Cisplatin resistance in testicular germ cell tumors (TGCTs) is a clinical challenge. We investigated the underlying mechanisms associated with cancer stem cell (CSC) markers and modalities circumventing the chemoresistance. Chemoresistant models (designated as CisR) of human embryonal carcinoma cell lines NTERA-2 and NCCIT were derived and characterized using flow cytometry, gene expression, functional and protein arrays. Tumorigenicity was determined on immunodeficient mouse model. Disulfiram was used to examine chemosensitization of resistant cells. ALDH1A3 isoform expression was evaluated by immunohistochemistry in 216 patients’ tissue samples. Chemoresistant cells were significantly more resistant to cisplatin, carboplatin and oxaliplatin compared to parental cells. NTERA-2 CisR cells exhibited altered morphology and increased tumorigenicity. High ALDH1A3 expression and increased ALDH activity were detected in both refractory cell lines. Disulfiram in combination with cisplatin showed synergy for NTERA-2 CisR and NCCIT CisR cells and inhibited growth of NTERA-2 CisR xenografts. Significantly higher ALDH1A3 expression was detected in TGCTs patients’ tissue samples compared to normal testicular tissue. We characterized novel clinically relevant model of chemoresistant TGCTs, for the first time identified the ALDH1A3 as a therapeutic target in TGCTs and more importantly, showed that disulfiram represents a viable treatment option for refractory TGCTs.

Recent research studies are showing breast tissues as a place where various species of microorganisms can thrive and cannot be considered sterile, as previously thought. We analysed the microbial composition of primary tumour tissue and normal breast tissue and found differences between them and between multiple breast cancer phenotypes. We sequenced the transcriptome of breast tumours and normal tissues (from cancer-free women) of 23 individuals from Slovakia and used bioinformatics tools to uncover differences in the microbial composition of tissues. To analyse our RNA-seq data (rRNA depleted), we used and tested Kraken2 and Metaphlan3 tools. Kraken2 has shown higher reliability for our data. Additionally, we analysed 91 samples obtained from SRA database, originated in China and submitted by Sichuan University. In breast tissue, the most enriched group were Proteobacteria, then Firmicutes and Actinobacteria for both datasets, in Slovak samples also Bacteroides, while in Chinese samples Cyanobacteria were more frequent. We have observed changes in the microbiome between cancerous and healthy tissues and also different phenotypes of diseases, based on the presence of circulating tumour cells and few other markers.

Patients with ovarian cancer represent a heterogeneous population with a variable prognosis and response to chemotherapy. Plasma DNA has been shown to have a prognostic value in different types of cancer including ovarian carcinoma. Whether total circulating DNA, which can be assessed much easier without knowing the tumor-specific mutations, has similar informative value is currently unknown. The aim of this study was to evaluate the prognostic value of extracellular DNA in advanced ovarian cancer. This prospective study included 67 patients (pts) with ovarian cancer treated with 1st line paclitaxel and carboplatin (25 pts) and paclitaxel, carboplatin and bevacizumab (42 pts). Thirty-five patients had optimal surgical debulking before chemotherapy. Extracellular DNA was quantified using real time PCR before administration of chemotherapy (67 pts) and after 6 cycles of chemotherapy (44 pts). Total extracellular DNA (ecDNA), as well as extracellular DNA of nuclear (nDNA) and mitochondrial origin (mtDNA) significantly (p < 0.05) decreased after 6 cycles of chemotherapy (by 54%, 63% and 52%, respectively. Patients with stage I disease had significantly lower mtDNA compared to patients with stage II-IV (8604 vs. 16, 984 ge/mL, p = 0.03). Patients with lower baseline nDNA had superior progression-free (HR = 0.35 (0.14–0.86)) and overall survival (HR = 0.18 (0.04–0.77). The prognostic value of nDNA was confirmed independent of tumor stage and confirmed in multivariate analysis. Our data suggest that ecDNA of both, nuclear and mitochondrial origin could be added to prognostic markers in ovarian cancer. Analysis of ecDNA does not require the knowledge of tumor-specific mutations in contrast to the quantification of tumor-derived ecDNA. Study of the dynamics and cell type-specific source of the ecDNA could shed light on its biology in cancer and might help to direct the treatment of ovarian cancer.

