Edyta Borkowska

Identification of Differentially Expressed Long Noncoding RNAs in Bladder Cancer

Purpose: Loss of epigenetic gene regulation through altered long noncoding RNA (lncRNA) expression seems important in human cancer. LncRNAs have diagnostic and therapeutic potential, and offer insights into the biology disease, but little is known of their expression in urothelial cancer. Here, we identify differentially expressed lncRNAs with potential regulatory functions in urothelial cancer. Experimental Design: The expression of 17,112 lncRNAs and 22,074 mRNAs was determined using microarrays in 83 normal and malignant urothelial (discovery) samples and selected RNAs with qPCR in 138 samples for validation. Significantly differentially expressed RNAs were identified and stratified according to tumor phenotype. siRNA knockdown, functional assays, and whole-genome transcriptomic profiling were used to identify potential roles of selected lncRNAs. Results: We observed upregulation of many lncRNAs in urothelial cancer that was distinct to corresponding, more balanced changes for mRNAs. In general, lncRNA expression reflected disease phenotype. We identified 32 lncRNAs with potential roles in disease progression. Focusing upon a promising candidate, we implicate upregulation of AB074278 in apoptosis avoidance and the maintenance of a proproliferative state in cancer through a potential interaction with EMP1, a tumor suppressor and a negative regulator of cell proliferation. Conclusions: We report differential expression profiles for numerous lncRNA in urothelial cancer. We identify phenotype-specific expression and a potential mechanistic target to explain this observation. Further studies are required to validate lncRNAs as prognostic biomarkers in this disease. Clin Cancer Res; 20(20); 5311–21. ©2014 AACR.

Concentration of RGDS-containing degradation products in uremic plasma is correlated with progression in renal failure

A concentration of protein degradation products containing the RGDS sequence, which could contribute to a lower reactivity of uremic platelets, has been estimated in both uremic (n = 16) and control (n = 7) plasmas. Degradation products and other small molecules were separated from plasma by filtration through AMICON YM-10 filter. RGDS antigen was determined in filtered material using the radioimmunoassay method based on monospecific anti-RGDS rabbit polyclonal antibodies. The concentration of RGDS-containing degradation products in uremic plasma ranged from 0.8 to 353 nM with mean value 58.6 +/- 24.9 nM and was higher than in control (0.7 to 5.9 nM, mean value 2.1 +/- 0.9 nM). Moreover, the level of RGDS-antigen positively correlated with plasma creatinine concentration (R = 0.87, p < 0.001). The filtered material showed an inhibitory effect on fibrinogen binding to control platelets in respect to RGDS-antigen concentration. We conclude that the elevated concentration of RGDS-containing degradation products in uremic plasma is partially responsible for bleeding tendency in renal failure.

Platelet membrane fluidity and intraplatelet Ca2+ mobilization are affected in uraemia

Abstract:  In present investigations, platelet membrane fluidity and intraplatelet Ca2+ mobilization were analysed in uraemic platelets by fluorescence techniques. Thirteen non‐dialyzed uraemic patients and 16 control subjects were examined. Anisotropy of DPH‐probe, measured at 37°C, was significantly higher in control (0.2236 ± 0.0050) than in uraemic platelets (0.1969 ± 0.0082; p < 0.01). There was no difference between control (109.8 ± 6.0 nm) and uraemic platelets (100.0 ± 7.3 nm) when the basal [Ca2+]i in resting platelets was determined. Activation of platelets by ADP (12.5 μm) or by thrombin (0.1 U/ml) resulted in an increase in [Ca2+]i. It was significantly higher (p* < 0.003 for ADP and p* < 0.009 for thrombin, respectively) in control platelets (383.6 ± 56.3 nm and 2031.0 ± 298.8 nm, respectively) than in uraemic ones (191.0 ± 21.3 nm and 838.7 ± 144.1 nm, respectively). The amount of released Ca2+ was higher in control platelets activated by both ADP and thrombin (157.6 ± 21.4 nm and 409.3 ± 71.0 nm, respectively) than in uraemic platelets (76.7 ± 15.7 nm and 203.0 ± 29.3 nm, respectively) and the differences were significant (p < 0.01 and p* < 0.01, respectively). These results indicate an abnormal intracellular Ca2+ mobilization in uraemic platelets. Both increased membrane fluidity and decreased Ca2+ mobilization should be considered as a possible reason of reduced fibrinogen receptor exposure on uraemic platelets.

EORTC risk tables - their usefulness in the assessment of recurrence and progression risk in non-muscle-invasive bladder cancer in Polish patients.

