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Advances in Anatomic Pathology:
doi: 10.1097/PAP.0b013e3182976c94
Review Articles

Ras, Raf, and MAP Kinase in Melanoma

Solus, Jason F. MD; Kraft, Stefan MD

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Author Information

Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA

The authors have no funding or conflicts of interest to disclose.

Reprints: Stefan Kraft, MD, Department of Pathology, Massachusetts General Hospital, 55 Fruit Street, Warren 831, Boston, MA 02114 (e-mail: skraft@partners.org). All figures can be viewed online in color at http://www.anatomicpathology.com.

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Abstract

A growing understanding of the biology and molecular mechanisms of melanoma has led to the identification of a number of driver mutations for this aggressive tumor. The most common mutations affect signaling of the Ras/Raf/MAPK (mitogen-activated protein kinase) pathway. This review will focus on mutations in genes encoding proteins that play a role in the MAPK pathway and that have been implicated in melanoma biology, such as BRAF, NRAS, and MEK (MAPK kinase), and detail the current understanding of their role in melanoma progression from a molecular biology perspective. Furthermore, this review will also consider some additional mutations in genes such as KIT, GNAQ, and GNA11, which can be seen in certain subtypes of melanoma and whose gene products interact with the MAPK pathway. In addition, the association of these molecular changes with clinical and classical histopathologic characteristics of melanoma will be outlined and their role in diagnosis of melanocytic lesions discussed. Finally, a basic overview of the current targeted therapy landscape, as far as relevant to the pathologist, will be provided.

Melanoma is a highly aggressive malignancy that is still increasing in incidence. The wealth of new knowledge regarding the biology of melanoma and its molecular mechanisms have led to new investigations of targeted therapies that hope to change the dismal outcomes currently seen in patients with advanced melanoma. Among presumed driver mutations (ie, mutations providing a growth advantage of tumor cells), mutations in components of the Ras/Raf/MAPK (mitogen-activated protein kinase) pathway have proven to be essential in the majority of melanomas. This review will focus on the role of some of the MAPK pathway–associated genes (ie, BRAF, NRAS, etc.), with additional consideration of genes encoding for products that feed into the MAPK pathway such as KIT and GNAQ/GNA11.

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Ras/Raf/MAPK PATHWAY ALTERATIONS IN MELANOMA

A large proportion of cutaneous melanomas show mutations in various genes encoding for proteins that are part of the Ras/Raf/MAPK signaling pathway (Fig. 1), which regulates proliferation of melanocytes.1,2 In general, this signaling pathway originates from receptor-linked tyrosine kinases, such as epidermal growth factor receptor, fibroblast growth factor receptor, hepatocyte growth factor, and stem cell factor (SCF), which are activated by extracellular ligands. Ligand binding activates the tyrosine kinase activity of the cytoplasmic domain of the receptor, leading to phosphorylation of tyrosine residues. These changes result in recruitment of GRB2 and SOS adaptor proteins. When these 3 components associate, SOS changes to an activated state that in turn leads to activation of a Ras protein (ie, H-Ras, N-Ras, K-Ras) by binding GTP.

FIGURE 1
FIGURE 1
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Activated GTP-bound Ras, in addition to activating other pathways such as the phosphoinositide-3 kinase (PI3K)/Akt pathway, is able to switch on the activity of the Raf serine/threonine kinase (in melanocytes predominantly B-Raf), which phosphorylates and activates MEK1 and MEK2 (also known as MAPK kinase or MAPKK) tyrosine/threonine kinases. Finally, MEK activates the MAPK’s Erk1 and Erk2.3 Those Erk MAPKs are then able to play an important role in regulating transcriptional factors, which ultimately determine cellular events such as proliferation, senescence, apoptosis/survival, and differentiation.

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BRAF

A majority of cutaneous melanomas show either BRAF or NRAS mutations, with approximately 40% to 50% exhibiting BRAF mutations and 15% to 20% exhibiting NRAS mutations (Table 1).4–12 The BRAF mutations appear at an early stage in tumorigenesis and are preserved through progression.8 These mutations lead to increased activity of the MAPK pathway downstream. The most common mutation, a T to A nucleotide transversion leading to a V600E amino acid substitution within the activation segment of the Raf serine/threonine kinase gene product, increases the catalytic activity of B-Raf and leads to subsequent activation of MEK and ERK MAPKs.4,11,13 Interestingly, this T to A transversion is not considered a “UV signature,”—that is, it is not a type of mutation that is typically seen in the setting of chronic ultraviolet (UV) exposure.

