Abstract 2212: The analytic performance of a real-time PCR-based assay for the BRAF V600E mutation used as the companion diagnostic test for the novel BRAF inhibitor RG7204 (PLX4032) in metastatic melanoma
Authors: Halait Harkanwal , DeMartin Kelli , Shah Sweta , Soviero Stephen , Langland Rachel , Cheng Suzanne , Hillman Grantland , Wu Lin , Lawrence Jeffrey 
Published: June 2012

Abstract
Approximately 50% of human melanomas harbor activating mutations in the BRAF oncogene, and a large majority are of the V600E (1799T&gt;A) type. RG7204 (PLX4032), a highly selective small molecule that targets V600E-mutated BRAF, has shown striking efficacy in Phase 1 and Phase 2 clinical trials in metastatic melanoma. These findings create a critical need for a robust well-validated companion diagnostic test that is both sensitive and specific for the mutation. Here we describe analytic performance characteristics of the cobas BRAF V600 Mutation Test, a real-time PCR assay, which was designed to detect the V600E (1799T&gt;A) mutation in formalin-fixed paraffin-embedded (FFPE) samples of malignant melanoma, and has been used to select patients in clinical trials of RG7204. Analytic sensitivity was assessed using DNA blends from melanoma cell lines, DNA blends from FFPE tumor sections of melanoma, and individual FFPE tumor specimens with low levels of BRAF mutant alleles, as determined by 454 sequencing (a quantitative “deep” sequencing method). A &gt; 96% hit rate was obtained across all specimen types with 5% mutant sequences at a DNA input of 125 ng per PCR reaction, a DNA amount readily obtained from a single 5-micron FFPE tissue section. The calculated PROBIT values (95% probability) for the cobas test ranged from 0.6 to 30 ng per PCR genomic DNA input for specimens with 5% mutant sequences. The cobas test was compared to bidirectional Sanger sequencing using a set of 219 FFPE melanoma samples. The cobas test detected 14 V600E mutations among the 120 samples (11.7%) that were called mutation-negative by Sanger sequencing, and in all 14 cases a V600E mutation was confirmed by 454 sequencing. Conversely the cobas test detected no V600E mutation in 5 of 99 samples (5%) that were called mutant-positive by Sanger, and in all 5 cases 454 sequencing detected no mutation. The cobas test showed no cross reactivity with BRAF homologs, including RAF1, ARAF and the BRAF pseudogene; however, some cross-reactivity with V600K and V600D was observed with the cobas test. When a panel of tumor samples (including samples with low levels of mutant alleles and low tumor content) was repeatedly tested by two operators, using different instruments and reagents lots, a correct mutation call was obtained in 98.75% of tests. These analytic studies demonstrate that the cobas BRAF V600 Mutation Test is a reproducible assay that is more sensitive and more specific than conventional Sanger sequencing for the detection of the V600E (1799T&gt;A) mutation and detects some non-V600E mutations.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2212. doi:10.1158/1538-7445.AM2011-2212