BRAF V600E Mutation Test in Fine Needle Aspiration (FNA) Specimens of Thyroid Nodules by Competitive Allele-Specific TaqMan PCR (castPCR)

Abstract

Background: BRAF V600E mutation has been confirmed as a significant molecular indicator for the diagnosis and prognosis of papillary thyroid carcinoma (PTC). Sanger sequencing is the recognized approach for BRAF V600E mutation analysis, yet it is not proficient at detecting mutations that occur at low frequencies, especially in thyroid fine needle aspiration (FNA) specimens. This study aimed to evaluate the diagnostic value of Competitive Allele-Specific TaqMan PCR (castPCR) as a new reliable method for BRAF V600E mutation test in thyroid FNA specimens compared to Sanger sequencing. Methods: Thirty patients with thyroid nodules were enrolled prospectively at Dharmais Cancer Hospital Jakarta from 2013–2014. The BRAF V600E mutation was analyzed from FNA specimens using castPCR and Sanger Sequencing methods. The sensitivity and specificity of those methods were compared with routine diagnostic histopathological examination as a gold standard for PTC assessment.Results: A total of 66.6% (20/30) cases were confirmed as PTC by postsurgical histopathological examination. Among these, the BRAF V600E mutation was found in 60.0% (12/20) by castPCR and 40.0% (8/20) by Sanger sequencing. Neither castPCR nor Sanger sequencing detected BRAF V600E mutation in other nodular thyroid diseases. The sensitivity and specificity of castPCR were 60.0% and 100.0%, while Sanger sequencing was 40.0% and 100.0%, respectively. Conclusions: This study indicated that castPCR has a higher sensitivity for the BRAF V600E mutation test than Sanger Sequencing, especially in samples with a low abundance of mutant alleles. A sensitive, rapid, and easy castPCR method is reliable for evaluating BRAF V600E mutation and can improve PTC preoperative diagnosis in clinical settings.