Melanoma: Molecular Pathogenesis and Therapeutic Management

Abstract

Malignant melanoma remains one of the fastest growing cancers worldwide. Although the primary cutaneous melanoma can be managed by surgery, the advanced metastatic melanoma cannot be managed by surgery alone and thus, requires better therapeutic approaches. In view of high mortality rates due to metastatic melanoma, better understanding of the molecular pathogenesis of malignant melanoma is urgently needed. Such information is expected to prove very valuable in early detection of potential metastatic lesions and developing newer therapeutic approaches in order to better manage this malignancy. This article reviews the available information on the molecular changes associated with malignant melanoma and discusses the potential of such information in facilitating the development of newer anti-melanoma therapeutics. Current state of knowledge and the future of traditional and newly approved anti-melanoma therapeutics are also discussed.

Introduction

Cutaneous melanoma is a malignant neoplasm of the melanocytes. Melanocytes, found in the basal layer of the epidermis, produce the pigment melanin that is responsible for skin color. It is estimated that in the US for the year 2014, about 76,100 new cases of melanoma of the skin will be diagnosed and about 9,710 people will die due to this disease this year alone (1). Among the Caucasian population, the incidence of malignant melanoma has increased 3–6% over the last few decades (2), making this one of the fastest growing cancers worldwide (3).

As one of the three major types of skin cancer, with basal cell carcinoma and squamous cell carcinoma being the other two, melanoma remains the deadliest, causing the vast majority of the skin cancer-related deaths even though it accounts for approximately 5% of the skin cancer cases (4, 5). Primary melanoma patients demonstrate approximately an 11% mortality rate (2, 6), whereas the mortality rate due to metastatic melanoma is significantly higher. Metastatic melanoma patients typically have a low survival rate due to the poor efficacies of current cancer therapies. The extreme difficulties encountered in therapeutic management of melanoma patients have prompted large scale efforts to elucidate the molecular pathogenesis of malignant melanoma in hopes to finding more effective treatment options.

Classification and Staging

There are four major forms of melanoma including (i) superficial spreading, (ii) nodular, (iii) lentigo maligna and (iv) acral lentiginous melanomas. Of these, the superficial spreading form remains the most common and accounts for about 70% of melanomas followed by nodular form that represents about 15–30% of melanoma cases. The lentigo maligna and acral lentiginous forms represent less than 10% of melanoma cases (7, 8). In terms of staging, four systems are followed including (i) the Clark scale, (ii) the Breslow scale, (iii) TNM staging and (iv) Number stages. The Clark scale evaluates the depth of lesion in terms of it affecting various skin layers. The Breslow scale evaluates as to how thick the melanoma is in the skin. The TNM (Tumor, Node, Metastases) staging is based on thickness of the lesion and evaluation of its spread to lymph nodes and different tissues in the body and is also used for clinical staging per the American Joint Committee on Cancer (AJCC). The number staging system (Stage 0 to Stage 4) couples information on depth of the lesion and the TNM staging. For example, Stage 0 implies that the lesion is confined to epidermis (in situ) without spread to deeper layers such as dermis whereas Stage 4, the other extreme, indicates spread to lymph nodes and metastases to distant parts of the body such as lung, liver or brain (7, 9).

Diagnosis

Diagnosis of melanoma can be accomplished through clinician assessment of the skin lesion with the unaided eye. Clinicians frequently assess lesions based on the “ABCDE rule” that is meant to indicate A: asymmetry, B: irregular border, C: color variations, D: diameter >6 mm, and E: elevated surface (5, 7, 10). However, diagnosis with the unaided eye is not always accurate as seen in the approximately 80% accurate diagnosis rate demonstrated amongst dermatologists, and approximately 30% rate for non-dermatological specialists (5). Detection processes have become more technical over time in order to improve efficacy and limit the number of false negative cases that would allow undetected melanoma to develop to dangerous stages. The use of a skin surface microscope or a dermoscope allows improved visualization of the lesion (11, 12). Development of advanced digital systems have allowed for the development of an automated melanoma diagnosis system termed MEDS, which integrates multiple classification algorithms and uses them to analyze different measurements and characteristics of the patient lesion to produce effective diagnoses (5).

