**Meet the editor**

Mandi M. Murph is an Associate Professor in the Department of Pharmaceutical and Biomedical Sciences in the College of Pharmacy at the University of Georgia. She holds an American Cancer Society Research Scholar award for research on melanoma. Her laboratory works on pre-clinical testing of novel therapeutic compounds for use against BRAF wild-type V600 melanoma and

researches molecular means to exploit melanoma signaling pathways. She has grant funding from the Georgia Research Alliance and the National Cancer Institute, which is part of the National Institutes of Health. Before becoming a professor at the University of Georgia, Murph received training at the University of Texas M.D. Anderson Cancer Center in Houston. She teaches cancer therapy courses to University of Georgia students.

## Contents

**Preface XI**


Chapter 6 **Changing Perceptions of Lymphadenectomy and Sentinel Lymph Node Biopsy in Melanoma 135** Antonio Sommariva, Camilla Cona and Carlo Riccardo Rossi

### Chapter 7 **Current Insights Into Canine Cutaneous Melanocytic Tumours Diagnosis 159**

Luis Resende, Joana Moreira, Justina Prada, Felisbina Luisa Queiroga and Isabel Pires

#### **Section 3 Therapeutic Approaches Against Disease 197**

	- **Section 4 Emerging Research on Melanoma 295**

## Preface

Chapter 7 **Current Insights Into Canine Cutaneous Melanocytic Tumours**

Luis Resende, Joana Moreira, Justina Prada, Felisbina Luisa

**Diagnosis 159**

**VI** Contents

**Response 225**

**Management 279**

Paola Savoia and Paolo Fava

**Section 4 Emerging Research on Melanoma 295**

Chapter 13 **RAGE and Its Ligands in Melanoma 325**

Chapter 14 **Gangliosides and Antigangliosides in Malignant**

Corina-Daniela Ene (Nicolae) and Ilinca Nicolae

Estelle Leclerc

**Melanoma 361**

Queiroga and Isabel Pires

**Section 3 Therapeutic Approaches Against Disease 197**

Jin Wang, Duane D. Miller and Wei Li

Monica Neagu and Carolina Constantin

Jennifer Makalowski and Hinrich Abken

Chapter 12 **Autotaxin – An Enzymatic Augmenter of Malignant Progression Linked to Inflammation 297**

Chapter 9 **New Insights in Cutaneous Melanoma Immune-Therapy —**

Chapter 10 **Can Redirected T Cells Outsmart Aggressive Melanoma? The Promise and Challenge of Adoptive Cell Therapy 247**

Chapter 11 **Toxicities of New Drugs for Melanoma Treatment and their**

**Tackling Immune-Suppression and Specific Anti-Tumoral**

David N. Brindley, Matthew G.K. Benesch and Mandi M. Murph

Chapter 8 **Emerging Drug Combination Approaches in Melanoma Therapy 199**

> There has never been a more rapid period of formulary expansion for melanoma treatment than what has transpired in recent years. This rapid expansion comprises various choices for monoclonal antibody immunotherapy, or immune system checkpoint therapy, and targeted, small molecule inhibitors against BRAF and MEK. Since these options have entered the clin‐ ic, the entire melanoma research landscape and disease management has undergone restruc‐ turing. Other types of cancers have even followed suit with immunotherapy indication expansions that were initially intended for melanoma.

> Thus, Melanoma – Current Clinical Management and Future Therapeutics offers readers an advanced course on revamped melanoma, but it can also function as an addendum to polish expertise on this changing field. The first section of the book includes a thorough introduc‐ tion to melanoma, with a chapter that describes disease epidemiology, intrinsic versus ex‐ trinsic risk factors and the role of pigmentation genetics in incidence. In addition to cutaneous superficial spreading melanoma, the most common type of melanoma, a separate introductory chapter discusses acral lentiginous melanoma, which is more common among individuals with dark pigmentation, and provides visual clinical presentations of disease. The last introductory chapter provides a molecular primer of signaling mechanisms driving melanoma progression.

> Othersections of the book deal with clinical management and therapeutic treatment of mela‐ noma. For example, clinical chapters discuss melanoma surgery and staging of disease among cutaneous and acral melanoma, including canine melanocytic tumors, and include a plethora of surgical images. Among these chapters, surgical removal of extensive disease and skin grafting for facial reconstruction are visually depicted in numerous figures. In ad‐ dition, sentinel lymph node biopsy and lymphadenectomy are portrayed in a detailed clini‐ cal chapter, which exhibits multiple photographs of the surgical excision technique performed among those with advanced disease.

> Immunotherapy is the major therapeutic approach discussed in this book, with several chapters outlining the applications for immune system checkpoints in melanoma. Clinical trial data is discussed combining small molecule inhibitors of MAPK, PI3K or mTOR, with and without immunotherapy, reflecting the successes and challenges with this approach, as well as with inhibitors of angiogenesis Adverse events and treatment side effects have a chapter devoted to this topic, since toxicities are an unfortunate but integral component of treatment. Finally, emerging research about potential targets in melanoma comprises the last section where readers will find chapters on autotaxin, gangliosides and RAGE.

#### XII Preface

This book would not have been possible without the help of Iva Simcic, whose help guided me through this process. I thank her for her time, energy and patience. I hope that the read‐ ership enjoys this collection of work as much as I did.

> **Prof. Mandi Murph** University of Georgia USA

## **Introduction to Melanoma**

This book would not have been possible without the help of Iva Simcic, whose help guided me through this process. I thank her for her time, energy and patience. I hope that the read‐

> **Prof. Mandi Murph** University of Georgia

> > USA

ership enjoys this collection of work as much as I did.

VIII Preface

## **Melanoma — Epidemiology, Risk Factors, and the Role of Adaptive Pigmentation**

Erin M. Wolf Horrell, Kalin Wilson and John A. D'Orazio

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58994

## **1. Introduction**

Malignant cutaneous melanoma is an aggressive form of skin cancer that affects well over 100,000 individuals world-wide each year. Melanoma results from uncontrolled proliferation of melanocytes and can occur throughout the body including skin, mucosal surfaces, and the retina. This chapter will focus on cutaneous melanoma because it is the most common site of the disease. Cutaneous melanoma has a high association with exposure to UV radiation and is most commonly found on sun exposed surfaces [5]. If diagnosed in its early stages, resection of cutaneous melanoma is associated with favorable five-year survival rates. As melanoma progresses, however, it has a tendency to metastasize beyond its primary site. It expands both radially and vertically through the skin and eventually spreads throughout the body via hematogenous or lymphatic routes. Long-term prognosis correlates strongly with the stage of disease, and after melanoma metastasizes, survival rates markedly decline. In general, fiveyear survival rates for metastatic melanoma are under 20%. Thus, early identification and treatment are essential clinical tools to minimize mortality.

For a variety of reasons, the incidence of melanoma has increased faster than any other cancer over the last several decades [6] (Figure 1), and the estimated healthcare cost in 2020 is predicted to be 4.58 billion dollars [7]. Considering the deadly nature of metastatic melanoma along with the steady increase in incidence throughout the past century, appropriate measures to prevent the development of the disease are important and will require a more complete knowledge of the risk factors associated with this disease. This chapter reviews the epidemio‐ logic and genetic risk factors associated with the development of malignant cutaneous melanoma, with an emphasis on mechanisms of melanocyte resistance to UV damage in the skin.

© 2015 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 eproduction in any medium, provided the original work is properly cited.

**Melanoma — Epidemiology, Risk Factors, and the Role of Adaptive Pigmentation**

University of Kentucky College of Medicine, Markey Cancer Center, Department of Pediatrics, the Graduate Center for Toxicology and the

Malignant cutaneous melanoma is an aggressive form of skin cancer that affects well over 100,000 individuals world-wide each year. Melanoma results from uncontrolled proliferation of melanocytes and can occur throughout the body including skin, mucosal surfaces, and the retina. This chapter will focus on cutaneous melanoma because it is the most common site of the disease. Cutaneous melanoma has a high association with exposure to UV radiation and is traditionally found on sun exposed surfaces [5]. If diagnosed in its early stages, resection of cutaneous melanoma is associated with favorable five-year survival rates. As melanoma progresses, however, it has a tendency to metastasize beyond its primary site. It expands both radially and vertically through the skin and eventually spreads throughout the body via hematogenous or lymphatic routes. Long-term prognosis correlates strongly with the stage of disease, and after melanoma metastasizes, survival rates markedly decline. In general, five-year survival rates for metastatic melanoma are under 20%. Thus, early identification and treatment are essential clinical tools to minimize mortality.

 For a variety of reasons, the incidence of melanoma has increased faster than any other cancer over the last several decades [6] (Figure 1), and the estimated healthcare cost in 2020 is predicted to be 4.58 billion dollars [7]. Considering the deadly nature of metastatic melanoma along with the steady increase in incidence throughout the past century, appropriate measures to prevent the development of the disease are important and will

associated with the development of malignant cutaneous melanoma, with an emphasis on mechanisms of melanocyte resistance to UV damage in the

Erin M. Wolf Horrell, Kalin Wilson and John A. D'Orazio

Department of Physiology, Lexington

USA

jdorazio@uky.edu.

**1. Introduction**

Figure 1. Lifetime risk of melanoma has increased in the United States from 1935-2011. (Adapted from [4]). **Figure 1.** Lifetime risk of melanoma has increased in the United States from 1935-2011. (Adapted from [4]).

#### This section will address the epidemiology of melanoma in reference to risk factors, incidence and mortality, gender differences, and variations **2. Epidemiology**

skin.

between ethnicities. **2.1. Incidence and mortality** This section will address the epidemiology of melanoma in reference to risk factors, incidence and mortality, gender differences, and variations between ethnicities.

#### **2.1. Incidence and mortality**

**2. Epidemiology**

The Surveillance, Epidemiology, and End Results (SEER) database predicts a diagnosis of melanoma in over 76,000 individuals in the United States for 2014. Though it can affect patients of any age, melanoma traditionally affects older individuals with an average age at diagnosis and death of 62 and 69 years respectively. While many cancer incidence rates have plateaued over the last century, the incidence of melanoma has steadily increased [6]. In the early 1930's, the lifetime risk of developing melanoma for an American was 1 in 1500, while in 2002, it was reported to be 1 in 68 [8]. The melanoma incidence rate increased an additional 1.8% per year between 2002 and 2012, such that 21.3 per 100,000 individuals were diagnosed with melanoma between 2007 and 2011 [1]. Though some portion of this rise is due to enhanced awareness and improvements in diagnosis, the causes underlying the increased incidence may be diverse. An important factor may be that life span has increased during this time period. As melanoma incidence correlates with age, we would expect more cases in a population of individuals that live longer [9]. However, chief among the potential contributive factors is increased exposure to ultraviolet (UV) radiation, either solar or artificial. The popularization of a tanned physique in Western cultures beginning the early 1900's has led to intentional exposures to UV. Many individuals believe they look better, feel healthier, appear younger and are happier with tanned skin [10]. The desire for UV exposure coupled with increased recreational and occu‐ pational opportunities, results in significantly more time devoted to sun exposure or in artificial tanning beds.

Although incidence rates have been increasing steadily, melanoma mortality rates have stabilized over the past 20 years due to advances in medical, surgical and supportive care [11]. The overall five-year survival rate is currently above 90%, likely related to diagnosis at curable stages for the majority of cases [1]. From 1975 until the 1990's, the mortality rate climbed 1.9% per year. From 2006-2010, there were 2.7 deaths per 100,000 individuals, and 9,700 individuals are predicted to die from melanoma in 2014 [1] (Figure 2). The mortality rate for women has actually decreased by 0.6% from 1989-2007. The mortality rate for men has increase 0.2% during the same time frame [1].

**Figure 2.** A. Five year relative survival rate for melanoma in the United States from 1975-2009. B. Mortality rate for melanoma in the United States from 1975-2009 (Adapted from [1]).

#### **2.2. Gender**

**Melanoma — Epidemiology, Risk Factors, and the Role of Adaptive Pigmentation**

Figure 1. Lifetime risk of melanoma has increased in the United States from 1935-2011. (Adapted from [4]).

**Figure 1.** Lifetime risk of melanoma has increased in the United States from 1935-2011. (Adapted from [4]).

This section will address the epidemiology of melanoma in reference to risk factors, incidence

The Surveillance, Epidemiology, and End Results (SEER) database predicts a diagnosis of melanoma in over 76,000 individuals in the United States for 2014. Though it can affect patients of any age, melanoma traditionally affects older individuals with an average age at diagnosis and death of 62 and 69 years respectively. While many cancer incidence rates have plateaued over the last century, the incidence of melanoma has steadily increased [6]. In the early 1930's, the lifetime risk of developing melanoma for an American was 1 in 1500, while in 2002, it was reported to be 1 in 68 [8]. The melanoma incidence rate increased an additional 1.8% per year between 2002 and 2012, such that 21.3 per 100,000 individuals were diagnosed with melanoma between 2007 and 2011 [1]. Though some portion of this rise is due to enhanced awareness and improvements in diagnosis, the causes underlying the increased incidence may be diverse. An important factor may be that life span has increased during this time period. As melanoma incidence correlates with age, we would expect more cases in a population of individuals that live longer [9]. However, chief among the potential contributive factors is increased exposure to ultraviolet (UV) radiation, either solar or artificial. The popularization of a tanned physique in Western cultures beginning the early 1900's has led to intentional exposures to UV. Many individuals believe they look better, feel healthier, appear younger and are happier with tanned skin [10]. The desire for UV exposure coupled with increased recreational and occu‐ pational opportunities, results in significantly more time devoted to sun exposure or in

University of Kentucky College of Medicine, Markey Cancer Center, Department of Pediatrics, the Graduate Center for Toxicology and the

Malignant cutaneous melanoma is an aggressive form of skin cancer that affects well over 100,000 individuals world-wide each year. Melanoma results from uncontrolled proliferation of melanocytes and can occur throughout the body including skin, mucosal surfaces, and the retina. This chapter will focus on cutaneous melanoma because it is the most common site of the disease. Cutaneous melanoma has a high association with exposure to UV radiation and is traditionally found on sun exposed surfaces [5]. If diagnosed in its early stages, resection of cutaneous melanoma is associated with favorable five-year survival rates. As melanoma progresses, however, it has a tendency to metastasize beyond its primary site. It expands both radially and vertically through the skin and eventually spreads throughout the body via hematogenous or lymphatic routes. Long-term prognosis correlates strongly with the stage of disease, and after melanoma metastasizes, survival rates markedly decline. In general, five-year survival rates for metastatic melanoma are under 20%. Thus, early identification and treatment are essential clinical tools to minimize mortality.

 For a variety of reasons, the incidence of melanoma has increased faster than any other cancer over the last several decades [6] (Figure 1), and the estimated healthcare cost in 2020 is predicted to be 4.58 billion dollars [7]. Considering the deadly nature of metastatic melanoma along with the steady increase in incidence throughout the past century, appropriate measures to prevent the development of the disease are important and will

Erin M. Wolf Horrell, Kalin Wilson and John A. D'Orazio

Department of Physiology, Lexington

4 Melanoma – Current Clinical Management and Future Therapeutics

USA

skin.

**2. Epidemiology**

between ethnicities.

**2.1. Incidence and mortality**

artificial tanning beds.

**2. Epidemiology**

**2.1. Incidence and mortality**

and mortality, gender differences, and variations between ethnicities.

jdorazio@uky.edu.

**1. Introduction**

Melanoma affects men and women differently. Before the age of 40, women are more suscep‐ tible to melanoma (1 in 391 women versus 1 in 691 men diagnosed each year), however, after the age of 40, the rates reverse, and 1 in 35 men will develop melanoma versus 1 in 54 women [12]. Overall men are more susceptible to melanoma, with 27.7 new cases per 100,000 men versus 16.7 new cases per 100,000 women [1]. While the mortality rate for women is decreasing, it is actually increasing for men [13]. Men account for 60% of deaths due to melanoma [14]. Although both genders are experiencing a rise in melanoma rates, there has been a massive increase in the incidence rate for women under 40, presumably due to recreational UV exposure and the popularization of having a tanned complexion [15]. In women between the ages of 20-29, melanoma is the 2nd most common cancer, trailing behind breast cancer [16]. In fact, there has been a 50% increase in melanoma incidence in young adult women since 1980 such that it is now the leading cause of cancer death among women ages 20 – 25 years old. Melanoma also is the second-most common cancer in adolescents and young adults (men and women) between the ages of 15 and 29 years. Melanoma presentation appears to differ somewhat between genders. In women, melanoma often presents on the extremities while in men, it most frequently presents on the trunk [17].

#### **2.3. Ethnicity**

The incidence, mortality rates, and presentation for melanoma differ markedly by ethnicity (Figure 3). Caucasians are the most likely to develop melanoma, however the overall five year survival is lower for African Americans than Caucasians (77% and 91% respectively) [13]. The initial diagnosis is generally at a later stage in individuals with darker skin pigmentation than in Caucasians [18] and tends to be a different subtype. Caucasians frequently present with superficial spreading melanoma while individuals with darker pigmentation often present with acral lentigenous melanoma[19]. In African Americans, Asians, Filipinos, Indonesians, and Native Hawaiians, melanoma often presents on areas that are not sun exposed including the palms and soles of hands and feet respectively, mucous membranes, and nail beds while melanoma in Caucasians generally presents on sun-exposed areas [20].

**Figure 3.** Melanoma incidence rate by ethnicity in the United States from 1975-2011. There is a discrepancy in the inci‐ dence rate between the different ethnicities. Ethnicities with fairer complexions have a higher incidence rate than eth‐ nicities with darker complexions (Adapted from [1]).

## **3. Risk factors**

it is actually increasing for men [13]. Men account for 60% of deaths due to melanoma [14]. Although both genders are experiencing a rise in melanoma rates, there has been a massive increase in the incidence rate for women under 40, presumably due to recreational UV exposure and the popularization of having a tanned complexion [15]. In women between the ages of 20-29, melanoma is the 2nd most common cancer, trailing behind breast cancer [16]. In fact, there has been a 50% increase in melanoma incidence in young adult women since 1980 such that it is now the leading cause of cancer death among women ages 20 – 25 years old. Melanoma also is the second-most common cancer in adolescents and young adults (men and women) between the ages of 15 and 29 years. Melanoma presentation appears to differ somewhat between genders. In women, melanoma often presents on the extremities while in

The incidence, mortality rates, and presentation for melanoma differ markedly by ethnicity (Figure 3). Caucasians are the most likely to develop melanoma, however the overall five year survival is lower for African Americans than Caucasians (77% and 91% respectively) [13]. The initial diagnosis is generally at a later stage in individuals with darker skin pigmentation than in Caucasians [18] and tends to be a different subtype. Caucasians frequently present with superficial spreading melanoma while individuals with darker pigmentation often present with acral lentigenous melanoma[19]. In African Americans, Asians, Filipinos, Indonesians, and Native Hawaiians, melanoma often presents on areas that are not sun exposed including the palms and soles of hands and feet respectively, mucous membranes, and nail beds while

**Figure 3.** Melanoma incidence rate by ethnicity in the United States from 1975-2011. There is a discrepancy in the inci‐ dence rate between the different ethnicities. Ethnicities with fairer complexions have a higher incidence rate than eth‐

melanoma in Caucasians generally presents on sun-exposed areas [20].

men, it most frequently presents on the trunk [17].

6 Melanoma – Current Clinical Management and Future Therapeutics

nicities with darker complexions (Adapted from [1]).

**2.3. Ethnicity**

Risk factors forthe development of melanoma can be divided into extrinsic and intrinsic factors and include exposure to UV radiation either via sunlight or indoor tanning salons, medica‐ tions, chemical exposures, presence of nevi, family history of cancer, and pigment of skin [21] (Table 1).


**Table 1.** Major melanoma risk factors.

#### **3.1. Extrinsic**

#### *3.1.1. Ultraviolet radiation*

Ultraviolet radiation, probably the single-most important environmental carcinogen with respect to melanoma, is found in natural sunlight and in artificial tanning sources. UV energy is conventionally divided into three wavelengths: UVA (320-400 nm), UVB (290-320 nm), and UVC (100-280 nm). Each type of UV energy has its own distinctive energy profile, biophysical characteristics, and effects on biologic tissue. Although solar energy contains all three UV subtypes, absorption of the higher-energy UV components by atmospheric ozone results in ambient sunlight being mainly (>90%) UVA and the remainder UVB. Both UVA and UVB are bioactive and cause DNA damage and cellular injury that contribute to carcinogenesis. In fact, exposure to UV radiation may be responsible for over 80% of all melanomas [22].

UVA and UVB contribute to the pathogenesis of melanoma via distinct but overlapping mechanisms [23]. UVA (and to a lesser extent UVB) causes DNA damage indirectly through the generation of highly reactive free radicals and oxidative injury [24, 25]. Free radicals change DNA in ways that ultimately affect base-pairing, which can lead to mutation. Oxidation of

**Figure 4.** Generation of 6,4-photoproducts and cyclobutane dimers from adjacent pyrimidines. UVB radiation is absor‐ bed by thymine and cytosine residues, resulting in the formation of mutagenic photoproducts that contribute to malig‐ nancy.

guanine, for example, is well-known to cause formation of 8-oxo-dG which base pairs with A insteadofC;this changeleads totransversionpointmutations ifnotrepairedinatimelymanner. Indeed, some estimate that up to 80% of malignant cutaneous melanomas may result from indirect DNA damage, highlighting the importance of "lower UV energy" in the pathogene‐ sis ofmelanoma [26].UVBradiation,incontrast,ishigherinenergy anddirectly affects adjacent pyrimidines in the double helix to cause a photochemical reaction. Through direct absorption of UVB energy by pyrimidines, two major types of photoproducts are generated: cyclopyrimi‐ dine dimers (CPD's) and 6,4 photoproducts (6,4-PP) [27-30] (Figure 4). Both lesions distort the DNA double helix and, if left unrepaired, lead to characteristic "UV signature" transition mutations (G →T and G→C mutations) [30]. It is largely through the identification of these UV signature mutations that we know that UV is a major risk factor for skin cancer and melano‐ ma [31]. Although UVC, the UV component with the highest energy per photon, can cause substantial damage to cells, the ozone layer absorbs the majority of UVC emitted by the sun, thereforethiscomponentisnotthoughttobeasignificantcontributortomostcasesofmelanoma. Melanomas classically occur on sun exposed skin [32], and exposure to UV radiation corre‐ lates with not only the risk for melanoma [33] but also mortality rates [34]. The correlation between melanoma risk is particularly strong with the UVA component, providing further evidence that UVA may be a significant causative factor in melanomagenesis [35].

#### *3.1.1.1. Geographic factors*

The global distribution of melanoma also demonstrates the importance of UV radiation in the pathogenesis of this disease. Countries that are located on latitudes closer to the equator have increased UV intensity and higher rates of melanoma among fair-skinned persons [4, 36]. Australia and New Zealand, which are near the equator, have the highest incidence of melanoma in the world with a risk of 1 in 50 individuals [21]. The World Heath Organization reports that Australia and New Zealand have an age adjusted incidence rate of 35.1 individuals per 100,000 individuals [2]. The incidence of melanoma in Australia has doubled from 1986-2006 [37]. Norway, despite also having a fair-skinned, UV-sensitive population predom‐ inantly of Scandinavian descent, has a relatively low incidence of melanoma, presumably due to its high latitude (with comparatively weak ambient solar energy) and low UV exposure [38]. High altitudes are also a risk factor for development of melanoma, presumably because UV strength is higher due to less interference between solar energy and particulate matter present in the atmosphere [39, 40] (Figure 5).

Although latitude and altitude do play a major role in melanoma risk, skin complexion is also an important component to explain the variations in melanoma incidence through‐ out the world. Central America, despite being closer to the equator than North America has a significantly lower age adjusted incidence rates (1.5 and 13.8 individuals per 100,000 individuals) presumably due to having a predominantly dark-skinned complexion [2].

#### *3.1.1.2. UV exposure patterns*

Intermittent sun exposure confers a higher risk for developing melanoma than continual exposure [41-43], but the age at which the exposure causes the greatest damage is still controversial. Some studies suggest that sun exposure for a younger individual is more likely to be associated with the development of melanoma [44], while others suggest age of exposure is less important than the cumulative dose of UV [45]. Regardless of age, sunburn is a major risk factor in the development of melanoma, and the risk doubles with more than 5 sunburns [45, 46] or one or more blistering sunburns [43].

#### *3.1.1.3. Sunscreen*

guanine, for example, is well-known to cause formation of 8-oxo-dG which base pairs with A insteadofC;this changeleads totransversionpointmutations ifnotrepairedinatimelymanner. Indeed, some estimate that up to 80% of malignant cutaneous melanomas may result from indirect DNA damage, highlighting the importance of "lower UV energy" in the pathogene‐ sis ofmelanoma [26].UVBradiation,incontrast,ishigherinenergy anddirectly affects adjacent pyrimidines in the double helix to cause a photochemical reaction. Through direct absorption of UVB energy by pyrimidines, two major types of photoproducts are generated: cyclopyrimi‐ dine dimers (CPD's) and 6,4 photoproducts (6,4-PP) [27-30] (Figure 4). Both lesions distort the DNA double helix and, if left unrepaired, lead to characteristic "UV signature" transition mutations (G →T and G→C mutations) [30]. It is largely through the identification of these UV signature mutations that we know that UV is a major risk factor for skin cancer and melano‐ ma [31]. Although UVC, the UV component with the highest energy per photon, can cause substantial damage to cells, the ozone layer absorbs the majority of UVC emitted by the sun, thereforethiscomponentisnotthoughttobeasignificantcontributortomostcasesofmelanoma. Melanomas classically occur on sun exposed skin [32], and exposure to UV radiation corre‐

**Figure 4.** Generation of 6,4-photoproducts and cyclobutane dimers from adjacent pyrimidines. UVB radiation is absor‐ bed by thymine and cytosine residues, resulting in the formation of mutagenic photoproducts that contribute to malig‐

8 Melanoma – Current Clinical Management and Future Therapeutics

nancy.

The effect of sunscreen on melanoma prevalence is also controversial for a variety of reasons. The widespread use of broad-spectrum sunscreen has not decreased the incidence despite blocking both UVA and UVB radiation. Early sunscreens were designed to prevent sunburn and only blocked UVB radiation. Individuals who used UVB blocking sunscreen were able to stay out in the sun for longer periods of time and were exposed to greater doses of UVA radiation. A delay may exist between the advent of broad-spectrum sunscreen and its effect on melanoma incidence due to the latency between sun exposure and development of permission from [2]).

highest in countries populated by fair-skinned persons living in high-UV environments (adapted with

*Sunscreen* 

[45, 49].

*Indoor tanning bed use*  There has been an explosive increase in the widespread use of indoor tanning beds since their invention in the early 1970's. Over 30 million individuals use indoor tanning salons [50] and over 70% are female [51]. Overall, 2-3 million of salon-users are teenagers [52].

The effect of sunscreen on melanoma prevalence is also controversial for a variety of reasons. The widespread use of broad-spectrum sunscreen has not decreased the incidence despite blocking both UVA and UVB radiation. Early sunscreens were designed to prevent sunburn and only blocked UVB radiation. Individuals who used UVB blocking sunscreen were able to stay out in the sun for longer periods of time and were exposed to greater doses of UVA radiation. A delay may exist between the advent of broadspectrum sunscreen and its effect on melanoma incidence due to the latency between sun exposure and development of melanoma. In addition, the use of sunscreen can also promote cellular damage by increasing the reactive oxygen species [47] and if absorbed past the epidermis, can cause photosensitization in melanocytes [48]. Despite some negative effects, the American Society for Clinical Oncology states that the use of broad-spectrum sunscreen with an SPF of 15 or greater decreases an individual's risk of melanoma by up to 50% and should be worn daily to prevent damage from UV radiation

among young adolescents. The earlier tanning begins, the more potential latency there is for carcinogenesis and malignant transformation. In individuals between 18-29 years of age, 76% diagnosed with malignant melanoma had previously used artificial tanning devices [54]. There is a strong positive correlation not only between the use of indoor tanning beds and the development of melanoma [55] but also with death due to **Figure 5.** Geographical variation in melanoma incidence (A) and mortality (B). Melanoma incidence is highest in coun‐ tries populated by fair-skinned persons living in high-UV environments (adapted with permission from [2]).

At age 17, 35% of American females admit to use of an artificial tanning device [53]. The prevalence of artificial tanning is concerning, especially

melanoma. In addition, the use of sunscreen can also promote cellular damage by increasing the reactive oxygen species [47] and if absorbed past the epidermis, can cause photosensitiza‐ tion in melanocytes [48]. Despite some negative effects, the American Society for Clinical Oncology states that the use of broad-spectrum sunscreen with an SPF of 15 or greater decreases an individual's risk of melanoma by up to 50% and should be worn daily to prevent damage from UV radiation [45, 49]. melanoma [56]. One tanning session increases the chance an individual will develop melanoma by 20%, and each additional session per year increases risk by another 2% [54]. The International Agency for Research on Cancer classifies UV tanning devices in Group1, most dangerous oncogenic substances [57]. Among salon establishments, artificial tanning beds vary in their delivered dose, and the amount of UVA and UVB radiation is unregulated [10] making artificial tanning incredibly dangerous. State legislative statues vary as to whether minors have restricted access. Many experts hypothesize that the burgeoning use of artificial tanning salons over the last three decades may be a major contributing factor to the continued increase in melanoma incidence.

#### *3.1.1.4. Indoor tanning bed use*

There has been an explosive increase in the widespread use of indoor tanning beds since their invention in the early 1970's. Over 30 million individuals use indoor tanning salons [50] and over 70% are female [51]. Overall, 2-3 million of salon-users are teenagers [52]. At age 17, 35% of American females admit to use of an artificial tanning device [53]. The prevalence of artificial tanning is concerning, especially among young adolescents. The earlier tanning begins, the more potential risk there is for carcinogenesis and malignant transformation. In individuals between 18-29 years of age, 76% diagnosed with malignant melanoma had previously used artificial tanning devices [54]. There is a strong positive correlation not only between the use of indoor tanning beds and the development of melanoma [55] but also with death due to melanoma [56]. One tanning session increases the chance an individual will develop melanoma by 20%, and each additional session per year increases risk by another 2% [54]. The Interna‐ tional Agency for Research on Cancer classifies UV tanning devices in Group1, most dangerous oncogenic substances [57]. Among salon establishments, artificial tanning beds vary in their delivered dose, and the amount of UVA and UVB radiation is unregulated [10] making artificial tanning incredibly dangerous. State legislative statues vary as to whether minors have restricted access. Many experts hypothesize that the burgeoning use of artificial tanning salons over the last three decades may be a major contributing factor to the continued increase in melanoma incidence.

#### *3.1.2. Medications*

*Sunscreen* 

[45, 49].

*Indoor tanning bed use*  There has been an explosive increase in the widespread use of indoor tanning beds since their invention in the early 1970's. Over 30 million individuals use indoor tanning salons [50] and over 70% are female [51]. Overall, 2-3 million of salon-users are teenagers [52].

The effect of sunscreen on melanoma prevalence is also controversial for a variety of reasons. The widespread use of broad-spectrum sunscreen has not decreased the incidence despite blocking both UVA and UVB radiation. Early sunscreens were designed to prevent sunburn and only blocked UVB radiation. Individuals who used UVB blocking sunscreen were able to stay out in the sun for longer periods of time and were exposed to greater doses of UVA radiation. A delay may exist between the advent of broadspectrum sunscreen and its effect on melanoma incidence due to the latency between sun exposure and development of melanoma. In addition, the use of sunscreen can also promote cellular damage by increasing the reactive oxygen species [47] and if absorbed past the epidermis, can cause photosensitization in melanocytes [48]. Despite some negative effects, the American Society for Clinical Oncology states that the use of broad-spectrum sunscreen with an SPF of 15 or greater decreases an individual's risk of melanoma by up to 50% and should be worn daily to prevent damage from UV radiation

melanoma. In addition, the use of sunscreen can also promote cellular damage by increasing the reactive oxygen species [47] and if absorbed past the epidermis, can cause photosensitiza‐ tion in melanocytes [48]. Despite some negative effects, the American Society for Clinical Oncology states that the use of broad-spectrum sunscreen with an SPF of 15 or greater decreases an individual's risk of melanoma by up to 50% and should be worn daily to prevent

**Figure 5.** Geographical variation in melanoma incidence (A) and mortality (B). Melanoma incidence is highest in coun‐

tries populated by fair-skinned persons living in high-UV environments (adapted with permission from [2]).

At age 17, 35% of American females admit to use of an artificial tanning device [53]. The prevalence of artificial tanning is concerning, especially among young adolescents. The earlier tanning begins, the more potential latency there is for carcinogenesis and malignant transformation. In individuals between 18-29 years of age, 76% diagnosed with malignant melanoma had previously used artificial tanning devices [54]. There is a strong positive correlation not only between the use of indoor tanning beds and the development of melanoma [55] but also with death due to melanoma [56]. One tanning session increases the chance an individual will develop melanoma by 20%, and each additional session per year increases risk by another 2% [54]. The International Agency for Research on Cancer classifies UV tanning devices in Group1, most dangerous oncogenic substances [57]. Among salon establishments, artificial tanning beds vary in their delivered dose, and the amount of UVA and UVB radiation is unregulated [10] making artificial tanning incredibly dangerous. State legislative statues vary as to whether minors have restricted access. Many experts hypothesize that the burgeoning use of artificial tanning salons over the last three decades may be a major contributing

Figure 5: Geographical variation in melanoma incidence (A) and mortality (B). Melanoma incidence is highest in countries populated by fair-skinned persons living in high-UV environments (adapted with

There has been an explosive increase in the widespread use of indoor tanning beds since their invention in the early 1970's. Over 30 million individuals use indoor tanning salons [50] and

damage from UV radiation [45, 49].

factor to the continued increase in melanoma incidence.

*3.1.1.4. Indoor tanning bed use*

permission from [2]).

10 Melanoma – Current Clinical Management and Future Therapeutics

A

B

Many medications that have great benefit for disease treatment also increase patient suscept‐ ibility to cancer. Psoralen and ultraviolet A radiation (PUVA) is an effective treatment for psoriasis and other dermatologic conditions. Psoralen increases reactivity to UV radiation, therefore the combination of psoralen and UVA causes a substantial degree of cellular damage. Patients who receive PUVA have a 10 fold increase in risk of developing melanoma 15 years after treatment, and the risk increases with number of treatment sessions (>250) and time following treatments [58].

Neonatal blue light phototherapy (NBLP) is another example of light therapy that may lead to an increased risk for the development of melanoma. NBLP is a treatment for neonates with elevated bilirubin levels and risk of kernicterus. The therapy is associated with short term side effects that are treatable and/or reversible; however, it may also increase the risk of develop‐ ment of melanoma in later years. The blue lamps used for treatment generally emit a combi‐ nation of blue light and wavelengths in the UVA region of the spectrum [59]. UVA is known to cause DNA damage as explained above, and some studies have demonstrated that visual light can also cause damage through increased activity along the cytokine and oxidant signaling pathways [60]. Reports disagree as to whether exposure to NBLP causes an increase in nevi number and melanoma susceptibility. Bauer et al. assessed 8112 Caucasian children and showed no correlation between nevi number and exposure to NBLP [61]. However, Matichard et al. and Csoma et al. demonstrated that treatment with NBLP correlated with size of nevus (nevi > 2mm in diameter significantly correlated with exposure to NBLP) [62] or with presence of atypical nevi respectively [63]. A study of twins in 2011 demonstrated that NBLP treatment correlated with a higher prevalence of both clinically normal and dysplastic nevi [64] suggesting that NBLP does increase the risk of development of melanoma in adulthood, and children who receive NBLP should be monitored throughout life.

#### *3.1.3. Heavy metals and chemical exposure*

Exposure to heavy metals and certain chemicals are associated with an increased risk of melanoma, presumably through mutagenic changes to DNA in melanocytes. Fortes and de Vries suggest that exposure to polycyclic hydrocarbons (for example, individuals who work in industries associated with petroleum, printing, and electronics), ionizing radiation, poly‐ vinyl chloride (a substance present in clothing dye), heavy metals, and pesticides all increase the risk of developing melanoma [65]. Both polycyclic aromatic hydrocarbons [66] and heavy metals [67] react with UVA to generate free radicals which can subsequently damage DNA, but a variety of molecular mechanisms may be involved in carcinogenesis with these agents.

#### **3.2. Intrinsic**

#### *3.2.1. History of skin cancer*

Personal or family history of skin cancers is associated with higher melanoma risk. UVinitiated malignancies such as squamous cell carcinoma, basal cell carcinoma or actinic keratosis [68, 69] may indicate cumulative UV exposure, however other skin malignancies not thought to be UV-related (e.g. mycosis fungoides) also increase risk of melanoma [70]. Increased risk from skin cancers such as mycosis fungoides may be due to the immunosup‐ pression associated with the disease (immunodeficiency discussed below). First degree relatives of an individual with melanoma also have a higher risk of developing melanoma than the general population [71], and if a first degree relative has had multiple melanomas, the relative risk of an individual developing melanoma is increased to 61.78 [72]. A past medical history of cutaneous melanoma also substantially increases the risk of subsequently develop‐ ing another [73, 74]. Although the majority of melanomas are sporadic, 10% of diagnoses are in the setting of familial syndromes [75]. For example, individuals diagnosed with Dysplastic Nevus Syndrome, also known as the Familial Atypical Multiple-Mole Melanoma Syndrome, have a 48.9% risk of developing melanoma by age 50 and an 82% risk by age 72 [76]. One of the most common causes of a familial melanoma syndrome is a mutation in the cyclin dependent kinase inhibitor 2A (*CDKN2A*) gene [75] which regulates cell cycle progression. However it is important to note that increased melanoma prevalence within a family may also represent shared environmental factors such as geography and chemical exposure rather than genetic mutations.

#### *3.2.2. Nevi*

Nevi can foreshadow the development of melanoma [77-79]. In 1978 two independent studies reported an association between nevi and melanoma for individuals with familial melanoma syndromes. Reimer et al. reported there was "a syndrome of pigmented lesions in melanomaprone families" [80] while Lynch et al. described melanomas that were linked to individuals with a large number of "moles of variable size and color" [81]. A majority of benign nevi and melanomas share a common mutation in the BRAF gene (V600E) which results in a gain of function in BRAF signaling [26, 82-84]. This mutation activates the mitogen activated protein kinase cascade leading to the deregulation of the cell cycle and an increase in cell division. While the BRAF mutation may be sufficient for the formation of a benign nevus, additional mutations are needed (e.g. PTEN loss) for the nevus to convert to a malignant melanoma.

The number of nevi, the presence of atypical or large nevi, and the development of new nevi all correlate with melanoma risk [85, 86]. *De novo* nevi formation is a result of exposure to UV radiation, and sunscreen may not influence this process [87-89]. While the risk of melanoma rises with an increased number of total body nevi, malignant degeneration of any particular nevus is rare. Rather, melanomas generally arise from dysplastic nevi, [90], and the risk of a normal nevus converting to melanoma is very low [91]. The presence of only one dysplastic nevus increases risk by 2 fold, however, >10 dysplastic nevi can increase the risk up to 12 fold [92] [93]. Dysplastic nevi are present in 34-56% of melanoma cases [94].

Risk associated with congenital nevi varies with size and quantity. Small congenital nevi are not associated with an increase in risk [92] while large congenital nevi covering over 5% of the body surface area confers an increased risk [95]. Individual large congenital nevi >20 cm in diameter increase an individual's lifetime risk of melanoma to 10% [96]. Reports suggest that if a melanoma is going to arise from a congenital nevi, most will occur by the age of 10 pointing to the importance for screening the pediatric population [97].

#### *3.2.3. Medical history*

*3.1.3. Heavy metals and chemical exposure*

12 Melanoma – Current Clinical Management and Future Therapeutics

**3.2. Intrinsic**

*3.2.1. History of skin cancer*

genetic mutations.

*3.2.2. Nevi*

Exposure to heavy metals and certain chemicals are associated with an increased risk of melanoma, presumably through mutagenic changes to DNA in melanocytes. Fortes and de Vries suggest that exposure to polycyclic hydrocarbons (for example, individuals who work in industries associated with petroleum, printing, and electronics), ionizing radiation, poly‐ vinyl chloride (a substance present in clothing dye), heavy metals, and pesticides all increase the risk of developing melanoma [65]. Both polycyclic aromatic hydrocarbons [66] and heavy metals [67] react with UVA to generate free radicals which can subsequently damage DNA, but a variety of molecular mechanisms may be involved in carcinogenesis with these agents.

Personal or family history of skin cancers is associated with higher melanoma risk. UVinitiated malignancies such as squamous cell carcinoma, basal cell carcinoma or actinic keratosis [68, 69] may indicate cumulative UV exposure, however other skin malignancies not thought to be UV-related (e.g. mycosis fungoides) also increase risk of melanoma [70]. Increased risk from skin cancers such as mycosis fungoides may be due to the immunosup‐ pression associated with the disease (immunodeficiency discussed below). First degree relatives of an individual with melanoma also have a higher risk of developing melanoma than the general population [71], and if a first degree relative has had multiple melanomas, the relative risk of an individual developing melanoma is increased to 61.78 [72]. A past medical history of cutaneous melanoma also substantially increases the risk of subsequently develop‐ ing another [73, 74]. Although the majority of melanomas are sporadic, 10% of diagnoses are in the setting of familial syndromes [75]. For example, individuals diagnosed with Dysplastic Nevus Syndrome, also known as the Familial Atypical Multiple-Mole Melanoma Syndrome, have a 48.9% risk of developing melanoma by age 50 and an 82% risk by age 72 [76]. One of the most common causes of a familial melanoma syndrome is a mutation in the cyclin dependent kinase inhibitor 2A (*CDKN2A*) gene [75] which regulates cell cycle progression. However it is important to note that increased melanoma prevalence within a family may also represent shared environmental factors such as geography and chemical exposure rather than

Nevi can foreshadow the development of melanoma [77-79]. In 1978 two independent studies reported an association between nevi and melanoma for individuals with familial melanoma syndromes. Reimer et al. reported there was "a syndrome of pigmented lesions in melanomaprone families" [80] while Lynch et al. described melanomas that were linked to individuals with a large number of "moles of variable size and color" [81]. A majority of benign nevi and melanomas share a common mutation in the BRAF gene (V600E) which results in a gain of function in BRAF signaling [26, 82-84]. This mutation activates the mitogen activated protein kinase cascade leading to the deregulation of the cell cycle and an increase in cell division. Medical conditions associated with immunodeficiency or that use immunosuppressive therapies can trigger melanoma. Patients diagnosed with human immunodeficiency virus/ acquired immunodeficiency syndrome have an increased prevalence of melanoma with a 50% increased risk of the disease [98] [99]. Because antiretroviral treatment for HIV/AIDS has increased patient's lifespan, these individuals should be closely monitored and obtain regular screening throughout their life. Patients who receive an organ transplant not only have 2.4 greater risk of developing melanoma [100], they also have a more aggressive cancer [101] and a worse prognosis [102] than the general population. Transfer of melanoma from donor to recipient is possible if the donor was previously afflicted with the condition [103].

A previous medical history of noncutaneous skin cancer is also associated with an increased risk of developing melanoma. Individuals who were previously diagnosed with Kaposi sarcoma, breast cancer, lymphoma, prostate cancer, thyroid cancer, and leukemia had an increased risk of subsequently developing melanoma [104]. Childhood cancer survivors have a 2.5 fold increased risk of developing melanoma [105] and are diagnosed at a younger age than the general population (32 years) [106]. Studies speculate that the increased risk following malignancy may be due to either germline mutations in oncogenes or due to the chemotherapy and radiation to treat the prior malignancy [105].

Although melanoma is associated with the production of female hormones, no increased risk of melanoma-associated pathogenesis can be attributed to pregnancy [107]. A majority of women who are pregnant experience a phenomenon known as melasma, an increase in pigment due to increases in melanocyte activity [108]; however, the increase in pigment is not associated with an increase in melanoma incidence.

#### *3.2.4. Nucleotide excision repair*

Nucleotide excision repair (NER) is the molecular process by which bulky DNA lesions are recognized, excised, and repaired by the coordinated actions of multiple factors [109-111]. As described above, UVB radiation and to a lesser extent UVA radiation promote the formation of photoproducts that distort the double helix and prevent transcription [112, 113]. Without accurate repair, these photoproducts may cause transition mutations and lead to unregulated cellular proliferation and carcinogenesis. diagnosed at a younger age than the general population (32 years) [106]. Studies speculate that the increased risk following malignancy may be due to either germline mutations in oncogenes or due to the chemotherapy and radiation to treat the prior malignancy [105]. Although melanoma is associated with the production of female hormones, no increased risk of melanoma-associated pathogenesis can be attributed to pregnancy [107]. A majority of women who are pregnant experience a phenomenon known as melasma, an increase in pigment due to increases in melanocyte activity [108]; however, the increase in pigment is not associated with an increase in melanoma incidence.

There are two primary mechanisms of NER, global genome NER (GGR) and transcription coupled NER (TCR), which differ in their initiation site (Figure 6). GGR recognizes damage in non-transcribed regions of the genome. Xeroderma pigmentosum (XP) complementation group C (XPC) and HR23B heterodimerize, recognize distortions within the DNA double helix [114, 115], and recruit the TFIIH complex. TFIIH is a multiprotein complex composed of nine proteins including the helicases XPB and XPD. XPB and XPD unwind 20-30 nucleotides surrounding the damaged base in the 3'-5' and 5'-3' direction respectively [116]. The opened DNA structure is stabilized by recruitment of XPA and RPA [117, 118]. After the structure is stabilized, XPF and XPG endonucleases remove the damaged base [119, 120] and the gap is repaired by polymerase δ and ε [121]. **3.2.4. Nucleotide excision repair** Nucleotide excision repair (NER) is the molecular process by which bulky DNA lesions are recognized, excised, and repaired by the coordinated actions of multiple factors [109-111]. As described above, UVB radiation and to a lesser extent UVA radiation promote the formation of photoproducts that distort the double helix and prevent transcription [112, 113]. Without accurate repair, these photoproducts may cause transition mutations and lead to unregulated cellular proliferation and carcinogenesis. There are two primary mechanisms of NER, global genome NER (GGR) and transcription coupled NER (TCR), which differ in their initiation site (Figure 6). GGR recognizes damage in non-transcribed regions of the genome. Xeroderma pigmentosum (XP) complementation group C (XPC) and HR23B heterodimerize, recognize distortions within the DNA double helix [114, 115], and recruit the TFIIH complex. TFIIH is a multiprotein complex composed of nine proteins including the helicases XPB and XPD. XPB and XPD unwind 20-30 nucleotides surrounding the damaged base in the 3'-5' and 5'-3' direction respectively [116]. The opened DNA structure is stabilized by recruitment of XPA and RPA [117, 118]. After the structure is

stabilized, XPF and XPG endonucleases remove the damaged base [119, 120] and the gap is repaired by polymerase δ and ε [121].

Figure 6. The nucleotide excision repair (NER) pathway is the major way cells rid themselves of bulky DNA lesions such as UV photoproducts. NER is accomplished

stalled RNA polymerase II [124-126]. After damage recognition, either by GGR or TCR, many NER factors work in concert to unwind the double helix

through the cooperative action of a variety of proteins, working in concert to (1) recognize DNA damage, (2) access and unwind the DNA in the region of the lesion, (3) incise and remove the damage, and (4) repair the gap with a high degree of fidelity using the undamaged strand as a template. Without effective NER, UV mutations accumulate and skin cancers of all kinds occur with high incidence. (Adapted from[1] [3]). TCR recognizes damage in transcribed regions of the genome after transcription by RNA polymerase II has stalled [122, 123]. Following exposure to UV radiation, Cockayne syndrome B (CSB), Cockayne syndrome A (CSA), and the core NER factors (excluding XPC) are recruited to the sites of **Figure 6.** The nucleotide excision repair (NER) pathway is the major way cells rid themselves of bulky DNA lesions such as UV photoproducts. NER is accomplished through the cooperative action of a variety of proteins, working in concert to (1) recognize DNA damage, (2) access and unwind the DNA in the region of the lesion, (3) incise and re‐ move the damage, and (4) repair the gap with a high degree of fidelity using the undamaged strand as a template. Without effective NER, UV mutations accumulate and skin cancers of all kinds occur with high incidence. (Adapted from [3]).

diagnosed at a younger age than the general population (32 years) [106]. Studies speculate that the increased risk following malignancy may be due Although melanoma is associated with the production of female hormones, no increased risk of melanoma-associated pathogenesis can be attributed to pregnancy [107]. A majority of women who are pregnant experience a phenomenon known as melasma, an increase in pigment due to increases in TCR recognizes damage in transcribed regions of the genome after transcription by RNA polymerase II has stalled [122, 123]. Following exposure to UV radiation, Cockayne syndrome B (CSB), Cockayne syndrome A (CSA), and the core NER factors (excluding XPC) are recruited to the sites of stalled RNA polymerase II [124-126]. After damage recognition, either by GGR or TCR, many NER factors work in concert to unwind the double helix in the area of photo‐ damage, excise the damaged strand, and repair the gap using the undamaged sister strand as a template. In this way, NER corrects UV photodamage with a high degree of fidelity and minimizes the chances for UV-induced mutagenesis..

Nucleotide excision repair (NER) is the molecular process by which bulky DNA lesions are recognized, excised, and repaired by the coordinated actions of multiple factors [109-111]. As described above, UVB radiation and to a lesser extent UVA radiation promote the formation of photoproducts that distort the double helix and prevent transcription [112, 113]. Without accurate repair, these photoproducts may cause transition There are two primary mechanisms of NER, global genome NER (GGR) and transcription coupled NER (TCR), which differ in their initiation site (Figure 6). GGR recognizes damage in non-transcribed regions of the genome. Xeroderma pigmentosum (XP) complementation group C (XPC) and HR23B heterodimerize, recognize distortions within the DNA double helix [114, 115], and recruit the TFIIH complex. TFIIH is a multiprotein complex composed of nine proteins including the helicases XPB and XPD. XPB and XPD unwind 20-30 nucleotides surrounding the damaged base in the 3'-5' and 5'-3' direction respectively [116]. The opened DNA structure is stabilized by recruitment of XPA and RPA [117, 118]. After the structure is The importance of DNA repair in preventing melanoma is evident in individuals diagnosed with xeroderma pigmentosum (XP) [127, 128]. They are highly sensitive to UV radiation and develop epidermal thinning, telangiectasias, and altered pigmentation in addition to increased prevalence of skin malignancies [129], with a large number of UV-induced mutations in oncogenes and tumor suppressors [130]. These patients have defective DNA repair due to mutations in one of 8 factors associated with NER [131]. Since DNA repair is not possible, patients with XP are encouraged to limit exposure to UV radiation in order to prevent cellular damage. Individuals diagnosed with XP have a 1000 fold increase in skin cancer risk compared to the average population and are often diagnosed with melanoma in the second decade (on average over 40 years before the general public) [132].

#### *3.2.5. Skin complexion*

*3.2.4. Nucleotide excision repair*

cellular proliferation and carcinogenesis.

14 Melanoma – Current Clinical Management and Future Therapeutics

**3.2.4. Nucleotide excision repair**

repaired by polymerase δ and ε [121].

from [3]).

Nucleotide excision repair (NER) is the molecular process by which bulky DNA lesions are recognized, excised, and repaired by the coordinated actions of multiple factors [109-111]. As described above, UVB radiation and to a lesser extent UVA radiation promote the formation of photoproducts that distort the double helix and prevent transcription [112, 113]. Without accurate repair, these photoproducts may cause transition mutations and lead to unregulated

to either germline mutations in oncogenes or due to the chemotherapy and radiation to treat the prior malignancy [105].

melanocyte activity [108]; however, the increase in pigment is not associated with an increase in melanoma incidence.

stabilized, XPF and XPG endonucleases remove the damaged base [119, 120] and the gap is repaired by polymerase δ and ε [121].

There are two primary mechanisms of NER, global genome NER (GGR) and transcription coupled NER (TCR), which differ in their initiation site (Figure 6). GGR recognizes damage in non-transcribed regions of the genome. Xeroderma pigmentosum (XP) complementation group C (XPC) and HR23B heterodimerize, recognize distortions within the DNA double helix [114, 115], and recruit the TFIIH complex. TFIIH is a multiprotein complex composed of nine proteins including the helicases XPB and XPD. XPB and XPD unwind 20-30 nucleotides surrounding the damaged base in the 3'-5' and 5'-3' direction respectively [116]. The opened DNA structure is stabilized by recruitment of XPA and RPA [117, 118]. After the structure is stabilized, XPF and XPG endonucleases remove the damaged base [119, 120] and the gap is

mutations and lead to unregulated cellular proliferation and carcinogenesis.

accumulate and skin cancers of all kinds occur with high incidence. (Adapted from[1] [3]).

**Figure 6.** The nucleotide excision repair (NER) pathway is the major way cells rid themselves of bulky DNA lesions such as UV photoproducts. NER is accomplished through the cooperative action of a variety of proteins, working in concert to (1) recognize DNA damage, (2) access and unwind the DNA in the region of the lesion, (3) incise and re‐ move the damage, and (4) repair the gap with a high degree of fidelity using the undamaged strand as a template. Without effective NER, UV mutations accumulate and skin cancers of all kinds occur with high incidence. (Adapted The amount of melanin pigment present in the skin determines skin complexion, and low basal pigmentation (having a fair-skinned phenotype) constitutes a major risk factor for the devel‐ opment of melanoma. Melanin pigments, all derived from the amino acid tyrosine, are synthesized by melanocytes, transported to the keratinocytes where they absorb UV radiation, and prevent damage to the sensitive layers of the skin. In fact, melanocytes produce two distinct forms of melanin. Eumelanin is a dark brown/black chemically inert pigment that potently blocks penetration of UV energy into the skin. Pheomelanin, in contrast, is a lightercolored pigment that is much less effective at blocking UV penetration and that may even potentiate oxidative UV damage [133] (Figure 7).

Figure 6. The nucleotide excision repair (NER) pathway is the major way cells rid themselves of bulky DNA lesions such as UV photoproducts. NER is accomplished through the cooperative action of a variety of proteins, working in concert to (1) recognize DNA damage, (2) access and unwind the DNA in the region of the lesion, (3) incise and remove the damage, and (4) repair the gap with a high degree of fidelity using the undamaged strand as a template. Without effective NER, UV mutations TCR recognizes damage in transcribed regions of the genome after transcription by RNA polymerase II has stalled [122, 123]. Following exposure to UV radiation, Cockayne syndrome B (CSB), Cockayne syndrome A (CSA), and the core NER factors (excluding XPC) are recruited to the sites of stalled RNA polymerase II [124-126]. After damage recognition, either by GGR or TCR, many NER factors work in concert to unwind the double helix The amount of eumelanin in the epidermis largely determines skin complexion. The Fitzpa‐ trick skin phototype was developed by a Harvard University Medical School dermatologist to classify an individual's UV susceptibility based on basal pigment levels, tendency to burn, and ability to tan [134]. Individuals with a lower Fitzpatrick score have fair skin (less pigment), red or blonde hair, burn easily and are unable to tan, while individuals with a higher Fitzpatrick score have a darker complexion (more pigment), do not burn, and tan easily (Table 2). Compared to individuals with a Fitzpatrick score of IV, individuals with a Fitzpatrick score of I have a relative risk of 2.09 developing melanoma [71]. A recent study demonstrated that pigment and race were not sufficient to predict an individual's Fitzpatrick score and sun sensitivity; this finding highlights the importance of physician-based counseling to all patients regarding sun safety and prevention of sun-induced malignancies [135].

**Figure 7.** Synthesis of eumelanin and pheomelanin is regulated by the melanocortin 1 receptor (MC1R). Eumelanin, a dark brown/black pigment with excellent UV protective properties, is produced when melanocytes are stimulated through MC1R signaling and cytoplasmic cAMP levels are increased. In contrast, pheomelanin, a sulfated pigment with poorer UV-blocking properties is made when MC1R is inactive.


**Table 2.** The Fitzpatrick scale of skin complexion. MED represents the minimal erythematous dose (the least amount of UV required to cause a sunburn).

#### **4. Pigmentation and MC1R**

The melanocortin 1 receptor (MC1R) is a major determinant of skin pigmentation and UV sensitivity. UV exposure activates the MC1R, which directly controls not only the adaptive tanning response (UV-induced pigmentation) but regulates melanocyte DNA repair and, thus, the mutagenic risk as well.

#### **4.1. Eumelanin versus pheomelanin**

Melanocytes, derived from neural crest cells, produce pigment in the skin. As described above, lack of basal skin pigmentation is a major risk factor for the development of melanoma. The two major types of pigment produced by the melanocyte are eumelanin and pheomelanin. Pigmentation depends on the type and amount of melanin produced in addition to its cellular distribution rather than number of melanocytes present in the skin [136-138]. Eumelanin is a dark insoluble polymer that absorbs UV light [139, 140] and oxidants [141], protecting DNA from the damaging effects of these factors. Pheomelanin is a soluble red/yellow polymer containing cysteine, which provides little protection from UV light and reports demonstrate that pheomelanin can promote oxidative damage [133, 142]. Synthesis of both eumelanin and pheomelanin begins with the conversion of tyrosine to DOPA and then to DOPAquinone via the enzyme tyrosinase [143]. Incorporation of a cysteine into DOPAquinone molecule even‐ tually leads to the production of pheomelanin rather than eumelanin. Individuals without a functional tyrosinase are unable to produce any pigment and have a condition known as albinism [140]. Control of the ratio of pheomelanin to eumelanin in a cell is determined by multiple factors including pH of the cellular milieu and levels of the tyrosinase enzyme [133, 144]. Higher levels of tyrosinase and neutral pH favor eumelanin production and darker pigmentation [144, 145]. In addition to pH and tyrosinase, the melanocortin 1 receptor is one of the major factors controlling the pigment ratio.

#### **4.2. MC1R and pigment switch**

**Table 2.** The Fitzpatrick scale of skin complexion. MED represents the minimal erythematous dose (the least amount of

The melanocortin 1 receptor (MC1R) is a major determinant of skin pigmentation and UV sensitivity. UV exposure activates the MC1R, which directly controls not only the adaptive tanning response (UV-induced pigmentation) but regulates melanocyte DNA repair and, thus,

UV required to cause a sunburn).

the mutagenic risk as well.

**4. Pigmentation and MC1R**

**HO**

**HO HO N H**

**COOH NH2 Tyrosine**

16 Melanoma – Current Clinical Management and Future Therapeutics

**COOH**

with poorer UV-blocking properties is made when MC1R is inactive.

**DOPAquinone**

**Melanocortin 1 Receptor**

**COOH NH2**

Eumelanin Pheomelanin

**Figure 7.** Synthesis of eumelanin and pheomelanin is regulated by the melanocortin 1 receptor (MC1R). Eumelanin, a dark brown/black pigment with excellent UV protective properties, is produced when melanocytes are stimulated through MC1R signaling and cytoplasmic cAMP levels are increased. In contrast, pheomelanin, a sulfated pigment

**HO HO**

**Albinism (OCA1)**

**Cysteine**

**COOH NH2 S H2N COOH**

**O O**

**(MC1R) cAMP cAMP cAMP** *(no cAMP)*

**Tyrosinase**

The melanocortin 1 receptor (MC1R) is one of the major proteins controlling the switch between the production of eumelanin and pheomelanin, and therefore is a one of the major control points of pigment production. Increased activation of MC1R by melanotropic hor‐ mones leads to an increase in tyrosinase expression and eumelanin production [146]. MC1R is a G-coupled protein receptor that is activated by alpha-melanocyte stimulating hormone (α-MSH) leading to the activation of adenylate cyclase and an accumulation of cAMP. cAMP promotes two pathways: 1) it activates protein kinase A (PKA) and 2) it up-regulates cAMP responsible binding element (CREB) and microphthalmia transcription factor (MITF); these factors ultimately cause an increased expression of enzymes involved in pigment production [136, 140, 147-149]. MC1R is a highly polymorphic protein with over 100 variants reported [150-152]. Five specific variants, D84E, R142H, R151C, R160W, and D294H, are associated with a decrease in pigment production and the red hair/fair skin phenotype [153-155]. These individuals are more susceptible to melanoma due to a decrease in eumelanin production coupled with inefficient DNA repair; this latter point suggests that MC1R plays a role not only in pigment production, but also in nucleotide excision repair [156, 157].

Studies using agouti signaling protein (ASIP) confirm the role of MC1R in the production of eumelanin. ASIP is a competitive antagonist of MSH and binds to MC1R causing an increase in the production of pheomelanin [158]. The effects of ASIP on melanocyte pigment production require a functional MC1R [158, 159]. The reduction in eumelanogenesis and increase in pheomelanogensis accompanied by ASIP signaling is only partially due to inhibition of MSH binding to MC1R. Binding of ASIP to MC1R causes a decrease in tyrosinase activity as well as tyrosinase related protein 1 and 2 protein levels, thus promoting pheomelanin synthesis [158-161]. ASIP also signals through a cAMP independent pathway via attractin and mahoguin to influence MC1R signaling and increase pheomelanin levels [162].

#### **4.3. MC1R and adaptive pigmentation**

The inability to have an adequate adaptive tanning response is a major risk factor for the development of melanoma. As explained above, the tanning response increases the produc‐ tion of pigment in the skin and serves as a protective barrier from the damaging effects of UV radiation. Adaptive pigmentation is dependent upon MC1R signaling [163]. UV radiation promotes cellular damage in keratinocytes, activating the damage response protein p53. p53 activation increases the transcription of proopiomelanocortin, which is processed and cleaved to MSH [164]. MSH is secreted from the keratinocytes and diffuses to the melanocyte mem‐ brane where it binds to and activates MC1R promoting the synthesis of eumelanin [146, 165]. IndividualswithdefectiveMC1Rsignaling,whetherfrominabilityofMC1Rtobindtoitsligands or from an inert response upon binding, cannot increase their pigment production following exposure to UV radiation and instead are highly susceptible to burning [163] (Figure 8).

**Figure 8.** Cutaneous response to UV radiation. UV radiation induces the secretion of MSH and agouti signaling pro‐ tein. MSH binds to MC1R and activates adenylyl cyclase (AC) which leads to the accumulation of cAMP. cAMP signal‐ ing to promote pathways responsible for cutaneous UV protection. Agouti signaling protein binds to MC1R and prevents activation of adenylyl cyclase inhibiting the adaptive tanning pathways.

Current research is investigating pharmacological methods to increase the tanning response in order to prevent damage, particularly in those individuals who have defective MC1R signaling. Forskolin is an activator of adenylyl cyclase and functions downstream of MC1R leading to an increase in the production of cAMP. Forskolin applied to a transgenic mouse model with humanized skin promoted the synthesis of eumelanin and adaptive pigmentation [166]. Phosphodiesterase inhibitors including rolipram prevent cAMP degradation and is hypothesized to have similar effects to forskolin. Rolipram is currently an FDA-approved drug and may have clinical applications for this condition.

## **5. Conclusion**

tyrosinase related protein 1 and 2 protein levels, thus promoting pheomelanin synthesis [158-161]. ASIP also signals through a cAMP independent pathway via attractin and mahoguin

The inability to have an adequate adaptive tanning response is a major risk factor for the development of melanoma. As explained above, the tanning response increases the produc‐ tion of pigment in the skin and serves as a protective barrier from the damaging effects of UV radiation. Adaptive pigmentation is dependent upon MC1R signaling [163]. UV radiation promotes cellular damage in keratinocytes, activating the damage response protein p53. p53 activation increases the transcription of proopiomelanocortin, which is processed and cleaved to MSH [164]. MSH is secreted from the keratinocytes and diffuses to the melanocyte mem‐ brane where it binds to and activates MC1R promoting the synthesis of eumelanin [146, 165]. IndividualswithdefectiveMC1Rsignaling,whetherfrominabilityofMC1Rtobindtoitsligands or from an inert response upon binding, cannot increase their pigment production following exposure to UV radiation and instead are highly susceptible to burning [163] (Figure 8).

**Figure 8.** Cutaneous response to UV radiation. UV radiation induces the secretion of MSH and agouti signaling pro‐ tein. MSH binds to MC1R and activates adenylyl cyclase (AC) which leads to the accumulation of cAMP. cAMP signal‐ ing to promote pathways responsible for cutaneous UV protection. Agouti signaling protein binds to MC1R and

prevents activation of adenylyl cyclase inhibiting the adaptive tanning pathways.

to influence MC1R signaling and increase pheomelanin levels [162].

**4.3. MC1R and adaptive pigmentation**

18 Melanoma – Current Clinical Management and Future Therapeutics

The incidence of melanoma has dramatically increased throughout the past century. Although the cause for the increase is unknown, it is clear there are a number of environmental and genetic factors responsible for melanoma risk. The major environmental risk factor is exposure to UV radiation via ambient sunlight or artificial tanning beds. The intensity of the ambient sunlight varies with geography throughout the world. Countries located on latitudes closer to the equator have a higher incidence rate compared to countries located further away from the equator. Exposure to UV radiation, however, does not entirely explain the increase in mela‐ noma diagnoses. Other environmental factors also play a role including certain medications (PUVA, NBLP) and exposure to heavy metals. Genetic factors also play a major role in melanoma risk. As melanoma often originates from pre-existing nevi, the presence of a large number of nevi, nevi with large diameters, or the presence of dysplastic nevi increase the risk. A past medical history of cutaneous or non-cutaneous cancer also increases the risk of subsequently developing melanoma. Although there are many intrinsic factors which play a role in determining one's risk of developing melanoma, the most important is the ability to produce pigment. Eumelanin protects the skin from UV induced damage. Individuals with fair skin and a low Fitzpatrick phototype are highly susceptible to melanoma. A subset of individuals with fair skin also has defective MC1R signaling and is unable to promote the adaptive tanning pathway. Clearly, much is known about the risk factors for developing melanoma, and hopefully as we better understand the pathogenesis of the disease, we will develop therapeutics and strategies to prevent melanoma from occurring.

## **Acknowledgements**

We are indebted to Dr. Catherine Anthony of the Markey Cancer Center Research Communi‐ cations Office for useful edits. This work was supported by the following NIH grants: R01 CA131075, UL1TR000117, ES07266, T32 ES007266, and T32CA165990. We also thank the Drury Pediatric Research Endowed Chair Fund, Wendy Will Case Cancer Research Fund, Markey Cancer Foundation, Children's Miracle Network, and Jennifer and David Dickens Melanoma Research Foundation.

### **Author details**

Erin M. Wolf Horrell, Kalin Wilson and John A. D'Orazio\*

\*Address all correspondence to: jdorazio@uky.edu.

University of Kentucky College of Medicine, Markey Cancer Center, Department of Pediatrics, the Graduate Center for Toxicology and the Department of Physiology, Lexington, USA

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## **Acral Melanoma — A Distinct Molecular and Clinical Subtype**

R. Barra-Martínez, N.E. Herrera-González,

F. Fernández –Ramírez and L.A. Torres

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59093

## **1. Introduction**

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Skin cancer represents one-third of all cancers that are diagnosed every year worldwide. From all types of skin cancer, cutaneous melanoma is the least frequent among this group of malignant tumors, it is however, the most highly invasive and metastatic tumor. It has also shown an important growth of around 150% in its appearance since 1971, having an estimation of 76,250 new cases diagnosed in the USA in 2012. In 1971 there was only 1 case in 600 white people. In the period of 1992 to 2004, its annual growth was of 3.1%. This led to an estimated incidence of 1 in every 90 white people by 2000. By 2010 it was 1 in every 50, for people above 50 years old in the USA. Its epidemiological importance lies in its high mortality rate, since it is the cause of 90% of deaths for skin cancer and it is potentially the most dangerous form of skin tumor [1]. The outlook for patients with advanced melanoma is often fatal due to a lack of effective treatments.

In Mexico, melanoma incidence is around 1.7 per 100 000 people, and the Histopathologic Record of Malignancies in 2001 reported that it is the second most frequent. Women are the gender most affected by Melanoma (1.6/1). The median age of presentation in this country is 54 years and in 77% of the cases there is no relation to sun exposure. On the other hand, it is important to point out that there is a lack of formal registration of neoplasias in health institutions of our nation [1, 2, 3].

There are four clinicopathological subtypes that Melanoma presents, which usually are correlated to ethnic differences and to exposition to UV radiation: Superficial spreading melanoma (SSM), Lentigo maligna melanoma (LMM), Nodular melanoma (NM) and Acral lentiginous melanoma (ALM) [1, 4]. SSM and LMM, are the most common among Caucasians

© 2015 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.

and their presence has been directly related to sun exposure. Nodular melanoma is less frequent (10%) than SSM (70%), but both melanomas share pigmentary characteristics in patients. It is probable, however, that these two types of melanoma have a direct relation with sun exposure. However, this risk factor has not been proven in NM, given that this type can be found in any place on the body, not just in skin areas frequently exposed to sun light.

The most frequent subtypes of Melanoma in our population are, NM and ALM [4, 5] (Figure 1). Studies supporting the hypothesis that ALM may be a biological and genetically different subtype, will be mentioned in the following sections. Its distinct development pattern suggests that it may possess molecular and cellular uniqueness.

This may be because of different genetic alterations that control the transformation of mela‐ nocytes to acral melanoma, leading to the need for a specific-therapeutic target towards this subtype of melanoma with a high rate among Mexicans and Latin-Americans.

**Figure 1.** An indolent ALM subtype on the foot of a 58-year-old male patient, with 8 months of evolution. Nodular melanoma (NM) and Acral Lentiginous Melanoma (ALM) are the most frequent subtypes of Melanoma in our popula‐ tion. The lower extremities (mainly on the feet) are the most common anatomic locations reported for ALM.

## **2. Risk factors**

Several studies have identified certain variables associated with an increased risk of develop‐ ing melanoma: European ancestry, phenotype, UV radiation exposure, personal or family history of malignant melanoma, and molecular alterations are some of the most relevant [6]. Genetic alterations, will receive particularly emphasis later in this review. It is important to mention, that the risk factors traditionally associated with melanoma are related principally to Caucasians, where most clinical and basic studies are carried out. However, there are not enough studies supporting the predisposition of such factors for the subtypes of melanoma occuring in Mexican or Latin-American population.

Yamaguchi et al, reported that the risk factors traditionally described such as UV expo‐ sure, have a small influence in the pathogenesis of dark skin population. It has been postulated that UV radiation plays a smaller role in the pathogenesis of melanoma in the darker-skinned population. This is due to the fact that with an increase in melanin content, the larger melanosomes (in darker skin), absorb and scatter more energy than the smaller ones in lighter skin [7].

## **3. Sun exposure**

and their presence has been directly related to sun exposure. Nodular melanoma is less frequent (10%) than SSM (70%), but both melanomas share pigmentary characteristics in patients. It is probable, however, that these two types of melanoma have a direct relation with sun exposure. However, this risk factor has not been proven in NM, given that this type can be found in any place on the body, not just in skin areas frequently exposed to sun light.

The most frequent subtypes of Melanoma in our population are, NM and ALM [4, 5] (Figure 1). Studies supporting the hypothesis that ALM may be a biological and genetically different subtype, will be mentioned in the following sections. Its distinct development pattern suggests

This may be because of different genetic alterations that control the transformation of mela‐ nocytes to acral melanoma, leading to the need for a specific-therapeutic target towards this

**Figure 1.** An indolent ALM subtype on the foot of a 58-year-old male patient, with 8 months of evolution. Nodular melanoma (NM) and Acral Lentiginous Melanoma (ALM) are the most frequent subtypes of Melanoma in our popula‐

Several studies have identified certain variables associated with an increased risk of develop‐ ing melanoma: European ancestry, phenotype, UV radiation exposure, personal or family history of malignant melanoma, and molecular alterations are some of the most relevant [6]. Genetic alterations, will receive particularly emphasis later in this review. It is important to

tion. The lower extremities (mainly on the feet) are the most common anatomic locations reported for ALM.

**2. Risk factors**

subtype of melanoma with a high rate among Mexicans and Latin-Americans.

that it may possess molecular and cellular uniqueness.

32 Melanoma – Current Clinical Management and Future Therapeutics

Although various phenotypic characteristics enhance or reduce the risk of developing melanoma, sun exposure has been reported as the main cause of SSM, LMM and NM subtypes. The incidence of this disease is much higher in people who tend to burn rather than tan. (8)

UVA rays constitute 90% of solar radiation. However, UVB rays are greater risks for those who traditionally work or have recreational and daily activities outside. Among those with SSM and LMM, 75% of the affected individuals are sunburned by UVB rays. However, ALM has not been related to this risk factor. In addition, it is known that excessive sun exposure at early ages (childhood and adolescence) has a negative outcome in DNA reparation. The most important exogenous factor for melanoma is UV radiation exposure, particularly intermittent exposition, and it is known as the only etiological agent that can be modified. The increase in sun exposure and the damage to higher levels of the atmosphere because of contamination has resulted in an increase of the amount of radiation [9,10,11].

Sun exposure increases the expression of α-melanocyte stimulating hormone (α-MSH) and of the peptides of pro-opiomelanocortin in the skin. Melanocyte- stimulating hormone belongs to a group called the melanocortins. α-MSH is the most important melanocortin for pigmen‐ tation and shows a high affinity for melanocyte stimulating hormone receptor (MSHR). Once α-MSH binds to MSHR, cellular cAMP is activated, as well as other signal transduction pathways. The final result is the production of eumelanin.

Eumelanin, the primary pigment that colors the skin, hair and eyes in humans, also protects the body from UV and other hazardous radiations that can damage skin cells. When eumelanin has been crystalized, it forms both a geometric and a chemical disorder at the same time. It turns out that both kinds of disorders, may play a complementary role in producing eumela‐ nin's broadband absorption. The tiny crystals of eumelanin have a chemically ordered state with intrinsic randomness: the orientations of the stacked molecules are arbitrary and the sizes of the crystals are different. That combination of order and disorder contributes to eumelanin's broadband absorption (12). Recently, it was reported that eumelanin is a naturally existing nanocomposite that has very critical macroscopic properties as a result of its nanostructure properties. All eumelanin molecules share very similar chemistry, although there are more than 100 variations. It has been proposed that the small variations from one molecule to another may contribute to the disorder that broadens the ability to absorb UV light (13).

Additionally, different alleles for melanocortin 1 receptor (MC1R) gene have been identified. The subtypes related to loss of function reduce the production of cAMP after stimulation of α-MSH, and thus increase the expression of pheomelanin. This is observed in individuals of light skin, increasing the risk of developing melanoma by the formation of free radicals after UV ray exposure.

Recently, a study was performed in 789 patients with diagnosis of melanoma. In order to characterize these patients according to their levels of sun exposure, three groups were established in the study: intermittent, chronic, and absence of exposure [14]. The vast majority of patients were in the first group. These melanomas were present in skin locations exposed to sun radiation in an intermittent manner. SSM and NM belong to this group. Multiple common and atypical nevi may constitute a marker of risk of these melanomas. The second group was formed by patients with melanomas in skin areas with chronic sun exposure, showing all the damage and premise typical of this location. LMM belongs to this group. Finally, the third group corresponds to melanomas present in skin areas with no sun exposure, usually diagnosed in late stages. This group includes ALM and mucosa melanoma, which usually are thicker at diagnosis and have worse prognosis [15, 16, 17]

## **4. Acral lentiginous melanoma**

Acral lentiginous melanoma (ALM) distinguishes itself from the other subtypes for many characteristics, mainly histological and clinical-prognostic. A huge controversy regarding the cause of its worse prognosis, has been going on since its description.

ALM usually occurs on palmoplantar or subungual areas (Fig 2), lacking hair. Similar to NM, it is very aggressive when its proliferative vertical phase develops. It is more commonly found in individuals with dark skin (35-60%), oriental population and Afro-Caribbean. Considering its localization, it is thought to be UV-protected by the thickened stratum corneum and the nail matrix. ALM is rare in Caucasian populations (1-5%), but has a higher incidence among our population and Latin-Americans with dark skin(8, 9, 18). However, for individuals with lighter skin in our population, SSM is the most frequent.

According to the data of The National Cancer Institute Surveillance, Epidemiology and End Results, ALM is the least frequent histological subtype in the USA, with a 2-3% frequency. However, it shows a higher percentage among afroamerican people and Asians.

Reed described for the first time ALM as pigmented lesions of the extremities, mainly of the plantar and palmar regions, that were characterized by a phase of lentiginous (radial) growth, which evolved in months or years to the invasive vertical phase.(19)

Arrington was the first to notice that this type of melanoma was more prevalent among the African American population and that their prognosis was less favorable due to being diagnosed at an advanced stage. Yet, it was not documented by the SEER as a different histological subtype until 1986 [20]. Usually, ALM is not associated with nevi, family history or gene susceptibility known to melanoma. In its great majority, ALM are diagnosed as thick melanomas which in most cases are usually presented in their late phase, with a worse prognosis and a lower survival rate than the most common subtypes of melanoma associated with chronic sun exposure [20].

properties. All eumelanin molecules share very similar chemistry, although there are more than 100 variations. It has been proposed that the small variations from one molecule to another

Additionally, different alleles for melanocortin 1 receptor (MC1R) gene have been identified. The subtypes related to loss of function reduce the production of cAMP after stimulation of α-MSH, and thus increase the expression of pheomelanin. This is observed in individuals of light skin, increasing the risk of developing melanoma by the formation of free radicals after

Recently, a study was performed in 789 patients with diagnosis of melanoma. In order to characterize these patients according to their levels of sun exposure, three groups were established in the study: intermittent, chronic, and absence of exposure [14]. The vast majority of patients were in the first group. These melanomas were present in skin locations exposed to sun radiation in an intermittent manner. SSM and NM belong to this group. Multiple common and atypical nevi may constitute a marker of risk of these melanomas. The second group was formed by patients with melanomas in skin areas with chronic sun exposure, showing all the damage and premise typical of this location. LMM belongs to this group. Finally, the third group corresponds to melanomas present in skin areas with no sun exposure, usually diagnosed in late stages. This group includes ALM and mucosa melanoma, which

Acral lentiginous melanoma (ALM) distinguishes itself from the other subtypes for many characteristics, mainly histological and clinical-prognostic. A huge controversy regarding the

ALM usually occurs on palmoplantar or subungual areas (Fig 2), lacking hair. Similar to NM, it is very aggressive when its proliferative vertical phase develops. It is more commonly found in individuals with dark skin (35-60%), oriental population and Afro-Caribbean. Considering its localization, it is thought to be UV-protected by the thickened stratum corneum and the nail matrix. ALM is rare in Caucasian populations (1-5%), but has a higher incidence among our population and Latin-Americans with dark skin(8, 9, 18). However, for individuals with

According to the data of The National Cancer Institute Surveillance, Epidemiology and End Results, ALM is the least frequent histological subtype in the USA, with a 2-3% frequency.

Reed described for the first time ALM as pigmented lesions of the extremities, mainly of the plantar and palmar regions, that were characterized by a phase of lentiginous (radial) growth,

Arrington was the first to notice that this type of melanoma was more prevalent among the African American population and that their prognosis was less favorable due to being

However, it shows a higher percentage among afroamerican people and Asians.

which evolved in months or years to the invasive vertical phase.(19)

may contribute to the disorder that broadens the ability to absorb UV light (13).

34 Melanoma – Current Clinical Management and Future Therapeutics

usually are thicker at diagnosis and have worse prognosis [15, 16, 17]

cause of its worse prognosis, has been going on since its description.

lighter skin in our population, SSM is the most frequent.

**4. Acral lentiginous melanoma**

UV ray exposure.

Pereda et al, reported the Clinical presentation of ALM in patients from Spain. In seventeen of them, ALM was found on a foot and in six on a hand. Four ALMs of the hand were subungueal. Most of the foot ALMs were located on the sole (twelve cases) [22].

**Figure 2.** A subungueal tumor of a 71-year-old male patient with an advanced ALM (thick Breslow), on the thumb of the left hand. ALM is the most prevalent subtype in Mexicans and LatinAmericans. It is usually found on hands and feet: palms and soles, wrists and heels, and under the nails. ALM and NM are usually diagnosed in later stages, unlike SSM and LMM. The most affected sites are by far the feet and nails. Initially, they may be misdiagnosed as a mole, an ulcer, an abscess, a wart, a nail bed dystrophy or as the result of a trauma.

The most frequent location of ALM is in the feet (Figure 1), mainly the plantar region and the first finger, as in subungual areas in the majority of non caucasians racial groups. This has led to the conclusion that trauma may be an important factor of ALM, and not sun exposure [22]. Even when the superficial region of the palms and plantar region are similar, in the plantar region there is a continuous exposure to pressure, friction, maceration and irritation. Never‐ theless, there are counterarguments towards this theory, including that the hands are more exposed to UV radiation and acute trauma. In addition, it has not been possible to prove changes in the incidence of plantar melanoma when some African tribes began urbanization and the use of shoes.

Another important factor in the predominance of plantar ALM, is the fact that there is a 50% greater amount of melanocytes there than in the palms [23, 24]. The research of Ghadially proposed that trauma during less than 12 months, did not increase the risk of melanoma in the Xiphophorus fish model [25]. Troyanova [26] described mechanical trauma on previous pigmented skin lesions as a risk factor for the formation of melanoma and remarks that trauma has to be carefully checked. Recently, an amelanotic subungueal melanoma arising after trauma was reported by Rangwala [27]. Melanomas formed on burns scars and tattoos have also been frequently publishedin recent years.Of 687 Chi‐ nese melanoma patients, 15.2% showed a strong association between trauma in the extremities and melanoma [28].

Clinical experience coming from medical attention given to patients of melanoma treated in two of the major oncological centers of our nation, shows that AM occurs in no photo exposed areas [5], mainly in lower extremities (more than 80% of the cases) and in subungual regions (Figure 3). This information has been reported by the Mixed Tumor Unit from the Oncologic Service (General Hospital of Mexico) and the Dermatologic Center "Ladislao de La Pascua", Mexico City [18]

**Figure 3.** ALM on the foot of a 61-year-old male patient, with indolent growth. The Oncology Unit of the General Hos‐ pital of Mexico reports that more than 50% of ALMs are presented on the lower extremities (mainly on the soles and subungueal areas. Being the toe by far, the most affected).

#### Acral Melanoma — A Distinct Molecular and Clinical Subtype http://dx.doi.org/10.5772/59093 37

region there is a continuous exposure to pressure, friction, maceration and irritation. Never‐ theless, there are counterarguments towards this theory, including that the hands are more exposed to UV radiation and acute trauma. In addition, it has not been possible to prove changes in the incidence of plantar melanoma when some African tribes began urbanization

Another important factor in the predominance of plantar ALM, is the fact that there is a 50% greater amount of melanocytes there than in the palms [23, 24]. The research of Ghadially proposed that trauma during less than 12 months, did not increase the risk of melanoma in the Xiphophorus fish model [25]. Troyanova [26] described mechanical trauma on previous pigmented skin lesions as a risk factor for the formation of melanoma and remarks that trauma has to be carefully checked. Recently, an amelanotic subungueal melanoma arising after trauma was reported by Rangwala [27]. Melanomas formed on burns scars and tattoos have also been frequently publishedin recent years.Of 687 Chi‐ nese melanoma patients, 15.2% showed a strong association between trauma in the

Clinical experience coming from medical attention given to patients of melanoma treated in two of the major oncological centers of our nation, shows that AM occurs in no photo exposed areas [5], mainly in lower extremities (more than 80% of the cases) and in subungual regions (Figure 3). This information has been reported by the Mixed Tumor Unit from the Oncologic Service (General Hospital of Mexico) and the Dermatologic Center "Ladislao de La Pascua",

**Figure 3.** ALM on the foot of a 61-year-old male patient, with indolent growth. The Oncology Unit of the General Hos‐ pital of Mexico reports that more than 50% of ALMs are presented on the lower extremities (mainly on the soles and

and the use of shoes.

36 Melanoma – Current Clinical Management and Future Therapeutics

extremities and melanoma [28].

subungueal areas. Being the toe by far, the most affected).

Mexico City [18]

**Figure 4.** A subungueal tumor of a 54-year-old male patient with an advanced amelanic melanoma. ALMs are usually diagnosed in the later stages unlike SSM and LMM. It was misdiagnosed as a bleeding ulcer of progressive growth.

**Figure 5.** A plantar tumor of a 65-year-old male patient. The lower extremity is the most common anatomic location reported for mexicans. Initially this case was diagnosed as a pigmented lesion of a fast vertical phase progression.

### **5. Molecular alterations in melanoma**

Melanoma arises through a complex process of cellular mutations and a loss of keratinocyte control over growth and differentiation. As malignant melanoma progresses, it develops through interaction between dysfunctional melanocytes and the tumor microenvironment. This in turn, allows the formation of nevocyte nests at the demal-epidermal junction and is accompanied with changes in both, keratinocytes and local adhesion molecules (29). The progression from healthy melanocyte to melanoma occurs through mutations within the tumor and through alterations of the cellular environment.

In the skin, tissue homeostasis is critical in cellular regulation and melanoma breaks this regulation through multiple processes. Defining intercellular molecular dialogues in human skin promises to provide key information about the transformation of melanocyte to melanoma. A great number of genes and proteins have been reported to play an essential role in this transformation. The most important are: B-RAF, c-KIT, PTEN, p16, p53, cyclin1, ARF, K-RAS (30).

#### **6. Familial melanoma**

It has been reported by a meta-analysis that the presence of at least one-first degree relative with melanoma increases to double or more, the risk of developing this disease.

Many genetic studies in melanoma-prone families lead to the identification of *CDKN2A* as the main familial melanoma gene. This gene is located at the chromosome 9p21 region. *CDKN2A* encodes two different proteins: INK4A (p16) and ARF (p14). In order to pro‐ duce these two proteins, the use of alternative promoters and different first exons is necessary. Exon 1α is used for INK4A and 1β for ARF. The second exons of the two transcripts are translated in distinct reading frames, encoding two completely different proteins (with no amino acid homology). Nevertheless, both proteins share potent anticanc‐ er activities. *INK4A* inhibits the G1 cyclin-dependent kinases (CDKs) 4/6, which phosphor‐ ylate and inactivates the retinoblastoma protein (RB). After that, the S-phase is allowed to occur. Loss of INK4A function promotes RB inactivation resulting in cell cycle progres‐ sion. *ARF* (Alternative Reading Frame*)* inhibits MDM2 mediated ubiquitination and degradation of p53. Loss of ARF, inactivates p53. Alterations of p16 are associated to familial melanoma in 20% and in 40% to sporadic melanoma [31].

#### **7. Melanomas with intermitent sun exposure**

As it was mentioned earlier, more than 90% of melanomas are diagnosed in white and light skinned populations. The majority of the international studies have addressed these popula‐ tions, since they are at higher risk of developing melanoma. Thus, most reports are drawn from data of white and light skinned populations. The main alterations reported in SSM and NM, are B-RAF and N-RAS mutations.

#### **7.1. BRAF**

**5. Molecular alterations in melanoma**

38 Melanoma – Current Clinical Management and Future Therapeutics

ARF, K-RAS (30).

**6. Familial melanoma**

tumor and through alterations of the cellular environment.

melanoma in 20% and in 40% to sporadic melanoma [31].

**7. Melanomas with intermitent sun exposure**

Melanoma arises through a complex process of cellular mutations and a loss of keratinocyte control over growth and differentiation. As malignant melanoma progresses, it develops through interaction between dysfunctional melanocytes and the tumor microenvironment. This in turn, allows the formation of nevocyte nests at the demal-epidermal junction and is accompanied with changes in both, keratinocytes and local adhesion molecules (29). The progression from healthy melanocyte to melanoma occurs through mutations within the

In the skin, tissue homeostasis is critical in cellular regulation and melanoma breaks this regulation through multiple processes. Defining intercellular molecular dialogues in human skin promises to provide key information about the transformation of melanocyte to melanoma. A great number of genes and proteins have been reported to play an essential role in this transformation. The most important are: B-RAF, c-KIT, PTEN, p16, p53, cyclin1,

It has been reported by a meta-analysis that the presence of at least one-first degree relative

Many genetic studies in melanoma-prone families lead to the identification of *CDKN2A* as the main familial melanoma gene. This gene is located at the chromosome 9p21 region. *CDKN2A* encodes two different proteins: INK4A (p16) and ARF (p14). In order to pro‐ duce these two proteins, the use of alternative promoters and different first exons is necessary. Exon 1α is used for INK4A and 1β for ARF. The second exons of the two transcripts are translated in distinct reading frames, encoding two completely different proteins (with no amino acid homology). Nevertheless, both proteins share potent anticanc‐ er activities. *INK4A* inhibits the G1 cyclin-dependent kinases (CDKs) 4/6, which phosphor‐ ylate and inactivates the retinoblastoma protein (RB). After that, the S-phase is allowed to occur. Loss of INK4A function promotes RB inactivation resulting in cell cycle progres‐ sion. *ARF* (Alternative Reading Frame*)* inhibits MDM2 mediated ubiquitination and degradation of p53. Loss of ARF, inactivates p53. Alterations of p16 are associated to familial

As it was mentioned earlier, more than 90% of melanomas are diagnosed in white and light skinned populations. The majority of the international studies have addressed these popula‐ tions, since they are at higher risk of developing melanoma. Thus, most reports are drawn from

with melanoma increases to double or more, the risk of developing this disease.

It is a tyrosine and threonine kinase that participates in the signal transduction known as Ras/ Raf/MEK/ERK/MAP kinase. *BRAF* functions to regulate the MAPK/ERK pathway, which is conserved in all eukaryotes. This pathway acts as a signal transducer between the extracellular environment and the nucleus, activating downstream transcription factors to induce a range of biochemical processes including growth and differentiation, proliferation and migration associated to the ability of tissue invading [32, 33, 34]. Through direct secuentiation of PCR products, three substitutions of simple bases have been demonstrated. The most prevalent (95% of the cases) is constituted by the transversion of T1799A, occurring on exon 15 of *BRAF*, and causes the substitution of the amino acid valine for glutamic acid in position 599 (V599E). Such position has recently been modified to V600E.

Other *BRAF* mutations that cause carcinogenesis exist, such as K601E or those affecting exon 11, but they rarely occur in melanoma. A fusion of *BRAF* to *AKAP9*, by a paracentromeric inversion of the long arm of chromosome 7 has been observed in cases associated to ionized radiation exposure. This produces an oncogene, *AKAP9-BRAF*, which also results in a constitutive activation of the MAP kinase pathway [35, 36, 37].

Exon 15 BRAF mutations in melanoma were first reported in 2002. The number of reports of BRAF mutations in primary melanoma tissue increases continually. Varying prevalences in primary melanoma tissue have been detected, from 41% to 88% in SSM. These BRAF alterations alone, are not capable of demonstrating neither the immortalization of malignant cells, nor the progression of the disease further more than a nevus [37].

A pilot BRAF mutation study in ALM patients was carried out by collaboration between the Genetic Service of the General Hospital of Mexico and the Molecular Oncology Laboratory of the School of Medicine at the National Polytechnic Institute. DNA sequencing of seven ALM samples of Mexican patients (General Hospital of Mexico) showed no T1799A mutation in any of the studied samples (M. Sc. Thesis, 2012) [38].

#### **7.2. NRAS**

*RAS* are among the most frequently mutated oncogenes in human cancers. They show different mutation patterns and spectrums in all members: *NRAS*, *HRAS* and *KRAS.* The isoforms share a high degree of similarity, although each one displays preferential coupling to particular cancer types.

*RAS* play a fundamental role in the signaling pathway of MAP kinase. Extracellular signals such as hormones, cytokines, and various growth factors interact with their receptors to activate the small G-proteins of the RAS family. This pathway includes *BRAF* and it contributes to the control of cellular proliferation, particularly malignant cell proliferation. *RAS* also controls apoptosis through the PI3K-PTEN-Akt pathway.

SSM shows mutations almost exclusively in *NRAS*. A prevalence of 5% to 10% *NRAS* mutation has been reported. However for hereditary melanomas, the prevalence increases to 80%. Most cases of *NRAS* mutation are in codon 61. *NRAF* and *BRAF* mutations are mutually exclusive [39].

### **8. Melanoma with chronic sun exposure**

There are similarities between this type of melanoma and those without sun exposure. However, there are differences with ALM since they show much lower mutational characteristics.

*KIT* (CD117) encodes a tyrosine kinase receptor for stem cells factor and plays a key role in melanocyte development, migration and proliferation. *KIT* is found on chromosome 4 and may be altered in a variety of different types of cancers. It is mutated in 28% of LMM, 36% in ALM, and is absent in melanomas with intermittent sun exposure. KIT over expression during the vertical growth phase of melanoma development supports the hypothesis of its participa‐ tion in late stages of the disease. It is the target of several small molecules inhibitors such as imatinib, being a therapeutic target for this type of melanoma. Different roles of KIT among different types of melanoma, give additional evidence that each subtype can be considered different biologically and genetically [40].

## **9. Melanoma without sun exposure**

ALM and mucosal melanomas have an inverse correlation among mutations of *BRAF* and *NRAS*. The increase in the copy number of Cyclin D1, CDK4, KIT and ABCB5 has also been reported.

#### **9.1. CYCLIN D1**

It is an important regulator of transition of the cell cycle for phases G1/S. It also participates in the phosphorylation of RB protein by binding kinase 4, cyclin dependent. Many studies have revealed a highly organized sequence of events indispensable for promotion of continued proliferation. The decision to continue cell cycle progression occurs when cellular RAS induces the elevation of cyclin D1. These levels are maintained through G1 phase and are necessary for the initiation of S phase, thus resulting in immediately reduced cyclin D1 levels. One requirement for DNA synthesis, is the reduction of cyclin D1 to low levels during the S phase. This forces the cell when it enters G2 phase, to induce high cyclin D1 levels once more. Thus, cyclin D1 is proposed to be a switch activator in the regulation of continued cell cycle pro‐ gression [41, 42].

Cyclin D1 frequent amplification was reported in 44.4% of ALM by Sauter et al, Cyclin D1 was overexpressed in all cases with amplifications and in 20% of cases without amplification. Cyclin D1 may be an oncogene in melanoma and that targeting its expression can be thera‐ peutically useful in the future for ALM [42].

#### **9.2. CDK4**

SSM shows mutations almost exclusively in *NRAS*. A prevalence of 5% to 10% *NRAS* mutation has been reported. However for hereditary melanomas, the prevalence increases to 80%. Most cases of *NRAS* mutation are in codon 61. *NRAF* and *BRAF* mutations are

There are similarities between this type of melanoma and those without sun exposure. However, there are differences with ALM since they show much lower mutational

*KIT* (CD117) encodes a tyrosine kinase receptor for stem cells factor and plays a key role in melanocyte development, migration and proliferation. *KIT* is found on chromosome 4 and may be altered in a variety of different types of cancers. It is mutated in 28% of LMM, 36% in ALM, and is absent in melanomas with intermittent sun exposure. KIT over expression during the vertical growth phase of melanoma development supports the hypothesis of its participa‐ tion in late stages of the disease. It is the target of several small molecules inhibitors such as imatinib, being a therapeutic target for this type of melanoma. Different roles of KIT among different types of melanoma, give additional evidence that each subtype can be considered

ALM and mucosal melanomas have an inverse correlation among mutations of *BRAF* and *NRAS*. The increase in the copy number of Cyclin D1, CDK4, KIT and ABCB5 has also been

It is an important regulator of transition of the cell cycle for phases G1/S. It also participates in the phosphorylation of RB protein by binding kinase 4, cyclin dependent. Many studies have revealed a highly organized sequence of events indispensable for promotion of continued proliferation. The decision to continue cell cycle progression occurs when cellular RAS induces the elevation of cyclin D1. These levels are maintained through G1 phase and are necessary for the initiation of S phase, thus resulting in immediately reduced cyclin D1 levels. One requirement for DNA synthesis, is the reduction of cyclin D1 to low levels during the S phase. This forces the cell when it enters G2 phase, to induce high cyclin D1 levels once more. Thus, cyclin D1 is proposed to be a switch activator in the regulation of continued cell cycle pro‐

Cyclin D1 frequent amplification was reported in 44.4% of ALM by Sauter et al, Cyclin D1 was overexpressed in all cases with amplifications and in 20% of cases without amplification.

mutually exclusive [39].

characteristics.

reported.

**9.1. CYCLIN D1**

gression [41, 42].

**8. Melanoma with chronic sun exposure**

40 Melanoma – Current Clinical Management and Future Therapeutics

different biologically and genetically [40].

**9. Melanoma without sun exposure**

It is a union protein of cyclin D1, found in chromosome 12q14. During early stages of phase G1 of the cell cycle, Cyclin D binds to kinase dependent cyclin 4 or 6 (CDK4 or CDK6) and the resulting complex "frees the brake" that limits the progression towards late G1, going into phase S. The cyclin D-CDK4/6 complex releases a potent inhibitor of the cell cycle progression: the one formed by protein pRB and inactive transcription factors. The types of melanoma having an increase in the number of copies of CyclinD1 and CDK4 have less possibilities of responding to the therapeutic target used in BRAF, such as Vemurafenib.

#### **9.3. ABCB5**

Human ATP-binding cassette transporter, also known as P-glycoprotein, is related to the subfamily of multidrug resistant genes (MDR). The ABCB subfamily includes eleven members that have different expression patterns. Whereas the functions of several transport‐ ers of the ABCB family are not known, there is some information about ABCB5 in relation to its pattern of expression or its associated function [43]. It has been described by Frank et al [44, 45] described it and it was implicated in the regulation of progenitor cell fusion (located in chromosome 7p21-15). Their study used an enriched culture of human epider‐ mal melanocytes isolated from the foreskins of healthy donors and melanoma cell lines. They showed a higher and more intense ABCB5 expression in ALM than in SSM. Herrera-Gonzalez and her team [46], reported a 90% overexpression of ABCB5 in ALM samples of Mexican patients by using RT-PCR. A direct relationship between mRNA expression and the aggressiveness of ALM was also shown.

## **10. Conclusion**

In regard to the progression of human cancer, it is universally thought, that it develops from a single mutated cell, followed by malignant clonal expansion secondary to additional genomic and genetic alterations. As the malignant cells continue to acquire these alterations, tumor subclones with distinct phenotypic advantages (47) for its progression may be produced: for example invasion, proliferation, ability to colonize different organs, etc. In many cancers, regulation of specific signaling molecules also goes awry, affecting a host of other proteins and cellular processes.

In recent years, the histological and phenotypic characteristics of ALM combined with its high proportion among melanomas in Afro-Americans, Asian and Latin Americans, has confirmed the thought that this histological type of melanoma may differ biological and molecularly from its most common counterparts, recognized by its sun exposure and its greater frequency among Caucasians. While ALM is characterized by a high frequency of focal amplifications (mainly involving CCND1, CDK4) and deletions, the most common cutaneous melanomas exhibit few changes in the number of genetic copies. In a similar way, while *BRAF* and *NRAS* are mutated in 50% and 20% of the cases respectively, mutation in *KIT* (which codifies the tyrosine kinase receptor) seems to be absent in the most common cutaneous melanoma. However, BRAF mutations in ALM constitute approximately 16% of the cases reported by Curtin et al. [40, 41]

A systematic sequencing was used for the human genome sequence. New technologies allow sequencing of randomly generated DNA fragments from cancer genomes and thus detect alterations and copy number variation as well as base substitutions, to identify most somatic mutations in an individual cancer genome. Pleasance et al, sequenced the complete genome of the COLO-829 cell line, an immortal and cancer derived from a metastasis of a melanoma from a 43 year-old-male. In their study they identified 33,345 somatic base substitutions [48, 49]

The study of Turajlic et al. [50] is the first to characterize the mutational spectra in ALM. They used a whole genome sequencing to characterize punctual somatic mutations and structural variation in a primary acral melanoma and its lymph node metastasis. Evidence of transcrip‐ tion-coupled repair was suggested by the lower mutational rate in the transcribed regions and expressed genes. Primary melanoma and metastasis, at the level of global gene copy number alterations, loss of heterozygosity and single expressed genes are very similar[50].

These results may be controversial since their sequenced results were on the genome of only one patient. They cite that despite the perception that acral skin is sun protected, the dominant mutational signature reveals sun damage. We judge that there is no solid data regarding the presence of solar exposure on sites where ALM is developed.

