BRAF^V600E immunohistochemistry can reliably substitute BRAF molecular testing in the Lynch syndrome screening algorithm in colorectal cancer

Aims: The Lynch syndrome (LS) screening algorithm requires BRAF testing as a fundamental step to distinguish sporadic from LS-associated colorectal carcinomas (CRC). BRAF testing by immunohistochemistry (IHC) has shown variable results in the literature. Our aim was to analyse concordance between BRAF^V600E IHC and BRAF molecular analysis in a large, mono-institutional CRC whole-slide, case series with laboratory validation.

Methods and results: MisMatch repair (MMR) protein (hMLH1, hPMS2, hMSH2, and hMSH6) and BRAF^V600E IHC were performed on all unselected cases of surgically resected CRCs (2018-2023). An in-house validation study for BRAF^V600E IHC was performed in order to obtain optimal IHC stains. BRAFV^V600E IHC was considered negative (score 0), positive (scores 2-3), and equivocal (score 1). Interobserver differences in BRAF^V600E IHC scoring were noted in the first 150 cases prospectively collected. Nine-hundred and ninety CRCs cases (830 proficient (p)MMR/160 deficient (d)MMR) were included and all cases performed BRAF^V600E IHC (BRAF^V600E IHC-positive 13.5% of all series; 66.3% dMMR cases; 3.4% pMMR cases), while 333 also went to BRAF mutation analysis. Optimal agreement in IHC scoring between pathologists (P < 0.0001) was seen; concordance between BRAF^V600E IHC and BRAF molecular analysis was extremely high (sensitivity 99.1%, specificity 99.5%; PPV 99.1%, and NPV 99.5%). Discordant cases were reevaluated; 1 score 3 + IHC/wildtype case was an interpretation error and one score 0 IHC/mutated case was related to heterogenous BRAF^V600E IHC expression. Among the 12 IHC-equivocal score 1+ cases (which require BRAF molecular analysis), three were BRAF-mutated and nine BRAF-wildtype.

Conclusion: BRAF^V600E IHC can be used as a reliable surrogate of molecular testing after stringent in-house validation.