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Introduction and objective Conventional serum tumor markers (STMs) for testicular germ cell tumors (GCTs) offer limited performance with particularly poor sensitivity in cases of minimal residual disease and pure seminoma. While growing evidence has indicated miR‐371a‐3p to be a superior biomarker, its utility in detecting pure seminoma at recurrence has not been extensively explored. This study's objective was to explore miR‐371a‐3p's utility in detecting metastatic pure seminoma at retroperitoneal lymph node dissection (RPLND). Methods RNA was isolated from patient serum samples collected pre‐RPLND. Fifteen patients were assigned to our ‘benign’ (n = 6) or ‘seminoma’ (n = 9) group based on pathological confirmation of viable seminoma. Five of the patients received chemotherapy before RPLND (PC‐RPLND), and 10 were chemotherapy naïve. MiR‐371a‐3p expression was quantified via RT‐quantitative polymerase chain reaction. The Cq values were statistically evaluated to obtain performance measurements. Results Median relative expression of miR‐371a‐3p was higher in the Seminoma group than the Benign, but this difference was not statistically significant (Rq = 3705 and 241, respectively, p = 0.2844). Of the 10 chemotherapy naïve patients, nine had viable seminoma at RPLND, and seven had elevated miR‐371a‐3p expression. Among the five postchemotherapy (PC) patients, zero had viable GCT at RPLND, and two had elevated miR‐371a‐3p expression. The primary RPLND group presented 78% sensitivity and 100% specificity. Specificity in the PC‐RPLND group was 60%. An optimal Rq threshold of 28.62 was determined by Youden's J statistic, yielding 78% sensitivity and 67% specificity. Receiver operating characteristic analysis provided an AUC of 0.704 (95% CI: 0.43–0.98, p = 0.1949). Despite modest performance, miR‐371a‐3p exhibited improved sensitivity and specificity compared with conventional STMs. Conclusions MiR‐371a‐3p outperformed STMs in the primary RPLND settings. However, miR‐371a‐3p was not a robust predictor of pathology in the PC setting. These results suggest that pure seminoma at RPLND is a clinical context, wherein the miRNA assay may require further refinement.

location. Hilar and retrocaval suspicious lymphadenopathies are associated with higher concordance and increased risk of LNI. In consequence, performing a more extended LND for staging purposes in these patients may allow for a better risk classification and for a better planning of adjuvant treatments.

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Circulating miR-371a-3p has excellent performance in the detection of viable (non-teratoma) germ cell tumor (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 vs. 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.