Mitochondrial DNA variability and Covid-19 in the Slovak population.

Recent studies have shown that mitochondria are involved in the pathogenesis of Covid-19. Mitochondria play a role in production of reactive oxygen species and induction of an innate immune response, both important during infections. Common variability of mitochondrial DNA (mtDNA) can affect oxidative phosphorylation and the risk or lethality of cardiovascular, neurodegenerative diseases and sepsis. However, it is unclear whether susceptibility of severe Covid-19 might be affected by mtDNA variation. Thus, we have analyzed mtDNA in a sample of 446 Slovak patients hospitalized due to Covid-19 and a control population group consisting of 1874 individuals. MtDNA variants in the HVRI region have been analyzed and classified into haplogroups at various phylogenetic levels. Binary logistic regression was used to assess the risk of Covid-19. Haplogroups T1, H11, K and variants 16256C > T, 16265A > C, 16293A > G, 16311 T > C and 16399A > G were associated with an increased Covid-19 risk. On contrary, Haplogroup J1, haplogroup clusters H + U5b and T2b + U5b, and the mtDNA variant 16189 T > C were associated with decreased risk of Covid-19. Following the application of the Bonferroni correction, statistical significance was observed exclusively for the cluster of haplogroups H + U5b. Unsurprisingly, the most significant factor contributing to the mortality of patients with Covid-19 is the age of patients. Our findings suggest that mtDNA haplogroups can play a role in Covid-19 pathogenesis, thus potentially useful in identifying susceptibility to its severe form. To confirm these associations, further studies taking into account the nuclear genome or other non-biological influences are needed.

Integrated Microarray-Based Data Analysis of miRNA Expression Profiles: Identification of Novel Biomarkers of Cisplatin-Resistance in Testicular Germ Cell Tumours.

Testicular germ cell tumours (TGCTs) are the most common solid malignancy among young men, and their incidence is still increasing. Despite good curability with cisplatin (CDDP)-based chemotherapy, about 10% of TGCTs are non-responsive and show a chemoresistant phenotype. To further increase TGCT curability, better prediction of risk of relapse and early detection of refractory cases is needed. Therefore, to diagnose this malignancy more precisely, stratify patients more accurately and improve decision-making on treatment modality, new biomarkers are still required. Numerous studies showed association of differential expressions of microRNAs (miRNAs) with cancer. Using microarray analysis followed by RT-qPCR validation, we identified specific miRNA expression patterns that discriminate chemoresistant phenotypes in TGCTs. Comparing CDDP-resistant vs. -sensitive TGCT cell lines, we identified miR-218-5p, miR-31-5p, miR-125b-5p, miR-27b-3p, miR-199a-5p, miR-214-3p, let-7a and miR-517a-3p as significantly up-regulated and miR-374b-5p, miR-378a-3p, miR-20b-5p and miR-30e-3p as significantly down-regulated. In patient tumour samples, we observed the highest median values of relative expression of miR-218-5p, miR-31-5p, miR-375-5p and miR-517a-3p, but also miR-20b-5p and miR-378a-3p, in metastatic tumour samples when compared with primary tumour or control samples. In TGCT patient plasma samples, we detected increased expression of miR-218-5p, miR-31-5p, miR-517a-3p and miR-375-5p when compared to healthy individuals. We propose that miR-218-5p, miR-31-5p, miR-375-5p, miR-517-3p, miR-20b-5p and miR-378a-3p represent a new panel of biomarkers for better prediction of chemoresistance and more aggressive phenotypes potentially underlying metastatic spread in non-seminomatous TGCTs. In addition, we provide predictions of the targets and functional and regulatory networks of selected miRNAs.

The results of multigene panel sequencing in Slovak HBOC families.

