FormalPara Key Points

Genetic aberrations are present in the great majority of patients with melanoma.

The emergence of effective molecular-targeted therapy for melanoma mandates accurate and widespread molecular testing for patients with metastatic melanoma.

Emerging technology is being used to develop tissue- and blood-based molecular analysis that is expected to improve the care of patients with metastatic melanoma.

1 Introduction

The elucidation of how melanoma cells utilize molecular signaling pathways to promote growth and survival, and the identification of agents that target mediators of this pathway, has led to major clinical benefits for patients suffering from this devastating disease [1]. Additionally, an improved understanding of immune activation and tumor immunology has led to the approval of transformative immunotherapies (ipilimumab, nivolumab, and pembrolizumab) that have been shown to improve patient survival [2–4]. In fact, over the past 4 years, the US FDA have approved six agents for the treatment of metastatic/unresectable melanoma, marking an unprecedented advancement in the standard of care. Three of these agents, vemurafenib, dabrafenib, and trametinib, are drugs that block mediators in the most important signaling pathway in melanoma, the mitogen-activated protein kinase (MAPK) pathway. Vemurafenib and dabrafenib are BRAF inhibitors, while trametinib is a MEK1/2 inhibitor. Each of these drugs has been shown to improve outcomes in BRAF-mutant melanoma compared with chemotherapy, and more recently, combined BRAF/MEK-inhibitor therapy (dabrafenib plus trametinib, vemurafenib plus cobimetinib) has been shown to be more effective than single-agent BRAF-inhibitor therapy; which has led to the FDA approval of the dabrafenib/trametinib combination and the filing of the vemurafenib/cobimetinib combination to the FDA for consideration of approval [5–9]. While a number of questions remain regarding the optimal sequencing and combination of molecularly targeted and immune-targeted therapies, it is clear that molecular analysis remains a key component in the care of every patient with metastatic melanoma.

Since BRAF mutations are present in approximately 50 % of metastatic cutaneous melanomas, lead to constitutive activation of the MAPK pathway, and may be effectively targeted by BRAF and MEK inhibitors, BRAF mutation analysis has been part of the standard care approach to patients with metastatic melanoma for over 5 years [10, 11]. Other oncogenic mutations have also been described, including NRAS, NF1, and CKIT, which may also be targeted by small molecule inhibitors [11, 12]. While definitive benefit of therapeutic targeting of these mutations has not been established, phase II clinical trial data suggest improvement with MEK inhibitor therapy in uveal melanoma and NRAS mutant melanoma as well as with CKIT inhibitors in CKIT-mutant or amplified melanoma [13–17]. Thus, genetic evaluation beyond BRAF mutational analysis should be a crucial part of the work up of metastatic melanoma. In this review, the standard techniques of molecular evaluation of metastatic melanoma are described, their molecularly targeted therapeutic implications are discussed, and future directions in this evolving field are presented.

2 Key Molecular Pathways in Cutaneous Melanoma

The MAPK pathway is activated in the majority of the metastatic melanomas [18]. Signaling through the MAPK pathway may occur through canonical activation by growth factor—growth factor receptor [receptor tyrosine kinase (RTK) or g-protein coupled receptor (GPCR)] interaction, hyperactivation/expression of RTK, oncogenic mutation of one of its mediators (RAS and BRAF most commonly), or loss of function mutations of key MAPK pathway regulators [1]. The RAS family consists of three isoforms (H-, K-, N-) that, when activated, have a GTP-bound kinase domain compared with the GDP-bound inactive kinase [19]. One of the protein products of the neurofibromatosis gene, NF1 is a tumor-suppressor protein that negatively regulates RAS signaling [20]. In physiological conditions, NF1 stimulates the GTPase activity of RAS that leads to accumulation of GDP-bound (inactive state) NRAS. The loss of function of NF1 leads to the loss of regulatory control of RAS, which ultimately leads to increased downstream activity [21]. Once activated, RAS can mediate signaling through a number of signaling [including the MAPK and phosphoinositol-3-kinase (PI3K)] pathways. RAS-binding domains (RBD) are found on one of the effectors of RAS, namely RAF family members, and RAS interaction with the RBD leads to homo- and hetero-dimerization of RAF isoforms (A-, B-, and C-) [22]. RAF dimerization leads to RAF kinase activation and serine phosphorylation of MEK1/2, which subsequently phosphorylates the extracellular signal-regulated kinase (ERK), also known as MAPK, which is a major regulator of proliferation and survival [22].

