Division of Molecular and Genomic Pathology, Department of Pathology, University of Pittsburgh, 200 Lothrop Street, Pittsburgh, Pennsylvania 15261.
J Clin Endocrinol Metab. 2013 Nov;98(11):E1852-60. doi: 10.1210/jc.2013-2292. Epub 2013 Aug 26.
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.
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.
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.
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.
下一代测序（NGS）允许对人类基因组的大片段进行高通量测序分析。我们探索了使用靶向 NGS 同时检测甲状腺癌中的多种突变。
设计了一个定制面板（ThyroSeq），以针对 12 个癌症基因的 284 个突变热点进行靶向检测。对 228 个甲状腺肿瘤和非肿瘤样本的 DNA 进行测序分析，包括 105 个冷冻样本、72 个福尔马林固定样本和 51 个细针穿刺样本，代表所有主要类型的甲状腺癌。
仅需 5-10ng 的输入 DNA 即可成功分析 99.6%的样本。突变检测的分析准确性为 100%，突变等位基因的灵敏度为 3%-5%。ThyroSeq DNA 检测在 27 个经典乳头状甲状腺癌（PTC）中的 19 个（70%）、30 个滤泡状变异 PTC 中的 25 个（83%）、18 个传统（78%）和 18 个嗜酸细胞滤泡状癌中的 7 个（39%）、10 个低分化癌中的 3 个（30%）、27 个间变性（ATC）中的 20 个（74%）和 15 个髓样甲状腺癌中的 11 个（73%）中发现了突变。相比之下，83 个良性结节中有 5 个（6%）为阳性。大多数肿瘤仅有一个突变，而一些 ATC 和 PTC 则显示有两个或三个突变。最常见的检测到的突变是 BRAF 和 RAS，其次是 PIK3CA、TP53、TSHR、PTEN、GNAS、CTNNB1 和 RET。PTC 中的 BRAF 突变等位基因频率为 18%-48%，在 ATC 中较低。
ThyroSeq NGS 面板允许以高精度和灵敏度同时检测多种突变，仅需少量 DNA，可用于各种甲状腺组织和细针穿刺样本，并提供突变等位基因的定量评估。使用这种方法，在特定类型的甲状腺癌中检测到 30%-83%的点突变，在良性甲状腺结节中仅检测到 6%，并且在肿瘤结节内的大多数细胞中均存在这些突变。
