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Q&A Column, 3/14

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Editor: Frederick L. Kiechle, MD, PhD

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Molecular testing for actionable mutations in melanoma

Written authorizations for oncologic prognostic/therapuetic tests

Molecular testing for high-grade gliomas/glioblastomas

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Q. What are the actionable mutations in melanoma that warrant routine molecular testing? Who should be tested, and what specimen should be tested? Is there a role for stat BRAF testing and for next-generation sequencing?

A. Somatic activating mutations in BRAF, NRAS, or KIT are common and generally mutually exclusive in melanoma. Mutations in at least one of these genes can be found in approximately 90 percent of melanomas and each may render a patient eligible for systemic targeted therapy. BRAF- and KIT-directed agents are FDA approved. While there are no direct NRAS inhibitors, patients with NRAS-mutant melanomas may be eligible for MEK and/or PI3K inhibitors available in clinical trials, as the RAS GTPases are known to be upstream activators of both MAPK and PI3K pathways.

BRAF: Approximately 50 percent of melanomas harbor activating mutations in BRAF. Mutations at codon 600, including V600E, V600K, V600D, and V600R, are likely to respond to BRAF-targeted agents such as vemurafenib and dabrafenib. Mutations outside of codon 600 should not be treated with a BRAF inhibitor. Likewise, vemurafenib and related agents are contraindicated in patients with wild-type BRAF melanomas. Also worth noting is that BRAF mutation is not useful for diagnostic purposes because it occurs at high frequency in nevi and does not, therefore, distinguish benign melanocytic lesions from malignant.

NRAS: NRAS mutations are detectable in 15 to 25 percent of melanomas, primarily in codon 61 and less frequently in codons 12 and 13. Outside of the clinical trial setting, it is a marker we don’t know yet what to do with. Nevertheless, it is advisable to evaluate this gene because the mutation frequency is significant and therapeutic options for melanoma patients are limited.

KIT: Clinical trials demonstrate that the overall response rate to imatinib in KIT-mutated melanoma is 16 to 23 percent.1-3 These studies demonstrate the value of KIT-mutation testing in melanoma. Approximately five to 30 percent of acral and mucosal melanomas, and melanomas arising on chronically sun-damaged skin, harbor KIT mutations. Mutations occur primarily in exon 11 and less frequently in exons 13, 17, and 18.

The simple answer to the question about whom to test is any stage IV melanoma patient who is being considered for systemic therapy. At present there is little or no role for systemic targeted therapy in patients with disease limited to stages I–II (limited to primary site in skin) or III (lymph node involvement). However, clinical trials are ongoing in resected stage III disease, results of which may expand the scope of pretreatment testing to the adjuvant setting.

Regarding what specimen should be tested, in theory a metastatic tumor should be tested because this is the disease that will be treated with systemic therapy and followed for a treatment response. Indeed, there are data to suggest that testing of primary tumors and metastases may yield distinct results. Colombino M, et al.,4 showed a relatively high concordance of BRAF or NRAS mutation between primary tumor and lymph node or visceral metastasis (93 percent and 96 percent, respectively). Concordance dropped to 80 percent for brain metastasis and 75 percent for skin metastasis.

When a metastatic focus is not available for testing, the primary tumor is certainly acceptable as long as there is sufficient tumor. Because melanoma metastasizes early (Breslow depth >1 mm), it is not uncommon to see a very thin, small primary tumor submitted for testing. These early melanomas are particularly challenging to test because there is very little tumor and it is often admixed with a significant amount of inflammatory, epidermal, and dermal cells. In these cases a wild-type result may be misleading as there may be reduced sensitivity due to insufficient tumor relative to non-tumor cells. The same is true for microscopic lymph node metastases, another common specimen submitted for testing. Thus, it may be valuable to biopsy presumed metastatic foci for the purpose of confirming stage IV disease and for obtaining tumor-rich material for testing.