NRAS mutations have been identified in approximately 15–25 % of melanomas and play an essential role in oncogenesis [11, 18, 23]. Somatic mutations in the NRAS gene cause constitutive activity of the NRAS protein, which leads to activation of serine/threonine kinases, thereby promoting cell cycle progression, cellular transformation, and increased cell survival [18]. BRAF and CRAF are the two most important downstream mediators of activated RAS, and while direct inhibition of NRAS has been challenging, downstream targeting of BRAF/CRAF, MEK, and ERK is a valid strategy supported by preclinical and emerging clinical data [24–29]. Tumors with loss of function mutations of NF1 are expected to be similarly sensitive to strategies that are being developed to treat NRAS mutant melanoma [30]. In patients with NRAS mutant melanoma, treatment with MEK inhibitors is associated with responses in 20 % of patients and a progression-free survival (PFS) of approximately 4 months [13]. Given this promising response rate and disappointing PFS, efforts have been made to build upon this modest efficacy signal. Preclinical work to identify the major mediator of resistance of MEK inhibitors in NRAS mutant melanoma showed that cell cycle mediators generally, and CDK4 specifically, are implicated, and cytotoxicity is enhanced with the combination of MEK inhibitors plus CDK4/6 inhibitors [31]. In patients, the combination of binimetinib (MEK inhibitor) and LEE011 (CDK4/6 inhibitor) was associated with a 33 % response rate in a phase I trial, but was associated with significant toxicity [32]. A similar phase I/II trial of trametinib and the CDK4/6 inhibitor palbociclib is ongoing (NCT02065063).

As described above, mutations in BRAF are the most commonly identified mutations in metastatic melanoma, occurring in 40–50 % of cases [10, 11]. Among BRAF mutations, the most common is a substitution of valine with glutamic acid (V600E), though other mutations at the 600 position include V600K, V600D, and V600R and represent 5–25 % of BRAFV600 mutations in melanoma [10, 33–36]. Additionally, mutations at other positions, including G466, G469, D594, D597, and K601, have also been reported [37]. The most compelling clinical data with BRAF and MEK inhibitors has been in patients with either BRAF V600E or BRAF V600K mutations, and vemurafenib, dabrafenib, trametinib, and the combination of dabrafenib and trametinib are FDA approved only for these patients [5, 6, 38, 39]. There have been both clinical and preclinical reports of responses to BRAF and/or MEK inhibitors with the other BRAF V600 mutations, as well as for some non-V600 mutations [40–42]. Resistance to BRAF-targeted therapy is the single major problem with single-agent BRAF inhibitors and BRAF/MEK inhibitor combinations, with patients receiving these therapies experiencing PFS ranging from 5–7 to 9–11 months, respectively [5–9]. While no single mechanism of resistance has been described, dozens of genetic-, epigenetic-, post-translational-, and protein-level (e.g., increased expression of RTKs, BCL-2 family members, etc.) events have been implicated in mediating resistance to these agents [43–50]. To date, no obvious strategy has emerged to effectively treat patients who have developed BRAF-targeted therapy resistance.

Other oncogenic mutations are more frequently seen in patients with non-cutaneous subtypes of melanoma, such as those melanomas that arise from acral skin, mucosal sites, or the uvea [51]. One such gene is CKIT, which is rarely mutated in cutaneous melanoma (3 %) but frequently mutated in acral and mucosal melanoma (~20 %) [11, 12]. This is clinically relevant since the treatment of patients with CKIT mutant melanoma has been associated with responses to the CKIT inhibitors imatinib and dasatinib. Specifically, in three phase II studies, response rates in patients with either mutation or amplification of CKIT, especially in exons 11 or 13, had response rates in the 15–35 % range [14–16]. Uveal melanoma is associated with mutations in GNAQ and GNA11, oncogenes that encode members of the G protein alpha subunit [52, 53]. These mutations are mutually exclusive, present in over 80 % of patients with uveal melanoma (and rarely in non-uveal melanoma), and lead to the activation of the MAPK pathway, leading to enhanced cell division and tumor growth [54]. Not surprisingly, MEK inhibitors have been evaluated in these patients, and a randomized phase II trial has investigated the MEK inhibitor selumetinib compared with chemotherapy (temozolomide). In this trial, patients treated with selumetinib had better response rates and PFS than those treated with chemotherapy, though overall survival was similar in both cohorts [17]. Based on these data, in April 2015, the FDA granted approval with orphan drug designation to selumetinib for the treatment of patients with metastatic uveal melanoma.

