Mutation Tests in Melanoma Crucial for Treatment

April 15, 2014
Special Reports, Melanoma (Issue 2), Volume 2, Issue 1

The American Cancer Society estimates that more than 75,000 new cases of melanoma will be diagnosed in 2014, and that nearly 10,000 people will die of melanoma this year.

The American Cancer Society estimates that more than 75,000 new cases of melanoma will be diagnosed in 2014, and that nearly 10,000 people will die of melanoma this year.1While new and promising treatment approaches continue to emerge, melanoma is a diverse disease, and improved diagnostics for classifying the disease are required to use these new treatments most effectively.

The predominant mutation associated with melanoma, reported in about 50% of cases, occurs at position 600 in BRAF, a protein that is part of the mitogen-activated protein (MAP) kinase signal transduction pathway. Approximately 80% to 90% of theseBRAFmutations are known as V600E, a T>A switch that results in an amino acid change of valine to glutamic acid at position 600, within the activation portion of the BRAF kinase domain. Another 5% to 10% of mutations at the same position are known as V600K, which results in a lysine substituted for valine at the same position.2,3

About 15% to 25% of melanomas display mutations in NRAS, a signaling molecule upstream of BRAF in the MAPK pathway,4while approximately 15% of melanomas show a mutation in KIT, a cell surface receptor involved in the same signaling pathway.5

Targeting Tumor Mutations

It is important to determine tumor genotype to choose the most effective therapy for the patient. For example, BRAF inhibitors have proven to be effective, at least initially, in extending progression-free survival (PFS) and overall survival (OS).6Keith T. Flaherty, MD, director of developmental therapeutics at the Massachusetts General Hospital Cancer Center, Boston, was lead author of the 2010 New England Journal of Medicine article that announced results from a phase I clinical trials for the first approved BRAF inhibitor, vemurafenib.7

Keith T. Flaherty, MD

As Flaherty commented at the time,8“Until now, we’ve never had a credible first-treatment option for metastatic melanoma, so this has completely transformed how we approach treatment for patients with theBRAFmutation.” However, use of BRAF inhibitors in tumors with wild-type BRAF can have the opposite of the desired effect, leading to increased proliferation, highlighting the importance of accurate genotyping before using these agents.9

It is also useful to monitor tumor genotype during treatment. Tumors can be resourceful when subjected to selectional pressure from inhibitors targeting specific signaling pathway components. Tumor cells will often develop secondary mutations that activate alternate pathways to circumvent targeted therapies and allow the tumor to again grow. Typically, BRAF itself does not mutate additionally in response to inhibitor treatment; rather other upstream or downstream elements in MAPK pathway mutate, such as NRAS10or MEK.11

The melanoma field has long awaited additional advanced diagnostic tools for characterizing tumors to facilitate a personalized medicine approach. Tools have emerged, with more on the horizon, although some may not be immediately available for clinical use.

Mutation Detection—BRAFV600E

The first US Food and Drug Administration (FDA)-approved clinical diagnostic assay for melanoma genotyping, and the current standard, is the Roche cobas® 4800 BRAF V600 Mutation test. The Roche test is a real-time polymerase chain reaction (PCR) assay that can detect the V600E mutation in formalin-fixed, paraffin-embedded (FFPE) melanoma tissue samples. The assay has demonstrated >99% sensitivity in detection of theBRAFV600Emutation when compared with Sanger sequencing, with a lower limit of detection of mutant alleles <4% to 5%.12

In 2013, a United Kingdom panel of melanoma experts recommended that all high-risk melanoma patients (ie, those with stage IIb and higher) receive the V600E test.13Despite the utility of the Roche BRAF Mutation Test for V660E, however, 50% of melanoma patients are wild-type for BRAF and will need additional testing.