The majority of patients with testicular germ cell tumors (GCTs) can be cured with cisplatin-based chemotherapy. However, for a subset of patients present with cisplatin-refractory disease, which confers a poor prognosis, the treatment options are limited. Novel therapies are therefore urgently needed to improve outcomes in this challenging patient population. It has previously been shown that Wnt/β-catenin signaling is active in GCTs suggesting that its inhibitors LGK974 and PRI-724 may show promise in the management of cisplatin-refractory GCTs. We herein investigated whether LGK-974 and PRI-724 provide a treatment effect in cisplatin-resistant GCT cell lines. Taking a genoproteomic approach and utilizing xenograft models we found the increased level of β-catenin in 2 of 4 cisplatin-resistant (CisR) cell lines (TCam-2 CisR and NCCIT CisR) and the decreased level of β-catenin and cyclin D1 in cisplatin-resistant NTERA-2 CisR cell line. While the effect of treatment with LGK974 was limited or none, the NTERA-2 CisR exhibited the increased sensitivity to PRI-724 in comparison with parental cell line. Furthermore, the pro-apoptotic effect of PRI-724 was documented in all cell lines. Our data strongly suggests that a Wnt/β-catenin signaling is altered in cisplatin-resistant GCT cell lines and the inhibition with PRI-724 is effective in NTERA-2 CisR cells. Further evaluation of Wnt/β-catenin pathway inhibition in GCTs is therefore warranted.

Germ cell neoplasia in situ (GCNIS), is preinvasive stage of testicular germ cell tumours (TGCTs). Fibrillins, which are integral components of microfibrils are suggested to be involved in cancer pathogenesis and maintenance of embryonic stem cells pluripotency. The aim of this study was to examine fibrillin-1 (FBN-1) expression in TGCTs patients. Surgical specimens from 203 patients with TGCTs were included into the translational study. FBN-1 expression was evaluated in the tumour tissue, in GCNIS and in adjacent non-neoplastic testicular tissue in all available cases. Tissue samples were processed by the tissue microarray method. FBN-1 was detected by immunohistochemistry using goat polyclonal antibody and the expression was evaluated by the multiplicative quickscore (QS). The highest FBN-1 positivity was detected in GCNIS (mean QS = 11.30), with overexpression of FBN-1 (QS >9) in the majority (77.1 %) of cases. Expression of FBN-1 in all subtypes of TGCTs was significantly lower in comparison to expression in GCNIS (all p <0.001). Seminoma had significantly higher expression compared to EC, ChC and TER (all p <0.05), but not to YST (p = 0.84). In non-neoplastic testicular tissue the FBN-1 positivity was very low (mean QS = 0.02). Sensitivity, specificity, positive and negative predictive value of FBN-1 expression for diagnosis of GCNIS were 97.1, 98.8, 98.6 and 97.7 %. FBN-1 is overexpressed in TGCTs and especially in GCNIS when compared to non-neoplastic testicular tissue in patients with germ cell tumors and could be involved in germ cell neoplasia in situ development.

WNT/βcatenin (WNTβ) pathway is activated in early stages of embryonic development. We aimed to evaluate the significance of βcatenin in germ cell tumors (GCTs) and explore associations with the inflamed environment. Surgical specimens from 247 patients were analyzed. Βcatenin expression was detected in the tumor tissue by immunohistochemistry and correlated with clinical characteristics, outcome, PD-L1 expression and systemic immune-inflammation index (SII). The Ingenuity Pathway Analysis (IPA) was used to investigate the immune-cell related effects of βcatenin and PD-L1 encoding genes. βcatenin was expressed in 86.2% of GCTs. The expression in seminomas was significantly lower compared to all subtypes of non-seminoma (all P < 0.0001). A high expression (weighted histoscore > 150) was associated with primary mediastinal non-seminoma (P = 0.035), intermediate/poor risk disease (P = 0.033) and high tumor markers (P = 0.035). We observed a positive correlation with the PD-L1 in tumor and an inverse correlation with the SII. IPA uncovered relationships of CTNNB (βcatenin) and CD274 (PD-L1) genes and their effects on differentiation, proliferation and activation of lymphocyte subtypes. Herein, we showed that βcatenin is associated with male adult GCT characteristics as well as supressed immune environment.