipilimumab-treated patients, and objective responses may be of prolonged duration. In some patients clinical improvement manifests as stable disease, which may also extend for months or years. One of the three studies failed to meet its pri‐ mary goal of shrinking tumors in at least 10% of the study's 155 patients. Overall the medi‐

In the meantime, the side effect profile was high in the ipilimumab treated group, with the generation of autoimmune-like effects, such as diarrhea, dermatitis and effects upon the thy‐ roid and pituitary glands. Several patients also experienced vitiligo, indicative of anti-mela‐ nocyte autoimmunity. However, the majority of the side effects were noted to be transient (except the vitiligo), improving or disappearing after the completion of therapy. Early clini‐ cal data suggest a correlation between these side effects and response to ipilimumab treat‐ ment and most likely reflect the drug mechanism of action and corresponding effects on the

In 2011, the first-line pivotal trial data for ipilimumab was released. The median survival of patients treated with ipilimumab at a dose of 10 mg/kg in combination with dacarbazine was 11.2 months. Patients treated with dacarbazine alone showed a median survival of 9.1 months. The improvement in the median survival was 2.1 months. The estimated survival rates in the two groups of, respectively, 47.3% and 36.3% at 1 year, 28.5% and 17.9% at 2 years, and 20.8% and 12.2% at 3 years. These results are not better than those observed with the 3 mg/kg dose in second-line treatment. One possible reason is that a significant and un‐ expectedly high rate of hepatitis in the dacarbazine + ipilimumab arm did take a significant percentage of patients off treatment before the third or especially the fourth dose of ipilimu‐ mab could be administered, thus limiting both the number of administrations of dacarba‐ zine as well as of ipilimumab in the combination arm. Other immune-related adverse events were not increased compared to those with the 3 mg/kg dose experience. The overall inter‐

cation produced weaker-than-anticipated efficacy on melanoma patients.

response of the host to tumor cells.

lives of 2– 4 weeks.

immune system [42].

The first randomized comparison of high-dose IFN versus observation found the median re‐ lapse-free survival was 1.72 years in the high-dose IFN arm versus 0.98 year in the observa‐ tion arm (P=0.0023) and the median overall survival was 3.82 versus 2.78 years (P=0.0237) respectively [37]. But in a later pooled analysis of more patients in more clinical trials, the relapse-free survival benefit was maintained but no overall survival benefit was seen [38].

The exact mechanism of IFN IFN-α's anti-tumor efficacy is still unknown. But it was found that the STAT1/STAT3 expression ratios rose in association with IFN treatment. The clinical effects of IFN-α2b in human melanoma are also found to be inversely related to STAT3 ex‐ pression (41). Induction of apoptosis has been shown to be important *in vitro*, if not *in vivo*. IFN-α can induce apoptosis in transformed cell lines as well as primary tumor cells [39].

High-dose IFN is the standard of care for high-risk melanoma patients in the adjuvant set‐ ting. However, it is associated with significant toxicity. The incidence and severity of these adverse events is clearly dose-related. Consequently, there has been a great deal of interest in intermediate- and low-dose regimens administered through subcutaneous injection. However, none of the trials using intermediate or low dosing so far have been able to dem‐ onstrate any reliable benefit in terms of relapse-free survival or overall survival [40].

#### **3.3. Pegylated interferon-α2b**

Pegylated interferon-α2b gained its approval from the FDA in March 2011 in the adjuvant setting for melanoma patients with lymph-node-positive disease (stage III) after lymphnode dissection. The approval was based on a randomized controlled phase-III trial in 1256 stage-III melanoma patients. This trial compared treatment with pegylated interferon- α2b for up to 5 years with observation. The results revealed a significant and sustained impact on relapse free survival (RFS) in the intention-to-treat (ITT) population. This trial also showed that interferon-α2b treatment didn't significantly improve distant metastasis-free survival (DMFS) or overall survival (OS) [41]. Pegylated interferon-α2b also showed much better effect in patients with sentinel-node-positive disease (stage III-N1: microscopic in‐ volvement only) compared with patients with palpable nodal disease (stage III-N2). It also significantly improved DMFS in sentinel-node-positive patients in contrast to a marginal ef‐ fect in patients with palpable nodes. The authors identified tumor stage as a predictive fac‐ tor in trials. One very important finding from clinical trials was that ulceration of the primary melanoma indicated a distinct biology that was clearly IFN sensitive in contrast to the non-ulcerated type of melanoma.

#### **3.4. Anti-CTLA4 antibodies: Ipilimumab and tremelimumab**

Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) also known as CD152 (Cluster of differentia‐ tion 152) is a member of the immunoglobulin super family, which is expressed on the sur‐ face of Helper T cells and transmits an inhibitory signal to T cells that eventually shuts off the activated state. The rationale for involving this in treatment of metastatic melanoma is to block the negative signal sending by CTLA4 by using anti-CTLA4 antibodies, thus reduce the sensitivity of activated T cells to negative regulatory signals and enhance the immune response of the host to tumor cells.

next 48 weeks for a full year's. But IFN-α2b can also be used one month before definitive

The first randomized comparison of high-dose IFN versus observation found the median re‐ lapse-free survival was 1.72 years in the high-dose IFN arm versus 0.98 year in the observa‐ tion arm (P=0.0023) and the median overall survival was 3.82 versus 2.78 years (P=0.0237) respectively [37]. But in a later pooled analysis of more patients in more clinical trials, the relapse-free survival benefit was maintained but no overall survival benefit was seen [38].

The exact mechanism of IFN IFN-α's anti-tumor efficacy is still unknown. But it was found that the STAT1/STAT3 expression ratios rose in association with IFN treatment. The clinical effects of IFN-α2b in human melanoma are also found to be inversely related to STAT3 ex‐ pression (41). Induction of apoptosis has been shown to be important *in vitro*, if not *in vivo*. IFN-α can induce apoptosis in transformed cell lines as well as primary tumor cells [39].

High-dose IFN is the standard of care for high-risk melanoma patients in the adjuvant set‐ ting. However, it is associated with significant toxicity. The incidence and severity of these adverse events is clearly dose-related. Consequently, there has been a great deal of interest in intermediate- and low-dose regimens administered through subcutaneous injection. However, none of the trials using intermediate or low dosing so far have been able to dem‐

Pegylated interferon-α2b gained its approval from the FDA in March 2011 in the adjuvant setting for melanoma patients with lymph-node-positive disease (stage III) after lymphnode dissection. The approval was based on a randomized controlled phase-III trial in 1256 stage-III melanoma patients. This trial compared treatment with pegylated interferon- α2b for up to 5 years with observation. The results revealed a significant and sustained impact on relapse free survival (RFS) in the intention-to-treat (ITT) population. This trial also showed that interferon-α2b treatment didn't significantly improve distant metastasis-free survival (DMFS) or overall survival (OS) [41]. Pegylated interferon-α2b also showed much better effect in patients with sentinel-node-positive disease (stage III-N1: microscopic in‐ volvement only) compared with patients with palpable nodal disease (stage III-N2). It also significantly improved DMFS in sentinel-node-positive patients in contrast to a marginal ef‐ fect in patients with palpable nodes. The authors identified tumor stage as a predictive fac‐ tor in trials. One very important finding from clinical trials was that ulceration of the primary melanoma indicated a distinct biology that was clearly IFN sensitive in contrast to

Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) also known as CD152 (Cluster of differentia‐ tion 152) is a member of the immunoglobulin super family, which is expressed on the sur‐ face of Helper T cells and transmits an inhibitory signal to T cells that eventually shuts off the activated state. The rationale for involving this in treatment of metastatic melanoma is to

onstrate any reliable benefit in terms of relapse-free survival or overall survival [40].

**3.3. Pegylated interferon-α2b**

544 Melanoma - From Early Detection to Treatment

the non-ulcerated type of melanoma.

**3.4. Anti-CTLA4 antibodies: Ipilimumab and tremelimumab**

surgical lymphadenectomy. This is called 'neoadjuvant' treatment [36].

As of October 2007 there are two fully human monoclonal anti-CTLA4 antibodies in ad‐ vanced clinical trials, one from Medarex, Inc. (Princeton, NJ) and Bristol-Myers Squibb (New York), called ipilimumab (MDX-010), and one from Pfizer (New York), called tremelimumab (formerly ticilimumab, CP-675,206) [42]. These antibodies were produced using different types of mice with engineered immune systems, and are thus fully human, with long halflives of 2– 4 weeks.

Ipilimumab (MDX-010) is an IgG1 monoclonal antibody. Preclinical and early clinical stud‐ ies of patients with metastatic melanoma show that ipilimumab promotes antitumor activity as monotherapy and in combination with treatments such as chemotherapy, vaccines, or cy‐ tokines. The initial success with these antibodies has encouraged the rapid development of new agonistic and antagonistic antibodies that alter immune regulation, such as anti-PD-1, anti-4-1BB, anti-CD40, and anti-OX-40. On December 10, 2007, Bristol-Myers Squibb and Medarex released the results of three studies on ipilimumab [42]. The three studies tested 487 patients with metastatic melanoma. Short-term tumor progression prior to delayed re‐ gression has been observed in ipilimumab-treated patients, and objective responses may be of prolonged duration. In some patients clinical improvement manifests as stable disease, which may also extend for months or years. One of the three studies failed to meet its pri‐ mary goal of shrinking tumors in at least 10% of the study's 155 patients. Overall the medi‐ cation produced weaker-than-anticipated efficacy on melanoma patients.

In the meantime, the side effect profile was high in the ipilimumab treated group, with the generation of autoimmune-like effects, such as diarrhea, dermatitis and effects upon the thy‐ roid and pituitary glands. Several patients also experienced vitiligo, indicative of anti-mela‐ nocyte autoimmunity. However, the majority of the side effects were noted to be transient (except the vitiligo), improving or disappearing after the completion of therapy. Early clini‐ cal data suggest a correlation between these side effects and response to ipilimumab treat‐ ment and most likely reflect the drug mechanism of action and corresponding effects on the immune system [42].

In 2011, the first-line pivotal trial data for ipilimumab was released. The median survival of patients treated with ipilimumab at a dose of 10 mg/kg in combination with dacarbazine was 11.2 months. Patients treated with dacarbazine alone showed a median survival of 9.1 months. The improvement in the median survival was 2.1 months. The estimated survival rates in the two groups of, respectively, 47.3% and 36.3% at 1 year, 28.5% and 17.9% at 2 years, and 20.8% and 12.2% at 3 years. These results are not better than those observed with the 3 mg/kg dose in second-line treatment. One possible reason is that a significant and un‐ expectedly high rate of hepatitis in the dacarbazine + ipilimumab arm did take a significant percentage of patients off treatment before the third or especially the fourth dose of ipilimu‐ mab could be administered, thus limiting both the number of administrations of dacarba‐ zine as well as of ipilimumab in the combination arm. Other immune-related adverse events were not increased compared to those with the 3 mg/kg dose experience. The overall inter‐ pretation therefore is that dacarbazine did not help, but may rather have mitigated the re‐ sults in the dacarbazine plus ipilimumab arm. Based on all the data available, the large cumulative phase-II experience, and the two phase-III trials, the FDA approved treatment of melanoma with ipilimumab alone at the 3mg/kg dose [43].

**3.6. Vaccines based on tumor cells: Canvaxin, melacine, and MVax**

within the skin [49].

melanoma base on a similar reason [51].

benefit from Melacine in patients with melanoma [52].

The basic idea is to use tumor cell-based vaccine to stimulate and activate the host immune system to recognize, contain and eliminate cancer cells. This effect may be based on the fol‐ lowing two pathways: direct migration of the tumor cells to the draining lymph node basin after injection, or uptake of apoptotic or necrotic tumor cells by host dendritic cells located

Therapeutic Agents for Advanced Melanoma

http://dx.doi.org/10.5772/53632

547

The most extensively studied tumor cell-based vaccine is a polyvalent, antigen-rich whole cell vaccine called Canvaxin (CancerVax Corp., Carlsbad, CA). It is comprised of three mela‐ noma cell lines that contain over 20 immunogenic melanoma tumor antigens, given intra‐ dermally every two weeks for 3 to 5 doses, followed by monthly injections for the remainder of the first year. However, several small, single-institution phase 1 and 2 clinical trials of Canvaxin have not yielded a striking clinical benefit in most patients when administered with BCG as an immunoadjuvant [50]. But the rare complete responder to Canvaxin therapy has prompted the initiation of two multicenter phase 3 randomized trials of Canvaxin thera‐ py in 1998. In these trials, patients who have undergone complete resection of regional (stage III) or distant (stage IV) metastatic melanoma receive postoperative adjuvant immu‐ notherapy with Canvaxin plus Bacillus of Calmette and Guerin (BCG) or BCG alone. In April 2005, CancerVax announced the discontinuation of their phase 3 clinical trial of Can‐ vaxin in patients with Stage IV melanoma based upon the clinical funding that it was un‐ likely that the trial would provide significant evidence of a survival benefit for Canvaxintreated patients versus those receiving placebo. On October 3, 2005, CancerVax announced the discontinuation of another phase 3 clinical trial of Canvaxin in patients with Stage III

The second tumor cell-based vaccine that has been well studied since 1988 is Melacine. It is an allogeneic melanoma cell lysate combined with an immunologic adjuvant which is composed of a mixture of detoxified endotoxin, cell wall cytoskeleton and monophos‐ phoryl lipid A. Early phase 1 and 2 clinical trials in 1987 and 1988 revealed some prom‐ ising results, with one complete and three partial responses seen in 25 patients treated with Melacine. These results prompted the completion of seven open-label phase 2 trials involving 139 patients with stage III/IV melanoma and a multicenter phase 3 clinical trial of Melacine versus the Dartmouth regimen. The objective response rates for all of the above studies have been between 5 and 10%. Based largely upon these former results and the clinical results of other phase 3 trials, a phase 3 observation controlled trial of Melacine in melanoma patients was conducted. But the results revealed no evidence of a

One very promising autologous cell vaccine is MVax which is now in active phase 3 clinical trial sponsored by AVAX Technologies, Inc. This vaccine is derived from autologous tumor cells that have been irradiated and then modified with the hapten dinitrophenyl (DNP) [53]. In February 2004 the Journal of Clinical Oncology published an article by Dr. David Berd on the treatment of 214 Stage IIIb and IIIc melanoma patients that showed a five-year survival rate of 44%. Comparison to published results of similar patients treated with surgery alone showed five-year survival figures of 22%. In stage IV patients MVax has demonstrated sig‐

Tremelimumab is an IgG2 monoclonal antibody produced by Pfizer. It blocks the binding of the antigen-presenting cell ligands B7.1 and B7.2 to CTLA-4, resulting in inhibition of B7- CTLA-4-mediated down-regulation of T-cell activation. Subsequently, B7.1 or B7.2 may in‐ teract with another T-cell surface receptor protein, CD28, resulting in a B7-CD28-mediated T-cell activation unopposed by B7-CTLA-4-mediated inhibition. Tremelimumab is thought to stimulate patients' immune systems to attack their tumors. It has been shown to induce durable tumor responses in patients with metastatic melanoma in phase 1 and phase 2 clini‐ cal studies [44].

On April 2, 2008, Pfizer announced that it has discontinued a phase 3 clinical trial for pa‐ tients with metastatic melanoma after the review of interim data showed that the trial would not demonstrate superiority to standard chemotherapy [45]. Studies for other tumors are planned as of October 2009, namely for prostate cancer and bladder cancer.

## **3.5. Anti-integrin antibody: Etaracizumab**

Etaracizumab (also known as etaratuzumab, MEDI-522, trade name Abegrin) is an IgG1 humanized monoclonal antibody directed against the αVβ3 integrin. αVβ3 is essential for endothelial cell proliferation, maturation, and survival. When it is blocked, proliferating en‐ dothelial cells undergo apoptosis and regress. In addition, αVβ3 is highly expressed in mela‐ nomas and is associated with tumor growth and invasion. In preclinical studies using αVβ3 antagonists, inhibition of melanoma tumor growth independent of its antiangiogenic effects was reported [46]. Etaracizumab has been investigated in 3 phase 1, dose-escalation studies in patients with refractory melanoma. In the phase 2 trial, 57 patients received etaracizumab alone, and 55 patients received etaracizumab plus DTIC. Etaracizumab with or without DTIC generally was well tolerated and was active in patients with metastatic melanoma. The median survival was 12.6 months for the group that received etaracizumab with DTIC and 9.4 months for the group that received etaracizumab without DTIC [47]. These results encouraged people to further test this antibody in more clinical trials.

Early 2010, a study by the Etaracizumab Melanoma Study Group was reported. In this study, 112 patients were randomized to receive etaracizumab alone or etaracizumab plus DTIC. None of the patients in the etaracizumab alone study arm and 12.7% of patients in the etaracizumab plus DTIC study arm achieved an objective response. Stable disease occurred in 45.6% of patients in the etaracizumab alone study arm and 40.0% of patients in the etara‐ cizumab plus DTIC study arm. Despite a modest increase in survival, 12.6 months in the etaracizumab alone arm, versus 9.4 months in the etaracizumab plus DTIC arm, the re‐ searchers concluded that the survival results in both treatment arms of this study were con‐ sidered unlikely to result in clinically meaningful improvement over DTIC alone [48]. At the present time, clinical development of etaracizumab has been interrupted.

#### **3.6. Vaccines based on tumor cells: Canvaxin, melacine, and MVax**

pretation therefore is that dacarbazine did not help, but may rather have mitigated the re‐ sults in the dacarbazine plus ipilimumab arm. Based on all the data available, the large cumulative phase-II experience, and the two phase-III trials, the FDA approved treatment of

Tremelimumab is an IgG2 monoclonal antibody produced by Pfizer. It blocks the binding of the antigen-presenting cell ligands B7.1 and B7.2 to CTLA-4, resulting in inhibition of B7- CTLA-4-mediated down-regulation of T-cell activation. Subsequently, B7.1 or B7.2 may in‐ teract with another T-cell surface receptor protein, CD28, resulting in a B7-CD28-mediated T-cell activation unopposed by B7-CTLA-4-mediated inhibition. Tremelimumab is thought to stimulate patients' immune systems to attack their tumors. It has been shown to induce durable tumor responses in patients with metastatic melanoma in phase 1 and phase 2 clini‐

On April 2, 2008, Pfizer announced that it has discontinued a phase 3 clinical trial for pa‐ tients with metastatic melanoma after the review of interim data showed that the trial would not demonstrate superiority to standard chemotherapy [45]. Studies for other tumors are

Etaracizumab (also known as etaratuzumab, MEDI-522, trade name Abegrin) is an IgG1 humanized monoclonal antibody directed against the αVβ3 integrin. αVβ3 is essential for endothelial cell proliferation, maturation, and survival. When it is blocked, proliferating en‐ dothelial cells undergo apoptosis and regress. In addition, αVβ3 is highly expressed in mela‐ nomas and is associated with tumor growth and invasion. In preclinical studies using αVβ3 antagonists, inhibition of melanoma tumor growth independent of its antiangiogenic effects was reported [46]. Etaracizumab has been investigated in 3 phase 1, dose-escalation studies in patients with refractory melanoma. In the phase 2 trial, 57 patients received etaracizumab alone, and 55 patients received etaracizumab plus DTIC. Etaracizumab with or without DTIC generally was well tolerated and was active in patients with metastatic melanoma. The median survival was 12.6 months for the group that received etaracizumab with DTIC and 9.4 months for the group that received etaracizumab without DTIC [47]. These results

Early 2010, a study by the Etaracizumab Melanoma Study Group was reported. In this study, 112 patients were randomized to receive etaracizumab alone or etaracizumab plus DTIC. None of the patients in the etaracizumab alone study arm and 12.7% of patients in the etaracizumab plus DTIC study arm achieved an objective response. Stable disease occurred in 45.6% of patients in the etaracizumab alone study arm and 40.0% of patients in the etara‐ cizumab plus DTIC study arm. Despite a modest increase in survival, 12.6 months in the etaracizumab alone arm, versus 9.4 months in the etaracizumab plus DTIC arm, the re‐ searchers concluded that the survival results in both treatment arms of this study were con‐ sidered unlikely to result in clinically meaningful improvement over DTIC alone [48]. At the

planned as of October 2009, namely for prostate cancer and bladder cancer.

encouraged people to further test this antibody in more clinical trials.

present time, clinical development of etaracizumab has been interrupted.

melanoma with ipilimumab alone at the 3mg/kg dose [43].

546 Melanoma - From Early Detection to Treatment

**3.5. Anti-integrin antibody: Etaracizumab**

cal studies [44].

The basic idea is to use tumor cell-based vaccine to stimulate and activate the host immune system to recognize, contain and eliminate cancer cells. This effect may be based on the fol‐ lowing two pathways: direct migration of the tumor cells to the draining lymph node basin after injection, or uptake of apoptotic or necrotic tumor cells by host dendritic cells located within the skin [49].

The most extensively studied tumor cell-based vaccine is a polyvalent, antigen-rich whole cell vaccine called Canvaxin (CancerVax Corp., Carlsbad, CA). It is comprised of three mela‐ noma cell lines that contain over 20 immunogenic melanoma tumor antigens, given intra‐ dermally every two weeks for 3 to 5 doses, followed by monthly injections for the remainder of the first year. However, several small, single-institution phase 1 and 2 clinical trials of Canvaxin have not yielded a striking clinical benefit in most patients when administered with BCG as an immunoadjuvant [50]. But the rare complete responder to Canvaxin therapy has prompted the initiation of two multicenter phase 3 randomized trials of Canvaxin thera‐ py in 1998. In these trials, patients who have undergone complete resection of regional (stage III) or distant (stage IV) metastatic melanoma receive postoperative adjuvant immu‐ notherapy with Canvaxin plus Bacillus of Calmette and Guerin (BCG) or BCG alone. In April 2005, CancerVax announced the discontinuation of their phase 3 clinical trial of Can‐ vaxin in patients with Stage IV melanoma based upon the clinical funding that it was un‐ likely that the trial would provide significant evidence of a survival benefit for Canvaxintreated patients versus those receiving placebo. On October 3, 2005, CancerVax announced the discontinuation of another phase 3 clinical trial of Canvaxin in patients with Stage III melanoma base on a similar reason [51].

The second tumor cell-based vaccine that has been well studied since 1988 is Melacine. It is an allogeneic melanoma cell lysate combined with an immunologic adjuvant which is composed of a mixture of detoxified endotoxin, cell wall cytoskeleton and monophos‐ phoryl lipid A. Early phase 1 and 2 clinical trials in 1987 and 1988 revealed some prom‐ ising results, with one complete and three partial responses seen in 25 patients treated with Melacine. These results prompted the completion of seven open-label phase 2 trials involving 139 patients with stage III/IV melanoma and a multicenter phase 3 clinical trial of Melacine versus the Dartmouth regimen. The objective response rates for all of the above studies have been between 5 and 10%. Based largely upon these former results and the clinical results of other phase 3 trials, a phase 3 observation controlled trial of Melacine in melanoma patients was conducted. But the results revealed no evidence of a benefit from Melacine in patients with melanoma [52].

One very promising autologous cell vaccine is MVax which is now in active phase 3 clinical trial sponsored by AVAX Technologies, Inc. This vaccine is derived from autologous tumor cells that have been irradiated and then modified with the hapten dinitrophenyl (DNP) [53]. In February 2004 the Journal of Clinical Oncology published an article by Dr. David Berd on the treatment of 214 Stage IIIb and IIIc melanoma patients that showed a five-year survival rate of 44%. Comparison to published results of similar patients treated with surgery alone showed five-year survival figures of 22%. In stage IV patients MVax has demonstrated sig‐ nificant response rates as a monotherapy and in published reports MVax plus adjuvant IL-2 have reported response rate of 35% (13% Complete Response, 22% Partial Response). This compares to published response rates in low dose IL-2 of 3% [54].

MDX-1379 vaccine consists of two gp100 melanoma peptides. These peptides are part of a protein normally found on melanocytes, or pigmented skin cells, and on melanoma cells. These melanoma peptides are recognized by cytotoxic T cells in melanoma patients that are positive for HLA-A2, a human immune system compatibility antigen that is expressed in approximately half of the melanoma population. Phase II data show limited evidence of MDX-1379's clinical activity although there is strong proof-of-concept for therapeutic vac‐ cines based on gp100 in melanoma. Medarex is currently conducting a phase 3 clinical trial with ipilimumab and MDX-1379 combination therapy in stage III and IV melanoma at mul‐ tiple sites within the United States. Preliminary data showed MDX-1379 plus ipilimumab in‐ duced a modest percentage of durable response in stage IV melanoma. But autoimmune events could make the risk/benefit ratio for MDX-1379 plus ipilimumab unfavorable [58].

Therapeutic Agents for Advanced Melanoma

http://dx.doi.org/10.5772/53632

549

Astuprotimut-R (also called recombinant MAGE-A3 antigen-specific cancer immunothera‐ peutic GSK1203486A) is a cancer vaccine consisting of a recombinant form of human mela‐ noma antigen A3 (MAGE-A3) combined with a proprietary adjuvant with potential immunostimulatory and antineoplastic activities. Upon administration, astuprotimut-R may stimulate a cytotoxic T-lymphocyte response against tumor cells expressing the MAGE-A3 antigen, resulting in tumor cell death. MAGE-A3, a tumor-associated antigen (TAA) origi‐ nally discovered in melanoma cells, is expressed by various tumor types including melano‐ ma, non-small cell lung cancer, head and neck cancer, bladder cancer, with no expression in normal cells. MAGE-A3 protein has been in-licensed by GlaxoSmithKline (GSK) from the Ludwig Institute for Cancer Research. The proprietary immunostimulating adjuvant in this agent is composed of a specific combination of immunostimulating compounds selected to increase the anti-tumor immune response to MAGE-A3. Using this vaccine as intramuscular administration together with GSK's two proprietary adjuvant systems, AS15 or AS02B, they have developed a treatment regimen for cancer patients called Antigen-Specific Cancer Im‐

In 2008, GSK reported a randomized, open-label phase 2 study designed to evaluate Astu‐ protimut-R. A total of 72 patients with measurable metastatic MAGE-A3-positive cutaneous melanoma (unresectable or in transit stage III or stage IV M1a) were randomized to receive immunization with MAGE-A3 protein combined with either AS15 or AS02B as first-line metastatic treatment. Patients were to receive a maximum of 24 immunizations over four years. Clinical activity is assessed by the Response Evaluation Criteria In Solid Tumors (RE‐ CIST) criteria, the international standards for evaluation of solid tumors. Complete response (CR) and partial response (PR) *i.e.*, disappearance or significant reduction of tumor, were re‐ ported in 4 patients in the AS15 group (3 CR and 1 PR) with two of these ongoing for more than two years; in the AS02B arm, 1 patient showed a partial response which lasted for 6 months. The safety profile was similar in both groups with the majority of reported adverse events being mild or moderate local or systemic reactions [59]. Currently this agent still is

under phase 2 clinical development for progressive metastatic cutaneous melanoma.

Because melanoma tumors are heterogeneous in their antigenic profile, it is very difficult to make vaccines that can elicit cytotoxic T-cell responses universally in all the host immune systems. Rosenberg's group analyzed 28 different peptide-based vaccines utilized in stage

munotherapeutic (ASCI).

In October 2006, AVAX obtained a Special Protocol Assessment (SPA) agreement with the FDA for its phase 3 protocol. The SPA allows for the start of the phase 3 registration clinical trial for MVax for the treatment of patients with metastatic melanoma. In addition, the SPA addressed AVAX's ability to use a surrogate endpoint as a basis for accelerated approval. Based on this SPA, a phase 3 trial for stage IV melanoma was started on May 2007. AVAX plans to enroll up to 387 patients who will be assigned in a double-blind fashion at a 2:1 ra‐ tio to MVax or placebo vaccine. The MVax arm will consist of an initial dose of MVax fol‐ lowed by cyclophosphamide and then six weekly doses of MVax administered with BCG. Following vaccine administration patients will receive a specific schedule of low dose IL-2. Patients assigned to the control group will receive a treatment identical to the MVax group, except that a placebo vaccine will replace MVax. The primary endpoints of the study are best overall anti-tumor response rate and the percentage of patients surviving at least 2 years. Secondary endpoints of the study will include overall survival time, response dura‐ tion, percentage complete and partial responses, progression free survival and treatment re‐ lated adverse events [55].

#### **3.7. Vaccines based on peptides: MDX-1379, astuprotimut-R, and others**

The identification of tumor antigens that are present on the surface of melanoma cells is the basis for developing cancer vaccines that utilize peptide based immunotherapy. There are several melanoma differentiation antigens known involved in the synthesis of melanin and recognized by melanoma-reactive T cells, for example, gp100, MART-1/Melan-A, tyrosinase, TRP-1 and TRP-2, NY-ESO-1and the melanoma-associated antigen (MAGE) *etc.* One big ad‐ vantage of peptide based-vaccination is that it has few toxic side effects or adverse reactions. Data suggests that most tumor cell lines established from fine needle aspiration biopsies of patients with metastatic melanoma exhibit a relatively homogeneous co-expression of MART-1 and tyrosinase, with a much more heterogeneous expression of other tumor anti‐ gens, such as gp100, NY-ESO 1 and the MAGE antigens [56].

Rosenberg and his colleagues developed a with a peptide based-vaccine using modified immunodominant peptide of the gp100 antigen, g209-2M. They used this agent vaccinat‐ ed stage IV melanoma patients subcutaneously every three weeks. Following two immu‐ nizations, 10 of 11 (91%) of patients showed a consistently high level of immunization against the native g209~217 peptide, but not against the control peptide g280~288. This study also demonstrated that the majority of patients immunized with the g209-2M pep‐ tide in incomplete Freund's adjuvant (IFA) consistently developed high levels of circulat‐ ing immune precursors reactive against the native g209~217 peptide. Clinically, one of nine patients who received the g209~217 peptide in IFA experienced an objective cancer regression that lasted 4 months. Three of the eleven patients exhibited mixed responses with complete or partial regression of several lesions. However, all patients eventually developed progressive disease [57].

MDX-1379 vaccine consists of two gp100 melanoma peptides. These peptides are part of a protein normally found on melanocytes, or pigmented skin cells, and on melanoma cells. These melanoma peptides are recognized by cytotoxic T cells in melanoma patients that are positive for HLA-A2, a human immune system compatibility antigen that is expressed in approximately half of the melanoma population. Phase II data show limited evidence of MDX-1379's clinical activity although there is strong proof-of-concept for therapeutic vac‐ cines based on gp100 in melanoma. Medarex is currently conducting a phase 3 clinical trial with ipilimumab and MDX-1379 combination therapy in stage III and IV melanoma at mul‐ tiple sites within the United States. Preliminary data showed MDX-1379 plus ipilimumab in‐ duced a modest percentage of durable response in stage IV melanoma. But autoimmune events could make the risk/benefit ratio for MDX-1379 plus ipilimumab unfavorable [58].

nificant response rates as a monotherapy and in published reports MVax plus adjuvant IL-2 have reported response rate of 35% (13% Complete Response, 22% Partial Response). This

In October 2006, AVAX obtained a Special Protocol Assessment (SPA) agreement with the FDA for its phase 3 protocol. The SPA allows for the start of the phase 3 registration clinical trial for MVax for the treatment of patients with metastatic melanoma. In addition, the SPA addressed AVAX's ability to use a surrogate endpoint as a basis for accelerated approval. Based on this SPA, a phase 3 trial for stage IV melanoma was started on May 2007. AVAX plans to enroll up to 387 patients who will be assigned in a double-blind fashion at a 2:1 ra‐ tio to MVax or placebo vaccine. The MVax arm will consist of an initial dose of MVax fol‐ lowed by cyclophosphamide and then six weekly doses of MVax administered with BCG. Following vaccine administration patients will receive a specific schedule of low dose IL-2. Patients assigned to the control group will receive a treatment identical to the MVax group, except that a placebo vaccine will replace MVax. The primary endpoints of the study are best overall anti-tumor response rate and the percentage of patients surviving at least 2 years. Secondary endpoints of the study will include overall survival time, response dura‐ tion, percentage complete and partial responses, progression free survival and treatment re‐

compares to published response rates in low dose IL-2 of 3% [54].

**3.7. Vaccines based on peptides: MDX-1379, astuprotimut-R, and others**

gens, such as gp100, NY-ESO 1 and the MAGE antigens [56].

developed progressive disease [57].

The identification of tumor antigens that are present on the surface of melanoma cells is the basis for developing cancer vaccines that utilize peptide based immunotherapy. There are several melanoma differentiation antigens known involved in the synthesis of melanin and recognized by melanoma-reactive T cells, for example, gp100, MART-1/Melan-A, tyrosinase, TRP-1 and TRP-2, NY-ESO-1and the melanoma-associated antigen (MAGE) *etc.* One big ad‐ vantage of peptide based-vaccination is that it has few toxic side effects or adverse reactions. Data suggests that most tumor cell lines established from fine needle aspiration biopsies of patients with metastatic melanoma exhibit a relatively homogeneous co-expression of MART-1 and tyrosinase, with a much more heterogeneous expression of other tumor anti‐

Rosenberg and his colleagues developed a with a peptide based-vaccine using modified immunodominant peptide of the gp100 antigen, g209-2M. They used this agent vaccinat‐ ed stage IV melanoma patients subcutaneously every three weeks. Following two immu‐ nizations, 10 of 11 (91%) of patients showed a consistently high level of immunization against the native g209~217 peptide, but not against the control peptide g280~288. This study also demonstrated that the majority of patients immunized with the g209-2M pep‐ tide in incomplete Freund's adjuvant (IFA) consistently developed high levels of circulat‐ ing immune precursors reactive against the native g209~217 peptide. Clinically, one of nine patients who received the g209~217 peptide in IFA experienced an objective cancer regression that lasted 4 months. Three of the eleven patients exhibited mixed responses with complete or partial regression of several lesions. However, all patients eventually

lated adverse events [55].

548 Melanoma - From Early Detection to Treatment

Astuprotimut-R (also called recombinant MAGE-A3 antigen-specific cancer immunothera‐ peutic GSK1203486A) is a cancer vaccine consisting of a recombinant form of human mela‐ noma antigen A3 (MAGE-A3) combined with a proprietary adjuvant with potential immunostimulatory and antineoplastic activities. Upon administration, astuprotimut-R may stimulate a cytotoxic T-lymphocyte response against tumor cells expressing the MAGE-A3 antigen, resulting in tumor cell death. MAGE-A3, a tumor-associated antigen (TAA) origi‐ nally discovered in melanoma cells, is expressed by various tumor types including melano‐ ma, non-small cell lung cancer, head and neck cancer, bladder cancer, with no expression in normal cells. MAGE-A3 protein has been in-licensed by GlaxoSmithKline (GSK) from the Ludwig Institute for Cancer Research. The proprietary immunostimulating adjuvant in this agent is composed of a specific combination of immunostimulating compounds selected to increase the anti-tumor immune response to MAGE-A3. Using this vaccine as intramuscular administration together with GSK's two proprietary adjuvant systems, AS15 or AS02B, they have developed a treatment regimen for cancer patients called Antigen-Specific Cancer Im‐ munotherapeutic (ASCI).

In 2008, GSK reported a randomized, open-label phase 2 study designed to evaluate Astu‐ protimut-R. A total of 72 patients with measurable metastatic MAGE-A3-positive cutaneous melanoma (unresectable or in transit stage III or stage IV M1a) were randomized to receive immunization with MAGE-A3 protein combined with either AS15 or AS02B as first-line metastatic treatment. Patients were to receive a maximum of 24 immunizations over four years. Clinical activity is assessed by the Response Evaluation Criteria In Solid Tumors (RE‐ CIST) criteria, the international standards for evaluation of solid tumors. Complete response (CR) and partial response (PR) *i.e.*, disappearance or significant reduction of tumor, were re‐ ported in 4 patients in the AS15 group (3 CR and 1 PR) with two of these ongoing for more than two years; in the AS02B arm, 1 patient showed a partial response which lasted for 6 months. The safety profile was similar in both groups with the majority of reported adverse events being mild or moderate local or systemic reactions [59]. Currently this agent still is under phase 2 clinical development for progressive metastatic cutaneous melanoma.

Because melanoma tumors are heterogeneous in their antigenic profile, it is very difficult to make vaccines that can elicit cytotoxic T-cell responses universally in all the host immune systems. Rosenberg's group analyzed 28 different peptide-based vaccines utilized in stage IV melanoma patients. A total of 381 patients were treated with 370 patients showing no re‐ sponse, 9 patients showing a partial response and 2 patients with a complete response, for an overall objective response rate of only 2.9%. This suggested the lack of effectiveness with this single peptide based vaccination approach [60].

treatment of over 1,000 patients with DC-based vaccines, the record of effectiveness have

Therapeutic Agents for Advanced Melanoma

http://dx.doi.org/10.5772/53632

551

One very successful DC-based trial for patients with advanced, metastatic melanoma was reported by Nestle *et al*. He used plastic adherent monocytes matured with a xenogeneicbased 10% fetal calf serum, subsequently pulsed with either tumor cell lysate or multiple HLA-matched peptides injected intranodally. This trial involved 16 patients who were im‐ munized on an outpatient basis. Overall, 5 of 16 patients experienced an objective response, 2 complete and 3 partial responses. The side effects were noted to be minimal in all cases, with the development of vitiligo in a few patients. One dramatic feature of this treatment was the durability of the clinical responses, with the 2 complete responders remaining free

One phase 3 clinical trial about using DC-based vaccine to treat metastatic melanoma was report by Schadendorf and colleagues recently [65]. The trial was a prospective, randomized trial that analyzed the therapeutic effects of an autologous peptide-pulsed DC-based vaccine in patients with stage IV melanoma compared to standard chemotherapy with DTIC alone. The results revealed that the overall response in the vaccine group was 3.8% compared to 5.5% in the DTIC group, with no statistically significant differences noted in response, toxici‐ ty, overall and progression-free survival between the two groups. The median time to pro‐ gression was 2.8 months versus 3.2 months respectively and the median survival was 11

Although disappointed by many trials, several new avenues of DC-based immunotherapy are actively being pursued and in various stages of development, focusing on different ways to enhance the therapeutic efficacy of DC in combination with various immunoadjuvants

One very promising approach to treat metastatic melanoma is to use fully activated anti-tu‐ mor T-cells as warhead. This regimen involves the adoptive autologous transfer of highly selective tumor-reactive T-cells directed against over-expressed self-derived differentiation antigens after lymphodepleting chemotherapy. Rosenberg group reported in 2004 a clinical trial using this method. Cancer regression in patients with refractory metastatic melanoma with large, vascularized tumors was noted in a remarkable 18 of 35 patients (51% response rate), including four patients with a complete regression of all metastatic disease. Such re‐ sults may stem from the ability to infuse a large number of fully activated tumor infiltrating lymphocytes with anti-tumor activity into a host that is depleted of regulatory T-cells [66].

The recent developments in the field of gene transfer have advanced the use of gene therapy as a novel strategy against a variety of human malignancies. Because of its unique set of

months for the DTIC arm but only 9 months for the vaccine arm [65].

been disappointing [63].

of disease for over 15 months [64].

and other anticancer agents.

**4. Gene therapy agents**

**3.9. Individual therapy based on activated T-cells**

Next logical step is to make vaccines with multiple peptides to overcome tumor cell antigen‐ ic heterogeneity. A recent randomized phase 2 trial was performed in 26 patients with meta‐ static melanoma, vaccinating with four melanoma peptides. Although a high level of specific T-cell responses were noted (in 42% of the peripheral blood, 80% of sentinel lymph nodes), only three patients had a clinical response [61].

Here is the biggest issue in this area, actually many peptide based-vaccinations have result‐ ed in a significant increase in the number of lymphocyte precursors reactive against a varie‐ ty of tumor differentiation antigens by immunization with native or modified peptides. However, such immunological responses to peptide-based therapy have not translated into meaningful clinical responses for the vast majority of patients. To date, there is no study that has clearly shown a direct correlation between an immunologic response to therapy (im‐ mune cell activation) and a clinical response (regression of established tumor).

## **3.8. Vaccines based on dendritic cells**

In the normal human epidermis and dermis, dendritic cells (DC) are present as relatively immature antigen presenting cells, exhibiting relatively low levels of class II major histo‐ compatibility complex (MHC) molecules and co-stimulatory molecules. But these immature DC are quite capable of capturing various soluble protein antigens, such as apoptotic and necrotic tumor cells and then cross-presenting such tumor-associated antigens to cytotoxic CD8+ T cells. When relatively immature DC in the skin is triggered to enter afferent lym‐ phatic channels, this migrating pathway also initiates a phenotypic conversion that has pro‐ found immunological consequences [30]. When the DC arrives in the lymph node, it is characterized by an abundant levels of class II MHC antigens, as well as high surface levels of costimulatory molecules, such as CD40, CD54, CD80, CD83, and CD86. The matured DC is then capable of forming stable MHC class II-peptide complexes available to activate anti‐ gen specific CD4+ T cells [62].

To make the dendritic cell-based vaccine, the monocyte-derived, autologous DC can be pulsed *in vitro* with either whole irradiated, autologous tumor cells or tumor cell lysate. Once the tumor cells are "fed" to the DC *in vitro*, the apoptotic or necrotic cells are then processed and tumor-specific peptide antigens are then transported to the surface in both an MHC class I- and II-restricted fashion. Both immature and mature DC can be administered to patients as vaccine safely with few adverse side effects. The administration of DC via var‐ ious routes of vaccination (intradermal, intranodal and intravenous) is also feasible. The first published clinical trial of DC vaccination was in 1995 and has since been followed by 98 ad‐ ditional clinical trials describing more than 1,000 DC-based vaccines performed in 15 differ‐ ent countries. Twenty-eight trials focused on patients with various advanced stages of melanoma. The safety profile was again noted to be quite remarkable, however, despite the treatment of over 1,000 patients with DC-based vaccines, the record of effectiveness have been disappointing [63].

One very successful DC-based trial for patients with advanced, metastatic melanoma was reported by Nestle *et al*. He used plastic adherent monocytes matured with a xenogeneicbased 10% fetal calf serum, subsequently pulsed with either tumor cell lysate or multiple HLA-matched peptides injected intranodally. This trial involved 16 patients who were im‐ munized on an outpatient basis. Overall, 5 of 16 patients experienced an objective response, 2 complete and 3 partial responses. The side effects were noted to be minimal in all cases, with the development of vitiligo in a few patients. One dramatic feature of this treatment was the durability of the clinical responses, with the 2 complete responders remaining free of disease for over 15 months [64].

One phase 3 clinical trial about using DC-based vaccine to treat metastatic melanoma was report by Schadendorf and colleagues recently [65]. The trial was a prospective, randomized trial that analyzed the therapeutic effects of an autologous peptide-pulsed DC-based vaccine in patients with stage IV melanoma compared to standard chemotherapy with DTIC alone. The results revealed that the overall response in the vaccine group was 3.8% compared to 5.5% in the DTIC group, with no statistically significant differences noted in response, toxici‐ ty, overall and progression-free survival between the two groups. The median time to pro‐ gression was 2.8 months versus 3.2 months respectively and the median survival was 11 months for the DTIC arm but only 9 months for the vaccine arm [65].

Although disappointed by many trials, several new avenues of DC-based immunotherapy are actively being pursued and in various stages of development, focusing on different ways to enhance the therapeutic efficacy of DC in combination with various immunoadjuvants and other anticancer agents.

#### **3.9. Individual therapy based on activated T-cells**

One very promising approach to treat metastatic melanoma is to use fully activated anti-tu‐ mor T-cells as warhead. This regimen involves the adoptive autologous transfer of highly selective tumor-reactive T-cells directed against over-expressed self-derived differentiation antigens after lymphodepleting chemotherapy. Rosenberg group reported in 2004 a clinical trial using this method. Cancer regression in patients with refractory metastatic melanoma with large, vascularized tumors was noted in a remarkable 18 of 35 patients (51% response rate), including four patients with a complete regression of all metastatic disease. Such re‐ sults may stem from the ability to infuse a large number of fully activated tumor infiltrating lymphocytes with anti-tumor activity into a host that is depleted of regulatory T-cells [66].

## **4. Gene therapy agents**

IV melanoma patients. A total of 381 patients were treated with 370 patients showing no re‐ sponse, 9 patients showing a partial response and 2 patients with a complete response, for an overall objective response rate of only 2.9%. This suggested the lack of effectiveness with

Next logical step is to make vaccines with multiple peptides to overcome tumor cell antigen‐ ic heterogeneity. A recent randomized phase 2 trial was performed in 26 patients with meta‐ static melanoma, vaccinating with four melanoma peptides. Although a high level of specific T-cell responses were noted (in 42% of the peripheral blood, 80% of sentinel lymph

Here is the biggest issue in this area, actually many peptide based-vaccinations have result‐ ed in a significant increase in the number of lymphocyte precursors reactive against a varie‐ ty of tumor differentiation antigens by immunization with native or modified peptides. However, such immunological responses to peptide-based therapy have not translated into meaningful clinical responses for the vast majority of patients. To date, there is no study that has clearly shown a direct correlation between an immunologic response to therapy (im‐

In the normal human epidermis and dermis, dendritic cells (DC) are present as relatively immature antigen presenting cells, exhibiting relatively low levels of class II major histo‐ compatibility complex (MHC) molecules and co-stimulatory molecules. But these immature DC are quite capable of capturing various soluble protein antigens, such as apoptotic and necrotic tumor cells and then cross-presenting such tumor-associated antigens to cytotoxic CD8+ T cells. When relatively immature DC in the skin is triggered to enter afferent lym‐ phatic channels, this migrating pathway also initiates a phenotypic conversion that has pro‐ found immunological consequences [30]. When the DC arrives in the lymph node, it is characterized by an abundant levels of class II MHC antigens, as well as high surface levels of costimulatory molecules, such as CD40, CD54, CD80, CD83, and CD86. The matured DC is then capable of forming stable MHC class II-peptide complexes available to activate anti‐

To make the dendritic cell-based vaccine, the monocyte-derived, autologous DC can be pulsed *in vitro* with either whole irradiated, autologous tumor cells or tumor cell lysate. Once the tumor cells are "fed" to the DC *in vitro*, the apoptotic or necrotic cells are then processed and tumor-specific peptide antigens are then transported to the surface in both an MHC class I- and II-restricted fashion. Both immature and mature DC can be administered to patients as vaccine safely with few adverse side effects. The administration of DC via var‐ ious routes of vaccination (intradermal, intranodal and intravenous) is also feasible. The first published clinical trial of DC vaccination was in 1995 and has since been followed by 98 ad‐ ditional clinical trials describing more than 1,000 DC-based vaccines performed in 15 differ‐ ent countries. Twenty-eight trials focused on patients with various advanced stages of melanoma. The safety profile was again noted to be quite remarkable, however, despite the

mune cell activation) and a clinical response (regression of established tumor).

this single peptide based vaccination approach [60].

550 Melanoma - From Early Detection to Treatment

nodes), only three patients had a clinical response [61].

**3.8. Vaccines based on dendritic cells**

gen specific CD4+ T cells [62].

The recent developments in the field of gene transfer have advanced the use of gene therapy as a novel strategy against a variety of human malignancies. Because of its unique set of characteristics, melanoma represents a suitable target for gene therapy. Several strategies have been used by gene therapy to treat melanoma. First is to target melanoma cells to intro‐ duce "suicide" genes. Second is to transfer tumor suppressor genes. Third is to inactivate aberrant oncogene expression. Fourth is to introduce genes encoding immunologically rele‐ vant molecules. Last is to target the host's immune cells to redirect immune responses against melanoma. Clinical trials have shown the feasibility and safety of gene therapy against malignant melanoma. Although no major successes have been reported, the positive results observed in some patients support the potential for gene therapy in the management of this disease. To make gene therapy as an effective modality of treatment for malignant melanoma, better vector technology as well as increased understanding of the "bystander ef‐ fect" triggered by gene transfer approaches are needed [67].

pared with 6.8% in the latter group (P=0.007) [72]. Median progression-free survival for the oblimersan group was 2.4 months as compared with 1.6 months for the DTIC group, with a relative risk reduction of 27% (P=0.0003). The median survival was increased from 7.8 months in the DTIC arm to 9 months in the oblimersan arm with a P value of 0.077, which became significant when the patients with normal baseline LDH were analyzed. In terms of toxicity, no new or unexpected adverse events were observed in this study, which had not

Therapeutic Agents for Advanced Melanoma

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553

However, in May 2004, a new drug application (NDA) based on 6-months of minimum fol‐ low-up data from this trial failed to receive an affirmative vote for approval by an advisory committee to the FDA. Genta subsequently withdrew that application, and the Company

Allovectin-7 is a bicistronic plasmid formulated with a cationic lipid system containing the DNA sequences encoding HLA-B7 and beta-2 microglobulin, which together form a MHC1 antigen. Injection of Allovectin-7 directly into tumors is designed to stimulate an immune response against both local and distant metastatic tumors. Allovectin-7 is a nov‐ el gene therapy approach for cancer with a unique mechanism of action that is funda‐ mentally different from currently approved treatments. The following three mechanisms were believed to play roles in this agent's efficacy. Mechanism one, in HLA-B7 negative patients, a vigorous allogeneic immune response may be initiated against the foreign MHC class I antigen. Mechanism two, in all patients, ß2 microglobulin may reconstitute normal class I antigen presentation and/or increase tumor antigen presentation to the im‐ mune system. Mechanism three, in some patients, an innate pro-inflammatory response may occur that induces tumor responses following intralesional injection of the DNA/ lipid complex. The final outcome of all these mechanisms is to initially cause recognition of the tumor at the local site to allow a then sensitized immune response to recognize

In 2001, Dr. Richards and his colleagues began a high-dose, 2 mg, phase 2 trial evaluat‐ ing the Allovectin-7 immunotherapeutic alone for patients with stage III or stage IV mel‐ anoma, who have few other treatment options. The high-dose phase 2 trial completed enrollment in 2003. The data showed that the trial had a total of 15 responders among the 127 patients receiving the high dose (11.8%), with four of the patients having com‐ plete responses and 11 having partial responses. The Kaplan-Meier estimated median du‐ ration of response was 13.8 months. The Kaplan-Meier median survival was 18 months. The safety profile was excellent with no reported Grade 3 or Grade 4 adverse events as‐

Allovectin-7 has been granted orphan drug designation for the treatment of invasive and metastatic melanoma by the FDA's Office of Orphan Products Development. Orphan drug designation provides U.S. marketing exclusivity for seven years if marketing approval is re‐

has not yet made a decision regarding re-filing the U.S. application [73].

been seen with DTIC alone.

**4.2. DNA Plasmid-lipid complex allovectin-7**

un-injected tumors at distant metastatic sites [74].

sociated with Allovectin-7 [75].

ceived from the FDA

The gene therapy in our discussion is to introduce oligonucleotide or DNA sequence into host body thus to stimulate immune response to tumor cells. So it is also called DNA vacci‐ nation. This approach has been shown to induce long-lasting immunity against infectious agents and protection from tumor outgrowth in several animal models [68]. Likewise, intra‐ muscular injections of DNA (composed of naked DNA expression plasmids) into humans have also resulted in the development of an immunologic response [69]. It is hypothesized that one mechanism of tumor antigen expression may involve the DNA introducing the ap‐ propriate genes into dendritic cells for subsequent processing and presentation to the host immune system. One of the obvious advantages of DNA vaccinations is that they can be ad‐ ministered to patients regardless of HLA-phenotype and without identifying immunogenic epitopes.

#### **4.1. Anti-BCL2 antisense oligonucleotide genasense**

Genasense (Oblimersan sodium developed by Genta Inc. which is a biopharmaceutical com‐ pany based in Berkeley Heights, New Jersey) is a phosphorothioate antisense oligonucleo‐ tide directed against the first six codons of the Bcl-2 messenger RNA. Binding of the drug to the mRNA recruits RNAse H, resulting in cleavage of the mRNA. As a result, further trans‐ lation is halted and intracellular protein concentrations of Bcl-2 decrease with time. Melano‐ ma cell lines having Bcl-2 overexpression have been shown to enhance activity of metastasis-related proteinases, *in vitro* cell invasion, and *in vivo* tumor growth [70]. Many *in vitro* studies have demonstrated increased sensitivity of melanoma cells to chemotherapy when combined with antisense Bcl-2 therapy [71]. Genasense is the first oncology drug of its kind to directly target the biochemical pathway (known as apoptosis) whereby cancer cells are ultimately killed by chemotherapy. Genasense is believed to inhibit the production of Bcl-2, a protein that is believed to be a fundamental cause of resistance to anticancer thera‐ py. By inhibiting Bcl-2, Genasense may greatly improve the activity of anticancer therapy.

Encouraged by previous data, numerous clinical trials were started to evaluate the addition of oblimersan to chemotherapy in various solid tumors, including melanoma. Updated anal‐ ysis from a randomized phase 3 trial, comparing DTIC combined with oblimersan, with DTIC alone in 771 patients with Stage IV or unresectable Stage III melanoma who had not previously received chemotherapy has shown a response rate of 12.4% in the former com‐ pared with 6.8% in the latter group (P=0.007) [72]. Median progression-free survival for the oblimersan group was 2.4 months as compared with 1.6 months for the DTIC group, with a relative risk reduction of 27% (P=0.0003). The median survival was increased from 7.8 months in the DTIC arm to 9 months in the oblimersan arm with a P value of 0.077, which became significant when the patients with normal baseline LDH were analyzed. In terms of toxicity, no new or unexpected adverse events were observed in this study, which had not been seen with DTIC alone.

However, in May 2004, a new drug application (NDA) based on 6-months of minimum fol‐ low-up data from this trial failed to receive an affirmative vote for approval by an advisory committee to the FDA. Genta subsequently withdrew that application, and the Company has not yet made a decision regarding re-filing the U.S. application [73].

## **4.2. DNA Plasmid-lipid complex allovectin-7**

characteristics, melanoma represents a suitable target for gene therapy. Several strategies have been used by gene therapy to treat melanoma. First is to target melanoma cells to intro‐ duce "suicide" genes. Second is to transfer tumor suppressor genes. Third is to inactivate aberrant oncogene expression. Fourth is to introduce genes encoding immunologically rele‐ vant molecules. Last is to target the host's immune cells to redirect immune responses against melanoma. Clinical trials have shown the feasibility and safety of gene therapy against malignant melanoma. Although no major successes have been reported, the positive results observed in some patients support the potential for gene therapy in the management of this disease. To make gene therapy as an effective modality of treatment for malignant melanoma, better vector technology as well as increased understanding of the "bystander ef‐

The gene therapy in our discussion is to introduce oligonucleotide or DNA sequence into host body thus to stimulate immune response to tumor cells. So it is also called DNA vacci‐ nation. This approach has been shown to induce long-lasting immunity against infectious agents and protection from tumor outgrowth in several animal models [68]. Likewise, intra‐ muscular injections of DNA (composed of naked DNA expression plasmids) into humans have also resulted in the development of an immunologic response [69]. It is hypothesized that one mechanism of tumor antigen expression may involve the DNA introducing the ap‐ propriate genes into dendritic cells for subsequent processing and presentation to the host immune system. One of the obvious advantages of DNA vaccinations is that they can be ad‐ ministered to patients regardless of HLA-phenotype and without identifying immunogenic

Genasense (Oblimersan sodium developed by Genta Inc. which is a biopharmaceutical com‐ pany based in Berkeley Heights, New Jersey) is a phosphorothioate antisense oligonucleo‐ tide directed against the first six codons of the Bcl-2 messenger RNA. Binding of the drug to the mRNA recruits RNAse H, resulting in cleavage of the mRNA. As a result, further trans‐ lation is halted and intracellular protein concentrations of Bcl-2 decrease with time. Melano‐ ma cell lines having Bcl-2 overexpression have been shown to enhance activity of metastasis-related proteinases, *in vitro* cell invasion, and *in vivo* tumor growth [70]. Many *in vitro* studies have demonstrated increased sensitivity of melanoma cells to chemotherapy when combined with antisense Bcl-2 therapy [71]. Genasense is the first oncology drug of its kind to directly target the biochemical pathway (known as apoptosis) whereby cancer cells are ultimately killed by chemotherapy. Genasense is believed to inhibit the production of Bcl-2, a protein that is believed to be a fundamental cause of resistance to anticancer thera‐ py. By inhibiting Bcl-2, Genasense may greatly improve the activity of anticancer therapy.

Encouraged by previous data, numerous clinical trials were started to evaluate the addition of oblimersan to chemotherapy in various solid tumors, including melanoma. Updated anal‐ ysis from a randomized phase 3 trial, comparing DTIC combined with oblimersan, with DTIC alone in 771 patients with Stage IV or unresectable Stage III melanoma who had not previously received chemotherapy has shown a response rate of 12.4% in the former com‐

fect" triggered by gene transfer approaches are needed [67].

552 Melanoma - From Early Detection to Treatment

**4.1. Anti-BCL2 antisense oligonucleotide genasense**

epitopes.

Allovectin-7 is a bicistronic plasmid formulated with a cationic lipid system containing the DNA sequences encoding HLA-B7 and beta-2 microglobulin, which together form a MHC1 antigen. Injection of Allovectin-7 directly into tumors is designed to stimulate an immune response against both local and distant metastatic tumors. Allovectin-7 is a nov‐ el gene therapy approach for cancer with a unique mechanism of action that is funda‐ mentally different from currently approved treatments. The following three mechanisms were believed to play roles in this agent's efficacy. Mechanism one, in HLA-B7 negative patients, a vigorous allogeneic immune response may be initiated against the foreign MHC class I antigen. Mechanism two, in all patients, ß2 microglobulin may reconstitute normal class I antigen presentation and/or increase tumor antigen presentation to the im‐ mune system. Mechanism three, in some patients, an innate pro-inflammatory response may occur that induces tumor responses following intralesional injection of the DNA/ lipid complex. The final outcome of all these mechanisms is to initially cause recognition of the tumor at the local site to allow a then sensitized immune response to recognize un-injected tumors at distant metastatic sites [74].

In 2001, Dr. Richards and his colleagues began a high-dose, 2 mg, phase 2 trial evaluat‐ ing the Allovectin-7 immunotherapeutic alone for patients with stage III or stage IV mel‐ anoma, who have few other treatment options. The high-dose phase 2 trial completed enrollment in 2003. The data showed that the trial had a total of 15 responders among the 127 patients receiving the high dose (11.8%), with four of the patients having com‐ plete responses and 11 having partial responses. The Kaplan-Meier estimated median du‐ ration of response was 13.8 months. The Kaplan-Meier median survival was 18 months. The safety profile was excellent with no reported Grade 3 or Grade 4 adverse events as‐ sociated with Allovectin-7 [75].

Allovectin-7 has been granted orphan drug designation for the treatment of invasive and metastatic melanoma by the FDA's Office of Orphan Products Development. Orphan drug designation provides U.S. marketing exclusivity for seven years if marketing approval is re‐ ceived from the FDA

Vical is conducting the AIMM (Allovectin-7 Immunotherapeutic for Metastatic Melanoma) trial, a phase 3 pivotal trial of Allovectin-7 as first-line therapy in approximately 375 patients with Stage III or IV recurrent metastatic melanoma in accordance with a SPA agreement completed with the FDA. The trial is being conducted at approximately 60 clinical sites worldwide. They designed the trial to include patients most likely to benefit from our treat‐ ment, and specifically excluded patients with brain or liver metastases, patients previously treated with chemotherapy, and patients with elevated lactate dehydrogenase (LDH) levels.

national, open label, randomized study designed to assess the efficacy and safety of treat‐ ment with OncoVEX (GM-CSF) as compared to subcutaneously administered GM-CSF in patients with unresectable stage III (b-c) and stage IV (M1a-c) disease. Patients will have re‐ ceived at least one prior therapy for active disease which includes any type of therapy in‐ cluding investigational drugs. A total of 360 patients will be enrolled (240 to the OncoVEX (GM-CSF) arm and 120 to the control arm). The study design was agreed with the FDA un‐

Therapeutic Agents for Advanced Melanoma

http://dx.doi.org/10.5772/53632

555

**5. Possible reasons for extremely high resistance of metastatic melanoma**

Despite an epic number of clinical trials to test a wide variety of anticancer strategies, the average survival rate for patients with metastatic melanoma remains unimproved during the past 30 years (41). Though constant clinical trials effort, although some approaches showed promising intermediate results, still no agent has been granted FDA approval for the treatment of metastatic melanoma. There are several reasons that may account for the

Melanoma cells are quite resistant to most chemotherapy reagents. This is associated with the specific feature of melanoma cells. In nature, these cells have low levels of spontaneous apoptosis *in vivo* compared with other tumor cell types, and they are relatively resistant to drug-induced apoptosis *in vitro* [78]*.* The natural role of melanocytes is to protect inner or‐ gans from UV light, a potent DNA damaging agent. Therefore, it is not surprising that mela‐ noma cells may have special DNA damage repair systems and enhanced survival properties [79]. Moreover, recent studies showed that, during melanoma progression, it acquired com‐ plex genetic alterations that led to hyperactivation of efflux pumps, detoxification enzymes, and a multifactorial alteration of survival and apoptotic pathways. All these have been pro‐

The major barrier is immunosuppressive effects activated by tumors. Tumor cell can escape immune rejection and induce immunosuppression through the following five major paths. Firstly, tumor cells may lose or down-regulate either the melanoma associated antigens or MHC molecules. Secondly, tumor cells may produce a plethora of immunosuppressive fac‐ tors such as interleukin-10, VEGF and transforming growth factor. These factors create an inherently unfavorable microenvironment that limits the host immune response, in addition to tolerating the T-cell response to established tumor. Third possible reason is intrinsic inef‐ ficiency of DC whereby the appropriate co-stimulatory molecules are not being presented on the cell surface. Fourth possible reason is tumor-related alterations in T-cell signaling and a skewing of the immune response from a Th1 (immunoactivating) to a Th2 response (im‐ munotolerant). Lastly, the concept of tumor cell escape and immune tolerance is an exceed‐

extremely high resistance of metastatic melanoma to current treatment modalities.

posed to mediate the multi-drug resistant phenotype of melanoma [80].

der the special protocol assessment process [77].

**5.1. Reasons for chemotherapy resistance**

**5.2. Barriers for successful immunotherapy**

In January 2010 Vical announced that the company has completed enrollment of the plan‐ ned 375 subjects in its multinational phase 3 trial of Allovectin-7 in patients with metastatic melanoma. Allovectin-7®'s safety profile is excellent with no drug-related serious adverse events reported to date in the phase 3 trial [74].

#### **4.3. Herpes simplex virus based oncoVEX**

OncoVEX (GM-CSF) is an enhanced potency, immuneenhanced oncolytic herpes simplex vi‐ rus type 1 (HSV-1). It is deleted for infected-cell protein gene 34.5 (ICP34.5), providing tu‐ mor selective replication, and ICP47 gene which otherwise blocks antigen presentation. In addition, ICP47 deletion increases unique short region protein 11 (US11) gene expression thereby enhancing virus growth and replication in tumor cells. The coding sequence for hu‐ man granulocyte-macrophage colony-stimulating factor (GM-CSF) is inserted, replacing ICP34.5, to enhance the immune response to tumor antigens released following virus repli‐ cation.