Introduction The assessment of risk of recurrence and progression of bladder cancer (BC) is still rather difficult. We decided to check the rates of the changes mentioned above in the group of the Polish patients after a year–long observation and next to compare them with the results calculated in the European Organisation of Research and Treatment of Cancer (EORTC) risk tables. Methods The tested group consisted of 91 patients who underwent transurethral resection of bladder tumour (TURBT). When being diagnosed, 60 cases were in the pTa clinical stage, whereas 30 cases were in T1. The coexisting carcinoma in situ (CIS) was observed in four cases. On the basis of the scores obtained from the EORTC tables, the patients were divided into the groups of low, intermediate or high risk of disease recurrence and progression. Results Recurrence was noticed in 23 patients (25%), while progression was observed in 11 patients (12.1%). The rate of the observed recurrences proved to be lower than it had been predicted in all the groups, except for one of the intermediate–risk group (score 1– 4). Moreover, the rate of the progressions predicted according to the EORTC risk tables was higher in all the risk groups. Conclusions It can be noticed that the rate of real recurrences is lower than expected, whereas the rate of the observed progressions is overestimated. Partly, it could be the result of using a relatively small group of patients for observation and applying a different method of treatment.

P53 mutations in urinary bladder cancer patients from Central Poland

The present study aimed at detection ofP53 gene mutations in cells of urinary bladder neoplasms, as the mutations may be regarded as an independent prognostic factor for progression and recurrence of tumours. In the study, 82 patients with clinically diagnosed urinary bladder tumour were included. The control was composed of DNA samples from urine and blood of 202 healthy patients. Exons 5–8 of theP53 gene were screened for mutations by using multitemperature single-strand conformational polymorphism (MSSCP) analysis. Samples with abnormal MSSCP patterns were subjected to direct sequencing. The frequency of mutations in exons 5–8 of theP53 gene in patients with bladder cancer was lower (3.3% in grade G1, 24% in G2, and 39% in G3) than the data reported in the literature. We found a higher percentage of polymorphism at codon 213 of theP53 gene in bladder cancer patients (6%), compared with the values in the reference group (2.5%). These results were matched with those of the loss of heterozygosity (LOH) analysis. In conclusion, mutations were found mainly in more advanced histopathological and clinical stages of the disease and at the CIS stage (carcinoma in situ). It cannot be excluded that the observed polymorphism at codon 213 may be a predisposing factor for urinary bladder carcinoma development.

Application of multiplex FISH, CGH and MSSCP techniques for cytogenetic and molecular analysis of transitional cell carcinoma (TCC) cells in voided urine specimens

Multiplex FISH (UroVysion), Comparative Genomic Hybridization (CGH), and Multitemperature Single-Strand Conformation Polymorphism (MSSCP) were applied for non-invasive diagnosis and prognosis of bladder cancer. The Uro Vysion test was positive in 80% of patients with pT1 and in 100% of patients with either pT2 or pT3 tumours. Tumours with pT3T4 stages were characterized by high numbers of chromosomal imbalances, detected by CGH. The mutation of the p53 gene was detected in 16% of patients, but only in those with pT2 or pT3 tumours.

Detection of bladder cancer in urine sediments by a hypermethylation panel of selected tumor suppressor genes

BACKGROUND Promoter hypermethylation can be a useful biomarker for early detection and prognosis of bladder cancer, monitoring response to treatment and complement classical diagnostic procedures. OBJECTIVE The molecular test was performed on DNA from bladder cancer cells in voided urine samples, tumor tissue DNA and normal control DNAs. We aimed to assess the diagnostic potential of epigenetic changes in urine DNA from bladder cancer cases at various clinico-pathological stages of the disease. METHODS The methylation status of 5 genes (p14ARF, p16INK4A, RASSF1A, DAPK, APC) in 113 tumor samples paired with voided urine specimens was analyzed by MSP. We compared the results of methylation analysis with UroVysion test. RESULTS The methylation profile in tumor/urine DNA was significantly correlated (p ≤ 0,05) with tumor grade in p14ARF, RASSF1a, APC/p14ARF, APC genes, respectively and with stage in p14ARF, RASSF1a/p14ARF genes, respectively. The results of UroVysion test were in correlation with hypermethylation both in tumor and urine DNA in p14ARF, RASSF1a and APC genes (p = 0,008; 0,02 and 0,04, respectively). CONCLUSIONS Promoter hypermethylation of tumor suppressor genes is a frequent mechanism in bladder cancer. We found promoter hypermethylation in all grades and stages of all cases examined. Methylation profile of selected suppressor genes may be a potential useful biomarker and enhance early detection of bladder cancer using a noninvasive urine test.