TABLE 1
TABLE 1
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It is noteworthy that a high BRAF mutation frequency has also been found in benign nevi, suggesting that BRAF mutations occur early in the development of melanoma and that additional molecular defects are necessary for malignant transformation.13,14 Although nevi show high levels of the p16INK4a tumor-suppressor gene product, which may protect from progression toward melanoma by p16-driven cell cycle arrest and senescence, inactivating mutations or deletions of the encoding CDKN2A gene (which is also the major gene involved in familial melanoma15) may account for progression to melanoma.11,16–18 However, senescent melanocytes exhibit a mosaic staining pattern of p16, suggesting that p16 may not be the only factor playing a role in this process.17 In addition, although p16INK4a loss leads to a shorter latency period and more BRAFV600E-induced melanomas in a mouse model, it is not required.19

Another possible driver of melanoma in BRAF-mutated melanocytic lesions may be activation of the PI3K/Akt pathway, which promotes survival and cell cycle entry in melanoma cells (Fig. 1). Approximately 20% to 40% of melanomas show loss or altered expression of the PTEN (phosphatase and tensin homologue) tumor-suppressor gene, which acts as an upstream inhibitor of the Akt-PI3K pathway because of its phosphatase activity.6,20–22 Although inactivating mutations in PTEN were initially believed to be relatively uncommon,20,23 recent data (eg, Hodis et al24) suggest a higher rate. There is also a correlation between PTEN mutations and BRAF mutations in human melanoma cell lines, suggesting cooperation between the Ras/Raf/MAPK and PI3K/Akt pathways.25 Since then, this has been further supported by data from mouse models and human melanoma. Although BRAFV600E expression in melanocytes of mice results in melanocytic hyperplasia, frank invasive and metastatic melanoma development is only seen upon additional PTEN silencing.26 Decreased PTEN expression or increased Akt expression is seen only in human melanomas associated with nevi and not in benign nevi alone, in contrast to BRAF (or NRAS) mutations, which are found in nevi and melanoma.27 A similar cooperative role for malignant transformation has been suggested for BRAF mutations and germline mutations or loss of the TP53 gene, which leads to invasive melanoma.28,29 However, TP53 is not as often mutated in human melanoma as in other cancers.30 A recent study showed that overexpression of its negative regulator MDM4 inhibits p53-mediated apoptosis in melanoma.31 However, MDM4 overexpression in melanomas appears to occur irrespective of BRAF mutational status. Finally, microphthalmia transcription factor was found to cooperate with BRAF in melanoma formation.32

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NRAS

Melanomas with normal BRAF often show activating mutations in the NRAS protooncogene, which is upstream of BRAF, predominantly in codon 61 (eg, Ko and Fisher11; Fig. 1).6,33–37NRAS mutations leave the molecule in its active guanosine triphosphate-bound state, which results in increased downstream signaling through the MAPK pathway mediated through Raf,7 predominantly C-Raf.38 However, N-Ras can also activate other pathways such as the PI3K pathway.2 Although mutations at codon 61 of NRAS lead to loss of GTPase activity and to a permanently activated conformation of Ras, the codon 12 and 13 mutations prevent inactivation by GTPase-activating proteins.7,39 In addition to Raf and MAPK, a recent study has shown that the NRAS-mutant melanocytes also rely on Rac1, a Rho GTPase, for their survival and invasive qualities.40

Similar to BRAF mutations, NRAS mutations per se do not appear to be sufficient for malignant transformation and are likely an early event.7,8,41,42 Although they are seen in a lower fraction of benign nevi when compared with BRAF mutations,13 they are frequently present in congenital nevi.43 Similar to BRAF-mutated melanoma, a cooperative effect of NRAS mutations, with loss, inactivating mutations, or promoter methylation of the CDKN2A gene for the p16INK4a (and p14ARF) tumor suppressors, or p53 deletion has been suggested.44–47 As opposed to BRAF-mutated melanoma, alterations of the PI3K/Akt pathway such as PTEN mutations, or decreased PTEN expression with associated increased Akt activity, are not typically seen in NRAS-mutated melanoma.25,48

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Other Ras/Raf/MAPK Alterations

Recently, several studies involving next-generation sequencing techniques have identified multiple additional recurrently mutated somatic genes—that is, possible additional driver mutations.24,49–54 Most of these studies show that the majority of mutated genes carry a “UV signature,” with more of these mutations seen in melanomas with chronic sun exposure. However, many of these may represent UV-induced silent passenger mutations.