OncoVEX is developed by BioVex (Woburn, MA). It is a first-in-class oncolytic, or cancer de‐ stroying virus, that works by replicating and spreading within solid tumors (leaving healthy cells unaffected), thereby causing cancer cell death and stimulating the immune system to destroy un-injected metastatic deposits. Both modes of action have been clearly validated in the clinic, where multiple patients with metastatic disease progressing at enrollment have been declared disease free.

BioVex recently concluded a 50-patient phase 2 trial for OncoVEX (GM-CSF) as a standalone therapy in patients with Stage IIIc and Stage IV melanoma. The trial was designed to measure overall objective response, which is defined as a complete response, where disease is completely eliminated, or partial response, where there is a >50% reduction in disease bur‐ den. 74% of patients who entered the study were progressing after having failed prior thera‐ py. 13 objective systemic responses (26% objective response rate) were achieved including eight CRs, seven of which remain free of disease. 12 responses have so far continued for more than 6 months (ranging from 6 to more than 29 months). Responses were observed in patients with all stages of disease, including the complete resolution of un-injected visceral deposits. Adverse effects were primarily limited to transient flu-like symptoms [76].

In April 2009, BioVex Inc. announced that its OPTiM (OncoVEX Pivotal Trial in Melanoma) phase 3 study with OncoVEX (GM-CSF) in previously treated patients with Stage III and Stage IV melanoma had initiated. The study has commenced recruiting patients in the U.S. and with sites in the United Kingdom, Germany and Australia. The OPTiM trial is a multinational, open label, randomized study designed to assess the efficacy and safety of treat‐ ment with OncoVEX (GM-CSF) as compared to subcutaneously administered GM-CSF in patients with unresectable stage III (b-c) and stage IV (M1a-c) disease. Patients will have re‐ ceived at least one prior therapy for active disease which includes any type of therapy in‐ cluding investigational drugs. A total of 360 patients will be enrolled (240 to the OncoVEX (GM-CSF) arm and 120 to the control arm). The study design was agreed with the FDA un‐ der the special protocol assessment process [77].

## **5. Possible reasons for extremely high resistance of metastatic melanoma**

Despite an epic number of clinical trials to test a wide variety of anticancer strategies, the average survival rate for patients with metastatic melanoma remains unimproved during the past 30 years (41). Though constant clinical trials effort, although some approaches showed promising intermediate results, still no agent has been granted FDA approval for the treatment of metastatic melanoma. There are several reasons that may account for the extremely high resistance of metastatic melanoma to current treatment modalities.

#### **5.1. Reasons for chemotherapy resistance**

Vical is conducting the AIMM (Allovectin-7 Immunotherapeutic for Metastatic Melanoma) trial, a phase 3 pivotal trial of Allovectin-7 as first-line therapy in approximately 375 patients with Stage III or IV recurrent metastatic melanoma in accordance with a SPA agreement completed with the FDA. The trial is being conducted at approximately 60 clinical sites worldwide. They designed the trial to include patients most likely to benefit from our treat‐ ment, and specifically excluded patients with brain or liver metastases, patients previously treated with chemotherapy, and patients with elevated lactate dehydrogenase (LDH) levels.

In January 2010 Vical announced that the company has completed enrollment of the plan‐ ned 375 subjects in its multinational phase 3 trial of Allovectin-7 in patients with metastatic melanoma. Allovectin-7®'s safety profile is excellent with no drug-related serious adverse

OncoVEX (GM-CSF) is an enhanced potency, immuneenhanced oncolytic herpes simplex vi‐ rus type 1 (HSV-1). It is deleted for infected-cell protein gene 34.5 (ICP34.5), providing tu‐ mor selective replication, and ICP47 gene which otherwise blocks antigen presentation. In addition, ICP47 deletion increases unique short region protein 11 (US11) gene expression thereby enhancing virus growth and replication in tumor cells. The coding sequence for hu‐ man granulocyte-macrophage colony-stimulating factor (GM-CSF) is inserted, replacing ICP34.5, to enhance the immune response to tumor antigens released following virus repli‐

OncoVEX is developed by BioVex (Woburn, MA). It is a first-in-class oncolytic, or cancer de‐ stroying virus, that works by replicating and spreading within solid tumors (leaving healthy cells unaffected), thereby causing cancer cell death and stimulating the immune system to destroy un-injected metastatic deposits. Both modes of action have been clearly validated in the clinic, where multiple patients with metastatic disease progressing at enrollment have

BioVex recently concluded a 50-patient phase 2 trial for OncoVEX (GM-CSF) as a standalone therapy in patients with Stage IIIc and Stage IV melanoma. The trial was designed to measure overall objective response, which is defined as a complete response, where disease is completely eliminated, or partial response, where there is a >50% reduction in disease bur‐ den. 74% of patients who entered the study were progressing after having failed prior thera‐ py. 13 objective systemic responses (26% objective response rate) were achieved including eight CRs, seven of which remain free of disease. 12 responses have so far continued for more than 6 months (ranging from 6 to more than 29 months). Responses were observed in patients with all stages of disease, including the complete resolution of un-injected visceral

deposits. Adverse effects were primarily limited to transient flu-like symptoms [76].

In April 2009, BioVex Inc. announced that its OPTiM (OncoVEX Pivotal Trial in Melanoma) phase 3 study with OncoVEX (GM-CSF) in previously treated patients with Stage III and Stage IV melanoma had initiated. The study has commenced recruiting patients in the U.S. and with sites in the United Kingdom, Germany and Australia. The OPTiM trial is a multi-

events reported to date in the phase 3 trial [74].

**4.3. Herpes simplex virus based oncoVEX**

554 Melanoma - From Early Detection to Treatment

cation.

been declared disease free.

Melanoma cells are quite resistant to most chemotherapy reagents. This is associated with the specific feature of melanoma cells. In nature, these cells have low levels of spontaneous apoptosis *in vivo* compared with other tumor cell types, and they are relatively resistant to drug-induced apoptosis *in vitro* [78]*.* The natural role of melanocytes is to protect inner or‐ gans from UV light, a potent DNA damaging agent. Therefore, it is not surprising that mela‐ noma cells may have special DNA damage repair systems and enhanced survival properties [79]. Moreover, recent studies showed that, during melanoma progression, it acquired com‐ plex genetic alterations that led to hyperactivation of efflux pumps, detoxification enzymes, and a multifactorial alteration of survival and apoptotic pathways. All these have been pro‐ posed to mediate the multi-drug resistant phenotype of melanoma [80].

#### **5.2. Barriers for successful immunotherapy**

The major barrier is immunosuppressive effects activated by tumors. Tumor cell can escape immune rejection and induce immunosuppression through the following five major paths. Firstly, tumor cells may lose or down-regulate either the melanoma associated antigens or MHC molecules. Secondly, tumor cells may produce a plethora of immunosuppressive fac‐ tors such as interleukin-10, VEGF and transforming growth factor. These factors create an inherently unfavorable microenvironment that limits the host immune response, in addition to tolerating the T-cell response to established tumor. Third possible reason is intrinsic inef‐ ficiency of DC whereby the appropriate co-stimulatory molecules are not being presented on the cell surface. Fourth possible reason is tumor-related alterations in T-cell signaling and a skewing of the immune response from a Th1 (immunoactivating) to a Th2 response (im‐ munotolerant). Lastly, the concept of tumor cell escape and immune tolerance is an exceed‐ ingly complex process. We need to further understand these mechanisms before we can have successful immunotherapy to melanoma [32].

temic way based on the understanding about melanoma molecular pathology seems to be a

Therapeutic Agents for Advanced Melanoma

http://dx.doi.org/10.5772/53632

557

Individualized T-cell-based therapy is a very promising approach. Combined with other suitable tumor killing agents, it could improve the patient survival rate and time. Unfortu‐ nately, the selective tumor-reactive T-cells isolated from a patient can only be used for this same patient. Thus the cost associated with this treatment method is very high. Such an ex‐

In learning from our efforts in the past, we must continue to challenge the current para‐ digms of treatment as we forge new paths to more effective treatment options. This will like‐ ly involve a multimodal approach to therapy utilizing all of the available tools in our arsenal. Several agents given in unique combinations may then synergize with standard che‐ motherapeutic regimens resulting in prolonged clinical responses and long term survival. Take Sorafenib as an example, its failure maybe largely due to the fact that it only blocks the RAF-MEK-ERK signaling pathway. Melanoma cells can still survive by compensatory upregulation in other survival pathways such as the PI3K-AKT pathway. Melanoma can also develop drug resistance with time by over-expressing MDR genes. Ideally, if we can use drugs to synergistically block all the major survival pathways in melanoma cells and then educate our immune system to fight the tumor cells, we will have a much better chance to

Department of Pharmaceutical Sciences, University of Tennessee Health Science Center,

[1] Eggermont, A.M. and C. Robert, *New drugs in melanoma: It's a whole new world.* Eur J

[2] Kahler, K.C. and A. Hauschild, *Treatment and side effect management of CTLA-4 anti‐ body therapy in metastatic melanoma.* J Dtsch Dermatol Ges, 2011. 9(4): p. 277-86.

[3] Roukos, D.H., *PLX4032 and melanoma: resistance, expectations and uncertainty.* Expert

[4] Williams, D.A. and T.L. Lemke, *Foye's Principles of Medicinal Chemistry*. Fifth Edition

pensive treatment may not be available to all the patients in the near future.

and Duane D. Miller

\*Address all correspondence to: wli@uthsc.edu

Cancer, 2011. 47(47): p. 2150-2157.

Rev Anticancer Ther, 2011. 11(3): p. 325-8.

ed2002, Philadelphia, PA: Lippincott Williams & Wilkins.

reasonable way to fight this disease.

conquer this deadly disease.

**Author details**

Zhao Wang, Wei Li\*

Memphis, USA

**References**

Specifically for cancer vaccines, there are some further barriers. First is the characterization of vaccines potency and toxicity. This is especially important in the transition from phase 2 to phase 3 trials. To select a meaningful and validated end point for trials is a big challenge most of the time. Second barrier is selection of the maximum tolerated dose of cancer vac‐ cine, particularly compared with traditional anticancer agents. Cancer vaccines are typically not very toxic. So the optimum dose often has to be based on the immune response of pa‐ tients. But if the patients have previously been heavily treated with other anticancer agents, this can lead to a compromised immune system that makes it difficult to detect an evoked immune response. The third barrier is appropriate trial design and statistical data process. This is also a key part and can substantially affect final trial outcome [53].

## **6. Future directions**

With the rapidly rising incidence and the high resistance to current therapeutic agents, de‐ veloping more effective drugs for metastatic melanoma is urgently needed. But before we can thoroughly understand all the major molecular pathological changes associated with melanoma malignancy, it is very difficult to reach a cure for it.

Melanoma is an extremely complicated disease, with many gene mutation and signaling pathway changes. Elevated signaling pathway in melanoma including mitogen-activated protein kinase (MAPK) pathway, phosphatidylinositol 3 kinase (PI3K)-AKT pathway, Wnt-Frizzled-β-catenin pathway, JAK/Stat pathway and α-MSH-MC1R or microphthalmia-asso‐ ciated transcription factor (MITF) pathway. The first two are crucial pathway accounting for melanoma malignance. Gene mutation that are involved in melanoma include the following oncogenes: BRAF, N-ras, akt3; tumor suppressors: CDKN2A, PTEN, p53, APAF-1, p16, p15, p19; others: Cyclin D1, MITF etc [81].

The binding of growth factors to their respective receptors leads to activation of RAS pro‐ teins. Ras will then activate Raf. Raf activate mitogen-activated protein kinase (MEK), which then act on extracellular-related kinase (ERK). Phosporylated ERK kinases (ERK-P) translo‐ cate to the nucleus and activate transcription factors, which promote cell cycle progression and proliferation. The PI3K-AKT pathway mediates cell survival signaling via growth fac‐ tors. Phosphatase and tensin homolog (PTEN) inhibits growth factor signaling by inactivat‐ ing phosphatidylinositol triphosphate (PIP3) generated by PI3K. Activated PI3K converts the plasma membrane lipid phosphatidylinositol 4,5-bisphosphonate to PIP3, which acts as a second messenger leading to the phosphorylation AKT and subsequent up-regulation of cell cycle, growth, and survival proteins. AKT can also up-regulate mTOR (mammalian tar‐ get of rapamycin), S6K, and NFκb leading to cell growth and inhibition of apoptosis.

Knowing the huge complexity of melanoma, it's easy to understand why so many random trials of single agents or combinational treatment have failed. So targeted therapy in a sys‐ temic way based on the understanding about melanoma molecular pathology seems to be a reasonable way to fight this disease.

Individualized T-cell-based therapy is a very promising approach. Combined with other suitable tumor killing agents, it could improve the patient survival rate and time. Unfortu‐ nately, the selective tumor-reactive T-cells isolated from a patient can only be used for this same patient. Thus the cost associated with this treatment method is very high. Such an ex‐ pensive treatment may not be available to all the patients in the near future.

In learning from our efforts in the past, we must continue to challenge the current para‐ digms of treatment as we forge new paths to more effective treatment options. This will like‐ ly involve a multimodal approach to therapy utilizing all of the available tools in our arsenal. Several agents given in unique combinations may then synergize with standard che‐ motherapeutic regimens resulting in prolonged clinical responses and long term survival. Take Sorafenib as an example, its failure maybe largely due to the fact that it only blocks the RAF-MEK-ERK signaling pathway. Melanoma cells can still survive by compensatory upregulation in other survival pathways such as the PI3K-AKT pathway. Melanoma can also develop drug resistance with time by over-expressing MDR genes. Ideally, if we can use drugs to synergistically block all the major survival pathways in melanoma cells and then educate our immune system to fight the tumor cells, we will have a much better chance to conquer this deadly disease.

## **Author details**

ingly complex process. We need to further understand these mechanisms before we can

Specifically for cancer vaccines, there are some further barriers. First is the characterization of vaccines potency and toxicity. This is especially important in the transition from phase 2 to phase 3 trials. To select a meaningful and validated end point for trials is a big challenge most of the time. Second barrier is selection of the maximum tolerated dose of cancer vac‐ cine, particularly compared with traditional anticancer agents. Cancer vaccines are typically not very toxic. So the optimum dose often has to be based on the immune response of pa‐ tients. But if the patients have previously been heavily treated with other anticancer agents, this can lead to a compromised immune system that makes it difficult to detect an evoked immune response. The third barrier is appropriate trial design and statistical data process.

With the rapidly rising incidence and the high resistance to current therapeutic agents, de‐ veloping more effective drugs for metastatic melanoma is urgently needed. But before we can thoroughly understand all the major molecular pathological changes associated with

Melanoma is an extremely complicated disease, with many gene mutation and signaling pathway changes. Elevated signaling pathway in melanoma including mitogen-activated protein kinase (MAPK) pathway, phosphatidylinositol 3 kinase (PI3K)-AKT pathway, Wnt-Frizzled-β-catenin pathway, JAK/Stat pathway and α-MSH-MC1R or microphthalmia-asso‐ ciated transcription factor (MITF) pathway. The first two are crucial pathway accounting for melanoma malignance. Gene mutation that are involved in melanoma include the following oncogenes: BRAF, N-ras, akt3; tumor suppressors: CDKN2A, PTEN, p53, APAF-1, p16, p15,

The binding of growth factors to their respective receptors leads to activation of RAS pro‐ teins. Ras will then activate Raf. Raf activate mitogen-activated protein kinase (MEK), which then act on extracellular-related kinase (ERK). Phosporylated ERK kinases (ERK-P) translo‐ cate to the nucleus and activate transcription factors, which promote cell cycle progression and proliferation. The PI3K-AKT pathway mediates cell survival signaling via growth fac‐ tors. Phosphatase and tensin homolog (PTEN) inhibits growth factor signaling by inactivat‐ ing phosphatidylinositol triphosphate (PIP3) generated by PI3K. Activated PI3K converts the plasma membrane lipid phosphatidylinositol 4,5-bisphosphonate to PIP3, which acts as a second messenger leading to the phosphorylation AKT and subsequent up-regulation of cell cycle, growth, and survival proteins. AKT can also up-regulate mTOR (mammalian tar‐

get of rapamycin), S6K, and NFκb leading to cell growth and inhibition of apoptosis.

Knowing the huge complexity of melanoma, it's easy to understand why so many random trials of single agents or combinational treatment have failed. So targeted therapy in a sys‐

This is also a key part and can substantially affect final trial outcome [53].

melanoma malignancy, it is very difficult to reach a cure for it.

have successful immunotherapy to melanoma [32].

556 Melanoma - From Early Detection to Treatment

**6. Future directions**

p19; others: Cyclin D1, MITF etc [81].

Zhao Wang, Wei Li\* and Duane D. Miller

\*Address all correspondence to: wli@uthsc.edu

Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, USA

## **References**


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**Chapter 21**

**Update in Ocular Melanoma**

Victoria de los Ángeles Bustuoabad, Lucia Speroni and Arturo Irarrázabal

http://dx.doi.org/10.5772/53633

**1. Introduction**

**1.1. Ocular anatomy**

anterior to posterior:

miosis).

ing the same embryologic origin.

pathetic innervation: longitudinal (outermost), radial and circular.

anatomy.

Additional information is available at the end of the chapter

In order to understand the pathophysiology of this condition we will describe the *uvea*

*The Iris* is a contractile diaphragm that controls the degree of retinal illumination, it has a central aperture, the pupil, located slightly nasally. It consists of the following layers from

**1.** Stroma: a thin avascular layer with fibroblasts and melanocytes. It is heavily pigment‐ ed in persons with brown eyes less pigmented in green and hazel irises and least in blue. Posteriorly, the stroma contains the sphincter pupillae muscle (parasympathetic,

**2.** Pigment epithelium: consists of 2 layers of cells: -anterior layer, which intermingles with the dilator pupillae muscle (sympathetic, mydriasis) -posterior layer, which is con‐ tinuous with the pigment epithelium of the ciliary body and RPE of the retina, all hav‐

*The cilliary body* is one of the three parts of the uvea and extends for 6 mm from the end of the retina (ora serrata) till the scleral spur. Its epithelial portion (adjacent to vitreous) con‐ sists of a posterior portion (pars plana) and an anterior portion (pars plicata). The latter has 60-70 folds called the ciliary processes which secrete the aqueous humor into the posterior chamber. Its uveal portion contains the ciliary muscle which has 3 parts, all under parasym‐

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

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

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

## **Chapter 21**

## **Update in Ocular Melanoma**

Victoria de los Ángeles Bustuoabad, Lucia Speroni and Arturo Irarrázabal

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53633

**1. Introduction**

#### **1.1. Ocular anatomy**

In order to understand the pathophysiology of this condition we will describe the *uvea* anatomy.

*The Iris* is a contractile diaphragm that controls the degree of retinal illumination, it has a central aperture, the pupil, located slightly nasally. It consists of the following layers from anterior to posterior:


*The cilliary body* is one of the three parts of the uvea and extends for 6 mm from the end of the retina (ora serrata) till the scleral spur. Its epithelial portion (adjacent to vitreous) con‐ sists of a posterior portion (pars plana) and an anterior portion (pars plicata). The latter has 60-70 folds called the ciliary processes which secrete the aqueous humor into the posterior chamber. Its uveal portion contains the ciliary muscle which has 3 parts, all under parasym‐ pathetic innervation: longitudinal (outermost), radial and circular.

*The choroid is a* dark brown vascular sheet, 0.25mm thick, lying between the sclera and the retina. The outer vascular bed has large vessels (layer of Haller) and the inner bed consists of an extensive network of fenestrated vessels, the choriocapillaris which is the major blood supply to the outer layers of the retina and to the whole macula The inner layers, up to the middle of the Pigmented Epithelium are supplied by the central retinal artery.

The inhibition of macrophage action was expressed by an insufficient production of nitric oxide in the ocular tissue, which is known to modulate the tumoricidal activity of cytotoxic T lymphocytes [3]. It has also been shown that tumor rejection in mice by the CD8 + CTL is

Update in Ocular Melanoma http://dx.doi.org/10.5772/53633 567

The study of the mechanisms that facilitate the growth of an immunogenic tumor in the anterior chamber, demonstrated an influx of CD8 + CTL infiltrating the tumor. This phe‐ nomenon was preceded by intratumoral accumulation of CD11 + myeloid cells B, which exert a powerful immunosuppressive activity on the CTL, facilitating tumor escape from the immune system. Regulatory T cells, myeloid suppressor cells and stroma cells could also reduce the delayed type hypersensitivity reaction to induce apoptosis of CD8 + and

The *racial* background of the patient is an important factor. Whites have been shown to be eight times more likely to develop choroidal melanoma than African-Americans. This trend is also observed in skin melanoma, whites are six times. More likely to develop this malig‐ nancy than African-Americans [6-7]. The individuals with light iris are at increased risk of developing uveal melanoma. This is a finding that implicates *sunlight exposure* as an impor‐ tant environmental risk factor. The protective effect of melanin may be particularly impor‐ tant in the iris as it is the only part of the uveal tract positioned in front of the lens, which

The median *age* at diagnosis is about 55-65 years and the incidence decreases after 70 years of age. With regards to incidence depending on *sex*, there is a slight predominance

In a study involving 4500 patients with uveal melanoma, only 0.6% of the cases had a family history of this disease [9]. Thus *heredity* does not seem to be a significant determinant of uveal melanoma. With respect to *occupational and chemical exposures*, the only specific occu‐ pational exposure that has been linked to uveal melanoma is welding. Ocular melanomas have been induced in laboratory animals after administration of *radium, methylcholanthrene,*

Accuracy in the early diagnosis of ocular melanoma is crucial to improve the prognosis. Currently the diagnosis of ocular melanoma is based on both the clinical experience of the

The rate of misdiagnosis for eyes enucleated for choroidal melanoma was 20 % until the

mediated by TNF-α [4].

**4. Demographic and epidemiological data**

serves as an effective ultraviolet filter.

*N-2fluorenylacetamide, ethionine and nickel subsulfide.*

specialists and on the use of modern diagnostic techniques.

1970's but it has decreased to 1% since then. [10-16]

**5. Diagnosis of ocular melanoma**

NK cells [5]

of males [8].

#### **1.2. Difference between ocular melanoma and cutaneous melanoma**

Ocular and cutaneous melanomas show several differences despite they both derive from melanocytes. Both malignancies show a high tendency to metastasize though they display different preferential sites. Skin melanomas spread to distant skin sites, lung, liver, central nervous system and bone. However uveal melanoma, the most frequently diagnosed of the ocular melanomas, gives rise to metastases almost exclusively in the liver which is affected in 90% of the cases.

Interestingly, both malignancies display similar chromosomal aberrations as well as a simi‐ lar gene expression profile [1]. The similarity in this aspect, despite the difference in tumor behaviour, serves as a proof of the role of the microenvironment in tumor development.

With respect to the early diagnosis, in the skin melanoma the suspected diagnosis and the subsequent clinical follow-up are based on the ABCDE rule.On the other hand, in the diag‐ nosis of ocular melanoma the most relevant information comes from the ophthalmoscopy and the ultrasonography. Finally, consulting times for patients and prognosis are different for both types of melanoma.

## **2. Objectives**

In this chapter we will focus in the clinical management of ocular melanoma from the diag‐ nosis to the treatment.

## **3. Immunopathology**

The eye is an immunologically privileged site; from an evolutionary point of view this con‐ dition helps to control or eliminate pathogens while generating the least inflammatory dam‐ age to the ocular tissues. However, the counterpart of this phenomenon is that it favors the escape of the tumor cells from the controls of the immune system, facilitating the growth of the uveal melanoma and its metastatic dissemination. Experiments in mice it have shown that cytotoxic cell activity in the ocular tissue might be modulated by two mechanisms:


The inhibition of macrophage action was expressed by an insufficient production of nitric oxide in the ocular tissue, which is known to modulate the tumoricidal activity of cytotoxic T lymphocytes [3]. It has also been shown that tumor rejection in mice by the CD8 + CTL is mediated by TNF-α [4].

The study of the mechanisms that facilitate the growth of an immunogenic tumor in the anterior chamber, demonstrated an influx of CD8 + CTL infiltrating the tumor. This phe‐ nomenon was preceded by intratumoral accumulation of CD11 + myeloid cells B, which exert a powerful immunosuppressive activity on the CTL, facilitating tumor escape from the immune system. Regulatory T cells, myeloid suppressor cells and stroma cells could also reduce the delayed type hypersensitivity reaction to induce apoptosis of CD8 + and NK cells [5]

## **4. Demographic and epidemiological data**

*The choroid is a* dark brown vascular sheet, 0.25mm thick, lying between the sclera and the retina. The outer vascular bed has large vessels (layer of Haller) and the inner bed consists of an extensive network of fenestrated vessels, the choriocapillaris which is the major blood supply to the outer layers of the retina and to the whole macula The inner layers, up to the

Ocular and cutaneous melanomas show several differences despite they both derive from melanocytes. Both malignancies show a high tendency to metastasize though they display different preferential sites. Skin melanomas spread to distant skin sites, lung, liver, central nervous system and bone. However uveal melanoma, the most frequently diagnosed of the ocular melanomas, gives rise to metastases almost exclusively in the liver which is affected

Interestingly, both malignancies display similar chromosomal aberrations as well as a simi‐ lar gene expression profile [1]. The similarity in this aspect, despite the difference in tumor behaviour, serves as a proof of the role of the microenvironment in tumor development.

With respect to the early diagnosis, in the skin melanoma the suspected diagnosis and the subsequent clinical follow-up are based on the ABCDE rule.On the other hand, in the diag‐ nosis of ocular melanoma the most relevant information comes from the ophthalmoscopy and the ultrasonography. Finally, consulting times for patients and prognosis are different

In this chapter we will focus in the clinical management of ocular melanoma from the diag‐

The eye is an immunologically privileged site; from an evolutionary point of view this con‐ dition helps to control or eliminate pathogens while generating the least inflammatory dam‐ age to the ocular tissues. However, the counterpart of this phenomenon is that it favors the escape of the tumor cells from the controls of the immune system, facilitating the growth of the uveal melanoma and its metastatic dissemination. Experiments in mice it have shown that cytotoxic cell activity in the ocular tissue might be modulated by two mechanisms:

**1.** By direct interference of the specific effector function of CD8 + lymphocytes.

**2.** Indirectly affected by stimulation of macrophages [2].

middle of the Pigmented Epithelium are supplied by the central retinal artery.

**1.2. Difference between ocular melanoma and cutaneous melanoma**

in 90% of the cases.

566 Melanoma - From Early Detection to Treatment

for both types of melanoma.

**2. Objectives**

nosis to the treatment.

**3. Immunopathology**

The *racial* background of the patient is an important factor. Whites have been shown to be eight times more likely to develop choroidal melanoma than African-Americans. This trend is also observed in skin melanoma, whites are six times. More likely to develop this malig‐ nancy than African-Americans [6-7]. The individuals with light iris are at increased risk of developing uveal melanoma. This is a finding that implicates *sunlight exposure* as an impor‐ tant environmental risk factor. The protective effect of melanin may be particularly impor‐ tant in the iris as it is the only part of the uveal tract positioned in front of the lens, which serves as an effective ultraviolet filter.

The median *age* at diagnosis is about 55-65 years and the incidence decreases after 70 years of age. With regards to incidence depending on *sex*, there is a slight predominance of males [8].

In a study involving 4500 patients with uveal melanoma, only 0.6% of the cases had a family history of this disease [9]. Thus *heredity* does not seem to be a significant determinant of uveal melanoma. With respect to *occupational and chemical exposures*, the only specific occu‐ pational exposure that has been linked to uveal melanoma is welding. Ocular melanomas have been induced in laboratory animals after administration of *radium, methylcholanthrene, N-2fluorenylacetamide, ethionine and nickel subsulfide.*

## **5. Diagnosis of ocular melanoma**

Accuracy in the early diagnosis of ocular melanoma is crucial to improve the prognosis. Currently the diagnosis of ocular melanoma is based on both the clinical experience of the specialists and on the use of modern diagnostic techniques.

The rate of misdiagnosis for eyes enucleated for choroidal melanoma was 20 % until the 1970's but it has decreased to 1% since then. [10-16]

## **5.1. Clinical**

The most common symptoms include *visual loss, photopsias and visual field defects*. None of these symptoms are specific of choroidal melanoma. Pain is very atypical in ocular melano‐ ma, except in those cases that present massive extraocular extension, inflammation or neo‐ vascular glaucoma.

Other studies like Optical Coherence Tomography and Indocyanine Green angiography may be useful in the diagnosis of this pathology. Magnetic resonance imaging, nuclear mag‐ netic resonance spectography, color Doppler ultrasonography, electrophysiologic testing

Update in Ocular Melanoma http://dx.doi.org/10.5772/53633 569

Currently much effort is directed toward understanding uveal melanoma genetics and ge‐ nomics [27], hoping that this knowledge will contribute to the development of effective mo‐

Inhibitors of B.Raf and MEK kinases hold promise for treatment of cutaneous melanomas harboring BRAF mutations. BRAF are rare in ocular melanomas, but somatic mutations in the G protein alfa subunits G alfa q and G alfa 11 (encoded by *Gnaq* and *Gna11*, respectively) occur, in a mutually exclusive pattern, in 80% of uveal melanomas. The impact of the B-Raf inhibitor PLX4720 and the MEK inhibitor AZD6244, the AKT inhibitor MK2206 and the PKC

A randomized phase II study compared MEK inhibition (AZD6244) to temozomide in ad‐ vanced uveal melanoma. MEK inhibition seems to be a rationale therapeutic strategy in

Enucleation is indicated when the tumor size exceeds 16 mm of base and 10 mm of height, is diffuse and with bad prognosis; however it is very important to emphasize that there is no scientific evidence of increased survival after enucleation. Also, enucleation does not pre‐

A novel minimally invasive surgical technique for resection of selected cases of small iris tu‐ mours has been described. This technique avoids the potential morbidity associated with a

Radiotherapy, *Brachytherapy* (BT: I125, 103 Pd, 131 Cs, Ru) and Proton Beam Radiotherapy

The most commonly employed form of radiotherapy has been the application of an epiescl‐ eral radioactive plaque and the most frequently employed isotopes include *60 Co (Cobalt),*

It is extremely important to highlight the conservative treatment of melanoma, proposed by *Irarrazabal A. et al* using brachytherapy. This procedure has shown positive results in pre‐ serving the eye, without increase in mortality. Moreover useful vision was retained in more

**6. Current medical management of patients with Ocular Melanoma**

and inmmunologic testing do not offer reliable results. [20-26]

inhibitors bisindolylmaleimide I (GF109203X) has been assessed [28].

large corneoescleral incision allowing for rapid visual recovery [30].

*106 Ru (Ruthenium), 192 Ir ( Iridium) and 125 I (Iodine)* [31-32]*.*

uveal melanoma, using Gnaq/11 as a potential predictor of sensitivity [29].

**a.** Cytogenetic: Personalized Targeted Therapy

**b.** Surgery: Resection/Enucleation

than half of the treated patients [33].

lecular therapies

vent metastases.

(PBRT)

*Indirect Ophthalmoscopy* through a well-dilated pupil is the most important examination in the diagnosis of choroidal melanoma. The classic image is a pigmented, dome-shaped or col‐ lar button-shaped tumor in a minority of cases and an associated exudative retinal detach‐ ment, orange tumor pigmentation (Lipofuscin) and sentinel vessels (prominent epiescleral vessels especially in those involving ciliary body). Scleral transillumination has been advo‐ cated by Reese. [17]

The lesions most commonly mistaken for choroidal melanoma are choroidal nevus (49%), periphereal exudative hemorrhagic chorioretinopathy (8%), congenital hypertrophy of the retinal pigment epithelium (6%), hemorrhagic detachment of the retina or pigment epithe‐ lium (5%), circumscribed choroidal hemangioma (8%) and age related macular degenera‐ tion (4%) [18]

#### **5.2. Complementary studies**

*Ultrasonography:* The most important ancillary test in the evaluation of a patient with intra‐ ocular mass lesions is the combination of both A-mode and B-mode ultrasonography (see Box 1). For tumors larger than 3 mm in thickness, a combination of both scans in skilled hands can diagnose choroidal melanomas with greater than 95% accuracy [19].

*A-mode:*

*2. vascular pulsations within the tumor*

*B-mode: 3 classic futures*

*1. Low to medium reflectivity within the melanoma.*

*2. Choroidal excavation.*

*3. Shadowing in the orbit.*

#### **Box 1.** Ultrasonography

*Fluorescein angiography:* Early hyperfluorescence with late leakage and multifocal punctate hyperfluorescence. This study is of major importance in order to distinguish lesions that simulate choroidal melanoma.

*<sup>1.</sup> medium to low internal echoes with smooth attenuation.*

Other studies like Optical Coherence Tomography and Indocyanine Green angiography may be useful in the diagnosis of this pathology. Magnetic resonance imaging, nuclear mag‐ netic resonance spectography, color Doppler ultrasonography, electrophysiologic testing and inmmunologic testing do not offer reliable results. [20-26]

## **6. Current medical management of patients with Ocular Melanoma**

### **a.** Cytogenetic: Personalized Targeted Therapy

**5.1. Clinical**

vascular glaucoma.

568 Melanoma - From Early Detection to Treatment

cated by Reese. [17]

tion (4%) [18]

*A-mode:*

**5.2. Complementary studies**

*2. vascular pulsations within the tumor*

*B-mode: 3 classic futures*

*2. Choroidal excavation.*

*3. Shadowing in the orbit.*

**Box 1.** Ultrasonography

simulate choroidal melanoma.

*1. medium to low internal echoes with smooth attenuation.*

*1. Low to medium reflectivity within the melanoma.*

The most common symptoms include *visual loss, photopsias and visual field defects*. None of these symptoms are specific of choroidal melanoma. Pain is very atypical in ocular melano‐ ma, except in those cases that present massive extraocular extension, inflammation or neo‐

*Indirect Ophthalmoscopy* through a well-dilated pupil is the most important examination in the diagnosis of choroidal melanoma. The classic image is a pigmented, dome-shaped or col‐ lar button-shaped tumor in a minority of cases and an associated exudative retinal detach‐ ment, orange tumor pigmentation (Lipofuscin) and sentinel vessels (prominent epiescleral vessels especially in those involving ciliary body). Scleral transillumination has been advo‐

The lesions most commonly mistaken for choroidal melanoma are choroidal nevus (49%), periphereal exudative hemorrhagic chorioretinopathy (8%), congenital hypertrophy of the retinal pigment epithelium (6%), hemorrhagic detachment of the retina or pigment epithe‐ lium (5%), circumscribed choroidal hemangioma (8%) and age related macular degenera‐

*Ultrasonography:* The most important ancillary test in the evaluation of a patient with intra‐ ocular mass lesions is the combination of both A-mode and B-mode ultrasonography (see Box 1). For tumors larger than 3 mm in thickness, a combination of both scans in skilled

*Fluorescein angiography:* Early hyperfluorescence with late leakage and multifocal punctate hyperfluorescence. This study is of major importance in order to distinguish lesions that

hands can diagnose choroidal melanomas with greater than 95% accuracy [19].

Currently much effort is directed toward understanding uveal melanoma genetics and ge‐ nomics [27], hoping that this knowledge will contribute to the development of effective mo‐ lecular therapies

Inhibitors of B.Raf and MEK kinases hold promise for treatment of cutaneous melanomas harboring BRAF mutations. BRAF are rare in ocular melanomas, but somatic mutations in the G protein alfa subunits G alfa q and G alfa 11 (encoded by *Gnaq* and *Gna11*, respectively) occur, in a mutually exclusive pattern, in 80% of uveal melanomas. The impact of the B-Raf inhibitor PLX4720 and the MEK inhibitor AZD6244, the AKT inhibitor MK2206 and the PKC inhibitors bisindolylmaleimide I (GF109203X) has been assessed [28].

A randomized phase II study compared MEK inhibition (AZD6244) to temozomide in ad‐ vanced uveal melanoma. MEK inhibition seems to be a rationale therapeutic strategy in uveal melanoma, using Gnaq/11 as a potential predictor of sensitivity [29].

**b.** Surgery: Resection/Enucleation

Enucleation is indicated when the tumor size exceeds 16 mm of base and 10 mm of height, is diffuse and with bad prognosis; however it is very important to emphasize that there is no scientific evidence of increased survival after enucleation. Also, enucleation does not pre‐ vent metastases.

A novel minimally invasive surgical technique for resection of selected cases of small iris tu‐ mours has been described. This technique avoids the potential morbidity associated with a large corneoescleral incision allowing for rapid visual recovery [30].

Radiotherapy, *Brachytherapy* (BT: I125, 103 Pd, 131 Cs, Ru) and Proton Beam Radiotherapy (PBRT)

The most commonly employed form of radiotherapy has been the application of an epiescl‐ eral radioactive plaque and the most frequently employed isotopes include *60 Co (Cobalt), 106 Ru (Ruthenium), 192 Ir ( Iridium) and 125 I (Iodine)* [31-32]*.*

It is extremely important to highlight the conservative treatment of melanoma, proposed by *Irarrazabal A. et al* using brachytherapy. This procedure has shown positive results in pre‐ serving the eye, without increase in mortality. Moreover useful vision was retained in more than half of the treated patients [33].

In a study comparing patients treated with *Ruthenium* brachytherapy with patients undergo‐ ing simultaneous thermotherapy or BT alone, combined treatment provided higher local control, eye globes preservation, better recurrence-free survival rates, lower rates of metasta‐ ses and prolonged survival than treatment with BT alone.

body, *ipilimumab*, improved overall survival of patients with advanced cutaneous melanoma in a phase 3 trial. However, uveal melanoma patients were excluded from this study. A sub‐ analysis, performed by the ipilimumab-ocular melanoma expanded access program (I-OMEAP) study group, aimed at assessing the activity and safety of ipilimumab in patients with uveal melanoma in a setting similar to daily clinical practice. The results indicated that uveal melanoma is a potential target for ipilimumab treatment and that it should be further

Update in Ocular Melanoma http://dx.doi.org/10.5772/53633 571

The *R24 monoclonal antibody,* that recognizes the disialoganglioside GD3 expressed on the surface of malignant melanoma cells, could mediate destruction of these cells. A combina‐ tion of R24 with a low dose of IL-2 was found to promote destruction of cultured melanoma

Choroidal melanomas should be diagnosed and treated at the very early stage as the initial spread of metastases is thought to occur during the proliferative stage of tumor develop‐

TTT is recomended for the management of posterior choroidal nevi suspected for malignant transformation or small choroidal melanomas that are less than 2-5 mm in thickness [41]. TTT might be the treatment of choice for selected, very small melanomas. However, studies with long follow-up and large number of patients are needed to evaluate its effectiveness.

Choroidal melanomas treated with TTT as stand-alone procedure need a close monitoring since these tumors developed a significant rate of local recurrences and ocular side-effects in

Anti-angiogenic therapy is based on the assumption that a tumor cannot grow beyond the limits of diffusion (about 1-2 mm) of oxygen and nutrients from capillaries, unless angiogen‐ esis takes place. VEGF plays a key role in angiogenesis, regulating vasopermeability and the proliferation and migration of endothelial cells. VEGF levels are significantly elevated in uveal melanoma patients with metastatic disease compared to patients without metastases. Anti-angiogenic therapy, such as bevacizumab, is currently used for the treatment of meta‐

Bevacizumab may be used as an adjuvant agent when used following plaque brachyther‐ apy in the treatment of choroidal melanoma. The combination of this treatments was as‐ sessed in an interventional case series of 100 patients treated from 2006-2008 for choroidal melanoma and the results were satisfactory. Melanoma specific mortality was 0% at 9 months after treatment. Mean visual acuity for combined treatment at 6 months

The *bevacizumab - radiotherapy combination* could be a promising clinical approach for the management of human uveal melanoma, since it may allow the use of lower doses of radio‐

cells and it can be safely administered to patients with metastatic melanoma [40].

investigated in clinical trials [39].

ment.

the long run.

was 20/30 [42]

**d.** Transpupillary Thermotherapy (TTT):

**e.** Antiangiogenic drugs (Bevacizumab)

stases of several malignancies. [43].

therapy without compromising the antitumor effect [44].

*I125* episcleral brachytherapy in uveal melanoma is effective in tumor control, allowing preservation of the eye and useful visual function for the majority of patients [34].

It has been suggested that length of remaining life after diagnosis of uveal melanoma is sim‐ ilar following enucleation (removal of the eye) to local eye-conserving radiotherapy. The multidisciplinary COMS Group emphasized that there were no differences in survival out‐ comes and a small difference in quality-of-life outcomes between patients in the brachyther‐ apy arm and those in the enucleation arm [35].

Radiation treatment was found to reduce the tumor in 94% of the cases. Mean tumor thickness decreased from 3.7 to 2.5 and 2.1 after 3 and 5 years respectively. Recurrence oc‐ cured in 6% of the treated patients. Although this therapy is associated with complica‐ tions like radiation optic neuropathy in 81% and vitreous bleeding in 30% of cases, it is a promising treatment given that enucleation was necessary in only 3% of patients and metastasis developed in 15% during follow up. Even though the visual acuity decreases considerably after optic disc irradiation with proton beam therapy, the rates of tumor con‐ trol and eye retention are favourable.

The second most frequent method of radiotherapy is the use of heavy ions such as *Proton Beam Radiotherapy* [36]. In a comparison of the efficacy of PBRT and Ruthenium-106 notched plaque radiotherapy with or without TTT for the treatment of juxtapapillary choroidal mela‐ noma, it was found that the tumors were successfully treated using either proton beam or notched plaque combined with adjuvant TTT [37].However, vision is often sacrificed. On the other hand, Notched plaque alone is not as efficient in reducing the tumor but results in improved visual outcome [37].

Proton beam irradiation of uveal melanoma has great advantages over brachytherapy be‐ cause of the homogenous dose delivered to the tumor and the possibility of sparing normal tissue close to the tumor. Complications such as retinal detachment, maculopathy, papillop‐ athy, cataract, glaucoma, vitreous hemorrhage and dryness are described. The severest com‐ plication that usually leads to secondary enucleation is neovascular glaucoma and it is encountered after irradiation of large to extra-large tumors. It is hypothesized that the resid‐ ual tumor scar may produce proinflammatory cytokines and Vascular endothelial growth factor- VEGF (toxic tumor syndrome) leading to intraocular inflammation and neovascular glaucoma. Additional treatments after proton beam such as transpupillary thermotherapy, endoresection of the tumor scar or intravitreal injections of anti-VEGF may reduce the rate of these complications [38].

**c.** Monoclonal Antibodies

Current systemic treatments for metastatic uveal melanoma have not improved overall sur‐ vival. The fully human anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) monoclonal anti‐ body, *ipilimumab*, improved overall survival of patients with advanced cutaneous melanoma in a phase 3 trial. However, uveal melanoma patients were excluded from this study. A sub‐ analysis, performed by the ipilimumab-ocular melanoma expanded access program (I-OMEAP) study group, aimed at assessing the activity and safety of ipilimumab in patients with uveal melanoma in a setting similar to daily clinical practice. The results indicated that uveal melanoma is a potential target for ipilimumab treatment and that it should be further investigated in clinical trials [39].

The *R24 monoclonal antibody,* that recognizes the disialoganglioside GD3 expressed on the surface of malignant melanoma cells, could mediate destruction of these cells. A combina‐ tion of R24 with a low dose of IL-2 was found to promote destruction of cultured melanoma cells and it can be safely administered to patients with metastatic melanoma [40].

**d.** Transpupillary Thermotherapy (TTT):

In a study comparing patients treated with *Ruthenium* brachytherapy with patients undergo‐ ing simultaneous thermotherapy or BT alone, combined treatment provided higher local control, eye globes preservation, better recurrence-free survival rates, lower rates of metasta‐

*I125* episcleral brachytherapy in uveal melanoma is effective in tumor control, allowing

It has been suggested that length of remaining life after diagnosis of uveal melanoma is sim‐ ilar following enucleation (removal of the eye) to local eye-conserving radiotherapy. The multidisciplinary COMS Group emphasized that there were no differences in survival out‐ comes and a small difference in quality-of-life outcomes between patients in the brachyther‐

Radiation treatment was found to reduce the tumor in 94% of the cases. Mean tumor thickness decreased from 3.7 to 2.5 and 2.1 after 3 and 5 years respectively. Recurrence oc‐ cured in 6% of the treated patients. Although this therapy is associated with complica‐ tions like radiation optic neuropathy in 81% and vitreous bleeding in 30% of cases, it is a promising treatment given that enucleation was necessary in only 3% of patients and metastasis developed in 15% during follow up. Even though the visual acuity decreases considerably after optic disc irradiation with proton beam therapy, the rates of tumor con‐

The second most frequent method of radiotherapy is the use of heavy ions such as *Proton Beam Radiotherapy* [36]. In a comparison of the efficacy of PBRT and Ruthenium-106 notched plaque radiotherapy with or without TTT for the treatment of juxtapapillary choroidal mela‐ noma, it was found that the tumors were successfully treated using either proton beam or notched plaque combined with adjuvant TTT [37].However, vision is often sacrificed. On the other hand, Notched plaque alone is not as efficient in reducing the tumor but results in

Proton beam irradiation of uveal melanoma has great advantages over brachytherapy be‐ cause of the homogenous dose delivered to the tumor and the possibility of sparing normal tissue close to the tumor. Complications such as retinal detachment, maculopathy, papillop‐ athy, cataract, glaucoma, vitreous hemorrhage and dryness are described. The severest com‐ plication that usually leads to secondary enucleation is neovascular glaucoma and it is encountered after irradiation of large to extra-large tumors. It is hypothesized that the resid‐ ual tumor scar may produce proinflammatory cytokines and Vascular endothelial growth factor- VEGF (toxic tumor syndrome) leading to intraocular inflammation and neovascular glaucoma. Additional treatments after proton beam such as transpupillary thermotherapy, endoresection of the tumor scar or intravitreal injections of anti-VEGF may reduce the rate

Current systemic treatments for metastatic uveal melanoma have not improved overall sur‐ vival. The fully human anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) monoclonal anti‐

preservation of the eye and useful visual function for the majority of patients [34].

ses and prolonged survival than treatment with BT alone.

apy arm and those in the enucleation arm [35].

570 Melanoma - From Early Detection to Treatment

trol and eye retention are favourable.

improved visual outcome [37].

of these complications [38]. **c.** Monoclonal Antibodies Choroidal melanomas should be diagnosed and treated at the very early stage as the initial spread of metastases is thought to occur during the proliferative stage of tumor develop‐ ment.

TTT is recomended for the management of posterior choroidal nevi suspected for malignant transformation or small choroidal melanomas that are less than 2-5 mm in thickness [41]. TTT might be the treatment of choice for selected, very small melanomas. However, studies with long follow-up and large number of patients are needed to evaluate its effectiveness.

Choroidal melanomas treated with TTT as stand-alone procedure need a close monitoring since these tumors developed a significant rate of local recurrences and ocular side-effects in the long run.

**e.** Antiangiogenic drugs (Bevacizumab)

Anti-angiogenic therapy is based on the assumption that a tumor cannot grow beyond the limits of diffusion (about 1-2 mm) of oxygen and nutrients from capillaries, unless angiogen‐ esis takes place. VEGF plays a key role in angiogenesis, regulating vasopermeability and the proliferation and migration of endothelial cells. VEGF levels are significantly elevated in uveal melanoma patients with metastatic disease compared to patients without metastases. Anti-angiogenic therapy, such as bevacizumab, is currently used for the treatment of meta‐ stases of several malignancies. [43].

Bevacizumab may be used as an adjuvant agent when used following plaque brachyther‐ apy in the treatment of choroidal melanoma. The combination of this treatments was as‐ sessed in an interventional case series of 100 patients treated from 2006-2008 for choroidal melanoma and the results were satisfactory. Melanoma specific mortality was 0% at 9 months after treatment. Mean visual acuity for combined treatment at 6 months was 20/30 [42]

The *bevacizumab - radiotherapy combination* could be a promising clinical approach for the management of human uveal melanoma, since it may allow the use of lower doses of radio‐ therapy without compromising the antitumor effect [44].

## **f.** Chemotherapy

There is no current evidence that chemotherapy has a significant role in the primary man‐ agement of uveal melanoma. Such treatment may prolong survival for a few months but it is unlikely that it will be curative.

This classification might be helpful for the prognosis in three aspects: in the screening tar‐ geted to metastasis, in the earlier diagnosis of the metastasis and for an earlier preventing treatment of the metastasis in high risk cases. In this regard, it is important to highlight that there is no scientific evidence about increased survival due to metastasis treatment [47].

Update in Ocular Melanoma http://dx.doi.org/10.5772/53633 573

**8. Uveal melanoma TNM staging and survival: Implications in patient**

*Damato, B; Eleuteri, A* [48] support the idea that Kaplan-Meier survival curves based only in tumour size and extend do not provide a true indication of prognosis. This is because the survival prognosis in uveal melanoma correlates not only with clinical stage but also with histologic grade, genetic type and competing causes of death. They propose an online pre‐

**management and prognosis**

dictor tool using the following data:

**8.1. Parameters**

Large ultrasound diameter Cilliary body involvement

Extraocular extension Years since treatment

Closed PAS+ ve loops

Regional Lymph nodes

Threshold for next scan (number)

Distant metastasis First scan (years)

**8.2. TNM Stage**

C.

Survival Controls

Epithelloid Cells

Mitotic rate/40 Monosomy 3

Age Sex

Uveal melanoma metastases develop in 6.5-35% of patients, most commonly to the liver. Metastatic uveal melanoma survival is poor, with 5-7 months of median survival. A ret‐ rospective study including 58 patients with uveal melanoma metastases showed that the median overall survival (OS) for all the patients was 10.83 months. Patients who had un‐ dergone chemotherapy presented 10.83 months of median OS whereas the patients who did not undergo this treatment had an OS of 8.033 months. Patients with metastatic uveal melanoma should be included in clinical trials evaluating other options with new‐ er agents [45].

## **7. Medical prognosis: Mortality (Hepatic metastasis), loss of the eye, loss of vision**

These three variables will affect directly the patient survival:


**Prognosis: GEP (Gene Expression Profile)** In a prospective evaluation involving 514 uveal melanoma patients [46], the gene expression profile prognostic assay helped in classifying the primary tumor into two prognostic subgroups:

Class I (60% of the cases)

Low metastatic risk :

IA (87%) almost without metastasis (0.8% of the patients).

IB (13%) few metastasis (10.8%) + disomy cr3 few metastasis.

Class II (40% of the cases)

*High metastatic risk:* metastasis (29.8%) + monosomy or pseudodisomy cr3 metastasis is not sure, + Trisomy cr6: 80 % of patients will show metastasis 4 years after diagnosis.

**g.** Others (Adjuvant therapy with interferon, Imatinilo Mesylate, Paclitaxeldocosahexae‐ noico Acid, Factionated Radiosurgery Cyberknife, aflibercept, vaccine).

This classification might be helpful for the prognosis in three aspects: in the screening tar‐ geted to metastasis, in the earlier diagnosis of the metastasis and for an earlier preventing treatment of the metastasis in high risk cases. In this regard, it is important to highlight that there is no scientific evidence about increased survival due to metastasis treatment [47].

## **8. Uveal melanoma TNM staging and survival: Implications in patient management and prognosis**

*Damato, B; Eleuteri, A* [48] support the idea that Kaplan-Meier survival curves based only in tumour size and extend do not provide a true indication of prognosis. This is because the survival prognosis in uveal melanoma correlates not only with clinical stage but also with histologic grade, genetic type and competing causes of death. They propose an online pre‐ dictor tool using the following data:

#### **8.1. Parameters**

Age

**f.** Chemotherapy

er agents [45].

**of vision**

unlikely that it will be curative.

572 Melanoma - From Early Detection to Treatment

There is no current evidence that chemotherapy has a significant role in the primary man‐ agement of uveal melanoma. Such treatment may prolong survival for a few months but it is

Uveal melanoma metastases develop in 6.5-35% of patients, most commonly to the liver. Metastatic uveal melanoma survival is poor, with 5-7 months of median survival. A ret‐ rospective study including 58 patients with uveal melanoma metastases showed that the median overall survival (OS) for all the patients was 10.83 months. Patients who had un‐ dergone chemotherapy presented 10.83 months of median OS whereas the patients who did not undergo this treatment had an OS of 8.033 months. Patients with metastatic uveal melanoma should be included in clinical trials evaluating other options with new‐

**g.** Others (Adjuvant therapy with interferon, Imatinilo Mesylate, Paclitaxeldocosahexae‐

**7. Medical prognosis: Mortality (Hepatic metastasis), loss of the eye, loss**

**Variable Importance for prognosis**

**Prognosis: GEP (Gene Expression Profile)** In a prospective evaluation involving 514 uveal melanoma patients [46], the gene expression profile prognostic assay helped in classifying

*High metastatic risk:* metastasis (29.8%) + monosomy or pseudodisomy cr3 metastasis is not

sure, + Trisomy cr6: 80 % of patients will show metastasis 4 years after diagnosis.

noico Acid, Factionated Radiosurgery Cyberknife, aflibercept, vaccine).

These three variables will affect directly the patient survival:

*A Size (Base more than 16 mm and altura more than 10 mm).* + *B Cell Type (Epithelloid Cells)* ++ *C Genetic Type (GEP: Gene Expression Profile)* +++

the primary tumor into two prognostic subgroups:

IA (87%) almost without metastasis (0.8% of the patients).

IB (13%) few metastasis (10.8%) + disomy cr3 few metastasis.

Class I (60% of the cases)

Class II (40% of the cases)

Low metastatic risk :

Sex

Large ultrasound diameter

Cilliary body involvement

Extraocular extension

Years since treatment

Epithelloid Cells

Closed PAS+ ve loops

Mitotic rate/40

Monosomy 3

Regional Lymph nodes

Distant metastasis

First scan (years)

Threshold for next scan (number)

#### **8.2. TNM Stage**

C. Survival Controls Subjects

Difference

Relative

## **9. Conclusion**

The uveal melanoma, which arises from melanocytes residing in the stroma, is the most common primary intraocular tumour in adults. More than 90% involve the choroid, the re‐ mainder being confined to the ciliary body and iris.

ase (MEK) inhibitors, ipilimumab and AEB071 are candidate drugs, and studies are under‐

Currently, the aim is to improve the detection of uveal melanoma so as to maximize the op‐ portunities for conserving the eye and vision, as well as preventing metastatic spread. Pa‐ tient management has been enhanced by the formation of multidisciplinary teams in

way to determine the therapeutic effects of these drugs in uveal melanoma [51].

This publication was supported by grants from Raymos S.A.C.I. laboratory.

, Lucia Speroni2

[1] Van den Bosch T, Kilic E, Paridaens D, de Klein A. Genetics of uveal melanoma and cutaneous melanoma: two of a kind? Dermatol Res Pract. 2010;2010:360136.

[2] Vicetti Miguel RD, Cherpes TL, Watson LJ, Mc Kenna KC: CTL induction of tumori‐ cidal nitric oxide production by intratumoral macrophages is critical for tumor elimi‐

[3] Dace DS, Chen PW, Niederkorn JY: CD8+ Tcells circumvent privilege in the eye and mediate intraocular tumor rejection by aTNF-alfa dependent mechanism. J Immunol,

[4] McKenna KC, Kapp JA: Accumulation of immunosuppressive CD11B+ myeloid cells correlates with the failure to prevent tumor grouth in the anterior chamber of the

[5] Poggi A, Zocchi MR: Mechanisms of tumor escape: role of tumor microenviroment inducing apoptosis of cytolytic effector cells. Arch. Immunol The Exp (Warsz) 2006,

2 Department of Anatomy and Cellular Biology, Tufts University, Boston, USA

3 Consultores Oftalmológicos Institute, Montevideo, Buenos Aires, Argentina

and Arturo Irarrázabal3

Update in Ocular Melanoma http://dx.doi.org/10.5772/53633 575

specialized ocular oncology centers all over the world.

**Acknowledgements**

**Author details**

**References**

Victoria de los Ángeles Bustuoabad1

1 German Hospital, Buenos Aires, Argentina

nation. J Immunol 2010, 185:6706-18.

eye. J Immunol, 2006, 177:1599-608.

2007, 178:6115-22

54:323-33.

The most common symptoms in uveal melanoma include visual loss, photopsias and visual field defects but none of these symptoms are specific of this malignancy. Diagnosis is based on slit-lamp biomicroscopy and/or ophthalmoscopy, with ultrasonography, autofluores‐ cence photography. Although each day we count with more variety and helpful comple‐ mentary studies, suspicious lesions should be closely monitored. Uveal melanomas are diverse in their clinical features and behaviour. Despite ocular treatment almost 50% of pa‐ tients with primary uveal melanoma will develop distance metastasis [50]. The metastatic disease occurs almost exclusively in patients whose tumour show chromosome 3 loss and/or class 2 gene expression profile. When the tumour shows such lethal genetic changes, the sur‐ vival time depends on the anatomical stage and the histological grade of the malignancy.

*Prognostication* has improved as a result of progress in multivariate analysis including all the major risk factors.

Screening for metastases is more sensitive as a consequence of the advances in liver scan‐ ning with magnetic resonance imaging and other methods. More patients with metastases are living longer, benefiting from therapies such as: partial hepatectomy; radiofrequency ablation; ipilumumab immunotherapy; selective internal radiotherapy; intra-hepatic chemo‐ therapy, possibly with isolated liver perfusion; and systemic chemotherapy [48].

*Conservation of the eye* with useful vision has improved thanks to the advances in brachyther‐ apy, proton beam radiotherapy, transpupillary thermotherapy. The current trend is to try to preserve the affected eye by all means, as there is no scientific evidence that shows that re‐ moving the affected eye will improve survival.. This is a great difference in the treatment of ocular vs cutaneous melanoma. The specialists must take into consideration the need to pro‐ tect the eye with melanoma and preserve as much vision as possible as the other eye may be affected by another pathology in the future with the consequent loss of vision.

On the basis of the currently available information it appears that patients treated with radi‐ otherapy have a survival rate at least as good, if not better than those treated with enuclea‐ tion [36].

Several drugs, such as bortezomib, celecoxib, dacarbazine, anti-angiogenic agents (such as bevacizumab, sorafenib and sunitinib), temsirolimus, mitogen-activated protein kinase kin‐ ase (MEK) inhibitors, ipilimumab and AEB071 are candidate drugs, and studies are under‐ way to determine the therapeutic effects of these drugs in uveal melanoma [51].

Currently, the aim is to improve the detection of uveal melanoma so as to maximize the op‐ portunities for conserving the eye and vision, as well as preventing metastatic spread. Pa‐ tient management has been enhanced by the formation of multidisciplinary teams in specialized ocular oncology centers all over the world.

## **Acknowledgements**

Subjects

Relative

Difference

**9. Conclusion**

574 Melanoma - From Early Detection to Treatment

major risk factors.

tion [36].

mainder being confined to the ciliary body and iris.

The uveal melanoma, which arises from melanocytes residing in the stroma, is the most common primary intraocular tumour in adults. More than 90% involve the choroid, the re‐

The most common symptoms in uveal melanoma include visual loss, photopsias and visual field defects but none of these symptoms are specific of this malignancy. Diagnosis is based on slit-lamp biomicroscopy and/or ophthalmoscopy, with ultrasonography, autofluores‐ cence photography. Although each day we count with more variety and helpful comple‐ mentary studies, suspicious lesions should be closely monitored. Uveal melanomas are diverse in their clinical features and behaviour. Despite ocular treatment almost 50% of pa‐ tients with primary uveal melanoma will develop distance metastasis [50]. The metastatic disease occurs almost exclusively in patients whose tumour show chromosome 3 loss and/or class 2 gene expression profile. When the tumour shows such lethal genetic changes, the sur‐ vival time depends on the anatomical stage and the histological grade of the malignancy.

*Prognostication* has improved as a result of progress in multivariate analysis including all the

Screening for metastases is more sensitive as a consequence of the advances in liver scan‐ ning with magnetic resonance imaging and other methods. More patients with metastases are living longer, benefiting from therapies such as: partial hepatectomy; radiofrequency ablation; ipilumumab immunotherapy; selective internal radiotherapy; intra-hepatic chemo‐

*Conservation of the eye* with useful vision has improved thanks to the advances in brachyther‐ apy, proton beam radiotherapy, transpupillary thermotherapy. The current trend is to try to preserve the affected eye by all means, as there is no scientific evidence that shows that re‐ moving the affected eye will improve survival.. This is a great difference in the treatment of ocular vs cutaneous melanoma. The specialists must take into consideration the need to pro‐ tect the eye with melanoma and preserve as much vision as possible as the other eye may be

On the basis of the currently available information it appears that patients treated with radi‐ otherapy have a survival rate at least as good, if not better than those treated with enuclea‐

Several drugs, such as bortezomib, celecoxib, dacarbazine, anti-angiogenic agents (such as bevacizumab, sorafenib and sunitinib), temsirolimus, mitogen-activated protein kinase kin‐

therapy, possibly with isolated liver perfusion; and systemic chemotherapy [48].

affected by another pathology in the future with the consequent loss of vision.

This publication was supported by grants from Raymos S.A.C.I. laboratory.

## **Author details**

Victoria de los Ángeles Bustuoabad1 , Lucia Speroni2 and Arturo Irarrázabal3


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[8] Gragoudas ES: First 1000 patients with uveal melanoma treated by proton beam irra‐ diation. Presented at the second International Meeting in the Diagnosis and Treat‐

[9] Singh AD, Shields CL, De Potter P, Shields JA, Trock B, Cater J, Pastore D. Familial uveal melanoma. Clinical observations on 56 patients. Arch Ophthalmol.1996 Apr;

[10] Ferry AP: Lesions mistaken for malignant melanoma of the posterior uvea: a clinico‐ pathologic analysis of 100 cases with ophthalmoscopically vivible lesions. Arch, Oph‐

[11] Shields JA, Zimmerman LE: Lesions simulating malignant melanoma of the posterior

[12] Zimmerman LE: Bedell Lecture. Problems in the diagnosis of malignant melanoma of

[13] Chang M, Zimmerman LE. Mc Lean IW: The persisting pseudomelanoma problem.

[14] Shields JA, Augsburger JJ, Brown GC, Stephens RF: The Differential diagnosis of pos‐

[15] Shields JA, Mashayekhi A, Ra S, Shields CL: Pseudomelanomas of the posterior

[16] Char DH, Stone RD, Irvine AR, et al: Diagnostic modalities in choroidal melanoma.

[17] Reese AB: Tumors of the eye 3rd edn. Hagerstown, MD: Harper & Row; 1976:174-262. [18] Shields JA, Mashayekhi A, Ra S, Shields CL: Pseudomelanomas of the posterior

[19] Char DH, Stone RD, Irvine AR, et al: Diagnostic modalities in choroidal melanoma.

[20] Char DH: Inhibition of leukocyte migration with melanoma-associated antigens in

[21] Brownstein S, Phillips TM, Lewis MG: Specificity of tumor-associated antibodies in serum of patients with uveal melanoma. Can J Ophthalmol 1978; 13:190-193.

