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.
The Journal of molecular diagnostics : JMD. 2022 Aug [Epub]
Fei Ye, Darren R Feldman, Alisa Valentino, Rachel So, Maria Bromberg, Shah Khan, Samuel A Funt, Joel Sheinfeld, David B Solit, Melissa S Pessin, Ellinor I Peerschke
Department of Pathology and Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, New York. Electronic address: ., Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York; Department of Medicine, Weil Cornell Medical College, New York, New York., Department of Pathology and Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, New York., Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York., Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, New York., Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York; Department of Medicine, Weil Cornell Medical College, New York, New York; Kravis Center for Molecular Oncology, Memorial Sloan Kettering Cancer Center, New York, New York., Department of Pathology and Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, New York. Electronic address: .