Immunohistochemistry Using a BRAF V600E Mutation-Specific Monoclonal Antibody Is Not a Useful Surrogate for Genotyping in Colorectal Adenocarcinoma
Cheryl A Adackapara, Lynette M Sholl, Justine A Barletta, Jason L Hornick. Brigham and Women's Hospital, Harvard Medical School, Boston, MA
Background: Chemotherapy combined with anti-EGFR monoclonal antibodies prolongs survival in patients with advanced colorectal carcinoma (CRC). BRAF V600E and KRAS activating mutations are identified in around 20% and 50% of CRC, respectively, leading to constitutive EGFR pathway activation and lack of response to anti-EGFR therapy. Therefore, predictive mutation screening is routinely performed in patients with metastatic CRC. BRAF testing is also performed on CRC that demonstrate high-level microsatellite instability, since the presence of a BRAF mutation indicates the tumor is sporadic (i.e., not associated with Lynch syndrome). A novel BRAF V600E-specific monoclonal antibody has recently become commercially available. The aim of this study was to determine whether immunohistochemistry (IHC) for BRAF V600E can predict BRAF mutations in CRC.
Design: Whole tissue sections from 52 genotyped cases of CRC (46 primary tumors and 6 metastases) were examined. BRAF codon 600 and KRAS codons 12 and 13 were genotyped by pyrosequencing in a CLIA-certified laboratory. Cases included 17 tumors with BRAF V600E, 18 with KRAS mutations, and 17 wild-type for BRAF and KRAS. IHC was performed following pressure cooker antigen retrieval with an anti-BRAF V600E monoclonal antibody (Spring Bioscience; clone VE1; 1:50 dilution with overnight incubation). Cytoplasmic staining was scored as negative, weak, moderate or strong, with a known BRAF V600E-mutant melanoma serving as a positive control. Two authors scored the slides, with discrepancies resolved across the microscope.
Results: Cytoplasmic staining was observed in 71% of tumors with BRAF V600E mutation. Staining was moderate or strong in 50% of the positive cases (35% of BRAF-mutant cases overall). Weak cytoplasmic staining was detected in 17% of KRAS-mutant cases and 35% of wild-type cases. Non-specific nuclear staining in both tumor cells and normal colonic epithelium was a common finding. The sensitivity and specificity of BRAF V600E IHC for BRAF V600E mutation is 71% and 74%; if only moderate or strong staining is considered positive, the specificity is 100%, but the sensitivity is only 35%.
Conclusions: Overall, BRAF V600E mutation-specific IHC is not specific for BRAF mutations in CRC. While moderate or strong cytoplasmic staining is specific for BRAF V600E mutations, this antibody is insufficiently sensitive to serve as an effective screening tool for CRC. Weak staining lacks specificity, as it is observed in BRAF-mutant, KRAS-mutant, and wild-type tumors.
Monday, March 4, 2013 1:00 PM
Poster Session II # 122, Monday Afternoon