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.

FIG 1.

Study profile. The diagram shows the selection process of patient enrollment. GCT, germ cell tumor.

FIG 2.

Relative expression of microRNA (miR)-371a-3p in patients with germ cell tumors (GCTs) and controls. (A) Box plots that represent miR-371a-3p expression in controls, patients with GCTs stratified for clinical stages (CSs) I, II, and III. (B) Differential expression of miR-371a-3p in seminoma (S) and nonseminoma (NS) in various CSs. (C) Marker expression in various histologic subtypes for CS I and CS II/III patients. Box plots represent S, mixed NS (mNS), embryonal carcinoma (EC), yolk sac tumor (YST), and teratoma (T). (D) Expression of miR-371a-3p in controls and patients with GCT recurrence. The y-axis is arranged in a logarithmic scale in all panels. (*) P < .05; (†) P < .01; (‡) P < .001.

FIG 3.

Discriminative ability of microRNA (miR)-371a-3p. (A) Receiver operating characteristic curves that discriminate controls (n = 258) from all patients with germ cell tumors (GCTs; n = 522; area under the curve [AUC], 0.966), clinical stage (CS) I only (n = 371; AUC, 0.953), or CS II/III only (n = 151; AUC, 0.996). (B) Sensitivity of miR-371a-3p in all GCTs (n = 522) compared with the classic GCT markers β-human chorionic gonadotropin (bHCG), α-fetoprotein (AFP), and lactate dehydrogenase (LDH) and all three classic markers combined. (C) Same comparison for CS I GCT only (n = 371). (D) Same comparison for CS II/III GCT only (n = 151). (E) Same comparison for seminoma only (n = 323). (F) Same comparison for nonseminoma only (n = 199). Error bars represent the 95% CI. (*) P < .001.

FIG 4.

Dependency of microRNA (miR)-371a-3p expression and sensitivity on tumor diameter. (A) Scatterplot that represents the relationship between tumor diameter and the miR-371a-3p expression of clinical stage (CS) I seminoma (circles; n = 259), CS I mixed nonseminoma (squares; n = 74), and CS I embryonal carcinomas (triangles; n = 29). Regression lines are depicted for all three groups in the corresponding color. The dashed line represents the cutoff value. (B) Sensitivity in various categories of tumor diameter of CS I seminoma. (C) Sensitivity in various categories of tumor diameter of CS I nonseminoma. Error bars indicate 95% CI.

FIG 5.

Post-treatment decrease of microRNA (miR)-371a-3p. (A) Decrease of miR-371a-3p expression after surgical removal of the primary tumor in clinical stage (CS) I (n = 316), CS II (n = 80), and CS III (n = 28) patients. (B) miR-371a-3p serum levels over the course of chemotherapy in CS II patients (n = 70). (C) miR-371a-3p serum levels over the course of chemotherapy in CS III patients (n = 46). (D) Decrease of miR-371a-3p expression in recurrent germ cell tumors (GCTs) after treatment (n = 29). Lines in panels B and C are interpolated through missing values. The y-axis in all panels is arranged on a logarithmic scale. (*) P < .001. (†) Patient with known disease progression and subsequent death.

FIG A1.

Receiver operating characteristic curve that discriminates clinical stage I patients (n = 371) from patients with systemic disease (n = 151) at an area under the curve of 0.76. A relative quantity cutoff value of 308 (highest Youden index) resulted in a sensitivity of 83.4% and a specificity of 60.1%.

FIG A2.

Linear regression analysis revealed an association of microRNA (miR)-371a-3p expression with pathologic tumor (pT) stage (R² = 0.664; P < .001).

FIG A3.

Diagram showing results of paired measurements of microRNA (miR)-371-a-3p levels (before and after orchiectomy) in clinical stage (CS) I patients. (*) Two of the nine patients had elevated levels of classic markers before and after surgery.

FIG A4.

Box plots of microRNA (miR)-371a-3p expression in patients with systemic disease before chemotherapy. Patients with International Germ Cell Cancer Collaborative Group (IGCCCG) poor prognosis have significantly higher levels than patients with good prognosis (P = .04).