[22] Felberg NT, Donoso LA, Federman JL: Tumor-associated antibodies in the serum of

choroidal tumors. Invest Ophthalmol Vis Sci 1977; 16:176-179.

patients with ocular melanoma. Ophthalmology 1980; 87:529-533.

the choroid and ciliary body. Am J Phthalmol 1973; 75:917-929.

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ment of Intraocular Tumors in Nyon, Switzerland, November, 1987.

black patiens. Arch Ophthalmol 1984; 102:77-79.

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114(4):392-9.

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thalmol 1964; 72:463-469.

uvea. Arch Ophthalmol 1973; 89:466-471.

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Am J Ophthalmol 1980; 89:223-230.

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Am J Ophthalmol 1980; 89:223-230.


[36] Gragoudas ES, Goiten M, Verhey L, Munzenreider J, Suit HD, Kohler A: Proton Beam irradiation, an alternative to Enucleation for intraocular melanoma. Ophthal‐ mology 87:571-581, 1980.

[47] Augsburger JJ, Correa ZM, Shaikh AH: Quality of evidence about effectiveness of treatments for metastatic uveal melanoma, Trans Am Ophthalmol Soc, 2008;

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[48] Damato B, Eleuteri A, Taktak AF, Coupland SE: Estimating prognosis for survival af‐ ter treatment of choroidal melanoma. Prog Rtin Eye Res, 2011 Sep; 30(5):285-95. Epub

[50] Spagnolo F, Caltabiano G, Queirolo P: Uveal Melanoma. Cancer Treat Rev 2012 Aug;

[51] Velho TR, Kapiteijn E, Jager MJ: New therapeutic agents in uveal melanoma. Anti‐

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106:128-35; discussion 135-7.

38(5):549-53. Epub 2012 Jan 24.

cancer Res. 2012 Jul;32(7):2591-8.

2011 May 30.


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[37] Tsimpida M, Hungerford J, Cohen V: Treatment of juxtapapillary choroidal melano‐ ma. XVth Biannual Meeting ISOO International Society of Ocular Oncology, 2011,

[38] Damato B: Progress in the management of patients with uveal melanoma. Eye

[39] Danielli R, Ridolfi R, Chiarion-Sileni V, Queirolo P, Testori A, Plummer R, Boitano M, Calabró L, Rossi CD, Giacomo AM, Ferrucci Pf, Ridolfi L, Altomonte M, Miracco C, Balestrazzi A, Maio M: Ipilimumab in pretreated patients with metastatic uveal melanoma.: safety and clinical efficacy. Cancer Immunol Immunother, 2012 Jan; 61(!):

[40] Soiffer RJ, Chapman PB, Murray C, Williams L, Unger P, Collins H, Houghton AN, Ritz J: Administration of R24 monoclonal antibody and low-dose interleukin 2 for

[41] Mordechai R, Moroz I, Moisseiev J, Vishnevskia-Dai V: Our experience with transpu‐ pillary thermo therapy for suspected or small choroidal melanomas. XVth Biannual

[42] Houston SK, Murray T, Markoe A, Pina Y, Decataur C: Intravitreal bevacizumab as an adjuvant agent when used immediately after treatment with plaque brachythera‐ py. XVth Biannual Meeting ISOO International Society of Ocular Oncology, 2011,

[43] el Filali M, van der Velden PA, Luyten GP, Jager MJ: Anti-angiogenic therapy in

[44] Sudaka A, Susini A, Lo Nigro C, Fischel JL, Toussan N, Formento P, Tonissi F, Lat‐ tanzio L, Russi E, Etienne-Grimaldi MC, Merlano M, Milano G: Combination of beva‐ cizumab and irradiation on uveal melanoma: an in vitro and in vivo preclinical

[45] Pons F, Plana M, Caminal JM, Pera J, Fernandes I, Perez J, Garcia-Del-Muro X, Mar‐ coval J, Penin R, Fabra A, Piulats JM: Metastatic uveal melanoma: is there a role for conventional chemotherapy? - A single center study based on 58 patients. Melanoma

[46] Harbour JW, Onken M, Worley L, Augsburger J, Correa Z, Devron HC, Nudleman E, Aaberg TJr, Altaweel MM, Bardenstein D, Finger P, Gallie B, Harocopos GJ, Hovland PG, McGowan H, Milman T, Mruthyunjaya P, Simpson ER, Smith M, Wilson D, Wir‐ ostko WJ: Prospective evaluation of a gene expression profile prognostic assay for uveal melanoma in 514 patients. XVth Biannual Meeting ISOO International Society

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578 Melanoma - From Early Detection to Treatment

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of Ocular Oncology, 2011, Nov 14-17.


**Section 3**

**Melanoma Related Features**

## **Melanoma Related Features**

**Chapter 22**

**The Menace of Melanoma: A Photodynamic**

Metastatic malignant melanoma (MMM) remains one of the most dreaded skin cancers worldwide. Numerous factors contribute to its resistance to hosts of treatment regimes and despite significant scientific advances over the last decade in the field of chemotherapeutics and melanocytic targets, there still remains the need for improved therapeutic modalities. Photodynamic therapy (PDT), a minimally invasive therapeutic modality has been shown to be effective in a number of oncologic and non-oncologic conditions. Using second-genera‐ tion stable, lipophillic photosensitizers with optimised activation wavelengths, PDT may be a promising tool for adjuvant therapy and even pre-treatment in combating melanoma. Po‐ tential targets for PDT in melanoma eradication include cell proliferation inhibition, activa‐ tion of cell death and reduction in pro-survival autophagy, a decrease in the cellular melanocytic antioxidant system and a disruption in the endogenous multi-drug resistant (MDR) cellular machinery. This chapter highlights the current knowledge with respect to these characteristics and suggests that PDT be considered as a good candidate for adjuvant treatment in post-resected malignant metastatic melanoma. Furthermore, it suggests that primary consideration must be given to organelle-specific destruction in melanoma specifi‐ cally targeting the melanosomes – the one organelle that is specific to cells of the melanocyt‐ ic lineage that houses the toxic compound, melanin. We believe that using this combined knowledge may eventually lead to an effective therapeutic tool to combat this highly intract‐

Melanoma accounts for 4% of all dermatologic cancers but remains responsible for 80% of deaths from skin cancer with the average patient diagnosed with disseminated metastases

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

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

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

**Approach to Adjunctive Cancer Therapy**

Additional information is available at the end of the chapter

L.M. Davids and B. Kleemann

http://dx.doi.org/10.5772/53676

**1. Introduction**

able disease.

**1.1. Melanoma clinical statistics**

## **The Menace of Melanoma: A Photodynamic Approach to Adjunctive Cancer Therapy**

L.M. Davids and B. Kleemann

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53676

## **1. Introduction**

Metastatic malignant melanoma (MMM) remains one of the most dreaded skin cancers worldwide. Numerous factors contribute to its resistance to hosts of treatment regimes and despite significant scientific advances over the last decade in the field of chemotherapeutics and melanocytic targets, there still remains the need for improved therapeutic modalities. Photodynamic therapy (PDT), a minimally invasive therapeutic modality has been shown to be effective in a number of oncologic and non-oncologic conditions. Using second-genera‐ tion stable, lipophillic photosensitizers with optimised activation wavelengths, PDT may be a promising tool for adjuvant therapy and even pre-treatment in combating melanoma. Po‐ tential targets for PDT in melanoma eradication include cell proliferation inhibition, activa‐ tion of cell death and reduction in pro-survival autophagy, a decrease in the cellular melanocytic antioxidant system and a disruption in the endogenous multi-drug resistant (MDR) cellular machinery. This chapter highlights the current knowledge with respect to these characteristics and suggests that PDT be considered as a good candidate for adjuvant treatment in post-resected malignant metastatic melanoma. Furthermore, it suggests that primary consideration must be given to organelle-specific destruction in melanoma specifi‐ cally targeting the melanosomes – the one organelle that is specific to cells of the melanocyt‐ ic lineage that houses the toxic compound, melanin. We believe that using this combined knowledge may eventually lead to an effective therapeutic tool to combat this highly intract‐ able disease.

## **1.1. Melanoma clinical statistics**

Melanoma accounts for 4% of all dermatologic cancers but remains responsible for 80% of deaths from skin cancer with the average patient diagnosed with disseminated metastases

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

## surviving for an average of 5 years (Cancer facts and figures, 2003, Atlanta, American Can‐ cer Society, 2003]. According to the World Health Organization (WHO) melanoma skin can‐ cer has been increasing over the past decades with a global estimation of 132 000 melanomarelated skin cancers reported to occur each year. Over the past 50 years, melanoma incidence has risen by 3–8% per year in most people of European background, with the greatest increases in elderly men [1]. In Europe, the current estimates at 15-20 per 100 000 people predominating in the 20-35 year old age group in Caucasians [2]. South Africa, next to Australia, has one of the highest incidences of malignant melanoma in the world. Reliable statistics for South Africa are lacking, however currently an estimate figure for the South Af‐ rican Cape region is 69 new cases per year per population of 100 000 Caucasians (Australia is 65 per 100 000). This means that 1 in 1429 people will develop malignant melanoma. The age-standardised incidence of melanoma was 27.2 per 100 000 for males and 22.2 per 100 000 for females from 1990-1999 but this increased to 36.9 for males and 33.5 per 100 000 for fe‐ males (2000-2003) (CANSA association of South Africa www.melanoma.co.za/ D\_doccnr\_MFS.asp) (Table 1).

Despite extensive research and clinical trials, the prognosis and survival of metastatic mela‐ noma remains dismal. Early detection of localized melanoma may be cured through surgery however there is no therapy for metastatic melanoma or melanoma with metastatic poten‐ tial. In addition, recurrence rates of resected melanoma remain high. Because melanoma is inherently resistant to traditional forms of chemotherapy and radiotherapy [3], various strat‐ egies have been developed for treatments which include immunotherapy eg. interleukin-2 (IL-2) [4], radiotherapy [5] biochemotherapy [6-9] and gene therapy [4,10]. A limited number of these therapies have progressed to human clinical trials but their outcomes remain negli‐ gible. One promising therapy is high-dose interferon (IFN) alpha-2b therapy which has just recently been approved as the only adjuvant therapy for melanoma approved by the US Food and Drug Administration [11]. The other is the use of BRAF kinase inhibitors such as vemurafenib [12]. Despite convincing evidence of improved disease-free survival associated with this therapy, the overall survival remains negligible or very small [13-15]. In addition, a number of melanoma-specific and melanoma-associated tumor antigens such as gp100, MART-1 and MAGE3 have been cloned [16] and the hope is that these potential antigens may be developed to stimulate tumor-specific T cells to eliminate melanoma cells [17]. De‐ spite these advances, there remains the need for the development of novel and effective ap‐ proaches to treat melanoma and this review explores the possibility of using photodynamic therapy (PDT) as an adjuvant therapy alone or in combination with current therapeutics to

The Menace of Melanoma: A Photodynamic Approach to Adjunctive Cancer Therapy

http://dx.doi.org/10.5772/53676

585

Melanoma represents the malignant phenotype of a skin melanocyte. Melanoma occurs most frequently after intermittent exposure to UV radiation and in people with chronic sun‐ burns. Epidemiologic data suggest that chronic or low-grade exposures to UV induce pro‐ tection against DNA damage, whereas acute, intense UV exposure leads to DNA damage and concomitant genetic alterations in the melanocyte genome [18]. It develops as a result of accumulated abnormalities in genetic pathways within the melanocyte which give way to increased cell proliferation and prevent normal pathways of apoptosis in response to DNA damage. Furthermore, this damage results in the selection for genetic mutations that allow all aspects of the malignant phenotype, including stimulation of blood vessel growth, eva‐ sion of the immune response, tumour invasion, and metastasis [19]. Although the mecha‐ nisms of differential cancer cell killing are poorly understood [20], selection of cells that are resistant to apoptotic mechanisms might contribute to the resistance of melanoma cells to the cytotoxic effects of chemotherapy, radiotherapy, and immunotherapy, especially through the expression of apoptosis inhibitors such as B-cell lymphoma derived protein 2

Melanocytes progress through a series of steps toward malignant transformation by the ac‐ quisition of various phenotypic features. The particular histological features characterising each step of progression are the visible manifestations of underlying genetic changes [22].

combat melanoma.

**1.2. Melanoma origins**

(Bcl-2) and BclxL [21].


**Table 1.** Melanoma statistics in 2 southern and northern hemisphere countries

Despite extensive research and clinical trials, the prognosis and survival of metastatic mela‐ noma remains dismal. Early detection of localized melanoma may be cured through surgery however there is no therapy for metastatic melanoma or melanoma with metastatic poten‐ tial. In addition, recurrence rates of resected melanoma remain high. Because melanoma is inherently resistant to traditional forms of chemotherapy and radiotherapy [3], various strat‐ egies have been developed for treatments which include immunotherapy eg. interleukin-2 (IL-2) [4], radiotherapy [5] biochemotherapy [6-9] and gene therapy [4,10]. A limited number of these therapies have progressed to human clinical trials but their outcomes remain negli‐ gible. One promising therapy is high-dose interferon (IFN) alpha-2b therapy which has just recently been approved as the only adjuvant therapy for melanoma approved by the US Food and Drug Administration [11]. The other is the use of BRAF kinase inhibitors such as vemurafenib [12]. Despite convincing evidence of improved disease-free survival associated with this therapy, the overall survival remains negligible or very small [13-15]. In addition, a number of melanoma-specific and melanoma-associated tumor antigens such as gp100, MART-1 and MAGE3 have been cloned [16] and the hope is that these potential antigens may be developed to stimulate tumor-specific T cells to eliminate melanoma cells [17]. De‐ spite these advances, there remains the need for the development of novel and effective ap‐ proaches to treat melanoma and this review explores the possibility of using photodynamic therapy (PDT) as an adjuvant therapy alone or in combination with current therapeutics to combat melanoma.

#### **1.2. Melanoma origins**

surviving for an average of 5 years (Cancer facts and figures, 2003, Atlanta, American Can‐ cer Society, 2003]. According to the World Health Organization (WHO) melanoma skin can‐ cer has been increasing over the past decades with a global estimation of 132 000 melanomarelated skin cancers reported to occur each year. Over the past 50 years, melanoma incidence has risen by 3–8% per year in most people of European background, with the greatest increases in elderly men [1]. In Europe, the current estimates at 15-20 per 100 000 people predominating in the 20-35 year old age group in Caucasians [2]. South Africa, next to Australia, has one of the highest incidences of malignant melanoma in the world. Reliable statistics for South Africa are lacking, however currently an estimate figure for the South Af‐ rican Cape region is 69 new cases per year per population of 100 000 Caucasians (Australia is 65 per 100 000). This means that 1 in 1429 people will develop malignant melanoma. The age-standardised incidence of melanoma was 27.2 per 100 000 for males and 22.2 per 100 000 for females from 1990-1999 but this increased to 36.9 for males and 33.5 per 100 000 for fe‐ males (2000-2003) (CANSA association of South Africa www.melanoma.co.za/

D\_doccnr\_MFS.asp) (Table 1).

584 Melanoma - From Early Detection to Treatment

**Australia (2001)**

Men Women

**South Africa (2000)**

Men Women

Men Women

Men Women

**USA (2001)**

**UK (2000)**

**Age**-**standardised incidence(105** /**yr)**

> 41.4 (world) 31.1 (world)

> 36.9 (world) 33.5 (world)

> 21.4 (world) 13.8 (world)

> 9.7 (world) 11.2 (world)

**Table 1.** Melanoma statistics in 2 southern and northern hemisphere countries

**Lifetime risk (incidence)**

> 1 in 25 1 in 35

> 1 in 29 1 in 40

> 1 in 53 1 in 78

1 in 147 1 in 117 **Incidence trend over 10 years**

22% increase 12% increase

33% increase 27% increase

31% increase 25% increase

59% increase 41% incraese **Mortality trend over 10 years**

> 2% increase 0% increase

1.5% increase 1% increase

0% increase 1% decrease

20% increase 3% increase

**Most common cancer (ranking)**

> 4th 3rd

> 4th 3rd

> 5th 7th

12th 7th

Melanoma represents the malignant phenotype of a skin melanocyte. Melanoma occurs most frequently after intermittent exposure to UV radiation and in people with chronic sun‐ burns. Epidemiologic data suggest that chronic or low-grade exposures to UV induce pro‐ tection against DNA damage, whereas acute, intense UV exposure leads to DNA damage and concomitant genetic alterations in the melanocyte genome [18]. It develops as a result of accumulated abnormalities in genetic pathways within the melanocyte which give way to increased cell proliferation and prevent normal pathways of apoptosis in response to DNA damage. Furthermore, this damage results in the selection for genetic mutations that allow all aspects of the malignant phenotype, including stimulation of blood vessel growth, eva‐ sion of the immune response, tumour invasion, and metastasis [19]. Although the mecha‐ nisms of differential cancer cell killing are poorly understood [20], selection of cells that are resistant to apoptotic mechanisms might contribute to the resistance of melanoma cells to the cytotoxic effects of chemotherapy, radiotherapy, and immunotherapy, especially through the expression of apoptosis inhibitors such as B-cell lymphoma derived protein 2 (Bcl-2) and BclxL [21].

Melanocytes progress through a series of steps toward malignant transformation by the ac‐ quisition of various phenotypic features. The particular histological features characterising each step of progression are the visible manifestations of underlying genetic changes [22]. Originating from a benign nevus, melanocytes undergo aberrant growth within the lesion subsequently displaying irregular borders, a change in colour and often an associated aller‐ gic response. At this stage the lesion is considered dysplastic. At a molecular level, these changes are associated with abnormal activation of the mitogen-activated protein kinase (MAPK) signalling pathway resulting in somatic mutations in the N-RAS and BRAF genes which are associated with about 15 and 50% of melanomas, respectively [23,24]. There is complementarity between the presence of *NRAS* and *BRAF* mutations in any individual melanoma since each has the same effect of causing unrestrained cell proliferation.

sons contributing to the high mortality rates associated with cutaneous melanoma. Each of

The Menace of Melanoma: A Photodynamic Approach to Adjunctive Cancer Therapy

http://dx.doi.org/10.5772/53676

587

PDT is a minimally invasive therapeutic modality which has been shown to be effective in several types of cancer including non-melanoma skin cancer (NMSC) and other skin tumors such as lymphoma as well as non-oncological conditions such as psoriasis vulgaris, acne vulgaris and human papilloma virus-induced skin disease [36,37]. The basis of PDT is the systemic or topical application and preferential uptake of a photosensitizer (PS). The PS is then activated at a specific wavelength of light and in the presence of oxygen, produces re‐ active oxygen species (ROS). The accumulative presence of these cytotoxic photoproducts start a cascade of molecular and biochemical events resulting in cell death via apoptotic or

The main advantage of PDT over conventional cancer treatments are i) it has a very low sys‐ temic cumulative toxicity allowing repeated dosing, ii) its ability to destroy tumors selec‐ tively (this seems to be related to the lipophillic nature of photosensitizers). Due to this selectivity, damage to normal surrounding cells is minimal. Finally, iii) PDT can be applied alone or in combination as an adjuvant therapeutic modality with chemotherapy, surgery, radiotherapy and immunotherapy [40,41]. These properties have led to PDT receiving in‐ creased support from preclinical research [42,43]. PDT requires three elements to be effica‐ cious - a good PS, a coherent light source and the presence of molecular oxygen. A large amount of data with regard to these three elements over the last few years have resulted in the development of more naturally-derived, efficacious, second-generation photosensitizers.

Photosensitizers are critical to the successful eradication of malignant cells and numerous first and second-generation photosensitizers have been tested both clinically (in vivo) and in vitro over the past years (for a detailed summary of melanoma-PDT research see Table 2). The structure of many PS is based on the tetrapyrrol ring eg. protoporphyrin IX, Photofrin and chlorines related to it eg. phthalocyanines. Newer, more stable second-generation PS in‐ clude natural hydroxyquinone chromophores such as hypericins and porphycenes [44-47]. It is now accepted that a good PS for PDT is – i) chemically pure with good stability, ii) prefer‐ entially accumulated and retained by target tissue, iii) minimal toxicity in the absence of

ed high molecular extinction coefficient [40]. Due to these properties, a number of synthetic or natural compounds have thus far been studied for a variety of cancers however these have been limited to porphycenes (structural isomers of porphyrins) such as aminolevulinic acid (ALA, trade name, Levulan®) and methylaminolevulinic acid (MAL, trade name, Met‐ vix® ) for the treatment of squamous cell (SCC) and basal cell carcinomas (BCC) as well as

O2 with an associat‐

light with maximal efficacy upon activation, iv) high quantum yield of 1

these topics will be dealt with in the context of targeting them with PDT.

**1.3. Photodynamic Therapy (PDT) as a cancer treatment**

necrotic mechanisms [38,39].

*1.3.1. Photosensitizers and melanoma-PDT*

actinic keratoses [48-50] (Table 2).

In addition, mutations in both the cyclin-dependent kinase inhibitor 2A (CDKN2A) and the phosphatase and tensin homologue (PTEN) gene increases the probability of dysplastic nae‐ vi becoming malignant [25]. This genetic locus is frequently targeted for disruption in mela‐ nomas [26]. When defective, p16 is unable to inactivate CDK4 and CDK6, which phosphorylate Rb, releasing the transcription factor E2F and leading to cell cycle progres‐ sion [27].The molecule that is usually central to protection against DNA damage, p53, is rarely mutated early in melanoma, which is possibly one of several adaptations to permit survival of cells responsible for generating sun-protective pigment, melanin [28]. Interest‐ ingly, by-products of melanin biosynthesis can themselves cause oxidative stress and con‐ tribute to malignant change.

Further progression of melanoma is associated with decreased differentiation and clonal proliferation leading to the radial growth phase (RGP). Clinically, RGP presents as patches or plaques which can measure up to 2.5cm. Superficial spreading melanoma lesions are slightly raised and show striking variations of red, blue, white, brown, and black coloration. In RGP, melanoma mitoses are frequently seen in the epidermis but rarely in the dermis. Af‐ ter complete surgical excision of the tumor, RGP melanomas are usually associated with longterm metastasis-free survival [29-33]. RGP cells can progress to vertical growth phase (VGP) cells which breach the basement membrane and invade the dermis as nodules or nests of cells. Vertical growth phase (VGP) melanomas usually present as gray-black, blueblack, or even amelanotic nodules. In late or developed VGP, melanomas form expansile nodules in the dermis with cytology different from melanoma cells in the overlying epider‐ mis. Mitotic figures are variably present, and tumor aggregates may extend into the reticular dermis or even subcutaneous fat. Dermal tumoral nests are larger in VGP than in RGP. Moreover, these cells are considered to have metastatic potential. Interestingly, not all mela‐ nomas pass through each of these individual phases – RGP and VGP can both develop di‐ rectly from melanocytes or naevi and both can progress directly to metastatic malignant melanoma [34]. Moreover, the transition from RGP to VGP in cutaneous melanoma isassoci‐ ated with the loss of c-KIT expression and the gain of the melanoma cell adhesion molecule (MCAM/MUC18) [35].

Increased proliferation and survival, chemoresistance, the ability to resist apoptosis, the in‐ duction of autophagy and the presence of the pigment melanin have all been listed as rea‐ sons contributing to the high mortality rates associated with cutaneous melanoma. Each of these topics will be dealt with in the context of targeting them with PDT.

### **1.3. Photodynamic Therapy (PDT) as a cancer treatment**

Originating from a benign nevus, melanocytes undergo aberrant growth within the lesion subsequently displaying irregular borders, a change in colour and often an associated aller‐ gic response. At this stage the lesion is considered dysplastic. At a molecular level, these changes are associated with abnormal activation of the mitogen-activated protein kinase (MAPK) signalling pathway resulting in somatic mutations in the N-RAS and BRAF genes which are associated with about 15 and 50% of melanomas, respectively [23,24]. There is complementarity between the presence of *NRAS* and *BRAF* mutations in any individual

In addition, mutations in both the cyclin-dependent kinase inhibitor 2A (CDKN2A) and the phosphatase and tensin homologue (PTEN) gene increases the probability of dysplastic nae‐ vi becoming malignant [25]. This genetic locus is frequently targeted for disruption in mela‐ nomas [26]. When defective, p16 is unable to inactivate CDK4 and CDK6, which phosphorylate Rb, releasing the transcription factor E2F and leading to cell cycle progres‐ sion [27].The molecule that is usually central to protection against DNA damage, p53, is rarely mutated early in melanoma, which is possibly one of several adaptations to permit survival of cells responsible for generating sun-protective pigment, melanin [28]. Interest‐ ingly, by-products of melanin biosynthesis can themselves cause oxidative stress and con‐

Further progression of melanoma is associated with decreased differentiation and clonal proliferation leading to the radial growth phase (RGP). Clinically, RGP presents as patches or plaques which can measure up to 2.5cm. Superficial spreading melanoma lesions are slightly raised and show striking variations of red, blue, white, brown, and black coloration. In RGP, melanoma mitoses are frequently seen in the epidermis but rarely in the dermis. Af‐ ter complete surgical excision of the tumor, RGP melanomas are usually associated with longterm metastasis-free survival [29-33]. RGP cells can progress to vertical growth phase (VGP) cells which breach the basement membrane and invade the dermis as nodules or nests of cells. Vertical growth phase (VGP) melanomas usually present as gray-black, blueblack, or even amelanotic nodules. In late or developed VGP, melanomas form expansile nodules in the dermis with cytology different from melanoma cells in the overlying epider‐ mis. Mitotic figures are variably present, and tumor aggregates may extend into the reticular dermis or even subcutaneous fat. Dermal tumoral nests are larger in VGP than in RGP. Moreover, these cells are considered to have metastatic potential. Interestingly, not all mela‐ nomas pass through each of these individual phases – RGP and VGP can both develop di‐ rectly from melanocytes or naevi and both can progress directly to metastatic malignant melanoma [34]. Moreover, the transition from RGP to VGP in cutaneous melanoma isassoci‐ ated with the loss of c-KIT expression and the gain of the melanoma cell adhesion molecule

Increased proliferation and survival, chemoresistance, the ability to resist apoptosis, the in‐ duction of autophagy and the presence of the pigment melanin have all been listed as rea‐

melanoma since each has the same effect of causing unrestrained cell proliferation.

tribute to malignant change.

586 Melanoma - From Early Detection to Treatment

(MCAM/MUC18) [35].

PDT is a minimally invasive therapeutic modality which has been shown to be effective in several types of cancer including non-melanoma skin cancer (NMSC) and other skin tumors such as lymphoma as well as non-oncological conditions such as psoriasis vulgaris, acne vulgaris and human papilloma virus-induced skin disease [36,37]. The basis of PDT is the systemic or topical application and preferential uptake of a photosensitizer (PS). The PS is then activated at a specific wavelength of light and in the presence of oxygen, produces re‐ active oxygen species (ROS). The accumulative presence of these cytotoxic photoproducts start a cascade of molecular and biochemical events resulting in cell death via apoptotic or necrotic mechanisms [38,39].

The main advantage of PDT over conventional cancer treatments are i) it has a very low sys‐ temic cumulative toxicity allowing repeated dosing, ii) its ability to destroy tumors selec‐ tively (this seems to be related to the lipophillic nature of photosensitizers). Due to this selectivity, damage to normal surrounding cells is minimal. Finally, iii) PDT can be applied alone or in combination as an adjuvant therapeutic modality with chemotherapy, surgery, radiotherapy and immunotherapy [40,41]. These properties have led to PDT receiving in‐ creased support from preclinical research [42,43]. PDT requires three elements to be effica‐ cious - a good PS, a coherent light source and the presence of molecular oxygen. A large amount of data with regard to these three elements over the last few years have resulted in the development of more naturally-derived, efficacious, second-generation photosensitizers.

## *1.3.1. Photosensitizers and melanoma-PDT*

Photosensitizers are critical to the successful eradication of malignant cells and numerous first and second-generation photosensitizers have been tested both clinically (in vivo) and in vitro over the past years (for a detailed summary of melanoma-PDT research see Table 2). The structure of many PS is based on the tetrapyrrol ring eg. protoporphyrin IX, Photofrin and chlorines related to it eg. phthalocyanines. Newer, more stable second-generation PS in‐ clude natural hydroxyquinone chromophores such as hypericins and porphycenes [44-47]. It is now accepted that a good PS for PDT is – i) chemically pure with good stability, ii) prefer‐ entially accumulated and retained by target tissue, iii) minimal toxicity in the absence of light with maximal efficacy upon activation, iv) high quantum yield of 1 O2 with an associat‐ ed high molecular extinction coefficient [40]. Due to these properties, a number of synthetic or natural compounds have thus far been studied for a variety of cancers however these have been limited to porphycenes (structural isomers of porphyrins) such as aminolevulinic acid (ALA, trade name, Levulan®) and methylaminolevulinic acid (MAL, trade name, Met‐ vix® ) for the treatment of squamous cell (SCC) and basal cell carcinomas (BCC) as well as actinic keratoses [48-50] (Table 2).


*in vitro/ in vivo*

*in vitro/ in*

*in vitro/ in vivo*

*in vitro*

*vivo* melanoma bearing mice

*in vitro* SK-MEL-188 (human melanoma) cells

*in vitro* C57 mice bearing a sub-cutaneously

B78H1 amelanotic mouse melanoma cells

A375 human melanoma cells B16F10

and C57BL/6 mice bearing a subcutaneously transplanted B78H1

amelanotic melanoma.

*in vitro* G361 human melanoma cells

*in vitro* melanoma cells

**Tumour/Cell line Photosensitizer Ref**

(ZnPPIX)

*in vivo* mice bearing mouse melanomas verteporfin [72]

*in vivo* transplanted B16 melanoma novel derivatives of chlorin e[6] [75]

*in vitro* B16 melanoma cells IPL and IPL plus 5-ALA [78] *in vitro* melanoma cells bacteriochlorins and photofrin [79]

*in vitro* WM 1552C human melanoma cells liposomes (LP) and nanocapsules (NC) containing

*in vitro* A375 melanoma cells 5,15-Diarylporphyrins (1-5) and Photofrin [82] *in vivo* B16 melanoma tumours on mice 2 doses of photosensitizer [83] *in vitro* B16 mouse melanoma cells chlordiazepoxide (CDZ) [84]

fullerene)

transplanted melanoma Zn(II)-phthalocyanine disulphide (C11Pc) [80]

S91 mouse melanoma cells and DBA mice 5,10,15,20-tetrakis[2-chloro-5-

*in vitro* A375 melanoma cells 5-aminolevulinic acid [5-ALA) and novel

*in vitro* WM451LU melanoma cells photosensitizers and heme oxygenase I (HO-I) and

PDT and Lycopene, β-carotene, vitamin C, Nacetylcysteine, trolox, N-tert-butyl-α-phenylnitrone and HO-1 activity inhibitor zinc protoporphyrine IX

The Menace of Melanoma: A Photodynamic Approach to Adjunctive Cancer Therapy

sulfophenyl)bacteriochlorin (TCPBSO3H) [66]

poly(ADP-ribose) polymerase (PARP) inhibitors [73]

metallophthalocyanine (MPc) [74]

Chloroaluminum phthalocyanine (CIAIPc) [81]

octabutoxy-naphthalocyanines [85]

zinc-5,10,15,20-tetrakis(4-sulphonatophenyl) porphyrine (ZnTPPS(4)), chloraluminium phtalocyanine disulfonate (ClAlPcS(2)) and 5-

aminolevulinic acid (ALA)

mouse melanoma cells Pc4 encapsulated in silica nanoparticles [87]

C(60)-(Glc)1 (D-glucose residue pendant fullerene) and C(60)-(6Glc)1 (a maltohexaose residue pendant

chlorin and bacteriochlorin derivatives of

5,10,15,20-tetrakis[2-chloro-5 sulfophenyl)porphyrin

[71]

589

http://dx.doi.org/10.5772/53676

[76]

[77]

[86]


*in vivo*

*in vitro/ in vivo*

*in vivo*

*in vitro*

*in vitro/ in vivo*

*in vitro* B16-F10 melanoma cells

588 Melanoma - From Early Detection to Treatment

*in vivo* mice carrying B16-F10 melanoma xenografts

> subcutaneous amelanotic melanoma transplanted in C57/BL6 mice

B16F10 mouse melanoma cells and lung

B16-F1 and Cloudman S9 melanoma-

S91 Cloudman melanoma cells and DBA

Melanoma cells and xenograft melanoma

*in vitro* melanoma, keratinocyte and fibroblast

melanomas in C57BL/6 mice

*in vitro* Melanoma, keratinocyte and fibroblast

bearing mice

*in vitro* melanoma cells

model

**Tumour/Cell line Photosensitizer Ref**

(CB) (CB:ZnPc-MLs)

*in vitro* A375, UCT Mel-1 human melanoma cells hypericin and kojic acid (depigmenting agent) [54]

*in vitro* C32 human melanoma cells Ficus carica L. cultivar Dottato extracts [56]

*in vivo* malignant melanoma mouse model methylene blue [59] *in vitro* A549 and S91 melanoma cells halogenated sulfonamide bacteriochlorins [60]

*in vitro* A375 melanoma cells Cachrys pungens Jan extracts from Italy [63] *in vitro* B16F10 murine melanoma indocyanine green (ICG) and hyperthermia [64] *in vitro* B78-H1 murine melanoma cells pheophorbide a [65]

*in vitro* M21 human melanoma cells Hedyotis corymbosa extracts [68]

*in vitro* A375, UCT Mel-1 human melanoma cells hypericin and phenylthiourea (depigmenting

mice synthetic chlorin derivative (TCPCSO₃H) [66]

cells zinc tetrasulfophthalocyanines (ZnTSPc) [70]

*in vitro* A375 melanoma cells carotenoids (neoxanthin, fucoxanthin and

luciferase

cells aluminum tetrasulfophthalocyanines [57]

magnetoliposomes (MLs) loaded with zinc phthalocyanine (ZnPc) complexed with cucurbituril

butadiyne-linked conjugated porphyrin dimer (Oxdime) [52]

polyethylene glycol-Pba [53]

pheophorbide a (Pba) and monomethoxy-

aminolevulic acid, gaussia luciferase, and its' substrate coelenterazine; murine neural stem cells (NSCs) and rat umbilical cord matrix-derived stem cells (RUCMSCs) with a plasmid expressing gaussia

chlorin e(6) and modular nanotransporters targeted to α-melanocyte-stimulating hormone (αMSH) and

siphonaxanthin) [61]

photosensitizers and chemotherapeutics [62]

phenanthroline) rhodium(III) chloride (OCTBP) [67]

agent) [69]

epidermal growth factor (EGF) receptor

2 cationic octanuclear metalla-cubes dual

cis-Dichlorobis [3,4,7,8-tetramethyl-1,10-

[51]

[55]

[58]


*in vitro*

*in vitro*

*in vivo*

*in vitro*

*in vivo*

*in vitro/ in vivo*

**Tumour/Cell line Photosensitizer Ref**

The Menace of Melanoma: A Photodynamic Approach to Adjunctive Cancer Therapy

5,10,15,20-tetrakis[2-chloro-3-

cells phthalocyanine ClAlPcS(2) [106]

albino rabbit eyes hematoporphyrin monomethyl ether [108]

line porfimer sodium [110]

C57BL/6 mice BPD, ce6, Photofrin, and mTHPC and gamma-inulin [111]

line porfirmer sodium (photofrin II) [117]

*in vitro* A375 human melanoma cells acridine orange [112]

*in vitro* Cloudman S91/I3 mouse melanoma cells photofrin II (PfII- porfirmer sodium), verteporfin,

*in vivo* B-16 melanoma-bearing C57BL/6 mice ATX-S10 No (II) and intratumoral injection of naïve

*in vitro* G361 human melanoma cells ZnTPPS(4) sensitizer bound to cyclodextrin

*in vitro* G361 human melanoma cells ZnTPPS(4) sensitizer bound to cyclodextrin

*in vitro* B78H1 melanoma cells liposome-delivered Ni(II)-octabutoxy-

*in vitro* M2R mouse melanoma cells O-[Pd-bacteriochlorophyllide]-serine methyl ester

*in vitro* B78H1 mouse melanoma cells Ni(II)-octabutoxy-naphthalocyanine (NiNc) [119]

*in vitro* human choroidal melanoma (CM) cells tetrahydroporphyrin tetratosylat (THPTS) [122]

mPEG-b-p(HPMAm-Lac2) micelles [105]

http://dx.doi.org/10.5772/53676

591

sulfophenyl)porphyrin (TCPPSO(3)H), [107]

PdTPPS(4)) [109]

and merocyanine 540 (MC540) [113]

5-aminolaevulinic acid (ALA) [114]

dendritic cells (IT-DC) [115]

hpbetaCD [118].

hpbetaCD [120]

(Pd-Bchl-Ser) [121]

naphthalocyanine [119]

Zn(ii)-phthalocyanine derivative bearing four 10Benriched o-carboranyl units [10B-ZnB4Pc) [116]

*in vitro* B16F10 mouse melanoma cells solketal-substituted phthalocyanine (Si(sol)2Pc in

*in vitro* G361 human melanoma cells 3 porphyrin sensitizers (TPPS(4), ZnTPPS[4] and

B19 mouse and G361 human melanoma

choroidal melanomas in 46 New Zealand

YUSAC2/T34A-C4 human melanoma cell

A-Mel-3 melanomas implanted in the dorsal skin fold chamber of Syrian Golden

B16F1 mouse melanoma cells and C57BL6 mice bearing a subcutaneously

injected B16F1 melanoma.

*in vitro* human Beidegröm Melanoma (BM) cell

*in vivo* subcutaneous B16BL6 melanoma-bearing

hamsters

S91 mouse and SKMEL 188 human

melanoma cells


590 Melanoma - From Early Detection to Treatment

*in vitro*

*in vitro/ in vivo*

*in vitro/ in vivo*

*in vitro/ in vivo*

*in vitro/ in vivo*

*in vitro*

*in vitro*

*in vitro*

**Tumour/Cell line Photosensitizer Ref**

carboranyl-containing chlorin

ultrasound [90]

methylene blue [92]

(TPFC) [93]

porfimer sodium and antibodies neutralizing decayaccelerating factor (DAF), complement-receptor-1-

5,10,15,20-tetraphenylporphin-loaded PEG-PE micelles [97]

Five 5,10,15,20-tetra[4-pyridyl)porphyrin (TPP) areneruthenium(II) derivatives and a p-

pentamethylcyclopentadienyliridium and -rhodium

depigmentation with violet light [100]

chloride complexes (TiO2/PtCl4) [101]

porphyrine (ZnTPPS4) and atomic force microscopy [102]

(H2TCP) [104]

[95]

[99]

related protein y (Crry), and protectin

*in vitro* Sk-Mel-28 human skin melanoma cells indocyanine green [88]

*in vitro* A375 human melanoma cells 5-aminolevulinic acid (ALA) [91]

*in vitro* G361 human melanoma cells porphyrines (TPPS4, ZnTPPS4 and PdTPPS4 [96]

*in vitro* G361 human melanoma cells chloroaluminum phthalocyanine (ClAlPc) and

cells hypericin [89]

(MMCs) porfimer sodium [94]

melanoma cells hypericin [98]

analogues

cells 5-aminolevulinic acid (ALA) [103]

*in vitro* S-91 mouse melanoma cells titanium dioxide modified with platinum(IV)

*in vitro* G361 human melanoma cells zinc-5,10,15,20-tetrakis(4-sulphonatophenyl)

*in vitro* B16F1 mouse melanoma cells meso-tetra[4-nido-carboranylphenyl)porphyrin

cymeneosmium and two

methyl 5-aminolevulinate (MAL) and

UCT Mel-1 and A375 human melanoma

B16F1 mouse melanoma cells and C57BL6 mice bearing a subcutaneously

B16F1 mouse melanoma cells and C57BL6 mice bearing a subcutaneously

human malignant melanoma cells

B-16 mouse melanoma cells and subcutaneous B-16 melanoma-bearing

UCT Mel-1 and UCT Mel-3 human

B16F10 melanotic melanomas transplanted to nude mice

WM451Lu metastatic human melanoma

injected B16F1 melanoma.

injected B16F1 melanoma.

*in vivo* B57BL/6 mice bearing a B16BL6 melanoma

C57BL/6 mice

*in vitro* Me300 human melanoma cells


SK-23 mouse melanoma and SK-Mel 28

Syrian Golden hamsters fitted with dorsal skinfold chambers containing A-Mel-3

melanoma cells

*in vivo* C57/BL6 mice bearing a subcutaneously transplanted B1 melanoma

*in vivo* C57/BL6 mice bearing a subcutaneously

*in vivo* C57/BL6 mice bearing a subcutaneously

*in vivo* C57/BL6 mice bearing a subcutaneously

*in vivo* C57/BL6 mice bearing a subcutaneously

*in vivo* M2R mouse melanoma tumors implanted

*in vivo* C57/BL6 mice bearing a subcutaneously

melanoma cells

melanoma cells

melanoma cells

Syrian Golden hamsters fitted with dorsal skinfold chambers containing A-Mel-3

Syrian Golden hamsters fitted with dorsal skinfold chambers containing A-Mel-3

Syrian Golden hamsters fitted with dorsal skinfold chambers containing A-Mel-3

*in vitro*

*in vivo*

*in vivo*

*in vivo*

*in vivo*

**Tumour/Cell line Photosensitizer Ref**

The Menace of Melanoma: A Photodynamic Approach to Adjunctive Cancer Therapy

naphthalocyanine (NiNc), [144]

http://dx.doi.org/10.5772/53676

593

5-aminolaevulinic acid (ALA) [146]

n-hexylsiloxy) silicon phthalocyanine (HexSiPc) [147]

(verteporfin, BPD-MA) [148]

benzoporphyrin derivative monoacid ring A

9-acetoxy-2,7,12,17-tetrakis-(beta-methoxyethyl) porphycene (ATMPn) [153].

5-aminolaevulinic acid (ALA) [155]

5-aminolaevulinic acid (ALA) [159]

human melanoma methylene blue [143]

*in vitro* B78H1 melanoma cells liposome-incorporated Ni(II)-octabutoxy-

*in vitro* B78H1 melanoma cells Cu(II)-hematoporphyrin (CuHp) [145]

*in vitro* M6 human melanoma cells dichlorosilicon phthalocyanine (Cl2SiPc) and bis(tri-

*in vitro* SkMel-23 melanoma cells 5-aminolaevulinic acid (ALA) [149]

transplanted B16 melanoma Si(i.v.)-naphthalocyanine (isoBO-SiNc) [150]

transplanted B16 melanoma aluminum phthalocyanine (AlpcS4 [151]

transplanted B16F10 melanoma lutetium texaphyrin (PCI-0123), [152]

transplanted B16 melanoma Si(IV)-methoxyethylene-glycol-naphthalocyanine [154]

in CD1 nude mice bacteriochlorophyll-serine (Bchl-Ser), [157]

transplanted B16 melanoma Zn(II)-2,3 naphthalocyanine (ZnNc) [160]

*in vivo* 10 choroidal melanomas in rabbits liposomal preparation of benzoporphyrin derivative[156]

*in vitro* uveal melanoma cells hematoporphyrin esters (HPE) [158]


*in vivo*

*in vivo*

*in vitro*

*in vivo*

*in vivo* C57BL6 mice bearing a subcutaneously

syrian Golden hamsters fitted with dorsal skinfold chambers containing A-Mel-3

UB900518 human melanoma cells

S91 mouse and SKMEL 188 human

Syrian Golden hamsters fitted with dorsal skinfold chambers containing A-Mel-3

*in vivo* pigmented choroidal melanoma 44 New Zealand albino rabbit eyes

*in vitro* Me45 human melanoma cells

592 Melanoma - From Early Detection to Treatment

melanoma cells

melanoma cells

*in vivo* Nude CD1 mice bearing malignant M2R

**Tumour/Cell line Photosensitizer Ref**

heterodimer 2 [126]

aluminium Pc (ClAlPc), [129]

(TPPS4) and zinc metallocomplex (ZnTPPS4) [131]

5-aminolaevulinic acid (ALA) [133]

liposomal preparation of benzoporphyrin derivative (BPD), verteporfin [136]

meta(tetrahydroxyphenyl)chlorin or m-THPC [138]

Forskolin, DSF, and Z.VAD.fmk [47]

5-aminolaevulinic acid (ALA) [141]

[130]

meso-tetra-4-N-methylpyridyl-porphyrin iodide and 5,10-di-[4-acetamidophenyl)-15,20-di-[4-N-

*in vivo* M2R mouse melanoma xenografts WST11 [123] *in vitro* B16 mouse melanoma cells 5-aminolevulinic acid [5-ALA) ester derivatives [124] *in vivo* B-16 melanoma-bearing C57BL/6 mice metal-free sulfonated phthalocyanine (H(2)PcS(2.4))[125]

*in vitro* B16 mouse melanoma cells 5-aminolevulinic acid (ALA) [127]

injected B16F10 melanoma silkworm excreta (SPbalpha) porfirmer sodium [128]

methylpyridyl) porphyrin

*in vitro* A375 human melanoma cells alpha-methylene-gamma-butyrolactone-psoralen

*in vitro* M3Dau human melanoma cells silicon-phthalocyanines (SiPc) and chloro-

*in vitro* G361 human melanoma cells meso-tetrakis[4-sulphonatophenyl)porphine

*in vitro* B16A45 (B16) mouse melanoma cells delta-aminolevulinic acid (ALA) and

*in vitro* B16 mouse melanoma cells m-THPC and four apoptosis inhibitors: BAPTA-AM,

*in vitro* Bro, SKMel-23, SKMel-28 5-aminolevulinic acid (ALA) [139] *In vitro* SKMEL 188 human melanoma cells tritolylporphyrin dimer (T-D). [140]

*in vitro* G361 human melanoma cells ATX-S10(Na) [132]

*in vitro* B16 mouse melanoma cells 5-aminolaevulinic acid (ALA) [134]

transplanted on nude (*nu/nu*) CD-1 mice Liposomal meso-tetrakis-phenylporphyrin (TPP) [135]

melanoma cells indocyanine green (ICG) [137]

melanoma xenografts bacteriochlorophyll-serine (Bchl-Ser), [142]


> 1 duodenal metastatic melanoma

2 late-stage melanoma. Patient 1 had the primary tumour and local metastases on the left arm and metastatic tumours in the lungs. Patient 2 had a head and neck melanoma with multiple local metastases, which had failed repeated attempts at surgical resection and highdose radiation therapy.

4 uveal melanoma, PDT on actual tumour site

25 small and medium choroidal melanomas

6 brain metastasis of malignant melanoma

clinical

clinical

clinical

clinical

clinical

**Tumour Pigmentary**

clinical melanoma in situ unigmented methyl aminolevulinate

clinical choroidal melanomaunpigmented benzoporphyrin derivative

pigmented

mildly to heavily pigmented

ND

**phenotype**

ND porfirmer sodium

(MAL)

(BPD)

indocyanine

green (ICG) + imiquimod (toll-like receptor agonist)

benzoporphyrin derivative

indocyanine green and transpupillary thermotherapy

(BPD)

**Photosensitizer Outcome of study Ref**

The Menace of Melanoma: A Photodynamic Approach to Adjunctive Cancer Therapy

died of unrelated causes.

during 24-month follow-up.

months after PDT.

All 6 patients [100%) remained free of brain disease till death, 50% died of malignant melanoma elsewhere and 50%

Recurrence at the original tumour site 4

The tumor fully disappeared 1 month after the treatment, the visual acuity improved from 4/16 to 4/4. The disease did not recur

Patient 1 free of all clinically detectable tumours (including the lung metastases) "/>20 months after the first treatment cycle. Patient 2 has been free of any clinical evidence of the tumour for over 6 months.

Vascular occlusion and thrombosis in mildly pigmented melanoma but no response in

After a mean of 2.4 treatments (range, 1 to 5 treatments), all of the tumors but one showed a significant volume reduction without clinical evidence of recurrences. Complications included retinal vascular occlusions, edema and superficial scarring of the macula, and rhegmatogenous retinal

pigmented ones.

detachment.

[172]

595

http://dx.doi.org/10.5772/53676

[173]

[174]

[175]

[176]

[177]

ND porfirmer sodium Successful treatment. [171]

**Table 2.** Comprehensive update of in vivo and in vitro photodynamic therapy studies from 1996-present.

For melanoma treatment, where PDT will be more effective as a post-operative adjunctive treat‐ ment, very few reports highlight its effectiveness even though laboratory studies using melano‐ ma cells show promise. Clinically, PDT has shown promise in the treatment of both ocular amelanotic melanomas [164] and skin metastases [165] however, more extensive clinical studies need to be conducted before PDT is accepted as the adjunctive therapy of choice [166] (Table 3).



32 choroidal tumours in New Zealand

**Tumour Pigmentary**

clinical choroidal melanomaunpigmented verteporfin

melanomas pigmented

**phenotype**

unpigmented verteporfin

*in vitro* G361,M18 and M6 human melanoma

594 Melanoma - From Early Detection to Treatment

*in vivo*

**Type of study**

clinical

clinical

clinical

3 choroidal

9 posteriorly located choroidal melanomas

11 late stage melanomas (cutaneous metastases)

ND

**Tumour/Cell line Photosensitizer Ref**

**Photosensitizer Outcome of study Ref**

The tumors treated with PDT and bevacizumab showed a marked reduction in tumor vascularity. The tumors receiving PDT as a primary treatment were followed by progressive tumor growth that led to

Eight tumors demonstrated apparent complete regression over 1 month to 14 months with no recurrence during followup of between 34 months and 81 months. One case developed 2 separate local recurrences at 21 months and 34 months.

Complete response was observed in 6 patients. All lesions in the treatment area of the patients responded to photoimmunotherapy, 8 of which achieved complete local response (CLR). CLR was observed in the non-treatment site (regional) lesions in four patients. Five patients were still alive at the time of last

enucleation years after.

follow-up.

follow-up.

Dramatic tumor regression over 2 months to a completely flat scar [1.3 mm thickness), and remained stable at 50 months of

[167]

[168]

[169]

[170]

albino rabbit eyes. benzoporphyrin derivative [161]

cells hypericin [162]

*in vitro* melanoma cell lines hypericin [163]

For melanoma treatment, where PDT will be more effective as a post-operative adjunctive treat‐ ment, very few reports highlight its effectiveness even though laboratory studies using melano‐ ma cells show promise. Clinically, PDT has shown promise in the treatment of both ocular amelanotic melanomas [164] and skin metastases [165] however, more extensive clinical studies need to be conducted before PDT is accepted as the adjunctive therapy of choice [166] (Table 3).

> PDT and intravitreal bevacizumab

indocyanine green and imiquimod (immune modifier)

**Table 2.** Comprehensive update of in vivo and in vitro photodynamic therapy studies from 1996-present.


type of PDT is more convenient for patients and clinicians and causes less pain. It poses a particularly interesting avenue to explore for hospitals in developing countries where space is limited and budgets are inadequate. Daylight-mediated PDT is an effective treatment for thin actinic keratosis, as shown in three randomized controlled clinical studies (reviewed in Wiegell et al., 2011) [183]. The potential of hypericin in clinical practice has been highlighted by reports on its use to treat squamous and basal cell carcinomas [184-187], pancreatic tu‐ mors [188], bladder carcinomas [189-193], nasophyrangeal tumors [194,195] and recently

The Menace of Melanoma: A Photodynamic Approach to Adjunctive Cancer Therapy

http://dx.doi.org/10.5772/53676

597

**Figure 1.** Absorbance spectrum of hypericin. Box, the wavelength of light used in our studies [98,197] representing

Despite these promising studies, very few reports have highlighted hypericin's role in tar‐ geting melanoma. For the most part, cytotoxicity testing of new photosensitizers are tested on cell lines in vitro using assays such as the 3-[4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra‐ zolium bromide (MTT), which is a colorimetric assays for measuring the activity of enzymes that reduce MTT to formazan dyes, giving a purple color [198,199]. Other tests for cytotoxic‐ ity include dead cell protease tests. Despite being used as the "gold standard" for cytotoxici‐ ty testing, it must be borne in mind that these assays are based on cellular metabolic activity and could result in a false positive result were the treatment to produce a cytostatic effect in cells. Moreover, as these are colorimetric-based tests, the photosensitizer used itself may in‐

One of the first few reports testing 1 to 20mg/ml hypericin efficacy on squamous carcinoma, sarcoma and melanoma cell lines found that a combination of activating laser light sources resulted in a reduction in cell viability of 90% [163]. Following this, Hadjur et al. (1996) ex‐ posed human pigmented and unpigmented melanoma cell lines to hypericin and showed minimal cytotoxicity on uptake but upon activation with white light, increased cell death in

*1.3.3. Melanoma cell death and biological mechanisms induced by hypericin-PDT*

one of the two activation peaks. Inset, chemical structure of hypericin.

terfere with the wavelength at which these tests are read.

melanomas [196].

**Table 3.** Clinical reports and outcomes of photodynamic effectiveness of photodynamic therapy protocols including the melanoma pigmentary phenotype.

#### *1.3.2. Hypericin, a second generation photosensitizer for PDT*

Hypericin, a second generation PS isolated from the plant *Hypericum perforatum,* is a phenan‐ throperylenequinone with two broad peaks of absorption – 300-400nm (ultraviolet) and 500-600nm (white light) (Figure 1). This may be considered as a disadvantage as a number of current second-generation photosensitizers have absorption peaks beyond 630nm allow‐ ing for increased penetration into tissues [182]. However, white light, used to activate hyper‐ icin, does penetrate deep into the dermis of the skin. Moreover, activation with ultraviolet light could be a distinct advantage for the use of hypericin in daylight-mediated PDT. This type of PDT is more convenient for patients and clinicians and causes less pain. It poses a particularly interesting avenue to explore for hospitals in developing countries where space is limited and budgets are inadequate. Daylight-mediated PDT is an effective treatment for thin actinic keratosis, as shown in three randomized controlled clinical studies (reviewed in Wiegell et al., 2011) [183]. The potential of hypericin in clinical practice has been highlighted by reports on its use to treat squamous and basal cell carcinomas [184-187], pancreatic tu‐ mors [188], bladder carcinomas [189-193], nasophyrangeal tumors [194,195] and recently melanomas [196].

**Type of study**

clinical

clinical

clinical

clinical

**Tumour Pigmentary**

14 skin metastasis from malignant melanoma, despite multiple courses of chemotherapy

596 Melanoma - From Early Detection to Treatment

38 choroidal melanomas

4 choroidal melanomas

3 uveal melanoma, PDT on unaffected areas before enucleation

the melanoma pigmentary phenotype.

clinical 36 uveal melanomasvarious hematoporphyrin

*1.3.2. Hypericin, a second generation photosensitizer for PDT*

**phenotype**

pigmented chlorin e6

ND indocyanine green (ICG)

ND benzoporphyrin derivative (BPD)

ND benzoporphyrin derivative (BPD)

derivative

**Table 3.** Clinical reports and outcomes of photodynamic effectiveness of photodynamic therapy protocols including

Hypericin, a second generation PS isolated from the plant *Hypericum perforatum,* is a phenan‐ throperylenequinone with two broad peaks of absorption – 300-400nm (ultraviolet) and 500-600nm (white light) (Figure 1). This may be considered as a disadvantage as a number of current second-generation photosensitizers have absorption peaks beyond 630nm allow‐ ing for increased penetration into tissues [182]. However, white light, used to activate hyper‐ icin, does penetrate deep into the dermis of the skin. Moreover, activation with ultraviolet light could be a distinct advantage for the use of hypericin in daylight-mediated PDT. This

**Photosensitizer Outcome of study Ref**

Complete regression after the first PDT treatment in eight cases and complete regression after multiple treatments in six cases. 11 of 14 patients died due to the progression of the melanoma, the median survival time after surgery was 883 days.

Changes in microcirculation 6 months after PDT, as well as significant decrease of tumors thickness in ultrasonography (mean 38%), were detected in all cases. Complete regression of intrinsic vessels was demonstrated by ICGA in 26 cases, and partial regression of pathological vascularization was found in 12 patients.

One tumor decreased in size and remained stable for 18 months. One tumor had no growth for 11 months. Two melanomas continued to grow, necessitating

Vascular thrombosis. No damage to the

76 % of tumours were not growing at the end of the first year, 62 % after the second year and 38% after the fifth year. No eyes were lost as a result of PDT. The degree of tumour pigmentation and patient age at therapy significantly influence the tumour

enucleation.

photoreceptors.

response to PDT.

[165]

[178]

[179]

[180]

[181]

**Figure 1.** Absorbance spectrum of hypericin. Box, the wavelength of light used in our studies [98,197] representing one of the two activation peaks. Inset, chemical structure of hypericin.

## *1.3.3. Melanoma cell death and biological mechanisms induced by hypericin-PDT*

Despite these promising studies, very few reports have highlighted hypericin's role in tar‐ geting melanoma. For the most part, cytotoxicity testing of new photosensitizers are tested on cell lines in vitro using assays such as the 3-[4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra‐ zolium bromide (MTT), which is a colorimetric assays for measuring the activity of enzymes that reduce MTT to formazan dyes, giving a purple color [198,199]. Other tests for cytotoxic‐ ity include dead cell protease tests. Despite being used as the "gold standard" for cytotoxici‐ ty testing, it must be borne in mind that these assays are based on cellular metabolic activity and could result in a false positive result were the treatment to produce a cytostatic effect in cells. Moreover, as these are colorimetric-based tests, the photosensitizer used itself may in‐ terfere with the wavelength at which these tests are read.

One of the first few reports testing 1 to 20mg/ml hypericin efficacy on squamous carcinoma, sarcoma and melanoma cell lines found that a combination of activating laser light sources resulted in a reduction in cell viability of 90% [163]. Following this, Hadjur et al. (1996) ex‐ posed human pigmented and unpigmented melanoma cell lines to hypericin and showed minimal cytotoxicity on uptake but upon activation with white light, increased cell death in all three cell lines. Their findings thus suggest that amelanotic melanomas may be more sus‐ ceptible to hypericin-PDT than pigmented melanomas. Their possible reasons for this relat‐ ed to the presence of melanin and antioxidant status of melanomas [162].

**A**

**%**

**B**

**%**

**C**

**%**

**30' 1 4 7 24**

The Menace of Melanoma: A Photodynamic Approach to Adjunctive Cancer Therapy

**Time after treatment (h)**

**30' 1 4 7 24**

**Time after treatment (h)**

**30' 1 4 7 24**

**Time after treatment (h)**

**Figure 2.** Graphs representing fluorescent activated cell sorting (FACS) analyses of melanoma cells at 30min, 1, 4, 7 and 24h after hypericin-PDT treatment (3µM hypericin with 1 J/cm2 UVA). A: unpigmented A375, B: mildly pigmented 501mel and C: pigmented UCT Mel-1. Cells were stained for early apoptosis (FITC Annexin V, BD Biosciences) and ne‐ crosis (LIVE/DEAD Fixable Violet stain, Invitrogen). Different modes of cell death are represented as proportional percen‐ tages normalised to the control, black: late apoptotic/ necrotic, dark grey: necrotic, light grey: apoptotic, white: live; n=3.

**Live Early Apoptotic Necrotic**

**Live Early Apoptotic Necrotic**

**Live Early Apoptotic Necrotic**

**Late Apoptotic/ Necrotic**

**Late Apoptotic/ Necrotic**

**Late Apoptotic/ Necrotic**

599

http://dx.doi.org/10.5772/53676

Work from our laboratory has shown a pigmentation dependant susceptibility of mela‐ noma cells to hypericin-PDT, with pigmented cells being less susceptible than unpig‐ mented cells [54,98,200,201]. Upon depigmentation with tyrosinase inhibitors, kojic acid and phenylthiourea, pigmented melanoma cells become more susceptible to hypericin-PDT [54,69]. Moreover, 72 hours after hypericin-PDT the cell viability of the depigment‐ ed melanoma cells remained significantly less than the control cells. Over the same time period the cells not treated with kojic acid approached a cell viability similar to the control.

Melanin is a potent antioxidant which could be a reason for the increased resistance of pig‐ mented melanoma cells to PDT due to the scavenging of ROS produced by this therapy. In‐ deed we have shown that after depigmenting melanoma cells with kojic acid more ROS is produced upon treatment with hypericin-PDT compared to pigmented melanoma cells which were not depigmented [54]. We did not find a difference between the caspase 3, 7 ac‐ tivity after hypericin-PDT for both the depigmented and pigmented melanoma cells, which was lower than control. This suggests that pigmented melanoma cells might induce a cas‐ pase-independent mode of cell death such as the activation of apoptosis-inducing factor (AIF). Moreover, these cells might also undergo necrosis, necroptosis or autophagy in re‐ sponse to hypericin-PDT. We have further shown induction of autophagy at 4hours after hypericin-PDT in both pigmented and unpigmented melanoma cells [197]. Interestingly, pigmented melanoma cells (UCT Mel-1) show higher levels of externalisation of Annexin V, an early apoptotic event, compared to mildy and unpigmented melanoma cells [501mel and A375, respectively) after hypericin-PDT (Figure 2). However, the cell death response of pig‐ mented and unpigmented melanoma cells is very complex and does seem to be cell type de‐ pendant. A possible explanation for this may be that the cell lines used in our studies are from different genetic origins and they thus might differ in various biochemical characteris‐ tics, including their antioxidant systems. The subcellular localisation of the photosensitizer is another factor determining the cell death mode initiated by PDT. Upon activation by light, photosensitizers produce ROS which are short-lived species acting directly in their vicinity of production. Localisation to different cellular compartments thus induces different modes of cell death.

Note: Since the discovery of programmed cell death in the 1960's the cell death field has evolved immensely. Researchers have shifted from morphological classifications to using more biochemical criteria. The increase in cell death studies necessitated a systemic classifica‐ tion of cell death modalities, which led to the formation of the Nomenclature Committee on Cell Death (NCCD). The main mission of this committee is 'to provide a forum in which names describing distinct modalities of cell death are critically evaluated and recommendations on their definition and use are formulated, hoping that a non-rigid, yet uniform nomenclature will facilitate the communication among scientists and ultimately accelerate the pace of discovery' [202-204].

