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Analytical performance of a real-time PCR-based assay for V600 mutations in the BRAF gene, used as the companion diagnostic test for the novel BRAF inhibitor vemurafenib in metastatic melanoma. | LitMetric

AI Article Synopsis

Article Abstract

Melanomas frequently harbor BRAFV600 mutations. Vemurafenib (RG7204/PLX4032), a small-molecule inhibitor of mutant BRAF, has shown striking clinical efficacy in BRAFV600 mutant melanoma, creating the need for a well-validated companion diagnostic to select patients for treatment. We describe analytic performance characteristics of the cobas 4800 BRAF V600 Mutation Test, the test used to select patients for the pivotal vemurafenib trials. This real-time polymerase chain reaction assay was designed to detect the V600E (1799T>A) mutation DNA from formalin-fixed paraffin-embedded tissue samples. Sensitivity was assessed using blends of cell lines or tumor DNA, and tumor specimens with low levels of mutant alleles, as determined by 454 sequencing (a quantitative next-generation pyrosequencing method). A >96% hit rate was obtained across all specimen types with 5% mutant alleles at a DNA input of 125 ng, an amount readily obtained from one 5-μm section. The cobas test showed a higher sensitivity and specificity than direct bidirectional sequencing in a panel of 219 melanoma specimens. Cross reactivity with V600K and V600D was observed. Repeated testing of 5 specimens by 2 operators, using different instruments and reagent lots, yielded correct calls in 158/160 tests (98.8%). A set of 26 highly pigmented samples were identified that gave invalid test results. A simple 1:2 dilution resulted in a valid test result of 76% in such cases. The cobas test is a reproducible assay that detects some non-V600E mutations and is more accurate than direct sequencing in detecting BRAFV600E.

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http://dx.doi.org/10.1097/PDM.0b013e31823b216fDOI Listing

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