Hereditary breast and ovarian cancer (HBOC) is primarily associated with mutations in the BRCA1/2 genes. However, causal variants in other high, moderate, and low penetrance genes proportionally increase the risk of breast/ovarian cancer. This study aims to provide data about the mutation spectrum of HBOC-associated genes in Slovak HBOC families and estimate the ratio of BRCA versus non-BRCA causal variants. We used panel sequencing containing 22 high/moderate-risk susceptibility genes and parallel MLPA analysis of BRCA1/2, CHEK2 genes, to analyze 94 individuals with a strong family/personal history of breast and/or ovarian cancer. The analyzed group consisted of 80 patients diagnosed with cancer (85.1%) and 14 healthy individuals (14.9%) with a positive family history of HBOC syndrome. In total, we have identified 22 causal DNA variants (23.4%) showing 15 primary findings in BRCA1/2 genes (68.2%) and 7 positive secondary findings in CHEK2, PALB2, CDH1, and MUTYH genes (31.8%). The most frequent pathogenic alterations were BRCA1 mutations c.181T>G and CNV variant (c.5573-?_c.5701+?)del, known as deletion of exons 21-22. Besides known mutations, the BRCA1 variant c.2794del (p.Val932Leufs*68) and variant c.2480dup (p.Tyr827*) in the CDH1 gene represent the novel, previously unpublished variants that might be population-specific. In conclusion, we provide the first report of multigene panel testing in Slovak HBOC families demonstrating that almost one-third of pathogenic mutations are situated in susceptibility genes other than BRCA1/2. Although multigene panel testing requires precise data filtration and interpretation, it might bring the relevant data for clinical management of the patients.

miR-497-5p Decreased Expression Associated with High-Risk Endometrial Cancer.

The current guidelines for diagnosis, prognosis, and treatment of endometrial cancer (EC), based on clinicopathological factors, are insufficient for numerous reasons; therefore, we investigated the relevance of miRNA expression profiles for the discrimination of different EC subtypes. Among the miRNAs previously predicted to allow distinguishing of endometrioid ECs (EECs) according to different grades (G) and from serous subtypes (SECs), we verified the utility of miR-497-5p. In ECs, we observed downregulated miR-497-5p levels that were significantly decreased in SECs, clear cell carcinomas (CCCs), and carcinosarcomas (CaSas) compared to EECs, thereby distinguishing EEC from SEC and rare EC subtypes. Significantly reduced miR-497-5p expression was found in high-grade ECs (EEC G3, SEC, CaSa, and CCC) compared to low-grade carcinomas (EEC G1 and mucinous carcinoma) and ECs classified as being in advanced FIGO (International Federation of Gynecology and Obstetrics) stages, that is, with loco-regional and distant spread compared to cancers located only in the uterus. Based on immunohistochemical features, lower miR-497-5p levels were observed in hormone-receptor-negative, p53-positive, and highly Ki-67-expressing ECs. Using a machine learning method, we showed that consideration of miR-497-5p expression, in addition to the traditional clinical and histopathologic parameters, slightly improves the prediction accuracy of EC diagnosis. Our results demonstrate that changes in miR-497-5p expression influence endometrial tumorigenesis and its evaluation may contribute to more precise diagnoses.

Discriminating miRNA Profiles between Endometrioid Well- and Poorly-Differentiated Tumours and Endometrioid and Serous Subtypes of Endometrial Cancers.

The discrimination of different subtypes of endometrial carcinoma (EC) is frequently problematic when using the current histomorphological classification; therefore, new markers for this differentiation are needed. Here, we examined differences in miRNA expression between well- and poorly-differentiated (grades 1 and 3) endometrioid endometrial carcinoma (EEC) and between EEC and serous endometrial carcinoma (SEC). The expression of 84 tumour-suppressor miRNAs was analysed by real-time polymerase chain reactions in 62 EC and 20 non-neoplastic endometrial specimens. The potential functions of the differentially expressed miRNAs were determined by bioinformatics analyses. The expression of let-7c-5p, miR-125b-5p, miR-23b-3p, and miR-99a-5p in grade 3 EEC was decreased compared to grade 1 EEC. To discriminate between EEC and SEC, let-7g-5p, miR-195-5p, miR-34a-5p, and miR-497-5p expression was significantly downregulated in SEC. In bioinformatic analyses, miRNAs that could discriminate grade 1 from grade 3 mainly targeted genes involved in PI3K-AKT signaling, whereas miRNAs that could discriminate EEC from SEC targeted genes involved in several signaling pathways, but mainly MAPK signaling. Taken collectively, our results indicate that the activation of certain signaling pathways can be useful in the molecular characterization of EEC and SEC.

Differential gene expression in cisplatin-resistant and -sensitive testicular germ cell tumor cell lines.