P53 plays a critical role in regulating the cell cycle. TP53 is the most commonly mutated tumor-suppressor gene in cancer, though it is only mutated in approximately 20 % of patients with melanoma [11]. However, MDM4 and MDM2, which are proteins that contain a p53 binding domain and lead to p53 ubiquitination and proteasomal degradation, are overexpressed in at least 50 % of melanomas [55, 56]. Specifically, mutations or amplifications of MDM4 and/or MDM2 can functionally inactivate p53 and provide a growth and survival advantage to these malignant cells [55–57]. The inhibition of the interaction between p53 and MDM2/4 could theoretically restore the function of p53 in melanoma cells and increase the sensitivity of these cells to targeted therapy, such as MEK inhibitors [58]. There are preclinical data to support this approach, and clinical trials are ongoing to determine the feasibility and effectiveness of combined MDM2 and MEK inhibition in p53 wild-type melanoma (NCT02110355).

The CDKN2A gene is located on chromosome 9p21 and encodes two proteins, p16INK4a, which is a cyclin-dependent kinase (CDK) inhibitor, and p14ARF, which functions to inhibit MDM2 and thus preserve p53 levels [59]. In patients with a family history of melanoma, CDKN2A gene mutations are 7–10 times more common, and represent the dominant inherited genetic mutation associated with the development of melanoma [60]. However, the overwhelming majority of patients with melanoma do not have an inherited mutation in CDKN2A, though over 70 % have one or more of the following genetic aberrations: deletion of CDKN2A (~40 %), mutations in p16INK4a or p14ARF (19 and 12 %, respectively), CCND1 (Cyclin D) amplification (11 %), or CDK4 amplification (3 %) [11]. Since all of these events lead to dysregulation of the cell cycle and act through activation of CDK4 (typically upstream), it makes sense that CDK4 inhibitors would be evaluated in melanoma. In fact, CDK4/6 inhibitors have been shown to inhibit growth in cell lines with the above-mentioned aberrancies and are now being evaluated in patients with advanced melanoma as single agents and in combination with MAPK inhibitors [61] (NCT01841463, NCT01820364, NCT02065063, NCT01781572). Figure 1 summarizes the molecular signaling of key molecular pathways relevant to melanoma development and progression described above.

Fig. 1
figure 1

Summary of molecular signaling in melanoma. Multiple mediators and regulators of the MAPK and PI3K pathways have been linked to melanoma. Key oncogenic mutations (blue) and tumor-suppressor genes (red) are shown. CDK4 cyclin-dependent kinase 4, GPCR g-protein coupled receptor, PI3K phosphoinositol-3-kinase, PTEN phosphatase and tensin homolog, RTK receptor tyrosine kinase, TORC1/2 target of rapamycin complex 1/2

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3 Molecular Testing in Melanoma