The Menace of Melanoma: A Photodynamic Approach to Adjunctive Cancer Therapy http://dx.doi.org/10.5772/53676 599

**Live Early Apoptotic Necrotic**

**Live Early Apoptotic Necrotic**

**Live Early Apoptotic Necrotic**

**Late Apoptotic/ Necrotic**

**Late Apoptotic/ Necrotic**

**Late Apoptotic/ Necrotic**

all three cell lines. Their findings thus suggest that amelanotic melanomas may be more sus‐ ceptible to hypericin-PDT than pigmented melanomas. Their possible reasons for this relat‐

Work from our laboratory has shown a pigmentation dependant susceptibility of mela‐ noma cells to hypericin-PDT, with pigmented cells being less susceptible than unpig‐ mented cells [54,98,200,201]. Upon depigmentation with tyrosinase inhibitors, kojic acid and phenylthiourea, pigmented melanoma cells become more susceptible to hypericin-PDT [54,69]. Moreover, 72 hours after hypericin-PDT the cell viability of the depigment‐ ed melanoma cells remained significantly less than the control cells. Over the same time period the cells not treated with kojic acid approached a cell viability similar to the

Melanin is a potent antioxidant which could be a reason for the increased resistance of pig‐ mented melanoma cells to PDT due to the scavenging of ROS produced by this therapy. In‐ deed we have shown that after depigmenting melanoma cells with kojic acid more ROS is produced upon treatment with hypericin-PDT compared to pigmented melanoma cells which were not depigmented [54]. We did not find a difference between the caspase 3, 7 ac‐ tivity after hypericin-PDT for both the depigmented and pigmented melanoma cells, which was lower than control. This suggests that pigmented melanoma cells might induce a cas‐ pase-independent mode of cell death such as the activation of apoptosis-inducing factor (AIF). Moreover, these cells might also undergo necrosis, necroptosis or autophagy in re‐ sponse to hypericin-PDT. We have further shown induction of autophagy at 4hours after hypericin-PDT in both pigmented and unpigmented melanoma cells [197]. Interestingly, pigmented melanoma cells (UCT Mel-1) show higher levels of externalisation of Annexin V, an early apoptotic event, compared to mildy and unpigmented melanoma cells [501mel and A375, respectively) after hypericin-PDT (Figure 2). However, the cell death response of pig‐ mented and unpigmented melanoma cells is very complex and does seem to be cell type de‐ pendant. A possible explanation for this may be that the cell lines used in our studies are from different genetic origins and they thus might differ in various biochemical characteris‐ tics, including their antioxidant systems. The subcellular localisation of the photosensitizer is another factor determining the cell death mode initiated by PDT. Upon activation by light, photosensitizers produce ROS which are short-lived species acting directly in their vicinity of production. Localisation to different cellular compartments thus induces different modes

Note: Since the discovery of programmed cell death in the 1960's the cell death field has evolved immensely. Researchers have shifted from morphological classifications to using more biochemical criteria. The increase in cell death studies necessitated a systemic classifica‐ tion of cell death modalities, which led to the formation of the Nomenclature Committee on Cell Death (NCCD). The main mission of this committee is 'to provide a forum in which names describing distinct modalities of cell death are critically evaluated and recommendations on their definition and use are formulated, hoping that a non-rigid, yet uniform nomenclature will facilitate the communication among scientists and ultimately accelerate the pace of discovery'

ed to the presence of melanin and antioxidant status of melanomas [162].

control.

598 Melanoma - From Early Detection to Treatment

of cell death.

[202-204].

**Time after treatment (h)**

**Figure 2.** Graphs representing fluorescent activated cell sorting (FACS) analyses of melanoma cells at 30min, 1, 4, 7 and 24h after hypericin-PDT treatment (3µM hypericin with 1 J/cm2 UVA). A: unpigmented A375, B: mildly pigmented 501mel and C: pigmented UCT Mel-1. Cells were stained for early apoptosis (FITC Annexin V, BD Biosciences) and ne‐ crosis (LIVE/DEAD Fixable Violet stain, Invitrogen). Different modes of cell death are represented as proportional percen‐ tages normalised to the control, black: late apoptotic/ necrotic, dark grey: necrotic, light grey: apoptotic, white: live; n=3.

## **1.4. PDT targets to treat melanoma**

### *1.4.1. Cell proliferation and survival*

It is now well established that one of the chief characteristics of cancer cells is their ability to overcome cellular control of proliferation [205]. In melanocytes, proliferation is caused by a combination of several mitogenic growth factors such as stem cell factor (SCF), epidermal growth factor (EGF), fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) which cause a sustained extracellular receptor kinase (ERK) activity [206]. In melanoma, the RAS/Raf/MEK/ERK pathway is a key regulating pathway in proliferation with ERK being hyperactivated in up to 90% of human melanomas [207]. BRAF and PTEN mutations (see above) are co-incident in about 20% of cases [208]. The most common mutation in BRAF is a glutamic acid for valine substitution at position 600 (V600EBRAF) [209]. This mutation leads to constitutive ERK signalling resulting in hyperproliferation and cell survival [210]. This path‐ way, through the EGF receptor as an extracellular ligand, has been a worthwhile target for PDT in that sustained activation of the ERK pathway protected cells from photofrin-based PDT as well as a reduction in the Raf protein levels in treated cells [211].

Cancer cells are known to resist cell death by upregulation of anti-apoptotic proteins, muta‐ tions in pro-apoptotic proteins, inhibition of cell senescence or through protective mecha‐ nisms such as autophagy. PDT that is targeted for cancer therapy aims to invoke cell cytoxicity through attacking these characteristics – topics of a number of recent reviews

The Menace of Melanoma: A Photodynamic Approach to Adjunctive Cancer Therapy

http://dx.doi.org/10.5772/53676

601

Intriguingly, the emergence of a defined 'immunogenic apoptosis' seems to be a new 'subset' of apoptosis and autophagic cell death which has been shown to have the ability to release/expose damage-associated membrane proteins (DAMPs) [224-227]. Therapeuti‐ cally, the immunogenicity of apoptosis is preferable for application rather than necrosis (or for that matter autophagic cell death) since necrosis can lead to harmful immunologi‐ cal reactions [228] (on the other hand, the extent of immunological impact of autophagic cell death is as yet uncharacterized, thereby making it an uncertain modality to use in the context of 'immunochemotherapy') [229]. The cell killing effectiveness is however de‐ pendent on parameters such as the PS used, the light dose and most importantly, the subcellular localization of the PS. It is crucial for photosensitizers to effectively enter the cell and accumulate in specific intracellular organelles in order to be efficient in their killing ability. Clearly, the final destination of the PS and its immediate vicinity will lead to different modes of cell death and consequently different efficiencies. Recent reports have highlighted that hypericin not only localises to different subcellular organelles but that this localization is exposure and dose-dependent in addition to being tumour cellspecific [230-234]. As a start however, the lipophillic nature of hypericin dictates its asso‐ ciation with cellular membranes [235]. The fact that hypericin has been shown to associate with serum proteins (LDL and HDL lipoproteins [236] ensues that it enters cells quickly and is preferentially taken up by cancer cells in the 3-dimensional milieu as recent reports showed that these cells have high levels of LDL surface receptors [237]. This is further supported by a recent report showing that cholesterol serves as a key de‐

terminant for the uptake of hypericin into cellular membranes [238].

Noteworthy however is that even though high levels of hydrophobicity ensues, high lev‐ els of intracellular accumulation of the photosensitizer, changes in the physical structure of the PS due to aggregation and other modifications, may lead to reduced PDT efficien‐ cy [239]. Overall, the consensus emerging is that hypericin localises to three intracellular organelles namely, the endoplasmic reticulum (ER)-Golgi network [230,231,240,241], mito‐ chondria (Mt) [242-245] and lysosomes [237,246] where through synergistic action, apop‐ tosis is induced. More recent work by the Agostinis group show that hypericin-based PDT would produce photo-oxidative ER (p-ox ER stress) stress while 5-ALA (localizes in the mitochondria)-based PDT would produce photo-oxidative mitochondrial stress [36,247]. They also observed that Hyp-PDT induces 'pre-apoptotic' active exo-ATP secre‐ tion and late stage passive release of DAMPs like HSP70, HSP90 and CRT [223]. Overall they suggest that the potential of Hyp-PDT in causing exposure/secretion of 'critical' DAMPs add to the apoptotic cell death modality in a rather 'small club' of anti-cancer‐ ous therapeutic agents/modalities capable of exposing immunogenic signals like ecto-

[36,221-223].

CRT [227,248].

Nuclear factor kappa beta (NF-κβ) signalling leads to transcriptional regulation of a number of genes involved in responses ranging from proliferation, metastasis, and survival to in‐ flammation. It therefore is an important target in PDT to stop aberrant cell proliferation. PDT-induced oxidative stress through increased ROS production has been shown to activate (NF-κβ) [212] and inactivate its inhibitor (Iκβ). Moreover, Ryter and Gomer showed in‐ creased NF-κβ binding in response to PDT stress in mouse cancer cells leading to a reduc‐ tion in proliferation [213].

#### *1.4.2. Inhibition of apoptosis*

Apoptosis, a controlled mode of cell death, is characterised by cell shrinkage, chromatin condensation, DNA fragmentation, membrane blebbing and activation of caspases [214]. It is now well established that activation of the caspase cascade occurs through death receptor activation (extrinsic pathway) or through mitochondrial outer membrane permeabilization (intrinsic pathway). Both of these pathways have been shown to be activated through PDT. Several biochemical studies have established that PDT with different photosensitizers, in‐ cluding hypericin, utilise the mitochondrial-mediated pathway of caspase activation [215,216] although PDT has recently been shown to also engage caspase-independent path‐ ways [217]. It is further known that several anti-cancer agents induce apoptosis and may share common pathways leading to cell killing with Fas/APO-1/CD95 [218,219]. Ali et al. (2002) elegantly showed that hypericin-PDT induces human nasopharyngeal cancer cells to undergo apoptosis through the Fas/FasL system. Moreover, they showed that the upregula‐ tion of Fas/FasL results in the release of cytochrome c into the cytoplasm with subsequent caspase induction – results that suggest that although apoptosis is considered a product of either an extrinsic or intrinsic mechanism; the overall response to PDT may be a combina‐ tion of mechanisms [220].

Cancer cells are known to resist cell death by upregulation of anti-apoptotic proteins, muta‐ tions in pro-apoptotic proteins, inhibition of cell senescence or through protective mecha‐ nisms such as autophagy. PDT that is targeted for cancer therapy aims to invoke cell cytoxicity through attacking these characteristics – topics of a number of recent reviews [36,221-223].

**1.4. PDT targets to treat melanoma**

600 Melanoma - From Early Detection to Treatment

*1.4.1. Cell proliferation and survival*

tion in proliferation [213].

*1.4.2. Inhibition of apoptosis*

tion of mechanisms [220].

It is now well established that one of the chief characteristics of cancer cells is their ability to overcome cellular control of proliferation [205]. In melanocytes, proliferation is caused by a combination of several mitogenic growth factors such as stem cell factor (SCF), epidermal growth factor (EGF), fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) which cause a sustained extracellular receptor kinase (ERK) activity [206]. In melanoma, the RAS/Raf/MEK/ERK pathway is a key regulating pathway in proliferation with ERK being hyperactivated in up to 90% of human melanomas [207]. BRAF and PTEN mutations (see above) are co-incident in about 20% of cases [208]. The most common mutation in BRAF is a glutamic acid for valine substitution at position 600 (V600EBRAF) [209]. This mutation leads to constitutive ERK signalling resulting in hyperproliferation and cell survival [210]. This path‐ way, through the EGF receptor as an extracellular ligand, has been a worthwhile target for PDT in that sustained activation of the ERK pathway protected cells from photofrin-based

Nuclear factor kappa beta (NF-κβ) signalling leads to transcriptional regulation of a number of genes involved in responses ranging from proliferation, metastasis, and survival to in‐ flammation. It therefore is an important target in PDT to stop aberrant cell proliferation. PDT-induced oxidative stress through increased ROS production has been shown to activate (NF-κβ) [212] and inactivate its inhibitor (Iκβ). Moreover, Ryter and Gomer showed in‐ creased NF-κβ binding in response to PDT stress in mouse cancer cells leading to a reduc‐

Apoptosis, a controlled mode of cell death, is characterised by cell shrinkage, chromatin condensation, DNA fragmentation, membrane blebbing and activation of caspases [214]. It is now well established that activation of the caspase cascade occurs through death receptor activation (extrinsic pathway) or through mitochondrial outer membrane permeabilization (intrinsic pathway). Both of these pathways have been shown to be activated through PDT. Several biochemical studies have established that PDT with different photosensitizers, in‐ cluding hypericin, utilise the mitochondrial-mediated pathway of caspase activation [215,216] although PDT has recently been shown to also engage caspase-independent path‐ ways [217]. It is further known that several anti-cancer agents induce apoptosis and may share common pathways leading to cell killing with Fas/APO-1/CD95 [218,219]. Ali et al. (2002) elegantly showed that hypericin-PDT induces human nasopharyngeal cancer cells to undergo apoptosis through the Fas/FasL system. Moreover, they showed that the upregula‐ tion of Fas/FasL results in the release of cytochrome c into the cytoplasm with subsequent caspase induction – results that suggest that although apoptosis is considered a product of either an extrinsic or intrinsic mechanism; the overall response to PDT may be a combina‐

PDT as well as a reduction in the Raf protein levels in treated cells [211].

Intriguingly, the emergence of a defined 'immunogenic apoptosis' seems to be a new 'subset' of apoptosis and autophagic cell death which has been shown to have the ability to release/expose damage-associated membrane proteins (DAMPs) [224-227]. Therapeuti‐ cally, the immunogenicity of apoptosis is preferable for application rather than necrosis (or for that matter autophagic cell death) since necrosis can lead to harmful immunologi‐ cal reactions [228] (on the other hand, the extent of immunological impact of autophagic cell death is as yet uncharacterized, thereby making it an uncertain modality to use in the context of 'immunochemotherapy') [229]. The cell killing effectiveness is however de‐ pendent on parameters such as the PS used, the light dose and most importantly, the subcellular localization of the PS. It is crucial for photosensitizers to effectively enter the cell and accumulate in specific intracellular organelles in order to be efficient in their killing ability. Clearly, the final destination of the PS and its immediate vicinity will lead to different modes of cell death and consequently different efficiencies. Recent reports have highlighted that hypericin not only localises to different subcellular organelles but that this localization is exposure and dose-dependent in addition to being tumour cellspecific [230-234]. As a start however, the lipophillic nature of hypericin dictates its asso‐ ciation with cellular membranes [235]. The fact that hypericin has been shown to associate with serum proteins (LDL and HDL lipoproteins [236] ensues that it enters cells quickly and is preferentially taken up by cancer cells in the 3-dimensional milieu as recent reports showed that these cells have high levels of LDL surface receptors [237]. This is further supported by a recent report showing that cholesterol serves as a key de‐ terminant for the uptake of hypericin into cellular membranes [238].

Noteworthy however is that even though high levels of hydrophobicity ensues, high lev‐ els of intracellular accumulation of the photosensitizer, changes in the physical structure of the PS due to aggregation and other modifications, may lead to reduced PDT efficien‐ cy [239]. Overall, the consensus emerging is that hypericin localises to three intracellular organelles namely, the endoplasmic reticulum (ER)-Golgi network [230,231,240,241], mito‐ chondria (Mt) [242-245] and lysosomes [237,246] where through synergistic action, apop‐ tosis is induced. More recent work by the Agostinis group show that hypericin-based PDT would produce photo-oxidative ER (p-ox ER stress) stress while 5-ALA (localizes in the mitochondria)-based PDT would produce photo-oxidative mitochondrial stress [36,247]. They also observed that Hyp-PDT induces 'pre-apoptotic' active exo-ATP secre‐ tion and late stage passive release of DAMPs like HSP70, HSP90 and CRT [223]. Overall they suggest that the potential of Hyp-PDT in causing exposure/secretion of 'critical' DAMPs add to the apoptotic cell death modality in a rather 'small club' of anti-cancer‐ ous therapeutic agents/modalities capable of exposing immunogenic signals like ecto-CRT [227,248].

#### *1.4.3. Induction of autophagy*

A recent finding is the induction of the cytoprotective programme of autophagy in mela‐ nomas in response to PDT-induced oxidative stress [89]. In addition, recent reports showed that cancer cells may respond to chemotherapeutics or other forms of oxidative stress such as PDT, through the induction of autophagy initially but continued stress leads to an overwhelming of the endogenous antioxidant enzymes along with a shift from autophagy to a possible senescent phenotype in an attempt to prolong cellular sur‐ vival. Consequently however, the cell enters an apoptotic or necrotic mode of cell death [249-251]. Autophagy, defined as a cellular response to nutrient deprivation with conse‐ quent organelle breakdown, could converge with PDT at a number of cellular locations. Although more work relating to this aspect in melanomas is needed, reports on other cancer cells have shown that autophagy can be induced if the lysosomal system, needed for the clearance of ROS-damaged organelles, is affected by PDT [252]. Another cellular location is the mitochondria, where the PDT-induced loss of anti-apoptotic protein Bcl-2, may lead to an initation of autophagy [253].

*1.4.5. Melanin and melanosomes as pro-survival agents*

target organelle in the fight against melanoma [268].

regulate melanosomal formation [274].

All the potential intracellular organelle targets for PDT mentioned above are consistent with most cancer cells. However, the one aspect that sets melanoma apart from other cancers is the presence of its cell-specific organelle called the melanosome and its associated product, melanin pigment. It is thus not inconceivable to believe that the intractability of this skin disease may in some way be related to this organelle and its function [266,267]. It follows logically then, that treatment regimes need to consider the melanosome as another potential

The Menace of Melanoma: A Photodynamic Approach to Adjunctive Cancer Therapy

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603

Melanosomes are membrane-bound organelles in melanocytic cells which house the path‐ way that results in the formation of the polymeric pigment, melanin [269,270]. The enzymes which participate in this pathway are translated in the cytoplasm and chaperoned to the melanosomes. Tyrosinase (TYR), the rate-limiting enzyme of the pathway, and its related proteins tyrosinase-related proteins 1 and 2 (TYRP-1 and TYRP-2) act in concert to first con‐ vert tyrosine to 3,4-dihydroxy-phenylalanine (DOPA) via tyrosine hydroxylase activity and then convert DOPA to DOPAquinone via dopa oxidase activity. Both of these activities oc‐ cur via separate tyrosinase catalytic sites. During melanin synthesis toxic intermediates such as 5, 6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid are produced. Structural‐ ly, the melanosomes are designed to compartmentalise these cytotoxic melanin intermedi‐ ates from spilling into the cytoplasm [271]. Melanosomal biogenesis progresses through four distinct stages of maturation where the first two stages contain no melanin and the later stages constitute intermediates required to generate a matrix favourable for the formation of melanin [269,272]. The Pmel17/gp100/Silv/ME20 protein, a product of the Silver locus in melanocytic cells [273], is capable of polymerizing into fibrillar arrays that form the back‐ bone of melanosomes. As a major component of the fibrillar matrix of early stage melano‐ somes, Pmel-17 serves as the best marker to follow intracellular trafficking steps that

Moreover, as Pmel-17 facilitates melanin deposition and plays a pivotal role in melanosome biogenesis, it remains a strategic target when trying to combat melanoma through the fact that melanosomes are involved in scavenging endogenous cytotoxic metabolites and storing their waste products - a function that has been suggested to be a key in creating multi-drug resistance [267]. With the premise that melanosomes may be acting as cytotoxic drug "sinks" through the sequestration of chemotherapeutic drugs [267], it would be considered an effective therapy for a PS to enter the melanosomal membrane and damage the wall of the melanosome thus allowing the leakage of toxic melanin intermediates resulting in cell death. The drawback is that melanosomes, which are classified into stages along their bio‐ genesis [269,270] only produce the toxic intermediates during their final maturing stages III and IV [268-270,275-277]. Most pigmented melanomas do however present with a majority of these end-stage melanosomes in their cytoplasm making the melanosomal membrane an attractive target for PDT. On the basis of this information, one may imagine that pigmented melanomas are therefore more susceptible to PDT-induced cell death. In contrast, our work has shown that pigmented melanomas are much less susceptible to hypericin-PDT than un‐ pigmented/amelanotic melanomas despite hypericin readily entering the melanosomes [89].

#### *1.4.4. Chemoresistance due to increased antioxidants*

Cancer cells are considered to be under continuous oxidative stress which has been sug‐ gested to aid in tumor progression [254]. In support, several studies have shown tumor cell lines producing higher levels of ROS compared to their normal counterparts [255,256]. Due to this increased level of ROS and hence constitutive increased level of ox‐ idative stress, it is not surprising that cancer cells have an extensive and advanced intra‐ cellular antioxidant network – a characteristic which further increases their chemoresistant property. Interestingly, the antioxidant status of melanomas differs from that of other skin cancers such as basal and squamous cell carcinomas in that their anti‐ oxidant activity levels (i.e. catalase, glutathione peroxidise, superoxide dismutase) are much higher [257]. In contrast, melanocytes, their normal untransformed phenotype, have lower levels of antioxidant activities and associated lower levels of resistance to oxi‐ dative stress [258]. It is therefore reasonable to postulate whether breaking this tolerance to oxidative stress may increase therapeutic efficacy in targeting melanoma. A number of studies have therefore suggested that treating melanoma by inhibiting cellular antioxi‐ dants may be efficacious [259-261]. One example of this was the addition of the superox‐ ide dismutase (SOD) activity inhibitor, 2-methoxyestradiol (2-ME2), to a mouse transplant model which induced growth arrest of melanoma cells after injection [262]. Paradoxical‐ ly, several studies have suggested that antioxidants can enhance the action of cancer che‐ motherapeutics drugs in their in vitro models through inhibition of a variety of factors which contribute to the malignant phenotype [263,264]. A recent study however, using six different combinations of antioxidants and chemotherapeutic drugs in combination, failed to identify a single combination in which an antioxidant reduced the survival of malignant breast carcinoma cells [265]. To our knowledge the use of PDT as an inhibitor of antioxidants has not been tested in cancer cells.

#### *1.4.5. Melanin and melanosomes as pro-survival agents*

*1.4.3. Induction of autophagy*

602 Melanoma - From Early Detection to Treatment

may lead to an initation of autophagy [253].

*1.4.4. Chemoresistance due to increased antioxidants*

of antioxidants has not been tested in cancer cells.

A recent finding is the induction of the cytoprotective programme of autophagy in mela‐ nomas in response to PDT-induced oxidative stress [89]. In addition, recent reports showed that cancer cells may respond to chemotherapeutics or other forms of oxidative stress such as PDT, through the induction of autophagy initially but continued stress leads to an overwhelming of the endogenous antioxidant enzymes along with a shift from autophagy to a possible senescent phenotype in an attempt to prolong cellular sur‐ vival. Consequently however, the cell enters an apoptotic or necrotic mode of cell death [249-251]. Autophagy, defined as a cellular response to nutrient deprivation with conse‐ quent organelle breakdown, could converge with PDT at a number of cellular locations. Although more work relating to this aspect in melanomas is needed, reports on other cancer cells have shown that autophagy can be induced if the lysosomal system, needed for the clearance of ROS-damaged organelles, is affected by PDT [252]. Another cellular location is the mitochondria, where the PDT-induced loss of anti-apoptotic protein Bcl-2,

Cancer cells are considered to be under continuous oxidative stress which has been sug‐ gested to aid in tumor progression [254]. In support, several studies have shown tumor cell lines producing higher levels of ROS compared to their normal counterparts [255,256]. Due to this increased level of ROS and hence constitutive increased level of ox‐ idative stress, it is not surprising that cancer cells have an extensive and advanced intra‐ cellular antioxidant network – a characteristic which further increases their chemoresistant property. Interestingly, the antioxidant status of melanomas differs from that of other skin cancers such as basal and squamous cell carcinomas in that their anti‐ oxidant activity levels (i.e. catalase, glutathione peroxidise, superoxide dismutase) are much higher [257]. In contrast, melanocytes, their normal untransformed phenotype, have lower levels of antioxidant activities and associated lower levels of resistance to oxi‐ dative stress [258]. It is therefore reasonable to postulate whether breaking this tolerance to oxidative stress may increase therapeutic efficacy in targeting melanoma. A number of studies have therefore suggested that treating melanoma by inhibiting cellular antioxi‐ dants may be efficacious [259-261]. One example of this was the addition of the superox‐ ide dismutase (SOD) activity inhibitor, 2-methoxyestradiol (2-ME2), to a mouse transplant model which induced growth arrest of melanoma cells after injection [262]. Paradoxical‐ ly, several studies have suggested that antioxidants can enhance the action of cancer che‐ motherapeutics drugs in their in vitro models through inhibition of a variety of factors which contribute to the malignant phenotype [263,264]. A recent study however, using six different combinations of antioxidants and chemotherapeutic drugs in combination, failed to identify a single combination in which an antioxidant reduced the survival of malignant breast carcinoma cells [265]. To our knowledge the use of PDT as an inhibitor All the potential intracellular organelle targets for PDT mentioned above are consistent with most cancer cells. However, the one aspect that sets melanoma apart from other cancers is the presence of its cell-specific organelle called the melanosome and its associated product, melanin pigment. It is thus not inconceivable to believe that the intractability of this skin disease may in some way be related to this organelle and its function [266,267]. It follows logically then, that treatment regimes need to consider the melanosome as another potential target organelle in the fight against melanoma [268].

Melanosomes are membrane-bound organelles in melanocytic cells which house the path‐ way that results in the formation of the polymeric pigment, melanin [269,270]. The enzymes which participate in this pathway are translated in the cytoplasm and chaperoned to the melanosomes. Tyrosinase (TYR), the rate-limiting enzyme of the pathway, and its related proteins tyrosinase-related proteins 1 and 2 (TYRP-1 and TYRP-2) act in concert to first con‐ vert tyrosine to 3,4-dihydroxy-phenylalanine (DOPA) via tyrosine hydroxylase activity and then convert DOPA to DOPAquinone via dopa oxidase activity. Both of these activities oc‐ cur via separate tyrosinase catalytic sites. During melanin synthesis toxic intermediates such as 5, 6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid are produced. Structural‐ ly, the melanosomes are designed to compartmentalise these cytotoxic melanin intermedi‐ ates from spilling into the cytoplasm [271]. Melanosomal biogenesis progresses through four distinct stages of maturation where the first two stages contain no melanin and the later stages constitute intermediates required to generate a matrix favourable for the formation of melanin [269,272]. The Pmel17/gp100/Silv/ME20 protein, a product of the Silver locus in melanocytic cells [273], is capable of polymerizing into fibrillar arrays that form the back‐ bone of melanosomes. As a major component of the fibrillar matrix of early stage melano‐ somes, Pmel-17 serves as the best marker to follow intracellular trafficking steps that regulate melanosomal formation [274].

Moreover, as Pmel-17 facilitates melanin deposition and plays a pivotal role in melanosome biogenesis, it remains a strategic target when trying to combat melanoma through the fact that melanosomes are involved in scavenging endogenous cytotoxic metabolites and storing their waste products - a function that has been suggested to be a key in creating multi-drug resistance [267]. With the premise that melanosomes may be acting as cytotoxic drug "sinks" through the sequestration of chemotherapeutic drugs [267], it would be considered an effective therapy for a PS to enter the melanosomal membrane and damage the wall of the melanosome thus allowing the leakage of toxic melanin intermediates resulting in cell death. The drawback is that melanosomes, which are classified into stages along their bio‐ genesis [269,270] only produce the toxic intermediates during their final maturing stages III and IV [268-270,275-277]. Most pigmented melanomas do however present with a majority of these end-stage melanosomes in their cytoplasm making the melanosomal membrane an attractive target for PDT. On the basis of this information, one may imagine that pigmented melanomas are therefore more susceptible to PDT-induced cell death. In contrast, our work has shown that pigmented melanomas are much less susceptible to hypericin-PDT than un‐ pigmented/amelanotic melanomas despite hypericin readily entering the melanosomes [89]. We hypothesize that the reason for this is due to the presence of the pigment melanin. In support of this, pigmented human xenograft melanotic melanoma in mice, was shown to be far less responsive to PDT than amelanotic melanoma [278].

ABCG2 transporter and thus create an intracellular pool of ROS resulting in efficacious cell

The Menace of Melanoma: A Photodynamic Approach to Adjunctive Cancer Therapy

http://dx.doi.org/10.5772/53676

605

Overall, the ability to halt melanoma cancer progression through targeting melanoma stem cells could be extremely advantageous. However, with such a large number of potential markers and their interaction with PDT unknown, it may be a better option to focus on ABC transporters and investigate their susceptibility to second generation PS-based PDT as a

There is no doubt that our understanding of the molecular and cellular basis of melanoma has grown substantially over the past decade. However, due to its multifunctional nature, the nee d for better, improved therapies to combat or target melanoma remain essential. In addition, better understanding of the heterogenous nature of this diverse disease will likely lead to re-evaluation of the basic concepts underlying melanoma therapeutics development and clinical trial design. Till then however, novel adjuvant treatment modalities such as PDT using photostable, second-generation photosensitizers such as hypericin remain an op‐ tion and need to be investigated further. Moreover, optimization of this type of therapy with regard to subcellular localization and its effect on cell death mechanisms within melanoma cells is needed. Targeting the integrity of melanocytes-specific organelles such as the mela‐ nosomes and producing an over-riding increase in ROS with consequent cytotoxicity re‐ mains a good therapeutic option but needs a systematic, scientific approach. Intriguingly, as more avenues of therapeutic targets such as melanoma stem cells and ABC transporters be‐ come illuminated, the ability to invoke cell death modalities in combination with PDT be‐ come more evident. Finally, it is clear that all these factors need to be considered in synergy

if progress is to be made toward combating the menace that is metastatic melanoma.

This work was supported by the University of Cape Town Research Committee, the Nation‐ al Research Foundation of South Africa (LMD) and Postgraduate funding (BK) from the

Redox Laboratory, Department of Human Biology; Faculty of Health Sciences; University of

death. This is definitely an avenue for exploration.

means to an end for melanoma progression.

**2. Conclusion and future directions**

**Acknowledgements**

NRF and DAAD.

**Author details**

L.M. Davids and B. Kleemann

Cape Town Medical School; Cape Town, South Africa

Melanin has been shown to act as both an oxidant and antioxidant [266,279] and in parallel studies, its presence in melanomas have been linked to chemoresistance. In support, further studies have shown that a lack of pigment in melanomas decreases their resistance to cell death. Our ongoing investigation into susceptibility to PDT-induced cell death in depig‐ mented melanomas supports this hypothesis [54,69].

#### *1.4.6. Cancer stem cells as future PDT targets*

The cancer stem cell hypothesis purports the idea that a subset of cancer cells is capable of maintaining and driving disease progression [280,281]. With the identification of cancer stem cell populations in colon, breast and brain tumors [282-285], it is believed that these cells are in‐ tegrally related to tumor formation, resistance to chemotherapy and escape from remission [286]. While the qualifications for melanoma stem cells have generally been defined as tumori‐ genicity in xenograft spheroid formation and self-renewal in non-adherent cultures, the mark‐ ers used to identify these cells from the general tumor population remain debatable. A brief summary of these markers and their potential as targets for novel PDT-based therapy, follow.

ATP-binding cassette (ABC) transporters are a vast family of transmembrane proteins that have been studied for their ability to actively transport cytotoxic substances out of cells [287]. Intriguingly, some of these transporters have been demonstrated to be highly expressed in highly tumorigenic subpopulations of melanoma suggesting that they may be markers of mel‐ anoma stem cells [286]. One of these includes the ABCB5 transporter. Known for increased ex‐ pression during melanoma progression in human tumor samples, ABCB5+ cells were able to resist treatment with doxorubicin [288]. While ABCB5+ cells were not able to renew in culture (a "stemness trait"), a subpopulation of cells that were indeed able to renew expressed the ABC transporter, Multi-drug Resistant-1 (MDR1) [289]. In vitro, MDR1+ cells exhibited less pigmen‐ tation than MDR1- cells, possessed the ability to continuously self-renew in soft agar and ex‐ pressed the pluripotency and self-renewal regulators, human telomerase reverse transcriptase (hTERT) – all characteristics pointing towards "stemness". Interestingly, while MDR+ cells did exhibit cancer stem-cell like properties in vitro, they also co-expressed ABCB5 and ABCC2 mRNAs suggesting that a number of ABC transporters may be expressed in sub-populations [289]. To further add to the complication of delineating melanoma stem cell markers as poten‐ tial targets for PDT is the fact that a number of recent markers are co-expressed with ABC trans‐ porters. These include CD133/prominin-1/AC133, which is co-expressed with ABCB5 and ABCG2 [288,290] and Nestin ([286].

Accumulating evidence suggests that another transporter, ABCG2, has physiological rele‐ vance in terms of photosensitivity and hence, PDT [291,292]. It has been shown that clinical photosensitizers and chemotherapeutic drugs have been transported out of cells by ABCG2 whereas this effect was abrogated by co-administration of its inhibitor, imatinib mesylate [293]. It is fascinating to speculate that a PDT protocol using a new, more stable photosensi‐ tizer such as hypericin may, through optimized concentrations, inhibit the action of the ABCG2 transporter and thus create an intracellular pool of ROS resulting in efficacious cell death. This is definitely an avenue for exploration.

Overall, the ability to halt melanoma cancer progression through targeting melanoma stem cells could be extremely advantageous. However, with such a large number of potential markers and their interaction with PDT unknown, it may be a better option to focus on ABC transporters and investigate their susceptibility to second generation PS-based PDT as a means to an end for melanoma progression.

## **2. Conclusion and future directions**

We hypothesize that the reason for this is due to the presence of the pigment melanin. In support of this, pigmented human xenograft melanotic melanoma in mice, was shown to be

Melanin has been shown to act as both an oxidant and antioxidant [266,279] and in parallel studies, its presence in melanomas have been linked to chemoresistance. In support, further studies have shown that a lack of pigment in melanomas decreases their resistance to cell death. Our ongoing investigation into susceptibility to PDT-induced cell death in depig‐

The cancer stem cell hypothesis purports the idea that a subset of cancer cells is capable of maintaining and driving disease progression [280,281]. With the identification of cancer stem cell populations in colon, breast and brain tumors [282-285], it is believed that these cells are in‐ tegrally related to tumor formation, resistance to chemotherapy and escape from remission [286]. While the qualifications for melanoma stem cells have generally been defined as tumori‐ genicity in xenograft spheroid formation and self-renewal in non-adherent cultures, the mark‐ ers used to identify these cells from the general tumor population remain debatable. A brief summary of these markers and their potential as targets for novel PDT-based therapy, follow.

ATP-binding cassette (ABC) transporters are a vast family of transmembrane proteins that have been studied for their ability to actively transport cytotoxic substances out of cells [287]. Intriguingly, some of these transporters have been demonstrated to be highly expressed in highly tumorigenic subpopulations of melanoma suggesting that they may be markers of mel‐ anoma stem cells [286]. One of these includes the ABCB5 transporter. Known for increased ex‐ pression during melanoma progression in human tumor samples, ABCB5+ cells were able to resist treatment with doxorubicin [288]. While ABCB5+ cells were not able to renew in culture (a "stemness trait"), a subpopulation of cells that were indeed able to renew expressed the ABC transporter, Multi-drug Resistant-1 (MDR1) [289]. In vitro, MDR1+ cells exhibited less pigmen‐ tation than MDR1- cells, possessed the ability to continuously self-renew in soft agar and ex‐ pressed the pluripotency and self-renewal regulators, human telomerase reverse transcriptase (hTERT) – all characteristics pointing towards "stemness". Interestingly, while MDR+ cells did exhibit cancer stem-cell like properties in vitro, they also co-expressed ABCB5 and ABCC2 mRNAs suggesting that a number of ABC transporters may be expressed in sub-populations [289]. To further add to the complication of delineating melanoma stem cell markers as poten‐ tial targets for PDT is the fact that a number of recent markers are co-expressed with ABC trans‐ porters. These include CD133/prominin-1/AC133, which is co-expressed with ABCB5 and

Accumulating evidence suggests that another transporter, ABCG2, has physiological rele‐ vance in terms of photosensitivity and hence, PDT [291,292]. It has been shown that clinical photosensitizers and chemotherapeutic drugs have been transported out of cells by ABCG2 whereas this effect was abrogated by co-administration of its inhibitor, imatinib mesylate [293]. It is fascinating to speculate that a PDT protocol using a new, more stable photosensi‐ tizer such as hypericin may, through optimized concentrations, inhibit the action of the

far less responsive to PDT than amelanotic melanoma [278].

mented melanomas supports this hypothesis [54,69].

*1.4.6. Cancer stem cells as future PDT targets*

604 Melanoma - From Early Detection to Treatment

ABCG2 [288,290] and Nestin ([286].

There is no doubt that our understanding of the molecular and cellular basis of melanoma has grown substantially over the past decade. However, due to its multifunctional nature, the nee d for better, improved therapies to combat or target melanoma remain essential. In addition, better understanding of the heterogenous nature of this diverse disease will likely lead to re-evaluation of the basic concepts underlying melanoma therapeutics development and clinical trial design. Till then however, novel adjuvant treatment modalities such as PDT using photostable, second-generation photosensitizers such as hypericin remain an op‐ tion and need to be investigated further. Moreover, optimization of this type of therapy with regard to subcellular localization and its effect on cell death mechanisms within melanoma cells is needed. Targeting the integrity of melanocytes-specific organelles such as the mela‐ nosomes and producing an over-riding increase in ROS with consequent cytotoxicity re‐ mains a good therapeutic option but needs a systematic, scientific approach. Intriguingly, as more avenues of therapeutic targets such as melanoma stem cells and ABC transporters be‐ come illuminated, the ability to invoke cell death modalities in combination with PDT be‐ come more evident. Finally, it is clear that all these factors need to be considered in synergy if progress is to be made toward combating the menace that is metastatic melanoma.

## **Acknowledgements**

This work was supported by the University of Cape Town Research Committee, the Nation‐ al Research Foundation of South Africa (LMD) and Postgraduate funding (BK) from the NRF and DAAD.

## **Author details**

L.M. Davids and B. Kleemann

Redox Laboratory, Department of Human Biology; Faculty of Health Sciences; University of Cape Town Medical School; Cape Town, South Africa

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1;100(7):3983-3988.

106-110.

56-60.


[277] Valencia JC, Hoashi T, Pawelek JM, Solano F, Hearing VJ. Pmel17: controversial in‐ deed but critical to melanocyte function. Pigment Cell Res 2006 Jun;19(3):250-2; au‐ thor reply 253-7.

[264] Tyagi AK, Singh RP, Agarwal C, Chan DC, Agarwal R. Silibinin strongly synergizes human prostate carcinoma DU145 cells to doxorubicin-induced growth Inhibition,

[265] De Larco JE, Park CA, Dronava H, Furcht LT. Paradoxical roles for antioxidants in

[266] Farmer PJ, Gidanian S, Shahandeh B, Di Bilio AJ, Tohidian N, Meyskens FL,Jr. Mela‐ nin as a target for melanoma chemotherapy: pro-oxidant effect of oxygen and metals

[267] Chen KG, Valencia JC, Lai B, Zhang G, Paterson JK, Rouzaud F, et al. Melanosomal sequestration of cytotoxic drugs contributes to the intractability of malignant mela‐

[268] Chen KG, Leapman RD, Zhang G, Lai B, Valencia JC, Cardarelli CO, et al. Influence of melanosome dynamics on melanoma drug sensitivity. J Natl Cancer Inst 2009 Sep

[269] Raposo G, Marks MS. The dark side of lysosome-related organelles: specialization of the endocytic pathway for melanosome biogenesis. Traffic 2002 Apr;3(4):237-248.

[270] Hearing VJ. Biogenesis of pigment granules: a sensitive way to regulate melanocyte

[271] Baldea I. Melanocyte pigmentation – friend or foe on the route to melanoma. In: Ta‐ naka Y, editor. Breakthroughs in Melanoma Research Rijeka, Croatia: InTech; 2011. p.

[272] Solano F, Martinez-Esparza M, Jimenez-Cervantes C, Hill SP, Lozano JA, Garcia-Bor‐ ron JC. New insights on the structure of the mouse silver locus and on the function of

[273] Kwon BS, Chintamaneni C, Kozak CA, Copeland NG, Gilbert DJ, Jenkins N, et al. A melanocyte-specific gene, Pmel 17, maps near the silver coat color locus on mouse chromosome 10 and is in a syntenic region on human chromosome 12. Proc Natl

[274] Theos AC, Truschel ST, Raposo G, Marks MS. The Silver locus product Pmel17/ gp100/Silv/ME20: controversial in name and in function. Pigment Cell Res 2005 Oct;

[275] Orlow SJ. Melanosomes are specialized members of the lysosomal lineage of organ‐

[276] Raposo G, Tenza D, Murphy DM, Berson JF, Marks MS. Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melano‐

the silver protein. Pigment Cell Res 2000;13 Suppl 8:118-124.

G2-M arrest, and apoptosis. Clin Cancer Res 2002 Nov;8(11):3512-3519.

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[291] Robey RW, Steadman K, Polgar O, Bates SE. ABCG2-mediated transport of photo‐ sensitizers: potential impact on photodynamic therapy. Cancer Biol Ther 2005 Feb; 4(2):187-194.

**Chapter 23**

**Study of the Anti-Photoaging Effect**

Hideaki Matsuda, Megumi Masuda, Kazuya Murata,

During the aging process, morphological changes in the human skin appear most noticeably in areas of frequent exposure to ultraviolet (UV) light from the sun, such as the face and hands. Chronic UV exposure induces photoaging, characterized by pigmented spots and wrinkles in the skin. Gradual destruction of the ozonosphere has raised photoaging risk. This has led to rapid growth of the anti-photoaging cosmetic market, especially among women with young and fair skin. Sunscreen agents are a first choice for protection against photoaging. However, a certain amount of UV irradiation penetrates skin dermis, and adverse effect may occur with use of these agents. Because of this, a current trend is the development of safer cosmetic ingre‐

Therefore, in this chapter we discuss searching for novel cosmetic ingredients from natu‐ ral resources which prevent the generation of pigmented spots and wrinkles *via* antago‐ nistic activities against UV signaling pathways. We focused this search on plants growing along the coasts of South Pacific islands, as they may have developed specific self-defense systems against harmful UV radiation. Following this strategy, we selected "noni" (*Morin‐*

Selected *M. citrifolia* as a subject is commonly called "noni", which is a subtropical plant dis‐ tributed widely in the tropical/ subtropical zone, including Tahiti and Hawaii. The tree is a

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

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

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

rapidly growing evergreen that is resistant to drought and poor soil conditions.

dients that effectively inhibit the UV signaling pathways leading to photoaging [1].

Additional information is available at the end of the chapter

**of Noni (***Morinda citrifolia***)**

Yumi Abe and Akemi Uwaya

http://dx.doi.org/10.5772/53621

**1. Introduction**

*da citrifolia* L., Rubiaceae).

**2. Subtropical plant noni**


**Chapter 23**

## **Study of the Anti-Photoaging Effect of Noni (***Morinda citrifolia***)**

Hideaki Matsuda, Megumi Masuda, Kazuya Murata, Yumi Abe and Akemi Uwaya

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53621

## **1. Introduction**

[291] Robey RW, Steadman K, Polgar O, Bates SE. ABCG2-mediated transport of photo‐ sensitizers: potential impact on photodynamic therapy. Cancer Biol Ther 2005 Feb;

[292] Tamura A, An R, Hagiya Y, Hoshijima K, Yoshida T, Mikuriya K, et al. Drug-induced phototoxicity evoked by inhibition of human ABC transporter ABCG2: development of in vitro high-speed screening systems. Expert Opin Drug Metab Toxicol 2008 Mar;

[293] Liu W, Baer MR, Bowman MJ, Pera P, Zheng X, Morgan J, et al. The tyrosine kinase inhibitor imatinib mesylate enhances the efficacy of photodynamic therapy by inhib‐

iting ABCG2. Clin Cancer Res 2007 Apr 15;13(8):2463-2470.

4(2):187-194.

628 Melanoma - From Early Detection to Treatment

4(3):255-272.

During the aging process, morphological changes in the human skin appear most noticeably in areas of frequent exposure to ultraviolet (UV) light from the sun, such as the face and hands. Chronic UV exposure induces photoaging, characterized by pigmented spots and wrinkles in the skin. Gradual destruction of the ozonosphere has raised photoaging risk. This has led to rapid growth of the anti-photoaging cosmetic market, especially among women with young and fair skin. Sunscreen agents are a first choice for protection against photoaging. However, a certain amount of UV irradiation penetrates skin dermis, and adverse effect may occur with use of these agents. Because of this, a current trend is the development of safer cosmetic ingre‐ dients that effectively inhibit the UV signaling pathways leading to photoaging [1].

Therefore, in this chapter we discuss searching for novel cosmetic ingredients from natu‐ ral resources which prevent the generation of pigmented spots and wrinkles *via* antago‐ nistic activities against UV signaling pathways. We focused this search on plants growing along the coasts of South Pacific islands, as they may have developed specific self-defense systems against harmful UV radiation. Following this strategy, we selected "noni" (*Morin‐ da citrifolia* L., Rubiaceae).

## **2. Subtropical plant noni**

Selected *M. citrifolia* as a subject is commonly called "noni", which is a subtropical plant dis‐ tributed widely in the tropical/ subtropical zone, including Tahiti and Hawaii. The tree is a rapidly growing evergreen that is resistant to drought and poor soil conditions.

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

Noni has been utilized for various reasons in many areas. The root was used as dye in Ja‐ pan, and the leaves and the seeds, as well as the fruit, were eaten frequently in Southeast Asia and the Pacific islands. Currently, the fruit juice and leaf tea are sold in the function‐ al foods market. Noni is also used as an herbal medicine to promote health and beauty. The whole plant, from the root to fruit, has been used without waste for more than 2000 years as a panacea [2]. Therefore, noni was considered a "gift from God" by generations of Pacific Islanders.

*3.1.2. DPPH radical scavenging activities*

tioxidant activity than Fruit-ext and Leaf-ext.

**Samples Concentration OD (×1000) a)**

Control 472±4

As oxidative reactions contribute to melanogenesis, the DPPH assay was performed to measure the antioxidant activity of noni. As shown in Table 2, Seed-ext exhibited potent DPPH radical scavenging activity, with an IC50 value of 12 μg/ml. Leaf-ext and Fruit-ext ex‐ hibited weaker antioxidant activities, with IC50 values of 113 and 240 μg/ml, respectively [8].

The results of the two assays reveal that Seed-ext has stronger tyrosinase inhibitory and an‐

Fruit-ext 20 (µg/ml) 471±3 0

Leaf-ext 20 (µg/ml) 471±2 0

Seed-ext 20 (µg/ml) 449±5i 5

Kojic acid 10 (µM) 207±3i 56

represents the mean±S.E. of 3 experiments. Significantly different from control group, i

**Samples Concentration OD (×1000) a)**

Control 974±21

**Table 1.** Tyrosinase Inhibitory Activities of Fruit-ext, Leaf-ext, Seed-ext and Kojic Acid (a) OD: optical density. Each value

**at 520 nm**

Fruit-ext 100 (µg/ml) 759±3i 22 240 (µg/ml) 200 (µg/ml) 536±5i 45 400 (µg/ml) 188±3i 81 Leaf-ext 50 (µg/ml) 765±4i 22 113 (µg/ml) 100 (µg/ml) 570±3i 42 200 (µg/ml) 301±6i 69 Seed-ext 5 (µg/ml) 771±2i 21 12 (µg/ml) 10 (µg/ml) 537±7i 45 20 (µg/ml) 121±5i 88 L-Ascorbic acid 20 (µM) 570±4i 41 23 (µM) 50 (µM) 79±2i 92

**Table 2.** DPPH Radical Scavenging Activities of Fruit-ext, Leaf-ext, Seed-ext and L-Ascorbic Acid (a) OD: optical density.

Each value represents the mean±S.E. of 3 experiments. Significantly different from control group, i

represents the concentration of sample required to scavenge 50% of DPPH free radical.)

**at 475 nm**

Study of the Anti-Photoaging Effect of Noni (*Morinda citrifolia*)

100 (µg/ml) 451±2 5 500 (µg/ml) 419±9i 11

100 (µg/ml) 460±2 3 500 (µg/ml) 442±1i 6

100 (µg/ml) 394±4i 17 500 (µg/ml) 365±4i 23

50 (µM) 77±3i 84

: *p*<0.01.)

**Inhibition (%)**

**Inhibition (%)**

http://dx.doi.org/10.5772/53621

631

**IC <sup>50</sup> value b)**

: *p*<0.01. b) IC50 value

In the last decade, many papers have reported the chemical constituents and biological ac‐ tivities of noni including hypotensive [3], hypoglycemic [4], and anticancer activities [5], which scientifically support traditional claims of noni. Noni has been used traditionally for the treatment of dermatoses such as ringworm, dry skin, acne, pustule, and other skin trou‐ bles. Moreover, ripe noni fruit juice has been drunk for cosmetic reasons. In some areas, the dried immature noni fruit, the leaves, or the seeds mixed with coconut oil have been used as an external treatment [6]. But studies reporting potential cosmetic uses of noni, such as in‐ hibition of melanogenesis and reduction of wrinkles, have only recently been completed. Noni fruit contains a large number of seeds throughout its flesh. But during the production of noni fruit juice, the seeds are removed and discarded. Considering the potential utility of all parts of the plant, we investigated extracts from the fruit flesh, leaves, and seeds for ac‐ tive anti-photoaging agents.

## **3. Skin whitening effect of noni**

#### **3.1. Screening tests for melanogenesis inhibitory effect of noni**

During photoaging, UV rays trigger melanogenesis and chromatosis. These processes gener‐ ate pigmented spots by UV activation of melanocyte tyrosinase (a melanin synthesis en‐ zyme) which then converts L-tyrosine to L-DOPA, followed by conversion to dopaquinone. Dopaquinone subsequently forms melanin through several steps, including auto-oxidation. As such, tyrosinase inhibitors may be useful for prevention of pigmented spots.

Initial evaluation of the anti-melanogenesis activity of noni was carried out using an *in vitro* tyrosinase inhibition assay with 50% ethanol extracts of fruit flesh (Fruit-ext), leaves (Leafext), and seeds (Seed-ext). As oxidative reactions also contribute to melanogenesis [7], the 1 diphenyl-2-picrylhydrazyl (DPPH) assay was also performed to find whether noni has antioxidant activity.

#### *3.1.1. Tyrosinase inhibitory activities*

The results of the *in vitro* tyrosinase inhibition assay are shown in Table 1. At 20 to 500 μg/ml, Seed-ext inhibited tyrosinase activity, in a concentration-dependent manner. Fruitext exhibited weak activity only at 500 μg/ml, and Leaf-ext did not inhibit enzyme activity at any concentration [8].

## *3.1.2. DPPH radical scavenging activities*

Noni has been utilized for various reasons in many areas. The root was used as dye in Ja‐ pan, and the leaves and the seeds, as well as the fruit, were eaten frequently in Southeast Asia and the Pacific islands. Currently, the fruit juice and leaf tea are sold in the function‐ al foods market. Noni is also used as an herbal medicine to promote health and beauty. The whole plant, from the root to fruit, has been used without waste for more than 2000 years as a panacea [2]. Therefore, noni was considered a "gift from God" by generations of

In the last decade, many papers have reported the chemical constituents and biological ac‐ tivities of noni including hypotensive [3], hypoglycemic [4], and anticancer activities [5], which scientifically support traditional claims of noni. Noni has been used traditionally for the treatment of dermatoses such as ringworm, dry skin, acne, pustule, and other skin trou‐ bles. Moreover, ripe noni fruit juice has been drunk for cosmetic reasons. In some areas, the dried immature noni fruit, the leaves, or the seeds mixed with coconut oil have been used as an external treatment [6]. But studies reporting potential cosmetic uses of noni, such as in‐ hibition of melanogenesis and reduction of wrinkles, have only recently been completed. Noni fruit contains a large number of seeds throughout its flesh. But during the production of noni fruit juice, the seeds are removed and discarded. Considering the potential utility of all parts of the plant, we investigated extracts from the fruit flesh, leaves, and seeds for ac‐

During photoaging, UV rays trigger melanogenesis and chromatosis. These processes gener‐ ate pigmented spots by UV activation of melanocyte tyrosinase (a melanin synthesis en‐ zyme) which then converts L-tyrosine to L-DOPA, followed by conversion to dopaquinone. Dopaquinone subsequently forms melanin through several steps, including auto-oxidation.

Initial evaluation of the anti-melanogenesis activity of noni was carried out using an *in vitro* tyrosinase inhibition assay with 50% ethanol extracts of fruit flesh (Fruit-ext), leaves (Leafext), and seeds (Seed-ext). As oxidative reactions also contribute to melanogenesis [7], the 1 diphenyl-2-picrylhydrazyl (DPPH) assay was also performed to find whether noni has

The results of the *in vitro* tyrosinase inhibition assay are shown in Table 1. At 20 to 500 μg/ml, Seed-ext inhibited tyrosinase activity, in a concentration-dependent manner. Fruitext exhibited weak activity only at 500 μg/ml, and Leaf-ext did not inhibit enzyme activity at

As such, tyrosinase inhibitors may be useful for prevention of pigmented spots.

Pacific Islanders.

630 Melanoma - From Early Detection to Treatment

tive anti-photoaging agents.

antioxidant activity.

any concentration [8].

*3.1.1. Tyrosinase inhibitory activities*

**3. Skin whitening effect of noni**

**3.1. Screening tests for melanogenesis inhibitory effect of noni**

As oxidative reactions contribute to melanogenesis, the DPPH assay was performed to measure the antioxidant activity of noni. As shown in Table 2, Seed-ext exhibited potent DPPH radical scavenging activity, with an IC50 value of 12 μg/ml. Leaf-ext and Fruit-ext ex‐ hibited weaker antioxidant activities, with IC50 values of 113 and 240 μg/ml, respectively [8].

The results of the two assays reveal that Seed-ext has stronger tyrosinase inhibitory and an‐ tioxidant activity than Fruit-ext and Leaf-ext.


**Table 1.** Tyrosinase Inhibitory Activities of Fruit-ext, Leaf-ext, Seed-ext and Kojic Acid (a) OD: optical density. Each value represents the mean±S.E. of 3 experiments. Significantly different from control group, i : *p*<0.01.)


**Table 2.** DPPH Radical Scavenging Activities of Fruit-ext, Leaf-ext, Seed-ext and L-Ascorbic Acid (a) OD: optical density. Each value represents the mean±S.E. of 3 experiments. Significantly different from control group, i : *p*<0.01. b) IC50 value represents the concentration of sample required to scavenge 50% of DPPH free radical.)

## **3.2. Inhibitory effect of noni seeds on melanogenesis and its active compounds**

According to the *in vitro* the screenings, Seed-ext may have melanogenesis inhibitory prop‐ erties. Further examination of Seed-ext involved the use of B16 murine melanoma cells as an *in vitro* melanogenesis test model. In this assay, cells were stimulated by *α*-melanocyte stim‐ ulating hormone (*α*-MSH) and incubated for 72 hrs with the vehicle or test material [9]. As shown in Table 3, vehicle control treated cells, stimulated with *α*–MSH, significantly pro‐ moted melanogenesis compared to control that was not stimulated with *α*-MSH. At concen‐ trations ranging from 12.5 to 200 μg/ml, Seed-ext inhibited *α*-MSH-stimulated melanogenesis in a concentration dependent manner without any significant effects on cell proliferation [10].

**Samples**

Run 1

Run 2

tyrosinase itself.

**inhibitory melanogenesis activity**

**Concentration (µM)**

**α-MSH (µM)**

Control 4.5±0.8 55.2±1.3 Vehicle control 1 37.5±1.5ii 100.0±1.1ii **1** 1.25 1 29.9±0.6i 97.8±0.4

Kojic acid 100 1 17.2±1.1i 97.1±1.3

Control 7.3±2.5 58.8±2.4 Vehicle control 1 39.0±0.9ii 100.0±5.1ii **2** 12.5 1 39.3±0.7 101.6±1.0

Kojic acid 100 1 15.7±1.0i 101.9±3.8

Compared to kojic acid, which is a common skin whitening ingredient, **1** exhibited more po‐ tent inhibition of melanogenesis. This suggested that the anti-melanogenesis effect of **1** may be due to the suppression of tyrosinase protein expression in the cells, rather than inhibiting

As the anti-melanogenesis of Seed-ext may be due to **1** and **2**, the inhibitory mechanism of

**Table 4.** Effects of 3,3'-Bisdemethylpinoresinol (1), Americanin A (2) and Kojic Acid on α-MSH-Stimulated Melanogenesis in B16 Melanoma Cells (Each value in melanin content represents the mean±S.E. of 3 experiments. Significantly different from the control group, ii: *p*<0.01. Significantly different from the vehicle control group, i

**3.3. Effects of noni compounds; 3,3'-bisdemethylpinoresinol and americanin A on**

these two lignans was studied with *α*-MSH stimulated B16 melanoma cells.

*p*<0.01. Each value in cell proliferation represents the mean±S.E. of 3 experiments.)

**Melanin content (µg/well)**

Study of the Anti-Photoaging Effect of Noni (*Morinda citrifolia*)

2.5 1 21.6±0.8i 101.0±1.7 1 18.5±1.2i 99.1±0.7 1 15.2±0.2i 88.8±1.2i 1 13.2±0.8i 88.5±0.3i

200 1 6.4±0.4i 98.2±1.4

 1 36.7±0.7 107.1±0.4 1 33.9±0.8 103.3±0.9 1 25.5±1.5i 99.4±4.5 1 13.9±1.0i 102.8±1.4

200 1 8.6±0.6i 98.4±2.7

**Cell proliferation (%)**

633

http://dx.doi.org/10.5772/53621

:

The tyrosinase inhibitory activity of Seed-ext was not as potent as that of other well known skin whitening agents. But in the B16 melanoma cells culture system, it inhibited melanin production. Thus, noni seed may be useful as an anti-photoaging cosmetic ingredient which prevents pigmented spots by interacting with a different active site than existing general skin whitening agents.


**Table 3.** Effects of Seed-ext and Kojic Acid on α-MSH-Stimulated Melanogenesis in B16 Melanoma Cells (Each value in melanin content represents the mean±S.E. of 3 experiments. Significantly different from the control group, ii: *p*<0.01. Significantly different from the vehicle control group, <sup>i</sup> : *p*<0.01. Each value in cell proliferation represents the mean ±S.E. of 3 experiments.)

As Seed-ext was confirmed to potently inhibit melanogenesis, activity guided isolation of the active compounds was carried out. Two lignans, 3,3'-bisdemethylpinoresinol (**1**) and americanin A (**2**), were isolated from noni seeds and found to be active constituents. As shown in Table 4, 10 and 20 μM of **1** displayed weak inhibition of cell proliferation. But 1.25 to 5 μM of **1** inhibited melanogenesis in a concentration dependent without any significant effects on cell proliferation. Also, 100 and 200 μM of **2** inhibited melanogenesis [10]. **1** (IC50 value: 0.3 mM) and **2** (IC50 value: 2.7 mM) exhibited tyrosinase inhibition, and **1** (IC50 value: 4 μM) and **2** (IC50 value: 11 μM) exhibited potent DPPH radical scavenging properties [8].


**3.2. Inhibitory effect of noni seeds on melanogenesis and its active compounds**

proliferation [10].

632 Melanoma - From Early Detection to Treatment

skin whitening agents.

**Samples Concentration <sup>α</sup>-MSH**

Significantly different from the vehicle control group, <sup>i</sup>

±S.E. of 3 experiments.)

According to the *in vitro* the screenings, Seed-ext may have melanogenesis inhibitory prop‐ erties. Further examination of Seed-ext involved the use of B16 murine melanoma cells as an *in vitro* melanogenesis test model. In this assay, cells were stimulated by *α*-melanocyte stim‐ ulating hormone (*α*-MSH) and incubated for 72 hrs with the vehicle or test material [9]. As shown in Table 3, vehicle control treated cells, stimulated with *α*–MSH, significantly pro‐ moted melanogenesis compared to control that was not stimulated with *α*-MSH. At concen‐ trations ranging from 12.5 to 200 μg/ml, Seed-ext inhibited *α*-MSH-stimulated melanogenesis in a concentration dependent manner without any significant effects on cell

The tyrosinase inhibitory activity of Seed-ext was not as potent as that of other well known skin whitening agents. But in the B16 melanoma cells culture system, it inhibited melanin production. Thus, noni seed may be useful as an anti-photoaging cosmetic ingredient which prevents pigmented spots by interacting with a different active site than existing general

**(µM)**

50 (µg/ml) 1 21.1±0.6i 105.9±1.2 200 (µg/ml) 1 11.9±0.6i 103.5±2.5

200 (µM) 1 6.9±0.4i 103.2±2.4

Control 1.6±0.4 61.9±1.2 Vehicle control 1 42.8±1.6ii 100.0±1.2ii Seed-ext 12.5 (µg/ml) 1 29.0±0.6i 100.6±1.1

Kojic acid 100 (µM) 1 13.5±0.4i 107.4±1.9

**Table 3.** Effects of Seed-ext and Kojic Acid on α-MSH-Stimulated Melanogenesis in B16 Melanoma Cells (Each value in melanin content represents the mean±S.E. of 3 experiments. Significantly different from the control group, ii

As Seed-ext was confirmed to potently inhibit melanogenesis, activity guided isolation of the active compounds was carried out. Two lignans, 3,3'-bisdemethylpinoresinol (**1**) and americanin A (**2**), were isolated from noni seeds and found to be active constituents. As shown in Table 4, 10 and 20 μM of **1** displayed weak inhibition of cell proliferation. But 1.25 to 5 μM of **1** inhibited melanogenesis in a concentration dependent without any significant effects on cell proliferation. Also, 100 and 200 μM of **2** inhibited melanogenesis [10]. **1** (IC50 value: 0.3 mM) and **2** (IC50 value: 2.7 mM) exhibited tyrosinase inhibition, and **1** (IC50 value: 4 μM) and **2** (IC50 value: 11 μM) exhibited potent DPPH radical scavenging properties [8].

**Melanin content (µg/well)**

: *p*<0.01. Each value in cell proliferation represents the mean

**Cell proliferation (%)**

: *p*<0.01.

**Table 4.** Effects of 3,3'-Bisdemethylpinoresinol (1), Americanin A (2) and Kojic Acid on α-MSH-Stimulated Melanogenesis in B16 Melanoma Cells (Each value in melanin content represents the mean±S.E. of 3 experiments. Significantly different from the control group, ii: *p*<0.01. Significantly different from the vehicle control group, i : *p*<0.01. Each value in cell proliferation represents the mean±S.E. of 3 experiments.)

Compared to kojic acid, which is a common skin whitening ingredient, **1** exhibited more po‐ tent inhibition of melanogenesis. This suggested that the anti-melanogenesis effect of **1** may be due to the suppression of tyrosinase protein expression in the cells, rather than inhibiting tyrosinase itself.

#### **3.3. Effects of noni compounds; 3,3'-bisdemethylpinoresinol and americanin A on inhibitory melanogenesis activity**

As the anti-melanogenesis of Seed-ext may be due to **1** and **2**, the inhibitory mechanism of these two lignans was studied with *α*-MSH stimulated B16 melanoma cells.

### *3.3.1. Effects of 3,3'-bisdemethylpinoresinol and americanin A on tyrosinase expression in B16 melanoma cells*

First, the effect of **1** and **2** on tyrosinase expression in *α*-MSH stimulated B16 melanoma cells was investigated by using Western blot analysis. As shown in Fig. 1, the tyrosinase expres‐ sion of the control at 72 hrs was enhanced remarkably. But after 72 hrs of treatment with **1** (5 μM) or **2** (200 μM), the enhancement of expression was notably suppressed without any sig‐ nificant effect on cell proliferation [10].


**Figure 1.** Effects of 3,3'-Bisdemethylpinoresinol (1), Americanin A (2) and Kojic Acid on Tyrosinase Expression in α-MSH-Stimulated B16 Melanoma Cells (The cells were treated with α-MSH (1 µM) in the presence of **1** (5 µM), **2** (200 µM) or kojic acid (200 µM) for 72 hrs. The level of tyrosinase expression was examined by Western blot analysis using specific antibody. Equal protein loading was confirmed by β-actin expression.)

