Molecular Platforms Utilized to Detect BRAF V600E Mutation in Melanoma

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.
Semin Cutan Med Surg 31:267-273 © 2012 Frontline Medical Communications

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