Many chemotherapy drugs block tumor cell division by damaging DNA. DNA polymerases eta (Pol η), iota (Pol ι), kappa (Pol κ), REV1 of the Y-family and zeta (Pol ζ) of the B-family efficiently incorporate nucleotides opposite a number of DNA lesions during translesion DNA synthesis. Primase-polymerase PrimPol and the Pol α-primase complex reinitiate DNA synthesis downstream of the damaged sites using their DNA primase activity. These enzymes can decrease the efficacy of chemotherapy drugs, contribute to the survival of tumor cells and to the progression of malignant diseases. DNA polymerases are promising targets for increasing the effectiveness of chemotherapy, and mutations and polymorphisms in some DNA polymerases can serve as additional prognostic markers in a number of oncological disorders.

Tens of thousands of DNA lesions are formed in mammalian cells each day. DNA translesion synthesis is the main mechanism of cell defense against unrepaired DNA lesions. DNA polymerases iota (Pol ι), eta (Pol η), kappa (Pol κ), and zeta (Pol ζ) have active sites that are less stringent toward the DNA template structure and efficiently incorporate nucleotides opposite DNA lesions. However, these polymerases display low accuracy of DNA synthesis and can introduce mutations in genomic DNA. Impaired functioning of these enzymes can lead to an increased risk of cancer.

Aim. To determine the influence of the polymorphic variants of the base (OGG1 and XRCC1) and nucleotide (ERRC2/XPD and ERRC6/CSB) excision repair genes on the mutational and epigenetic status of bladder tumors. Methods. For this study, we used previously obtained data on the polymorphism of DNA repair genes in bladder cancer (BC) patients, whose tumor tissue samples were analyzed for the presence of key mutational and epigenetic changes. Results. Genotypes containing at least one minor OGG1 rs1052133 allele were significantly associated with an increased frequency of RAS family gene mutations and a reduced frequency of PIK3CA mutations. The polymorphisms of both base excision repair genes influenced the epigenetic variability of urothelial carcinomas, the modifying effect of OGG1 rs1052133 manifesting in ISL1 methylation and XRCC1 rs25487 impacting p16 or TIMP3 methylation. The minor ERRC6/CSB rs2228526 allele was associated with RAS mutations and lack of TIMP3 methylation. Likewise, carriers of the genotypes containing at least one minor ERRC2/XPD rs1799793 allele were less frequently found to have methylated RUNX3 gene in tumor tissues. Conclusions. The polymorphisms of the base (OGG1, XRCC1) and nucleotide (ERRC2/XPD, ERRC6/CSB) excision repair genes modify the mutational and epigenetic variability of a number of key BC genes. The study of the mechanisms of such interactions is necessary for understanding the molecular basis of BC pathogenesis. Keywords: bladder cancer, OGG1, XRCC1, ERRC2/XPD, ERRC6/CSB gene polymorphism, mutation, methylation.

AIM Base excision repair (BER) gene polymorphisms are known to play an independent role in predisposition to developing different cancers as well as to be associated with clinicopathological traits of the disease modifying its clinical outcomes. One of the underlying mechanisms is presumed to include interplay between BER gene polymorphisms and key mutational, epigenetic and chromosomal events in tumor tissues. The present study was aimed at elucidating potential gene-gene interaction and assessing their mutual effects in bladder cancer (BC). MATERIALS AND METHODS The earlier obtained data on genotyping patients with verified diagnosis of BC for OGG1 rs1052133 (Ser326Cys) and XRCC1 rs25487 (Arg399Gln) polymorphisms were used for this study. The tumor tissue samples from the same patients were analyzed for mutations, epigenetic variations and losses of heterozygosity in some key genes involved in divergent pathogenic pathways of BC. RESULTS It was shown that the OGG1 (326 codon) heterozygous genotype as well as the minor 326Cys allele can intensify a mutational response of the RAS locus in urothelial carcinomas in the total cohort of patients simultaneously decreasing the mutation rates in the PIK3CA locus in smokers. The XRCC1 (399 codon) heterozygous genotype as well as the minor 399Gln allele reduced the frequency of LOH in the PTEN and TNKS genes, but did not affect the mutational variability in any locus tested. Both polymorphisms influenced the methylation status, carriers of OGG1 326Ser/Cys or Ser/Cys+Cys/Cys genotypes demonstrating increased frequency of methylated RUNX3 and ISL1 genes whereas the similar effect of XRCC1 polymorphism concerning methylation of p16 and TIMP3 genes. When dividing the total cohort into groups based on the extent of tumor spread, the observed associations were characteristic of non-muscle invasive BC. CONCLUSION The BER gene polymorphisms contributed to modification of key molecular events in urothelial carcinomas. Their mutual effects mainly manifested in non-muscle invasive BC. The underlying mechanisms as well as possible clinical outcomes need to be further explored to propose novel prognostic biomarkers for BC.

