Rapidly advancing genomic sequencing technologies are changing all areas of cancer, from diagnosis to surveillance, and prognostication to treatment. The role of genomic testing in melanoma is expanding, and multiple genomically based tests are available, including somatic tumor sequencing for actionable genetic alterations and tumor mutational burden, prognostic gene expression profiling from tumor tissue, and germline genetic testing from blood. The available testing options have varying levels of supporting data, from robust to preliminary. Here we summarize the available genomic and genetic tests for melanoma, and the level of evidence supporting each of these. We also discuss the current impact of genomic sequencing on the management of melanoma, as well as roles it may play in the near future.
Introduction
The last decade has seen rapid advances in the utilization and understanding of immunotherapy; these advances have been largely due to groundbreaking work in the immunobiology of melanoma, a cancer well known to have one of the highest mutational burdens among common cancers.[1] During the same period of time, tumor mutation profiling to inform targeted therapy use has gained considerable traction and, in a more limited form, has become essential to clinical decision making for melanoma treatment in the context of highly effective BRAF and MEK inhibition. Because of its very considerable genetic mutational burden, melanoma is fertile ground for the development of genetic technology for treatment and surveillance. Multiple clinical tests that involve genetic profiling of melanoma are already commercially available and are being used with increasing frequency in both the postoperative and metastatic settings (Table 1). Here we describe the current clinical uses of genomic sequencing in melanoma for surveillance, identification of inherited cancer predispositions, and therapeutic decision making.
In the Metastatic Setting: Flavors of Somatic Sequencing
Because of the high response rates and long-term survival gains possible with the combination of BRAF and MEK inhibitors, somatic tumor profiling has become essential to initial medical decision making in metastatic melanoma. Given recently published data showing the efficacy of dabrafenib and trametinib in the adjuvant setting,[2] BRAF status is reflexively determined in all patients with stage III or stage IV disease. BRAF status may be ascertained in several ways, with each mode of ascertainment having certain limitations. For rapid turnaround, as might be required in a rapidly declining patient with stage IV melanoma, we routinely obtain immunohistochemistry (IHC) staining of BRAF V600E protein. Results are available in 24 to 48 hours. An important limitation of this test is that IHC is uninformative with regard to other actionable V600 mutations. While the V600E mutation is by far the most common, the V600K mutation accounts for 10% to 30% of all V600 mutations[3] and is equally sensitive to BRAF-MEK inhibition. Thus, IHC cannot be used alone as a comprehensive diagnostic test for BRAF status, and somatic tumor sequencing is required to fully characterize BRAF mutation status.
Somatic tumor sequencing is available from a variety of different vendors at different price points and with varying utility. For characterization solely of melanoma genes of clinical interest, a targeted panel should include at least BRAF, KIT, and NRAS (Table 2). GNAQ and GNA11 are often included in commercial melanoma targeted panels because of their ability to identify a melanoma as one of ocular origin; however, the identification of mutations in these genes does not yet have therapeutic implications. NF1 is the third most commonly mutated gene in melanoma but is not normally tested due to its lack of clinical actionability; however, mutations in this gene confer a worse prognosis.[4,5] Mutations in the most commonly tested genes-BRAF, KIT, and NRAS-have at least phase II data supporting the efficacy of targeted agents in melanomas carrying the corresponding mutant genes. For BRAF V600-mutant melanomas, multiple phase III studies have demonstrated response rates of 70% to 80% and 5-year survival rates approaching 25% with combination BRAF-MEK inhibition; combinations currently approved by the US Food and Drug Administration (FDA) include dabrafenib/trametinib[6,7] and cobimetinib/vemurafenib.[8] Phase II data demonstrate moderate efficacy exist for KIT inhibitors in KIT-mutant melanomas[9,10] and for MEK inhibitors in NRAS-mutant melanomas,[11] with overall response rates of approximately 20% for both classes of drugs.