Research in melanoma diagnostics has also focused on detecting melanoma-specific biological markers that may help predict the course of the disease. Assaying the blood of melanoma patients who had been deemed cancer free for melanoma cells and other mRNA markers helps predict the probability that the patient will experience remission of metastatic melanoma (13). Another molecular marker that can be used for diagnostic purposes is Microphthalmia transcription factor (Mitf) (14). Mitf has been found to be uniquely expressed in melanoma melanocytes. In addition, Mitf antibody has been reported to detect and stain melanocytic lesions that conventional markers HMB-45 or S-100 failed to stain (14).

Etiology and Pathogenesis

Ultraviolet Radiation

Epidemiological studies have done much to shed light on the possible causes of malignant melanoma. Studies have shown that a major risk factor for melanoma development is exposure to Ultraviolet (UV) radiation exposure. One or more blistering sunburn during childhood or adolescence more than doubles the risk for melanoma in later life (15). This suggests that UV exposure plays an important role in melanoma tumorigenesis. Additional experiments have shown that UV radiation frequently leads to DNA mutations, such as the formation of pyrimidine dimers or deamination of cytosine into thymidine (16, 17). Cutaneous melanoma samples demonstrate a high base mutation rate that exceeds that of almost every other form of solid cancer, which may be attributed to the potency of UV mutagenic effects (18–20). Individuals who have history of familial melanoma, which contributes to 8–12% of melanoma cases, demonstrate a high sensitivity to UV radiation (21–24). These individuals are more likely to develop melanoma earlier in age and to develop multiple melanoma lesions.

Melanocytic Nevi

Another risk factor for malignant melanoma is the melanocytic nevus. Nevi are benign lesions composed of a concentrated amount of melanocytes. This leads to a dark pigmented spot on the skin due to the large amount of melanin that is produced. Nevi are colloquially known as a mole or birthmark. Melanocytic nevi can be congenital or appear later in life. Atypical or excessive amounts of nevi acquired later in life are a potent risk factor or diagnostic feature indicating melanoma development (25). If nevi begin to change in shape, color, or texture then it becomes a strong indicator for malignant melanoma. In one study, about 81% of melanoma patients observed a changing nevus in the location of the malignant lesion (26). However, it is important to note that not all changing nevi lead to or are indicative of melanoma, and that the exact mechanism and cause for the changes are still not understood. The indication of changing nevi allows for early melanoma detection, and with the proper surgical excision, the cancer free survival rate is extremely high compared to metastatic melanoma.

Molecular Changes Associated with Malignant Melanoma

After the melanoma spreads or metastasizes from its origin into other cutaneous or subcutaneous tissues, the response rate to treatment plummets to approximately 5–20%, and the 10-year survival rate becomes only about 10% (27, 28). Metastatic melanoma has been especially difficult to treat, showing low cure and survival rates after surgical resection and radiation therapy (27). At the cellular level, cancer cells possess distinguishing molecular properties that allow for apoptosis evasion, limitless growth potential without the need for growth factors, angiogenesis, and metastasis (29). Identifying the specific molecular changes that allow melanoma cells to have a growth and survival advantage over others may allow for the development of more effective targeted therapies that can improve the prognosis of melanoma patients.

BRAF

Studies have shown that around 40–60% of all melanoma cases exhibit an activated BRAF mutation (30). BRAF is a proto-oncogene that encodes a serine/threonine protein kinase as part of the RAS-RAF-MEK-ERK kinase pathway, which promotes cell growth and proliferation (31, 32). Normally, BRAF participates in homo- or heterodimerization with another RAF kinase in response to growth signals. However, an activating mutation leads BRAF to act as a self-sufficient and continuously activated monomer (33). The result is uncontrolled cell proliferation and that may play a role in tumor development and growth. The V600E missense mutation, that changes valine into glutamic acid, is the most common BRAF alteration and constitutes about 90% of BRAF activating mutations or about 50% of all metastatic melanomas (30, 34, 35). The V600K is the second most common mutation that results in valine to lysine change. Interestingly, BRAF mutations, such as the V600E amino acid substitution are also exhibited in 68% of benign nevi (36). This evidence raises the possibility that BRAF mutations might not be involved in melanoma carcinogenesis. Because nevi are usually stable after their formation, some researchers instead suspect that BRAF mutations instead play a part in melanocytic neoplasm formation (36).

