PURPOSE: Previous studies suggested that serum levels of microRNA (miR)-371a-3p (so-called M371 test) have a much higher sensitivity and specificity than the classic markers of testicular germ cell tumors (GCTs) and are applicable toward both seminoma and nonseminoma. We sought to confirm the usefulness of this test as a novel biomarker for GCT. PATIENTS AND METHODS: In a prospective, multicentric study, serum samples of 616 patients with testicular GCTs and 258 male controls were examined for serum levels of miRNA-371a-3p (miR levels) by quantitative polymerase chain reaction. The GCT population encompassed 359 patients with seminoma and 257 with nonseminoma; 371 had clinical stage I disease, 201 had systemic disease, and 46 had relapses. Paired measurements before and after orchiectomy were performed in 424 patients; 118 with systemic disease had serial measurements during treatment. miR levels were compared with those of β-human chorionic gonadotropin, α-fetoprotein, and lactate dehydrogenase. RESULTS: For the primary diagnosis of GCT, the M371 test showed a sensitivity of 90.1%, a specificity of 94.0%, an area under the curve of 0.966 upon receiver operating characteristic analysis, and a positive predictive value of 97.2%. α-Fetoprotein, β-human chorionic gonadotropin, and lactate dehydrogenase had sensitivities of less than 50% in seminoma and slightly higher sensitivities in nonseminomas. miR levels were significantly associated with clinical stage, primary tumor size, and response to treatment. Relapses had elevated miR levels that subsequently dropped to normal upon remission. Teratoma did not express miR-371a-3p. CONCLUSION: The M371 test outperforms the classic markers of GCT with both a sensitivity and a specificity greater than 90%. All histologic subgroups, except teratoma, express this marker. The test could be considered for clinical implementation after further validation.

Clinical management of germ cell tumours (GCTs) relies on monitoring of serum tumour markers. However, the markers α-fetoprotein (AFP), the β-subunit of human chorionic gonadotropin (bHCG), and lactate dehydrogenase (LDH) are expressed in <60% of GCT cases. To test the utility of the microRNAs (miRNAs) miR-371a-3p, miR-372-3p, miR-373-3p, and miR-367-3p as sensitive and specific GCT serum biomarkers. Serum levels of miRNAs were measured in 166 consecutive patients with GCT before and after treatment and in 106 male controls. In the first 50 consecutive patients, all four miRNAs were measured. In the main study, only the most sensitive miRNA was further analysed. The specificity and sensitivity of the four miRNAs were studied using receiver operating characteristic curves. miRNA sensitivities were compared to those of classical markers. Statistical cross-comparisons of miRNA levels for GCT subgroups and controls were performed at various time points during treatment. Overall, miR-371a-3p performed best, with 88.7% sensitivity (95% confidence interval [CI] 82.5-93.3%) and 93.4% specificity (95% CI 86.9-97.3%) and an area under the curve of 0.94, outperforming AFP, bHCG, and LDH (combined sensitivity 50%). According to Kernel density estimation, the sensitivity and specificity were 86.3% and 92.5%, respectively. miR-371a-3p levels dropped to normal after completion of treatment. The miRNA levels correlated with treatment failure and relapse. Teratoma did not express miR-371a-3p. The miRNA miR-371a-3p is a specific and sensitive novel serum GCT biomarker that accurately correlates with disease activity. Validation of this test in a large-scale prospective study is needed. PATIENT SUMMARY: miR-371a-3p is a novel serum marker for germ cell tumours that is expressed by 88.7% of patients and thus is far more sensitive and specific than classical serum markers. It correlates with tumour burden and treatment results. Validation in a large patient cohort is needed.

Background: miRNAs are small noncoding RNA molecules that can be released into body fluids. Germ cell tumours (GCTs) overexpress miRNAs of the miR-371-3 cluster. Thus, serum levels of these miRNAs may correlate with tumour load. Methods: miRNAs of the miR-371-3 cluster were quantified in cubital vein blood samples of 20 GCT patients with clinical stage 1, and of 4 patients with advanced stages before and after treatment. In six patients testicular vein blood (TVB) was examined additionally. Seventeen healthy males served as controls. Likewise, expression of miRNAs in 15 matching tumour specimens was measured. Results: In all patients, serum levels of miRNAs 371-3 were much higher than in controls. In stage 1, levels decreased postoperatively 336.7-fold, 7.4-fold, and 7.7-fold for miRNAs 371a-3p, 372, and 373-3p, respectively (P<0.01). Also, in those cases with advanced disease levels dropped to the normal range after completion of treatment. miR-371-3 levels in TVB exceeded those in peripheral blood in all cases. Expression of miR-371a-3p was also documented in tumour tissue. However, no correlation was found regarding the extent of miRNA expression in tissue and the values measured in matching serum. Conclusion: Thus, miR-371a-3p serum level appears to be a useful biomarker in GCTs.

Introduction. Although serum tumor markers beta human chorionic gonadotropin (bHCG), alpha-fetoprotein (AFP), and lactate dehydrogenase (LDH) are well-established tools for the management of testicular germ cell tumours (GCTs), there are only few data from contemporary cohorts of primary GCT patients regarding these biomarkers. Our aim was to evaluate marker elevations in testicular GCTs and to document their associations with various clinical characteristics.Patients and Methods. A total of 422 consecutive patients with GCTs were retrospectively analysed regarding serum levels of bHCG, AFP, and LDH during the course of treatment. Additionally, the following characteristics were recorded: histology, age, laterality, clinical stage (CS), pT-stage, and tumour size. Marker elevations were first tabulated in dichotomized way (elevated: yes/no) in various subgroups and second as continuous measured serum values. Descriptive statistical methods were employed to look for differences among subgroups and for associations of elevations with clinical parameters.Results. In all GCT patients, the frequencies of elevated levels of bHCG, AFP, LDH, and bHCG or AFP were 37.9%, 25.6%, 32.9%, and 47.6%; in pure seminomas 28%, 2.8%, 29.1%, and 30.3%; and in nonseminoma 53.0%, 60.1%, 38.7%, and 73.8%. Significant associations were noted with pT-stages >pT1, clinical stages >CS1, tumour size, and younger age. Frequencies of marker elevations dropped significantly after treatment, but LDH levels remained elevated in 30.5%-34.1%. Relapsing patients (n=27) had elevated levels of bHCG, AFP, and LDH in 25.9%, 22.2%, and 29.6%, respectively, thirteen of whom with a changed marker pattern.Conclusions. The classical GCT-biomarkers correlate with treatment success. Clinical utility is limited due to proportions of < 50% of patients with elevated levels and the low specificity of LDH. The elevation rates are significantly associated with histology, clinical and pT-stages, tumour size, and younger age. Individual marker patterns may change upon relapse. Clinically, ideal biomarkers are yet to be found.