Molecular subtyping of bladder cancer using Kohonen self-organizing maps.

Kohonen self‐organizing maps (SOMs) are unsupervised Artificial Neural Networks (ANNs) that are good for low‐density data visualization. They easily deal with complex and nonlinear relationships between variables. We evaluated molecular events that characterize high‐ and low‐grade BC pathways in the tumors from 104 patients. We compared the ability of statistical clustering with a SOM to stratify tumors according to the risk of progression to more advanced disease. In univariable analysis, tumor stage (log rank P = 0.006) and grade (P < 0.001), HPV DNA (P < 0.004), Chromosome 9 loss (P = 0.04) and the A148T polymorphism (rs 3731249) in CDKN2A (P = 0.02) were associated with progression. Multivariable analysis of these parameters identified that tumor grade (Cox regression, P = 0.001, OR.2.9 (95% CI 1.6–5.2)) and the presence of HPV DNA (P = 0.017, OR 3.8 (95% CI 1.3–11.4)) were the only independent predictors of progression. Unsupervised hierarchical clustering grouped the tumors into discreet branches but did not stratify according to progression free survival (log rank P = 0.39). These genetic variables were presented to SOM input neurons. SOMs are suitable for complex data integration, allow easy visualization of outcomes, and may stratify BC progression more robustly than hierarchical clustering.

polymorphic variants of H-raS protooncogene and their possible role in bladder cancer etiology

Introduction H-RAS gene is a protooncogene encoding p21ras, a small protein with GTPase activity. This protein is a component of many signaling cascades, while mutations in H-RAS gene are often found in urinary bladder cancer and leads to continuous transmission of signals stimulating cancer cell growth and proliferation. The T81C polymorphism of H-RAS gene is a SNP, which, although does not seem to impair p21ras protein structure and function, may contribute to the development of bladder cancer. Objectives The aim of our study was to characterize the prevalence and clinical significance of T81C polymorphism in patients with diagnosed bladder cancer. Materials and methods 132 patients with diagnosed urinary bladder cancer were included in this study. The control group consisted of 106 healthy individuals. The experimental material was DNA, isolated from tumor tissue and peripheral blood lymphocytes. T81C polymorphism was detected using the MSSCP method and DNA sequencing. Results In the examined DNA samples, frequent polymorphic variations were found in codon 27 of H-RAS gene. In order to assess the clinical relevance of the polymorphism, the results were compared with those for the control group. The homozygous CC variant occurred more frequently in bladder cancer patients than in healthy individuals. Conclusions DNA polymorphisms start to play an important role in evaluation of disease risk and progression. The occurrence of multiple variants of the same gene may contribute to differences in reactions to specific medications and sensitivity to carcinogens or DNA repair capacity. Our study demonstrated T81C polymorphism of H-RAS gene to have seemingly been associated with an increased risk of bladder cancer development.

detection of loss of heterozygosity in patients with urinary bladder carcinoma: neoplastic tissue vs. urine sediment cells

Introduction Loss of heterozygosity (LOH) is frequently observed in urinary bladder neoplasms. In the reported study, an attempt was undertaken to determine the loss of heterozygosity of TP53(17p13), RB1(13q14), CDKN2A/ ARF(9p21) genes in DNA from neoplastic tissue, collected from patients with diagnosed urinary bladder carcinoma, and to compare the results with those of LOH evaluation in DNA isolated from urine sediment cells. Material and methods After isolation, DNA was amplified (PCR) by means of primers to five polymorphic microsatellite markers, the products being then separated on agarose gel. Following the method, a total of 125 DNA samples were obtained, isolated from neoplastic cells, together with 125 corresponding DNA samples, isolated from urine sediment cells. Results The loss of heterozygosity in at least one marker was identified in 39.2%. (49/125) of DNA from studied tumors and in 34.3% (43/125) of DNA samples, isolated from urine sediment cells. An analysis of LOH from the DNA, isolated from urine sediment cells, allowed for identification of 81.8% of neoplastic tumors with 99.7% specificity. Conclusions Our observations have demonstrated that LOH within 13q14, 17p13 and 9p21 loci is more often observed in clinically more advanced neoplasms. LOH in 17p13 locus is more frequently found in tumors at high histopathological stage, while in low-stage neoplasms, LOH is most often observed on chromosome 9. The high sensitivity (81.8%) and specificity (99.7%) of LOH studies in DNA, isolated from urine sediment cells, make this technique an advantageous, non-invasive method for detection and screening of bladder cancer.