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

Dorina Ylli,John Costello,Kirk Jensen,Cristiane Jeyce Gomes-Lima,Aneeta Patel,Leonard Wartofsky,Kenneth Dale Burman,Zhao-Zhang Li,Vasyl V Vasko,Maria Cecilia Mendonca-Torres

doi:10.3390/cancers11121916

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.

Highlights

Papillary thyroid cancer (PTC) is the most common endocrine malignancy and is increasing at the fastest incidence rate of any malignancy [1]
Amplification conditions were optimized by testing a range of annealing temperatures (50–65 ◦ C), and optimal amplifications for BRAFV600E, TERTC228T, and TERTC250T were achieved at 60 ◦ C, 55 ◦ C, and 55 ◦ C, respectively
Digital PCRs were performed at a gradient of annealing temperatures ranging from 50 ◦ C to 65 ◦ C, and optimal segregations for BRAFV600E, TERTC228T, and TERTC250T were at 60 ◦ C, 55 ◦ C, and 55 ◦ C, respectively

Summary

Introduction

Papillary thyroid cancer (PTC) is the most common endocrine malignancy and is increasing at the fastest incidence rate of any malignancy [1]. Somatic alterations of genes involved in the mitogen-activated protein kinase (MAPK) pathway were frequently detected in PTC, with RET (rearranged during transfection) rearrangements accounting for 10–15%, Ras point mutations (rat sarcoma viral oncogene homolog) for. The vast majority of BRAF alterations are characterized by a single amino acid substitution of valine by glutamic acid in a mutational hotspot at amino acid position 600 (BRAF c.1799T>A (p.Val600Glu), referred to hereafter as BRAFV600E). This exchange mimics the phosphorylation of amino acid residues T599 and S602 and induces a conformational change of the activation segment leading to a constitutive kinase activity of BRAF and phosphorylation of downstream targets [8]

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