Evaluation of BRAF V600E mutation detection assays for purpose of clinical tumor sample analysis
Authors: Walker Russell M. , Lichter David I. , Trepicchio William L. , Izmailova Elena 
Published: May 2015

Abstract
Background: The RAS-RAF-MEK-ERK protein kinase pathway regulates multiple processes required for cell survival and proliferation. Aberrant activation of this pathway via somatic mutations is frequently found in tumors and leads to uncontrolled cell growth. Inhibiting MEK has been shown to impair proliferation and induce apoptosis of tumor cells, thus making MEK an attractive therapeutic target. Consequently, there are several MEK inhibitors currently under clinical investigation, including TAK-733, a novel non-ATP-competitive allosteric selective inhibitor of MEK. A common mutation has been identified in the BRAF gene, resulting in a valine &gt; glutamic acid substitution (A&gt;T nucleotide change) at amino acid 600 (V600E). This specific aberration accounts for ~90% of BRAF mutations, and leads to constitutive activation of the BRAF kinase. Studies have demonstrated that cell lines and xenograft models with the BRAF V600E mutation are more sensitive to MEK inhibition than wild-type tumor cells. Thus, it is important to be able to accurately assess patients’ BRAF V600E mutation status as part of a MEK-inhibitor drug development program.
Aims: The goal of this study was to assess the relative sensitivity of three different genotyping technology platforms for BRAF V600E mutation detection and to evaluate the performance of the genotyping assays on clinical melanoma samples.
Materials and Methods: DNA was extracted from cell pellets obtained from Millennium Pharmaceuticals’ cell bank of genetically characterized lines. The cell line DNA was diluted to produce samples with known ratios of V600E mutant (both homozygous and heterozygous) and wild-type alleles. These samples were tested in order to assess the sensitivity of three mutation detection technology platforms: direct sequencing, allelic discrimination PCR, and the real-time PCR based B-RAF Mutation Test Kit from DxS Ltd (Manchester, UK). To evaluate assay performance in clinically relevant samples, 15 formalin fixed paraffin embedded (FFPE) clinical melanoma samples were obtained from ProteoGenex, Inc. (Culver City, CA). The melanoma tumor blocks were cut and mounted onto slides in 5 µM sections. DNA was extracted, quantitated, and subsequently tested for the BRAF V600E mutation using both the DxS and allelic discrimination assays.
Results: The DxS Test Kit was able to detect the homozygous V600E mutation in samples with 1% or greater mutant DNA and the heterozygous mutation in samples with 5% or greater mutant DNA. The allelic discrimination assay was able to detect the homozygous and heterozygous V600E mutation in samples with 10% and 25% mutant DNA, respectively. Sanger sequencing was able to detect the homozygous and heterozygous V600E mutation in samples with 25% and 100% mutant DNA, respectively. Results from both the DxS and allelic discrimination assays were in complete concordance when testing clinical melanoma samples, and indicated that 9/15 samples (60%) contained the BRAF V600E mutation.
Summary: The DxS research-only assay for BRAF V600E mutation detection is the most sensitive genotyping method assessed in this study. Both the allelic discrimination and the DxS BRAF V600E mutation detection assays are fit for the purpose of analyzing clinical melanoma samples.