Further research has revealed that the early stage of melanoma formation, known as the radial growth phase, exhibits a low 10% BRAF mutation rate, which supports the hypothesis that BRAF does not play a part in melanoma initiation (37). At the same time, 60–70% of vertical growth lesions and metastasized melanomas possess BRAF mutations, which suggests that this oncogenic mutation may be involved in cancer progression (36–39). The existence of activating BRAF in metastasized melanoma is consistent with the proposed BRAF function of cell growth promotion. The high prevalence of the BRAF^V600E mutation in melanoma patients makes this a prime target for anti-melanoma therapeutics.

NRAS

Another source of molecular changes that may allow for melanoma initiation or propagation is found in the NRAS GTPase or neuroblastoma RAS oncogene. NRAS GTPase mutations are found in 15–20% of melanoma patients and activating mutations are usually found in the Q60/61 and G12/13 codons (40–42). NRAS is also a constituent of the RAS-RAF-mitogen activating kinase pathway, and is specifically part of the class of Ras proteins that modulate Raf protein activity (32, 35). Therefore, both NRAS and BRAF are found on the same pathway. Interestingly, concurrent mutations in both BRAF and NRAS are rarely found in the same patient, although some melanoma patients who harbored BRAF mutations and failed BRAF inhibitor treatment did display NRAS mutations in melanomas that had progressed (43). These findings suggest that the underlying mechanisms of melanoma development may be redundant particularly in context to BRAF and NRAS.

The patients with NRAS mutations possess thick vertical growth tumors that researchers attribute to the heightened cell proliferations rates induced by the NRAS mutation (40). These characteristics that NRAS mutations confer to melanoma tumors are similar those of BRAF. Like BRAF mutations, NRAS mutations are also found in high rates in benign nevi, which may limit the possibility that a NRAS mutation acts in melanoma initiation (36, 41, 44). Mutation analysis of primary and metastatic tumor samples show that metastatic tumors exhibited a higher incidence of BRAF and NRAS mutations compared to primary tumors (32). In addition, BRAF and NRAS mutations in metastatic melanomas were correlated with lower survival rates (32). Collectively, these findings seem to suggest that both of these mutations in the MAPK pathway may be involved in promoting the transition of primary melanoma into the metastatic variant. Because NRAS mutations are also linked to uncontrolled proliferation, NRAS can also serve as a favorable target to develop anti-melanoma therapeutics.

PI3K-ATK/PTEN

The PI3K-AKT pathway is a separate mechanistic route that also plays a role in cell proliferation and survival (45). Similarly to the RAS-RAF-MEK-ERK pathway, constitutive activation of PI3K-AKT leads to a competitive growth advantage that makes way for melanoma proliferation and metastasis (46). PI3K-AKT pathway obtains advantageous gain of function changes through point mutations or changes in chromosome copy number for genes that encode key proteins in the pathway as seen in PIK3CA and AKT1(41). Analysis of melanoma tumor samples has identified about 3% PI3K missense mutation rate (41, 47, 48). Such a low rate makes this pathway appear as an unlikely contributor to melanoma development and progressions.

However, there are other ways the PI3K-AKT pathway can achieve hyper-activation. For example, high levels of PI3K-AKT activity can also result from activation via an NRAS mutation or a lack of inhibition due to PTEN inactivation (41). An NRAS-driven constitutive activation indicates that not only is the PI3K-AKT pathway upregulated, but the RAS-RAF-MEK-ERK pathway as well, which requires separate inhibitors to block both pathways because there remains a paucity of NRAS inhibitors. PTEN, or phosphatase and tensin homologues, is a tumor suppressor gene that produces a protein that dephosphorylates and inactivates the PI3K (45). One of PI3K-AKT pathway’s functions to maintain cell viability is to inhibit apoptosis. Thus, when this pathway is hyper-active or has lost its negative feedback regulation system, then chemotherapy and radiation therapy may lose their death inducing therapeutic potencies (49).

p53

Tumor suppressor gene p53 has been implicated in a variety of human malignancies, such as breast, colorectal, lung, and prostate cancer (50–60). The p53 protein is activated by cell stress or DNA damage and induces cell death (61). The role of p53 in melanoma is disputed. Immunohistochemistry analysis shows varying prevalence of p53 mutation, some reporting an 11% altered expression rate of p53, while other reporting as high as 85% (50, 62). Sequence analysis shows that the p53 gene locus does not appear to possess mutations and expression of wild type p53 is preserved (50). Despite the lack of p53 mutations, melanoma cells generally do not undergo apoptosis in response to gamma radiation or chemotoxic drugs, which suggests that perhaps p53 might not be functioning properly. Another hypothesis deduces that other protein(s) is/are inactivating p53, preventing it from carrying out its tumor suppression functions.