Studies suggest that ungual matrix does not provide a complete protection against UV radiation and it has previously been suggested that UVB can penetrate the human nail [51]. There are substantial variations in the number and pattern of mutations in individual cancers reflecting different exposures, DNA repair defects and cellular origins [52, 53]. It is also possible that characteristics of different mutations are induced by the inefficiency of pyrimidine dimers repaired by excision of nucleotide due to the presence of a mutation in ERCC5 [54, 55].

Several studies support the hypothesis that ALM is a different genetic subtype and its inherent heterogeneity represents a challenge in the era of directed therapy [56].

## **Author details**

R. Barra-Martínez1 , N.E. Herrera-González2 , F. Fernández –Ramírez3 and L.A. Torres1

1 Oncology Unit of General Hospital of Mexico, Mexico

2 Molecular Oncology Laboratory. Postgraduate Section. Superior School of Medicine, IPN, Mexico

3 Genetic Unit of General Hospital of Mexico, Mexico

### **References**

exhibit few changes in the number of genetic copies. In a similar way, while *BRAF* and *NRAS* are mutated in 50% and 20% of the cases respectively, mutation in *KIT* (which codifies the tyrosine kinase receptor) seems to be absent in the most common cutaneous melanoma. However, BRAF mutations in ALM constitute approximately 16% of the cases reported by

A systematic sequencing was used for the human genome sequence. New technologies allow sequencing of randomly generated DNA fragments from cancer genomes and thus detect alterations and copy number variation as well as base substitutions, to identify most somatic mutations in an individual cancer genome. Pleasance et al, sequenced the complete genome of the COLO-829 cell line, an immortal and cancer derived from a metastasis of a melanoma from a 43 year-old-male. In their study they identified 33,345 somatic base substitutions [48, 49] The study of Turajlic et al. [50] is the first to characterize the mutational spectra in ALM. They used a whole genome sequencing to characterize punctual somatic mutations and structural variation in a primary acral melanoma and its lymph node metastasis. Evidence of transcrip‐ tion-coupled repair was suggested by the lower mutational rate in the transcribed regions and expressed genes. Primary melanoma and metastasis, at the level of global gene copy number

alterations, loss of heterozygosity and single expressed genes are very similar[50].

presence of solar exposure on sites where ALM is developed.

heterogeneity represents a challenge in the era of directed therapy [56].

, N.E. Herrera-González2

1 Oncology Unit of General Hospital of Mexico, Mexico

3 Genetic Unit of General Hospital of Mexico, Mexico

These results may be controversial since their sequenced results were on the genome of only one patient. They cite that despite the perception that acral skin is sun protected, the dominant mutational signature reveals sun damage. We judge that there is no solid data regarding the

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## **Signaling Pathways Altered During the Metastatic Progression of Melanoma**

Ana Carolina Monteiro, Mariana Toricelli and Miriam Galvonas Jasiulionis

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59747

## **1. Introduction**

Metastasis is one of the most important parameters that affect cancer patient's prognosis. It is associated with resistance to treatments, high recurrence rates and poor cancer patient survival rates [1]. Although melanoma is an aggressive type of skin cancer that carries significant morbidity and mortality, the prognosis for patients with localized tumor is quite good, the aggravation being the metastatic capacity of the tumor. When the process of metastasis takes place, the prognosis is generally very poor, with the metastatic melano‐ ma being rarely curable [2-3].

The establishment of metastatic lesions is a complex process and requires a series of sequential steps, each of which is rate limiting, and although substantial progress has been made in understanding the molecular mechanisms of metastasis, new data suggest this process is perhaps even more complicated than originally suspected [4]. Another issue in defining the factors involved in the melanoma metastasis is that melanoma is highly heterogeneous and for this its classification and staging is until today a challenge for specialists [1]. Because of that, there is still a lot unclear understanding about the progression of the disease, making it difficult to ensure which molecular pathways are involved in each step of the disease devel‐ opment. Therefore, for the development of new prognostic and therapeutic targets of mela‐ noma metastasis, it is important to elucidate which pathways are involved specifically in the progression of the localized tumor to its metastasis [2]. For that, a deep review of the literature, analysing carefully which pathologic classification each study considered, may clarify the molecular alterations involved specifically in the most aggressive step in melanoma progres‐ sion.

© 2015 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 eproduction in any medium, provided the original work is properly cited.

Signaling pathways, such as the canonical Wnt pathway, the c-kit receptor and the MITF transcription fator are among pathways found altered during melanoma [5]. The reactivation of melanocyte-specific programs seems to contribute, in combination with other oncogenic changes, with melanoma aggressiveness. Others pathways that are classically altered in melanoma metastasis are the MAPK and PI3K pathways, with gain-of-function mutations leading to melanoma metastasis progression [1].

Besides these pathways that are consistently involved in the metastasis of melanoma, there are some other potentially altered pathways in this process. For example, it is known that the tumor microenvironment has an important role in determining melanoma progression to metastasis [6], and in this scope the importance of metalloproteinases is being highlighted. In addition, numerous studies report modulation of the metastatic properties of melanoma cells by microRNAs, suggesting this as a common occurrence. MicroRNAs regulate tumor sup‐ pressor genes and oncogenes, each miRNA being able to regulate a great number of genes. Besides, due to its stable and lineage-specific expression nature, microRNAs are attractive candidate as biomarkers and therapeutic targets [7].

Although there are a great number of studies trying to elucidate the molecular mechanisms of melanoma progression, the process of melanoma metastasis is still unclear. Finding core mediators of different processes in metastasis is a formidable challenge, and may provide opportunities for developing new prevention and treatment strategies. This chapter will discuss basic concepts of malignant melanoma metastasis, focusing on the pathways that the literature indicate as consistently altered in this process and will describe how the host's environment influences the biological behavior of metastatic cells.

## **2. Melanoma metastasis**

Cutaneous melanoma is the most aggressive type of skin cancer, and the high mortality rates worldwide caused by this disease is due to its great ability to form metastases and resist to current therapies [8,9,10]. Genetic and epigenetic alterations contribute to the development of cells able to invade and metastasize [11]. These changes ensure the features that allow cells to modulate the microenvironment, and change their interactions with the extracellular matrix and other cells [8]. Although our knowledgement about the molecular pathogenesis of melanoma has increased, the molecular changes occuring during the malignant transformation of melanocytes are not very well described.

## **3. Adhesion molecules**

One of the main characteristics of metastatic melanoma is cell heterogeneity [10]. This characteristic provides the cell ability to invade and colonize different tissues [11]. Local invasion and metastasis processes are responsible for the morbidity and mortality associated with melanoma. The development of invasive cells occurs in the vertical growth phase (VGP) when melanoma cells are able to penetrate the basement membrane, grow in the dermis and metastasize [12]. Moreover, these cells have many cytogenetic abnormalities, suggesting a significant genomic instability [13]. The development of metastatic melanoma from primary VGP melanomas occurs when these cells dissociate from the primary tumor, migrate through the adjacent stroma and invade the lymphatic and/or blood vessels to form tumors at distant sites [13]. The invasion and migration of melanoma are related to changes in cell adhesion. Typically, cell adhesion controls cell migration, organization, organogenesis and tissue architecture [14]. Disturbances in adhesion contribute to tumor invasion, tumor-stroma interactions and signaling between tumor cells and normal cells [14]. There are numerous cell adhesion molecules, which are sub-grouped based on their structural similarity and are categorized as integrins, cadherins, immunoglobulin superfamily or selectins. The expression of these molecules is influenced by the environment, microenvironment and genetic/epigenetic factors [14]. Therefore, determining changes in the expression of these molecules during metastasis may help to define future therapeutic targets. Several adhesion molecules have been reported to play a role in melanoma progression.

Signaling pathways, such as the canonical Wnt pathway, the c-kit receptor and the MITF transcription fator are among pathways found altered during melanoma [5]. The reactivation of melanocyte-specific programs seems to contribute, in combination with other oncogenic changes, with melanoma aggressiveness. Others pathways that are classically altered in melanoma metastasis are the MAPK and PI3K pathways, with gain-of-function mutations

Besides these pathways that are consistently involved in the metastasis of melanoma, there are some other potentially altered pathways in this process. For example, it is known that the tumor microenvironment has an important role in determining melanoma progression to metastasis [6], and in this scope the importance of metalloproteinases is being highlighted. In addition, numerous studies report modulation of the metastatic properties of melanoma cells by microRNAs, suggesting this as a common occurrence. MicroRNAs regulate tumor sup‐ pressor genes and oncogenes, each miRNA being able to regulate a great number of genes. Besides, due to its stable and lineage-specific expression nature, microRNAs are attractive

Although there are a great number of studies trying to elucidate the molecular mechanisms of melanoma progression, the process of melanoma metastasis is still unclear. Finding core mediators of different processes in metastasis is a formidable challenge, and may provide opportunities for developing new prevention and treatment strategies. This chapter will discuss basic concepts of malignant melanoma metastasis, focusing on the pathways that the literature indicate as consistently altered in this process and will describe how the host's

Cutaneous melanoma is the most aggressive type of skin cancer, and the high mortality rates worldwide caused by this disease is due to its great ability to form metastases and resist to current therapies [8,9,10]. Genetic and epigenetic alterations contribute to the development of cells able to invade and metastasize [11]. These changes ensure the features that allow cells to modulate the microenvironment, and change their interactions with the extracellular matrix and other cells [8]. Although our knowledgement about the molecular pathogenesis of melanoma has increased, the molecular changes occuring during the malignant transformation

One of the main characteristics of metastatic melanoma is cell heterogeneity [10]. This characteristic provides the cell ability to invade and colonize different tissues [11]. Local invasion and metastasis processes are responsible for the morbidity and mortality associated with melanoma. The development of invasive cells occurs in the vertical growth phase (VGP)

leading to melanoma metastasis progression [1].

50 Melanoma – Current Clinical Management and Future Therapeutics

candidate as biomarkers and therapeutic targets [7].

**2. Melanoma metastasis**

**3. Adhesion molecules**

of melanocytes are not very well described.

environment influences the biological behavior of metastatic cells.

As previously stated, the extracellular matrix (ECM) surrounding the cell provides a physical support for the cell adhesion. The anchorage of cell in the ECM is not just structural, consid‐ ering that this binding stimulates signal transduction cascades that mediate signaling required for the proliferation, migration, differentiation and cell survival [15]. The type of apoptosis triggered by the loss of anchorage is called "*anoikis*". Currently, the role of anchorage in the cell survival is widely accepted and studied in numerous adherent cell types, such as fibroblasts, endothelial, bronchial epithelial, liver, intestine, prostate cells and in keratinocytes [16]. The acquisition of *anoikis*-resistant phenotype is one of the critical steps during tumor progression. In melanoma, we and others [17-19] demonstrated that malignant transformation is associated with resistance to *anoikis* [16]. One of the key molecules involved in *anoikis* are integrins. The physical connection between extracellular matrix and the cytoskeleton of actin is mainly mediated by receptors of the integrin family. Besides being involved in the interaction of the cell with the extracellular matrix, integrins are also responsible for signaling between the cell and its microenvironment. The integrin family is among the best-characterized adhesion molecules. Currently, 18 types of α subunits and 8 types of β subunits are known, which combine to form at least 24 integrins already described [20]. The expression pattern of integrins on cell surface causes the cell to fit perfectly into its microenvironment. In this regard, integrins have altered interactions with their microenvironment may give drastic consequences for cell fate as provide cell tend to lose their original adhesion, recognizing a different substrate and reconfigure it with characteristics that enable metastasize [21-22]. It is known that the increase and alteration of integrin expression is indicative of progression of melanoma [23]. Many integrins are found altered in metastatic melanoma (TABLE 1). Thus, a large number of studies have shown that expression of αvβ3 and αvβ1 integrins is related with malignant transfor‐ mation of melanocytes or melanoma progression [24-28]. αvβ3 integrin is an integrin expressed only in melanoma cells and not in benign melanocytes [24]. β3 integrins have also been associated with angiogenesis [29]. αvβ3 and αvβ1 integrins are expressed in metastatic melanoma and late melanoma compared with early melanoma and nevi [28-30]. The αvβ3 integrin is associated with melanoma progression, acting as a receptor for vitronectin, which self-regulates the expression of matrix metalloproteinase-2 (MMP-2] and increases invasive proteins [24-25]. Alpha-v integrin antibodies block the growth of human melanoma trans‐ plants in mice and a new inhibitor of αvβ3 integrin blocks *anoikis* and metastasis in human melanoma cell line M21[31-32].


**Table 1.** Main integrins found altered in metastatic melanomas

Immunoglobulin superfamily adhesion molecules are cell surface glycoproteins that express a variable number of loops in its extracellular domain. Most of these molecules has a trans‐ membrane domain but is linked to the cell surface only by a glycophosphatidilinositol anchor [35]. Heterophilic interactions with members of the immunoglobulin superfamily, integrins, cadherins and extracellular matrix components may occur, as well as homophilic interactions, which are essential in Ca2+-dependent cell adhesion [36-37]. The family of immunoglobulins has an important relevance in the pathophysiology of melanoma, among which we can highlight the CD54 (ICAM), CD66 (CEACAM), CD146 (MCAM or Mel-CAM) and CD166 (ALCAM). These molecules appear to be important in the melanoma progression, although many of their functions are still uncertain. Through literature review, it is possible to note their participation in advanced cases of melanoma and metastatic disease [14, 38]. Particularly, CD54 (ICAM) has been associated with melanoma progression and risk of metastasis [39]. Its expression is evident in melanoma when compared with nevi [26, 40-42]. The CD66 (CEA‐ CAM) is a glycoprotein surface molecule involved in intercellular adhesion and associated with diverse cellular functions that regulate growth and differentiation and play an important role in insulin homeostasis, vasculogenesis and immunomodulation. Furthermore, it implies in many intracellular signaling mediated processes involved in the growth and differentiation of tumor cells, performing thus a key role in the modulation of many types of cancer. A strong correlation between the expression of this molecule in primary tumors and subsequent development of metastatic disease was observed. An apparently gradual increase in CD66 expression in cutaneous melanocytic lesions in more advanced stages of neoplastic progression was observed, indicating that CD66 may play an important role in the development and progression of melanoma. Furthermore, this molecule interacts with integrins (especially with beta-3 subunit) and this interaction appears to be important in cell migration and metastasis [43]. The CD146 molecule was first identified as a cell adhesion molecule specific for melanoma and capable of providing homologous and heterologous interactions between neoplastic melanocytes and endothelial cells in a calcium-independent manner [14]. Also known as MELCAM, MCAM and MUC18 [44], CD146 plays a pro-migratory key role in the vascular system, normal development and tumor progression, displaying overexpression in many tumors, including melanoma, prostate cancer and cancer breast [45-47]. The expression of CD146 in melanocytes, nevi and melanoma cells from radial growth phase is environmentally regulated through direct contact cell-cell with keratinocytes, but the mechanisms of this regulation are not well-established [48]. Moreover, in melanoma progression, the expression of this molecule increases gradually and reaches its peak in metastatic disease [14, 20, 49]. It is reported that CD146 displays strictly correlation with cadherins and experimental situations where an increased expression of cadherins is stimulated, CD146 levels return to normal levels [50]. The CD166, also named ALCAM, was first identified on activated leukocytes, hemato‐ poietic stem cells and myeloid progenitors. Furthermore, it is possible to observe its expression in neuronal cells, mesenchymal stem cells, stromal cells from bone marrow, but also in cultured metastatic melanoma cells [51]. Like other adhesion molecules, CD166 shows homophilic and heterophilic interactions and has represented a marker of metastatic development similarly to beta-3 integrin, since its expression reaches peak levels in metastatic melanoma cells [52].

self-regulates the expression of matrix metalloproteinase-2 (MMP-2] and increases invasive proteins [24-25]. Alpha-v integrin antibodies block the growth of human melanoma trans‐ plants in mice and a new inhibitor of αvβ3 integrin blocks *anoikis* and metastasis in human

> **Integrins Reference** Alpha1 beta1 [26] Alpha2 beta1 [26] Alpha3 beta1 [25] Alpha4 beta1 [26] Alpha5 beta1 [33] Alpha6 beta1 [14, 26] Alpha7 beta1 [34] alphaV beta3 [14] alphaV beta5 [34]

Immunoglobulin superfamily adhesion molecules are cell surface glycoproteins that express a variable number of loops in its extracellular domain. Most of these molecules has a trans‐ membrane domain but is linked to the cell surface only by a glycophosphatidilinositol anchor [35]. Heterophilic interactions with members of the immunoglobulin superfamily, integrins, cadherins and extracellular matrix components may occur, as well as homophilic interactions, which are essential in Ca2+-dependent cell adhesion [36-37]. The family of immunoglobulins has an important relevance in the pathophysiology of melanoma, among which we can highlight the CD54 (ICAM), CD66 (CEACAM), CD146 (MCAM or Mel-CAM) and CD166 (ALCAM). These molecules appear to be important in the melanoma progression, although many of their functions are still uncertain. Through literature review, it is possible to note their participation in advanced cases of melanoma and metastatic disease [14, 38]. Particularly, CD54 (ICAM) has been associated with melanoma progression and risk of metastasis [39]. Its expression is evident in melanoma when compared with nevi [26, 40-42]. The CD66 (CEA‐ CAM) is a glycoprotein surface molecule involved in intercellular adhesion and associated with diverse cellular functions that regulate growth and differentiation and play an important role in insulin homeostasis, vasculogenesis and immunomodulation. Furthermore, it implies in many intracellular signaling mediated processes involved in the growth and differentiation of tumor cells, performing thus a key role in the modulation of many types of cancer. A strong correlation between the expression of this molecule in primary tumors and subsequent development of metastatic disease was observed. An apparently gradual increase in CD66 expression in cutaneous melanocytic lesions in more advanced stages of neoplastic progression was observed, indicating that CD66 may play an important role in the development and

melanoma cell line M21[31-32].

52 Melanoma – Current Clinical Management and Future Therapeutics

**Table 1.** Main integrins found altered in metastatic melanomas

Another major family of adhesion molecules found to be altered in metastatic melanoma is the cadherin family. Cadherins are calcium-dependent cell adhesion molecules with important functions in the formation and maintenance of normal tissue architecture [11]. Cadherins are a superfamily of at least 30 different molecules, whose expression is temporally and spatially controlled. Classic cadherins are divided into three subtypes: N (neural), E (epithelial) and P (placental) [53]. The E-cadherin molecule, which is expressed by epithelial cells, is the one most frequently altered in tumors. Different studies have shown that E-cadherin is frequently inactivated in the development of human carcinomas, including carcinomas of breast, colon, prostate, stomach, liver, esophagus, skin, kidney and lung and is associated with invasion and metastases formation in lymph nodes [53-54]. The inhibition of the E-cadherin function may occur by several mechanisms, including mutation or deletion of *CDH1* gene, chromosomal rearrangement or promoter hypermethylation [53]. In fact, deletions or hypermethylation of 16q22 region, where the E-cadherin gene is located, are common in carcinomas but, in melanoma cells, deletions, mutations and methylation of the E-cadherin gene are apparently not involved [54]. Three transcriptional factors, AP-2, Snail and SIP1, have recently been shown to be important in the transcriptional silencing of the E-cadherin gene in melanomas [55-57]. Loss of AP-2 expression in metastatic melanoma cells results in the deregulation of MCAM/ MUC18, c-Kit and E-cadherin expression, all of which are involved in melanoma progression [57]. In experimental models, inhibition of E-cadherin expression in carcinoma cells facilitates tumor invasion, while the reestablishment of the expression results in proliferation inhibition and invasion and metastasis reduction [58]. E-cadherin is partially responsible for the phe‐ nomenon of contact inhibition, a characteristic of normal epithelial cells, associated with the proliferation blockade when cells come into contact with each other [53]. This feature is essential for maintaining epithelia architecture. In animal models, functional loss of E-cadherin is associated with acceleration of tumor progression. Adenocarcinomas and metastatic lesions appear earlier in animals that do not express E-cadherin function [59]. These E-cadherin properties allowed classifying it as a metastasis suppressor molecule. Loss E-cadherin expression appears to be a critical step in the melanoma progression, allowing the tumor cells to be released from the epidermis and to invade the dermis [60]. The cadherin switch from Ecadherin to N-cadherin results in disassociation of melanoma cells from keratinocytes and promotes melanoma cell invasion through the dermis. The N-cadherin expression in melano‐ ma cells correlates with increased motility and invasion, suggesting that N-cadherin potenti‐ ates the interaction between tumor cells and stromal cells, including fibroblasts and endothelial cells [61]. Anti-N-cadherin antibodies can delay the trans-endothelial migration of melanoma cells and induce apoptosis of melanoma cells [61]. The E-cadherin cytoplasmic portion interacts with alpha-and ß-catenin. Besides the ß-catenin be part of E-cadherin adhesion complex, it plays an essential role as a mediator of the signal transduction pathway of Wnt/Wingless (glycoprotein that plays a role in embryogenesis), which activates the transcription factors LEF/ Tcf [53]. The transcription factors LEF/Tcf are responsible for controlling expression of genes encoding cyclin D1, MYC and metalloproteinases [54]. In a simplified manner, the cytoplasmic pool of ß-catenin can be considered regulatory elements of epithelial cells proliferation and invasion. In tissues where there is interaction between cells and formation of adherent junctions mediated by E-cadherin, ß-catenin molecules are recruited to the sub-membrane region. The lack of degradation mechanism or the functional loss of E-cadherin leads to the ßcatenin cytoplasmic accumulation and its translocation to the nucleus. The ß-catenin also plays a role in the control of proliferation and apoptosis and is also increased in some cancers. Data show that E-cadherin suppresses the growth of metastatic melanoma cells by inhibiting betacatenin signaling pathway/Wnt [54]. Therefore, these data suggest that E-cadherin may play an important role early in the metastatic cascade.

#### **4. Matrix metalloproteinases**

Proteolytic enzymes, by their ability to degrade ECM proteins, become important components in the process of tumor progression. With the sequencing of the human genome, more than 500 genes were identified as encoding proteases or protease-like proteins, with a large number being associated with the tumor process [62]. Among these, the matrix metalloproteinases (MMPs)-a group of 24 enzymes that degrade ECM components and the basal membrane [63] have been the focus of much research on cancer [63-64]. This family of glycoproteins is secreted as a latent pro-enzyme, activated by proteolysis of a conserved region present in the N-terminal domain and is divided into six groups depending on the type of substrate that degrades: collagenases, gelatinases, stromelysins and extracellular matrix metalloproteinases [62]. MMPs are regulated both at transcriptional and post-transcriptional level. These regulation mechanisms operate to ensure their coordinated expression [63]. MMPs activity occurs only where proteolysis is required. Cytokines and peptide factors, such as interleukin (IL)-4 and IL-10, growth factors (transforming growth factor (TGF-α), basic fibroblast growth factor (bFGF), and TGF-beta1, induce the expression of different members of MMPs family [65]. However, malignant tumors have strategies that avoid these regulatory mechanisms and provide proteolytic activity that accompanies cancer development and metastasis [65]. Increased MMP activity is associated with the stages of tumor invasion and metastasis and is frequently overexpressed in different cancers. Upon activation, MMP activity is modulated by common endopeptidase inhibitors, such as α2-macroglobulins (present in plasma and tissue fluids) and, more specifically, by the inhibitors of matrix metalloproteases (TIMPs) [63-65]. MMPs mediate the extracellular matrix and basement membrane degradation during early stages of tumorigenesis process, contributing to the formation of a microenvironment that promotes tumor growth. MMPs also participate in later stages of cancer development, promoting the sustained growth of both primary tumors and metastases by activating growth factors, inactivating protein binding to growth factors or releasing mitogenic molecules residing on the extracellular matrix. One of the first steps in the carcinoma invasion is the disruption of the basement membrane and subsequent migration through extracellular matrix [9]. The basement membrane is composed of molecules such as laminin, type IV collagen and proteoglycans containing heparan sulfate [66]. More recently, it was shown that laminin-5 (molecule present in basement membranes of epithelium) is associated with the control of melanoma cells migratory phenotype [67]. The cleavage of laminin-5 by MMP-2 or MMP-14 shows a cryptic site of laminin that triggers cell motility [67]. This laminin-5 cleaved form is found in experimental tumors. In human cancers, MMP-14 co-localizes with laminin-5, which suggests that it is associated with microinvasive cancers [68]. E-cadherin is cleaved by MMP-3 or 7, and the release of E-cadherin promotes the invasion of tumor cells in a paracrine manner *in vitro*, possibly acting as a competitive inhibitor of other E-cadherin [69]. Cleavage of Ecadherin also triggers epithelial to mesenchymal transition associated with the invasive behavior of tumors, including metastatic melanomas [69]. Other molecules such as CD44, also regulate this process [70]. MMPs activation is also among the mechanisms that tumor cells use to escape the "surveillance" of the immune system. MMPs also activate TGF-ß, a suppressor factor of T lymphocytes in the response against tumors. Genetically modified animals that do not express some MMPs have smaller tumors than normal animals, and develop tumors later [71]. These molecules, which have been considered tumor-associated molecules, are not produced only by the tumor cells themselves. Studies using the technique of *in situ* hybridi‐ zation showed that several MMPs mRNA are also produced by stromal fibroblasts and inflammatory cells present in the tumor microenvironment [72-74]. The inflammatory cells such as mast cells, macrophages and neutrophils, as well as producing MMPs also produce cytokines that may act as positive modulators of this process [65]. This creates a tissue network of transcriptional activation, synthesis, secretion and MMPs activation. The phenomenon discussed above clearly illustrates the concept that the tumor behavior depends not only on tumor cells, but also on their interactions with elements of the host. Likewise, the tumor cells invade the host tissues and endothelial cells are recruited by the tumor, forming vascular structures (blood vessels and lymphatics) that comprise an important element of the tumor microenvironment. The tumor vascularization occurs by angiogenesis. At the same time it creates vascular routes inflow nutrients necessary for the tumor mass growth. The newly

essential for maintaining epithelia architecture. In animal models, functional loss of E-cadherin is associated with acceleration of tumor progression. Adenocarcinomas and metastatic lesions appear earlier in animals that do not express E-cadherin function [59]. These E-cadherin properties allowed classifying it as a metastasis suppressor molecule. Loss E-cadherin expression appears to be a critical step in the melanoma progression, allowing the tumor cells to be released from the epidermis and to invade the dermis [60]. The cadherin switch from Ecadherin to N-cadherin results in disassociation of melanoma cells from keratinocytes and promotes melanoma cell invasion through the dermis. The N-cadherin expression in melano‐ ma cells correlates with increased motility and invasion, suggesting that N-cadherin potenti‐ ates the interaction between tumor cells and stromal cells, including fibroblasts and endothelial cells [61]. Anti-N-cadherin antibodies can delay the trans-endothelial migration of melanoma cells and induce apoptosis of melanoma cells [61]. The E-cadherin cytoplasmic portion interacts with alpha-and ß-catenin. Besides the ß-catenin be part of E-cadherin adhesion complex, it plays an essential role as a mediator of the signal transduction pathway of Wnt/Wingless (glycoprotein that plays a role in embryogenesis), which activates the transcription factors LEF/ Tcf [53]. The transcription factors LEF/Tcf are responsible for controlling expression of genes encoding cyclin D1, MYC and metalloproteinases [54]. In a simplified manner, the cytoplasmic pool of ß-catenin can be considered regulatory elements of epithelial cells proliferation and invasion. In tissues where there is interaction between cells and formation of adherent junctions mediated by E-cadherin, ß-catenin molecules are recruited to the sub-membrane region. The lack of degradation mechanism or the functional loss of E-cadherin leads to the ßcatenin cytoplasmic accumulation and its translocation to the nucleus. The ß-catenin also plays a role in the control of proliferation and apoptosis and is also increased in some cancers. Data show that E-cadherin suppresses the growth of metastatic melanoma cells by inhibiting betacatenin signaling pathway/Wnt [54]. Therefore, these data suggest that E-cadherin may play

Proteolytic enzymes, by their ability to degrade ECM proteins, become important components in the process of tumor progression. With the sequencing of the human genome, more than 500 genes were identified as encoding proteases or protease-like proteins, with a large number being associated with the tumor process [62]. Among these, the matrix metalloproteinases (MMPs)-a group of 24 enzymes that degrade ECM components and the basal membrane [63] have been the focus of much research on cancer [63-64]. This family of glycoproteins is secreted as a latent pro-enzyme, activated by proteolysis of a conserved region present in the N-terminal domain and is divided into six groups depending on the type of substrate that degrades: collagenases, gelatinases, stromelysins and extracellular matrix metalloproteinases [62]. MMPs are regulated both at transcriptional and post-transcriptional level. These regulation mechanisms operate to ensure their coordinated expression [63]. MMPs activity occurs only where proteolysis is required. Cytokines and peptide factors, such as interleukin (IL)-4 and IL-10, growth factors (transforming growth factor (TGF-α), basic fibroblast growth factor

an important role early in the metastatic cascade.

54 Melanoma – Current Clinical Management and Future Therapeutics

**4. Matrix metalloproteinases**

formed vessels may give way to the efflux of tumor cells to the hematogenic or lymphatic circulation, thus resulting in systemic tumor dissemination [9].

#### **5. Tissue inhibitors of metalloproteinases**

Inhibitors of matrix metalloproteinases have been identified in various species, such as *C. elegans*, *Drosophila*, zebrafish and humans, suggesting that these genes are present from the start of the evolutionary process [75]. Recent studies have shown developmental defects in Timps-deficient organisms in both mammals and in non-mammals, revealing the importance of these proteins during embryonic development. In mammals, the family of tissue inhibitors of metalloproteases (Timps) consists of four distinct members: TIMP1, 2, 3 and 4, which share substantial sequence homology and structural identity at the protein level [75]. TIMPs have basically two structural domains: an N-terminal domain containing 6 conserved cysteine residues forming three "loops", having the inhibitory activity of MMPs; and a C-terminal, which also contains six conserved cysteine residues and form three "loops". The balance between activities of the protease inhibitor and the proteolytic potential determines the tumor progression [75]. Thus, increases in expression and activity of MMPs are found in almost all human cancers. Interestingly, the expression of their inhibitors, TIMPs, is also generally increased in several cancers. Among them, TIMP1 has been associated with poor prognosis in metastatic melanoma, suggesting promising value of TIMP1 as a prognostic marker of the tumor [76-77]. In our laboratory, a model that allows us to study different stages of melanoma progression was developed. Murine melan-a melanocytes surviving after 1, 2, 3 and 4 cycles of adhesion impediment (named respectively 1C, 2C, 3C and 4C cell lines) showed changes in morphology and growth independent of phorbol myristate acetate (PMA). Different melano‐ ma cell lines (such as 4C3-, 4C3+, 4C11-, and 4C11+) were established after subjecting the surviving spheroids formed after blocking the adhesion of 4C cell line to a limiting dilution [17]. Previous data from our laboratory show a progressive increase in the TIMP1 expression along the melanoma genesis, and this increase is related to resistance to *anoikis* and to a more aggressive phenotype *in vivo* [76]. We also observed that soluble TIMP1 in non-tumorigenic lineage melan-a confers *anoikis* resistance and it is differentially associated with CD63 and β1 integrin along the melanoma genesis [77]. CD63, β1-integrin and TIMP1 are not interacting in the murine melan-a melanocytes. The 4C cell line, corresponding to pre-malignant melano‐ cytes, shows interaction between CD63 and β1-integrins, and CD63 and TIMP1, which could initiate the signaling pathways for cell survival, since 4C cell line is more *anoikis*-resistant when compared with its parental melan-a cell line. In 4C11- and 4C11+ melanomacell lines, a tighter CD63/β1integrins/TIMP1 complex seem to be formed, which could result in a more efficient activation of cell survival signals, giving to these cells a higher resistance to *anoikis* (Figure 1). However, the mechanisms regulating the functions of TIMP1 and signaling pathways activated by this complex along the tumorigenesis are still unclear. Studies are in progress to further elucidate the role of TIMP1 in metastatic melanoma.

**Figure 1. Schematic representation of the interaction among CD63, β1-integrin and TIMP1 along melanoma pro‐ gression. A.** In the melanocyte lineage melan-a, there is no interaction among CD63, β1-integrin and TIMP1. In the premalignant 4C cell line, TIMP1 associates with CD63, but not with β1-integrins, and CD63 interacts with β1-integrins. In 4C11- and 4C11+ melanoma cell lines, the formation of CD63/β1-integrin/TIMP1 complex occurs, which could be relat‐ ed to a more efficient activation of cell survival signals [77].

## **6. Pathways classically involved in melanoma and their involvement in melanoma metastasis**

#### **6.1. BRAF**

formed vessels may give way to the efflux of tumor cells to the hematogenic or lymphatic

Inhibitors of matrix metalloproteinases have been identified in various species, such as *C. elegans*, *Drosophila*, zebrafish and humans, suggesting that these genes are present from the start of the evolutionary process [75]. Recent studies have shown developmental defects in Timps-deficient organisms in both mammals and in non-mammals, revealing the importance of these proteins during embryonic development. In mammals, the family of tissue inhibitors of metalloproteases (Timps) consists of four distinct members: TIMP1, 2, 3 and 4, which share substantial sequence homology and structural identity at the protein level [75]. TIMPs have basically two structural domains: an N-terminal domain containing 6 conserved cysteine residues forming three "loops", having the inhibitory activity of MMPs; and a C-terminal, which also contains six conserved cysteine residues and form three "loops". The balance between activities of the protease inhibitor and the proteolytic potential determines the tumor progression [75]. Thus, increases in expression and activity of MMPs are found in almost all human cancers. Interestingly, the expression of their inhibitors, TIMPs, is also generally increased in several cancers. Among them, TIMP1 has been associated with poor prognosis in metastatic melanoma, suggesting promising value of TIMP1 as a prognostic marker of the tumor [76-77]. In our laboratory, a model that allows us to study different stages of melanoma progression was developed. Murine melan-a melanocytes surviving after 1, 2, 3 and 4 cycles of adhesion impediment (named respectively 1C, 2C, 3C and 4C cell lines) showed changes in morphology and growth independent of phorbol myristate acetate (PMA). Different melano‐ ma cell lines (such as 4C3-, 4C3+, 4C11-, and 4C11+) were established after subjecting the surviving spheroids formed after blocking the adhesion of 4C cell line to a limiting dilution [17]. Previous data from our laboratory show a progressive increase in the TIMP1 expression along the melanoma genesis, and this increase is related to resistance to *anoikis* and to a more aggressive phenotype *in vivo* [76]. We also observed that soluble TIMP1 in non-tumorigenic lineage melan-a confers *anoikis* resistance and it is differentially associated with CD63 and β1 integrin along the melanoma genesis [77]. CD63, β1-integrin and TIMP1 are not interacting in the murine melan-a melanocytes. The 4C cell line, corresponding to pre-malignant melano‐ cytes, shows interaction between CD63 and β1-integrins, and CD63 and TIMP1, which could initiate the signaling pathways for cell survival, since 4C cell line is more *anoikis*-resistant when compared with its parental melan-a cell line. In 4C11- and 4C11+ melanomacell lines, a tighter CD63/β1integrins/TIMP1 complex seem to be formed, which could result in a more efficient activation of cell survival signals, giving to these cells a higher resistance to *anoikis* (Figure 1). However, the mechanisms regulating the functions of TIMP1 and signaling pathways activated by this complex along the tumorigenesis are still unclear. Studies are in progress to

circulation, thus resulting in systemic tumor dissemination [9].

**5. Tissue inhibitors of metalloproteinases**

56 Melanoma – Current Clinical Management and Future Therapeutics

further elucidate the role of TIMP1 in metastatic melanoma.

The MAPK pathway is an important intracellular signal transduction pathway, which regulates cellular proliferation, differentiation, gene expression, cell survival and apoptosis [78]. This pathway is activated by different factors through different receptors, as tyrosine kinases and G-protein-coupled receptors. The activation of those membrane receptors promotes RAS activation, which activates several effector proteins, as the ones in RAF family. RAF then activate the kinase cascade (MEK1/2 and ERK1/2), which can phosphorylate nuclear and cytoplasmic substrates involved in several cellular processes [1].

The most mutated gene in this pathway is the *BRAF* gene, with the most common mutation in melanoma being the BRAFV600E mutation. This mutation involves the change of valine to glutamic acid at codon 600 (V600E) in the exon 15, which causes a conformational change in the protein structure leading to its activation. Therefore, since the BRAF is constitutively activated, cells with BRAFV600E present hyperactivation of the MAPK pathway and are able to signal through it without activation by RAS [78-79].

Since the first report by Davies and colleagues in 2002, several studies have confirmed that activating BRAF mutations are present in approximately 60% of melanoma, and over 90% of them are BRAFV600E [80]. *BRAF* mutations are not only present in melanoma but also in nevi. Some shows that the incidence of this mutation is higher in nevi (up to 80%) than in melanoma (up to 65%) and this brought the question if mutations in BRAF can be acquired during primary tumor development or even during metastasis [78, 81]. Several results suggest that BRAF mutation occurs early in the malignant transformation of melanocytes but is not sufficient to cause melanoma and so this mutation is probably acquired during the melanoma progression [78].

It is predicted that the constitutive activation of the MAPK pathway can lead to oncogenic transformation of cells by promoting cell proliferation, invasion, metastasis, migration, survival and angiogenesis [82]. Specifically, MAPK is predicted to mediate melanoma metastasis by inducing proteolytic enzymes, as MMPs, which leads to degradation of basement membrane, and by regulating genes involved in cell migration, cell survival, and growth [83]. However, this has been poorly studied.

In 2012, Colombino and colleagues analyzed primary and metastatic melanoma samples and found that overall 43% of primary melanomas have mutated BRAF with no significant frequency increase in metastatic lesions [84]. However, Shinozaki and collaborators in 2004 analyzed the BRAF mutation in tumor specimens and showed that this mutation is more frequent in melanoma metastasis (57%) than in primary melanomas (31%), which suggests that it can be acquired during metastasis. They also analyzed 13 pairs of primary melanomas with their respective metastases. Four pairs were not mutated in the primary or the metastatic tumor, other four pairs were mutated in both stages and five pairs (38%) present the wild type gene in the primary tumors and the mutated one in the metastatic tumors. This suggests that BRAF is not a key factor for the development of metastasis, although it can be acquired during this process. However, the frequency of mutation found in the study was lower than the report in the literature [81].

Another report that analyzed metastatic tumors in an Australian cohort demonstrated that 48% of the patients had a BRAF mutation, with 74% being the BRAFV600E. Interestingly, the presence of the BRAF mutation led to a poorer survival unless patients were treated with BRAF inhibitor [85].

Another study shows that inhibition of BRAFV600E expression causes a significant decrease in the metastatic ability of the metastatic cell lines Lu1205 and UACC 903M. This impaired ability to metastasize was due the reduction of cell extravasation through the endothelium, process mediated by IL-8 and ICAM [79]. Mutated BRAF can also participate of metastasis develop‐ ment by the generation of new blood vessels by promoting macrophage inhibitory cytokine-1 (MIC-1) and vascular endothelial growth factors (VEGF) secretion [82]. MIC-1 was shown to be upregulated in metastatic melanoma cell lines and patient biopsies in comparison with melanocytes. A trend was also observed in comparison with primary melanomas [86]. A RNA interference targeting BRAFV600E blocks melanoma cell invasion *in vitro* and decreases MMP-2 activity while BRAFV600E induces activity of MMP-1, also suggesting that this mutation can be involved in the metastatic process [82].

Mutation in BRAF can also activate ERK1/2, which was seen activated by Jorgensen and collaborators in 54% of primary melanomas and 33% of metastatic tumors [87]. However, Mirmohammadsadegh and collaborators analyzed the expression of phosphorylated ERK1/2 (pERK) in human cells and specimens and saw that it levels was low in melanocytes, upregu‐ lated in melanoma cell lines and abundant in melanoma metastasis. Yet, this study did not distinguish between non-metastatic and metastatic cell lines and did not analyze samples of primary tissue [88].

The successful treatment of metastatic melanomas with BRAFV600E inhibitors [89] and the events here forehead mentioned suggests that this mutation is involved somehow in the process of metastasis, but the exact mechanisms involved are still unclear [82].

#### **6.2. MITF**

RAF then activate the kinase cascade (MEK1/2 and ERK1/2), which can phosphorylate nuclear

The most mutated gene in this pathway is the *BRAF* gene, with the most common mutation in melanoma being the BRAFV600E mutation. This mutation involves the change of valine to glutamic acid at codon 600 (V600E) in the exon 15, which causes a conformational change in the protein structure leading to its activation. Therefore, since the BRAF is constitutively activated, cells with BRAFV600E present hyperactivation of the MAPK pathway and are able to

Since the first report by Davies and colleagues in 2002, several studies have confirmed that activating BRAF mutations are present in approximately 60% of melanoma, and over 90% of them are BRAFV600E [80]. *BRAF* mutations are not only present in melanoma but also in nevi. Some shows that the incidence of this mutation is higher in nevi (up to 80%) than in melanoma (up to 65%) and this brought the question if mutations in BRAF can be acquired during primary tumor development or even during metastasis [78, 81]. Several results suggest that BRAF mutation occurs early in the malignant transformation of melanocytes but is not sufficient to cause melanoma and so this mutation is probably acquired during

It is predicted that the constitutive activation of the MAPK pathway can lead to oncogenic transformation of cells by promoting cell proliferation, invasion, metastasis, migration, survival and angiogenesis [82]. Specifically, MAPK is predicted to mediate melanoma metastasis by inducing proteolytic enzymes, as MMPs, which leads to degradation of basement membrane, and by regulating genes involved in cell migration, cell survival, and growth [83].

In 2012, Colombino and colleagues analyzed primary and metastatic melanoma samples and found that overall 43% of primary melanomas have mutated BRAF with no significant frequency increase in metastatic lesions [84]. However, Shinozaki and collaborators in 2004 analyzed the BRAF mutation in tumor specimens and showed that this mutation is more frequent in melanoma metastasis (57%) than in primary melanomas (31%), which suggests that it can be acquired during metastasis. They also analyzed 13 pairs of primary melanomas with their respective metastases. Four pairs were not mutated in the primary or the metastatic tumor, other four pairs were mutated in both stages and five pairs (38%) present the wild type gene in the primary tumors and the mutated one in the metastatic tumors. This suggests that BRAF is not a key factor for the development of metastasis, although it can be acquired during this process. However, the frequency of mutation found in the study was lower than the report

Another report that analyzed metastatic tumors in an Australian cohort demonstrated that 48% of the patients had a BRAF mutation, with 74% being the BRAFV600E. Interestingly, the presence of the BRAF mutation led to a poorer survival unless patients were treated with BRAF

Another study shows that inhibition of BRAFV600E expression causes a significant decrease in the metastatic ability of the metastatic cell lines Lu1205 and UACC 903M. This impaired ability

and cytoplasmic substrates involved in several cellular processes [1].

signal through it without activation by RAS [78-79].

58 Melanoma – Current Clinical Management and Future Therapeutics

the melanoma progression [78].

However, this has been poorly studied.

in the literature [81].

inhibitor [85].

Microphthalmia-associated transcription factor (MITF) is a master regulator of melanocyte development and regulates survival, growth, differentiation and pigmentation of these cells [90]. There are several isoforms of MITF described, but in melanocytes and melanoma there is a predominance of the isoform MITF-M [91].

The levels of MITF expression are very important to cell fate. High levels of MITF induce cell cycle arrest and differentiation, while low levels promote cell cycle arrest, apoptosis and even invasion and metastasis. If MITF levels are depleted for long time, melanoma can enter quiescence or senescence. Thus, only an intermediate level of MITF favors cell proliferation [1, 90]. Wherefore, levels of MITF have to be tightly regulated during melanocytic development and even during melanoma progression.

Several reports indicate that most human melanomas express MITF [1], and that MITF is expressed in high levels in benign and primary tumors [91]. MITF is genomically amplified in 10% of primary melanomas and 21% of metastatic melanoma, and the amplification in the metastatic samples correlates with a significant decrease in the 5-year survival rate of patients [92]. However, expression of MITF in metastatic lesion is variable. Beyond the specimens that present MITF amplification and therefore high levels of expression, other metastatic lesions present predominantly downregulation of MITF [93]. A report demonstrated that depletion of MITF expression in mouse and human cells is sufficient to induce experimental lung metastasis [91]. Consistently, high levels of MITF in melanoma patients are associated with low invasiveness and fewer metastases [90]. Because some metastatic lesions present low expression of MITF while others present MITF amplification, it is proposed that MITF can have paradoxical effects in different sub-groups of melanomas. For example, the decrease in MITF expression in metastatic melanomas can be beneficial for tumor growth because it reduces pigmentation, and therefore the cellular energy utilized for pigment production [93]. However, in specimens with high expression of MITF, it can regulate c-Met expression, which upregu‐ lation appears to have a functional role in metastatization of melanoma [94].

Because MITF is part of a complex pathway, alterations in factors upstream or downstream to MITF can affect melanoma development [90]. However, this has not been properly investi‐ gated in metastatic melanoma. Until now, the expression and functional role of MITF in metastatic melanoma is not determined.

#### **6.3. TGF-β**

The transforming growth factor-β (TGF-β) is a cytokine implies in several cellular processes, as cell proliferation, differentiation and survival. TGF-β interacts with its receptor, leading to the receptor phosphorylation, which then phosphorylates the cytoplasmatic proteins SMAD. The SMADs proteins accumulate in the nucleus and acts as transcription factors. TGF-β can also signal through a non-canonical pathway. In this respect, TGF-β can activate phosphati‐ dylinositol-3-kinase (PI3K) and several mitogen activated protein kinases (MAPKs), leading to cell signaling independent of SMAD [95].

There are three isoforms of TGF-β in mammalian, TGF-β1, TGF-β2, TGF-β3. *In vitro*, normal melanocytes and malignant melanomas express TGF-β1 and TGF-β3, but TGF-β2 is only found in melanoma cells. The expression of TGF-β2 correlates with the deepness of melanoma. Thus, metastatic and highly invasive tumors present TGF-β2 expression, while a minority of primary melanomas minimally invasive express TGF-β and *in situ* tumors does not express it [95]. In addition, melanoma secretes large amounts of TGF-β, and high amounts of TGF-β in plasma are associated with advanced tumor stages [96] since it foments tumor growth, angiogenesis, invasiveness and dissemination.

The overexpression of an inhibitory SMAD, SMAD 7, is effective in reducing secretion of MMPs and impairing bone metastasis development. The blockage of TGF-β receptor has the same effect. TGF-β also promotes the epithelial to mesenchymal transition (EMT), an event associated with increase in metastasis, increase in MMP2 and MMP9 expression, and angio‐ genesis by modulating IL-8, MMPs, VEGF, among other important factors [95, 97].

TGF-β also promotes metastasis through expression induction of the transcription factor GLI2. High expression of GLI2 correlated with increased cell invasiveness and bone metastasis emergence. In a murine model of induced metastasis, cells with elevated expression of GLI2 caused bone metastasis, while cells with low level of this factor have reduced and heterogenic capacity to form metastasis. In addition, the GLI2 knockdown in an aggressive melanoma cell line (1205Lu) dramatically diminishes their ability to form bone metastasis [96].

#### **6.4. Wnt (β-catenin and WNTs)**

expression of MITF while others present MITF amplification, it is proposed that MITF can have paradoxical effects in different sub-groups of melanomas. For example, the decrease in MITF expression in metastatic melanomas can be beneficial for tumor growth because it reduces pigmentation, and therefore the cellular energy utilized for pigment production [93]. However, in specimens with high expression of MITF, it can regulate c-Met expression, which upregu‐

Because MITF is part of a complex pathway, alterations in factors upstream or downstream to MITF can affect melanoma development [90]. However, this has not been properly investi‐ gated in metastatic melanoma. Until now, the expression and functional role of MITF in

The transforming growth factor-β (TGF-β) is a cytokine implies in several cellular processes, as cell proliferation, differentiation and survival. TGF-β interacts with its receptor, leading to the receptor phosphorylation, which then phosphorylates the cytoplasmatic proteins SMAD. The SMADs proteins accumulate in the nucleus and acts as transcription factors. TGF-β can also signal through a non-canonical pathway. In this respect, TGF-β can activate phosphati‐ dylinositol-3-kinase (PI3K) and several mitogen activated protein kinases (MAPKs), leading

There are three isoforms of TGF-β in mammalian, TGF-β1, TGF-β2, TGF-β3. *In vitro*, normal melanocytes and malignant melanomas express TGF-β1 and TGF-β3, but TGF-β2 is only found in melanoma cells. The expression of TGF-β2 correlates with the deepness of melanoma. Thus, metastatic and highly invasive tumors present TGF-β2 expression, while a minority of primary melanomas minimally invasive express TGF-β and *in situ* tumors does not express it [95]. In addition, melanoma secretes large amounts of TGF-β, and high amounts of TGF-β in plasma are associated with advanced tumor stages [96] since it foments tumor growth, angiogenesis,

The overexpression of an inhibitory SMAD, SMAD 7, is effective in reducing secretion of MMPs and impairing bone metastasis development. The blockage of TGF-β receptor has the same effect. TGF-β also promotes the epithelial to mesenchymal transition (EMT), an event associated with increase in metastasis, increase in MMP2 and MMP9 expression, and angio‐

TGF-β also promotes metastasis through expression induction of the transcription factor GLI2. High expression of GLI2 correlated with increased cell invasiveness and bone metastasis emergence. In a murine model of induced metastasis, cells with elevated expression of GLI2 caused bone metastasis, while cells with low level of this factor have reduced and heterogenic capacity to form metastasis. In addition, the GLI2 knockdown in an aggressive melanoma cell

genesis by modulating IL-8, MMPs, VEGF, among other important factors [95, 97].

line (1205Lu) dramatically diminishes their ability to form bone metastasis [96].

lation appears to have a functional role in metastatization of melanoma [94].

metastatic melanoma is not determined.

60 Melanoma – Current Clinical Management and Future Therapeutics

to cell signaling independent of SMAD [95].

invasiveness and dissemination.

**6.3. TGF-β**

β-catenin is an adherent junction protein and a transcriptional coativator. It mediates cell adhesion, proliferation, survival and migration [1]. It participates in the WNT pathway and the interaction between WNT ligands and receptors leads to the accumulation of β-catenin in the nucleus, where it promotes transactivation of target genes [98].

The role of β-catenin in melanoma it is still unclear. Some show β-catenin is frequently constitutively activated in melanomas and that its accumulation promotes metastasis, while others show its high expression suppresses invasion and indicates a better prognosis for patients [1, 99].

A report shows that β-catenin is an important regulator of melanoma metastasis to lymph nodes and lungs. In this case, β-catenin cooperates with PTEN loss and BRAFV600E to promote primary tumor and metastasis, but the mechanism was not enlightened [1]. In other study, activation of β-catenin signaling increased metastatic potential in NRAS-driven melanoma in mice, although it repressed cell migration, indicating that β-catenin inhibits the initial steps of metastasis, as cell migration, but can be involved in the latest steps of this process [100].

However, another study that analyzed β-catenin expression by immunohistochemistry in melanocytic samples identified that β-catenin was strongly stained in 96% of melanocytic nevi, in 94% of radial growth phase primary melanoma, in 65% of vertical growth phase primary melanoma and in only 38% of metastatic melanomas [101]. The involvement of β-catenin decrease in melanoma metastasis was also seen indirectly by decrease in WLS, an important component to WNT ligands secretion. The reduction of WLS expression by shRNA in the melanoma cell line A375 caused spontaneous metastasis in the lung of mice. Of 36 animals injected with WLS shRNA cells, 10 developed lung metastasis while none of the 18 animals injected with control shRNA cell presented metastasis. This was due to the inhibition of the WNT/ β-catenin pathway [98].

Therefore, β-catenin effects on melanoma metastasis are still not fully understood and may depend on cellular context [100]. Some state that WNT signaling via the canonical pathway, which involves β-catenin, is associated with a less metastatic phenotype, while the noncanonical pathway, involving Wnt5A, would be related with increased malignancy [6]. The overexpression of Wnt5A appears to be consistently associated with a more aggressive disease and metastasis development [1] and this is probably due the upregulation of metastatic markers, as CD44 and Snail, and the promotion of EMT [6].

Wnt5 activity is regulated by heparan sulfate proteoglycans (HSPGs), glycoproteins that are categorized based on the structure of their glycosaminoglycan (GAG) chains and although has been poorly studied in melanomas, has been recently associated with melanoma metastasis. For example, HSPGs are important to cell signaling through the non-canonical Wnt pathway. The HSPGs syndecans 1 and 4 are necessary to the presentation of Wnt5A to its receptor ROR2 and its consequent internalization and signaling. Cleavage of the syndecans GAGs result in less Wnt5A at the cell surface and a consequent decrease in the cell metastatic potential [6].

### **6.5. PI3K**

The phosphoinositide-3 kinase (PI3K) pathway is another key signaling cascade that controls cell survival, proliferation and motility. PI3K is activated by receptor tyrosine kinases or RAS leading to the release of second messenger PIP3, which can activate several downstream effectors, as AKT. PTEN (Phosphatase and Tensin Homolog) is an important negative regulator of this pathway [1].

PI3K pathway is commonly hyperactivated in melanomas. A report that analyzed 68 meta‐ static melanomas showed that 41% presented a mutation in this pathway [102]. Besides 3% of metastatic melanomas presents activating mutation of PI3K and 5-20% of late-stage melanoma present mutations that leads to PTEN loss, which is the protein most commonly mutated in this pathway [1]. Another study that analyzed PI3K protein expression demonstrated that PI3K expression was low in nevi and high in melanomas. Interestingly this expression was even higher in metastatic specimens [103].

Inactivation of PTEN by mutation leads to a constitutive activation of PI3K pathway. Because PTEN mutations are rarely found in primary melanomas, it is proposed that the activation of the PI3K pathway may be important to the late events of melanoma progression, as invasion and metastasis [103]. An interesting study demonstrated that mice with PTEN loss and BRAFV600E develop malignant melanoma that is able to metastasize to lymph nodes and distant organs, while mice with only BRAFV600E produced benign melanocytic hyperplasia, showing that PTEN loss cooperate with BRAFV600E to induce metastasis [104].

AKT is also able to induce tumor cell invasion and metastasis to the lung in a mouse model by inhibiting the small GTPase RhoB [105]. Moreover, a higher percentage of biopsies present strong phospho-AKT (the activated form of AKT) staining in metastatic melanoma than in primary melanomas and nevi and high levels of phospho-AKT are associated with poor patient survival rates (Dai 2005).

## **7. Other pathways**

#### **7.1. Telomerase**

Telomerase is a ribonucleoprotein complex and its main function is to maintain the telomeric repeats that cap the ends of eukaryotic chromosomes and therefore preserve their integrity by preventing end-to-end fusions [106]. In melanocytic lesions, is seen that nevi have low or even absent telomerase activity, primary melanomas have intermediate levels of activity and metastatic melanomas present increased telomerase activity. Telomerase promoter seems to be frequently mutated in melanoma and two mutational hotspots were found in 85% of melanoma metastases [107].

The inhibition of the RNA portion of the telomerase, called TER (telomerase RNA), results in severe decrease in metastatic tumor development. In an experiment, mice that received injection of melanoma cells with TER downregulated presented 70% fewer metastases in the lung then mice injected with control cells. The inhibition of TER also led to the downregulation of several genes, including genes involved in transcriptional regulation, cell proliferation and adhesion, chromatin assembly and others. Interestingly, it was also seen a regulation of genes of the glycolytic pathway, as aldolase, suggesting that telomerase can mediate its metastatic properties through activation of glycolysis in cancer [106].

#### **7.2. ACP5**

**6.5. PI3K**

regulator of this pathway [1].

62 Melanoma – Current Clinical Management and Future Therapeutics

higher in metastatic specimens [103].

survival rates (Dai 2005).

**7. Other pathways**

melanoma metastases [107].

**7.1. Telomerase**

The phosphoinositide-3 kinase (PI3K) pathway is another key signaling cascade that controls cell survival, proliferation and motility. PI3K is activated by receptor tyrosine kinases or RAS leading to the release of second messenger PIP3, which can activate several downstream effectors, as AKT. PTEN (Phosphatase and Tensin Homolog) is an important negative

PI3K pathway is commonly hyperactivated in melanomas. A report that analyzed 68 meta‐ static melanomas showed that 41% presented a mutation in this pathway [102]. Besides 3% of metastatic melanomas presents activating mutation of PI3K and 5-20% of late-stage melanoma present mutations that leads to PTEN loss, which is the protein most commonly mutated in this pathway [1]. Another study that analyzed PI3K protein expression demonstrated that PI3K expression was low in nevi and high in melanomas. Interestingly this expression was even

Inactivation of PTEN by mutation leads to a constitutive activation of PI3K pathway. Because PTEN mutations are rarely found in primary melanomas, it is proposed that the activation of the PI3K pathway may be important to the late events of melanoma progression, as invasion and metastasis [103]. An interesting study demonstrated that mice with PTEN loss and BRAFV600E develop malignant melanoma that is able to metastasize to lymph nodes and distant organs, while mice with only BRAFV600E produced benign melanocytic hyperplasia, showing

AKT is also able to induce tumor cell invasion and metastasis to the lung in a mouse model by inhibiting the small GTPase RhoB [105]. Moreover, a higher percentage of biopsies present strong phospho-AKT (the activated form of AKT) staining in metastatic melanoma than in primary melanomas and nevi and high levels of phospho-AKT are associated with poor patient

Telomerase is a ribonucleoprotein complex and its main function is to maintain the telomeric repeats that cap the ends of eukaryotic chromosomes and therefore preserve their integrity by preventing end-to-end fusions [106]. In melanocytic lesions, is seen that nevi have low or even absent telomerase activity, primary melanomas have intermediate levels of activity and metastatic melanomas present increased telomerase activity. Telomerase promoter seems to be frequently mutated in melanoma and two mutational hotspots were found in 85% of

The inhibition of the RNA portion of the telomerase, called TER (telomerase RNA), results in severe decrease in metastatic tumor development. In an experiment, mice that received injection of melanoma cells with TER downregulated presented 70% fewer metastases in the

that PTEN loss cooperate with BRAFV600E to induce metastasis [104].

The analysis of the expression of ACP5 (Tartrate-resistant acid phosphatase 5), a protein involved in bone development [108], in a melanoma tissue microarray comparing primary and metastatic specimens demonstrated that ACP5 is upregulated in the metastatic lesions. To confirm the role of ACP5 in metastasis, the authors overexpressed ACP5 in a human poorly metastatic melanoma cell line (1205Lu) and observed an increase in the metastatic ability of those cells *in vivo*. Animals that received injection of the parental 1205Lu cell did not develop spontaneous metastasis, while 40% of the animals that were injected with the cells overex‐ pressing ACP5 developed metastasis to the lung and lymph nodes [109].

#### **7.3. Chemokines**

Chemokines are secreted chemotactic cytokines that were first identified as modulators of leukocytes trafficking to inflammatory sites. Chemokines realize its biological function through the binding in chemokine receptors, G-protein coupled receptors. It has been seen by several authors that melanoma cells have a high expression of chemokines and that its interaction with its receptors stimulates tumor growth, angiogenesis and metastasis [110].

The expression of CXCL8, a potent chemokine also known as IL-8, has been associated with tumor angiogenesis, progression and metastasis in some mouse models. In one case, the induction of CXCL8 expression by ultraviolet-B radiation potentiated tumor and metastasis development. In addition, metastatic melanoma cell lines express higher levels of CXCL8 than its non-metastatic variants [110]. Moreover, in an analysis of tumor specimens, it was seen that radial growth tumors do not present CXCL8 expression, while half of the vertical growth tumors present it. A significant increase in CXCL8 expression was also seen in metastatic samples in comparison with thin melanomas [111]. The CXCL8 receptor, CXCR2, was also seen to be involved in melanoma metastasis. *In vivo* murine studies using knockout models demonstrated that CXCR2 played a major role in melanoma metastasis to the lung [112].

Chemokines receptors also appear to be important to specific organ metastasis. CXCR4 was identified as the most frequent expressed chemokine receptor in liver metastasis of paraffinembedded tissue, with 89% of the samples expressing it [113]. CCR9 was observed as expressed in a great majority of intestine metastasis of melanoma, but not in other organs [114].

#### **7.4. NEDD9**

NEDD9 (neural precursor expressed, developmentally downregulated 9) is an adaptor protein that belong to the Cas family of signal transduction molecules. NEDD9 is genomi‐ cally amplified in melanomas and its high expression correlates with melanoma progres‐ sion and metastasis. NEDD9 is frequently overexpressed in human metastatic tumor in comparison to primary ones and its expression enhances invasion *in vitro* and metastasis *in vivo* of both normal and transformed melanocytes. NEDD9 appears to mediate melano‐ ma invasive behavior through interaction with focal adhesion kinase and modulation of focal contact formation [115].

### **7.5. MicroRNAs**

MicroRNAs (miRNAs) are small non-coding RNA Molecules - 17 to 22 nucleotides - that regulate gene expression post-transcriptionally. MiRNAs are negative regulators of gene expression. These regulatory RNAs act by binding to mRNA molecules of specific gene targets and inducing their cleavage or translation repression. Because just approximately seven nucleotides of the miRNA – the so-called seed region - interact with the target mRNA, each miRNA may have a large number of targets. Therefore, a unique miRNA can influence the expression of even hundreds of proteins and, for that, miRNAs participate of several processes in the cell as development, cell proliferation, differentiation and metabolism [116-117].

Because of the critical role miRNAs have in the cell biology, changes in their expression patterns have impact in different disorders, as cancer itself [116]. After miRNAs being related with tumorigenesis, it did not take long until they were specifically related with metastasis. The first microRNA linked with metastasis was described in 2007 by Ma, Weinberg and colleagues. In this paper, the authors demonstrated that microRNA-10b is highly expressed in metastatic breast cancer cells and that its overexpression in non-metastatic breast tumors leads to metastasis [91, 118]. Two years later, in 2009, Hurst termed the miRNAs involved in the metastasis process, metastamiRs. The metastamiRs modulate key signaling pathways and can promote or suppress metastasis [117].

Recently, miRNAs have been linked with melanoma metastasis, but there is still limited information about it. Most time, there is only one study analyzing the involvement of certain miRNA with melanoma aggressiveness. Here, we will present some of miRNAs that are consistently associated with melanoma metastasis, but are not necessarily defined as meta‐ stamiRs.

A miRNA that appears to act as a metastamiR in melanoma is the miR-214. MiR-214 expression was evaluated in the poorly metastatic lineage A375 and in its highly metastat‐ ic variant cell lines, originated from several passages *in vivo*. It was seen an upregulation of miR-214 in the highly metastatic melanoma cells lines in comparison with the parental one. Beyond that, the overexpression of miR-214 in a metastatic lineage that expressed intermediate levels of this miRNA resulted in a higher number of lung metastases than the control in a metastasis formation assay *in vivo*. In addition, miR-214 silencing in a highly metastatic lineage, which expressed high levels of this miRNA, caused a significantly reduction of lung metastases formation. Furthermore, as primary tumor growth was not influenced by miR-214, these results indicate miR-214 as a metastamiR with an important role in the melanoma progression [119].

Other metastamiRs which involvement with melanoma has been recently discovered are miR-30b and miR-30d. These clustered miRNAs (miRNAs that are transcribed together) are upregulated in melanoma metastasis samples related to primary melanomas and nevi. Overexpression and underexpression assays *in vivo* proved the importance of this cluster expression to metastasis occurrence. In addition to the increased metastatic potential, the upregulation of these miRNAs correlates with shorter time to recurrence and reduced overall survival. Overexpression of miR-30b/30d promotes melanoma metastasis by modifying the glycosylation pattern of transmembrane proteins, more specifically by directly repressing GalNAc transferase GALNT7, which culminates in pro-invasive and immunosuppressive effects [120].

sion and metastasis. NEDD9 is frequently overexpressed in human metastatic tumor in comparison to primary ones and its expression enhances invasion *in vitro* and metastasis *in vivo* of both normal and transformed melanocytes. NEDD9 appears to mediate melano‐ ma invasive behavior through interaction with focal adhesion kinase and modulation of

MicroRNAs (miRNAs) are small non-coding RNA Molecules - 17 to 22 nucleotides - that regulate gene expression post-transcriptionally. MiRNAs are negative regulators of gene expression. These regulatory RNAs act by binding to mRNA molecules of specific gene targets and inducing their cleavage or translation repression. Because just approximately seven nucleotides of the miRNA – the so-called seed region - interact with the target mRNA, each miRNA may have a large number of targets. Therefore, a unique miRNA can influence the expression of even hundreds of proteins and, for that, miRNAs participate of several processes in the cell as development, cell proliferation, differentiation and metabolism [116-117].

Because of the critical role miRNAs have in the cell biology, changes in their expression patterns have impact in different disorders, as cancer itself [116]. After miRNAs being related with tumorigenesis, it did not take long until they were specifically related with metastasis. The first microRNA linked with metastasis was described in 2007 by Ma, Weinberg and colleagues. In this paper, the authors demonstrated that microRNA-10b is highly expressed in metastatic breast cancer cells and that its overexpression in non-metastatic breast tumors leads to metastasis [91, 118]. Two years later, in 2009, Hurst termed the miRNAs involved in the metastasis process, metastamiRs. The metastamiRs modulate key signaling pathways and can

Recently, miRNAs have been linked with melanoma metastasis, but there is still limited information about it. Most time, there is only one study analyzing the involvement of certain miRNA with melanoma aggressiveness. Here, we will present some of miRNAs that are consistently associated with melanoma metastasis, but are not necessarily defined as meta‐

A miRNA that appears to act as a metastamiR in melanoma is the miR-214. MiR-214 expression was evaluated in the poorly metastatic lineage A375 and in its highly metastat‐ ic variant cell lines, originated from several passages *in vivo*. It was seen an upregulation of miR-214 in the highly metastatic melanoma cells lines in comparison with the parental one. Beyond that, the overexpression of miR-214 in a metastatic lineage that expressed intermediate levels of this miRNA resulted in a higher number of lung metastases than the control in a metastasis formation assay *in vivo*. In addition, miR-214 silencing in a highly metastatic lineage, which expressed high levels of this miRNA, caused a significantly reduction of lung metastases formation. Furthermore, as primary tumor growth was not influenced by miR-214, these results indicate miR-214 as a metastamiR with an important

focal contact formation [115].

64 Melanoma – Current Clinical Management and Future Therapeutics

promote or suppress metastasis [117].

role in the melanoma progression [119].

stamiRs.

**7.5. MicroRNAs**

The miR-1908, miR-199a-5p and miR-199a-3p also promote human melanoma metastasis. These miRNAs are overexpressed in highly metastatic cell lines in comparison with their poorly metastatic parental cells. Ectopic expression of these miRNAs in the parental cell lines (MeWo) caused increased metastatic potential and individual inhibition of these miRNAs in the metastatic cells suppressed metastatic colonization. Interestingly, the expression of these miRNAs are upregulated in primary melanomas that had metastasized in comparison with those that had not, indicating that the expression of them in primary tumors is an early event that can be predictive of melanoma metastasis. Another indicator that those are metastamiRs is that they do not affect tumor growth [121].

An important genomic region involved in melanoma development is the 7q31-34 region, because it is commonly amplified in melanomas. This region includes the BRAF and c-MET oncogenes, and the miR-182. It was observed a higher expression of this miRNA in metastatic melanoma tumors compared with primary melanomas and nevi, and this is caused, at least in part, by gene amplification. It was seen that this overexpression promotes metastatic properties as cell ability to extravasate or to seed at a distant site, through the downregulation of FOX3 and MITF-M [122].

Another miRNA that is upregulated in metastatic melanoma in comparison with primary melanoma and dysplastic nevi is the miR-21. MiR-21 expression also correlates positively with Breslow thickness, advanced clinical stage and metastatic behavior [123-124]. miR-532-5p also presents higher expression levels in metastatic melanoma tumors than in primary melanomas. An important target of miR-532-5p in metastatic tumor is the transcription factor RUNX3, which may act through β-catenin [125].

miR-221/222 are also upregulated in metastatic cells relative to primary melanomas or melanocytes. They are not expressed in compound nevus, but are progressively increased in the melanoma progression, with the higher level being in the metastases samples. Although they have increased expression in metastatic melanomas, they increase during tumorigenesis, which suggests that they are not metastamiRs *per se* [126].

In comparison with miRNAs that are overexpressed in melanoma, little is known about miRNAs that are diminished during melanoma progression [127], so even less is known about miRNAs downregulated in melanoma metastasis. Fortunately, there is some information in this area and we will present it here.

The miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) is important in melanomadevelopment,howeverits functionis controversial,withsomeshowingthatmiR-200 is upregulated during melanoma progression and some showing that it is downregulated. Importantly for metastasis, miR-200c was shown to be downregulated in metastatic melano‐ ma compared with primary melanoma and melanocytic nevi[128].In addition, miR-200c levels were different in melanoma samples located at primary versus distant metastatic sites [129]. The role in suppressing metastasis was confirmed by a miR-200c overexpression assay, which showed inhibition of melanoma growth and metastasis *in vivo*.It appears that one pathway that this miRNA act is through Bmi-1 (a Polycomb group protein) and E-cadherin, which, togeth‐ er, regulate proliferation and motility [128]. Interestingly, the combination of miR-200c overexpression with the tumor vaccine B16F10/GPI-IL-21 potentiated the metastasis inhibi‐ tioneffectofthevaccine,whichhaspreviouslyshownsomeprotectiveanti-melanomaimmunity but failed to completely inhibit tumor and metastases development [130].

One of the first discovered miRNAs, let-7b, is also related with the aggressiveness of melano‐ ma. This miRNA has been implicated as a tumor suppressorin several tumors and its levels are diminished in metastatic melanoma cell lines compared to primary melanoma cell lines [131]. The ectopic expressionoflet-7binthehighlymetastatic cellsB16-F10originatedagreatdecrease in the number of tumor nodules formed in the lung. The authors also suggested that the low expression of miR-7b facilitates metastasis through the upregulation of its target Basigin (an extracellular matrix metalloproteinase inducer), that consequently increases MMPs produc‐ tion [132].

MiR-9 is also frequently lost in metastatic melanoma tissues related to primary melanoma tissues. In addition, the same expression was observed in metastatic melanoma cell lines compared with cells from radial growth phase melanomas and vertical growth phase mela‐ nomas. The low expression of miR-9 correlates with high cell proliferation, migration, and metastasis development *in vivo*. The low levels of this miRNA stimulate the metastatic phenotype through the upregulation of NF-kB (a direct target of miR-9) and consequent high levels of Snail and downregulation of E-cadherin [133].

Others miRNAs that are downregulated in metastatic melanoma in comparison with primary melanoma are the miR-20b and miR-145, however to confirm their role in metastasis it would be important to do other experiments beyond its expression analysis. The mechanism of action of miR-145 is still unknown [134], however miR-20b seems to act through the regulation of the proteinase-activated receptor 1 (PAR-1), a thrombin receptor that it is involved in thrombosis and hemostasis and appears to have a key role in the progress of malignant melanoma [135].

Although there are still limited studies about the role of miRNAs in the process of melanoma metastasis, it is already a fact that the miRNA circuit is altered in this process and therefore the miRNA processing machinery may be deregulated. However, this has been poorly studied. A study shows that TARBP2 e SND1 (two RISC components) are overexpressed in cutaneous malignant melanoma metastases related to primary cutaneous malignant melanoma. This study also shows that Dicer, Drosha and other molecules of the miRNA machinery are not altered in the metastasis process [136]. In another study with tissue samples, it was observed Dicer upregulation in metastatic melanoma in comparison with *in situ* melanoma. Further‐ more, high expression of Dicer correlated with aggressive cancer features as increased tumor mitotic index, Breslow's depth of invasion and nodal metastasis and with metastasis to the non-sentinel lymph node. High levels of Dicer expression also appears to be related with elevate rates of metastasis to organs and to the sentinel lymph node, but this relation was not statistically significant [131]. Both studies were performed with a restrict number of samples, so further studies are important to confirm which enzymes of the miRNA machinery are altered in the melanoma metastasis process.

## **8. Conclusion**

The miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) is important in melanomadevelopment,howeverits functionis controversial,withsomeshowingthatmiR-200 is upregulated during melanoma progression and some showing that it is downregulated. Importantly for metastasis, miR-200c was shown to be downregulated in metastatic melano‐ ma compared with primary melanoma and melanocytic nevi[128].In addition, miR-200c levels were different in melanoma samples located at primary versus distant metastatic sites [129]. The role in suppressing metastasis was confirmed by a miR-200c overexpression assay, which showed inhibition of melanoma growth and metastasis *in vivo*.It appears that one pathway that this miRNA act is through Bmi-1 (a Polycomb group protein) and E-cadherin, which, togeth‐ er, regulate proliferation and motility [128]. Interestingly, the combination of miR-200c overexpression with the tumor vaccine B16F10/GPI-IL-21 potentiated the metastasis inhibi‐ tioneffectofthevaccine,whichhaspreviouslyshownsomeprotectiveanti-melanomaimmunity

One of the first discovered miRNAs, let-7b, is also related with the aggressiveness of melano‐ ma. This miRNA has been implicated as a tumor suppressorin several tumors and its levels are diminished in metastatic melanoma cell lines compared to primary melanoma cell lines [131]. The ectopic expressionoflet-7binthehighlymetastatic cellsB16-F10originatedagreatdecrease in the number of tumor nodules formed in the lung. The authors also suggested that the low expression of miR-7b facilitates metastasis through the upregulation of its target Basigin (an extracellular matrix metalloproteinase inducer), that consequently increases MMPs produc‐

MiR-9 is also frequently lost in metastatic melanoma tissues related to primary melanoma tissues. In addition, the same expression was observed in metastatic melanoma cell lines compared with cells from radial growth phase melanomas and vertical growth phase mela‐ nomas. The low expression of miR-9 correlates with high cell proliferation, migration, and metastasis development *in vivo*. The low levels of this miRNA stimulate the metastatic phenotype through the upregulation of NF-kB (a direct target of miR-9) and consequent high

Others miRNAs that are downregulated in metastatic melanoma in comparison with primary melanoma are the miR-20b and miR-145, however to confirm their role in metastasis it would be important to do other experiments beyond its expression analysis. The mechanism of action of miR-145 is still unknown [134], however miR-20b seems to act through the regulation of the proteinase-activated receptor 1 (PAR-1), a thrombin receptor that it is involved in thrombosis and hemostasis and appears to have a key role in the progress of malignant melanoma [135]. Although there are still limited studies about the role of miRNAs in the process of melanoma metastasis, it is already a fact that the miRNA circuit is altered in this process and therefore the miRNA processing machinery may be deregulated. However, this has been poorly studied. A study shows that TARBP2 e SND1 (two RISC components) are overexpressed in cutaneous malignant melanoma metastases related to primary cutaneous malignant melanoma. This study also shows that Dicer, Drosha and other molecules of the miRNA machinery are not altered in the metastasis process [136]. In another study with tissue samples, it was observed Dicer upregulation in metastatic melanoma in comparison with *in situ* melanoma. Further‐

but failed to completely inhibit tumor and metastases development [130].

66 Melanoma – Current Clinical Management and Future Therapeutics

levels of Snail and downregulation of E-cadherin [133].

tion [132].

Cutaneous melanoma is a melanocytic tumor whose incidence and mortality are on the rise worldwide. Prognosis of patients with melanoma depends on the stage of the tumor and is usually based on microstaging and radiological evaluation of metastases. Among the numer‐ ous prognostic parameters, tumor thickness is the most sensitive in predicting the risk of metastasis. However, it is still difficult to determine the prognosis for melanoma patients individually. More specific prognostic indicators for metastatic melanoma are needed.

The homeostasis of skin melanocytes is controlled by the keratinocytes of the epidermis and the balance between these cells is maintained by regulating the division of melanocytes. Mutations in genes regulating growth, changes in the production of paracrine growth factors and loss of adhesion molecules (integrins, cadherins, selectins and family of immunoglobulins) contribute to the disruption of cell signaling. Thus, the melanocytes may escape regulation, spread and proliferate and thereby form melanoma.

Several genes are involved in the pathogenesis of malignant melanoma. The best known in metastatic melanoma are the mutations in BRAF, MITF and changes in WNT and PI3K pathways. Due to the heterogeneity of metastatic lesions many changes have been described for metastatic melanoma, thus leading to greater difficulty of classifying major pathways altered in melanoma. Therefore, studies on the molecular biology of melanoma seeking to identify molecular markers for prognosis are interesting. Immunohistochemical (Mel-CAM), enzyme (Tyrosinase), protein (integrins, ICAM-1, cyclin D1), microRNAs and genetic (CDKN2A, p53) markers could be used for this purpose.

In this way, although there are several molecular alterations described in melanoma metasta‐ sis, there are some inconsistent data, probably due driver factors, as the heterogeneity of metastatic lesions, difficulty of correctly classifying the different stages of the tumor and difficulty in obtaining appropriate samples. Moreover, there is complicacy in the classification of cell lines and use of inappropriate technique. For example, today it is well established that metastatic characteristics can only be analyzed *in vivo*, but there are cases that only *in vitro* assays, as migration and invasion assays, are performed and the authors quote an involvement with metastasis. In addition, the same cell line is considered as highly aggressive in some studies and as poorly metastatic in others. These issues complicate a proper understanding of the melanoma metastasis biology.

## **Acknowledgements**

This work was supported by FAPESP (2012/08776-5 to MGJ, 2013/04829-0 to ACM, and 2010/18715-8 to MT) and CNPq (470681/2012-8 to MGJ).

### **Author details**

Ana Carolina Monteiro, Mariana Toricelli and Miriam Galvonas Jasiulionis\*

\*Address all correspondence to: mjasiulionis@gmail.com

Pharmacology Department, Universidade Federal de São Paulo, São Paulo, SP, Brazil

Ana Carolina Monteiro and Mariana Toricelli share the first authorship

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## **Chapter 4**

## **Surgery and Staging of Melanoma**

Rolland Gyulai, Zsolt Kádár and Zsuzsanna Lengyel

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59471

## **1. Introduction**

Surgical management of melanoma encompasses primary biopsy and complete wide local excision (WLE) of the tumor, as well as the surgical treatment of metastatic melanoma, including both cutaneous and internal metastases. The management of a melanoma suspect lesion starts with the initial biopsy of the lesion. A great variety of techniques can be used for the biopsy of melanomas, and the choice of the technique is dependent on multiple factors (patient's age, strength of clinical suspicion of melanoma, thickness of the tumor, localization, etc.). Once the clinical diagnosis of melanoma is confirmed histologically, local tumor control is established by wide local excision. The margins for WLE are determined by the T stage of the tumor, and are between 0.5-2.0 cm. Certain clinical melanoma types (e.g. lentigo maligna) require special attention, as the recommended margins can differ from those generally advised for other melanoma types.

Important surgical procedures include wide local excision with safety margins, sentinel lymph node biopsy, regional lymph node dissection and reconstruction of defects after melanoma excision. A plastic surgeon of the appropriate specialty should perform the excision and reconstruction.

Performing surgery of locoregional and distant metastases depends on various factors. However, in highly selected patients, complete surgical resection of the metastases may result in prolonged survival. In addition, combination of surgery with novel immuno-, and targeted therapies, may result in an even better outcome in the future for patientswith stage IV disease.

© 2015 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.

## **2. Primary treatment of suspicious pigmented lesion/melanoma**

Early diagnosis and complete removal of the malignant cells are of paramount importance in the treatment of malignant melanoma. This usually requires a two-step approach. First, pigmented or amelanotic lesions suspicious for melanoma should be promptly biopsied and submitted to pathological evaluation, and second, the tumor should be subsequently excised with adequate surgical margins. The margins of the final excision are determined with the tumor characteristics in mind, as determined by the histopathological analysis of the biopsy specimen. Thus, removal of appropriate biopsy sample containing the fragment with the worst prognostic characteristics, is of substantial importance. As extensive loss of tissues may potentially influence the feasibility of further surgical interventions, such as the sentinel lymph node biopsy, the use of proper biopsy techniques is essential during the primary treatment of melanoma.

Recommendations regarding the width of the surgical margin of excision are nowadays clearly defined for primary melanoma, and are based on the histopathological features of the mela‐ noma. These recommendations, however, are sometimes difficult or impossible to follow, like in the case of specially localized melanomas, or certain melanoma subtypes. This chapter summarizes the available evidence regarding different biopsy techniques and the surgical management of primary melanoma.

#### **2.1. Biopsy of melanoma suspect lesions**

The primary aim of performing biopsy in the case of a melanoma suspect lesion is to establish or exclude the diagnosis of melanoma. An additional goal is to ensure accurate pathological staging of the tumor in order to enable adequate surgical management by performing wide local excision (WLE). Excisional, incisional and shave biopsy techniques are used in the surgical treatment of melanoma.

#### *2.1.1. Excisional biopsy*

The preferred biopsy technique for most melanomas is excisional biopsy.[1,2] This means that the entire lesion is removed with an additional 1-3 mm margin of normal-appearing skin. Wider excisions, however, should be avoided, to permit subsequent lymphatic mapping for sentinel lymph node biopsy. Generally, the excised tissue sample should contain part of the subcutaneous fat as well, and should be oriented to aid subsequent histopathological evalua‐ tion. The positioning of the excision also should possibly allow for subsequent wider excisions. The excisional biopsy technique can be used in most melanomas, when primary closure of the wound is feasible. Although the lowest frequency of positive margins is reported when excisional biopsy is used, positive margins and even residual melanoma on WLE do occur.[3]

#### *2.1.2. Incisional biopsy*

The reported frequency of excisional biopsy technique used for diagnosing melanoma varies significantly with centers, countries, and individuals, and ranges between 10 and 86 percent. [1,3-6] Thus, in a significant portion of melanoma suspect lesions, a biopsy technique other than excisional biopsy are used. Even guidelines that emphasize the importance of excisional biopsy in melanoma management state that incisional biopsy may be appropriate in certain clinical circumstances. Such clinical scenarios may include cases when excisional biopsy is not feasible due to the large size or the location (nose, ear, face, palm, and sole) of the lesion, concerns about cosmesis or low clinical suspicion of melanoma.

In the case of incisional biopsy only a portion of the lesion is removed, either by a punch biopsy or using a scalpel (Figure 1.a-c). As incisional biopsy specimens contain only part of the lesion, concerns regarding misdiagnosis, staging inaccuracy or diagnostic uncertainty may arise. Careful selection of the biopsy site is therefore crucial to ensure that the biopsy best represents the entire lesion, both in terms of the type and the T stage of the tumor. This is usually achieved by sampling the thickest, most raised area of the tumor, or the darkest part of flat lesions. Complex lesions containing multiple suspicious foci may require more than one simultaneous biopsy sampling. Both in case of punch biopsy and incisional biopsy adequate depth (reaching to the subcutaneous fat) of the sampling should be guaranteed.

The theory that incisional biopsy, by cutting through the neoplastic tissue, represents an increased risk for lymphatic or hematologous metastatisation, and thus it should be regarded as a harmful procedure, has been refuted by several earlier studies.[3,7] Moreover, Molenkamp et al. reported slightly better survival in patients with residual tumor cells in their re-excision samples, compared to patients without residual cells in their re-excision specimen.[3] The authors speculated that immunity against residual tumor cells, triggered by biopsy induced wound healing, might be responsible for this finding.

### *2.1.3. Shave biopsy*

**2. Primary treatment of suspicious pigmented lesion/melanoma**

82 Melanoma – Current Clinical Management and Future Therapeutics

melanoma.

management of primary melanoma.

surgical treatment of melanoma.

*2.1.1. Excisional biopsy*

*2.1.2. Incisional biopsy*

**2.1. Biopsy of melanoma suspect lesions**

Early diagnosis and complete removal of the malignant cells are of paramount importance in the treatment of malignant melanoma. This usually requires a two-step approach. First, pigmented or amelanotic lesions suspicious for melanoma should be promptly biopsied and submitted to pathological evaluation, and second, the tumor should be subsequently excised with adequate surgical margins. The margins of the final excision are determined with the tumor characteristics in mind, as determined by the histopathological analysis of the biopsy specimen. Thus, removal of appropriate biopsy sample containing the fragment with the worst prognostic characteristics, is of substantial importance. As extensive loss of tissues may potentially influence the feasibility of further surgical interventions, such as the sentinel lymph node biopsy, the use of proper biopsy techniques is essential during the primary treatment of

Recommendations regarding the width of the surgical margin of excision are nowadays clearly defined for primary melanoma, and are based on the histopathological features of the mela‐ noma. These recommendations, however, are sometimes difficult or impossible to follow, like in the case of specially localized melanomas, or certain melanoma subtypes. This chapter summarizes the available evidence regarding different biopsy techniques and the surgical

The primary aim of performing biopsy in the case of a melanoma suspect lesion is to establish or exclude the diagnosis of melanoma. An additional goal is to ensure accurate pathological staging of the tumor in order to enable adequate surgical management by performing wide local excision (WLE). Excisional, incisional and shave biopsy techniques are used in the

The preferred biopsy technique for most melanomas is excisional biopsy.[1,2] This means that the entire lesion is removed with an additional 1-3 mm margin of normal-appearing skin. Wider excisions, however, should be avoided, to permit subsequent lymphatic mapping for sentinel lymph node biopsy. Generally, the excised tissue sample should contain part of the subcutaneous fat as well, and should be oriented to aid subsequent histopathological evalua‐ tion. The positioning of the excision also should possibly allow for subsequent wider excisions. The excisional biopsy technique can be used in most melanomas, when primary closure of the wound is feasible. Although the lowest frequency of positive margins is reported when excisional biopsy is used, positive margins and even residual melanoma on WLE do occur.[3]

The reported frequency of excisional biopsy technique used for diagnosing melanoma varies significantly with centers, countries, and individuals, and ranges between 10 and 86 percent. In the case of shave biopsy a superficial, a few mm deep flat section of the skin is removed (Figure 1.d). Shave biopsy is ideally performed at the level of the deep dermis, however, the depth of excision is often compromised with the desire to provide a cosmetically good result. Shave biopsy of primary melanoma, as it often results in incomplete removal of the tumor and thus compromises pathological staging of the tumor, is not recommended in most of the cases for melanoma biopsy. However, if the index of melanoma suspicion is low, shave biopsy may be performed. When performing shave biopsy in pigmented lesions, deep scoop shave biopsy is the preferred technique.

#### *2.1.4. The effect of biopsy techniques on staging, prognosis and treatment of melanoma*

The biopsy of the pigmented lesion should not only establish or exclude the diagnosis of melanoma, but also provide information on the T stage of the tumor. Ideally, this initial T stage will be the same as the one achieved after the wide local excision of the tumor. This will ensure that the original treatment plan regarding the width of the surgical margin and the requirement for sentinel lymph node biopsy, does not need subsequent adjustment. As these treatment parameters are primarily determined by Breslow's depth of the tumor, achieving appropriate deep margin sampling is of paramount importance during melanoma biopsy.

Theoretically, sampling errors may stem from several scenarios (Figure 1.a-d). Tumor depth determination may be compromised by not representative tissue sampling (Figure 1a), positive deep biopsy margin (Figure 1b) or not representative tumor depth (Figure 1c). Diagnostic inaccuracy, as a consequence of inappropriate tumor depth measurement, may (Figure 1b) or may not (Figure 1c) lead to upstaging after wide local excision. In a recent study, positive deep margins were found in 12%, 32%, 17% and 24% of cases undergoing excisional, shave, punch and incisional biopsies, respectively.[4] After wide local excision, tumor depth was more than the biopsy depth in 44% of the cases with residual tumors in WLE, and resulted in T-stage reclassification in 22% of cases. Reclassification was necessary in 2%, 7%, 24% and 24% of cases when the initial diagnosis was established by excisional, shave, punch or incisional biopsy, respectively. Diagnostic inaccuracy led to subsequent treatment change in 2%, 5%, 18% and 18% of cases when excisional, shave, punch or incisional biopsy, respectively, was used for initial sampling. Although there is no data regarding the tumor thickness in the individual biopsy groups, it is likely that shave biopsy was more frequently used for thinner and incisional techniques for thicker melanomas. This may explain why initial punch and incisional biopsy so frequently required later reclassification in this study.

**Figure 1.** Diagnostic inaccuracy arising from different forms of sampling error using incisional (punch) biopsy or shave biopsy techniques. Inadequate punch biopsy sampling of pigmented lesion (a) may result in non-representative tissue sampling. Tissue sample is removed from the benign part of the pigmented lesion, while missing the malignant part leading to false negative diagnosis. Inadequate depth of punch biopsy sampling (b) results in positive deep mar‐ gin on histology. Accurate T stage cannot be established, and histological reevaluation after WLE will result in the up‐ staging of the tumor from T1 to T2. Inadequate assessment of melanoma depth due to failure in sampling the deepest part of the tumor (c). Accurate T stage cannot be established, however, reevaluation will not result in upstaging. Posi‐ tive deep margin after shave biopsy sampling of melanoma (d). Proper T stage cannot be established, and diagnostic inaccuracy may result in upstaging after WLE.

It must also be emphasized that a rather substantial part of excised melanomas are clinically not suspected to be melanoma, and vice versa, in a smaller, but significant portion of pig‐ mented lesions, the clinical diagnosis of melanoma is not confirmed by the histopathological examination. The mean number of pigmented lesions to be excised to detect one melanoma was 29 (range 11-83) in a study among general practitioners in Perth, Australia.[8] Thus, in cases of clinical uncertainty, shave or incisional biopsies may help to establish early melanoma diagnosis, and biopsies may also help to avoid unnecessary wide excisions in case of benign lesions.

Several studies have compared the effect of biopsy techniques on the prognosis of melanoma. [9-13] While in some studies a decreased survival rate was associated with incisional, shave or needle aspiration biopsy compared to those who had excisional biopsy, these studies included either low number of patients or significant age differences among patient groups. [12,13] Other, more recent studies, involving significantly higher numbers of patients, found no negative effect of non-radical diagnostic techniques on the survival of melanoma patients. [10,11] Moreover, Molenkamp et al. reported slightly better survival in patients with residual tumor cells in their re-excision samples,[3] and speculated that biopsy induced wound healing may theoretically trigger immunity against residual tumor cells. In summary, incisional (punch or scalpel) and shave biopsies may be used for the initial diagnosis of melanoma, although, excisional biopsy, when feasible, is recommended as the first choice.

#### *2.1.5. Biopsy techniques in different melanoma types and special locations*

Theoretically, sampling errors may stem from several scenarios (Figure 1.a-d). Tumor depth determination may be compromised by not representative tissue sampling (Figure 1a), positive deep biopsy margin (Figure 1b) or not representative tumor depth (Figure 1c). Diagnostic inaccuracy, as a consequence of inappropriate tumor depth measurement, may (Figure 1b) or may not (Figure 1c) lead to upstaging after wide local excision. In a recent study, positive deep margins were found in 12%, 32%, 17% and 24% of cases undergoing excisional, shave, punch and incisional biopsies, respectively.[4] After wide local excision, tumor depth was more than the biopsy depth in 44% of the cases with residual tumors in WLE, and resulted in T-stage reclassification in 22% of cases. Reclassification was necessary in 2%, 7%, 24% and 24% of cases when the initial diagnosis was established by excisional, shave, punch or incisional biopsy, respectively. Diagnostic inaccuracy led to subsequent treatment change in 2%, 5%, 18% and 18% of cases when excisional, shave, punch or incisional biopsy, respectively, was used for initial sampling. Although there is no data regarding the tumor thickness in the individual biopsy groups, it is likely that shave biopsy was more frequently used for thinner and incisional techniques for thicker melanomas. This may explain why initial punch and incisional biopsy

**Figure 1.** Diagnostic inaccuracy arising from different forms of sampling error using incisional (punch) biopsy or shave biopsy techniques. Inadequate punch biopsy sampling of pigmented lesion (a) may result in non-representative tissue sampling. Tissue sample is removed from the benign part of the pigmented lesion, while missing the malignant part leading to false negative diagnosis. Inadequate depth of punch biopsy sampling (b) results in positive deep mar‐ gin on histology. Accurate T stage cannot be established, and histological reevaluation after WLE will result in the up‐ staging of the tumor from T1 to T2. Inadequate assessment of melanoma depth due to failure in sampling the deepest part of the tumor (c). Accurate T stage cannot be established, however, reevaluation will not result in upstaging. Posi‐ tive deep margin after shave biopsy sampling of melanoma (d). Proper T stage cannot be established, and diagnostic

It must also be emphasized that a rather substantial part of excised melanomas are clinically not suspected to be melanoma, and vice versa, in a smaller, but significant portion of pig‐ mented lesions, the clinical diagnosis of melanoma is not confirmed by the histopathological examination. The mean number of pigmented lesions to be excised to detect one melanoma

so frequently required later reclassification in this study.

84 Melanoma – Current Clinical Management and Future Therapeutics

inaccuracy may result in upstaging after WLE.

Although, when feasible, excisional biopsy is the recommended technique for initial diagnosis of melanoma, there are certainly significant differences among melanoma subtypes, which, consequently, require different surgical approaches. The most critical factors when choosing the appropriate biopsy technique are clinical estimate of depth and size and localization of the lesion. Although there are no general rules regarding biopsy techniques for different clinical types of melanoma, some practical recommendations can be formulated. It must be empha‐ sized as well that in case of lesions requiring amputation of the anatomical unit (e.g. digit or ear), histological confirmation of the diagnosis of melanoma is necessary before performing the final procedure.


excisional biopsy. Therefore, especially when surgical management involves amputation, an initial incisional biopsy is essential to confirm the clinical diagnosis of melanoma.


#### **2.2. Wide Local Excision (WLE) of malignant melanoma**

Once the histopathological examination of the biopsy sample established the diagnosis of melanoma, the entire tumor should be surgically removed with adequate safety margins from the surrounding healthy-appearing skin. The wide local excision is intended to provide adequate surgical control of the tumor spread by removing all tumor cells from the primary tumor bed and the potential satellite lesions from the immediate vicinity of the tumor. Additionally, WLE provides tissue samples for the final T staging of melanoma.

#### *2.2.1. General recommendations for surgical margins for wide local excision of melanoma*

Current recommendations for surgical management of melanoma are based on randomized clinical trials completed several years ago.[14-20] The margin of wide local excision depends on the T stage of the melanoma, which is primarily determined by the depth of tumor invasion (see Table 1. for staging). While there has been considerable debate regarding the radicality of surgery, current guidelines recommend 0.5 – 2.0 cm surgical margins (see Table 1. for recom‐ mended surgical margins).[1]

#### *2.2.2. Surgical margins for wide local excision in clinical types and special anatomical regions*

Most melanomas on the trunk and the proximal part of the extremities may be surgically managed according to the generally recommended margins for re-excision. Certain melanoma types, however, owing to their unique localization (face, acral region of the extremities) or type (lentigo maligna), require special approach, and allow only compromised excisional margins.

#### *2.2.2.1. Melanoma in situ (lentigo maligna and non lentigo maligna type)*

In general, the NCCN guideline recommends 0.5-1.0 mm margins for in situ melanomas.[1] These recommendations, however, are based on expert consensus, as there are no randomized prospective studies that have examined the surgical margins for melanoma in situ. Recently it has been shown that almost all (99%) melanoma in situ lesions are completely removed with a 0.9 mm margin, and a margin of 0.6 mm provides negative resection margins in 86% of cases.


**Table 1.** Summary of T staging and current National Comprehensive Cancer Network (NCCN) recommended wide local excision margins for melanoma.

[21] Another recent study found that in situ melanoma lesions that were not lentigo maligna type, were unlikely to recur if completely removed, even with narrow margins (Figure 2.a).[22] On the other hand, a significantly higher incomplete excision rate was found in the lentigo maligna group (Figure 2.b), compared with the non-lentigo maligna type in situ melanomas (29.3% vs. 5.9%, respectively). Thus, the authors propose more aggressive treatment, if possible, for in situ melanomas of lentigo maligna type.

#### *2.2.2.2. Eyelid melanoma*

excisional biopsy. Therefore, especially when surgical management involves amputation, an initial incisional biopsy is essential to confirm the clinical diagnosis of melanoma.

**•** *Ulcerated and/or regressive melanomas* should be managed with special attention, as these clinical features are usually associated with poorer outcome. Furthermore, both ulceration and regression can interfere with histopathological evaluation, and can obscure the establishment of proper diagnosis. Therefore, excisional biopsy sampling is highly recom‐

**•** *Melanomas in special anatomical regions* (eyelid, ear, nose, genitoanal) usually require individual approach, as excisional biopsy sampling is often not feasible with primary wound closure. For that reason, it is recommended that an incisional biopsy is taken before

Once the histopathological examination of the biopsy sample established the diagnosis of melanoma, the entire tumor should be surgically removed with adequate safety margins from the surrounding healthy-appearing skin. The wide local excision is intended to provide adequate surgical control of the tumor spread by removing all tumor cells from the primary tumor bed and the potential satellite lesions from the immediate vicinity of the tumor.

Current recommendations for surgical management of melanoma are based on randomized clinical trials completed several years ago.[14-20] The margin of wide local excision depends on the T stage of the melanoma, which is primarily determined by the depth of tumor invasion (see Table 1. for staging). While there has been considerable debate regarding the radicality of surgery, current guidelines recommend 0.5 – 2.0 cm surgical margins (see Table 1. for recom‐

mended in these cases to avoid false negative diagnosis or understaging.

Additionally, WLE provides tissue samples for the final T staging of melanoma.

*2.2.1. General recommendations for surgical margins for wide local excision of melanoma*

*2.2.2. Surgical margins for wide local excision in clinical types and special anatomical regions*

*2.2.2.1. Melanoma in situ (lentigo maligna and non lentigo maligna type)*

Most melanomas on the trunk and the proximal part of the extremities may be surgically managed according to the generally recommended margins for re-excision. Certain melanoma types, however, owing to their unique localization (face, acral region of the extremities) or type (lentigo maligna), require special approach, and allow only compromised excisional margins.

In general, the NCCN guideline recommends 0.5-1.0 mm margins for in situ melanomas.[1] These recommendations, however, are based on expert consensus, as there are no randomized prospective studies that have examined the surgical margins for melanoma in situ. Recently it has been shown that almost all (99%) melanoma in situ lesions are completely removed with a 0.9 mm margin, and a margin of 0.6 mm provides negative resection margins in 86% of cases.

final management of pigmented lesions in these locations.

**2.2. Wide Local Excision (WLE) of malignant melanoma**

86 Melanoma – Current Clinical Management and Future Therapeutics

mended surgical margins).[1]

There is no generally accepted consensus regarding the appropriate surgical margins for eyelid melanomas. In this melanoma group, guidelines for WLE are impractical, and cannot be used in the majority of cases. In a recent retrospective study, local, nodal and distant metastases occurred in 21%, 11% and 4% of 56 cases with eyelid melanomas, respectively.[23] Pathological margins of >2 mm were associated with increased disease-free survival, compared with margins ≤2 mm. Lower eyelid melanomas were found to have significantly higher recurrence rate than upper eyelid tumors.