Alternately, many cancer centers have adopted the use of comprehensive tumor genomic sequencing with targeted gene panels that include from tens to hundreds of genes. Many of these genomic profiling tests assess tumor mutational burden (TMB), a measure of the mutational complexity of a tumor. The first indication that higher TMB correlates with greater likelihood of response to immunotherapy was discovered within a cohort of ipilimumab-treated melanomas, and this trend was later identified in multiple tumor types.[12,13] As with programmed death ligand 1 expression, the correlation between TMB and immunotherapy response does not have perfect positive predictive value, and thus is not used in our clinical decision making at this time. More comprehensive panels also provide alternative means of assessing microsatellite instability (MSI), traditionally measured with a polymerase chain reaction (PCR)-based test or measured by proxy with IHC staining for the loss of mismatch repair gene expression. MSI status is now an FDA-approved companion diagnostic for the use of immunotherapy in mismatch repair–deficient tumors, regardless of histology. Because immunotherapy is already FDA-approved for melanoma, obtaining MSI status does not add to therapeutic options for melanoma patients. However, the finding of MSI in a melanoma tumor sample is suggestive of underlying mismatch repair deficiency and should prompt a referral to medical genetics.
For the time being, the added value of comprehensive tumor sequencing over limited melanoma-specific sequencing is in the identification of infrequent genomic alterations that would qualify a patient for a clinical trial. In our experience at a large academic medical center, 11% of all cancer patients who undergo genomic sequencing ultimately receive treatment based on sequencing results, either on-trial or off-trial.[14] In melanoma, putative targets that may be identified and for which matched agents are available within clinical trials include CDK4/6 mutations, PI3K/AKT mutations,[15] and NTRK/ROS1/ALK fusions. While some lament the use of sequencing technology in the clinic as the commercialization of a highly expensive research technology before its utility has been defined,[16] the responses that can be achieved with appropriately matched therapy can be remarkable-for example, an 11-month ongoing response to a NTRK/ROS1/ALK inhibitor in a heavily pretreated metastatic acral melanoma found to carry a ROS1 fusion.[17]
There has been a good deal of publicity in the past year regarding so-called "liquid biopsies," a reference to the sequencing of circulating tumor DNA (ctDNA) for therapeutic drug matching. Much like comprehensive tumor sequencing, this genetic testing technology is used to identify therapeutic targeted agents, many of which are investigational and accessible only by enrolling in a clinical trial. However, in a tumor type such as melanoma, which most often only has limited tissue available from a very small primary or from a fine-needle aspirate, ctDNA testing may be of value if no tissue is available or accessible for essential BRAF testing. In such cases, we use ctDNA testing as an alternative means of determining BRAF status.
In the Adjuvant Setting: Genetic Prognostication Profiles and ctDNA Studies
Commercial genetic prognostication testing in melanoma began with the discovery that the somatic loss of chromosome 3 portends a poor prognosis in uveal melanoma.[18] Additionally, aberrations in chromosome 1, 6, and 8 further refine prognostication,[19] and chromosomal copy number testing now includes the combination of all clinically relevant chromosomal aberrations. In addition to chromosome-based prognostication, two different gene expression profiling (GEP) tests are commercially available for melanoma in the postoperative setting—one for uveal melanoma, and the second for cutaneous melanoma. These GEP tests aim to prognosticate risk of metastatic disease beyond what is provided by American Joint Committee on Cancer (AJCC) staging. Importantly, neither of these tests is designed to predict response to therapy, which critics say make the tests clinically irrelevant. GEP tests divide tumors into one of two possible categories: low risk or high risk for distant metastases.
KEY POINTS
- Somatic characterization of melanoma can identify targeted therapy options for advanced disease, including standard-of-care BRAF/MEK inhibition, as well as phase II–supported KIT inhibitors and MEK inhibitors for KIT- and NRAS-mutated melanomas, respectively.
- Genetic prognostication can help delineate risk for uveal melanoma recurrence, while for cutaneous melanomas, genetic prognostication using gene expression profiling and circulating DNA show promise but are not yet in routine use.
- Multiple new genes have been associated with familial melanoma; germline sequencing for individuals with multiple melanomas or a family history of cancer is an area of active investigation and should be considered on a case-by-case basis.
Proponents of the testing argue that increased surveillance can be undertaken for high-risk individuals who might not otherwise be recommended for intensive surveillance based on staging alone.[20] Others cautiously propose that these tests may have future utility in determining management based on risk classification, while acknowledging that the data are currently too immature to use such testing for this purpose routinely.[21] For uveal melanoma, a rare tumor with little data on surveillance or treatment options, we routinely obtain GEP testing to clarify risk beyond traditional Collaborative Ocular Melanoma Study staging, and intensify surveillance based on risk categorization. For cutaneous melanoma, the advantage of risk stratification is less clear, given the strong and well-characterized correlation of metastatic risk to AJCC stage. A preliminary retrospective analysis suggests a benefit of GEP testing over AJCC in some cases of early-stage melanoma, and the potential utility of AJCC and GEP testing in combination.[22] In light of the lack of supporting prospective data, we currently do not routinely obtain GEP testing in cutaneous melanoma, but consider such testing on a case-by-case basis.