CDK4/CDKN2A

Oncogene Cyclin-dependent kinase 4 (CDK4) and tumor suppressor gene CDKN2A, which encodes p16INK4a have been implicated in familial melanoma development (63). In the case of familial melanoma, there is a genetic susceptibility to develop this disease (64). It has been observed that about 5–10% of melanoma patients have a relative who has also suffered from this cancer (64, 65). The proposed form of genetic inheritance of this predisposition is through autosomal dominant inheritance, with a 53% penetrance rate by the 8th decade (64, 66). Along with other D-type cylcins, CDK6, and pRb, CDK4 and p16INK4a regulate cell cycle progression form the G1 to S phase (67–72). Upon activation by Cyclin D1, CDK4 acts as a proto-oncogene that promotes the progression from G1 to S phase, allowing cell proliferation, whereas, p16INK4a inhibits CDK4 action, arresting cell division (24, 67). Studies have shown that about 25–60% of melanoma patients with a family history of melanoma have an inactivated p16INKA mutation found on 9p21 (63, 64, 73–80), while mutations found in the CDK4 gene occur at a much lower rate (81). In view of these molecular changes, the logical step would be to inhibit CDK4 to prevent further cell cycle progression and limit the uncontrolled growth in melanoma.

c-KIT

About 2–5% of all melanoma cases consist of the subtype acral lentiginous melanoma (8, 82, 83). Acral melanoma is characterized as cancerous lesions found on the palms and the soles of the feet and affects Blacks and Asian/Pacific Islanders at higher rates than Caucasian populations (82, 84). Mutations or chromosomal duplication in the c-KIT gene have been detected in about 23% of acral melanomas and in less than 2% of cutaneous melanomas (85). c-KIT encodes a tyrosine kinase transmembrane receptor, and in the case of melanocytes it is believed to contribute to melanogenesis and melanocyte cell division (86). Point mutations and gene duplications of c-KIT frequently lead to increased expression of the KIT protein, which may alter melanocyte proliferation, cell death, chemotaxis and adhesion, and contribute to tumorigenesis (86–88). Thus, c-KIT may serve as target to develop anti-melanoma therapeutics.

MC1R

Melanocortin 1 receptor (MC1R) acts as a G-protein coupled receptor in melanocytes and plays a critical role in determining skin pigmentation (84, 89). Exposure to UV radiation leads to generation of alpha-melanocyte stimulating hormone, which activates MC1R to generate melanin via the process of melanogenesis (89). Melanin is a light-absorbing polymer derived from oxidized tyrosine that determines the darkness and pigmentation of human skin in response to UV radiation (17, 90). Melanin is made up of two classes of dark brown-black eumelanin and red yellow pheomelanin that are both found in the skin (91). Studies have found that a high pheomelanin to eumelanin ratio along with an overall low level of melanin contributes to fair skin, red hair color (RHC), and the inability to tan phenotypes (92).

The full functions of melanin remain to be elucidated. Originally believed to shield from photo-radiation and protect the skin from DNA damage, melanin also possesses properties of a “thermoregulator, free radical sink, cation chelator, and an antioxidant” (90, 93). Fair skin individuals demonstrate lower amounts of melanin containing granules called melanosomes in their epidermis, which is believed to allow more penetration of UV radiation, leading to increased rates of “sun-induced” DNA damage in melanocytes and the formation of malignant neoplasms (92, 94). UV radiation, in addition to DNA mutagenesis, also leads to the production of reactive oxygen species (ROS), which can lead to oxidative stress within melanocytes and genetic mutations (93). In normal conditions, melanin along with the glutathione pathway acts as antioxidants that neutralize the destructive effects of ROS. However, in MC1R variant individuals, the decreased melanin production would lead to an increased state of oxidative stress and thus, DNA damage that may trigger mechanisms of melanoma initiation (93).

