Accuracy comparison of direct Sanger sequencing, immunohistochemistry, and droplet digital polymerase chain reaction in detecting BRAF mutations in papillary thyroid carcinoma: a retrospective diagnostic accuracy study.

Purpose: The BRAFV600E mutation is a common genetic alteration in papillary thyroid carcinoma (PTC) and is associated with poor prognostic factors. Accurate detection is crucial for risk stratification. This study compares the performance of direct Sanger sequencing (SS), immunohistochemistry (IHC), and droplet digital PCR (ddPCR) in detecting the BRAFV600E mutation in PTC. Methods: Tumor samples from patients undergoing thyroidectomy were analyzed for BRAFV600E using SS, IHC, and ddPCR. A mutant allele fraction >1% was considered ddPCR positive. Sensitivity and concordance rates were evaluated. Results: A total of 48 PTC and 9 benign samples were tested. All benign samples were negative for BRAFV600E by both SS and ddPCR. Among PTC cases, the mutation was detected in 72.9% by SS, 89.6% by IHC, and 83.3% by ddPCR. Both IHC and ddPCR were significantly more sensitive than SS (P = 0.001 and P < 0.001, respectively). Concordant results across all 3 methods were seen in 83.3% of PTC cases. Among the 8 discordant samples (all SS-negative), 5 were positive by both IHC and ddPCR, and 3 were IHC-positive only. Of these, 6 showed adenine peaks on SS chromatograms, with a mean ddPCR mutant allele fraction of 14.5%, compared to 0.36% in the 2 without adenine peaks. Conclusion: IHC and ddPCR demonstrated superior sensitivity compared to SS for detecting BRAFV600E mutations in PTC. These findings support the use of IHC and ddPCR as more reliable alternatives to SS in clinical practice.