Cut-offs for relapse detection in men with stage I testicular germ cell tumors during active surveillance within a prospective multicentre cohort study using either raw or housekeeper normalized miR-371a-3p serum levels

Purpose

MiR-371a-3p represents a novel liquid biomarker that detects all histologies of germ-cell tumors (GCT) except teratoma. However, it is currently unclear whether miR-371a-3p results obtained directly from RT-PCR (raw Cq) or normalized for housekeeper genes and transformed into the relative quantity (RQ) value should be used and at what cut-off level. The purpose of this research was to evaluate, which values should be used, and a potential cut-off level for relapse-detection to inform subsequent studies.

Materials and Methods

We applied a CE-certified qRT-PCR test to measure miR-371a-3p at each follow-up visit during active surveillance in 34 men with stage I testicular GCT. MiR-371a-3p levels were calculated by the ΔΔ method.

Results

About 18 Patients had pure seminoma and 16 had mixed or nonseminomatous testicular GCT. Recurrences were detected in 10 patients and were correctly identified by both raw and housekeeper-normalized miR-371a-3p serum levels. The raw Cq, with a cut-off value of <28, resulted in only 1 false positive (3%), whereas RQ, with a cut-off value of >15, produced 6 false positive results (17%). Most of these false positive results normalized in subsequent measurements. The RQ approach detected recurrence in 1 patient 6 months earlier than the raw Cq approach.

Conclusion

Our preliminary data suggest that this CE-certified assay, using previously suggested cut-off values, is a promising method for detecting disease recurrence, provided a confirmatory second test is conducted to identify false positive results. To avoid unnecessary scans or overtreatment, we are currently validating this assay and cut-offs in a prospective cohort study.