Immunohistochemistry with the anti-BRAF V600E (VE1) antibody: impact of pre-analytical conditions and concordance with DNA sequencing in colorectal and papillary thyroid carcinoma

Abstract

The most common of all activating BRAF mutations (T1799A) leads to a substitution of valine (V) to glutamic acid (E) at the position 600 of the amino acid sequence. The major goal of this study was to compare detection of the BRAF V600E mutation by DNA sequencing with immunohistochemistry (IHC) using the anti-BRAF V600E (VE1) antibody. Archival formalin fixed, paraffin embedded tissues from 352 patients with colon adenocarcinoma (n = 279) and papillary thyroid carcinoma (n = 73) were evaluated for the BRAF V600E mutation by sequencing and IHC. The discordant cases were re-evaluated by repeat IHC, SNaPshot and next-generation sequencing (NGS). Furthermore, the effect of pre-analytical variables on the utility of this antibody was evaluated in two xenograft mouse models.After resolving 15 initially discordant cases, 212 cases were negative for the BRAF V600E mutation by IHC. Of these, 210 cases (99.1%) were also negative by sequencing and two cases (0.9%) remained discordant. Of the 140 cases that were IHC positive for BRAF V600E, 138 cases were confirmed by sequencing (98.6%) and two cases remained discordant (1.4%). Overall, the negative predictive value was 99.1%, positive predictive value 98.6%, sensitivity 98.6%, specificity 99.1% and overall percentage agreement 98.9% (348/352 cases). Tissue fixation studies indicated that tissues should be fixed for 12-24 h within 2 h of tissue collection with 10% neutral buffered formalin.

Fig. 1

Representative images of colon cancer and thyroid cancer tissues positive/negative for BRAF V600E mutation stained with the anti-BRAF V600E (VE1) mouse monoclonal antibody, negative mouse monoclonal control and H&E. Scale bar = 200 μm.

Fig. 2

Mutation analysis of BRAF gene in colon cancer tissues. Example of sequencing electropherogram with BRAF codon wild-type (A) and V600E heterozygous (B). The corresponding images of tissues stained with anti-BRAF V600E (VE1) mouse monoclonal antibody are included. Brown colour indicates positive staining with anti-BRAF V600E (VE1) antibody.

Fig. 3

Representative images of LS411N and A2058 xenograft tissues stained with anti-BRAF V600E (VE1) mouse monoclonal antibody after fixation with different fixatives for 24 h. Scale bar = 200 μm.