A Multi-institutional Pooled Analysis Demonstrates That Circulating miR-371a-3p Alone is Sufficient for Testicular Malignant Germ Cell Tumor Diagnosis

Published:September 14, 2021DOI:



      Circulating microRNAs have clear potential for improving malignant germ-cell-tumor (MGCT) diagnosis. Here, we address the central issue of whether measurement of a single microRNA is sufficient for detecting testicular MGCTs, or whether there is added benefit in quantifying other members of the 4-microRNA panel previously identified (miR-371a-3p/miR-372-3p/miR-373-3p and miR-367-3p).

      Patients and Methods

      We performed a pooled analysis of available published raw data where all 4 panel miRNAs had been assessed using pre-amplification PCR technology (4 studies; total 329 patients). Two studies using identical methodology (and identical normalization using endogenous miR-30b-5p) were used in the discovery phase (n = 51 patients: 17 MGCT, 34 controls). The 2 other studies (n = 278 patients: 140 MGCT, 138 controls), which assessed the same test panel but with different normalization approaches (endogenous miR-93-5p, exogenous cel-miR-39-3p), were used for the validation phase. We derived sensitivity, specificity, positive- and negative-predictive-values (PPV/NPV) for the detection thresholds that maximised the Youden Index (YI).


      In the discovery-phase, the YI was 0.97 for miR-371a-3p (sensitivity = 1, specificity = 0.97), 0.71 (miR-367-3p), 0.68 (miR-372-3p), and 0.50 (miR-373-3p). These findings were confirmed in the validation-phase, with YI of 0.75 for miR-371a-3p (sensitivity = 0.90, specificity 0.85), 0.55 (miR-367-3p), 0.47 (miR-372-3p), and 0.51 (miR-373-3p). Importantly, no combination of markers added additional diagnostic benefit to miR-371a-3p alone, in either the discovery or the validation phase.


      Quantifying circulating miR-371a-3p alone is sufficient for testicular MGCT diagnosis. PCR measurement of this single miRNA marker will be more cost-effective and easier to interpret, facilitating future incorporation into routine clinical practice.