It has been reported that certain MC1R allele variants confer an increased susceptibility to melanoma development especially among some Caucasian populations (89). A case control study analyzing MC1R variants detected in melanoma patients and matched control subjects, found that 72% of melanoma patients possessed a MC1R polymorphism compared to only 56% of the controls (95). There are over 60 MC1R polymorphisms that have been grouped based upon their “strength of” correlation to the red hair phenotype (96). Each MC1R allele variants nearly doubles the risk for melanoma, but does not appear to be sufficient to initiate melanoma development (96). Instead, MC1R variants that cause reduced or complete loss of MC1R signaling capabilities increase the propensity to acquire additional genetic mutations that may promote melanoma development (89). Individuals with MC1R variant alleles that lead to low melanin production are more vulnerable to UV-induced DNA mutagenesis making them susceptible to acquiring mutations in genetic loci of BRAF, NRAS, or CDKN2A (89, 97, 98). Such variants have been found with constitutively activated BRAF melanoma patients who typically do not possess a history of chronic-sun damage of the skin (89). Rarely have wild-type MC1R alleles been found in BRAF mutant melanomas (89). BRAF mutations were seen to be 6.0 times more likely to occur in American individuals who possessed a MC1R polymorphism in non-chronic-sun-damaged melanomas typically found in young patients (96).

Analysis also found that 84% of melanoma tumors with MC1R allele variants also possessed CDKN2A mutations (99). CDKN2A mutations are frequently germ line mutations, present at birth and transmitted between parent and offspring, causing familial melanoma, thus it cannot be theorized that MC1R variants increase the chances of melanocytes to develop the CDKN2A mutation (95). Instead, it has been found that the MC1R variants may influence the penetrance, or the rate at which CDKN2A mutation patients develop melanoma, by increasing the penetrance as seen from the fact that 50% of MC1R wild type individuals with CDKN2A mutations developed cancer, whereas 83% of MC1R variant alleles with CDKN2A mutations developed melanoma (95). MC1R variants (Arg151Cys, Arg160Trp, and Asp284His) that were frequently implicated in these analyses were the same alleles that have been correlated with the RHC, pale skin, and inability to tan characteristics (89, 95, 96). Taking advantage of these trends may in the future allow for improved diagnostic procedures that utilize MC1R variant allele analyses or melanin measurements to provide more information on an individual’s likelihood of developing melanoma (100).

Cadherin

A distinguishing feature of malignant neoplasms is the ability to metastasize to other organs and tissues throughout the body. It can be envisioned that transitioning from vertical growth melanoma into metastatic melanoma may require new gain of function mutations. This is supported by the fact that analyses of primary and metastatic tumors reveal acquisition of larger number of mutations and chromosomal aberrations by metastatic tumors when compared to the primary tumors (101). In normal conditions, melanocytes are connected to undifferentiated basal keratinocytes via cell-cell adhesion molecules, such as transmembrane glycoprotein E-cadherin (102–104). This interaction allows for melanocyte homeostatic regulations as the keratinocyte acts to mediate cell growth, dendrite growth, and expression of cell surface receptors (102). When melanoma is transitioning from a primary tumor into a metastatic variant, it appears to undergo epithelial to mesenchymal transition (EMT) (105). EMT involves changes in cellular cytoskeleton and adhesion molecules that may facilitate cancer cell transition towards a metastatic variant.

One of the changes characterized in the EMT is the switch in the expression of E-cadherin to N-cadherin by the melanoma cells (105). The mutations accumulated seek to knock out the pathways that would induce apoptosis in melanoma cells, conferring a growth advantage and releasing growth restrictions on the tumor (102, 105). The loss of E-cadherin prevents melanocyte regulation by keratinocytes. The newly increased N-cadherin expression is believed to facilitate melanoma migration into the dermis and interactions with dermal fibroblasts and vascular endothelial cells that express the same cadherin. This sequence of events are believed to allow the melanoma to spread out of its epidermal origin (103). N-cadherin expression also leads to the activation of the PI3K-AKT pathway, which appears to inhibit the pro-apoptotic action of Bad and promoting the anti-apoptotic activity of beta-catenin. The N-cadherin-containing melanoma cells also adhere to vascular endothelial cells, which may provide the melanoma access to enter blood vessel and metastasize throughout the body (103).