Melanoma Panel IncludingNRAS

Until recently, no test was available for the 15% to 25% of patients with melanoma andNRASmutations. However, a California company, DiaCarta, recently received CE/IVD status for a melanoma mutation detection panel that includes not onlyBRAFat position 600, but alsoNRAS, detecting mutations at codons 12 and 13 (exon 2), codons 59 and 61 (exon 3), and codons 117 and 146 (exon 4) of theNRASproto-oncogene.14The assay, based on real-time PCR, also tests for a mutation of c-KIT, a protein that acts upstream ofNRASin the MAPK pathway, at codon 816 (exon 17) in thec-KITproto-oncogene.

According to DiaCarta’s chief scientific officer, Michael J. Powell, PhD, the assay is both fast and sensitive. “Our assays take less than 2.5 hours from receipt of sample to result and are capable of detecting as little as 0.1% mutant DNA from tumor biopsies or FFPE samples.” When asked to comment about the source of the speed advantage, Powell said, “Our sample prep method is carried out in a single tube, the contents of which are then used directly in our assays. This feature not only gets results sooner, but also greatly reduces error rates in processing DNA from clinical samples. The worst thing about such errors is that they provide mainly false-negative results.”

Informed Immunotherapy

Ipilimumab, a monoclonal antibody to cytotoxic T-lymphocyte antigen-4 (CTLA-4), stimulates the immune system and is approved for use in unresectable or metastatic melanoma.15Some of the most promising current therapies for melanoma involve combining targeted inhibitors such as vemurafenib with immunotherapy agents.16

With the success of immunotherapies for the treatment of melanoma, interest in immunologic biomarkers has grown as tumor classification has been seen to be important for immunotherapy treatment. In a recent study, patients with immunity to the antigen, NY-ESO-1, were seen to be 50% more likely to receive benefit from immunotherapy CTLA-4 antibody ipilimumab.17

According to an analyses presented at the 2014 AACR annual meeting, PD-L1 levels adequately predict response and clinical outcomes for the PD-1 inhibitor MK-3475 in patients with melanoma (and non-small cell lung cancer). Patients with melanoma, whose tumors expressed PD-L1 saw greater immune responses, slower disease progression, and higher survival rates, compared with patients whose tumors had no PD-L1. In both studies, patients with low-levels of PD-L1 still experienced some level of response to treatment.18

S6 Phosphorylation as Biomarker for Inhibitor Resistance

In terms of proteomic assays to characterize melanoma, a potential clinical diagnostic assay involves detection of the phosphorylation state of ribosomal protein S6. In xenograft studies, a decrease in P-S6 (detected by quantitative immunofluorescence microscopy) correlated with sensitivity to combined treatment with the BRAF inhibitor vemurafenib and the MEK inhibitor selumetinib. Tumors that did not display a decrease in P-S6 protein could be predicted to be resistant to the combined therapy.19

Although measuring a change in P-S6 after initiating treatment would require a second tumor biopsy from the patient, melanoma cells for the technique can be collected by fine needle aspiration, which is less invasive and safer than surgically obtained biopsies. For an individual patient, such an indication of dual resistance would not yield a positive prognosis; Corcoran et al stated that, “if the information obtained through this approach can more effectively predict who is most or least likely to benefit from therapy, the ability to spare patients the added cost and potential toxicity of an ineffective therapy and the opportunity to switch to a potentially more active agent or combination of agents may justify the additional procedure.”

Future Possibilities

Microarrays continue to hold promise as tools that can take a genome-wide view to support personalized medicine in the treatment of malignant melanoma, but remain at the experimental stage. Research in the emerging field of epigenetics has offered tantalizing glimpses of the potential of detecting changes in DNA methylation state or chromatin remodeling, not only across the entire genome, but particularly in association with genes known to be involved in the progression of melanoma. Noncoding RNAs of various lengths may also eventually have direct clinical relevance.20

Urine-based cell-free (cf) DNA BRAF V600 mutational testing is currently being investigated for detection and monitoring. Early trials have shown preliminary concordance between mutational tissue testing and droplet digital PCR in urinary cfDNA from patients with advanced cancers, including melanoma.21