#### *2.2.2.3. External ear melanoma*

Although external ear melanoma had been considered to be a more aggressive type of melanoma, this hypothesis is not supported by more recent evidence. Histologically, melano‐ mas arising on the external ear are most frequently superficial spreading melanomas (33-46%), followed almost equally by lentigo maligna (19.6%-26%) and nodular (16-22%) types. While narrower excisional margins and Mohs surgery are gaining acceptance in the treatment of ear 2.2.2.5. Mucosal melanoma of the female genitalia

melanoma as well, the use of these techniques are associated with significant (30%) recurrence rates.[24] Therefore, management should follow standard melanoma treatment recommen‐ dations in external ear melanoma cases, if feasible (Figure 2.c). Although 70% of patients present with clinically localized disease, the overall prognosis of this melanoma type is poor. Surgical management of vulvar melanoma (Figure 2.e) consist of wide excision with a 1-cm margin for melanomas with a thickness of <1 mm, and a 2-cm margin for thicker lesions.[27,28]

#### *2.2.2.4. Mucosal melanoma of the head and neck* 2.2.2.6. Mucosal melanoma of the male genitalia Melanoma or the glans, preputium or urethra is certainly an uncommon entity. Therefore, standard

Achieving melanoma-free resection margins is often difficult in this melanoma type (Figure 2.d). This may be attributed to the close proximity of critical anatomic structures, the presence of satellite formation, multifocality, angiolymphatic invasion, and submucosal spread, which are common features in oral cavity and sinonasal melanoma. The 2-year and 5-year survival rates for mucosal melanoma of the head and neck are 54% and 32%, respectively. Taking into account the high recurrence rate in this melanoma subtype, even apparently localized lesions may require radical surgery with planned reconstruction.[25,26] WLE provided effective local control for low stage penile (Figure 2.f) or urethral melanomas and all scrotal lesions.[29] 2.2.2.7. Anorectal melanoma Because of the rarity and the advanced stage at which most patients present, a standard surgical intervention has not been established to date for anorectal melanoma. Usually, wide local excision (with negative margin) is the preferred surgrical management in most patients. Extensive disease that is not amenable to local excision, may require abdominoperineal resection.[30]

recommendations are not available for the management of this subtype of melanoma. Partial penectomy or

Figure 2. Melanomas in special anatomical regions require individual surgical approach. In case of in situ melanoma not lentigo maligna type (a), a 5-6 mm surgigal margin is sufficient to ensure clear resection margins. In case of lentigo maligna type in situ melanomas (b), a wider, 10 mm margin is recommended, if feasible. Management of external ear melanomas (c) should follow standard melanoma treatment recommendations, if feasible. Mucosal melanomas of the head and neck region (d) require radical surgery with planned reconstruction in most cases. For mucosal melanoma of the female genitalia (e) wide excision with a 1-2 cm margin is recommended. Melanoma or the glans, preputium or urethra (f) wide local excision or penectomy provides effective local control. **Figure 2.** Melanomas in special anatomical regions require individual surgical approach. In case of in situ melanoma not lentigo maligna type (a), a 5-6 mm surgical margin is sufficient to ensure clear resection margins. In case of lentigo maligna type in situ melanomas (b), a wider, 10 mm margin is recommended, if feasible. Management of external ear melanomas (c) should follow standard melanoma treatment recommendations, if feasible. Mucosal melanomas of the head and neck region (d) require radical surgery with planned reconstruction in most cases. For mucosal melanoma of the female genitalia (e) wide excision with a 1-2 cm margin is recommended. Melanoma of the glans, preputium or urethra (f) wide local excision or penectomy provides effective local control.

### *2.2.2.5. Mucosal melanoma of the female genitalia*

Although 70% of patients present with clinically localized disease, the overall prognosis of this melanoma type is poor. Surgical management of vulvar melanoma (Figure 2.e) consist of wide excision with a 1-cm margin for melanomas with a thickness of <1 mm, and a 2-cm margin for thicker lesions.[27,28]

#### *2.2.2.6. Mucosal melanoma of the male genitalia*

Melanoma of the glans, preputium or urethra is certainly an uncommon entity. Therefore, standard recommendations are not available for the management of this subtype of melanoma. Partial penectomy or WLE provided effective local control for low stage penile (Figure 2.f) or urethral melanomas and all scrotal lesions.[29]

#### *2.2.2.7. Anorectal melanoma*

melanoma as well, the use of these techniques are associated with significant (30%) recurrence rates.[24] Therefore, management should follow standard melanoma treatment recommen‐

melanomas with a thickness of <1 mm, and a 2-cm margin for thicker lesions.[27,28]

localized lesions may require radical surgery with planned reconstruction.[25,26]

32%, respectively. Taking into account the high recurrence rate in this melanoma subtype, even apparently

Although 70% of patients present with clinically localized disease, the overall prognosis of this melanoma type is poor. Surgical management of vulvar melanoma (Figure 2.e) consist of wide excision with a 1-cm margin for

Melanoma or the glans, preputium or urethra is certainly an uncommon entity. Therefore, standard recommendations are not available for the management of this subtype of melanoma. Partial penectomy or WLE provided effective local control for low stage penile (Figure 2.f) or urethral melanomas and all scrotal

 Because of the rarity and the advanced stage at which most patients present, a standard surgical intervention has not been established to date for anorectal melanoma. Usually, wide local excision (with negative margin) is the preferred surgrical management in most patients. Extensive disease that is not amenable to local

b) c)

Figure 2. Melanomas in special anatomical regions require individual surgical approach. In case of in situ melanoma not lentigo maligna type (a), a 5-6 mm surgigal margin is sufficient to ensure clear resection margins. In case of lentigo maligna type in situ melanomas (b), a wider, 10 mm margin is recommended, if feasible. Management of external ear melanomas (c) should follow standard melanoma treatment recommendations, if feasible. Mucosal melanomas of the head and neck region (d) require radical surgery with planned reconstruction in most cases. For mucosal melanoma of the female genitalia (e) wide excision with a 1-2 cm margin is recommended. Melanoma or the glans, preputium or urethra (f) wide local excision or penectomy provides

**Figure 2.** Melanomas in special anatomical regions require individual surgical approach. In case of in situ melanoma not lentigo maligna type (a), a 5-6 mm surgical margin is sufficient to ensure clear resection margins. In case of lentigo maligna type in situ melanomas (b), a wider, 10 mm margin is recommended, if feasible. Management of external ear melanomas (c) should follow standard melanoma treatment recommendations, if feasible. Mucosal melanomas of the head and neck region (d) require radical surgery with planned reconstruction in most cases. For mucosal melanoma of the female genitalia (e) wide excision with a 1-2 cm margin is recommended. Melanoma of the glans, preputium or

f)

Achieving melanoma-free resection margins is often difficult in this melanoma type (Figure 2.d). This may be attributed to the close proximity of critical anatomic structures, the presence of satellite formation, multifocality, angiolymphatic invasion, and submucosal spread, which are common features in oral cavity and sinonasal melanoma. The 2-year and 5-year survival rates for mucosal melanoma of the head and neck are 54% and 32%, respectively. Taking into account the high recurrence rate in this melanoma subtype, even apparently localized lesions

a) c) d)

dations in external ear melanoma cases, if feasible (Figure 2.c).

2.2.2.6. Mucosal melanoma of the male genitalia

2.2.2.5. Mucosal melanoma of the female genitalia

88 Melanoma – Current Clinical Management and Future Therapeutics

may require radical surgery with planned reconstruction.[25,26]

excision, may require abdominoperineal resection.[30]

*2.2.2.4. Mucosal melanoma of the head and neck*

2.2.2.7. Anorectal melanoma

d) e)

lesions.[29]

effective local control.

urethra (f) wide local excision or penectomy provides effective local control.

Because of the rarity and the advanced stage at which most patients present, a standard surgical intervention has not been established to date for anorectal melanoma. Usually, wide local excision (with negative margin) is the preferred surgical management in most patients. Extensive disease that is not amenable to local excision, may require abdominoperineal resection.[30]

#### **2.3. Surgical techniques**

#### *2.3.1. Neck and face*

The face is an important area because it encompasses the eyes, nose, mouth and it is in proximity to the ears. The surgical radicality of extension must often be compromised to avoid injury to these structures. These structures limit the excision margins for surgical treatment of melanomas occurring on the face. In the management of cutaneous melanoma the first step is the wide local excision.

The first goal is to treat the cancer with maximal protection of function and aesthetics. We need take notice of the size and the localization of the defect, what kind of tissues are missing (bone, muscle, fat, skin, cartilage, etc.), the base of the wound, the acceptable functional impairment, the morbidity of the donor area, the moveable area around the defect, the history of the patient (previous operation or irradiation) and the expectations of the patient too. Ideally incisions should be within the relaxed skin tension lines (RSTL) or parallel to them, so the scars will be functionally and aesthetically superior. The RSTL is a complex interaction of the external and internal factors, which contains the skin as well.

If there is a melanoma on the neck, middle or lower area of the face, we can perform an elliptical excision parallel to the RSTL with a 3 to 5 mm safety border, undermining the surrounding area and closing the wound primarily.[17] If the tumor size is bigger we need to prepare local flaps to cover the defect. Local flaps' blood supply are very reliable, random pattern from the surrounding tissues or axial pattern from a named source artery. The laxity, quality and texture surrounding areas, due to irradiation or previous operations.

2.3. Surgical techniques

2.3.1. Neck and Face

of the surrounding skin is the best to prepare local flaps and it is the nearest approach to the defect's skin. the base of the wound, the acceptable functional impairment, the morbidity of the donor area, the moveable area around the defect, the history of the patient (previous operation or irradiation) and the expectations of the patient too. Ideally incisions should be within the relaxed skin tension lines (RSTL) or parallel to them, so the scars will be

The face is an important area because it encompasses the eyes, nose, mouth and it is in proximity to the ears. The surgical radicality of extension must often be compromised to avoid injury to these structures. These structures limit the excision margins for surgical treatment of melanomas occurring on the face. In the

The first goal is to treat the cancer with maximal protection of function and aesthetics. We need take notice of the size and the localization of the defect, what kind of tissues are missing (bone, muscle, fat, skin, cartilage, etc.),

If the size of the defect is too big and there is no possibility for local flaps,skin grafting can be performed. There are split-thickness and full-thickness skin grafting. The split-thickness skin is0.25-0.75 mm thick, the procedure is simple, fast, and not demanding. The donor area heals spontaneously and is fit to be used again as a donor area, although the graft may contract and become hypo-or hyperpigmented. The full-thickness skin is 0.8-1.1 mm thick, it rarely contracts and/or gets pigmented, it is more resistant against external cues compared to split-thickness grafts, and the subcutaneous layer may regenerate. On the other hand, this surgical technique ismore demanding for the patient, and it requires a good blood supply of therecipient area, which limits the size of the graft. Lastly, the donor area need to be sutured /closed primarily. functionally and aesthetically superior. The RSTL is a complex interaction of the external and internal factors, which contains the skin as well. If there is a melanoma on the neck, middle or lower area of the face, we can perform an elliptical excision parallel to the RSTL with a 3 to 5 mm safety border, undermining the surrounding area and closing the wound primarily.[17] If the tumor size is bigger we need to cut local flaps to cover the defect. Local flaps' blood supply are very reliable, random pattern from the surrounding tissues or axial pattern from a named source artery. The laxity, quality and texture of the surrounding skin is the best to prepare local flaps and it is the nearest approach to the defect's skin. If the size of the defect is too big and there is no possiblity for local flaps, we can choose skin grafting. There are split-thickness and full-thickness skin grafting. The split-thickness skin is between 0.25-0.75 mm thick, it is single, fast, not so demanding, the donor area heals spontaneously and is fit to be used again as a donor area, but the

Recommended flaps are: rotational facial flaps, bilobed flaps, transpositonal flaps, V-Y advancement flaps (Figure 3.). It is uncommon to use distant flaps on the face except for some cases, when there is not enough skin in the surrounding areas, due to irradiation or previous operations. graft could be scaring and hypo- or hyperpigmented. The full-thickness skin is between 0.8-1.1 mm thick, it will be rarely scaring and/or pigmented, it is more resistant against external insult, the subcutaneous layer can develop, but it is more demanding for the patient, it requires a good blood supply of the recipient area, that is why the size of the graft is limited and the donor area need to be sutured. Recommended flaps are: rotational facial flaps, bilobed flaps, transpositonal flaps, V-Y advancement flaps. It is uncommon to use distant flaps on the face except for some cases, when there is not enough skin in the

 Figure 3. Removal of extensive melanoma from the face and reconstruction with combined local flaps. Melanoma (lentigo maligna type) on the face. Note markings of planned flaps (a). Extensive tissue defect after excision of melanoma with surgical margins. Reconstruction with combined local flaps (b). Good cosmetical result on 10th postoperative day (c). **Figure 3.** Removal of extensive melanoma from the face and reconstruction with combined local flaps. Melanoma (lenti‐ go maligna type) on the face. Note markings of planned flaps (a). Extensive tissue defect after excision of melanoma with surgical margins. Reconstruction with combined local flaps (b). Good cosmetical result on 10th postoperative day (c).

#### 2.3.2. Eye After primary melanoma excision the choice of repair depends on the size and location of the defect, surrounding tissue mobility, degree of vascular compromise, extent of lamella loss, skin texture and color match. It is not *2.3.2. Eye*

common to excise and primarily close the wounds around the eyes.[31] In general it is done, if the size of the melanoma is very small, the melanoma is in situ, there is a sufficient skin laxity, or the patient's general health status does not permit more intensive surgical intervention. In the periocular region a 5 mm margin of excision for thin eyelid melanomas is recommended.[32,33] Care must be taken to avoid placing too much tension on the eyelid. If the defect involves one third of the eyelid margins, we can perform pentagonal wedge closure after the excision. After primary melanoma excision the choice of repair depends on the size and location of the defect, surrounding tissue mobility, degree of vascular compromise, extent of lamella loss, skin texture and color match. It is not common to excise and primarily close the wounds around the eyes.[31] In general it is done, if the size of the melanoma is very small, the melanoma is in situ, there is a sufficient skin laxity, or the patient's general health status does not permit more intensive surgical intervention. In the periocular region a 5 mm margin of excision for thin eyelid melanomas is recommended.[32,33] Care must be taken to avoid placing too much tension on the eyelid. If the defect involves one third of the eyelid margins, we can perform pentagonal wedge closure after the excision.

We have to pay attention to the canthal regions and the lower eyelid to avoid their injuries, leading to dryness and ectropium. We should also pay attention to the eyebrows and try to reconstruct them.

of the surrounding skin is the best to prepare local flaps and it is the nearest approach to the

management of cutaneous melanoma the first step is the wide local excision.

90 Melanoma – Current Clinical Management and Future Therapeutics

of the graft is limited and the donor area need to be sutured.

surrounding areas, due to irradiation or previous operations.

pentagonal wedge closure after the excision.

a) b) c)

The face is an important area because it encompasses the eyes, nose, mouth and it is in proximity to the ears. The surgical radicality of extension must often be compromised to avoid injury to these structures. These structures limit the excision margins for surgical treatment of melanomas occurring on the face. In the

The first goal is to treat the cancer with maximal protection of function and aesthetics. We need take notice of the size and the localization of the defect, what kind of tissues are missing (bone, muscle, fat, skin, cartilage, etc.), the base of the wound, the acceptable functional impairment, the morbidity of the donor area, the moveable area around the defect, the history of the patient (previous operation or irradiation) and the expectations of the patient too. Ideally incisions should be within the relaxed skin tension lines (RSTL) or parallel to them, so the scars will be functionally and aesthetically superior. The RSTL is a complex interaction of the external and internal factors,

If the size of the defect is too big and there is no possibility for local flaps,skin grafting can be performed. There are split-thickness and full-thickness skin grafting. The split-thickness skin is0.25-0.75 mm thick, the procedure is simple, fast, and not demanding. The donor area heals spontaneously and is fit to be used again as a donor area, although the graft may contract and become hypo-or hyperpigmented. The full-thickness skin is 0.8-1.1 mm thick, it rarely contracts and/or gets pigmented, it is more resistant against external cues compared to split-thickness grafts, and the subcutaneous layer may regenerate. On the other hand, this surgical technique ismore demanding for the patient, and it requires a good blood supply of therecipient area, which limits the size of the graft. Lastly, the donor area need to be sutured /closed primarily.

If there is a melanoma on the neck, middle or lower area of the face, we can perform an elliptical excision parallel to the RSTL with a 3 to 5 mm safety border, undermining the surrounding area and closing the wound primarily.[17] If the tumor size is bigger we need to cut local flaps to cover the defect. Local flaps' blood supply are very reliable, random pattern from the surrounding tissues or axial pattern from a named source artery. The laxity, quality and texture of the surrounding skin is the best to prepare local flaps and it is the nearest approach to the

If the size of the defect is too big and there is no possiblity for local flaps, we can choose skin grafting. There are split-thickness and full-thickness skin grafting. The split-thickness skin is between 0.25-0.75 mm thick, it is single, fast, not so demanding, the donor area heals spontaneously and is fit to be used again as a donor area, but the graft could be scaring and hypo- or hyperpigmented. The full-thickness skin is between 0.8-1.1 mm thick, it will be rarely scaring and/or pigmented, it is more resistant against external insult, the subcutaneous layer can develop, but it is more demanding for the patient, it requires a good blood supply of the recipient area, that is why the size

Recommended flaps are: rotational facial flaps, bilobed flaps, transpositonal flaps, V-Y advancement flaps (Figure 3.). It is uncommon to use distant flaps on the face except for some cases, when there is not enough skin in the surrounding areas, due to irradiation or previous

Recommended flaps are: rotational facial flaps, bilobed flaps, transpositonal flaps, V-Y advancement flaps. It is uncommon to use distant flaps on the face except for some cases, when there is not enough skin in the

 Figure 3. Removal of extensive melanoma from the face and reconstruction with combined local flaps. Melanoma (lentigo maligna type) on the face. Note markings of planned flaps (a). Extensive tissue defect after excision of melanoma with surgical

**Figure 3.** Removal of extensive melanoma from the face and reconstruction with combined local flaps. Melanoma (lenti‐ go maligna type) on the face. Note markings of planned flaps (a). Extensive tissue defect after excision of melanoma with surgical margins. Reconstruction with combined local flaps (b). Good cosmetical result on 10th postoperative day (c).

After primary melanoma excision the choice of repair depends on the size and location of the defect, surrounding tissue mobility, degree of vascular compromise, extent of lamella loss, skin texture and color match. It is not common to excise and primarily close the wounds around the eyes.[31] In general it is done, if the size of the melanoma is very small, the melanoma is in situ, there is a sufficient skin laxity, or the patient's general health status does not permit more intensive surgical intervention. In the periocular region a 5 mm margin of excision for thin eyelid melanomas is recommended.[32,33] Care must be taken to avoid placing too much tension on the eyelid. If the defect involves one third of the eyelid margins, we can perform pentagonal wedge closure after the

After primary melanoma excision the choice of repair depends on the size and location of the defect, surrounding tissue mobility, degree of vascular compromise, extent of lamella loss, skin texture and color match. It is not common to excise and primarily close the wounds around the eyes.[31] In general it is done, if the size of the melanoma is very small, the melanoma is in situ, there is a sufficient skin laxity, or the patient's general health status does not permit more intensive surgical intervention. In the periocular region a 5 mm margin of excision for thin eyelid melanomas is recommended.[32,33] Care must be taken to avoid placing too much tension on the eyelid. If the defect involves one third of the eyelid margins, we can perform

margins. Reconstruction with combined local flaps (b). Good cosmetical result on 10th postoperative day (c).

defect's skin.

which contains the skin as well.

2.3. Surgical techniques

2.3.1. Neck and Face

operations.

2.3.2. Eye

*2.3.2. Eye*

excision.

defect's skin.

In this region primarily we perform local flaps and skin grafting or cartilage grafting to cover the defects after melanoma excision. The local flaps can be skin, skin-subcutaneous and skinmuscles flaps from the surrounding area, where the laxity, quality and texture are the best suited to cover these defects. Their blood supply can show random pattern from the subcuta‐ neous layer or axial pattern from a named source artery. In these cases it is advisable to do cantopexy to prevent ectropium evolution.

Recommended flaps are: V-Y advancement flap, Tenzel and Mustarde rotation flaps, Romboid-Limberg transpositional flaps, Median and Paramedian forehead flaps and Glabellar flap.[31,33] We have to pay attention to the canthal regions and the lower eyelid to avoid their injuries, leading to dryness and ectropium. We should also pay attention to the eyebrows and try to reconstruct them. In this region firstly we perform local flaps and skin grafting or cartilage grafting to cover the defects after melanoma excision. The local flaps can be skin, skin-subcutaneous and skin-muscles flaps from the surrounding

Full-thickness skin graft is used in this region to minimize scaring and pigmentation. It protects against extrinsic factors and the subcutaneous layer may regenerate. Disadvantages of fullthickness skin grafting include demanding surgery, the need for good blood supply in the recipient area and the need for suturing in the donor area. The size of this graft is also limited (Figure 4.). area, where the laxity, quality and texture are the best suited to cover these defects. Their blood supply can show random pattern from the subcutaneous layer or axial pattern from a named source artery. In these cases it is advisable to do cantopexy to prevent ectropium evaluation. Recommended flaps are: V-Y advancement flap, Tenzel and Mustarde rotation flaps, Romboid-Limberg transpositional flaps, Median and Paramedian forehead flaps and Glabellar flap.[31,33]

If the tumor involves the tarsal plates and after the excision of the melanoma a tarsal defect develops, auricular or nasal free cartilage graft can be used to cover the defect. Generally, the cartilage graft can be covered with a local flap to reconstruct the total eyelid layers. In these cases composite grafts may be used as well, which contain skin, cartilage and, if necessary, conjunctiva. The skin graft is full thickness skin to minimize scaring and pigmenttation. It guards against extrinsic factors and a subcutaneous layer can develop. Disadvantages of full thickness skin grafting include demanding surgery, the need for good blood supply in the recipient area, the need for suturing in donor area. The size of this graft is also limited. If the tumor involves the tarsal plates and after the excision of the melanoma a tarsal defect develops, auricular or nasal free cartilage graft can be used to cover the defect. Generally, the cartilage graft can be covered with a

Occasionally, if the patient's general health status is poor, it is acceptable in the medial canthal area to leave the defect open, and let it heal by second-intention. Adequate wound manage‐ ment and dressing should be applied to help the granulation and epithelisation. Another use of second-intention healing is to delay skin grafting. After granulation the skin graft procedure can be performed.[31,33] local flap to reconstruct the total eyelid layers. In these cases composite grafts may be used as well, which contain skin, cartilage and, if necessary, conjunctiva. Occasionally, if the patient's general health status is bad, it is acceptable in the medial canthal area to leave the defect open, and let it heal by second-intention. Adequate wound management and dressing should be applied to help the granulation and epithelisation. Another use of second-intention healing is to delay skin grafting. After granulation the skin graft procedure can be performed.[31,33]

defect (a) and full-thickness skin graft covering of the defect (b). 2.3.3. Nose **Figure 4.** Full-thickness skin grafting for the reconstruction of lower eyelid defect after excision of melanoma. Lower eyelid defect (a) and full-thickness skin graft covering of the defect (b).

Figure 4. Full-thickness skin grafting for the reconstruction of lower eyelid defect after excision of melanoma. Lower eyelid

The nose is a three-layered structure of a skin and fibrofatty covering, a bony and cartilaginous framework and an inner lining of vestibular skin and nasal mucosa. Nasal defect may involve these layers alone or in combination.[34,35] The nose is divided into topographic subunits, as tip, dorsum, sidewalls, alar lobules, soft triangles and columella.[34,35] A wide range of techniques including defect and subunit reconstruction – using simple as well as more complex flap and multistaged procedures – must be in the surgeon's armamentarium. When a defect involves greater than 50% of a subunit, replacement of the entire subunit should be considered. We can perform primary wound closure after elliptical excision with undermining the surrounding area only on the dorsal region if the size of the tumor is not too large and the skin is loose. Second-intention healing is good only for superficial wounds on concave surfaces.[34] If the tumor size is bigger we can use local flaps from the middle face and the frontal region. (Figure 5. and 6.) The nasal tip, columella and alar regions are more difficult, and primary closure is usually not possible. Instead we use local flaps, skin grafting and composite grafting in these regions. The advantages of local flaps over skin grafts include better contour, color and texture match and less scar contracture. The most common local flap is the nasolabial flap, while the forehead flap remains the workhorse for major nasal reconstruction with numerous modifications.[34] Some others are the dorsonasal flap, glabellar flap, advancement flap from the middle face, and flaps from the upper perioral region. These local flaps are useful for defects of about 2 cm or less. If the tumor is large enough to require a tip and alar nose amputation, we can use reconstructive surgical methods or prosthetic devices. Full-thickness skin grafting is easy to perform and it is useful in the reconstruction of superficial defects in the areas of the tip and alar lobules (Figure 7.). On

#### *2.3.3. Nose*

The nose is a three-layered structure of a skin and fibrofatty covering, a bony and cartilaginous framework and an inner lining of vestibular skin and nasal mucosa. Nasal defect may involve these layers alone or in combination.[34,35] The nose is divided into topographic subunits, as tip, dorsum, sidewalls, alar lobules, soft triangles and columella.[34,35] A wide range of techniques including defect and subunit reconstruction – using simple as well as more complex flap and multistaged procedures – must be in the surgeon's armamentarium. When a defect involves greater than 50% of a subunit, replacement of the entire subunit should be considered. We can perform primary wound closure after elliptical excision with undermining the surrounding area only on the dorsal region if the size of the tumor is not too large and the skin is loose. Second-intention healing is good only for superficial wounds on concave surfaces.[34] If the tumor size is bigger we can use local flaps from the middle face and the frontal region. (Figure 5. and 6.) The nasal tip, columella and alar regions are more difficult, and primary closure is usually not possible. Instead we use local flaps, skin grafting and composite grafting in these regions. The advantages of local flaps over skin grafts include better contour, color and texture match and less scar contracture. The most common local flap is the nasolabial flap, while the forehead flap remains the workhorse for major nasal reconstruction with numerous modifications.[34] Some others are the dorsonasal flap, glabellar flap, advancement flap from the middle face, and flaps from the upper perioral region. These local flaps are useful for defects of about 2 cm or less. If the tumor is large enough to require a tip and alar nose amputation, we can use reconstructive surgical methods or prosthetic devices. Full-thickness skin grafting is easy to perform and it is useful in the reconstruction of superficial defects in the areas of the tip and alar lobules (Figure 7.). On the other hand, it may heal with a contrasting flattened and shiny appearance.[34,35] The term composite graft means that skin and cartilage are grafted together from the conchal region, helical rim or helical root.

#### *2.3.4. Ear*

On the ear after wide excision, which includes skin and subcutaneous tissue removing, primary closure is usually not performed. There are many options for the reconstruction of aurical defects including direct closure, second-intention healing, full thickness skin grafts and local flaps.[36,37] The defects of the posterior wall of the ear, where the skin is more abundant and loose, can often be closed primarily. If the defect is in the central and anterior region with intact cartilage, most defects will do well by second intention healing or full thickness skin grafting.[37] If the defect involves the cartilage the most common surgical procedure is wedge excision, which means excising the skin and cartilage together in a V or W form. After these excisions we should reconstruct the cartilage and then the skin. If larger excisions are necessary, we need to use local flaps, which are from the earlobe, pre-and retroauricular regions. These flaps iclude direct advancement flap, rotational flaps, transposition and subcutaneous island flaps. If more than one third of the ear is involved by the tumor, we need to perform partial amputation. If the tumor is in an advanced stage, it may be necessary to amputate the whole ear. Amputation requires more complex reconstructive surgical methods to restore the ear or prosthetic devices can be used.

**Figure 5.** Glabellar flap for the reconstruction of a defect on the dorsum of the nose after wide excision of melanoma. Extensive melanoma on the dorsum of the nose (a). The defect and preparing glabellar flap (b). Suturing the flap (c) and postoperative results 3 weeks after operation (d).

**Figure 6.** Forehead flap for the resonstruction of nasal and glabellar defect after melanoma excision. Melanoma on the nose and glabellar region (a). After ecxision and performing forehead flap (b) Suturing the flap. Good cosmetical result (c).

#### *2.3.5. Mouth and perioral region*

*2.3.3. Nose*

92 Melanoma – Current Clinical Management and Future Therapeutics

*2.3.4. Ear*

prosthetic devices can be used.

The nose is a three-layered structure of a skin and fibrofatty covering, a bony and cartilaginous framework and an inner lining of vestibular skin and nasal mucosa. Nasal defect may involve these layers alone or in combination.[34,35] The nose is divided into topographic subunits, as tip, dorsum, sidewalls, alar lobules, soft triangles and columella.[34,35] A wide range of techniques including defect and subunit reconstruction – using simple as well as more complex flap and multistaged procedures – must be in the surgeon's armamentarium. When a defect involves greater than 50% of a subunit, replacement of the entire subunit should be considered. We can perform primary wound closure after elliptical excision with undermining the surrounding area only on the dorsal region if the size of the tumor is not too large and the skin is loose. Second-intention healing is good only for superficial wounds on concave surfaces.[34] If the tumor size is bigger we can use local flaps from the middle face and the frontal region. (Figure 5. and 6.) The nasal tip, columella and alar regions are more difficult, and primary closure is usually not possible. Instead we use local flaps, skin grafting and composite grafting in these regions. The advantages of local flaps over skin grafts include better contour, color and texture match and less scar contracture. The most common local flap is the nasolabial flap, while the forehead flap remains the workhorse for major nasal reconstruction with numerous modifications.[34] Some others are the dorsonasal flap, glabellar flap, advancement flap from the middle face, and flaps from the upper perioral region. These local flaps are useful for defects of about 2 cm or less. If the tumor is large enough to require a tip and alar nose amputation, we can use reconstructive surgical methods or prosthetic devices. Full-thickness skin grafting is easy to perform and it is useful in the reconstruction of superficial defects in the areas of the tip and alar lobules (Figure 7.). On the other hand, it may heal with a contrasting flattened and shiny appearance.[34,35] The term composite graft means that skin and cartilage are grafted

On the ear after wide excision, which includes skin and subcutaneous tissue removing, primary closure is usually not performed. There are many options for the reconstruction of aurical defects including direct closure, second-intention healing, full thickness skin grafts and local flaps.[36,37] The defects of the posterior wall of the ear, where the skin is more abundant and loose, can often be closed primarily. If the defect is in the central and anterior region with intact cartilage, most defects will do well by second intention healing or full thickness skin grafting.[37] If the defect involves the cartilage the most common surgical procedure is wedge excision, which means excising the skin and cartilage together in a V or W form. After these excisions we should reconstruct the cartilage and then the skin. If larger excisions are necessary, we need to use local flaps, which are from the earlobe, pre-and retroauricular regions. These flaps iclude direct advancement flap, rotational flaps, transposition and subcutaneous island flaps. If more than one third of the ear is involved by the tumor, we need to perform partial amputation. If the tumor is in an advanced stage, it may be necessary to amputate the whole ear. Amputation requires more complex reconstructive surgical methods to restore the ear or

together from the conchal region, helical rim or helical root.

Melanomas in the perioral regions are not common. The tumor can involve the skin, the oral orbicular muscle and the mucosal layer alone or together.[37] In this region we should pay

**Figure 7.** Split-thickness skin grafting for the reconstruction of extensive defect on the tip of the nose after excision of melanoma. Melanoma involving the tip and alar lobules of the nose (a-b) Split-thickness skin graft covering of the de‐ fect (c).

attention to protect the muscular-, sensorial-, and the closing function of the mouth, the adequate oral access for eating and to the use of dentures, to the symmetry, the upper/lower lip ratio and the quality of scars.[37] After tumor excision, if the defect is less than one half of the lip width, we can use primary wound closure after undermining the surrounding layers. Primary closure offers the best aesthetic result and should be prioritized. In the upper lip we can perform wedge excision while in the lower lip W-shape excision is used. In cases of large tumor size the defect can be covered by local skin-, and skin-mucosal flaps originating from the perioral region or from the lips and the inner surface of the mouth. The ideal donor areas for labial reconstruction are the remaining labial tissue and the opposing lip.[37] The lip is elastic and can be elongated, which is a very useful for reconstruction.

Recommended local flaps are: V-Y advancement flap, Rotational flap, Nasolabial flap, Forehead flap, Abby flap, Estlander flap, Karapandzic flap (Figure 8.).

#### *2.3.6. Digits*

The skin of the hand and toe is specialized and structurally unique, balancing the need for sensing, mobility for complex motor skills on the hand, durability to withstand wear and tear on the toe.[38] Earlier melanomas arising on the skin and/or nail bed of the digits were mostfrequently managed with amputation at the proximal joint from the tumor. Recently tissue- sparing excision is performed increasingly.[38] When excising melanomas on the toes, amputations should be limited to preserve as much length and function of the digit as possible without compromising the necessary safety border. It requires a more conservative surgery, wide excision and only partial resection of the affected phalanx.[38] The excision is done in the subcutaneous layer and on the fingertip, the entire nail complex needs to be removed. In these cases the defect can be covered with a local flap, like V-Y advancement flap, or we can use local flaps from the neighboring digits. If the bone is not directly involved it is not necessary to remove the total phalanx or metacarpus or metatarsus, since the removal of bone in these localisations does not have oncological benefit (Figure 9.).

On the ear after wide excision, which includes skin and subcutaneous tissue removing, primary closure is usually not performed. There are many options for the reconstruction of aurical defects including direct closure, secondintention healing, full thickness skin grafts and local flaps.[36,37] The defects of the posterior wall of the ear, where the skin is more abundant and loose, can often be closed primarily. If the defect is in the central and anterior region with intact cartilage, most defects will do well by second intention healing or full thickness skin grafting.[37] If the defect involves the cartilage the most common surgical procedure is wedge excision, which means excising the skin and cartilage together in a V or W form. After these excisions we should reconstruct the cartilage and then the skin, too. If larger excisions are necessary, we need to use local flaps, which are from the earlobe, pre- and retroauricular regions. These flaps iclude direct advancement flap, rotational flaps, transposition and subcutaneous island flaps. If more than one third of the ear is involved by the tumor, we need to perform partial amputation. If the tumor is in a progressive status, it may be necessary to amputate the whole ear. Amputation requires more difficult reconstructive surgical methods to restore the ear or we can use prosthetic

Melanomas in the perioral regions are not so common, but the tumor can involve the skin, the oral orbicular muscle and the mucosal layer alone or together.[37] In this region we should pay attention to protect the muscular function, sensorial function and the closing function of the mouth, an adequate oral access for eating and using dentures, symmetry, upper/lower lip ratio and quality of scars.[37] After tumor excision, if the defect is less than one half of the lip width, we can use primary wound closure after undermining the surrounding layers. Primary closure offers the best aesthetic result and should be prioritized. In the upper lip we can perform wedge excision while in the lower lip W-shape excision is used. In cases of large tumor size, after the excision we make local skin flaps and skin-mucosal flaps from the perioral region or from the lips and the inside of the mouth. The ideal donor areas for labial reconstruction are the remaining labial tissue and the opposing lip.[37] The lip is elastic and can

be elongated, which is a very useful characteristic for reconstructive surgery.

2.3.4. Ear

devices.

2.3.5. Mouth and perioral region

Estlander flap, Karapandzic flap.

attention to protect the muscular-, sensorial-, and the closing function of the mouth, the adequate oral access for eating and to the use of dentures, to the symmetry, the upper/lower lip ratio and the quality of scars.[37] After tumor excision, if the defect is less than one half of the lip width, we can use primary wound closure after undermining the surrounding layers. Primary closure offers the best aesthetic result and should be prioritized. In the upper lip we can perform wedge excision while in the lower lip W-shape excision is used. In cases of large tumor size the defect can be covered by local skin-, and skin-mucosal flaps originating from the perioral region or from the lips and the inner surface of the mouth. The ideal donor areas for labial reconstruction are the remaining labial tissue and the opposing lip.[37] The lip is

**Figure 7.** Split-thickness skin grafting for the reconstruction of extensive defect on the tip of the nose after excision of melanoma. Melanoma involving the tip and alar lobules of the nose (a-b) Split-thickness skin graft covering of the de‐

Recommended local flaps are: V-Y advancement flap, Rotational flap, Nasolabial flap,

The skin of the hand and toe is specialized and structurally unique, balancing the need for sensing, mobility for complex motor skills on the hand, durability to withstand wear and tear on the toe.[38] Earlier melanomas arising on the skin and/or nail bed of the digits were mostfrequently managed with amputation at the proximal joint from the tumor. Recently tissue- sparing excision is performed increasingly.[38] When excising melanomas on the toes, amputations should be limited to preserve as much length and function of the digit as possible without compromising the necessary safety border. It requires a more conservative surgery, wide excision and only partial resection of the affected phalanx.[38] The excision is done in the subcutaneous layer and on the fingertip, the entire nail complex needs to be removed. In these cases the defect can be covered with a local flap, like V-Y advancement flap, or we can use local flaps from the neighboring digits. If the bone is not directly involved it is not necessary to remove the total phalanx or metacarpus or metatarsus, since the removal of bone in these

elastic and can be elongated, which is a very useful for reconstruction.

94 Melanoma – Current Clinical Management and Future Therapeutics

Forehead flap, Abby flap, Estlander flap, Karapandzic flap (Figure 8.).

localisations does not have oncological benefit (Figure 9.).

*2.3.6. Digits*

fect (c).

advancement flap, or we can use local flaps from the neighboring digits. If the bone is not directly involved it is not necessary to remove the total phalanx or metacarpus or metatarsus, since the removal of bone in these localisations does not have oncological benefit (Figure 9.). Figure 8. Reconstruction of facial defect with local flap after removal of melanoma. Large defect in the nasolabial region after melanoma excision (a). Preparing the local flap (b). Closing the wound with sutures (c). Good postoperative result 2 weeks after the operation (d). **Figure 8.** Reconstruction of facial defect with local flap after removal of melanoma. Large defect in the nasolabial re‐ gion after melanoma excision (a). Preparing the local flap (b). Closing the wound with sutures (c). Good postoperative result 2 weeks after the operation (d).

entire nail complex needs to be removed. In these cases the defect can be covered with a local flap, like V-Y

Figure 9. Surgical management of amelanotic melanoma on the toe. Melanoma present on the distal phalanx of the toe (a).

Melanomas arising between the digits is difficult to treat. Covering to these defects can be done with full thickness skin grafts and local flaps. It may be necessary to remove the phalanx and metacarpuses and/or metatarsuses

Acral lentiginous melanoma tends to be diagnosed at later stages due to medical diagnostic mistakes and patients' poor attention to lesions arising on extremities.[38] The reconstruction of the defects after melanoma excision on the dorsal or plantar region of the hand is challenging. Generally we can use full thickness skin grafts or local flaps and we should pay attention to retaining function. First, it is important to cover the joints, tendons and bones. If the defect is large we can use distal flaps with microsurgical techniques. It is important to mind the weight-bearing regions of the foot, because reconstructing these defects requires a flap from the adjacent area or a free flap with microvascular anastomoses that contain adequate soft tissue to cover the defect and to supply the

Locoregional recurrence can occur as regional nodal disease or as satellite or in-transit metastases.

In-transit metastases are locoregional relapses found between the primary melanoma and the draining lymphatic basin. By definition lesions that occur more than 2 cm from the primary melanoma are termed in-transit metastases. The ones that are located closer (≤ 2 cm) are regarded as satellite lesions. The risk factors for the development of in-transit metastases include lymph node involvement and was confirmed as the most important prognostic factor by Weide et al.[39] Furthermore the risk of local recurrence increases significantly as the thickness of the primary melanoma increases and with the presence of ulceration.[15,39] Both satellite and intransit metastases are regarded as stage IIIB (without regional nodal metastases) or stage IIIC (with regional nodal metastases) disease by the 2009 American Joint Committee on Cancer staging system and are associated with worse prognosis than local recurrence.[40] Patient with locoregional recurrences should undergo staging procedures (e.g. PET, CT scans) to rule out presence of distant metastatic disease. If there is no evidence of

**Figure 9.** Surgical management of amelanotic melanoma on the toe. Melanoma present on the distal phalanx of the toe (a). Amputation at the distal phalanx (b). Suturing of the wound (c).

Amputation at the distal phalanx (b). Suturing of the wound (c).

depending on the progression of the tumor.

3. Surgery of locoregional recurrence of melanoma

2.3.7. Interdigital spaces

2.3.8. Hand and foot

function too.

#### *2.3.7. Interdigital spaces*

Melanomas arising between the digits is difficult to treat. Covering to these defects can be done with full thickness skin grafts and local flaps. It may be necessary to remove the phalanx and metacarpuses and/or metatarsuses depending on the progression of the tumor.

#### *2.3.8. Hand and foot*

Acral lentiginous melanoma tends to be diagnosed at later stages due to medical diagnostic mistakes and patients' poor attention to lesions arising on extremities.[38] The reconstruction of the defects after melanoma excision on the dorsal or plantar region of the hand is challeng‐ ing. Generally we can use full thickness skin grafts or local flaps and we should pay attention to retaining function. First, it is important to cover the joints, tendons and bones. If the defect is large we can perform distal flaps with microsurgical techniques. It is important to mind the weight-bearing regions of the foot, because reconstructing these defects requires a flap from the adjacent area or a free flap with microvascular anastomoses that contain adequate soft tissue to cover the defect and to supply the function too.

## **3. Surgery of locoregional recurrence of melanoma**

Locoregional recurrence can occur as regional nodal disease or as satellite or in-transit metastases.

In-transit metastases are locoregional relapses found between the primary melanoma and the draining lymphatic basin. By definition lesions that occur more than 2 cm from the primary melanoma are termed in-transit metastases. The ones that are located closer (≤ 2 cm) are regarded as satellite lesions (Figure 10). The risk factors for the development of in-transit metastases include lymph node involvement and was confirmed as the most important prognostic factor by Weide et al.[39] Furthermore the risk of local recurrence increases significantly as the thickness of the primary melanoma increases and with the presence of ulceration.[15,39] Both satellite and in-transit metastases are regarded as stage IIIB (without regional nodal metastases) or stage IIIC (with regional nodal metastases) disease by the 2009 American Joint Committee on Cancer staging system and are associated with worse prognosis than local recurrence.[40] Patient with locoregional recurrences should undergo staging procedures (e.g. PET, CT scans) to rule out presence of distant metastatic disease. If there is no evidence of extraregional disease, the treatment strategies for in-transit metastatic disease depend on the size, number and location of the lesions.

In case of solitar lesion or limited disease surgical excision of the metastases with histologically negative margins is the adequate treatment. The precise width of surgical margin is not determined. The resection should be with generous margin depending on the anatomic site involved. Multifocal metastases within a circumscribed area may be resected en-bloc. Primary closure is prefered if possible, however skin grafting or flaps may be done for skin coverege. In patients, who have surgical resectable in-tranist metastases and have not had a lymphane‐

*2.3.7. Interdigital spaces*

*2.3.8. Hand and foot*

metastases.

Melanomas arising between the digits is difficult to treat. Covering to these defects can be done with full thickness skin grafts and local flaps. It may be necessary to remove the phalanx and

Acral lentiginous melanoma tends to be diagnosed at later stages due to medical diagnostic mistakes and patients' poor attention to lesions arising on extremities.[38] The reconstruction of the defects after melanoma excision on the dorsal or plantar region of the hand is challeng‐ ing. Generally we can use full thickness skin grafts or local flaps and we should pay attention to retaining function. First, it is important to cover the joints, tendons and bones. If the defect is large we can perform distal flaps with microsurgical techniques. It is important to mind the weight-bearing regions of the foot, because reconstructing these defects requires a flap from the adjacent area or a free flap with microvascular anastomoses that contain adequate soft

Locoregional recurrence can occur as regional nodal disease or as satellite or in-transit

In-transit metastases are locoregional relapses found between the primary melanoma and the draining lymphatic basin. By definition lesions that occur more than 2 cm from the primary melanoma are termed in-transit metastases. The ones that are located closer (≤ 2 cm) are regarded as satellite lesions (Figure 10). The risk factors for the development of in-transit metastases include lymph node involvement and was confirmed as the most important prognostic factor by Weide et al.[39] Furthermore the risk of local recurrence increases significantly as the thickness of the primary melanoma increases and with the presence of ulceration.[15,39] Both satellite and in-transit metastases are regarded as stage IIIB (without regional nodal metastases) or stage IIIC (with regional nodal metastases) disease by the 2009 American Joint Committee on Cancer staging system and are associated with worse prognosis than local recurrence.[40] Patient with locoregional recurrences should undergo staging procedures (e.g. PET, CT scans) to rule out presence of distant metastatic disease. If there is no evidence of extraregional disease, the treatment strategies for in-transit metastatic disease

In case of solitar lesion or limited disease surgical excision of the metastases with histologically negative margins is the adequate treatment. The precise width of surgical margin is not determined. The resection should be with generous margin depending on the anatomic site involved. Multifocal metastases within a circumscribed area may be resected en-bloc. Primary closure is prefered if possible, however skin grafting or flaps may be done for skin coverege. In patients, who have surgical resectable in-tranist metastases and have not had a lymphane‐

metacarpuses and/or metatarsuses depending on the progression of the tumor.

tissue to cover the defect and to supply the function too.

96 Melanoma – Current Clinical Management and Future Therapeutics

depend on the size, number and location of the lesions.

**3. Surgery of locoregional recurrence of melanoma**

**Figure 10.** Satelitte metastases (red arrows) around primary melanoma (a) and multiple cutaneous and subcutaneous in transit metastases on the lower leg (b).

dectomy previously a sentinel lymph node biopsy (SLNB) may be considered.[41,42] Some authors recommend performing SLNB even for patients who had undergone SLN biopsy earlier or lymph node dissection suggesting a potential benefit for proper staging and for administering the adequate therapy.[43,44]

In the presence of multiple, inoperable, locoregional cutaneous metastases on the extremity isolated limb perfusion (ILP) should be considered.[45,46] A systematic review of twenty two studies, including 2 018 patients[47] who had isolated limb perfusion concluded that the median complete response rate to ILP was of 58.20%, with a median overall response rate of 90.35%. Amputation for extensive regional recurrence is rarely indicated, as patients in such cases have a high risk of development of metastases in distant organs and no survival benefit can be achived.

For refractory-, recurrent and for anatomically unresectable lesions intralesional (interferon, interleukin-2) or topical (imiquimod, diphencyprone) therapy, cryosurgery, electrochemo‐ therapy, laser-, radio-, and systemic therapy may also be an effective treatment opition.[48-51] Electrochemotherapy combines inravenous or intralesional cytotoxic drug, most commonly cisplatin or belomycin and intralesional electric pulses (Figure 11.). The electric pulse creates cell membrane poration resulting in a better penetration of the chemotherapeutic agent.[52,53] A study has reported a 72% objective response rate of the total of 54 lesions treated with electrochemotherapy.[53] However, superiority of one over the other has not been proven and the choice of the method depends on individual factors.

Patients suspicious for regional lymphnode recurrence should have a fine needle biopsy to confirm the diagnosis and a workup (PET CT or CT scans) to rule out distant metastases. Then lymphadenectomy should be performed in patients who did not have one or the lymphnode disscetion was uncompleted. For patients who have undergone previous lymphadenectomy, excision of the recurrent tumor is still indicated, if feasible. In this case the marking of the lymphnodes by ultrasound prior the surgery makes the surgeon's job easier. In patients with recurrent disease limited to the regional lymph node basin, completion lymphadenectomy offers the best potentially curative treatment option and can provide excellent long-term survival for selected patients.[54]

**Figure 11.** Electrochemoterapy (bleomycin) of multiple cutaneous and subcutaneous melanoma metastases. Before (a) and 10 days after (b) therapy. Photos courtesy of Erika Kis MD, PhD, Department of Dermatology and Allergology, University of Szeged, Hungary.

## **4. Surgery of stage IV disease**

Metastatic melanoma has a poor prognosis and a median survival of 6-10 months depending on the site of metastasis.[40] These patients are classified as stage IV according to the American Joint Committee on Cancer (AJCC 2009) staging manual and seperated into three groups (Table 2.). Stage IV patients with M1a disease have higher survival rates than patients with lung metastases (M1b), who have a better prognosis than those with M1c disease with or without elevated lactate dehydrogenase serum levels (LDH).[55]


**Table 2.** M classification of distant metastases in melanoma according to AJCC 2009.

Currently there is no gold standard care for treatment of stage IV disease. The therapeutic landscape for melanoma is rapidly changing. The first novel agent showing overall survival benefit in unresectable stage III or metastatic melanoma was an anti-CTLA4 blocking mono‐ clonal antibody (ipilimumab) approved by the FDA in 2011.[56] Since then target therapies have been approved for the treatment of metastatic melanoma (BRAF inhibitors: dabrafenib, vemurafenib, MEK inhibitor: trametinib).[57-59] Moreover new immun (e.g. PD-1 inhibitors) and target therapies are on their way. The impact of these drugs on survival rates are clear and promising, but surgery of distant metastases could increase this rate.

Numerous studies, mainly retrospective, showed that patients in whom complete surgical excision of metastases was carried out have a 5-year survival rate of 15–28 % vs. 5-10% in patients who received systemic therapy alone.[60-64] The prospective trial of the Southwest Oncology Group showed a median overall survival of 21 months (overall survival at 3 and 4 years were 36% and 31% respectively) in 64 patients whose metastases had been completely resected. The majority of the patients had one disease site (n=50) and skin and soft tissue sites were present in more than 50% of the cases. The authors concluded that aggressive surgical therapy with follow up adjuvant therapy can be an appropriate cure for these selected patients. [65] International MMAIT-IV trial further supported the role of surgery for stage IV melanoma. In this prospective trial patients who had undergone complete resection of their metatstatic disease were treated with two types of immunotherapy. The 5-year survival was 40–45%.[66] The Multicenter Selective Lymphadenectomy Trial (MSLT-I) also suggested that patients with complete resection exhibit an improved survival compared to patients receiving systemic therapy alone, regardless of site and number of metastases.[67]

Despite that these data are persuasive for surgery in patients with distant metastases, surgery is rarley used in stage IV melanoma except for palliation. Many oncologists believe that once melanoma has spread to a distant site, surgery is not helpful because patients already have occult micrometastases and circulating tumor cells. In the report of Koyanagi et al[68,69] 52% of stage IV patients had detectable circulating tumor cells. However the presence of tumor cells in the blood do not obviously generate metastatic lesions. Most of stage IV melanoma patients at first have disease progression in one organ and the number of metastases in the site can vary.

**4. Surgery of stage IV disease**

98 Melanoma – Current Clinical Management and Future Therapeutics

University of Szeged, Hungary.

nodes

M1c Distant metastases at other location or

elevated lactate dehydrogenase serum levels (LDH).[55]

Metastatic melanoma has a poor prognosis and a median survival of 6-10 months depending on the site of metastasis.[40] These patients are classified as stage IV according to the American Joint Committee on Cancer (AJCC 2009) staging manual and seperated into three groups (Table 2.). Stage IV patients with M1a disease have higher survival rates than patients with lung metastases (M1b), who have a better prognosis than those with M1c disease with or without

**Figure 11.** Electrochemoterapy (bleomycin) of multiple cutaneous and subcutaneous melanoma metastases. Before (a) and 10 days after (b) therapy. Photos courtesy of Erika Kis MD, PhD, Department of Dermatology and Allergology,

**M classification Site of distant metastases LDH level**

M1b Lung metastases normal

Distant metastases at any location with elevated serum levels of LDH

Currently there is no gold standard care for treatment of stage IV disease. The therapeutic landscape for melanoma is rapidly changing. The first novel agent showing overall survival benefit in unresectable stage III or metastatic melanoma was an anti-CTLA4 blocking mono‐ clonal antibody (ipilimumab) approved by the FDA in 2011.[56] Since then target therapies have been approved for the treatment of metastatic melanoma (BRAF inhibitors: dabrafenib, vemurafenib, MEK inhibitor: trametinib).[57-59] Moreover new immun (e.g. PD-1 inhibitors) and target therapies are on their way. The impact of these drugs on survival rates are clear and

normal

normal elevated

M1a Metastases of the skin, subcutis or lymph nodes beyond regional lymph

**Table 2.** M classification of distant metastases in melanoma according to AJCC 2009.

promising, but surgery of distant metastases could increase this rate.

The advantage of surgical resection of metastatic melanoma is that it may delay disease progression by interrupting the metastatic cascade associated with hematogenous seeding of cells to other sites.[70,71] Surgical resection also decreases tumor burden thus reducing tumorinduced immunosuppression. Metastases greater than 2 cm are eradicated easier with surgery than with systemic treatments.[72] In addition, surgical resection has less side-effects than systemic therapeutic agents.

The recent development of imaging techniques has led to more accurate detection of metasta‐ ses (size of 5-10mm), aiding surgeons in improved delineation of the extent of the disease and planing for the operation. In line with this development in surgical techniques, anesthesia and intensive supportive care have reduced operative mortality and morbidity rate even for multiple metastectomy.[71] It is evident that appropriate patient selection is essential for a good outcome.

Surgery in metastatic diseases is most effective in patients with small number of metastases and/or few metastatic organ sites.[73] Based on the MSLT-I study and Wevers et al, the percent of stage IV patients eligible for surgery can range widely from more than half to only 22%.[67, 74] In deciding about surgery, one should consider underlying co-morbidities, performance status and life expectany. If no survival benefit and/or advantage in quality of life can be achieved with surgical metastectomy, it may be disregarded. It has been shown by numerous studies that complete (R0) resection is associated with a better survial and in all cases com‐ drug for a couple of months[84-86] (Figure 12).

tumour-volume doubling time of >60 days.[73,79,80] (Table 3.).

Feasibility of complete surgical resection (R0)

Table 3. Factors to consider prior to surgical resection in stage IV melanoma

recurrence were the two most important prognostic factors for survival after recurrence.

essential for a good outcome.

 Number of metastases Site of metastases Tumor doubling time Disease free interval Other therapy modalities Acceptable functional deficit

Co-morbidities

The recent development of imaging techniques has led to more accurate detection of metastases (size of 5- 10mm), aiding surgeons in improved delineation of the extent of the disease and planing for the operation. In line with this development in surgical techniques, anesthesia and intensive supportive care have reduced operative mortality and morbidity rate even for multiple metastectomy.[71] It is evident that appropriate patient selection is

Surgery in metastatic diseases is most effective in patients with small number of metastases and/or few metastatic organ sites.[73] Based on the MSLT-I study and Wevers et al, the percent of stage IV patients eligible for surgery can range widely from more than half to only 22%.[67,74] In deciding about surgery, one should consider underlying co-morbidities, performance status and life expectany. If no survival benefit and/or advantage in quality of life can be achieved with surgical metastectomy, it may be disregarded. It has been shown by numerous studies that complete (R0) resection is associated with a better survial and in all cases completness should be strained by the surgeon.[75-78] Further prognostic factors are prolonged disease-free survival and a

Multiple disease sites are not a contraindication to surgical resection but all of the factors mentioned above should be considered before procedure. Reccurent disease can be treated with repeated metastectomy.[81-83] Ollila et al points out that prolonged disease-free interval prior to recurrence and complete surgical metastasectomy of the

In the case of large tumor masses, when surgery can not be carried out, effective systemic treatment prior to surgery is advisable in order to treat the initially unresectable disease. Neoadjuvant setting has been successfully applied in several solid tumors (e.g. breats, head and neck cancer) but it has not been used in advanced cutaneous melanoma, because no effective systemic treatments were available for this disease. The presence of

inoperable metastasis in the left axillary region (a). BRAF inhibitor (vemurafenib) was initiated. After 3 months of BRAF inhibitor treatment the tumor almost completly regressed (b). Subsequently, the patient underwent surgery for the remnant disease (c). In contrast to BRAF inhibitors, ipilimumab seems to be a less effective agent in neoadjuvant setting because of its mechanism of action and relatively slow pattern of time response. However the surgical excision of lesions that **Figure 12.** Neoadjuvant BRAFi in the treatment of melanoma. The 52 year-old patient with unknown primary melano‐ ma and inoperable metastases in the left axillary region (a). BRAF inhibitor (vemurafenib) was initiated. After 3 months of BRAF inhibitor treatment the tumor almost completly regressed (b). Subsequently, the patient underwent surgery for the remnant disease (c).

Figure 12. Neoadjuvant BRAFi in the treatment of melanoma. The 52 year-old patient with unknown primary melanoma and

pletness should be strained by the surgeon.[75-78] Further prognostic factors are prolonged disease-free survival and a tumour-volume doubling time of >60 days.[73,79,80] (Table 3.). are resistant to treatment with ipilimumab may improve outcomes for some patients. Other immuntherapies are in development (PD-1 inhibitors) which show an earlier tumor response compared to ipilimumab.[87] Overall,


**Table 3.** Factors to consider prior to surgical resection in stage IV melanoma

Multiple disease sites are not a contraindication to surgical resection but all of the factors mentioned above should be considered before procedure. Reccurent disease can be treated with repeated metastectomy.[81-83] Ollila et al points out that prolonged disease-free interval prior to recurrence and complete surgical metastasectomy of the recurrence were the two most important prognostic factors for survival after recurrence.

In the case of large tumor masses, when surgery can not be carried out, effective systemic treatment prior to surgery is advisable in order to treat the initially unresectable disease. Neoadjuvant setting has been successfully applied in several solid tumors (e.g. breats, head and neck cancer) but it has not been used in advanced cutaneous melanoma, because no effective systemic treatments were available for this disease. The presence of new systemic therapies (biological and target) may change this. Several case reports have shown the beneficial effects of BRAF inhibitors. Patients with unresectable bulky disease regained surgical suitabilty after taking the drug for a couple of months[84-86] (Figure 12).

Figure 13. Multiple distant soft tissue metastases (a) and multiple distant skin metastases (b).

4.1. Surgery of distant skin, soft tissue and lymphnode metastases (Stage IV M1a)

surgical resection of metastatic lesion in highly selected patients appears to offer a survival advantage over systemic treatment modalities alone. The decision for surgery of stage IV melanoma patients should be discussed at an interdisciplinary tumor board. After complete metastectomy, adjuvant therapy may be indicated as melanoma is likely to recur. Surgical resection should be considered more often than it is currently practiced, since the combined advances in imaging techniques and promising novel systemic agents can improve patients'

Almost 40% of patients with stage IV melanoma have M1a disease.[88] Median survival of patients in this group is 18-40 months. Skin and soft tissue metastases are usually associated with a better prognosis than distant lymph node disease. Positive prognostic factors for M1a disease are fewer lesions, longer disease-free interval, and smaller size of tumors.[61] Skin and soft tissue metastases should be resected as soon as possible before the metastasis becomes large and, if applicable, with wide margins (2 cm). In case of lymphnode metastases regional

infection, and decreased quality of life. Surgical resection for palliation may be indicated in these situations.

**Figure 13.** Multiple distant soft tissue metastases (a) and multiple distant skin metastases (b).

quality of life and clinical outcomes.

pletness should be strained by the surgeon.[75-78] Further prognostic factors are prolonged disease-free survival and a tumour-volume doubling time of >60 days.[73,79,80] (Table 3.).

Figure 12. Neoadjuvant BRAFi in the treatment of melanoma. The 52 year-old patient with unknown primary melanoma and inoperable metastasis in the left axillary region (a). BRAF inhibitor (vemurafenib) was initiated. After 3 months of BRAF inhibitor treatment the tumor almost completly regressed (b). Subsequently, the patient underwent surgery for the remnant disease (c). In contrast to BRAF inhibitors, ipilimumab seems to be a less effective agent in neoadjuvant setting because of its mechanism of action and relatively slow pattern of time response. However the surgical excision of lesions that are resistant to treatment with ipilimumab may improve outcomes for some patients. Other immuntherapies are in development (PD-1 inhibitors) which show an earlier tumor response compared to ipilimumab.[87] Overall,

**Figure 12.** Neoadjuvant BRAFi in the treatment of melanoma. The 52 year-old patient with unknown primary melano‐ ma and inoperable metastases in the left axillary region (a). BRAF inhibitor (vemurafenib) was initiated. After 3 months of BRAF inhibitor treatment the tumor almost completly regressed (b). Subsequently, the patient underwent

The recent development of imaging techniques has led to more accurate detection of metastases (size of 5- 10mm), aiding surgeons in improved delineation of the extent of the disease and planing for the operation. In line with this development in surgical techniques, anesthesia and intensive supportive care have reduced operative mortality and morbidity rate even for multiple metastectomy.[71] It is evident that appropriate patient selection is

Surgery in metastatic diseases is most effective in patients with small number of metastases and/or few metastatic organ sites.[73] Based on the MSLT-I study and Wevers et al, the percent of stage IV patients eligible for surgery can range widely from more than half to only 22%.[67,74] In deciding about surgery, one should consider underlying co-morbidities, performance status and life expectany. If no survival benefit and/or advantage in quality of life can be achieved with surgical metastectomy, it may be disregarded. It has been shown by numerous studies that complete (R0) resection is associated with a better survial and in all cases completness should be strained by the surgeon.[75-78] Further prognostic factors are prolonged disease-free survival and a

Multiple disease sites are not a contraindication to surgical resection but all of the factors mentioned above should be considered before procedure. Reccurent disease can be treated with repeated metastectomy.[81-83] Ollila et al points out that prolonged disease-free interval prior to recurrence and complete surgical metastasectomy of the

In the case of large tumor masses, when surgery can not be carried out, effective systemic treatment prior to surgery is advisable in order to treat the initially unresectable disease. Neoadjuvant setting has been successfully applied in several solid tumors (e.g. breats, head and neck cancer) but it has not been used in advanced cutaneous melanoma, because no effective systemic treatments were available for this disease. The presence of new systemic therapies (biological and target) may change this. Several case reports have shown the beneficial effects of BRAF inhibitors. Patients with unresectable bulky disease regained surgical suitabilty after taking the

Multiple disease sites are not a contraindication to surgical resection but all of the factors mentioned above should be considered before procedure. Reccurent disease can be treated with repeated metastectomy.[81-83] Ollila et al points out that prolonged disease-free interval prior to recurrence and complete surgical metastasectomy of the recurrence were the two most

In the case of large tumor masses, when surgery can not be carried out, effective systemic treatment prior to surgery is advisable in order to treat the initially unresectable disease. Neoadjuvant setting has been successfully applied in several solid tumors (e.g. breats, head and neck cancer) but it has not been used in advanced cutaneous melanoma, because no effective systemic treatments were available for this disease. The presence of new systemic therapies (biological and target) may change this. Several case reports have shown the beneficial effects of BRAF inhibitors. Patients with unresectable bulky disease regained

surgical suitabilty after taking the drug for a couple of months[84-86] (Figure 12).

• Feasibility of complete surgical resection (R0)

surgery for the remnant disease (c).

**Table 3.** Factors to consider prior to surgical resection in stage IV melanoma

important prognostic factors for survival after recurrence.

• Number of metastases • Site of metastases • Tumor doubling time • Disease free interval • Other therapy modalities • Acceptable functional deficit

essential for a good outcome.

 Number of metastases Site of metastases Tumor doubling time Disease free interval Other therapy modalities Acceptable functional deficit

drug for a couple of months[84-86] (Figure 12).

100 Melanoma – Current Clinical Management and Future Therapeutics

Co-morbidities

tumour-volume doubling time of >60 days.[73,79,80] (Table 3.).

Feasibility of complete surgical resection (R0)

Table 3. Factors to consider prior to surgical resection in stage IV melanoma

recurrence were the two most important prognostic factors for survival after recurrence.

a) b) c)

• Co-morbidities

In contrast to BRAF inhibitors, ipilimumab seems to be a less effective agent in neoadjuvant setting because of its mechanism of action and relatively slow pattern of time response. However the surgical excision of lesions that are resistant to treatment with ipilimumab may improve outcomes for some patients. Other immuntherapies are in development (PD-1 inhibitors) which show an earlier tumor response compared to ipilimumab.[87] Overall, surgical resection of metastatic lesion in highly selected patients appears to offer a survival advantage over systemic treatment modalities alone. The decision for surgery of stage IV melanoma patients should be discussed at an interdisciplinary tumor board. After complete metastectomy, adjuvant therapy may be indicated as melanoma is likely to recur. Surgical resection should be considered more often than it is currently practiced, since the combined advances in imaging techniques and promising novel systemic agents can improve patients' quality of life and clinical outcomes. 4.2. Surgery of pulmonary metastases (Stage IV M1b) The lung is the most typical site of visceral metastases (40%) for melanoma. Pulmonary metastases are associated with a longer survival than metastases to other visceral sites.[40] A growing number of studies have shown that pulmonary metastectomy improves survival [89-96] (Table 4). Tafra et al reported that of 984 melanoma patients with lung metastases, the 106 patients that underwent metastectomy had better 5 year survival than patients treated with non-surgical methods (27% vs. 3%, respectively).[79] Chua et al conducted a large single center study with 1737 patients.[89] 292 patients had surgery for lung metastases and the 5 -year survival for this patient group was 38%. According to the various reports, factors predictive of improved survival are: ability to achieve a complete resection, prolonged disease-free interval (>36 months), 2 or fewer pulmonary nodules,[89-91] size of the largest metastasis <2 cm, prior response to chemotherapy/immunotherapy, and male sex.[92] While the disease may recurr, most of the data demonstrate that long term survival can be achieved with repeated metastectomy (in cases of extra-thoracic lesions also) in suitable patients.[91] The presence of multiple and even bilateral pulmonary nodules is not a contraindication to surgery.[89,97] Interestingly, hilar or mediastinal lymph node involvement did not have an effect on survival.[93] In most cases pulmonary metastectomy involves wedge resection and segmentectomy with occasional indication for lobectomy.

#### **4.1. Surgery of distant skin, soft tissue and lymphnode metastases (Stage IV M1a)**

Almost 40% of patients with stage IV melanoma have M1a disease.[88] Median survival of patients in this group is 18-40 months. Skin and soft tissue metastases are usually associated with a better prognosis than distant lymph node disease (Figure 13.). Positive prognostic factors for M1a disease are fewer lesions, longer disease-free interval, and smaller size of tumors.[61] Skin and soft tissue metastases should be resected as soon as possible before the metastases becomes large and, if applicable, with wide margins (2 cm). In case of lymphnode metastases regional lymphnode dissection is performed (for details see section on lymph node dissection). Factors to consider prior to surgical resection in stage IV melanoma are summar‐ ized in Table 3. Complete surgical resection of M1a disease can promote survival up to 60 months, even after recurrence.[81] Metastases can ulcerate causing pain, bleeding, infection, and decreased quality of life. Surgical resection for palliation may be indicated in these situations.

### **4.2. Surgery of pulmonary metastases (Stage IV M1b)**

The lung is the most typical site of visceral metastases (40%) for melanoma. Pulmonary metastases are associated with a longer survival than metastases to other visceral sites.[40] A growing number of studies have shown that pulmonary metastectomy improves survival [89-96] (Table 4). Tafra et al reported that of 984 melanoma patients with lung metastases, the 106 patients that underwent metastectomy had better 5 year survival than patients treated with non-surgical methods (27% vs. 3%, respectively).[79] Chua et al conducted a large single center study with 1737 patients.[89] 292 patients had surgery for lung metastases and the 5-year survival for this patient group was 38%.

According to the various reports, factors predictive of improved survival are: ability to achieve a complete resection, prolonged disease-free interval (>36 months), 2 or fewer pulmonary nodules,[89-91] size of the largest metastasis <2 cm, prior response to chemotherapy/immu‐ notherapy, and male sex.[92] While the disease may recurr, most of the data demonstrate that long term survival can be achieved with repeated metastectomy (in cases of extra-thoracic lesions also) in suitable patients.[91]

The presence of multiple and even bilateral pulmonary nodules is not a contraindication to surgery.[89,97] Interestingly, hilar or mediastinal lymph node involvement did not have an effect on survival.[93] In most cases pulmonary metastectomy involves wedge resection and segmentectomy with occasional indication for lobectomy.


**Table 4.** Studies of pulmonary metastectomy in patients with lung metastases from melanoma

As mentioned earlier, patient selection in Stage IV disease is very important. Tumor doubling time (TDT), an index calculated on the basis of tumour growth rate as detected on the chest radiographs, is one of the major factors predictive of survival and should be used as a consideration in the decision of whether or not to operate.[80] Patients undergoing surgical managment of lung metastases should have pulmonary function and clinical condition

5 year survival (%)

suitable for the operation, controlled primary lesion, metastases that appeared technically resectable on diagnostic imaging, and preoperative biopsy consistent with melanoma. calculated on the basis of tumour growth rate as detected on the chest radiographs, is one of the major factors predictive of survival and should be used as a consideration in the decision of whether or not to operate.[80] Patients undergoing surgical managment of lung metastases should have pulmonary function and clinical

As mentioned earlier, patient selection in Stage IV disease is very important. Tumor doubling time (TDT), an index

Table 4. Studies of pulmonary metastectomy in patients with lung metastases from melanoma

Median OS (months)

The spread of advanced imaging techniques (CT, PET) contribute to the earlier detection of melanoma metastases and give a more precise preoperative image of the location, thus aiding the accurate selection of surgery candidates (Figure 14.). condition suitable for the operation, controlled primary lesion, metastases that appeared technically resectable on diagnostic imaging, and preoperative biopsy consistent with melanoma. The spread of advanced imaging techniques (CT, PET) contribute to the earlier detection of melanoma metastases and give a more precise preoperative image of the location, thus aiding the accurate selection of

Figure 14. Solitary pulmonary metastases in a 40 year-old patient (red arrow). b. Post surgical scarring at the site of the metastases (blue arrow). **Figure 14.** Solitary pulmonary metastases in a 40 year-old patient (red arrow). b. Post surgical scarring at the site of the metastases (blue arrow).

Upper aerodigestive tract metastases of melanoma are extremily rare. The patients are usually symptomatic with hemoptysis or cough. Treatments with approriate aggressive multimodal therapies are needed in such cases (metastectomy, segmental resection, laser excision, external beam radiation).[98-100] Upper aerodigestive tract metastases of melanoma are extremily rare. The patients are usually symptomatic with hemoptysis or cough. Treatments with approriate aggressive multimodal therapies are needed in such cases (metastectomy, segmental resection, laser excision, external beam radiation).[98-100]

In conclusions, findings are suggestive that pulmonary metastectomy for carefully selected patients warrant a favourable outcome. In conclusions, findings are suggestive that pulmonary metastectomy for carefully selected patients warrant a favourable outcome.

#### **4.3. Surgery of visceral metastases**

OS: overall survival

Author Number of patients

undergoing surgery

Andrews et al[91] 86 35 33 Chua et al[89] 292 23 34 Leo et al[93] 282 19 22 Neuman et al[94] 26 40 29 Ollila et al[61] 45 23.1 15.6 Petersen et al[90] 318 19 21 Schunan et al[92] 30 18.3 35.1 Tafra et al[79] 106 23 27 Younes et al[96] 48 32 36

surgery candidates (Figure 14.).

**4.2. Surgery of pulmonary metastases (Stage IV M1b)**

102 Melanoma – Current Clinical Management and Future Therapeutics

survival for this patient group was 38%.

lesions also) in suitable patients.[91]

**Author Number of patients**

OS: overall survival

segmentectomy with occasional indication for lobectomy.

**undergoing surgery**

Andrews et al[91] 86 35 33 Chua et al[89] 292 23 34 Leo et al[93] 282 19 22 Neuman et al[94] 26 40 29 Ollila et al[61] 45 23.1 15.6 Petersen et al[90] 318 19 21 Schunan et al[92] 30 18.3 35.1 Tafra et al[79] 106 23 27 Younes et al[96] 48 32 36

**Table 4.** Studies of pulmonary metastectomy in patients with lung metastases from melanoma

As mentioned earlier, patient selection in Stage IV disease is very important. Tumor doubling time (TDT), an index calculated on the basis of tumour growth rate as detected on the chest radiographs, is one of the major factors predictive of survival and should be used as a consideration in the decision of whether or not to operate.[80] Patients undergoing surgical managment of lung metastases should have pulmonary function and clinical condition

The lung is the most typical site of visceral metastases (40%) for melanoma. Pulmonary metastases are associated with a longer survival than metastases to other visceral sites.[40] A growing number of studies have shown that pulmonary metastectomy improves survival [89-96] (Table 4). Tafra et al reported that of 984 melanoma patients with lung metastases, the 106 patients that underwent metastectomy had better 5 year survival than patients treated with non-surgical methods (27% vs. 3%, respectively).[79] Chua et al conducted a large single center study with 1737 patients.[89] 292 patients had surgery for lung metastases and the 5-year

According to the various reports, factors predictive of improved survival are: ability to achieve a complete resection, prolonged disease-free interval (>36 months), 2 or fewer pulmonary nodules,[89-91] size of the largest metastasis <2 cm, prior response to chemotherapy/immu‐ notherapy, and male sex.[92] While the disease may recurr, most of the data demonstrate that long term survival can be achieved with repeated metastectomy (in cases of extra-thoracic

The presence of multiple and even bilateral pulmonary nodules is not a contraindication to surgery.[89,97] Interestingly, hilar or mediastinal lymph node involvement did not have an effect on survival.[93] In most cases pulmonary metastectomy involves wedge resection and

**Median OS (months) 5 year survival (%)**

#### *4.3.1. Liver metastases (Stage IV M1c)*

Liver metastases can occur both in cases of cutaneous and/or ocular melanoma. It is important to distinguish them, as in metastastic ocular melanoma the liver is the predominant metastatic site (89% of cases) and often the first and only site of metastases.[100,101] In contrast, cutaneous melanoma metastases can occur in the lungs, lymph nodes, brain and soft tissue. Only few patients develop liver (15%-20%) and bowel metastases.[102,103] The difference in metastatic presentation is most likely driven by the absence of lymphatics in the uveal tract, therefore melanoma spreads hematogenously.[104]

Studies evaluating the role of surgery in the treatment of hepatic metastases from melanoma are mainly retrospective case series from single institutions. Some of these studies included non-surgical (chemotherapy, best supportive care etc) comparator arm, while others did not have a control group.[76-78,105-108] (Table 5.)

The comparative studies showed a longer median overall survival in patients who underwent hepatic resection compared to non-surgical treatment in both ocular and cutaneous melano‐ mas. Overall survival was 2-4 months for patients with unresected hepatic metastases versus 28 months for those with completly resected liver metastases.[78] A recent metaanalysis of five studies also revealed a significant improvment in overall survival after surgery compared to non-surgical procedures.[109] The majority of noncomparative studies also reported benefit from resection of metastases.[66,75,110-114]

All studies observed that R0 resection was associated with longer overall survival than R1 or R2 surgery.[75-78]


**Table 5.** Studies of hepatic resection in patients with liver metastases from melanoma

The outcome of surgery is also influenced by the number of metastases, length of disease-free interval and limited disease distribution.[78,115,116] However, eligibility for surgery upon the extent of disease and the number of metastases varied in the studies. In the Kodjikian study[116], the cut-off number for resection liver metastases was 10 or less lesions.

Recurrence rates following hepatic resection in the studies ranged between 72% to 75%. [78,111] All in all the hepatic resection for malignant melanoma is a safe operation.

60-day mortality rates were recorded in some reviews at 1.9% and 2.3% and postoperative complications after metastectomy occured in 15% to 20% of cases.[78,111]

In the treatment of hepatic metastases, systemic and /or non-surgical therapies can be applied in the form of adjuvant or neoadjuvant settings that supplement surgical resection. There are no clear data yet to determine the most efficacious time for the administration of systemic therapy secondary to surgery. Pawlik et al found that patients who had received adjuvant systemic therapy prior the hepatic resection had increased survival compared to patients having resection alone.[114] Adam et al reported increased survival in patients responsive to neoadjuvant chemotherapy.[110]

The data mentioned above indicate that both ocular and cutaneous metastatic melanoma patients with liver metastases benefit from surgery. To achieve this outcome, accurate patient selection is crucial. Only patients with limited disease/metastases who can be rendered surgically free of disease should be considered as candidates for hepatic resection. For patients with unresectable metastatic melanoma, systemic and /or regional (hepatic intra-arterial chemotherapy, hepatic arterial embolization, isolated/percutaneous hepatic perfusion) therapies should be taken into consideration.

#### *4.3.2. Gastrointestinal metastases (Stage IV M1c)*

The comparative studies showed a longer median overall survival in patients who underwent hepatic resection compared to non-surgical treatment in both ocular and cutaneous melano‐ mas. Overall survival was 2-4 months for patients with unresected hepatic metastases versus 28 months for those with completly resected liver metastases.[78] A recent metaanalysis of five studies also revealed a significant improvment in overall survival after surgery compared to non-surgical procedures.[109] The majority of noncomparative studies also reported benefit

All studies observed that R0 resection was associated with longer overall survival than R1 or

Adam et al[110] Cutaneous and ocular 1452 19 21 Caralt et al[117] Cutaneous and ocular NR 26.3 NR Chua et al[115] Cutaneous and ocular 23 21 NR

Faries et al[107] Cutaneous and ocular 1078 24.8 30 Frenkel et al[76] Ocular 74 23 NR Groeschl et al[111] NR NR 39 36 Herman et al[118] Cutaneous and ocular 367 22 NR Kim et al[119] Cutaneous and GI tract NR 9.5 4 Kodjikian et al[116] Ocular 63 20.5 24 Mariani et al[77] Ocular 798 23.0 NR Marshall et al[105] Ocular 188 24.0 NR

Pawlik et al[114] Cutaneous and ocular 40 29.4 21 for ocular

Ocular 470 21 NR

Pilati et al[66] Cutaneous and ocular 36 15 NR

Ripley et al[112] Cutaneous and ocular 539 36 53 Rivoire et al[106] Ocular 63 25 NR Rose et al[78] Cutaneous 1750 28 29 Ryu et al[113] Cutaneous and ocular 33 29 42

**Table 5.** Studies of hepatic resection in patients with liver metastases from melanoma

**Median OS**

32 29 3

**(months) 5 year survival (%)**

from resection of metastases.[66,75,110-114]

104 Melanoma – Current Clinical Management and Future Therapeutics

de Ridder et al[75] Ocular, cutanous and

unknown

**Author Melanoma type Number of patients**

R2 surgery.[75-78]

Piperno-Neumann et

OS: overall survival; NR: not reported

al[108]

Gastrointestinal (GI) tract is an uncommon metastatic site for melanoma malignum occuring in only 2-5% of patients. However, more than a quarter of patients with melanoma at autopsy revealed GI metastases.[120,121] Patients with metastases to the GI are often symptomatic with pain (29–64%), obstruction (27%), bleeding (27%), palpable mass (12%) or weight loss (9%). [122] In addition, the high incidence of metastases of melanoma in the small intestine has been recently assigned to the presence of functionally active chemokine CCR9 on melanoma cells that facilitate metastases to the small bowel.[123] In a large number of cases palliative surgery is needed to alleviate bleeding and /or obstruction. Looking at the survival benefit of surgery, some studies found significantly improved survival in patients who underwent surgery and had a complete resection.[121,124,125] Ollila et al. reported a 5-year survival rate of 41% after complete resection.[121]

In conclusion, metastatic melanoma of the gastrointestinal tract is very rare, but should be suspected in any patient with a history of cutaneous melanoma and new gastrointestinal symptoms. Surgical interventions for symptomatic patients with melanoma of the gastroin‐ testinal tract significantly relieve pain and improve quality of life and may confer a survival advantage.

**Figure 15.** Duodenal melanoma metastasis.

#### *4.3.3. Metastases to the spleen, the pancreas, or the adrenal glands (Stage IV M1c)*

Isolated metastases to the spleen, pancreas or the adrenal glands are extremely rare. Few reports have demonstrated that surgical resection improves the 5-year survival.[62,126] Analysis of patients with solitary metastases to the adrenal glands yielded median survival times of 60 months.[127] In the study of Wood et al, sixty patients underwent adrenalectomy, hepatectomy, splenectomy, or pancreatectomy for melanoma metastases. The reported 5-year survival in the group after complete resection was 24%, whereas in the incomplete resection group, there were no 5-year survivors.[62,126]

#### **4.4. Surgery of brain metastases (Stage IV M1c)**

Metastatic disease to the brain is a frequent manifestation of melanoma with cerebral meta‐ stases accounting for 20-54% of deaths from melanoma.[128] It is associated with significant morbidity and mortality and poor prognosis. The median survival upon diagnosis of the cranial metastases is approximately 4 months.[129] Non-systemic treatment options are surgery, and stereotactic or palliative whole-brain radiotherapy.[130,131]

Usually patients present with symptoms such as seizures, vertigo, nausea, and vision altera‐ tion. In patients with good performance status and controlled primary disease, the surgical resection of solitary cerebral metastases is preferred. Positive prognostic factors in cases of brain metastases are younger age, good performance status, lack of neurologic symptoms, lack of extracranial disease and single focus of disease. Surgery might also be indicated for palliative reasons. If the solitary lesion is unresectable due to localization or extracranial disease, the determination of BRAF status is essential, since the efficacy of BRAF inhibitor dabrafenib in cerebral metastatic disease has been shown.[132] If no mutation is detected in BRAF, ipilimu‐ mab might be a treatment option[133] (for details see chapter 'Treatment of Brain metastases').

 Figure 16. Pre-operative CT scan with multiple melanoma metastases. Brain metastases are indicated with red arrows (a). Post **Figure 16.** Pre-operative CT scan with multiple melanoma metastases. Brain metastases are indicated with red arrows (a). Post surgical CT scan (b).

#### 4.5. Bone metastases (Stage IV M1c) **4.5. Bone metastases (Stage IV M1c)**

surgical CT scan (b).

Skelatal metastases are present in 5-17% of stage IV patients and have a poor prognosis. [134] Colman et al conducted the largest retrospective analysis of melanoma patients with bone metastases.[135] The study compared the survival rate of the group of patients who underwent surgery with wide resection of metastases with the group who received other surgery or were treated without operation. The observed 1-year overall survival rate in the resection group was twice as high as that of matched historical controls (50.0 vs. 24.8%). They found that overall survival may be improved in carefully selected patients where all known macroscopic tumor can be resected.[134-137] Skelatal metastases are present in 5-17% of stage IV patients and have a poor prognosis. [134] Colman et al conducted the largest retrospective analysis of melanoma patients with bone metastases.[135] The study compared the survival rate of the group of patients who underwent surgery with wide resection of metastases with the group who received other surgery or were treated without operation. The observed 1-year overall survival rate in the resection group was twice as high as that of matched historical controls (50.0 vs. 24.8%). They found that overall survival may be improved in carefully selected patients where all known macroscopic tumor can be resected.[134-137]

## **Author details**

**Figure 15.** Duodenal melanoma metastasis.

group, there were no 5-year survivors.[62,126]

106 Melanoma – Current Clinical Management and Future Therapeutics

**4.4. Surgery of brain metastases (Stage IV M1c)**

*4.3.3. Metastases to the spleen, the pancreas, or the adrenal glands (Stage IV M1c)*

surgery, and stereotactic or palliative whole-brain radiotherapy.[130,131]

Isolated metastases to the spleen, pancreas or the adrenal glands are extremely rare. Few reports have demonstrated that surgical resection improves the 5-year survival.[62,126] Analysis of patients with solitary metastases to the adrenal glands yielded median survival times of 60 months.[127] In the study of Wood et al, sixty patients underwent adrenalectomy, hepatectomy, splenectomy, or pancreatectomy for melanoma metastases. The reported 5-year survival in the group after complete resection was 24%, whereas in the incomplete resection

Metastatic disease to the brain is a frequent manifestation of melanoma with cerebral meta‐ stases accounting for 20-54% of deaths from melanoma.[128] It is associated with significant morbidity and mortality and poor prognosis. The median survival upon diagnosis of the cranial metastases is approximately 4 months.[129] Non-systemic treatment options are

Usually patients present with symptoms such as seizures, vertigo, nausea, and vision altera‐ tion. In patients with good performance status and controlled primary disease, the surgical resection of solitary cerebral metastases is preferred. Positive prognostic factors in cases of brain metastases are younger age, good performance status, lack of neurologic symptoms, lack of extracranial disease and single focus of disease. Surgery might also be indicated for palliative Rolland Gyulai\* , Zsolt Kádár and Zsuzsanna Lengyel

\*Address all correspondence to: gyulai.rolland@pte.hu

Department of Dermatology, Venerology and Oncodermatology, University of Pécs, Pécs, Hungary

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## **Current Surgical Management of Acral Lentiginous Melanoma**

Yasuhiro Nakamura, Yukiko Teramoto, Sayuri Sato and Akifumi Yamamoto

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59133

## **1. Introduction**

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118 Melanoma – Current Clinical Management and Future Therapeutics

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Malignant melanoma (MM) is the most common cause of death from skin cancer in Caucasians. The incidence of MM has considerably increased in most countries in the recent decades. However, the incidence rate of melanoma demonstrates a racial difference. The incidence rates range between 20 and 40 per 100,000 people in Australia and United States each year [1, 2]. In contrast, the incidence in Asians is clearly lower, approximately one per 100,000 [3, 4]. However, the most common clinical subtype of melanoma in Asians is acral lentiginous melanoma (ALM), which occurs at a rate of approximately 40%–65% of all cutaneous mela‐ nomas [4, 5] compared with only 2%–3% in Caucasians [6]. This subtype of melanoma, first described by Reed in 1976, is characterized by its predilection for the acral regions such as the soles, palms, and nail apparatus and by a pattern of radial lentiginous growth phase that evolves over months or years without any solar elastosis to a dermal (vertical) invasive stage. This subgroup has been designated as "plantar lentiginous melanoma," which corresponds to ALM [7]. Thereafter, MM was classified into the following four subtypes by Clark et al. in 1986 according to histological features; nodular melanoma (NM), superficial spreading melanoma (SSM), lentigo maligna melanoma (LMM), and ALM [8].

The prognosis of ALM is generally considered to be poorer than other subtypes such as SMM and LMM. In particular, the lesions of the sole are often overlooked by patients. In addition, ALM has a likelihood of being misdiagnosed as a benign melanocytic nevus, which leads to the development of tumor and delay in treatment [9]. This problem has improved with the advent of dermoscopy, which enables the dermatologists to distinguish between the early stages of ALM and benign melanocytic nevus.

© 2015 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 eproduction in any medium, provided the original work is properly cited.

Acral sites such as the palms and soles are not sun-exposured areas, and therefore overexpo‐ sure to ultraviolet light has not been shown to be a risk factor for the development of ALM [10]. On the other hand, ultraviolet light can play an important role in the development of LMM [11]. A recent study showed that 81% of non-chronic sun-damaged melanomas such as SSM had a high likelihood of BRAF or N-RAS mutations and the other subtypes, including ALM, had a low rate of mutation in either gene. In contrast, ALM had a frequent mutation or amplification of the KIT gene [12].

Because of the biological and unique anatomical specificity of ALM, there are some contro‐ versies in the treatment of ALM. In this chapter, we review the published articles regarding the surgical treatment of primary ALM lesions, including our paper and experiences, assess the current role of surgery, and discuss several major controversies of ALM surgical treatment.

## **2. Problems of excision margins in ALM**

The World Health Organization Melanoma Program undertook a randomized trial and compared lateral margins of 1 and 3 cm for 612 melanoma patients with thicknesses of <2 mm [13]. Disease-free and overall survival rates did not differ between the two groups. Two subsequent trials in Europe compared the results of treating melanomas with lateral margins of 2 or 5 cm. The Scandinavian Melanoma Group Study and French Cooperative Group Trial compared lateral margins of 2 cm with 5 cm for patients with 0.8–2.0 mm-thick and <2.1 mmthick melanomas, respectively [14, 15]. Neither study showed any evidence that lateral margins of 5 cm reduced the local recurrence rate or improved survival rates. The Intergroup Melanoma Surgical Trial also reported the results of a randomized prospective trial that compared lateral margins of 2 cm with 4 cm for 740 melanoma patients with thicknesses of 1.01–4.0 mm [16]. This trial also demonstrated that the local recurrence and survival rates were similar for the two groups. This evidence suggests that lateral margins of at least 2 cm are suitable for patients with melanoma with thicknesses of >2 mm.

Although some uncertainties remain and further trial-based evidence is required for clarifi‐ cation, the general consensus is that at least margins of 1 cm should be adequate for melanomas with a thickness of ≤1 mm [17]. For 1.01–2mm-thick tumors, some evidence suggests that lateral margins > 1 cm are desirable [18]. As for *in situ* lesions, a lateral margin of 5 mm is generally recommended in some national guidelines, including Japan. However, a recent prospective, comparative study demonstrated that 86% of 1120 *in situ* lesions were successfully excised with lateral margins of 6 mm and 98.9% were successful with lateral margins of 9 mm and concluded that lateral margins of 9 mm were appropriate for *in situ* lesions [19].

The appropriate depth of excision for melanoma is also still controversial. The depth of excision has been recommended to be at least to the level of muscle fascia; deeper excisions have not been shown to improve outcomes [20, 21]. On the other hand, a recent study demonstrated that there was no advantage for a resection of the deep muscular fascia but a potential for an increased risk of intralymphatic recurrences [22].

Based on the evidence described above, current major melanoma guidelines such as the National Comprehensive Cancer Network Guideline recommends adequate lateral margins alone, depending on the maximum thickness of the tumor. However, most of the available evidence has been obtained from clinical trials that included melanomas that were located mainly on the trunk or proximal extremities. Most studies and trials have not included melanomas in the head and neck region and on distal extremities, including ALMs. Acral sites are locations where more complex reconstructive procedures may be required if wider and deeper excision margins are applied. It is unclear whether the application of current recom‐ mended excision margins to the treatment of ALM that is biologically different from other subtypes is appropriate.

## **3. Surgical management of subungual melanoma (SUM)**

SUM is a rare manifestation of MM. The incidence of SUM also has a racial difference. It represents approximately 2%–3% and 20% of all cutaneous MMs in Caucasians [23-26] and Asians [4, 27], respectively.

The anatomy of the nail apparatus is complex and is composed of the proximal nail fold, nail matrix, nail bed, and hyponychium. The proximal nail fold is continuous with the dorsal aspect of the nail matrix distally and the extensor aspect of the digital skin proximally. The cuticle separates the ventral portion of the proximal nail fold from the underlying nail plate. The nail plate is the horny end product of the nail matrix. The hyponychium is the distal portion of the nail bed, which represents the site of union between the nail bed and tip of the digits [28].

#### **3.1. Invasive SUM**

Acral sites such as the palms and soles are not sun-exposured areas, and therefore overexpo‐ sure to ultraviolet light has not been shown to be a risk factor for the development of ALM [10]. On the other hand, ultraviolet light can play an important role in the development of LMM [11]. A recent study showed that 81% of non-chronic sun-damaged melanomas such as SSM had a high likelihood of BRAF or N-RAS mutations and the other subtypes, including ALM, had a low rate of mutation in either gene. In contrast, ALM had a frequent mutation or

Because of the biological and unique anatomical specificity of ALM, there are some contro‐ versies in the treatment of ALM. In this chapter, we review the published articles regarding the surgical treatment of primary ALM lesions, including our paper and experiences, assess the current role of surgery, and discuss several major controversies of ALM surgical treatment.

The World Health Organization Melanoma Program undertook a randomized trial and compared lateral margins of 1 and 3 cm for 612 melanoma patients with thicknesses of <2 mm [13]. Disease-free and overall survival rates did not differ between the two groups. Two subsequent trials in Europe compared the results of treating melanomas with lateral margins of 2 or 5 cm. The Scandinavian Melanoma Group Study and French Cooperative Group Trial compared lateral margins of 2 cm with 5 cm for patients with 0.8–2.0 mm-thick and <2.1 mmthick melanomas, respectively [14, 15]. Neither study showed any evidence that lateral margins of 5 cm reduced the local recurrence rate or improved survival rates. The Intergroup Melanoma Surgical Trial also reported the results of a randomized prospective trial that compared lateral margins of 2 cm with 4 cm for 740 melanoma patients with thicknesses of 1.01–4.0 mm [16]. This trial also demonstrated that the local recurrence and survival rates were similar for the two groups. This evidence suggests that lateral margins of at least 2 cm are suitable for patients

Although some uncertainties remain and further trial-based evidence is required for clarifi‐ cation, the general consensus is that at least margins of 1 cm should be adequate for melanomas with a thickness of ≤1 mm [17]. For 1.01–2mm-thick tumors, some evidence suggests that lateral margins > 1 cm are desirable [18]. As for *in situ* lesions, a lateral margin of 5 mm is generally recommended in some national guidelines, including Japan. However, a recent prospective, comparative study demonstrated that 86% of 1120 *in situ* lesions were successfully excised with lateral margins of 6 mm and 98.9% were successful with lateral margins of 9 mm and

The appropriate depth of excision for melanoma is also still controversial. The depth of excision has been recommended to be at least to the level of muscle fascia; deeper excisions have not been shown to improve outcomes [20, 21]. On the other hand, a recent study demonstrated that there was no advantage for a resection of the deep muscular fascia but a potential for an

Based on the evidence described above, current major melanoma guidelines such as the National Comprehensive Cancer Network Guideline recommends adequate lateral margins

concluded that lateral margins of 9 mm were appropriate for *in situ* lesions [19].

amplification of the KIT gene [12].

**2. Problems of excision margins in ALM**

120 Melanoma – Current Clinical Management and Future Therapeutics

with melanoma with thicknesses of >2 mm.

increased risk of intralymphatic recurrences [22].

In the surgical treatment of SUM, amputation has been traditionally performed [29]. Compa‐ rative studies of various amputation levels have not shown any advantages for metacarpal amputations over metacarpophalangeal (MP), proximal interphalangeal (PIP), or distal interphalangeal (DIP) amputations [30]; this means that the prognosis of SUM does not depend on the amputation level but on the clinical stage [31]. Therefore, the recent trend of surgery is to utilize more distal amputations, without compromising recurrence or survival [25, 26]. In particular, in the thumb, a more distal amputation is preferable (Fig. 1). Although amputation at the interphalangeal joint of the thumb results in only a 10% loss of function of a useful hand, amputation at the MP joint of the thumb results in a 40% loss of function [32]. In contrast, from the viewpoint of the functional and cosmetic aspects, ray amputation may be more appropriate than MP, PIP, or DIP amputation for the treatment of the other fingers (Fig. 2).

#### **3.2.** *In situ* **or minimally-invasive SUM**

Recently, several authors have proposed conservative surgery, which involves a narrow surgical margin without amputation followed by skin grafting for *in situ* SUM and minimallyinvasive SUM (Fig. 3), which is defined as a Breslow tumor thickness (TT) ≤0.5 mm [33-38]. There have also been a few case reports of two-step surgeries using artificial dermis tissue for the tentative coverage of the defect after local excision, followed by skin graft coverage after

**Figure 1. Minimal amputation of subungual melanoma of the thumb for avoidance of functional loss.** (A) Subun‐ gual melanoma on the left thumb. (B) Amputation at the interphalangeal joint.

**Figure 2. Cosmetic and functional advantages of ray amputation for the treatment of SUM of the finger.** (A) Con‐ spicuous amputated stump of a case of subungual melanoma on the fourth finger after proximal interphalangeal am‐ putation. (B) A case of ray amputation for melanoma of the third fingernail. (C) Inconspicuous cosmetic result with no stump after ray amputation. (D) Hand position to "scoop up water."

the histopathological confirmation of negative surgical margins [39, 40]. These strategies are becoming more common, although the total number of such surgeries still remains small.

#### **3.3. Possibility of conservative surgery for invasive SUM**

**Figure 1. Minimal amputation of subungual melanoma of the thumb for avoidance of functional loss.** (A) Subun‐

**Figure 2. Cosmetic and functional advantages of ray amputation for the treatment of SUM of the finger.** (A) Con‐ spicuous amputated stump of a case of subungual melanoma on the fourth finger after proximal interphalangeal am‐ putation. (B) A case of ray amputation for melanoma of the third fingernail. (C) Inconspicuous cosmetic result with no

gual melanoma on the left thumb. (B) Amputation at the interphalangeal joint.

122 Melanoma – Current Clinical Management and Future Therapeutics

stump after ray amputation. (D) Hand position to "scoop up water."

The unique anatomy of the nail apparatus described above also demonstrates that there is scant soft tissue between the nail unit and bony surface of the distal phalanx. Haneke [41] studied the distance from the tip of the nail matrix to the extensor tendon insertion of the middle finger of a young male, and the measured distance was 0.8 mm. Kim et al. [42] also studied the shortest distances between the deepest base of the nail matrix and surface of the distal phalanx in cadavers. The mean distances were 0.90 mm for thumbs, 0.72 mm for fifth fingers, 0.87 mm for first toes, and 1.09 mm for fifth toes. The mean distance of all digits was 0.90 mm. These studies have suggested that wide local excision was insufficient for the eradication of invasive SUM with a safe, deep surgical margin and appeared to be sufficient for only *in situ* or minimally-invasive SUM.

Dermatologists sometimes encounter patients with invasive SUMs that do not invade or attach to the distal phalanx. Therefore, we measured the shortest distances of 30 surgical specimens of invasive SUM between the deepest base of the tumor and the surface of the distal phalanx [43]. There were no bone invasion cases with 4 mm-thick SUMs and the shortest tumor-tobone distances exceeded 0.9 mm in all specimens with thicknesses of <4 mm (Fig. 4). In statistical analysis, the Pearson chi-square test showed that there was a higher likelihood of bone attachment or invasion when TT exceeded 4 mm, which was statistically significant (*P*=0.009). Both univariate and multivariate analyses also revealed that thick TT alone had a statistically significant effect [odds ratio 1.807 and 1.865 (95% CI 1.1085–3.008 and 1.111–3.13, *P*=0.023 and 0.018)]. This study only focused on the histologic evaluation of deep margins and has limitations including the lack of prognostic information; however, there have also been several reports of patients with >0.5-mm-thick SUMs who underwent non-amputative surgeries and who had no local recurrence or metastasis [36, 40, 44].

These studies indicate that the patients with invasive SUMs of intermediate thicknesses may be candidates for non-amputative surgery. However, the main problem is that it is still difficult to evaluate tumor thickness by inspection or dermoscopy preoperatively. There is a possibility of the presence of an unexpectedly thicker area in the tumor than the thickness identified from an incisional biopsy, which can consequently change the surgical strategy. High-resolution ultrasound is a noninvasive examination that can evaluate tumor thickness preoperatively, and there have been several studies that reported its accuracy for the determination of tumor thickness [45-47]. However, these studies excluded SUM; therefore, its accuracy for tumor thickness evaluation in SUM remains unclear. Furthermore, an ultrasound at 20 MHz tends to overestimate thickness because of lymphocytic infiltration around the tumor or nevus remnant [48, 49]. Reflectance confocal microscopy permits the acquisition of dynamic images of the epidermis and papillary dermis with resolution to a cellular level [50] and has been experimentally used for intraoperative imaging of the nail matrix [51]. However, its limitation is the imaging depth, which cannot be deeper than 200–300 μm [50].

**Figure 3. Non-amputative wide local excision followed by skin grafting for** *in situ* **subngual melanoma.** (A) *In situ* subungual melanoma on the left second toe. (B) Nail apparatus excision including the periosteum of the distal pha‐ lanx. (C) Defect coverage by skin grafting. (D) Postoperative findings 8 months after surgery.

**Figure 4.** The relationship between shortest distance from tumor to bone and tumor thickness of invasive SUM.

For other surgical procedures, Chow et al. has reported on wide local excision procedures involving the removal of the nail apparatus with a layer of underlying bone of 1-mm depth running parallel to the nail bed before reconstruction with skin grafting [32]. Moehrle et al. reported on the excision of SUM with the distal part of the distal phalanx (processus ungui‐ natus), which has led to its denotation as a "functional" surgery [52].

## **4. Surgical management of ALM on the volar skin**

In general, ALMs start as *in situ* lesions, with brown macules that enlarge slowly and form irregularly pigmented, asymmetric macular lesions over the years, corresponding to the radial growth phase. Thereafter, indurated nodules appear within the macular lesions and sometimes the nodules ulcerate in the so-called vertical growth phase.

#### **4.1. Problems of excision margins**

**Figure 3. Non-amputative wide local excision followed by skin grafting for** *in situ* **subngual melanoma.** (A) *In situ* subungual melanoma on the left second toe. (B) Nail apparatus excision including the periosteum of the distal pha‐

**Figure 4.** The relationship between shortest distance from tumor to bone and tumor thickness of invasive SUM.

lanx. (C) Defect coverage by skin grafting. (D) Postoperative findings 8 months after surgery.

124 Melanoma – Current Clinical Management and Future Therapeutics

Because of this very slow clinical course, ALMs on the volar skin often contain *in situ* lesions at the periphery (Fig. 5). Based on the recommended lateral margins described above, the dermatological surgeons are often confused regarding the decision for lateral margins. It is still unclear whether lateral margins of 0.5–1 cm from the peripheral border of the lesion should be suitable as the peripheral lesion is considered an *in situ* lesion, or lateral margins of 2 cm from the periphery of the lesion should be selected as the entire tumor itself is regarded as an invasive lesion.

**Figure 5.** Confusion regarding excision margin for acral lentiginous melanoma which has *in situ* lesion. The arrow in‐ dicates the invasive lesion. Blue dotted line indicates the peripheral border of the in situ lesion. Red and black dotted lines indicate lateral margins of 0.5 cm and 2 cm from the border of the lesion, respectively.

#### **4.2. ALM of the palm**

In general, most ALMs on the palm are diagnosed at an early stage because the palm is the site where the patient easily notices the lesion at an earlier stage. Therefore, wide local excision is easily accomplished and the full-or split-thickness skin graft is adequate for the coverage of the surgical defect. The main point that the surgeon should pay attention to is avoiding intraoperative injuries of the palmar digital nerves, which are easily exposed during tumor excision because of the scant subcutaneous fatty tissue in some areas of the palm (Fig. 6). As for skin grafting, the skin obtained from non-weight-bearing areas of the foot is preferable because of the similarity to the quality of the palmar skin; however, the skin quality difference between the palmar skin and the skin obtained from other areas may be inconspicuous in elderly patients (Fig. 6).