Upreguated expression of VE-cadherin is another change that appears to promote the metastatic potential of melanoma cells (106). Compared to primary tumors that express VE-cadherin at very low levels, metastatic melanomas demonstrate high levels of expression, which is correlated to the phenomenon of vasculogenic mimicry. In three-dimensional culture, aggressive melanoma cells form tube-like extensions that are similar to embryonic vascularization networks (106). Vasculogenic mimicry has been correlated with VE-cadherin expression due to the fact that when VE-cadherin expression is limited, the formation of tubular networks decreases (106). Melanoma vasculogenic mimicry sheds light on the ability for melanoma tumors to gain access to blood supplies or their means of vascularization, which may lead to a major competitive growth advantage for the tumor (106, 107). Expression of VE-cadherin on melanoma cell membranes is believed to allow tumor cells to adhere to endothelial cells as well, which may facilitate for blood vessel penetration (106).

Melanoma Therapeutics

Traditional Therapies

Primary melanomas are typically treated with surgical excision, which yields a high survival rate. However, following metastasis, surgical excision of the tumor only yields about 10% five-year survival rate (27). Radiation therapy has also proven to be ineffective for treating metastatic melanoma (27). Thus, metastatic melanomas are medically managed by chemotherapeutics and dacarbazine has been used for this purpose. Dacarbazine, dimethyltriazeno-imidazol carboxamide (DTIC), is an alkylating agent and the only monochemotherapy approved by the FDA for treating melanomas (27, 108, 109). After being activated in the liver, DITC generates the metabolite MITC that possesses cytotoxic effects (110). MITC alkylates DNA bases, which prevents DNA replication and can induce mutations (111). Dacarbazine treatment demonstrates about 15–20% response rate that is sustained for an average of 3 months until resistance to dacarbazine develops (108, 111). Although this appears to be a relatively low success rate, dacarbazine is actually considered to be the best option for treating metastatic melanoma (112). In addition to dacarbazine, other single agent chemotherapies include antimicrotubule agents taxane and vindesine, platinum analog cisplatin, and nitrosoureaeas. Such alternatives all demonstrate a response rate of less than 20% when used alone (27, 108, 112). A combination of single agent chemotherapies, such as cisplatin, vindesine, and dacarbazine (CVD), fail to yield an increased response rate among metastatic patients (108, 113). Two major immunotherapies including interleukin 2 (IL-2) and interferon alpha (IFN-α) were developed in hopes to manipulate patients’ own immune activity to target melanoma cells (108, 114). Intravenous infusions of the cytokine IL-2, also called Proleukin and aldesleukin, promote natural killer lymphocytes to differentiate into lymphokine-activated killer cells (LAK) (108, 115, 116). LAK cells attack and lyse tumor cells by seeking out cells that do not display MHC I molecules (108). IL-2 approach yields a 16–17% response rate but is associated with increased morbidity and toxicities, the most frequent being capillary leak syndrome (117). IFN-α is an FDA approved adjuvant therapy, which is administered after surgery in order to help prevent any remaining melanoma cells from proliferating (118). IFN-α treatment leads to a long-lasting immune response by continuously activating CD4+ T-helper-1 lymphocytes, which activate macrophages and natural killer cells via the secretion of IFN-γ and IL-2 (108, 119). It has been noted that combination of immunotherapeutic agents, such as IL-2 and IFN-α, and cytotoxic chemotherapeutics, such as CVD, offers an increased response rate of about 31% (113).

Newer Therapeutic Approaches

Vemurafenib

Because BRAF is mutated and activated in a large number of melanoma cases, and linked to promoting cell proliferation, it was a logical target to develop therapeutics. Accordingly, Vemurafenib (PLX4032) was developed as BRAF kinase inhibitor. Vemurafenib (Figure 1) inhibits the mutant BRAF and thus, confers a degree of specificity towards melanoma harboring mutant form BRAF. In an international multicenter Phase III trial (BRIM-3), vemurafenib was compared with dacarbazine for unresectable stage IIIC and stage IV metastatic BRAF^V600E positive melanomas. Previously untreated 675 patients were enrolled in the trial. Patients were given oral vemurafenib 960 mg BID and those on dacarbazine arm received dacarbazine 1000 mg/m2 IV every three weeks. The primary endpoints were progression-free survival (PFS) and overall survival (OS). The secondary endpoints were best overall response rate (BORR), time to response, safety and tolerability. It was concluded that vemurafenib treatment led to improvement in OS, PFS and BORR versus dacarbazine (34, 120).

Figure 1.

Structure of vemurafenib. From PubChem.