  1. American Cancer Society. Accessed March 20, 2014.
  2. Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer.Nature. 2002;417(6892):949-954.
  3. Rubinstein JC, Sznol M, Pavlick AC, et al. Incidence of the V600K mutation among melanoma patients with BRAF mutations, and potential therapeutic response to the specific BRAF inhibitor PLX4032.J Transl Med. 2010;8:67.
  4. Curtin JA, Fridlyand J, Kageshita T, et al. Distinct sets of genetic alterations in melanoma.N Engl J Med. 2005;353(20):2135-2147.
  5. Wallander ML, Layfield LJ, Emerson LL, et al. KIT mutations in ocular melanoma: frequency and anatomic distribution.Mod Pathol. 2011;24(8):1031-1035.
  6. Flaherty KT, Puzanov I, Kim KB, et al. Inhibition of mutated, activated BRAF in metastatic melanoma.N Engl J Med. 2010;363(9):809-819.
  7. Bollag G, Tsai J, Zhang J, et al. Vemurafenib: the first drug approved for BRAF-mutant cancer.Nat Rev Drug Discov. 2012;11(11):873-886.
  8. Massachusetts General Hospital News Release. Accessed March 23, 2014.
  9. Poulikakos PI, Zhang C, Bollag G, et al. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF.Nature. 2010;464:427-430.
  10. Nazarian R, Shi H, Wang Q, et al. Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation.Nature. 2010;468(7326):973-977.
  11. Wagle N, Emery C, Berger MF, et al. Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling.J Clin Oncol. 2011;29(22):3085-3096.
  12. Curry JL, Torres-Cabala CA, Tetzlaff MT, Bowman C, Prieto VG. Molecular platforms utilized to detect BRAF V600E mutation in melanoma.Semin Cutan Med Surg. 2012;31(4):267-273.
  13. Gonzalez D, Fearfield L, Nathan P, et al. BRAF mutation testing algorithm for vemurafenib treatment in melanoma: recommendations from an expert panel.Br J Dermatol. 2013;168(4):700-707.
  14. QClamp&trade; Melanoma Mutation Panel, BRAF, NRAS and c-KIT. DiaCarta, Inc, Hayward, CA.
  15. Lipson EJ, Drake CG. Ipilimumab: an anti-CTLA-4 antibody for metastatic melanoma.Clin Cancer Res. 2011;17(22):6958-6962.
  16. Koya RC, Mok S, Otte N, et al. BRAF inhibitor vemurafenib improves the antitumor activity of adoptive cell immunotherapy.Cancer Res. 2012;72(16):3928-37.
  17. Yuan J, Adamow M, Ginsberg BA, et al. Integrated NY-ESO-1 antibody and CD8+ T-cell responses correlate with clinical benefit in advanced melanoma patients treated with ipilimumab. ProcNatl Acad Sci U S A. 2011;108(40):16723-16728.
  18. Daud AI, Hamid O, Ribas A, et al. Antitumor activity of the anti-PD-1 monoclonal antibody MK-3475 in melanoma (MEL): Correlation of tumor PD-L1 expression with outcome. Presented at the American Association for Cancer Research annual meeting; April 6, 2014; San Diego, CA. Abstract CT104.
  19. Corcoran RB, Rothenberg, SM, Aaron N, et al. TORC1 suppression predicts responsiveness to RAF and MEK inhibition in BRAF-mutant melanoma.Sci Transl Med. 2013;5(196);196ra98.
  20. Joyce CW, Murphy IG, Rafferty M, et al. Tumor profiling using protein biomarker panels in malignant melanoma; application of tissue microarrays and beyond .Expert Rev Proteomics. 2012;9(4):415-423.
  21. Janku F, Falchook GS, Piha-Paul SA, et al, Droplet digital PCR mutant and longitudinal monitoring of BRAF mutations in cell-free urinary DNA of patients metastatic cancers or Erdheim-Chester disease. Presented at the American Association for Cancer Research annual meeting; April 8, 2014; San Diego, CA. Abstract LB170