Group, North Central Cancer Treatment

3576 Background: BRAF encodes a serine-threonine kinase that is a downstream effector of activated RAS. A point mutation (V600E) in BRAF occurs in a subset of colorectal cancers (CRCs) and is associated with adverse outcome and may predict non response to anti-EGFR antibodies. Detection of a BRAFV600E mutation in a CRC with microsatellite instability indicates a sporadic origin and excludes Lynch Syndrome. While BRAFV600E mutation status is determined using a DNA-based assay, antibodies against the BRAFV600E protein have recently been developed. We examined mutant BRAFV600E protein expression and its concordance with mutation status. Methods: Primary stage III colon carcinomas (50 BRAFV600E mutation carriers and 25 wild-type cases) were studied from a completed phase III adjuvant trial comparing FOLFOX +/- cetuximab (NCCTG N0147). In archival resection specimens, immunohistochemistry (IHC) was performed using a pan-BRAF antibody and a V600E mutation-specific antibody raised against an immunogenic synthetic peptide derived from the internal region of the BRAFV600E protein. BRAFV600E mutations in codon 15 were analyzed in extracted DNA using a multiplex, allele specific PCR–based assay. BRAF staining was scored independently by two pathologists blinded to mutation status. Results: In primary colon carcinomas stained with a pan-BRAF antibody, diffuse cytoplasmic staining for BRAF proteins was detected in 74 of 75 carcinomas with one case deemed non-evaluable. Using the mutation-specific BRAFV600E antibody, diffuse cytoplasmic staining was detected in 49 of 74 tumors without appreciable heterogeneity of expression. Among these 49 tumors expressing mutant BRAFV600E proteins, all (100%) were found to carry a BRAFV600E mutation according to a DNA-based assay. In contrast, absent BRAFV600E staining was observed in all 25 tumors that were found to have wild-type copies of BRAFV600E detected using a PCR-based assay. Conclusions: For the detection of mutant BRAFV600E, complete concordance was found between IHC and a DNA-based method in colon carcinomas. This finding supports the use of IHC as a simplified strategy to screen CRCs for mutant BRAFV600E proteins in routine clinical practice to inform clinical decision-making.