Abstract C27: Detection of the BRAF V600E mutation in colon cancer and thyroid cancer by immunohistochemistry.
Authors: Dvorak Katerina , Palting John , Waring Paul M. 
Published: August 2014

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. 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 antibody is mutation specific and can distinguish the V600E mutation in BRAF from the wild type BRAF protein.

Methods: Tissues from 352 patients with colon cancer (N=279) and thyroid cancer (N=73) were evaluated for the BRAF V600E mutation. Genomic DNA was extracted from 20 μm tissue sections and analyzed by PCR amplification followed by DNA sequencing. Immunohistochemical staining using anti-BRAF V600E (VE1) antibody was done on VENTANA BenchMark XT platform with OptiView DAB IHC Detection Kit. Furthermore, the effect of six different fixatives, time of fixation and ischemia on BRAF V600E staining intensity was evaluated in two mouse xenograft models since the BRAF V600E antigen may be affected by fixation conditions or delay to fixation,.

Results: In our cohort 212 cases were negative for BRAF V600E mutation by IHC. Out of these 212 cases 206 cases (97.2%) were also negative for BRAF V600E mutation by sequencing and 6 cases (2.8%) were discordant. Of the 140 cases that were IHC positive for BRAF V600E mutation, 133 cases were confirmed by sequencing (95.0%) and 7 cases were discordant (5.0%). Overall, there was high concordance between IHC and DNA sequencing: negative predictive value was 97.2%, positive predictive value was 95.0%, sensitivity was 95.7%, and specificity was 96.7%. The overall percentage agreement across all cases was 96.6% (339/352 cases). In addition, tissue fixation studies indicate that 10% neutral buffered formalin or zinc formalin are the only appropriate fixatives and tissues should be fixed within 2 hours following tissue collection.

Conclusion: In summary, our data indicate that there is high concordance between sequencing and IHC using anti-BRAF V600E (VE1) antibody for the detection of the BRAF V600E mutation in colorectal cancer and thyroid cancer in FFPE tissues. In addition, these studies demonstrated that the appropriate tissue fixation protocol is critical for BRAF V600E immunohistochemical studies. Overall, IHC with anti-BRAF V600E (VE1) antibody is highly sensitive and specific detection method for determination of BRAF V600E mutation.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C27.

Citation Format: Katerina Dvorak, John Palting, Paul M. Waring. Detection of the BRAF V600E mutation in colon cancer and thyroid cancer by immunohistochemistry. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C27.