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## **Meet the editor**

Guy Huynh Thien Duc is Research Director emeritus from the CNRS (Centre National de la Recherche Scientifique). He started his career in the Pasteur Institute where he prepared his Ph.D in the field of Immunopathology. Thereafter, as researcher in the CNRS, he has been mainly involved in fundamental aspects of Immunology, focusing on Transplantation Immunity and

Immunomodulation. In the last two decades, his work at the University Paris XI in the southern of Paris, presently inside the structure of INSERM U-1014 Groupe Hospitalier Paul Brousse – Villejuif, is essentially devoted to Cancer Immunotherapy.

Contents

**Preface IX**

Jianli Dong

**Section 1 Fundamental Aspects of the Melanoma Biology 1**

Chapter 1 **Overcoming Resistance to BRAF and MEK Inhibitors by Simultaneous Suppression of CDK4 3**

Chapter 2 **Targeted Therapies in Melanoma: Successes and Pitfalls 29**

Antonio Ascierto, Amelia Lissia and Antonio Cossu

Chapter 4 **Melanoma Genetics: From Susceptibility to Progression 83**

Chapter 5 **Diagnosis, Histopathologic and Genetic Classification of Uveal**

Chapter 6 **Recombinant DNA Technology in Emerging Modalities for**

Vitali Alexeev, Alyson Pidich, Daria Marley Kemp and Olga

**Melanoma Immunotherapy 175**

Chapter 3 **Low-Penetrance Variants and Susceptibility to Sporadic**

**Malignant Melanoma 59**

Martinez-Cadenas

**Melanoma 137**

and E. Kilic

Igoucheva

Giuseppe Palmieri, Maria Colombino, Maria Cristina Sini, Paolo

G. Ribas, M. Ibarrola-Villava, M.C. Peña-Chilet, L.P. Fernandez and C.

Guilherme Francisco, Priscila Daniele Ramos Cirilo, Fernanda Toledo Gonçalves, Tharcísio Citrângulo Tortelli Junior and Roger Chammas

J.G.M. van Beek, A.E. Koopmans, R.M. Verdijk, N.C. Naus, A. de Klein

## Contents

#### **Preface XIII**




Chapter 8 **Pars Plana Vitrectomy Associated with or Following Plaque Brachytherapy for Choroidal Melanoma 219** John O. Mason and Sara Mullins

Chapter 17 **Management of Acral Lentiginous Melanoma 475** Yoshitaka Kai and Sakuhei Fujiwara

Yasuhiro Nakamura and Fujio Otsuka

Chapter 19 **Cutaneous Melanoma − Surgical Treatment 523**

Chapter 20 **Therapeutic Agents for Advanced Melanoma 537** Zhao Wang, Wei Li and Duane D. Miller

Chapter 22 **The Menace of Melanoma: A Photodynamic Approach to**

Chapter 23 **Study of the Anti-Photoaging Effect of Noni (Morinda**

**Adjunctive Cancer Therapy 583** L.M. Davids and B. Kleemann

Chapter 24 **Inhibiting S100B in Malignant Melanoma 649**

Chapter 21 **Update in Ocular Melanoma 565**

**Section 3 Melanoma Related Features 581**

Irarrázabal

**citrifolia) 629**

Akemi Uwaya

J. Weber

Chapter 25 **Immunomodulation 669**

Thomas Kieber-Emmons

Chapter 18 **Sentinel Lymph Node Biopsy for Melanoma and Surgical Approach to Lymph Node Metastasis 499**

> Mario Santinami, Roberto Patuzzo, Roberta Ruggeri, Gianpiero Castelli, Andrea Maurichi, Giulia Baffa and Carlotta Tinti

Contents **VII**

Victoria de los Ángeles Bustuoabad, Lucia Speroni and Arturo

Hideaki Matsuda, Megumi Masuda, Kazuya Murata, Yumi Abe and

Kira G. Hartman, Paul T. Wilder, Kristen Varney, Alexander D. Jr. MacKerell, Andrew Coop, Danna Zimmer, Rena Lapidus and David

Konstantinos Arnaoutakis, Dorothy A. Graves, Laura F. Hutchins and


Chapter 14 **Cellular and Molecular Mechanisms of Methotrexate Resistance in Melanoma 391** Luis Sanchez del-Campo, Maria F. Montenegro, Magali Saez-Ayala, María Piedad Fernández-Pérez, Juan Cabezas-Herrera and Jose Neptuno Rodriguez-Lopez


Chapter 17 **Management of Acral Lentiginous Melanoma 475** Yoshitaka Kai and Sakuhei Fujiwara

Chapter 7 **Acquired Resistance to Targeted MAPK Inhibition in**

John O. Mason and Sara Mullins

**Section 2 Melanoma Treatment Approaches 253**

Lee D. Cranmer\*

**Pediatric Age 329**

**in Melanoma 391**

Neptuno Rodriguez-Lopez

Chapter 15 **Surgery and the Staging of Melanoma 411**

Chapter 16 **Melanoma: Treatments and Resistance 439**

Ascanio

Kavitha Gowrishankar, Matteo S. Carlino and Helen Rizos

Chapter 8 **Pars Plana Vitrectomy Associated with or Following Plaque Brachytherapy for Choroidal Melanoma 219**

Chapter 9 **Combination Therapies to Improve Delivery of Protective T Cells into the Melanoma Microenvironment 231** Devin B. Lowe, Jennifer L. Taylor and Walter J. Storkus

Chapter 10 **Management of In-Transit Malignant Melanoma 255** Paul J. Speicher, Douglas S. Tyler and Paul J. Mosca

Chapter 12 **Surgical Treatment of Nevi and Melanoma in the**

**Targeting the Beating Heart 365** Jennifer Makalowski and Hinrich Abken

Chapter 13 **Adoptive Cell Therapy of Melanoma: The Challenges of**

Chapter 14 **Cellular and Molecular Mechanisms of Methotrexate Resistance**

Jonathan Castillo Arias and Miriam Galvonas Jasiulionis

Chapter 11 **Management of Brain Metastasis in Melanoma Patients 275**

Sherif S. Morgan\*, Joanne M. Jeter, Evan M. Hersh, Sun K. Yi and

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

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

Z. Al-Hilli, D. Evoy, J.G. Geraghty, E.W. McDermott and R.S. Prichard

**Melanoma 197**

**VI** Contents


Preface

pheomelanin/eumelanin ratio.

types in general.

lated features.

The link of melanoma risk to ultraviolet (UV) radiation exposure is widely recognized, but UV radiation independent events account also for a significant number of cases which high‐ lights the need for analysing further the mechanism(s) underlying melanomagenesis. There‐ fore, the essential aspects to be considered would be related to the balance between Mc1R (melanocortin 1 receptor)-inherited background and the mutated BRAF (BRAF V600E) con‐ veying stresses caused either by UV radiation or oxydative damage in the context of defined

Concerning the treatment of metastatic melanoma, overall results so far obtained still re‐ mained poor, although significant response rate has been observed with vemurafenib (PLX4032). However resistance to this remarkable small molecule is beginning to emerge

In this context, it is worth noting the development of new technologies, following the advent of human genome sequencing allowing to identify important somatic driver mutations that harness most aggressive cancer types. Progress gained in sequencing thousands of individu‐ al cancer genomes has already provided an invaluable insight into activating mutations and surrogate signalling pathways sustaining deregulated proliferation, invasiveness and resist‐ ance to apoptosis as well as to inhibitors. On the other hand, the throughout deep sequenc‐ ing will also help development of other active inhibitors like PLX4032 specifically adapted for targeting defined activating mutations. Needless to say that personalized medicine based on patient's genetic background represents also important aspect for taking in consid‐ eration. Overall, the huge effort provided by scientists in many areas along with that of physicians recently will open, beyond doubt, the ways to development of appropriate and efficient strategies in the treatment of metastatic melanoma in particular and other cancer

As such, the book "Melanoma - From Early Detection to Treatment" assembles data and knowledge from most experienced experts in the field. It covers sections from fundamental aspects of the melanoma biology to various treatment approaches including melanoma re‐

Research Director emeritus from the CNRS (Centre National de la Recherche Scientifique),

INSERM, U-1014, Université Paris XI – Groupe hospitalier Paul-Brousse,

**Guy Huynh Thien Duc**

Villejuif, France

Acknowledgements: We thank Chaobin Zhu for his helpful assistance.

and it is known that only patients with relevant mutation respond to this agent.

## Preface

The link of melanoma risk to ultraviolet (UV) radiation exposure is widely recognized, but UV radiation independent events account also for a significant number of cases which high‐ lights the need for analysing further the mechanism(s) underlying melanomagenesis. There‐ fore, the essential aspects to be considered would be related to the balance between Mc1R (melanocortin 1 receptor)-inherited background and the mutated BRAF (BRAF V600E) con‐ veying stresses caused either by UV radiation or oxydative damage in the context of defined pheomelanin/eumelanin ratio.

Concerning the treatment of metastatic melanoma, overall results so far obtained still re‐ mained poor, although significant response rate has been observed with vemurafenib (PLX4032). However resistance to this remarkable small molecule is beginning to emerge and it is known that only patients with relevant mutation respond to this agent.

In this context, it is worth noting the development of new technologies, following the advent of human genome sequencing allowing to identify important somatic driver mutations that harness most aggressive cancer types. Progress gained in sequencing thousands of individu‐ al cancer genomes has already provided an invaluable insight into activating mutations and surrogate signalling pathways sustaining deregulated proliferation, invasiveness and resist‐ ance to apoptosis as well as to inhibitors. On the other hand, the throughout deep sequenc‐ ing will also help development of other active inhibitors like PLX4032 specifically adapted for targeting defined activating mutations. Needless to say that personalized medicine based on patient's genetic background represents also important aspect for taking in consid‐ eration. Overall, the huge effort provided by scientists in many areas along with that of physicians recently will open, beyond doubt, the ways to development of appropriate and efficient strategies in the treatment of metastatic melanoma in particular and other cancer types in general.

As such, the book "Melanoma - From Early Detection to Treatment" assembles data and knowledge from most experienced experts in the field. It covers sections from fundamental aspects of the melanoma biology to various treatment approaches including melanoma re‐ lated features.

Acknowledgements: We thank Chaobin Zhu for his helpful assistance.

#### **Guy Huynh Thien Duc**

Research Director emeritus from the CNRS (Centre National de la Recherche Scientifique), INSERM, U-1014, Université Paris XI – Groupe hospitalier Paul-Brousse, Villejuif, France

**Section 1**

**Fundamental Aspects of the Melanoma Biology**

**Fundamental Aspects of the Melanoma Biology**

**Chapter 1**

**Overcoming Resistance to BRAF and MEK Inhibitors by**

Melanoma is one of the most prevalent malignancies and has a very poor prognosis. Muta‐ tions in v-raf murine sarcoma viral oncogene homolog B1 (*BRAF*) occur in approximately 50% of melanomas [4]. While the response to selective BRAF inhibitors (BRAFi) in *BRAF*mutant melanoma is encouraging, virtually all patients rapidly develop secondary resist‐ ance [6, 7]. Based on the finding that the mitogen activated protein kinase/ERK kinase (MEK)-extracellular signal regulated kinase (ERK) pathway is frequently reactivated by var‐ ious BRAFi resistance mechanisms, a combination trial of a selective mutant BRAF inhibitor (dabrafenib, GSK2118436) with a MEK inhibitor (trametinib, GSK1120212) is underway and has achieved clinical responses in 17% and disease control in 67% in patients who failed pri‐ or single-agent treatment with a BRAF inhibitor [9]. While these results are promising, there is a critical need to overcome resistance to BRAF and MEK inhibitors. The clinical efficacy of BRAFi and MEKi therapy is believed to rely on a functional retinoblastoma (RB) axis to in‐ hibit cell proliferation. The inhibitor of cyclin-dependent kinase 4A (*INK4A*) gene encode the p16 protein, a critical cell cycle regulator that interacts with cyclin dependent kinase (CDK) 4, inhibiting its ability to phosphorylate and inactivate RB [12, 13]. Genetic disruption of *INK4A* occurs in approximately 50% of melanomas irrespective of *BRAF* mutation and has been detected in melanoma cells that developed resistance to BRAFi. Of note, cyclin D is still expressed even in the setting of maximum tolerance dosing of BRAF inhibitor [7]. We have reported that combination of BRAFi or MEKi with the expression of wild-type *INK4A* or a CDK4 inhibitor (CDK4i) significantly suppresses growth and enhances apoptosis in melano‐ ma cells [1-3]. Currently, CDK4 inhibitors are in active clinical development (http://clinical‐ trials.gov/). Based on our previous work and recent insights into molecular mechanisms of resistance to BRAF and MEK inhibitors, we hypothesize that simultaneous suppression of

> © 2013 Dong; 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.

**Simultaneous Suppression of CDK4**

Additional information is available at the end of the chapter

Jianli Dong

**1. Introduction**

http://dx.doi.org/10.5772/53620

## **Overcoming Resistance to BRAF and MEK Inhibitors by Simultaneous Suppression of CDK4**

Jianli Dong

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53620

## **1. Introduction**

Melanoma is one of the most prevalent malignancies and has a very poor prognosis. Muta‐ tions in v-raf murine sarcoma viral oncogene homolog B1 (*BRAF*) occur in approximately 50% of melanomas [4]. While the response to selective BRAF inhibitors (BRAFi) in *BRAF*mutant melanoma is encouraging, virtually all patients rapidly develop secondary resist‐ ance [6, 7]. Based on the finding that the mitogen activated protein kinase/ERK kinase (MEK)-extracellular signal regulated kinase (ERK) pathway is frequently reactivated by var‐ ious BRAFi resistance mechanisms, a combination trial of a selective mutant BRAF inhibitor (dabrafenib, GSK2118436) with a MEK inhibitor (trametinib, GSK1120212) is underway and has achieved clinical responses in 17% and disease control in 67% in patients who failed pri‐ or single-agent treatment with a BRAF inhibitor [9]. While these results are promising, there is a critical need to overcome resistance to BRAF and MEK inhibitors. The clinical efficacy of BRAFi and MEKi therapy is believed to rely on a functional retinoblastoma (RB) axis to in‐ hibit cell proliferation. The inhibitor of cyclin-dependent kinase 4A (*INK4A*) gene encode the p16 protein, a critical cell cycle regulator that interacts with cyclin dependent kinase (CDK) 4, inhibiting its ability to phosphorylate and inactivate RB [12, 13]. Genetic disruption of *INK4A* occurs in approximately 50% of melanomas irrespective of *BRAF* mutation and has been detected in melanoma cells that developed resistance to BRAFi. Of note, cyclin D is still expressed even in the setting of maximum tolerance dosing of BRAF inhibitor [7]. We have reported that combination of BRAFi or MEKi with the expression of wild-type *INK4A* or a CDK4 inhibitor (CDK4i) significantly suppresses growth and enhances apoptosis in melano‐ ma cells [1-3]. Currently, CDK4 inhibitors are in active clinical development (http://clinical‐ trials.gov/). Based on our previous work and recent insights into molecular mechanisms of resistance to BRAF and MEK inhibitors, we hypothesize that simultaneous suppression of

© 2013 Dong; 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.

CDK4 is an effective strategy to overcome resistance to BRAF and MEK inhibitors. BRAF mutation assays have been used to guide treatment with BRAF and MEK inhibitors, devel‐ opment of sensitive and specific INK4A/p16 assays may serve as predictive biomarkers for treatment with CDK4 inhibitors.

clinical and traditional histological methods as superficial spreading melanoma, lentigo ma‐ ligna melanoma, acral lentiginous melanoma, and nodular melanoma. In early stages of melanomas, neoplastic cells are confined to the epidermis or with microinvasion into the dermis. In advanced melanomas, cancer cells expand in the dermis and generate tumor nod‐ ules and have a high potential for metastatic spread. In the metastatic phase, cancer cells dis‐ seminate to lymph nodes or distant organs [34, 35]. For the early diagnosed and localized melanomas, surgery is the choice of treatment. But there is currently no effective treatment for invasive and metastatic melanomas. Patients with late stage melanomas have a high mortality rate and life expectancy averages approximately 6-8 months after diagnosis.

Overcoming Resistance to BRAF and MEK Inhibitors by Simultaneous Suppression of CDK4

http://dx.doi.org/10.5772/53620

5

**Figure 1.** p16-cyclin D/CDK4 modifies the outcome of RAS/RAF/MEK/ERK signaling activation. RAF relays RAS signals through MEK to ERK. The activation of this pathway has multiple effects on cell proliferation, differentiation, and sur‐ vival depending on the cellular contexts [5]. Constitutive activation of growth factor signaling pathways or NRAS and BRAF activating mutations can trigger over-expression of p16 leading to proliferative senescence, which manifest as benign nevus [10, 11]. Loss of p16 by genetic and epigenetic changes allows activation of cyclin D/CDK4 and inactiva‐ tion of RB, leading to E2F activation, cell cycle progression from G1 to S phase, cell proliferation and tumor formation [12, 13]. Further genetic changes cause tumor progression to malignant melanoma. Of the three *RAS* and three *RAF*

Of note, in addition to melanomas, *BRAF* mutations are found at high frequencies (70-80%) in benign melanocytic nevi [36, 37]. There are a large numbers of melanocytic nevi in the general population compared to the relatively low incidence of melanomas [34, 35]. Clinical‐ ly, it is known that nevi often regress over time. This suggests that BRAF mutations alone are insufficient to induce malignant transformation in nevus cells. The growth arrest of nevi is believed to result from oncogene-induced senescence, which is known as a protective mechanism against unlimited proliferation that could result from *BRAF* mutations and acti‐ vation of the ERK signaling pathway (nevus in Fig. 1) [10, 11]. Tumor suppressor genes have been found to be involved in senescence process. For example, p16 is one tumor suppressor

genes, *NRAS* and *BRAF* are mutated in melanoma [4].

## **2. Body**

**Constitutive activation of RAS-RAF-MEK-ERK signaling pathway in melanomas.***NRAS* and *BRAF* mutations were found respectively in 10-20% and 60-80% melanomas [4]. NRAS and BRAF are components of the RAS-RAF-mitogen activated protein kinase/ERK kinase (MEK)-extracellular signal regulated kinase (ERK) signaling pathway (Fig. 1) [5]. This sig‐ naling pathway plays an essential role in cell proliferation, differentiation and survival [5, 14, 15]. Constitutive activation of the ERK pathway has been shown to mediate the trans‐ forming activity of mutant BRAF in melanoma cells [16-18]. Suppression of mutant *BRAF* expression has been shown to inhibit ERK pathway activation and subsequent suppression of melanoma cell proliferation and survival *in vitro* and *in vivo* [19-21]. Our previous data revealed that the inhibition of mutant BRAF decreased levels of phospho-ERK (p-ERK), a marker of ERK pathway activation in melanoma cells [5, 14, 15].

The high frequency of *BRAF* mutation in melanomas makes it an ideal target for therapy. Because normal cells require wild-type *BRAF* for survival [22], specifically inhibiting mu‐ tant, but not wild-type *BRAF* in tumor cells could avoid toxic side effects generated by tar‐ geting normal cells. The finding that mutations in v-raf murine sarcoma viral oncogene homolog B1 (*BRAF*) occur in approximately 50% of melanomas led to extensive investiga‐ tion of targeting BRAF for melanoma treatment, resulting in the first approved mutant spe‐ cific BRAF inhibitor for treatment of advanced melanoma.

**Combine BRAF and MEK inhibitors with chemotherapeutic agents.** Intrinsic therapy re‐ sistance is a major limitation in the treatment of malignant melanomas. The mechanisms in‐ volved in the resistance of melanomas are largely unknown [23, 24]. It is believed that apoptosis and cytostasis (growth arrest/differentiation) are two of the main cellular respons‐ es to anticancer agents and loss of either process promotes treatment failure [25-27]. Activat‐ ing *BRAF* mutations could drive cell proliferation and increase the cell death threshold through ERK pathway or alternative mechanisms [28-30], resulting in the blockage of both cytotoxic and cytostatic effects of therapeutic drugs [14, 31, 32]. It has been shown that inhib‐ ition of ERK pathway sensitizes melanoma cells to apoptosis induced by DNA damaging agents including cisplatin and ultra-violate (UV) irradiation [32, 33]. Rational combination of BRAF and MEK inhibitors with selective chemotherapeutic agents, for example, dacarbazine (DTIC), may generate additive/synergistic therapeutic effects.

**ERK pathway activation and p16 in melanocytic lesions.** Melanocytic lesions can be group‐ ed into two main categories: nevi and melanomas. Nevi are divided into several different types based on histology. These include acquired melanocytic nevi, congenital melanocytic nevi, blue nevi, Spitz nevi, and dysplastic nevi. Melanoma can be further divided based on clinical and traditional histological methods as superficial spreading melanoma, lentigo ma‐ ligna melanoma, acral lentiginous melanoma, and nodular melanoma. In early stages of melanomas, neoplastic cells are confined to the epidermis or with microinvasion into the dermis. In advanced melanomas, cancer cells expand in the dermis and generate tumor nod‐ ules and have a high potential for metastatic spread. In the metastatic phase, cancer cells dis‐ seminate to lymph nodes or distant organs [34, 35]. For the early diagnosed and localized melanomas, surgery is the choice of treatment. But there is currently no effective treatment for invasive and metastatic melanomas. Patients with late stage melanomas have a high mortality rate and life expectancy averages approximately 6-8 months after diagnosis.

CDK4 is an effective strategy to overcome resistance to BRAF and MEK inhibitors. BRAF mutation assays have been used to guide treatment with BRAF and MEK inhibitors, devel‐ opment of sensitive and specific INK4A/p16 assays may serve as predictive biomarkers for

**Constitutive activation of RAS-RAF-MEK-ERK signaling pathway in melanomas.***NRAS* and *BRAF* mutations were found respectively in 10-20% and 60-80% melanomas [4]. NRAS and BRAF are components of the RAS-RAF-mitogen activated protein kinase/ERK kinase (MEK)-extracellular signal regulated kinase (ERK) signaling pathway (Fig. 1) [5]. This sig‐ naling pathway plays an essential role in cell proliferation, differentiation and survival [5, 14, 15]. Constitutive activation of the ERK pathway has been shown to mediate the trans‐ forming activity of mutant BRAF in melanoma cells [16-18]. Suppression of mutant *BRAF* expression has been shown to inhibit ERK pathway activation and subsequent suppression of melanoma cell proliferation and survival *in vitro* and *in vivo* [19-21]. Our previous data revealed that the inhibition of mutant BRAF decreased levels of phospho-ERK (p-ERK), a

The high frequency of *BRAF* mutation in melanomas makes it an ideal target for therapy. Because normal cells require wild-type *BRAF* for survival [22], specifically inhibiting mu‐ tant, but not wild-type *BRAF* in tumor cells could avoid toxic side effects generated by tar‐ geting normal cells. The finding that mutations in v-raf murine sarcoma viral oncogene homolog B1 (*BRAF*) occur in approximately 50% of melanomas led to extensive investiga‐ tion of targeting BRAF for melanoma treatment, resulting in the first approved mutant spe‐

**Combine BRAF and MEK inhibitors with chemotherapeutic agents.** Intrinsic therapy re‐ sistance is a major limitation in the treatment of malignant melanomas. The mechanisms in‐ volved in the resistance of melanomas are largely unknown [23, 24]. It is believed that apoptosis and cytostasis (growth arrest/differentiation) are two of the main cellular respons‐ es to anticancer agents and loss of either process promotes treatment failure [25-27]. Activat‐ ing *BRAF* mutations could drive cell proliferation and increase the cell death threshold through ERK pathway or alternative mechanisms [28-30], resulting in the blockage of both cytotoxic and cytostatic effects of therapeutic drugs [14, 31, 32]. It has been shown that inhib‐ ition of ERK pathway sensitizes melanoma cells to apoptosis induced by DNA damaging agents including cisplatin and ultra-violate (UV) irradiation [32, 33]. Rational combination of BRAF and MEK inhibitors with selective chemotherapeutic agents, for example, dacarbazine

**ERK pathway activation and p16 in melanocytic lesions.** Melanocytic lesions can be group‐ ed into two main categories: nevi and melanomas. Nevi are divided into several different types based on histology. These include acquired melanocytic nevi, congenital melanocytic nevi, blue nevi, Spitz nevi, and dysplastic nevi. Melanoma can be further divided based on

marker of ERK pathway activation in melanoma cells [5, 14, 15].

cific BRAF inhibitor for treatment of advanced melanoma.

(DTIC), may generate additive/synergistic therapeutic effects.

treatment with CDK4 inhibitors.

4 Melanoma - From Early Detection to Treatment

**2. Body**

**Figure 1.** p16-cyclin D/CDK4 modifies the outcome of RAS/RAF/MEK/ERK signaling activation. RAF relays RAS signals through MEK to ERK. The activation of this pathway has multiple effects on cell proliferation, differentiation, and sur‐ vival depending on the cellular contexts [5]. Constitutive activation of growth factor signaling pathways or NRAS and BRAF activating mutations can trigger over-expression of p16 leading to proliferative senescence, which manifest as benign nevus [10, 11]. Loss of p16 by genetic and epigenetic changes allows activation of cyclin D/CDK4 and inactiva‐ tion of RB, leading to E2F activation, cell cycle progression from G1 to S phase, cell proliferation and tumor formation [12, 13]. Further genetic changes cause tumor progression to malignant melanoma. Of the three *RAS* and three *RAF* genes, *NRAS* and *BRAF* are mutated in melanoma [4].

Of note, in addition to melanomas, *BRAF* mutations are found at high frequencies (70-80%) in benign melanocytic nevi [36, 37]. There are a large numbers of melanocytic nevi in the general population compared to the relatively low incidence of melanomas [34, 35]. Clinical‐ ly, it is known that nevi often regress over time. This suggests that BRAF mutations alone are insufficient to induce malignant transformation in nevus cells. The growth arrest of nevi is believed to result from oncogene-induced senescence, which is known as a protective mechanism against unlimited proliferation that could result from *BRAF* mutations and acti‐ vation of the ERK signaling pathway (nevus in Fig. 1) [10, 11]. Tumor suppressor genes have been found to be involved in senescence process. For example, p16 is one tumor suppressor found to be induced by ERK activation and telomere attrition involving cell senescence [8, 10, 11, 38]. The tumor suppressor p16 is encoded by *INK4A* (Fig. 2) and is often inactivated in a variety of human cancers, including 30-70% in melanomas [39, 40]. Most melanomas, but not nevi, have lost the expression of wild-type *INK4A*, either through DNA deletion/ mutation or promoter hypermethylation [41-45]. It is possible that the loss-of-function of p16 in melanomas may make it possible to bypass the cellular senescence mechanism and func‐ tion as an anti-tumor mechanism against ERK signal activation triggered by *NRAS* and *BRAF* oncogenic mutation (Fig. 1) [11, 46, 47].

the p16-cyclin D-CDK4-RB axis that harbor genetic changes at low frequency in melano‐ mas (e.g., *CDK4* and *RB* each approximately 3%) [59], and may not overlap with *BRAF* mutation (e.g., amplification of cyclin D1 gene *CCND1* and *CDK4*) [60], *INK4A* lesions are frequently detected in melanomas (~50%) irrespective of *BRAF* mutation [59-61]; therefore, abnormal p16 is a major mechanism of RB-axis attenuation in *BRAF*-mutant melanoma cells. p16 binds to and inhibits the catalytic activity of CDK4, representing a crucial gatekeeper at the G1>S checkpoint [62, 63]. The relative abundance of CDK4-cy‐ clin D and p16 can determine the activity of the CDK4 kinase, thus regulate RB and cellcycle progression [62, 63]. BRAF-MEK-ERK signaling pathway upregulates/activates the cyclin D-CDK4 enzyme, which phosphorylates and inactivates RB leading to cell cycle progression in melanoma cells; such an effect can be blocked by tumor suppressor p16 [2, 3, 61]. Several pathways that confer BRAFi resistance, including COT, RAF splicing variants, RAF dimerization, NRAS, IGF-1R, and RTK can reactivate cyclin D-CDK4 through signaling pathways including MEK-ERK as well as PI3K-AKT [51-53, 55, 56, 64]. Although the addition of MEKi to BRAFi may suppress reactivation of MEK-ERK-cyclin D-CDK4, alternative resistance mechanisms, including growth factor receptors and PI3K-AKT pathway can activate cyclin D-CDK4 [52, 55, 64-66] *in the absence of a functional p16*, adding CDK4 inhibitor may help overcoming resistance to BRAFi and MEKi (Fig. 3). *BRAF* mutation assays have been used to direct BRAFi treatment. There is significant genotypic heterogeneity of *INK4A* including bi- and mono-allelic deletions, nonsense and missense mutations, and also different levels of epigenetic modification by promoter hy‐ per-methylation. Characterization of whether these *INK4A* changes correlate with differ‐ ent treatment resistance to BRAFi/MEKi/CDK4i may lead to companion molecular tests

**Simultaneous Suppression of CDK4**

**Author(s) Name(s): Jianli Dong** 

**Page No.** 

11 Table 2

Notice:

**Line** 

**Proof Corrections Form** 

**Chapter Title: Overcoming Resistance to BRAF and MEK Inhibitors by** 

Overcoming Resistance to BRAF and MEK Inhibitors by Simultaneous Suppression of CDK4

**SAMPLE PROOF CORRECTIONS FORM** 

**No. Delete Replace with** 

5 3 chest anatomy and feature artery origin anatomic features of chest and LMCA origin





**PROOF CORRECTIONS FORM** 

RB-p

dust

http://dx.doi.org/10.5772/53620

7

3 18 <10-13 cm/s <10-13 cm/s first hour

4 5 forth of fourth- or

4 15 set at 15-20 cm set at 10-15 cm

to better manage melanoma patients under BRAFi, MEKi, and CDK4i therapy.

2nd row, 1st column:

dutst

corrections form.

with' column.

BRAFi + MEKi

BRAF/MEK/ERK

melanomas that develop resistance mechanisms un-opposed by BRAFi + MEKi treatment.

(Pfizer) (http://clinicaltrials.gov/).

As shown in Fig. 4, in addition to BRAF and MEK inhibitors, several drugs designed to in‐ hibit the activity of CDK4 are in active clinical trials for melanoma and other cancers includ‐ ing LEE011 (Novartis Pharmaceuticals ), LY2835219 (Eli Lilly and Company), PD-0332991

new, corrected references list after the proofreading form table.

Cyclin D-CDK4

Other resistant mechanisms including activation of growth factor receptors and PI3K-AKT

**Figure 3.** The presence of functional p16 may offset resistance mechanisms that lead to activation of cyclin D-CDK4 in melanomas that progressed under BRAFi/MEKi treatment, whereas abnormal p16 may predict treatment failure in

**Combined inhibition of CDK4 potentiate the effect of MEKi.** In order to design better strategies for the treatment of this devastating disease a better understanding of melanoma biology is necessary. Multiple genetic and environmental factors have been linked to the de‐

p16/CDK4i

12 Fig. 7 Could you please reduce the size of figure by ~20%?

13 5 . Third or fifth one . Fifth one

why such formatting was removed from the original manuscript.



1

Indirect evidence from cultured cells and animal models reveal that there may be a coop‐ erative role between the constitutive activation of ERK pathway and the loss of p16 in tumor progression. Daniotti et al. [48] reported the co-existence of *BRAF* mutations and *INK4A* mutations/deletions/loss-of-expression in 26 of 41 (63%) short-term cell lines ob‐ tained from melanoma biopsies. Recent evidence suggests that growth arrest in benign nevi is due to cell senescence and that p16 at least partially contributes to the process of senescence in nevi [11, 46, 47].

**Figure 2.** *INK4A* and *ARF* share sequences in the *CDKN2A* locus. Exons are shown as rectangles. Alternative first exons (1α and 1β) are transcribed from different promoters (arrows). Exons 1α and 1β are spliced to the same splicing ac‐ ceptor site in exon 2 but are translated in alternative reading frames. *INK4A* coding sequences in exons 1α, 2, and 3 and *ARF* coding sequences in exons 1β and 2 are indicated by different shading patterns. Adopted from Sherr [8]. *INK4A* lesions detected by FISH and Sanger sequencing may also affect *ARF*.

**Resistance of melanoma to BRAF and MEK inhibitors.** The finding that mutations in *BRAF* occur in melanomas led to extensive investigation of targeting BRAF for melanoma treat‐ ment. While the response to selective mutant BRAF inhibitors (BRAFi) in *BRAF*-mutant mel‐ anoma is encouraging, virtually all patients rapidly develop secondary resistance. Based on the finding that the mitogen activated protein kinase/ERK kinase (MEK)-extracellular signal regulated kinase (ERK) pathway is frequently reactivated by various BRAFi resistance mechanisms, the first combination trial of a selective BRAF inhibitor (dabrafenib, GSK2118436) with a MEK inhibitor (trametinib, GSK1120212) is underway and has achieved clinical responses in 17% and disease control in 67% in patients who failed prior single-agent treatment with a BRAF inhibitor [9]. While these results are promising, again, the treatment response is short-lived; there is a critical need for additional strategies to overcome this deadly disease [49, 50].

There is evidence that treatment response to BRAFi and MEKi relies on a functional p16 cyclin D-CDK4-retinoblastoma (RB) axis. *INK4A* mutations/deletions occur in most of the melanoma cells that demonstrated resistance to BRAFi (e.g.; 451Lu, Mel1617, WM983, WM902, A375, M238, SKMEL28, and A2058) [51-57]. Over-expression of cyclin D and de‐ letion of *RB* confer treatment resistance to BRAFi [56, 58]. Unlike other components of

**Proof Corrections Form** 

**Chapter Title: Overcoming Resistance to BRAF and MEK Inhibitors by** 

the p16-cyclin D-CDK4-RB axis that harbor genetic changes at low frequency in melano‐ mas (e.g., *CDK4* and *RB* each approximately 3%) [59], and may not overlap with *BRAF* mutation (e.g., amplification of cyclin D1 gene *CCND1* and *CDK4*) [60], *INK4A* lesions are frequently detected in melanomas (~50%) irrespective of *BRAF* mutation [59-61]; therefore, abnormal p16 is a major mechanism of RB-axis attenuation in *BRAF*-mutant melanoma cells. p16 binds to and inhibits the catalytic activity of CDK4, representing a crucial gatekeeper at the G1>S checkpoint [62, 63]. The relative abundance of CDK4-cy‐ clin D and p16 can determine the activity of the CDK4 kinase, thus regulate RB and cellcycle progression [62, 63]. BRAF-MEK-ERK signaling pathway upregulates/activates the cyclin D-CDK4 enzyme, which phosphorylates and inactivates RB leading to cell cycle progression in melanoma cells; such an effect can be blocked by tumor suppressor p16 [2, 3, 61]. Several pathways that confer BRAFi resistance, including COT, RAF splicing variants, RAF dimerization, NRAS, IGF-1R, and RTK can reactivate cyclin D-CDK4 through signaling pathways including MEK-ERK as well as PI3K-AKT [51-53, 55, 56, 64]. Although the addition of MEKi to BRAFi may suppress reactivation of MEK-ERK-cyclin D-CDK4, alternative resistance mechanisms, including growth factor receptors and PI3K-AKT pathway can activate cyclin D-CDK4 [52, 55, 64-66] *in the absence of a functional p16*, adding CDK4 inhibitor may help overcoming resistance to BRAFi and MEKi (Fig. 3). *BRAF* mutation assays have been used to direct BRAFi treatment. There is significant genotypic heterogeneity of *INK4A* including bi- and mono-allelic deletions, nonsense and missense mutations, and also different levels of epigenetic modification by promoter hy‐ per-methylation. Characterization of whether these *INK4A* changes correlate with differ‐ ent treatment resistance to BRAFi/MEKi/CDK4i may lead to companion molecular tests to better manage melanoma patients under BRAFi, MEKi, and CDK4i therapy. **Simultaneous Suppression of CDK4 SAMPLE PROOF CORRECTIONS FORM Page No. Line No. Delete Replace with**  3 18 <10-13 cm/s <10-13 cm/s first hour 4 5 forth of fourth- or 4 15 set at 15-20 cm set at 10-15 cm 5 3 chest anatomy and feature artery origin anatomic features of chest and LMCA origin 11 Table 2 2nd row, 1st column: dutst dust 12 Fig. 7 Could you please reduce the size of figure by ~20%? 13 5 . Third or fifth one . Fifth one Notice: - Make sure to read InTech's manuscript preparation guidelines before filling out the proof corrections form. Some types of formatting (e.g. underline or bold and italic used together) are not allowed. These modifications can not be performed even if listed on the proof corrections form. It also may be the reason why such formatting was removed from the original manuscript. - If you are only inserting text, please fill in both columns, as seen in the 1st row of the sample proof corrections form. - Please enter the changes in order in which they appear in the text. - If there are figures to be replaced, feel free to paste them into the 'Replace with' column. - If there are multiple corrections in one sentence or a few sentences in a row, please list them as one entry.

found to be induced by ERK activation and telomere attrition involving cell senescence [8, 10, 11, 38]. The tumor suppressor p16 is encoded by *INK4A* (Fig. 2) and is often inactivated in a variety of human cancers, including 30-70% in melanomas [39, 40]. Most melanomas, but not nevi, have lost the expression of wild-type *INK4A*, either through DNA deletion/ mutation or promoter hypermethylation [41-45]. It is possible that the loss-of-function of p16 in melanomas may make it possible to bypass the cellular senescence mechanism and func‐ tion as an anti-tumor mechanism against ERK signal activation triggered by *NRAS* and

Indirect evidence from cultured cells and animal models reveal that there may be a coop‐ erative role between the constitutive activation of ERK pathway and the loss of p16 in tumor progression. Daniotti et al. [48] reported the co-existence of *BRAF* mutations and *INK4A* mutations/deletions/loss-of-expression in 26 of 41 (63%) short-term cell lines ob‐ tained from melanoma biopsies. Recent evidence suggests that growth arrest in benign nevi is due to cell senescence and that p16 at least partially contributes to the process of

**Figure 2.** *INK4A* and *ARF* share sequences in the *CDKN2A* locus. Exons are shown as rectangles. Alternative first exons (1α and 1β) are transcribed from different promoters (arrows). Exons 1α and 1β are spliced to the same splicing ac‐ ceptor site in exon 2 but are translated in alternative reading frames. *INK4A* coding sequences in exons 1α, 2, and 3 and *ARF* coding sequences in exons 1β and 2 are indicated by different shading patterns. Adopted from Sherr [8].

**Resistance of melanoma to BRAF and MEK inhibitors.** The finding that mutations in *BRAF* occur in melanomas led to extensive investigation of targeting BRAF for melanoma treat‐ ment. While the response to selective mutant BRAF inhibitors (BRAFi) in *BRAF*-mutant mel‐ anoma is encouraging, virtually all patients rapidly develop secondary resistance. Based on the finding that the mitogen activated protein kinase/ERK kinase (MEK)-extracellular signal regulated kinase (ERK) pathway is frequently reactivated by various BRAFi resistance mechanisms, the first combination trial of a selective BRAF inhibitor (dabrafenib, GSK2118436) with a MEK inhibitor (trametinib, GSK1120212) is underway and has achieved clinical responses in 17% and disease control in 67% in patients who failed prior single-agent treatment with a BRAF inhibitor [9]. While these results are promising, again, the treatment response is short-lived; there is a critical need for additional strategies to overcome this

There is evidence that treatment response to BRAFi and MEKi relies on a functional p16 cyclin D-CDK4-retinoblastoma (RB) axis. *INK4A* mutations/deletions occur in most of the melanoma cells that demonstrated resistance to BRAFi (e.g.; 451Lu, Mel1617, WM983, WM902, A375, M238, SKMEL28, and A2058) [51-57]. Over-expression of cyclin D and de‐ letion of *RB* confer treatment resistance to BRAFi [56, 58]. Unlike other components of

*BRAF* oncogenic mutation (Fig. 1) [11, 46, 47].

6 Melanoma - From Early Detection to Treatment

*INK4A* lesions detected by FISH and Sanger sequencing may also affect *ARF*.

senescence in nevi [11, 46, 47].

deadly disease [49, 50].

As shown in Fig. 4, in addition to BRAF and MEK inhibitors, several drugs designed to in‐ hibit the activity of CDK4 are in active clinical trials for melanoma and other cancers includ‐ ing LEE011 (Novartis Pharmaceuticals ), LY2835219 (Eli Lilly and Company), PD-0332991 (Pfizer) (http://clinicaltrials.gov/). Enter all the sentences you want changed in the 'Delete' column, and the corrected sentences to the 'Replace with' column. - If extensive changes are made to the references section, please replace the whole section. You can provide the new, corrected references list after the proofreading form table. **PROOF CORRECTIONS FORM** 

**Figure 3.** The presence of functional p16 may offset resistance mechanisms that lead to activation of cyclin D-CDK4 in melanomas that progressed under BRAFi/MEKi treatment, whereas abnormal p16 may predict treatment failure in melanomas that develop resistance mechanisms un-opposed by BRAFi + MEKi treatment.

**Combined inhibition of CDK4 potentiate the effect of MEKi.** In order to design better strategies for the treatment of this devastating disease a better understanding of melanoma biology is necessary. Multiple genetic and environmental factors have been linked to the de‐ 1

velopment and aggressive behavior of melanomas [49, 50]. *BRAF* mutations have been iden‐ tified in approximately 60–80% of human melanomas, while *NRAS* mutations occur in about 10% of melanomas [4, 67]. Both NRAS and BRAF are components of the RAS-RAF-mitogen activated protein kinase/ERK kinase (MEK)-extracellular signal regulated kinase (ERK) sig‐ naling pathway. Apart from *NRAS* and *BRAF* mutation, other factors have been identified leading to constitutive activation of the ERK signaling, for example, amplification and so‐ matic mutations of KIT and constitutive expression of hepatocyte growth factor (HGF) and fibroblast growth factor (FGF) [49, 50]. ERK signaling pathway controls cell proliferation, differentiation, and survival, and has been shown to be a targetable pathway in melanoma treatment [5, 14, 15, 68].

mas [27, 72]. Therefore, we tested PD98059 and 219476, commercially available inhibitors of

Overcoming Resistance to BRAF and MEK Inhibitors by Simultaneous Suppression of CDK4

http://dx.doi.org/10.5772/53620

9

**Figure 4.** BRAF, MEK and CDK4 inhibitors are in active clinical development and may be used in combination to in‐ crease treatment efficacy. Melanoma cells acquire resistance to BRAF and MEK inhibitors by mechanisms including ac‐ tivation of growth factor receptors and RAS signaling pathways. Activation of growth factor receptors and RAS pathways can cause overexpression of cyclin D and activation CDK4 kinase, leading to cell cycle proliferation, which is believed to play major roles in the emergence of treatment resistance. Adding CDK4 inhibitors may overcome resist‐ ance to treatment targeting BRAF and MEK. Apart from Vemurafenib (PLX4032, RO5185426) (Hoffmann-La Roche) that has been U.S. Food and Drug Administration (FDA) approved for treatment of melanoma, other mutant BRAF inhibitors including PLX3603 (RO5212054) (Hoffmann-La Roche) and GSK2118436 (dabrafenib) (GlaxoSmithKline) are in active clinical trials. There are clinical trials of MEK inhibitors PD-325901 (Pfizer), GSK1120212 (GlaxoSmithKline), MSC1936369B (EMD Serono), ARRY-438162 (MEK162) (Array BioPharma), AZD6244 (AstraZeneca), and BAY86-9766 (Bayer). Several drugs designed to inhibit the activity of CDK4 are also in active clinical trials for melanoma and other cancers including PD-0332991 (Pfizer), LY2835219 (Eli Lilly and Company), LEE011 (Novartis Pharmaceuticals) (http://

MEK inhibitor PD98059 (Calbiochem, San Diego, CA) was dissolved in dimethyl sulfoxide (DMSO) as a 50 mM stock solution, aliquoted and stored at -20C. CDK4 inhibitor 219476 (Cat. # 219476, Calbiochem, San Diego, CA) was dissolved in DMSO as a 2 mM stock solu‐ tion and stored at 4C. Human melanoma cell lines 624Mel, A101D, and OM431 were kindly provided by Dr. Stuart Aaronson (Mount Sinai School of Medicine, New York, NY). Cells were maintained in Dulbecco's modified Eagle medium (DMEM) (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (FBS; Sigma, St. Louis, MO) and 50 units/mL

MEK and CDK4, respectively, in human melanoma cells.

clinicaltrials.gov/).

Deregulation of the p16-cyclin D:cyclin-dependent kinases (CDK) 4/6-retinoblastoma (RB) pathway is a common paradigm in malignancy including melanoma [12, 13, 39] and rep‐ resents another attractive target in melanoma treatment. The great majority of melanoma cells have lost or reduced expression of wild-type *INK4A* caused by genetic and epigenet‐ ic changes including mutation, deletion, and promoter hypermethylation [69, 70]. Loss of p16 leaves cyclin D:CDK4 unoppressed to phosphorylate and inactivate RB and cell cycle progression [8, 13, 49, 50, 69, 70]. Amplification of cyclin D1 and CDK4 genes have also been identified, mostly in melanomas that harbor wild-type *NRAS* and *BRAF* [58]. A germ-line Arg24Cys (R24C) mutation in CDK4 was identified in familial melanoma pa‐ tients [40, 58]. This mutation abolishes CDK4 inhibition by p16 and thus is believed to be a functional equivalent to p16 loss. Both ERK signaling and CDK4 kinase have been shown to regulate RB protein and cell cycle progression [58, 61]. Activation of BRAF-MEK-ERK signaling pathway can cause upregulation of cyclin D resulting in the activa‐ tion of CDK4 [61]. Activated CDK4 phosphorylates and inactivated RB proteins result in the liberation of E2F transcription factors and cell cycle progression. It has been shown that in advanced melanoma cells, RB is highly phosphorylated and inactive, and E2F transcriptional activity is constitutively high ([5, 12].

Various resistance mechanisms have been identified that contribute to treatment failure of melanoma patients to BRAFi and MEKi therapy. Loss of p16 may represent a common gate‐ way permitting the phenotypic expression of several resistance mechanisms to BRAFi and MEKi (Figs. 1 and 3), a hypothesis that has not been and is waiting to be tested in clinical trials. We reported that simultaneous expression of *BRAF* siRNA and *INK4A* cDNA in mela‐ noma cells leads to dramatically increased apoptosis (17), suggesting that correcting the two most common genetic lesions could be effective in melanoma treatment. It is unclear wheth‐ er the effect is specific to *BRAF* and *INK4A* or can be generalized to other components of the ERK and RB pathways. It has been shown that *BRAF* and *INK4A* may have activities inde‐ pendent of the corresponding canonical ERK and RB pathways, and the two pathways also mediate cellular signals independent of aberrant *BRAF* and *INK4A*. For example, RAF can act through apoptosis signal-regulating kinase-1 (ASK1)/c-Jun-NH2-kinase or mammalian sterile 20- like-kinase 2 (MST2) pathways ([71]; cyclin D:CDK4 can be activated by enhanced phosphatidylinositol 3-kinase (PI3K) and wingless (WNT) signaling pathways in melano‐ mas [27, 72]. Therefore, we tested PD98059 and 219476, commercially available inhibitors of MEK and CDK4, respectively, in human melanoma cells.

velopment and aggressive behavior of melanomas [49, 50]. *BRAF* mutations have been iden‐ tified in approximately 60–80% of human melanomas, while *NRAS* mutations occur in about 10% of melanomas [4, 67]. Both NRAS and BRAF are components of the RAS-RAF-mitogen activated protein kinase/ERK kinase (MEK)-extracellular signal regulated kinase (ERK) sig‐ naling pathway. Apart from *NRAS* and *BRAF* mutation, other factors have been identified leading to constitutive activation of the ERK signaling, for example, amplification and so‐ matic mutations of KIT and constitutive expression of hepatocyte growth factor (HGF) and fibroblast growth factor (FGF) [49, 50]. ERK signaling pathway controls cell proliferation, differentiation, and survival, and has been shown to be a targetable pathway in melanoma

Deregulation of the p16-cyclin D:cyclin-dependent kinases (CDK) 4/6-retinoblastoma (RB) pathway is a common paradigm in malignancy including melanoma [12, 13, 39] and rep‐ resents another attractive target in melanoma treatment. The great majority of melanoma cells have lost or reduced expression of wild-type *INK4A* caused by genetic and epigenet‐ ic changes including mutation, deletion, and promoter hypermethylation [69, 70]. Loss of p16 leaves cyclin D:CDK4 unoppressed to phosphorylate and inactivate RB and cell cycle progression [8, 13, 49, 50, 69, 70]. Amplification of cyclin D1 and CDK4 genes have also been identified, mostly in melanomas that harbor wild-type *NRAS* and *BRAF* [58]. A germ-line Arg24Cys (R24C) mutation in CDK4 was identified in familial melanoma pa‐ tients [40, 58]. This mutation abolishes CDK4 inhibition by p16 and thus is believed to be a functional equivalent to p16 loss. Both ERK signaling and CDK4 kinase have been shown to regulate RB protein and cell cycle progression [58, 61]. Activation of BRAF-MEK-ERK signaling pathway can cause upregulation of cyclin D resulting in the activa‐ tion of CDK4 [61]. Activated CDK4 phosphorylates and inactivated RB proteins result in the liberation of E2F transcription factors and cell cycle progression. It has been shown that in advanced melanoma cells, RB is highly phosphorylated and inactive, and E2F

Various resistance mechanisms have been identified that contribute to treatment failure of melanoma patients to BRAFi and MEKi therapy. Loss of p16 may represent a common gate‐ way permitting the phenotypic expression of several resistance mechanisms to BRAFi and MEKi (Figs. 1 and 3), a hypothesis that has not been and is waiting to be tested in clinical trials. We reported that simultaneous expression of *BRAF* siRNA and *INK4A* cDNA in mela‐ noma cells leads to dramatically increased apoptosis (17), suggesting that correcting the two most common genetic lesions could be effective in melanoma treatment. It is unclear wheth‐ er the effect is specific to *BRAF* and *INK4A* or can be generalized to other components of the ERK and RB pathways. It has been shown that *BRAF* and *INK4A* may have activities inde‐ pendent of the corresponding canonical ERK and RB pathways, and the two pathways also mediate cellular signals independent of aberrant *BRAF* and *INK4A*. For example, RAF can act through apoptosis signal-regulating kinase-1 (ASK1)/c-Jun-NH2-kinase or mammalian sterile 20- like-kinase 2 (MST2) pathways ([71]; cyclin D:CDK4 can be activated by enhanced phosphatidylinositol 3-kinase (PI3K) and wingless (WNT) signaling pathways in melano‐

treatment [5, 14, 15, 68].

8 Melanoma - From Early Detection to Treatment

transcriptional activity is constitutively high ([5, 12].

**Figure 4.** BRAF, MEK and CDK4 inhibitors are in active clinical development and may be used in combination to in‐ crease treatment efficacy. Melanoma cells acquire resistance to BRAF and MEK inhibitors by mechanisms including ac‐ tivation of growth factor receptors and RAS signaling pathways. Activation of growth factor receptors and RAS pathways can cause overexpression of cyclin D and activation CDK4 kinase, leading to cell cycle proliferation, which is believed to play major roles in the emergence of treatment resistance. Adding CDK4 inhibitors may overcome resist‐ ance to treatment targeting BRAF and MEK. Apart from Vemurafenib (PLX4032, RO5185426) (Hoffmann-La Roche) that has been U.S. Food and Drug Administration (FDA) approved for treatment of melanoma, other mutant BRAF inhibitors including PLX3603 (RO5212054) (Hoffmann-La Roche) and GSK2118436 (dabrafenib) (GlaxoSmithKline) are in active clinical trials. There are clinical trials of MEK inhibitors PD-325901 (Pfizer), GSK1120212 (GlaxoSmithKline), MSC1936369B (EMD Serono), ARRY-438162 (MEK162) (Array BioPharma), AZD6244 (AstraZeneca), and BAY86-9766 (Bayer). Several drugs designed to inhibit the activity of CDK4 are also in active clinical trials for melanoma and other cancers including PD-0332991 (Pfizer), LY2835219 (Eli Lilly and Company), LEE011 (Novartis Pharmaceuticals) (http:// clinicaltrials.gov/).

MEK inhibitor PD98059 (Calbiochem, San Diego, CA) was dissolved in dimethyl sulfoxide (DMSO) as a 50 mM stock solution, aliquoted and stored at -20C. CDK4 inhibitor 219476 (Cat. # 219476, Calbiochem, San Diego, CA) was dissolved in DMSO as a 2 mM stock solu‐ tion and stored at 4C. Human melanoma cell lines 624Mel, A101D, and OM431 were kindly provided by Dr. Stuart Aaronson (Mount Sinai School of Medicine, New York, NY). Cells were maintained in Dulbecco's modified Eagle medium (DMEM) (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (FBS; Sigma, St. Louis, MO) and 50 units/mL penicillin–streptomycin (Invitrogen, Carlsbad, CA) in a humidified incubator at 37C with 5% CO2. CellTiter 96® R AQueous One Solution Cell Proliferation Assay (MTS) kit (Prome‐ ga Corporation, Madison, WI) was used to measure dehydrogenase enzyme activity found in metabolically active cells. Melanoma cells were seeded in a 96 well plate at a density of 2 ×104 cells/well in DMEM with 5% FBS. On the second day, the culture medium in each well was changed to 150 *μ*L DMEM without phenol red and supplemented with 0.5% FBS. Cells were treated in triplicate for 24 and 48 hr with either vehicle solvent (control), 25 *μ*M PD98059, 1 *μ*M 219476, or their combination for 624Mel; control solvent, 50 *μ*M PD98059, 1 *μ*M 219476, or their combination for A101D; and control solvent, 50 *μ*M PD98059, 2 *μ*M 219476, or their combination for OM431 cells. CellTiter 96® AQueous One Solution Reagent (30 *μ*L) was then added per well and cell cultures were returned to the incubator for another 4 hr. Subsequently, the absorbance of each well was measured at 450 nm with a Vmax Kinet‐ ic Microplate Reader (Molecular Devices, Sunnyvale, CA). The absorbance of the well with only medium and CellTiter 96® AQueous One Solution Regent was background and sub‐ tracted from each sample well. The average and standard deviation of three wells with the same treatment were calculated.

regulators cyclin-dependent kinase inhibitor p27 kinase interacting protein 1 (KIP1) and RB, cells were treated with the chemicals in medium with 5% FBS for 24 hr and then harvested. For apoptosis-related protein B-cell chronic lymphocytic leukemia (CLL)/lymphoma 2 (BCL2), BCL2-like 1 (BCL2L1 or bcl-xL), inhibitor of apoptosis family (IAP) protein baculo‐ viral IAP repeat-containing 5 (BIRC5 or survivin), apoptosis facilitator BCL2 interacting me‐ diator (BIM), cysteine-aspartic acid protease (caspase) 3, and poly (ADP-ribose) polymerase (PARP), cells were treated with the various chemicals in DMEM with 5% FBS for 48 hr and then harvested. For phospho- and total-ERK, cells were treated with the chemicals in medi‐ um with 0.5% FBS for 18 hr and then harvested. Western blots were performed as described [1-3]. Briefly, harvested cells were lysed in Lysis Solution (Cell Signaling, Danvers, MA) sup‐ plemented with Complete Mini Protease Inhibitor Cocktail Tablets (Roche Diagnostics Cor‐ poration, Indianapolis, IN). Protein concentration of lysates was determined using the Quick Start Bradford 1 × Dye Reagent (Bio-Rad, Hercules, CA). Lysates were separated in either 12 or 15% SDS-polyacrylamide gel, electrophoretically transferred to Immobilon-P membrane (Millipore Corp, Billerica, MA), and probed with primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The following antibodies were used: BCL2 and tubulin, beta (Sigma-Aldrich, St. Louis, MO); BCL2L1 and BIRC5 (San‐ ta Cruz Biotechnology, Santa Cruz, CA); phosphor-ERK, total ERK, Caspase 3, PARP, and PhosphoPlus(R) RB (Ser780, Ser795, Ser807/811) Antibody Kit (Cell Signaling, Boston, MA); p27KIP1 (BD Biosciences, San Jose, CA); and peroxidase-conjugated antimouse and antirab‐ bit secondary antibodies (Calbiochem, San Diego, CA). Immunoreactive bands were visual‐ ized with SuperSignal chemiluminescence substrate (Pierce, Rockford, IL). The blots were

Overcoming Resistance to BRAF and MEK Inhibitors by Simultaneous Suppression of CDK4

http://dx.doi.org/10.5772/53620

11

exposed to blue sensitive blue X-ray film (Phenix Research, Candler, NC) [1-3].

**Figure 5.** Regulation of ERK phosphorylation, RB phosphorylation, and p27KIP1 expression by PD98059 and 219476, alone and in combination. Human melanoma cell lines 624Mel, A101D, and OM431 were treated with either vehicle solvent (Con), PD98059 (PD), 219476 (CD), or PD98059 plus 219476 (PC) as described in Materials and methods. Western blot was performed using 20 μg total cell lysates, tubulin was used as loading control, as

described previously [1-3].

Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling of DNA fragments (TUNEL) method using *in situ* Cell Death Detection Kit, Fluorescein (Roche Applied Science, Indianapolis, IN). Melanoma cells were seeded in triplicate in a 6 well plate at a density of 2 × 105 cells/well in DMEM with 5% FBS and antibiotics. On the second day, cells were treated with PD98059 and 219476 under the same conditions as the MTS assay. After treatment with the respective chemicals for 48 hr, cells were harvested to detect apoptotic cells using the TUNEL assay according to the manufacturer's instructions (Roche Applied Science, Indianapolis, IN). Using a cytospin, cells were placed onto Polysine glass slides (Fisher Scientific, Fair lawn, NJ), fixed in 4% paraformaldehyde (Fisher Scientific, Fair lawn, NJ) at room temperature for 1 hr, then permeabilized with a fresh prepared mixture of 0.1% Triton X-100 (MP Biomedicals, Inc. Solon, OH) and 0.1% sodium citrate (Fisher Scientific, Fair lawn, NJ) for 5 min at room temperature. Slides were rinsed with phosphate buffered saline (PBS), air dried, and in‐ cubated with 50 *μ*L of TUNEL reaction mixture, containing terminal deoxynucleotidyl transferase (TdT)- and fluorescein isothiocyanate (FITC)-labeled dUTP, in a dark humidi‐ fied atmosphere at 37C for 2 hr. For nuclei counterstaining, slides were cover-slipped with Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA). Fluoresce positive cells were viewed with a Nikon Eclipse TE 2000-U inverted mi‐ croscope (Nikon Corp., Tokyo, Japan) equipped with a FITC filter and a DAPI filter. The percentage of apoptotic cells was determined for each sample in a blind fashion by counting the number of green fluorescent nuclei (TUNEL positive) among a total of 300 or more 4'-6-diamidino-2-phenylindole (DAPI)-stained blue nuclei in three random fields at magnification of 20/0.5 (objective) as described previously [1-3].

For Western blotting, 1 × 106 melanoma cells were seeded in a cell culture dish (10 cm) in DMEM containing 5% FBS and antibiotics. On the second day, cells were treated with PD98059 and 219476 at the same concentration as described in the MTS assay. For cell cycle regulators cyclin-dependent kinase inhibitor p27 kinase interacting protein 1 (KIP1) and RB, cells were treated with the chemicals in medium with 5% FBS for 24 hr and then harvested. For apoptosis-related protein B-cell chronic lymphocytic leukemia (CLL)/lymphoma 2 (BCL2), BCL2-like 1 (BCL2L1 or bcl-xL), inhibitor of apoptosis family (IAP) protein baculo‐ viral IAP repeat-containing 5 (BIRC5 or survivin), apoptosis facilitator BCL2 interacting me‐ diator (BIM), cysteine-aspartic acid protease (caspase) 3, and poly (ADP-ribose) polymerase (PARP), cells were treated with the various chemicals in DMEM with 5% FBS for 48 hr and then harvested. For phospho- and total-ERK, cells were treated with the chemicals in medi‐ um with 0.5% FBS for 18 hr and then harvested. Western blots were performed as described [1-3]. Briefly, harvested cells were lysed in Lysis Solution (Cell Signaling, Danvers, MA) sup‐ plemented with Complete Mini Protease Inhibitor Cocktail Tablets (Roche Diagnostics Cor‐ poration, Indianapolis, IN). Protein concentration of lysates was determined using the Quick Start Bradford 1 × Dye Reagent (Bio-Rad, Hercules, CA). Lysates were separated in either 12 or 15% SDS-polyacrylamide gel, electrophoretically transferred to Immobilon-P membrane (Millipore Corp, Billerica, MA), and probed with primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The following antibodies were used: BCL2 and tubulin, beta (Sigma-Aldrich, St. Louis, MO); BCL2L1 and BIRC5 (San‐ ta Cruz Biotechnology, Santa Cruz, CA); phosphor-ERK, total ERK, Caspase 3, PARP, and PhosphoPlus(R) RB (Ser780, Ser795, Ser807/811) Antibody Kit (Cell Signaling, Boston, MA); p27KIP1 (BD Biosciences, San Jose, CA); and peroxidase-conjugated antimouse and antirab‐ bit secondary antibodies (Calbiochem, San Diego, CA). Immunoreactive bands were visual‐ ized with SuperSignal chemiluminescence substrate (Pierce, Rockford, IL). The blots were exposed to blue sensitive blue X-ray film (Phenix Research, Candler, NC) [1-3].

penicillin–streptomycin (Invitrogen, Carlsbad, CA) in a humidified incubator at 37C with 5% CO2. CellTiter 96® R AQueous One Solution Cell Proliferation Assay (MTS) kit (Prome‐ ga Corporation, Madison, WI) was used to measure dehydrogenase enzyme activity found in metabolically active cells. Melanoma cells were seeded in a 96 well plate at a density of 2

 cells/well in DMEM with 5% FBS. On the second day, the culture medium in each well was changed to 150 *μ*L DMEM without phenol red and supplemented with 0.5% FBS. Cells were treated in triplicate for 24 and 48 hr with either vehicle solvent (control), 25 *μ*M PD98059, 1 *μ*M 219476, or their combination for 624Mel; control solvent, 50 *μ*M PD98059, 1 *μ*M 219476, or their combination for A101D; and control solvent, 50 *μ*M PD98059, 2 *μ*M 219476, or their combination for OM431 cells. CellTiter 96® AQueous One Solution Reagent (30 *μ*L) was then added per well and cell cultures were returned to the incubator for another 4 hr. Subsequently, the absorbance of each well was measured at 450 nm with a Vmax Kinet‐ ic Microplate Reader (Molecular Devices, Sunnyvale, CA). The absorbance of the well with only medium and CellTiter 96® AQueous One Solution Regent was background and sub‐ tracted from each sample well. The average and standard deviation of three wells with the

Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling of DNA fragments (TUNEL) method using *in situ* Cell Death Detection Kit, Fluorescein (Roche Applied Science, Indianapolis, IN). Melanoma cells were seeded in triplicate in a 6 well plate at a density of 2 × 105 cells/well in DMEM with 5% FBS and antibiotics. On the second day, cells were treated with PD98059 and 219476 under the same conditions as the MTS assay. After treatment with the respective chemicals for 48 hr, cells were harvested to detect apoptotic cells using the TUNEL assay according to the manufacturer's instructions (Roche Applied Science, Indianapolis, IN). Using a cytospin, cells were placed onto Polysine glass slides (Fisher Scientific, Fair lawn, NJ), fixed in 4% paraformaldehyde (Fisher Scientific, Fair lawn, NJ) at room temperature for 1 hr, then permeabilized with a fresh prepared mixture of 0.1% Triton X-100 (MP Biomedicals, Inc. Solon, OH) and 0.1% sodium citrate (Fisher Scientific, Fair lawn, NJ) for 5 min at room temperature. Slides were rinsed with phosphate buffered saline (PBS), air dried, and in‐ cubated with 50 *μ*L of TUNEL reaction mixture, containing terminal deoxynucleotidyl transferase (TdT)- and fluorescein isothiocyanate (FITC)-labeled dUTP, in a dark humidi‐ fied atmosphere at 37C for 2 hr. For nuclei counterstaining, slides were cover-slipped with Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA). Fluoresce positive cells were viewed with a Nikon Eclipse TE 2000-U inverted mi‐ croscope (Nikon Corp., Tokyo, Japan) equipped with a FITC filter and a DAPI filter. The percentage of apoptotic cells was determined for each sample in a blind fashion by counting the number of green fluorescent nuclei (TUNEL positive) among a total of 300 or more 4'-6-diamidino-2-phenylindole (DAPI)-stained blue nuclei in three random fields

at magnification of 20/0.5 (objective) as described previously [1-3].

DMEM containing 5% FBS and antibiotics. On the second day, cells were treated with PD98059 and 219476 at the same concentration as described in the MTS assay. For cell cycle

melanoma cells were seeded in a cell culture dish (10 cm) in

×104

same treatment were calculated.

10 Melanoma - From Early Detection to Treatment

For Western blotting, 1 × 106

**Figure 5.** Regulation of ERK phosphorylation, RB phosphorylation, and p27KIP1 expression by PD98059 and 219476, alone and in combination. Human melanoma cell lines 624Mel, A101D, and OM431 were treated with either vehicle solvent (Con), PD98059 (PD), 219476 (CD), or PD98059 plus 219476 (PC) as described in Materials and methods. Western blot was performed using 20 μg total cell lysates, tubulin was used as loading control, as described previously [1-3].

PD98059 and 219476 inhibit tumor cell growth in a dose dependent manner [1, 2]. In order to make it possible to monitor the additional therapeutic effects of the combinatorial treat‐ ment, both chemicals were used at dosages lower than that which would lead to maximal effect by either agent. The cytotoxicity of PD98059 and 219476 was examined 24 and 48 hr after treatment using the MTS assay that measures the dehydrogenase enzyme activity found in metabolically active cells. After 24-hr treatment, there was no significant difference in cell viability between control, single, and combined treatment groups of 624Mel cells (*p* = . 05, R-square 0.57320, ANOVA). Small but significant differences were observed in A101D and OM431 cells (*p* = .05, R-square 0.7136 and 0.8091 in A101D and OM431 cells, respective‐ ly, ANOVA); the differences were between the combined treatment vs. control and PD98059 in A101D cells, and between the combined treatment vs. control and single treatment of ei‐ ther PD98059 or 219476 in OM431 cells (Figure 2(a), HSD Test at 0.05 significance level). Af‐ ter 48-hr treatment, a significant difference in MTS counts existed for the control, PD98059, 219476, and PD98059 plus 219476 groups in all the three cell lines (*p <*.0001, R-square 0.981444, 0.956956, and 0.991102 in 624Mel, A101D, and OM431, respectively, ANOVA). Further analysis showed that simultaneous treatment with PD98059 and 219476 after 48-hr treatment resulted in significantly reduced numbers of cell survival than control-treatment or monotreatment as measured by MTS in all the three cell lines (Fig. 6B, HSD Test at 0.05

Overcoming Resistance to BRAF and MEK Inhibitors by Simultaneous Suppression of CDK4

http://dx.doi.org/10.5772/53620

13

Next, we performed the TUNEL DNA fragmentation assay to identify loss of viability due to programmed cell death after 48-hr treatment. As shown in Figure 3, at the drug concen‐ trations used, significantly different levels of apoptosis exist among control for PD98059, 219476, and combinatorial treatment groups (*p <* .0001, R-square 0.973862, 0.990697, and 0.987900 in 624Mel, A101D, and OM431, respectively, ANOVA). Treatment with PD98059 alone resulted in no difference in apoptosis over controls in all three cell lines; 219476 en‐ hanced apoptosis in OM431 but not in the other two cell lines; However, combined treat‐ ment dramatically increased apoptosis over that seen for the control-treatment and

As apoptosis was the major effect observed when melanoma cells were exposed simultane‐ ously to MEK and CDK4 inhibitors, we examined the expression of several pro-apoptotic and anti-apoptotic proteins. Mono-treatment with PD98059 or 219476 caused a decreased or no change in the expression of anti-apoptotic proteins BCL2, BCL2L1, and BIRC5. While there were variations in the patterns of expression of BCL2, BCL2L1, and BIRC5 among the different cell lines (Fig. 8), combinatorial treatment caused a comprehensive down-regula‐ tion of the proteins in all three cell lines (Fig. 8). In addition, apoptosis facilitator BIM-EL was increased following treatment with PD98059 and PD98059 plus 219476 in all three cell lines. It was also increased in OM431 cells following treatment with 219476. Consistent with increased apoptosis, caspase 3 was activated by simultaneous treatment with PD98059 plus 219476 in all three cell lines, as shown by decreased procaspase 3, increased levels of the ac‐ tive form of caspase 3 (cleaved caspase 3), and degradation of PARP, a direct substrate of

monotreatment (Fig. 7. HSD Test at 0.05 significance level).

significance level).

active caspase 3 (Fig. 8).

**Figure 6.** Cytotoxicity by PD98059, 219476, and combinatorial treatment. MTS cytotoxicity assay was performed in 624Mel, A101D, and OM431 cells after (A) 24-hr and (B) 48-hr treatment in medium supplemented with 0.5% FBS. The results are given as means ± SD from three independent tests, as described previously [1-3].

We have shown previously that human melanoma cell lines 624Mel, A101D, and OM431 cell lines harbor heterozygous *BRAF* T1799A mutation and loss of wild-type *INK4A* [1, 61]. Cells were treated, alone or in combination, with MEK inhibitor PD98059 (22) and CDK4 inhibitor 219476 (23). As anticipated, ERK phosphorylation was reduced in cells treated with PD98059, and PD98059 plus 219476 (Fig. 4A). Phosphorylation of S780, S795, and S807/811 of RB, known cyclin D:CDK4 and cyclin E:cyclin dependent kinase 2 (CDK2) targets (7),was decreased in cells treated with either PD98059 or 219476 (except S780 and S807/811 in OM431 cells), and further reduced in cells with combinatorial treatment (Fig. 4B). Of note, total RB was decreased under combinatorial treatment with PD98059 and 219476 in all three melanoma cells (Fig. 4B). Levels of p27KIP1, a negative regulator of cyclin E:CDK2, were in‐ creased in cells treated with either PD98059 or 219476, and further increased in cells with combinatorial treatment (Fig. 4C).

PD98059 and 219476 inhibit tumor cell growth in a dose dependent manner [1, 2]. In order to make it possible to monitor the additional therapeutic effects of the combinatorial treat‐ ment, both chemicals were used at dosages lower than that which would lead to maximal effect by either agent. The cytotoxicity of PD98059 and 219476 was examined 24 and 48 hr after treatment using the MTS assay that measures the dehydrogenase enzyme activity found in metabolically active cells. After 24-hr treatment, there was no significant difference in cell viability between control, single, and combined treatment groups of 624Mel cells (*p* = . 05, R-square 0.57320, ANOVA). Small but significant differences were observed in A101D and OM431 cells (*p* = .05, R-square 0.7136 and 0.8091 in A101D and OM431 cells, respective‐ ly, ANOVA); the differences were between the combined treatment vs. control and PD98059 in A101D cells, and between the combined treatment vs. control and single treatment of ei‐ ther PD98059 or 219476 in OM431 cells (Figure 2(a), HSD Test at 0.05 significance level). Af‐ ter 48-hr treatment, a significant difference in MTS counts existed for the control, PD98059, 219476, and PD98059 plus 219476 groups in all the three cell lines (*p <*.0001, R-square 0.981444, 0.956956, and 0.991102 in 624Mel, A101D, and OM431, respectively, ANOVA). Further analysis showed that simultaneous treatment with PD98059 and 219476 after 48-hr treatment resulted in significantly reduced numbers of cell survival than control-treatment or monotreatment as measured by MTS in all the three cell lines (Fig. 6B, HSD Test at 0.05 significance level).

Next, we performed the TUNEL DNA fragmentation assay to identify loss of viability due to programmed cell death after 48-hr treatment. As shown in Figure 3, at the drug concen‐ trations used, significantly different levels of apoptosis exist among control for PD98059, 219476, and combinatorial treatment groups (*p <* .0001, R-square 0.973862, 0.990697, and 0.987900 in 624Mel, A101D, and OM431, respectively, ANOVA). Treatment with PD98059 alone resulted in no difference in apoptosis over controls in all three cell lines; 219476 en‐ hanced apoptosis in OM431 but not in the other two cell lines; However, combined treat‐ ment dramatically increased apoptosis over that seen for the control-treatment and monotreatment (Fig. 7. HSD Test at 0.05 significance level).

**Figure 6.** Cytotoxicity by PD98059, 219476, and combinatorial treatment. MTS cytotoxicity assay was performed in 624Mel, A101D, and OM431 cells after (A) 24-hr and (B) 48-hr treatment in medium supplemented with 0.5% FBS.

We have shown previously that human melanoma cell lines 624Mel, A101D, and OM431 cell lines harbor heterozygous *BRAF* T1799A mutation and loss of wild-type *INK4A* [1, 61]. Cells were treated, alone or in combination, with MEK inhibitor PD98059 (22) and CDK4 inhibitor 219476 (23). As anticipated, ERK phosphorylation was reduced in cells treated with PD98059, and PD98059 plus 219476 (Fig. 4A). Phosphorylation of S780, S795, and S807/811 of RB, known cyclin D:CDK4 and cyclin E:cyclin dependent kinase 2 (CDK2) targets (7),was decreased in cells treated with either PD98059 or 219476 (except S780 and S807/811 in OM431 cells), and further reduced in cells with combinatorial treatment (Fig. 4B). Of note, total RB was decreased under combinatorial treatment with PD98059 and 219476 in all three melanoma cells (Fig. 4B). Levels of p27KIP1, a negative regulator of cyclin E:CDK2, were in‐ creased in cells treated with either PD98059 or 219476, and further increased in cells with

The results are given as means ± SD from three independent tests, as described previously [1-3].

combinatorial treatment (Fig. 4C).

12 Melanoma - From Early Detection to Treatment

As apoptosis was the major effect observed when melanoma cells were exposed simultane‐ ously to MEK and CDK4 inhibitors, we examined the expression of several pro-apoptotic and anti-apoptotic proteins. Mono-treatment with PD98059 or 219476 caused a decreased or no change in the expression of anti-apoptotic proteins BCL2, BCL2L1, and BIRC5. While there were variations in the patterns of expression of BCL2, BCL2L1, and BIRC5 among the different cell lines (Fig. 8), combinatorial treatment caused a comprehensive down-regula‐ tion of the proteins in all three cell lines (Fig. 8). In addition, apoptosis facilitator BIM-EL was increased following treatment with PD98059 and PD98059 plus 219476 in all three cell lines. It was also increased in OM431 cells following treatment with 219476. Consistent with increased apoptosis, caspase 3 was activated by simultaneous treatment with PD98059 plus 219476 in all three cell lines, as shown by decreased procaspase 3, increased levels of the ac‐ tive form of caspase 3 (cleaved caspase 3), and degradation of PARP, a direct substrate of active caspase 3 (Fig. 8).

In this study, we simultaneously inhibited MEK and CDK4 kinases using pharmacological inhibitors PD98059 and 219476 and observed significantly increased apoptosis compared to control and single agent treatment. This is consistent with our previous report that simulta‐ neous knockdown of BRAF using small interfering RNA (siRNA) and expression of *INK4A* cDNA in melanoma cells leads to a significant increase in apoptosis [1, 3]. These results demonstrate that an increase in apoptosis can be achieved through combinatorial targeting of ERK and RB pathways. It has been well established that constitutive activation of the ERK signaling induces the expression of cyclin D [1, 2, 61], which binds to and activates CDK4 leading to the phosphorylation of RB protein facilitating cell cycle entry [1, 2, 61]. Consistent with an epistatic regulation between ERK pathway and cyclin D:CDK4, amplification of cy‐ clin D1, and CDK4 genes have been identified mainly in melanomas that harbor wild-type *NRAS* and *BRAF* [58, 60]. Additionally, cyclin D:CDK4 mediates resistance to inhibitors of the ERK signaling pathway [58]. Therefore, the enhanced apoptosis and decreased prolifera‐ tion by simultaneously inhibiting ERK and RB pathways could result from the double hit‐ ting of ERK-cyclin D:CDK4-RB that regulate cell cycle progression and cell survival. Alternatively, in support of our previous results that *BRAF* and *INK4A* have a nonlinear functional interaction [1, 61], additional cellular processes could be affected when cells are exposed to both PD98059 and 219476. ERK pathway has pleiopotent activities that regulate cell proliferation, survival, and differentiation through both cyclin D:CDK4 dependent and independent routes [5, 61]. Likewise, cyclin D:CDK4 can be regulated and converges multi‐ ple cellular signals. For example, while PI3K signaling can activate CDK4 through downre‐ gulation of *INK4A* and upregulation of cyclin D [73], WNT signaling can turn on CDK4 through suppression of *INK4A* transcription [72], It is conceivable that inhibition of MEK and CDK4 not only affects ERK and RB pathways, but also PI3K, WNT, and other ERK sig‐ naling activities not mediated through the RB pathway. Therefore, simultaneous targeting of both ERK and RB pathways can generate enhanced effects by targeting both linear and non‐

Overcoming Resistance to BRAF and MEK Inhibitors by Simultaneous Suppression of CDK4

http://dx.doi.org/10.5772/53620

15

Apoptosis resistance is a critical factor for therapy failure in melanoma patients. Encourag‐ ingly, combined treatment with PD98059 and 219476 leads to significant apoptosis in all the three melanoma cell lines studied (Fig. 7). The apoptotic rate caused by the combined treat‐ ment is higher than the combined apoptosis by monotreatment, suggesting that MEK and CDK4 kinases mediate each other's pro-survival effect. The apoptotic effect is associated with changes of apoptosis-related proteins (Fig. 8). PD98059 and 219476 combined treatment leads to significant down-regulation of the pro-survival proteins BCL2, BCL2L1, and BIRC5, and up-regulation of the pro-apoptotic protein BIM. We showed previously that BCL2 and BIM were regulated by *BRAF* and *INK4A* [1, 61]. BCL2L1 and BIRC5 are highly expressed in melanoma cells, and increased expression correlates with tumor progression [74, 75]. A straightforward explanation for the observed apoptosis is that the changes in the pro-apop‐ totic and anti-apoptotic factors offset the balance and lead to apoptosis [1]. Sequencing anal‐ ysis of TP53 cDNA [1, 3] showed that 624Mel and OM431cells respectively harbor a T1076G (Cys275Trp) and a G1048A (Gly266Glu) mutations in the DNA binding domain that is likely to compromise the transcription and apoptosis function of p53 [76]. No TP53 mutation has been detected in A101D cells. Although apoptosis is enhanced in all the three cell lines, it is

overlapping activities.

**Figure 7.** MEK and CDK4 inhibitors induce apoptosis of melanoma cells. TUNEL Assay was performed in 624Mel, A101D and OM431 cells after 48h treatment with vehicle solvent, PD98059, 219476, or PD98059 plus 219476 in medium with 0.5% FBS. The results were given as means ± SD from three independent assays, as described pre‐ viously [1, 2].

**Figure 8.** Changes in the expression of pro-survival and pro-apoptotic proteins. Cells were treated with solvent vehicle control (1), PD98059 (2), 219476 (3), and PD98059 plus 219476 (4) for 48 h in medium containing 5% FBS. Western blotting of 20 μg total cell extracts from 624Mel, A101D and OM431 cells using BCL2, BCL2L1, BIRC5, BIM, caspase-3, and PARP antibodies; tubulin was used as loading control, as described previously [1, 2].

In this study, we simultaneously inhibited MEK and CDK4 kinases using pharmacological inhibitors PD98059 and 219476 and observed significantly increased apoptosis compared to control and single agent treatment. This is consistent with our previous report that simulta‐ neous knockdown of BRAF using small interfering RNA (siRNA) and expression of *INK4A* cDNA in melanoma cells leads to a significant increase in apoptosis [1, 3]. These results demonstrate that an increase in apoptosis can be achieved through combinatorial targeting of ERK and RB pathways. It has been well established that constitutive activation of the ERK signaling induces the expression of cyclin D [1, 2, 61], which binds to and activates CDK4 leading to the phosphorylation of RB protein facilitating cell cycle entry [1, 2, 61]. Consistent with an epistatic regulation between ERK pathway and cyclin D:CDK4, amplification of cy‐ clin D1, and CDK4 genes have been identified mainly in melanomas that harbor wild-type *NRAS* and *BRAF* [58, 60]. Additionally, cyclin D:CDK4 mediates resistance to inhibitors of the ERK signaling pathway [58]. Therefore, the enhanced apoptosis and decreased prolifera‐ tion by simultaneously inhibiting ERK and RB pathways could result from the double hit‐ ting of ERK-cyclin D:CDK4-RB that regulate cell cycle progression and cell survival. Alternatively, in support of our previous results that *BRAF* and *INK4A* have a nonlinear functional interaction [1, 61], additional cellular processes could be affected when cells are exposed to both PD98059 and 219476. ERK pathway has pleiopotent activities that regulate cell proliferation, survival, and differentiation through both cyclin D:CDK4 dependent and independent routes [5, 61]. Likewise, cyclin D:CDK4 can be regulated and converges multi‐ ple cellular signals. For example, while PI3K signaling can activate CDK4 through downre‐ gulation of *INK4A* and upregulation of cyclin D [73], WNT signaling can turn on CDK4 through suppression of *INK4A* transcription [72], It is conceivable that inhibition of MEK and CDK4 not only affects ERK and RB pathways, but also PI3K, WNT, and other ERK sig‐ naling activities not mediated through the RB pathway. Therefore, simultaneous targeting of both ERK and RB pathways can generate enhanced effects by targeting both linear and non‐ overlapping activities.

**Figure 7.** MEK and CDK4 inhibitors induce apoptosis of melanoma cells. TUNEL Assay was performed in 624Mel, A101D and OM431 cells after 48h treatment with vehicle solvent, PD98059, 219476, or PD98059 plus 219476 in medium with 0.5% FBS. The results were given as means ± SD from three independent assays, as described pre‐

**Figure 8.** Changes in the expression of pro-survival and pro-apoptotic proteins. Cells were treated with solvent vehicle control (1), PD98059 (2), 219476 (3), and PD98059 plus 219476 (4) for 48 h in medium containing 5% FBS. Western blotting of 20 μg total cell extracts from 624Mel, A101D and OM431 cells using BCL2, BCL2L1, BIRC5, BIM, caspase-3,

and PARP antibodies; tubulin was used as loading control, as described previously [1, 2].

viously [1, 2].

14 Melanoma - From Early Detection to Treatment

Apoptosis resistance is a critical factor for therapy failure in melanoma patients. Encourag‐ ingly, combined treatment with PD98059 and 219476 leads to significant apoptosis in all the three melanoma cell lines studied (Fig. 7). The apoptotic rate caused by the combined treat‐ ment is higher than the combined apoptosis by monotreatment, suggesting that MEK and CDK4 kinases mediate each other's pro-survival effect. The apoptotic effect is associated with changes of apoptosis-related proteins (Fig. 8). PD98059 and 219476 combined treatment leads to significant down-regulation of the pro-survival proteins BCL2, BCL2L1, and BIRC5, and up-regulation of the pro-apoptotic protein BIM. We showed previously that BCL2 and BIM were regulated by *BRAF* and *INK4A* [1, 61]. BCL2L1 and BIRC5 are highly expressed in melanoma cells, and increased expression correlates with tumor progression [74, 75]. A straightforward explanation for the observed apoptosis is that the changes in the pro-apop‐ totic and anti-apoptotic factors offset the balance and lead to apoptosis [1]. Sequencing anal‐ ysis of TP53 cDNA [1, 3] showed that 624Mel and OM431cells respectively harbor a T1076G (Cys275Trp) and a G1048A (Gly266Glu) mutations in the DNA binding domain that is likely to compromise the transcription and apoptosis function of p53 [76]. No TP53 mutation has been detected in A101D cells. Although apoptosis is enhanced in all the three cell lines, it is more pronounced in A101D than 624Mel and OM431 cells (Fig. 7), suggesting that TP53 sta‐ tus may influence the magnitude of apoptosis. Combinatorial-treated cells have further in‐ hibited phosphorylation of ERK and RB, reduced total RB, and increased expression of p27KIP1 (Fig. 5). We observed similar effects on ERK and p27KIP1 in a previous report of simultaneous expression of *BRAF* siRNA, and *INK4A* cDNA in melanoma cells [1, 3]. Yu et al. demonstrated that loss of Rb causes apoptosis without effect on cell proliferation [77], and Wang et al. found that overexpression of p27KIP1 leads to apoptosis in melanoma cells [78]. The mechanisms of these changes in relationship to each other and to the observed co‐ operative effects need to be further investigated. To our knowledge, this study is the first to demonstrate that combined inhibition of MEK and CDK4 using pharmacological inhibitors can cooperate to trigger significant apoptosis in melanoma cells. Deregulation of the RAS-RAF-MEK-ERK and p16-cycylin D:CDK4-RB pathways are common in human malignancies and appears to be important for melanoma development. There has been significant effort to target components of these pathways in cancer treatment. Pharmacologic agents targeting components of the ERK and RB pathways have been developed. However, clinical studies as monotherapy showed that the clinical responses have failed expectations and maximum tolerated doses are often reached before reaching clinical efficacy. Our current study further reinforces the notion that combination targeting of ERK and RB pathways is a promising strategy for melanoma treatment and should encourage further in-depth investigations.

the combination treatment [9]. We hypothesize that clinical response to combination therapy of BRAFi and MEKi correlates with status of *INK4A*/p16 (Table 2). The development of clini‐ cally useful *INK4A* assays requires an understanding of the underlying biology and access to technology that allows high quality assay performance. Recent advances in molecular technology enable accurate, rapid, and cost-effective *INK4A* molecular testing that can be performed routinely on tumor specimens. However, validation of the technical performance characteristics of *INK4A* assays and understanding of assay limitations are necessary for the

Overcoming Resistance to BRAF and MEK Inhibitors by Simultaneous Suppression of CDK4

As examples, Table 2 is a list of molecular assays to comprehensively examine *INK4A*/p16 lesions in melanoma specimens. Technical and clinical validation studies are necessary be‐

Test Method Reference

*INK4A* mutation Sanger sequencing [86, 87]

p16 expression Immunohistochemical staining (IHC) [90, 91]

These assays need to be validated both technically and clinically with defined cut-off values. There should be correlation of results among assay methods; for example, cells with bi-allel‐ ic *INK4A* deletion show negative p16 IHC staining and cells with mono-allelic *INK4A* dele‐ tion show mutations with loss of heterozygosity (LOH), and p16 expression inversely correlates with levels of *INK4A* promoter methylation. The major obstacles in testing tumor specimens are the presence of non-tumor cells in the samples, the cellular heterogeneity within tumor specimens, and degradation/damage of nucleic acid and protein during sam‐ ple processing. To ensure accurate testing results, SOPs need to be established with clearly

centromeric probe (CEP9) SpectrumGreen Probe, Abbott Molecular, Des

Pyrosequencing (PyroMark Q24 CpG p16 Kit, Qiagen, Valencia, CA) [82, 88, 89]

[85, 86]

http://dx.doi.org/10.5772/53620

17

*INK4A* deletion fluorescent *in situ* hybridization (FISH) (p16 SpectrumOrange/ chromosome 9

*INK4A status* **p16 protein sequence and expression**

Various mutations Heterogeneous sequence changes

Wild-type Normal sequence

Promoter hypermethylation Lower levels of p16

Bi-allelic deletion Protein null

**Table 1.** Heterogeneity of *INK4A* and p16 in melanoma specimens

fore the routine use of these assays in the clinic.

Plaines, IL)

*INK4A* promoter methylation

**Table 2.** Summary of molecular assays

accurate interpretation of test results.

**Development of biomarkers to predict treatment response to BRAF, MEK, and CDK4 in‐ hibitors.** Apart from *BRAF* mutation, there is no other validated molecular assay to direct BRAFi and MEKi treatment. Comprehensive and standardized *INK4A* molecular assays have not been established in the context of BRAFi and MEKi treatment. Technical and clini‐ cal validation of *INK4A* molecular assays may lead to the clinical use of new molecular com‐ panion biomarkers to accurately predict clinical response to BRAF and MEK inhibitors, and may also direct future combination treatment that includes CDK4 inhibitors for metastatic melanoma. Because CDK4 is important in both normal and cancerous cells, CDK4 inhibitors are expected to decrease the ability of the bone marrow to make white blood cells, platelets, and red blood cells. Although these effects are expected to be reversible, they can increase the risk of infection, bleeding and fatigue. Like BRAF inhibitors, these drugs are also expect‐ ed to be expensive. Therefore, development of predictive molecular markers, as in the case of *BRAF* mutation assay for BRAFi, should help selecting patients that are likely to response to the treatment, therefore to maximize efficacy and avoid unnecessary side-effect and treat‐ ment cost [79, 80].

Genetic and epigenetic changes of *INK4A* have been identified in 30-70% of melanomas irre‐ spective of *BRAF* mutation [59, 70, 81]. Bi-allelic deletion of *INK4A* (p16 null) occurs in 10-27% of melanomas [60, 82]. Other changes include mono-allelic deletion, point mutation, or promoter hypermethylation, resulting in various levels of p16 expression/activity (Table 1) [57, 60, 81-83]. It is believed that the acquisition of p16 lesions allows melanoma cells to bypass senescence/growth arrest during melanoma development [84]. Although preliminary results with combination therapy of BRAFi and MEKi are encouraging with better clinical response over single agent BRAFi treatment [9], levels of treatment responses vary under the combination treatment [9]. We hypothesize that clinical response to combination therapy of BRAFi and MEKi correlates with status of *INK4A*/p16 (Table 2). The development of clini‐ cally useful *INK4A* assays requires an understanding of the underlying biology and access to technology that allows high quality assay performance. Recent advances in molecular technology enable accurate, rapid, and cost-effective *INK4A* molecular testing that can be performed routinely on tumor specimens. However, validation of the technical performance characteristics of *INK4A* assays and understanding of assay limitations are necessary for the accurate interpretation of test results.


**Table 1.** Heterogeneity of *INK4A* and p16 in melanoma specimens

As examples, Table 2 is a list of molecular assays to comprehensively examine *INK4A*/p16 lesions in melanoma specimens. Technical and clinical validation studies are necessary be‐ fore the routine use of these assays in the clinic.


**Table 2.** Summary of molecular assays

more pronounced in A101D than 624Mel and OM431 cells (Fig. 7), suggesting that TP53 sta‐ tus may influence the magnitude of apoptosis. Combinatorial-treated cells have further in‐ hibited phosphorylation of ERK and RB, reduced total RB, and increased expression of p27KIP1 (Fig. 5). We observed similar effects on ERK and p27KIP1 in a previous report of simultaneous expression of *BRAF* siRNA, and *INK4A* cDNA in melanoma cells [1, 3]. Yu et al. demonstrated that loss of Rb causes apoptosis without effect on cell proliferation [77], and Wang et al. found that overexpression of p27KIP1 leads to apoptosis in melanoma cells [78]. The mechanisms of these changes in relationship to each other and to the observed co‐ operative effects need to be further investigated. To our knowledge, this study is the first to demonstrate that combined inhibition of MEK and CDK4 using pharmacological inhibitors can cooperate to trigger significant apoptosis in melanoma cells. Deregulation of the RAS-RAF-MEK-ERK and p16-cycylin D:CDK4-RB pathways are common in human malignancies and appears to be important for melanoma development. There has been significant effort to target components of these pathways in cancer treatment. Pharmacologic agents targeting components of the ERK and RB pathways have been developed. However, clinical studies as monotherapy showed that the clinical responses have failed expectations and maximum tolerated doses are often reached before reaching clinical efficacy. Our current study further reinforces the notion that combination targeting of ERK and RB pathways is a promising strategy for melanoma treatment and should encourage further in-depth investigations.

**Development of biomarkers to predict treatment response to BRAF, MEK, and CDK4 in‐ hibitors.** Apart from *BRAF* mutation, there is no other validated molecular assay to direct BRAFi and MEKi treatment. Comprehensive and standardized *INK4A* molecular assays have not been established in the context of BRAFi and MEKi treatment. Technical and clini‐ cal validation of *INK4A* molecular assays may lead to the clinical use of new molecular com‐ panion biomarkers to accurately predict clinical response to BRAF and MEK inhibitors, and may also direct future combination treatment that includes CDK4 inhibitors for metastatic melanoma. Because CDK4 is important in both normal and cancerous cells, CDK4 inhibitors are expected to decrease the ability of the bone marrow to make white blood cells, platelets, and red blood cells. Although these effects are expected to be reversible, they can increase the risk of infection, bleeding and fatigue. Like BRAF inhibitors, these drugs are also expect‐ ed to be expensive. Therefore, development of predictive molecular markers, as in the case of *BRAF* mutation assay for BRAFi, should help selecting patients that are likely to response to the treatment, therefore to maximize efficacy and avoid unnecessary side-effect and treat‐

Genetic and epigenetic changes of *INK4A* have been identified in 30-70% of melanomas irre‐ spective of *BRAF* mutation [59, 70, 81]. Bi-allelic deletion of *INK4A* (p16 null) occurs in 10-27% of melanomas [60, 82]. Other changes include mono-allelic deletion, point mutation, or promoter hypermethylation, resulting in various levels of p16 expression/activity (Table 1) [57, 60, 81-83]. It is believed that the acquisition of p16 lesions allows melanoma cells to bypass senescence/growth arrest during melanoma development [84]. Although preliminary results with combination therapy of BRAFi and MEKi are encouraging with better clinical response over single agent BRAFi treatment [9], levels of treatment responses vary under

ment cost [79, 80].

16 Melanoma - From Early Detection to Treatment

These assays need to be validated both technically and clinically with defined cut-off values. There should be correlation of results among assay methods; for example, cells with bi-allel‐ ic *INK4A* deletion show negative p16 IHC staining and cells with mono-allelic *INK4A* dele‐ tion show mutations with loss of heterozygosity (LOH), and p16 expression inversely correlates with levels of *INK4A* promoter methylation. The major obstacles in testing tumor specimens are the presence of non-tumor cells in the samples, the cellular heterogeneity within tumor specimens, and degradation/damage of nucleic acid and protein during sam‐ ple processing. To ensure accurate testing results, SOPs need to be established with clearly defined instructions on the selection and handling of tumor specimens. For example, FISH assay requires fixation time between 6-48 hrs [92]. Alterations in *INK4A* may also affect the overlapping *ARF* gene (Fig. 2). Although the proposed study focuses on *INK4A*, changes in *INK4A* may also affect *ARF*, which may also be analyzed. Assay clinical sensitivity, clinical specificity, positive predictive value, and negative predictive value of *INK4A* biomarkers for a given treatment response can be calculated as described in Table 4.

tide [98] or of flavopiridol, a pan-CDK inhibitor [99], and combination of BRAFi or MEKi with the expression of wild-type *INK4A* or a CDK4 inhibitor significantly suppresses growth and enhances apoptosis in melanoma cells [2, 3]. Therefore, melanoma combination treat‐ ments that include CDK4 inhibitors may overcome treatment resistance and enhance effica‐ cy. There is a critical need to identify predictive markers for therapies not only to improve treatment outcomes, but to help avoid ineffective toxic therapies, also because of the likely high cost of combination regimens. Like *BRAF* mutation assay, testing of *INK4A*-p16 may predict which patients will response to BRAF, MEK, and CDK4 inhibitors. Therefore, *INK4A* biomarkers may also have great potential to guide future melanoma combination treatments

Overcoming Resistance to BRAF and MEK Inhibitors by Simultaneous Suppression of CDK4

http://dx.doi.org/10.5772/53620

19

that include CDK4 inhibitors.

ASK1: apoptosis signal-regulating kinase-1

BCL2: B-cell chronic lymphocytic leukemia/lymphoma 2

*BRAF*: v-raf murine sarcoma viral oncogene homolog B1

BIRC5: baculoviral IAP repeat-containing 5, also known as survivin

ARF: alternative open reading frame

BIM: BCL2 interacting mediator

Caspase: cysteine-aspartic acid protease

CEP9: chromosome 9 centromeric probe

DMEM: Dulbecco's modified Eagle medium

ERK: extracellular-signal-regulated kinase

CLL: chronic lymphocytic leukemia DAPI: 4'-6-diamidino-2-phenylindole

CDK2: cyclin-dependent kinase 2 CDK4: cyclin-dependent kinase 4

**Nomenclature**

BCL2L1: BCL2-like 1

BRAFi: BRAF inhibitor

CDK4i: CDK4 inhibitor

DMSO: dimethyl sulfoxide

DTIC: dacarbazine

FBS: fetal bovine serum


**Table 3.** Calculation of clinical sensitivity, clinical specificity and predictive values

## **3. Conclusion**

Patients with metastatic melanoma have a median survival of 6-8 months [93]. Recently, ipi‐ limumab (Yervoy, Bristol-Myers Squibb), an inhibitor of cytotoxic T-lymphocyte antigen 4 (CTLA-4) and vemurafenib (PLX4032, Zelboraf, Plexxikon/Roche), an inhibitor of mutant BRAF, gained FDA approval to treat patients with metastatic melanoma. Although both drugs offer new approaches to the treatment of advanced melanoma, their therapeutic effi‐ cacy is limited. Both drugs typically lengthen life by only several months in patients that ini‐ tially responded to the treatment [94, 95]. There is mounting evidence that acquired resistance to BRAFi frequently correlates with reactivation of the RAS-RAF-MEK-ERK sig‐ naling pathway [52, 53, 64]. This finding led to clinical trials combining BRAFi and MEKi in patients with *BRAF*-mutant metastatic melanoma who progressed on a prior BRAFi treat‐ ment regimen [94]. Dabrafenib (GSK2118436, GlaxoSmithKline) is a potent and selective in‐ hibitor of mutant BRAF and is comparable in safety and efficacy to vemurafenib. In phase I testing, it achieved a 67% response rate in metastatic melanoma patients with BRAF V600 mutations [96]. Trametinib (GSK1120212, GlaxoSmithKline) is a potent and selective inhibi‐ tor of MEK1/2, achieved a clinical response of 40% in patients with an activating *BRAF* mu‐ tation in phase I study [97]. A multicenter phase I/II trial of combined treatment with dabrafenib and trametinib demonstrated a disease control rate of 67% (12/18) in patients who failed prior single-agent treatment with a BRAFi [9]. We hypothesize that although re‐ activation of MEK-ERK-cyclin D-CDK4 in tumors previously treatment with BRAFi may be suppressed by the combination of dabrafenib and trametinib, cyclin D-CDK4 can also be re‐ activated by alternative resistance mechanisms that cannot be suppressed by the addition of MEKi (e.g.; activation of growth factor receptor and PI3K-AKT pathway) [51-53, 55, 56, 65, 66], if unopposed by p16, can lead to resistance to the BRAFi and MEKi combination thera‐ py (Fig. 1). It has been shown that melanoma cells that harbor abnormal *INK4A* are more sensitive than *INK4A* wild-type cells to the growth inhibitory effect of a p16-mimicking pep‐ tide [98] or of flavopiridol, a pan-CDK inhibitor [99], and combination of BRAFi or MEKi with the expression of wild-type *INK4A* or a CDK4 inhibitor significantly suppresses growth and enhances apoptosis in melanoma cells [2, 3]. Therefore, melanoma combination treat‐ ments that include CDK4 inhibitors may overcome treatment resistance and enhance effica‐ cy. There is a critical need to identify predictive markers for therapies not only to improve treatment outcomes, but to help avoid ineffective toxic therapies, also because of the likely high cost of combination regimens. Like *BRAF* mutation assay, testing of *INK4A*-p16 may predict which patients will response to BRAF, MEK, and CDK4 inhibitors. Therefore, *INK4A* biomarkers may also have great potential to guide future melanoma combination treatments that include CDK4 inhibitors.

## **Nomenclature**

defined instructions on the selection and handling of tumor specimens. For example, FISH assay requires fixation time between 6-48 hrs [92]. Alterations in *INK4A* may also affect the overlapping *ARF* gene (Fig. 2). Although the proposed study focuses on *INK4A*, changes in *INK4A* may also affect *ARF*, which may also be analyzed. Assay clinical sensitivity, clinical specificity, positive predictive value, and negative predictive value of *INK4A* biomarkers for

Lesion +ve A B Positive predictive value = A / (A + B) Lesion -ve C D Negative predictive value = D / (C + D)

Patients with metastatic melanoma have a median survival of 6-8 months [93]. Recently, ipi‐ limumab (Yervoy, Bristol-Myers Squibb), an inhibitor of cytotoxic T-lymphocyte antigen 4 (CTLA-4) and vemurafenib (PLX4032, Zelboraf, Plexxikon/Roche), an inhibitor of mutant BRAF, gained FDA approval to treat patients with metastatic melanoma. Although both drugs offer new approaches to the treatment of advanced melanoma, their therapeutic effi‐ cacy is limited. Both drugs typically lengthen life by only several months in patients that ini‐ tially responded to the treatment [94, 95]. There is mounting evidence that acquired resistance to BRAFi frequently correlates with reactivation of the RAS-RAF-MEK-ERK sig‐ naling pathway [52, 53, 64]. This finding led to clinical trials combining BRAFi and MEKi in patients with *BRAF*-mutant metastatic melanoma who progressed on a prior BRAFi treat‐ ment regimen [94]. Dabrafenib (GSK2118436, GlaxoSmithKline) is a potent and selective in‐ hibitor of mutant BRAF and is comparable in safety and efficacy to vemurafenib. In phase I testing, it achieved a 67% response rate in metastatic melanoma patients with BRAF V600 mutations [96]. Trametinib (GSK1120212, GlaxoSmithKline) is a potent and selective inhibi‐ tor of MEK1/2, achieved a clinical response of 40% in patients with an activating *BRAF* mu‐ tation in phase I study [97]. A multicenter phase I/II trial of combined treatment with dabrafenib and trametinib demonstrated a disease control rate of 67% (12/18) in patients who failed prior single-agent treatment with a BRAFi [9]. We hypothesize that although re‐ activation of MEK-ERK-cyclin D-CDK4 in tumors previously treatment with BRAFi may be suppressed by the combination of dabrafenib and trametinib, cyclin D-CDK4 can also be re‐ activated by alternative resistance mechanisms that cannot be suppressed by the addition of MEKi (e.g.; activation of growth factor receptor and PI3K-AKT pathway) [51-53, 55, 56, 65, 66], if unopposed by p16, can lead to resistance to the BRAFi and MEKi combination thera‐ py (Fig. 1). It has been shown that melanoma cells that harbor abnormal *INK4A* are more sensitive than *INK4A* wild-type cells to the growth inhibitory effect of a p16-mimicking pep‐

a given treatment response can be calculated as described in Table 4.

Sensitivity = A / (A + C) Specificity = D / (B + D)

**Table 3.** Calculation of clinical sensitivity, clinical specificity and predictive values

**3. Conclusion**

*INK4A result* **Treatment resistant case Treatment sensitive case**

18 Melanoma - From Early Detection to Treatment

ASK1: apoptosis signal-regulating kinase-1 ARF: alternative open reading frame BCL2: B-cell chronic lymphocytic leukemia/lymphoma 2 BCL2L1: BCL2-like 1 BIM: BCL2 interacting mediator BIRC5: baculoviral IAP repeat-containing 5, also known as survivin *BRAF*: v-raf murine sarcoma viral oncogene homolog B1 BRAFi: BRAF inhibitor Caspase: cysteine-aspartic acid protease CDK2: cyclin-dependent kinase 2 CDK4: cyclin-dependent kinase 4 CDK4i: CDK4 inhibitor CEP9: chromosome 9 centromeric probe CLL: chronic lymphocytic leukemia DAPI: 4'-6-diamidino-2-phenylindole DMEM: Dulbecco's modified Eagle medium DMSO: dimethyl sulfoxide DTIC: dacarbazine ERK: extracellular-signal-regulated kinase FBS: fetal bovine serum

#### FDA: Food and Drug Administration

FGF: fibroblast growth factor

FISH: fluorescent *in situ* hybridization

FITC: fluorescein isothiocyanate

HGF: hepatocyte growth factor

IAP: inhibitor of apoptosis family

IHC: immunohistochemical staining

*INK4A*: inhibitor of cyclin-dependent kinase 4A; part of cyclin-dependent kinase inhibitor 2A gene (*CDKN2A*), also known as multiple tumor suppressor 1 (*MTS1*)

**Acknowledgments**

**Author details**

Jianli Dong\*

veston, USA

**References**

Leukemia Group B Foundation (to J.D.).

Address all correspondence to: jidong@utmb.edu

Commun, 2008. 370(3): p. 509-13.

vation. Biol Cell, 2001. 93: p. 53-62.

N Engl J Med, 2010. 363(9): p. 809-19.

417(6892): p. 949-54.

Med Rep, 2011. 3: p. 8.

2001. 2: p. 731-7.

We thank Dr. Stuart Aaronson for human melanoma cell lines. This work was supported by Bill Walter III Melanoma Research Fund, Harry J. Lloyd Charitable Trust, and Cancer and

Overcoming Resistance to BRAF and MEK Inhibitors by Simultaneous Suppression of CDK4

http://dx.doi.org/10.5772/53620

21

Molecular Diagnostics Laboratory, University of Texas Medical Branch at Galveston, Gal‐

[1] Dong, J. and C.L. Schwab, Simultaneous knockdown of mutant BRAF and expression of INK4A in melanoma cells leads to potent growth inhibition and apoptosis. Treat‐

[2] Li, J., et al., Simultaneous inhibition of MEK and CDK4 leads to potent apoptosis in

[3] Zhao, Y., et al., Simultaneous knockdown of BRAF and expression of INK4A in mela‐ noma cells leads to potent growth inhibition and apoptosis. Biochem Biophys Res

[4] Davies, H., et al., Mutations of the BRAF gene in human cancer. Nature, 2002.

[5] Peyssonnaux, C. and A. Eychene, The Raf/MEK/ERK pathway: new concepts of acti‐

[6] Flaherty, K.T., Next generation therapies change the landscape in melanoma. F1000

[7] Flaherty, K.T., et al., Inhibition of mutated, activated BRAF in metastatic melanoma.

[8] Sherr, C.J., The INK4a/ARF network in tumour suppression. Nat Rev Mol Cell Biol,

[9] Flaherty, K., et al., Phase I/II expansion cohort of BRAF inhibitor GSK2118436 + MEK inhibitor GSK1120212 in patients with BRAF mutant metastatic melanoma who pro‐

ment of Metastatic Melanoma, ed. R.M. R. 2011: InTech. 149-182.

human melanoma cells. Cancer Invest, 2010. 28(4): p. 350-6.

KIP1: kinase interacting protein 1

LOH: loss of heterozygosity

MEK: mitogen-activated protein kinase/ERK kinase

MEKi: MEK inhibitor

MST2: sterile 20- like-kinase 2

PAGE: polyacrylamide gel electrophoresis

PARP: poly (ADP-ribose) polymerase

PBS: phosphate buffered saline

PI3K: phosphatidylinositol 3-kinase

p-ERK: phopho-ERK

RAF: v-raf murine sarcoma viral oncogene homolog. Human has three RAF: CRAF, BRAF, and ARAF

RAS: rat sarcoma viral oncogene homolog. Human has three RAS: HRAS, NRAS, and KRAS (KRAS4A and KRAS4B proteins arise from alternative splicing)

RB: retinoblastoma proteins including pRB, p107, and p103

SDS: sodium dodecyl sulfate

siRNA: small interfering RNA

TdT: terminal deoxynucleotidyl transferase

TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling

UV: ultra violate

WNT: wingless

## **Acknowledgments**

We thank Dr. Stuart Aaronson for human melanoma cell lines. This work was supported by Bill Walter III Melanoma Research Fund, Harry J. Lloyd Charitable Trust, and Cancer and Leukemia Group B Foundation (to J.D.).

## **Author details**

Jianli Dong\*

FDA: Food and Drug Administration

20 Melanoma - From Early Detection to Treatment

FISH: fluorescent *in situ* hybridization

FGF: fibroblast growth factor

FITC: fluorescein isothiocyanate

HGF: hepatocyte growth factor

IAP: inhibitor of apoptosis family

KIP1: kinase interacting protein 1

MEK: mitogen-activated protein kinase/ERK kinase

LOH: loss of heterozygosity

MST2: sterile 20- like-kinase 2

PBS: phosphate buffered saline

SDS: sodium dodecyl sulfate

siRNA: small interfering RNA

UV: ultra violate

WNT: wingless

TdT: terminal deoxynucleotidyl transferase

PAGE: polyacrylamide gel electrophoresis

PARP: poly (ADP-ribose) polymerase

PI3K: phosphatidylinositol 3-kinase

MEKi: MEK inhibitor

p-ERK: phopho-ERK

and ARAF

IHC: immunohistochemical staining

*INK4A*: inhibitor of cyclin-dependent kinase 4A; part of cyclin-dependent kinase inhibitor

RAF: v-raf murine sarcoma viral oncogene homolog. Human has three RAF: CRAF, BRAF,

RAS: rat sarcoma viral oncogene homolog. Human has three RAS: HRAS, NRAS, and KRAS

TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling

(KRAS4A and KRAS4B proteins arise from alternative splicing)

RB: retinoblastoma proteins including pRB, p107, and p103

2A gene (*CDKN2A*), also known as multiple tumor suppressor 1 (*MTS1*)

Address all correspondence to: jidong@utmb.edu

Molecular Diagnostics Laboratory, University of Texas Medical Branch at Galveston, Gal‐ veston, USA

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[98] Noonan, D.M., et al., In vitro and in vivo tumor growth inhibition by a p16-mimick‐ ing peptide in p16INK4A-defective, pRb-positive human melanoma cells. J Cell

[99] Robinson, W.A., et al., The effect of flavopiridol on the growth of p16+ and p16- mel‐

metastatic melanoma. Drug Des Devel Ther, 2011. 5: p. 489-95.

in melanoma. Nat Rev Clin Oncol, 2011. 8(7): p. 426-33.

GSK1120212. Mol Cancer Ther, 2011. 11(3): p. 720-9.

anoma cell lines. Melanoma Res, 2003. 13: p. 231-8.

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35(4): p. 438-43.

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Physiol, 2005. 202(3): p. 922-8.

33-9.

Clin Oncol, 2009. 27(36): p. 6199-206.


[89] Zainuddin, N., et al., Quantitative evaluation of p16(INK4a) promoter methylation using pyrosequencing in de novo diffuse large B-cell lymphoma. Leuk Res, 2011. 35(4): p. 438-43.

[74] Piras, F., et al., Nuclear survivin is associated with disease recurrence and poor sur‐ vival in patients with cutaneous malignant melanoma. Histopathology, 2007. 50(7):

[75] Zhuang, L., et al., Mcl-1, Bcl-XL and Stat3 expression are associated with progression of melanoma whereas Bcl-2, AP-2 and MITF levels decrease during progression of

[76] Petitjean, A., et al., Impact of mutant p53 functional properties on TP53 mutation pat‐ terns and tumor phenotype: lessons from recent developments in the IARC TP53 da‐

[77] Yu, B.D., et al., Distinct and nonoverlapping roles for pRB and cyclin D:cyclin-de‐ pendent kinases 4/6 activity in melanocyte survival. Proc Natl Acad Sci U S A, 2003.

[78] Wang, X., et al., p27Kip1 overexpression causes apoptotic death of mammalian cells.

[79] Engstrom, P.F., et al., NCCN molecular testing white paper: effectiveness, efficiency,

[80] Ong, F.S., et al., Personalized medicine and pharmacogenetic biomarkers: progress in molecular oncology testing. Expert Rev Mol Diagn, 2012. 12(6): p. 593-602.

[81] Grafstrom, E., et al., Biallelic deletions in INK4 in cutaneous melanoma are common and associated with decreased survival. Clin Cancer Res, 2005. 11(8): p. 2991-7.

[82] Jonsson, A., et al., High frequency of p16(INK4A) promoter methylation in NRASmutated cutaneous melanoma. J Invest Dermatol, 2010. 130(12): p. 2809-17.

[83] Miller, P.J., et al., Classifying variants of CDKN2A using computational and labora‐

[84] Bennett, D.C., How to make a melanoma: what do we know of the primary clonal

[85] Chung, C.T., et al., FISH assay development for the detection of p16/CDKN2A dele‐ tion in malignant pleural mesothelioma. J Clin Pathol, 2010. 63(7): p. 630-4.

[86] Gerami, P., et al., Fluorescence in situ hybridization (FISH) as an ancillary diagnostic tool in the diagnosis of melanoma. Am J Surg Pathol, 2009. 33(8): p. 1146-56.

[87] Straume, O., et al., Significant impact of promoter hypermethylation and the 540 C>T polymorphism of CDKN2A in cutaneous melanoma of the vertical growth phase.

[88] Tellez, C.S., et al., CpG island methylation profiling in human melanoma cell lines.

and reimbursement. J Natl Compr Canc Netw, 2011. 9 Suppl 6: p. S1-16.

melanoma. Mod Pathol, 2007. 20(4): p. 416-26.

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p. 835-42.

26 Melanoma - From Early Detection to Treatment

100(25): p. 14881-6.


**Chapter 2**

**Targeted Therapies in Melanoma:**

**Successes and Pitfalls**

Giuseppe Palmieri, Maria Colombino,

Amelia Lissia and Antonio Cossu

http://dx.doi.org/10.5772/53624

**1. Introduction**

Maria Cristina Sini, Paolo Antonio Ascierto,

Additional information is available at the end of the chapter

pecially dysplastic nevi, also known as atypical moles) [7-8].

Incidence of melanoma is steadily rising worldwide [1]. Lifetime risk of developing melano‐ ma in Caucasians is estimated as 1 in 50 individuals [2-3]. The incidence of melanoma varies according to the geographical origins of the population and the extent of sun exposure. In Australia and United States, an incidence of melanoma higher than observed in the Europe‐ an countries (with the notable exception of Sweden) has been reported [4-5]. There is a gra‐ dient of melanoma incidence from north to south in Europe, with highest frequencies in the northern counties. This suggests that initiation and development of melanoma is due to a combination of the damaging effects of UV and a predisposing genetic background [5].

Melanoma arises from melanocytes, neural crest-derived cells that are located in the basal layer of the epidermis and skin appendages in humans. Melanocytes, by synthesizing mela‐ nin pigments and exporting them to adjacent keratonocytes play a key role in protecting the skin from the damaging effects of ultraviolet (UV) and other solar radiation [6]. Melanocytes can proliferate to form nevi (common moles), initially in the basal epidermis (junctional ne‐ vus) and later by limited local dermal infiltration (compound nevus). Nevi develop during embryonic life (congenital nevus) and in children and adults, (acquired nevus) partly as a result of solar exposure in the latter two populations. Further progression of melanocytic tu‐ mors relates to factors that include intermittent exposure to UV radiation (though a direct relationship between risk of melanoma and UV exposure remains somehow unclear), a his‐ tory of sunburn and endogenous factors such as skin type and elevated numbers of nevi (es‐

> © 2013 Palmieri 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,

**Chapter 2**

## **Targeted Therapies in Melanoma: Successes and Pitfalls**

Giuseppe Palmieri, Maria Colombino, Maria Cristina Sini, Paolo Antonio Ascierto, Amelia Lissia and Antonio Cossu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53624

## **1. Introduction**

Incidence of melanoma is steadily rising worldwide [1]. Lifetime risk of developing melano‐ ma in Caucasians is estimated as 1 in 50 individuals [2-3]. The incidence of melanoma varies according to the geographical origins of the population and the extent of sun exposure. In Australia and United States, an incidence of melanoma higher than observed in the Europe‐ an countries (with the notable exception of Sweden) has been reported [4-5]. There is a gra‐ dient of melanoma incidence from north to south in Europe, with highest frequencies in the northern counties. This suggests that initiation and development of melanoma is due to a combination of the damaging effects of UV and a predisposing genetic background [5].

Melanoma arises from melanocytes, neural crest-derived cells that are located in the basal layer of the epidermis and skin appendages in humans. Melanocytes, by synthesizing mela‐ nin pigments and exporting them to adjacent keratonocytes play a key role in protecting the skin from the damaging effects of ultraviolet (UV) and other solar radiation [6]. Melanocytes can proliferate to form nevi (common moles), initially in the basal epidermis (junctional ne‐ vus) and later by limited local dermal infiltration (compound nevus). Nevi develop during embryonic life (congenital nevus) and in children and adults, (acquired nevus) partly as a result of solar exposure in the latter two populations. Further progression of melanocytic tu‐ mors relates to factors that include intermittent exposure to UV radiation (though a direct relationship between risk of melanoma and UV exposure remains somehow unclear), a his‐ tory of sunburn and endogenous factors such as skin type and elevated numbers of nevi (es‐ pecially dysplastic nevi, also known as atypical moles) [7-8].

© 2013 Palmieri 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.

Considering the growth patterns, four histological types of melanoma have been historically recognized: superficial spreading melanoma (SSM), lentigo maligna melanoma (LMM), nod‐ ular melanoma (NM), and acral lentiginous melanoma (ALM) [9]. Comparative genomic hy‐ bridization revealed that several genomic regions (mostly, 11q13, 22q11-13, and 5p15) were abnormally amplified in ALM [10]; such regions were different from those found altered in superficial SSM or NM (mainly, 9p21 and 1p22) [11]. Recently, a new classification of mela‐ noma including the site of primary tumour and the degree of chronic sun-induced damage of the surrounding skin has been introduced [12]. Based on these criteria, melanomas are classified into four groups; melanoma on skin with chronic sun-damage (CSD melanoma), melanoma on skin without chronic sun-damage (non-CSD melanoma), melanoma on palms, soles and nail bed (acral melanoma), and melanoma on mucous membrane (mucosal mela‐ noma) [12]. Non-CSD melanomas are characterized by high frequency of BRAF or NRAS mutations (which are mutually exclusive), while CSD, acral, and mucosal melanomas show a low frequency of BRAF/NRAS mutations but a high incidence of alterations in additional genes, such as mutations of receptor tyrosine kinase KIT gene, amplifications of cyclin D1 (CCND1) and cyclin-dependent kinase 4 (CDK4) genes [7, 12-13]. All genes affected into the different types of melanoma are involved in regulating cell-cycle progression and cell sur‐ vival [12-13]. On the other hand, such a difference of genetic alterations indicates distinct ge‐ netic pathways in the pathogenesis of melanoma depending on the anatomical site of the primary lesion. Trying to merge the two classifications, it could be affirmed that non-CSD melanoma roughly corresponds to SSM, CSD melanoma to LMM, and acral melanoma to ALM. Since NM may arise at any anatomical site, this histological type can not be included in any of the subgroups of the latter classification (indeed, no distinct genetic pathway has been so far correlated with NM).

During recent past years, melanocytic transformation is being demonstrated to occur as a sequential accumulation of genetic and molecular alterations [13-14]. In this sense, it is be‐ coming an unquestionable certainty that molecular classification of melanoma patients could be achieved through the assessment of the molecular profile of primary tumors and/or the correspondent metastases, by unveiling which gene or pathway is truly affect‐ ed. Although pathogenetic mechanisms underlying melanoma development are still large‐ ly unknown, several genes and metabolic pathways have been shown to carry molecular alterations in melanoma.

**Figure 1. Main pathways involved in melanomagenesis.** Arrows, activation signals. Interrupted lines, inhibition signals.

Targeted Therapies in Melanoma: Successes and Pitfalls

http://dx.doi.org/10.5772/53624

31

RAS proteins are small GTPases thatare activated by extracellular stimuli and regulate sig‐ nal transduction of the BRAF-MEK1/2-ERK1/2 and AKT/PI3K pathways, controlling crucial cellular processes such as proliferation, differentiation, cell adhesion, apoptosis, and cell mi‐ gration [14-16]. RAS gene is mutated in an estimated 20-30% of all cancers [17]. RAS proteins are constituted by three main isoforms - NRAS, KRAS, and HRAS, which present a similar function but a different specificity for tissue distribution [18]. On this regard, KRAS altera‐ tions mostly occur in gastrointestinal cancer [17-18], HRAS alterations are frequently ob‐ served in bladder cancer, and NRAS is mutated in 15-25% of melanomas [17-18]. Despite the huge amount of knowledge implicating RAS in tumour initiation and promotion, RAS itself

The RAF kinase family consists of three members - ARAF, BRAF, and CRAF, all of which can activate a series of protein kinases; such a signaling cascade culminate in the phosphory‐ lation and activation of the extracellular signal-regulated kinase (ERK) downstream protein [19]. In melanoma, the most commonly mutated component of this pathway is the *BRAF* gene; the prevalent *BRAF* mutation (in nearly, 90% of cases) being a substitution of valine with glutamic acid at position 600 (V600E) [20]. Mutated *BRAF* induces constitutive ERK ac‐ tivation; activated ERK then translocates to the nucleus and initiates the transcription of a variety of growth-related genes, stimulating cell proliferation and survival [20]. Indeed, the

has not become a successful target of therapy.

## **2. Main genes and related pathways**

#### **2.1.** *BRAF* **and MAPK pathway**

The *mitogen-activated protein kinase* (MAPK) signal transduction pathway regulates cell growth, survival, and invasion. MAPK signaling is initiated at the cell membrane, either by receptor tyrosine kinases (RTKs) binding ligand or integrin adhesion to extracellular matrix, which transmits activation signals via RAS on the cell membrane inner surface (Figure 1).

Considering the growth patterns, four histological types of melanoma have been historically recognized: superficial spreading melanoma (SSM), lentigo maligna melanoma (LMM), nod‐ ular melanoma (NM), and acral lentiginous melanoma (ALM) [9]. Comparative genomic hy‐ bridization revealed that several genomic regions (mostly, 11q13, 22q11-13, and 5p15) were abnormally amplified in ALM [10]; such regions were different from those found altered in superficial SSM or NM (mainly, 9p21 and 1p22) [11]. Recently, a new classification of mela‐ noma including the site of primary tumour and the degree of chronic sun-induced damage of the surrounding skin has been introduced [12]. Based on these criteria, melanomas are classified into four groups; melanoma on skin with chronic sun-damage (CSD melanoma), melanoma on skin without chronic sun-damage (non-CSD melanoma), melanoma on palms, soles and nail bed (acral melanoma), and melanoma on mucous membrane (mucosal mela‐ noma) [12]. Non-CSD melanomas are characterized by high frequency of BRAF or NRAS mutations (which are mutually exclusive), while CSD, acral, and mucosal melanomas show a low frequency of BRAF/NRAS mutations but a high incidence of alterations in additional genes, such as mutations of receptor tyrosine kinase KIT gene, amplifications of cyclin D1 (CCND1) and cyclin-dependent kinase 4 (CDK4) genes [7, 12-13]. All genes affected into the different types of melanoma are involved in regulating cell-cycle progression and cell sur‐ vival [12-13]. On the other hand, such a difference of genetic alterations indicates distinct ge‐ netic pathways in the pathogenesis of melanoma depending on the anatomical site of the primary lesion. Trying to merge the two classifications, it could be affirmed that non-CSD melanoma roughly corresponds to SSM, CSD melanoma to LMM, and acral melanoma to ALM. Since NM may arise at any anatomical site, this histological type can not be included in any of the subgroups of the latter classification (indeed, no distinct genetic pathway has

During recent past years, melanocytic transformation is being demonstrated to occur as a sequential accumulation of genetic and molecular alterations [13-14]. In this sense, it is be‐ coming an unquestionable certainty that molecular classification of melanoma patients could be achieved through the assessment of the molecular profile of primary tumors and/or the correspondent metastases, by unveiling which gene or pathway is truly affect‐ ed. Although pathogenetic mechanisms underlying melanoma development are still large‐ ly unknown, several genes and metabolic pathways have been shown to carry molecular

The *mitogen-activated protein kinase* (MAPK) signal transduction pathway regulates cell growth, survival, and invasion. MAPK signaling is initiated at the cell membrane, either by receptor tyrosine kinases (RTKs) binding ligand or integrin adhesion to extracellular matrix, which transmits activation signals via RAS on the cell membrane inner surface (Figure 1).

been so far correlated with NM).

30 Melanoma - From Early Detection to Treatment

alterations in melanoma.

**2.1.** *BRAF* **and MAPK pathway**

**2. Main genes and related pathways**

**Figure 1. Main pathways involved in melanomagenesis.** Arrows, activation signals. Interrupted lines, inhibition signals.

RAS proteins are small GTPases thatare activated by extracellular stimuli and regulate sig‐ nal transduction of the BRAF-MEK1/2-ERK1/2 and AKT/PI3K pathways, controlling crucial cellular processes such as proliferation, differentiation, cell adhesion, apoptosis, and cell mi‐ gration [14-16]. RAS gene is mutated in an estimated 20-30% of all cancers [17]. RAS proteins are constituted by three main isoforms - NRAS, KRAS, and HRAS, which present a similar function but a different specificity for tissue distribution [18]. On this regard, KRAS altera‐ tions mostly occur in gastrointestinal cancer [17-18], HRAS alterations are frequently ob‐ served in bladder cancer, and NRAS is mutated in 15-25% of melanomas [17-18]. Despite the huge amount of knowledge implicating RAS in tumour initiation and promotion, RAS itself has not become a successful target of therapy.

The RAF kinase family consists of three members - ARAF, BRAF, and CRAF, all of which can activate a series of protein kinases; such a signaling cascade culminate in the phosphory‐ lation and activation of the extracellular signal-regulated kinase (ERK) downstream protein [19]. In melanoma, the most commonly mutated component of this pathway is the *BRAF* gene; the prevalent *BRAF* mutation (in nearly, 90% of cases) being a substitution of valine with glutamic acid at position 600 (V600E) [20]. Mutated *BRAF* induces constitutive ERK ac‐ tivation; activated ERK then translocates to the nucleus and initiates the transcription of a variety of growth-related genes, stimulating cell proliferation and survival [20]. Indeed, the increased activity of ERK seems to be implicated in rapid melanoma cell growth, enhanced cell survival and resistance to apoptosis [21]. High levels of activated ERK may further in‐ duce the metastatic potential of melanoma by increasing the expression of invasion-promot‐ ing integrins [22-23]. Presence of *BRAF* mutations in benign and dysplastic nevi supports the hypothesis that activation of the RAF/MEK/ERK pathway is an early event in melanoma progression [24-25]. In other words, *BRAF* activation is necessary but not sufficient for the development of melanoma and additional co-operating genetic events are required to ach‐ ieve full malignancy.

cyclin/CDK complexes and, in this way, ensuring the control of the cell replication [33]. In particular, p16CDKN2A is part of the G1–S cell cycle checkpoint mechanism that involves the retinoblastoma-susceptibility tumor suppressor protein (pRb). The p16CDKN2A inhibits the Cyclin D/CDK4 complex, which, in turn, phosphorylates pRb and allows progression through the G1–S checkpoint (Figure 1) [33]. The *Cyclin D* (*CCND1*) and *CDK4* genes are found altered in less than 5% of total melanomas [12], though with an heterogeneous prevalence according to the distinct types of melanoma (see above). Somatic *CDK4* ampli‐ fication is relatively common in acral and mucosal melanomas [12], whereas germline *CDK4* mutations are observed in a limited fraction of melanoma-prone families [34]. The *CCND1* gene amplifications is primarily found in ALM lesions (more than one third of cases) and to a lesser degree in other types (11% for LMM and 6% for SSM) [35]. Regard‐ ing the alternative *CDKN2A* gene product, p14CDKN2A is an antagonist of the murine dou‐ ble minute 2 (MDM2) protein, which targets p53 to degradation by ubiquitination and proteasome processing, thus abrogating p53 control of cell growth (Figure 1) [32-33]. In particular, the p14CDKN2A protein exerts a tumor suppressor effect by inhibiting the onco‐ genic actions of the downstream MDM2 protein, whose direct interaction with p53 blocks any p53-mediated activity and targets the p53 protein for rapid degradation [32-33]. The p53 is a transcription factor that functions as a major negative regulator of cell prolifera‐ tion and survival, being activated by different adverse signals (i.e. growth factors with‐ drawal, DNA damage, oncogenic aberrations, hypoxia, etc.) and driving cells to either interrupt progression into the cell-cycle or enter apoptosis program, in order to avoid re‐ production of altered cells [33, 36]. In normal conditions, expression levels of p53 within cells are low. In response to DNA damage, p53 accumulates and prevents cell division. Therefore, inactivation of the *TP53* gene results in an intracellular accumulation of genetic damage which promotes tumor formation [36]. In melanoma, such an inactivation is most‐ ly due to functional gene silencing since the frequency of *TP53* mutations is low (less than 10% of cases) [37]. Impairment of the p14CDKN2A-MDM2-p53 cascade, whose final effectors are the Bax/Bcl-2 proteins, has been implicated in defective apoptotic responses to geno‐ toxic damage and, thus, to anticancer agents (in most cases, high expression levels of Bcl-2 protein have been demonstrated to reduce apoptosis and sensitivity of melanoma cells to proapoptotic stimuli, contributing to further increase tumor aggressiveness and refractori‐

Targeted Therapies in Melanoma: Successes and Pitfalls

http://dx.doi.org/10.5772/53624

33

More in general, genetic loss or rearrangement in the CDKN2A locus may result in im‐ pairing or silencing p16CDKN2A, p14CDKN2A or both genes, with the consequence of losing the mechanisms controlling cell proliferation and/or survival. In melanoma, the *CDKN2A* gene is somatically inactivated by genomic deletions (approximately 50% of cases) or point mutations (about 10% of cases); in addition, this gene is often transcriptionally si‐ lenced by promoter hypermethylation [38]. A reduced expression of the p16CDKN2A protein seems to be strictly associated with malignant tumor invasion, varying from 5% to about 15% in benign melanocytic lesions, from 10% to about 50% in primary melanomas, and from 50% to about 60% in melanoma metastases [39]. The *CDKN2A* gene is frequently mutated at germline level in patients with a strong familial history of melanoma (three or more affected family members), indicating that it represents a key susceptibility gene for

ness to therapy) [33].

In a study aimed to better define the role of BRAF in melanomagenesis, a transgenic zebra fish expressing V600EBRAF showed dramatic development of patches of ectopic melanocytes (designated as fish-nevi) [26]. Remarkably, activated *BRAF* in p53-deficient zebra fish in‐ duced the formation of melanocytic lesions that rapidly developed into invasive melanomas that resembled human melanomas in terms of their histology and biological behaviors [26]. These data provide direct evidence that the p53 and BRAF pathways interact functionally during melanomagenesis.

The *BRAF* gene also cooperates with the cyclin-dependent kinase inhibitor p16CDKN2A (see be‐ low). Activating *BRAF* mutations have been reported to constitutively induce up-regulation of *p16*CDKN2A and cell cycle arrest (this phenomenon appears to be a protective response to an inappropriate mitogenic signal). In particular, mutant BRAF protein induces cell senescence by increasing the expression levels of the p16CDKN2A protein, which, in turn, may limit hyper‐ plastic growth caused by *BRAF* mutations [25]. Therefore, inactivation of *p16*CDKN2A gene may promote the melanocytic proliferation depending on oncogenic *BRAF*. In this sense, several factors seem to be able to induce the arrest of the cell cycle and cell senescence caused by *BRAF* activation [27-28].

Finally, it has been showed that primary melanomas arising from chronically sun-damages skin and from mucosal sites, which typically do not harbour *BRAF* and *NRAS* mutations, have increased copy number of the *CCND1/Cyclin D1* gene [12]. In contrast to primary mela‐ nomas, a subset (>15%) of metastatic melanoma samples with *BRAF* mutations also exhibit amplification of *CCND1/Cyclin D1*. These melanomas are resistant to BRAF inhibitors high‐ lighting the need for combination therapy [29-30].

#### **2.2.** *CDKN2A* **and senescence/apoptosis pathways**

The cyclin-dependent kinase inhibitor 2 (*CDKN2A*; at chromosome 9p21) gene encodes two proteins, p16CDKN2A and p14CDKN2A (a product of an alternative splicing), that are known to function as tumor suppressors [31-33]. The cyclin proteins are regulatory effec‐ tors able to bind and activate the cyclin-dependent kinases (CDKs) that bear catalytic kin‐ ase activity. Several distinct cyclin/CDK complexes have been identified and functionally assigned to specific phases of the cell cycle: Cyclin D/CDK4 complex leads the passage from the pre-replicative (G1) to the DNA duplication (S) phase; the Cyclin E/CDK2 com‐ plex promotes the progression through the S phase and the Cyclin B/CDK1 complex indu‐ ces cells to enter mitosis [31-32]. In such a functional network, proteins like p16CDKN2A and p14CDKN2A act as inhibitors of the cell cycle, negatively interfering with the activity of the cyclin/CDK complexes and, in this way, ensuring the control of the cell replication [33]. In particular, p16CDKN2A is part of the G1–S cell cycle checkpoint mechanism that involves the retinoblastoma-susceptibility tumor suppressor protein (pRb). The p16CDKN2A inhibits the Cyclin D/CDK4 complex, which, in turn, phosphorylates pRb and allows progression through the G1–S checkpoint (Figure 1) [33]. The *Cyclin D* (*CCND1*) and *CDK4* genes are found altered in less than 5% of total melanomas [12], though with an heterogeneous prevalence according to the distinct types of melanoma (see above). Somatic *CDK4* ampli‐ fication is relatively common in acral and mucosal melanomas [12], whereas germline *CDK4* mutations are observed in a limited fraction of melanoma-prone families [34]. The *CCND1* gene amplifications is primarily found in ALM lesions (more than one third of cases) and to a lesser degree in other types (11% for LMM and 6% for SSM) [35]. Regard‐ ing the alternative *CDKN2A* gene product, p14CDKN2A is an antagonist of the murine dou‐ ble minute 2 (MDM2) protein, which targets p53 to degradation by ubiquitination and proteasome processing, thus abrogating p53 control of cell growth (Figure 1) [32-33]. In particular, the p14CDKN2A protein exerts a tumor suppressor effect by inhibiting the onco‐ genic actions of the downstream MDM2 protein, whose direct interaction with p53 blocks any p53-mediated activity and targets the p53 protein for rapid degradation [32-33]. The p53 is a transcription factor that functions as a major negative regulator of cell prolifera‐ tion and survival, being activated by different adverse signals (i.e. growth factors with‐ drawal, DNA damage, oncogenic aberrations, hypoxia, etc.) and driving cells to either interrupt progression into the cell-cycle or enter apoptosis program, in order to avoid re‐ production of altered cells [33, 36]. In normal conditions, expression levels of p53 within cells are low. In response to DNA damage, p53 accumulates and prevents cell division. Therefore, inactivation of the *TP53* gene results in an intracellular accumulation of genetic damage which promotes tumor formation [36]. In melanoma, such an inactivation is most‐ ly due to functional gene silencing since the frequency of *TP53* mutations is low (less than 10% of cases) [37]. Impairment of the p14CDKN2A-MDM2-p53 cascade, whose final effectors are the Bax/Bcl-2 proteins, has been implicated in defective apoptotic responses to geno‐ toxic damage and, thus, to anticancer agents (in most cases, high expression levels of Bcl-2 protein have been demonstrated to reduce apoptosis and sensitivity of melanoma cells to proapoptotic stimuli, contributing to further increase tumor aggressiveness and refractori‐ ness to therapy) [33].

increased activity of ERK seems to be implicated in rapid melanoma cell growth, enhanced cell survival and resistance to apoptosis [21]. High levels of activated ERK may further in‐ duce the metastatic potential of melanoma by increasing the expression of invasion-promot‐ ing integrins [22-23]. Presence of *BRAF* mutations in benign and dysplastic nevi supports the hypothesis that activation of the RAF/MEK/ERK pathway is an early event in melanoma progression [24-25]. In other words, *BRAF* activation is necessary but not sufficient for the development of melanoma and additional co-operating genetic events are required to ach‐

In a study aimed to better define the role of BRAF in melanomagenesis, a transgenic zebra fish expressing V600EBRAF showed dramatic development of patches of ectopic melanocytes (designated as fish-nevi) [26]. Remarkably, activated *BRAF* in p53-deficient zebra fish in‐ duced the formation of melanocytic lesions that rapidly developed into invasive melanomas that resembled human melanomas in terms of their histology and biological behaviors [26]. These data provide direct evidence that the p53 and BRAF pathways interact functionally

The *BRAF* gene also cooperates with the cyclin-dependent kinase inhibitor p16CDKN2A (see be‐ low). Activating *BRAF* mutations have been reported to constitutively induce up-regulation of *p16*CDKN2A and cell cycle arrest (this phenomenon appears to be a protective response to an inappropriate mitogenic signal). In particular, mutant BRAF protein induces cell senescence by increasing the expression levels of the p16CDKN2A protein, which, in turn, may limit hyper‐ plastic growth caused by *BRAF* mutations [25]. Therefore, inactivation of *p16*CDKN2A gene may promote the melanocytic proliferation depending on oncogenic *BRAF*. In this sense, several factors seem to be able to induce the arrest of the cell cycle and cell senescence

Finally, it has been showed that primary melanomas arising from chronically sun-damages skin and from mucosal sites, which typically do not harbour *BRAF* and *NRAS* mutations, have increased copy number of the *CCND1/Cyclin D1* gene [12]. In contrast to primary mela‐ nomas, a subset (>15%) of metastatic melanoma samples with *BRAF* mutations also exhibit amplification of *CCND1/Cyclin D1*. These melanomas are resistant to BRAF inhibitors high‐

The cyclin-dependent kinase inhibitor 2 (*CDKN2A*; at chromosome 9p21) gene encodes two proteins, p16CDKN2A and p14CDKN2A (a product of an alternative splicing), that are known to function as tumor suppressors [31-33]. The cyclin proteins are regulatory effec‐ tors able to bind and activate the cyclin-dependent kinases (CDKs) that bear catalytic kin‐ ase activity. Several distinct cyclin/CDK complexes have been identified and functionally assigned to specific phases of the cell cycle: Cyclin D/CDK4 complex leads the passage from the pre-replicative (G1) to the DNA duplication (S) phase; the Cyclin E/CDK2 com‐ plex promotes the progression through the S phase and the Cyclin B/CDK1 complex indu‐ ces cells to enter mitosis [31-32]. In such a functional network, proteins like p16CDKN2A and p14CDKN2A act as inhibitors of the cell cycle, negatively interfering with the activity of the

ieve full malignancy.

32 Melanoma - From Early Detection to Treatment

during melanomagenesis.

caused by *BRAF* activation [27-28].

lighting the need for combination therapy [29-30].

