Refining the serum miR-371a-3p test for viable germ cell tumor detection: identification and definition of an indeterminate range

Abstract

Circulating miR-371a-3p has excellent performance in the detection of viable (non-teratoma) GCT pre-orchiectomy; however, its ability to detect occult disease is understudied. To refine the serum miR-371a-3p assay in the minimal residual disease setting we compared performance of raw (Cq) and normalized (∆Cq, RQ) values from prior assays, and validated interlaboratory concordance by aliquot swapping. Revised assay performance was determined in a cohort of 32 patients suspected of occult retroperitoneal disease. Assay superiority was determined by comparing resulting receiver-operator characteristic (ROC) curves using the Delong method. Pairwise t-tests were used to test for interlaboratory concordance. Performance was comparable when thresholding based on raw Cq vs. normalized values. Interlaboratory concordance of miR-371a-3p was high, but reference genes miR-30b-5p and cel-miR-39-3p were discordant. Introduction of an indeterminate range of Cq 28-35 with a repeat run for any indeterminate improved assay accuracy from 0.84 to 0.92 in a group of patients suspected of occult GCT. We recommend that serum miR-371a-3p test protocols are updated to a) utilize threshold-based approaches using raw Cq values, b) continue to include an endogenous (e.g., miR-30b-5p) and exogenous non-human spike-in (e.g., cel-miR-39-3p) microRNA for quality control, and c) to re-run any sample with an indeterminate result.

Figure 1. Raw miR-371a-3p Cq is concordant across laboratory sites.

A: Comparison of raw Cq values for cel-miR-39-3p (external spike-in control), miR-30b-5p (internal reference gene), and miR-371a-3p at the US and UK sites. B: Boxplot of Cq differences (US – UK).

Figure 2. Establishment of indeterminate range in the serum miR-371a-3p test.

A-B: Density plots of serum miR-371a-3p Cq values (n = 81) under original (A) or revised (B) method. Shaded region corresponds to indeterminate range (Cq 28-35). C: ROC plot showing performance of original (solid line) and revised (dashed line) methodology. D: Comparison of sensitivity, specificity, positive predictive value, and negative predictive value across all measured Cq thresholds using original (solid line) and revised (dashed line) methodology.

Figure 3. Revised method improves performance of serum miR-371a-3p test in patients with minimal residual disease.

A-B: Cq values of serum miR-371a-3p using original (A) and revised (B) methodology. C: ROC plot showing performance of original (solid line) and revised (dashed line) methodology. D: Comparison of sensitivity, specificity, positive predictive value, and negative predictive value across all measured Cq thresholds using original (solid line) and revised (dashed line) methodology.

Figure 4. Decision-making flowchart for revised serum miR-371a-3p method.

First, results of miR-30b-5p are evaluated for quality control. If Cq > 30, insufficient RNAs may have been isolated from the serum sample, and this sample should be reextracted. If Cq < 30, proceed to evaluation of miR-371a-3p. If Cq < 28, accept positive result. If Cq > 35, accept negative result. If 28 < Cq < 35, assay should be repeated from reverse transcription (RT) step. If Cq < 28 or Cq > 35, accept results as above. If 28 < Cq < 35 again, report indeterminate and recommend short interval follow up.