A multicenter phase II single arm trial was conducted on 132 previously treated patients. Patients had melanoma that harbored BRAF^V600E or BRAF^V600K mutation and had been treated with one or more prior therapies. Vemurafenib was given at 960 mg BID orally. The median duration of follow-up was12.9 months. The results achieved 53% response rate with 6% showing complete response and 47% showing partial response. The median progression-free survival was noted to be 6.8 months and the median overall survival 15.9 months (121). The results from the clinical trials indicated vemurafenib to be effective and well tolerated agent. Vermurafenib was duly approved by the FDA in August 2011 for unresectable stage III and IV or metastatic melanomas that harbor BRAF^V600 mutations. It is prescribed at a dose of 960 mg BID orally and the duration of treatment is until disease progresses or unacceptable side effects develop. The side effects associated with vemurafenib include arthralgia, fatigue, photosensitivity, alopecia, nausea and diarrhea. Cutaneous squamous cell carcinoma (SCC) and keratoacanthoma or both can also occur. It can cause QT prolongation and thus, enhanced risk of ventricular arrhythmias and can also lead to new primary cutaneous melanoma. Vemurafenib is contraindicated for melanomas that harbor wild type BRAF. It is also contraindicated in electrolyte abnormalities, long QT syndrome with drugs that cause prolongation of QT interval.

Dabrafenib

Dabrafenib (Figure 2) is considered as a next generation agent and has a mechanism of action similar to that of vemurafenib. A phase III (BREAK-3) trial compared dabrafenib and dacarbazine in BRAF^V600E-positive unresectable or metastatic (stage III or IV) previously untreated melanomas. Dabrafenib was given at 150 mg BID orally and dacarbazine at 1000 mg/m2 IV every three weeks. The trial enrolled 250 patients and the results indicted dabrafenib to have acceptable safety profile and showed improvement in PFS over dacarbazine. The FDA approved dabrafenib in 2013 for BRAF^V600-positive unresectable or metastatic melanomas and the recommended dose is 150 mg BID orally. Dabrafenib is contraindicated in melanoma harboring wild type BRAF. The most common side effects include hyperkeratosis, headache, pyrexia, arthralgia, papilloma, alopecia, and palmar-plantar erhthrodysesthesia syndrome. The serious adverse events include the development of new primary squamous cell carcinoma, melanomas, and keratoacanthomas, febrile drug reactions, hyperglycemia, and uveitis, iritis (122, 123).

Figure 2.

Structure of dabrafenib. From PubChem.

Trametinib

Trametinib (Figure 3) was approved by the FDA in May 2013 for BRAF^V600E or BRAF^V600K positive unresectable or metastatic melanomas. Its mechanism of action is somewhat different from that of vemurafenib or dabrafenib. Trametinib inhibits MEK i.e. the extraceullular signal-regulated kinase that is downstream of BRAF. Its recommended dose is 2 mg orally once daily and the duration of treatment remains until disease progression or unacceptable side effects develop. The contraindications include melanoma harboring wild type BRAF and patients previously treated with BRAF inhibitors. The side effects associated with trametinib are cardiomyopathy, retinal pigment epithelial detachment, retinal vein occlusion, interstitial lung disease, serious skin toxicity involving rash, dermatitis, acneiform rash, palmar-plantar erythrodysesthesia syndrome, and erythema. Other common side effects are rash, diarrhea, lymphedema, dermatitis acneiform, stomatitis, hypertension, abdominal pain, hemorrhage, dry skin, pruritus and paronychia. It may also inhibit fertility in females.

Figure 3.

Structure of trametinib. From PubChem.

Although BRAF inhibitors have shown promise, acquired resistance does occur and believed to involve activation of MAPK pathway (124). BRAF inhibitors are also associated with other cutaneous malignancies that are thought to occur due to paradoxical activation of MAPK pathway in unaffected skin cells that do not harbor BRAF mutation (124). MEK resides downstream of BRAF and combination of BRAF inhibitor with MEK inhibitor seem to suggest a better approach to overcome the limitations of BRAF inhibitors as single agent therapeutics in this setting. In keeping with this idea, FDA granted (January 2014) accelerated approval to combination of dabrafenib and trametinib for unresectable or metastatic melanoma with BRAF^V600 mutations. The accelerated approval was granted with the contingency that an ongoing phase III trial investigating the combination of dabrafenib and trametinib would be successfully completed. More recently, a study reported the results of a phase III trial comparing the effects of dabrafenib and trametinib combination with those of vemurafenib. The results indicated that dabrafenib and trametinib in combination improved OS significantly compared to vemurafenib alone in BRAF^V600 mutant metastatic melanomas. Interestingly, only 1% of patients on combination therapy reported cutaneous squamous cell carcinoma and keratoacanthoma, while 18% of those on vemurafenib reported such abnormalities (124).

