Division of Otolaryngology-Head and Neck Surgery, University of Alberta, 8440-112 st, 1E4 Walter Mackenzie Centre, Edmonton, AB, T6G 2B7, Canada.
Alberta Head and Neck Centre for Oncology and Reconstruction, Walter MacKenzie Health Sciences Centre, Edmonton, AB, Canada.
J Otolaryngol Head Neck Surg. 2018 Sep 24;47(1):60. doi: 10.1186/s40463-018-0299-2.
Recent guidelines for the management of thyroid nodules incorporate mutation testing as an adjunct for surgical decision-making, however current tests are costly with limited accuracy. Droplet digital PCR (ddPCR) is an ultrasensitive method of nucleic acid detection that is particularly useful for identifying gene mutations. This study aimed to assess the analytic and clinical validity of RAS and BRAF ddPCR mutational testing as a diagnostic tool for thyroid fine needle aspirate biopsy (FNAB).
Patients with thyroid nodules meeting indication for FNAB were prospectively enrolled from March 2015 to September 2017. In addition to clinical protocol, an additional FNAB was obtained for ddPCR. Optimized ddPCR probes were used to detect mutations including HRASG12 V, HRASQ61K, HRASQ61R, NRASQ61R, NRASQ61K and BRAFV600E. The diagnostic performance of BRAF and RAS mutations was assessed individually or in combination with Bethesda classification against final surgical pathology.
A total of 208 patients underwent FNAB and mutational testing with the following Bethesda cytologic classification: 26.9% non-diagnostic, 55.2% benign, 5.3% FLUS/AUS, 2.9% FN/SPN, 2.4% SFM and 7.2% malignant. Adequate RNA was obtained from 91.3% (190) FNABs from which mutations were identified in 21.1% of HRAS, 11.5% of NRAS and 7.4% of BRAF. Malignant cytology or BRAFV600E was 100% specific for malignancy. Combining cytology with ddPCR BRAF600E mutations testing increased the sensitivity of Bethesda classification from 41.7 to 75%. Combined BRAFV600E and Bethesda results had a positive predictive value (PPV) of 100% and negative predictive value (NPV) of 89.7% for thyroid malignancy in our cohort.
DdPCR offers a novel and ultrasensitive method of detecting RAS and BRAF mutations from thyroid FNABs. BRAFV600E mutation testing by ddPCR may serve as a useful adjunct to increase sensitivity and specificity of thyroid FNAB.
最近的甲状腺结节管理指南将突变检测作为手术决策的辅助手段，但目前的检测方法成本高，准确性有限。液滴数字 PCR（ddPCR）是一种核酸检测的超灵敏方法，特别适用于鉴定基因突变。本研究旨在评估 RAS 和 BRAF ddPCR 突变检测作为甲状腺细针抽吸活检（FNAB）诊断工具的分析和临床有效性。
2015 年 3 月至 2017 年 9 月，前瞻性纳入符合 FNAB 适应证的甲状腺结节患者。除临床方案外，还额外获得了一份用于 ddPCR 的 FNAB。使用优化的 ddPCR 探针检测包括 HRASG12V、HRASQ61K、HRASQ61R、NRASQ61R、NRASQ61K 和 BRAFV600E 在内的突变。评估 BRAF 和 RAS 突变的诊断性能，分别或联合 Bethesda 分类与最终手术病理进行比较。
共 208 例患者接受 FNAB 和突变检测，以下是 Bethesda 细胞学分类：26.9%非诊断性，55.2%良性，5.3%FLUS/AUS，2.9%FN/SPN，2.4%SFM 和 7.2%恶性。91.3%（190 例）FNAB 获得了足够的 RNA，其中 HRAS 突变率为 21.1%，NRAS 为 11.5%，BRAF 为 7.4%。恶性细胞学或 BRAFV600E 对恶性肿瘤具有 100%的特异性。将细胞学与 ddPCR BRAF600E 突变检测相结合，可将 Bethesda 分类的敏感性从 41.7%提高至 75%。在本队列中，联合 BRAFV600E 和 Bethesda 结果的阳性预测值（PPV）为 100%，阴性预测值（NPV）为 89.7%，对甲状腺恶性肿瘤有意义。
ddPCR 为甲状腺 FNAB 提供了一种新颖的超灵敏的 RAS 和 BRAF 突变检测方法。ddPCR 的 BRAFV600E 突变检测可作为增加甲状腺 FNAB 敏感性和特异性的有用辅助手段。