3.1 Tissue-Based Molecular Analysis

The assessment of the mutational status of the BRAF is extremely important because V600 mutations have predictive value for use of BRAF inhibitors in melanomas [62]. BRAF mutations are present in approximately 50 % of all melanomas, wherein V600E and V600K represent the great majority (in excess of 90 %) [11]. Recently, a BRAFV600E mutation-specific monoclonal antibody (VE1) has shown very high sensitivity and specificity when compared with DNA-based sequencing [63, 64]. The use of such a specific and convenient marker as VE-1 has potential clinical utility due to rapidity of results, though the clinical standard of BRAFV600 mutational analysis to date remains more sophisticated molecular analysis as BRAF IHC staining has not been predictive of outcome with BRAF inhibitors and only detects V600E mutations [65]. These other methods of BRAF mutation assessment that are available include Sanger sequencing, pyrosequencing, real-time polymerase chain reaction (PCR), and high-resolution melting analysis [66–68]. The two FDA-approved, commercially available methods for evaluation of BRAF mutation are Cobas 4800 and THxID. The Cobas 4800 test is a real-time PCR base assay that uses a probe-based methodology that primarily detects the V600E mutation, with mutational variants such as V600K, V600R, and V6000D detected inconsistently [35, 66]. This assay was used in the pivotal trials of vemurafenib and has a 97.3 % positive agreement in detecting BRAFV600E and is more sensitive than Sanger sequencing [5, 69]. The THxIDTM-BRAF assay, which was developed as a companion diagnostic to dabrafenib and trametinib, incorporates amplification refractory mutation system (ARMS)-PCR that utilizes primers designed to quantify both V600E and V600K, making it a more accurate test than the Cobas assay for V600K mutations [70].

Other mutational detection platforms utilize different technologies that allow for the detection of mutations in many genes. The Sequenom mutation detection assay uses a massARRAY system-based test that combines mass spectrometry, PCR, and primer extension to determine each allele, and it is able to analyze dozens of hotspot mutations in a single run [71, 72]. An assay with similar capabilities is SNaPshot, which is a multiplex mutational profiling based on the applied biosystems SNaPshot platform that utilizes a fluorescence technology to detect hotspot mutations of multiple genes simultaneously [73]. Both of these platforms can detect mutations when mutant allelic frequency is as low as 5 % of the total DNA, though they are limited by their inability to detect oncogene amplification and tumor suppressor gene deletions. Massive parallel sequencing, also commonly referred to as next-generation sequencing (NGS), uses a testing platform that can sequence 1 million to billions of short reads per instrument run rather than single DNA fragments. This enables rapid sequencing of large stretches of DNA base pairs, spanning entire exomes or genomes. A number of institutions and private companies have developed either targeted whole exome sequencing (WES) or whole genome sequencing (WGS) platforms, which can detect point mutations, insertions, deletions, chromosomal rearrangements, and genomic amplification. One such example is the FoundationOne® Panel from Foundation Medicine, which assesses over 275 cancer-related genes and is able to detect single nucleotide variants, short insertions and deletions, copy number alterations (e.g., amplification), and selected fusions [74].

While the standard of care for patients with solid tumor malignancies generally, and melanoma specifically, does not mandate this type of analysis, as a greater understanding of the prognostic and predictive impact of genetic aberrations is gained and the cost of these technologies decreases, it is expected that these types of analyses will become standardized for routine oncologic care. An important aspect of this type of testing in patients with melanoma is the high mutational burden associated with cutaneous melanoma, which complicates the interpretation of which alterations are biologically relevant (driver mutations) and which are not (passenger mutations) [11]. Additionally, wide-scale genomic analysis has the capacity to identify inherited, germline mutations of uncertain significance that may be interpreted as a tumor-specific, somatic mutation without tumor/normal sequencing comparison; a technique that offers an ability to distinguish germline from somatic mutations but also increases the cost of the analysis [75]. It remains to be seen whether WES/WGS becomes widely adopted for patients with melanoma, due to the challenges of interpreting the individual contributions of individual mutations in the background of a high mutational burden, but targeted NGS assays are being used more broadly and provide important information about the identified genes that have been determined to be therapeutically relevant to date. Table 1 summarizes the tissue-based analytics techniques currently in use and/or in development.

Table 1 Summary of tissue-based, mutational detection assays
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3.2 Blood-Based Analysis

Analysis of the peripheral blood has been implemented to assist in the management of malignant disease. The most obvious use of blood analysis is the identification and quantification of leukemic cells in the setting of acute or chronic leukemia. However, the last several decades have seen an emerging use of circulating biomarkers, typically serum or plasma protein levels such as carcinoembryonic antigen (CEA) or prostate-specific antigen (PSA), which are followed serially in patients with known solid tumor malignancies as a ‘tumor marker’ to help approximate disease activity or tumor burden. While these types of assays remain useful in a number of diseases, recent advances in molecular analytics now offer the potential for multiplexed proteomic analysis of serum, sophisticated analysis of circulating nucleic acids such as circulating free DNA (cfDNA) or RNA, isolation and analysis of shed tumor factors (e.g., exosomes), and capture and testing of circulating tumor cells (CTCs). Presently, there are few if any reliable blood-based biomarkers routinely in use for patients with melanoma, though a number of potential assays are being developed that may prove to be useful.

