BRAF V600E immunohistochemistry is reliable in primary and metastatic colorectal carcinoma regardless of treatment status and shows high intratumoral homogeneity

Abstract

In colorectal carcinoma the evaluation of BRAF mutation status is increasingly being performed given its utility as a prognostic and predictive biomarker. However, there are conflicting reports of the sensitivity and specificity of BRAF V600E immunohistochemistry (IHC), and little is known about its reliability in tissues collected from metastatic sites or after chemotherapy, radiation therapy and/or targeted therapy. The degree of intratumoral staining heterogeneity is also not well established. We performed IHC for BRAF V600E (VE1) on 204 cases of colorectal carcinoma including 59 with the BRAF V600E mutation. These included primary (n=147) and metastatic/recurrent (n=57) tumors, collected before (n=133) or after (n=71) chemotherapy, radiation therapy and/or targeted therapy. Evaluation of a test cohort (39 cases) with knowledge of mutation status established a specific staining pattern for the mutation: diffuse cytoplasmic staining of near-uniform intensity, regardless of strength of staining. Using this pattern, pathologists at 3 levels of training independently performed blinded evaluation of the remaining cases. BRAF V600E staining was 96.3% sensitive and 98.5% specific for the mutation, including both pretreatment and posttreatment specimens. Fleiss κ for interobserver agreement was 0.96. Staining of whole sections of the BRAF mutants showed diffuse staining in all cases and uniform or near-uniform intensity in 91%. In 20 cases with both pretreatment and posttreatment specimens, there was 100% accuracy and agreement in staining between samples. We conclude that BRAF V600E IHC is reliable for the evaluation of mutational status in colorectal carcinoma regardless of site or prior treatment history, and staining shows a high degree of intratumoral homogeneity.

Figure 1

BRAF V600E (VE1) immunohistochemistry in normal colonic mucosa and BRAF mutant colorectal carcinoma. Non-specific staining of normal mucosa was heterogeneous with negative staining in the crypt base and conspicuous nuclear and weak cytoplasmic staining of the luminal aspect of the crypts (A). Absolute intensity of staining in BRAF mutants varied from strong (B) to moderate (C) to weak (D) but was notably uniform in all cases. Nuclear staining occurred occasionally (C) in BRAF mutants, especially in the luminal aspect, but should be regarded as non-specific.

Figure 2

Staining for BRAF V600E (VE1) in pre-treatment (A) and post-treatment (B) tumors from the same patient showed nearly identical intensity and uniformity in the six BRAF mutants examined. One case of interest (C) carried a BRAF V600E mutation and had undergone treatment with a mutation specific BRAF-inhibitor prior to sampling. The VE1 antibody demonstrated convincingly positive staining (D) despite prior targeted therapy.

Figure 3

False negative interpretations of BRAF V600E (VE1) immunohistochemistry in BRAF mutant colorectal carcinoma (A–B, C–D, and E–F). Cytoplasmic staining in signet-ring cells is confounded by the presence of intracytoplasmic mucin (B). Very weak but uniform staining was present in a case with medullary morphology that was called negative by two of three observers (D). Weak cytoplasmic staining (as in D and F) should not be disregarded. Rather, uniform cytoplasmic staining, even if dim, should be taken as positive.

Figure 4

False positive interpretations of BRAF V600E (VE1) immunohistochemistry in BRAF wild-type colorectal carcinoma. In two non-BRAF mutant cases (A-B and C-D) interpreted as positive by one of three observers, tumorous tissue was scant and staining of malignant cells was weak and, in retrospect, not uniform across the tumor cells. High level background staining of stromal cells (B and D) was seen occasionally, particularly in smooth muscle (B), and in one case in an adjacent ganglion (D, top left).

Figure 5

This case had no detectable BRAF mutation by molecular analysis, but multiple tissue cores (A, C) showed scant groups of tumor cells that fulfilled all positive BRAF V600E staining criteria (B, D): strong and uniform cytoplasmic staining with or without membranous accentuation. Repeat molecular analysis was performed and was positive for the V600E mutation, consistent with an initial false negative molecular result, likely due to the limited tumor purity and low tumor cellularity.