Immunohistochemical Investigation of BRAF P.V600E Mutations in Thyroid Carcinoma Using 2 Separate BRAF Antibodies

Background: Approximately 45% of papillary thyroid carcinomas harbor BRAF p.V600E mutations and current practice algorithms endorse molecular testing for BRAF p.V600E. We assessed the utility of immunohistochemistry to detect BRAF p.V600E mutations in thyroid carcinomas using 2 separate BRAF monoclonal antibodies: one that detects both mutant and wild-type protein (pan-BRAF) and another that detects only the mutant protein (mut-BRAF).

Methods: We selected 41 formalin-fixed paraffin-embedded thyroid carcinomas (29 papillary, 1 follicular, 7 medullary, and 4 anaplastic) from 37 thyroidectomies and 4 fine-needle aspirations. Immunohistochemistry was performed using a pan-BRAF (clone EP152Y) or a mut-BRAF (clone VE1) monoclonal antibody. Tumors were considered positive if >10% of neoplastic cells showed moderate (2+) or strong (3+) cytoplasmic staining. BRAF p.V600E mutations were confirmed by molecular pyrosequencing, the gold standard for statistical analysis.

Results: pan-BRAF reactivity was observed in 80.5% (n=33) of cases: 34.1% (n=14) harbored BRAF p.V600E mutations and 46.3% (n=19) were wild type. mut-BRAF reactivity was observed in 46.3% (n=19) of cases: 34.1% (n=14) harbored BRAF p.V600E mutations and 12.2% (n=5) were wild type. The pan-BRAF antibody detected 14 more false positives (specificity: 29.6%, PPV: 42.4%) compared with the mut-BRAF antibody (specificity: 61.5%, PPV: 73.7%), but both antibodies detected the same 5 false positives. No false negatives were detected with either antibody (sensitivity and NPV 100.0% for both).

Conclusions: The suboptimal specificity and PPV limits the diagnostic utility of both antibodies to reliably detect BRAF p.V600E mutations in thyroid carcinoma. However, both antibodies provide excellent sensitivity and NPV and either could be used to exclude BRAF wild-type thyroid carcinomas before molecular testing.