**2.2.** *CDKN2A* **and senescence/apoptosis pathways**

More in general, genetic loss or rearrangement in the CDKN2A locus may result in im‐ pairing or silencing p16CDKN2A, p14CDKN2A or both genes, with the consequence of losing the mechanisms controlling cell proliferation and/or survival. In melanoma, the *CDKN2A* gene is somatically inactivated by genomic deletions (approximately 50% of cases) or point mutations (about 10% of cases); in addition, this gene is often transcriptionally si‐ lenced by promoter hypermethylation [38]. A reduced expression of the p16CDKN2A protein seems to be strictly associated with malignant tumor invasion, varying from 5% to about 15% in benign melanocytic lesions, from 10% to about 50% in primary melanomas, and from 50% to about 60% in melanoma metastases [39]. The *CDKN2A* gene is frequently mutated at germline level in patients with a strong familial history of melanoma (three or more affected family members), indicating that it represents a key susceptibility gene for familial melanoma [40]. In melanoma, CDKN2A mutations typically occur in the p16CDKN2A gene, either alone or in combination with p14CDKN2A gene (some families harbor however mutations only in this latter gene) [33, 40].

unable to stimulate phosphorylation of the PI3K protein; this in turn maintains suppression of cell cycle progression and cell growth. In other words, there is a balance between PIP2 and PIP3 which is maintained by the opposite activities of PTEN and PI3K, which instead converts PIP2 into PIP3 [50]. Upon growth stimulation, mainly obtained by triggering the RAS kinase, PI3K is constitutively activated (Figure 1), resulting in an increase of intracellu‐ lar levels of PIP3 and a consequent activation of AKT by phosphorylation [50-51]. Activated AKT in turn phosphorylates its substrate, the serine/threonine kinase mTOR, leading to in‐ creased synthesis of target proteins that promote cell division and survival as well as apop‐ totic escape [51]. The mechanisms associated with the ability of AKT to suppress apoptosis include the phosphorylation and inactivation of many pro-apoptotic proteins, such as BAD (Bcl-2 antagonist of cell death) and MDM2, as well as the activation of NF-kB [52] (Figure 1). Three *AKT* genes have been described in humans: *AKT1*, which is involved in apoptosis and protein synthesis; *AKT2*, which is involved in controlling the glucose metabolism; and *AKT3*, whose increased activity (often associated with the amplification of the *AKT3* locus at chromosome 1q43-44) is mainly involved in stimulating cell growth and has been implicated in many cancers including melanoma [51-52]. More than two thirds of primary and meta‐ static melanomas exhibit higher levels of phosphorylated AKT [52], suggesting that such an

Targeted Therapies in Melanoma: Successes and Pitfalls

http://dx.doi.org/10.5772/53624

35

alteration might be considered as an early event in melanoma pathogenesis.

sion of phosphorylated AKT [37].

spreading and migration [33, 50].

**2.4.** *MITF* **and melanocytic differentiation**

Overall, PI3K expression is higher in malignant melanomas, as compared to nevi, and seems to correlate with a worse prognosis [53]. In primary melanomas, since activating mutations of PI3K are quite rare (about 1%)*,* and comparative genomic hybridization did not reveal amplification at this gene locus [12, 37], activation of the PI3K pathway is most‐ ly due to functional silencing of the tumour suppressor gene PTEN. Inactivation of *PTEN* gene is mainly due to hypermethylation-based epigenetic mechanisms, with a low inci‐ dence (less than 10%) of somatic mutations and/or allelic deletions; loss of (or reduced) PTEN protein is observed by immunohistochemistry in 20-40% of melanoma tissues [54-55]. Consistent with its role in the PI3K-AKT pathway, vast majority (more than 80%) of melanoma samples with loss of PTEN protein presents a significant increase in expres‐

*PTEN* inactivation has been mostly observed as a late event in melanoma, although a dosedependent down-regulation of PTEN expression has been implicated in early stages of tu‐ morigenesis, often occurring in conjunction with mutations in *BRAF* gene (which have been demonstrated to indeed play a role in induction of the melanocytic proliferation and early steps of melanoma development) [50]. PTEN downregulation In addition, alterations of the BRAF-MAPK pathway are frequently associated with PTEN-AKT impairment [7, 56]. In summary, the combined effects of the inactivation of *PTEN* gene and activation of the PI3K-AKT effectors may result in aberrant cell growth, apoptosis escape, and abnormal cell

The microphthalmia-associated transcription factor (MITF) is a transcription factor that is in‐ volved in differentiation and maintenance of melanocytes, playing a role in melanoma de‐

A recent meta-analysis of studies conducted in independent populations indicated that mul‐ tiple variants of the melanocortin-1 receptor (*MC1R*) gene increase the melanoma risk in *CDKN2A* mutation carriers [41]. The *MC1R* gene encodes a G-protein coupled receptor. In the skin, two types of melanin pigment, dark-protective eumelanin and red-photo reactive pheomelanin, are present [42]. MC1R plays an important role in determining the ratio of eu‐ melanin and pheomelanin production. After stimulation by UV, keratinocytes produce al‐ pha melanocyte stimulating hormone (MSH) that binds to the MC1R on melanocytes and shifts the balance of these two pigments in the direction of eumelanin [42]. In particular, stimulation of MC1R by MSH mediates activation of adenylate cyclase, subsequent eleva‐ tion of cAMP levels, and activation of the microphthalmia transcription factor (MITF; see be‐ low). Activated MITF binds to a conserved region found in the promoters of the *tyrosinase* (*TYR*), *tyrosinase-related protein 1* (*TYRP1*), and *DOPAchrome tautomerase* (*DCT*) genes, stimu‐ lating the transcriptional up-regulation of these proteins and inducing maturation of the melanosomes [43]. This ultimately results in increased eumelanin production and darkening of the skin or hair.

New findings have shed light on the mechanisms by which MC1R contributes to melanoma risk. In vitro studies showed that acute UV irradiation of melanocytes with impaired MC1R results in an increased production of free radicals [44]. Melanomas that arise on body sites only intermittently exposed to sun, and which therefore lack marked signs of chronic solar damage, were found to have a high frequency of *BRAF* mutations [12]. One could speculate that induction of *BRAF* mutations may occur only when solar exposure is not sufficiently prolonged to induce the striking tissue changes that generate the hallmark signs of solar damage. Several *MC1R* variants, that impairing relevant protein function, have been associ‐ ated with *BRAF* mutation in melanoma arising in Caucasian populations from United States and Europe [45-48]. On the basis of such indications, it is possible that increased production of free radicals following UV exposure is combination with impairment of *MC1R* may in‐ duce mutations in the *BRAF* gene.

Additional mechanisms promoting susceptibility to pathogenetic mutations of the *BRAF* gene may however exist since there is no demonstrable association between germ line *MC1R* status and the prevalence of somatic *BRAF* mutations in melanomas from Australian popu‐ lation, even after classifying the melanomas by their location relative to intermittent and chronic sun-exposure [49].

#### **2.3.** *PTEN* **and mTORC pathways**

Phosphatase and tensin homolog deleted in chromosome ten (PTEN) has a key role in cellu‐ lar signal transduction by decreasing intracellular phosphatidylinositol [3,4-bisphosphate (PIP2) and 3,4,5-triphosphate (PIP3)] that are produced by the activation of phoshoinosite 3 kinase (PI3K) [50]. In the absence of extracellular growth stimuli mediated by cell surface re‐ ceptors and G-proteins, PTEN dephosphorylates PIP3 generating the PIP2 phospholipid, unable to stimulate phosphorylation of the PI3K protein; this in turn maintains suppression of cell cycle progression and cell growth. In other words, there is a balance between PIP2 and PIP3 which is maintained by the opposite activities of PTEN and PI3K, which instead converts PIP2 into PIP3 [50]. Upon growth stimulation, mainly obtained by triggering the RAS kinase, PI3K is constitutively activated (Figure 1), resulting in an increase of intracellu‐ lar levels of PIP3 and a consequent activation of AKT by phosphorylation [50-51]. Activated AKT in turn phosphorylates its substrate, the serine/threonine kinase mTOR, leading to in‐ creased synthesis of target proteins that promote cell division and survival as well as apop‐ totic escape [51]. The mechanisms associated with the ability of AKT to suppress apoptosis include the phosphorylation and inactivation of many pro-apoptotic proteins, such as BAD (Bcl-2 antagonist of cell death) and MDM2, as well as the activation of NF-kB [52] (Figure 1).

Three *AKT* genes have been described in humans: *AKT1*, which is involved in apoptosis and protein synthesis; *AKT2*, which is involved in controlling the glucose metabolism; and *AKT3*, whose increased activity (often associated with the amplification of the *AKT3* locus at chromosome 1q43-44) is mainly involved in stimulating cell growth and has been implicated in many cancers including melanoma [51-52]. More than two thirds of primary and meta‐ static melanomas exhibit higher levels of phosphorylated AKT [52], suggesting that such an alteration might be considered as an early event in melanoma pathogenesis.

Overall, PI3K expression is higher in malignant melanomas, as compared to nevi, and seems to correlate with a worse prognosis [53]. In primary melanomas, since activating mutations of PI3K are quite rare (about 1%)*,* and comparative genomic hybridization did not reveal amplification at this gene locus [12, 37], activation of the PI3K pathway is most‐ ly due to functional silencing of the tumour suppressor gene PTEN. Inactivation of *PTEN* gene is mainly due to hypermethylation-based epigenetic mechanisms, with a low inci‐ dence (less than 10%) of somatic mutations and/or allelic deletions; loss of (or reduced) PTEN protein is observed by immunohistochemistry in 20-40% of melanoma tissues [54-55]. Consistent with its role in the PI3K-AKT pathway, vast majority (more than 80%) of melanoma samples with loss of PTEN protein presents a significant increase in expres‐ sion of phosphorylated AKT [37].

*PTEN* inactivation has been mostly observed as a late event in melanoma, although a dosedependent down-regulation of PTEN expression has been implicated in early stages of tu‐ morigenesis, often occurring in conjunction with mutations in *BRAF* gene (which have been demonstrated to indeed play a role in induction of the melanocytic proliferation and early steps of melanoma development) [50]. PTEN downregulation In addition, alterations of the BRAF-MAPK pathway are frequently associated with PTEN-AKT impairment [7, 56]. In summary, the combined effects of the inactivation of *PTEN* gene and activation of the PI3K-AKT effectors may result in aberrant cell growth, apoptosis escape, and abnormal cell spreading and migration [33, 50].

#### **2.4.** *MITF* **and melanocytic differentiation**

familial melanoma [40]. In melanoma, CDKN2A mutations typically occur in the p16CDKN2A gene, either alone or in combination with p14CDKN2A gene (some families harbor however

A recent meta-analysis of studies conducted in independent populations indicated that mul‐ tiple variants of the melanocortin-1 receptor (*MC1R*) gene increase the melanoma risk in *CDKN2A* mutation carriers [41]. The *MC1R* gene encodes a G-protein coupled receptor. In the skin, two types of melanin pigment, dark-protective eumelanin and red-photo reactive pheomelanin, are present [42]. MC1R plays an important role in determining the ratio of eu‐ melanin and pheomelanin production. After stimulation by UV, keratinocytes produce al‐ pha melanocyte stimulating hormone (MSH) that binds to the MC1R on melanocytes and shifts the balance of these two pigments in the direction of eumelanin [42]. In particular, stimulation of MC1R by MSH mediates activation of adenylate cyclase, subsequent eleva‐ tion of cAMP levels, and activation of the microphthalmia transcription factor (MITF; see be‐ low). Activated MITF binds to a conserved region found in the promoters of the *tyrosinase* (*TYR*), *tyrosinase-related protein 1* (*TYRP1*), and *DOPAchrome tautomerase* (*DCT*) genes, stimu‐ lating the transcriptional up-regulation of these proteins and inducing maturation of the melanosomes [43]. This ultimately results in increased eumelanin production and darkening

New findings have shed light on the mechanisms by which MC1R contributes to melanoma risk. In vitro studies showed that acute UV irradiation of melanocytes with impaired MC1R results in an increased production of free radicals [44]. Melanomas that arise on body sites only intermittently exposed to sun, and which therefore lack marked signs of chronic solar damage, were found to have a high frequency of *BRAF* mutations [12]. One could speculate that induction of *BRAF* mutations may occur only when solar exposure is not sufficiently prolonged to induce the striking tissue changes that generate the hallmark signs of solar damage. Several *MC1R* variants, that impairing relevant protein function, have been associ‐ ated with *BRAF* mutation in melanoma arising in Caucasian populations from United States and Europe [45-48]. On the basis of such indications, it is possible that increased production of free radicals following UV exposure is combination with impairment of *MC1R* may in‐

Additional mechanisms promoting susceptibility to pathogenetic mutations of the *BRAF* gene may however exist since there is no demonstrable association between germ line *MC1R* status and the prevalence of somatic *BRAF* mutations in melanomas from Australian popu‐ lation, even after classifying the melanomas by their location relative to intermittent and

Phosphatase and tensin homolog deleted in chromosome ten (PTEN) has a key role in cellu‐ lar signal transduction by decreasing intracellular phosphatidylinositol [3,4-bisphosphate (PIP2) and 3,4,5-triphosphate (PIP3)] that are produced by the activation of phoshoinosite 3 kinase (PI3K) [50]. In the absence of extracellular growth stimuli mediated by cell surface re‐ ceptors and G-proteins, PTEN dephosphorylates PIP3 generating the PIP2 phospholipid,

mutations only in this latter gene) [33, 40].

34 Melanoma - From Early Detection to Treatment

of the skin or hair.

duce mutations in the *BRAF* gene.

**2.3.** *PTEN* **and mTORC pathways**

chronic sun-exposure [49].

The microphthalmia-associated transcription factor (MITF) is a transcription factor that is in‐ volved in differentiation and maintenance of melanocytes, playing a role in melanoma de‐ velopment and pathogenesis [43, 57]. MITF is activated by the MAPK pathway as well as by the cAMP pathway (Figure 1), and leads to transcription of genes involved in pigmentation (TYR, TYRP1, and DCT; see above) as well as cell cycle progression and survival [43]. The *MITF* gene is amplified in melanoma (about 20% of cases); *MITF* amplification correlated with increased resistance to chemotherapy and decreased overall survival [57].

cytoplasm. NF-κB exists as cytoplasmic hetero- or homodimers associated with members of the inhibitor-of-kB (IkB) proteins (IkB*a*, Ik*Bb* and IkB*e*), which form complexes seques‐ tering NF-kB into the cytoplasm [74]. Upon appropriate stimulation, the phosphorylation of IkB proteins is promoted, triggering their ubiquitination and degradation in the protea‐ some [74]. As a consequence, NF-kB may translocate to the nucleus where it binds to tar‐ get DNA loci and induces transcription of several genes - including *iNOS* - associated with immune and inflammatory response, angiogenesis, cell proliferation, tumor promo‐

Targeted Therapies in Melanoma: Successes and Pitfalls

http://dx.doi.org/10.5772/53624

37

Regarding the role of NF-kB in tumorigenesis, there are compelling evidence that activation of NF-kB controls multiple cellular processes in cancer due to its ability to promote cell pro‐ liferation, suppress apoptosis, promote cell migration, and suppress cell differentiation, opening the way for new therapeutic approaches against such a target [75-76]. In melanoma, NF-kB is constitutively activated since expression of the IkB proteins seems to be significant‐

cKIT is a member of the transmenbrane receptor tyrosine kinase family that comprised five immunoglobin-like motifs, a single transmembrane region, an inhibitory cytoplasmic juxta‐ menbrane domain, and a split cytoplasmic kinase domain separated by a kinase insert seg‐ ment [78]. Under physiological conditions, binding of the cKIT ligand stem-cell factor (SCF) to the extracellular domain of the receptor leads its dimerization, activation of the intracellu‐ lar tyrosine kinase domain through autophosporylation of specific tyrosine residue [78]. The intracellular signaling through cKIT plays a critical role in the development of several mam‐ malian cells, including growth, differentiation, migration, and proliferation of melanocytes [79]. It has been defined that cKIT recruits and activates a number of intracellular signaling pathways implicated in tumor progression, such as MAPK, PI3K/AKT, Src, activators of

Although the role of cKIT in melanomagenesis is still controversial, several studies have reported its downregulation during melanoma growth and invasion (in vertical growth phase of melanoma and metastatic lesions) [81-83]. Indeed, the majority of highly meta‐ static human melanomas do not express detectable levels of the cKIT receptor [83]. As a confirmation of this, over-expression of cKIT in metastatic melanoma cell lines led to im‐ portant reduction in tumor growth, while cKIT activation through exposure to cKIT li‐

Specific mutations within the cKIT gene cause constitutive phosphorylation and activation of the kinase domain resulting in uncontrolled cell proliferation. [84]. Although such muta‐ tions seem to be more rare than *BRAF* and *NRAS* mutations, these may reflect the important role of cKIT tyrosine kinase in melanocyte development [84]. Sequencing of cKIT exons 11, 13, 17, and 18 revealed the most prevalent mutation to be K642, L576P, D816H-V, V559A [84]. The cKIT mutations are more common in mucosal and acral melanomas compared with cutaneous melanomas and are in most cases accompanied by an increase in gene copy number (40% mucosal or acral melanomas - frequently, associated with amplification of *cy‐*

tion, and apoptosis [73-74].

ly reduced in comparison to nevi [77].

gands induced apoptosis [83].

**2.6.** *cKIT* **and tyrosinase kinase receptors**

transcription (STAT), and phospholipase-C (PLC) [79-80].

The connection between MITF and melanoma development is complex because it plays a double role of inducer/repressor of cellular proliferation. High levels of MITF expression lead to G1 cell-cycle arrest and differentiation, through induction of the cell cycle inhibitors p16CDKN2A and p21 [58-59] (Figure 1). Very low or null MITF expression levels predispose to apoptosis whereas inter-mediate MITF expression levels promote cell proliferation [57-59]. Therefore, it is thought that melanoma cells have developed strategies to maintain MITF lev‐ els in the range compatible with tumorigenesis. It has been shown that constitutive ERK ac‐ tivity, stimulated by V600EBRAF in melanoma cells, is associated with MITF ubiquitindependent degradation [60]. Nevertheless, continued expression of MITF is necessary for proliferation and survival of melanoma cells, because it also regulates CDK2 and Bcl-2 genes [61-62]. It has been recently shown that oncogenic BRAF may control intracellular levels of the MITF protein through a fine balance of two opposite mechanisms: a direct reduction of MITF levels, by inducing protein degradation, and an indirect increase of MITF levels, by stimulating transcription factors which increase protein expression levels [63]. Oncogenic *BRAF* mutations are associated with *MITF* amplification in a low fraction (10-15%) of mela‐ nomas [63], suggesting that other mechanisms are likely to be involved in ERK-dependent degradation of MITF.

#### **2.5.** *iNOS* **and** *NF-kB* **pathways**

Human melanoma cells are known to express the inducible nitric oxide synthase (iNOS) enzyme, which is responsible for synthesis of nitric oxide (NO), a free radical involved in several physiological processes such as neurotransmission, vasodilation, and regulation of immune responses [64]. The iNOS enzyme has been found to be frequently expressed in melanoma [65-66] and the subsequent increased concentrations of NO have been demon‐ strated to contribute to melanomagenesis through a sustained protection of the tumour from apoptosis [67]. However, the role of iNOS in melanoma progression remains contro‐ versial. Higher levels of iNOS have been found in subcutaneous and lymph node metasta‐ ses of non-progressive melanoma as compared to metastases of progressive melanoma [68], however, iNOS was found to be expressed to a lesser extent in metastases as com‐ pared with nevi and primary melanomas [69]. Nevertheless, the expression of iNOS in pa‐ tients with lymph nodes and in-transit metastases (stage III disease) has been proposed as an indicator of poor prognosis [70].

Recently, it has been reported that the constitutive iNOS expression in melanoma cells might be induced by activation of the MAPK pathway through stimulation of the activity of the Nuclear Factor-kB (NF-kB) [71-72]. NF-kB is a protein complex that acts as a tran‐ scriptional factor and regulates the transcription of several genes involved in many critical pathways [73]. In a quiescent status, proteins of the NF-kB complex are localized into the cytoplasm. NF-κB exists as cytoplasmic hetero- or homodimers associated with members of the inhibitor-of-kB (IkB) proteins (IkB*a*, Ik*Bb* and IkB*e*), which form complexes seques‐ tering NF-kB into the cytoplasm [74]. Upon appropriate stimulation, the phosphorylation of IkB proteins is promoted, triggering their ubiquitination and degradation in the protea‐ some [74]. As a consequence, NF-kB may translocate to the nucleus where it binds to tar‐ get DNA loci and induces transcription of several genes - including *iNOS* - associated with immune and inflammatory response, angiogenesis, cell proliferation, tumor promo‐ tion, and apoptosis [73-74].

Regarding the role of NF-kB in tumorigenesis, there are compelling evidence that activation of NF-kB controls multiple cellular processes in cancer due to its ability to promote cell pro‐ liferation, suppress apoptosis, promote cell migration, and suppress cell differentiation, opening the way for new therapeutic approaches against such a target [75-76]. In melanoma, NF-kB is constitutively activated since expression of the IkB proteins seems to be significant‐ ly reduced in comparison to nevi [77].

#### **2.6.** *cKIT* **and tyrosinase kinase receptors**

velopment and pathogenesis [43, 57]. MITF is activated by the MAPK pathway as well as by the cAMP pathway (Figure 1), and leads to transcription of genes involved in pigmentation (TYR, TYRP1, and DCT; see above) as well as cell cycle progression and survival [43]. The *MITF* gene is amplified in melanoma (about 20% of cases); *MITF* amplification correlated

The connection between MITF and melanoma development is complex because it plays a double role of inducer/repressor of cellular proliferation. High levels of MITF expression lead to G1 cell-cycle arrest and differentiation, through induction of the cell cycle inhibitors p16CDKN2A and p21 [58-59] (Figure 1). Very low or null MITF expression levels predispose to apoptosis whereas inter-mediate MITF expression levels promote cell proliferation [57-59]. Therefore, it is thought that melanoma cells have developed strategies to maintain MITF lev‐ els in the range compatible with tumorigenesis. It has been shown that constitutive ERK ac‐ tivity, stimulated by V600EBRAF in melanoma cells, is associated with MITF ubiquitindependent degradation [60]. Nevertheless, continued expression of MITF is necessary for proliferation and survival of melanoma cells, because it also regulates CDK2 and Bcl-2 genes [61-62]. It has been recently shown that oncogenic BRAF may control intracellular levels of the MITF protein through a fine balance of two opposite mechanisms: a direct reduction of MITF levels, by inducing protein degradation, and an indirect increase of MITF levels, by stimulating transcription factors which increase protein expression levels [63]. Oncogenic *BRAF* mutations are associated with *MITF* amplification in a low fraction (10-15%) of mela‐ nomas [63], suggesting that other mechanisms are likely to be involved in ERK-dependent

Human melanoma cells are known to express the inducible nitric oxide synthase (iNOS) enzyme, which is responsible for synthesis of nitric oxide (NO), a free radical involved in several physiological processes such as neurotransmission, vasodilation, and regulation of immune responses [64]. The iNOS enzyme has been found to be frequently expressed in melanoma [65-66] and the subsequent increased concentrations of NO have been demon‐ strated to contribute to melanomagenesis through a sustained protection of the tumour from apoptosis [67]. However, the role of iNOS in melanoma progression remains contro‐ versial. Higher levels of iNOS have been found in subcutaneous and lymph node metasta‐ ses of non-progressive melanoma as compared to metastases of progressive melanoma [68], however, iNOS was found to be expressed to a lesser extent in metastases as com‐ pared with nevi and primary melanomas [69]. Nevertheless, the expression of iNOS in pa‐ tients with lymph nodes and in-transit metastases (stage III disease) has been proposed as

Recently, it has been reported that the constitutive iNOS expression in melanoma cells might be induced by activation of the MAPK pathway through stimulation of the activity of the Nuclear Factor-kB (NF-kB) [71-72]. NF-kB is a protein complex that acts as a tran‐ scriptional factor and regulates the transcription of several genes involved in many critical pathways [73]. In a quiescent status, proteins of the NF-kB complex are localized into the

with increased resistance to chemotherapy and decreased overall survival [57].

degradation of MITF.

**2.5.** *iNOS* **and** *NF-kB* **pathways**

36 Melanoma - From Early Detection to Treatment

an indicator of poor prognosis [70].

cKIT is a member of the transmenbrane receptor tyrosine kinase family that comprised five immunoglobin-like motifs, a single transmembrane region, an inhibitory cytoplasmic juxta‐ menbrane domain, and a split cytoplasmic kinase domain separated by a kinase insert seg‐ ment [78]. Under physiological conditions, binding of the cKIT ligand stem-cell factor (SCF) to the extracellular domain of the receptor leads its dimerization, activation of the intracellu‐ lar tyrosine kinase domain through autophosporylation of specific tyrosine residue [78]. The intracellular signaling through cKIT plays a critical role in the development of several mam‐ malian cells, including growth, differentiation, migration, and proliferation of melanocytes [79]. It has been defined that cKIT recruits and activates a number of intracellular signaling pathways implicated in tumor progression, such as MAPK, PI3K/AKT, Src, activators of transcription (STAT), and phospholipase-C (PLC) [79-80].

Although the role of cKIT in melanomagenesis is still controversial, several studies have reported its downregulation during melanoma growth and invasion (in vertical growth phase of melanoma and metastatic lesions) [81-83]. Indeed, the majority of highly meta‐ static human melanomas do not express detectable levels of the cKIT receptor [83]. As a confirmation of this, over-expression of cKIT in metastatic melanoma cell lines led to im‐ portant reduction in tumor growth, while cKIT activation through exposure to cKIT li‐ gands induced apoptosis [83].

Specific mutations within the cKIT gene cause constitutive phosphorylation and activation of the kinase domain resulting in uncontrolled cell proliferation. [84]. Although such muta‐ tions seem to be more rare than *BRAF* and *NRAS* mutations, these may reflect the important role of cKIT tyrosine kinase in melanocyte development [84]. Sequencing of cKIT exons 11, 13, 17, and 18 revealed the most prevalent mutation to be K642, L576P, D816H-V, V559A [84]. The cKIT mutations are more common in mucosal and acral melanomas compared with cutaneous melanomas and are in most cases accompanied by an increase in gene copy number (40% mucosal or acral melanomas - frequently, associated with amplification of *cy‐* *clin D1* - as well as 30% of melanomas on skin with chronic sun-induced damage) [84]. High expression levels of cKIT and CDK4 proteins have been identified in another subset of mela‐ nomas lacking *BRAF* mutations [85].

gp100 vaccine alone [median OS 10.0 vs 6.4 mesi; Hazard Ratio (HR) for death, 0.68; p<0,001) [91]. In the second study, Dacarbazine was administered as standard chemotherapeutic drug for melanoma patients, in association with Ipilimumab or placebo; the OS rate was significantly higher in the group of patients treated with Ipilimumab + Dacarbazine (11.2 *vs*. 9.1 months), with even more significant percentages of survival for such an association after one year (47.3% *vs*. 36.3%), two years (28.5% *vs*. 17.9%), or three years (20.8% *vs*. 12.2%) of follow-up [92]. The DFS and OS curves from the two studies have been indicated as largely overlapping, strongly dem‐ onstrating, for the first time in the history of medical treatments for the advanced disease, a clear survival benefit in metastatic (disease stage IV) melanoma. For these reasons, Ipilimumab has

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39

been recently approved by FDA and EMEA for the treatment of metastatic melanoma.

The different molecular *pathways* involved into the pathogenesis of melanoma are function‐ ally linked each other (Figure 1). There is thus a need to consider such biological cascades as part of a functional web, and the alterations detected in distinct components of the various pathways must be globally considered for the effects determining in such a functional web. This new vision helps in clarifying the reasons by which some alterations may coexist or not in specific melanoma subtypes. As an example, *BRAF* mutations may be observed in con‐ junction with *PI3K* alterations, but none of them may coexist with *NRAS* mutations; since BRAF and PI3K kinases act downstream NRAS protein, occurrence of *NRAS* mutations acti‐ vating both MAPK and PI3K-AKT pathways makes unnecessary the further activation of BRAF and PI3K (upstream effectors of the MAPK and PI3K-AKT pathways, respectively). Analogously, oncogenic *BRAF* mutations are able to more intensively activate ERK protein, main last effector downstream the MAPK pathway, when inactivation of the mechanisms

In an attempt to simplify such complex processes underlying the different phases of devel‐ opment and progression of melanoma, the main pathogenetic molecular alterations may be

**•** oncogenic *BRAF* mutations, genomic rearrangements (mainly represented by allelic dele‐ tions) at the 9p21 chromosome, and increased expression levels of the AKT3 protein are the main alterations involved into the phase of stimulation of the proliferation for normal

**•** impairments of the mechanisms controlling the cell senescence, apoptosis and cell surviv‐ al (which particularly include alterations in the different components of *CDKN2A* path‐ ways: functional deficit of *p16CDKN2A* gene, amplification of *CDK4*-*Cyclin D1*/*CCND1* loci, inattivation of *TP53* gene through a deregulation of the p14CDKN2A-MDM2-p53 functional cascade), oncogenic mutations in *NRAS* gene, activating mutations and, to a lesser extent, gene amplifications of *cKIT* are the main alterations involved into the phase of acquisition of the malignant phenotype which underlay the development of melanoma (*intermediate*

**3. Melanoma subtypes and targeted therapeutic options**

controlling senescence and apoptosis concomitantly occurs.

grouped in the following way:

*neoplastic phase*);

melanocytes (*initial preneoplastic phase*);

### **2.7. CTLA-4 and T-cell activation**

The above-mentioned main intracellular molecular pathways are thus involved in tumor growth and survival, actively participating to the different phases of development and progres‐ sion of melanoma. Additional extracellular factors, mainly represented by different compo‐ nents of the tumor microenvironment, have been implied to play a role in melanoma tissue invasion and metastatic dissemination. As an example, changes in the expression of adhesion molecules such as MCAM/MUC18, E-cadherin, and integrins occur in the transition from the ra‐ dial growth phase (RGP) to the vertical growth phase (VGP) of melanoma; they are induced by both intracellular modifications [i.e., activation of the focal adhesion kinase (FAK) and integrin linked kinase (ILK) patways or high levels of activated ERK (phospoERK1-2)] and biological sig‐ nals directly generated by the extracellular matrix (ECM), which is composed of proteins, glyco‐ proteins, proteoglycans, and glycosaminoglycans in complex arrangements [22-23].

Among others, a block of the anti-tumor immune response induced by changes in pericellular microenvironment has been demonstrated to contribute to melanoma progression [86]. In re‐ cent past years, research has tried to better define the molecular mechanisms underlying the downregulation of the immune system by such pericellular components, in order to develop new therapeutic targets [87]. Actually, two immunomodulant antibodies, such as anti-CTLA4 and anti-PD1, have been demonstrated to be effective in inhibiting some down-regulators of the anti-tumor immune response [30, 88]. Moreover, drugs able to interfere with the differentiation of the myeloid-derived suppressor cells (MDSC) and T regulatory cells (Treg), which are both physiologically involved in controlling an abnormal immune response during the inflammato‐ ry processes and pathologically favoring tumor progression through suppression of T-cell acti‐ vation, represent additional therapeutic strategies to be exploited [89].

For T-cell activation, melanoma antigens that are bound to the major histocompatibility com‐ plex (MHC) on antigen-presenting cells (APCs) require the costimulation of CD28 receptor on Tcells by CD80 or CD86 ligands on APCs [90]. The cytotoxic T-lymphocyte antigen-4 (CTLA-4) can bind with greater affinity to CD80 and CD86, and thus disrupt the necessary costimulatory signal provided by APCs [88, 90]. This led to the hypothesis that blockade of CTLA-4 function may allow for optimal costimulation of CD28 receptors on T-cells by APC CD80/86, and en‐ hanced T-cell activation [88, 90]. Ipilimumab (Yervoy™ Bristol-Myers Squibb, New York, NY) blocks the costimulatory signal required for T-cell activation [30, 88]. In particular, Ipilimumab is a recombinant human IgG1 monoclonal antibody that binds to CTLA-4 and blocks binding to CD80 or CD86 on APCs, thus increasing activation and proliferation of T cells [88]. Two random‐ ized phase III trials have indicated a significant advantage in disease-free survival (DFS) and overall survival (OS) in either monotherapy or combination therapy [91-92]. The first trial com‐ pared monotherapy with Ipilimumab 3 mg/kg, combination of Ipilimumab with gp100 vaccine, and gp100 vaccine alone; the study demonstrated a significant advantage in OS for patients treated with Ipilimumab (regardless the addition off gp100) in comparison to those receiving the gp100 vaccine alone [median OS 10.0 vs 6.4 mesi; Hazard Ratio (HR) for death, 0.68; p<0,001) [91]. In the second study, Dacarbazine was administered as standard chemotherapeutic drug for melanoma patients, in association with Ipilimumab or placebo; the OS rate was significantly higher in the group of patients treated with Ipilimumab + Dacarbazine (11.2 *vs*. 9.1 months), with even more significant percentages of survival for such an association after one year (47.3% *vs*. 36.3%), two years (28.5% *vs*. 17.9%), or three years (20.8% *vs*. 12.2%) of follow-up [92]. The DFS and OS curves from the two studies have been indicated as largely overlapping, strongly dem‐ onstrating, for the first time in the history of medical treatments for the advanced disease, a clear survival benefit in metastatic (disease stage IV) melanoma. For these reasons, Ipilimumab has been recently approved by FDA and EMEA for the treatment of metastatic melanoma.

## **3. Melanoma subtypes and targeted therapeutic options**

*clin D1* - as well as 30% of melanomas on skin with chronic sun-induced damage) [84]. High expression levels of cKIT and CDK4 proteins have been identified in another subset of mela‐

The above-mentioned main intracellular molecular pathways are thus involved in tumor growth and survival, actively participating to the different phases of development and progres‐ sion of melanoma. Additional extracellular factors, mainly represented by different compo‐ nents of the tumor microenvironment, have been implied to play a role in melanoma tissue invasion and metastatic dissemination. As an example, changes in the expression of adhesion molecules such as MCAM/MUC18, E-cadherin, and integrins occur in the transition from the ra‐ dial growth phase (RGP) to the vertical growth phase (VGP) of melanoma; they are induced by both intracellular modifications [i.e., activation of the focal adhesion kinase (FAK) and integrin linked kinase (ILK) patways or high levels of activated ERK (phospoERK1-2)] and biological sig‐ nals directly generated by the extracellular matrix (ECM), which is composed of proteins, glyco‐

proteins, proteoglycans, and glycosaminoglycans in complex arrangements [22-23].

vation, represent additional therapeutic strategies to be exploited [89].

Among others, a block of the anti-tumor immune response induced by changes in pericellular microenvironment has been demonstrated to contribute to melanoma progression [86]. In re‐ cent past years, research has tried to better define the molecular mechanisms underlying the downregulation of the immune system by such pericellular components, in order to develop new therapeutic targets [87]. Actually, two immunomodulant antibodies, such as anti-CTLA4 and anti-PD1, have been demonstrated to be effective in inhibiting some down-regulators of the anti-tumor immune response [30, 88]. Moreover, drugs able to interfere with the differentiation of the myeloid-derived suppressor cells (MDSC) and T regulatory cells (Treg), which are both physiologically involved in controlling an abnormal immune response during the inflammato‐ ry processes and pathologically favoring tumor progression through suppression of T-cell acti‐

For T-cell activation, melanoma antigens that are bound to the major histocompatibility com‐ plex (MHC) on antigen-presenting cells (APCs) require the costimulation of CD28 receptor on Tcells by CD80 or CD86 ligands on APCs [90]. The cytotoxic T-lymphocyte antigen-4 (CTLA-4) can bind with greater affinity to CD80 and CD86, and thus disrupt the necessary costimulatory signal provided by APCs [88, 90]. This led to the hypothesis that blockade of CTLA-4 function may allow for optimal costimulation of CD28 receptors on T-cells by APC CD80/86, and en‐ hanced T-cell activation [88, 90]. Ipilimumab (Yervoy™ Bristol-Myers Squibb, New York, NY) blocks the costimulatory signal required for T-cell activation [30, 88]. In particular, Ipilimumab is a recombinant human IgG1 monoclonal antibody that binds to CTLA-4 and blocks binding to CD80 or CD86 on APCs, thus increasing activation and proliferation of T cells [88]. Two random‐ ized phase III trials have indicated a significant advantage in disease-free survival (DFS) and overall survival (OS) in either monotherapy or combination therapy [91-92]. The first trial com‐ pared monotherapy with Ipilimumab 3 mg/kg, combination of Ipilimumab with gp100 vaccine, and gp100 vaccine alone; the study demonstrated a significant advantage in OS for patients treated with Ipilimumab (regardless the addition off gp100) in comparison to those receiving the

nomas lacking *BRAF* mutations [85].

**2.7. CTLA-4 and T-cell activation**

38 Melanoma - From Early Detection to Treatment

The different molecular *pathways* involved into the pathogenesis of melanoma are function‐ ally linked each other (Figure 1). There is thus a need to consider such biological cascades as part of a functional web, and the alterations detected in distinct components of the various pathways must be globally considered for the effects determining in such a functional web. This new vision helps in clarifying the reasons by which some alterations may coexist or not in specific melanoma subtypes. As an example, *BRAF* mutations may be observed in con‐ junction with *PI3K* alterations, but none of them may coexist with *NRAS* mutations; since BRAF and PI3K kinases act downstream NRAS protein, occurrence of *NRAS* mutations acti‐ vating both MAPK and PI3K-AKT pathways makes unnecessary the further activation of BRAF and PI3K (upstream effectors of the MAPK and PI3K-AKT pathways, respectively). Analogously, oncogenic *BRAF* mutations are able to more intensively activate ERK protein, main last effector downstream the MAPK pathway, when inactivation of the mechanisms controlling senescence and apoptosis concomitantly occurs.

In an attempt to simplify such complex processes underlying the different phases of devel‐ opment and progression of melanoma, the main pathogenetic molecular alterations may be grouped in the following way:


**•** complete silencing of *p16CDKN2A* gene, functional loss of PTEN, activation of the PI3K-AKT pathway, and amplification of the *MITF* gene are the main alterations involved into the phase of acquisition of a more aggressive and invasive phenotype which underlay the progression and dissemination of melanoma (*final metasticase phase*).

Nevertheless, all these evidence represent a strong indication that the different molecular pathways associated with the melanomagenesis does correspond to different subsets of mel‐ anoma patients, with distinguished biological and clinical behavior of the disease. Identifica‐ tion of such different patients' subsets should be introduced in clinical trials, in order to better assess the classification of all predictive and prognostic factors associated with the disease as well as more accurately address patients to the most effective therapeutic inter‐

Targeted Therapies in Melanoma: Successes and Pitfalls

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41

On the basis of the presence of the specific molecular features, a discrimination of the main subtypes of melanoma, along with the more appropriate therapeutic option for each sub‐

Prevalence of *BRAF* mutations, with tendency to increased expression level of activated ERK (phospoERK1-2) in melanoma tissues. This subtype benefits by the treatment with inhibitors

After failure of BAY 43-9006 (which is not specific for mutated BRAF, but suppresses activi‐ ty of several different kinases [95]), a second generation BRAF inhibitor (Vemurafenib, also known as PLX4032 or RO5185426) was highly specific for V600EBRAF mutation and appeared very promising from the clinical point of view [96]. Very recently, results from a phase III study comparing the vemurafenib to dacarbazine have been indeed reported, indicating a relative reduction of 63% in the risk of death and of 74% in the risk of either death or disease progression, as compared with dacarbazine [97]. Preliminary data using an additional V600EBRAF inhibitor compound, Dabrafenib (previously known as GSK2118436), seemed to point out that this molecule is also active on V600KBRAF and V600DBRAF mutations [98]; actual‐ ly, treatment with such a compound is under evaluation in a phase III study among BRAF

However, preliminary data seem to indicate that a large variety of induced alterations may drive resistance to BRAF inhibitors: upregulation of the receptor tyrosine kinase (RTK) effec‐ tors [99], mutation in *NRAS* gene and platelet-derived growth factor receptor β (PDGFRβ) [99], amplification of the *CCND1/Cyclin D1* gene or lack of phosphatase-and-tensin homo‐ logue (PTEN) function [100], mutations in downstream *MEK* gene [101], activation of MAPK pathway agonists such as COT kinase [102], or enhancement of the IGF-1R/PI3K signaling

The MEK inhibitors (AS703026, E6201, GSK1120212, GDC0973, MEK162) as single agents have activity against melanoma, in patients either carrying BRAF mutations and unexposed to prior BRAF inhibitor therapy or presenting NRAS mutations [30]. A new combination of MEK and BRAF inhibitors as first line therapy for BRAF mutated melanoma patients naïve

The *BRAF* mutations may coexist with additional molecular alterations, with subsequent

constitution of further biological and molecular subgroups of melanoma patients:

vention according to their biological and molecular status.

type, could be schematically reported.

mutation positive stage III-IV melanoma patients.

[103]. These findings highlight the need for combination therapy.

to prior anti-BRAF treatment is showing great promise [30].

**1.** Subtype MAPK

of BRAF.

All these findings clearly indicate the existence of a complex molecular machinery that pro‐ vides checks and balances in normal melanocytes, allowing a physiologically controlled cell proliferation. Progression from normal melanocytes to malignant metastatic cell in melano‐ ma patients is the result of a combination of down- or up-regulations of the various effectors acting into the different molecular pathways. According to this, it has been proposed a line‐ ar model of pathogenesis of melanoma based on the sequential accumulation of most of the previously-described molecular alterations (Figure 2A) [7, 13, 93]. In a limited fraction of cases, it has been recently hypothesized a second non-linear model of melanomagenesis based on accumulation of the same genetic alterations in tissue stem cells, with generation of malignant cells directly forming RGP or VGP or metastatic melanoma lesions (Figure 2B) [94]. This latter hypothesis has been derived by the evidence of some inconsistencies of the linear model in subgroups of melanomas (i.e., incidence of *BRAF* mutations higher in VGP lesions than that found in RGP lesions [94]).

**Figure 2.** Models of development and progression for melanoma. A, sequential model. B, non-linear model.

Nevertheless, all these evidence represent a strong indication that the different molecular pathways associated with the melanomagenesis does correspond to different subsets of mel‐ anoma patients, with distinguished biological and clinical behavior of the disease. Identifica‐ tion of such different patients' subsets should be introduced in clinical trials, in order to better assess the classification of all predictive and prognostic factors associated with the disease as well as more accurately address patients to the most effective therapeutic inter‐ vention according to their biological and molecular status.

On the basis of the presence of the specific molecular features, a discrimination of the main subtypes of melanoma, along with the more appropriate therapeutic option for each sub‐ type, could be schematically reported.

### **1.** Subtype MAPK

**•** complete silencing of *p16CDKN2A* gene, functional loss of PTEN, activation of the PI3K-AKT pathway, and amplification of the *MITF* gene are the main alterations involved into the phase of acquisition of a more aggressive and invasive phenotype which underlay the

All these findings clearly indicate the existence of a complex molecular machinery that pro‐ vides checks and balances in normal melanocytes, allowing a physiologically controlled cell proliferation. Progression from normal melanocytes to malignant metastatic cell in melano‐ ma patients is the result of a combination of down- or up-regulations of the various effectors acting into the different molecular pathways. According to this, it has been proposed a line‐ ar model of pathogenesis of melanoma based on the sequential accumulation of most of the previously-described molecular alterations (Figure 2A) [7, 13, 93]. In a limited fraction of cases, it has been recently hypothesized a second non-linear model of melanomagenesis based on accumulation of the same genetic alterations in tissue stem cells, with generation of malignant cells directly forming RGP or VGP or metastatic melanoma lesions (Figure 2B) [94]. This latter hypothesis has been derived by the evidence of some inconsistencies of the linear model in subgroups of melanomas (i.e., incidence of *BRAF* mutations higher in VGP

**Figure 2.** Models of development and progression for melanoma. A, sequential model. B, non-linear model.

progression and dissemination of melanoma (*final metasticase phase*).

lesions than that found in RGP lesions [94]).

40 Melanoma - From Early Detection to Treatment

Prevalence of *BRAF* mutations, with tendency to increased expression level of activated ERK (phospoERK1-2) in melanoma tissues. This subtype benefits by the treatment with inhibitors of BRAF.

After failure of BAY 43-9006 (which is not specific for mutated BRAF, but suppresses activi‐ ty of several different kinases [95]), a second generation BRAF inhibitor (Vemurafenib, also known as PLX4032 or RO5185426) was highly specific for V600EBRAF mutation and appeared very promising from the clinical point of view [96]. Very recently, results from a phase III study comparing the vemurafenib to dacarbazine have been indeed reported, indicating a relative reduction of 63% in the risk of death and of 74% in the risk of either death or disease progression, as compared with dacarbazine [97]. Preliminary data using an additional V600EBRAF inhibitor compound, Dabrafenib (previously known as GSK2118436), seemed to point out that this molecule is also active on V600KBRAF and V600DBRAF mutations [98]; actual‐ ly, treatment with such a compound is under evaluation in a phase III study among BRAF mutation positive stage III-IV melanoma patients.

However, preliminary data seem to indicate that a large variety of induced alterations may drive resistance to BRAF inhibitors: upregulation of the receptor tyrosine kinase (RTK) effec‐ tors [99], mutation in *NRAS* gene and platelet-derived growth factor receptor β (PDGFRβ) [99], amplification of the *CCND1/Cyclin D1* gene or lack of phosphatase-and-tensin homo‐ logue (PTEN) function [100], mutations in downstream *MEK* gene [101], activation of MAPK pathway agonists such as COT kinase [102], or enhancement of the IGF-1R/PI3K signaling [103]. These findings highlight the need for combination therapy.

The MEK inhibitors (AS703026, E6201, GSK1120212, GDC0973, MEK162) as single agents have activity against melanoma, in patients either carrying BRAF mutations and unexposed to prior BRAF inhibitor therapy or presenting NRAS mutations [30]. A new combination of MEK and BRAF inhibitors as first line therapy for BRAF mutated melanoma patients naïve to prior anti-BRAF treatment is showing great promise [30].

The *BRAF* mutations may coexist with additional molecular alterations, with subsequent constitution of further biological and molecular subgroups of melanoma patients:

**a.** Impairment of the p16CDKN2A-CDK4/CCND1-RB or p14CDKN2A-MDM2-TP53 pathways, with reduced expression of the p16CDKN2A protein and tendency to amplification of the *CDK4*/*CCND1* gene loci or inactivation of the TP53 gene (with consequent functional loss of the p53 protein), respectively. This subtype benefits by the treatment with inhibi‐ tors of the cyclin-dependent kinases.

To date, two approaches have been considered in developing drugs against RAS. The first is based on the block of farnesylation. A small clinical trial using an inhibitor of the farnesyl transferase enzyme failed to be efficacious in a melanoma cohort; however, patients includ‐ ed into such a study were not selected on the basis of their *NRAS* mutation status [112-113]. In the light of recent successes of the target therapies based on anti-BRAF or anti-cKIT inhib‐ itors, a more stringently selected cohort carrying alterations in *NRAS* gene would have in‐ creased responsiveness. On the other hand, a direct targeting of RAS has been demonstrated to be very difficult [114]; this is the reason why therapeutic strategies have focused on inhib‐ iting downstream effectors into the pathways activated by RAS (i.e., MEK inhibitors for the MAPK pathway - see above - and mTORC inhibitors for the PI3K-AKT pathway - see be‐

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Prevalence of *cKIT* mutations ± gene amplification and/or increased protein expression lev‐ els. This subtype benefits by the treatment with inhibitors of cKIT (in particular, patients carrying gene mutations, with some sequence variants - such as K642E e L576P - which are

Activating cKIT mutations have been implicated in a variety of cancers, mainly represented by gastrointestinal stromal tumors (GIST) and chronic myelogenous leukemia (CML). This is the reason why several drugs targeting cKIT have been developed and tested in clinical trials, including Imatinib (approved for Philadelphia chromosome–positive CML and cKITpositive GIST) and Sunitib (approved for advanced kidney cancer and Imatinib-resistant GIST) as well as Nilotinib and Dasatinib (approved for CML and Philadelphia chromosome-

The inhibitors of cKIT that may have a therapeutic benefit in melanoma, by inducing cell cy‐ cle arrest and apoptosis as well as significantly inhibiting cell migration and invasion of tu‐

Imatinib mesylate, formerly known as STI571, is designated chemically as 4-benzamide methanesulfonate. The efficacy of imatinib varies with the site of cKIT mutation; moreover, this drug can inhibit both the wild-type receptor activated by ligand and mutated receptor in the absence of ligand. However, imatinib is less effective at inhibiting the receptor with

Nilotinib (AMN107), which has been rationally designed based on the imatinib mesylate scaffold to have a more selective action. On this regard, Nilotinib inhibits both wild-type and cKIT mutants in exon 11 (V560del and V560G), exon 13 (K642E), double mutants in‐ volving exons 11, 13, and 17 as well as imatinib-resistant cKIT mutant (V560del/V654A)

Dasatinib, which is a piperazinyl ethanol exhibiting increased potency but reduced selectivi‐ ty compared with imatinib mesylate, has been demonstrated to inhibit both wild type and mutant cKIT in a dose-dependent manner, causing inhibition of cell migration and invasion through reduction of the phosphorylation of either Src kinase and FAK pathway [116-117].

low), which represent the second treatment approach against RAS.

**3.** Subtype cKIT

highly responsive).

mor cells, are:

cells [115];

positive acute lymphoblastic leukemia).

mutations in the enzymatic site (exon 17 mutations) [79];

Melanoma patients carrying genetic alterations affecting p16CDKN2A could potentially be treated with inhibitors of CDK4/CDK6. There are currently no validated therapeutic options for melanoma with mutated p14CDKN2A. Conversely, several CDK4 inhibitors (Alvocidib, AT-7519, P1446A-05, PD-0332991, Flavopiridol/alvocidib/HMR 1275, P276-00, R547, SNS-032/ BMS-387032, UCN-01, ZK 304709/MTGI) are currently under investigation for a variety of cancer types, including metastatic melanoma, and results are awaited. For p53, there are currently no drugs, approved or in trials, against such a target. Conversely, an an‐ ti-sense agent (Oblimersen) targeted at nuclear Bcl-2 has been evaluated in trials, failing to demonstrate a significant clinical benefit among patients with melanoma [104].

**b.** Amplification of *MITF* ± associated with reduction of the protein expression levels.

No drug targeting MITF has been developed; however, expression of MITF has been dem‐ onstrated to be reduced by compounds inhibiting the multiple histone deacetylase (HDAC) complex [105]. Ongoing trials based on HDAC-inhibitors [LBH589 (Panobinostat) or Valpro‐ ic acid (Vorinostat)] will elucidate whether a clinical benefit could be obtained by down-reg‐ ulating intracellular level of MITF protein.

**c.** Activation of NF-kB.

Proteasome inhibitors, such as Bortezomib (Velcade, previously known as PS-341), represent a new class of anticancer therapeutic agents which inhibit degradation of important cell cy‐ cle and/or regulatory proteins, including IkB [106-107]. Bortezomid has been demonstrated to contribute in maintaining integrity of the complexes sequestering NF-kB into the cyto‐ plasm, thus reducing the NF-kB activity [106-107]. Phase 2 studies combinating Bortezomib with other chemotherapeutic agents, including paclitaxel, carboplatin, or temozolomide equally have been established [108-109]. A compound that more directly targets the NF-kB pathway is BMS-345541 (4(2'-aminoethyl) amino-1,8-dimethylimidazo(1,2-a)quinoxaline), identified as a selective inhibitor of the catalytic subunits of IKK that binds at an allosteric site of the enzyme [110].

Since mutational activation of BRAF in human melanomas has been demonstrated to con‐ tribute to constitutive induction of NF-kB activity through an increase of the IKK activity [111], inhibition of BRAF signaling using the above mentioned inhibitors may decrease the NF-kB transcriptional activity and sensitize melanoma cells to apoptosis.

#### **2.** Subtype NRAS

Prevalence of *NRAS* mutation, with markedly increased expression level of activated ERK (phospoERK1-2) and eventual activation of AKT. This subtype benefits by the treatment with inhibitors of MEK or mTORC.

To date, two approaches have been considered in developing drugs against RAS. The first is based on the block of farnesylation. A small clinical trial using an inhibitor of the farnesyl transferase enzyme failed to be efficacious in a melanoma cohort; however, patients includ‐ ed into such a study were not selected on the basis of their *NRAS* mutation status [112-113]. In the light of recent successes of the target therapies based on anti-BRAF or anti-cKIT inhib‐ itors, a more stringently selected cohort carrying alterations in *NRAS* gene would have in‐ creased responsiveness. On the other hand, a direct targeting of RAS has been demonstrated to be very difficult [114]; this is the reason why therapeutic strategies have focused on inhib‐ iting downstream effectors into the pathways activated by RAS (i.e., MEK inhibitors for the MAPK pathway - see above - and mTORC inhibitors for the PI3K-AKT pathway - see be‐ low), which represent the second treatment approach against RAS.

## **3.** Subtype cKIT

**a.** Impairment of the p16CDKN2A-CDK4/CCND1-RB or p14CDKN2A-MDM2-TP53 pathways, with reduced expression of the p16CDKN2A protein and tendency to amplification of the *CDK4*/*CCND1* gene loci or inactivation of the TP53 gene (with consequent functional loss of the p53 protein), respectively. This subtype benefits by the treatment with inhibi‐

Melanoma patients carrying genetic alterations affecting p16CDKN2A could potentially be treated with inhibitors of CDK4/CDK6. There are currently no validated therapeutic options for melanoma with mutated p14CDKN2A. Conversely, several CDK4 inhibitors (Alvocidib, AT-7519, P1446A-05, PD-0332991, Flavopiridol/alvocidib/HMR 1275, P276-00, R547, SNS-032/ BMS-387032, UCN-01, ZK 304709/MTGI) are currently under investigation for a variety of cancer types, including metastatic melanoma, and results are awaited. For p53, there are currently no drugs, approved or in trials, against such a target. Conversely, an an‐ ti-sense agent (Oblimersen) targeted at nuclear Bcl-2 has been evaluated in trials, failing to

demonstrate a significant clinical benefit among patients with melanoma [104].

**b.** Amplification of *MITF* ± associated with reduction of the protein expression levels.

No drug targeting MITF has been developed; however, expression of MITF has been dem‐ onstrated to be reduced by compounds inhibiting the multiple histone deacetylase (HDAC) complex [105]. Ongoing trials based on HDAC-inhibitors [LBH589 (Panobinostat) or Valpro‐ ic acid (Vorinostat)] will elucidate whether a clinical benefit could be obtained by down-reg‐

Proteasome inhibitors, such as Bortezomib (Velcade, previously known as PS-341), represent a new class of anticancer therapeutic agents which inhibit degradation of important cell cy‐ cle and/or regulatory proteins, including IkB [106-107]. Bortezomid has been demonstrated to contribute in maintaining integrity of the complexes sequestering NF-kB into the cyto‐ plasm, thus reducing the NF-kB activity [106-107]. Phase 2 studies combinating Bortezomib with other chemotherapeutic agents, including paclitaxel, carboplatin, or temozolomide equally have been established [108-109]. A compound that more directly targets the NF-kB pathway is BMS-345541 (4(2'-aminoethyl) amino-1,8-dimethylimidazo(1,2-a)quinoxaline), identified as a selective inhibitor of the catalytic subunits of IKK that binds at an allosteric

Since mutational activation of BRAF in human melanomas has been demonstrated to con‐ tribute to constitutive induction of NF-kB activity through an increase of the IKK activity [111], inhibition of BRAF signaling using the above mentioned inhibitors may decrease the

Prevalence of *NRAS* mutation, with markedly increased expression level of activated ERK (phospoERK1-2) and eventual activation of AKT. This subtype benefits by the treatment with

NF-kB transcriptional activity and sensitize melanoma cells to apoptosis.

tors of the cyclin-dependent kinases.

42 Melanoma - From Early Detection to Treatment

ulating intracellular level of MITF protein.

**c.** Activation of NF-kB.

site of the enzyme [110].

**2.** Subtype NRAS

inhibitors of MEK or mTORC.

Prevalence of *cKIT* mutations ± gene amplification and/or increased protein expression lev‐ els. This subtype benefits by the treatment with inhibitors of cKIT (in particular, patients carrying gene mutations, with some sequence variants - such as K642E e L576P - which are highly responsive).

Activating cKIT mutations have been implicated in a variety of cancers, mainly represented by gastrointestinal stromal tumors (GIST) and chronic myelogenous leukemia (CML). This is the reason why several drugs targeting cKIT have been developed and tested in clinical trials, including Imatinib (approved for Philadelphia chromosome–positive CML and cKITpositive GIST) and Sunitib (approved for advanced kidney cancer and Imatinib-resistant GIST) as well as Nilotinib and Dasatinib (approved for CML and Philadelphia chromosomepositive acute lymphoblastic leukemia).

The inhibitors of cKIT that may have a therapeutic benefit in melanoma, by inducing cell cy‐ cle arrest and apoptosis as well as significantly inhibiting cell migration and invasion of tu‐ mor cells, are:

Imatinib mesylate, formerly known as STI571, is designated chemically as 4-benzamide methanesulfonate. The efficacy of imatinib varies with the site of cKIT mutation; moreover, this drug can inhibit both the wild-type receptor activated by ligand and mutated receptor in the absence of ligand. However, imatinib is less effective at inhibiting the receptor with mutations in the enzymatic site (exon 17 mutations) [79];

Nilotinib (AMN107), which has been rationally designed based on the imatinib mesylate scaffold to have a more selective action. On this regard, Nilotinib inhibits both wild-type and cKIT mutants in exon 11 (V560del and V560G), exon 13 (K642E), double mutants in‐ volving exons 11, 13, and 17 as well as imatinib-resistant cKIT mutant (V560del/V654A) cells [115];

Dasatinib, which is a piperazinyl ethanol exhibiting increased potency but reduced selectivi‐ ty compared with imatinib mesylate, has been demonstrated to inhibit both wild type and mutant cKIT in a dose-dependent manner, causing inhibition of cell migration and invasion through reduction of the phosphorylation of either Src kinase and FAK pathway [116-117].

## **4.** Subtype mTORC

Prevalence of PTEN loss (± *PI3K* mutations, which are mostly infrequent) and phosphoryla‐ tion of AKT, with absence of concurrent mutations in *BRAF* gene. This subtype mainly bene‐ fits by the treatment with inhibitors of mTOR.

**•** Acral melanoma

damage (CSD)

(non-CSD)

**•** Mucosal melanoma

**•** Uveal melanoma

cases).

and/or 5p15 genomic loci.

± amplification of *cKIT* (in about 5% of cases).

Mutation ± amplification of *cKIT*; amplification of *CDK4* or *CCND1* (associated with in‐ creased expression levels of the related proteins); amplification of the 11q13, 22q11-13,

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**•** Melanoma of the head and neck district and melanoma on skin with chronic sun-induced

Amplification of *CDK4* and/or *CCND1*; increased expression levels of p53 protein; mutation

**•** Melanoma of the trunk and melanoma on skin without chronic sun-induced damage

Mutation of *BRAF* or, alternatively, *NRAS* (with eventual coexistence of molecolar altera‐ tions which may ba associated with BRAF mutations; see above); tendency to reduced ex‐

Mutation ± amplification of *cKIT*; amplification of *CDK4* or *CCND1* (associated with in‐ creased expression levels of the related proteins); mutation of *BRAF* (in less than 10% of

Knowledge of the principal molecular alterations to be tested in patients with such distinct subtypes of melanoma will be of great clinical importance, because it is likely to result in separate targeted therapeutic approaches and prevention strategies. To date, it has been al‐ ready developed a panel of molecular tests to be performed in patients with melanoma from different anatomical locations (Figure 3). This initial "*flow-chart*" will surely become more detailed and enriched on the basis of the progressive identification and validation of addi‐

Finally, recent meta-analyses tried to define the prognostic role of majority of molecular al‐

loss of p53; over-expression of iNOS, AP-2, MMP-2 and metallothioneine; increased prolifer‐

reduced expression or loss of p16CDKN2A; over-expression of Bcl-2 and ATF-2 (± associated

with simultaneous increases expression of beta-catenin, fibronectin and p21 proteins).

pression of the p53 protein; occurrence of specific polymorphisms in *MC1R* gene.

Mutation ± amplification of *cKIT*; mutation of *GNAQ* and/or *GNA11*.

tional genetics and molecular alterations correlated with the disease.

terations previously described [124-125]:

ation index (high expression levels of Ki-67);

**•** negative prognostic factors

**•** positive prognostic factors

In melanoma cells, three potential targets may be considered for therapeutic intervention against this pathway: AKT, PI3K and mTOR. Restoration of functional PTEN or interfering with AKT and PI3K activity would increase chemosensitivity to apoptotic agents and im‐ prove the efficacy of anti-tumor treatment. Several inhibitors of PI3K (BKM120, BEZ235, BGT226, GDC0941, PX-866, SF1126, and XL147) and AKT (GSK690693, MK2206, and VQD-002) have been developed; results of ongoing trials are thus awaited. To date, clinical trials using agents against the PI3K/AKT pathway have failed to demonstrate significant ef‐ ficacy [118]. However, one therapeutic approach which seems to inhibit this pathway is based on the use of mTOR inhibitors [119]: rapamycin, Temsirolimus (CCI-779), Everolimus (RAD001), Sirolimus and AZD8055. While controversial data have been reported for rapa‐ mycin (suppressing disease progression in some patients with glioblastoma but ineffective in controlling the disease in others) [120], a limited advantage in response rates has been so far described for Temsirolimus [121]. It is to be underlined that none of the trials with mTOR inhibitors included patients specifically selected for alterations in the AKT/PI3K pathway. Several clinical trials are investigating specific combinations of mTOR inhibitors and chemo‐ therapy drugs in the treatment of melanoma.

**5.** Subtype GNAQ/GNA11

Prevalence of *GNAQ/GNA11* mutations, with increased expression level of phosphoERK. This subtype benefits by the treatment with inhibitors of MEK.

The *GNAQ* and *GNA11* genes encodes specific GTP binding proteins that mediate signal transduction from the inner cell surface to the MAPK pathway through activation of the protein kinase C (PKC) enzyme [122]. Somatic mutations in *GNAQ* gene have been observed in about 90% of blue naevi, 50% of malignant blue naevi and 50% of primary uveal melano‐ ma; conversely, the *GNA11* mutations have been found in less than 10% of blue nevi, about one third of primary uveal melanomas, and about 60% of metastatic uveal melanoma [122]. Since mutations in these two genes have non been detected among all the remaining types of melanoma (cutaneous, acral, mucosal), a clinical trial aimed at testing efficacy of a MEK inhibitor, Dabrafenib/GSK1120212, has been focused on patients with metastatic uveal mela‐ noma carrying *GNAQ* and/or *GNA11* mutations [123].

## **4. Diagnostic panel of molecular alterations**

The most prevalent molecular alterations within the heterogeneous patterns of biological features which characterize the distinct subtypes of melanoma are here summarized, accord‐ ing to the anatomical site of melanoma onset, the degree of exposure to the sun, and the his‐ tologic characteristics of the tumor lesions.

## **•** Acral melanoma

**4.** Subtype mTORC

44 Melanoma - From Early Detection to Treatment

fits by the treatment with inhibitors of mTOR.

therapy drugs in the treatment of melanoma.

This subtype benefits by the treatment with inhibitors of MEK.

noma carrying *GNAQ* and/or *GNA11* mutations [123].

**4. Diagnostic panel of molecular alterations**

tologic characteristics of the tumor lesions.

**5.** Subtype GNAQ/GNA11

Prevalence of PTEN loss (± *PI3K* mutations, which are mostly infrequent) and phosphoryla‐ tion of AKT, with absence of concurrent mutations in *BRAF* gene. This subtype mainly bene‐

In melanoma cells, three potential targets may be considered for therapeutic intervention against this pathway: AKT, PI3K and mTOR. Restoration of functional PTEN or interfering with AKT and PI3K activity would increase chemosensitivity to apoptotic agents and im‐ prove the efficacy of anti-tumor treatment. Several inhibitors of PI3K (BKM120, BEZ235, BGT226, GDC0941, PX-866, SF1126, and XL147) and AKT (GSK690693, MK2206, and VQD-002) have been developed; results of ongoing trials are thus awaited. To date, clinical trials using agents against the PI3K/AKT pathway have failed to demonstrate significant ef‐ ficacy [118]. However, one therapeutic approach which seems to inhibit this pathway is based on the use of mTOR inhibitors [119]: rapamycin, Temsirolimus (CCI-779), Everolimus (RAD001), Sirolimus and AZD8055. While controversial data have been reported for rapa‐ mycin (suppressing disease progression in some patients with glioblastoma but ineffective in controlling the disease in others) [120], a limited advantage in response rates has been so far described for Temsirolimus [121]. It is to be underlined that none of the trials with mTOR inhibitors included patients specifically selected for alterations in the AKT/PI3K pathway. Several clinical trials are investigating specific combinations of mTOR inhibitors and chemo‐

Prevalence of *GNAQ/GNA11* mutations, with increased expression level of phosphoERK.

The *GNAQ* and *GNA11* genes encodes specific GTP binding proteins that mediate signal transduction from the inner cell surface to the MAPK pathway through activation of the protein kinase C (PKC) enzyme [122]. Somatic mutations in *GNAQ* gene have been observed in about 90% of blue naevi, 50% of malignant blue naevi and 50% of primary uveal melano‐ ma; conversely, the *GNA11* mutations have been found in less than 10% of blue nevi, about one third of primary uveal melanomas, and about 60% of metastatic uveal melanoma [122]. Since mutations in these two genes have non been detected among all the remaining types of melanoma (cutaneous, acral, mucosal), a clinical trial aimed at testing efficacy of a MEK inhibitor, Dabrafenib/GSK1120212, has been focused on patients with metastatic uveal mela‐

The most prevalent molecular alterations within the heterogeneous patterns of biological features which characterize the distinct subtypes of melanoma are here summarized, accord‐ ing to the anatomical site of melanoma onset, the degree of exposure to the sun, and the his‐ Mutation ± amplification of *cKIT*; amplification of *CDK4* or *CCND1* (associated with in‐ creased expression levels of the related proteins); amplification of the 11q13, 22q11-13, and/or 5p15 genomic loci.

**•** Melanoma of the head and neck district and melanoma on skin with chronic sun-induced damage (CSD)

Amplification of *CDK4* and/or *CCND1*; increased expression levels of p53 protein; mutation ± amplification of *cKIT* (in about 5% of cases).

**•** Melanoma of the trunk and melanoma on skin without chronic sun-induced damage (non-CSD)

Mutation of *BRAF* or, alternatively, *NRAS* (with eventual coexistence of molecolar altera‐ tions which may ba associated with BRAF mutations; see above); tendency to reduced ex‐ pression of the p53 protein; occurrence of specific polymorphisms in *MC1R* gene.

**•** Mucosal melanoma

Mutation ± amplification of *cKIT*; amplification of *CDK4* or *CCND1* (associated with in‐ creased expression levels of the related proteins); mutation of *BRAF* (in less than 10% of cases).

**•** Uveal melanoma

Mutation ± amplification of *cKIT*; mutation of *GNAQ* and/or *GNA11*.

Knowledge of the principal molecular alterations to be tested in patients with such distinct subtypes of melanoma will be of great clinical importance, because it is likely to result in separate targeted therapeutic approaches and prevention strategies. To date, it has been al‐ ready developed a panel of molecular tests to be performed in patients with melanoma from different anatomical locations (Figure 3). This initial "*flow-chart*" will surely become more detailed and enriched on the basis of the progressive identification and validation of addi‐ tional genetics and molecular alterations correlated with the disease.

Finally, recent meta-analyses tried to define the prognostic role of majority of molecular al‐ terations previously described [124-125]:

**•** negative prognostic factors

loss of p53; over-expression of iNOS, AP-2, MMP-2 and metallothioneine; increased prolifer‐ ation index (high expression levels of Ki-67);

**•** positive prognostic factors

reduced expression or loss of p16CDKN2A; over-expression of Bcl-2 and ATF-2 (± associated with simultaneous increases expression of beta-catenin, fibronectin and p21 proteins).

resistance to BRAF inhibitors [131-134]. BRAF and MEK targeted synergic therapy is cur‐ rently tested in a phase I clinical trial (NCT01072175) which combines the selective RAF in‐ hibitor GSK2118436 with the MEK inhibitor GSK1120212 in patients with BRAF mutant

After decades without perspective, the history of medical treatment for the advanced mela‐ noma is rapidly changing. Combined therapeutic approaches do represent the next chal‐

Authors are always grateful to patients for their cooperation into the various studies. Work was funded by Regione Autonoma della Sardegna and Ricerca Finalizzata Ministero della

, Maria Cristina Sini1

1 Unit of Cancer Genetics, Institute of Biomolecular Chemistry (ICB), National Research

2 Unit of Medical Oncology and Innovative Therapy, Istituto Nazionale Tumori Fondazione

3 University Hospital Health Unit - Azienda Ospedaliero Universitaria (AOU), Via Matteot‐

[1] Curado MP, Edwards B, Shin HR, Storm H, Ferlay J, Heanue M, Boyle P (eds). Can‐ cer Incidence in Five Continents, Vol. IX, International Agency for Research on Can‐

[2] Linos E, Swetter S, Cockburn MG, et al. Increasing burden of melanoma in the Unit‐

[3] Rigel DS. Epidemiology of melanoma. Semin. Cutan. Med. Surg. 2010; 29: 204-209

, Paolo Antonio Ascierto2

Targeted Therapies in Melanoma: Successes and Pitfalls

http://dx.doi.org/10.5772/53624

47

,

tumors [135].

Salute.

**Acknowledgments**

**Author details**

Amelia Lissia3

ti, Sassari, Italy

**References**

Giuseppe Palmieri1\*, Maria Colombino1

and Antonio Cossu3

"G. Pascale", Via Mariano Semmola, Napoli, Italy

\*Address all correspondence to: gpalmieri@yahoo.com

Council (CNR), Traversa La Crucca, 3 - Baldinca Li Punti, Sassari, Italy

cer (IARC) Scientific Publications, No. 160, Lyon, IARC, 2007.

ed States. J Invest Derm. 2009;129: 1666-74

lenge for treatment of patients with such a disease.

**Figure 3. Principal genetic and molecular tests on tumor tissues for the different types of melanoma.** Amp, gene amplification detected by *fluorescence in situ hybridization* (FISH) analysis. Exp, protein expression level detected by immunohistochemistry. Mut, gene sequence variation detected by mutation analysis. In red, tests for less prevalent alterations.

## **5. Conclusion**

Taken together all the described molecular mechanisms involved in melanoma genesis and progression, data seem to emphasize the fact that in melanoma, but probably in all types of cancer, it is unlikely that targeting a single component in the signalling pathway will yield significant anti-tumour responses. For this purpose, molecular analyses could help clinicians to define the prognosis (prognostic value) as well as to make a prediction, identifying the subsets of patients who would be expected to be more or less likely to respond to specific therapeutic interventions (predictive value).

In other words, it is becoming evident that combination therapies targeting simultaneously several signaling pathways might be a winning therapeutic strategy to treat melanoma pa‐ tients. Preclinical studies using combination of anti BRAF and AKT3 siRNA demonstrated a significantly higher reduction of tumor growth compared to single agent administration [126-127]. There is also evidence of synergism among MEK and PI3K inhibitors as well as promising results have been obtained by combinations of the mTOR inhibitors and sorafe‐ nib or MEK inhibitors [121, 128-129]. In contrast to single agent activity, these combinations of target drugs resulted in recovering of apoptosis by complete down-regulation of the antiapoptotic proteins Bcl-2 and Mcl-1. Cooperation between BRAF and MEK inhibitors has also been demonstrated in preclinical studies with a consistent increase of apoptosis and abroga‐ tion of ERK activation compared to BRAF inhibitor alone [130]. Such cooperation was based on the observation that MEK activation was not abrogated in melanoma cells that develop resistance to BRAF inhibitors [131-134]. BRAF and MEK targeted synergic therapy is cur‐ rently tested in a phase I clinical trial (NCT01072175) which combines the selective RAF in‐ hibitor GSK2118436 with the MEK inhibitor GSK1120212 in patients with BRAF mutant tumors [135].

After decades without perspective, the history of medical treatment for the advanced mela‐ noma is rapidly changing. Combined therapeutic approaches do represent the next chal‐ lenge for treatment of patients with such a disease.

## **Acknowledgments**

Authors are always grateful to patients for their cooperation into the various studies. Work was funded by Regione Autonoma della Sardegna and Ricerca Finalizzata Ministero della Salute.

## **Author details**

**Figure 3. Principal genetic and molecular tests on tumor tissues for the different types of melanoma.** Amp, gene amplification detected by *fluorescence in situ hybridization* (FISH) analysis. Exp, protein expression level detected by immunohistochemistry. Mut, gene sequence variation detected by mutation analysis. In red, tests for less prevalent

Taken together all the described molecular mechanisms involved in melanoma genesis and progression, data seem to emphasize the fact that in melanoma, but probably in all types of cancer, it is unlikely that targeting a single component in the signalling pathway will yield significant anti-tumour responses. For this purpose, molecular analyses could help clinicians to define the prognosis (prognostic value) as well as to make a prediction, identifying the subsets of patients who would be expected to be more or less likely to respond to specific

In other words, it is becoming evident that combination therapies targeting simultaneously several signaling pathways might be a winning therapeutic strategy to treat melanoma pa‐ tients. Preclinical studies using combination of anti BRAF and AKT3 siRNA demonstrated a significantly higher reduction of tumor growth compared to single agent administration [126-127]. There is also evidence of synergism among MEK and PI3K inhibitors as well as promising results have been obtained by combinations of the mTOR inhibitors and sorafe‐ nib or MEK inhibitors [121, 128-129]. In contrast to single agent activity, these combinations of target drugs resulted in recovering of apoptosis by complete down-regulation of the antiapoptotic proteins Bcl-2 and Mcl-1. Cooperation between BRAF and MEK inhibitors has also been demonstrated in preclinical studies with a consistent increase of apoptosis and abroga‐ tion of ERK activation compared to BRAF inhibitor alone [130]. Such cooperation was based on the observation that MEK activation was not abrogated in melanoma cells that develop

alterations.

**5. Conclusion**

46 Melanoma - From Early Detection to Treatment

therapeutic interventions (predictive value).

Giuseppe Palmieri1\*, Maria Colombino1 , Maria Cristina Sini1 , Paolo Antonio Ascierto2 , Amelia Lissia3 and Antonio Cossu3

\*Address all correspondence to: gpalmieri@yahoo.com

1 Unit of Cancer Genetics, Institute of Biomolecular Chemistry (ICB), National Research Council (CNR), Traversa La Crucca, 3 - Baldinca Li Punti, Sassari, Italy

2 Unit of Medical Oncology and Innovative Therapy, Istituto Nazionale Tumori Fondazione "G. Pascale", Via Mariano Semmola, Napoli, Italy

3 University Hospital Health Unit - Azienda Ospedaliero Universitaria (AOU), Via Matteot‐ ti, Sassari, Italy

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JE, Wright JJ, Bassett RL, Hwu P, Kim KB. Phase I study of the combination of sorafe‐ nib and temsirolimus in patients with metastatic melanoma. Clin Cancer Res. 2012;18:1120-8

**Chapter 3**

**Low-Penetrance Variants and**

G. Ribas, M. Ibarrola-Villava, M.C. Peña-Chilet,

Additional information is available at the end of the chapter

L.P. Fernandez and C. Martinez-Cadenas

http://dx.doi.org/10.5772/53097

**1. Introduction**

susceptibility [14-17].

may be associated with cancer risk [25-29].

**Susceptibility to Sporadic Malignant Melanoma**

Although malignant melanoma (MM) is mainly a sporadic disease, about 3 to 15% of the cases may show familial aggregation [1, 2]. The diagnosis of melanoma in different members of the same families does indeed suggest there is a genetically-based hereditary predisposi‐ tion in a significant percentage of the cases. However, this predisposition has proven to be genetically heterogeneous. Only two high-penetrance genes had been described so far: *CDKN2A* and *CDK4* [1]. Yet mutations in these genes are found in only 30–40% of melano‐ ma kindreds, indicating the existence of additional genes involved in melanoma predisposi‐ tion [1]. Also, common low-penetrance alleles of the human pigmentation *MC1R* gene have been implicated in melanoma predisposition as well [3-13]. More recently, several other pig‐ mentation genes, such as *ASIP*, *TYR*, *TYRP1*, *SLC45A2* and *OCA2* have also emerged as be‐ ing potentially important in both normal human pigmentation variation and in melanoma

Other putative low-penetrance genes involved in melanoma predisposition are DNA repair genes belonging to the base excision repair (BER) and the nucleotide excision repair (NER) mechanisms. BER and NER pathways eliminate a wide variety of DNA damage, including ultraviolet (UV) photoproducts. Therefore, the ability of each individual to repair DNA damage following different causes might explain at least in part the variability in melanoma susceptibility. Although several studies have investigated the association between polymor‐ phisms in NER genes and risk of melanoma, most of the study sizes were relatively small, and the results were not consistent [18-24]. On the other hand, genetic polymorphisms have been identified in several BER genes and studies suggest that some of these polymorphisms

> © 2013 Ribas 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.


## **Low-Penetrance Variants and Susceptibility to Sporadic Malignant Melanoma**

G. Ribas, M. Ibarrola-Villava, M.C. Peña-Chilet, L.P. Fernandez and C. Martinez-Cadenas

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53097

## **1. Introduction**

JE, Wright JJ, Bassett RL, Hwu P, Kim KB. Phase I study of the combination of sorafe‐ nib and temsirolimus in patients with metastatic melanoma. Clin Cancer Res.

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58 Melanoma - From Early Detection to Treatment

464:427-30

2010;464:431-5

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BRAF(V600E). Nature 2011; 480: 387-90

Although malignant melanoma (MM) is mainly a sporadic disease, about 3 to 15% of the cases may show familial aggregation [1, 2]. The diagnosis of melanoma in different members of the same families does indeed suggest there is a genetically-based hereditary predisposi‐ tion in a significant percentage of the cases. However, this predisposition has proven to be genetically heterogeneous. Only two high-penetrance genes had been described so far: *CDKN2A* and *CDK4* [1]. Yet mutations in these genes are found in only 30–40% of melano‐ ma kindreds, indicating the existence of additional genes involved in melanoma predisposi‐ tion [1]. Also, common low-penetrance alleles of the human pigmentation *MC1R* gene have been implicated in melanoma predisposition as well [3-13]. More recently, several other pig‐ mentation genes, such as *ASIP*, *TYR*, *TYRP1*, *SLC45A2* and *OCA2* have also emerged as be‐ ing potentially important in both normal human pigmentation variation and in melanoma susceptibility [14-17].

Other putative low-penetrance genes involved in melanoma predisposition are DNA repair genes belonging to the base excision repair (BER) and the nucleotide excision repair (NER) mechanisms. BER and NER pathways eliminate a wide variety of DNA damage, including ultraviolet (UV) photoproducts. Therefore, the ability of each individual to repair DNA damage following different causes might explain at least in part the variability in melanoma susceptibility. Although several studies have investigated the association between polymor‐ phisms in NER genes and risk of melanoma, most of the study sizes were relatively small, and the results were not consistent [18-24]. On the other hand, genetic polymorphisms have been identified in several BER genes and studies suggest that some of these polymorphisms may be associated with cancer risk [25-29].

© 2013 Ribas 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.

Further candidate low-penetrance genes allegedly linked to melanoma predisposition are members of the glutathione S-transferase (*GST*) gene family. GSTs are multifunctional en‐ zymes involved in the detoxification of a wide range of reactive oxygen species (ROS) which, together with inflammatory response to UV exposure, contribute to skin carcinogen‐ esis by oxidative stress mechanisms [30-32]. Since UV radiation can also indirectly induce oxidative stress via ROS, several *GST* genes have been considered as possible low-pene‐ trance melanoma predisposition genes. Among the *GST* genes, *GSTM1*, *GSTT1* and *GSTP1* commonly harbor functional polymorphisms in the general population [33]. The frequencies of variants in the *GSTM1* and *GSTT1* genes have been studied in relation to susceptibility to melanoma; however, conflicting results have been reported [34-39].

used. DNA concentration was quantified in samples prior to genotyping by using Quant-iT

Low-Penetrance Variants and Susceptibility to Sporadic Malignant Melanoma

http://dx.doi.org/10.5772/53097

61

*MC1R* variants were detected by automated gene sequencing. The *MC1R* coding region was amplified by PCR using two overlapping pairs of primers previously described [9]. PCR products were 671 and 610 bp in length, respectively, and they overlapped by 104 bp. PCR amplification was performed according to Matichard and cols [9]. Sequence analysis was performed on the ABI Prism system (Life Technology, Foster city, CA) using the BigDye Terminator Cycle Sequencing kit and the ABI 3700 automated DNA sequencer according to the manufacturer's instructions. The sequence results were analyzed using Polyphred, Phred Phrap and Consed software [45-47] or SeqScape in order to detect all possible

The rest of the genes in the study were analyzed by genotyping selected SNPs. Public data‐ bases were used to collect information about single nucleotide polymorphisms (SNPs): NCBI (http://www.ncbi.nlm.nih.gov), Ensembl (http://www.ensembl.org/index.html) and HapMap (http://www.hapmap.org). SNPs selected were located in exons, in putative pro‐ moter regions or had been reported to be associated with cancer in previous studies. All SNPs had a minor allele frequency (MAF) greater than or equal to 5%. As a quality control measure we included two sample duplicates and a non-template sample per 96-well plate. For some high-throughput platforms three DNA duplicates (two intra-assays and one interassay) were added. For all the studies performed genotypes were scored by two different

The PCR primers and probes were designed by Life Technology (Foster City, CA) using their Custom Taqman SNP genotyping assays or KASPAR SNP Genotyping System KBio‐ sciences (Hoddesdon, Herts UK). The primer and allele-specific probe sequences for Taq‐ man as well as those used for Kaspar assays are detailed elsewhere [6, 7, 14, 16, 17, 27].

PCR conditions used were according to the manufacturer's protocol (Life technology, Foster City, CA). After PCR, the genotype of each sample was automatically determined by meas‐ uring allele-specific final fluorescence in the ABI Prism 7900HT Detection System, using the

Genotyping assays were designed according to the Sequenom MassARRAY Assay De‐ sign software (version 3.0.0; Sequenom Inc., San Diego, CA, USA). Assay primers are

SDS 2.1 software for allele discrimination (Life technology, Foster City, CA).

PicoGreen dsDNA Reagent (Invitrogen, Eugene, OR, USA).

changes. All detected changes were confirmed manually.

personnel in the laboratory and no discrepancies were observed.

**2.2. SNP genotyping**

*2.2.1. MC1R sequencing*

*2.2.2. Gene and SNP selection*

*2.2.3. Taqman and kaspar assays*

*2.2.4. Sequenom*

In recent years, several genome-wide association studies (GWAS) have identified novel ge‐ nomic *loci* associated with pigmentation and skin cancer [40-44]. GWAS are the ideal strat‐ egy to identify common, low-penetrance susceptibility *loci* without prior hypothesis bias due to gene role knowledge. Some of the associations detected were already known, such as *MC1R* with pigmentation and skin cancer, *ASIP*, *TYR*, *OCA2,* etc. However, several novel chromosomal regions and genes have been revealed using large cohorts of samples, such as *IRF4, PARP1, CASP8, CCND1* and others.

What follows is a summary of the results obtained in our laboratory after the screening of genes belonging to three different genetic pathways: pigmentation, DNA repair and oxida‐ tive stress. For the past few years, our group has been studying melanoma candidate genes in Spain, a southern European population, displaying a considerably darker skin than most of the other well-studied Caucasian populations, including Australian, North American and Northern Europe populations.

## **2. Research methods**

#### **2.1. Study subjects and data collection**

A case-control collection of 946 non-related MM cases from several Spanish Hospitals and 353 volunteer cancer-free controls were recruited from 1 September 2004 to January 2011. All participants were Caucasians of Spanish origin. A standardized questionnaire was used to collect information on pigmentation characteristics (eye, hair and skin color, number of nevi, presence of solar lentigines, sun exposure habits and presence of childhood sunburns), Fitzpatrick's classification of skin type (extracted from the medical record of cases only), tu‐ mor localization, Breslow index (depth index), and personal or family history of cancer. All study subjects gave informed consent and the study was approved by the Ethics Committee of Gregorio Marañón General University hospital and Clínico University Hospital.

Genomic DNA from cases and controls was isolated from peripheral blood lymphocytes and diluted to a final solution of 50ng/ml. MagNA Pure LC Instrument DNA extracción was used according to the manufacturer's protocol (Roche Applied Science, Mannheim, Germa‐ ny); the DNAzol procedure (Invitrogen, Eugene, OR, USA) or traditional saline method was used. DNA concentration was quantified in samples prior to genotyping by using Quant-iT PicoGreen dsDNA Reagent (Invitrogen, Eugene, OR, USA).

## **2.2. SNP genotyping**

Further candidate low-penetrance genes allegedly linked to melanoma predisposition are members of the glutathione S-transferase (*GST*) gene family. GSTs are multifunctional en‐ zymes involved in the detoxification of a wide range of reactive oxygen species (ROS) which, together with inflammatory response to UV exposure, contribute to skin carcinogen‐ esis by oxidative stress mechanisms [30-32]. Since UV radiation can also indirectly induce oxidative stress via ROS, several *GST* genes have been considered as possible low-pene‐ trance melanoma predisposition genes. Among the *GST* genes, *GSTM1*, *GSTT1* and *GSTP1* commonly harbor functional polymorphisms in the general population [33]. The frequencies of variants in the *GSTM1* and *GSTT1* genes have been studied in relation to susceptibility to

In recent years, several genome-wide association studies (GWAS) have identified novel ge‐ nomic *loci* associated with pigmentation and skin cancer [40-44]. GWAS are the ideal strat‐ egy to identify common, low-penetrance susceptibility *loci* without prior hypothesis bias due to gene role knowledge. Some of the associations detected were already known, such as *MC1R* with pigmentation and skin cancer, *ASIP*, *TYR*, *OCA2,* etc. However, several novel chromosomal regions and genes have been revealed using large cohorts of samples, such as

What follows is a summary of the results obtained in our laboratory after the screening of genes belonging to three different genetic pathways: pigmentation, DNA repair and oxida‐ tive stress. For the past few years, our group has been studying melanoma candidate genes in Spain, a southern European population, displaying a considerably darker skin than most of the other well-studied Caucasian populations, including Australian, North American and

A case-control collection of 946 non-related MM cases from several Spanish Hospitals and 353 volunteer cancer-free controls were recruited from 1 September 2004 to January 2011. All participants were Caucasians of Spanish origin. A standardized questionnaire was used to collect information on pigmentation characteristics (eye, hair and skin color, number of nevi, presence of solar lentigines, sun exposure habits and presence of childhood sunburns), Fitzpatrick's classification of skin type (extracted from the medical record of cases only), tu‐ mor localization, Breslow index (depth index), and personal or family history of cancer. All study subjects gave informed consent and the study was approved by the Ethics Committee

Genomic DNA from cases and controls was isolated from peripheral blood lymphocytes and diluted to a final solution of 50ng/ml. MagNA Pure LC Instrument DNA extracción was used according to the manufacturer's protocol (Roche Applied Science, Mannheim, Germa‐ ny); the DNAzol procedure (Invitrogen, Eugene, OR, USA) or traditional saline method was

of Gregorio Marañón General University hospital and Clínico University Hospital.

melanoma; however, conflicting results have been reported [34-39].

*IRF4, PARP1, CASP8, CCND1* and others.

60 Melanoma - From Early Detection to Treatment

Northern Europe populations.

**2.1. Study subjects and data collection**

**2. Research methods**

## *2.2.1. MC1R sequencing*

*MC1R* variants were detected by automated gene sequencing. The *MC1R* coding region was amplified by PCR using two overlapping pairs of primers previously described [9]. PCR products were 671 and 610 bp in length, respectively, and they overlapped by 104 bp. PCR amplification was performed according to Matichard and cols [9]. Sequence analysis was performed on the ABI Prism system (Life Technology, Foster city, CA) using the BigDye Terminator Cycle Sequencing kit and the ABI 3700 automated DNA sequencer according to the manufacturer's instructions. The sequence results were analyzed using Polyphred, Phred Phrap and Consed software [45-47] or SeqScape in order to detect all possible changes. All detected changes were confirmed manually.

### *2.2.2. Gene and SNP selection*

The rest of the genes in the study were analyzed by genotyping selected SNPs. Public data‐ bases were used to collect information about single nucleotide polymorphisms (SNPs): NCBI (http://www.ncbi.nlm.nih.gov), Ensembl (http://www.ensembl.org/index.html) and HapMap (http://www.hapmap.org). SNPs selected were located in exons, in putative pro‐ moter regions or had been reported to be associated with cancer in previous studies. All SNPs had a minor allele frequency (MAF) greater than or equal to 5%. As a quality control measure we included two sample duplicates and a non-template sample per 96-well plate. For some high-throughput platforms three DNA duplicates (two intra-assays and one interassay) were added. For all the studies performed genotypes were scored by two different personnel in the laboratory and no discrepancies were observed.

#### *2.2.3. Taqman and kaspar assays*

The PCR primers and probes were designed by Life Technology (Foster City, CA) using their Custom Taqman SNP genotyping assays or KASPAR SNP Genotyping System KBio‐ sciences (Hoddesdon, Herts UK). The primer and allele-specific probe sequences for Taq‐ man as well as those used for Kaspar assays are detailed elsewhere [6, 7, 14, 16, 17, 27].

PCR conditions used were according to the manufacturer's protocol (Life technology, Foster City, CA). After PCR, the genotype of each sample was automatically determined by meas‐ uring allele-specific final fluorescence in the ABI Prism 7900HT Detection System, using the SDS 2.1 software for allele discrimination (Life technology, Foster City, CA).

#### *2.2.4. Sequenom*

Genotyping assays were designed according to the Sequenom MassARRAY Assay De‐ sign software (version 3.0.0; Sequenom Inc., San Diego, CA, USA). Assay primers are detailed elsewhere [15, 27]. One duplicate sample, one father–mother–child trio and two negative controls were included across the plates to assess the accuracy of genotyping. SNPs were genotyped using iPLEXTM chemistry on a MALDI-TOF Mass Spectrometer (Sequenom Inc, San Diego, CA, USA). PCR reactions were carried out according to their own instructions (Sequenom Inc.).

**3. Results**

**3.1. MC1R**

identified.

Of the 946 individuals studied, 559 (59.15%) carried at least one *MC1R* variant, including 388 (65.43%) of 593 cases and 171 (48.4%) of the 353 controls. A total of 36 *MC1R* variants were

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63

Among these, 25 variants were non-synonymous changes, 20 of which had been described previously [5] and 5 were identified for the first time: S41F, M128T, P268R, A285V and N281S. Six variants of the receptor have been traditionally associated with red hair color

Similarly, another three variants have not been associated with RHC phenotype and have been designated as NRHC (V60L, V92M and R163Q). These amino acid changes have been

The other variants detected in the *MC1R* gene have frequencies lower than 1% in control samples and included C35Y, F45L, a trinucleotide deletion that results in a new amino acid in position 54 (c.161delTGG, V54E), S83P, G89R, V92L, T95M, a nonsense change (Y152X), two nucleotide insertions (c.537insC, p.ins179C; and c.537insT, p.ins179T), R213W, R272M, K278E, and T308M, plus all novel ones described before. These rare variants have been des‐ ignated as "ns\_rare SNPs" in Figure 1. The synonymous variants included the most com‐ mon change, T314T (A>G), and the rare changes I63I, Q233Q, I264I, F300F as well as I180I and S316S, the last two described for the first time. These synonymous variants are called "s\_rare" with or without T314T in Figure 1. The estimated frequency of common *MC1R* var‐ iants and some combinations such as all synonymous changes with and without the com‐ mon T314T and all non-synonymous rare variants, as well as the corresponding estimated

Among the 36 changes detected, five were individually associated with melanoma risk: V60L, R151C, I155T, R160W and D294H (P< 0.05). The highest OR was estimated for I155T (OR 3.65, 95% CI: 1.40–9.52; P=0.006). The estimated OR associated with carrying one nonsynonymous variant was 1.58 (95% CI: 1.19–2.097; P =0.0013); however, the OR for carrying two non-synonymous variants was 4.38 (95% CI: 2.72–7.05; P =1.33x 10-9). The MM associat‐ ed OR among those Spanish patients carrying one RHC variant was 2.36 (95% CI: 1.71–3.26; P =1.86 x10-7). However if we consider individuals homozygous or compound heterozygous

We considered blue ⁄green eye color, blond ⁄red hair color, solar lentigines and childhood sunburns as confounders in a multivariate model. *MC1R* variant analysis retained statistical‐ ly significant results when adjusted for all potential confounders (OR: 1.77, 95% CI: 1.37– 2.27; P =9.57x 10-6). Hair color, solar lentigines and childhood sunburns were independently associated with MM (OR: 2.11, 95% CI: 1.22–3.66, P=0.008; OR: 2.28 95% CI: 1.61–3.23,

for two RHC variants, the OR increased to 12.76 (95% CI: 3.06–53.29; P =1.9 x10-5).

P=3.54x10-6; OR: 4.77, 95% CI: 3.37–6.77, P =9.57x 10-6, respectively).

studied in different populations because their frequency is greater than 1%.

(RHC): D84E, R142H, R151C, R160W, I155T and D294H.

OR for MM and associated P-values are shown in Figure 1.

#### *2.2.5. Illumina*

A total of 384 SNPs were genotyped using the GoldenGate Genotyping Assay system ac‐ cording to the manufacturer's protocol (Illumina, San Diego, CA, USA) [16]. Genotyping was carried out using 350 ng of DNA per reaction. In addition, cases and control samples were always included in the same run. Genotypes were called using the proprietary soft‐ ware supplied by Illumina (BeadStudio, version 3.1.3.).

#### *2.2.6. Taqman quantitative real-time PCR*

The *GSTM1* and *GSTT1* copy number polymorphisms were determined using the TaqMan Quantitative real-time PCR. Assay designs were Hs02575461\_cn (*GSTM1)* and Hs000100004\_cn (*GSTT1)* and were used according to Life Technology instructions. After PCR, the genotype of each sample was automatically determined by measuring allele-specif‐ ic final fluorescence in the ABI Prism 7900HT Detection System, using the SDS 2.3 software for allele discrimination (Life Technology, Foster City, Ca, USA). Analysis of PCR products was done using the COPY CALLER Software v1.0 (Life Technology, Foster City, USA) that allowed the classification of unambiguous homozygous (zero copies), heterozygous (one copy) and homozygous (two copies) *GSTM1* or *GSTT1* carrier individuals.

#### **2.3. Statistical analysis**

Associations between *MC1R* variants and melanoma risk were initially assessed individual‐ ly using Fisher's exact test. Associations with melanoma were assessed using logistic regres‐ sion. Estimating odds ratios (ORs), their associated 95% confidence intervals (CIs) and Pvalues were obtained using SPSS v19. Multivariate logistic regression was also applied, including age, sex, hair color, skin color, solar lentigines and childhood sunburn as covari‐ ates. Associations between the number of variants carried and various individual and tumor characteristics were assessed via logistic regression.

To study the effect of combined protective and risk genotypes, we reduced the sample set to 528 samples successfully genotyped for all the associated SNPs. We used a 2x2 contingency table and a t-student test between *SLC45A2* (rs35414) and both *SILV* (rs2069398) and *NOS1* (rs2682826), as well as with all three genes together. In addition, we studied the results be‐ tween the risk alleles, *TYR* (rs17793678), *ADAMTS20* (rs1510521), *GSTP1* (rs1695) and *OCA2/ HERC2* (rs12913832). Finally, we analyzed possible interactions between *MC1R* (0, 1 or 2 variants) and all previous risk and protective alleles

## **3. Results**

detailed elsewhere [15, 27]. One duplicate sample, one father–mother–child trio and two negative controls were included across the plates to assess the accuracy of genotyping. SNPs were genotyped using iPLEXTM chemistry on a MALDI-TOF Mass Spectrometer (Sequenom Inc, San Diego, CA, USA). PCR reactions were carried out according to

A total of 384 SNPs were genotyped using the GoldenGate Genotyping Assay system ac‐ cording to the manufacturer's protocol (Illumina, San Diego, CA, USA) [16]. Genotyping was carried out using 350 ng of DNA per reaction. In addition, cases and control samples were always included in the same run. Genotypes were called using the proprietary soft‐

The *GSTM1* and *GSTT1* copy number polymorphisms were determined using the TaqMan Quantitative real-time PCR. Assay designs were Hs02575461\_cn (*GSTM1)* and Hs000100004\_cn (*GSTT1)* and were used according to Life Technology instructions. After PCR, the genotype of each sample was automatically determined by measuring allele-specif‐ ic final fluorescence in the ABI Prism 7900HT Detection System, using the SDS 2.3 software for allele discrimination (Life Technology, Foster City, Ca, USA). Analysis of PCR products was done using the COPY CALLER Software v1.0 (Life Technology, Foster City, USA) that allowed the classification of unambiguous homozygous (zero copies), heterozygous (one

Associations between *MC1R* variants and melanoma risk were initially assessed individual‐ ly using Fisher's exact test. Associations with melanoma were assessed using logistic regres‐ sion. Estimating odds ratios (ORs), their associated 95% confidence intervals (CIs) and Pvalues were obtained using SPSS v19. Multivariate logistic regression was also applied, including age, sex, hair color, skin color, solar lentigines and childhood sunburn as covari‐ ates. Associations between the number of variants carried and various individual and tumor

To study the effect of combined protective and risk genotypes, we reduced the sample set to 528 samples successfully genotyped for all the associated SNPs. We used a 2x2 contingency table and a t-student test between *SLC45A2* (rs35414) and both *SILV* (rs2069398) and *NOS1* (rs2682826), as well as with all three genes together. In addition, we studied the results be‐ tween the risk alleles, *TYR* (rs17793678), *ADAMTS20* (rs1510521), *GSTP1* (rs1695) and *OCA2/ HERC2* (rs12913832). Finally, we analyzed possible interactions between *MC1R* (0, 1 or 2

copy) and homozygous (two copies) *GSTM1* or *GSTT1* carrier individuals.

their own instructions (Sequenom Inc.).

62 Melanoma - From Early Detection to Treatment

*2.2.6. Taqman quantitative real-time PCR*

**2.3. Statistical analysis**

ware supplied by Illumina (BeadStudio, version 3.1.3.).

characteristics were assessed via logistic regression.

variants) and all previous risk and protective alleles

*2.2.5. Illumina*

## **3.1. MC1R**

Of the 946 individuals studied, 559 (59.15%) carried at least one *MC1R* variant, including 388 (65.43%) of 593 cases and 171 (48.4%) of the 353 controls. A total of 36 *MC1R* variants were identified.

Among these, 25 variants were non-synonymous changes, 20 of which had been described previously [5] and 5 were identified for the first time: S41F, M128T, P268R, A285V and N281S. Six variants of the receptor have been traditionally associated with red hair color (RHC): D84E, R142H, R151C, R160W, I155T and D294H.

Similarly, another three variants have not been associated with RHC phenotype and have been designated as NRHC (V60L, V92M and R163Q). These amino acid changes have been studied in different populations because their frequency is greater than 1%.

The other variants detected in the *MC1R* gene have frequencies lower than 1% in control samples and included C35Y, F45L, a trinucleotide deletion that results in a new amino acid in position 54 (c.161delTGG, V54E), S83P, G89R, V92L, T95M, a nonsense change (Y152X), two nucleotide insertions (c.537insC, p.ins179C; and c.537insT, p.ins179T), R213W, R272M, K278E, and T308M, plus all novel ones described before. These rare variants have been des‐ ignated as "ns\_rare SNPs" in Figure 1. The synonymous variants included the most com‐ mon change, T314T (A>G), and the rare changes I63I, Q233Q, I264I, F300F as well as I180I and S316S, the last two described for the first time. These synonymous variants are called "s\_rare" with or without T314T in Figure 1. The estimated frequency of common *MC1R* var‐ iants and some combinations such as all synonymous changes with and without the com‐ mon T314T and all non-synonymous rare variants, as well as the corresponding estimated OR for MM and associated P-values are shown in Figure 1.

Among the 36 changes detected, five were individually associated with melanoma risk: V60L, R151C, I155T, R160W and D294H (P< 0.05). The highest OR was estimated for I155T (OR 3.65, 95% CI: 1.40–9.52; P=0.006). The estimated OR associated with carrying one nonsynonymous variant was 1.58 (95% CI: 1.19–2.097; P =0.0013); however, the OR for carrying two non-synonymous variants was 4.38 (95% CI: 2.72–7.05; P =1.33x 10-9). The MM associat‐ ed OR among those Spanish patients carrying one RHC variant was 2.36 (95% CI: 1.71–3.26; P =1.86 x10-7). However if we consider individuals homozygous or compound heterozygous for two RHC variants, the OR increased to 12.76 (95% CI: 3.06–53.29; P =1.9 x10-5).

We considered blue ⁄green eye color, blond ⁄red hair color, solar lentigines and childhood sunburns as confounders in a multivariate model. *MC1R* variant analysis retained statistical‐ ly significant results when adjusted for all potential confounders (OR: 1.77, 95% CI: 1.37– 2.27; P =9.57x 10-6). Hair color, solar lentigines and childhood sunburns were independently associated with MM (OR: 2.11, 95% CI: 1.22–3.66, P=0.008; OR: 2.28 95% CI: 1.61–3.23, P=3.54x10-6; OR: 4.77, 95% CI: 3.37–6.77, P =9.57x 10-6, respectively).

Allele frequencies for each SNP and the P-value for their comparisons between case and con‐ trol subjects are detailed in Figure 2. After discarding two of the selected SNPs, one in the *OCA2* gene due to its monomorphic nature in our sample collection, and one in the *SLC45A2* gene due to its departure from HWE, we observed evidence of differences in allele frequency for one SNP in the *SLC45A2* gene, corresponding to F374L (NCBI dbSNP rs16891982). The estimated OR per minor allele copy was 0.41 (95% CI, 0.24–0.70; P =0.001) with the minor allele being more

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**Figure 2.** Odds ratios (OR), 95% confidence interval (95% CI), P-values and Minor Allele Frequency (MAF) for the best seven genetic variants in genes belonging to pigmentation, DNA repair and oxidative stress pathways. Grey bars rep‐ resent control data while black bars represent MM cases. Dark circles denote statistically significant values. Exact P-

In a second study we genotyped 384 SNPs from 65 genes belonging mainly to the pigmenta‐ tion pathway [16]. Ten SNPs located on six individual chromosomes (one in each of *ADAMTS20*, *TYR* and *SILV/CDK2*; two in each of *KIT* and *MYO7A*; and three in *SLC45A2*) constituted the top ten MM phase I associated SNPs in our sample after establishing a re‐

values are indicated in number.

frequent in controls than cases (16% vs. 7%; adjusted P=0.001).

**Figure 1.** a) Summary of all *MC1R* variants associated with MM. Odds Ratios (OR), 95% confidence interval (95% CI) and P-values. b) Minor Allele Frequencies (MAF) of *MC1R* genetic variants. ns: non-synonymous variants :V60L, V92M, S83P, D84E,V122M, R142H, R151C, I155T R160W, R163Q and D294H; s\_rare SNPs: synonymous variants with MAFs in controls lower than 1%: I63I, I180I, Q233Q, I264I, F300F and S316S; s\_rare +T314T: same variants as before plus the addition of T314T; ns\_rare SNPs: non-synonymous variants with less than 1% frequency in controls: C35Y;S41F, F45L, 54delTGG, G89R, V92L,T95M, M128T, Y152X, 179insTor C, R213W, P268R, T272M, K278E, N281S, A285V and T308M. Grey squares represent control data whereas black bars represent MM cases. Dark circles denote statistically signifi‐ cant values.

#### **3.2. Other genes from pathways associated to melanoma**

Several studies have been performed in order to evaluate other pigmentation-related genes and their relationship to MM susceptibility. The first results generated by Fernandez and cols. [4] analyzed the oculocutaneous albinism (OCA) genes: *TYR* (MIM#606933), *OCA2* (MIM#611409), *TYRP1* (MIM#115501) and *SLC45A2* (MIM#606202); the melanocyte protein *SILV* (MIM#155550) and *MC1R* inverse agonist *ASP* (MIM#600201).

Allele frequencies for each SNP and the P-value for their comparisons between case and con‐ trol subjects are detailed in Figure 2. After discarding two of the selected SNPs, one in the *OCA2* gene due to its monomorphic nature in our sample collection, and one in the *SLC45A2* gene due to its departure from HWE, we observed evidence of differences in allele frequency for one SNP in the *SLC45A2* gene, corresponding to F374L (NCBI dbSNP rs16891982). The estimated OR per minor allele copy was 0.41 (95% CI, 0.24–0.70; P =0.001) with the minor allele being more frequent in controls than cases (16% vs. 7%; adjusted P=0.001).

**Figure 1.** a) Summary of all *MC1R* variants associated with MM. Odds Ratios (OR), 95% confidence interval (95% CI) and P-values. b) Minor Allele Frequencies (MAF) of *MC1R* genetic variants. ns: non-synonymous variants :V60L, V92M, S83P, D84E,V122M, R142H, R151C, I155T R160W, R163Q and D294H; s\_rare SNPs: synonymous variants with MAFs in controls lower than 1%: I63I, I180I, Q233Q, I264I, F300F and S316S; s\_rare +T314T: same variants as before plus the addition of T314T; ns\_rare SNPs: non-synonymous variants with less than 1% frequency in controls: C35Y;S41F, F45L, 54delTGG, G89R, V92L,T95M, M128T, Y152X, 179insTor C, R213W, P268R, T272M, K278E, N281S, A285V and T308M. Grey squares represent control data whereas black bars represent MM cases. Dark circles denote statistically signifi‐

Several studies have been performed in order to evaluate other pigmentation-related genes and their relationship to MM susceptibility. The first results generated by Fernandez and cols. [4] analyzed the oculocutaneous albinism (OCA) genes: *TYR* (MIM#606933), *OCA2* (MIM#611409), *TYRP1* (MIM#115501) and *SLC45A2* (MIM#606202); the melanocyte protein

**3.2. Other genes from pathways associated to melanoma**

*SILV* (MIM#155550) and *MC1R* inverse agonist *ASP* (MIM#600201).

cant values.

64 Melanoma - From Early Detection to Treatment

**Figure 2.** Odds ratios (OR), 95% confidence interval (95% CI), P-values and Minor Allele Frequency (MAF) for the best seven genetic variants in genes belonging to pigmentation, DNA repair and oxidative stress pathways. Grey bars rep‐ resent control data while black bars represent MM cases. Dark circles denote statistically significant values. Exact Pvalues are indicated in number.

In a second study we genotyped 384 SNPs from 65 genes belonging mainly to the pigmenta‐ tion pathway [16]. Ten SNPs located on six individual chromosomes (one in each of *ADAMTS20*, *TYR* and *SILV/CDK2*; two in each of *KIT* and *MYO7A*; and three in *SLC45A2*) constituted the top ten MM phase I associated SNPs in our sample after establishing a re‐ strictive P-value threshold of 0.01. A phase II validation study was conducted to analyze the most significant SNP of each of the 6 candidate genes. One SNP, rs35414 in the *SLC45A2* gene, had an unadjusted P=0.002 in this phase II and overall OR of 0.75 (95% CI 0.67–0.84, P=0.0001) (Figure 2). None of the other five SNPs tested in phase II reached statistical signifi‐ cance at this stage. However, three of them, located in *TYR*, *SILV/CDK2* and *ADAMTS20* had an overall P<0.05 when phase I and II were considered together.

Evidence of association with phenotypic characteristics for two *KIT* SNPs, rs759083 and rs13135792, were also present. Both SNPs appeared to be associated with both light hair col‐

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Two different SNPs in *MYO7A* were associated with dark hair color (rs948970, P=0.04), and childhood sunburn (rs3758708, P=0.0474). One SNP in the *TYR* gene (rs17793678) was associ‐ ated with light eye color (P=0.0239). Likewise, the *ADAMTS20* gene was associated with light eye color (P=0.0339), blond or red hair color (P=0.0353) and with number of nevi (rs1510521; P=0.0338). Finally, *SILV/CDK2* SNP rs2069398 was associated with absence of

We explored the combined effects of the individually associated SNPs located in the six rele‐ vant genes studied: *SLC45A2*, *SILV*, *NOS1* (protective associated genes) and *TYR*, *ADAMTS20* and *GSTP1* (risk associated genes). Although in our series it does not show in‐ dividual association with MM, we added rs12913832 from the *OCA2/HERC2* gene due to its strong association with eye color [6, 48] and the fact that it has been demonstrated to have

Two SNPs, rs16891982 and rs35414, located in the *SLC45A2* gene were associated with MM. Both of them could have been used to perform the interaction analyses; however, rs35414 was chosen due to its higher MAF. SNPs rs2069398, located in the *SILV/CDK2* gene region, and rs2682826 in *NOS1* were also included in the analyses. Interaction results are shown in

We observed some degree of epistatic protective interaction between rs35414 (*SLC45A2* gene) and rs2069398 (*SILV/CDK2* gene region) when considering rare allele carriers at both *loci*. A significant decrease in MM associated OR was observed when two (heterozygotes for both *SLC45A2* and *SILV*) or three (heterozygous for *SLC45A2* and minor homozygotes for *SILV*) rare alleles were present at both *loci* (OR: 0.54, 95% CI: 0.39–0.75, P=0.0003). Similarly, when three (minor homozygotes for *SLC45A2* and heterozygous for *SILV*) or four (minor ho‐ mozygous for both *SLC45A2* and *SILV*) rare alleles were carried, a greater decrease in MM risk was observed (OR: 0.31, 95% CI: 0.18–0.55, P=0.0001). Results including joint genotypes for rs35414 and rs2069398 SNPs, individual status (heterozygous or minor homozygous),

In addition, we observed some degree of epistatic protective interaction between rs35414 (*SLC45A2*) and rs2682826 (*NOS1*) when considering heterozygote alleles at both *loci* (OR 0.23, 95% CI 0.16–0.55, P=0.0001). However, a trend toward protective effect was detected when both homozygous rare alleles were compared (OR: 0.50, 95% CI 0.18–1.20; P=0.1). This lack of statistically significant results is most probably due to the reduced number of sam‐

ORs with their corresponding 95% CIs and P-values are shown in Figure 3a.

ples in this category (21 MM cases vs. 17 controls). Results are shown in Figure 3b.

or (P=0.0021 and P=0.0072) and childhood sunburns (P=0.0112 and P=0.0167).

childhood sunburns (P=0.0353).

epistatic effects with the *MC1R* gene [49].

*3.4.1. Interaction between protective alleles*

**3.4. Gene-gene interactions**

Figure 3.

After analysis of genes in the pigmentation pathway, we conducted two studies where 16 genes belonging to both base excision repair (BER) and nucleotide excision repair (NER) pathways, as well as 14 genes involved in oxidative stress, including *GSTT1* and *GSTM1*, were screened for copy number variation [17, 27]. Two statistically significant re‐ sults suggested a putative role of oxidative stress processes in the genetic predisposition to melanoma.

First of all, a novel variant in the *NOS1* oxidative stress gene (rs2682826) was detected (P=0.01). A second association pointed to *GSTP1* polymorphism rs1695, encoding the amino acid change I105V, and individually associated with MM (OR: 1.32, 95%CI: 1.06–1.63, P=0.01) found associated with melanoma for the first time. The best seven SNPs associated with MM in our population are shown in Figure 2. We could not detect any association be‐ tween *GSTM1* or *GSTT1* deletions and MM risk.

#### **3.3. Phenotypic characteristics**

If we take into account *MC1R* and the associated phenotypic characteristics we detect the estimated ORs for melanoma associated with various phenotypic characteristics based on univariate analyses. MM risk was associated with the presence of blond or RHC (OR: 4.86, 95% CI: 2.35–10.03, P=2x10-5), solar lentigines (OR: 1.71, 95% CI: 1.04–2.81, P =0.032) and childhood sunburn (OR: 10.41, 95%CI: 5.81–18.65, P=3x10-13). No association with melanoma risk was observed for eye color, skin color or number of nevi.

The number of *MC1R* variants was statistically significantly associated with blond or RHC (OR: 1.80, 95% CI: 1.26–2.58, P=0.001), fair skin (OR: 1.42, 95% CI: 1.06–1.89, P=0.018) and with the presence of childhood sunburn (OR: 1.71, 95% CI: 1.28–2.27, P= 2x10-4). The corre‐ sponding ORs for the number of functional *MC1R* variants were 2.32 (95% CI: 1.42–3.78, P=0.001) for blond or RHC, 1.58 (95% CI: 1.09–2.3, P= 0.014) for fair skin and 2.35 (95% CI: 1.6–3.45, P=5 x10-5) for the presence of childhood sunburn.

We assessed whether *SLC45A2* polymorphisms were associated with various phenotypic characteristics. The F374L variant allele was associated with dark eye color, dark hair color, darker skin and absence of both solar lentigines and childhood sunburns. Finally, we tested for associations between *SLC45A2* SNPs and phototype, tumor location, and tumor depth among cases only (Table 5). The minor (G) allele of the F374L variant was found to be associ‐ ated with phototypes III/IV (per allele OR, 3.25; 95% CI, 1.05–10.03; P=0.04). Additionally, *SLC45A2* SNPs rs35414 and rs35415 were also associated with dark skin color (P=0.028 and P=0.0485) and only rs35414 with dark hair color (P=0.0183).

Evidence of association with phenotypic characteristics for two *KIT* SNPs, rs759083 and rs13135792, were also present. Both SNPs appeared to be associated with both light hair col‐ or (P=0.0021 and P=0.0072) and childhood sunburns (P=0.0112 and P=0.0167).

Two different SNPs in *MYO7A* were associated with dark hair color (rs948970, P=0.04), and childhood sunburn (rs3758708, P=0.0474). One SNP in the *TYR* gene (rs17793678) was associ‐ ated with light eye color (P=0.0239). Likewise, the *ADAMTS20* gene was associated with light eye color (P=0.0339), blond or red hair color (P=0.0353) and with number of nevi (rs1510521; P=0.0338). Finally, *SILV/CDK2* SNP rs2069398 was associated with absence of childhood sunburns (P=0.0353).

## **3.4. Gene-gene interactions**

strictive P-value threshold of 0.01. A phase II validation study was conducted to analyze the most significant SNP of each of the 6 candidate genes. One SNP, rs35414 in the *SLC45A2* gene, had an unadjusted P=0.002 in this phase II and overall OR of 0.75 (95% CI 0.67–0.84, P=0.0001) (Figure 2). None of the other five SNPs tested in phase II reached statistical signifi‐ cance at this stage. However, three of them, located in *TYR*, *SILV/CDK2* and *ADAMTS20*

After analysis of genes in the pigmentation pathway, we conducted two studies where 16 genes belonging to both base excision repair (BER) and nucleotide excision repair (NER) pathways, as well as 14 genes involved in oxidative stress, including *GSTT1* and *GSTM1*, were screened for copy number variation [17, 27]. Two statistically significant re‐ sults suggested a putative role of oxidative stress processes in the genetic predisposition

First of all, a novel variant in the *NOS1* oxidative stress gene (rs2682826) was detected (P=0.01). A second association pointed to *GSTP1* polymorphism rs1695, encoding the amino acid change I105V, and individually associated with MM (OR: 1.32, 95%CI: 1.06–1.63, P=0.01) found associated with melanoma for the first time. The best seven SNPs associated with MM in our population are shown in Figure 2. We could not detect any association be‐

If we take into account *MC1R* and the associated phenotypic characteristics we detect the estimated ORs for melanoma associated with various phenotypic characteristics based on univariate analyses. MM risk was associated with the presence of blond or RHC (OR: 4.86, 95% CI: 2.35–10.03, P=2x10-5), solar lentigines (OR: 1.71, 95% CI: 1.04–2.81, P =0.032) and childhood sunburn (OR: 10.41, 95%CI: 5.81–18.65, P=3x10-13). No association with melanoma

The number of *MC1R* variants was statistically significantly associated with blond or RHC (OR: 1.80, 95% CI: 1.26–2.58, P=0.001), fair skin (OR: 1.42, 95% CI: 1.06–1.89, P=0.018) and with the presence of childhood sunburn (OR: 1.71, 95% CI: 1.28–2.27, P= 2x10-4). The corre‐ sponding ORs for the number of functional *MC1R* variants were 2.32 (95% CI: 1.42–3.78, P=0.001) for blond or RHC, 1.58 (95% CI: 1.09–2.3, P= 0.014) for fair skin and 2.35 (95% CI:

We assessed whether *SLC45A2* polymorphisms were associated with various phenotypic characteristics. The F374L variant allele was associated with dark eye color, dark hair color, darker skin and absence of both solar lentigines and childhood sunburns. Finally, we tested for associations between *SLC45A2* SNPs and phototype, tumor location, and tumor depth among cases only (Table 5). The minor (G) allele of the F374L variant was found to be associ‐ ated with phototypes III/IV (per allele OR, 3.25; 95% CI, 1.05–10.03; P=0.04). Additionally, *SLC45A2* SNPs rs35414 and rs35415 were also associated with dark skin color (P=0.028 and

had an overall P<0.05 when phase I and II were considered together.

tween *GSTM1* or *GSTT1* deletions and MM risk.

risk was observed for eye color, skin color or number of nevi.

1.6–3.45, P=5 x10-5) for the presence of childhood sunburn.

P=0.0485) and only rs35414 with dark hair color (P=0.0183).

**3.3. Phenotypic characteristics**

66 Melanoma - From Early Detection to Treatment

to melanoma.

We explored the combined effects of the individually associated SNPs located in the six rele‐ vant genes studied: *SLC45A2*, *SILV*, *NOS1* (protective associated genes) and *TYR*, *ADAMTS20* and *GSTP1* (risk associated genes). Although in our series it does not show in‐ dividual association with MM, we added rs12913832 from the *OCA2/HERC2* gene due to its strong association with eye color [6, 48] and the fact that it has been demonstrated to have epistatic effects with the *MC1R* gene [49].

#### *3.4.1. Interaction between protective alleles*

Two SNPs, rs16891982 and rs35414, located in the *SLC45A2* gene were associated with MM. Both of them could have been used to perform the interaction analyses; however, rs35414 was chosen due to its higher MAF. SNPs rs2069398, located in the *SILV/CDK2* gene region, and rs2682826 in *NOS1* were also included in the analyses. Interaction results are shown in Figure 3.

We observed some degree of epistatic protective interaction between rs35414 (*SLC45A2* gene) and rs2069398 (*SILV/CDK2* gene region) when considering rare allele carriers at both *loci*. A significant decrease in MM associated OR was observed when two (heterozygotes for both *SLC45A2* and *SILV*) or three (heterozygous for *SLC45A2* and minor homozygotes for *SILV*) rare alleles were present at both *loci* (OR: 0.54, 95% CI: 0.39–0.75, P=0.0003). Similarly, when three (minor homozygotes for *SLC45A2* and heterozygous for *SILV*) or four (minor ho‐ mozygous for both *SLC45A2* and *SILV*) rare alleles were carried, a greater decrease in MM risk was observed (OR: 0.31, 95% CI: 0.18–0.55, P=0.0001). Results including joint genotypes for rs35414 and rs2069398 SNPs, individual status (heterozygous or minor homozygous), ORs with their corresponding 95% CIs and P-values are shown in Figure 3a.

In addition, we observed some degree of epistatic protective interaction between rs35414 (*SLC45A2*) and rs2682826 (*NOS1*) when considering heterozygote alleles at both *loci* (OR 0.23, 95% CI 0.16–0.55, P=0.0001). However, a trend toward protective effect was detected when both homozygous rare alleles were compared (OR: 0.50, 95% CI 0.18–1.20; P=0.1). This lack of statistically significant results is most probably due to the reduced number of sam‐ ples in this category (21 MM cases vs. 17 controls). Results are shown in Figure 3b.

The interaction analyses between *NOS1* rs2682826 SNP and *SILV* rs20693989 SNP revealed a trend toward significance when both rare alleles are considered (OR: 0.19, 95% CI: 0.03–1.07, P=0.055). Results are shown in Figure 3c.

tained an OR of 1.54 (95% CI 0.96–2.48) with a trend toward significance (P=0.088). Results

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**Figure 4.** Interactions between risk allele variants and their effect on MM susceptibility. a) *TYR* and *ADAMTS20*, b) *GSTP1* and *TYR*, c) *GSTP1* and *OCA2/HERC2* and d) *TYR* and *OCA2/HERC2*. OR: odds ratios per minor allele; 95% CI: 95% confidence intervals; REF: reference value; HET: heterozygotes; H MIN: minor allele homozygotes; and MIN: only minor homozygotes for *TYR* and *GSTP1* respectively. For genes labeled in vertical we joined heterozygous and minor

A similar effect was observed when we compared rs1695 (*GSTP1*) with both rs1510521 (*ADAMTS20*) and rs12913832 (*OCA2/HERC2*) polymorphisms. We detected some degree of epistatic risk interaction when considering homozygous rare alleles at both *loci*. Results for rs1695 and rs1510521 showed an OR of 2.53 (95% CI 1.005–6.49, P=0.04, data shown in Fig‐ ure 4b), while the interaction between rs1695 and rs12913832 had an OR of 2.81 (95% CI

An additional comparison with rs17793678 (*TYR*) and rs12913832 (*HERC2/OCA2)* showed increasing epistasis as the number of risk alleles augmented, showing a trend toward signifi‐ cance only when homozygous minor alleles were considered at both *loci*, OR=2.95 (95% CI 0.84–11.326] (p=0.077). See Figure 4d. All other complex comparisons did not add further in‐

For the second type of analyses we included the *MC1R locus* together with the previous four polymorphisms. As *MC1R* had already been associated with MM, it seemed biologically plausible that genetic interactions would be detected between risk variants within *MC1R*

homozygous status. Dark circles denote statistically significant results.

1.11–7.27, P=0.018, see Figure 4c).

formation.

are shown in Figure 4a.

**Figure 3.** Interaction between protective allele variants and their effect on MM susceptibility. a) *SLC45A*2 and *SILV*, b) *SLC45A2* and *NOS1*, c) *NOS1* and *SILV* and d) *SLC45A2*, *NOS1* and *SILV* all together. OR: odds ratios per minor allele; 95% CI: 95% confidence intervals; REF: reference value; HET: heterozygotes; and HMIN: minor homozygotes. For the genes labeled in vertical, we joined heterozygotes and minor homozygotes. Dark circles denote statistically significant results.

Finally, when the effect of the interaction between the three *loci* protectively associated with MM, rs35414 (*SLC45A2*), rs2682826 (*NOS1*) and rs2069398 (*SILV*) were analyzed*,* statistically significant results were observed. Individuals heterozygous for the three genes were more common in controls than in MM cases (OR: 0.09, 95% CI: 0.022–0.316, P=0.0001), showing a cumulative protective effect. The comparison with rare homozygous alleles in all three *loci* was statistically not relevant due to the small number of samples found (Figure 3d).

#### *3.4.2. Interaction between risk alleles*

In order to show the distinct combinations of MM risk alleles we performed two different analyses. The first comparison studied the effect in MM susceptibility by taking together four genotypes: rs17793678 (*TYR*), rs1510521 (*ADAMTS20]*; rs1695 (*GSTP1*), and rs12913832 (*OCA2/HERC2*). Results are shown in Figure 4a-d.

Some degree of epistatic risk interaction was seen between rs17793678 (*TYR*) and rs1510521 (*ADAMTS20*) when considering either heterozygous or rare alleles at both *loci*. We observed an increased risk effect when two or three rare alleles were present at both *loci* (rs17793678 heterozygotes and rs1510521 heterozygous, and minor homozygous carriers; OR: 1.41, 95% CI: 1.12–1.78, P=0.004). When three or four rare alleles (rs17793678 minor homozygotes and rs1510521 heterozygotes, and minor homozygous carriers) were analyzed together, we ob‐ tained an OR of 1.54 (95% CI 0.96–2.48) with a trend toward significance (P=0.088). Results are shown in Figure 4a.

The interaction analyses between *NOS1* rs2682826 SNP and *SILV* rs20693989 SNP revealed a trend toward significance when both rare alleles are considered (OR: 0.19, 95% CI: 0.03–1.07,

**Figure 3.** Interaction between protective allele variants and their effect on MM susceptibility. a) *SLC45A*2 and *SILV*, b) *SLC45A2* and *NOS1*, c) *NOS1* and *SILV* and d) *SLC45A2*, *NOS1* and *SILV* all together. OR: odds ratios per minor allele; 95% CI: 95% confidence intervals; REF: reference value; HET: heterozygotes; and HMIN: minor homozygotes. For the genes labeled in vertical, we joined heterozygotes and minor homozygotes. Dark circles denote statistically significant

Finally, when the effect of the interaction between the three *loci* protectively associated with MM, rs35414 (*SLC45A2*), rs2682826 (*NOS1*) and rs2069398 (*SILV*) were analyzed*,* statistically significant results were observed. Individuals heterozygous for the three genes were more common in controls than in MM cases (OR: 0.09, 95% CI: 0.022–0.316, P=0.0001), showing a cumulative protective effect. The comparison with rare homozygous alleles in all three *loci*

In order to show the distinct combinations of MM risk alleles we performed two different analyses. The first comparison studied the effect in MM susceptibility by taking together four genotypes: rs17793678 (*TYR*), rs1510521 (*ADAMTS20]*; rs1695 (*GSTP1*), and rs12913832

Some degree of epistatic risk interaction was seen between rs17793678 (*TYR*) and rs1510521 (*ADAMTS20*) when considering either heterozygous or rare alleles at both *loci*. We observed an increased risk effect when two or three rare alleles were present at both *loci* (rs17793678 heterozygotes and rs1510521 heterozygous, and minor homozygous carriers; OR: 1.41, 95% CI: 1.12–1.78, P=0.004). When three or four rare alleles (rs17793678 minor homozygotes and rs1510521 heterozygotes, and minor homozygous carriers) were analyzed together, we ob‐

was statistically not relevant due to the small number of samples found (Figure 3d).

P=0.055). Results are shown in Figure 3c.

68 Melanoma - From Early Detection to Treatment

results.

*3.4.2. Interaction between risk alleles*

(*OCA2/HERC2*). Results are shown in Figure 4a-d.

**Figure 4.** Interactions between risk allele variants and their effect on MM susceptibility. a) *TYR* and *ADAMTS20*, b) *GSTP1* and *TYR*, c) *GSTP1* and *OCA2/HERC2* and d) *TYR* and *OCA2/HERC2*. OR: odds ratios per minor allele; 95% CI: 95% confidence intervals; REF: reference value; HET: heterozygotes; H MIN: minor allele homozygotes; and MIN: only minor homozygotes for *TYR* and *GSTP1* respectively. For genes labeled in vertical we joined heterozygous and minor homozygous status. Dark circles denote statistically significant results.

A similar effect was observed when we compared rs1695 (*GSTP1*) with both rs1510521 (*ADAMTS20*) and rs12913832 (*OCA2/HERC2*) polymorphisms. We detected some degree of epistatic risk interaction when considering homozygous rare alleles at both *loci*. Results for rs1695 and rs1510521 showed an OR of 2.53 (95% CI 1.005–6.49, P=0.04, data shown in Fig‐ ure 4b), while the interaction between rs1695 and rs12913832 had an OR of 2.81 (95% CI 1.11–7.27, P=0.018, see Figure 4c).

An additional comparison with rs17793678 (*TYR*) and rs12913832 (*HERC2/OCA2)* showed increasing epistasis as the number of risk alleles augmented, showing a trend toward signifi‐ cance only when homozygous minor alleles were considered at both *loci*, OR=2.95 (95% CI 0.84–11.326] (p=0.077). See Figure 4d. All other complex comparisons did not add further in‐ formation.

For the second type of analyses we included the *MC1R locus* together with the previous four polymorphisms. As *MC1R* had already been associated with MM, it seemed biologically plausible that genetic interactions would be detected between risk variants within *MC1R* and other associated genes. Indeed, increased risks appeared when heterozygotes or rare al‐ leles at *MC1R* were combined with heterozygotes plus rare homozygous at *GSTP1* (OR: 5.3, 95% CI: 2.80–417.42; P =1x10-4, see Figure 5a), at *TYR* (OR: 6.42; 95% CI 2.32–18.64; P=0.0001, see Figure 5b), and at *OCA2/HERC2* (OR: 7.163; 95% CI 2.659–20.05; P=0.0001, see Figure 5c).

**Figure 5.** Interactions between risk allele variants on *MC1R* together with other risk associated genes alone or in mul‐ tiple combinations, and their effect on MM susceptibility. a) *MC1R* and *GSTP1*, b) *MC1R* and *TYR*, c) *MC1R* and *OCA2/ HERC2* and d) *MC1R*, *TYR* and *OCA2/HERC2,* e) *MC1R, GSTP1, TYR* and *OCA2/HERC2.* OR: odds ratios per minor allele; 95% CI: 95% confidence intervals; REF: reference value; HET: heterozygotes; H MIN: minor allele homozygotes. For the genes labeled in vertical we joined heterozygotes and minor homozygotes. Dark circles denote statistically significant results.

**Figure 6.** Interactions between risk variants on *MC1R* compared with protective alleles in a) *SLC45A2*, b) *SLC45A2*, *NOS1* and *SILV*, and their effect on MM susceptibility. OR: odds ratios per minor allele; 95% CI: 95% confidence inter‐ vals; WT: wild type alleles; *MC1R* 0: normal sequence in the *MC1R* gene; *MC1R* 1: carriers of 1 non-synonymous variant in *MC1R*; *MC1R* 2/+: carriers of two or more non-synonymous variants in the *MC1R* gene. For the genes labeled in vertical we have joined heterozygotes and minor homozygotes. Red circles represent carriers of risk alleles in *MC1R* whereas green circles correspond to carriers of risk alleles in *MC1R* and protective alleles either in *SLC45A2* alone or in

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The role of *SLC45A2* and the other protective genes (*NOS1* and *SILV*) in melanoma predis‐ position was further analyzed in relation to *MC1R*, the main low-penetrance gene associated to melanoma. Since all these genes have been studied by our group, we analyzed the inter‐ action effects between both *MC1R* and *SLC45A*2 *loci* (see Figure 6a) and between *MC1R* and

A great reduction of risk was detected when the rare protective alleles at *SLC45A2* were car‐ ried in individuals with two *MC1R* variants (OR: 2.20, 95% CI 1.14–4.23, P=0.02), in compari‐ son to individuals carrying only two *MC1R* variants (OR: 4.64; 95% CI 1.85–11.58, P=0.001). Individuals having only one *MC1R* mutation plus the rare protective *SLC45A2* allele also

combination.

*3.4.3. Complex interactions*

all the protective alleles (see Figure 6b).

The last group of comparisons was done taking into consideration three and four genes at the same time. Firstly, we showed that combining *MC1R* genotypes with both *TYR* and *GSTP1* resulted in the highest MM associated risk (OR: 11.56, 95% CI 2.25–79.54, P=0.0001; data shown in Figure 5d). Secondly, we performed a final analysis taking into account only rare homozygotes of the four risk alleles and compared them, for MM association, with wild type genotype individuals. We obtained an OR of 4.008 (95% CI 1.25–13.2) with a P-value of 0.016 (see Figure 5e). There is not enough power to consider any other comparison with stat‐ istical significance.

**Figure 6.** Interactions between risk variants on *MC1R* compared with protective alleles in a) *SLC45A2*, b) *SLC45A2*, *NOS1* and *SILV*, and their effect on MM susceptibility. OR: odds ratios per minor allele; 95% CI: 95% confidence inter‐ vals; WT: wild type alleles; *MC1R* 0: normal sequence in the *MC1R* gene; *MC1R* 1: carriers of 1 non-synonymous variant in *MC1R*; *MC1R* 2/+: carriers of two or more non-synonymous variants in the *MC1R* gene. For the genes labeled in vertical we have joined heterozygotes and minor homozygotes. Red circles represent carriers of risk alleles in *MC1R* whereas green circles correspond to carriers of risk alleles in *MC1R* and protective alleles either in *SLC45A2* alone or in combination.

#### *3.4.3. Complex interactions*

and other associated genes. Indeed, increased risks appeared when heterozygotes or rare al‐ leles at *MC1R* were combined with heterozygotes plus rare homozygous at *GSTP1* (OR: 5.3, 95% CI: 2.80–417.42; P =1x10-4, see Figure 5a), at *TYR* (OR: 6.42; 95% CI 2.32–18.64; P=0.0001, see Figure 5b), and at *OCA2/HERC2* (OR: 7.163; 95% CI 2.659–20.05; P=0.0001, see Figure 5c).

70 Melanoma - From Early Detection to Treatment

**Figure 5.** Interactions between risk allele variants on *MC1R* together with other risk associated genes alone or in mul‐ tiple combinations, and their effect on MM susceptibility. a) *MC1R* and *GSTP1*, b) *MC1R* and *TYR*, c) *MC1R* and *OCA2/ HERC2* and d) *MC1R*, *TYR* and *OCA2/HERC2,* e) *MC1R, GSTP1, TYR* and *OCA2/HERC2.* OR: odds ratios per minor allele; 95% CI: 95% confidence intervals; REF: reference value; HET: heterozygotes; H MIN: minor allele homozygotes. For the genes labeled in vertical we joined heterozygotes and minor homozygotes. Dark circles denote statistically significant

The last group of comparisons was done taking into consideration three and four genes at the same time. Firstly, we showed that combining *MC1R* genotypes with both *TYR* and *GSTP1* resulted in the highest MM associated risk (OR: 11.56, 95% CI 2.25–79.54, P=0.0001; data shown in Figure 5d). Secondly, we performed a final analysis taking into account only rare homozygotes of the four risk alleles and compared them, for MM association, with wild type genotype individuals. We obtained an OR of 4.008 (95% CI 1.25–13.2) with a P-value of 0.016 (see Figure 5e). There is not enough power to consider any other comparison with stat‐

results.

istical significance.

The role of *SLC45A2* and the other protective genes (*NOS1* and *SILV*) in melanoma predis‐ position was further analyzed in relation to *MC1R*, the main low-penetrance gene associated to melanoma. Since all these genes have been studied by our group, we analyzed the inter‐ action effects between both *MC1R* and *SLC45A*2 *loci* (see Figure 6a) and between *MC1R* and all the protective alleles (see Figure 6b).

A great reduction of risk was detected when the rare protective alleles at *SLC45A2* were car‐ ried in individuals with two *MC1R* variants (OR: 2.20, 95% CI 1.14–4.23, P=0.02), in compari‐ son to individuals carrying only two *MC1R* variants (OR: 4.64; 95% CI 1.85–11.58, P=0.001). Individuals having only one *MC1R* mutation plus the rare protective *SLC45A2* allele also showed a reduction in MM risk, although this decline does not seem to reach significant val‐ ues (data shown in Figure 6a). These results confirmed the protective role of the rs35414 var‐ iant in *SLC45A2* regarding MM risk. Similar effects are observed when we included in the calculations the protective SNPs located in *NOS1* and *SILV*, however less statistically signifi‐ cant results were obtained due to the small number of samples in each class.

ing that risk increased with the number of non-synonymous changes carried, regardless of whether they were RHC or NRHC. The presence of two non-synonymous changes implies that both copies of the MC1R protein are compromised. In addition, the presence of two NRHC increases by more than five times the risk of only one non-synonymous variant (P=1.9x10-5). All these results taken together strongly support the role of the *MC1R* gene as highly linked to the susceptibility of developing MM in Mediterranean countries such as

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In recent years, *SLC45A2* has joined the group of genes (including *MC1R*, *OCA2*, and *ASP*) identified as being related to pigmentation, and it is now considered integral to pig‐ mentation variation. Mutations in the *SLC45A2* or *MATP* gene (MIM #606202), which enc‐ odes the membrane-associated transporter, have recently been associated with the *OCA4* albinism subtype [57]. The non-synonymous variant F374L (rs16891982) has been reported to have a strong association with dark hair, skin and eye color in Europeans [58]. These phenotypic correlations were replicated in our analysis on the Spanish population and we also established for the first time its role in MM susceptibility [14]. We also found that other polymorphisms on *SLC45A2* other than rs16891982 were also associated with dark phenotypic characteristics [16], confirming the role of *SLC45A2* in pigmentation. A paral‐ lel work by Guedj and cols. [59] also detected association of this variant with melanoma in a French population, supporting our previous results. It has been proposed that the F374 allele causes a reduction of protein function that alters the intracellular trafficking of melanosomal proteins, creating an environment for decreased melanin production [58]. More than 90% of European genes carry the F374 allele, which is rare or absent in Afri‐ cans. The remaining 10% of people of European descent with the Leucine ancestral allele (the most common in Africans) appear to have significantly more pigmented skin. There is a clear evidence of selective sweep of the chromosomal segment around the *SLC45A2* gene in the European population, which is consistent with our data [60]. The derived L374 allele shows unusually large allele frequency differences between Europeans and other populations, and has reduced haplotype diversity. These patterns are consistent with the

The *NOS1* gene is located on chromosome 12q24.2 and consists of 29 exons, encompassing more than 160kb of genomic DNA [61] and it is the main NO synthesizing enzyme in the central nervous system [62, 63]. The rs2682826 SNP is located in the 3' UTR of exon 29 of the *NOS1* gene and was selected as the tag SNP of one of the most frequent haplotypes. Howev‐ er, rs2682826 seems to be the most likely functional SNP due to its location close to several miRNAs binding sites in 3'UTR region. Possibly, differences in protein translation might be elicited depending on the allele present in the mRNA of this gene. No other regulatory ele‐ ment close to this region seems to modulate this gene. We propose this SNP as a novel var‐

Despite not being able to detect association between MM and the absence of either *GSTM1* or *GSTT1* copies, minor homozygotes for rs1695 in the *GSTP1* gene appeared to be strongly associated with MM. The latter SNP encodes an amino acid change, I105V, that was descri‐ bed for the first time associated with MM (P =0.01) in our population. This finding is consis‐

action of recent natural selection on these genes in Europeans.

iant related to melanoma (P=0.01).

Spain [3, 5, 10, 52].

