ASCO GU 2022: Optimization of Serum miR-371a-3p for the Detection of Chemotherapy-Naïve Minimal Residual Germ Cell Tumor

( In a session on the third day of the American Society for Clinical Oncology (ASCO) Genitourinary Cancer Symposium 2022, Poster Session C focused on Renal Cell Cancer; Adrenal, Penile, Urethral, and Testicular Cancers. In this session, Dr. Lafin presented a poster highlighting approaches to maximize the utility of serum miR-371a-3p for the detection of chemotherapy-naïve minimal residual germ cell tumor.

Currently utilized serum tumors markers such as alpha-fetoprotein, beta-HCG, and LDH show relatively poor sensitivity and specificity for the detection of germ cell tumors (GCTs). However, while not yet in clinical use yet, circulating miRNAs, particularly miR-371a-3p, have exhibited strong performance characteristics in the pre-orchiectomy setting. Their ability to detect chemotherapy-naïve occult disease is less clear. In a small cohort of patients, these same authors showed that miR-371a-3p outperforms conventional markers in the context of chemotherapy-naïve minimal residual disease.

In this analysis, the authors examined over 250 assays to compare performance between classification based on raw Cq, Cq normalized to miR-30b-5p (∆Cq), or relative quantification to normal serum (RQ). We prospectively collected pre-surgical serum samples and clinical information from GCT patients undergoing primary retroperitoneal lymph node dissection (RPLND) at our institution.


Patients were classified based on histology as “Control” (pure teratoma or no GCT) or “GCT”. RNA was extracted from these samples, and miR-371a-3p (target) and miR-30b-5p (reference gene) were detected by qPCR. The test performance was quantified using sensitivity, specificity, and area under the ROC curve (AUC).

The authors found that the sensitivity, specificity, and AUC of miR-371a-3p did not significantly change based on thresholding metric (Cq, ∆Cq, or RQ).


However, they found that there was a 25% chance that any given control sample would return a false positive result based in part on a bimodal distribution of Cq values, and to combat this, the authors estimated an indeterminate range of Cq 28-35. Further, repeating any indeterminate sample once substantially reduced the size of the first peak in control samples and improved performance.


We then compared the performance of this updated process to a simple binary threshold in a cohort of patients with occult disease. 32 patients were enrolled in the study (15 Control, 17 GCT). 12 samples were indeterminate on first run (6 Control, 6 GCT). 8 remained indeterminate on a second run, all of which harbored teratoma or viable GCT. Inclusion of a second run prevented 3 false positives, improving specificity from 86% to 100% and yielding a sensitivity of 92% and AUC of 0.96 (95% CI: 0.89-1).


The authors, therefore, conclude that use of Cq instead of RQ to classify results for the miR-371a-3p test eliminates the need for a normalizing sample in each run without injuring assay performance. Further, in this setting of chemotherapy-naïve minimal residual germ cell tumor, consideration of an indeterminate range may improve the performance of the miR-371a-3p test.

Presented by: John T Lafin, PhD, University of Texas Southwestern Medical Center, Dallas, TX