Assessment of BRAF V600E Mutation-Specific Antibody (VE1) in Colorectal Cancers
Shih-Fan Kuan, Reetesh K Pai, Sarah Navina, Marina N Nikiforova, Kristi L Cressman. University of Pittsburgh, Pittsburgh, PA
Background: BRAF V600E mutation occurs in about 10% of colorectal cancers (CRCs). Most of them are CRCs with high-levels of microsatellite instability (MSI-H CRCs) arising in sessile serrated adenoma (SSAs). BRAF mutation is an unfavorable prognostic factor and predicts resistance to anti-EGFR therapy. BRAF mutation status also helps to further subclassify of MSI-H CRCs into Lynch syndrome-associated CRCs and sporadic MSI-H CRCs. Clinical testing of BRAF mutation by sequencing usually takes days to complete. An antibody targeting the BRAF V600E mutation (VE1) has recently become available (Spring Bioscience, CA). We tested the feasibility of this antibody to expedite clinical testing of BRAF mutation.
Design: Formalin-fixed, paraffin-embedded tissues from 76 colectomy cases for CRC were used to validate antibody VE1. BRAF V600E mutation status was first tested by sequencing. Then, immunohistochemical staining using VE1 was performed by manual methods as well as on Ventana Benchmark Ultra automated stainer after optimization of the procedure.
Results: The VE1 antibody was very sensitive to factors such as tissue fixation, antigen retrieval, and antibody incubation and had a narrow range for optimal conditions. Repeat attempts to optimize the staining by manual processing did not generate reproducible results. However, we were able to optimize the staining results using Ventana Benchmark Ultra instrument following the recommendations by the vendor. The slides were independently reviewed by 3 GI pathologists (SFK, RKP, and SN) and had 100% concordant readings. Discrete and recognizable positive staining for BRAF mutation was identified in the cytoplasm of all 31 CRCs with known BRAF mutation by sequencing. All 45 BRAF wild-type CRCs exhibited negative staining. The specificity and sensitivity of VE1 antibody were 100% in CRC. Three of 5 associated sessile serrated adenomas (SSAs) in this series showed weak positive VE1 staining while all 3 dysplastic foci within the SSAs were strongly positive. Nonspecific background staining was occasionally noted in smooth muscle, mucinous granules, and some nuclei on the surface crypts along with the edge artifacts.
Conclusions: Immunohistochemical staining using BRAF mutation-specific antibody in colon cancer had results completely concordant with those by sequencing. It is a useful ancillary test to assess the status of BRAF mutation. Clinical application requires careful optimization of conditions on automated stainers to minimize nonspecific staining. The VE1 antibody may also be useful in cellular localization and first-line screening of colon cancers for research purposes.
Monday, March 4, 2013 1:00 PM
Poster Session II # 123, Monday Afternoon