## **4. Discussion**

Since *MC1R* genetic variability is strongly associated with the RHC phenotype [12], a large number of studies have investigated the involvement of this gene in MM susceptibility. *MC1R* is highly polymorphic, with more than 100 variants described in Caucasian popula‐ tions [13]. Despite of its high variability, the synonymous changes are greatly reduced, with only three described in the literature: T314T (A>G), F300F (C>T) and C273C (C>T) [50, 51]. Results obtained from our laboratory confirmed the association between five *MC1R* poly‐ morphic variants and MM risk in the Spanish population. We found 36 *MC1R* variants, this number being quite similar to the number found in other Mediterranean population studies (16 in France, 26–29 in Italy and 18 in Greece) [9, 11]. The most frequent Spanish variant is V60L with a frequency of 12.03%. This value is close to that reported in other populations (15.7% among Northern Italians, 12.4% among fair-skinned Australians and 15.0% among Northern Europeans) [52]. The RHC phenotype-associated variants (R151C, R160W and D294H) were present at frequencies of 0,71, 0.71 and 1.42%, respectively, in our population, compared to 9.9, 8.7 and 3.6%, respectively, reported in Northern European populations [52]. There were very few red-haired individuals among the control sample, and only 36 (6%) of MM cases had red hair. This finding is consistent with other results from Mediterra‐ nean populations and is at odds with red hair frequencies found in Northern European pop‐ ulations [12]. Red-haired subjects with no *MC1R* variants are not uncommon and have been seen in a Northern European population as well [53].

RHC variants have been consistently associated with MM in Northern European popula‐ tions [3, 10, 12] and also in the Northern French population [9]. In Spain, we detected statis‐ tically significant individual associations for R151C, R160W and D294H. These three variants have been detected in the Northern French and Central Italian populations [9, 54]. We did not observe any MM risk associated with the rare RHC variant D84E (OR: 1.63, 95% CI: 0.02–128, P= 0.99), as detected in Northern Europeans [32, 55, 56], probably due to its low prevalence in Spain (0.28% in controls). The I155T variant has not been associated with MM in other populations to date, but this may also be due to its low frequency. However, our results clearly suggest that this rare variant increases risk of MM, at least in the Spanish pop‐ ulation (OR: 3.51, 95% CI: 1.35–9.12, P= 0.006). While the associations of RHC with MM were expected, the case of V60L (an NRHC variant) was more intriguing, since its involvement in MM pathology has been generally unclear in Caucasian populations. However, V60L could play a role in MM susceptibility only in darker skinned populations since it has been found associated with MM in other Mediterranean populations such as France and Greece [9, 11]. The fact that NRHC variants could be important in MM risk is also supported by our find‐ ing that risk increased with the number of non-synonymous changes carried, regardless of whether they were RHC or NRHC. The presence of two non-synonymous changes implies that both copies of the MC1R protein are compromised. In addition, the presence of two NRHC increases by more than five times the risk of only one non-synonymous variant (P=1.9x10-5). All these results taken together strongly support the role of the *MC1R* gene as highly linked to the susceptibility of developing MM in Mediterranean countries such as Spain [3, 5, 10, 52].

showed a reduction in MM risk, although this decline does not seem to reach significant val‐ ues (data shown in Figure 6a). These results confirmed the protective role of the rs35414 var‐ iant in *SLC45A2* regarding MM risk. Similar effects are observed when we included in the calculations the protective SNPs located in *NOS1* and *SILV*, however less statistically signifi‐

Since *MC1R* genetic variability is strongly associated with the RHC phenotype [12], a large number of studies have investigated the involvement of this gene in MM susceptibility. *MC1R* is highly polymorphic, with more than 100 variants described in Caucasian popula‐ tions [13]. Despite of its high variability, the synonymous changes are greatly reduced, with only three described in the literature: T314T (A>G), F300F (C>T) and C273C (C>T) [50, 51]. Results obtained from our laboratory confirmed the association between five *MC1R* poly‐ morphic variants and MM risk in the Spanish population. We found 36 *MC1R* variants, this number being quite similar to the number found in other Mediterranean population studies (16 in France, 26–29 in Italy and 18 in Greece) [9, 11]. The most frequent Spanish variant is V60L with a frequency of 12.03%. This value is close to that reported in other populations (15.7% among Northern Italians, 12.4% among fair-skinned Australians and 15.0% among Northern Europeans) [52]. The RHC phenotype-associated variants (R151C, R160W and D294H) were present at frequencies of 0,71, 0.71 and 1.42%, respectively, in our population, compared to 9.9, 8.7 and 3.6%, respectively, reported in Northern European populations [52]. There were very few red-haired individuals among the control sample, and only 36 (6%) of MM cases had red hair. This finding is consistent with other results from Mediterra‐ nean populations and is at odds with red hair frequencies found in Northern European pop‐ ulations [12]. Red-haired subjects with no *MC1R* variants are not uncommon and have been

RHC variants have been consistently associated with MM in Northern European popula‐ tions [3, 10, 12] and also in the Northern French population [9]. In Spain, we detected statis‐ tically significant individual associations for R151C, R160W and D294H. These three variants have been detected in the Northern French and Central Italian populations [9, 54]. We did not observe any MM risk associated with the rare RHC variant D84E (OR: 1.63, 95% CI: 0.02–128, P= 0.99), as detected in Northern Europeans [32, 55, 56], probably due to its low prevalence in Spain (0.28% in controls). The I155T variant has not been associated with MM in other populations to date, but this may also be due to its low frequency. However, our results clearly suggest that this rare variant increases risk of MM, at least in the Spanish pop‐ ulation (OR: 3.51, 95% CI: 1.35–9.12, P= 0.006). While the associations of RHC with MM were expected, the case of V60L (an NRHC variant) was more intriguing, since its involvement in MM pathology has been generally unclear in Caucasian populations. However, V60L could play a role in MM susceptibility only in darker skinned populations since it has been found associated with MM in other Mediterranean populations such as France and Greece [9, 11]. The fact that NRHC variants could be important in MM risk is also supported by our find‐

cant results were obtained due to the small number of samples in each class.

seen in a Northern European population as well [53].

**4. Discussion**

72 Melanoma - From Early Detection to Treatment

In recent years, *SLC45A2* has joined the group of genes (including *MC1R*, *OCA2*, and *ASP*) identified as being related to pigmentation, and it is now considered integral to pig‐ mentation variation. Mutations in the *SLC45A2* or *MATP* gene (MIM #606202), which enc‐ odes the membrane-associated transporter, have recently been associated with the *OCA4* albinism subtype [57]. The non-synonymous variant F374L (rs16891982) has been reported to have a strong association with dark hair, skin and eye color in Europeans [58]. These phenotypic correlations were replicated in our analysis on the Spanish population and we also established for the first time its role in MM susceptibility [14]. We also found that other polymorphisms on *SLC45A2* other than rs16891982 were also associated with dark phenotypic characteristics [16], confirming the role of *SLC45A2* in pigmentation. A paral‐ lel work by Guedj and cols. [59] also detected association of this variant with melanoma in a French population, supporting our previous results. It has been proposed that the F374 allele causes a reduction of protein function that alters the intracellular trafficking of melanosomal proteins, creating an environment for decreased melanin production [58]. More than 90% of European genes carry the F374 allele, which is rare or absent in Afri‐ cans. The remaining 10% of people of European descent with the Leucine ancestral allele (the most common in Africans) appear to have significantly more pigmented skin. There is a clear evidence of selective sweep of the chromosomal segment around the *SLC45A2* gene in the European population, which is consistent with our data [60]. The derived L374 allele shows unusually large allele frequency differences between Europeans and other populations, and has reduced haplotype diversity. These patterns are consistent with the action of recent natural selection on these genes in Europeans.

The *NOS1* gene is located on chromosome 12q24.2 and consists of 29 exons, encompassing more than 160kb of genomic DNA [61] and it is the main NO synthesizing enzyme in the central nervous system [62, 63]. The rs2682826 SNP is located in the 3' UTR of exon 29 of the *NOS1* gene and was selected as the tag SNP of one of the most frequent haplotypes. Howev‐ er, rs2682826 seems to be the most likely functional SNP due to its location close to several miRNAs binding sites in 3'UTR region. Possibly, differences in protein translation might be elicited depending on the allele present in the mRNA of this gene. No other regulatory ele‐ ment close to this region seems to modulate this gene. We propose this SNP as a novel var‐ iant related to melanoma (P=0.01).

Despite not being able to detect association between MM and the absence of either *GSTM1* or *GSTT1* copies, minor homozygotes for rs1695 in the *GSTP1* gene appeared to be strongly associated with MM. The latter SNP encodes an amino acid change, I105V, that was descri‐ bed for the first time associated with MM (P =0.01) in our population. This finding is consis‐ tent with the hypothesis that patients with the *GSTP1* V105 variant enzyme have a reduced ability to detoxify compounds, which results in lower clearance and reduced efficacy. The *GSTP1* V105 variant is associated with a lower thermal stability and altered catalytic activity to a variety of substrates compared with those for *GSTP1* I105 [34].

**Acknowledgements**

Medicine at the University of Valencia.

, M. Ibarrola-Villava1

Genet, 68: 884-94, 2001

**Author details**

G. Ribas1

**References**

All these works were supported by several grants from the Ministerio de Educación y Cien‐ cia (MEC) (SAF2007-65542-C02-01), Fundación Mutua Madrileña (FMMA 2009), and Minis‐ terio Salud Carlos III (CP08/00069 and FI10\_0405). LPF was funded by the Ministerio de Ciencia y Tecnología (MCT) and a grant from the Fondo de Investigación Sanitaria (FIS) FI05/00918; MI-V and MP-Ch were funded by the Spanish Ministerio de Educación y Cien‐ cia under a grant FPI (BES-2008-009234) and by Generalitat Valenciana ValI+D under a grant (ACIF[2011/207) respectively. GR is funded by Ministerio de Salud Carlos III (MS08/00069). We thank the staff at the Spanish National Genotyping Centre in Santiago de Compostela and Madrid for their expert technical support with Sequenom and Illumina. Quantitative re‐ al-time PCR and Taqman was performed at the Unidad Central de Investigacion Medica (UCIM) of the Faculty of Medicine at the University of Valencia. Sequencing was done at the Sequencing Unit at CNIO and the Diagnostic and Genotyping Unit at UCIM, Faculty of

Low-Penetrance Variants and Susceptibility to Sporadic Malignant Melanoma

, M.C. Peña-Chilet1

1 Health Research Institute-INCLIVA, Av. Blasco Ibañez, Valencia, Spain

2 IIB Instituto de Investigaciones Biomédicas "Alberto Sols", Madrid, Spain

3 Department of Medicine, University of Castellon Jaume I, Castellon, Spain

[1] Bataille, V. Genetic epidemiology of melanoma. Eur J Cancer, 39: 1341-7, 2003

[2] Ferlay, J., Autier, P., Boniol, M., Heanue, M., Colombet, M., and Boyle, P. Estimates of the cancer incidence and mortality in Europe in 2006. Ann Oncol, 18: 581-92, 2007.

[3] Bastiaens, M. T., ter Huurne, J. A., Kielich, C., Gruis, N. A., Westendorp, R. G., Ver‐ meer, B. J., and Bavinck, J. N. Melanocortin-1 receptor gene variants determine the risk of nonmelanoma skin cancer independently of fair skin and red hair. Am J Hum

[4] Fernandez, L., Milne, R., Bravo, J., Lopez, J., Aviles, J., Longo, M., Benitez, J., Lazaro, P., and Ribas, G. MC1R: three novel variants identified in a malignant melanoma as‐

sociation study in the Spanish population. Carcinogenesis, 28: 1659-64, 2007

, L.P. Fernandez2

and C. Martinez-Cadenas3

http://dx.doi.org/10.5772/53097

75

It seemed biologically plausible that genetic interactions would be detected between sever‐ al of the SNPs identified in our studies, and for that reason we grouped protective alleles located in the genes *SLC45A2, SILV/CDK2* and *NOS1*. On one hand, a great reduction of risk was detected when rare alleles at these *loci* were combined. The strongest protective combination was between *SLC45A2* and *SILV/CDK2*, followed by the combination of het‐ erozygote samples at *SLC45A2* and *NOS1*. Other protective effects were seen, although they did not reach statistical significance due to the low number of samples in the ana‐ lyzed categories.

In order to determine possible genetic interactions between *MC1R* and the rest of the genes studied, we compared individuals carrying risk variants in *TYR*, *ADAMTS20* or *OCA2/ HERC2* with the *MC1R locus*. Indeed, increasing risk effects were observed in all compari‐ sons when as more risk alleles accumulated in the same individuals. However, when *MC1R* was taken into consideration, the epistatic effect was far stronger, increasing from an OR of 4.38 when combined with *GSTP1* up to an OR of 11.56 when *MC1R*, *TYR* and *GSTP1* hetero‐ zygotes and homozygotes are considered together, and achieving risk effect values similar to having two *MC1R* RHC alleles. It is worth mentioning here that some genetic interactions, for instance between *MC1R* and *OCA2,* have already been described among pigmentation genes [49, 64].

Finally, we tried to see whether protective alleles were going to be able to reduce the risk induced by the accumulation of risk alleles. Some comparisons were not possible due to the absence of individuals in some of the categories. Therefore, we present only the results be‐ tween *MC1R* risk alleles and the more robust protective gene, *SLC45A2*, alone or in combi‐ nation with *NOS1* and *SILV*. We could indeed observe a great reduction in risk in individuals carrying two risk alleles in *MC1R* when simultaneously carrying protective al‐ leles in the other genes. These results are important because the overall risk for an individu‐ al does not rely in only one gene but in the interaction of all his/hers genetic background and this should be considered in the future.

In summary, we found that five *MC1R* variants (V60L, R151C, I155T, R160W and D294H) are individually associated with MM risk in the Spanish population. Carrying two non-syn‐ onymous *MC1R* variants was associated with even higher risk, more than doubling the risk of carrying a single variant and having a five time higher risk than when carrying an NRHC variant. We described for the first time an association with the F374L variant, located on the *SLC45A2* gene, which appears to be a novel protective low-penetrance melanoma suscepti‐ bility gene. We therefore propose an integral study when trying to assert the MM risk of an individual, because the combination of rare alleles at several *loci* modulates the final risk/ protective value that predisposes him/her to MM.

## **Acknowledgements**

tent with the hypothesis that patients with the *GSTP1* V105 variant enzyme have a reduced ability to detoxify compounds, which results in lower clearance and reduced efficacy. The *GSTP1* V105 variant is associated with a lower thermal stability and altered catalytic activity

It seemed biologically plausible that genetic interactions would be detected between sever‐ al of the SNPs identified in our studies, and for that reason we grouped protective alleles located in the genes *SLC45A2, SILV/CDK2* and *NOS1*. On one hand, a great reduction of risk was detected when rare alleles at these *loci* were combined. The strongest protective combination was between *SLC45A2* and *SILV/CDK2*, followed by the combination of het‐ erozygote samples at *SLC45A2* and *NOS1*. Other protective effects were seen, although they did not reach statistical significance due to the low number of samples in the ana‐

In order to determine possible genetic interactions between *MC1R* and the rest of the genes studied, we compared individuals carrying risk variants in *TYR*, *ADAMTS20* or *OCA2/ HERC2* with the *MC1R locus*. Indeed, increasing risk effects were observed in all compari‐ sons when as more risk alleles accumulated in the same individuals. However, when *MC1R* was taken into consideration, the epistatic effect was far stronger, increasing from an OR of 4.38 when combined with *GSTP1* up to an OR of 11.56 when *MC1R*, *TYR* and *GSTP1* hetero‐ zygotes and homozygotes are considered together, and achieving risk effect values similar to having two *MC1R* RHC alleles. It is worth mentioning here that some genetic interactions, for instance between *MC1R* and *OCA2,* have already been described among pigmentation

Finally, we tried to see whether protective alleles were going to be able to reduce the risk induced by the accumulation of risk alleles. Some comparisons were not possible due to the absence of individuals in some of the categories. Therefore, we present only the results be‐ tween *MC1R* risk alleles and the more robust protective gene, *SLC45A2*, alone or in combi‐ nation with *NOS1* and *SILV*. We could indeed observe a great reduction in risk in individuals carrying two risk alleles in *MC1R* when simultaneously carrying protective al‐ leles in the other genes. These results are important because the overall risk for an individu‐ al does not rely in only one gene but in the interaction of all his/hers genetic background

In summary, we found that five *MC1R* variants (V60L, R151C, I155T, R160W and D294H) are individually associated with MM risk in the Spanish population. Carrying two non-syn‐ onymous *MC1R* variants was associated with even higher risk, more than doubling the risk of carrying a single variant and having a five time higher risk than when carrying an NRHC variant. We described for the first time an association with the F374L variant, located on the *SLC45A2* gene, which appears to be a novel protective low-penetrance melanoma suscepti‐ bility gene. We therefore propose an integral study when trying to assert the MM risk of an individual, because the combination of rare alleles at several *loci* modulates the final risk/

to a variety of substrates compared with those for *GSTP1* I105 [34].

lyzed categories.

74 Melanoma - From Early Detection to Treatment

genes [49, 64].

and this should be considered in the future.

protective value that predisposes him/her to MM.

All these works were supported by several grants from the Ministerio de Educación y Cien‐ cia (MEC) (SAF2007-65542-C02-01), Fundación Mutua Madrileña (FMMA 2009), and Minis‐ terio Salud Carlos III (CP08/00069 and FI10\_0405). LPF was funded by the Ministerio de Ciencia y Tecnología (MCT) and a grant from the Fondo de Investigación Sanitaria (FIS) FI05/00918; MI-V and MP-Ch were funded by the Spanish Ministerio de Educación y Cien‐ cia under a grant FPI (BES-2008-009234) and by Generalitat Valenciana ValI+D under a grant (ACIF[2011/207) respectively. GR is funded by Ministerio de Salud Carlos III (MS08/00069). We thank the staff at the Spanish National Genotyping Centre in Santiago de Compostela and Madrid for their expert technical support with Sequenom and Illumina. Quantitative re‐ al-time PCR and Taqman was performed at the Unidad Central de Investigacion Medica (UCIM) of the Faculty of Medicine at the University of Valencia. Sequencing was done at the Sequencing Unit at CNIO and the Diagnostic and Genotyping Unit at UCIM, Faculty of Medicine at the University of Valencia.

## **Author details**


## **References**


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Low-Penetrance Variants and Susceptibility to Sporadic Malignant Melanoma

http://dx.doi.org/10.5772/53097

77

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**Chapter 4**

**Melanoma Genetics: From Susceptibility to Progression**

Melanoma genetics has been for a long time a great challenge to cancer biologists, in part due to a complete lack of a single candidate gene to melanoma development. Differ‐ ent from breast and colorectal cancers, where BRCA-1/2 and *APC*/mismatch repair genes, respectively, characterize familial clusters of cancer susceptibility (reaching penetrance rates as high as 90% in some cases), in melanomas, the mutation rate of the most com‐ monly altered genes associated with disease progression do not exceed 60% of the cases in familial clusters. Among the "classical melanoma genes" are those coded at the *CDKN2A locus* (coding for p14 and p16, both related to cell cycle arrest), BRAF (specially the V600E mutation), a downstream transducer of the RAS signaling pathway and criti‐ cal for the cellular response to growth signals, and mutations in NRAS, somewhat relat‐

However, alterations in those genes, either by mutations or by epigenetic alterations do not account for all melanoma cases. Moreover, the mutations found in the classical mela‐ noma genes are not typical UV signature mutations (such as C to T transitions). This ob‐ servation poses an interesting problem in melanoma biology. Extensive epidemiological data indicates that intermittent exposure to UV radiation, mainly UVB is a major etiolog‐ ic factor for melanoma development. On the other hand, genes commonly mutated in melanomas lack UV signature mutations. Thus, evidence so far for the presence of UVBgenerated signature mutations in melanoma that could be defined as driver mutations has been less than compelling. Two critical questions need therefore to be answered; (1) If the classical melanoma genes do not account for the majority of cases, what other

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

© 2013 Francisco 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,

Guilherme Francisco, Priscila Daniele Ramos Cirilo,

Fernanda Toledo Gonçalves,

http://dx.doi.org/10.5772/54143

Roger Chammas

**1. Introduction**

Tharcísio Citrângulo Tortelli Junior and

ed to initiation and progression of melanoma.

Additional information is available at the end of the chapter

## **Melanoma Genetics: From Susceptibility to Progression**

Guilherme Francisco, Priscila Daniele Ramos Cirilo, Fernanda Toledo Gonçalves, Tharcísio Citrângulo Tortelli Junior and Roger Chammas

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54143

## **1. Introduction**

[64] Duffy, D. L., Zhao, Z. Z., Sturm, R. A., Hayward, N. K., Martin, N. G., and Montgom‐

82 Melanoma - From Early Detection to Treatment

ery, G. W. Multiple pigmentation gene polymorphisms account for a substantial pro‐ portion of risk of cutaneous malignant melanoma. J Invest Dermatol, 130: 520-8, 2010.

> Melanoma genetics has been for a long time a great challenge to cancer biologists, in part due to a complete lack of a single candidate gene to melanoma development. Differ‐ ent from breast and colorectal cancers, where BRCA-1/2 and *APC*/mismatch repair genes, respectively, characterize familial clusters of cancer susceptibility (reaching penetrance rates as high as 90% in some cases), in melanomas, the mutation rate of the most com‐ monly altered genes associated with disease progression do not exceed 60% of the cases in familial clusters. Among the "classical melanoma genes" are those coded at the *CDKN2A locus* (coding for p14 and p16, both related to cell cycle arrest), BRAF (specially the V600E mutation), a downstream transducer of the RAS signaling pathway and criti‐ cal for the cellular response to growth signals, and mutations in NRAS, somewhat relat‐ ed to initiation and progression of melanoma.

> However, alterations in those genes, either by mutations or by epigenetic alterations do not account for all melanoma cases. Moreover, the mutations found in the classical mela‐ noma genes are not typical UV signature mutations (such as C to T transitions). This ob‐ servation poses an interesting problem in melanoma biology. Extensive epidemiological data indicates that intermittent exposure to UV radiation, mainly UVB is a major etiolog‐ ic factor for melanoma development. On the other hand, genes commonly mutated in melanomas lack UV signature mutations. Thus, evidence so far for the presence of UVBgenerated signature mutations in melanoma that could be defined as driver mutations has been less than compelling. Two critical questions need therefore to be answered; (1) If the classical melanoma genes do not account for the majority of cases, what other

genes are involved in melanomagenesis? And, (2) what is the real relationship between the mutagenic potential of UV radiation and melanoma genetics?

(320-400 nm), UVB (280-320 nm) and UVC (200-280 nm). Despite the fact that UVA is more abundant in sunlight (90 %), UVB is about 1000-fold more efficient to cause sunburns and DNA damage than UVA [2]. Skin exposure to UV light affects epidermal and dermal cell survival and proliferation, besides other cutaneous functions [3]. Acute effects of UV exposure are usually the most harmful, including DNA damage, apoptosis, erythema, immunosuppression,

Melanoma Genetics: From Susceptibility to Progression

http://dx.doi.org/10.5772/54143

85

One of the main effects of UV exposure on cancer development is direct damages to DNA. Photoreactions due to absorption of UV (mainly UVB) by DNA lead to the estab‐ lishment of covalent linkages of adjacent pyrimidine bases (cytosine or thymine) thus forming cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts. CPDs are consti‐ tuted by the ligation of C-4 and C-5 carbons in both pyrimidines, whereas 6-4 photo‐ products are produced between C-6 and C-4 carbons of two adjacent pyrimidines, more frequently between TC and CC residues. CPDs are also considered more carcinogenic than 6-4 photoproducts, the frequency of CPD formation is three times higher and are less efficiently repaired [5]. If not repaired, both photoproducts lead to genetic mutations such as C→T and CC→TT transitions, besides single and double strand breaks on DNA [6]. Other genotoxic agents associated with excessive exposure to UV are reactive oxygen species (ROS), characterizing an indirect effect of radiation [7]. ROS can promote deani‐ mation and adduct formation, leading to errors in base pairing and, thus, mutations and

Epidemiologic studies have indicated that a pattern of intense and intermittent expo‐ sure to sunlight is a major risk factor for developing melanoma [8]. History of skin sunburns have been frequently used as measure to intermittent exposure and can be a marker for high risk of melanoma development. One or more severe sunburns on younger ages increase the risk of melanoma [9-11]. Besides the clear risk attributed to sunlight and thus UV exposure in melanomagenesis, the lack of a typical signature of UV mutation in genes classically related to melanoma development and progression had intrigued researchers for years. First, *CDKN2A* and *CDK4* genes had inheritable mutations and thus not showed typical UV mutations. Even somatic alterations in *CDKN2A* show very low levels of UV mutations and the majority of *CDKN2A* altera‐ tions in melanomas are epigenetic silencing and homozygous deletions [12]. Regarding *NRAS*, another "old gene" involved with melanoma progression, the most commonly described mutation lies in codon 61 and does not correspond to typical UV mutations (CAA(Gln) to AAA(Lys) or CAA(Gln) to CGA(Arg)) and can be found in about 10% of somatic melanomas [13]. Regarding *BRAF* mutation, the most prevalent V600E (found in about 40% of melanomas – [14]), shows a transversion of thymine substitut‐ ed by adenine. Finally, regarding the *PTEN* gene, increased allelic loss can be detected ranging to 40 to 60% of melanoma cases; less than 10% of melanoma samples show mutations in *PTEN*, the extensive majority consisting of frameshift mutations [15]. All those cited reports are the opposite to the high frequency of typical UV mutations in critical genes related to the other two skin cancers, namely basal cell carcinomas and

all factors contributing to aging and skin cancer [4].

chromosomal reorganization, contributing to carcinogenic process.

squamous cell carcinomas, such as *TP53* [16].

In the following pages, we will discuss the new findings about the biology of this neoplasia, besides discussing the known genes involved in melanomagenesis. A systematic review of to date GWAS data, deep-sequencing data and functional genomics will serve as the background for this discussion. As examples, GWAS studies have identified genetic variations in genes related to pigmentation that confer susceptibility to melanomas. The importance of these studies resides in the identification of new variants that can represent low penetrance sus‐ ceptibility genes. Other classes of genes that have emerged as critical genes to melanoma are DNA repair genes, especially NER genes (Nucleotide Excision Repair – a pathway that repair typical UV DNA damages). New studies have identified polymorphisms in those genes that confer higher risk to melanoma development. This susceptibility, in an interesting manner, seems to be influenced by the UV index of a certain region. On the other hand, microarray studies have suggested that DNA repair genes may be critical to metastasis sucess of melano‐ mas, through stabilization of a "metastatic genome". Deep-sequencing studies of melanoma cells have also identified genes and patterns of mutational status that correlate with UV signatures, bringing new clues to melanoma genetics. Are these driver or passenger mutations? The importance of other genes and pathways is also highlighted. One good example of a gene involved with melanoma progression is the *Microphthalmia*-associated *transcription factor* (*MITF*). *MITF* has been found to be expressed in several melanomas and its function is related to a diversity of cell processes, contributing to melanoma progression. The importance of *TP53* gene and its pathways in melanocyte/melanoma biology is also discussed. The *TP53* gene has intrigued biologists for a long time, since its mutational frequency is very low in melanomas, differently from other skin cancers, which harbor a high frequency of p53 mutations, which in turn are UV-type mutations. Functional data indicate however that the p53 pathway is dysfunctional in melanomas. What are the bases for this malfunction in this critical pathway for genome stability?

Thus, in this chapter we discuss both the "old" and the "new" genetics of melanoma suscept‐ ibility and progression. A discussion that will allow for the readers a systematic overview of what is known about the classical melanoma genetics, at the same time that may provide the basis to explore the new concepts that are emerging in this field.

## **2. UV exposure, deep-sequencing and melanomas – Understanding the melanoma development in depth**

Skin constitutes the first defense barrier in protection of internal environment and it is therefore subjected to several aggressions by pathogenic microorganisms or by chemical or physical damaging agents. Among these several agents, sunlight ultraviolet radiation (UV) is consid‐ ered the most potent carcinogenic factor for skin cancers, although the precise relationship between dose, time and nature of sunlight exposure to skin cancer development remains controversial [1]. Ultraviolet radiation can be classified according to its wavelength in UVA (320-400 nm), UVB (280-320 nm) and UVC (200-280 nm). Despite the fact that UVA is more abundant in sunlight (90 %), UVB is about 1000-fold more efficient to cause sunburns and DNA damage than UVA [2]. Skin exposure to UV light affects epidermal and dermal cell survival and proliferation, besides other cutaneous functions [3]. Acute effects of UV exposure are usually the most harmful, including DNA damage, apoptosis, erythema, immunosuppression, all factors contributing to aging and skin cancer [4].

genes are involved in melanomagenesis? And, (2) what is the real relationship between

In the following pages, we will discuss the new findings about the biology of this neoplasia, besides discussing the known genes involved in melanomagenesis. A systematic review of to date GWAS data, deep-sequencing data and functional genomics will serve as the background for this discussion. As examples, GWAS studies have identified genetic variations in genes related to pigmentation that confer susceptibility to melanomas. The importance of these studies resides in the identification of new variants that can represent low penetrance sus‐ ceptibility genes. Other classes of genes that have emerged as critical genes to melanoma are DNA repair genes, especially NER genes (Nucleotide Excision Repair – a pathway that repair typical UV DNA damages). New studies have identified polymorphisms in those genes that confer higher risk to melanoma development. This susceptibility, in an interesting manner, seems to be influenced by the UV index of a certain region. On the other hand, microarray studies have suggested that DNA repair genes may be critical to metastasis sucess of melano‐ mas, through stabilization of a "metastatic genome". Deep-sequencing studies of melanoma cells have also identified genes and patterns of mutational status that correlate with UV signatures, bringing new clues to melanoma genetics. Are these driver or passenger mutations? The importance of other genes and pathways is also highlighted. One good example of a gene involved with melanoma progression is the *Microphthalmia*-associated *transcription factor* (*MITF*). *MITF* has been found to be expressed in several melanomas and its function is related to a diversity of cell processes, contributing to melanoma progression. The importance of *TP53* gene and its pathways in melanocyte/melanoma biology is also discussed. The *TP53* gene has intrigued biologists for a long time, since its mutational frequency is very low in melanomas, differently from other skin cancers, which harbor a high frequency of p53 mutations, which in turn are UV-type mutations. Functional data indicate however that the p53 pathway is dysfunctional in melanomas. What are the bases for this malfunction in this critical pathway

Thus, in this chapter we discuss both the "old" and the "new" genetics of melanoma suscept‐ ibility and progression. A discussion that will allow for the readers a systematic overview of what is known about the classical melanoma genetics, at the same time that may provide the

**2. UV exposure, deep-sequencing and melanomas – Understanding the**

Skin constitutes the first defense barrier in protection of internal environment and it is therefore subjected to several aggressions by pathogenic microorganisms or by chemical or physical damaging agents. Among these several agents, sunlight ultraviolet radiation (UV) is consid‐ ered the most potent carcinogenic factor for skin cancers, although the precise relationship between dose, time and nature of sunlight exposure to skin cancer development remains controversial [1]. Ultraviolet radiation can be classified according to its wavelength in UVA

basis to explore the new concepts that are emerging in this field.

**melanoma development in depth**

the mutagenic potential of UV radiation and melanoma genetics?

84 Melanoma - From Early Detection to Treatment

for genome stability?

One of the main effects of UV exposure on cancer development is direct damages to DNA. Photoreactions due to absorption of UV (mainly UVB) by DNA lead to the estab‐ lishment of covalent linkages of adjacent pyrimidine bases (cytosine or thymine) thus forming cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts. CPDs are consti‐ tuted by the ligation of C-4 and C-5 carbons in both pyrimidines, whereas 6-4 photo‐ products are produced between C-6 and C-4 carbons of two adjacent pyrimidines, more frequently between TC and CC residues. CPDs are also considered more carcinogenic than 6-4 photoproducts, the frequency of CPD formation is three times higher and are less efficiently repaired [5]. If not repaired, both photoproducts lead to genetic mutations such as C→T and CC→TT transitions, besides single and double strand breaks on DNA [6]. Other genotoxic agents associated with excessive exposure to UV are reactive oxygen species (ROS), characterizing an indirect effect of radiation [7]. ROS can promote deani‐ mation and adduct formation, leading to errors in base pairing and, thus, mutations and chromosomal reorganization, contributing to carcinogenic process.

Epidemiologic studies have indicated that a pattern of intense and intermittent expo‐ sure to sunlight is a major risk factor for developing melanoma [8]. History of skin sunburns have been frequently used as measure to intermittent exposure and can be a marker for high risk of melanoma development. One or more severe sunburns on younger ages increase the risk of melanoma [9-11]. Besides the clear risk attributed to sunlight and thus UV exposure in melanomagenesis, the lack of a typical signature of UV mutation in genes classically related to melanoma development and progression had intrigued researchers for years. First, *CDKN2A* and *CDK4* genes had inheritable mutations and thus not showed typical UV mutations. Even somatic alterations in *CDKN2A* show very low levels of UV mutations and the majority of *CDKN2A* altera‐ tions in melanomas are epigenetic silencing and homozygous deletions [12]. Regarding *NRAS*, another "old gene" involved with melanoma progression, the most commonly described mutation lies in codon 61 and does not correspond to typical UV mutations (CAA(Gln) to AAA(Lys) or CAA(Gln) to CGA(Arg)) and can be found in about 10% of somatic melanomas [13]. Regarding *BRAF* mutation, the most prevalent V600E (found in about 40% of melanomas – [14]), shows a transversion of thymine substitut‐ ed by adenine. Finally, regarding the *PTEN* gene, increased allelic loss can be detected ranging to 40 to 60% of melanoma cases; less than 10% of melanoma samples show mutations in *PTEN*, the extensive majority consisting of frameshift mutations [15]. All those cited reports are the opposite to the high frequency of typical UV mutations in critical genes related to the other two skin cancers, namely basal cell carcinomas and squamous cell carcinomas, such as *TP53* [16].

In a seminal study, a comparison of four distinct sets of melanomas at the genomic level gave important clues about the role of UV in melanomagenesis [17]. The authors com‐ pared the number of copies of DNA and the mutational status of two critical genes to melanoma development, *BRAF* and *NRAS* in a panel consisting of 126 melanomas from four groups differing among them according the degree of exposure to ultraviolet light: 30 melanomas from skin with chronic sun-induced damage; 40 melanomas from skin without such damage; 36 melanomas from palms, soles, and subungual (acral) sites; and 20 mucosal melanomas. The results indicated that melanomas from sun-protected areas (acral and mucosal) had more frequent chromosomal aberrations including amplifications and losses compared to sun-exposed melanomas. Frequent amplification was identified in *CCND1* gene (cyclin D1 gene) and *CDK4* gene (more frequent in acral and mucosal melanomas). Moreover, deletions of the *CDKN2A* locus in were found in 50 percent of all melanomas, making it the most commonly lost genomic region, being also more fre‐ quent in acral and mucosal melanomas). Mutations in *BRAF* gene were significantly more common in the group of melanomas that were on skin without chronic sun-in‐ duced damage than in the other three groups. Therefore, there are distinct patterns of genetic alterations in the four groups of primary melanomas. The differences in both chromosomal aberrations and the frequency of mutations of specific genes suggest that these tumors develop through different mechanistic routes, and likely respond to differ‐ ent selective influences.

in *SPDEF* gene, which codes to an ETS transcription factor family, described as associated with some cancers types [20]. Moreover, mutations in *MMP28* gene (a member of matrix metallo‐ proteinases) and in *UVRAG* (a putative tumor suppressor gene – [21]) were found. In addition,

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Of the total number of mutations found (33345), almost 25000 were C to T mutations, and of the 510 dinucleotide substitutions, 360 were CC to TT changes [19]. The mutational spectrum observed is consistent with UV-associated mutations, fact that denotes the influence of UV on melanoma development. C to T and CC to TT changes were significantly more frequent in CpG dinucleotides than the expected by chance. The mutational pattern also indicated a strong relation of UV mutations with the nucleotide excision DNA repair pathway (NER) due to the high frequency of mutations in non-transcribed strands when compared to mutational frequency in transcribed strands. The transcription-coupled repair (a sub-pathway of NER system), which operates in transcribed strands, is credited to be more efficiently in repair UV lesions when compared to NER system that operates in non-transcribed strands. Finally, besides the majority of C to T mutations, the second commonest mutation frequency was substitution of G to T. High production of ROS can lead to oxidized guanines and in turn causes G to T changes. As UV exposure can also lead to ROS production, is tempting to suggest that besides the direct DNA damage caused by UV, contributing to C to T changes, indirect effects such as ROS production also may contribute to melanoma carcinogenesis. Thus, this first entire catalogue of mutations in melanoma by whole genome sequencing supports the notion that

A third whole genome sequencing study also confirmed the elevated mutational rate in melanomas, that in mean was about 30 mutations per Mb, and the C to T mutations were the most frequent, once again reinforcing the role of UV irradiation in melanomas [22]. However, the great advantage of the study was using metastatic melanoma samples and also including melanomas from different body areas. Thus, the authors could present an interesting panel of mutational rate across the different melanoma subtypes. As example, acral melanomas showed mutational rates comparable to other solid tumor types (3 mutations per Mb), whereas melanomas from the trunk showed higher mutational rates. The sequencing of a melanoma from an individual with history of chronic sun exposure exhibited the higher mutational rate across the samples analyzed (111 somatic mutations per Mb). Moreover, that melanoma with the higher mutational rate showed 93% of C to T substitutions, while acral melanomas showed only 36% of such mutations. These data strongly support the contribution of sun exposure in melanoma etiology. From the most significantly genes, the authors identified frequent mutations in *PREX2* gene, mutated in 11 of the 25 melanomas samples. PREX2 is involved with PTEN pathway modulating its function [23]. Functional analysis by expressing the mutant forms of PREX2 in melanocytes injected in immunodeficient significantly accelerated *in vivo* tumorigenesis, suggesting that PREX2 mutations contribute to melanoma progression. Whole genome sequence of acral melanomas also identified low frequency of mutations (2.16

a 12-kilobase internal homozygous deletion was found in PTEN gene.

UV exposure plays a critical role in melanoma development.

per Mb in the primary tumor and 1.95 per Mb in metastasis sample) [24].

Additional studies from exome sequencing have identified new genes related to melanoma development and also chemoresistance. From exome sequencing study performed in seven

The unanswered question about the real impact of UV light on melanoma genetics began to be solved with the development of new technologies in DNA sequencing, the so called "deep-sequencing method". With this technology, the researchers could perform large-scale sequencing, covering the whole genome. In one of the first studies using deep-sequencing methods, the authors reported more than 1000 mutations using 210 di‐ verse human cancers, including melanomas [18]. This study covered 274 megabases (Mb) and was restricted to 518 protein kinase genes. The results showed that melanomas (in that case, melanoma cell lines), had a high prevalence of mutations showing a mean number of 18.54 mutations per Mb of DNA. The main result from this first study was that of 144 mutations in melanomas, more than 90% was C to T mutations, the typical transition of UV-related mutations. Most somatic mutations found were classified as "passengers" mutations, i.e. those which do not contribute directly to carcinogenesis. "Driver" mutations, those mutations that contribute to carcinogenesis, were found in ap‐ proximately 120 genes.

A second study, in this time a comprehensive catalogue of the whole genome of a melanoma cell line and a lymphoblastoid cell line from the same person, provided the first catalogue of somatic mutations from an individual cancer [19]. The numbers generated by the deepsequencing are impressive. The study identified 33345 somatic mutations, where 32325 were single base mutations and 510 were double-base mutations. A total of 292 somatic base substitutions were in protein-coding sequences and of these, 187 were non-synonymous mutations leading to amino acid changes, including 172 missense mutations and 15 nonsense. Several individual substitutions highlighted novel candidate cancer genes such as mutations in *SPDEF* gene, which codes to an ETS transcription factor family, described as associated with some cancers types [20]. Moreover, mutations in *MMP28* gene (a member of matrix metallo‐ proteinases) and in *UVRAG* (a putative tumor suppressor gene – [21]) were found. In addition, a 12-kilobase internal homozygous deletion was found in PTEN gene.

In a seminal study, a comparison of four distinct sets of melanomas at the genomic level gave important clues about the role of UV in melanomagenesis [17]. The authors com‐ pared the number of copies of DNA and the mutational status of two critical genes to melanoma development, *BRAF* and *NRAS* in a panel consisting of 126 melanomas from four groups differing among them according the degree of exposure to ultraviolet light: 30 melanomas from skin with chronic sun-induced damage; 40 melanomas from skin without such damage; 36 melanomas from palms, soles, and subungual (acral) sites; and 20 mucosal melanomas. The results indicated that melanomas from sun-protected areas (acral and mucosal) had more frequent chromosomal aberrations including amplifications and losses compared to sun-exposed melanomas. Frequent amplification was identified in *CCND1* gene (cyclin D1 gene) and *CDK4* gene (more frequent in acral and mucosal melanomas). Moreover, deletions of the *CDKN2A* locus in were found in 50 percent of all melanomas, making it the most commonly lost genomic region, being also more fre‐ quent in acral and mucosal melanomas). Mutations in *BRAF* gene were significantly more common in the group of melanomas that were on skin without chronic sun-in‐ duced damage than in the other three groups. Therefore, there are distinct patterns of genetic alterations in the four groups of primary melanomas. The differences in both chromosomal aberrations and the frequency of mutations of specific genes suggest that these tumors develop through different mechanistic routes, and likely respond to differ‐

The unanswered question about the real impact of UV light on melanoma genetics began to be solved with the development of new technologies in DNA sequencing, the so called "deep-sequencing method". With this technology, the researchers could perform large-scale sequencing, covering the whole genome. In one of the first studies using deep-sequencing methods, the authors reported more than 1000 mutations using 210 di‐ verse human cancers, including melanomas [18]. This study covered 274 megabases (Mb) and was restricted to 518 protein kinase genes. The results showed that melanomas (in that case, melanoma cell lines), had a high prevalence of mutations showing a mean number of 18.54 mutations per Mb of DNA. The main result from this first study was that of 144 mutations in melanomas, more than 90% was C to T mutations, the typical transition of UV-related mutations. Most somatic mutations found were classified as "passengers" mutations, i.e. those which do not contribute directly to carcinogenesis. "Driver" mutations, those mutations that contribute to carcinogenesis, were found in ap‐

A second study, in this time a comprehensive catalogue of the whole genome of a melanoma cell line and a lymphoblastoid cell line from the same person, provided the first catalogue of somatic mutations from an individual cancer [19]. The numbers generated by the deepsequencing are impressive. The study identified 33345 somatic mutations, where 32325 were single base mutations and 510 were double-base mutations. A total of 292 somatic base substitutions were in protein-coding sequences and of these, 187 were non-synonymous mutations leading to amino acid changes, including 172 missense mutations and 15 nonsense. Several individual substitutions highlighted novel candidate cancer genes such as mutations

ent selective influences.

86 Melanoma - From Early Detection to Treatment

proximately 120 genes.

Of the total number of mutations found (33345), almost 25000 were C to T mutations, and of the 510 dinucleotide substitutions, 360 were CC to TT changes [19]. The mutational spectrum observed is consistent with UV-associated mutations, fact that denotes the influence of UV on melanoma development. C to T and CC to TT changes were significantly more frequent in CpG dinucleotides than the expected by chance. The mutational pattern also indicated a strong relation of UV mutations with the nucleotide excision DNA repair pathway (NER) due to the high frequency of mutations in non-transcribed strands when compared to mutational frequency in transcribed strands. The transcription-coupled repair (a sub-pathway of NER system), which operates in transcribed strands, is credited to be more efficiently in repair UV lesions when compared to NER system that operates in non-transcribed strands. Finally, besides the majority of C to T mutations, the second commonest mutation frequency was substitution of G to T. High production of ROS can lead to oxidized guanines and in turn causes G to T changes. As UV exposure can also lead to ROS production, is tempting to suggest that besides the direct DNA damage caused by UV, contributing to C to T changes, indirect effects such as ROS production also may contribute to melanoma carcinogenesis. Thus, this first entire catalogue of mutations in melanoma by whole genome sequencing supports the notion that UV exposure plays a critical role in melanoma development.

A third whole genome sequencing study also confirmed the elevated mutational rate in melanomas, that in mean was about 30 mutations per Mb, and the C to T mutations were the most frequent, once again reinforcing the role of UV irradiation in melanomas [22]. However, the great advantage of the study was using metastatic melanoma samples and also including melanomas from different body areas. Thus, the authors could present an interesting panel of mutational rate across the different melanoma subtypes. As example, acral melanomas showed mutational rates comparable to other solid tumor types (3 mutations per Mb), whereas melanomas from the trunk showed higher mutational rates. The sequencing of a melanoma from an individual with history of chronic sun exposure exhibited the higher mutational rate across the samples analyzed (111 somatic mutations per Mb). Moreover, that melanoma with the higher mutational rate showed 93% of C to T substitutions, while acral melanomas showed only 36% of such mutations. These data strongly support the contribution of sun exposure in melanoma etiology. From the most significantly genes, the authors identified frequent mutations in *PREX2* gene, mutated in 11 of the 25 melanomas samples. PREX2 is involved with PTEN pathway modulating its function [23]. Functional analysis by expressing the mutant forms of PREX2 in melanocytes injected in immunodeficient significantly accelerated *in vivo* tumorigenesis, suggesting that PREX2 mutations contribute to melanoma progression. Whole genome sequence of acral melanomas also identified low frequency of mutations (2.16 per Mb in the primary tumor and 1.95 per Mb in metastasis sample) [24].

Additional studies from exome sequencing have identified new genes related to melanoma development and also chemoresistance. From exome sequencing study performed in seven melanomas the authors found a total of 4933 somatic mutations, 3611 of which were located in protein-coding regions in 2586 genes [25]. Confirming previous results, C to T transitions were the most representative mutations (ranging from 73 to 87% of all mutations). In order to get a more comprehensive view of melanoma genome, the authors looked to genes involved with MAPK pathway, which includes NRAS and BRAF. Two of seven melanomas analyzed showed a somatic G to A transition at homologous site in the *MAP2K1* and *MAP2K2* genes, kinases that are downstream targets of BRAF. In an independent set of 127 melanomas, 8% confirmed the existence of damaging mutations in either gene. Following functional studies with either gene demonstrated a constitutive activation and resulted in ERK1/2 phosphoryla‐ tion and the oncogenic activity of such mutations was also evaluated in transformation assays. Moreover, in four of the seven melanomas, mutations were found in *FAT4*, *DSC1* and *LRP1B* genes, which might be candidate genes, as suggested by the authors [25].

index regions to high-UV index regions in the globe will help us understanding more about

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When we talk about susceptibility genes to diseases, especially to cancer, we are talking about inheritable genetic alterations. Such alterations in critical genes related to tumor suppression contribute to modulate the susceptibility to certain tumors. Inheritable alterations can be classified as mutations or polymorphisms (also known as single nucleotide polymorphism – SNP). Both genetic alterations have different features such as: (i) related to population allelic frequency (mutations < 1% and polymorphisms > 1%); (ii) related to its impact to gene functionality, where mutations cause deleterious alterations to the function while polymor‐ phisms may modify the function, however not in a deleterious manner; (iii) related to pene‐ trance, where mutations exerts its deleterious function in a high penetrance to development of the disease. Conversely, polymorphisms exert its function in a low penetrance to disease and may be more susceptible to environmental influence; (iv) age of tumor onset, where high penetrance mutated genes contribute to disease development in younger ages while poly‐ morphisms are related to older ages to cancer development. Temporally, high susceptibility genes to melanoma were well established through the years, however low susceptibility genes have been identified recently. Appreciation of high penetrance genes came from multiple studies of melanoma-predisposed families studies; in which linkage analysis, cytogenetic and candidate gene studies helped to identify those genes. However, the high-penetrance genes account for 5 to 10% melanoma cases, indicating that other genes, including low penetrance genes may modulate the susceptibility. The development of new technologies has contributed to identify new susceptible genes and understand their roles to melanoma. In this section we

the genes involved in melanomagenesis.

**3. Melanoma genetics: Susceptibility genes**

discuss the "old" and the "new" genetics for melanoma susceptibility.

The best-established gene for melanoma susceptibility is the *CDKN2A* (cyclin-dependent kinase inhibitor 2A gene) locus, which is located in chromosome 9p21. Involvement of a 9p locus in melanomas was first indicated by cytogenetically detectable loss or translocation of this region. Subsequent loss of heterozigosity (LOH) studies and later studies indicated the existence of a tumor suppressor gene in this region. Germline mutations in this locus have been described among melanoma-predisposed families since 1995, and approximately 40% of familial melanomas cases harbor *CDKN2A* mutations [29]. The *CDKN2A* locus encodes for two different proteins, which are related to cell cycle control and tumor suppression. The two proteins are produced by alternative reading frame of four exons [30]. The proteins produced by *CDKN2A* locus, p16/Ink4a and p14/Arf, are involved with regulation of cell cycle from G1 to S phase, besides the ability of p14/Arf to induce apoptosis [30]. Regarding p16/Ink4a, its main function is to bind to CDK4 and to inhibit its kinase activity. By inhibiting CDK4 activity,

**3.1. High penetrance genes: "The old genetics" for melanoma**

*3.1.1. CDKN2A – The classical susceptibility gene*

In an independent study [26], other melanoma exome sequencing also identified mutations in genes participating of MAPK pathway, more precisely *MAP3K5* and *MAP3K9*. Validation of such data indicated mutations in *MAP3K5* in 8 of 85 melanoma cell lines and mutation in 13 of 85 cell lines to *MAP3K9*. Functional analysis of such mutations indicated a significantly reduction in kinase activity of both proteins. Moreover, such mutations in both genes resulted in decreased levels of phosphorylated MEK-ERK and JNK, pathways involved with apoptosis, differentiation, survival and senescence. Interestingly, decreased expression of MAP3K5 and MAP3K9 by siRNA method led to chemoresistance to temozolomide [26]. A third exome sequencing study, using a large sample size (147 melanomas from sun-exposed areas), identified a recurrent UV-signature in *RAC1* gene in 9.2% of cases. Biochemical and functional analysis of mutated RAC1 showed that such alteration promotes melanocyte proliferation and migration [27].

All of these genome sequence studies identified a great number of mutations, however most mutations are passenger mutations. In order to differentiate passenger from driver mutations, Linda Chin coordinated an effort to sequence exons and introns of melanoma samples, comparing their frequency in order to identify positively selected genes, based on enrichment of mutations in exons [28]. The authors identified positive selection in melanoma genes including well-know genes such as *BRAF, NRAS, PTEN, TP53, p16* and also indentify new candidate genes, such as *PPP6C*, *RAC1* (previously described in [26]), *SNX31*, *TACC1* and *STK19*. Noteworthy, to *PPP6C* (a subunit of PP6C protein phosphatase), a candidate to tumor suppressor gene, showed 60% of mutations clustered within a 12 amino acid region flanking an arginine at codon 264. Regarding *RAC1*, the mutant forms also indicated gain-of-function. The study also indicated the role of UV in the advent of melanoma driver mutations. Of 262 driver mutations found in 21 genes identified by the study, 46% were caused by C to T mutations (37%) or G to T (9%), alterations characteristics of UVB/UVA-induced mutations. These numbers increased to 67% by excluding mutations in *BRAF* or *NRAS* genes.

Innovative strategies exploiting deep sequencing will contribute to the understanding of the diversity of pathways involved with melanoma. We anticipate that studies of melanomas arising in different ethnic groups, and mainly from individuals who migrated from low-UV index regions to high-UV index regions in the globe will help us understanding more about the genes involved in melanomagenesis.

## **3. Melanoma genetics: Susceptibility genes**

melanomas the authors found a total of 4933 somatic mutations, 3611 of which were located in protein-coding regions in 2586 genes [25]. Confirming previous results, C to T transitions were the most representative mutations (ranging from 73 to 87% of all mutations). In order to get a more comprehensive view of melanoma genome, the authors looked to genes involved with MAPK pathway, which includes NRAS and BRAF. Two of seven melanomas analyzed showed a somatic G to A transition at homologous site in the *MAP2K1* and *MAP2K2* genes, kinases that are downstream targets of BRAF. In an independent set of 127 melanomas, 8% confirmed the existence of damaging mutations in either gene. Following functional studies with either gene demonstrated a constitutive activation and resulted in ERK1/2 phosphoryla‐ tion and the oncogenic activity of such mutations was also evaluated in transformation assays. Moreover, in four of the seven melanomas, mutations were found in *FAT4*, *DSC1* and *LRP1B*

In an independent study [26], other melanoma exome sequencing also identified mutations in genes participating of MAPK pathway, more precisely *MAP3K5* and *MAP3K9*. Validation of such data indicated mutations in *MAP3K5* in 8 of 85 melanoma cell lines and mutation in 13 of 85 cell lines to *MAP3K9*. Functional analysis of such mutations indicated a significantly reduction in kinase activity of both proteins. Moreover, such mutations in both genes resulted in decreased levels of phosphorylated MEK-ERK and JNK, pathways involved with apoptosis, differentiation, survival and senescence. Interestingly, decreased expression of MAP3K5 and MAP3K9 by siRNA method led to chemoresistance to temozolomide [26]. A third exome sequencing study, using a large sample size (147 melanomas from sun-exposed areas), identified a recurrent UV-signature in *RAC1* gene in 9.2% of cases. Biochemical and functional analysis of mutated RAC1 showed that such alteration promotes melanocyte proliferation and

All of these genome sequence studies identified a great number of mutations, however most mutations are passenger mutations. In order to differentiate passenger from driver mutations, Linda Chin coordinated an effort to sequence exons and introns of melanoma samples, comparing their frequency in order to identify positively selected genes, based on enrichment of mutations in exons [28]. The authors identified positive selection in melanoma genes including well-know genes such as *BRAF, NRAS, PTEN, TP53, p16* and also indentify new candidate genes, such as *PPP6C*, *RAC1* (previously described in [26]), *SNX31*, *TACC1* and *STK19*. Noteworthy, to *PPP6C* (a subunit of PP6C protein phosphatase), a candidate to tumor suppressor gene, showed 60% of mutations clustered within a 12 amino acid region flanking an arginine at codon 264. Regarding *RAC1*, the mutant forms also indicated gain-of-function. The study also indicated the role of UV in the advent of melanoma driver mutations. Of 262 driver mutations found in 21 genes identified by the study, 46% were caused by C to T mutations (37%) or G to T (9%), alterations characteristics of UVB/UVA-induced mutations.

These numbers increased to 67% by excluding mutations in *BRAF* or *NRAS* genes.

Innovative strategies exploiting deep sequencing will contribute to the understanding of the diversity of pathways involved with melanoma. We anticipate that studies of melanomas arising in different ethnic groups, and mainly from individuals who migrated from low-UV

genes, which might be candidate genes, as suggested by the authors [25].

migration [27].

88 Melanoma - From Early Detection to Treatment

When we talk about susceptibility genes to diseases, especially to cancer, we are talking about inheritable genetic alterations. Such alterations in critical genes related to tumor suppression contribute to modulate the susceptibility to certain tumors. Inheritable alterations can be classified as mutations or polymorphisms (also known as single nucleotide polymorphism – SNP). Both genetic alterations have different features such as: (i) related to population allelic frequency (mutations < 1% and polymorphisms > 1%); (ii) related to its impact to gene functionality, where mutations cause deleterious alterations to the function while polymor‐ phisms may modify the function, however not in a deleterious manner; (iii) related to pene‐ trance, where mutations exerts its deleterious function in a high penetrance to development of the disease. Conversely, polymorphisms exert its function in a low penetrance to disease and may be more susceptible to environmental influence; (iv) age of tumor onset, where high penetrance mutated genes contribute to disease development in younger ages while poly‐ morphisms are related to older ages to cancer development. Temporally, high susceptibility genes to melanoma were well established through the years, however low susceptibility genes have been identified recently. Appreciation of high penetrance genes came from multiple studies of melanoma-predisposed families studies; in which linkage analysis, cytogenetic and candidate gene studies helped to identify those genes. However, the high-penetrance genes account for 5 to 10% melanoma cases, indicating that other genes, including low penetrance genes may modulate the susceptibility. The development of new technologies has contributed to identify new susceptible genes and understand their roles to melanoma. In this section we discuss the "old" and the "new" genetics for melanoma susceptibility.

#### **3.1. High penetrance genes: "The old genetics" for melanoma**

#### *3.1.1. CDKN2A – The classical susceptibility gene*

The best-established gene for melanoma susceptibility is the *CDKN2A* (cyclin-dependent kinase inhibitor 2A gene) locus, which is located in chromosome 9p21. Involvement of a 9p locus in melanomas was first indicated by cytogenetically detectable loss or translocation of this region. Subsequent loss of heterozigosity (LOH) studies and later studies indicated the existence of a tumor suppressor gene in this region. Germline mutations in this locus have been described among melanoma-predisposed families since 1995, and approximately 40% of familial melanomas cases harbor *CDKN2A* mutations [29]. The *CDKN2A* locus encodes for two different proteins, which are related to cell cycle control and tumor suppression. The two proteins are produced by alternative reading frame of four exons [30]. The proteins produced by *CDKN2A* locus, p16/Ink4a and p14/Arf, are involved with regulation of cell cycle from G1 to S phase, besides the ability of p14/Arf to induce apoptosis [30]. Regarding p16/Ink4a, its main function is to bind to CDK4 and to inhibit its kinase activity. By inhibiting CDK4 activity, p16/Ink4a avoids the phosphorylation of retinoblastoma (*Rb*) tumor suppressor gene, acting therefore as a negative regulator of E2F function. Thus, loss-of-function mutations or loss of p16/Ink4a expression, allow for CDK4 to phosphorylate Rb, thereby releasing E2F activity in the transition of G1 to S phase.

therefore CDK4 activity is not inhibited. The functional consequence is then phosphorylation of Rb, leading to Rb inactivity and thus allowing the cell to progress on cell cycle. Only in a few reports the whole *CDK4* gene was sequenced. Expansion of *CDK4* sequencing, including the whole gene, instead of only codon 2, might help to identify new mutations in non-9p-linked

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*3.1.3. Evidence of new susceptibility locus and other critical genes that confer risk to melanoma*

region, however, sequence analysis has not identified any mutations [37].

melanoma was published elsewhere [38].

studies in melanoma.

**3.2. Low penetrance genes: The "new genetics" of melanoma**

*3.2.1. MC1R gene – Coloring the knowledge of melanoma susceptibility*

Different from other familial cancer, such as breast and HNPCC colon cancers, a unique candidate gene seems not responsible to all familial cases of melanoma. As cited above, up to 40% of familial melanomas could be attributed to *CDKN2A* mutations. This fact opens the possibility to other susceptibility genes with high penetrance. A study performed in families with no *CDKN2A* and *CDK4* mutations identified a possible candidate locus in 1p22 chromo‐ some [36]. Subsequent analysis of this locus in additional pedigrees supported this previous evidence. Moreover, LOH studies also indicated a putative tumor suppressor gene is this

Other germline mutations in critical genes responsible for cancer susceptibility, which melanoma is not a clinically feature, also increase the risk for melanoma, where some mela‐ noma cases have been reported. Individuals harboring germline mutations in *RB1* gene (Retinoblastoma); *TP53* and *CHEK2* genes (Li-Fraumeni and Li-Fraumeni Like syndrome respectively); *NF1* gene (Neurofibromatosis type 1); *Xeroderma Pigmentosum* genes (XP) and *BRCA2* were also associated with melanoma. Even melanomas cases were reported in such syndromes. The absolute low number of melanomas reported in these syndromes, especially in Li-Fraumeni syndrome, creates a debate regarding whether melanomas could be a rare manifestation of these cancer syndromes. A detailed discussion on the role of these genes and

The great development in low penetrance genes search for melanoma risk came with the development of genome-wide association studies (GWAS). With GWAS, several hundreds of thousands DNA variants can be detected and larger samples sizes can be used, thus increasing the power of analysis. A great advantage of using GWAS is the possibility to identify variants that are not located in protein coding regions. Coupled with the development of GWAS, the use of meta-analysis has also contributed to identify new low penetrance genes. Meta-analysis is a widely accepted method that summarizes the results from multiple published studies, then producing results with larger sample size and increasing statistical power. We discuss below the main findings regarding low-penetrance genes and melanoma of GWAS and meta-analysis

The *MC1R* (melanocortin 1 receptor) is a critical gene related to human skin pigmentation. *MC1R* codes for a transmembrane protein receptor that binds to α-melanocyte stimulating

melanoma families.

The role of p14/Arf in tumor suppression is related to regulation of p53 pathway. Its function is related to binding to HDM-2 protein and inhibition of its activity. The MDM-2 protein is a key regulator of p53 protein due to its ability to ubiquitinate p53, leading to p53 degradation. Thus, by p14/Arf function, MDM-2 is depleted and p53 is stabilized. Inactivation of p14/Arf functions is associated with MDM-2 accumulation, which in turn leads to p53 degradation and consequently loss of its tumor suppressor function. In summary, loss-of-function alterations in *CDKN2A* simultaneously impair two of the most critical pathways in tumor suppression, the Rb and p53 pathways. Most germline mutations in *CDKN2A* locus are missense mutations, usually found in exons 1α and exon 2, although mutations in 5´ UTR and intron regions are also found, affecting thus translation initiation and splicing events [31]. Overall, *CDKN2A* mutations have been found in 20 – 40% of families with 3 or more affected members and in 10% of fam‐ ilies with 2 melanoma cases. However the frequency can vary according to different pop‐ ulations, fact that can be explained by different founder mutations in some of those populations. The low mutation detection rate has suggested that other susceptibility genes exist in melanomas. Moreover, the penetrance of mutations in *CDKN2A* shows geographical variations [32].

Some studies have suggested that the penetrance of the *CDKN2A* mutations may be modulated by other genetic risk modifiers. Certain MC1R variants (discussed below) in‐ crease melanoma risk in familial melanomas harboring mutations in *CDKN2A* [33]. As MC1R, a gene strongly related to skin color, plays a role as a modifier gene, it seems logical that other pigmentation genes might similarly act as genetic modifiers to *CDKN2A* penetrance. Environmental factors, such as relative exposure to UV radiation may contribute to the variability in penetrance of *CDKN2A* mutations according to geo‐ graphical reasons, as suggested by previous studies [32].

#### *3.1.2. CDK4 – The second line in melanoma susceptibility*

Another well known gene associated with melanoma susceptibility is the *CDK*4 gene. The gene is located in chromosome 12q13 and codes for cyclin-dependent kinase. Mutations in the *CDK4* gene were just described in 15 families with melanoma predisposition [34], where just two known mutations are described and located in codon, Arg24Cys e Arg24His. Curiously, these mutations can be also found in sporadic melanomas. Although less frequent than the inherit‐ able mutations in *CDKN2A* locus, patients harboring mutations in *CDK4* usually show the same clinical characteristics as patients with mutations in *CDKN2A* such as mean age at diagnostics, mean number of melanomas and mean number of nevi [35]. These clinical similarities (phenocopies) shown by mutations in different genes may be explained by the same pathway that *CDK4* and p16 protein share together the Rb (retinoblastoma) tumor suppressor pathway. Both CDK4 mutant variants described above are unable to bind p16, and therefore CDK4 activity is not inhibited. The functional consequence is then phosphorylation of Rb, leading to Rb inactivity and thus allowing the cell to progress on cell cycle. Only in a few reports the whole *CDK4* gene was sequenced. Expansion of *CDK4* sequencing, including the whole gene, instead of only codon 2, might help to identify new mutations in non-9p-linked melanoma families.

## *3.1.3. Evidence of new susceptibility locus and other critical genes that confer risk to melanoma*

p16/Ink4a avoids the phosphorylation of retinoblastoma (*Rb*) tumor suppressor gene, acting therefore as a negative regulator of E2F function. Thus, loss-of-function mutations or loss of p16/Ink4a expression, allow for CDK4 to phosphorylate Rb, thereby releasing E2F activity in

The role of p14/Arf in tumor suppression is related to regulation of p53 pathway. Its function is related to binding to HDM-2 protein and inhibition of its activity. The MDM-2 protein is a key regulator of p53 protein due to its ability to ubiquitinate p53, leading to p53 degradation. Thus, by p14/Arf function, MDM-2 is depleted and p53 is stabilized. Inactivation of p14/Arf functions is associated with MDM-2 accumulation, which in turn leads to p53 degradation and consequently loss of its tumor suppressor function. In summary, loss-of-function alterations in *CDKN2A* simultaneously impair two of the most critical pathways in tumor suppression, the Rb and p53 pathways. Most germline mutations in *CDKN2A* locus are missense mutations, usually found in exons 1α and exon 2, although mutations in 5´ UTR and intron regions are also found, affecting thus translation initiation and splicing events [31]. Overall, *CDKN2A* mutations have been found in 20 – 40% of families with 3 or more affected members and in 10% of fam‐ ilies with 2 melanoma cases. However the frequency can vary according to different pop‐ ulations, fact that can be explained by different founder mutations in some of those populations. The low mutation detection rate has suggested that other susceptibility genes exist in melanomas. Moreover, the penetrance of mutations in *CDKN2A* shows

Some studies have suggested that the penetrance of the *CDKN2A* mutations may be modulated by other genetic risk modifiers. Certain MC1R variants (discussed below) in‐ crease melanoma risk in familial melanomas harboring mutations in *CDKN2A* [33]. As MC1R, a gene strongly related to skin color, plays a role as a modifier gene, it seems logical that other pigmentation genes might similarly act as genetic modifiers to *CDKN2A* penetrance. Environmental factors, such as relative exposure to UV radiation may contribute to the variability in penetrance of *CDKN2A* mutations according to geo‐

Another well known gene associated with melanoma susceptibility is the *CDK*4 gene. The gene is located in chromosome 12q13 and codes for cyclin-dependent kinase. Mutations in the *CDK4* gene were just described in 15 families with melanoma predisposition [34], where just two known mutations are described and located in codon, Arg24Cys e Arg24His. Curiously, these mutations can be also found in sporadic melanomas. Although less frequent than the inherit‐ able mutations in *CDKN2A* locus, patients harboring mutations in *CDK4* usually show the same clinical characteristics as patients with mutations in *CDKN2A* such as mean age at diagnostics, mean number of melanomas and mean number of nevi [35]. These clinical similarities (phenocopies) shown by mutations in different genes may be explained by the same pathway that *CDK4* and p16 protein share together the Rb (retinoblastoma) tumor suppressor pathway. Both CDK4 mutant variants described above are unable to bind p16, and

the transition of G1 to S phase.

90 Melanoma - From Early Detection to Treatment

geographical variations [32].

graphical reasons, as suggested by previous studies [32].

*3.1.2. CDK4 – The second line in melanoma susceptibility*

Different from other familial cancer, such as breast and HNPCC colon cancers, a unique candidate gene seems not responsible to all familial cases of melanoma. As cited above, up to 40% of familial melanomas could be attributed to *CDKN2A* mutations. This fact opens the possibility to other susceptibility genes with high penetrance. A study performed in families with no *CDKN2A* and *CDK4* mutations identified a possible candidate locus in 1p22 chromo‐ some [36]. Subsequent analysis of this locus in additional pedigrees supported this previous evidence. Moreover, LOH studies also indicated a putative tumor suppressor gene is this region, however, sequence analysis has not identified any mutations [37].

Other germline mutations in critical genes responsible for cancer susceptibility, which melanoma is not a clinically feature, also increase the risk for melanoma, where some mela‐ noma cases have been reported. Individuals harboring germline mutations in *RB1* gene (Retinoblastoma); *TP53* and *CHEK2* genes (Li-Fraumeni and Li-Fraumeni Like syndrome respectively); *NF1* gene (Neurofibromatosis type 1); *Xeroderma Pigmentosum* genes (XP) and *BRCA2* were also associated with melanoma. Even melanomas cases were reported in such syndromes. The absolute low number of melanomas reported in these syndromes, especially in Li-Fraumeni syndrome, creates a debate regarding whether melanomas could be a rare manifestation of these cancer syndromes. A detailed discussion on the role of these genes and melanoma was published elsewhere [38].

#### **3.2. Low penetrance genes: The "new genetics" of melanoma**

The great development in low penetrance genes search for melanoma risk came with the development of genome-wide association studies (GWAS). With GWAS, several hundreds of thousands DNA variants can be detected and larger samples sizes can be used, thus increasing the power of analysis. A great advantage of using GWAS is the possibility to identify variants that are not located in protein coding regions. Coupled with the development of GWAS, the use of meta-analysis has also contributed to identify new low penetrance genes. Meta-analysis is a widely accepted method that summarizes the results from multiple published studies, then producing results with larger sample size and increasing statistical power. We discuss below the main findings regarding low-penetrance genes and melanoma of GWAS and meta-analysis studies in melanoma.

#### *3.2.1. MC1R gene – Coloring the knowledge of melanoma susceptibility*

The *MC1R* (melanocortin 1 receptor) is a critical gene related to human skin pigmentation. *MC1R* codes for a transmembrane protein receptor that binds to α-melanocyte stimulating hormone (α-MSH), upon binding the activation of adenylate cyclase is triggered and conse‐ quently intracellular cAMP levels increases, then leading to a switch in melanin production from pheomelanin pigments to eumelanin (a photoprotective pigment). The activation of MC1R is an integral part of the tanning response following UV irradiation.

*3.2.2. MITF*

As it is well established, melanin is one of the major protective factors against ultraviolet radiation DNA damage that results in melanoma development. The formation of this pigment is triggered by melanocyte-stimulating hormone, a peptide hormone coded by the the proopiomelancortin gene (*POMC*). Melanocyte-stimulating hormone binding to *MC1R* also results in the induction of microphthalmia-associated transcription factor (MITF) [47;48]. This transcription factor is coded by *MITF* gene located in chromosome 3 (3p14.2-p14.1) and it regulates a suite of genes involved in cell cycle control and melanogenesis [49]. These functions allow MITF to mediate differentiation and survival of melanocytes while limiting their uncontrolled progression. It was observed by Cheli et al. (2010) [49] that loss of *MITF* in the germline abolishes melanocyte formation in mice, whereas its loss in established melanocyte gives rise to their expansion [49]. MITF achieves this partly via inducing senescence through expression of p16INK4a, p21, and anti-apoptosis genes such as B-cell lymphoma 2 (*BCL2*) and apex nuclease 1 (*APEX1*) [49]. Recently, two independent groups identified a rare functional non-synonymous SNP (E318K) in *MITF* gene that alters MITF transcriptional activity, and it is associated with a large population-wide melanoma risk estimated between odds ratio 2.19 (95% CI 1.41, 3.45) and 4.78 (95% CI 2.05, 11.75) ([50],[51]; respectively). *MITF* gene is also associated with increased nevus count and non-blue eye color, consistent with its enhanced transcriptional ability. Adjusting for these traits reduced (odds ratio 1.82, 95% CI 0.85, 3.92)

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There is a positive feedback loop in melanocytes caused by UV radiation damage, which increases melanin production and blocks cell cycle progression via MITF until DNA damage is no longer detected [48]. Given its protective nature, melanoma researchers have spent significant effort testing skin coloration genes derived from animal studies or genetic association studies identified as targets of *MITF*, or highlighted by human pig‐

Genetic epidemiological studies have recently identified a subset of other pigmentation genes that are associated with risk for melanoma and other cutaneous malignancies as well as photosensitivity for MITF-regulated solute carrier family 45 member 2 gene - *SLC45A2* [54;55]. This gene is located in chromosome 5p, comprised of seven exons spanning 40 kb, and encodes a 530 amino acid protein presumably located in the melanosome membrane [56-57]. The protein SLC45A2 probably directs the traffic of melanosomal proteins and other substances to the melanosomes [57]. The mutation 1122C>G in *SLC45A2*gene, which results in non-synon‐ ymous amino acid change (Phe374Leu) has been related with pigmentation variation and ethnic ancestry in different populations [58]. However, according to meta-analysis that summarize some association studies [54;55], this mutation confers protection from cutaneous melanoma in individuals with a fair phenotype in populations from South European regions (France, Italy and Spain) – OR =0.41 (95% CI: 0.33–0.50). This meta-analysis may explain the

but did not abolish E318K association with melanoma [50].

mentation GWAS [52;53].

**SLC45A2**

*3.2.3. Other pigmentation genes*

The *MC1R* gene is highly polymorphic, a fact that denotes the huge variation in pigmentation phenotypes and skin colors in humans. This huge variation can create different haplotypes (many of them with amino acid substitutions) which have been show to modify the receptor functions altering the ratios of pheomelanin and eumelanin. The high levels of pheomelanin associated with some *MC1R* variants cause the red hair and fair skin phenotype. In European and Asian populations, there is considerable diversity of *MC1R* haplotypes, while in African populations the variation is less common, indicating an evolutional pressure to keep the high levels of eumelanin [39]. Germline variants that compromise the signaling of MC1R are present in about 80% of red hair and fair skin individuals; about 20% in individuals with brown or black hair and less than 4% in persons with a robust tanning response [40].

Epidemiological studies have indicated that red hair and fair skin are host characteristics predisposing to melanoma [11]. This phenotype is known to be more sensitive to harmful effects of UV exposure, mainly because the low capacity of tanning in red hair and fair skin individuals. As certain *MC1R* variants are strongly associated with skin color, and the type of skin color is associated with melanoma risk, it is not surprising therefore than some *MC1R* polymorphisms could influence susceptibility to melanoma development. Molecular epidemi‐ ology studies have reported melanoma patients as significantly harboring some *MC1R*variants more than control healthy subjects. Individuals that carry *MC1R* variants present a 2.2-to-3.9 fold risk to develop melanomas. Notably, there is an additive effect on having multiple variants, for example carriers of two *MC1R* variants have a 4.1-to-4.8 fold risk of developing melanoma [41-44].

In a recent meta-analysis, of 9 *MC1R* variants analyzed (V60L, D84E, V92M, R142H, R151C, I155T, R160W, R163Q, D294H), all variants were associated with melanoma risk. The odds ratio (OR) and the 95% confidence interval (95% CI), ranged from 1.18 (95% CI 1.04 – 1.35) to V60L to 2.40 (95% CI 1.64 – 3.51) to R142H [45]. Besides the risk values, the study showed a critical variation of a certain polymorphism among control and case populations. As example, to V60L variant, the frequency ranged from 5% in controls to 19.75 in cases, while to R160W, this variation was from 3.95% in controls to 11.64% in cases. The meta-analysis also validated the risk of melanoma associated with the so-called RHC and NRHC phenotypes [45]. The RHC phenotype (from red hair color) is defined by a nonfunctional melanocortin receptor, which leads to accumulation of pheomelanin, phenotype associated with fair skin, red hair, freckles and poor tanning ability [46]. Conversely, variants giving rise to receptors with a weak or without loss of function are called NRHC (from nonred hair color) convert pheomelanin into eumelanin less efficiently than control individuals. The RHC is composed by the variants R151C, R160W and D294H, a dominant effect of these variants is observed and the odds ratio to development of melanoma is 2.44 (95% CI 1.72 – 3.45) while to NRHC variants, the attributed odds ratio is 1.29 (95% CI 1.10 – 1.51) [45].

## *3.2.2. MITF*

hormone (α-MSH), upon binding the activation of adenylate cyclase is triggered and conse‐ quently intracellular cAMP levels increases, then leading to a switch in melanin production from pheomelanin pigments to eumelanin (a photoprotective pigment). The activation of

The *MC1R* gene is highly polymorphic, a fact that denotes the huge variation in pigmentation phenotypes and skin colors in humans. This huge variation can create different haplotypes (many of them with amino acid substitutions) which have been show to modify the receptor functions altering the ratios of pheomelanin and eumelanin. The high levels of pheomelanin associated with some *MC1R* variants cause the red hair and fair skin phenotype. In European and Asian populations, there is considerable diversity of *MC1R* haplotypes, while in African populations the variation is less common, indicating an evolutional pressure to keep the high levels of eumelanin [39]. Germline variants that compromise the signaling of MC1R are present in about 80% of red hair and fair skin individuals; about 20% in individuals with brown or

Epidemiological studies have indicated that red hair and fair skin are host characteristics predisposing to melanoma [11]. This phenotype is known to be more sensitive to harmful effects of UV exposure, mainly because the low capacity of tanning in red hair and fair skin individuals. As certain *MC1R* variants are strongly associated with skin color, and the type of skin color is associated with melanoma risk, it is not surprising therefore than some *MC1R* polymorphisms could influence susceptibility to melanoma development. Molecular epidemi‐ ology studies have reported melanoma patients as significantly harboring some *MC1R*variants more than control healthy subjects. Individuals that carry *MC1R* variants present a 2.2-to-3.9 fold risk to develop melanomas. Notably, there is an additive effect on having multiple variants, for example carriers of two *MC1R* variants have a 4.1-to-4.8 fold risk of developing

In a recent meta-analysis, of 9 *MC1R* variants analyzed (V60L, D84E, V92M, R142H, R151C, I155T, R160W, R163Q, D294H), all variants were associated with melanoma risk. The odds ratio (OR) and the 95% confidence interval (95% CI), ranged from 1.18 (95% CI 1.04 – 1.35) to V60L to 2.40 (95% CI 1.64 – 3.51) to R142H [45]. Besides the risk values, the study showed a critical variation of a certain polymorphism among control and case populations. As example, to V60L variant, the frequency ranged from 5% in controls to 19.75 in cases, while to R160W, this variation was from 3.95% in controls to 11.64% in cases. The meta-analysis also validated the risk of melanoma associated with the so-called RHC and NRHC phenotypes [45]. The RHC phenotype (from red hair color) is defined by a nonfunctional melanocortin receptor, which leads to accumulation of pheomelanin, phenotype associated with fair skin, red hair, freckles and poor tanning ability [46]. Conversely, variants giving rise to receptors with a weak or without loss of function are called NRHC (from nonred hair color) convert pheomelanin into eumelanin less efficiently than control individuals. The RHC is composed by the variants R151C, R160W and D294H, a dominant effect of these variants is observed and the odds ratio to development of melanoma is 2.44 (95% CI 1.72 – 3.45) while to NRHC variants, the attributed

MC1R is an integral part of the tanning response following UV irradiation.

92 Melanoma - From Early Detection to Treatment

black hair and less than 4% in persons with a robust tanning response [40].

melanoma [41-44].

odds ratio is 1.29 (95% CI 1.10 – 1.51) [45].

As it is well established, melanin is one of the major protective factors against ultraviolet radiation DNA damage that results in melanoma development. The formation of this pigment is triggered by melanocyte-stimulating hormone, a peptide hormone coded by the the proopiomelancortin gene (*POMC*). Melanocyte-stimulating hormone binding to *MC1R* also results in the induction of microphthalmia-associated transcription factor (MITF) [47;48]. This transcription factor is coded by *MITF* gene located in chromosome 3 (3p14.2-p14.1) and it regulates a suite of genes involved in cell cycle control and melanogenesis [49]. These functions allow MITF to mediate differentiation and survival of melanocytes while limiting their uncontrolled progression. It was observed by Cheli et al. (2010) [49] that loss of *MITF* in the germline abolishes melanocyte formation in mice, whereas its loss in established melanocyte gives rise to their expansion [49]. MITF achieves this partly via inducing senescence through expression of p16INK4a, p21, and anti-apoptosis genes such as B-cell lymphoma 2 (*BCL2*) and apex nuclease 1 (*APEX1*) [49]. Recently, two independent groups identified a rare functional non-synonymous SNP (E318K) in *MITF* gene that alters MITF transcriptional activity, and it is associated with a large population-wide melanoma risk estimated between odds ratio 2.19 (95% CI 1.41, 3.45) and 4.78 (95% CI 2.05, 11.75) ([50],[51]; respectively). *MITF* gene is also associated with increased nevus count and non-blue eye color, consistent with its enhanced transcriptional ability. Adjusting for these traits reduced (odds ratio 1.82, 95% CI 0.85, 3.92) but did not abolish E318K association with melanoma [50].

There is a positive feedback loop in melanocytes caused by UV radiation damage, which increases melanin production and blocks cell cycle progression via MITF until DNA damage is no longer detected [48]. Given its protective nature, melanoma researchers have spent significant effort testing skin coloration genes derived from animal studies or genetic association studies identified as targets of *MITF*, or highlighted by human pig‐ mentation GWAS [52;53].

#### *3.2.3. Other pigmentation genes*

#### **SLC45A2**

Genetic epidemiological studies have recently identified a subset of other pigmentation genes that are associated with risk for melanoma and other cutaneous malignancies as well as photosensitivity for MITF-regulated solute carrier family 45 member 2 gene - *SLC45A2* [54;55]. This gene is located in chromosome 5p, comprised of seven exons spanning 40 kb, and encodes a 530 amino acid protein presumably located in the melanosome membrane [56-57]. The protein SLC45A2 probably directs the traffic of melanosomal proteins and other substances to the melanosomes [57]. The mutation 1122C>G in *SLC45A2*gene, which results in non-synon‐ ymous amino acid change (Phe374Leu) has been related with pigmentation variation and ethnic ancestry in different populations [58]. However, according to meta-analysis that summarize some association studies [54;55], this mutation confers protection from cutaneous melanoma in individuals with a fair phenotype in populations from South European regions (France, Italy and Spain) – OR =0.41 (95% CI: 0.33–0.50). This meta-analysis may explain the incidence of melanoma in cases with skin phototypes III–IV, dark eye and hair color, absence of ephelides, lentigines and with a low number of nevi [57].

pigmentation genes. These findings emphasize the contribution of pigmentation pathways to melanoma predisposition and tumorigenesis through gene-environment interactions. Since pigmentation genes in the melanin synthesis pathway also confer risk for cutaneous malig‐ nancy, a better understanding of the operative molecular mechanisms involved in this relationship has the potential to impact individual risk assessment for cutaneous malignant

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Epidemiological and experimental data suggest that UV radiation is the main carcinogenic agent responsible for melanoma development. While UV-B radiation (290–320 nm) induces critical damage to DNA in the form of cyclobutane pyrimidine dimers (CPD) and pyrimidine photoproducts, UV-A radiation (320–400 nm) induces single strand breaks and generates free radicals that cause oxidative damage [69]. While UV-induced DNA damage often activates distinct DNA repair pathways that maintain genome integrity, the main processes involve the Base Excision Repair (BER), which operates mainly to repair damage caused by oxidative stress and single strand breaks and Nucleotide Excision Repair (NER) that acts to neutralize

The differences in DNA repair capacity among individuals are genetically determined in function of mutations and polymorphisms in many genes implicated in these pathways and it has been examined in relation to cutaneous malignant melanoma. According with a recent review [34], some studies found significant association between variations in DNA repair genes and melanoma. The gene *XPD*, located in 19q13.3, codes a protein that is involved in transcription-coupled nucleotide excision repair and is an integral member of the basal transcription factor BTF2/TFIIH complex. The SNP 13181 A>C in exon 23 of the gene, with amino acid change in protein (Lys751Gln) was described as a risk factor for cutaneous melanoma susceptibility, with an OR of 1.12 (95% CI, 1.03-1.21) [70]. Other polymorphisms in members of XP family genes involved with NER pathway were also described. Another recent study found melanoma protection for the *XPG* (13q33) 1104 His/His genotype (OR 0.32; 95% CI 0.13-0.75), and increased risk for three polymorphisms in chromosome 3p25 at *XPC* gene (OR 3.64; 95% CI 1.77-7.48) (PAT+;

In other repair pathways significant association has been described, for example variants in exon 7 of *XRCC3 (*14q32.3)*.* This gene encodes a member of the RecA/Rad51-related protein family that participates in homologous recombination to maintain chromosome stability and repair DNA damage. T241M *XRCC3* was associated with an increased risk for cutaneous melanoma [72]. Individuals who carry variant alleles had a decreased risk of cutaneous melanoma (OR 0.83, 95% CI, 0.79-0.98) [72]. Same results were found in a previous study [73]. An additional study reported a significant association between *MGMT* haplotypes and cutaneous melanoma risk, with a greater risk observed among 84Phe or 143Val carriers, who

A summary of the main founds regarding low penetrance genes and melanoma risk can be

*3.2.4. DNA repair genes – Polymorphisms contributing to a mutator phenotype?*

melanoma in the future [68].

found in Table 1.

photoproducts such as CPD and 6–4 dimers [2].

IV-6A and 939Gln), which represent a haplotype for *XPC* [71].

have a lower alkylation-damage repair capacity due to the variant alleles [74].

#### **ASIP**

Another pigmentation gene extensively studied in melanoma is *ASIP,* located in chromosome 20q11.22, which encodes agouti signaling protein. Agouti signalling protein (ASIP) was first described to inhibit eumelanogenesis in human melanocytes [59]. The protein ASIP is a MC1R ligand of 132 amino acids that antagonises the function of the transmembrane receptor [60]. According with a recent review [34], in a large study of European population descendants, a significant association was found between two SNP haplotype (rs1015362 and rs4911414), at the *ASIP* locus and cutaneous melanoma, with a modest OR =1.45 [61]. In another study [62], the haplotype near *ASIP* with same SNPs was associated with fair skin color (OR, 2.28; 95% CI, 1.46-3.57) as well as the risks of melanoma (OR 1.68; 95% CI 1.18-2.39). Similar results were described [63] in a German population study with increased risk to melanoma development in carriers of the rs4911414 variant (OR 1.27; 95% CI 1.03–1.57). An Australian genome-wide association study [64] also indicated the presence of a melanoma susceptibility locus on chromosome 20q11.22, with an OR of 1.72 for *ASIP* SNPs, (rs910873 and rs1885120). As the *ASIP* gene encodes the antagonist melanocortin receptor, polymorphisms of this gene can alter the protein conformation or decreased level of ASIP mRNA in melanocytes. As a consequence of low ASIP protein levels, its inhibiting effect is diminished, while eumelanogenesis is increased. If there are some altered ratio of pheomelanin and eumelanin caused by huge variation in *MC1R* gene, the high level of pheomelanin synthesis will increase, resulting in phenotypes with increased risk of cutaneous melanoma (red hair and fair skin phenotype).

#### **TYR**

The gene *TYR*, located in 11q14-q21, coded tyrosinase, which is a copper-dependent enzyme that catalyzes the first two steps during melanogenesis. The protein is required for the synthesis of both types of melanin, eumelanin and pheomelanin. While a basal activity of the enzyme leads to pheomelanin synthesis, a switch to eumelanogensis occurs upon increased protein activity. It has been reported that TYR presents higher enzymatic activity in a neutral environment than in acidic conditions. This formed the basis for the assumption that a neutral environment is required for the formation of eumelanosomes instead of pheomelanosomes and that the pH value is a control mechanism for melanin synthesis [65]. In a recent review of the literature [34] polymorphisms in *TYR* gene has also been implicated in cutaneous malig‐ nant melanoma susceptibility, where variants in coding region (rs1126809) of the gene increased melanoma risk (OR 1.27; 95% CI 1.16: 1.40 and OR 1.22 ; 95% CI 1.14 : 1.31) ([66] [67]; respectively).

Genome-wide association studies (GWAS) have unveiled single nucleotide polymorphisms (SNPs) or genetic variants in other genes involved with pigmentation pathways that can contribute to melanoma susceptibility. Examples follow; two pore segment channel 2 *(TPCN2*), KIT ligand (*KITLG*), solute carrier family 24, member 5 (*SLC24A5*), interferon regulatory factor 4 (*IRF4*), oculocutaneous albinism II (*OCA2*), HECT and RLD domain containing E3 ubiquitin protein ligase 2 (*HERC2*) and tyrosinase-related protein 1 (*TYRP1*) pigmentation genes. These findings emphasize the contribution of pigmentation pathways to melanoma predisposition and tumorigenesis through gene-environment interactions. Since pigmentation genes in the melanin synthesis pathway also confer risk for cutaneous malig‐ nancy, a better understanding of the operative molecular mechanisms involved in this relationship has the potential to impact individual risk assessment for cutaneous malignant melanoma in the future [68].

### *3.2.4. DNA repair genes – Polymorphisms contributing to a mutator phenotype?*

incidence of melanoma in cases with skin phototypes III–IV, dark eye and hair color, absence

Another pigmentation gene extensively studied in melanoma is *ASIP,* located in chromosome 20q11.22, which encodes agouti signaling protein. Agouti signalling protein (ASIP) was first described to inhibit eumelanogenesis in human melanocytes [59]. The protein ASIP is a MC1R ligand of 132 amino acids that antagonises the function of the transmembrane receptor [60]. According with a recent review [34], in a large study of European population descendants, a significant association was found between two SNP haplotype (rs1015362 and rs4911414), at the *ASIP* locus and cutaneous melanoma, with a modest OR =1.45 [61]. In another study [62], the haplotype near *ASIP* with same SNPs was associated with fair skin color (OR, 2.28; 95% CI, 1.46-3.57) as well as the risks of melanoma (OR 1.68; 95% CI 1.18-2.39). Similar results were described [63] in a German population study with increased risk to melanoma development in carriers of the rs4911414 variant (OR 1.27; 95% CI 1.03–1.57). An Australian genome-wide association study [64] also indicated the presence of a melanoma susceptibility locus on chromosome 20q11.22, with an OR of 1.72 for *ASIP* SNPs, (rs910873 and rs1885120). As the *ASIP* gene encodes the antagonist melanocortin receptor, polymorphisms of this gene can alter the protein conformation or decreased level of ASIP mRNA in melanocytes. As a consequence of low ASIP protein levels, its inhibiting effect is diminished, while eumelanogenesis is increased. If there are some altered ratio of pheomelanin and eumelanin caused by huge variation in *MC1R* gene, the high level of pheomelanin synthesis will increase, resulting in phenotypes with increased risk of cutaneous melanoma (red hair and fair skin phenotype).

The gene *TYR*, located in 11q14-q21, coded tyrosinase, which is a copper-dependent enzyme that catalyzes the first two steps during melanogenesis. The protein is required for the synthesis of both types of melanin, eumelanin and pheomelanin. While a basal activity of the enzyme leads to pheomelanin synthesis, a switch to eumelanogensis occurs upon increased protein activity. It has been reported that TYR presents higher enzymatic activity in a neutral environment than in acidic conditions. This formed the basis for the assumption that a neutral environment is required for the formation of eumelanosomes instead of pheomelanosomes and that the pH value is a control mechanism for melanin synthesis [65]. In a recent review of the literature [34] polymorphisms in *TYR* gene has also been implicated in cutaneous malig‐ nant melanoma susceptibility, where variants in coding region (rs1126809) of the gene increased melanoma risk (OR 1.27; 95% CI 1.16: 1.40 and OR 1.22 ; 95% CI 1.14 : 1.31) ([66]

Genome-wide association studies (GWAS) have unveiled single nucleotide polymorphisms (SNPs) or genetic variants in other genes involved with pigmentation pathways that can contribute to melanoma susceptibility. Examples follow; two pore segment channel 2 *(TPCN2*), KIT ligand (*KITLG*), solute carrier family 24, member 5 (*SLC24A5*), interferon regulatory factor 4 (*IRF4*), oculocutaneous albinism II (*OCA2*), HECT and RLD domain containing E3 ubiquitin protein ligase 2 (*HERC2*) and tyrosinase-related protein 1 (*TYRP1*)

of ephelides, lentigines and with a low number of nevi [57].

94 Melanoma - From Early Detection to Treatment

**ASIP**

**TYR**

[67]; respectively).

Epidemiological and experimental data suggest that UV radiation is the main carcinogenic agent responsible for melanoma development. While UV-B radiation (290–320 nm) induces critical damage to DNA in the form of cyclobutane pyrimidine dimers (CPD) and pyrimidine photoproducts, UV-A radiation (320–400 nm) induces single strand breaks and generates free radicals that cause oxidative damage [69]. While UV-induced DNA damage often activates distinct DNA repair pathways that maintain genome integrity, the main processes involve the Base Excision Repair (BER), which operates mainly to repair damage caused by oxidative stress and single strand breaks and Nucleotide Excision Repair (NER) that acts to neutralize photoproducts such as CPD and 6–4 dimers [2].

The differences in DNA repair capacity among individuals are genetically determined in function of mutations and polymorphisms in many genes implicated in these pathways and it has been examined in relation to cutaneous malignant melanoma. According with a recent review [34], some studies found significant association between variations in DNA repair genes and melanoma. The gene *XPD*, located in 19q13.3, codes a protein that is involved in transcription-coupled nucleotide excision repair and is an integral member of the basal transcription factor BTF2/TFIIH complex. The SNP 13181 A>C in exon 23 of the gene, with amino acid change in protein (Lys751Gln) was described as a risk factor for cutaneous melanoma susceptibility, with an OR of 1.12 (95% CI, 1.03-1.21) [70]. Other polymorphisms in members of XP family genes involved with NER pathway were also described. Another recent study found melanoma protection for the *XPG* (13q33) 1104 His/His genotype (OR 0.32; 95% CI 0.13-0.75), and increased risk for three polymorphisms in chromosome 3p25 at *XPC* gene (OR 3.64; 95% CI 1.77-7.48) (PAT+; IV-6A and 939Gln), which represent a haplotype for *XPC* [71].

In other repair pathways significant association has been described, for example variants in exon 7 of *XRCC3 (*14q32.3)*.* This gene encodes a member of the RecA/Rad51-related protein family that participates in homologous recombination to maintain chromosome stability and repair DNA damage. T241M *XRCC3* was associated with an increased risk for cutaneous melanoma [72]. Individuals who carry variant alleles had a decreased risk of cutaneous melanoma (OR 0.83, 95% CI, 0.79-0.98) [72]. Same results were found in a previous study [73]. An additional study reported a significant association between *MGMT* haplotypes and cutaneous melanoma risk, with a greater risk observed among 84Phe or 143Val carriers, who have a lower alkylation-damage repair capacity due to the variant alleles [74].

A summary of the main founds regarding low penetrance genes and melanoma risk can be found in Table 1.


**Figure 1.** A schematic view of the main genes and pathways related to melanoma progression. The genes and path‐ ways described are the here called "old genetics" of melanoma progression. Arrows indicate activation and blunt ar‐

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*4.1.1. Mitogen-Activated Protein Kinases (MAPK) pathway – MAPing the first melanoma progression*

Several molecular pathways are activated/deactivated during tumor formation and some of them are responsible for the development of specific phases of tumor progression. Among them, is the Mitogen-activated protein kinases (MAPK) pathway. The pathway consists in a chain-like activation cascade of serine/threonine-specific protein kinases, where one protein must be phosphorylated to activate another. The proteins involved in this pathway are the RAS oncogene, discovered in the early 80s, with three known isoforms (H-Ras, K-RAS and N-RAS); RAF kinase, with also three isoforms (A-RAF, B-RAF and C-RAF or RAF-1); MEK kinase and ERK kinase, which have cytoplasmic targets or can phosphorylate transcription factors in the nucleus. The MAPK pathway is one of the most well-known pathways involved not only in melanoma formation, but probably in most types of tumors. The pathway is responsible to conduct an extracellular signal, like growth signal, from receptors in cell surface towards cell nucleus. After activation of RAS, the first protein of the cascade, a multitude of cellular responses, like protein synthesis, regulation of cell survival, differentiation and proliferation can be observed, showing the importance of this pathway for melanoma progression. Muta‐ tions in MAPK pathway are necessary for the development of early stages melanomas, as the transfection of constitutively activated MEK into immortalized melanocytes is sufficient to

rows indicate inhibition.

*pathway*

**Table 1.** Summary of low-penetrance candidate melanoma susceptibility genes

## **4. Melanoma genetics: Progression genes**

#### **4.1. "Old genetics" of melanoma progression**

The here called "old-genetics" of melanoma progression consist of known genes which its functions are well described and are also related to several other cancer types, mainly due to its function in controlling survival and proliferation pathways. An overview of such "oldgenetics" of melanoma is shown in Figure 1.

**Gene/ Polymorphism rs OR (95% CI) References** *MC1R -* V60L 1805005 1.10 (1.04-1.35) [45] *MC1R -* D84E 1805006 1.67 (1.21-2.30) [45] *MC1R -* V92M 2228479 1.32 (1.04-1.68) [45] *MC1R -* R142H 11547464 2.40 (1.64-3.51) [45] *MC1R -* R151C 1805007 1.93 (1.54-2.41) [45] *MC1R -* I155T 1110400 1.39 (1.05-1.83) [45] *MC1R -* R160W 1805008 1.55 (1.21-1.97) [45] *MC1R -* R163Q 885479 1.21 (1.02-1.42) [45] *MC1R -* D294H 1805009 1.89 (1.39-2.56) [45]

*SLC45A2* - F374L 16891982 0.41 (0.33-0.50) [57]

*ASIP -* haplotype G;G 910873/ 1885120 1.72 (1.53, 2.01) [64]

*XPD -* K751Q 1052559 1.12 (1.03-1.21) [70] *XPG -* D1104H 17655 0.32 (0.13-0.75) [71] *XPC* - IV11-6C/A 3.10 (1.65–5.83) [71] *XPC* - K939Q 2228001 2.89 (1.52–5.50) [71] *XPC -* PAT(-/+) 3.27 (1.75–6.12) [71]

PAT+; 6A,Gln allele 3.64 (1.77–7.48) [71]

The here called "old-genetics" of melanoma progression consist of known genes which its functions are well described and are also related to several other cancer types, mainly due to its function in controlling survival and proliferation pathways. An overview of such "old-

12917/ 2308321

1015362/ 4911414

2.19 (1.41-3.45) 4.78 (2.05-11.75)

1.45 (P = 1.2 x 10-9) 1.68 (1.18-2.39) 1.27 (1.03-1.57)

1.27 (1.16-1.40) 1.22 (1.14-1.31)

0.83 (0.79-0.98) 2.36 (1.44–3.86)

1.75 (1.11-2.76) [74]

[50] [51]

[61] [62] [63]

[66] [67]

[72] [73]

*MITF -* E318K 149617956

*TYR -* R402Q 1126809

*XRCC3 -* T241M 861539

**4. Melanoma genetics: Progression genes**

**4.1. "Old genetics" of melanoma progression**

genetics" of melanoma is shown in Figure 1.

**Table 1.** Summary of low-penetrance candidate melanoma susceptibility genes

*ASIP -* haplotype G;T

96 Melanoma - From Early Detection to Treatment

XPC haplotype

*MGMT* haplotype L84F/ I143V

**Figure 1.** A schematic view of the main genes and pathways related to melanoma progression. The genes and path‐ ways described are the here called "old genetics" of melanoma progression. Arrows indicate activation and blunt ar‐ rows indicate inhibition.

#### *4.1.1. Mitogen-Activated Protein Kinases (MAPK) pathway – MAPing the first melanoma progression pathway*

Several molecular pathways are activated/deactivated during tumor formation and some of them are responsible for the development of specific phases of tumor progression. Among them, is the Mitogen-activated protein kinases (MAPK) pathway. The pathway consists in a chain-like activation cascade of serine/threonine-specific protein kinases, where one protein must be phosphorylated to activate another. The proteins involved in this pathway are the RAS oncogene, discovered in the early 80s, with three known isoforms (H-Ras, K-RAS and N-RAS); RAF kinase, with also three isoforms (A-RAF, B-RAF and C-RAF or RAF-1); MEK kinase and ERK kinase, which have cytoplasmic targets or can phosphorylate transcription factors in the nucleus. The MAPK pathway is one of the most well-known pathways involved not only in melanoma formation, but probably in most types of tumors. The pathway is responsible to conduct an extracellular signal, like growth signal, from receptors in cell surface towards cell nucleus. After activation of RAS, the first protein of the cascade, a multitude of cellular responses, like protein synthesis, regulation of cell survival, differentiation and proliferation can be observed, showing the importance of this pathway for melanoma progression. Muta‐ tions in MAPK pathway are necessary for the development of early stages melanomas, as the transfection of constitutively activated MEK into immortalized melanocytes is sufficient to induce tumorigenesis in nude mice, activation of the angiogenic switch, and increased production of the pro-angiogenic factor, vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs) [75].

but can also induce DNA repair, regulates autophagy and MMPs expression. The link between MAPK pathway and E2F transcription factor family may provide new strategies for melanoma treatment. New drugs using the B-RafV600E mutation as a target is current‐ ly being used in the clinics. Vemurafenib (PLX-4032) is a novel treatment for metastatic disease for melanomas with the V600E mutation. Vemurafenib treatment has demonstrat‐ ed improved progression-free and prolonging overall survival in three months, com‐ pared with chemotherapy in a randomized trial, and represents a new standard of care in patients with advanced melanoma harboring a BRAF-V600 mutation [83]. However, Vemurafenib treatment induces several resistance pathways in B-RafV600E cells and is ex‐ pected to failure after a few months, but it is the best treatment for melanoma disease so far. Among the resistance pathway induced by the drug are MEK activation by MAP3K8 [84], up regulation of N-RAS [85] and activation of fibroblast growth factor receptor 3

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RAS can also activate other effectors pathways rather than RAF. RAS can interact directly with phosphatidylinositol 3-kinases (PI3Ks), activating other molecular pathways. One of the pathways activated by PI3Ks is the AKT/PKB pathway, which has a strong anti-apoptotic function by phosphorylating various targets and seems to be an important part of the survival

MAPK activation is necessary for early stages melanomas, but is not sufficient for the devel‐ opment of advanced disease. Other molecular mechanisms are necessary for melanoma invade other tissues and survive in different microenvironments. AKT/PKB seems to be important for the development of radial growth melanomas, from cell lines which are characterized as radial growth melanomas. In this model, AKT overexpression induced VEGF expression and switched to a more glycolytic metabolism [87]. The AKT family consists of three members, AKT1–3 and 43–50% of melanomas have a selective constitutively active AKT3. AKT3 overexpression may occur as a result of copy number increases in the long arm of chromosome 1. Another mechanism for PI3K/AKT pathway activation in melanoma is through the acquis‐ ition of activating E17K mutations in AKT3. AKT has a critical role in cancer development through its ability to block apoptosis through the direct phosphorylation of BAD as well as through its effects in many other pathways, including the inhibition of forkhead signaling and the inhibition of glycogen synthase kinase-3. One of the most critical regulators of AKT is the phosphatase and tensin homolog (PTEN), which degrades the products of PI3K, preventing AKT activation. The mechanism by which the PI3K/AKT pathway is activated in melanoma may involve the loss of expression or functional inactivation of PTEN [88]. However, PI3K pathway mutations, though more heterogeneous, were present in 41% of the melanoma, with

PTEN being the highest mutated gene of the PI3K pathway in melanomas (22%) [89].

*PTEN* is a tumor suppressor gene located in chromosome 10q23.3 and is a dual specificity phosphatase capable of dephosphorylating both tyrosine phosphate and serine/threonine phosphate residues in proteins. It also functions as a major lipid phosphatase, counteracting PI3K by dephosphorylating the second messengers phosphatidylinositol-3,4,5-triphosphate

*4.1.2. PI3K pathway – Supporting MAPK pathway to melanoma progression*

signal that is generated by RAS activation.

(FGFR3) [86].

The most common mutations found in MAPK proteins in melanomas are in RAS protein, more specific in N-RAS and in RAF proteins, in B-RAF. The RAS proteins are members of a large superfamily of low molecular-weight GTP-binding proteins. The activation state of RAS proteins depends on whether they are bound to GTP (in which case, they are active and are able to engage downstream target enzyme) or GDP (in which case, they are inactive and fail to interact with these effectors). In normal cells, the activity of RAS proteins is controlled by the ratio of bound GTP to GDP [76]. N-RAS mutations can be found in over 15% of all melanoma cases and are most commonly the result of the substitution from leucine to glutamine at position 61 [77]. It is correlated to the vertical growth phase of melanoma progression. Although initially thought to occur mainly at the plasma membrane, there is increasing evidence that isoform-specific RAS signaling can take place at different cellular compartments and within different regions of the plasma membrane. Such compartmentali‐ zation and trafficking of endogenous RAS oncogenes is likely to play an important role in regulating downstream signaling processes involved in tumorigenesis [78]. For its activation and function as a signal transducer, N-RAS needs to be modified by a farnesylation near its Cterminal domain. Several farnesylation inhibitors were tested in the clinics and all results were disappointing [79]. In part, the failure of the clinical trials can be explained due to the fact that the farnesylation inhibitors may work in Rho (a subfamily of RAS superfamily) rather than RAS, or the fact that the inhibitors works on normal and mutated RAS.

Other important component of MAPK pathway that is mutated in melanomas is the RAF kinase B-RAF, the primer mediator of RAS protein. Some reports have shown that over 60% of all melanoma cases have mutation in B-RAF [80]. RAF mutations occur in the kinase domains and the most common mutation found in melanomas, approximately 80%, is the substitution of valine at position 600 with glutamic acid also called B-RAFV600E mutation. This mutation creates a constitutively active status for B-RAF, independently of a previous activation by RAS oncogene and extracellular stimulus and it is more frequently found in skin of individuals with intermittent sun exposure than unexposed or chronically sun-damaged skin. Interestingly, B-RAF mutation frequency in benign melanocytic nevi seems to be equal or even higher than in that for melanomas. The frequency also varies, like melanomas, from 0% in Spitz nevi up to 90% in intradermal nevi. These differences, between B-RAF mutation in nevi and melanomas make the assessment of the impact of these mutations on prognosis difficult to determine [38]. B-RAF mutation in nevi might be a critical step in melanoma development, suggesting its importance in early stages of the disease.

Melanomas usually do not have B-RafV600E mutation at the same time they have muta‐ tions in any RAS isotype. However, some small proportions of cases carry mutation in both B-RAF and any RAS isoform, but in these cases, B-RAF mutation almost never is in V600E locus [81]. Recently, a link between B-RAF and the cell cycle controller E2F has been shown. B-RAF is able to phosphorylate the retinoblastoma (Rb) protein and release E2F transcription factor family to work [82]. E2F family is a classic cell cycle controller, but can also induce DNA repair, regulates autophagy and MMPs expression. The link between MAPK pathway and E2F transcription factor family may provide new strategies for melanoma treatment. New drugs using the B-RafV600E mutation as a target is current‐ ly being used in the clinics. Vemurafenib (PLX-4032) is a novel treatment for metastatic disease for melanomas with the V600E mutation. Vemurafenib treatment has demonstrat‐ ed improved progression-free and prolonging overall survival in three months, com‐ pared with chemotherapy in a randomized trial, and represents a new standard of care in patients with advanced melanoma harboring a BRAF-V600 mutation [83]. However, Vemurafenib treatment induces several resistance pathways in B-RafV600E cells and is ex‐ pected to failure after a few months, but it is the best treatment for melanoma disease so far. Among the resistance pathway induced by the drug are MEK activation by MAP3K8 [84], up regulation of N-RAS [85] and activation of fibroblast growth factor receptor 3 (FGFR3) [86].

#### *4.1.2. PI3K pathway – Supporting MAPK pathway to melanoma progression*

induce tumorigenesis in nude mice, activation of the angiogenic switch, and increased production of the pro-angiogenic factor, vascular endothelial growth factor (VEGF), and

The most common mutations found in MAPK proteins in melanomas are in RAS protein, more specific in N-RAS and in RAF proteins, in B-RAF. The RAS proteins are members of a large superfamily of low molecular-weight GTP-binding proteins. The activation state of RAS proteins depends on whether they are bound to GTP (in which case, they are active and are able to engage downstream target enzyme) or GDP (in which case, they are inactive and fail to interact with these effectors). In normal cells, the activity of RAS proteins is controlled by the ratio of bound GTP to GDP [76]. N-RAS mutations can be found in over 15% of all melanoma cases and are most commonly the result of the substitution from leucine to glutamine at position 61 [77]. It is correlated to the vertical growth phase of melanoma progression. Although initially thought to occur mainly at the plasma membrane, there is increasing evidence that isoform-specific RAS signaling can take place at different cellular compartments and within different regions of the plasma membrane. Such compartmentali‐ zation and trafficking of endogenous RAS oncogenes is likely to play an important role in regulating downstream signaling processes involved in tumorigenesis [78]. For its activation and function as a signal transducer, N-RAS needs to be modified by a farnesylation near its Cterminal domain. Several farnesylation inhibitors were tested in the clinics and all results were disappointing [79]. In part, the failure of the clinical trials can be explained due to the fact that the farnesylation inhibitors may work in Rho (a subfamily of RAS superfamily) rather than

RAS, or the fact that the inhibitors works on normal and mutated RAS.

Other important component of MAPK pathway that is mutated in melanomas is the RAF kinase B-RAF, the primer mediator of RAS protein. Some reports have shown that over 60% of all melanoma cases have mutation in B-RAF [80]. RAF mutations occur in the kinase domains and the most common mutation found in melanomas, approximately 80%, is the substitution of valine at position 600 with glutamic acid also called B-RAFV600E mutation. This mutation creates a constitutively active status for B-RAF, independently of a previous activation by RAS oncogene and extracellular stimulus and it is more frequently found in skin of individuals with intermittent sun exposure than unexposed or chronically sun-damaged skin. Interestingly, B-RAF mutation frequency in benign melanocytic nevi seems to be equal or even higher than in that for melanomas. The frequency also varies, like melanomas, from 0% in Spitz nevi up to 90% in intradermal nevi. These differences, between B-RAF mutation in nevi and melanomas make the assessment of the impact of these mutations on prognosis difficult to determine [38]. B-RAF mutation in nevi might be a critical step in melanoma development, suggesting its

Melanomas usually do not have B-RafV600E mutation at the same time they have muta‐ tions in any RAS isotype. However, some small proportions of cases carry mutation in both B-RAF and any RAS isoform, but in these cases, B-RAF mutation almost never is in V600E locus [81]. Recently, a link between B-RAF and the cell cycle controller E2F has been shown. B-RAF is able to phosphorylate the retinoblastoma (Rb) protein and release E2F transcription factor family to work [82]. E2F family is a classic cell cycle controller,

matrix metalloproteinases (MMPs) [75].

98 Melanoma - From Early Detection to Treatment

importance in early stages of the disease.

RAS can also activate other effectors pathways rather than RAF. RAS can interact directly with phosphatidylinositol 3-kinases (PI3Ks), activating other molecular pathways. One of the pathways activated by PI3Ks is the AKT/PKB pathway, which has a strong anti-apoptotic function by phosphorylating various targets and seems to be an important part of the survival signal that is generated by RAS activation.

MAPK activation is necessary for early stages melanomas, but is not sufficient for the devel‐ opment of advanced disease. Other molecular mechanisms are necessary for melanoma invade other tissues and survive in different microenvironments. AKT/PKB seems to be important for the development of radial growth melanomas, from cell lines which are characterized as radial growth melanomas. In this model, AKT overexpression induced VEGF expression and switched to a more glycolytic metabolism [87]. The AKT family consists of three members, AKT1–3 and 43–50% of melanomas have a selective constitutively active AKT3. AKT3 overexpression may occur as a result of copy number increases in the long arm of chromosome 1. Another mechanism for PI3K/AKT pathway activation in melanoma is through the acquis‐ ition of activating E17K mutations in AKT3. AKT has a critical role in cancer development through its ability to block apoptosis through the direct phosphorylation of BAD as well as through its effects in many other pathways, including the inhibition of forkhead signaling and the inhibition of glycogen synthase kinase-3. One of the most critical regulators of AKT is the phosphatase and tensin homolog (PTEN), which degrades the products of PI3K, preventing AKT activation. The mechanism by which the PI3K/AKT pathway is activated in melanoma may involve the loss of expression or functional inactivation of PTEN [88]. However, PI3K pathway mutations, though more heterogeneous, were present in 41% of the melanoma, with PTEN being the highest mutated gene of the PI3K pathway in melanomas (22%) [89].

*PTEN* is a tumor suppressor gene located in chromosome 10q23.3 and is a dual specificity phosphatase capable of dephosphorylating both tyrosine phosphate and serine/threonine phosphate residues in proteins. It also functions as a major lipid phosphatase, counteracting PI3K by dephosphorylating the second messengers phosphatidylinositol-3,4,5-triphosphate (PIP3) and phosphatidylinositol-3,4-diphosphate (PIP2), which are required for the activation of AKT/PKB [17]. PTEN can work in other pathways than AKT/PKB. PTEN is involved in cell migration, spreading, and focal adhesion formation through direct dephosphorylation and inactivation of focal adhesion kinase (FAK). Also, PTEN inhibits Shc phosphorylation, preventing the association of Shc with Grb2/Sos and activation of the Ras/Raf/MEK1/MAPK pathway. PTEN suppresses the stabilization of hypoxia-mediated HIF-1*α*, which when stabilized through the PI3K/AKT pathway, upregulates VEGF expression suggesting a possible role for PTEN in angiogenesis [88]. An interesting study sequenced the PTEN gene from melanomas from patients harboring the *Xeroderma Pigmentosum* syndrome [90]. A total of 59 melanomas from 8 XP patients showed a mutation rate of 56% in *PTEN* gene. A detailed look for the mutational spectrum revealed that 91% of the melanomas with mutations had 1 to 4 UV type mutations (C to T changes) occurring at adjacent pyrimidines. Functional analyses also indicated impared PTEN function caused by the mutations. The study showed critical data to the understanding of melanoma progression in XP patients.

**4.2. "New genetics" of melanoma progression**

melanoma progression are represented in Figure 2.

rows indicate inhibition.

Melanoma is a complex genetic disease. Recent studies have begun to characterize the mechanisms underlying melanoma plasticity, relating to intratumoral switching between varying malignant capacities, such as proliferation, invasion, or tumorigenesis. The rate at which somatic and germline genetic alterations have been cataloged in melanoma has accelerated greatly in recent years. The ability to modulate genes and proteins of interest, even when pharmacologic agents are not available, has provided preclinical evidence that many putative oncogenes represent potential therapeutic targets [93]. At the same time, the notorious resistance of melanoma to treatments with its strong potential to metastasize represents the major clinical obstacle in the treatment of these tumors. These observations allow the scientists to improve staging and subtype classification and lead them to design better therapeutic agents and approaches. New insights about genetics of melanoma, including high-throughput strategies such as gene expression microarrays, comparative genomic hybridization, mutation analysis by deep sequencing and microRNAs gene regulation have helped researchers to elucidate the crucial cell-signaling pathways or validate the already postulated pathways as modified in melanomas. The genes and pathways discussed below for the "new genetics" of

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**Figure 2.** A schematic view of the main genes and pathways related to melanoma progression. The genes and path‐ ways described are the here called "new genetics" of melanoma progression. Arrows indicate activation and blunt ar‐

#### *4.1.3. WNT5A – Progression to the edges, leading to melanoma metastasis*

The metastatic disease does not have fixed histopathological subclasses. That is why there is a need to look for genetic profiles that could predict a behavior in advanced stages. WNT5A, a protein of Wnt family, was identified as the gene that best defined the new subclasses of tumors. The Wnt family of proteins has over 19 members, all of which are secreted, that are very closely structurally related. The activation of Wnt signaling can have very different results depending on which members of the family are involved. Wnt proteins work through three different pathways: the *β*-catenin pathway, the Wnt/Ca2+ pathway and the planar cell polarity pathway. The activation of WNT5A in melanomas uses the non-canonical pathway Wnt/Ca2+ together with Frizzled receptors, activating phospholipase C, which translocate to the membrane and hydrolyzes membrane phospholipids, initiating phosphatidylinositol signal‐ ing [91]. *In vitro* analysis of melanoma cell lines differing in WNT5A expression levels showed that WNT5A overexpression is correlated with increased motility and invasiveness of the cell [91]. WNT5A correlates with high aggressive metastatic disease and its activation is mediated through PKC pathways which are associated to cytoskeletal organization and invasion. WNT5A protein expression in human melanoma biopsies directly correlates with increasing tumor grade while inversely correlating with patient survival [92]. Members of the Wnt pathways have been identified in melanoma. WNT5A and others members like Rho pathway and frizzled 7 may play an important role in transition of melanoma from VGP to metastases. It is very likely that the temporal activation of Wnt pathways is very important for melanoma development and progression. It would not be surprising if *β*-catenin expression was an early event, and metastatic cells need to down regulate expression of this protein prior to invading, and escaping the immune system. WNT5A may provide a survival advantage to melanoma cells, despite the fact that in others tumor it may act as a tumor suppressor. Thus, its early expression may result in suppression of tumorigenesis, whereas if it is expressed at a later stage, it becomes a potent inducer of migration and motility. Wnt signaling and its effects on melanoma establishment and progression are complex, and surely temporal and context dependent [92].

#### **4.2. "New genetics" of melanoma progression**

(PIP3) and phosphatidylinositol-3,4-diphosphate (PIP2), which are required for the activation of AKT/PKB [17]. PTEN can work in other pathways than AKT/PKB. PTEN is involved in cell migration, spreading, and focal adhesion formation through direct dephosphorylation and inactivation of focal adhesion kinase (FAK). Also, PTEN inhibits Shc phosphorylation, preventing the association of Shc with Grb2/Sos and activation of the Ras/Raf/MEK1/MAPK pathway. PTEN suppresses the stabilization of hypoxia-mediated HIF-1*α*, which when stabilized through the PI3K/AKT pathway, upregulates VEGF expression suggesting a possible role for PTEN in angiogenesis [88]. An interesting study sequenced the PTEN gene from melanomas from patients harboring the *Xeroderma Pigmentosum* syndrome [90]. A total of 59 melanomas from 8 XP patients showed a mutation rate of 56% in *PTEN* gene. A detailed look for the mutational spectrum revealed that 91% of the melanomas with mutations had 1 to 4 UV type mutations (C to T changes) occurring at adjacent pyrimidines. Functional analyses also indicated impared PTEN function caused by the mutations. The study showed critical

The metastatic disease does not have fixed histopathological subclasses. That is why there is a need to look for genetic profiles that could predict a behavior in advanced stages. WNT5A, a protein of Wnt family, was identified as the gene that best defined the new subclasses of tumors. The Wnt family of proteins has over 19 members, all of which are secreted, that are very closely structurally related. The activation of Wnt signaling can have very different results depending on which members of the family are involved. Wnt proteins work through three different pathways: the *β*-catenin pathway, the Wnt/Ca2+ pathway and the planar cell polarity pathway. The activation of WNT5A in melanomas uses the non-canonical pathway Wnt/Ca2+ together with Frizzled receptors, activating phospholipase C, which translocate to the membrane and hydrolyzes membrane phospholipids, initiating phosphatidylinositol signal‐ ing [91]. *In vitro* analysis of melanoma cell lines differing in WNT5A expression levels showed that WNT5A overexpression is correlated with increased motility and invasiveness of the cell [91]. WNT5A correlates with high aggressive metastatic disease and its activation is mediated through PKC pathways which are associated to cytoskeletal organization and invasion. WNT5A protein expression in human melanoma biopsies directly correlates with increasing tumor grade while inversely correlating with patient survival [92]. Members of the Wnt pathways have been identified in melanoma. WNT5A and others members like Rho pathway and frizzled 7 may play an important role in transition of melanoma from VGP to metastases. It is very likely that the temporal activation of Wnt pathways is very important for melanoma development and progression. It would not be surprising if *β*-catenin expression was an early event, and metastatic cells need to down regulate expression of this protein prior to invading, and escaping the immune system. WNT5A may provide a survival advantage to melanoma cells, despite the fact that in others tumor it may act as a tumor suppressor. Thus, its early expression may result in suppression of tumorigenesis, whereas if it is expressed at a later stage, it becomes a potent inducer of migration and motility. Wnt signaling and its effects on melanoma establishment and progression are complex, and surely temporal and context

data to the understanding of melanoma progression in XP patients.

100 Melanoma - From Early Detection to Treatment

*4.1.3. WNT5A – Progression to the edges, leading to melanoma metastasis*

dependent [92].

Melanoma is a complex genetic disease. Recent studies have begun to characterize the mechanisms underlying melanoma plasticity, relating to intratumoral switching between varying malignant capacities, such as proliferation, invasion, or tumorigenesis. The rate at which somatic and germline genetic alterations have been cataloged in melanoma has accelerated greatly in recent years. The ability to modulate genes and proteins of interest, even when pharmacologic agents are not available, has provided preclinical evidence that many putative oncogenes represent potential therapeutic targets [93]. At the same time, the notorious resistance of melanoma to treatments with its strong potential to metastasize represents the major clinical obstacle in the treatment of these tumors. These observations allow the scientists to improve staging and subtype classification and lead them to design better therapeutic agents and approaches. New insights about genetics of melanoma, including high-throughput strategies such as gene expression microarrays, comparative genomic hybridization, mutation analysis by deep sequencing and microRNAs gene regulation have helped researchers to elucidate the crucial cell-signaling pathways or validate the already postulated pathways as modified in melanomas. The genes and pathways discussed below for the "new genetics" of melanoma progression are represented in Figure 2.

**Figure 2.** A schematic view of the main genes and pathways related to melanoma progression. The genes and path‐ ways described are the here called "new genetics" of melanoma progression. Arrows indicate activation and blunt ar‐ rows indicate inhibition.

## *4.2.1. Activating Transcription Factor 2 (ATF2) – Helping melanoma progression activation*

exhibit higher levels of phosphorylated ATF2 compared to immortalized non-malignant mouse melanocytes. Following treatment with retinoic acid, ATF2 phosphorylation was reduced, resulting in c-Jun dimerization with c-Fos and promoting a shift from proliferation towards differentiation [103]. Additional experiments showed that delivery of ATF2 inhibitory

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Even with these encouraging results, one question remains unanswered: how ATF2 inhibition induces apoptosis in melanoma cells? It was demonstrated that ATF250–100 induced apoptosis by sequestering ATF2 to the cytoplasm, thereby inhibiting its transcriptional activities [105]. In addition, mutations within the c-Jun N-terminal kinases (JNK) binding region of ATF250– <sup>100</sup> or expression of TAM67, a dominant negative of the Jun family of transcription factors, or JunD-RNA interference attenuate inhibition of melanoma tumorigenicity by ATF250–100. The JNKs are kinases responsive to stress stimuli, such as ultraviolet irradiation used in this study. These results were crucial to show that inhibition of ATF2 in concert with increased JNK/Jun activities is central for the sensitization of melanoma cells to apoptosis and inhibition of their tumorigenicity. Furthermore, ATF250–100 increases ATF2 localization within the cytoplasm. Indeed, one study evaluating the ATF2 as a prognostic marker among patients with melano‐ mas validated this result. A study to determine the prognostic value of ATF2 evaluating the pattern and level of its expression in a tissue microarray was conducted [106]. Cytoplasmic ATF2 expression was associated with primary tumor rather than metastases and with better patient survival whereas nuclear ATF2 expression was associated with metastatic tumor and with poor survival. Nuclear ATF2 seems to be transcriptionally active while cytoplasmic ATF2 probably represents an inactive form. These findings support one preclinical finding in which transcriptionally active ATF2 is involved in tumor progression-proliferation in melanoma, suggesting that ATF2 might be a useful prognostic marker in early-stage melanoma. Although the use peptide ATF250–100 have shown good results to sensitize melanoma cells to treatments, Ronai group´s continued investigating peptides with smaller size but producing the same effect. In 2004, Bhoumik *et al*. [107] presented one peptide with only 10aa - ATF251-60. This peptide sensitizes melanoma cells to spontaneous apoptosis and inhibits the *in vivo* growth. Furthermore, the ATF251-60 expression coincides with activation of caspase 9, an important molecule activated during apoptosis. This study points to mechanisms underlying the

peptides elicited efficient inhibition of melanoma tumor growth [104].

activities of the ATF2 peptide while highlighting its possible use in drug design.

Based on these findings, ATF2 present oncogenic action, but could it act as one tumor sup‐ pressor molecule? Although genetic changes in ATF2 have not been identified in human tumors, many data sustain the notion that ATF2 is not only oncogenic, whereas its altered expression and sub cellular localization is associated with tumor stage and prognosis in melanomas, but it also acts as a tumor suppressor molecule, under specific conditions. This hypothesis arose from independent studies with skin and mammary tumors. Studies from a mouse mammary tumor model revealed that loss of ATF2 *per se*, does not promote mammary tumor formation, but heterozygous mouse *ATF2* mutants developed mammary tumors when crossed with p53 mutant mice, indicating that ATF2 may have a suppressor function only when combined with a p53 mutant background [108]. Likewise, loss of ATF2 transcriptional activities in keratinocytes promotes faster development of skin papillomas. Deletion of

The ATF2 (Activating Transcription Factor-2 or cAMP response element [CRE]) it was first identified as an inducible enhancer of genes that can be transcribed in response to increased cAMP levels and mediates various transcriptional regulatory effects, for example, ATF2/Jun complex is implicated in multiple cellular processes [94,95]. The ATF2 transcriptional targets genes is divided into (a) regulation of transcription factors and proteins engaged in stress and DNA damage response (b) regulation of genes associated with growth and tumorigenesis (c) regulation of genes important for maintenance and physiological homeostasis [94]. In addition to its function as a transcription factor, ATF2 was found to play an important role in DNA damage response. After damage occurs, ATF2 is phosphorylated by ATM and its results in rapid localization of ATF2 to ionizing radiation (IR) induced foci (IRIF), which contain DNA repair proteins and chromatin-modifying enzymes. Furthermore, ATF2 phosphorylated is required for an intact intra-S-phase checkpoint response necessary to stop DNA replication [96]. In recent years, the study of ATF2 activity in melanoma cells has revealed a probably oncogenic function. In the early '90s, Ronai and Weinstein [97] elucidated the cellular response to UV irradiation. The authors characterized a UV-responsive element (URE;TGACAACA) and soon after, its binding proteins, AP1 and ATF family members [98]. Interesting, the URE was found within the promoter sequences of stress-responsive genes, including c-jun, DNA polymerase B, and cyclin A, as well as on regulatory regions of viruses that respond to UV irradiation [99]. Differences in transcriptional activities of URE-bound proteins were found after UV-irradiation of keratinocytes, melanocytes and melanoma, and also in repair deficient cells of patients with *Xeroderma pigmentosum*, or Cockayne syndrome [for review 9]. In 1998, the Ronai´s group investigated which of the CREB-associated proteins is directly involved in modifying specific characteristics of melanoma phenotypes. They demonstrate that ATF2 is the primary binding protein and regulator of URE-mediated transcription and it contributes specifically to radiation resistance of human melanoma cells.

An approach to selectively inhibit ATF2 activity in human melanoma was designed, based on peptides derived from ATF2 trans-activating domain which affect ATF2 transcriptional activity. In an attempt to sensitize melanoma cells to UV irradiation, Ronai *et al*. investigated the ability of cells to enter in apoptosis competing by endogenous ATF2 expression with ATF2 derived peptide(s) alone and/or combined with inhibition of p38 activity (one Mitogenactivated Protein Kinases that is responsive to stress stimuli) via its pharmacological inhibitor (SB203580) [100]. The expression of a 50-amino acid peptide derived from the NH2-terminal domain of ATF2 (ATF250–100) was sufficient to sensitize melanoma cells to radiation. Combi‐ nation of this peptide with SB203580 induced programmed cell death in late stage melanoma cells via Fas signaling, whereas Fas ligand/receptor interactions play an important role in the progression of cancer. In 2002, experimental mouse models validated the expression of this peptide. The ATF250–100 not only sensitized melanoma cells to apoptosis but efficiently inhibited tumor growth and metastasis [101]. Analysis of mouse cell lines derived from melanomas formed in the HGF/SF (Hepatocyte Growth Factor/Scatter Factor) transgenic mouse, revealed that the proliferation rate in culture increased with increased ATF-2 activity [102], confirming the role of ATF2 in melanoma development. Along these lines, B16 mouse melanoma cells exhibit higher levels of phosphorylated ATF2 compared to immortalized non-malignant mouse melanocytes. Following treatment with retinoic acid, ATF2 phosphorylation was reduced, resulting in c-Jun dimerization with c-Fos and promoting a shift from proliferation towards differentiation [103]. Additional experiments showed that delivery of ATF2 inhibitory peptides elicited efficient inhibition of melanoma tumor growth [104].

*4.2.1. Activating Transcription Factor 2 (ATF2) – Helping melanoma progression activation*

102 Melanoma - From Early Detection to Treatment

specifically to radiation resistance of human melanoma cells.

The ATF2 (Activating Transcription Factor-2 or cAMP response element [CRE]) it was first identified as an inducible enhancer of genes that can be transcribed in response to increased cAMP levels and mediates various transcriptional regulatory effects, for example, ATF2/Jun complex is implicated in multiple cellular processes [94,95]. The ATF2 transcriptional targets genes is divided into (a) regulation of transcription factors and proteins engaged in stress and DNA damage response (b) regulation of genes associated with growth and tumorigenesis (c) regulation of genes important for maintenance and physiological homeostasis [94]. In addition to its function as a transcription factor, ATF2 was found to play an important role in DNA damage response. After damage occurs, ATF2 is phosphorylated by ATM and its results in rapid localization of ATF2 to ionizing radiation (IR) induced foci (IRIF), which contain DNA repair proteins and chromatin-modifying enzymes. Furthermore, ATF2 phosphorylated is required for an intact intra-S-phase checkpoint response necessary to stop DNA replication [96]. In recent years, the study of ATF2 activity in melanoma cells has revealed a probably oncogenic function. In the early '90s, Ronai and Weinstein [97] elucidated the cellular response to UV irradiation. The authors characterized a UV-responsive element (URE;TGACAACA) and soon after, its binding proteins, AP1 and ATF family members [98]. Interesting, the URE was found within the promoter sequences of stress-responsive genes, including c-jun, DNA polymerase B, and cyclin A, as well as on regulatory regions of viruses that respond to UV irradiation [99]. Differences in transcriptional activities of URE-bound proteins were found after UV-irradiation of keratinocytes, melanocytes and melanoma, and also in repair deficient cells of patients with *Xeroderma pigmentosum*, or Cockayne syndrome [for review 9]. In 1998, the Ronai´s group investigated which of the CREB-associated proteins is directly involved in modifying specific characteristics of melanoma phenotypes. They demonstrate that ATF2 is the primary binding protein and regulator of URE-mediated transcription and it contributes

An approach to selectively inhibit ATF2 activity in human melanoma was designed, based on peptides derived from ATF2 trans-activating domain which affect ATF2 transcriptional activity. In an attempt to sensitize melanoma cells to UV irradiation, Ronai *et al*. investigated the ability of cells to enter in apoptosis competing by endogenous ATF2 expression with ATF2 derived peptide(s) alone and/or combined with inhibition of p38 activity (one Mitogenactivated Protein Kinases that is responsive to stress stimuli) via its pharmacological inhibitor (SB203580) [100]. The expression of a 50-amino acid peptide derived from the NH2-terminal domain of ATF2 (ATF250–100) was sufficient to sensitize melanoma cells to radiation. Combi‐ nation of this peptide with SB203580 induced programmed cell death in late stage melanoma cells via Fas signaling, whereas Fas ligand/receptor interactions play an important role in the progression of cancer. In 2002, experimental mouse models validated the expression of this peptide. The ATF250–100 not only sensitized melanoma cells to apoptosis but efficiently inhibited tumor growth and metastasis [101]. Analysis of mouse cell lines derived from melanomas formed in the HGF/SF (Hepatocyte Growth Factor/Scatter Factor) transgenic mouse, revealed that the proliferation rate in culture increased with increased ATF-2 activity [102], confirming the role of ATF2 in melanoma development. Along these lines, B16 mouse melanoma cells

Even with these encouraging results, one question remains unanswered: how ATF2 inhibition induces apoptosis in melanoma cells? It was demonstrated that ATF250–100 induced apoptosis by sequestering ATF2 to the cytoplasm, thereby inhibiting its transcriptional activities [105]. In addition, mutations within the c-Jun N-terminal kinases (JNK) binding region of ATF250– <sup>100</sup> or expression of TAM67, a dominant negative of the Jun family of transcription factors, or JunD-RNA interference attenuate inhibition of melanoma tumorigenicity by ATF250–100. The JNKs are kinases responsive to stress stimuli, such as ultraviolet irradiation used in this study. These results were crucial to show that inhibition of ATF2 in concert with increased JNK/Jun activities is central for the sensitization of melanoma cells to apoptosis and inhibition of their tumorigenicity. Furthermore, ATF250–100 increases ATF2 localization within the cytoplasm. Indeed, one study evaluating the ATF2 as a prognostic marker among patients with melano‐ mas validated this result. A study to determine the prognostic value of ATF2 evaluating the pattern and level of its expression in a tissue microarray was conducted [106]. Cytoplasmic ATF2 expression was associated with primary tumor rather than metastases and with better patient survival whereas nuclear ATF2 expression was associated with metastatic tumor and with poor survival. Nuclear ATF2 seems to be transcriptionally active while cytoplasmic ATF2 probably represents an inactive form. These findings support one preclinical finding in which transcriptionally active ATF2 is involved in tumor progression-proliferation in melanoma, suggesting that ATF2 might be a useful prognostic marker in early-stage melanoma. Although the use peptide ATF250–100 have shown good results to sensitize melanoma cells to treatments, Ronai group´s continued investigating peptides with smaller size but producing the same effect. In 2004, Bhoumik *et al*. [107] presented one peptide with only 10aa - ATF251-60. This peptide sensitizes melanoma cells to spontaneous apoptosis and inhibits the *in vivo* growth. Furthermore, the ATF251-60 expression coincides with activation of caspase 9, an important molecule activated during apoptosis. This study points to mechanisms underlying the activities of the ATF2 peptide while highlighting its possible use in drug design.

Based on these findings, ATF2 present oncogenic action, but could it act as one tumor sup‐ pressor molecule? Although genetic changes in ATF2 have not been identified in human tumors, many data sustain the notion that ATF2 is not only oncogenic, whereas its altered expression and sub cellular localization is associated with tumor stage and prognosis in melanomas, but it also acts as a tumor suppressor molecule, under specific conditions. This hypothesis arose from independent studies with skin and mammary tumors. Studies from a mouse mammary tumor model revealed that loss of ATF2 *per se*, does not promote mammary tumor formation, but heterozygous mouse *ATF2* mutants developed mammary tumors when crossed with p53 mutant mice, indicating that ATF2 may have a suppressor function only when combined with a p53 mutant background [108]. Likewise, loss of ATF2 transcriptional activities in keratinocytes promotes faster development of skin papillomas. Deletion of functional *ATF2* in keratinocytes was achieved using a K14-cre mouse which was crossed with mutant ATF2 mice. Exposure of K14-ATF2 mutant mice to DMBA (a carcinogen that causes Ras mutation) followed by application of TPA (a tumor promoter) resulted in faster formation of papillomas which were bigger, compared with mice bearing wild-type (WT) *ATF2* in their keratinocytes [109]. Importantly, mice in which *ATF2* was deleted only in keratinocytes did not develop papillomas, differently from WT mice when treated with the carcinogens DMBA or TPA alone. Therefore, ATF2 can limit tumor development by cooperating with existing oncogenes and inactivated tumor suppressor genes.

prolonged MITF depletion leads melanomas to either quiescence or senescence [121]. So, MITF regulates distinct functions in melanocytic cells at different levels of expression. While MITF lower levels are commonly observed in melanoma cells rather than in mela‐ nocytes, high levels of MITF activate the expression of differentiation-associated genes implicated on melanosome function and promote a differentiation-associated cell cycle arrest via up regulation of the p16 (*CDKN2A*) and p21 (*CDKN1A*) cyclin-dependent kin‐ ase inhibitors ([122-124]. Chromatin immunoprecipitation of MITF from 501 melanoma cell line followed by high-throughput deep sequencing and RNA sequencing from MITFdepleted cells, showed *TYROSINASE*, *MET*, *LIG1*, *BRCA1*, *CCND1*, and *CCNB1* genes transcriptionally-regulated by *MITF*. Thus, MITF-depleted cells exhibit diminished capaci‐ ty to passage through S-phase and repair DNA damage. These data highlight the multitasking role of MITF that, in addition to differentiation, survival, and its antiproliferative roles, also includes a role in the S phase, controling mitosis and suppressing senescence. In an opposite way, increased MITF levels reduce melanoma cell prolifera‐ tion even in the presence of oncogenic BRAF [124]. MITF can cooperate with BRafV600E to transform immortalized melanocytes by expression of telomerase (TERT), dominant-nega‐ tive p53 and activated Cdk4 [121]. These data indicates that, although MITF alone cannot transform normal human melanocytes, it can cooperate with BRafV600E to contribute to the transformation process, functioning as a "lineage-specific oncogene", because it provides essential survival functions and contributes to proliferation. In this context, and bearing in mind that ERK is hyperactivated in melanoma and required for proliferation and sur‐ vival, it is striking that MITF is targeted for degradation after its phosphorylation by ERK [125]. Indeed, constitutive activation of ERK by BRafV600E in melanocytes results in

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One interesting example from different levels of MITF action came from an elegant transla‐ tional study [127]. The authors demonstrated that the transcription factor ATF2 negatively regulates *MITF* transcription in melanocytes and around 50% of melanoma cells. Increased MITF expression (upon inhibition of ATF2), effectively attenuated the ability of BRafV600Emelanocytes to exhibit a transformed phenotype. This effect was partially rescued when MITF expression was also blocked. The development of melanoma in mice carrying genetic changes seen in human tumors was inhibited upon inactivation of ATF2 in melanocytes. Melanocytes from mice lacking active ATF2 increased levels of MITF, confirming that ATF2 negatively regulates MITF and implicating this newly discovered regulatory link in melanomagenesis. Additionally, primary melanoma specimens that exhibit a high nuclear ATF2-to-MITF ratio were found to be associated with metastatic disease and poor prognosis, substantiating the significance of MITF control by ATF2. Taken together, these findings provide a genetic evidence for the role of ATF2 in melanoma development and indicate an ATF2 function as a regulator of MITF expression, which is central to understanding MITF control at the early

Another possible mechanism that could explain the different levels of expression of *MITF* observed in melanoma cells is DNA copy number alterations (CNAs). Copy num‐ ber alterations involving "driver genes" can modulate substantially their expression

constant down regulation of MITF [for review 126].

phases of melanocyte transformation.

Present knowledge positions ATF2 as important transcription factor and DNA damage response protein, which is also implicated in the regulation of cellular growth control. Along the growing complexity of understanding ATF2 regulation and function are the observations that point to its ability to elicit oncogenes or tumor suppressor functions, depending on the tissue type. Based on these findings, it was proposed one model for ATF2 oncogenic *versus* tumor suppressor functions. Future studies will reveal the nature of these major differences, and further delineate the important role ATF2 plays in cellular growth control prior and following DNA damage, as in transformation and cancer development. In addition, the ATF2 function findings highlight the importance of transcriptional regulation, which enables the sensitization of melanoma to treatment and inhibits their growth and metastasis *in vivo*.

#### *4.2.2. Microphthalmia-associated Transcription Factor (MITF) the conductor of melanoma players*

*Microphthalmia* locus displays important roles for biology and pathology of pigmentation of the skin, as well as eye development and degeneration. Ever since, many other mutant alleles of the locus have been found in mice and other vertebrates [for review 110].The human *MITF* gene (3p14.2-p14.1) was cloned in 1994 [111] and so far, *MITF-A*, *MITF-B*, *MITF-H*, and *MITF-M* splice variants were described [112;113]. MITF contains a basic DNA binding domain and binds to DNA sequences primarily consisting of a 5´-CATGTG-3´ or 5´-CACGTG-3´ motif [114-116]. Ten isoforms of MITF have been described [117], but the m-MITF isoform is exclusively expressed in melanocytes. All MITF isoforms have a central transcriptional activation domain. MITF acts as a transcription factor which controls proliferation and apoptosis and plays a central role in the differentiation, growth, and survival of cells of the melanocytic lineage [118]. MITF is the main transcription activator for key genes involved in melanogenesis (*TYR*, *TYRP1*, *MLANA*, *SILV*), but its function can switch, in balance with *POU3F2*, to activate proliferation and inhibit invasion [119].