      AFP (alpha-fetoprotein), AUC (area-under-the-curve), CNS (central nervous system), Cq (cycle threshold), HCG (human-chorionic-gonadotrophin), LDH (lactate dehydrogenase), MGCT (malignant germ-cell-tumor), miRNA (microRNA), NPV (negative-predictive-value), PCR (polymerase chain reaction), PPV (positive-predictive-value), UTSW (University of Texas Southwestern Medical Center, Dallas, TX, US), Youden Index (YI)
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        • Murray M.J.
        • Huddart R.A.
        • Coleman N.
        The present and future of serum diagnostic tests for testicular germ cell tumours.
        Nat Rev Urol. 2016; 13: 715-725
        • Howlader N.
        • Noone A.M.
        • Krapcho M.
        • et al.
        SEER Cancer Statistics Review, 1975-2017.
        National Cancer Institute, BethesdaMD2020 (Based on November 2019 SEER data submission]. Available from)
        • Murray M.J.
        • Coleman N.
        Can circulating microRNAs solve clinical dilemmas in testicular germ cell malignancy?.
        Nat Rev Urol. 2019; 16: 505-506
        • Gilligan T.D.
        • Seidenfeld J
        • Basch EN
        • et al.
        American society of clinical oncology clinical practice guideline on uses of serum tumor markers in adult males with germ cell tumors.
        J Clin Oncol. 2010; 28: 3388-3404
        • Almstrup K.
        • Lobo J
        • Mørup N
        • et al.
        Application of miRNAs in the diagnosis and monitoring of testicular germ cell tumours.
        Nat Rev Urol. 2020; 17: 201-213
        • Mitchell P.S.
        • Parkin RK
        • Kroh EM
        • et al.
        Circulating microRNAs as stable blood-based markers for cancer detection.
        Proc Natl Acad Sci U S A,. 2008; 105: 10513-10518
        • Palmer R.D.
        • Murray MJ
        • Sani H
        • et al.
        Malignant germ cell tumors display common microRNA profiles resulting in global changes in expression of messenger RNA targets.
        Cancer Res. 2010; 70: 2911-2923
        • Murray M.J.
        • Halsall DJ
        • Hook CE
        • et al.
        Identification of MicroRNAs from the miR-371∼373 and miR-302 clusters as potential serum biomarkers of malignant germ cell tumors.
        Am J Clin Pathol. 2011; 135: 119-125
        • Gillis A.J.
        • Rijlaarsdam MA
        • Eini R
        • et al.
        Targeted serum miRNA (TSmiR) test for diagnosis and follow-up of (testicular) germ cell cancer patients: a proof of principle.
        Mol Oncol. 2013; 7: 1083-1092
        • Syring I.
        • Bartels J
        • Holdenrieder S
        • et al.
        Circulating Serum miRNA (miR-367-3p, miR-371a-3p, miR-372-3p and miR-373-3p) as biomarkers in patients with testicular germ cell cancer.
        J Urol. 2015; 193: 331-337
        • Murray M.J.
        • Bell E
        • Raby KL
        • et al.
        A pipeline to quantify serum and cerebrospinal fluid microRNAs for diagnosis and detection of relapse in paediatric malignant germ-cell tumours.
        Br J Cancer. 2016; 114: 151-162
        • Dieckmann K.P.
        • Radtke A
        • Spiekermann M
        • et al.
        Serum levels of MicroRNA miR-371a-3p: a sensitive and specific new biomarker for germ cell tumours.
        Eur Urol. 2017; 71: 213-220
        • Lafin J.T.
        • Singla N
        • Woldu SL
        • et al.
        Serum microRNA-371a-3p levels predict viable germ cell tumor in chemotherapy-naive patients undergoing retroperitoneal lymph node dissection.
        Eur Urol. 2020; 77: 290-292
        • Dieckmann K.P.
        • Radtke A
        • Geczi L
        • et al.
        Serum levels of microRNA-371a-3p (M371 Test) as a new biomarker of testicular germ cell tumors: results of a prospective multicentric study.
        J Clin Oncol. 2019; 37: 1412-1423
        • Ricardo Leão R
        • van Agthoven T
        • Figueiredo A
        • et al.
        Serum miRNA Predicts Viable Disease after Chemotherapy in Patients with Testicular Nonseminoma Germ Cell Tumor.
        J Urol. 2018; 200: 126-135
        • Mego M.
        • van Agthoven T
        • Gronesova P
        • et al.
        Clinical utility of plasma miR-371a-3p in germ cell tumors.
        J Cell Mol Med. 2019; 23: 1128-1136
        • Nappi L.
        • Thi M
        • Lum A
        • et al.
        Developing a highly specific biomarker for germ cell malignancies: plasma miR371 expression across the germ cell malignancy spectrum.
        J Clin Oncol. 2019; 37: 3090-3098
        • Daniel Charytonowicz D.
        • Aubrey H
        • Bell C
        • et al.
        Cost analysis of noninvasive blood-based microRNA testing versus CT scans for follow-up in patients with testicular germ-cell tumors.
        Clin Genitourin Cancer. 2019; 17: e733-e744
        • Aditya Bagrodia A.
        • Savelyeva A
        • Lafin JT
        • et al.
        Impact of circulating microRNA test (miRNA-371a-3p) on appropriateness of treatment and cost outcomes in patients with Stage I non-seminomatous germ cell tumours.
        BJU Int. 2021; 128: 57-64
        • Fern L.A.
        • Greenwood M
        • Smith S
        • et al.
        Pre-implementation assessment of the acceptability of using circulating microRNAs for follow-up of malignant germ-cell tumors.
        Clin Genitourin Cancer. 2021 Mar 17; (S1558-7673(21)00068-9Online ahead of print)
        • Youden W.J.
        Index for rating diagnostic tests.
        Cancer. 1950; 3: 32-35
        • Hindson C.M.
        • Chevillet JR
        • Briggs HA
        • et al.
        Absolute quantification by droplet digital PCR versus analog real-time PCR.
        Nat Methods. 2013; 10: 1003-1005