Another prospective and highly anticipated use of genomic testing in melanoma is ctDNA surveillance in detection of disease recurrence, adjuvant surveillance, and assessment of metastatic disease response. In the adjuvant AVAST-M trial, a negative trial of adjuvant bevacizumab, ctDNA levels of known BRAF and NRAS mutants were monitored post resection, and those patients with detectable ctDNA after surgery had significantly worse metastasis-free and overall survival than those who had undetectable ctDNA.[23] In the setting of treatment for metastatic melanoma, data from four large randomized trials of combination BRAF-MEK inhibition suggest that undetectable ctDNA prior to treatment initiation is an independent prognostic factor for improved progression-free survival.[24] In a small retrospective cohort treated with immunotherapy, combination BRAF-MEK inhibition, or both therapies, early reduction in ctDNA levels during treatment correlated with better progression-free survival, and provided complementary data alongside functional imaging.[25] Importantly, melanomas in this cohort were serially monitored with ctDNA for mutations in genes other than BRAF, including in NRAS and in the TERT promoter region. Serial ctDNA monitoring is not yet commercially available, and further work needs to be done regarding the appropriate platform for ctDNA monitoring for clinical use-whether this should be deep sequencing, digital droplet PCR, or a combinations of these two genomic surveillance modalities.
Heritable Melanoma: Germline Sequencing
As somatic genomic sequencing and testing matures, awareness is growing that germline mutations that confer heritable predispositions to cancer are more frequent in certain cancer types than we had previously recognized. Evidence of apparently sporadic cancers having been found to be associated with inherited mutations in known cancer susceptibility genes has been recently published for several cancer types: colon cancer,[26] renal cell carcinoma,[27] and bladder cancer.[28] Moreover, in a cohort of unselected patients with advanced cancers, paired sequencing of tumor and normal tissue revealed a 17.5% mutation rate of clinically actionable inherited mutations, half of which would not have been referred for targeted germline testing based on current guidelines.[29] In a large Norwegian twin study of cancer concordance, melanoma was found to be the cancer with the highest heritability.[30] Given the high hereditability of melanoma, it is unsurprising that eight new genes have been linked to inherited melanoma predisposition in the last 7 years (BAP1[31] and MITF[32] in 2011; PTEN[33] and RB1[34] in 2012; TERT promoter[35] in 2013; and POT1,[36] ACD,[37] and TERF2IP[37] in 2014. Correspondingly, six commercial germline testing companies now offer hereditary melanoma and skin cancer testing panels that vary in coverage from 6 to 19 genes. Given the rapidly changing landscape of genes known to be responsible for hereditary melanoma (Table 3), with implications for both patients and their family members, we agree with current guidelines, which suggest referral to a genetic counselor if more than three melanomas, or a mix of melanoma, pancreatic cancer, and/or astrocytoma, occur in a family or individual.[38,39]
Summary
Because melanoma is one of the most heavily somatically mutated cancers, its high mutational burden is a rich resource for unraveling the genetic underpinnings of cancers of all types. Not only does melanoma's mutational burden confer a higher sensitivity to immunotherapy, but it also implies a greater capacity for genetically-based targeted therapies, for genomic characterization and risk assessment, and for surveillance using genetic technologies such as ctDNA; in addition, under certain conditions, it portends a high likelihood of hereditability. We currently suggest that all patients with melanoma in both the adjuvant and metastatic settings undergo BRAF characterization for therapeutic decision making; more extended panel testing can also be considered, especially if a clinical trial might be a possibility in the future. Genomic prognostication of uveal melanomas should be pursued to determine risk and the optimal intensity of surveillance intensity; however, we consider data for GEP testing in cutaneous melanoma too preliminary to recommend routine testing. ctDNA surveillance is promising but the data need maturation. Lastly, we recommend referral to genetic counseling if multiple melanomas or other cancers are present in an individual or family.
Financial Disclosure:Dr. Tarhini has served as a consultant to Array Biopharma, Bristol-Myers Squibb, Genentech, Incyte, Merck, NewLink Genetics, Novartis, Regeneron, and Sanofi-Genzyme. Dr. Funchain has no significant financial interest in or other relationship with the manufacturer of any product or provider of any service mentioned in this article.
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