Recent observations of reversible phenotypic heterogeneity in melanoma have proposed a novel "phenotypic plasticity model" of cancer, whereas MITF seems to be one of the central players in melanoma phenotypic plasticity. The "dynamic epigenetic model" or rheostat model proposes that variations in the tumor microenvironment result in epige‐ netic lesions, leading to alterations observed in melanomas [for review 120]. In this mod‐ el, high expression levels of MITF regulate genes involved with differentiation and cell cycle arrest. When MITF is expressed at average levels, melanoma cells proceed through cell cycle, while reduction of MITF to low levels switches off the cell proliferation pro‐ gram, inducing cell cycle arrest, and promotes invasion and metastasis. For example, prolonged MITF depletion leads melanomas to either quiescence or senescence [121]. So, MITF regulates distinct functions in melanocytic cells at different levels of expression. While MITF lower levels are commonly observed in melanoma cells rather than in mela‐ nocytes, high levels of MITF activate the expression of differentiation-associated genes implicated on melanosome function and promote a differentiation-associated cell cycle arrest via up regulation of the p16 (*CDKN2A*) and p21 (*CDKN1A*) cyclin-dependent kin‐ ase inhibitors ([122-124]. Chromatin immunoprecipitation of MITF from 501 melanoma cell line followed by high-throughput deep sequencing and RNA sequencing from MITFdepleted cells, showed *TYROSINASE*, *MET*, *LIG1*, *BRCA1*, *CCND1*, and *CCNB1* genes transcriptionally-regulated by *MITF*. Thus, MITF-depleted cells exhibit diminished capaci‐ ty to passage through S-phase and repair DNA damage. These data highlight the multitasking role of MITF that, in addition to differentiation, survival, and its antiproliferative roles, also includes a role in the S phase, controling mitosis and suppressing senescence. In an opposite way, increased MITF levels reduce melanoma cell prolifera‐ tion even in the presence of oncogenic BRAF [124]. MITF can cooperate with BRafV600E to transform immortalized melanocytes by expression of telomerase (TERT), dominant-nega‐ tive p53 and activated Cdk4 [121]. These data indicates that, although MITF alone cannot transform normal human melanocytes, it can cooperate with BRafV600E to contribute to the transformation process, functioning as a "lineage-specific oncogene", because it provides essential survival functions and contributes to proliferation. In this context, and bearing in mind that ERK is hyperactivated in melanoma and required for proliferation and sur‐ vival, it is striking that MITF is targeted for degradation after its phosphorylation by ERK [125]. Indeed, constitutive activation of ERK by BRafV600E in melanocytes results in constant down regulation of MITF [for review 126].

functional *ATF2* in keratinocytes was achieved using a K14-cre mouse which was crossed with mutant ATF2 mice. Exposure of K14-ATF2 mutant mice to DMBA (a carcinogen that causes Ras mutation) followed by application of TPA (a tumor promoter) resulted in faster formation of papillomas which were bigger, compared with mice bearing wild-type (WT) *ATF2* in their keratinocytes [109]. Importantly, mice in which *ATF2* was deleted only in keratinocytes did not develop papillomas, differently from WT mice when treated with the carcinogens DMBA or TPA alone. Therefore, ATF2 can limit tumor development by cooperating with existing

Present knowledge positions ATF2 as important transcription factor and DNA damage response protein, which is also implicated in the regulation of cellular growth control. Along the growing complexity of understanding ATF2 regulation and function are the observations that point to its ability to elicit oncogenes or tumor suppressor functions, depending on the tissue type. Based on these findings, it was proposed one model for ATF2 oncogenic *versus* tumor suppressor functions. Future studies will reveal the nature of these major differences, and further delineate the important role ATF2 plays in cellular growth control prior and following DNA damage, as in transformation and cancer development. In addition, the ATF2 function findings highlight the importance of transcriptional regulation, which enables the sensitization of melanoma to treatment and inhibits their growth and metastasis *in vivo*.

*4.2.2. Microphthalmia-associated Transcription Factor (MITF) the conductor of melanoma players*

*Microphthalmia* locus displays important roles for biology and pathology of pigmentation of the skin, as well as eye development and degeneration. Ever since, many other mutant alleles of the locus have been found in mice and other vertebrates [for review 110].The human *MITF* gene (3p14.2-p14.1) was cloned in 1994 [111] and so far, *MITF-A*, *MITF-B*, *MITF-H*, and *MITF-M* splice variants were described [112;113]. MITF contains a basic DNA binding domain and binds to DNA sequences primarily consisting of a 5´-CATGTG-3´ or 5´-CACGTG-3´ motif [114-116]. Ten isoforms of MITF have been described [117], but the m-MITF isoform is exclusively expressed in melanocytes. All MITF isoforms have a central transcriptional activation domain. MITF acts as a transcription factor which controls proliferation and apoptosis and plays a central role in the differentiation, growth, and survival of cells of the melanocytic lineage [118]. MITF is the main transcription activator for key genes involved in melanogenesis (*TYR*, *TYRP1*, *MLANA*, *SILV*), but its function can switch, in balance with

Recent observations of reversible phenotypic heterogeneity in melanoma have proposed a novel "phenotypic plasticity model" of cancer, whereas MITF seems to be one of the central players in melanoma phenotypic plasticity. The "dynamic epigenetic model" or rheostat model proposes that variations in the tumor microenvironment result in epige‐ netic lesions, leading to alterations observed in melanomas [for review 120]. In this mod‐ el, high expression levels of MITF regulate genes involved with differentiation and cell cycle arrest. When MITF is expressed at average levels, melanoma cells proceed through cell cycle, while reduction of MITF to low levels switches off the cell proliferation pro‐ gram, inducing cell cycle arrest, and promotes invasion and metastasis. For example,

oncogenes and inactivated tumor suppressor genes.

104 Melanoma - From Early Detection to Treatment

*POU3F2*, to activate proliferation and inhibit invasion [119].

One interesting example from different levels of MITF action came from an elegant transla‐ tional study [127]. The authors demonstrated that the transcription factor ATF2 negatively regulates *MITF* transcription in melanocytes and around 50% of melanoma cells. Increased MITF expression (upon inhibition of ATF2), effectively attenuated the ability of BRafV600Emelanocytes to exhibit a transformed phenotype. This effect was partially rescued when MITF expression was also blocked. The development of melanoma in mice carrying genetic changes seen in human tumors was inhibited upon inactivation of ATF2 in melanocytes. Melanocytes from mice lacking active ATF2 increased levels of MITF, confirming that ATF2 negatively regulates MITF and implicating this newly discovered regulatory link in melanomagenesis. Additionally, primary melanoma specimens that exhibit a high nuclear ATF2-to-MITF ratio were found to be associated with metastatic disease and poor prognosis, substantiating the significance of MITF control by ATF2. Taken together, these findings provide a genetic evidence for the role of ATF2 in melanoma development and indicate an ATF2 function as a regulator of MITF expression, which is central to understanding MITF control at the early phases of melanocyte transformation.

Another possible mechanism that could explain the different levels of expression of *MITF* observed in melanoma cells is DNA copy number alterations (CNAs). Copy num‐ ber alterations involving "driver genes" can modulate substantially their expression [128]. Melanoma genomes frequently contain somatic copy number alterations that can significantly perturb the expression level of affected genes. Recently, accurate strategies have been used to identify new genes and/or focus on molecular pathways already de‐ scribed as affected in melanomas (*BRAF*, *PTEN* and *MITF* alterations) [129]. By using in‐ tegrative strategy of SNP (Single Nucleotide Polymorphism) array-based genetic, which has higher genomic resolution than CGH arrays, with gene expression signatures de‐ rived from NCI60 cell lines identified *MITF* as the target of novel melanoma amplifica‐ tion [121]. *MITF* amplification was more prevalent in metastatic disease and correlated with decreased overall patient survival. *BRAF* mutation and p16 inactivation was accom‐ panied by *MITF* amplification in melanoma cell lines. Moreover, it was described that ec‐ topic MITF expression in conjunction with the BRAFV600E mutant transformed primary human melanocytes, reinforcing the MITF as a melanoma oncogene. Although *MITF* am‐ plification (10–100-fold) is observed around 10–16% of metastatic melanomas (in which *BRAF* is mutated), *MITF* levels are only increased about 1.5-fold compared with cells without amplification [121], again suggesting that MITF levels must be maintained with‐ in narrow limits. However, because only 10–16% of *BRAF*-mutated melanomas have *MITF* amplification, this raises the crucial question of how the remaining 84–90% coun‐ teracts MITF degradation mediated by hyperactivated ERK. One mechanism could in‐ volve β-catenin (molecule which regulates cell growth and adhesion between cells). βcatenin can induce MITF expression through a LEF-1/TCF binding site in the MITF promoter [130]. Although mutations in β-catenin are rare in melanoma [131], nuclear and/or cytoplasmic localization of β-catenin was found in 28% of metastatic melanoma [132]. Therefore, regardless the mechanism of activation, MITF was shown to be a key mediator of switching between the slow-growing invasive phenotype and the prolifera‐ tive phenotype in melanoma cells.

profiling from RCC cell line identified a Mi-E318K signature related to cell growth, proliferation and inflammation. Therefore, the mutant MITF present all features of a

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The understanding of the tumor stage, microenvironment, and mechanisms employed in phenotype switching have significant implications for clinical strategies in melanoma man‐ agement. The description of *BRAF* and *KIT* mutations provided the first basis for a molecular classification of cutaneous melanoma and brought up insights about therapeutic approaches. Therapies based on BRAF moves on direction of regulatory approval and incorporation as standard therapy for patients with metastatic disease, as well as targeting mutated *KIT* has also been established for melanoma patients. *NRAS* mutations have been known to be present in a subset of melanomas and represent a complicated subgroup for targeted therapies. Matching patient subgroups defined by genetic aberrations in the phosphoinositide 3-kinase (PI3K) and p16/cyclin dependent kinase 4 (CDK4) pathways with appropriate targeted therapies has not yet been realized. So, an increasing understanding of lineage-specific transcriptional regulators, as MITF, and how they could play a role in melanoma pathophysi‐ ology provided other clues to therapies. Modulating MITF in a direct way with pharmacologic inhibitors would be challenging, particularly if the interaction of MITF with certain promoter regions on specific genes is desired. Reduction of MITF activity sensitizes melanoma cells to different chemotherapeutic agents [for review 121]. Targeting MITF combined with BRAF or cyclin-dependent kinase inhibitors is an exciting therapeutic strategy for melanoma patients.

One therapeutic strategy is target one or more of the post-translational processes that deter‐ mine MITF activity, stability, or degradation. Another approach is targeting the melanocytespecific mechanisms controlling MITF expression. Nonspecific histone deacetylases seem to function in such a manner [134]. Furthermore, MITF and its target genes have been used as diagnostic markers for melanoma [135]. As cited above, MITF-M isoform is involved in the *in vivo* growth control and contribute to the phenotype of human melanoma whereas MITF-M may qualify as a marker capable of identifying subgroups of melanoma patients with different tumor biology and prognosis [136]. Many MITF transcriptional targets are emerging, and it is likely that their identification may bring therapeutic strategies based on lineage-specific conditions. One candidate is cyclin dependent kinase 2 (CDK2). This molecule seems to contribute to deregulate cell cycle control via its transcriptional control by MITF, which is unique in the melanocyte lineage due to its genomic location adjacent to a *MITF*. Another molecule that seems to be regulated by MITF is BCL2 and it may contribute to resistance to

*4.2.4 DNA repair genes – Dual effect of DNA repair genes in melanoma progression*

Exposure to UV radiation from sunlight induces DNA damage, which can lead to melanocyte carcinogenesis when not efficiently corrected. UV radiation may induce direct alterations through formation of pyrimidine dimmers, indirect alterations through formation of reactive oxygen species that may oxidize DNA bases and also induce DNA breaks. In a scenario where

gain-of-function variant associated with tumorigenesis.

*4.2.3. MITF as therapeutic strategy?*

apoptosis in melanomas [134].

Recent studies have shown the role of germline mutations associated with MITF func‐ tion. Evidence for germline mutations in melanomas comes from studies with relatives of patients with melanoma with increased risk of melanoma development, indicating the presence of mutations in genes with high penetrance [for review 133]. A study conducted by Bertolotto et al. involving patients with melanoma and renal cell carcino‐ ma (RCC) supports the hypothesis of genetic predisposition for both cancers [51]. MITF stimulates the transcription of HIF1A, the pathway of which is targeted by kid‐ ney cancer susceptibility genes, indicating that MITF might have a role in conferring a genetic predisposition to co-occurring melanoma and RCC. A germline missense substi‐ tution in *MITF* (Mi-E318K) was identified occurring at a significantly higher frequency in genetically enriched patients affected with melanoma, RCC or both cancers. Overall, patients bearing the Mi-E318K genotype had more than fivefold increased risk of de‐ veloping melanoma, RCC or both cancers. The E318K variant was significantly associ‐ ated with melanoma in a large case–control sample. The variant allele was significantly over-represented in cases with a family history of melanoma, multiple pri‐ mary melanomas, or both. The variant allele was also associated with increased nevus count and no blue eye colour. In addition, Mi-E318K enhanced MITF protein binding to the HIF1A promoter and increased its transcriptional activity. Gene expression profiling from RCC cell line identified a Mi-E318K signature related to cell growth, proliferation and inflammation. Therefore, the mutant MITF present all features of a gain-of-function variant associated with tumorigenesis.

#### *4.2.3. MITF as therapeutic strategy?*

[128]. Melanoma genomes frequently contain somatic copy number alterations that can significantly perturb the expression level of affected genes. Recently, accurate strategies have been used to identify new genes and/or focus on molecular pathways already de‐ scribed as affected in melanomas (*BRAF*, *PTEN* and *MITF* alterations) [129]. By using in‐ tegrative strategy of SNP (Single Nucleotide Polymorphism) array-based genetic, which has higher genomic resolution than CGH arrays, with gene expression signatures de‐ rived from NCI60 cell lines identified *MITF* as the target of novel melanoma amplifica‐ tion [121]. *MITF* amplification was more prevalent in metastatic disease and correlated with decreased overall patient survival. *BRAF* mutation and p16 inactivation was accom‐ panied by *MITF* amplification in melanoma cell lines. Moreover, it was described that ec‐ topic MITF expression in conjunction with the BRAFV600E mutant transformed primary human melanocytes, reinforcing the MITF as a melanoma oncogene. Although *MITF* am‐ plification (10–100-fold) is observed around 10–16% of metastatic melanomas (in which *BRAF* is mutated), *MITF* levels are only increased about 1.5-fold compared with cells without amplification [121], again suggesting that MITF levels must be maintained with‐ in narrow limits. However, because only 10–16% of *BRAF*-mutated melanomas have *MITF* amplification, this raises the crucial question of how the remaining 84–90% coun‐ teracts MITF degradation mediated by hyperactivated ERK. One mechanism could in‐ volve β-catenin (molecule which regulates cell growth and adhesion between cells). βcatenin can induce MITF expression through a LEF-1/TCF binding site in the MITF promoter [130]. Although mutations in β-catenin are rare in melanoma [131], nuclear and/or cytoplasmic localization of β-catenin was found in 28% of metastatic melanoma [132]. Therefore, regardless the mechanism of activation, MITF was shown to be a key mediator of switching between the slow-growing invasive phenotype and the prolifera‐

Recent studies have shown the role of germline mutations associated with MITF func‐ tion. Evidence for germline mutations in melanomas comes from studies with relatives of patients with melanoma with increased risk of melanoma development, indicating the presence of mutations in genes with high penetrance [for review 133]. A study conducted by Bertolotto et al. involving patients with melanoma and renal cell carcino‐ ma (RCC) supports the hypothesis of genetic predisposition for both cancers [51]. MITF stimulates the transcription of HIF1A, the pathway of which is targeted by kid‐ ney cancer susceptibility genes, indicating that MITF might have a role in conferring a genetic predisposition to co-occurring melanoma and RCC. A germline missense substi‐ tution in *MITF* (Mi-E318K) was identified occurring at a significantly higher frequency in genetically enriched patients affected with melanoma, RCC or both cancers. Overall, patients bearing the Mi-E318K genotype had more than fivefold increased risk of de‐ veloping melanoma, RCC or both cancers. The E318K variant was significantly associ‐ ated with melanoma in a large case–control sample. The variant allele was significantly over-represented in cases with a family history of melanoma, multiple pri‐ mary melanomas, or both. The variant allele was also associated with increased nevus count and no blue eye colour. In addition, Mi-E318K enhanced MITF protein binding to the HIF1A promoter and increased its transcriptional activity. Gene expression

tive phenotype in melanoma cells.

106 Melanoma - From Early Detection to Treatment

The understanding of the tumor stage, microenvironment, and mechanisms employed in phenotype switching have significant implications for clinical strategies in melanoma man‐ agement. The description of *BRAF* and *KIT* mutations provided the first basis for a molecular classification of cutaneous melanoma and brought up insights about therapeutic approaches. Therapies based on BRAF moves on direction of regulatory approval and incorporation as standard therapy for patients with metastatic disease, as well as targeting mutated *KIT* has also been established for melanoma patients. *NRAS* mutations have been known to be present in a subset of melanomas and represent a complicated subgroup for targeted therapies. Matching patient subgroups defined by genetic aberrations in the phosphoinositide 3-kinase (PI3K) and p16/cyclin dependent kinase 4 (CDK4) pathways with appropriate targeted therapies has not yet been realized. So, an increasing understanding of lineage-specific transcriptional regulators, as MITF, and how they could play a role in melanoma pathophysi‐ ology provided other clues to therapies. Modulating MITF in a direct way with pharmacologic inhibitors would be challenging, particularly if the interaction of MITF with certain promoter regions on specific genes is desired. Reduction of MITF activity sensitizes melanoma cells to different chemotherapeutic agents [for review 121]. Targeting MITF combined with BRAF or cyclin-dependent kinase inhibitors is an exciting therapeutic strategy for melanoma patients.

One therapeutic strategy is target one or more of the post-translational processes that deter‐ mine MITF activity, stability, or degradation. Another approach is targeting the melanocytespecific mechanisms controlling MITF expression. Nonspecific histone deacetylases seem to function in such a manner [134]. Furthermore, MITF and its target genes have been used as diagnostic markers for melanoma [135]. As cited above, MITF-M isoform is involved in the *in vivo* growth control and contribute to the phenotype of human melanoma whereas MITF-M may qualify as a marker capable of identifying subgroups of melanoma patients with different tumor biology and prognosis [136]. Many MITF transcriptional targets are emerging, and it is likely that their identification may bring therapeutic strategies based on lineage-specific conditions. One candidate is cyclin dependent kinase 2 (CDK2). This molecule seems to contribute to deregulate cell cycle control via its transcriptional control by MITF, which is unique in the melanocyte lineage due to its genomic location adjacent to a *MITF*. Another molecule that seems to be regulated by MITF is BCL2 and it may contribute to resistance to apoptosis in melanomas [134].

#### *4.2.4 DNA repair genes – Dual effect of DNA repair genes in melanoma progression*

Exposure to UV radiation from sunlight induces DNA damage, which can lead to melanocyte carcinogenesis when not efficiently corrected. UV radiation may induce direct alterations through formation of pyrimidine dimmers, indirect alterations through formation of reactive oxygen species that may oxidize DNA bases and also induce DNA breaks. In a scenario where such alterations may facilitate the carcinogenic process, DNA repair systems are critical to suppress malignant transformation. There are different DNA repair systems inside the cells, which may repair a variety of DNA lesions, since mismatch base pairing formation during replication process, oxidized DNA bases, bulky addictions, intra and interstrand damages and single and double strand breaks. The main DNA repair systems are: Base excision repair (BER), Nucleotide Excision Repair (NER), Mismatch Repair (MMR), Homologous Repair (HR) and Non-Homologous End-Joinning Repair (NHEJ) [137].

MMR repair systems was also observed. Since the inactivation of the MMR system leads to DNA damage hypersensitivity, it is likely that over-expression of the MMR system could improve the cellular resistance to DNA lesions. However, the main results indicate an overexpression of genes related to rescue of stalled DNA replication forks, DNA double strand breaks and interstrand crosslink repair. These processes, acting on the S-phase checkpoint and post-replicative repair mechanisms, are essential for cell proliferation and survival by correct‐ ing eventual damages and replicative stress, such as cancer cells may exhibit higher rate of DNA synthesis. Thus, by maintaining an elevate activity of such DNA repair systems and checkpoints in S-phase during replicative stress, metastatic cancer cells can grow and survive,

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As example, a gene overexpressed in M+ melanomas was *TOP2A*, an enzyme that plays a role in replication and chromosome segregation by solving torsional stress [140]. Moreover, cells over-expressing *TOP2A* were more resistant to chemotherapeutic drugs such as alkylating agents [141]. Finally, over-expression of genes related to FANC-BRCA pathways (genes acting in double strand breaks repair and rescue of blocked replication forks) in melanomas M+ suggest the critical role that these process exerts on keeping the genetic stability in cancer cells with metastatic fitness. These findings demonstrate an important duality of DNA repair genes in tumor progression. First, the development of malignant cells from normal cells has been credited to a reduction or lack of DNA repair genes, thereby allowing the accumulation of mutations and subsequent transformation of the cells. This concept is well documented, since the relationship of individuals with genetic predisposition to certain tumors, where such predisposition may be is attributed to genes related to DNA repair or pathways that do support to DNA repair pathways [142]. However, at some point in the progression of melanoma, genetic stability appears to be a crucial factor to ensure that the tumor cells maintain the genetic repertoire that guarantees the ability of invasion and metastasis. Thus, melanoma cells with higher expression of DNA repair genes, would have greater capacity for metastasis due to maintenance of genetic capability. The genetic stability suggested [139] is not limited to the repair genes. Genes linked to telomere stability, as well as genes that ensure proper chromo‐ some separations were also highly expressed in melanomas M+. Another important implica‐ tion based on the results is that high expression of repair gene may be responsible for the characteristic low response of metastatic melanoma to chemotherapy, since many of the chemotherapeutic agents used to treat melanoma act causing DNA damage. It was also observed a higher expression of genes correlated to resistance to chemotherapeutic agents such as cisplatin and dacarbazine (e.g., *BRCA1, XRCC5, XRCC6*). In addition, other genes related to the maintenance of DNA replication machinery were also highly expressed, leading to translesion replication, thereby preventing the apoptosis signal being secondary to the arrest

Following studies, confirmed the high expression of FANC DNA repair genes in melanoma samples when compared to normal skin and non-melanoma skin cancers [143]. Moreover, there is a positive correlation regarding FANC genes and melanoma thickness by Breslow index. Conversely, NER genes were significantly decreased in melanomas, albeit its expression was not correlated with melanoma thickness. Immunohistochemistry of independent mela‐

and further be resistant to chemotherapy.

of DNA/RNA polymerase.

The critical role of DNA repair systems in cancer suppression is observed in a diversity of cancer predisposition syndromes which the main cause is due to mutations in DNA repair genes. Mutations in genes of nucleotide excision repair (NER), which preferentially corrects UV damages, caused the so-called *Xeroderma Pigmentosum* syndrome. The affected individuals have a one thousand fold greater chances of developing skin cancer under the age of 20 years [138], including melanomas, compared to DNA repair proficient individuals.

As discussed above, genetic variants that may alter the functionality of DNA repair genes, mainly genes from NER repair systems, may also modulate the susceptibility for melanoma. DNA repair systems were pointed as a functional network that could contrib‐ ute to melanocyte carcinogenesis process by complete inactivating (such as in XP pa‐ tients) or by differential functionality due to genetic variants associated with environmental factors such as UV exposure. However, this intuitive thought regarding the role of DNA repair systems restrict to the initials steps of melanoma development has changed in the last years. A study published in 2008 [139] has suggested a new role of DNA repair systems in melanoma progression and metastasis. Aiming at the better understanding of primary melanoma to metastasis progression, the authors used a collec‐ tion of frozen primary melanomas to study their gene expression by microarray. Those patients that had primary melanomas included in the study had a follow up for four years. After that, 26 of 60 patients showed metastasis while the other 34 patients did not. Gene expression of primary melanomas that originated metastasis (called M+ by the au‐ thors) was compared with the gene expression of primary melanomas that did not origi‐ nate metastasis (M-). The results indicated a high and robust significant expression of genes involved with DNA replication (*p* = 4.02 x 10-14) and DNA repair genes (*p* = 1.4 x 10-16) in those M+ primary melanomas. Besides the high expression of such class of genes, a strong correlation with Breslow index was also observed. To certain genes, its high expression was positively correlated to tumor thickness. To genes with low expres‐ sion in M+ primary melanomas compared to M- melanomas, a negative correlation with tumor thickness was observed.

The study indentified a total of 48 genes with higher expression in M+, which are related to DNA repair genes and genes related to maintenance of genetic stability in replication process [139]. Among those genes, genes from the BER repair systems (a repair system strongly related to repair oxidized bases and single strand breaks) such as *OGG1* and *EXO1* were high expressed. A possible biological interpretation is that the high expression of these genes could facilitate tumor growth and invasiveness, since base oxidation is the most frequent spontane‐ ous and deleterious lesions observed in actively replicating cells. Overexpression of genes of MMR repair systems was also observed. Since the inactivation of the MMR system leads to DNA damage hypersensitivity, it is likely that over-expression of the MMR system could improve the cellular resistance to DNA lesions. However, the main results indicate an overexpression of genes related to rescue of stalled DNA replication forks, DNA double strand breaks and interstrand crosslink repair. These processes, acting on the S-phase checkpoint and post-replicative repair mechanisms, are essential for cell proliferation and survival by correct‐ ing eventual damages and replicative stress, such as cancer cells may exhibit higher rate of DNA synthesis. Thus, by maintaining an elevate activity of such DNA repair systems and checkpoints in S-phase during replicative stress, metastatic cancer cells can grow and survive, and further be resistant to chemotherapy.

such alterations may facilitate the carcinogenic process, DNA repair systems are critical to suppress malignant transformation. There are different DNA repair systems inside the cells, which may repair a variety of DNA lesions, since mismatch base pairing formation during replication process, oxidized DNA bases, bulky addictions, intra and interstrand damages and single and double strand breaks. The main DNA repair systems are: Base excision repair (BER), Nucleotide Excision Repair (NER), Mismatch Repair (MMR), Homologous Repair (HR) and

The critical role of DNA repair systems in cancer suppression is observed in a diversity of cancer predisposition syndromes which the main cause is due to mutations in DNA repair genes. Mutations in genes of nucleotide excision repair (NER), which preferentially corrects UV damages, caused the so-called *Xeroderma Pigmentosum* syndrome. The affected individuals have a one thousand fold greater chances of developing skin cancer under the age of 20 years

As discussed above, genetic variants that may alter the functionality of DNA repair genes, mainly genes from NER repair systems, may also modulate the susceptibility for melanoma. DNA repair systems were pointed as a functional network that could contrib‐ ute to melanocyte carcinogenesis process by complete inactivating (such as in XP pa‐ tients) or by differential functionality due to genetic variants associated with environmental factors such as UV exposure. However, this intuitive thought regarding the role of DNA repair systems restrict to the initials steps of melanoma development has changed in the last years. A study published in 2008 [139] has suggested a new role of DNA repair systems in melanoma progression and metastasis. Aiming at the better understanding of primary melanoma to metastasis progression, the authors used a collec‐ tion of frozen primary melanomas to study their gene expression by microarray. Those patients that had primary melanomas included in the study had a follow up for four years. After that, 26 of 60 patients showed metastasis while the other 34 patients did not. Gene expression of primary melanomas that originated metastasis (called M+ by the au‐ thors) was compared with the gene expression of primary melanomas that did not origi‐ nate metastasis (M-). The results indicated a high and robust significant expression of genes involved with DNA replication (*p* = 4.02 x 10-14) and DNA repair genes (*p* = 1.4 x 10-16) in those M+ primary melanomas. Besides the high expression of such class of genes, a strong correlation with Breslow index was also observed. To certain genes, its high expression was positively correlated to tumor thickness. To genes with low expres‐ sion in M+ primary melanomas compared to M- melanomas, a negative correlation with

The study indentified a total of 48 genes with higher expression in M+, which are related to DNA repair genes and genes related to maintenance of genetic stability in replication process [139]. Among those genes, genes from the BER repair systems (a repair system strongly related to repair oxidized bases and single strand breaks) such as *OGG1* and *EXO1* were high expressed. A possible biological interpretation is that the high expression of these genes could facilitate tumor growth and invasiveness, since base oxidation is the most frequent spontane‐ ous and deleterious lesions observed in actively replicating cells. Overexpression of genes of

[138], including melanomas, compared to DNA repair proficient individuals.

Non-Homologous End-Joinning Repair (NHEJ) [137].

108 Melanoma - From Early Detection to Treatment

tumor thickness was observed.

As example, a gene overexpressed in M+ melanomas was *TOP2A*, an enzyme that plays a role in replication and chromosome segregation by solving torsional stress [140]. Moreover, cells over-expressing *TOP2A* were more resistant to chemotherapeutic drugs such as alkylating agents [141]. Finally, over-expression of genes related to FANC-BRCA pathways (genes acting in double strand breaks repair and rescue of blocked replication forks) in melanomas M+ suggest the critical role that these process exerts on keeping the genetic stability in cancer cells with metastatic fitness. These findings demonstrate an important duality of DNA repair genes in tumor progression. First, the development of malignant cells from normal cells has been credited to a reduction or lack of DNA repair genes, thereby allowing the accumulation of mutations and subsequent transformation of the cells. This concept is well documented, since the relationship of individuals with genetic predisposition to certain tumors, where such predisposition may be is attributed to genes related to DNA repair or pathways that do support to DNA repair pathways [142]. However, at some point in the progression of melanoma, genetic stability appears to be a crucial factor to ensure that the tumor cells maintain the genetic repertoire that guarantees the ability of invasion and metastasis. Thus, melanoma cells with higher expression of DNA repair genes, would have greater capacity for metastasis due to maintenance of genetic capability. The genetic stability suggested [139] is not limited to the repair genes. Genes linked to telomere stability, as well as genes that ensure proper chromo‐ some separations were also highly expressed in melanomas M+. Another important implica‐ tion based on the results is that high expression of repair gene may be responsible for the characteristic low response of metastatic melanoma to chemotherapy, since many of the chemotherapeutic agents used to treat melanoma act causing DNA damage. It was also observed a higher expression of genes correlated to resistance to chemotherapeutic agents such as cisplatin and dacarbazine (e.g., *BRCA1, XRCC5, XRCC6*). In addition, other genes related to the maintenance of DNA replication machinery were also highly expressed, leading to translesion replication, thereby preventing the apoptosis signal being secondary to the arrest of DNA/RNA polymerase.

Following studies, confirmed the high expression of FANC DNA repair genes in melanoma samples when compared to normal skin and non-melanoma skin cancers [143]. Moreover, there is a positive correlation regarding FANC genes and melanoma thickness by Breslow index. Conversely, NER genes were significantly decreased in melanomas, albeit its expression was not correlated with melanoma thickness. Immunohistochemistry of independent mela‐ noma and non-melanoma skin cancers, confirmed the results previous discovered in gene expression regarding FANC genes and melanomas. Interestingly, down regulation of NER genes may have contributed to initial steps of melanomagenesis, however, the high expression of gene products of DNA repair pathways, mainly those regarding to solve double strand breaks, may be related to melanoma progression.

significantly less frequently in benign pigmented lesions (9/75). However, the following studies found PAX3 expression in melanoblasts localized in hair follicles and also in mature melanocytes in hair follicles, in 100% of the nevi examined, 94% of primary melanomas and

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A most complete study performed in melanocytic lesions [152], analyzed PAX3 expression in normal skin, nevi, primary melanoma and melanoma metastases by immunohistochemistry. PAX3 was expressed in all samples and in normal cells. PAX3 expression showed a pattern of distribution characteristic of melanocytes (at epidermal-dermal boundary and along the hair follicle). Moreover, PAX3-positive cells were fewer and had a weaker staining in normal skin, as compared to nevi and melanomas. Co-expression of PAX3 with MITF was also observed in all samples, however, in normal skin some cells expressed only MITF, highlighting the differences in melanocyte phenotype. PAX3-positive cells were also co-stained with markers of less or more melanocyte differentiation, such as HES1 and Melan-A respectively. The samples indicated PAX3-positive cells co-stained with either markers, showing then a variable differentiation status of epidermal and follicular melanocytes, however a higher proportion of PAX3 and Melan-A positive cells. Finally, to further describe the phenotype of PAX3 positive melanocytes and melanoma cells, antiapoptotic factor BCL2L1 and melanoma progression marker MCAM were also analyzed in those cells. Regarding BCL2L1, a high similar proportion of PAX3-positive cells were also BCL2L1 positive cells, in all samples, with exception of melanoma metastases. These results suggest a role for PAX3 in regulation of survival of melanocytes and melanomas. Regarding MCAM, all melanocytic lesions showed its expression. Co-staining of MCAM and PAX3 increased in proportion from nevi to primary melanoma to melanomas metastases. As suggested above, PAX3 also plays a role in regulating genes involved in protecting cancer cells from apoptosis, as indicated by studies where the down-regulation of PAX3 increased the levels of apoptosis [153;154]. One of the mechanisms by which PAX3 may be involved with resistance to apoptosis resides in the fact that PAX3 interacts with the enhancer element of Bcl-XL gene, triggering its activation [155]. Another mechanism described for the anti-apoptotic role of PAX3 is via the regulation of tumor supressor PTEN [156]. In melanoma cells, the down regulation of PAX3 showed a dosedependent reduction of proliferation and induction of apoptosis when cells were treated with cisplatin [157]. Indeed, PAX3 down-regulation lead to increase in p53 protein and also caspase3

Functional studies have clarified the PAX3 function on melanocytes/melanomas [158]. PAX3, acting synergistically with SOX10, play a role in the regulation of MET expression. MET is a transmembrane receptor tyrosine kinase activated by Hepatocyte Growth Factor (HGF) and plays a role in normal development and in cell migration, growth, survival, differentiation, angiogenesis [159]. The HGF-MET pathway is involved in melanocyte biology acting on survival and maintenance of specific genes. MET is commonly over-expressed in melanoma and is associated with a more aggressive phenotype in terms of invasion and metastasis [160;161]. A strong correlation of expression of MET with PAX3 and SOX10 in primary melanomas was observed [158]. Thus, the expression of PAX3 may facilitate melanoma

in 90% of metastatic melanomas examined [151;152].

(a critical protein involved with apoptosis).

In another study, expression of DNA repair genes was associated with prognosis, disease relapse, tumor thickness and response to chemotherapy in melanomas [144]. In that study, high expression of genes *RAD51, RAD52 and TOP2A* was significantly associated to poor relapse-free survival. Expression of *RAD51* was 1.22 times greater in tumors from patients who relapsed versus those who did not; the fold changes between tumors from relapsers and nonrelapsers for *RAD52* and *TOP2A* were 1.16 and 1.12 respectively. *RAD54B, RAD52, TOP2A* and *RAD51* were also overexpressed in tumours from patients who died versus surviving patients. As reported by the studies cited above [139;143], expression of DNA repair genes was also correlated with tumor thickness and to mitotic rate. Finally, when the chemotherapy response was analyzed, *RAD51* and *TOP2A* had significantly higher expression in tumors from nonresponders compared to responders [144]. Finally, the results described point to new methods for melanoma treatment, where in addition to chemotherapy and radiotherapy for melanoma cells, the development of new drugs capable to modify the activity of proteins related to DNA repair, may increase the efficiency of treatment.

#### *4.2.5. PAX3 – Back to stemness?*

The *PAX* family genes (from Paired Box) consist of transcriptional factors highly conserved and also essential to development of different tissues during embryogenesis as well as essential to maintenance of stem cells in the adult organism. Indeed, *PAX* genes are related to regulation of several processes such as proliferation, migration, avoiding apoptosis and sustaining stemness phenotype in undifferentiated cells. There are nine PAX proteins, of which PAX3 is a particularly interesting protein for its function in regulating the development of melanocytes and other cell types.

Together with SOX10, PAX3 regulate transcription of MITF [145] and c-RET [146] in melano‐ cytes. PAX3 is a key transcription factor during the development of the neural crest and its derivatives in the embryo. The neural crest cells detach from the dorsal neural epithelium and give origin to a diverse set of cells, including melanocytes. PAX3 starts its expression in neural crest precursors that are further committed with melanocytic cells lineage, such as melano‐ blasts [147]. PAX3 exerts its activity by expressing MITF and repressing Dct (Dopachrometautomerase), thus leading to an undifferentiated cell state [148]. When MITF levels reach a threshold, a complex consisting of MITF and β-catenin binds to Dct promoter, abolishing PAX3 inhibition, which leads to Dct expression and melanocyte differentiation. It is thought that upon terminal differentiation, the expression of PAX3 is reduced as suggested by initial studies that reported no expression of PAX3 in normal skin melanocytes [148;149] PAX3 expression has been described in nevi, in most primary melanoma tumors, melanoma cell lines [150-152]. The first study described the expression of PAX3 in 8/8 melanoma cell lines [150]. The study also showed that PAX3 was commonly expressed in primary melanoma samples (21/58) but significantly less frequently in benign pigmented lesions (9/75). However, the following studies found PAX3 expression in melanoblasts localized in hair follicles and also in mature melanocytes in hair follicles, in 100% of the nevi examined, 94% of primary melanomas and in 90% of metastatic melanomas examined [151;152].

noma and non-melanoma skin cancers, confirmed the results previous discovered in gene expression regarding FANC genes and melanomas. Interestingly, down regulation of NER genes may have contributed to initial steps of melanomagenesis, however, the high expression of gene products of DNA repair pathways, mainly those regarding to solve double strand

In another study, expression of DNA repair genes was associated with prognosis, disease relapse, tumor thickness and response to chemotherapy in melanomas [144]. In that study, high expression of genes *RAD51, RAD52 and TOP2A* was significantly associated to poor relapse-free survival. Expression of *RAD51* was 1.22 times greater in tumors from patients who relapsed versus those who did not; the fold changes between tumors from relapsers and nonrelapsers for *RAD52* and *TOP2A* were 1.16 and 1.12 respectively. *RAD54B, RAD52, TOP2A* and *RAD51* were also overexpressed in tumours from patients who died versus surviving patients. As reported by the studies cited above [139;143], expression of DNA repair genes was also correlated with tumor thickness and to mitotic rate. Finally, when the chemotherapy response was analyzed, *RAD51* and *TOP2A* had significantly higher expression in tumors from nonresponders compared to responders [144]. Finally, the results described point to new methods for melanoma treatment, where in addition to chemotherapy and radiotherapy for melanoma cells, the development of new drugs capable to modify the activity of proteins related to DNA

The *PAX* family genes (from Paired Box) consist of transcriptional factors highly conserved and also essential to development of different tissues during embryogenesis as well as essential to maintenance of stem cells in the adult organism. Indeed, *PAX* genes are related to regulation of several processes such as proliferation, migration, avoiding apoptosis and sustaining stemness phenotype in undifferentiated cells. There are nine PAX proteins, of which PAX3 is a particularly interesting protein for its function in regulating the development of melanocytes

Together with SOX10, PAX3 regulate transcription of MITF [145] and c-RET [146] in melano‐ cytes. PAX3 is a key transcription factor during the development of the neural crest and its derivatives in the embryo. The neural crest cells detach from the dorsal neural epithelium and give origin to a diverse set of cells, including melanocytes. PAX3 starts its expression in neural crest precursors that are further committed with melanocytic cells lineage, such as melano‐ blasts [147]. PAX3 exerts its activity by expressing MITF and repressing Dct (Dopachrometautomerase), thus leading to an undifferentiated cell state [148]. When MITF levels reach a threshold, a complex consisting of MITF and β-catenin binds to Dct promoter, abolishing PAX3 inhibition, which leads to Dct expression and melanocyte differentiation. It is thought that upon terminal differentiation, the expression of PAX3 is reduced as suggested by initial studies that reported no expression of PAX3 in normal skin melanocytes [148;149] PAX3 expression has been described in nevi, in most primary melanoma tumors, melanoma cell lines [150-152]. The first study described the expression of PAX3 in 8/8 melanoma cell lines [150]. The study also showed that PAX3 was commonly expressed in primary melanoma samples (21/58) but

breaks, may be related to melanoma progression.

110 Melanoma - From Early Detection to Treatment

repair, may increase the efficiency of treatment.

*4.2.5. PAX3 – Back to stemness?*

and other cell types.

A most complete study performed in melanocytic lesions [152], analyzed PAX3 expression in normal skin, nevi, primary melanoma and melanoma metastases by immunohistochemistry. PAX3 was expressed in all samples and in normal cells. PAX3 expression showed a pattern of distribution characteristic of melanocytes (at epidermal-dermal boundary and along the hair follicle). Moreover, PAX3-positive cells were fewer and had a weaker staining in normal skin, as compared to nevi and melanomas. Co-expression of PAX3 with MITF was also observed in all samples, however, in normal skin some cells expressed only MITF, highlighting the differences in melanocyte phenotype. PAX3-positive cells were also co-stained with markers of less or more melanocyte differentiation, such as HES1 and Melan-A respectively. The samples indicated PAX3-positive cells co-stained with either markers, showing then a variable differentiation status of epidermal and follicular melanocytes, however a higher proportion of PAX3 and Melan-A positive cells. Finally, to further describe the phenotype of PAX3 positive melanocytes and melanoma cells, antiapoptotic factor BCL2L1 and melanoma progression marker MCAM were also analyzed in those cells. Regarding BCL2L1, a high similar proportion of PAX3-positive cells were also BCL2L1 positive cells, in all samples, with exception of melanoma metastases. These results suggest a role for PAX3 in regulation of survival of melanocytes and melanomas. Regarding MCAM, all melanocytic lesions showed its expression. Co-staining of MCAM and PAX3 increased in proportion from nevi to primary melanoma to melanomas metastases. As suggested above, PAX3 also plays a role in regulating genes involved in protecting cancer cells from apoptosis, as indicated by studies where the down-regulation of PAX3 increased the levels of apoptosis [153;154]. One of the mechanisms by which PAX3 may be involved with resistance to apoptosis resides in the fact that PAX3 interacts with the enhancer element of Bcl-XL gene, triggering its activation [155]. Another mechanism described for the anti-apoptotic role of PAX3 is via the regulation of tumor supressor PTEN [156]. In melanoma cells, the down regulation of PAX3 showed a dosedependent reduction of proliferation and induction of apoptosis when cells were treated with cisplatin [157]. Indeed, PAX3 down-regulation lead to increase in p53 protein and also caspase3 (a critical protein involved with apoptosis).

Functional studies have clarified the PAX3 function on melanocytes/melanomas [158]. PAX3, acting synergistically with SOX10, play a role in the regulation of MET expression. MET is a transmembrane receptor tyrosine kinase activated by Hepatocyte Growth Factor (HGF) and plays a role in normal development and in cell migration, growth, survival, differentiation, angiogenesis [159]. The HGF-MET pathway is involved in melanocyte biology acting on survival and maintenance of specific genes. MET is commonly over-expressed in melanoma and is associated with a more aggressive phenotype in terms of invasion and metastasis [160;161]. A strong correlation of expression of MET with PAX3 and SOX10 in primary melanomas was observed [158]. Thus, the expression of PAX3 may facilitate melanoma progression and metastasis through the expression of MET, a classical proto-oncogene involved in invasion, metastasis, resistance of apoptosis, and tumor cell expansion.

of mutagenic agents to TP53, sun exposure is a potent mechanism of induction of *TP53* mutations as suggested by the frequent occurrence of such mutations in skin cancers such as basal cell carcinomas (BCC) and squamous cell carcinomas (SCC). In such skin cancers, UVrelated mutations (C to T and CC to TT transitions) are frequently described in *TP53* and in other genes, confirming then the role of UV exposure in skin cancers. As melanocytes from exposed skin areas have UV-exposure as the major environmental factor to its tumorigenesis, one could expect a high frequency of UV-related mutations in *TP53* in melanomas, as those

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However, the proportion of primary melanomas harboring *TP53* mutations is frequently low, around 7% of melanoma samples, although ranging from 0 to 24% between individual studies [38]. Data from meta-analysis of 645 melanoma specimens showed that only 13.2% were *TP53* mutants, and more than half were UV signature changes [164]. Curiously, *TP53* mutations have been described in some nevi and in melanomas from XP patients [165]. In fact, on one hand *TP53* inactivating mutations play a role in cancer progression, however, on the other hand, TP53 mutations in melanomas are frequently low. With this duality in mind, interesting question arises: What is the function of *TP53* in melanoma initiation and progression? Moreover, is there a positive pressure to keep wild-type *TP53* in melanomas? Recent functional studies start to address these questions. Indeed, inactivation of p53 pathway may be relevant for melanocyte transformation [166]. Study from melanocytes indicated that murine cells engineered to have high levels of p53 developed less pigmented lesions, primary melanomas and metastases [167]. Besides this feature, melanomas from elevated p53 levels had lower size and growth rate, indicating a role for p53 as a suppressor of tumor development. Regarding human melanocytes and melanoma cells, pharmacological activation of p53 by a specific inhibitor of HDM-2, led cells to cell cycle arrest in low doses and to apoptosis in high doses. In addition, chemical activation of p53 in primary human melanocytes and melanoma cells demonstrated that these cells were far more sensitive to cell cycle arrest than to apoptosis. Moreover, *CDKN1A* (also known as p21) was identified as the predominant network operating in such tumor suppressor activity in melanocytes and melanomas [167]. In summary, such study indicated an anti-proliferative role of p53, both *in vivo* and *in vitro*, as the preferential mode for tumor suppression in melanocytes, indicating then the "need" for p53 supression to allow melanomagenesis. However, as mutations are infrequent in *TP5*3 from melanomas, a possible way to inactivate p53 pathway is by regulating its activity. One possible mechanism for the inactivation of the p53 pathway in melanoma may be attributed to mutations in the *CDKN2A* locus. As discussed above, *CDKN2A* is frequently mutated, lost or even epigeneti‐ cally silenced in melanomas. Besides p16 protein, the locus also codes for p14ARF protein, which is regulates HDM-2 protein, the classical negative regulator of p53. Thus, one manner to contribute to melanoma progression through inactivation of p53-dependent pathways is by inactivation of p14 protein, or even by amplification of *HDM-2* gene. Under both circumstan‐ ces, abundance of p53 protein decreases. However, p14 mutations are frequently associated with familial melanomas, which does not explain the somatic cases, *HDM-2* amplification in melanomas occurs in a very low frequency (ranging 3 to 5% - [168]) and high-level expression

found in the melanoma genome [19] and *PTEN* gene [90].

of wild-type p53 can be found in melanoma tissues and cell lines [169].

PAX3 activities as a transcription factor were also analyzed by comparing melanocytic and melanoma cell lines [162]. Initially, PAX3 binding to promoter regions of specific genes was analyzed and a enrichment of binding in melanoma cells was observed in genes such as HES1, SOX9 and NES (genes related to maintenance of stemness phenotype), CCNA2 and TPD52 (genes related to proliferation), BCL2L1, PTEN and TGFB1 (genes related to survival) and MCAM, CSPG4 and CXCR4 (genes related to migration). Conversely, in melanocytic cell lines, enrichment of PAX3 binding was just observed in HES1, SOX9, MCAM, TGFB1 and CSPG4, however quantitative analysis indicated lower PAX3 binding activity in melanocyte promot‐ ers, as compared to melanomas. Finally, a correlation of PAX3 promoter binding levels in melanocyte/melanoma cell line with gene expression of those genes indicated up-regulation of SOX9, NES, CCNA2, TPD52, TGFB1, MCAM, CSPG4 and CXCR4 in melanoma. Regarding BCL2L1 and PTEN, lower levels were observed in melanoma. In general, the study described a correlation between PAX3 binding to the target gene and its expression level, identified possible PAX3-regulated genes and also suggested the differential activity of PAX3 in transcriptional activity in melanocytes and melanoma cells. The interpretation of the results indicates critical features of the PAX3 function. Those genes up-regulated are genes related to cancer progression (SOX9 and NES), genes involved with cell motility, spread and metastatic potential (MCAM, CSPG4 and CXCR4) and with proliferation (TPD52). Moreover, down regulation of PTEN also contributes to melanoma progression due to tumor suppression activity of PTEN. Decreased of CDK2, BCL2 and MelanA (a melanocyte differentiation marker) gene expression and inhibition of cell growth was observed with PAX3 knock-down in melanoma cell lines, although the results were strongly cell line dependent [157]. Moreover, an induced cell cycle arrest in S and G2/M phases and increase in apoptosis was also observed in PAX3 knock-down melanoma cells, and in one cell line. Silencing of PAX3 induced terminal differentiation.

In general, there is convincing evidence that PAX3 is expressed in melanomas and in mela‐ nocytic lesions, such as nevi. Indeed, PAX3 expression in melanomas may play a role in progression regulating processes such as survival, proliferation, metastases and participating in the maintenance of stemness. However, PAX3 seems expressed in a subset of differentiated melanocytes. Further clarification of PAX3 function in these cells is necessary. Environmental stimuli may be related to PAX3 expression in melanocytic lesions, as reported by up-regulation of PAX3 under UV-induced loss of TFG-β signaling from keratinocytes [163]. Thus, PAX3 may be a good target gene to understanding the melanomagenesis process and more studies regarding its function are required.

#### *4.2.6 TP53 gene and melanoma – What is its function?*

The *TP53* gene is thought to be the "guardian of the genome" due to its pleiotropic function in protecting cells from genotoxic events, acting on cell cycle control, DNA repair and also triggering apoptosis. In general, *TP53* is frequently mutated in a diversity of cancer types and its inactivation confers advantage to tumor initiation and progression. Regarding the sources of mutagenic agents to TP53, sun exposure is a potent mechanism of induction of *TP53* mutations as suggested by the frequent occurrence of such mutations in skin cancers such as basal cell carcinomas (BCC) and squamous cell carcinomas (SCC). In such skin cancers, UVrelated mutations (C to T and CC to TT transitions) are frequently described in *TP53* and in other genes, confirming then the role of UV exposure in skin cancers. As melanocytes from exposed skin areas have UV-exposure as the major environmental factor to its tumorigenesis, one could expect a high frequency of UV-related mutations in *TP53* in melanomas, as those found in the melanoma genome [19] and *PTEN* gene [90].

progression and metastasis through the expression of MET, a classical proto-oncogene

PAX3 activities as a transcription factor were also analyzed by comparing melanocytic and melanoma cell lines [162]. Initially, PAX3 binding to promoter regions of specific genes was analyzed and a enrichment of binding in melanoma cells was observed in genes such as HES1, SOX9 and NES (genes related to maintenance of stemness phenotype), CCNA2 and TPD52 (genes related to proliferation), BCL2L1, PTEN and TGFB1 (genes related to survival) and MCAM, CSPG4 and CXCR4 (genes related to migration). Conversely, in melanocytic cell lines, enrichment of PAX3 binding was just observed in HES1, SOX9, MCAM, TGFB1 and CSPG4, however quantitative analysis indicated lower PAX3 binding activity in melanocyte promot‐ ers, as compared to melanomas. Finally, a correlation of PAX3 promoter binding levels in melanocyte/melanoma cell line with gene expression of those genes indicated up-regulation of SOX9, NES, CCNA2, TPD52, TGFB1, MCAM, CSPG4 and CXCR4 in melanoma. Regarding BCL2L1 and PTEN, lower levels were observed in melanoma. In general, the study described a correlation between PAX3 binding to the target gene and its expression level, identified possible PAX3-regulated genes and also suggested the differential activity of PAX3 in transcriptional activity in melanocytes and melanoma cells. The interpretation of the results indicates critical features of the PAX3 function. Those genes up-regulated are genes related to cancer progression (SOX9 and NES), genes involved with cell motility, spread and metastatic potential (MCAM, CSPG4 and CXCR4) and with proliferation (TPD52). Moreover, down regulation of PTEN also contributes to melanoma progression due to tumor suppression activity of PTEN. Decreased of CDK2, BCL2 and MelanA (a melanocyte differentiation marker) gene expression and inhibition of cell growth was observed with PAX3 knock-down in melanoma cell lines, although the results were strongly cell line dependent [157]. Moreover, an induced cell cycle arrest in S and G2/M phases and increase in apoptosis was also observed in PAX3 knock-down melanoma cells, and in one cell line. Silencing of PAX3 induced terminal

In general, there is convincing evidence that PAX3 is expressed in melanomas and in mela‐ nocytic lesions, such as nevi. Indeed, PAX3 expression in melanomas may play a role in progression regulating processes such as survival, proliferation, metastases and participating in the maintenance of stemness. However, PAX3 seems expressed in a subset of differentiated melanocytes. Further clarification of PAX3 function in these cells is necessary. Environmental stimuli may be related to PAX3 expression in melanocytic lesions, as reported by up-regulation of PAX3 under UV-induced loss of TFG-β signaling from keratinocytes [163]. Thus, PAX3 may be a good target gene to understanding the melanomagenesis process and more studies

The *TP53* gene is thought to be the "guardian of the genome" due to its pleiotropic function in protecting cells from genotoxic events, acting on cell cycle control, DNA repair and also triggering apoptosis. In general, *TP53* is frequently mutated in a diversity of cancer types and its inactivation confers advantage to tumor initiation and progression. Regarding the sources

involved in invasion, metastasis, resistance of apoptosis, and tumor cell expansion.

differentiation.

regarding its function are required.

112 Melanoma - From Early Detection to Treatment

*4.2.6 TP53 gene and melanoma – What is its function?*

However, the proportion of primary melanomas harboring *TP53* mutations is frequently low, around 7% of melanoma samples, although ranging from 0 to 24% between individual studies [38]. Data from meta-analysis of 645 melanoma specimens showed that only 13.2% were *TP53* mutants, and more than half were UV signature changes [164]. Curiously, *TP53* mutations have been described in some nevi and in melanomas from XP patients [165]. In fact, on one hand *TP53* inactivating mutations play a role in cancer progression, however, on the other hand, TP53 mutations in melanomas are frequently low. With this duality in mind, interesting question arises: What is the function of *TP53* in melanoma initiation and progression? Moreover, is there a positive pressure to keep wild-type *TP53* in melanomas? Recent functional studies start to address these questions. Indeed, inactivation of p53 pathway may be relevant for melanocyte transformation [166]. Study from melanocytes indicated that murine cells engineered to have high levels of p53 developed less pigmented lesions, primary melanomas and metastases [167]. Besides this feature, melanomas from elevated p53 levels had lower size and growth rate, indicating a role for p53 as a suppressor of tumor development. Regarding human melanocytes and melanoma cells, pharmacological activation of p53 by a specific inhibitor of HDM-2, led cells to cell cycle arrest in low doses and to apoptosis in high doses. In addition, chemical activation of p53 in primary human melanocytes and melanoma cells demonstrated that these cells were far more sensitive to cell cycle arrest than to apoptosis. Moreover, *CDKN1A* (also known as p21) was identified as the predominant network operating in such tumor suppressor activity in melanocytes and melanomas [167]. In summary, such study indicated an anti-proliferative role of p53, both *in vivo* and *in vitro*, as the preferential mode for tumor suppression in melanocytes, indicating then the "need" for p53 supression to allow melanomagenesis. However, as mutations are infrequent in *TP5*3 from melanomas, a possible way to inactivate p53 pathway is by regulating its activity. One possible mechanism for the inactivation of the p53 pathway in melanoma may be attributed to mutations in the *CDKN2A* locus. As discussed above, *CDKN2A* is frequently mutated, lost or even epigeneti‐ cally silenced in melanomas. Besides p16 protein, the locus also codes for p14ARF protein, which is regulates HDM-2 protein, the classical negative regulator of p53. Thus, one manner to contribute to melanoma progression through inactivation of p53-dependent pathways is by inactivation of p14 protein, or even by amplification of *HDM-2* gene. Under both circumstan‐ ces, abundance of p53 protein decreases. However, p14 mutations are frequently associated with familial melanomas, which does not explain the somatic cases, *HDM-2* amplification in melanomas occurs in a very low frequency (ranging 3 to 5% - [168]) and high-level expression of wild-type p53 can be found in melanoma tissues and cell lines [169].

Some reports have indicated that high expression of p53 can be found in both melanoma samples and melanoma cell lines. In addition, others reports have also indicated that this high expression does not correlate with p53 functionality. Melanoma cell lines harboring wild-type p53 showed transcriptional inactivity [169], a feature of melanoma cell lines that corroborates with data showing different gene expression of p53 targets in melanomas compared with nevi, strongly suggesting a dysfunctional p53 [166]. Moreover, melanoma cell lines with wild-type p53 shows an absent p53 DNA-binding activity [170]. All these reports indicate that down‐ stream mechanisms could be operating to down-regulate p53 pathway in melanomas. One of the challenges of melanoma genetics in the coming years is to identify and characterize those downstream mechanisms, which certainly will improve our knowledge about p53 dysfunction in melanoma biology as well as identifying possible windows for melanoma treatment. There are at this moment critical candidates genes to act as negative regulator of p53 activity. Proteins such as iASPP (Inhibitor of apoptosis-stimulating protein of p53) [171], delta Np73 [172], YB-1 [173] and Parc protein [174] has been described as p53 inhibitors. Alternatively, posttransla‐ tional modifications may also be responsible to p53 transcriptional silencing, such as phos‐ phorylation, acetylation, methylation, sumoylation and neddlyation. Some findings have suggested that accumulation and increase in wild type p53 expression during melanoma progression may be indicative of dysfunctional p53 activity, reflecting posttranslational p53 modifications. Cytoplasmatic functions of transcriptionally inactive p53 have also emerged as a good hypothesis to a new p53 activity in either limit or promote tumor growth [175].

pro-apoptotic proteins PUMA and NOXA. Under ER stress, melanoma cells accumulate p53, which in turn (even in basal activity) induces the transcription of the microRNA miR149\* [180]. The p53-dependent expression of miR149\* decrease the activity of GSK3α, resulting in Mcl-1 increase and consequent resistance to apoptosis. Moreover, decrease of miR149\* elevated the rate of cell death in these melanoma cells and inhibited melanoma growth in a xenograft model. Finally, elevated expression of miR149 was found in mela‐

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Other elegant functional study indicated critical features of p53 role in melanocytes and melanoma cells [177]. First, the study indicated that p53 may be dispensable for melanoma cells due to lack of increase in DNA damage and enhanced proliferative potential in p53 depleted cells. Conversely, depletion of p53 in melanocytes increased mitotic defects. This last result is consistent with animal models in which genetic depletion of p53 cooperates with cell transformation [167]. Indeed, in melanoma cells p53 is kept in a basal state of functionality. This basal activity showed to be critical to melanoma growth, as: (i) basal p53 activity leads to HDM-2 expression, which in turn keeps the basal levels of p53; (ii) this basal level of p53 avoids the activation of a p53-dependent pro-senescence program; (iii) in a basal state, p53 does not induce expression of p21, which in turn does not inhibits E2F1. The following E2F1 activation contributes to melanoma cell proliferation; (iv) expression of HMD-2 leads to activation of E2F1 in a p53-independet manner, contributing to melanoma cell proliferation. Instead, the "so-called" HDM-2 addiction in melanoma cells seems not to be related to melanocytes due to maintenance of viability and absence of senescence when p53 is activated by MDM-2 depletion. In summary, this study [177] elucidates new functions of the p53-HDM-2 axis in melanomas. Besides, the p53-HDM-2 axis in melanomas is now suggested as a promising target for melanoma treatment, since the use of specific HDM-2 antagonist rescues the p53

noma samples, associated with decrease of GSK3α and increase of Mcl-1.

activity, leading to melanoma growth suppression and melanoma cell death [181].

*4.2.7. MicroRNAs and melanoma – Another level of gene expression in melanomas*

The identification of negative p53 regulators that keep p53 pathway dysfunctional seems critical for a better understanding of the involvement of p53-dependent pathways in melano‐ magenesis and progression. Further functional studies will elucidate the intriguing questions regarding the real function of p53 to melanoma biology: Why has TP53 low frequency of mutations? How is p53 basal state maintained? What are p53 functions in melanomas?

MicroRNAs (miRNAs) are small non-coding RNAs (21–23 nucleotides) encoded in the genome of plants, invertebrates, and vertebrates. These small molecules bind imperfectly to the 3´ untranslated (3´UTR) regions of target messenger RNAs (mRNAs) thus, miRNAs are central regulators of gene expression and can act both in a positive and a negative way to control protein levels in the cell. More than a thousand miRNAs exist in the human genome and each one can potentially regulate hundreds of mRNAs. Target prediction algorithms can be helpful in identifying potential mRNA targets of the miRNA of interest and further they should be validated by functional studies [182]. MicroRNAs play an important role in many cellular processes, such as differentiation, proliferation, apoptosis, and stress response. Additionally, they are key regulators in many diseases, including cancer [183]. These molecules regulate

Additional reports have also confirmed the p53 transcriptional inability in melanomas [176]. The results from such study showed that p53 downstream genes involved in apoptosis have low expression in melanoma metastases and melanoma cell lines. Conversely, genes involved with cell cycle were over-expressed in melanoma cell lines. Curiously, little difference between melanomas with wild-type p53 and mutant p53 could be observed regarding expression of p53 target genes, which confirm the notion that possible negative regulators are involved in the suppression of the p53 pathway. Even with down-regulation of p53 by using short-harpin method, there was limited effect on p53 target genes in p53 wild-type melanomas, however to melanocytes, p53 inhibition leads to alteration of several p53-dependent transcripts. An interesting feature observed was related to the proliferative capacity in melanocytes and melanomas, down regulation of p53 in melanocytes resulted in a gene expression similar to melanomas and increased proliferation rates while in melanomas, down regulation of p53 contributed to decreased proliferation, corroborating the results described by an independent study [177] (discussed below).

Although melanomas may have an inability to exert p53 full transcriptional capability, the p53 accumulation observed in such melanomas may still have basal activity. A cen‐ tral question is to understand the role of this basal p53 transcriptional activity in pro‐ gression of melanomas. Recent functional studies start to address this interesting question. Melanoma cells are described as largely adapted to certain stress such as endo‐ plasmic reticulum (ER) stress [178], a situation where melanomas acquire resistance to ER stress-induced apoptosis as well as resistance to chemotherapy [179]. This adaptative response may be attributed to expression of Mcl-1 protein, which acts antagonizing the pro-apoptotic proteins PUMA and NOXA. Under ER stress, melanoma cells accumulate p53, which in turn (even in basal activity) induces the transcription of the microRNA miR149\* [180]. The p53-dependent expression of miR149\* decrease the activity of GSK3α, resulting in Mcl-1 increase and consequent resistance to apoptosis. Moreover, decrease of miR149\* elevated the rate of cell death in these melanoma cells and inhibited melanoma growth in a xenograft model. Finally, elevated expression of miR149 was found in mela‐ noma samples, associated with decrease of GSK3α and increase of Mcl-1.

Some reports have indicated that high expression of p53 can be found in both melanoma samples and melanoma cell lines. In addition, others reports have also indicated that this high expression does not correlate with p53 functionality. Melanoma cell lines harboring wild-type p53 showed transcriptional inactivity [169], a feature of melanoma cell lines that corroborates with data showing different gene expression of p53 targets in melanomas compared with nevi, strongly suggesting a dysfunctional p53 [166]. Moreover, melanoma cell lines with wild-type p53 shows an absent p53 DNA-binding activity [170]. All these reports indicate that down‐ stream mechanisms could be operating to down-regulate p53 pathway in melanomas. One of the challenges of melanoma genetics in the coming years is to identify and characterize those downstream mechanisms, which certainly will improve our knowledge about p53 dysfunction in melanoma biology as well as identifying possible windows for melanoma treatment. There are at this moment critical candidates genes to act as negative regulator of p53 activity. Proteins such as iASPP (Inhibitor of apoptosis-stimulating protein of p53) [171], delta Np73 [172], YB-1 [173] and Parc protein [174] has been described as p53 inhibitors. Alternatively, posttransla‐ tional modifications may also be responsible to p53 transcriptional silencing, such as phos‐ phorylation, acetylation, methylation, sumoylation and neddlyation. Some findings have suggested that accumulation and increase in wild type p53 expression during melanoma progression may be indicative of dysfunctional p53 activity, reflecting posttranslational p53 modifications. Cytoplasmatic functions of transcriptionally inactive p53 have also emerged as a good hypothesis to a new p53 activity in either limit or promote tumor growth [175].

Additional reports have also confirmed the p53 transcriptional inability in melanomas [176]. The results from such study showed that p53 downstream genes involved in apoptosis have low expression in melanoma metastases and melanoma cell lines. Conversely, genes involved with cell cycle were over-expressed in melanoma cell lines. Curiously, little difference between melanomas with wild-type p53 and mutant p53 could be observed regarding expression of p53 target genes, which confirm the notion that possible negative regulators are involved in the suppression of the p53 pathway. Even with down-regulation of p53 by using short-harpin method, there was limited effect on p53 target genes in p53 wild-type melanomas, however to melanocytes, p53 inhibition leads to alteration of several p53-dependent transcripts. An interesting feature observed was related to the proliferative capacity in melanocytes and melanomas, down regulation of p53 in melanocytes resulted in a gene expression similar to melanomas and increased proliferation rates while in melanomas, down regulation of p53 contributed to decreased proliferation, corroborating the results described by an independent

Although melanomas may have an inability to exert p53 full transcriptional capability, the p53 accumulation observed in such melanomas may still have basal activity. A cen‐ tral question is to understand the role of this basal p53 transcriptional activity in pro‐ gression of melanomas. Recent functional studies start to address this interesting question. Melanoma cells are described as largely adapted to certain stress such as endo‐ plasmic reticulum (ER) stress [178], a situation where melanomas acquire resistance to ER stress-induced apoptosis as well as resistance to chemotherapy [179]. This adaptative response may be attributed to expression of Mcl-1 protein, which acts antagonizing the

study [177] (discussed below).

114 Melanoma - From Early Detection to Treatment

Other elegant functional study indicated critical features of p53 role in melanocytes and melanoma cells [177]. First, the study indicated that p53 may be dispensable for melanoma cells due to lack of increase in DNA damage and enhanced proliferative potential in p53 depleted cells. Conversely, depletion of p53 in melanocytes increased mitotic defects. This last result is consistent with animal models in which genetic depletion of p53 cooperates with cell transformation [167]. Indeed, in melanoma cells p53 is kept in a basal state of functionality. This basal activity showed to be critical to melanoma growth, as: (i) basal p53 activity leads to HDM-2 expression, which in turn keeps the basal levels of p53; (ii) this basal level of p53 avoids the activation of a p53-dependent pro-senescence program; (iii) in a basal state, p53 does not induce expression of p21, which in turn does not inhibits E2F1. The following E2F1 activation contributes to melanoma cell proliferation; (iv) expression of HMD-2 leads to activation of E2F1 in a p53-independet manner, contributing to melanoma cell proliferation. Instead, the "so-called" HDM-2 addiction in melanoma cells seems not to be related to melanocytes due to maintenance of viability and absence of senescence when p53 is activated by MDM-2 depletion. In summary, this study [177] elucidates new functions of the p53-HDM-2 axis in melanomas. Besides, the p53-HDM-2 axis in melanomas is now suggested as a promising target for melanoma treatment, since the use of specific HDM-2 antagonist rescues the p53 activity, leading to melanoma growth suppression and melanoma cell death [181].

The identification of negative p53 regulators that keep p53 pathway dysfunctional seems critical for a better understanding of the involvement of p53-dependent pathways in melano‐ magenesis and progression. Further functional studies will elucidate the intriguing questions regarding the real function of p53 to melanoma biology: Why has TP53 low frequency of mutations? How is p53 basal state maintained? What are p53 functions in melanomas?

#### *4.2.7. MicroRNAs and melanoma – Another level of gene expression in melanomas*

MicroRNAs (miRNAs) are small non-coding RNAs (21–23 nucleotides) encoded in the genome of plants, invertebrates, and vertebrates. These small molecules bind imperfectly to the 3´ untranslated (3´UTR) regions of target messenger RNAs (mRNAs) thus, miRNAs are central regulators of gene expression and can act both in a positive and a negative way to control protein levels in the cell. More than a thousand miRNAs exist in the human genome and each one can potentially regulate hundreds of mRNAs. Target prediction algorithms can be helpful in identifying potential mRNA targets of the miRNA of interest and further they should be validated by functional studies [182]. MicroRNAs play an important role in many cellular processes, such as differentiation, proliferation, apoptosis, and stress response. Additionally, they are key regulators in many diseases, including cancer [183]. These molecules regulate pathways in cancer by targeting various oncogenes and tumor suppressors and there is an increasing body of evidence suggesting that genomic instability regions harbor miRNA genes [184]. The first study to associate genomic instability regions, miRNAs and cancer was published in 2002 [185]. The authors found frequent deletions at 13q14 involving miR-15 and miR-16 genes in B-cell from chronic lymphocytic leukaemia. Since then, hundreds miRNAs have been reported acting as oncogenes or tumour suppressor genes in a wide variety of cancers [for review 183]. The first miRNAs described as involved in cancer formation was miRlet-7 [186] and further the family of miRs let 7a and let 7b were reported to play a role in melanomas [for review 187]. For example, miR-let 7-b acts as a negative regulator of melanoma cell proliferation via regulation of cyclin D1, whereas miR-let-7a was demonstrated to regulate the expression of integrin-β3 and the Ras [188]. So, modulation of miRNA expression is increasingly thought to be an important mechanism by which tumour suppressor proteins and oncoproteins exert some of their effects. Studies assessing the role of miRNAs in melanomas are still very recent and many efforts have been made to identify the 'melano-miRs'. Despite the increasing number of studies (NCBI searching in September 2012 retrieved 162 results) a small number of miRNAs were identified to regulate genes involved specifically in melano‐ magenesis and some of them will be discussed here.

one study linking three miRNAs to BRafV600E [196]. Recently, [197] a network of 420 miRNAs deregulated in B-Raf/MKK/ERK pathway in melanoma cells whereas majority of which modulate the expression of key cancer regulatory genes and functions was identified. In addition to MEK/ERK pathway, new insights about miRNAs and p16INK4A-CDK4-RB pathway have been described. The mains senescence pathway associated with miRNAs are p53/p21 and p16/Rb pathways [for review 198]. Several miRNAs have been shown to be involved in the regulation of pathways involved in cellular senescence exerting negative effects on cell cycle progression, such as E2F family of transcription factors acting in cell cycle [198-200]. Recent studies reported that E2F1 to E2F3 are targets of several miRNAs, such as miR-34a [201]. In addition, miR-205 in human melanoma cells induces senescence by targeting E2F1 [202] and miR-203 also induces senescence by targeting E2F3 in melanoma cells [203]. Therefore, miRNA/E2F interaction is an important mechanism that leads melanomas cells to senescence.

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117

Other studies have identified a cluster of miRNAs that are either involved in melanomagenesis or predictors of survival. A study has identified the miR-506–514 cluster as a transforming oncogene that regulates melanoma progression and melanocyte transformation [204]. More‐ over, the authors showed that ectopic expression of this cluster in melanocytes was sufficient to transform them, activating cell growth, cell proliferation and migration/invasion along with inhibiting apoptosis. Although this study did not identify any direct gene targets of the miRNAs, further investigation is necessary because this cluster may reveal pathways that contribute to both the initiation and the maintenance of melanoma. As presented above, studies showed the increased expression of the miR-221/222 cluster associated with melanoma progression [for review 205]. A cascade involving *PLZF* transcription factor as a repressor of miR-221 and miR-222 by direct binding to their putative regulatory region was described [206]. These miRNAs regulate directly *KIT* and *CDKN1B*, respectively resulting in cell cycle inhibi‐ tion and differentiation. Thus, over-expression of these miRNAs cluster increases proliferation

Approaches investigating miRNAs expression are also based on gene silencing by CpG methylation. Since miRNAs precursor genes are usually within regions of coding genes (intron sequences, for example), dysfunction of these protein-coding genes by epigenetic mechanisms may also be expected to cause aberrant regulation of the miRNA target genes [207]. For example, miRNA-34a is highly methylated in melanoma cell lines and primary tumors and additionally, it was described that *MET* transcript is miRNA-34a target [for review 188]. Besides miR-34a, the miR-34b, belonging to the same family, seems to have an important effect on melanomas. A group of epigenetically regulated miRNA genes in melanoma cells, and confirmed the upstream hypermethylated CpG island sequences of several miRNAs genes in cell lines derived from different stages of melanoma, but not in melanocytes and keratinocytes was identifies [208]. Among them, miR-34b expression reduced cell invasion and motility rates of melanoma cell lines. After deep sequencing, the authors identified network modules that are potentially regulated by miR-34b, and which suggest a mechanism for the role of miR-34b in regulating normal cell motility and cytokinesis. Additionally, this same group identified the epigenetic regulation of miR-375 in human melanomas. Melanoma cells were treated with one demethylating agent (5-aza-2'-deoxycytidine) and it was identified the miR-375 highly

and tumourigenesis and activates invasion/migration in melanomas.

The linking between expression of miR-137 and *MITF* expression, a crucial gene involved in melanomas and already presented above have been described [189]. However, *MITF* seems to be also regulated by miR-182, miR-148, and miR-340, respectively [190;191]. Additionally, melanoma tumors preferentially express *MITF* mRNA isoforms with shorter 3´UTR, "to avoid" miRNA post-transcriptional regulation. Although the translation of the transcripts can be regulated by miRNAs the transcriptional regulation of miRNAs is still poorly known [192]. Some studies have searched for miRNA promoters that are specific to melanoma progression [193]. In an opposite way, the authors identified miRNAs that are specifically regulated by *MITF* transcription factor/oncoprotein and identified miR-146a, miR-221/222 and miR-363 as MITF-regulated. This high-throughput identification of miRNA promoters and enhancer regulatory elements sheds light on evolution of miRNA transcription and permits rapid identification of transcriptional networks of miRNAs, inclusive in melanomas. Moreover, expression of *MITF* has been recently shown to be involved in the regulation of DICER, the central regulator of miRNA maturation and key enzyme involved in the formation of the RNAinduced silencing complex. MITF binds and activates a conserved regulatory element up‐ stream of DICER's transcriptional start site upon melanocyte differentiation [194]. Moreover, when DICER was knocked out, melanocytes failed to survive [194].

Besides miRNAs "*MITF* regulators" or miRNAs "regulated by MITF", other molecules with known target genes in melanoma are also regulated by miRNAs. Recently an interesting review focusing on miRNAs that act in major pathways of formation of melanomas: RAS-RAF-MEK-ERK, p16INK4A-CDK4-RB, PIK3-AKT and the MITF pathway was published [195]. As cited, mutation BRafV600E occurs in 50–70% of sporadic melanomas which active constitutively the MEK/ERK signaling pathway, promoting tumor progression and metastasis through enhanced cell proliferation, survival, motility and invasion. Two studies have investigated the correlation between B-Raf mutational status and miRNA expression in melanomas and only one study linking three miRNAs to BRafV600E [196]. Recently, [197] a network of 420 miRNAs deregulated in B-Raf/MKK/ERK pathway in melanoma cells whereas majority of which modulate the expression of key cancer regulatory genes and functions was identified. In addition to MEK/ERK pathway, new insights about miRNAs and p16INK4A-CDK4-RB pathway have been described. The mains senescence pathway associated with miRNAs are p53/p21 and p16/Rb pathways [for review 198]. Several miRNAs have been shown to be involved in the regulation of pathways involved in cellular senescence exerting negative effects on cell cycle progression, such as E2F family of transcription factors acting in cell cycle [198-200]. Recent studies reported that E2F1 to E2F3 are targets of several miRNAs, such as miR-34a [201]. In addition, miR-205 in human melanoma cells induces senescence by targeting E2F1 [202] and miR-203 also induces senescence by targeting E2F3 in melanoma cells [203]. Therefore, miRNA/E2F interaction is an important mechanism that leads melanomas cells to senescence.

pathways in cancer by targeting various oncogenes and tumor suppressors and there is an increasing body of evidence suggesting that genomic instability regions harbor miRNA genes [184]. The first study to associate genomic instability regions, miRNAs and cancer was published in 2002 [185]. The authors found frequent deletions at 13q14 involving miR-15 and miR-16 genes in B-cell from chronic lymphocytic leukaemia. Since then, hundreds miRNAs have been reported acting as oncogenes or tumour suppressor genes in a wide variety of cancers [for review 183]. The first miRNAs described as involved in cancer formation was miRlet-7 [186] and further the family of miRs let 7a and let 7b were reported to play a role in melanomas [for review 187]. For example, miR-let 7-b acts as a negative regulator of melanoma cell proliferation via regulation of cyclin D1, whereas miR-let-7a was demonstrated to regulate the expression of integrin-β3 and the Ras [188]. So, modulation of miRNA expression is increasingly thought to be an important mechanism by which tumour suppressor proteins and oncoproteins exert some of their effects. Studies assessing the role of miRNAs in melanomas are still very recent and many efforts have been made to identify the 'melano-miRs'. Despite the increasing number of studies (NCBI searching in September 2012 retrieved 162 results) a small number of miRNAs were identified to regulate genes involved specifically in melano‐

The linking between expression of miR-137 and *MITF* expression, a crucial gene involved in melanomas and already presented above have been described [189]. However, *MITF* seems to be also regulated by miR-182, miR-148, and miR-340, respectively [190;191]. Additionally, melanoma tumors preferentially express *MITF* mRNA isoforms with shorter 3´UTR, "to avoid" miRNA post-transcriptional regulation. Although the translation of the transcripts can be regulated by miRNAs the transcriptional regulation of miRNAs is still poorly known [192]. Some studies have searched for miRNA promoters that are specific to melanoma progression [193]. In an opposite way, the authors identified miRNAs that are specifically regulated by *MITF* transcription factor/oncoprotein and identified miR-146a, miR-221/222 and miR-363 as MITF-regulated. This high-throughput identification of miRNA promoters and enhancer regulatory elements sheds light on evolution of miRNA transcription and permits rapid identification of transcriptional networks of miRNAs, inclusive in melanomas. Moreover, expression of *MITF* has been recently shown to be involved in the regulation of DICER, the central regulator of miRNA maturation and key enzyme involved in the formation of the RNAinduced silencing complex. MITF binds and activates a conserved regulatory element up‐ stream of DICER's transcriptional start site upon melanocyte differentiation [194]. Moreover,

Besides miRNAs "*MITF* regulators" or miRNAs "regulated by MITF", other molecules with known target genes in melanoma are also regulated by miRNAs. Recently an interesting review focusing on miRNAs that act in major pathways of formation of melanomas: RAS-RAF-MEK-ERK, p16INK4A-CDK4-RB, PIK3-AKT and the MITF pathway was published [195]. As cited, mutation BRafV600E occurs in 50–70% of sporadic melanomas which active constitutively the MEK/ERK signaling pathway, promoting tumor progression and metastasis through enhanced cell proliferation, survival, motility and invasion. Two studies have investigated the correlation between B-Raf mutational status and miRNA expression in melanomas and only

magenesis and some of them will be discussed here.

116 Melanoma - From Early Detection to Treatment

when DICER was knocked out, melanocytes failed to survive [194].

Other studies have identified a cluster of miRNAs that are either involved in melanomagenesis or predictors of survival. A study has identified the miR-506–514 cluster as a transforming oncogene that regulates melanoma progression and melanocyte transformation [204]. More‐ over, the authors showed that ectopic expression of this cluster in melanocytes was sufficient to transform them, activating cell growth, cell proliferation and migration/invasion along with inhibiting apoptosis. Although this study did not identify any direct gene targets of the miRNAs, further investigation is necessary because this cluster may reveal pathways that contribute to both the initiation and the maintenance of melanoma. As presented above, studies showed the increased expression of the miR-221/222 cluster associated with melanoma progression [for review 205]. A cascade involving *PLZF* transcription factor as a repressor of miR-221 and miR-222 by direct binding to their putative regulatory region was described [206]. These miRNAs regulate directly *KIT* and *CDKN1B*, respectively resulting in cell cycle inhibi‐ tion and differentiation. Thus, over-expression of these miRNAs cluster increases proliferation and tumourigenesis and activates invasion/migration in melanomas.

Approaches investigating miRNAs expression are also based on gene silencing by CpG methylation. Since miRNAs precursor genes are usually within regions of coding genes (intron sequences, for example), dysfunction of these protein-coding genes by epigenetic mechanisms may also be expected to cause aberrant regulation of the miRNA target genes [207]. For example, miRNA-34a is highly methylated in melanoma cell lines and primary tumors and additionally, it was described that *MET* transcript is miRNA-34a target [for review 188]. Besides miR-34a, the miR-34b, belonging to the same family, seems to have an important effect on melanomas. A group of epigenetically regulated miRNA genes in melanoma cells, and confirmed the upstream hypermethylated CpG island sequences of several miRNAs genes in cell lines derived from different stages of melanoma, but not in melanocytes and keratinocytes was identifies [208]. Among them, miR-34b expression reduced cell invasion and motility rates of melanoma cell lines. After deep sequencing, the authors identified network modules that are potentially regulated by miR-34b, and which suggest a mechanism for the role of miR-34b in regulating normal cell motility and cytokinesis. Additionally, this same group identified the epigenetic regulation of miR-375 in human melanomas. Melanoma cells were treated with one demethylating agent (5-aza-2'-deoxycytidine) and it was identified the miR-375 highly methylated. Ectopic expression of miR-375 inhibited melanoma cell proliferation, invasion, and cell motility, and induced cell shape changes, suggesting that miR-375 may have an important function in the development and progression of human melanomas [209].

genes involved in invasion and angiogenesis. Histological examination of skin biopsies remains the standard method for melanoma diagnosis and prognosis. Significant morpholog‐ ical overlap between benign and malignant lesions complicates diagnosis, and tumour thickness is not always an accurate predictor of prognosis. For purpose of clinical management, the microRNA profiling of clinical melanoma subtypes samples considering the somatic and inherited mutations associated with melanomas, including the presence of one variant in a miRNA binding site in the 3'UTR of the *KRAS* oncogene has been evaluated [218]. The authors showed that miR-142-3p, miR-486, miR-214, miR-218, miR-362, miR-650 and miR-31 were significantly correlated with acral as compared to non-acral melanomas. In addition, the *KRAS*-variant was enriched in non-acral melanoma and that miR-137 under expression was significantly associated with melanomas with the *KRAS*-variant. Recently, it was develop one *in situ* measurement methodology to evaluate the miR-205 in a cohort of human melanomas [219]. Based on previous findings, the authors hypothesized that decreased miR-205 would be associated with more aggressive tumors. So, multiplexing miR-205 qISH (quantitative *in situ* hybridization) with immunofluorescent assessment of S100/GP100 (melanocytic markers), the authors evaluated quantitatively the miR-205 expression. Outcome was assessed on the Yale Melanoma Discovery Cohort consisting of 105 primary melanoma specimens and then, validated the results on an independent set of 206 primary melanomas. Measurement of melanoma cell miR-205 levels shows a significantly shorter melanoma-specific survival in patients with their low expression and it was significantly independent of stage, age, gender, and Breslow depth. Low levels of miR-205 expression in melanoma cell quantified by ISH show worse outcome, supporting the role of miR-205 as a tumor suppressor miRNA. This promising result indicates that the quantification of miR-205 *in situ* suggests its potential use for future prognostic or predictive models. Studies investigating the various roles of miRNAs in melanocytes and melanoma are gaining momentum and should continue to provide fertile

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119

In this chapter we proposed to discuss the melanoma genetics, starting from the genes that may confer susceptibility to the genes that may be involved with progression. Moreover, we addressed the already known genes (here called as "old genetics") as well as new genes that have been discovered as involved in melanoma (here called as "new genetics). It is noteworthy that the new technologies such as GWAS and deep-sequencing have improved our knowledge about melanoma genetics. Nowadays we have critical information about the disease, such as the clear involvement of UV in carcinogenic process and the many pathways that contribute significantly to it. As could be observed, conversely to other cancer types where single genes has great impact on susceptibility and progression, such as BRCA in breast and ovarian, mismatch repair pathways and colorectal cancer or *TP53* to Li-Fraumeni syndrome, a single gene cannot be pointed as "the melanoma gene". Huge amplitude of genetic pathways may be related to melanoma progression and this same amplitude may be responsible for melano‐ ma metastasis and chemoresistance, making this neoplasia of complex management. However,

ground for both clinical and basic research.

**5. Conclusions**

All of these studies investigated the biological functions of miRNAs and their contribution to melanomagenesis. Other studies have attempted to identify miRNAs signatures for diagnostic and prognostic, melanoma progression by comparing the expression profiles in different stages of transformation, and others focused on specific pathways. Some of these studies will be presented here. In 2007, assays were performed using the well established NCI-60 cancer cell line panel and normal tissue [210]. The study was able to discriminate between the malignancies, including melanomas cell lines whereas miR-146, miR-204 and miR-211 miRNAs shown to be highly expressed in melanomas. Large cohorts of miRNAs associated with malignant transformation as well as with the progression and with metastatic coloniza‐ tion in melanocytes and subsets of melanoma cell lines also was identified [211]. Subsequently, down regulation of miRNA-200c in melanocytes, melanoma cell lines, and patient samples could be reported, whereas miRNA-205 and miRNA-23b were markedly reduced among patient samples [212]. In contrast, miR-146a and miR-155 were upregulated in all analyzed patients but none of the cell lines. Using deep sequencing approach of a diverse set of mela‐ noma and pigment cell libraries it was identified 539 known mature sequences along with the prediction of 279 novel miRNAs candidates [213]. Some of these novel candidate miRNAs may be specific to the melanocytic lineage and as such could be used as biomarkers in the early detection of distant metastases by measuring the circulating levels in blood. The expression of 611 miRNAs in 59 metastatic specimens was profiled and the authors were able to identify a "miRNA classifier" consisting of miR-150, miR-342-3p, miR-455-3p, miR-145, miR-155 and miR-497 that were considered predictors of post-recurrence survival [214]. Similarly the analyses of the miRNA expression profiling from melanoma lymph node metastases reported a unique signature consisting of down regulation of miR-191, combined with up regulation of miR-193a, miR-193b, miR-365, miR-338 and let-7. Together, this miRNAs also serves as predictors of short-term survival in melanoma patients [215]. These findings indicate that miRNAs are differentially expressed in melanoma subtypes and that their dysfunction can be impacted by inherited gene variants, supporting the hypothesis that miRNA dysfunction reflects biological differences in melanoma. Recently, the use of microarray analysis of formalin-fixed and paraffin-embedded samples from different stages of melanomagenesis identified differentially expressed microRNAs [216]. The miR-203 and miR-205 were differ‐ entially expressed between primary and metastatic melanomas and functional *in vitro* assays validated this found. So, these results indicated that differentially expressed miRNAs that could be useful as diagnostic or prognostic markers for melanoma.

As such, miRNAs represent a new class of molecules that might prove to be powerful cancer biomarkers useful in future staging systems and used as stratification criteria in clinical trials as well as treatment of patients with disseminated disease. It was demonstrated that miR-214 is over-expressed in metastatic melanoma cell lines as well as tumor specimens. MiR-214 regulates the expression of two transcription factors, AP-2c and AP-2a [217]. These molecules have been previously shown to play major roles in melanoma metastasis via regulation of genes involved in invasion and angiogenesis. Histological examination of skin biopsies remains the standard method for melanoma diagnosis and prognosis. Significant morpholog‐ ical overlap between benign and malignant lesions complicates diagnosis, and tumour thickness is not always an accurate predictor of prognosis. For purpose of clinical management, the microRNA profiling of clinical melanoma subtypes samples considering the somatic and inherited mutations associated with melanomas, including the presence of one variant in a miRNA binding site in the 3'UTR of the *KRAS* oncogene has been evaluated [218]. The authors showed that miR-142-3p, miR-486, miR-214, miR-218, miR-362, miR-650 and miR-31 were significantly correlated with acral as compared to non-acral melanomas. In addition, the *KRAS*-variant was enriched in non-acral melanoma and that miR-137 under expression was significantly associated with melanomas with the *KRAS*-variant. Recently, it was develop one *in situ* measurement methodology to evaluate the miR-205 in a cohort of human melanomas [219]. Based on previous findings, the authors hypothesized that decreased miR-205 would be associated with more aggressive tumors. So, multiplexing miR-205 qISH (quantitative *in situ* hybridization) with immunofluorescent assessment of S100/GP100 (melanocytic markers), the authors evaluated quantitatively the miR-205 expression. Outcome was assessed on the Yale Melanoma Discovery Cohort consisting of 105 primary melanoma specimens and then, validated the results on an independent set of 206 primary melanomas. Measurement of melanoma cell miR-205 levels shows a significantly shorter melanoma-specific survival in patients with their low expression and it was significantly independent of stage, age, gender, and Breslow depth. Low levels of miR-205 expression in melanoma cell quantified by ISH show worse outcome, supporting the role of miR-205 as a tumor suppressor miRNA. This promising result indicates that the quantification of miR-205 *in situ* suggests its potential use for future prognostic or predictive models. Studies investigating the various roles of miRNAs in melanocytes and melanoma are gaining momentum and should continue to provide fertile ground for both clinical and basic research.

## **5. Conclusions**

methylated. Ectopic expression of miR-375 inhibited melanoma cell proliferation, invasion, and cell motility, and induced cell shape changes, suggesting that miR-375 may have an

All of these studies investigated the biological functions of miRNAs and their contribution to melanomagenesis. Other studies have attempted to identify miRNAs signatures for diagnostic and prognostic, melanoma progression by comparing the expression profiles in different stages of transformation, and others focused on specific pathways. Some of these studies will be presented here. In 2007, assays were performed using the well established NCI-60 cancer cell line panel and normal tissue [210]. The study was able to discriminate between the malignancies, including melanomas cell lines whereas miR-146, miR-204 and miR-211 miRNAs shown to be highly expressed in melanomas. Large cohorts of miRNAs associated with malignant transformation as well as with the progression and with metastatic coloniza‐ tion in melanocytes and subsets of melanoma cell lines also was identified [211]. Subsequently, down regulation of miRNA-200c in melanocytes, melanoma cell lines, and patient samples could be reported, whereas miRNA-205 and miRNA-23b were markedly reduced among patient samples [212]. In contrast, miR-146a and miR-155 were upregulated in all analyzed patients but none of the cell lines. Using deep sequencing approach of a diverse set of mela‐ noma and pigment cell libraries it was identified 539 known mature sequences along with the prediction of 279 novel miRNAs candidates [213]. Some of these novel candidate miRNAs may be specific to the melanocytic lineage and as such could be used as biomarkers in the early detection of distant metastases by measuring the circulating levels in blood. The expression of 611 miRNAs in 59 metastatic specimens was profiled and the authors were able to identify a "miRNA classifier" consisting of miR-150, miR-342-3p, miR-455-3p, miR-145, miR-155 and miR-497 that were considered predictors of post-recurrence survival [214]. Similarly the analyses of the miRNA expression profiling from melanoma lymph node metastases reported a unique signature consisting of down regulation of miR-191, combined with up regulation of miR-193a, miR-193b, miR-365, miR-338 and let-7. Together, this miRNAs also serves as predictors of short-term survival in melanoma patients [215]. These findings indicate that miRNAs are differentially expressed in melanoma subtypes and that their dysfunction can be impacted by inherited gene variants, supporting the hypothesis that miRNA dysfunction reflects biological differences in melanoma. Recently, the use of microarray analysis of formalin-fixed and paraffin-embedded samples from different stages of melanomagenesis identified differentially expressed microRNAs [216]. The miR-203 and miR-205 were differ‐ entially expressed between primary and metastatic melanomas and functional *in vitro* assays validated this found. So, these results indicated that differentially expressed miRNAs that

important function in the development and progression of human melanomas [209].

118 Melanoma - From Early Detection to Treatment

could be useful as diagnostic or prognostic markers for melanoma.

As such, miRNAs represent a new class of molecules that might prove to be powerful cancer biomarkers useful in future staging systems and used as stratification criteria in clinical trials as well as treatment of patients with disseminated disease. It was demonstrated that miR-214 is over-expressed in metastatic melanoma cell lines as well as tumor specimens. MiR-214 regulates the expression of two transcription factors, AP-2c and AP-2a [217]. These molecules have been previously shown to play major roles in melanoma metastasis via regulation of

In this chapter we proposed to discuss the melanoma genetics, starting from the genes that may confer susceptibility to the genes that may be involved with progression. Moreover, we addressed the already known genes (here called as "old genetics") as well as new genes that have been discovered as involved in melanoma (here called as "new genetics). It is noteworthy that the new technologies such as GWAS and deep-sequencing have improved our knowledge about melanoma genetics. Nowadays we have critical information about the disease, such as the clear involvement of UV in carcinogenic process and the many pathways that contribute significantly to it. As could be observed, conversely to other cancer types where single genes has great impact on susceptibility and progression, such as BRCA in breast and ovarian, mismatch repair pathways and colorectal cancer or *TP53* to Li-Fraumeni syndrome, a single gene cannot be pointed as "the melanoma gene". Huge amplitude of genetic pathways may be related to melanoma progression and this same amplitude may be responsible for melano‐ ma metastasis and chemoresistance, making this neoplasia of complex management. However, in a biologist point of view such huge amplitude makes this neoplasia fascinating to under‐ stand, challenging researchers to approach the problem in creative ways.

[4] Brash DE, Ziegler A, Jonason AS, Simon JA, Kunala S, Leffell DJ. Sunlight and sunburn in human skin cancer: p53, apoptosis, and tumor promotion. J Investig Dermatol Symp

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[5] You YH, Lee DH, Yoon JH, Nakajima S, Yasui A, Pfeifer GP. Cyclobutane pyrimidine dimers are responsible for the vast majority of mutations induced by UVB irradiation

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[7] Sander CS, Chang H, Hamm F, Elsner P, Thiele JJ. Role of oxidative stress and the antioxidant network in cutaneous carcinogenesis. Int J Dermatol 2004; 43:326-35.

[8] Nelemans PJ, Groenendal H, Kiemeney LA, Rampen FH, Ruiter DJ, Verbeek AL. Effect of intermittent exposure to sunlight on melanoma risk among indoor workers and sun-

[9] Weinstock MA, Colditz GA, Willett WC, Stampfer MJ, Bronstein BR, Mihm MC Jr, Speizer FE. Nonfamilial cutaneous melanoma incidence in women associated with sun

[10] Elwood JM. Melanoma and sun exposure: contrasts between intermittent and chronic

[11] Luiz OC, Gianini RJ, Gonçalves FT, Francisco G, Festa-Neto C, Sanches JA, et al. Ethnicity and cutaneous melanoma in the city of Sao Paulo, Brazil: a case-control study.

[12] Law MH, Macgregor S, Hayward NK. Melanoma genetics: recent findings take us

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It is tempting to assume that the more we know about melanoma biology, including melanoma genetics, much more efficacious melanoma prevention and treatment will be. Heterogeneity within the very same tumor will certainly hamper treatment. We will need to take it in account in the days of personalized medicine. To this, improvement of technologies, coordinated studies of gene-environment interactions, allied to functional studies and critical clinical trials, will be necessary for the adequate translation of this body of information into patient benefit.

## **Acknowledgements**

The authors thank Cristina Grandal for helping with figures editing.

## **Author details**

Guilherme Francisco1,2, Priscila Daniele Ramos Cirilo1,2, Fernanda Toledo Gonçalves3 , Tharcísio Citrângulo Tortelli Junior1,2 and Roger Chammas1,2

\*Address all correspondence to: guilherme@lim24.fm.usp.br

\*Address all correspondence to: rchammas@usp.br

1 Department of Radiology and Oncology, School of Medicine, University of São Paulo, Brazil

2 Center of Translational Research in Oncology, São Paulo State Cancer Institute (ICESP), São Paulo, Brazil

3 Department of Ethical and Legal Medicine, School of Medicine, University of São Paulo, Brazil

## **References**


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in a biologist point of view such huge amplitude makes this neoplasia fascinating to under‐

It is tempting to assume that the more we know about melanoma biology, including melanoma genetics, much more efficacious melanoma prevention and treatment will be. Heterogeneity within the very same tumor will certainly hamper treatment. We will need to take it in account in the days of personalized medicine. To this, improvement of technologies, coordinated studies of gene-environment interactions, allied to functional studies and critical clinical trials, will be necessary for the adequate translation of this body of information into patient benefit.

stand, challenging researchers to approach the problem in creative ways.

The authors thank Cristina Grandal for helping with figures editing.

Tharcísio Citrângulo Tortelli Junior1,2 and Roger Chammas1,2

\*Address all correspondence to: guilherme@lim24.fm.usp.br

\*Address all correspondence to: rchammas@usp.br

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Guilherme Francisco1,2, Priscila Daniele Ramos Cirilo1,2, Fernanda Toledo Gonçalves3

1 Department of Radiology and Oncology, School of Medicine, University of São Paulo, Brazil

2 Center of Translational Research in Oncology, São Paulo State Cancer Institute (ICESP),

3 Department of Ethical and Legal Medicine, School of Medicine, University of São Paulo,

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**Acknowledgements**

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**Author details**

São Paulo, Brazil

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**Chapter 5**

**Diagnosis, Histopathologic and Genetic Classification**

Uveal melanoma (UM) is the most common cause of primary eye cancer in the western world. During embryogenesis neural crest cells migrate to the neural tract where they devel‐ op into melanocytes. Melanomas of the uvea are derived from these melanocytes. UM may arise in the iris (5%), ciliary body (23%) or choroid (72%). Choroidal melanomas are the most common and usually display a discoid, dome-shaped or mushroom shaped growth pattern. Approximately 80% of the primary intraocular tumours are diagnosed as UM in patients above the age of 20 years, with a mean age of 60 years (Singh & Topham, 2003). Despite a shift towards more conservative eye treatments, survival has not improved during 1973 to 2008 (Singh et al, 2011). Growth of the primary tumour is related with histopathological fea‐ tures, as well as the genetic changes within these tumours. In this chapter we will not dis‐ cuss iris melanoma, as this shows a different clinical and genetic behaviour, compared to ciliary body and choroidal melanoma. The clinical features, histopathological profile and ge‐ netic alterations of UM, as well as therapeutic options for primary tumours and metastases

The incidence of UM ranges from 4.3 to 10.9 per million (Singh et al, 2009). For the past fifty years, the incidence has remained stable, unlike trends indicating a higher incidence of cuta‐ neous melanoma. The incidence in Europe and United States is comparable to that in Aus‐ tralia and New Zealand. In Europe, a lower incidence is reported in Spain and the south of

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

© 2013 van Beek 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,

**of Uveal Melanoma**

http://dx.doi.org/10.5772/53631

**1. Introduction**

will be discussed.

**2. Epidemiology**

N.C. Naus, A. de Klein and E. Kilic

J.G.M. van Beek, A.E. Koopmans, R.M. Verdijk,

Additional information is available at the end of the chapter


## **Diagnosis, Histopathologic and Genetic Classification of Uveal Melanoma**

J.G.M. van Beek, A.E. Koopmans, R.M. Verdijk, N.C. Naus, A. de Klein and E. Kilic

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53631

## **1. Introduction**

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[217] Penna E, Orso F, Cimino D, Tenaglia E, Lembo A, Quaglino E, et al. microRNA-214 Contributes to Melanoma Tumour Progression Through Suppression of TFAP2C. The

[218] Chan E, Patel R, Nallur S, Ratner E, Bacchiocchi A, Hoyt K, et al. MicroRNA Signatures

[219] Hanna JA, Hahn L, Agarwal S, Rimm DL. In Situ Measurement of miR-205 in Malignant Melanoma Tissue Supports its Role as a Tumor Suppressor microRNA. Laboratory

Differentiate Melanoma Subtypes. Cell Cycle 2011;10(11):1845-1852.

melanoma. The Journal of Investigative Dermatology 2010;130(8):2062-2070.

Lines. Cancer Research 2007;67(6):2456-2468.

Research 2010;70(10):4163–4173.

Research 2010;16(5):1577-1586.

EMBO Journal 2011;30(10):1990-2007.

Investigation 2012, doi: 10.1038/labinvest.2012.119.

2010;12(5):e9685.

136 Melanoma - From Early Detection to Treatment

553-561.

1751.

Uveal melanoma (UM) is the most common cause of primary eye cancer in the western world. During embryogenesis neural crest cells migrate to the neural tract where they devel‐ op into melanocytes. Melanomas of the uvea are derived from these melanocytes. UM may arise in the iris (5%), ciliary body (23%) or choroid (72%). Choroidal melanomas are the most common and usually display a discoid, dome-shaped or mushroom shaped growth pattern. Approximately 80% of the primary intraocular tumours are diagnosed as UM in patients above the age of 20 years, with a mean age of 60 years (Singh & Topham, 2003). Despite a shift towards more conservative eye treatments, survival has not improved during 1973 to 2008 (Singh et al, 2011). Growth of the primary tumour is related with histopathological fea‐ tures, as well as the genetic changes within these tumours. In this chapter we will not dis‐ cuss iris melanoma, as this shows a different clinical and genetic behaviour, compared to ciliary body and choroidal melanoma. The clinical features, histopathological profile and ge‐ netic alterations of UM, as well as therapeutic options for primary tumours and metastases will be discussed.

## **2. Epidemiology**

The incidence of UM ranges from 4.3 to 10.9 per million (Singh et al, 2009). For the past fifty years, the incidence has remained stable, unlike trends indicating a higher incidence of cuta‐ neous melanoma. The incidence in Europe and United States is comparable to that in Aus‐ tralia and New Zealand. In Europe, a lower incidence is reported in Spain and the south of

© 2013 van Beek 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.

Italy, about 2 per million, whereas registries in France, the Netherlands, Switzerland and Germany has intermediate values around 4 to 5 per million. The United Kingdom registered over 6 per million, and the highest incidence is up to > 8 per million in Norway and Den‐ mark (Virgili et al, 2007).

## **3. Predisposing factors**

Men and women with UM are more or less affected equally (Damato & Coupland, 2012; Singh et al, 2011). Iris melanoma is more common in women than in men (Damato & Coup‐ land, 2012). Several phenotypes, like blue or grey eyes and fair skin have been suggested to predispose for UM (Schmidt-Pokrzywniak et al, 2009). This might explain why Caucasians are approximately 150 times more frequently affected than Africans (Margo et al, 1998; Singh et al, 2005a). In Asians UM is less common (Biswas et al, 2002).

**Figure 1.** A large amelanotic uveal melanoma leads to a visual field defect.

when in doubt, an intraocular biopsy is taken of the tumour.

mimicking lesion (Eskelin & Kivelä, 2002; Khan & Damato, 2007).

Diagnosis of UM is based on a combination of clinical examination with slit lamp biomicro‐ scopy, indirect ophthalmoscopy (figure 1, 2a, 3a) and ultrasonography (US) (figure 2b, 3b). Iris melanomas are readily detectable by slit lamp biomicroscopy, whereas ciliary body tu‐ mours are hidden behind the iris and can be visualized by US. Choroidal tumours, depend‐ ing on their location, are diagnosed by dilated indirect ophthalmoscopy and US. In suspect cases of intravenous fluorescein angiography can be helpful in differentiating melanomas from other diagnoses. Also optical coherence tomography (OCT) and autofluorescence can provide additional information (Lavinsky et al, 2007; Shields et al, 2008). In selected cases,

Diagnosis, Histopathologic and Genetic Classification of Uveal Melanoma

http://dx.doi.org/10.5772/53631

139

Indirect ophthalmoscopy through a dilated pupil provides a correct diagnosis in more than 95% of the cases (Char et al, 1980). Accuracy of the right diagnosis is established to be over 99% by experienced clinicians with US, ophthalmoscopy, and fluorescein angiography and confirmed by histopathology (Collaborative Ocular Melanoma Study Group, 1990). The abil‐ ity to differentiate melanoma from other lesions has improved over the last decades. When comparing studies of 1964 and 1973, in 19% of the enucleated patients with the clinical diag‐ nosis melanoma no histopathological evidence of a melanoma was found (Ferry, 1964; Shields, 1973). The accuracy in diagnosing medium to small sized tumours is quite challeng‐ ing. Nine percent of presumed melanomas are found to have another diagnosis by fine nee‐ dle aspiration biopsy (Char & Miller, 1995). Most important is to minimise the delay in referring patients with melanoma to a specialised centre. It is reported that in 29% of the pa‐ tients a melanoma is missed during the first visit by an ophthalmologist, and that 31.5% of the patients referred to an oncology centre with the diagnosis of melanoma actually had a

**5. Diagnosis**

From all the parts of the uvea the iris is most exposed to ultraviolet light, because of filtering effects of the lens and retinal pigment epithelium (RPE), the choroid receives less light (Singh et al, 2004). Although several epidemiologic and case control studies have been per‐ formed to investigate the influence of sunlight exposure on UM, the results are not conclu‐ sive (Guenel et al, 2001; Holly et al, 1990; Pane & Hirst, 2000; Shah et al, 2005; Vajdic et al, 2002). UM may occur as a part of familial syndromes, like xeroderma pigmentosa, Li-Frau‐ meni syndrome and familial breast and ovarian cancer. Of all UM 0.6% is considered to be familial (Singh et al, 1996). In a retrospective study 0.0017% of the primary UM patients were in the setting of familial atypical mole and melanoma syndrome (FAMM). These pa‐ tients were relatively young with a mean age of 40 years (Singh et al, 1995). Furthermore, an association of neurofibromatosis type 1 and UM has been suggested, since both are of neural crest origin, however this association remains unclear (Honavar et al, 2000). Ocular and ocu‐ lodermal melanocytosis (Nevus of Ota), dysplastic nevi and cutaneous melanoma are corre‐ lated with an increased risk of UM development (Carreno et al, 2012; Gonder et al, 1982; Hammer et al, 1995; Richtig et al, 2004; Singh et al, 1998; Toth-Molnar et al, 2000; van Hees et al, 1994). Additionally, in UM patients ocular and oculodermal melanocytosis are about 35 to 70 times more common (Carreno et al, 2012; Singh et al, 1998).

## **4. Clinical presentation**

Depending on de location and size of the tumour, patients can present with visual com‐ plaints. Most UMs are detected during a routine ophthalmic examination. Approximately 30% of the patients have no symptoms at time of diagnosis, and if there are any complaints these consist mostly of blurred vision, floaters, photopsias and visual field loss (Damato, 2010) (figure 1). Usually patients do not present with severe ocular pain, however, this can occur secondary to inflammation or neovascular glaucoma.

**Figure 1.** A large amelanotic uveal melanoma leads to a visual field defect.

## **5. Diagnosis**

Italy, about 2 per million, whereas registries in France, the Netherlands, Switzerland and Germany has intermediate values around 4 to 5 per million. The United Kingdom registered over 6 per million, and the highest incidence is up to > 8 per million in Norway and Den‐

Men and women with UM are more or less affected equally (Damato & Coupland, 2012; Singh et al, 2011). Iris melanoma is more common in women than in men (Damato & Coup‐ land, 2012). Several phenotypes, like blue or grey eyes and fair skin have been suggested to predispose for UM (Schmidt-Pokrzywniak et al, 2009). This might explain why Caucasians are approximately 150 times more frequently affected than Africans (Margo et al, 1998;

From all the parts of the uvea the iris is most exposed to ultraviolet light, because of filtering effects of the lens and retinal pigment epithelium (RPE), the choroid receives less light (Singh et al, 2004). Although several epidemiologic and case control studies have been per‐ formed to investigate the influence of sunlight exposure on UM, the results are not conclu‐ sive (Guenel et al, 2001; Holly et al, 1990; Pane & Hirst, 2000; Shah et al, 2005; Vajdic et al, 2002). UM may occur as a part of familial syndromes, like xeroderma pigmentosa, Li-Frau‐ meni syndrome and familial breast and ovarian cancer. Of all UM 0.6% is considered to be familial (Singh et al, 1996). In a retrospective study 0.0017% of the primary UM patients were in the setting of familial atypical mole and melanoma syndrome (FAMM). These pa‐ tients were relatively young with a mean age of 40 years (Singh et al, 1995). Furthermore, an association of neurofibromatosis type 1 and UM has been suggested, since both are of neural crest origin, however this association remains unclear (Honavar et al, 2000). Ocular and ocu‐ lodermal melanocytosis (Nevus of Ota), dysplastic nevi and cutaneous melanoma are corre‐ lated with an increased risk of UM development (Carreno et al, 2012; Gonder et al, 1982; Hammer et al, 1995; Richtig et al, 2004; Singh et al, 1998; Toth-Molnar et al, 2000; van Hees et al, 1994). Additionally, in UM patients ocular and oculodermal melanocytosis are about 35

Depending on de location and size of the tumour, patients can present with visual com‐ plaints. Most UMs are detected during a routine ophthalmic examination. Approximately 30% of the patients have no symptoms at time of diagnosis, and if there are any complaints these consist mostly of blurred vision, floaters, photopsias and visual field loss (Damato, 2010) (figure 1). Usually patients do not present with severe ocular pain, however, this can

Singh et al, 2005a). In Asians UM is less common (Biswas et al, 2002).

to 70 times more common (Carreno et al, 2012; Singh et al, 1998).

occur secondary to inflammation or neovascular glaucoma.

mark (Virgili et al, 2007).

138 Melanoma - From Early Detection to Treatment

**3. Predisposing factors**

**4. Clinical presentation**

Diagnosis of UM is based on a combination of clinical examination with slit lamp biomicro‐ scopy, indirect ophthalmoscopy (figure 1, 2a, 3a) and ultrasonography (US) (figure 2b, 3b). Iris melanomas are readily detectable by slit lamp biomicroscopy, whereas ciliary body tu‐ mours are hidden behind the iris and can be visualized by US. Choroidal tumours, depend‐ ing on their location, are diagnosed by dilated indirect ophthalmoscopy and US. In suspect cases of intravenous fluorescein angiography can be helpful in differentiating melanomas from other diagnoses. Also optical coherence tomography (OCT) and autofluorescence can provide additional information (Lavinsky et al, 2007; Shields et al, 2008). In selected cases, when in doubt, an intraocular biopsy is taken of the tumour.

Indirect ophthalmoscopy through a dilated pupil provides a correct diagnosis in more than 95% of the cases (Char et al, 1980). Accuracy of the right diagnosis is established to be over 99% by experienced clinicians with US, ophthalmoscopy, and fluorescein angiography and confirmed by histopathology (Collaborative Ocular Melanoma Study Group, 1990). The abil‐ ity to differentiate melanoma from other lesions has improved over the last decades. When comparing studies of 1964 and 1973, in 19% of the enucleated patients with the clinical diag‐ nosis melanoma no histopathological evidence of a melanoma was found (Ferry, 1964; Shields, 1973). The accuracy in diagnosing medium to small sized tumours is quite challeng‐ ing. Nine percent of presumed melanomas are found to have another diagnosis by fine nee‐ dle aspiration biopsy (Char & Miller, 1995). Most important is to minimise the delay in referring patients with melanoma to a specialised centre. It is reported that in 29% of the pa‐ tients a melanoma is missed during the first visit by an ophthalmologist, and that 31.5% of the patients referred to an oncology centre with the diagnosis of melanoma actually had a mimicking lesion (Eskelin & Kivelä, 2002; Khan & Damato, 2007).

## **5.1. Characteristics**

Melanoma are generally pigmented, but one fourth are relatively non-pigmented or amela‐ notic (figure 1). Melanoma can develop into two different directions: towards the vitreous and outwards, through the underlying sclera. Having broken through Bruch's membrane, into the vitreous, UMs achieve a characteristic shape, even pathognomonic, like a 'collar but‐ ton' or 'mushroom'. Small melanomas can appear flat or dome shaped.

## **5.2. Clinical prognostic factor**

Well-known clinical prognostic factors are age and location of the tumour. Older patients tend to have a worse prognosis (Shields et al, 2012). One study found that UMs were located predominantly posterior and temporal or had a preference for macular zone, while others found a more equal distribution of melanoma (Krohn et al, 2008; Li et al, 2000; Shields et al, 2009b). Patients with larger tumours, tumours that ruptured through Bruch membrane and in patients who have developed metastasis, the tumours were significantly more often locat‐ ed anterior to the equator (Krohn et al, 2008).

**Figure 3.** a: Pigmented uveal melanoma with orange pigment (lipofuscin); 3b: A homogeneous grey scale in the tu‐ mour and choroidal excavation on B-scan ultrasonography; 3c: Optical coherence tomography of the same tumour

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141

The most important clinical prognostic factor is tumour size, and is often used for selection of the treatment. There are several treatment options, which will be discussed later in this chapter. UM are subdivided into different categories depending on the apical size and diam‐ eter, however, many centres use their own definition. Most widely used definition is sug‐ gested by the COMS study. Small melanomas are 1.0 - 2.5 mm in apical height and > 5.0 mm in largest basal dimension (Collaborative Ocular Melanoma Study Group, 1997). Medium tumours are defined as tumours 2.5 to 10 mm in apical height and ≤ 16 mm in largest basal diameter. Large tumours are ≥ 2 mm in apical height and > 16 mm in maximal basal diame‐ ter, or a melanoma > 10 mm in apical height, regardless of the basal diameter (Collaborative Ocular Melanoma Study Group, 2003). One large study described that each increase in milli‐ meter of tumour thickness increased the risk for metastasis by 5% (Shields et al, 2009b). The mortality rate for small (< 2 - 3 mm height), medium (3 - 8 mm height) and large (> 8 mm height) melanoma was 16%, 32% and 53% in 5 years, respectively, and has not changed in recent years (Diener-West et al, 1992). This supports the model of tumour doubling time of melanoma and its' related metastasis. The model suggests that micrometastasis already exist

with subretinal fluid.

**Figure 2.** a: A dark pigmented uveal melanoma with orange pigment; 2b: On B-scan ultrasonography acoustic hol‐ lowing and choroidal excavation is present, 2c: Subretinal fluid and retinal pigment epithelial alterations are visible on optical coherence tomography scan at the top of the tumour.

**5.1. Characteristics**

**5.2. Clinical prognostic factor**

140 Melanoma - From Early Detection to Treatment

ed anterior to the equator (Krohn et al, 2008).

optical coherence tomography scan at the top of the tumour.

Melanoma are generally pigmented, but one fourth are relatively non-pigmented or amela‐ notic (figure 1). Melanoma can develop into two different directions: towards the vitreous and outwards, through the underlying sclera. Having broken through Bruch's membrane, into the vitreous, UMs achieve a characteristic shape, even pathognomonic, like a 'collar but‐

Well-known clinical prognostic factors are age and location of the tumour. Older patients tend to have a worse prognosis (Shields et al, 2012). One study found that UMs were located predominantly posterior and temporal or had a preference for macular zone, while others found a more equal distribution of melanoma (Krohn et al, 2008; Li et al, 2000; Shields et al, 2009b). Patients with larger tumours, tumours that ruptured through Bruch membrane and in patients who have developed metastasis, the tumours were significantly more often locat‐

**Figure 2.** a: A dark pigmented uveal melanoma with orange pigment; 2b: On B-scan ultrasonography acoustic hol‐ lowing and choroidal excavation is present, 2c: Subretinal fluid and retinal pigment epithelial alterations are visible on

ton' or 'mushroom'. Small melanomas can appear flat or dome shaped.

**Figure 3.** a: Pigmented uveal melanoma with orange pigment (lipofuscin); 3b: A homogeneous grey scale in the tu‐ mour and choroidal excavation on B-scan ultrasonography; 3c: Optical coherence tomography of the same tumour with subretinal fluid.

The most important clinical prognostic factor is tumour size, and is often used for selection of the treatment. There are several treatment options, which will be discussed later in this chapter. UM are subdivided into different categories depending on the apical size and diam‐ eter, however, many centres use their own definition. Most widely used definition is sug‐ gested by the COMS study. Small melanomas are 1.0 - 2.5 mm in apical height and > 5.0 mm in largest basal dimension (Collaborative Ocular Melanoma Study Group, 1997). Medium tumours are defined as tumours 2.5 to 10 mm in apical height and ≤ 16 mm in largest basal diameter. Large tumours are ≥ 2 mm in apical height and > 16 mm in maximal basal diame‐ ter, or a melanoma > 10 mm in apical height, regardless of the basal diameter (Collaborative Ocular Melanoma Study Group, 2003). One large study described that each increase in milli‐ meter of tumour thickness increased the risk for metastasis by 5% (Shields et al, 2009b). The mortality rate for small (< 2 - 3 mm height), medium (3 - 8 mm height) and large (> 8 mm height) melanoma was 16%, 32% and 53% in 5 years, respectively, and has not changed in recent years (Diener-West et al, 1992). This supports the model of tumour doubling time of melanoma and its' related metastasis. The model suggests that micrometastasis already exist several years before diagnosis of the primary tumour (Eskelin et al, 2000). This emphasizes the importance of identifying small melanoma and reducing the risk of metastases.

### **5.3. Clinical predictive factors of small melanoma**

In general, choroidal nevi have a less than 5 mm basal diameter and are minimal in height (< 2 mm), although several definitions of nevi have been proposed. Due to differ‐ ent examination methods and definitions, the prevalence of nevi is between 0.2% and 30% (Gass, 1977; Wilder, 1946). Overall in a Caucasian population the incidence is 6.5% (Sumich et al, 1998). Whenever, growth of a nevus is measured on US in a short time a transformation into a small melanoma is suspected. On the other hand benign nevi can also grow slowly. Mashayekhi *et al* observed in 31% of nevi a slight growth, without evi‐ dence of development into a melanoma over a mean follow up of 15 years (Mashayekhi et al, 2011). As described by Singh and co-workers, assuming that all melanoma result from nevi, 1 out of 8845 choroidal nevi can undergo malignant transformation into mela‐ noma in the Caucasian population in the USA (Singh et al, 2005b). In Australia this is es‐ timated 1 out of 4300 nevi (Sumich et al, 1998).

**Figure 4.** a: Peripapillary nevus, barely elevated, with margin located < 3 mm to the optic disc in the right eye of a 72

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143

US is a non-invasive tool and helps to establish the diagnosis of UM, despite media opacities or whether the tumour is located far peripherally. UM shows characteristic low to medium internal reflectivity on A-scan. B-scan US is primarily used to plan therapy based on the first measurement, and to periodically measure tumour prominence (height) and basal diameter for follow-up. The B-scan can identify possible extraocular extension as an empty area be‐ hind the sclera. On B-scan US the internal structure of the tumour is typically seen as a rela‐ tive homogeneous grey scale, although this pattern is not specifically diagnostic (figure 3b). At the base of the tumour an acoustically silent zone (called acoustic hollowing) is seen, as well as choroidal excavation and shadowing in the orbit (figure 2b). Eighty-eight percent of the UM show US hollowness or low acoustic reflectivity (Boldt et al, 2008). Choroidal exca‐ vation is not observed in all melanomas and varies from 42% to 70% (Coleman et al, 1974; Sobottka et al, 1998; Verbeek, 1985). US provides accurate measurements with an interob‐

The diagnostic value of fluorescein angiography in UM is limited. Fluorescein angiography does not show pathognomonic patterns and is especially helpful in differentiating lesions, which simulate melanoma. The pigmentation, size and effect on the RPE of the tumour in‐ fluence the fluorescein angiogram. It is of little help in some medium to large melanomas that have an intrinsic tumour circulation. This 'double circulation' (simultaneous visualiza‐ tion of retinal and choroidal circulation) consists of late staining of the lesion and multiple pin-point leaks at the level of the RPE, which is evident in the early phase of the angiogram. Blockage of background fluorescence and late staining, when fluorescein leaks from the ves‐ sels can be seen on an angiogram as well (Atmaca et al, 1999). Characteristic signs are hypo‐

year-old man; 4b: High reflectivity on B-scan ultrasonography.

server variability of 0.5 mm (Char et al, 1990).

*5.4.2. Fluorescein angiography*

**5.4. Ancillary testing**

*5.4.1. Ultrasonography*

It is important to differentiate melanoma form other choroidal pathologies, such as choroi‐ dal nevi, by identifying indicators of potential malignancy which may differentiate nevi from small UM. Shields *et al* constructed a mnemonic "TFSOM", i.e. "to find small ocular melanoma" to assist in identifying small choroidal melanoma at risk for growth (Shields et al, 1995). The letters of the mnemonic indicate: Thickness > 2 mm, subretinal Fluid, Symp‐ toms, Orange pigment and Margin to the optic disc. Tumours with no, one or more than two factors have 4%, 36% or > 45% chance of growth, respectively (Shields et al, 2000). A tumour with a thickness of more than 2 mm is considered suspect of being a melanoma rather than a nevus. Subretinal fluid is the strongest indicator of malignancy. Exudative retinal detach‐ ment, overlying or adjacent to the tumour, is associated with tumour growth (Augsburger et al, 1989). Presence of symptoms, as mentioned earlier or a change in symptoms is a risk fac‐ tor for malignancy. Orange pigment is formed on melanomas of the posterior pole, although this can also be seen on the surface of presumed benign nevi and haemangioma. Orange pigment is an accumulation of lipofuscin within the RPE. In amelanotic tumours it appears brown-black of colour. Besides orange pigment as a risk factor, a tumour margin within 3 mm of the optic disc is also suspect for malignant potential (figure 4a).

Later "Using Helpful Hints Daily" was added to "TFSOM" mnemonic (Shields et al, 2009a). These features indicate a low acoustic profile or Ultrasound Hollowness, absence of a Halo around the tumour and absence of Drusen over the tumour. US hollowness is shown in 25% of nevi that transformed into melanoma, compared to the 4% with growth without US hollowness (Shields et al, 2009a). A halo around a tumour is a pigmented le‐ sion with a surrounding depigmentation, as can also be noticed in dysplastic nevi. Dru‐ sen suggest a chronic lesion and usually indicate that the tumour is benign, however this is not conclusive.

**Figure 4.** a: Peripapillary nevus, barely elevated, with margin located < 3 mm to the optic disc in the right eye of a 72 year-old man; 4b: High reflectivity on B-scan ultrasonography.

#### **5.4. Ancillary testing**

several years before diagnosis of the primary tumour (Eskelin et al, 2000). This emphasizes

In general, choroidal nevi have a less than 5 mm basal diameter and are minimal in height (< 2 mm), although several definitions of nevi have been proposed. Due to differ‐ ent examination methods and definitions, the prevalence of nevi is between 0.2% and 30% (Gass, 1977; Wilder, 1946). Overall in a Caucasian population the incidence is 6.5% (Sumich et al, 1998). Whenever, growth of a nevus is measured on US in a short time a transformation into a small melanoma is suspected. On the other hand benign nevi can also grow slowly. Mashayekhi *et al* observed in 31% of nevi a slight growth, without evi‐ dence of development into a melanoma over a mean follow up of 15 years (Mashayekhi et al, 2011). As described by Singh and co-workers, assuming that all melanoma result from nevi, 1 out of 8845 choroidal nevi can undergo malignant transformation into mela‐ noma in the Caucasian population in the USA (Singh et al, 2005b). In Australia this is es‐

It is important to differentiate melanoma form other choroidal pathologies, such as choroi‐ dal nevi, by identifying indicators of potential malignancy which may differentiate nevi from small UM. Shields *et al* constructed a mnemonic "TFSOM", i.e. "to find small ocular melanoma" to assist in identifying small choroidal melanoma at risk for growth (Shields et al, 1995). The letters of the mnemonic indicate: Thickness > 2 mm, subretinal Fluid, Symp‐ toms, Orange pigment and Margin to the optic disc. Tumours with no, one or more than two factors have 4%, 36% or > 45% chance of growth, respectively (Shields et al, 2000). A tumour with a thickness of more than 2 mm is considered suspect of being a melanoma rather than a nevus. Subretinal fluid is the strongest indicator of malignancy. Exudative retinal detach‐ ment, overlying or adjacent to the tumour, is associated with tumour growth (Augsburger et al, 1989). Presence of symptoms, as mentioned earlier or a change in symptoms is a risk fac‐ tor for malignancy. Orange pigment is formed on melanomas of the posterior pole, although this can also be seen on the surface of presumed benign nevi and haemangioma. Orange pigment is an accumulation of lipofuscin within the RPE. In amelanotic tumours it appears brown-black of colour. Besides orange pigment as a risk factor, a tumour margin within 3

Later "Using Helpful Hints Daily" was added to "TFSOM" mnemonic (Shields et al, 2009a). These features indicate a low acoustic profile or Ultrasound Hollowness, absence of a Halo around the tumour and absence of Drusen over the tumour. US hollowness is shown in 25% of nevi that transformed into melanoma, compared to the 4% with growth without US hollowness (Shields et al, 2009a). A halo around a tumour is a pigmented le‐ sion with a surrounding depigmentation, as can also be noticed in dysplastic nevi. Dru‐ sen suggest a chronic lesion and usually indicate that the tumour is benign, however this

mm of the optic disc is also suspect for malignant potential (figure 4a).

the importance of identifying small melanoma and reducing the risk of metastases.

**5.3. Clinical predictive factors of small melanoma**

142 Melanoma - From Early Detection to Treatment

timated 1 out of 4300 nevi (Sumich et al, 1998).

is not conclusive.

#### *5.4.1. Ultrasonography*

US is a non-invasive tool and helps to establish the diagnosis of UM, despite media opacities or whether the tumour is located far peripherally. UM shows characteristic low to medium internal reflectivity on A-scan. B-scan US is primarily used to plan therapy based on the first measurement, and to periodically measure tumour prominence (height) and basal diameter for follow-up. The B-scan can identify possible extraocular extension as an empty area be‐ hind the sclera. On B-scan US the internal structure of the tumour is typically seen as a rela‐ tive homogeneous grey scale, although this pattern is not specifically diagnostic (figure 3b). At the base of the tumour an acoustically silent zone (called acoustic hollowing) is seen, as well as choroidal excavation and shadowing in the orbit (figure 2b). Eighty-eight percent of the UM show US hollowness or low acoustic reflectivity (Boldt et al, 2008). Choroidal exca‐ vation is not observed in all melanomas and varies from 42% to 70% (Coleman et al, 1974; Sobottka et al, 1998; Verbeek, 1985). US provides accurate measurements with an interob‐ server variability of 0.5 mm (Char et al, 1990).

#### *5.4.2. Fluorescein angiography*

The diagnostic value of fluorescein angiography in UM is limited. Fluorescein angiography does not show pathognomonic patterns and is especially helpful in differentiating lesions, which simulate melanoma. The pigmentation, size and effect on the RPE of the tumour in‐ fluence the fluorescein angiogram. It is of little help in some medium to large melanomas that have an intrinsic tumour circulation. This 'double circulation' (simultaneous visualiza‐ tion of retinal and choroidal circulation) consists of late staining of the lesion and multiple pin-point leaks at the level of the RPE, which is evident in the early phase of the angiogram. Blockage of background fluorescence and late staining, when fluorescein leaks from the ves‐ sels can be seen on an angiogram as well (Atmaca et al, 1999). Characteristic signs are hypo‐ fluorescence in the early phase followed by diffuse hyperfluorescence and hyperfluorescent spots (due to changes in RPE). In the late phase the dye accumulates in the tumour tissue and hyperfluorescents (figure 5b). Hypofluorescent spots correspond with deposits of or‐ ange pigment on the surface of the tumour.

teristic or subtle patterns of autofluorescence were observed (Lavinsky et al, 2007; Shields et al, 2008). Choroidal melanoma and related retinal and RPE changes, show different auto‐ fluorescence patterns, and secondary changes, such as subclinical retinal detachments asso‐ ciated with presence of small amounts of subretinal fluid can discriminate small choroidal melanoma and nevi at risk for growth (Muscat et al, 2004). Like some nevi UM show bright‐ er hyperautofluorescence in overlying orange pigment, RPE detachment and subsequently decreased brightness in subretinal fluid and drusen (Shields et al, 2008) (figures 2c and 3c).

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Magnetic resonance imaging (MRI) and computed tomography (CT) can be of additional value in the differential diagnosis of UM. On CT an UM appears as a hyperdense lesion with moderate contrast enhancement. Tumours thinner than 2 mm are not detectable on CT. Besides that, CT is less accurate than US in differentiating melanoma and is more ex‐ pensive (Mafee et al, 1986; Peyster et al, 1985). For extrascleral extension CT is inferior to US (Scott et al, 1998). On the other hand, MRI seems more sensitive and more specific than US for detection of extraocular extension of UM (Hosten et al, 1997). A choroidal melanoma appears hyperintense on a T1 and hypointense on a T2 weighted scan. As this can also be the appearance of a melanocytoma, MRI is not specific for uveal melanoma. Due to the higher expenses of CT and MRI and the superiority of US, both techniques

About 54 different conditions are able to simulate UM. The most frequent diagnosis is cho‐ roidal nevus, accounting for 49% of the approximately 1739 presumed melanoma patients referred to a large tertiary Oncology Department in the USA (Shields et al, 2005b). The dif‐ ferentiation between small melanomas and choroidal nevi remains a clinical challenge. Clin‐ ical features that are more prevalent in *choroidal nevi* than in melanomas are drusen and RPE changes, whereas retinal detachment, choroidal neovascularisation or haemorrhagic retinal detachment can occur in both. On B-scan US, nevi have a high internal reflectivity (figure 4b). Also orange pigment and subretinal fluid, which are features of potential malignancy as mentioned previously, can be present in nevi. Ten percent of the nevi have orange pigment

*Congenital hypertrophy of the retinal pigment epithelium* (CHRPE) has sharper edges than mela‐ noma and usually sharply bordered nonpigmented areas (lacunae), or a depigmentated or pigmented halo within. The lesions might be slightly elevated and are black or grey of col‐ our. CHRPE is a benign lesion and is typically located in the peripheral fundus. On the other hand, adenocarcinomas arising from a CHRPE have been reported (Shields et al, 2009e).

*Optic disc melanocytoma* is a heavily pigmented benign lesion with a fibrillated or feathery margin. Although it can occur anywhere in the uveal tract, the tumour is most often located unilateral and on or nearby the optic disc. Optic disc melanocytoma is a variant of melano‐ cytic nevus. Most patients (75%) have no visual complaints, whereas patients with visual

*5.4.5. Magnetic resonance imaging and computed tomography*

are not routinely used for diagnostic evaluation.

**5.5. Differential diagnosis**

and 18% have subretinal fluid.

**Figure 5.** a: A partly pigmented and non-pigmented uveal melanoma; 5b: Fluorescein angiogram with blockage of the background and fluorescein leaking from the vessels.

#### *5.4.3. Indocyanine green angiography*

Indocyanine green angiography is designed to visualize the choroidal vessels and provides more information than fluorescein angiography. Whether an evident pattern can be seen on an angiogram depends on the pigmentation, thickness, disruption through Bruch's mem‐ brane and vascularisation of the tumour (Atmaca et al, 1999). More fluorescence is seen in less pigmented and larger tumours. The choroidal vasculature can be better visualised with indocyanine green than fluorescein. On indocyanine green late staining is observed, because of the leaking of indocyanine green in the extracellular space of the tumour (Frenkel et al, 2008; Guyer et al, 1993; Stanga et al, 2003).

#### *5.4.4. Optical coherence tomography and fundus autofluorescence*

OCT and fundus autofluorescence imaging have limited use in detecting changes in the cho‐ roid, however, both techniques are non-invasive and of help in identifying subtle changes in the RPE, retina and vitreoretinal interface. By means of an OCT subretinal fluid can be vi‐ sualized and quantified, small tumours can be measured, whereas with fundus autofluores‐ cence orange pigment can be shown. Spectral domain OCT can be useful in the detection of subretinal deposits, vitreous seeding and transretinal tumour extension (Heindl et al, 2009).

Although OCT itself is not useful in diagnosing uveal melanoma, it aids in differentiating other pigmented lesions from melanomas (Schaudig et al, 1998). For example, melanocyto‐ ma tend to have a high reflective signal anteriorly, corresponding with the nerve fibre layer, and an optical shadowing posteriorly (Muscat et al, 2001). In most choroidal nevi no charac‐ teristic or subtle patterns of autofluorescence were observed (Lavinsky et al, 2007; Shields et al, 2008). Choroidal melanoma and related retinal and RPE changes, show different auto‐ fluorescence patterns, and secondary changes, such as subclinical retinal detachments asso‐ ciated with presence of small amounts of subretinal fluid can discriminate small choroidal melanoma and nevi at risk for growth (Muscat et al, 2004). Like some nevi UM show bright‐ er hyperautofluorescence in overlying orange pigment, RPE detachment and subsequently decreased brightness in subretinal fluid and drusen (Shields et al, 2008) (figures 2c and 3c).

#### *5.4.5. Magnetic resonance imaging and computed tomography*

Magnetic resonance imaging (MRI) and computed tomography (CT) can be of additional value in the differential diagnosis of UM. On CT an UM appears as a hyperdense lesion with moderate contrast enhancement. Tumours thinner than 2 mm are not detectable on CT. Besides that, CT is less accurate than US in differentiating melanoma and is more ex‐ pensive (Mafee et al, 1986; Peyster et al, 1985). For extrascleral extension CT is inferior to US (Scott et al, 1998). On the other hand, MRI seems more sensitive and more specific than US for detection of extraocular extension of UM (Hosten et al, 1997). A choroidal melanoma appears hyperintense on a T1 and hypointense on a T2 weighted scan. As this can also be the appearance of a melanocytoma, MRI is not specific for uveal melanoma. Due to the higher expenses of CT and MRI and the superiority of US, both techniques are not routinely used for diagnostic evaluation.

#### **5.5. Differential diagnosis**

fluorescence in the early phase followed by diffuse hyperfluorescence and hyperfluorescent spots (due to changes in RPE). In the late phase the dye accumulates in the tumour tissue and hyperfluorescents (figure 5b). Hypofluorescent spots correspond with deposits of or‐

**Figure 5.** a: A partly pigmented and non-pigmented uveal melanoma; 5b: Fluorescein angiogram with blockage of

Indocyanine green angiography is designed to visualize the choroidal vessels and provides more information than fluorescein angiography. Whether an evident pattern can be seen on an angiogram depends on the pigmentation, thickness, disruption through Bruch's mem‐ brane and vascularisation of the tumour (Atmaca et al, 1999). More fluorescence is seen in less pigmented and larger tumours. The choroidal vasculature can be better visualised with indocyanine green than fluorescein. On indocyanine green late staining is observed, because of the leaking of indocyanine green in the extracellular space of the tumour (Frenkel et al,

OCT and fundus autofluorescence imaging have limited use in detecting changes in the cho‐ roid, however, both techniques are non-invasive and of help in identifying subtle changes in the RPE, retina and vitreoretinal interface. By means of an OCT subretinal fluid can be vi‐ sualized and quantified, small tumours can be measured, whereas with fundus autofluores‐ cence orange pigment can be shown. Spectral domain OCT can be useful in the detection of subretinal deposits, vitreous seeding and transretinal tumour extension (Heindl et al, 2009).

Although OCT itself is not useful in diagnosing uveal melanoma, it aids in differentiating other pigmented lesions from melanomas (Schaudig et al, 1998). For example, melanocyto‐ ma tend to have a high reflective signal anteriorly, corresponding with the nerve fibre layer, and an optical shadowing posteriorly (Muscat et al, 2001). In most choroidal nevi no charac‐

ange pigment on the surface of the tumour.

144 Melanoma - From Early Detection to Treatment

the background and fluorescein leaking from the vessels.

2008; Guyer et al, 1993; Stanga et al, 2003).

*5.4.4. Optical coherence tomography and fundus autofluorescence*

*5.4.3. Indocyanine green angiography*

About 54 different conditions are able to simulate UM. The most frequent diagnosis is cho‐ roidal nevus, accounting for 49% of the approximately 1739 presumed melanoma patients referred to a large tertiary Oncology Department in the USA (Shields et al, 2005b). The dif‐ ferentiation between small melanomas and choroidal nevi remains a clinical challenge. Clin‐ ical features that are more prevalent in *choroidal nevi* than in melanomas are drusen and RPE changes, whereas retinal detachment, choroidal neovascularisation or haemorrhagic retinal detachment can occur in both. On B-scan US, nevi have a high internal reflectivity (figure 4b). Also orange pigment and subretinal fluid, which are features of potential malignancy as mentioned previously, can be present in nevi. Ten percent of the nevi have orange pigment and 18% have subretinal fluid.

*Congenital hypertrophy of the retinal pigment epithelium* (CHRPE) has sharper edges than mela‐ noma and usually sharply bordered nonpigmented areas (lacunae), or a depigmentated or pigmented halo within. The lesions might be slightly elevated and are black or grey of col‐ our. CHRPE is a benign lesion and is typically located in the peripheral fundus. On the other hand, adenocarcinomas arising from a CHRPE have been reported (Shields et al, 2009e).

*Optic disc melanocytoma* is a heavily pigmented benign lesion with a fibrillated or feathery margin. Although it can occur anywhere in the uveal tract, the tumour is most often located unilateral and on or nearby the optic disc. Optic disc melanocytoma is a variant of melano‐ cytic nevus. Most patients (75%) have no visual complaints, whereas patients with visual loss were related to neuroretinitis from tumour necrosis and secondary subretinal fluid of the fovea (Shields et al, 2006; Shields et al, 2004). In addition, visual field defects have been described (Meyer et al, 1999; Shields et al, 2006). Ocular melanocytosis is associated with melanocytoma in 8% of cases, and melanocytoma enlargement is noticed in 57% within 8 years (Lee et al, 2010) and 32% within 10 years (Shields et al, 2004). Although malignant transformation is extremely rare, it has been reported (Meyer et al, 1999; Shields et al, 2004).