Some of these studies identified novel mutations in genes encoding for Ras/Raf/MAPK pathway molecules. For example, 8% of melanomas showed gain-of-function mutations in the MAP2K1 [encoding for MEK1 (MAPKK1)] and MAP2K2 [encoding MEK2 (MAPKK2)] genes.52 These mutations result in constitutive activation of downstream Erk MAPK phosphorylation and also higher resistance to MEK inhibitors. In addition, 24% of melanomas were found to exhibit MAP3K5 [MAP kinase kinase kinase 5 (MAPKKK5); synonyms ASK1, MEKK5] and MAP3K9 [MAP kinase kinase kinase 9 (MAPKKK9); synonyms MLK1, MEKK5] mutations.54 Although these mutations lead to decreased kinase activity, which may seem paradoxical at first glance, these molecules appear to signal through other downstream MAPK from MAP3K9, such as JNK, leading to increased survival and chemoresistance. All of the above MAPK mutations did not correlate with BRAF or NRAS mutation status.

Two recent studies that were carried out on a larger scale introduced new somatic mutations in other genes, which code for products that interact with the MAPK pathway. This was done in conjunction with an effort to exclude silent passenger mutations, which occur at a high rate especially in sun-exposed melanomas due to UV damage. Both studies identified the small Ras-related Rho family GTPase Rac1 as a novel mutated melanoma gene, in addition to other mutations such as those in the serine phosphatase gene PPP6C.24,50 Rac1 regulates cytoskeletal rearrangement and is important for migration, adhesion, and invasion.55 Of the melanomas 3.9% (or 9.2% of sun-exposed nonmucosal nonacral nonuveal melanomas in the other study) were found to show an RAC1 P29S mutation, which keeps Rac1 in its active, GTP-bound state and promotes melanocyte proliferation and migration, probably through MLK3 and Erk MAPK activation.24,50 Interestingly, both studies found a recurrent mutation with a strong UVB signature in RAC1 (among other novel driver mutations showing UV signatures in PPP6C and STK19). This is one of the first observations of a direct UV-induced potential driver mutation, especially as NRAS and BRAF do not show UV-induced mutations.

In addition, one of the studies24 found mutations with a low prevalence in SNX31 and TACC1, 2 proteins with potential Ras pathway interactions. Melanomas negative for NRAS or BRAF mutations may still affect MAPK signaling through other mutations in oncoproteins: 25% to 30% of these “double-negative” melanomas show mutations with putative loss of function in NF1,24,50 which is a negative regulator of Ras and may lead to MAPK activation.56 In addition, occasional mutations in HRAS, CRAF, MAP2K1, or KIT were found in these double-negative melanomas.24

Finally, both studies provided further evidence for the importance of additional mutations for MAPK-dependent tumor progression in BRAF-driven or NRAS-driven melanoma: Hodis et al24 showed that 44% of BRAF-driven melanomas (but only 4% of NRAS-driven melanomas, in confirmation of the earlier studies mentioned above) show PTEN mutations or focal deletions; Krauthammer et al50 showed that BRAF-driven or NRAS-driven melanomas often have losses of PTEN and/or CDKN2A mutations (which codes for p16INK4a and p14ARF). Irrespective of BRAF or NRAS status, Hodis et al24 found retinoblastoma (RB) gene pathway mutations such as those observed in CDKN2A in 24% of melanomas and TP53 gene mutations in 19% of melanomas.

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KITANDGNAQ/GNA11MUTATIONS IN MELANOMA

In an initial study, 39% of mucosal, 36% of acral, and 28% of melanomas arising in chronically sun-damaged skin were found to exhibit mutations or amplifications in the KIT gene, which encodes for the SCF receptor tyrosine kinase, with only 6% showing BRAF mutations.57,58 Upon ligand binding, Kit dimerization results in autophosphorylation and downstream activation of the MAPK, PI3K/Akt, and JAK/STAT pathways.59 This often correlates with increased Kit positivity as assessed by immunohistochemistry,60 although no correlation has been found in some studies.58 However, another recent study showed that much lower fractions of melanomas arising in chronically sun-damaged skin show KIT mutations.61KIT mutations often affect the region coding for the juxtamembrane domain of the receptor, which are often similar to the mutations found in gastrointestinal stromal tumors, and putatively lead to its dimerization and constitutive activation in the absence of SCF.11