FNA smears may be suitable for testing as long as the test being considered is highly sensitive and appropriate for small quantities of DNA. While the nucleic acid yields are low from these slide scrapings, the DNA is often high quality because the sample was not formalin-fixed. Allele-specific PCR assays are reliable at detecting mutations in smears but are limited by the mutation-specific primers included in the assay. Approximately five to 10 percent of melanomas with activating BRAF mutations will harbor non-V600E alterations. These less common mutations could be missed in an allele-specific PCR assay that amplifies only the V600E allele. KIT testing is not appropriate for allele-specific PCR as the mutations are too variable and include indels that require alternative methods of detection. We have found that FNA smears of melanoma are amenable to amplification-based next-generation sequencing techniques. The Ion Torrent AmpliSeq system can readily amplify alleles from very small quantities of DNA such as those rendered from a smear. The advantage of testing a melanoma smear with this approach is that multiple genes can be assayed with high sensitivity from one sample. This method can detect both point mutations and small indels and, therefore, is useful for BRAF, NRAS, and KIT.

Yes, there is a role for stat BRAF testing. In fact this is a perfectly legitimate request for critical clinical scenarios. Patients with rapidly progressive disease who are highly symptomatic can benefit from prompt initiation of BRAF inhibitors when there is a detectable activating mutation. Melanoma is a cancer capable of growing rapidly in critical organs such as the brain, heart/pericardium, and GI tract (causing obstruction, bleeding). The BRAF inhibitors are capable of reducing symptoms rapidly, sometimes within days, and almost always within weeks (in responding patients). For these requests, we can process new biopsies with a stat protocol (six hours) and then triage the slides to the molecular oncology service for selection of tumor for the extraction. Our molecular laboratory prioritizes this sample and generates a pyrosequencing result by the next morning. This has been an effective strategy for preventing potentially catastrophic events such as a brain stem progression, as was imminent in one of our patients recently. Six months later his tumor was not detectable by imaging and he was symptom-free.

Similar to lung adenocarcinoma, melanoma is an ideal cancer for multigene panels. At present, some next-generation-sequencing–based oncology panels become cost-effective once three or more genes become necessary to test. Furthermore, multigene panel testing by NGS can render a timely result, such as within a 10-day turnaround time. It is quite difficult to reach that TAT with sequential, single-gene reflex testing. As with lung cancer, it is prudent to initiate systemic therapy in a stage IV melanoma patient as soon as possible, as this disease is extremely aggressive. NGS hotspot mutation panels provide the added benefit of deep sequencing, that is, detection of low frequency alleles. For KIT this is particularly important as traditional Sanger sequencing, a relatively insensitive assay, is routinely used to detect the indels that are a common mechanism of KIT mutation. NGS can also detect the more rare actionable mutations that occur in melanoma, such as PI3K and AKT, depending on what genes are included in the panel. Indeed, we recently detected a PDGFRα mutation in a tumor wild type for BRAF, NRAS, and KIT, rendering the patient eligible for imatinib or related drugs. All of these genes were sequenced in one NGS test. The PDGFRα mutation would not have been detected by the routine single-gene assays. Thus, in terms of targeted oncology panels, NGS platforms can be useful for melanoma. The role of whole genome or whole exome sequencing is not clear at this point.

  1. Hodi FS, Corless CL, Giobbie-Hurder A, et al. Imatinib for melanomas harboring mutationally activated or amplified KIT arising on mucosal, acral, and chronically sun-damaged skin. J Clin Oncol. 2013;31(26):3182–3190.
  2. Carvajal RD, Antonescu CR, Wolchok JD, et al. KIT as a therapeutic target in metastatic melanoma. JAMA. 2011;305(22):2327–2334.
  3. Guo J, Si L, Kong Y, et al. Phase II, open-label, single-arm trial of imatinib mesylate in patients with metastatic melanoma harboring c-Kit mutation or amplification. J Clin Oncol. 2011; 29(21):2904–2909.
  4. Colombino M, Capone M, Lissia A, et al. BRAF/NRAS mutation frequencies among primary tumors and metastases in patients with melanoma. J Clin Oncol. 2012;30(20):2522–2529.

Allie H. Grossmann, MD, PhD
Department of Pathology, University of Utah
ARUP Laboratories, Salt Lake City
Member, CAP Personalized Health Care Committee Work Group

Kenneth F. Grossmann, MD, PhD
Division of Oncology, Department of Medicine, Huntsman Cancer Center
University of Utah, Salt Lake City

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