*Peripheral exudative hemorrhagic chorioretinopathy* (PEHC) lesions, unilateral and often bilater‐ al, have peripheral (> 3 mm outside the fovea) subretinal or sub-RPE haemorrhage that aris‐ es from choroidal neovascularisation. In the periphery signs of macular degeneration, such as lipid exudation, subretinal fluid and fibrosis can be observed (Mantel et al, 2009; Shields et al, 2009c). Also in the macula drusen, RPE alterations or choroidal neovascularisation can be present, which is then consistent with macular degeneration (Shields et al, 2009c). On Bscan internal lesion characteristics show a solid or hollow acoustic quality and no choroidal excavation (Mantel et al, 2009; Shields et al, 2009d). The majority of the peripheral lesions

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*Choroidal haemorrhage* may be distinguished from UM by partially or totally resorption of the haemorrhage over a few weeks, and on US an after-movement can be noticed by kinetic evaluation. Key features are elevated eye pressure, forward movement of diaphragm com‐

*Posterior nodular scleritis* is rare, but often underdiagnosed. It is twice as common in women as in men, and in 35% of the patients it occurs in both eyes. The most common symptoms are periocular pain, pain with eye movement and decreased vision. The differentiation be‐ tween scleritis and melanoma can be made by US. On B-scan echogenic scleral nodules, flu‐ id in Tenon's capsule, swelling of the optic disc and serous retinal detachment are found

*Intraocular leiomyoma* is a rare benign amelanotic tumour of the uvea and mimics an UM. It presents as a dome-shaped lesion, showing light translucency and often contains dilated episcleral vessels, with a predilection in young females (Shields et al, 1994). Sometimes the diagnosis cannot be made by non-invasive examination and intraocular biopsy is necessary

*Adenoma of the RPE* is infrequently diagnosed before enucleation. RPE adenoma is domeshaped and has in contrast to melanoma a higher internal reflectivity on A-scan US (Naka‐ mura et al, 2012). Compared to UM, RPE adenoma has more frequently retinal feeder vessels, retinal or subretinal exudates and exudative retinal detachment (Wei et al, 2010).

UMs develop from melanocytes of the uvea that are derived from neural crest cells. Initially Callender and colleagues described several melanoma cell types, (Callender, 1931) currently three histopathological uveal melanoma categories are being recognised: spindle, epithelioid and mixed cell type (Campbell et al, 1998). Haematoxylin and eosin (H&E) staining is used to differentiate between cell types. Spindle cells exhibit elongated nuclei that may contain eosinophilic nucleoli. In general, Ums containing spindle cells grow slowly and might be as‐ sociated with better prognosis. On the other hand, UMs consisting of faster growing epithe‐ lioid cells, have a more aggressive behaviour, and are therefore associated with poor clinical

resolve spontaneously over time, leaving a scar.

bined with severe pain (Yang et al, 2003).

**6. Classification and histopathologic features**

(McCluskey et al, 1999).

(Richter et al, 2003).

*Hemorrhagic detachment of the retina and RPE* may also simulate melanoma.

*Hyperplasia of the RPE* is a common ocular finding, which is idiopathic or develops in re‐ sponse to trauma, inflammation, haemorrhage and retinal detachment. It is characterised as a black irregular usually small retinal lesion consisting of proliferated RPE cells. Intraretinal pigmented spicules can be seen, and when it manifests as a subretinal localized mass, a mel‐ anoma can be suspected.

*Choroidal haemangioma* is a benign tumour consisting of blood vessels with a typical red to orange colour. Some areas of increased pigmentation can be observed, which makes it diffi‐ cult to differentiate from melanoma. On angiography typical early hyperfluorescence is shown and on US a characteristic high internal reflectivity is present.

*Choroidal metastases* are the most common intraocular malignancies. The prevalence of uveal metastases from all forms of carcinoma is between 2% and 9%, with a mean of 7% for breast cancer and 5% for lung cancer (Kanthan et al, 2007). The origin of choroidal metastases is predominantly breast cancer in woman and lung cancer in man. Less fre‐ quently patients are diagnosed with other primary tumours, such as gastrointestinal tract, kidney, skin and prostate carcinoma (Shields et al, 1997). Choroidal metastases typi‐ cally develop after the diagnosis of breast cancer and in some cases systemic metastases have already been detected. In 66% to 97% of lung cancer patients, choroidal metastases are detected after the primary tumour has been diagnosed (Kanthan et al, 2007). In con‐ clusion, uveal metastases can also be observed before the diagnosis of breast or lung can‐ cer (Demirci et al, 2003; Singh et al, 2012). The median interval between diagnosis of the primary tumour and uveal metastasis is 1 - 4.5 years (Amer et al, 2004; Ratanatharathorn et al, 1991; Rosset et al, 1998; Rottinger et al, 1976; Tsina et al, 2005). Choroidal metasta‐ ses are creamy yellow, flat or elevated and often multilobulated lesions that can occur bi‐ lateral. More than half of the patients may develop subretinal fluid (Demirci et al, 2003). The lesion can show clumps of brown pigmentation, known as leopard spots and RPE al‐ terations. Metastases grow in a different fashion than primary UMs, they infiltrate and replace the normal choroidal architecture more diffusely. On US metastases from breast carcinoma show a higher internal reflectivity than UM (Sobottka et al, 1998).

*Choroidal osteoma* is a rare ossifying benign lesion of the choroid that appears as a yellowish to orange well-defined, juxtapapillary or macular choroidal tumour. These lesions mostly occur in young women with a mean age of 26 years; usually it occurs unilateral, although in 20-30% of cases it appears to be bilateral. Over time an osteoma may enlarge and decalcify partially or totally (Ross & Kemp, 2009; Shields et al, 2005a). There is a 31% chance of devel‐ oping choroidal neovascularisation after 10 years (Shields et al, 2005a). On B-scan US a high‐ ly reflective lesion that shadows the orbit can be seen.

*Peripheral exudative hemorrhagic chorioretinopathy* (PEHC) lesions, unilateral and often bilater‐ al, have peripheral (> 3 mm outside the fovea) subretinal or sub-RPE haemorrhage that aris‐ es from choroidal neovascularisation. In the periphery signs of macular degeneration, such as lipid exudation, subretinal fluid and fibrosis can be observed (Mantel et al, 2009; Shields et al, 2009c). Also in the macula drusen, RPE alterations or choroidal neovascularisation can be present, which is then consistent with macular degeneration (Shields et al, 2009c). On Bscan internal lesion characteristics show a solid or hollow acoustic quality and no choroidal excavation (Mantel et al, 2009; Shields et al, 2009d). The majority of the peripheral lesions resolve spontaneously over time, leaving a scar.

*Hemorrhagic detachment of the retina and RPE* may also simulate melanoma.

loss were related to neuroretinitis from tumour necrosis and secondary subretinal fluid of the fovea (Shields et al, 2006; Shields et al, 2004). In addition, visual field defects have been described (Meyer et al, 1999; Shields et al, 2006). Ocular melanocytosis is associated with melanocytoma in 8% of cases, and melanocytoma enlargement is noticed in 57% within 8 years (Lee et al, 2010) and 32% within 10 years (Shields et al, 2004). Although malignant transformation is extremely rare, it has been reported (Meyer et al, 1999; Shields et al, 2004).

*Hyperplasia of the RPE* is a common ocular finding, which is idiopathic or develops in re‐ sponse to trauma, inflammation, haemorrhage and retinal detachment. It is characterised as a black irregular usually small retinal lesion consisting of proliferated RPE cells. Intraretinal pigmented spicules can be seen, and when it manifests as a subretinal localized mass, a mel‐

*Choroidal haemangioma* is a benign tumour consisting of blood vessels with a typical red to orange colour. Some areas of increased pigmentation can be observed, which makes it diffi‐ cult to differentiate from melanoma. On angiography typical early hyperfluorescence is

*Choroidal metastases* are the most common intraocular malignancies. The prevalence of uveal metastases from all forms of carcinoma is between 2% and 9%, with a mean of 7% for breast cancer and 5% for lung cancer (Kanthan et al, 2007). The origin of choroidal metastases is predominantly breast cancer in woman and lung cancer in man. Less fre‐ quently patients are diagnosed with other primary tumours, such as gastrointestinal tract, kidney, skin and prostate carcinoma (Shields et al, 1997). Choroidal metastases typi‐ cally develop after the diagnosis of breast cancer and in some cases systemic metastases have already been detected. In 66% to 97% of lung cancer patients, choroidal metastases are detected after the primary tumour has been diagnosed (Kanthan et al, 2007). In con‐ clusion, uveal metastases can also be observed before the diagnosis of breast or lung can‐ cer (Demirci et al, 2003; Singh et al, 2012). The median interval between diagnosis of the primary tumour and uveal metastasis is 1 - 4.5 years (Amer et al, 2004; Ratanatharathorn et al, 1991; Rosset et al, 1998; Rottinger et al, 1976; Tsina et al, 2005). Choroidal metasta‐ ses are creamy yellow, flat or elevated and often multilobulated lesions that can occur bi‐ lateral. More than half of the patients may develop subretinal fluid (Demirci et al, 2003). The lesion can show clumps of brown pigmentation, known as leopard spots and RPE al‐ terations. Metastases grow in a different fashion than primary UMs, they infiltrate and replace the normal choroidal architecture more diffusely. On US metastases from breast

shown and on US a characteristic high internal reflectivity is present.

carcinoma show a higher internal reflectivity than UM (Sobottka et al, 1998).

ly reflective lesion that shadows the orbit can be seen.

*Choroidal osteoma* is a rare ossifying benign lesion of the choroid that appears as a yellowish to orange well-defined, juxtapapillary or macular choroidal tumour. These lesions mostly occur in young women with a mean age of 26 years; usually it occurs unilateral, although in 20-30% of cases it appears to be bilateral. Over time an osteoma may enlarge and decalcify partially or totally (Ross & Kemp, 2009; Shields et al, 2005a). There is a 31% chance of devel‐ oping choroidal neovascularisation after 10 years (Shields et al, 2005a). On B-scan US a high‐

anoma can be suspected.

146 Melanoma - From Early Detection to Treatment

*Choroidal haemorrhage* may be distinguished from UM by partially or totally resorption of the haemorrhage over a few weeks, and on US an after-movement can be noticed by kinetic evaluation. Key features are elevated eye pressure, forward movement of diaphragm com‐ bined with severe pain (Yang et al, 2003).

*Posterior nodular scleritis* is rare, but often underdiagnosed. It is twice as common in women as in men, and in 35% of the patients it occurs in both eyes. The most common symptoms are periocular pain, pain with eye movement and decreased vision. The differentiation be‐ tween scleritis and melanoma can be made by US. On B-scan echogenic scleral nodules, flu‐ id in Tenon's capsule, swelling of the optic disc and serous retinal detachment are found (McCluskey et al, 1999).

*Intraocular leiomyoma* is a rare benign amelanotic tumour of the uvea and mimics an UM. It presents as a dome-shaped lesion, showing light translucency and often contains dilated episcleral vessels, with a predilection in young females (Shields et al, 1994). Sometimes the diagnosis cannot be made by non-invasive examination and intraocular biopsy is necessary (Richter et al, 2003).

*Adenoma of the RPE* is infrequently diagnosed before enucleation. RPE adenoma is domeshaped and has in contrast to melanoma a higher internal reflectivity on A-scan US (Naka‐ mura et al, 2012). Compared to UM, RPE adenoma has more frequently retinal feeder vessels, retinal or subretinal exudates and exudative retinal detachment (Wei et al, 2010).

## **6. Classification and histopathologic features**

UMs develop from melanocytes of the uvea that are derived from neural crest cells. Initially Callender and colleagues described several melanoma cell types, (Callender, 1931) currently three histopathological uveal melanoma categories are being recognised: spindle, epithelioid and mixed cell type (Campbell et al, 1998). Haematoxylin and eosin (H&E) staining is used to differentiate between cell types. Spindle cells exhibit elongated nuclei that may contain eosinophilic nucleoli. In general, Ums containing spindle cells grow slowly and might be as‐ sociated with better prognosis. On the other hand, UMs consisting of faster growing epithe‐ lioid cells, have a more aggressive behaviour, and are therefore associated with poor clinical outcome. Epithelioid cells have more polygonal cytoplasm and contain eccentric placed large pleomorphic nuclei and prominent eosinophilic nucleoli (figure 6). The mixed-cell type melanoma has variable proportion of spindle and epithelioid cells with a minimum of 10% of any one type (Edge & American Joint Committee on Cancer, 2010). Other inter-tumour factors, like the presence of certain extracellular matrix patterns (three closed loops located back to back identified by Periodic-acid Schiff (PAS) staining) and increased mitotic figures (number of mitoses per 50 high-power fields equal to 8mm2) can both provide additional adverse prognostic information (Folberg et al, 1993; Mooy et al, 1995). Other histological fea‐ tures associated with mortality and metastases are mean diameter of ten largest nucleoli, de‐ gree of pigmentation, presence of inflammation and tumour necrosis (Gill & Char, 2012). Extrascleral extension by perineural, perivascular, intravascular or direct scleral invasion is correlated with a worse prognosis, especially when the orbital fat resection margin is posi‐ tive (Collaborative Ocular Melanoma Study Group, 1998).

anomalies on either the short arm (p) and or long arm (q) of chromosomes 1, 3, 6 and 8,

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To examine chromosomal changes in UM tissue several cytogenetic and molecular techni‐ ques are available. UMs are quite suitable for cytogenetic analysis because of their relatively simply karyotype. Large chromosomal gains, deletions and translocations can be visualized with conventional karyotyping and spectral karyotyping (SKY) (figure 7a). However, for the detection of smaller abnormalities other techniques are necessary, such as FISH (figure 7b), comparative genomic hybridization (CGH) or quantitative polymerase chain reaction (qPCR) based techniques. An approach is the multiplex ligation probe amplification (MLPA) which allows the relative quantification of multiple loci in one single reaction. MLPA can detect patients at risk for metastatic disease using the results for chromosome 3 and 8 with similar accuracy as FISH *(*Damato et al, 2009*;* Vaarwater et al, 2012*).* MLPA and other qPCRbased techniques as Multiplex Amplicon Quantification (MAQ) fill the gap between more expensive genome-wide screening assays and cheaper methods that only provide informa‐ tion on a single locus (Kumps et al, 2010). A different technique is microsatellite analysis (MSA). Microsatellites are tandem repeats of polymorphic sequences located in the non-cod‐ ing regions of DNA. An extreme form of microsatellite instability was first described in he‐ reditary nonpolyposis colorectal cancer syndrome (Thibodeau et al, 1993). This technique is used to study loss of heterozygosity (LOH) as an indicator of chromosomal loss. A draw‐ back of MSA is that only a limited number of markers can be analyzed in one experiment.

**Figure 7.** a: Example of a karyogram showing monosomy 3 and trisomy of chromosome 8; 7b: FISH analysis of a tu‐ mour demonstrates 1 signal for the probe on centromere 3 (green signals) and 3 to 4 signals of the probe on centro‐

After completion of the human genome project, genome-wide DNA assays became availa‐ ble. Micro-assay based CGH, single nucleotide polymorphism (SNP) analysis and gene ex‐ pression profiling (GEP) analysis are the frequently applied techniques. With the

which can serve as prognostic markers.

mere 8 (red signals).

**7.1. Cytogenetic and molecular techniques in UM research**

Immunohistochemistry may be of diagnostic value. S-100 is expressed by cells of neuroecto‐ dermal origin. HMB-45 binds to gp100, an antigen expressed by melanocytes that can be useful in differentiating UM from nonmelanocytic tumours (Burnier et al, 1991).

**Figure 6.** a: Haematoxylin and eosin staining of formalin fixed and paraffin embedded eye sample with a typical mushroom shaped melanoma.; 6b: Uveal melanoma tissue with spindle cell type characterised by elongated nuclei; 6c: Uveal melanoma tissue with epithelioid cells containing large pleomorphic nuclei and prominent eosinophilic nu‐ cleoli.

## **7. Genetic classification**

Cytogenetic studies in solid tumours have been a greater challenge than in haematological malignancies since metaphase chromosome spreads of good quality are more difficult to ob‐ tain. Solid tumours frequently have highly complex chromosome alterations and are more heterogeneous. Despite this, UM has been well studied since the late eighties with different techniques, such as cytogenetic and fluorescent in situ hybridization (FISH) analysis. Over the years, we have learned that the majority of UMs contain non-random chromosomal anomalies on either the short arm (p) and or long arm (q) of chromosomes 1, 3, 6 and 8, which can serve as prognostic markers.

#### **7.1. Cytogenetic and molecular techniques in UM research**

outcome. Epithelioid cells have more polygonal cytoplasm and contain eccentric placed large pleomorphic nuclei and prominent eosinophilic nucleoli (figure 6). The mixed-cell type melanoma has variable proportion of spindle and epithelioid cells with a minimum of 10% of any one type (Edge & American Joint Committee on Cancer, 2010). Other inter-tumour factors, like the presence of certain extracellular matrix patterns (three closed loops located back to back identified by Periodic-acid Schiff (PAS) staining) and increased mitotic figures (number of mitoses per 50 high-power fields equal to 8mm2) can both provide additional adverse prognostic information (Folberg et al, 1993; Mooy et al, 1995). Other histological fea‐ tures associated with mortality and metastases are mean diameter of ten largest nucleoli, de‐ gree of pigmentation, presence of inflammation and tumour necrosis (Gill & Char, 2012). Extrascleral extension by perineural, perivascular, intravascular or direct scleral invasion is correlated with a worse prognosis, especially when the orbital fat resection margin is posi‐

Immunohistochemistry may be of diagnostic value. S-100 is expressed by cells of neuroecto‐ dermal origin. HMB-45 binds to gp100, an antigen expressed by melanocytes that can be

**Figure 6.** a: Haematoxylin and eosin staining of formalin fixed and paraffin embedded eye sample with a typical mushroom shaped melanoma.; 6b: Uveal melanoma tissue with spindle cell type characterised by elongated nuclei; 6c: Uveal melanoma tissue with epithelioid cells containing large pleomorphic nuclei and prominent eosinophilic nu‐

Cytogenetic studies in solid tumours have been a greater challenge than in haematological malignancies since metaphase chromosome spreads of good quality are more difficult to ob‐ tain. Solid tumours frequently have highly complex chromosome alterations and are more heterogeneous. Despite this, UM has been well studied since the late eighties with different techniques, such as cytogenetic and fluorescent in situ hybridization (FISH) analysis. Over the years, we have learned that the majority of UMs contain non-random chromosomal

useful in differentiating UM from nonmelanocytic tumours (Burnier et al, 1991).

tive (Collaborative Ocular Melanoma Study Group, 1998).

148 Melanoma - From Early Detection to Treatment

cleoli.

**7. Genetic classification**

To examine chromosomal changes in UM tissue several cytogenetic and molecular techni‐ ques are available. UMs are quite suitable for cytogenetic analysis because of their relatively simply karyotype. Large chromosomal gains, deletions and translocations can be visualized with conventional karyotyping and spectral karyotyping (SKY) (figure 7a). However, for the detection of smaller abnormalities other techniques are necessary, such as FISH (figure 7b), comparative genomic hybridization (CGH) or quantitative polymerase chain reaction (qPCR) based techniques. An approach is the multiplex ligation probe amplification (MLPA) which allows the relative quantification of multiple loci in one single reaction. MLPA can detect patients at risk for metastatic disease using the results for chromosome 3 and 8 with similar accuracy as FISH *(*Damato et al, 2009*;* Vaarwater et al, 2012*).* MLPA and other qPCRbased techniques as Multiplex Amplicon Quantification (MAQ) fill the gap between more expensive genome-wide screening assays and cheaper methods that only provide informa‐ tion on a single locus (Kumps et al, 2010). A different technique is microsatellite analysis (MSA). Microsatellites are tandem repeats of polymorphic sequences located in the non-cod‐ ing regions of DNA. An extreme form of microsatellite instability was first described in he‐ reditary nonpolyposis colorectal cancer syndrome (Thibodeau et al, 1993). This technique is used to study loss of heterozygosity (LOH) as an indicator of chromosomal loss. A draw‐ back of MSA is that only a limited number of markers can be analyzed in one experiment.

**Figure 7.** a: Example of a karyogram showing monosomy 3 and trisomy of chromosome 8; 7b: FISH analysis of a tu‐ mour demonstrates 1 signal for the probe on centromere 3 (green signals) and 3 to 4 signals of the probe on centro‐ mere 8 (red signals).

After completion of the human genome project, genome-wide DNA assays became availa‐ ble. Micro-assay based CGH, single nucleotide polymorphism (SNP) analysis and gene ex‐ pression profiling (GEP) analysis are the frequently applied techniques. With the development of Next Generation Sequencing (NGS) technologies, the genome can be ana‐ lyzed at base pair level. Genome-wide mutation analysis of tumour samples led to the dis‐ covery of a subset of genes in UM such as *GNAQ* and *BAP1*.

6p is frequently found in tumours with disomy 3 (Ehlers et al, 2008; Hoglund et al, 2004; Sisley et al, 1997). However, this combination of gain of 6p with disomy 3 could not be con‐ firmed by others (Mensink et al, 2009). Aberrations resulting in a relative increase of 6p have been found to be related with both a longer survival (White et al, 1998) or a decreased sur‐ vival (Aalto et al, 2001). The effect of chromosome 6 aberrations on patient outcome is not

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**Figure 8.** Single nucleotide polymorphism (SNP) array of an uveal melanoma. The upper panel (LogR ratio) shows loss of chromosome 3, partial loss of chromosome 8p and gain of chromosome 8q. The lower panel depicts the B-allele

In cutaneous melanoma rearrangements on the short arm of chromosome 1 are a common abnormality, occurring in about 80% of all cases (Fountain et al, 1990; Zhang et al, 1999). In UM this region on 1p is also frequently affected, giving rise to a deletion of 1p. However, these anomalies on chromosome 1 are less common than those in skin melanomas with a frequency of approximately 30% (Horsman & White, 1993; Parrella et al, 1999; Prescher et al,

Aberrations on other chromosomes have been explored, such as chromosome 9p21 (Scholes et al, 2001), chromosome 11q23 (Sisley et al, 2000), chromosome 18q22 (Mensink et al, 2008;

frequency representing allelic imbalance at these chromosomes.

1990; Prescher et al, 1995; Sisley et al, 2000).

*7.2.4. Chromosome 1*

conclusive.

## **7.2. Chromosomal anomalies**

## *7.2.1. Monosomy 3*

Monosomy of chromosome 3 is observed in approximately 50% of the cases of UM and is strongly associated with clinical and histopathological prognostic factors and with metastat‐ ic death (Horsman et al, 1990; Prescher et al, 1990; Sisley et al, 1990). Prescher and associates were the first to find a strong correlation between loss of chromosome 3 and a poor progno‐ sis of the patient (Prescher et al, 1996). Since then several groups have confirmed the prog‐ nostic value of monosomy 3 (Kilic et al, 2006; Sisley et al, 2000; Sisley et al, 1997; White et al, 1998). It is assumed that loss of chromosome 3 is a primary event, as it often occurs with other chromosomal aberrations in UM such as 1p loss, and gain of 6p and 8q (Prescher et al, 1995). Kiliç and colleagues established that tumours with concurrent loss of chromosome 1p and 3 are at higher risk of metastasizing than the tumours with other aberrations (Kilic et al, 2005). Mostly one entire copy of chromosome 3 is lost, although in some cases, isodisomy of chromosome 3 is acquired (Aalto et al, 2001; Scholes et al, 2001; White et al, 1998). Partial deletions or translocations have rarely been described on this chromosome making it diffi‐ cult to map putative tumour suppressor genes. However, recently a mutation in the *BAP1* gene, located on chromosome 3, has been identified in UMs and this gene seems to play an important role in the tumour progression (Harbour et al, 2010). This gene will be discussed in more detail later in this chapter.

#### *7.2.2. Chromosome 8*

Abnormalities in chromosome 8, and in particular gain of 8q or an isochromosome 8q, are thought to be a secondary event in UM as variable copy numbers can be present in one mel‐ anoma (Horsman & White, 1993; Prescher et al, 1994). Gain of chromosome 8q is frequently found in tumours that also have loss of chromosome 3, and this is associated with a poor patient outcome (Aalto et al, 2001; Prescher et al, 1995; White et al, 1998). A SNP array analy‐ sis with this chromosome status is depicted in figure 8. The relationship between the percen‐ tages of aberrant copy numbers within UM cells and patient outcome has been investigated. A higher percentage of monosomy 3 and chromosome 8q gain in primary UM cells shows a strong relation with poor disease-free survival compared to low percentage aberrations (van den Bosch et al, 2012).

#### *7.2.3. Chromosome 6*

Rearrangements on chromosome 6 affect both arms of the chromosome, resulting in dele‐ tions of 6q and gains of 6p. The relative gain of chromosome 6p can occur either through an isochromosome of 6p or a deletion of 6q. Tumours with gain of 6p are thought to be a sepa‐ rate group within UM with an alternative genetic pathway in carcinogenesis, since gain of 6p is frequently found in tumours with disomy 3 (Ehlers et al, 2008; Hoglund et al, 2004; Sisley et al, 1997). However, this combination of gain of 6p with disomy 3 could not be con‐ firmed by others (Mensink et al, 2009). Aberrations resulting in a relative increase of 6p have been found to be related with both a longer survival (White et al, 1998) or a decreased sur‐ vival (Aalto et al, 2001). The effect of chromosome 6 aberrations on patient outcome is not conclusive.

**Figure 8.** Single nucleotide polymorphism (SNP) array of an uveal melanoma. The upper panel (LogR ratio) shows loss of chromosome 3, partial loss of chromosome 8p and gain of chromosome 8q. The lower panel depicts the B-allele frequency representing allelic imbalance at these chromosomes.

#### *7.2.4. Chromosome 1*

development of Next Generation Sequencing (NGS) technologies, the genome can be ana‐ lyzed at base pair level. Genome-wide mutation analysis of tumour samples led to the dis‐

Monosomy of chromosome 3 is observed in approximately 50% of the cases of UM and is strongly associated with clinical and histopathological prognostic factors and with metastat‐ ic death (Horsman et al, 1990; Prescher et al, 1990; Sisley et al, 1990). Prescher and associates were the first to find a strong correlation between loss of chromosome 3 and a poor progno‐ sis of the patient (Prescher et al, 1996). Since then several groups have confirmed the prog‐ nostic value of monosomy 3 (Kilic et al, 2006; Sisley et al, 2000; Sisley et al, 1997; White et al, 1998). It is assumed that loss of chromosome 3 is a primary event, as it often occurs with other chromosomal aberrations in UM such as 1p loss, and gain of 6p and 8q (Prescher et al, 1995). Kiliç and colleagues established that tumours with concurrent loss of chromosome 1p and 3 are at higher risk of metastasizing than the tumours with other aberrations (Kilic et al, 2005). Mostly one entire copy of chromosome 3 is lost, although in some cases, isodisomy of chromosome 3 is acquired (Aalto et al, 2001; Scholes et al, 2001; White et al, 1998). Partial deletions or translocations have rarely been described on this chromosome making it diffi‐ cult to map putative tumour suppressor genes. However, recently a mutation in the *BAP1* gene, located on chromosome 3, has been identified in UMs and this gene seems to play an important role in the tumour progression (Harbour et al, 2010). This gene will be discussed

Abnormalities in chromosome 8, and in particular gain of 8q or an isochromosome 8q, are thought to be a secondary event in UM as variable copy numbers can be present in one mel‐ anoma (Horsman & White, 1993; Prescher et al, 1994). Gain of chromosome 8q is frequently found in tumours that also have loss of chromosome 3, and this is associated with a poor patient outcome (Aalto et al, 2001; Prescher et al, 1995; White et al, 1998). A SNP array analy‐ sis with this chromosome status is depicted in figure 8. The relationship between the percen‐ tages of aberrant copy numbers within UM cells and patient outcome has been investigated. A higher percentage of monosomy 3 and chromosome 8q gain in primary UM cells shows a strong relation with poor disease-free survival compared to low percentage aberrations (van

Rearrangements on chromosome 6 affect both arms of the chromosome, resulting in dele‐ tions of 6q and gains of 6p. The relative gain of chromosome 6p can occur either through an isochromosome of 6p or a deletion of 6q. Tumours with gain of 6p are thought to be a sepa‐ rate group within UM with an alternative genetic pathway in carcinogenesis, since gain of

covery of a subset of genes in UM such as *GNAQ* and *BAP1*.

**7.2. Chromosomal anomalies**

150 Melanoma - From Early Detection to Treatment

in more detail later in this chapter.

*7.2.2. Chromosome 8*

den Bosch et al, 2012).

*7.2.3. Chromosome 6*

*7.2.1. Monosomy 3*

In cutaneous melanoma rearrangements on the short arm of chromosome 1 are a common abnormality, occurring in about 80% of all cases (Fountain et al, 1990; Zhang et al, 1999). In UM this region on 1p is also frequently affected, giving rise to a deletion of 1p. However, these anomalies on chromosome 1 are less common than those in skin melanomas with a frequency of approximately 30% (Horsman & White, 1993; Parrella et al, 1999; Prescher et al, 1990; Prescher et al, 1995; Sisley et al, 2000).

Aberrations on other chromosomes have been explored, such as chromosome 9p21 (Scholes et al, 2001), chromosome 11q23 (Sisley et al, 2000), chromosome 18q22 (Mensink et al, 2008; White et al, 2006), and chromosome 16q (Kilic et al, 2006; Vajdic et al, 2003). The impact on the prognosis, however, remains unclear due to contradictory findings.

overall these mutations are rare (Cohen et al, 2003; Kilic et al, 2004; Mooy et al, 1991; Sal‐

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With the recent discovery of activating *GNAQ* and *GNA11* mutations new light has been shed on the MAPK pathway. Van Raamsdonk and co-workers demonstrated an alterna‐ tive route to MAPK activation through G-protein signalling in melanocytic neoplasia in‐ cluding UMs. They reported a *GNAQ* mutation in 83% of blue naevi and in 46% of UM cases (Van Raamsdonk et al, 2009). Other studies confirmed these results, *GNAQ* muta‐ tions were found in half of the UM cases (Bauer et al, 2009; Onken et al, 2008). *GNAQ* and its paralog *GNA11* encode the heterotrimeric guanine nucleotide-binding protein G subunit alpha q and 11, respectively. Through mutations these subunits become activated and abrogate their intrinsic GTPase activity, which is required to return them to an inac‐ tive state. This oncogenic conversion is suggested to be the cause of constitutive MAPK pathway activation. A subsequent study reported that 83% of UM samples harboured Gα-protein mutations (*GNAQ* or *GNA11* mutations) affecting specific regions on either exon 4 or 5 (codon R183 or Q209, respectively) in a mutually exclusive pattern (Van Raamsdonk et al, 2010). There is no relation between *GNAQ* mutations and prognosis (Bauer et al, 2009). Hence, the presence of Gα-protein mutations in tumours at all stages of malignant progression and in melanocytic lesions of the choroid, suggests that they

are early events in UM (Onken et al, 2008; Van Raamsdonk et al, 2009).

Exome genome sequencing led to the discovery of the BRCA1 associated protein 1 (*BAP1*) gene in UM (Harbour et al, 2010). *BAP1,* a nuclearly localized enzyme, was origi‐ nally identified as an ubiquitin hydrolase that binds to the RING finger domain of BRCA1 (Farmer et al, 2005; Jensen et al, 1998). It has de-ubiquitinating activity and is in‐ volved in several biological processes, including regulation of cell cycle and cell growth, chromatin dynamics and DNA damage response (Farmer et al, 2005). *BAP1* is located on chromosome 3p21.1 and is thought to be a tumour suppressor gene (Ventii et al, 2008). Mutations in this gene first have been reported in a small number of breast and lung cancer cell lines (Jensen et al, 1998). Recently, inactivating somatic mutations were found in 84% of the metastasizing UMs. These mutations were only found in 1 out of 26 inves‐ tigated class 1 tumours against 26 out of 31 class 2 tumours, implicating that *BAP1* muta‐ tions occur late in the UM progression (Harbour et al, 2010). In addition, co-segregating germline *BAP1* mutations have been described in several families with different range of diseases, such as cutaneous melanomas (Wiesner et al, 2011), malignant pleural mesothe‐ liomas (Testa et al, 2011), and other cancers such as meningioma (Abdel-Rahman et al, 2011). Given the functional complexity of *BAP1*, different germline mutations in *BAP1* may predispose to divergent tumour types. To understand more about the impact of *BAP1* mutations on UM and other types of cancers, more extensive clinical, molecular ge‐

danha et al, 2004).

*7.3.3. BAP1 gene*

netic, and functional studies are ongoing.

*7.3.2. GNAQ and GNA11 gene*

#### *7.2.5. Gene expression profiling*

Using GEP UMs can be classified into two classes of tumours that correspond remarka‐ bly well with the ability of the tumour to metastasize. In a study of 25 UMs, class 1 tu‐ mours had a low risk of metastasizing and class 2 tumours had a high risk of developing metastasis (Onken et al, 2004). This molecular classification strongly predicts metastatic death and outperforms other clinical, histopathological and cytogenetic prognostic indica‐ tors (Petrausch et al, 2008; van Gils et al, 2008; Worley et al, 2007). Class 1 tumours pre‐ dominantly show disomy of chromosome 3, whereas class 2 tumours consist mostly of monosomy 3 (Worley et al, 2007).

#### **7.3. Candidate genes**

After identifying the non-random chromosomal alterations in UM, the search for potential oncogenes and tumour suppressor genes followed. By narrowing down altered regions on chromosomes, researchers have tried to identify genes involved in tumourigenesis or pro‐ gression towards metastasis. This way, studies have been conducted on chromosome 8q re‐ vealing potential oncogenes such as *MYC*, which is amplified in about 30% of the UMs (Parrella et al, 2001). Other oncogenes on chromosome 8q have been described, such as *DDEF1* and *NBS1* (now referred to as *ASAP1* and *NBN*, respectively) (Ehlers & Harbour, 2005; Ehlers et al, 2005). Yet, no specific oncogenic mutations on this region have been re‐ ported thus far. Other candidate genes were proposed, such as *HDM2*, *BCL-2* and *CCND1*. However, the pathogenic significance for any of these genes has not been established.

Mutations in certain genes have been well described for cutaneous melanoma. Examples of such genes are the oncogenes *NRAS*, *BRAF* and *AKT3*, and the tumour suppressors *CDKN2A*, *PTEN* and *TP53*. In contrast to skin melanomas, *PTEN* mutations were not ob‐ served in a study of nine cell lines (Naus et al, 2000). Nevertheless, in 15% of the UM cases mutations in *PTEN* were found resulting in activation of *AKT* and overexpression of the PI3K-PTEN-AKT pathway preventing apoptosis (Abdel-Rahman et al, 2006; Ehlers et al, 2008; Ibrahim & Haluska, 2009).

#### *7.3.1. The RAS-RAF-MEK-ERK pathway*

In a large proportion of the UMs the RAS-RAF-MEK-ERK pathway or mitogen-activated protein kinase (MAPK) pathway is constitutionally activated, leading to excessive cell proliferation and suggesting the presence of activating mutations upstream in the path‐ way (Weber et al, 2003; Zuidervaart et al, 2005). Mutation analysis on potential mutation sites in the *BRAF* gene were performed, since a single substitution (p.V600E) in *BRAF* oc‐ curs frequently in benign and premalignant cutaneous nevi (Davies et al, 2002; Pollock et al, 2003). However, *NRAS* and *BRAF* mutations have been reported in a few UMs but overall these mutations are rare (Cohen et al, 2003; Kilic et al, 2004; Mooy et al, 1991; Sal‐ danha et al, 2004).

#### *7.3.2. GNAQ and GNA11 gene*

White et al, 2006), and chromosome 16q (Kilic et al, 2006; Vajdic et al, 2003). The impact on

Using GEP UMs can be classified into two classes of tumours that correspond remarka‐ bly well with the ability of the tumour to metastasize. In a study of 25 UMs, class 1 tu‐ mours had a low risk of metastasizing and class 2 tumours had a high risk of developing metastasis (Onken et al, 2004). This molecular classification strongly predicts metastatic death and outperforms other clinical, histopathological and cytogenetic prognostic indica‐ tors (Petrausch et al, 2008; van Gils et al, 2008; Worley et al, 2007). Class 1 tumours pre‐ dominantly show disomy of chromosome 3, whereas class 2 tumours consist mostly of

After identifying the non-random chromosomal alterations in UM, the search for potential oncogenes and tumour suppressor genes followed. By narrowing down altered regions on chromosomes, researchers have tried to identify genes involved in tumourigenesis or pro‐ gression towards metastasis. This way, studies have been conducted on chromosome 8q re‐ vealing potential oncogenes such as *MYC*, which is amplified in about 30% of the UMs (Parrella et al, 2001). Other oncogenes on chromosome 8q have been described, such as *DDEF1* and *NBS1* (now referred to as *ASAP1* and *NBN*, respectively) (Ehlers & Harbour, 2005; Ehlers et al, 2005). Yet, no specific oncogenic mutations on this region have been re‐ ported thus far. Other candidate genes were proposed, such as *HDM2*, *BCL-2* and *CCND1*.

However, the pathogenic significance for any of these genes has not been established.

Mutations in certain genes have been well described for cutaneous melanoma. Examples of such genes are the oncogenes *NRAS*, *BRAF* and *AKT3*, and the tumour suppressors *CDKN2A*, *PTEN* and *TP53*. In contrast to skin melanomas, *PTEN* mutations were not ob‐ served in a study of nine cell lines (Naus et al, 2000). Nevertheless, in 15% of the UM cases mutations in *PTEN* were found resulting in activation of *AKT* and overexpression of the PI3K-PTEN-AKT pathway preventing apoptosis (Abdel-Rahman et al, 2006; Ehlers et al,

In a large proportion of the UMs the RAS-RAF-MEK-ERK pathway or mitogen-activated protein kinase (MAPK) pathway is constitutionally activated, leading to excessive cell proliferation and suggesting the presence of activating mutations upstream in the path‐ way (Weber et al, 2003; Zuidervaart et al, 2005). Mutation analysis on potential mutation sites in the *BRAF* gene were performed, since a single substitution (p.V600E) in *BRAF* oc‐ curs frequently in benign and premalignant cutaneous nevi (Davies et al, 2002; Pollock et al, 2003). However, *NRAS* and *BRAF* mutations have been reported in a few UMs but

the prognosis, however, remains unclear due to contradictory findings.

*7.2.5. Gene expression profiling*

152 Melanoma - From Early Detection to Treatment

monosomy 3 (Worley et al, 2007).

2008; Ibrahim & Haluska, 2009).

*7.3.1. The RAS-RAF-MEK-ERK pathway*

**7.3. Candidate genes**

With the recent discovery of activating *GNAQ* and *GNA11* mutations new light has been shed on the MAPK pathway. Van Raamsdonk and co-workers demonstrated an alterna‐ tive route to MAPK activation through G-protein signalling in melanocytic neoplasia in‐ cluding UMs. They reported a *GNAQ* mutation in 83% of blue naevi and in 46% of UM cases (Van Raamsdonk et al, 2009). Other studies confirmed these results, *GNAQ* muta‐ tions were found in half of the UM cases (Bauer et al, 2009; Onken et al, 2008). *GNAQ* and its paralog *GNA11* encode the heterotrimeric guanine nucleotide-binding protein G subunit alpha q and 11, respectively. Through mutations these subunits become activated and abrogate their intrinsic GTPase activity, which is required to return them to an inac‐ tive state. This oncogenic conversion is suggested to be the cause of constitutive MAPK pathway activation. A subsequent study reported that 83% of UM samples harboured Gα-protein mutations (*GNAQ* or *GNA11* mutations) affecting specific regions on either exon 4 or 5 (codon R183 or Q209, respectively) in a mutually exclusive pattern (Van Raamsdonk et al, 2010). There is no relation between *GNAQ* mutations and prognosis (Bauer et al, 2009). Hence, the presence of Gα-protein mutations in tumours at all stages of malignant progression and in melanocytic lesions of the choroid, suggests that they are early events in UM (Onken et al, 2008; Van Raamsdonk et al, 2009).

#### *7.3.3. BAP1 gene*

Exome genome sequencing led to the discovery of the BRCA1 associated protein 1 (*BAP1*) gene in UM (Harbour et al, 2010). *BAP1,* a nuclearly localized enzyme, was origi‐ nally identified as an ubiquitin hydrolase that binds to the RING finger domain of BRCA1 (Farmer et al, 2005; Jensen et al, 1998). It has de-ubiquitinating activity and is in‐ volved in several biological processes, including regulation of cell cycle and cell growth, chromatin dynamics and DNA damage response (Farmer et al, 2005). *BAP1* is located on chromosome 3p21.1 and is thought to be a tumour suppressor gene (Ventii et al, 2008). Mutations in this gene first have been reported in a small number of breast and lung cancer cell lines (Jensen et al, 1998). Recently, inactivating somatic mutations were found in 84% of the metastasizing UMs. These mutations were only found in 1 out of 26 inves‐ tigated class 1 tumours against 26 out of 31 class 2 tumours, implicating that *BAP1* muta‐ tions occur late in the UM progression (Harbour et al, 2010). In addition, co-segregating germline *BAP1* mutations have been described in several families with different range of diseases, such as cutaneous melanomas (Wiesner et al, 2011), malignant pleural mesothe‐ liomas (Testa et al, 2011), and other cancers such as meningioma (Abdel-Rahman et al, 2011). Given the functional complexity of *BAP1*, different germline mutations in *BAP1* may predispose to divergent tumour types. To understand more about the impact of *BAP1* mutations on UM and other types of cancers, more extensive clinical, molecular ge‐ netic, and functional studies are ongoing.

## **8. Metastases**

Irrespective of primary treatment of the UM nearly half of the patients develop metastases (Gilissen et al, 2011). UM spreads haematogenous, with a high tendency to metastasize to the liver in 90-95% of the patients. One explanation for the development of new distant metastasis years after the control of primary tumour is the presence of circulating tumour cells at time of the initial diagnosis (Manschot et al, 1995). In other words, the disease is of‐ ten already disseminated at time of tumour diagnosis. Several pathways have been implicat‐ ed in the preferential homing of tumour cells to the liver, such as hepatocyte growth factor (HGF) and it's corresponding receptor c-Met, insulin-like growth factor 1 (IGF-1), and che‐ mokine CXCL12 (Bakalian et al, 2008). In case of liver metastasis prognosis is poor with a median survival of approximately 8 months (Eskelin et al, 2003).

survival and metastasis-free survival (Seddon et al, 1985; Seddon et al, 1990). Brachytherapy is the most common method for treating UM, and currently the ruthenium-106 (Ru-106) and iodine-125 (I-125) applicators are the most frequently used. Brachytherapy can be used in combination with other methods of treatment of UM, such as local resection or transpupil‐ lary thermotherapy (Pe'er, 2012). Local control with plaque radiotherapy has provided over‐ all survival comparable to enucleation. Radiation-induced side effects have necessitated secondary enucleation in 10-22% of the patients (Bell & Wilson, 2004; Char et al, 1993; Fin‐ ger, 1997; Garretson et al, 1987; Gunduz et al, 1999; Lommatzsch et al, 2000; Packer et al, 1992; Shields et al, 1991; Tjho-Heslinga et al, 1999; Vrabec et al, 1991). Local recurrences after brachytherapy are reported between 4 - 28%, depending on the size of the tumour and the follow up time (Damato & Foulds, 1996; Gragoudas, 1997; Karlsson et al, 1989; Seregard et al, 1997; Tjho-Heslinga et al, 1999; Wilson & Hungerford, 1999; Zografos et al, 1992). Radia‐ tion-induced complications include radiation retinopathy, radiation maculopathy, radiation opticopathy as well as recurrences (Gragoudas et al, 1999; Kinyoun et al, 1996; Summanen et al, 1996). Heavy particle radiation with positive charged particles (protons or helium-ions) enables treatment of small, medium- and large-choroidal melanomas. The local recurrence rate for proton beam irradiation is similar to brachytherapy and at 10 years is usually around 5% (Gragoudas, 1997; Zografos et al, 1992). Secondary enucleation is performed in 10 - 15% of patients either due to complications or local recurrence. Other complications, such as maculopathy, opticopathy, cataract, glaucoma, vitreous haemorrhage, retinal de‐ tachment and dryness have also been described (Desjardins et al, 2012). In concordance with proton beam irradiation radiogenic side effects are also reported after SRT. Side effects, such as radiation retinopathy, opticopathy and neovascular glaucoma are responsible for the ma‐ jority of secondary visual loss and secondary enucleations after SRT (Mueller et al, 2000; Ze‐ hetmayer et al, 2000). The efficacy of SRT for UM has been proven in different studies with local tumour control rates reported over 90%, 5 and 10 years after treatment (Zehetmayer, 2012). Local resection (endoresection and exoresection) of UM aims to conserve the eye and remain a useful vision. The tumour can be removed in several manners, through the vitrous and retinal with a vitreous cutter, endoresection, or through a scleral opening exoresection. Variations of exoresection include iridectomy, iridocyclectomy, cyclochoroidectomy, and choroidectomy. Endoresection as well as exoresection can be used as a primary procedure, after another conservative therapy as a treatment option for recurrences or toxic tumour syndrome. An advantage of local resection is that eyes that would otherwise be inoperable can be preserved, while relative large tumour samples are available for prognostication and

Diagnosis, Histopathologic and Genetic Classification of Uveal Melanoma

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155

research (Damato & Foulds, 1996; Damato, 2012; Robertson, 2001).

Although treatment options for small to medium sized melanoma improves visual out‐ come, there has not been any standardized therapy that improves survival in metastatic disease. Systemic treatment options, such as intravenous chemotherapy and immunother‐ apy do not seem to give promising results or survival benefit (Augsburger et al, 2009).

**10. Treatment of liver metastases**

Despite the fact that there a no therapeutic options for metastatic UM that improve sur‐ vival or quality of life, the following methods can be used for screening of liver metasta‐ sis: liver function tests (gamma-glutamyl transpeptidase (γGT) and lactate dehydrogenase (LDH) from blood), liver imaging with US, CT and MRI. Although screening annually or semi-annually for liver metastasis by liver function tests are being widely used, there are reports of disseminated liver metastases and normal liver function tests (Donoso et al, 1985; Eskelin et al, 1999).

Patients have 97.5% chance or more of having no metastasis in the case of normal liver func‐ tion tests, because of the high negative predictive value. However, isolated or combined liv‐ er function tests for aspartate aminotransferase (AST), alanine transaminase (ALT), yGT, LDH and phosphatidic acid (PA) are not indicated for detection of early liver metastasis (Mouriaux et al, 2012). Other upcoming screening options make use of serum markers, Among which S-100β (neural crest marker), melanoma inhibitory activity (MIA), tissue pol‐ ypeptide specific antigen (TPS) and osteopontin (OPN). MIA and S-100β showed significant increase in levels before clinical diagnosis of metastasis (Barak et al, 2011). In a lead time of more than 6 months before clinical metastasis a significant increase in OPN and steeper trendlines in MIA and S-100β levels were demonstrated (Hendler et al, 2011).

## **9. Treatment of primary UM**

Conservation of the eye in UM with useful vision has improved with advances in local irra‐ diation (brachytherapy), heavy particle radiation techniques (proton and helium ion beam), stereotactic radiotherapy (SRT), endoresection, exoresection, transpupillary thermotherapy and photodynamic therapy (Spagnolo et al, 2012). If the tumours are larger, advanced and, in particular, if there is evidence of extraocular extension enucleation is advised (Spagnolo et al, 2012). In addition, enucleation is also performed after serious treatment induced com‐ plications (Hungerford, 1993; Shields et al, 1991). Choice of treatment depends on the loca‐ tion and size of the tumour and goals of therapy. Even though enucleation is sometimes required, eye-preserving approaches have shown to be equally successful regarding overall survival and metastasis-free survival (Seddon et al, 1985; Seddon et al, 1990). Brachytherapy is the most common method for treating UM, and currently the ruthenium-106 (Ru-106) and iodine-125 (I-125) applicators are the most frequently used. Brachytherapy can be used in combination with other methods of treatment of UM, such as local resection or transpupil‐ lary thermotherapy (Pe'er, 2012). Local control with plaque radiotherapy has provided over‐ all survival comparable to enucleation. Radiation-induced side effects have necessitated secondary enucleation in 10-22% of the patients (Bell & Wilson, 2004; Char et al, 1993; Fin‐ ger, 1997; Garretson et al, 1987; Gunduz et al, 1999; Lommatzsch et al, 2000; Packer et al, 1992; Shields et al, 1991; Tjho-Heslinga et al, 1999; Vrabec et al, 1991). Local recurrences after brachytherapy are reported between 4 - 28%, depending on the size of the tumour and the follow up time (Damato & Foulds, 1996; Gragoudas, 1997; Karlsson et al, 1989; Seregard et al, 1997; Tjho-Heslinga et al, 1999; Wilson & Hungerford, 1999; Zografos et al, 1992). Radia‐ tion-induced complications include radiation retinopathy, radiation maculopathy, radiation opticopathy as well as recurrences (Gragoudas et al, 1999; Kinyoun et al, 1996; Summanen et al, 1996). Heavy particle radiation with positive charged particles (protons or helium-ions) enables treatment of small, medium- and large-choroidal melanomas. The local recurrence rate for proton beam irradiation is similar to brachytherapy and at 10 years is usually around 5% (Gragoudas, 1997; Zografos et al, 1992). Secondary enucleation is performed in 10 - 15% of patients either due to complications or local recurrence. Other complications, such as maculopathy, opticopathy, cataract, glaucoma, vitreous haemorrhage, retinal de‐ tachment and dryness have also been described (Desjardins et al, 2012). In concordance with proton beam irradiation radiogenic side effects are also reported after SRT. Side effects, such as radiation retinopathy, opticopathy and neovascular glaucoma are responsible for the ma‐ jority of secondary visual loss and secondary enucleations after SRT (Mueller et al, 2000; Ze‐ hetmayer et al, 2000). The efficacy of SRT for UM has been proven in different studies with local tumour control rates reported over 90%, 5 and 10 years after treatment (Zehetmayer, 2012). Local resection (endoresection and exoresection) of UM aims to conserve the eye and remain a useful vision. The tumour can be removed in several manners, through the vitrous and retinal with a vitreous cutter, endoresection, or through a scleral opening exoresection. Variations of exoresection include iridectomy, iridocyclectomy, cyclochoroidectomy, and choroidectomy. Endoresection as well as exoresection can be used as a primary procedure, after another conservative therapy as a treatment option for recurrences or toxic tumour syndrome. An advantage of local resection is that eyes that would otherwise be inoperable can be preserved, while relative large tumour samples are available for prognostication and research (Damato & Foulds, 1996; Damato, 2012; Robertson, 2001).

### **10. Treatment of liver metastases**

**8. Metastases**

154 Melanoma - From Early Detection to Treatment

1985; Eskelin et al, 1999).

**9. Treatment of primary UM**

Irrespective of primary treatment of the UM nearly half of the patients develop metastases (Gilissen et al, 2011). UM spreads haematogenous, with a high tendency to metastasize to the liver in 90-95% of the patients. One explanation for the development of new distant metastasis years after the control of primary tumour is the presence of circulating tumour cells at time of the initial diagnosis (Manschot et al, 1995). In other words, the disease is of‐ ten already disseminated at time of tumour diagnosis. Several pathways have been implicat‐ ed in the preferential homing of tumour cells to the liver, such as hepatocyte growth factor (HGF) and it's corresponding receptor c-Met, insulin-like growth factor 1 (IGF-1), and che‐ mokine CXCL12 (Bakalian et al, 2008). In case of liver metastasis prognosis is poor with a

Despite the fact that there a no therapeutic options for metastatic UM that improve sur‐ vival or quality of life, the following methods can be used for screening of liver metasta‐ sis: liver function tests (gamma-glutamyl transpeptidase (γGT) and lactate dehydrogenase (LDH) from blood), liver imaging with US, CT and MRI. Although screening annually or semi-annually for liver metastasis by liver function tests are being widely used, there are reports of disseminated liver metastases and normal liver function tests (Donoso et al,

Patients have 97.5% chance or more of having no metastasis in the case of normal liver func‐ tion tests, because of the high negative predictive value. However, isolated or combined liv‐ er function tests for aspartate aminotransferase (AST), alanine transaminase (ALT), yGT, LDH and phosphatidic acid (PA) are not indicated for detection of early liver metastasis (Mouriaux et al, 2012). Other upcoming screening options make use of serum markers, Among which S-100β (neural crest marker), melanoma inhibitory activity (MIA), tissue pol‐ ypeptide specific antigen (TPS) and osteopontin (OPN). MIA and S-100β showed significant increase in levels before clinical diagnosis of metastasis (Barak et al, 2011). In a lead time of more than 6 months before clinical metastasis a significant increase in OPN and steeper

Conservation of the eye in UM with useful vision has improved with advances in local irra‐ diation (brachytherapy), heavy particle radiation techniques (proton and helium ion beam), stereotactic radiotherapy (SRT), endoresection, exoresection, transpupillary thermotherapy and photodynamic therapy (Spagnolo et al, 2012). If the tumours are larger, advanced and, in particular, if there is evidence of extraocular extension enucleation is advised (Spagnolo et al, 2012). In addition, enucleation is also performed after serious treatment induced com‐ plications (Hungerford, 1993; Shields et al, 1991). Choice of treatment depends on the loca‐ tion and size of the tumour and goals of therapy. Even though enucleation is sometimes required, eye-preserving approaches have shown to be equally successful regarding overall

trendlines in MIA and S-100β levels were demonstrated (Hendler et al, 2011).

median survival of approximately 8 months (Eskelin et al, 2003).

Although treatment options for small to medium sized melanoma improves visual out‐ come, there has not been any standardized therapy that improves survival in metastatic disease. Systemic treatment options, such as intravenous chemotherapy and immunother‐ apy do not seem to give promising results or survival benefit (Augsburger et al, 2009). Several locoregional techniques are available, for example immunoembolization, chemo‐ embolization, isolated liver perfusion and hepatic intra-arterial chemotherapy. In highly selected patients, surgical resection of liver metastases can be beneficial. Operating on pa‐ tients with a time from diagnosis of the primary tumour to liver metastases of > 24 months, ≤ 4 liver metastatic lesions and absence of 'miliary' disease (multiple, diffuse, millimetre-sized, dark punctuate lesions on CT) is associated with prolonged survival. A median survival of 27 months has been described in patients with microscopically com‐ plete liver resection versus 14 months in patients with microscopically or macroscopically incomplete liver resection (Mariani et al, 2009).

RPE: retina pigment epithelia

CT: computed tomography

H&E: haematoxylin and eosin

FISH: fluorescent in situ hybridization

CGH: comparative genomic hybridization

MAQ: multiplex amplicon quantification

SNP: single nucleotide polymorphism

MAPK: mitogen-activated protein kinase

qPCR: quantitative polymerase chain reaction MLPA: multiplex ligation probe amplification

PAS: Periodic-acid Schiff

SKY: spectral karyotyping

MSA: microsatellite analysis LOH: loss of heterozygosity

GEP: gene expression profiling NGS: next generation sequencing

HGF: hepatocyte growth factor

LDH: lactate dehydrogenase

ALT : alanine transaminase

PA: phosphatidic acid

AST: aspartate aminotransferase

MIA: melanoma inhibitory activity

IGF-1: insulin-like growth factor 1

γGT: gamma-glutamyl transpeptidase

OCT: optical coherence tomography MRI: magnetic resonance imaging

US: ultrasonography

FAMM: familial atypical mole and melanoma syndrome

CHRPE: congenital hypertrophy of the retinal pigment epithelium

Diagnosis, Histopathologic and Genetic Classification of Uveal Melanoma

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157

PEHC: peripheral exudative hemorrhagic chorioretinopathy

## **11. Future prospects**

With the discovery of *GNAQ* and *BAP1* mutations, new therapeutic strategies based on the specific mutated gene content seem promising. For tumours with Gα-protein mutations, the therapeutic goal is to inhibit downstream signalling molecules in the MAPK pathway that are activated. Preclinical studies show that inhibition of MAPK pathway in UM cell lines re‐ sults in decreased cell proliferation (Van Raamsdonk et al, 2009). There are several key mol‐ ecules in the MAPK pathway, which have been explored as potential therapeutic targets. One of such is MEK, and Gα-protein mutant UM cells showed to be mildly sensitive to the MEK inhibitor AZD6244 (Gill & Char, 2012). Another recent preclinical study proposed to target both the MAPK and PI3K/AKT pathway since both pathways are activated in UM. A combination of MEK and PI3K inhibition treatment resulted in induction of apoptosis in a Gα-mutant UM cells (Khalili et al, 2012). Other potential targets in the MAPK pathway are currently being investigated, including protein kinase C, which is a component of signalling from *GNAQ* to Erk1/2 (Wu et al, 2012).

Therapeutically targeting UMs with a *BAP1* mutation works in a different manner than the Gα-protein mutations, since *BAP1* acts as a tumour suppressor gene. Regaining lost func‐ tions of suppressor genes are in general more challenging than inhibiting an overactive on‐ cogene. Nevertheless, ongoing studies show that histone deacetylase (HDAC) inhibitors may have therapeutic potential in UM. Landreville and colleagues established that HDAC inhibitors can reverse the histone H2A hyperubiquitination that occurs in cultured UM cells depleted of *BAP1*, and it induces morphologic differentiation, cell-cycle exit, and shifts to a differentiated, melanocytic GEP (Landreville et al, 2012). Examples of HDAC inhibitors are valproic acid, trichostatin A, LBH-589, and suberoylanilide hydroxamic acid. Clinical trials are needed to evaluate the effect of these compounds in UM patients, and hopefully UM specific treatment based on mutational content will lead to improved patient survival.

## **Abbreviations**

UM: uveal melanoma

RPE: retina pigment epithelia

FAMM: familial atypical mole and melanoma syndrome

US: ultrasonography

Several locoregional techniques are available, for example immunoembolization, chemo‐ embolization, isolated liver perfusion and hepatic intra-arterial chemotherapy. In highly selected patients, surgical resection of liver metastases can be beneficial. Operating on pa‐ tients with a time from diagnosis of the primary tumour to liver metastases of > 24 months, ≤ 4 liver metastatic lesions and absence of 'miliary' disease (multiple, diffuse, millimetre-sized, dark punctuate lesions on CT) is associated with prolonged survival. A median survival of 27 months has been described in patients with microscopically com‐ plete liver resection versus 14 months in patients with microscopically or macroscopically

With the discovery of *GNAQ* and *BAP1* mutations, new therapeutic strategies based on the specific mutated gene content seem promising. For tumours with Gα-protein mutations, the therapeutic goal is to inhibit downstream signalling molecules in the MAPK pathway that are activated. Preclinical studies show that inhibition of MAPK pathway in UM cell lines re‐ sults in decreased cell proliferation (Van Raamsdonk et al, 2009). There are several key mol‐ ecules in the MAPK pathway, which have been explored as potential therapeutic targets. One of such is MEK, and Gα-protein mutant UM cells showed to be mildly sensitive to the MEK inhibitor AZD6244 (Gill & Char, 2012). Another recent preclinical study proposed to target both the MAPK and PI3K/AKT pathway since both pathways are activated in UM. A combination of MEK and PI3K inhibition treatment resulted in induction of apoptosis in a Gα-mutant UM cells (Khalili et al, 2012). Other potential targets in the MAPK pathway are currently being investigated, including protein kinase C, which is a component of signalling

Therapeutically targeting UMs with a *BAP1* mutation works in a different manner than the Gα-protein mutations, since *BAP1* acts as a tumour suppressor gene. Regaining lost func‐ tions of suppressor genes are in general more challenging than inhibiting an overactive on‐ cogene. Nevertheless, ongoing studies show that histone deacetylase (HDAC) inhibitors may have therapeutic potential in UM. Landreville and colleagues established that HDAC inhibitors can reverse the histone H2A hyperubiquitination that occurs in cultured UM cells depleted of *BAP1*, and it induces morphologic differentiation, cell-cycle exit, and shifts to a differentiated, melanocytic GEP (Landreville et al, 2012). Examples of HDAC inhibitors are valproic acid, trichostatin A, LBH-589, and suberoylanilide hydroxamic acid. Clinical trials are needed to evaluate the effect of these compounds in UM patients, and hopefully UM specific treatment based on mutational content will lead to improved patient survival.

incomplete liver resection (Mariani et al, 2009).

**11. Future prospects**

156 Melanoma - From Early Detection to Treatment

from *GNAQ* to Erk1/2 (Wu et al, 2012).

**Abbreviations**

UM: uveal melanoma

OCT: optical coherence tomography

MRI: magnetic resonance imaging

CT: computed tomography

CHRPE: congenital hypertrophy of the retinal pigment epithelium

PEHC: peripheral exudative hemorrhagic chorioretinopathy

H&E: haematoxylin and eosin

PAS: Periodic-acid Schiff

FISH: fluorescent in situ hybridization

SKY: spectral karyotyping

CGH: comparative genomic hybridization

qPCR: quantitative polymerase chain reaction

MLPA: multiplex ligation probe amplification

MAQ: multiplex amplicon quantification

MSA: microsatellite analysis

LOH: loss of heterozygosity

SNP: single nucleotide polymorphism

GEP: gene expression profiling

NGS: next generation sequencing

MAPK: mitogen-activated protein kinase

HGF: hepatocyte growth factor

IGF-1: insulin-like growth factor 1

γGT: gamma-glutamyl transpeptidase

LDH: lactate dehydrogenase

AST: aspartate aminotransferase

ALT : alanine transaminase

PA: phosphatidic acid

MIA: melanoma inhibitory activity

TPS: tissue polypeptide specific antigen OPN: osteopontin SRT: stereotactic radiotherapy Ru-106: ruthenium-106 I-125: iodine-125 HDAC: histone deacetylase

## **Author details**

J.G.M. van Beek1 , A.E. Koopmans1,2, R.M. Verdijk3 , N.C. Naus1 , A. de Klein2 and E. Kilic1 [7] Atmaca LS, Batioğlu F & Atmaca P. (1999). Fluorescein and indocyanine green video‐ angiography of choroidal melanomas. In *Jpn J Ophthalmol*, Vol. 43. pp. 25-30.

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2 Department of Clinical Genetics, The Netherlands

3 Department of Pathology, The Netherlands

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**Chapter 6**

**Recombinant DNA Technology in Emerging Modalities**

The history of immunotherapy of cancer dates back to 1890s when New York surgeon William Coley used Streptococcus and Serratia bacterial extracts to treat cancer. Up to the mid-1930s 'Coley's mixed toxins,'' were used to treat various tumors. Better understanding of the human immune system led to the identification of a number of tumor-associated antigens (TAAs) in the 1980s [1] and development of various immunotherapeutic approaches. Of particular relevance to melanoma immunotherapy was the identification of various antigens expressed specifically in melanocytes and, respectively, in the majority of melanomas. These melanomaassociated antigens include tyrosinase (Tyr), a key enzyme in melanin biosynthesis, tyrosinaserelated proteins 1 and 2 (TRP1, TRP2), gp100 (aka pmel17), Melan-a, and MART1. These and several other melanoma-associated antigens formed the basis for the immunologic targeting of the tumor. Up to date, multiple peptide, dendritic cell, adjuvant, lymphocyte, antibody, DNA and virus-based strategies were tested in pre-clinical and clinical studies with varying degrees of success. In recent years, identification of the specific antigenic MHC class I epitopes, advancements in genetic engineering, gene delivery, and cell-based therapeutic approaches

allowed development of the novel melanoma-targeting immuno-therapeutics.

Identification of the tumor-reactive T cells among a population of the tumor-infiltrating lymphocytes led to the development of the T cell-based therapies, particularly to the strategy

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

© 2013 Alexeev 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,

**2. Genetic engineering of antigen-specific T cells**

**2.1. Recombinant T cell receptors**

**for Melanoma Immunotherapy**

Daria Marley Kemp and Olga Igoucheva

Additional information is available at the end of the chapter

Vitali Alexeev, Alyson Pidich,

http://dx.doi.org/10.5772/55357

**1. Introduction**

## **Recombinant DNA Technology in Emerging Modalities for Melanoma Immunotherapy**

Vitali Alexeev, Alyson Pidich, Daria Marley Kemp and Olga Igoucheva

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55357

## **1. Introduction**

The history of immunotherapy of cancer dates back to 1890s when New York surgeon William Coley used Streptococcus and Serratia bacterial extracts to treat cancer. Up to the mid-1930s 'Coley's mixed toxins,'' were used to treat various tumors. Better understanding of the human immune system led to the identification of a number of tumor-associated antigens (TAAs) in the 1980s [1] and development of various immunotherapeutic approaches. Of particular relevance to melanoma immunotherapy was the identification of various antigens expressed specifically in melanocytes and, respectively, in the majority of melanomas. These melanomaassociated antigens include tyrosinase (Tyr), a key enzyme in melanin biosynthesis, tyrosinaserelated proteins 1 and 2 (TRP1, TRP2), gp100 (aka pmel17), Melan-a, and MART1. These and several other melanoma-associated antigens formed the basis for the immunologic targeting of the tumor. Up to date, multiple peptide, dendritic cell, adjuvant, lymphocyte, antibody, DNA and virus-based strategies were tested in pre-clinical and clinical studies with varying degrees of success. In recent years, identification of the specific antigenic MHC class I epitopes, advancements in genetic engineering, gene delivery, and cell-based therapeutic approaches allowed development of the novel melanoma-targeting immuno-therapeutics.

## **2. Genetic engineering of antigen-specific T cells**

## **2.1. Recombinant T cell receptors**

Identification of the tumor-reactive T cells among a population of the tumor-infiltrating lymphocytes led to the development of the T cell-based therapies, particularly to the strategy

known as adoptive T cells transfer. This strategy is based on the isolation of the tumorinfiltrating lymphocytes following analysis of their ability to target tumor cells and clonal expansion of tumor-reactive T cells via stimulation of cell proliferation with anti-CD3 and antiCD28 antibodies in the presence of IL-2. Upon obtaining a large quantity (> 108 cells), these cells are infused back to a tumor-bearing patient along with the lymphodepleting chemother‐ apy to temporary knock down circulating immunocytes and repetitive administration of the IL-2 (Fig. 1).

up to 70 % with up to 30% complete remission lasting for up to 3 years was reported when radiation sensitization was used in conjunction with the transfer of the tumor-reactive TILs.

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177

Despite the success of the pioneering studies at the Surgery Branch of the US National Cancer Institute and the consequent clinical trials, this approach, although holding much promise in treating melanoma, is facing several challenges that limit broad application of the TIL-based immunotherapy. As TILs are isolated from resected tumors, the first and foremost requirement is the eligibility for surgery, which should be conducted, preferably, in the facility equipped for the isolation of TILs, identification and expansion of the tumor-reactive T cells. *Ex vivo* stimulation and propagation of TILs to large quantities required for the effective immuno‐ therapy is time-consuming, labor-retaining, and expensive. Although recent clinical studies showed that infusion of the minimally cultured TILs without pre-selection for tumor reactivity provide a rather high response rate [3], the search for a better melanoma-targeting strategy is

Nevertheless, isolation of the individual melanoma-reactive T cell clones allowed the devel‐ opment of another immunotherapeutic approach – generation of the T cells expressing recombinant antigen specific T cell receptors (TCRs). TCRs are members of the immunoglo‐ bulin family proteins. Each TCR consists of 2 different membrane-anchored chains that are joined by the disulfide bridges to form heterodimer. About 95% of the T cells are characterized by the expression of the α and β chains, whereas the remaining 5% express γ and δ chains. Respectively, T cells expressing these receptors are often referred to as α/β and γ/δ T cells. Each chain is comprised of the variable and constant regions. The variable domain of both α- and β-chains have three hypervariable regions also known as complementarity determining regions (CDR), however, the β-chain has an additional area of hypervariability that is not involved in antigen binding. TCR α and γ chains are generated within T cells by VJ recombi‐ nation, whereas β and δ chains by the V(D)J recombination. Currently, the majority of the TILs selected for the ability to target tumors are α/β T cells expressing respective TCR chains that determine T cell specificity to an antigenic peptides presented by the major histocompatability complex (MHC) proteins. Therefore, it was proposed that sequences encoding tumor antigen recognizing TCR chains can be obtained from tumor-reactive T cells and then used for the gene transfer into patient-derived lymphocytes, thereby creating large quantities of tumor-reactive T cells. The first TCRs specific to melanocytic antigens MART-1 and gp-100 were cloned in 1990s. Pioneering clinical studies using human peripheral blood lymphocytes transduced with these TCRs demonstrated melanoma regression in lymphodepleted patients [4] (Fig. 1). Although these and other initial clinical studies demonstrated a feasibility of the recombinant T cells-based approach, they also revealed multiple challenges. For example, the ability of recombinant TCR chains to interact and pair with the endogenous chains could lead to the generation of 4 different TCRs in a single cell (Fig. 2). Chain misparing decreases the expression of the function, tumor-reactive TCRs and therefore reduces T cell-mediated tumor targeting. To overcome misparing, several strategies were proposed. Recent pre-clinical and clinical studies demonstrated that replacement of the human TCR constant region with murine counterpart reduced misparing and allowed enhanced expression of the functional TCRs and improved T cell functional activity [5]. It was also reported that targeted mutagenesis and generation of the additional cystein residues in recombinant α and β chains permitted stronger

on-going.

**Figure 1.** Clinical application of the T cell-mediated tumor immunotherapy. Diagram on the top depicts application of the Tumor-Infiltrating Lymphocytes (TILs). Diagram on the bottom illustrates application of the genetically engineered (TCR and CAR-modified) T cells.

Presently, 87 clinical trials using TIL are completed or on-going. These clinical trials are aimed at treatment of multiple cancers including: Malignant Melanoma, Nasopharyngeal Carcinoma, Hepatocellular Carcinoma, Breast Carcinoma, Leukemia, Lymphoma, Multiple Myeloma, Plasma Cell Neoplasm, Kidney Cancer, Metastatic Colorectal Cancer, Metastatic Gastric Cancer, Metastatic Pancreatic Cancer, Metastatic Hepatocellular Carcinoma, Cervical Cancer, Oropharyngeal Cancer, Vaginal Cancer, Anal Cancer, Penile Cancer, Non-Small Cell Lung Cancer, Brain and Central Nervous System Tumors. Several completed clinical trials on malignant melanoma clearly demonstrated that infusing TILs along with IL-2 and preconditioning with reduced-intensity circulating lymphocyte-depleting chemotherapy medi‐ ates tumor-targeting immune response in up to 50% of patients [2]. The highest response rate up to 70 % with up to 30% complete remission lasting for up to 3 years was reported when radiation sensitization was used in conjunction with the transfer of the tumor-reactive TILs.

known as adoptive T cells transfer. This strategy is based on the isolation of the tumorinfiltrating lymphocytes following analysis of their ability to target tumor cells and clonal expansion of tumor-reactive T cells via stimulation of cell proliferation with anti-CD3 and

cells are infused back to a tumor-bearing patient along with the lymphodepleting chemother‐ apy to temporary knock down circulating immunocytes and repetitive administration of the

**Figure 1.** Clinical application of the T cell-mediated tumor immunotherapy. Diagram on the top depicts application of the Tumor-Infiltrating Lymphocytes (TILs). Diagram on the bottom illustrates application of the genetically engineered

Presently, 87 clinical trials using TIL are completed or on-going. These clinical trials are aimed at treatment of multiple cancers including: Malignant Melanoma, Nasopharyngeal Carcinoma, Hepatocellular Carcinoma, Breast Carcinoma, Leukemia, Lymphoma, Multiple Myeloma, Plasma Cell Neoplasm, Kidney Cancer, Metastatic Colorectal Cancer, Metastatic Gastric Cancer, Metastatic Pancreatic Cancer, Metastatic Hepatocellular Carcinoma, Cervical Cancer, Oropharyngeal Cancer, Vaginal Cancer, Anal Cancer, Penile Cancer, Non-Small Cell Lung Cancer, Brain and Central Nervous System Tumors. Several completed clinical trials on malignant melanoma clearly demonstrated that infusing TILs along with IL-2 and preconditioning with reduced-intensity circulating lymphocyte-depleting chemotherapy medi‐ ates tumor-targeting immune response in up to 50% of patients [2]. The highest response rate

cells), these

antiCD28 antibodies in the presence of IL-2. Upon obtaining a large quantity (> 108

IL-2 (Fig. 1).

176 Melanoma - From Early Detection to Treatment

(TCR and CAR-modified) T cells.

Despite the success of the pioneering studies at the Surgery Branch of the US National Cancer Institute and the consequent clinical trials, this approach, although holding much promise in treating melanoma, is facing several challenges that limit broad application of the TIL-based immunotherapy. As TILs are isolated from resected tumors, the first and foremost requirement is the eligibility for surgery, which should be conducted, preferably, in the facility equipped for the isolation of TILs, identification and expansion of the tumor-reactive T cells. *Ex vivo* stimulation and propagation of TILs to large quantities required for the effective immuno‐ therapy is time-consuming, labor-retaining, and expensive. Although recent clinical studies showed that infusion of the minimally cultured TILs without pre-selection for tumor reactivity provide a rather high response rate [3], the search for a better melanoma-targeting strategy is on-going.

Nevertheless, isolation of the individual melanoma-reactive T cell clones allowed the devel‐ opment of another immunotherapeutic approach – generation of the T cells expressing recombinant antigen specific T cell receptors (TCRs). TCRs are members of the immunoglo‐ bulin family proteins. Each TCR consists of 2 different membrane-anchored chains that are joined by the disulfide bridges to form heterodimer. About 95% of the T cells are characterized by the expression of the α and β chains, whereas the remaining 5% express γ and δ chains. Respectively, T cells expressing these receptors are often referred to as α/β and γ/δ T cells. Each chain is comprised of the variable and constant regions. The variable domain of both α- and β-chains have three hypervariable regions also known as complementarity determining regions (CDR), however, the β-chain has an additional area of hypervariability that is not involved in antigen binding. TCR α and γ chains are generated within T cells by VJ recombi‐ nation, whereas β and δ chains by the V(D)J recombination. Currently, the majority of the TILs selected for the ability to target tumors are α/β T cells expressing respective TCR chains that determine T cell specificity to an antigenic peptides presented by the major histocompatability complex (MHC) proteins. Therefore, it was proposed that sequences encoding tumor antigen recognizing TCR chains can be obtained from tumor-reactive T cells and then used for the gene transfer into patient-derived lymphocytes, thereby creating large quantities of tumor-reactive T cells. The first TCRs specific to melanocytic antigens MART-1 and gp-100 were cloned in 1990s. Pioneering clinical studies using human peripheral blood lymphocytes transduced with these TCRs demonstrated melanoma regression in lymphodepleted patients [4] (Fig. 1). Although these and other initial clinical studies demonstrated a feasibility of the recombinant T cells-based approach, they also revealed multiple challenges. For example, the ability of recombinant TCR chains to interact and pair with the endogenous chains could lead to the generation of 4 different TCRs in a single cell (Fig. 2). Chain misparing decreases the expression of the function, tumor-reactive TCRs and therefore reduces T cell-mediated tumor targeting. To overcome misparing, several strategies were proposed. Recent pre-clinical and clinical studies demonstrated that replacement of the human TCR constant region with murine counterpart reduced misparing and allowed enhanced expression of the functional TCRs and improved T cell functional activity [5]. It was also reported that targeted mutagenesis and generation of the additional cystein residues in recombinant α and β chains permitted stronger pairing of these chains, higher expression of functional TCRs and improved T cell function [6, 7]. Recent studies also showed that targeting of the endogenous chains by siRNA allows higher expression of the functional recombinant TCR. Of particular interest is the proposed approach to encode siRNA along with the TCR chains to concurrently express recombinant and inhibit translation of the endogenous chains [8]. Protein engineering was also employed to improve pairing of the recombinant chains. Thus, substitution of specific amino acids within constant regions of the antigen-specific TCRs supported paring and enhanced functional activity of these receptors [9]. It remains to be determined which of these recombinant DNA-based methods will provide better targeting of melanoma (Fig. 2). Nevertheless, recent studies using chimeric murine-human hybrid highly avid tyrosinase-specific TCR demonstrated a favorable clinical outcome [10].

It is apparent that both α and β chains of the antigen-specific TCR should be expressed in each individual T cell. To date, internal ribosomal entry site (IRES) elements [11], double promoters [12], or co-infection with several viral vectors [13] were used to express several heterologous proteins in cells. However, these methods have their imperfections. For instance, in IRESmediated co-expression, the upstream protein is usually more strongly transcribed than the downstream protein. Expression of the proteins from two different or biscistronic promoters or the use of multiples viruses also do not provide equal concurrent expression of multiple transgenes. A more promising approach involves the use of the self-processing viral peptide bridges such as 2A or 2A-like peptides described in Picornaviridae [14]. In picornavirus, these sequences share a highly conservative 18 amino acids motif mediating cleavage between Cterminal glycine and N-terminal proline of the 2B peptide. At present 2a and 2A-like sequences are commonly refer to as *cis*-acting hydrolase elements that allows ribosome skipping and cellular expression of multiple, discrete proteins in essentially equimolar quantities derived from a single ORF. To ensure concurrent expression of both α and β chains of the transgenic TCR an A2 sequence is most commonly used for quantitative co-expression of these heterol‐

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179

Transfer of the recombinant TCR genes into the T cells is another somewhat limiting factor for the broad application of the genetically engineered T cells for melanoma immuno-targeting. Currently, for human applications, a gene transfer platform that can mediate stable transfer of the TCR genes is retroviral system [15]. Lentiviral vectors and transposons were also tested [16, 17]. Use of retroviruses provided several advantages including a rather high infectivity and rapid integration of the transgene into host genome. With multiple vector backbones, virus packaging cell lines, and well-established GMP protocols, a retroviral system offers relative simplicity of viral vector construction and production of viruses. Since retroviruses can infect only dividing cells, stimulation of the T cell proliferation must be done prior to the gene transfer. Also, these viruses have limited capacity for the packaging. For instance, high virus titers cannot be obtained with larger retroviral vectors. Although an average size of a viral vector encoding typical α/β TCR is around 7 kb, this limits possible alternative approaches such as inclusion of various regulatory elements or another transgenes that may enhance T cell activation. Use of the viral system also presents certain safety concerns relevant to the random integration of the transgenes into the host genome that may result in the activation of oncogenes or inactivation of tumor suppressors. This may lead to the various adverse invents including development of a lymphoproliferative disease resembling leukemia due, in part, to the integration of the retroviral gene transfer vehicle near an oncogene [18, 19]. Thus far, the development of lymphoma-like symptoms has not been reported in patients treated with recombinant T cells. It is also essential to note that production of the TCR-encoding cGMP virus substantially increases the cost of the treatment with recombinant T cells. On the contrary to the retrovirus-based gene transfer, lentiviruses can infect non-dividing cells and therefore can be used for the gene transfer into quiescent T cells. Although "safe" lentiviral systems are developed to minimize the chance of producing replication-competent virus (eg. ViaraSafe from Cell Biolabs), transduction of patient-derived T cells for the adoptive transfer will always

ogous proteins.

present some degree of risk.

**Figure 2.** Strategies aimed at the improvement of the recombinant TCR pairing. Expression of the recombinant TCR may lead to the generation of 4 different TCRs within a cell (center). Different strategies designed to improve tumorspecific recombinant TCR pairing and activity include: generation of hybrid molecules containing the constant region from murine TCR, addition of disulfide bonds, alteration of the amino acid sequence within the TCR chains, and siRNAmediated inhibition of the endogenous TCR gene expression (see text for details).

It is apparent that both α and β chains of the antigen-specific TCR should be expressed in each individual T cell. To date, internal ribosomal entry site (IRES) elements [11], double promoters [12], or co-infection with several viral vectors [13] were used to express several heterologous proteins in cells. However, these methods have their imperfections. For instance, in IRESmediated co-expression, the upstream protein is usually more strongly transcribed than the downstream protein. Expression of the proteins from two different or biscistronic promoters or the use of multiples viruses also do not provide equal concurrent expression of multiple transgenes. A more promising approach involves the use of the self-processing viral peptide bridges such as 2A or 2A-like peptides described in Picornaviridae [14]. In picornavirus, these sequences share a highly conservative 18 amino acids motif mediating cleavage between Cterminal glycine and N-terminal proline of the 2B peptide. At present 2a and 2A-like sequences are commonly refer to as *cis*-acting hydrolase elements that allows ribosome skipping and cellular expression of multiple, discrete proteins in essentially equimolar quantities derived from a single ORF. To ensure concurrent expression of both α and β chains of the transgenic TCR an A2 sequence is most commonly used for quantitative co-expression of these heterol‐ ogous proteins.

pairing of these chains, higher expression of functional TCRs and improved T cell function [6, 7]. Recent studies also showed that targeting of the endogenous chains by siRNA allows higher expression of the functional recombinant TCR. Of particular interest is the proposed approach to encode siRNA along with the TCR chains to concurrently express recombinant and inhibit translation of the endogenous chains [8]. Protein engineering was also employed to improve pairing of the recombinant chains. Thus, substitution of specific amino acids within constant regions of the antigen-specific TCRs supported paring and enhanced functional activity of these receptors [9]. It remains to be determined which of these recombinant DNA-based methods will provide better targeting of melanoma (Fig. 2). Nevertheless, recent studies using chimeric murine-human hybrid highly avid tyrosinase-specific TCR demonstrated a favorable

**Figure 2.** Strategies aimed at the improvement of the recombinant TCR pairing. Expression of the recombinant TCR may lead to the generation of 4 different TCRs within a cell (center). Different strategies designed to improve tumorspecific recombinant TCR pairing and activity include: generation of hybrid molecules containing the constant region from murine TCR, addition of disulfide bonds, alteration of the amino acid sequence within the TCR chains, and siRNA-

mediated inhibition of the endogenous TCR gene expression (see text for details).

clinical outcome [10].

178 Melanoma - From Early Detection to Treatment

Transfer of the recombinant TCR genes into the T cells is another somewhat limiting factor for the broad application of the genetically engineered T cells for melanoma immuno-targeting. Currently, for human applications, a gene transfer platform that can mediate stable transfer of the TCR genes is retroviral system [15]. Lentiviral vectors and transposons were also tested [16, 17]. Use of retroviruses provided several advantages including a rather high infectivity and rapid integration of the transgene into host genome. With multiple vector backbones, virus packaging cell lines, and well-established GMP protocols, a retroviral system offers relative simplicity of viral vector construction and production of viruses. Since retroviruses can infect only dividing cells, stimulation of the T cell proliferation must be done prior to the gene transfer. Also, these viruses have limited capacity for the packaging. For instance, high virus titers cannot be obtained with larger retroviral vectors. Although an average size of a viral vector encoding typical α/β TCR is around 7 kb, this limits possible alternative approaches such as inclusion of various regulatory elements or another transgenes that may enhance T cell activation. Use of the viral system also presents certain safety concerns relevant to the random integration of the transgenes into the host genome that may result in the activation of oncogenes or inactivation of tumor suppressors. This may lead to the various adverse invents including development of a lymphoproliferative disease resembling leukemia due, in part, to the integration of the retroviral gene transfer vehicle near an oncogene [18, 19]. Thus far, the development of lymphoma-like symptoms has not been reported in patients treated with recombinant T cells. It is also essential to note that production of the TCR-encoding cGMP virus substantially increases the cost of the treatment with recombinant T cells. On the contrary to the retrovirus-based gene transfer, lentiviruses can infect non-dividing cells and therefore can be used for the gene transfer into quiescent T cells. Although "safe" lentiviral systems are developed to minimize the chance of producing replication-competent virus (eg. ViaraSafe from Cell Biolabs), transduction of patient-derived T cells for the adoptive transfer will always present some degree of risk.

Besides viral approaches, non-viral gene transfer may also be used for the expression of the TCRs in T cells. Recently, a Sleeping Beauty Transposon System was tested for the transduction of the T cells [17]. Sleeping Beauty Transposon System consists of two components - the transposon, composed of inverted terminal repeat sequences (IRs) with desired genetic material in between, and a SB transposase enzyme. Most recently, a number of IRs and hyperactive transposases with increasing enzymatic activities were developed to mediate transposition of transposon-encoding proteins into the genomic DNA [20]. Although trans‐ position of SB transposons appears to be unregulated, it has certain advantages over viral based approaches. For instance, expression of transgenes, TCRs in particular, could be regulated by specific promoters that provide either T cell specific expression (eg. CD3 promoter; [21, 22]), or high level of expression (eg. elongation factor 1 promoter; [23,24]. Promoters may also be selected for further specific applications (discussed below). On the contrary to the viral gene transfer, non-viral systems also permit significantly simpler production of the cGMP-grade material (plasmid DNA) and lesser safety testing. Up to date, the Sleeping Beauty transposonmediated approach was shown to mediate a long-term stable integration of the T-cell receptor genes targeting melanoma-derived antigen, MART-1, in laboratory settings (Fig. 3b). This system provided 50% efficiency of the TCR integration into the genome of the T cells and sustained functional reactivity of lymphocytes to the antigen-positive melanoma [25].

Other non-viral strategies could be useful in genetic engineering of the T cells. For example, integrase-mediated insertion of the genetic material may provide stable, site-directed integra‐ tion of the transgenes (TCRs) into T cell genome. This strategy involves integrase from the *Streptomyces* phage ΦC31 that catalyzes unidirectional recombination between attP motifs in phage and attB sites in bacterial genomes. Usually attP and attB sites are cleaved and joined to each other, generating two hybrid sequences (attL and attR) that flank the integrated phage genome. However, ΦC31 integrase can also recognize several endogenous sequences in eukaryotic chromosomes as attP sites and integrate attB-bearing transgenes into them (Fig. 3c). Such pseudo attP sites were found in every mammalian genome with more than 100 ΦC31 integration sites identified in human cells. Thus far, only three preferred sites located in human Xq22.1, 8p22, 19q13.31 loci are commonly used by this enzyme [26, 27]. Therefore, ΦC31 integrase-based system is somewhat similar to the SB transposone system (Fig. 3b, c). Yet, it provides better specificity of the transgene integration. We recently tested whether ΦC31 can efficiently integrate transgenes into the T cells. Our initial data using GFP-encoding reporter plasmid with short (34bp) attB site demonstrated that nucleofection reaction provides rather efficient transduction of the transgene and ΦC31 integrase-encoding plasmids into T cells (Fig. 4a) and stable, integrase-dependent insertion of the reporter into both CD4+ and CD8+ T cells (Fig. 4b). Transduction of the T cells with tyrosinase-specific TCR (described in 10) ligated into the attB-harboring mammalian expression vector also resulted in the sustained expression of this melanoma-specific TCR and the ability of the T cells to target antigen-positive melanoma cells *in vitro* (Fig. 4c)

Collectively, viral and non-viral strategies for the genetic engineering of the T cells expressing melanoma-specific TCRs are suitable for the *ex vivo* production of large quantities (more than 108 cells) of the tumor-specific T cells that can be used for the adoptive T cell transfer. However, clinical utility of the non-viral approaches remains to be elucidated. In spite of T cell trans‐

duction strategy, it is clear that the ability to generate melanoma-specific recombinant T cell receptors allowed significant advancement in the development of the clinically-applicable TCR-based approach for melanoma immunotherapy. Its primary advantages are in the use of

**Figure 3.** Schematic diagram depicting genetic engineering of the tumor-targeting T cells expressing recombinant TCR. Diagrams depict generation of the recombinant T cells via (a) retrovirus-mediated gene transfer, (b) Sleeping Beauty trans‐

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poson-mediated gene transposition, and (c) ΦC31 integrase-mediated gene insertion (see text for details).

Recombinant DNA Technology in Emerging Modalities for Melanoma Immunotherapy http://dx.doi.org/10.5772/55357 181

Besides viral approaches, non-viral gene transfer may also be used for the expression of the TCRs in T cells. Recently, a Sleeping Beauty Transposon System was tested for the transduction of the T cells [17]. Sleeping Beauty Transposon System consists of two components - the transposon, composed of inverted terminal repeat sequences (IRs) with desired genetic material in between, and a SB transposase enzyme. Most recently, a number of IRs and hyperactive transposases with increasing enzymatic activities were developed to mediate transposition of transposon-encoding proteins into the genomic DNA [20]. Although trans‐ position of SB transposons appears to be unregulated, it has certain advantages over viral based approaches. For instance, expression of transgenes, TCRs in particular, could be regulated by specific promoters that provide either T cell specific expression (eg. CD3 promoter; [21, 22]), or high level of expression (eg. elongation factor 1 promoter; [23,24]. Promoters may also be selected for further specific applications (discussed below). On the contrary to the viral gene transfer, non-viral systems also permit significantly simpler production of the cGMP-grade material (plasmid DNA) and lesser safety testing. Up to date, the Sleeping Beauty transposonmediated approach was shown to mediate a long-term stable integration of the T-cell receptor genes targeting melanoma-derived antigen, MART-1, in laboratory settings (Fig. 3b). This system provided 50% efficiency of the TCR integration into the genome of the T cells and sustained functional reactivity of lymphocytes to the antigen-positive melanoma [25].

Other non-viral strategies could be useful in genetic engineering of the T cells. For example, integrase-mediated insertion of the genetic material may provide stable, site-directed integra‐ tion of the transgenes (TCRs) into T cell genome. This strategy involves integrase from the *Streptomyces* phage ΦC31 that catalyzes unidirectional recombination between attP motifs in phage and attB sites in bacterial genomes. Usually attP and attB sites are cleaved and joined to each other, generating two hybrid sequences (attL and attR) that flank the integrated phage genome. However, ΦC31 integrase can also recognize several endogenous sequences in eukaryotic chromosomes as attP sites and integrate attB-bearing transgenes into them (Fig. 3c). Such pseudo attP sites were found in every mammalian genome with more than 100 ΦC31 integration sites identified in human cells. Thus far, only three preferred sites located in human Xq22.1, 8p22, 19q13.31 loci are commonly used by this enzyme [26, 27]. Therefore, ΦC31 integrase-based system is somewhat similar to the SB transposone system (Fig. 3b, c). Yet, it provides better specificity of the transgene integration. We recently tested whether ΦC31 can efficiently integrate transgenes into the T cells. Our initial data using GFP-encoding reporter plasmid with short (34bp) attB site demonstrated that nucleofection reaction provides rather efficient transduction of the transgene and ΦC31 integrase-encoding plasmids into T cells (Fig. 4a) and stable, integrase-dependent insertion of the reporter into both CD4+ and CD8+ T cells (Fig. 4b). Transduction of the T cells with tyrosinase-specific TCR (described in 10) ligated into the attB-harboring mammalian expression vector also resulted in the sustained expression of this melanoma-specific TCR and the ability of the T cells to target antigen-positive melanoma

Collectively, viral and non-viral strategies for the genetic engineering of the T cells expressing melanoma-specific TCRs are suitable for the *ex vivo* production of large quantities (more than

 cells) of the tumor-specific T cells that can be used for the adoptive T cell transfer. However, clinical utility of the non-viral approaches remains to be elucidated. In spite of T cell trans‐

cells *in vitro* (Fig. 4c)

180 Melanoma - From Early Detection to Treatment

108

**Figure 3.** Schematic diagram depicting genetic engineering of the tumor-targeting T cells expressing recombinant TCR. Diagrams depict generation of the recombinant T cells via (a) retrovirus-mediated gene transfer, (b) Sleeping Beauty trans‐ poson-mediated gene transposition, and (c) ΦC31 integrase-mediated gene insertion (see text for details).

duction strategy, it is clear that the ability to generate melanoma-specific recombinant T cell receptors allowed significant advancement in the development of the clinically-applicable TCR-based approach for melanoma immunotherapy. Its primary advantages are in the use of

**2.2. Chimeric antigen receptors**

effector function and anergic status of the T cells.

CD3ξ, CD137 (4-1BB), and CD28 signaling domains (see text for details).

Independently, an alternative approach involving recombinant DNA technology was devel‐ oped to generate tumor-targeting T cells. It utilizes fusion of the variable chain of the tumorantigen-specific antibody, TCR constant region, and intracellular signaling domains. Initially, these structures were called T-bodies [31]. They are comprise of the single-chain antibody (sFv), TCR transmembrane domain and the intracellular signaling domain of the TCR-ζ. One of the first tumor associated antigens targeted by T cells expressing T-bodies was erbB2 (HER2/neu) receptor that is over-expressed in multiple cancers [32]. Later, a more general term – chimeric antigen receptors or CARs emerged. As compared to the TCRs, CARs allow overcoming dependency on HLA type, antigen presentation, and restricted intracellular signaling of the recombinant α/β TCRs. Initial studies with T-bodies (and recombinant TCRs) demonstrated a rather short lifespan of the engineered T cells and the inability of the recombinant receptors to fully support persistence of the T cell. To address this issue, several studies were conducted to identify the most potent CAR structures by testing several signaling molecules involved in T cell activation (Fig. 5). It was demonstrated that fusion of TCR-ζ with the intracellular domain of CD28 can augment cytokine production by CAR-expressing T cells upon encountering antigen and enhance antitumor efficacy [33]. Inclusion of CD134 (OX-40) into CAR structure also led to the elevated tumoricidal activity of the recombinant T cells [34]. Comparative analysis of the different CARs comprised of TCR-ζ signal transduction domain, CD28 and/or CD137 (4-1BB) intracellular domains demonstrated that addition of the CD137 supports T cell function to a greater extent as compared to other constructs [35]. Collectively, addition of these signaling domains to the CAR structure allowed overcoming (to certain extent) inefficient

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**Figure 5.** Recombinant TCR and CAR structures. (a) Diagram depicting recombinant TCR structure. (b) Diagram illus‐ trating molecular interactions involved in TCR-mediated pro-proliferation and pro-survival intracellular signaling in‐ cluding engagement of the CD28, CD3, and CD137 (4-1BB). (c) Diagram depicting recombinant CAR structure with

As recognition of target cells by CAR depends on the antibody, CARs can recognize not only polypeptides but also non-protein molecules such as tumor-associated glycolipids and carbohydrates. However, antibody-mediated binding require surface expression of an antigen and strict selection of TAAs to avoid autoimmune side effects. Also, use of the mouse mono‐ clonal antibody sequences in CAR design may lead to the unwanted immune recognition of

**Figure 4.** ΦC31 integrase-mediated genetic modification of the T cells. (a) Example showing typical nucleofection re‐ action of the reporter gene (GFP) into freshly isolated T cells 24 hours post Nucleofection (representative FACS pro‐ files). (b) Analysis of the GFP expression after Nucleofection of the T cells in the presence of absence of the ΦC31 integrase-encoding plasmid (representative profiles and the direct fluorescent microscopy) 3 and 14 days after Nucle‐ ofection. (c) FACS-based analysis of the CTL activity of the recombinant T cells generated to express tyrosinase-specific TCR against human melanoma in vitro (representative profile). Times and percentages are indicated in the panels; Shaded Profiles – GFP-positive cells (see text for details).

a natural and a rather well-understood mechanism of the T cell function and the ability to select/generate multiple melanoma-reactive TCRs that can be used alone or in combination. Currently, several melanoma-targeting TCRs specific to tyrosinase, MART-1, and gp100 were cloned. One can envision generation of TCR-encoding cDNA banks that could be utilized for the generation of different melanoma-reacting T cells from the pool of patient-derived T cells to target several TAAs. However, this strategy has several disadvantages including restriction of specific TCRs to one HLA type, dependence from the expression and presentation of an antigen, limited intracellular signaling from the recombinant α/β TCRs, misparing of TCR chains, and the inability to target non-protein tumor antigens.

At present time, about 20 clinical studies involving melanoma-specific T cells expressing recombinant TCR were conducted in US alone (some of them reviewed in [28]). The result of some of the completed trials opened new perspectives for the improvement of the TCR-based strategies. For instance, adoptive transfer of the T cells genetically engineered to express highly avid MART-1-specific TCR has achieved objective clinical responses in a 13% of treated patients [29]. Analysis of CTL-resistant tumor cell revealed that these resistant clones exhibited hyperactivation of the NF-κB survival pathway and overexpression of the antiapoptotic Bcl-2, Bcl-x, Bcl-xL, and Mcl-1 genes [30]. These studies suggest that sensitivity of melanoma to the recombinant T cells could be increased by the pharmacological inhibition of the NF-κB pathway and/or Bcl-2 family members. Multiple investigative studies are on-going to further improve recombinant TCR-based approach.

#### **2.2. Chimeric antigen receptors**

a natural and a rather well-understood mechanism of the T cell function and the ability to select/generate multiple melanoma-reactive TCRs that can be used alone or in combination. Currently, several melanoma-targeting TCRs specific to tyrosinase, MART-1, and gp100 were cloned. One can envision generation of TCR-encoding cDNA banks that could be utilized for the generation of different melanoma-reacting T cells from the pool of patient-derived T cells to target several TAAs. However, this strategy has several disadvantages including restriction of specific TCRs to one HLA type, dependence from the expression and presentation of an antigen, limited intracellular signaling from the recombinant α/β TCRs, misparing of TCR

**Figure 4.** ΦC31 integrase-mediated genetic modification of the T cells. (a) Example showing typical nucleofection re‐ action of the reporter gene (GFP) into freshly isolated T cells 24 hours post Nucleofection (representative FACS pro‐ files). (b) Analysis of the GFP expression after Nucleofection of the T cells in the presence of absence of the ΦC31 integrase-encoding plasmid (representative profiles and the direct fluorescent microscopy) 3 and 14 days after Nucle‐ ofection. (c) FACS-based analysis of the CTL activity of the recombinant T cells generated to express tyrosinase-specific TCR against human melanoma in vitro (representative profile). Times and percentages are indicated in the panels;

At present time, about 20 clinical studies involving melanoma-specific T cells expressing recombinant TCR were conducted in US alone (some of them reviewed in [28]). The result of some of the completed trials opened new perspectives for the improvement of the TCR-based strategies. For instance, adoptive transfer of the T cells genetically engineered to express highly avid MART-1-specific TCR has achieved objective clinical responses in a 13% of treated patients [29]. Analysis of CTL-resistant tumor cell revealed that these resistant clones exhibited hyperactivation of the NF-κB survival pathway and overexpression of the antiapoptotic Bcl-2, Bcl-x, Bcl-xL, and Mcl-1 genes [30]. These studies suggest that sensitivity of melanoma to the recombinant T cells could be increased by the pharmacological inhibition of the NF-κB pathway and/or Bcl-2 family members. Multiple investigative studies are on-going to further

chains, and the inability to target non-protein tumor antigens.

improve recombinant TCR-based approach.

Shaded Profiles – GFP-positive cells (see text for details).

182 Melanoma - From Early Detection to Treatment

Independently, an alternative approach involving recombinant DNA technology was devel‐ oped to generate tumor-targeting T cells. It utilizes fusion of the variable chain of the tumorantigen-specific antibody, TCR constant region, and intracellular signaling domains. Initially, these structures were called T-bodies [31]. They are comprise of the single-chain antibody (sFv), TCR transmembrane domain and the intracellular signaling domain of the TCR-ζ. One of the first tumor associated antigens targeted by T cells expressing T-bodies was erbB2 (HER2/neu) receptor that is over-expressed in multiple cancers [32]. Later, a more general term – chimeric antigen receptors or CARs emerged. As compared to the TCRs, CARs allow overcoming dependency on HLA type, antigen presentation, and restricted intracellular signaling of the recombinant α/β TCRs. Initial studies with T-bodies (and recombinant TCRs) demonstrated a rather short lifespan of the engineered T cells and the inability of the recombinant receptors to fully support persistence of the T cell. To address this issue, several studies were conducted to identify the most potent CAR structures by testing several signaling molecules involved in T cell activation (Fig. 5). It was demonstrated that fusion of TCR-ζ with the intracellular domain of CD28 can augment cytokine production by CAR-expressing T cells upon encountering antigen and enhance antitumor efficacy [33]. Inclusion of CD134 (OX-40) into CAR structure also led to the elevated tumoricidal activity of the recombinant T cells [34]. Comparative analysis of the different CARs comprised of TCR-ζ signal transduction domain, CD28 and/or CD137 (4-1BB) intracellular domains demonstrated that addition of the CD137 supports T cell function to a greater extent as compared to other constructs [35]. Collectively, addition of these signaling domains to the CAR structure allowed overcoming (to certain extent) inefficient effector function and anergic status of the T cells.

**Figure 5.** Recombinant TCR and CAR structures. (a) Diagram depicting recombinant TCR structure. (b) Diagram illus‐ trating molecular interactions involved in TCR-mediated pro-proliferation and pro-survival intracellular signaling in‐ cluding engagement of the CD28, CD3, and CD137 (4-1BB). (c) Diagram depicting recombinant CAR structure with CD3ξ, CD137 (4-1BB), and CD28 signaling domains (see text for details).

As recognition of target cells by CAR depends on the antibody, CARs can recognize not only polypeptides but also non-protein molecules such as tumor-associated glycolipids and carbohydrates. However, antibody-mediated binding require surface expression of an antigen and strict selection of TAAs to avoid autoimmune side effects. Also, use of the mouse mono‐ clonal antibody sequences in CAR design may lead to the unwanted immune recognition of the CAR-expressing T cells and limit long-term clinical use [36, 37]. Nevertheless, existence of a large number of the tumor antigen-specific antibodies and robust anti-tumor response by CAR-expressing T cells suggest great clinical utility of these recombinant molecules. Currently, in US alone there are 18 clinical trials aimed at treatment of various malignancies with CARengineered leukocytes, with 16 trials in the recruitment phase. Eight of them are aimed at targeting different B cell malignancies with anti-CD19-CAR. Three trials are intended to test HER2-specific CAR-modified T cells for the treatment of sarcoma, glioblastoma, and advanced Her2-positive lung malignancy.

Thus, using an animal model, it was shown that after systemic transplantation, anti–VEGFR-2 CAR and IL-12–co-transduced T cells infiltrated the tumors, expanded and persisted within tumor mass leading to tumor regression [47]. The anti-tumor effect was dependent on targeting of IL-12–responsive host cells via activation of anti–VEGFR-2 CAR-T cells and release of IL-12. Based on this data, one clinical trial aimed at the assessment of safety and effectiveness of cell therapy was initiated to treat recurrent and relapsed cancer by using anti-VEGFR2 CAR-

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Presently, there is an accumulating body of evidence suggesting clinical utility of the T cell genetically engineered to express melanoma antigen-specific CARs. It is likely that in the near future CAR-mediated targeting of different melanoma antigens will evolve into general

Another immunotherapeutic approach directly relevant to recombinant DNA is genetic or DNA vaccination. The original idea of DNA vaccination emanated from the observations that intramuscular injection of DNA encoding influenza A virus protein resulted in the robust activation of the immune responses that protected the host from viral challenge [48]. Generally, DNA-mediated activation of immune response involves multiple processes. First, plasmid DNA should be delivered intracellularly and expressed in the host cells. Next, in most of cases the antigen has to be secreted from the cells and picked up by the dendritic cells (DC), processed and presented in the context of the MHC class II to the CD4+ T helper (Th) cells. Alternatively, if the antigen is expressed directly in the DCs, it could be processed intracellularly and presented via MHC class I molecules, leading to the activation of the CD8+ T cells and induction of the cytotoxic immune responses. Initial studies on DNA vaccination were carried out using an intramuscular route of vaccine administration (Fig. 6). This allowed high levels of antigen expression and secretion from the elongated muscle cells into perimysium, the resident site of the intramuscular DCs. Later, DNA vaccination through the skin was suggested to be superior over the intramuscular route. Skin has evolved as a barrier to prevent the entry of pathogens, with efficient immune surveillance complex including Langerhans cells, dendritic cells, lymphocytes, and other leukocytes. Skin is also rich in lymphatic vasculature network that provides an efficient route for DC and T cell trafficking. Depending on the physical methods of into-skin DNA delivery, DNA-based vaccines can be targeted to specific locations in the

The DNA vaccination approach has several advantages over other types of vaccinations: (i) multiple expression vectors coding for different antigen and co-stimulatory molecules can be concurrently delivered into the skin (or the muscle); (ii) the use of cell-type-specific promoters can provide specificity of protein expression; (iii) protein expression from designed plasmids can be controlled by inducible promoters, the use of ubiquitous chromatin opening elements (UCOE), or chemically (e.g. sodium butyrate). Also of note is the relative simplicity and inexpensiveness of the cGMP grade DNA vaccine production and pre-clinical testing. These

modified T cells.

practice of cancer immunotherapy.

**3. DNA vaccination**

skin [49].

With regard to melanoma, several CAR designs were tested for the ability to target this malignancy. Thus, recent studies demonstrated that treatment of melanoma xenografts in nude mice using engineered T cells expressing tandem CAR (CD28/TCRζ) specific to ganglio‐ side GD3 with IL2 supplementation led to complete remissions of the established tumors in 50 % of treated animals [38]. As GD3 is often over-expressed in melanoma, this approach could be potent in eliminating melanoma in human patients.

Another attractive target for the CAR-mediated T cell therapy for melanoma is a high molec‐ ular weight melanoma-associated antigen (HMW-MAA) encoded by CSPG4 gene. This is a cell-surface proteoglycan expressed on more than 90% of the tumors. Recent studies on targeting of this antigen using CAR that is comprised of the anti- HMW-MAA antibody chain and intracellular signaling domains of the CD28, CD137, and CD3ζ demonstrated that T cell genetically modified to express this CAR were cytolytic to the HMW-MAA–positive melano‐ ma cells, produced cytokine and proliferate *in vitro* [39]. The potential clinical utility of the CAR-mediated HMW-MAA targeting was emphasized by another recent animal study [40]. Analysis of a few human melanoma biopsies revealed the presence of less than 2% of specific tumor cells co-expressing CD20 and HMW-MAA. Implantation of tumors containing these CD20+ HMW-MAA+ cells into immuno-deficient mice resulted in a rapid growth of tumors. Targeting of these pre-established lesions with T cells expressing either CD20 or HMW-MAAdirected CAR showed elimination of lesions in nearly 90% of treated animals. CD20-specific engineered T cells were unable to eradicated melanoma lesions artificially expressing CD20 suggesting that native expression of the antigen is required for effective targeting. These studies provided additional evidence that direction of the T cells toward HMW-MAA via genetic engineering can permit effective elimination of tumor lesions.

As progression of most tumors including melanoma depends on the microenvironment, T-cell mediated targeting of the microenvironmental components could also be a viable strategy for melanoma immunotherapy. Particularly, tumor survival was shown to be dependent on the *de novo* formation of the intratumoral blood vessels characterised by high levels of the vascular endothelial growth factor receptor 2 (VEGFR2/KDR). Also, a number of studies associated high levels of VEGFR2 expression with various tumor stroma cells including subsets of macro‐ phages, immature monocytes, immature dendritic cells and immuno-suppressive CD4+CD25+ regulatory T cells (Treg) [41-46]. Therefore, it was suggested that targeting of VEGFR2 – positive cells in tumor stroma may provide clinical benefits and tumor regression. In support of this notion, recent studies demonstrated that the direction of the T cells toward VEGFR-2 via CAR provide an effective means to eliminate pre-established experimental melanoma. Thus, using an animal model, it was shown that after systemic transplantation, anti–VEGFR-2 CAR and IL-12–co-transduced T cells infiltrated the tumors, expanded and persisted within tumor mass leading to tumor regression [47]. The anti-tumor effect was dependent on targeting of IL-12–responsive host cells via activation of anti–VEGFR-2 CAR-T cells and release of IL-12. Based on this data, one clinical trial aimed at the assessment of safety and effectiveness of cell therapy was initiated to treat recurrent and relapsed cancer by using anti-VEGFR2 CARmodified T cells.

Presently, there is an accumulating body of evidence suggesting clinical utility of the T cell genetically engineered to express melanoma antigen-specific CARs. It is likely that in the near future CAR-mediated targeting of different melanoma antigens will evolve into general practice of cancer immunotherapy.

## **3. DNA vaccination**

the CAR-expressing T cells and limit long-term clinical use [36, 37]. Nevertheless, existence of a large number of the tumor antigen-specific antibodies and robust anti-tumor response by CAR-expressing T cells suggest great clinical utility of these recombinant molecules. Currently, in US alone there are 18 clinical trials aimed at treatment of various malignancies with CARengineered leukocytes, with 16 trials in the recruitment phase. Eight of them are aimed at targeting different B cell malignancies with anti-CD19-CAR. Three trials are intended to test HER2-specific CAR-modified T cells for the treatment of sarcoma, glioblastoma, and advanced

With regard to melanoma, several CAR designs were tested for the ability to target this malignancy. Thus, recent studies demonstrated that treatment of melanoma xenografts in nude mice using engineered T cells expressing tandem CAR (CD28/TCRζ) specific to ganglio‐ side GD3 with IL2 supplementation led to complete remissions of the established tumors in 50 % of treated animals [38]. As GD3 is often over-expressed in melanoma, this approach could

Another attractive target for the CAR-mediated T cell therapy for melanoma is a high molec‐ ular weight melanoma-associated antigen (HMW-MAA) encoded by CSPG4 gene. This is a cell-surface proteoglycan expressed on more than 90% of the tumors. Recent studies on targeting of this antigen using CAR that is comprised of the anti- HMW-MAA antibody chain and intracellular signaling domains of the CD28, CD137, and CD3ζ demonstrated that T cell genetically modified to express this CAR were cytolytic to the HMW-MAA–positive melano‐ ma cells, produced cytokine and proliferate *in vitro* [39]. The potential clinical utility of the CAR-mediated HMW-MAA targeting was emphasized by another recent animal study [40]. Analysis of a few human melanoma biopsies revealed the presence of less than 2% of specific tumor cells co-expressing CD20 and HMW-MAA. Implantation of tumors containing these CD20+ HMW-MAA+ cells into immuno-deficient mice resulted in a rapid growth of tumors. Targeting of these pre-established lesions with T cells expressing either CD20 or HMW-MAAdirected CAR showed elimination of lesions in nearly 90% of treated animals. CD20-specific engineered T cells were unable to eradicated melanoma lesions artificially expressing CD20 suggesting that native expression of the antigen is required for effective targeting. These studies provided additional evidence that direction of the T cells toward HMW-MAA via

As progression of most tumors including melanoma depends on the microenvironment, T-cell mediated targeting of the microenvironmental components could also be a viable strategy for melanoma immunotherapy. Particularly, tumor survival was shown to be dependent on the *de novo* formation of the intratumoral blood vessels characterised by high levels of the vascular endothelial growth factor receptor 2 (VEGFR2/KDR). Also, a number of studies associated high levels of VEGFR2 expression with various tumor stroma cells including subsets of macro‐ phages, immature monocytes, immature dendritic cells and immuno-suppressive CD4+CD25+ regulatory T cells (Treg) [41-46]. Therefore, it was suggested that targeting of VEGFR2 – positive cells in tumor stroma may provide clinical benefits and tumor regression. In support of this notion, recent studies demonstrated that the direction of the T cells toward VEGFR-2 via CAR provide an effective means to eliminate pre-established experimental melanoma.

Her2-positive lung malignancy.

184 Melanoma - From Early Detection to Treatment

be potent in eliminating melanoma in human patients.

genetic engineering can permit effective elimination of tumor lesions.

Another immunotherapeutic approach directly relevant to recombinant DNA is genetic or DNA vaccination. The original idea of DNA vaccination emanated from the observations that intramuscular injection of DNA encoding influenza A virus protein resulted in the robust activation of the immune responses that protected the host from viral challenge [48]. Generally, DNA-mediated activation of immune response involves multiple processes. First, plasmid DNA should be delivered intracellularly and expressed in the host cells. Next, in most of cases the antigen has to be secreted from the cells and picked up by the dendritic cells (DC), processed and presented in the context of the MHC class II to the CD4+ T helper (Th) cells. Alternatively, if the antigen is expressed directly in the DCs, it could be processed intracellularly and presented via MHC class I molecules, leading to the activation of the CD8+ T cells and induction of the cytotoxic immune responses. Initial studies on DNA vaccination were carried out using an intramuscular route of vaccine administration (Fig. 6). This allowed high levels of antigen expression and secretion from the elongated muscle cells into perimysium, the resident site of the intramuscular DCs. Later, DNA vaccination through the skin was suggested to be superior over the intramuscular route. Skin has evolved as a barrier to prevent the entry of pathogens, with efficient immune surveillance complex including Langerhans cells, dendritic cells, lymphocytes, and other leukocytes. Skin is also rich in lymphatic vasculature network that provides an efficient route for DC and T cell trafficking. Depending on the physical methods of into-skin DNA delivery, DNA-based vaccines can be targeted to specific locations in the skin [49].

The DNA vaccination approach has several advantages over other types of vaccinations: (i) multiple expression vectors coding for different antigen and co-stimulatory molecules can be concurrently delivered into the skin (or the muscle); (ii) the use of cell-type-specific promoters can provide specificity of protein expression; (iii) protein expression from designed plasmids can be controlled by inducible promoters, the use of ubiquitous chromatin opening elements (UCOE), or chemically (e.g. sodium butyrate). Also of note is the relative simplicity and inexpensiveness of the cGMP grade DNA vaccine production and pre-clinical testing. These

Melanoma DNA Vaccine. Up to date, ONCEPT is the first and only USDA-approved thera‐ peutic vaccine for the treatment of cancer in either animals or humans. (The first DNA vaccine was licensed by the USDA in 2005 for prevention of West Nile virus infection in horses).

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However, presently only a few human clinical trials on DNA vaccination were conducted. One of such study, aimed at the evaluation of the immune response in patients with hormonerefractory prostate cancer showed that DNA vaccination with a prostate-specific antigen (PSA) encoding plasmid given with GM-CSF and IL-2 is safe in doses of up to 900 μg, and that the

Similar to the reference above canine studies, DNA vaccines were shown to be effective in mouse melanoma models when mice were vaccinated with heterologous DNA encoding human melanoma-associated antigen gp-100 [53]. This vaccination regimen was augmented by the GM-CSF and was most effective in the prophylactic setting. It was also effective in suppressing pre-established melanoma. However, vaccinations with autologous mouse melanoma antigens were less successful. Nevertheless, the relative simplicity of modifying recombinant DNA allowed testing of various genetic alterations aimed at breaking the immunologic tolerance and enhancing immune responses to DNA vaccines. For example, concurrent vaccination with DNA encoding several melanoma-specific epitopes can be used. This approach was tested in several studies with different degree of success. As a result, vaccination of mice with gp10025–33 and TRP-2181–188 encoding minigene was effective in preventing melanoma development [54]. As many of the melanoma MHC class I epitopes were characterized for melanoma including those derived from tyrosinase, TRP1, TRP2, gp-100, MART-1, and MC-1R (some of them shared between mouse and human MHC molecules [55], one can envision generation of an ultimate genetic immunogen capable of targeting several

Recombinant DNA technology has also allowed introduction of immuno-augmentation sequences into the DNA vaccine composition. Identified universal pan HLA DR helper binding *e*pitope (PADRE; KXVAAWYLKA) was shown to enhance immunogenicity of both peptide and DNA vaccines [56, 57]. Other studies demonstrated augmentation of melanoma-specific immune responses via direct fusion of the DNA vaccine with the VP22 protein of the herpes

Besides introducing immuno-enhancing alteration to the DNA vaccine, other strategies could be employed to enhance DNA vaccination efficacy including addition of the immunoenhancing molecules to vaccine composition, alteration of the microenvironment at vaccine administration site, and use of the prime-boost immunization regimens. Recent studies demonstrated that antibody-mediated inhibition of the cytotoxic T lymphocyte antigen 4 (CTLA-4) enhances melanoma-specific immune response. This strategy was recently tested in treatment of stage III-IV melanoma and the drug (Ipilimumam) was approved by the FDA as first anti-melanoma immunotherapeutic [59, 60]. CTLA-4 presents its immuno-inhibitory function during activation of the T cells by the antigen-presenting cells. It also inhibits TCRmediated intracellular signaling in activated T cells and down-modulating T cell mediated immunity. Therefore, it is possible that inhibition of CTLA-4 in conjunction with DNA vaccination may provide significant enhancement of the vaccine-mediated immune response

vaccination can induce cellular and humoral immune responses [52].

melanoma-associated antigens.

simplex virus-1 [58].

**Figure 6.** Intramuscular and Intradermal DNA vaccination. Intramuscular and intradermal sites are used for DNA vacci‐ nation. The former allows high level of antigen (Ag) expression in muscle cells and MHC class II Ag processing and presentation whereas the latter permits expression of the Ag in the Antigen-Presenting Cells (APC) and direct presen‐ tation of the antigenic peptides to the CD8+ cytotoxic T cells (see text for details).

attractive characteristics of DNA vaccines have prompted extensive research within the past 10 years.

Multiple studies on pre-clinical animal models of melanoma and other cancers have been conducted. Studies on the canine model of aggressive and metastatic melanoma (stages II-IV) demonstrated that xenogeneic vaccination of dogs with DNA vaccine coding for human tyrosinase led to an excellent clinical response in the majority of vaccinated dogs. A long-term survival of dogs with advanced stage IV disease with bulky lung metastases (on average 400 days) was observed [50]. Vaccinated dogs with stage II/III disease also had long-term survivals (on average 500 days) with no evidence of melanoma on necropsy. Overall, median survival time for all treated dogs was 389 days. Another canine model study [51] showed that xenoge‐ neic DNA vaccination induces melanoma-specific antibody response, which coincides with observed clinical responses. As a result, in 2010 Merial, an animal health company has gained full-licensure from the U.S. Department of Agriculture (USDA) for ONCEPT™ Canine Melanoma DNA Vaccine. Up to date, ONCEPT is the first and only USDA-approved thera‐ peutic vaccine for the treatment of cancer in either animals or humans. (The first DNA vaccine was licensed by the USDA in 2005 for prevention of West Nile virus infection in horses).

However, presently only a few human clinical trials on DNA vaccination were conducted. One of such study, aimed at the evaluation of the immune response in patients with hormonerefractory prostate cancer showed that DNA vaccination with a prostate-specific antigen (PSA) encoding plasmid given with GM-CSF and IL-2 is safe in doses of up to 900 μg, and that the vaccination can induce cellular and humoral immune responses [52].

Similar to the reference above canine studies, DNA vaccines were shown to be effective in mouse melanoma models when mice were vaccinated with heterologous DNA encoding human melanoma-associated antigen gp-100 [53]. This vaccination regimen was augmented by the GM-CSF and was most effective in the prophylactic setting. It was also effective in suppressing pre-established melanoma. However, vaccinations with autologous mouse melanoma antigens were less successful. Nevertheless, the relative simplicity of modifying recombinant DNA allowed testing of various genetic alterations aimed at breaking the immunologic tolerance and enhancing immune responses to DNA vaccines. For example, concurrent vaccination with DNA encoding several melanoma-specific epitopes can be used. This approach was tested in several studies with different degree of success. As a result, vaccination of mice with gp10025–33 and TRP-2181–188 encoding minigene was effective in preventing melanoma development [54]. As many of the melanoma MHC class I epitopes were characterized for melanoma including those derived from tyrosinase, TRP1, TRP2, gp-100, MART-1, and MC-1R (some of them shared between mouse and human MHC molecules [55], one can envision generation of an ultimate genetic immunogen capable of targeting several melanoma-associated antigens.

Recombinant DNA technology has also allowed introduction of immuno-augmentation sequences into the DNA vaccine composition. Identified universal pan HLA DR helper binding *e*pitope (PADRE; KXVAAWYLKA) was shown to enhance immunogenicity of both peptide and DNA vaccines [56, 57]. Other studies demonstrated augmentation of melanoma-specific immune responses via direct fusion of the DNA vaccine with the VP22 protein of the herpes simplex virus-1 [58].

attractive characteristics of DNA vaccines have prompted extensive research within the past

tation of the antigenic peptides to the CD8+ cytotoxic T cells (see text for details).

**Figure 6.** Intramuscular and Intradermal DNA vaccination. Intramuscular and intradermal sites are used for DNA vacci‐ nation. The former allows high level of antigen (Ag) expression in muscle cells and MHC class II Ag processing and presentation whereas the latter permits expression of the Ag in the Antigen-Presenting Cells (APC) and direct presen‐

Multiple studies on pre-clinical animal models of melanoma and other cancers have been conducted. Studies on the canine model of aggressive and metastatic melanoma (stages II-IV) demonstrated that xenogeneic vaccination of dogs with DNA vaccine coding for human tyrosinase led to an excellent clinical response in the majority of vaccinated dogs. A long-term survival of dogs with advanced stage IV disease with bulky lung metastases (on average 400 days) was observed [50]. Vaccinated dogs with stage II/III disease also had long-term survivals (on average 500 days) with no evidence of melanoma on necropsy. Overall, median survival time for all treated dogs was 389 days. Another canine model study [51] showed that xenoge‐ neic DNA vaccination induces melanoma-specific antibody response, which coincides with observed clinical responses. As a result, in 2010 Merial, an animal health company has gained full-licensure from the U.S. Department of Agriculture (USDA) for ONCEPT™ Canine

10 years.

186 Melanoma - From Early Detection to Treatment

Besides introducing immuno-enhancing alteration to the DNA vaccine, other strategies could be employed to enhance DNA vaccination efficacy including addition of the immunoenhancing molecules to vaccine composition, alteration of the microenvironment at vaccine administration site, and use of the prime-boost immunization regimens. Recent studies demonstrated that antibody-mediated inhibition of the cytotoxic T lymphocyte antigen 4 (CTLA-4) enhances melanoma-specific immune response. This strategy was recently tested in treatment of stage III-IV melanoma and the drug (Ipilimumam) was approved by the FDA as first anti-melanoma immunotherapeutic [59, 60]. CTLA-4 presents its immuno-inhibitory function during activation of the T cells by the antigen-presenting cells. It also inhibits TCRmediated intracellular signaling in activated T cells and down-modulating T cell mediated immunity. Therefore, it is possible that inhibition of CTLA-4 in conjunction with DNA vaccination may provide significant enhancement of the vaccine-mediated immune response induction. Although providing CTLA-4 inhibiting antibodies like Ipilimumab along with DNA vaccination is not feasible, other options could be explored. For example, recently characterized genetically engineered lipocalin (LCN2) exhibits a strong cross-species antago‐ nistic activity to CTLA-4 [61]. It is likely that this molecule could be included into DNA vaccine composition to enhance DC-mediated activation of the T cells. Other immuno-modulatory strategies may include addition of CD40 ligand, which was shown to stimulate expression of maturation markers CD80, CD86 and IL-12 in APC [62, 63] and its ability to activate CD8+ T cells and increase cell-mediated immunity [64, 65]. Addition of different cytokines and growth factors including GM-CSF, IL-2, IL12 for stimulation/support of the T cells was also tested in several studies (as exemplified in preceding paragraphs) and could be further explored. Alteration of microenvironment via application chemokines to recruit specific sets of the leukocytes to the vaccine administration site may also provide a favorable milieu for the launch of the effective DNA-vaccine induced immune response [66, 67]. These and many other strategies can be proposed; however, the clinical utility of the DNA vaccination combination with other approaches remains to be determined. Nevertheless, presently in the US alone, 10 clinical studies utilizing xenogenic (mouse) or human DNA vaccines coding for melanoma associated antigens have been completed. In these trials, tyrosinase, gp75, gp100, and TRP2 were used as antigens. Although most of these studies are already completed, currently no study results are posted nor are follow-up reports available on patient survival and charac‐ terization of immune response. Nevertheless, DNA vaccination remains to be a promising modality that could provide cost-effective and generic immunotherapy for patients with melanoma and other cancers.

using CCL21 transduced DCs pulsed with MART-1 and gp100 was completed in 2012. Altogether, a total of 64 clinical trials aimed at targeting of melanoma using dendritic cells are listed. Thirty nine of them are completed with no reports yet available. The majority of these

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During last decade, various melanoma-specific immunotherapeutics that utilize recombinant DNA have been developed and tested in pre-clinical and clinical studies with varying degrees of clinical success. Many of these approaches, including recombinant TCRs and CARs, have already demonstrated promising clinical results, thus providing us with the hope that in the

Presented here data regarding clinical trials was obtained through registry and results database of publicly and privately supported clinical studies (clinicaltrials.gov). Experimental data presented on Fig. 4 was obtained in Dr. Alexeev's laboratory. Plasmid DNA encoding tyrosinase-specific TCR was kindly provided by Dr. S.A. Rosenberg (NCI, NIH). ΦC31 integrase-encoding plasmid was obtained from Dr. M.P. Calos (Stanford University, CA).

near future melanoma immunotherapy will become curable for melanoma patients.

, Daria Marley Kemp1

2 Temple University School of Medicine, Philadelphia, Pennsylvania, USA

*Academy of Sciences of the United States of America*, 86, 2804-2808.

1 Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas

[1] Knuth, A., Wolfel, T., Klehmann, E., Boon, T. and Meyer zum Buschenfelde, K.H. (1989) Cytolytic T-cell clones against an autologous human melanoma: specificity study and definition of three antigens by immunoselection. *Proceedings of the National*

\*Address all correspondence to: vitali.alexeev@jefferson.edu

Jefferson University, Philadelphia, Pennsylvania, USA

and Olga Igoucheva1

trials in some way utilize recombinant DNA technology.

**5. Conclusion**

**Acknowledgements**

**Author details**

**References**

Vitali Alexeev1\*, Alyson Pidich2

## **4. Other strategies involving recombinant DNA technology**

At the present time, almost every immuno-therapeutic approach utilizes recombinant DNA in one way or another. Understanding of the immuno-regulatory functions of DCs and the molecular mechanisms involved in the capture, processing and presentation of antigens by DCs allowed the development of the DC-based vaccines. Initially, in the mid 1990's, several pre-clinical and clinical studies were conducted using autologous DCs pulsed with melanomaassociated antigens. These studies demonstrated that antigen-loaded DC can trigger active melanoma specific immune responses [68, 69]. However, it became apparent that enforced expression of the antigens in DC rather than loading of these cells with peptides allows for presentation of the tumor-derived antigens via MHC class I complex and priming of the CD8+ T cells to elicit cytotoxic immune response. Moreover, to provide DC specific expression of the antigens, long and short CD11c promoters were characterized and used in several studies [70, 71]. These promoters allow effective and cell type specific expression of the antigens in DCs, as well as more efficient priming and activation of the T cells *in vitro* and *in vivo*. Considering a necessity of the direct interaction of the DC with T lymphocytes, application of T cell recruiting chemokines was also explored recently. These pioneering studies demonstrate that forced expression of the secondary lymphoid chemokine, CCL21, in antigen loaded DCs enhances their ability to recruit and activate T cells [72, 73]. One clinical phase I clinical trial using CCL21 transduced DCs pulsed with MART-1 and gp100 was completed in 2012. Altogether, a total of 64 clinical trials aimed at targeting of melanoma using dendritic cells are listed. Thirty nine of them are completed with no reports yet available. The majority of these trials in some way utilize recombinant DNA technology.

## **5. Conclusion**

induction. Although providing CTLA-4 inhibiting antibodies like Ipilimumab along with DNA vaccination is not feasible, other options could be explored. For example, recently characterized genetically engineered lipocalin (LCN2) exhibits a strong cross-species antago‐ nistic activity to CTLA-4 [61]. It is likely that this molecule could be included into DNA vaccine composition to enhance DC-mediated activation of the T cells. Other immuno-modulatory strategies may include addition of CD40 ligand, which was shown to stimulate expression of maturation markers CD80, CD86 and IL-12 in APC [62, 63] and its ability to activate CD8+ T cells and increase cell-mediated immunity [64, 65]. Addition of different cytokines and growth factors including GM-CSF, IL-2, IL12 for stimulation/support of the T cells was also tested in several studies (as exemplified in preceding paragraphs) and could be further explored. Alteration of microenvironment via application chemokines to recruit specific sets of the leukocytes to the vaccine administration site may also provide a favorable milieu for the launch of the effective DNA-vaccine induced immune response [66, 67]. These and many other strategies can be proposed; however, the clinical utility of the DNA vaccination combination with other approaches remains to be determined. Nevertheless, presently in the US alone, 10 clinical studies utilizing xenogenic (mouse) or human DNA vaccines coding for melanoma associated antigens have been completed. In these trials, tyrosinase, gp75, gp100, and TRP2 were used as antigens. Although most of these studies are already completed, currently no study results are posted nor are follow-up reports available on patient survival and charac‐ terization of immune response. Nevertheless, DNA vaccination remains to be a promising modality that could provide cost-effective and generic immunotherapy for patients with

**4. Other strategies involving recombinant DNA technology**

At the present time, almost every immuno-therapeutic approach utilizes recombinant DNA in one way or another. Understanding of the immuno-regulatory functions of DCs and the molecular mechanisms involved in the capture, processing and presentation of antigens by DCs allowed the development of the DC-based vaccines. Initially, in the mid 1990's, several pre-clinical and clinical studies were conducted using autologous DCs pulsed with melanomaassociated antigens. These studies demonstrated that antigen-loaded DC can trigger active melanoma specific immune responses [68, 69]. However, it became apparent that enforced expression of the antigens in DC rather than loading of these cells with peptides allows for presentation of the tumor-derived antigens via MHC class I complex and priming of the CD8+ T cells to elicit cytotoxic immune response. Moreover, to provide DC specific expression of the antigens, long and short CD11c promoters were characterized and used in several studies [70, 71]. These promoters allow effective and cell type specific expression of the antigens in DCs, as well as more efficient priming and activation of the T cells *in vitro* and *in vivo*. Considering a necessity of the direct interaction of the DC with T lymphocytes, application of T cell recruiting chemokines was also explored recently. These pioneering studies demonstrate that forced expression of the secondary lymphoid chemokine, CCL21, in antigen loaded DCs enhances their ability to recruit and activate T cells [72, 73]. One clinical phase I clinical trial

melanoma and other cancers.

188 Melanoma - From Early Detection to Treatment

During last decade, various melanoma-specific immunotherapeutics that utilize recombinant DNA have been developed and tested in pre-clinical and clinical studies with varying degrees of clinical success. Many of these approaches, including recombinant TCRs and CARs, have already demonstrated promising clinical results, thus providing us with the hope that in the near future melanoma immunotherapy will become curable for melanoma patients.

## **Acknowledgements**

Presented here data regarding clinical trials was obtained through registry and results database of publicly and privately supported clinical studies (clinicaltrials.gov). Experimental data presented on Fig. 4 was obtained in Dr. Alexeev's laboratory. Plasmid DNA encoding tyrosinase-specific TCR was kindly provided by Dr. S.A. Rosenberg (NCI, NIH). ΦC31 integrase-encoding plasmid was obtained from Dr. M.P. Calos (Stanford University, CA).

## **Author details**

Vitali Alexeev1\*, Alyson Pidich2 , Daria Marley Kemp1 and Olga Igoucheva1

\*Address all correspondence to: vitali.alexeev@jefferson.edu

1 Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA

2 Temple University School of Medicine, Philadelphia, Pennsylvania, USA

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**Chapter 7**

**Acquired Resistance to Targeted**

Kavitha Gowrishankar, Matteo S. Carlino and

Additional information is available at the end of the chapter

tions in the BRAF kinase or its upstream regulator, N-RAS [5, 6].

The worldwide incidence of cutaneous melanoma has steadily increased in fair-skinned in‐ dividuals over recent decades with estimates suggesting a doubling of melanoma incidence every 10-20 years [1]. Melanoma remains the major cause of skin cancer related deaths [2], with survival rates averaging less than six months for patients with metastases in visceral organs [3]. Conventional systemic therapies, including single agent dacarbazine and temo‐ zolamide, produce response rates of less than 10%, and are not proven to improve survival (reviewed in [4]). Recently, however, the treatment of melanoma has been revolutionized by therapies targeting the RAF-MEK-ERK mitogen activated protein kinase (MAPK) pathway. This pathway is constitutively activated in the majority of melanomas via oncogenic muta‐

Most BRAF mutations produce a single amino acid substitution of valine by glutamic acid at amino acid 600 (V600E), and this leads to a 500-fold increase in kinase activity [5, 7]. Target‐ ing this mutant BRAF with the highly specific inhibitors, vemurafenib (PLX4032) and dabra‐ fenib (GSK2118436) has produced response rates above 50% and improved progression-free survival in patients with BRAF-mutant metastatic melanoma [8-12]. Both BRAF inhibitors are active against melanoma brain metastases [13, 14] and vemurafenib treatment prolongs overall survival compared with dacarbazine [11]. Despite the marked initial responses to BRAF inhibitors, tumor re-growth occurs in most patients with a median progression-free

The U.S Food and Drug Administration (FDA) approved the use of vemurafenib for the treatment of BRAF-mutant melanoma in 2011, and submissions for the use of dabrafenib in

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

© 2013 Gowrishankar 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,

Helen Rizos

**1. Introduction**

http://dx.doi.org/10.5772/53629

survival of 5 to 6 months [8, 11, 15].

**MAPK Inhibition in Melanoma**


**Chapter 7**

## **Acquired Resistance to Targeted MAPK Inhibition in Melanoma**

Kavitha Gowrishankar, Matteo S. Carlino and Helen Rizos

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53629

## **1. Introduction**

with a recipient (autologous) vaccine expressing transgenic human CD40L and IL-2 after chemotherapy and allogeneic stem cell transplantation. *Blood*, 107, 1332-1341. [66] Guo, J.H., Fan, M.W., Sun, J.H. and Jia, R. (2009) Fusion of antigen to chemokine CCL20 or CXCL13 strategy to enhance DNA vaccine potency. *International immuno‐*

[67] Novak, L., Igoucheva, O., Cho, S. and Alexeev, V. (2007) Characterization of the CCL21-mediated melanoma-specific immune responses and in situ melanoma eradi‐

[68] Nestle, F.O., Alijagic, S., Gilliet, M., Sun, Y., Grabbe, S., Dummer, R., Burg, G. and Schadendorf, D. (1998) Vaccination of melanoma patients with peptide- or tumor ly‐

[69] 69.Thurner, B., Haendle, I., Roder, C., Dieckmann, D., Keikavoussi, P., Jonuleit, H., Bender, A., Maczek, C., Schreiner, D., von den Driesch, P. *et al.* (1999) Vaccination with mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced

[70] Brocker, T., Riedinger, M. and Karjalainen, K. (1997) Targeted expression of major histocompatibility complex (MHC) class II molecules demonstrates that dendritic cells can induce negative but not positive selection of thymocytes in vivo. *The Journal*

[71] Ni, J., Nolte, B., Arnold, A., Fournier, P. and Schirrmacher, V. (2009) Targeting antitumor DNA vaccines to dendritic cells via a short CD11c promoter sequence. *Vaccine*,

[72] Mule, J.J. (2009) Dendritic cell-based vaccines for pancreatic cancer and melanoma.

[73] Terando, A., Roessler, B. and Mule, J.J. (2004) Chemokine gene modification of hu‐ man dendritic cell-based tumor vaccines using a recombinant adenoviral vector. *Can‐*

stage IV melanoma. *The Journal of experimental medicine*, 190, 1669-1678.

*pharmacology*, 9, 925-930.

196 Melanoma - From Early Detection to Treatment

cation. *Molecular cancer therapeutics*, 6, 1755-1764.

*of experimental medicine*, 185, 541-550.

*cer gene therapy*, 11, 165-173.

27, 5480-5487.

sate-pulsed dendritic cells. *Nature medicine*, 4, 328-332.

*Annals of the New York Academy of Sciences*, 1174, 33-40.

The worldwide incidence of cutaneous melanoma has steadily increased in fair-skinned in‐ dividuals over recent decades with estimates suggesting a doubling of melanoma incidence every 10-20 years [1]. Melanoma remains the major cause of skin cancer related deaths [2], with survival rates averaging less than six months for patients with metastases in visceral organs [3]. Conventional systemic therapies, including single agent dacarbazine and temo‐ zolamide, produce response rates of less than 10%, and are not proven to improve survival (reviewed in [4]). Recently, however, the treatment of melanoma has been revolutionized by therapies targeting the RAF-MEK-ERK mitogen activated protein kinase (MAPK) pathway. This pathway is constitutively activated in the majority of melanomas via oncogenic muta‐ tions in the BRAF kinase or its upstream regulator, N-RAS [5, 6].

Most BRAF mutations produce a single amino acid substitution of valine by glutamic acid at amino acid 600 (V600E), and this leads to a 500-fold increase in kinase activity [5, 7]. Target‐ ing this mutant BRAF with the highly specific inhibitors, vemurafenib (PLX4032) and dabra‐ fenib (GSK2118436) has produced response rates above 50% and improved progression-free survival in patients with BRAF-mutant metastatic melanoma [8-12]. Both BRAF inhibitors are active against melanoma brain metastases [13, 14] and vemurafenib treatment prolongs overall survival compared with dacarbazine [11]. Despite the marked initial responses to BRAF inhibitors, tumor re-growth occurs in most patients with a median progression-free survival of 5 to 6 months [8, 11, 15].

The U.S Food and Drug Administration (FDA) approved the use of vemurafenib for the treatment of BRAF-mutant melanoma in 2011, and submissions for the use of dabrafenib in

© 2013 Gowrishankar 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 treatment of BRAF-mutant melanoma were made in late 2012. We are now beginning to understand the complex pathways regulating the response and side-effect profiles of these targeted inhibitors. The challenge is to define the molecular drivers and pathways of resist‐ ance and response and to translate these molecular findings into rational strategies for clini‐ cal testing and improved therapies. In the following chapter we describe the molecular mechanisms that contribute to BRAF inhibitor resistance *in vitro* and *in vivo*. We also high‐ light the current strategies employed to dissect resistance drivers and explore the future of targeted therapies in the long-term treatment of melanoma.

observed with trastuzumab in HER2-amplified breast cancer, imatinib in gastrointestinal stromal tumors (GISTs) and chronic myelogenous leukemia (CML), epidermal growth factor receptor (EGFR) inhibitors in lung cancer and hedgehog inhibitors in medulloblastoma [27]. Resistance mechanisms to these drugs are complex but include the acquisition of secondary mutations in the target oncogene that prevent drug binding, up-regulation of signalling pathways downstream of the target and the induction of alternate, secondary survival path‐ ways. Defining the mechanisms of melanoma resistance to targeted inhibitors is a high pri‐ ority, as it can guide the selection of appropriate drug combinations and advance the development of new and improved drugs. This is best demonstrated for imatinib-resistant leukemias. The identification of secondary Bcr-Abl mutations in these resistant cancers pro‐ moted the development of the potent, next-generation receptor tyrosine kinase inhibitors

Acquired Resistance to Targeted MAPK Inhibition in Melanoma

http://dx.doi.org/10.5772/53629

199

**Figure 1. MAPK signalling cascade.** Activation of the RAS GTPase promotes the kinase activity of the RAF serine/ threonine protein kinases, ARAF, BRAF and CRAF. Activated RAF kinases promote the sequential phosphorylation and activation of the MEK1/2 and ERK1/2 kinases. The ERK proteins translocate into the nucleus and stimulate the transla‐ tion of proteins and the activities of many transcription factors. This leads to a series of gene expression changes, in‐ cluding elevated CCND1 that promotes cell proliferation and survival. Specific inhibitors to RAF and MEK kinases are indicated. ARAF, v-raf murine sarcoma 3611 viral oncogene homolog; BRAF, v-raf murine sarcoma viral oncogene B1; CCND1, cyclin D1; CRAF, v-raf-1 murine leukemia viral oncogene homolog 1; ERK, extracellular signal-regulated kin‐

dasatanib and nilotinib [28].

ase; MEK mitogen-activated protein kinase kinase.