**Figure 6. Surgery for the acral lentiginous melanoma on the palm.** (A) The purple line indicates a lateral margin of 1 cm from the border of the lesion. (B) After wide local excision. The palmar digital nerves are safely preserved (taped by yellow vessel loops). (C) Defect coverage by full-thickness skin grafting taken from the inguinal area. (D) Postopera‐ tive findings 24 months after surgery.

#### **4.3. ALM of the sole**

ALM of the sole has been traditionally difficult to find and diagnose because of its "out-of sight" location. The lesion often precludes primary closure because of the lack of mobility of the skin on the sole and horizontal-growth of the tumor, which increases the reconstructive complexity. Therefore, after wide local excision, substantial defects have to be repaired using various methods such as skin grafting, local and distant flaps, and secondary intention healing depending on the size and site of the defect and the medical condition and lifestyle of the patient [53]. Unlike the palm of the hands, reconstruction of the sole of the foot is difficult because of its anatomy. The sole has weight-bearing areas such as the heel. In addition, a defect on the foot may result in insufficient vascular flow [54].

#### *4.3.1. Split-thickness or full-thickness skin graft*

**4.2. ALM of the palm**

126 Melanoma – Current Clinical Management and Future Therapeutics

elderly patients (Fig. 6).

tive findings 24 months after surgery.

**4.3. ALM of the sole**

In general, most ALMs on the palm are diagnosed at an early stage because the palm is the site where the patient easily notices the lesion at an earlier stage. Therefore, wide local excision is easily accomplished and the full-or split-thickness skin graft is adequate for the coverage of the surgical defect. The main point that the surgeon should pay attention to is avoiding intraoperative injuries of the palmar digital nerves, which are easily exposed during tumor excision because of the scant subcutaneous fatty tissue in some areas of the palm (Fig. 6). As for skin grafting, the skin obtained from non-weight-bearing areas of the foot is preferable because of the similarity to the quality of the palmar skin; however, the skin quality difference between the palmar skin and the skin obtained from other areas may be inconspicuous in

**Figure 6. Surgery for the acral lentiginous melanoma on the palm.** (A) The purple line indicates a lateral margin of 1 cm from the border of the lesion. (B) After wide local excision. The palmar digital nerves are safely preserved (taped by yellow vessel loops). (C) Defect coverage by full-thickness skin grafting taken from the inguinal area. (D) Postopera‐

ALM of the sole has been traditionally difficult to find and diagnose because of its "out-of sight" location. The lesion often precludes primary closure because of the lack of mobility of the skin on the sole and horizontal-growth of the tumor, which increases the reconstructive complexity. Therefore, after wide local excision, substantial defects have to be repaired using various methods such as skin grafting, local and distant flaps, and secondary intention healing depending on the size and site of the defect and the medical condition and lifestyle of the

Although split-thickness skin grafting is considered to be adequate for non-weight-bearing areas, it has been considered to be inappropriate for the heel and distal plantar area and could lead to dismal results [55]. If the surgical defect is located on the non-weight-bearing areas such as the foot and distal plantar area, or if the patient has severe medical comorbidities or is less active, the defect may be covered by split-or full-thickness skin grafting. Although skin grafting has advantages such as being much simpler than flap reconstruction, it is considered that defects of the weight-bearing areas should be repaired using local or distant flaps in ambulatory patients.

By authors' experience, the indication for skin grafting on the heel depends on the amount of the remaining subcutaneous fat pad after tumor excision. If the primary tumor is a thinner lesion and there is no need to excise the enormous subcutaneous fat pad together with the tumor, both split-or full-thickness skin grafts may be acceptable for weight-bearing recon‐ struction (Fig. 7A). In contrast, neither split-nor full-thickness skin grafting is appropriate when wide local excision including the subcutaneous fat beneath the tumor is required for oncologic purposes, which may lead to exposure of the calcaneum. Insufficient cushion because of the paucity of the fat pad eventually produces an erosion or ulceration of the skin graft (Fig. 7B).

As for the distal plantar area, the weight pressure on this area is not as great as on the heel. Therefore, we think that skin grafting for this area is more acceptable than that for the heel. Even through a large amount of the subcutaneous fat may be dissected with the tumor, fullthickness skin grafting may be durable for weight pressure over a long-term postoperative course (Fig. 7C). When there is remaining subcutaneous fat, reconstruction using the splitthickness skin grafting is also acceptable (Fig. 7D).

Skin grafts are generally taken from the contralateral limb because the melanoma cells were thought to primarily metastasize via lymphatic routes [56]. In contrast, a recent study has shown that there was no difference in the rates of donor site recurrence between the ipsilateral and contralateral limbs [57].

#### *4.3.2. Local or distant flaps*

A flap reconstruction is usually recommended for the closure of weight-bearing areas of the feet. The coverage of weight-bearing areas provides well-padded tissue superior to skin grafting. Among the various flaps, the medial plantar flap is the optimal reconstructive procedure. The plantar flap can provide the same quality of skin cover for weight-bearing areas and can also provide some sensation. In particular, using heel reconstruction, the donor site is located distally and away from the lymphatics of the primary site (Fig. 8).

The distally-based sural artery neurocutaneous flap is an optional procedure for heel recon‐ struction. Because the donor site is the posterior aspect of the lower leg, the quality of the skin is different from that of the sole. A comparative study between the medial plantar flap and distally-based sural artery flap demonstrated that the postoperative complications were higher in patients who underwent sural artery neurocutaneous flap procedures [58].

**Figure 7. Skin grafting on the weight-bearing areas after excision of acral lentiginous melanoma.** (A) Split-thickness skin grafting on the remaining subcutaneous fat pad of the heel. (B) Split-thickness skin grafting on the heel after exci‐ sion of the tumor with a large amount of subcutaneous fat pad. The ulceration is visible on the skin graft. (C) Fullthickness skin grafting (arrows) on the plantar aponeurosis, 10 years after surgery. (D) Split-thickness skin grafting on the remaining subcutaneous fat, 2 years after surgery.

#### *4.3.3. Secondary intention healing*

Secondary intention healing requires a long-term healing period, prolonged care such as regular and frequent dressing changes, and careful observation. According to previous reports, it took approximately 12–18 weeks to close the defects (mean defect size: 32.6–36.5 cm2 ) [54, 59]. Despite these drawbacks, it has the advantages of avoiding a secondary wound for tissue harvesting, a smaller scar because of the natural contraction of the wound, and granulation tissue that acts as a cushion to absorb impact while walking [54]. Recently, the effect of negative pressure wound therapy in addition to secondary intention healing has been investigated [59]. Compared with secondary intention healing alone, there was no difference in time to complete wound healing. However, the vascularity score and height of the scars was significantly better, and no wound infections during the course of treatment had occurred in the former group.

These methods clearly require a more extended period of healing time than skin grafting or flaps. Therefore, it would be appropriate to apply these methods to small-to medium-sized surgical defects.

**Figure 8. Reconstructive surgery of the acral lentiginous melanoma on the heel using medial plantar flap.** (A) The flap design and excision margin of the tumor on the heel. (B) Elevation of the medial plantar flap. (C) Defect coverage by flap and the coverage of the donor site by skin grafting. (D) Postoperative findings 9 days after surgery.

## **5. Conclusions**

) [54,

The distally-based sural artery neurocutaneous flap is an optional procedure for heel recon‐ struction. Because the donor site is the posterior aspect of the lower leg, the quality of the skin is different from that of the sole. A comparative study between the medial plantar flap and distally-based sural artery flap demonstrated that the postoperative complications were higher

**Figure 7. Skin grafting on the weight-bearing areas after excision of acral lentiginous melanoma.** (A) Split-thickness skin grafting on the remaining subcutaneous fat pad of the heel. (B) Split-thickness skin grafting on the heel after exci‐ sion of the tumor with a large amount of subcutaneous fat pad. The ulceration is visible on the skin graft. (C) Fullthickness skin grafting (arrows) on the plantar aponeurosis, 10 years after surgery. (D) Split-thickness skin grafting on

Secondary intention healing requires a long-term healing period, prolonged care such as regular and frequent dressing changes, and careful observation. According to previous reports, it took approximately 12–18 weeks to close the defects (mean defect size: 32.6–36.5 cm2

59]. Despite these drawbacks, it has the advantages of avoiding a secondary wound for tissue harvesting, a smaller scar because of the natural contraction of the wound, and granulation tissue that acts as a cushion to absorb impact while walking [54]. Recently, the effect of negative pressure wound therapy in addition to secondary intention healing has been investigated [59]. Compared with secondary intention healing alone, there was no difference in time to complete wound healing. However, the vascularity score and height of the scars was significantly better, and no wound infections during the course of treatment had occurred in the former group.

the remaining subcutaneous fat, 2 years after surgery.

*4.3.3. Secondary intention healing*

in patients who underwent sural artery neurocutaneous flap procedures [58].

128 Melanoma – Current Clinical Management and Future Therapeutics

Increased understanding of the molecular biology and pathogenesis of melanoma may lead to the development of novel therapeutic agents and treatment plans for melanoma. However, surgery is still the mainstay of treatment as there are no proven effective adjuvant systemic treatments.

Although several reliable national guidelines for melanoma have been produced using the evidence-based method, there are still some controversies in the treatment of ALM. Because of the biological and unique anatomical specificity of ALM, It is still unclear whether the thickness of ALM and recommended lateral margins are the same as that with other subtypes. Large randomized prospective studies with long-term follow up are necessary to fully evaluate surgery for ALM in the future. The accumulation of evidence produced by such studies will lead to the development of novel ALM treatments in the melanoma guidelines.

### **Acknowledgements**

This work was partly supported by the National Cancer Center Research and Development Fund (26-A-4).

## **Author details**

Yasuhiro Nakamura\* , Yukiko Teramoto, Sayuri Sato and Akifumi Yamamoto

\*Address all correspondence to: ynakamur@saitama-med.ac.jp

Department of Skin Oncology/Dermatology, Comprehensive Cancer Center, Saitama Medi‐ cal University International Medical Center, Saitama, Japan

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**Acknowledgements**

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Fund (26-A-4).

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Yasuhiro Nakamura\*

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## **Changing Perceptions of Lymphadenectomy and Sentinel Lymph Node Biopsy in Melanoma**

Antonio Sommariva, Camilla Cona and Carlo Riccardo Rossi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59195

## **1. Introduction**

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Lymph nodes (LN) represent the most frequent site of metastases for melanoma and the main purpose of lymphadenectomy (LND) is to provide loco-regional control of disease and accurate staging as well as to eventually cure patients with AJCC stage III melanoma. Cur‐ rently, lymph node involvement is mostly diagnosed after sentinel lymph node biopsy (SLNB). However, although SLNB in melanoma patients at risk for lymph node metastasis is routinely performed almost everywhere, the role of completion lymphadenectomy (CLND) after positive SLNB remains controversial, as only 15-20% of the patients operated show additional lymph node metastases in the dissected basin. The MSLT-1 trial, which evaluated the impact of SLNB and immediate LND versus simple observation and LND after clinical evidence of metastases only, did not show any survival benefit between the two randomized groups of patients [1]. Moreover, other studies have shown that some of the patients with positive sentinel nodes seem at lower risk for additional lymph node metastasis, and will probably never develop additional non sentinel lymph node metastases. On the other hand, the final analysis of the MSLT-1 trial confirmed a longer disease free survival and a gain in survival only in the patients with positive nodes in the CLND group.

Until the results of the two ongoing prospective studies (MSLT-2 and MINITUB) investigating the role of CLND after SLNB positivity are available, radical lymphadenectomy should be considered the standard of care in patients with lymph node metastases, as suggested by the NCCN guidelines, which recommend lymphadenectomy in presence of positivity of SLNB or histological/cytological confirmed clinical lymph node metastases [2].

© 2015 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 eproduction in any medium, provided the original work is properly cited.

Despite this recommendation, adherence to clinical practice guidelines remains low among melanoma surgeons. In the USA, 50% of patients with positive SLNB do not undergo com‐ pletion lymphadenectomy [3]. The still evident degree of confusion on the optimal surgical treatment of AJCC stage III melanoma has been confirmed by a recent international survey on lymphadenectomy in melanoma patients with positive sentinel nodes (SN), which showed an extremely heterogeneous approach to the extent of the completion lymph node dissection (CLND), especially for SN located in the neck or groin [4]. Aside from the indication and the levels of dissection, no agreement has yet been reached on the criteria to define lymphade‐ nectomy as adequate and, even if the minimum number of lymph nodes that should be excised achieves a reasonable consensus, other quality assurance (QA) parameters for lymphadenec‐ tomy are far from being accepted.

In this context, a consensus process on indications, technical aspects and QA parameters for SLNB and, in particular, for lymphadenectomy is desirable among melanoma surgeons and pathologists. Providing new evidence-based results in these controversial fields might help to achieve better standards of treatment. The purpose of this chapter is to critically review the most recent literature on the lymph node surgical treatment in melanoma patients providing, wherever available, new evidence and contributing to standardize the current management of this tumor.

## **2. State of the art on SLNB and lymphadenectomy**

### **2.1. Rationale for indication**

Cutaneous melanoma presents an increasing annual incidence worldwide and despite several advances in understanding the molecular mechanism of tumour progression and the devel‐ opment of more selective therapeutic strategies, a significant proportion of patients remain incurable. Lymph nodes represent the most frequent site of metastases from melanoma and the main purposes of lymphadenectomy are to provide loco-regional control of disease, accurate staging as well as to eventually cure patients with AJCC stage III melanoma. A significant number of patients with localized melanoma harbor clinical occult metastases in the regional node basin, which, if left untreated, will lead to palpable metastatic nodes (macroscopic disease). In the past, the common approach to lymph node in melanoma patients ranged between two different strategies; observation followed by therapeutic lymph node dissection (TLND) when clinical disease became evident and elective lymph node dissection (ELND) at the time of treatment of the primary in absence of macroscopic disease. However, removing lymph node metastases before they become evident is potentially a better strategy to prevent local failure and could potentially prevent systemic failure in a significant portion of patients. The drawback of ELND is that 15 to 20% of patients only have microscopic disease in the regional lymph nodes; therefore, the majority of patients will have no benefit from elective surgery and will receive surgical overtreatment. Four randomized controlled trials (RCT) [5] and one meta-analysis [6] have compared these two treatment strategies with, none demonstrating a survival advantage. The negative result in survival of ELND can be explained by the fact that more than 80% of patients in these trials will never develop lymph nodes metastases, making the studies underpowered to discover a statistical difference between the two groups. However, subgroup analysis from the WHO Melanoma Group Trial [7] showed a significant survival advantage in patients with clinically occult disease who underwent ELND compared to patients who underwent TLND for positive nodal recurrence (5-years survival 48.2 versus 26.6, P=0.04 respectively). These data suggest that there may be an improved survival in patients with occult disease, highlighting the importance of identification of early nodal disease. The difficulty in showing a survival advantage after ELND, unnecessary in 80% of patients and frequently associated with a high morbidity rate (including wound complications, lymphedema and pain), has made it, over the years, unappealing.

Despite this recommendation, adherence to clinical practice guidelines remains low among melanoma surgeons. In the USA, 50% of patients with positive SLNB do not undergo com‐ pletion lymphadenectomy [3]. The still evident degree of confusion on the optimal surgical treatment of AJCC stage III melanoma has been confirmed by a recent international survey on lymphadenectomy in melanoma patients with positive sentinel nodes (SN), which showed an extremely heterogeneous approach to the extent of the completion lymph node dissection (CLND), especially for SN located in the neck or groin [4]. Aside from the indication and the levels of dissection, no agreement has yet been reached on the criteria to define lymphade‐ nectomy as adequate and, even if the minimum number of lymph nodes that should be excised achieves a reasonable consensus, other quality assurance (QA) parameters for lymphadenec‐

In this context, a consensus process on indications, technical aspects and QA parameters for SLNB and, in particular, for lymphadenectomy is desirable among melanoma surgeons and pathologists. Providing new evidence-based results in these controversial fields might help to achieve better standards of treatment. The purpose of this chapter is to critically review the most recent literature on the lymph node surgical treatment in melanoma patients providing, wherever available, new evidence and contributing to standardize the current management

Cutaneous melanoma presents an increasing annual incidence worldwide and despite several advances in understanding the molecular mechanism of tumour progression and the devel‐ opment of more selective therapeutic strategies, a significant proportion of patients remain incurable. Lymph nodes represent the most frequent site of metastases from melanoma and the main purposes of lymphadenectomy are to provide loco-regional control of disease, accurate staging as well as to eventually cure patients with AJCC stage III melanoma. A significant number of patients with localized melanoma harbor clinical occult metastases in the regional node basin, which, if left untreated, will lead to palpable metastatic nodes (macroscopic disease). In the past, the common approach to lymph node in melanoma patients ranged between two different strategies; observation followed by therapeutic lymph node dissection (TLND) when clinical disease became evident and elective lymph node dissection (ELND) at the time of treatment of the primary in absence of macroscopic disease. However, removing lymph node metastases before they become evident is potentially a better strategy to prevent local failure and could potentially prevent systemic failure in a significant portion of patients. The drawback of ELND is that 15 to 20% of patients only have microscopic disease in the regional lymph nodes; therefore, the majority of patients will have no benefit from elective surgery and will receive surgical overtreatment. Four randomized controlled trials (RCT) [5] and one meta-analysis [6] have compared these two treatment strategies with, none demonstrating a survival advantage. The negative result in survival of ELND can be explained

**2. State of the art on SLNB and lymphadenectomy**

tomy are far from being accepted.

136 Melanoma – Current Clinical Management and Future Therapeutics

**2.1. Rationale for indication**

of this tumor.

The controversy on performing ELND disappeared with the advent of SLNB, which deter‐ mined a consistent paradigm shift in melanoma patients at risk of lymph node metastasis. After the pioneering studies of Donald Morton, who first hypothesized the role of sentinel lymph node as the first lymph node receiving lymphatic drainage from the primary tumour, SLNB emerged as a minimal invasive staging procedure for determining the nodal status in patients with melanoma [8]. The intra-operative use of a combined technique based on blue dye and radiotracer has been demonstrated to be feasible and accurate for nodal staging of patients with melanoma [9]. A recent meta-analysis of 71 studies, which includes 25240 melanoma patients who underwent SLNB in the period 1998-2009, showed that SLNB is highly accurate in melanoma with a proportion of patients successfully mapped (a least one sentinel lymph node removed) of 98.1%, a rate which tends to increase with the year of publication and quality score of the studies, female sex, ulceration and age [10]. The same study reported a false negative rate of SLNB of 12.5% (i.e. the proportion of patients with nodal recurrence in un-dissected nodal basins after a negative SLNB over the total positive patients and the false negative patients), which is inversely associated with the proportion of patients successfully mapped. The ability of SLNB to predict the negative status of the lymph node basin is expressed by the post-test probability negative (PTPN, the ratio of patients with negative SNB who recurred to all patients with negative SLNB). The PTNB in this study is 3.4%, which represents the proportion of patients with negative SLNB who recur. This risk seems inversely related to the proportion of patients successfully mapped and positively associated with the length of follow-up, younger patient age, the proportion of females, the mean Breslow thickness and the proportion of ulcerated tumours. The overall data analysis showed that, after a negative SLNB, the chances of nodal recurrence can be estimated to be equal to or lower than 5% providing reassurance that SLNB is a feasible and reliable method for accurately predicting the lymph node status of melanoma patients and is now considered a reliable staging proce‐ dure for melanoma.

SLNB status has been identified as the most important prognostic factor for overall survival in melanoma patients with no clinical evidence of metastatic disease [11] and has been included in the AJCC TNM staging system since the 6th edition in 2001 [12]. For this reason, a general consensus on performing SLNB in patients with intermediate thickness melanomas (Breslow 1-4mm)) [13] has been reached, as SLNB gives important prognostic information that can be used for planning follow-up protocols and adjuvant treatments [14]. Although the use of SLNB in thick melanomas remains uncertain, the procedure is recommended in this sub-group of patients mainly for staging purposes and local control of disease [14]. For thin melanoma (≤1mm), the role of SLNB is more controversial. It is known that the risk of node positivity in thin melanoma patients is less than 5%, but we should consider that this group accounts for the majority of patients with melanoma (about 65%) and therefore a large number of patients with microscopic disease might be left under staged and possibly undertreated. A sufficient level of evidence exists to also consider SLNB in patients with thin melanoma, particularly in presence of ulceration and/or mitotic rate ≥1 (AJCC T1b melanomas) [15]. Several studies have investigated the optimal cut-off value to consider SLNB cost-effective in thin melanomas. In patients with Breslow ≤0.50 mm, SLNB positivity is very unlikely, with a reported incidence of positive nodes of 0% [16]. Between 0.51 and 0.99 mm, the risk tends to increase and in a subgroup of patients with thin melanoma of at least 0.76 mm in depth and 1 or more mitosis, a 12.5% incidence of SN metastases has been reported [17].

It has been calculated that only 50-60% of patients with positive SLNB underwent CLND in USA [3] and Europe [18]. This proportion is probably higher among surgeons normally dealing with melanoma, as reported by a recent survey [4]. In this study, mainly involving surgeons working in melanoma or surgical oncology units, 91.8% of responders recommend CLND in patients with positive SLNB. However, the role of CLND in the presence of positive SLNB, remains uncertain. The Multicenter Selective Lymphadenectomy (MSTL-1) trial was started in 1994 and evaluated over 8 years the outcome of 2001 patients with primary cutaneous melanoma randomly assigned to undergo wide excision and nodal observation (observation group) or wide excision and SLNB, with immediate lymphadenectomy in presence of nodal metastases detected on biopsy (biopsy group). The prognostic value of SLNB was overall confirmed in patients with intermediate-thickness (1.2 to 3.5 mm) melanoma; 10-years Melanoma-Specific survival was 85.1±1.5 in negative SLNB and 62.1± 4.8 in positive SLNB [1]. Moreover, the MSLT-1 confirms that, among other established prognostic factors (Breslow thickness and ulceration), SLNB status is the most powerful indicator for disease recurrence (HR=2.64) and death from melanoma (HR=2.40). Considering survival analysis of patients with intermediate-thickness melanomas, a better 10-year disease free survival was detected in the biopsy group (71.3±1.8% versus 64.7±2.3%, HR for recurrence and metastasis=0.76, P=0.01), even though no difference was detected in the 10-year melanoma-specific survival among the two arms (81.4±1.5 and 78.3±2.0%, P=0.18). Even if no impact on overall survival has been observed in the biopsy group, at this level of evidence the present data suggest performing CLND for all patients with positive SLNB, mainly for achieving better regional control [2, 14]. Furthermore, a complete LND with therapeutical intent is recommended in presence of clinically evident, cyto/histologically proven lymph node metastasis.

#### **2.2. Surgical techniques**

SLNB involves preoperative lymphoscintigraphy, obtained through the injection of human albumin nanocolloid labelled with technetium 99mTc. The injection is in the intradermal layer, close to the scar of the removed melanoma or to the tumor if still present, and followed by scintigraphic scans (early and late) in the likely locations of lymphatic drainage [9]. Once the basin and location of the sentinel node has been identified, cutaneous projection area of each single node is marked on the skin. Immediately prior to surgery, the primary site is further injected intradermally with 0.5 to 1 mL of a vital dye (patent Blu), to increase the sensitivity of the method and to facilitate the finding of the lymph node (figure 1). SLNB is performed through a small skin incision, which should take into consideration the incision necessary for a subsequent radical lymphadenectomy [14]. Under the guidance of a radioisotope probe and following the blue lymphatic channels, the sentinel lymph node(s) is identified and removed (figure 1). Care should be taken not to disrupt or cauterize the lymph node capsule. Each SLN removed is checked ex vivo for radioactivity and the nodal basin is rescanned. Drains are seldom required and most patients are operated in one day-surgery regimen. The incidence of post-operative complications is relatively low, mainly related to wound (dehiscence/ infection or lymphatic collection), although limb lymphedema occurs not so un-frequently as generally supposed [19].

**Figure 1.** Surgical technique of SLNB

in thick melanomas remains uncertain, the procedure is recommended in this sub-group of patients mainly for staging purposes and local control of disease [14]. For thin melanoma (≤1mm), the role of SLNB is more controversial. It is known that the risk of node positivity in thin melanoma patients is less than 5%, but we should consider that this group accounts for the majority of patients with melanoma (about 65%) and therefore a large number of patients with microscopic disease might be left under staged and possibly undertreated. A sufficient level of evidence exists to also consider SLNB in patients with thin melanoma, particularly in presence of ulceration and/or mitotic rate ≥1 (AJCC T1b melanomas) [15]. Several studies have investigated the optimal cut-off value to consider SLNB cost-effective in thin melanomas. In patients with Breslow ≤0.50 mm, SLNB positivity is very unlikely, with a reported incidence of positive nodes of 0% [16]. Between 0.51 and 0.99 mm, the risk tends to increase and in a subgroup of patients with thin melanoma of at least 0.76 mm in depth and 1 or more mitosis,

It has been calculated that only 50-60% of patients with positive SLNB underwent CLND in USA [3] and Europe [18]. This proportion is probably higher among surgeons normally dealing with melanoma, as reported by a recent survey [4]. In this study, mainly involving surgeons working in melanoma or surgical oncology units, 91.8% of responders recommend CLND in patients with positive SLNB. However, the role of CLND in the presence of positive SLNB, remains uncertain. The Multicenter Selective Lymphadenectomy (MSTL-1) trial was started in 1994 and evaluated over 8 years the outcome of 2001 patients with primary cutaneous melanoma randomly assigned to undergo wide excision and nodal observation (observation group) or wide excision and SLNB, with immediate lymphadenectomy in presence of nodal metastases detected on biopsy (biopsy group). The prognostic value of SLNB was overall confirmed in patients with intermediate-thickness (1.2 to 3.5 mm) melanoma; 10-years Melanoma-Specific survival was 85.1±1.5 in negative SLNB and 62.1± 4.8 in positive SLNB [1]. Moreover, the MSLT-1 confirms that, among other established prognostic factors (Breslow thickness and ulceration), SLNB status is the most powerful indicator for disease recurrence (HR=2.64) and death from melanoma (HR=2.40). Considering survival analysis of patients with intermediate-thickness melanomas, a better 10-year disease free survival was detected in the biopsy group (71.3±1.8% versus 64.7±2.3%, HR for recurrence and metastasis=0.76, P=0.01), even though no difference was detected in the 10-year melanoma-specific survival among the two arms (81.4±1.5 and 78.3±2.0%, P=0.18). Even if no impact on overall survival has been observed in the biopsy group, at this level of evidence the present data suggest performing CLND for all patients with positive SLNB, mainly for achieving better regional control [2, 14]. Furthermore, a complete LND with therapeutical intent is recommended in presence of

a 12.5% incidence of SN metastases has been reported [17].

138 Melanoma – Current Clinical Management and Future Therapeutics

clinically evident, cyto/histologically proven lymph node metastasis.

SLNB involves preoperative lymphoscintigraphy, obtained through the injection of human albumin nanocolloid labelled with technetium 99mTc. The injection is in the intradermal layer, close to the scar of the removed melanoma or to the tumor if still present, and followed by scintigraphic scans (early and late) in the likely locations of lymphatic drainage [9]. Once the

**2.2. Surgical techniques**

Radical lymphadenectomy for melanoma involves the "en bloc" excision of lymph nodes with surrounding fat tissue. In the axilla, a radical lymphadenectomy should include dissection of levels I, II and III lymph nodes around the axillary vein [20]. A section of the pectoralis minor muscle is suggested by some for a better access to level II lymph nodes or in presence of bulky level II and III nodes. Long thoracic and thoracodorsal nerves should be preserved and sectioned only if directly involved by the tumor. In case of metastasis to the inguinal lymph nodes, the standard approach involves the removal of the inguinal, external iliac and obturator lymph nodes [21]. In the classic description of inguinal dissection, a longitudinal or lazy-Sshaped skin incision is employed, extending a few centimeters cranially to the superior anterior iliac spine up to the apex of the femoral triangle (figure 2). The incision should include the SLNB scar. The cutaneous flaps are created medially and laterally, up to the pubic tubercle, the anterior margin of the gracilis and abductor muscles and up to the superior anterior iliac spine and Sartorius muscle, respectively. Deep dissection continues through the fascia lata, over the underlining muscles and femoral vessels. The saphena magna vein is generally sectioned at the apex of the femoral triangle and at the level of the saphenofemoral junction. In case of the risk of femoral vessel exposition after wound dehiscence, transposition of the sartorious muscle is warranted. Iliac and obturatory dissection is obtained through an extraperitoneal approach. After sectioning of the oblique muscles and the inguinal ligament, the pelvic area is reached and the external iliac and obturatory lymph nodes removed, after identification of the urether and the obturator nerve.

For cervical lymph node metastastis, clear indications on the thoroughness of dissection are lacking and all the recommendations are supported by a low level of evidence and are obtained from opinions of experts in this field [22]. In case of clinically evident cervical lymph node metastasis, surgery is aimed at the removal of all five levels of lymph nodes (submandibular, jugular and supraclavicular), preserving sternocleidomastoid muscle, internal giugular vein and accessory spinal nerve (figure 3). Removal of the superficial part of the parotid is recom‐ mended only if clinically involved, because of the high risk of nerve damage observed.

Although general principles and technical details to perform adequate SLNB and LND are diffusely reported, surgeons in the clinical setting find many controversial aspects, regarding, in particular, the extent of given lymphadenectomy [22]. The question on what can be considered an adequate lymphadenectomy for metastatic melanoma is therefore largely unanswered. National guidelines are vague in defining this issue and simply suggest describing the anatomical limits of dissection [2]. This level of indeterminateness affects the attitude of melanoma surgeons in performing lymph node dissection [4]. A general agreement emerges in the presence of clinical evident lymph node metastatic disease, where a full regional lymphadenectomy is considered by most surgeons. More controversial is the thoroughness of lymphadenectomy in SLNB positive patients, in which, due to the significant risk, the approach is heterogeneous and controversial. For neck dissection, considering the risk of nerve damage as well as for the anatomic complexity, a consensus seems to emerge on performing CLND selectively and to remove the levels likely to be involved, depending on the site of the primary tumour, the site of the sentinel node and the lymphatic drainage highlighted at lymphoscin‐ tigraphy. Meanwhile, a superficial parotidectomy is associated only in presence of clinically evident metastasis [22]. For axilla, a general agreement exists on performing, in all cases, a three level dissection as the risk of lymphedema seems not affected by a more extensive lymphadenectomy. In fact, despite the risk of metastases of the third level is quite low [23], Changing Perceptions of Lymphadenectomy and Sentinel Lymph Node Biopsy in Melanoma http://dx.doi.org/10.5772/59195 141

**Figure 2.** Intraoperative view of inguinal LND.

muscle is suggested by some for a better access to level II lymph nodes or in presence of bulky level II and III nodes. Long thoracic and thoracodorsal nerves should be preserved and sectioned only if directly involved by the tumor. In case of metastasis to the inguinal lymph nodes, the standard approach involves the removal of the inguinal, external iliac and obturator lymph nodes [21]. In the classic description of inguinal dissection, a longitudinal or lazy-Sshaped skin incision is employed, extending a few centimeters cranially to the superior anterior iliac spine up to the apex of the femoral triangle (figure 2). The incision should include the SLNB scar. The cutaneous flaps are created medially and laterally, up to the pubic tubercle, the anterior margin of the gracilis and abductor muscles and up to the superior anterior iliac spine and Sartorius muscle, respectively. Deep dissection continues through the fascia lata, over the underlining muscles and femoral vessels. The saphena magna vein is generally sectioned at the apex of the femoral triangle and at the level of the saphenofemoral junction. In case of the risk of femoral vessel exposition after wound dehiscence, transposition of the sartorious muscle is warranted. Iliac and obturatory dissection is obtained through an extraperitoneal approach. After sectioning of the oblique muscles and the inguinal ligament, the pelvic area is reached and the external iliac and obturatory lymph nodes removed, after

For cervical lymph node metastastis, clear indications on the thoroughness of dissection are lacking and all the recommendations are supported by a low level of evidence and are obtained from opinions of experts in this field [22]. In case of clinically evident cervical lymph node metastasis, surgery is aimed at the removal of all five levels of lymph nodes (submandibular, jugular and supraclavicular), preserving sternocleidomastoid muscle, internal giugular vein and accessory spinal nerve (figure 3). Removal of the superficial part of the parotid is recom‐ mended only if clinically involved, because of the high risk of nerve damage observed.

Although general principles and technical details to perform adequate SLNB and LND are diffusely reported, surgeons in the clinical setting find many controversial aspects, regarding, in particular, the extent of given lymphadenectomy [22]. The question on what can be considered an adequate lymphadenectomy for metastatic melanoma is therefore largely unanswered. National guidelines are vague in defining this issue and simply suggest describing the anatomical limits of dissection [2]. This level of indeterminateness affects the attitude of melanoma surgeons in performing lymph node dissection [4]. A general agreement emerges in the presence of clinical evident lymph node metastatic disease, where a full regional lymphadenectomy is considered by most surgeons. More controversial is the thoroughness of lymphadenectomy in SLNB positive patients, in which, due to the significant risk, the approach is heterogeneous and controversial. For neck dissection, considering the risk of nerve damage as well as for the anatomic complexity, a consensus seems to emerge on performing CLND selectively and to remove the levels likely to be involved, depending on the site of the primary tumour, the site of the sentinel node and the lymphatic drainage highlighted at lymphoscin‐ tigraphy. Meanwhile, a superficial parotidectomy is associated only in presence of clinically evident metastasis [22]. For axilla, a general agreement exists on performing, in all cases, a three level dissection as the risk of lymphedema seems not affected by a more extensive lymphadenectomy. In fact, despite the risk of metastases of the third level is quite low [23],

identification of the urether and the obturator nerve.

140 Melanoma – Current Clinical Management and Future Therapeutics

surgical management of recurrent disease in the apex of axilla appears more difficult. More controversial is the approach to the groin, in particular for CLND after a positive SLNB, where two distinct lymphatic basin (inguinal and pelvic) are involved. Several national guidelines suggest combining pelvic dissection out of the inguinal, only in presence of radiological evidence of pelvic metastases, >3 positive inguinal nodes and metastases to the Cloquet's lymph node (so called sentinel lymph node of the pelvis). However, the evidence is low to sustain this surgical approach and a randomized controlled trial from the Australian and New Zealand Melanoma Trial Group comparing inguinal and pelvic CLND in SLNB-positive patients with negative PET/CT pelvic scan is about to start (EAGLE FM Study, ClinicalTri‐ als.gov Identifier, NCT02166788) [24].

## **3. New evidence-based results**

Referring to the most recent literature on SLNB and LND in melanoma patients, new evidencebased results are now available which can contribute to answer to (and find consensus on) the three main questions still pending: 1) How can we make surgeons more confident with indications to SLNB and Lymphadenectomy (in SN positive patients)? 2) How can we get them convinced that completeness of lymphadenectomy is an important issue?, and 3) are new, more convincing, evidence-based referral values for the minimum number of lymph node to be excised now available?

#### **3.1. Indication to lymphadenectomy in SLNB positive patients**

The role of completion lymphadenectomy (CLND) after positive SLNB remains uncertain as additional non sentinel nodes (i.e. identified within lymph node dissection after SLNB) have been identified in 9 to 25% of patients. This rate is probably underestimated because, unlike the pathologic protocols normally applied for SLNB specimens, those for therapeutic lym‐ phadenectomy are routinely limited to bisecting lymph nodes without any immunohisto‐ chemical stains. Aimed at investigating the prognostic and therapeutic impact of CLND in sentinel positive nodes two prospective trials have been undertaken: The Multicenter Selective Lymphadenectomy Trial-2 (MSTL-2) and the MiniTub trial. The MSTL-2 trial randomized patients with at least one positive SLN to observation or CLND. MSTL-2, whose patient accrual was completed in 2014, was designed to verify the incidence of nodal recurrence after removal of positive SLN(s) without CLND, the incidence and predictors of additional lymph nodes in the SLNB basin after CLND and the survival impact of CLND in SLNB positive patients. The MiniTub trial is a prospective registry investigating the outcome of patients with a T2-T3 primary melanoma and minimal SN tumour burden treated with CLND or nodal observation. While waiting for the results of these important studies, other important scientific reports have recently appeared in the literature, supporting the indication for CLND. For instance, the strength of the indication for CLND in these patients has been recently increased by the longterm results of the MSLT-1 trial [1, 25] Despite the lack of survival benefit of performing SLNB in the whole group, in a sub-group analysis, which excluded the SLNB negative patients, the node positive patients with intermediate thickness melanoma showed a 21% higher 10-year survival compared to patients who underwent lymphadenectomy for metastases discovered during follow-up. Of note is that the mean number of tumour involved nodes was significantly lower in the biopsy group with respect to the observation group (1.4 versus 3.3, P>0.001) [25]. The therapeutic effect of immediate CLND over lymphadenectomy at the time of clinically evident disease in patients with microscopic disease is confirmed by an interesting study which shows that immediate CLND is associated with a 10-year survival of 60% compared to delayed lymphadenectomy (around 45%), despite patients with early lymphadenectomy presenting worse adverse prognostic factors [26]. Moreover, a meta-analysis of non randomized studies encompassing 2633 patients, demonstrates, in patients with clinically undetectable lymph node metastasis, a 20% survival benefit after with SLNB followed by CLND [27].

Another argument in favour of the appropriateness of CLND comes from the important observation that patients with <11excised nodes were not adequately staged [28]. Furthermore, in case of sentinel node positivity, non sentinel lymph node status has an independent prognostic value in melanoma patients. The value of this observation is re-inforced by a metaanalysis suggesting the use of this new and easily reproducible prognostic factor as risk stratification criteria for clinical trials investigating adjuvant therapies and its inclusion in the future edition of the AJCC staging system [29]. Thus, not performing CLND in a SL positive patient today means lack of knowledge about his staging work-up, depriving him of important clinical information.

Taken together, all this information strongly supports that an early diagnosis of lymph node metastases (SLNB) and the removal of the affected lymphatic basin (CLND) can more effec‐ tively cure melanoma patients. Likely, some immunological events within the SL environment precede melanoma sentinel spread, suggesting that melanoma is preparing the sentinel lymph nodes to receive metastatic melanoma cells [30]. Removing SLNs and non sentinel LNs at an early stage, when probably the loco-regional immunosuppressive changes are not fully active, could explain in part the different prognostic impact of sentinel and non sentinel metastatic lymph node and opens the door to future investigations on the mechanisms of tumor response and on the immunological role of sentinel lymph nodes in melanoma.

#### **3.2. Adequacy of lymphadenectomy and quality control**

three main questions still pending: 1) How can we make surgeons more confident with indications to SLNB and Lymphadenectomy (in SN positive patients)? 2) How can we get them convinced that completeness of lymphadenectomy is an important issue?, and 3) are new, more convincing, evidence-based referral values for the minimum number of lymph node to be

The role of completion lymphadenectomy (CLND) after positive SLNB remains uncertain as additional non sentinel nodes (i.e. identified within lymph node dissection after SLNB) have been identified in 9 to 25% of patients. This rate is probably underestimated because, unlike the pathologic protocols normally applied for SLNB specimens, those for therapeutic lym‐ phadenectomy are routinely limited to bisecting lymph nodes without any immunohisto‐ chemical stains. Aimed at investigating the prognostic and therapeutic impact of CLND in sentinel positive nodes two prospective trials have been undertaken: The Multicenter Selective Lymphadenectomy Trial-2 (MSTL-2) and the MiniTub trial. The MSTL-2 trial randomized patients with at least one positive SLN to observation or CLND. MSTL-2, whose patient accrual was completed in 2014, was designed to verify the incidence of nodal recurrence after removal of positive SLN(s) without CLND, the incidence and predictors of additional lymph nodes in the SLNB basin after CLND and the survival impact of CLND in SLNB positive patients. The MiniTub trial is a prospective registry investigating the outcome of patients with a T2-T3 primary melanoma and minimal SN tumour burden treated with CLND or nodal observation. While waiting for the results of these important studies, other important scientific reports have recently appeared in the literature, supporting the indication for CLND. For instance, the strength of the indication for CLND in these patients has been recently increased by the longterm results of the MSLT-1 trial [1, 25] Despite the lack of survival benefit of performing SLNB in the whole group, in a sub-group analysis, which excluded the SLNB negative patients, the node positive patients with intermediate thickness melanoma showed a 21% higher 10-year survival compared to patients who underwent lymphadenectomy for metastases discovered during follow-up. Of note is that the mean number of tumour involved nodes was significantly lower in the biopsy group with respect to the observation group (1.4 versus 3.3, P>0.001) [25]. The therapeutic effect of immediate CLND over lymphadenectomy at the time of clinically evident disease in patients with microscopic disease is confirmed by an interesting study which shows that immediate CLND is associated with a 10-year survival of 60% compared to delayed lymphadenectomy (around 45%), despite patients with early lymphadenectomy presenting worse adverse prognostic factors [26]. Moreover, a meta-analysis of non randomized studies encompassing 2633 patients, demonstrates, in patients with clinically undetectable lymph

node metastasis, a 20% survival benefit after with SLNB followed by CLND [27].

Another argument in favour of the appropriateness of CLND comes from the important observation that patients with <11excised nodes were not adequately staged [28]. Furthermore, in case of sentinel node positivity, non sentinel lymph node status has an independent prognostic value in melanoma patients. The value of this observation is re-inforced by a meta-

**3.1. Indication to lymphadenectomy in SLNB positive patients**

142 Melanoma – Current Clinical Management and Future Therapeutics

excised now available?

Although the extent of each LND is still argued, it has recently been demonstrated in melanoma patients that the so-called lymph node ratio (i.e. the number of positive lymph nodes over the total number of excised lymph nodes) is associated with survival [31-33]. Patients with low ratio present better prognosis independently of the number of the positive nodes, indirectly confirming the prognostic value of the number of lymph node removed during surgery. Moreover, a recent multicentric Italian study showed, in the largest caseload so far available, that patients who had a higher number of excised lymph nodes after lymphadenectomy have a better prognosis, independently of AJCC T stage, ulceration, LN tumor burden and N stage [28] (figure 3). A clear and univocal explanation of this data is not available. The association between the number of lymph nodes and prognosis can find different explanations; 1) more lymph nodes means a better immunological control of melanoma, 2) a more accurate patient staging of patients 3) a therapeutic role of more extensive surgery. The latter lends support to the hypothesis that a thorough lymphadenectomy might have a therapeutic effect in melanoma patients with lymph node metastases, in particular in those who underwent CLND for a positive SLNB with intermediate thickness primary tumour.

Once the need of a CLND in melanoma patients with lymph node metastases is accepted and the anatomical extent of a given procedure is established, how can we provide surgeons with parameters and referral values for QA? Unfortunately, shared parameters for QA of lymph node dissections for melanoma are still lacking, with the exception of the minimum number of retrieved lymph nodes, for which a general consensus seems to exist for its simplicity, reproducibility and comparability [4]. However, the benchmark values of this quality param‐ eter (minimum number of lymph nodes for each dissected field) and the method to obtain these benchmark values are still matter of study. The minimum number values proposed in the literature are quite heterogeneous, reflecting the different method adopted for proposing it (table 1).

**Figure 3.** Patient survival according to the number of excised lymph nodes categorized as follow: ≤ 10 LNs, 11-20 LNs, 21-30 LNs and > 30 LNs. From IMI (Italian Melanoma Intergroup) caseload.


**Table 1.** Minimum number values of excised lymph node for single lymphatic basin.

The most recently proposed cut-offs to deem a lymph-node dissection adequate are evidencebased (obtained by the 10th percentile/survival method) and come from two independent caseloads: one from Australia [38, 39] and the other from Italy [28, 40]. As reported in table 1, the results are similar and should prompt surgeons (and pathologists) to adopt them as referral standards to measure their own performance, making a through revision of the procedure necessary when the reported numbers are below these thresholds. Another interesting evidence-based observation in this field comes from an already cited study which, besides a correlation between the absolute number of excised lymph nodes and survival, shows that, an adequate sub-staging of AJCC stage III melanoma patients is not possible below the cut-offs reported in the table [28] (figure 4).

**Figure 4.** Loss of prognostic significance of AJCC TNM N substages according to the number of lymph nodes (< or > 11). From IMI (Italian Melanoma Intergroup) caseload.

The implementation of QA programs at institutional/multi-institutional levels needs to define other parameters for monitoring quality and the relative benchmark values. Beyond the minimum number of lymph nodes, complication rates and local recurrence rates have been suggested as QA parameters for lymphadenectomy in a recent national consensus [22]. In the near future, standardization and implementation of effective QA programs for major surgical procedures in melanoma should increase patients' standard of care as well as the likelihood of reliable results from clinical trials testing new treatments in the adjuvant setting.

### **4. Perspectives**

**Minimum number of excised lymph nodes**

**Neck Groin**

**≤3 levels ≥4 levels Inguinal Inguinal and pelvic**

**Reference Method Axilla**

144 Melanoma – Current Clinical Management and Future Therapeutics

21-30 LNs and > 30 LNs. From IMI (Italian Melanoma Intergroup) caseload.

**Table 1.** Minimum number values of excised lymph node for single lymphatic basin.

Balch et al. [34] Expert opinion 10 20 20 5 5 MSLT-2 [1] Expert opinion 15 30 6 6

**Figure 3.** Patient survival according to the number of excised lymph nodes categorized as follow: ≤ 10 LNs, 11-20 LNs,

Eggermont et al [35] Expert opinion 10 15 15 5 5 Galliot-Repkat et al. [36] Survival analysis 10 10 10 10 10 Xing et al. [33] Survival analysis 8 15 6 6 Billimoira et al. [37] Expert opinion 10 15 15 5 5 Spillane et al. [38, 39] 10th percentile 10 6 20 7 14 Rossi et al. [40] 10th percentile 12 7 14 6 13 Rossi et al. [28] Survival analysis 11 14 9 12

The most recently proposed cut-offs to deem a lymph-node dissection adequate are evidencebased (obtained by the 10th percentile/survival method) and come from two independent

#### **4.1. Improving patient selection for SLNB and CLND**

Almost 80% of patients who undergo SLNB do not harbor node metastases. Having no benefit from the procedure, they are considered to receive a surgical over-treatment. Moreover, SLNB represents a surgical procedure associated with a defined morbidity rate (10%) [41] and significant cost for the health care system [42]. For these reasons, a series of clinical pathological variables associated with SLN status has been widely studied in the literature, but the statistical predictive power of each single factor on SLNB positivity remains poorly defined (table 2).


**Table 2.** Factors associated with risk of metastases in sentinel nodes

The development of statistical predictive models which analyse independent variables seems able to spare an unnecessary SLNB in between 18 to 30% of cases with an estimated error rate (i.e. patient with sentinel negative prediction even if they are sentinel positive at pathological examination) of 0.5-2.1% [43]. However, these tools need to be tested and validated in pro‐ spective studies and eventually implemented in the clinical setting. On the other hand, new markers of biologic behaviour can overcome and define the metastatic phenotype in primary melanoma and quantify the true risk of nodal metastases, but additional studies are needed to identify a subgroup of patients (in particular for thin melanomas) with defined clinicalpathological parameters at risk of SL positivity.

Identification of clinical and pathological parameters predictive of non sentinel nodes posi‐ tivity represents a crucial point to improve selection of patient candidates for CLND, as it is possible to spare un-necessary CLND in a defined quota of patients [44]. Several predictors of additional non-sentinel positive LNs have been identified, including those associated with primary tumor (i.e., melanoma thickness) or sentinel nodes (i.e. metastatic burden) [45, 46]. For instance, in patients with thin melanoma, the risk of additional lymph nodes in CLND is calculated 0.1% suggesting that the potential benefit of lymph node dissection after SLNB in this group should be balanced with the morbidity of CLND [47]. Histo-pathological parame‐ ters reflecting the pattern and amount of melanoma involvement in the SNs and the related risk-assessment systems able to predict the risk of additional non sentinel lymph node metastases in CLND are reported (table 3).

As well as for predictors of SL positivity, these parameters need to be validated prospectively, and the ongoing research on new biological markers might predict the pathological status of the additional nodes, even more precisely in the near future.

#### **4.2. Lowering the false negative rate of SLNB**

The false negative rate (FNR) of SLNB probably represents the most important drawback for this procedure. The values reported in literature are wide ranging between 8.6 and 21% [10]. The main reason for this variability resides in the different methods to calculate this proportion after SLNB. In the past, many authors have erroneously considered the FNR as the ratio between the FN cases and the truly negative plus the truly positive instead the of truly positive


**Table 3.** Risk assessment systems of Non-SLN involvement

variables associated with SLN status has been widely studied in the literature, but the statistical predictive power of each single factor on SLNB positivity remains poorly defined (table 2).

The development of statistical predictive models which analyse independent variables seems able to spare an unnecessary SLNB in between 18 to 30% of cases with an estimated error rate (i.e. patient with sentinel negative prediction even if they are sentinel positive at pathological examination) of 0.5-2.1% [43]. However, these tools need to be tested and validated in pro‐ spective studies and eventually implemented in the clinical setting. On the other hand, new markers of biologic behaviour can overcome and define the metastatic phenotype in primary melanoma and quantify the true risk of nodal metastases, but additional studies are needed to identify a subgroup of patients (in particular for thin melanomas) with defined clinical-

Identification of clinical and pathological parameters predictive of non sentinel nodes posi‐ tivity represents a crucial point to improve selection of patient candidates for CLND, as it is possible to spare un-necessary CLND in a defined quota of patients [44]. Several predictors of additional non-sentinel positive LNs have been identified, including those associated with primary tumor (i.e., melanoma thickness) or sentinel nodes (i.e. metastatic burden) [45, 46]. For instance, in patients with thin melanoma, the risk of additional lymph nodes in CLND is calculated 0.1% suggesting that the potential benefit of lymph node dissection after SLNB in this group should be balanced with the morbidity of CLND [47]. Histo-pathological parame‐ ters reflecting the pattern and amount of melanoma involvement in the SNs and the related risk-assessment systems able to predict the risk of additional non sentinel lymph node

As well as for predictors of SL positivity, these parameters need to be validated prospectively, and the ongoing research on new biological markers might predict the pathological status of

The false negative rate (FNR) of SLNB probably represents the most important drawback for this procedure. The values reported in literature are wide ranging between 8.6 and 21% [10]. The main reason for this variability resides in the different methods to calculate this proportion after SLNB. In the past, many authors have erroneously considered the FNR as the ratio between the FN cases and the truly negative plus the truly positive instead the of truly positive

Breslow thickness Ulceration Mitotic rate

Clarke level IV Young age

Lymphovascular invasion

**Table 2.** Factors associated with risk of metastases in sentinel nodes

146 Melanoma – Current Clinical Management and Future Therapeutics

pathological parameters at risk of SL positivity.

metastases in CLND are reported (table 3).

**4.2. Lowering the false negative rate of SLNB**

the additional nodes, even more precisely in the near future.

plus false negative and, only recently, a standard definition has been adopted [53]. However, a recent meta-analysis shows a FNR of 12.5% [10]. This means that considering melanoma patients harbouring node metastases, approximately one out of ten of these patients has a negative SLNB. Furthermore, FNR tends to increase with the duration of follow-up and the quality of the study and is inversely correlated with the identification rate. Reasons for FNR after SLNB can involve different specialists at different steps of the SLNB procedure; lympho‐ schintigraphy evaluation (nuclear medicine physician), lymph node detection during surgery (surgeon) and node's pathological examination (pathologist) [54]. The number of peri-tumoral injection seems to influence the outcome of lymphoscintigraphy but controlled studies are needed to confirm the real impact on FNR [9]. Failure in lymphoscintigraphy interpretation has been demonstrated to lead to in one third of false negative results after SLNB [54]. One third of cases, a FN result is explained by the failure of surgery to remove all the nodes identified at pre-operative lymphoscintigraphy, especially in neck and groin lymphatic basin. It should be noted that the ratio of marked on lymphoscintigraphy and excised sentinel lymph nodes is equal in only 38% of patients who underwent SLNB and that 20% of patients have fewer lymph nodes removed then those marked during lymphoscintigraphy [55]. FNR after SLNB seems higher in head and neck melanomas, confirming a greater complexity of SLNB in this body district, mainly for the proximity of primary tumor and lymphatic basin, the complexity of lymphatic drainage of neck and the higher risk of complications [56]. Moreover, a lack of standardization exists between centres on the threshold beyond which radioactivity of residual lymph nodes should indicate their excision. The 10 % rule (i.e., SN defined as all the lymph nodes with >10% radioactivity of the hottest SN removed) was proposed as standardization criteria and it was demonstrated to be able to reduce the rate of missing positive nodes, but a clear consensus on this is still lacking and further research is needed in this field [57, 58].

Pathologists can also contribute to FNR. In fact, the most appropriate pathologic protocol for SN examinations is still a matter of discussion. The number of sections to be stained and the optimal distance between them can significantly influence the metastases detection in SNs. Two main protocols have been popularized which seems to reach an acceptable compromise between diagnostic accuracy and costs [59, 60]. Although some evidence suggests that ultrastaging with polymerase chain reaction (PCR) of SNs represents an appealing prognostic tool and seems to improve melanoma cell detection in SNs, the clinical and prognostic value of molecular biology-based detection of melanoma cells in SNs needs to be further verified and supported by additional investigation in this field [61]

#### **4.3. Videoscopic approach to lymphadenectomy**

Fear of complication often influences surgeons' and patients' decision on whether or not to perform a lymphadenectomy in melanoma patients. Thus, to reduce morbidity is an impor‐ tant issue for surgical oncologists and forthis purpose video-assisted surgery has recently been proposed for lymph node dissection. Considering the principal lymphatic basins, groin is indubitably associated with the greater incidence of wound complications. Wound infection, dehiscence/necrosis and seroma/lymphocele after traditional lymphadenectomy ranges between 15-55, 7-53 and 2-46%, respectively [62]. Videoscopic lymphadenectomy (VL) of the groin appears to be a promising tool in lowering the incidence of wound complication.Inguinal and iliac-obturator VL consists of two different surgical times. The inguinal part is performed using three trocars, placed at a variable distance from the apex ofthe femoral triangle (figure 5).

**Figure 5.** Trocar position for inguinal lymphadenectomy VL.

The working space is obtained after a skin incision and blunt dissection of the area under the Camper fascia. The creation of a working space using high pressure CO2 levels (25 mm/Hg) at the beginning of the procedure make dissection easier. The saphenous vein is generally sectioned with endostaplers or endoclips. Removal of the surgical specimen is performed using endobag.

For iliac and obturator VL (figure 6), the access is extraperitoneal with the first trocar being placed infraumbilical and two trocars between the umbilicus and the pubic symphysis.

**Figure 6.** Intraoperative view of iliac and obturator VL

Two main protocols have been popularized which seems to reach an acceptable compromise between diagnostic accuracy and costs [59, 60]. Although some evidence suggests that ultrastaging with polymerase chain reaction (PCR) of SNs represents an appealing prognostic tool and seems to improve melanoma cell detection in SNs, the clinical and prognostic value of molecular biology-based detection of melanoma cells in SNs needs to be further verified and

Fear of complication often influences surgeons' and patients' decision on whether or not to perform a lymphadenectomy in melanoma patients. Thus, to reduce morbidity is an impor‐ tant issue for surgical oncologists and forthis purpose video-assisted surgery has recently been proposed for lymph node dissection. Considering the principal lymphatic basins, groin is indubitably associated with the greater incidence of wound complications. Wound infection, dehiscence/necrosis and seroma/lymphocele after traditional lymphadenectomy ranges between 15-55, 7-53 and 2-46%, respectively [62]. Videoscopic lymphadenectomy (VL) of the groin appears to be a promising tool in lowering the incidence of wound complication.Inguinal and iliac-obturator VL consists of two different surgical times. The inguinal part is performed using three trocars, placed at a variable distance from the apex ofthe femoral triangle (figure 5).

The working space is obtained after a skin incision and blunt dissection of the area under the Camper fascia. The creation of a working space using high pressure CO2 levels (25 mm/Hg) at the beginning of the procedure make dissection easier. The saphenous vein is generally

supported by additional investigation in this field [61]

**4.3. Videoscopic approach to lymphadenectomy**

148 Melanoma – Current Clinical Management and Future Therapeutics

**Figure 5.** Trocar position for inguinal lymphadenectomy VL.

The greatest advantage of VL is probably the virtual elimination of inguinal incision and (in case of iliac lymphadenectomy) the avoidance of the parietal abdominal muscles section. This potentially leads to a significant reduction of the post-operative wound related morbidity and pain. In one uncontrolled comparative study, the incidence of complications (infection and wound dehiscence) was significantly lower after VL (47.5% versus 80%, P=0.002) [63]. In another comparative study, although the incidence of infection and wound dehiscence was not statistically different after VL compared to open lymphadenectomy, in the open group wound infections appear more serious, requiring hospital readmission and intravenous antibiotics in five of the eight patients (62 %) [64]. Although the experiences are limited and the level of evidence is low, VL for melanoma is technically feasible, seems associated with a lower post-operative morbidity profile with comparable oncological outcomes (i.e. number of excised lymph nodes, loco-regional recurrence) (table 4). Before VL becomes suitable for routine clinical practice, the lower post-operative morbidity and safe oncological profile shown in retrospective and prospective series needs to be investigated within prospective RCTs.


**Table 4.** Summary of results on videoscopic groin lymphadenectomy for melanoma

## **5. Conclusion**

Even in the present exciting era of discovering new drugs to cure patients with advanced melanoma, surgery still represents the most performed and effective treatment for this potentially lethal disease. Nevertheless, the effort to solve many controversies related to this important subject has been so far insufficient and the ongoing clinical practice guidelines often lack clear indications for an adequate clinical approach, in particular dealing with patients at high risk for or with lymph node metastasis. While waiting for the conclusion of the ongoing controlled clinical trials (MLST-2 and MiniTub), surgeons should look for new evidence based results strengthening support for indication of SLNB and lymph node dissection, completeness of the latter and QA parameters on which the surgical performance should be measured.

Indication to SLNB is accepted almost everywhere as a staging procedure. Moreover, a recent meta-analysis of retrospective studies and the last report on the long-term results of the MLST-1 controlled trial reinforce its curative value in patients with positive nodes who undergo immediate CLND. In perspective, a more precise patient selection, based on valida‐ tion new statistical tools and/or identification of new molecular markers, and lowering its false negative rate might improve its efficiency and make this procedure even more appealing.

At present, LND represents the most controversial subject in the surgical treatment of mela‐ noma, particularly in SN positive patients. Its indication can be further warranted by, besides the long-term results of the MLST-1, the demonstration of its essential role as staging proce‐ dure. A recent study shows that below the threshold of 11 excised lymph nodes an accurate sub-staging is impossible, and another one demonstrates that the status of the additional lymph nodes is an independent prognostic factor in stage III melanoma patients. These evidence-based results also prompt for their inclusion in the surgical QA process (the former), and in the forthcoming melanoma staging system (the latter). Even if the extension of each lymphadenectomy is still a matter of discussion, further evidence has been recently added to the need of its completeness, such as the demonstration that the lymph node ratio and the absolute number of excised lymph nodes are independently associated with survival. As for other solid tumours in which LND has an impact on staging and survival, melanoma surgeons are in search of simple and reproducible parameters to deem the procedure adequate. The minimum number of lymph nodes to be excised seems to meet this requirement, and the reproducible numbers provided as benchmark values by the 10th percentile method, for each type of LND, are likely to make this parameter the most reliable. Looking at the future, statistical tools and molecular markers for a better patient selection, randomized trials for devising the LND extent, and the mini-invasive surgical approach to reduce the fear of complication and improve patients' quality of life, will probably fulfil the present lack of knowledge and make surgical treatment of melanoma more standardized and cost-effective. Nevertheless, since now surgeons can be helped by the new evidence-based results in the difficult process of building consensus on some important issues in melanoma surgery.

## **Author details**

**Videoscopic Lymphadenectomy**

150 Melanoma – Current Clinical Management and Future Therapeutics

Sommariva, et al.

**5. Conclusion**

**Procedures (N)**

Trias M, et al [65] Iliac 12 0 16.7 10.2 Not reported Schneider C, et al [66] Iliac 31 0 9.7 4.6 9.7 Abbott AM, et al [64] Inguinal 13 7.7 1.8 13 Not reported Martin BM, et al [63] Inguinal 40 10 15 12.6 2.5

[unpublished data] Inguinal and Iliac <sup>24</sup> 16.5 <sup>4</sup> 20.4 <sup>0</sup>

Even in the present exciting era of discovering new drugs to cure patients with advanced melanoma, surgery still represents the most performed and effective treatment for this potentially lethal disease. Nevertheless, the effort to solve many controversies related to this important subject has been so far insufficient and the ongoing clinical practice guidelines often lack clear indications for an adequate clinical approach, in particular dealing with patients at high risk for or with lymph node metastasis. While waiting for the conclusion of the ongoing controlled clinical trials (MLST-2 and MiniTub), surgeons should look for new evidence based results strengthening support for indication of SLNB and lymph node dissection, completeness of the latter and QA parameters on which the surgical performance should be measured.

Indication to SLNB is accepted almost everywhere as a staging procedure. Moreover, a recent meta-analysis of retrospective studies and the last report on the long-term results of the MLST-1 controlled trial reinforce its curative value in patients with positive nodes who undergo immediate CLND. In perspective, a more precise patient selection, based on valida‐ tion new statistical tools and/or identification of new molecular markers, and lowering its false negative rate might improve its efficiency and make this procedure even more appealing.

At present, LND represents the most controversial subject in the surgical treatment of mela‐ noma, particularly in SN positive patients. Its indication can be further warranted by, besides the long-term results of the MLST-1, the demonstration of its essential role as staging proce‐ dure. A recent study shows that below the threshold of 11 excised lymph nodes an accurate sub-staging is impossible, and another one demonstrates that the status of the additional lymph nodes is an independent prognostic factor in stage III melanoma patients. These evidence-based results also prompt for their inclusion in the surgical QA process (the former), and in the forthcoming melanoma staging system (the latter). Even if the extension of each lymphadenectomy is still a matter of discussion, further evidence has been recently added to the need of its completeness, such as the demonstration that the lymph node ratio and the

**Table 4.** Summary of results on videoscopic groin lymphadenectomy for melanoma

**Conversion rate (%)**

**Wound complication rate (%)**

**Lymph node excised (N)**

**Local recurrence rate (%)**

> Antonio Sommariva1 , Camilla Cona1,2 and Carlo Riccardo Rossi1,2

1 Melanoma and Sarcoma Unit, Veneto Institute of Oncology IOV-IRCCS, Padova, Italy

2 Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy

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## **Current Insights Into Canine Cutaneous Melanocytic Tumours Diagnosis**

Luis Resende, Joana Moreira, Justina Prada, Felisbina Luisa Queiroga and Isabel Pires

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59480

## **1. Introduction**

Skin melanoma is a devastating disease, frequently diagnosed in human and dogs, accounting for 0,8% - 2% of all skin tumors in latter species [1].

Melanocytic tumours are common neoplasms in dogs, accounting for 4 to 7% of neoplastic lesions in general, and up to 7% of all malignant tumours [2,3]. They generally arise on the oral cavity (Figure 1), lip, skin (Figure 2) and digit, amongst other locations (Figure 3) [4].

**Figure 1.** Canine oral melanoma (courtesy of Dr. Abel Fernandes).

© 2015 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.

**Figure 2.** Canine cutaneous melanoma.

**Figure 3.** Canine ocular melanoma.

Biologic behaviour of these tumours is often related to its location. Over 85% of melanocytic lesions located on haired skin are described as of benign behaviour. The majority of oral and mucocutaneous junction melanomas, with the exception of the eyelid, and 50% of digital melanomas originated from the nail bed are reported as malignant [5,6].

Cutaneous canine melanocytic lesions are usually benign [7,8]. They are generally detected at a late stage, when excision is rarely curative and metastasis is often detectable in regional lymph nodes [7]. Malignant tumors are found most frequently on the head, ventral abdomen, and scrotum [7]; in the last one, they represent 3,1% of all cutaneous malignant melanomas and 4,7% of scrotal tumours [9]. Amelanotic lesions can occur as cutaneous neoplasms, but are more frequent in the oral cavity, and tend to be behaviorally malignant [10].

Metastasis are often found on regional lymph nodes and lungs, but organs as brain, heart and spleen are also commonly affected [11].

Veterinary nomenclature of canine melanocytic tumours has been subjected to many contro‐ versies and changes over time. Through this article, in accordance with the revised World Health Organization classification system, and in order to simplify and avoid confusion, the authors describe benign lesions as melanocytoma, whereas malignant lesions are referred to as melanoma [12].

Etiology of this neoplasms is still uncertain, and several factors may be related, such as consanguinity, trauma, chemical exposure, hormones and genetic susceptibility [10]. Unlike humans, ionizing solar radiation exposure doesn't seem to be related to canine melanoma initiation [4].

Canine melanocytic tumour diagnosis often represents a challenge for the pathologist, since a high number of neoplastic lesions are quite similar in terms of its clinical and histologic appearance, including carcinoma, sarcoma, lymphoma and plasmocitoma, amongst others [10]. Indeed, cutaneous neoplasms with malignant behavior are more difficult to distinguish histologically from benign neoplasms than oral or lip neoplasms [13].

Diagnosis is usually based on fine-needle aspiration citology, but biopsy for histopathological examination is essential to determine its malignant potential [4,14]. The most reliable histologic criteria is the mitotic index, defined as the total number of mitotic figures observed per ten high-power light microscopic fields, which is known to be 90% accurate [5,6,15].

**Figure 2.** Canine cutaneous melanoma.

160 Melanoma – Current Clinical Management and Future Therapeutics

**Figure 3.** Canine ocular melanoma.

spleen are also commonly affected [11].

Biologic behaviour of these tumours is often related to its location. Over 85% of melanocytic lesions located on haired skin are described as of benign behaviour. The majority of oral and mucocutaneous junction melanomas, with the exception of the eyelid, and 50% of digital

Cutaneous canine melanocytic lesions are usually benign [7,8]. They are generally detected at a late stage, when excision is rarely curative and metastasis is often detectable in regional lymph nodes [7]. Malignant tumors are found most frequently on the head, ventral abdomen, and scrotum [7]; in the last one, they represent 3,1% of all cutaneous malignant melanomas and 4,7% of scrotal tumours [9]. Amelanotic lesions can occur as cutaneous neoplasms, but are

Metastasis are often found on regional lymph nodes and lungs, but organs as brain, heart and

Veterinary nomenclature of canine melanocytic tumours has been subjected to many contro‐ versies and changes over time. Through this article, in accordance with the revised World

melanomas originated from the nail bed are reported as malignant [5,6].

more frequent in the oral cavity, and tend to be behaviorally malignant [10].

Immunochemistry has arised as an extremely useful tool, for both diagnostic and prognostic purposes. A positive diagnostic for melanocytic neoplasms is obtained with a positive labelling of S100 protein, vimentine, Neuron Specific Enolase (*NSE*) and a simoultaneous negative labelling with cytokeratine [16].

The treatment of choice for local cutaneous melanomas is surgical excision; tumours with benign histopathology criteria have an excellent prognostic after this surgery. However, for malignant tumors, the prognostic is guarded, since metastatic rates of 30-75% have been reported [5,6].

Alternative therapy methods described in the literature include systemic chemotherapy, radiotherapy [17], photodynamic therapy [18], local hyperthermia [19,20] and intralesional injection of cisplatin or carboplatin.

The poor responses to the conventional therapy are leading to a development of new immunotherapy procedures – including intralesional adenoviral vector-mediated transfer of CD40L, a tumor necrosis factor gene [21], therapy with a plasmid DNA encoding staphylococcal enterotoxin B [22], and systemic tratment with liposome-encapsulated muramyl tripeptide [23].

At last, the most promising therapy appears to be a xenogenetic human tyrosinase DNA vaccine, with minimal local reaction and no systemic toxicity signs, and a great clinical responde with significant increasing of the survival time [24].

In this paper, the authors aim to contribute to the understanding of melanocytic tumours in the dog, making a critical review of the literature and discussing the parameters currently considered valid for diagnosis use in canine melanocytic neoplams.

## **2. Signalment**

Cutaneous melanocytic tumours are most common in older dogs (mean age of 9 years old) [25], with a higher mean age for dogs with malignant melanocytic tumours (12 years). However, age has not been related with the patient clinical outcome and survival time [26].

Although all breeds of dog (and crossbred animals) may be affected, some breeds are reported as predisposed, including Schnauzer, Doberman, Scottish Terrier, Irish Setter, Golden Retriever, Chow Chow, Cocker Spaniel, German Shepherd and Rottweiler [4,27]. Breed predisposition is thought to be related to an underlying genetic risk and/or increased pigmen‐ tation in the described above breeds [4].

One study [6] has also established a relationship between patient breed and tumour behavior, likely due to genetic susceptibility, as prior described. In the referred work, melanocytic lesions tended to be behaviorally benign in Doberman pinschers and miniature schnauzers, while miniature poodles were the mostly affected breed with malignant melanoma. However, it must be noted that oral neoplasms were also included in that study.

An early report described a higher frequency of these lesions in male dogs [28], but recent literature denies gender predisposition [6,27,29-31].

## **3. Pathogenesis**

Melanin is a dark-brown pigment synthetized by melanocytes, dendritic cells found within the basal layer of the epidermis. These cells are dispersed from each other, located between basal keratinocytes, forming adherent and regulatory junctions mediated by epithelial cadherin (E-cadherin) molecules. After its synthesis, melanin is retained in melanosomas and transferred to the adjacent keratinocytes [32].

Conversion of normal melanocytes to clusters of neoplastic melanocytes is a process composed by a series of events: initiation, promotion, transformation and metastasis [4].

Little is known about initiation on most animal melanomas, but ionizing solar radiation exposure – the main initiator factor in Human melanomas [33] - doesn't seem to be related to canine melanomas [4]. A higher incidence of spontaneously mutated cells due to familiar clustering through inbreeding may be a critical initiation factor in domestic animals.

Malignant transformation of canine cutaneous melanocytomas is very uncommon. Regard‐ ing cutaneous melanomas, there are a few published case reports, including one by Valentine and team [34], which have described a single case of malignant transformation of a congenital melanocytic nevus is a Golden retriever. Conroy [35] described two cases of melanoma originated from junctional or dermal hamartomas and a single case of a primary melanomas originated from a subcutaneous melanocytoma have also been reported [36]. In summary, canine cutaneous melanomas are thought to arise *de novo* from epider‐ mal and dermal melanocytes.

Promotion phase is related to mutated cells proliferation, with subsequent amplification of cell population and origin of additional mutations [4]. Melanoma promoters include chronic trauma, chemical exposure, drugs and hormones [10], and its action results in reactive hyperplasia of the epithelium, with disruption of regular keratinocyte-melanocyte interactions and proliferation of initiated cells.

The next step in carcinogenesis involves a series of transformation events. Recent develop‐ ments in genetic and molecular study techniques have identified the role of a few tumour suppressors in melanoma cell lines, giving new insights on the importance of these molecules in canine melanoma development. A reduction or loss of *p16* expression was one of the most commonly found changes, in both benign and malignant tumours, suggesting that inactivation of this pathway is a critical step in the pathogenesis of melanoma [37]. Altered expression of *PTEN*, *TP53*, *Rb* and *p21* have also been related to its progression [38], as well as the presence of various oncogenes (as a result of proto-oncogenes mutation), such as *c-myc*, *c-erb-B-2*, *cyes*, *c-kit* and *ras* [4].

After local proliferation phase, neoplastic cells may acquire a malignant behavior, and disseminate through hematic or lymphatic vessels to various other organs, originating secondary neoplasms known as metastasis. This complex process has it start with loss of adhesion and detachment of neoplastic cells from the primary mass, hematic and lymphatic vessels intravasion and attachment and proliferation within a secondary location [4].

Metastasis process is dependent of various adhesion molecules regulation by neoplastic cells. Several studies have shown an association between decreased and altered expression of Ecadherin, a calcium-dependent adhesion molecule responsible for melanocyte-keratinocyte interaction, and canine cutaneous melanoma progression [39,40]. CD44, a second transmem‐ brane glycoprotein which facilitates metastasis, is required for several processes, including hyaluronate degradation, cell aggregation and migration, angiogenesis and hematopoiesis [4]. Down-regulation of regular CD44 plus up-regulation of CD44v5 has also been associated with melanoma metastasis, particularly with lymph node metastasis [41].

Autonomous growth is a key requirement for both primary and secondary neoplastic devel‐ opment. The most important autocrine growth factors in animal melanoma include basic fibroblast growth factor (*bFGF*), melanoma growth stimulatory activity or growth regulated proteins, platelet-derive growth factor-A, α-melanocyte stimulating hormone, and a series of interleukins (*IL-8*, *IL-10* and *IL-18*) [4].

## **4. Gross morphologic features**

**2. Signalment**

**3. Pathogenesis**

tation in the described above breeds [4].

162 Melanoma – Current Clinical Management and Future Therapeutics

Cutaneous melanocytic tumours are most common in older dogs (mean age of 9 years old) [25], with a higher mean age for dogs with malignant melanocytic tumours (12 years). However,

Although all breeds of dog (and crossbred animals) may be affected, some breeds are reported as predisposed, including Schnauzer, Doberman, Scottish Terrier, Irish Setter, Golden Retriever, Chow Chow, Cocker Spaniel, German Shepherd and Rottweiler [4,27]. Breed predisposition is thought to be related to an underlying genetic risk and/or increased pigmen‐

One study [6] has also established a relationship between patient breed and tumour behavior, likely due to genetic susceptibility, as prior described. In the referred work, melanocytic lesions tended to be behaviorally benign in Doberman pinschers and miniature schnauzers, while miniature poodles were the mostly affected breed with malignant melanoma. However, it

An early report described a higher frequency of these lesions in male dogs [28], but recent

Melanin is a dark-brown pigment synthetized by melanocytes, dendritic cells found within the basal layer of the epidermis. These cells are dispersed from each other, located between basal keratinocytes, forming adherent and regulatory junctions mediated by epithelial cadherin (E-cadherin) molecules. After its synthesis, melanin is retained in melanosomas and

Conversion of normal melanocytes to clusters of neoplastic melanocytes is a process composed

Little is known about initiation on most animal melanomas, but ionizing solar radiation exposure – the main initiator factor in Human melanomas [33] - doesn't seem to be related to canine melanomas [4]. A higher incidence of spontaneously mutated cells due to familiar

Malignant transformation of canine cutaneous melanocytomas is very uncommon. Regard‐ ing cutaneous melanomas, there are a few published case reports, including one by Valentine and team [34], which have described a single case of malignant transformation of a congenital melanocytic nevus is a Golden retriever. Conroy [35] described two cases of melanoma originated from junctional or dermal hamartomas and a single case of a primary melanomas originated from a subcutaneous melanocytoma have also been reported [36]. In summary, canine cutaneous melanomas are thought to arise *de novo* from epider‐

clustering through inbreeding may be a critical initiation factor in domestic animals.

by a series of events: initiation, promotion, transformation and metastasis [4].

age has not been related with the patient clinical outcome and survival time [26].

must be noted that oral neoplasms were also included in that study.

literature denies gender predisposition [6,27,29-31].

transferred to the adjacent keratinocytes [32].

mal and dermal melanocytes.

Macroscopically, canine malignant melanoma cannot be differentiated from melanocytoma [42]. Melanomas in dogs tend to be dermal in location, unlike Human melanomas – which are intraepidermal with some degree of dermal invasion. Prognostic schemes, such as Clark's level or Breslow thickness, built nased on depht of dermal invasion, are not applicable on canine lesions [43].

Canine melanocytomas share some aspects with Human benign melanocytic lesions, in terms of clinical evolution most common metastatic locations [42], and genetic alterations [44].

Cutaneous melanocytomas are usually symmetrical, circumscribed, but encapsulated [43], solitary, black, brown, or gray cutaneous alopecic nodules [43,45] with a variable size with range of 1-4 cm in diameter (Figure 4 and Figure 5) [43]. Epidermis is usually intact, and alopecia is frequent. Epidermal cells may be hyperpigmented, and the majority of dermal cells are replaced by the tumoral ones, which in larger masses might also extends into the subcu‐ taneous tissue. The tumors may have a varieated appearance, with areas of pigmentation intermingled with no pigmented regions [42].

Canine cutaneous malignant melanomas can vary considerably in appearance, regardless of the location. Melanomas tend to be asymmetrical. The asymmetry may be most readily recognizable in the epidermal component of junctional tumors [43]. Melanomas size vary from some milimiteres to as large as 10 centimeters in diameter (mean range being 1 to 3 cm in diameter) [43], but this is not a reliable indicator of malignancy [13,42]. The color is variable, ranging from gray or brown to black, red, or even dark blue [7]. Cutaneous melanoma presentation includes smooth domes, sessile nodules, polypoid, plaquelike [7,43], or even lobulated masses [7]. The larger ones are often ulcerated [7,43]. The tumors may invade deeply into the subcutaneous tissue and along fascial planes [42].

**Figure 4.** Canine cutaneous melanoma.

**Figure 5.** Canine cutaneous melanoma.

## **5. Cytological diagnosis**

Canine melanocytomas share some aspects with Human benign melanocytic lesions, in terms of clinical evolution most common metastatic locations [42], and genetic alterations [44].

Cutaneous melanocytomas are usually symmetrical, circumscribed, but encapsulated [43], solitary, black, brown, or gray cutaneous alopecic nodules [43,45] with a variable size with range of 1-4 cm in diameter (Figure 4 and Figure 5) [43]. Epidermis is usually intact, and alopecia is frequent. Epidermal cells may be hyperpigmented, and the majority of dermal cells are replaced by the tumoral ones, which in larger masses might also extends into the subcu‐ taneous tissue. The tumors may have a varieated appearance, with areas of pigmentation

Canine cutaneous malignant melanomas can vary considerably in appearance, regardless of the location. Melanomas tend to be asymmetrical. The asymmetry may be most readily recognizable in the epidermal component of junctional tumors [43]. Melanomas size vary from some milimiteres to as large as 10 centimeters in diameter (mean range being 1 to 3 cm in diameter) [43], but this is not a reliable indicator of malignancy [13,42]. The color is variable, ranging from gray or brown to black, red, or even dark blue [7]. Cutaneous melanoma presentation includes smooth domes, sessile nodules, polypoid, plaquelike [7,43], or even lobulated masses [7]. The larger ones are often ulcerated [7,43]. The tumors may invade deeply

intermingled with no pigmented regions [42].

164 Melanoma – Current Clinical Management and Future Therapeutics

**Figure 4.** Canine cutaneous melanoma.

**Figure 5.** Canine cutaneous melanoma.

into the subcutaneous tissue and along fascial planes [42].

Microscopic examination of a cytological specimen obtained through fine needle aspiration has become a valuable technique to obtain a preliminary, and often definitive, diagnosis [46]. Being a quick, non-evasive and inexpensive procedure, it can also provide information on the stage, prognosis and metastasis evidence. Its main limitation reside on the fact that nonpigmented melanomas may strongly resemble other neoplastic lesions, and the amount of cytological sample might be very small and not fully representative of the lesion [4,46].

Several studies in Human cancer have described a strong accuracy in cytological examination in comparison with histopathological findings [47,48], but there are only few studies on the subject in Veterinary Medicine [49]. For instance, Ghisleni and others [50] evaluated a series of cutaneous and subcutaneous masses from dogs and cats through histopathology and cytology, describing an agreement between both techniques in 90.9% of the samples.

Melanocytic tumours are characterized by the presence of cells with abundant cytoplasmatic melanin granules. Neoplastic cells may appear with an epithelial (cohesive cells), mesenchy‐ mal (single oval or spindle-shaped cells) or round cell morphology. Nuclei may present a central or eccentric location, and is often solitary, though multinucleated forms are occasionally found. Nucleoli tends to be very prominent, with variable shapes such as round, oval or angular. These cells have a light basophilic cytoplasm, with a moderate to high nuclearcytoplasmatic ratio. Varying degrees of pigmentation might be found within the same tumour smear [4,46], (Figure 6 and Figure 7).

Malignant criteria consist, most importantly, of marked anisokaryosis and nuclear pleomor‐ phism, but also of the presence of large and atypical nucleoli [46]. Mitotic index, the most reliable criteria in histopathological evaluation, has no use in cytology. Regional lymph nodes are the most commonly evaluated site while monitoring for metastasis [4].

**Figure 6.** Canine cutaneous melanoma (Wright, 100x).

**Figure 7.** Canine cutaneous melanoma (Wright, 400x).

## **6. Histological diagnosis**

Histological characteristics of canine melanocytic neoplasms were defined by World Health Organization, in *International Histological Classification of Tumours of Domestic Animals*, back in 1974 (Figure 8 and Figure 9) [13].

**Figure 8.** Canine cutaneous melanocytoma (H&E, 200x).

Histological appearance does not always correlate well with biological behavior [8]. More recent studies have provided a numerical ''tumor score'' taking into consideration mitotic index, nuclear atypia, inflammation, necrosis and volume which appears to have improved correlation between histology and behavior [8].

The term nevus, commonly used in describing pigmented melanocytic lesions of the epidermis and dermis in humans, is not used in veterinary dermatopathology [7].

**Figure 9.** Canine cutaneous melanoma (H&E, 200x).

In this chapter we review several histological parameters on their ability to diagnose and predict prognosis (i.e., prediction of mortality) of canine melanocytic neoplasms. Malignancy celular features include a characteristic large nucleous, nuclear atypia, hyperchromasia, abnormal chromatin clumping and anomalous mitotic figures [51].

#### **6.1. Predominant cell type**

**Figure 7.** Canine cutaneous melanoma (Wright, 400x).

166 Melanoma – Current Clinical Management and Future Therapeutics

Histological characteristics of canine melanocytic neoplasms were defined by World Health Organization, in *International Histological Classification of Tumours of Domestic Animals*, back in

Histological appearance does not always correlate well with biological behavior [8]. More recent studies have provided a numerical ''tumor score'' taking into consideration mitotic index, nuclear atypia, inflammation, necrosis and volume which appears to have improved

The term nevus, commonly used in describing pigmented melanocytic lesions of the epidermis

and dermis in humans, is not used in veterinary dermatopathology [7].

**6. Histological diagnosis**

1974 (Figure 8 and Figure 9) [13].

**Figure 8.** Canine cutaneous melanocytoma (H&E, 200x).

correlation between histology and behavior [8].

Melanocytic neoplasms are generally composed of one of the following cell types: epithelioid (Figure 10), spindle, mixed (Figure 11), dendritic [51,52], and round cells [43]. Other less commonly described cell types include signet ring and ballon cells [51]. All these cell types may occur either alone or in combination [43]. In melanomas, ganglion cell and multinucleated giant cell forms also may be observed [43]. The epithelioid cell type is the most common type in all locations, whereas a mixture of cell types is seen with less frequency [51].

The epithelioid cells are round, with discrete cell borders, abundant glassy cytoplasm, appearing arranged in sheets and larger nests [43]. Similar to their spindle-shaped counterparts melanomas may exhibit large ovoid nuclei and prominent nucleoli [7,43], marked anisokar‐ yosis and variable chromatin patterns [43].

The spindle cell tumors are arranged in streams and interweaving bundles, resembling fibrosarcoma or neurofibrosarcoma presentation. In malignant lesions, the nuclei are large and fusiform with prominent nucleoli [7,43], and moderate to marked nuclear pleomorphism is seen [43]. Spindle cell predominant morphology was statistically associated with benignity in one study (in 71% melanocytomas in contrast to 29% melanomas) [43]. The mixed type consists of both cell morphologies and patterns [7].

Dendritic melanocytes have a highly angular shape, sometimes with long cytoplasmic processes, and are usually arranged in small nests [43] or organized in tightly swirling streams, often with a fingerprint pattern [7]. The dendritic or whorled forms occurs only in the skin [53]. Round cells tumors have round to polygonal cells arranged in sheets as dense packets of cells, the packets being separated by a scant stroma. The nuclei is large and round in melanomas [43].

The signet-ring cell tumors consist of compact neoplastic clusters, with round to ovoid cells presenting a faintly-eosinophilic cytoplasm and an intensely-stained periphery. A vesicu‐ lar nuclei, crescent-shaped, and with a peripheral location, gives the cell a signet-ring appearance [52].

**Figure 10.** Epithelioid cell canine cutaneous melanoma (H&E, 400x).

**Figure 11.** Mixed cell canine cutaneous melanoma (H&E, 400x).

Balloon cells are found organized in groups, separated by collagenous septa [52]. The cells are round to polyhedral and have clear or faintly eosinophilic cytoplasm [43,52]. In melanomas, the nuclei are round situated mainly at the periphery of the tumor cells [52]. Usually contains one central prominent nucleolus [7], which sometimes is difficult to detect. Heterochromatin, which is sparse, is dispersed throughout the nuclei [52].

Benign melanocytic cells present an enlarged vesicular nuclei with small nucleoli. Nuclear shape vary on according to the predominant tumour cell type. Mitotic figures are rare, and mitotic atypia is rarely observed. [43].

Although cell type appears to play some role in the prognosis of ocular melanocytic neoplasms, this feature lacked significance in prognosis of melanocytic tumors occurring in the mouth, feet, buccal mucosa and skin of dogs in several studies [8]. On the contrary, the epithelioid shape was associated with an unfavorable course, for 8/15 cases. The difference seems to be significant (p = 0,03) [12].

### **6.2. Nuclear atypia**

Round cells tumors have round to polygonal cells arranged in sheets as dense packets of cells, the packets being separated by a scant stroma. The nuclei is large and round in melanomas [43].

The signet-ring cell tumors consist of compact neoplastic clusters, with round to ovoid cells presenting a faintly-eosinophilic cytoplasm and an intensely-stained periphery. A vesicu‐ lar nuclei, crescent-shaped, and with a peripheral location, gives the cell a signet-ring

appearance [52].

**Figure 10.** Epithelioid cell canine cutaneous melanoma (H&E, 400x).

168 Melanoma – Current Clinical Management and Future Therapeutics

**Figure 11.** Mixed cell canine cutaneous melanoma (H&E, 400x).

One of the major criteria of malignancy in melanocytic neoplasms arising at any pigmented anatomic site is nuclear atypia. This feature is more valuable in epithelioid tumours than in spindle, whorled type or signet-ring cells, due to the insufficient nuclear detail associated with the later neoplasms [8,44,54]. However, not all studies are consensual [12].

Well-differentiated tumoral melanocytes have a small nucleus with one central nucleolus [8]. In contrary, undifferentiated tumours generally present cells with multiple, large, irregular and eccentrically nucleoli [8].

Several criteria are used to estimate nuclear atypia, including the percentage of nuclei involved [8]. Figures 12 and 13 (below) present moderate and severe nuclear atypia, respectively.

#### **6.3. Mitotic index**

In animal cutaneous and eye melanocytic neoplasms, mitotic index (MI) is the most reliable histological feature for distinguishing malignant from benign tumors [44,51], and also in predicting the clinical course of the disease [8]. In cutaneous melanoma, an MI of ≥3/10 hpf is significantly correlated with decreased survival [55].

The number of mitoses is usually lower in melanocytomas [<3 mitotic figures per 10 high power fields (hpf)] than melanomas [42,44,53].

In one study, the mitotic index was strongly correlated with the clinical outcome of tumors. For tumors with a favorable outcome, the mean value of the number of mitosis (on 10 randomly selected high power fields) was 1,98 (from 0 to 27). For tumors with a malignant behavior, it was 18,53 (from 0 to 75) [12].

The evaluation of the MI in conjunction with nuclear atypia classification (Figure 13) offers a more precise value the histological diagnosis [8].

**Figure 12.** Canine cutaneous melanoma with moderate diferenciation (H&E, 400x).

**Figure 13.** Canine cutaneous melanoma with evident nuclear atypia and numerous mitosis (arrow), (H&E, 400x).

### **6.4. Cellular pleomorphism**

Cellular pleomorphism criteria include several features, such as cell size and shape, pigmen‐ tation degree and nuclear features (including prominence of nucleoli and chromatin pattern). The usefulness of these parameters as individual prognostic factors is doubtful; however, it increases when these are used together [8,27].

### **6.5. Degree of pigmentation**

Degree of pigmentation is highly variable, even within a single smear (Figures 14, 15 and 16). Even on histological evaluation of amelanotic-classified tumours is common to detect a few very fine pigment granules in some cells. These are generally punctuate, spherical or elongated, as observed in pigmented keratinocytes. Cells with a very fine pigment may present a dusty gray appearance, instead of the typical granulation image [7]. In summary, it can be difficult to accurately diagnose an amelanotic melanocytic neoplasm and to define the degree of pigmentation [13].

Individual tumoral cells possess a different amount of melanin, which granules are generally small and uniform in size within the same cell. Splindle, round to polygonal, and balloon cells have sparsely-distributed melanin granules, while large epithelioid and dendritic cells are known to have a higher degree of pigmentation [43].

The majority of canine melanocytomas (except for ballon cell ones) have marked to moderate melanin pigmentation overall, especially in the superficial aspect of the tumors [43]. Similarly to cellular morphology, the degree of pigmentation of neoplastic cells was not an indicator of prognosis [12].

**Figure 14.** Canine cutaneous melanoma with marked pigmentation (H&E, 400x).

**Figure 12.** Canine cutaneous melanoma with moderate diferenciation (H&E, 400x).

170 Melanoma – Current Clinical Management and Future Therapeutics

**6.4. Cellular pleomorphism**

**6.5. Degree of pigmentation**

increases when these are used together [8,27].

**Figure 13.** Canine cutaneous melanoma with evident nuclear atypia and numerous mitosis (arrow), (H&E, 400x).

Cellular pleomorphism criteria include several features, such as cell size and shape, pigmen‐ tation degree and nuclear features (including prominence of nucleoli and chromatin pattern). The usefulness of these parameters as individual prognostic factors is doubtful; however, it

Degree of pigmentation is highly variable, even within a single smear (Figures 14, 15 and 16). Even on histological evaluation of amelanotic-classified tumours is common to detect a few very fine pigment granules in some cells. These are generally punctuate, spherical or elongated,

**Figure 15.** Canine cutaneous melanoma with moderate pigmentation (H&E, 400x).

**Figure 16.** Amelanotic melanoma (H&E, 400x).

#### **6.6. Junctional activity**

Junctional activity refers to the proliferation of neoplastic melanocytes at the interface between the epidermis and dermis or epithelium and submucosa [53].

The presence or absence of junctional activity is not specific to melanoma and often occurs in melanocytomas [7], however, malignant melanomas arising in the skin often show marked junctional activity, (Figure 17 and Figure 18) [42].

Junctional activity was not statistically associated with survival for skin neoplasms in one study [8]. In contrast, another work considered junctional activity as an independent prog‐ nostic factor (p =0,0239) for cutaneous melanocytic neoplasms, and found that its occurrence was associated with a longer survival time (p = 0,0046) [55].

**Figure 17.** Canine cutaneous melanoma without junctional activity (H&E, 200x).

**Figure 18.** Canine cutaneous melanoma with junctional activity (H&E, 400x).

### **6.7. Intraepithelial neoplastic cells**

The presence of intraepidermal tumoural cells can be graded as absent, slight (25% of neoplastic melanocytes in epidermis), moderate or prominent (more than 50% of tumour cells are present in the epidermis) [44]. However, this feature appears not to be of prognos‐ tic significance [8].

In a recent study was found that the samples of canine melanomas presented medium to prominent scatter of intraepidermal melanocytes, lower pigmentation, and higher nesting of intraepidermal melanocytes, in comparison with melanocytomas [44].

In most melanocytomas, the overlying epidermis is hyperpigmented, regardless of the presence or absence of intraepidermal clusters of neoplastic melanocytes [43].

#### **6.8. Ulceration**

**Figure 16.** Amelanotic melanoma (H&E, 400x).

172 Melanoma – Current Clinical Management and Future Therapeutics

the epidermis and dermis or epithelium and submucosa [53].

was associated with a longer survival time (p = 0,0046) [55].

**Figure 17.** Canine cutaneous melanoma without junctional activity (H&E, 200x).

junctional activity, (Figure 17 and Figure 18) [42].

Junctional activity refers to the proliferation of neoplastic melanocytes at the interface between

The presence or absence of junctional activity is not specific to melanoma and often occurs in melanocytomas [7], however, malignant melanomas arising in the skin often show marked

Junctional activity was not statistically associated with survival for skin neoplasms in one study [8]. In contrast, another work considered junctional activity as an independent prog‐ nostic factor (p =0,0239) for cutaneous melanocytic neoplasms, and found that its occurrence

**6.6. Junctional activity**

Canine melanomas are most frequently ulcerated [44] and particularly larger masses [43] than melanocytomas.

According to Laprie and team [55], ulceration might be taken as a prognostic marker for this neoplasms. In the referred work, the presence of an ulcerated epidermis was associated with a shorter survival time (p = 0,0023) and shown to be an independent prognostic factor (p = 0,0065) [55]. However, two other studies found no correlation between ulceration and clinical evolution of lip, nail bed [27] or cutaneous melanocytic neoplasms [12,27].

#### **6.9. Level of infiltration/invasion**

Melanocytic tumours strictly limited to the dermis, with a shallow depth, are associated with a greater survival time (p < 0,0001), and deep level of infiltration has been shown to be a significant prognostic factor (p = 0,0012) [55]. One other study that evaluated the level of invasion (in cutaneous and subcutaneous tissues) concluded that tumors confined to the superficial dermis were associated with a benign course in 94% of cases. On the other hand, tumors reaching the deep dermis and the subcutis showed a malignant behavior; however, the sample number were too low to allow for a conclusion [12].

#### **6.10. Necrosis**

Necrosis is a common feature, particularly in larger masses [43]. The presence of necrosis was correlated with malignancy and with a short survival time in a study set of 389 melanocytic neoplasms containing both benign and malignant lesions from various locations (mouth, feet and lip, skin) [8]. In other report with a set of 38 malignant melanomas from various locations, no correlation was found with survival time [54]. In summary, necrosis is considered of limited prognostic value in animals [7].

#### **6.11. Morphologic classification**

Melanocytomas include dermal, compound, and balloon cell tumors, as well as multiple dysplastic melanocytoma syndrome in dogs [43].

Dermal melanocytoma are strictly intradermal in location and larger tumours may extend into the subcutis [43]. Melanocytomas are generally composed by spindle cells disposed in bundles, nests and whorles, with a moderate cellular concentration and a lack of stromal collagen [43,45]. Melanophages might be dispersed throughout the tumoral nodule or in aggregates [43]. Mitotic figures are ocasionally seen (inferior to 1 per 10 high power fields), and mitotic atypia is not observed [43].

Some dermal melanocytomas are composed of epithelioid or dendritic cells that are heavily pigmented [43]. Nuclear morphology may be obscured by the large amount of pigment [43]. Although nuclei may be large, there is minimal nuclear pleomorphism [43].

A compound melanocytoma has a wedge-shaped configuration, and its description refers to the fact of including both junctional and dermal components. A numerous and densely packed tumor cell population is present in the dermis, while a variable amount of tumor cells accu‐ mulate in clusters and nests within the epidermis, along the dermal–epidermal junction and in the outer follicular wall – this pattern is referred to as 'junctional activity' [43].g

Balloon cell melanocytoma have a dermal location, and are predominantly composed of large round cells [43,45], although some fusiform or polygonal melanocytes might be present [42], with an abundant, pale eosinophilic and finely granular cytoplasm [42,43,45]. These lesions often lack readily visible pigmentation; however, dust-like melanin granules may be detected in small numbers [42,43]. Nuclei are small, uniform, and ovoid [43,45] and mitotic figures are rarely observed [42,43].

Multiple dysplastic melanocytoma syndrome mostly resemble compound melanocytomas on low magnification. However, their incidence increases in larger lesions. Also, cytologic atypia and mitotic figures are present and some of the proliferating melanocytes have irregularly shaped and enlarged hyperchromatic nuclei [43].

Canine melanocytoma–acanthomas are mixed tumors that are composed of a benign melano‐ cytic proliferation, resembling compound melanocytoma, and a benign epithelial proliferation [43,45]. The epithelial component usually appears follicular and resembles an isthmus-type tricholemmoma or infundibular keratinizing acanthoma [43]. The epithelial population forms a mass in the dermis composed of cords and nests with occasional small cystic structures containing keratin [45]. Melanocytic cells form nests in the epidermis and sometimes in the cords of epithelial cells within the dermal mass; melanocytic spindle cells can form whorls and bundles between the epithelial cords and nests [45]. A dermal melanocytoma– acanthoma has been immunophenotyped in a German Shepherd dog identifying the presence of keratinocytes and melanocytes [56].

superficial dermis were associated with a benign course in 94% of cases. On the other hand, tumors reaching the deep dermis and the subcutis showed a malignant behavior; however,

Necrosis is a common feature, particularly in larger masses [43]. The presence of necrosis was correlated with malignancy and with a short survival time in a study set of 389 melanocytic neoplasms containing both benign and malignant lesions from various locations (mouth, feet and lip, skin) [8]. In other report with a set of 38 malignant melanomas from various locations, no correlation was found with survival time [54]. In summary, necrosis is considered of limited

Melanocytomas include dermal, compound, and balloon cell tumors, as well as multiple

Dermal melanocytoma are strictly intradermal in location and larger tumours may extend into the subcutis [43]. Melanocytomas are generally composed by spindle cells disposed in bundles, nests and whorles, with a moderate cellular concentration and a lack of stromal collagen [43,45]. Melanophages might be dispersed throughout the tumoral nodule or in aggregates [43]. Mitotic figures are ocasionally seen (inferior to 1 per 10 high power fields), and mitotic

Some dermal melanocytomas are composed of epithelioid or dendritic cells that are heavily pigmented [43]. Nuclear morphology may be obscured by the large amount of pigment [43].

A compound melanocytoma has a wedge-shaped configuration, and its description refers to the fact of including both junctional and dermal components. A numerous and densely packed tumor cell population is present in the dermis, while a variable amount of tumor cells accu‐ mulate in clusters and nests within the epidermis, along the dermal–epidermal junction and

Balloon cell melanocytoma have a dermal location, and are predominantly composed of large round cells [43,45], although some fusiform or polygonal melanocytes might be present [42], with an abundant, pale eosinophilic and finely granular cytoplasm [42,43,45]. These lesions often lack readily visible pigmentation; however, dust-like melanin granules may be detected in small numbers [42,43]. Nuclei are small, uniform, and ovoid [43,45] and mitotic figures are

Multiple dysplastic melanocytoma syndrome mostly resemble compound melanocytomas on low magnification. However, their incidence increases in larger lesions. Also, cytologic atypia and mitotic figures are present and some of the proliferating melanocytes have irregularly

Although nuclei may be large, there is minimal nuclear pleomorphism [43].

in the outer follicular wall – this pattern is referred to as 'junctional activity' [43].g

the sample number were too low to allow for a conclusion [12].

174 Melanoma – Current Clinical Management and Future Therapeutics

**6.10. Necrosis**

prognostic value in animals [7].

**6.11. Morphologic classification**

atypia is not observed [43].

rarely observed [42,43].

shaped and enlarged hyperchromatic nuclei [43].

dysplastic melanocytoma syndrome in dogs [43].

Dermal melanomas have no junctional activity, but in some cases the tumour might extend deeply into the subcutaneous tissue. There might be a predominance of a certain cell type, but most tumors reveal a mixture of spindle cells, round to polygonal cells, and/or epithelioid cells. While spindle cells are poorly pigmented, more round and epithelioid cells tend to have a moderate to abundant amont of melanin granule [43]. Other important features in this kind of neoplastic lesions include a marked nuclear pleomorphism, nucleolar prominence, a moderate mitotic rate (3 or greater per 10 high power fields), atypical mitotic figures, asymmetry of the tumor nodule and a lymphoplasmacytic cell population [43].

Melanomas have an obvious 'junctional activity' pattern, with tumor cells distributed through the dermal–epidermal junction, as well as at higher levels of the epidermis, particularly in those of the nail bed and lip [43]. The intraepidermal element is mainly composed of epithelioid melanocytes, disposed individually or arranged in nests and clusters [43]. Tumours with numerous melanocytes distributed through all levels of the epidermis are referred to as lesions with a 'pagetoid' pattern [43]. Other melanomas present numerous individual neoplastic melanocytes present within the basal cell layer only, which is also referred to as an 'atypical lentiginous infiltrate' [43].