3.2.1 Lactate Dehydrogenase and S100

There is no uniformly accepted blood-based ‘tumor marker’ used to follow patients with either high-risk, resected melanoma or those receiving therapy for unresectable and/or metastatic melanoma. Two markers occasionally used are serum lactate dehydrogenase (LDH) and S100. LDH is an enzyme that is detectable in the blood and that can be elevated for a number of reasons, though typically is increased in the setting of intravascular or extravascular cellular destruction (e.g., hemolysis, intravascular mechanical red cell shearing) or tissue ischemia and necrosis (e.g., myocardial infarction, ischemic bowel). In melanoma, serum LDH has long been described as a marker of poor prognosis in stage III and IV disease (independent of therapy) and is part of the American Joint Committee on Cancer (AJCC) staging system for patients with stage IV disease [76–79]. It is of uncertain to little value as a traditional tumor marker that can be followed serially, such as PSA in men with prostate cancer. Similarly, S100 offers prognostic value, but is of low utility in the stage III or IV setting when followed serially [79–83].

3.2.2 Circulating Tumor Cells

The isolation and quantification of CTCs from solid tumor malignancies has been an area of intense investigation. For epithelial malignancies, capture techniques have targeted the surface marker epithelial cell adhesion molecule (EPCAM), which is highly expressed on circulating malignant cells [84]. With the isolation of cells, a wide variety of testing can be performed, including mutation genotyping prior to targeted therapy, quantification as a surrogate for tumor burden, analysis for acquired changes in the setting of a specific therapy, and even the establishment of CTC cultures that may be used to screen for effective therapies [85, 86]. Unfortunately, circulating melanoma cells rarely express EPCAM and heterogeneously express classic melanoma antigens such as MART1, MELAN-A, and MAGE3 [87]. As such, progress has been limited, though techniques utilizing multiple antibody cocktails as well as negative selection microfluidics have recently demonstrated that melanoma cells can be successfully captured, quantified, and analyzed from peripheral blood [88, 89]. While these techniques require validation in larger sample sets and across multiple institutions, these findings serve as a proof of principle that melanoma CTC analysis may be a potential ‘liquid biopsy’ technique that will help manage melanoma patients in the future.

Another potentially useful analysis of CTCs is the evaluation of tumor cell gene expression profiling from isolated peripheral blood mononuclear cells (PBMCs). Since tumor cells differentially express certain genes compared with PBMCs, it is possible to analyze messenger RNA (mRNA) from the isolated PBMCs (where CTCs are found during standard isolation techniques). One such assay is a four mRNA reverse-transcriptase quantitative PCR (RT-qPCR) assay (MART-1, GalNAc-T, PAX-3, and MAGE-A3) that shows increasing signal (higher percentage detected) in patients with higher AJCC stage (15, 30, 75, 85 % for stages I–IV, respectively) [90, 91]. Another RT-qPCR technique has shown high sensitivity and specificity to determine BRAFV600E mutation status in patients with metastatic melanoma [92].

3.2.3 Circulating Nucleic Analysis and Multiplex Assays

With the development of effective targeted therapies for molecular-defined subsets of solid tumor malignancies, including melanoma, the widespread use of blood-based assays that determine mutation status is expected in the near future. Additionally, since many of the techniques being developed are quantitative, it is safe to assume that serial measurement of mutation levels with blood-based assays will allow providers to monitor the effectiveness of therapies in patients with metastatic disease and to determine disease status (recurrence, remission, progression) in patients with both high-risk and metastatic disease. To date, detection and quantification of oncogenic mutations in the blood of cancer patients has been well described for mutations in PI3K, KRAS, EGFR, and BRAF, using a number of techniques, including ARMS, digital droplet PCR (ddPCR), allele-specific quantitative PCR (AS qPCR), cold PCR, and the use of a combination of emulsion-based digital PCR and flow cytometry (a technique knows as beads, emulsion, amplification, and magnetics or ‘BEAMing’) [93–99]. Additionally, emerging technology to isolate shed exosomes and analyze exosomal DNA and RNA has been used to detect, quantify, and follow BRAFV600 levels.