## **2. The BRAF kinase and the MAPK pathway**

Aberrant activation of the MAPK pathway is present in over 80% of primary cutaneous mel‐ anomas [16]. MAPK signalling is driven by mutated N-RAS and activating mutations in the downstream RAS effector, BRAF, in 20% and 60% of melanomas, respectively (Figure 1) [16]. Of cutaneous melanomas with no mutations in BRAF or N-RAS, many activate MAPK signalling via oncogenic mutations in the receptor tyrosine kinase, c-Kit [17], activating mu‐ tations in the Rac1 GTPase or inactivating mutations in the N-RAS inhibitor NF1 [18].

Among the BRAF mutations identified in melanoma, over 80% involve a single nucleotide mutation resulting in the substitution of valine for glutamic acid at amino acid 600. This mu‐ tation is also present in up to 80% of benign, growth-arrested nevi [19], implicating BRAF as an initiating event that co-operates with additional genetic lesions to promote melanoma. Over 60 other mutations in BRAF have been described in melanoma; most affect codon 600 (V600E, V600K, V600R and V600D), lie within the kinase domain and show elevated kinase activity. In particular, alterations affecting codon 600 show 150- (BRAFV600K) to 700- (BRAFV600D) fold more kinase activity than the wild type BRAF protein [7].

A wealth of preclinical data has demonstrated the critical role of BRAFV600E as an oncogene in melanoma. The specific silencing of BRAF with short interfering (si)RNA resulted in de‐ creased ERK signalling, diminished proliferation and regression of BRAF mutant melano‐ mas [20-23]. More importantly, class I RAF inhibitors, which target the activated form of RAF kinases, show remarkable antitumor activity; both vemurafenib and dabrafenib have shown response rates of 50% in patients with BRAF-mutant melanoma [8-12]. In addition, the selective inhibition of the BRAF target proteins, MEK1/2, with trametinib (GSK11202212) improved rates of progression-free and overall survival amongst patients with BRAF mu‐ tant melanoma when compared to dacarbazine [9, 15, 24, 25].

## **3. Mechanisms of acquired BRAF inhibitor resistance in melanoma**

Despite the marked initial responses to single-agent BRAF inhibitors, tumor re-growth oc‐ curs in most patients and 5-20% of individuals fail to respond early during treatment [8, 10, 11, 26]. The acquisition of resistance to targeted therapy is common and resistance has been observed with trastuzumab in HER2-amplified breast cancer, imatinib in gastrointestinal stromal tumors (GISTs) and chronic myelogenous leukemia (CML), epidermal growth factor receptor (EGFR) inhibitors in lung cancer and hedgehog inhibitors in medulloblastoma [27]. Resistance mechanisms to these drugs are complex but include the acquisition of secondary mutations in the target oncogene that prevent drug binding, up-regulation of signalling pathways downstream of the target and the induction of alternate, secondary survival path‐ ways. Defining the mechanisms of melanoma resistance to targeted inhibitors is a high pri‐ ority, as it can guide the selection of appropriate drug combinations and advance the development of new and improved drugs. This is best demonstrated for imatinib-resistant leukemias. The identification of secondary Bcr-Abl mutations in these resistant cancers pro‐ moted the development of the potent, next-generation receptor tyrosine kinase inhibitors dasatanib and nilotinib [28].

the treatment of BRAF-mutant melanoma were made in late 2012. We are now beginning to understand the complex pathways regulating the response and side-effect profiles of these targeted inhibitors. The challenge is to define the molecular drivers and pathways of resist‐ ance and response and to translate these molecular findings into rational strategies for clini‐ cal testing and improved therapies. In the following chapter we describe the molecular mechanisms that contribute to BRAF inhibitor resistance *in vitro* and *in vivo*. We also high‐ light the current strategies employed to dissect resistance drivers and explore the future of

Aberrant activation of the MAPK pathway is present in over 80% of primary cutaneous mel‐ anomas [16]. MAPK signalling is driven by mutated N-RAS and activating mutations in the downstream RAS effector, BRAF, in 20% and 60% of melanomas, respectively (Figure 1) [16]. Of cutaneous melanomas with no mutations in BRAF or N-RAS, many activate MAPK signalling via oncogenic mutations in the receptor tyrosine kinase, c-Kit [17], activating mu‐

Among the BRAF mutations identified in melanoma, over 80% involve a single nucleotide mutation resulting in the substitution of valine for glutamic acid at amino acid 600. This mu‐ tation is also present in up to 80% of benign, growth-arrested nevi [19], implicating BRAF as an initiating event that co-operates with additional genetic lesions to promote melanoma. Over 60 other mutations in BRAF have been described in melanoma; most affect codon 600 (V600E, V600K, V600R and V600D), lie within the kinase domain and show elevated kinase activity. In particular, alterations affecting codon 600 show 150- (BRAFV600K) to 700-

A wealth of preclinical data has demonstrated the critical role of BRAFV600E as an oncogene in melanoma. The specific silencing of BRAF with short interfering (si)RNA resulted in de‐ creased ERK signalling, diminished proliferation and regression of BRAF mutant melano‐ mas [20-23]. More importantly, class I RAF inhibitors, which target the activated form of RAF kinases, show remarkable antitumor activity; both vemurafenib and dabrafenib have shown response rates of 50% in patients with BRAF-mutant melanoma [8-12]. In addition, the selective inhibition of the BRAF target proteins, MEK1/2, with trametinib (GSK11202212) improved rates of progression-free and overall survival amongst patients with BRAF mu‐

**3. Mechanisms of acquired BRAF inhibitor resistance in melanoma**

Despite the marked initial responses to single-agent BRAF inhibitors, tumor re-growth oc‐ curs in most patients and 5-20% of individuals fail to respond early during treatment [8, 10, 11, 26]. The acquisition of resistance to targeted therapy is common and resistance has been

tations in the Rac1 GTPase or inactivating mutations in the N-RAS inhibitor NF1 [18].

(BRAFV600D) fold more kinase activity than the wild type BRAF protein [7].

tant melanoma when compared to dacarbazine [9, 15, 24, 25].

targeted therapies in the long-term treatment of melanoma.

198 Melanoma - From Early Detection to Treatment

**2. The BRAF kinase and the MAPK pathway**

**Figure 1. MAPK signalling cascade.** Activation of the RAS GTPase promotes the kinase activity of the RAF serine/ threonine protein kinases, ARAF, BRAF and CRAF. Activated RAF kinases promote the sequential phosphorylation and activation of the MEK1/2 and ERK1/2 kinases. The ERK proteins translocate into the nucleus and stimulate the transla‐ tion of proteins and the activities of many transcription factors. This leads to a series of gene expression changes, in‐ cluding elevated CCND1 that promotes cell proliferation and survival. Specific inhibitors to RAF and MEK kinases are indicated. ARAF, v-raf murine sarcoma 3611 viral oncogene homolog; BRAF, v-raf murine sarcoma viral oncogene B1; CCND1, cyclin D1; CRAF, v-raf-1 murine leukemia viral oncogene homolog 1; ERK, extracellular signal-regulated kin‐ ase; MEK mitogen-activated protein kinase kinase.

## **4. Alterations affecting BRAF**

Drug resistance is often associated with the acquisition of so-called gatekeeper mutations in the target oncogene that prevent drug binding. In a series of detailed reports, deep sequenc‐ ing of melanoma biopsies derived from patients progressing on vemurafenib treatment did not find any secondary BRAF mutations. Moreover, immunoprecipitated BRAF from ve‐ murafenib-resistant melanomas retained drug sensitivity in an *in vitro* kinase assay, confirm‐ ing drug-target binding was maintained [29].

**4.2. BRAF splicing variants**

In other melanomas, resistance to vemurafenib was acquired via the expression of splice variant isoforms of BRAFV600E. Three of five vemurafenib-resistant clones of the SKMEL-238 melanoma cells expressed a novel 61kDa variant of BRAFV600E. This p61BRAFV600E splice var‐ iant, lacked exons 4-8, a region encoding the RAS binding domain, and was sufficient to ren‐ der MEK activation resistant to vemurafenib (Figure 3). The variant appears to arise from a splicing defect as no intragenic somatic deletions within the BRAF gene were detected [33].

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201

**Figure 2. Mechanisms of resistance to BRAF inhibition**. MAPK re-activation, in the presence of RAF inhibitors, can occur via A. the mutational activation and amplification of RAS, B. the upregulation of RTKs such as PDGFRß and IGF-1R, C. elevated expression of CRAF, COT or CCND1, D. MEK mutations, or E. the expression and dimerization of BRAFV600E splice variants, such as p61BRAFV600E. Mutant RAS and upregulated RTKs also activate the PI3K/mTOR sur‐ vival pathway, which is further activated by the loss of PTEN (adapted from [87]). AKT, v-akt murine thymoma viral oncogene; BRAF, v-raf murine sarcoma viral oncogene B1; CCND1, cyclin D1; CRAF, v-raf-1 murine leukemia viral onco‐ gene homolog 1; ERK, extracellular signal-regulated kinase; COT, mitogen-activated protein kinase kinase kinase 8; MEK mitogen-activated protein kinase kinase; mTOR, mechanistic target of rapamycin; PI3K, phosphoinositide-3-kin‐

ase PTEN, phosphatase and tensin homolog; RTK, receptor tyrosine kinase.

#### **4.1. BRAF copy number gain**

The amplification and overexpression of BRAFV600E is associated with BRAF inhibitor re‐ sistance in melanoma [30] (Figure 2). In a sequencing screen of 20 pairs of patient-match‐ ed baseline (before BRAF inhibitor therapy) and progressing (acquired resistance to BRAF inhibition) melanoma tissue, 20% showed BRAFV600E copy-number gains, ranging from 2- to 14- fold. These copy-number gains, which are likely underestimates due to non-tumor cell contamination, correlated with increased BRAF protein expression in tu‐ mor specimens. Moreover, preclinical melanoma cell models with ectopically expressed BRAFV600E confirmed that cells overexpressing mutant BRAF developed resistance to ve‐ murafenib and that this resistance could be overcome by increasing the dose of vemura‐ fenib, applying MEK inhibitors (AZD6244) or concurrently inhibiting both MEK and BRAF (Figure 1) [30].

Unlike melanoma cell models [30], BRAF-mutant colorectal cancer cells with amplification of the BRAF gene (2- to 7- fold) were resistance to the MEK inhibitor AZD6244 [31]. In these colon cancer cells, the increased expression of mutant BRAF resulted in excess activation of MEK and ERK, rendering cells unresponsive to MEK inhibition. In the presence of the BRAF inhibitor, AZ628, however, the abundance of activated MEK was reduced and the allosteric MEK inhibitor AZD6244 prevented ERK phosphorylation [31]. Thus, the concurrent inhibi‐ tion of MEK and BRAF overcomes resistance mediated by BRAF amplification in both mela‐ noma and colorectal cancers.

Intriguingly, BRAF copy-number gains (3- to 4-fold) were also identified in baseline (drugnaive) melanoma and colorectal tumor samples. In one such colorectal tumor only 28% of cells showed BRAF amplification and 10% of these tumor cells had more than 10 copies of BRAF [30, 31]. These data indicate that cell sensitivity to MEK and BRAF inhibition is likely to reflect the level of BRAF amplification and resistance may arise from the expansion of a limited number of cells with pre-existing BRAF gains. This notion is consistent with a recent study showing that K-RAS mutations conferring resistance to EGFR inhibitors were likely to be present in a clonal subpopulation of the colorectal tumor cells prior to the initiation of targeted therapy. These results may explain resistance to RAF inhibitors and other targeted therapies occurs in a highly reproducible fashion within 5 to 6 months [32].

## **4.2. BRAF splicing variants**

**4. Alterations affecting BRAF**

200 Melanoma - From Early Detection to Treatment

ing drug-target binding was maintained [29].

**4.1. BRAF copy number gain**

BRAF (Figure 1) [30].

noma and colorectal cancers.

Drug resistance is often associated with the acquisition of so-called gatekeeper mutations in the target oncogene that prevent drug binding. In a series of detailed reports, deep sequenc‐ ing of melanoma biopsies derived from patients progressing on vemurafenib treatment did not find any secondary BRAF mutations. Moreover, immunoprecipitated BRAF from ve‐ murafenib-resistant melanomas retained drug sensitivity in an *in vitro* kinase assay, confirm‐

The amplification and overexpression of BRAFV600E is associated with BRAF inhibitor re‐ sistance in melanoma [30] (Figure 2). In a sequencing screen of 20 pairs of patient-match‐ ed baseline (before BRAF inhibitor therapy) and progressing (acquired resistance to BRAF inhibition) melanoma tissue, 20% showed BRAFV600E copy-number gains, ranging from 2- to 14- fold. These copy-number gains, which are likely underestimates due to non-tumor cell contamination, correlated with increased BRAF protein expression in tu‐ mor specimens. Moreover, preclinical melanoma cell models with ectopically expressed BRAFV600E confirmed that cells overexpressing mutant BRAF developed resistance to ve‐ murafenib and that this resistance could be overcome by increasing the dose of vemura‐ fenib, applying MEK inhibitors (AZD6244) or concurrently inhibiting both MEK and

Unlike melanoma cell models [30], BRAF-mutant colorectal cancer cells with amplification of the BRAF gene (2- to 7- fold) were resistance to the MEK inhibitor AZD6244 [31]. In these colon cancer cells, the increased expression of mutant BRAF resulted in excess activation of MEK and ERK, rendering cells unresponsive to MEK inhibition. In the presence of the BRAF inhibitor, AZ628, however, the abundance of activated MEK was reduced and the allosteric MEK inhibitor AZD6244 prevented ERK phosphorylation [31]. Thus, the concurrent inhibi‐ tion of MEK and BRAF overcomes resistance mediated by BRAF amplification in both mela‐

Intriguingly, BRAF copy-number gains (3- to 4-fold) were also identified in baseline (drugnaive) melanoma and colorectal tumor samples. In one such colorectal tumor only 28% of cells showed BRAF amplification and 10% of these tumor cells had more than 10 copies of BRAF [30, 31]. These data indicate that cell sensitivity to MEK and BRAF inhibition is likely to reflect the level of BRAF amplification and resistance may arise from the expansion of a limited number of cells with pre-existing BRAF gains. This notion is consistent with a recent study showing that K-RAS mutations conferring resistance to EGFR inhibitors were likely to be present in a clonal subpopulation of the colorectal tumor cells prior to the initiation of targeted therapy. These results may explain resistance to RAF inhibitors and other targeted

therapies occurs in a highly reproducible fashion within 5 to 6 months [32].

In other melanomas, resistance to vemurafenib was acquired via the expression of splice variant isoforms of BRAFV600E. Three of five vemurafenib-resistant clones of the SKMEL-238 melanoma cells expressed a novel 61kDa variant of BRAFV600E. This p61BRAFV600E splice var‐ iant, lacked exons 4-8, a region encoding the RAS binding domain, and was sufficient to ren‐ der MEK activation resistant to vemurafenib (Figure 3). The variant appears to arise from a splicing defect as no intragenic somatic deletions within the BRAF gene were detected [33].

**Figure 2. Mechanisms of resistance to BRAF inhibition**. MAPK re-activation, in the presence of RAF inhibitors, can occur via A. the mutational activation and amplification of RAS, B. the upregulation of RTKs such as PDGFRß and IGF-1R, C. elevated expression of CRAF, COT or CCND1, D. MEK mutations, or E. the expression and dimerization of BRAFV600E splice variants, such as p61BRAFV600E. Mutant RAS and upregulated RTKs also activate the PI3K/mTOR sur‐ vival pathway, which is further activated by the loss of PTEN (adapted from [87]). AKT, v-akt murine thymoma viral oncogene; BRAF, v-raf murine sarcoma viral oncogene B1; CCND1, cyclin D1; CRAF, v-raf-1 murine leukemia viral onco‐ gene homolog 1; ERK, extracellular signal-regulated kinase; COT, mitogen-activated protein kinase kinase kinase 8; MEK mitogen-activated protein kinase kinase; mTOR, mechanistic target of rapamycin; PI3K, phosphoinositide-3-kin‐ ase PTEN, phosphatase and tensin homolog; RTK, receptor tyrosine kinase.

**Figure 4. BRAF dimerization, RAF inhibitor binding and MAPK signalling.** Mutant BRAF functions as a monomer and is effectively inhibited at low RAF inhibitor concentrations. In cells with activated RAS, the binding of RAS to RAF kinases promotes the homo- and heterodimerization (not shown) of wild type RAF proteins. In the presence of low BRAF inhibitor concentrations, one protomer of the RAF dimer binds inhibitor and this promotes the transactivation of the second, inhibitor-free RAF protomer. Thus in RAS-activated cells, BRAF inhibitor can induce the activation of RAF dimers and promote elevated MAPK signalling. Similarly, dimers of p61BRAFV600E splicing variant are resistant to BRAF inhibition because binding of the drug to one protomer, allosterically alters the second protomer and diminishes its affinity for the RAF inhibitor. Much higher concentrations of RAF inhibitor are required to bind both protomers in a

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203

In the normal physiological setting, activated RAS signalling promotes the dimerization and activation of RAF proteins. In the presence of BRAF inhibitors and RAS signalling, the bind‐ ing of drug to one molecule in a non-mutated RAF dimer can promote activation of the sec‐ ond RAF molecule (Figure 4). Thus, in the presence of RAS activation, the activity of homoand heterodimeric RAF complexes can be paradoxically activated by RAF inhibitors [35-37].

In melanomas with BRAFV600E, any alterations promoting RAF dimerization are predicted to confer resistance to RAF inhibitors. As expected, activating N-RAS mutations mediate resistance to vemurafenib [29] and dabrafenib [38]. Oncogenic N-RASQ61K was detected in a single vemurafenib-resistant clone derived from the M249 melanoma cells. This resist‐ ant subclone maintained ERK activation in the presence of vemurafenib, presumably via a kinase switch from BRAF to CRAF (Figure 1) [39, 40]. These cells were also sensitive to inhibition with the MEK inhibitor, AZD6244 in the presence or absence of vemurafenib, suggesting that in this cell clone oncogenic N-RAS confers resistance by principally en‐ gaging the MAPK cascade. Oncogenic N-RAS mutations were also detected in a patient with acquired resistance to vemurafenib; separate N-RAS mutations (Q61K and Q61R) were detected in two melanoma biopsies taken on initial progression and six months af‐ ter initial progression. Both mutations were associated with copy-number amplification

RAF dimer and inhibit ERK signalling.

**5. N-RAS mutations**

and N-RAS overexpression [29].

**Figure 3. Aberrant splicing of BRAFV600E confers RAF inhibitor resistance.** Several in frame BRAFV600E splice variants lacking the RAS binding domain have been detected in vemurafenib-resistant melanoma tumor specimens. The exon structure of full-length and spice variant forms of BRAF are shown. The location of the activating V600E mutation, the RAS binding and kinase domains are highlighted (adapted from [33]).

It is known that the amino terminus of BRAF negatively regulates its kinase activity by masking the carboxy-terminal catalytic domain. Upon binding to activated RAS, the aminoterminal regulatory domain of RAF proteins unfolds to expose the carboxy terminal sites that are required for dimerization and full kinase activity. The in-frame deletion in the p61BRAFV600E variant leads to the constitutive dimerization of BRAF in the absence of acti‐ vated RAS [34]. Dimerization of p61BRAFV600E was shown to be critical for mediating BRAFinhibitor resistance, as the R509H dimerization-deficient mutant form of p61BRAFV600E was sensitive to vemurafenib and monomeric p61BRAFV600E was inhibited by vemurafenib *in vi‐ tro*. Thus, it has been proposed that binding of vemurafenib to one p61BRAFV600E protomer elicits an allosteric change in the other, drug-free protomer, thereby decreasing its affinity for the drug (Figure 4). Four independent BRAF splicing variants were also detected in tu‐ mors derived from six of nineteen patients with acquired resistance to vemurafenib (Figure 3). In each case, the alternative splice variants were in frame, lacked the RAS binding do‐ main and were confined to the mutant allele [33]. This indicates that BRAF missplicing is likely due to a mutation or epigenetic change that specifically impacts the BRAFV600E allele. Importantly, no splice BRAF variants were detected in 27 melanomas resected from vemura‐ fenib-naive patients [33].

**Figure 4. BRAF dimerization, RAF inhibitor binding and MAPK signalling.** Mutant BRAF functions as a monomer and is effectively inhibited at low RAF inhibitor concentrations. In cells with activated RAS, the binding of RAS to RAF kinases promotes the homo- and heterodimerization (not shown) of wild type RAF proteins. In the presence of low BRAF inhibitor concentrations, one protomer of the RAF dimer binds inhibitor and this promotes the transactivation of the second, inhibitor-free RAF protomer. Thus in RAS-activated cells, BRAF inhibitor can induce the activation of RAF dimers and promote elevated MAPK signalling. Similarly, dimers of p61BRAFV600E splicing variant are resistant to BRAF inhibition because binding of the drug to one protomer, allosterically alters the second protomer and diminishes its affinity for the RAF inhibitor. Much higher concentrations of RAF inhibitor are required to bind both protomers in a RAF dimer and inhibit ERK signalling.

## **5. N-RAS mutations**

**Figure 3. Aberrant splicing of BRAFV600E confers RAF inhibitor resistance.** Several in frame BRAFV600E splice variants lacking the RAS binding domain have been detected in vemurafenib-resistant melanoma tumor specimens. The exon structure of full-length and spice variant forms of BRAF are shown. The location of the activating V600E mutation, the

It is known that the amino terminus of BRAF negatively regulates its kinase activity by masking the carboxy-terminal catalytic domain. Upon binding to activated RAS, the aminoterminal regulatory domain of RAF proteins unfolds to expose the carboxy terminal sites that are required for dimerization and full kinase activity. The in-frame deletion in the p61BRAFV600E variant leads to the constitutive dimerization of BRAF in the absence of acti‐ vated RAS [34]. Dimerization of p61BRAFV600E was shown to be critical for mediating BRAFinhibitor resistance, as the R509H dimerization-deficient mutant form of p61BRAFV600E was sensitive to vemurafenib and monomeric p61BRAFV600E was inhibited by vemurafenib *in vi‐ tro*. Thus, it has been proposed that binding of vemurafenib to one p61BRAFV600E protomer elicits an allosteric change in the other, drug-free protomer, thereby decreasing its affinity for the drug (Figure 4). Four independent BRAF splicing variants were also detected in tu‐ mors derived from six of nineteen patients with acquired resistance to vemurafenib (Figure 3). In each case, the alternative splice variants were in frame, lacked the RAS binding do‐ main and were confined to the mutant allele [33]. This indicates that BRAF missplicing is likely due to a mutation or epigenetic change that specifically impacts the BRAFV600E allele. Importantly, no splice BRAF variants were detected in 27 melanomas resected from vemura‐

RAS binding and kinase domains are highlighted (adapted from [33]).

202 Melanoma - From Early Detection to Treatment

fenib-naive patients [33].

In the normal physiological setting, activated RAS signalling promotes the dimerization and activation of RAF proteins. In the presence of BRAF inhibitors and RAS signalling, the bind‐ ing of drug to one molecule in a non-mutated RAF dimer can promote activation of the sec‐ ond RAF molecule (Figure 4). Thus, in the presence of RAS activation, the activity of homoand heterodimeric RAF complexes can be paradoxically activated by RAF inhibitors [35-37].

In melanomas with BRAFV600E, any alterations promoting RAF dimerization are predicted to confer resistance to RAF inhibitors. As expected, activating N-RAS mutations mediate resistance to vemurafenib [29] and dabrafenib [38]. Oncogenic N-RASQ61K was detected in a single vemurafenib-resistant clone derived from the M249 melanoma cells. This resist‐ ant subclone maintained ERK activation in the presence of vemurafenib, presumably via a kinase switch from BRAF to CRAF (Figure 1) [39, 40]. These cells were also sensitive to inhibition with the MEK inhibitor, AZD6244 in the presence or absence of vemurafenib, suggesting that in this cell clone oncogenic N-RAS confers resistance by principally en‐ gaging the MAPK cascade. Oncogenic N-RAS mutations were also detected in a patient with acquired resistance to vemurafenib; separate N-RAS mutations (Q61K and Q61R) were detected in two melanoma biopsies taken on initial progression and six months af‐ ter initial progression. Both mutations were associated with copy-number amplification and N-RAS overexpression [29].

In a second study, oncogenic N-RASQ61H was detected in two of six dabrafenib resistant subclones, generated from the MelRMu cell line. In contrast to the initial report [29], these two N-RAS mutant, MelRMu sublines showed diminished sensitivity to MEK inhibitor, trameti‐ nib and to the combined inhibition of BRAF and MEK, when compared to the parental cells. Moreover, ectopic expression of N-RASQ61K in the MelRMu cells diminished the efficacy of combined MEK and BRAF inhibition [38]. A third report also identified N-RAS mutations (N-RASQ61K and N-RASA146T) in two melanoma sublines with acquired resistance to dabrafe‐ nib. These mutations were shown to confer dabrafenib resistance, and induced the heterodi‐ merization of BRAFV600E with C-RAF in the presence of drug [41]. These N-RAS mutant clones showed partial sensitivity to trametinib and to the concurrent inhibition of BRAF and MEK proteins [41]. It is known that mutant N-RAS can signal via multiple pathways includ‐ ing the PI3K/AKT/mTOR survival cascade [42] and consequently, N-RAS mutant dabrafe‐ nib-resistant melanoma cells were responsive to the simultaneous inhibition of MEK and the PI3K/mTOR pathway [41].

**7. MEK mutations**

inhibitor PLX4720 [50] (Figure 5).

conferred vemurafenib resistance in melanoma cells.

Mutations in mitogen activated protein kinase, MEK1 have also been shown to confer resist‐ ance to MAPK inhibitors. A random mutagenesis screen of MEK1 revealed that mutations interfering with target-drug binding (e.g. I99T, G128D, L215P) and mutations that upregu‐ late MEK1 intrinsic activity (e.g. Q56P, P124S) conferred resistance to the allosteric MEK in‐ hibitor AZD6244 [49]. The G128D MEK1 mutation also conferred resistance to the BRAF

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**Figure 5. MEK1 mutations associated with MAPK inhibitor resistance.** Allosteric MEK inhibitors binds to the MEK1 hydrophobic pocket that includes residues from helix C and the activation loop. Primary MEK1 mutations affect this drug-binding pocket (e.g. I99T, I111N/S, L115P/R, G128D, F129L, V211D and L215P) and can directly perturb the allosteric binding of the MEK inhibitor. Secondary MEK1 mutations reside outside the drug-binding region and include mutations near the amino terminus (e.g. Q56P) and proximal to the helix C (C121S, P124S/L). These secondary MEK1 mutations increase MEK1 kinase activity. The C121S and P124L MEK1 mutation have been detected in MEK inhibitor resistant patient tumors [50], whereas P124S and I111S MEK1 mutations were identified in pre-treatment melanomas [54]. Shi et al. found that of the P124S, I111S and C121S, only C121S

Deep sequencing of tumors from five patients progressing on AZD6244 treatment, identified the MEK1P124L mutation in the progressing, but not pre-treatment tumor sample from one patient. The existence of this MEK1 mutation was independently verified in *ex vivo* cell lines established from tumor material, and its activity in conferring MEK- and BRAF-inhibitor re‐

There are some discrepancies in the literature regarding the role of activated RAS in selec‐ tively sensitizing cancer cells to MEK inhibition. Certainly, N-RAS mutation status did not predict MEK inhibitor sensitivity in melanoma cell lines [43], and MEK inhibitors show only modest clinical activity in patients with RAS-mutant tumors [9, 44]. It seems likely that the impact of mutant N-RAS on MEK inhibitor responses reflects its expression and activity and ultimately the network of activated N-RAS-dependent effectors. This is in agreement with a recent report demonstrating that K-RAS13D-mutant HCT116 colorectal cancer cells became resistant to MEK inhibition upon amplification of the driving K-RAS13D oncogene [45].

## **6. CRAF overexpression**

Increased expression of the CRAF kinase has also been associated with BRAF inhibitor re‐ sistance (Figure 2). Villanueva et al. (2010) observed increased CRAF protein levels in mela‐ noma cells chronically treated with the BRAF inhibitor SB-590885. In this cell model MAPK signalling driven by persistent insulin growth factor receptor (IGF-1R) activity, was rewired to utilise both CRAF and ARAF (Figure 1), and the inhibition of all three RAF isoforms was required to inhibit the proliferation of these 885-resistant cells [46]. This is in contrast to mel‐ anoma sublines rendered resistant to the pan-RAF inhibitor AZ628. These AZ628-resistant cells showed elevated basal levels of CRAF protein, but the knockdown of CRAF alone strongly inhibited cell proliferation, in the absence of AZ628 treatment [47]. These cells switched from BRAF to CRAF dependence, and the precise mechanism of CRAF-mediated AZ628 resistance remains unclear, as this inhibitor strongly suppresses both BRAFV600E and CRAF [48]. The role of CRAF in conferring RAF-inhibitor resistance may reflect the distinct genetic profiles of the melanoma cells used, the pathway rewiring involved in resistance, the mechanism of drug action and its impact on the RAF protein dimerization.

## **7. MEK mutations**

In a second study, oncogenic N-RASQ61H was detected in two of six dabrafenib resistant subclones, generated from the MelRMu cell line. In contrast to the initial report [29], these two N-RAS mutant, MelRMu sublines showed diminished sensitivity to MEK inhibitor, trameti‐ nib and to the combined inhibition of BRAF and MEK, when compared to the parental cells. Moreover, ectopic expression of N-RASQ61K in the MelRMu cells diminished the efficacy of combined MEK and BRAF inhibition [38]. A third report also identified N-RAS mutations (N-RASQ61K and N-RASA146T) in two melanoma sublines with acquired resistance to dabrafe‐ nib. These mutations were shown to confer dabrafenib resistance, and induced the heterodi‐ merization of BRAFV600E with C-RAF in the presence of drug [41]. These N-RAS mutant clones showed partial sensitivity to trametinib and to the concurrent inhibition of BRAF and MEK proteins [41]. It is known that mutant N-RAS can signal via multiple pathways includ‐ ing the PI3K/AKT/mTOR survival cascade [42] and consequently, N-RAS mutant dabrafe‐ nib-resistant melanoma cells were responsive to the simultaneous inhibition of MEK and the

There are some discrepancies in the literature regarding the role of activated RAS in selec‐ tively sensitizing cancer cells to MEK inhibition. Certainly, N-RAS mutation status did not predict MEK inhibitor sensitivity in melanoma cell lines [43], and MEK inhibitors show only modest clinical activity in patients with RAS-mutant tumors [9, 44]. It seems likely that the impact of mutant N-RAS on MEK inhibitor responses reflects its expression and activity and ultimately the network of activated N-RAS-dependent effectors. This is in agreement with a recent report demonstrating that K-RAS13D-mutant HCT116 colorectal cancer cells became resistant to MEK inhibition upon amplification of the driving K-RAS13D oncogene [45].

Increased expression of the CRAF kinase has also been associated with BRAF inhibitor re‐ sistance (Figure 2). Villanueva et al. (2010) observed increased CRAF protein levels in mela‐ noma cells chronically treated with the BRAF inhibitor SB-590885. In this cell model MAPK signalling driven by persistent insulin growth factor receptor (IGF-1R) activity, was rewired to utilise both CRAF and ARAF (Figure 1), and the inhibition of all three RAF isoforms was required to inhibit the proliferation of these 885-resistant cells [46]. This is in contrast to mel‐ anoma sublines rendered resistant to the pan-RAF inhibitor AZ628. These AZ628-resistant cells showed elevated basal levels of CRAF protein, but the knockdown of CRAF alone strongly inhibited cell proliferation, in the absence of AZ628 treatment [47]. These cells switched from BRAF to CRAF dependence, and the precise mechanism of CRAF-mediated AZ628 resistance remains unclear, as this inhibitor strongly suppresses both BRAFV600E and CRAF [48]. The role of CRAF in conferring RAF-inhibitor resistance may reflect the distinct genetic profiles of the melanoma cells used, the pathway rewiring involved in resistance, the

mechanism of drug action and its impact on the RAF protein dimerization.

PI3K/mTOR pathway [41].

204 Melanoma - From Early Detection to Treatment

**6. CRAF overexpression**

Mutations in mitogen activated protein kinase, MEK1 have also been shown to confer resist‐ ance to MAPK inhibitors. A random mutagenesis screen of MEK1 revealed that mutations interfering with target-drug binding (e.g. I99T, G128D, L215P) and mutations that upregu‐ late MEK1 intrinsic activity (e.g. Q56P, P124S) conferred resistance to the allosteric MEK in‐ hibitor AZD6244 [49]. The G128D MEK1 mutation also conferred resistance to the BRAF inhibitor PLX4720 [50] (Figure 5).

**Figure 5. MEK1 mutations associated with MAPK inhibitor resistance.** Allosteric MEK inhibitors binds to the MEK1 hydrophobic pocket that includes residues from helix C and the activation loop. Primary MEK1 mutations affect this drug-binding pocket (e.g. I99T, I111N/S, L115P/R, G128D, F129L, V211D and L215P) and can directly perturb the allosteric binding of the MEK inhibitor. Secondary MEK1 mutations reside outside the drug-binding region and include mutations near the amino terminus (e.g. Q56P) and proximal to the helix C (C121S, P124S/L). These secondary MEK1 mutations increase MEK1 kinase activity. The C121S and P124L MEK1 mutation have been detected in MEK inhibitor resistant patient tumors [50], whereas P124S and I111S MEK1 mutations were identified in pre-treatment melanomas [54]. Shi et al. found that of the P124S, I111S and C121S, only C121S conferred vemurafenib resistance in melanoma cells.

Deep sequencing of tumors from five patients progressing on AZD6244 treatment, identified the MEK1P124L mutation in the progressing, but not pre-treatment tumor sample from one patient. The existence of this MEK1 mutation was independently verified in *ex vivo* cell lines established from tumor material, and its activity in conferring MEK- and BRAF-inhibitor re‐ sistance validated in transfected melanoma cells. As with BRAF truncation and amplifica‐ tion, alterations in MEK1 protein did not alter the sensitivity of melanoma cells to the combined inhibition of BRAF and MEK inhibitors. A MEK1C121S mutation was detected via mutational profiling in a melanoma sample from a patient with acquired resistance to ve‐ murafenib. This mutation was not detected in the pre-treatment biopsy, showed increased intrinsic kinase activity and conferred resistance to BRAF and MEK inhibition *in vitro* [50]*.* BRAFi-resistant YUSIT1 melanoma cells also acquired a MEK1 mutation with increased kin‐ ase activity (K59del). These cells were dependent on MEK1 for proliferation and displayed higher ERK phosphorylation following treatment with dabrafenib [41].


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An emerging theme in BRAF inhibitor resistance is the upregulation and activation of recep‐ tor tyrosine kinases. Garraway and co-workers demonstrated that ectopically expressed re‐ ceptor tyrosine kinases Axl and ERBB2, circumvented vemurafenib activity [55], and two independent reports detected increased expression and activity of the platelet derived growth factor (PDGFRß) and IGF-1R in vemurafenib-resistant melanoma sublines [29, 46] (Figure 2). Vemurafenib-resistant tumor biopsies derived from patients also showed overex‐ pression of PDGFRß (4 of 11 patients) and IGF-1R (2 of 5 patients) compared to the corre‐ sponding pre-treatment tumor specimens [29, 46]. The knockdown or inhibition of PDGFRß and IGF-1R overcame vemurafenib resistance in cell lines, but resistance was not due to acti‐ vation of ERK alone [29, 46]. Instead, receptor tyrosine kinase-upregulated, vemurafenib re‐ sistant melanoma cell lines, showed phosphorylation of both ERK and PI3K/AKT (Figure 2), and the concurrent and sustained inhibition of the MAPK and PI3K/AKT/mTOR pathways was required to overcome PDGFRß- and IGF-1R-mediated vemurafenib resistance [46, 56]. The upregulation and activation of these receptors was not due to gene amplification or ge‐

These studies predict that RTK activation via increased autocrine tumor cell ligand secre‐ tion, or paracrine ligand production from stromal cells may confer resistance to MAPK in‐ hibition. A recent report found increased activation of the fibroblast growth factor (FGF) receptor 3 was associated with elevated levels of autocrine secreted FGF2 ligand in vemura‐ fenib resistant melanoma sublines [57]. Moreover the secretion of growth factors from cocultured fibroblast cells conferred vemurafenib resistance to BRAF-mutant melanoma cell lines. Stromal cell secretion of the hepatocyte growth factor (HGF) correlated best with ve‐ murafenib resistance in this cell screen, and HGF was detected in tumor-associated stromal cells in 23 of 34 melanoma tumors resected from patients prior to MAPK inhibitor treatment. Critically, these 23 patients also showed activation of the HGF receptor MET in their tumor biopsies, and had a poorer response to MAPK-inhibitor treatment compared to patients whose stromal cells lacked HGF expression [58]. Increased plasma HGF levels in 126 meta‐ static melanoma patients, prior to treatment with vemurafenib, was also associated with a reduction in the progression-free and overall survival rates [59]. The stromal cell secretion of HGF resulted in reactivation of the MAPK and PI3K/mTOR signalling pathways and imme‐ diate (innate) resistance to RAF inhibition. Whether activation of MET also has a role in ac‐

creased ERK phosphorylation and suppressed cell viability [55].

**9. Activation of receptor tyrosine kinases**

netic alterations within the coding sequence [29, 46].

Resistance to the allosteric MEK inhibitor PD0325901 in breast and colorectal cancer cell lines was also consistently associated with MEK mutations in the allosteric binding domain. MEK-inhibitor resistant sublines derived from the MDA-MB-231 breast and HCT-116 colon cancer cells gained the MEK1L115P and MEK1F129L mutations, respectively and the MEK-inhib‐ itor resistant LoVo colorectal cells acquired a MEK2V215E mutation (homologous to V211D mutations in MEK1) (Figure 5). The L115P and V211D mutations abrogated MEK inhibitor binding, while F129L increased the intrinsic activity of MEK and showed enhanced interac‐ tion with CRAF [51, 52]. Cell lines expressing mutant MEK1K57N, which was identified in two lung adenocarcinomas, also showed decreased sensitivity to MEK inhibition [53].

A recent study found that MEK1 mutations identified in resistant melanoma lesions might not predict BRAF-inhibitor sensitivity. Shi *et al* found that five of 31 melanomas excised pre-BRAF inhibitor treatment carried concurrent somatic BRAF and MEK1 (MEK1P124S and MEKI111S) activating mutations and that three of these five patients showed objective tumor responses. When the P124S, I111S and C121S MEK1 mutants were stably introduced into a series of melanoma cell lines, only the MEK1C121S mutant restored p-ERK levels in the presence of vemurafenib, even though all mutants showed intrinsically enhanced kinase activity [54] (Figure 5). Thus, the relative impact of MEK1 mutations may vary depending on the type of mutation, tumor genetic background and the dependence on BRAF. For instance, YUSIT1 cells were dependent on the MEK1K59del for proliferation, and the MEK1F129L mutant may induce a BRAF to CRAF kinase switch [41, 51]. Finally, a more detailed tumor profile, correlating tumor response with the rela‐ tive proportion of double-BRAF/MEK1 mutant cells within metastases, will help clarify the precise role of MEK1 in mediating BRAF-inhibitor resistance.

## **8. COT overexpression**

A recent gain of function screen tested the activity of 597 kinases (75% of the annotated hu‐ man kinases) in conferring vemurafenib resistance in the A375 melanoma cell line. Nine can‐ didates, including receptor tyrosine kinases (Axl, ERBB2), conferred significant resistance with the mitogen activated protein kinase kinase kinase 8 (the gene encoding COT/Tpl2) emerging as the top candidate. Overexpression of COT resulted in constitutive ERK activa‐ tion in the presence of vemurafenib (Figure 2). COT activated ERK via MEK-dependent and -independent mechanisms and ectopic COT expression conferred decreased sensitivity to the MEK inhibitors CI-1040 and AZD6244. COT expression was also elevated in two of three patient samples obtained early in the course of treatment and further increased in a relaps‐ ing specimen relative to its pre-treatment and on-treatment controls. Considering that inhib‐ ition of BRAFV600E increases COT expression, it is possible that COT accumulation may reflect secondary responses to BRAF inhibition and resistance. Nevertheless, the silencing and inhibition of COT in the RPMI-7951 melanoma cells, which express increased COT, de‐ creased ERK phosphorylation and suppressed cell viability [55].

## **9. Activation of receptor tyrosine kinases**

sistance validated in transfected melanoma cells. As with BRAF truncation and amplifica‐ tion, alterations in MEK1 protein did not alter the sensitivity of melanoma cells to the combined inhibition of BRAF and MEK inhibitors. A MEK1C121S mutation was detected via mutational profiling in a melanoma sample from a patient with acquired resistance to ve‐ murafenib. This mutation was not detected in the pre-treatment biopsy, showed increased intrinsic kinase activity and conferred resistance to BRAF and MEK inhibition *in vitro* [50]*.* BRAFi-resistant YUSIT1 melanoma cells also acquired a MEK1 mutation with increased kin‐ ase activity (K59del). These cells were dependent on MEK1 for proliferation and displayed

Resistance to the allosteric MEK inhibitor PD0325901 in breast and colorectal cancer cell lines was also consistently associated with MEK mutations in the allosteric binding domain. MEK-inhibitor resistant sublines derived from the MDA-MB-231 breast and HCT-116 colon cancer cells gained the MEK1L115P and MEK1F129L mutations, respectively and the MEK-inhib‐ itor resistant LoVo colorectal cells acquired a MEK2V215E mutation (homologous to V211D mutations in MEK1) (Figure 5). The L115P and V211D mutations abrogated MEK inhibitor binding, while F129L increased the intrinsic activity of MEK and showed enhanced interac‐ tion with CRAF [51, 52]. Cell lines expressing mutant MEK1K57N, which was identified in two

A recent study found that MEK1 mutations identified in resistant melanoma lesions might not predict BRAF-inhibitor sensitivity. Shi *et al* found that five of 31 melanomas excised pre-BRAF inhibitor treatment carried concurrent somatic BRAF and MEK1 (MEK1P124S and MEKI111S) activating mutations and that three of these five patients showed objective tumor responses. When the P124S, I111S and C121S MEK1 mutants were stably introduced into a series of melanoma cell lines, only the MEK1C121S mutant restored p-ERK levels in the presence of vemurafenib, even though all mutants showed intrinsically enhanced kinase activity [54] (Figure 5). Thus, the relative impact of MEK1 mutations may vary depending on the type of mutation, tumor genetic background and the dependence on BRAF. For instance, YUSIT1 cells were dependent on the MEK1K59del for proliferation, and the MEK1F129L mutant may induce a BRAF to CRAF kinase switch [41, 51]. Finally, a more detailed tumor profile, correlating tumor response with the rela‐ tive proportion of double-BRAF/MEK1 mutant cells within metastases, will help clarify

A recent gain of function screen tested the activity of 597 kinases (75% of the annotated hu‐ man kinases) in conferring vemurafenib resistance in the A375 melanoma cell line. Nine can‐ didates, including receptor tyrosine kinases (Axl, ERBB2), conferred significant resistance with the mitogen activated protein kinase kinase kinase 8 (the gene encoding COT/Tpl2) emerging as the top candidate. Overexpression of COT resulted in constitutive ERK activa‐ tion in the presence of vemurafenib (Figure 2). COT activated ERK via MEK-dependent and

lung adenocarcinomas, also showed decreased sensitivity to MEK inhibition [53].

the precise role of MEK1 in mediating BRAF-inhibitor resistance.

**8. COT overexpression**

206 Melanoma - From Early Detection to Treatment

higher ERK phosphorylation following treatment with dabrafenib [41].

An emerging theme in BRAF inhibitor resistance is the upregulation and activation of recep‐ tor tyrosine kinases. Garraway and co-workers demonstrated that ectopically expressed re‐ ceptor tyrosine kinases Axl and ERBB2, circumvented vemurafenib activity [55], and two independent reports detected increased expression and activity of the platelet derived growth factor (PDGFRß) and IGF-1R in vemurafenib-resistant melanoma sublines [29, 46] (Figure 2). Vemurafenib-resistant tumor biopsies derived from patients also showed overex‐ pression of PDGFRß (4 of 11 patients) and IGF-1R (2 of 5 patients) compared to the corre‐ sponding pre-treatment tumor specimens [29, 46]. The knockdown or inhibition of PDGFRß and IGF-1R overcame vemurafenib resistance in cell lines, but resistance was not due to acti‐ vation of ERK alone [29, 46]. Instead, receptor tyrosine kinase-upregulated, vemurafenib re‐ sistant melanoma cell lines, showed phosphorylation of both ERK and PI3K/AKT (Figure 2), and the concurrent and sustained inhibition of the MAPK and PI3K/AKT/mTOR pathways was required to overcome PDGFRß- and IGF-1R-mediated vemurafenib resistance [46, 56]. The upregulation and activation of these receptors was not due to gene amplification or ge‐ netic alterations within the coding sequence [29, 46].

These studies predict that RTK activation via increased autocrine tumor cell ligand secre‐ tion, or paracrine ligand production from stromal cells may confer resistance to MAPK in‐ hibition. A recent report found increased activation of the fibroblast growth factor (FGF) receptor 3 was associated with elevated levels of autocrine secreted FGF2 ligand in vemura‐ fenib resistant melanoma sublines [57]. Moreover the secretion of growth factors from cocultured fibroblast cells conferred vemurafenib resistance to BRAF-mutant melanoma cell lines. Stromal cell secretion of the hepatocyte growth factor (HGF) correlated best with ve‐ murafenib resistance in this cell screen, and HGF was detected in tumor-associated stromal cells in 23 of 34 melanoma tumors resected from patients prior to MAPK inhibitor treatment. Critically, these 23 patients also showed activation of the HGF receptor MET in their tumor biopsies, and had a poorer response to MAPK-inhibitor treatment compared to patients whose stromal cells lacked HGF expression [58]. Increased plasma HGF levels in 126 meta‐ static melanoma patients, prior to treatment with vemurafenib, was also associated with a reduction in the progression-free and overall survival rates [59]. The stromal cell secretion of HGF resulted in reactivation of the MAPK and PI3K/mTOR signalling pathways and imme‐ diate (innate) resistance to RAF inhibition. Whether activation of MET also has a role in ac‐ quired resistance to RAF inhibitors remains to be determined, but activating somatic MET mutations and amplifications have been detected in human cancers [60-62]. Regardless, of the mechanism of MET activation, the sensitivity of MET-activated melanoma cells can be restored by the simultaneous inhibition of RAF and either HGF or MET [58]. Finally, it is worth noting that although activation of PDGFRß and IGF-1R are associated with vemurafe‐ nib resistance [29, 46], the ligand activation of these two receptors appears insufficient to drive sustained pathway activation or vemurafenib resistance [58, 59].

should behave as cells with elevated cyclinD1/CDK4 overexpression. BRAFV600E cells with concurrent loss of both pRb and PTEN were completely resistant to RAF inhibition, and these cells continued proliferating in the presence of this RAF inhibitor [68]. The clinical significance of pRb loss in conferring MAPK inhibitor resistance is uncertain, however,

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Finally, activation of the STAT3 pathway was found to be associated with AZD6244 resist‐ ance in a panel of lung cancer cell lines. STAT3 activity was shown to decrease BIM accumu‐ lation through the upregulation of miR-17, and the inhibition of STAT3 or miR-17

Irrespective of the precise mechanisms of resistance to class I RAF inhibitors, tumors that acquire resistance or are inherently insensitive to these inhibitors often maintain some dependency on the MAPK pathway [29, 33, 38, 55, 57, 74]. These data suggest that fur‐ ther inhibition of the MAPK cascade at the downstream MEK or ERK nodes may be ef‐ fective in treating resistance to single agent BRAF inhibitors. Despite the preclinical evidence of MEK-inhibitor sensitivity in cells with acquired resistance to BRAF inhibitors [38], clinical trials applying this strategy have been disappointing. The MEK inhibitor tra‐ metinib showed minimal activity (response rates of 3%) in patients previously treated with a BRAF inhibitor [75]. Clinical benefit was observed, however, when patients who progressed on prior BRAF inhibitor were treated with a combination of BRAF and MEK inhibitors. Partial responses were observed in 17% of patients, suggesting that dual MAPK blockade can abrogate some BRAF inhibitor resistance mechanisms [76]. The tri‐ aging of patients, based on BRAF inhibitor resistance drivers, may also improve the clin‐ ical benefit of second line MAPK inhibitor therapies. For instance, melanoma cells expressing BRAF splice variants are sensitive to MEK inhibition [33], whereas cells with BRAF copy number gains respond to the concurrent inhibition of BRAF and MEK [30, 31]. Finally, specific inhibitors of ERK have recently become available, and these show anti-proliferative activity in MEK-inhibitor resistant cells and synergise with MEK inhibi‐

Sustained and significant responses have also been observed when RAF-inhibitor resistant cell lines are treated with combination MAPK and PI3K/mTOR inhibitors. For instance, in RTK-expressing vemurafenib-resistant cells, inhibition of PI3K/mTOR activity in combina‐ tion with vemurafenib showed potent synergy. Compensatory signalling via MEK permit‐ ted survival in the presence of PI3K/mTOR/MAPK inhibition, but cytotoxicity was restored using a combination of MEK inhibitor with the dual PI3K/mTOR inhibitor BEZ235 [56]. A number of combinations of MEK and PI3K/mTOR pathway inhibitors combinations have entered early phase clinical trials, however their benefit in the setting of BRAF/MEK inhibi‐

upregulated BIM and sensitized resistant cells to MEK inhibition [73].

**11. Therapies to overcome MAPK inhibitor resistance**

tors to prevent or delay the emergence of acquired resistance [52].

tor resistance remains untested.

as pRb loss is uncommon in melanoma [72].

## **10. Other regulators of response to MAPK inhibitors**

Typically, the suppression of MAPK signalling promotes cell cycle arrest that is associated with increased expression of the CDK inhibitor p27Kip1 and inhibition of cyclin D1 expres‐ sion (Figure 1). Cyclin D1 is a regulator of the cyclin dependent kinases (CDKs) 4 and 6 and the formation of binary cyclin D-CDK4/6 complexes promote the phosphorylation of the ret‐ inoblastoma protein (pRb) and cell cycle progression [63-65]. Cyclin D1 is commonly ampli‐ fied in melanoma and often in conjunction with mutated BRAF [66, 67]. The clinical significance of this genotype was demonstrated in BRAF-mutant melanoma cell lines with increased cyclin D1 protein expression. These cells showed intrinsic resistance to the growth-arresting effects of the RAF inhibitor, SB590885 and the ectopic expression of cyclin D1 conferred RAF-inhibitor resistance, which was enhanced by the dual overexpression of CDK4 and cyclin D1 [67]. These data confirm that the MAPK-independent expression of critical MAPK downstream targets will regulate RAF-inhibitor response and may diminish the dependence of cells to oncogenic BRAF.

Several independent studies have shown that loss of the phosphatase and tensin homo‐ log (PTEN) tumor suppressor, which occurs in over 10% of melanoma tumors, is predic‐ tive of attenuated RAF-inhibitor mediated cytotoxicity [68, 69]. Cells lacking PTEN remain dependent on MAPK for proliferation but utilise increased AKT signalling for survival (Figure 2). Elevated AKT promotes the nuclear exclusion of the FOXO3a tran‐ scription factor, which leads to the downstream suppression of the FOXO3a pro-apoptot‐ ic target BIM [69, 70]. Predictably, ectopic expression of activated AKT3 also prevented BRAF inhibitor induced BIM and apoptosis [71] and MEK inhibitor-sensitive cancer cell lines show significantly higher FOXO3a and BIM protein levels compared to resistant cell lines [70]. Similar to RTK-induced resistance, the simultaneous inhibition of the MAPK and AKT pathways is required to restore PTEN-null cell sensitivity to MAPK in‐ hibitors [68]. Finally, homozygous PTEN loss and increased pAKT levels were associated with vemurafenib resistance in a progressing biopsy derived from a single patient [46].

Considering the independent roles of cyclin D and PTEN in diminishing dependence on MAPK signalling and engaging the AKT survival cascade, it is anticipated that the con‐ current alteration of these cell cycle regulators would confer increased levels of resist‐ ance to MAPK inhibitors. In a recent study, vemurafenib was shown to have purely cytostatic effects in melanoma cells with either PTEN or pRb loss; pRb deleted cells should behave as cells with elevated cyclinD1/CDK4 overexpression. BRAFV600E cells with concurrent loss of both pRb and PTEN were completely resistant to RAF inhibition, and these cells continued proliferating in the presence of this RAF inhibitor [68]. The clinical significance of pRb loss in conferring MAPK inhibitor resistance is uncertain, however, as pRb loss is uncommon in melanoma [72].

Finally, activation of the STAT3 pathway was found to be associated with AZD6244 resist‐ ance in a panel of lung cancer cell lines. STAT3 activity was shown to decrease BIM accumu‐ lation through the upregulation of miR-17, and the inhibition of STAT3 or miR-17 upregulated BIM and sensitized resistant cells to MEK inhibition [73].

## **11. Therapies to overcome MAPK inhibitor resistance**

quired resistance to RAF inhibitors remains to be determined, but activating somatic MET mutations and amplifications have been detected in human cancers [60-62]. Regardless, of the mechanism of MET activation, the sensitivity of MET-activated melanoma cells can be restored by the simultaneous inhibition of RAF and either HGF or MET [58]. Finally, it is worth noting that although activation of PDGFRß and IGF-1R are associated with vemurafe‐ nib resistance [29, 46], the ligand activation of these two receptors appears insufficient to

Typically, the suppression of MAPK signalling promotes cell cycle arrest that is associated with increased expression of the CDK inhibitor p27Kip1 and inhibition of cyclin D1 expres‐ sion (Figure 1). Cyclin D1 is a regulator of the cyclin dependent kinases (CDKs) 4 and 6 and the formation of binary cyclin D-CDK4/6 complexes promote the phosphorylation of the ret‐ inoblastoma protein (pRb) and cell cycle progression [63-65]. Cyclin D1 is commonly ampli‐ fied in melanoma and often in conjunction with mutated BRAF [66, 67]. The clinical significance of this genotype was demonstrated in BRAF-mutant melanoma cell lines with increased cyclin D1 protein expression. These cells showed intrinsic resistance to the growth-arresting effects of the RAF inhibitor, SB590885 and the ectopic expression of cyclin D1 conferred RAF-inhibitor resistance, which was enhanced by the dual overexpression of CDK4 and cyclin D1 [67]. These data confirm that the MAPK-independent expression of critical MAPK downstream targets will regulate RAF-inhibitor response and may diminish

Several independent studies have shown that loss of the phosphatase and tensin homo‐ log (PTEN) tumor suppressor, which occurs in over 10% of melanoma tumors, is predic‐ tive of attenuated RAF-inhibitor mediated cytotoxicity [68, 69]. Cells lacking PTEN remain dependent on MAPK for proliferation but utilise increased AKT signalling for survival (Figure 2). Elevated AKT promotes the nuclear exclusion of the FOXO3a tran‐ scription factor, which leads to the downstream suppression of the FOXO3a pro-apoptot‐ ic target BIM [69, 70]. Predictably, ectopic expression of activated AKT3 also prevented BRAF inhibitor induced BIM and apoptosis [71] and MEK inhibitor-sensitive cancer cell lines show significantly higher FOXO3a and BIM protein levels compared to resistant cell lines [70]. Similar to RTK-induced resistance, the simultaneous inhibition of the MAPK and AKT pathways is required to restore PTEN-null cell sensitivity to MAPK in‐ hibitors [68]. Finally, homozygous PTEN loss and increased pAKT levels were associated with vemurafenib resistance in a progressing biopsy derived from a single patient [46].

Considering the independent roles of cyclin D and PTEN in diminishing dependence on MAPK signalling and engaging the AKT survival cascade, it is anticipated that the con‐ current alteration of these cell cycle regulators would confer increased levels of resist‐ ance to MAPK inhibitors. In a recent study, vemurafenib was shown to have purely cytostatic effects in melanoma cells with either PTEN or pRb loss; pRb deleted cells

drive sustained pathway activation or vemurafenib resistance [58, 59].

**10. Other regulators of response to MAPK inhibitors**

the dependence of cells to oncogenic BRAF.

208 Melanoma - From Early Detection to Treatment

Irrespective of the precise mechanisms of resistance to class I RAF inhibitors, tumors that acquire resistance or are inherently insensitive to these inhibitors often maintain some dependency on the MAPK pathway [29, 33, 38, 55, 57, 74]. These data suggest that fur‐ ther inhibition of the MAPK cascade at the downstream MEK or ERK nodes may be ef‐ fective in treating resistance to single agent BRAF inhibitors. Despite the preclinical evidence of MEK-inhibitor sensitivity in cells with acquired resistance to BRAF inhibitors [38], clinical trials applying this strategy have been disappointing. The MEK inhibitor tra‐ metinib showed minimal activity (response rates of 3%) in patients previously treated with a BRAF inhibitor [75]. Clinical benefit was observed, however, when patients who progressed on prior BRAF inhibitor were treated with a combination of BRAF and MEK inhibitors. Partial responses were observed in 17% of patients, suggesting that dual MAPK blockade can abrogate some BRAF inhibitor resistance mechanisms [76]. The tri‐ aging of patients, based on BRAF inhibitor resistance drivers, may also improve the clin‐ ical benefit of second line MAPK inhibitor therapies. For instance, melanoma cells expressing BRAF splice variants are sensitive to MEK inhibition [33], whereas cells with BRAF copy number gains respond to the concurrent inhibition of BRAF and MEK [30, 31]. Finally, specific inhibitors of ERK have recently become available, and these show anti-proliferative activity in MEK-inhibitor resistant cells and synergise with MEK inhibi‐ tors to prevent or delay the emergence of acquired resistance [52].

Sustained and significant responses have also been observed when RAF-inhibitor resistant cell lines are treated with combination MAPK and PI3K/mTOR inhibitors. For instance, in RTK-expressing vemurafenib-resistant cells, inhibition of PI3K/mTOR activity in combina‐ tion with vemurafenib showed potent synergy. Compensatory signalling via MEK permit‐ ted survival in the presence of PI3K/mTOR/MAPK inhibition, but cytotoxicity was restored using a combination of MEK inhibitor with the dual PI3K/mTOR inhibitor BEZ235 [56]. A number of combinations of MEK and PI3K/mTOR pathway inhibitors combinations have entered early phase clinical trials, however their benefit in the setting of BRAF/MEK inhibi‐ tor resistance remains untested.

Many of the proteins involved in melanoma development and RAF-inhibitor resistance are targets of the heat shock protein (Hsp)-90 family of chaperones. Hsp90 proteins regu‐ late the conformation, stability and function of many RTKs and kinases, including IGF-1R, BRAF, CRAF, CDK4, AKT and cyclin D1 [77, 78]. The pharmacological inhibition of Hsp90 using the selective inhibitor, XL888 abrogated acquired and intrinsic vemurafe‐ nib resistance. XL888 induced apoptosis in melanoma cells with mutant N-RAS, elevated PDGFRß, COT, IGF-1R, CRAF and cyclin D1. Apoptosis was associated with diminished accumulation of the resistance driver, nuclear accumulation of FOXO3a and an increase in BIM expression. Moreover, Hsp90 inhibition was a more effective apoptotic inducer when combined with MEK and PI3K inhibition [79]. Hsp90 inhibitors have shown prom‐ ising results in ERBB2-amplified breast cancers [80], but lacked clinical activity in vemur‐ afenib-naive melanoma patients [81]. Evaluation of pre- and post treatment melanoma biopsies confirmed incomplete degradation of BRAFV600E, when the inhibitor was given on a weekly schedule. Whether Hsp90 inhibition will prove effective when administered more frequently, in RAF-inhibitor resistant melanoma patients, or in combination with MAPK inhibitors remains to be tested.

[29] and intra-tumoral heterogeneity has been observed in a progressing BRAF-mutant mel‐

Acquired Resistance to Targeted MAPK Inhibition in Melanoma

http://dx.doi.org/10.5772/53629

211

Nevertheless, defining the mechanisms of RAF-inhibitor resistance is a critical step in under‐ standing the signalling pathways required to circumvent therapy. At present, up to 40% of RAF-inhibitor resistant melanomas have undefined resistance drivers, and the role of MAPK and PI3K signalling needs to be assessed in this subgroup. The fact that half of all melanoma patients have wild type BRAF melanoma, further highlights the need for an inte‐ grated preclinical and clinical approach to guide rational design of effective initial and sec‐

This work is supported by Program Grant 633004 and project grants of the National Health and Medical Research Council of Australia (NHMRC) and an infrastructure grant to West‐ mead Millennium Institute by the Health Department of NSW through Sydney West Area Health Service. HR is a recipient of a Cancer Institute New South Wales, Research Fellow‐ ship and a NHMRC Senior Research Fellowship. MSC is supported by a Rotary Health Aus‐

Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium In‐

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

[2] Cummins DL, Cummins JM, Pantle H, Silverman MA, Leonard AL, Chanmugam A.

[3] Balch CM, Gershenwald JE, Soong SJ, Thompson JF, Atkins MB, Byrd DR, et al. Final version of 2009 AJCC melanoma staging and classification. J Clin Oncol. 2009;27(36):

stitute and Melanoma Institute Australia, Westmead Hospital, Westmead, Australia

Cutaneous malignant melanoma. Mayo Clin Proc. 2006;81(4):500-7.

anoma metastases from patients treated with BRAF inhibitors [85, 86].

Kavitha Gowrishankar, Matteo S. Carlino and Helen Rizos

\*Address all correspondence to: helen.rizos@sydney.edu.au

ond-line treatment options.

**Acknowledgements**

tralia scholarship

**Author details**

**References**

277-300.

6199-206..

## **12. Conclusions**

BRAF-targeted therapy has recently emerged as the standard treatment for patients with BRAF-mutant melanoma. Responses are not durable, however and studies of acquired re‐ sistance to BRAF inhibition reveal a diversity of resistance mechanism but a common resist‐ ance theme. Melanoma cells adapt by re-engaging MAPK signalling and activating parallel survival networks. The management and prevention of BRAF inhibitor resistance is likely to be achieved through combination therapies. The combination of BRAF and MEK inhibitors has shown better response than single agent therapy [25] and is currently being evaluated in phase III clinical trials compared to vemurafenib (NCT01597908) or dabrafenib (NCT01584648) in treatment naive patients with BRAFV600E mutant melanoma. Trials com‐ bining MEK with AKT inhibitors (NCT01021748), the pan-RAF inhibitor sorafenib and MEK inhibition (NCT0034999206), testing HDAC inhibition with vorinostat (NCT006670820) are also under way. Further, Phase I trials for inhibition of PDGFRβ, FGFR and other tyrosine kinases using Dovitinib in patients with advanced melanoma has shown promising results [82]. Finally, rechallenging patients with selective BRAF inhibitors after a treatment-free in‐ terval provided clinical benefit to two patients who had previously progressed on MAPK in‐ hibitors [83]. Additional studies are required to determine the significance of rechallenging patients after treatment interruption.

It has been suggested that a detailed catalogue of resistance mechanism in an individual's tumor should inform effective second line therapy [84]. This strategy may not prove suffi‐ cient, as it does not account for stromal-mediated resistance drivers, the heterogeneous na‐ ture of melanoma and the fact that melanoma tumors from a single patient may develop multiple mechanisms of resistance. For instance, two independent vemurafenib-resistant no‐ dal metastases derived from a single patient, harboured distinct N-RAS activating mutations [29] and intra-tumoral heterogeneity has been observed in a progressing BRAF-mutant mel‐ anoma metastases from patients treated with BRAF inhibitors [85, 86].

Nevertheless, defining the mechanisms of RAF-inhibitor resistance is a critical step in under‐ standing the signalling pathways required to circumvent therapy. At present, up to 40% of RAF-inhibitor resistant melanomas have undefined resistance drivers, and the role of MAPK and PI3K signalling needs to be assessed in this subgroup. The fact that half of all melanoma patients have wild type BRAF melanoma, further highlights the need for an inte‐ grated preclinical and clinical approach to guide rational design of effective initial and sec‐ ond-line treatment options.

## **Acknowledgements**

Many of the proteins involved in melanoma development and RAF-inhibitor resistance are targets of the heat shock protein (Hsp)-90 family of chaperones. Hsp90 proteins regu‐ late the conformation, stability and function of many RTKs and kinases, including IGF-1R, BRAF, CRAF, CDK4, AKT and cyclin D1 [77, 78]. The pharmacological inhibition of Hsp90 using the selective inhibitor, XL888 abrogated acquired and intrinsic vemurafe‐ nib resistance. XL888 induced apoptosis in melanoma cells with mutant N-RAS, elevated PDGFRß, COT, IGF-1R, CRAF and cyclin D1. Apoptosis was associated with diminished accumulation of the resistance driver, nuclear accumulation of FOXO3a and an increase in BIM expression. Moreover, Hsp90 inhibition was a more effective apoptotic inducer when combined with MEK and PI3K inhibition [79]. Hsp90 inhibitors have shown prom‐ ising results in ERBB2-amplified breast cancers [80], but lacked clinical activity in vemur‐ afenib-naive melanoma patients [81]. Evaluation of pre- and post treatment melanoma biopsies confirmed incomplete degradation of BRAFV600E, when the inhibitor was given on a weekly schedule. Whether Hsp90 inhibition will prove effective when administered more frequently, in RAF-inhibitor resistant melanoma patients, or in combination with

BRAF-targeted therapy has recently emerged as the standard treatment for patients with BRAF-mutant melanoma. Responses are not durable, however and studies of acquired re‐ sistance to BRAF inhibition reveal a diversity of resistance mechanism but a common resist‐ ance theme. Melanoma cells adapt by re-engaging MAPK signalling and activating parallel survival networks. The management and prevention of BRAF inhibitor resistance is likely to be achieved through combination therapies. The combination of BRAF and MEK inhibitors has shown better response than single agent therapy [25] and is currently being evaluated in phase III clinical trials compared to vemurafenib (NCT01597908) or dabrafenib (NCT01584648) in treatment naive patients with BRAFV600E mutant melanoma. Trials com‐ bining MEK with AKT inhibitors (NCT01021748), the pan-RAF inhibitor sorafenib and MEK inhibition (NCT0034999206), testing HDAC inhibition with vorinostat (NCT006670820) are also under way. Further, Phase I trials for inhibition of PDGFRβ, FGFR and other tyrosine kinases using Dovitinib in patients with advanced melanoma has shown promising results [82]. Finally, rechallenging patients with selective BRAF inhibitors after a treatment-free in‐ terval provided clinical benefit to two patients who had previously progressed on MAPK in‐ hibitors [83]. Additional studies are required to determine the significance of rechallenging

It has been suggested that a detailed catalogue of resistance mechanism in an individual's tumor should inform effective second line therapy [84]. This strategy may not prove suffi‐ cient, as it does not account for stromal-mediated resistance drivers, the heterogeneous na‐ ture of melanoma and the fact that melanoma tumors from a single patient may develop multiple mechanisms of resistance. For instance, two independent vemurafenib-resistant no‐ dal metastases derived from a single patient, harboured distinct N-RAS activating mutations

MAPK inhibitors remains to be tested.

210 Melanoma - From Early Detection to Treatment

patients after treatment interruption.

**12. Conclusions**

This work is supported by Program Grant 633004 and project grants of the National Health and Medical Research Council of Australia (NHMRC) and an infrastructure grant to West‐ mead Millennium Institute by the Health Department of NSW through Sydney West Area Health Service. HR is a recipient of a Cancer Institute New South Wales, Research Fellow‐ ship and a NHMRC Senior Research Fellowship. MSC is supported by a Rotary Health Aus‐ tralia scholarship

## **Author details**

Kavitha Gowrishankar, Matteo S. Carlino and Helen Rizos

\*Address all correspondence to: helen.rizos@sydney.edu.au

Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium In‐ stitute and Melanoma Institute Australia, Westmead Hospital, Westmead, Australia

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[63] Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase

[64] Lavoie JN, L'Allemain G, Brunet A, Muller R, Pouyssegur J. Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK

[65] Donovan JC, Milic A, Slingerland JM. Constitutive MEK/MAPK activation leads to p27(Kip1) deregulation and antiestrogen resistance in human breast cancer cells. J Bi‐

[66] Lazar V, Ecsedi S, Szollosi AG, Toth R, Vizkeleti L, Rakosy Z, et al. Characterization of candidate gene copy number alterations in the 11q13 region along with BRAF and

NRAS mutations in human melanoma. Mod Pathol. 2009;22(10):1367-78.

motility and invasion. Cancer Res. 2002;62(23):7025-30.

Nature. 2010;468:968-72.

216 Melanoma - From Early Detection to Treatment

2011;71(15):5067-74.

2012;287(33):28087-98.

Jul 4;487(7408):505-9.

Res. 2007;13(7):2246-53.

ing. Science. 2007;316(5827):1039-43.

ol Chem. 2001;276(44):40888-95.

progression. Genes Dev. 1999;13(12):1501-12.

pathway. J Biol Chem. 1996;271(34):20608-16.

Nature. 2012;487(7408):500-4.


[79] Paraiso KH, Haarberg E, Wood E, Rebecca VW, Chen YA, Xiang Y, et al. The heat shock protein-90 inhibitor XL888 overcomes BRAF inhibitor resistance mediated through diverse mechanisms. Clin Cancer Res. 2012;18(9):2502-14.

**Chapter 8**

**Pars Plana Vitrectomy Associated with or Following**

There are underlying concerns regarding the safety of vitrectomy surgery in eyes with intra‐ ocular malignancy. These concerns are associated with vitrectomy, both with or without pla‐ que brachytherapy for choroidal melanoma. These include the possibility of local tumor dissemination, extension of malignant cells to the ocular surface and orbit, and remote meta‐ stasis. The purpose of this chapter is to discuss the safety and efficacy of employing pars plana vitrectomy in the setting of choroidal melanoma, whether concurrent with treatment

Uveal melanomas are the most common primary intraocular malignancy and, besides the skin, the uvea is the area most commonly affected by melanoma. The incidence of ocular melanoma in the United States is approximately six cases per one million population each year with a median age of onset of 55 years. [1,2] Distant metastasis peaks two to three years after enucleation with uveal melanomas, and patients with remote metastasis seldom sur‐

Currently, treatments for remote metastasis include immunotherapy, hepatic chemoemboli‐ zation, as well as experimental treatment modalities. Unfortunately, survival has not dra‐ matically increased with any of the current treatment modalities for metastatic choroidal melanoma. Many factors have been suggested as being of prognostic value including larger tumor size, anterior tumor margin, cellular pleomorphism, extrascleral extension, and genet‐ ics including monosomy 3 and genetic expression profiling. These will be discussed in later

The indications for pars plana vitrectomy (PPV) have increased exponentially since its in‐ ception in the 1970s. Traditionally, 20-gauge PPV has caused delayed wound healing, re‐

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

© 2013 Mason and Mullins; 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,

**Plaque Brachytherapy for Choroidal Melanoma**

John O. Mason and Sara Mullins

http://dx.doi.org/10.5772/53630

**1. Introduction**

or post radiation treatment.

vive longer than one year. [3]

paragraphs.

Additional information is available at the end of the chapter


## **Pars Plana Vitrectomy Associated with or Following Plaque Brachytherapy for Choroidal Melanoma**

John O. Mason and Sara Mullins

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53630

## **1. Introduction**

[79] Paraiso KH, Haarberg E, Wood E, Rebecca VW, Chen YA, Xiang Y, et al. The heat shock protein-90 inhibitor XL888 overcomes BRAF inhibitor resistance mediated

[80] Modi S, Stopeck A, Linden H, Solit D, Chandarlapaty S, Rosen N, et al. HSP90 inhibi‐ tion is effective in breast cancer: a phase II trial of tanespimycin (17-AAG) plus tras‐ tuzumab in patients with HER2-positive metastatic breast cancer progressing on

[81] Solit DB, Osman I, Polsky D, Panageas KS, Daud A, Goydos JS, et al. Phase II trial of 17-allylamino-17-demethoxygeldanamycin in patients with metastatic melanoma.

[82] Nikolaou VA, Stratigos AJ, Flaherty KT, Tsao H. Melanoma: new insights and new

[83] Neyns B, Seghers AC, Wilgenhof S, Lebbe C. Successful rechallenge in two patients with BRAF-V600-mutant melanoma who experienced previous progression during

[84] Alcala AM, Flaherty KT. BRAF Inhibitors for the Treatment of Metastatic Melanoma: Clinical Trials and Mechanisms of Resistance. Clin Cancer Res. 2012;18(1):33-9.

[85] Wilmott JS, Tembe V, Howle JR, Sharma R, Thompson JF, Rizos H, et al. Intratumoral molecular heterogeneity in a BRAF-mutant, BRAF inhibitor-resistant melanoma: a case illustrating the challenges for personalized medicine. Molecular cancer thera‐

[86] Richtig E, Schrama D, Ugurel S, Fried I, Niederkorn A, Massone C, et al. BRAF muta‐ tion analysis of only one single metastatic lesion can restrict the treatment of melano‐

[87] Catalanotti F, Solit DB. Will Hsp90 inhibitors prove effective in BRAF-mutant mela‐

treatment with a selective BRAF inhibitor. Melanoma Res. 2012 (in press).

through diverse mechanisms. Clin Cancer Res. 2012;18(9):2502-14.

trastuzumab. Clin Cancer Res. 2011;17(15):5132-9.

therapies. J Invest Dermatol. 2012;132(3 Pt 2):854-63.

ma - a case report. Br J Dermatol. 2012;7(10):1365-2133.

nomas? Clin Cancer Res. 2012;18(9):2420-2.

Clin Cancer Res. 2008 Dec 15;14(24):8302-7.

peutics. 2012 (in press)

218 Melanoma - From Early Detection to Treatment

There are underlying concerns regarding the safety of vitrectomy surgery in eyes with intra‐ ocular malignancy. These concerns are associated with vitrectomy, both with or without pla‐ que brachytherapy for choroidal melanoma. These include the possibility of local tumor dissemination, extension of malignant cells to the ocular surface and orbit, and remote meta‐ stasis. The purpose of this chapter is to discuss the safety and efficacy of employing pars plana vitrectomy in the setting of choroidal melanoma, whether concurrent with treatment or post radiation treatment.

Uveal melanomas are the most common primary intraocular malignancy and, besides the skin, the uvea is the area most commonly affected by melanoma. The incidence of ocular melanoma in the United States is approximately six cases per one million population each year with a median age of onset of 55 years. [1,2] Distant metastasis peaks two to three years after enucleation with uveal melanomas, and patients with remote metastasis seldom sur‐ vive longer than one year. [3]

Currently, treatments for remote metastasis include immunotherapy, hepatic chemoemboli‐ zation, as well as experimental treatment modalities. Unfortunately, survival has not dra‐ matically increased with any of the current treatment modalities for metastatic choroidal melanoma. Many factors have been suggested as being of prognostic value including larger tumor size, anterior tumor margin, cellular pleomorphism, extrascleral extension, and genet‐ ics including monosomy 3 and genetic expression profiling. These will be discussed in later paragraphs.

The indications for pars plana vitrectomy (PPV) have increased exponentially since its in‐ ception in the 1970s. Traditionally, 20-gauge PPV has caused delayed wound healing, re‐

© 2013 Mason and Mullins; 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.

quirement of sclerotomy sutures, postoperative astigmatism, and patient discomfort. Recently, small gauge PPV has been introduced. The 25-gauge transconjunctival sutureless PPV system enables sutureless three-port PPV without the need for conjunctival peritomies, decreases mean operative times, reduces post surgical patient discomfort, and decreases sur‐ gically-induced trauma at sclerotomy sites. [4] Decreased traumatic conjunctival and scleral manipulation with less postop inflammation, as well as less induced astigmatism, allows for more rapid postoperative visual recovery. The self-sealing nature of the incisions in suture‐ less PPV, however, does pose potential concerns for the possibility of vitreous incarceration, postoperative endophthalmitis, and hypotony. [5-7]

sis of chromosomes including spectral karyotyping (SKY), fluorescence in situ hybridization

Pars Plana Vitrectomy Associated with or Following Plaque Brachytherapy for Choroidal Melanoma

http://dx.doi.org/10.5772/53630

221

The above techniques were superior to clinical and histopathologic variables alone in pre‐ dicting which patients will develop metastatic disease. However, these techniques had limi‐ tations including false positives and negatives, a high rate of failure due to the amount of tissue required, the variability of technique from center to center, and intratumoral hetero‐ geneity. Perhaps the most limiting factor in the accuracy of monosomy 3 analysis is due to heterogeneity. A single choroidal melanoma can be comprised of a mixture of cells, some of which contain one copy of chromosome 3 and others that contain the normal two copies.

Recently, thousands of genes could be monitored simultaneously for micro-RNA (mRNA) expression. [12] With the advent of newer software, massive amounts of data could be ana‐ lyzed and multidimensional analysis could provide new heights of biologic information that were previously unobtainable. This was seen in cancer, where gene expression profiling (GEP) revealed many types of cancer that were thought to be uniform based on their com‐ mon tissue source, but were instead composed of multiple subtypes of molecularly distinct cancers. This was the case with choroidal melanoma. GEP has helped to simplify our molec‐ ular understanding of choroidal melanoma. Rather than many different molecular subtypes, there are only two major choroidal melanoma subtypes, class 1 and class 2. GEP is extremely accurate for predicting patient metastatic rates. Class 1 tumors have a very low risk and

The superiority of GEP over monosomy 3 has been verified by multiple groups. [12] In the past, the major disadvantage of GEP was the expense and limited availability. [13] However, once the value of GEP became clear, considerable effort was devoted to optimizing it for ap‐ plication and use. With regards to choroidal melanoma, GEP evaluates 15 different genetic abnormalities resulting in a class 1 or class 2 classification. This small number of genes has allowed a commercially available, relatively inexpensive assay. This is a polymerase chain reaction based assay, which requires a much smaller biopsy sample and has a very low fail‐ ure rate. With this technology, we can now identify uveal melanoma patients who are likely to develop metastatic disease. But the question remains, what is the role of vitrectomy with

Obtaining cells for analysis requires a fine-needle aspiration biopsy (FNAB). Techniques for performing biopsy may be transvitreal or transscleral. Newer techniques even involve the use of small gauge vitrectomy. Transscleral biopsy involves the use of a 27- or 30-gauge nee‐ dle that is inserted tangentially through the sclera at the base of the tumor. Traditionally fol‐ lowing transscleral biopsy, a radioactive plaque is then placed. Transvitreal biopsy may also be performed with a 25- or 27-gauge needle via a pars plana approach. The needle is insert‐ ed into the tumor and tumor cells are aspirated. [14] Paul Finger, M.D. was a pioneer in in‐ troducing the 25-gauge vitrector to biopsy ocular melanomas. He first used the technique to aspirate cells from an iris melanoma. [13] More recently with the advent of sutureless vitrec‐ tomy, newer techniques include performing a 25-gauge vitrectomy followed by inserting a

Sampling one portion of the tumor can often produce the wrong test result.

(FISH), and comparative genomic hybridization (CGH).

class 2 have a very high risk of metastatic disease. [12]

regards to biopsying choroidal melanoma?

Posterior uveal melanomas can cause visual disturbances secondary to vitreous hemor‐ rhage, exudative retinal detachments, and radiation-related complications. Treatment con‐ sisted of enucleation prior to the Collaborative Ocular Melanoma Study (COMS), which found that at twelve years, there were no significant differences in survival between enu‐ cleation and plaque brachytherapy with regards to medium size choroidal melanomas. [8] Radiation has resulted in a new set of complications, some of which are amenable to the use of vitrectomy surgery in the setting of a treated choroidal melanoma. This chapter will dis‐ cuss the safety and efficacy of vitrectomy regarding diagnosis and biopsy, endoresection, and radiation-induced complications.

## **2. Pars plana vitrectomy for diagnostic biopsy as well as molecular genetic testing**

The diagnosis of posterior choroidal melanoma is often made clinically, as well as aided by ancillary testing, such as ultrasonography, optical coherence tomography, transillumination, and angiography. Choroidal melanoma rarely requires a biopsy to make the diagnosis. With the advent of cytogenetic and molecular genetic studies, there has been a recent effort to ob‐ tain fresh tumor tissue. Early cytogenetic studies suggested that certain chromosomes (chro‐ mosome 3, 6, and 8) abnormalities were associated with a higher likelihood for metastatic disease. In the early 1990s, it was recognized that a chromosomal 3 alteration was closely associated with metastatic disease. The most important was monosomy 3 (loss of one copy of chromosome 3), which is closely associated with metastatic disease. [9,10]

Other chromosomal changes have been associated with metastatic disease including loss of 1p and 8p as well as gain of 8q, loss of 6q, and gain of 6p. These have been linked statistical‐ ly to metastatic death in choroidal melanoma. [11]

This cytogenetic information has become increasingly accessible to physicians, and the risk of misinterpreting this information has also increased. Cytogenetic analysis for choroidal melanoma was first performed with standard karyotyping, in which direct visualization was used and chromosomal abnormalities identified by morphologic changes and chromo‐ some banding pattern and size. However, this technique required the need for a highly trained cytogeneticist, led to sampling error due to analysis of only a few tumor cells, and had an inability to detect small changes. There are other techniques that rely on direct analy‐ sis of chromosomes including spectral karyotyping (SKY), fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH).

quirement of sclerotomy sutures, postoperative astigmatism, and patient discomfort. Recently, small gauge PPV has been introduced. The 25-gauge transconjunctival sutureless PPV system enables sutureless three-port PPV without the need for conjunctival peritomies, decreases mean operative times, reduces post surgical patient discomfort, and decreases sur‐ gically-induced trauma at sclerotomy sites. [4] Decreased traumatic conjunctival and scleral manipulation with less postop inflammation, as well as less induced astigmatism, allows for more rapid postoperative visual recovery. The self-sealing nature of the incisions in suture‐ less PPV, however, does pose potential concerns for the possibility of vitreous incarceration,

Posterior uveal melanomas can cause visual disturbances secondary to vitreous hemor‐ rhage, exudative retinal detachments, and radiation-related complications. Treatment con‐ sisted of enucleation prior to the Collaborative Ocular Melanoma Study (COMS), which found that at twelve years, there were no significant differences in survival between enu‐ cleation and plaque brachytherapy with regards to medium size choroidal melanomas. [8] Radiation has resulted in a new set of complications, some of which are amenable to the use of vitrectomy surgery in the setting of a treated choroidal melanoma. This chapter will dis‐ cuss the safety and efficacy of vitrectomy regarding diagnosis and biopsy, endoresection,

**2. Pars plana vitrectomy for diagnostic biopsy as well as molecular**

of chromosome 3), which is closely associated with metastatic disease. [9,10]

ly to metastatic death in choroidal melanoma. [11]

The diagnosis of posterior choroidal melanoma is often made clinically, as well as aided by ancillary testing, such as ultrasonography, optical coherence tomography, transillumination, and angiography. Choroidal melanoma rarely requires a biopsy to make the diagnosis. With the advent of cytogenetic and molecular genetic studies, there has been a recent effort to ob‐ tain fresh tumor tissue. Early cytogenetic studies suggested that certain chromosomes (chro‐ mosome 3, 6, and 8) abnormalities were associated with a higher likelihood for metastatic disease. In the early 1990s, it was recognized that a chromosomal 3 alteration was closely associated with metastatic disease. The most important was monosomy 3 (loss of one copy

Other chromosomal changes have been associated with metastatic disease including loss of 1p and 8p as well as gain of 8q, loss of 6q, and gain of 6p. These have been linked statistical‐

This cytogenetic information has become increasingly accessible to physicians, and the risk of misinterpreting this information has also increased. Cytogenetic analysis for choroidal melanoma was first performed with standard karyotyping, in which direct visualization was used and chromosomal abnormalities identified by morphologic changes and chromo‐ some banding pattern and size. However, this technique required the need for a highly trained cytogeneticist, led to sampling error due to analysis of only a few tumor cells, and had an inability to detect small changes. There are other techniques that rely on direct analy‐

postoperative endophthalmitis, and hypotony. [5-7]

and radiation-induced complications.

220 Melanoma - From Early Detection to Treatment

**genetic testing**

The above techniques were superior to clinical and histopathologic variables alone in pre‐ dicting which patients will develop metastatic disease. However, these techniques had limi‐ tations including false positives and negatives, a high rate of failure due to the amount of tissue required, the variability of technique from center to center, and intratumoral hetero‐ geneity. Perhaps the most limiting factor in the accuracy of monosomy 3 analysis is due to heterogeneity. A single choroidal melanoma can be comprised of a mixture of cells, some of which contain one copy of chromosome 3 and others that contain the normal two copies. Sampling one portion of the tumor can often produce the wrong test result.

Recently, thousands of genes could be monitored simultaneously for micro-RNA (mRNA) expression. [12] With the advent of newer software, massive amounts of data could be ana‐ lyzed and multidimensional analysis could provide new heights of biologic information that were previously unobtainable. This was seen in cancer, where gene expression profiling (GEP) revealed many types of cancer that were thought to be uniform based on their com‐ mon tissue source, but were instead composed of multiple subtypes of molecularly distinct cancers. This was the case with choroidal melanoma. GEP has helped to simplify our molec‐ ular understanding of choroidal melanoma. Rather than many different molecular subtypes, there are only two major choroidal melanoma subtypes, class 1 and class 2. GEP is extremely accurate for predicting patient metastatic rates. Class 1 tumors have a very low risk and class 2 have a very high risk of metastatic disease. [12]

The superiority of GEP over monosomy 3 has been verified by multiple groups. [12] In the past, the major disadvantage of GEP was the expense and limited availability. [13] However, once the value of GEP became clear, considerable effort was devoted to optimizing it for ap‐ plication and use. With regards to choroidal melanoma, GEP evaluates 15 different genetic abnormalities resulting in a class 1 or class 2 classification. This small number of genes has allowed a commercially available, relatively inexpensive assay. This is a polymerase chain reaction based assay, which requires a much smaller biopsy sample and has a very low fail‐ ure rate. With this technology, we can now identify uveal melanoma patients who are likely to develop metastatic disease. But the question remains, what is the role of vitrectomy with regards to biopsying choroidal melanoma?

Obtaining cells for analysis requires a fine-needle aspiration biopsy (FNAB). Techniques for performing biopsy may be transvitreal or transscleral. Newer techniques even involve the use of small gauge vitrectomy. Transscleral biopsy involves the use of a 27- or 30-gauge nee‐ dle that is inserted tangentially through the sclera at the base of the tumor. Traditionally fol‐ lowing transscleral biopsy, a radioactive plaque is then placed. Transvitreal biopsy may also be performed with a 25- or 27-gauge needle via a pars plana approach. The needle is insert‐ ed into the tumor and tumor cells are aspirated. [14] Paul Finger, M.D. was a pioneer in in‐ troducing the 25-gauge vitrector to biopsy ocular melanomas. He first used the technique to aspirate cells from an iris melanoma. [13] More recently with the advent of sutureless vitrec‐ tomy, newer techniques include performing a 25-gauge vitrectomy followed by inserting a 25- or 27-gauge needle through the 25-gauge cannula and into the center of the tumor, fol‐ lowed by aspiration of tumor cells. [15]

ranging from 2% to 9.4%. [19-22] A very large risk and potential drawback has been the question of whether this procedure results in liberating active tumor causing a potential in‐ crease in orbital recurrence and metastatic disease. This question will be answered by the

Pars Plana Vitrectomy Associated with or Following Plaque Brachytherapy for Choroidal Melanoma

http://dx.doi.org/10.5772/53630

223

Kertes et al, in the *British Journal of Ophthalmology,* followed 32 consecutive patients that were treated with endoresection. The patient's choroidal melanoma was pathologically confirmed and all patients were followed for a mean of 40.1 months. At the time, this was the longest follow up and largest series reporting on endoresection for posterior uveal melanoma. [19] The authors found that only three patients developed distant metastasis and died of malignant melanoma. In one case, distant metastasis developed in association with an intraocular recurrence. The most common complications the authors encountered were vitreous hemorrhage in 37.5% of patients, cataract in eight of 32 eyes, and three cas‐ es of retinal detachment. With an average of almost three and one-half years follow up, the authors concluded that their results, with regards to metastatic disease, were no worse than patients treated with plaque brachytherapy. They stated that their experience did not support the contention that surgical manipulation of malignant choroidal melanoma pro‐ motes metastasis. Furthermore, endoresection is a reasonable and safe alternative to the management of posterior uveal melanoma. Approximately 50% of uveal melanomas occur less than 3mm from the optic nerve or fovea, and the authors felt that their technique was particularly well suited to the treatment of choroidal melanomas that are in close proximi‐

Damato and associates, in a 1998 *British Journal of Ophthalmology* article, reported on 52 pa‐ tients undergoing endoresection for choridal melanoma. Their technique involved vitrecto‐ my, retinal incision, hemostasis by raising intraocular pressure and by moderate hypotensive anesthesia, endoresection using the vitrector, endodiathermy, endolaser, and fluid-air exchange to reattach the retina. [20] They used adjunctive ruthenium plaque radio‐ therapy in selected cases. Their patients had a mean tumor thickness of 3.9 mm. Most of the choroidal melanomas extended to within two disc diameters of the optic nerve. Follow up was a median of 20 months. The main complications included retinal detachment in 16 of 52 patients and cataract in 25 of 52 patients. Twenty-three of the 52 patients had 20/200 or bet‐ ter vision postoperatively. No patients developed local tumor recurrence. Only one of the 52 patients undergoing endoresection developed metastatic disease, 41 months postoperative‐ ly. The authors concluded that endoresection did not increase the rate of metastatic disease.

Garcia-Arumi and associates reported on 25 consecutive patients undergoing vitreoretinal surgery with endoresection for high posterior choroidal melanomas. [21] The tumor thick‐ ness ranged from 9.1 mm to 12.8 mm. The authors employed standard endoresection techni‐ que, but did use a 120-degree anterior retinotomy prior to endoresecting the melanoma and reattaching the retina. The postoperative complications included cataract in 40%, retinal de‐ tachment in 16%, epiretinal macular proliferation in 8%, and submacular hemorrhage in 4%. The final visual acuity postoperatively ranged from hand motion to 20/30, with a mean of 20/100. Remarkably, no tumors recurred, and there was no evidence of metastatic disease in follow up, which ranged from 12 to 72 months. The authors state that the reason for having

studies cited below.

ty to the optic nerve and fovea.

Potential complications of biopsy include vitreous hemorrhage, retinal detachment, and the potential for intraocular or extraocular tumor dissemination. In a series of 500 fine needle biopsy procedures, there were no cases of local recurrence or intraocular dissemination. [16] However, follow up was only three years. There were no cases of extrascleral extension due to FNAB. In another publication by Shields et al, they focused on the outcome of each pa‐ tient after FNAB. [17] Each patient was treated with plaque brachytherapy at the time of FNAB, and there were no enucleations as all biopsy specimens were obtained by needle sampling. There were no complications in this study as well. Twenty-five-gauge PPV using the vitrector to obtain cells has also resulted in no intraocular dissemination or increased metastasis to date. [13,15]

Most recently, investigators at the Jules Stein Eye Institute performed an Institutional Re‐ view Board approved retrospective study to evaluate local and systemic outcomes in pa‐ tients undergoing FNAB at the time of plaque surgery for choroidal melanoma. Included were all patients with choroidal melanoma treated with Iodine-125 brachytherapy and intra‐ operative transscleral FNAB from 2005 to 2010. [18] The study included 170 consecutive pa‐ tients. The technique used for FNAB involved transscleral approach using a 30-gauge needle. They found no cases of treatment failure and there were no cases of orbital dissemi‐ nation. Metastatic disease developed in 14 of the 170 patients. The metastatic rate in their study was similar to the metastatic rate in the COMS. The COMS did not include FNAB of tumors. In this study, when compared with the largest multicenter prospective study ever performed in ocular oncology, performing FNAB did not increase the risk of developing metastatic disease from choroidal melanoma.

## **3. Pars plana vitrectomy for treatment of choroidal melanoma using endoresection**

Removal of a tumor using an internal approach (endoresection) was first investigated for posterior choroidal melanomas in the 1980s, primarily for small juxtapapillary melanomas that were not amenable to other forms of treatment including brachytherapy at that time. Endoresection has never gained widespread popularity. However, studies have investigated the use of endoresection as an alternative to plaque brachytherapy to avoid radiation related complications such as radiation retinopathy, optic neuropathy, and retinal ischemia. Surgi‐ cal techniques vary dependent upon surgeon, however the basic principles remain. A threeport PPV is performed with posterior hyaloid dissection. Diathermy or endolaser is used around the periphery of the tumor, followed by creation of a retinotomy. The vitreous cutter is used to excise the tumor to bare sclera. Photocoagulation is then used followed by gas or silicone oil tamponade to flatten the retina. This surgical technique has many complications including retinal detachment, proliferative vitreoretinopathy, severe bleeding, and cataract. The results for local tumor control and metastasis have been favorable, with complications ranging from 2% to 9.4%. [19-22] A very large risk and potential drawback has been the question of whether this procedure results in liberating active tumor causing a potential in‐ crease in orbital recurrence and metastatic disease. This question will be answered by the studies cited below.

25- or 27-gauge needle through the 25-gauge cannula and into the center of the tumor, fol‐

Potential complications of biopsy include vitreous hemorrhage, retinal detachment, and the potential for intraocular or extraocular tumor dissemination. In a series of 500 fine needle biopsy procedures, there were no cases of local recurrence or intraocular dissemination. [16] However, follow up was only three years. There were no cases of extrascleral extension due to FNAB. In another publication by Shields et al, they focused on the outcome of each pa‐ tient after FNAB. [17] Each patient was treated with plaque brachytherapy at the time of FNAB, and there were no enucleations as all biopsy specimens were obtained by needle sampling. There were no complications in this study as well. Twenty-five-gauge PPV using the vitrector to obtain cells has also resulted in no intraocular dissemination or increased

Most recently, investigators at the Jules Stein Eye Institute performed an Institutional Re‐ view Board approved retrospective study to evaluate local and systemic outcomes in pa‐ tients undergoing FNAB at the time of plaque surgery for choroidal melanoma. Included were all patients with choroidal melanoma treated with Iodine-125 brachytherapy and intra‐ operative transscleral FNAB from 2005 to 2010. [18] The study included 170 consecutive pa‐ tients. The technique used for FNAB involved transscleral approach using a 30-gauge needle. They found no cases of treatment failure and there were no cases of orbital dissemi‐ nation. Metastatic disease developed in 14 of the 170 patients. The metastatic rate in their study was similar to the metastatic rate in the COMS. The COMS did not include FNAB of tumors. In this study, when compared with the largest multicenter prospective study ever performed in ocular oncology, performing FNAB did not increase the risk of developing

**3. Pars plana vitrectomy for treatment of choroidal melanoma using**

Removal of a tumor using an internal approach (endoresection) was first investigated for posterior choroidal melanomas in the 1980s, primarily for small juxtapapillary melanomas that were not amenable to other forms of treatment including brachytherapy at that time. Endoresection has never gained widespread popularity. However, studies have investigated the use of endoresection as an alternative to plaque brachytherapy to avoid radiation related complications such as radiation retinopathy, optic neuropathy, and retinal ischemia. Surgi‐ cal techniques vary dependent upon surgeon, however the basic principles remain. A threeport PPV is performed with posterior hyaloid dissection. Diathermy or endolaser is used around the periphery of the tumor, followed by creation of a retinotomy. The vitreous cutter is used to excise the tumor to bare sclera. Photocoagulation is then used followed by gas or silicone oil tamponade to flatten the retina. This surgical technique has many complications including retinal detachment, proliferative vitreoretinopathy, severe bleeding, and cataract. The results for local tumor control and metastasis have been favorable, with complications

lowed by aspiration of tumor cells. [15]

222 Melanoma - From Early Detection to Treatment

metastatic disease from choroidal melanoma.

metastasis to date. [13,15]

**endoresection**

Kertes et al, in the *British Journal of Ophthalmology,* followed 32 consecutive patients that were treated with endoresection. The patient's choroidal melanoma was pathologically confirmed and all patients were followed for a mean of 40.1 months. At the time, this was the longest follow up and largest series reporting on endoresection for posterior uveal melanoma. [19] The authors found that only three patients developed distant metastasis and died of malignant melanoma. In one case, distant metastasis developed in association with an intraocular recurrence. The most common complications the authors encountered were vitreous hemorrhage in 37.5% of patients, cataract in eight of 32 eyes, and three cas‐ es of retinal detachment. With an average of almost three and one-half years follow up, the authors concluded that their results, with regards to metastatic disease, were no worse than patients treated with plaque brachytherapy. They stated that their experience did not support the contention that surgical manipulation of malignant choroidal melanoma pro‐ motes metastasis. Furthermore, endoresection is a reasonable and safe alternative to the management of posterior uveal melanoma. Approximately 50% of uveal melanomas occur less than 3mm from the optic nerve or fovea, and the authors felt that their technique was particularly well suited to the treatment of choroidal melanomas that are in close proximi‐ ty to the optic nerve and fovea.

Damato and associates, in a 1998 *British Journal of Ophthalmology* article, reported on 52 pa‐ tients undergoing endoresection for choridal melanoma. Their technique involved vitrecto‐ my, retinal incision, hemostasis by raising intraocular pressure and by moderate hypotensive anesthesia, endoresection using the vitrector, endodiathermy, endolaser, and fluid-air exchange to reattach the retina. [20] They used adjunctive ruthenium plaque radio‐ therapy in selected cases. Their patients had a mean tumor thickness of 3.9 mm. Most of the choroidal melanomas extended to within two disc diameters of the optic nerve. Follow up was a median of 20 months. The main complications included retinal detachment in 16 of 52 patients and cataract in 25 of 52 patients. Twenty-three of the 52 patients had 20/200 or bet‐ ter vision postoperatively. No patients developed local tumor recurrence. Only one of the 52 patients undergoing endoresection developed metastatic disease, 41 months postoperative‐ ly. The authors concluded that endoresection did not increase the rate of metastatic disease.

Garcia-Arumi and associates reported on 25 consecutive patients undergoing vitreoretinal surgery with endoresection for high posterior choroidal melanomas. [21] The tumor thick‐ ness ranged from 9.1 mm to 12.8 mm. The authors employed standard endoresection techni‐ que, but did use a 120-degree anterior retinotomy prior to endoresecting the melanoma and reattaching the retina. The postoperative complications included cataract in 40%, retinal de‐ tachment in 16%, epiretinal macular proliferation in 8%, and submacular hemorrhage in 4%. The final visual acuity postoperatively ranged from hand motion to 20/30, with a mean of 20/100. Remarkably, no tumors recurred, and there was no evidence of metastatic disease in follow up, which ranged from 12 to 72 months. The authors state that the reason for having such a low rate of retinal detachment was due to modifying their technique, including trim‐ ming the vitreous base, examining the peripheral retina carefully for breaks, and avoiding high infusion pressure. These authors also concluded that endoresection was efficacious and did not increase the rate of metastatic disease.

Gibran and Kapoor reported on six consecutive patients with choroidal melanoma and secondary exudative retinal detachment that underwent radiation therapy, transretinal bi‐ opsy with the 25-gauge vitrector, and surgical treatment of the exudative retinal detach‐ ment, including vitrectomy and drainage of subretinal fluid with retinal tamponade. [23] All patients had a successful reattachment of the retina with significant restoration of vi‐ sion. There were no recurrences of exudative retinal detachment. Five of the six patients had 20/40 or better vision postoperatively. More importantly, there were no cases of extra‐

Pars Plana Vitrectomy Associated with or Following Plaque Brachytherapy for Choroidal Melanoma

http://dx.doi.org/10.5772/53630

225

**5. Pars plana vitrectomy for complications after brachytherapy for**

Posterior choroidal melanoma treated by brachytherapy can often result in decreased visual acuity due to retinal detachment, vitreous hemorrhage, radiation retinopathy, radiation op‐ tic neuropathy, radiation macular ischemia, epimacular proliferation, vitreous debris, and cataract. Potential indications for vitrectomy following melanoma brachytherapy include exudative retinal detachment, tractional and rhegmatogenous retinal detachment, vitreous

There is controversy regarding surgical treatment of eyes harboring a melanoma, whether treated or untreated, and whether there are viable tumor cells that may increase intraocular recurrence, extraocular recurrence, and metastatic disease. This question will be answered by the articles discussed below, as well as our data at Retina Consultants of Alabama/The University of Alabama at Birmingham School of Medicine, Department of Ophthalmology. Foster and associates reported on nine patients undergoing 20-gauge PPV in eyes contain‐ ing a treated posterior uveal melanoma. [24] In their series, vitrectomy was performed for vitreous hemorrhage in five patients, macular pucker in two patients, macular hole in one patient, and rhegmatogenous retinal detachment in one patient. Vitrectomy was per‐ formed at a mean 24.7 months after melanoma radiation treatment. Dispersion of tumor cells during vitrectomy was not observed in any patient. One patient had melanoma cells detected in the vitreous aspirate. This patient had intratumoral and vitreous hemorrhage before plaque brachytherapy, underwent combined cataract extraction and vitrectomy, and developed intraocular tumor dissemination 56 months after vitrectomy. No other pa‐ tients developed intraocular tumor dissemination. None of the nine patients developed

More recently, Bansal et al reported on the author's experience with vitrectomy for vitreous hemorrhage in eyes with posterior choroidal melanoma. [25] They reviewed the medical re‐ cords of 47 patients who underwent vitrectomy for vitreous hemorrhage following Io‐ dine-125 brachytherapy for posterior choroidal melanoma. The primary outcome of their analysis included rates of intraocular tumor dissemination, extrascleral extension, local tu‐ mor recurrence, and systemic metastasis. The average time to develop vitreous hemorrhage was 22 months following brachytherapy. With a mean follow up of five years, only four of

scleral extension or metastatic disease.

hemorrhage, vitreous debris, and epimacular proliferation.

**choroidal melanoma**

systemic metastatic disease.

Most recently, Karkhaneh and associates reported on 20 patients undergoing endoresection for medium size posterior choroidal melanoma. [22] Tumor thickness ranged from 5.5 mm to 11 mm. Preoperative visual acuity ranged from hand motion to 20/40, while postoperative visual acuity ranged from no light perception to 20/30. The authors stated that for tumors with thickness less than 9 mm in their study, they could have been treated with radiothera‐ py, but endoresection of the tumor may be an alternative approach in some parts of the world where radiotherapy is not readily available. Of the authors' patients, 6.7% had 20/40 or better vision at three years, while 73% had 20/200 or less. Only one in 20 patients died of metastatic disease in a mean follow up of 89.5 months. This is the longest follow up of all case series of patients undergoing endoresection for choroidal melanoma. This rate of meta‐ static disease is certainly lower than the metastatic rate seen in the COMS.

In contrast to enucleation, endoresection of posterior choroidal melanoma is designed to preserve vision and maintain a cosmetically acceptable eye. In contrast to brachytherapy, endoresection has fewer long-term complications such as radiation optic neuropathy or ra‐ diation retinopathy. Immediate complications of endoresection can be severe, including vit‐ reous hemorrhage, retinal detachment, cataract, and proliferative vitreoretinopathy. [19-22] The primary goal of endoresection is to eradicate the tumor while preserving vision. How‐ ever, the question of cutting into a malignant tumor and liberating cells, some of which may lead to local tumor recurrence or orbital tumor recurrence and/or distant metastasis, needed to be answered. There is no current evidence that endoresection of posterior choroidal mela‐ noma is different from enucleation or brachytherapy with regard to patient survival and metastatic disease, seen in the above detailed literature. [19-22]

## **4. Pars plana vitrectomy for exudative retinal detachment secondary to choroidal melanoma**

Exudative retinal detachment is the most common etiology of vision loss from untreated, recently diagnosed choroidal melanoma. Historically, management has been conservative, as the exudative retinal detachment will often resolve following brachytherapy for choroi‐ dal melanoma. However, large exudative retinal detachments secondary to choroidal mel‐ anoma often will not resolve and can lead to irreversible vision loss from photoreceptor damage during the several months needed for post brachytherapy resolution. The consis‐ tency of subretinal fluid associated with exudative retinal detachment from choroidal mel‐ anoma is found to be more viscous compared to subretinal fluid in rhegmatogenous retinal detachments, which can explain the longer duration of reabsorption leading to lim‐ ited visual recovery. [23]

Gibran and Kapoor reported on six consecutive patients with choroidal melanoma and secondary exudative retinal detachment that underwent radiation therapy, transretinal bi‐ opsy with the 25-gauge vitrector, and surgical treatment of the exudative retinal detach‐ ment, including vitrectomy and drainage of subretinal fluid with retinal tamponade. [23] All patients had a successful reattachment of the retina with significant restoration of vi‐ sion. There were no recurrences of exudative retinal detachment. Five of the six patients had 20/40 or better vision postoperatively. More importantly, there were no cases of extra‐ scleral extension or metastatic disease.

## **5. Pars plana vitrectomy for complications after brachytherapy for choroidal melanoma**

such a low rate of retinal detachment was due to modifying their technique, including trim‐ ming the vitreous base, examining the peripheral retina carefully for breaks, and avoiding high infusion pressure. These authors also concluded that endoresection was efficacious and

Most recently, Karkhaneh and associates reported on 20 patients undergoing endoresection for medium size posterior choroidal melanoma. [22] Tumor thickness ranged from 5.5 mm to 11 mm. Preoperative visual acuity ranged from hand motion to 20/40, while postoperative visual acuity ranged from no light perception to 20/30. The authors stated that for tumors with thickness less than 9 mm in their study, they could have been treated with radiothera‐ py, but endoresection of the tumor may be an alternative approach in some parts of the world where radiotherapy is not readily available. Of the authors' patients, 6.7% had 20/40 or better vision at three years, while 73% had 20/200 or less. Only one in 20 patients died of metastatic disease in a mean follow up of 89.5 months. This is the longest follow up of all case series of patients undergoing endoresection for choroidal melanoma. This rate of meta‐

In contrast to enucleation, endoresection of posterior choroidal melanoma is designed to preserve vision and maintain a cosmetically acceptable eye. In contrast to brachytherapy, endoresection has fewer long-term complications such as radiation optic neuropathy or ra‐ diation retinopathy. Immediate complications of endoresection can be severe, including vit‐ reous hemorrhage, retinal detachment, cataract, and proliferative vitreoretinopathy. [19-22] The primary goal of endoresection is to eradicate the tumor while preserving vision. How‐ ever, the question of cutting into a malignant tumor and liberating cells, some of which may lead to local tumor recurrence or orbital tumor recurrence and/or distant metastasis, needed to be answered. There is no current evidence that endoresection of posterior choroidal mela‐ noma is different from enucleation or brachytherapy with regard to patient survival and

**4. Pars plana vitrectomy for exudative retinal detachment secondary to**

Exudative retinal detachment is the most common etiology of vision loss from untreated, recently diagnosed choroidal melanoma. Historically, management has been conservative, as the exudative retinal detachment will often resolve following brachytherapy for choroi‐ dal melanoma. However, large exudative retinal detachments secondary to choroidal mel‐ anoma often will not resolve and can lead to irreversible vision loss from photoreceptor damage during the several months needed for post brachytherapy resolution. The consis‐ tency of subretinal fluid associated with exudative retinal detachment from choroidal mel‐ anoma is found to be more viscous compared to subretinal fluid in rhegmatogenous retinal detachments, which can explain the longer duration of reabsorption leading to lim‐

static disease is certainly lower than the metastatic rate seen in the COMS.

metastatic disease, seen in the above detailed literature. [19-22]

**choroidal melanoma**

ited visual recovery. [23]

did not increase the rate of metastatic disease.

224 Melanoma - From Early Detection to Treatment

Posterior choroidal melanoma treated by brachytherapy can often result in decreased visual acuity due to retinal detachment, vitreous hemorrhage, radiation retinopathy, radiation op‐ tic neuropathy, radiation macular ischemia, epimacular proliferation, vitreous debris, and cataract. Potential indications for vitrectomy following melanoma brachytherapy include exudative retinal detachment, tractional and rhegmatogenous retinal detachment, vitreous hemorrhage, vitreous debris, and epimacular proliferation.

There is controversy regarding surgical treatment of eyes harboring a melanoma, whether treated or untreated, and whether there are viable tumor cells that may increase intraocular recurrence, extraocular recurrence, and metastatic disease. This question will be answered by the articles discussed below, as well as our data at Retina Consultants of Alabama/The University of Alabama at Birmingham School of Medicine, Department of Ophthalmology.

Foster and associates reported on nine patients undergoing 20-gauge PPV in eyes contain‐ ing a treated posterior uveal melanoma. [24] In their series, vitrectomy was performed for vitreous hemorrhage in five patients, macular pucker in two patients, macular hole in one patient, and rhegmatogenous retinal detachment in one patient. Vitrectomy was per‐ formed at a mean 24.7 months after melanoma radiation treatment. Dispersion of tumor cells during vitrectomy was not observed in any patient. One patient had melanoma cells detected in the vitreous aspirate. This patient had intratumoral and vitreous hemorrhage before plaque brachytherapy, underwent combined cataract extraction and vitrectomy, and developed intraocular tumor dissemination 56 months after vitrectomy. No other pa‐ tients developed intraocular tumor dissemination. None of the nine patients developed systemic metastatic disease.

More recently, Bansal et al reported on the author's experience with vitrectomy for vitreous hemorrhage in eyes with posterior choroidal melanoma. [25] They reviewed the medical re‐ cords of 47 patients who underwent vitrectomy for vitreous hemorrhage following Io‐ dine-125 brachytherapy for posterior choroidal melanoma. The primary outcome of their analysis included rates of intraocular tumor dissemination, extrascleral extension, local tu‐ mor recurrence, and systemic metastasis. The average time to develop vitreous hemorrhage was 22 months following brachytherapy. With a mean follow up of five years, only four of the 47 patients (8%) developed metastatic disease, and no cases had intraocular tumor dis‐ semination or extraocular extension.

Fortunately, all literature to date has shown PPV to be very efficacious and safe in the set‐ ting of a patient with choroidal melanoma. Furthermore, there has been no increased rate of metastasis of choroidal melanoma when vitrectomy has been employed prior to or fol‐

Pars Plana Vitrectomy Associated with or Following Plaque Brachytherapy for Choroidal Melanoma

http://dx.doi.org/10.5772/53630

227

1 Retina Consultants of Alabama, P.C. and the University of Alabama at Birmingham School

2 University of Alabama at Birmingham School of Medicine, Department of Ophthalmology,

[1] Scotto J, Fraumeni JF Jr, Lee JAH. Melanomas of the Eye and Other Noncutaneous Sites: Epidemiologic Aspects. Journal of National Cancer Institute 1976;56 489-491.

[2] Seddon JM, Egan KM, Gragoudas ES. Epidemiology of Uveal Melanoma. In: Ryan SJ. (ed.) Retina. Volume 1. Second Edition. St. Louis: CV Mosby; 1994. p717-724.

[3] Zimmerman LE, McLean IW. An Evaluation of Enucleation in the Management of

[4] Fujii GY, de Juan E Jr, Humayun MS, et al. Initial Experience Using the Transconjuc‐ tival Sutureless Vitrectomy System for Vitreoretinal Surgery. Ophthalmology

[5] Taylor SR, Aylward GW. Endophthalmitis Following 25-Gauge Vitrectomy. Eye

[6] Taban M, Ufret-Vincenty RL, Sears JE. Endophthalmitis After 25-Gauge Transcon‐

[7] Magasso L, Maia A, Maia Mauricio, et al. Endophthalmitis After 25-Gauge Pars Pla‐

[8] The Collaborative Ocular Melanoma Study Randomized Trial of Iodine 125 Brachy‐ therapy for Choroidal Melanoma: V. Twelve-Year Mortality Rates and Prognostic Factors: COMS Report No. 28. Archives of Ophthalmology 2006;124 1684-1693.

junctival Sutureless Vitrectomy. Retina 2006;26 830-831.

na Vitrectomy. Retinal Cases and Brief Reports 2007;1 185-187.

Uveal Melanomas. American Journal of Ophthalmology 1979;87 741-760.

lowing treatment of the tumor.

John O. Mason1\* and Sara Mullins2

Birmingham, Alabama, USA

2002;109 1814-1820.

2005;19 1228-1229.

**References**

\*Address all correspondence to: masonallmason@yahoo.com

of Medicine, Department of Ophthalmology, Birmingham, Alabama, USA

**Author details**

In 2012, Sisk and Murray reported on combined phacoemulsification and sutureless vitrec‐ tomy for complex vitreoretinal diseases. [26] In their retrospective review of 114 eyes that had vitrectomy and cataract extraction, 72 of these eyes had a diagnosis of melanoma post radiation treatment. The authors' primary outcome measures were visual acuity and perio‐ perative complications. They did not report on tumor recurrence or metastatic disease.

We at Retina Consultants of Alabama/The University of Alabama at Birmingham School of Medicine, Department of Ophthalmology reviewed the medical records of 155 consecutive patients with choroidal melanoma treated with Iodine-125 brachytherapy. [15] We identified 20 patients that subsequently underwent 25-gauge PPV following brachytherapy. The aver‐ age age was 64. The etiology for 25-gauge PPV was epimacular proliferation in one patient, vitreous debris in two patients, rhegmatogenous retinal detachment in three patients, exu‐ dative retinal detachment in one patient, and vitreous hemorrhage in 13 patients. The aver‐ age interval from plaque brachytherapy to 25-gauge vitrectomy was 59 months (range 16 to 98 months). Patients were followed after their 25-gauge PPV an average of 36 months (range nine to 100 months). We found one of 20 patients to have local intraocular recurrence. One patient subsequently was enucleated for a blind painful eye secondary to neovascular glau‐ coma. Sixteen of the 20 patients had improvement in vision following PPV. Most important‐ ly, no systemic metastatic disease was reported in any of the 20 patients at their last visit, which was a mean of 36 months following their 25-gauge PPV.

In summary, the reports by Foster et al, [24] Bansal et al, [25] and Mason and Mullins [15] reveal the safety of performing vitrectomy in patients who have been treated with brachy‐ therapy for choroidal melanoma. The combined reports by Foster and Mason found zero of 29 patients developed metastatic disease following vitrectomy, while Bansal found only four of 47 patients to have developed metastatic disease following vitrectomy in patients with treated choroidal melanoma. This meta-analysis certainly reveals a much lower incidence of metastatic disease than previous reports, including the COMS (which did not include PPV following brachytherapy). [8] Our current findings based on the above literature, are the fol‐ lowing: sutureless vitrectomy may be performed safely in patients having post brachythera‐ py complications, sutureless vitrectomy does not increase the rate of metastatic disease, and, patients may take comfort that vitrectomy following brachytherapy for choroidal melanoma may result in an increase in vision with no threat of increasing metastatic disease.

## **6. Conclusion**

With the advent of globe salvaging techniques regarding management of choroidal mela‐ noma, ocular oncologists face challenges in the care of these patients. Tumor biopsy is gaining widespread acceptance to obtain tissue for genetic analyses, allowing for more precise determination of metastatic disease prognosis. The use of vitrectomy with regards to tumor biopsy, endoresection, and post radiation complications has expanded rapidly. Fortunately, all literature to date has shown PPV to be very efficacious and safe in the set‐ ting of a patient with choroidal melanoma. Furthermore, there has been no increased rate of metastasis of choroidal melanoma when vitrectomy has been employed prior to or fol‐ lowing treatment of the tumor.

## **Author details**

the 47 patients (8%) developed metastatic disease, and no cases had intraocular tumor dis‐

In 2012, Sisk and Murray reported on combined phacoemulsification and sutureless vitrec‐ tomy for complex vitreoretinal diseases. [26] In their retrospective review of 114 eyes that had vitrectomy and cataract extraction, 72 of these eyes had a diagnosis of melanoma post radiation treatment. The authors' primary outcome measures were visual acuity and perio‐ perative complications. They did not report on tumor recurrence or metastatic disease.

We at Retina Consultants of Alabama/The University of Alabama at Birmingham School of Medicine, Department of Ophthalmology reviewed the medical records of 155 consecutive patients with choroidal melanoma treated with Iodine-125 brachytherapy. [15] We identified 20 patients that subsequently underwent 25-gauge PPV following brachytherapy. The aver‐ age age was 64. The etiology for 25-gauge PPV was epimacular proliferation in one patient, vitreous debris in two patients, rhegmatogenous retinal detachment in three patients, exu‐ dative retinal detachment in one patient, and vitreous hemorrhage in 13 patients. The aver‐ age interval from plaque brachytherapy to 25-gauge vitrectomy was 59 months (range 16 to 98 months). Patients were followed after their 25-gauge PPV an average of 36 months (range nine to 100 months). We found one of 20 patients to have local intraocular recurrence. One patient subsequently was enucleated for a blind painful eye secondary to neovascular glau‐ coma. Sixteen of the 20 patients had improvement in vision following PPV. Most important‐ ly, no systemic metastatic disease was reported in any of the 20 patients at their last visit,

In summary, the reports by Foster et al, [24] Bansal et al, [25] and Mason and Mullins [15] reveal the safety of performing vitrectomy in patients who have been treated with brachy‐ therapy for choroidal melanoma. The combined reports by Foster and Mason found zero of 29 patients developed metastatic disease following vitrectomy, while Bansal found only four of 47 patients to have developed metastatic disease following vitrectomy in patients with treated choroidal melanoma. This meta-analysis certainly reveals a much lower incidence of metastatic disease than previous reports, including the COMS (which did not include PPV following brachytherapy). [8] Our current findings based on the above literature, are the fol‐ lowing: sutureless vitrectomy may be performed safely in patients having post brachythera‐ py complications, sutureless vitrectomy does not increase the rate of metastatic disease, and, patients may take comfort that vitrectomy following brachytherapy for choroidal melanoma

may result in an increase in vision with no threat of increasing metastatic disease.

With the advent of globe salvaging techniques regarding management of choroidal mela‐ noma, ocular oncologists face challenges in the care of these patients. Tumor biopsy is gaining widespread acceptance to obtain tissue for genetic analyses, allowing for more precise determination of metastatic disease prognosis. The use of vitrectomy with regards to tumor biopsy, endoresection, and post radiation complications has expanded rapidly.

which was a mean of 36 months following their 25-gauge PPV.

semination or extraocular extension.

226 Melanoma - From Early Detection to Treatment

**6. Conclusion**

John O. Mason1\* and Sara Mullins2

\*Address all correspondence to: masonallmason@yahoo.com

1 Retina Consultants of Alabama, P.C. and the University of Alabama at Birmingham School of Medicine, Department of Ophthalmology, Birmingham, Alabama, USA

2 University of Alabama at Birmingham School of Medicine, Department of Ophthalmology, Birmingham, Alabama, USA

## **References**


[9] Kilic E, Naus NC, van Gils W, et al. Concurrent Loss of Chromosome Arm 1p and Chromosome 3 Predicts a Decreased Disease-Free Survival in Uveal Melanoma Pa‐ tients. Investigative Ophthalmology and Visual Science 2005;46(7) 2253-2257.

[21] Garcia-Arumi J, Sararols L, Martinez V, Corcostegui B. Vitreoretinal Surgery and En‐ doresection in High Posterior Choroidal Melanomas. Retina 2001;21(5) 445-452.

Pars Plana Vitrectomy Associated with or Following Plaque Brachytherapy for Choroidal Melanoma

http://dx.doi.org/10.5772/53630

229

[22] Karkhaneh R, Chams H, Amoli FA, Riazi-Esfahani M, et al. Long-Term Surgical Out‐ come of Posterior Choroidal Melanoma Treated by Endoresection. Retina 2007;27(7)

[23] Gibran SK, Kapoor KG. Management of Exudative Retinal Detachment in Choroidal

[24] Foster WJ, Harbour W, Holekamp NM, Shah GK, Thomas MA. Pars Plana Vitrecto‐ my in Eyes Containing a Treated Posterior Uveal Melanoma. American Journal of

[25] Bansal AS, Bianciotto C, Maguire JI, Regillo CD, Shields JA, Shields CA. Pars Plana Vitrectomy in Eyes with Treated Posterior Uveal Melanoma. Retina Today

[26] Sisk RA, Murray TG. Combined Phacoemulsification and Sutureless 23-Gauge Pars Plana Vitrectomy for Complex Vitreoretinal Diseases. British Journal of Ophthalmol‐

Melanoma. Clinical and Experimental Ophthalmology 2009;37 654-659.

908-914.

Ophthalmology 2003;136 471-476.

2011;Nov/Dec 54-56.

ogy 2010;94 1028-1032.


[21] Garcia-Arumi J, Sararols L, Martinez V, Corcostegui B. Vitreoretinal Surgery and En‐ doresection in High Posterior Choroidal Melanomas. Retina 2001;21(5) 445-452.

[9] Kilic E, Naus NC, van Gils W, et al. Concurrent Loss of Chromosome Arm 1p and Chromosome 3 Predicts a Decreased Disease-Free Survival in Uveal Melanoma Pa‐

tients. Investigative Ophthalmology and Visual Science 2005;46(7) 2253-2257.

2007;131(1) 91-96.

228 Melanoma - From Early Detection to Treatment

[10] Maat W, Jordanova ES, van Zelderen-Bhola SL, et al. The Heterogeneous Distribu‐ tion of Monosomy 3 in Uveal Melanomas: Implications for Prognostication Based on Fine-Needle Aspiration Biopsies. Archives of Pathology and Laboratory Medicine

[11] Sisley K, Rennie IG, Cottam DW, Potter AM, Potter CW, Rees RC. Cytogenetic Find‐ ings in Six Posterior Uveal Melanomas: Involvement of Chromosomes 3, 6, and 8.

[12] Harbour JW, Onken MD, Roberson ED, et al. Frequent Mutation of BAP1 in Metasta‐

[13] Finger PT. A New Technique for 25-Gauge Iridotomy, Iridectomy, and Tumor Biop‐ sy: The Finger Iridectomy Technique May be a Flexible and Safe Way to Biopsy Tu‐

[14] Faulkner-Jones BE, Foster WJ, Harbour JW, Smith ME, Davila RM. Fine Needle Aspi‐ ration Biopsy with Adjunct Immunohistochemistry in Intraocular Tumor Manage‐

[15] Mason JO III, Mullins S. Risk and Benefit of Pars Plana Vitrectomy in Eyes Treated with Plaque Therapy for Choroidal Melanoma: Proceedings of the University of Ala‐ bama at Birmingham School of Medicine, Department of Ophthalmology Annual Clinical and Research Symposium 2012, 27-28 April 2012, Birmingham, Alabama,

[16] Shields CL, Ganguly A, Bianciotto CG, Turaka K, Tavallali A, Shields JA. Prognosis of Uveal Melanoma in 500 Cases Using Genetic Testing of Fine-Needle Aspiration Bi‐

[17] Shields CL, Ganguly A, Materin MA, Teixeira L, Mashayekhi A, Swanson LA, Marr BP, Shields JA. Chromosome 3 Analysis of Uveal Melanoma Using Fine-Needle As‐ piration Biopsy at the Time of Plaque Radiotherapy in 140 Consecutive Cases. Trans‐

[18] Johansson CC, Mougiakakos D, Trocme E, et al. Expression and Prognostic Signifi‐ cance of iNO8 in Uveal Melanoma. International Journal of Cancer 2010;126

[19] Kertes PJ, Johnson JC, Peyman GA. Internal Resection of Posterior Uveal Melanomas.

[20] Damato B, Groenewald C, McGalliard J, Wong D. Endoresection of Choroidal Mela‐

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United States of America.

2682-2689.


**Chapter 9**

**Combination Therapies to Improve Delivery of**

Incidence rates for melanoma continue to rise between 2-3% each year in the United States [1]. Although melanoma accounts for 5% of new cancer cases, the disease is responsible for most deaths resulting from skin cancer. Five-year survival rates for localized disease have historically been greater than 95% after successfully excising tumors that are less than 1 mm thick [2]. Yet, despite intense efforts in the field, the ability to improve patient survival with invasive forms of the disease has changed little over the past two decades. Current five-year prognostic rates for regional and metastatic melanoma are approximately 66% and 15%, re‐

FDA-approved agents dacarbazine (DTIC), interferon-α, and high-dose IL-2 have long been employed as palliative therapies in advanced-stage melanoma patients (albeit with significant adverse side effects) [3]. Recent exciting data from large multicenter clinical trials has helped usher in the FDA approval of two new therapies that significantly im‐ prove upon the efficacy of existing first-line treatments such as DTIC. Ipilimumab is a humanized monoclonal antibody that functionally blocks the CTLA-4 molecule involved in suppressing T cell activation. In a randomized, double-blind phase III study, metastat‐ ic melanoma patients with unresectable stage III or IV disease were administered ipili‐ mumab, ipilimumab plus a peptide vaccine specific to the melanosomal antigen gp100, or gp100 vaccine alone [4]. Ipilimumab therapy resulted in at least a 10 month median overall survival compared to 6.4 months for the gp100 vaccine treatment arm, but no statistical differences were observed between the ipilimumab treatment groups. In a fol‐

> © 2013 Lowe 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.

**Protective T Cells into the Melanoma**

**Microenvironment**

http://dx.doi.org/10.5772/53634

Walter J. Storkus

**1. Introduction**

spectfully [1].

Devin B. Lowe, Jennifer L. Taylor and

Additional information is available at the end of the chapter

## **Combination Therapies to Improve Delivery of Protective T Cells into the Melanoma Microenvironment**

Devin B. Lowe, Jennifer L. Taylor and Walter J. Storkus

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53634

## **1. Introduction**

Incidence rates for melanoma continue to rise between 2-3% each year in the United States [1]. Although melanoma accounts for 5% of new cancer cases, the disease is responsible for most deaths resulting from skin cancer. Five-year survival rates for localized disease have historically been greater than 95% after successfully excising tumors that are less than 1 mm thick [2]. Yet, despite intense efforts in the field, the ability to improve patient survival with invasive forms of the disease has changed little over the past two decades. Current five-year prognostic rates for regional and metastatic melanoma are approximately 66% and 15%, re‐ spectfully [1].

FDA-approved agents dacarbazine (DTIC), interferon-α, and high-dose IL-2 have long been employed as palliative therapies in advanced-stage melanoma patients (albeit with significant adverse side effects) [3]. Recent exciting data from large multicenter clinical trials has helped usher in the FDA approval of two new therapies that significantly im‐ prove upon the efficacy of existing first-line treatments such as DTIC. Ipilimumab is a humanized monoclonal antibody that functionally blocks the CTLA-4 molecule involved in suppressing T cell activation. In a randomized, double-blind phase III study, metastat‐ ic melanoma patients with unresectable stage III or IV disease were administered ipili‐ mumab, ipilimumab plus a peptide vaccine specific to the melanosomal antigen gp100, or gp100 vaccine alone [4]. Ipilimumab therapy resulted in at least a 10 month median overall survival compared to 6.4 months for the gp100 vaccine treatment arm, but no statistical differences were observed between the ipilimumab treatment groups. In a fol‐

© 2013 Lowe 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.

low-up phase III trial, patients with treatment-naive stage III or IV melanoma received DTIC alone or combined ipilimumab and DTIC therapy [5]. Although there was an im‐ provement to median overall survival (9.1 versus 11.2 months, respectively), treatment with ipilimumab/DTIC significantly improved survival rates in patients at 3 years of fol‐ low-up. Vemurafenib is a small molecule drug that inhibits the activity of mutant BRAF (BRAF V600E) molecules in melanoma cells that constitutively signal via the MAPK pathway, promoting tumor cell proliferation and preventing cancer cell apoptosis [6]. Pa‐ tients with previously untreated metastatic melanoma were first screened for the BRAF V600E mutation and then randomized to receive vemurafenib or DTIC in a phase III clinical trial [7]. At 6 months post therapy, vemurafenib resulted in an improved overall survival rate of 84% relative to 64% for DTIC treatment. Objective responses were also observed in 48% of vemurafenib-treated patients compared to 5% confirmed responses in the DTIC treatment arm. Although these preliminary findings are promising, the fol‐ low-up time of the study was inadequate to address the final objective and evaluation of progression-free survival rates for these patients is currently ongoing [8]. In a similar‐ ly structured phase II trial, vemurafenib administration in previously-treated BRAF V600E-selected melanoma patients led to a median overall survival of 15.9 months, which exceeds that previously observed for standard first-line treatments in patients with metastatic melanoma [9]. Unfortunately, the current level of care for metastatic mel‐ anoma remains far below the general expectations of wide-spread durable responses since most patients relapse from the above mentioned therapeutic interventions and eventually succumb to disease.

the MAPK and PI3K-AKT signaling pathways, promoting cell growth and migration and preventing apoptosis [15]. Additional common melanoma defects are cells disrupted/defi‐ cient in the gene encoding PTEN. Under normal physiologic conditions, growth factors bind their respective cell surface receptor tyrosine kinase (RTK) and induce PI3K activity. PTEN serves to block PI3K function by preventing phosphorylation of PIP2 to PIP3, which ultimately drives signaling events through the PI3K-AKT pathway. In the absence of the phosphatase activity of PTEN, the AKT signaling cascade is unrestrained, driving the cell into a pro-survival mode. Simultaneous PTEN and BRAF alterations are two of the more widely documented correlative markers in late-stage melanoma patients and highlight the importance of the overlapping and non-overlapping functions of the AKT and MAPK pathways, respectively, in maintaining a malignant state. The common mela‐ noma genetic aberrations (e.g., BRAF, KIT, PTEN) are not currently utilized for clinical di‐ agnosis or prognosis, though, considering the seemingly paradoxical instances where gene markers do not correlate with independent classifiers of tumorigenesis [2]. For example, PTEN expression profiles have been reported to predict more aggressive forms of melano‐ ma in cases of PTEN gene disruption [16] or activation [17] alongside clinico-pathological results. Drug-candidate discovery and testing has instead flourished with the improved knowledge of recurring primary genetic aberrations that appear to induce melanoma, as highlighted above for the FDA-approved BRAF inhibitor vemurafenib. Many other poten‐ tial therapies have entered into clinical trials and have been well-described in a recent re‐ view [6]. One such promising drug is the RTK inhibitor dasatinib. With regard to melanoma, dasatinib targets KIT (and a limited range of alternate RTKs), leading to the disruption of the MAPK and PI3K signaling pathways. In a recently completed phase I trial, unselected patients with stage III or IV metastatic melanoma were administered da‐ satinib along with DTIC [18]. Combined treatment resulted in an objective response rate of 13.8% and median progression free survival of 13.4 weeks and appeared to be more ac‐ tive than either agent applied alone based on historical controls. Although these results are promising and support follow-up studies with this TKI, clinical evidence suggests that dasatinib preferably inhibits mutated KIT (occurring at exon 11 or 13) versus overex‐ pressed wild-type KIT in melanoma patients [19-21]. It will, therefore, be of interest to closely monitor the differential anti-tumor efficacy of dasatinib treatment in melanoma patients harboring KIT mutations in future trials in order to select the most suitable pa‐

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Monotherapeutic use of drugs specific to the more commonly disrupted signaling path‐ ways in melanoma has several drawbacks. At best, known drug/molecular target combi‐ nations are available for no more than 50% of melanoma patients (as in the case with vemurafenib and mutated BRAF), which severely limits treatment options for excluded patients. Drug resistance also presents a major concern in melanoma patients treated un‐ der these regimes. Tumor cells are capable of thwarting the benefits of targeted molecu‐ lar approaches based on a number of innate and acquired mechanisms that include utilizing compensatory cell signaling pathways [22] and survival signals provided by the supportive TME [23]. In the instance of vemurafenib treatment, most BRAF-V600E-select‐ ed patients respond to therapy in the short-term (~80%) but fail to maintain durable re‐

tient population for clinical trial accrual.

## **2. Supposed barriers to effective treatment**

Improving tumor stage classification, candidate drug/therapy selection, and prediction of a patient's outcome to treatment could result from defining molecular events involved in the transformation of normal melanocytes into melanomas [2]. The delineation of these molecular patterns has proven difficult, however, since melanoma contains high frequen‐ cies of dissimilar gene mutations, deletions, duplications, and translocations across the range of patients evaluated [10]. A number of inherited events have been illuminated (transmissible through genetic or epigenetic means) that appear directly involved in ini‐ tiating a melanocyte's pathway to malignancy by first inducing the clonal selection and outgrowth of cells [11]. Examples include alterations in the kinases BRAF and KIT and the tumor suppressor protein PTEN. The activating BRAF (BRAF-V600E) point mutation occurs in approximately 50% of melanomas (more commonly in cutaneous melanomas) and constitutively drives the MAPK pathway - without upstream RAS activation - lead‐ ing to cell proliferation and survival [12]. The frequency of BRAF mutations is also pre‐ served among primary and metastatic melanoma lesions, supporting the hypothesis that genetic disruption of BRAF is an early event that does not drive metastasis alone [12, 13]. KIT alterations account for up to 25% of acral and mucosal melanoma subtypes [6, 14]. The most common genetic change in KIT is an activating point mutation that stimulates the MAPK and PI3K-AKT signaling pathways, promoting cell growth and migration and preventing apoptosis [15]. Additional common melanoma defects are cells disrupted/defi‐ cient in the gene encoding PTEN. Under normal physiologic conditions, growth factors bind their respective cell surface receptor tyrosine kinase (RTK) and induce PI3K activity. PTEN serves to block PI3K function by preventing phosphorylation of PIP2 to PIP3, which ultimately drives signaling events through the PI3K-AKT pathway. In the absence of the phosphatase activity of PTEN, the AKT signaling cascade is unrestrained, driving the cell into a pro-survival mode. Simultaneous PTEN and BRAF alterations are two of the more widely documented correlative markers in late-stage melanoma patients and highlight the importance of the overlapping and non-overlapping functions of the AKT and MAPK pathways, respectively, in maintaining a malignant state. The common mela‐ noma genetic aberrations (e.g., BRAF, KIT, PTEN) are not currently utilized for clinical di‐ agnosis or prognosis, though, considering the seemingly paradoxical instances where gene markers do not correlate with independent classifiers of tumorigenesis [2]. For example, PTEN expression profiles have been reported to predict more aggressive forms of melano‐ ma in cases of PTEN gene disruption [16] or activation [17] alongside clinico-pathological results. Drug-candidate discovery and testing has instead flourished with the improved knowledge of recurring primary genetic aberrations that appear to induce melanoma, as highlighted above for the FDA-approved BRAF inhibitor vemurafenib. Many other poten‐ tial therapies have entered into clinical trials and have been well-described in a recent re‐ view [6]. One such promising drug is the RTK inhibitor dasatinib. With regard to melanoma, dasatinib targets KIT (and a limited range of alternate RTKs), leading to the disruption of the MAPK and PI3K signaling pathways. In a recently completed phase I trial, unselected patients with stage III or IV metastatic melanoma were administered da‐ satinib along with DTIC [18]. Combined treatment resulted in an objective response rate of 13.8% and median progression free survival of 13.4 weeks and appeared to be more ac‐ tive than either agent applied alone based on historical controls. Although these results are promising and support follow-up studies with this TKI, clinical evidence suggests that dasatinib preferably inhibits mutated KIT (occurring at exon 11 or 13) versus overex‐ pressed wild-type KIT in melanoma patients [19-21]. It will, therefore, be of interest to closely monitor the differential anti-tumor efficacy of dasatinib treatment in melanoma patients harboring KIT mutations in future trials in order to select the most suitable pa‐ tient population for clinical trial accrual.

low-up phase III trial, patients with treatment-naive stage III or IV melanoma received DTIC alone or combined ipilimumab and DTIC therapy [5]. Although there was an im‐ provement to median overall survival (9.1 versus 11.2 months, respectively), treatment with ipilimumab/DTIC significantly improved survival rates in patients at 3 years of fol‐ low-up. Vemurafenib is a small molecule drug that inhibits the activity of mutant BRAF (BRAF V600E) molecules in melanoma cells that constitutively signal via the MAPK pathway, promoting tumor cell proliferation and preventing cancer cell apoptosis [6]. Pa‐ tients with previously untreated metastatic melanoma were first screened for the BRAF V600E mutation and then randomized to receive vemurafenib or DTIC in a phase III clinical trial [7]. At 6 months post therapy, vemurafenib resulted in an improved overall survival rate of 84% relative to 64% for DTIC treatment. Objective responses were also observed in 48% of vemurafenib-treated patients compared to 5% confirmed responses in the DTIC treatment arm. Although these preliminary findings are promising, the fol‐ low-up time of the study was inadequate to address the final objective and evaluation of progression-free survival rates for these patients is currently ongoing [8]. In a similar‐ ly structured phase II trial, vemurafenib administration in previously-treated BRAF V600E-selected melanoma patients led to a median overall survival of 15.9 months, which exceeds that previously observed for standard first-line treatments in patients with metastatic melanoma [9]. Unfortunately, the current level of care for metastatic mel‐ anoma remains far below the general expectations of wide-spread durable responses since most patients relapse from the above mentioned therapeutic interventions and

Improving tumor stage classification, candidate drug/therapy selection, and prediction of a patient's outcome to treatment could result from defining molecular events involved in the transformation of normal melanocytes into melanomas [2]. The delineation of these molecular patterns has proven difficult, however, since melanoma contains high frequen‐ cies of dissimilar gene mutations, deletions, duplications, and translocations across the range of patients evaluated [10]. A number of inherited events have been illuminated (transmissible through genetic or epigenetic means) that appear directly involved in ini‐ tiating a melanocyte's pathway to malignancy by first inducing the clonal selection and outgrowth of cells [11]. Examples include alterations in the kinases BRAF and KIT and the tumor suppressor protein PTEN. The activating BRAF (BRAF-V600E) point mutation occurs in approximately 50% of melanomas (more commonly in cutaneous melanomas) and constitutively drives the MAPK pathway - without upstream RAS activation - lead‐ ing to cell proliferation and survival [12]. The frequency of BRAF mutations is also pre‐ served among primary and metastatic melanoma lesions, supporting the hypothesis that genetic disruption of BRAF is an early event that does not drive metastasis alone [12, 13]. KIT alterations account for up to 25% of acral and mucosal melanoma subtypes [6, 14]. The most common genetic change in KIT is an activating point mutation that stimulates

eventually succumb to disease.