Spindle cell and desmoplastic melanomas, a subgroup of dermal melanomas, is composed predominantly of spindled melanocytes densely packed, or arranged loosely within abundant pale stroma [43]. Occasionally, there is a prominent fibroblastic component associated with the spindle shaped melanocytes, and collagen may become more abundant than the tumor cells [43]. The vast majority are amelanotic, thus a Fontana–Masson stain usually is necessary to detect the presence of melanin granules; cells are usually arranged in bundles or palisades, mimicking tumors of neural origin [43].

Balloon cell melanoma (clear cell melanoma), possess large cells with a clear eosinophilic cytoplasm [43]. The majority of balloon cell melanomas are amelanotic [43]. Some dust-like melanin granules may be present in a few tumor cells, and Fontana–Masson staining may be required for their demonstration [43,45]. These cells have a large vesicular nuclei with a prominent nucleoli [7,43]. Mitotic activity is generally low [43,45] and these dermal masses exhibit no junctional activity [45].

Signet-ring melanomas are composed of round to polygonal cells [45,52], with a pale eosino‐ philic cytoplasm and a darker periphery [52]. The nuclei are vesicular, crescent shaped and located at the periphery, giving the cells the appearance of signet-rings [52]. Nucleoli are prominent and occasional multinucleated cells may be present [45].

## **7. Histochemical diagnosis**

The histological diagnosis of melanoma can be a challenge for the pathologist, especially in amelanotic tumours. On the other hand, in tumours heavily pigmented the observation of cellular features could be very difficult, requiring the use of bleaching, a histochemical method where melanin is extracted [7].

In cases where the diagnosis of a melanocytic tumor is not evident, histochemical methods specific for the cells producing melanin, such as DOPA (dihydroxyphenylalanine) reaction can be used [7,43].

## **8. Molecular diagnostic methods**

A diagnosis of melanocytic tumors in dogs is not always easy to obtain only by conventional histological methods. Melanoma is often similar to other tumours types and has a highly variable histologic pattern which implies an accurate differential diagnosis. Furthermore, the distinction between benign and malignant tumors is not always easy [7]. Thus, it is essential the research of additional tools that can be used in melanocytic tumours diagnosis and to achieve a more accurate prognosis [57].

#### **8.1. Classical diagnostic markers**

Several markers are used to evaluate the presence of proteins normally found in melanocytes or in cells of neuroectoderm origin. The most common antibodies used are S-100 protein, *melanoma-associated antigen* (Melan-A)/MART-1, HMB-45, MEL-1, vimentin, *Neuron Specific Enolase* (NSE), microphthalmia transcription factor (MiTF), PNL2, tyrosinase, and tyrosinaserelated proteins 1 and 2 (TRP-1 and TRP-2). Tumor cells are usually positive for vimentin, S100, neuron-specific enolase, and Melan-A, and negative for cytokeratin [7]. However, there is a heterogeneneity of antigen expression. The majority of melanomas are S100 positive, but other tumour types are also positive to this protein. NSE is also positive in smooth muscle and neuroendocrine tumours. Melan-A and HMB-45 expression is not a constant in every canine melanocytic tumours. Vimentin is expressed in other tumours as sarcomas [7,58-61]. HMB-45, tyrosinase, and tyrosine hydroxylase showed 100% specific but low sensitivities. One study refers that PNL2, TRP-1, and TRP-2 seems to be highly sensitive and specific for the diagnosis of canine amelanotic tumours, but it was only performed in oral melanomas [62].

In the absence of an ideal marker that excludes definitively other tumour types, confirms a diagnosis of canine melanoma and positively reacts with tumour cells in all melanomas, a diagnostic panel must be performed, including different antibodies [58-61].

#### **8.2. Growth fraction**

located at the periphery, giving the cells the appearance of signet-rings [52]. Nucleoli are

The histological diagnosis of melanoma can be a challenge for the pathologist, especially in amelanotic tumours. On the other hand, in tumours heavily pigmented the observation of cellular features could be very difficult, requiring the use of bleaching, a histochemical method

In cases where the diagnosis of a melanocytic tumor is not evident, histochemical methods specific for the cells producing melanin, such as DOPA (dihydroxyphenylalanine) reaction can

A diagnosis of melanocytic tumors in dogs is not always easy to obtain only by conventional histological methods. Melanoma is often similar to other tumours types and has a highly variable histologic pattern which implies an accurate differential diagnosis. Furthermore, the distinction between benign and malignant tumors is not always easy [7]. Thus, it is essential the research of additional tools that can be used in melanocytic tumours diagnosis and to

Several markers are used to evaluate the presence of proteins normally found in melanocytes or in cells of neuroectoderm origin. The most common antibodies used are S-100 protein, *melanoma-associated antigen* (Melan-A)/MART-1, HMB-45, MEL-1, vimentin, *Neuron Specific Enolase* (NSE), microphthalmia transcription factor (MiTF), PNL2, tyrosinase, and tyrosinaserelated proteins 1 and 2 (TRP-1 and TRP-2). Tumor cells are usually positive for vimentin, S100, neuron-specific enolase, and Melan-A, and negative for cytokeratin [7]. However, there is a heterogeneneity of antigen expression. The majority of melanomas are S100 positive, but other tumour types are also positive to this protein. NSE is also positive in smooth muscle and neuroendocrine tumours. Melan-A and HMB-45 expression is not a constant in every canine melanocytic tumours. Vimentin is expressed in other tumours as sarcomas [7,58-61]. HMB-45, tyrosinase, and tyrosine hydroxylase showed 100% specific but low sensitivities. One study refers that PNL2, TRP-1, and TRP-2 seems to be highly sensitive and specific for the diagnosis

of canine amelanotic tumours, but it was only performed in oral melanomas [62].

diagnostic panel must be performed, including different antibodies [58-61].

In the absence of an ideal marker that excludes definitively other tumour types, confirms a diagnosis of canine melanoma and positively reacts with tumour cells in all melanomas, a

prominent and occasional multinucleated cells may be present [45].

**7. Histochemical diagnosis**

176 Melanoma – Current Clinical Management and Future Therapeutics

where melanin is extracted [7].

**8. Molecular diagnostic methods**

achieve a more accurate prognosis [57].

**8.1. Classical diagnostic markers**

be used [7,43].

The proliferative activity has provided valuable information on cell growth kinetics and consequently tumour behavior in melanocytic tumours [63]. There are some markers that estimate tumor proliferation, by identifying steps associated with cell cycle [64].

MIB-1 is the monoclonal antibody which is reactive against Ki-67 nuclear antigen, a protein present in all active phases of the cell cycle (G1, S, G2 and M) while being absent in resting phase (G0) [65,66].

In canine melanocytic tumours, Ki-67 may be useful in distinction between benign and malignant tumors: melanocytomas seem to have a significant lower growth fraction than malignant melanomas [67]. Furthermore, KI-67 could be an important prognosis factor in canine cutaneous melanocytic tumours [64,68].

#### **8.3. DNA ploidy**

Changes in DNA content may reflect chromosomal alterations and represent tumour genetic instability [69-72]. Changes in the DNA ploidy constitute an early event in carcinogenesis [73,74] and detection of aneuploid cell population of pre-neoplastic lesions can be considered a factor risk of the emergence of malignant tumours [75]. Although usually benign tumors were diploid [76] and aneuploidy were malignant [77], diploidy is not synonymous of a benign behavior [78]. Moreover, not all malignant tumors are aneuploid [79,80].

There are few studies about the ploidy assessment in canine melanocytic tumors [81,82] and its usefulness is still discussed. DNA index and ploidy balance seem to provide an additional tool to evaluate melanocytic tumors, being useful in the distinction of benign and malignant melanocytic tumours, mainly in amelanotic lesions [82]. Flow cytometry apparently has a limited utility for predicting the biological behavior of pigmented canine melanomas. DNA content and nuclear morphometric variables have little value in predicting survival time [83].

#### **8.4. c-kit expression**

The c-Kit protein (CD117), a transmembrane receptor that belongs to RTK III family, is a growth factor for melanocyte migration and proliferation. A loss-of-function KIT mutation are usually related with human melanocytic tumors [84,85].

In canine cutaneous melanocytic tumours, c-kit immunolabeling (both extension and intensity) were generally higher in melanocytomas than in malignant melanomas. The lack of c-kit expression in canine cutaneous malignant melanomas might be used as a criteria of tumor aggressiveness, helping to achieve a proper diagnosis [86].

#### **8.5. Matrix metalloproteinase 2 and 9 expression**

MMPs are zinc- and calcium-dependent proteases that promove not only the disruption and remodeling of structural barriers [87-89] but also a response to signaling molecules, acting as ligand for cellular adhesion receptors [90-92].

MMP-2 is widely distributed and constitutively expressed by most cells [93,94]. This protease has major roles reducing cell adhesion, stimulating cell migration and differentiation, and acting as an anti-inflammatory factor [95]. MMP-9 expression is normally induced, while almost MMPs are constitutively secreted after their translation [93,96], and may act as anti- or pro-inflammatory factors [95].

MMPs play a pivotal role in cancer development and progression [92,94,96-98] contributing to tumour proliferation, invasion, intravasation into circulation, extravasation, migration to metastatic sites and angiogenesis [90,94,99-101], deregulate the balance between growth and antigrowth signals in the tumour microenvironment [97,102,103], orchestrate inflammation [94,102,104], and evade apoptosis [89,91,102].

In canine cutaneous melanocytic tumours, MMP-2 and MMP-9 may be taken as a complement to histology in tumour diagnosis, especially in borderline lesions. Both MMP-2 and MMP-9 were expressed in the majority of canine cutaneous melanocytic tumours. MMP-2 is most commonly expressed in melanocytomas than in melanomas [105]. MMP-9 was overexpressed in malignant melanomas, compared with its expression in melanocytomas [106].

Additionally, in canine malignant melanomas a switch may occur in the MMP expression profile during tumour progression; meaning that the aggressiveness, evaluated by nucle‐ ar grade, seems to be associated with a decrease of MMP-9 and an increase of MMP-2 expression [105].

#### **8.6. Inflammatory cells associated to tumour**

The tumour associated inflammatory infiltrate may be modulate and determine tumor behavior [107]. The most studied cells in CCMT are macrophages and T-lymphocyte, however, studies on the matter are scarce.

#### *8.6.1. Tumour Associated Macrophages (TAMs)*

Macrophages constitute the most abundant leukocytes in the tumor environment, recruited by a number of chemoattractants that are produced by the tumor cells and tumor-associated stroma [108,109]. TAMs play a critical role in tumour progression and invasion by inducing neovascularization, suppressing immunocompetent cells and supporting cancer stem cells [110-113].

One study published by our group found that canine cutaneous melanocytomas present a lower number of TAMs than malignant melanomas. TAMs could constitute an important marker of canine melanocytic aggressiveness, being implicated in the progression of melanocytic precursor lesions to malignant melanoma [114].

#### *8.6.2. T-Lymphocytic Infiltrate (TLI)*

In spite of the fact that the role of the T-lymphocytic infiltrate in cancer tumourigenesis remains controversial, several studies showed that the presence of TILs are related to tumoural behavior in different tumour types [115,116].

Preliminary studies of our group showed that there is a difference between TILs in benign and malignant melanocytic neoplasms, whereas all melanocytomas presented little or even absence of TILs and melanomas had a more intense TILs, (Figure 19), [117].

**Figure 19.** Abundant CD3+TILs in canine cutaneous melanoma (IHC, 100x), courtesy of Dr. Patricia Monteiro.

### **8.7. E-cadherin/β-catenin**

MMP-2 is widely distributed and constitutively expressed by most cells [93,94]. This protease has major roles reducing cell adhesion, stimulating cell migration and differentiation, and acting as an anti-inflammatory factor [95]. MMP-9 expression is normally induced, while almost MMPs are constitutively secreted after their translation [93,96], and may act as anti- or

MMPs play a pivotal role in cancer development and progression [92,94,96-98] contributing to tumour proliferation, invasion, intravasation into circulation, extravasation, migration to metastatic sites and angiogenesis [90,94,99-101], deregulate the balance between growth and antigrowth signals in the tumour microenvironment [97,102,103], orchestrate inflammation

In canine cutaneous melanocytic tumours, MMP-2 and MMP-9 may be taken as a complement to histology in tumour diagnosis, especially in borderline lesions. Both MMP-2 and MMP-9 were expressed in the majority of canine cutaneous melanocytic tumours. MMP-2 is most commonly expressed in melanocytomas than in melanomas [105]. MMP-9 was overexpressed

Additionally, in canine malignant melanomas a switch may occur in the MMP expression profile during tumour progression; meaning that the aggressiveness, evaluated by nucle‐ ar grade, seems to be associated with a decrease of MMP-9 and an increase of MMP-2

The tumour associated inflammatory infiltrate may be modulate and determine tumor behavior [107]. The most studied cells in CCMT are macrophages and T-lymphocyte,

Macrophages constitute the most abundant leukocytes in the tumor environment, recruited by a number of chemoattractants that are produced by the tumor cells and tumor-associated stroma [108,109]. TAMs play a critical role in tumour progression and invasion by inducing neovascularization, suppressing immunocompetent cells and supporting cancer stem cells

One study published by our group found that canine cutaneous melanocytomas present a lower number of TAMs than malignant melanomas. TAMs could constitute an important marker of canine melanocytic aggressiveness, being implicated in the progression of

In spite of the fact that the role of the T-lymphocytic infiltrate in cancer tumourigenesis remains controversial, several studies showed that the presence of TILs are related to tumoural

in malignant melanomas, compared with its expression in melanocytomas [106].

pro-inflammatory factors [95].

expression [105].

[110-113].

[94,102,104], and evade apoptosis [89,91,102].

178 Melanoma – Current Clinical Management and Future Therapeutics

**8.6. Inflammatory cells associated to tumour**

however, studies on the matter are scarce.

*8.6.1. Tumour Associated Macrophages (TAMs)*

*8.6.2. T-Lymphocytic Infiltrate (TLI)*

behavior in different tumour types [115,116].

melanocytic precursor lesions to malignant melanoma [114].

Epithelial cadherin (E-cadherin) is a transmembranar glycoprotein which belongs to a family of cell to cell adhesion molecules dependent of the present of calcium molecules to bind cytoskeleton proteins through catenins. A decreased or altered expression of E-cadherin molecules often represents an increased invasiveness of tumour cells and ultimately malig‐ nancy in animal and human epithelial tumours [118,119].

In canine melanocytic tumours, the benign lesions present a membranous labelling (Figure 20) and the malignant ones an erroneous labelling, with a cytoplasmatic predominant immu‐ nostaining. Additionally, a loss of E-cadherin expression is noted in melanoma [120]. A loss of membrane E-cadherin/β-catenin complex is also detected in canine melanoma showing that a disruption of E-cadherin/β-catenin complexes and a increase of β-catenin may be associated with canine melanocytic tumours progression and aggressiveness [121].

#### **8.8. Cox-1 and Cox-2 expression**

Cyclooxygenase (COX), also known as the prostaglandin H-synthase, is an enzyme envolved on prostanoids biosynthesis. Cox-1 and Cox-2 are the two cyclooxigenase isoforms identified to date, similar in its structure but produced by different genes. The biological functions are also different: Cox-1, constitutively expressed in many tissues, plays an important role in the regulation of normal physiological, while Cox-2 is usually absent from normal cells but induced by growth factors, inflammatory reactions, tumour promoters and oncogenes [122-125].

**Figure 20.** Strong membranar E-cadherin expression (IHC, 200x), courtesy of Dr. Mariana Santos.

Cox-1 and Cox-2 expression was recently described in canine melanocytic tumours [126-128]. Cox-1 is expressed in almost every tumours, both benign and malignant melanocytic skin lesions. Regarding Cox-2, melanocytomas did not present a positive immunolabelling, but in melanomas Cox-2 expression was present in more than 50% of the tumours [114,127]. The differences observed suggest that Cox-2 expression could be a useful tool in canine melanoma diagnosis, particularly in borderline lesions.

COX-2 expression was also observed in tumours with epithelium ulceration, necrosis, high mitotic index and nuclear grade and in less pigmented neoplasms, which could represent the higher aggressiveness of Cox-2 positive melanocytic tumours. In canine malignant melano‐ mas, Cox-2 is associated with a higher cellular proliferation [114]. Besides the relation with tumour behavior [127], Cox-2 over-expression relates with a poor overall survival [129].

#### **8.9. Angiogenesis**

Vascular system is essential in oxygen and nutrients supply, elimination of metabolism products and promoting efficient access of leukocytes [130].

Angiogenesis is a complex process by which new blood vessels develop from pre-existing vasculature [131-133]. It is a fundamental requirement for organs development. Angiogenesis is also implicated in the pathogenesis of different pathological alterations, such as cancer and inflammation [134-136].

The tumor-associated neovascularization, by establishing continuity with the systemic circulation, allows tumor cells expressing their critical advantage in growth and facilitates metastization [137-139].

#### *8.9.1. Vascular endothelial growth factor*

Angiogenesis is a delicate process, tightly regulated by the balance of pro- and anti-angiogenic factors [140]. Among the angiogenic factors, VEGF is the more powerful and ubiquitous vascular endothelial growth factor, capable of inducing proliferation, migration, specialization and survival of endothelial cells [141,142]. Functions of VEGF family members related to neoplastic pathogenesis are linked not only to its angiogenic capacity [143], but also with the lymphangiogenesis [144,145], immunosuppression [146-148], stimulation and recruitment of endothelial and hematopoietic precursors of the bone marrow [149,150] and anti-apoptotic activity [150].

In canine melanomas, the only study published on VEGF expression is in oral melanomas. High blood concentrations of VEGF were correlated to a shorter survival time in dogs receiving definitive therapy and were associated with tumour stage [151].

This is a promising area, since VEGF may be a good indicator of preneoplastic change in melanocytic lesions [137,152]. VEGF plays a role in human melanoma progression [153,154] with a strong involvment in the switch from the radial to the vertical growth phase and the metastatic phase. So, anti-angiogenic agents might even interfere with or block melanoma progression [155].

Preliminary studies performed by Gomes J and Pires I (data unpublished) show that VEGF may be useful as a discriminating factor between malignant melanoma and benign, since it is more intensely expressed in melanomas (Figure 21) than in melanocytomas.

**Figure 21.** Canine cutaneous melanoma with a strong and diffuse VEGF expression (IHC, 200x), courtesy of Dr. Joana Gomes.

#### *8.9.2. Microvessel Density (MVD)*

Cox-1 and Cox-2 expression was recently described in canine melanocytic tumours [126-128]. Cox-1 is expressed in almost every tumours, both benign and malignant melanocytic skin lesions. Regarding Cox-2, melanocytomas did not present a positive immunolabelling, but in melanomas Cox-2 expression was present in more than 50% of the tumours [114,127]. The differences observed suggest that Cox-2 expression could be a useful

COX-2 expression was also observed in tumours with epithelium ulceration, necrosis, high mitotic index and nuclear grade and in less pigmented neoplasms, which could represent the higher aggressiveness of Cox-2 positive melanocytic tumours. In canine malignant melano‐ mas, Cox-2 is associated with a higher cellular proliferation [114]. Besides the relation with tumour behavior [127], Cox-2 over-expression relates with a poor overall survival [129].

Vascular system is essential in oxygen and nutrients supply, elimination of metabolism

Angiogenesis is a complex process by which new blood vessels develop from pre-existing vasculature [131-133]. It is a fundamental requirement for organs development. Angiogenesis is also implicated in the pathogenesis of different pathological alterations, such as cancer and

The tumor-associated neovascularization, by establishing continuity with the systemic circulation, allows tumor cells expressing their critical advantage in growth and facilitates

Angiogenesis is a delicate process, tightly regulated by the balance of pro- and anti-angiogenic factors [140]. Among the angiogenic factors, VEGF is the more powerful and ubiquitous

tool in canine melanoma diagnosis, particularly in borderline lesions.

**Figure 20.** Strong membranar E-cadherin expression (IHC, 200x), courtesy of Dr. Mariana Santos.

180 Melanoma – Current Clinical Management and Future Therapeutics

products and promoting efficient access of leukocytes [130].

**8.9. Angiogenesis**

inflammation [134-136].

metastization [137-139].

*8.9.1. Vascular endothelial growth factor*

Tumor angiogenesis can be estimated through a quantification of microvessel density (MVD). The most widely used method is the immunohistochemical methods, in which specific markers for endothelial cells are employed, as von Willebrand factor (Figure 22), CD34 and CD31 [156-158].

MVD seems to be important for diagnostic purposes in canine MT's. MVD is significantly higher in melanoma than in melanocytomas [159] and its expression has been associated with a high mitotic index, necrosis and ulceration (study performed by Gomes J and Pires I, data unpublished). However, its prognostic significance is still discussable [159,160].

**Figure 22.** Tumoural neovessels positive to von Willebrand factor (IHC, 400x), courtesy of Dr. Joana Gomes.

## **9. Conclusions**

The incidence of melanoma is increasing annually both in man and in dog. Given that dogs and humans share the same environment and similarities between human and canine melanoma it is urgent to discuss common mechanisms in melanoma development in both species.

Melanoma diagnosis in dogs can be challenging due to the variety of its histological appear‐ ances, especially when pathologists are facing amelanotic or metastatic lesions. Although the definitive diagnosis of a melanoma is often difficult by the lack of specific markers that can distinguish these lesions, immunohistochemistry plays a key role in the differential diagnosis with other neoplasms.

Additionally, the distinction between benign and malignant melanocytic tumours is not always easy, especially in borderline lesions, thus the importance of a strong knowledge of new markers of malignancy for the establishment of a definitive diagnosis and a correct therapy managment and prognosis establishment.

## **Author details**

MVD seems to be important for diagnostic purposes in canine MT's. MVD is significantly higher in melanoma than in melanocytomas [159] and its expression has been associated with a high mitotic index, necrosis and ulceration (study performed by Gomes J and Pires I, data

unpublished). However, its prognostic significance is still discussable [159,160].

182 Melanoma – Current Clinical Management and Future Therapeutics

**Figure 22.** Tumoural neovessels positive to von Willebrand factor (IHC, 400x), courtesy of Dr. Joana Gomes.

The incidence of melanoma is increasing annually both in man and in dog. Given that dogs and humans share the same environment and similarities between human and canine melanoma it is urgent to discuss common mechanisms in melanoma development in both

Melanoma diagnosis in dogs can be challenging due to the variety of its histological appear‐ ances, especially when pathologists are facing amelanotic or metastatic lesions. Although the definitive diagnosis of a melanoma is often difficult by the lack of specific markers that can distinguish these lesions, immunohistochemistry plays a key role in the differential diagnosis

Additionally, the distinction between benign and malignant melanocytic tumours is not always easy, especially in borderline lesions, thus the importance of a strong knowledge of new markers of malignancy for the establishment of a definitive diagnosis and a correct

**9. Conclusions**

with other neoplasms.

therapy managment and prognosis establishment.

species.

Luis Resende1 , Joana Moreira2 , Justina Prada3 , Felisbina Luisa Queiroga4 and Isabel Pires3\*

\*Address all correspondence to: ipires@utad.pt

1 Veterinary Faculty, Lusófona University, Lisbon, Portugal

2 Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro, Portugal

3 Animal and Veterinary Research Centre (CECAV), Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro, Portugal

4 Center for Research and Technology of Agro-Environment and Biological Sciences (CIT‐ AB), Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro, Por‐ tugal

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**Therapeutic Approaches Against Disease**

## **Emerging Drug Combination Approaches in Melanoma Therapy**

Jin Wang, Duane D. Miller and Wei Li

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59360

## **1. Introduction**

The FDA approvals of ipilimumab targeting the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), pembrolizumab targeting the programmed cell death protein 1 (PD-1), BRAF inhibitors vemurafenib and dabrafenib, and MEK inhibitor trametinib represent significant milestones in more effective treatment of advanced melanoma. However, it is clear that the use of these single-agent therapies have limitation clinically. For example, ipilimumab only showed 4.5% objective response rate when used alone in a Phase II clinical trial [1]. The efficacy of vemurafenib lasts only 6.7 months before the disease relapses especially in patients with metastatic melanoma [2]. Therefore, rational combination approaches are strongly preferred in order to improve the overall patient progression-free survival (PFS), overcome or delay the development of multi-drug resistance and reduce the incidents of side effects [3-6].

In this chapter, we will summarize the emerging combination therapy approaches from both clinical trial and preclinical research in the past five years.

## **2. Combination of kinase inhibitors for melanoma treatment**

#### **2.1. Combined inhibitions targeting components within the Mitogen-Activated Protein Kinase (MAPK) signaling pathway**

#### *2.1.1. Targeting BRAF: Mechanism of action, toxicity and drug resistance*

BRAF is a serine/threonine growth signal transduction protein kinase from RAF family which plays important roles in the RAS/RAF/MEK/ERK pathway and directs cell division, prolifer‐

© 2015 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 eproduction in any medium, provided the original work is properly cited.

ation and secretion [7]. BRAF inhibitors (BRAFi) are ATP-competitive ligands which inactivate the function of BRAF protein by either stabilizing the inactive form of kinase domain (sorafe‐ nib) or preferentially inhibit the active form of the kinase (vemurafenib, dabrafenib) [8, 9]. Various mutations of *BRAF* gene have been identified in cancers including melanoma, colorectal and ovarian cancer. Around 60% of human melanoma adopted the T1799A trans‐ version in exon 15, which lead to BRAFV600E mutation and the over-activated monomer phosphorylation for BRAFV600E [9, 10]. The two FDA approved BRAFi (Vemurafenib and dabrafenib) selectively and potently block the activation of BRAFV600E and thus inhibit the MAPK signaling pathway. These drugs show very high clinical efficacy in metastatic mela‐ noma patients harboring the BRAFV600E mutation [11-13]. Interestingly, in a clinical study which treated 43 patients with any V600 BRAF mutation including the rare V600R variant, five out of the six melanoma patients having V600R mutation had clinical response to the therapy of vemurafenib or dabrafenib (response rate 86%) [14].

**Figure 1.** The mechanisms of BRAF inhibitor vemurafenib (Vem) action, toxicity and the interaction between melano‐ ma cells with T lymphocytes.

However, wide type BRAF melanoma tumors do not respond to vemurafenib or dabrafenib inhibition, although they are sensitive to the MEK inhibitors [9]. Paradoxically, in cells with RAS mutation and wild-type BRAF, treatment with vemurafenib or dabrafenib will promote the formation of BRAF-CRAF heterodimer and lead to the activation of subsequent MEK/ERK signaling and cell proliferation as shown in Figure 1 [5]. This mechanism is used to explain the observation of typical clinical side effects associated with the use of vemurafenib: nearly 25% of patients developed skin lesions and even cutaneous squamous cell carcinoma (CSCC). In addition, *in vitro* study has revealed that vemurafenib inhibits multiple off-target kinases including c-Jun N-terminal kinase (JNK), suppresses JNK-dependent apoptosis, and generates CSCC toxicity [15].

#### *2.1.2. Mechanisms of resistance to BRAF inhibition*

ation and secretion [7]. BRAF inhibitors (BRAFi) are ATP-competitive ligands which inactivate the function of BRAF protein by either stabilizing the inactive form of kinase domain (sorafe‐ nib) or preferentially inhibit the active form of the kinase (vemurafenib, dabrafenib) [8, 9]. Various mutations of *BRAF* gene have been identified in cancers including melanoma, colorectal and ovarian cancer. Around 60% of human melanoma adopted the T1799A trans‐ version in exon 15, which lead to BRAFV600E mutation and the over-activated monomer phosphorylation for BRAFV600E [9, 10]. The two FDA approved BRAFi (Vemurafenib and dabrafenib) selectively and potently block the activation of BRAFV600E and thus inhibit the MAPK signaling pathway. These drugs show very high clinical efficacy in metastatic mela‐ noma patients harboring the BRAFV600E mutation [11-13]. Interestingly, in a clinical study which treated 43 patients with any V600 BRAF mutation including the rare V600R variant, five out of the six melanoma patients having V600R mutation had clinical response to the therapy of

**Figure 1.** The mechanisms of BRAF inhibitor vemurafenib (Vem) action, toxicity and the interaction between melano‐

vemurafenib or dabrafenib (response rate 86%) [14].

200 Melanoma – Current Clinical Management and Future Therapeutics

ma cells with T lymphocytes.

In general, due to alternative pathway activations and inter-and intra-patients melanoma genetic heterogeneity, various mechanisms of resistance to BRAF inhibition have been identified [10, 16-19]. As we mentioned before, melanoma tumors bearing wide type BRAF are intrinsically resistant to vemurafenib and dabrafenib. Tumor micro-environment also con‐ tributes to the innate resistance to BRAF inhibition in melanoma. For example, stromal cells secrete hepatocyte growth factor (HGF), which activates the HGF-receptor MET, MAPK and PI3K-AKT pathways [20].

Eventually, nearly all BRAF mutated melanoma tumors develop acquired drug resistance upon treatment with BRAF inhibitors. The disease progression arises as early as two-month continuous treatment [18, 19]. The mechanisms of acquired resistance to BRAF inhibition can be generalized into two categories: BRAFV600E-bypass mechanisms and MAPK-bypass mech‐ anisms.

First, the BRAFV600E-bypass mechanisms reactivate MAPK signaling and lead to ERK-depend‐ ent tumor cell survival and proliferation (Figure 2A). COT, which is coded by gene *MAP3K8*, is a MEK kinase. The overexpression of COT or amplification of *MAP3K8* directly activates MEK signaling without the participation of RAF protein [21]. The mutant of MEK1C121S increases catalytic capability and circumvents BRAF to activate basal level of ERK phosphor‐ ylation [22]. Before the treatment of vemurafenib or dabrafenib, melanoma cells with BRAFV600E mutation have over-activated monomer BRAF/MEK/ERK cascade which forms an ERK-dependent negative feedback loop. This negative feedback loop reduces the expression of the active RAS-GTP. In the presence of vemurafenib or dabrafenib, ERK phosphorylation level is rapidly reduced and the feed-back suppression on RAS activation is abolished (Figure 1). Therefore, eventually the ERK cascade level is restored through RAS over-activation. NRAS mutants including NRASQ61K and NRASQ61R can drive ERK activation through ARAF or CRAF homo-or hetero-dimers which are alternative MEK activators [23]. The combinations of BRAF inhibition plus MEK or ERK inhibition have showed efficacy of overcoming the resistance through these BRAF V600E-bypass mechanisms [24-26], leading to the recent FDA approval of dabrafenib plus trametinib combination therapy for advanced melanoma.

Second, the MAPK-bypass mechanisms allow melanoma cells to escape from the cytotoxicity of BRAF or MEK inhibition through the activation of ERK-independent survival pathways (Figure 2B). The PI3K-AKT signaling pathway can be activated through the overexpression of receptor tyrosine kinases (RTKs), for example, insulin-like growth factor 1 (IGF-1) receptor (IGF-1R) and platelet-derived growth factor receptor beta (PDGFRβ) [27]. The elevated levels of IGF-1R, PDGFRβ or HGF can also stimulate another receptor tyrosine kinase, MET, and increase the activity of PI3K. Phosphatase tensin (PTEN) is a negative regulator of PI3K. The PTEN loss-of-function mutation induces the resistance of BRAF inhibition and reduces the PFS of dabrafenib therapy in melanoma patients due to the PI3K activation [28]. Moreover, the upregulation of cyclin D1 can activate cyclin-dependent kinase 4 (CDK4) and 6 (CDK6) and make melanoma cells less dependent on MAPK signaling in cell cycle progressing [29].

**Figure 2.** The mechanisms of acquired resistance to BRAF inhibition.

Additionally, Jaehyuk Choi *et al* has reported a BRAFL505H mutation which changes an amino acid residue in BRAF-vemurafenib interface and causes the resistance to vemurafenib treat‐ ment *in vitro* [30]. Since vemurafenib is a substrate of the ATP-binding cassette sub-family G member 2 (ABCG2), the overexpression of ABCG2 in BRAFV600E melanoma cell lines has caused the increasing of vemurafenib efflux *in vitro* [31]. The elucidation on the mechanism of acquired-resistance to BRAFi opens a door to rationally design and explore the proper combination strategies to overcome or delay the development of BRAFi resistance.

#### *2.1.3. Targeting MEK: Mechanism of action, toxicity and resistance*

Trametinib, which is approved by FDA in May 2013 as a monotherapy agent against advanced melanoma with BRAFV600E and BRAFV600K mutations, is a first-in-class, orally available, allosteric (non-ATP-competitive) MEK1/MEK2 inhibitor (MEKi) [32, 33]. It selectively inhibits MEK, the down-stream kinase protein of RAF in the RAS-RAF-MEK-ERK pathway. As a result, melanoma cells with acquired resistance to BRAFi are commonly cross-resistant to MEKi such as trametinib or selumetinib, another selective allosteric MEKi [24, 34]. This mechanism explains the clinical trial results in which trametinib monotherapy fails to significantly benefit patients who have already developed acquired BRAFi resistance [35]. In contrast to the use of a BRAFi, no CSCC side effects are observed among the patients received trametinib treatment in clinical trials [13, 32]. However, similar to the use of vemurafenib, disease progression occurs within 6-7 months in patients receiving single-agent trametinib treatment [36]. Nevertheless, a retrospective analysis of 23 patients, who were first treated with MEKi and upon progression with a selective BRAFi, shows that the median time to progression (TTP) has been prolonged to 8.9 months from 4.8 months using a single-agent MEKi or 4.4 months for a single-agent BRAFi treatment, respectively [37]. However, a recent clinical trial indicated that if melanoma patients were treated with a BRAFi first then MEKi therapy, no confirmed response was observed [35]. This indicates that optimal treatment schedule and sequence is important for the melanoma therapy targeting the MAPK pathway.

#### *2.1.4. Drug combination targeting MAPK pathway: From lab bench to clinical practice*

(Figure 2B). The PI3K-AKT signaling pathway can be activated through the overexpression of receptor tyrosine kinases (RTKs), for example, insulin-like growth factor 1 (IGF-1) receptor (IGF-1R) and platelet-derived growth factor receptor beta (PDGFRβ) [27]. The elevated levels of IGF-1R, PDGFRβ or HGF can also stimulate another receptor tyrosine kinase, MET, and increase the activity of PI3K. Phosphatase tensin (PTEN) is a negative regulator of PI3K. The PTEN loss-of-function mutation induces the resistance of BRAF inhibition and reduces the PFS of dabrafenib therapy in melanoma patients due to the PI3K activation [28]. Moreover, the upregulation of cyclin D1 can activate cyclin-dependent kinase 4 (CDK4) and 6 (CDK6) and make melanoma cells less dependent on MAPK signaling in cell cycle progressing [29].

Additionally, Jaehyuk Choi *et al* has reported a BRAFL505H mutation which changes an amino acid residue in BRAF-vemurafenib interface and causes the resistance to vemurafenib treat‐ ment *in vitro* [30]. Since vemurafenib is a substrate of the ATP-binding cassette sub-family G member 2 (ABCG2), the overexpression of ABCG2 in BRAFV600E melanoma cell lines has caused the increasing of vemurafenib efflux *in vitro* [31]. The elucidation on the mechanism of acquired-resistance to BRAFi opens a door to rationally design and explore the proper

Trametinib, which is approved by FDA in May 2013 as a monotherapy agent against advanced melanoma with BRAFV600E and BRAFV600K mutations, is a first-in-class, orally available, allosteric (non-ATP-competitive) MEK1/MEK2 inhibitor (MEKi) [32, 33]. It selectively inhibits

combination strategies to overcome or delay the development of BRAFi resistance.

**Figure 2.** The mechanisms of acquired resistance to BRAF inhibition.

202 Melanoma – Current Clinical Management and Future Therapeutics

*2.1.3. Targeting MEK: Mechanism of action, toxicity and resistance*

Given that the mechanisms of tumor cells develop resistance to BRAFi partially by reactivating the ERK cascade and side effects such as CSCC are RAF-dependent, combining BRAFi with MEKi has attracted lots of research interest in order to further block the MAPK signaling pathway. *In vitro* and murine models first show the synergistic anti-proliferation and antitumor growth effects using the combined BRAFi and MEKi treatment [9, 27, 38, 39]. Further, this combination overcomes the acquired resistance to BRAFi [27, 38] in both cellular based assay and mouse xenograft models. In addition, the combined inhibition of BRAF-MEK suppresses the paradoxical BRAFi-induced MAPK signal elevation in melanoma cells and reduces the incidences of skin lesions in a rat model [9].

When it comes to the clinical trial data, the combined inhibition of BRAF-MEK has presented significant improvements of major patient benefits (PFS and overall survival). A phase I/II trial (ClinicalTrials.gov, NCT1072175) investigated the combination of oral dabrafenib (150 mg twice per day) plus oral trametinib (1 or 2 mg daily) (combination 150/1 and 150/2) versus monotherapy of dabrafenib (150 mg twice per day) over 108 metastatic melanoma patients bearing either V600E (92 patients) or V600K (16 patients) BRAF mutation [12, 36]. Median PFS in combination 150/2 group reached 9.4 months, compared to 5.8 months in the dabrafenib monotherapy group (hazard ratio 0.39, 95% confidence interval 0.25 to 0.62). The incidence of CSCC adverse events among combination 150/2 group is non-significantly lower than that among monotherapy group (7% versus 19%, *P*=0.09). But more frequent cases of pyrexia which is not common in trametinib single treatment have been reported in combination 150/2 group (71%, with recurrent rate 79%), as compared with dabrafenib monotherapy group (26%) [40]. These promising data lead to an accelerated FDA approval of the combination of dabrafenib (BRAFi) and trametinib (MEKi) for the treatment of unresectable or metastatic melanoma patients with BRAF V600E or V600K mutation, although further phase III studies with recruitment of more patients comparing the combination therapy with dabrafenib or vemur‐ afenib single treatment (ClinicalTrials.gov, NCT01584648, NCT01597908) are still being assessed.

In addition, several ongoing phase I/II clinical trials now have shown that generally the combination of other BRAFi and MEKi is well tolerated in patients with or without receiving BRAFi treatment before (ClinicalTrials.gov, NCT01271803 vemurafenib (BRAFi)+cobimetinib (MEKi), NCT01543698 LGX818 (BRAFi)+MEK162 (MEKi)) [41-43] and overall response rate has increased comparing to the monotherapy groups, although the anti-tumor efficacy data haven't been released.

#### **2.2. Combination targeted therapy using Phosphatidylinositol 3-Kinase (PI3K)/AKT/ mammalian Target of Rapamycin (mTOR) inhibitors**

The activation of PI3K/AKT/mTOR pathway have been widely proved to be one of the major mechanisms of intrinsic or acquired resistance to both DNA-methylation agents (e.g. dacar‐ bazine) and targeted BRAF inhibitor therapy (Figure 2). Some cell lines that are cross-resistant to both BRAFi and MEKi, are still sensitive to the inhibition of AKT/mTOR [34]. On the other hand, mechanistic study revealed evidences of a negative crosstalk between RAF/MEK/ERK and PI3K/AKT/mTOR pathways through RAS kinase. Therefore, when the downstream mTOR function is blocked, PI3K will be able to activate MAPK pathway via a switch of RAS [44, 45]. These investigations suggest a promising combination strategy of targeting MAPK pathway together with PI3K/AKT/mTOR cascade. Several preclinical studies widely proved that in MAPK inhibition sensitive melanoma cell lines, co-targeting PI3K/AKT/mTOR effectively induces cancer cell apoptosis with down-regulated anti-apoptotic BCL-2 family proteins [34, 46-48]. Such a co-targeting strategy can also postpone the emergence of acquired resistance to BRAFi dabrafenib mediated by PTEN mutation or disruption [49, 50]. Further, the dual inhibition of two pathways has successfully overcome NRAS mutation mediated resistance to MAPK blockade *in vitro* and induced xenograft tumor regression *in vivo* [34, 38, 51]. Finally, the combination of vemurafenib (BRAFi) or selumetinib (MEKi) with BEZ235 (dual PI3K and mTOR1/2 inhibitor) has been shown to overcome the PDGFRβ-driven resistance to MAPK pathway inhibition [52].

A series of Phase I studies have evaluated the clinical relevance of the combination therapy which co-targets PI3K/AKT/mTOR and RAF/MEK/ERK pathways in terms of the incidence on severe side effect and anti-tumor efficacy in 236 patients. These patients have advanced cancers including melanoma, colorectal, pancreatic and non-small cell lung cancers. Results from three combination groups (AKTi MK2206+MEKi selumetinib, NCT01021748; AKTi GSK2141795+MEKi trametinib, NCT01138085; mTOR inhibitor everolimus+MEKi trametinib, NCT 00955773) are compared to the single treatment groups [53]. Overall, the combination therapy did not provide significant increase of tumor control rate (64.6% for combination, 52.7% for monotherapy, *P*=0.16), although all five colorectal patients with co-activation of both pathways in combination group achieved tumor regression to varied extent between 2% and 64%. However, this combination strategy causes significant higher rates of drug-related grade III and above side effects (53.9% for combination, 18.1% for monotherapy, *P* < 0.001). Further‐ more, two clinical trials which involve the combination therapy of BRAFi or MEKi with AKTi DNE3 recently have been terminated due to the safety concerns of the toxic properties of DNE3 (ClinicalTrials.gov, NCT02087254 and NCT02095652). Nevertheless, in another ongoing phase I/II trial which measures the safety and efficacy of a well-tolerated pan-PI3K inhibitor BKM120 combined with vemurafenib therapy, preliminary data reveals that a vemurafenib-refractory melanoma patient with PTEN expression achieved a 35.9% reduction in target tumor (Clini‐ calTrials.gov, NCT01512251) [54]. In general, drug-related toxicity is one of the major issues for this cross-pathway targeted combination therapy and patients genetic profiling is very important to achieve the maximum objective response.

#### **2.3. Combining targeted therapy with anti-angiogenic agents**

afenib single treatment (ClinicalTrials.gov, NCT01584648, NCT01597908) are still being

In addition, several ongoing phase I/II clinical trials now have shown that generally the combination of other BRAFi and MEKi is well tolerated in patients with or without receiving BRAFi treatment before (ClinicalTrials.gov, NCT01271803 vemurafenib (BRAFi)+cobimetinib (MEKi), NCT01543698 LGX818 (BRAFi)+MEK162 (MEKi)) [41-43] and overall response rate has increased comparing to the monotherapy groups, although the anti-tumor efficacy data

**2.2. Combination targeted therapy using Phosphatidylinositol 3-Kinase (PI3K)/AKT/**

The activation of PI3K/AKT/mTOR pathway have been widely proved to be one of the major mechanisms of intrinsic or acquired resistance to both DNA-methylation agents (e.g. dacar‐ bazine) and targeted BRAF inhibitor therapy (Figure 2). Some cell lines that are cross-resistant to both BRAFi and MEKi, are still sensitive to the inhibition of AKT/mTOR [34]. On the other hand, mechanistic study revealed evidences of a negative crosstalk between RAF/MEK/ERK and PI3K/AKT/mTOR pathways through RAS kinase. Therefore, when the downstream mTOR function is blocked, PI3K will be able to activate MAPK pathway via a switch of RAS [44, 45]. These investigations suggest a promising combination strategy of targeting MAPK pathway together with PI3K/AKT/mTOR cascade. Several preclinical studies widely proved that in MAPK inhibition sensitive melanoma cell lines, co-targeting PI3K/AKT/mTOR effectively induces cancer cell apoptosis with down-regulated anti-apoptotic BCL-2 family proteins [34, 46-48]. Such a co-targeting strategy can also postpone the emergence of acquired resistance to BRAFi dabrafenib mediated by PTEN mutation or disruption [49, 50]. Further, the dual inhibition of two pathways has successfully overcome NRAS mutation mediated resistance to MAPK blockade *in vitro* and induced xenograft tumor regression *in vivo* [34, 38, 51]. Finally, the combination of vemurafenib (BRAFi) or selumetinib (MEKi) with BEZ235 (dual PI3K and mTOR1/2 inhibitor) has been shown to overcome the PDGFRβ-driven resistance to

A series of Phase I studies have evaluated the clinical relevance of the combination therapy which co-targets PI3K/AKT/mTOR and RAF/MEK/ERK pathways in terms of the incidence on severe side effect and anti-tumor efficacy in 236 patients. These patients have advanced cancers including melanoma, colorectal, pancreatic and non-small cell lung cancers. Results from three combination groups (AKTi MK2206+MEKi selumetinib, NCT01021748; AKTi GSK2141795+MEKi trametinib, NCT01138085; mTOR inhibitor everolimus+MEKi trametinib, NCT 00955773) are compared to the single treatment groups [53]. Overall, the combination therapy did not provide significant increase of tumor control rate (64.6% for combination, 52.7% for monotherapy, *P*=0.16), although all five colorectal patients with co-activation of both pathways in combination group achieved tumor regression to varied extent between 2% and 64%. However, this combination strategy causes significant higher rates of drug-related grade III and above side effects (53.9% for combination, 18.1% for monotherapy, *P* < 0.001). Further‐ more, two clinical trials which involve the combination therapy of BRAFi or MEKi with AKTi

**mammalian Target of Rapamycin (mTOR) inhibitors**

204 Melanoma – Current Clinical Management and Future Therapeutics

assessed.

haven't been released.

MAPK pathway inhibition [52].

Melanoma is a vascular tumor. The abnormal expression of the epidermal growth factor (EGF) family protein and the up-regulation of EGFR-mediated alternative survival pathway have critically shaped the response of melanoma to the current chemotherapy agents [55-58]. In a recent study by Sun *et al*, six out of sixteen melanoma cell lines display acquired EGFR expression after the development of resistance to BRAFi and MEKi [59]. Even before the FDA approval of BRAFi and MEKi, the combination of bevacizumab, a recombinant human monoclonal antibody VEGF inhibitor, with a specific chemotherapy agent (for example, fluorouracil [60] or fotemustine[61]), has become a first-line treatment for metastatic melanoma patients. Clinical trials that study the combination of anti-angiogenic agents with cytotoxic agents have achieved promising anti-tumor activity, although tolerability issues exist [62]. VEGF blockage has been shown to enhance the efficacy of a GM-CSF-secreting immunother‐ apy *in vitro* [63]. In addition, a VEGF receptor-2 inhibitor, semaxanib, prolonged both the complete and partial response time of an immunomodulatory drug, thalidomide, over 10 recurrent metastasis melanoma patients without showing significant drug-drug interaction toxicity in a phase II trial [64].

Along with the rapid development of targeted melanoma therapeutics, the combined inhibi‐ tion of VEGFR plus PDGFR or mTOR has shown synergy anti-tumor effects on mouse models of B16 metastatic melanoma without increasing toxicity [65, 66]. A large-scale, unbiased drug screening study, which aims to discover effective genotype selective combinatorial therapeu‐ tics of vemurafenib-resistant BRAF and RAS mutant melanoma, identifies a triple BRAF+EGFR +AKT inhibition as highly effective approach [3]. In the year of 2010, combination of bevaci‐ zumab with an mTOR inhibitor, everolimus, was evaluated in a phase II trial for patients with metastatic melanoma [67]. The treatment was well tolerated in most patients. Seven out of fiftyseven patients (12%) receiving combination therapy have shown major responses, although the median PFS was only 4 months. This year (2014), in a phase II trial that combines bevaci‐ zumab and sorafenib, which is an inhibitor of both RAF kinase and VEGFR-2/PDGFRβ signaling, no objective tumor responses are seen in all the fourteen patients receiving treatment [68, 69]. Interestingly, the median TTP of patients with low VEGF (<300 pg/ml) was longer than that of patients with high VEGF (50 weeks versus 15 weeks, *P*=0.02). Therefore, the levels of VEGF in patients do influence the tumor progression profile (ClinicalTrials.gov, NCT00387751).

### **2.4. Combination therapy using targeted therapy with versatile chemotherapy agents**

Since the abnormally activated (phosphorylation) of ERK and AKT constitutively exist in melanoma cells and promote the disease progression especially metastasis, blocking ERK or AKT pathway can sensitize the metastatic melanoma to the apoptosis induced by chemother‐ apeutic agents including cisplatin, temozolomide, DTIC and arsenite [70-72]. With the understanding of tumor biology about the programmed cell apoptosis and the rapid devel‐ opment of agents that can trigger the cell death process in melanoma, the combination of a MAPK inhibitor with a BCL-2 inhibitor (ABT-737 [73] or navitoclax [74]), or a MDM2 antago‐ nist nutlin-3 [75], has synergistically induced apoptosis of melanoma *in vitro* and suppressed xenograft tumor growth *in vivo*. A comparative analysis on the samples collected from patients receiving vemurafenib or dabrafenib/trametinib combination treatment showed that BCL-2 expression level is closely related to the onset of MAPK inhibition resistance [74]. Clinical trials are being conducted to investigate the combination of BCL-2 inhibitor (BH3 mimetics) navitoclax and vemurafenib [74].

Due to the heterogenetic characteristics of melanoma disease, Vultur A *et al* [76] recently report that MEK or BRAF inhibition can potentially strengthen the invasion property of human melanoma cells by about 20%. As a result, co-inhibiting kinases that are actively involved in cell invasion process, such as RTK, STAT3 and Src, together with MEK inhibition has effectively abolished the invasive phenotype and further caused the tumor cell death in a 3D matrix model.

Metformin, a biguanide oral anti-diabetic drug, has been discovered with antitumor activity in various cancer types including melanoma. Although the exact mechanisms remain to be elucidated, accumulating data suggest that metformin can activate AMP-activated protein kinase (AMPK) and thus increase the activities of VEGF and ERK in BRAFV600E mutated melanoma cells [77]. AMPK negatively regulates malignant cell proliferation and viability [78]. The combination of vemurafenib and metformin has shown synergistic anti-proliferative effects on six out of eleven tested BRAFV600E melanoma cell lines [79]. Pilot clinical studies that evaluate the safety and efficacy of metformin combination therapies (plus dabrafenib or trametinib) are now recruiting patients (ClinicalTrials.gov, NCT0184000, NCT02143050).

Unlike the cutaneous melanoma, over-activation of MAPK pathway in uveal melanoma is associated GNAQ or GNA11 mutations instead of BRAF or RAS mutations [80]. Protein kinase C (PKC) inhibitors such as enzastaurin or AEB071 induce apoptosis in GNAQ-mutant but not in GNAQ wild type uveal melanoma cells [81]. The level of ERK phosphorylation also decreases in these cells when they are treated using PKC inhibitors [81]. Chen *et al.* has recently confirmed the synergy of the combination using a PKC inhibitor with a MEKi (PD0325901 or MEK162) in GNAQ/11 mutant uveal melanoma cells [82].

Understanding the mechanisms of resistance to MAPK inhibition in melanoma can lead to rational combination designs in order to overcome acquired drug resistance to BRAF inhibitors. For example, our lab recently identified a synergistic combination in which a novel tubulin inhibitor ABI-274 combined with vemurafenib could overcome the acquired vemurafenib-resistance [83]. This combination treatment effectively arrested the vemurafe‐ nib-resistant melanoma cells in both G0/G1 and G2/M phases and induced strong apopto‐ sis through the down-regulation of AKT phosphorylation. In addition, the combination of a MEKi (TAK-733) with an Hsp90 inhibitor (ganetespib) induces tumor regressions in vemurafenib-resistant xenograft models also through the depletion of AKT signaling [84]. With the finding that up-regulated cyclin D1 expression is critical for the survival of vemurafenib-resistant cells, a selective inhibitor of cyclin dependent kinase (CDK) 4/6, LY2835219, has been reported to overcome the reactivation of MAPK signaling in vemura‐ fenib-resistant BRAFV600E melanoma [85].

### **3. Combinations involving immunotherapy in melanoma treatment**

#### **3.1. Combined blockade of immuno-checkpoints**

**2.4. Combination therapy using targeted therapy with versatile chemotherapy agents**

navitoclax and vemurafenib [74].

206 Melanoma – Current Clinical Management and Future Therapeutics

model.

Since the abnormally activated (phosphorylation) of ERK and AKT constitutively exist in melanoma cells and promote the disease progression especially metastasis, blocking ERK or AKT pathway can sensitize the metastatic melanoma to the apoptosis induced by chemother‐ apeutic agents including cisplatin, temozolomide, DTIC and arsenite [70-72]. With the understanding of tumor biology about the programmed cell apoptosis and the rapid devel‐ opment of agents that can trigger the cell death process in melanoma, the combination of a MAPK inhibitor with a BCL-2 inhibitor (ABT-737 [73] or navitoclax [74]), or a MDM2 antago‐ nist nutlin-3 [75], has synergistically induced apoptosis of melanoma *in vitro* and suppressed xenograft tumor growth *in vivo*. A comparative analysis on the samples collected from patients receiving vemurafenib or dabrafenib/trametinib combination treatment showed that BCL-2 expression level is closely related to the onset of MAPK inhibition resistance [74]. Clinical trials are being conducted to investigate the combination of BCL-2 inhibitor (BH3 mimetics)

Due to the heterogenetic characteristics of melanoma disease, Vultur A *et al* [76] recently report that MEK or BRAF inhibition can potentially strengthen the invasion property of human melanoma cells by about 20%. As a result, co-inhibiting kinases that are actively involved in cell invasion process, such as RTK, STAT3 and Src, together with MEK inhibition has effectively abolished the invasive phenotype and further caused the tumor cell death in a 3D matrix

Metformin, a biguanide oral anti-diabetic drug, has been discovered with antitumor activity in various cancer types including melanoma. Although the exact mechanisms remain to be elucidated, accumulating data suggest that metformin can activate AMP-activated protein kinase (AMPK) and thus increase the activities of VEGF and ERK in BRAFV600E mutated melanoma cells [77]. AMPK negatively regulates malignant cell proliferation and viability [78]. The combination of vemurafenib and metformin has shown synergistic anti-proliferative effects on six out of eleven tested BRAFV600E melanoma cell lines [79]. Pilot clinical studies that evaluate the safety and efficacy of metformin combination therapies (plus dabrafenib or trametinib) are now recruiting patients (ClinicalTrials.gov, NCT0184000, NCT02143050).

Unlike the cutaneous melanoma, over-activation of MAPK pathway in uveal melanoma is associated GNAQ or GNA11 mutations instead of BRAF or RAS mutations [80]. Protein kinase C (PKC) inhibitors such as enzastaurin or AEB071 induce apoptosis in GNAQ-mutant but not in GNAQ wild type uveal melanoma cells [81]. The level of ERK phosphorylation also decreases in these cells when they are treated using PKC inhibitors [81]. Chen *et al.* has recently confirmed the synergy of the combination using a PKC inhibitor with a MEKi (PD0325901 or

Understanding the mechanisms of resistance to MAPK inhibition in melanoma can lead to rational combination designs in order to overcome acquired drug resistance to BRAF inhibitors. For example, our lab recently identified a synergistic combination in which a novel tubulin inhibitor ABI-274 combined with vemurafenib could overcome the acquired vemurafenib-resistance [83]. This combination treatment effectively arrested the vemurafe‐

MEK162) in GNAQ/11 mutant uveal melanoma cells [82].

Given the unsatisfactory results of cytokine-based melanoma immunotherapy (recombinant interferon-α 2b and high dose interleukin-2) in the past decade, the development and approval of ipilimumab (anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) monoclonal antibody) in 2013 have marked a breakthrough of immune-checkpoints blockade therapy [86]. CTLA-4 (CD152) expresses on the surface of active T-lymphocytes and inhibits the initial Tcell proliferation and migration to the tumor tissue [87]. CTLA-4 antibodies preferentially target the suppressive regulatory T cells and prevent them from being hijacked by tumors [88]. In a double-blinded phase III study in 676 patients with pretreated and refractory metastatic melanoma, ipilimumab at the dose of 3 mg/kg achieved a median OS of 10 months [86]. In a meta-Kaplan-Meier-analysis of data collected from 1,861 melanoma patients in a clinical trial, a plateau of survival curve starts from around 3 years after ipilimumab treatment with followup extends as long as ten years, indicating a long-term survival benefits of ipilimumab therapy (ClinicalTrials.gov, NCT01844505). In addition, ipilimumab showed good tolerance and efficacy in several other clinical trials in which it was combined with a standard chemotherapy agent such as dacarbazine, fotemustine or temozolomide [89].

Another success of immune-check point blockade strategy is the development of antiprogrammed death-1 (PD-1) antibodies, represented by pembrolizumab (MK-3475) and nivolumab [90, 91]. Pembrolizumab, as the first-in-class PD-1 inhibitor, has obtained FDA approval in September 2014 for patients with advanced or unresectable melanoma. The cDNA of PD-1 (CD279) is first cloned in programmed death T cells although PD-1 itself does not directly induce apoptosis. PD-1 is over-expressed on the surface of dysfunctional activated Tcells and contributes to the maintenance of T cell dysfunction (exhaust) phenotype and proliferation disability in the tumor site [92]. Two counter receptors of PD-1 have been identified: PD-L1 and PD-L2. PD-L1 is more frequently and exclusively expressed in various tumor cells; therefore, antibodies targeting PD-L1 (MPDL3280A and BMS-936559) also have anti-tumor activity in advanced cancer including melanoma [91, 93]. The PD-1-PD-L1 ligation retards the recognition and destroying of tumor cells by CD8+cytotoxic T-lymphocytes [87]. As a result, blocking PD-1 or PD-L1 will reverse the cancer cell immune escape. Because both CTLA-4 and PD-1 are key negative receptors that cooperatively modulate the adaptive immune response in tumor progression, their combination has been shown to be synergistic in B16 melanoma tumors without overt toxicity [94].

In a cohort phase I trial that studied the concurrent administration of ipilimumab and nivolumab to 53 patients with advanced, treatment-resistant melanoma, more than 80% tumor reduction was observed in 30% patients after 12 weeks treatment at the maximum tolerated dose. Twenty-one out of fifty-three patients had objective responses and over 80% of these patients had tumor regression. Grade 3/4 adverse events are diagnosed in 53% patients but the toxicities are manageable with immune-suppressants [95]. Consequential trials with more enrollment number of patients are necessary to further evaluate the safety and efficacy of this promising double immune-checkpoints blockage therapy comparing with each of its mono‐ therapy regiments.

Finally, combinatorial clinical trials using ipilimumab with other immunotherapy agents have shown some favorable therapeutic benefits. For example, combination of ipilimumab with peginterferon α-2b (pegylated interferon α-2b) in patients with unresectable melanoma both demonstrated significant increase of response rate and OS comparing with the monotherapy arm [96, 97] in recent phase I trials.

### **3.2. Combined therapy inhibiting both immuno-checkpoint and MAPK signaling pathway**

Checkpoint blockade immunotherapy and MAPK targeted chemotherapy have distinct clinical profiles. For example, targeted therapy has relative higher initial response rate (~60% for BRAFi) with rapid onset of effect, but its efficacy restrictively rely on the continuous treatment and the therapeutic response is usually not durable due to the quick development of acquired drug resistance. In contrast, immunotherapy has much a lower response rate (4.5% for ipilimumab), delayed onset of effect and difficulty in predicting patient outcome, but it has shown potentially durable responses and long-term survival benefit even off treatment. In addition, since the MAPK pathway is not required in the process of anti-tumor immune response, blocking MAPK signaling should not interfere with the efficacy of checkpoint blockade immunotherapy. Therefore, it seems very rational that the combination of a MAPKi and an immunotherapy agent such as ipilimumab or pembrolizumab can maximize the therapeutic benefits in advance melanoma.

Interestingly, BRAF and MEK inhibition displayed an "endogenous vaccine-like" effects in melanoma cells [98]. Cytotoxic agents like BRAFi induce tumor cell death and promote the uptake and presentation of tumor antigens to the effector immune cells (T cells and B cells) through antigen-presenting cells [54]. MEK inhibition, BRAFV600E RNA silencing or BRAF inhibition by PLX4720 increases the CD4+ and CD8+ lymphocytes mediated T-cell infiltration and reduce the level of immune-suppressants including IL-6, IL-10 or VEGF [99-101] in mice. The expression of PD-L1 is found to be elevated in BRAFi-resistant melanoma cells and it is mediated through the off-target activity of BRAFi in JUN and STAT3 signaling [102]. However, Vella *et al* has published a paper in 2014 and stated that they have not found any impact of dabrafenib treatment on T lymphocytes. trametinib alone or in combination with dabrafenib has suppressed T lymphocyte proliferation, cytokine secretion and antigen-specific expansion in their isolated T lymphocyte and monocyte-derived dendritic cells. These findings should be carefully tested *in vivo* to evaluate the clinical relevance [103].

As for the clinical practice, dose-limiting hepatotoxicity issues have led to the premature termination of the first phase I study on combination of ipilimumab with vemurafenib (ClinicalTrials.gov, NCT01400451). This signified the complexity of adverse effect in combined therapy of immune-regulating agents and kinase inhibitors. Another phase I study of ipili‐ mumab plus dabrafenib, or ipilimumab plus the combination of dabrafenib with trametinib is still active and a phase II study is exploring the safety and efficacy of sequential administration of vemurafenib followed by ipilimumab (ClinicalTrials.gov, NCT01767454, NCT01673854). The data of these most recent trials will be released in the near future.

## **4. Conclusions**

immune response in tumor progression, their combination has been shown to be synergistic

In a cohort phase I trial that studied the concurrent administration of ipilimumab and nivolumab to 53 patients with advanced, treatment-resistant melanoma, more than 80% tumor reduction was observed in 30% patients after 12 weeks treatment at the maximum tolerated dose. Twenty-one out of fifty-three patients had objective responses and over 80% of these patients had tumor regression. Grade 3/4 adverse events are diagnosed in 53% patients but the toxicities are manageable with immune-suppressants [95]. Consequential trials with more enrollment number of patients are necessary to further evaluate the safety and efficacy of this promising double immune-checkpoints blockage therapy comparing with each of its mono‐

Finally, combinatorial clinical trials using ipilimumab with other immunotherapy agents have shown some favorable therapeutic benefits. For example, combination of ipilimumab with peginterferon α-2b (pegylated interferon α-2b) in patients with unresectable melanoma both demonstrated significant increase of response rate and OS comparing with the monotherapy

**3.2. Combined therapy inhibiting both immuno-checkpoint and MAPK signaling pathway**

Checkpoint blockade immunotherapy and MAPK targeted chemotherapy have distinct clinical profiles. For example, targeted therapy has relative higher initial response rate (~60% for BRAFi) with rapid onset of effect, but its efficacy restrictively rely on the continuous treatment and the therapeutic response is usually not durable due to the quick development of acquired drug resistance. In contrast, immunotherapy has much a lower response rate (4.5% for ipilimumab), delayed onset of effect and difficulty in predicting patient outcome, but it has shown potentially durable responses and long-term survival benefit even off treatment. In addition, since the MAPK pathway is not required in the process of anti-tumor immune response, blocking MAPK signaling should not interfere with the efficacy of checkpoint blockade immunotherapy. Therefore, it seems very rational that the combination of a MAPKi and an immunotherapy agent such as ipilimumab or pembrolizumab can maximize the

Interestingly, BRAF and MEK inhibition displayed an "endogenous vaccine-like" effects in melanoma cells [98]. Cytotoxic agents like BRAFi induce tumor cell death and promote the uptake and presentation of tumor antigens to the effector immune cells (T cells and B cells) through antigen-presenting cells [54]. MEK inhibition, BRAFV600E RNA silencing or BRAF inhibition by PLX4720 increases the CD4+ and CD8+ lymphocytes mediated T-cell infiltration and reduce the level of immune-suppressants including IL-6, IL-10 or VEGF [99-101] in mice. The expression of PD-L1 is found to be elevated in BRAFi-resistant melanoma cells and it is mediated through the off-target activity of BRAFi in JUN and STAT3 signaling [102]. However, Vella *et al* has published a paper in 2014 and stated that they have not found any impact of dabrafenib treatment on T lymphocytes. trametinib alone or in combination with dabrafenib has suppressed T lymphocyte proliferation, cytokine secretion and antigen-specific expansion

in B16 melanoma tumors without overt toxicity [94].

208 Melanoma – Current Clinical Management and Future Therapeutics

therapy regiments.

arm [96, 97] in recent phase I trials.

therapeutic benefits in advance melanoma.

Extensive efforts and remarkable progresses have been made to discover and investigate rational approaches in combination melanoma therapy since the recent approval of MAPKi and immune checkpoints blockade antibodies. A number of new targeted or immune drugs for metastatic melanoma are currently under commercial development or late stage clinical trials, some of which will likely be approved in the next few years. Quality of life for many melanoma patients has been dramatically increased. However, significant challenges still remain. While some clinical evidence has really raised the expectation of survivals for patients with advanced melanoma, the benefits of combination therapy are usually accompanied by limitations. Comprehensive genetic profile and tailored patient matching is essential for targeted therapy, while biomarkers are critical to predict the patient immunotherapy response. Drug-related toxicity for combination treatment usually is not a simple one-plus-one situation, and potential drug-drug interactions, especially the combination of a targeted agent with an immunotherapeutic agent must be carefully evaluated in order to achieve both fast and durable responses. Adverse effects should be closely monitored and potential alternative dosing regiments is worth further exploration. Optimized dose schedule may help to delay the resistance development and reduce the frequency of adverse effect. For example, inter‐ mittent doses of BRAFi was able to enhance the tolerance in combination with immunotherapy, decrease the paradoxical MAPK activation, which might be the main cause of severe toxicity in clinical trial [104]. Solid evidence of synergistic combination in preclinical research must be established before clinical trial conduction. In fact, with the relatively large number of available targeted agents and immunotherapeutic agents for metastatic melanoma, the huge number of possible drug combinations coupled with dosing sequences or schedules already presents a significant challenge in designing proper clinical trials. To test all the possible drug combina‐ tions along with different dosing sequences clinically will not only have low benefits to patients, but is also a huge financial burden to the society. Carefully designed, predictive preclinical studies will be essential to provide critical supports for rational prioritization of clinical trials using drug combinations. Finally, clear understandings of various combination mechanisms and patient genetic profiles are critically important for the development of new combination approaches, prediction of expected therapy response and potential side effects. With the rapid advances in this field, it is likely that optimal combination treatments will great improve the management of advanced melanoma in cancer patients.

## **Abbreviations**

AMPK: 5' adenosine monophosphate-activated protein kinase BRAF: B-Raf protein BRAFi: BRAF inhibitor CDK: Cyclin dependent kinase CR: Complete response CTLA-4: Cytotoxic T lymphocyte-associated antigen 4 ERK: Extracellular signal-regulated kinase HR: Hazard ratio JNK: c-Jun N-terminal kinase MAPK: Mitogen-activated protein kinase MEKi: MEK inhibitor MHC: Membrane histocompatibility complex mTOR: Mammalian target of rapamycin ORR: Overall response rate OS: Overall survival PD-1: Programmed cell death 1 PD-L1: Programmed cell death 1 ligand 1 PDGFR: Platete-derived growth factor receptor PFS: Progression-free survival PI3K: Phosphoinositide 3-kinase PKC: Protein kinase C PR: Partial response RTKs: Receptor tyrosine kinases TCR: T cell receptor VEGF: Vascular endothelial growth factor

VEGFR: Vascular endothelial growth factor receptor

## **Acknowledgements**

With the rapid advances in this field, it is likely that optimal combination treatments will great

improve the management of advanced melanoma in cancer patients.

210 Melanoma – Current Clinical Management and Future Therapeutics

AMPK: 5' adenosine monophosphate-activated protein kinase

CTLA-4: Cytotoxic T lymphocyte-associated antigen 4

ERK: Extracellular signal-regulated kinase

MAPK: Mitogen-activated protein kinase

mTOR: Mammalian target of rapamycin

PD-L1: Programmed cell death 1 ligand 1

PDGFR: Platete-derived growth factor receptor

MHC: Membrane histocompatibility complex

**Abbreviations**

BRAF: B-Raf protein

BRAFi: BRAF inhibitor

CR: Complete response

HR: Hazard ratio

MEKi: MEK inhibitor

OS: Overall survival

CDK: Cyclin dependent kinase

JNK: c-Jun N-terminal kinase

ORR: Overall response rate

PD-1: Programmed cell death 1

PFS: Progression-free survival

PKC: Protein kinase C PR: Partial response

TCR: T cell receptor

PI3K: Phosphoinositide 3-kinase

RTKs: Receptor tyrosine kinases

VEGF: Vascular endothelial growth factor

This work was supported by NIH grants R01CA148706. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Jin Wang acknowledges the support of the Alma and Hal Reagan Fellowship.

## **Author details**

Jin Wang, Duane D. Miller\* and Wei Li

\*Address all correspondence to: dmiller@uthsc.edu or wli@uthsc.edu

Department of Pharmaceutical Sciences, College of Pharmacy. The University of Tennessee Health Science Center, Memphis, TN, USA

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## **New Insights in Cutaneous Melanoma Immune-Therapy — Tackling Immune-Suppression and Specific Anti-Tumoral Response**

Monica Neagu and Carolina Constantin

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59494

## **1. Introduction**

In this chapter the maturity of immune-therapy in cancer, with emphasis on melanoma will be discussed.

The heterogeneity of melanoma tumour regarding primary and metastatic variants will be argued. Therefore the mutational heterogeneity of this type of tumor triggers complex immune-therapy approach. Notions such as *Immune-therapy* will be tackled, meaning targeting immune elements like immune suppression and using immune drugs like monoclonal antibodies against targets that can or not be immune elements. The chapter will end with the importance of designing complex immune-therapies like abolishing the immune-suppression and enhancing the specific anti-tumoral effect.

Physiologically, the immune system can recognize cells that display an aberrant proliferation like neoplasia. The immune system is equipped with cells that can destroy cancer cells them during their early development. Years ago, when the theory of immunoediting was initiated [1], immunosurveillance was defined as a complex pathway that supervises and controls the elimination of transformed self cells/tissue. When the immune system cannot properly control these aberrantly proliferating cells, and the equilibrium is deregulated, tumour cells escape and form a clinically significant tumoral tissue [2].

In cancer immune-therapy, several approaches that aim to start and sustain the immune response and eventually elicit an immunological memory were lately tackled. The therapy armentarium that started the "bumpy road" from bench to bedside comprises cancer vaccines, adoptive T cell therapy, anti-tumor antibodies, immune checkpoint blockade and/or various immune combinations. Some of the conclusions of the last Congress of SITC (*Society for*

*Immunotherapy of Cancer*) is that combining these above mentioned immune approaches with other immunomodulators (e.g. cytokines, cyclic dinucleotides) and/or indoleamine 2,3 dioxygenase (IDO) inhibitors can increase the efficacy of immunotherapy [3] and hopefully replace in the future routine approaches such as chemotherapy/radiotherapy.

Early diagnosed stages of melanoma are resolved mainly by surgery and large margin excision, but for advanced stages, systemic therapies, whether chemotherapy, immune-therapy or combined ones have had very low efficacy. Advanced melanoma remains a continuous clinical provocation for the physicians who use therapeutical approaches with low response rates, unmanageable toxicities, and reduced efficacy.

One of the main *molecular hurdles* in cutaneous melanoma is the heterogeneity of tumors. A tumoral tissue has cells with different characteristics in terms of proliferation, invasive‐ ness and pheno/genotype. In melanoma, aggressiveness has a distinct cellular genotype and in the tumor dynamic development, cells go through several phenotype switching [4]. Specific genetic expression studies have identified more than 100 genes over-expressed in cells with a higher proliferative capacity or in cells that were committed to invade tissues [5].

Recently, a study was published on single melanoma cells and the report shows that there are 114 genes expression that could distinguish the proliferative and invasive phenotype of cells. Among these genes, regulatory networks were found along with genes that encode for pluripotency factor (e.g. POUF51); all these genes were found associated with cell's tumori‐ genic potential. Authors report that among the regulatory network genes, MITF (microphthal‐ mia-associated transcription factor) is one of the key players in the heterogeneous character of tumour cells populations. Moreover, the heterogeneity of cells depends on the 2D or 3D status of the cell cultures, thus TPBG (trophoblast glycoprotein) is expressed in a melanoma cell line, 501Mel, only in 3D cultures [4]. In the same way, in 1205Lu melanospheres, PI3K/AKT (phosphatidylinositol 3' –kinase/protein kinase B) signaling pathway is enhanced [4], while DAPK1 (death-associated protein kinase 1) expression is decreased in 501Mel experimental tumors, finding that is in line with the hypermethylated gene promoter associated to melano‐ ma [6]. This study emphasizes that when transformed melanocytes start to organize in growing tumors, the heterogeneity of the cellular populations' increases, the tumoral tissue having MITF-low/negative cells [4, 7]. In experimental tumour spheres, cells that grow on the exterior layers have an active proliferation, while in the interior of the tumour, due to hypoxic conditions, cells are arrested in the G1 phase [8]. The overall mechanism is that 3D growth enhances tumour-initiating properties [9].

Cell's heterogeneity is important from the immunological point of view. Tumour heteroge‐ neous tissues have different expression of tumor antigens, thus any type of therapy that addresses only one tumour epitope is proned to have low efficacy. This is the rationale to investigate tumor antigens and evaluate the patient's immune responses prior to any immune-therapy [10].

Extensive studies performed by large groups of researchers and extended networks like *Melanoma Research Networks* established in Europe, Canada, and New Zeeland have provided seminal scientific information regarding melanoma's immune biology. All these insights have led to an actual scientific leap by the development of the first immune-therapies that proved efficacy in advanced melanoma treatment. Therefore drugs that aim intracellular pathways such as mitogen-activated protein kinase (MAPK) pathway, or antibodies that aim CTLA4 (cytotoxic T lymphocyte-associated protein 4), or PD1/PD1-L (programmed cell death 1/ programmed cell death 1 ligand) have recently followed the bench to bedside path [11]. Building upon the early success of these therapies, trials involving new classes of drugs and combinations of these drugs are underway [12].

Starting from 2011, metastatic melanoma beneficiated from four new approved drugs, all these drugs proving in clinical trials the improvement of patient's survival. From this four drugs, one is a B-Raf enzyme inhibitor (vemurafenib), one is an inhibitor of the associated enzyme B-Raf (dabrafenib), and one is a MEK inhibitor (trametinib); the only one that is an actual immune-therapy, is an anti-CTLA-4 (ipilimumab) antibody [13]. Therefore searching new efficacious immune-therapies in this disease is still an open subject of intense research.

This chapter summarizes the main achievements gathered in the last 3 years regarding immune-therapy as the ultimate approach for melanoma treatment.

## **2. Reducing specific immune-suppression**

*Immunotherapy of Cancer*) is that combining these above mentioned immune approaches with other immunomodulators (e.g. cytokines, cyclic dinucleotides) and/or indoleamine 2,3 dioxygenase (IDO) inhibitors can increase the efficacy of immunotherapy [3] and hopefully

Early diagnosed stages of melanoma are resolved mainly by surgery and large margin excision, but for advanced stages, systemic therapies, whether chemotherapy, immune-therapy or combined ones have had very low efficacy. Advanced melanoma remains a continuous clinical provocation for the physicians who use therapeutical approaches with low response rates, un-

One of the main *molecular hurdles* in cutaneous melanoma is the heterogeneity of tumors. A tumoral tissue has cells with different characteristics in terms of proliferation, invasive‐ ness and pheno/genotype. In melanoma, aggressiveness has a distinct cellular genotype and in the tumor dynamic development, cells go through several phenotype switching [4]. Specific genetic expression studies have identified more than 100 genes over-expressed in cells with a higher proliferative capacity or in cells that were committed to invade tissues [5]. Recently, a study was published on single melanoma cells and the report shows that there are 114 genes expression that could distinguish the proliferative and invasive phenotype of cells. Among these genes, regulatory networks were found along with genes that encode for pluripotency factor (e.g. POUF51); all these genes were found associated with cell's tumori‐ genic potential. Authors report that among the regulatory network genes, MITF (microphthal‐ mia-associated transcription factor) is one of the key players in the heterogeneous character of tumour cells populations. Moreover, the heterogeneity of cells depends on the 2D or 3D status of the cell cultures, thus TPBG (trophoblast glycoprotein) is expressed in a melanoma cell line, 501Mel, only in 3D cultures [4]. In the same way, in 1205Lu melanospheres, PI3K/AKT (phosphatidylinositol 3' –kinase/protein kinase B) signaling pathway is enhanced [4], while DAPK1 (death-associated protein kinase 1) expression is decreased in 501Mel experimental tumors, finding that is in line with the hypermethylated gene promoter associated to melano‐ ma [6]. This study emphasizes that when transformed melanocytes start to organize in growing tumors, the heterogeneity of the cellular populations' increases, the tumoral tissue having MITF-low/negative cells [4, 7]. In experimental tumour spheres, cells that grow on the exterior layers have an active proliferation, while in the interior of the tumour, due to hypoxic conditions, cells are arrested in the G1 phase [8]. The overall mechanism is that 3D growth

Cell's heterogeneity is important from the immunological point of view. Tumour heteroge‐ neous tissues have different expression of tumor antigens, thus any type of therapy that addresses only one tumour epitope is proned to have low efficacy. This is the rationale to investigate tumor antigens and evaluate the patient's immune responses prior to any

Extensive studies performed by large groups of researchers and extended networks like *Melanoma Research Networks* established in Europe, Canada, and New Zeeland have provided seminal scientific information regarding melanoma's immune biology. All these insights have

replace in the future routine approaches such as chemotherapy/radiotherapy.

manageable toxicities, and reduced efficacy.

226 Melanoma – Current Clinical Management and Future Therapeutics

enhances tumour-initiating properties [9].

immune-therapy [10].

Until recently, the approved therapy armentarium in advanced melanoma was comprising only dacarbazine (DTIC), hydroxyurea, while the only approved immune agent was high-dose interleukin-2 (IL-2) [14]. These drugs cannot provide satisfactory overall survival (OS) rates in advanced stages. After searching various combination of immune-therapies, from vaccination [15] to drugs that inhibit immune-signaling pathways [16], in 2011 FDA approved, ipilimumab and vemurafenib, agents that significantly increased OS and long-term improvement in advanced melanoma [17].

In this case, monoclonal antibodies (mAbs) as immune-therapy agents have the intrinsic role to establish an antineoplastic action through stimulation of a specific immune response. This action can be performed by inducing *de novo* primary response and/or by eliciting an already existing antitumor action, but repressed in these patients.

The most advanced, in terms of positive research results, were the immune-related drugs that abrogated the immune –suppression such as CTLA4 or PD-1. Other mAbs were designed to aim toward stimulation of co-stimulatory receptors, molecules that are expressed by antigen presenting cells (APC); the aimed molecules were CD40 or OX40 (member 4 tumor necrosis factor receptor superfamily) or GITR (TNFRSF18), expressed on activated T lymphocytes.

Tremelimumab, also an anti CTLA4 mAb, is being evaluated in solid tumors. Nivolumab, an anti PD-1 mAb is also in the evaluation phase. This year (2014) clinical trials assessing OX40 and GITR-activating mAbs were initiated as well [18].

### **2.1. Anti-CTLA-4 antibodies**

Activated T lymphocytes express transiently CTLA-4 transmembrane protein, while on T regulatory lymphocytes (Tregs) this protein is expressed constitutively. Two possible mecha‐ nisms are known accounting for CTLA-4 immune-suppressive effect. One of the mechanisms is the competitive binding to B7-1 and B7-2 in the detriment of the normal binding to CD28, delivering thus an immune-suppressive signal [19]. Actually CTLA-4 competes with the binding of CD28 to B7, thus hindering a normal activation. The other possible mechanism is that cells expressing CTLA-4, endocytose the appropriate ligands of other cells, as such, CD28 cannot trigger activation [20].

Overall in cancer research, drugs that aim B7 family can enhance the therapeutic panel and thorough studies are further needed for elucidating these regulatory pathways [21].

The particular action of monoclonal anti-CTLA-4 antibody is to link to CTLA-4, blocking the inhibitory immune-suppressive signal, T cell can perform thus its activation pathways, proliferate while infiltrating the tumors, and in the end, set off tumour apoptosis. Designed to bind to CTLA-4, ipilimumab was approved by FDA in 2011 for advanced melanoma [22] and in the same year the European Commission issued a marketing authorization for ipilimumab [23].This type of immune-drug aims to downregulate the inhibitory activity of T-lymphocytes leading to the normal activation of T cells by allowing the binding of CD28 to B7, this costi‐ mulatory process participates to the main coupling of MHC (major histocompatibility complex) that presents the tumour antigen to TCR (T cell receptor). Re-establishing these physiological molecular interactions, T-cells can mount an efficient antitumor immune response (Figure 1) [24]. CTLA-4 expressed on T cells is omnipresent and is not dependent upon the tumour's particularities, thus the drug's action should not be dependent on tumor's characteristics.

EMA recommendation after analyzing the post-phase III results extended clinical trials [25,17] is a dose of intravenously 3 mg/kg ipilimumab over a 90 min period, this procedure needs to be repeated every 3 weeks; overall four doses should be administered [23]. If the patients are treated in combination with DTIC it is recommended that a dose of 10 mg/kg should be used [25,17].

The actual clinical results of using this immune-therapy showed that patients with advanced melanoma increased their median OS to 10 months when compared to the 6 months OS in gp100 melanoma vaccine. When comparing OS in patients treated with ipilimumab in combination with DTIC, OS was 11 months, while DTIC monotherapy just 9 months.

As stated above ipilimumab's action is not dependent on specific and individual tumor cell mutations, hence it can be efficient in different patients and stages [26, 27]. The beneficial clinical results regarding OS were registered as independent of various parameters such as age, gender, stage and/or previous therapy regimens.

Investigating the patients survival curves it has been shown that there are groups surviving more than 4 years [28], and that their clinical response was durable during the follow-up [29]. Durability is an important differentiating criterion when recommending first-line therapy with immunomodulators in comparison to the duration obtained in kinase inhibitors treatment.

**2.1. Anti-CTLA-4 antibodies**

228 Melanoma – Current Clinical Management and Future Therapeutics

cannot trigger activation [20].

characteristics.

[25,17].

Activated T lymphocytes express transiently CTLA-4 transmembrane protein, while on T regulatory lymphocytes (Tregs) this protein is expressed constitutively. Two possible mecha‐ nisms are known accounting for CTLA-4 immune-suppressive effect. One of the mechanisms is the competitive binding to B7-1 and B7-2 in the detriment of the normal binding to CD28, delivering thus an immune-suppressive signal [19]. Actually CTLA-4 competes with the binding of CD28 to B7, thus hindering a normal activation. The other possible mechanism is that cells expressing CTLA-4, endocytose the appropriate ligands of other cells, as such, CD28

Overall in cancer research, drugs that aim B7 family can enhance the therapeutic panel and

The particular action of monoclonal anti-CTLA-4 antibody is to link to CTLA-4, blocking the inhibitory immune-suppressive signal, T cell can perform thus its activation pathways, proliferate while infiltrating the tumors, and in the end, set off tumour apoptosis. Designed to bind to CTLA-4, ipilimumab was approved by FDA in 2011 for advanced melanoma [22] and in the same year the European Commission issued a marketing authorization for ipilimumab [23].This type of immune-drug aims to downregulate the inhibitory activity of T-lymphocytes leading to the normal activation of T cells by allowing the binding of CD28 to B7, this costi‐ mulatory process participates to the main coupling of MHC (major histocompatibility complex) that presents the tumour antigen to TCR (T cell receptor). Re-establishing these physiological molecular interactions, T-cells can mount an efficient antitumor immune response (Figure 1) [24]. CTLA-4 expressed on T cells is omnipresent and is not dependent upon the tumour's particularities, thus the drug's action should not be dependent on tumor's

EMA recommendation after analyzing the post-phase III results extended clinical trials [25,17] is a dose of intravenously 3 mg/kg ipilimumab over a 90 min period, this procedure needs to be repeated every 3 weeks; overall four doses should be administered [23]. If the patients are treated in combination with DTIC it is recommended that a dose of 10 mg/kg should be used

The actual clinical results of using this immune-therapy showed that patients with advanced melanoma increased their median OS to 10 months when compared to the 6 months OS in gp100 melanoma vaccine. When comparing OS in patients treated with ipilimumab in

As stated above ipilimumab's action is not dependent on specific and individual tumor cell mutations, hence it can be efficient in different patients and stages [26, 27]. The beneficial clinical results regarding OS were registered as independent of various parameters such as

Investigating the patients survival curves it has been shown that there are groups surviving more than 4 years [28], and that their clinical response was durable during the follow-up [29]. Durability is an important differentiating criterion when recommending first-line therapy with immunomodulators in comparison to the duration obtained in kinase inhibitors treatment.

combination with DTIC, OS was 11 months, while DTIC monotherapy just 9 months.

age, gender, stage and/or previous therapy regimens.

thorough studies are further needed for elucidating these regulatory pathways [21].

**Figure 1.** Main immune-suppression molecular mechanisms that can be overridden by therapeutical monoclonal anti‐ bodies targeting CTLA-4 and PD1.

As we are focusing on an immune therapy and as *immune memory* is a characteristic of this process, it was a logic approach to see what the effects are when ipilimumab gets another round of therapy. The National Comprehensive Cancer Network (NCCN) recommendation show that ipilimumab re-treatment can be done when the first round induced intolerable toxicity and/or for patients relapsing after the first therapy or proving at least 3 months stable disease [30]. Adverse reaction during re-treatment are similar to those for the first approach (see below) and no actual predisposition was noted regarding first encountered toxicity with the retreatment one. Authors report that retreatment with ipilimumab is a feasible therapy and, currently a phase II trial (http://trialsunited.com/studies/NCT01709162) focuses on the immune response parameters in ipilimumab re-treatment [31].

Non-cutaneous melanomas were also therapeutic targets for ipilimumab therapy. Thus, in an Australian study published in 2014 over 100 patients were followed after ipilimumab therapy. Median OS for mucosal and uveal melanoma patients was half of that registered in cutaneous melanoma patients. This report underlines the severities of adverse effects, even death-related to therapy cases, thus administration and follow-up by an experienced clinical team is extremely necessary in this type of clinical trials [32].

Stage IV patients presenting brain metastasis were another therapy target group as bloodbrain barrier is permeable to activated T lymphocytes, cells capable of inducing a local immune response [33]. In a large study comprising over 800 patients with brain metasta‐ sis treated with ipilimumab, up to 25% survived at least 1 year, as the un-treated median OS is only 5 months [34].

Adverse reactions in ipilimumab therapy are associated with hyper-immune reactions, but these can be solved by the physician using additional classic therapies. Over 10% of patients experienced gastrointestinal deregulations (e.g. diarrhea, nausea, vomiting, decreased appetite and abdominal pain), rash, pruritus, fatigue. Side-effects are manageable by the physician and, seldom, the severity can lead to treatment discontinuation (www.ema.euro‐ pa.eu). These immune-related adverse effects (irAEs) are mild to moderate toxicity and are experienced by around 60% of the patients, while around 15% developed grade 3 or 4 toxicity [25]. Endocrine system – related adverse effects were also reported in this therapy; in patients group receiving ipilimumab, 8% have experienced hypophysitis and 6% hypothyroidism/ thyroiditis. Combined therapy, ipilimumab and nivolumab, induce in 22% of the patients thyroiditis or hypothyroidism and in 9% hypophysitis. Authors report hormone replacement as adjuvant therapy and immediate initiation of this therapy reverses symptoms [35]. If the ipilimumab therapy is combined with vemurafenib, important hepatotoxicity was reported, thus caution should be taken when combining immune-therapy with this B-Raf enzyme inhibitor [36].

#### **2.2. Anti-PD-1 or PD-1L antibodies**

#### *2.2.1. Nivolumab*

In July 2014, the first human monoclonal antibody against programmed death receptor-1 was announced as approved in Japan-*Nivolumab* [37]. As shown above in Figure 1, it targets a negative regulatory molecule that sustains immunosuppression. After accomplishing phase I and II clinical trials, around 25% of stage III and IV patients had a good clinical response when 2 mg/kg intravenous nivolumab was administered every 3 weeks. The clinical outcome was very optimistic in this study, patients did not progress in their disease for a median of 172 days, and at the time of publication (July 2014) median OS was still not achieved.

Nivolumab displayed a good tolerability profile; grade 3 or 4 adverse effects were reported in les than 18 % of patients, mainly an increased γ-glutamyl transferase [38].

Another group studying T lymphocyte interaction with tumour cell, interaction mediated by PD-1 receptor linking to PD-L 1, has shown that in phase I/II studies this antibody can lead to tumor regression and can enhance OS in various cancers including skin melanoma. Studying antibodies that target PD-1 or PD-1L (e.g. nivolumab, MK-3475, pidilizumab, MPDL3280A, BMS-936559, MEDI4736, MSB0010718C) the authors show that the positive clinical response goes to a maximum of 50% response rate when antibodies against PD-1 combined with anti-CTLA-4 were used. The clinical responses start early upon treatment and continue after the treatment is finished [39].

Another study published in 2014 searched to evaluate the survival of patients upon discon‐ tinuation of the therapy. After enrolling over 100 patients with advanced melanoma, the authors concluded that the median OS was 16.8 months [40].

Melanoma patients along with other cancer diagnosed patients were treated with anti-PD-1 (nivolumab). This early-phase clinical trial published in 2014 aimed to elucidate the link between PD-1, PD-L1, and PD-L2 expression, immune cell infiltration and the clinical efficacy of this therapy. The degree of PD-L1 expression depicted on tumor cells was associated with its receptor PD-1 expressed by lymphocytes. The other ligand of PD-1, PD-L2 corroborated with PD-L1 expression. The expression of PD-L1 on the tumors was correlated with the clinical efficacy of the anti-PD-1 therapy, and the found best correlation when compared to other studied factors, such as PD-1 expression and/or TIL (tumor infiltrating lymphocytes). The study concludes that, when achieving maximum efficacy with novilumab, tumor PD-L1 expression is the base of anti-PD-1 therapeutical blockade [41].

In 2013, results from phase I clinical trials, of nivolumab and MK-3475 (anti-PD-1 and anti-PD-L1 antibodies) were released. For MK-3475 an objective response of 38 % with only 13 % of the patients reporting grade 3/4 toxicities was shown [42]. These results were probably the ground for further approval (see below).

Phase III clinical trails are on-going and, it seems that PD-1-PD-L1 triggers a sequence of intracellular signaling that brings important clinical benefits [43].

Adverse effects in this type of therapy are of low grade, the physician can impose a good patient management [39] and long-term safety is acceptable [40].

### *2.2.2. Pembrolizumab*

sis treated with ipilimumab, up to 25% survived at least 1 year, as the un-treated median

Adverse reactions in ipilimumab therapy are associated with hyper-immune reactions, but these can be solved by the physician using additional classic therapies. Over 10% of patients experienced gastrointestinal deregulations (e.g. diarrhea, nausea, vomiting, decreased appetite and abdominal pain), rash, pruritus, fatigue. Side-effects are manageable by the physician and, seldom, the severity can lead to treatment discontinuation (www.ema.euro‐ pa.eu). These immune-related adverse effects (irAEs) are mild to moderate toxicity and are experienced by around 60% of the patients, while around 15% developed grade 3 or 4 toxicity [25]. Endocrine system – related adverse effects were also reported in this therapy; in patients group receiving ipilimumab, 8% have experienced hypophysitis and 6% hypothyroidism/ thyroiditis. Combined therapy, ipilimumab and nivolumab, induce in 22% of the patients thyroiditis or hypothyroidism and in 9% hypophysitis. Authors report hormone replacement as adjuvant therapy and immediate initiation of this therapy reverses symptoms [35]. If the ipilimumab therapy is combined with vemurafenib, important hepatotoxicity was reported, thus caution should be taken when combining immune-therapy with this B-Raf enzyme

In July 2014, the first human monoclonal antibody against programmed death receptor-1 was announced as approved in Japan-*Nivolumab* [37]. As shown above in Figure 1, it targets a negative regulatory molecule that sustains immunosuppression. After accomplishing phase I and II clinical trials, around 25% of stage III and IV patients had a good clinical response when 2 mg/kg intravenous nivolumab was administered every 3 weeks. The clinical outcome was very optimistic in this study, patients did not progress in their disease for a median of 172

Nivolumab displayed a good tolerability profile; grade 3 or 4 adverse effects were reported in

Another group studying T lymphocyte interaction with tumour cell, interaction mediated by PD-1 receptor linking to PD-L 1, has shown that in phase I/II studies this antibody can lead to tumor regression and can enhance OS in various cancers including skin melanoma. Studying antibodies that target PD-1 or PD-1L (e.g. nivolumab, MK-3475, pidilizumab, MPDL3280A, BMS-936559, MEDI4736, MSB0010718C) the authors show that the positive clinical response goes to a maximum of 50% response rate when antibodies against PD-1 combined with anti-CTLA-4 were used. The clinical responses start early upon treatment and continue after the

Another study published in 2014 searched to evaluate the survival of patients upon discon‐ tinuation of the therapy. After enrolling over 100 patients with advanced melanoma, the

days, and at the time of publication (July 2014) median OS was still not achieved.

les than 18 % of patients, mainly an increased γ-glutamyl transferase [38].

authors concluded that the median OS was 16.8 months [40].

OS is only 5 months [34].

230 Melanoma – Current Clinical Management and Future Therapeutics

inhibitor [36].

*2.2.1. Nivolumab*

treatment is finished [39].

**2.2. Anti-PD-1 or PD-1L antibodies**

In September 2014, FDA granted accelerated approval to pembrolizumab (formerly known as MK-3475), an antibody targeting PD-1, to be used following ipilimumab therapy. A recently published report showed the efficacy and safety results of this antibody at two doses (2 mg/kg and 10 mg/kg) given every 21 days. The enrolled patients that received the therapy were refractory to ipilimumab therapy. Similar safety profiles were reported whether patients were treated with 2 mg/kg or 10 mg/kg and authors show that no drug-related deaths were registered [44]. Another published study had similar results with the difference that the response rate between patients with or without prior ipilimumab treatment were not statisti‐ cally different. Positive clinical outcome was registered with the overall median progressionfree survival exceeding 7 months. Patients with advanced melanoma, prior refractory to ipilimumab, proved in this study a high rate of tumor regression [45].

Drug-related adverse effects were fatigue in one third of the patients and around 20% of them experienced pruritus and rash. Grade 3 fatigue was the single drug-related grade 3 to 4 adverse effect in 3% of the patients [44].

The positive results in a difficult to manage patients, like the ones refractory to ipilimumab, probably accelerated this drug authorization with two months ahead of its planned approval and the clinical study is still on going [46].

After ipilimumab approval, finding another antibody that could be used as immune-therapy for blocking an immune checkpoint like PD-1 and PD-1L gained an intense research frenzy in the last years.Therapeutical approaches that use immunomodulatory drugs have completely different mode of action in comparison to the well-known chemotherapeutical procedures. From this point of view investigating the intimate mechanisms that underlie their effect is of outmost importance because it can reveal new signaling molecules, future to be drug targets. Moreover biomarkers that can clinically predict the patient response could optimize the approach and personalize the immune-therapy [47].

#### **2.3. Biomarkers for clinical benefit prediction**

In the last couple of years there is a less abundant literature focusing on predictive and/or prognostic biomarkers in the immune-therapy of cutaneous melanoma. Biomarkers that were published lately range from classic serum LDH, to membrane molecules and circulating cells without any *clear-cut biomarker* that could predict the immune-therapy efficacy.

In the last year researchers were focusing on biomarkers that can predict immune-therapy with ipilimumab outcome. Thus some studies show that ipilimumab therapy was correlated with an increase in peripheral blood absolute lymphocyte count when patients had a good clinical outcome in terms of OS. More specifically OS was 11.9 months for patients that had more than 1,000 lymphocyte count / μL peripheral blood in comparison to OS of 1.4 months in patients with lower counts [48, 49]. These results were confirmed this year when increased absolute lymphocyte count was associated with increased progression free survival (PFS) but not with OS. Any other parameters including classic serum LDH did not relate to OS or PFS [32].

Another cell biomarker, forkhead box P3 (FoxP3) expressed by T-regs, was correlated with positive clinical outcome in advanced melanoma patients [50].

Correlationswereinvestigatedregardingcirculatorymyeloid-derivedsuppressorcells(MDSC) in treated patients. Authors report that circulatory MDSC with Lin(-) CD14(+) HLA-DR(-) phenotype are increasedinpatients comparedtonormal.After surgically removing the tumour and subjecting patients'to ipilimumab treatment,this immune parameter did not change.Then again, an interesting finding was that patients could be stratified in the ipilimumab-respond‐ ers and non-responders based on the lower and respectively higher concentration of circulato‐ ryMDSC,thuspinpointingthese cellsaspossiblepredictivemarkersofresponse toipilimumab. This candidate immune-marker did not correlate with baseline serum LDH, but showed higher values inseveremetastasis comparedto localizedmetastasis to skinand/orto lymphnodes [51].

As to the possible efficacy biomarkers for nivolumab therapy, in a phase I clinical trial, stage III or IV patients were followed after this therapy by several biomarkers evaluation. This recent study reports that high circulatory T lymphocytes with NY-ESO-1 and MART-1-specific CD8(+) phenotype are associated with disease progression. After therapy, increased circula‐ tory Tregs and decreased antigen-specific T cells are the two immune biomarkers that were found associated with disease progression. The expression of PD-L1 on the tumor did not correlate with the clinical response [52].

#### **2.4. Animal models studying immune-therapy mechanisms**

There are few studies focusing on animal models that bring new data regarding the intimate cellular mechanisms in immune-therapy. Having in mind the fact that these recent immunetherapies are limited to certain groups of patients, the published animal models searched for the resistance mechanisms that could hinder this therapy.

different mode of action in comparison to the well-known chemotherapeutical procedures. From this point of view investigating the intimate mechanisms that underlie their effect is of outmost importance because it can reveal new signaling molecules, future to be drug targets. Moreover biomarkers that can clinically predict the patient response could optimize the

In the last couple of years there is a less abundant literature focusing on predictive and/or prognostic biomarkers in the immune-therapy of cutaneous melanoma. Biomarkers that were published lately range from classic serum LDH, to membrane molecules and circulating cells

In the last year researchers were focusing on biomarkers that can predict immune-therapy with ipilimumab outcome. Thus some studies show that ipilimumab therapy was correlated with an increase in peripheral blood absolute lymphocyte count when patients had a good clinical outcome in terms of OS. More specifically OS was 11.9 months for patients that had more than 1,000 lymphocyte count / μL peripheral blood in comparison to OS of 1.4 months in patients with lower counts [48, 49]. These results were confirmed this year when increased absolute lymphocyte count was associated with increased progression free survival (PFS) but not with OS. Any other parameters including classic serum LDH did not relate to OS or PFS [32].

Another cell biomarker, forkhead box P3 (FoxP3) expressed by T-regs, was correlated with

Correlationswereinvestigatedregardingcirculatorymyeloid-derivedsuppressorcells(MDSC) in treated patients. Authors report that circulatory MDSC with Lin(-) CD14(+) HLA-DR(-) phenotype are increasedinpatients comparedtonormal.After surgically removing the tumour and subjecting patients'to ipilimumab treatment,this immune parameter did not change.Then again, an interesting finding was that patients could be stratified in the ipilimumab-respond‐ ers and non-responders based on the lower and respectively higher concentration of circulato‐ ryMDSC,thuspinpointingthese cellsaspossiblepredictivemarkersofresponse toipilimumab. This candidate immune-marker did not correlate with baseline serum LDH, but showed higher values inseveremetastasis comparedto localizedmetastasis to skinand/orto lymphnodes [51]. As to the possible efficacy biomarkers for nivolumab therapy, in a phase I clinical trial, stage III or IV patients were followed after this therapy by several biomarkers evaluation. This recent study reports that high circulatory T lymphocytes with NY-ESO-1 and MART-1-specific CD8(+) phenotype are associated with disease progression. After therapy, increased circula‐ tory Tregs and decreased antigen-specific T cells are the two immune biomarkers that were found associated with disease progression. The expression of PD-L1 on the tumor did not

There are few studies focusing on animal models that bring new data regarding the intimate cellular mechanisms in immune-therapy. Having in mind the fact that these recent immune-

without any *clear-cut biomarker* that could predict the immune-therapy efficacy.

positive clinical outcome in advanced melanoma patients [50].

correlate with the clinical response [52].

**2.4. Animal models studying immune-therapy mechanisms**

approach and personalize the immune-therapy [47].

