MicroRNA profiling of testicular Leydig cell tumors identifies a microRNA signature associated with malignancy and miR-196b-5p as a potentially useful biomarker

: Approximately 10% of testicular Leydig cell tumors (LCTs) are clinically malignant and unresponsive to systemic treatment. Predicting their clinical behavior can be problematic because there are no biomarkers that can consistently discriminate between benign and malignant LCTs. We assessed microRNA expression profiles of LCTs to identify differentially expressed microRNAs that could potentially distinguish benign from malignant neoplasms. The study consisted of two phases. In the first (discovery) phase, we interrogated 768 microRNAs in a series of 11 LCTs (six malignant and five benign) using Taqman Low-Density Array (TLDA) microRNA profiling. In the second phase, we validated the top differentially expressed microRNA targets with real-time quantitative PCR on a series of 35 LCTs (17 malignant and 18 benign), assessing their clinical performance for distinguishing malignant from benign LCTs. Target biologic pathways were analyzed using the miRTargetLink 2.0 tool. A total of 50 microRNAs were differentially regulated in malignant LCTs (27 upregulated, 23 downregulated). The top six microRNA candidates (top three upregulated and top three downregulated) were validated, showing good performance for discriminating between malignant and benign LCTs, with an area under the curve (AUC) ranging between 0.69 and 0.87. MiR-196b-5p showed the best performance, with sensitivity, specificity, negative predictive value, positive predictive value, and accuracy of 82%, 83%, 83%, 82%, and 83%, respectively. A panel (i.e. combined) analysis reached 100% sensitivity and 83% specificity. Pathway analysis revealed significant overlap in the biological process targeted by the upregulated microRNAs in malignant LCTs, including proliferation, development, metabolism, hormone synthesis, and cell death. Our results support the idea that malignant LCTs are associated with a distinct microRNA signature. MiR-196b-5p was identified as a potentially useful biomarker to distinguish benign from malignant tumors. The shared downstream targets of the top upregulated microRNAs suggest that dysregulation of cell proliferation and apoptosis underlie aggressive biologic behavior in LCTs and may offer opportunities for targeted therapies. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.