IHC for mutant BRAF V600E was performed on a BenchMark XT automated immunostainer (Ventana Medical Systems, Inc., Tuscon, Ariz). were unfavorable by IHC were also unfavorable by genetic analysis. Four cases that showed weak cytoplasmic staining and/or nuclear staining in the tumor cells were considered to be IHC equivocal; by genetic analysis, 2 of the 4 were positive and 2 were negative. The overall sensitivity and specificity of IHC for the detection of a V600E mutant tumor was 93.7% and 95.6%, respectively. Our results support the use of VE1 IHC for identification of colorectal neoplasms harboring the V600E mutation. Triptorelin Acetate Difficulties in IHC interpretation may arise in a small number of cases, and in those cases molecular testing is required. were first reported in 2002 and are present in a wide range of human tumors including melanoma, colorectal carcinoma (CRC), papillary thyroid carcinoma and ovarian carcinoma1-3. The vast majority of alterations are characterized by a T1799A transversion which results in a missense substitution of valine at amino acid position 600 by glutamic acid (V600E)4. This mutation causes constitutive activation of the RAS/RAF/MEK/ERK cell-signaling pathway (also known as the MAP kinase pathway) which regulates cell cycle, growth and survival. Approximately 10% of all CRCs harbor mutations with the frequency of mutations being lower among cases with stage IV disease5,6. The determination of the mutation status in CRC is currently clinically important for several reasons. Detection of the V600E mutation in tumors showing microsatellite instability (MSI-H) suggests somatic methylation of the promoter region instead of a germline mutation when screening for Lynch Syndrome7. In terms of prognosis, several studies have found the presence of a V600E mutation to predict for poor outcomes8-11. Finally, tumor genotype can help guide targeted therapy as stage IV mutant CRC may be resistant to epidermal growth factor receptor (EGFR) inhibitors12,13 and may be candidates for combined treatment with BRAF V600E and EGFR inhibitors14. Currently, evaluation of the mutational status is based solely on sequencing of the gene using a variety of methods including Sanger sequencing, pyrosequencing and mass spectrometry. Recently, a monoclonal Triptorelin Acetate antibody (VE1) has been generated which recognizes the mutant BRAF V600E protein15 using Western blot and immunohistochemistry (IHC) of routinely processed formalin-fixed paraffin embedded tissue (FFPE). Initial published studies have found IHC staining with this antibody to have a high sensitivity and specificity C11orf81 for identifying tumors harboring the V600E mutation in several tumor types15-20. However, experience in colorectal carcinomas is limited, with one group reporting disappointing results16,21-24. In this study we analyzed the IHC application of the VE1 antibody in colorectal neoplasms and compared the results with those obtained from sequencing of the gene. Materials and Methods Case selection The study was approved by the Institutional Review Board. Cases of CRC and adenomas that were Triptorelin Acetate previously tested for the presence of the V600E mutation were retrieved. The majority of the cases (63%) were tested in a CLIA certified lab, while the remaining were tested for research purposes using the same assay. Cases diagnosed from 1992 until 2011 were included in the study. Both biopsies and resections were analyzed. Immunohistochemistry Four micron thick sections were cut from FFPE tumor blocks. IHC for mutant BRAF V600E was performed on a BenchMark XT automated immunostainer (Ventana Medical Systems, Inc., Tuscon, Ariz). Sections were incubated with the anti-BRAF V600E antibody (clone VE1, SpringBio, Pleasanton, CA) diluted 1:50 for 48 minutes at room temperature. Antigen retrieval was performed by heating at 100C for 32 minutes, and primary antibody incubation was carried out at 37C for 32 minutes. Antigen detection was performed using the OptiView DAB Detection kit (Ventana Medical Systems, Inc.). Metastatic melanoma with a documented BRAF V600E mutation was used as a positive control. The immunostained slides were evaluated independently by two pathologists who were blinded to the molecular data and to each other’s IHC assessment. Cases were scored as positive when there was diffuse ( 80% of tumor cells) staining above any background staining. The intensity of cytoplasmic tumor cell staining in the positive cases was scored as weak, moderate and strong. Cases were considered as equivocal if 1. there was nuclear staining in the tumor cells in addition to cytoplasmic staining or 2. there was disagreement between the two pathologists. Mutation analysis For DNA preparation, FFPE tissue or, where available, frozen tissue was used. Serial sectioning of tissue was performed and the presence and extent of tumor was verified using hematoxylin and eosin stain (H&E). Macrodissection was performed if required to ensure 50% tumor content. Genomic DNA was extracted from 10 unstained sections 5 micron thick using the DNeasy Tissue kit (Qiagen) following the manufacturer’s protocol. Mutations were detected using the iPLEX assay.