Testicular germ cell tumors (TGCTs) represent a well curable malignity due to their exceptional response to cisplatin (CDDP). Despite remarkable treatment results, approximately 5% of TGCT patients develop CDDP resistance and die. Exceptional curability makes TGCTs a highly valuable model system for studying the molecular mechanisms of CDDP sensitivity. Our study was aimed at revealing difference in gene expression between the CDDP-resistant and -sensitive TGCT cell lines, and hence at identifying candidate genes that could serve as potential biomarkers of CDDP response. Using gene expression array, we identified 281 genes that are differentially expressed in CDDP-resistant compared to -sensitive TGCT cell lines. The expression of 25 genes with the highest fold change was validated by RT-qPCR. Of them, DNMT3L, GAL, IGFBP2, IGFBP7, L1TD1, NANOG, NTF3, POU5F1, SOX2, WNT6, ZFP42, ID2, PCP4, SLC40A1 and TRIB3, displayed comparable expression change in gene expression array and RT-qPCR, when all CDDP-resistant TGCT cell lines were pairwise combined with all -sensitive ones. Products of the identified genes are pluripotency factors, or are involved in processes, such as cell metabolism, proliferation or migration. We propose that, after clinical validation, these genes could serve as prognostic biomarkers for early detection of CDDP response in TGCT patients.

Probiotic Survey in Cancer Patients Treated in the Outpatient Department in a Comprehensive Cancer Center.

PURPOSE: Availability without prescription restriction, low cost, and simple oral administration allow cancer patients to use probiotics without knowledge of potential risks. We present a survey of probiotic use and the association with patient tumor characteristics in cancer patients treated at the outpatient department of the National Cancer Institute in Slovakia. PATIENTS AND METHODS: Between March and December 2014, 499 patients were asked to evaluate their overall experience with probiotics by questionnaire form, including the length and method of use relative to anticancer therapy, expectations, side-effect experiences, understanding of the possible risks, dietary supplement use, and others. The relevant data were statistically evaluated. RESULTS: The cohort consisted of 323 women (64.7%) and 176 men (35.3%); 91.6% were undergoing chemotherapy (2.6% together with radiotherapy) and 8.4% had no anticancer therapy. The prevalence of probiotic use was 28.5% and only 12 patients using probiotics (8.5%) described negative side effects. Most patients declared consideration of probiotic use based on recommendation from a physician (37.3%) or a pharmacist (14.8%). Nevertheless, up to 86.6% of patients declared no knowledge of possible risks. Statistically significant correlation was found between probiotic use and age of patients (P < .008), gender (P < .023), and taking other dietary supplements (P < .000002). CONCLUSIONS: In this prospective study, we present for the first time the prevalence, side-effect experience, and aspects that most likely influence probiotic use in cancer patients. Minimal knowledge of risks underlines the importance of an active approach by oncologists to inform patients about probiotic safety.

Internalization property of intestinal bacteria in colon cancer and HIV/AIDS patients.

OBJECTIVES: Bacteria from the intestinal tract of Slovak and American HIV/AIDS patients and Slovak colon cancer patients were tested for the capacity to be internalized by cells of the HL-60 cell line as well as by normal human lymphocytes. They were anticipated to possess a specific characteristic, i.e. a vigorous ability to be internalized by HL-60 cells and human lymphocytes. This assumption was confirmed by gentamicin protection assay. RESULTS: Internalization of bacteria from HIV/AIDS patients frequently resulted in partial (patients SKM1, SKM22) or complete lysis (patients SKK1-1, SKM12) of HL-60 cells. In comparison with intramucosal bacteria isolated from patients with colorectal cancer (TSG, 883, 660, 838, 536, MZRa), their capacity to internalize HL-60 cells was found to be 15-20 times higher (USP15/7, USP1/4, USP3/3, SK725/5). Partial lysis (patients USP15/7, USP3/3 and SKM22) and complete lysis (patients USP1/4, SKK1-1/1, SKM1/6, SKM12/5) were detected also after internalization of bacteria by normal human lymphocytes. Compared to the amount of intracellular bacteria isolated from patients with HIV/AIDS, the ability of bacteria from patients with colorectal cancer to internalize normal human lymphocytes was significantly lower (10-15 times), yet still higher than that of bacteria isolated from healthy people. CONCLUSIONS: Our results present the ability of bacteria of colon cancer patients and HIV/AIDS patients to internalize HL-60 cells and normal human lymphocytes. The findings underline the potentially important function of bacteria in the induction of colorectal cancer and immunodeficiency. The particularly high detection ability of bacteria from HIV/AIDS patients to internalize normal human cells emphasizes their potentially important role in the process of AIDS.