**2.3. Biomarkers for clinical benefit prediction**

232 Melanoma – Current Clinical Management and Future Therapeutics

In 2013, the role of IDO upon an experimental anti CTLA-4 blockade was shown. Authors used IDO knockout mice and showed that, upon treatment with anti-CTLA-4 antibody, B16 melanoma was growing more slowly and that, the animals' overall survival increased compared to normal mice expressing IDO. The mechanisms were similar when the animal model was treated with anti PD-1/ anti-PD-L1 and GITR. The authors show in this animal model that CTLA-4 and IDO inhibitors converge and that the inhibitory role of IDO can be the background mechanisms accounting for the resistance to anti-CTLA-4 therapy. Moreover, the process is T lymphocyte dependent and, if this resistance is overridden, effector T cells are found increased in tumour infiltration, the effector-to-regulatory T cell ratio increases as well [53]. The molecular mechanisms of IDO expression are intimately related to the immune response. Several years ago it was reported that IDO expression is controlled by T activated lymphocytes through their secreted cytokines. IL-13 can repress the induction of IDO mediated by IFN-gamma [54]. Regarding possible emerging therapies, authors report that fludarabine that hinders the up-regulation of IDO in a T lymphocytes dependent manner, can be tested as a pre-treatment drug for melanoma patients. These patients can receive afterwards immuno‐ therapies that would have been less efficient when IDO was over-expressed [55].

Using animal models, new emerging therapies can be discovered, overriding the resistance to immune-therapies in certain patients groups.

## **3. Dendritic cells pulsed with specific antigens as inductors of specific immune-response**

Treatment paradigms aim to include naturally occurring dendritic cells subsets in a single vaccine. The studies that are in the pre-clinical phases show synergistic effects between various antigen-presenting cells. We will present different types of methodologies to pulse dendritic cells, starting with mere cultivation of dendritic cells in total tumour lysates and ending with newer technologies such as electroporation mRNA-pulsed dendritic cells. Recently, the first clinical trials released their results and showed increased survival rates and broader anticancer immune responses. These new clinical findings will be presented.

In just a short period of time, the cancer immunotherapy field has gained new combatants through sipuleucel-T FDA approval, first DC immunovaccine for metastatic prostate cancer patients, followed closely by ipilimumab, an antibody specific to CTLA-4 as major target in metastatic melanoma [56]. Due to its clear tumor immunogenicity, melanoma treatment could be handled from an immunotherapeutic viewpoint. However, an important issue in melanoma immunovaccine success is the highly heterogeneous composition of antigens expressed within tumor site along with different genetic patterns of melanoma patients [57]. Deciphering this complex (intra)tumor heterogeneity of melanoma is directly linked to the possibility for clearly identifying, targeting and manageing drug-resistant cell subpopulations from the tumour site [58]. The genetic profile of melanoma patient is one of the foremost rationales for an autologous whole cell vaccine acting more efficiently in treating the micrometastasis than would an allogeneic designed one.

Revisiting the melanoma vaccines, it was pointed out that a preponderant cytokine-driven therapy activates a robust antitumoral T-cell mediated response representing a class of individualized auto-vaccination formula, surmounting thus the melanoma intratumoral heterogeneity [57]. Assembling a cancer vaccine aims to activate a specific anti-tumor immune response and/or to better access the tumor-associated antigens. In cutaneous melanoma, the pattern of tumor specific and tumor associated antigens is both large and heterogeneous, making melanoma immunogenicity exploitable in therapeutic approaches. Therefore, the plenty of discovered or yet hidden melanoma tumor antigens could open the way for vacci‐ nation of patients groups with the same vaccine type [59]. Being explored with synthetic peptides, whole tumor cells, cellular lysates or autologous immunovaccine, dendritic cells (DCs) take advantages in treatment or even in prevention approaches of cancer [60].

Dendritic cell in melanoma immunotherapy undergo a sequential number of actions. Thus, loading DCs with a tumor antigen and a specific adjuvant will induce the maturation state which involves antigens processing by proteasomal degradation and presenting the resulted peptides to T cells *via* MHC complex to stimulate further CD4+ cells, CD8+ cells as well as phagocytes and NK cells or, in certain activation conditions, induce Tregs that hamper antitumor responses [61].

#### **3.1. Dendritic cells subsets**

Inmalignantskin,theprincipalsubsetsofDCresponsibleforAg-specificTcellimmuneresponse comprise *epidermal* (Langerhans cells) and *dermal* cell populations. The primary tumor site and the sentinel lymph nodes endure the immune-suppression generated by melanoma, this immune site being the field where T cells should start the fight against melanoma while being armedbyactivatedDCtoengenderanti-melanomaimmunity[62].Asincaseoftumorassociated macrophages,underthe influenceoftumormilieuDCareversatileplayerswhichcouldbecome *tumor-associated DC* enhancing Immune-suppression by sustaining T cell regulatory activity [63](Table 1).Uponelectrochemotherapyoftumor cells,for example, a relativelynewapproach to deliver better an antitumor drug, melanoma inflammatory infiltrate contains beside dermal DC, *plasmacytoid* DC cells for capturing tumor antigens to further elicit, along with dermal and Langerhans cells, a T cell antitumor response [64].


**Table 1.** DCs subsets and receptors for immunovaccine (LC – Langerhans cell; pDC - plasmacytoid-derived DC; MoDC – monocyte-derived DC; DEC205 – C-type lectin receptor on DC; BDCA-2 – blood dendritic cell antigen, specific to pDC subset; DCIR - dendritic cell immunoreceptor; DC-SIGN - dendritic cell specific ICAM-3 grabbing non-integrin; MR – mannose receptor; TLR – toll-like receptor )

#### **3.2. Exploring dendritic cells in melanoma immunovaccine**

whole cell vaccine acting more efficiently in treating the micrometastasis than would an

Revisiting the melanoma vaccines, it was pointed out that a preponderant cytokine-driven therapy activates a robust antitumoral T-cell mediated response representing a class of individualized auto-vaccination formula, surmounting thus the melanoma intratumoral heterogeneity [57]. Assembling a cancer vaccine aims to activate a specific anti-tumor immune response and/or to better access the tumor-associated antigens. In cutaneous melanoma, the pattern of tumor specific and tumor associated antigens is both large and heterogeneous, making melanoma immunogenicity exploitable in therapeutic approaches. Therefore, the plenty of discovered or yet hidden melanoma tumor antigens could open the way for vacci‐ nation of patients groups with the same vaccine type [59]. Being explored with synthetic peptides, whole tumor cells, cellular lysates or autologous immunovaccine, dendritic cells

(DCs) take advantages in treatment or even in prevention approaches of cancer [60].

Dendritic cell in melanoma immunotherapy undergo a sequential number of actions. Thus, loading DCs with a tumor antigen and a specific adjuvant will induce the maturation state which involves antigens processing by proteasomal degradation and presenting the resulted peptides to T cells *via* MHC complex to stimulate further CD4+ cells, CD8+ cells as well as phagocytes and NK cells or, in certain activation conditions, induce Tregs that hamper

Inmalignantskin,theprincipalsubsetsofDCresponsibleforAg-specificTcellimmuneresponse comprise *epidermal* (Langerhans cells) and *dermal* cell populations. The primary tumor site and the sentinel lymph nodes endure the immune-suppression generated by melanoma, this immune site being the field where T cells should start the fight against melanoma while being armedbyactivatedDCtoengenderanti-melanomaimmunity[62].Asincaseoftumorassociated macrophages,underthe influenceoftumormilieuDCareversatileplayerswhichcouldbecome *tumor-associated DC* enhancing Immune-suppression by sustaining T cell regulatory activity [63](Table 1).Uponelectrochemotherapyoftumor cells,for example, a relativelynewapproach to deliver better an antitumor drug, melanoma inflammatory infiltrate contains beside dermal DC, *plasmacytoid* DC cells for capturing tumor antigens to further elicit, along with dermal and

**Antigen uptake receptors Unique receptors TLR receptors**

pDC FcR; DEC205; CD40; DCIR CD303; CD123; BDCA-2 TLR-7; TLR-9 specific to pDC

**Table 1.** DCs subsets and receptors for immunovaccine (LC – Langerhans cell; pDC - plasmacytoid-derived DC; MoDC – monocyte-derived DC; DEC205 – C-type lectin receptor on DC; BDCA-2 – blood dendritic cell antigen, specific to pDC subset; DCIR - dendritic cell immunoreceptor; DC-SIGN - dendritic cell specific ICAM-3 grabbing non-integrin;

allogeneic designed one.

234 Melanoma – Current Clinical Management and Future Therapeutics

antitumor responses [61].

**3.1. Dendritic cells subsets**

Langerhans cells, a T cell antitumor response [64].

MR – mannose receptor; TLR – toll-like receptor )

**DCs subsets Uptake receptors evaluated for immunotherapy of DC**

LC/dermal DC FcR; DEC205; CD40; DCIR Langerin TLR-3

MoDC FcR; DEC205; CD11c; CD40; DCIRDC-SIGN; MR TLR-4; 8; 3; 7.

As skin is an abundant cellular immune network and hence an accessible portal for thera‐ peutical approaches, DC cells remain an attractive target in melanoma therapy both as *exvivo*-generated or *in-vivo*-DC-targeting immunovaccine blueprint [65]. Peripheral blood and Langerhans cells are the main sources for DCs immunotherapy. Langerhans cells and mono‐ cyte-derived DCs elicit immune responses in comparable levels although the cytokine stimulation conditions are different. The initial therapeutical attempts with DCs vaccination was based on *ex-vivo* generated monocyte-derived DC pulsed with tumor lysates, peptide or tumor antigens which led to a tumor regression rate of 3-7%, having also a lower toxicity compared with standard therapeutical procedures [66]. In melanoma, DC derived from CD34+ progenitors prove better results compared with monocyte-DC vaccine in spite of the known heterogeneity of tumor antigens [10].

#### *3.2.1. Loading DC with tumor associated-antigens*

Loading effector immune cells with antigenic peptides or a whole tumor-associated antigen (TAA) was initially designed as an immune vaccination system with T lymphocytes *via* MHC molecules recognition. Due to possible issues related to molecule stability and/or delivery route, resulting in an ineffective antigen presentation, these approaches could fail in clinic due to a low response rate in patients. Using DC as tool for intracellular delivery of such tumor antigenic peptides, the process of antigen presentation to T cells could be improved [67]. Moreover, the Th1/Th2 balance could be regulated by such modified DC. Therefore, DCs loaded with MART-126-35 melanoma peptide were used in combination with anti-CTLA4 monoclonal antibody (tremelimumab) in advanced melanoma patients. Upon therapy, high levels of pro-inflammatory Th1 invariant natural killer T cells (iNKT) CD8(+) was associated with positive clinical responses, indicating that antitumor T cell activity could be immuno‐ modulated *via* iNKT cells by peptide-pulsed DCs [68]. In another recent study, high-risk stage III melanoma patients with lymph node resection were vaccinated with DC loaded with MHC I melanoma peptides respective to the patient's haplotype. The peptide pulsed DCs were well tolerated and elicited immune specific responses to melanoma antigens or/and IFN-γproducing CD8+ cell response to melanoma peptides in 15 of 22 patients. The three-year overall survival rate was 68.2% vs. 25.7% in the control patients group [69].

Monocyte-derived DCs could be loaded in different conditions with a mixture of peptides, tumor lysates or even with a single tumor peptide such as from Mage-3A1. For 8 of 11 patients enrolled in the study it was registered an increase of Mage-3A1-specific (CD8+) T cells, with regression of a few metastases for 6 advanced melanoma patients; a lack of Mage 3A1 expression was observed in some non-regressed areas of melanoma [70]. Even immature DCs could be exploited in vaccination, thus DCs generated from CD34+ progenitor cells were cytokines-stimulated and pulsed *in vitro* with a pool of melanoma derived peptides [71].

Engineered DCs loaded with peptides or antigens could be delivered by lymphatic nodes or intradermally; the last type being the optimum method for generating T cell antitumor immunity [72].

#### *3.2.2. DC electroporation with mRNA*

Cellular electroporation is a transfection method to efficiently introduce mRNA encoding for a certain biomolecule in order to express at high level that specific antigen. A main advantage of mRNA transfection is the prolongation of the exposure and an accurate antigens processing. This approach was translated for DCs as a promising opportunity to facilitate access to tumor antigens and thus priming the T cell specific antitumor melanoma response, being applicable even in advanced stages of melanoma [73].

In the last few years, mRNA was proposed as an innovative vehicle for antigen delivery appropriate for cancer vaccination purposes. DCs were evaluated as the most suitable immune cells for mRNA transfection due to their professional quality in processing and presenting antigens for inducing specific immune responses by T cells. mRNA as an antigen delivery tool could generate a whole antigenic protein with all epitopes ready to be viewed by MHC molecules; last but not least, the interest mRNA molecule could be produced in large quantities with high purity.

The successful use of *ex vivo* mRNA-modified DCs for melanoma immunotherapy rely on DCs cellular subtype involved, the proper cellular activation and path of delivery [74]. The DCs *subtype* electroporated with mRNA in cancer vaccination purposes accounts for vaccination efficiency. pDCs cells loaded with melanoma antigenic peptides and further activated at CD40L elicit T cytotoxic anti-melanoma responses [75]. The cellular *activation* method of DC counts for CTL full activity and subsequently inhibition of Tregs. Engagement of TLR in DC activation proves to be a good approach in immunovaccine. Moreover, transfection with mRNA and activation of DC cells is the fundament of the TriMix mRNA set encoding for CD40L, CD70 and active form of TLR4. Thus in one clinical study with TriMix-DCs intrader‐ mally administrated, the efficacy of procedure was registered by means of the skin infiltrating CD8+ lymphocytes monitored by IL-12p70 as a marker of successful inoculation of the DC product [76]. The *delivery way* of modified DCs was compared in several clinical studies. The best way to target the lymph nodes in melanoma site by modified mRNA DCs is intradermic inoculation associated with an increased number of T cells although intranodally delivery reveal a higher number of DC. One recent study refers to DC electroporated with mRNA for gp100/tyrosinase antigens and injected in regional lymph nodes of melanoma patients prior to local surgery. The mRNA for electroporated melanoma antigen was immunohistochemi‐ cally detected in T cells populations from both the primary and the adjacent lymph node concomitantly with a CD8+ T lymphocyte responses registered in 7 of 11 patients subjected to immunovaccination [77]. Some promising results in terms of safety, feasibility and immuno‐ genic properties were obtained in patients with advanced cutaneous melanoma in a pilot study were DC were co-electroporated with mRNA encoding for CD40L, TLR4 and CD70 as a autologous TriMix-DC formula combined with IFN-α-2b sequential administration [76].

The foremost parameter to be monitored for immunovaccine efficacy is the induction or the enhancement of melanoma anti-tumor immune response. Several technologies like ELISPOT technique allows quantifying the precise number of active immune cells by counting antigenspecific cells that secrete a particular anti-tumor cytokine e.g. IFN-γ [59]. Since 2006, clinical studies with tumor mRNA transfected DCs administrated usually intradermic in patients with metastatic melanoma, detect an immunological T cell response with promising results at least for disease stabilization. In addition, advanced melanoma patients were treated intradermic with DCs simultaneously co-electroporated for 4 tumor antigens – Mage A3, Mage C2, Tyrosinase and gp100/DC-LAMP followed by TriMix mRNA single or in combination with additional therapy like IFNa-2b. In both clinical set-ups, the CD4/CD8 T cell responses were enhanced [74].

*3.2.2. DC electroporation with mRNA*

236 Melanoma – Current Clinical Management and Future Therapeutics

even in advanced stages of melanoma [73].

with high purity.

Cellular electroporation is a transfection method to efficiently introduce mRNA encoding for a certain biomolecule in order to express at high level that specific antigen. A main advantage of mRNA transfection is the prolongation of the exposure and an accurate antigens processing. This approach was translated for DCs as a promising opportunity to facilitate access to tumor antigens and thus priming the T cell specific antitumor melanoma response, being applicable

In the last few years, mRNA was proposed as an innovative vehicle for antigen delivery appropriate for cancer vaccination purposes. DCs were evaluated as the most suitable immune cells for mRNA transfection due to their professional quality in processing and presenting antigens for inducing specific immune responses by T cells. mRNA as an antigen delivery tool could generate a whole antigenic protein with all epitopes ready to be viewed by MHC molecules; last but not least, the interest mRNA molecule could be produced in large quantities

The successful use of *ex vivo* mRNA-modified DCs for melanoma immunotherapy rely on DCs cellular subtype involved, the proper cellular activation and path of delivery [74]. The DCs *subtype* electroporated with mRNA in cancer vaccination purposes accounts for vaccination efficiency. pDCs cells loaded with melanoma antigenic peptides and further activated at CD40L elicit T cytotoxic anti-melanoma responses [75]. The cellular *activation* method of DC counts for CTL full activity and subsequently inhibition of Tregs. Engagement of TLR in DC activation proves to be a good approach in immunovaccine. Moreover, transfection with mRNA and activation of DC cells is the fundament of the TriMix mRNA set encoding for CD40L, CD70 and active form of TLR4. Thus in one clinical study with TriMix-DCs intrader‐ mally administrated, the efficacy of procedure was registered by means of the skin infiltrating CD8+ lymphocytes monitored by IL-12p70 as a marker of successful inoculation of the DC product [76]. The *delivery way* of modified DCs was compared in several clinical studies. The best way to target the lymph nodes in melanoma site by modified mRNA DCs is intradermic inoculation associated with an increased number of T cells although intranodally delivery reveal a higher number of DC. One recent study refers to DC electroporated with mRNA for gp100/tyrosinase antigens and injected in regional lymph nodes of melanoma patients prior to local surgery. The mRNA for electroporated melanoma antigen was immunohistochemi‐ cally detected in T cells populations from both the primary and the adjacent lymph node concomitantly with a CD8+ T lymphocyte responses registered in 7 of 11 patients subjected to immunovaccination [77]. Some promising results in terms of safety, feasibility and immuno‐ genic properties were obtained in patients with advanced cutaneous melanoma in a pilot study were DC were co-electroporated with mRNA encoding for CD40L, TLR4 and CD70 as a autologous TriMix-DC formula combined with IFN-α-2b sequential administration [76].

The foremost parameter to be monitored for immunovaccine efficacy is the induction or the enhancement of melanoma anti-tumor immune response. Several technologies like ELISPOT technique allows quantifying the precise number of active immune cells by counting antigenspecific cells that secrete a particular anti-tumor cytokine e.g. IFN-γ [59]. Since 2006, clinical studies with tumor mRNA transfected DCs administrated usually intradermic in patients with

Recently, a small study on seven melanoma patients involved DCs from peripheral blood mononuclear cells pulsed with gp100 peptides and maturated further *in vitro* with a cocktail of CD40L and INF-γ. It was noted a direct correlation between the clinical responses and levels of IL-12 produced by modified DCs. The highest levels of IL-12 were registered in one patient with complete remission. DCs from non-responding patients were unable to produce IL-12 without additional stimulation with TLR agonist, observation that induced a modification of the *in vitro* maturation protocol and thus contributed to the improvement of the clinical outcome [78].

## **4. Translating up-dated knowledge to clinics — Therapeutical future scenery**

There is a recent trend that advises as first therapy a BRAF inhibitor in *advanced* stages, because, time-wise, these patients cannot build an effective immune response while in patients with less advanced stages, immune-therapy will lead to a better outcome whether the patient presents or not a BRAF mutation [79].

In the clinical management of melanoma it is certain that immune-therapy is one of the pillars. Among this new therapeutical approaches, MEK inhibitors can overcome the resistance induced by BRAF inhibitors [79, 80]. has shown in phase 3 clinical studies, an improved [81].

Combination of these antibodies with the new anti-CTLA4 drug induces an astonishing 80% or more tumor regression in patients (http://www.clinicaltrials.gov/show/ NCT01783938; http://trialsunited.com/studies/NCT01024231/) [82]. Newer therapeutical combination of ipilimumab with oncolytic immunotherapy with GM-CSF-expressing engineered herpes simplex virus, have shown in phase 3 clinical studies an improved OS in advanced melanoma [83].

On-going studies seek to establish the clinical boundaries of NK adoptive transfer in melano‐ ma. Last year a study was published searching to rationalize the clinical trial framework [84] as the prior published results of a pilot trial showed no tumor regression in spite of the increased concentration of circulating autologous NK cells persistence [85].

Immunotherapy is the only therapeutical "light" that can increase OS in advanced melanomas. Eliciting an efficient immune response takes a certain amount of time in order to have an efficient anti-tumoral response, but as *immunological memory* is installed, the effect of immunetherapy persists in the absence of further immune-treatment.

We strongly believe, taking into account our experience in melanoma patients follow-up [86, 87] that immune-therapy is a major therapy weapon, increasing survival in advanced mela‐ nomas. The only draw-back is that, in order to develop its full efficacy, immune-therapy takes time, and this time interval can be up to 2–4 months. Thus, an early diagnostic in the metastatic stage would make a huge difference for a positive clinical outcome [88].

Besides imaging-based follow-up, immune parameters should complete the panel of investi‐ gation. New data regarding circulatory MDSC can enhance this panel and prospective clinical trials should soon validate them. Resistance to immune-therapy, like IDO expression opens new therapeutical avenues aiming at immune checkpoints and combining specific antibodies with IDO inhibitors.

## **5. Conclusion**

The immune system is still not fully explored in this type of skin cancer [89, 90], thus new insights in tumour microenvironment and the involvement of innate immunity cells could enhance the panel of new therapeutical targets.

Balancing antitumor efficacy and reconstitution of a proper functioning immune system are processes aimed by immune-therapy in cutaneous melanoma. Owing to cutaneous melanoma immunogenic outline, this disease treatment could be addressed from an immunotherapeutic viewpoint. As we are facing the great success of having the first immune-therapy approved drug in melanoma, there is an open research combat of targeting personalized/individual antigens or undifferentiating antigens-stem-like to tackle the aggressive character of this disease.

## **Acknowledgements**

The study was financed by the following Research Projects: PN-II-PT-PCCA-2013-4-1407, PN-II-ID-PCE-2011-3-0918 and PN.09.33-01.01/2009.

## **Author details**

Monica Neagu\* and Carolina Constantin

\*Address all correspondence to: neagu.monica@gmail.com

Immunobiology Laboratory, Victor Babes National Institute of Pathology, Bucharest, Romania

## **References**

We strongly believe, taking into account our experience in melanoma patients follow-up [86, 87] that immune-therapy is a major therapy weapon, increasing survival in advanced mela‐ nomas. The only draw-back is that, in order to develop its full efficacy, immune-therapy takes time, and this time interval can be up to 2–4 months. Thus, an early diagnostic in the metastatic

Besides imaging-based follow-up, immune parameters should complete the panel of investi‐ gation. New data regarding circulatory MDSC can enhance this panel and prospective clinical trials should soon validate them. Resistance to immune-therapy, like IDO expression opens new therapeutical avenues aiming at immune checkpoints and combining specific antibodies

The immune system is still not fully explored in this type of skin cancer [89, 90], thus new insights in tumour microenvironment and the involvement of innate immunity cells could

Balancing antitumor efficacy and reconstitution of a proper functioning immune system are processes aimed by immune-therapy in cutaneous melanoma. Owing to cutaneous melanoma immunogenic outline, this disease treatment could be addressed from an immunotherapeutic viewpoint. As we are facing the great success of having the first immune-therapy approved drug in melanoma, there is an open research combat of targeting personalized/individual antigens or undifferentiating antigens-stem-like to tackle the aggressive character of this

The study was financed by the following Research Projects: PN-II-PT-PCCA-2013-4-1407, PN-

Immunobiology Laboratory, Victor Babes National Institute of Pathology, Bucharest, Romania

stage would make a huge difference for a positive clinical outcome [88].

with IDO inhibitors.

**5. Conclusion**

disease.

**Acknowledgements**

**Author details**

Monica Neagu\*

enhance the panel of new therapeutical targets.

238 Melanoma – Current Clinical Management and Future Therapeutics

II-ID-PCE-2011-3-0918 and PN.09.33-01.01/2009.

and Carolina Constantin

\*Address all correspondence to: neagu.monica@gmail.com


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## **Can Redirected T Cells Outsmart Aggressive Melanoma? The Promise and Challenge of Adoptive Cell Therapy**

Jennifer Makalowski and Hinrich Abken

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59255

## **1. Introduction**

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#### **1.1. The challenge to induce lasting remission in late stage melanoma**

In early stages of the disease surgical resection of melanoma lesions is a curative option; a 10 year-survival rate of 75-85% can be achieved in stage I or II of the disease. However, stage III or IV melanoma is associated with low survival rates of less than 1 year upon diagnosis [1]. The poor prognosis in advanced stages of the disease is thought to be particularly due to the properties of melanoma cells to systemically spread into various organs, to form micrometastases beyond the detection limit of current imaging procedures [2, 3] and to give rise to relapse of the disease. This is even the case after initially complete response to therapy and after more than a decade from initial treatment. Durable remission is so far only achieved in pre-defined patient subsets despite the development of novel drugs and major improvements in therapeutic regimens [4-6]. This unsatisfactory situation is thought to be due to the extra‐ ordinary property of melanoma cells to persist in "dormancy" for long periods of time which is associated with their resistance to chemo-and radiotherapy [7-10]. Taken together, durable cure from melanoma requires eliminating single melanoma cells in a highly specific and efficient fashion even in dormant micro-metastatic lesions.

In this situation recruiting the cellular immune defense machinery to detect and destroy individual melanoma cells is a powerful alternative to conventional therapeutic regimens. The hope is sustained by the supportive effect of high dose interleukin-2 (IL-2) [11] and anticytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) antibody [12] as well as interferon (IFN) α-2b to prolong the disease-free survival even in late stages of the disease. However, the response rate is quite low and frequently not curative over time [13, 14].

© 2015 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.

A number of strategies for sharpening the immune cell response against melanoma are currently explored, some of these with remarkable success. In particular, the adoptive transfer of tumor infiltrating lymphocytes (TILs), isolated from melanoma biopsies and amplified to therapeutic relevant numbers ex vivo, produced encouraging phase II results [15, 16]. In a further development, patients' blood T cells are genetically engineered with pre-defined specificity for melanoma-associated antigens making adoptive cell therapy with melanoma specific T cells possible. In this contribution we will discuss the rationale for adoptive cell therapy of melanoma, evidence for efficacy and current challenges to achieve long-term remission. Upcoming strategies in melanoma stem cell targeting are also discussed.

## **2. Adoptive therapy with ex vivo amplified TILs can induce regression of melanoma**

An effective immune response can control melanoma. This notion is supported by the observation that spontaneous and complete melanoma regressions can occur and that immune compromised patients suffer from a higher frequency of melanoma [17, 18]. The conclusion is moreover sustained by the clinical observation that treatment with high dose IL-2 produces an objective response even in late stage melanoma, some patients with long-term complete response for years [11, 19]. Although about 16%, the response rate is remarkable compared to the low and short-lived response rates of classical therapeutic regimens.

First described in 1969 [20], melanoma is infiltrated by T cells of both effector and helper cell origin which can be expanded to high numbers ex vivo in the presence of IL-2. Pioneered by the NCI-Surgery Branch, such tumor infiltrating T cells (TILs) were selected for melanoma reactivity by incubation on feeder cells expressing melanoma-associated antigens [21] and readministered in substantial numbers together with high dose IL-2 to the patient (Figure 1). Initial trials produced an objective response rate of 11/20 patients [22] which is remarkable since TILs are obviously capable to fight melanoma even in late stage patients who experienced multiple lines of therapy. Responses, however, were of short duration and TILs did not persist for longer period in the peripheral blood after administration. Subsequent trials identified that the key to successful TIL therapy was the number of TILs administered to the patient, the activity of those cells against melanoma and the rapidity of T cell amplification ex vivo [23, 24]. During the subsequent years the initial protocols were optimized with respect to these and other issues and adopted according to GMP standards [25]; a number of trials are currently open in various centers (Table 1).

Persistence of administered TILs in circulation was substantially improved by depletion of the lymphoid compartment of the patient prior to adoptive cell therapy [26-28]. Such preconditioning by non-myeloablative chemotherapy had the effect that cytokines sustaining lymphocyte amplification including IL-7 and IL-15 were present in augmented levels ("cyto‐ kine sink"). Moreover, space for transferred lymphocytes was created and suppressor cells including regulatory T cells were depleted which additionally helped to improve engraftment of adoptively transferred T cells.


A number of strategies for sharpening the immune cell response against melanoma are currently explored, some of these with remarkable success. In particular, the adoptive transfer of tumor infiltrating lymphocytes (TILs), isolated from melanoma biopsies and amplified to therapeutic relevant numbers ex vivo, produced encouraging phase II results [15, 16]. In a further development, patients' blood T cells are genetically engineered with pre-defined specificity for melanoma-associated antigens making adoptive cell therapy with melanoma specific T cells possible. In this contribution we will discuss the rationale for adoptive cell therapy of melanoma, evidence for efficacy and current challenges to achieve long-term

248 Melanoma – Current Clinical Management and Future Therapeutics

remission. Upcoming strategies in melanoma stem cell targeting are also discussed.

the low and short-lived response rates of classical therapeutic regimens.

**melanoma**

open in various centers (Table 1).

of adoptively transferred T cells.

**2. Adoptive therapy with ex vivo amplified TILs can induce regression of**

An effective immune response can control melanoma. This notion is supported by the observation that spontaneous and complete melanoma regressions can occur and that immune compromised patients suffer from a higher frequency of melanoma [17, 18]. The conclusion is moreover sustained by the clinical observation that treatment with high dose IL-2 produces an objective response even in late stage melanoma, some patients with long-term complete response for years [11, 19]. Although about 16%, the response rate is remarkable compared to

First described in 1969 [20], melanoma is infiltrated by T cells of both effector and helper cell origin which can be expanded to high numbers ex vivo in the presence of IL-2. Pioneered by the NCI-Surgery Branch, such tumor infiltrating T cells (TILs) were selected for melanoma reactivity by incubation on feeder cells expressing melanoma-associated antigens [21] and readministered in substantial numbers together with high dose IL-2 to the patient (Figure 1). Initial trials produced an objective response rate of 11/20 patients [22] which is remarkable since TILs are obviously capable to fight melanoma even in late stage patients who experienced multiple lines of therapy. Responses, however, were of short duration and TILs did not persist for longer period in the peripheral blood after administration. Subsequent trials identified that the key to successful TIL therapy was the number of TILs administered to the patient, the activity of those cells against melanoma and the rapidity of T cell amplification ex vivo [23, 24]. During the subsequent years the initial protocols were optimized with respect to these and other issues and adopted according to GMP standards [25]; a number of trials are currently

Persistence of administered TILs in circulation was substantially improved by depletion of the lymphoid compartment of the patient prior to adoptive cell therapy [26-28]. Such preconditioning by non-myeloablative chemotherapy had the effect that cytokines sustaining lymphocyte amplification including IL-7 and IL-15 were present in augmented levels ("cyto‐ kine sink"). Moreover, space for transferred lymphocytes was created and suppressor cells including regulatory T cells were depleted which additionally helped to improve engraftment Aurora Health Care**; CHNHSFT**, Christie Hospital NHS Foundation Trust; **FHCRC**, Fred Hutchinson Cancer Research Center; **HMC**, Hadassah Medical Center; **HUH**, Herlev University Hospital (Copenhagen); **KUH,** Karolinska Universi‐ ty Hospital; **MDACC**, M.D. Anderson Cancer Center; **MOFFITT**, H. Lee Moffitt Cancer Center and Research Institute; **MUH**, Mie University Hospital; **NIH**, National Institutes of Health; **NUH**, Nantes University Hospital; **SMC**, Sheba Medical Center; **StLMC**, St. Luke's Medical Center; **UC**, University of California; **UHN,** University Health Network (Toronto)

**Table 1.** Adoptive cell therapy with tumor infiltrating lymphocytes (TILs) in patients with melanoma

There are still some issues to be addressed, for instance whether clinically most potent TILs can be defined by phenotype and whether these cells can be selectively expanded. There is a common sense that for therapeutic efficacy in the long-term the functional activity of T cells needs to be preserved without signs of exhaustion which is particularly crucial when T cells experienced extensive amplification ex vivo. In the further development of the procedure, TILs were only selected with respect to their proliferative capacities which is independent of their antigen specificity and represents a furthermore simplification of the standard protocol (Figure 1) [29-31]. Those so-called "young TILs" after short-term ex vivo expansions passed through fewer cell division cycles prior to infusion and are thereby in a maturation stage less prone to terminal differentiation and senescence [32]. Those protocols do not further select TILs for their melanoma reactivity based on the observa‐ tion that infusion of ex vivo activated, IFN-γ<sup>+</sup> TILs produced no superior therapeutic efficacy compared to non-responding TILs [16]. These modifications in the protocol resulted in improved persistence of young TILs [33] and about 50% response rates [27, 29, 34], so far in non-randomized trials (Table 1). A series of recent clinical trials with TILs following different lympho-conditioning regimes resulted in objective responses in 56% and com‐ plete responses in 22% of patients at the Surgery Branch [35]. Current TIL trials at various centers reproduced objective response rates of 40-50% in melanoma patients, a significant portion of patients free of disease 3-5 years after treatment [36, 37]. Of note, TILs can have anti-tumor activity also towards brain metastases as shown in a NCI trial with 7/17 complete and 6/17 partial remissions [38] sustaining the hope that adoptive cell therapy may be effective towards metastases which are otherwise not accessible.

While most trials apply non-separated TILs, administration of isolated CD8+ T cell clones with specificity for Melan-A and gp100 mediated only moderate benefit, required IL-2 and did not persist for longer times [39]. Those CD8+ T cells which persisted long-term acquired a pheno‐ type of central memory-type T cells in vivo [40]. It is therefore assumed that CD8+ TILs require help of CD4+ cells for prolonged persistence making application of non-separated T cell populations more suitable.

Not only the stage of maturation but also the recruitment of T cells through chemokine gradients is crucial for therapeutic success. A recent prospective-retrospective hypothesisdriven analysis revealed that coordinate over-expression of CXCL9, CXCL10, CXCL11, CCL5 in melanoma is associated with responsiveness to treatment after TIL therapy [41].

Melanoma-reactive T cells need to persist in circulation to ensure therapeutic success of TIL therapy [42, 43]. This is reflected by the median survival of patients treated with Melan-A specific TILs of 53.5 months compared to 3.5 months for patients who received TILs of unknown specificity [44]. Some trials are initiated using melanoma specific patient's T cells from the peripheral blood for adoptive cell therapy of melanoma (Table 2). MART-1 or gp100 specific T cell clones isolated and amplified ex vivo produced a 50% response rate [45], however, technical difficulties limit a broad application of such specific T cells since melanoma reactive T cells in the peripheral blood of melanoma patients are extremely rare.

There are still some issues to be addressed, for instance whether clinically most potent TILs can be defined by phenotype and whether these cells can be selectively expanded. There is a common sense that for therapeutic efficacy in the long-term the functional activity of T cells needs to be preserved without signs of exhaustion which is particularly crucial when T cells experienced extensive amplification ex vivo. In the further development of the procedure, TILs were only selected with respect to their proliferative capacities which is independent of their antigen specificity and represents a furthermore simplification of the standard protocol (Figure 1) [29-31]. Those so-called "young TILs" after short-term ex vivo expansions passed through fewer cell division cycles prior to infusion and are thereby in a maturation stage less prone to terminal differentiation and senescence [32]. Those protocols do not further select TILs for their melanoma reactivity based on the observa‐

compared to non-responding TILs [16]. These modifications in the protocol resulted in improved persistence of young TILs [33] and about 50% response rates [27, 29, 34], so far in non-randomized trials (Table 1). A series of recent clinical trials with TILs following different lympho-conditioning regimes resulted in objective responses in 56% and com‐ plete responses in 22% of patients at the Surgery Branch [35]. Current TIL trials at various centers reproduced objective response rates of 40-50% in melanoma patients, a significant portion of patients free of disease 3-5 years after treatment [36, 37]. Of note, TILs can have anti-tumor activity also towards brain metastases as shown in a NCI trial with 7/17 complete and 6/17 partial remissions [38] sustaining the hope that adoptive cell therapy may be

specificity for Melan-A and gp100 mediated only moderate benefit, required IL-2 and did not

Not only the stage of maturation but also the recruitment of T cells through chemokine gradients is crucial for therapeutic success. A recent prospective-retrospective hypothesisdriven analysis revealed that coordinate over-expression of CXCL9, CXCL10, CXCL11, CCL5

Melanoma-reactive T cells need to persist in circulation to ensure therapeutic success of TIL therapy [42, 43]. This is reflected by the median survival of patients treated with Melan-A specific TILs of 53.5 months compared to 3.5 months for patients who received TILs of unknown specificity [44]. Some trials are initiated using melanoma specific patient's T cells from the peripheral blood for adoptive cell therapy of melanoma (Table 2). MART-1 or gp100 specific T cell clones isolated and amplified ex vivo produced a 50% response rate [45], however, technical difficulties limit a broad application of such specific T cells since melanoma

cells for prolonged persistence making application of non-separated T cell

TILs produced no superior therapeutic efficacy

T cells which persisted long-term acquired a pheno‐

T cell clones with

TILs require

tion that infusion of ex vivo activated, IFN-γ<sup>+</sup>

250 Melanoma – Current Clinical Management and Future Therapeutics

persist for longer times [39]. Those CD8+

help of CD4+

populations more suitable.

effective towards metastases which are otherwise not accessible.

While most trials apply non-separated TILs, administration of isolated CD8+

type of central memory-type T cells in vivo [40]. It is therefore assumed that CD8+

in melanoma is associated with responsiveness to treatment after TIL therapy [41].

reactive T cells in the peripheral blood of melanoma patients are extremely rare.

**Figure 1. T cells used in adoptive cell therapy of melanoma.** (A) Tumor infiltrating T cells (TILs) are isolated from melanoma biopsies, selected for reactivity towards melanoma cells, amplified in the presence of IL-2 to clinically rele‐ vant numbers and infused to the patient. Alternatively, TILs are expanded without prior selection for melanoma reac‐ tivity using a short-term amplification protocol ("young TILs"). (B) T cells from the peripheral blood of melanoma patients are genetically modified by retro- or lentivirus transduction to express a recombinant T cell receptor (TCR) or a chimeric antigen receptor (CAR), specific for a melanoma associated antigen, amplified and administered to the pa‐ tient.


**CHUV**, Centre Hospitalier Universitaire Vaudois; **DFCI**, Dana-Farber Cancer Institute; **FHCRC**, Fred Hutchinson Can‐ cer Research Center; **NIH**, National Institutes of Health; **NUH**, Nantes University Hospital

**Table 2.** Adoptive cell therapy with autologous, antigen specific T cells in patients with melanoma

## **3. T cells with engineered anti-melanoma specificity**

The success of melanoma antigen specific T cells from peripheral blood strengthened efforts to obtain melanoma-specific T cell clones by genetic engineering of patient's T cells from the peripheral blood. In particular, the molecular cloning of the TCR from melanoma-reactive T cells enabled the engraftment of melanoma specificity to any T cell (Figure 1) [46-49]. A TCR with specificity for gp100 was cloned from melanoma reactive TILs and transferred by retrovirus-mediated gene transfer into blood T cells which thus obtained redirected specificity for gp100+ cells in addition to their parental specificity. TCR engineered T cells recognized gp100+ melanoma cells, secreted pro-inflammatory cytokines including IFN-γ and lysed gp100+ melanoma cells [50, 51]. By the same strategy, blood T cells were modified with the TCR specific for other melanoma associated antigens (Table 3). Using T cells modified with a gp100 specific TCR objective response was induced in 19% of patients, most responses were persistent [49]. Melanoma regression was also obtained in 5/11 melanoma patients after transfer of T cells modified with a TCR that recognizes NY-ESO-1, a protein encoded by a member of the cancer/ germline family of genes [52, 53].

Melanoma regression was obtained in about 30% of patients after cell therapy with MART-1 specific T cells [49, 52, 54-56]. As a side effect, patients suffered from vitiligo and destruction of melanocytes in the eye and ear indicating that T cells with engineered specificity can target rare and healthy cells even with the cognate antigen at low levels. In a recent trial, patients were treated with T cells engineered with an anti-MAGE-A3 TCR [57]. While 5/9 patients experienced melanoma regression, three of them had mental status changes and two lapsed into coma and died. Histology revealed necrotizing leukoencephalopathy which is likely due to the recognition of previously unknown epitopes of MAGE-A9/A12, the latter expressed in the brain.

Prolonged clinical remission was observed when engineered T cells persisted in the circulation for longer times; TCR modified T cells were recorded in the blood for more than a year after initiation of treatment [56, 58]. Moreover, TCR engineered T cells were capable to penetrate the blood-brain barrier and to induce regression of brain metastases [57] giving hope that patients with metastases at otherwise incurable sites may benefit from adoptive cell therapy. However, tumor cells may become invisible to TCR modified T cells due to repression of the MHC complex [60], β2 microglobulin mutation [61], and deficiencies in the antigen processing machinery [60, 62], all of them resulting in diminished antigen presentation and less TCRmediated T cell activation.

Engineering T cells with a recombinant TCR may produce a safety hazard when the transgenic αβ TCR forms hetero-dimers with the respective α and β TCR chains of the endogenous TCR. Such mis-pairing of TCR chains can induce severe auto-reactivity as a result in gain of an unpredictable specificity [63, 64]. The situation was technically solved by different means including replacing the human by the homologous murine constant moieties of the TCR [65] and by inserting additional cysteine bridges [66] to facilitate preferential pairing of the recombinant TCR αβ chains in the presence of the physiologic αβ TCR. These and other technical difficulties of the TCR strategy promoted the development of an artificial "all-in-one" receptor molecule to redirect T cells in an antigen-restricted fashion as summarized below.

**3. T cells with engineered anti-melanoma specificity**

252 Melanoma – Current Clinical Management and Future Therapeutics

for gp100+

germline family of genes [52, 53].

gp100+

gp100+

the brain.

mediated T cell activation.

The success of melanoma antigen specific T cells from peripheral blood strengthened efforts to obtain melanoma-specific T cell clones by genetic engineering of patient's T cells from the peripheral blood. In particular, the molecular cloning of the TCR from melanoma-reactive T cells enabled the engraftment of melanoma specificity to any T cell (Figure 1) [46-49]. A TCR with specificity for gp100 was cloned from melanoma reactive TILs and transferred by retrovirus-mediated gene transfer into blood T cells which thus obtained redirected specificity

cells in addition to their parental specificity. TCR engineered T cells recognized

melanoma cells, secreted pro-inflammatory cytokines including IFN-γ and lysed

melanoma cells [50, 51]. By the same strategy, blood T cells were modified with the TCR specific for other melanoma associated antigens (Table 3). Using T cells modified with a gp100 specific TCR objective response was induced in 19% of patients, most responses were persistent [49]. Melanoma regression was also obtained in 5/11 melanoma patients after transfer of T cells modified with a TCR that recognizes NY-ESO-1, a protein encoded by a member of the cancer/

Melanoma regression was obtained in about 30% of patients after cell therapy with MART-1 specific T cells [49, 52, 54-56]. As a side effect, patients suffered from vitiligo and destruction of melanocytes in the eye and ear indicating that T cells with engineered specificity can target rare and healthy cells even with the cognate antigen at low levels. In a recent trial, patients were treated with T cells engineered with an anti-MAGE-A3 TCR [57]. While 5/9 patients experienced melanoma regression, three of them had mental status changes and two lapsed into coma and died. Histology revealed necrotizing leukoencephalopathy which is likely due to the recognition of previously unknown epitopes of MAGE-A9/A12, the latter expressed in

Prolonged clinical remission was observed when engineered T cells persisted in the circulation for longer times; TCR modified T cells were recorded in the blood for more than a year after initiation of treatment [56, 58]. Moreover, TCR engineered T cells were capable to penetrate the blood-brain barrier and to induce regression of brain metastases [57] giving hope that patients with metastases at otherwise incurable sites may benefit from adoptive cell therapy. However, tumor cells may become invisible to TCR modified T cells due to repression of the MHC complex [60], β2 microglobulin mutation [61], and deficiencies in the antigen processing machinery [60, 62], all of them resulting in diminished antigen presentation and less TCR-

Engineering T cells with a recombinant TCR may produce a safety hazard when the transgenic αβ TCR forms hetero-dimers with the respective α and β TCR chains of the endogenous TCR. Such mis-pairing of TCR chains can induce severe auto-reactivity as a result in gain of an unpredictable specificity [63, 64]. The situation was technically solved by different means including replacing the human by the homologous murine constant moieties of the TCR [65] and by inserting additional cysteine bridges [66] to facilitate preferential pairing of the recombinant TCR αβ chains in the presence of the physiologic αβ TCR. These and other


LU, Loyola University (Chicago); MDACC, M.D. Anderson Cancer Center; MUH, Mie University Hospital; NIH, National Institutes of Health; TMUCIH, Tianjin Medical University Cancer Institute and Hospital; UC, University of California;

**Table 3.** Adoptive cell therapy with engineered antigen specific T cells in patients with melanoma

## **4. CAR T cells with engineered specificity for melanoma**

In order to link antigen recognition with the downstream signaling machinery of the TCR, Zelig Eshhar (Weizmann Institute of Science) reported a chimeric antigen receptor (CAR) molecule, also named immunoreceptor, which is composed in the extracellular moiety of a single chain fragment of variable region (scFv) antibody for binding and in the intracellular moiety of the CD3ζ endodomain to initiate T cell activation [67]. The coding sequence of such recombinant receptor molecule is transferred by retro- or lentiviral transduction into T cells in vitro (Figure 1) [68]. CAR engineered T cells, also nick-named "T-bodies", recognize their new target by CAR binding and become activated to secrete pro-inflammatory cytokines, to amplify and to lyse the cognate target cells. Since the binding domain is derived from an antibody the CAR recognizes the target in a MHC-independent fashion which makes major differences to TCR mediated T cell recognition. For instance, the CAR recognizes its target independently of the individual HLA subtype and CAR T cells are not affected by MHC repression and loss of HLA molecules on target cells which frequently occurs during tumor progression. However, recognition by CARs is restricted to target antigens on the cell surface; intracellular antigens like transcription factors are not visible to CAR T cells. Despite that limitation, a nearly infinite variety of targets can be recognized including those which are not classical T cell targets like carbohydrates and gangliosides [69].

Full and lasting T cell activation requires two complementary signals, one provided by the TCR/CD3 and the other by co-receptors the prototype of which is CD28. Prolonged T cell activation, however, requires costimulation and autocrine factors, in particular IL-2 which is only secreted upon TCR and simultaneous CD28 signaling. The lack of appropriate costimu‐ lation in the tumor lesion provides the rationale for combining the intracellular CD3ζ with the CD28 signaling domain in one polypeptide chain of a "second generation" CAR (Figure 2). A CAR with combined CD28-CD3ζ signaling domain provides both the primary CD3ζ and the required costimlatory signal when engaging the cognate target. CARs with a costimulatory domain clearly provide clinical benefit and improved T cell persistence compared to CARs with the CD3ζ domain only [70-72]. Other costimulatory moieties, such as 4-1BB (CD137) and OX40 (CD134), also provide full T cell activation when linked to CD3ζ in a CAR; the individual costimulory domains have different impact on T cell effector functions [73]. These and other costimulatory domains were furthermore combined in so-called "3rd generation" CARs which provide advantage for matured effector T cells in terminal differentiation but less in younger stages of T cell development [74]. A number of additional modifications of the CAR design were explored in order to improve T cell persistence and activation and finally the anti-tumor response [75, 76].

While the antibody domain defines the target specificity of the CAR, a plethora of antigens can potentially be used as target for the adoptive cell therapy of melanoma. T cells engineered for targeting melanoma-associated antigens include CARs with specificity for HMW-MAA, also known as MCSP [77, 78], melanotransferrin [79], and the gangliosides GD2 [80] and GD3 [81]. Trials are currently recruiting; to our best knowledge no published data are so far available.

During the last years spectacular efficacy was achieved with CAR T cells in phase I trials for the treatment of lymphoma/leukemia [82, 83]. Clinical response and prolonged T cell activation was accompanied by a "cytokine storm", which occurred even weeks after initial T cell administration; the side effect can clinically be managed by treatment with a neutralizing anti-IL-6 antibody without affecting the anti-tumor efficacy.

**4. CAR T cells with engineered specificity for melanoma**

254 Melanoma – Current Clinical Management and Future Therapeutics

carbohydrates and gangliosides [69].

response [75, 76].

available.

In order to link antigen recognition with the downstream signaling machinery of the TCR, Zelig Eshhar (Weizmann Institute of Science) reported a chimeric antigen receptor (CAR) molecule, also named immunoreceptor, which is composed in the extracellular moiety of a single chain fragment of variable region (scFv) antibody for binding and in the intracellular moiety of the CD3ζ endodomain to initiate T cell activation [67]. The coding sequence of such recombinant receptor molecule is transferred by retro- or lentiviral transduction into T cells in vitro (Figure 1) [68]. CAR engineered T cells, also nick-named "T-bodies", recognize their new target by CAR binding and become activated to secrete pro-inflammatory cytokines, to amplify and to lyse the cognate target cells. Since the binding domain is derived from an antibody the CAR recognizes the target in a MHC-independent fashion which makes major differences to TCR mediated T cell recognition. For instance, the CAR recognizes its target independently of the individual HLA subtype and CAR T cells are not affected by MHC repression and loss of HLA molecules on target cells which frequently occurs during tumor progression. However, recognition by CARs is restricted to target antigens on the cell surface; intracellular antigens like transcription factors are not visible to CAR T cells. Despite that limitation, a nearly infinite variety of targets can be recognized including those which are not classical T cell targets like

Full and lasting T cell activation requires two complementary signals, one provided by the TCR/CD3 and the other by co-receptors the prototype of which is CD28. Prolonged T cell activation, however, requires costimulation and autocrine factors, in particular IL-2 which is only secreted upon TCR and simultaneous CD28 signaling. The lack of appropriate costimu‐ lation in the tumor lesion provides the rationale for combining the intracellular CD3ζ with the CD28 signaling domain in one polypeptide chain of a "second generation" CAR (Figure 2). A CAR with combined CD28-CD3ζ signaling domain provides both the primary CD3ζ and the required costimlatory signal when engaging the cognate target. CARs with a costimulatory domain clearly provide clinical benefit and improved T cell persistence compared to CARs with the CD3ζ domain only [70-72]. Other costimulatory moieties, such as 4-1BB (CD137) and OX40 (CD134), also provide full T cell activation when linked to CD3ζ in a CAR; the individual costimulory domains have different impact on T cell effector functions [73]. These and other costimulatory domains were furthermore combined in so-called "3rd generation" CARs which provide advantage for matured effector T cells in terminal differentiation but less in younger stages of T cell development [74]. A number of additional modifications of the CAR design were explored in order to improve T cell persistence and activation and finally the anti-tumor

While the antibody domain defines the target specificity of the CAR, a plethora of antigens can potentially be used as target for the adoptive cell therapy of melanoma. T cells engineered for targeting melanoma-associated antigens include CARs with specificity for HMW-MAA, also known as MCSP [77, 78], melanotransferrin [79], and the gangliosides GD2 [80] and GD3 [81]. Trials are currently recruiting; to our best knowledge no published data are so far The enthusiasm in CAR T cell therapy, however, was dampened by reports on serious adverse events and fatalities after CAR T cell administration [84, 85]. Targeting ErbB2 produced respiratory failure which is thought to be due to low levels of antigen on a number of healthy cells which are sufficient to trigger "on-target-off-organ" T cell activation [86]. This and other serious adverse events emphasize a careful evaluation of potential targets and the necessity for T cell dose escalation studies to balance anti-tumor efficacy and auto-immunity [75, 87, 88].

**Figure 2. T cells with engineered specificity.** T cells physiologically recognize their target by the T cell receptor (TCR) complex which is composed of the TCR α and β chain for recognition and the CD3 chains for signaling. The variable regions of each TCR chain (Vα and Vβ) together bind to the MHC presented antigen, Cα and Cβ represent the constant domains. T cells can be genetically engineered with defined specificity by expression of recombinant TCR αβ chains of known specificity. In contrast to the TCR, the chimeric antigen receptor (CAR) is one polypeptide chain composed of a single chain fragment of variable region (scFv) antibody for antigen recognition, the extracellular spacer domain, a trans-membrane domain and the intracellular CD3ζ ("first generation" CAR), the CD28-CD3ζ ("second generation" CAR), or the CD28-OX40-CD3ζ ("third generation" CAR) signaling chain.

## **5. "Melanoma stem cells": Target cells to achieve long-term remission?**

Despite the tremendous cellular and phenotypic heterogeneity in tumor lesions, cancer is thought to be initiated and maintained by so-called cancer stem cells (CSCs). Such pluripotent stem cells are of low abundance, induce tumors upon transplantation under limiting dilution conditions, resist radiation and chemotherapy, and drive self-renewal and a-symmetric differentiation into a variety of cell types. Residual CSCs are thought to initiate cancer relapse even after years of "dormancy", which can be more than a decade after surgical treatment of the primary lesion [89]. While the concept of the hierarchical organization in driving tumor progression was initially drawn upon deciphering hematological malignancies, basically the same organization was subsequently reported for other solid cancers including mammary, prostate, pancreatic, colon carcinoma and glioma [90-94].

Transplantation of melanoma cell subsets into recipient mice under limiting dilution condi‐ tions also revealed that a defined subset of cancer cells, and not every cell from the same biopsy, can induce tumors of same histology as the parental tumor [90, 95-97]. One conclusion is that melanoma is organized in a hierarchical manner originating from a particular initiator cell, the cancer stem cell, which gives rise to the described diversity of cells in an established lesion. Melanoma initiating cells were described by various, but not common markers, including the transporter protein ABCB5 [95], CD20 [97], or the nerve growth factor receptor CD271 [98]. While CD271+ melanoma cells are present in a frequency of approximately 1/2000 cells [98], transplantation under more rigorous conditions, i.e., ideally of one single melanoma cell, revealed that nearly every fourth randomly taken melanoma cell (1/2-1/15) can induce tumors in the host animal. This observation, however, questioned the validity of the stem cell para‐ digm for melanoma [99, 100]. Subsequent studies made clear that the potential to induce melanoma is not closely associated with a particular phenotype and that the number of potential CSCs in melanoma may not necessarily be low. If nearly every melanoma cell is capable to re-program to a tumor initiating cell under certain conditions, blocking stem cell properties in melanoma will reduce tumor initiation and growth in a transplantation model finally resulting in melanoma ablation [101].

Once the tumor lesion is established, a minor subset of cancer cells seems to take over to control malignant progression. Evidence for this hypothesis was provided from a pre-clinical model [79] which asked whether all or a defined subset of melanoma cells in an established xenotransplanted lesion need to be eliminated to cause tumor regression. Such melanoma sustain‐ ing cell may be, but not must be identical to melanoma stem cells identified by transplantation assays.

Evidence for a particular targetable melanoma cell subset which sustains tumor progression was provided by the observation that elimination of CD20+ melanoma cells by adoptive transfer of CAR T cells completely eradicated xeno-transplanted melanoma. Those human melanoma biopsies contained a subset of CD20+ melanoma cells which constituted about 1-2% of mela‐ noma cells and which are present in different histological melanoma types and tumor stages. A caveat is that in approximately 20% of melanoma samples analyzed so far, no CD20+ melanoma cells were detected by histological screening; CD20-specific CAR T cells did not induce regression of those transplanted tumor lesions. Interestingly, CD20 re-expression in a random subpopulation of those tumor cells by genetic modification did not render the tumor lesion sensitive for eradication indicating that CD20 expression per se is not sufficient but requires additional capabilities to sustain melanoma progression [79].

**5. "Melanoma stem cells": Target cells to achieve long-term remission?**

prostate, pancreatic, colon carcinoma and glioma [90-94].

256 Melanoma – Current Clinical Management and Future Therapeutics

finally resulting in melanoma ablation [101].

biopsies contained a subset of CD20+

was provided by the observation that elimination of CD20+

While CD271+

assays.

CD20+

Despite the tremendous cellular and phenotypic heterogeneity in tumor lesions, cancer is thought to be initiated and maintained by so-called cancer stem cells (CSCs). Such pluripotent stem cells are of low abundance, induce tumors upon transplantation under limiting dilution conditions, resist radiation and chemotherapy, and drive self-renewal and a-symmetric differentiation into a variety of cell types. Residual CSCs are thought to initiate cancer relapse even after years of "dormancy", which can be more than a decade after surgical treatment of the primary lesion [89]. While the concept of the hierarchical organization in driving tumor progression was initially drawn upon deciphering hematological malignancies, basically the same organization was subsequently reported for other solid cancers including mammary,

Transplantation of melanoma cell subsets into recipient mice under limiting dilution condi‐ tions also revealed that a defined subset of cancer cells, and not every cell from the same biopsy, can induce tumors of same histology as the parental tumor [90, 95-97]. One conclusion is that melanoma is organized in a hierarchical manner originating from a particular initiator cell, the cancer stem cell, which gives rise to the described diversity of cells in an established lesion. Melanoma initiating cells were described by various, but not common markers, including the transporter protein ABCB5 [95], CD20 [97], or the nerve growth factor receptor CD271 [98].

transplantation under more rigorous conditions, i.e., ideally of one single melanoma cell, revealed that nearly every fourth randomly taken melanoma cell (1/2-1/15) can induce tumors in the host animal. This observation, however, questioned the validity of the stem cell para‐ digm for melanoma [99, 100]. Subsequent studies made clear that the potential to induce melanoma is not closely associated with a particular phenotype and that the number of potential CSCs in melanoma may not necessarily be low. If nearly every melanoma cell is capable to re-program to a tumor initiating cell under certain conditions, blocking stem cell properties in melanoma will reduce tumor initiation and growth in a transplantation model

Once the tumor lesion is established, a minor subset of cancer cells seems to take over to control malignant progression. Evidence for this hypothesis was provided from a pre-clinical model [79] which asked whether all or a defined subset of melanoma cells in an established xenotransplanted lesion need to be eliminated to cause tumor regression. Such melanoma sustain‐ ing cell may be, but not must be identical to melanoma stem cells identified by transplantation

Evidence for a particular targetable melanoma cell subset which sustains tumor progression

of CAR T cells completely eradicated xeno-transplanted melanoma. Those human melanoma

noma cells and which are present in different histological melanoma types and tumor stages. A caveat is that in approximately 20% of melanoma samples analyzed so far, no

melanoma cells were detected by histological screening; CD20-specific CAR T cells did

melanoma cells by adoptive transfer

melanoma cells which constituted about 1-2% of mela‐

melanoma cells are present in a frequency of approximately 1/2000 cells [98],

There are additionally clinical observations that sustain the notion of CD20+ cells in promoting melanoma progression. Firstly, a patient with stage III/IV metastatic, refractory melanoma and 2% CD20+ melanoma cells who received intra-lesional injections of the anti-CD20 therapeutic antibody rituximab experienced lasting remission accompanied by a decline of the melanoma serum marker S-100 to physiological levels and a switch of a T helper-2 to a more proinflammatory T helper-1 response [102]. Although anecdotic, data provide the first clinical evidence that targeted elimination of CD20+ melanoma cells can produce regression of chemotherapy-refractory melanoma. Secondly, in a small pilot trial, stage IV melanoma patients without evidence of disease by way of surgery, chemo-and/or radiation therapy received the anti-CD20 antibody systemically for a 2 year period [103]. Data suggest a benefit of anti-CD20 therapy in overall and recurrence-free survival; a caveat being that the number of patients is still small for definitive conclusions.

Currently, the hierarchical stem cell model in the maintenance of an established melanoma is supported by some experimental evidence [79], whereas a body of information on melanoma initiation by transplantation of single melanoma cells sustains the stochastic model [99, 100], although not confirmed by others [98]. The most determining proof of the stem cell hypothesis, however, will be the successful melanoma elimination by targeting stem cells or stem cell properties. For the development of such therapeutic strategies several aspects need to be taken into account.

First, standard therapy will rapidly de-bulk the tumor lesion and the remaining melanoma stem cells, which are more chemo-and radiation resistant, will drive relapse of the disease. Since those melanoma initiating cells are merely in a "dormant" state and replicate less frequently than the majority of melanoma cells in the same lesion, anti-proliferative strategies by classical chemotherapeutic drugs are unlikely efficient. Transporter systems including ABCB5, which is highly expressed by melanoma stem cells [95], additionally contribute to chemotherapy resistance; the chemotherapy and/or radiation itself may promote expression of those transporter systems and survival of those resistant cells which finally contributes to relapse of the disease.

Second, if clinical progression correlates with the prevalence of CD20+ melanoma cells, targeted elimination of those melanoma cells requires to meet the fact that those target cells are a small minority. Targeted elimination, e.g., by CD20 redirected cytotoxic T cells or by CD20-specific therapeutic antibodies like Rituxan™ (rituximab) or Arzerra™ (ofatumumab), will be required to obtain substantial efficacy.

Third, the extraordinary functional and phenotypic plasticity of melanoma cells may make it necessary to have the therapeutic agent in place for a longer time. In their pre-clinical model, Schmidt and colleagues [79] used CAR T cells which persist for long-term acting as an antigenspecific guardian as long as target cells are present. Since repetitive re-stimulation sustains the persistence and amplification of CAR T cells, cellular therapy has a major advantage compared to pharmaceutical drugs with a comparable short half-life. CAR T cells can moreover provide antigen-specific memory with defined specificity [104], potentially contributing to control melanoma in the long-term.

## **6. Production of engineered T cells for clinical application**

Application of adoptive cell therapy to clinical use requires efficient production of cells according to good manufacturing practice (GMP). This particularly applies to patient's T cells which are ex vivo genetically modified. The vector used for T cell modification is of major relevance with respect to the efficiency and stability in modification. Crucial steps in this process are the stable integration of the genetic vector, the site of integration to avoid insertion mutagenesis, and the resistance of the vector to genetic repression. To date, most clinical trials were performed employing retroviral or lentiviral vectors which fulfill some but not all of these requirements. Recently, other vector systems including RNA modification are alternatively utilized and it is expected that these systems will be explored in parallel in the near future.

The way of stimulating the T cells ex vivo for genetic modification and subsequent amplifica‐ tion is crucial for both the success in transduction and the functional capacities of modified cells. T cells are commonly activated by TCR/CD3 stimulation in addition to IL-2 [105]; most protocols use anti-CD3 and anti-CD28 magnetic beads [83, 106] which can be easily eliminated during the production process. IL-2 is replaced by other cytokines such as IL-7 and IL-15 to obtain a T cell population with a more naive and central memory phenotype [107]. Alterna‐ tively, cell lines were engineered, so-called "artificial APCs", which are modified with the various co-stimulating molecules to mimic the physiological stimulation and to provide the required signals [108]. However, difficulties in adopting those cells to GMP standard prevent their broad application in large scale production processes.

For the production itself, static culture systems in flasks or gas permeable bags are traditionally used. Due to their amplification at low cell densities (0.25-1x106 cells per ml), high culture volumes are required to obtain clinically relevant T cell numbers which is more easily achieved by non-static systems including the WAVE-Bioreactor or the G-Rex100 device [83, 106, 109]. In order to produce engineered T cells for a large number of patients it will be required to manufacture cells in a closed system and to produce multiple batches in parallel in the same clean room facility without the risk of batch contamination.

## **7. Challenges and promise in the adoptive cell therapy of melanoma**

To date, approximately half of the melanoma patients benefit from adoptive cell therapy with TILs. Specifically targeted T cells may further improve the therapeutic response. Despite substantial success, the strategy still has major challenges which need to be addressed in the near future.

Significant numbers of effector T cells have to accumulate in the targeted tumor lesion which is mediated by a network of chemokines. Adoptively transferred T cells use these networks to accumulate at the tumor site; melanoma cells secrete a number of chemokines including CXCL1 to attract lymphocytes. However, early imaging studies revealed that melanomaspecific T cells massively infiltrate the lungs, spleen and liver with only some accumulation at the tumor site before the cells decline to undetectable levels in circulation [110-112]. To improve tumor targeting TILs were engineered with CXCR2, the receptor for melanoma secreted CXCL1, which resulted in improved anti-tumor activity in a mouse model [113]. The strategy is currently being explored in an early phase I trial (Table 3) [113].

persistence and amplification of CAR T cells, cellular therapy has a major advantage compared to pharmaceutical drugs with a comparable short half-life. CAR T cells can moreover provide antigen-specific memory with defined specificity [104], potentially contributing to control

Application of adoptive cell therapy to clinical use requires efficient production of cells according to good manufacturing practice (GMP). This particularly applies to patient's T cells which are ex vivo genetically modified. The vector used for T cell modification is of major relevance with respect to the efficiency and stability in modification. Crucial steps in this process are the stable integration of the genetic vector, the site of integration to avoid insertion mutagenesis, and the resistance of the vector to genetic repression. To date, most clinical trials were performed employing retroviral or lentiviral vectors which fulfill some but not all of these requirements. Recently, other vector systems including RNA modification are alternatively utilized and it is expected that these systems will be explored in parallel in the near future. The way of stimulating the T cells ex vivo for genetic modification and subsequent amplifica‐ tion is crucial for both the success in transduction and the functional capacities of modified cells. T cells are commonly activated by TCR/CD3 stimulation in addition to IL-2 [105]; most protocols use anti-CD3 and anti-CD28 magnetic beads [83, 106] which can be easily eliminated during the production process. IL-2 is replaced by other cytokines such as IL-7 and IL-15 to obtain a T cell population with a more naive and central memory phenotype [107]. Alterna‐ tively, cell lines were engineered, so-called "artificial APCs", which are modified with the various co-stimulating molecules to mimic the physiological stimulation and to provide the required signals [108]. However, difficulties in adopting those cells to GMP standard prevent

For the production itself, static culture systems in flasks or gas permeable bags are traditionally

volumes are required to obtain clinically relevant T cell numbers which is more easily achieved by non-static systems including the WAVE-Bioreactor or the G-Rex100 device [83, 106, 109]. In order to produce engineered T cells for a large number of patients it will be required to manufacture cells in a closed system and to produce multiple batches in parallel in the same

**7. Challenges and promise in the adoptive cell therapy of melanoma**

To date, approximately half of the melanoma patients benefit from adoptive cell therapy with TILs. Specifically targeted T cells may further improve the therapeutic response. Despite substantial success, the strategy still has major challenges which need to be addressed in the

cells per ml), high culture

**6. Production of engineered T cells for clinical application**

their broad application in large scale production processes.

clean room facility without the risk of batch contamination.

near future.

used. Due to their amplification at low cell densities (0.25-1x106

melanoma in the long-term.

258 Melanoma – Current Clinical Management and Future Therapeutics

Since tumor eradication requires a beneficial T cell-to-target cell ratio, higher numbers of tumor-specific T cells applied per dose likely increase the clinical efficacy. The optimal dose of T cells, however, is still a matter of discussion and requires empiric evaluation. A number of trials, in particular applying TILs, administered up to 1010 cells per dose [27]. Such high doses in turn require extended expansions of T cells ex vivo with the risk of loss of the "young" phenotype and gain of more matured T cells. Highly expanded T cells become hypo-responsive to CD28 costimulation and rapidly enter activation induced cell death, in particular upon IL-2 driven expansion [114]. With respect to more potent effector functions short-term amplification protocols are envisioned for both TILs and engineered T cells. This may be achieved by T cell amplification in the presence of IL-15 and IL-21 and/or by 4-1BB co-stimulation [115].

On the other hand, administration of about 105 engineered T cells induced remarkable therapeutic efficacy in recent trials targeting CD19+ leukemia [83]. Since the T cells substantially amplify in vivo upon antigen encounter, the capacity of cells to amplify under appropriate conditions is more relevant than the applied cell number.

Once targeted in sufficient numbers to the tumor tissue, a major challenge is the tumor selectivity of redirected T cells. While the TCR and the CAR is specific for a particular target, in most cases the targeted antigen is not exclusively expressed by cancer but also by healthy cells, although sometimes at lower levels [116, 117]. MART-1, frequently expressed by the majority of melanoma cells, is also expressed by melanocytes. Targeting such type of antigen frequently produces vitiligo, sometimes also inner ear toxicity with a certain degree of deafness [49]. Since nearly all "tumor-associated antigens" which are frequently used as targets for adoptive cell therapy are self-antigens, strategies are needed to minimize such off-target toxicities. Among these, low-avidity TCRs or CARs or combinatorial antigen recognition by two CARs are currently explored.

Melanoma cells may become invisible to TILs or TCR modified T cells due to downregulation of their MHC components or due to deficiencies in antigen processing. Howev‐ er, melanoma cells may still be visible to CAR T cells which recognized their target by their antibody-derived binding domain in a MHC independent fashion. On the other hand, TCR T cells are capable to recognize cross-presented antigen, for instance tumor antigen presented by stroma cells, which is invisible to CAR T cells but helps to destroy the tumor lesion in the long-term [118, 119].

Consequently, a TCR-like CAR aims at combining the benefits of TCR and CAR redirected T cells. This is performed by using a single chain antibody with TCR-like specificity for recog‐ nizing MHC presented antigen. T cells with such a TCR-like CAR were successfully redirected in a MHC restricted fashion towards NY-ESO-1 and MAGE-A1, respectively [120, 121].

The redirected T cell activation depends on the amount of target antigen and binding affinity. Compared to TILs and TCR modified T cells, CAR T cells bind with extraordinary high affinity by their antibody-derived CAR binding domain. A furthermore increase in affinity by affinity maturation does not necessarily improve CAR redirected T cell activation [120, 122]; CD28 costimulation does not add to the affinity dependent activation threshold, however, prolongs T cell persistence and resistance to apoptosis [123]. Targeting cancer cells also depends on the amount of target antigen in addition to the binding affinity. Low affinity CARs require abundant antigen levels for efficient activation of engineered T cells while high affinity CARs are likewise effective against low antigen levels on target cells. In this context, the selectivity in targeting melanoma cells versus healthy cells needs to be discussed not only with respect to the targeted antigen itself but also to antigen amount and binding affinity.

Amplification and persistence of adoptively transferred cells correlates with clinical outcome in some trials [124]. T cells will persist in detectable numbers as long as targeted antigen is present, however, will contract to undetectable levels and disappear from circulation when no target is furthermore present. To enable survival of CAR T cells in the long-term, Epstein-Barr virus (EBV)-specific T cells were used as effector cells and modified with a tumor-specific CAR. The rationale is that EBV specific T cells are maintained in a sizable population in circulation by recognizing EBV antigens by their physiological TCR. The strategy is sustained by the first clinical observation that EBV-specific T cells engineered with an anti-GD2 CAR showed benefit over non-virus-specific, CAR engineered T cells in the treatment of neuroblastoma (NCT00085930) [124]. Other trials use EBV or CMV specific, autologous T cells engineered with a first or second generation CAR, for instance directed against HER2/neu (ErbB2) (NCT01109095), CD30 (NCT01192464), or CD19 (NCT00709033; NCT01475058; NCT01430390; NCT00840853; NCT01195480).

The T cell subset matters, adoptively transferred CD8+ T cell clones poorly persist [125] and need help of CD4+ cells. Prolonged T cell anti-tumor response also requires resistance to repression in the tumor tissue. A number of efforts are currently undertaken to counteract tumor associated T cell repression, in particular mediated by Treg cells and checkpoint mediators. In animal models, CD28 costimulation without induction of IL-2 secretion protects a CAR redirected T cell response from Treg cell repression [126]. On the other hand, repetitive T cell stimulation upregulates CTLA-4 which acts as negative regulator to return the T cell to a resting stage. Administration of a CTLA-4 blocker, e.g., ipilimumab antibody, may prolong the anti-tumor activation of transferred T cells, although it is not locally restricted and will likewise affect all T cells [127, 128]. Expression profiling of TCR-engineered T cells demon‐ strates overexpression of multiple inhibitory receptors in persisting lymphocytes, including PD-1 and CD160, the latter associated with decreased reactivity of TCR T cells in a ligand independent manner [129]. Essentially the same was observed for CAR T cells [130]. These analyses point to a multi-factorial T cell repression in the tumor tissue; there is more than one uni-directional strategy needed to sustain the T cell anti-tumor response in the long-term.

Consequently, a TCR-like CAR aims at combining the benefits of TCR and CAR redirected T cells. This is performed by using a single chain antibody with TCR-like specificity for recog‐ nizing MHC presented antigen. T cells with such a TCR-like CAR were successfully redirected in a MHC restricted fashion towards NY-ESO-1 and MAGE-A1, respectively [120, 121].

260 Melanoma – Current Clinical Management and Future Therapeutics

The redirected T cell activation depends on the amount of target antigen and binding affinity. Compared to TILs and TCR modified T cells, CAR T cells bind with extraordinary high affinity by their antibody-derived CAR binding domain. A furthermore increase in affinity by affinity maturation does not necessarily improve CAR redirected T cell activation [120, 122]; CD28 costimulation does not add to the affinity dependent activation threshold, however, prolongs T cell persistence and resistance to apoptosis [123]. Targeting cancer cells also depends on the amount of target antigen in addition to the binding affinity. Low affinity CARs require abundant antigen levels for efficient activation of engineered T cells while high affinity CARs are likewise effective against low antigen levels on target cells. In this context, the selectivity in targeting melanoma cells versus healthy cells needs to be discussed not only with respect

Amplification and persistence of adoptively transferred cells correlates with clinical outcome in some trials [124]. T cells will persist in detectable numbers as long as targeted antigen is present, however, will contract to undetectable levels and disappear from circulation when no target is furthermore present. To enable survival of CAR T cells in the long-term, Epstein-Barr virus (EBV)-specific T cells were used as effector cells and modified with a tumor-specific CAR. The rationale is that EBV specific T cells are maintained in a sizable population in circulation by recognizing EBV antigens by their physiological TCR. The strategy is sustained by the first clinical observation that EBV-specific T cells engineered with an anti-GD2 CAR showed benefit over non-virus-specific, CAR engineered T cells in the treatment of neuroblastoma (NCT00085930) [124]. Other trials use EBV or CMV specific, autologous T cells engineered with a first or second generation CAR, for instance directed against HER2/neu (ErbB2) (NCT01109095), CD30 (NCT01192464), or CD19 (NCT00709033; NCT01475058; NCT01430390;

repression in the tumor tissue. A number of efforts are currently undertaken to counteract tumor associated T cell repression, in particular mediated by Treg cells and checkpoint mediators. In animal models, CD28 costimulation without induction of IL-2 secretion protects a CAR redirected T cell response from Treg cell repression [126]. On the other hand, repetitive T cell stimulation upregulates CTLA-4 which acts as negative regulator to return the T cell to a resting stage. Administration of a CTLA-4 blocker, e.g., ipilimumab antibody, may prolong the anti-tumor activation of transferred T cells, although it is not locally restricted and will likewise affect all T cells [127, 128]. Expression profiling of TCR-engineered T cells demon‐ strates overexpression of multiple inhibitory receptors in persisting lymphocytes, including PD-1 and CD160, the latter associated with decreased reactivity of TCR T cells in a ligand independent manner [129]. Essentially the same was observed for CAR T cells [130]. These

T cell clones poorly persist [125] and

cells. Prolonged T cell anti-tumor response also requires resistance to

to the targeted antigen itself but also to antigen amount and binding affinity.

NCT00840853; NCT01195480).

need help of CD4+

The T cell subset matters, adoptively transferred CD8+

A major hurdle of specific immunotherapy in general is the tremendous heterogeneity of cancer cells within the same lesion. Low or loss of target antigen expression negatively affects the long-term therapeutic efficacy of an antigen-redirected approach. This is supported by several reports which document a relapse of antigen-loss tumor metastases after adoptive therapy with melanoma-reactive T cell clones [39,131, 132]. A solution may be the use of polyclonal T cells with specificities for various melanoma antigens or T cells modified with different CARs recognizing different antigens; however, target-negative tumor cells will not be recognized. On the other hand, pro-inflammatory cytokines secreted by redirected T cells upon activation can attract a second wave of innate immune cells which in turn may eradiate the antigen-negative tumor cells. At least in an animal model, antigen-negative melanoma cells are indeed eliminated when co-inoculated with antibody-targeted cytokines [133]. T cells engineered with induced expression of transgenic IL-12 can attract innate immune cells including macrophages into the tumor tissue which eliminate antigen-negative tumor cells in the same lesion, at least in an immune competent animal model [134]. Such "TRUCK" cells ("T cells redirected for unrestricted cytokine killing") may pave a novel way to deliver transgenic cell products to pre-defined, target lesions.

Combination of adoptive cell therapy with pathway inhibitors may improve the efficacy in melanoma cell elimination, in particular in disseminated stages of the disease. Metastatic melanoma patients with the B-raf activating mutation V600E benefit from a small molecule drug, PLX4032 or vemurafenib, which inhibits the mitogen-activated protein kinase (MAPK) pathway. Treatment with vemurafenib is accompanied by increased T cell infiltrations in the melanoma lesions [135, 136] which may contribute to the therapeutic effect and may be improved by co-administration of melanoma-specific T cells.

While adoptive cell therapy is mostly performed with modified or non-modified T cells, other cells like monocytes, macrophages as well as NK cells can also be redirected by CARs in an antigen-specific fashion [137-141, 144]. In contrast to T cells, NK cells can be rapidly activated and exhibit high cytotoxic potential and continuously growing NK cell lines such as NK-92 can be used for adoptive cancer immunotherapy [142]. CD3ζ chain CARs trigger cytolytic activities of NK cells which has been shown for CARs with various specificities [138, 141, 143-147]. Similar to T cells, the anti-tumor activity was improved by adding 4-1BB or 2B4 (CD244) costimulatory domains [148, 149]. Since NK cells cannot provide IL-2 or IL-15 required for amplification, CAR modified NK cells were additionally engineered to release IL-15 which sustains NK cell expansion and CAR-mediated cytotoxicity in the absence of IL-2 [150]. Despite these and other advances during the last years, experience with CAR engineered primary NK cells in clinical trials is still limited; whether redirected cells of the innate immune system are more advantageous in melanoma elimination than modified T cells has moreover to be explored in clinical trials.

## **Acknowledgements**

Work in the author's laboratory was supported by the Deutsche Forschungsgemeinschaft, Bonn, Deutsche Krebshilfe, Bonn, Else Kröner-Fresenius-Stiftung, Bad Homburg v.d.H., Wilhelm Sander-Stiftung, München, the European Union (European Regional Development Fund-Investing in your future), the German federal state North Rhine-Westphalia (NRW), and the Fortune program of the Medical Faculty of the University of Cologne.

## **Author details**

Jennifer Makalowski1,2 and Hinrich Abken1,2\*

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

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

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

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## **Toxicities of New Drugs for Melanoma Treatment and their Management**

Paola Savoia and Paolo Fava

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59860

## **1. Introduction**

Advanced melanoma is a highly aggressive tumor with a low response rate to the majority of pharmacological agents.

Among conventional cytotoxic chemotherapies, dacarbazine (DTIC) is the only FDA-ap‐ proved alkylating agent, at present; its clinical efficacy is pretty low with 5–10% responsive‐ ness, which is generally short-lived. Carmustine, temozolomide and other chemotherapic agents (taxanes and platinum-analogs) showed similar efficacy in metastatic settings [1, 2].

Adjuvant immunotherapy for stage III melanoma is mainly based on interferon-α2b (IFN-α2b) even if its efficacy is quite limited. In fact, only about the 20% of patients showed an improve‐ ment in relapse-free survival as demonstrated in randomized observation-controlled trials without a clear effect on overall survival [3]. FDA approved high-dose interleukin-2 (IL-2) for the treatment of advanced stage melanoma, on the basis of its ability to elicit durable responses in a small percentage of patients [4, 5]. However, the durable response rate is only 10-20% and toxicities associated with IL-2 are quite severe.

Recently, molecular targeted therapies have radically changed the management of metastat‐ ic melanoma. Anti-CTLA-4 monoclonal antibodies (Ipilimumab) and B-Raf inhibitors are the first examples of this new kind of drugs, the firsts approved with both overall survival and progression-free survival benefits in respect of the standard chemotherapic agent dacarbazine [6-10].

Ipilimumab targets the anti-cytotoxic T-lymphocyte antigen-4, a key immune-checkpoint molecule that down-regulates some pathways of T-cell activation. Ipilimumab inactivating the CTLA-4 inhibitory signal, enhance the immune system response against melanoma cells. Several randomized phases II and III trials demonstrated a statistically significant improve‐ ment in overall survival in patients with metastatic melanoma treated with Ipilimumab alone

© 2015 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 eproduction in any medium, provided the original work is properly cited.

or in combination with dacarbazine. The efficacy of Ipilimumab was confirmed both in treated and untreated metastatic melanoma patients [6, 9, 11]. Furthermore, re-treatment with Ipilimumab can re-establish disease control in a percentage of patients who progress after achieving an objective response or stable disease after the first treatment course [6, 9, 11-13].

As a consequence of this peculiar mechanism of action, Ipilimumab may determine the development of autoimmune conditions and exacerbate a series of immune-related adverse events, which will be described in the next section of this review.

Vemurafenib is a low molecular weight molecule (489.9 Da), orally available, which belongs to the new generation inhibitors of B-Raf as well as of other members of the RAF kinase family (including the products of ARAF, BRAF and CRAF genes). The BRAF protein is a part of the RAS/RAF/MEK/ERK signaling pathway, which is a key regulator of melano‐ ma cell growth. In cells expressing the pro-oncogenic BRAF-V600E, BRAF-V600D and BRAF-V600R genes, Vemurafenib inhibits both phosphorylated ERK (pERK) and pMEK in a dosedependent manner, resulting in a reduction of tumour growth and even in tumour regression in in vitro studies and xenograft transplant models. Several clinical trials have confirmed and extended these preclinical observations and, to date, RAF inhibitors represent the emerging standard of care for metastatic melanoma harboring the BRAF-V600E mutation, with clinical responsiveness in more than 90% of these patients [1, 14]. In particular, results from a phase III clinical trial of patiente expressing the BRAF-V600E mutated isoform affected by unresectable or metastatic melanoma showed a median overall survival significantly higher for Vemurafenib-treated patients,in comparison to those treated with dacarbazine (13.2 vs 9.9 months, respectively).

Even if the toxicity of this treatment is normally considered to be acceptable, Vemurafenib triggers the onset of a wide spectrum of systemic and cutaneous toxicities which can impact patient's quality of life in a significant way [15-17]. The adverse effects, which will be detailed later, are dose-dependent and related to the alteration of the cell-signaling pathway in response to B-Raf inhibition in cells expressing the wild-type BRAF gene [18].

## **2. Ipilimumab toxicities**

The onset of immune-related adverse events (irAEs) during the treatment with Ipilimumab is consequent to action on the immune system. Actually, CTLA-4 blockage removes CTLA4 mediated downregulation of the immune response, leading thus to a large spectrum of autoimmune–inflammatory side effects with a dose-dependent mechanism [19]. These irAEs are described both at the currently approved 3mg/kg dose and at the investigational 10mg/kg dose and may affect a number of organs and systems, including the eye, the skin, the gut and the endocrine system.

In a retrospective analysis of phase I–III Ipilimumab trials on patients with advanced mela‐ nomas, the occurrence of irAEs of any grade was a quite common phenomenon, regarding about 60% of the patients [20]. Nevertheless, Ipilimumab can be considered a safe drug: irAEsrelated deaths occurred only in about 1% of treated patients [20]. In these trials, the most common immune side effects were represented by enterocolitis, dermatitis, hepatitis, hypo‐ physitis, and uveitis, usually with an early onset. More recent data obtained on an Italian multicentric expanded-access cohort reported an occurrence of irAEs of any grade in 33% of treated patients, with a median time of onset of 5 weeks. Most irAEs were low grade, whereas grade 3/4 irAEs were described in 6% of the cases and were most commonly represented by diarrhea, liver toxicity and fatigue/asthenia [21].

or in combination with dacarbazine. The efficacy of Ipilimumab was confirmed both in treated and untreated metastatic melanoma patients [6, 9, 11]. Furthermore, re-treatment with Ipilimumab can re-establish disease control in a percentage of patients who progress after achieving an objective response or stable disease after the first treatment course [6, 9, 11-13]. As a consequence of this peculiar mechanism of action, Ipilimumab may determine the development of autoimmune conditions and exacerbate a series of immune-related adverse

Vemurafenib is a low molecular weight molecule (489.9 Da), orally available, which belongs to the new generation inhibitors of B-Raf as well as of other members of the RAF kinase family (including the products of ARAF, BRAF and CRAF genes). The BRAF protein is a part of the RAS/RAF/MEK/ERK signaling pathway, which is a key regulator of melano‐ ma cell growth. In cells expressing the pro-oncogenic BRAF-V600E, BRAF-V600D and BRAF-V600R genes, Vemurafenib inhibits both phosphorylated ERK (pERK) and pMEK in a dosedependent manner, resulting in a reduction of tumour growth and even in tumour regression in in vitro studies and xenograft transplant models. Several clinical trials have confirmed and extended these preclinical observations and, to date, RAF inhibitors represent the emerging standard of care for metastatic melanoma harboring the BRAF-V600E mutation, with clinical responsiveness in more than 90% of these patients [1, 14]. In particular, results from a phase III clinical trial of patiente expressing the BRAF-V600E mutated isoform affected by unresectable or metastatic melanoma showed a median overall survival significantly higher for Vemurafenib-treated patients,in comparison to those treated

Even if the toxicity of this treatment is normally considered to be acceptable, Vemurafenib triggers the onset of a wide spectrum of systemic and cutaneous toxicities which can impact patient's quality of life in a significant way [15-17]. The adverse effects, which will be detailed later, are dose-dependent and related to the alteration of the cell-signaling pathway in response

The onset of immune-related adverse events (irAEs) during the treatment with Ipilimumab is consequent to action on the immune system. Actually, CTLA-4 blockage removes CTLA4 mediated downregulation of the immune response, leading thus to a large spectrum of autoimmune–inflammatory side effects with a dose-dependent mechanism [19]. These irAEs are described both at the currently approved 3mg/kg dose and at the investigational 10mg/kg dose and may affect a number of organs and systems, including the eye, the skin, the gut and

In a retrospective analysis of phase I–III Ipilimumab trials on patients with advanced mela‐ nomas, the occurrence of irAEs of any grade was a quite common phenomenon, regarding about 60% of the patients [20]. Nevertheless, Ipilimumab can be considered a safe drug: irAEsrelated deaths occurred only in about 1% of treated patients [20]. In these trials, the most common immune side effects were represented by enterocolitis, dermatitis, hepatitis, hypo‐

events, which will be described in the next section of this review.

280 Melanoma – Current Clinical Management and Future Therapeutics

with dacarbazine (13.2 vs 9.9 months, respectively).

**2. Ipilimumab toxicities**

the endocrine system.

to B-Raf inhibition in cells expressing the wild-type BRAF gene [18].


Modified from Weber J, et al. J Clin Oncol 30 (21), 2012: 2691-2697[23].

**Table 1.** Most common Immunorelated Edverse events and their grade:


Modified from Weber J, et al. J Clin Oncol 30 (21), 2012: 2691-2697 [23].

**Table 2.** Most common immunorelated adverse events and their management:

In phase I-III studies, irAE resolution time varied from 4.3 to 7.7 weeks, whereas in patients included in the expanded-access projects ranged from 0.1 to 11.1 weeks (median 1.7 weeks). When grade 3/4 irAE were separately analyzed the median resolution time was 1.1 weeks (range, 0.1–3.4 weeks) [20, 21]. Despite some early findings, no relationship seems to exist between clinical benefit and irAEs onset in treated patients.

Grade 1/2 events take advantage from symptomatic treatments and the use of topical steroids, whereas early administration of high-dose systemic corticosteroids is mandatory for the right management of grade 3/4 irAEs. Specific guidelines to manage irAEs are available [19, 22]. In literature are available guidelines to manage Ipilimumab side effects (see Table 1 and 2) [23].

## **3. Cutaneous side effects from Ipilimumab**

**IrAE Grade 1 Grade 2 Grade 3 Grade 4**

topical

Antihistamines and

corticosteroids if no response, consider oral corticosteroids

Anti-diarrhea drugs Loperamide and diphenoxylate. Patient hydration

Withhold Ipilimumab dose and check LFTs every day for 3 consecutive days; if LFT improvement to grade 1, resume routine

If no improvement in the LFTs, administer corticosteroid treatment and skip the next Ipilimumab dose until event resolves

Symptoms suggestive of hypophysitis require

corticosteroid therapy. Temporary Ipilimumab

prompt

suspension

monitoring of LFTs and continue Ipilimumab

High-dose systemic corticosteroid therapy

Hospitalization, patients hydration and systemic corticosteroids

Withhold Ipilimumab dose and check LFTs every day for 3 consecutive days; if LFT improvement to grade 1, resume routine monitoring of LFTs and continue Ipilimumab If no improvement in LFTs, administer corticosteroid treatment and skip the next Ipilimumab dose until event resolves

Intravenous corticosteroids Hormone replacement

Hydration

suspension

Electrolyte replacement Temporary Ipilimumab High-dose systemic corticosteroid therapy; antibiotics if indicated and definitive discontinuation

of Ipilimumab

of Ipilimumab

Hospitalization, patient hydration, and systemic corticosteroids. Definitive discontinuation

High dose of intravenous corticosteroids

Definitive discontinuation

of Ipilimumab

Intravenous corticosteroids Hormone replacement

Hydration

of Ipilimumab

Electrolyte replacement Definitive discontinuation

Antihistamines and

282 Melanoma – Current Clinical Management and Future Therapeutics

corticosteroids; if no response, consider oral corticosteroids

Anti-diarrhea drugs, loperamide and diphenoxylate Patient hydration

Abnormal endocrine workup, grade 1 or 2 endocrine toxicity without

adrenal crisis May resolve spontaneously If no spontaneous resolution, consider low– moderate dose Of systemic corticosteroids Consider temporary Ipilimumab suspension

Modified from Weber J, et al. J Clin Oncol 30 (21), 2012: 2691-2697 [23].

**Table 2.** Most common immunorelated adverse events and their management:

topical

**Liver toxicity** Monitoring of LFTs

**Skin Toxicity**

**Diarrhea**

**Endocrine toxicity**

Systemic side effects from Ipilimumab are usually dose-related. However, the incidence and severity of pruritus or skin reactions appear independent of Ipilimumab dosage, as demon‐ strated by a meta-analysis of the studies in which Ipilimumab was administered as mono‐ therapy at different doses (3 mg/kg vs 10 mg/kg),

Commonly used targeted anticancer agents (e.g. erlotinib, cetuximab, panitumumab, vande‐ tanib, pertuzumab) usually induce a characteristic papulopustular (acneiform) rash in 68% to 75% of treated patients. Conversely, Ipilimumab-induced maculo-papular rash are more similar to those commonly seen with traditional drugs (ie, antibiotics, non steroidal antiinflammatory drugs) or shows clinical characteristics mimicking atopic dermatitis [22].

From a recent meta-analysis performed on 19 studies from 1998-2011, which included several trials testing Ipilimumab (as monotherapy or in combination at various doses in randomized multi-arm and single-arm studies) [24], emerged that the overall incidence of all-grade rash was 24.3%. The overall incidence of high-grade rash was 2.4%, with a relative risk ranging from 0.7% to 11.8%. Interestingly, among 320 patients receiving 3 mg/kg and 440 receiving 10 mg/kg, there was no significant difference in terms of incidence of all-grade or high-grade rash between doses [24]. Skin irAEs have a shorter time of onset than those affecting other sites and usually develop 3-4 weeks after Ipilimumab initiation.

In our series of 57 patients treated with Ipilimumab, the incidence of cutaneous side effects was 10% (any grade). Ipilimumab-related skin lesion were erythematous, edematous or maculo-papular, often located on the trunk and extremities (Figure 1); vasculitic and purpuric lesions were also observed. Pruritus was present in almost half of the patients who showed skin reactions [unpublished data]. Some literature reports suggest that rash can coincide with the regression of subcutaneous disease and may be especially pronounced around nevi, suggesting the presence of an underlying inflammatory response against melanocytes [25-27]. The onset of vitiligo-like lesions during Ipilimumab treatment is also a finding related to an immune activation status of the host, and was a relatively common event in our experience [personal unpublished data], as well as in other case reports [28].

From a histological point of view, skin biopsies of Ipilimumab-related skin lesions showed a perivascular inflammatory infiltrate in the superficial dermis that extend to the epidermis with CD4+ and CD8+ T cells that are CD3+. Eosinophils may also be present [24-27].

## **4. Vemurafenib toxicities**

Even if Vemurafenib is generally a safe and well-tolerated drug, a wide spectrum of toxic effects has been described [14-18, 29, 30]. The most common one is represented by arthralgia, which occurs in about 60% of patients. Most cases are mild to moderate, but about 5% of patients treated with Vemurafenib experienced a grade 3 arthralgia [14, 29]. These latter cases can be managed conservatively with non-steroidal anti-inflammatory drugs and acetamino‐ phen; however, severe cases may require a short course of steroid treatment [31]

**Figure 1.** Erythemato-edematous rash in a patient treated with Ipilimumab

Liver enzymes should be monitored, since elevated liver enzymes have been documented in about 20% of treated patients;, usually resolving with a treatment suspension [14, 29, 31].

Only a relatively small percentage of patients (approximately 20%) experience nausea, which is normally responsive to oral antiemetics [31].

Quite unusual but potentially life-threatening side effects are related to alterations of the cardiac rhythm, including the prolongation of cardiac repolarization and arrhythmia, which occurs in about 8% of patients. Hence, the monitoring of cardiac activity is mandatory in all Vemurafenib-treated patients; however, the occurrence of absolute QTc values >500 ms [29, 31, 32] that require prompt clinical management is quite rare.

## **5. Cutaneous side effects from Vemurafenib**

Cutaneous reactions are the most common side effects described during Vemurafenib treatment and impact significantly on patient's quality of life [15-17, 29]. As expected from the experience collected with other molecular targeted therapies, skin toxicity is related to the alteration of the wild-type BRAF signaling [18].

The cutaneous adverse reactions affect a percentage of treated patients from 75 to 90%, without difference of age and sex and can be classified according to the reaction pattern and time of appearance as follows:

#### **5.1. Rash**

From a histological point of view, skin biopsies of Ipilimumab-related skin lesions showed a perivascular inflammatory infiltrate in the superficial dermis that extend to the epidermis with

Even if Vemurafenib is generally a safe and well-tolerated drug, a wide spectrum of toxic effects has been described [14-18, 29, 30]. The most common one is represented by arthralgia, which occurs in about 60% of patients. Most cases are mild to moderate, but about 5% of patients treated with Vemurafenib experienced a grade 3 arthralgia [14, 29]. These latter cases can be managed conservatively with non-steroidal anti-inflammatory drugs and acetamino‐

CD4+ and CD8+ T cells that are CD3+. Eosinophils may also be present [24-27].

phen; however, severe cases may require a short course of steroid treatment [31]

**Figure 1.** Erythemato-edematous rash in a patient treated with Ipilimumab

Liver enzymes should be monitored, since elevated liver enzymes have been documented in about 20% of treated patients;, usually resolving with a treatment suspension [14, 29, 31].

**4. Vemurafenib toxicities**

284 Melanoma – Current Clinical Management and Future Therapeutics

Literature data report the onset of a maculo-papular eruption in about 50% of treated patients [14, 16, 18, 29]. Also in our experience, rash was the earliest and most frequent cutaneous side effect during Vemurafenib treatment (48% of treated patients of our series). From a clinical point of view, this rash could be similar to other drug-related exanthemas and it is character‐ ized by the onset of maculae and follicular papules mainly distributed on the trunk and limbs (Figure 2); the head region is generally spared. Rush appears after a median time of 11 days (range 7-55 days), it is generally self-limiting and spontaneously resolve after a median of 18 days from the onset (range 2-95). In the majority of cases, it is asymptomatic, even if some patients reported pruritus [personal unpublished data].

Cases that underwent skin biopsy show an inflammatory lympho-histiocytic lichenoid infiltrate, even if keratinocytes activation should be observed [18]. The origin of this maculopapular eruption is still unclear; however these features can explain the usefulness of topical steroids, as well as the anecdotic finding that rush did not occurred in patients receiving concomitant steroids for medical treatments related to other diseases.

Because of the self-limiting nature of this side effect, we recommend the routinely use of topical emollients; steroids should be limited to symptomatic cases. Patients also have to be informed about the frequency and benignity of this rash; however, persistent or clinically atypical exanthemas should be referred to an experienced dermatologist to avoid the risk of Steven-Johnson Syndrome /Toxic Epidermal Necrolysis (SJS/TEN) [33, 34].

**Figure 2.** Maculae and follicular papules distributed on limbs in a patients treated with Vemurafenib

#### **5.2. Warts**

Viral warts represent the second most frequent cutaneous side effect of Vemurafenib [15-17] which affects about 41% of patients in our experience; median time of onset is 50 days from the initiation of treatment. Warts affect mainly the regions of the head and neck, less frequently the trunk and limbs (Figure 3). In our casistics, viral warts were the first cutaneous side effect in 13.7% of patients [unpublished data].

Histologically, Vemurafenib-dependent warts were indistinguishable from common viral warts [15, 19]; standard wart treatments (e.g. cryosurgery, keratolytic solutions, and diather‐ mic coagulation) usually are very effective.

#### **5.3. Hyperkeratosis**

In a percentage of Vemurafenib-treated patients, the induction of a keratinocytic hyperprolif‐ eration without signs of apoptosis results in an increased epidermal thickness [28]. Plantar hyperkeratosis occurs mainly in areas under physical pressure, whereas diffuse hyperkeratotic follicular papules are observed mainly in the lower limbs and forearms (Figure 4). In our

**Figure 3.** Disseminated viral warts in a patient treated with Vemurafenib

experience, the median onset time of localized and diffuse hyperkeratosis is 44 and 31 days, respectively.

Topical keratolytic and emollient treatment can reduce hyperkeratosis, even if a complete resolution of this side effect was observed only after Vemurafenib discontinuation.

**Figure 4.** Plantar hyperkeratosis occurred in areas under physical pressure during Vemurafenib treatment

#### **5.4. Photosensitivity**

**Figure 2.** Maculae and follicular papules distributed on limbs in a patients treated with Vemurafenib

Viral warts represent the second most frequent cutaneous side effect of Vemurafenib [15-17] which affects about 41% of patients in our experience; median time of onset is 50 days from the initiation of treatment. Warts affect mainly the regions of the head and neck, less frequently the trunk and limbs (Figure 3). In our casistics, viral warts were the first cutaneous side effect

Histologically, Vemurafenib-dependent warts were indistinguishable from common viral warts [15, 19]; standard wart treatments (e.g. cryosurgery, keratolytic solutions, and diather‐

In a percentage of Vemurafenib-treated patients, the induction of a keratinocytic hyperprolif‐ eration without signs of apoptosis results in an increased epidermal thickness [28]. Plantar hyperkeratosis occurs mainly in areas under physical pressure, whereas diffuse hyperkeratotic follicular papules are observed mainly in the lower limbs and forearms (Figure 4). In our

**5.2. Warts**

**5.3. Hyperkeratosis**

in 13.7% of patients [unpublished data].

286 Melanoma – Current Clinical Management and Future Therapeutics

mic coagulation) usually are very effective.

Photosensitivity is another common phenomenon in Vemurafenib-treated patients. Even if this side effect does not represent a life-threatening condition, it can impact on patients' quality of life and could be difficult to manage [14, 17, 29, 30]. Painful sunburns are an early phenom‐ enon and may occur after few minutes of sun exposure; UVA seems to play a more prominent role than UVB. In our experience, sunburns were observed in 14% of patients, also after a few days of treatment (Figure 5). Patient phototype and intensity of sun exposure can concur in the onset of this phenomenon. In our series, all patients who developed sunburns during Vemurafenib treatment showed a photo type II [personal unpublished data].

Sun protection is mandatory in Vemurafenib-treated patients, and should be started together with BRAF inhibitor.

F**igure 5:** Painful sunburns developed after a few days of Vemurafenib treatment

Actinic conjunctivitis is also described as an early as well as a very late side effect.

**Figure 5.** Painful sunburns developed after a few days of Vemurafenib treatment

#### *Effluvium and hair changes*  **5.5. Effluvium and hair changes**

In our experience, effluvium occur in 17% of patient, after a median time of 88 days, usually without complete hair loss. Moreover, some patients experience a curling and ticking of the hair. Hair changes belong to the late onset side effects. All these phenomena could be explained by a paradoxical up-regulation of MAPK signaling [35-37]. In our experience, effluvium occur in 17% of patient, after a median time of 88 days, usually without complete hair loss. Moreover, some patients experience a curling and ticking of the hair. Hair changes belong to the late onset side effects. All these phenomena could be explained by a paradoxical up-regulation of MAPK signaling [35-37].