Promoter hypermethylation of tumor suppressor genes is one of the mechanisms of epigenetic regulation disturbance of gene expression and is often observed in different cancer types. The profile of mutational and epigenetic changes characterizes a malignant potential of a tumor, as well as its ability to invade and metastasize.The aim of the study was to determine a prognostic value of p16, p14ARF and TIMP3 gene methylation in the group of 158 bladder cancer patients. Epigenetic changes in these genes were observed with a frequency of 11.4, 0 and 10.8 %, respectively, and did not depend on clinic-morphological characteristics.A statistically significant association of p16 and TIMP3 abnormal methylation with smoking was found, indicating a possible influence of tobacco smoke carcinogens on the occurrence of these epigenetic changes. In the multivariate Cox regression analysis, p16 promoter hypermethylation was an independent predictor for bladder cancer progression (HR 6.84; 95 % CI 1.6–29.9; р = 0.011).The use of the data on the p16 methylation status may improve the accuracy of prognosis of the bladder cancer clinical course and the selection of appropriate treatment strategy.

The analysis of the RUNX3 gene methylation status was conducted in the prospective group of 262 bladder cancer patients. In comparison to normal urothelium, a high frequency (63 %) of RUNX3 promoter hypermethylation was observed in tumor tissues. А statistically significant association of RUNX3 epigenetic abnormalities with a more aggressive tumor phenotype – an advanced tumor stage and grade, as well as a large tumor size – was found. We have shown that RUNX3 hypermethylation is an independent risk factor for progression and cancer-specific survival in the group of patients with non-muscle invasive bladder cancer.

The HRAS, KRAS, and NRAS gene products belong to the superfamily of small GTPases. These proteins regulate the cellular response to extracellular stimuli through the activation of different signaling pathways. Although the role of the RAS gene mutations in the pathogenesis of various human cancers has been established, the clinical significance of these molecular alterations in bladder cancer remains unclear. The aim of this study was to determine the frequency and spectrum of the HRAS, KRAS, and NRAS mutations, to analyze their association with the clinicopathological variables, and to determine the prognostic value of these alterations in terms of recurrence, progression and mortality, in a prospective cohort of 249 bladder cancer patients. The frequency of the RAS mutations, detected by the SNaPshot method, was 11.2%, of which the HRAS mutations accounted for 64.3%, KRAS, for 28.6%, and NRAS, for 7.1%. We failed to find any correlation between all the RAS gene mutations and pathomorphological characteristics. However, when analyzed separately, the opposite association of the HRAS and KRAS mutations with the clinical parameters of bladder cancer was for the first time shown: the HRAS mutations were significantly associated with low stage low grade papillary tumors of a small size (p < 0.05), whereas the KRAS mutations were associated with non-papillary urothelial carcinomas and the presence of metastases (p < 0.05). Analysis of the prognostic value of the molecular alterations revealed the association of the KRAS mutations with decreased cancer-specific survival in both the total group of patients and the subgroup with nonmuscle invasive disease. The data obtained suggest that the HRAS and KRAS gene mutations may characterize alternative pathways of bladder cancer pathogenesis: the HRAS mutations indicating benign and KRAS mutations, aggressive disease course.

A prospective study was conducted to assess the prognostic value of FGFR3 gene mutation status in patients with non-muscle invasive bladder cancer. A total of 265 patients were included in the study. FGFR3 gene mutations were found in 168 (63.4 %) cases. FGFR3 mutation rate was significantly higher in low-grade tumors (p = 0.00 004). With a median follow-up of 34 months hazard ratio of progression in FGFR3 mutant cases compared to FGFR3 wild type was 0.50 (95 % CI 0.17–1.49; p = 0.21). In the subgroup analysis, it was found that FGFR3 mutations in patients with T1 high grade tumors (n = 41) were associated with a significantly better prognosis: 3-year progression-free survival (PFS) in FGFR3 mutant cases (n = 17) was 100 % compared to 71.2 % (95 % CI 42.8–99.6 %) in the absence of mutations (n = 24). For other subgroups (Ta, T1 low grade) no statistically significant difference in PFS by FGFR3 mutation status was noted.

AIM:The aim of this study was to determine the frequencies of FGFR3 and TP53 mutations in a prospective cohort of 150 bladder cancer patients and to assess the relationship between their mutational status and clinicopathological variables.MATERIALS AND METHODS:The FGFR3 and TP53 mutations were detected by the SNaPshot method and PCR-single-strand conformational polymorphism analysis followed by DNA sequencing.RESULTS:The activating FGFR3 mutations were found in 71 (47.3%) whereas TP53 mutations were observed in 31 (20.7%) urothelial carcinomas. FGFR3-mutant tumors significantly correlated with lower tumor stage and grade, papillary form of bladder cancer and the absence of metastases while TP53-mutant tumors were strongly associated with higher tumor stage and grade as well as the presence of metastasis. We also found significant inverse correlation between FGFR3 mutations and TP53 alterations in urothelial carcinomas (p=0.03). Four possible genotypes were observed in the whole studied cohort, namely FGFR3mut/TP53wt (41.3%), FGFR3wt/TP53wt (38%), FGFR3wt/TP53mut (14.7%), and FGFR3mut/TP53mut (6%). Tumors with FGFR3wt/TP53wt genotype comprised the subgroup, in which all stages and grades were equally distributed.CONCLUSIONS:Our findings confirm the alternative role of FGFR3 and TP53 mutations in the development of bladder cancer. Together these two genetic markers are attributed to 62% of the tumors studied. Tumors with both wild type genes included urothelial carcinomas of all stages and grades and may develop through another genetic pathway. To elucidate complete molecular profile of bladder tumors further additional studies are needed.