Serum Levels of MicroRNA-371a-3p (M371 Test) as a New Biomarker of Testicular Germ Cell Tumors: Results of a Prospective Multicentric Study

Abstract

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.

Graded expression of microRNA-371a-3p in tumor tissues, contralateral testes, and in serum of patients with testicular germ cell tumor

“…As in advanced clinical stages the marker substance is released from both the primary tumor and metastatic seeds it is rational that the correlation of tissue expression with serum levels is only significant in cases confined to the testis. The serum level of circulating miR-371a-3p is obviously a product of the number of miR-producing tumor cells (tumor bulk) as shown previously [5] and of the specific secreting capacity of the individual GCT as shown herein. Most probably, additional biological determinants e. g. direct vascular invasion of the tumor and other hitherto unknown factors do also affect the serum level of miR-371a-3p.…”

“…Of note, all subtypes of GCT except teratoma stained positive for the miR-371a-3p probe. The absence of miR-371a-p staining in teratoma cells is consistent with the non-expression of this miR in serum of GCT patients [4,5,16]. Likewise, in an evaluation of miR-371a-3p expression levels in GCT subtypes by RNA extraction from formalin fixed paraffin embedded orchiectomy specimens, Vilela-Salgueiro et al revealed a strong expression in all GCT subtypes except teratoma [25].…”

“…As miR-371 has been shown to be specifically associated with human stem cells [28][29][30] and as it is found in seminal plasma, too [31,32], it is rational to assume that this miR is specifically generated by normal testicular germ cells and most probably even more by the cells of GCT. In serum, patients with nonseminomas showed significantly higher miR-371a-3p expression, and CS2/3 patients had higher levels than those with CS1 [5]. In tissue no difference was detectable between the miR-371a-3p expression of seminomas and nonseminomas nor between tissues of CS1 and CS 2/3 cases.…”

“…(4) In patients with localized disease (CS1), the GCT tissue miR-levels significantly correlate with the corresponding serum levels. (5) There is a baseline miR-371a-3p expression in tissues of contralateral testes of GCT patients and also in normal testes.…”

“…Serum levels of microRNAs (miRs) of the clusters miR-371-373 and miR-302/367 have been suggested as novel biomarkers of testicular germ cell tumors (GCTs) [1][2][3]. Of the candidate miRs, miR-371a-3p appears to be the most promising serum marker of GCT with a sensitivity of 90.1% and specificity of 94.1% [4,5] outperforming the classical markers (alpha fetoprotein, beta human chorionic gonadotropin, lactate dehydrogenase) with their sensitivities of less than 50% [6]. Apparently, miR-371a-3p features almost all of the qualities a valuable tumor marker is supposed to have [7] since it correlates with clinical stages, and tumor sizes, it highlights response (or non-response) to therapy, and it is present in cases with relapsing GCT suggesting a prominent role of this miR upon follow-up examinations [8][9][10][11][12][13][14][15].…”