232 Melanoma - From Early Detection to Treatment

**2. Supposed barriers to effective treatment**

Monotherapeutic use of drugs specific to the more commonly disrupted signaling path‐ ways in melanoma has several drawbacks. At best, known drug/molecular target combi‐ nations are available for no more than 50% of melanoma patients (as in the case with vemurafenib and mutated BRAF), which severely limits treatment options for excluded patients. Drug resistance also presents a major concern in melanoma patients treated un‐ der these regimes. Tumor cells are capable of thwarting the benefits of targeted molecu‐ lar approaches based on a number of innate and acquired mechanisms that include utilizing compensatory cell signaling pathways [22] and survival signals provided by the supportive TME [23]. In the instance of vemurafenib treatment, most BRAF-V600E-select‐ ed patients respond to therapy in the short-term (~80%) but fail to maintain durable re‐ sponses [24]. Such clinical observations are not specific to melanoma but describe a wider phenomenon of eventually developing resistance to molecularly-targeted approaches in solid tumors [25, 26]. It has been hypothesized that therapy administration actually pro‐ motes the natural selection of a resistant tumor mass in the host [27]. These problematic corollaries will have to be overcome through the prudent use of combinational strategies that coordinately attack tumor cells and/or the tumor stroma at multiple, non-redundant levels. As one example, tyrosine kinase inhibitor (TKI) drugs (e.g., sunitinib, axitinib, da‐ satinib) remain attractive front-line agents to improve the efficacy of other co-applied strategies such as immunotherapy since these small molecule inhibitors may enable heightened responses to immune intervention based on the removal of suppression path‐ ways inherent in the TME (as discussed in subsequent sections).

utes to a reduced efficacy of drug function such as in the case of radiotherapies. Lastly, con‐ ventional strategies that incorporate cytotoxic drugs have a diminished effect on tumor cells

Combination Therapies to Improve Delivery of Protective T Cells into the Melanoma Microenvironment

http://dx.doi.org/10.5772/53634

235

Correcting deficiencies in the tumor vasculature could potentially circumvent many of the problems that serve to limit the effective treatment of late-stage metastatic melanoma pa‐ tients as outlined above. Historically, vasculature disruption was hypothesized to starve tu‐ mors, leading to apoptosis/necrosis and lesional regression. In reality, anti-vasculature measures appear to primarily modulate the overall tumor blood vessel architecture through actions on immature endothelial cells [32]. These effects lead to transient improvements in blood flow (thereby, diminishing hypoxia and acidosis) and reduced interstitial pressure in the tumor mass [31]. In phase II clinical trials, patients with either metastatic melanoma or colorectal cancer have experienced improved response rates when bevacizumab (an anti-VEGF monoclonal antibody therapy) was combined with a standard of care treatment such as chemotherapy [33-37]. Although bevacizumab monotherapy exhibits minimal clinical im‐ pact [38], the antibody appears to exert a helper action by improving the bioavailability/ activity of co-delivered cytotoxic drugs via its disruption of the melanoma-associated vascu‐ lature. This overarching paradigm has been formally tested in a number of preclinical mod‐ els showing the improved distribution and efficacy of anti-tumor agents subsequent to tumor blood vessel "normalization" [29]. One caveat to this strategy is the need to consider the optimal schedule for application of each modality to yield superior anti-tumor efficacy. Our laboratory has recently reported that delayed TKI administration in a therapeutic mela‐ noma mouse model negated protection from a dendritic cell (DC) vaccine based on subcuta‐ neous tumor growth kinetics [39]. These studies and others indicate a window of therapeutic opportunity where anti-vasculature measures are highly effective in enhancing co-administered anti-tumor therapies. Melanomas, however, would be expected to become refractory to the action of anti-vascular drugs based on the selection of mature blood vessels that are effectively stabilized by pericytes [32]. As noted with molecular targeting strategies, tumor cells are also likely selected based on their ability to induce angiogenesis via alternate signaling pathways that do not overlap those sensitive to the originally-administered agents. In the absence of an effective second line strategy, increased tumor growth following

The immune system provides a promising platform for consideration of inclusion in com‐ bined anti-melanoma therapies as it holds many theoretical advantages over standard treatment options such as chemotherapy or bulk cytokine (biologic modifier) administra‐ tion. Namely, immunotherapies can be tailored to specifically target and kill tumor cells

selected for growth under hypoxic and acidic conditions.

anti-vasculature monotherapy may instead occur [40].

**3.2. Immunotherapy and melanoma**

**3. Improving treatment strategies**

**3.1. Vascular reconditioning hypothesis**

The initial driver mutations occurring in a melanocyte (e.g., BRAF, KIT, PTEN) are directly implicated in arresting cell cycle control points and promoting the clonal selection and ex‐ pansion of cells that may disseminate systemically [11]. These primary genetic aberrations also induce an array of secondary events – all of which may contribute to molecular intraand inter-patient heterogeneity. The pattern of tumor growth typically follows a course, whereby, melanomas transition from a benign radial phase in the epidermis (i.e., nevus) to vertical growth into the dermis and eventual systemic spread [28]. Upon reaching a size of 1-2 mm, a primary tumor nodule is growth-limited based on the need to develop a blood supply capable of providing sufficient nutrients to cells and effectively discharging metabol‐ ic waste [29]. To progress beyond this 1-2 mm limit, molecular signals in the tumor must be initiated to promote neovascularization. Hypoxia serves as one stimulus to initiate the ex‐ pression of vascular endothelial growth factor (VEGF) by melanoma cells [30]. VEGF secre‐ tion by tumor cells can also result from inflammatory cytokines derived from infiltrating immunosuppressive cell populations such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). In general terms, locoregional VEGF production recruits endothelial precursor cells by binding its cognate high affinity receptor VEGFR2 [29, 30]. Endothelial cells in turn promote pericyte trafficking and coverage via elaboration of platelet-derived growth factor (PDGF). The effects of angiogenic pathways induced under conditions of tumor growth, however, do not resemble normal physiologic conditions. There is no hierarchical structure of arterioles to venules to capillaries. Instead, the tumor blood supply consists of a chaotic distribution of immature and mature endothelial cells, which are partly due to continued VEGF signaling by melanoma and endothelial cells and pericytes. Chronic VEGF expression serves to antagonize the interaction of endothelial cells and pericytes (by inhibiting PDGF/PDGFR binding) as well as to promote an ongoing cycle of endothelial cell recruitment and proliferation. The end-results are blood vessels com‐ prised of loosely connected endothelial cells with little-to-no pericyte coverage. Consequent‐ ly, blood flow is severely restricted in areas of the tumor while fluid build-up (e.g., plasma protein extravasation) occurs in the tumor interstitium, all of which contributes to height‐ ened hypoxia, acidosis, and interstitial pressure. These TME dynamics in late-stage disease may help account for melanoma's intrinsic resistance to chemo/radiotherapies [31]. First, the delivery of anti-tumor strategies is impaired due to deficiencies in the tumor-derived blood supply and increased interstitial pressure. The hypoxic environment also directly contrib‐ utes to a reduced efficacy of drug function such as in the case of radiotherapies. Lastly, con‐ ventional strategies that incorporate cytotoxic drugs have a diminished effect on tumor cells selected for growth under hypoxic and acidic conditions.

## **3. Improving treatment strategies**

sponses [24]. Such clinical observations are not specific to melanoma but describe a wider phenomenon of eventually developing resistance to molecularly-targeted approaches in solid tumors [25, 26]. It has been hypothesized that therapy administration actually pro‐ motes the natural selection of a resistant tumor mass in the host [27]. These problematic corollaries will have to be overcome through the prudent use of combinational strategies that coordinately attack tumor cells and/or the tumor stroma at multiple, non-redundant levels. As one example, tyrosine kinase inhibitor (TKI) drugs (e.g., sunitinib, axitinib, da‐ satinib) remain attractive front-line agents to improve the efficacy of other co-applied strategies such as immunotherapy since these small molecule inhibitors may enable heightened responses to immune intervention based on the removal of suppression path‐

The initial driver mutations occurring in a melanocyte (e.g., BRAF, KIT, PTEN) are directly implicated in arresting cell cycle control points and promoting the clonal selection and ex‐ pansion of cells that may disseminate systemically [11]. These primary genetic aberrations also induce an array of secondary events – all of which may contribute to molecular intraand inter-patient heterogeneity. The pattern of tumor growth typically follows a course, whereby, melanomas transition from a benign radial phase in the epidermis (i.e., nevus) to vertical growth into the dermis and eventual systemic spread [28]. Upon reaching a size of 1-2 mm, a primary tumor nodule is growth-limited based on the need to develop a blood supply capable of providing sufficient nutrients to cells and effectively discharging metabol‐ ic waste [29]. To progress beyond this 1-2 mm limit, molecular signals in the tumor must be initiated to promote neovascularization. Hypoxia serves as one stimulus to initiate the ex‐ pression of vascular endothelial growth factor (VEGF) by melanoma cells [30]. VEGF secre‐ tion by tumor cells can also result from inflammatory cytokines derived from infiltrating immunosuppressive cell populations such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). In general terms, locoregional VEGF production recruits endothelial precursor cells by binding its cognate high affinity receptor VEGFR2 [29, 30]. Endothelial cells in turn promote pericyte trafficking and coverage via elaboration of platelet-derived growth factor (PDGF). The effects of angiogenic pathways induced under conditions of tumor growth, however, do not resemble normal physiologic conditions. There is no hierarchical structure of arterioles to venules to capillaries. Instead, the tumor blood supply consists of a chaotic distribution of immature and mature endothelial cells, which are partly due to continued VEGF signaling by melanoma and endothelial cells and pericytes. Chronic VEGF expression serves to antagonize the interaction of endothelial cells and pericytes (by inhibiting PDGF/PDGFR binding) as well as to promote an ongoing cycle of endothelial cell recruitment and proliferation. The end-results are blood vessels com‐ prised of loosely connected endothelial cells with little-to-no pericyte coverage. Consequent‐ ly, blood flow is severely restricted in areas of the tumor while fluid build-up (e.g., plasma protein extravasation) occurs in the tumor interstitium, all of which contributes to height‐ ened hypoxia, acidosis, and interstitial pressure. These TME dynamics in late-stage disease may help account for melanoma's intrinsic resistance to chemo/radiotherapies [31]. First, the delivery of anti-tumor strategies is impaired due to deficiencies in the tumor-derived blood supply and increased interstitial pressure. The hypoxic environment also directly contrib‐

ways inherent in the TME (as discussed in subsequent sections).

234 Melanoma - From Early Detection to Treatment

#### **3.1. Vascular reconditioning hypothesis**

Correcting deficiencies in the tumor vasculature could potentially circumvent many of the problems that serve to limit the effective treatment of late-stage metastatic melanoma pa‐ tients as outlined above. Historically, vasculature disruption was hypothesized to starve tu‐ mors, leading to apoptosis/necrosis and lesional regression. In reality, anti-vasculature measures appear to primarily modulate the overall tumor blood vessel architecture through actions on immature endothelial cells [32]. These effects lead to transient improvements in blood flow (thereby, diminishing hypoxia and acidosis) and reduced interstitial pressure in the tumor mass [31]. In phase II clinical trials, patients with either metastatic melanoma or colorectal cancer have experienced improved response rates when bevacizumab (an anti-VEGF monoclonal antibody therapy) was combined with a standard of care treatment such as chemotherapy [33-37]. Although bevacizumab monotherapy exhibits minimal clinical im‐ pact [38], the antibody appears to exert a helper action by improving the bioavailability/ activity of co-delivered cytotoxic drugs via its disruption of the melanoma-associated vascu‐ lature. This overarching paradigm has been formally tested in a number of preclinical mod‐ els showing the improved distribution and efficacy of anti-tumor agents subsequent to tumor blood vessel "normalization" [29]. One caveat to this strategy is the need to consider the optimal schedule for application of each modality to yield superior anti-tumor efficacy. Our laboratory has recently reported that delayed TKI administration in a therapeutic mela‐ noma mouse model negated protection from a dendritic cell (DC) vaccine based on subcuta‐ neous tumor growth kinetics [39]. These studies and others indicate a window of therapeutic opportunity where anti-vasculature measures are highly effective in enhancing co-administered anti-tumor therapies. Melanomas, however, would be expected to become refractory to the action of anti-vascular drugs based on the selection of mature blood vessels that are effectively stabilized by pericytes [32]. As noted with molecular targeting strategies, tumor cells are also likely selected based on their ability to induce angiogenesis via alternate signaling pathways that do not overlap those sensitive to the originally-administered agents. In the absence of an effective second line strategy, increased tumor growth following anti-vasculature monotherapy may instead occur [40].

#### **3.2. Immunotherapy and melanoma**

The immune system provides a promising platform for consideration of inclusion in com‐ bined anti-melanoma therapies as it holds many theoretical advantages over standard treatment options such as chemotherapy or bulk cytokine (biologic modifier) administra‐ tion. Namely, immunotherapies can be tailored to specifically target and kill tumor cells while leaving the surrounding normal tissue intact. Immune memory (recall) can also aid in sustained therapeutic action as a result of active vaccination, allowing for the mainte‐ nance of sub-clinical residual disease (in the adjuvant setting) or the prevention of recur‐ rent tumor variants (i.e., through mechanisms of immune cross-priming and epitope spreading in the protective T cell repertoire). Several clinical studies have highlighted the proof-of-principle for immunotherapy in mediating objective clinical responses in melano‐ ma patients. Therapies incorporating ipilimumab and bevacizumab have been discussed in preceding sections. Impressive clinical results have also been obtained using *ex vivo* ex‐ panded tumor infiltrating lymphocytes (TIL; T cells) in combination with rhIL-2 and nonlethal irradiation therapy, although this is a highly specialized process limited to a few locations worldwide [41, 42]. Durable complete responses (CR) (RECIST) have been ob‐ served in 22% of patients undergoing this form of treatment and most responses have been durable for > 3 years irrespective of prior treatments. Not all patients are suited to this approach, however, due to the technical constraints of resecting and culturing TIL (approximately 45% of patients are eligible at this stage) and severe toxicities associated with IL-2 administration and lymphodepletion.

immunosuppression toward inflammatory Type-1 immunity, one can envision improved clinical benefits for coordinately-applied cancer vaccines. Yet, the optimization of the vac‐

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http://dx.doi.org/10.5772/53634

237

DCs provide a theoretical advantage over other vaccine types since they potently stimu‐ late antigen-specific *de novo* and memory recall T cell responses [47]. Under steady-state conditions, a mature DC first migrates out of the periphery and into the TME where an‐ tigen is sampled and processed/presented in the form of MHC class I/II-peptide com‐ plexes [53]. After upregulating CCR7 expression, antigen-loaded DC become competent to migrate to tissue-draining lymph nodes, where it may provide an antigenic target, costimulation (e.g., DC CD80/86 binding the T cell receptor CD28), and cytokines to al‐ low for the activation of antigen-specific CD4+ and/or CD8+ T cells. These educated ef‐ fector T cells then return to the blood circulation where a portion of these cells may

Effective vaccination against melanoma antigens (many of which are non-mutated and ex‐ pressed by normal melanocytes) presents a formidable challenge. Indeed, most tumor-asso‐ ciated antigens are on the whole less immunogenic than tumor specific antigens that arise as a result of viral infection (e.g., HPV induced cervical cancer). Assuming that host central and peripheral tolerance mechanisms have not deleted the appropriate T cell repertoire, the ma‐ turation status of the DC may be key to whether specific anti-melanoma T cell responses can be invoked at all. For example, improperly matured DCs may engage responder T cells and induce either anergy or death rather than T cell activation, expansion, and differentiation in‐ to effector cells. In addition, the immunosuppressive TME can adversely condition both en‐ dogenous DC and T cell survival/function. Immuno-oncologists have attempted to tackle these confounding issues by adoptive transfer of *ex vivo* manipulated DCs (and T cells) that exhibit preferred (normal) bioactivity. In the case of DCs, these cells may be harvested as blood precursors from cancer patients and subsequently polarized to a Type-1 phenotype through genetic manipulation or exposure to a cocktail of inflammatory-prone soluble medi‐ ators in culture. After further loading with target antigens associated with tumor cell growth and progression, this cellular vaccine may be reinfused back into the patient. Fully-mature DCs generated in this fashion are able to efficiently home to draining lymph nodes and acti‐ vate/instruct resident effector-prone T cells while remaining functionally-resistant to TME inhibitory factors such as IL-10, TGF-β, VEGF, IL-6, and PGE2 [54]. The framework for the autologous DC delivery strategy in cancer patients has been validated to some degree with the FDA-approved cellular immunotherapy designated sipuleucel-T. In this protocol, pe‐ ripheral blood mononuclear cells (PBMCs) are harvested from men with castration-resistant prostate cancer and incubated with a fusion protein containing prostatic acid phosphatase and GM-CSF, a cytokine important for DC maturation [55, 56]. The stimulated PBMCs are then delivered back into patients every two weeks for a total of three injections. In a phase III double-blind multicenter trial, sipuleucel-T resulted in a median survival advantage of 4.1 months in 22% of individuals versus the placebo group [55]. Sipuleucel-T promoted

cine sub-component of such regimens remains an area of intense study [52].

**3.3. Focus on dendritic cell vaccination**

enter the tumor and perform anti-tumor activities.

The general failure of immunotherapeutic strategies to date likely involves a number of issues. As noted, melanoma is a vascularized cancer that maintains an aberrant blood vessel system. Immunologic strategies that rely on the anti-tumor properties of effector cells such as CD8+ T cells or antibodies may be unable to penetrate areas of the tumor based on the abnormal dynamics of blood flow and high interstitial pressure. Other mel‐ anoma characteristics such as reduced oxygen content and low pH serve to further re‐ duce the function of cytotoxic CD8+ T cells if they should even be recruited into the TME. First-line strategies that recondition the melanoma-associated vasculature would be expected to overcome such obstacles and allow for the improved delivery and cytotoxic action of immunotherapeutic moieties.

Melanoma is an inherently immunogenic tumor, given the anti-tumor properties of resected TIL *in vitro* [43] and clinical observations that patients with higher frequencies of TIL have improved overall survival [43, 44]. However, the late-stage TME is also quite immunosup‐ pressive. Due in part to the hypoxic nature of the TME, immunosuppressive cells such as regulatory T cells (Tregs), TAMs, and MDSCs become enriched within the tumor and rein‐ force their own survival/function while coordinately opposing the survival/function of pro‐ tective T effector cells and Type-1 polarized DCs via soluble mediators and direct cell-to-cell contact [45, 46]. Elaboration of cytokines such as IL-10 and TGF-β sustain Tregs and inhibit T cell Type-1 polarization and DC maturation [47-49]. T cells are further suppressed by MDSC secretion of reactive oxygen and nitrogen species, TGF-β, VEGF, and arginase (i.e., through L-arginine depletion) [50]. Additionally, melanoma cells can express inhibitory molecules such as PD-L1 on their cell surface that interacts with T cell-expressed PD1, lead‐ ing to T cell dysfunction and death [51]. Melanoma cells might also prevent DC processing/ presentation or T cell targeting through defects in the antigen presenting machinery and/or antigen loss. Therefore, combined immunotherapies must counteract the suppressive TME at some level (e.g., ipilimumab's anti-CTLA-4 mode of action). By reversing the balance of immunosuppression toward inflammatory Type-1 immunity, one can envision improved clinical benefits for coordinately-applied cancer vaccines. Yet, the optimization of the vac‐ cine sub-component of such regimens remains an area of intense study [52].

## **3.3. Focus on dendritic cell vaccination**

while leaving the surrounding normal tissue intact. Immune memory (recall) can also aid in sustained therapeutic action as a result of active vaccination, allowing for the mainte‐ nance of sub-clinical residual disease (in the adjuvant setting) or the prevention of recur‐ rent tumor variants (i.e., through mechanisms of immune cross-priming and epitope spreading in the protective T cell repertoire). Several clinical studies have highlighted the proof-of-principle for immunotherapy in mediating objective clinical responses in melano‐ ma patients. Therapies incorporating ipilimumab and bevacizumab have been discussed in preceding sections. Impressive clinical results have also been obtained using *ex vivo* ex‐ panded tumor infiltrating lymphocytes (TIL; T cells) in combination with rhIL-2 and nonlethal irradiation therapy, although this is a highly specialized process limited to a few locations worldwide [41, 42]. Durable complete responses (CR) (RECIST) have been ob‐ served in 22% of patients undergoing this form of treatment and most responses have been durable for > 3 years irrespective of prior treatments. Not all patients are suited to this approach, however, due to the technical constraints of resecting and culturing TIL (approximately 45% of patients are eligible at this stage) and severe toxicities associated

The general failure of immunotherapeutic strategies to date likely involves a number of issues. As noted, melanoma is a vascularized cancer that maintains an aberrant blood vessel system. Immunologic strategies that rely on the anti-tumor properties of effector cells such as CD8+ T cells or antibodies may be unable to penetrate areas of the tumor based on the abnormal dynamics of blood flow and high interstitial pressure. Other mel‐ anoma characteristics such as reduced oxygen content and low pH serve to further re‐ duce the function of cytotoxic CD8+ T cells if they should even be recruited into the TME. First-line strategies that recondition the melanoma-associated vasculature would be expected to overcome such obstacles and allow for the improved delivery and cytotoxic

Melanoma is an inherently immunogenic tumor, given the anti-tumor properties of resected TIL *in vitro* [43] and clinical observations that patients with higher frequencies of TIL have improved overall survival [43, 44]. However, the late-stage TME is also quite immunosup‐ pressive. Due in part to the hypoxic nature of the TME, immunosuppressive cells such as regulatory T cells (Tregs), TAMs, and MDSCs become enriched within the tumor and rein‐ force their own survival/function while coordinately opposing the survival/function of pro‐ tective T effector cells and Type-1 polarized DCs via soluble mediators and direct cell-to-cell contact [45, 46]. Elaboration of cytokines such as IL-10 and TGF-β sustain Tregs and inhibit T cell Type-1 polarization and DC maturation [47-49]. T cells are further suppressed by MDSC secretion of reactive oxygen and nitrogen species, TGF-β, VEGF, and arginase (i.e., through L-arginine depletion) [50]. Additionally, melanoma cells can express inhibitory molecules such as PD-L1 on their cell surface that interacts with T cell-expressed PD1, lead‐ ing to T cell dysfunction and death [51]. Melanoma cells might also prevent DC processing/ presentation or T cell targeting through defects in the antigen presenting machinery and/or antigen loss. Therefore, combined immunotherapies must counteract the suppressive TME at some level (e.g., ipilimumab's anti-CTLA-4 mode of action). By reversing the balance of

with IL-2 administration and lymphodepletion.

236 Melanoma - From Early Detection to Treatment

action of immunotherapeutic moieties.

DCs provide a theoretical advantage over other vaccine types since they potently stimu‐ late antigen-specific *de novo* and memory recall T cell responses [47]. Under steady-state conditions, a mature DC first migrates out of the periphery and into the TME where an‐ tigen is sampled and processed/presented in the form of MHC class I/II-peptide com‐ plexes [53]. After upregulating CCR7 expression, antigen-loaded DC become competent to migrate to tissue-draining lymph nodes, where it may provide an antigenic target, costimulation (e.g., DC CD80/86 binding the T cell receptor CD28), and cytokines to al‐ low for the activation of antigen-specific CD4+ and/or CD8+ T cells. These educated ef‐ fector T cells then return to the blood circulation where a portion of these cells may enter the tumor and perform anti-tumor activities.

Effective vaccination against melanoma antigens (many of which are non-mutated and ex‐ pressed by normal melanocytes) presents a formidable challenge. Indeed, most tumor-asso‐ ciated antigens are on the whole less immunogenic than tumor specific antigens that arise as a result of viral infection (e.g., HPV induced cervical cancer). Assuming that host central and peripheral tolerance mechanisms have not deleted the appropriate T cell repertoire, the ma‐ turation status of the DC may be key to whether specific anti-melanoma T cell responses can be invoked at all. For example, improperly matured DCs may engage responder T cells and induce either anergy or death rather than T cell activation, expansion, and differentiation in‐ to effector cells. In addition, the immunosuppressive TME can adversely condition both en‐ dogenous DC and T cell survival/function. Immuno-oncologists have attempted to tackle these confounding issues by adoptive transfer of *ex vivo* manipulated DCs (and T cells) that exhibit preferred (normal) bioactivity. In the case of DCs, these cells may be harvested as blood precursors from cancer patients and subsequently polarized to a Type-1 phenotype through genetic manipulation or exposure to a cocktail of inflammatory-prone soluble medi‐ ators in culture. After further loading with target antigens associated with tumor cell growth and progression, this cellular vaccine may be reinfused back into the patient. Fully-mature DCs generated in this fashion are able to efficiently home to draining lymph nodes and acti‐ vate/instruct resident effector-prone T cells while remaining functionally-resistant to TME inhibitory factors such as IL-10, TGF-β, VEGF, IL-6, and PGE2 [54]. The framework for the autologous DC delivery strategy in cancer patients has been validated to some degree with the FDA-approved cellular immunotherapy designated sipuleucel-T. In this protocol, pe‐ ripheral blood mononuclear cells (PBMCs) are harvested from men with castration-resistant prostate cancer and incubated with a fusion protein containing prostatic acid phosphatase and GM-CSF, a cytokine important for DC maturation [55, 56]. The stimulated PBMCs are then delivered back into patients every two weeks for a total of three injections. In a phase III double-blind multicenter trial, sipuleucel-T resulted in a median survival advantage of 4.1 months in 22% of individuals versus the placebo group [55]. Sipuleucel-T promoted heightened Type-I T cell and antibody responses against the vaccine fusion protein in a ma‐ jority of patients presumably due to the enhanced maturation state of infused activated DCs [56]. Overall survival correlated with improved specific immunity in responding patients suggesting that sipuleucel-T's mechanism of action includes immune targeting of prostate carcinoma cells by vaccine-induced T cells.

tients receiving the α-DC1 vaccine. The ability of α-DC1 to produce IL-12 (and, hence, stim‐ ulate CD4+ and CD8+ T cell function) correlated to prolonged progression free survival. Based on the safety profile and relative success of this trial, the α-DC1 generation protocol is currently being evaluated in a phase I trial in patients with metastatic melanoma

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239

Another way to improve the immunogenicity of autologous DC-based therapy involves the choice of antigenic target for presentation to T cells (i.e., therapeutic selection of the re‐ sponding anti-tumor T cell repertoire for expansion). Most DC-based vaccine trials have in‐ corporated melanoma-associated antigens such as gp100, tyrosinase, Melan-A/MART1 and MAGE in the vaccine formulation. Despite the surprisingly high immunogenic nature of these "self" antigens in vaccinated patients, tumor cells can continue to grow progressively by evading the effector T cell system via various well-described mechanisms [46, 48, 53]. For instance, the tumor mass is composed of a heterogeneous mixture of cancer cells that exhibit a range of defects/deficiencies in the antigen presentation machinery that limits effective presentation of tumor antigen-derived peptides in MHC complexes and leads to poor recog‐ nition by the immune system. As such, a fraction of tumor cells may become "invisible" to the adaptive immune system, resulting in the negative selection of treatment-resistant tumor cells in progressor lesions [68]. This scenario can be avoided in part by the use of vaccines incorporating antigens that represent proteins required for maintenance of the transformed state, progressive growth, or metastasis. Alternatively, one may consider the inclusion of an‐ tigens expressed not by tumor cells themselves but by the supportive stromal cells (whose phenotype is uniquely modified by the TME) that enable the formation of large bulk tumors. We hypothesize that peptides associated with tumor angiogenesis (summarized in Table 1) may provide an ideal source of targets for DC/peptide vaccine design. In effect, targeting the underlying tumor stroma (e.g., vascular cells, pericytes) would disrupt melanoma growth and promote tumor-specific immunity and protection. Our laboratory has previously dem‐ onstrated the ability to successfully treat HLA-A2+ transgenic mice bearing established co‐ lon carcinoma or melanomas using DC-based vaccines containing antigens differentially associated with the tumor vasculature [69]. Animals administered peptide-loaded DC vac‐ cines displayed enhanced protection from established tumor growth and ability, in instances of complete regression, to provide durable protection from dormant disease. Interestingly, active vaccination against tumor stromal antigens led to the corollary cross-priming of T cell responses directed against alternate vascular-associated antigens that were not originally comprised in the vaccine therapy as well as *bona fide* tumor cell-associated antigens. Normal donors and melanoma patients also exhibited immune reactivity to many of the stromal an‐ tigens upon *in vitro* sensitization, indicating that operational tolerance to such "self" anti‐ gens may be broken using a DC/peptide-based vaccination approach [70]. Importantly, this vaccine strategy appears safe in treated mice since we have not observed deleterious immu‐ nologic responses against the normal tissue vasculature, disruptions to the normal cutane‐ ous wound healing process, or aberrations in the fertility/litter size of pre-vaccinated female

(NCT00390338).

animals [69, 70].

Many clinical studies have highlighted the ability of DC-based adoptive therapy to boost resident anti-tumor T cell responses and to mediate corollary clinical activity in patients with melanoma [57-65]. In one of the first reported DC-based therapy trials in the melanoma setting, DCs were harvested from patients (regardless of their HLA type), cultured in the presence of rhGM-CSF and rhIL-4 for one week, and pulsed with melanoma-associated pep‐ tides (e.g., HLA-A2 restricted gp100, tyrosinase, and Melan-A/MART1 peptides) or autolo‐ gous tumor lysates [59]. The cellular vaccines were delivered into tumor uninvolved inguinal lymph nodes at least 4 times at weekly intervals. Eleven out of 16 (69%) enrolled patients developed DTH reactions to intradermal injections of DCs loaded with either vac‐ cine-derived peptides or tumor lysates following DC vaccine therapy. Subsequent analysis of infiltrating T cells in representative biopsied DTH sites revealed peptide-specific reactivi‐ ty to antigenic components of the vaccine. Overall, 2 CR and 3 PR were observed with these same patients also exhibiting vaccine-specific reactivity as evidenced in DTH testing. In a separate phase I clinical trial reported by Ribas and colleagues, GM-CSF/IL-4 *ex vivo* cul‐ tured DCs were loaded with a Melan-A/MART1 peptide and delivered intradermally into metastatic melanoma patients a total of 3 times every 2 weeks alongside tremelimumab (an‐ ti-CTLA-4) treatment [66]. Tetramer and ELISPOT analysis revealed increases in the fre‐ quency of peripheral Melan-A/MART1-reactive CD8+ T cells as a consequence of specific vaccination in 9 of 15 (60%) individuals, although tremelimumab therapy did not appear to enhance Melan-A/MART1 T cell frequency and function. Four vaccinated patients experi‐ enced objective clinical responses (2 CR, 2 PR) with 3 individuals also displaying an im‐ proved MART-1 T cell response post-DC vaccination. Although such studies provide proofof principle, major improvements are still needed in order to achieve durable clinical responses and prolonged survival rates in a majority of patients undergoing autologous DC therapy. A potential improvement to DC activity *in vivo* may reside with how DCs are ma‐ nipulated *ex vivo* following leukopheresis. In cases where DCs are stimulated to an under‐ whelmed (use of GM-CSF/IL-4) or exhausted (use of PGE2) Type-1 state, effector T cells suffer from an inability to effectively mediate anti-tumor responses [49]. One promising DC polarizing method incorporates IL-1β, TNF-α, IFN-α, IFN-γ, and poly-I:C in the *ex vivo* cul‐ turing phase to effectively mature DCs (designated α-DC1). Twenty-two patients with re‐ current malignant glioma were administered up to 4 vaccinations intranodally of α-DC1 loaded with glioma associated antigens at 2 week intervals [67]. At the conclusion of the im‐ munization cycle, 58% of evaluable patients demonstrated a response to at least one antigen‐ ic component of the vaccine based on PBMC specific activity through IFN-γ ELISPOT or tetramer analysis. Upregulated gene expression profiles of Type-1 cytokines (e.g., IFN-α, IFN-γ) and chemokines (e.g., CXCL10) were also observed in PBMCs from α-DC1 treated patients, suggesting that the vaccine therapy enhanced the cytolytic activity and trafficking ability of immune cells. Progression free survival was extended to 12 months in 9 of 22 pa‐ tients receiving the α-DC1 vaccine. The ability of α-DC1 to produce IL-12 (and, hence, stim‐ ulate CD4+ and CD8+ T cell function) correlated to prolonged progression free survival. Based on the safety profile and relative success of this trial, the α-DC1 generation protocol is currently being evaluated in a phase I trial in patients with metastatic melanoma (NCT00390338).

heightened Type-I T cell and antibody responses against the vaccine fusion protein in a ma‐ jority of patients presumably due to the enhanced maturation state of infused activated DCs [56]. Overall survival correlated with improved specific immunity in responding patients suggesting that sipuleucel-T's mechanism of action includes immune targeting of prostate

Many clinical studies have highlighted the ability of DC-based adoptive therapy to boost resident anti-tumor T cell responses and to mediate corollary clinical activity in patients with melanoma [57-65]. In one of the first reported DC-based therapy trials in the melanoma setting, DCs were harvested from patients (regardless of their HLA type), cultured in the presence of rhGM-CSF and rhIL-4 for one week, and pulsed with melanoma-associated pep‐ tides (e.g., HLA-A2 restricted gp100, tyrosinase, and Melan-A/MART1 peptides) or autolo‐ gous tumor lysates [59]. The cellular vaccines were delivered into tumor uninvolved inguinal lymph nodes at least 4 times at weekly intervals. Eleven out of 16 (69%) enrolled patients developed DTH reactions to intradermal injections of DCs loaded with either vac‐ cine-derived peptides or tumor lysates following DC vaccine therapy. Subsequent analysis of infiltrating T cells in representative biopsied DTH sites revealed peptide-specific reactivi‐ ty to antigenic components of the vaccine. Overall, 2 CR and 3 PR were observed with these same patients also exhibiting vaccine-specific reactivity as evidenced in DTH testing. In a separate phase I clinical trial reported by Ribas and colleagues, GM-CSF/IL-4 *ex vivo* cul‐ tured DCs were loaded with a Melan-A/MART1 peptide and delivered intradermally into metastatic melanoma patients a total of 3 times every 2 weeks alongside tremelimumab (an‐ ti-CTLA-4) treatment [66]. Tetramer and ELISPOT analysis revealed increases in the fre‐ quency of peripheral Melan-A/MART1-reactive CD8+ T cells as a consequence of specific vaccination in 9 of 15 (60%) individuals, although tremelimumab therapy did not appear to enhance Melan-A/MART1 T cell frequency and function. Four vaccinated patients experi‐ enced objective clinical responses (2 CR, 2 PR) with 3 individuals also displaying an im‐ proved MART-1 T cell response post-DC vaccination. Although such studies provide proofof principle, major improvements are still needed in order to achieve durable clinical responses and prolonged survival rates in a majority of patients undergoing autologous DC therapy. A potential improvement to DC activity *in vivo* may reside with how DCs are ma‐ nipulated *ex vivo* following leukopheresis. In cases where DCs are stimulated to an under‐ whelmed (use of GM-CSF/IL-4) or exhausted (use of PGE2) Type-1 state, effector T cells suffer from an inability to effectively mediate anti-tumor responses [49]. One promising DC polarizing method incorporates IL-1β, TNF-α, IFN-α, IFN-γ, and poly-I:C in the *ex vivo* cul‐ turing phase to effectively mature DCs (designated α-DC1). Twenty-two patients with re‐ current malignant glioma were administered up to 4 vaccinations intranodally of α-DC1 loaded with glioma associated antigens at 2 week intervals [67]. At the conclusion of the im‐ munization cycle, 58% of evaluable patients demonstrated a response to at least one antigen‐ ic component of the vaccine based on PBMC specific activity through IFN-γ ELISPOT or tetramer analysis. Upregulated gene expression profiles of Type-1 cytokines (e.g., IFN-α, IFN-γ) and chemokines (e.g., CXCL10) were also observed in PBMCs from α-DC1 treated patients, suggesting that the vaccine therapy enhanced the cytolytic activity and trafficking ability of immune cells. Progression free survival was extended to 12 months in 9 of 22 pa‐

carcinoma cells by vaccine-induced T cells.

238 Melanoma - From Early Detection to Treatment

Another way to improve the immunogenicity of autologous DC-based therapy involves the choice of antigenic target for presentation to T cells (i.e., therapeutic selection of the re‐ sponding anti-tumor T cell repertoire for expansion). Most DC-based vaccine trials have in‐ corporated melanoma-associated antigens such as gp100, tyrosinase, Melan-A/MART1 and MAGE in the vaccine formulation. Despite the surprisingly high immunogenic nature of these "self" antigens in vaccinated patients, tumor cells can continue to grow progressively by evading the effector T cell system via various well-described mechanisms [46, 48, 53]. For instance, the tumor mass is composed of a heterogeneous mixture of cancer cells that exhibit a range of defects/deficiencies in the antigen presentation machinery that limits effective presentation of tumor antigen-derived peptides in MHC complexes and leads to poor recog‐ nition by the immune system. As such, a fraction of tumor cells may become "invisible" to the adaptive immune system, resulting in the negative selection of treatment-resistant tumor cells in progressor lesions [68]. This scenario can be avoided in part by the use of vaccines incorporating antigens that represent proteins required for maintenance of the transformed state, progressive growth, or metastasis. Alternatively, one may consider the inclusion of an‐ tigens expressed not by tumor cells themselves but by the supportive stromal cells (whose phenotype is uniquely modified by the TME) that enable the formation of large bulk tumors. We hypothesize that peptides associated with tumor angiogenesis (summarized in Table 1) may provide an ideal source of targets for DC/peptide vaccine design. In effect, targeting the underlying tumor stroma (e.g., vascular cells, pericytes) would disrupt melanoma growth and promote tumor-specific immunity and protection. Our laboratory has previously dem‐ onstrated the ability to successfully treat HLA-A2+ transgenic mice bearing established co‐ lon carcinoma or melanomas using DC-based vaccines containing antigens differentially associated with the tumor vasculature [69]. Animals administered peptide-loaded DC vac‐ cines displayed enhanced protection from established tumor growth and ability, in instances of complete regression, to provide durable protection from dormant disease. Interestingly, active vaccination against tumor stromal antigens led to the corollary cross-priming of T cell responses directed against alternate vascular-associated antigens that were not originally comprised in the vaccine therapy as well as *bona fide* tumor cell-associated antigens. Normal donors and melanoma patients also exhibited immune reactivity to many of the stromal an‐ tigens upon *in vitro* sensitization, indicating that operational tolerance to such "self" anti‐ gens may be broken using a DC/peptide-based vaccination approach [70]. Importantly, this vaccine strategy appears safe in treated mice since we have not observed deleterious immu‐ nologic responses against the normal tissue vasculature, disruptions to the normal cutane‐ ous wound healing process, or aberrations in the fertility/litter size of pre-vaccinated female animals [69, 70].


mentioned, the aberrant dynamics of the tumor vascular architecture and enrichment of reg‐ ulatory cell populations (e.g., MDSC, Treg) in the TME consort to diminish the recruitment, vitality, and tumoricidal activity of immune cells *in situ*. Therefore, the conditional abroga‐ tion of the negative attributes of the TME would be predicted to improve infiltration and function of vaccine-expanded T cell populations, leading to more durable objective clinical responses in melanoma patients as diagramed in Figure 1. What follows are examples of three FDA-approved TKI drugs that could be utilized in DC-based vaccine combination im‐

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241

**Abnormal Tumor Vasculature**

content)

**Reconditioned TME** 



**Figure 1.** Paradigm for effective combination treatment of melanoma. Established vascularized cancers such as mela‐ noma are entrenched with a chaotic blood vessel network and immunosuppressive cell populations. These TME prop‐ erties serve to prevent the intratumoral delivery and function of single-agent cytotoxic therapies, including specific active vaccination. In cases of combined therapeutic strategies where the melanoma-associated vasculature is first modulated through TKI drug sensitization, for example, immature blood vessels (i.e., endothelial cells loosely decorat‐ ed by or absent in pericyte coverage) may be disrupted, resulting in a normoxic TME with reduced interstitial pressure and acidity. Frequencies of MDSC and Treg cells are also minimized through mechanisms that are not entirely clear.

↑ Hypoxia (Low O<sup>2</sup>

↑ Acidity

↑ Interstitial pressure

MDSC Treg Mature DC Effector T cell Blood vessel

munotherapies to achieve this goal.

TKI therapy

DC vaccination

+

**Table 1.** Candidate melanoma-associated vascular peptides for DC vaccine design. CD8+ T cell response summaries are provided from previous work by our laboratory [69, 70]. Naïve HLA-A2+ transgenic mice were vaccinated biweekly with DCs pre-pulsed with the appropriate antigen-derived peptide. One week following the second DC vaccine, splenic CD8+ T cells were harvested and co-cultured 48 hours with the HLA-A2+ T2 cell line pulsed with the relevant peptide. CD8+ T cell elaboration of IFN-γ (as a read-out for Type-1 activity) was then determined through ELISA. Human CD8+ T cell responses to stromal peptides were determined by first isolating PBMCs and stimulating cells in the presence of antigen-loaded autologous DCs for 1 week. Normal donor samples underwent 2 rounds of IVS while PBMCs obtained from melanoma patients were subjected to 1 round of IVS. CD8+ T cell IFN-γ expression was assessed as similarly described for HLA-A2+ transgenic mice. Abbreviations used: AA, amino acid; P, pericyte; VEC, vascular endothelial cell; -, No observed activity; +, low activity; ++, medium activity; +++, high activity; IVS, *in vitro* sensitization

#### **3.4. Combining small molecule drugs with DC vaccination**

In addition to empirically improving DC vaccine design (e.g., via the *ex vivo* conditioning of the APC and a rationale selection of the included antigenic targets), the effectiveness of such treatments would be expected to improve by mitigating the functional constraints on vac‐ cine-induced T effector cells imposed by the generally suppressive TME. As previously mentioned, the aberrant dynamics of the tumor vascular architecture and enrichment of reg‐ ulatory cell populations (e.g., MDSC, Treg) in the TME consort to diminish the recruitment, vitality, and tumoricidal activity of immune cells *in situ*. Therefore, the conditional abroga‐ tion of the negative attributes of the TME would be predicted to improve infiltration and function of vaccine-expanded T cell populations, leading to more durable objective clinical responses in melanoma patients as diagramed in Figure 1. What follows are examples of three FDA-approved TKI drugs that could be utilized in DC-based vaccine combination im‐ munotherapies to achieve this goal.

**Stromal antigen**

sensitization

**Cell**

240 Melanoma - From Early Detection to Treatment

**expression AA positions Peptide sequence**

DLK1 P 269-277 RLTPGVHEL ++ + ++

EphA2 VEC 883-891 TLADFDPRV +++ + ++ HBB P 31-39 RLLVVYPWT + + ++

NG2 P 770-778 TLSNLSFPV - - ++

NP1 P 331-339 GLLRFVTAV + + +++

NP2 P 214-222 DIWDGIPHV - - ++

PDGFRβ P 890-898 ILLWEIFTL +++ + + PSMA VEC 441-450 LLQERGVAYI + - + RGS5 P 5-13 LAALPHSCL + - ++ TEM1 VEC/P 691-700 LLVPTCVFLV + ++ ++ VEGFR1 VEC/P 770-778 TLFWLLLTL ++ + +

**Table 1.** Candidate melanoma-associated vascular peptides for DC vaccine design. CD8+ T cell response summaries are provided from previous work by our laboratory [69, 70]. Naïve HLA-A2+ transgenic mice were vaccinated biweekly with DCs pre-pulsed with the appropriate antigen-derived peptide. One week following the second DC vaccine, splenic CD8+ T cells were harvested and co-cultured 48 hours with the HLA-A2+ T2 cell line pulsed with the relevant peptide. CD8+ T cell elaboration of IFN-γ (as a read-out for Type-1 activity) was then determined through ELISA. Human CD8+ T cell responses to stromal peptides were determined by first isolating PBMCs and stimulating cells in the presence of antigen-loaded autologous DCs for 1 week. Normal donor samples underwent 2 rounds of IVS while PBMCs obtained from melanoma patients were subjected to 1 round of IVS. CD8+ T cell IFN-γ expression was assessed as similarly described for HLA-A2+ transgenic mice. Abbreviations used: AA, amino acid; P, pericyte; VEC, vascular endothelial cell; -, No observed activity; +, low activity; ++, medium activity; +++, high activity; IVS, *in vitro*

In addition to empirically improving DC vaccine design (e.g., via the *ex vivo* conditioning of the APC and a rationale selection of the included antigenic targets), the effectiveness of such treatments would be expected to improve by mitigating the functional constraints on vac‐ cine-induced T effector cells imposed by the generally suppressive TME. As previously

**3.4. Combining small molecule drugs with DC vaccination**

**CD8+ T cell response**

**HLA-A2+ normal donors**

**HLA-A2+ melanoma patients**

**HLA-A2+ transgenic mice**

310-318 ILGVLTSLV ++ + ++ 328-336 FLNKCETWV +++ + ++

105-114 RLLGNVLVCV + + +

2238-2246 LILPLLFYL + - ++

433-441 GMLGMVSGL ++ + +++ 869-877 VLLGAVCGV +++ + +

328-336 YLQVDLRFL - - ++

**Figure 1.** Paradigm for effective combination treatment of melanoma. Established vascularized cancers such as mela‐ noma are entrenched with a chaotic blood vessel network and immunosuppressive cell populations. These TME prop‐ erties serve to prevent the intratumoral delivery and function of single-agent cytotoxic therapies, including specific active vaccination. In cases of combined therapeutic strategies where the melanoma-associated vasculature is first modulated through TKI drug sensitization, for example, immature blood vessels (i.e., endothelial cells loosely decorat‐ ed by or absent in pericyte coverage) may be disrupted, resulting in a normoxic TME with reduced interstitial pressure and acidity. Frequencies of MDSC and Treg cells are also minimized through mechanisms that are not entirely clear.

Consequently, vaccine-initiated effector T cells can better traffick into tumors and exert their anti-tumor functions. Mature DCs are also able to infiltrate the tumor lesion and sample material from dying cells or necrotic tissue for crosspresentation purposes to unknown/untargeted tumor associated antigens, leading to activation of a broad T cell rep‐ ertoire that is competent to promote durable anti-tumor immunity. Abbreviations used: TKI, tyrosine kinase inhibitor; MDSC, myeloid-derived suppressor cell; Treg, regulatory T cell; DC, dendritic cell; TME, tumor microenvironment

nib on DC/peptide-based vaccination on established melanoma growth in murine models [82]. Melanoma-bearing mice administered axitinib and specific vaccines were protected from tumor growth and displayed enhanced survival for up to 80 days following melano‐ ma implantation. Axitinib-sensitization improved the trafficking and retention of vaccineinduced CD8+ T cells in the TME, with the Type-1 functionality (as assessed by IFNγ expression) of CD8+ T cells elevated in both the tumor site and the TDLN. Similar to our observations with sunitinib [39], axitinib reduced systemic frequencies of MDSCs and Tregs and promoted a Type-1 TME, as evidenced by the upregulated expression of Tbet,

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Dasatinib has already been reported to selectively abrogate mutated KIT activity in human melanomas [19, 83]. This TKI also inhibits other tyrosine kinases such as the Src family of kinases (impacting PI3K-AKT signaling) involved in melanoma adhesion, motility, and in‐ vasion [84, 85]. As a monotherapy, dasatinib was well-tolerated in melanoma patients, yield‐ ing an objective response rate comparable to alternate current first-line treatment options [18]. Dasatinib diminishes tumor angiogenesis by inhibiting the tyrosine kinases EphA2 and PDGFR that play significant roles in endothelial and pericyte biology, respectively [84]. In unpublished results from our laboratory, dasatinib mediates anti-TME effects that are simi‐ lar to sunitinib and axitinib in melanoma-bearing mice [39, 82]. Animals treated with dasati‐ nib undergo a restructuring of the tumor vasculature in association with reduced hypoxia and MDSC/Treg frequencies and increased accumulation of T effector cells in the TME, par‐ ticularly when combined with a DC/peptide-based vaccine. The combined therapy also yielded greatest objective clinical benefit when compared with either monotherapeutic ap‐ proach. Overall, these studies have supported the design of a pilot phase II trial (dasatinib + DC/tumor stromal antigen-based vaccine) at the University of Pittsburgh planned to begin enrolling patients in Q4 2012. In this trial, HLA-A2+ patients with advanced-stage melano‐ ma will be administered dasatinib and an autologous αDC1/peptide vaccine, with frequen‐ cies of antigen-specific T cells monitored in patient blood and tumor biopsies over time

The emergence of ipilimumab and vemurafenib as treatment alternatives to the long-stand‐ ing DTIC-, IL-2-, and IFN-α-based therapies attests to progress made in treating patients with metastatic melanoma. Although the genetic heterogeneity of melanoma cells has con‐ founded high-throughput sequencing technologies, patterns of molecular aberrations are be‐ coming clearer and help support the clinical application of FDA-approved small molecule drugs (such as TKIs) as therapeutic options in eligible patients. Select TKIs (e.g., sunitinib, axitinib, dasatinib) not only directly inhibit melanoma growth and progression by specifical‐ ly disrupting cell intrinsic signaling pathways, but these drugs indirectly perturb tumori‐ genesis based on their "normalizing" effects on the TME. Central to this therapeutic paradigm is the ability of the drugs to recondition the chaotic architecture and fluid dynam‐ ics of the blood vasculature in the TME. The short-term consequences of TKI sensitization

IFN-γ, CXCR3, and CXCL10 gene transcripts.

along with objective clinical responses.

**4. Conclusions**

Sunitinib binds to and inhibits a range of tyrosine kinases including the vascular associated molecules VEGFR and PDGFR. The drug is approved for use in patients with metastatic re‐ nal cell carcinoma (mRCC) or gastrointestinal stromal tumors, where most patients respond favorably to treatment in the short-term [71, 72]. In one recently reported phase I trial, meta‐ static melanoma patients harboring KIT mutations were administered sunitinib using the FDA-approved regimen of 50 mg/day for 4 weeks followed by 2 weeks off drug [73]. Out of 10 evaluable patients, 1 individual had a CR that lasted 15 months while 2 PR endured be‐ tween 1-7 months. A separate clinical study, reported on the ability of sunitinib to work in concert with docetaxel therapy in patients with solid tumors including melanoma [74]. Two PR were confirmed in a total of 12 metastatic melanoma patients treated with the combina‐ tion regimen, supporting a potential tumor vascular "reconditioning" role of sunitinib in im‐ proving the delivery and function of cytotoxic therapies within the TME. Our own animal studies support a similar paradigm for combination immunotherapies [39]. Protection from established melanoma progression (based on tumor growth kinetics and survival) were en‐ hanced in mice receiving both sunitinib and DC/peptide-based vaccination versus either agent administered as a monotherapy. Sunitinib co-treatment facilitated the recruitment of DC-"primed" Type-1 CD8+ T cells into melanoma lesions based in part on the upregulated expression of VCAM-1 (on vascular endothelial cells) and CXCR3 ligand chemokines (e.g., CXCL9, CXCL10, CXCL11) within the TME. This TKI also reduced frequencies of immuno‐ suppressive cell populations such as MDSC and Tregs in the tumor and tumor draining lymph node (TDLN), which was associated with increased cytotoxic potential mediated by vaccine-induced CD8+ T cells. Sunitinib therapy has similarly been reported to prevent the peripheral accumulations of MDSCs and Tregs in mRCC patients [75-77]. Although the mo‐ lecular mechanism underlying these alterations remains an open question, sunitinib inhibits STAT3 activation (via inhibition of upstream tyrosine kinases) which may prove core to its perceived anti-tumor actions [39, 75].

Axitinib is a potent TKI targeting VEGFRs (VEGFR1, 2, and 3) that support tumor angio‐ genesis [30, 78]. Following the completion of a recent phase III trial [79], axitinib was granted approval by the FDA as a second-line therapy for mRCC patients refractory to first-line treatment options including sunitinib. Axitinib has also been used to treat pa‐ tients with melanoma. Pre-clinical studies have supported a role for axitinib monothera‐ py to disrupt angiogenesis and tumor formation in xenograft melanoma models [80]. A multicenter phase II trial also justified the continued use of axitinib-based treatment in metastatic melanoma patients [81]. Individuals receiving this TKI experienced reductions of VEGFR2 and VEGFR3 and increased levels of soluble VEGF in their plasma. Treat‐ ment with axitinib was associated with an overall objective response rate of 18.8%, which is comparable to historical response rates for chemotherapy and IL-2-based therapies. Giv‐ en the relative clinical success for axitinib monotherapy, we assessed the impact of axiti‐ nib on DC/peptide-based vaccination on established melanoma growth in murine models [82]. Melanoma-bearing mice administered axitinib and specific vaccines were protected from tumor growth and displayed enhanced survival for up to 80 days following melano‐ ma implantation. Axitinib-sensitization improved the trafficking and retention of vaccineinduced CD8+ T cells in the TME, with the Type-1 functionality (as assessed by IFNγ expression) of CD8+ T cells elevated in both the tumor site and the TDLN. Similar to our observations with sunitinib [39], axitinib reduced systemic frequencies of MDSCs and Tregs and promoted a Type-1 TME, as evidenced by the upregulated expression of Tbet, IFN-γ, CXCR3, and CXCL10 gene transcripts.

Dasatinib has already been reported to selectively abrogate mutated KIT activity in human melanomas [19, 83]. This TKI also inhibits other tyrosine kinases such as the Src family of kinases (impacting PI3K-AKT signaling) involved in melanoma adhesion, motility, and in‐ vasion [84, 85]. As a monotherapy, dasatinib was well-tolerated in melanoma patients, yield‐ ing an objective response rate comparable to alternate current first-line treatment options [18]. Dasatinib diminishes tumor angiogenesis by inhibiting the tyrosine kinases EphA2 and PDGFR that play significant roles in endothelial and pericyte biology, respectively [84]. In unpublished results from our laboratory, dasatinib mediates anti-TME effects that are simi‐ lar to sunitinib and axitinib in melanoma-bearing mice [39, 82]. Animals treated with dasati‐ nib undergo a restructuring of the tumor vasculature in association with reduced hypoxia and MDSC/Treg frequencies and increased accumulation of T effector cells in the TME, par‐ ticularly when combined with a DC/peptide-based vaccine. The combined therapy also yielded greatest objective clinical benefit when compared with either monotherapeutic ap‐ proach. Overall, these studies have supported the design of a pilot phase II trial (dasatinib + DC/tumor stromal antigen-based vaccine) at the University of Pittsburgh planned to begin enrolling patients in Q4 2012. In this trial, HLA-A2+ patients with advanced-stage melano‐ ma will be administered dasatinib and an autologous αDC1/peptide vaccine, with frequen‐ cies of antigen-specific T cells monitored in patient blood and tumor biopsies over time along with objective clinical responses.

## **4. Conclusions**

Consequently, vaccine-initiated effector T cells can better traffick into tumors and exert their anti-tumor functions. Mature DCs are also able to infiltrate the tumor lesion and sample material from dying cells or necrotic tissue for crosspresentation purposes to unknown/untargeted tumor associated antigens, leading to activation of a broad T cell rep‐ ertoire that is competent to promote durable anti-tumor immunity. Abbreviations used: TKI, tyrosine kinase inhibitor; MDSC, myeloid-derived suppressor cell; Treg, regulatory T cell; DC, dendritic cell; TME, tumor microenvironment

Sunitinib binds to and inhibits a range of tyrosine kinases including the vascular associated molecules VEGFR and PDGFR. The drug is approved for use in patients with metastatic re‐ nal cell carcinoma (mRCC) or gastrointestinal stromal tumors, where most patients respond favorably to treatment in the short-term [71, 72]. In one recently reported phase I trial, meta‐ static melanoma patients harboring KIT mutations were administered sunitinib using the FDA-approved regimen of 50 mg/day for 4 weeks followed by 2 weeks off drug [73]. Out of 10 evaluable patients, 1 individual had a CR that lasted 15 months while 2 PR endured be‐ tween 1-7 months. A separate clinical study, reported on the ability of sunitinib to work in concert with docetaxel therapy in patients with solid tumors including melanoma [74]. Two PR were confirmed in a total of 12 metastatic melanoma patients treated with the combina‐ tion regimen, supporting a potential tumor vascular "reconditioning" role of sunitinib in im‐ proving the delivery and function of cytotoxic therapies within the TME. Our own animal studies support a similar paradigm for combination immunotherapies [39]. Protection from established melanoma progression (based on tumor growth kinetics and survival) were en‐ hanced in mice receiving both sunitinib and DC/peptide-based vaccination versus either agent administered as a monotherapy. Sunitinib co-treatment facilitated the recruitment of DC-"primed" Type-1 CD8+ T cells into melanoma lesions based in part on the upregulated expression of VCAM-1 (on vascular endothelial cells) and CXCR3 ligand chemokines (e.g., CXCL9, CXCL10, CXCL11) within the TME. This TKI also reduced frequencies of immuno‐ suppressive cell populations such as MDSC and Tregs in the tumor and tumor draining lymph node (TDLN), which was associated with increased cytotoxic potential mediated by vaccine-induced CD8+ T cells. Sunitinib therapy has similarly been reported to prevent the peripheral accumulations of MDSCs and Tregs in mRCC patients [75-77]. Although the mo‐ lecular mechanism underlying these alterations remains an open question, sunitinib inhibits STAT3 activation (via inhibition of upstream tyrosine kinases) which may prove core to its

Axitinib is a potent TKI targeting VEGFRs (VEGFR1, 2, and 3) that support tumor angio‐ genesis [30, 78]. Following the completion of a recent phase III trial [79], axitinib was granted approval by the FDA as a second-line therapy for mRCC patients refractory to first-line treatment options including sunitinib. Axitinib has also been used to treat pa‐ tients with melanoma. Pre-clinical studies have supported a role for axitinib monothera‐ py to disrupt angiogenesis and tumor formation in xenograft melanoma models [80]. A multicenter phase II trial also justified the continued use of axitinib-based treatment in metastatic melanoma patients [81]. Individuals receiving this TKI experienced reductions of VEGFR2 and VEGFR3 and increased levels of soluble VEGF in their plasma. Treat‐ ment with axitinib was associated with an overall objective response rate of 18.8%, which is comparable to historical response rates for chemotherapy and IL-2-based therapies. Giv‐ en the relative clinical success for axitinib monotherapy, we assessed the impact of axiti‐

perceived anti-tumor actions [39, 75].

242 Melanoma - From Early Detection to Treatment

The emergence of ipilimumab and vemurafenib as treatment alternatives to the long-stand‐ ing DTIC-, IL-2-, and IFN-α-based therapies attests to progress made in treating patients with metastatic melanoma. Although the genetic heterogeneity of melanoma cells has con‐ founded high-throughput sequencing technologies, patterns of molecular aberrations are be‐ coming clearer and help support the clinical application of FDA-approved small molecule drugs (such as TKIs) as therapeutic options in eligible patients. Select TKIs (e.g., sunitinib, axitinib, dasatinib) not only directly inhibit melanoma growth and progression by specifical‐ ly disrupting cell intrinsic signaling pathways, but these drugs indirectly perturb tumori‐ genesis based on their "normalizing" effects on the TME. Central to this therapeutic paradigm is the ability of the drugs to recondition the chaotic architecture and fluid dynam‐ ics of the blood vasculature in the TME. The short-term consequences of TKI sensitization are impressive and include a reversal of hypoxia, acidosis, and interstitial pressure in the TME, which allows for a corollary improvement in the accumulation and action of co-ap‐ plied cytotoxic therapies (including immunotherapies).

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Combinational immunotherapies hold great promise in minimizing/preventing the emer‐ gence and progression of (same) therapy-resistant melanoma populations, as has typically been observed in cases of single-agent treatment strategies. These approaches also have po‐ tential to result in a state of perpetual disease dormancy which may extend patient overall survival [69]. The current challenge to the field is to determine the best combination (dosing and scheduling) of agents to best affect a state of durable clinical benefit in the advancestage disease setting. From our work, and that of many others, immunotherapy represents one promising component of such combined treatment strategies, particularly when inte‐ grated with agents that act as immune adjuvants, inhibitors of immune regulatory cells, and "normalizers" of the TME. Preclinical studies have clearly justified the combined strategy of TKI drug therapy alongside specific DC/peptide-based vaccination. In particular, TKI ad‐ ministration essentially serves as an "immune adjuvant" by reversing the inherent immuno‐ suppression of the TME upon diminishing frequencies of suppressive cell populations and physically manipulating the tumor vasculature architecture. Vaccine-initiated effector T cells are then able to more effectively infiltrate a tumor lesion in order to perform their clini‐ cally-beneficial cytolytic functions. Prospective clinical trials will test the validity of this op‐ erational biologic paradigm on patient outcome and define a series of safe and effective combination treatment options for melanoma patients.

## **Acknowledgments**

This work was supported by NIH grants P01 CA100327, R01 CA114071, R01 CA140375 and P50 CA121973 (to W.J.S.) and the University of Pittsburgh Cancer Center Support Grant (CCSG; P30 CA047904). D.B.L. was supported by a Postdoctoral Fellowship (PF-11-151-01- LIB) from the American Cancer Society.

## **Author details**

Devin B. Lowe1 , Jennifer L. Taylor1 and Walter J. Storkus1,2\*

\*Address all correspondence to: storkuswj@upmc.edu

1 Dermatology and Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

2 University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

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are impressive and include a reversal of hypoxia, acidosis, and interstitial pressure in the TME, which allows for a corollary improvement in the accumulation and action of co-ap‐

Combinational immunotherapies hold great promise in minimizing/preventing the emer‐ gence and progression of (same) therapy-resistant melanoma populations, as has typically been observed in cases of single-agent treatment strategies. These approaches also have po‐ tential to result in a state of perpetual disease dormancy which may extend patient overall survival [69]. The current challenge to the field is to determine the best combination (dosing and scheduling) of agents to best affect a state of durable clinical benefit in the advancestage disease setting. From our work, and that of many others, immunotherapy represents one promising component of such combined treatment strategies, particularly when inte‐ grated with agents that act as immune adjuvants, inhibitors of immune regulatory cells, and "normalizers" of the TME. Preclinical studies have clearly justified the combined strategy of TKI drug therapy alongside specific DC/peptide-based vaccination. In particular, TKI ad‐ ministration essentially serves as an "immune adjuvant" by reversing the inherent immuno‐ suppression of the TME upon diminishing frequencies of suppressive cell populations and physically manipulating the tumor vasculature architecture. Vaccine-initiated effector T cells are then able to more effectively infiltrate a tumor lesion in order to perform their clini‐ cally-beneficial cytolytic functions. Prospective clinical trials will test the validity of this op‐ erational biologic paradigm on patient outcome and define a series of safe and effective

This work was supported by NIH grants P01 CA100327, R01 CA114071, R01 CA140375 and P50 CA121973 (to W.J.S.) and the University of Pittsburgh Cancer Center Support Grant (CCSG; P30 CA047904). D.B.L. was supported by a Postdoctoral Fellowship (PF-11-151-01-

and Walter J. Storkus1,2\*

1 Dermatology and Immunology, University of Pittsburgh School of Medicine, Pittsburgh,

plied cytotoxic therapies (including immunotherapies).

244 Melanoma - From Early Detection to Treatment

combination treatment options for melanoma patients.

**Acknowledgments**

**Author details**

Devin B. Lowe1

PA, USA

LIB) from the American Cancer Society.

, Jennifer L. Taylor1

\*Address all correspondence to: storkuswj@upmc.edu

2 University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA


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**Section 2**

**Melanoma Treatment Approaches**

**Melanoma Treatment Approaches**

**Chapter 10**

**Management of In-Transit Malignant Melanoma**

In-transit melanoma is a unique pattern of recurrence that occurs in up to ten percent of pa‐ tients with melanoma. In-transit disease denotes multifocal tumor deposits occurring be‐ tween the site of the primary lesion and its regional draining lymph node basin [1, 2]. It is an independent adverse prognostic factor and is frequently associated with distant metastasis. This pattern of recurrence represents a challenging management problem, but provides unique treatment modalities as well. In addition, studying in-transit melanoma has the po‐ tential to shed additional light on melanoma biology. The goal of this chapter is to discuss the presentation, underlying disease biology, and various current treatment strategies for

The nomenclature used for in-transit melanoma can be confusing, in part because a number of different terms have traditionally been used in the literature to describe what is most like‐ ly the same oncologic process. Historically, terms such as locoregional recurrence, satellito‐ sis, and in-transit disease have all been used with varying definitions and intentions. Historically, satellitosis has been defined as locoregional recurrence, not lying within the re‐ gional nodal basin, that is located within either 5cm of the initial lesion or 2cm of the exci‐ sion scar, whereas the term in-transit disease has been defined as such a recurrence occurring at greater distances from the initial lesion or scar, respectively. In either case, such lesions likely represent tumor deposits growing along routes of lymphatic drainage. More recently, it has become apparent that for locoregional recurrence, distance from the primary

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

© 2013 Speicher 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,

Paul J. Speicher, Douglas S. Tyler and Paul J. Mosca

Additional information is available at the end of the chapter

this unique pattern of recurrence in melanoma.

http://dx.doi.org/10.5772/53618

**1. Introduction**

**2. Background**

**2.1. Nomenclature and staging**

## **Management of In-Transit Malignant Melanoma**

Paul J. Speicher, Douglas S. Tyler and Paul J. Mosca

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53618

## **1. Introduction**

In-transit melanoma is a unique pattern of recurrence that occurs in up to ten percent of pa‐ tients with melanoma. In-transit disease denotes multifocal tumor deposits occurring be‐ tween the site of the primary lesion and its regional draining lymph node basin [1, 2]. It is an independent adverse prognostic factor and is frequently associated with distant metastasis. This pattern of recurrence represents a challenging management problem, but provides unique treatment modalities as well. In addition, studying in-transit melanoma has the po‐ tential to shed additional light on melanoma biology. The goal of this chapter is to discuss the presentation, underlying disease biology, and various current treatment strategies for this unique pattern of recurrence in melanoma.

## **2. Background**

## **2.1. Nomenclature and staging**

The nomenclature used for in-transit melanoma can be confusing, in part because a number of different terms have traditionally been used in the literature to describe what is most like‐ ly the same oncologic process. Historically, terms such as locoregional recurrence, satellito‐ sis, and in-transit disease have all been used with varying definitions and intentions. Historically, satellitosis has been defined as locoregional recurrence, not lying within the re‐ gional nodal basin, that is located within either 5cm of the initial lesion or 2cm of the exci‐ sion scar, whereas the term in-transit disease has been defined as such a recurrence occurring at greater distances from the initial lesion or scar, respectively. In either case, such lesions likely represent tumor deposits growing along routes of lymphatic drainage. More recently, it has become apparent that for locoregional recurrence, distance from the primary

© 2013 Speicher 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.

lesion to the site of recurrence does not carry significant prognostic value [3-6]. Accordingly, the most recent AJCC staging system for melanoma does not differentiate between in-transit lesions and satellitosis in the assignment of stage, both being designated as N2 or N3 dis‐ ease, depending on regional node status [7]. Thus, in an effort to address the ambiguity aris‐ ing from nomenclature, many authors have advocated for eliminating the term satellitosis, instead referring to all regional non-nodal metastatic disease as in-transit disease.

the primary tumor and its lymphatic drainage basin. For non-extremity disease, the distribu‐ tion can be even more variable, with widespread tumor burden on the head, neck or trunk,

Management of In-Transit Malignant Melanoma

http://dx.doi.org/10.5772/53618

257

**Figure 1.** Examples of in-transit melanoma of the arm (left) and leg (right). Note the distribution and extent of dis‐ ease, making these presentations very poor candidates for surgical excision. On the left, there is evidence of in-transit metastases both within the area of previous skin flap, as well as extending more proximally along its course of lym‐

In-transit melanoma is a relatively uncommon phenomenon, with fewer than 10% of mela‐ nomas recurring as in-transit disease [1, 11]. This accounts for approximately 12-22% of all recurrences, although this number is difficult to determine with accuracy due to ambiguity regarding terminology used to describe local recurrence versus regional in-transit disease [12-14]. Stage of disease appears to be the most important factor that predicts the develop‐ ment of in-transit metastasis. The presence of associated nodal disease significantly increas‐ es risk of in-transit recurrence, with one study reporting incidence as high as 31% when three or more positive nodes were present [12]. Location itself also appears to be a factor, with a higher incidence of in-transit disease in the lower extremities compared to the upper extremities [15]. Of note, some earlier authors observed that surgical lymph node dissection may lead to increased risk of recurrence as in-transit disease, an area of some debate. This is postulated to be a result of lymphatic trapping, whereby dissection of the draining lymph node basin removes the potential outflow of lymphatic tumor deposits, possibly leading to increased likelihood of in-transit disease. In larger, more recent studies, however, neither sentinel lymph node biopsy nor lymphadenectomy were found to have any effect on the in‐

phatic drainage. On the right, there is extensive disease extending up to the inguinal crease.

**2.3. Incidence**

cidence of in-transit metastases [16-19].

depending on the location of the primary melanoma.


**Table 1.** Breakdown of AJCC staging for stage III melanoma [7].

An additional and equally important point of clarification is the distinction between actual local recurrence and in-transit disease. True local recurrence is defined as a primary tumor that recurs as a result of incomplete primary excision, and is confined to or contiguous with an excision scar and bearing an in situ component [8]. As this carries a much better progno‐ sis, it must be distinguished from potentially similar appearing in-transit disease found in close proximity to a prior excisional scar.

#### **2.2. Presentation**

By definition, in-transit melanoma represents advanced stage disease, and such recurrences are typically discovered months after the initial management of a primary lesion. In most series, this disease-free interval to recurrence as in-transit disease ranges from 12-16 months [9, 10]. The clinical presentation can be quite variable, but usually involves anywhere from one to upwards of one-hundred small cutaneous or subcutaneous nodules. The lesions themselves can differ significantly in size, ranging from sub-millimeter diameter to well over one centimeter. They may take the form of superficial cutaneous (also called epidermo‐ tropic) or deeper subcutaneous tumors. For extremity-based disease, the lesions may be clustered near the primary lesion, or may involve the entire extremity extending between the primary tumor and its lymphatic drainage basin. For non-extremity disease, the distribu‐ tion can be even more variable, with widespread tumor burden on the head, neck or trunk, depending on the location of the primary melanoma.

**Figure 1.** Examples of in-transit melanoma of the arm (left) and leg (right). Note the distribution and extent of dis‐ ease, making these presentations very poor candidates for surgical excision. On the left, there is evidence of in-transit metastases both within the area of previous skin flap, as well as extending more proximally along its course of lym‐ phatic drainage. On the right, there is extensive disease extending up to the inguinal crease.

#### **2.3. Incidence**

lesion to the site of recurrence does not carry significant prognostic value [3-6]. Accordingly, the most recent AJCC staging system for melanoma does not differentiate between in-transit lesions and satellitosis in the assignment of stage, both being designated as N2 or N3 dis‐ ease, depending on regional node status [7]. Thus, in an effort to address the ambiguity aris‐ ing from nomenclature, many authors have advocated for eliminating the term satellitosis,

instead referring to all regional non-nodal metastatic disease as in-transit disease.

**Stage T N M**

1-3 nodes (clinically detectable), *OR*

1-3 nodes (clinically detectable), *OR*

greater than 4 positive nodes

An additional and equally important point of clarification is the distinction between actual local recurrence and in-transit disease. True local recurrence is defined as a primary tumor that recurs as a result of incomplete primary excision, and is confined to or contiguous with an excision scar and bearing an in situ component [8]. As this carries a much better progno‐ sis, it must be distinguished from potentially similar appearing in-transit disease found in

By definition, in-transit melanoma represents advanced stage disease, and such recurrences are typically discovered months after the initial management of a primary lesion. In most series, this disease-free interval to recurrence as in-transit disease ranges from 12-16 months [9, 10]. The clinical presentation can be quite variable, but usually involves anywhere from one to upwards of one-hundred small cutaneous or subcutaneous nodules. The lesions themselves can differ significantly in size, ranging from sub-millimeter diameter to well over one centimeter. They may take the form of superficial cutaneous (also called epidermo‐ tropic) or deeper subcutaneous tumors. For extremity-based disease, the lesions may be clustered near the primary lesion, or may involve the entire extremity extending between

any combination of positive nodes and in-transit

in-transit lesions

in-transit lesions, *OR*

disease, *OR*

**Table 1.** Breakdown of AJCC staging for stage III melanoma [7].

close proximity to a prior excisional scar.

1-3 nodes (not clinically detectable) No distant disease

1-3 nodes (not clinically detectable) No distant disease

No distant disease

No distant disease

IIIA Any depth,

IIIB Any depth,

IIIC Any depth,

**2.2. Presentation**

*Without* ulceration

256 Melanoma - From Early Detection to Treatment

*With* ulceration

*With* ulceration

Any depth, *Without* ulceration

> In-transit melanoma is a relatively uncommon phenomenon, with fewer than 10% of mela‐ nomas recurring as in-transit disease [1, 11]. This accounts for approximately 12-22% of all recurrences, although this number is difficult to determine with accuracy due to ambiguity regarding terminology used to describe local recurrence versus regional in-transit disease [12-14]. Stage of disease appears to be the most important factor that predicts the develop‐ ment of in-transit metastasis. The presence of associated nodal disease significantly increas‐ es risk of in-transit recurrence, with one study reporting incidence as high as 31% when three or more positive nodes were present [12]. Location itself also appears to be a factor, with a higher incidence of in-transit disease in the lower extremities compared to the upper extremities [15]. Of note, some earlier authors observed that surgical lymph node dissection may lead to increased risk of recurrence as in-transit disease, an area of some debate. This is postulated to be a result of lymphatic trapping, whereby dissection of the draining lymph node basin removes the potential outflow of lymphatic tumor deposits, possibly leading to increased likelihood of in-transit disease. In larger, more recent studies, however, neither sentinel lymph node biopsy nor lymphadenectomy were found to have any effect on the in‐ cidence of in-transit metastases [16-19].

#### **2.4. Outcomes**

The presence of in-transit metastases indicates either N2 or N3 status under the current AJCC TNM system, and is classified as stage IIIB or C disease, respectively. In-transit mela‐ noma carries a poor prognosis, with 5-year survival rates ranging from 25% to 30% in most reports [12, 20, 21]. Additionally, the presence or absence of regional lymph node disease is of significant prognostic value; the combination of nodal metastasis and in-transit melano‐ ma comprise stage IIIC disease, which is associated with a poorer outcome than stage IIIB (40% vs. 59% five-year survival, respectively) [7]. There is a high incidence of occult distant metastasis in the presence of in-transit melanoma, but this is not universally the case. Stud‐ ies examining the outcomes of major amputation for the treatment of this pattern of recur‐ rence have identified a number of patients who experience a complete and durable response and have demonstrated five-year survival rates ranging from 21-32% [22-26]. This indicates that a significant minority of patients with in-transit metastases have disease that is truly limited to the extremity at the time of detection. Nonetheless, it is essential that distant metastases be ruled out when staging patients with in-transit melanoma, since treatment op‐ tions and prognosis may differ substantially when measurable distant disease is present.

**4.1. Local management**

surgical margins is usually all that is required.

mains suboptimal in many situations.

**4.2. Radiation therapy**

local surgical excision regarding indications and prognosis.

Distinguishing in-transit disease from true local recurrence is of great importance, as the management and prognosis differ substantially. Local recurrence, or tumor confined to or contiguous with an excision scar and bearing an in situ component, should be managed sim‐ ilarly to the primary lesion with wide local excision. For in-transit disease, however, it is generally accepted that the wide local excision margin guidelines applicable to primary mel‐ anomas need not be applied. In-transit metastases are generally very clearly demarcated his‐ tologically from surrounding tissue, and complete macroscopic excision with negative

Management of In-Transit Malignant Melanoma

http://dx.doi.org/10.5772/53618

259

In addition to wide local excision, there has been significant interest in other forms of local therapy for melanoma lesions, including laser ablation, external beam radiation, and intrale‐ sional injections. Irrespective of modality, these should all be thought of as equivalents to

Laser therapy was first described in 1973, and has gained favor in the local treatment of intransit disease that is not amenable to surgical excision, such as when the disease is too ex‐ tensive [29]. It is most useful in patients with a large number of small in-transit lesions, but its advantages and utility decrease as lesions increase in size [30]. For tumors smaller than approximately 3mm, the entire lesion can be ablated using a carbon dioxide laser, though larger lesions must be circumscribed using the laser and subsequently excised with forceps.

Intralesional injections have also been used in the treatment of in-transit melanoma with some success. The most commonly used therapies include bacillus Calmette-Guérin (BCG), dinitrochlorobenzene (DNCB), and interferon-alpha (INF-α), and IL-2. Small stud‐ ies have demonstrated complete response rates of 31-63% (overall response 45-91%), al‐ though long-term survival, when reported, remained unfortunately low [31-33]. This suggests that if surgical excision is not a viable option, intralesional injection is a reasona‐ ble alternative. More recently, electrochemotherapy (ECT) has gained popularity as local alternative to radiotherapy and laser ablation. This technique relies on using high intensi‐ ty electric pulses to allow intracellular delivery of cytotoxic drugs, such as cisplatin and bleomycin, via intralesional injection [34]. Complete response rates have been reported as 53-89% (overall response 84-99%), with minimal systemic toxicity [35-37]. Unfortunately, regardless of which method is employed, local management of in-transit melanoma re‐

Early in-vitro and clinical studies suggested that melanoma tumors exhibited significant in‐ trinsic resistance to ionizing radiation, and as such, radiotherapy has not traditionally been considered to have a major role in the treatment of in-transit melanoma [38, 39]. More recent studies, however, have suggested radiotherapy may be of value in certain subsets of indi‐ viduals, particularly those with one or few metastatic lesions that are not amenable to surgi‐ cal excision [40]. As a primary treatment, radiotherapy is largely reserved for palliation of patients with incurable symptomatic lesions, particularly in cases that are not amenable to

### **3. Biology of in-transit disease**

The underlying biology of in-transit melanoma is believed to be related to lymphatic dis‐ semination of small tumor emboli along the lymphatic drainage from the primary tumor. It is generally accepted that these migrating tumor cells become trapped in the dermal and subdermal lymphatics, typically, though not always, somewhere between the primary le‐ sion and the draining regional lymph nodes. These cells are thought to remain static along this route, eventually progressing to a clinically detectable lesion. Consistent with this theo‐ ry, in-transit melanoma is often described as an ongoing process, with increasing disease burden over time. Although the lymphatic route is the most likely biological explanation, some authors have suggested other mechanisms. One alternate explanation describes intransit disease as a manifestation of systemic disease resulting from hematogenous spread, similar to distant metastases [27, 28]. Proponents of this argue that in the lymphatic theory, wider margins of primary excision would be expected to include more static occult cells, with subsequent improved clinical outcomes, yet this has not been shown to be the case. It is difficult to reconcile this theory, however, with the significant differences in survival ob‐ served in stage III versus stage IV melanoma.

### **4. Therapy for in-transit disease**

Treatment of locoregionally recurrent melanoma depends on a number of important factors, including tumor size, multiplicity, and anatomic location. Although in-transit melanoma is often followed by metastatic disease, it is important that the surgeon choose an appropriate therapy based on clinical presentation, history, technical experience, and patient preference.

#### **4.1. Local management**

**2.4. Outcomes**

258 Melanoma - From Early Detection to Treatment

**3. Biology of in-transit disease**

served in stage III versus stage IV melanoma.

**4. Therapy for in-transit disease**

The presence of in-transit metastases indicates either N2 or N3 status under the current AJCC TNM system, and is classified as stage IIIB or C disease, respectively. In-transit mela‐ noma carries a poor prognosis, with 5-year survival rates ranging from 25% to 30% in most reports [12, 20, 21]. Additionally, the presence or absence of regional lymph node disease is of significant prognostic value; the combination of nodal metastasis and in-transit melano‐ ma comprise stage IIIC disease, which is associated with a poorer outcome than stage IIIB (40% vs. 59% five-year survival, respectively) [7]. There is a high incidence of occult distant metastasis in the presence of in-transit melanoma, but this is not universally the case. Stud‐ ies examining the outcomes of major amputation for the treatment of this pattern of recur‐ rence have identified a number of patients who experience a complete and durable response and have demonstrated five-year survival rates ranging from 21-32% [22-26]. This indicates that a significant minority of patients with in-transit metastases have disease that is truly limited to the extremity at the time of detection. Nonetheless, it is essential that distant metastases be ruled out when staging patients with in-transit melanoma, since treatment op‐ tions and prognosis may differ substantially when measurable distant disease is present.

The underlying biology of in-transit melanoma is believed to be related to lymphatic dis‐ semination of small tumor emboli along the lymphatic drainage from the primary tumor. It is generally accepted that these migrating tumor cells become trapped in the dermal and subdermal lymphatics, typically, though not always, somewhere between the primary le‐ sion and the draining regional lymph nodes. These cells are thought to remain static along this route, eventually progressing to a clinically detectable lesion. Consistent with this theo‐ ry, in-transit melanoma is often described as an ongoing process, with increasing disease burden over time. Although the lymphatic route is the most likely biological explanation, some authors have suggested other mechanisms. One alternate explanation describes intransit disease as a manifestation of systemic disease resulting from hematogenous spread, similar to distant metastases [27, 28]. Proponents of this argue that in the lymphatic theory, wider margins of primary excision would be expected to include more static occult cells, with subsequent improved clinical outcomes, yet this has not been shown to be the case. It is difficult to reconcile this theory, however, with the significant differences in survival ob‐

Treatment of locoregionally recurrent melanoma depends on a number of important factors, including tumor size, multiplicity, and anatomic location. Although in-transit melanoma is often followed by metastatic disease, it is important that the surgeon choose an appropriate therapy based on clinical presentation, history, technical experience, and patient preference.

Distinguishing in-transit disease from true local recurrence is of great importance, as the management and prognosis differ substantially. Local recurrence, or tumor confined to or contiguous with an excision scar and bearing an in situ component, should be managed sim‐ ilarly to the primary lesion with wide local excision. For in-transit disease, however, it is generally accepted that the wide local excision margin guidelines applicable to primary mel‐ anomas need not be applied. In-transit metastases are generally very clearly demarcated his‐ tologically from surrounding tissue, and complete macroscopic excision with negative surgical margins is usually all that is required.

In addition to wide local excision, there has been significant interest in other forms of local therapy for melanoma lesions, including laser ablation, external beam radiation, and intrale‐ sional injections. Irrespective of modality, these should all be thought of as equivalents to local surgical excision regarding indications and prognosis.

Laser therapy was first described in 1973, and has gained favor in the local treatment of intransit disease that is not amenable to surgical excision, such as when the disease is too ex‐ tensive [29]. It is most useful in patients with a large number of small in-transit lesions, but its advantages and utility decrease as lesions increase in size [30]. For tumors smaller than approximately 3mm, the entire lesion can be ablated using a carbon dioxide laser, though larger lesions must be circumscribed using the laser and subsequently excised with forceps.

Intralesional injections have also been used in the treatment of in-transit melanoma with some success. The most commonly used therapies include bacillus Calmette-Guérin (BCG), dinitrochlorobenzene (DNCB), and interferon-alpha (INF-α), and IL-2. Small stud‐ ies have demonstrated complete response rates of 31-63% (overall response 45-91%), al‐ though long-term survival, when reported, remained unfortunately low [31-33]. This suggests that if surgical excision is not a viable option, intralesional injection is a reasona‐ ble alternative. More recently, electrochemotherapy (ECT) has gained popularity as local alternative to radiotherapy and laser ablation. This technique relies on using high intensi‐ ty electric pulses to allow intracellular delivery of cytotoxic drugs, such as cisplatin and bleomycin, via intralesional injection [34]. Complete response rates have been reported as 53-89% (overall response 84-99%), with minimal systemic toxicity [35-37]. Unfortunately, regardless of which method is employed, local management of in-transit melanoma re‐ mains suboptimal in many situations.

#### **4.2. Radiation therapy**

Early in-vitro and clinical studies suggested that melanoma tumors exhibited significant in‐ trinsic resistance to ionizing radiation, and as such, radiotherapy has not traditionally been considered to have a major role in the treatment of in-transit melanoma [38, 39]. More recent studies, however, have suggested radiotherapy may be of value in certain subsets of indi‐ viduals, particularly those with one or few metastatic lesions that are not amenable to surgi‐ cal excision [40]. As a primary treatment, radiotherapy is largely reserved for palliation of patients with incurable symptomatic lesions, particularly in cases that are not amenable to surgical excision. Generally speaking, when unresectable in-transit melanoma is amenable to regional chemotherapy, this should be considered prior to employing radiotherapy.

**4.4. Regional chemotherapy agents**

effective dose is achieved without systemic toxicity.

patients at Duke University Medical Center [56].

**4.5. Isolated limb perfusion**

Melphalan is typically the drug of choice for regional chemotherapy. It is an alkylating agent derived from phenylalanine, an amino acid preferentially taken up by melanocytes due to its key role in melanin synthesis. Theoretically, melphalan should produce selective toxicity in melanocytes and melanin-containing melanoma cells. As a systemic agent, how‐ ever, melphalan is ineffective despite its theoretical benefits, as its allowable dose is signifi‐ cantly less than its effective dose. For regional therapy, in contrast, this much higher

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261

Other agents have been employed either alone or in combination with melphalan in the treatment of in-transit melanoma. An essential quality of any agent considered for regional therapy is the constraint that it must not require metabolic transformation to take on a bio‐ logically active form. Cisplatin is another alkylating agent that held significant promise in preclinical studies of regional chemotherapy. Early clinical reports were favorable regarding response rates, but were plagued by concerns over toxicity [49-51]. Subsequent studies con‐ firmed significant limb-threatening toxicity with the use of cisplatin, and as such most au‐ thors recommend against its routine use in regional therapy [52, 53]. Similarly, TNFα has exhibited some potential, particularly when combined with interferon-gamma, but wide‐ spread use of TNFα-based regimens have been tempered by significant concerns regarding toxicity [54]. The 2006 ACOSOG Z0020 trial comparing melphalan with melphalan plus TNFα was terminated early after interim analysis demonstrated a significant increase in tox‐ icity with the addition of TNFα and yet a similar clinical response rate compared to melpha‐ lan alone [55]. Temozolomide is a newer alkylating agent that could have potential application in regional chemotherapy, as it also does not require hepatic conversion to be‐ come active. Early results in animal models reported superior tumor growth delay com‐ pared to regional melphalan, and a phase 1 clinical trial is currently underway, enrolling

Isolated limb perfusion (ILP) was first described in Creech and colleagues in 1958, basing their technique on advances in cardiopulmonary bypass developed for cardiac surgery in the 1950s [57]. They utilized an extracorporeal oxygenator as part of the isolated limb circuit to deliver high dose chemotherapy while maintaining normal oxygen tension and pH of the treated limb. Ten years later, Stehlin and coworkers added the effects of hyperthermia to the treatment protocol, now called hyperthermic isolated limb perfusion (HILP), enhancing the cytotoxicity of the chemotherapy and increasing efficacy [58]. The technical aspects of HILP

The procedure is performed under general anesthesia, and the vasculature supplying the af‐ fected limb is exposed and cannulated. During this exposure, one typically performs a re‐ gional lymphadenectomy, which aids vascular exposure (particularly in the case of the iliac vessels) and is often indicated from an oncologic standpoint. The target limb is isolated from the systemic circulation using a proximal tourniquet. Perfusion is then initiated via the can‐ nulated vessels, utilizing a membrane oxygenator and cardiopulmonary bypass apparatus

vary somewhat among surgeons and institutions, but the basic technique is similar.

While some studies have demonstrated potential benefit of adjuvant radiation therapy in patients with nodal melanoma metastases, there are very little data regarding the use of ad‐ juvant radiation therapy in the setting of in-transit disease [41, 42]. Treatment depends on area and location of involvement. While not routine practice, adjuvant radiotherapy should be considered in patients with head and neck disease, and in those with positive margins that are not amenable to re-excision [43-45].

## **4.3. Regional therapy**

Given the high rate of local treatment failure and frequently increased burden of in-transit dis‐ ease, regionally focused modalities offer potential strategies to obtain more durable treatment responses. Regional chemotherapy is a promising therapeutic option for suitable patients with extremity in-transit melanoma and is currently the focus of exciting research. This modality in‐ volves vascular isolation of the affected area, after which chemotherapy is then delivered at doses 10-20 times higher than doses that can be achieved and tolerated systemically, with dos‐ ing based on affected limb volume. As regional therapy requires complete vascular isolation of the affected body area, obvious anatomic limitations are involved. The inflow and outflow ves‐ sels to the area of interest must be selectively cannulated, and the treatment region must then be isolated from the systemic circulation, usually by means of a tourniquet.

There remains significant debate as to whether regional chemotherapy produces an overall survival benefit over other therapeutic modalities, but studies have demonstrated a survival benefit in patients who exhibited a clinical response [46-48]. Originally described in the 1950s, two primary forms of regional chemotherapy have evolved: hyperthermic isolated limb perfusion (HILP) and isolated limb infusion (ILI).


**Table 2.** Comparison of technique and parameters between hyperthermic isolated limb perfusion (HILP) and isolated limb infusion (ILI).

### **4.4. Regional chemotherapy agents**

surgical excision. Generally speaking, when unresectable in-transit melanoma is amenable to regional chemotherapy, this should be considered prior to employing radiotherapy.

While some studies have demonstrated potential benefit of adjuvant radiation therapy in patients with nodal melanoma metastases, there are very little data regarding the use of ad‐ juvant radiation therapy in the setting of in-transit disease [41, 42]. Treatment depends on area and location of involvement. While not routine practice, adjuvant radiotherapy should be considered in patients with head and neck disease, and in those with positive margins

Given the high rate of local treatment failure and frequently increased burden of in-transit dis‐ ease, regionally focused modalities offer potential strategies to obtain more durable treatment responses. Regional chemotherapy is a promising therapeutic option for suitable patients with extremity in-transit melanoma and is currently the focus of exciting research. This modality in‐ volves vascular isolation of the affected area, after which chemotherapy is then delivered at doses 10-20 times higher than doses that can be achieved and tolerated systemically, with dos‐ ing based on affected limb volume. As regional therapy requires complete vascular isolation of the affected body area, obvious anatomic limitations are involved. The inflow and outflow ves‐ sels to the area of interest must be selectively cannulated, and the treatment region must then

There remains significant debate as to whether regional chemotherapy produces an overall survival benefit over other therapeutic modalities, but studies have demonstrated a survival benefit in patients who exhibited a clinical response [46-48]. Originally described in the 1950s, two primary forms of regional chemotherapy have evolved: hyperthermic isolated

Drug delivery Cardiopulmonary bypass Manual pump with three-way stopcock Circuit pressure High; with significant risk for systemic leak Low; significantly reduced risk of systemic

Limb oxygenation Active membrane oxygenation No external oxygenation; profound hypoxia

Technical demand Technically complex, difficult re-operation Technically simpler, re-do operation without

**Table 2.** Comparison of technique and parameters between hyperthermic isolated limb perfusion (HILP) and isolated

**HILP ILI**

leak

difficulty

Percutaneous access under fluoroscopic guidance, smaller diameter cannulas

be isolated from the systemic circulation, usually by means of a tourniquet.

limb perfusion (HILP) and isolated limb infusion (ILI).

Vessel access Open surgical exposure; large diameter cannulas

Limb pH Physiologic Acidotic

Temperature 39-40°C 37.8-38.5°C Duration of treatment 60 minutes 30 minutes

that are not amenable to re-excision [43-45].

260 Melanoma - From Early Detection to Treatment

**4.3. Regional therapy**

limb infusion (ILI).

Melphalan is typically the drug of choice for regional chemotherapy. It is an alkylating agent derived from phenylalanine, an amino acid preferentially taken up by melanocytes due to its key role in melanin synthesis. Theoretically, melphalan should produce selective toxicity in melanocytes and melanin-containing melanoma cells. As a systemic agent, how‐ ever, melphalan is ineffective despite its theoretical benefits, as its allowable dose is signifi‐ cantly less than its effective dose. For regional therapy, in contrast, this much higher effective dose is achieved without systemic toxicity.

Other agents have been employed either alone or in combination with melphalan in the treatment of in-transit melanoma. An essential quality of any agent considered for regional therapy is the constraint that it must not require metabolic transformation to take on a bio‐ logically active form. Cisplatin is another alkylating agent that held significant promise in preclinical studies of regional chemotherapy. Early clinical reports were favorable regarding response rates, but were plagued by concerns over toxicity [49-51]. Subsequent studies con‐ firmed significant limb-threatening toxicity with the use of cisplatin, and as such most au‐ thors recommend against its routine use in regional therapy [52, 53]. Similarly, TNFα has exhibited some potential, particularly when combined with interferon-gamma, but wide‐ spread use of TNFα-based regimens have been tempered by significant concerns regarding toxicity [54]. The 2006 ACOSOG Z0020 trial comparing melphalan with melphalan plus TNFα was terminated early after interim analysis demonstrated a significant increase in tox‐ icity with the addition of TNFα and yet a similar clinical response rate compared to melpha‐ lan alone [55]. Temozolomide is a newer alkylating agent that could have potential application in regional chemotherapy, as it also does not require hepatic conversion to be‐ come active. Early results in animal models reported superior tumor growth delay com‐ pared to regional melphalan, and a phase 1 clinical trial is currently underway, enrolling patients at Duke University Medical Center [56].

#### **4.5. Isolated limb perfusion**

Isolated limb perfusion (ILP) was first described in Creech and colleagues in 1958, basing their technique on advances in cardiopulmonary bypass developed for cardiac surgery in the 1950s [57]. They utilized an extracorporeal oxygenator as part of the isolated limb circuit to deliver high dose chemotherapy while maintaining normal oxygen tension and pH of the treated limb. Ten years later, Stehlin and coworkers added the effects of hyperthermia to the treatment protocol, now called hyperthermic isolated limb perfusion (HILP), enhancing the cytotoxicity of the chemotherapy and increasing efficacy [58]. The technical aspects of HILP vary somewhat among surgeons and institutions, but the basic technique is similar.

The procedure is performed under general anesthesia, and the vasculature supplying the af‐ fected limb is exposed and cannulated. During this exposure, one typically performs a re‐ gional lymphadenectomy, which aids vascular exposure (particularly in the case of the iliac vessels) and is often indicated from an oncologic standpoint. The target limb is isolated from the systemic circulation using a proximal tourniquet. Perfusion is then initiated via the can‐ nulated vessels, utilizing a membrane oxygenator and cardiopulmonary bypass apparatus to maintain limb oxygen tension and pH at physiologic levels. The perfusion treatment is generally continued for 60 to 90 minutes, depending on the protocol. External warming blankets and heated melphalan perfusate are used to achieve hyperthermia. During HILP, it is important to monitor for leakage of the perfusate into the systemic circulation, particular‐ ly when high dose TNF-alpha is employed, as systemic leakage can lead to significant mor‐ bidity or mortality. Traditionally this monitoring was performed using intravenous fluorescein and watching for staining proximal to the tourniquet. A more precise method in‐ volves the administration of radiolabeled tracer into the HILP circuit, followed by continu‐ ously monitored systemic radiation exposure using a gamma probe placed over the chest. After completion of chemotherapy perfusion, a 30-minute washout period with crystalloids follows to remove the active agents.

response rates of 39-82% [46, 48, 59-62] have been reported. However, the previously men‐ tioned multi-center ACOSOG Z0020 study demonstrated complete response rates of only 25%, significantly lower than what had been previously reported [55]. Overall, recurrence rates are 50-60% within one year, and overall 5-year survival rates remain in the 30-40% range [63]. As such, while HILP may be the best treatment option for suitable patients with in-transit extremity melanoma, there remains significant room for therapeutic improvement.

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**Study (year) [ref] Patients (n) CR (%) PR (%) OR (%)**

Minor (1985) [60] 18 82 18 100 Storm (1985) [62] 26 50 31 81 Di Filippo (1989) [59] 69 39 43 82 Cornett (2006) [55] 58 25 39 64 Sanki (2007) [48] 120 69 15 84 Raymond (2011) [61] 62 55 26 81

**Table 3.** Response rates following HILP in patients with in-transit melanoma. Adapted with permission from Coleman et al., Expert Rev. Anticancer Ther. 2009;9(11):1599-1602. CR: Complete response; PR: Partial response; OR: Overall

Isolated limb infusion (ILI) was developed by Thompson and coworkers at the Sydney Mel‐ anoma Unit as a less invasive alternative to HILP. This technique employs percutaneous catheters inserted under fluoroscopic guidance as a means to cannulate the target limb ves‐ sels. An external tourniquet is used to isolate the limb, which is then wrapped in heating blankets. The key difference with ILI as compared to HILP is the lack of a perfusion pump and membrane oxygenator. The melphalan solution is instead manually circulated via the arterial catheter using a syringe and three-way stopcock. Consequently, during ILI the limb is not maintained at normal pH and oxygen tension, and becomes markedly hypoxic and acidotic during the course of the procedure. Some authors propose that the acidosis and hy‐ poxia may serve to augment melphalan action [64]. In addition, while external and internal warming are performed in ILI, limb temperatures achieved with ILI are lower than those in

From a technical standpoint, ILI is appreciably simpler and easier to perform and learn. The infusion treatment is continued for about 30 minutes, followed by a similar washout period with crystalloid. Lower doses of melphalan are typically used, often in combina‐ tion with dactinomycin, and regional morbidity is reduced, particularly with respect to in‐ cidence of severe toxicity. In light of these factors, ILI is generally well tolerated, and is often offered to frail patients with multiple comorbidities who would not tolerate the lon‐ ger and more invasive groin exposure required for HILP. Along similar lines, due to its simplicity and lower morbidity, ILI can be safely offered as a repeat procedure. Although theoretically attractive as a means of obtaining fractionated regional chemotherapy, elec‐

HILP and generally do not exceed 38.5 degrees centigrade [65, 66].

response.

**4.6. Isolated limb infusion**

**Figure 2.** Hyperthermic isolated limb perfusion. Surgical exposure of the proximal vasculature is followed by cannula‐ tion and circulation of chemotherapy perfusate. Acid-base status and oxygenation is maintained throughout the pro‐ cedure. Reproduced with permission.

Results of HILP vary widely, perhaps depending on the patient population and adjunctive agents employed. In single-center studies, overall response rates of 81-100% and complete response rates of 39-82% [46, 48, 59-62] have been reported. However, the previously men‐ tioned multi-center ACOSOG Z0020 study demonstrated complete response rates of only 25%, significantly lower than what had been previously reported [55]. Overall, recurrence rates are 50-60% within one year, and overall 5-year survival rates remain in the 30-40% range [63]. As such, while HILP may be the best treatment option for suitable patients with in-transit extremity melanoma, there remains significant room for therapeutic improvement.


**Table 3.** Response rates following HILP in patients with in-transit melanoma. Adapted with permission from Coleman et al., Expert Rev. Anticancer Ther. 2009;9(11):1599-1602. CR: Complete response; PR: Partial response; OR: Overall response.

#### **4.6. Isolated limb infusion**

to maintain limb oxygen tension and pH at physiologic levels. The perfusion treatment is generally continued for 60 to 90 minutes, depending on the protocol. External warming blankets and heated melphalan perfusate are used to achieve hyperthermia. During HILP, it is important to monitor for leakage of the perfusate into the systemic circulation, particular‐ ly when high dose TNF-alpha is employed, as systemic leakage can lead to significant mor‐ bidity or mortality. Traditionally this monitoring was performed using intravenous fluorescein and watching for staining proximal to the tourniquet. A more precise method in‐ volves the administration of radiolabeled tracer into the HILP circuit, followed by continu‐ ously monitored systemic radiation exposure using a gamma probe placed over the chest. After completion of chemotherapy perfusion, a 30-minute washout period with crystalloids

**Figure 2.** Hyperthermic isolated limb perfusion. Surgical exposure of the proximal vasculature is followed by cannula‐ tion and circulation of chemotherapy perfusate. Acid-base status and oxygenation is maintained throughout the pro‐

Results of HILP vary widely, perhaps depending on the patient population and adjunctive agents employed. In single-center studies, overall response rates of 81-100% and complete

follows to remove the active agents.

262 Melanoma - From Early Detection to Treatment

cedure. Reproduced with permission.

Isolated limb infusion (ILI) was developed by Thompson and coworkers at the Sydney Mel‐ anoma Unit as a less invasive alternative to HILP. This technique employs percutaneous catheters inserted under fluoroscopic guidance as a means to cannulate the target limb ves‐ sels. An external tourniquet is used to isolate the limb, which is then wrapped in heating blankets. The key difference with ILI as compared to HILP is the lack of a perfusion pump and membrane oxygenator. The melphalan solution is instead manually circulated via the arterial catheter using a syringe and three-way stopcock. Consequently, during ILI the limb is not maintained at normal pH and oxygen tension, and becomes markedly hypoxic and acidotic during the course of the procedure. Some authors propose that the acidosis and hy‐ poxia may serve to augment melphalan action [64]. In addition, while external and internal warming are performed in ILI, limb temperatures achieved with ILI are lower than those in HILP and generally do not exceed 38.5 degrees centigrade [65, 66].

From a technical standpoint, ILI is appreciably simpler and easier to perform and learn. The infusion treatment is continued for about 30 minutes, followed by a similar washout period with crystalloid. Lower doses of melphalan are typically used, often in combina‐ tion with dactinomycin, and regional morbidity is reduced, particularly with respect to in‐ cidence of severe toxicity. In light of these factors, ILI is generally well tolerated, and is often offered to frail patients with multiple comorbidities who would not tolerate the lon‐ ger and more invasive groin exposure required for HILP. Along similar lines, due to its simplicity and lower morbidity, ILI can be safely offered as a repeat procedure. Although theoretically attractive as a means of obtaining fractionated regional chemotherapy, elec‐ tive repeat ILI has not been shown to improve survival compared to single ILI [67]. How‐ ever, repeat ILI can be very valuable in the management of recurrent or progressive intransit disease after primary regional therapy.

**Study (year) [ref] Patients (n) CR (%) PR (%) OR (%)**

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Mian (2001) [70] 9 44 56 100 Lindner (2002) [66] 128 41 44 85 Brady (2006) [69] 22 23 27 50 Kroon (2008) [47] 185 38 46 84 Beasley (2009) [68] 128 31 33 64 Raymond (2011) [61] 126 30 13 43

**Table 4.** Response rates following ILI in patients with in-transit melanoma. Adapted with permission from Coleman et al., Expert Rev. Anticancer Ther. 2009;9(11):1599-1602. CR: Complete response; PR: Partial response; OR: Overall

As a result of the high concentration of chemotherapies administered in regional therapy, some degree of tissue toxicity is often seen. Multiple grading systems have been devel‐ oped to score regional toxicity after treatment, with one of the most prominent being that developed by Wieberdink and colleagues. In this system scores range from Grade I, or no evidence of significant reaction, to Grade V, representing reaction severe enough to war‐ rant possible amputation [72]. Up to 85% of patients will exhibit Grade I or II level of tox‐ icity, but as a result of careful drug dosing based on limb volume rather than total body weight, fortunately overall less than 1% of patients develop Grade V toxicity [73]. While the spectrum of toxicity is similar between patients undergoing ILI and HILP, the risk of significant toxicity is greater among those undergoing HILP. Furthermore, HILP carries a higher risk of limb loss from amputation as compared to ILI. Regardless of modality, most adverse reactions are transient, with almost all patients demonstrating some skin er‐ ythema and edema that peaks in the first month post-operatively. Rare but more serious complications include severe muscle toxicity and the development of compartment syn‐

Amputation is almost never indicated in the standard treatment of in-transit melanoma. As mentioned previously, historical treatment of in-transit disease by means of limb amputa‐ tion has led to long-term survival rates of 20-30 percent, which would suggest that a signifi‐ cant minority of patients with locoregional disease have recurrence that is in fact confined entirely to the affected extremity. Recent advancements in aggressive local management, re‐ gional therapy and systemic treatment have rendered extremity amputation obsolete except for the most intractable disease, particularly in light of comparable five-year survival rates among patients undergoing these therapies. Thus, amputation should generally only be of‐ fered with palliative intent or in patients who refuse or are not candidates for regional che‐

response.

**4.7. Post-treatment complications**

drome, necessitating fasciotomy.

motherapy or other less morbid therapies [22, 26].

**4.8. Amputation**

**Figure 3.** Isolated limb infusion. Catheters are placed percutaneously, and chemotherapy is circulated by hand with‐ out active oxygenation, leading to profound hypoxia and acidosis.

Outcomes after ILI are generally inferior to HILP, with complete response ranging from 23-44% and overall response ranging from 43-100% [47, 61, 65, 66, 68-70]. In one of the larg‐ est studies explicitly comparing patterns of recurrence, ILI was found to have both signifi‐ cantly higher probability of recurrence (85% vs. 65%) and shorter time to first recurrence (8 months vs. 23 months) as compared to HILP [71]. Notably, there was no statistically signifi‐ cant difference in overall survival between the two groups, although there was a trend in favor of HILP.


**Table 4.** Response rates following ILI in patients with in-transit melanoma. Adapted with permission from Coleman et al., Expert Rev. Anticancer Ther. 2009;9(11):1599-1602. CR: Complete response; PR: Partial response; OR: Overall response.

#### **4.7. Post-treatment complications**

tive repeat ILI has not been shown to improve survival compared to single ILI [67]. How‐ ever, repeat ILI can be very valuable in the management of recurrent or progressive in-

**Figure 3.** Isolated limb infusion. Catheters are placed percutaneously, and chemotherapy is circulated by hand with‐

Outcomes after ILI are generally inferior to HILP, with complete response ranging from 23-44% and overall response ranging from 43-100% [47, 61, 65, 66, 68-70]. In one of the larg‐ est studies explicitly comparing patterns of recurrence, ILI was found to have both signifi‐ cantly higher probability of recurrence (85% vs. 65%) and shorter time to first recurrence (8 months vs. 23 months) as compared to HILP [71]. Notably, there was no statistically signifi‐ cant difference in overall survival between the two groups, although there was a trend in

out active oxygenation, leading to profound hypoxia and acidosis.

favor of HILP.

transit disease after primary regional therapy.

264 Melanoma - From Early Detection to Treatment

As a result of the high concentration of chemotherapies administered in regional therapy, some degree of tissue toxicity is often seen. Multiple grading systems have been devel‐ oped to score regional toxicity after treatment, with one of the most prominent being that developed by Wieberdink and colleagues. In this system scores range from Grade I, or no evidence of significant reaction, to Grade V, representing reaction severe enough to war‐ rant possible amputation [72]. Up to 85% of patients will exhibit Grade I or II level of tox‐ icity, but as a result of careful drug dosing based on limb volume rather than total body weight, fortunately overall less than 1% of patients develop Grade V toxicity [73]. While the spectrum of toxicity is similar between patients undergoing ILI and HILP, the risk of significant toxicity is greater among those undergoing HILP. Furthermore, HILP carries a higher risk of limb loss from amputation as compared to ILI. Regardless of modality, most adverse reactions are transient, with almost all patients demonstrating some skin er‐ ythema and edema that peaks in the first month post-operatively. Rare but more serious complications include severe muscle toxicity and the development of compartment syn‐ drome, necessitating fasciotomy.

#### **4.8. Amputation**

Amputation is almost never indicated in the standard treatment of in-transit melanoma. As mentioned previously, historical treatment of in-transit disease by means of limb amputa‐ tion has led to long-term survival rates of 20-30 percent, which would suggest that a signifi‐ cant minority of patients with locoregional disease have recurrence that is in fact confined entirely to the affected extremity. Recent advancements in aggressive local management, re‐ gional therapy and systemic treatment have rendered extremity amputation obsolete except for the most intractable disease, particularly in light of comparable five-year survival rates among patients undergoing these therapies. Thus, amputation should generally only be of‐ fered with palliative intent or in patients who refuse or are not candidates for regional che‐ motherapy or other less morbid therapies [22, 26].

#### **4.9. Systemic treatment**

While a comprehensive discussion regarding systemic therapy for the treatment of mela‐ noma is beyond the scope of this chapter, when appropriate this modality should be con‐ sidered in the management of in-transit disease. Systemic therapy is typically applied in cases of in-transit disease in the presence of distant metastases – that is, stage IV disease [74]. Similarly, patients with non-extremity in-transit metastases – such as in-transit dis‐ ease involving the head and neck, truncal or genitalia – present a difficult management problem and are often palliated best with systemic treatment options. Systemic therapy should also be considered for in-transit metastases in patients with recurrent or progres‐ sive disease who are not candidates for repeat local or regional therapy. Unfortunately, systemic therapy for the treatment of patients with advanced melanoma has historically been quite poor. A large meta-analysis of 42 trials of systemic treatments demonstrating a median progression free survival of 1.7 months with only 14.5% of patients being progres‐ sion-free at 6 months [75]. Despite this poor track record, newer approaches to systemic treatment of regional disease may hold promise, including vascular regulating agents, sig‐ nal targeting therapies and immune modulation therapy.

dures and hospitalizations. As such, management of this disease can be challenging and frustrating to clinicians as well. Similar to systemic melanoma, in-transit disease is notori‐ ously resistant to chemotherapy, and treatment outcomes remain unsatisfactorily poor. Lo‐ cal therapies often tout impressive initial response rates, but are plagued by recurrence. Over the past half-century, advances have been made in regional approaches to chemothera‐ py, including isolated limb perfusion and isolated limb infusion. While some of these meth‐ ods have demonstrated limited success, significant improvements in patient outcomes will

Management of In-Transit Malignant Melanoma

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267

Division of Surgical Oncology, Department of Surgery, Duke University Medical Center,

[1] Pawlik TM, Ross MI, Johnson MM, Schacherer CW, McClain DM, Mansfield PF, et al. Predictors and natural history of in-transit melanoma after sentinel lymphadenecto‐

[2] Meier F, Will S, Ellwanger U, Schlagenhauff B, Schittek B, Rassner G, et al. Metastatic pathways and time courses in the orderly progression of cutaneous melanoma. The

[3] Singletary SE, Tucker SL, Boddie AW, Jr. Multivariate analysis of prognostic factors in regional cutaneous metastases of extremity melanoma. Cancer. 1988;61(7):1437-40.

[4] Karakousis CP, Temple DF, Moore R, Ambrus JL. Prognostic parameters in recurrent

[5] Roses DF, Karp NS, Oratz R, Dubin N, Harris MN, Speyer J, et al. Survival with re‐ gional and distant metastases from cutaneous malignant melanoma. Surgery, gyne‐

[6] Haffner AC, Garbe C, Burg G, Buttner P, Orfanos CE, Rassner G. The prognosis of primary and metastasising melanoma. An evaluation of the TNM classification in

[7] Balch CM, Gershenwald JE, Soong SJ, Thompson JF, Atkins MB, Byrd DR, et al. Final version of 2009 AJCC melanoma staging and classification. Journal of clinical oncolo‐

2,495 patients. British journal of cancer. 1992;66(5):856-61. Epub 1992/11/01.

my. Annals of surgical oncology. 2005;12(8):587-96. Epub 2005/07/16.

British journal of dermatology. 2002;147(1):62-70. Epub 2002/07/09.

malignant melanoma. Cancer. 1983;52(3):575-9. Epub 1983/08/01.

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require further advances in both regional and systemic treatment of melanoma.

Paul J. Speicher, Douglas S. Tyler and Paul J. Mosca

**Author details**

Durham, USA

**References**

Epub 1988/04/01.

Current strategies have focused on attempting to increase tumor sensitivity to chemother‐ apeutics, improve local drug delivery, or target apoptotic pathways in an attempt to aug‐ ment response to regional therapy. The BRAF enzyme inhibitor vemurafenib, as well as the immune modulating anti-CTLA-4 antibody ipilimumab, have recently shown promise in phase III trials, although neither is likely to provide durable disease-free survival [76, 77]. Another newer agent is bevacizumab, a monoclonal antibody to vascular endothelial growth factor (VEGF), which is believed to normalize immature and shunt-dominated tu‐ mor vasculature, leading to improved delivery of chemotherapeutics to tumor cells. A re‐ cent preclinical animal study demonstrated that systemic treatment with bevacizumab prior to regional therapy increased delivery of melphalan to the tumors of interest [78]. Another vascular targeting agent of recent interest is ADH-1, a pentapeptide that targets and disrupts N-cadherin adhesion complexes, which are predominantly expressed by mel‐ anocytes after malignant transition into melanoma [79, 80]. ADH-1 is believed to increase blood vessel permeability, increasing chemotherapy drug delivery [81]. A recent phase II clinical trial studying pre-treatment systemic ADH-1 administration prior to ILI with mel‐ phalan demonstrated a reassuring complete response rate of 38% and an overall response rate of 60%, although no significant progression free survival was appreciated [82]. The role of all of these agents as systemic adjuncts to regional chemotherapy remains to be seen, and is being defined in ongoing trials.

## **5. Conclusions**

In-transit melanoma is a distinctive form of tumor recurrence, and is an indicator of latestage disease. It is very distressing to patients, often requiring multiple treatments, proce‐ dures and hospitalizations. As such, management of this disease can be challenging and frustrating to clinicians as well. Similar to systemic melanoma, in-transit disease is notori‐ ously resistant to chemotherapy, and treatment outcomes remain unsatisfactorily poor. Lo‐ cal therapies often tout impressive initial response rates, but are plagued by recurrence. Over the past half-century, advances have been made in regional approaches to chemothera‐ py, including isolated limb perfusion and isolated limb infusion. While some of these meth‐ ods have demonstrated limited success, significant improvements in patient outcomes will require further advances in both regional and systemic treatment of melanoma.

## **Author details**

**4.9. Systemic treatment**

266 Melanoma - From Early Detection to Treatment

While a comprehensive discussion regarding systemic therapy for the treatment of mela‐ noma is beyond the scope of this chapter, when appropriate this modality should be con‐ sidered in the management of in-transit disease. Systemic therapy is typically applied in cases of in-transit disease in the presence of distant metastases – that is, stage IV disease [74]. Similarly, patients with non-extremity in-transit metastases – such as in-transit dis‐ ease involving the head and neck, truncal or genitalia – present a difficult management problem and are often palliated best with systemic treatment options. Systemic therapy should also be considered for in-transit metastases in patients with recurrent or progres‐ sive disease who are not candidates for repeat local or regional therapy. Unfortunately, systemic therapy for the treatment of patients with advanced melanoma has historically been quite poor. A large meta-analysis of 42 trials of systemic treatments demonstrating a median progression free survival of 1.7 months with only 14.5% of patients being progres‐ sion-free at 6 months [75]. Despite this poor track record, newer approaches to systemic treatment of regional disease may hold promise, including vascular regulating agents, sig‐

Current strategies have focused on attempting to increase tumor sensitivity to chemother‐ apeutics, improve local drug delivery, or target apoptotic pathways in an attempt to aug‐ ment response to regional therapy. The BRAF enzyme inhibitor vemurafenib, as well as the immune modulating anti-CTLA-4 antibody ipilimumab, have recently shown promise in phase III trials, although neither is likely to provide durable disease-free survival [76, 77]. Another newer agent is bevacizumab, a monoclonal antibody to vascular endothelial growth factor (VEGF), which is believed to normalize immature and shunt-dominated tu‐ mor vasculature, leading to improved delivery of chemotherapeutics to tumor cells. A re‐ cent preclinical animal study demonstrated that systemic treatment with bevacizumab prior to regional therapy increased delivery of melphalan to the tumors of interest [78]. Another vascular targeting agent of recent interest is ADH-1, a pentapeptide that targets and disrupts N-cadherin adhesion complexes, which are predominantly expressed by mel‐ anocytes after malignant transition into melanoma [79, 80]. ADH-1 is believed to increase blood vessel permeability, increasing chemotherapy drug delivery [81]. A recent phase II clinical trial studying pre-treatment systemic ADH-1 administration prior to ILI with mel‐ phalan demonstrated a reassuring complete response rate of 38% and an overall response rate of 60%, although no significant progression free survival was appreciated [82]. The role of all of these agents as systemic adjuncts to regional chemotherapy remains to be

In-transit melanoma is a distinctive form of tumor recurrence, and is an indicator of latestage disease. It is very distressing to patients, often requiring multiple treatments, proce‐

nal targeting therapies and immune modulation therapy.

seen, and is being defined in ongoing trials.

**5. Conclusions**

Paul J. Speicher, Douglas S. Tyler and Paul J. Mosca

Division of Surgical Oncology, Department of Surgery, Duke University Medical Center, Durham, USA

## **References**


gy : official journal of the American Society of Clinical Oncology. 2009;27(36): 6199-206. Epub 2009/11/18.

tients with primary cutaneous melanoma. Annals of surgical oncology. 2005;12(8):

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[22] Jaques DP, Coit DG, Brennan MF. Major amputation for advanced malignant mela‐ noma. Surgery, gynecology & obstetrics. 1989;169(1):1-6. Epub 1989/07/01.

[23] Karakousis CP, Choe KJ, Holyoke ED. Biologic behavior and treatment of intransit metastasis of melanoma. Surgery, gynecology & obstetrics. 1980;150(1):29-32. Epub

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[25] Pack GT, Gerber DM, Scharnagel IM. End results in the treatment of malignant mela‐ noma; a report of 1190 cases. Annals of surgery. 1952;136(6):905-11. Epub 1952/12/01.

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**Chapter 11**

**Management of Brain Metastasis in Melanoma Patients**

The American Cancer Society estimates that 76,250 Americans will be diagnosed with malig‐ nant melanoma and 9,180 will die from the disease in 2012 [1]. The incidence is increasing both in the United States and worldwide [2]. Brain metastasis is a common problem in this population with 45-60% of those with metastatic melanoma developing brain metastases during the course of their illness [3]. Post-mortem studies demonstrate that brain lesions are present in 70-90% of patients who die of melanoma [3]. Development of brain metastases may have adverse impact bothonapatient'sprognosisand,ifsymptomatic,severeeffectsonquality-of-life(QOL)[4].Ifleft

The literature pertaining to the treatment of brain metastasis from melanoma is scant when compared to brain metastases from more common solid tumors. In particular, brain metastases from non-small cell lung cancer (NSCLC) and breast cancer have been the subject of a larger number of investigative efforts. This chapter will extrapolate relevant results from other common solid tumors to the treatment of melanoma. In addition, systemic treatment ap‐ proaches that may be useful in managing intracranial disease will be presented. Leptomenin‐ geal involvement of the central nervous system, a less common form of central nervous system

Three randomized trials have investigated treatment of a single brain metastasis with whole brain radiation therapy (WBRT) alone or combined with surgical resection (Table 1) [5-7]. In

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

© 2013 Morgan\* 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,

untreated, symptomatic brain lesions may be fatal within several weeks [3].

(CNS) invasion by melanoma, will not be discussed.

**2. Treatment modalities**

**2.1. Surgery**

Sherif S. Morgan\*, Joanne M. Jeter, Evan M. Hersh,

Additional information is available at the end of the chapter

Sun K. Yi and Lee D. Cranmer\*

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

**1. Introduction**


## **Management of Brain Metastasis in Melanoma Patients**

Sherif S. Morgan\*, Joanne M. Jeter, Evan M. Hersh, Sun K. Yi and Lee D. Cranmer\*

Additional information is available at the end of the chapter

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

## **1. Introduction**

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pathway. Molecular biology of the cell. 2005;16(9):4386-97. Epub 2005/07/01.

2012;18(12):3328-39. Epub 2012/04/13.

274 Melanoma - From Early Detection to Treatment

pathology. 2000;156(5):1515-25. Epub 2000/05/04.

cology. 2011;29(9):1210-5. Epub 2011/02/24.

ment. Cancer research. 2008;68(10):3777-84. Epub 2008/05/17.

The American Cancer Society estimates that 76,250 Americans will be diagnosed with malig‐ nant melanoma and 9,180 will die from the disease in 2012 [1]. The incidence is increasing both in the United States and worldwide [2]. Brain metastasis is a common problem in this population with 45-60% of those with metastatic melanoma developing brain metastases during the course of their illness [3]. Post-mortem studies demonstrate that brain lesions are present in 70-90% of patients who die of melanoma [3]. Development of brain metastases may have adverse impact bothonapatient'sprognosisand,ifsymptomatic,severeeffectsonquality-of-life(QOL)[4].Ifleft untreated, symptomatic brain lesions may be fatal within several weeks [3].

The literature pertaining to the treatment of brain metastasis from melanoma is scant when compared to brain metastases from more common solid tumors. In particular, brain metastases from non-small cell lung cancer (NSCLC) and breast cancer have been the subject of a larger number of investigative efforts. This chapter will extrapolate relevant results from other common solid tumors to the treatment of melanoma. In addition, systemic treatment ap‐ proaches that may be useful in managing intracranial disease will be presented. Leptomenin‐ geal involvement of the central nervous system, a less common form of central nervous system (CNS) invasion by melanoma, will not be discussed.

## **2. Treatment modalities**

## **2.1. Surgery**

Three randomized trials have investigated treatment of a single brain metastasis with whole brain radiation therapy (WBRT) alone or combined with surgical resection (Table 1) [5-7]. In

all three, overall survival (OS) was the primary endpoint. In one study, the addition of surgery to WBRT achieved better control at the target lesion site than did WBRT alone [7]. Two of the trials indicated a survival benefit conferred by surgical treatment when added to WBRT, compared to WBRT alone. Differences in the proportion of patients with NSCLC, percentages of patients with extracranial disease, treatment of patients with non-metastatic intracranial disease, and cross-over from one treatment arm to the other may explain why the study of Mintz and co-workers did not indicate a survival benefit [6]. Extent of extracranial disease status was a consistent predictor of survival.

Since these studies primarily enrolled patients with primary NSCLC or breast cancer, their applicability to melanoma is uncertain. No prospective trials of surgery for melanoma patients with brain metastases have been published to date. However, a number of large retrospective studies have been reported (Table 2) [8-13]. Surgical treatment is consistently reported as a factor strongly associated with prolonged survival over those treated with WBRT alone. Selection biases are inherent in retrospective studies. Indeed, two of the studies specifically identified factors predicting patient selection for more or less aggressive treatment and followup based on the presumed severity of CNS involvement [10, 12]. Given that these retrospective reports in melanoma concur with the randomized trials of surgical therapy in non-melanoma brain metastases, similar randomized trials of surgery for melanoma brain metastases are

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277

Traditionally, surgical management of brain metastases was restricted to individuals with a single accessible lesion. Bindal and co-workers found that individuals with a variety of primary solid tumors (n=56; melanoma=25/45%) undergoing resection of 2-3 brain metastases had survival rates equivalent to those undergoing resection of a single lesion [14]. In patients with complete resection of all known lesions, median survival was 14 months, equivalent to that for patients treated surgically for a single CNS lesion. Patients who could not undergo complete resection of CNS disease demonstrated inferior median overall survival of 6 months. Thus, presence of multiple CNS metastases is not a contra-indication to surgical treatment, although the advent of stereotactic radiosurgery (SRS) has made this approach less common.

Stereotactic radiosurgery (SRS) has become a major modality in the local treatment of brain metastases. When compared to conventional techniques, SRS allows for safe and effective dose escalation. This is achieved through use of multiple modulated beamlets from a variety of angles, allowing optimized conformality and avoidance of normal tissues. SRS is minimally or non-invasive and allows targeting multiple CNS lesions including those that may be surgically accessible. Treatment is often performed on an outpatient basis and over a short

The Radiation Therapy Oncology Group (RTOG) conducted a large randomized study of SRS combined with WBRT (n=164) versus WBRT alone (n=167) (Table 3) [15]. The study enrolled patients with a variety of tumor types, although NSCLC patients comprised the largest proportion. The addition of SRS to WBRT resulted in a survival benefit for patients with a single brain lesion (6.5 months for combination therapy versus 4.9 months for WBRT alone, p=0.0393), but not for patients with multiple lesions (5.8 months for combination therapy versus 6.7 months for WBRT alone, p=0.9776) or all patients combined (6.5 months for the combination versus 5.7 months for WBRT alone, p=0.1356). At 6 months, SRS-treated patients required lower doses of corticosteroids and were more likely to discontinue steroid use altogether (52% in SRS+WBRT decreased their dose compared to 33% in the WBRT only group, p<0.0158). Patients receiving SRS also were more likely to improve their performance status (13% improved vs. 4% improved in WBRT group, p=0.0331). Local control of targeted tumors

was better with SRS. Disease control at distant sites within the brain was equivalent.

time duration. Retreatment of the same or of new lesions is possible.

probably unnecessary.

**2.2. Stereotactic Radiosurgery (SRS)**


KPS: Karnofsky performance status; Mel: Melanoma patients; NR: Not reported; NSCLC: Non-small cell lung cancer; OS: Overall survival; QOL: Quality of life; R: Refers to treatment arm receiving WBRT alone; S: Refers to treatment arm combining surgery and WBRT

**Table 1.** Randomized trials of surgical resection of a single brain metastasis combined with WBRT versus WBRT alone

Since these studies primarily enrolled patients with primary NSCLC or breast cancer, their applicability to melanoma is uncertain. No prospective trials of surgery for melanoma patients with brain metastases have been published to date. However, a number of large retrospective studies have been reported (Table 2) [8-13]. Surgical treatment is consistently reported as a factor strongly associated with prolonged survival over those treated with WBRT alone. Selection biases are inherent in retrospective studies. Indeed, two of the studies specifically identified factors predicting patient selection for more or less aggressive treatment and followup based on the presumed severity of CNS involvement [10, 12]. Given that these retrospective reports in melanoma concur with the randomized trials of surgical therapy in non-melanoma brain metastases, similar randomized trials of surgery for melanoma brain metastases are probably unnecessary.

Traditionally, surgical management of brain metastases was restricted to individuals with a single accessible lesion. Bindal and co-workers found that individuals with a variety of primary solid tumors (n=56; melanoma=25/45%) undergoing resection of 2-3 brain metastases had survival rates equivalent to those undergoing resection of a single lesion [14]. In patients with complete resection of all known lesions, median survival was 14 months, equivalent to that for patients treated surgically for a single CNS lesion. Patients who could not undergo complete resection of CNS disease demonstrated inferior median overall survival of 6 months. Thus, presence of multiple CNS metastases is not a contra-indication to surgical treatment, although the advent of stereotactic radiosurgery (SRS) has made this approach less common.

### **2.2. Stereotactic Radiosurgery (SRS)**

all three, overall survival (OS) was the primary endpoint. In one study, the addition of surgery to WBRT achieved better control at the target lesion site than did WBRT alone [7]. Two of the trials indicated a survival benefit conferred by surgical treatment when added to WBRT, compared to WBRT alone. Differences in the proportion of patients with NSCLC, percentages of patients with extracranial disease, treatment of patients with non-metastatic intracranial disease, and cross-over from one treatment arm to the other may explain why the study of Mintz and co-workers did not indicate a survival benefit [6]. Extent of extracranial disease

> **Median Survival**

40 w-S 15 w-R P<0.01

10 m-S 6 m-R P=0.04

5.6 m-S 6.3 m-R P=0.24

KPS: Karnofsky performance status; Mel: Melanoma patients; NR: Not reported; NSCLC: Non-small cell lung cancer; OS: Overall survival; QOL: Quality of life; R: Refers to treatment arm receiving WBRT alone; S: Refers to treatment arm

**Table 1.** Randomized trials of surgical resection of a single brain metastasis combined with WBRT versus WBRT alone

**Recurrence/ Progression in CNS**

**Distant**

20%-S 13%-R P=0.52

**Treated Site**

20%-S 52%-R P<0.02 **Comments**

NR NR Survival benefit in those with

NR NR 45% of enrolled patients

analysis.

37% of enrolled patients with metastatic disease at enrollment. Extracranial disease and older age predicted decreased survival in multivariate analysis.

stable extra-cranial disease (12 m-S vs. 7 m-R, p=0.02) and patients younger than 61 y (19 m-S vs. 9 m-R, p=0.003). No survival benefit for surgery in patients with progressive extracranial disease or age"/>60.

with metastatic disease at enrollment. Only extracranial disease status predicted survival in multivariate

status was a consistent predictor of survival.

1 48

5 63

8

combining surgery and WBRT

**Patients (#)**

> 25-S 23-R

> 32-S 31-R

> 84 41-S 43-R

**Disease Types # (%)**

37 NSCLC (77%) 3 Mel. (6%)

33 NSCLC (52%) 6 Mel. (10%)

45 NSCLC (53%)

**Study Centers**

Patchell *et al*., 1990 [7]

Noordijk *et al.*, 1994 [5]

Mintz *et al.,* 1996 [6]

**(#)**

276 Melanoma - From Early Detection to Treatment

Stereotactic radiosurgery (SRS) has become a major modality in the local treatment of brain metastases. When compared to conventional techniques, SRS allows for safe and effective dose escalation. This is achieved through use of multiple modulated beamlets from a variety of angles, allowing optimized conformality and avoidance of normal tissues. SRS is minimally or non-invasive and allows targeting multiple CNS lesions including those that may be surgically accessible. Treatment is often performed on an outpatient basis and over a short time duration. Retreatment of the same or of new lesions is possible.

The Radiation Therapy Oncology Group (RTOG) conducted a large randomized study of SRS combined with WBRT (n=164) versus WBRT alone (n=167) (Table 3) [15]. The study enrolled patients with a variety of tumor types, although NSCLC patients comprised the largest proportion. The addition of SRS to WBRT resulted in a survival benefit for patients with a single brain lesion (6.5 months for combination therapy versus 4.9 months for WBRT alone, p=0.0393), but not for patients with multiple lesions (5.8 months for combination therapy versus 6.7 months for WBRT alone, p=0.9776) or all patients combined (6.5 months for the combination versus 5.7 months for WBRT alone, p=0.1356). At 6 months, SRS-treated patients required lower doses of corticosteroids and were more likely to discontinue steroid use altogether (52% in SRS+WBRT decreased their dose compared to 33% in the WBRT only group, p<0.0158). Patients receiving SRS also were more likely to improve their performance status (13% improved vs. 4% improved in WBRT group, p=0.0331). Local control of targeted tumors was better with SRS. Disease control at distant sites within the brain was equivalent.


**Study Dates, Patient Source**

center

~1978-1998

1979-1991 All surgically treated melanoma patients with a single brain metastasis at two centers. No active non-CNS metastases

present.

1972-1987 All surgically treated melanoma patients with a single brain metastasis at a single center

Sampson *et al.,* 1998 [8]

Skibber *et al.,* 1996 [53]

Hagen *et al.,* 1990 [54]

**Population, Institution**

All surgically treated brain metastasis patients (n=702) at a single

All melanoma patients (n=6953) treated at a single center

**Melanoma Patients Studied and Treatment**

29 S

13 died within 62 d of surgery

524 total 87 S/R 52 S 180 R 205 C

34 total 22 S/R 12 S

35 total 16 S 19 S/R

with whole brain radiotherapy; SRS: Stereotactic radiosurgery; Supp: Supportive care;

**Table 2.** Retrospective case series of surgery as treatment for brain metastasis in melanoma

**Median Survival CNS**

S/R (9.5 m) vs. S (8.3 m), p=0.67

Surgical therapy (NR) vs. R (120 d), p<0.0001 S/R (268 d) vs. S (195 d), p=0.9998 R (120 d) vs. C (39 d), p<0.0006

S/R (18 m) vs. S(6 m), p=0.002

S (8.3 m) vs. S/R (6.4

m), p=NS

C: Chemotherapy; Local Therapy: Treatment of CNS lesions with either surgery or stereotactic radiosurgery; KPS: Karnofsky performance status; NR: Not reported; NS: not significant; OS: Overall survival; QOL: Quality of life; R: Whole brain radiotherapy; S: Refers to treatment arm using surgery alone; Sig: Statistically significant; S/R: Surgery combined

A smaller study used the same design, but its primary endpoint was local disease control in patients with 2-4 brain lesions (Table 3) [16]. The study was halted at 60% of planned accrual due to meeting its primary endpoint (27 patients; 5 with melanoma). SRS-treated patients had significantly improved local disease control (p=0.0016). Median time-to-progression at SRStreated sites was 6 months in patients treated with WBRT alone, versus 36 months in those

**Recurrence Rates Based on Therapy Received**

Management of Brain Metastasis in Melanoma Patients

Overall CNS relapse rate: 30% S/R vs. 90% S, p=0.02

Median time to CNS relapse: S/R (26.6 m) vs. S (5.7 m), p<0.05

NR No sig.

**QOL**

279

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

difference in symptomati c results between patients treated with surgery and those treated with radiation (p=0.138)

NR

NR


**Study Dates, Patient Source**

278 Melanoma - From Early Detection to Treatment

1991-2001

1985-2000

1984-1998 All brain metastasis patients (n=1154) at a single center

1979-1999 All surgically treated melanoma patients with brain metastasis at a single center

1974-1994 91 total

All patients with brain metastasis from melanoma (n=1137) at a single center

Raizer *et al.,* 2008 [13]

Fife *et al.,* 2004 [12]

Buchsbaum *et al.,* 2002 [10]

Zacest *et al.,* 2002 [11]

Wronski and Arbit, 2000 [9] **Population, Institution**

All metastatic melanoma patients (n=1114) at a single center

**Melanoma Patients Studied and Treatment**

355 total 12 S/R +SRS 20 S + SRS 58 S/R 20 R + SRS 36 S 26 SRS 100 R 83 Supp.

686 total 158 S/R 47 S 236 R 210 Supp.

74 total 14 S/R 19 R + SRS 3 S/R + SRS 10 S or SRS 25 R 3 Supp.

147 total 9 S 102 S/R 33 S/R/C 3 S/C

49 S/R

**Median Survival CNS**

Surgery (9 m) vs. no surgery (4 m), p<0.0001 R 4.0 m Supp. 2.0 m

All pts. 4.1 m S/R 8.9 m S 8.7 m R 3.4 m Supp. 2.1 m S= S/R, p=0.21 S or S/R >R > Supp.,

p<0.001

All pts. 5.5m (S or SRS) + R 8.8 m S or SRS 4.8 m R 2.3 m Supp. 1.1 m (S or SRS) + R vs. other groups, p<0.0001 S/R =R + SRS, p=0.5128

All pts. 8.5 m 50% overall

All pts. 6.7 m 56% S/R vs.

**Recurrence Rates Based on Therapy Received**

NR NR

NR NR

49% Local + R 17% R 20% S or SRS

recurrence rate

46% S, p=NR

NR

Neurological symptoms after treatment: Resolved 52% Improved 26% Unchanged 9% N/A 13%

NR

**QOL**

C: Chemotherapy; Local Therapy: Treatment of CNS lesions with either surgery or stereotactic radiosurgery; KPS: Karnofsky performance status; NR: Not reported; NS: not significant; OS: Overall survival; QOL: Quality of life; R: Whole brain radiotherapy; S: Refers to treatment arm using surgery alone; Sig: Statistically significant; S/R: Surgery combined with whole brain radiotherapy; SRS: Stereotactic radiosurgery; Supp: Supportive care;

**Table 2.** Retrospective case series of surgery as treatment for brain metastasis in melanoma

A smaller study used the same design, but its primary endpoint was local disease control in patients with 2-4 brain lesions (Table 3) [16]. The study was halted at 60% of planned accrual due to meeting its primary endpoint (27 patients; 5 with melanoma). SRS-treated patients had significantly improved local disease control (p=0.0016). Median time-to-progression at SRStreated sites was 6 months in patients treated with WBRT alone, versus 36 months in those treated with SRS and WBRT (p=0.0005). Extracranial disease status was the major survival determinant in a *post hoc* analysis.

**Study Study Design Patient**

Randomized Multi-institution, cooperative group study 1996-2001 Primary endpoint: median survival

Randomized Single institution

Primary endpoint: local control at SRStreated site

Single-arm Multi-institution, cooperative group study 1998-2003 Primary endpoint: 3m and 6m intracranial progression rate

Single-arm Multi-institution 1998-2003 Primary

survival

endpoint: Overall

with whole brain radiotherapy; SRS: Stereotactic radiosurgery

Andrews *et al.,* 2004 [15]

Kondziolka *et al.,* 1999 [16]

Manon *et al.,* 2005 [17]

Friehs *et al.,* 1998 [18]

**Numbers & Tumor Types**

331 total 64% Lung 10% Breast 5% Melanoma 21% Other 1-3 CNS metastases

27 total: 44% Lung 19% Melanoma 15% Renal 15% Breast 7% Other 2-4 CNS metastases

31 total 45% Melanoma 45% Renal 10% Sarcoma 1-3 CNS metastases

45 total 100% Mel. 1-6 CNS metastases

KPS: Karnofsky performance status; Mel: Melanoma; Met: Metastasis/metastases; MMSE: Mini-Mental Status Examina‐ tion; NR: Not reported; QOL: Quality of life; R: Whole brain radiotherapy; SRS+R: Stereotactic radiosurgery combined

**Table 3.** Prospective trials of stereotactic radiosurgery as treatment for brain metastases in melanoma

164 SRS+R 167 R

13 SRS+R 14 R

**Treatment Median Survival CNS Recurrence**

Management of Brain Metastasis in Melanoma Patients

Time to intra-cranial progression SRS+R=R,

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281

Local control at 1 yr 82% SRS+R vs. 71% R, p=0.01

Median time to CNS

Local: 36 m SRS+R vs. 6 m

Any: 34 m SRS+R vs. 5 m

p=0.13

failure:

R, p=0.0005

R, p=0.002

3 m: Any 25.8% SRS-treated 19.3% Outside SRS 16.2%

6 m: Any 48.3% SRS-treated 32.2% Outside SRS field32.2%

tumors controlled at

13 (29%) with known distant failure in CNS.

follow-up.

31 SRS 8.3 m Intra-cranial Failure Rates

45 SRS 4.2 m 86% of SRS-treated

Overall 6.5 m S+R vs. 5.7 m R, p=0.14 Single met. 6.5 m SRS+R vs. 4.9 m R,

Multiple met. 5.8 m SRS+R vs. 6.7 m R, p=0.98

11 m SRS+R vs. 7.5 m R, p=0.22.

p=0.39

Only two relatively small, single-arm prospective studies of SRS in melanoma have been published. One of these studies enrolled 31 patients, including 14 (45%) with melanoma (Table 3) [17]. Patients received only SRS as CNS therapy. Overall intracranial failure rate was 50% at 6 months. About one-third of patients failed within the SRS-treated tumor volume. The second study enrolled 45 melanoma patients receiving SRS at one of two treatment centers (Table 3) [18]. Up to 6 metastases were treated. Use of WBRT in conjunction with this therapy was not reported. Median survival of all patients was 4.2 months. The local control rate with SRS was 86%, although the follow-up period was not defined. Follow-up imaging was available for only 71 out of 86 treated lesions.

Numerous retrospective studies have reported the results of SRS therapy in melanoma (Table 4) [19-41]. These are quite variable in design. While some studied melanoma patients exclu‐ sively, others enrolled patients with other tumor types. Several studies appear to include the same set of patients treated at a given institution during overlapping time periods (noted in Table 4). Treatment and follow-up plans were not pre-specified or standardized. Although all patients received SRS, they often received a wide array of other therapies, including immediate or delayed WBRT, concurrent or delayed surgery, and partial brain irradiation. Patients received SRS both as primary brain metastasis therapy and as salvage therapy after failure of prior treatment. Some patients received therapy for a single metastasis, while others were treated for multiple brain metastases. Several studies specifically identify selection bias in the treated population, with more aggressive therapy being reserved for patients with more severe CNS disease [37, 38]. Collectively, the study heterogeneity limits the conclusions that can be reached from these retrospective analyses.

