Developing a Highly Specific Biomarker for Germ Cell Malignancies: Plasma miR371 Expression Across the Germ Cell Malignancy Spectrum

Abstract

Purpose: Our objective was to evaluate operating characteristics, particularly specificity and positive predictive value (PPV), by mapping plasma miR371 expression to actual clinical events in patients with a history of germ cell tumor.

Patients and methods: One hundred eleven male patients with a history of or newly diagnosed germ cell tumors were evaluable. Biospecimens obtained before confirmed clinical events were analyzed for miR371 expression with blinding of providers and laboratory personnel to analytic results or clinical status, respectively. Cases (patients with clinically confirmed active germ cell malignancy [aGCM]) and controls (patients with no clinically confirmed aGCM) were assigned over the course of the management. Patients were assigned risk status (high, low, or moderate) based on the composite clinical picture at time points in management.

Results: Considering all cases and controls and results of prospectively obtained biosamples analyzed for miR371 expression, 46 (35%) of 132 samples had clinically confirmed aGCM over the course of management; 44 (96%) of these 46 patients had plasma miR371 expression (true positives) with no false positives. Two (4%) of 46 patients had no miRNA expression despite pathologic confirmation of aGCM (false negatives). Plasma miR371 expression in confirmed aGCM had a specificity, sensitivity, positive predictive value, and negative predictive value of 100%, 96%, 100%, and 98%, respectively. Interpretation of sensitivity and negative predictive value is limited by modest follow-up. Specificity and sensitivity were 100% and 98%, 100% and 92%, and 100% and 97% in the low-, moderate-, and high-risk groups, respectively, with a median follow-up time of 15 months.

Conclusion: Plasma miR371 expression predicts aGCM with high specificity and positive predictive value. Although other operating characteristics of miR371 await longer follow-up for more complete definition, the findings of a highly specific liquid biopsy strongly support moving forward with large-scale, real-world clinical trials to further define full operating characteristics and to identify clinical utility and areas of patient benefit.

FIG 1.

Clinical details of patients with true-negative microRNA results. Three patients presented with obvious clinical signs of germ cell tumor but no clinical confirmation of active germ cell malignancy (aGCM) and true-negative miR371 results. The first patient (from the top) was older, had pathologically confirmed testicular seminoma, and developed enlarged retroperitoneal lymph nodes (stage IIA) with negative tumor markers and confirmed growing pattern of the tumor by two sequential computed tomography scans. miR371 was checked after orchiectomy and was negative. A biopsy of the retroperitoneal lymph nodes revealed the presence of a follicular lymphoma. The second patient was diagnosed with extensive metastatic germ cell tumor. The pathology of the primary testis cancer showed a transformed teratoma; lactate dehydrogenase (LDH) was the only elevated tumor marker, and the miR371 was negative. After the chemotherapy, the residual tumor was resected, and the pathology showed only transformed teratoma. The third patient had a large mediastinal mass. A biopsy showed fibrosis, and he had low positive α-fetoprotein (AFP; 13 µg/L). miR371 was negative. After chemotherapy, his AFP was stably low (16 µg/L), and the pathology of the residual mass showed fibrosis only. RPLND, retroperitoneal lymph node dissection.

FIG 2.

Schema of the study. Patients with germ cell malignancy from BC Cancer in Vancouver and the Huntsman Cancer Institute in Salt Lake City, Utah, were enrolled in the study. One patient had blood collected for miR371 before starting chemotherapy but not before surgery for residual viable disease and was considered unevaluable. The patient samples were divided into the following three risk categories, according to the probability of harboring active germ cell malignancy (aGCM): low risk (5% to 25% risk), which included patients with postorchiectomy clinical stage (CS) I seminoma and CS IA nonseminoma with no suspicious signs of relapse on surveillance and patients with or without residual radiologic disease after chemotherapy and normal tumor markers (TMs); moderate risk (25% to 50% risk), which included patients with postorchiectomy CS IB nonseminoma with no suspicious finding of relapse, patients with CS I seminoma and nonseminoma with clinical signs of suspicious relapse on surveillance, and patients with low positive TMs after chemotherapy; and high risk (90% to 100% risk), which included patients with gross clinical metastatic germ cell tumors before starting chemotherapy, patients with testicular mass before orchiectomy, patients with CS IS, and patients with obvious viable (high positive TMs) residual disease after chemotherapy.

FIG 3.

miR371 is expressed in patients with clear evidence of active germ cell malignancy. Plasma miR371 was evaluated in the 132 specimens divided as cases and controls, as reported in Table 1. (A) miR371 cycle threshold (Ct) raw values were used to qualitatively assess miR371 expression, using a cutoff value of 40 Ct (dashed line). (B) miR371 expression was quantified as fold of expression increase relative to the negative controls, after normalization for miR-30b-5p, miR39-3p, and miR-451. The black arrows indicate the two patients with false-negative results. aGCM, active germ cell malignancy.

FIG 4.

Areas under the receiver operating characteristic curve of miR371, computed tomography (CT) scan, and the classic germ cell tumor markers α-fetoprotein (AFP), β-human chorionic gonadotropin (β-HCG), and lactate dehydrogenase (LDH) of (A) the whole cohort of samples (n = 132) and (B) samples from the moderate-risk group. The patients were classified as cases or controls, as reported in Table 1.