#### **5.6. Hands oedema and urticaria**

13

*Hands oedema and urticaria*  A less frequent skin toxicity is represented by localized hand oedema, that developed in a few patients as an early side effect, usually within a month from the beginning of the treatment, in the absence of other signs of localized or diffuse oedema. In these patients, laboratory tests did not show renal toxicity or hypoalbuminemia. Morevover, cases of urticarial episodes during the Vemurafenib treatment are described, particularly in patients with a personal history of atopia; normally, these episodes spontaneously resolve without drug suspension, and, hence, the relationship between Vemurafenib and urticaria remains to be ascertained [15-17, 29].

#### **5.7. Skin cancer**

Sun protection is mandatory in Vemurafenib-treated patients, and should be started together

F**igure 5:** Painful sunburns developed after a few days of Vemurafenib treatment

these phenomena could be explained by a paradoxical up-regulation of MAPK signaling [35-37].

In our experience, effluvium occur in 17% of patient, after a median time of 88 days, usually without complete hair loss. Moreover, some patients experience a curling and ticking of the hair. Hair changes belong to the late onset side effects. All these phenomena could be explained

A less frequent skin toxicity is represented by localized hand oedema, that developed in a few patients as an early side effect, usually within a month from the beginning of the treatment, in

Actinic conjunctivitis is also described as an early as well as a very late side effect.

with BRAF inhibitor.

288 Melanoma – Current Clinical Management and Future Therapeutics

13

*Effluvium and hair changes* 

**5.5. Effluvium and hair changes**

**5.6. Hands oedema and urticaria**

**Figure 5.** Painful sunburns developed after a few days of Vemurafenib treatment

by a paradoxical up-regulation of MAPK signaling [35-37].

*Hands oedema and urticaria* 

The first reports of skin toxicity obtained from phase I-III clinical trials and expanded access studies showed the onset of squamous cell carcinomas (SCCs) in up to 31% of Vemurafenibtreated patients [30]. However, a pathology review of all lesions excised in phase II study revealed that 90% of reported SCCs were keratoacanthomas and the remaining 10%, welldifferentiated squamous cell carcinomas. More recent reports stated that incidence of SCCs and keratoacanthomas is about 14-18%, respectively [18].

Literature data hypothesize that keratoacanthomas and SCCs develop as a consequence of preexisting precancerous RAS mutations in keratinocytes of sun-exposed areas that are then activated by Vemurafenib through a paradoxical up-regulation of MAPK signaling [30, 35]. This mechanism could explain the keratinocytes proliferation that leads to keratosis pilarislike lesions, palmo-plantar hyperkeratosis and hair changes in Vemurafenib-treated patients; notably, the same side effects are also observed during treatment with sorafenib and MEK inhibitors. Along this line, chemoprevention of cutaneous SCCs by the subministration of systemic retinoids has been reported to be successful in Vemurafenib-treated [28]. In our experience, also topical retinoid can significantly reduce the hyperkeratosis, with lower side effects (unpublished data).

## **6. Toxicity profiles of emerging BRAF inhibitors**

The second-generation BRAFV600 inhibitor Dabrafenib has an acceptable safety profile. The percentage of patients that experience treatment-related side toxicities is lower respect to Vemurafenib and drug-related adverse events of grade ≥2 occur in about 5% of patients.

In our experience, effluvium occur in 17% of patient, after a median time of 88 days, usually without complete hair loss. Moreover, some patients experience a curling and ticking of the hair. Hair changes belong to the late onset side effects. All Clinical trials with Dabrafenib and Vemurafenib show several differences in type, grade and frequencies of toxicities [15-18, 38, 39]. Cutaneous toxicities such as rash, hyperkeratosis and the development of non-melanoma skin cancers are less frequent in Dabrafenib-treated patients than inr those treated with Vemurafenib. In particular, skin carcinomas occur in 19% of patients treated with Vemurafenib as opposed to 5% during treatment with dabrafenib. Patients included in the phase I and II trials with Dabrafenib do not experienced photosensi‐ tivity, which could therefore be considered a Vemurafenib-specific toxicity.

> Non-cutaneous toxicities such as arthralgia and fatigue also occur at an increased rate and grade for patients treated with Vemurafenib, whereas pyrexia is a specific toxicity seen with dabrafenib. The mechanisms underlying Dabrafenib-associated pyrexia are poorly under‐ stood and require further investigation. However, this condition can be successfully treated

with steroids. No patient included in clinical trials with Dabrafenib experience liver toxicity [38, 39].

The increased incidence of high class toxicities scored with Vemurafenib than with Dabrafenib is likely to be explained by a number of factors, including differences in drug dosage (the administered dose for Dabrafenib is lower than for Vemurafenib), RAF inhibitor potency, histopathologic assessment of cutaneous lesions, classification and reporting of toxicity. Moreover, the differences in phototype and exposure to exogenous risk factors for skin carcinomas of the different geographic populations enrolled in these studies could also play an important role.

## **7. Conclusions**

The efficacy of new drugs for the treatment of metastatic melanoma is accompanied by a new spectrum of toxicities, very different from those caused by conventional chemotherapy, but not less important. Therefore, it is crucial that clinicians develop the necessary skills for the early detection and management of these toxicities, in order to limit the need of interruption or suspension of these treatment and to offer the best chance of disease control.

## **Author details**

Paola Savoia\* and Paolo Fava

\*Address all correspondence to: paola.savoia@unito.it

Department of Medical Science, University of Turin, Italy

### **References**


[4] Atkins, M.B., Lotze, M.T., Dutcher, et al. High-dose recombinant interleukin 2 thera‐ py for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and 1993. J Clin. Oncol 1999; 17: 2105-16.

with steroids. No patient included in clinical trials with Dabrafenib experience liver toxicity

The increased incidence of high class toxicities scored with Vemurafenib than with Dabrafenib is likely to be explained by a number of factors, including differences in drug dosage (the administered dose for Dabrafenib is lower than for Vemurafenib), RAF inhibitor potency, histopathologic assessment of cutaneous lesions, classification and reporting of toxicity. Moreover, the differences in phototype and exposure to exogenous risk factors for skin carcinomas of the different geographic populations enrolled in these studies could also play

The efficacy of new drugs for the treatment of metastatic melanoma is accompanied by a new spectrum of toxicities, very different from those caused by conventional chemotherapy, but not less important. Therefore, it is crucial that clinicians develop the necessary skills for the early detection and management of these toxicities, in order to limit the need of interruption

[1] Chapman, P.B., Einhorn, L.H., Meyers, M.L., Saxman, S., Destro, A.N., Panageas, K.S., Begg, C.B., Agarwala, S.S., Schuchter, L.M., Ernstoff, M.S., Houghton, A.N., Kirkwood, J.M. Phase III multicenter randomized trial of the Dartmouth regimen versus dacarbazine in patients with metastatic melanoma. J. Clin. Oncol. 1999; 17,

[2] Tarhini, A.A., Agarwala, S.S. Cutaneous melanoma: available therapy for metastatic

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[38, 39].

an important role.

**7. Conclusions**

**Author details**

and Paolo Fava

290 Melanoma – Current Clinical Management and Future Therapeutics

\*Address all correspondence to: paola.savoia@unito.it

disease. Dermatol. Ther. 2006; 19: 19–25

Department of Medical Science, University of Turin, Italy

Paola Savoia\*

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[21] Ascierto P, Simeone E, Chiarion Sileni V et al. Clinical experience with Ipilimumab 3 mg/kg: real-world efficacy and safety data from an expanded access programme co‐

[22] Lacouture, M, Wolchok J, Yosipovitch G, et al. Ipilimumab in patients with cancer and thecmanagement of dermatologic adverse events. J Am Acad Dermatol 2014;

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[26] Klein O, Ebert LM, Nicholaou T, et al. Melan-A-specific cytotoxic T cells are associat‐ ed with tumor regression and autoimmunity following treatment with anti-CTLA-4.

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**Emerging Research on Melanoma**

## **Autotaxin – An Enzymatic Augmenter of Malignant Progression Linked to Inflammation**

David N. Brindley, Matthew G.K. Benesch and Mandi M. Murph

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59013

## **1. Introduction**

Malignant melanoma cells are incredibly hardy, stemming from their intrinsic defensive nature. These cells inherit unique characteristics, which allowed their non-malignant prede‐ cessors, melanocytes, to survive solar ultraviolet radiation and simultaneously provide protection to neighboring cells. Most other cell types would die after such harsh exposure. Unsurprisingly, melanoma cells, which survive ultraviolet radiation, are intrinsically resistant to most chemotherapy.

Although there are numerous molecular reasons for this reality, herein we focus on the causal role of autotaxin (ATX) in melanoma. ATX is a 125 kDa glycoprotein enzyme that was initially discovered in the serum-free medium of A2058 human melanoma cells by Stracke et al. in 1992 [1] (Figure 1). Today, we know much more about this glycoprotein enzyme and how it affects melanoma. Indeed, ATX is highly overexpressed among primary melanomas and metastatic melanomas, in comparison to melanoma *in situ* and in normal skin [2].

In fact, "autotaxin" derived its name based on its initial property as an "autocrine motility factor". The rationale is that A2058 melanoma cells secrete ATX into culture medium and then respond to it with self-stimulated random and directed motility. Even though the amount of ATX in conditioned medium is less than 0.005% of total protein, only miniscule amounts, detected in picomolar and nanomolar concentrations, are needed to promote motility. [1] This suggests a large role for a low-abundant secreted enzyme.

There are five alternatively-spliced isoforms of ATX that are catalytically active [3,4]. The original ATX protein described in 1992 is termed ATXα, whereas the most abundant isoform is ATXβ and is the same isoform responsible for plasma lysoPLD activity [5]. Full length ATX

is synthesized as a pre-proenzyme and is secreted by the classical secretory pathway [6,7]. Secreted ATX binds to cell surface integrin or heparan sulfates through its somatomedin-Blike (SMB) domain. This surface binding is believed to localize LPA production adjacent to LPA receptors [8-12].

**Figure 1. Major milestones in autotaxin (ATX) research.** After the discovery of ATX in the conditioned medium of melanoma cells, it was not until a decade later that the enzyme was connected to lysophosphatidate. The elucidation of the crystal structure by two different groups caused a surge of novel inhibitors.

## **2. Multiple functions of ATX**

In addition to the ability of ATX to stimulate motility, ATX also has ATPase, phosphodiesterase and ATP pyrophosphatase activities [13]. In other words, ATX releases nucleoside-5'-mono‐ phosphates from phosphodiester and pyrophosphate bonds [14], which is why it belongs to the family of ENPP (ectonucleotide pyrophosphatase/ phosphodiesterase) enzymes with ENPP1/PC-1, ENPP3/B10 [15], ENPP4 [14,16], ENPP6 [17], ENPP7 [18], and is also named ENPP2 (Table 1). Interestingly, all of the phosphodiesterase catalytic activity of ATX resides in one amino acid, threonine 210. Losing this phosphorylatable residue in the catalytic site results in a loss of phosphodiesterase activity and motility, but not ATP binding [19]. Theo‐ retically, GTP, NAD, FAD, AMP and PPi are all susceptible to hydrolysis by ATX. However, the preferred substrate for ATX is lysophosphatidylcholine (LPC), which it converts to lysophosphatidate (LPA) (Figure 2). Since LPC concentrations in human plasma are greater than 200 μM, this should outcompete the hydrolysis of nucleotide phosphates and pyrophos‐ phate, which are present in much lower concentrations.


is synthesized as a pre-proenzyme and is secreted by the classical secretory pathway [6,7]. Secreted ATX binds to cell surface integrin or heparan sulfates through its somatomedin-Blike (SMB) domain. This surface binding is believed to localize LPA production adjacent to

**Figure 1. Major milestones in autotaxin (ATX) research.** After the discovery of ATX in the conditioned medium of melanoma cells, it was not until a decade later that the enzyme was connected to lysophosphatidate. The elucidation of

In addition to the ability of ATX to stimulate motility, ATX also has ATPase, phosphodiesterase and ATP pyrophosphatase activities [13]. In other words, ATX releases nucleoside-5'-mono‐ phosphates from phosphodiester and pyrophosphate bonds [14], which is why it belongs to the family of ENPP (ectonucleotide pyrophosphatase/ phosphodiesterase) enzymes with ENPP1/PC-1, ENPP3/B10 [15], ENPP4 [14,16], ENPP6 [17], ENPP7 [18], and is also named ENPP2 (Table 1). Interestingly, all of the phosphodiesterase catalytic activity of ATX resides in one amino acid, threonine 210. Losing this phosphorylatable residue in the catalytic site results in a loss of phosphodiesterase activity and motility, but not ATP binding [19]. Theo‐ retically, GTP, NAD, FAD, AMP and PPi are all susceptible to hydrolysis by ATX. However, the preferred substrate for ATX is lysophosphatidylcholine (LPC), which it converts to lysophosphatidate (LPA) (Figure 2). Since LPC concentrations in human plasma are greater than 200 μM, this should outcompete the hydrolysis of nucleotide phosphates and pyrophos‐

the crystal structure by two different groups caused a surge of novel inhibitors.

phate, which are present in much lower concentrations.

**2. Multiple functions of ATX**

LPA receptors [8-12].

298 Melanoma – Current Clinical Management and Future Therapeutics

**Table 1.** Comparison between enzymes among the Ectonucleotide Pyrophosphatase/ Phosphodiesterase (ENPP) family

**Figure 2. Autotaxin (ATX) catalyzes the biosynthesis of lysophosphatidiate (LPA).** Since the lysophosphatidylcholine (LPC) concentrations in human circulation are >200 μM, ATX has abundantly available lipids to produce LPA. ATX hydrolyzes the choline head group from LPC to yield LPA. As a consequence of LPA signaling on cells, a multitude of various responses result, including motility, viability, growth, proliferation and contraction.

The ability of ATX to stimulate motility in melanoma cells can be modulated by chemical methods and genetic engineering. For example, ATX-meditated motility is inhibited by the PI3K inhibitors, wortmannin and LY294002, along with the catalytically inactive mutant of PI3K, PI3KK832R [20]. In addition, *ras-*transformed NIH3T3 cells become more motile and invasive in the presence of ATX. These cells also show enhanced colony formation in soft agar *in vitro* and produce significantly larger and metastatic tumors *in vivo* [21]. This suggests that while ATX alone may be insufficient to transform cells to a malignant phenotype, the presence of ATX significantly enhances malignant transformation among 'primed' cells.

## **3. Role as the main enzyme for the generation of extracellular LPA**

The majority of LPA in the circulation is generated by ATX from the abundant LPC (>200 μM in human plasma) in the circulation. In fact, LPC is the major plasma phospholipid and it is bound to albumin [22]. Extracellular LPC is derived from two major routes of metabolism. The first is through the action of lecithin:cholesterol acyltransferase, which is present in plasma high-density lipoproteins. Lecithin:cholesterol acyltransferase, preferentially transfers unsaturated fatty acids from postion-2 of phosphatidylcholine to cholesterol, producing cholesterol ester and a mainly saturated LPC. However, a large proportion of circulating LPC is polyunsaturated [22] and this indicates another route for the production of extracellular LPC. Part of the polyunsaturated LPC is derived from secretion by hepatocytes, but it is possible that other cell types could produce polyunsaturated LPC. Since hepatocytes secrete a large quantity of arachidonoyl-LPC [22-26], it was originally postulated that this might represent a novel transport system for delivering choline and polyunsaturated fatty acids to the brain [22]. Although, this could still be true, we now know that LPC is an important substrate for ATX and that this is the major route for the production of extracellular LPA [27].

This predominant role of ATX in generating LPA is confirmed by circulating LPA concentra‐ tions that are 50% of normal levels among ATX heterozygous mice for a null-mutation for ATX [28,29]. Also, ATX inhibition produces a rapid decrease in plasma LPA of >95% [30-32]. The effects of ATX inhibition are more dramatic for the unsaturated species [33]. This is compatible with the substrate preference of ATX for unsaturated and polyunsaturated LPCs [34]. The crystal structures of ATX:LPA complexes show a hydrophobic pocket in the catalytic domain that is slightly U-shaped. This accommodates the kinked acyl chains of unsaturated fatty acids better than the linear conformations of saturated fatty acids [34].

Even though ATX is the major enzyme that generates LPA, other enzymes have a minor role in its biosynthesis. For example, saturated LPA species can also be derived through secretory phospholipase A2, which hydrolyzes phosphatidate in microvesicles that are shed from cells during inflammation [35] and platelet aggregation [36]. In addition, LPA production by the Group VIA phospholipase A2 (Ca2+-independent) appears to be involved in the development of prostate cancers [37].

## **4. Crystal structure of ATX**

The elucidation of the crystal structure of mouse and rat ATX revealed that the enzyme is composed of four domains [9,34]. The most important among these domains is the slightly Ushaped catalytic domain, which contains the enzyme's active site, a hydrophobic pocket and hydrophobic channel. Although ATX can hydrolyze both nucleotides and lysophospholipid substrates, when the unique hydrophobic pocket engages lysophospholipids, nucleotides are unable to bind to the active site. In addition, lipid acyl chains form further interactions with this pocket that nucleotides do not, which is also why ATX has a higher affinity for lysophos‐ pholipids [38]. The unique shape of the catalytic domain also accommodates the kinked acyl chains of unsaturated fatty acids better than the linear conformations of saturated fatty acids [34], which further explains substrate specificity for ATX.

ATX is the only member of the ENPP enzyme family that contains a hydrophobic pocket for lysophospholipids, thus giving it a unique capability over other ENPPs. Interestingly, one amino acid, asparagine 230, is required for ATX to recognize phosphate moieties and produce lysophosphatidate. Mutating asparagine to alanine inhibited all production. [34] Other singlepoint mutations deep within the hydrophobic pocket are capable of altering binding selectivity and reducing some of ATX's activity [9].

Perhaps the most exciting biological implication arising from the structural resolution of ATX is its proposed role as a directed transporter of LPA. In other words, ATX does not appear to arbitrarily release lysophospholipids from its hydrophobic pocket and channel them into solution. Rather, ATX is hypothesized to directly transfer LPA to its receptors along the plasma membrane. The flat surface of ATX on the side of the channel entrance facing the cell presum‐ ably facilitates this purpose [34]. This insinuates a role for ATX as an indirect initiator of cell signaling through its guided transport of an agonist to its receptor. It also further explains early observations of ATX as a motility-stimulating factor, even though it was actually due to LPA.

## **5. ATX Inhibitors**

*in vitro* and produce significantly larger and metastatic tumors *in vivo* [21]. This suggests that while ATX alone may be insufficient to transform cells to a malignant phenotype, the presence

The majority of LPA in the circulation is generated by ATX from the abundant LPC (>200 μM in human plasma) in the circulation. In fact, LPC is the major plasma phospholipid and it is bound to albumin [22]. Extracellular LPC is derived from two major routes of metabolism. The first is through the action of lecithin:cholesterol acyltransferase, which is present in plasma high-density lipoproteins. Lecithin:cholesterol acyltransferase, preferentially transfers unsaturated fatty acids from postion-2 of phosphatidylcholine to cholesterol, producing cholesterol ester and a mainly saturated LPC. However, a large proportion of circulating LPC is polyunsaturated [22] and this indicates another route for the production of extracellular LPC. Part of the polyunsaturated LPC is derived from secretion by hepatocytes, but it is possible that other cell types could produce polyunsaturated LPC. Since hepatocytes secrete a large quantity of arachidonoyl-LPC [22-26], it was originally postulated that this might represent a novel transport system for delivering choline and polyunsaturated fatty acids to the brain [22]. Although, this could still be true, we now know that LPC is an important substrate for ATX and that this is the major route for the production of extracellular LPA [27]. This predominant role of ATX in generating LPA is confirmed by circulating LPA concentra‐ tions that are 50% of normal levels among ATX heterozygous mice for a null-mutation for ATX [28,29]. Also, ATX inhibition produces a rapid decrease in plasma LPA of >95% [30-32]. The effects of ATX inhibition are more dramatic for the unsaturated species [33]. This is compatible with the substrate preference of ATX for unsaturated and polyunsaturated LPCs [34]. The crystal structures of ATX:LPA complexes show a hydrophobic pocket in the catalytic domain that is slightly U-shaped. This accommodates the kinked acyl chains of unsaturated fatty acids

Even though ATX is the major enzyme that generates LPA, other enzymes have a minor role in its biosynthesis. For example, saturated LPA species can also be derived through secretory phospholipase A2, which hydrolyzes phosphatidate in microvesicles that are shed from cells during inflammation [35] and platelet aggregation [36]. In addition, LPA production by the Group VIA phospholipase A2 (Ca2+-independent) appears to be involved in the development

The elucidation of the crystal structure of mouse and rat ATX revealed that the enzyme is composed of four domains [9,34]. The most important among these domains is the slightly Ushaped catalytic domain, which contains the enzyme's active site, a hydrophobic pocket and

of ATX significantly enhances malignant transformation among 'primed' cells.

300 Melanoma – Current Clinical Management and Future Therapeutics

better than the linear conformations of saturated fatty acids [34].

of prostate cancers [37].

**4. Crystal structure of ATX**

**3. Role as the main enzyme for the generation of extracellular LPA**

ATX, as an extracellular enzyme, is a very attractive drug candidate for reducing the abun‐ dance of extracellular LPA and subsequent signaling. Indeed, even a transient knockdown of ATX using siRNA is sufficient to significantly reduce melanoma cell viability [2]. Furthermore, the advantage of inhibiting ATX is the attenuation of signaling by all LPA receptors. This is considerably advantageous over the use of individual receptor antagonists because there are at least six and possibly eight confirmed LPA receptors with extensive redundancy. The design of ATX inhibitors has been reviewed previously [39-41] and so we will focus only those inhibitors that show utility *in vivo*.

Among the first ATX inhibitors was L-histidine, which was reported to have an ATX binding affinity (K*<sup>i</sup>* ) of about 1 mM *in vitro*, with 10 mM concentrations required to block ATXstimulated migration in melanoma cells by 90% [42]. L-histidine inhibits ATX non-competi‐ tively by chelating cations such as Zn2+, which are required for optimum catalytic rates [42]. Later work showed L-histidine administered intraperitoneally to rats limits thioacetamideinduced liver fibrosis [43]. Although L-histidine is required mM concentrations for activity *in vitro*, the authors cited its importance because it was the first "proof-of-principle" that inhibiting ATX was achievable.

Few studies have focused on inhibiting ATX transcription. However, one study showed that cholera toxin inhibits the proliferation of human hepatocellular carcinoma cells *in vitro* by suppressingtheproductionofATXthroughaTNF-α-dependentmechanism[44].Cholera toxin increases the expression of anti-inflammatory cytokines (IL-4 and IL-10), which suppress ATX mRNA levels [45]. Knockdown of ATXexpression with siRNA decreased the effects of choleratoxin in suppressing the growth of Hep3B and Huh7 HCC cells [44]. Also, oral administration of the anti-inflammatory steroid, prednisolone, decreased serum ATX levels in a dose-depend‐ ent manner in patients treated for autoimmune skin diseases [46]. It is uncertain if suppress‐ ing ATX activity contributed to symptom relief since tapering the prednisolone dosage caused a rebound in serum ATX levels independently of disease severity. Significantly, the authors proposed that serum ATX levels could be used as a marker of compliance and/or efficacy of steroid therapy [46]. In addition, rifampicin, which is an inhibitor of DNA-dependent RNA polymerase, can be used to lower ATX mRNA transcription in the treatment of cholestatic pruritus. Cessation of rifampicin treatment led to both reoccurrence of pruritus and a re‐ boundofATXlevels topretreatmentlevels [47].All ofthe treatment options fordecreasingATX expression appear to be effective in only specific diseases. By contrast, ATX inhibitors are designed to block LPA production and therefore this approach should have greater utility in multiple pathologies.

Major criteria for the efficacy of any therapeutic agent is a favorable therapeutic index and good bioavailability and potency. Reports on competitive ATX inhibitors began around 2006. These consisted of carba analogs of cyclic phosphatidate (cPA) with a K*<sup>i</sup>* of approximately 100 nM (Table 2). These compounds inhibited metastasis of melanoma cells, which were injected in the tail vein in mouse models [48] and they inhibited chronic inflammation-induced C-fiber stimulation in rat neuropathic pain models [49]. However, cyclic phosphatidate analogs are also weak agonists of LPA receptors, which limits their utility [39].


**Table 2.** ATX inhibitors used to effect a therapeutic response *in vivo*

A series of lipid-mimetic ATX inhibitors were developed in 2009 based on α-bromophospho‐ nates (BrP-LPA). The *anti*-BrP-LPA isomer had IC50 of 22 nM in plasma, which was more potent than for *syn*-BrP-LPA [50] (Table 2). BrP-LPA is also a pan-antagonist of LPA1-3, which is effective in decreasing to tumor growth in orthotopic xenograft models of breast cancer using MDA-MB-231 cells [50] and in metastasis of A549 lung cancer cells in nude mice [51]. We have also previously demonstrated that BrP-LPA is more effective than dacarbazine in reducing the viability of melanoma cells [59].

Few studies have focused on inhibiting ATX transcription. However, one study showed that cholera toxin inhibits the proliferation of human hepatocellular carcinoma cells *in vitro* by suppressingtheproductionofATXthroughaTNF-α-dependentmechanism[44].Cholera toxin increases the expression of anti-inflammatory cytokines (IL-4 and IL-10), which suppress ATX mRNA levels [45]. Knockdown of ATXexpression with siRNA decreased the effects of choleratoxin in suppressing the growth of Hep3B and Huh7 HCC cells [44]. Also, oral administration of the anti-inflammatory steroid, prednisolone, decreased serum ATX levels in a dose-depend‐ ent manner in patients treated for autoimmune skin diseases [46]. It is uncertain if suppress‐ ing ATX activity contributed to symptom relief since tapering the prednisolone dosage caused a rebound in serum ATX levels independently of disease severity. Significantly, the authors proposed that serum ATX levels could be used as a marker of compliance and/or efficacy of steroid therapy [46]. In addition, rifampicin, which is an inhibitor of DNA-dependent RNA polymerase, can be used to lower ATX mRNA transcription in the treatment of cholestatic pruritus. Cessation of rifampicin treatment led to both reoccurrence of pruritus and a re‐ boundofATXlevels topretreatmentlevels [47].All ofthe treatment options fordecreasingATX expression appear to be effective in only specific diseases. By contrast, ATX inhibitors are designed to block LPA production and therefore this approach should have greater utility in

Major criteria for the efficacy of any therapeutic agent is a favorable therapeutic index and good bioavailability and potency. Reports on competitive ATX inhibitors began around 2006. These consisted of carba analogs of cyclic phosphatidate (cPA) with a K*<sup>i</sup>* of approximately 100 nM (Table 2). These compounds inhibited metastasis of melanoma cells, which were injected in the tail vein in mouse models [48] and they inhibited chronic inflammation-induced C-fiber stimulation in rat neuropathic pain models [49]. However, cyclic phosphatidate analogs are

cPA Lipid • inhibits B16F10 melanoma metastasis in mouse tail vein model [48]

BrP-LPA Lipid • reduces MDA-MB-231 orthotopic breast tumor growth in mice [50]

PF-8380 Non-Lipid • inhibits inflammatory hyperalgesia in rat air-pouch models [30]

ONO-8430506 Non-Lipid • reduces tumor growth and metastasis in 4T1/Balb/c syngeneic and

• inhibits C-fiber stimulation by chronic inflammation in rat neuropathic pain

• inhibits A549 lung metastasis in engineered 3D mouse xenografts [51]

• radiosensitizes glioblastoma multiforme heteotropic mouse models [56]

orthotopic mouse model and is synergistic with doxorubicin [33,57] • reduces urethral tension in rat benign prostatic hyperplasia models [58]

• radiosensitizes GL-261 mouse glioma models [52] reduces collagen-induced arthritis in mice [53] GWJ-A-23 Lipid • reduces allergen-induced asthmatic phenotype in ATX-transgenic mice [54]

• reduces fibrosis in bleomycin-treated mice [55]

also weak agonists of LPA receptors, which limits their utility [39].

models [49]

**Compound Class** *In vivo therapeutic effects*

302 Melanoma – Current Clinical Management and Future Therapeutics

**Table 2.** ATX inhibitors used to effect a therapeutic response *in vivo*

multiple pathologies.

In addition to its efficacy against cancer cells, BrP-LPA had a radio-sensitizing effect on the tumor vasculature and delayed tumor growth by 7 days compared to radiation alone in a heterotopic murine glioma model using GL-261 cells [52]. This study was one of the first to demonstrate that ATX inhibitors can potential be used as an adjuvant therapy for cancer. A later report showed that BrP-LPA attenuated disease symptoms by diminishing synovial LPA signaling in a collagen-induced arthritis mouse model. Histological analysis of the joints showed a marked decrease in inflammation and synovial hyperplasia [53].

Several lipid-analogs of LPC, which have been used as ATX inhibitors, have relatively poor bioavailability, mainly because of the hydrophobic acyl tail [41]. This is illustrated by S32826, which is a benzyl phosphonic acid derivative (Figure 3). Despite having a very low nM IC50 *in vitro*, the long chain contributes to a very high lipophilicity and it cannot suppress circulating ATX activity for more than a few minutes [32]. Analogs of S32826, such as GWJ-A-23, were developed by shortening the hydrophobic chain and adding α-halo-or α-hydroxy-substituents to increase solubility. However, most of these compounds are less potent inhibitors of ATX compared to S32826 [60], but have been used in pulmonary studies of ATX [54,55]. Hence, much of the recent effort in discovering ATX inhibitors for use *in vivo* has concentrated on compounds that are more soluble and not based on lipid analogs.

There are numerous, small, non-lipid inhibitors of ATX that have been modified to increase potency [40,41]. These inhibitors tend to have better bioavailability because of decreased hydrophobicity and they are unlikely to be rapidly degraded by endogenous hydrolytic pathways [61]. One of these is PF-8380, which is a piperazinylbenzoxazolone derivative that was developed by Pfizer from compound library screening and optimization (Table 2). PF-8380 has an IC50 of 2.8 nM against recombinant human ATX and 101 nM for ATX in human whole blood. It was the first ATX inhibitor that was reported to decrease plasma LPA levels *in vivo* for an extended period [30]. In rat air-pouch models, 30 mg/kg PF-8380 inhibited inflammatory hyperalgesia with the same efficacy as 30 mg/kg naproxen, a routinely used nonsteroidal antiinflammatory drug [30]. This dosage of PF-8380 produced a maximum decrease in LPA concentrations in plasma and also at the site of inflammation. Like BrP-LPA, PF-8380 had radio-sensitizing effects in a heterotopic mouse model of glioblastoma multiforme, delaying tumor growth by at least 20 days [52, 56]. In this study, inhibition of ATX by PF-8380 abrogated radiation-induced activation of Akt and subsequently decreased tumor vascularity and tumor growth [56]. Although we have *repeatedly* tested 30 mg/kg PF-8380 in animal models of melanoma, we have not observed a reduction of tumor growth (results not shown).

We have worked recently with another potent ATX inhibitor, which is a tetrahydrocarboline derivative (ONO-8430506) developed by Ono Pharmaceuticals Ltd. (Patent WO20120052227). Oral dosing with 10 mg/kg ONO-8430506 suppressed plasma ATX activity as measured by choline release assay in the presence of 3 mM LPC by at least 90% at 6 h after administration in mice [33]. Plasma LPA levels were suppressed, especially the unsaturated species. C16:1- LPA and C20:5-LPA remained non-detectable at 6 and 24 h after ONO-8430506 administration. ONO-8430506 decreased the initial rate of tumor growth and subsequent lung metastasis by up to 60% in a syngeneic orthotopic model of breast cancer in BALB/c mice. This was accom‐ panied by decreased concentrations of unsaturated LPA species in the breast tumors. These findings again confirm that ATX produces most of the extracellular LPA and that decreases in LPA concentrations in tissues mirror the decreases in plasma LPA levels following ATX inhibition [30, 33].

In other work, ONO-8430506 (30 mg/kg/day) decreased intra-urethral pressure and this was ascribed to urethral relaxation [58]. This work demonstrates the potential of ATX inhibition to decrease smooth muscle contraction by LPA. It also shows that ATX inhibition ameliorates urethral obstructive disease, such as benign prostatic hyperplasia.

## **6. Strategies to identify novel ATX inhibitors**

So far, the most common technique for ATX inhibitor discovery and design is to screen libraries of compounds by using assays with ATX substrates such as Fluorescent Substrate-3 (FS-3). Inhibitors are then modified to increase potency. FS-3 is a fluorogenic substrate that is a doublylabeled analogue of LPC. The fluorophore is quenched through intra-molecular energy transfer. Hydrolysis of FS-3 by ATX increases the fluorescent signal by removing the quencher [62]. The initial studies with this technique identified several inhibitors with an IC50 in the μM range [63, 64]. Later work with compounds designed from these studies developed nine pharmacophores for ATX inhibition and the results of these analyses were used to screen the National Cancer Institute's open chemical repository database to prioritize screening efforts [61]. This lead to the identification of several novel compounds with an IC50 in the high to low μM range [61, 65].

The identification of the crystal structure of ATX is also enabling structure-activity relation‐ ships to be established and more rational-design approaches towards optimizing inhibitor structures are now possible. The first study to report this approach was by Kawaguchi *et al.* who identified a thiazolone derivative from a 81,600-compound library, which inhibited ATX activity with an IC50 of 180 nM [66]. The authors proposed a series of side-chain modifications from the crystal structure of ATX complexed with this inhibitor. This led to the synthesis of a derivative compound, 3BoA, which has an IC50 of 13 nM [66]. More recent studies by Fells *et al.* combined large library screening results using the crystal structure of ATX with virtual screening tools to discover additional novel ATX inhibitors [67, 68]. This technique identified a common aromatic sulfonamide structural motif among potent inhibitors that targets the hydrophobic pocket of ATX. This accommodates the hydrocarbon tail of LPC/LPA [67, 68]. Similar techniques are expected to lead to the rapid development of ATX inhibitors for subsequent testing and development *in vivo*.

## **7. Physiological functions of ATX and LPA signaling**

### **7.1. Vasculature system**

Oral dosing with 10 mg/kg ONO-8430506 suppressed plasma ATX activity as measured by choline release assay in the presence of 3 mM LPC by at least 90% at 6 h after administration in mice [33]. Plasma LPA levels were suppressed, especially the unsaturated species. C16:1- LPA and C20:5-LPA remained non-detectable at 6 and 24 h after ONO-8430506 administration. ONO-8430506 decreased the initial rate of tumor growth and subsequent lung metastasis by up to 60% in a syngeneic orthotopic model of breast cancer in BALB/c mice. This was accom‐ panied by decreased concentrations of unsaturated LPA species in the breast tumors. These findings again confirm that ATX produces most of the extracellular LPA and that decreases in LPA concentrations in tissues mirror the decreases in plasma LPA levels following ATX

In other work, ONO-8430506 (30 mg/kg/day) decreased intra-urethral pressure and this was ascribed to urethral relaxation [58]. This work demonstrates the potential of ATX inhibition to decrease smooth muscle contraction by LPA. It also shows that ATX inhibition ameliorates

So far, the most common technique for ATX inhibitor discovery and design is to screen libraries of compounds by using assays with ATX substrates such as Fluorescent Substrate-3 (FS-3). Inhibitors are then modified to increase potency. FS-3 is a fluorogenic substrate that is a doublylabeled analogue of LPC. The fluorophore is quenched through intra-molecular energy transfer. Hydrolysis of FS-3 by ATX increases the fluorescent signal by removing the quencher [62]. The initial studies with this technique identified several inhibitors with an IC50 in the μM range [63, 64]. Later work with compounds designed from these studies developed nine pharmacophores for ATX inhibition and the results of these analyses were used to screen the National Cancer Institute's open chemical repository database to prioritize screening efforts [61]. This lead to the identification of several novel compounds with an IC50 in the high to low

The identification of the crystal structure of ATX is also enabling structure-activity relation‐ ships to be established and more rational-design approaches towards optimizing inhibitor structures are now possible. The first study to report this approach was by Kawaguchi *et al.* who identified a thiazolone derivative from a 81,600-compound library, which inhibited ATX activity with an IC50 of 180 nM [66]. The authors proposed a series of side-chain modifications from the crystal structure of ATX complexed with this inhibitor. This led to the synthesis of a derivative compound, 3BoA, which has an IC50 of 13 nM [66]. More recent studies by Fells *et al.* combined large library screening results using the crystal structure of ATX with virtual screening tools to discover additional novel ATX inhibitors [67, 68]. This technique identified a common aromatic sulfonamide structural motif among potent inhibitors that targets the hydrophobic pocket of ATX. This accommodates the hydrocarbon tail of LPC/LPA [67, 68]. Similar techniques are expected to lead to the rapid development of ATX inhibitors for

urethral obstructive disease, such as benign prostatic hyperplasia.

**6. Strategies to identify novel ATX inhibitors**

304 Melanoma – Current Clinical Management and Future Therapeutics

inhibition [30, 33].

μM range [61, 65].

subsequent testing and development *in vivo*.

Although studies *in vitro* detected no difference in the growth rate of *ras-*transformed NIH3T3 cells with or without ATX in culture, the growth rates in animals were significantly different, which suggested a role for ATX in blood vessel formation [21]. Indeed, Matrigel plugs of cells with ATX displayed extensive micro-aneurysms filled with red blood cells and more tumor cells, further corroborating this role. Furthermore, the ability of ATX to stimulate blood vessel formation in Matrigel plugs after 6 days was equivalent to VEGF, thus solidifying ATX as an angiogenesis-stimulating factor in the circulation [69].

Developmentally, the expression of ATX is indispensible. Beginning at 8.5 days, ATX expres‐ sion is detectable in the early mouse embryo, within the floor plate of the neural tube [70]. Knocking out of ATX in mice causes embryonic lethality around 9.5 – 10.5 days with embryos exhibiting open, kinky neural tubes [28,71]. Although heartbeats are detected until 10.5 days *in utero*, contractions are weak, irregular and clearly abnormal. In addition, even the yolk sacs of ATX knockout embryos are irregular with a complete absence of blood vessels. [72] The role of ATX in vascular and neural development is also observed in zebrafish [72, 73]. ATX regulates the differentiation of oligodendrocytes in the hindbrain [74] and the correct left-right asym‐ metry for normal organ morphogenesis through Wnt-dependent pathways [75]. Taken together, these results definitively prove that the absence of ATX results in severe defects of the vasculature system and embryonic lethality. Organisms cannot develop without functional ATX expression.

Other groups have explored the parallel correlation by investigating the vasculature system when ATX is overexpressed in transgenic mice. Interestingly, ATX transgenic mice have an unusual susceptibility to bleeding and impaired platelet-dependent thrombus formation after injury [10]. Further studies have confirmed an important role for ATX in platelet activation through the production of LPA [76]. In addition, ATX binds to platelet β1-and β3-integrins, which localizes ATX to the cell surface of the appropriate microenvironment [11].

## **8. Adipose tissue regulation**

An alternative, yet highly interesting function of ATX *in vivo* occurs in adipogenesis, obesity and the regulation of glucose homeostasis [77]. Foremost, ATX is expressed and secreted by adipose tissue. Its expression is also significantly upregulated during pre-adipocyte differen‐ tiation into adipocytes (adipogenesis) as well as in the adipose tissue of genetically obese diabetic mice [78]. In support of this finding, transgenic ATX mice accumulate more fat than littermates when they are fed a high-fat diet and thus, are more susceptible to diet-induced obesity [79]. Other work showed that ATX mRNA is upregulated in differentiating adipocytes but downregulated in hypertrophied adipocytes from obese mice [80]. Further, in humans, serum ATX levels and ATX mRNA expression in subcutaneous fat were negatively correlated with body mass index [80]. However this does not appear to be true if the subject is diabetic. Instead, these patients tend to have higher serum ATX levels [80]. In regards to the regulation of glucose metabolism, LPA produced by ATX is able to dose-dependently inhibit glucoseinduced insulin secretion [77, 81]. Furthermore, knocking out the expression of ATX in adipose tissue results in a mouse with improved glucose tolerance [82].

## **9. Wound healing, tissue remodeling and inflammation**

ATX and LPA facilitate critical processes necessary for skin re-epithelialization and wound healing. For example, among blister fluids, both ATX and LPA are produced and detected, originating *de novo* in the blister fluid and not from plasma. LPA is a potent activator of platelet aggregation and promotes keratinocyte migration, proliferation and differentiation. Thus, ATX and LPA facilitate critical processes necessary for skin re-epithelialization and wound healing [83]. ATX expression and LPA production are increased in rabbit aqueous humor following wounding by corneal freezing [84].

The range ofphysiologicalfunctions requiringATXisquitediverse.For example,ATXandLPA signaling are involvedinlutealtissue remodeling ofregressing corpora lutea inrat ovaries.This occurs by recruiting phagocytes and proliferating fibroblasts, which are ultimately the factors involved in remodeling [85]. Otherroles for ATX include hairfollicle morphogenesis [86], bone mineralization [87] and myeloid differentiation in human bone marrow [88].

Another unique role of ATX occurs in response to oxidative stress in microglia, whereby ATX expression is increased. This protects microglia cells from damage by H2O2 and this effect is partially reversed by the mixed LPA1/3 antagonist, Ki16425 [89]. Microglia cells overexpressing ATX show suppressed production of the pro-inflammatory cytokines, TNF-α and IL-6, and increases in the anti-inflammatory cytokine IL-10 upon treatment with lipopolysaccharide [90]. ATX is expressed in high endothelial venules in lymph nodes and other secondary lymphoid tissues [91]. This mediates lymphocyte extravasation, a process required for maintaining immune homeostasis [92, 93]. However, in chronically inflamed tissues, ATX mediates lymphocyte trafficking and increases cytokine production in response to repeated microinjuries and incomplete tissue repair [94-96].

Interestingly, the catalytic activity of ATX has a dualistic role in wound healing. In this way, an ATX-like enzyme, SMaseD, is responsible for the pathology associated with venomous poisons through its dermonecrotic and hemolytic activities [97, 98]. In other words, the aberrant over-production of LPA by ATX cultivates an inappropriate immune response, similar to a wound that never heals, whereby overabundant inflammatory cytokines and chemokines are released. The damage is manifested in several ways, including the presence of severe dermonecrosis with blackened or missing skin appearing at the wounded site. The dermonecrosis can occur after envenomation by either *Loxosceles reclusa,* the brown recluse spider [97], or *Hemiscorpius lepturus,* a venomous scorpion [98]. Intriguingly, susceptibility to SMaseD is conferred by the LPA1 receptor [99], suggesting a possible role for LPA receptor antagonists in this pathology.

As mentioned above, one of the main functions of ATX in adults is to repair damaged tissue. ATX is secreted in this situation partly in response to inflammation and the release of inflam‐ matory cytokines. In normal wound healing, the production of LPA by ATX causes cells to migrate into the area of damage to effect wound repair and the formation of new blood vessels. In cases where the inflammation is not resolved, the process can result in tissue damage and fibrosis as in rheumatoid arthritis, atherosclerosis, organ fibrosis, diabetes and even obesity [100]. Cancer can be added to this list since it has been likened to "a wound that does not heal" [101]. The role of inflammatory cytokines in tumor progression [102-106] explains why inflammatory bowel disease and viral hepatitis can progress to cancer [107].

## **10. ATX in malignancy: Metastasis and angiogenesis**

with body mass index [80]. However this does not appear to be true if the subject is diabetic. Instead, these patients tend to have higher serum ATX levels [80]. In regards to the regulation of glucose metabolism, LPA produced by ATX is able to dose-dependently inhibit glucoseinduced insulin secretion [77, 81]. Furthermore, knocking out the expression of ATX in adipose

ATX and LPA facilitate critical processes necessary for skin re-epithelialization and wound healing. For example, among blister fluids, both ATX and LPA are produced and detected, originating *de novo* in the blister fluid and not from plasma. LPA is a potent activator of platelet aggregation and promotes keratinocyte migration, proliferation and differentiation. Thus, ATX and LPA facilitate critical processes necessary for skin re-epithelialization and wound healing [83]. ATX expression and LPA production are increased in rabbit aqueous humor

The range ofphysiologicalfunctions requiringATXisquitediverse.For example,ATXandLPA signaling are involvedinlutealtissue remodeling ofregressing corpora lutea inrat ovaries.This occurs by recruiting phagocytes and proliferating fibroblasts, which are ultimately the factors involved in remodeling [85]. Otherroles for ATX include hairfollicle morphogenesis [86], bone

Another unique role of ATX occurs in response to oxidative stress in microglia, whereby ATX expression is increased. This protects microglia cells from damage by H2O2 and this effect is partially reversed by the mixed LPA1/3 antagonist, Ki16425 [89]. Microglia cells overexpressing ATX show suppressed production of the pro-inflammatory cytokines, TNF-α and IL-6, and increases in the anti-inflammatory cytokine IL-10 upon treatment with lipopolysaccharide [90]. ATX is expressed in high endothelial venules in lymph nodes and other secondary lymphoid tissues [91]. This mediates lymphocyte extravasation, a process required for maintaining immune homeostasis [92, 93]. However, in chronically inflamed tissues, ATX mediates lymphocyte trafficking and increases cytokine production in response to repeated micro-

Interestingly, the catalytic activity of ATX has a dualistic role in wound healing. In this way, an ATX-like enzyme, SMaseD, is responsible for the pathology associated with venomous poisons through its dermonecrotic and hemolytic activities [97, 98]. In other words, the aberrant over-production of LPA by ATX cultivates an inappropriate immune response, similar to a wound that never heals, whereby overabundant inflammatory cytokines and chemokines are released. The damage is manifested in several ways, including the presence of severe dermonecrosis with blackened or missing skin appearing at the wounded site. The dermonecrosis can occur after envenomation by either *Loxosceles reclusa,* the brown recluse spider [97], or *Hemiscorpius lepturus,* a venomous scorpion [98]. Intriguingly, susceptibility to SMaseD is conferred by the LPA1 receptor [99], suggesting a possible role for LPA receptor

mineralization [87] and myeloid differentiation in human bone marrow [88].

tissue results in a mouse with improved glucose tolerance [82].

306 Melanoma – Current Clinical Management and Future Therapeutics

following wounding by corneal freezing [84].

injuries and incomplete tissue repair [94-96].

antagonists in this pathology.

**9. Wound healing, tissue remodeling and inflammation**

ATX is among the top 40 upregulated genes in metastatic cancer [108] and this is explained by the effects of LPA, which signals through at least six and putatively eight G-protein-coupled receptors. Through these receptors, LPA stimulates cell motility, cell survival/viability, cell proliferation, morphological changes, contraction, wound healing and invasion [109-118]. LPA achieves these effects by signaling through the relative activations of phosphatidylinositol 3 kinase (PI3K), ERK1/2, mTOR, Ca2+-transients, Rac, Rho and Ras [119].

The involvement of ATX and LPA in tumor progression affects multiple malignant processes and stages of tumor progression. For example, LPA increases the production of vascular endothelial growth factor, which stimulates angiogenesis [51, 120], a process required for tumor growth beyond 1 mm. However, in order for tumors to arise at all, tumor suppressors must be made ineffective. LPA levels can rise to 10 μM in the ascites fluid from advanced ovarian cancer patients [121]. Interestingly, LPA also decreases the abundance of the tumor suppressor, p53 [122], thus increasing cancer cell survival and proliferation, even in the presence of actinomycin D.

Pre-clinical models of disease and clinical pathology provide insight into the role of ATX and LPA receptors in cancer. For example, transgenic multiparous mice designed to overexpress ATX, LPA1, LPA2 or LPA3 in mammary epithelium develop spontaneous metastatic mammary tumors as they age [123]. Women who express high levels of LPA3 receptors in epithelial cells, or ATX in stromal cells, have larger breast tumors, nodal involvement, and higher stages of disease [124]. Since many early stage breast cancer patients are able to be cured using current treatment modalities, this suggests that the presence of ATX and/or LPA receptors has the ability to alter outcomes of malignancy.

The involvement of ATX and LPA in tumor progression can be understood in terms of a dysfunctional wound healing response. As mentioned above, one of the main functions of ATX in adults is to repair damaged tissue. ATX is secreted in this situation partly in response to inflammation and the release of inflammatory cytokines. In normal wound healing, the production of LPA by ATX causes cells to migrate into the area of damage to effect wound repair and the formation of new blood vessels. Cancer can be considered to be a case of unresolved inflammation and it has been likened to "a wound that does not heal" [101]. In fact, inflammation is now considered to be one of the "Hallmarks of Cancer" [125]. The secretion of ATX and increased LPA signaling should now be included as one of the inflam‐ matory factors that drives tumor progression.

The role of ATX and LPA in this process is well illustrated in the case of work with mouse models of breast cancer. Most breast cancer cells do not themselves express ATX, but rather this is produced by fibroblasts within the breast tissue or by the surrounding adipose tissue [33] (Figure 3). The development of the breast tumor causes the release of inflammatory cytokines, which stimulate fibroblasts and adipose tissue to secrete ATX in a syngeneic orthotopic mouse model of breast cancer [33, 107]. This is part of a vicious cycle since LPA in turn stimulates the production of inflammatory cytokines [126-128]. This inflammatory cycle can be effectively blocked in a mouse by inhibiting ATX activity with ONO-8430506, which results in about a 60% decrease in tumor growth and lung metastasis.

Although breast cancer cells do not do not express significant levels of ATX activity, this is not typical of other tumors where ATX activity is expressed in the cancer cells themselves. These cancers include thyroid [129], neuroblastomas [71, 130, 131] and melanomas [132-134]. However, in the case of melanomas, their normal predecessor cells, melanocytes, do not express ATX. Thus, the data suggests that ATX expression is acquired during the transition to malignancy in melanoma [2]. The secretion of inflammatory cytokines by cells in the tumor also produces a vicious cycle of ATX secretion and LPA production, which is a driving force in tumor progression [107]. In the case of breast tumors, we propose that the ATX come from surrounding adipose tissue, whereas in thyroid tumors, neuroblastomas and melanomas, ATX is secreted by the cancer cells (Figure 3).

## **11. ATX in chemo-resistance**

fact, inflammation is now considered to be one of the "Hallmarks of Cancer" [125]. The secretion of ATX and increased LPA signaling should now be included as one of the inflam‐

The role of ATX and LPA in this process is well illustrated in the case of work with mouse models of breast cancer. Most breast cancer cells do not themselves express ATX, but rather this is produced by fibroblasts within the breast tissue or by the surrounding adipose tissue [33] (Figure 3). The development of the breast tumor causes the release of inflammatory cytokines, which stimulate fibroblasts and adipose tissue to secrete ATX in a syngeneic orthotopic mouse model of breast cancer [33, 107]. This is part of a vicious cycle since LPA in turn stimulates the production of inflammatory cytokines [126-128]. This inflammatory cycle can be effectively blocked in a mouse by inhibiting ATX activity with ONO-8430506, which

**Figure 3. Proposed inflammatory-mediated models of ATX secretion.** A) In cancer cells that overexpress ATX such as thyroid cancers, neuroblastomas and melanomas, autocrine-secreted ATX produces LPA, which signals through LPA re‐ ceptors (LPARs) on the cancer cell surface. This signaling further increases the production of pro-inflammatory signals in‐ cluding growth factors, cytokines and chemokines. These molecules in turn can signal through specific receptors to increase ATX production. B) In breast cancer, a paracrine model of ATX secretion is possible since breast adipose tissue se‐ cretes high levels of ATX, whereas breast cancer cells normally produce negligible ATX. As the tumor grows, pro-inflam‐ matory signals secreted by the tumor create an inflammatory environment within the surrounding adipose tissue (green arrows). This signaling increases ATX secretion and LPA production, which in turn can establish an autocrine feedback loop of increased pro-inflammatory signaling and ATX production (red arrows) within the adipose tissue. Increased ATX and LPA production can in turn contribute to tumor progression (green arrows). A combination of both autocrine and

Although breast cancer cells do not do not express significant levels of ATX activity, this is not typical of other tumors where ATX activity is expressed in the cancer cells themselves. These cancers include thyroid [129], neuroblastomas [71, 130, 131] and melanomas [132-134]. However, in the case of melanomas, their normal predecessor cells, melanocytes, do not express ATX. Thus, the data suggests that ATX expression is acquired during the transition to malignancy in melanoma [2]. The secretion of inflammatory cytokines by cells in the tumor also produces a vicious cycle of ATX secretion and LPA production, which is a driving force

paracrine production of ATX is also possible when cancer cells produce significant quantities of ATX.

results in about a 60% decrease in tumor growth and lung metastasis.

matory factors that drives tumor progression.

308 Melanoma – Current Clinical Management and Future Therapeutics

Another important role of ATX expression and LPA signaling in malignancy occurs during the acquisition and manifestation of chemo-resistance. LPA facilitates chemo-resistance to the cytotoxic effects mediated by Taxol [119, 135, 136], doxorubicin [57], actinomycin D [122] and carboplatin [137]. These effects are mediated by LPA partly through activation of survival and viability pathways, such as ERK and PI3K. In addition, we previously demonstrated that LPA signaling does not encompass the entire molecular mechanism and there are other proteins, like the Regulators of G-protein Signaling proteins, which play a more dominant role [138]. Indeed, in the absence of appropriate Regulators of G-protein Signaling proteins, cells exposed to LPA have increased capacity to acquire chemo-resistance.

As chemo-resistance is a complex process, there are other molecular mechanisms involved. For example, among chemo-resistant cells, increased expression of multidrug resistance transporters enables toxins, like chemotherapeutic drugs, to be exported out of cancer cells. This is particularly problematic in the case of renal cell carcinomas, for which cytotoxic chemotherapy is largely ineffective, but also occurs widely in malignancy.

Recent work shows that the activation of PI3K by through LPA1 receptors increases the stability of the transcription factor Nrf2, which increases the expression of antioxidant genes and multidrug resistant transporters [57]. The expression of antioxidant genes protects cancer cells against the oxidative damage caused by chemotherapeutic agents. Also, the expression of multidrug resistant transporters enables toxic oxidative products and chemotherapeutic drugs to be exported out of cancer cells. These effects explain why inhibiting ATX activity and blocking LPA signaling improves the efficacy of doxorubicin as a chemotherapeutic agent [57]. Thus blocking ATX activity can provide a novel adjuvant therapy for improving the efficacy of existing chemotherapeutic agents.

ATX inhibition could also have a beneficial effect as an adjuvant for improving the effects of radiotherapy as discussed above. This is possible since LPA, through activation of LPA2 receptors, also protects against radiation-induced cell death. This action depends on the depletion Siva-1, which is a pro-apoptotic signaling protein [139].

The function of ATX in aggravating resistance to chemotherapy and radiotherapy can be understood in terms of the vicious cycle of inflammation caused by repeated bouts of therapy as described above [140] (Figure 3). Cancer therapy itself causes damage to the tumor and surrounding tissue, which responds by producing inflammatory cytokines resulting in increased ATX production [107]. This explains why blocking this cycle by inhibiting LPA formation can improve the sensitivity to chemotherapy by attenuating the effects of increased Nrf2 expression.

## **12. ATX in melanoma**

Although accumulating studies suggest that inhibiting ATX activity could provide a novel adjuvant therapy for improving the efficacy of existing chemotherapeutic agents, we have previously demonstrated a role for ATX inhibitors as monotherapy against advanced cutane‐ ous melanoma [2,48,59]. After injecting B16F10 metastatic melanoma cells into the tail veins of C57/Bl6 mice, we observed a significant reduction in the number of lung nodules, which represent metastatic melanoma tumors, after treatment with a phosphonothionate analogue of carba cyclic phosphatidic acid, thio-ccPA 18:1 [2]. This compound was synthesized for im‐ proved metabolic stability and activity, based on our previous results [48]. In addition to being an inhibitor of ATX, thio-ccPA 18:1 is a direct antagonist of LPA1 and LPA3 receptors [141].

As mentioned previously, melanomas are notoriously resistant to chemotherapy. In light of the role of ATX/LPA signaling in melanoma, perhaps it should not be surprising that mela‐ noma cells, which produce high quantities of ATX, are resistant to chemotherapy, since excessive LPA signaling contributes to this phenotype. Only a few chemotherapy agents are approved options against melanoma, these include dacarbazine and temozolomide. Thus, we compared these single agents against both the anti-BrP-LPA and the mixed diastereomers BrP-LPA on the viability of MeWo melanoma cells. Indeed, both BrP-LPA compounds were more effective single-agents at 10 μM and 100 μM than either dacarbazine or temozolomide, at concentration ranging from 10-1000 μM [59]. This suggests that targeted approaches against ATX in melanoma have potential and further results will be reported in due time.

Besides cutaneous melanoma, in a study on uveal melanoma, ATX was the only gene among 32 candidate genes whose expression was sufficient to distinguish classes representing metastasis and prognosis. Paradoxically, "underexpression" of ATX correlated with poor prognosis and metastatic death among 27 samples [132]. Based on the discussion provided above it is tempting to propose that melanocytes evolved to survive solar ultraviolet radiation and simultaneously provide protection to neighboring cells by producing ATX and thus providing LPA. However, with repeated DNA damage and incomplete repair from excessive UV radiation, melanocytes are malignantly transformed into melanoma cells. At this point, the increased production of ATX and signaling by inflammatory cytokines, which are meant to facilitate repair, could be subverted into promoting cancer progression.

## **13. Summary and conclusions**

Advanced metastatic melanoma is an incurable disease in dire need of additional therapeutic options. Although many newly targeted inhibitors have extended the life of patients with *BRAF* mutations, they do not achieve cure due to chemo-resistance, and they are not applicable to patients with Wild-type B-Raf. Thus, additional therapeutics are desperately needed to treat this growing population. Herein we have summarized the current state of ATX inhibitors and what we currently know about the role of ATX in melanoma and malignancy.

The original ATX inhibitors had little utility *in vivo* because of the very low bioavailability. However, as described above, potent ATX inhibitors are now being developed, which are effective *in vivo* for longer than 24 h. These inhibitors appear to be well tolerated by animals and the next stage is to take such inhibitors through Phase 1 clinical trials. These ATX inhibitors should be effective in improving the outcomes for which the ATX/LPA axis is involved. These include various forms of cancer whereby an ATX inhibitor could be used as a monotherapy or as an adjuvant to improve existing chemotherapies or radiation treatment. The ATX inhibitors should also be effective in improving the treatment of a variety of inflammatory conditions. These compounds deserve further examination.

## **Acknowledgements**

**12. ATX in melanoma**

310 Melanoma – Current Clinical Management and Future Therapeutics

Although accumulating studies suggest that inhibiting ATX activity could provide a novel adjuvant therapy for improving the efficacy of existing chemotherapeutic agents, we have previously demonstrated a role for ATX inhibitors as monotherapy against advanced cutane‐ ous melanoma [2,48,59]. After injecting B16F10 metastatic melanoma cells into the tail veins of C57/Bl6 mice, we observed a significant reduction in the number of lung nodules, which represent metastatic melanoma tumors, after treatment with a phosphonothionate analogue of carba cyclic phosphatidic acid, thio-ccPA 18:1 [2]. This compound was synthesized for im‐ proved metabolic stability and activity, based on our previous results [48]. In addition to being an inhibitor of ATX, thio-ccPA 18:1 is a direct antagonist of LPA1 and LPA3 receptors [141]. As mentioned previously, melanomas are notoriously resistant to chemotherapy. In light of the role of ATX/LPA signaling in melanoma, perhaps it should not be surprising that mela‐ noma cells, which produce high quantities of ATX, are resistant to chemotherapy, since excessive LPA signaling contributes to this phenotype. Only a few chemotherapy agents are approved options against melanoma, these include dacarbazine and temozolomide. Thus, we compared these single agents against both the anti-BrP-LPA and the mixed diastereomers BrP-LPA on the viability of MeWo melanoma cells. Indeed, both BrP-LPA compounds were more effective single-agents at 10 μM and 100 μM than either dacarbazine or temozolomide, at concentration ranging from 10-1000 μM [59]. This suggests that targeted approaches against

ATX in melanoma have potential and further results will be reported in due time.

to facilitate repair, could be subverted into promoting cancer progression.

what we currently know about the role of ATX in melanoma and malignancy.

**13. Summary and conclusions**

Besides cutaneous melanoma, in a study on uveal melanoma, ATX was the only gene among 32 candidate genes whose expression was sufficient to distinguish classes representing metastasis and prognosis. Paradoxically, "underexpression" of ATX correlated with poor prognosis and metastatic death among 27 samples [132]. Based on the discussion provided above it is tempting to propose that melanocytes evolved to survive solar ultraviolet radiation and simultaneously provide protection to neighboring cells by producing ATX and thus providing LPA. However, with repeated DNA damage and incomplete repair from excessive UV radiation, melanocytes are malignantly transformed into melanoma cells. At this point, the increased production of ATX and signaling by inflammatory cytokines, which are meant

Advanced metastatic melanoma is an incurable disease in dire need of additional therapeutic options. Although many newly targeted inhibitors have extended the life of patients with *BRAF* mutations, they do not achieve cure due to chemo-resistance, and they are not applicable to patients with Wild-type B-Raf. Thus, additional therapeutics are desperately needed to treat this growing population. Herein we have summarized the current state of ATX inhibitors and

The original ATX inhibitors had little utility *in vivo* because of the very low bioavailability. However, as described above, potent ATX inhibitors are now being developed, which are

This work was supported by research grants from the National Institutes of Health (1R15CA151006-01 American Recovery and Reinvestment Act and 1R15CA176653-01A1), a Research Scholar Grant 120634-RSG-11-269-01-CDD from the American Cancer Society and a Distinguished Scientist award from the Georgia Research Alliance to MMM. MGKB received a Vanier Canada Graduate Scholarship from the Government of Canada, a Killam Trust Award and an MD/PhD scholarship from Alberta Innovates-Health Solutions. DNB was supported by grants from the Canadian Breast Cancer Foundation, Women and Children's Health Research Institute of the University of Alberta and CIHR with the Alberta Cancer Foundation. DNB declares a conflict of interest in having received a consulting fee from Ono Pharmaceuticals.

## **Author details**

David N. Brindley1 , Matthew G.K. Benesch1 and Mandi M. Murph2\*

\*Address all correspondence to: mmurph@uga.edu

1 Signal Transduction Research Group, Department of Biochemistry, University of Alberta, Heritage Medical Research Center, Edmonton, Alberta, Canada

2 Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, College of Pharmacy, Athens, Georgia, USA

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## **Chapter 13**

## **RAGE and Its Ligands in Melanoma**

## Estelle Leclerc

in Response to Radio/Chemotherapy. Mol Cancer Res: doi:

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324 Melanoma – Current Clinical Management and Future Therapeutics

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59172

## **1. Introduction**

Melanoma is a complex disease with both genetic and epigenetic components [1-3]. Once melanoma has formed distant metastases, melanoma patients have generally poor prognoses; less than 10% of these patients will survive 10 years [4]. For many years, treatment with the cytotoxic drug dacarbazine was the standard treatment for patients with metastatic melanoma, but the response rates were low and varied from 5-20% [4]. Intense research efforts on understanding the molecular mechanisms of melanoma progression led to the discovery and the approval by the FDA of two new drugs in 2011, vemurafenib and ipilimumab, which raised great hopes among melanoma patients [5-12]. Although treatment with vemurafenib has resulted in high overall responses rates [11], resistance against the drug appeared in treated melanoma patients within a year of treatment, leading to tumor regrowth [13-16]. On the other hand, treatment with ipilimumab does not result in resistance but can produce life threatening autoimmune adverse effects [17]. In addition, ipilimumab works best in patients whose tumors present abundant tumor infiltrated immune cells [18]. It is therefore essential to identify new therapeutic targets in melanoma.

One potential therapeutic target is the receptor for advanced glycation end products (RAGE). A possible role of RAGE in melanoma is emerging and has been the topic of a growing number of studies in the past decade [19-25]. These studies will be reviewed here and we will also discuss the RAGE ligands that play important roles in melanoma progression.

## **2. The Receptor for Advanced Glycation End Products (RAGE)**

### **2.1. RAGE function**

RAGE is an immunoglobulin-like cell-surface receptor that is involved in a large number of pathologies, including Alzheimer's disease, cancer, infectious diseases and complications of

© 2015 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.

diabetes [31-38]. The ligands of RAGE are numerous (Table 1) and belong to distinct families of molecules. However, they also share structural features such as the propensity to form oligomers [39]. It is believed that RAGE recognizes similar structural elements or patterns within its numerous ligands and therefore RAGE is described as a pattern recognition receptor [40, 41].

**Figure 1.** Schematic representation of RAGE. The extracellular part of RAGE is composed of three distinct immunoglo‐ bulin-like domains (V, C1 and C2). The V and C1 domains form a structural and functional unit [26]. Most ligands interact with the V-C1 domain. Four adaptor molecules have been suggested to bind to the intracellular domain of RAGE: ERK1/2 [27], diaphanous-1 (Dia-1) [28], TIRAP and MyD88 [29]. Following activation of RAGE by its ligands, multiple signaling pathways (MAPK, PI3K/Akt) or molecules (NADPH oxidase, Rac-1, cdc42) are activated and lead to the transcription of genes related to inflammation, cell proliferation and migration, and to the expression of RAGE it‐ self, resulting in a positive feedback loop [30].

The physiological role of RAGE is not yet fully understood. Studies have shown that RAGE plays a role in the innate but not adaptive immunity and has been shown to play an important function in peripheral nerve repair [42-44]. A characteristic of RAGE is that it is expressed at low basal level in most tissues except in lungs where it has been suggested to exert a protective effect [45-48]. RAGE has also been shown to modulate the auditory system in mice [49].

Several spliced isoforms of RAGE have been described. Besides full-length RAGE, presented in Figure 1, RAGE exists as a soluble form (sRAGE) lacking the intracellular and transmem‐ brane domains. The soluble form can result either from a splicing event or from the action of a metalloprotease such as ADAM 10 [50-58]. Other major identified spliced isoforms lack the N-terminal domain or present a deletion in the intracellular domain, resulting in abnormal ligand interaction and cell signaling activities, respectively [51, 56, 59]. sRAGE has been used in many animal experiments to validate the role of RAGE in various diseases such as Alz‐ heimer's disease, atherosclerosis or encephalomyelitis [60-62]. Two different models explain‐ ing the mechanism of action of sRAGE have been proposed. The "classic" mechanism suggests that sRAGE acts as a decoy receptor and interacts with circulating RAGE ligands, which results in the absence of RAGE/ligand complex formation and RAGE activation. Recently, the group of Fritz has proposed a second mechanism of action for sRAGE: Fritz et al. suggest that sRAGE could form a complex with the extracellular part of another full-length receptor and that this receptor complex would not be functional because it possesses only one intracellular domain [63]. According to this hypothesis, RAGE can only transmit a signal if two intracellular domains form a dimer [63].

The role of sRAGE as biomarker has also been investigated in many diseases but has resulted in contradictory data for certain pathologies. In Alzheimer's disease patients, the circulating plasma levels of sRAGE have been shown to be significantly reduced compared to healthy individuals [64]. A more detailed immunohistological examination of AD brain showed that the reduction in sRAGE was more pronounced in the hippocampus of AD brains than normal brains [65]. Systemic low levels of sRAGE have also been found in patients with emphysema, a form of chronic obstructive pulmonary disease (COPD) and sRAGE is suggested to be a biomarker for this lung disease [66, 67]. However, in diabetes, the situation is more complex and in different studies the levels of sRAGE have been either positively or negatively correlated with the disease (reviewed and discussed in [68])

In cancer, an association between sRAGE and the progression of cancer has also been shown. Significantly lower levels of sRAGE have been found in lung, breast, liver or pancreatic cancer patients than in normal individuals [69-72]. Our study of 40 melanoma human samples also showed significantly lower transcript levels of the spliced form of RAGE in 90% of melanoma stage III and stage IV tissue samples compared to normal samples [73].

#### **2.2. RAGE structure and signaling**

diabetes [31-38]. The ligands of RAGE are numerous (Table 1) and belong to distinct families of molecules. However, they also share structural features such as the propensity to form oligomers [39]. It is believed that RAGE recognizes similar structural elements or patterns within its numerous ligands and therefore RAGE is described as a pattern recognition receptor

**Figure 1.** Schematic representation of RAGE. The extracellular part of RAGE is composed of three distinct immunoglo‐ bulin-like domains (V, C1 and C2). The V and C1 domains form a structural and functional unit [26]. Most ligands interact with the V-C1 domain. Four adaptor molecules have been suggested to bind to the intracellular domain of RAGE: ERK1/2 [27], diaphanous-1 (Dia-1) [28], TIRAP and MyD88 [29]. Following activation of RAGE by its ligands, multiple signaling pathways (MAPK, PI3K/Akt) or molecules (NADPH oxidase, Rac-1, cdc42) are activated and lead to the transcription of genes related to inflammation, cell proliferation and migration, and to the expression of RAGE it‐

The physiological role of RAGE is not yet fully understood. Studies have shown that RAGE plays a role in the innate but not adaptive immunity and has been shown to play an important function in peripheral nerve repair [42-44]. A characteristic of RAGE is that it is expressed at low basal level in most tissues except in lungs where it has been suggested to exert a protective effect [45-48]. RAGE has also been shown to modulate the auditory system in mice [49].

Several spliced isoforms of RAGE have been described. Besides full-length RAGE, presented in Figure 1, RAGE exists as a soluble form (sRAGE) lacking the intracellular and transmem‐ brane domains. The soluble form can result either from a splicing event or from the action of

self, resulting in a positive feedback loop [30].

[40, 41].

326 Melanoma – Current Clinical Management and Future Therapeutics

RAGE is a single transmembrane receptor with a large extracellular part comprising of three domains that share structural features with immunoglobulin domains: a variable type (V) domain (residues 23-119), and two constant type domains (C1: residues 120-233, and C2: residues 234-325) (Figure 1) [74]. The V and C1 domains form a structural and functional unit and are the site of binding of most ligands [26].

Recent studies have suggested that RAGE structure is as complex as RAGE signaling is, and multiple conformational forms of RAGE in complex with its ligands have been proposed [75]. Recent evidence suggests that RAGE exists as a dimer on the surface of cells, in the absence of ligand [76, 77]. It has been suggested that the interaction of RAGE with its ligands occurs mainly through electrostatic interactions between the positive charges present on the V and C1 domains of RAGE and the negative charges present on RAGE ligands [39, 63]. Because both RAGE and its ligands can form oligomers, several oligomeric models of RAGE in complex with its ligands have been proposed to reflect the possible RAGE/ligand interactions [39, 63, 75-79]. RAGE oligomerization could occur through contacts between the V domain [80], C1 domain [80], [77] or C2 domain, [75, 79], in a ligand dependent manner [79].

RAGE signaling is complex and RAGE/ligand interaction results in the activation of multiple signaling pathways that are ligand and tissue specific (Figure 1) [34, 81-84]. In general, RAGE engagement by its ligand leads to the activation of key elements of the PI3K/Akt pathways or the mitogen-activated protein (MAP) kinase signaling pathways which include ERK1/2, p38 and JNK. Small GTPases such as p21-Ras, Rac-1 or cdc42 can also be activated as a result of RAGE activation [28, 81, 85-87]. In many cases, RAGE activation by its ligands leads to the initiation of, and sustained inflammation through the activation of the transcription factor NFκB, but also AP-1, STAT-3 and CREB (Figure 1) [36-38, 68, 88].

#### **2.3. RAGE in melanoma**

A role of RAGE in melanoma progression was first suggested by Huttunen et al [19]. In their study, the authors generated tumors in mice using the mouse melanoma B16F10 cell-line expressing either full-length RAGE or a form of RAGE lacking the cytoplasmic domain [19]. Comparison of the number of metastases formed in the two groups of mice revealed that mice that were implanted with cells expressing the mutant form of RAGE generated significantly less tumors than mice implanted with cells expressing full-length RAGE. In a different study, Abe et al. showed that the growth of xenograft tumor in mice could be reduced using anti-RAGE antibodies [21]. We showed that RAGE overexpression in the WM115 human melanoma cell line resulted in a significant decrease in cell proliferation, but also in a significant increase in cell migration and invasion suggesting that RAGE can modulate different aspects of cancer [24]. We demonstrated that the decrease in cell proliferation was accompanied with a decrease in the activity of ERK1/2 [24] and p38 (Meghnani et al. 2014, International Journal of Biochem‐ istry and Cell Biology, in press). The recent report of Popa et al. also suggests the presence of different oligomeric forms of RAGE with different roles and subcellular location during melanoma progression [25].

Studies in human melanoma tissue samples have shown that RAGE was not present in all melanoma samples but rather in a subset of samples. Hsieh et al. showed that only 20% tissue samples showed RAGE staining [20]. Our analysis of 40 samples of human melanoma tissue samples also showed large variations in RAGE transcripts levels, with up to 50-fold differences between samples [22].

Other studies have identified RAGE ligands as important elements in melanoma development. S100B has been studied in details and its role as a diagnostic marker for melanoma patients is well established (see section 3.1.1 on S100B). Abe et al. showed that AGEs could promote the proliferation of melanoma cells in culture [21]. Saha et al. showed that S100A8/A9 could attract B16F10 melanoma cells to a metastatic site, in a RAGE dependent manner [23]. In addition, our recent study showed a strong correlation between the expression of RAGE and that of its S100 protein ligands, in mouse xenograft melanoma tumors (Meghnani et al. 2014, Interna‐ tional Journal of Biochemistry and Cell Biology, in press).

## **3. RAGE ligands**

with its ligands have been proposed to reflect the possible RAGE/ligand interactions [39, 63, 75-79]. RAGE oligomerization could occur through contacts between the V domain [80], C1

RAGE signaling is complex and RAGE/ligand interaction results in the activation of multiple signaling pathways that are ligand and tissue specific (Figure 1) [34, 81-84]. In general, RAGE engagement by its ligand leads to the activation of key elements of the PI3K/Akt pathways or the mitogen-activated protein (MAP) kinase signaling pathways which include ERK1/2, p38 and JNK. Small GTPases such as p21-Ras, Rac-1 or cdc42 can also be activated as a result of RAGE activation [28, 81, 85-87]. In many cases, RAGE activation by its ligands leads to the initiation of, and sustained inflammation through the activation of the transcription factor NF-

A role of RAGE in melanoma progression was first suggested by Huttunen et al [19]. In their study, the authors generated tumors in mice using the mouse melanoma B16F10 cell-line expressing either full-length RAGE or a form of RAGE lacking the cytoplasmic domain [19]. Comparison of the number of metastases formed in the two groups of mice revealed that mice that were implanted with cells expressing the mutant form of RAGE generated significantly less tumors than mice implanted with cells expressing full-length RAGE. In a different study, Abe et al. showed that the growth of xenograft tumor in mice could be reduced using anti-RAGE antibodies [21]. We showed that RAGE overexpression in the WM115 human melanoma cell line resulted in a significant decrease in cell proliferation, but also in a significant increase in cell migration and invasion suggesting that RAGE can modulate different aspects of cancer [24]. We demonstrated that the decrease in cell proliferation was accompanied with a decrease in the activity of ERK1/2 [24] and p38 (Meghnani et al. 2014, International Journal of Biochem‐ istry and Cell Biology, in press). The recent report of Popa et al. also suggests the presence of different oligomeric forms of RAGE with different roles and subcellular location during

Studies in human melanoma tissue samples have shown that RAGE was not present in all melanoma samples but rather in a subset of samples. Hsieh et al. showed that only 20% tissue samples showed RAGE staining [20]. Our analysis of 40 samples of human melanoma tissue samples also showed large variations in RAGE transcripts levels, with up to 50-fold differences

Other studies have identified RAGE ligands as important elements in melanoma development. S100B has been studied in details and its role as a diagnostic marker for melanoma patients is well established (see section 3.1.1 on S100B). Abe et al. showed that AGEs could promote the proliferation of melanoma cells in culture [21]. Saha et al. showed that S100A8/A9 could attract B16F10 melanoma cells to a metastatic site, in a RAGE dependent manner [23]. In addition, our recent study showed a strong correlation between the expression of RAGE and that of its S100 protein ligands, in mouse xenograft melanoma tumors (Meghnani et al. 2014, Interna‐

tional Journal of Biochemistry and Cell Biology, in press).

domain [80], [77] or C2 domain, [75, 79], in a ligand dependent manner [79].

κB, but also AP-1, STAT-3 and CREB (Figure 1) [36-38, 68, 88].

328 Melanoma – Current Clinical Management and Future Therapeutics

**2.3. RAGE in melanoma**

melanoma progression [25].

between samples [22].

Since the identification of AGEs as ligands of RAGE, the list of RAGE ligands has grown to more than ten ligands or groups of ligands (Table 1). In this section, we will discuss the current knowledge on ligands that have been shown to play a role in melanoma progression, with a focus on S100 proteins.


**Table 1.** Non-exhaustive list of the different ligands of RAGE. The S100 proteins that are ligands of RAGE will be described in details in section 3.1

#### **3.1. S100 proteins**

Members of the S100 protein family have amino-acid sequence and structure similarities [111, 112]. They are small calcium binding proteins and belong to the superfamily of EF-hand proteins [111]. Restricted to vertebrates, S100 proteins show strong tissue and cell specificity [111, 113]. Most S100 genes (S100A1 to S100A16) are located on chromosome 1 in a region that is prone to chromosomal rearrangement, linking these S100 proteins to cancer [113]. The presence of EF-hands within the structure of S100 proteins enable them to bind calcium with moderate micromolar affinity [111, 112, 114, 115]. In addition to calcium, S100 proteins can also bind zinc and copper [116, 117]. For most S100 proteins, binding to calcium results in conformational rearrangements that allow the S100 proteins to interact with their targets [115, 117, 118]. Certain target proteins are S100 specific whereas others are shared among multiple S100s (Reviewed in [119]).

Most S100 target proteins are intracellular [111, 112, 120]. An example of such target that plays a role in cancer is the tumor suppressor p53 protein [121-126]. Several members of the S100 protein family (S100B, S100A1, S100A2, S100A4, S100A6 and S100A10] bind to p53 but the outcome of this interaction differs depending of the S100 [121-126]. S100 proteins can also be secreted into the extracellular space through mechanisms that are not clearly understood [127, 128]. When secreted, many S100 proteins have been shown to interact with RAGE [82, 93, 129].

Many S100 proteins are found in cells constituting the epidermis [130]. S100B and S100A6 have been found in both melanocytes and Langerhan's cells [131-133]. S100A2, A7, A10, A11, A12 and S100A15 have been described in normal keratinocytes [131-138]. In pathophysiological conditions, such as in inflamed keratinocytes and melanoma, many S100 proteins have been found overexpressed [130, 136]. The next section will describe the role of these S100 proteins in melanoma.

#### *3.1.1. S100B*

S100B is used as a prognostic marker for stage IV malignant melanoma patients [139-141]. Serum concentration of S100B increases during the disease and high levels of S100B in the serum is indicative of a poor prognosis [139-141]. In melanoma tumors, melanoma cells are the main cells responsible for secreting S100B [141]. Within the cells, the role of S100B is not clearly understood but the main target of S100B appears to be the tumor suppressor p53 protein. Indeed, several studies from the group of Weber have shown that S100B interacts with p53 in melanoma cells and tumors, resulting in p53 inhibition and increased expression of S100B, in a negative feedback loop [142-144]. His group is currently investigating small interfering antisense RNA inhibitors of S100B as inhibitors of melanoma tumor growth [145].

Other intracellular target proteins of S100B could also contribute to increases in cellular proliferation and tumor growth in melanoma. For example, S100B interacts with and activates the glycolytic enzyme fructose-1,6-biphosphate aldolase, [146]. Consequences of this activa‐ tion could be an increase in melanoma cell metabolism and glycolysis. Inhibiting melanoma cell metabolism and glycolysis is currently been considered in clinical trials [147, 148]. Inhibiting S100B/ fructose-1,6-biphosphate aldolase could be an approach to further reduce glycolytic activity in melanoma cells. S100B also interacts with many components of the cytoskeleton such as tubulin [149, 150], the actin binding protein caldesmon [151] or the small GTPase Rac1 and the cdc42 effector IQGAP1 [152]. All these proteins play important function in malignant melanoma [153-155] and increases in S100B levels could therefore favor increases in cell proliferation, migration and invasion through their modulation. S100B could also play a role in melanoma cell growth through the activation of the Nuclear Dbf2 related (ndr) kinase [156-158] and the interaction with the phosphoprotein AHNAK/desmoyokin [159-161].

As mentioned earlier, S100B is secreted from melanoma cells. The mechanisms of S100B secretion are still poorly understood but recent studies have suggested that RAGE participates in the translocation and secretion of several S100s including S100B [127, 128]. Similarly, the role of RAGE/S100B in melanoma is slowly being unraveled. S100B has been shown to signal through RAGE in a large number of diseases (reviewed in [162]) and we studied in details the *in vitro* interaction of RAGE with S100B [26, 81, 163]. For example, we showed that not only dimeric S100B but also tetrameric and hexameric S100B could interact with RAGE and signal in cells [163]. In addition, we recently showed that overexpression of RAGE, in human WM115 melanoma cells, was accompanied by the up-regulation of S100B in these cells [24], suggesting a strong association between RAGE and S100B in melanoma. When the RAGE transfected WM115 cells were implanted in mice as xenografts, we also observed higher levels of S100B in the serum of these animals, compared to animals implanted with control WM115 cells (Meghnani et al. 2014, International Journal of Biochemistry and Cell Biology, in press).

#### *3.1.2. S100A2*

outcome of this interaction differs depending of the S100 [121-126]. S100 proteins can also be secreted into the extracellular space through mechanisms that are not clearly understood [127, 128]. When secreted, many S100 proteins have been shown to interact with RAGE [82, 93, 129]. Many S100 proteins are found in cells constituting the epidermis [130]. S100B and S100A6 have been found in both melanocytes and Langerhan's cells [131-133]. S100A2, A7, A10, A11, A12 and S100A15 have been described in normal keratinocytes [131-138]. In pathophysiological conditions, such as in inflamed keratinocytes and melanoma, many S100 proteins have been found overexpressed [130, 136]. The next section will describe the role of these S100 proteins

S100B is used as a prognostic marker for stage IV malignant melanoma patients [139-141]. Serum concentration of S100B increases during the disease and high levels of S100B in the serum is indicative of a poor prognosis [139-141]. In melanoma tumors, melanoma cells are the main cells responsible for secreting S100B [141]. Within the cells, the role of S100B is not clearly understood but the main target of S100B appears to be the tumor suppressor p53 protein. Indeed, several studies from the group of Weber have shown that S100B interacts with p53 in melanoma cells and tumors, resulting in p53 inhibition and increased expression of S100B, in a negative feedback loop [142-144]. His group is currently investigating small interfering antisense RNA inhibitors of S100B as inhibitors of melanoma tumor growth [145]. Other intracellular target proteins of S100B could also contribute to increases in cellular proliferation and tumor growth in melanoma. For example, S100B interacts with and activates the glycolytic enzyme fructose-1,6-biphosphate aldolase, [146]. Consequences of this activa‐ tion could be an increase in melanoma cell metabolism and glycolysis. Inhibiting melanoma cell metabolism and glycolysis is currently been considered in clinical trials [147, 148]. Inhibiting S100B/ fructose-1,6-biphosphate aldolase could be an approach to further reduce glycolytic activity in melanoma cells. S100B also interacts with many components of the cytoskeleton such as tubulin [149, 150], the actin binding protein caldesmon [151] or the small GTPase Rac1 and the cdc42 effector IQGAP1 [152]. All these proteins play important function in malignant melanoma [153-155] and increases in S100B levels could therefore favor increases in cell proliferation, migration and invasion through their modulation. S100B could also play a role in melanoma cell growth through the activation of the Nuclear Dbf2 related (ndr) kinase [156-158] and the interaction with the phosphoprotein AHNAK/desmoyokin [159-161].

As mentioned earlier, S100B is secreted from melanoma cells. The mechanisms of S100B secretion are still poorly understood but recent studies have suggested that RAGE participates in the translocation and secretion of several S100s including S100B [127, 128]. Similarly, the role of RAGE/S100B in melanoma is slowly being unraveled. S100B has been shown to signal through RAGE in a large number of diseases (reviewed in [162]) and we studied in details the *in vitro* interaction of RAGE with S100B [26, 81, 163]. For example, we showed that not only dimeric S100B but also tetrameric and hexameric S100B could interact with RAGE and signal in cells [163]. In addition, we recently showed that overexpression of RAGE, in human WM115 melanoma cells, was accompanied by the up-regulation of S100B in these cells [24], suggesting

in melanoma.

330 Melanoma – Current Clinical Management and Future Therapeutics

*3.1.1. S100B*

S100A2 is mainly located in the nuclei of cells [164]. The function of S100A2 in cancer is not clear. In certain cancers such as prostate, oral, lung and breast cancers, S100A2 has been shown to play the role of tumor suppressor [22, 165-169], whereas in other cancers such as esophageal squamous carcinoma, gastric, and ovarian cancer, studies indicate that it acts as tumor promoter [170-172]. In addition, in certain cancers, such as in non-small cell lung cancer (NSCLC), studies are contradictory since both down-and up-regulation of S100A2 have been reported [173-175].

In melanoma, most studies reported that S100A2 plays the role of tumor suppressor [130, 132, 165, 176]. We also showed a significant reduction in S100A2 mRNA level in both stage III and stage IV melanoma patients samples compared to control samples [22]. Additional evidences of a tumor suppressor effect of S100A2 comes from several studies that show positive corre‐ lations between the levels of S100A2 and anti-proliferative effects of chemotherapeutic agents in melanoma cells [177, 178]. However, our most recent study shows that xenograft melanoma tumors overexpressing RAGE, presented a growth advantage over control tumors, and exhibited significant higher levels of S100A2 than control tumors (Meghnani et al. 2014, International Journal of Biochemistry and Cell Biology, in press). We observed that S100A2 was up-regulated, both at the transcript and protein levels, in the RAGE overexpressing tumors (Meghnani et al. unpublished results). These data suggest a complex role of S100A2 in promoting or suppressing melanoma tumor growth.

As mentioned above, S100A2 is located in the nuclei of cells and has been shown to interact with the tumor suppressor p53, in two oral cancer cell lines (FADU and SCC-25), thereby modulating the transcriptional activity of p53 [123]. *In vitro* binding studies have confirmed the interaction between S100A2 and p53 and has shown differences in binding of p53 to S100B and S100A2; for example, although both S100B and S100A2 could bind to the monomeric form of p53, only S100B was able to disrupt the oligomerization of p53, suggesting different mode of transcriptional modulation of p53 by the S100 proteins [124, 126]. In addition to interacting with p53, S100A2 transcriptional activity is regulated by other members of the large p53 family, such as p63 and p73, suggesting an additional level of complexity of the S100A2/p53 regulation [179-181]. In breast cancer tissues and cells, it was shown that both the p53 homologue Np63, and BRCA1 were important for the transcriptional activity of S100A2 [182]. These recent data could have a large impact in understanding the molecular mechanism of melanoma because positive correlations have been found between the BRCA1 associated protein (BAP1] and malignant uveal and cutaneous melanoma [183-185].

We recently demonstrated that *in vitro*, S100A2 could interact with RAGE [22] and our recent study showed an association between RAGE overexpression and the levels of S100A2 in melanoma xenograft tumors (Meghnani et al. 2014, International Journal of Biochemistry and Cell Biology, in press). However, a direct interaction of S100A2/RAGE in melanoma cells and tissue has yet to be demonstrated.

#### *3.1.3. S100A4*

S100A4 was initially identified from metastatic cells and was hence named metastasin [186]. Overexpression of S100A4 in multiple cell lines has been shown to increase cancer cell invasiveness and motility [187, 188]. The role of S100A4 in cancer was further demonstrated in S100A4-/-mice that showed delayed tumor growth, compared to control mice, when implanted with highly metastatic mammary carcinoma cells [189, 190].

In melanoma, the role of S100A4 is complex and appears to vary during the progression of the disease. An early study showed no significant differences in S100A4 mRNA levels between melanoma tissue samples and control samples [165]. Our analysis of 40 samples of stage III and stage IV melanoma tissue samples showed a significantly reduction of S100A4 mRNA in stage IV compared to control samples [22]. Another study also reported different correlation between the levels of S100A4 and patient survival rates during the progression of melanoma: high levels of S100A4 in primary melanoma tumors were associated with low patient survival rates whereas high S100A4 levels in metastatic tumors were not associated with differences in patient survival rates, suggesting a role of S100A4 at a early stage of the disease [191].

S100A4 exerts both intra-and extracellular functions (reviewed in [192]). Inside the cells, S100A4 can be found in the nuclei and cytoplasm [192]. S100A4 has been shown to interact with p53 and, as described for S100B, to disrupt p53 oligomerization [193, 194]. *In vivo*, interaction of S100A4 with p53 has been shown to promote degradation of p53, resulting in increases in tumor growth [195]. In the cytoplasm, S100A4 interacts with a large number of proteins of the cytoskeleton such as non-muscle myosin II [196] and tropomyosin [197], resulting in cytoskeletal reorganization, which occurs during cell migration and invasion [198, 199].

An important property of S100A4 is that it has been found secreted in the extracellular medium of cells and is present in the milieu of many tumor types such as breast cancer [200], ovarian carcinoma [201], osteosarcoma [202] or adenocarcinoma tumors [203]. Many cells from normal tissues and from tumors have been shown to release S100A4. These cells include fibroblasts, leukocytes, and endothelial cells [192, 204]. Extracellular S100A4 has been shown to promote tumor growth and metastasis [192], neovascularization and angiogenesis [205-207].

Two main targets of extracellular S100A4 with relevance to tumor growth and metastasis have been identified: Annexin II and RAGE [208, 209]. Interaction of S100A4 with annexin II has been associated with increased mechanisms of angiogenesis such as the formation of capillarylike tubes by endothelial cells [208]. S100A4 has been shown to enhance motility of pulmonary artery smooth muscle cells in a RAGE dependent manner [210]. Similarly, S100A4 was shown to promote prostate and colorectal cancer tumorigenesis and metastasis in a RAGE dependent manner [211, 212]. In melanoma, a recent study has demonstrated that S100A4 derived from macrophages could promote lung colonization of B16F10 melanoma cells in mice, in a RAGE dependent manner as well [213]. We have also observed that RAGE overexpression in the WM115 melanoma cells resulted in the up-regulation of S100A4, by the melanoma cells, in xenograft tumors implanted in mice (Meghnani et al. 2014, International Journal of Biochem‐ istry and Cell Biology, in press).

#### *3.1.4. S100A6*

Cell Biology, in press). However, a direct interaction of S100A2/RAGE in melanoma cells and

S100A4 was initially identified from metastatic cells and was hence named metastasin [186]. Overexpression of S100A4 in multiple cell lines has been shown to increase cancer cell invasiveness and motility [187, 188]. The role of S100A4 in cancer was further demonstrated in S100A4-/-mice that showed delayed tumor growth, compared to control mice, when

In melanoma, the role of S100A4 is complex and appears to vary during the progression of the disease. An early study showed no significant differences in S100A4 mRNA levels between melanoma tissue samples and control samples [165]. Our analysis of 40 samples of stage III and stage IV melanoma tissue samples showed a significantly reduction of S100A4 mRNA in stage IV compared to control samples [22]. Another study also reported different correlation between the levels of S100A4 and patient survival rates during the progression of melanoma: high levels of S100A4 in primary melanoma tumors were associated with low patient survival rates whereas high S100A4 levels in metastatic tumors were not associated with differences in patient survival rates, suggesting a role of S100A4 at a early stage of the disease [191].

S100A4 exerts both intra-and extracellular functions (reviewed in [192]). Inside the cells, S100A4 can be found in the nuclei and cytoplasm [192]. S100A4 has been shown to interact with p53 and, as described for S100B, to disrupt p53 oligomerization [193, 194]. *In vivo*, interaction of S100A4 with p53 has been shown to promote degradation of p53, resulting in increases in tumor growth [195]. In the cytoplasm, S100A4 interacts with a large number of proteins of the cytoskeleton such as non-muscle myosin II [196] and tropomyosin [197], resulting in cytoskeletal reorganization, which occurs during cell migration and invasion

An important property of S100A4 is that it has been found secreted in the extracellular medium of cells and is present in the milieu of many tumor types such as breast cancer [200], ovarian carcinoma [201], osteosarcoma [202] or adenocarcinoma tumors [203]. Many cells from normal tissues and from tumors have been shown to release S100A4. These cells include fibroblasts, leukocytes, and endothelial cells [192, 204]. Extracellular S100A4 has been shown to promote

Two main targets of extracellular S100A4 with relevance to tumor growth and metastasis have been identified: Annexin II and RAGE [208, 209]. Interaction of S100A4 with annexin II has been associated with increased mechanisms of angiogenesis such as the formation of capillarylike tubes by endothelial cells [208]. S100A4 has been shown to enhance motility of pulmonary artery smooth muscle cells in a RAGE dependent manner [210]. Similarly, S100A4 was shown to promote prostate and colorectal cancer tumorigenesis and metastasis in a RAGE dependent manner [211, 212]. In melanoma, a recent study has demonstrated that S100A4 derived from macrophages could promote lung colonization of B16F10 melanoma cells in mice, in a RAGE dependent manner as well [213]. We have also observed that RAGE overexpression in the

tumor growth and metastasis [192], neovascularization and angiogenesis [205-207].

implanted with highly metastatic mammary carcinoma cells [189, 190].

tissue has yet to be demonstrated.

332 Melanoma – Current Clinical Management and Future Therapeutics

*3.1.3. S100A4*

[198, 199].

S100A6 is another member of the S100 family with a link to cancer. S100A6 was identified from melanoma tissue samples by comparing melanocytic lesions from normal nevi [214]. S100A6 is most abundant in epithelial cells and fibroblasts but is also found, in smaller amounts, in other cell-types such as neurons, glial cells, smooth muscle cells, cardiac myocytes, platelets and lymphocytes [215, 216](reviewed in [217]). S100A6 has been found elevated in a large number of cancer types which include colorectal cancer, pancreatic, hepato-cellular carcinoma, melanoma, lung cancer and gastric cancer [165, 218-224](reviewed in [217]). In melanoma tumor samples, a positive correlation was found between the levels of S100A6 transcripts and the severity of the disease [165]. In animal models, S100A6 was found to correlate with the metastatic behavior of the melanoma cells [214, 225]. We also observed higher levels of S1006, at the transcript level, in 43% of stage III melanoma samples examined, compared to control samples [22]. In agreement with our study, examination of another set of melanoma tissue samples showed that 33% of the samples stained positive for S100A6 [133]. However, exami‐ nation of melanoma tumor biopsies by immunohistochemistry often shows that the expression of S100A6 is weak and patchy, and that other non-melanoma cutaneous lesions also stain positive for S100A6, making it difficult to use S100A6 as a diagnostic marker for melanoma [176, 226, 227].

S100A6 has been shown to interact with both nuclear and cytoplasmic proteins. S100A6 interacts with the tumor suppressor p53 but this interaction is different than the interaction of p53 with S100B, suggesting different regulation of the transcriptional activity of p53 by the two S100 proteins [124, 228]. S100A6 was shown to interact with a peptide derived from cell membrane associated annexin I, but the physiological relevance of this interaction has yet to be demonstrated [229]. We showed that S100A6 could interact with both the VC1 and the C2 domain of RAGE, but that in human neuroblastoma, signaling was transduced through the C2 domain [81]. The interaction of the C3S mutant form of S100A6 with the V domain of RAGE has recently been studied in details [230]. Similarly to what we observed with S100B, S100A2 and S100A4, RAGE overexpression in the WM115 melanoma cells resulted in the up-regulation of S100A6, by the melanoma cells, when the cells were forming xenograft tumors in nude mice, compared to control tumors (Meghnani et al. 2014, International Journal of Biochemistry and Cell Biology, in press).

#### *3.1.5. S100A8/A9*

S100A8 and S100A9 are mainly expressed by cells of myeloid origin such as monocytes and macrophages, and were first described as cytokine-like proteins for their up-regulation in inflamed tissues and inflammatory disorders [231]. Recent studies have also shown that they play significant roles in cancer where they promote tumor growth and metastasis in a large number of cancers such as in breast, prostate and pancreatic cancer [232-238]. In tumors, recent studies have shown that S100A8/A9 regulates the accumulation of myeloid-derived suppres‐ sor cells (MDSC), and also promote the expansion of these cells, resulting in increased tumor growth [239-243].

Neither S100A8 nor S100A9 is present in significant amounts in the cells of a normal epidermis, including melanocytes [130, 244]. Although a study described that S100A8/A9 was absent from melanocytic lesions [244], other studies have suggested that they participate in melanoma progression. S100A9 has been shown to promote melanoma metastases formation in a mouse model [245]. In a different mouse model of melanoma, the levels of MDSCs correlated with the levels of S100A9, [246]. In addition, Saha et al. recently demonstrated that tail-vein injected B16F10 melanoma cells, that do not express S100A8 or S100A9, could migrate to S100A8/A9 abondant lungs of uteroglobin-knockout mice [23]. The migration of the B16F10 cells occurred along a concentration gradient of S100A8/A9, and resulted in the formation of secondary tumors in the lungs [23].

S100A8/A9 mediates their effect through the interaction with cell surface receptors or mole‐ cules. The two pattern recognition receptors, toll-like receptor 4 (TLR-4) and RAGE, have been shown to transmit RAGE signaling [23, 247, 248]. In certain conditions, signaling through RAGE has been shown to require carboxylated glycans that are covalently attached to RAGE [249, 250]. The importance of RAGE glycosylation for RAGE signaling has also been demon‐ strated for another member of the S100 protein family, S100A12 [251]. S100A8/A9 has been shown to interact with other glycans, such as heparin or heparin sulfate glycosaminoglycans [137]. In addition, S100A9 has also been shown to interact with and transmit signal through EMMPIRIN [245].

#### *3.1.6. Other S100 proteins*

Other S100 proteins are found in cells of the epidermis (reviewed by [130]), and their associ‐ ation with melanoma will be briefly described in this paragraph.

S100A7 or "psoriasin" has been linked to psoriasis because of its up-regulation in this disease [252, 253]. Although no direct role of S100A7 in melanoma has been described yet, in one study, significantly higher levels of S100A7 were found in the urine of melanoma patients compared to that of healthy patients [254].

S100A10 has been found expressed at various levels in melanoma tumor samples and mela‐ nocytes [22, 244]. Our recent study where we compared the levels of S100 proteins in WM115 xenografts, generated from either control WM115 cells or from RAGE overexpressing WM115 cells, showed that S100A10 was up-regulated in the RAGE overexpressing tumors (unpub‐ lished data). Comparison of the WM115-RAGE cells with control cells did not reveal any difference in S100A10 levels between the two cell lines, suggesting that RAGE overexpression was responsible for the up-regulation of S100A10 in the tumors. S100A10 could therefore play a role in melanoma progression.

S100A11 can either promote tumor growth or play the role of tumor suppressor, depending of the type of cancer (reviewed in [255]). S100A11 has been suggested to play a role in uveal melanoma, but has not yet been linked to cutaneous melanoma [256].

### **3.2. Other ligands of RAGE**

sor cells (MDSC), and also promote the expansion of these cells, resulting in increased tumor

Neither S100A8 nor S100A9 is present in significant amounts in the cells of a normal epidermis, including melanocytes [130, 244]. Although a study described that S100A8/A9 was absent from melanocytic lesions [244], other studies have suggested that they participate in melanoma progression. S100A9 has been shown to promote melanoma metastases formation in a mouse model [245]. In a different mouse model of melanoma, the levels of MDSCs correlated with the levels of S100A9, [246]. In addition, Saha et al. recently demonstrated that tail-vein injected B16F10 melanoma cells, that do not express S100A8 or S100A9, could migrate to S100A8/A9 abondant lungs of uteroglobin-knockout mice [23]. The migration of the B16F10 cells occurred along a concentration gradient of S100A8/A9, and resulted in the formation of secondary

S100A8/A9 mediates their effect through the interaction with cell surface receptors or mole‐ cules. The two pattern recognition receptors, toll-like receptor 4 (TLR-4) and RAGE, have been shown to transmit RAGE signaling [23, 247, 248]. In certain conditions, signaling through RAGE has been shown to require carboxylated glycans that are covalently attached to RAGE [249, 250]. The importance of RAGE glycosylation for RAGE signaling has also been demon‐ strated for another member of the S100 protein family, S100A12 [251]. S100A8/A9 has been shown to interact with other glycans, such as heparin or heparin sulfate glycosaminoglycans [137]. In addition, S100A9 has also been shown to interact with and transmit signal through

Other S100 proteins are found in cells of the epidermis (reviewed by [130]), and their associ‐

S100A7 or "psoriasin" has been linked to psoriasis because of its up-regulation in this disease [252, 253]. Although no direct role of S100A7 in melanoma has been described yet, in one study, significantly higher levels of S100A7 were found in the urine of melanoma patients compared

S100A10 has been found expressed at various levels in melanoma tumor samples and mela‐ nocytes [22, 244]. Our recent study where we compared the levels of S100 proteins in WM115 xenografts, generated from either control WM115 cells or from RAGE overexpressing WM115 cells, showed that S100A10 was up-regulated in the RAGE overexpressing tumors (unpub‐ lished data). Comparison of the WM115-RAGE cells with control cells did not reveal any difference in S100A10 levels between the two cell lines, suggesting that RAGE overexpression was responsible for the up-regulation of S100A10 in the tumors. S100A10 could therefore play

S100A11 can either promote tumor growth or play the role of tumor suppressor, depending of the type of cancer (reviewed in [255]). S100A11 has been suggested to play a role in uveal

melanoma, but has not yet been linked to cutaneous melanoma [256].

ation with melanoma will be briefly described in this paragraph.

growth [239-243].