*3.3.2. Inhibition of tyrosinase in B16 melanoma cells by 3,3'-bisdemethyl-pinoresinol and americanin A*

**Figure 2.** Effects of 3,3'-Bisdemethylpinoresinol (1) and Americanin A (2) on Melanogenesis and Tyrosinase Activity in α-MSH-Stimulated B16 Melanoma Cells (The cells were treated with α-MSH (1 µM, black column), **1** (5 µM, white col‐ umn) and **2** (200 µM, slashed column) for the indicated times. (A) The melanin content was determined. (B) Tyrosinase activity was determined by measuring the formation of dopachrome. Data represent mean±S.E. of two different ex‐

Study of the Anti-Photoaging Effect of Noni (*Morinda citrifolia*)

http://dx.doi.org/10.5772/53621

635

*3.3.3. Effect of 3,3'-bisdemethylpinoresinol and americanin A on phosphorylation of p38 MAPK*

During melanogenesis in melanocytes, it is known that microphthalmia-associated tran‐ scription (MITF) is a transcription factor that regulates expression of the tyrosinase gene, and that melanins are produced by the activation of MITF [11]. Since mitogen-activated pro‐ tein kinases (MAPKs) pathway is one of the intracellular signals that activates MITF, further research on this pathway was performed. It has been reported that phosphorylation of p38 MAPK activates MITF, whereas that of ERK1/2 and p70 S6K suppress MITF [12, 13]. The ef‐ fects of **1** and **2** on the MAPK signaling activities of *α*-MSH-stimulated B16 melanoma cells

periments each carried out in triplicate.)

were examined.

With suppressed expression of the enzyme, the activity of tyrosinase in the cell may de‐ crease. Secondly, *α*-MSH stimulated B16 melanoma cells were cultivated during treatment with **1** or **2**. Next, the amount of melanin and tyrosinase activity in the cells were measured.

As shown in Fig. 2A, the intracellular melanin content of control group increased remarka‐ bly after cultivation for 24 to 72 hrs, whereas the content in cells treated with **1** (5 μM) or **2** (200 μM) decreased after 72 hrs. As shown in Fig. 2B, the tyrosinase activity in the *α*-MSH stimulated cells was also significantly inhibited by addition of **1** (5 μM) or **2** (200 μM) after 24 to 72 hrs incubation [10].

As just described, lignans **1** (5 μM) and **2** (200 μM) inhibited intracellular melanin contents induced by *α*-MSH. The results of Western blot analysis demonstrated that **1** and **2** remarka‐ bly inhibit tyrosinase stimulated by *α*-MSH. Moreover, as **1** and **2** reduced intracellular tyro‐ sinase activity, it became clear that their melanogenesis inhibitory activity involved suppression of tyrosinase expression.

*3.3.1. Effects of 3,3'-bisdemethylpinoresinol and americanin A on tyrosinase expression in B16*

First, the effect of **1** and **2** on tyrosinase expression in *α*-MSH stimulated B16 melanoma cells was investigated by using Western blot analysis. As shown in Fig. 1, the tyrosinase expres‐ sion of the control at 72 hrs was enhanced remarkably. But after 72 hrs of treatment with **1** (5 μM) or **2** (200 μM), the enhancement of expression was notably suppressed without any sig‐

**Figure 1.** Effects of 3,3'-Bisdemethylpinoresinol (1), Americanin A (2) and Kojic Acid on Tyrosinase Expression in α-MSH-Stimulated B16 Melanoma Cells (The cells were treated with α-MSH (1 µM) in the presence of **1** (5 µM), **2** (200 µM) or kojic acid (200 µM) for 72 hrs. The level of tyrosinase expression was examined by Western blot analysis using

*3.3.2. Inhibition of tyrosinase in B16 melanoma cells by 3,3'-bisdemethyl-pinoresinol and americanin*

With suppressed expression of the enzyme, the activity of tyrosinase in the cell may de‐ crease. Secondly, *α*-MSH stimulated B16 melanoma cells were cultivated during treatment with **1** or **2**. Next, the amount of melanin and tyrosinase activity in the cells were measured.

As shown in Fig. 2A, the intracellular melanin content of control group increased remarka‐ bly after cultivation for 24 to 72 hrs, whereas the content in cells treated with **1** (5 μM) or **2** (200 μM) decreased after 72 hrs. As shown in Fig. 2B, the tyrosinase activity in the *α*-MSH stimulated cells was also significantly inhibited by addition of **1** (5 μM) or **2** (200 μM) after

As just described, lignans **1** (5 μM) and **2** (200 μM) inhibited intracellular melanin contents induced by *α*-MSH. The results of Western blot analysis demonstrated that **1** and **2** remarka‐ bly inhibit tyrosinase stimulated by *α*-MSH. Moreover, as **1** and **2** reduced intracellular tyro‐ sinase activity, it became clear that their melanogenesis inhibitory activity involved

specific antibody. Equal protein loading was confirmed by β-actin expression.)

*melanoma cells*

*A*

24 to 72 hrs incubation [10].

suppression of tyrosinase expression.

nificant effect on cell proliferation [10].

634 Melanoma - From Early Detection to Treatment

**Figure 2.** Effects of 3,3'-Bisdemethylpinoresinol (1) and Americanin A (2) on Melanogenesis and Tyrosinase Activity in α-MSH-Stimulated B16 Melanoma Cells (The cells were treated with α-MSH (1 µM, black column), **1** (5 µM, white col‐ umn) and **2** (200 µM, slashed column) for the indicated times. (A) The melanin content was determined. (B) Tyrosinase activity was determined by measuring the formation of dopachrome. Data represent mean±S.E. of two different ex‐ periments each carried out in triplicate.)

#### *3.3.3. Effect of 3,3'-bisdemethylpinoresinol and americanin A on phosphorylation of p38 MAPK*

During melanogenesis in melanocytes, it is known that microphthalmia-associated tran‐ scription (MITF) is a transcription factor that regulates expression of the tyrosinase gene, and that melanins are produced by the activation of MITF [11]. Since mitogen-activated pro‐ tein kinases (MAPKs) pathway is one of the intracellular signals that activates MITF, further research on this pathway was performed. It has been reported that phosphorylation of p38 MAPK activates MITF, whereas that of ERK1/2 and p70 S6K suppress MITF [12, 13]. The ef‐ fects of **1** and **2** on the MAPK signaling activities of *α*-MSH-stimulated B16 melanoma cells were examined.

The levels of phosphorylation of p38 MAPK were compared, at 6 and 12 hrs after stimula‐ tion of B16 melanoma cells by *α*-MSH (1 μM). As shown in Fig. 3A, the analysis at 6 and 12 hrs reveals that the levels of phosphorylation of p38 MAPK in B16 cells were enhanced by *α*-MSH treatment in comparison to those without *α*-MSH. The treatment with lignan **1** (5 μM) or lignan **2** (200 μM) suppressed phosphorylation of p38 MAPK and enhanced that of ERK1/2 at 6 and 12 hrs after stimulated by *α*-MSH (Fig. 3B). However, both lignans had no effect on p70 S6K phosophorylation (Fig. 3B) [10].

As the inhibitory mechanism of melanogenesis in α-MSH-stimulated B16 melanoma cells, These results strongly suggest that the lignans inhibit tyrosinase expression by suppressing p38 MAPK phosphorylation and enhancing ERK1/2, which then prevents activation of MITF.

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637

It is thought that wrinkles form mainly by aging, but they are also caused by photoaging. The causes are degradation of moisture-retaining property of epidermis and transformation of dermal configuration due to altered elastic or collagen fibers in the cutis. Anti-wrinkle cosmetics are useful for comparatively slight wrinkles and fine lines. The main functions of anti-wrinkle cosmetics included normalization of moisture retention by the cornified layer, acceleration of keratinocyte turnover, and acceleration of collagen and elastin synthesis through proliferation and stimulation of fibroblasts. Recently, retinol and its analogs, socalled retinoids, have received increased attention and utilization as a cosmetic ingredient because they promote fibroblastic collagen production in the cutis and improve skin tension. Also, vitamin A reportedly accelerates turnover, which reduces hyperpigmented skin spots caused by melanin excreted by the epidermis, even though it has no inhibitory effects on ac‐ tivation of tyrosinase and melanogenesis. But retinoids irritate the skin and have adverse ef‐

fects such as dermatitis. Therefore, safer reducing wrinkle ingredients are needed [1].

Thus, HLE inhibitors may be useful for the prevention of wrinkle formation.

*4.1.1. Inhibitory effect of noni on HLE and its active compounds*

and Leaf-ext had no inhibitory activity at 1.0 mg/ml [8].

more potent than the positive control (data not shown) [8].

kle formation during photoaging.

One cause of photoaging wrinkle formation is the degradation of collagen, a main component of corium connective tissue [14]. The degradation of collagen is promoted by release of human leukocyte elastase (HLE) from infiltrated neutrophils into the skin by UV irradiation [15]. HLE cleaves the triple helix structure of type I collagen and degrades elastic fiber in human skin [16].

The inhibitory effect of noni on wrinkle formation was investigated by measuring HLE in‐ hibiting activity *in vitro*. As shown in Table 5, concentrations raging from 0.5 to 1.0 mg/ml of Seed-ext displayed HLE inhibitory activity in a concentration-dependent manner. Fruit-ext

As Seed-ext exhibited potent HLE inhibitory activity compared with Fruit-ext and Leaf-ext, noni seeds appear to be the most source of cosmetic ingredients capable of preventing wrin‐

The active compounds in Seed-ext were isolated using the HLE inhibition bioassay as a frac‐ tionation guide. Ursolic acid (**3**) was isolated from Seed-ext and found to the active constitu‐ ent. The IC50 value of **3** in the HLE inhibition assay was 0.07 mM. The IC50 value of phenylmethanesulfonyl fluoride (PMSF), the positive control, was 0.14 mM. Thus, **3** was

**4. Inhibitory effect of noni on wrinkle formation**

**4.1. Wrinkle inhibition screening test of noni**

**Figure 3.** (A) Effect of α-MSH on Phosphorylation of p38 MAPK and ERK1/2 in B16 Melanoma Cells. (B) Effects of 3,3'-Bisde‐ methylpinoresinol (1) and Americanin A (2) on Phosphorylation of p38 MAPK, ERK1/2 and p70 S6K in α-MSH-Stimulated B16 Melanoma Cells ((A) The cells were treated with or without α-MSH (1 µM) for the indicated times. (B) The cells were treated with α-MSH (1 µM) in the presence of **1** (5 µM) or **2** (200 µM) for the indicated times. Phosphorylation of p38 MAPK, ERK1/2, and p70 S6K was assessed by Western blot analysis with using specific antibody for phosphorylated forms of p38 MAPK, ERK1/2, and p70 S6K. Equal protein loading was confirmed by β-actin expression.)

As the inhibitory mechanism of melanogenesis in α-MSH-stimulated B16 melanoma cells, These results strongly suggest that the lignans inhibit tyrosinase expression by suppressing p38 MAPK phosphorylation and enhancing ERK1/2, which then prevents activation of MITF.

## **4. Inhibitory effect of noni on wrinkle formation**

The levels of phosphorylation of p38 MAPK were compared, at 6 and 12 hrs after stimula‐ tion of B16 melanoma cells by *α*-MSH (1 μM). As shown in Fig. 3A, the analysis at 6 and 12 hrs reveals that the levels of phosphorylation of p38 MAPK in B16 cells were enhanced by *α*-MSH treatment in comparison to those without *α*-MSH. The treatment with lignan **1** (5 μM) or lignan **2** (200 μM) suppressed phosphorylation of p38 MAPK and enhanced that of ERK1/2 at 6 and 12 hrs after stimulated by *α*-MSH (Fig. 3B). However, both lignans had no

**Figure 3.** (A) Effect of α-MSH on Phosphorylation of p38 MAPK and ERK1/2 in B16 Melanoma Cells. (B) Effects of 3,3'-Bisde‐ methylpinoresinol (1) and Americanin A (2) on Phosphorylation of p38 MAPK, ERK1/2 and p70 S6K in α-MSH-Stimulated B16 Melanoma Cells ((A) The cells were treated with or without α-MSH (1 µM) for the indicated times. (B) The cells were treated with α-MSH (1 µM) in the presence of **1** (5 µM) or **2** (200 µM) for the indicated times. Phosphorylation of p38 MAPK, ERK1/2, and p70 S6K was assessed by Western blot analysis with using specific antibody for phosphorylated forms of p38

MAPK, ERK1/2, and p70 S6K. Equal protein loading was confirmed by β-actin expression.)

effect on p70 S6K phosophorylation (Fig. 3B) [10].

636 Melanoma - From Early Detection to Treatment

It is thought that wrinkles form mainly by aging, but they are also caused by photoaging. The causes are degradation of moisture-retaining property of epidermis and transformation of dermal configuration due to altered elastic or collagen fibers in the cutis. Anti-wrinkle cosmetics are useful for comparatively slight wrinkles and fine lines. The main functions of anti-wrinkle cosmetics included normalization of moisture retention by the cornified layer, acceleration of keratinocyte turnover, and acceleration of collagen and elastin synthesis through proliferation and stimulation of fibroblasts. Recently, retinol and its analogs, socalled retinoids, have received increased attention and utilization as a cosmetic ingredient because they promote fibroblastic collagen production in the cutis and improve skin tension. Also, vitamin A reportedly accelerates turnover, which reduces hyperpigmented skin spots caused by melanin excreted by the epidermis, even though it has no inhibitory effects on ac‐ tivation of tyrosinase and melanogenesis. But retinoids irritate the skin and have adverse ef‐ fects such as dermatitis. Therefore, safer reducing wrinkle ingredients are needed [1].

#### **4.1. Wrinkle inhibition screening test of noni**

One cause of photoaging wrinkle formation is the degradation of collagen, a main component of corium connective tissue [14]. The degradation of collagen is promoted by release of human leukocyte elastase (HLE) from infiltrated neutrophils into the skin by UV irradiation [15]. HLE cleaves the triple helix structure of type I collagen and degrades elastic fiber in human skin [16]. Thus, HLE inhibitors may be useful for the prevention of wrinkle formation.

## *4.1.1. Inhibitory effect of noni on HLE and its active compounds*

The inhibitory effect of noni on wrinkle formation was investigated by measuring HLE in‐ hibiting activity *in vitro*. As shown in Table 5, concentrations raging from 0.5 to 1.0 mg/ml of Seed-ext displayed HLE inhibitory activity in a concentration-dependent manner. Fruit-ext and Leaf-ext had no inhibitory activity at 1.0 mg/ml [8].

As Seed-ext exhibited potent HLE inhibitory activity compared with Fruit-ext and Leaf-ext, noni seeds appear to be the most source of cosmetic ingredients capable of preventing wrin‐ kle formation during photoaging.

The active compounds in Seed-ext were isolated using the HLE inhibition bioassay as a frac‐ tionation guide. Ursolic acid (**3**) was isolated from Seed-ext and found to the active constitu‐ ent. The IC50 value of **3** in the HLE inhibition assay was 0.07 mM. The IC50 value of phenylmethanesulfonyl fluoride (PMSF), the positive control, was 0.14 mM. Thus, **3** was more potent than the positive control (data not shown) [8].


Since Seed-ext displayed strong HLE inhibitory activity, its ability to inhibit MMP-1 secre‐ tion was investigated in UV-irradiated normal human dermal fibroblasts (NHDFs). The amount of MMP-1 protein secreted from NHDFs into culture media was analyzed by West‐

vehicle control group at 9 to 48 hrs incubation, as compared to the control group without UVA-irradiation (Fig. 4). The group which was treated with Seed-ext (10 μg/ml) after UVAirradiated NHDFs inhibited the secretion MMP-1 at 24 to 48 hrs, when compared to the ve‐

**Figure 4.** Effect of Seed-ext on MMP-1 Secretion from UVA-Irradiated NHDFs (Fibroblasts were exposed to UVA (5 J/cm2) and then cultured in the serum free medium containing test samples for 9, 24, and 48 hrs. MMP-1 protein levels in the cultured medium at the indicated times were assessed by Western blot analysis using the antibody against hu‐

The active compounds responsible for the inhibitory effect of Seed-ext on MMP-1 secretion were searched for by following bioassay guided fractionation. As shown in Fig. 5A and 5B, 3,3'-bisdemethylpinoresinol (**1**), which is an active anti-melanogenesis compound, signifi‐ cantly inhibited the secretion MMP-1 at 3 μM. On the other hand, ursolic acid (**3**), which is an active anti-HLE compound, had no effect on MMP-1 secretion (data not shown). There

It is clear that ursolic acid (**3**) inhibits HLE, whereas 3,3'-bisdemethylpinoresinol (**1**) inhibits MMP-1 secretion. Further, Seed-ext inhibits both MMP-1 secretion and HLE activity. There are very few plant extracts or compounds that have both inhibitory effects. Thus, noni seeds

In UV irradiated skin, MMP-1 is a major collagenolytic enzyme responsible for collagen damage [21]. It has been reported that UV irradiation promotes expression of MMP-1 in NHDFs [22] and secretion into culture media [23]. It is also known that UV irradiation acti‐ vates intracellular fibroblast signals, c-Jun-N-terminal kinase (JNK) and p38 mitogen-acti‐ vated protein kinase (p38 MAPK) cascades, promotes phosphorylation of JNK and p38, enhances expression of c-Jun and c-Fos, followed by activation of activation protein-1 (AP-1), and, in the end, enhances expression of MMP-1 [14, 24]. In order to find the mecha‐ nism of inhibition of MMP-1, compound **1** was investigated for effects on intracellular

man MMP-1. The blot is representative of three separate experiments and represents a single immunoblot.)

was no cytotoxicity from **1** and **3** at 0.03, 0.1 and 0.3 μM [20].

may be an ideal cosmetic ingredient to prevent wrinkle formation.

**4.3. Inhibitory effect of 3,3'-bisdemethylpinoresinol on MMP-1 secretion**

MMP-1 expression and activation of MAPKs in UVA-irradiated NHDFs.

hicle control (Fig. 4) [20]. Seed-ext was not cytotoxic at 3 to 30 μg/ml.

) enhanced the secretion of MMP-1 from NHDFs in the

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639

ern blotting. UVA irradiation (5 J/cm2

**Table 5.** HLE Inhibitory Activities of Fruit-ext, Leaf-ext, Seed-ext and PMSF (a) OD: optical density. b) IC50 value represents the concentration of sample required to inhibit 50% of HLE activity. c) Control A is a control for extracts. d) Control B is a control for PMSF. Each value represents the mean±S.E. of 3 experiments. Significantly different from control A group, i : *p*<0.01. Significantly different from control B group, ii: *p*<0.01.)

#### **4.2. Inhibitory effect of noni seeds on matrix metalloproteinase-1 (MMP-1) secretion and its active compounds**

Matrix metalloproteinases (MMPs) are matrix degrading enzymes associated with destruc‐ tive processes including inflammation, tumor invasion and skin aging [1]. More than 20 subtypes of MMPs have been reported [17]. Among these, MMP-1, secreted from human skin fibroblasts, is mainly responsible for the degradation of dermal type I collagen in the photoaging process [18]. Also, HLE activates MMP-1 [19]. Thus, it is expected that MMP-1 and HLE inhibitors may be useful for the prevention of photoaging and subsequent wrin‐ kle formation.

Since Seed-ext displayed strong HLE inhibitory activity, its ability to inhibit MMP-1 secre‐ tion was investigated in UV-irradiated normal human dermal fibroblasts (NHDFs). The amount of MMP-1 protein secreted from NHDFs into culture media was analyzed by West‐ ern blotting. UVA irradiation (5 J/cm2 ) enhanced the secretion of MMP-1 from NHDFs in the vehicle control group at 9 to 48 hrs incubation, as compared to the control group without UVA-irradiation (Fig. 4). The group which was treated with Seed-ext (10 μg/ml) after UVAirradiated NHDFs inhibited the secretion MMP-1 at 24 to 48 hrs, when compared to the ve‐ hicle control (Fig. 4) [20]. Seed-ext was not cytotoxic at 3 to 30 μg/ml.

**Samples**

control A group, i

kle formation.

**its active compounds**

**Concentration OD (×1000) a)**

Fruit-ext 0.1 (mg/ml) 994±38i -8

Leaf-ext 0.1 (mg/ml) 1014±16i -10

Control A c) 946±23

638 Melanoma - From Early Detection to Treatment

Control B d) 925±9

**at 405 nm**

0.5 (mg/ml) 1014±9i -10

1.0 (mg/ml) 1039±9i -12

0.5 (mg/ml) 1023±18i -11

1.0 (mg/ml) 1042±36i -13

Seed-ext 0.1 (mg/ml) 1052±20i -14 1.0 (mg/ml)

0.5 (mg/ml) 722±21i 22

1.0 (mg/ml) 467±30i 50

PMSF 0.08 (mM) 676±7ii <sup>29</sup> 0.14 (mM)

0.15 (mM) 383±14ii 60

0.50 (mM) 100±1ii 90

**4.2. Inhibitory effect of noni seeds on matrix metalloproteinase-1 (MMP-1) secretion and**

Matrix metalloproteinases (MMPs) are matrix degrading enzymes associated with destruc‐ tive processes including inflammation, tumor invasion and skin aging [1]. More than 20 subtypes of MMPs have been reported [17]. Among these, MMP-1, secreted from human skin fibroblasts, is mainly responsible for the degradation of dermal type I collagen in the photoaging process [18]. Also, HLE activates MMP-1 [19]. Thus, it is expected that MMP-1 and HLE inhibitors may be useful for the prevention of photoaging and subsequent wrin‐

: *p*<0.01.)

**Table 5.** HLE Inhibitory Activities of Fruit-ext, Leaf-ext, Seed-ext and PMSF (a) OD: optical density. b) IC50 value represents the concentration of sample required to inhibit 50% of HLE activity. c) Control A is a control for extracts. d) Control B is a control for PMSF. Each value represents the mean±S.E. of 3 experiments. Significantly different from

: *p*<0.01. Significantly different from control B group, ii

**Inhibition (%)**

**IC 50 value b)**

**Figure 4.** Effect of Seed-ext on MMP-1 Secretion from UVA-Irradiated NHDFs (Fibroblasts were exposed to UVA (5 J/cm2) and then cultured in the serum free medium containing test samples for 9, 24, and 48 hrs. MMP-1 protein levels in the cultured medium at the indicated times were assessed by Western blot analysis using the antibody against hu‐ man MMP-1. The blot is representative of three separate experiments and represents a single immunoblot.)

The active compounds responsible for the inhibitory effect of Seed-ext on MMP-1 secretion were searched for by following bioassay guided fractionation. As shown in Fig. 5A and 5B, 3,3'-bisdemethylpinoresinol (**1**), which is an active anti-melanogenesis compound, signifi‐ cantly inhibited the secretion MMP-1 at 3 μM. On the other hand, ursolic acid (**3**), which is an active anti-HLE compound, had no effect on MMP-1 secretion (data not shown). There was no cytotoxicity from **1** and **3** at 0.03, 0.1 and 0.3 μM [20].

It is clear that ursolic acid (**3**) inhibits HLE, whereas 3,3'-bisdemethylpinoresinol (**1**) inhibits MMP-1 secretion. Further, Seed-ext inhibits both MMP-1 secretion and HLE activity. There are very few plant extracts or compounds that have both inhibitory effects. Thus, noni seeds may be an ideal cosmetic ingredient to prevent wrinkle formation.

#### **4.3. Inhibitory effect of 3,3'-bisdemethylpinoresinol on MMP-1 secretion**

In UV irradiated skin, MMP-1 is a major collagenolytic enzyme responsible for collagen damage [21]. It has been reported that UV irradiation promotes expression of MMP-1 in NHDFs [22] and secretion into culture media [23]. It is also known that UV irradiation acti‐ vates intracellular fibroblast signals, c-Jun-N-terminal kinase (JNK) and p38 mitogen-acti‐ vated protein kinase (p38 MAPK) cascades, promotes phosphorylation of JNK and p38, enhances expression of c-Jun and c-Fos, followed by activation of activation protein-1 (AP-1), and, in the end, enhances expression of MMP-1 [14, 24]. In order to find the mecha‐ nism of inhibition of MMP-1, compound **1** was investigated for effects on intracellular MMP-1 expression and activation of MAPKs in UVA-irradiated NHDFs.

**Figure 5.** Effects of 3,3'-Bisdemethylpinoresinol (1) and and epigallocatechin-3-*O*-gallate (EGCG) on MMP-1 Secretion from UVA-Irradiated NHDFs (Fibroblasts were exposed to UVA (5 J/cm2) and then cultured in the serum free medium containing test samples for 48 hrs. MMP-1 protein levels in the cultured medium were assessed by Western blot analy‐ sis using human MMP-1 specific antibody. (A) The blot is representative of three separate experiments and represents a single immunoblot. (B) All data is reported as mean±S.E. of three separate experiments. Significantly different from the vehicle control group, \*: *p*<0.05, \*\*: *p*<0.01.)

**Figure 6.** (A) Effect of 3,3'-Bisdemethylpinoresinol (1) on the Intracellular MMP-1 Expression in UVA-Irradiated NHDFs (B) Effect of 3,3'-Bisdemethylpinoresinol (1) on MMP-1 Secretion from UVA-Irradiated NHDFs ((A) Fibroblasts were ex‐ posed to UVA (5 J/cm2) and then cultured in the serum free medium containing test samples for 4, 8, 24, and 48 hrs. Intracellular MMP-1 protein levels, at the indicated times, were assessed by Western blot analysis using human MMP-1 specific antibody. The blot is representative of two separate experiments and represents a single immunoblot. (B) Fi‐ broblasts were exposed to UVA (5 J/cm2) and then cultured in serum free medium containing test samples for 24 to 48 hrs. MMP-1 protein levels in cultured medium were assessed by Western blot analysis using human MMP-1 specific

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641

*4.3.2. Effect of 3,3'-bisdemethylpinoresinol on MAPKs phosphorylation in UVA-irradiated NHDFs*

The effect of **1** on activation of MAPKs was investigated by Western blot analysis. JNK and p38 MAPK phosophorylation in UVA irradiated NHDFs versus incubation time is shown in Fig. 7A. JNK phosophorylation was enhanced in a more rapid and transient manner at 0.5 to 1 hr af‐ ter UVA irradiation, whereas that of p38 MAPK was enhanced at 0 to 1 hr. Therefore, the effect of **1** (0.1 and 0.3 μM) on JNK and p38 MAPK phosophorylation in UVA-irradiated NHDFs was examined at 0.5 hr after irradiation. As shown in Fig. 7B, **1** (0.1 μM) had no inhibitory effect of

The results suggest that 1 inhibits MMP-1 secretion in UVA-irradiated NHDFs by decreas‐ ing JNK and p38 phosphorylation and suppressing c-Jun and c-Fos expression. This, subse‐ quently, inhibits AP-1 and MMP-1 expression, resulting in a reduction in MMP- secretion.

antibody. The blot is representative of two separate experiments and represents a single immunoblot.)

JNK and p38 MAPK, whereas **1** (0.3 μM) inhibited phosphorylation of both [20].

#### *4.3.1. Effect of 3,3'-bisdemethylpinoresinol (1) on intracellular MMP-1 expression in UVA irradiation NHDFs*

First, the effect of **1** on intracellular MMP-1 expression in UVA-irradiation was examined. In the vehicle control group, UVA irradiation enhanced the levels of intracellular MMP-1 ex‐ pression at 24 and 48 hrs, with most of the MMP-1 expression being detected at 48 hrs (Fig. 6). But in the treatment group (addition of **1**, 0.3 μM), the expression was down-regulated at 48 hrs (Fig. 6A). The levels of MMP-1 secretion from UVA-irradiated NHDF into the culture medium increased at 48 hrs in the vehicle control group, whereas the treatment group (addi‐ tion of **1**, 0.3 μM) obviously inhibited MMP-1 secretion (Fig. 6B) [20].

**Figure 6.** (A) Effect of 3,3'-Bisdemethylpinoresinol (1) on the Intracellular MMP-1 Expression in UVA-Irradiated NHDFs (B) Effect of 3,3'-Bisdemethylpinoresinol (1) on MMP-1 Secretion from UVA-Irradiated NHDFs ((A) Fibroblasts were ex‐ posed to UVA (5 J/cm2) and then cultured in the serum free medium containing test samples for 4, 8, 24, and 48 hrs. Intracellular MMP-1 protein levels, at the indicated times, were assessed by Western blot analysis using human MMP-1 specific antibody. The blot is representative of two separate experiments and represents a single immunoblot. (B) Fi‐ broblasts were exposed to UVA (5 J/cm2) and then cultured in serum free medium containing test samples for 24 to 48 hrs. MMP-1 protein levels in cultured medium were assessed by Western blot analysis using human MMP-1 specific antibody. The blot is representative of two separate experiments and represents a single immunoblot.)

#### *4.3.2. Effect of 3,3'-bisdemethylpinoresinol on MAPKs phosphorylation in UVA-irradiated NHDFs*

**Figure 5.** Effects of 3,3'-Bisdemethylpinoresinol (1) and and epigallocatechin-3-*O*-gallate (EGCG) on MMP-1 Secretion from UVA-Irradiated NHDFs (Fibroblasts were exposed to UVA (5 J/cm2) and then cultured in the serum free medium containing test samples for 48 hrs. MMP-1 protein levels in the cultured medium were assessed by Western blot analy‐ sis using human MMP-1 specific antibody. (A) The blot is representative of three separate experiments and represents a single immunoblot. (B) All data is reported as mean±S.E. of three separate experiments. Significantly different from

First, the effect of **1** on intracellular MMP-1 expression in UVA-irradiation was examined. In the vehicle control group, UVA irradiation enhanced the levels of intracellular MMP-1 ex‐ pression at 24 and 48 hrs, with most of the MMP-1 expression being detected at 48 hrs (Fig. 6). But in the treatment group (addition of **1**, 0.3 μM), the expression was down-regulated at 48 hrs (Fig. 6A). The levels of MMP-1 secretion from UVA-irradiated NHDF into the culture medium increased at 48 hrs in the vehicle control group, whereas the treatment group (addi‐

*4.3.1. Effect of 3,3'-bisdemethylpinoresinol (1) on intracellular MMP-1 expression in UVA*

tion of **1**, 0.3 μM) obviously inhibited MMP-1 secretion (Fig. 6B) [20].

the vehicle control group, \*: *p*<0.05, \*\*: *p*<0.01.)

640 Melanoma - From Early Detection to Treatment

*irradiation NHDFs*

The effect of **1** on activation of MAPKs was investigated by Western blot analysis. JNK and p38 MAPK phosophorylation in UVA irradiated NHDFs versus incubation time is shown in Fig. 7A. JNK phosophorylation was enhanced in a more rapid and transient manner at 0.5 to 1 hr af‐ ter UVA irradiation, whereas that of p38 MAPK was enhanced at 0 to 1 hr. Therefore, the effect of **1** (0.1 and 0.3 μM) on JNK and p38 MAPK phosophorylation in UVA-irradiated NHDFs was examined at 0.5 hr after irradiation. As shown in Fig. 7B, **1** (0.1 μM) had no inhibitory effect of JNK and p38 MAPK, whereas **1** (0.3 μM) inhibited phosphorylation of both [20].

The results suggest that 1 inhibits MMP-1 secretion in UVA-irradiated NHDFs by decreas‐ ing JNK and p38 phosphorylation and suppressing c-Jun and c-Fos expression. This, subse‐ quently, inhibits AP-1 and MMP-1 expression, resulting in a reduction in MMP- secretion.

group (LPS-induced) significantly prolonged blood passage time, in comparison with the

Platelet count, fibrinogen and fibrin degradation products (FDP) were also measured. Seed-

**Platelets (×104/μl)** **Fibrinogen (mg/dl)**

Study of the Anti-Photoaging Effect of Noni (*Morinda citrifolia*)

: *p*<0.01. a) For 7 successive days, 0.2% CMC-Na was

**FDP (µg/ml)** 643

http://dx.doi.org/10.5772/53621

**(s) c)**

Control *p.o.* 17±1 81.1±2.5 198.0±5.2 0.43±0.06

Vehicle control *p.o.* 1813±34ii 18.4±1.1ii 96.6±3.9ii 2.06±0.17ii

Seed-ext 200 (mg/kg) *p.o.* 1722±16iii 26.7±0.5i 96.7±2.9 1.86±0.11

Heparin 500 (U/kg) i.v. 26±1i 46.1±2.8i 194.1±7.2i 0.52±0.15iii

**Table 6.** Effects of Seed-ext and Heparin on Blood Passage Time, Platelet Count, Fibrinogen, and FDP in Rats, after LPS Injection (Each value represents the mean±S.E. (*n*=7). Significantly different from the control group, ii: *p*<0.01.

The results from the DIC rat model experiments suggest that Seed-ext improves poor blood flu‐ idity. Therefore, noni seeds may help reduce pigmentation associated with blood stagnation.

Blood fluidity, as measured by using MC-FAN, is influenced by erythroid deformability, leukocytic adherence ability and thrombocytic agglutinability [28, 29]. Blood flow regulating factors in microcirculation have vascular systemic and blood component functions. Circula‐ tory system function deteriorates with platelet aggregation induced coagulation, degrada‐ tion of erythrocyte deformability, hemagglutination, elevation of plasma viscosity, and by

In order to examine the inhibitory effect of Seed-ext on blood coagulation, collagen-induced platelet aggregation [30] and polybrene-induced erythrocyte aggregation [31], *in vitro* tests were carried out. Seed-ext did not inhibit platelet aggregation (data not shown). But it did inhibit hemagglutination at concentrations ranging from 50 to 500 μg/ml (Table 7). As

**5.2. Inhibitory effect of noni seeds, and active consituents, on platelet aggregation**

administered orally to control and vehicle control groups. Each extract (200 and 500 mg/kg) was suspended with CMC-Na and administrated orally to each test group once daily for 7 successive days (Day 1-7). 1 hr after the final daily dose on Day 7, LPS (1 mg/kg, dissolved in saline, i.v.) was injected into the tail vein. Heparin (500 U/kg, dissolved in saline, i.v.) was administered intravenously to the rats 1 hr before LPS injection on Day 7 to the heparin group. b) *p.o.*: oral administration, i.v.: intravenous administration c) Blood passage time measured by MC-FAN as the time taken for

the flow of 50 µl of sample mixture (1.8 ml of blood and 0.2 ml of 3.8% sodium citrate solution).

500 (mg/kg) *p.o.* 1413±19i 22.5±0.8 105.2±2.7 1.75±0.07

control group. However, Seed-ext dose-dependently reduced passage time [27].

ext had no effect on any of these hematological parameters (Table 6) [27].

**Samples Dose a) Route b) Blood passage time**

Significantly different from the vehicle control group, iii: *p*<0.05, i

degradation of fibrinolytic system activation [28, 29].

**Figure 7.** Time Course of MAPKs Phosphorylation in UVA-Irradiated NHDFs (B) Effect of 3,3'-Bisdemethylpinoresinol (1) on MAPKs Phosphorylation in UVA-Irradiated NHDFs ((A) Fibroblasts were exposed to UVA (5 J/cm2) and then cul‐ tured in the serum free medium for the indicated time. Total cellular proteins were prepared for Western blot analysis of MAPKs and phospho-MAPKs proteins using the antibodies against phospho-form and total-form of JNK and p38. βactin was used as an internal control. The blot is representative of three separate experiments and represents a single immunoblot. (B) Fibroblasts were exposed to UVA (5 J/cm2) and then cultured in the serum free medium for 0.5 hr. Total cellular proteins were prepared for Western blot analysis of MAPKs and phospho-MAPKs proteins using the anti‐ bodies against phospho-form and total-form of JNK and p38. β-actin was used for internal control. The blot is repre‐ sentative of three separate experiments and represents a single immunoblot.)

## **5. Inhibitory effect of noni seeds on poor blood fluidity**

In addition to melanogenesis and degradation of collagen by HLE and MMP-1, blood stag‐ nation may also contribute to hyperpigmented spots on the skin. In oriental medicine, the clinical state of blood stasis in the venous system is called "Oketsu" in Japanese. Accumula‐ tion of wastes caused by blood stagnation in the skin leads to pigmentation. Degradation of blood flow, leading to dry skin and atrophy, also leads to wrinkle formation. Therefore, im‐ proved blood fluidity is likely to prevent formation of pigmented spots and wrinkles. As cosmetic ingredients that improve blood fluidity are desired for use in anti-photoaging products, the effect of noni seeds on poor blood fluidity was investigated.

#### **5.1. Antithrombotic effect of noni seeds in a disseminated intravascular coagulation (DIC) rat model**

The effect of noni seeds on excessive increased blood coagulation, accompanying poor blood fluidity, was examined by using a DIC rat model, induced by lipopolysaccharide (LPS) [25, 26].

Each Seed-ext (200 and 500 mg/kg) was administrated orally once a day to the rats for 7 suc‐ cessive days. LPS was injected on Day 7. Four hrs after injection, blood samples were collect‐ ed from the abdominal great vein (vena cava). Whole blood passage time was measured by a micro channel array flow analyzer (MC-FAN). As shown in Table 6, the vehicle control group (LPS-induced) significantly prolonged blood passage time, in comparison with the control group. However, Seed-ext dose-dependently reduced passage time [27].

Platelet count, fibrinogen and fibrin degradation products (FDP) were also measured. Seedext had no effect on any of these hematological parameters (Table 6) [27].


**Figure 7.** Time Course of MAPKs Phosphorylation in UVA-Irradiated NHDFs (B) Effect of 3,3'-Bisdemethylpinoresinol (1) on MAPKs Phosphorylation in UVA-Irradiated NHDFs ((A) Fibroblasts were exposed to UVA (5 J/cm2) and then cul‐ tured in the serum free medium for the indicated time. Total cellular proteins were prepared for Western blot analysis of MAPKs and phospho-MAPKs proteins using the antibodies against phospho-form and total-form of JNK and p38. βactin was used as an internal control. The blot is representative of three separate experiments and represents a single immunoblot. (B) Fibroblasts were exposed to UVA (5 J/cm2) and then cultured in the serum free medium for 0.5 hr. Total cellular proteins were prepared for Western blot analysis of MAPKs and phospho-MAPKs proteins using the anti‐ bodies against phospho-form and total-form of JNK and p38. β-actin was used for internal control. The blot is repre‐

In addition to melanogenesis and degradation of collagen by HLE and MMP-1, blood stag‐ nation may also contribute to hyperpigmented spots on the skin. In oriental medicine, the clinical state of blood stasis in the venous system is called "Oketsu" in Japanese. Accumula‐ tion of wastes caused by blood stagnation in the skin leads to pigmentation. Degradation of blood flow, leading to dry skin and atrophy, also leads to wrinkle formation. Therefore, im‐ proved blood fluidity is likely to prevent formation of pigmented spots and wrinkles. As cosmetic ingredients that improve blood fluidity are desired for use in anti-photoaging

**5.1. Antithrombotic effect of noni seeds in a disseminated intravascular coagulation (DIC)**

The effect of noni seeds on excessive increased blood coagulation, accompanying poor blood fluidity, was examined by using a DIC rat model, induced by lipopolysaccharide (LPS) [25, 26].

Each Seed-ext (200 and 500 mg/kg) was administrated orally once a day to the rats for 7 suc‐ cessive days. LPS was injected on Day 7. Four hrs after injection, blood samples were collect‐ ed from the abdominal great vein (vena cava). Whole blood passage time was measured by a micro channel array flow analyzer (MC-FAN). As shown in Table 6, the vehicle control

sentative of three separate experiments and represents a single immunoblot.)

642 Melanoma - From Early Detection to Treatment

**5. Inhibitory effect of noni seeds on poor blood fluidity**

products, the effect of noni seeds on poor blood fluidity was investigated.

**rat model**

**Table 6.** Effects of Seed-ext and Heparin on Blood Passage Time, Platelet Count, Fibrinogen, and FDP in Rats, after LPS Injection (Each value represents the mean±S.E. (*n*=7). Significantly different from the control group, ii: *p*<0.01. Significantly different from the vehicle control group, iii: *p*<0.05, i : *p*<0.01. a) For 7 successive days, 0.2% CMC-Na was administered orally to control and vehicle control groups. Each extract (200 and 500 mg/kg) was suspended with CMC-Na and administrated orally to each test group once daily for 7 successive days (Day 1-7). 1 hr after the final daily dose on Day 7, LPS (1 mg/kg, dissolved in saline, i.v.) was injected into the tail vein. Heparin (500 U/kg, dissolved in saline, i.v.) was administered intravenously to the rats 1 hr before LPS injection on Day 7 to the heparin group. b) *p.o.*: oral administration, i.v.: intravenous administration c) Blood passage time measured by MC-FAN as the time taken for the flow of 50 µl of sample mixture (1.8 ml of blood and 0.2 ml of 3.8% sodium citrate solution).

The results from the DIC rat model experiments suggest that Seed-ext improves poor blood flu‐ idity. Therefore, noni seeds may help reduce pigmentation associated with blood stagnation.

## **5.2. Inhibitory effect of noni seeds, and active consituents, on platelet aggregation**

Blood fluidity, as measured by using MC-FAN, is influenced by erythroid deformability, leukocytic adherence ability and thrombocytic agglutinability [28, 29]. Blood flow regulating factors in microcirculation have vascular systemic and blood component functions. Circula‐ tory system function deteriorates with platelet aggregation induced coagulation, degrada‐ tion of erythrocyte deformability, hemagglutination, elevation of plasma viscosity, and by degradation of fibrinolytic system activation [28, 29].

In order to examine the inhibitory effect of Seed-ext on blood coagulation, collagen-induced platelet aggregation [30] and polybrene-induced erythrocyte aggregation [31], *in vitro* tests were carried out. Seed-ext did not inhibit platelet aggregation (data not shown). But it did inhibit hemagglutination at concentrations ranging from 50 to 500 μg/ml (Table 7). As shown in Table 7, **3** inhibited platelet aggregation at 10 to 50 μM. Lingnans **1** and **2** had weak effects of platelet aggregation, when comparison to **3** [27].

conducted. ELT is the time required for the disappearance of a fibrin clot produced by the addition of thrombin to the eugloblin fraction obtained from blood samples [32]. A reduc‐ tion in ELT reveals activation of fibrinolysis activity, whereas an extension in ELT implies

Seed-ext was administrated orally, and 1 hr later, blood samples were collected. Then ELT was measured using eugloblin fractions from the sample. As shown in Table 8, Seed-ext sig‐ nificantly reduced ELT at dosages from 50 and 200 mg/kg in dose-dependent manner. This

> **ELT (min)**

http://dx.doi.org/10.5772/53621

645

reveals that Seed-ext may have an enhancing effect on fibrinolysis activity [27].

Control A a) *p.o.* 98±2 Seed-ext 50 (mg/kg) *p.o.* 55±5i

Control B b) i.v. 97±2 Dextran sulphate sodium salt 5 (mg/kg) i.v. 32±3ii

**Table 8.** Effects of Seed-ext and Dextran Sulphate Sodium Salt on ELT in Rats (a) Control A is a control for extracts. b) Control B is a control for dextran sulphate sodium salt. Each value represents the mean±S.E. of 7 rats. Significantly

Seed-ext has an inhibitory effect on hemagglutination. But it also activates fibrinolysis, sug‐ gesting that it may improve blood flow through anti-coagulation and fibrinolysis systems. As such, noni seeds may be a useful supplementary ingredient for the prevention of both

We are the first to investigate and find 4 inhibitory effects—namely tyrosinase, melanogene‐ sis, HLE, and MMP-1—for noni seeds related to prevention of pigmented spots and wrin‐ kles by photoaging. As a desirable anti-photoaging agent that is antagonistic to the UV signaling pathways of photoaging, Seed-ext may be a useful novel cosmetic ingredient for the prevention or treatment for pigmented spots and wrinkles. Since we found Seed-ext may improve blood fluidity, it may also be a useful supplemental ingredient aimed for beauty.

Noni fruit flesh and leaves have been used as functional foods, but the seeds have been dis‐ carded without utilization in most cases. Production of a cosmetic ingredient from noni

: *p*<0.01. Significantly different from control B group, ii: *p*<0.01.)

200 (mg/kg) *p.o.* 42±7i

Study of the Anti-Photoaging Effect of Noni (*Morinda citrifolia*)

**Samples Dose Route**

pigmented spots and wrinkles caused by venous blood stagnation.

However, further research, including clinical trials, is needed.

seeds adds significant value to this largely unused natural resource.

reduced activity [32].

different from control A group, i

**6. Conclusion**

The results suggest that one inhibitory mechanisms behind the effect of Seed-ext on poor blood fluidity in the DIC rat model may be anti-hemaggulatination by 1, 2, and 3.


**Table 7.** Effects of Seed-ext, **1**, **2**, **3** and Neuraminidase on Polybrene-Induced Erythrocyte Aggregation (Each value represents the mean±S.E. of 3 experiments. Significantly different from control group, iii: *p*<0.05, i : *p*<0.01.)

#### **5.3. Fibrinolytic activity of noni seeds in rats**

Activation of the fibrinolytic system improves blood flow by promoting the lysis of thrombi in blood vessel walls. To better understand the fibrinolytic potential of Seed-ext, as it relates to degradation of blood fluidity, the euglobulin lysis time (ELT) assay in normal rats was conducted. ELT is the time required for the disappearance of a fibrin clot produced by the addition of thrombin to the eugloblin fraction obtained from blood samples [32]. A reduc‐ tion in ELT reveals activation of fibrinolysis activity, whereas an extension in ELT implies reduced activity [32].

Seed-ext was administrated orally, and 1 hr later, blood samples were collected. Then ELT was measured using eugloblin fractions from the sample. As shown in Table 8, Seed-ext sig‐ nificantly reduced ELT at dosages from 50 and 200 mg/kg in dose-dependent manner. This reveals that Seed-ext may have an enhancing effect on fibrinolysis activity [27].


**Table 8.** Effects of Seed-ext and Dextran Sulphate Sodium Salt on ELT in Rats (a) Control A is a control for extracts. b) Control B is a control for dextran sulphate sodium salt. Each value represents the mean±S.E. of 7 rats. Significantly different from control A group, i : *p*<0.01. Significantly different from control B group, ii: *p*<0.01.)

Seed-ext has an inhibitory effect on hemagglutination. But it also activates fibrinolysis, sug‐ gesting that it may improve blood flow through anti-coagulation and fibrinolysis systems. As such, noni seeds may be a useful supplementary ingredient for the prevention of both pigmented spots and wrinkles caused by venous blood stagnation.

## **6. Conclusion**

shown in Table 7, **3** inhibited platelet aggregation at 10 to 50 μM. Lingnans **1** and **2** had

The results suggest that one inhibitory mechanisms behind the effect of Seed-ext on poor

**(%)**

200 (µg/ml) 8±1i 82 500 (µg/ml) 9±1i 81

20 (µM) 39±1 13 50 (µM) 34±1i 24 100 (µM) 27±1i 41

20 (µM) 41±1 9 50 (µM) 38±1 16 100 (µM) 32±1i 29

10 (µM) 31±2i 31 20 (µM) 10±5i 78 50 (µM) 7±2i 84

15.6 (mU/ml) 33±3i 27 31.3 (mU/ml) 23±1i 48 62.5 (mU/ml) 17±1i 63 125 (mU/ml) 8±0i 82

**Inhibition (%)**

: *p*<0.01.)

blood fluidity in the DIC rat model may be anti-hemaggulatination by 1, 2, and 3.

Seed-ext 50 (µg/ml) 27±1i 40

**1** 10 (µM) 42±1 7

**2** 10 (µM) 42±2 7

**3** 5 (µM) 40±0 11

Neuraminidase 7.8 (mU/ml) 36±2 19

**Table 7.** Effects of Seed-ext, **1**, **2**, **3** and Neuraminidase on Polybrene-Induced Erythrocyte Aggregation (Each value

Activation of the fibrinolytic system improves blood flow by promoting the lysis of thrombi in blood vessel walls. To better understand the fibrinolytic potential of Seed-ext, as it relates to degradation of blood fluidity, the euglobulin lysis time (ELT) assay in normal rats was

represents the mean±S.E. of 3 experiments. Significantly different from control group, iii: *p*<0.05, i

**5.3. Fibrinolytic activity of noni seeds in rats**

weak effects of platelet aggregation, when comparison to **3** [27].

644 Melanoma - From Early Detection to Treatment

**Samples Concentration Aggregation**

Control 45±1

We are the first to investigate and find 4 inhibitory effects—namely tyrosinase, melanogene‐ sis, HLE, and MMP-1—for noni seeds related to prevention of pigmented spots and wrin‐ kles by photoaging. As a desirable anti-photoaging agent that is antagonistic to the UV signaling pathways of photoaging, Seed-ext may be a useful novel cosmetic ingredient for the prevention or treatment for pigmented spots and wrinkles. Since we found Seed-ext may improve blood fluidity, it may also be a useful supplemental ingredient aimed for beauty. However, further research, including clinical trials, is needed.

Noni fruit flesh and leaves have been used as functional foods, but the seeds have been dis‐ carded without utilization in most cases. Production of a cosmetic ingredient from noni seeds adds significant value to this largely unused natural resource.

## **Author details**

Hideaki Matsuda1 , Megumi Masuda1 , Kazuya Murata1 , Yumi Abe2 and Akemi Uwaya2 [12] Kim D.-S., Hwang E.-S., Lee J.-E., Kim S.-Y., Kwon S.-B., Park K.-C., Sphingosine-1 phosphate decreases melanin synthesis via sustained ERK activation and subsequent

Study of the Anti-Photoaging Effect of Noni (*Morinda citrifolia*)

http://dx.doi.org/10.5772/53621

647

[13] Smalley K., Eisen T., The involvement of p38 mitogen-activated protein kinase in the alpha-melanocyte stimulating hormone (alpha-MSH)-induced melanogenic and antiproliferative effects in B16 murine melanoma cells., FEBS Letters, 2000; 476(3),

[14] Rittié L., Fisher G. J., UV-light-induced signal cascades and skin aging., Ageing Re‐

[15] Rijken F., Kiekens R. C. M., Bruijnzeel P. L. B., Skin-infiltrating neutrophils following exposure to solar-simulated radiation could play an important role in photoageing of

[16] Kafienah W., Buttle D. J., Burnett D., Hollander A. P., Cleavage of native type I colla‐ gen by human neutrophil elastase., The Biochemical Journal, 1998; 330, 897-902. [17] Grant G. M., Giambernardi T. A., Grant A. M., Klebe R. J., Overview of expression of matrix metalloproteinases (MMP-17, MMP-18, and MMP-20) in cultured human

[18] Wang X.-Y., Bi Z.-G., UVB-irradiated human keratinocytes and interleukin-1alpha in‐ directly increase MAP kinase/AP-1 activation and MMP-1 production in UVA-irradi‐

[19] Takeuchi H., Gomi T., Shishido M., Watanabe H., Suenobu N., Neutrophil elastase contributes to extracellular matrix damage induced by chronic low-dose UV irradia‐ tion in a hairless mouse photoaging model., Journal of Dermatological Science, 2010;

[20] Masuda M., Murata K., Naruto S., Uwaya A., Isami F., Matsuda H., Matrix metallo‐ proteinase-1 inhibitory activities of *Morinda citrifolia* seed extract and its constituents in UVA-irradiated human dermal fibroblasts., Biological and Pharmaceutical Bulle‐

[21] Brennan M., Bhatti H., Nerusu K. C., Bhagavathula N., Kang S., Fisher G. J., Varani J., Voorhees J. J., Matrix metalloproteinase-1 is the major collagenolytic enzyme respon‐ sible for collagen damage in UV-irradiated human skin., Photochemistry and Photo‐

[22] Suganuma K., Nakajima H., Ohtsuki M., Imokawa G., Astaxanthin attenuates the UVA-induced up-regulation of matrix metalloproteinase-1 and skin fibroblast elas‐ tase in human dermal fibroblasts., Journal of Dermatological Science, 2010; 58(2),

[23] Oh H.-I., Shim J.-S., Gwon S.-H., Kwon H.-J., Hwang J.-K., The effect of xanthorrhizol on the expression of matrix metalloproteinase-1 and type-I procollagen in ultravioletirradiated human skin fibroblasts., Phytotherapy Research, 2009; 23(9), 1299-1302.

ated dermal fibroblasts., Chinese Medical Journal, 2006; 119(10), 827-831.

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1 Faculty of Pharmacy, Kinki University, Japan

2 Research & Development, Morinda Worldwide, Inc., Japan

## **References**


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**Author details**

646 Melanoma - From Early Detection to Treatment

Hideaki Matsuda1

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1 Faculty of Pharmacy, Kinki University, Japan

, Kazuya Murata1

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**Chapter 24**

**Inhibiting S100B in Malignant Melanoma**

The development of new therapies for patients diagnosed with malignant melanoma is in high need. In this chapter, the design and testing of inhibitors are discussed for S100B, a calciumbinding protein that down-regulates the tumor suppressor p53. Because p53 is wild type in many malignant melanoma patients, the restoration of p53 with S100B inhibitors (SBiXs) represents a new and potentially effective strategy for sensitizing melanoma cells to p53 dependent apoptosis pathways and for targeting this deadly cancer. Such a strategy requires blocking of the S100B-p53 protein-protein interaction (PPI) and involves methods including computer aided drug design (CADD), screening technologies, nuclear magnetic resonance (NMR), X-ray crystallography, and medicinal chemistry approaches. The ultimate goal is to

The S100 family of EF-hand calcium-binding proteins has more than 20 members, with the genes encoding these proteins present only in vertebrates [7]. S100 proteins (S100s) are expressed in both a cell type and tissue-specific manner to provide diverse functional roles including calcium homeostasis, cell-cell communication, cell proliferation, differentiation, cytoskeletal dynamics, and cell morphology [7-10]. On the other hand, dysregulation of S100 expression is observed in several types of cancers, including malignant melanoma [7-9]. They are also problematic when elevated in several cognitive disorders including those arising from traumatic brain injuries [12-16]. While S100 proteins themselves have no inherent enzymatic

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

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

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

design a highly specific and potent inhibitor of S100B that has clinical value.

Kira G. Hartman, Paul T. Wilder, Kristen Varney,

Danna Zimmer, Rena Lapidus and David J. Weber

Alexander D. Jr. MacKerell, Andrew Coop,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55176

**2. The S100 protein family**

**1. Introduction**


## **Inhibiting S100B in Malignant Melanoma**

Kira G. Hartman, Paul T. Wilder, Kristen Varney, Alexander D. Jr. MacKerell, Andrew Coop, Danna Zimmer, Rena Lapidus and David J. Weber

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55176

## **1. Introduction**

[24] Fisher G. J., Talwar H. S., Lin J., Lin P., McPhillips F., Wang Z., Li X., Wan Y., Kang S., Voorhees J. J., Retinoic acid inhibits induction of c-Jun protein by ultraviolet radia‐ tion that occurs subsequent to activation of mitogen-activated protein kinase path‐ ways in human skin *in vivo*., The Journal of Clinical Investigation, 1998; 101(6),

[25] Schoendorf T. H., Rosenberg M., Beller F. K., Endotoxin-induced disseminated intra‐ vascular coagulation in nonpregnant rats. A new experimental model., The American

[26] Chang G.-T., Min S.-Y., Kim J.-H., Kim S.-H., Kim J.-K., Kim C.-H., Anti-thrombic ac‐ tivity of Korean herbal medicine, Dae-Jo-Whan and its herbs., Vascular Pharmacolo‐

[27] Masuda M., Murata K., Itoh K., Naruto S., Uwaya A., Isami F., Matsuda H., Effects of *Morinda citrifolia* extract and its constituents on blood fluidity., Journal of Traditional

[28] Seki K., Sumino H., Murakami M., Study on blood rheology measured by MC-FAN.,

[29] Itoh K., Masuda M., Naruto S., Murata K., Matsuda H., Effects of unripe *Citrus hassa‐ ku* fruits extract and its flavanone glycosides on blood fluidity., Biological and Phar‐

[30] Born G. V. R., Cross M. J., The Aggregation of Blood Platelets., The Journal of Physi‐

[31] Sabio H., Levien M., Atkinson S., Continuous recording of red cell interactions.,

[32] Sakuragawa N., Euglobulin lysis time., Nihon Rinsho, 1997; 55, 139-144.

1432-1440.

648 Melanoma - From Early Detection to Treatment

Journal of Pathology, 1971; 65(1), 51-58.

gy, 2005; 43(4), 283-288.

Medicines, 2011, 28(2), 47-54.

ology, 1963; 168, 178-195.

Rinsho Byori, 2003; 51(8), 770-775.

maceutical Bulletin, 2010; 33(4), 659-664.

Thrombosis Research, 1983; 29(5), 537-540.

The development of new therapies for patients diagnosed with malignant melanoma is in high need. In this chapter, the design and testing of inhibitors are discussed for S100B, a calciumbinding protein that down-regulates the tumor suppressor p53. Because p53 is wild type in many malignant melanoma patients, the restoration of p53 with S100B inhibitors (SBiXs) represents a new and potentially effective strategy for sensitizing melanoma cells to p53 dependent apoptosis pathways and for targeting this deadly cancer. Such a strategy requires blocking of the S100B-p53 protein-protein interaction (PPI) and involves methods including computer aided drug design (CADD), screening technologies, nuclear magnetic resonance (NMR), X-ray crystallography, and medicinal chemistry approaches. The ultimate goal is to design a highly specific and potent inhibitor of S100B that has clinical value.

## **2. The S100 protein family**

The S100 family of EF-hand calcium-binding proteins has more than 20 members, with the genes encoding these proteins present only in vertebrates [7]. S100 proteins (S100s) are expressed in both a cell type and tissue-specific manner to provide diverse functional roles including calcium homeostasis, cell-cell communication, cell proliferation, differentiation, cytoskeletal dynamics, and cell morphology [7-10]. On the other hand, dysregulation of S100 expression is observed in several types of cancers, including malignant melanoma [7-9]. They are also problematic when elevated in several cognitive disorders including those arising from traumatic brain injuries [12-16]. While S100 proteins themselves have no inherent enzymatic

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

activity, they regulate important biological processes via specific Ca2+-dependent proteinprotein interactions [17,18].

**3. S100B structure & interactions with ions**

binding affinities [28,36].

brain (Table 1).

As with most S100 proteins, each 91 amino acid subunit of S100B has four alpha helices arranged into two helix-loop-helix (HLH) calcium-binding motifs connected by the flexible "hinge" region. Helix 1 and 2 make up the S100 EF-hand, while helix 3 and 4 form the canonical EF-hand (Figure 1). Each S100B subunit, therefore, binds two molecules of calcium, though with significantly different affinities [27]. While the canonical EF-hand binds Ca2+ with moderate affinity (*KD* = 20 – 50 μM), the weaker S100 EF-hand binds Ca2+ with a much lower affinity (*KD* = 200 – 500 μM) [27-30]. Calcium binding induces a dramatic conformational change in S100B where helix 3 rotates by 90º to become perpendicular to helix 4 (Figure 1). This change in conformation exposes a unique binding pocket, which in turn, binds to targets specific for each S100 protein generating a biological response [23]. However, it is clear that the dissocia‐ tion constants of calcium from most S100s *in vitro*, including S100B, are too weak to compete on their own for free Ca2+ typically present in the cytosol (100 to 500 nM). Interestingly, it is now understood that the affinity of S100s for Ca2+ is increased by as much as 300-fold when bound to biologically relevant target proteins (i.e. at 100 nM free Ca2+) suggesting that S100s typically only sequester free Ca2+ when their biologically relevant targets are present at optimal levels within the cell [31-34]. While the mechanism for this allosteric tightening of Ca2+ is not completely understood, it is known that when a target peptide derived from CapZ (termed TRTK-12) is bound to S100B, a loss in μs-ms motions occurs throughout the protein including in the side chain of a Ca2+-coordinating residues. These results were consistent with the hypotheses that stabilizing motions, particularly for Ca2+-coordinating residue(s) in EF2, could be responsible for the significant increase in Ca2+-ion binding affinity observed when a target is bound to Ca2+-S100B [35]. Likewise, the binding of S100B to Zn2+ (*KD* = 90 ± 20 nM) stabilizes residues in the C-terminus of the protein, resulting in an increase in both Ca2+- and target-

Inhibiting S100B in Malignant Melanoma http://dx.doi.org/10.5772/55176 651

Thus, as with many EF-hand proteins, S100 signaling proteins do not bind Ca2+ with high affinity unless they are bound to their biologically relevant protein target(s) [32-34,37,38]. In other words, in the absence of a bound target, the Ca2+-binding affinity for most S100 proteins is relatively low (i.e. in the μM range [1,17,27,39], but when bound to peptides (i.e. TRTK-12) or full-length targets, the Ca2+-binding affinity can be increased by 5- to 300-fold, respective‐ ly [32-34,37,38,40]. This property is physiologically necessary because while there are over 600 EF-hand Ca2+-binding domains within any given cell, Ca2+ homeostasis must be main‐ tained with sufficient free Ca2+ ion concentrations for proper signaling (i.e. 100 to 500 nM). Thus, as a physiological control mechanism, S100s and many other EF-hand proteins do not sequester significant amounts of free Ca2+ unless their functionally relevant molecular target is available [29,34,38]. It is especially important for drug design that we continue to investi‐ gate and understand this phenomenon at the molecular level because S100 inhibitor bind‐ ing must mimic the EF-hand-target complex and allosterically tighten Ca2+ ion binding affinity upon complex formation to be effective inside the cell [35,37]. For S100B, this includes targets such as p53, hdm2, hdm4, Rsk1 and RAGE, among others, which subsequently contributes to a Ca2+-mediated growth response in a cell-specific manner, including those in skin and

The first members of the S100 family were discovered in 1965 in a subcellular fraction from bovine brain tissue and were named based on their solubility in 100% saturated ammoni‐ um sulfate. When this protein fraction was examined in detail, two similar, but distinct proteins were discovered and designated S100α and S100β that are now referred to as S100A1 and S100B, respectively [17,19]. As with S100A1 and S100B, other S100s have a similar molecular weight (9-12 kDa), have homologous amino acid sequences (>40%), and typically exist as symmetric homodimers, or as heterodimers, held together by noncovalent interac‐ tions as pairs of four-helix bundles [20,21]. Two EF-hand helix-loop-helix calcium-binding structural motifs, first defined using the "E" and "F" helices from the X-ray crystal struc‐ ture of parvalbumin, are present in each S100 subunit [22]. The N-terminal "*S100"* or "*pseudo*" EF-hand (EF1) is comprised of 14 rather than the original 12 residues and this 14 amino acid sequence readily distinguishes S100s from other EF-hand calcium signaling proteins. The canonical EF-hand (EF2) is found at the C-terminus of each subunit and typically binds Ca2+ with a higher affinity than EF1. The two EF-hand domains are connected by a stretch of amino acid residues (<25 residues) termed the "hinge region". This "hinge" together with the Cterminal loop of the S100 protein contains the least amount of sequence homology and, therefore, represents the two regions that give each family member their individual targetbinding specificity [7,8,23]. In addition to binding Ca2+, several S100s bind Zn2+ at a sepa‐ rate site from the EF-hand calcium-binding domains. The Zn2+ site can also bind other metals (i.e. Mn2+, Cu2+, and others) and has two ligating residues contributed from each subunit at the dimer interface to provide tetrahedral coordination that is typical for Zn2+ [24]. Howev‐ er, for S100B, Zn2+-binding is not sufficient to induce target binding on its own, but rather functions by increasing the affinity S100B has for Ca2+ and its target proteins [25,26].