3.2.4 Future Directions of Circulating Analysis

As noted above, it is clearly possible to isolate tumor cells and shed factors (exosomes) from the peripheral blood as well as to detect and analyze circulating nucleic acids. To date, none of these techniques have been adopted in the clinical practice of those caring for patients with melanoma; however, a number of potential applications exist for these types of analyses. First and foremost, the mere ability to accurately determine a patient’s mutational status would greatly simplify clinical management. In current practice, a patient who presents with recurrent or newly diagnosed metastatic disease must have a biopsy to establish the diagnosis of metastatic disease, and then the tumor must be analyzed to determine mutational status. While techniques such as the BRAF IHC have a relatively quick turnaround (~48 h from biopsy), it does not provide information about non-V600E, BRAF V600 mutations and obviously does not evaluate for non-BRAF mutations, such as NRAS or CKIT. Other mutational detection platforms (Table 1) have turnaround times of approximately 1 week. Thus, clinicians are often in the uncomfortable position of waiting 1–2 weeks to learn the mutational status of patients prior to making a treatment recommendation. While many clinical situations do not require BRAF status to be known at the time therapy begins (such as with front-line immune checkpoint inhibitors), scenarios exist in which a rapid and predictable response is necessary for the patient to survive in the short term. It is this type of case that highlights the unmet need for rapid and accurate mutational analysis. A blood-based assay would have the advantages of rapid sample acquisition (compared with biopsy) and a quick turnaround time (likely within 24–48 h).

A second utility of a blood-based assay, particularly one that provides quantitative data, would be the ability to follow mutational load over time. In the high-risk setting, such as stage IIB–IIIC melanoma, mutation levels could theoretically be followed regularly in place of body imaging, which would have the benefit of lowering cost, reducing radiation exposure, and potentially improving the positive predictive value of positron emission tomography/computerized tomography (PET/CT) scans. In the stage IV melanoma setting, serial monitoring of mutational load would be of great clinical value, particularly if, as some preliminary testing has shown, mutation levels may increase in advance of radiographic evidence of progression in a subset of patients [100, 101]. In an ideal scenario, patients would be followed with multiplexed assays that allow for the detection and quantification of a number of mutations, which would allow for the potential detection of emerging subclones that would better inform providers and provide the opportunity to make a change in systemic therapy prior to the development of obvious clinical or radiographic progression; all in the absence of performing repeat tissue biopsies.

4 Conclusion

The mainstays of treatment for metastatic melanoma include immune-targeted and molecular-targeted therapy. The latter requires reliable mutational analysis to accurately identify the subpopulations of patients most likely to benefit from these treatments. The first example of this in melanoma was the identification that only patients with BRAF mutations at the V600 position reliably respond to potent and specific inhibitors of BRAF. This led to the development of companion diagnostics that were FDA approved alongside vemurafenib (Cobas 4800) and dabrafenib (THxIDTM-BRAF). Now, with the development of potentially effective therapies for other molecular subgroups, such as NRAS and/or non-V600 BRAF mutations, there is a need to expand the mutational analysis beyond a single-nucleotide substation at the 600 position in BRAF. With the advent of a new generation of multiplexed assays that utilize a wide variety of qualitative and quantification strategies, in both tissue and blood, the field is in a good position to identify molecular subsets of patients most likely to respond to molecular-targeted therapy. Further, recent data from molecular analysis of samples obtained from melanoma patients prior to treatment with immune-checkpoint inhibitors suggest certain features may define which patients are most likely to respond to these types of therapies and include higher mutational burden, expression of neo-antigens, NRAS mutational status, and evidence of immune infiltration [102–106]. As these data mature, it will be critical to determine whether these features are predictive of treatment response to both molecularly targeted and immune-targeted therapy, or are only predictive of immune-targeted therapy (perhaps only to specific immunotherapies). As the standard of care for advanced melanoma continues to evolve, the field must continue to take a comprehensive approach to biomarker development that attempts to build models of predicting response to therapy and tools to more effectively follow patients over time.