Targeted next-generation sequencing panel (ThyroSeq) for detection of mutations in thyroid cancer

J Clin Endocrinol Metab. 2013 Nov;98(11):E1852-60. doi: 10.1210/jc.2013-2292. Epub 2013 Aug 26.


Objectives: Next-generation sequencing (NGS) allows for high-throughput sequencing analysis of large regions of the human genome. We explored the use of targeted NGS for simultaneous testing for multiple mutations in thyroid cancer.

Design: A custom panel (ThyroSeq) was designed to target 12 cancer genes with 284 mutational hot spots. Sequencing was performed to analyze DNA from 228 thyroid neoplastic and nonneoplastic samples including 105 frozen, 72 formalin-fixed, and 51 fine-needle aspiration samples representing all major types of thyroid cancer.

Results: Only 5-10 ng of input DNA was sufficient for successful analysis of 99.6% of samples. The analytical accuracy for mutation detection was 100% with the sensitivity of 3%-5% of mutant allele. ThyroSeq DNA assay identified mutations in 19 of 27 of classic papillary thyroid carcinomas (PTCs) (70%), 25 of 30 follicular variant PTCs (83%), 14 of 18 conventional (78%) and 7 of 18 oncocytic follicular carcinomas (39%), 3 of 10 poorly differentiated carcinomas (30%), 20 of 27 anaplastic (ATCs) (74%), and 11 of 15 medullary thyroid carcinomas (73%). In contrast, 5 of 83 benign nodules (6%) were positive for mutations. Most tumors had a single mutation, whereas several ATCs and PTCs demonstrated two or three mutations. The most common mutations detected were BRAF and RAS followed by PIK3CA, TP53, TSHR, PTEN, GNAS, CTNNB1, and RET. The BRAF mutant allele frequency was 18%-48% in PTCs and was lower in ATCs.

Conclusions: The ThyroSeq NGS panel allows simultaneous testing for multiple mutations with high accuracy and sensitivity, requires a small amount of DNA and can be performed in a variety of thyroid tissue and fine-needle aspiration samples, and provides quantitative assessment of mutant alleles. Using this approach, the point mutations were detected in 30%-83% of specific types of thyroid cancer and in only 6% of benign thyroid nodules and were shown to be present in the majority of cells within the cancer nodule.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenocarcinoma, Follicular / genetics*
  • Adenocarcinoma, Follicular / pathology
  • Biopsy, Fine-Needle
  • Carcinoma / genetics*
  • Carcinoma / pathology
  • Carcinoma, Neuroendocrine
  • Carcinoma, Papillary
  • Female
  • Gene Expression Regulation, Neoplastic
  • High-Throughput Nucleotide Sequencing / methods*
  • High-Throughput Nucleotide Sequencing / standards
  • Humans
  • Male
  • Neoplasms / genetics*
  • Neoplasms / pathology
  • Point Mutation
  • Reproducibility of Results
  • Thyroid Cancer, Papillary
  • Thyroid Gland / pathology
  • Thyroid Neoplasms / genetics*
  • Thyroid Neoplasms / pathology

Supplementary concepts

  • Thyroid cancer, medullary