**Figure 1.** Structures of apo- versus holo-S100B. Ribbon diagrams comparing the NMR solution structures of calciumfree (left) and calcium-bound S100B (right), illustrating the 90° reorientation of helices 3 and 3' in each subunit of S100B. Shown in yellow on one subunit is the S100B target binding site that is exposed after the Ca2+-dependent con‐ formational change.

## **3. S100B structure & interactions with ions**

activity, they regulate important biological processes via specific Ca2+-dependent protein-

The first members of the S100 family were discovered in 1965 in a subcellular fraction from bovine brain tissue and were named based on their solubility in 100% saturated ammoni‐ um sulfate. When this protein fraction was examined in detail, two similar, but distinct proteins were discovered and designated S100α and S100β that are now referred to as S100A1 and S100B, respectively [17,19]. As with S100A1 and S100B, other S100s have a similar molecular weight (9-12 kDa), have homologous amino acid sequences (>40%), and typically exist as symmetric homodimers, or as heterodimers, held together by noncovalent interac‐ tions as pairs of four-helix bundles [20,21]. Two EF-hand helix-loop-helix calcium-binding structural motifs, first defined using the "E" and "F" helices from the X-ray crystal struc‐ ture of parvalbumin, are present in each S100 subunit [22]. The N-terminal "*S100"* or "*pseudo*" EF-hand (EF1) is comprised of 14 rather than the original 12 residues and this 14 amino acid sequence readily distinguishes S100s from other EF-hand calcium signaling proteins. The canonical EF-hand (EF2) is found at the C-terminus of each subunit and typically binds Ca2+ with a higher affinity than EF1. The two EF-hand domains are connected by a stretch of amino acid residues (<25 residues) termed the "hinge region". This "hinge" together with the Cterminal loop of the S100 protein contains the least amount of sequence homology and, therefore, represents the two regions that give each family member their individual targetbinding specificity [7,8,23]. In addition to binding Ca2+, several S100s bind Zn2+ at a sepa‐ rate site from the EF-hand calcium-binding domains. The Zn2+ site can also bind other metals (i.e. Mn2+, Cu2+, and others) and has two ligating residues contributed from each subunit at the dimer interface to provide tetrahedral coordination that is typical for Zn2+ [24]. Howev‐ er, for S100B, Zn2+-binding is not sufficient to induce target binding on its own, but rather

functions by increasing the affinity S100B has for Ca2+ and its target proteins [25,26].

**Figure 1.** Structures of apo- versus holo-S100B. Ribbon diagrams comparing the NMR solution structures of calciumfree (left) and calcium-bound S100B (right), illustrating the 90° reorientation of helices 3 and 3' in each subunit of S100B. Shown in yellow on one subunit is the S100B target binding site that is exposed after the Ca2+-dependent con‐

protein interactions [17,18].

650 Melanoma - From Early Detection to Treatment

formational change.

As with most S100 proteins, each 91 amino acid subunit of S100B has four alpha helices arranged into two helix-loop-helix (HLH) calcium-binding motifs connected by the flexible "hinge" region. Helix 1 and 2 make up the S100 EF-hand, while helix 3 and 4 form the canonical EF-hand (Figure 1). Each S100B subunit, therefore, binds two molecules of calcium, though with significantly different affinities [27]. While the canonical EF-hand binds Ca2+ with moderate affinity (*KD* = 20 – 50 μM), the weaker S100 EF-hand binds Ca2+ with a much lower affinity (*KD* = 200 – 500 μM) [27-30]. Calcium binding induces a dramatic conformational change in S100B where helix 3 rotates by 90º to become perpendicular to helix 4 (Figure 1). This change in conformation exposes a unique binding pocket, which in turn, binds to targets specific for each S100 protein generating a biological response [23]. However, it is clear that the dissocia‐ tion constants of calcium from most S100s *in vitro*, including S100B, are too weak to compete on their own for free Ca2+ typically present in the cytosol (100 to 500 nM). Interestingly, it is now understood that the affinity of S100s for Ca2+ is increased by as much as 300-fold when bound to biologically relevant target proteins (i.e. at 100 nM free Ca2+) suggesting that S100s typically only sequester free Ca2+ when their biologically relevant targets are present at optimal levels within the cell [31-34]. While the mechanism for this allosteric tightening of Ca2+ is not completely understood, it is known that when a target peptide derived from CapZ (termed TRTK-12) is bound to S100B, a loss in μs-ms motions occurs throughout the protein including in the side chain of a Ca2+-coordinating residues. These results were consistent with the hypotheses that stabilizing motions, particularly for Ca2+-coordinating residue(s) in EF2, could be responsible for the significant increase in Ca2+-ion binding affinity observed when a target is bound to Ca2+-S100B [35]. Likewise, the binding of S100B to Zn2+ (*KD* = 90 ± 20 nM) stabilizes residues in the C-terminus of the protein, resulting in an increase in both Ca2+- and targetbinding affinities [28,36].

Thus, as with many EF-hand proteins, S100 signaling proteins do not bind Ca2+ with high affinity unless they are bound to their biologically relevant protein target(s) [32-34,37,38]. In other words, in the absence of a bound target, the Ca2+-binding affinity for most S100 proteins is relatively low (i.e. in the μM range [1,17,27,39], but when bound to peptides (i.e. TRTK-12) or full-length targets, the Ca2+-binding affinity can be increased by 5- to 300-fold, respective‐ ly [32-34,37,38,40]. This property is physiologically necessary because while there are over 600 EF-hand Ca2+-binding domains within any given cell, Ca2+ homeostasis must be main‐ tained with sufficient free Ca2+ ion concentrations for proper signaling (i.e. 100 to 500 nM). Thus, as a physiological control mechanism, S100s and many other EF-hand proteins do not sequester significant amounts of free Ca2+ unless their functionally relevant molecular target is available [29,34,38]. It is especially important for drug design that we continue to investi‐ gate and understand this phenomenon at the molecular level because S100 inhibitor bind‐ ing must mimic the EF-hand-target complex and allosterically tighten Ca2+ ion binding affinity upon complex formation to be effective inside the cell [35,37]. For S100B, this includes targets such as p53, hdm2, hdm4, Rsk1 and RAGE, among others, which subsequently contributes to a Ca2+-mediated growth response in a cell-specific manner, including those in skin and brain (Table 1).

## **4. S100B pathology**

The protein S100B is found in melanocytes, glial cells, chondrocytes, and adipocytes, exhibiting both intra- and extracellular functionality. The cellular responses elicited by S100B can vary depending on several factors, including concentration (nM or μM), cell type, and cellular location [8,9]. Of particular concern is the role of elevated S100B in melanoma (Figure 2), the most deadly of all skin cancers, notorious for its resistence to chemotherapy and radiation. Clinical studies have established S100B as an effective biomarker for melanoma; however, this is only the case when highly specific S100B antibodies are used [12]. For example, in one study, samples from 412 melanoma patients at varying stages were com‐ pared to those diagnosed with non-melanoma skin cancers and inflammatory cutaneous diseases. Using a cutoff value of 0.2 μg/l serum S100B, a positive correlation was observed for patients having S100B levels above the cutoff level and advancement of tumor stage, indicative of a contribution by S100B to micro- and/or macro-metastases [41-43]. Though elevated S100B cannot be used to identify tumor thickness or lympth node status, it is predictive of poor patient prognosis, increased tumor recurrence, and low overall survival [9,41-44]. Subsequent studies reinforce these findings and consistently show elevated levels of S100B to be a sensitive and specific marker of melanoma progression with the ability to detect metastases or relapse at much earlier timepoints. S100B levels can also be used to monitor treatment strategies for rapid identification of whether a particular therapy is promising or for deciding to take an alternative approach [9]. While S100B is a useful prognostic indicator for melanoma, its use as a biomarker for several other cancers with elevated S100B is still under investigation; including colorectal cancer [45-47], several gliomas [48,49], mengiomas [50], non-small cell lung cancer (NSCLC) [51], renal cell carcinoma (RCC) [52], and thyroid carcinoma [53]. In addition, these clinical observations underscore the need to fully understand the role of elevated S100B in cancer, which is ongoing [2-4,54].

are associated with pathological states including Alzheimer's disease (AD), Down's syndrome (DS), and schizophrenia [55-59]. One mechanism for this pathology is that elevated intracel‐ lular levels of S100B present in glial cells are excreted and regulate neighboring neuronal cell activity. At low levels, the presence of this extracellular S100B is sufficient to promote neurite extension and growth, while elevated S100B levels are toxic and lead to neuronal cell apoptosis [9,60]. As with skin cancer, the clinical utility of S100B as a marker to identify and characterize neurological diseases and traumas is complicated by overlapping expression of S100B and other S100s in several cell types, its multiple mechanisms of secretion, and its association with more than one neurodegenerative disorder [14]. However, as found for melanoma, lowering S100B levels upon drug delivery is one means used to evaluate drug efficacy for treating schizophrenia [16,61]. Furthermore, the development of S100B inhibitors themselves may be useful for the treatment of these neuropathies, making the identification of such compounds important for advancing efforts towards understanding and treating cancers and cognitive

disorders in which S100B levels are at pathologically high levels [62].

NDR

Energy Metabolism Fructose 1,6 bisphosphate aldolase

Calponin CapZα GFAP IQGAP1 MARCKS\* Src kinase τ-protein\* Tubulin

Cell Cycle Regulation Hdm2, Hdm4

Cytoskeletal Regulation Caldesmon\*

\*PKC-mediated phosphorylation target proteins

**Table 1.** Targets of S100B

**5. S100B targets**

**Cellular Activity Protein References**

[63] [64,65]

Inhibiting S100B in Malignant Melanoma http://dx.doi.org/10.5772/55176 653

[66] [67] [68] [69] [70] [71] [26] [72] [73]

[74] [75]

Ca2+ Homeostasis AHNAK\* [26]

Phosphoglucomutase

Growth & Survival p53\* [1,28,76,77]

The ability of S100B to bind a diverse array of protein and enzyme targets is attributable to its broad consensus target-binding sequence [63]. S100B targets include proteins involved in calcium homeostasis, cell-cycle regulation, cytoskeletal regulation, energy metabolism, and

**Figure 2.** Staining for elevated S100B in a human malignant melanoma biopsy. Elevated S100B is stained brown in this human biopsy recorded for a patient before entering a S100B inhibitor clinical trial (SBi1). Patients are also tested for their p53 status and S100B:p53 ratio as recommended by Lin et al [2-4].

Although not considered in detail here, S100B also plays an important role in the brain, and as with cancer, several cognitive disorders show over-expression of S100B in brain tissue and are associated with pathological states including Alzheimer's disease (AD), Down's syndrome (DS), and schizophrenia [55-59]. One mechanism for this pathology is that elevated intracel‐ lular levels of S100B present in glial cells are excreted and regulate neighboring neuronal cell activity. At low levels, the presence of this extracellular S100B is sufficient to promote neurite extension and growth, while elevated S100B levels are toxic and lead to neuronal cell apoptosis [9,60]. As with skin cancer, the clinical utility of S100B as a marker to identify and characterize neurological diseases and traumas is complicated by overlapping expression of S100B and other S100s in several cell types, its multiple mechanisms of secretion, and its association with more than one neurodegenerative disorder [14]. However, as found for melanoma, lowering S100B levels upon drug delivery is one means used to evaluate drug efficacy for treating schizophrenia [16,61]. Furthermore, the development of S100B inhibitors themselves may be useful for the treatment of these neuropathies, making the identification of such compounds important for advancing efforts towards understanding and treating cancers and cognitive disorders in which S100B levels are at pathologically high levels [62].


**Table 1.** Targets of S100B

**4. S100B pathology**

652 Melanoma - From Early Detection to Treatment

The protein S100B is found in melanocytes, glial cells, chondrocytes, and adipocytes, exhibiting both intra- and extracellular functionality. The cellular responses elicited by S100B can vary depending on several factors, including concentration (nM or μM), cell type, and cellular location [8,9]. Of particular concern is the role of elevated S100B in melanoma (Figure 2), the most deadly of all skin cancers, notorious for its resistence to chemotherapy and radiation. Clinical studies have established S100B as an effective biomarker for melanoma; however, this is only the case when highly specific S100B antibodies are used [12]. For example, in one study, samples from 412 melanoma patients at varying stages were com‐ pared to those diagnosed with non-melanoma skin cancers and inflammatory cutaneous diseases. Using a cutoff value of 0.2 μg/l serum S100B, a positive correlation was observed for patients having S100B levels above the cutoff level and advancement of tumor stage, indicative of a contribution by S100B to micro- and/or macro-metastases [41-43]. Though elevated S100B cannot be used to identify tumor thickness or lympth node status, it is predictive of poor patient prognosis, increased tumor recurrence, and low overall survival [9,41-44]. Subsequent studies reinforce these findings and consistently show elevated levels of S100B to be a sensitive and specific marker of melanoma progression with the ability to detect metastases or relapse at much earlier timepoints. S100B levels can also be used to monitor treatment strategies for rapid identification of whether a particular therapy is promising or for deciding to take an alternative approach [9]. While S100B is a useful prognostic indicator for melanoma, its use as a biomarker for several other cancers with elevated S100B is still under investigation; including colorectal cancer [45-47], several gliomas [48,49], mengiomas [50], non-small cell lung cancer (NSCLC) [51], renal cell carcinoma (RCC) [52], and thyroid carcinoma [53]. In addition, these clinical observations underscore the need

to fully understand the role of elevated S100B in cancer, which is ongoing [2-4,54].

**Figure 2.** Staining for elevated S100B in a human malignant melanoma biopsy. Elevated S100B is stained brown in this human biopsy recorded for a patient before entering a S100B inhibitor clinical trial (SBi1). Patients are also tested

Although not considered in detail here, S100B also plays an important role in the brain, and as with cancer, several cognitive disorders show over-expression of S100B in brain tissue and

for their p53 status and S100B:p53 ratio as recommended by Lin et al [2-4].

#### **5. S100B targets**

The ability of S100B to bind a diverse array of protein and enzyme targets is attributable to its broad consensus target-binding sequence [63]. S100B targets include proteins involved in calcium homeostasis, cell-cycle regulation, cytoskeletal regulation, energy metabolism, and growth/survival (Table 1). One common theme among several S100B-target interactions is that they regulate protein phosphorylation [78]. For example, S100B associates with nuclear Dbf2 related (NDR) protein by binding a region distinct from the active site and inducing a confor‐ mational change, which stimulates autophosphorylation, and ultimately activates the protein [64]. S100B also regulates phosphorylation by binding to kinase substrates such as those of protein kinase C (PKC) and sterically blocking phosphorylation [76,77,79] (Table 1). This includes the myristoylated alanine-rich C-kinase substrate (MARCKS), τ-protein, and caldes‐ mon to name a few [66,80,81]. One noteable S100B target is the PKC substrate, p53, which is activated by phosphorylation in the C-terminal negative regulatory domain (NRD). In addition to blocking PKC-dependent phosphorylation, the S100B-p53 complex formation shifts the p53 tetramer to dimer to monomer equilibrium towards oligomer dissociation [76,78]. Thus, for p53, when S100B levels are too high, PKC-mediated activation of p53 is inhibited and p53 tetramers are dissociated. Consequently, p53 cannot bind DNA, which affects its transcrip‐ tional activity [2,28,76,77,82,83] and inhibits its ability to control cell cycle progression and apoptosis [2-4]. Other S100B targets include the E3 that designates p53 for ubiquitination, Hdm2, and the Hdm2 regulator, Hdm4 [63]. Thus, studies are underway to understand how S100B complexes involving Hdm2/Hdm4 contribute to lowering p53 levels in melanoma. Complicating this is the fact that both of these negative regulators of p53, Hdm2 and S100B, are themselves transcriptionally regulated by p53 [4,63]. This feedback regulatory mechanism results in time-dependent regulation of p53 that depends on having correct levels of all four proteins for proper regulation of cell cycle growth arrest and apoptosis [63]. Since elevated S100B disrupts the maintenance of p53 levels and promotes a cancerous phenotype, the development of small molecule inhibitors designed to target Ca2+-bound S100B has become a high priority. Specifically, investigations are focused on the identification of compounds capable of blocking the Ca2+-dependent S100B-p53 interaction in malignant melanoma (Figure 3). Ideally, administration of such compounds would reactivate p53 in malignant melanoma, as found for siRNAS100B, to induce normal apoptosis pathways and reduce proliferation/ survival of the cancer cells [2-4].

**6. Targeting the S100B-p53 interaction**

it for use in malignant melanoma a fairly quick transition.

Binding of S100B to p53 blocks PKC-dependent phosphorylation, p53 tetramerization, and p53-dependent transcription activation [28,63,76,82,83]. Therefore, efforts to restore wild-type p53 activities in malignant melanoma are underway as part of a drug design strategy [28]. A combination of approaches is being used, including those involving target validation and screening, computer aided drug design, structural biology, medicinal chemistry, and *in vivo* biology and drug testing methods (Figure 4). In one case, a previously FDA approved drug was discovered to block the S100B-p53 interaction. The wealth of available data associated with this compound, including its use in animals and human clinical trials made repurposing

Inhibiting S100B in Malignant Melanoma http://dx.doi.org/10.5772/55176 655

**Figure 4.** Summary of strategies for advancing hits from screening studies to leads, modified leads, and then ad‐ vanced lead compounds. This schematic is a general guideline for the early stages of drug development. The data from these approaches is compiled and used to choose "candidate" compounds that are studied more extensively in humans for their toxicology and effectiveness in treating cancer. It is oftern advantageous to consider repurposing

Screening for S100B inhibitors was initiated using computer-aided drug design methods (CADD) [28,84], and in all steps of identifying and prioritizing "hits" during these and other screens, the pharmacological activity of compounds was evaluated semi-quantitatively, providing an unbiased means of eliminating compounds that do not fulfill specific "drug-like" criteria [84,85]. Compounds identified in screens are also evaluated regarding their potential for absorption, distribution, and metabolism/excretion (ADME) properties [86]. Among many CADD approaches, a recent structure-based technique termed Site Identification by Ligand Competitive Saturation (SILCS) is now used extensively [87-89]. The simultaneous presence of benzene, propane and water in MD simulations of the target protein (ie. S100B) in this fragment-based computational approach identifies potential binding regions for aliphatic moieties, aromatic moieties and hydrogen bond donors and acceptors, while simultaneously allowing for increased flexibility and conformational changes to occur within the drug-binding

compounds that are already known to be safe in humans as a means to advance this process more quickly.

**Figure 3.** Illustration of the p53-binding site on S100B. Shown are ribbon diagrams Ca2+-bound S100B (NMR), p53367-388-bound Ca2+-bound S100B (NMR, PDB entry 1DT7). The helices of S100B are colored in blue, while the p53367-388 peptide is shown in red. Gray spheres represent the two calcium molecules per subunit. S100B inhibitors (SBiXs) are being developed to inhibit the Ca2+-dependent formation of the S100B-p53 complex.

## **6. Targeting the S100B-p53 interaction**

growth/survival (Table 1). One common theme among several S100B-target interactions is that they regulate protein phosphorylation [78]. For example, S100B associates with nuclear Dbf2 related (NDR) protein by binding a region distinct from the active site and inducing a confor‐ mational change, which stimulates autophosphorylation, and ultimately activates the protein [64]. S100B also regulates phosphorylation by binding to kinase substrates such as those of protein kinase C (PKC) and sterically blocking phosphorylation [76,77,79] (Table 1). This includes the myristoylated alanine-rich C-kinase substrate (MARCKS), τ-protein, and caldes‐ mon to name a few [66,80,81]. One noteable S100B target is the PKC substrate, p53, which is activated by phosphorylation in the C-terminal negative regulatory domain (NRD). In addition to blocking PKC-dependent phosphorylation, the S100B-p53 complex formation shifts the p53 tetramer to dimer to monomer equilibrium towards oligomer dissociation [76,78]. Thus, for p53, when S100B levels are too high, PKC-mediated activation of p53 is inhibited and p53 tetramers are dissociated. Consequently, p53 cannot bind DNA, which affects its transcrip‐ tional activity [2,28,76,77,82,83] and inhibits its ability to control cell cycle progression and apoptosis [2-4]. Other S100B targets include the E3 that designates p53 for ubiquitination, Hdm2, and the Hdm2 regulator, Hdm4 [63]. Thus, studies are underway to understand how S100B complexes involving Hdm2/Hdm4 contribute to lowering p53 levels in melanoma. Complicating this is the fact that both of these negative regulators of p53, Hdm2 and S100B, are themselves transcriptionally regulated by p53 [4,63]. This feedback regulatory mechanism results in time-dependent regulation of p53 that depends on having correct levels of all four proteins for proper regulation of cell cycle growth arrest and apoptosis [63]. Since elevated S100B disrupts the maintenance of p53 levels and promotes a cancerous phenotype, the development of small molecule inhibitors designed to target Ca2+-bound S100B has become a high priority. Specifically, investigations are focused on the identification of compounds capable of blocking the Ca2+-dependent S100B-p53 interaction in malignant melanoma (Figure 3). Ideally, administration of such compounds would reactivate p53 in malignant melanoma, as found for siRNAS100B, to induce normal apoptosis pathways and reduce proliferation/

**Figure 3.** Illustration of the p53-binding site on S100B. Shown are ribbon diagrams Ca2+-bound S100B (NMR), p53367-388-bound Ca2+-bound S100B (NMR, PDB entry 1DT7). The helices of S100B are colored in blue, while the p53367-388 peptide is shown in red. Gray spheres represent the two calcium molecules per subunit. S100B inhibitors

(SBiXs) are being developed to inhibit the Ca2+-dependent formation of the S100B-p53 complex.

survival of the cancer cells [2-4].

654 Melanoma - From Early Detection to Treatment

Binding of S100B to p53 blocks PKC-dependent phosphorylation, p53 tetramerization, and p53-dependent transcription activation [28,63,76,82,83]. Therefore, efforts to restore wild-type p53 activities in malignant melanoma are underway as part of a drug design strategy [28]. A combination of approaches is being used, including those involving target validation and screening, computer aided drug design, structural biology, medicinal chemistry, and *in vivo* biology and drug testing methods (Figure 4). In one case, a previously FDA approved drug was discovered to block the S100B-p53 interaction. The wealth of available data associated with this compound, including its use in animals and human clinical trials made repurposing it for use in malignant melanoma a fairly quick transition.

**Figure 4.** Summary of strategies for advancing hits from screening studies to leads, modified leads, and then ad‐ vanced lead compounds. This schematic is a general guideline for the early stages of drug development. The data from these approaches is compiled and used to choose "candidate" compounds that are studied more extensively in humans for their toxicology and effectiveness in treating cancer. It is oftern advantageous to consider repurposing compounds that are already known to be safe in humans as a means to advance this process more quickly.

Screening for S100B inhibitors was initiated using computer-aided drug design methods (CADD) [28,84], and in all steps of identifying and prioritizing "hits" during these and other screens, the pharmacological activity of compounds was evaluated semi-quantitatively, providing an unbiased means of eliminating compounds that do not fulfill specific "drug-like" criteria [84,85]. Compounds identified in screens are also evaluated regarding their potential for absorption, distribution, and metabolism/excretion (ADME) properties [86]. Among many CADD approaches, a recent structure-based technique termed Site Identification by Ligand Competitive Saturation (SILCS) is now used extensively [87-89]. The simultaneous presence of benzene, propane and water in MD simulations of the target protein (ie. S100B) in this fragment-based computational approach identifies potential binding regions for aliphatic moieties, aromatic moieties and hydrogen bond donors and acceptors, while simultaneously allowing for increased flexibility and conformational changes to occur within the drug-binding site [87-89]. In addition, SILCS is very useful for strategically modifying "hits" or "lead compounds" to span a larger area of the protein surface [87,88]. CADD methods such as these are particularly important for blocking protein-protein interactions (PPIs) such as that for the S100B-p53 complex since at least three distinct target binding pockets have been identified on S100B (Figure 5) [27,29,30,37,63,68,90,91]. As a result, the drug pentamidine diisethionate (Pnt), which is referred to as SBi1 (designated SBiX, where 'X' is an arbitrary compound number), was identified at a very early stage of the screening process as an effective inhibitor of the S100B-p53 complex [84]. Pnt was approved by the FDA as an antimicrobial agent for the treatment of Pneumocystis carinii pneumonia (PCP), which allowed for repurposing of this drug for *in vivo* testing for efficacy in treating malignant melanoma (Figure 4). To this end, a clinical trial is ongoing at the University of Maryland Medical Center (UMMC) to determine the efficacy of Pnt in melanoma patients *(0794GCC:* "*Treatment of melanoma with wild-type p53 and detectable S100B using pentamidine (SBi1): a Phase II trial with correlative biomarker end‐ points";* CA135624; PI: Dr. Ed Sausville, M.D.; Co-PI: Dr. David J. Weber). Although, there are promising results for the use of Pnt for the treatment of malignant melanoma, efforts have continued with the goal of engineering a compound with higher efficacy and more specificity for targeting S100B (versus other S100 proteins).

While it is important to show that an S100B-compound complex forms *in vitro*, it is also important to demonstrate that the SBiX has anti-cancer activity in cellular assays (i.e. growth inhibition, reduction in survival, increase apoptosis activity etc). In addition to showing compound efficacy in cells, these assays are important for providing information about other properties of the compound(s) including its membrane permeability and overall toxicity. One reliable method for identifying inhibitors of S100B-dependent pathways is via the creation of matched cell lines that only differ in S100B expression level. For example, MALME-3M melanoma cells were selected because they express elevated S100B protein levels and retain wild-type p53. Small interfering RNA (siRNA) was then stably transfected into the MALME-3M cells to give a scrambled siRNA control with high S100B levels and an isogenet‐ ically matched siRNAS100B construct resulting in low S100B (unpublished data). These matched cell lines provided a means for a large-scale screen of compound libraries to identify SBiXs with potency and specificity towards pathways involving S100B. The "hits" in this cellular assay were then routinely tested for direct binding and secondary cellular assays were completed by comparing the effect of these hits on primary malignant melanoma cells sideby-side with normal melanocytes [84]. Compounds that result in indiscriminate cell death were considered toxic rather than from specific inhibition of S100B or an S100B-dependent pathway and highlighted the importance of including normal melanocytes in every screen. Preferential growth inhibition of melanoma with high S100B as compared to little or no effect on the melanocytes or cells containing siRNAS100B was considered to be an early indication that the compound may have promising therapeutic value. This is exemplified with pentamidine, since treatment of C8146A primary melanoma cells resulted in significant cell growth inhibition, but

Inhibiting S100B in Malignant Melanoma http://dx.doi.org/10.5772/55176 657

little or no effect was noticed from this drug on normal melanocytes [84].

One of the most important requirements of any drug development program is to obtain physiological data at an early stage in the process to help determine whether a lead compound is effective and/or shows unanticipated toxicities *in vivo [93]*. This is particularly important for S100 inhibitors since there are over 20 structurally similar proteins in the S100 protein family, and they each regulate specific cellular pathway(s) [94,95]. To address these issues as quickly as possible, an *in vivo* screening assay was developed to test potential lead compounds for melanoma at an early stage in the drug development process. Once promising hits are designated as lead compounds via *in vitro* and cellular testing and secondary validation protocols, they need to be tested *in vivo*. Leads to be tested *in vivo* are chosen based on binding affinities (KDs) and/or specificity both in biochemical assays and in cellular assays. For the cellular assays, specificity and potential off-target effects are evaluated by comparing IC50 values of isogenic cell lines with or without S100B over-expression [96,97]. The vast majority of lead SBiXs are new chemical entities that require extensive optimization prior to entering *in vivo* testing, with the exception of those compounds that have been repurposed from other studies and trials. Animal models play a fundamental role in such *in vivo* testing. For example, a compound may need to be more lipophilic to pass through the cell membrane and reach its target or side groups may need to be added to allow for oral delivery or brain penetration. In the case of accessible tumors such as melanoma, drugs can be delivered directly to the tumor (intratumoral) without optimization for systemic delivery [93]. In addition, intratumoral delivery can achieve significantly higher drug concentrations at the site of action than can be

**Figure 5.** Three binding pockets are targeted on S100B. Surface representation of the structures of Ca2+-S100B bound to (a) the C-terminal negative regulatory domain of p53 (PDB ID: 1DT7)(1), (b) TRTK-12 (PDB ID: 1MWN) [5], and (c) pentamidine, also referred to as SBi1 (PDB ID: 3CR4) [6]. Sites 1, 2, and 3 are labeled. The protein is depicted as a blue surface and the regions within 3 Å of the bound peptide or small molecule are colored yellow. TRTK-12, p53 peptide, and pentamidine are shown in red.

In addition to CADD, biochemical and cellular screening methods are continuously ongoing to identify "hits" that can be considered further as scaffolds for drug development (Figure 4). One sensitive method done *in vitro* is a fluorescence polarization competition assay (FPCA), which can identify PPI inhibitors in a high-throughput manner. This competition assay takes advantage of a small molecule inhibitor causing the dissociation of a small peptide-bound fluorophore from a larger protein-peptide complex. Specifically, SBiX inhibitors can readily be identified since the smaller, faster rotating free fluorophore-bound peptide will exhibit lower polarization when competed away by the SBiX and compared to the larger S100Bpeptide complex. Importantly, the labeled-peptide must differ in molecular weight from the S100B-peptide complex to provide a reasonable dynamic range for the assay [92].

While it is important to show that an S100B-compound complex forms *in vitro*, it is also important to demonstrate that the SBiX has anti-cancer activity in cellular assays (i.e. growth inhibition, reduction in survival, increase apoptosis activity etc). In addition to showing compound efficacy in cells, these assays are important for providing information about other properties of the compound(s) including its membrane permeability and overall toxicity. One reliable method for identifying inhibitors of S100B-dependent pathways is via the creation of matched cell lines that only differ in S100B expression level. For example, MALME-3M melanoma cells were selected because they express elevated S100B protein levels and retain wild-type p53. Small interfering RNA (siRNA) was then stably transfected into the MALME-3M cells to give a scrambled siRNA control with high S100B levels and an isogenet‐ ically matched siRNAS100B construct resulting in low S100B (unpublished data). These matched cell lines provided a means for a large-scale screen of compound libraries to identify SBiXs with potency and specificity towards pathways involving S100B. The "hits" in this cellular assay were then routinely tested for direct binding and secondary cellular assays were completed by comparing the effect of these hits on primary malignant melanoma cells sideby-side with normal melanocytes [84]. Compounds that result in indiscriminate cell death were considered toxic rather than from specific inhibition of S100B or an S100B-dependent pathway and highlighted the importance of including normal melanocytes in every screen. Preferential growth inhibition of melanoma with high S100B as compared to little or no effect on the melanocytes or cells containing siRNAS100B was considered to be an early indication that the compound may have promising therapeutic value. This is exemplified with pentamidine, since treatment of C8146A primary melanoma cells resulted in significant cell growth inhibition, but little or no effect was noticed from this drug on normal melanocytes [84].

site [87-89]. In addition, SILCS is very useful for strategically modifying "hits" or "lead compounds" to span a larger area of the protein surface [87,88]. CADD methods such as these are particularly important for blocking protein-protein interactions (PPIs) such as that for the S100B-p53 complex since at least three distinct target binding pockets have been identified on S100B (Figure 5) [27,29,30,37,63,68,90,91]. As a result, the drug pentamidine diisethionate (Pnt), which is referred to as SBi1 (designated SBiX, where 'X' is an arbitrary compound number), was identified at a very early stage of the screening process as an effective inhibitor of the S100B-p53 complex [84]. Pnt was approved by the FDA as an antimicrobial agent for the treatment of Pneumocystis carinii pneumonia (PCP), which allowed for repurposing of this drug for *in vivo* testing for efficacy in treating malignant melanoma (Figure 4). To this end, a clinical trial is ongoing at the University of Maryland Medical Center (UMMC) to determine the efficacy of Pnt in melanoma patients *(0794GCC:* "*Treatment of melanoma with wild-type p53 and detectable S100B using pentamidine (SBi1): a Phase II trial with correlative biomarker end‐ points";* CA135624; PI: Dr. Ed Sausville, M.D.; Co-PI: Dr. David J. Weber). Although, there are promising results for the use of Pnt for the treatment of malignant melanoma, efforts have continued with the goal of engineering a compound with higher efficacy and more specificity

**Figure 5.** Three binding pockets are targeted on S100B. Surface representation of the structures of Ca2+-S100B bound to (a) the C-terminal negative regulatory domain of p53 (PDB ID: 1DT7)(1), (b) TRTK-12 (PDB ID: 1MWN) [5], and (c) pentamidine, also referred to as SBi1 (PDB ID: 3CR4) [6]. Sites 1, 2, and 3 are labeled. The protein is depicted as a blue surface and the regions within 3 Å of the bound peptide or small molecule are colored yellow. TRTK-12, p53 peptide,

In addition to CADD, biochemical and cellular screening methods are continuously ongoing to identify "hits" that can be considered further as scaffolds for drug development (Figure 4). One sensitive method done *in vitro* is a fluorescence polarization competition assay (FPCA), which can identify PPI inhibitors in a high-throughput manner. This competition assay takes advantage of a small molecule inhibitor causing the dissociation of a small peptide-bound fluorophore from a larger protein-peptide complex. Specifically, SBiX inhibitors can readily be identified since the smaller, faster rotating free fluorophore-bound peptide will exhibit lower polarization when competed away by the SBiX and compared to the larger S100Bpeptide complex. Importantly, the labeled-peptide must differ in molecular weight from the

S100B-peptide complex to provide a reasonable dynamic range for the assay [92].

for targeting S100B (versus other S100 proteins).

656 Melanoma - From Early Detection to Treatment

and pentamidine are shown in red.

One of the most important requirements of any drug development program is to obtain physiological data at an early stage in the process to help determine whether a lead compound is effective and/or shows unanticipated toxicities *in vivo [93]*. This is particularly important for S100 inhibitors since there are over 20 structurally similar proteins in the S100 protein family, and they each regulate specific cellular pathway(s) [94,95]. To address these issues as quickly as possible, an *in vivo* screening assay was developed to test potential lead compounds for melanoma at an early stage in the drug development process. Once promising hits are designated as lead compounds via *in vitro* and cellular testing and secondary validation protocols, they need to be tested *in vivo*. Leads to be tested *in vivo* are chosen based on binding affinities (KDs) and/or specificity both in biochemical assays and in cellular assays. For the cellular assays, specificity and potential off-target effects are evaluated by comparing IC50 values of isogenic cell lines with or without S100B over-expression [96,97]. The vast majority of lead SBiXs are new chemical entities that require extensive optimization prior to entering *in vivo* testing, with the exception of those compounds that have been repurposed from other studies and trials. Animal models play a fundamental role in such *in vivo* testing. For example, a compound may need to be more lipophilic to pass through the cell membrane and reach its target or side groups may need to be added to allow for oral delivery or brain penetration. In the case of accessible tumors such as melanoma, drugs can be delivered directly to the tumor (intratumoral) without optimization for systemic delivery [93]. In addition, intratumoral delivery can achieve significantly higher drug concentrations at the site of action than can be obtained via systemic delivery. For this purpose a multi-allelic genetically engineered mouse model was chosen to test SBiX compounds since this model mimics spontaneous tumoriogen‐ esis and heterogeneity as well as provide additional information necessary for additional target validation [98]. One such melanoma model is the RAS-induced INK4a/ARF-/- mouse [99], which was chosen for *in vivo* SBiX screening because it has: (i) an intact S100B-p53 signaling pathway (elevated S100B and wild type p53), (ii) an intact immune system, (iii) tumors which are amenable to intratumoral delivery, and (iv) a proven record in developing new melanoma therapies [99,100]. This screen utilizes 2-3 month old experimental Tyr::RASG12V/INK4a/ ARF-/- male mice that develop spontaneous cutaneous melanomas in the pinna of the ears (30%), torso (23%), and tail (20%) without distant metastasis [99] and uses tumor proliferation rate as the primary outcome. Although the screen is not optimized for obtaining tolerability, PK or PD information, the gross/histological pathology, SBiX levels and p53 pathway reacti‐ vation in the tumors are monitored as is necessary to select more advanced leads that have potential for proceeding to pre-clinical testing (Table 2).

**7. SBiX lead optimization**

SBiX leads are typically optimized using structure-based drug design and by examining structure/activity relationships (SAR) using traditional medicinal chemistry approaches. Modified leads are also tested using cellular and *in vivo* assays described above to determine whether the modification improved efficacy, specificity, and other criteria listed in Table 2. Although several leads were identified for S100B and are undergoing optimization via a structure-based drug design approach, diverse scaffolds remain essential at this stage of

Inhibiting S100B in Malignant Melanoma http://dx.doi.org/10.5772/55176 659

**Figure 6.** SAR by NMR [11]. NMR screens identify compounds that bind pockets "all over" the protein, which can be

The availability of 3D structures of S100B-SBiX complexes allow for CADD to be used to select compounds from 3D chemical databases with an enhanced potential for binding to S100B [101-105] and/or to engineer compounds de novo via *in silico* methods [106,107]. In an iterative process, new S100B-drug complexes are solved, lead modifications to improve affinity predicted via CADD, predicted compounds synthesized and the resulting compounds

linked synthetically to obtain high affinity and specific SBiX inhibitors.

development in case the existing lead compounds become intractable.

In the case of modified leads that have ADME properties favorable for systemic administra‐ tion, concurrent tolerability (MTD) and pharmacokinetic (PK) assays are also conducted. MTD and PK trials are also performed to determine if a lead is suitable for pre-clinical testing or if it requires additional medicinal chemistry optimization and/or further evaluation prior to preclinical testing. If the compound is found to be toxic, then it is eliminated from further consider‐ ation. Should successful tumor shrinkage be observed in mice treated with the well-tolerated S100B inhibitors, an effort is then put in place to consider phase 1 or 2b human clinical trials.


**Table 2.** Some criteria for leads, modified leads, and advanced leads

## **7. SBiX lead optimization**

obtained via systemic delivery. For this purpose a multi-allelic genetically engineered mouse model was chosen to test SBiX compounds since this model mimics spontaneous tumoriogen‐ esis and heterogeneity as well as provide additional information necessary for additional target validation [98]. One such melanoma model is the RAS-induced INK4a/ARF-/- mouse [99], which was chosen for *in vivo* SBiX screening because it has: (i) an intact S100B-p53 signaling pathway (elevated S100B and wild type p53), (ii) an intact immune system, (iii) tumors which are amenable to intratumoral delivery, and (iv) a proven record in developing new melanoma therapies [99,100]. This screen utilizes 2-3 month old experimental Tyr::RASG12V/INK4a/ ARF-/- male mice that develop spontaneous cutaneous melanomas in the pinna of the ears (30%), torso (23%), and tail (20%) without distant metastasis [99] and uses tumor proliferation rate as the primary outcome. Although the screen is not optimized for obtaining tolerability, PK or PD information, the gross/histological pathology, SBiX levels and p53 pathway reacti‐ vation in the tumors are monitored as is necessary to select more advanced leads that have

In the case of modified leads that have ADME properties favorable for systemic administra‐ tion, concurrent tolerability (MTD) and pharmacokinetic (PK) assays are also conducted. MTD and PK trials are also performed to determine if a lead is suitable for pre-clinical testing or if it requires additional medicinal chemistry optimization and/or further evaluation prior to preclinical testing. If the compound is found to be toxic, then it is eliminated from further consider‐ ation. Should successful tumor shrinkage be observed in mice treated with the well-tolerated S100B inhibitors, an effort is then put in place to consider phase 1 or 2b human clinical trials.

**Parameter Leads Modified Leads Advanced Leads**

KD <10 µM <50 nM <50 nM

IC50 in cells <10 µM <50 nM <50 nM

Off target effects KD≈IC<sup>50</sup> KD≈IC<sup>50</sup> KD≈IC<sup>50</sup>

Activity in target (-/-) cells <50% <20% <10%

P450 CYP induction Not determined <50% at 30 mM <50% at 30 mM

Bioavailability Not determined Preferred oral Preferred oral

BSA Ligand KD Not determined KD> 10 mM KD> 10 mM

Specificity >5:1 >50:1 >500:1

**Table 2.** Some criteria for leads, modified leads, and advanced leads

Metabolic stability Not determined >80% after 1 hour >80% after 1 hour

CYP2D6 Metabolism Not determined No No

potential for proceeding to pre-clinical testing (Table 2).

658 Melanoma - From Early Detection to Treatment

SBiX leads are typically optimized using structure-based drug design and by examining structure/activity relationships (SAR) using traditional medicinal chemistry approaches. Modified leads are also tested using cellular and *in vivo* assays described above to determine whether the modification improved efficacy, specificity, and other criteria listed in Table 2. Although several leads were identified for S100B and are undergoing optimization via a structure-based drug design approach, diverse scaffolds remain essential at this stage of development in case the existing lead compounds become intractable.

**Figure 6.** SAR by NMR [11]. NMR screens identify compounds that bind pockets "all over" the protein, which can be linked synthetically to obtain high affinity and specific SBiX inhibitors.

The availability of 3D structures of S100B-SBiX complexes allow for CADD to be used to select compounds from 3D chemical databases with an enhanced potential for binding to S100B [101-105] and/or to engineer compounds de novo via *in silico* methods [106,107]. In an iterative process, new S100B-drug complexes are solved, lead modifications to improve affinity predicted via CADD, predicted compounds synthesized and the resulting compounds experimentally evaluated. Work is also underway to develop novel inhibitors of S100B via a fragment-based approach that targets multiple binding sites on the protein identified by NMR, X-ray crystallography, and CADD techniques, including the new SILCS technique [87]. The SAR by NMR approach is now a standard method for quickly identifying 1 H-15N and/or 1 H-13C chemical shift perturbations in HSQC and/or TROSY spectra as a result of an S100B-small molecule interaction; this enables rapid, qualitative identification of binding site(s) on S100B for fragment-based design of new compounds that take advantage of the well-established "chelate effect" involved in linking fragments that bind neighboring sites [108,109] (Figure 6). For fast exchanging inhibitors, saturation transfer differences are collected to identify protons of the lead at the S100B-lead interface [84,110,111]. Thus, progress using a combination of functional-group optimization and the fragment-based approaches offers the potential for improvement in affinities/specificity, and for identifying novel leads. As new lead compounds are identified and structurally characterized (NMR, X-ray crystallography), SBiX affinity is considered as is scaffold diversity due to potential unforeseen problems with compounds that can occur during later-stage preclinical development (e.g. pharmacokinetic limitations, toxicity in humans). Therefore, as many as 3-6 chemical scaffolds are under consideration for development, and this number will be reduced as the project proceeds based on ADME properties, synthetic feasibility, and other pre-clinical/clinical information (Table 2). Such criteria include physiochemical properties related to bioavailability (molecular weight, clogP, TPSA, pKa, nitrogen atoms, carboxylates, H-bond donors, H-bond acceptors, rotatable bonds, H-bonds), and dose-limiting toxicities that may be predictive of the therapeutic index. Importantly, promising leads are routinely tested *in vivo* as early as possible to avoid wasted effort on toxic/ineffective compounds.

**Author details**

Kira G. Hartman1

Medicine, Baltimore, MD, USA

Baltimore, MD, USA

Baltimore, MD, USA

27487-27498

322, 1111-1122

417-429

MD, USA

**References**

, Paul T. Wilder1,3,4, Kristen Varney1,2,3, Alexander D. Jr. MacKerell2,3,4,

Inhibiting S100B in Malignant Melanoma http://dx.doi.org/10.5772/55176 661

1 Department of Biochemistry and Molecular Biology, University of Maryland School of

2 Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy,

3 Center for Biomolecular Therapeutics, University of Maryland School of Medicine,

4 University of Maryland Marlene and Stewart Greenebaum NCI Cancer Center, Baltimore,

[1] Rustandi, R. R., Baldisseri, D. M., and Weber, D. J. (2000) *Nat Struct Biol* 7, 570-574

[2] Lin, J., Blake, M., Tang, C., Zimmer, D., Rustandi, R. R., Weber, D. J., and Carrier, F.

[3] Lin, J., Yang, Q., Wilder, P. T., Carrier, F., and Weber, D. J. (2010) *J Biol Chem* 285,

[4] Lin, J., Yang, Q., Yan, Z., Markowitz, J., Wilder, P. T., Carrier, F., and Weber, D. J.

[5] Rustandi, R. R., Baldisseri, D. M., Inman, K. G., Nizner, P., Hamilton, S. M., Landar,

[6] Charpentier, T. H., Wilder, P. T., Liriano, M. A., Varney, K. M., Pozharski, E., MacK‐ erell, A. D., Jr., Coop, A., Toth, E. A., and Weber, D. J. (2008) *J Mol Biol* 382, 56-73

[7] Marenholz, I., Heizmann, C. W., and Fritz, G. (2004) *Biochem Biophys Res Commun*

[10] Zimmer, D. B., Cornwall, E. H., Landar, A., and Song, W. (1995) *Brain Res Bull* 37,

A., Zimmer, D. B., and Weber, D. J. (2002) *Biochemistry* 41, 788-796

Andrew Coop2,4, Danna Zimmer1,3, Rena Lapidus3,4 and David J. Weber1,3,4\*

\*Address all correspondence to: dweber@som.umaryland.edu

(2001) *J Biol Chem* 276, 35037-35041

(2004) *J Biol Chem* 279, 34071-34077

[8] Donato, R. (2001) *Int J Biochem Cell Biol* 33, 637-668

[9] Harpio, R., and Einarsson, R. (2004) *Clin Biochem* 37, 512-518

## **8. Summary**

Ongoing collaborative efforts involving biology, structure determination, CADD and synthetic chemistry have lead to the development of a collection of inhibitors of S100B. These efforts include identification of the FDA approved compound pentamidine, which is currently being evaluated in human clinical trials. In addition, the work has identified several novel chemical scaffolds that are undergoing optimization and have laid the foundation for the application of fragment-based approaches to design additional novel scaffolds. Notably, while the goal of this research is to develop a potent inhibitor of S100B for the treatment of malignant melanoma, we anticipate that the knowledge gained to date will be of utility in designing specific inhibitors of other members of the S100 protein family for the treatment of a range of S100 associated disease states.

## **Acknowledgements**

Support from the NIH (CA107331; to DJW), The Center for Biomolecular Therapeutics (CBT), and the University of Maryland Computer-Aided Drug Design Center is appreciated.

## **Author details**

experimentally evaluated. Work is also underway to develop novel inhibitors of S100B via a fragment-based approach that targets multiple binding sites on the protein identified by NMR, X-ray crystallography, and CADD techniques, including the new SILCS technique [87]. The

chemical shift perturbations in HSQC and/or TROSY spectra as a result of an S100B-small molecule interaction; this enables rapid, qualitative identification of binding site(s) on S100B for fragment-based design of new compounds that take advantage of the well-established "chelate effect" involved in linking fragments that bind neighboring sites [108,109] (Figure 6). For fast exchanging inhibitors, saturation transfer differences are collected to identify protons of the lead at the S100B-lead interface [84,110,111]. Thus, progress using a combination of functional-group optimization and the fragment-based approaches offers the potential for improvement in affinities/specificity, and for identifying novel leads. As new lead compounds are identified and structurally characterized (NMR, X-ray crystallography), SBiX affinity is considered as is scaffold diversity due to potential unforeseen problems with compounds that can occur during later-stage preclinical development (e.g. pharmacokinetic limitations, toxicity in humans). Therefore, as many as 3-6 chemical scaffolds are under consideration for development, and this number will be reduced as the project proceeds based on ADME properties, synthetic feasibility, and other pre-clinical/clinical information (Table 2). Such criteria include physiochemical properties related to bioavailability (molecular weight, clogP, TPSA, pKa, nitrogen atoms, carboxylates, H-bond donors, H-bond acceptors, rotatable bonds, H-bonds), and dose-limiting toxicities that may be predictive of the therapeutic index. Importantly, promising leads are routinely tested *in vivo* as early as possible to avoid wasted

Ongoing collaborative efforts involving biology, structure determination, CADD and synthetic chemistry have lead to the development of a collection of inhibitors of S100B. These efforts include identification of the FDA approved compound pentamidine, which is currently being evaluated in human clinical trials. In addition, the work has identified several novel chemical scaffolds that are undergoing optimization and have laid the foundation for the application of fragment-based approaches to design additional novel scaffolds. Notably, while the goal of this research is to develop a potent inhibitor of S100B for the treatment of malignant melanoma, we anticipate that the knowledge gained to date will be of utility in designing specific inhibitors of other members of the S100 protein family for the treatment of a range of S100 associated

Support from the NIH (CA107331; to DJW), The Center for Biomolecular Therapeutics (CBT), and the University of Maryland Computer-Aided Drug Design Center is appreciated.

H-15N and/or 1

H-13C

SAR by NMR approach is now a standard method for quickly identifying 1

effort on toxic/ineffective compounds.

660 Melanoma - From Early Detection to Treatment

**8. Summary**

disease states.

**Acknowledgements**

Kira G. Hartman1 , Paul T. Wilder1,3,4, Kristen Varney1,2,3, Alexander D. Jr. MacKerell2,3,4, Andrew Coop2,4, Danna Zimmer1,3, Rena Lapidus3,4 and David J. Weber1,3,4\*

\*Address all correspondence to: dweber@som.umaryland.edu

1 Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA

2 Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, USA

3 Center for Biomolecular Therapeutics, University of Maryland School of Medicine, Baltimore, MD, USA

4 University of Maryland Marlene and Stewart Greenebaum NCI Cancer Center, Baltimore, MD, USA

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**Chapter 25**

**Immunomodulation**

http://dx.doi.org/10.5772/53667

**1. Introduction**

and disappointment.

mune basis.

Konstantinos Arnaoutakis, Dorothy A. Graves, Laura F. Hutchins and Thomas Kieber-Emmons

Additional information is available at the end of the chapter

Immune treatment for melanoma has roots in clinical observations surrounding regression of normal nevi, the appearance of halo nevi, the correlation of vitiligo with outcome, the oc‐ currence of spontaneous regression of primary melanomas, nodal metastases of unknown primary lesions and the occurrence of metastases many years after resection of a primary le‐ sion. Coupling these observations with the observations on the power of the immune system to reject transplanted organs and control leukemia after allogeneic bone marrow transplant, the possibility of harnessing and directing this power has been a source of both excitement

In examining the natural history of normal nevi, it is noted that they undergo a life cycle in which growth occurs during childhood in the border of the epidermis and dermis (junction‐ al nevi). With increasing age the melanocytes move deeper into the dermis. As adult life continues they regress in old age. It is not evident that this type of regression has an im‐

Another type of regression called halo nevi is more definitively tied to the immune system [1]. Akasu, et. al have described halo nevi regression in four stages characterized initially by pan-T lymphocytes in stage one and the addition of KP-1 positive cells as well as FX IIIapositive cells in stage two. Stage three continues with increased numbers of FX IIIa-positive cells and the addition of Langerhans cells. Finally upon complete regression in stage four there is a moderate mononuclear infiltrate comprised predominantly of T cells [2]. The role of natural killer (NK) cells has been studied in normal and malignant melanocytic lesions. The highest concentration of NK cells was seen in regressing malignant lesions followed by regressing normal nevi [3]. This type of "spontaneous" regression is observed in other be‐

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

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

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

**Chapter 25**

## **Immunomodulation**

Konstantinos Arnaoutakis, Dorothy A. Graves, Laura F. Hutchins and Thomas Kieber-Emmons

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53667

**1. Introduction**

Immune treatment for melanoma has roots in clinical observations surrounding regression of normal nevi, the appearance of halo nevi, the correlation of vitiligo with outcome, the oc‐ currence of spontaneous regression of primary melanomas, nodal metastases of unknown primary lesions and the occurrence of metastases many years after resection of a primary le‐ sion. Coupling these observations with the observations on the power of the immune system to reject transplanted organs and control leukemia after allogeneic bone marrow transplant, the possibility of harnessing and directing this power has been a source of both excitement and disappointment.

In examining the natural history of normal nevi, it is noted that they undergo a life cycle in which growth occurs during childhood in the border of the epidermis and dermis (junction‐ al nevi). With increasing age the melanocytes move deeper into the dermis. As adult life continues they regress in old age. It is not evident that this type of regression has an im‐ mune basis.

Another type of regression called halo nevi is more definitively tied to the immune system [1]. Akasu, et. al have described halo nevi regression in four stages characterized initially by pan-T lymphocytes in stage one and the addition of KP-1 positive cells as well as FX IIIapositive cells in stage two. Stage three continues with increased numbers of FX IIIa-positive cells and the addition of Langerhans cells. Finally upon complete regression in stage four there is a moderate mononuclear infiltrate comprised predominantly of T cells [2]. The role of natural killer (NK) cells has been studied in normal and malignant melanocytic lesions. The highest concentration of NK cells was seen in regressing malignant lesions followed by regressing normal nevi [3]. This type of "spontaneous" regression is observed in other be‐

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

nign skin lesions such as keratoachanthomas, and the pathologic studies give insight into the possibility of stimulating similar immune action against malignancies [1, 2].

standardized by various systems. The World Health Organization (WHO) developed the most commonly used system called response criteria for solid tumors (RECIST) [19]. Recent developments in assessing response during trials utilizing the drug, ipilimumab, have served to highlight differences in direct cytotoxic chemotherapy responses and those seen with immunotherapy. The responses in tumor measurement seen in the ipilimumab trials did not correspond to the survival endpoints. Subjects had prolonged survival with delayed or no tumor measurement changes. These observations have led to a new system for assess‐

Immunomodulation

671

http://dx.doi.org/10.5772/53667

Biomarkers can serve to assess efficacy, but it is difficult to find results that consistently cor‐ relate with clinical response. Some assays can serve as immunologic endpoints in addition to or instead of tumor regression, PFS and OS in clinical studies. The most frequent meas‐ urements include: antibody titers, delayed type hypersensitivity (DTH) skin tests, enzymelinked immunosorbent assay (ELISA), enzyme-linked immunospot assay (ELISPOT), tetramer markers of antigen specific T cells and other assays indicating that the therapy in‐

All of these issues should be considered in assessing the past studies and in the planning of future studies. As we will discuss, progress has been made and enthusiasm is at an

Predicting response to treatment is probably the best way to increase benefit from a variety of different immunotherapy agents that are currently approved or under investigation for the treatment of early stage (as adjuvant therapy) or advanced melanoma. Identifying pa‐ tients that are likely to respond to these agents would spare patients unnecessary toxicities

Response to treatment may be determined by clinical characteristics or by the presence or level of biomarkers in the serum or tissue that predict a robust response to immunotherapy agents. Clinical characteristics such as ulcerated lesions appear to get more benefit from in‐ terferon (IFN) in the adjuvant setting [26]. In addition, Eastern Cooperative Oncology Group (ECOG) performance status, the number of involved organs and sites of metastases appear to be treatment factors predicting response to interleukin 2 (IL-2) in some studies [27].

The appearance of serum autoantibodies or clinical manifestations of autoimmunity during treatment with IFNα-2b was associated with improved outcomes in patients with melanoma [28]. In addition, multiplexed analysis of serum cytokines appeared to be potentially useful as a predictive marker of response to IFNα-2b in patients with high-risk operable melanoma [29]. A serum proteomic analysis has been found to possibly predict response to IL-2 treat‐ ment [30]. In this study, high pretreatment serum vascular endothelial growth factor (VEGF) and fibronectin levels were predictive of resistance to treatment. A marker of potential inter‐ est in ipilimumab therapy appears to be the absolute lymphocyte count [31]. Absolute lym‐

/L at the time of the third ipilimumab dose were

ing immune based therapies called immune-related response criteria (irRC) [20].

duced a response toward its target [21-25].

and encourage more research in the field.

phocyte counts that exceeded 1 x 109

all-time high.

**2.1. Patient selection**

The autoimmune condition of vitiligo has been associated with regression of metastases as well as better outcomes associated with immunotherapy. Vitiligo is mediated through auto‐ antibodies. Antibodies against tyrosinase have been observed in patients with melanoma as well as vitiligo not associated with melanoma. Other autoimmune effects are well docu‐ mented with immunotherapeutic treatments, indicating the ability of the therapy to break tolerance to self-antigens [4-7].

Spontaneous regression is observed in primary melanoma [8, 9]. Statistics vary on the inci‐ dence but may be as high as 20%, especially if cases of unknown primary are included. When remnants of primary lesions are found, they frequently are partially regressed and show histologic evidence of infiltration by lymphocytes. Although some have observed a worse clinical outcome with partially regressed primary lesions, patients with nodal meta‐ stases and unknown primaries tend to have a better outcome. The latter observation is at‐ tributed to improved immune surveillance compared to patients with intact primaries [10-14]. Regression of metastases is less common and has anecdotally been tied to infections or surgeries. Regression of metastases seems to predict a better overall outcome [15, 16].

The issue of late metastases from primary melanoma is well documented but much harder to explain. Issues within the tumor microenvironment remain incompletely explored, but high on the list of explanations is the possibility that the immune system is able to control proliferation until some as yet undocumented effect allows escape [17, 18].

Early studies with nonspecific therapies described below produced enough positive re‐ sults to keep interest in melanoma immunotherapy alive. However, progress in the clinic has been slow until very recently. Many issues have presented challenges to progress. Among them is an incomplete understanding of the normal immune system, including immune tolerance and the effects of the tumor microenvironment on the immune re‐ sponse. Drug development required technology to produce biologic agents, and that ca‐ pability has only recently been perfected. Issues of study design also need to be kept in mind. Subject selection can be difficult since these studies require immune competence but disease advanced enough to answer the question in a reasonable time frame with a reasonable number of subjects.

## **2. Biomarkers and endpoints**

Biomarkers and surrogate endpoints are tools to obtain information about disease status or response to interventions. The mainstay of efficacy determination in cancer therapeutic clini‐ cal trials has been the regression of known tumor masses listed as complete response (CR), partial response (PR), stable disease (SD) or progressive disease (PD) along with various sur‐ vival endpoints: overall survival (OS), disease free survival (DFS) and progression free sur‐ vival (PFS). Each of these endpoints is defined by the study. The response criteria have been standardized by various systems. The World Health Organization (WHO) developed the most commonly used system called response criteria for solid tumors (RECIST) [19]. Recent developments in assessing response during trials utilizing the drug, ipilimumab, have served to highlight differences in direct cytotoxic chemotherapy responses and those seen with immunotherapy. The responses in tumor measurement seen in the ipilimumab trials did not correspond to the survival endpoints. Subjects had prolonged survival with delayed or no tumor measurement changes. These observations have led to a new system for assess‐ ing immune based therapies called immune-related response criteria (irRC) [20].

Biomarkers can serve to assess efficacy, but it is difficult to find results that consistently cor‐ relate with clinical response. Some assays can serve as immunologic endpoints in addition to or instead of tumor regression, PFS and OS in clinical studies. The most frequent meas‐ urements include: antibody titers, delayed type hypersensitivity (DTH) skin tests, enzymelinked immunosorbent assay (ELISA), enzyme-linked immunospot assay (ELISPOT), tetramer markers of antigen specific T cells and other assays indicating that the therapy in‐ duced a response toward its target [21-25].

All of these issues should be considered in assessing the past studies and in the planning of future studies. As we will discuss, progress has been made and enthusiasm is at an all-time high.

#### **2.1. Patient selection**

nign skin lesions such as keratoachanthomas, and the pathologic studies give insight into

The autoimmune condition of vitiligo has been associated with regression of metastases as well as better outcomes associated with immunotherapy. Vitiligo is mediated through auto‐ antibodies. Antibodies against tyrosinase have been observed in patients with melanoma as well as vitiligo not associated with melanoma. Other autoimmune effects are well docu‐ mented with immunotherapeutic treatments, indicating the ability of the therapy to break

Spontaneous regression is observed in primary melanoma [8, 9]. Statistics vary on the inci‐ dence but may be as high as 20%, especially if cases of unknown primary are included. When remnants of primary lesions are found, they frequently are partially regressed and show histologic evidence of infiltration by lymphocytes. Although some have observed a worse clinical outcome with partially regressed primary lesions, patients with nodal meta‐ stases and unknown primaries tend to have a better outcome. The latter observation is at‐ tributed to improved immune surveillance compared to patients with intact primaries [10-14]. Regression of metastases is less common and has anecdotally been tied to infections or surgeries. Regression of metastases seems to predict a better overall outcome [15, 16].

The issue of late metastases from primary melanoma is well documented but much harder to explain. Issues within the tumor microenvironment remain incompletely explored, but high on the list of explanations is the possibility that the immune system is able to control

Early studies with nonspecific therapies described below produced enough positive re‐ sults to keep interest in melanoma immunotherapy alive. However, progress in the clinic has been slow until very recently. Many issues have presented challenges to progress. Among them is an incomplete understanding of the normal immune system, including immune tolerance and the effects of the tumor microenvironment on the immune re‐ sponse. Drug development required technology to produce biologic agents, and that ca‐ pability has only recently been perfected. Issues of study design also need to be kept in mind. Subject selection can be difficult since these studies require immune competence but disease advanced enough to answer the question in a reasonable time frame with a

Biomarkers and surrogate endpoints are tools to obtain information about disease status or response to interventions. The mainstay of efficacy determination in cancer therapeutic clini‐ cal trials has been the regression of known tumor masses listed as complete response (CR), partial response (PR), stable disease (SD) or progressive disease (PD) along with various sur‐ vival endpoints: overall survival (OS), disease free survival (DFS) and progression free sur‐ vival (PFS). Each of these endpoints is defined by the study. The response criteria have been

proliferation until some as yet undocumented effect allows escape [17, 18].

the possibility of stimulating similar immune action against malignancies [1, 2].

tolerance to self-antigens [4-7].

670 Melanoma - From Early Detection to Treatment

reasonable number of subjects.

**2. Biomarkers and endpoints**

Predicting response to treatment is probably the best way to increase benefit from a variety of different immunotherapy agents that are currently approved or under investigation for the treatment of early stage (as adjuvant therapy) or advanced melanoma. Identifying pa‐ tients that are likely to respond to these agents would spare patients unnecessary toxicities and encourage more research in the field.

Response to treatment may be determined by clinical characteristics or by the presence or level of biomarkers in the serum or tissue that predict a robust response to immunotherapy agents. Clinical characteristics such as ulcerated lesions appear to get more benefit from in‐ terferon (IFN) in the adjuvant setting [26]. In addition, Eastern Cooperative Oncology Group (ECOG) performance status, the number of involved organs and sites of metastases appear to be treatment factors predicting response to interleukin 2 (IL-2) in some studies [27].

