Evaluation of Circulating miRNA Biomarkers of Testicular Germ Cell Tumors during Therapy and Follow-up-A Copenhagen Experience

Abstract

New microRNA-based serum biomarkers (miRNA-367-3p, -371a-3p, -372-3p, and -373-3p) have shown great potential for the detection of testicular germ cell tumors (TGCTs), but few studies have investigated the clinical utility and performance of these tests in treatment monitoring. In this study, circulating miRNA levels were measured, together with serum tumor markers alpha-fetoprotein (AFP), β-subunit of human chorionic gonadotropin (β-HCG) and lactate dehydrogenase (LDH) in 406 consecutive blood samples obtained during the treatment and follow-up of 52 TGCT patients at the Copenhagen University Hospital. After testing three different methods of RNA isolation from peripheral blood and PCR quantification in a subset of samples (n = 15), the best performing setup of targeted isolation of miRNAs inside and outside exosomes was selected to analyze all samples. At primary diagnosis, the miRNAs significantly outperformed the serum tumor markers, with a sensitivity and specificity of 78% and 100% (based on 40 patients), respectively. The picture was not as clear when patient trajectories were investigated, with both positive and negative signals for miRNAs and serum tumor markers. To establish whether measuring miRNAs adds value beyond the primary diagnosis, large prospective clinical trials comparing miRNAs and classical tumor markers during the treatment and follow-up of TGCT patients are needed.

Keywords: AFP; HCG; TGCT; chemotherapy; follow-up; miR-371a; miRNA; testicular germ cell cancer.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Methodological comparison. (a) Comparison of three different methods for detection of circulating miRNAs: Exiqon total RNA purification (miRCURY RNA Isolation Kit), cDNA synthesis (Universal cDNA Synthesis Kit II) and qPCR (PCR ExiLENT SYBR® Green master mix) (Exiqon total RNA); Exiqon exosome purification (miRCURY™ Exosome Isolation Kit—Serum and plasma) followed by total RNA purification (miRCURY RNA Isolation Kit), cDNA synthesis (Universal cDNA Synthesis Kit II) and qPCR (PCR ExiLENT SYBR® Green master mix) (Exiqon Exosome); and TaqMan® miRNA ABC purification kit—Human Panel A, cDNA synthesis (Advanced miRNA cDNA synthesis kit) and qPCR (TaqMan Fast Advanced qPCR Mastermix with corresponding advanced miRNA TaqMan probes) (LifeTech TaqMan ABC). Fifteen samples were analyzed, representing four controls, one GCNIS, five seminoma, two mixed TGCTs, two embryonal carcinomas, and one teratoma. A colored cell indicates that the specific miRNA was detected in that sample by use of the specific method. For sensitivity and specificity calculations, the sample from the teratoma patient was excluded, since teratomas are not expressing the miRNAs. Sensitivity and specificity are shown for each miRNA in each setup, as well as a combined sensitivity and specificity, assuming that the detection of one or more miRNA gives a positive signal. (b) Bar plot of the mean Ct value for miR-20a-5p in all samples using the three different methods. The mean Ct values are significantly different from each other (p <0.0001), using a one-way Anova. The mean Ct of all three methods is shown with a dotted line, and the error bars reflect the standard deviation. (c) Heatmap showing the miR-20a-5p Ct values in serum and plasma samples using the LifeTech method. The CV in serum was 3.19% and in plasma 11.08%. Abbreviations used: GCNIS; germ cell neoplasia in situ, TGCT; testicular germ cell tumor, EC; embryonal carcinoma, T; teratoma, CV; coefficient of variation.

Figure 2

Clinical validation of the LifeTech method. (a) Relative miRNA expression normalized to miR-20a-5p in serum from 40 patients, before orchiectomy compared with 22 controls without TGCTs. (b–e) matched serum from 8 patients before and after orchiectomy. Blood samples were drawn 1–12 days before orchiectomy (median 3 days) and again 2–21 days after orchiectomy (median 18 days). Red lines represent patients with CSI and blue lines represent patients with CSII. Abbreviations used: TGCT; testicular germ cell tumor, CSI; clinical stage I, and CSII; clinical stage II.

Figure 3

Examples of evolution of miRNAs and serum tumor markers (STMs) at primary diagnosis and during treatment in eight representative patients with TGCT. The number of patients belonging to each group (n) is given under the group symbol (a-h). (a,b) Patients with measurable levels of both STMs (AFP, β-HCG, and LDH) and miRNAs. (c,d) Only measurable levels of miRNAs, non-elevated STM levels. (e,f) Only elevated levels of STM, unmeasurable miRNA levels. (g,h) Non-elevated levels of STMs and miRNAs. (a,c,e,g) Thirty-nine patients had information on both miRNAs and STM at primary diagnosis/before orchiectomy. (b,d,f,h) Twenty-five patients had information on both miRNAs and STMs during chemotherapy treatment. Abbreviations used: STM; serum tumor markers, NS; non-seminoma, S; seminoma, BS; blood sample, Chemo; chemotherapy, LDH; lactate dehydrogenase, HCG; beta-subunit of human chorionic gonadotropin, AFP; alpha-fetoprotein, miRs; miRNAs.