AUTHOR=da Costa Vitor Rodrigues , Bim Larissa Valdemarin , Pacheco e Silva Luiza Dornelles Penteado , Colloza-Gama Gabriel Avelar , Bastos André Uchimura , Delcelo Rosana , Oler Gisele , Cerutti Janete Maria 

TITLE=Advances in Detecting Low Prevalence Somatic TERT Promoter Mutations in Papillary Thyroid Carcinoma

JOURNAL=Frontiers in Endocrinology

VOLUME=Volume 12 - 2021

YEAR=2021

URL=https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2021.643151

DOI=10.3389/fendo.2021.643151

ISSN=1664-2392

ABSTRACT=Background: Two recurrent TERT (telomerase reverse transcriptase) promoter mutations, named C228T and C250T, were reported in thyroid carcinomas and were correlated with high-risk clinicopathological features and a worse prognosis. Although they are far more frequent in the poorly differentiated and undifferentiated thyroid cancer, the TERT promoter mutations play a significant role on PTC recurrence and disease-specific mortality. However, the prevalence varies considerably through studies and it is uncertain if these differences are due to population variation or the methodology used to detect TERT mutations. In this study we aim to compare three different strategies to detect TERT promoter mutations in PTC. 
Methods: DNA was isolated from formalin-fixed paraffin-embedded (FFPE) specimens from 89 PTC and 40 paired lymph node metastases. The prevalence of the hot spots TERT C228T and C250T mutations were assessed in FFPE samples using TaqMan SNP genotyping assays. Random samples were tested by Sanger Sequencing and droplet digital PCR (ddPCR).
Results: In general, 16 out of 89 (18%) PTC samples and 14 out of 40 (35%) lymph node metastases harbored TERT promoter mutations by TaqMan assay. Sanger sequencing, performed in random selected samples, failed to detect TERT mutations in 4 samples that were positive by TaqMan SNP genotyping assay. Remarkable, ddPCR assay allowed detection of TERT promoter mutations in 6 samples that harbor very low mutant allele frequency (≤ 2%) and were negative by both genotype assay and Sanger Sequencing.
Conclusion: This study observed a good concordance among the methodologies used to detect TERT promoter mutations when a high percentage of mutated alleles present. Sanger analysis demonstrated a limit of detection of mutated alleles. Therefore, the prevalence of TERT promoter mutations in PTC may be higher than previously reported, since most studies have conventionally used Sanger sequencing. The efficient characterization of genetic alterations that are used as preoperative or postoperative diagnostic, risk stratification of the patient and individualized treatment decisions, mainly in highly heterogeneous tumors, require highly sensitive and specific approaches.