BackgroundEarly diagnosis of thyroid cancer is hampered by the inability of fine-needle aspiration biopsy (FNAB) to accurately classify ∼30% of cases while preoperative cancer staging detects lymph nodal involvement in only half of cases. Liquid biopsy may present an accurate, non-invasive alternative for preoperative thyroid nodule assessment. Thyrotropin receptor (TSHR) mRNA, a surrogate marker for circulating cancer cells (CTC), may be an option for early detection of malignancy from peripheral blood, but requires methodological improvements. We aimed to investigate if TSHR mRNA can be detected in low sample volumes by employing an ultrasensitive method – droplet digital PCR (ddPCR).MethodsLess than 5 mL of blood was collected from 47 patients with thyroid nodules (25 benign and 22 malignant). RNA was isolated from the fraction of mononuclear cells where CTCs segregate. Samples were analysed for the presence of TSHR mRNA by ddPCR.ResultsThyrotropin receptor mRNA was detectable in 4 mL sample volumes, with the test having good specificity (80%) but modest diagnostic accuracy (68.1%). Combining TSHR mRNA with ultrasound features and FNAB diagnosis, the test reaches high rule-out performances (sensitivity = 90% and NPV = 88.2%). Strikingly, TSHR mRNA correctly classified all samples with thyroid capsule invasion, lymph node metastasis and extrathyroidal extension. If aggressiveness is defined using these parameters, TSHR mRNA test reaches 100% sensitivity and 100% NPV for detecting high-risk cases.ConclusionsEmploying ddPCR for TSHR mRNA improves its measurement by enabling detection in sample volumes common for laboratory testing. The test displays high prognostic performance, showing potential in preoperative risk assessment.

中文翻译：

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检测分化型甲状腺癌患者外周血促甲状腺素受体 mRNA 排除非侵袭性病例

背景甲状腺癌的早期诊断因细针抽吸活检 (FNAB) 无法准确分类约 30% 的病例而受到阻碍，而术前癌症分期仅在一半病例中检测到淋巴结受累。液体活检可能为术前甲状腺结节评估提供准确、无创的替代方案。促甲状腺素受体 (TSHR) mRNA 是循环癌细胞 (CTC) 的替代标志物，可能是早期检测外周血恶性肿瘤的一种选择，但需要方法学改进。我们的目的是研究是否可以通过采用超灵敏方法——微滴式数字 PCR (ddPCR) 在低样本量中检测 TSHR mRNA。方法从 47 名甲状腺结节患者（25 名良性和 22 名恶性）中采集了不到 5 mL 的血液。从 CTC 分离的单核细胞部分中分离出 RNA。通过 ddPCR 分析样品中 TSHR mRNA 的存在。结果在 4 mL 样品体积中可检测到促甲状腺素受体 mRNA，该测试具有良好的特异性 (80%)，但诊断准确性较低 (68.1%)。该测试将 TSHR mRNA 与超声特征和 FNAB 诊断相结合，达到了很高的排除性能（灵敏度 = 90%，NPV = 88.2%）。引人注目的是，TSHR mRNA 正确分类了所有甲状腺被膜侵犯、淋巴结转移和甲状腺外扩散的样本。如果使用这些参数定义侵袭性，则 TSHR mRNA 测试可达到 100% 的灵敏度和 100% NPV，用于检测高风险病例。 结论 对 TSHR mRNA 采用 ddPCR，可在实验室测试常用的样本量中进行检测，从而改善其测量结果。该测试显示出较高的预后性能，显示出术前风险评估的潜力。