Analytical Validation and Performance Characteristics of Molecular Serum Biomarkers, miR-371a-3p and miR-372-3p, for Male Germ Cell Tumors, in a Clinical Laboratory Setting.

Detection of serum embryonic miRNAs miR-371a-3p and miR-372-3p has been proposed to aid in diagnosis, prognosis, and management of patients with testicular germ cell tumors (GCTs). This study describes the analytical validation and performance of a laboratory-developed test to detect these miRNA targets by stem loop real-time quantitative RT-PCR (RT-qPCR) in serum from patients with GCTs. The assay was standardized using an exogenous spike-in control of nonhuman miRNA from Caenorhabditis elegans (cel-miR-39-3p) to assess extraction efficiency, and an endogenous housekeeping miRNA, miR-30b-5p, to control for miRNA normalization. miRNA results were expressed as relative expression level, using the comparative threshold cycle method (2ΔΔCT). Analytical sensitivity of miR-371a-3p and miR-372-3p was 12.5 and 1.25 copies/μL, respectively. Clinical accuracy was evaluated using GCT patients with (n = 34) and without (n = 17) active disease. Positive/negative cutoffs and indeterminate findings were established on the basis of results from healthy volunteers (n = 25) and assay precision. miR-371a-3p and miR-372-3p exhibited a sensitivity of 81.8% and 87.5%, respectively, and a specificity of 100% and 94%, respectively, and an area under the receiver operating characteristic curve of 0.93 and 0.95, respectively. Taken together, RT-qPCR testing for serum miR-371a-3p and miR-372-3p represents a robust, sensitive, and specific clinical assay to aid in the clinical management of patients with GCT.