Reported median survival of melanoma patients in these series ranged from 4.4 to 11.1 months. These values approximate ranges reported in patients with brain metastases from other primary tumor types, in which median survival is estimated to be 6.5 to 10.5 months [10, 42, 43]. Several factors predicted shorter survival in multiple studies: decreased performance status or its surrogate indicators, multiple CNS lesions, greater intracranial tumor volume, infratentorial lesion location, and active extracranial disease.

Some studies did not find that the initial number of lesions predicted survival [28, 29, 34, 37, 39]. This contradicts the results of the only randomized trial of SRS with survival as the primary endpoint, in which a survival benefit was observed only in patients with a single CNS lesion (OS was 6.5 months in patients with SRS+WRBT compared to 4.9 months in the WBRT group alone; p=0.0393) [15]. This may be due to inadequate statistical power in the retrospective studies, given the heterogeneity of the populations under study.

CNS disease control was reported in most of these retrospective studies as 1-year actuari‐ al control rates. At SRS-treated sites, reported in most of the studies, this was 47-87%. Oneyear control at non-SRS treated sites was 24-57%. The overall CNS control at one year was only 24-38%.


treated with SRS and WBRT (p=0.0005). Extracranial disease status was the major survival

Only two relatively small, single-arm prospective studies of SRS in melanoma have been published. One of these studies enrolled 31 patients, including 14 (45%) with melanoma (Table 3) [17]. Patients received only SRS as CNS therapy. Overall intracranial failure rate was 50% at 6 months. About one-third of patients failed within the SRS-treated tumor volume. The second study enrolled 45 melanoma patients receiving SRS at one of two treatment centers (Table 3) [18]. Up to 6 metastases were treated. Use of WBRT in conjunction with this therapy was not reported. Median survival of all patients was 4.2 months. The local control rate with SRS was 86%, although the follow-up period was not defined. Follow-up imaging was

Numerous retrospective studies have reported the results of SRS therapy in melanoma (Table 4) [19-41]. These are quite variable in design. While some studied melanoma patients exclu‐ sively, others enrolled patients with other tumor types. Several studies appear to include the same set of patients treated at a given institution during overlapping time periods (noted in Table 4). Treatment and follow-up plans were not pre-specified or standardized. Although all patients received SRS, they often received a wide array of other therapies, including immediate or delayed WBRT, concurrent or delayed surgery, and partial brain irradiation. Patients received SRS both as primary brain metastasis therapy and as salvage therapy after failure of prior treatment. Some patients received therapy for a single metastasis, while others were treated for multiple brain metastases. Several studies specifically identify selection bias in the treated population, with more aggressive therapy being reserved for patients with more severe CNS disease [37, 38]. Collectively, the study heterogeneity limits the conclusions that can be

Reported median survival of melanoma patients in these series ranged from 4.4 to 11.1 months. These values approximate ranges reported in patients with brain metastases from other primary tumor types, in which median survival is estimated to be 6.5 to 10.5 months [10, 42, 43]. Several factors predicted shorter survival in multiple studies: decreased performance status or its surrogate indicators, multiple CNS lesions, greater intracranial tumor volume,

Some studies did not find that the initial number of lesions predicted survival [28, 29, 34, 37, 39]. This contradicts the results of the only randomized trial of SRS with survival as the primary endpoint, in which a survival benefit was observed only in patients with a single CNS lesion (OS was 6.5 months in patients with SRS+WRBT compared to 4.9 months in the WBRT group alone; p=0.0393) [15]. This may be due to inadequate statistical power in the retrospective

CNS disease control was reported in most of these retrospective studies as 1-year actuari‐ al control rates. At SRS-treated sites, reported in most of the studies, this was 47-87%. Oneyear control at non-SRS treated sites was 24-57%. The overall CNS control at one year was

determinant in a *post hoc* analysis.

280 Melanoma - From Early Detection to Treatment

available for only 71 out of 86 treated lesions.

reached from these retrospective analyses.

only 24-38%.

infratentorial lesion location, and active extracranial disease.

studies, given the heterogeneity of the populations under study.

KPS: Karnofsky performance status; Mel: Melanoma; Met: Metastasis/metastases; MMSE: Mini-Mental Status Examina‐ tion; NR: Not reported; QOL: Quality of life; R: Whole brain radiotherapy; SRS+R: Stereotactic radiosurgery combined with whole brain radiotherapy; SRS: Stereotactic radiosurgery

**Table 3.** Prospective trials of stereotactic radiosurgery as treatment for brain metastases in melanoma


**Study Study Design Patient**

All patients with mel., renal cancer or sarcoma receiving SRS as therapy

1999-2003 Single institution All mel. patients receiving GK as initial

therapy

1996-2001 Single institution All mel. patients receiving GK

1991-2001 Single institution All mel. patients receiving LA-SRS

1986-2000 Two institutions All mel. patients treated

with LA-SRS

1990-2000 Single institution All patients with mel., renal cancer or sarcoma receiving SRS.

1996-2002 Single institution All mel. patients receiving GK

Koc *et al.,* 2005 [29]

Radbill *et al.,* 2004 [35]

Selek *et al.,* 2004 [38]

Herfarth *et al.,* 2003 [28]

Brown *et al.,* 2002 [19]

Gonzalez-Martinez *et al.,* 2002 [25]

**Numbers & Tumor Types**

26 total 100% mel.

64 total 100% mel.

103 total 100% mel.

64 total 100% mel.

41 total 23 mel. 16 renal 2 sarcoma

24 total 100% mel. 20 SRS 4 R with SRS sal. 8 Surg. with SRS sal. 9 SRS + surg.

10 SRS 14 SRS+R

**Treatment Median**

9 sarcoma 24% 1-y

12 SRS 14 SRS+R

32 SRS 13 SRS sal. 8 SRS+R 8 SRS+surg. 2 SRS + R + surg. 1 NR

61 SRS 12 SRS + R 30 SRS sal. **Survival**

survival for mel.

6 m 25% 1-yr survival

26 wk Single CNS met. (77 wk) vs. multiple (20 wk), p=0.003

Overall 6.7 m SRS 7.5 m SRS+R 3.7 m SRS sal. 5.4 m 25% 1-y survival

All SRS 10.6 m SRS-treated sites

14.2 m No difference in survival SRS+R vs. SRS, p=0.54

5.5 m NR

**CNS Control Rates (1-yr actuarial unless otherwise stated)**

Management of Brain Metastasis in Melanoma Patients

Distant 24%

SRS-treated sites

SRS-treated sites

NR

56% Distant 25%

48% SRS alone 60% SRS+R 0% SRS sal. 5% Distant 24% SRS alone 18% SRS+R 0% SRS sal. 51%

81% Lesions ≥2 cm (64%) vs. <2 cm (88%), p=0.05 Distant CNS control

NR

6 m CNS control SRS-treated sites: SRS+R (100%) vs. SRS (85%), p=0.02 Distant: SRS+R (91%) vs. SRS (35%), p=0.004 **Comments (Prognostic factors multivariate unless otherwise noted)**

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283

Adjuvant R did not decrease distant failure, although small population receiving it.

Patients with more aggressive disease were more likely to receive R

after SRS.


**Study Study Design Patient**

282 Melanoma - From Early Detection to Treatment

1987-2008 Single institution All patients with mel. receiving GK

1999-2005 Single institution All pts. receiving Cyberknife therapy for mel. or renal cancer

1998-2007 Single institution All patients receiving

1998-2006 Single institution All mel. pts. receiving

1999-2004 Single institution All mel. patients receiving LA-SRS

1998-2004 Single institution All mel. patients receiving GK

1997-2003 Single institution All mel. patients receiving GK

1991-2002 Single Institution

GK

GK

Liew *et al.,* 2011 [31]

Hara *et al.,* 2009 [27]

Powell *et al.,* 2008 [34]

Redmond *et al.,* 2008 [36]

Samlowski *et al.,* 2007 [37]

Christopoul ou *et al.,* 2006 [22]

Gaudy-Marqueste *et al.,* 2006 [23]

Chang *et al.,* 2005 [20]

**Numbers & Tumor Types**

344 total 100% mel.

62 total 44 mel. 18 renal

76 total 50 mel. 23 renal 3 sarcoma

59 total 100% mel.

44 total 100% mel.

29 total 100% mel.

106 total 100% mel.

189 total 103 mel. 77 renal

**Treatment Median**

163 SRS 118 SRS+R 63 SRS + other

33 SRS 17 SRS sal. 5 SRS+R 7 SRS + Surg.

39 SRS 37 SRS+R

32 SRS 27 SRS+R

19 SRS 4 SRS +partial R 14 SRS+R 16% SRS with salvage

R

All SRS 4 with prior R 2 with prior surg.

130 SRS 16 SRS + R 43 SRS sal.

106 SRS 5.1 m

**Survival**

5.6 m after SRS 8.3 m from diagnosis of brain met

8.3 m 5.6 m for mel.

5.1 m Histology does not predict outcome

11.1 m from brain metastasis diagnosis 48% 1-yr survival 18% 2-yr survival

5.7 m NR

13% 1-yr survival

**CNS Control Rates (1-yr actuarial unless otherwise stated)**

SRS treated sites

SRS-treated sites

Local and distant

SRS-treated sites

SRS-treated sites

SRS-treated sites

SRS-treated sites

69%

47%

7.5 m For mel.

4.4 m NR Timing between SRS and

63% Distant 33%

87%

38%

78% Distant 37% Local and distant

26%

47%

**Comments (Prognostic factors multivariate unless otherwise noted)**

recurrence.

1993.

R not sig. for survival or

Population may overlap with that of Mori et al., 1998 and Somoza et al.,

Local control higher for renal than mel . (94% vs. 63%, p=0.001)

Patients receiving SRS+R had a higher mean number of presented metastases (3.8) than those receiving salvage R after SRS failure (1.6). 22 (50%) treated with surgery at some point. Multiple lesions treated

R undefined.

in 22 (50%).

No patients received planned R

Inadequate patients treated with R to assess

effects.


**Study Study Design Patient**

1991-1995 Single institution (UCSF)

1992-1994 Single institution All mel. patients receiving LA-SRS

1988-1992 Single institution All mel. patients receiving GK

All melanoma patients receiving GK

Seung *et al.,* 1998 [39]

Gieger *et al.,* 1997 [24]

Somoza *et al.,* 1993 [40]

resection

**Numbers & Tumor Types**

55 total 100% melanoma

12 total 100% mel.

23 total 100% mel.

**Table 4.** Retrospective studies of stereotactic radiosurgery for brain metastasis in melanoma

**2.3. Comparative benefit of SRS versus surgery**

28 SRS 11 SRS+ R 16 SRS sal.

1 SRS 10 SRS+R 1 SRS sal.

19 SRS + R 4 SRS with R 3-12 m later

CNS: Central nervous system; GK: Gamma knife-based stereotactic radiosurgery; KPS: Karnofsky performance status; LA-SRS: Linear accelerator-based stereotactic radiosurgery; Mel: Melanoma; Met: Metastasis/metastases; NR: Not reported; QOL: Quality of life; Partial R: Partial brain irradiation; R: Whole brain radiotherapy; RPA: Recursive Partitioning Analysis; Sig: Statistically significant; SIR: Score Index for Radiosurgery; SRS: Stereotactic radiosurgery; SRS+R: Stereotactic radiosurgery combined with whole brain radiotherapy; SRS sal: Stereotactic radiosurgery salvage after failure of prior therapy; SRS+Surg: Combination therapy of stereotactic radiosurgery and conventional surgical resection; Surg: Surgical

The relative benefit of SRS versus surgery has not been tested in randomized clinical trials to date. One small randomized trial indirectly addressed this question, although not specifically for melanoma (Table 5) [44]. Sixty-four subjects with a single, surgically accessible brain lesion were randomly assigned to surgical excision and adjuvant WBRT or to SRS alone. A direct comparison of surgery and SRS is not possible due to the inclusion of adjuvant WBRT for all surgical patients and its omission in SRS-treated patients. Nine (14%) of the subjects had

8 m 36% 1-y survival

7 m 26% 1-y survival

**Treatment Median**

**Survival**

**CNS Control Rates (1-yr actuarial unless otherwise stated)**

Management of Brain Metastasis in Melanoma Patients

SRS+R 80% SRS 100% SRS sal. 86% Distant: Overall 70% SRS+R 77% SRS 56% SRS sal. 57%

35 wk SRS-treated sites 77% Distant 36% Entire CNS 24%

> At least 6 patients with at least one SRS-treated lesions progressing. At least 3 (25%) developing new distant CNS lesions

**Comments (Prognostic factors multivariate unless otherwise noted)**

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285

Imaging follow-up not

with that of Mathieu et al., 2007 and Mori *et al.,*

consistent.

NR Population may overlap

1998.


CNS: Central nervous system; GK: Gamma knife-based stereotactic radiosurgery; KPS: Karnofsky performance status; LA-SRS: Linear accelerator-based stereotactic radiosurgery; Mel: Melanoma; Met: Metastasis/metastases; NR: Not reported; QOL: Quality of life; Partial R: Partial brain irradiation; R: Whole brain radiotherapy; RPA: Recursive Partitioning Analysis; Sig: Statistically significant; SIR: Score Index for Radiosurgery; SRS: Stereotactic radiosurgery; SRS+R: Stereotactic radiosurgery combined with whole brain radiotherapy; SRS sal: Stereotactic radiosurgery salvage after failure of prior therapy; SRS+Surg: Combination therapy of stereotactic radiosurgery and conventional surgical resection; Surg: Surgical resection

**Table 4.** Retrospective studies of stereotactic radiosurgery for brain metastasis in melanoma

#### **2.3. Comparative benefit of SRS versus surgery**

**Study Study Design Patient**

284 Melanoma - From Early Detection to Treatment

1989-1999 Single institution All mel. patients receiving GK

1994-1999 Single institution All mel. patients receiving GK

1994-1999 Single institution All patients receiving

1994-1997 Single institution All mel. patients receiving GK

1993-1996 Single institution All mel. patients receiving GK

1988-1996 Single institution All mel. patients receiving GK

GK

Mingione *et al.,* 2002 [32]

Yu *et al.,* 2002 [41]

Chen *et al.,* 1999 [21]

Lavine *et al.,* 1999 [30]

Grob *et al.,* 1998 [26]

Mori *et al.,* 1998 [33]

**Numbers & Tumor Types**

45 total 100% mel.

122 total 100% mel.

199 total 88 mel. 40 NSCLC 5 SCLC 12 Renal 12 Breast 9 Colon 24 Other

45 total 100% mel.

35 total 100% mel.

60 total 100% mel. 12 SRS 36 SRS+R 12 SRS sal.

43 SRS. 2 SRS + R

**Treatment Median**

29 SRS 16 SRS+R

83 SRS 12 SRS + R 10 SRS/R >1.5 m before SRS 17 SRS/R >1.5 m after SRS

**Survival**

10.4 m 31% 1-yr survival

7 m 26% 1-yr survival

199 SRS 8.5 m

7 m for mel.

**CNS Control Rates (1-yr actuarial unless otherwise stated)**

SRS-treated sites

89% of lesions with follow-up "controlled for the lifetime of the patient"

84% Distant 57%

8 m 3 m CNS control

35 SRS 7 m Actuarial control

7 m median survival 21% 1-yr survival

97% Distant 81%

SRS-treated sites

rate of evaluable, treated lesions: 3 m 98% 6 m 100% 9 m 95% 12 m 87% Distant CNS control not reported

Control in 46 pts. receiving SRS +/-R: SRS-treated sites: Overall 85%

**Comments (Prognostic factors multivariate unless otherwise noted)**

impact on survival or CNS recurrence rate.

Population may overlap with that of Lavine et al., 1999 and Chen et al.,

Use of R reported, but not defined.

Population may overlap with that of Yu et al., 2002 and Lavine et al.,

Follow-up available for only 69% of lesions.

Population may overlap with that of Yu et al., 1999 and Chen et al.,

Other therapies in addition to SRS depending on clinical condition. Only 2 (4%) received SRS+R

Population may overlap with that of Mathieu et al., 2007 and Somoza et

al., 1993.

NR Adjuvant R had no

1999.

1999.

1999.

The relative benefit of SRS versus surgery has not been tested in randomized clinical trials to date. One small randomized trial indirectly addressed this question, although not specifically for melanoma (Table 5) [44]. Sixty-four subjects with a single, surgically accessible brain lesion were randomly assigned to surgical excision and adjuvant WBRT or to SRS alone. A direct comparison of surgery and SRS is not possible due to the inclusion of adjuvant WBRT for all surgical patients and its omission in SRS-treated patients. Nine (14%) of the subjects had melanoma. No difference in overall survival was observed (9.5 months for surgery versus 10.3 months for SRS, p=0.8). A statistically non-significant improvement in local tumor control favored SRS (82% for surgery plus WBRT versus 97% for SRS, p=0.06). The one-year recurrence rate at distant CNS sites was significantly higher in the group receiving SRS alone (3% for surgery plus WBRT versus 26% for SRS alone, p<0.05). Thus, this study perhaps served to highlight the risks of omitting adjuvant treatment, rather than the relative merits of SRS versus surgery.

ancies might explain the differences in results when contrasted with the results of the two other

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287

Collectively these studies do not indicate whether surgery or SRS is superior. There are no easily detectable differences in local control rates. Logistic differences therefore are important in selecting therapy. Unless a clinical situation arises in which surgery provides clear superi‐ ority (e.g. rapid control of symptomatic lesions; histological diagnosis), SRS will likely be the

Adjuvant therapy of the CNS is that which is administered in conjunction with definitive local therapy (surgery or SRS) of radiologically evident tumors to treat co-existing micrometastatic disease. This is distinguished from prophylactic cranial irradiation (PCI). PCI is administered in patients with systemic cancer after responses to systemic therapy, and has proven benefit in several conditions, such as small cell lung cancer (SCLC) [47, 48]. In melanoma, PCI has not been adequately assessed to recommend. Adjuvant CNS therapy has traditionally relied on WBRT. Although new systemic agents with proven anti-melanoma activity and CNS pene‐ tration may come to be used for this purpose as well, such use is experimental at present. Adjuvant WBRT is a controversial topic in metastatic brain tumor management, primarily due

Three factors must be considered in determining whether or not to use adjuvant WBRT: (a) the effectiveness of WBRT in preventing emergence of new brain tumors; (b) the adverse effects of WBRT; and (c) the competing adverse effect of foregoing WBRT, namely an increased rate of CNS tumor progression. As new systemic therapies are proposed for this purpose, the same considerations apply. The relevant adverse effects relate to deterioration of neurocognitive function (NCF) and QOL, which could result from either WBRT itself or from progressive brain tumors. It is in balancing these factors that a rational decision regarding the use, or non-use,

Despite the frequency of brain metastasis in melanoma patients, no prospective trials have been conducted to assess adjuvant WBRT in this population. Data from the treatment of brain metastases focusing on other tumor types must be reviewed to come to any conclusions (Table 5). Five randomized trials of adjuvant WBRT have been reported. Four of these are multiinstitutional efforts, reflecting the difficulty in conducting this type of study [49-52]. A fifth study, discussed earlier, compared outcome in patients with a single brain metastasis treated with surgery and WBRT or with SRS alone [44]. The majority of patients in all of the studies were those with NSCLC primary tumors. Relatively few melanoma patients were enrolled.

Only one study used intracranial recurrence rate as the planned primary endpoint [52]. Ninetyfive patients were enrolled after surgical resection of an isolated brain metastasis. Sixty percent had NSCLC. Forty-nine patients were randomized to receive adjuvant WBRT (50.4 Gy administered as 28-1.8 Gy fractions). The remaining forty-six patients were observed. Only

predominant modality employed to treat macroscopic melanoma lesions in the CNS.

studies described [44, 45].

**2.4. Adjuvant Whole Brain Radiotherapy (WBRT)**

to questions of efficacy and of neurocognitive toxicity.

*2.4.1. Randomized trials of adjuvant WBRT in solid tumor patients*

of adjuvant WBRT can be made.

Two retrospective studies compared SRS to surgery [45, 46]. Melanoma patients were in the minority in both studies. O'Neill and co-workers analyzed patients seen from 1991-1999 at the Mayo Clinic who underwent either SRS or surgery for a solitary brain metastasis [45]. Eligible patients were candidates for either procedure: all had solitary lesions measuring less than 35 mm (maximum size conventionally treated with SRS), none of the lesions were surgically inaccessible, and none required immediate surgical decompression. Ninety-seven patients met these criteria, of whom only seven were melanoma patients. Seventy-four were treated surgically and twenty-three were treated with SRS. Although not achieving statistical signif‐ icance, more SRS-treated patients received WBRT (96% SRS vs. 82% surgery, p=0.172). The treatment groups differed at baseline in performance status (worse in the SRS group, p=0.0016). Overall survival was similar between the two groups and was predicted by age, performance status, and systemic extracranial disease status rather than the type of brain metastasis treatment. Similar proportions of patients had CNS recurrence (29% SRS versus 30% surgery), but patients receiving surgery were more likely to have local recurrence at treated sites (58% of recurrences in 19 patients vs. 0% out of 6 recurrences in SRS-treated patients, p=0.02). Although this study suggests that local recurrences are more common after surgery, the retrospective nature of the study and the small number of patients limits its applicability.

In contrast, another single institution, retrospective study from a similar time period (1991-1994) suggested that SRS led to higher local recurrence rates than surgery [46]. Thirtyone patients were treated with SRS and sixty-two with surgery for brain metastases. Twentyone patients (23%) had melanoma. Patients were matched with regard to histology, extracranial disease status, performance status, time from initial diagnosis to CNS metastasis, number of CNS metastases, age, and gender. Patients in the two groups were equally likely to have received WBRT. Patients receiving surgical treatment survived significantly longer than those treated with SRS (16.4 months surgery vs. 7.5 months SRS, p=0.0018). This improvement in survival was attributable to decreased rates of death from neurological causes in the surgical group (19% surgery vs. 50% SRS, p=0.037); deaths due to systemic disease were equivalent (p=0.28). Surgery yielded lower local tumor recurrence rates than SRS (8.1% surgery vs. 38.7% SRS). There was no statistically significant different in distant CNS recurrence rates between the two groups.

This retrospective study is subject to the biases inherent in such an undertaking. The authors matched patients for a variety of known relevant parameters, but the differences in local control may reflect the use of older SRS technology, high quality neurosurgical treatment at the referral center where the study was undertaken, or a combination of both. These discrep‐ ancies might explain the differences in results when contrasted with the results of the two other studies described [44, 45].

Collectively these studies do not indicate whether surgery or SRS is superior. There are no easily detectable differences in local control rates. Logistic differences therefore are important in selecting therapy. Unless a clinical situation arises in which surgery provides clear superi‐ ority (e.g. rapid control of symptomatic lesions; histological diagnosis), SRS will likely be the predominant modality employed to treat macroscopic melanoma lesions in the CNS.

## **2.4. Adjuvant Whole Brain Radiotherapy (WBRT)**

melanoma. No difference in overall survival was observed (9.5 months for surgery versus 10.3 months for SRS, p=0.8). A statistically non-significant improvement in local tumor control favored SRS (82% for surgery plus WBRT versus 97% for SRS, p=0.06). The one-year recurrence rate at distant CNS sites was significantly higher in the group receiving SRS alone (3% for surgery plus WBRT versus 26% for SRS alone, p<0.05). Thus, this study perhaps served to highlight the risks of omitting adjuvant treatment, rather than the relative merits of SRS versus

Two retrospective studies compared SRS to surgery [45, 46]. Melanoma patients were in the minority in both studies. O'Neill and co-workers analyzed patients seen from 1991-1999 at the Mayo Clinic who underwent either SRS or surgery for a solitary brain metastasis [45]. Eligible patients were candidates for either procedure: all had solitary lesions measuring less than 35 mm (maximum size conventionally treated with SRS), none of the lesions were surgically inaccessible, and none required immediate surgical decompression. Ninety-seven patients met these criteria, of whom only seven were melanoma patients. Seventy-four were treated surgically and twenty-three were treated with SRS. Although not achieving statistical signif‐ icance, more SRS-treated patients received WBRT (96% SRS vs. 82% surgery, p=0.172). The treatment groups differed at baseline in performance status (worse in the SRS group, p=0.0016). Overall survival was similar between the two groups and was predicted by age, performance status, and systemic extracranial disease status rather than the type of brain metastasis treatment. Similar proportions of patients had CNS recurrence (29% SRS versus 30% surgery), but patients receiving surgery were more likely to have local recurrence at treated sites (58% of recurrences in 19 patients vs. 0% out of 6 recurrences in SRS-treated patients, p=0.02). Although this study suggests that local recurrences are more common after surgery, the retrospective nature of the study and the small number of patients limits its applicability.

In contrast, another single institution, retrospective study from a similar time period (1991-1994) suggested that SRS led to higher local recurrence rates than surgery [46]. Thirtyone patients were treated with SRS and sixty-two with surgery for brain metastases. Twentyone patients (23%) had melanoma. Patients were matched with regard to histology, extracranial disease status, performance status, time from initial diagnosis to CNS metastasis, number of CNS metastases, age, and gender. Patients in the two groups were equally likely to have received WBRT. Patients receiving surgical treatment survived significantly longer than those treated with SRS (16.4 months surgery vs. 7.5 months SRS, p=0.0018). This improvement in survival was attributable to decreased rates of death from neurological causes in the surgical group (19% surgery vs. 50% SRS, p=0.037); deaths due to systemic disease were equivalent (p=0.28). Surgery yielded lower local tumor recurrence rates than SRS (8.1% surgery vs. 38.7% SRS). There was no statistically significant different in distant CNS recurrence rates between

This retrospective study is subject to the biases inherent in such an undertaking. The authors matched patients for a variety of known relevant parameters, but the differences in local control may reflect the use of older SRS technology, high quality neurosurgical treatment at the referral center where the study was undertaken, or a combination of both. These discrep‐

surgery.

286 Melanoma - From Early Detection to Treatment

the two groups.

Adjuvant therapy of the CNS is that which is administered in conjunction with definitive local therapy (surgery or SRS) of radiologically evident tumors to treat co-existing micrometastatic disease. This is distinguished from prophylactic cranial irradiation (PCI). PCI is administered in patients with systemic cancer after responses to systemic therapy, and has proven benefit in several conditions, such as small cell lung cancer (SCLC) [47, 48]. In melanoma, PCI has not been adequately assessed to recommend. Adjuvant CNS therapy has traditionally relied on WBRT. Although new systemic agents with proven anti-melanoma activity and CNS pene‐ tration may come to be used for this purpose as well, such use is experimental at present. Adjuvant WBRT is a controversial topic in metastatic brain tumor management, primarily due to questions of efficacy and of neurocognitive toxicity.

Three factors must be considered in determining whether or not to use adjuvant WBRT: (a) the effectiveness of WBRT in preventing emergence of new brain tumors; (b) the adverse effects of WBRT; and (c) the competing adverse effect of foregoing WBRT, namely an increased rate of CNS tumor progression. As new systemic therapies are proposed for this purpose, the same considerations apply. The relevant adverse effects relate to deterioration of neurocognitive function (NCF) and QOL, which could result from either WBRT itself or from progressive brain tumors. It is in balancing these factors that a rational decision regarding the use, or non-use, of adjuvant WBRT can be made.

#### *2.4.1. Randomized trials of adjuvant WBRT in solid tumor patients*

Despite the frequency of brain metastasis in melanoma patients, no prospective trials have been conducted to assess adjuvant WBRT in this population. Data from the treatment of brain metastases focusing on other tumor types must be reviewed to come to any conclusions (Table 5). Five randomized trials of adjuvant WBRT have been reported. Four of these are multiinstitutional efforts, reflecting the difficulty in conducting this type of study [49-52]. A fifth study, discussed earlier, compared outcome in patients with a single brain metastasis treated with surgery and WBRT or with SRS alone [44]. The majority of patients in all of the studies were those with NSCLC primary tumors. Relatively few melanoma patients were enrolled.

Only one study used intracranial recurrence rate as the planned primary endpoint [52]. Ninetyfive patients were enrolled after surgical resection of an isolated brain metastasis. Sixty percent had NSCLC. Forty-nine patients were randomized to receive adjuvant WBRT (50.4 Gy administered as 28-1.8 Gy fractions). The remaining forty-six patients were observed. Only


one patient in each group had melanoma. The CNS recurrence rate was 18% (9/49) in those receiving adjuvant WBRT. This contrasted sharply with a 70% (32/46) CNS recurrence rate in the observation group (p<0.001). The median time-to-CNS recurrence was markedly pro‐ longed in those receiving adjuvant WBRT (220 weeks versus 26 weeks observation, p<0.001) due to decreased recurrence rates both at resection sites (10% WBRT versus 46% observation, p<0.001) and at distant sites within the brain (14% WBRT versus 37% observation, p<0.01). There was no difference in median survival (49 weeks WBRT versus 43 weeks observation, p=0.39) or in maintenance of independent function (maintenance of KPS >60%). A decreased rate of neurologic cause of death was evident in the WBRT-treated group (14% WBRT versus 44% observation, p=0.003), although the determination of this was less objective than deter‐

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Another randomized trial tested adjuvant WBRT (30 Gy in 10 fractions) in conjunction with SRS [49]. The study enrolled 132 patients with one-to-four metastases measuring less than 3 cm in maximal dimension. Sixty-five patients received SRS and WBRT; sixty-seven received SRS alone. Two-thirds of those enrolled had NSCLC. The majority of the remainder had breast, colon or renal primary sites. The primary endpoint was overall survival. The researchers initially estimated that 89 evaluable patients per group would be required to detect a 30% difference in median survival time. A planned interim analysis, performed after 122 patients enrolled, led to early study termination. Four-to-five-fold more patients would have been

Although underpowered to detect a survival advantage, a number of secondary endpoints yielded significant results. CNS progression at 1 year was 47% in the combination therapy group and 76% in the SRS monotherapy group (p<0.001). WBRT improved control at one year for both SRS-treated sites (89% WBRT versus 72% without, p=0.002) and distant CNS sites (58% WBRT versus 36% without, p=0.003). No differences were observed in median survival, neurological cause of death, and acute or late neurological toxicity. Rates of systemic functional preservation (assessed by KPS), neurological preservation, and neurocognitive preservation

A third trial randomized patients with 1-3 brain metastases to either SRS or SRS combined with adjuvant WBRT (30 Gy in 12 fractions) [50]. A novel endpoint for the study was chosen: change in performance on the Hopkins Verbal Learning Test-Revised (HVLT) at 4 months after primary therapy. The majority of enrolled patients were those with NSCLC primary tumors (55%), with melanoma in the minority (12%). The study was stopped early after accrual of 58 patients (28 SRS+WBRT, 30 SRS) due to its achieving the primary endpoint. Patients treated with the combination demonstrated a 52% decline in HVLT score at 4 months, versus a 24% decline in those receiving SRS only (p=0.04). This difference persisted at 6 months. Significant differences in performance on a panel of other neurocognitive tests were not detected. The study was stopped early and may have therefore been underpowered to detect other important differences in outcome. Decreased HVLT performance occurred despite decreased rates of CNS progression at one year in the combination therapy group (SRS-treated sites: 0% SRS +WBRT vs. 33% SRS, p=0.01; distant CNS: 27% vs. 55%, p=0.02). The authors also reported

(assessed by the Mini-Mental Status Examination, MMSE) were also not different.

mination of intracranial recurrence by imaging.

required to detect a significant difference in the primary endpoint.

**Table 5.** Randomized trials of adjuvant WBRT with surgery or stereotactic radiosurgery for brain metastases

one patient in each group had melanoma. The CNS recurrence rate was 18% (9/49) in those receiving adjuvant WBRT. This contrasted sharply with a 70% (32/46) CNS recurrence rate in the observation group (p<0.001). The median time-to-CNS recurrence was markedly pro‐ longed in those receiving adjuvant WBRT (220 weeks versus 26 weeks observation, p<0.001) due to decreased recurrence rates both at resection sites (10% WBRT versus 46% observation, p<0.001) and at distant sites within the brain (14% WBRT versus 37% observation, p<0.01). There was no difference in median survival (49 weeks WBRT versus 43 weeks observation, p=0.39) or in maintenance of independent function (maintenance of KPS >60%). A decreased rate of neurologic cause of death was evident in the WBRT-treated group (14% WBRT versus 44% observation, p=0.003), although the determination of this was less objective than deter‐ mination of intracranial recurrence by imaging.

**Study Evaluable**

Patchell *et al.,* 1998 [52]

Aoyama *et al.,* 2006 [49]

Muacevic *et al.,* 2008 [44]

Chang EL *et al.,* 2009 [50]

Kocher *et al.,* 2011 [51]

**Patients (#)**

288 Melanoma - From Early Detection to Treatment

95 total 49 S+R 46 S

132 total 65 SRS+R 67 SRS

64 total 31 SRS 33 S+R

58 28 SRS+R 30 SRS

359 total SRS 100 SRS+R 99 S 79 S+R 81

**Disease types #**

57 NSCLC 9 Breast 29 Other

88 NSCLC 9 Breast 11 GI 10 Renal 14 Other

22 NSCLC 10 GU 11 Breast 9 Mel. 4 GI 8 Other

32 NSCLC 8 Breast 7 Mel. 4 Renal 7 Other

190 NSCLC 29 Renal 42 Breast 18 Mel. 30 Colon 50 Other

**Primary Endpoint**

CNS recurrence rate

OS SRS+R

OS 9.5 m S+R

HVLT at 4 m vs. baseline

Duration of functional independenc e (measured as deterioration of WHO PS to >2)

**Table 5.** Randomized trials of adjuvant WBRT with surgery or stereotactic radiosurgery for brain metastases

**Median Survival**

S+R (48 wk) vs. S (43 wk), p=0.39

(7.5m) vs. SRS (8 .0 m), p=0.42

10.3 m SRS P=0.8

SRS+R (5.7 m) vs. SRS (15.2m), p=0.003

WBRT (10.7 m) vs. No WBRT (10.9 m), p=0.89

**Recurrence/Progression**

**S/SRS-site Distant**

14% S+R 37% S P<0.01

1-y rate 42% SRS+R 64% SRS P=0.003

1-y rate 26% SRS 3% S+R P=0.04

1-y rate 27% SRS+R 55% SRS P=0.02

2-y rate S – 42% S+R – 23% (p = 0.008) SRS – 48% SRS+R – 33% (p = 0.023) **Comments**

Single brain metastasis. Overall CNS recurrence rate (primary endpoint) significantly less in R-treated patients (18% S+R vs. 70% S, p<0.001). Decreased rate of neurological cause of death in group receiving R (14% S

+R vs. 44% S, p=0.003)

1-4 brain metastases. Overall CNS recurrence rate at 1-y:

Salvage treatment required:

Single, surgically accessible brain

Study stopped early due to poor

Up to 3 brain metastases. Decline in function at 4 m (primary

Study accrual stopped early due to achieving primary endpoint.

1-3 brain metastasis eligible. Either stable extracranial disease for 3 months or no extracranial metastases. Median Survival with WHO PS ≤2 (primary endpoint) 9.5m WBRT vs. 10.0

Overall rate of CNS progression at 6 and 24 m: 15.2% and 31.4% WBRT vs. 39.7 and 54% no WBRT, p<0.0001 Neurological cause of death 25% WBRT vs. 43% no WBRT, p=NR

m no WBRT, p=0.709

47% SRS+R 76% SRS P<0.001

15% SRS+R 43% SRS P<0.001

metastasis.

accrual.

endpoint): 48% SRS+R 24% SRS P=0.04

**in CNS**

10% S+R 46% S P<0.001

1-y rate 11% SRS +R 38% SRS P=0.002

1-y rate 3% SRS 18% S+R P=0.06

1-y rate 0% SRS+R 33% SRS P=0.01

2-y rate S – 59% S+R – 27% (p < 0.001) SRS – 31% SRS+R – 19% (p = 0.040) Another randomized trial tested adjuvant WBRT (30 Gy in 10 fractions) in conjunction with SRS [49]. The study enrolled 132 patients with one-to-four metastases measuring less than 3 cm in maximal dimension. Sixty-five patients received SRS and WBRT; sixty-seven received SRS alone. Two-thirds of those enrolled had NSCLC. The majority of the remainder had breast, colon or renal primary sites. The primary endpoint was overall survival. The researchers initially estimated that 89 evaluable patients per group would be required to detect a 30% difference in median survival time. A planned interim analysis, performed after 122 patients enrolled, led to early study termination. Four-to-five-fold more patients would have been required to detect a significant difference in the primary endpoint.

Although underpowered to detect a survival advantage, a number of secondary endpoints yielded significant results. CNS progression at 1 year was 47% in the combination therapy group and 76% in the SRS monotherapy group (p<0.001). WBRT improved control at one year for both SRS-treated sites (89% WBRT versus 72% without, p=0.002) and distant CNS sites (58% WBRT versus 36% without, p=0.003). No differences were observed in median survival, neurological cause of death, and acute or late neurological toxicity. Rates of systemic functional preservation (assessed by KPS), neurological preservation, and neurocognitive preservation (assessed by the Mini-Mental Status Examination, MMSE) were also not different.

A third trial randomized patients with 1-3 brain metastases to either SRS or SRS combined with adjuvant WBRT (30 Gy in 12 fractions) [50]. A novel endpoint for the study was chosen: change in performance on the Hopkins Verbal Learning Test-Revised (HVLT) at 4 months after primary therapy. The majority of enrolled patients were those with NSCLC primary tumors (55%), with melanoma in the minority (12%). The study was stopped early after accrual of 58 patients (28 SRS+WBRT, 30 SRS) due to its achieving the primary endpoint. Patients treated with the combination demonstrated a 52% decline in HVLT score at 4 months, versus a 24% decline in those receiving SRS only (p=0.04). This difference persisted at 6 months. Significant differences in performance on a panel of other neurocognitive tests were not detected. The study was stopped early and may have therefore been underpowered to detect other important differences in outcome. Decreased HVLT performance occurred despite decreased rates of CNS progression at one year in the combination therapy group (SRS-treated sites: 0% SRS +WBRT vs. 33% SRS, p=0.01; distant CNS: 27% vs. 55%, p=0.02). The authors also reported improved survival in the group treated with SRS alone (5.7 months SRS+WBRT vs. 15.2 months SRS, p=0.003).

uncontrolled extracranial disease is unclear, but likely has a lesser effect. If seeding from extracranial disease was a dominant mechanism leading to CNS failure, adjuvant WBRT

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No trial to date evaluating the omission of adjuvant WBRT after local therapy has demonstrated a survival benefit to WBRT. The study by Chang and co-workers indicated that the use of adjuvant WBRT after local therapy might be associated with a decrement in survival. It is difficult to draw firm conclusions about these data, as the study was stopped early, was not powered to detect a survival benefit, and contradicted the surviv‐ al results of the other four larger randomized studies presented above. This includes the study by Aoyama and co-workers [50], which evaluated overall survival as its primary endpoint and was unable to detect a survival difference between its treatment arms, without a marked increase in sample size to over 800. The study by Kocher and coworkers demonstrated an improvement in PFS associated with adjuvant WBRT [51]. This study excluded patients with uncontrolled or progressive primary disease, mitigating

The studies presented here represent the best assessment of the efficacy of adjuvant WBRT therapy in treatment of solid tumor brain metastases. This therapy is clearly able to decrease intracranial recurrence rates, both at locally treated and distant sites within the CNS. The effect of this therapy on survival and the relative benefits versus the cognitive effects of the therapy are less clear. Melanoma patients formed a small fraction of the patients enrolled in these trials and one might therefore question whether these results even apply in the melanoma setting. To do so requires examination of the rather imperfect retrospective dataset regarding adjuvant

The randomized studies discussed above primarily enrolled patients diagnosed with NSCLC. There have been no prospective studies evaluating the role of adjuvant WBRT specifically in the melanoma patient population. Many retrospective studies have been reported; unsurpris‐ ingly, these have indicated that adjuvant WBRT confers no survival benefit (see Tables 2, 4) [8-10, 12, 19, 29, 31, 32, 34, 35, 38, 39, 41, 53]. Since most melanoma patients with brain lesions present with active extracranial disease, any potential survival benefit due to adjuvant WBRT after local CNS therapy is probably undermined: extracranial disease serves as a competing

It is difficult to make firm conclusions based on the numerous melanoma case series on whether adjuvant WBRT actually decreases the rate of intracranial recurrence after local therapy. Selection and ascertainment biases are major concerns. Patients with clinically advanced disease are often selected for more aggressive therapy. Groups receiving aggressive therapy

Several retrospective studies identify such biases. In the study of Buchsbaum and co-workers a paradoxically *higher* rate of CNS recurrence (49%) was identified in patients having received combined local CNS lesion therapy and adjuvant WBRT versus local therapy alone (20%) [10].

are likely to undergo more frequent and detailed surveillance for recurrence.

would not be predicted to decrease its occurrence.

extracranial disease burden as a competing risk for death

WBRT specifically in melanoma.

*2.4.2. Adjuvant WBRT in melanoma patients*

cause of death, diluting any study's statistical power.

Patients who were treated only with SRS required salvage therapy for intracranial progression in 87% of cases. Ten (33%) of the patients treated with SRS alone required craniotomy, ten (33%) received salvage WBRT and six (20%) received salvage SRS. In the group treated with SRS and adjuvant WBRT, two patients (7%) received salvage WBRT, and three (11%) pro‐ gressed intracranially, but received no salvage therapy.

This study provides convincing evidence that the addition of adjuvant WBRT to SRS therapy for brain metastases impairs HVLT performance. This occurs despite a decreased rate of intracranial progression in those receiving WBRT. Salvage therapy for intracranial progression was required in the majority of patients treated with SRS alone, including salvage craniotomy in one-third of the patients. The clinical significance of HVLT deterioration due to adjuvant WBRT, vis a vis that of frequently needed salvage therapy for CNS disease was not addressed.

A fourth randomized trial assessing adjuvant WBRT enrolled patients with 1-3 brain meta‐ stases and stable or absent extracranial disease [51]. The majority of patients had NSCLC (53%); only 5% were melanoma patients. Patients received SRS or surgery as primary therapy and were then randomized to receive adjuvant WBRT (30 Gy in 10 fractions) or no additional therapy. The composite primary endpoint was median overall survival in patients with KPS of 0-2. In the intent-to-treat analysis, 180 patients were assigned to receive WBRT and 179 to observation. At the end of the study, per protocol, 164 patients received WBRT and 166 patients were on observation. Analysis was by intention-to-treat.

No differences were detected in the primary endpoint of survival with functional independ‐ ence (9.5 months WBRT versus 10.0 months observation, p=0.709) or median overall survival (10.7 months WBRT versus 10.9 months observation, p=0.891). Intracranial recurrence rates were markedly suppressed by adjuvant WBRT. Overall intracranial progression occurred in 48% of WBRT-treated patients and in 78% of the observation group (p<0.001). This translated to improved progression-free survival (PFS) in the WBRT-treated group (4.6 months vs. 3.4 months observation, p=0.002). Two years after surgery, WBRT reduced the probability of relapse at intial site from 59% (observation) to 27% (p<0.001) and at distant CNS sites from 42% (observation) to 23% (p=0.008). Similarly, after SRS, WBRT reduced the probability of relapse at SRS-treated site from 31% (observation) to 19% (p=0.040) and at distant CNS sites from 48% (observation) to 33% (p=0.023). Neurological cause of death was suppressed by adjuvant WBRT (28% WBRT versus 44% observation; p<0.002). Extracranial disease progression rates at 24 months were identical (65% WBRT and 63% observation, p=0.73).

All four randomized trials showed decreased intracranial recurrence rates when adjuvant WBRT was administered, both at the site of treatment and at distant sites within the brain. Similar effects from adjuvant WBRT on distant CNS recurrence were reported by the trial of Muacevic and co-workers, in which patients were randomized to surgery with adjuvant WBRT versus SRS alone, discussed above [44]. The impact on the reduction in distant CNS recurrence with the use of adjuvant WBRT is likely from the eradication of subclinical microscopic disease present at the time of brain metastasis diagnosis. The effect of WBRT on CNS seeding from uncontrolled extracranial disease is unclear, but likely has a lesser effect. If seeding from extracranial disease was a dominant mechanism leading to CNS failure, adjuvant WBRT would not be predicted to decrease its occurrence.

No trial to date evaluating the omission of adjuvant WBRT after local therapy has demonstrated a survival benefit to WBRT. The study by Chang and co-workers indicated that the use of adjuvant WBRT after local therapy might be associated with a decrement in survival. It is difficult to draw firm conclusions about these data, as the study was stopped early, was not powered to detect a survival benefit, and contradicted the surviv‐ al results of the other four larger randomized studies presented above. This includes the study by Aoyama and co-workers [50], which evaluated overall survival as its primary endpoint and was unable to detect a survival difference between its treatment arms, without a marked increase in sample size to over 800. The study by Kocher and coworkers demonstrated an improvement in PFS associated with adjuvant WBRT [51]. This study excluded patients with uncontrolled or progressive primary disease, mitigating extracranial disease burden as a competing risk for death

The studies presented here represent the best assessment of the efficacy of adjuvant WBRT therapy in treatment of solid tumor brain metastases. This therapy is clearly able to decrease intracranial recurrence rates, both at locally treated and distant sites within the CNS. The effect of this therapy on survival and the relative benefits versus the cognitive effects of the therapy are less clear. Melanoma patients formed a small fraction of the patients enrolled in these trials and one might therefore question whether these results even apply in the melanoma setting. To do so requires examination of the rather imperfect retrospective dataset regarding adjuvant WBRT specifically in melanoma.

#### *2.4.2. Adjuvant WBRT in melanoma patients*

improved survival in the group treated with SRS alone (5.7 months SRS+WBRT vs. 15.2 months

Patients who were treated only with SRS required salvage therapy for intracranial progression in 87% of cases. Ten (33%) of the patients treated with SRS alone required craniotomy, ten (33%) received salvage WBRT and six (20%) received salvage SRS. In the group treated with SRS and adjuvant WBRT, two patients (7%) received salvage WBRT, and three (11%) pro‐

This study provides convincing evidence that the addition of adjuvant WBRT to SRS therapy for brain metastases impairs HVLT performance. This occurs despite a decreased rate of intracranial progression in those receiving WBRT. Salvage therapy for intracranial progression was required in the majority of patients treated with SRS alone, including salvage craniotomy in one-third of the patients. The clinical significance of HVLT deterioration due to adjuvant WBRT, vis a vis that of frequently needed salvage therapy for CNS disease was not addressed.

A fourth randomized trial assessing adjuvant WBRT enrolled patients with 1-3 brain meta‐ stases and stable or absent extracranial disease [51]. The majority of patients had NSCLC (53%); only 5% were melanoma patients. Patients received SRS or surgery as primary therapy and were then randomized to receive adjuvant WBRT (30 Gy in 10 fractions) or no additional therapy. The composite primary endpoint was median overall survival in patients with KPS of 0-2. In the intent-to-treat analysis, 180 patients were assigned to receive WBRT and 179 to observation. At the end of the study, per protocol, 164 patients received WBRT and 166 patients

No differences were detected in the primary endpoint of survival with functional independ‐ ence (9.5 months WBRT versus 10.0 months observation, p=0.709) or median overall survival (10.7 months WBRT versus 10.9 months observation, p=0.891). Intracranial recurrence rates were markedly suppressed by adjuvant WBRT. Overall intracranial progression occurred in 48% of WBRT-treated patients and in 78% of the observation group (p<0.001). This translated to improved progression-free survival (PFS) in the WBRT-treated group (4.6 months vs. 3.4 months observation, p=0.002). Two years after surgery, WBRT reduced the probability of relapse at intial site from 59% (observation) to 27% (p<0.001) and at distant CNS sites from 42% (observation) to 23% (p=0.008). Similarly, after SRS, WBRT reduced the probability of relapse at SRS-treated site from 31% (observation) to 19% (p=0.040) and at distant CNS sites from 48% (observation) to 33% (p=0.023). Neurological cause of death was suppressed by adjuvant WBRT (28% WBRT versus 44% observation; p<0.002). Extracranial disease progression rates

All four randomized trials showed decreased intracranial recurrence rates when adjuvant WBRT was administered, both at the site of treatment and at distant sites within the brain. Similar effects from adjuvant WBRT on distant CNS recurrence were reported by the trial of Muacevic and co-workers, in which patients were randomized to surgery with adjuvant WBRT versus SRS alone, discussed above [44]. The impact on the reduction in distant CNS recurrence with the use of adjuvant WBRT is likely from the eradication of subclinical microscopic disease present at the time of brain metastasis diagnosis. The effect of WBRT on CNS seeding from

gressed intracranially, but received no salvage therapy.

were on observation. Analysis was by intention-to-treat.

at 24 months were identical (65% WBRT and 63% observation, p=0.73).

SRS, p=0.003).

290 Melanoma - From Early Detection to Treatment

The randomized studies discussed above primarily enrolled patients diagnosed with NSCLC. There have been no prospective studies evaluating the role of adjuvant WBRT specifically in the melanoma patient population. Many retrospective studies have been reported; unsurpris‐ ingly, these have indicated that adjuvant WBRT confers no survival benefit (see Tables 2, 4) [8-10, 12, 19, 29, 31, 32, 34, 35, 38, 39, 41, 53]. Since most melanoma patients with brain lesions present with active extracranial disease, any potential survival benefit due to adjuvant WBRT after local CNS therapy is probably undermined: extracranial disease serves as a competing cause of death, diluting any study's statistical power.

It is difficult to make firm conclusions based on the numerous melanoma case series on whether adjuvant WBRT actually decreases the rate of intracranial recurrence after local therapy. Selection and ascertainment biases are major concerns. Patients with clinically advanced disease are often selected for more aggressive therapy. Groups receiving aggressive therapy are likely to undergo more frequent and detailed surveillance for recurrence.

Several retrospective studies identify such biases. In the study of Buchsbaum and co-workers a paradoxically *higher* rate of CNS recurrence (49%) was identified in patients having received combined local CNS lesion therapy and adjuvant WBRT versus local therapy alone (20%) [10]. Follow-up scans were more frequent in the combined therapy group, possibly explaining the increased detection of progression and therefore higher documented recurrence rates. Samlowski and co-workers indicated that patients having received combined SRS and adjuvant WBRT had a higher mean number of CNS lesions at presentation than those selected for SRS alone [37]. Not surprisingly, more aggressive upfront therapy is apparently adminis‐ tered to patients with a greater initial disease burden.

WBRT. Death due to neurological causes was more common in the group that did not receive

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Another study during approximately the same time period (1979-1991) examined adjuvant WBRT after surgery in patients with a solitary CNS metastasis from melanoma (Table 2) [54]. Patients had no active extracranial disease and underwent resection of a single metastasis. Of the 34 subjects, 22 received WBRT. Median survival was improved in the combination therapy group (18 months versus 6 months with surgery alone, p=0.002), but CNS relapse rates were similar (30% surgery+WBRT *vs.* 22% surgery only; p=0.65). This study evaluated a highly selected patient group. This study and that of Hagen also suffer from being older studies, with more limited CNS imaging capabilities [53]. Nevertheless, the results tend to echo those of Patchell's randomized trial, suggesting a decreased CNS recurrence rate in CNS melanoma

Another report reviewed a single institution's experience with SRS in the treatment of 41 patients with radioresistant tumors, including 23 with melanoma [19]. Adjuvant WBRT improved local control (100% control with SRS and WBRT versus 85% with SRS alone at 6 months) and distant brain failure rates (17% failure with SRS and WBRT versus 64% failure with SRS alone). As might be predicted, adjuvant WBRT did not affect overall survival.

In summary, retrospective case series in melanoma indicate that adjuvant WBRT does not convey an overall survival benefit. This is consistent with the results of the randomized trials of WBRT primarily conducted in non-melanoma brain metastases. It is therefore reasonable to conclude that the addition of adjuvant WBRT does not improve the overall survival of the

As regards the effect of adjuvant WBRT on the prevention of CNS recurrence in melanoma, this collection of retrospective studies provides conflicting data. Some have shown no effect, others have shown decreased intracranial recurrence rates with the addition of WBRT, and still others have indicated that WBRT is associated with increased recurrence rates. Biases in

An ongoing randomized phase 3 trial is currently accruing for the comparison of distant intracranial control with the addition of adjuvant WBRT to observation following surgery and/ or SRS in melanoma patients with 1-3 brain lesions (NCT01503827) [55]. Secondary endpoints will include the effects on OS, QOL, and NCF. This prospective, randomized, melanomaspecific trial will hopefully reconcile the contradictory observations reported in the retrospec‐ tive studies discussed above. With improving systemic therapy, including agents able to penetrate the CNS at clinically relevant concentrations, even this randomized trial may not be

An alternative strategy to managing CNS metastases involves the use of "salvage" SRS. After patients receive initial local therapy with SRS alone, WBRT is omitted to spare normal brain tissues from unnecessary radiation doses and avoid potential adverse neurocognitive effects. Patients undergo CNS imaging at planned intervals or if symptoms suggest progression. SRS

treatment selection and ascertainment are strong confounders in many of the studies.

able to answer its major questions about adjuvant WBRT in melanoma patient.

WBRT (24% WBRT versus 85% observation, p<0.01).

patients treated with adjuvant WBRT after local therapy [52].

majority of melanoma patients with brain metastases.

**Salvage SRS: An alternative to WBRT?**

is then used to treat new lesions.

Another study reported that patients receiving SRS with WBRT had 0% 1-year actuarial control within the CNS versus 60% for those treated with SRS alone (p=0.0005), strongly suggesting selection bias [38]. Those patients with initially more advanced disease were more likely to be treated with the combined modality technique. Advanced disease was found as a strong predictor for poorer outcomes. Therefore local control rates were likely confounded by the level of disease burden at presentation and not necessarily by the choice of treatment modality.

Other studies indicate similar paradoxical results in patients treated with adjuvant WBRT. Wronski and Arbit reported an increased risk of CNS recurrence (56%) in patients treated with surgery and WBRT versus 46% in those treated with surgery alone [9]. Another study reported a 20% failure rate at SRS-treated sites in patients receiving adjuvant WBRT versus 0% in those treated with SRS alone [33]. Perhaps indicative of a possible beneficial effect from adjuvant WBRT, failure at distant sites within the CNS was only 23% in the combination therapy group versus 44% in those treated with SRS alone. Those failing at the local site after combined modality treatment had larger initial volumes of disease compared with those treated with SRS alone. The additional fractionated dose contributed from WBRT at the site of failure may not have adequately addressed the increased tumor burden initially present. This was likely a significant confounder in local control outcomes.

Several studies concluded that WBRT does not significantly impact CNS recurrence rates. In one study of 333 melanoma patients, WBRT before or after SRS did not alter the intracranial recurrence rates [31]. The same study also showed that patient survival was significantly shorter with WBRT (4.5 months) compared to SRS alone (6.4 months, p=0.05). Again, selection bias for patients with more lesions or more aggressive disease could explain this result. Radbill *et al.*reported that adjuvant WBRT did not decrease the rate of failure at non-SRS-treated sites in the CNS (p=0.13) [35]. However, the number of patients treated with adjuvant WBRT (13%) was potentially too small to detect a benefit. Mingione *et al.*, studying 45 melanoma patients, of whom 16 received adjuvant WBRT, concluded that WBRT had no impact on outcomes [32]. Yu *et al.* also found that adjuvant WBRT did not decrease distant CNS recurrence; this conclusion was again limited by the small proportion of WBRT-treated patients (32/122 patients; 32%) [41].

Three studies have suggested a benefit from WBRT in preventing CNS recurrence in the melanoma population. One retrospective study of 35 melanoma patients undergoing resection of a single brain metastasis at a single institution from 1972 to 1987 documented a CNS recurrence rate of 37% in those treated with adjuvant WBRT, versus 69% in those not receiving this therapy (Table 2) [53]. Median time to CNS relapse was 26.6 months in the group receiving adjuvant WBRT, as compared to 5.7 months in those not receiving such therapy (p<0.05). Survival was predicted by the extracranial disease status, rather than receipt of adjuvant WBRT. Death due to neurological causes was more common in the group that did not receive WBRT (24% WBRT versus 85% observation, p<0.01).

Another study during approximately the same time period (1979-1991) examined adjuvant WBRT after surgery in patients with a solitary CNS metastasis from melanoma (Table 2) [54]. Patients had no active extracranial disease and underwent resection of a single metastasis. Of the 34 subjects, 22 received WBRT. Median survival was improved in the combination therapy group (18 months versus 6 months with surgery alone, p=0.002), but CNS relapse rates were similar (30% surgery+WBRT *vs.* 22% surgery only; p=0.65). This study evaluated a highly selected patient group. This study and that of Hagen also suffer from being older studies, with more limited CNS imaging capabilities [53]. Nevertheless, the results tend to echo those of Patchell's randomized trial, suggesting a decreased CNS recurrence rate in CNS melanoma patients treated with adjuvant WBRT after local therapy [52].

Another report reviewed a single institution's experience with SRS in the treatment of 41 patients with radioresistant tumors, including 23 with melanoma [19]. Adjuvant WBRT improved local control (100% control with SRS and WBRT versus 85% with SRS alone at 6 months) and distant brain failure rates (17% failure with SRS and WBRT versus 64% failure with SRS alone). As might be predicted, adjuvant WBRT did not affect overall survival.

In summary, retrospective case series in melanoma indicate that adjuvant WBRT does not convey an overall survival benefit. This is consistent with the results of the randomized trials of WBRT primarily conducted in non-melanoma brain metastases. It is therefore reasonable to conclude that the addition of adjuvant WBRT does not improve the overall survival of the majority of melanoma patients with brain metastases.

As regards the effect of adjuvant WBRT on the prevention of CNS recurrence in melanoma, this collection of retrospective studies provides conflicting data. Some have shown no effect, others have shown decreased intracranial recurrence rates with the addition of WBRT, and still others have indicated that WBRT is associated with increased recurrence rates. Biases in treatment selection and ascertainment are strong confounders in many of the studies.

An ongoing randomized phase 3 trial is currently accruing for the comparison of distant intracranial control with the addition of adjuvant WBRT to observation following surgery and/ or SRS in melanoma patients with 1-3 brain lesions (NCT01503827) [55]. Secondary endpoints will include the effects on OS, QOL, and NCF. This prospective, randomized, melanomaspecific trial will hopefully reconcile the contradictory observations reported in the retrospec‐ tive studies discussed above. With improving systemic therapy, including agents able to penetrate the CNS at clinically relevant concentrations, even this randomized trial may not be able to answer its major questions about adjuvant WBRT in melanoma patient.

#### **Salvage SRS: An alternative to WBRT?**

Follow-up scans were more frequent in the combined therapy group, possibly explaining the increased detection of progression and therefore higher documented recurrence rates. Samlowski and co-workers indicated that patients having received combined SRS and adjuvant WBRT had a higher mean number of CNS lesions at presentation than those selected for SRS alone [37]. Not surprisingly, more aggressive upfront therapy is apparently adminis‐

Another study reported that patients receiving SRS with WBRT had 0% 1-year actuarial control within the CNS versus 60% for those treated with SRS alone (p=0.0005), strongly suggesting selection bias [38]. Those patients with initially more advanced disease were more likely to be treated with the combined modality technique. Advanced disease was found as a strong predictor for poorer outcomes. Therefore local control rates were likely confounded by the level of disease burden at presentation and not necessarily by the choice of treatment modality. Other studies indicate similar paradoxical results in patients treated with adjuvant WBRT. Wronski and Arbit reported an increased risk of CNS recurrence (56%) in patients treated with surgery and WBRT versus 46% in those treated with surgery alone [9]. Another study reported a 20% failure rate at SRS-treated sites in patients receiving adjuvant WBRT versus 0% in those treated with SRS alone [33]. Perhaps indicative of a possible beneficial effect from adjuvant WBRT, failure at distant sites within the CNS was only 23% in the combination therapy group versus 44% in those treated with SRS alone. Those failing at the local site after combined modality treatment had larger initial volumes of disease compared with those treated with SRS alone. The additional fractionated dose contributed from WBRT at the site of failure may not have adequately addressed the increased tumor burden initially present. This was likely

Several studies concluded that WBRT does not significantly impact CNS recurrence rates. In one study of 333 melanoma patients, WBRT before or after SRS did not alter the intracranial recurrence rates [31]. The same study also showed that patient survival was significantly shorter with WBRT (4.5 months) compared to SRS alone (6.4 months, p=0.05). Again, selection bias for patients with more lesions or more aggressive disease could explain this result. Radbill *et al.*reported that adjuvant WBRT did not decrease the rate of failure at non-SRS-treated sites in the CNS (p=0.13) [35]. However, the number of patients treated with adjuvant WBRT (13%) was potentially too small to detect a benefit. Mingione *et al.*, studying 45 melanoma patients, of whom 16 received adjuvant WBRT, concluded that WBRT had no impact on outcomes [32]. Yu *et al.* also found that adjuvant WBRT did not decrease distant CNS recurrence; this conclusion was again limited by the small proportion of WBRT-treated patients (32/122

Three studies have suggested a benefit from WBRT in preventing CNS recurrence in the melanoma population. One retrospective study of 35 melanoma patients undergoing resection of a single brain metastasis at a single institution from 1972 to 1987 documented a CNS recurrence rate of 37% in those treated with adjuvant WBRT, versus 69% in those not receiving this therapy (Table 2) [53]. Median time to CNS relapse was 26.6 months in the group receiving adjuvant WBRT, as compared to 5.7 months in those not receiving such therapy (p<0.05). Survival was predicted by the extracranial disease status, rather than receipt of adjuvant

tered to patients with a greater initial disease burden.

292 Melanoma - From Early Detection to Treatment

a significant confounder in local control outcomes.

patients; 32%) [41].

An alternative strategy to managing CNS metastases involves the use of "salvage" SRS. After patients receive initial local therapy with SRS alone, WBRT is omitted to spare normal brain tissues from unnecessary radiation doses and avoid potential adverse neurocognitive effects. Patients undergo CNS imaging at planned intervals or if symptoms suggest progression. SRS is then used to treat new lesions.

This strategy has not yet been tested in a randomized trial for patients with brain metastases from melanoma. There are limited data that have included melanoma pa‐ tients in the prospective evaluation of this treatment paradigm. For example, one prospec‐ tive study assessed SRS as a single treatment modality in 41 patients with no more than 4 brain metastases [56]. Seven (16%) of the patients had melanoma primary tumors. Twenty-three of the enrolled patients (56%) experienced intracranial progression. Nine received salvage treatment with additional SRS and one with surgery and WBRT for persistent tumor. Eleven patients were treated with salvage WBRT due to an excessive number of new CNS lesions and two patients received non-radiotherapy palliative therapy. Intracranial recurrences were common in the absence of upfront WBRT; less than half of recurring patients (9/23) were eligible for salvage SRS therapy due to excessive number of new lesions, limited life expectancy or decreased performance status.

illness." Neither baseline neurocognitive data information for the identified cases nor infor‐ mation regarding the source populations was provided. Among the 12 cases identified, a variety of radiation dose and fractionation schemes were employed. The authors suggested that the incidence of radiation-related leukoencephalopathy might have been underestimated due to lack of sensitive tools for identifying neurocognitive dysfunction. Baseline neurocog‐ nitive dysfunction in patients with primary or secondary brain malignancy, however, is present in as many as 90% of patients prior to treatment [61] due to the general debility of patients with metastatic cancer, the neurocognitive effects of systemic chemotherapy and supportive therapies, and the age of the patients. Thus, the results of this relatively old study do not provide a clear picture of neurocognitive dysfunction associated with radiotherapeutic

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Fairly good evidence shows that radiation therapy of the brain leads to neurocognitive dysfunction, which in some cases can be severe. A variety of patient-related factors play a role in the development of risk for developing radiation-associated neurocognitive dysfunction. These include patient age (children or those more than 50 years of age), other therapies received (chemotherapy and/or anti-convulsants), and length of survival post radiation therapy (as seen in survivors diagnosed with more favorable and indolent diseases, e.g., low-grade glioma) [62-68]. Factors related to radiation therapy delivery include total dose received, dose per

More rigorous prospective assessments suggest that the neurocognitive impact of WBRT may be modest. Data from the study of primary brain tumor patients, in which extracranial disease and its treatment are not factors, may be relevant. For example, one study examined the dosedependency of radiotherapy-associated neurocognitive dysfunction in patients treated for primary brain tumors [71]. Neuropsychological testing was undertaken up to 12 months after completion of radiotherapy. No dysfunction was observed in patients receiving up to 30 Gy,

Another setting to examine the effects of WBRT is in diseases for which PCI is of proven benefit, such as SCLC. In two large studies evaluating the role of PCI for good responders with SCLC, there was no difference in NCF between those randomized to receive WBRT or not (24 Gy in 12 fractions-36 Gy in 18 fractions) [47, 48]. In the study by Gregor *et al.,* both groups of patients demonstrated baseline neurocognitive impairment versus normal controls, likely reflecting effects of prior treatment. Among those without baseline impairment, impairment in cognitive test performance was evident at 6 months and 1 year, but no obvious differences were seen when comparing PCI-treated and –untreated patients. The authors did not, however, describe

Another prospective, non-randomized study showed no difference in cognitive function after 30-40 Gy of radiation therapy with 2-34 months of follow-up [72]. Again, a high degree of preexisting neurocognitive deficit was already present. This may have been attributable to

A non-randomized, prospective study of PCI was undertaken in NSCLC patients [73]. Seventyfive patients received induction radiochemotherapy for locally advanced NSCLC. Forty-seven

rigorous statistical assessment of the longitudinal neurocognitive testing data [48].

treatment of brain metastases.

fraction, and amount of cerebral volume irradiated [68-71].

chemotherapy given prior to radiation therapy.

a typical dose used for adjuvant WBRT. Fraction size was not reported.

Data from a large, multi-institutional, retrospective study of 569 patients (16% with melanoma) support the feasibility of salvage SRS in replacement of upfront WBRT [57]. Of 268 patients treated initially with SRS alone, 98 received salvage therapy for CNS recur‐ rence. Sixty-three (64%) of those needing salvage therapy received WBRT as part of the salvage regimen (which included SRS and/or surgery) and forty-seven (48%) received WBRT as the sole salvage therapy.

One retrospective study examined 45 patients (20 with melanoma, 44%) receiving SRS as salvage therapy [58]. Excellent local control at treated sites was achieved (92.4% at 52 weeks). Patients who received upfront WBRT were significantly less likely to require salvage therapy (p=0.008), although no survival benefit was reported.

A CNS metastasis management strategy in which SRS is used as sole initial therapy warrants continued evaluation, particularly for patients diagnosed with melanoma. The existing studies of this approach suggest that intracranial recurrence rates remain high with the omission of WBRT. Although salvage therapy with SRS may be planned initially, a large fraction of patients will require WBRT in the salvage setting to treat macroscopic recurrences, when WBRT is likely to be *least* effective

#### *2.4.3. Neurocognitive effects of WBRT*

A major argument against the use of adjuvant WBRT relates to its impact on NCF and higher executive neurologic functions, including learning, memory, calculation, and task planning. A variety of standardized neuropsychological tests measure global NCF, such as the MMSE. NCF impairment has a direct impact on overall QOL, affecting patients' ability to carry out activities of daily living, medical treatment compliance, and higher order planning and function [59].

One widely cited retrospective study examined patients treated at a single center for brain metastasis by either WBRT alone (n=370) or surgical metastectomy combined with WBRT (n=118) [60]. Radiation-associated dementia was reported at a rate of 1.9 (n=7) and 5.1% (n=5), respectively. Cases were defined as those patients treated for brain metastases with WBRT without evident CNS recurrence who subsequently developed "…a progressive dementing illness." Neither baseline neurocognitive data information for the identified cases nor infor‐ mation regarding the source populations was provided. Among the 12 cases identified, a variety of radiation dose and fractionation schemes were employed. The authors suggested that the incidence of radiation-related leukoencephalopathy might have been underestimated due to lack of sensitive tools for identifying neurocognitive dysfunction. Baseline neurocog‐ nitive dysfunction in patients with primary or secondary brain malignancy, however, is present in as many as 90% of patients prior to treatment [61] due to the general debility of patients with metastatic cancer, the neurocognitive effects of systemic chemotherapy and supportive therapies, and the age of the patients. Thus, the results of this relatively old study do not provide a clear picture of neurocognitive dysfunction associated with radiotherapeutic treatment of brain metastases.

This strategy has not yet been tested in a randomized trial for patients with brain metastases from melanoma. There are limited data that have included melanoma pa‐ tients in the prospective evaluation of this treatment paradigm. For example, one prospec‐ tive study assessed SRS as a single treatment modality in 41 patients with no more than 4 brain metastases [56]. Seven (16%) of the patients had melanoma primary tumors. Twenty-three of the enrolled patients (56%) experienced intracranial progression. Nine received salvage treatment with additional SRS and one with surgery and WBRT for persistent tumor. Eleven patients were treated with salvage WBRT due to an excessive number of new CNS lesions and two patients received non-radiotherapy palliative therapy. Intracranial recurrences were common in the absence of upfront WBRT; less than half of recurring patients (9/23) were eligible for salvage SRS therapy due to excessive number of

Data from a large, multi-institutional, retrospective study of 569 patients (16% with melanoma) support the feasibility of salvage SRS in replacement of upfront WBRT [57]. Of 268 patients treated initially with SRS alone, 98 received salvage therapy for CNS recur‐ rence. Sixty-three (64%) of those needing salvage therapy received WBRT as part of the salvage regimen (which included SRS and/or surgery) and forty-seven (48%) received

One retrospective study examined 45 patients (20 with melanoma, 44%) receiving SRS as salvage therapy [58]. Excellent local control at treated sites was achieved (92.4% at 52 weeks). Patients who received upfront WBRT were significantly less likely to require salvage therapy

A CNS metastasis management strategy in which SRS is used as sole initial therapy warrants continued evaluation, particularly for patients diagnosed with melanoma. The existing studies of this approach suggest that intracranial recurrence rates remain high with the omission of WBRT. Although salvage therapy with SRS may be planned initially, a large fraction of patients will require WBRT in the salvage setting to treat macroscopic recurrences, when WBRT is likely

A major argument against the use of adjuvant WBRT relates to its impact on NCF and higher executive neurologic functions, including learning, memory, calculation, and task planning. A variety of standardized neuropsychological tests measure global NCF, such as the MMSE. NCF impairment has a direct impact on overall QOL, affecting patients' ability to carry out activities of daily living, medical treatment compliance, and higher order

One widely cited retrospective study examined patients treated at a single center for brain metastasis by either WBRT alone (n=370) or surgical metastectomy combined with WBRT (n=118) [60]. Radiation-associated dementia was reported at a rate of 1.9 (n=7) and 5.1% (n=5), respectively. Cases were defined as those patients treated for brain metastases with WBRT without evident CNS recurrence who subsequently developed "…a progressive dementing

new lesions, limited life expectancy or decreased performance status.

WBRT as the sole salvage therapy.

294 Melanoma - From Early Detection to Treatment

*2.4.3. Neurocognitive effects of WBRT*

planning and function [59].

to be *least* effective

(p=0.008), although no survival benefit was reported.

Fairly good evidence shows that radiation therapy of the brain leads to neurocognitive dysfunction, which in some cases can be severe. A variety of patient-related factors play a role in the development of risk for developing radiation-associated neurocognitive dysfunction. These include patient age (children or those more than 50 years of age), other therapies received (chemotherapy and/or anti-convulsants), and length of survival post radiation therapy (as seen in survivors diagnosed with more favorable and indolent diseases, e.g., low-grade glioma) [62-68]. Factors related to radiation therapy delivery include total dose received, dose per fraction, and amount of cerebral volume irradiated [68-71].

More rigorous prospective assessments suggest that the neurocognitive impact of WBRT may be modest. Data from the study of primary brain tumor patients, in which extracranial disease and its treatment are not factors, may be relevant. For example, one study examined the dosedependency of radiotherapy-associated neurocognitive dysfunction in patients treated for primary brain tumors [71]. Neuropsychological testing was undertaken up to 12 months after completion of radiotherapy. No dysfunction was observed in patients receiving up to 30 Gy, a typical dose used for adjuvant WBRT. Fraction size was not reported.

Another setting to examine the effects of WBRT is in diseases for which PCI is of proven benefit, such as SCLC. In two large studies evaluating the role of PCI for good responders with SCLC, there was no difference in NCF between those randomized to receive WBRT or not (24 Gy in 12 fractions-36 Gy in 18 fractions) [47, 48]. In the study by Gregor *et al.,* both groups of patients demonstrated baseline neurocognitive impairment versus normal controls, likely reflecting effects of prior treatment. Among those without baseline impairment, impairment in cognitive test performance was evident at 6 months and 1 year, but no obvious differences were seen when comparing PCI-treated and –untreated patients. The authors did not, however, describe rigorous statistical assessment of the longitudinal neurocognitive testing data [48].

Another prospective, non-randomized study showed no difference in cognitive function after 30-40 Gy of radiation therapy with 2-34 months of follow-up [72]. Again, a high degree of preexisting neurocognitive deficit was already present. This may have been attributable to chemotherapy given prior to radiation therapy.

A non-randomized, prospective study of PCI was undertaken in NSCLC patients [73]. Seventyfive patients received induction radiochemotherapy for locally advanced NSCLC. Forty-seven received PCI (30 Gy over 3 weeks), while twenty-eight others did not. PCI reduced the overall rate of brain relapse from 54% to 13% at 3-4 years. In fifteen long-term survivors (10 PCI, 5 without PCI), no significant differences were noted in a battery of neuropsychological tests undertaken at a median of 47 (PCI) and 70 (no PCI) months.

functions [79]. In studies of patients with brain metastases, the test is part of a battery of administered tests intended to develop a general overview of neurocognitive function [80, 81].

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In the Chang study, a battery of neurocognitive function tests was administered, along with HVLT. Differences in performance on these other tests were not different between the two groups. The authors cautioned that the wide confidence intervals in the results of non-HVLT tests did not exclude a difference between the two test groups, but they also did not demon‐

Studies of the effects of brain radiotherapy presented here vary in quality. They do not however give a clear picture suggesting severe adverse consequences of brain radiotherapy. Adverse effects are certainly identified in several studies, although their clinical significance is not certain and its cause is not clearly attributable to CNS radiotherapy. Intuitively, radiation therapy in and of itself is not beneficial for the nervous system. In the setting of brain metastasis treatment, however, the adverse effects of radiation therapy must be balanced against those

While little melanoma-specific data are available, the primary brain tumor literature reveals that there are significant negative cognitive effects from tumor progression. This literature is particularly useful, as cognitive deterioration in primary brain tumor patients is due entirely to incracranial disease and CNS treatment effects, as opposed to extracranial disease progres‐ sion. Deterioration in MMSE was a strong predictor of impending intracranial tumor progres‐ sion in a study of 1,244 glioma patients [82]. A change in MMSE score was seen even *prior* to radiographic progression. Decreased MMSE score also strongly correlated with performance

Another study in 445 brain metastasis patients (25 with melanoma) compared the drop in MMSE score before and after treatment with WBRT [83, 84]. The study was designed to assess the effect of the radiation fractionation schedules on survival, for which no effect was found. Tumor control was the primary factor in determining MMSE scores at 3 months. A 6.2 point drop (out of 30 possible) was seen in those with radiographic evidence of progression, compared to a 0.5 point drop in those with controlled tumors. In multivariate analysis, control

Another prospective brain metastasis study assessed a novel radiosensitizing agent combined with WBRT [85]. A detailed neurocognitive battery assessed NCF before and after therapy. Patients in the control arm, receiving WBRT alone, were subdivided into "good responders" (at least a 45% reduction in tumor size) and "poor responders" (less than a 45% reduction). Good responders had better NCF preservation rate, as well as a modest survival advantage

These studies indicate that CNS tumor progression has adverse effects on neurocognitive status and QOL (reflected by performance status deterioration). While not melanomaspecific, there is no reason to believe that CNS progression of melanoma tumors would be

strate a specific difference between the groups.

*2.4.4. Neurocognitive effects of brain tumor progression*

of brain metastases was the only factor affecting MMSE score.

(median survival 300 days versus 240 days; p=0.03).

of CNS tumor recurrence.

status deterioration.

A study recently presented short term follow-up of longitudinal NCF in patients having received PCI (small cell lung cancer; n=13), therapeutic cranial irradiation (TCI; brain meta‐ stases; n=16) or non-cranial irradiation (control: breast cancer; n=15) [74]. NCF was assessed prior to and during radiation treatment and 6-8 weeks after its completion. At 6-8 weeks after treatment, only verbal memory scores were lower in patients receiving cranial irradiation versus controls. Visual memory and attention were not affected. Pre-treatment verbal memory performance score was the major predictor of post-treatment outcome in univariate analysis, with a lesser contribution attributable to cranial irradiation. The data from this admittedly small study suggest that WBRT can have a negative impact on verbal memory, although other factors contributing to the baseline status seem dominant.

Aoyama and co-workers conducted a randomized trial of SRS with or without WBRT, discussed in detail above [75]. Baseline and follow-up MMSE scores were available for 110 and 92 of the 132 patients enrolled in the trial, respectively. Baseline MMSE scores were predicted by patient age, performance status, tumoral edema and total tumor volume, but not by the initial number of tumors.

Deterioration in MMSE occurred in equal proportions of each group (14/36 SRS + WBRT versus 12/46 SRS alone, p=0.21). Average time-to-deterioration was longer in the combined therapy group (13.6 months versus 6.8 months SRS alone, p=0.05). In the 14 members of the combined therapy group, the adjudged cause of deterioration was brain tumor progression in 3, toxic effects of radiotherapy in 5 and indeterminate in 6; in the group treated only with SRS, MMSE deterioration was due to brain tumor progression in 11 and indeterminate in 1 (combined vs. SRS, p<0.0001). The temporal trends in NCF between the two arms suggest that SRS-related cognitive decline may be associated with tumor recurrence, which may or may not be rever‐ sible with salvage therapy. Later dysfunction with WBRT is more variable in cause. Some may be attributable to CNS tumor recurrence, but other cases being attributable to late effects of radiation on normal brain tissue. Such treatment-associated damage would not be amenable to corrective therapy with further tumor-specific therapy.

The study of Chang and co-workers, discussed earlier, prospectively addressed NCF in the setting of adjuvant WBRT [50]. This study is notable in that the score on a specific neurocog‐ nitive test, HVLT, was the primary endpoint. Patients receiving adjuvant WBRT experienced greater rates of decline in their HVLT performance than those treated with SRS alone, despite decreased intracranial progression in the WBRT-treated patients.

The HVLT tests basic verbal learning capacity and is proposed as a screening test for mild dementia [76, 77]. The HVLT may have somewhat greater sensitivity for mild dementia than the MMSE, as well some logistical advantages [78]. In isolation, however, results from the HVLT must be judged cautiously, as it does not assess other more complex neurocognitive functions [79]. In studies of patients with brain metastases, the test is part of a battery of administered tests intended to develop a general overview of neurocognitive function [80, 81].

In the Chang study, a battery of neurocognitive function tests was administered, along with HVLT. Differences in performance on these other tests were not different between the two groups. The authors cautioned that the wide confidence intervals in the results of non-HVLT tests did not exclude a difference between the two test groups, but they also did not demon‐ strate a specific difference between the groups.

Studies of the effects of brain radiotherapy presented here vary in quality. They do not however give a clear picture suggesting severe adverse consequences of brain radiotherapy. Adverse effects are certainly identified in several studies, although their clinical significance is not certain and its cause is not clearly attributable to CNS radiotherapy. Intuitively, radiation therapy in and of itself is not beneficial for the nervous system. In the setting of brain metastasis treatment, however, the adverse effects of radiation therapy must be balanced against those of CNS tumor recurrence.

#### *2.4.4. Neurocognitive effects of brain tumor progression*

received PCI (30 Gy over 3 weeks), while twenty-eight others did not. PCI reduced the overall rate of brain relapse from 54% to 13% at 3-4 years. In fifteen long-term survivors (10 PCI, 5 without PCI), no significant differences were noted in a battery of neuropsychological tests

A study recently presented short term follow-up of longitudinal NCF in patients having received PCI (small cell lung cancer; n=13), therapeutic cranial irradiation (TCI; brain meta‐ stases; n=16) or non-cranial irradiation (control: breast cancer; n=15) [74]. NCF was assessed prior to and during radiation treatment and 6-8 weeks after its completion. At 6-8 weeks after treatment, only verbal memory scores were lower in patients receiving cranial irradiation versus controls. Visual memory and attention were not affected. Pre-treatment verbal memory performance score was the major predictor of post-treatment outcome in univariate analysis, with a lesser contribution attributable to cranial irradiation. The data from this admittedly small study suggest that WBRT can have a negative impact on verbal memory, although other

Aoyama and co-workers conducted a randomized trial of SRS with or without WBRT, discussed in detail above [75]. Baseline and follow-up MMSE scores were available for 110 and 92 of the 132 patients enrolled in the trial, respectively. Baseline MMSE scores were predicted by patient age, performance status, tumoral edema and total tumor volume, but not by the

Deterioration in MMSE occurred in equal proportions of each group (14/36 SRS + WBRT versus 12/46 SRS alone, p=0.21). Average time-to-deterioration was longer in the combined therapy group (13.6 months versus 6.8 months SRS alone, p=0.05). In the 14 members of the combined therapy group, the adjudged cause of deterioration was brain tumor progression in 3, toxic effects of radiotherapy in 5 and indeterminate in 6; in the group treated only with SRS, MMSE deterioration was due to brain tumor progression in 11 and indeterminate in 1 (combined vs. SRS, p<0.0001). The temporal trends in NCF between the two arms suggest that SRS-related cognitive decline may be associated with tumor recurrence, which may or may not be rever‐ sible with salvage therapy. Later dysfunction with WBRT is more variable in cause. Some may be attributable to CNS tumor recurrence, but other cases being attributable to late effects of radiation on normal brain tissue. Such treatment-associated damage would not be amenable

The study of Chang and co-workers, discussed earlier, prospectively addressed NCF in the setting of adjuvant WBRT [50]. This study is notable in that the score on a specific neurocog‐ nitive test, HVLT, was the primary endpoint. Patients receiving adjuvant WBRT experienced greater rates of decline in their HVLT performance than those treated with SRS alone, despite

The HVLT tests basic verbal learning capacity and is proposed as a screening test for mild dementia [76, 77]. The HVLT may have somewhat greater sensitivity for mild dementia than the MMSE, as well some logistical advantages [78]. In isolation, however, results from the HVLT must be judged cautiously, as it does not assess other more complex neurocognitive

undertaken at a median of 47 (PCI) and 70 (no PCI) months.

factors contributing to the baseline status seem dominant.

to corrective therapy with further tumor-specific therapy.

decreased intracranial progression in the WBRT-treated patients.

initial number of tumors.

296 Melanoma - From Early Detection to Treatment

While little melanoma-specific data are available, the primary brain tumor literature reveals that there are significant negative cognitive effects from tumor progression. This literature is particularly useful, as cognitive deterioration in primary brain tumor patients is due entirely to incracranial disease and CNS treatment effects, as opposed to extracranial disease progres‐ sion. Deterioration in MMSE was a strong predictor of impending intracranial tumor progres‐ sion in a study of 1,244 glioma patients [82]. A change in MMSE score was seen even *prior* to radiographic progression. Decreased MMSE score also strongly correlated with performance status deterioration.

Another study in 445 brain metastasis patients (25 with melanoma) compared the drop in MMSE score before and after treatment with WBRT [83, 84]. The study was designed to assess the effect of the radiation fractionation schedules on survival, for which no effect was found. Tumor control was the primary factor in determining MMSE scores at 3 months. A 6.2 point drop (out of 30 possible) was seen in those with radiographic evidence of progression, compared to a 0.5 point drop in those with controlled tumors. In multivariate analysis, control of brain metastases was the only factor affecting MMSE score.

Another prospective brain metastasis study assessed a novel radiosensitizing agent combined with WBRT [85]. A detailed neurocognitive battery assessed NCF before and after therapy. Patients in the control arm, receiving WBRT alone, were subdivided into "good responders" (at least a 45% reduction in tumor size) and "poor responders" (less than a 45% reduction). Good responders had better NCF preservation rate, as well as a modest survival advantage (median survival 300 days versus 240 days; p=0.03).

These studies indicate that CNS tumor progression has adverse effects on neurocognitive status and QOL (reflected by performance status deterioration). While not melanomaspecific, there is no reason to believe that CNS progression of melanoma tumors would be any less adverse. These adverse effects of tumor progression must be balanced against those of adjuvant WBRT.

discontinuing corticosteroids due to symptom improvement served as a surrogate marker for palliative effects of WBRT. Upon completion of WBRT, 52% of all patients and 48% of symp‐ tomatic patients discontinued steroids. The same study demonstrated a small measurable response in tumor size following WBRT. Out of 87 patients, 65 had measurable disease at baseline; only 28 had at least one follow-up MRI scan to assess response. This may reflect a bias favoring follow-up scans being undertaken in those with responding disease. In these 28 patients at a median follow-up of 7 weeks, 75 tumors showed a median reduction in tumor size of 17%. The median OS of all patients evaluated in this study was 19 weeks. The median OS for patients who had undergone surgical resection prior to WBRT (22 patients) was 45 weeks, versus 16 weeks for those who did not undergo surgical resection (p<0.0001). Absence of extracranial disease (in 14 patients) was associated with higher median OS of 54 weeks,

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Two prospective studies have combined WBRT with either temozolomide or fotemustine in melanoma patients with brain metastases [88, 89]. With temozolomide in a phase 2 study of 31 patients, only 3 (10%) demonstrated a response in the CNS, with median PFS in the CNS and OS of 2 and 6 months, respectively. In the phase 3 study of the combination with fote‐ mustine, objective response rate (ORR) was 10% with median time-to-CNS-progression of 56 days and median OS of 105 days. These studies provide estimates of the clinical effect of WBRT, even though the relative contributions of WBRT and chemotherapy drug cannot be quanti‐

The use of WBRT in a patient with advanced CNS melanoma probably yields a modest survival benefit over supportive care alone. Symptom palliation is also probably a benefit of this therapy. There are many holes in the WBRT data set, many of which will never be answered definitively as melanoma treatment evolves. WBRT as a monotherapy has several signifcicant disadvantages, including its modest benefit at best, inability to undertake retreatment, and lack of effect on extracranial disease. These limitations will likely be overcome only with the design of systemic therapy regimens, to be administered concur‐

Until the recent approvals in 2011 of ipilimumab [90, 91] and vemurafenib [92], no therapy tested in a randomized trial demonstrated an improvement in overall survival for metastatic melanoma patients. Dacarbazine had been the standard first-line systemic treatment since it was approved in the United States in 1975. Metastatic melanoma patients with intracranial or meningeal metastases were generally excluded from clinical trial participation for three reasons: 1) brain metastases were thought to portend a poor prognosis; 2) systemic therapies that were tested were not very effective in intracranial disease; and 3) it was presumed that most agents would not cross the blood-brain barrier. In this section, we will cover efforts to use chemotherapy, molecularly-targeted therapy, and immunotherapy for the management

compared to 17 weeks in patients who had extracranial disease (p<0.0001).

tated.

rently with, or in lieu of, WBRT.

of melanoma brain metastases (Table 6) [88, 89, 93-99].

**2.5. Systemic therapy**

## *2.4.5. WBRT in advanced CNS melanoma*

In some patients, disease in the CNS cannot reasonably be controlled using local treatment of brain metastases with surgery or SRS. At some point, lesion number becomes excessive, or lesions are present in locations that are not amenable to local treatment. Alternatively, a patient's extracranial disease may be so extensive that it is likely to be life-limiting, and the goal of CNS disease treatment is primarily symptom palliation. WBRT is often used in this circumstance, with the twin goals of improving survival and providing symptoms palliation.

No randomized, prospective studies are available to quantitate the benefit of WBRT, especially when compared to supportive care alone. A number of large retrospective case series have examined the questions specifically of survival, although these suffer from heterogeneous patient populations. In the study of Sampson and co-workers, 205 melanoma patients with brain metastases received systemic palliative chemotherapy, with median OS of 39 days, versus 120 days among the 180 patients treated only with whole brain radiotherapy (p=0.0006) [8]. Receipt of radiotherapy treatment was statistically significant in the multivariate analysis of another large retrospective study, with radiotherapy demonstrating median OS of 3.6 months, versus 1.3 months for those treated with corticosteroids alone (HR=0.38; p<0.001). In the study of Raizer and co-workers, 83 patients received no specific therapy for brain meta‐ stases, vesus 100 receiving WBRT alone [13]. Median OS was 2.0 and 4.0 months, respectively. The statistical significance of this difference was not reported.

The study of Fife and co-workers examined patients treated at a single center in Australia in the 1952-2000 date range [12]. For the 1985-2000 cohort, 210 patients received supportive care, versus 236 receiving radiotherapy alone. Median OS was 2.1 and 3.4 months in these two groups; in multi-variate Cox regression analysis, radiotherapy was associated with a decreased hazard ratio for death (HR-0.851; p=0.111). This may not have achieved statistical significance due to the heterogeneity of the patients in these two groups. In addition to treatment modality, other significant factors associated with survival were the presence of concurrent metastases at diagnosis, older age, and a longer time from initial melanoma diagnosis.

An older retrospective study identified 60 melanoma patients with cerebral melanoma metastases that were enrolled in two Radiation Therapy Oncology Group (RTOG) studies [86]. The study sought to determine the effects of WBRT on performance status, neurologic function, and neurologic symptoms. In the analysis, this study demonstrated that WBRT provided improvement of neurologic symptoms (including headache, motor loss, convulsion) in 76% of patients. Median survival in this uncontrolled report was 10-14 weeks, although the baseline clinical characteristics of the study population were quite variable.

Another retrospective study identified 87 patients who had received WBRT, of whom 46 (53%) had 3 or more metastases [87]. The majority of patients were already receiving dexamethasone before initiating radiation, and therefore it was difficult to isolate the effects of WBRT, since CNS signs and symptoms can be alleviated by corticosteroid treatment. The fraction of patients discontinuing corticosteroids due to symptom improvement served as a surrogate marker for palliative effects of WBRT. Upon completion of WBRT, 52% of all patients and 48% of symp‐ tomatic patients discontinued steroids. The same study demonstrated a small measurable response in tumor size following WBRT. Out of 87 patients, 65 had measurable disease at baseline; only 28 had at least one follow-up MRI scan to assess response. This may reflect a bias favoring follow-up scans being undertaken in those with responding disease. In these 28 patients at a median follow-up of 7 weeks, 75 tumors showed a median reduction in tumor size of 17%. The median OS of all patients evaluated in this study was 19 weeks. The median OS for patients who had undergone surgical resection prior to WBRT (22 patients) was 45 weeks, versus 16 weeks for those who did not undergo surgical resection (p<0.0001). Absence of extracranial disease (in 14 patients) was associated with higher median OS of 54 weeks, compared to 17 weeks in patients who had extracranial disease (p<0.0001).

Two prospective studies have combined WBRT with either temozolomide or fotemustine in melanoma patients with brain metastases [88, 89]. With temozolomide in a phase 2 study of 31 patients, only 3 (10%) demonstrated a response in the CNS, with median PFS in the CNS and OS of 2 and 6 months, respectively. In the phase 3 study of the combination with fote‐ mustine, objective response rate (ORR) was 10% with median time-to-CNS-progression of 56 days and median OS of 105 days. These studies provide estimates of the clinical effect of WBRT, even though the relative contributions of WBRT and chemotherapy drug cannot be quanti‐ tated.

The use of WBRT in a patient with advanced CNS melanoma probably yields a modest survival benefit over supportive care alone. Symptom palliation is also probably a benefit of this therapy. There are many holes in the WBRT data set, many of which will never be answered definitively as melanoma treatment evolves. WBRT as a monotherapy has several signifcicant disadvantages, including its modest benefit at best, inability to undertake retreatment, and lack of effect on extracranial disease. These limitations will likely be overcome only with the design of systemic therapy regimens, to be administered concur‐ rently with, or in lieu of, WBRT.

#### **2.5. Systemic therapy**

any less adverse. These adverse effects of tumor progression must be balanced against

In some patients, disease in the CNS cannot reasonably be controlled using local treatment of brain metastases with surgery or SRS. At some point, lesion number becomes excessive, or lesions are present in locations that are not amenable to local treatment. Alternatively, a patient's extracranial disease may be so extensive that it is likely to be life-limiting, and the goal of CNS disease treatment is primarily symptom palliation. WBRT is often used in this circumstance, with the twin goals of improving survival and providing symptoms palliation.

No randomized, prospective studies are available to quantitate the benefit of WBRT, especially when compared to supportive care alone. A number of large retrospective case series have examined the questions specifically of survival, although these suffer from heterogeneous patient populations. In the study of Sampson and co-workers, 205 melanoma patients with brain metastases received systemic palliative chemotherapy, with median OS of 39 days, versus 120 days among the 180 patients treated only with whole brain radiotherapy (p=0.0006) [8]. Receipt of radiotherapy treatment was statistically significant in the multivariate analysis of another large retrospective study, with radiotherapy demonstrating median OS of 3.6 months, versus 1.3 months for those treated with corticosteroids alone (HR=0.38; p<0.001). In the study of Raizer and co-workers, 83 patients received no specific therapy for brain meta‐ stases, vesus 100 receiving WBRT alone [13]. Median OS was 2.0 and 4.0 months, respectively.

The study of Fife and co-workers examined patients treated at a single center in Australia in the 1952-2000 date range [12]. For the 1985-2000 cohort, 210 patients received supportive care, versus 236 receiving radiotherapy alone. Median OS was 2.1 and 3.4 months in these two groups; in multi-variate Cox regression analysis, radiotherapy was associated with a decreased hazard ratio for death (HR-0.851; p=0.111). This may not have achieved statistical significance due to the heterogeneity of the patients in these two groups. In addition to treatment modality, other significant factors associated with survival were the presence of concurrent metastases

An older retrospective study identified 60 melanoma patients with cerebral melanoma metastases that were enrolled in two Radiation Therapy Oncology Group (RTOG) studies [86]. The study sought to determine the effects of WBRT on performance status, neurologic function, and neurologic symptoms. In the analysis, this study demonstrated that WBRT provided improvement of neurologic symptoms (including headache, motor loss, convulsion) in 76% of patients. Median survival in this uncontrolled report was 10-14 weeks, although the baseline

Another retrospective study identified 87 patients who had received WBRT, of whom 46 (53%) had 3 or more metastases [87]. The majority of patients were already receiving dexamethasone before initiating radiation, and therefore it was difficult to isolate the effects of WBRT, since CNS signs and symptoms can be alleviated by corticosteroid treatment. The fraction of patients

The statistical significance of this difference was not reported.

at diagnosis, older age, and a longer time from initial melanoma diagnosis.

clinical characteristics of the study population were quite variable.

those of adjuvant WBRT.

298 Melanoma - From Early Detection to Treatment

*2.4.5. WBRT in advanced CNS melanoma*

Until the recent approvals in 2011 of ipilimumab [90, 91] and vemurafenib [92], no therapy tested in a randomized trial demonstrated an improvement in overall survival for metastatic melanoma patients. Dacarbazine had been the standard first-line systemic treatment since it was approved in the United States in 1975. Metastatic melanoma patients with intracranial or meningeal metastases were generally excluded from clinical trial participation for three reasons: 1) brain metastases were thought to portend a poor prognosis; 2) systemic therapies that were tested were not very effective in intracranial disease; and 3) it was presumed that most agents would not cross the blood-brain barrier. In this section, we will cover efforts to use chemotherapy, molecularly-targeted therapy, and immunotherapy for the management of melanoma brain metastases (Table 6) [88, 89, 93-99].

## *2.5.1. Chemotherapy*

Several chemotherapeutic regimens failed to demonstrate benefit in melanoma brain meta‐ stasis, including regimens containing platinum-based compounds, dacarbazine, etoposide, and others [87, 100-109]. This may be largely due to the low efficacy of many of the tested agents in melanoma generally. It is probably unreasonable to expect agents with limited activity against extracranial disease to have activity in the CNS, with the added barrier of CNS penetration. Three chemotherapy agents with defined CNS activity in non-melanoma neo‐ plastic settings, namely temozolomide, thalidomide, and fotemustine have been investigated in some detail in melanoma [110, 111].

hemorrhage (n=7), pulmonary embolism (n=2), deep vein thrombosis (n=1), and grade 3 rash (n=1). Of 15 evaluable patients, 3 (12% of the intent-to-treat population) achieved CR or PR, while 7 patients had minor response or SD in the brain. Of the 10 patients who derived benefit, however, 5 patients progressed at extracranial sites. Overall OS was 5 months in all 26 patients, while it was 6 months in the 15 evaluable patients. Given the limited efficacy and the toxicity associated with the temozolomide/thalidomide combination, its use in melanoma is not

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Temozolomide has also been evaluated in the adjuvant setting. A multicenter phase 3 study compared temozolomide to dacarbazine in the time to develop CNS metastasis [94]. The study randomized 150 patients to receive either oral temozolomide or intravenous dacarbazine in combination with cisplatin and interleukin-2. Compared to dacarbazine, temozolomide reduced the 1-year CNS failure from 31.1% to 20.6%, but was not statistically significant (p=0.22). The median OS was not different between the two arms. Even though temozolomide penetrates the CNS, it did not delay incidence of CNS failure. Thus it appears that temozolo‐

Fotemustine is a chloroethyl-nitrosurea approved in Europe for the treatment of metastatic melanoma. Fotemustine demonstrates high CNS penetration; its efficacy in melanoma patients with intracranial disease has been evaluated in three major studies. A French multicenter phase 2 study evaluated 153 metastatic melanoma patients for response to single-agent fotemustine [97]. Previously treated patients were allowed in the study. Since fotemustine crosses the blood

Out of the 153 evaluable patients, 36 (23.5%) had cerebral metastasis as the dominant disease site. In patients with cerebral metastases, the drug yielded an ORR of 25% in the CNS, similar to the 24.2% ORR observed in extracranial disease. The median OS of all patients was 85 weeks, but survival of the brain metastases patients was not reported. This study suggests that fotemustine has activity in melanoma, including CNS metastases. The magnitude of the benefit

To confirm the observed activity, a phase 3 trial randomized 229 patients with metastatic melanoma to receive either fotemustine or dacarbazine [113]. Dacarbazine is a useful and interesting comparator in this study, as prior studies had failed to demonstrate any significant activity in the CNS [100, 102, 104]. This study enrolled patients with and without pre-existing brain metastases. Forty-three patients with brain metastases enrolled, of whom 22 received

Among all patients, fotemustine yielded an ORR of 15% versus dacarbazine's 7% (p=0.043). The authors reported a trend to improved survival among fotemustine-treated patients, with median OS of 7.3 months versus 5.6 months in the dacarbazine arm (p=0.067). In the brain metastases sub-group, fotemustine yielded a 6% ORR, while dacarbazine produced no responses. While myelosuppression was the most common adverse event observed in both arms, fotemustine-induced myelosuppression was more frequent and severe. In the fotemus‐ tine arm, 71% (vs. 14% with dacarbazine) of patients experienced neutropenia, and 51% (vs. 5% with dacarbazine) of patients experienced grade 3- 4 neutropenia. Similarly, thrombocy‐

warranted, outside the setting of a clinical trial.

mide may not be very effective in the adjuvant setting.

is similar in the CNS and at extracranial sites.

fotemustine, while 21 patients received dacarbazine.

brain barrier, patients with intracranial metastases were enrolled.

Temozolomide is metabolized to the same active metabolite as dacarbazine. It is orally bioavailable and penetrates the blood-brain barrier at clinically significant concentrations [111]. The drug is approved for the treatment of primary brain tumors, confirming its clinically significant penetration of the CNS. Since temozolomide is as effective as dacarbazine in treatment of metastatic melanoma and yields similar patient survival [112], several clinical trials evaluated its efficacy in melanoma patients with brain metastases.

A multicenter, open label, single-arm phase 2 study aimed to determine the efficacy and safety (both as primary endpoints) of temozolomide in metastatic melanoma patients who had developed brain metastasis [93]. The study enrolled 151 patients, comprising of 117 chemo‐ therapy-naïve and 34 previously treated. The clinical condition of the enrollees did not require immediate surgery or radiation therapy, justifying chemotherapy as the sole therapy.

For chemotherapy-naïve patients, eight patients (7%) achieved response, including one complete (CR) and seven partial responses (PR); 34 patients (29%) achieved stable disease (SD) in brain lesions for at least 4 weeks. Median OS was 3.5 months. In previously treated patients, 1 patient (3%) achieved PR, 6 (18%) had SD, and the median OS was 2.2 months. Notably, 25% of the chemotherapy-naïve and 21% of previously treated patients had extensive intracranial disease, defined as more than 4 radiologically evident brain lesions. The authors concluded that further evaluation was warranted, particularly in combination with other treatment modalities, but activity as a single agent in this setting was limited.

The combination of temozolomide and WBRT has been evaluated. A prospective phase 2 trial evaluated the combination in patients with CNS melanoma [88]. In 31 evaluable patients, temozolomide and WBRT combination yielded an overall ORR of 9.7%, comprising of one CR in the CNS lasting 4.5 months and two PR in the CNS lasting 2 months and 7 months. Although the combination of temozolomide and WBRT could be safely administered, its efficacy was limited.

Thalidomide, an anti-angiogenic agent crossing the blood-brain barrier, has been tested in combination with temozolomide to treat melanoma patients with brain metastases. In a phase 2 study, the combination of temozolomide and thalidomide was tested in chemotherapy-naïve patients [96]. The primary endpoint was ORR in the brain assessed every 8 weeks. Of the 26 patients treated, 16 patients were symptomatic and 25 had extracranial metastases. Treatmentassociated toxicity, especially hemorrhage and thromboembolism was a problem: eleven patients discontinued treatment before completing one cycle of treatment due to intracranial hemorrhage (n=7), pulmonary embolism (n=2), deep vein thrombosis (n=1), and grade 3 rash (n=1). Of 15 evaluable patients, 3 (12% of the intent-to-treat population) achieved CR or PR, while 7 patients had minor response or SD in the brain. Of the 10 patients who derived benefit, however, 5 patients progressed at extracranial sites. Overall OS was 5 months in all 26 patients, while it was 6 months in the 15 evaluable patients. Given the limited efficacy and the toxicity associated with the temozolomide/thalidomide combination, its use in melanoma is not warranted, outside the setting of a clinical trial.

*2.5.1. Chemotherapy*

300 Melanoma - From Early Detection to Treatment

limited.

in some detail in melanoma [110, 111].

Several chemotherapeutic regimens failed to demonstrate benefit in melanoma brain meta‐ stasis, including regimens containing platinum-based compounds, dacarbazine, etoposide, and others [87, 100-109]. This may be largely due to the low efficacy of many of the tested agents in melanoma generally. It is probably unreasonable to expect agents with limited activity against extracranial disease to have activity in the CNS, with the added barrier of CNS penetration. Three chemotherapy agents with defined CNS activity in non-melanoma neo‐ plastic settings, namely temozolomide, thalidomide, and fotemustine have been investigated

Temozolomide is metabolized to the same active metabolite as dacarbazine. It is orally bioavailable and penetrates the blood-brain barrier at clinically significant concentrations [111]. The drug is approved for the treatment of primary brain tumors, confirming its clinically significant penetration of the CNS. Since temozolomide is as effective as dacarbazine in treatment of metastatic melanoma and yields similar patient survival [112], several clinical

A multicenter, open label, single-arm phase 2 study aimed to determine the efficacy and safety (both as primary endpoints) of temozolomide in metastatic melanoma patients who had developed brain metastasis [93]. The study enrolled 151 patients, comprising of 117 chemo‐ therapy-naïve and 34 previously treated. The clinical condition of the enrollees did not require

For chemotherapy-naïve patients, eight patients (7%) achieved response, including one complete (CR) and seven partial responses (PR); 34 patients (29%) achieved stable disease (SD) in brain lesions for at least 4 weeks. Median OS was 3.5 months. In previously treated patients, 1 patient (3%) achieved PR, 6 (18%) had SD, and the median OS was 2.2 months. Notably, 25% of the chemotherapy-naïve and 21% of previously treated patients had extensive intracranial disease, defined as more than 4 radiologically evident brain lesions. The authors concluded that further evaluation was warranted, particularly in combination with other treatment

The combination of temozolomide and WBRT has been evaluated. A prospective phase 2 trial evaluated the combination in patients with CNS melanoma [88]. In 31 evaluable patients, temozolomide and WBRT combination yielded an overall ORR of 9.7%, comprising of one CR in the CNS lasting 4.5 months and two PR in the CNS lasting 2 months and 7 months. Although the combination of temozolomide and WBRT could be safely administered, its efficacy was

Thalidomide, an anti-angiogenic agent crossing the blood-brain barrier, has been tested in combination with temozolomide to treat melanoma patients with brain metastases. In a phase 2 study, the combination of temozolomide and thalidomide was tested in chemotherapy-naïve patients [96]. The primary endpoint was ORR in the brain assessed every 8 weeks. Of the 26 patients treated, 16 patients were symptomatic and 25 had extracranial metastases. Treatmentassociated toxicity, especially hemorrhage and thromboembolism was a problem: eleven patients discontinued treatment before completing one cycle of treatment due to intracranial

immediate surgery or radiation therapy, justifying chemotherapy as the sole therapy.

trials evaluated its efficacy in melanoma patients with brain metastases.

modalities, but activity as a single agent in this setting was limited.

Temozolomide has also been evaluated in the adjuvant setting. A multicenter phase 3 study compared temozolomide to dacarbazine in the time to develop CNS metastasis [94]. The study randomized 150 patients to receive either oral temozolomide or intravenous dacarbazine in combination with cisplatin and interleukin-2. Compared to dacarbazine, temozolomide reduced the 1-year CNS failure from 31.1% to 20.6%, but was not statistically significant (p=0.22). The median OS was not different between the two arms. Even though temozolomide penetrates the CNS, it did not delay incidence of CNS failure. Thus it appears that temozolo‐ mide may not be very effective in the adjuvant setting.

Fotemustine is a chloroethyl-nitrosurea approved in Europe for the treatment of metastatic melanoma. Fotemustine demonstrates high CNS penetration; its efficacy in melanoma patients with intracranial disease has been evaluated in three major studies. A French multicenter phase 2 study evaluated 153 metastatic melanoma patients for response to single-agent fotemustine [97]. Previously treated patients were allowed in the study. Since fotemustine crosses the blood brain barrier, patients with intracranial metastases were enrolled.

Out of the 153 evaluable patients, 36 (23.5%) had cerebral metastasis as the dominant disease site. In patients with cerebral metastases, the drug yielded an ORR of 25% in the CNS, similar to the 24.2% ORR observed in extracranial disease. The median OS of all patients was 85 weeks, but survival of the brain metastases patients was not reported. This study suggests that fotemustine has activity in melanoma, including CNS metastases. The magnitude of the benefit is similar in the CNS and at extracranial sites.

To confirm the observed activity, a phase 3 trial randomized 229 patients with metastatic melanoma to receive either fotemustine or dacarbazine [113]. Dacarbazine is a useful and interesting comparator in this study, as prior studies had failed to demonstrate any significant activity in the CNS [100, 102, 104]. This study enrolled patients with and without pre-existing brain metastases. Forty-three patients with brain metastases enrolled, of whom 22 received fotemustine, while 21 patients received dacarbazine.

Among all patients, fotemustine yielded an ORR of 15% versus dacarbazine's 7% (p=0.043). The authors reported a trend to improved survival among fotemustine-treated patients, with median OS of 7.3 months versus 5.6 months in the dacarbazine arm (p=0.067). In the brain metastases sub-group, fotemustine yielded a 6% ORR, while dacarbazine produced no responses. While myelosuppression was the most common adverse event observed in both arms, fotemustine-induced myelosuppression was more frequent and severe. In the fotemus‐ tine arm, 71% (vs. 14% with dacarbazine) of patients experienced neutropenia, and 51% (vs. 5% with dacarbazine) of patients experienced grade 3- 4 neutropenia. Similarly, thrombocy‐ topenia was observed in 94% of patients receiving fotemustine (vs. 57% with dacarbazine) and grade 3- 4 occurred in 43% of patients (vs. 6% with dacarbazine).

disease. For example, NCT01378975 is an open-label single-arm phase 2 study enrolling metastatic melanoma patients with BRAF V600 and measurable brain metastases (sympto‐ matic or asymptomatic). Patients are enrolled regardless of prior systemic treatment history for brain metastases (except for previous treatment with BRAF or MEK inhibitors). The high response rate of patients harboring V600E mutations in melanoma (~50% vs. ~5% for dacar‐ bazine) suggests that vemurafenib, and potentially other BRAF-targeted therapies, might be useful in post-surgical/SRS adjuvant therapy as an alternative to WBRT. This hypothesis

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303

Dabrafenib is another potent and selective BRAF V600E inhibitor that inhibits growth of B-Raf mutant melanoma and mutant B-Raf colorectal xenografts in mice [116]. In a phase 1 study, 184 patients with metatastic melanoma, untreated brain metastases, or other solid tumors received dabrafenib [117]. Only three patients with wildtype B-Raf were evaluated, with no evidence of benefit; such patients were subsequently excluded. The study included 156 metastatic melanoma patients, of whom 10 had pre-existing brain metastases. For patients with intracranial disease due to melanoma, 9 out of the 10 patients had reductions in the size of their brain lesions as well as their extracranial disease, with 4 of them achieving complete resolution

A phase 2 study specifically assessing the response to dabrafenib in melanoma patients with intracranial disease harboring a V600E or V600K mutation was recently published [98]. The study enrolled 172 patients, of whom 89 patients had not received previous local treatment for brain metastases (cohort A) and 83 patients who progressed following previous local treatment (cohort B). In cohort A, the overall intracranial response rate (OIRR), which is the primary endpoint of this study, was 39.2% (29/74) in patients with the V600E mutation and 6.7% (1/15) in patients with the V600K mutation. In cohort B, the OIRR was 30.8% (20/65) in patients with the V600E mutation and 22.2% (4/18) in patients harboring the V600K mutation. These data suggest clinical activity in melanoma brain metastases patients harboring the V600E mutation and some activity in V600K patients, whether or not they received prior therapy for their brain

Given the limited activity of agents available up to this time, such as temozolomide, findings of CNS activity may not require formal confirmation in a phase 3 randomized trial. It is difficult to imagine what the comparator agent of such a trial would be. However, a study of the combination of either WBRT or SRS with concurrent B-Raf inhibitors (vemurafenib or dabra‐ fenib) or with B-Raf inhibitors following radiotherapy would be important in the development

Following the success, and subsequent FDA approval, of ipilimumab in the management of metastatic melanoma [90, 91], several anecdotal case reports highlighted the activity of ipilimumab in melanoma patients with brain metastases [118, 119]. For example, a retrospec‐ tive analysis assessing the activity of ipilimumab in melanoma patients with brain metastasis who were enrolled in a phase 2 trial [120] identified 12 patients, of whom 2 achieved PR and

of optimal therapy for patients with CNS metastases of melanoma.

*2.5.3. Immunotherapy of melanoma in the central nervous system*

should be tested, especially if CNS activity is confirmed.

of the CNS lesions.

metastases.

The responses of patients who had brain metastases in this study were not as impressive as previously reported in the phase 2 study discussed above, although this might be expected in a more rigorous phase 3 study setting. Although not quite statistically significant, fotemustine delayed the median time-to-develop first brain metastasis among those without pre-existing brain lesions to 22.7 months, versus 7.2 months for patients treated with dacarbazine (p=0.059), suggesting that fotemustine might have activity as an adjuvant treatment after surgical management of CNS metastases. This has not been tested, as of 2012.

A multicenter phase 3 trial randomized 76 patients to receive fotemustine alone or in combi‐ nation with WBRT in brain metastasis patients and sought to determine the cerebral response and time-to-cerebral-progression [89]. The primary endpoints of this study was to compare the CNS ORR (CR+PR), CNS control rate (CR+PR+SD), and the time to CNS progression. Compared to fotemustine alone, the combination did not significantly improve the ORR or the control rate. The addition of WBRT, however, delayed CNS progression; it was 49 days (range 11–539 days) in the fotemustine-only arm and 56 days (range 19–348 days) in patients treated with fotemustine and WBRT (Wilcoxon test, p=0.028). The combination did not, however, significantly improve the clinical CNS control rate (after 7 weeks) or OS. In regards to safety, myelosuppression was similar in both arms, but alopecia was much higher in the combination arm (40% compared to 2.6% in the fotemustine-only arm).

#### *2.5.2. Targeted agents*

Approximately 40 to 50% of all melanomas harbor a mutation in *BRAF* [114]. Notably, 95% of *BRAF* mutations are at the valine in the amino acid position 600, and over 90% of these are substitutions to aspartic acid (depicted as V600E). *In vitro*, the V600E mutation causes a 500 fold increase in the activity of B-Raf kinase; its expression is sufficient to cause tumor formation by normal melanocytes injected into nude mice [114].

Vemurafenib, a small molecule inhibitor of the V600E-mutant, was approved in the United States in 2011 for the treatment of metastatic melanoma in patients harboring the V600E mutation [92]. Clinical trials leading up to its approval excluded patients who had active intracranial disease. Thus, the efficacy of vemurafenib is not well studied in patients with preexisting intracranial disease.

A single-arm, open-label, pilot study was conducted in metastatic melanoma patients with the V600E mutation and unresectable brain metastases, who failed previous treatments of temozolomide and/or WBRT. Four patients, with extensive disease (3 to 10+ brain metastases), were enrolled. At the time of the abstract presentation, the staging reports for two of the four patients were available. The first patient had a confirmed PR in both intracranial and extrac‐ ranial lesions, while the second patient had minor responses in intracranial and extracranial metastases. Although very limited data, vemurafenib exhibits preliminary evidence of activity in melanoma patients with brain metastases who failed prior therapy [115]. Additional studies are in progress to demonstrate efficacy of vemurafenib in melanoma patients with intracranial disease. For example, NCT01378975 is an open-label single-arm phase 2 study enrolling metastatic melanoma patients with BRAF V600 and measurable brain metastases (sympto‐ matic or asymptomatic). Patients are enrolled regardless of prior systemic treatment history for brain metastases (except for previous treatment with BRAF or MEK inhibitors). The high response rate of patients harboring V600E mutations in melanoma (~50% vs. ~5% for dacar‐ bazine) suggests that vemurafenib, and potentially other BRAF-targeted therapies, might be useful in post-surgical/SRS adjuvant therapy as an alternative to WBRT. This hypothesis should be tested, especially if CNS activity is confirmed.

topenia was observed in 94% of patients receiving fotemustine (vs. 57% with dacarbazine) and

The responses of patients who had brain metastases in this study were not as impressive as previously reported in the phase 2 study discussed above, although this might be expected in a more rigorous phase 3 study setting. Although not quite statistically significant, fotemustine delayed the median time-to-develop first brain metastasis among those without pre-existing brain lesions to 22.7 months, versus 7.2 months for patients treated with dacarbazine (p=0.059), suggesting that fotemustine might have activity as an adjuvant treatment after surgical

A multicenter phase 3 trial randomized 76 patients to receive fotemustine alone or in combi‐ nation with WBRT in brain metastasis patients and sought to determine the cerebral response and time-to-cerebral-progression [89]. The primary endpoints of this study was to compare the CNS ORR (CR+PR), CNS control rate (CR+PR+SD), and the time to CNS progression. Compared to fotemustine alone, the combination did not significantly improve the ORR or the control rate. The addition of WBRT, however, delayed CNS progression; it was 49 days (range 11–539 days) in the fotemustine-only arm and 56 days (range 19–348 days) in patients treated with fotemustine and WBRT (Wilcoxon test, p=0.028). The combination did not, however, significantly improve the clinical CNS control rate (after 7 weeks) or OS. In regards to safety, myelosuppression was similar in both arms, but alopecia was much higher in the combination

Approximately 40 to 50% of all melanomas harbor a mutation in *BRAF* [114]. Notably, 95% of *BRAF* mutations are at the valine in the amino acid position 600, and over 90% of these are substitutions to aspartic acid (depicted as V600E). *In vitro*, the V600E mutation causes a 500 fold increase in the activity of B-Raf kinase; its expression is sufficient to cause tumor formation

Vemurafenib, a small molecule inhibitor of the V600E-mutant, was approved in the United States in 2011 for the treatment of metastatic melanoma in patients harboring the V600E mutation [92]. Clinical trials leading up to its approval excluded patients who had active intracranial disease. Thus, the efficacy of vemurafenib is not well studied in patients with pre-

A single-arm, open-label, pilot study was conducted in metastatic melanoma patients with the V600E mutation and unresectable brain metastases, who failed previous treatments of temozolomide and/or WBRT. Four patients, with extensive disease (3 to 10+ brain metastases), were enrolled. At the time of the abstract presentation, the staging reports for two of the four patients were available. The first patient had a confirmed PR in both intracranial and extrac‐ ranial lesions, while the second patient had minor responses in intracranial and extracranial metastases. Although very limited data, vemurafenib exhibits preliminary evidence of activity in melanoma patients with brain metastases who failed prior therapy [115]. Additional studies are in progress to demonstrate efficacy of vemurafenib in melanoma patients with intracranial

grade 3- 4 occurred in 43% of patients (vs. 6% with dacarbazine).

302 Melanoma - From Early Detection to Treatment

management of CNS metastases. This has not been tested, as of 2012.

arm (40% compared to 2.6% in the fotemustine-only arm).

by normal melanocytes injected into nude mice [114].

*2.5.2. Targeted agents*

existing intracranial disease.

Dabrafenib is another potent and selective BRAF V600E inhibitor that inhibits growth of B-Raf mutant melanoma and mutant B-Raf colorectal xenografts in mice [116]. In a phase 1 study, 184 patients with metatastic melanoma, untreated brain metastases, or other solid tumors received dabrafenib [117]. Only three patients with wildtype B-Raf were evaluated, with no evidence of benefit; such patients were subsequently excluded. The study included 156 metastatic melanoma patients, of whom 10 had pre-existing brain metastases. For patients with intracranial disease due to melanoma, 9 out of the 10 patients had reductions in the size of their brain lesions as well as their extracranial disease, with 4 of them achieving complete resolution of the CNS lesions.

A phase 2 study specifically assessing the response to dabrafenib in melanoma patients with intracranial disease harboring a V600E or V600K mutation was recently published [98]. The study enrolled 172 patients, of whom 89 patients had not received previous local treatment for brain metastases (cohort A) and 83 patients who progressed following previous local treatment (cohort B). In cohort A, the overall intracranial response rate (OIRR), which is the primary endpoint of this study, was 39.2% (29/74) in patients with the V600E mutation and 6.7% (1/15) in patients with the V600K mutation. In cohort B, the OIRR was 30.8% (20/65) in patients with the V600E mutation and 22.2% (4/18) in patients harboring the V600K mutation. These data suggest clinical activity in melanoma brain metastases patients harboring the V600E mutation and some activity in V600K patients, whether or not they received prior therapy for their brain metastases.

Given the limited activity of agents available up to this time, such as temozolomide, findings of CNS activity may not require formal confirmation in a phase 3 randomized trial. It is difficult to imagine what the comparator agent of such a trial would be. However, a study of the combination of either WBRT or SRS with concurrent B-Raf inhibitors (vemurafenib or dabra‐ fenib) or with B-Raf inhibitors following radiotherapy would be important in the development of optimal therapy for patients with CNS metastases of melanoma.

#### *2.5.3. Immunotherapy of melanoma in the central nervous system*

Following the success, and subsequent FDA approval, of ipilimumab in the management of metastatic melanoma [90, 91], several anecdotal case reports highlighted the activity of ipilimumab in melanoma patients with brain metastases [118, 119]. For example, a retrospec‐ tive analysis assessing the activity of ipilimumab in melanoma patients with brain metastasis who were enrolled in a phase 2 trial [120] identified 12 patients, of whom 2 achieved PR and 3 had SD in brain metastases. The median OS of all 12 patients was 14 months (range was 2.7 to 56.4 months).

study provides preliminary evidence that the combination of ipilimumab and fotemustine is active in patients with metastatic melanoma, including those with intracranial disease. To confirm the activity of the combination, a randomized phase 3 trial is planned and will compare the activity of the combination versus fotemustine alone in patients with advanced melanoma

> No prior CTx: ORR 7% SD 29% Prior CTx: PR 3% SD 18%

(1 CR and 2 PR)

3 CR or PR (12% by intent-totreat)

CNS failure: CTI - 24/57 pts CDI 34/61 pts *P* = 0.22 1y CNS failure rate CTI – 21% CDI – 31%

ORR in CNS 15 evaluable pts

**Response Median Survival Comments**

3.5m Prior CTx: 2.2m

PFS 2m OS 6m

OS 5m OS 6m (for evaluable pts)

PFS CTI – 4.1m CDI – 3.9m *P*=0.90 OS CTI – 8.4m CDI – 8.7m 1y OS CTI – 31% CDI – 42%

ORR ORR 25% CNS NR for CNS pts. The overall ORR in all

OS

86 days (arm A) 105 days (arm B). *P* = 0.561

15 pts completed ≥ 1 cycle. 11 discontinued before completing 1 cycle: 7 for intracranial hemorrhage, 2 for pulmonary embolism, 1 deep vein thrombosis, and 1 for Grade 3 rash

pts was 24%

Treatment Naïve:

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305

**Primary Endpoint**

ORR in brain and toxicity

Time to CNS mets

CNS ORR on day 50 CNS Control Rate (CR+PR +SD) on day 50 ORR 7.4% (arm A) 10.0% (arm B) *P* = 0.73 Control Rate 30% (arm A) 47% (arm B)

and brain metastases (NIBIT-M2; CA184-192).

TMZ 151 total

**Patients**

26 pts 16 symptomatic 25 extensive extracranial mets

150 pts 118 evaluable (57 in CTI and 61 in CDI)

Fotemustine 153 evaluable

pts; 36 (23.5%) had CNS disease

76 pts Arm A: 39 pts Arm B: 37 pts

117 treatment naive 35 pts prior CTx

TMZ and WBRT 31 pts ORR CNS ORR 10%

**Study Treatment Evaluable**

TMZ + Thalidomide

CTI (TMZ + Cisplatin + IL2)

vs. CDI (DTIC + Cisplatin + IL2 Phase 3

Arm A: Fotemustine vs. Arm B: Fotemustine + WBRT Phase 3

Agarwala *et al.*, 2004 [93]

Margolin *et al.,* 2002 [88]

Hwu *et al.,* 2005 [96]

Chiarion-Sileni *et al.,* 2011 [94]

Jacquillat *et al.,* 1990 [97]

Mornex *et al.,* 2003 [89]

Another retrospective study evaluated the outcome of 77 patients who underwent radiosurgery between 2002 and 2010 for melanoma brain metastases, of whom 27 (35%) received ipilimumab [121]. Ipilimumab–treated patients displayed a median OS of 21.3 months, versus 4.9 months for those not treated with ipilimumab. Even when adjusted for performance status, ipilimumab treatment was associated with a higher survival probabil‐ ity (HR 0.48, p=0.03). The median survival of ipilimumab-treated patients with poor prognosis (11/27 patients), who had Diagnosis-Specific Graded Prognostic Assessment (DS-GPA; discussed in more detail below) score of 0-2 was 15.7 months, while those with better prognosis (16/27 patients), DS-GPA score of 3-4 had a median survival of 25.2 months. The survival of patients who received ipilimumab was similar whether they received ipilimu‐ mab before or after developing brain metastases.

To determine the efficacy of ipilimumab prospectively, Margolin and colleagues de‐ signed a phase 2 study to assess the activity of ipilimumab in melanoma patients with brain metastasis [99]. The study segregated patients into two cohorts; cohort A included 51 patients who were neurologically asymptomatic, while cohort B included 21 patients with symptoms requiring corticosteroids, which continued during the course of the study, if necessary. The overall ORR in cohort A was 18% (9/51) and 5% (1/21) in cohort B. When assessing response in brain lesions alone, the ORR in cohort A was 24% (12/51) and 10% (2/21) in cohort B. The ORR of extracranial disease was similar in each group to the intracranial response: ORR was 27% (14/51) and 5% (1/21) in cohorts A and B, respective‐ ly. The median OS was 7 months for cohort A and 3.7 months in cohort B. Since the study did not specifically address the cause of deaths for patients enrolled in the study, it is not clear whether the variation in OS between the two cohorts was due to progression of intracranial or extracranial disease, or additional complications associated with symptomat‐ ic intracranial disease.

A number of observations can be made from the results of this study: a) the response of brain lesions was similar to responses in extracranial metastases; and b) patients with asymptomatic intracranial disease, not on corticosteroid treatment, tended to respond better. The authors of the study present two hypotheses that may explain the difference in response between the two cohorts: i) as suggested by survival data, patients with sympto‐ matic intracranial disease requiring corticosteroids have inherently poorer prognosis; or ii) corticosteroids may potentially interfere with the effector lymphocyte activation induced by ipilimumab. The authors did contend that corticosteroid use with ipilimumab did not entirely abrogate its efficacy.

A single-arm phase 2 study conducted in seven Italian centers assessed the combination of ipilimumab and fotemustine in patients with metastatic melanoma, including patients with asymptomatic brain metastases [95]. The open-label, single-arm phase 2 study enrolled 86 metastatic melanoma patients, of whom 20 had brain metastases at baseline. The overall study population disease control rate (defined as immune-related CR, PR, or SD) was 46.5% (40/86 patients). Similarly, ten of the brain metastasis patients (50%) achieved disease control. This study provides preliminary evidence that the combination of ipilimumab and fotemustine is active in patients with metastatic melanoma, including those with intracranial disease. To confirm the activity of the combination, a randomized phase 3 trial is planned and will compare the activity of the combination versus fotemustine alone in patients with advanced melanoma and brain metastases (NIBIT-M2; CA184-192).

3 had SD in brain metastases. The median OS of all 12 patients was 14 months (range was 2.7

Another retrospective study evaluated the outcome of 77 patients who underwent radiosurgery between 2002 and 2010 for melanoma brain metastases, of whom 27 (35%) received ipilimumab [121]. Ipilimumab–treated patients displayed a median OS of 21.3 months, versus 4.9 months for those not treated with ipilimumab. Even when adjusted for performance status, ipilimumab treatment was associated with a higher survival probabil‐ ity (HR 0.48, p=0.03). The median survival of ipilimumab-treated patients with poor prognosis (11/27 patients), who had Diagnosis-Specific Graded Prognostic Assessment (DS-GPA; discussed in more detail below) score of 0-2 was 15.7 months, while those with better prognosis (16/27 patients), DS-GPA score of 3-4 had a median survival of 25.2 months. The survival of patients who received ipilimumab was similar whether they received ipilimu‐

To determine the efficacy of ipilimumab prospectively, Margolin and colleagues de‐ signed a phase 2 study to assess the activity of ipilimumab in melanoma patients with brain metastasis [99]. The study segregated patients into two cohorts; cohort A included 51 patients who were neurologically asymptomatic, while cohort B included 21 patients with symptoms requiring corticosteroids, which continued during the course of the study, if necessary. The overall ORR in cohort A was 18% (9/51) and 5% (1/21) in cohort B. When assessing response in brain lesions alone, the ORR in cohort A was 24% (12/51) and 10% (2/21) in cohort B. The ORR of extracranial disease was similar in each group to the intracranial response: ORR was 27% (14/51) and 5% (1/21) in cohorts A and B, respective‐ ly. The median OS was 7 months for cohort A and 3.7 months in cohort B. Since the study did not specifically address the cause of deaths for patients enrolled in the study, it is not clear whether the variation in OS between the two cohorts was due to progression of intracranial or extracranial disease, or additional complications associated with symptomat‐

A number of observations can be made from the results of this study: a) the response of brain lesions was similar to responses in extracranial metastases; and b) patients with asymptomatic intracranial disease, not on corticosteroid treatment, tended to respond better. The authors of the study present two hypotheses that may explain the difference in response between the two cohorts: i) as suggested by survival data, patients with sympto‐ matic intracranial disease requiring corticosteroids have inherently poorer prognosis; or ii) corticosteroids may potentially interfere with the effector lymphocyte activation induced by ipilimumab. The authors did contend that corticosteroid use with ipilimumab did not

A single-arm phase 2 study conducted in seven Italian centers assessed the combination of ipilimumab and fotemustine in patients with metastatic melanoma, including patients with asymptomatic brain metastases [95]. The open-label, single-arm phase 2 study enrolled 86 metastatic melanoma patients, of whom 20 had brain metastases at baseline. The overall study population disease control rate (defined as immune-related CR, PR, or SD) was 46.5% (40/86 patients). Similarly, ten of the brain metastasis patients (50%) achieved disease control. This

to 56.4 months).

304 Melanoma - From Early Detection to Treatment

ic intracranial disease.

entirely abrogate its efficacy.

mab before or after developing brain metastases.



patients for enrollment in clinical trials. Its clinically available factors are useful to consider in

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RPA's initial description included 1200 patients, 200 of whom were affected by melano‐ ma. Histology and tissue of origin were significant prognostic factors, with melanoma being unfavorable. The validity of RPA has since been confirmed in the melanoma subgroup [10,

While originally intended for stratification of patients in radiation therapy trials, RPA class also stratifies risk in patients undergoing surgical metastectomy [125, 126]. In 2004, the RTOG study enrolled 333 patients between 1996 and 2001, of whom 167 were assigned to WBRT and SRS and 164 received WBRT alone [15]. Median survival was longer in patients with a single brain metastasis for patients receiving WBRT+SRS combination compared to patients who only received WBRT (6.5 months *vs.* 4.9 months, p=0.0393). This study shed light on a limitation of

The Diagnosis-Specific Graded Prognostic Assessment (DS-GPA) was developed by retro‐ spective analysis of 4,259 patients newly diagnosed with brain metastases [127]. In addition to the factors in RPA, it includes number of brain metastases and the underlying disease giving rise to brain metastases. In the melanoma subset, the analysis identified two significant prognostic factors: performance status (represented by KPS) and number of radiologically evident brain metastases. For KPS, a score of 90-100 is 2 points, 70-80 is 1 point, and less than 70 is 0 points. A single brain metastasis is 2 points, 2 to 3 metastasis is 1 point, and more than 3 metastases is 0 points. The DS-GPA score, calculated by adding the point values from a patient's KPS score and number of metastases, ranges from 0 (worst prognosis) to 4 (best prognosis). Median OS for melanoma patients ranges from 3.4 months (GS-GPA score of 0 to

Several other systems have been developed for use in specific sub-populations. The Basic Score for Brain Metastases (BS-BM) was developed by analyzing results from 110 SRS-treated patients [128]. The system generates a score based on KPS, control of primary tumor site, and extracranial disease status. Only 19 patients (17%) of the initial group of patients had mela‐ noma. The system has not yet been studied in melanoma patients specifically and focuses on SRS treatment. Its applicability to other treatment modalities remains to be established.

The Score Index for Radiosurgery (SIR) was developed from the study of 65 SRS-treated pa‐ tients with brain metastases from a variety of primary tumor types [129]. SIR derives a score from patient age, performance status, systemic disease status, maximum CNS lesion volume, and number of CNS lesions. In the population initially studied, SIR was more accurate in predicting survival than RPA. A retrospective study confirmed its utility in melanoma patients [38].

The Malignant Melanoma-Gamma Knife Radiosurgery score (MM-GKR) also assesses outcomes in metastatic melanoma patients treated with SRS [23]. Scoring is based on perform‐ ance status, age, and CNS lesion location. The authors claim greater prognostic accuracy than with either RPA or SIR, particularly in identifying patients with an especially poor prognosis.

The Prognostic Index (PI) score estimates prognosis in patients treated with palliative WBRT [43]. Factors used in this system include number of extracranial metastatic sites, RPA class,

RPA: it does not take into account the number of brain metastases present.

a discussion of brain metastasis patients.

1.0) to 13.2 months (GS-GPA score of 3.5 to 4.0).

19, 35, 42, 43].

CNS: Central Nervous System; CR: Complete response; CTx: Chemotherapy; DC: Disease control; DCR: Disease control rate; DTIC: Dacarbazine; NR: Not reported; OIRR: Overall intracranial response rate; ORR: Objective response rate; OS: Overall survival; PFS: Progression-free survival; PR: Partial response; SD: Stable disease; TMZ: Temozolomide; WBRT: Whole-brain radiation therapy

**Table 6.** Prospective trials of systemic therapy treatments for melanoma brain metastases

## **3. Risk stratification**

Several systems estimate risk of recurrence and death in patients with brain metastases, including some with melanoma-specific data (Table 7). Recursive Partitioning Analysis (RPA) is one such system [122-124]. This combines age, performance status, and extracranial disease status to assign a class from I to III that estimates survival. Its original intention was to stratify patients for enrollment in clinical trials. Its clinically available factors are useful to consider in a discussion of brain metastasis patients.

**Study Treatment Evaluable**

306 Melanoma - From Early Detection to Treatment

Dabrafenib 172 pts

Ipilimumab 72 pts

Long *et al.,* 2012 [98]

Margolin *et al.,* 2012 [99]

Di Giacoma *et al.,* 2012 [95]

**Patients**

Cohort A: no prior CNS therapy, 89 pts

Cohort B: Prior CNS therapy, 83 pts

51 Cohort A (no CNS symptoms) 21 Cohort B (CNS symptoms requiring corticosteroids

) Phase 2

86 pts total 20 CNS disease at baseline

Fotemustine + Ipilimumab

Whole-brain radiation therapy

**3. Risk stratification**

**Primary Endpoint**

Time to objective CNS progression

DCR (CR, PR, SD) at 12 wks

Immunerelated DCR (CR, PR, SD) at 24 weeks

**Table 6.** Prospective trials of systemic therapy treatments for melanoma brain metastases

*P* = 0.19 Time to CNS progression 49 days (arm A) 56 days (arm B) *P* = 0.028

V600E 39% V600K 6.7% Cohort B V600E 31% V600K 22%

DCR Cohort A 18% Cohort B 5% DCR in CNS Cohort A – 24% Cohort B – 10%

DCR Overall 46.5% CNS 50%

CNS: Central Nervous System; CR: Complete response; CTx: Chemotherapy; DC: Disease control; DCR: Disease control rate; DTIC: Dacarbazine; NR: Not reported; OIRR: Overall intracranial response rate; ORR: Objective response rate; OS: Overall survival; PFS: Progression-free survival; PR: Partial response; SD: Stable disease; TMZ: Temozolomide; WBRT:

Several systems estimate risk of recurrence and death in patients with brain metastases, including some with melanoma-specific data (Table 7). Recursive Partitioning Analysis (RPA) is one such system [122-124]. This combines age, performance status, and extracranial disease status to assign a class from I to III that estimates survival. Its original intention was to stratify

OIRR Cohort A

**Response Median Survival Comments**

At 6 months, Cohort A: 27% Cohort B: 41%

OS Cohort A 7m Cohort B 3.7m

CNS PFS 4.5m CNS OS 13.4m At 1-yr, 54% of CNS pts alive

Study limited to V600E and V600K BRAF mutated melanoma

Out of the 10 brain responses 5 PR or SD 5 CR

RPA's initial description included 1200 patients, 200 of whom were affected by melano‐ ma. Histology and tissue of origin were significant prognostic factors, with melanoma being unfavorable. The validity of RPA has since been confirmed in the melanoma subgroup [10, 19, 35, 42, 43].

While originally intended for stratification of patients in radiation therapy trials, RPA class also stratifies risk in patients undergoing surgical metastectomy [125, 126]. In 2004, the RTOG study enrolled 333 patients between 1996 and 2001, of whom 167 were assigned to WBRT and SRS and 164 received WBRT alone [15]. Median survival was longer in patients with a single brain metastasis for patients receiving WBRT+SRS combination compared to patients who only received WBRT (6.5 months *vs.* 4.9 months, p=0.0393). This study shed light on a limitation of RPA: it does not take into account the number of brain metastases present.

The Diagnosis-Specific Graded Prognostic Assessment (DS-GPA) was developed by retro‐ spective analysis of 4,259 patients newly diagnosed with brain metastases [127]. In addition to the factors in RPA, it includes number of brain metastases and the underlying disease giving rise to brain metastases. In the melanoma subset, the analysis identified two significant prognostic factors: performance status (represented by KPS) and number of radiologically evident brain metastases. For KPS, a score of 90-100 is 2 points, 70-80 is 1 point, and less than 70 is 0 points. A single brain metastasis is 2 points, 2 to 3 metastasis is 1 point, and more than 3 metastases is 0 points. The DS-GPA score, calculated by adding the point values from a patient's KPS score and number of metastases, ranges from 0 (worst prognosis) to 4 (best prognosis). Median OS for melanoma patients ranges from 3.4 months (GS-GPA score of 0 to 1.0) to 13.2 months (GS-GPA score of 3.5 to 4.0).

Several other systems have been developed for use in specific sub-populations. The Basic Score for Brain Metastases (BS-BM) was developed by analyzing results from 110 SRS-treated patients [128]. The system generates a score based on KPS, control of primary tumor site, and extracranial disease status. Only 19 patients (17%) of the initial group of patients had mela‐ noma. The system has not yet been studied in melanoma patients specifically and focuses on SRS treatment. Its applicability to other treatment modalities remains to be established.

The Score Index for Radiosurgery (SIR) was developed from the study of 65 SRS-treated pa‐ tients with brain metastases from a variety of primary tumor types [129]. SIR derives a score from patient age, performance status, systemic disease status, maximum CNS lesion volume, and number of CNS lesions. In the population initially studied, SIR was more accurate in predicting survival than RPA. A retrospective study confirmed its utility in melanoma patients [38].

The Malignant Melanoma-Gamma Knife Radiosurgery score (MM-GKR) also assesses outcomes in metastatic melanoma patients treated with SRS [23]. Scoring is based on perform‐ ance status, age, and CNS lesion location. The authors claim greater prognostic accuracy than with either RPA or SIR, particularly in identifying patients with an especially poor prognosis.

The Prognostic Index (PI) score estimates prognosis in patients treated with palliative WBRT [43]. Factors used in this system include number of extracranial metastatic sites, RPA class, 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.

**System Prognostic Factors Prognostic Classification Median OS**

≥3 2 2

Age ≤60 "/>60 - -

No - Yes -

No Yes

Factor 0 1 2 0.0-1.0

>3 2-3 1

ECM Yes No
