Molecular platforms utilized to detect BRAF V600E mutation in melanoma

Semin Cutan Med Surg. 2012 Dec;31(4):267-73. doi: 10.1016/j.sder.2012.07.007.


Metastatic melanoma (MM) is a deadly skin disease refractory to standard chemotherapy. Despite numerous clinical and pathological parameters derived to guide patient management, clinical outcomes in melanoma patients remain difficult to predict. There is a critical need to delineate the important biomarkers typical of this disease. These biomarkers will ideally illuminate those key biochemical pathways responsible for the aggressive behavior of melanoma and, in the process, unveil new opportunities for the design of rational therapeutic interventions in high-risk patients. The most common recurring mutation in cutaneous melanoma is the prooncogenic BRAF V600E mutation that drives melanoma cell proliferation. The development of RAF inhibitors targeted against BRAF V600E mutant melanoma cells has revolutionized the treatment of MM. Clinical trials with BRAF inhibitor vemurafenib have shown objective clinical response and improved survival in patients with MM; therefore, knowledge of the molecular signature of melanoma in patients will be important in directing management decisions. Several molecular platforms exist to analyze the mutation status of melanoma. These include Sanger sequencing, pyrosequencing, allele-specific reverse transcriptase polymerase chain reaction, mass spectrometry base sequencing (Sequenom), high-resolution melting curve analysis, and next-generation sequencing methods using microfluidics technology. The Food and Drug Administration has approved the cobas BRAF V600 Mutation Test developed by Roche to analyze BRAF mutation status in formalin-fixed paraffin-embedded tumor samples. The cobas Mutation Test has been designed specifically to detect BRAF V600E mutations, and the analytic performance of this assay has demonstrated >99% sensitivity in the detection of BRAF V600E mutation when compared with the Sanger sequencing method and confirmed with the next-generation sequencing 454-pyrosequencing technology. The lower limit of detection of the percentage of mutant alleles in a tissue sample for the cobas test is less than 4%-5%. Some cross-reactivity with other variants of mutant BRAF was seen with the cobas V600 platform; however, this clinical test offers highly sensitive reproducible BRAF V600E mutation analysis in formalin-fixed paraffin-embedded tumor samples.

MeSH terms

  • Antineoplastic Agents / therapeutic use
  • DNA Mutational Analysis / methods
  • Humans
  • Indoles / therapeutic use
  • Mass Spectrometry / methods
  • Melanoma / drug therapy
  • Melanoma / genetics*
  • Melanoma / pathology
  • Melanoma / secondary
  • Molecular Diagnostic Techniques*
  • Mutation / genetics*
  • Neoplasm Proteins / antagonists & inhibitors
  • Neoplasm Proteins / genetics*
  • Paraffin Embedding
  • Proto-Oncogene Proteins B-raf / antagonists & inhibitors
  • Proto-Oncogene Proteins B-raf / genetics*
  • Real-Time Polymerase Chain Reaction / methods
  • Sequence Analysis, DNA / methods
  • Skin Neoplasms / drug therapy
  • Skin Neoplasms / genetics*
  • Skin Neoplasms / pathology
  • Sulfonamides / therapeutic use
  • Vemurafenib


  • Antineoplastic Agents
  • Indoles
  • Neoplasm Proteins
  • Sulfonamides
  • Vemurafenib
  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf