Mutation‐specific antibody detects mutant BRAF^V600E protein expression in human colon carcinomas

Abstract

Background A point mutation (V600E) in the BRAF oncogene is a prognostic biomarker and may predict for non response to anti-EGFR antibody therapy in colon carcinomas. BRAFV600E mutations are also frequently detected in tumors with microsatellite instability and indicate a sporadic origin. Using a mutation-specific antibody, we examined mutant BRAFV600E protein expression and its concordance with BRAFV600E mutation data. Materials/Methods Resected, primary stage III colon carcinomas were analyzed for BRAFV600E mutations in exon 15, and 50 BRAFV600E mutation carriers and 25 wild-type cases were selected for immunohistochemistry (IHC). IHC was performed in archival tissue specimens using a pan-BRAF antibody and a mutation-specific antibody against BRAFV600E proteins. Staining was scored by two pathologists blinded to clinical and mutation data. Results Using a pan-BRAF antibody, total BRAF protein expression was observed in the tumor cell cytoplasm in 74 of 75 colon carcinomas. A mutation-specific antibody identified diffuse cytoplasmic staining of mutant BRAFV600E proteins in 49 of 74 cancers. All 49 of these cases were shown to carry BRAFV600E mutations by a PCR-based assay. In contrast, BRAFV600E staining was absent in all 25 tumors found to carry wild-type copies of BRAFV600E. Conclusion A BRAF mutation-specific (V600E) antibody detects tumors with BRAFV600E mutations and shows complete concordance with a DNA-based method. These results support the use of IHC as a simplified strategy to screen colorectal cancers for mutant BRAFV600E proteins in clinical practice.

Assessment of BRAF V600E Status in Colorectal Carcinoma: Tissue-Specific Discordances between Immunohistochemistry and Sequencing

“…Multiple studies have suggested that cases in which VE1 IHC results are positive but sequencing results are negative might be attributable to the lower sensitivity of molecular analysis (24,26,27). Specifically, the contention is that low tumor volumes and dilution with nontumoral tissue can produce a false-negative molecular result.…”

“…Previous studies on the suitability of VE1 IHC for the detection of BRAF V600E mutation in colorectal carcinoma found sensitivity ranging from 59% to 100% and specificity ranging from 51% to 100% with use of different IHC techniques, including a wide spectrum of antibody conditions, and different sequencing techniques as the comparator (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). In the largest such series to date, investigators used whole tissue sections to analyze 113 BRAF wild-type and 52 BRAF V600E-mutated colorectal carcinomas in the validation cohort (21) and reported sensitivity of 96% and specificity of 99%.…”

The current value of determining the mismatch repair status of colorectal cancer: A rationale for routine testing

“…This requires the use of laser-capture microdissection or an alternate specimen in some instances (Geiersbach and Samowitz, 2011). While the majority of studies incorporating the BRAF antibody have reported a high sensitivity and specificity (Affolter et al, 2013;Rossle et al, 2013;Sinicrope et al, 2013b) there have been some contradictory results (Adackapara et al, 2013;Lasota et al, 2014). If proven to be reliable, the relatively inexpensive tests of reflex IHC for dMMR and BRAF may refine the cohort of patients that go on to further genetic testing, thereby improving the feasibility of universal screening for Lynch syndrome Toon et al, 2013).…”

Application of Immunohistochemistry in Gastrointestinal and Liver Neoplasms: New Markers and Evolving Practice

“…53,54 Although it has been shown that BRAF V600E mutation predicts a poor prognosis in right-sided microsatellite-stable CRC, this prognostic indication has not been widely explored clinically. 102 Traditionally, BRAF mutation is detected by DNA sequencing or polymerase chain reaction-based mutation detection methods. Recently, antibodies specific to BRAF V600E have been developed, and their use in IHC on formalin-fixed, paraffin-embedded tumor tissue has become popular.…”