The appearance of serum autoantibodies or clinical manifestations of autoimmunity during treatment with IFNα-2b was associated with improved outcomes in patients with melanoma [28]. In addition, multiplexed analysis of serum cytokines appeared to be potentially useful as a predictive marker of response to IFNα-2b in patients with high-risk operable melanoma [29]. A serum proteomic analysis has been found to possibly predict response to IL-2 treat‐ ment [30]. In this study, high pretreatment serum vascular endothelial growth factor (VEGF) and fibronectin levels were predictive of resistance to treatment. A marker of potential inter‐ est in ipilimumab therapy appears to be the absolute lymphocyte count [31]. Absolute lym‐ phocyte counts that exceeded 1 x 109 /L at the time of the third ipilimumab dose were associated with a survival benefit at one year. Immune responses to NY-ESO-1, a cancer-tes‐ tis antigen, also appear to correlate with clinical benefit from ipilimumab [32].

in the proliferation of antibody-producing plasma cells and CTLs to result in direct kill‐ ing of the tumor cells. NK cells and macrophages can also directly kill tumor cells alone

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The goal of cancer immunotherapy is to provoke the immune system to generate a tumor cell rejection strength response and to prevent recurrence of cancer by establishing longterm effector cell memory. In order for the immune system to mount an attack against mela‐ noma, it must first recognize the involved tumor cells as foreign or in need of clearing (a danger signal); it can then target them for killing. Tumor cells, like all cells, display a variety of proteins on their cell surface, and when antigen is presented in the context of MHC, the cell may be recognized by the T cell receptor (TCR) on an effector T lymphocyte. Tumor cells in general and melanoma tissues specifically, are antigenically diverse, and their ability to survive correlates with the ability of the tumor antigens to avoid detection by the immune system [40, 41]. Highly antigenic tumor cells are killed off rather quickly, due to the immune system's ability to recognize the tumor cells and mount an effective immune response, while poorly antigenic tumor cells thrive. Tumor specific transplantation antigens (TSTAs) gener‐ ally convey strong immunogenicity. These are antigens expressed on the surface of tumor cells that are specific to that tumor or type of tumor. However, the majority of antigens asso‐ ciated with melanoma cells are tumor associated transplantation antigens, or TATAs. TA‐ TAs are antigens that are associated with tumor cells, but not unique to tumor cells. TATAs are far better at preserving a tumor cell under the radar of the immune system, because

Within the tumor microenvironment, tolerance may be naturally overcome by antigen ex‐ pression levels or the timing of antigen expression. Melanomas overexpress many antigens that are present in normal melanocytes but at lower levels, and expression of these antigens suggest a progression of differentiation from normal melanocytes to melanomas. For exam‐ ple, a melanoma expressing a mutant triosephosphate isomerase protein was discovered to bind MHC class II at five times greater affinity than the wild type oligopeptide, resulting in both a significant increase in surface expression and an increase in immunogenicity [42]. Some melanoma cells overexpress the transferrin receptor by a factor of 100 [43]. Some hu‐ man melanomas overexpress the gangliosides relative to levels seen in normal melanocytes, illustrating that overexpression of carbohydrates can attract the attention of the immune sys‐

Much of melanoma's antigenicity comes from the more than 100 identified melanoma TA‐ TAs. Melan-A/MART-1, gp100 and tyrosinase are well studied differentiation antigens ex‐

Melanoma cells may also express oncofetal antigens which are normally displayed dur‐ ing embryogenesis but only expressed in select tissues, if at all, in adults. These include the cancer germ-line/cancer-testis (CT) antigens. MAGE-A family members and NY-ESO-1 are the most significant members of this group to date, and expression of MAGE-A1 and MAGE-A4 increases with tumor progression [47, 54, 55]. NY-ESO-1 is only

or with the help of antibodies or complement.

**3.1. Melanoma antigenicity**

these antigens are not danger signals.

tem, similar to protein antigens [44].

pressed in both primary and metastatic melanoma [45-53].

Gene signatures within the tumor have also shown some correlation with clinical benefit both for IL-2 and vaccination [33, 34]. Little is known about some of the newer drugs such as the CTLA-4 antagonists (ipilimumab) or other checkpoint inhibitors. It has been suggested that the presence of PD-L1 expression detected by immunohistochemistry may predict re‐ sponse to PD-1 antibody therapy [35].

Undoubtedly, utilizing serum and tissue biomarkers for response to treatment is much more challenging for immunotherapy than in the field of molecular targeted therapies. For exam‐ ple V600E BRAF mutations predict treatment response in patients who receive vemurafenib, a BRAF inhibitor. In melanoma immunotherapy, however, no serum or tissue biomarkers have yet been prospectively studied in the context of a clinical trial.

## **3. Immunoregulatory barriers, immune tolerance and tumor microenvironment**

The immune system is designed to protect our bodies from foreign agents. This protec‐ tion is selective, such that host tissues are recognized as self and preserved (termed im‐ munologic tolerance), while other agents are recognized as foreign and targeted for killing. Cancer cells, however, present the immune system with a unique challenge. While some, such as virally transformed cells, express foreign viral proteins on their sur‐ face, most tumors express normal proteins and carbohydrates. Efforts to understand how tumors survive immunosurveillance versus how and when they are targeted for killing have preoccupied scientists for well over half a century. This section explores what we currently know about the complex interplay between the immune system and cancer cells as it relates to immunotherapy.

We know now that tumor cells are immunogenic but efficacy is limited due to the lack of robustness of the response. There are two primary reasons for this: 1) due to the na‐ ture of the immunogens (self antigens) and 2) the active role-played by tumors to sup‐ press the response. The mammalian anti-tumor response engages both the humoral and cell-mediated arms of the immune system through both specific (adaptive) and non-spe‐ cific (innate) effectors. While cytotoxic T lymphocytes (CTLs), NK cells and T helper (TH) cells are viewed as the most significant players in the anti-tumor response, they are not alone [36]. Antigen presenting cells (APCs - macrophages and dendritic cells) are ab‐ solutely essential to stimulate a variety of anti-tumor responses across tumor types, and anti-tumor antibodies are often easily found in patients with melanoma and many other solid tumors, indicating a strong humoral response following stimulation by antigen spe‐ cific TH cells [37]. There is accumulating evidence that the CD4+ T cell population is far more involved in the anti-tumor response than previously thought [38, 39]. When APCs present antigen to TH cells in the context of a major histocompatibility complex (MHC) molecule on their surface, TH cells become activated and can stimulate B cells to result in the proliferation of antibody-producing plasma cells and CTLs to result in direct kill‐ ing of the tumor cells. NK cells and macrophages can also directly kill tumor cells alone or with the help of antibodies or complement.

## **3.1. Melanoma antigenicity**

associated with a survival benefit at one year. Immune responses to NY-ESO-1, a cancer-tes‐

Gene signatures within the tumor have also shown some correlation with clinical benefit both for IL-2 and vaccination [33, 34]. Little is known about some of the newer drugs such as the CTLA-4 antagonists (ipilimumab) or other checkpoint inhibitors. It has been suggested that the presence of PD-L1 expression detected by immunohistochemistry may predict re‐

Undoubtedly, utilizing serum and tissue biomarkers for response to treatment is much more challenging for immunotherapy than in the field of molecular targeted therapies. For exam‐ ple V600E BRAF mutations predict treatment response in patients who receive vemurafenib, a BRAF inhibitor. In melanoma immunotherapy, however, no serum or tissue biomarkers

The immune system is designed to protect our bodies from foreign agents. This protec‐ tion is selective, such that host tissues are recognized as self and preserved (termed im‐ munologic tolerance), while other agents are recognized as foreign and targeted for killing. Cancer cells, however, present the immune system with a unique challenge. While some, such as virally transformed cells, express foreign viral proteins on their sur‐ face, most tumors express normal proteins and carbohydrates. Efforts to understand how tumors survive immunosurveillance versus how and when they are targeted for killing have preoccupied scientists for well over half a century. This section explores what we currently know about the complex interplay between the immune system and cancer

We know now that tumor cells are immunogenic but efficacy is limited due to the lack of robustness of the response. There are two primary reasons for this: 1) due to the na‐ ture of the immunogens (self antigens) and 2) the active role-played by tumors to sup‐ press the response. The mammalian anti-tumor response engages both the humoral and cell-mediated arms of the immune system through both specific (adaptive) and non-spe‐ cific (innate) effectors. While cytotoxic T lymphocytes (CTLs), NK cells and T helper (TH) cells are viewed as the most significant players in the anti-tumor response, they are not alone [36]. Antigen presenting cells (APCs - macrophages and dendritic cells) are ab‐ solutely essential to stimulate a variety of anti-tumor responses across tumor types, and anti-tumor antibodies are often easily found in patients with melanoma and many other solid tumors, indicating a strong humoral response following stimulation by antigen spe‐ cific TH cells [37]. There is accumulating evidence that the CD4+ T cell population is far more involved in the anti-tumor response than previously thought [38, 39]. When APCs present antigen to TH cells in the context of a major histocompatibility complex (MHC) molecule on their surface, TH cells become activated and can stimulate B cells to result

tis antigen, also appear to correlate with clinical benefit from ipilimumab [32].

have yet been prospectively studied in the context of a clinical trial.

**3. Immunoregulatory barriers, immune tolerance and tumor**

sponse to PD-1 antibody therapy [35].

672 Melanoma - From Early Detection to Treatment

cells as it relates to immunotherapy.

**microenvironment**

The goal of cancer immunotherapy is to provoke the immune system to generate a tumor cell rejection strength response and to prevent recurrence of cancer by establishing longterm effector cell memory. In order for the immune system to mount an attack against mela‐ noma, it must first recognize the involved tumor cells as foreign or in need of clearing (a danger signal); it can then target them for killing. Tumor cells, like all cells, display a variety of proteins on their cell surface, and when antigen is presented in the context of MHC, the cell may be recognized by the T cell receptor (TCR) on an effector T lymphocyte. Tumor cells in general and melanoma tissues specifically, are antigenically diverse, and their ability to survive correlates with the ability of the tumor antigens to avoid detection by the immune system [40, 41]. Highly antigenic tumor cells are killed off rather quickly, due to the immune system's ability to recognize the tumor cells and mount an effective immune response, while poorly antigenic tumor cells thrive. Tumor specific transplantation antigens (TSTAs) gener‐ ally convey strong immunogenicity. These are antigens expressed on the surface of tumor cells that are specific to that tumor or type of tumor. However, the majority of antigens asso‐ ciated with melanoma cells are tumor associated transplantation antigens, or TATAs. TA‐ TAs are antigens that are associated with tumor cells, but not unique to tumor cells. TATAs are far better at preserving a tumor cell under the radar of the immune system, because these antigens are not danger signals.

Within the tumor microenvironment, tolerance may be naturally overcome by antigen ex‐ pression levels or the timing of antigen expression. Melanomas overexpress many antigens that are present in normal melanocytes but at lower levels, and expression of these antigens suggest a progression of differentiation from normal melanocytes to melanomas. For exam‐ ple, a melanoma expressing a mutant triosephosphate isomerase protein was discovered to bind MHC class II at five times greater affinity than the wild type oligopeptide, resulting in both a significant increase in surface expression and an increase in immunogenicity [42]. Some melanoma cells overexpress the transferrin receptor by a factor of 100 [43]. Some hu‐ man melanomas overexpress the gangliosides relative to levels seen in normal melanocytes, illustrating that overexpression of carbohydrates can attract the attention of the immune sys‐ tem, similar to protein antigens [44].

Much of melanoma's antigenicity comes from the more than 100 identified melanoma TA‐ TAs. Melan-A/MART-1, gp100 and tyrosinase are well studied differentiation antigens ex‐ pressed in both primary and metastatic melanoma [45-53].

Melanoma cells may also express oncofetal antigens which are normally displayed dur‐ ing embryogenesis but only expressed in select tissues, if at all, in adults. These include the cancer germ-line/cancer-testis (CT) antigens. MAGE-A family members and NY-ESO-1 are the most significant members of this group to date, and expression of MAGE-A1 and MAGE-A4 increases with tumor progression [47, 54, 55]. NY-ESO-1 is only expressed in adults in testis and placenta tissue, however, it is expressed in up to 40% of late stage melanomas and is highly immunogenic [56]. MAGE-6 is expressed in more than 70% of metastatic melanomas [57].

Tumors in general create microenvironments with depressed immune activity such that few functional cytotoxic cells are found near the developing tumor. One strategy for this in‐ volves the poorly understood regulation of lymphocyte types within the tumor microenvir‐ onment. Moderate to large numbers of tumor infiltrating lymphocytes (TILs) have been associated with improved survival in melanoma patients; however this has not been ob‐ served consistently [68, 69]. While most patients with melanoma have TILs, the mere pres‐ ence of TILs is obviously not sufficient to mount an effective anti-tumor response [70]. Tumor cells are able to attract a particular type of T cell that is immunosuppressive. Tregs can directly inhibit and kill CTLs and TH cells, and they functionally drive the tumor's T helper Type 2 (Th-2) immune environment by producing the immunosuppressive cytokines IL-10 and tumor growth factor beta (TGF-β) while suppressing CTL production of immu‐ nostimulatory T helper Type 1 (Th-1) cytokines interferon gamma (IFN γ) and IL-2 [71]. Tregs also negatively regulate effector dendritic cells and NK cells. In melanoma, depletion of Tregs prior to infusion with activated T lymphocytes (adoptive cell therapy) measurably

Immunomodulation

675

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Melanoma cells may also create an immunosuppressed microenvironment through galectin expression [73]. Deregulation of galectins is common in human tumors. Expression of galec‐ tin 3 correlates with melanoma metastasis and poorer disease outcomes, perhaps through induction of TIL apoptosis. Galectin 1 may also induce apoptosis of T cells and this may be an important mechanism of tumor evasion for melanoma. Additionally, galectins 1 and 3 convey resistance to apoptosis in tumor cells, though this is less studied in melanoma.

Immunosuppression similar to that found in the tumor microenvironment can also be found in the sentinel lymph node [71, 74]. Tregs are found in higher numbers in metastatic melano‐ ma sentinel lymph nodes, and as in the tumor microenvironment, this appears to be mediat‐ ed by Th-2 cytokines IL-6, IL-8, IL-10 and TGF-β, among others. This locoregional immunosupporession is thought to be necessary for metastasis and prepares the lymphatic environment for the arrival and survival of metastastic cells [75]. While dendritic cells are detectable in melanoma sentinel lymph nodes, they may be present in lower number and/or contain a higher percentage of immature dendritic cells that lack the costimulatory mole‐

IL-10 and TGF-β are immunosuppressive cytokines utilized by melanoma to create an im‐ munosuppressive microenvironment and progress disease toward metastasis [77, 78]. Both cytokines can induce T cells to undergo apoptosis; TGF-β can additionally induce apoptosis in dendritic cells and macrophages. Normal melanocytes are subject to TGF-β anti-prolifera‐ tive regulation, and loss of this phenotype is thought to be a crucial step toward melanoma development [77]. Neutrophils from patients with melanoma constitutively and spontane‐ ously synthesize IL-10 through activation by serum amyloid A 1 (SAA-1) which is enriched

There are a variety of other general tumor microenvironment conditions and immunoeva‐ sion strategies that melanomas employ to ensure their survival. Hypoxia occurs in solid tu‐ mor masses and is well known to create an immunosuppressive tumor microenvironment [80]. Tumor cells can also alter the expression of stress proteins that bind NK cells for target‐

improves response rates [72].

in melanoma tissue [79].

cules necessary for effective T cell activation [74, 76].

Identification of melanoma TATAs is crucial as a key strategy for immunotherapy. Adminis‐ tration of vaccines that deliver TATAs can push the immune system into overcoming toler‐ ance. T cells specific for TATAs have been identified in melanoma patients, and spontaneously occurring circulating T cells reactive to Melan-A and NY-ESO-1 were recent‐ ly found to be predictive of better survival [58]. A recent study reported an analysis of the human leukocyte antigen 1 (HLA-I) peptidomes from melanomas in four patients, and while finding that melanoma antigenicity was highly variable, the investigators also found that the peptidomes were highly immunogenic, identifying new potential peptides for mela‐ noma vaccines [41].

#### **3.2. Immunoevasion strategies**

Tumor cells increase their odds of survival by lowering their immunological profiles. TH cells, CTLs and antibodies specific for TATAs are readily detectable in the blood, lymph no‐ des and tumors of cancer patients. Despite tolerance, the immune system can mount an im‐ mune response to these antigens, but tumors and/or tumor cells may persist, so all the efforts of the immune system are not enough to clear tumor cells. The immunoevasion strat‐ egies utilized by melanomas are impressive and generally include down-regulation of TA‐ TAs on the tumor cell surface, secretion of immunosuppressive cytokines that affect APCs and shedding of material that promotes the stimulation of the inhibitory regulatory T cells (Tregs). Together, these strategies create a toleragenic tumor microenvironment that is both adaptive to immune pressures and predictive of clinical outcomes.

Many tumor cells stop displaying TATAs or TSTAs on their surface to escape immune rec‐ ognition [59]. Expression of MART-1, gp100 and tyrosinase generally decreases as melano‐ ma progresses [60]. Following immunization with gp100 or MART-1 peptides, melanoma metastases lost expression of the corresponding TATA, suggesting that TATAs can be downregulated in direct response to a specific CTL anti-tumor response [45]. This strategy is specifically adaptive to removing known CTL targets from the tumor population and selects for proliferation of tumor cells that do not bear antigens yet targeted by the CTL response.

Tumor cells may additionally repress expression of MHC class I proteins by repressing MHC I gene expression or posttranslational modifications [59]. In fact, many human tumors demonstrate a decreased expression of MHC I, and the loss of MHC I expression is often associated with more invasive and metastatic tumors [61, 62]. In melanoma, MHC I expres‐ sion correlates with disease progression, and the lack of HLA I expression and lack of re‐ sponse to T cell based immunotherapy may be linked to acquired β2-microglobulin gene defects [63-65]. C-myc oncogene overexpression in melanoma also correlates with HLA I downregulation [66]. The one caveat of this strategy, however, is that a total lack of MHC expression invites attack by NK cells [67]. To circumvent this, tumor cells often only lower MHC I expression, retaining some minimal expression to protect themselves while not alarming the immune system.

Tumors in general create microenvironments with depressed immune activity such that few functional cytotoxic cells are found near the developing tumor. One strategy for this in‐ volves the poorly understood regulation of lymphocyte types within the tumor microenvir‐ onment. Moderate to large numbers of tumor infiltrating lymphocytes (TILs) have been associated with improved survival in melanoma patients; however this has not been ob‐ served consistently [68, 69]. While most patients with melanoma have TILs, the mere pres‐ ence of TILs is obviously not sufficient to mount an effective anti-tumor response [70]. Tumor cells are able to attract a particular type of T cell that is immunosuppressive. Tregs can directly inhibit and kill CTLs and TH cells, and they functionally drive the tumor's T helper Type 2 (Th-2) immune environment by producing the immunosuppressive cytokines IL-10 and tumor growth factor beta (TGF-β) while suppressing CTL production of immu‐ nostimulatory T helper Type 1 (Th-1) cytokines interferon gamma (IFN γ) and IL-2 [71]. Tregs also negatively regulate effector dendritic cells and NK cells. In melanoma, depletion of Tregs prior to infusion with activated T lymphocytes (adoptive cell therapy) measurably improves response rates [72].

expressed in adults in testis and placenta tissue, however, it is expressed in up to 40% of late stage melanomas and is highly immunogenic [56]. MAGE-6 is expressed in more

Identification of melanoma TATAs is crucial as a key strategy for immunotherapy. Adminis‐ tration of vaccines that deliver TATAs can push the immune system into overcoming toler‐ ance. T cells specific for TATAs have been identified in melanoma patients, and spontaneously occurring circulating T cells reactive to Melan-A and NY-ESO-1 were recent‐ ly found to be predictive of better survival [58]. A recent study reported an analysis of the human leukocyte antigen 1 (HLA-I) peptidomes from melanomas in four patients, and while finding that melanoma antigenicity was highly variable, the investigators also found that the peptidomes were highly immunogenic, identifying new potential peptides for mela‐

Tumor cells increase their odds of survival by lowering their immunological profiles. TH cells, CTLs and antibodies specific for TATAs are readily detectable in the blood, lymph no‐ des and tumors of cancer patients. Despite tolerance, the immune system can mount an im‐ mune response to these antigens, but tumors and/or tumor cells may persist, so all the efforts of the immune system are not enough to clear tumor cells. The immunoevasion strat‐ egies utilized by melanomas are impressive and generally include down-regulation of TA‐ TAs on the tumor cell surface, secretion of immunosuppressive cytokines that affect APCs and shedding of material that promotes the stimulation of the inhibitory regulatory T cells (Tregs). Together, these strategies create a toleragenic tumor microenvironment that is both

Many tumor cells stop displaying TATAs or TSTAs on their surface to escape immune rec‐ ognition [59]. Expression of MART-1, gp100 and tyrosinase generally decreases as melano‐ ma progresses [60]. Following immunization with gp100 or MART-1 peptides, melanoma metastases lost expression of the corresponding TATA, suggesting that TATAs can be downregulated in direct response to a specific CTL anti-tumor response [45]. This strategy is specifically adaptive to removing known CTL targets from the tumor population and selects for proliferation of tumor cells that do not bear antigens yet targeted by the CTL response. Tumor cells may additionally repress expression of MHC class I proteins by repressing MHC I gene expression or posttranslational modifications [59]. In fact, many human tumors demonstrate a decreased expression of MHC I, and the loss of MHC I expression is often associated with more invasive and metastatic tumors [61, 62]. In melanoma, MHC I expres‐ sion correlates with disease progression, and the lack of HLA I expression and lack of re‐ sponse to T cell based immunotherapy may be linked to acquired β2-microglobulin gene defects [63-65]. C-myc oncogene overexpression in melanoma also correlates with HLA I downregulation [66]. The one caveat of this strategy, however, is that a total lack of MHC expression invites attack by NK cells [67]. To circumvent this, tumor cells often only lower MHC I expression, retaining some minimal expression to protect themselves while not

adaptive to immune pressures and predictive of clinical outcomes.

than 70% of metastatic melanomas [57].

674 Melanoma - From Early Detection to Treatment

noma vaccines [41].

**3.2. Immunoevasion strategies**

alarming the immune system.

Melanoma cells may also create an immunosuppressed microenvironment through galectin expression [73]. Deregulation of galectins is common in human tumors. Expression of galec‐ tin 3 correlates with melanoma metastasis and poorer disease outcomes, perhaps through induction of TIL apoptosis. Galectin 1 may also induce apoptosis of T cells and this may be an important mechanism of tumor evasion for melanoma. Additionally, galectins 1 and 3 convey resistance to apoptosis in tumor cells, though this is less studied in melanoma.

Immunosuppression similar to that found in the tumor microenvironment can also be found in the sentinel lymph node [71, 74]. Tregs are found in higher numbers in metastatic melano‐ ma sentinel lymph nodes, and as in the tumor microenvironment, this appears to be mediat‐ ed by Th-2 cytokines IL-6, IL-8, IL-10 and TGF-β, among others. This locoregional immunosupporession is thought to be necessary for metastasis and prepares the lymphatic environment for the arrival and survival of metastastic cells [75]. While dendritic cells are detectable in melanoma sentinel lymph nodes, they may be present in lower number and/or contain a higher percentage of immature dendritic cells that lack the costimulatory mole‐ cules necessary for effective T cell activation [74, 76].

IL-10 and TGF-β are immunosuppressive cytokines utilized by melanoma to create an im‐ munosuppressive microenvironment and progress disease toward metastasis [77, 78]. Both cytokines can induce T cells to undergo apoptosis; TGF-β can additionally induce apoptosis in dendritic cells and macrophages. Normal melanocytes are subject to TGF-β anti-prolifera‐ tive regulation, and loss of this phenotype is thought to be a crucial step toward melanoma development [77]. Neutrophils from patients with melanoma constitutively and spontane‐ ously synthesize IL-10 through activation by serum amyloid A 1 (SAA-1) which is enriched in melanoma tissue [79].

There are a variety of other general tumor microenvironment conditions and immunoeva‐ sion strategies that melanomas employ to ensure their survival. Hypoxia occurs in solid tu‐ mor masses and is well known to create an immunosuppressive tumor microenvironment [80]. Tumor cells can also alter the expression of stress proteins that bind NK cells for target‐ ed killing. Melanoma cells predominantly express the MICA and ULBP2 stress proteins, and a correlation has been found between poor prognosis and expression of soluble ULBP2 that is competitive for NK cell binding [81, 82]. Heat shock proteins are well known to promote tumor growth, invasion and metastasis through a variety of mechanisms [83]. Expression of heat shock proteins 90 and 40 (hsp90 and hsp40) in melanoma tissue correlates with ad‐ vanced disease and patient survival, in the case of hsp40 [84].

At present, the greatest promise for metastatic melanoma patients lies in immunomodulato‐ ry antibody therapy against immunological checkpoints. Immunotherapies that employ this targeting strategy are recent and have yielded some of the most promising clinical responses in decades. Immunological checkpoints are negative regulators of the immune system. Cyto‐ toxic T lymphocyte antigen 4 (CTLA4) is found on naïve T cells and Tregs; upon activation it turns off TCR signaling and serves to stop activation of targeted T cells. Antibodies to CTLA4 prevent this from happening and prolong and intensify T cell activation [36]. Ipili‐ mumab was approved by the U.S. Food and Drug Administration (FDA) in 2012 for the treatment of metastatic melanoma owing to the overall survival benefit observed in a phase III study that has now resulted in durable responses lasting 8 years and beyond [95]. Al‐ though ipilimumab is the only FDA-approved checkpoint inhibitor indicated for treatment of melanoma, there are others in the pipeline [96]. Tremelimumab is another CTLA4 anti‐ body, and there are several programmed death ligand 1 (PD-1) antibodies undergoing clini‐ cal development as well [97, 98]. Though both CTLA4 and PD-1 antibodies have demonstrated significant improvement in clinical outcome for metastatic melanoma pa‐ tients, they are not effective in all patients and cause a new and unique spectrum of side ef‐

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The success of anti-tumor and antiviral vaccines often requires the use of an adjuvant, a sub‐ stance that significantly enhances the immune response to a coadministered antigen. Only a handful of adjuvants have both sufficient potency and acceptable toxicity for clinical investi‐ gation. The critical roles of vaccine adjuvants lie in their ability to: (1) enable the use of oth‐ erwise impotent antigens; (2) extend the benefits of vaccination to poor responders (e.g., older or immune-compromised patients); and (3) effect dose-sparing of rare and expensive antigens in short supply (e.g., during an epidemic) [99]. Vaccine adjuvants for the most part can be evaluated as such only when they are associated with a vaccine. Early therapies were nonspecific and were thought to produce a general immune response. Many current vaccine

Adjuvant therapy of melanoma assumes that treatment will be more effective when the tu‐ mor burden is small. Mycobacterium bovis bacillus Calmette-Guérin (BCG) is an old vac‐ cine/adjuvant used in countries where tuberculosis is widespread. First used in humans in 1921, BCG is made from a strain of weakened bovine tuberculosis bacterium. The local and systemic effect of BCG has been known for decades and is an immunomodulating agent for melanoma. BCG therapy induces a massive local immune response characterized by the ex‐ pression of multiple cytokines. A significant correlation between a reduced risk of melano‐ ma and BCG and vaccinia vaccination in early childhood or infectious diseases later in life has already been reported from the FEBrile Infections and Melanoma (FEBIM) multicenter

fects termed "immune-related adverse events."

**4. Nonspecific immune therapy and adjuvants**

trials utilize nonspecific immune stimulants as adjuncts.

**4.1. BCG**

It is important to remember that while immune evasion and creation of an immunsuppres‐ sive tumor microenvironment is highly variable among melanomas, the ability of the tumor to effectively create a strong toleragenic microenvironment correlates with clinical outcome. Toleragenic tumor microenvironments are associated with sentinel lymph node involve‐ ment and more advanced disease [74]. Efforts to overcome this tolerance and re-capitulate the balance of immune system regulators to a state of anti-tumor effectiveness comprise the field of immunotherapy, and success in this therapeutic approach holds tremendous prom‐ ise for not only halting tumor progression but for turning back the clock to ultimately result in tumor clearance.

#### **3.3. Immunotherapy strategies**

Most immunotherapy efforts strive to activate T cells and specifically CTLs. Therapeutic melanoma vaccines may enhance antigen presentation directly through peptides or DNA. A synthetic peptide vaccine targeted to the melanoma gp100 TATA, for example, has resulted in good objective clinical responses [85]. Vaccines may also rely on the assistance of dendrit‐ ic cells, a key stimulator of immune cells [86-88]. The goal is to utilize TSTA or TATA anti‐ gens to provoke the development and proliferation of cytotoxic cells directed against tumor cells, thereby overcoming tolerance.

Adoptive cell transfer or therapy (ACT) is a passive immunotherapeutic approach in which a patient's antigen-specific cells are expanded and activated *ex vivo* and then reintroduced following radiation or chemotherapy [89]. TILs, autologous T cell clones, donor anti-tumor lymphocytes and genetically engineered lymphocytes have all been used in this strategy. Some encouraging results have been seen in melanoma patients with advanced disease [90]. In three separate trials, autologous TILs provided through ACT and administered with IL-2 to metastatic melanoma patients resulted in up to a 72% objective response rate, and 22% of the 93 subjects had complete tumor regression [91]. ACT employing autologous T cells tar‐ geting NY-ESO-1 resulted in objective responses in five of 11 metastatic melanoma patients and two complete regressions at one-year post-procedure [92]. While it is argued that ACT is more effective in metastatic melanoma than ipilimumab (see below), it is practically more complex to administer and less accessible for a majority of patients [90].

Cytokines have shown efficacy in high risk local and metastatic melanoma patients as well. IL-2 and IFNα-2b have been investigated the most. IL-2 alone produces a durable remission in some patients, though it is often associated with significant side effects, and better out‐ comes may be obtained by combining it with other therapeutic approaches [93]. A pooled analysis of nearly 2,000 stage IIB and III melanoma patients indicated that adjuvant high dose IFNα-2b prolongs relapse free survival in patients [94].

At present, the greatest promise for metastatic melanoma patients lies in immunomodulato‐ ry antibody therapy against immunological checkpoints. Immunotherapies that employ this targeting strategy are recent and have yielded some of the most promising clinical responses in decades. Immunological checkpoints are negative regulators of the immune system. Cyto‐ toxic T lymphocyte antigen 4 (CTLA4) is found on naïve T cells and Tregs; upon activation it turns off TCR signaling and serves to stop activation of targeted T cells. Antibodies to CTLA4 prevent this from happening and prolong and intensify T cell activation [36]. Ipili‐ mumab was approved by the U.S. Food and Drug Administration (FDA) in 2012 for the treatment of metastatic melanoma owing to the overall survival benefit observed in a phase III study that has now resulted in durable responses lasting 8 years and beyond [95]. Al‐ though ipilimumab is the only FDA-approved checkpoint inhibitor indicated for treatment of melanoma, there are others in the pipeline [96]. Tremelimumab is another CTLA4 anti‐ body, and there are several programmed death ligand 1 (PD-1) antibodies undergoing clini‐ cal development as well [97, 98]. Though both CTLA4 and PD-1 antibodies have demonstrated significant improvement in clinical outcome for metastatic melanoma pa‐ tients, they are not effective in all patients and cause a new and unique spectrum of side ef‐ fects termed "immune-related adverse events."

## **4. Nonspecific immune therapy and adjuvants**

The success of anti-tumor and antiviral vaccines often requires the use of an adjuvant, a sub‐ stance that significantly enhances the immune response to a coadministered antigen. Only a handful of adjuvants have both sufficient potency and acceptable toxicity for clinical investi‐ gation. The critical roles of vaccine adjuvants lie in their ability to: (1) enable the use of oth‐ erwise impotent antigens; (2) extend the benefits of vaccination to poor responders (e.g., older or immune-compromised patients); and (3) effect dose-sparing of rare and expensive antigens in short supply (e.g., during an epidemic) [99]. Vaccine adjuvants for the most part can be evaluated as such only when they are associated with a vaccine. Early therapies were nonspecific and were thought to produce a general immune response. Many current vaccine trials utilize nonspecific immune stimulants as adjuncts.

#### **4.1. BCG**

ed killing. Melanoma cells predominantly express the MICA and ULBP2 stress proteins, and a correlation has been found between poor prognosis and expression of soluble ULBP2 that is competitive for NK cell binding [81, 82]. Heat shock proteins are well known to promote tumor growth, invasion and metastasis through a variety of mechanisms [83]. Expression of heat shock proteins 90 and 40 (hsp90 and hsp40) in melanoma tissue correlates with ad‐

It is important to remember that while immune evasion and creation of an immunsuppres‐ sive tumor microenvironment is highly variable among melanomas, the ability of the tumor to effectively create a strong toleragenic microenvironment correlates with clinical outcome. Toleragenic tumor microenvironments are associated with sentinel lymph node involve‐ ment and more advanced disease [74]. Efforts to overcome this tolerance and re-capitulate the balance of immune system regulators to a state of anti-tumor effectiveness comprise the field of immunotherapy, and success in this therapeutic approach holds tremendous prom‐ ise for not only halting tumor progression but for turning back the clock to ultimately result

Most immunotherapy efforts strive to activate T cells and specifically CTLs. Therapeutic melanoma vaccines may enhance antigen presentation directly through peptides or DNA. A synthetic peptide vaccine targeted to the melanoma gp100 TATA, for example, has resulted in good objective clinical responses [85]. Vaccines may also rely on the assistance of dendrit‐ ic cells, a key stimulator of immune cells [86-88]. The goal is to utilize TSTA or TATA anti‐ gens to provoke the development and proliferation of cytotoxic cells directed against tumor

Adoptive cell transfer or therapy (ACT) is a passive immunotherapeutic approach in which a patient's antigen-specific cells are expanded and activated *ex vivo* and then reintroduced following radiation or chemotherapy [89]. TILs, autologous T cell clones, donor anti-tumor lymphocytes and genetically engineered lymphocytes have all been used in this strategy. Some encouraging results have been seen in melanoma patients with advanced disease [90]. In three separate trials, autologous TILs provided through ACT and administered with IL-2 to metastatic melanoma patients resulted in up to a 72% objective response rate, and 22% of the 93 subjects had complete tumor regression [91]. ACT employing autologous T cells tar‐ geting NY-ESO-1 resulted in objective responses in five of 11 metastatic melanoma patients and two complete regressions at one-year post-procedure [92]. While it is argued that ACT is more effective in metastatic melanoma than ipilimumab (see below), it is practically more

Cytokines have shown efficacy in high risk local and metastatic melanoma patients as well. IL-2 and IFNα-2b have been investigated the most. IL-2 alone produces a durable remission in some patients, though it is often associated with significant side effects, and better out‐ comes may be obtained by combining it with other therapeutic approaches [93]. A pooled analysis of nearly 2,000 stage IIB and III melanoma patients indicated that adjuvant high

complex to administer and less accessible for a majority of patients [90].

dose IFNα-2b prolongs relapse free survival in patients [94].

vanced disease and patient survival, in the case of hsp40 [84].

in tumor clearance.

**3.3. Immunotherapy strategies**

676 Melanoma - From Early Detection to Treatment

cells, thereby overcoming tolerance.

Adjuvant therapy of melanoma assumes that treatment will be more effective when the tu‐ mor burden is small. Mycobacterium bovis bacillus Calmette-Guérin (BCG) is an old vac‐ cine/adjuvant used in countries where tuberculosis is widespread. First used in humans in 1921, BCG is made from a strain of weakened bovine tuberculosis bacterium. The local and systemic effect of BCG has been known for decades and is an immunomodulating agent for melanoma. BCG therapy induces a massive local immune response characterized by the ex‐ pression of multiple cytokines. A significant correlation between a reduced risk of melano‐ ma and BCG and vaccinia vaccination in early childhood or infectious diseases later in life has already been reported from the FEBrile Infections and Melanoma (FEBIM) multicenter case-control study [100]. Such observations suggest that BCG can augment immune respons‐ es and be used in adjuvant therapy strategies.

processing and presentation of antigen to T-cells in draining lymph nodes. The Gram-nega‐ tive bacterial cell constituent lipopolysaccharide (LPS) is known to possess strong immunos‐ timulatory properties and has been evaluated as an adjuvant for promoting immune responses to minimally immunogenic antigens, including TATAs. The relatively recent dis‐ covery of TLRs and the identification of TLR4, in particular, as the signaling receptor for lip‐ id A have allowed for a better understanding of how this immunostimulant functions with regard to induction of innate and adaptive immune responses. Local TLR stimulation is an attractive approach to induce anti-tumor immunity. Tumor cells respond to TLR ligands with an increase in MHC class I expression and induce IL-6 secretion *in vitro*. Melanoma cells are typically characterized as having low expression of MHC I. Consequently TLR li‐ gands interacting with melanoma cells might enhance MHC class I expression, along with their targeting by melanoma specific CTLs. Although several lipid A species, including LPS and synthetic analogs, have been developed and tested as monotherapeutics for the treat‐ ment of cancer, monophosphoryl lipid A (MPL), a ligand for TLR4 has been evaluated as a cancer vaccine adjuvant in published human clinical trials. MPL comprises the lipid A por‐ tion of Salmonella minnesota LPS [109]. LPS and MPL induce similar cytokine profiles, but

Immunomodulation

679

http://dx.doi.org/10.5772/53667

DETOX, an adjuvant consisting of MPL and purified mycobacterial cell-wall skeleton (CWS) is another vaccine potentiating agent. MPL (Corixa Corp., Seattle, Washington, USA) adju‐ vant is a chemically modified LPS derivative that displays greatly reduced toxicity while maintaining most of the immunostimulatory activity of LPS [110] signaling through TLR4 to stimulate the innate immune system. MPL adjuvant has been used extensively in clinical tri‐ als as a component in prophylactic and therapeutic vaccines targeting infectious disease, cancer and allergies. MPL has been administered to more than 300,000 human subjects in studies of next-generation vaccines, emerging as a safe and effective vaccine adjuvant. In one study DETOX markedly potentiated antibody but had little effect on DTH responses to melanoma vaccine immunization. It did not appear to improve DFS in comparison to alum in this non-randomized study [111]. DETOX has been formulated into Melacine (Corixa Corp.), a vaccine prepared from the lysate of two melanoma cell lines adjuvanted with DE‐ TOX. In clinical trials with Melacine, tumor progression is delayed in the vaccine-treated pa‐

tients, although this was only observed in patients with certain HLA phenotypes.

The potency of local TLR treatment in therapy demonstrates that local treatment with TLR adjuvants like MPL might effectively restore anti-tumor immunity. Melacine is available for

Saponins are natural glycosides of steroid or triterpene which exhibit many different bio‐ logical and pharmacological activities [112]. Notably, saponins can activate the mammali‐ an immune system, and this has led to significant interest in their potential as vaccine adjuvants. The most widely used saponin-based adjuvants are Quil A and its derivative QS-21, isolated from the bark of the Quillaja saponaria Molina (Chilean soap bark tree); these have been evaluated in numerous clinical trials [113]. Their unique capacity to

MPL is at least 100-fold less toxic.

sale in Canada.

**4.3. QS-21**

Phase II trials indicate that active specific immunotherapy can alter the natural course of American Joint Committee on Cancer [AJCC] Stage III and IV melanoma following surgical resection of nodal or distant metastases. Initial adjuvant immunotherapy trials demonstrat‐ ed a greater disease-free interval in patients treated with BCG compared with historical con‐ trols [101]. In one study 149 patients at high risk of recurrence after surgical treatment of local or regional malignant melanoma were given BCG for 2 years and were followed up for a median of 28 months from the start of immunotherapy [101]. Studies such as these suggest that improved survival rates following recurrence might be explained by the pattern of re‐ currence; suggesting local or regional sites might be more responsive to treatment. Mecha‐ nistic studies suggest that tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is induced by BCG treatment. Subsequently, BCG and components of the mycobac‐ terial cell wall can directly stimulate the release of soluble TRAIL through toll-like recep‐ tor-2 (TLR2) recognition that is augmented by IFN. Based on the need for a Th-1 cytokine response to BCG therapy for therapeutic results, it might be proposed that cells migrating in response to BCG treatment release TRAIL. In addition, IFN acts to augment and prolong the amount of TRAIL released by effector cells, resulting in an effective therapeutic outcome.

Early trials of BCG-based immunotherapy for melanoma consistently showed a trend to‐ ward improved clinical outcomes in patients treated with BCG compared with observa‐ tion alone (reviewed by [102]). However, mature results of a phase III randomized trial of BCG versus observation and BCG plus dacarbazine versus BCG in the adjuvant thera‐ py of AJCC stage I-III melanoma (E1673: an ECOG trial) ascribes no benefit for BCG for this patient population [103]. As early as 1976 BCG was tested as an immunotherapy sys‐ temic adjunct to surgery in malignant melanoma [104]. In 1984 a polyvalent BCG formu‐ lation along with Canvaxin began testing in phase II trials as postsurgical adjuvant therapy for stage III melanoma [105], with results from such trials summarized as no ap‐ parent benefit from vaccination [106, 107].

Although experiments with animals have demonstrated that BCG mediates anti-tumor ac‐ tivity, most randomized adjuvant clinical trials have failed to show significant benefit to pa‐ tients with malignant melanoma. This may be because there is no accepted clinical technique for monitoring *in vivo* BCG activity. As a result the optimal route of administra‐ tion and dose of BCG have not been truly determined as well as the optimal BCG strain. At‐ tempts to improve the efficacy of BCG therapy have been made. One approach introduced the gene encoding the 65 kDa hsp of Mycobacterium tuberculosis into a mouse malignant melanoma cell line (B16) as proof of principle [108].The 65 kDa hsp was expressed after gene transduction and significantly enhanced the anti-tumor effect of BCG immunotherapy, fur‐ ther indicating that CD4+ T cells play an important role in this anti-tumor effect.

#### **4.2. DETOX**

Many adjuvants currently under evaluation for use in cancer vaccines activate relevant APCs, such as dendritic cells and macrophages, via TLRs and promote effective uptake, processing and presentation of antigen to T-cells in draining lymph nodes. The Gram-nega‐ tive bacterial cell constituent lipopolysaccharide (LPS) is known to possess strong immunos‐ timulatory properties and has been evaluated as an adjuvant for promoting immune responses to minimally immunogenic antigens, including TATAs. The relatively recent dis‐ covery of TLRs and the identification of TLR4, in particular, as the signaling receptor for lip‐ id A have allowed for a better understanding of how this immunostimulant functions with regard to induction of innate and adaptive immune responses. Local TLR stimulation is an attractive approach to induce anti-tumor immunity. Tumor cells respond to TLR ligands with an increase in MHC class I expression and induce IL-6 secretion *in vitro*. Melanoma cells are typically characterized as having low expression of MHC I. Consequently TLR li‐ gands interacting with melanoma cells might enhance MHC class I expression, along with their targeting by melanoma specific CTLs. Although several lipid A species, including LPS and synthetic analogs, have been developed and tested as monotherapeutics for the treat‐ ment of cancer, monophosphoryl lipid A (MPL), a ligand for TLR4 has been evaluated as a cancer vaccine adjuvant in published human clinical trials. MPL comprises the lipid A por‐ tion of Salmonella minnesota LPS [109]. LPS and MPL induce similar cytokine profiles, but MPL is at least 100-fold less toxic.

DETOX, an adjuvant consisting of MPL and purified mycobacterial cell-wall skeleton (CWS) is another vaccine potentiating agent. MPL (Corixa Corp., Seattle, Washington, USA) adju‐ vant is a chemically modified LPS derivative that displays greatly reduced toxicity while maintaining most of the immunostimulatory activity of LPS [110] signaling through TLR4 to stimulate the innate immune system. MPL adjuvant has been used extensively in clinical tri‐ als as a component in prophylactic and therapeutic vaccines targeting infectious disease, cancer and allergies. MPL has been administered to more than 300,000 human subjects in studies of next-generation vaccines, emerging as a safe and effective vaccine adjuvant. In one study DETOX markedly potentiated antibody but had little effect on DTH responses to melanoma vaccine immunization. It did not appear to improve DFS in comparison to alum in this non-randomized study [111]. DETOX has been formulated into Melacine (Corixa Corp.), a vaccine prepared from the lysate of two melanoma cell lines adjuvanted with DE‐ TOX. In clinical trials with Melacine, tumor progression is delayed in the vaccine-treated pa‐ tients, although this was only observed in patients with certain HLA phenotypes.

The potency of local TLR treatment in therapy demonstrates that local treatment with TLR adjuvants like MPL might effectively restore anti-tumor immunity. Melacine is available for sale in Canada.

#### **4.3. QS-21**

case-control study [100]. Such observations suggest that BCG can augment immune respons‐

Phase II trials indicate that active specific immunotherapy can alter the natural course of American Joint Committee on Cancer [AJCC] Stage III and IV melanoma following surgical resection of nodal or distant metastases. Initial adjuvant immunotherapy trials demonstrat‐ ed a greater disease-free interval in patients treated with BCG compared with historical con‐ trols [101]. In one study 149 patients at high risk of recurrence after surgical treatment of local or regional malignant melanoma were given BCG for 2 years and were followed up for a median of 28 months from the start of immunotherapy [101]. Studies such as these suggest that improved survival rates following recurrence might be explained by the pattern of re‐ currence; suggesting local or regional sites might be more responsive to treatment. Mecha‐ nistic studies suggest that tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is induced by BCG treatment. Subsequently, BCG and components of the mycobac‐ terial cell wall can directly stimulate the release of soluble TRAIL through toll-like recep‐ tor-2 (TLR2) recognition that is augmented by IFN. Based on the need for a Th-1 cytokine response to BCG therapy for therapeutic results, it might be proposed that cells migrating in response to BCG treatment release TRAIL. In addition, IFN acts to augment and prolong the amount of TRAIL released by effector cells, resulting in an effective therapeutic outcome. Early trials of BCG-based immunotherapy for melanoma consistently showed a trend to‐ ward improved clinical outcomes in patients treated with BCG compared with observa‐ tion alone (reviewed by [102]). However, mature results of a phase III randomized trial of BCG versus observation and BCG plus dacarbazine versus BCG in the adjuvant thera‐ py of AJCC stage I-III melanoma (E1673: an ECOG trial) ascribes no benefit for BCG for this patient population [103]. As early as 1976 BCG was tested as an immunotherapy sys‐ temic adjunct to surgery in malignant melanoma [104]. In 1984 a polyvalent BCG formu‐ lation along with Canvaxin began testing in phase II trials as postsurgical adjuvant therapy for stage III melanoma [105], with results from such trials summarized as no ap‐

Although experiments with animals have demonstrated that BCG mediates anti-tumor ac‐ tivity, most randomized adjuvant clinical trials have failed to show significant benefit to pa‐ tients with malignant melanoma. This may be because there is no accepted clinical technique for monitoring *in vivo* BCG activity. As a result the optimal route of administra‐ tion and dose of BCG have not been truly determined as well as the optimal BCG strain. At‐ tempts to improve the efficacy of BCG therapy have been made. One approach introduced the gene encoding the 65 kDa hsp of Mycobacterium tuberculosis into a mouse malignant melanoma cell line (B16) as proof of principle [108].The 65 kDa hsp was expressed after gene transduction and significantly enhanced the anti-tumor effect of BCG immunotherapy, fur‐

Many adjuvants currently under evaluation for use in cancer vaccines activate relevant APCs, such as dendritic cells and macrophages, via TLRs and promote effective uptake,

ther indicating that CD4+ T cells play an important role in this anti-tumor effect.

es and be used in adjuvant therapy strategies.

678 Melanoma - From Early Detection to Treatment

parent benefit from vaccination [106, 107].

**4.2. DETOX**

Saponins are natural glycosides of steroid or triterpene which exhibit many different bio‐ logical and pharmacological activities [112]. Notably, saponins can activate the mammali‐ an immune system, and this has led to significant interest in their potential as vaccine adjuvants. The most widely used saponin-based adjuvants are Quil A and its derivative QS-21, isolated from the bark of the Quillaja saponaria Molina (Chilean soap bark tree); these have been evaluated in numerous clinical trials [113]. Their unique capacity to stimulate both the Th-1 immune response and the production of CTLs against exogenous antigens makes them ideal for use in subunit vaccines and vaccines directed against in‐ tracellular pathogens as well as cancer.

51 IFA. The formulation is generally well-tolerated and induces transient local reactions. Some transient general reactions such as flu-like symptoms are also observed. The results suggest that numerous repeated vaccine doses can be safely administered. Immunization with tumor associated antigen peptides in combination with montanide expands tumor an‐

Immunomodulation

681

http://dx.doi.org/10.5772/53667

High-dose cyclophosphamide (CY) has long been used as an anti-cancer agent, a condition‐ ing regimen for hematopoietic stem cell transplantation and a potent immunosuppressive agent in autoimmune diseases including aplastic anemia. High-dose CY is highly toxic to lymphocytes but spares hematopoietic stem cells because of their abundant levels of alde‐ hyde dehydrogenase, the major mechanism of CY inactivation. CY has emerged as a clinical‐ ly feasible agent that can suppress Tregs and allow more effective induction of anti-tumor immune responses [123]. Tregs have become an important player in regulating anti-cancer

Studies using low dose CY in combination with vaccine components and IL-12 continue to suggest that CY is a viable addition to affect immune responses [125]. Low-dose CY is found to selectively deplete CD4+CD25+ T cells (Tregs) and impede tolerance allowing for a more active immune response. CY preconditioning can enhance the CD8+ T cell response to pep‐ tide vaccination, thus leading to enhanced anti-tumor effects against pre-existing tumors [126]. CY markedly enhanced the magnitude of secondary but not primary CTL response in‐ duced by vaccines and synergized with vaccine in therapy but not in prophylaxis tumor

The major issues that need to be addressed are designing more effective melanoma vaccines with a mix of melanoma-associated antigens that can stimulate clinically beneficial anti-tu‐ mor immune responses and finding an adjuvant that can safely, easily and powerfully boost

Cytokine therapy has had an important position in the treatment of melanoma in the adju‐ vant and metastatic settings. Various cytokines have been studied with variable success. The most important cytokines in melanoma treatment thus far have been IFN, IL-2, IL-21 and

Interferon IFN is a pleotropic cytokine that exerts anti-tumor activity through numerous mechanisms. High-dose IFNα-2b was approved by the FDA in 1995 for adjuvant therapy of

immune responses, with poor prognoses often ascribed to their action [124].

tigen-specific CD8+ T cells in melanoma patients [119-122].

**4.5. Cyclophosphamide**

models [127].

**4.6. Conclusions**

**5. Cytokine therapy**

GM-CSF.

**5.1. IFN**

the frequency and magnitude of these responses.

QS-21 possesses an ability to clinically augment significant antibody and T cell responses to vaccine antigens against a variety of infectious diseases, degenerative disorders and cancers. Currently, there exists no rapid *in vitro* biological screen for assessing the poten‐ tial efficacy of saponin vaccine adjuvants, given that the mechanism by which saponins augment the immune response is unknown. As a result, evaluation of novel saponins as immunostimulants typically proceed directly to preclinical studies involving mouse vacci‐ nation with antigens [99, 112-114].

QS-21 appears to augment both Th-1 and Th-2 type responses and to favor the *in vivo* pri‐ ming of antigen-specific CD8+ cytotoxic cells. QS-21 has been used in a variety of melanoma targeting vaccines [115]. QS-21 has been shown to be superior to some vaccine formulations such as GM2-KLH plus QS-21 vaccine compared to GM2/BCG vaccine [115]. Efforts to fur‐ ther advance QS-21 in the clinic, as well as to illuminate its unknown mechanism of action, require access to adjuvant-active samples of known composition [114]. The recent synthesis of active molecules of QS-21 has provided a robust method to produce this leading vaccine adjuvant in high purity as well as to produce novel synthetic QS-21 congeners designed to induce increased immune responsiveness and decreased toxicity [99, 112-114].

#### **4.4. Montanide**

Mineral oils are known to be very efficient adjuvants but can sometimes induce local reac‐ tions with reactive antigens. In contrast, non-mineral oils are well tolerated but less effective with poor immunogens. Mineral oils stay at the injection site and are progressively eliminat‐ ed by competent cells like the macrophages. They can also be partially metabolized into fat‐ ty acids, triglycerides, phospholipids or sterols. Water in oil emulsions represent one of the new promising generations of adjuvants for immunotherapy [116-118]. In this class both Montanide ISA 51 and 720 have been tested in animals and thousands of individuals and found to be safe. Nevertheless, the proper antigen concentration has yet to be established [116-118]. Adverse effects usually depend on the concentration and nature of the antigen.

The mechanistic premise of emulsions is the "depot" effect, in which the adjuvant protects the antigen from both dilution and rapid degradation and elimination by the host. By local‐ izing and slowly releasing intact antigen, the adjuvant permits a slow, prolonged exposure of the immune system cells to a low level of antigen. This prolonged exposure results in con‐ tinued stimulation of antibody producing cells, resulting in the production of high levels of antibody by the host.

MONTANIDE™ ISA 51 VG has been used in Phase I and II clinical trials for vaccines against malaria, HIV, and various cancers. MONTANIDE™ ISA 51 VG, has been tested in AIDS and cancer vaccine trials which together represent more than 10,000 patients and around 100,000 injections. A survey of ongoing clinical trials listed in ClinicalTrials.gov re‐ vealed 36 trials currently accruing patients that are using the olive-derived Montanide ISA 51 IFA. The formulation is generally well-tolerated and induces transient local reactions. Some transient general reactions such as flu-like symptoms are also observed. The results suggest that numerous repeated vaccine doses can be safely administered. Immunization with tumor associated antigen peptides in combination with montanide expands tumor an‐ tigen-specific CD8+ T cells in melanoma patients [119-122].

## **4.5. Cyclophosphamide**

stimulate both the Th-1 immune response and the production of CTLs against exogenous antigens makes them ideal for use in subunit vaccines and vaccines directed against in‐

QS-21 possesses an ability to clinically augment significant antibody and T cell responses to vaccine antigens against a variety of infectious diseases, degenerative disorders and cancers. Currently, there exists no rapid *in vitro* biological screen for assessing the poten‐ tial efficacy of saponin vaccine adjuvants, given that the mechanism by which saponins augment the immune response is unknown. As a result, evaluation of novel saponins as immunostimulants typically proceed directly to preclinical studies involving mouse vacci‐

QS-21 appears to augment both Th-1 and Th-2 type responses and to favor the *in vivo* pri‐ ming of antigen-specific CD8+ cytotoxic cells. QS-21 has been used in a variety of melanoma targeting vaccines [115]. QS-21 has been shown to be superior to some vaccine formulations such as GM2-KLH plus QS-21 vaccine compared to GM2/BCG vaccine [115]. Efforts to fur‐ ther advance QS-21 in the clinic, as well as to illuminate its unknown mechanism of action, require access to adjuvant-active samples of known composition [114]. The recent synthesis of active molecules of QS-21 has provided a robust method to produce this leading vaccine adjuvant in high purity as well as to produce novel synthetic QS-21 congeners designed to

Mineral oils are known to be very efficient adjuvants but can sometimes induce local reac‐ tions with reactive antigens. In contrast, non-mineral oils are well tolerated but less effective with poor immunogens. Mineral oils stay at the injection site and are progressively eliminat‐ ed by competent cells like the macrophages. They can also be partially metabolized into fat‐ ty acids, triglycerides, phospholipids or sterols. Water in oil emulsions represent one of the new promising generations of adjuvants for immunotherapy [116-118]. In this class both Montanide ISA 51 and 720 have been tested in animals and thousands of individuals and found to be safe. Nevertheless, the proper antigen concentration has yet to be established [116-118]. Adverse effects usually depend on the concentration and nature of the antigen.

The mechanistic premise of emulsions is the "depot" effect, in which the adjuvant protects the antigen from both dilution and rapid degradation and elimination by the host. By local‐ izing and slowly releasing intact antigen, the adjuvant permits a slow, prolonged exposure of the immune system cells to a low level of antigen. This prolonged exposure results in con‐ tinued stimulation of antibody producing cells, resulting in the production of high levels of

MONTANIDE™ ISA 51 VG has been used in Phase I and II clinical trials for vaccines against malaria, HIV, and various cancers. MONTANIDE™ ISA 51 VG, has been tested in AIDS and cancer vaccine trials which together represent more than 10,000 patients and around 100,000 injections. A survey of ongoing clinical trials listed in ClinicalTrials.gov re‐ vealed 36 trials currently accruing patients that are using the olive-derived Montanide ISA

induce increased immune responsiveness and decreased toxicity [99, 112-114].

tracellular pathogens as well as cancer.

680 Melanoma - From Early Detection to Treatment

nation with antigens [99, 112-114].

**4.4. Montanide**

antibody by the host.

High-dose cyclophosphamide (CY) has long been used as an anti-cancer agent, a condition‐ ing regimen for hematopoietic stem cell transplantation and a potent immunosuppressive agent in autoimmune diseases including aplastic anemia. High-dose CY is highly toxic to lymphocytes but spares hematopoietic stem cells because of their abundant levels of alde‐ hyde dehydrogenase, the major mechanism of CY inactivation. CY has emerged as a clinical‐ ly feasible agent that can suppress Tregs and allow more effective induction of anti-tumor immune responses [123]. Tregs have become an important player in regulating anti-cancer immune responses, with poor prognoses often ascribed to their action [124].

Studies using low dose CY in combination with vaccine components and IL-12 continue to suggest that CY is a viable addition to affect immune responses [125]. Low-dose CY is found to selectively deplete CD4+CD25+ T cells (Tregs) and impede tolerance allowing for a more active immune response. CY preconditioning can enhance the CD8+ T cell response to pep‐ tide vaccination, thus leading to enhanced anti-tumor effects against pre-existing tumors [126]. CY markedly enhanced the magnitude of secondary but not primary CTL response in‐ duced by vaccines and synergized with vaccine in therapy but not in prophylaxis tumor models [127].

## **4.6. Conclusions**

The major issues that need to be addressed are designing more effective melanoma vaccines with a mix of melanoma-associated antigens that can stimulate clinically beneficial anti-tu‐ mor immune responses and finding an adjuvant that can safely, easily and powerfully boost the frequency and magnitude of these responses.

## **5. Cytokine therapy**

Cytokine therapy has had an important position in the treatment of melanoma in the adju‐ vant and metastatic settings. Various cytokines have been studied with variable success. The most important cytokines in melanoma treatment thus far have been IFN, IL-2, IL-21 and GM-CSF.

#### **5.1. IFN**

Interferon IFN is a pleotropic cytokine that exerts anti-tumor activity through numerous mechanisms. High-dose IFNα-2b was approved by the FDA in 1995 for adjuvant therapy of resected stage IIB and III melanoma based on the results of ECOG E1684 [128]. This was a randomized controlled study of IFNα-2b administered at doses of 20 megaunits/m2 /d intra‐ venously (IV) 5 days per week for 1 month and 10 megaunits/m2 3 times per week subcuta‐ neously (s.c.) for 48 weeks versus observation in 287 patients. Through this study IFNα-2b was the first agent to show a significant benefit in relapse-free survival (RFS) and OS of high-risk melanoma patients in a randomized controlled trial. A subsequent study [129] (E1690) with a total of 642 patients evaluated the efficacy of high-dose IFNα-2b for 1 year (20 megaunits/m2 /d IV 5 days/week for 4 weeks; 10 megaunits/m2 s.c. TIW for 48 weeks) and low-dose IFNα-2b (3 megaunits/d TIW) for 2 years versus observation in high-risk (stage IIB and III) melanoma with RFS and OS as end points. The results of the intergroup E1690 trial demonstrated a RFS benefit of IFNα-2b that was dose-dependent and significant for the high-dose INFα-2b. Neither high-dose nor low dose IFNα-2b demonstrated an OS benefit compared with observation at the time.

are approximately 16% with IL-2 with a 6% CR rate [93]. Importantly, some patients ach‐ ieved durable CRs which led to the approval of high-dose IL-2 for patients with metastatic melanoma. Responses occurred with all sites of disease and in patients with large tumor burdens (unlike previously with IFN). Disease progression was not observed in any patient responding for longer than 30 months, and in some cases where disease progression was ob‐

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The use of high-dose IL-2, however, is limited by its severe toxicity; 2.2% of the patients in the National Cancer Institute (NCI) trial series died from treatment-related toxicities with bacterial sepsis being the predominant cause of death. No deaths were observed in the NCI series when antibiotic prophylaxis was implemented. However, the incidence of grade 3-4 toxicities remains high, ranging between 1-64%. Alternate regimens have been employed in‐ cluding low dose IL-2 alone or in combination with IFN-α or chemotherapy. However, there is evidence that suggests that high dose IL-2 is a more efficacious regimen. A phase II study showed durable CRs with high-dose bolus IL-2 in patients with metastatic melanoma who have experienced progression after biochemotherapy [135]. In addition, IL-2 based bioche‐ motherapy regimens have not shown significantly better results than chemotherapy alone,

The role of other cytokines has also been explored. IL-21 has recently emerged as a promising cytokine [137]. In an open-label, multicenter phase II study, IL-21 was given as a bolus injection on days 1 through 5 on alternate weeks using three different dosing regimens in 40 patients with malignant melanoma. Cohort 1 received 50 μg/kg per day by outpatient IV bolus injection for 5 days of each week during weeks 1, 3, and 5 of an 8-week cycle. Cohort 2 received 30 μg/kg per day on the same schedule, and cohort 3 re‐ ceived 50 μg/kg per day for 5 days of each week during weeks 1 and 3 of a 6-week cy‐ cle. The primary objective of the study was to assess efficacy (ORR and PFS) of IL-21 in this population. The ORR to IL-21 was 22.5%. The median PFS was 4.3 months and the median OS was 12.4 months, suggesting that this is an active agent that warrants further investigation. The 30 μg/kg per day dose and schedule was generally well tolerated as an outpatient regimen, with the most common adverse events being flu-like symptoms

Granulocyte macrophage-colony stimulating factor (GM-CSF) has also been studied mostly in the adjuvant setting. Forty-eight patients with stage III or IV melanoma were treated in a phase II trial with long-term, chronic, intermittent GM-CSF after complete surgical resection of disease [138]. The median survival duration was 37.5 months in the study patients versus 12.2 months in the matched controls. OS and DFS were significantly prolonged in patients who received GM-CSF compared with matched historical controls, and treatment was well tolerated with acceptable toxicity. A phase III prospective, randomized, placebo-controlled

served, durable disease free status was achieved with metastasectomy.

presumptively due to the fact that high dose IL-2 is not utilized [136].

and rash, most of which were grade 1 or 2.

**5.3. IL-21**

**5.4. GM-CSF**

Pooled data from E1684 and E1690 showed that RFS, but not OS, was significantly pro‐ longed for patients treated with high dose IFN versus observation [94]. Long term OS data from E1684 also shows a diminishing level of statistical significance (P=.02 at 7 years, but P=. 09 at 12.6 years' median follow-up) [94]. Additional studies by the Eastern Organisation for Research and Treatment of Cancer (EORTC) [130] with adjuvant pegylated IFNα-2b (6 μg/kg per week for 8 weeks followed by 3 μg/kg per week for an intended duration of 5 years) showed a similarly significant and sustained effect on RFS.

