Refers to Fischer, S. et al. The value of tumour markers in the detection of relapse—lessons learned from the Swiss Austrian German Testicular Cancer Cohort study. Eur. Urol. Open Sci. 50, 57–60 (2023).

For many years, concern has existed among clinicians regarding the limited performance characteristics for the conventional serum tumour markers (STMs) α-fetoprotein (AFP), human chorionic gonadotrophin (HCG) and lactate dehydrogenase (LDH) for the effective follow-up monitoring of patients with testicular germ cell malignancy. From a sensitivity perspective, for example, STMs have been shown to be positive in only 3% of patients with stage I seminoma experiencing relapse after undergoing active surveillance; hence the reliance on cross-sectional imaging1. Regarding specificity, a small testicular cancer study demonstrated false-positive elevations in both AFP (12%) and HCG (14%) more than four decades ago2. Other historical reports have subsequently highlighted this risk for AFP, for example, owing to liver dysfunction3, and HCG, for example, owing to hypogonadism4.

A recent prospective report has formally evaluated these conventional STMs for the detection of relapse in the large Swiss Austrian German Testicular Cancer Cohort study (SAG TCCS)5. The SAG TCCS was established in 2014, and in the report STMs were assessed in 793 patients with full clinical information available through to July 2021, with a median follow-up duration of 29 months (range 13–50 months)5. A ‘false positive’ was defined as patients remaining free from an imaging-proven relapse for a minimum of 6 months following an incident of elevated STMs. By this definition, 124 patients (16%) of the overall cohort experienced a false-positive marker event at any time during the follow-up period5 (Table 1). Unfortunately, 27 of these patients (22%) had additional imaging performed; in the other 78% of men, clinicians did not consider the marker event suspicious or relevant, refraining from further investigations5, but also highlighting the disregard with which these conventional STMs can be viewed for follow-up monitoring of testicular cancer.

Table 1 Performance of the conventional serum tumour markers AFP, HCG and LDH in the detection of relapse in the SAG TCCS
Full size table

Overall, 71 (9%) of the total cohort of 793 patients experienced a proven relapse, of whom 31 (44%) were STM-positive and 40 (56%) were STM-negative5 (Table 1). Importantly, in only 18% of this relapse group (13 of 71 patients) were STMs considered the single, first, most relevant indicator of relapse5. Positive predictive values in follow-up monitoring of just 10.7%, 33.8% and 9.4% were reported for AFP, HCG and LDH, respectively5 (Table 1). The authors conclude that they found a high rate of false-positive events and that more than half of proven relapses were STM-negative5. As a result, the authors appropriately recommend caution in the interpretation of STMs in follow-up monitoring for patients with testicular cancer. In particular, owing to the possibility of non-specific elevations, they advocate serial STM measurements rather than immediately suspecting relapse and initiating additional investigations such as imaging5.

This large, prospective study unequivocally highlights the limitations of sensitivity and specificity for the conventional STMs for follow-up monitoring of testicular germ cell malignancy. The authors also conclude by highlighting the importance of current prospective studies of circulating microRNAs for such a follow-up role in this space5. A review in 2016 highlighted the potential of circulating microRNAs for this purpose, based on predominantly retrospective studies6. Since that time, circulating microRNAs have been studied prospectively and embedded as end points in open clinical trials, such as P3BEP (NCT02582697), AGCT1531 (NCT03067181), SWOG-S1823 and DRKS00019223 (ref. 7). While awaiting accrual to, and analysis of the results of, these large trials, results of a small prospective study have recently been reported8. In this study, 33 patients undergoing active surveillance for stage I testicular cancer were included, of whom 10 had a recurrence using standard follow-up investigations8. All 10 patients who experienced recurrence exhibited increasing circulating miR-371a-3p levels during follow-up monitoring, whereas miR-371a-3p levels remained non-elevated in all but 1 patient without recurrence8. Furthermore, circulating miR-371a-3p quantification enabled detection of recurrences at a median of 2 months (range 0–5 months) earlier than standard follow-up investigations8. The authors concluded that the circulating miR-371a-3p assay enables reliable recurrence detection earlier than standard follow-up investigations. These promising preliminary data need confirmation in larger prospective cohorts, but offer the real prospect of replacement of surveillance imaging in testicular cancer follow-up, particularly for seminoma.

Accordingly, further improvements and refinements of the circulating miR-371a-3p assay have been taking place to pave the way for such clinical implementation, particularly for testicular cancer follow-up monitoring, for which the assay needs to demonstrate the ability to sensitively detect occult (minimal residual) disease. A very recent study has produced data supporting the continued use of assessment of exogenous (cel-miR-39-3p) and endogenous (miR-30b-5p) microRNAs for quality control purposes, but not for data normalization, instead using direct circulating raw PCR Cq values for miR-371a-3p quantification9. In this study, interlaboratory concordance of miR-371a-3p was also shown to be high9. Of note, minimizing any false-positive microRNA assay results in this setting is crucial to avoid potential over-treatment of these patient cohorts. To this end, the study authors recognized a random and stochastic positive PCR event in a minority proportion of negative control samples, occurring in two independent laboratories9. Consequently, the investigators recommended, in addition to reporting positive (Cq < 28) and negative (Cq > 35) results, the introduction of an ‘indeterminate’ range for samples with a raw miR-371a-3p Cq value falling between these two Cq values, with a repeat run for any such sample. The majority of such repeat samples returned a negative result, with the minority remaining in the indeterminate range, and being reported as such. This refined protocol improved assay accuracy from 0.84 to 0.92 in a group of patients suspected of having occult testicular germ cell malignancy9. Further confirmation of this indeterminate range in other germ cell malignancy and healthy patient cohorts will provide the further necessary evidence of the meticulous planning and preparation required to safely transfer this novel biomarker technology to the clinic. Such circulating microRNA follow-up monitoring has been welcomed by patient and public involvement groups10. Compared with the conventional need for multiple CT scans in follow-up monitoring, particularly for patients with stage I seminoma, owing to the pick-up rate of recurrence of just 3% using current STMs, circulating microRNA testing was shown to offer increased sensitivity and safety, reduced financial costs, reduced time for testing and results, and reduced patient anxiety10.

In summary, Fischer and colleagues5 use the large, prospective SAG TCCS resource to highlight the limitations of the conventional STMs AFP, HCG and LDH for the follow-up monitoring of patients with testicular germ cell malignancy5. Continued refinements of the circulating microRNA (miR-371a-3p) assay are likely to result in clinical implementation for this indication in the near future, following further validation in prospective studies and clinical trials.