The discovery of GNAQ (guanine nucleotide–binding protein Q polypeptide) and GNA11 (a paralogue of GNAQ) mutations in melanocytic neoplasms stems from a mice genetic screen that showed that hypermorphic mutations in GNAQ or GNA11 resulted in diffuse skin hyperpigmentation due to an increase in intradermal melanocytes.62 The histologic appearance of the skin lesions from these GNAQ-mutant or GNA11-mutant mice was reminiscent of human blue nevi, resulting in an investigation of these mutations in human melanocytic neoplasms.62 It was found in 2009 that approximately 50% of uveal melanomas and 83% of blue nevi exhibit mutations in GNAQ, which link G protein-coupled receptors to the intracellular pathways such as the MAPK pathway.63 More recent data illustrate that mutations in exon 5 (Q209) of GNAQ and GNA11 occur frequently in both neoplasms but in a mutually exclusive pattern, with enrichment of Q209 mutations of GNAQ in blue nevi (55% vs. 7%).64 Furthermore, the Q209 GNA11 mutation was more frequently seen in uveal melanoma metastases (57%) than was the Q209 GNAQ mutation (22%).64 To further corroborate the association of the Q209 GNA11 mutation with uveal melanoma metastases, a mouse model of uveal melanoma revealed spontaneous metastases and activation of the MAPK pathway with induction of this mutation.64

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CLINICAL CHARACTERISTICS OF MELANOMAS WITH MAPK PATHWAY MUTATIONS

As mentioned above, the BRAF and NRAS mutations seen in melanoma do not carry a “UV signature.”13 In studies correlating mutation status with chronic sun damage, BRAF mutations were less frequently found in melanomas occurring at sites of chronic sun damage, and more frequently in melanomas occurring in skin with intermittent acute sun damage.5,10 This finding has been further corroborated by additional studies that show that BRAF mutation–positive melanomas occur in younger patients at sites without prominent solar elastosis or freckles, more likely involve the trunk and lower extremities, and less frequently occur in chronically sun-exposed areas.65,66 In a meta-analysis of earlier studies,67 48% of melanomas arising in sites with intermittent sun exposure showed BRAF mutations, whereas 21% of melanomas arising in sites with chronic sun exposure showed BRAF mutations (the lower numbers may be due to inclusion of earlier studies, in which less sensitive methods may have been used). However, the frequency of NRAS mutations appears to be somewhat higher in melanomas from chronically sun-damaged skin compared with that of BRAF mutations (21% in melanomas from sites with intermittent sun exposure and 29% in melanomas from sites with chronic sun exposure). In line with these findings, NRAS-mutated melanoma was more frequently found on the extremities and in older patients.36,68,69

With regard to the relatively rare melanomas arising in non–hair-bearing skin or mucosal membranes, BRAF mutations are also less common in acral or mucosal melanomas.5,9,67,70 These melanoma types also appear to show a similar or slightly lower frequency of NRAS mutations when compared with cutaneous melanomas arising in hair-bearing skin. However, the frequency of NRAS mutations does appear to vary by site, with more frequent NRAS mutations in vaginal and esophageal tumors according to some studies, although case numbers in these studies were low.5,58,68,70–72 A large proportion of mucosal melanomas show evidence of Erk MAPK phosphorylation, which suggests that molecular alterations other than BRAF and NRAS mutations may lead to MAPK pathway activation in these tumors.70,72

With regard to clinical behavior, BRAF mutation–positive melanomas may preferentially metastasize to regional lymph nodes as opposed to non-nodal sites.73,74 The effect of BRAF and NRAS mutations on prognosis shows a somewhat complex picture. Whereas most studies on primary melanomas did not show a role for either mutation in survival,10,36,69,75–77 a recent meta-analysis showed a 1.7× increased mortality risk for BRAF-driven melanoma,78 and a recent large prospective study showed significantly decreased survival in all stages for NRAS-driven melanoma, with a trend toward decreased survival in BRAF-driven melanoma.79 For advanced-stage melanoma with regional or distant metastases (stage III and/or IV) the picture appears clearer; recent studies, some prospective, showed decreased survival for both NRAS-mutated and BRAF-mutated melanoma.68,79–82 For example, Jakob et al68 show a higher fraction of central nervous system involvement in stage IV disease both for NRAS-driven and BRAF-driven melanoma, with decreased survival in NRAS-mutated melanoma. In stage III disease, Moreau et al82 observed a worse overall survival in BRAF-driven melanoma, whereas Mann et al81 observed worse survival of patients with either BRAF-driven or NRAS-driven melanoma.

