Prevalence of well-characterized and putative marker mutations in benign and malignant thyroid neoplasms

Insufficient accuracy of preoperative differential diagnostics by cytological examination of fine-needle aspiration biopsy material and low proportion of cases of malignant neoplasms described by molecular genetic markers with known diagnostic significance determine the relevance of the search for new molecular genetic markers with an assessment of their association with cancer risk and clinical and morphological characteristics. The aim of the work was to study the prevalence of an expanded spectrum of known and new putative driver and secondary mutations in benign and malignant thyroid neoplasms. Material: fresh frozen surgical material of malignant (64 samples) and benign (16 samples) thyroid neoplasms. Methods: search for point variants, short insertions/deletions, and copy number variations was performed via targeted high-throughput sequencing using AmpliSeq technology on the Illumina NextSeq platform. Fusions were by the presence of fused transcript by targeted high-throughput sequencing using AmpliSeq technology on the Illumina MiSeq platform. Results: mutations in genes BRAF, KRAS, NRAS, HRAS were detected in 62% of cancer cases. In 12% of cancers we detected rearrangements of CCDC6-RET (3 cases) and PAX8-PPARG (2 cases), TPM3-NTRK1, ETV6-NTRK3, STRN-ALK - one case each. In all cases, the identified rearrangements were mutually exclusive with other known driver mutations. In benign neoplasms, no rearrangements were found. Mutations in the TERT gene promoter have been identified in 10% of thyroid cancers and have been associated with known driver mutations. The well-known missense mutation EIF1AX was detected in one case of a benign neoplasm of the thyroid gland; in malignant neoplasms, no EIF1AX mutation were detected. Putative driver mutations in the PPM1D, PDGFRA, KDR oncogenes were detected in thyroid cancer samples and were not detected in benign thyroid neoplasms. Conclusions: the work characterize the prevalence of driver mutations with a known diagnostic significance and mutations in genes described in the literature without a known diagnostic significance in benign and malignant thyroid neoplasms.

hyroid cancer, mutations, rearrangements, targeted high-throughput sequencing, differential diagnostics

1. Sanabria A., Kowalski L.P., Shah J.P., et al. Growing incidence of thyroid carcinoma in recent years: Factors underlying overdiagnosis. Head Neck 2018;40:855-66. doi:10.1002/hed.25029

2. Haugen B.R., Alexander E.K., Bible K.C., et al. 2015 American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer: The American Thyroid Association Guidelines Task Force on Thyroid Nodules and Differentiated Thyroid Cancer. Thyroid. 2016;26(1):1-133. doi:10.1089/thy.2015.0020

3. Cibas E.S., Ali S.Z. The 2017 Bethesda System for Reporting Thyroid Cytopathology. Thyroid. 2017;27(11):1341-1346. doi:10.1089/thy.2017.0500

4. Rogers W.A., Craig W.L., Entwistle V.A. Ethical issues raised by thyroid cancer overdiagnosis: A matter for public health?. Bioethics. 2017;31(8):590-598. doi:10.1111/bioe.12383

5. Rahman S.T., McLeod D.S.A., Pandeya N., et al. Understanding Pathways to the Diagnosis of Thyroid Cancer: Are There Ways We Can Reduce Over-Diagnosis?. Thyroid. 2019;29(3):341-348. doi:10.1089/thy.2018.0570

6. Cancer Genome Atlas Research Network. Integrated genomic characterization of papillary thyroid carcinoma. Cell. 2014;159(3):676-690. doi:10.1016/j.cell.2014.09.050

7. Kasaian K., Wiseman S.M., Walker B.A. et al. The genomic and transcriptomic landscape of anaplastic thyroid cancer: implications for therapy. BMC Cancer. 2015;15:984. Published 2015 Dec 18. doi:10.1186/s12885-015-1955-9

8. Kunstman J.W., Juhlin C.C., Goh G., et al. Characterization of the mutational landscape of anaplastic thyroid cancer via whole-exome sequencing. Hum Mol Genet. 2015;24(8):2318-2329. doi:10.1093/hmg/ddu749

9. Landa I., Ibrahimpasic T., Boucai L., et al. Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers. J Clin Invest. 2016;126(3):1052-1066. doi:10.1172/JCI85271

10. Swierniak M., Pfeifer A., Stokowy T., et al. Somatic mutation profiling of follicular thyroid cancer by next generation sequencing. Mol Cell Endocrinol. 2016;433:130-137. doi:10.1016/j.mce.2016.06.007

11. Yoo S.K., Lee S., Kim S.J., et al. Comprehensive Analysis of the Transcriptional and Mutational Landscape of Follicular and Papillary Thyroid Cancers. PLoS Genet. 2016;12(8):e1006239. Published 2016 Aug 5. doi:10.1371/journal.pgen.1006239

12. Pozdeyev N., Gay L.M., Sokol E.S., et al. Genetic Analysis of 779 Advanced Differentiated and Anaplastic Thyroid Cancers. Clin Cancer Res. 2018;24(13):3059-3068. doi:10.1158/1078-0432.CCR-18-0373

13. Li H., Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010; 26: 589-595.

14. Van der Auwera G.A., Carneiro M.O., Hartl C., et al. From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Curr Protoc Bioinformatics. 2013;43(1110):11.10.1-11.10.33. doi:10.1002/0471250953.bi1110s43

15. Boeva V., Popova T., Lienard M., et al. Multi-factor data normalization enables the detection of copy number aberrations in amplicon sequencing data. Bioinformatics. 2014;30(24):3443-3450. doi:10.1093/bioinformatics/btu436

16. Li M.M., Datto M., Duncavage E.J., et al. Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer: A Joint Consensus Recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists. J Mol Diagn. 2017;19(1):4-23. doi:10.1016/j.jmoldx.2016.10.002

17. Paschke R., Cantara S., Crescenzi A., et al. European Thyroid Association Guidelines regarding Thyroid Nodule Molecular Fine-Needle Aspiration Cytology Diagnostics. Eur Thyroid J. 2017;6(3):115-129. doi:10.1159/000468519

18. Melo M., da Rocha A.G., Vinagre J., et al. TERT promoter mutations are a major indicator of poor outcome in differentiated thyroid carcinomas. J Clin Endocrinol Metab 2014;99:E754-65. doi:10.1210/jc.2013-3734

19. Bournaud C., Descotes F., Decaussin-Petrucci M., et al. TERT promoter mutations identify a high-risk group in metastasis-free advanced thyroid carcinoma. Eur J Cancer. 2019;108:41-49. doi:10.1016/j.ejca.2018.12.003

20. Karunamurthy A., Panebianco F., Hsiao S.J., et al. Prevalence and phenotypic correlations of EIF1AX mutations in thyroid nodules. Endocr Relat Cancer. 2016;23(4):295-301. doi:10.1530/ERC-16-0043