Liquid biopsy-based biomarkers, such as microRNAs, represent valuable tools for patient management, but often do not make it to integration in the clinic. We aim to explore issues impeding this transition, in the setting of germ cell tumors, for which novel biomarkers are needed. We describe a model for identifying and validating clinically relevant microRNAs for germ cell tumor patients, using both in vitro, in vivo (mouse model) and patient-derived data. Initial wide screening of candidate microRNAs is performed, followed by targeted profiling of potentially relevant biomarkers. We demonstrate the relevance of appropriate (negative) controls, experimental conditions (proliferation), and issues related to sample origin (serum, plasma, cerebral spinal fluid) and pre-analytical variables (hemolysis, contaminants, temperature), all of which could interfere with liquid biopsy-based studies and their conclusions. Finally, we show the value of our identification model in a specific scenario, contradicting the presumed role of miR-375 as marker of teratoma histology in liquid biopsy setting. Our findings indicate other putative microRNAs (miR-885-5p, miR-448 and miR-197-3p) fulfilling this clinical need. The identification model is informative to identify the best candidate microRNAs to pursue in a clinical setting.

Inflammatory processes in the skin augment collagen degradation due to the up-regulation of matrix metalloproteinases (MMPs). The aim of the present project was to study the specific impact of MMP-3 on collagen loss in skin and its interplay with the collagenase MMP-13 under inflammatory conditions mimicked by the addition of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). Skin explants from MMP-3 knock-out (KO) mice or from transgenic (TG) mice overexpressing MMP-3 in the skin and their respective wild-type counterparts (WT and WTT) were incubated ex vivo for eight days. The rate of collagen degradation, measured by released hydroxyproline, was reduced (p < 0.001) in KO skin explants compared to WT control skin but did not differ (p = 0.47) between TG and WTT skin. Treatment with the MMP inhibitor GM6001 reduced hydroxyproline media levels from WT, WTT and TG but not from KO skin explants. TNF-α increased collagen degradation in the WT group (p = 0.0001) only. More of the active form of MMP-13 was observed in the three MMP-3 expressing groups (co-incubation with receptor-associated protein stabilized MMP-13 subforms and enhanced detection in the media). In summary, the innate level of MMP-3 seems responsible for the accelerated loss of cutaneous collagen under inflammatory conditions, possibly via MMP-13 in mice.

Testicular cancer is an important disease with increasing incidence and a high burden of morbidity and mortality in young men worldwide. Histological examination of the testicular tissue after orchiectomy plays an important role alongside patient history, imaging, clinical presentation and laboratory parameters. Surgical procedures and chemotherapeutic treatment provide a high chance of cure in early stages, though some patients in advanced stages belonging to a poor risk group experience cancer-related death. Though conventional serum-based tumor markers, including α-fetoprotein (AFP), the β-subunit of human chorionic gonadotropin (β-hCG), and lactate dehydrogenase (LDH), are useful as prognostic and diagnostic biomarkers, unfortunately, these tumor markers only have a sensitivity of about 60%, and in pure seminoma even lower with about 20%. Therefore, the development of new tumor markers is an important and intensively ongoing issue. The analysis of epigenetic modification and non-coding RNA microRNAs (miRNAs) are carrying most promising potential as tumor markers in future. miRNAs are small RNAs secreted by testicular tumor cells and circulate and be measurable in body fluids. In recent years, miRNAs of the miR-371-373 cluster in particular have been identified as potentially superior tumor markers in testicular cancer patients. Studies showed that miR-371a-3p and miR-302/367 expression significantly differ between testicular tumors and healthy testicular tissue. Several studies including high prospective multi-center trials clearly demonstrated that these miRNAs significantly exceed the sensitivity and specificity of conventional tumor markers and may help to facilitate the diagnosis, follow-up, and early detection of recurrences in testicular cancer patients. In addition, other miRNAs such as miR-223-3p, miR-449, miR-383, miR-514a-3p, miR-199a-3p, and miR-214 will be discussed in this review. However, further studies are needed to identify the value of these novel markers in additional clinical scenarios, including the monitoring in active surveillance or after adjuvant chemotherapy, but also to show the limitations of these tumor markers. The aim of this review is to give an overview on the current knowledge regarding the relevance of non-coding miRNAs as biomarkers in testicular cancer.

Background/Aim: Clinical management of testicular germ cell tumours (GCT) is based upon the measurement of serum tumour markers. Recent studies have shown that the microRNA-371a-3p is a sensitive and specific serum biomarker for all subgroups of GCT, except teratoma. To close the diagnostic gap relating to teratoma, serum levels of microRNA-375-3p have recently been suggested to represent a specific serum marker of this histological subgroup. In the present study, we tested this hypothesis. Materials and Methods: miRNA expression was analysed in serum of 21 GCT patients with teratoma, twelve patients with other GCT, and twelve male controls using the qPCR method. Results: The serum miR-375-3p levels of teratoma patients were not different from other GCT patients or controls. The ROC analysis revealed an AUC of 0.524 for the discrimination between teratoma and other pathologies. Conclusion: The miR-375-3p does probably not qualify for a useful serum biomarker to distinguish teratoma from other GCTs and from controls.

Background: The microRNAs of the miR‐371‐3 cluster are novel serum markers for testicular germ cell tumors. Sporadic reports suggested the expression of this miRNA in semen.Objectives: To verify the expression of miR‐371a‐3p in seminal plasma and unprocessed ejaculate; to compare seminal plasma miRNA levels in germ cell tumors patients with those of controls; to look for an association of miRNA levels with semen quality.Materials and methods: The miR‐371a‐3p expression was analyzed with qPCR. The study population consisted of 100 participants: seminal plasma samples from 20 germ cell tumors patients and 30 controls, serum samples from 12 healthy men, ejaculate samples from 38 men undergoing fertility testing.Results: The seminal plasma miR‐371a‐3p levels of germ cell tumors patients were not different from controls. The miRNA expression was very low in serum but much higher in seminal plasma. In ejaculate samples, the miRNA expression significantly correlated with sperm concentration and the total sperm count.Discussion: miR‐371‐a‐3p is present in sperm‐containing fluids. Seminal plasma levels cannot be used to distinguish germ cell tumors from controls. The correlation with sperm concentration in ejaculate samples suggests the spermatozoa as possible source of miR‐371a‐3p production.Conclusion: The miR‐371a‐3p levels in ejaculate could represent a novel biomarker for the non‐invasive evaluation of male infertility.