Cutaneous melanomas with KIT mutations, however, are sometimes found on the chronically sun-damaged skin of the head, neck, and distal extremities or in acral sites.57,58KIT mutations are often mutually exclusive with BRAF mutations. Cutaneous melanomas with KIT mutations on sun-damaged skin also less frequently arise in association with a precursor melanocytic nevus and instead are often encountered in patients with a history of actinic keratoses and nonmelanoma skin cancers.83KIT mutations in mucosal melanomas occur in 12.5% to 39% of cases.57,58,70,72 However, there appears to be variability depending on the anatomic site, with KIT mutations being more common in anogenital melanomas than head and neck melanomas.70,72,84

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HISTOPATHOLOGIC FEATURES OF MAPK PATHWAY–MUTATED MELANOMAS

Recent studies have uncovered histologic differences in melanomas depending on their mutational status. For example, the histologic hallmarks of BRAF mutation–positive melanomas include nest formation of intraepidermal melanocytes, pagetoid spread of melanocytes, thickening of epidermis, and sharp demarcation, which are features seen in superficial spreading melanoma.73,74 Thus, BRAF mutations are most commonly present in superficial spreading melanomas, whereas they are relatively rare in acral and lentigo maligna melanomas, all of which are characterized by a lentiginous growth pattern of solitary melanocytes with poor lateral circumscription and without marked pagetoid spread (Fig. 2).5,66 In contrast, KIT mutations in the skin are relatively more common in acral-lentiginous and lentigo maligna melanoma.5,36,57,58,68,85,86 In addition, BRAF-mutated melanomas are more frequently seen in association with nevi.36,86

FIGURE 2
FIGURE 2
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NRAS mutation–positive melanomas, in contrast, were not found to have such distinct histologic features in some studies but tend to have little or absent pagetoid spread.73,74 However, other studies have suggested that NRAS mutations are more frequently seen in nodular melanomas.33,35,66,68,85

The observations from the above studies and others were summarized in a study by Platz et al.67 According to these data, BRAF mutations are most commonly seen in superficial spreading melanomas (51%), followed by nodular melanomas (43%), with much lower percentages of acral-lentiginous (15%) and lentigo maligna melanomas (14%) showing BRAF mutations. NRAS mutations show a less distinct distribution and were most commonly seen in nodular melanomas (31%), followed by superficial spreading (21%), lentigo maligna (19%), and acral-lentiginous (8%) melanomas.

With regard to prognostic histopathologic parameters, the majority of studies found no correlation between Breslow thickness or mitotic activity and BRAF mutational status,8,68,75,77 although in some studies decreased thickness and decreased mitotic activity was found in BRAF-mutated melanomas.66,79 A correlation between tumor ulceration and tumor-infiltrating lymphocytes has also been suggested in some reports.36,69

In contrast, the majority of studies found a correlation between increased tumor thickness and NRAS mutation, which mirrors the higher percentage of NRAS mutations in nodular melanoma.36,69,79,87 In addition, some studies found increased mitotic activity in NRAS-mutated melanomas.69,79 However, no correlation with thickness and/or mitotic count was found in 3 studies.33,68,75

Desmoplastic melanoma is clinically and histologically distinct, with deeply infiltrative spindle cells in a collagenous stroma, showing high local recurrence rates with relatively low metastasis rates.88 Interestingly, it also appears to be distinct with regard to BRAF and NRAS status. Pure desmoplastic melanomas usually are negative for BRAF mutations, whereas data on NRAS mutations are scarce but also point to a low frequency.89–91

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DETECTION OFBRAFANDNRASMUTATIONS

