Division of Molecular and Genomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
Department of Physiology and Biophysics, Weill Cornell Medicine, New York, New York, USA.
Thyroid. 2021 Apr;31(4):589-595. doi: 10.1089/thy.2020.0307. Epub 2020 Oct 13.
Genetic profiling of resected tumor or biopsy samples is increasingly used for cancer diagnosis and therapy selection for thyroid and other cancer types. Although mutations occur in cell DNA and are typically detected using DNA sequencing, recent attempts focused on detecting pathogenic variants from RNA. The aim of this study was to determine the completeness of capturing mutations using RNA sequencing (RNA-Seq) in thyroid tissue and fine-needle aspiration (FNA) samples. To compare the detection rate of mutations between DNA sequencing and RNA-Seq, 35 tissue samples were analyzed in parallel by whole-exome DNA sequencing (WES) and whole-transcriptome RNA-Seq at two study sites. Then, DNA and RNA from 44 thyroid FNA samples and 47 tissue samples were studied using both targeted DNA sequencing and RNA-Seq. Of 162 genetic variants identified by WES of DNA in 35 tissue samples, 77 (48%) were captured by RNA-Seq, with a detection rate of 49% at site 1 and 46% at site 2 and no difference between thyroid and nonthyroid samples. Targeted DNA sequencing of 91 thyroid tissue and FNA samples detected 118 pathogenic variants, of which 57 (48%) were identified by RNA-Seq. For DNA variants present at >10% allelic frequency (AF), the detection rate of RNA-Seq was 62%, and for those at low (5-10%) AF, the detection rate of RNA-Seq was 7% ( < 0.0001). For common oncogenes ( and ), 94% of mutations present at >10% AF and 11% of mutations present at 5-10% AF were captured by RNA-Seq. As expected, none of promoter mutations were identified by RNA-Seq. The rate of mutation detection by RNA-Seq was lower in FNA samples than in tissue samples (32% vs. 49%, = 0.02). In this study, RNA-Seq analysis detected only 46-49% of pathogenic variants identifiable by sequencing of tumor DNA. Detection of mutations by RNA-Seq was more successful for mutations present at a high allelic frequency. Mutations were more often missed by RNA-Seq when present at low frequency or when tested on FNA samples. All mutations were missed by RNA-Seq. These data suggest that RNA-Seq does not detect a significant proportion of clinically relevant mutations and should be used with caution in clinical practice for detecting DNA mutations.
对切除肿瘤或活检样本的基因谱分析越来越多地用于甲状腺和其他癌症类型的癌症诊断和治疗选择。尽管突变发生在细胞 DNA 中，并且通常使用 DNA 测序来检测，但最近的尝试集中在从 RNA 检测致病性变体上。本研究的目的是确定使用 RNA 测序 (RNA-Seq) 在甲状腺组织和细针抽吸 (FNA) 样本中捕获突变的完整性。为了比较 DNA 测序和 RNA-Seq 之间的突变检测率，在两个研究地点，通过全外显子组 DNA 测序 (WES) 和全转录组 RNA-Seq 同时对 35 个组织样本进行了分析。然后，使用靶向 DNA 测序和 RNA-Seq 对 44 个甲状腺 FNA 样本和 47 个组织样本的 DNA 和 RNA 进行了研究。在 35 个组织样本的 WES DNA 中鉴定的 162 个遗传变异中，77 个(48%)被 RNA-Seq 捕获，在 1 号站点的检测率为 49%，在 2 号站点的检测率为 46%，甲状腺和非甲状腺样本之间无差异。对 91 个甲状腺组织和 FNA 样本的靶向 DNA 测序检测到 118 个致病性变体，其中 57 个(48%)被 RNA-Seq 检测到。对于等位基因频率 (AF) >10%的 DNA 变体，RNA-Seq 的检测率为 62%，而对于 AF 为 5-10%的变体，RNA-Seq 的检测率为 7%(<0.0001)。对于常见的致癌基因 (和)，在>10%AF 存在的突变中，94%的突变和在 5-10%AF 存在的突变中，11%的突变被 RNA-Seq 捕获。如预期的那样，没有 RNA-Seq 鉴定到 启动子突变。RNA-Seq 检测到的突变率在 FNA 样本中低于组织样本(32%比 49%，=0.02)。在这项研究中，RNA-Seq 分析仅检测到可通过肿瘤 DNA 测序识别的致病性变异的 46-49%。高等位基因频率存在的突变通过 RNA-Seq 检测的成功率更高。当存在于低频率或在 FNA 样本上进行测试时，RNA-Seq 更经常错过突变。所有的 突变都被 RNA-Seq 错过了。这些数据表明，RNA-Seq 并不能检测到相当比例的临床相关突变，在临床实践中应谨慎使用，以检测 DNA 突变。
