Comparison of droplet digital PCR and direct Sanger sequencing for the detection of the BRAF (V600E) mutation in papillary thyroid carcinoma

BACKGROUND: The BRAF (V600E) mutation status is a useful diagnostic and prognostic marker for papillary thyroid carcinoma (PTC). Although it is a commonly used method, Sanger sequencing has several limitations in detecting the BRAF (V600E) mutation. The aim of this study was to evaluate the efficiency of droplet digital PCR (ddPCR) as an alternative method for the detection of the BRAF (V600E) mutation in PTC patients. METHODS: Samples from a total of 120 patients with PTC and 30 patients with benign nodular thyroid disease who underwent thyroid surgery were collected. The BRAF (V600E) mutation status of the PTC patients was tested by Sanger sequencing and ddPCR. RESULTS: The BRAF (V600E) mutation was detected in 67 samples (44.67%) by Sanger sequencing and 92 samples (61.33%) by ddPCR. The detection of the mutation by the two methods was inconsistent in twenty‐five samples (16.67%). The sensitivity and specificity of the ddPCR method were 100% and 69.88%, respectively, and the positive predictive and negative predictive values were 72.83% and 100%, respectively. The concordance rate between the two methods in detecting the BRAF (V600E) mutation was 83.33%. Neither Sanger sequencing nor ddPCR detected BRAF (V600E) in 30 patients with benign nodular thyroid disease. The 92 samples with the BRAF (V600E) mutation were detected by ddPCR at a fractional abundance from 0.28% to 45.40% as follows: ≥10% (59 samples, 64.13%), 5%‐10% (8 samples, 8.70%), and ≤5% (25 samples, 27.17%). The BRAF (V600E) mutation was detected in all 59 samples at a fractional abundance ≥10% and in four samples at a fractional abundance from 5% to 10%, and no BRAF (V600E) mutation was detected at a fractional abundance ≤5% by Sanger sequencing. CONCLUSIONS: ddPCR was a reliable, highly sensitive alternative method for the detection of the BRAF (V600E) mutation in PTC patients.