Another study reported the results also of a phase III trial that compared vemurafenib and cobimetinib combination with vemurafenib and placebo group. Just like trametinib, cobimetinib is a MEK inhibitor. It was noted that the combination of vemurafenib and cobimetinib improved significantly the PFS in metastatic melanoma harboring BRAF^V600 mutation (125). Based on these studies, it is therefore, logical to think that the combination of BRAF and MEK inhibitors would appear to be superior to monotherapy going forward (Figure 4).

Figure 4. BRAF activation signaling events. Vemurafenib and dabrafenib inhibit BRAF^V600 mutant form, whereas trametinib and cobimetinib inhibit MEK.

Adapted from Ascierto et al. (141).

Ipilimumab

Ipilimumab is a humanized monoclonal antibody that prevents T-cell activation via inhibition of cytotoxic T-lymphocyte antigen-4 (CTLA-4) (126–129). It was approved by the FDA in March 2011 for late stage metastatic melanoma. In normal immunomodulation, T-Cell Receptor ligation to its respective MHC presented antigen leads to CTLA-4 co-receptor expression on T cells (130, 131). CTLA-4 is very similar in structure to co-receptor CD28, which allows both these receptors to bind to CD80 and CD86 ligands found on antigen presenting cells (131). CD28-CD80/86 interaction activates the T-cell and upregulates T-cell proliferation, differentiation and cytokine production, whereas, high affinity CTLA-4-CD80/86 binding inhibits T-cell activation, limiting the immune response (130–134). Ipilimumab augments T-cell activation by inhibiting the CTLA-4 receptor interaction with CD80 or CD86, which prevents T-cells from becoming inactivated (128). Ultimately, anti-tumor immunity is boosted through Ipilimumab. Previously treated stage III unresectable melanoma and stage IV metastatic melanoma patients receiving a 10 mg/kg dosage of Ipilimumab demonstrated an 11.1% response rate with lower response rates at lower dosage levels, demonstrating a dose dependent relationship (135). Ipilimumab yielded a median 10-month survival rate (136). However, Ipilimumab has also shown to lead to autoimmune side effects in 15% of patients receiving the highest dosage, 10mg/kg, of the drug (135). These side effects include fatigue, diarrhea, skin rash, endocrine deficiencies and intestinal inflammation. It may be noted that this therapeutic modality does not work in all patients.

Pembrolizumab

Pemrolizumab is a humanized monoclonal IgG4 antibody. It targets human cell surface receptor PD-1 (programmed cell death-1) and interferes with PD-1 ligand binding. This interference results in activation of T cell-mediated response against tumors (137). The FDA approved pembrolizumab for management of advanced or unresectable melanomas that are refractory to other therapeutics (138). The common side effects include fatigue, cough, nausea, pruritus, rash, decreased appetite, constipation, arthralgia and diarrhea.

Nivolumab

Nivolumab is a fully human monoclonal antibody that targets PD-1. It functions in manner similar to pemrolizumab. FDA granted approval in December 2014 for treatment of metastatic melanomas that do not respond to other forms of treatment (139). The common side effects include rash, itching, cough, upper respiratory tract infections, and edema. The serious side effects engaging the immune system can involve major organs (140).

Future Trends and Conclusions

Historical trends have shown that treatments for stage III and stage IV metastatic melanoma are becoming more targeted and utilize the information gained from improved understanding of cellular and immunological pathway changes that are associated with this malignancy. Pinpointing the mutations in the various pathways that melanoma patients have acquired would allow for the continued development of more efficacious treatments that strive to prevent drug resistance and cancer recurrence. The convergence of immunology with other disciplines of biomedical research would lead to further improvement in the development of newer and more efficacious therapeutic approaches to manage this debilitating and deadly disease. From the preceding information, it is clear that not only have treatments become more molecularly sophisticated, but diagnosis and early detection techniques also have the opportunity to become increasingly specific. Discovery of specific markers of melanoma risk can allow for early detection and limit the amount of cytotoxic treatment that a patient must receive.