For BRAF and NRAS mutational analysis, various different methods are currently being used, from traditional Sanger chain-termination sequencing over pyrosequencing to allele-specific standard polymerase chain reaction (PCR) techniques.92,93 Sanger chain-termination sequencing94 continues to be the gold standard and can detect all possible mutations but is relatively time-consuming and apparently shows a relatively low detection ratio of 1 in 5 mutant alleles compared with the 1 in 50 ratio in pyrosequencing.95 Allele-specific PCR is a real-time PCR method that allows for enrichment of known mutations through mutation-specific primers such as that for BRAFV600E, and as a consequence is very sensitive.96 The “Cobas 4800” test for formalin-fixed paraffin-embedded samples is a sensitive Food and drug Administration (FDA)-approved test for the detection of BRAFV600E mutations, and basically represents a modified allele-specific PCR with differentially labeled fluorescent probes for wild-type BRAF and mutant BRAFV600E.97 One potential limitation is cross-reactivity between V600K and V600D mutations. However, the clinical relevance of this cross-reactivity is uncertain. Pyrosequencing depends on the release of pyrophosphate during nucleotide binding in a synthesis reaction that uses a predetermined order of nucleotides.98,99 At present, a chemiluminescence-based detection method is being used. Pyrosequencing requires knowledge of the expected mutations and can only be performed along short stretches of DNA, but if designed well, pyrosequencing is a very rapid and sensitive method that can detect all possible mutations within a short stretch of DNA. As BRAF and NRAS show a limited number of mutations in small well-defined hot spots, pyrosequencing is very suitable for this purpose and shows superior sensitivity when compared with Sanger sequencing or allele-specific real-time PCR.100,101 In contrast, it would not be suitable for determining mutational status of a large gene with many different mutations in different areas.

Advanced highly sensitive technologies such as mass spectrometry sequencing, high-resolution melting analysis in combination with sequencing, coamplification at lower denaturation temperature-PCR, or advanced pyrosequencing techniques with microfluidic flow chambers have been tested,92,93,102–106 some of which also allow for analysis of multiple mutational hot spots in 1 sample or longer stretches of DNA such as whole exons. In practice, detection of rare mutant alleles may often not be necessary at present, given that less-abundant mutated genes may not be therapeutically relevant, although increased sensitivity would facilitate the detection of mutations in small samples such as in peripheral blood.93 As therapies for advanced melanoma may become increasingly personalized in the future, with combination therapies based on the mutational profile of a given melanoma, broader techniques assessing for multiple mutations in multiple genes, e.g. hybridization-based assays such as SNaPshot (multiplex PCR, multiplex single base primer extension, and capillary electrophoresis107) or next-generation sequencing techniques involving whole exome or genome sequencing (which would also detect unknown mutations), may become increasingly common tools.

Recently, a monoclonal antibody to the BRAFV600E protein has been characterized and studied in hairy cell leukemia, metastatic melanoma, and metastatic papillary thyroid cancer.108–112 This antibody was recently shown to exhibit excellent sensitivity and specificity for the detection of BRAFV600E mutations in metastatic melanoma by immunohistochemistry.113,114 In a small number of primary melanomas tested, positive labeling was limited to superficial spreading melanomas, which supports the previous reports mentioned above. Another study performed on 35 cases of primary cutaneous melanoma also showed excellent sensitivity and specificity of the BRAFV600E antibody.115 The VE1 antibody, however, is not able to detect other BRAF mutations at V600 or close by.101 A cost-effective combination strategy that uses the VE1 antibody for screening, with mutational analysis of negative or uninterpretable cases by pyrosequencing, has been suggested.101

One caveat is that the availability of an antibody to detect the BRAF mutation may lead to pathologists partly basing their diagnosis of melanoma on the presence of a BRAFV600E mutation. However, it is important to consider that, in the case of BRAFV600E mutation, the presence of the mutation does not prove that the lesion is a melanoma; as described above, a large proportion of nevi and other nonmelanocytic lesions also harbor the BRAFV600E mutation. It may, however, aid in the classification of poorly differentiated metastases from unknown primaries, although it is not entirely specific for melanocytic neoplasms.

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TARGETED THERAPEUTICS FOR MELANOMAS WITH MAPK PATHWAY MUTATIONS

Owing to the central importance of the MAPK pathway in the majority of melanomas, considerable research effort has led to many novel small molecule inhibitors being tested in clinical trials, or—in the case of vemurafenib—to FDA-approved therapies. Although therapy is not the main focus of this review article and other dedicated reviews are recommended for further reference (eg, Sullivan and Flaherty2), some basics will be discussed in the following portion.