The effect of IFNα on OS has been criticized strongly since only two studies (E1684 and E1694) have shown a survival benefit. In contrast, several other studies mentioned above have shown RFS as the only benefit. A recent meta-analysis [131] showed statistically signif‐ icant improvement in both RFS and OS. The meta-analysis included 14 randomized control‐ led trials published between 1990 and 2008 and involved 8,122 patients, of which 4,362 were allocated to the IFNα arm. Subgroup analysis and meta-regression did not identify an opti‐ mal IFNα dose, the optimal treatment duration or a subset of patients more responsive than others to the adjuvant therapy. Therefore, the role of IFN in the adjuvant setting remains controversial by many. The National Comprehensive Cancer Network (NCCN) has a 2B rec‐ ommendation for the use of IFN as an adjuvant treatment, and enrollment in clinical trials is encouraged.

Single-agent IFN has demonstrated modest activity in patients with metastatic malignant melanoma with response rates between 10%-20% [132]. Most of the responses were transient and usually restricted to cutaneous metastases [133]. Therefore, its use in the metastatic set‐ ting has been employed more frequently in combination with chemotherapy (biochemother‐ apy) with improved response rates but without a well documented survival benefit [134]. Other cytokines have been subsequently evaluated, and the interest in IFN has been shifted to the adjuvant setting as mentioned above.

#### **5.2. IL-2**

One of the most promising immune stimulating cytokines has been interleukin 2 (IL-2). High dose IL-2 produces not only PRs but also CRs. Overall objective response rates (ORR) are approximately 16% with IL-2 with a 6% CR rate [93]. Importantly, some patients ach‐ ieved durable CRs which led to the approval of high-dose IL-2 for patients with metastatic melanoma. Responses occurred with all sites of disease and in patients with large tumor burdens (unlike previously with IFN). Disease progression was not observed in any patient responding for longer than 30 months, and in some cases where disease progression was ob‐ served, durable disease free status was achieved with metastasectomy.

The use of high-dose IL-2, however, is limited by its severe toxicity; 2.2% of the patients in the National Cancer Institute (NCI) trial series died from treatment-related toxicities with bacterial sepsis being the predominant cause of death. No deaths were observed in the NCI series when antibiotic prophylaxis was implemented. However, the incidence of grade 3-4 toxicities remains high, ranging between 1-64%. Alternate regimens have been employed in‐ cluding low dose IL-2 alone or in combination with IFN-α or chemotherapy. However, there is evidence that suggests that high dose IL-2 is a more efficacious regimen. A phase II study showed durable CRs with high-dose bolus IL-2 in patients with metastatic melanoma who have experienced progression after biochemotherapy [135]. In addition, IL-2 based bioche‐ motherapy regimens have not shown significantly better results than chemotherapy alone, presumptively due to the fact that high dose IL-2 is not utilized [136].

#### **5.3. IL-21**

resected stage IIB and III melanoma based on the results of ECOG E1684 [128]. This was a

venously (IV) 5 days per week for 1 month and 10 megaunits/m2 3 times per week subcuta‐ neously (s.c.) for 48 weeks versus observation in 287 patients. Through this study IFNα-2b was the first agent to show a significant benefit in relapse-free survival (RFS) and OS of high-risk melanoma patients in a randomized controlled trial. A subsequent study [129] (E1690) with a total of 642 patients evaluated the efficacy of high-dose IFNα-2b for 1 year (20

low-dose IFNα-2b (3 megaunits/d TIW) for 2 years versus observation in high-risk (stage IIB and III) melanoma with RFS and OS as end points. The results of the intergroup E1690 trial demonstrated a RFS benefit of IFNα-2b that was dose-dependent and significant for the high-dose INFα-2b. Neither high-dose nor low dose IFNα-2b demonstrated an OS benefit

Pooled data from E1684 and E1690 showed that RFS, but not OS, was significantly pro‐ longed for patients treated with high dose IFN versus observation [94]. Long term OS data from E1684 also shows a diminishing level of statistical significance (P=.02 at 7 years, but P=. 09 at 12.6 years' median follow-up) [94]. Additional studies by the Eastern Organisation for Research and Treatment of Cancer (EORTC) [130] with adjuvant pegylated IFNα-2b (6 μg/kg per week for 8 weeks followed by 3 μg/kg per week for an intended duration of 5

The effect of IFNα on OS has been criticized strongly since only two studies (E1684 and E1694) have shown a survival benefit. In contrast, several other studies mentioned above have shown RFS as the only benefit. A recent meta-analysis [131] showed statistically signif‐ icant improvement in both RFS and OS. The meta-analysis included 14 randomized control‐ led trials published between 1990 and 2008 and involved 8,122 patients, of which 4,362 were allocated to the IFNα arm. Subgroup analysis and meta-regression did not identify an opti‐ mal IFNα dose, the optimal treatment duration or a subset of patients more responsive than others to the adjuvant therapy. Therefore, the role of IFN in the adjuvant setting remains controversial by many. The National Comprehensive Cancer Network (NCCN) has a 2B rec‐ ommendation for the use of IFN as an adjuvant treatment, and enrollment in clinical trials is

Single-agent IFN has demonstrated modest activity in patients with metastatic malignant melanoma with response rates between 10%-20% [132]. Most of the responses were transient and usually restricted to cutaneous metastases [133]. Therefore, its use in the metastatic set‐ ting has been employed more frequently in combination with chemotherapy (biochemother‐ apy) with improved response rates but without a well documented survival benefit [134]. Other cytokines have been subsequently evaluated, and the interest in IFN has been shifted

One of the most promising immune stimulating cytokines has been interleukin 2 (IL-2). High dose IL-2 produces not only PRs but also CRs. Overall objective response rates (ORR)

/d intra‐

s.c. TIW for 48 weeks) and

randomized controlled study of IFNα-2b administered at doses of 20 megaunits/m2

/d IV 5 days/week for 4 weeks; 10 megaunits/m2

years) showed a similarly significant and sustained effect on RFS.

megaunits/m2

encouraged.

**5.2. IL-2**

compared with observation at the time.

682 Melanoma - From Early Detection to Treatment

to the adjuvant setting as mentioned above.

The role of other cytokines has also been explored. IL-21 has recently emerged as a promising cytokine [137]. In an open-label, multicenter phase II study, IL-21 was given as a bolus injection on days 1 through 5 on alternate weeks using three different dosing regimens in 40 patients with malignant melanoma. Cohort 1 received 50 μg/kg per day by outpatient IV bolus injection for 5 days of each week during weeks 1, 3, and 5 of an 8-week cycle. Cohort 2 received 30 μg/kg per day on the same schedule, and cohort 3 re‐ ceived 50 μg/kg per day for 5 days of each week during weeks 1 and 3 of a 6-week cy‐ cle. The primary objective of the study was to assess efficacy (ORR and PFS) of IL-21 in this population. The ORR to IL-21 was 22.5%. The median PFS was 4.3 months and the median OS was 12.4 months, suggesting that this is an active agent that warrants further investigation. The 30 μg/kg per day dose and schedule was generally well tolerated as an outpatient regimen, with the most common adverse events being flu-like symptoms and rash, most of which were grade 1 or 2.

#### **5.4. GM-CSF**

Granulocyte macrophage-colony stimulating factor (GM-CSF) has also been studied mostly in the adjuvant setting. Forty-eight patients with stage III or IV melanoma were treated in a phase II trial with long-term, chronic, intermittent GM-CSF after complete surgical resection of disease [138]. The median survival duration was 37.5 months in the study patients versus 12.2 months in the matched controls. OS and DFS were significantly prolonged in patients who received GM-CSF compared with matched historical controls, and treatment was well tolerated with acceptable toxicity. A phase III prospective, randomized, placebo-controlled study (E4697) failed to show an OS benefit but improved DFS in patients with completely resected high-risk melanoma with minimal toxicity [139].

50 years [143]. In an expansion of this initial trial to 214 subjects, there was an improvement in OS in patients who developed a positive DTH response (59.3% vs 29.3%; p < 0.001). Forty-

Immunomodulation

685

http://dx.doi.org/10.5772/53667

Work done by the National Biotherapy Study Group utilizing patient specific autologous tu‐ mor cell lines in patients with melanoma has been summarized [145]. This series of studies utilized different adjuvants including BCG, IFNγ and GM-CSF. Once again, benefit was seen in groups developing a positive DTH response [145-147]. Additional non-randomized studies show positive outcomes as well, but without randomization, the results are difficult to weigh [148-151]. Some studies have genetically modified the tumor cells to secret cyto‐ kines. These trials have also been non-randomized but have shown positive results [152, 153]. A randomized trial using an autologous tumor vaccine processed to extract hsps com‐ pared to physician's choice was conducted in 322 patients with metastatic disease. There was no difference between the two groups overall, but subjects with M1a disease had longer

Allogeneic whole cell vaccines are produced by a cell line or cell lines. Other features are similar to the autologous cell vaccines. The advantage of this approach is that it is more readily available and would prevent delays in treatment that are necessitated by the autolo‐ gous vaccines. However the antigens may not match those of the patient's melanoma [141].

A vaccine developed by Dr. Donald Morton beginning in 1984 from three irradiated melano‐ ma cell lines has been studied the most extensively. It is named Canvaxin (CancerVax Corp., Carlsbad, California, USA). There were extensive phase II trials done in patients with stage IV disease demonstrating response to therapy. However, when tested in randomized multicenter trials in resected stage III and IV melanoma patients, there was no benefit noted. However, the trials were stopped prior to their planned accrual by the data safety monitor‐ ing board for futility [141, 155-157]. Other allogeneic vaccines including VACCIMEL (pro‐ duced from three melanoma cell lines) have had less mature study and similar biomarker

Tumor lysate vaccines have similar advantages and disadvantages of allogeneic vaccines. The vaccinia melanoma oncolysate (VMO) vaccine is prepared from four allogeneic cell lines infected with the vaccinia virus to increase immunogenicity. The cells are then lysed by sonication prior to administration. VMO yielded encouraging phase II results, however, there was no statistically significant increase in DFS when studied in an adju‐

A second tumor lysate vaccine, called Melacine (Corixa Corp., Seattle Washington, USA) is also a cell lysate vaccine which has been tested in two large randomized trials. One adjuvant trial conducted by the Southwest Oncology Group (SWOG) studied 600 eligible patients treated with Melacine along with the adjuvant DETOX versus observation. To be

seven percent of subjects had a DTH response [144].

survival when treated with vaccine [154].

**6.2. Allogeneic whole cell vaccines**

results [158].

**6.3. Tumor lysate vaccines**

vant setting [157, 159].

#### **5.5. Conclusions**

Immune stimulating cytokines have historically been an important part of the therapeutic armamentarium for early stage and metastatic melanoma due to the importance of the im‐ mune system in this disease. The currently approved IFN and IL-2 treatments in the adju‐ vant and metastatic settings, respectively, provide modest but reproducible clinical benefits. Their use is limited by toxicity and the lack of clearly defined predictive-to-treatment tools. In the near future, the development of novel molecular and immune treatments might limit their role. However, the durable responses that we see in some patients should not be ignor‐ ed, and the search for predictive biomarkers should continue.

## **6. Vaccine therapy**

The purpose of cancer vaccines is to evoke an immune response against malignant cells. One of the earliest approaches was taken more than 100 years ago when Dr. William Coley treat‐ ed patients with Coley's Toxin derived from bacteria [22]. Although clinical success in indi‐ vidual trials has been uncommon, a meta-analysis of 56 clinical trials showed that evidence of an immune response predicted a better outcome [140]. Vaccines are of various types, each with advantages and disadvantages. The following discussion will be divided by the vac‐ cine type, and when available, clinical data in advanced and adjuvant settings.

#### **6.1. Autologous whole cell vaccines**

Vaccines derived from the patient's own cancer should have the advantage of presenting the complete array of tumor antigens, both internal and external. There should be less chance of the remaining tumor mutating sufficiently to avoid detection. These vaccines should be able to produce both humoral and cellular immunity [141]. Autologous vaccines are produced by irradiating resected tumors or by establishing cell lines from resected specimens [22]. This approach is difficult from a technical and regulatory standpoint. It is further hampered by limiting eligible patients to those with accessible cancer and those who can wait for the vac‐ cine development [141]. These vaccines have used the cells themselves with adjuvants or cells modified to produce cytokines.

An autologous whole cell vaccine is exemplified in work done by Berd, et. al. Early reports demonstrated clinical response to an irradiated autologous tumor cell vaccine given with BCG as an adjuvant. Subsequently, low dose CY preceded vaccination. In that study there were 5 responses in the 40 subjects assessable for response. The responses were associated with DTH responses [142]. In the next series of studies the vaccine was modified by the hapten, dinitrophenyl (DNP), and BCG and CY were maintained. Sixty two subjects with re‐ sected nodal metastases were vaccinated and compared to historical controls. There was a perceived benefit in disease progression and survival, especially in subjects over the age of 50 years [143]. In an expansion of this initial trial to 214 subjects, there was an improvement in OS in patients who developed a positive DTH response (59.3% vs 29.3%; p < 0.001). Fortyseven percent of subjects had a DTH response [144].

Work done by the National Biotherapy Study Group utilizing patient specific autologous tu‐ mor cell lines in patients with melanoma has been summarized [145]. This series of studies utilized different adjuvants including BCG, IFNγ and GM-CSF. Once again, benefit was seen in groups developing a positive DTH response [145-147]. Additional non-randomized studies show positive outcomes as well, but without randomization, the results are difficult to weigh [148-151]. Some studies have genetically modified the tumor cells to secret cyto‐ kines. These trials have also been non-randomized but have shown positive results [152, 153]. A randomized trial using an autologous tumor vaccine processed to extract hsps com‐ pared to physician's choice was conducted in 322 patients with metastatic disease. There was no difference between the two groups overall, but subjects with M1a disease had longer survival when treated with vaccine [154].

### **6.2. Allogeneic whole cell vaccines**

study (E4697) failed to show an OS benefit but improved DFS in patients with completely

Immune stimulating cytokines have historically been an important part of the therapeutic armamentarium for early stage and metastatic melanoma due to the importance of the im‐ mune system in this disease. The currently approved IFN and IL-2 treatments in the adju‐ vant and metastatic settings, respectively, provide modest but reproducible clinical benefits. Their use is limited by toxicity and the lack of clearly defined predictive-to-treatment tools. In the near future, the development of novel molecular and immune treatments might limit their role. However, the durable responses that we see in some patients should not be ignor‐

The purpose of cancer vaccines is to evoke an immune response against malignant cells. One of the earliest approaches was taken more than 100 years ago when Dr. William Coley treat‐ ed patients with Coley's Toxin derived from bacteria [22]. Although clinical success in indi‐ vidual trials has been uncommon, a meta-analysis of 56 clinical trials showed that evidence of an immune response predicted a better outcome [140]. Vaccines are of various types, each with advantages and disadvantages. The following discussion will be divided by the vac‐

Vaccines derived from the patient's own cancer should have the advantage of presenting the complete array of tumor antigens, both internal and external. There should be less chance of the remaining tumor mutating sufficiently to avoid detection. These vaccines should be able to produce both humoral and cellular immunity [141]. Autologous vaccines are produced by irradiating resected tumors or by establishing cell lines from resected specimens [22]. This approach is difficult from a technical and regulatory standpoint. It is further hampered by limiting eligible patients to those with accessible cancer and those who can wait for the vac‐ cine development [141]. These vaccines have used the cells themselves with adjuvants or

An autologous whole cell vaccine is exemplified in work done by Berd, et. al. Early reports demonstrated clinical response to an irradiated autologous tumor cell vaccine given with BCG as an adjuvant. Subsequently, low dose CY preceded vaccination. In that study there were 5 responses in the 40 subjects assessable for response. The responses were associated with DTH responses [142]. In the next series of studies the vaccine was modified by the hapten, dinitrophenyl (DNP), and BCG and CY were maintained. Sixty two subjects with re‐ sected nodal metastases were vaccinated and compared to historical controls. There was a perceived benefit in disease progression and survival, especially in subjects over the age of

cine type, and when available, clinical data in advanced and adjuvant settings.

resected high-risk melanoma with minimal toxicity [139].

ed, and the search for predictive biomarkers should continue.

**5.5. Conclusions**

684 Melanoma - From Early Detection to Treatment

**6. Vaccine therapy**

**6.1. Autologous whole cell vaccines**

cells modified to produce cytokines.

Allogeneic whole cell vaccines are produced by a cell line or cell lines. Other features are similar to the autologous cell vaccines. The advantage of this approach is that it is more readily available and would prevent delays in treatment that are necessitated by the autolo‐ gous vaccines. However the antigens may not match those of the patient's melanoma [141].

A vaccine developed by Dr. Donald Morton beginning in 1984 from three irradiated melano‐ ma cell lines has been studied the most extensively. It is named Canvaxin (CancerVax Corp., Carlsbad, California, USA). There were extensive phase II trials done in patients with stage IV disease demonstrating response to therapy. However, when tested in randomized multicenter trials in resected stage III and IV melanoma patients, there was no benefit noted. However, the trials were stopped prior to their planned accrual by the data safety monitor‐ ing board for futility [141, 155-157]. Other allogeneic vaccines including VACCIMEL (pro‐ duced from three melanoma cell lines) have had less mature study and similar biomarker results [158].

#### **6.3. Tumor lysate vaccines**

Tumor lysate vaccines have similar advantages and disadvantages of allogeneic vaccines. The vaccinia melanoma oncolysate (VMO) vaccine is prepared from four allogeneic cell lines infected with the vaccinia virus to increase immunogenicity. The cells are then lysed by sonication prior to administration. VMO yielded encouraging phase II results, however, there was no statistically significant increase in DFS when studied in an adju‐ vant setting [157, 159].

A second tumor lysate vaccine, called Melacine (Corixa Corp., Seattle Washington, USA) is also a cell lysate vaccine which has been tested in two large randomized trials. One adjuvant trial conducted by the Southwest Oncology Group (SWOG) studied 600 eligible patients treated with Melacine along with the adjuvant DETOX versus observation. To be eligible the subjects had to have intermediate thickness lesions and negative nodes; how‐ ever, sentinel lymph node biopsy was not required, and therefore, the staging in this tri‐ al would be considered inadequate by today's standards. Survival in the overall analysis showed no difference between treatment and observation, however, a subgroup of pa‐ tients expressing certain MHC classes showed a five-year survival rate of 83% compared to 59% in the observation group. This subset analysis was statistically significant [160-163]. An Ad Hoc Melanoma Working Group reported a separate study in stage III resected patients comparing high dose IFN for one year versus low lose IFN plus Mela‐ cine with DETOX for 2 years. Six hundred subjects were registered. There was no differ‐ ence in outcome [164]. As described above, high dose IFN had been shown previously to be superior to observation.

ing a multi-epitope vaccine with GM-CSF and/or IFNα-2b showed that an immune re‐ sponse to the vaccine correlated with outcome but that the cytokines did not affect

Immunomodulation

687

http://dx.doi.org/10.5772/53667

One of the most studied peptide vaccines is a modified gp100 peptide antigen. This vac‐ cine has been studied in locally advanced stage III or stage IV patients comparing IL-2 to IL-2 plus vaccine. The results showed a statistically significant improvement in response rate and PFS in the vaccine arm [177]. This is in contrast to a report from the Cytokine Working Group analysis of three phase II trials looking at a similar vaccine with IL-2 showing no benefit to the addition of the vaccine [178]. Another phase III study compar‐ ing ipilimumab plus gp100 vaccine versus gp100 plus placebo versus ipilimumab plus placebo failed to show a benefit to the addition of the vaccine, and the vaccine alone

Anti-idiotype vaccines consist of monoclonal antibodies that mimic an antigen. The theoreti‐ cal hypothesis is sound [179-181], but trials have been limited [182-184]. These vaccines have

Viral vectors can boost the immunogenicity of the vaccines they carry [141, 185]. However the presence of neutralizing antibodies in the host could play a role [186]. Novel methods of utilizing this mode of immune stimulation are still being explored [187, 188]. A randomized trial in stage III resected patients utilizing a vaccinia viral lysate vaccine failed to show bene‐ fit [189]. Transduction of cell lines to produce expression of B7-1and IL-2 have been accom‐ plished and show promising immunostimulatory effects [190, 191]. These techniques are difficult to pursue from a technical and regulatory standpoint. Recombinant viral vaccines

DNA vaccines have the advantage of specificity for the target for which they encode, which can simplify monitoring, but in general they have not done well in breaking tolerance [141, 193-195]. Two groups have reported on vaccines that rely on production of GM-CSF. Dran‐ off has reported on a vaccine utilizing melanoma cell lines engineered to produce GM-CSF and has noted improved anti-tumor effects [196]. A different approach using an intralesional vaccination with an oncolytic herpesvirus encoding GM-CSF has been developed and has had initial positive results [197, 198]. Another agent called Allovectin-7 consists of a plasmid containing DNA encoding for the MHC class I gene, HLA-B7. It was administered by intra‐ lesional injection. Early studies showed evidence of biologic activity [199]. Subsequent phase II studies showed efficacy locally as well as systemically [200-203]. A phase III study has not

outcome [176].

was inferior [95].

**6.8. Viral vaccines**

**6.9. DNA vaccines**

yet been reported.

**6.7. Monoclonal antibodies**

not been tested prospectively.

have also been used to prime dendritic cells [192].

### **6.4. Protein vaccines**

Protein vaccines using purified proteins have the potential for a broader spectrum of anti‐ gens, but they can be more complex to manufacture and monitor for response [157]. The use of hsps, which have a normal function in chaperoning proteins as they are processed into peptides, has also been explored with peptide vaccines [154] and may have a role in identi‐ fying new antigenic targets [165]. A trial using NY-ESO was found to produce a strong im‐ munologic response after vaccination. There were better clinical outcomes compared to placebo in those given the protein vaccine compared to placebo. The adjuvant used in the trial was ISCOMATRIX [166].

### **6.5. Ganglioside antigen vaccines**

Gangliosides are non-protein antigens (glycosphingolipids containing sialic acids) that have been shown to elicit antibodies. They are present on melanoma cells (GM2, GD2, GD3) [157, 167]. The GM2 antigen plus BCG versus BCG alone was studied in stage III resected pa‐ tients. There was no difference in DFS, but subjects with IgM antibodies against the antigen had better outcome [167]. GM2 conjugated to a keyhole limpet hemocyanin (KLH) and ad‐ ministered with the adjuvant QS-21 had better immunogenicity [168]. This same vaccine was compared to high dose IFN in an Intergroup adjuvant trial in patients with resected stage IIB or III melanoma. The trial ended when an interim analysis showed therapeutic in‐ feriority in the vaccine arm [169].

#### **6.6. Peptide vaccines**

Peptide vaccines have the advantage of being easy to manufacture and have an excellent safety record. However, there are challenges that impact their effectiveness. These in‐ clude the identification of epitopes that stimulate a T cell response, selecting an appropri‐ ate adjuvant, breaking tolerance without causing limiting autoimmunity, handling MHC restriction and assessing the need for multi-epitope vaccines [170]. Peptide vaccines have been reported to increase survival following resection of metastatic lesions [171] and have been shown to have increased immune efficacy with various adjuvants [172, 173]. Use of multiple peptides has been another strategy [121, 174, 175]. An ECOG study test‐ ing a multi-epitope vaccine with GM-CSF and/or IFNα-2b showed that an immune re‐ sponse to the vaccine correlated with outcome but that the cytokines did not affect outcome [176].

One of the most studied peptide vaccines is a modified gp100 peptide antigen. This vac‐ cine has been studied in locally advanced stage III or stage IV patients comparing IL-2 to IL-2 plus vaccine. The results showed a statistically significant improvement in response rate and PFS in the vaccine arm [177]. This is in contrast to a report from the Cytokine Working Group analysis of three phase II trials looking at a similar vaccine with IL-2 showing no benefit to the addition of the vaccine [178]. Another phase III study compar‐ ing ipilimumab plus gp100 vaccine versus gp100 plus placebo versus ipilimumab plus placebo failed to show a benefit to the addition of the vaccine, and the vaccine alone was inferior [95].

## **6.7. Monoclonal antibodies**

Anti-idiotype vaccines consist of monoclonal antibodies that mimic an antigen. The theoreti‐ cal hypothesis is sound [179-181], but trials have been limited [182-184]. These vaccines have not been tested prospectively.

### **6.8. Viral vaccines**

eligible the subjects had to have intermediate thickness lesions and negative nodes; how‐ ever, sentinel lymph node biopsy was not required, and therefore, the staging in this tri‐ al would be considered inadequate by today's standards. Survival in the overall analysis showed no difference between treatment and observation, however, a subgroup of pa‐ tients expressing certain MHC classes showed a five-year survival rate of 83% compared to 59% in the observation group. This subset analysis was statistically significant [160-163]. An Ad Hoc Melanoma Working Group reported a separate study in stage III resected patients comparing high dose IFN for one year versus low lose IFN plus Mela‐ cine with DETOX for 2 years. Six hundred subjects were registered. There was no differ‐ ence in outcome [164]. As described above, high dose IFN had been shown previously to

Protein vaccines using purified proteins have the potential for a broader spectrum of anti‐ gens, but they can be more complex to manufacture and monitor for response [157]. The use of hsps, which have a normal function in chaperoning proteins as they are processed into peptides, has also been explored with peptide vaccines [154] and may have a role in identi‐ fying new antigenic targets [165]. A trial using NY-ESO was found to produce a strong im‐ munologic response after vaccination. There were better clinical outcomes compared to placebo in those given the protein vaccine compared to placebo. The adjuvant used in the

Gangliosides are non-protein antigens (glycosphingolipids containing sialic acids) that have been shown to elicit antibodies. They are present on melanoma cells (GM2, GD2, GD3) [157, 167]. The GM2 antigen plus BCG versus BCG alone was studied in stage III resected pa‐ tients. There was no difference in DFS, but subjects with IgM antibodies against the antigen had better outcome [167]. GM2 conjugated to a keyhole limpet hemocyanin (KLH) and ad‐ ministered with the adjuvant QS-21 had better immunogenicity [168]. This same vaccine was compared to high dose IFN in an Intergroup adjuvant trial in patients with resected stage IIB or III melanoma. The trial ended when an interim analysis showed therapeutic in‐

Peptide vaccines have the advantage of being easy to manufacture and have an excellent safety record. However, there are challenges that impact their effectiveness. These in‐ clude the identification of epitopes that stimulate a T cell response, selecting an appropri‐ ate adjuvant, breaking tolerance without causing limiting autoimmunity, handling MHC restriction and assessing the need for multi-epitope vaccines [170]. Peptide vaccines have been reported to increase survival following resection of metastatic lesions [171] and have been shown to have increased immune efficacy with various adjuvants [172, 173]. Use of multiple peptides has been another strategy [121, 174, 175]. An ECOG study test‐

be superior to observation.

686 Melanoma - From Early Detection to Treatment

trial was ISCOMATRIX [166].

**6.5. Ganglioside antigen vaccines**

feriority in the vaccine arm [169].

**6.6. Peptide vaccines**

**6.4. Protein vaccines**

Viral vectors can boost the immunogenicity of the vaccines they carry [141, 185]. However the presence of neutralizing antibodies in the host could play a role [186]. Novel methods of utilizing this mode of immune stimulation are still being explored [187, 188]. A randomized trial in stage III resected patients utilizing a vaccinia viral lysate vaccine failed to show bene‐ fit [189]. Transduction of cell lines to produce expression of B7-1and IL-2 have been accom‐ plished and show promising immunostimulatory effects [190, 191]. These techniques are difficult to pursue from a technical and regulatory standpoint. Recombinant viral vaccines have also been used to prime dendritic cells [192].

#### **6.9. DNA vaccines**

DNA vaccines have the advantage of specificity for the target for which they encode, which can simplify monitoring, but in general they have not done well in breaking tolerance [141, 193-195]. Two groups have reported on vaccines that rely on production of GM-CSF. Dran‐ off has reported on a vaccine utilizing melanoma cell lines engineered to produce GM-CSF and has noted improved anti-tumor effects [196]. A different approach using an intralesional vaccination with an oncolytic herpesvirus encoding GM-CSF has been developed and has had initial positive results [197, 198]. Another agent called Allovectin-7 consists of a plasmid containing DNA encoding for the MHC class I gene, HLA-B7. It was administered by intra‐ lesional injection. Early studies showed evidence of biologic activity [199]. Subsequent phase II studies showed efficacy locally as well as systemically [200-203]. A phase III study has not yet been reported.

## **7. Cellular therapy**

The transfer of immunologically competent white blood cells or their precursors into the host (cellular adoptive immunotherapy, adoptive cell treatment, adoptive cell therapy [ACT]) has been studied extensively in patients with melanoma over the last 30 years. Since it was thought that the effect of IL-2 is potentiated by this form of therapy, various studies examined the role of combination regimens with lymphokine-activated killer cells (LAK cells) or TILs with or without lymphodepletion with mixed results.

study that evaluated the role of dendritic cells pulsed with Mage-3A1 tumor peptide and a recall antigen, tetanus toxoid or tuberculin, 6 of 11 patients with advanced stage IV melanoma experienced significant regression of their metastases [210]. Resolution of skin metastases in two of the patients was accompanied by CD8+ T cell infiltration, whereas

Immunomodulation

689

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In another trial [211] 16 patients with metastatic stage IV melanoma were treated with den‐ dritic cells derived from incubation of peripheral blood mononuclear cells with IL-4 and GM-CSF and overnight pulsing with several peptides (tyrosinase, gp100 and MART-1). One patient had a complete remission of lung and pleural disease after two cycles of therapy. Two additional patients had SD, and two patients had mixed responses. In general, review‐ ing over 30 studies that employed dendritic cell-based treatments [212], it appears that clini‐ cal response (defined as CR, PR or SD) was significantly correlated with the use of peptide antigens, use of helper antigen or adjuvant and induction of tumor antigen specific T cells.

Although there appears to be a real effect of these treatments on tumor response in a subset of the treated patients, undoubtedly the success has not been universal and convincing [213]. This could be due to Tregs that counteract the effect of dendritic cells. In addition, melanoma can also mediate dendritic cell suppression possibly through the activation of the MEK1/2-p44/42 axis [214]. Finally, little is known about optimal dendritic cell generation,

Monoclonal antibodies targeted against a number of regulatory immune system checkpoints are being evaluated in patients with advanced melanoma. The recently approved ipilimu‐

Ipilimumab is a monoclonal antibody against cytotoxic T-lymphocyte antigen 4 (CTLA-4). In two phase III trials ipilimumab showed improved OS in patients with advanced melano‐ ma. In the first one [95], 676 HLA-A\*0201–positive patients with unresectable stage III or IV melanoma, whose disease had progressed while they were receiving therapy for metastatic disease were studied. More than 70% of the patients had M1c disease (presence of visceral metastases), and more than 36% had elevated lactate dehydrogenase levels. The patients were randomly assigned, in a 3:1:1 ratio, to receive ipilimumab plus gp100, ipilimumab alone or gp100 alone. Ipilimumab, at a dose of 3 mg/kg of body weight, was administered with or without gp100 every three weeks for up to four treatments. HLA-A\*0201–positivity was required because of the use of the gp100 vaccine. Certain patients were allowed to have

mab remains the prototype, but others are currently being evaluated in several trials.

another course of treatment upon progression. The primary end point was OS.

administration and immune monitoring which could hamper progress in this field.

nonregressing lesions lacked CD8+ T cells.

**8. Enhancement of cellular immunity**

**8.1. Checkpoint inhibitors**

**8.2. Ipilimumab**

## **7.1. TILs**

Earlier studies with LAK cells [204] showed promise, improving the responses with IL-2. A randomized study [205] with IL-2 and LAK cells compared to IL-2 alone failed to show sig‐ nificant improvement in survival which tempered the initial enthusiasm. Subsequent stud‐ ies with Tumor Infiltrating Lymphocytes (TILs) [206] showed some response to treatment (overall ORR in these patients was 34%) when combined with IL-2. Interestingly, there was no significant difference in the ORR in patients whose therapy with high-dose IL-2 had failed (32%) compared with patients not previously treated with IL-2 (34%). However, the responses appeared to be short-lived, probably due to the transient persistence of the trans‐ ferred TILs [207].

The addition of lymphodepletion has been thought to promote the persistence of the trans‐ ferred TILs by eliminating the regulatory cells. Pooled data from three clinical trials employ‐ ing three different lymphodepleting regimens [91] showed high responses between 49-72%. Ninety-five percent of these patients had progressive disease following a prior systemic treatment. Twenty of the 93 patients (22%) achieved complete tumor regression, and 19 have ongoing complete regressions beyond 3 years. The actuarial 3- and 5-year survival rates for the entire group were 36% and 29%, respectively, but for the 20 complete responders were 100% and 93%. Factors associated with objective response included longer telomeres of the infused cells, the number of CD8+ CD27+ cells infused and the persistence of the infused cells in the circulation at one month. This treatment appears to also be helpful in the treat‐ ment of intracranial disease [208].

At this point, there is reserved enthusiasm about the role of TILs with lymphodepletion reg‐ imens. There are several programs within the United States [209] outside of the NCI, where this work was pioneered, and internationally more groups are starting to employ similar strategies.

#### **7.2. Dendritic cells**

Another form of ACT is the infusion of dendritic cells. There is a lot of interest in devel‐ oping dendritic cell based immunotherapy strategies since the approval of sipuleucel-T, an autologous dendritic cell based immunotherapy in hormone refractory prostate can‐ cer. Dendritic cells are believed to induce a Th-1 response which activates CTLs through processing and presenting of peptides derived from the tumor protein antigens. In a study that evaluated the role of dendritic cells pulsed with Mage-3A1 tumor peptide and a recall antigen, tetanus toxoid or tuberculin, 6 of 11 patients with advanced stage IV melanoma experienced significant regression of their metastases [210]. Resolution of skin metastases in two of the patients was accompanied by CD8+ T cell infiltration, whereas nonregressing lesions lacked CD8+ T cells.

In another trial [211] 16 patients with metastatic stage IV melanoma were treated with den‐ dritic cells derived from incubation of peripheral blood mononuclear cells with IL-4 and GM-CSF and overnight pulsing with several peptides (tyrosinase, gp100 and MART-1). One patient had a complete remission of lung and pleural disease after two cycles of therapy. Two additional patients had SD, and two patients had mixed responses. In general, review‐ ing over 30 studies that employed dendritic cell-based treatments [212], it appears that clini‐ cal response (defined as CR, PR or SD) was significantly correlated with the use of peptide antigens, use of helper antigen or adjuvant and induction of tumor antigen specific T cells.

Although there appears to be a real effect of these treatments on tumor response in a subset of the treated patients, undoubtedly the success has not been universal and convincing [213]. This could be due to Tregs that counteract the effect of dendritic cells. In addition, melanoma can also mediate dendritic cell suppression possibly through the activation of the MEK1/2-p44/42 axis [214]. Finally, little is known about optimal dendritic cell generation, administration and immune monitoring which could hamper progress in this field.

## **8. Enhancement of cellular immunity**

## **8.1. Checkpoint inhibitors**

Monoclonal antibodies targeted against a number of regulatory immune system checkpoints are being evaluated in patients with advanced melanoma. The recently approved ipilimu‐ mab remains the prototype, but others are currently being evaluated in several trials.

## **8.2. Ipilimumab**

**7. Cellular therapy**

688 Melanoma - From Early Detection to Treatment

**7.1. TILs**

ferred TILs [207].

strategies.

**7.2. Dendritic cells**

ment of intracranial disease [208].

The transfer of immunologically competent white blood cells or their precursors into the host (cellular adoptive immunotherapy, adoptive cell treatment, adoptive cell therapy [ACT]) has been studied extensively in patients with melanoma over the last 30 years. Since it was thought that the effect of IL-2 is potentiated by this form of therapy, various studies examined the role of combination regimens with lymphokine-activated killer cells (LAK

Earlier studies with LAK cells [204] showed promise, improving the responses with IL-2. A randomized study [205] with IL-2 and LAK cells compared to IL-2 alone failed to show sig‐ nificant improvement in survival which tempered the initial enthusiasm. Subsequent stud‐ ies with Tumor Infiltrating Lymphocytes (TILs) [206] showed some response to treatment (overall ORR in these patients was 34%) when combined with IL-2. Interestingly, there was no significant difference in the ORR in patients whose therapy with high-dose IL-2 had failed (32%) compared with patients not previously treated with IL-2 (34%). However, the responses appeared to be short-lived, probably due to the transient persistence of the trans‐

The addition of lymphodepletion has been thought to promote the persistence of the trans‐ ferred TILs by eliminating the regulatory cells. Pooled data from three clinical trials employ‐ ing three different lymphodepleting regimens [91] showed high responses between 49-72%. Ninety-five percent of these patients had progressive disease following a prior systemic treatment. Twenty of the 93 patients (22%) achieved complete tumor regression, and 19 have ongoing complete regressions beyond 3 years. The actuarial 3- and 5-year survival rates for the entire group were 36% and 29%, respectively, but for the 20 complete responders were 100% and 93%. Factors associated with objective response included longer telomeres of the infused cells, the number of CD8+ CD27+ cells infused and the persistence of the infused cells in the circulation at one month. This treatment appears to also be helpful in the treat‐

At this point, there is reserved enthusiasm about the role of TILs with lymphodepletion reg‐ imens. There are several programs within the United States [209] outside of the NCI, where this work was pioneered, and internationally more groups are starting to employ similar

Another form of ACT is the infusion of dendritic cells. There is a lot of interest in devel‐ oping dendritic cell based immunotherapy strategies since the approval of sipuleucel-T, an autologous dendritic cell based immunotherapy in hormone refractory prostate can‐ cer. Dendritic cells are believed to induce a Th-1 response which activates CTLs through processing and presenting of peptides derived from the tumor protein antigens. In a

cells) or TILs with or without lymphodepletion with mixed results.

Ipilimumab is a monoclonal antibody against cytotoxic T-lymphocyte antigen 4 (CTLA-4). In two phase III trials ipilimumab showed improved OS in patients with advanced melano‐ ma. In the first one [95], 676 HLA-A\*0201–positive patients with unresectable stage III or IV melanoma, whose disease had progressed while they were receiving therapy for metastatic disease were studied. More than 70% of the patients had M1c disease (presence of visceral metastases), and more than 36% had elevated lactate dehydrogenase levels. The patients were randomly assigned, in a 3:1:1 ratio, to receive ipilimumab plus gp100, ipilimumab alone or gp100 alone. Ipilimumab, at a dose of 3 mg/kg of body weight, was administered with or without gp100 every three weeks for up to four treatments. HLA-A\*0201–positivity was required because of the use of the gp100 vaccine. Certain patients were allowed to have another course of treatment upon progression. The primary end point was OS.

The median OS was 10.0 months among patients receiving ipilimumab plus gp100, as com‐ pared with 6.4 months among patients receiving gp100 alone (hazard ratio for death, 0.68; P<0.001). The median OS with ipilimumab alone was 10.1 months (hazard ratio for death in comparison with gp100 alone, 0.66; P=0.003). No difference in OS was detected between the ipilimumab groups (hazard ratio with ipilimumab plus gp100, 1.04; P=0.76). The best OR or SD was seen in the ipilimumab-alone group (10.9%) and a disease control rate (the propor‐ tion of patients with a PR, CR or SD) of 28.5%. In the ipilimumab-alone group, 60.0% main‐ tained an OR for at least two years. Responses to ipilimumab continued to improve beyond week 24: in the ipilimumab-alone group, two patients with SD improved to a PR, and three with a PR improved to a CR. Interestingly, among 31 patients given reinduction therapy with ipilimumab, a PR, CR or SD was achieved by 21 weeks. Grade 3 or 4 immune-related adverse events occurred in 10 to 15% of patients treated with ipilimumab and in 3% treated with gp100 alone. There were 14 deaths related to the study drugs (2.1%), and seven were associated with immune-related adverse events.

The experience with ipilimumab has shown that a subgroup of patients may experience a late response and more interestingly, some patients exhibit apparent disease progression af‐ ter 12 weeks of ipilimumab followed by subsequent disease regression [218]. Therefore, tra‐ ditional criteria (e.g. RECIST) may not apply in the evaluation of patients who receive ipilimumab or similar treatments, and different criteria may need to be established in the in‐

Immunomodulation

691

http://dx.doi.org/10.5772/53667

Based on the favorable results from the ipilimumab studies, other anti-CTLA4 antibodies are currently being evaluated such as tremelimumab. Tremelimumab has a longer half-life than ipilimumab and is dosed less frequently. Phase II data showed results similar to ipilimumab with durable responses suggesting a potential role for tremelimumab in melanoma [219]. However, a phase III trial comparing tremelimumab and chemotherapy failed to demon‐

The toxicity of ipilimumab appears to be related to the increased activation of the immune system. A variety of immune mediated adverse events have been observed. Some of them are life-threatening and the most common are enterocolitis, hepatitis, dermatitis and endo‐ crinopathies, but others such as neurologic complications, ocular symptoms, hematologic manifestations, vasculitis, et al are also observed.The prompt administration of corticoste‐ roids is paramount when these are observed, and in some cases treatment interruption or permanent discontinuation is required. A relationship between the development of side ef‐

Another regulatory checkpoint is Programmed Death-1 receptor (PD-1). Its inhibition is cur‐ rently being evaluated in clinical trials. The PD-1 and PD ligand-1 (PD-L1) interaction is be‐ lieved to affect T cell anti-tumor immunity. Many tumors express high levels of PD-L1. When PD-L1 interacts with the PD-1 receptor on T cells, T cell function is impaired through a variety of mechanisms including induction of apoptosis, suppression of proliferation and inhibition of T cell cytokine production [96]. There are several ways to target PD-1; one way is targeting the PD-1 receptor and another is targeting the PD-1 ligand. There are several molecules currently under investigation that target the PD-1 receptor directly (BMS-936558, CT-011, MK-3475). In a recent study [98], BMS-936558 showed significant anti-tumor activity in a variety of solid tumors. In melanoma patients response rates were 28% and appeared to be durable. Interestingly, of 17 patients with PD-L1–negative tumors, none had an OR while 9 of 25 patients (36%) with PD-L1–positive tumors had an OR (P=0.006). CT-011 has demon‐ strated favorable results in hematologic malignancies but has not been well studied in mela‐ noma patients yet [221], although there is an ongoing phase I study being conducted at present. The role of MK-3475 in melanoma is also currently being evaluated in a phase I tri‐ al. Another molecule which is not probably directly targeting the PD-1 is AMP-224; it is a fusion protein of B7-DC and an antibody Fc portion. It is not a monoclonal antibody like the three checkpoint agents mentioned previously, and no data has been reported yet regarding

fects and anti-tumor activity has been proposed by several investigators [218].

terpretation of efficacy data in clinical trials [20].

strate an improvement in OS [220].

**8.3. Toxicity with ipilimumab**

**8.4. PD-1**

In the second phase III study [215], 502 patients with previously untreated metastatic mela‐ noma were assigned in a 1:1 ratio to receive 10 mg/kg ipilimumab plus 850 mg/m2 dacarba‐ zine or 850 mg/m2 dacarbazine plus placebo, given at weeks 1, 4, 7 and 10, followed by 850 mg/m2 dacarbazine alone every three weeks through week 22. Patients with SD or an OR and no dose-limiting toxic effects received ipilimumab or placebo every 12 weeks thereafter as maintenance therapy. Similarly with the previous study a significant number of patients had poor prognosis based on the presence of visceral metastases and increased lactate dehy‐ drogenase. The primary end point was OS.

OS was significantly longer in the group receiving ipilimumab plus dacarbazine than in the group receiving dacarbazine plus placebo (11.2 months vs. 9.1 months), with higher survival rates in the ipilimumab–dacarbazine group at one year (47.3% vs. 36.3%), two years (28.5% vs. 17.9%), and three years (20.8% vs. 12.2%) (hazard ratio for death, 0.72; P<0.001). The rate of disease control (PR, CR or SD) did not differ significantly between the two groups: 33.2% in the ipilimumab–dacarbazine group and 30.2% in the dacarbazine group (P=0.41). The rate of best OR (PR or CR) was 15.2% in the ipilimumab–dacarbazine group and 10.3% in the da‐ carbazine group (P=0.09). However, the median duration of response among all patients with a PR or CR was 19.3 months (95% CI, 12.1 to 26.1) in the ipilimumab–dacarbazine group and 8.1 months (95% CI, 5.19 to 19.8) in the dacarbazine group (P=0.03). In addition, some patients in the study who were receiving ipilimumab had an improvement from PR to CR after six months. Grade 3 or 4 adverse events occurred in 56.3% of patients treated with ipilimumab plus dacarbazine, as compared with 27.5% treated with dacarbazine and place‐ bo (P<0.001). No drug-related deaths or gastrointestinal perforations occurred in the ipili‐ mumab–dacarbazine group.

Patients with untreated brain metastases were excluded from both phase III studies. Howev‐ er, phase II data indicate that ipilimumab has activity in patients with brain metastases [216]. The currently approved dose is 3 mg/kg based on the registration trial [95], however other doses and schedules [217] have been used that do not appear to produce significantly different results but appear to increase toxicity.

The experience with ipilimumab has shown that a subgroup of patients may experience a late response and more interestingly, some patients exhibit apparent disease progression af‐ ter 12 weeks of ipilimumab followed by subsequent disease regression [218]. Therefore, tra‐ ditional criteria (e.g. RECIST) may not apply in the evaluation of patients who receive ipilimumab or similar treatments, and different criteria may need to be established in the in‐ terpretation of efficacy data in clinical trials [20].

Based on the favorable results from the ipilimumab studies, other anti-CTLA4 antibodies are currently being evaluated such as tremelimumab. Tremelimumab has a longer half-life than ipilimumab and is dosed less frequently. Phase II data showed results similar to ipilimumab with durable responses suggesting a potential role for tremelimumab in melanoma [219]. However, a phase III trial comparing tremelimumab and chemotherapy failed to demon‐ strate an improvement in OS [220].

## **8.3. Toxicity with ipilimumab**

The toxicity of ipilimumab appears to be related to the increased activation of the immune system. A variety of immune mediated adverse events have been observed. Some of them are life-threatening and the most common are enterocolitis, hepatitis, dermatitis and endo‐ crinopathies, but others such as neurologic complications, ocular symptoms, hematologic manifestations, vasculitis, et al are also observed.The prompt administration of corticoste‐ roids is paramount when these are observed, and in some cases treatment interruption or permanent discontinuation is required. A relationship between the development of side ef‐ fects and anti-tumor activity has been proposed by several investigators [218].

#### **8.4. PD-1**

The median OS was 10.0 months among patients receiving ipilimumab plus gp100, as com‐ pared with 6.4 months among patients receiving gp100 alone (hazard ratio for death, 0.68; P<0.001). The median OS with ipilimumab alone was 10.1 months (hazard ratio for death in comparison with gp100 alone, 0.66; P=0.003). No difference in OS was detected between the ipilimumab groups (hazard ratio with ipilimumab plus gp100, 1.04; P=0.76). The best OR or SD was seen in the ipilimumab-alone group (10.9%) and a disease control rate (the propor‐ tion of patients with a PR, CR or SD) of 28.5%. In the ipilimumab-alone group, 60.0% main‐ tained an OR for at least two years. Responses to ipilimumab continued to improve beyond week 24: in the ipilimumab-alone group, two patients with SD improved to a PR, and three with a PR improved to a CR. Interestingly, among 31 patients given reinduction therapy with ipilimumab, a PR, CR or SD was achieved by 21 weeks. Grade 3 or 4 immune-related adverse events occurred in 10 to 15% of patients treated with ipilimumab and in 3% treated with gp100 alone. There were 14 deaths related to the study drugs (2.1%), and seven were

In the second phase III study [215], 502 patients with previously untreated metastatic mela‐ noma were assigned in a 1:1 ratio to receive 10 mg/kg ipilimumab plus 850 mg/m2 dacarba‐

OS was significantly longer in the group receiving ipilimumab plus dacarbazine than in the group receiving dacarbazine plus placebo (11.2 months vs. 9.1 months), with higher survival rates in the ipilimumab–dacarbazine group at one year (47.3% vs. 36.3%), two years (28.5% vs. 17.9%), and three years (20.8% vs. 12.2%) (hazard ratio for death, 0.72; P<0.001). The rate of disease control (PR, CR or SD) did not differ significantly between the two groups: 33.2% in the ipilimumab–dacarbazine group and 30.2% in the dacarbazine group (P=0.41). The rate of best OR (PR or CR) was 15.2% in the ipilimumab–dacarbazine group and 10.3% in the da‐ carbazine group (P=0.09). However, the median duration of response among all patients with a PR or CR was 19.3 months (95% CI, 12.1 to 26.1) in the ipilimumab–dacarbazine group and 8.1 months (95% CI, 5.19 to 19.8) in the dacarbazine group (P=0.03). In addition, some patients in the study who were receiving ipilimumab had an improvement from PR to CR after six months. Grade 3 or 4 adverse events occurred in 56.3% of patients treated with ipilimumab plus dacarbazine, as compared with 27.5% treated with dacarbazine and place‐ bo (P<0.001). No drug-related deaths or gastrointestinal perforations occurred in the ipili‐

Patients with untreated brain metastases were excluded from both phase III studies. Howev‐ er, phase II data indicate that ipilimumab has activity in patients with brain metastases [216]. The currently approved dose is 3 mg/kg based on the registration trial [95], however other doses and schedules [217] have been used that do not appear to produce significantly

 dacarbazine alone every three weeks through week 22. Patients with SD or an OR and no dose-limiting toxic effects received ipilimumab or placebo every 12 weeks thereafter as maintenance therapy. Similarly with the previous study a significant number of patients had poor prognosis based on the presence of visceral metastases and increased lactate dehy‐

dacarbazine plus placebo, given at weeks 1, 4, 7 and 10, followed by 850

associated with immune-related adverse events.

690 Melanoma - From Early Detection to Treatment

drogenase. The primary end point was OS.

mumab–dacarbazine group.

different results but appear to increase toxicity.

zine or 850 mg/m2

mg/m2

Another regulatory checkpoint is Programmed Death-1 receptor (PD-1). Its inhibition is cur‐ rently being evaluated in clinical trials. The PD-1 and PD ligand-1 (PD-L1) interaction is be‐ lieved to affect T cell anti-tumor immunity. Many tumors express high levels of PD-L1. When PD-L1 interacts with the PD-1 receptor on T cells, T cell function is impaired through a variety of mechanisms including induction of apoptosis, suppression of proliferation and inhibition of T cell cytokine production [96]. There are several ways to target PD-1; one way is targeting the PD-1 receptor and another is targeting the PD-1 ligand. There are several molecules currently under investigation that target the PD-1 receptor directly (BMS-936558, CT-011, MK-3475). In a recent study [98], BMS-936558 showed significant anti-tumor activity in a variety of solid tumors. In melanoma patients response rates were 28% and appeared to be durable. Interestingly, of 17 patients with PD-L1–negative tumors, none had an OR while 9 of 25 patients (36%) with PD-L1–positive tumors had an OR (P=0.006). CT-011 has demon‐ strated favorable results in hematologic malignancies but has not been well studied in mela‐ noma patients yet [221], although there is an ongoing phase I study being conducted at present. The role of MK-3475 in melanoma is also currently being evaluated in a phase I tri‐ al. Another molecule which is not probably directly targeting the PD-1 is AMP-224; it is a fusion protein of B7-DC and an antibody Fc portion. It is not a monoclonal antibody like the three checkpoint agents mentioned previously, and no data has been reported yet regarding its efficacy in human trials. PD-L1 monoclonal antibodies have emerged as another strategy to affect the PD-1/PD L-1 pathway. A multi-center phase I study evaluating the role of BMS-936559 in patients with advanced cancers including melanoma showed a 17% response rate in melanoma patients with some of those being durable responses [97].

contemplating a neoadjuvant and adjuvant therapy that employs an anti-CTLA4 and IFN-α

Immunomodulation

693

http://dx.doi.org/10.5772/53667

A phase II study through ECOG combining ipilimumab and GM-CSF is currently ongoing. Previous studies [228] with periodic infusions of anti-CTLA-4 antibodies after vaccination with irradiated, autologous tumor cells engineered to secrete GM-CSF have yielded favora‐

A phase III trial evaluated the combination of IL-2 and the gp100 peptide vaccine in ad‐ vanced melanoma. This combination therapy increased the response rate (16% vs. 6%) with more CRs in the combination arm and a trend toward increased OS [177]. Interestingly, the single arm responses were lower than expected. A similarly designed study employing ipili‐

Immunotherapy holds the promise of "the cure" for cancer. Glimpses of this outcome have been seen throughout the past century but may be best exemplified in melanoma therapy. Although attempts in the past have not had satisfactory results, the knowledge gained along with the ability to develop biologically active drugs is bearing fruit in the current generation of clinical trials. The few positive results have kept the interest for cancer immunotherapy alive which has also helped us to achieve a better understanding of the immune system in relationship to cancer treatment. The improvement in outcome seen with gp100 plus IL-2 is impressive, and the power of the anti-cancer and autoimmune toxicities seen with ipilimu‐ mab is dramatic. The excitement stems not only from the clinical results that were obtained but also by our ability to successfully manipulate the complex immune system in a different way than before. Undoubtedly, there are a lot of unanswered questions including why are there still a large number of patients who do not achieve responses with immunotherapy and eventually die from advanced disease? However, unlike immunotherapy applied to other advanced setting solid tumors, immunotherapy in advanced melanoma has resulted

The future of immunotherapy in melanoma and other tumor types would ideally involve re‐ search in a broad range of directions. An optimization of the IL-2 based treatments is needed to improve the number of durable responses. Results from the current work at NCI aug‐ menting IL-2 treatment with lymphodepletion are quite encouraging. Identification of se‐ rum or tissue biomarkers is also urgently needed. Biomarker research is often complex, but this work should reveal a better understanding of the tumor itself as well as the host. The checkpoint inhibitors such as ipilimumab and tremelimumab have opened the door for re‐ search into other immunologic checkpoints such as PD-1 and co-stimulatory signals. The

mumab did not show significant benefit in the combination arm [95].

combination strategy.

**9.3. IL-2 and gp-100**

**10. Conclusion**

**9.2. Anti-CTLA4 and GM-CSF**

ble results with acceptable toxicity.

in some durable responses and possibly cures.

## **8.5. Co-stimulatory agonists**

4-1BB or CD137 is a member of the TNF receptor (TNFR) family and provides a costimulato‐ ry signal important to the effective generation of many types of T cell responses. A complet‐ ed phase I study in melanoma with BMS-663513, a fully human anti-CD137 agonist monoclonal antibody, showed that this agent was well tolerated and three PRs were seen [222].

OX-40 is another member of the TNFR family. An agonist molecule is also under investiga‐ tion, but mature data are not yet available [223].

## **8.6. Conclusions**

Checkpoint inhibitors and co-stimulatory agonists are improving anti-tumor cellular im‐ munity. In a similar way to the more non-specific activation through cytokines, responses are durable but are not seen in all patients. This brings up issues such as appropriate patient selection, biomarker development and optimization of the dose, frequency and administra‐ tion in order to optimize efficacy.

## **9. Combination approaches**

Combination approaches have been traditionally used in the treatment of melanoma (for ex‐ ample different cytokines together or cytokines with chemotherapy). It is quite interesting that the results of this strategy have not been yet as successful as expected. While new agents are developed and we understand more about their function, these strategies may be‐ come more successful. At this point there is a lot of interest combining checkpoint inhibitors with other checkpoint blocking agents, co-stimulatory agonists, chemotherapy, targeted agents (such as B-type Raf kinas [BRAF] inhibitors) and radiotherapy. Interestingly, the BRAF inhibitors appear to improve T cell recognition of melanoma [224] which reinforces a rational combination of targeted therapy and immunotherapy. The results are not yet ma‐ ture and studies are ongoing, but there is preclinical and retrospective data that support this model of treatment [224-227]. The following combination strategies have shown some recent benefit and promise.

#### **9.1. Anti-CTLA4 and IFNa**

A phase II study [227] combining tremelimumab and high dose IFN in patients with ad‐ vanced melanoma showed an ORR of 24% with 4 patients obtaining a CR. Toxicity was ac‐ ceptable and the median OS was 21 months. The University of Pittsburgh group is now contemplating a neoadjuvant and adjuvant therapy that employs an anti-CTLA4 and IFN-α combination strategy.

### **9.2. Anti-CTLA4 and GM-CSF**

A phase II study through ECOG combining ipilimumab and GM-CSF is currently ongoing. Previous studies [228] with periodic infusions of anti-CTLA-4 antibodies after vaccination with irradiated, autologous tumor cells engineered to secrete GM-CSF have yielded favora‐ ble results with acceptable toxicity.

### **9.3. IL-2 and gp-100**

its efficacy in human trials. PD-L1 monoclonal antibodies have emerged as another strategy to affect the PD-1/PD L-1 pathway. A multi-center phase I study evaluating the role of BMS-936559 in patients with advanced cancers including melanoma showed a 17% response

4-1BB or CD137 is a member of the TNF receptor (TNFR) family and provides a costimulato‐ ry signal important to the effective generation of many types of T cell responses. A complet‐ ed phase I study in melanoma with BMS-663513, a fully human anti-CD137 agonist monoclonal antibody, showed that this agent was well tolerated and three PRs were seen

OX-40 is another member of the TNFR family. An agonist molecule is also under investiga‐

Checkpoint inhibitors and co-stimulatory agonists are improving anti-tumor cellular im‐ munity. In a similar way to the more non-specific activation through cytokines, responses are durable but are not seen in all patients. This brings up issues such as appropriate patient selection, biomarker development and optimization of the dose, frequency and administra‐

Combination approaches have been traditionally used in the treatment of melanoma (for ex‐ ample different cytokines together or cytokines with chemotherapy). It is quite interesting that the results of this strategy have not been yet as successful as expected. While new agents are developed and we understand more about their function, these strategies may be‐ come more successful. At this point there is a lot of interest combining checkpoint inhibitors with other checkpoint blocking agents, co-stimulatory agonists, chemotherapy, targeted agents (such as B-type Raf kinas [BRAF] inhibitors) and radiotherapy. Interestingly, the BRAF inhibitors appear to improve T cell recognition of melanoma [224] which reinforces a rational combination of targeted therapy and immunotherapy. The results are not yet ma‐ ture and studies are ongoing, but there is preclinical and retrospective data that support this model of treatment [224-227]. The following combination strategies have shown some recent

A phase II study [227] combining tremelimumab and high dose IFN in patients with ad‐ vanced melanoma showed an ORR of 24% with 4 patients obtaining a CR. Toxicity was ac‐ ceptable and the median OS was 21 months. The University of Pittsburgh group is now

rate in melanoma patients with some of those being durable responses [97].

**8.5. Co-stimulatory agonists**

692 Melanoma - From Early Detection to Treatment

tion, but mature data are not yet available [223].

[222].

**8.6. Conclusions**

tion in order to optimize efficacy.

**9. Combination approaches**

benefit and promise.

**9.1. Anti-CTLA4 and IFNa**

A phase III trial evaluated the combination of IL-2 and the gp100 peptide vaccine in ad‐ vanced melanoma. This combination therapy increased the response rate (16% vs. 6%) with more CRs in the combination arm and a trend toward increased OS [177]. Interestingly, the single arm responses were lower than expected. A similarly designed study employing ipili‐ mumab did not show significant benefit in the combination arm [95].

## **10. Conclusion**

Immunotherapy holds the promise of "the cure" for cancer. Glimpses of this outcome have been seen throughout the past century but may be best exemplified in melanoma therapy. Although attempts in the past have not had satisfactory results, the knowledge gained along with the ability to develop biologically active drugs is bearing fruit in the current generation of clinical trials. The few positive results have kept the interest for cancer immunotherapy alive which has also helped us to achieve a better understanding of the immune system in relationship to cancer treatment. The improvement in outcome seen with gp100 plus IL-2 is impressive, and the power of the anti-cancer and autoimmune toxicities seen with ipilimu‐ mab is dramatic. The excitement stems not only from the clinical results that were obtained but also by our ability to successfully manipulate the complex immune system in a different way than before. Undoubtedly, there are a lot of unanswered questions including why are there still a large number of patients who do not achieve responses with immunotherapy and eventually die from advanced disease? However, unlike immunotherapy applied to other advanced setting solid tumors, immunotherapy in advanced melanoma has resulted in some durable responses and possibly cures.

The future of immunotherapy in melanoma and other tumor types would ideally involve re‐ search in a broad range of directions. An optimization of the IL-2 based treatments is needed to improve the number of durable responses. Results from the current work at NCI aug‐ menting IL-2 treatment with lymphodepletion are quite encouraging. Identification of se‐ rum or tissue biomarkers is also urgently needed. Biomarker research is often complex, but this work should reveal a better understanding of the tumor itself as well as the host. The checkpoint inhibitors such as ipilimumab and tremelimumab have opened the door for re‐ search into other immunologic checkpoints such as PD-1 and co-stimulatory signals. The need for a reliable melanoma biomarker is again paramount, and the significance of PD-L1 expression that is currently under investigation is anticipated with interest. Studying mech‐ anisms of resistance in all cancer immunotherapeutics is equally important.

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The combination of different immunotherapeutics with each other, with molecularly target‐ ed agents or with conventional treatments such as chemotherapy or radiation therapy, may not be the simple process that combination strategies with vaccines were in the past, but this approach is quite attractive and rational and will help us further understand the role of the immune system in this disease. In a similar fashion, sequential therapy, using different agents at different times might improve clinical response and survival. Appropriate first line selection is also quite challenging since there are currently three approved agents in BRAF V600E mutated patients (high does IL-2, ipilimumab and vemurafenib) and two for BRAF wild type patients (high dose IL-2 and ipilimumab). A randomized clinical trial of ipilimu‐ mab followed by vemurafenib versus vemurafenib followed by ipilimumab is planned through the NCI Cooperative Group mechanisms [218]. Future clinical trials will hopefully provide some answers to these questions. In the design of clinical trials, the importance of refining the RECIST criteria for immunotherapy agents cannot be overemphasized. Overall, this is an exciting time for cancer immunologists and clinicians who treat patients with mel‐ anoma. The future for innovative trials of new agents and combinations is brighter than ever before.

## **Acknowledgements**

The authors would like to thank Linda Ray for her invaluable administrative support.

## **Author details**

Konstantinos Arnaoutakis, Dorothy A. Graves, Laura F. Hutchins and Thomas Kieber-Emmons

Division of Hematology/Oncology and Department of Pathology University of Arkansas for Medical Sciences, Little Rock, AR, Winthrop P. Rockefeller Cancer Institute, USA

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The authors would like to thank Linda Ray for her invaluable administrative support.

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## *Edited by Guy Huynh Thien Duc*

The Book "Melanoma - From Early Detection To Treatment" is aiming to present data and knowledge from most experienced experts in the field. The book covers main topics from the fundamental aspects to multiple approaches in the disease treatment as well as related features. It offers a global view concerning one of the most frequent types of cancer to which a substantial high proportion of people worldwide is confronted at some time point in life.

Photo by Marwani22 / iStock

Melanoma - From Early Detection to Treatment

Melanoma

From Early Detection to Treatment

*Edited by Guy Huynh Thien Duc*