Analysis of bacteria from intestinal tract of FAP patients for the presence of APC-like sequences.

BACKGROUND: Familial adenomatous polyposis (FAP) is a hereditary disease induced by germ-line mutations in the tumor suppressor APC gene. These initiate the early stages of the adenoma-carcinoma sequence in familial, but also in sporadic (in 80% to 90%), colon tumorigenesis. We found the presence of APC-like sequences in bacteria of FAP patients. MATERIAL/METHODS: We analyzed bacteria isolated from FAP patients' rectal swabs. Total bacterial DNA was isolated and analyzed for detection of APC-like sequences using PCR. We also tested DNA homology rate and APC-like protein production. RESULTS: We collected blood samples and rectal swabs from patients with confirmed diagnosis of FAP. They were analyzed for presence of sections from exon 15 of the APC gene. Most positive results were found in sections located exactly in the area called the MCR (mutation cluster region), where the highest frequency of APC gene mutations were identified. By sequencing PCR products from bacteria in section F-G together with a patient's DNA sample and human APC gene, we found a more than 90% DNA homology rate. We also confirmed production of APC-like protein using Western blotting. CONCLUSIONS: Our results suggested two hypotheses. The APC-like protein might have same function as a truncated APC product, which is synthesized in most cases of mutations of APC gene in the MCR region in colorectal cancer cells. Alternatively, we can consider the possible existence of horizontal transfer of genetic information between eukaryotic and prokaryotic cells. Our study can be considered as a pilot project. For confirmation of our hypotheses, further research is needed.

Study of the APC gene function in the mouse APC+/APC1638N model.

OBJECTIVES: Familial adenomatous polyposis (FAP) is an autosomal dominant disease characterized by the presence of hundreds to thousands of benign polyps in the colon. If not removed prophylactically they represent a risk of developing malignant cancer with an almost 100% penentrance. FAP is induced by germline mutation in the APC gene. Tumorigenesis launched a second somatic mutation of APC gene allele, leading to synthesis of non-functional APC protein. One of the possibilities of cancer prevention could be an alternative gene therapy using bacteria as vectors for delivery of therapeutic protein molecules. MATERIAL AND METHODS: For this purpose mice model APC+/APC1638N with mutation in one allele murine homolog of the APC gene were used. Mice were fed orally commercial nutrition enriched with 0.5 ml PBS buffer with 5% milk containing 5×108 recombinant bacterial cells DE3plys6 bearing plasmid with cloned APC gene twice a week during 42 weeks. Afterwords mice were killed by thiopental, gastrointestinal tracts were removed, microscopically, macroscopically inspected for polyps/neoplastic lesions and immunohistochemically investigated with polyclonal rabbit antibody against APC protein. RESULTS: We have cloned full-lenght APC gene into vector for expression in bacterial cells Escherichia coli BL21(DE3) and BL21(DE3) pLysS. Expression of the APC protein, induced by IPTG, was detected in protein extracts of three bacterial clones: DE3104-11, DE3pLys5, DE3pLys6. APC protein was identified by Western blot analysis using monoclonal and polyclonal antibodies against the APC protein. Bacteria of clone DE3pLys6 were orally administered to APC+/APC1638N mice with mutations in the APC gene. All transgenic mice without therapy developed adenomatous polyps in the gastrointestinal tract. Transgenic mice treated by oral administration of bacteria expressing functional APC protein developed polyps in 33.3%. The remaining four mice 66.7% were without polyps development. CONCLUSION: Administration of APC gene expressing by bacteria to transgenic mice with mutation in APC gene leads to reduction in the number of mice developing polyps in the gastrointestinal tract. The effect of bacterially expressed APC protein in elimination of intestinal polyps or tumors has been monitored. These are our preliminary results and for possible confirmation of our hypotheses still more research is needed.