Parallel liquid biopsy analysis of miR-371a-3p and cell-free DNA in testicular germ cell tumors.

614 Background: miR-371a-3p has emerged as a novel biomarker in testicular germ cell tumors (TGCTs), showing strong potential to improve diagnosis and disease monitoring. Despite robust supporting data, miRNA testing has not yet been implemented in routine clinical practice. Cell-free DNA (cfDNA) represents another promising liquid biopsy biomarker that may provide complementary molecular information and enhance the clinical utility of miR-371a-3p. Methods: Prospectively collected samples from a representative unselected cohort of TGCT patients treated at a single center were evaluated for both miR-371a-3p and cfDNA levels. In total, 109 patient samples were analyzed: 48 at diagnosis, 6 at relapse, 33 during follow-up, and 22 during the disease and treatment course. Controls included 11 healthy adult donors. cfDNA concentrations were measured by qPCR using albumin as the housekeeping gene. miR-371a-3p was detected by an optimized commercial assay and normalized to miR-30b-5p as a control. Results: miR-30b-5p was detectable in 102 of 109 (94%) patient samples, which were then further analyzed for miR-371a-3p. miR-371a-3p was present in 19 of these 102 (19%) samples. It was significantly more frequently detected in patients with clinically manifest disease (15 of 38 samples, 39%) than in those without clinical evidence of disease (4 of 64 samples, 6%), p = 0.0001, representing the sensitivity of 39% and specificity of 94%, with ROC AUC 0.67. There was no difference in miR-371a-3p positivity between patients with seminomas vs. non-seminomas, but it was more frequently found in patients with elevated conventional serum tumor markers (10 of 29 samples, 34%) than in those with normal markers (9 of 73 samples, 12%), p 0.05). Conclusions: miR-371a-3p was associated with active or metastatic TGCT and was largely undetectable during remission. Despite its strong potential as a universal biomarker for TGCT—with the exception of mature teratoma—a considerable number of false-negative and false-positive results remain. Detailed analysis of these outliers may help identify biological factors underlying discordant results and guide further refinement of the assay. The lack of association between cfDNA concentration and miR-371a-3p relative expression suggests that levels of these biomarkers may provide independent information on TGCT biology.