Lu Sang-Yu, Chen Ying-Chao, Feng Jia-Lin, Zhou Qin-Yi, Chen Jing, Zhu Chen-Fang, Guo Miao-Miao, Zhang Man-Man, Zhang Qian-Yue, Lu Meng, Yang Liu, Wu Jing, Zhao Shuang-Xia, Song Huai-Dong, Ye Xiao-Ping
Department of Molecular Diagnostics, the Core Laboratory, Medical Center of Clinical Research, Department of Endocrinology, Shanghai Ninth People's Hospital, State Key Laboratory of Medical Genomics, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Institute and Department of Endocrinology and Metabolism, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Int J Endocrinol. 2022 Mar 29;2022:6243696. doi: 10.1155/2022/6243696. eCollection 2022.
exon 15 p.V600E ( V600E) mutation has been established as an important molecular marker for papillary thyroid carcinoma diagnosis by ultrasound-guided fine-needle aspiration biopsy (FNAB). Sanger sequencing is the gold standard for detecting V600E mutations but fails to identify low-frequency mutations. However, droplet digital PCR (ddPCR) is a popular new method for detecting low-frequency mutations. Here, we compare the efficiency of droplet digital PCR (ddPCR) and Sanger sequencing for detection of the V600E mutation in thyroid fine-needle aspiration (FNA) samples.
Thyroid fine-needle aspiration samples from 278 patients with 310 thyroid nodules were collected. Sanger sequencing and ddPCR were conducted to detect the V600E mutation.
The V600E mutation was found in 94 nodules (30.32%) by ddPCR and 40 nodules (12.90%) by Sanger sequencing in 310 FNA samples. A total of 119 nodules were confirmed PTC by postsurgical pathology. Among which the mutation was found in 80 (67.23%) nodules by ddPCR and 31 (26.05%) by Sanger sequencing. All nodules carrying the mutation detected by Sanger sequencing (SS+) were verified by ddPCR (ddPCR+). Also, all nodules with no mutation detected by ddPCR were interpreted as wild-type by Sanger sequencing (SS-). In addition. Almost all SS+/ddPCR + nodules (95.00%; 38/40) and SS-/ddPCR + nodules (100.00%; 54/54) displayed a mutation rate of >5% and <15%, respectively, indicating easy misdetection by Sanger sequencing when the mutation rate is between 5 and 15%.
ddPCR has higher sensitivity than Sanger sequencing and we propose ddPCR as a supplement to Sanger sequencing in molecular testing of using FNAB samples.
外显子15 p.V600E（V600E）突变已被确立为通过超声引导下细针穿刺活检（FNAB）诊断甲状腺乳头状癌的重要分子标志物。桑格测序是检测V600E突变的金标准，但无法识别低频突变。然而，液滴数字PCR（ddPCR）是一种检测低频突变的常用新方法。在此，我们比较液滴数字PCR（ddPCR）和桑格测序检测甲状腺细针穿刺（FNA）样本中V600E突变的效率。
收集278例患者310个甲状腺结节的甲状腺细针穿刺样本。进行桑格测序和ddPCR以检测V600E突变。
在310份FNA样本中，通过ddPCR在94个结节（30.32%）中发现V600E突变，通过桑格测序在40个结节（12.90%）中发现该突变。术后病理证实119个结节为PTC。其中，通过ddPCR在80个（67.23%）结节中发现该突变，通过桑格测序在31个（26.05%）结节中发现。桑格测序检测到携带突变的所有结节（SS+）均经ddPCR验证（ddPCR+）。此外，ddPCR未检测到突变的所有结节经桑格测序均判定为野生型（SS-）。另外，几乎所有SS+/ddPCR+结节（95.00%；38/40）和SS-/ddPCR+结节（100.00%；54/54）的突变率分别>5%和<15%，表明当突变率在5%至15%之间时，桑格测序容易出现误判。
ddPCR比桑格测序具有更高的灵敏度，我们建议在使用FNA样本进行V600E分子检测时，将ddPCR作为桑格测序的补充。
