Droplet-Digital and Reverse Transcription Quantitative 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 (MGCT). Here, the performance of two methods commonly used to measure miR-371a-3p levels was compared: reverse transcription quantitative PCR (RT-qPCR) and droplet-digital PCR (ddPCR). Samples from the University of Texas Southwestern (UTSW) Medical Center and the University of Cambridge (UoC) 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 patients, comprising 36 patients with MGCT and 34 controls (non-malignant GCT, non-GCT cancer, and non-cancer controls). The performance of the two assays was compared using receiver operating characteristic (ROC) curves and the area under the curve (AUC). Raw miR-371a-3p Cq values (RT-qPCR) were generally lower (i.e., miR-371a-3p was more abundant) and the number of positive droplets (ddPCR) higher for MGCT patients compared with controls. The AUC was 0.94 (95% CI: 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 current 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.