MicroRNA miR-371a-3p - A Novel Serum Biomarker of Testicular Germ Cell Tumors: Evidence for Specificity from Measurements in Testicular Vein Blood and in Neoplastic Hydrocele Fluid.

BACKGROUND: microRNAs (miRs)-371-3 are suggested to be novel biomarkers of germ cell tumors (GCTs), but their specificity is unresolved. We aimed at clarifying the origin of miR 371a-3p by measuring this miR in peripheral vein blood, and in fluids present in the vicinity of GCTs. METHODS: miR-371a-3p levels were measured by quantitative PCR in 9 tumor surrounding hydroceles and in cubital vein blood (CVB) and testicular vein blood (TVB) of 64 GCT patients, 51 with clinical stage (CS) 1, 13 with CS2-3. Thirty three CS1 cases had also postoperative CVB measurement. TVB miR levels were compared with those of CVB. Associations with clinical factors were analyzed statistically. RESULTS: TVB miR levels were 294-fold, 80-fold and 4.6-fold higher than those in CVB of CS1 patients, CS2-3 patients and controls, respectively. Neoplastic hydrocele fluid comprised of very high miR levels. In CS1, miR levels dropped to normal postoperatively. Statistically, CVB miR levels are significantly associated with tumor size (p = 0.0211) and testis length (p = 0.0493). TVB miR levels are associated with testis length (p = 0.0129). CONCLUSIONS: This study provides evidence for the origin of circulating miR 371a-3p molecules from GCT cells. miR-371a-3p represents a specific serum biomarker for germ cell cancer.

The expression of miRNA encoded by C19MC and miR-371-3 strongly varies among individual placentas but does not differ between spontaneous and induced abortions.

miRNAs of the largest human miRNA gene cluster at all, i.e., C19MC, are almost exclusively expressed in the placenta. Nevertheless, only little is known about the interindividual variation of their expression and even about possible influence of gestational age, conflicting data is reported as well as for miRNAs of the much smaller miR-371-3 cluster. Our present study aims at the analyses of the expression of miRNAs from both clusters at different times of pregnancy, possible differences between placenta samples obtained from spontaneous or induced abortions in the first trimester, and the possible variation of miRNA expression at different sites within same placentas. miR-371a-3p, miR-372-3p, miR-373-3p, miR-517a-3p, and miR-520c-3p were quantified in 85 samples and miR-371a-3p was quantified in maternal serum samples taken immediately before delivery. While for miRNA-517a-3p and miR-520c-3p the expression increased with increasing gestational age, the present study revealed strong interindividual differences in the expression of miR-371-3 in full-term placental tissue as well as for miRNAs of the C19MC cluster, where the levels differed to a much lesser extent than for the former microRNAs. Also, strong interindividual differences were noted between the serum samples but differences related to the site of the placenta where the sample has been taken from were excluded. For neither of the data from placental tissue, the study revealed differences between the spontaneous and induced abortion group. Thus, the differences do not in general seem to be related to first trimester abortion. It remains to be elucidated whether or not they affect other prenatal processes.

Serum Levels of MicroRNA miR-371a-3p: A Sensitive and Specific New Biomarker for Germ Cell Tumours.

BACKGROUND: Clinical management of germ cell tumours (GCTs) relies on monitoring of serum tumour markers. However, the markers α-fetoprotein (AFP), the ß-subunit of human chorionic gonadotropin (bHCG), and lactate dehydrogenase (LDH) are expressed in <60% of GCT cases. OBJECTIVE: 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. DESIGN, SETTING, AND PARTICIPANTS: 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. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: 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. RESULTS AND LIMITATIONS: 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. CONCLUSIONS: 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.

Expression of microRNAs of C19MC in Different Histological Types of Testicular Germ Cell Tumour.

BACKGROUND: Testicular germ cell tumours (TGCTs) are the most common tumours in men aged from 20 to 40 years, with a steadily increasing incidence. This study aimed to characterize the expression of the miRNA cluster C19MC in TGCT and to evaluate the suitability of a C19MC miRNA as a serum biomarker. MATERIALS AND METHODS: By quantitative reverse transcription PCR, we measured the expression of miR-517a-3p, miR-519a-3p, and miR-519c 3p in tissue samples of 25 TGCTs and the level of miR-517a-3p in serum samples obtained pre- and postoperatively from the same patients. RESULTS: We detected a significantly higher expression of C19MC miRNAs in non-seminomas than in seminomas and in clinical stages 2 and 3 than in stage 1 in both tissue and serum samples. CONCLUSION: miRNAs of C19MC are overexpressed in more aggressive types of TGCT, suggesting they contribute to malignancy. Furthermore, they might serve as serum biomarkers for these types of TGCT.

Is relative quantification dispensable for the measurement of microRNAs as serum biomarkers in germ cell tumors?

BACKGROUND: Classical biomarkers α-fetoprotein, ß-human chorionic gonadotropin and lactate dehydrogenase (AFP, bHCG and LDH) are elevated in only 60% of all testicular germ cell tumor (TGCT) patients. microRNAs (miRNAs) are a novel class of useful biomarkers in cancer and miRNAs of the miR-371-3 cluster were proven to be valuable markers for TGCT patients. MATERIALS AND METHODS: We compared the Ct and ΔCt values of miR-371-3 by real time PCR (qPCR) with and without 18S rRNA for normalization. Expression of miR-371a-3p, miR-372 and miR-373-3p was measured in 25 TGCTs, 4 non-TGCTs and 17 age-matched male controls. RESULTS: A highly positive correlation between Ct and ΔCt values was found in all samples. The highest correlation was found for miR-371a-3p (R2: 0.956). CONCLUSION: RESULTS show that qPCR can be used without endogenous control for analyzing miR-371-3 in the serum of patients with testicular cancer and male controls if the technical procedure is performed under controlled conditions.