The first selective BRAF inhibitor (vemurafenib; formerly PLX4032) for the treatment of metastatic melanoma exhibiting the BRAFV600E genotype was approved by the FDA in August 2011, because to marked results in 74% to 80% of patients with BRAFV600E-positive melanomas; however, tumor progression was seen in almost all patients 6 to 8 months after initial treatment.116 Another selective BRAF inhibitor (dabrafenib; formerly GSK2118436) showed promising results with an approximately 2-fold increase in progression-free survival in a phase III trial, which is in a similar range as that of vemurafenib.117

In addition, multiple clinical trials using small molecule inhibitors of Raf and its downstream effector MEK are ongoing, some showing promise as future therapeutic agents.2,7,118 For example, an MEK inhibitor (trametenib; GSK1120212) has recently completed a phase III trial and is effective in a subset of BRAF-mutant melanoma patients, whereas other MEK inhibitors have been less successful.2,119 Selumetinib, another MEK inhibitor, is effective in a small subset of BRAF-mutated melanomas.120 MEK162 appears to be effective in 20% to 30% of patients with BRAF-mutated or also with NRAS-mutated melanomas.2,7,121

As mentioned above, the response to BRAF inhibitors is typically short-lived, and a multitude of predominantly MAPK-dependent, but also MAPK-independent, resistance mechanisms have been described. Among these are copy number gains leading to increased BRAFV600E protein expression, upregulation of upstream receptor tyrosine kinases, increased C-Raf expression, Ras activation, NRAS/MEK/MAPK mutations, or increased activation of the PI3K/Akt pathway.2,7,9,122–129

Owing to this short-lived response with single-agent treatment, various combination regimens to overcome resistance mechanisms are also being tested.2,7 Very recently, a phase II trial combining the BRAF inhibitor dabrafenib and the MEK inhibitor trametinib showed increased response rates and progression-free survival in patients with BRAF-mutant melanoma when compared with monotherapy.130

Recent studies have also demonstrated that treatment with BRAF inhibitors leads to paradoxical C-Raf (RAF1) activation in cells with wild-type B-Raf through the formation of B-Raf/C-Raf heterodimers or C-Raf homodimers.131–134 C-Raf then activates the downstream MAPK pathway, providing proliferation signals, which may explain the resistance of BRAF wild-type melanomas to BRAF inhibitor therapy. Along with mutant RAS (predominantly HRAS), this molecular mechanism may be responsible for increased incidence of squamous neoplasms in patients on BRAF inhibitors.133,135

No direct drugs have been successful against mutated NRAS up to now,2,7,9 but early data from new MEK inhibitors such as the aforementioned MEK162 show some promise.7,121 Other studies with single-agent MEK inhibitors showed limited efficacy in NRAS-mutant melanomas overall.120,136 However, combination strategies may show success in the future, for example, by targeting MEK and CDK4, as recently shown in a mouse model of NRAS-mutant melanoma,137 or by targeting MEK and the PI3K/Akt pathway.7

KIT mutations also confer sensitivity to the imatinib tyrosine kinase inhibitor and other comparable agents.138–140 Although initial trials did not show a benefit in melanoma overall, newer trials on patients with melanomas showing KIT alterations show some promise for response to these targeted agents; however, the response rates are varied from complete response in a minority to transient, partial responses.59,141–143 The patients with the best responses had certain KIT mutations in exons 11 and 13, suggesting that these therapies may be suitable in a small subset of patients and underscoring the need for careful patient selection and diagnostic evaluation.

No direct anti-GNAQ or anti-GNA11 therapies currently exist for melanomas harboring GNAQ or GNA11 mutations, and there are currently no FDA-approved therapies for these patients. However, as GNAQ/GNA11 mutations lead to activation of downstream targets in the MAPK pathway, multiple MAPK-targeted therapies have been studied in a preclinical setting on uveal melanoma cell lines. MEK inhibitor combination therapy with a PI3K inhibitor or an mTOR inhibitor has shown increased cell death in uveal melanoma cell lines.144,145 In addition, protein kinase C inhibitors appear to have antitumor activity against uveal melanoma cells.146

In summary, it is evident that personalized therapies with a panel of molecular inhibitors will be increasingly performed, and as a consequence more sophisticated diagnostic techniques will be necessary.

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Keywords:

melanoma; mitogen-activated protein kinase (MAPK); Raf; Ras; molecular pathology

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