Lafin John, Scarpini Cinzia, Amini Armon, Konneh Bendu, Howard Jeffrey, Gerald Thomas, Nuno Michelle, Piao Jin, Savelyeva Anna, Wang Zhaohui, Gagan Jeffrey, Jia Liwei, Lewis Cheryl, Murray Sarah, Sawa Yun, Margulis Vitaly, Woldu Solomon, Strand Douglas, Coleman Nicholas, Amatruda James, Frazier Lindsay, Murray Matthew, Bagrodia Aditya
The University of Texas Southwestern Medical Center.
University of Cambridge.
Res Sq. 2023 Mar 21:rs.3.rs-2644890. doi: 10.21203/rs.3.rs-2644890/v1.
Circulating miR-371a-3p has excellent performance in the detection of viable (non-teratoma) GCT pre-orchiectomy; however, its ability to detect occult disease is understudied. To refine the serum miR-371a-3p assay in the minimal residual disease setting we compared performance of raw (Cq) and normalized (∆Cq, RQ) values from prior assays, and validated interlaboratory concordance by aliquot swapping. Revised assay performance was determined in a cohort of 32 patients suspected of occult retroperitoneal disease. Assay superiority was determined by comparing resulting receiver-operator characteristic (ROC) curves using the Delong method. Pairwise t-tests were used to test for interlaboratory concordance. Performance was comparable when thresholding based on raw Cq normalized values. Interlaboratory concordance of miR-371a-3p was high, but reference genes miR-30b-5p and cel-miR-39-3p were discordant. Introduction of an indeterminate range of Cq 28-35 with a repeat run for any indeterminate improved assay accuracy from 0.84 to 0.92 in a group of patients suspected of occult GCT. We recommend that serum miR-371a-3p test protocols are updated to a) utilize threshold-based approaches using raw Cq values, b) continue to include an endogenous (e.g., miR-30b-5p) and exogenous non-human spike-in (e.g., cel-miR-39-3p) microRNA for quality control, and c) to re-run any sample with an indeterminate result.
循环miR-371a-3p在睾丸切除术前行生殖细胞肿瘤（GCT，非畸胎瘤）活体检出中表现优异；然而，其对隐匿性疾病的检测能力研究不足。为优化微小残留病情况下的血清miR-371a-3p检测方法，我们比较了先前检测中的原始（Cq）值和标准化（∆Cq、RQ）值的性能，并通过样本交换验证了实验室间的一致性。在32例疑似隐匿性腹膜后疾病的患者队列中确定了修订后的检测性能。使用德龙方法比较所得的受试者工作特征（ROC）曲线来确定检测优势。采用配对t检验来检测实验室间的一致性。基于原始Cq值和标准化值进行阈值设定时，性能相当。miR-371a-3p的实验室间一致性较高，但内参基因miR-30b-5p和cel-miR-39-3p不一致。在一组疑似隐匿性GCT的患者中，引入Cq值在28 - 35之间的不确定范围并对任何不确定结果进行重复检测，将检测准确性从0.84提高到了0.92。我们建议更新血清miR-371a-3p检测方案，以：a）使用基于原始Cq值的阈值方法；b）继续纳入内源性（如miR-30b-5p）和外源性非人掺入（如cel-miR-39-3p）微小RNA进行质量控制；c）对任何结果不确定的样本进行重新检测。
