Microfluidic Droplet Digital PCR Is a Powerful Tool for Detection of BRAF and TERT Mutations in Papillary Thyroid Carcinomas

Abstract

We examined the utility of microfluidic digital PCR (dPCR) for detection of <i>BRAF</i> and <i>TERT</i> mutations in thyroid tumors. DNA extracted from 100 thyroid tumors (10 follicular adenomas, 10 follicular cancers, 5 medullary cancers, and 75 papillary thyroid cancer (PTC) were used for detection of <i>BRAF</i> and <i>TERT</i> mutations. Digital PCRs were performed using rare mutation SNP genotyping assays on QuantStudio 3D platform. In PTCs, <i>BRAF</i>V600E was detected by dPCR and Sanger sequencing in 42/75 (56%) and in 37/75 (49%), respectively. <i>BRAF</i>V600E was not detected in other tumors. The ratio of mutant/total <i>BRAF</i> alleles varied from 4.7% to 47.5%. These ratios were higher in classical PTCs (27.1%) as compared to follicular variant PTCs (9.4%) <i>p</i> = 0.001. In PTCs with and without metastases, the ratios of mutant/total <i>BRAF</i> alleles were 27.6% and 18.4%, respectively, (<i>p</i> = 0.03). In metastatic lesions percentages of mutant/total BRAF alleles were similar to those detected in primary tumors. <i>TERT</i>C228T and <i>TERT</i>C250T were found in two and one cases, respectively, and these tumors concomitantly harbored <i>BRAF</i>V600E. These tumors exhibited gross extra-thyroidal extension, metastases to lymph nodes, and pulmonary metastases (one case). Our results showed that dPCR allows quantitative assessment of druggable targets in PTCs and could be helpful in a molecular-based stratification of prognosis in patients with thyroid cancer.