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 BRAF and TERT 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 BRAF and TERT mutations. Digital PCRs were performed using rare mutation SNP genotyping assays on QuantStudio 3D platform. In PTCs, BRAFV600E was detected by dPCR and Sanger sequencing in 42/75 (56%) and in 37/75 (49%), respectively. BRAFV600E was not detected in other tumors. The ratio of mutant/total BRAF 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%) p = 0.001. In PTCs with and without metastases, the ratios of mutant/total BRAF alleles were 27.6% and 18.4%, respectively, (p = 0.03). In metastatic lesions percentages of mutant/total BRAF alleles were similar to those detected in primary tumors. TERTC228T and TERTC250T were found in two and one cases, respectively, and these tumors concomitantly harbored BRAFV600E. 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.

The ratio of BRAFV600E alleles can be used to assess the biological behavior of papillary thyroid carcinoma

“…The BRAFV600E mutation was undetectable by ARMS but detectable by ddPCR in 13 patients. Among the 329 PTC patients, the ratio of BRAFV600E alleles in 165 cases was less than 10%, which is usually beyond the range detected by the first-generation gene detection technology(26,41).…”

“…Detection of the BRAFV600E mutation by ddPCR has been demonstrated in melanoma samples and pancreas fine needle aspiration (FNA) specimens(23-25). A recent study showed that mutant BRAFV600E was detected in 56%(42/75) of PTC tumors by ddPCR and that the ratio of mutant/total BRAF alleles varied from 4.7% to 47.5%, suggesting that ddPCR detection of the BRAFV600E mutation should be a suitable assay for the molecular-based stratification of prognosis in patients with PTC(26).…”

Detection of BRAFV600E in Liquid Biopsy from Patients with Papillary Thyroid Cancer Is Associated with Tumor Aggressiveness and Response to Therapy

“…Microfluidic digital PCR analyses were performed on a QuantStudio™ 3D Digital PCR System (Life Technologies, Carlsbad, CA, USA) as per the previously published protocol [29]. The rare mutation SNP genotyping assays for BRAFV600E (Fluorescein-FAM) which was multiplexed with an assay for the detection of wild-type BRAF (2 -chloro-7 phenyl-1,4-dichloro-6-carboxy-fluorescein-VIC) were synthesized by Thermo Fisher Scientific.…”

“…We established a protocol to detect BRAFV600E in thyroid cancer samples by microfluidic digital PCR and described this technique in a previously published study [29]. In the present study, we applied a similar approach and in addition, used a co-amplification at COLD-PCR prior to digital PCR.…”

What Is New in Thyroid Cancer: The Special Issue of the Journal Cancers

“…In the first paper, Ylli et al analyzed the utility of digital PCR technique in detecting BRAF V600E and TERT promoter mutations ( C228T and C250T ) in thyroid cancer tissue samples. Interestingly, microfluidic digital PCR allowed for specific, sensitive and rapid detection of BRAF V600E and TERT C228T mutations in thyroid tissue samples and could be used for quantification of mutant alleles in an easy and cost-effective manner [ 6 ]. In the second paper, Capitoli et al applied, for the first time, Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry Imaging (MSI) on real thyroid Fine Needle Aspirations (FNAs) to test its possible complementary role in routine cytology in the diagnosis of thyroid nodules.…”