Droplet Digital PCR and Quantitative RT-PCR Comparison for Circulating miR-371a-3p in Malignant Germ Cell Tumors

Previous work has shown that circulating miR-371a-3p has higher sensitivity and specificity than current biomarkers for malignant germ cell tumors (MGCTs). Herein, the performance of two methods commonly used to measure miR-371a-3p levels was compared: quantitative RT-PCR (RT-qPCR) and droplet digital PCR (ddPCR). Samples from the University of Texas Southwestern Medical Center (Dallas, TX) and the University of Cambridge (Cambridge, UK) were evaluated using both RT-qPCR and ddPCR, as per current protocols (RT-qPCR) or standard manufacturer's recommendations (ddPCR). Data were available for 70 individuals: 36 patients with MGCT and 34 control participants (nonmalignant GCT, non-GCT cancer, and noncancer). The performance of the two assays was compared using receiver operating characteristic curves and the area under the curve. Raw miR-371a-3p Cq values (RT-qPCR) were generally lower (ie, miR-371a-3p was more abundant) and the number of positive droplets (ddPCR) higher for patients with MGCT compared with control participants. The area under the curve (95% CI) was 0.94 (0.90-0.99) and 0.82 (0.70-0.93) when using RT-qPCR and ddPCR, respectively. Thus, RT-qPCR had better classification performance compared with ddPCR in the present cohort, supporting the continued use of RT-qPCR in standard clinical practice. Further investigation is required to optimize ddPCR before it should be considered for clinical adoption.