Abstract 42: Evaluation of BRAF(V600E) mutation by immunohistochemical staining with anti-BRAF V600E (VE1) antibody: A comparison with Sanger sequencing.
Authors: Dvorak Katerina , Palting John D. , Aggeler Birte 
Published: August 2014

Abstract
Background: The most common of all activating BRAF mutations (T1799A point mutation) leads to a substitution of valine (V) to glutamic acid (E) at the position 600 of the amino acid sequence. This oncogenic mutation in the BRAF gene constitutively activates the MAPK signaling pathway and results in increased proliferation and inhibition of apoptosis. BRAF V600E mutation was described in approximately 8% of all solid tumors (43% melanomas, 39-67% papillary thyroid carcinomas and 12% colorectal adenocarcinomas). However, it is challenging to identify patients with this mutation. Currently, the gold standard is sequencing of the tumor DNA, but this method is not practical for routine practice. In contrast, immunohistochemistry (IHC) is a relatively easy diagnostic tool to detect proteins in paraffin-embedded tissues. The major goal of this study was to compare detection of BRAF V600E by sequencing and IHC using anti-BRAF V600E (VE1) antibody. This clone is mutation specific and can distinguish the V600E mutation in BRAF from the wild type BRAF protein.
Methods: Tissues from sixty-nine patients with colon cancer (N=40) and thyroid cancer (N=29) were evaluated for the BRAF V600E mutation. Genomic DNA was extracted from 20 μm tissue sections and analyzed by PCR amplification followed by Sanger sequencing. Primers were designed to cover the BRAF coding sequences at mutation site and a few nucleotides in the intron on both ends (BRAF-ex15F-TGCTTGCTCTGATAGGAAAATG; BRAF-ex15R-AGCATCTCAGGGCCAAAAAT). Both forward and reverse strands were sequenced on an Applied Biosystem's 3730xl DNA Analyzer. Immunohistochemical staining using anti-BRAF V600E (VE1) antibody was done on VENTANA BenchMark XT platform with OptiView DAB IHC Detection Kit.
Results: All cases (22/22, 100%) that were negative for BRAF V600E mutation by IHC were confirmed to be BRAF V600E negative by sequencing. Out of 47 cases staining positive for BRAF V600E (VE1) 43 cases were confirmed by sequencing. Of the four discordant cases, one case was weakly positive by IHC (score 1), two cases that were negative by sequencing showed stronger BRAF V600E (VE1) staining (score 2) and one case contained a different mutation at codon 600 (V600K). Overall, there was high concordance between IHC and DNA sequencing. Using the sequencing as the reference standard, negative predictive value was 100%, positive predictive value was 91% with a sensitivity of 100% and specificity of 85%. Overall percentage agreement across all cases was 94% (65/69 cases). Next-generation sequencing will be used to evaluate discordant cases for BRAF V600 mutation status.
Conclusion: Detection of the BRAF V600E mutation is emerging as an important biomarker with diagnostic, prognostic, and predictive potential. Importantly, our data suggests that there is high concordance between sequencing and IHC using anti-BRAF V600E (VE1) antibody.
Citation Format: Katerina Dvorak, John D. Palting, Birte Aggeler. Evaluation of BRAF(V600E) mutation by immunohistochemical staining with anti-BRAF V600E (VE1) antibody: A comparison with Sanger sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 42. doi:10.1158/1538-7445.AM2013-42