334 Melanoma – Current Clinical Management and Future Therapeutics

tumors in the lungs [23].

EMMPIRIN [245].

*3.1.6. Other S100 proteins*

to that of healthy patients [254].

a role in melanoma progression.

#### *3.2.1. Advanced Glycation End products (AGEs)*

Among RAGE ligands, AGEs were the first group of ligands to be identified [89]. AGEs form a group of very heterogeneous compounds since they are the result of condensation and oxidation reactions between proteins and sugars [257]. Although the term AGE was initially reserved for brown, fluorescent and cross-linked structures that were produced during the Maillard reaction, such as those found in glycated collagen, it is now used to describe other types of modifications and AGEs now include proteins containing carboxymethyllysine, carboxymethyl hydroxylysine or pyrraline [257]. Since many types of sugars (glucose, ribose…) can modify many proteins, the number of different structures produced by glycation is very large. The large heterogeneity in AGE compounds renders the comparison among studies also very difficult. When comparing the results of studies involving AGEs, it is therefore important to know the type of AGEs used in the study.

Melanoma cells have been shown to produce high levels of reactive carbonyl species such as glyoxal, methylglyoxal and malondialdehyde, which all can lead to the glycation of proteins, and which have been implicated in melanoma cell proliferation and formation of metastases [258-260]. Abe et al. investigated the effects of different AGEs (glucose-derived AGE, glycer‐ aldehyde-derived AGE, glycoaldehyde AGE, methylglyoxal-AGE, glyoxal-AGE and carbox‐ ymethyllysine-AGE) on melanoma cell proliferation, migration and invasion, and showed that all these AGEs were strongly present in human melanoma specimen whereas they were hardly detected in melanocytes [21]. However, the same authors showed that only certain types of AGE compounds (glyceraldehyde-derived AGE and glycoaldehyde AGE) could stimulate the proliferation, migration and invasion of G361 metastatic human melanoma cells *in vitro*. The role of RAGE in the *in vitro* AGE-dependent proliferation was demonstrated with anti-RAGE antibodies [21]. In addition, the authors showed that treatment of mice carrying G361 mela‐ noma tumors, with anti-RAGE antibodies, resulted in reduced tumor growth and formation of lungs metastases [21]. These results strongly suggested a role of AGE/RAGE in the devel‐ opment of melanoma tumors by the G361 human melanoma cell line [21]. Our detailed study on melanoma cell proliferation of a panel of 20 glycated proteins showed that many factors influenced the proliferation, such as the extent of lysine modification, the percentage of β-sheet and the oligomerization state of the glycated proteins [261]. We showed that glycated proteins that demonstrated higher percentage of oligomeric forms, β-sheet content and modification of lysine, promoted stronger cell proliferation that proteins that contained lower levels of oligomers, β-sheet or lysine modification.

#### *3.2.2. High Mobility Group Box 1 protein (HMGB1)*

HMGB-1 is present in most eukaryotic cells (reviewed by [97]). It functions both as a nuclear protein, where it binds to DNA and assists in the transcription of multiple genes, and as an extracellular protein, where it binds to the pattern recognition receptors TLR-2, TLR-4 and TLR-9 as well as to RAGE, thereby promoting inflammation, mediating response to infection and injury as well as promoting cell proliferation, migration or invasion [98, 262-266]. HMGB-1 is also described as an alarmin or damage-associated molecular pattern [267]. HMGB-1 interacts directly with a large number of transcription factors that are relevant in cancer, and include the tumor suppressor p53, p73, the retinoblastoma protein (RB), the p50 subunit of NF-κB and the estrogen receptor (reviewed by [268]). The release of HMGB-1 has been shown to be triggered by necrosis or apoptosis, such as following treatment of tumors with a chemo‐ therapeutic agent [264, 266, 267].

HMGB-1 has also been shown to be released from necrotic or apoptotic melanoma cells [269]. A recent study showed that HMGB-1 could be released from keratinocytes in culture, or from murine skin, following exposure to UV light [270]. Using a genetically engi‐ neered mouse model of melanoma, Bald et al. showed that UV exposure also promoted metastasis and angiogenesis, through HMGB-1, and TLR-4 signaling [271]. In a different mouse model, Tang et al. showed the role of the HMGB-1/RAGE axis in promoting melanoma tumor growth [272]. In melanoma patients, HMGB-1 levels have also been shown to predict patient survival rates [273].

#### *3.2.3. Glycosaminoglycans, β2 integrin Mac-1 and phosphatidylserine*

RAGE has been shown to interact with various glycosaminoglycans such as chondroitin sulfate, dermatan sulfate and heparin sulfate [108]. Many of these glycosaminoglycans are abundantly present in tumor stroma and have been shown to be key players of melano‐ ma progression and metastasis formation [274, 275]. RAGE also interacts with β2 integrin Mac-1 on leukocytes and has been shown to promote leukocyte recruitment at the site of inflammation [40]. The interaction between RAGE and Mac-1 is dependent of the pres‐ ence of cations, and has been shown to be significantly augmented by the presence of S100B. In a different study, S100A9 has also been shown to activate β2 integrin Mac-1 on neutro‐ phils suggesting that other S100 proteins could also participate to the complex between Mac-1 and RAGE [276]. Leukocyte recruitment at tumor sites is an important process that allows cytotoxic T-cells to infiltrate tumors and to help in the elimination of cancer cells [277]. In melanoma, success of the therapy with interleukin-2 (IL-2) is based on the infiltration of cytotoxic T-cells to the interior of the tumor [277]. The involvement of S100 proteins and RAGE in leukocyte recruitment suggests that therapeutic approaches target‐ ing RAGE should be carefully evaluated to avoid inhibiting the recruitment of cytotoxic Tcells at the site of melanoma tumors. On the other hand, targeting RAGE could also result in suppressing the recruitment of MDSCs, another form of leukocyte expressing β2 integrin Mac-1 [278], at the tumor site, which would be beneficial for patients [279, 280].

RAGE has also been shown to interact with the negatively charged phospholipid phosphati‐ dylserine (PS) [106]. This interaction has direct relevance in melanoma because PS has been shown to be exposed to the outer leaflet of the plasma membrane of cells forming melanoma metastases [281]. An association between malignancy of melanoma and PS exposure has also been described [281]. Further studies would be necessary to determine whether RAGE/PS could be targeted in melanoma.

## **4. Conclusion and therapeutic approaches**

In the last two decades, RAGE has emerged as a new therapeutic target in a large number of diseases. Because of the large number of ligands of RAGE that are relevant in melanoma, targeting RAGE/ligand appears to be a promising approach. Several molecules could be used as inhibitors and include the soluble form of the receptor (sRAGE), antibodies against RAGE or small molecules. Both soluble RAGE and anti-RAGE antibodies have been used to demon‐ strate the role of RAGE in experimental models of a large number of diseases such as athero‐ sclerosis, Alzheimer's disease or melanoma [21, 60, 282-286]. Two small molecule inhibitors of RAGE are currently available. One compound PFS-ZM1 has been recently used in an experi‐ mental model of Alzheimer's disease [287]. This inhibitor interacts with the V domain of RAGE and blocks the interaction of amyloid β-peptide with the receptor. The second compound (TPP488) has been evaluated for safety and efficacy, in a phase 2 study, in mild to moderate Alzheimer's disease patients. However, because of the large variation in the levels of RAGE observed in melanoma tumor samples, it is currently not known whether targeting RAGE would be efficacious in all melanoma tumors or only in the subset of tumors where it is overexpressed. Additional studies would be necessary to answer this important question.

## **Acknowledgements**

is also described as an alarmin or damage-associated molecular pattern [267]. HMGB-1 interacts directly with a large number of transcription factors that are relevant in cancer, and include the tumor suppressor p53, p73, the retinoblastoma protein (RB), the p50 subunit of NF-κB and the estrogen receptor (reviewed by [268]). The release of HMGB-1 has been shown to be triggered by necrosis or apoptosis, such as following treatment of tumors with a chemo‐

HMGB-1 has also been shown to be released from necrotic or apoptotic melanoma cells [269]. A recent study showed that HMGB-1 could be released from keratinocytes in culture, or from murine skin, following exposure to UV light [270]. Using a genetically engi‐ neered mouse model of melanoma, Bald et al. showed that UV exposure also promoted metastasis and angiogenesis, through HMGB-1, and TLR-4 signaling [271]. In a different mouse model, Tang et al. showed the role of the HMGB-1/RAGE axis in promoting melanoma tumor growth [272]. In melanoma patients, HMGB-1 levels have also been shown

RAGE has been shown to interact with various glycosaminoglycans such as chondroitin sulfate, dermatan sulfate and heparin sulfate [108]. Many of these glycosaminoglycans are abundantly present in tumor stroma and have been shown to be key players of melano‐ ma progression and metastasis formation [274, 275]. RAGE also interacts with β2 integrin Mac-1 on leukocytes and has been shown to promote leukocyte recruitment at the site of inflammation [40]. The interaction between RAGE and Mac-1 is dependent of the pres‐ ence of cations, and has been shown to be significantly augmented by the presence of S100B. In a different study, S100A9 has also been shown to activate β2 integrin Mac-1 on neutro‐ phils suggesting that other S100 proteins could also participate to the complex between Mac-1 and RAGE [276]. Leukocyte recruitment at tumor sites is an important process that allows cytotoxic T-cells to infiltrate tumors and to help in the elimination of cancer cells [277]. In melanoma, success of the therapy with interleukin-2 (IL-2) is based on the infiltration of cytotoxic T-cells to the interior of the tumor [277]. The involvement of S100 proteins and RAGE in leukocyte recruitment suggests that therapeutic approaches target‐ ing RAGE should be carefully evaluated to avoid inhibiting the recruitment of cytotoxic Tcells at the site of melanoma tumors. On the other hand, targeting RAGE could also result in suppressing the recruitment of MDSCs, another form of leukocyte expressing β2 integrin

Mac-1 [278], at the tumor site, which would be beneficial for patients [279, 280].

RAGE has also been shown to interact with the negatively charged phospholipid phosphati‐ dylserine (PS) [106]. This interaction has direct relevance in melanoma because PS has been shown to be exposed to the outer leaflet of the plasma membrane of cells forming melanoma metastases [281]. An association between malignancy of melanoma and PS exposure has also been described [281]. Further studies would be necessary to determine whether RAGE/PS

therapeutic agent [264, 266, 267].

336 Melanoma – Current Clinical Management and Future Therapeutics

to predict patient survival rates [273].

could be targeted in melanoma.

*3.2.3. Glycosaminoglycans, β2 integrin Mac-1 and phosphatidylserine*

The author was supported by the NDSU College of Pharmacy, Nursing and Allied Sciences and in part by the NDSU Advance FORWARD program sponsored by NSF HRD-0811239 and ND EPSCoR through grant #EPS-0814442.

## **Author details**

Estelle Leclerc\*

Address all correspondence to: Estelle.Leclerc@ndsu.edu

North Dakota State University/Department of Pharmaceutical Sciences, USA

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## **Gangliosides and Antigangliosides in Malignant Melanoma**

Corina-Daniela Ene (Nicolae) and Ilinca Nicolae

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59176

## **1. Introduction**

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2001;108(12):1853-63.

360 Melanoma – Current Clinical Management and Future Therapeutics

Cutaneous malignant melanoma is the most agressive skin cancer with rising incidence in the last years [1]. Nowadays, the only treatment to cure melanoma is the early diagnosis and surgical removal of the primer tumor. New research directions developed in order to discover markers for early detection and therapeutic response of melanoma, treatments that could improve the survival rate. Recent studies showed that melanoma is a heterogeneous group of complex molecular disorders [2-16]. The diversity of these alterations sustain the importance of, on one hand, an individualized diagnosis, prognosis and treatment of melanoma patients, and, on the other hand, the detection of new biomarkers and therapeutic approaches in these patients.

The development of new research and investigation techniques in the last years, offered information regarding pathogenesis of melanoma. The skin is considered a hypoxic organ and the low level of oxygen induces the transcription of some hypoxic markers. The cells respond to hypoxia by stimulating the synthesis of some heterodimeric factors, composed by alpha inductible subunit (hypoxia inducible factor alpha-HIF alpha) and beta subunit (aryl hydro‐ carbon receptor nuclear transducer ARNT). Cutaneous melanocytes, from the dermicepidermic junction, lay in a low oxygen medium [16, 17]. Tissue hypoxia can modify cellular behavior by direct influence on: cell cycle, cellular metabolism, differentiation, proliferation and survival, degradation and remodeling of extracellular matrix, tumor migration, invasion and metastasis, angiogenesis, apoptosis, cells sensitivity to antitumor therapy (Table 1) [16-29]. Cells ability to adapt to hypoxia is mediated by several transcription factors. A major role in cells response to hypoxia is played by HIF 1 alpha. Its expression and tissue distribution are influnenced by many factors [3, 30-38]: modulators of cellular degradation (EPF, UCP, VDU2, Sumoylation, DeSUMOylation, Prolyl hydroxylases, PVLH, OS-9, SSAT 1, SSAT 2, GSK3 beta,

© 2015 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 eproduction in any medium, provided the original work is properly cited.

FOXO 4, Calcineurin A), modulators of translation (RNA-binding protein, PTB and HuR, PtdIns3k and MAPK pathways, IRES-mediated translation, calcium signaling bng, miRNA).



FOXO 4, Calcineurin A), modulators of translation (RNA-binding protein, PTB and HuR,

PtdIns3k and MAPK pathways, IRES-mediated translation, calcium signaling bng, miRNA).

**Glucos**e - Adenylate kinase; Aldolase A,C (ALDA,C); Carbonic anhydrase-9,-12; Enolase- 1 (ENO1); Glucose transporter-,1,3 (GLU1,3); Glyceraldehyde phosphate dehydrogenase (GAPDH); Hexokinase1,2 (HK1,2); Lactate dehydrogenase-A (LDHA); Pyruvate kinase M

(PKM); Phosphofructokinase L (PFKL); Phosphoglycerate kinase 1(PGK1); 6-

phosphofructo-2-kinase/fructose-2,6-bisphosphonate-3 (PFKFB3).

Matrix metabolism Matrix metalloproteinases (MMPs) ; Plasminogen activator receptors and inhibitors (PAIs);

Mitochondrial respiration Pyruvate dehydrogenase kinase1 (PDK1) ; Monocarboxylate transporter (MCT4) ;

Transcriptional regulation Differentiated embryo-chondrocyte exppressed gene 1,2 (DEC1,20; Nuclear receptor 77

Vascular tone Nitric oxide synthase2 (NOS2); Heme oxigenase ; Endothelin 1 (ET1) ; Adrenomedullin

Cell proliferation/ survival Insulin-like growth factor-2 (IGF2) ; IGF-factor binding protein 1,2,3 (IGF-BP1,2,3);

Cell migration/invasion Collagen V; Autocrine motility factor/ glucose-6-phosphate isomerase (AMF/GPI);

Angiogenesis **Proangiogenic** - Vascular endothelial growth factor/receptors (VEGF, Flt-1 /VEGfF-R1,

Cell differentiation Notch signal activation; Tertiary complex p300/HIF1/STAT3/ROR gamma; Multistructural

(NUR77); v-ets erythroblastosis virus E26 oncogene homolog1 (ETS1).

Adrenomedulin (ADM); Coiled-coil-DIX1 (CCD1); Transforming growth factor alpha,beta (TGF alpha,beta); Cyclin G2; Survivin ; Notch-1; Nitric oxide synthase 2 (NOS2); P21;

Cathepsin D(CATHD); Integrin-linked kinase; Integrins; Lysyloxidase (LOX); Plasminogen activator receptor and inhibitor1 (PAI1); LDL receptor-related protein1 (LRP1); Microneme protein2/ CD99 (MIC2/CD99); Fibronectin; Urokinase plasminogen activator receptor (UPAR); Proto-oncogenes c-MET; Chemokine receptor type4 ( CXCR4) ; MMP2

Kdr/VEGF-R2); Endocrine-gland-derived VEGF (EG-VEGF); Leptin (LEP); Transforming growth factor –beta 3 (TGF-beta3); Angiopoietin 1,2 (Ang-1,2); TEK- tyrosine kinase endothelial (Tie-2); Adrenomedullin (ADM) ; Fibroblast growth factor (FGFs) ; Placenta

Cell metabolism **Iron** - Erythropoietin (EPO); Transferrin; Transferrin receptor (TfR); Ceruloplasmin

**Nucleotide** - Adenylate kinase; Ecto-5-nucleotidase

**Lipids-**Sphingosine kinase 1 (Sphk 1); Lpin 1.

**pH** - Carbonic anhydrase-9, 12

362 Melanoma – Current Clinical Management and Future Therapeutics

Collagen prolyl hidroxylase .

Cytochrome c oxidase (COX1-4).

Stem cells KLF4; Nanog; Oct-3/4; Oct-4A,SOX2; Wnt/beta-catenin.

Erythropoietin.

(ADM); Alpha-,beta-adrenergic receptor

complex Foxp3/pVHLE3/HIF1/nUb

Cytoskeletal structure Keratin 14,18,19 (KRT 14,18,19); Vimentin

**Aminoacids** - Transglutaminase 2;

**Function Gene**

**Table 1.** Representative target genes of Hypoxia-Inducible Factor1 (HIF1) and their functions [17-29].

Post-translational changes (polyubiquitination by PVHL-signaling for degradation, lysine acetylation by ARD1-facilitating PVHL binding) influence HIF activity. Biological disponibil‐ ity of HIF 1 alpha might be altered by enviromental regulators (nickel, cobalt, arsenite, chromium, cadmium, desferroxamine, cigarette smoking, UVB, cytokines, hormones, onco‐ genes). Alterations in HIF-signaling pathways have major role in initiation and progression of malignant tumors (Table 1) [17-38]. Recent studies confirmed that HIF 1 alpha controls the expression of some genes involved in melanoma biology [39, 40]. Changes in lipids biochem‐ istry associated with HIF overexpression were described in different types of neoplastic cells. Hypoxia promotes synthesis of free fatty acids, cholesterol, phospholipids, hormones, prostaglandins, leucotriens, sphingolipids. Cellular lipid uptake is increased in hypoxic conditions due to the interactions between lipids and hypoxia signaling pathways [41, 42]. These imbalances induce alteration of cell cycle, cellular proliferation, apoptosis, signal transduction, or alteration of antigenic structure of cellular membranes. Some studies reported the expression of an extensive adipose gene in pVHL cancer, in which HIF is constitutively activated [43]. Hypoxia inducible genes influence lipid droplet formation (hypoxia-inducible protein 2), prostaglandin biosynthesis (cyclooxygenase 2), lipid signaling systems (lipoxyge‐ nase 12-lox, sphingosine kinase, SphK1) and synthetic processes (stearoyl-CoA desaturase-1, a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids) [44-47]. Some of these genes are direct targets of HIF, while others are up-regulated by transcriptional factors HIF-dependent. Hypoxia interacts with transcriptional networks involved in lipid metabo‐ lism, including sterol response element binding proteins (SREBPs), DEC1/2 and GATA2/3 [9-11, 48-52].

Gangliosides form lipoproteic domains in cells membrane and affect series of fundamental biological processes like cell signal transduction, cell growth and differentiation, imune responses, cell transformation and degradation. Reference researches about gangliosides system in malignant tumors offer new data, useful for a better understanding of melanoma biology. Melanoma development needs high levels of oxygen and substances over the offer of the existent blood vessels. Angiogenesis is promoted under these conditions [10, 51]. The main stimuli of the angiogenic switch are: HIF 1alfa, low ph, hypoglycemia, presence of reactive oxygen/nitrogen species, inflammation. In hypoxic conditions, melanoma cells release proangiogenesis (VEGFs, FGFs, PDGF, HGF, TGF, TNF, PGF, Ang-1, IL8) and antiagiogenesis (trombospondin, angiostatin, endostatin, Ang2, IFNs, IL12, fibronectin, TIMPs, PAI-1, dopamine, retinoic acid, vitamin D) factors [56-58]. These molecules interact with RTKs signaling pathway from melanoma cells, endothelial cells, pericytes, immune cells. Ganglio‐ sides and their degradation products could influence angiogenesis in malignant melanoma by multiple mechanisms [59-61]. Monosialogangliosides and gangliosides in b series counteract the effects of proangiogenic growth factors, while complex gangliosides control endothelial cell response to the action of proangiogenic factors. Lisogangliosides and sphingosine inhibit the activity of protein-kinase associated growth factors, GM1 and GM3 inactivate PDGFR, GD1a and GD1b inactivate PKC, GM3 inhibits EGFR, GD1a and GQ1b intensify the activity of calcium/calmoduline dependent kinase [54, 61].

Gangliosides control cell differentiation and proliferation [62-64]. Monosialogangliosides suppress cell growth (GM1-MMP9, GM1-PDGF, GM1-NGF, GM3-EGF, GM3-IGFs, GM2 pFAK), while polisialogangliosides stimulate cell growth (GD3 binding integrin, p-FAK, pp130Cas, p=paxilin, p-Yes or GD2 binding p-FAK, p-p38, c-Met) [65]. The transduction mechanism is complex including complex signaling pathways (p38-MAPK, Erk1/2, p13k/AKT, JNK1/2/3) [65-70]. Gangliosides act as inhibitors or promoters of cell apoptosis in melanoma [71, 72]. GD3 induces apoptosis by activating caspases and stimulates production of reactive oxygen species (ROS). GD3 acetylation cancels apoptotic effect of GD3, conferring resistance to antitumor therapy [73, 74]. GM3 induces apoptosis by intracellular accumulation of ceramide; GM1 stimulates apoptosis by disrupting the flow of intramitochondrial calcium [73-75].

GD1b, GT1b, GQ1b have inhibitor effects on adenylate cyclase in peripheral T cells and suppress the production of cytokines, controlling humoral immune response (IL4, IL5) [76]. GM1 and GD1a inhibit the activity of protein-kinases involved in the immune response. GQ1b promotes production of immunoglobulins [74, 77, 78]. Anti-GD2 antibodies modify integrin conformation through signaling pathways FAK, ERK, p38/MAPK [74, 77, 78].

Gangliosides influence anticancerous therapy through several mechanisms [51, 79-85] i) overexpression of proangiogenic factors; ii) functional mutations in tumor suppression genes; iii) overexpression of some oncogenes conferring resistance to hypoxia, and though instability of genes and inhibition of apoptosis; iv) impaired detoxification and DNA repair mechanisms; v) mutations of endothelial cells; vi) selective activation of genes associated with melanoma resistance to apoptosis; vii) cells inability to accumulate sphingosine, sphingaanine and ceramide; viii) ability of melanoma cells to acetylate gangliosides.

protein 2), prostaglandin biosynthesis (cyclooxygenase 2), lipid signaling systems (lipoxyge‐ nase 12-lox, sphingosine kinase, SphK1) and synthetic processes (stearoyl-CoA desaturase-1, a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids) [44-47]. Some of these genes are direct targets of HIF, while others are up-regulated by transcriptional factors HIF-dependent. Hypoxia interacts with transcriptional networks involved in lipid metabo‐ lism, including sterol response element binding proteins (SREBPs), DEC1/2 and GATA2/3

Gangliosides form lipoproteic domains in cells membrane and affect series of fundamental biological processes like cell signal transduction, cell growth and differentiation, imune responses, cell transformation and degradation. Reference researches about gangliosides system in malignant tumors offer new data, useful for a better understanding of melanoma biology. Melanoma development needs high levels of oxygen and substances over the offer of the existent blood vessels. Angiogenesis is promoted under these conditions [10, 51]. The main stimuli of the angiogenic switch are: HIF 1alfa, low ph, hypoglycemia, presence of reactive oxygen/nitrogen species, inflammation. In hypoxic conditions, melanoma cells release proangiogenesis (VEGFs, FGFs, PDGF, HGF, TGF, TNF, PGF, Ang-1, IL8) and antiagiogenesis (trombospondin, angiostatin, endostatin, Ang2, IFNs, IL12, fibronectin, TIMPs, PAI-1, dopamine, retinoic acid, vitamin D) factors [56-58]. These molecules interact with RTKs signaling pathway from melanoma cells, endothelial cells, pericytes, immune cells. Ganglio‐ sides and their degradation products could influence angiogenesis in malignant melanoma by multiple mechanisms [59-61]. Monosialogangliosides and gangliosides in b series counteract the effects of proangiogenic growth factors, while complex gangliosides control endothelial cell response to the action of proangiogenic factors. Lisogangliosides and sphingosine inhibit the activity of protein-kinase associated growth factors, GM1 and GM3 inactivate PDGFR, GD1a and GD1b inactivate PKC, GM3 inhibits EGFR, GD1a and GQ1b intensify the activity

Gangliosides control cell differentiation and proliferation [62-64]. Monosialogangliosides suppress cell growth (GM1-MMP9, GM1-PDGF, GM1-NGF, GM3-EGF, GM3-IGFs, GM2 pFAK), while polisialogangliosides stimulate cell growth (GD3 binding integrin, p-FAK, pp130Cas, p=paxilin, p-Yes or GD2 binding p-FAK, p-p38, c-Met) [65]. The transduction mechanism is complex including complex signaling pathways (p38-MAPK, Erk1/2, p13k/AKT, JNK1/2/3) [65-70]. Gangliosides act as inhibitors or promoters of cell apoptosis in melanoma [71, 72]. GD3 induces apoptosis by activating caspases and stimulates production of reactive oxygen species (ROS). GD3 acetylation cancels apoptotic effect of GD3, conferring resistance to antitumor therapy [73, 74]. GM3 induces apoptosis by intracellular accumulation of ceramide; GM1 stimulates apoptosis by disrupting the flow of intramitochondrial calcium

GD1b, GT1b, GQ1b have inhibitor effects on adenylate cyclase in peripheral T cells and suppress the production of cytokines, controlling humoral immune response (IL4, IL5) [76]. GM1 and GD1a inhibit the activity of protein-kinases involved in the immune response. GQ1b promotes production of immunoglobulins [74, 77, 78]. Anti-GD2 antibodies modify integrin

conformation through signaling pathways FAK, ERK, p38/MAPK [74, 77, 78].

[9-11, 48-52].

[73-75].

of calcium/calmoduline dependent kinase [54, 61].

364 Melanoma – Current Clinical Management and Future Therapeutics

In a recent study the authors showed that malignant fenotype was associated with overex‐ pression of ganglioside sialic acid on the membranes of melanoma cells. The tissue level of ganglioside sialic acid was correlated with histological markers of melanoma (Breslow index, Clark level, presence/absence of ulceration) [53]. The gangliosides identified in melanoma were complex (Fig. 1) and they could be used as a differentiation marker between normal and malignant tissue [55]. The model of membranous gangliosides was characteristic for a cell type and for a tumor stage. The observed gangliosides in malignant melanoma were: *GD3>GM3>GD2>GM2>O-Ac GD3>GD1a>GT1b>GD1b>GQ1b>GM1*; in dysplastic nevi: *GD3>GM3>GD2>GM2>GM1*; in healthy tissue around the tumor: *GM3>GM2>GD3>GD2>GM1*. The transition from radial to vertical growth of melanoma was followed by high synthesis of polisialogangliosides [53].

The compositional analysis of gangliosides in melanocytic tumors showed that aberant glycosilation of sphingolipids could stimulate or inhibate invasion and metastasis of malignant tumor cells. High levels of monosialogangliosides could be found in benign tumors with slight evolution and in healthy tissue. Polisialogangliosides had high levels in malignant prolifera‐ tions with quick and irregular evolution. A special attention was given to gangliosides acetylation in malignant melanoma. Acetylated gliocsphigolipids were determined only in melanoma cells with vertical development. O-acetylation was selectively on disialoganglio‐ sides and was associated with metastasis of malignant melanoma [53].

Another research subject was the immunogenic potential of melanoma-associated ganglio‐ sides. Several tumor gangliosides induced the synthesis of antiganglioside antibodies in melanoma patients [9, 85-92]. The role of these antibodies is a subject of great current interest in medical research [9, 10, 78, 90, 93]. The data regarding endogenous immune response against gangliosides in melanoma patients and its pathophysiological relevance in management of melanoma were the main interests of the authors.

The **objective** of the study was to evaluate the role of gangliosides and antiganglioside antibodies in detection, staging and progression of cutaneous malignant melanoma. We aimed to determine the status of serum gangliosides and antiganglioside antibodies in patients with untreated malignant melanoma compared with patients with dysplastic nevi and control, their variation with surgical removal of the tumor and the relation between these parameters and some histological/biochemical factors used for melanoma staging (accepted by American Joint Committee of Cancer).

**Figure 1.** Metabolism of gangliosides in malignant melanoma. **GM1**=Gal-3GalNAc-4(Neu5Ac-3)Gal-4GlcCer; **GM2**=Gal‐ NAc-4(Neu5Ac-3)Gal-4GlcCer; **GM3**=Neu5Ac-3Gal-4GlcCer; **GD1a**=Neu5Ac-3Gal-3GalNAc-4(Neu5Ac-3)Gal-4GlcCer; **GD1b=**Gal-3GalNAc-4(Neu5Ac-8Neu5Ac-3)Gal-4GlcCer; **GD2**=GalNAc-4(Neu5Ac-8Neu5Ac-3)Gal-4GlcCer; **GD3**=Neu5Ac-8Neu5Ac-3Gal-4GlcCer; **GT1b**=Neu5Ac-3Gal-3GalNAc-4(Neu5Ac-8Neu5Ac-3)Gal-4GlcCer; **GQ1b**=Neu5Ac-8Neu5Ac-3Gal-3GalNAc-4(Neu5Ac-8Neu5Ac-3)Gal-4GlcCer; **O-acetyl-GD3**=Acetyl-O-Neu5Ac-8Neu5Ac-3Gal-4Glc1Cer; Glc=glucose; Gal=galactose; GalNAc=N-acetyl-galactosamine; NeuAc=neuraminicacid; Cer=ceramide; NANA=N-acetyl-neuraminic-acid.

## **2. Materials and method**

The study lasted five years and was based on the prospective-observational analysis of patients with melanocytic lesions. All the patients in the study signed the informed consent accordind with the Declaration from Helsinki in 1964. Our study included three groups: malignant melanoma group (128 adult patients diagnosed with malignant melanoma, with adequate nutritional status, without associated diseases, with no treatment for melanoma before the inclusion in the study), dysplastic nevi group (48 adult patients with dysplastic nevi without associated diseases) and control group (48 healthy subjects). The groups were similar for age, sex and nutritional characteristics.

No patients with age under 18 years, pregnancy, alcoholism or drug dependence, with any hormonal, antidepressive, antioxidant, with MAO inhibitors or blockers of dopaminergic receptors treatment or with associated diseases (neurological, psychiatric, digestive, endo‐ crine, cardiovascular, hepatic, renal, pulmonary, metabolic, autoimmune disorders, chronic infections/inflammation, others neoplastic diseases) were included in the study.

*The diagnosis protocol* was based on clinical examination, common haematological and bio‐ chemical determinations for all the patients in the study. The histological and immunohisto‐ chemical analysis were made for the patients with malignant melanoma and dysplastic nevi. After the diagnosis, in malignant melanoma and nevi groups the tumors were surgically removed. In all the patients included in the study we determined molecular markers that could indicate melanoma progression (lactate dehydrogenase, interleukin 8, C reactive protein); gangliosides and antigangliosides.

In nevi and melanoma groups, the variations of these factors were evaluated in six moments: T0-when included in the study; T1 – 8 weeks after surgical removal of the tumour, at 3(T2), 6 (T3), 12 (T4), 18 (T5) and 36 (T6) months after surgical removal of the tumour. In melanoma group, the variations of gangliosides and antigangliosides were analysed in relation to serum factors – lactate dehydrogenase, interleukin 8, C reactive proteins and histological factors – Breslow index, Clark level, presence/absence of ulceration.

#### **2.1. Clinical and biological characteristics of melanoma patients**

The clinical examination showed the following localization of cutaneous malignant melanoma: 64% on trunk, 21% on limbs, less than 10% on head and neck (Fig. 2). By histological charac‐ teristics, we found nodular melanoma in 60 patients (47%), superficial melanoma in 22 patients (17%), acral lenticular melanoma in 9 patients (7%) and unclassified melanoma in 22 patients (17%) (Fig. 3).

**Figure 2.** Anatomical site of melanomas

**2. Materials and method**

acid; Cer=ceramide; NANA=N-acetyl-neuraminic-acid.

366 Melanoma – Current Clinical Management and Future Therapeutics

sex and nutritional characteristics.

The study lasted five years and was based on the prospective-observational analysis of patients with melanocytic lesions. All the patients in the study signed the informed consent accordind with the Declaration from Helsinki in 1964. Our study included three groups: malignant melanoma group (128 adult patients diagnosed with malignant melanoma, with adequate nutritional status, without associated diseases, with no treatment for melanoma before the inclusion in the study), dysplastic nevi group (48 adult patients with dysplastic nevi without associated diseases) and control group (48 healthy subjects). The groups were similar for age,

**Figure 1.** Metabolism of gangliosides in malignant melanoma. **GM1**=Gal-3GalNAc-4(Neu5Ac-3)Gal-4GlcCer; **GM2**=Gal‐ NAc-4(Neu5Ac-3)Gal-4GlcCer; **GM3**=Neu5Ac-3Gal-4GlcCer; **GD1a**=Neu5Ac-3Gal-3GalNAc-4(Neu5Ac-3)Gal-4GlcCer; **GD1b=**Gal-3GalNAc-4(Neu5Ac-8Neu5Ac-3)Gal-4GlcCer; **GD2**=GalNAc-4(Neu5Ac-8Neu5Ac-3)Gal-4GlcCer; **GD3**=Neu5Ac-8Neu5Ac-3Gal-4GlcCer; **GT1b**=Neu5Ac-3Gal-3GalNAc-4(Neu5Ac-8Neu5Ac-3)Gal-4GlcCer; **GQ1b**=Neu5Ac-8Neu5Ac-3Gal-3GalNAc-4(Neu5Ac-8Neu5Ac-3)Gal-4GlcCer; **O-acetyl-GD3**=Acetyl-O-Neu5Ac-8Neu5Ac-3Gal-4Glc1Cer; Glc=glucose; Gal=galactose; GalNAc=N-acetyl-galactosamine; NeuAc=neuraminic-

> From patients with melanoma, 28 cases (22%) had Clark II, 47 cases (37%) had Clark III, 36 cases (28%) had Clark IV, 17 cases (13%) had Clark V. None of the patients included in the study had Clark I melanoma. From the patients with melanoma, 20.68% had Breslow under 1.00mm, 24.82% Breslow 1.01-2.0mm, 19.71% Breslow 2.01-3.0mm, 18.49% Breslow over 3.01mm. The ulceration was present in 12.5% patients with melanoma. After staging melano‐

**Figure 3.** Histological features of melanomas

**Figure 4.** Melanomas Clark levels

**Figure 5.** Clinical stages of melanomas

ma, we identified melanoma stage I – 51 cases, stage II – 54 cases, stage III – 54 cases, stage IV – 9 cases (Fig. 5).

The researchers in melanoma field considered lactatdehydeogenase (LDH), C reactive protein (CRP), interleukin 8 (IL8) useful for melanoma follow-up. High serum levels of those markers indicate the progression of melanoma. We presented their levels in the studied groups in Table 2.


**Table 2.** Serum level of LDH, CRP and IL8 in melanoma, dysplastic nevi and control groups

In melanoma group, LDH had the following levels at T0: in 0.5% cases smaller than 120U/L, in 73% between 120 and 450 U/L and in 26.5% over 450U/L. In dysplastic nevi patients and in control group, LDH level varied between 120 and 450 U/L. We considered the interval 120-450U/L as normal values for LDH. CRP was in 19% patients between 0 and 0.30mg/dl, in 47%patients between 0.30-0.60mg/dl and in 33% over 0.60mg/dl. We considered the interval 0.30-0.60mg/dl as normal level for CRP. IL8 is a marker of angiogenesis. We considered the normal level for IL8 5-42pg/ml. In melanoma group, IL8 had the following levels: in 16% patients under 15pg/ml, in 22% patients between 15 and 42pg/ml, and in 62% over 42pg/ml.

#### **2.2. Statistical analysis**

All the results were analysed using SPSS, a soft for statistic determinations. The results were presented as mean±standard deviation. The variations between groups were determined using t test or ANOVA test. The correlations between groups were calculated using linear regression and Pearson coefficient. p<0.05 was considered with statistical significance. We evaluated the relapse-free survival using Kaplan-Meier curves.

#### **2.3. Laboratory methods**

ma, we identified melanoma stage I – 51 cases, stage II – 54 cases, stage III – 54 cases, stage IV

39%

8% Stage I

47%

17%

22%

13%

28%

Stage II

Stage III

Stage IV

37%

12%

17%

7%

Nodular Superficial Lenticular Acral Others

368 Melanoma – Current Clinical Management and Future Therapeutics

Clark II Clark III Clark IV Clark V

42%

11%

**Figure 3.** Histological features of melanomas

**Figure 4.** Melanomas Clark levels

The researchers in melanoma field considered lactatdehydeogenase (LDH), C reactive protein (CRP), interleukin 8 (IL8) useful for melanoma follow-up. High serum levels of those markers indicate the progression of melanoma. We presented their levels in the

– 9 cases (Fig. 5).

studied groups in Table 2.

**Figure 5.** Clinical stages of melanomas

*Serum determination of gangliosides*. The assessment of gangliosidic acid (LASA) had the following steps: 50 microliters serum were diluted with 150 microliters of cold distilled water and the solution was shacked. There were added 3 ml of choloroform:methanol (v/v) 2:1, at 4-5 celsius degrees. The extraction and partition was finished after adding 0.5ml of cold distilled water. After separating the phases by centrifugation, sialic acid was titred with resorcinol-chlorhidric acid [94].

*Sialic acid assessment by resorcinol method*. Sialic acid, after the cleavage from glicoconju‐ gates through acid hydrolysis, reacted with resorcinol in acid solution, in the presence of divalent cooper ions. Thus, it formed a blue-violet complex. The complex was evaluated by photometric determination at 580nm, after extraction in a mixture of butanol-acetate and butyl-acetate [95, 96].

*Serum assessment of antiganglioside antibodies.* Antiganglioside antibodies were assessed using Euroline kits, by Immunoblot, a technique for in vivo determination (serum or plasma) of human antiganglioside antibodies Ig G and IgM type. We evaluated the immune response against seven types of ganglioside: GM1, GM2, GM3, GD1a, GD1b, GT1b and GQ1b. The test kit contained strips coated with parallel lines of purified antigens [97].

## **3. Results**

#### **3.1. Serum profile of gangliosides**

Serum gangliosides had increased levels in melanoma and nevi patients compared with control, before the surgical removal of the tumours. In metastatic melanoma, gangliosides levels were statistically significant higher than in melanoma without metastasis (Table 3). Serum gangliosides had a statistically significant decrease after the surgical removal of the tumour in patients with primary melanoma (39.82± 21.13 vs 57.29±17.61mg/dl, p<0.05). In patients with metastatic melanoma, respectively, dysplastic nevi, no statistically significant variations were determined with surgical removal of the tumour.


p1-melanoma, metastasis vs dysplastic nevi, p2-melanoma, metastasis, dysplastic nevi vs control group

**Table 3.** Serum levels of gangliosides in melanoma, dysplastic nevi and control groups

Serum gangliosides were analyzed in relation to age, sex, histological characteristics of melanoma (Breslow index, Clark level, histological type of the tumor, presence/absence of ulceration) (Table 4). Serum gangliosides did not vary significantly with age and sex, with tumor localization. Serum gangliosides had statistically significant increased values in patients with high Breslow index, respectively high Clark level. We analyzed serum level of ganglio‐ sides for the same Clark level in relation to Breslow index and histological type of melanoma (Fig 6).



p<0,05 was considered with statistically significance for IC=95%. p1-women vs men, p2-trunk vs limbs, p3-nodular melanoma vs extensive in surface melanoma, p4-Breslow<1.0mm vs Breslow>3.01mm, p5-Clark II vs Clark V, p6-ulcerated melanoma vs mela‐ noma without ulceration

**Table 4.** Serum gangliosides in relation to clinical and histological features of melanoma

**3. Results**

(Fig 6).

**Tumor localization**

**Histological type**

**3.1. Serum profile of gangliosides**

370 Melanoma – Current Clinical Management and Future Therapeutics

Serum gangliosides had increased levels in melanoma and nevi patients compared with control, before the surgical removal of the tumours. In metastatic melanoma, gangliosides levels were statistically significant higher than in melanoma without metastasis (Table 3). Serum gangliosides had a statistically significant decrease after the surgical removal of the tumour in patients with primary melanoma (39.82± 21.13 vs 57.29±17.61mg/dl, p<0.05). In patients with metastatic melanoma, respectively, dysplastic nevi, no statistically significant

Serum gangliosides were analyzed in relation to age, sex, histological characteristics of melanoma (Breslow index, Clark level, histological type of the tumor, presence/absence of ulceration) (Table 4). Serum gangliosides did not vary significantly with age and sex, with tumor localization. Serum gangliosides had statistically significant increased values in patients with high Breslow index, respectively high Clark level. We analyzed serum level of ganglio‐ sides for the same Clark level in relation to Breslow index and histological type of melanoma

p1=0.18

variations were determined with surgical removal of the tumour.

**Groups Gangliosides (mg/dL) p1 p2** Control 18.02±2.78 - 1 Dysplastic nevi 18.86±3.27 1 0.88 Malignant melanoma 57.29±17.61 0.04 0.03 Metastatic melanoma 80.14±19.26 0.00 0.00

p1-melanoma, metastasis vs dysplastic nevi, p2-melanoma, metastasis, dysplastic nevi vs control group

**Parameters Serum gangliosides (mg/dl) P**

Trunk 77.31±19.42 p2=0.30

Extensive in surface melanoma 36.42±18.55 p3=0.03

Women 19.06±2.66

Men 18.17±2.85

Head-neck 49.20±6.23

Limbs 66.81±12.51

Nodular melanoma 81.61±27.43

Lenticular malignant melanoma 72.53±27.51

**Table 3.** Serum levels of gangliosides in melanoma, dysplastic nevi and control groups

**Figure 6.** Graphical representation of serum levels of gangliosides in melanoma for the same Clark level in relation to Breslow index (p<0.05) and histological type of tumour (p>0.05) NM-nodular melanoma, MLM-lenticular melanoma, ALM-acral lenticular melanoma, SSM-extensive in surface melanoma

The correlation coefficient showed a positive relation between ganglioside levels and Clark level (r=0.60, CI=0.30-0.90, p<0.05), respectively, Breslow index (r=0.31, CI=0.19-0.58, p<0.05). For the same Clark level and Breslow index, the production of gangliosides was higher in nodular melanoma and in lentigous acral malignant melanoma compared with lenticular acral melanoma and extensive in surface melanoma (Figure 6).

#### **3.2. Serum profile of antiganglioside antibodies**

The immune response against gangliosides GM1, GM2, GM3, GD1a, GD1b, GT1b, GQ1b was evaluated by antiganglioside antibodies IgG and IgM type, before the surgical removal of the tumor. The types of antiganglioside antibodies in melanoma, dysplastic nevi and control group will be presented in this section only for moment 0. We considered antiganglioside antibodies negative when signal intensity was undetectable (0-5) or low (5-10) and positive when signal intensity was medium (11-50) or high (>50).

#### *3.2.1. Anti-GM1 prevalence in studied groups*

In patients with malignant melanoma, antiganglioside antibodies anti-GM1 IgG type were undetectable in 90.62% patients, with low intensity of the signal in 4.69% patients, with medium intensity of the signal in 4.68% patients. In dysplastic nevi group, 91.64% patients had undetectable IgG antibodies, in 8.33% the signal was low. In the control group we did not detect any positive anti-GM1 of IgG type. 72.65% patients with melanoma had signal of anti-GM1 IgM class negative, 11.71% low signal,13.29% medium signal and 2.34% high signal. 88.33% patients with dysplastic nevi had negative IgM anti-GM1 signal, in 10.42% the signal intensity was low, in 6.25% medium. In 93.75% healthy patients IgM antibodies had negative signal, in 4.17% the signal was low, in 2.08% medium. In melanoma patients anti-GM1 IgG type were positive in 4.68% cases and IgM in 15.63% cases. No statistical differences were observed in anti-GM1 IgG class status between melanoma, dysplastic nevi and control group. Anti-GM1 IgM class varied significantly between the studied groups (Table 5).


p-melanoma vs control, dysplastic vs control, NS=no statistical significance

**Table 5.** Anti-GM1 IgG and IgM class in patients with malignant melanoma, dysplastic nevi and control

Due to its statistically significant variation, we analysed IgM status in relation to clinical and histological features of melanoma. Anti-GM1 status was analyzed in relation to age, sex, histological characteristics of the tumor (Breslow index, Clark level, histological type of the tumor, presence/absence of ulceration) (Table 6).The intensity of the signal did not vary with sex and age. Anti-GM1 varied with histological type: in patients with nodular melanoma the intensity signal was significantly increased compared with acral lenticular melanoma (p<0.05), extensive in surface melanoma (p<0.05) or lenticular melanoma (p<0.05).

The correlation coefficient showed a positive relation between ganglioside levels and Clark level (r=0.60, CI=0.30-0.90, p<0.05), respectively, Breslow index (r=0.31, CI=0.19-0.58, p<0.05). For the same Clark level and Breslow index, the production of gangliosides was higher in nodular melanoma and in lentigous acral malignant melanoma compared with lenticular acral

The immune response against gangliosides GM1, GM2, GM3, GD1a, GD1b, GT1b, GQ1b was evaluated by antiganglioside antibodies IgG and IgM type, before the surgical removal of the tumor. The types of antiganglioside antibodies in melanoma, dysplastic nevi and control group will be presented in this section only for moment 0. We considered antiganglioside antibodies negative when signal intensity was undetectable (0-5) or low (5-10) and positive when signal

In patients with malignant melanoma, antiganglioside antibodies anti-GM1 IgG type were undetectable in 90.62% patients, with low intensity of the signal in 4.69% patients, with medium intensity of the signal in 4.68% patients. In dysplastic nevi group, 91.64% patients had undetectable IgG antibodies, in 8.33% the signal was low. In the control group we did not detect any positive anti-GM1 of IgG type. 72.65% patients with melanoma had signal of anti-GM1 IgM class negative, 11.71% low signal,13.29% medium signal and 2.34% high signal. 88.33% patients with dysplastic nevi had negative IgM anti-GM1 signal, in 10.42% the signal intensity was low, in 6.25% medium. In 93.75% healthy patients IgM antibodies had negative signal, in 4.17% the signal was low, in 2.08% medium. In melanoma patients anti-GM1 IgG type were positive in 4.68% cases and IgM in 15.63% cases. No statistical differences were observed in anti-GM1 IgG class status between melanoma, dysplastic nevi and control group.

Anti-GM1 IgM class varied significantly between the studied groups (Table 5).

**Groups Antibodies type Positive Negative P**

**Table 5.** Anti-GM1 IgG and IgM class in patients with malignant melanoma, dysplastic nevi and control

p-melanoma vs control, dysplastic vs control, NS=no statistical significance

IgG 6 122 NS IgM 20 108 0.00

IgG 0 48 NS IgM 3 45 NS

IgG 0 48 1 IgM 1 47 1

melanoma and extensive in surface melanoma (Figure 6).

**3.2. Serum profile of antiganglioside antibodies**

372 Melanoma – Current Clinical Management and Future Therapeutics

intensity was medium (11-50) or high (>50).

*3.2.1. Anti-GM1 prevalence in studied groups*

Melanoma

Control

Dysplastic nevi


p<0.05 was considered with statistically significance for IC=95%. p1-women vs men, p2-trunk vs limbs, p3-nodular mel‐ anoma vs extensive in surface melanoma, p4-Breslow<1.0mm vs Breslow>3.01mm, p5-Clark II vs Clark V, p6-ulcerated melanoma vs melanoma without ulceration

**Table 6.** Anti-GM1 antibodies IgM class in relation to clinical and histological features of melanoma

Anti-GM1 IgM intensity signal was significantly increased in melanoma on head and neck, compared with melanoma on trunk (p<0.05), respectively on limbs (p<0.05) (Table 6). Anti-GM1 signal varied statistically significant with Breslow index and Clark level. Compared with Breslow <1.0mm, signal intensity was significantly higher in patients with Breslow 2.01-3.0mm (p<0.05), respectively Breslow>3.01mm (p<0.05). Compared with Clark II, signal intensity was significantly higher in patients with Clark IV (p<0.05), respectively, Clark V (p<0.05). Signal intensity of anti-GM1 IgM type antibodies was statistically significant increased in melanoma with ulceration compared with melanoma without ulceration (p<0.05).

#### *3.2.2. Anti-GM2 prevalence in studied groups*

In patients with malignant melanoma, signal of antiganglioside antibodies anti-GM2 IgG type was undetectable in 96.87% patients, with low intensity in 1.57% patients, with medium intensity in 1.56% patients. In dysplastic nevi group, signal of antiganglioside antibodies anti-GM2 IgG type was undetectable in 97.92% cases, with low signal in 2.08% cases. In patients with malignant melanoma, signal of antiganglioside antibodies anti-GM2 IgM type was undetectable in 58.59% patients, with low intensity in 21.10% patients, with medium intensity in 4.68% patients and high intensity in 15.63% patients. In dysplastic nevi group, signal of antiganglioside antibodies anti-GM2 IgM type was undetectable in 70.83% cases, with low signal in 25.12% cases, and with medium intensity in 4.17% cases. In control group, anti-GM2 IgM type was undetectable in 91.67% volunteers, with weak signal in 6.25% and with medium signal in 2.08% volunteers. Only anti-GM2 IgM class varied significantly between the studied groups, therefore we analyzed IgM status by clinical and histological features of melanoma.


p-melanoma vs control, dysplastic vs control, NS=no statistical significance

**Table 7.** Anti-GM2 IgG and IgM class in patients with malignant melanoma, dysplastic nevi and control

Anti-GM2 IgM status was analyzed in relation to age, sex, histological characteristics of the tumor (Breslow index, Clark level, histological type of the tumor, presence/absence of ulceration) (Table 8). The intensity level did not vary with sex and age, with anatomical site of tumor or histological type of melanoma, Breslow index, Clark level, respectively presence/ absence of ulceration.


p<0.05 was considered with statistically significance for IC=95%. p1-women vs men, p2-trunk vs limbs, p3-nodular melanoma vs extensive in surface melanoma, p4-Breslow<1.0mm vs Breslow>3.01mm, p5-Clark II vs Clark V, p6-ulcerated melanoma vs melanoma without ulceration

**Table 8.** Anti-GM2 antibodies IgM class by clinical and histological features of melanoma

#### *3.2.3. Anti-GM3 prevalence in studied groups*

Anti-GM1 IgM intensity signal was significantly increased in melanoma on head and neck, compared with melanoma on trunk (p<0.05), respectively on limbs (p<0.05) (Table 6). Anti-GM1 signal varied statistically significant with Breslow index and Clark level. Compared with Breslow <1.0mm, signal intensity was significantly higher in patients with Breslow 2.01-3.0mm (p<0.05), respectively Breslow>3.01mm (p<0.05). Compared with Clark II, signal intensity was significantly higher in patients with Clark IV (p<0.05), respectively, Clark V (p<0.05). Signal intensity of anti-GM1 IgM type antibodies was statistically significant increased in melanoma

In patients with malignant melanoma, signal of antiganglioside antibodies anti-GM2 IgG type was undetectable in 96.87% patients, with low intensity in 1.57% patients, with medium intensity in 1.56% patients. In dysplastic nevi group, signal of antiganglioside antibodies anti-GM2 IgG type was undetectable in 97.92% cases, with low signal in 2.08% cases. In patients with malignant melanoma, signal of antiganglioside antibodies anti-GM2 IgM type was undetectable in 58.59% patients, with low intensity in 21.10% patients, with medium intensity in 4.68% patients and high intensity in 15.63% patients. In dysplastic nevi group, signal of antiganglioside antibodies anti-GM2 IgM type was undetectable in 70.83% cases, with low signal in 25.12% cases, and with medium intensity in 4.17% cases. In control group, anti-GM2 IgM type was undetectable in 91.67% volunteers, with weak signal in 6.25% and with medium signal in 2.08% volunteers. Only anti-GM2 IgM class varied significantly between the studied groups, therefore we analyzed IgM status by clinical and histological features of melanoma.

with ulceration compared with melanoma without ulceration (p<0.05).

**Groups Antibodies type Positive Negative P**

**Table 7.** Anti-GM2 IgG and IgM class in patients with malignant melanoma, dysplastic nevi and control

Anti-GM2 IgM status was analyzed in relation to age, sex, histological characteristics of the tumor (Breslow index, Clark level, histological type of the tumor, presence/absence of ulceration) (Table 8). The intensity level did not vary with sex and age, with anatomical site of tumor or histological type of melanoma, Breslow index, Clark level, respectively presence/

p-melanoma vs control, dysplastic vs control, NS=no statistical significance

IgG 2 126 NS IgM 26 102 <0.05

IgG 0 48 NS IgM 2 46 NS

IgG 0 48 1 IgM 1 47 1

*3.2.2. Anti-GM2 prevalence in studied groups*

374 Melanoma – Current Clinical Management and Future Therapeutics

Melanoma

Control

Dysplastic nevi

absence of ulceration.

In patients with malignant melanoma, signal of antiganglioside antibodies anti-GM3 IgG type was undetectable in 96.10% patients, with low intensity in 3.12% patients, with medium intensity in 0.78% patients. In dysplastic nevi group, signal of antiganglioside antibodies antiGM3 IgG type was undetectable in 87.50% cases, with low signal in 12.25% cases. IgG did not vary significantly between groups. In patients with malignant melanoma, signal of antigan‐ glioside antibodies anti-GM3 IgM type was undetectable in 74.22% patients, with low intensity in 8.59% patients, with medium intensity in 13.28% patients and high intensity in 3.91% patients. In dysplastic nevi group, signal of antiganglioside antibodies anti-GM3 IgM type was undetectable in 77.08% cases, with low signal in 18.75% cases, and with medium intensity in 4.17% cases. Anti-GM3 IgM class varied significantly between the studied groups (Table 9).


p-melanoma vs control, dysplastic vs control, NS=no statistical significance

**Table 9.** Anti-GM3 IgG and IgM class in patients with malignant melanoma, dysplastic nevi and control

Anti-GM3 IgM status was analyzed in relation to age, sex, histological characteristics of the tumor (Table 10). The intensity level did not vary with sex and age, anatomical site or histo‐ logical type of tumor, Breslow index or Clark level. Anti-GM3 antibodies had statistically significant higher intensity in ulcerated melanoma compared with melanoma without ulceration (Table 10).



p<0.05 was considered with statistically significance for IC=95%. p1-women vs men, p2-trunk vs limbs, p3-nodular mel‐ anoma vs extensive in surface melanoma, p4-Breslow<1.0mm vs Breslow>3.01mm, p5-Clark II vs Clark V, p6-ulcerated melanoma vs melanoma without ulceration

**Table 10.** Anti-GM3 antibodies IgM class by clinical and histological features

#### *3.2.4. Anti-GD1a prevalence in studied groups*

GM3 IgG type was undetectable in 87.50% cases, with low signal in 12.25% cases. IgG did not vary significantly between groups. In patients with malignant melanoma, signal of antigan‐ glioside antibodies anti-GM3 IgM type was undetectable in 74.22% patients, with low intensity in 8.59% patients, with medium intensity in 13.28% patients and high intensity in 3.91% patients. In dysplastic nevi group, signal of antiganglioside antibodies anti-GM3 IgM type was undetectable in 77.08% cases, with low signal in 18.75% cases, and with medium intensity in 4.17% cases. Anti-GM3 IgM class varied significantly between the studied groups (Table 9).

**Groups Antibodies type Positive Negative P**

**Table 9.** Anti-GM3 IgG and IgM class in patients with malignant melanoma, dysplastic nevi and control

**Parameters IgM signal intensity p**

Trunk 9.43±18.08 p2=0.68

Anti-GM3 IgM status was analyzed in relation to age, sex, histological characteristics of the tumor (Table 10). The intensity level did not vary with sex and age, anatomical site or histo‐ logical type of tumor, Breslow index or Clark level. Anti-GM3 antibodies had statistically significant higher intensity in ulcerated melanoma compared with melanoma without

p-melanoma vs control, dysplastic vs control, NS=no statistical significance

376 Melanoma – Current Clinical Management and Future Therapeutics

Women 10.42±19.74

Men 10.88.±19.57

Head-neck 11.85±15.87

Limbs 15.01±25.07

Nodular melanoma 7.73±12.84

Extensive in surface melanoma 14.81±24.57

IgG 1 127 NS IgM 6 122 <0.05

IgG 0 48 NS IgM 2 46 NS

IgG 0 48 1 IgM 0 48 1

p1=0.08

p3=0.57

Melanoma

Control

**Sex**

**Tumor site**

**Histological type**

Dysplastic nevi

ulceration (Table 10).

In patients with malignant melanoma, signal of antiganglioside antibodies anti-GD1a IgG type was undetectable in 89.06% patients, with low intensity in 10.16% patients, with medium intensity in 0.78% patients. In dysplastic nevi group, signal of antiganglioside antibodies anti-GD1a IgG type was undetectable in 95.83% cases, with low signal in 4.17% cases. No positive anti-GD1a IgG type were detected in nevi or control groups. In patients with malignant melanoma, signal of antiganglioside antibodies anti-GD1a IgM type was undetectable in 57.81% patients, with low intensity in 28.12% patients, with medium intensity in 3.12% patients and high intensity in 10.94% patients. In dysplastic nevi group, signal of antiganglioside antibodies anti-GD1a IgM type was undetectable in 77.08% cases, with low signal in 16.67% cases, and with medium intensity in 6.25% cases. No positive anti-GD1a IgM type were detected in nevi or control groups. No statistical differences were observed in anti-GD1a IgG class status between melanoma, dysplastic nevi and control group. Anti-GD1a IgM class varied significantly between the studied groups (Table 11). Due to this variation, we analyzed IgM status in relation to clinical and histological features of melanoma. In melanoma patients anti-GD1a IgG type were positive in 0,78% cases and IgM in 14,06% cases.


p-melanoma vs control, dysplastic vs control, NS=no statistical significance

**Table 11.** Anti-GD1a IgG and IgM class in patients with malignant melanoma, dysplastic nevi and control

Anti-GD1a IgM status was analyzed in relation to age, sex, histological characteristics of the tumor (Breslow index, Clark level, histological type of the tumor, presence/absence of ulceration) (Table 12). The intensity level did not vary with sex and age or histological type of melanoma. The antibodies varied with tumor site: the highest intensity was deter‐ mined in melanoma situated on trunk (p<0.05), followed by melanomas on head and neck (p<0.05) compared with melanomas on limbs. High intensity of anti-GD1a were observed in melanoma with Breslow 2.01-3.0mm (p<0.05), respectively Breslow>3.01mm (p<0.05) compared with Breslow 0.0-1.0mm. Increased intensity of anti-GD1a were determined in Clark IV (p<0.05), respectively Clark V (p<0.05) compared with Clark II melanomas. Anti-GD1a signal intensity varied significantly with ulceration, with higher levels in ulcerated melanomas (p<0.05) (Table 12).



p<0.05 was considered with statistically significance for IC=95%. p1-women vs men, p2-trunk vs limbs, p3-nodular mel‐ anoma vs extensive in surface melanoma, p4-Breslow<1.0mm vs Breslow>3.01mm, p5-Clark II vs Clark V, p6-ulcerated melanoma vs melanoma without ulceration

**Table 12.** Anti-GD1a antibodies IgM class in relation to clinical and histological features of melanoma

#### *3.2.5. Anti-GD1b prevalence in studied groups*

**Groups Antibodies type Positive Negative P**

**Table 11.** Anti-GD1a IgG and IgM class in patients with malignant melanoma, dysplastic nevi and control

**Parameters IgM signal intensity p**

Trunk 16.26±6.65 p2=0.03

1.01-2.0 mm 2.41±1.74 p4=0.03

Women 12.96±24.08

Men 13.32±23.34

Head-neck 10.42±8.31

Limbs 2.38±83.22

Nodular melanoma 25.25±30.38

Extensive in surface melanoma 2.37±1.76 Lenticular melanoma 2.54±1.76 Acral lenticular melanoma 2.33±1.38

0,0-1.0 mm 2.45±1.71

2.01 – 3.0mm 8.93±5.48

Anti-GD1a IgM status was analyzed in relation to age, sex, histological characteristics of the tumor (Breslow index, Clark level, histological type of the tumor, presence/absence of ulceration) (Table 12). The intensity level did not vary with sex and age or histological type of melanoma. The antibodies varied with tumor site: the highest intensity was deter‐ mined in melanoma situated on trunk (p<0.05), followed by melanomas on head and neck (p<0.05) compared with melanomas on limbs. High intensity of anti-GD1a were observed in melanoma with Breslow 2.01-3.0mm (p<0.05), respectively Breslow>3.01mm (p<0.05) compared with Breslow 0.0-1.0mm. Increased intensity of anti-GD1a were determined in Clark IV (p<0.05), respectively Clark V (p<0.05) compared with Clark II melanomas. Anti-GD1a signal intensity varied significantly with ulceration, with higher levels in ulcerated

p-melanoma vs control, dysplastic vs control, NS=no statistical significance

378 Melanoma – Current Clinical Management and Future Therapeutics

IgG 1 127 NS IgM 18 110 <0.05

IgG 0 48 NS IgM 3 45 NS

IgG 0 48 1 IgM 0 48 1

p1=0.93

p3=0.70

Melanoma

Control

**Sex**

**Tumor site**

**Histological type**

**Breslow index**

Dysplastic nevi

melanomas (p<0.05) (Table 12).

In patients with malignant melanoma, signal of antiganglioside antibodies anti-GD1b IgG type was undetectable in 61.71% patients, with low intensity in 34.37% patients, with medium intensity in 3.12% patients. In dysplastic nevi group, signal of antiganglioside antibodies anti-GD1b IgG type was undetectable in 81.25% cases, with low signal in 18.75% cases. In patients with malignant melanoma, signal of antiganglioside antibodies anti-GD1b IgM type was undetectable in 64.84% patients, with low intensity in 25.10% patients, with medium intensity in 4.68% patients and high intensity in 5.46% patients (Table 13).


p-melanoma vs control, dysplastic vs control, NS=no statistical significance

**Table 13.** Anti-GD1b IgG and IgM class in patients with malignant melanoma, dysplastic nevi and control

In dysplastic nevi group, signal of anti-GD1b IgM type was undetectable in 81.25% cases, with low signal in 5.46% cases, and with medium intensity in 1.56% cases. No positive anti-GD1b IgM type were detected in nevi or control groups. In melanoma patients anti-GD1b IgG type were positive in 3.12% cases, while IgM in 10.14% cases. Anti-GD1b IgG class had no significant variations between groups, while, IgM class modified significantly (Table 13).

Anti-GD1b IgM status was analyzed in relation to age, sex, histological characteristics of the tumor (Breslow index, Clark level, histological type of the tumor, presence/absence of ulceration) (Table 14). We did not determine significant variations of anti-GD1b except with the histological type of melanoma. Compared with nodular melanoma, we detected statisti‐ cally increased intensity in extensive in surface melanoma (p<0.05) and acral lentiguos melanoma (p<0.05).


p<0.05 was considered with statistically significance for IC=95%. p1-women vs men, p2-trunk vs limbs, p3-nodular mel‐ anoma vs extensive in surface melanoma, p4-Breslow<1,0mm vs Breslow>3,01mm, p5-Clark II vs Clark V, p6-ulcerated melanoma vs melanoma without ulceration

**Table 14.** Anti-GD1b antibodies IgM class in relation to clinical and histological features of melanoma

#### *3.2.6. Anti-GT1b prevalence in studied groups*

In patients with malignant melanoma, signal of anti-GT1b IgG type was undetectable in 83.59% patients, with low intensity in 16.40% patients. In dysplastic nevi group, signal of anti-GT1b IgG type was undetectable in 85.41% cases, with low signal in 14.58% cases. In patients with malignant melanoma, signal of anti-GT1b IgM type was undetectable in 53.24% patients, with low intensity in 33.59% patients, with medium intensity in 10.93% patients and high intensity in 3.12% patients. In dysplastic nevi group, signal of anti-GT1b IgM type was undetectable in 68.75% cases, with low signal in 27.08% cases, and with medium intensity in 4.16% cases. No positive anti-GT1b IgM or IgG type were detected in control group. The statistical analysis showed that IgM antibodies varied significantly between the studied groups (Table 15). We analyzed IgM status in relation to clinical and histological features of melanoma.


p-melanoma vs control, dysplastic vs control, NS=no statistical significance

Anti-GD1b IgM status was analyzed in relation to age, sex, histological characteristics of the tumor (Breslow index, Clark level, histological type of the tumor, presence/absence of ulceration) (Table 14). We did not determine significant variations of anti-GD1b except with the histological type of melanoma. Compared with nodular melanoma, we detected statisti‐ cally increased intensity in extensive in surface melanoma (p<0.05) and acral lentiguos

p<0.05 was considered with statistically significance for IC=95%. p1-women vs men, p2-trunk vs limbs, p3-nodular mel‐ anoma vs extensive in surface melanoma, p4-Breslow<1,0mm vs Breslow>3,01mm, p5-Clark II vs Clark V, p6-ulcerated

In patients with malignant melanoma, signal of anti-GT1b IgG type was undetectable in 83.59% patients, with low intensity in 16.40% patients. In dysplastic nevi group, signal of anti-GT1b

**Table 14.** Anti-GD1b antibodies IgM class in relation to clinical and histological features of melanoma

p1=0.52

p3=0.00

p4=0.14

p5=0.35

p6=0.23

**Parameters IgM signal intensity P**

Trunk 6.74±12.44 p2=0.14

Women 7.67±12.57

380 Melanoma – Current Clinical Management and Future Therapeutics

Men 9.36±16.02

Head-neck 10.57±21.36

Limbs 14.30±20.88

Nodular melanoma 6.00±11.40

Extensive in surface melanoma 13.72±18.44 Lenticular melanoma 7.36±15.95 Acral lenticular melanoma 16.55±19.00

0-1.0 mm 13.72±18.49

1.01-2.0 mm 11.52±18.17 2.01 – 3.0mm 6.43±14.07 >3.01 mm 5.65±8.33

II 12.44±16.85

III 9.29±15.93 IV 6.44±13.88 V 5.88±7.22

Melanom with ulceration 4.56±6.19

Melanom without ulceration 3.92±5.36

*3.2.6. Anti-GT1b prevalence in studied groups*

melanoma vs melanoma without ulceration

melanoma (p<0.05).

**Sex**

**Tumor site**

**Histological type**

**Breslow index**

**Clark level**

**Ulceration**

**Table 15.** Anti-GT1b IgG and IgM class in patients with malignant melanoma, dysplastic nevi and control

Anti-GT1b status was analyzed in relation to age, sex, histological characteristics of the tumor (Breslow index, Clark level, histological type of the tumor, presence/absence of ulceration) (Table 16). We did not determine significant variations of anti-GD1b except with the tumor site of melanoma. Compared with melanomas on head-neck, we detected statistically in‐ creased intensity in melanomas on trunk (p<0.05) or on limbs (p<0.05).



p<0.05 was considered with statistically significance for IC=95%. p1-women vs men, p2-trunk vs limbs, p3-nodular mel‐ anoma vs extensive in surface melanoma, p4-Breslow<1.0mm vs Breslow>3.01mm, p5-Clark II vs Clark V, p6-ulcerated melanoma vs melanoma without ulceration

**Table 16.** Anti-GT1b antibodies IgM class in relation to clinical and histological features of melanoma

#### *3.2.7. Anti-GQ1b prevalence in studied groups*

In all patients with malignant melanoma, dysplastic nevi and control group, antiganglioside antibodies anti-GQ1b IgG type were negative. In patients with malignant melanoma, signal of antiganglioside antibodies anti-GQ1b IgM type was undetectable in 90.62% patients, with low intensity in 4.68% patients, with medium intensity in 3.90% patients and high intensity in 0.78% patients. No positive anti-GT1b IgM type were detected in dysplastic nevi, respectively, control groups. No statistical differences were observed in anti-GQ1b IgG class status between melanoma, dysplastic nevi and control group. Anti-GQ1b IgM class varied significantly between the studied groups (Table 17). We analyzed IgM status by clinical and histological features of melanoma.


p-melanoma vs control, dysplastic vs control, NS=no statistical significance

**Histological type**

**Breslow index**

**Clark level**

**Ulceration**

Nodular melanoma 6.61±14.96

382 Melanoma – Current Clinical Management and Future Therapeutics

Extensive in surface melanoma 13.27±17.92 Lenticular melanoma 6.31±4.84 Acral lenticular melanoma 16.33±16.62

0-1.0 mm 13.27±17.92

1.01-2.0 mm 11.47±11.04 2.01 – 3.0mm 2.75±2.04 >3.01 mm 7.85±16.35

II 12.18±16.28

III 9.74±10.76 IV 5.33±13.25 V 9.44±19.85

Melanom with ulceration 2.75±1.69

Melanom without ulceration 9.86±15.03

melanoma vs melanoma without ulceration

features of melanoma.

*3.2.7. Anti-GQ1b prevalence in studied groups*

p<0.05 was considered with statistically significance for IC=95%. p1-women vs men, p2-trunk vs limbs, p3-nodular mel‐ anoma vs extensive in surface melanoma, p4-Breslow<1.0mm vs Breslow>3.01mm, p5-Clark II vs Clark V, p6-ulcerated

In all patients with malignant melanoma, dysplastic nevi and control group, antiganglioside antibodies anti-GQ1b IgG type were negative. In patients with malignant melanoma, signal of antiganglioside antibodies anti-GQ1b IgM type was undetectable in 90.62% patients, with low intensity in 4.68% patients, with medium intensity in 3.90% patients and high intensity in 0.78% patients. No positive anti-GT1b IgM type were detected in dysplastic nevi, respectively, control groups. No statistical differences were observed in anti-GQ1b IgG class status between melanoma, dysplastic nevi and control group. Anti-GQ1b IgM class varied significantly between the studied groups (Table 17). We analyzed IgM status by clinical and histological

**Table 16.** Anti-GT1b antibodies IgM class in relation to clinical and histological features of melanoma

p3=0.10

p4=0.07

p5=0.28

p6=0.23

**Table 17.** Anti-GQ1b IgG and IgM class in patients with malignant melanoma, dysplastic nevi and control

Anti-GQ1b IgM status was analyzed in relation to age, sex, histological characteristics of the tumor (Breslow index, Clark level, histological type of the tumor, presence/absence of ulceration) (Table 18) The intensity level did not vary with sex and age, tumor site or histo‐ logical type of melanoma, Clark level, Breslow index, presence/absence of ulceration.



p<0.05 was considered with statistically significance for IC=95%. p1-women vs men, p2-trunk vs limbs, p3-nodular mel‐ anoma vs extensive in surface melanoma, p4-Breslow<1.0mm vs Breslow>3.01mm, p5-Clark II vs Clark V, p6-ulcerated melanoma vs melanoma without ulceration

**Table 18.** Anti-GQ1b antibodies IgM class in relation to clinical and histological features of melanoma

### **3.3. The relationship between gangliosidic system and biochemical factors used for melanoma staging**

In this section we will present the statistical correlations between LDH, CRP and IL8 in melanoma patients, before the surgical removal of the tumor, and also, the relation between the biochemical factors and serum gangliosides/antigangliosides in melanoma patients (Table 19).


**Table 19.** The correlations between gangliosidic system and staging factors in malignant melanoma patients


In melanoma group positive statistical significant correlations were determined between LDH and Il8, LDH and CRP, respectively, CRP and IL8. A strong positive correlation was detected between serum gangliosides and LDH, CRP and IL8 in melanoma group.

1.01-2.0 mm 4.44±7.03 2.01 – 3.0mm 2.81±1.72 >3.01 mm 5.03±8.92

384 Melanoma – Current Clinical Management and Future Therapeutics

II 5.66±15.60

III 5.68±8.64 IV 2.97±1.62 V 4.72±10.68

Melanoma with ulceration 2.87±1.78

Melanoma without ulceration 5.05±10.33

r=0.36 p=0.00

r=0.40

r=0.44 p=0.00

melanoma vs melanoma without ulceration

**melanoma staging**

(Table 19).

IL8

CRP

Serum

gangliosides (LASA)

p<0.05 was considered with statistically significance for IC=95%. p1-women vs men, p2-trunk vs limbs, p3-nodular mel‐ anoma vs extensive in surface melanoma, p4-Breslow<1.0mm vs Breslow>3.01mm, p5-Clark II vs Clark V, p6-ulcerated

**Table 18.** Anti-GQ1b antibodies IgM class in relation to clinical and histological features of melanoma

**3.3. The relationship between gangliosidic system and biochemical factors used for**

In this section we will present the statistical correlations between LDH, CRP and IL8 in melanoma patients, before the surgical removal of the tumor, and also, the relation between the biochemical factors and serum gangliosides/antigangliosides in melanoma patients

**LDH CRP IL8**

r=0.92

p=0.00 - -

**Table 19.** The correlations between gangliosidic system and staging factors in malignant melanoma patients

r=0.43 p=0.00

p=0.00 -

r=0.47 p=0.00

p5=0.60

p6=0.82

**Clark level**

**Ulceration**

**Table 20.** The correlations between antigangliosides and staging factors in malignant melanoma patients

Anti-GM1 correlated positive, statistically significant with LDH, CRP and IL8. Anti-GM2 IgM type correlated weakly, statistically insignificant with the markers used for melanoma staging. Anti-GM3 was negatively associated before surgical intervention, with the markers that indicate the progression of melanoma: statistically insignificant with LDH, statistically significant with CRP respectively, IL8. Anti-GD1a was strongly positive associated with the markers for melanoma staging, before surgical intervention. The b serie of antiganglioside antibodies correlated weakly, negatively, with LDH, CRP and IL8.

#### **3.4. Variation of gangliosidic system in melanoma patients after surgical intervention**

From the 128 patients with malignant melanoma, we selected 30 cases with the same clinical stage (I or II), that were monitored for 36 months using the same investigation protocol. The decision to continue monitoring without receiving any treatment was based on oncologist decision, no signs of melanoma evolution, and also on patients attitude/agreement. The moments of evaluation were: T0-when included in the study; T1 – 8 weeks after surgical removal of the tumor, at 3(T2), 6 (T3), 12 (T4), 18 (T5) and 36 (T6) months after surgical removal of the tumor (Table 21).


1)=statistically significant variation compared with moment 1

**Table 21.** Variation of serum gangliosides and antiganglioside antibodies after surgical removal of melanoma

LASA increased after surgical intervention, having the biggest level at T6, its variation being statistically significant (p<0.05). Anti-GM1 decreased in the first six months after surgical removal of melanoma, afterwards its level increased, at T6 being statistically significant higher compared with its level at T1 (p<0.05). Anti-GM2 decreased during the 36 months of evalua‐ tion, but its variation was not statistically significant (p>0.05). Anti-GM3 decreased signifi‐ cantly at T2, T3, T4, T5, and respectively, at T6 compared with T1. Anti-GD1a increased significantly during the 36 months of evaluation compared with its level at T1. Antigangioside antibodies from b series had a sinuous variation during evaluation: anti-GD1b increased in the first 12 months after surgical removal of the tumor, afterwards, its intensity decreased compared with T1; anti-GT1b increased at T2, and then, its intensity decreased during evaluation; anti-GQ1b increased during evaluation.

After surgical removal of melanoma we detected anti-GM3 IgM type in 12 patients, data that could indicate the regression of melanoma. High pathological levels of serum gangliosides and detectable levels of anti-GD1a IgM were determined in 11 patients, data that could be associated with progression of melanoma and possible metastasis. The increase of serum gangliosides, during evaluation and the absence of antiganglioside antibodies in 7 patients suggest tumoral recurrence.

#### **3.5. Determination of relapse-free survival in malignant melanoma patients**

We determined the relapse free survival using Kaplan-Meier curves, in patients with operated malignant melanoma, monitored for 36 months. Survival rate was assessed in relation to serum levels of gangliosides, anti-GM3 IgM type and anti-GD1a IgM type. We choose a cut-off value suitable for estimating the prognosis.


**Table 22.** Free-relapse survival in patients with malignant melanoma

The relapse-free survival curves in relation to serum gangliosides showed an increase in survival in patients with serum gangliosides lower than 25mg/dl compared with those that had gangliosides over 25mg/dl (22.15±1.19 versus 16.33±1.72 months, p<0.05) (Table 22). The relapse-free survival curves in relation to serum anti-GM3 IgM type showed an increase in survival in patients with signal intensity over 14,20 compared with patients with lower anti-GM3 intensity signal (21.31±1.53 versus 17.82±0.94 months, p<0.05) (Table 22). The relapse-free survival curves in relation to serum anti-GD1a IgM type showed an increase in survival in patients with signal intensity under 15.35 compared with patients with higher anti-GM3 intensity signal (13.85±1.37 versus 21.67±1.04 months, p<0.05) (Table 22).

## **4. Discussions**

**T1 T2 T3 T4 T5 T6**

LASA 40.73±28.40 41.86±28.49 42.30±30.55 44.60±32.22 45.53±34.54 53.11±41.26(1)

Anti-GM1 8.14±12.16 7.80±11.40 6.22±7.12 8.15±9.30 9.33±7.24 13.10±10.25(1)

Anti-GM2 14.35±7.85 10.26±8.20 12.15±5.12 9.37±8.75 10.6±6.25 5.22±6.10

Anti-GM3 10.66±12.37 6,15±10.27(1) 4.06±3.86(1) 6.04±9.05(1) 4.60±8.15(1) 3.90±5.10(1)

Anti-GD1a 17.42±18.32 14,05±9.61 20.10±16.10 21.17±9.25 18.00±10.82 23.10±11.50(1)

Anti-GD1b 11.15±9,10 17.10±6.33 15.16±5.15 11,10±8.32 9.11±8.17 8.30±6.25

Anti-GT1b 11.15±18.10 16.22±17.02 10.61±8.33 8.62±6.55 7.14±8.09(1) 8.32±10.12

Anti-GQ1b 4.10±6,44 6.22±5.17 8.77±10.21(1) 9.40±7.25(1) 6.18±7.35 10.40±7.35

LASA increased after surgical intervention, having the biggest level at T6, its variation being statistically significant (p<0.05). Anti-GM1 decreased in the first six months after surgical removal of melanoma, afterwards its level increased, at T6 being statistically significant higher compared with its level at T1 (p<0.05). Anti-GM2 decreased during the 36 months of evalua‐ tion, but its variation was not statistically significant (p>0.05). Anti-GM3 decreased signifi‐ cantly at T2, T3, T4, T5, and respectively, at T6 compared with T1. Anti-GD1a increased significantly during the 36 months of evaluation compared with its level at T1. Antigangioside antibodies from b series had a sinuous variation during evaluation: anti-GD1b increased in the first 12 months after surgical removal of the tumor, afterwards, its intensity decreased compared with T1; anti-GT1b increased at T2, and then, its intensity decreased during

After surgical removal of melanoma we detected anti-GM3 IgM type in 12 patients, data that could indicate the regression of melanoma. High pathological levels of serum gangliosides and detectable levels of anti-GD1a IgM were determined in 11 patients, data that could be associated with progression of melanoma and possible metastasis. The increase of serum gangliosides, during evaluation and the absence of antiganglioside antibodies in 7 patients

We determined the relapse free survival using Kaplan-Meier curves, in patients with operated malignant melanoma, monitored for 36 months. Survival rate was assessed in relation to serum levels of gangliosides, anti-GM3 IgM type and anti-GD1a IgM type. We choose a cut-off value

**3.5. Determination of relapse-free survival in malignant melanoma patients**

**Table 21.** Variation of serum gangliosides and antiganglioside antibodies after surgical removal of melanoma

1)=statistically significant variation compared with moment 1

386 Melanoma – Current Clinical Management and Future Therapeutics

suggest tumoral recurrence.

suitable for estimating the prognosis.

evaluation; anti-GQ1b increased during evaluation.

There is little information about quantitative variations of serum gangliosides, their origin and progression of malignant melanoma in medical literature. Serum gangliosides are derived on one hand, from tumor microenvironment, and, on the other hand, from membranous compo‐ nents turn-over. The ability of melanoma cells to synthetize and release gangliosides in extracellular space are sustained by the results of this study.

Though, in systemic circulation, we detected high levels of gangliosides in patients with primary melanoma and metastatic melanoma compared with dysplastic nevi and control groups. The patients with metastatic melanoma had significantly increased levels of ganglio‐ sides compared with patients with primary tumor. The serum levels of gangliosides were similar in nevi and control group.

Important data regarding the origin of serum gangliosides were obtained by analyzing their levels before and after surgical removal of melanocytic tumors. The levels of serum ganglio‐ sides varied statistically significant with surgical intervention in patients with localized melanoma, and without statistical significance in patients with metastatic melanoma, respec‐ tively, dysplastic nevi. Therefore, serum concentration of gangliosides could give important data about tumor mass, tumor volume or tumor progression. We consider that low levels of gangliosides in multiple determinations after surgical removal of the tumor could indicate a correct surgical treatment of melanoma, while high levels in the same conditions could mean a progression of melanoma.

Our study showed a strong association between increased serum gangliosides and high Breslow index or high Clark level and presence of ulceration. Serum gangliosides correlat‐ ed strong and positive with biological factors used for melanoma staging – LDH, CRP, IL8. High levels of LDH, CRP, IL8 are markers for melanoma progression. These correlations show that the principle elements involved in melanoma progression are vascularization and neoangiogenesis.

Therefore, the levels of serum gangliosides could become a useful marker for clinical staging of melanoma, but its usage is limited by the lack of satisfactory criteria for interpretation. The problem becomes more complex, because there are multiple sources of serum gangloside, being difficult to know if the exact source are the tumoral cells or the host organism. This statement is sustained by the variability in composition of serum gangliosides and altered immunologic reactivity of melanoma patients. In the model of serum gangliosides before and after surgical treatment, we observed a tendency of the body to adjust the biosynthesis of gangliosides, mainly by normalizing glycosyltransferase activity in melanoma patients. To demonstrate a possible link between ganglioside system and prognosis of patients with cutaneous malignant melanoma, we calculated relapse-free survival of the disease according to serum levels of gangliosides. Based on this analysis, the authors found that low levels of circulating gangliosides are positive prognosis factors, in terms of increasing the relapse free survival in patients with malignant melanoma. These results justify the role of serum gan‐ gliosides as potential biomarkers in the management of patients with melanoma, a finding that supports previous researches [10, 11, 53, 102, 103].

Data in literature about the involvement of gangliosides in tumor processes are controversial. Some studies claim that overexpression of gangliosides on cells membrane and their accumu‐ lation in intercellular space and in serum of patients with cancer may play a role in tumor growth, neovascularization, and lack of immune response. Gangliosides influence tumor metastasis and angiogenesis by modulating the autocrine production of growth factors and thereby, protect the tumor from the host's immune system [67, 98, 100, 101,104, 105, 106].

In this sense, the relationship between ganglioside, tumor growth and progression have been the subject of several studies. A number of in vivo and in vitro studies have shown that gangliosides are involved in tumor suppression. Gangliosides metabolic products such as ceramides may be involved in apoptosis. Other studies argue that metastatic melanoma produces a variety of growth factors and interleukins that induce cell proliferation. Ganglio‐ sides can alter the growth of metastatic melanoma by modulation the activity of some growth factors, by regulating cAMP and some signaling pathways. Recently it has been demonstrated that gangliosides of human melanoma promote differentiation of dendritic cells from mono‐ cytes, maturation of Langerhans cells in the epidermis, and induce apoptosis of both cell types. Melanoma cells release active chemo attractants and other mediators to stimulate the migration and activation of macrophages, monocytes, granulocytes, keratinocytes, fibroblasts, platelets, and other components of the native immunity. Metastatic melanoma release these molecules in the tumor microenvironment [52, 82, 99, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117].

gangliosides in multiple determinations after surgical removal of the tumor could indicate a correct surgical treatment of melanoma, while high levels in the same conditions could mean

Our study showed a strong association between increased serum gangliosides and high Breslow index or high Clark level and presence of ulceration. Serum gangliosides correlat‐ ed strong and positive with biological factors used for melanoma staging – LDH, CRP, IL8. High levels of LDH, CRP, IL8 are markers for melanoma progression. These correlations show that the principle elements involved in melanoma progression are vascularization and

Therefore, the levels of serum gangliosides could become a useful marker for clinical staging of melanoma, but its usage is limited by the lack of satisfactory criteria for interpretation. The problem becomes more complex, because there are multiple sources of serum gangloside, being difficult to know if the exact source are the tumoral cells or the host organism. This statement is sustained by the variability in composition of serum gangliosides and altered immunologic reactivity of melanoma patients. In the model of serum gangliosides before and after surgical treatment, we observed a tendency of the body to adjust the biosynthesis of gangliosides, mainly by normalizing glycosyltransferase activity in melanoma patients. To demonstrate a possible link between ganglioside system and prognosis of patients with cutaneous malignant melanoma, we calculated relapse-free survival of the disease according to serum levels of gangliosides. Based on this analysis, the authors found that low levels of circulating gangliosides are positive prognosis factors, in terms of increasing the relapse free survival in patients with malignant melanoma. These results justify the role of serum gan‐ gliosides as potential biomarkers in the management of patients with melanoma, a finding that

Data in literature about the involvement of gangliosides in tumor processes are controversial. Some studies claim that overexpression of gangliosides on cells membrane and their accumu‐ lation in intercellular space and in serum of patients with cancer may play a role in tumor growth, neovascularization, and lack of immune response. Gangliosides influence tumor metastasis and angiogenesis by modulating the autocrine production of growth factors and thereby, protect the tumor from the host's immune system [67, 98, 100, 101,104, 105, 106].

In this sense, the relationship between ganglioside, tumor growth and progression have been the subject of several studies. A number of in vivo and in vitro studies have shown that gangliosides are involved in tumor suppression. Gangliosides metabolic products such as ceramides may be involved in apoptosis. Other studies argue that metastatic melanoma produces a variety of growth factors and interleukins that induce cell proliferation. Ganglio‐ sides can alter the growth of metastatic melanoma by modulation the activity of some growth factors, by regulating cAMP and some signaling pathways. Recently it has been demonstrated that gangliosides of human melanoma promote differentiation of dendritic cells from mono‐ cytes, maturation of Langerhans cells in the epidermis, and induce apoptosis of both cell types. Melanoma cells release active chemo attractants and other mediators to stimulate the migration and activation of macrophages, monocytes, granulocytes, keratinocytes, fibroblasts, platelets,

a progression of melanoma.

388 Melanoma – Current Clinical Management and Future Therapeutics

supports previous researches [10, 11, 53, 102, 103].

neoangiogenesis.

Other studies claim that metastatic melanoma produces a number of growth factors such as interleukin-8, alpha protein that regulates the growth/the activity of melanocytes. It has been suggested that gangliosides could alter the growth of metastatic melanoma by modulating the production of an autocrine growth factor and by adjusting the 3 '5'adenozin cyclic monophos‐ phate (cAMP) and its corresponding signaling paths. Other studies have suggested that soluble gangliosides could be involved in tumor-induced immunosuppression [98, 111, 112, 115, 116, 117, 118, 119].

Melanoma cells, especially those of metastatic melanoma overexpress a variety of ganglio‐ sides. Aberrant gangliosides and their high level could be a marker of malignancy. Serum levels of compounds with sialic acid did not vary in patients with precancerous pigmenta‐ ry lesions. We observed that in early diagnosed melanoma serum level of gangliosides was not significantly increased. Low levels of gangliosides in melanoma patients without ulceration or metastasis were associated with an increased relapse-free survival after surgical removal of the tumor. High levels of gangliosides in patients with metastatic melanoma were associated with progression of the disease and a decrease in relapse-free survival after surgical intervention [49, 53, 103].

The presence in the body of glicosphingolipids antigens, recognized by the immune system as nonself, determines the proliferation of some lymphocitary clones, that promote the synthesis of antibodies against these molecules. Glicosphigolipids associated with tumors, induce also, the synthesis of antibodies against gangliosides by a complex mechanism. It was accepted the idea of genetic similarity between some exogenous antigens and some compo‐ nents of nervous cells.

The umoral immune response in adult patients with untreated malignant melanoma, in dysplastic nevi and healthy volunteers was evaluated by the assessment of antigangliosides of IgG and IgM type against GM1, GM2, GM3, GD1a, GD1b, GT1b, GQ1b. In healthy individ‐ uals, antigangliosides antibodies IgG and IgM type were negative, in dysplastic nevi group, antibodies IgG type were absent, with the exception of anti-GM1 that were present in 4.17% cases. Antibodies IgM type had the following distribution: 6.25% anti-GM1; 4.17% anti-GM2; 4.17% anti-GM3; 6.25% anti-GD1a; 4.17% anti-GD1b; 4.17% anti-GT1b; 0% anti-GQ1b. In melanoma group, IgG antibodies had the following distribution: 4.68% anti-GM1; 1.56% anti-GM2; 0.78% anti-GM3; 0.78% anti-GD1a; 3.12% anti-GD1b, while the positive IgM were 15.63% anti-GM1; 20.31% anti-GM2; 17.19% anti-GM3; 14.06% anti-GD1a; 10,16% anti-GD1b; 14.06% anti-GT1b; 4.69% anti-GQ1b. No statistical differences were observed in IgG status between the studied groups. Significant variations of IgM antibodies were determined between melanoma and nevi, respectively control group.

Based on these results, we can appreciate that gangliosides expressed on melanoma cells induced the synthesis of antiganglioside antibodies. The presence of antiganglioside antibod‐ ies was associated with oncogenic transformation of melanocytes, but the moment of anti‐ bodies synthesis could not be determined. The antibodies identified in melanoma patients were mostly of IgM type.

To evaluate if the presence of IgM antibodies was associated with melanoma development, we determined their relation to clinical, histological and biological factors recommended by AJCC for melanoma staging. Anti-GM1 varied significantly with tumor site, histological site of melanoma, Breslow index, Clark level, presence of ulceration, anti-GM2 did not vary with histological characteristics of melanoma, anti-GM3 varied significantly with Clark level and presence of ulceration and anti-GD1a was influnced significanlty by tumor site, Breslow index, Clark level and presence of ulceration. Anti-GD1b was influenced significantly by histological type of melanoma, anti-GT1b only by tumor site, while anti-GQ1b was not influenced by histological characteristics of melanoma.

Positive correlation with statistical significance were determined between anti-GM1 and LDH, CRP, respectively, IL8, between anti-GD1a and LDH, CRP, respectively, IL8. Negative significant correlations were observed between anti-GM3, anti-GT1b and LDH, CRP, respec‐ tively, IL8. The transition from radial to vertical growth of melanoma marked by high levels of LDH, CRP and IL8 was associated with an increase in anti-GM1, anti-GD1a and a decrease in anti-GM3, anti-GM2 antibodies of IgM type. Therefore, the IgM antibodies against GM1 and GD1a might be useful in malignant melanoma staging and diagnosis. Also, they could facilitate tumor growth by promoting neovascularization, inflammation and angiogenesis.

Other important findings of our study are the potential protective role of anti-GM2 and anti-GM3 antibodies of IgM type in melanoma patients. Anti-GM2 and anti-GM3 could affect gangliosides expression on melanoma cells, and though, might influence indirectly cell proliferation, transmembrane signaling and cells interaction. IgM antibodies against GD1b, GT1b, GQ1b offered no data about melanoma progression in relation to analyzed histological factors. Their negative correlation with IL8, LDH and CRP suggest that they could suppress tumor growth and angiogenesis indirectly.

Ganglioside and antiganglioside antibodies could be used for melanoma staging and though, they could increase the precision of the outcome. High levels of gangliosides and anti-GM1 and GD1a before the surgical removal of the tumor were associated with advanced melanoma and poor prognosis. The presence of anti-GM2,-GM3,-GD1b,-GT1b,-GQ1b in patients with dysplastic nevi could be suggestive of malignant transformation. Assessment of gangliosides and antigangliosides before the surgical intervention could be an important item for the postsurgical follow-up.

Other studies in patients with prostate cancer or soft tissue sarcoma showed that anti-GM1 antibodies had no value for diagnose and prognosis. In patients with thyroid cancer well differentiated, the level of anti-GM1 IgG and IgM type was associated with carcinogenesis, with no diagnose value in thyroid cancer [120, 121, 122]. In a big cohort of patients with lupus systemic erythematous the presence of both anti-GM1 IgM and IgG type were associated with neuro-psychiatric disorders and depression. Anti-GM1 were identified in the serum of patients with chronic idiopathic hepatitis, in systemic infections, autoimmune disorders with neuro‐ logical involvement (encephalopathy, HIV neuropathy) and after parenteral administration of gangliosides [123, 124, 125, 126, 127, 128, 129, 130].

Manipulation of cellular growth dependent of GD1b, GT1b, GQ1b gangliosides was demon‐ strated in several cellular systems. In vitro growth of human metastatic melanoma WM266-4 was inhibited by GD1b, GT1b, GQ1b, while other gangliosides (GM 1, GM2, GM3, GD1a, GD2 and GD3) had no effect. The action of gangliosides from b series was inhibited by IL8. This phenomena could be antagonized by exogenous anti-IL8. No other growth factor (regulating of oncogene alpha growth factor, platelet regulating growth factor, interleukin 6) influenced melanoma evolution. A possible mechanism through which GD1b, GT1b and GQ1b inhibit melanoma growth could be the suppression of IL8 secretion, of ARNm expression and activation. Other studies suggest that GD1b could determine melanoma progression in vivo by stimulating angiogenesis. GT1b also influenced growth and motility of endothelial cells, while GM3 had an angiostatic effect. Multiple results proved the role of anti-GD1b,-GT1b,- GQ1b IgM type in soft tissue sarcoma Erlich subcutaneous solid tumor, Erlich carcinoma with ascitis [76, 121, 131, 132, 133, 134].

The ability of the human organism to promote an anti-GM1,-GM2,-GM3,-GD1a,-GD1b-GT1b,- GQ1b immune response could influence the evolution of patients with malignant tumors. Taking into account the immunogenic capacity of gangliosides in malignant melanoma and their effect on tumor development, we consider that an action on gangliosides metabolism could be a strategy of reducing tumor angiogenesis. Low gangliosides and increased antibod‐ ies against gangliosides from melanoma cells confer an advantage in survival of melanoma patients compared with patients without antiganglioside antibodies. Based on our results regarding gangliosides and antigangliosides profile in patients with cutaneous malignant melanoma, the authors consider that pharmacological modulation of ganglioside-antiganglio‐ side system could be a way to control melanoma development.

## **Author details**

To evaluate if the presence of IgM antibodies was associated with melanoma development, we determined their relation to clinical, histological and biological factors recommended by AJCC for melanoma staging. Anti-GM1 varied significantly with tumor site, histological site of melanoma, Breslow index, Clark level, presence of ulceration, anti-GM2 did not vary with histological characteristics of melanoma, anti-GM3 varied significantly with Clark level and presence of ulceration and anti-GD1a was influnced significanlty by tumor site, Breslow index, Clark level and presence of ulceration. Anti-GD1b was influenced significantly by histological type of melanoma, anti-GT1b only by tumor site, while anti-GQ1b was not influenced by

Positive correlation with statistical significance were determined between anti-GM1 and LDH, CRP, respectively, IL8, between anti-GD1a and LDH, CRP, respectively, IL8. Negative significant correlations were observed between anti-GM3, anti-GT1b and LDH, CRP, respec‐ tively, IL8. The transition from radial to vertical growth of melanoma marked by high levels of LDH, CRP and IL8 was associated with an increase in anti-GM1, anti-GD1a and a decrease in anti-GM3, anti-GM2 antibodies of IgM type. Therefore, the IgM antibodies against GM1 and GD1a might be useful in malignant melanoma staging and diagnosis. Also, they could facilitate

Other important findings of our study are the potential protective role of anti-GM2 and anti-GM3 antibodies of IgM type in melanoma patients. Anti-GM2 and anti-GM3 could affect gangliosides expression on melanoma cells, and though, might influence indirectly cell proliferation, transmembrane signaling and cells interaction. IgM antibodies against GD1b, GT1b, GQ1b offered no data about melanoma progression in relation to analyzed histological factors. Their negative correlation with IL8, LDH and CRP suggest that they could suppress

Ganglioside and antiganglioside antibodies could be used for melanoma staging and though, they could increase the precision of the outcome. High levels of gangliosides and anti-GM1 and GD1a before the surgical removal of the tumor were associated with advanced melanoma and poor prognosis. The presence of anti-GM2,-GM3,-GD1b,-GT1b,-GQ1b in patients with dysplastic nevi could be suggestive of malignant transformation. Assessment of gangliosides and antigangliosides before the surgical intervention could be an important item for the post-

Other studies in patients with prostate cancer or soft tissue sarcoma showed that anti-GM1 antibodies had no value for diagnose and prognosis. In patients with thyroid cancer well differentiated, the level of anti-GM1 IgG and IgM type was associated with carcinogenesis, with no diagnose value in thyroid cancer [120, 121, 122]. In a big cohort of patients with lupus systemic erythematous the presence of both anti-GM1 IgM and IgG type were associated with neuro-psychiatric disorders and depression. Anti-GM1 were identified in the serum of patients with chronic idiopathic hepatitis, in systemic infections, autoimmune disorders with neuro‐ logical involvement (encephalopathy, HIV neuropathy) and after parenteral administration of

Manipulation of cellular growth dependent of GD1b, GT1b, GQ1b gangliosides was demon‐ strated in several cellular systems. In vitro growth of human metastatic melanoma WM266-4

tumor growth by promoting neovascularization, inflammation and angiogenesis.

histological characteristics of melanoma.

390 Melanoma – Current Clinical Management and Future Therapeutics

tumor growth and angiogenesis indirectly.

gangliosides [123, 124, 125, 126, 127, 128, 129, 130].

surgical follow-up.

Corina-Daniela Ene (Nicolae)1\* and Ilinca Nicolae2

\*Address all correspondence to: koranik85@yahoo.com

1 University of Medicine and Pharmacy, Carol Davila, Bucharest, Romania

2 Victor Babes Hospital of Tropical and Infectious Diseases, Research in Dermatology, Bucharest, Romania

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## *Edited by Mandi Murph*

Melanoma - Current Clinical Management and Future Therapeutics serves as an advanced course in melanoma or an addendum to further polish expertise. Sections of the book include a thorough introduction on epidemiology and disease, the current surgical management of melanoma and lymph node dissection, immunotherapy along with drug toxicities and emerging research topics, like RAGE and autotaxin, that have potential therapeutic applications. The chapters in this book explore the most common subtype of melanoma, cutaneous disease, as well as a rare form, acral lentiginous melanoma and even canine tumors. Experts from around the globe contributed chapters, most of which have visual illustrations to depict aspects of disease management and therapy, allowing readers to grasp the advanced concepts presented.

Melanoma - Current Clinical Management and Future Therapeutics

Melanoma

Current Clinical Management

and Future Therapeutics

*Edited by Mandi Murph*

Photo by Ugreen / iStock