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

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.

Frequency and Spectrum of BRAF Mutations in a Retrospective, Single-Institution Study of 1112 Cases of Melanoma

“…Other non-p.V600E mutations involving codon 600 (p.V600R, p.V600E2, and p.V600D) occurred rarely (4.6%), which is in keeping with other reports. 7,8 We also detected rare exon 15 BRAF mutations occurring at codons 594, 597, and 601, as well as two exon 11 mutations (p.G466E and p.G469E) of unknown clinical significance in melanoma. Both of the exon 11 mutations occurred in lentigo maligna melanoma, but whether this has any biological or clinical significance is uncertain.…”

“…The p.V600K mutation is the most common non-p.V600E variant, with an overall frequency approaching 20%. Although some patients with p.V600K mutations have reportedly shown response to anti-BRAF targeted therapy, 2,5,7 the precise performance characteristics of these patients as a distinct subgroup when treated with these agents is not clear from the literature and warrants further study. Furthermore, the inadvertent inclusion of patients with the p.V600K mutation in clinical trials using FDA approved cobas 4800 mutation test could lead to its misclassification as p.V600E.…”

“…5 Others also have reported that the cobas 4800 BRAF V600 mutation test detects a subset of non-p.V600E BRAF mutations, most often p.V600K, and less often p.V600D and p.V600E2. 7,8 Although the proportion of non-p.V600E mutations that are detected using the FDA approved assay remains somewhat unclear, a recent study showed that the cobas 4800 mutation test detected 70% of 40 p.V600K mutations. 8 It therefore seems reasonable to conclude that the cobas 4800 BRAF V600 mutation test may miss a subset of patients who have non-p.V600E BRAF mutations but who may nonetheless respond to vemurafenib therapy.…”

“…2,5,6e9 Furthermore, variable cross-reactivity of the cobas 4800 BRAF V600 mutation test with non-p.V600E BRAF mutations has been reported. 7,8 We therefore sought to achieve a better understanding of the frequency and spectrum of mutations in hotspot regions of BRAF mutations in various types of melanoma. Here we report our experience testing for BRAF mutations involving exons 11 and 15 in a large number of different subtypes of melanoma in a clinical setting.…”

Comparison of Molecular Testing Methods for Detecting BRAF V600 Mutations in Melanoma Specimens with Challenging Attributes

“…Full descriptions of the assay and workflow have been described [8,9]. Although designed to detect the V600E mutation, the cobas test has some cross-reactivity with the V600K (GT>AA), V600D (TG>AC), and V600"E2" (GTG>GAA) mutations.…”