Refining the serum miR-371a-3p test for viable germ cell tumor detection: identification and definition of an indeterminate range

• Published in: Research square
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: United States 21.03.2023
• 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.