Zbigniew Jabłonowski

Hypermethylation of p16 and DAPK promoter gene regions in patients with non-invasive urinary bladder cancer

Introduction The aim of the study was to examine the frequency of methylation status in promoter regions of p16 and DAPK genes in patients with non-invasive bladder cancer. Material and methods Forty-two patients (92.9% men, 73.8% smokers, 71.4% T1G1, 19.1% T1G2, 9.5% T1G3) and 36 healthy controls were studied. Isolation of genomic DNA from blood serum and methylation-specific PCR (MSP) were applied. Methylation status – methylated and unmethylated promoter regions of p16 and DAPK genes were analysed. Results Seventeen out of 42 patients (40.5%) had the methylated p16 gene, while methylation of the DAPK gene was seen in 27 of 42 cases (64.3%). In 12 patients (28.6%) both analysed genes were methylated. A statistically significant (p = 0.046) higher frequency of DAPK gene methylation (71.4%) was observed in patients with lower grade (G1) bladder cancer. Conclusions Detection of the aberrant hypermethylation of DAPK and p16 genes in blood DNA from non-invasive bladder cancer patients might offer an effective means for earlier auxiliary diagnosis of the malignancy.

Level of selenoprotein transcripts in peripheral leukocytes of patients with bladder cancer and healthy individuals.

Abstract Background: Low concentrations of selenium (Se) in humans have been associated with risk of cancer. Selenoprotein mRNAs can potentially be regulated by Se status. Methods: Se status, GPx1 Pro198Leu and Sep15 1125G/A genetic polymorphism and human (h)GPx1, hGPx3, hSep15 and hSeP1 transcript levels in peripheral leukocytes of 33 males with bladder cancer and 47 healthy male controls were analysed. Results: All the subjects expressed detectable selenoprotein mRNA concentrations in leukocytes. Significantly lower expression of hGPx1, hGPx3, hSep15 and hSeP1 in leukocytes of bladder cancer patients compared to controls was observed. hGPx1, hGPx3 and hSep15 expression was significantly lower in non-smokers in the control group compared with smokers in the control group. A positive relationship between expression of all studied genes was also observed in non-smoking controls. Expression of hGPx3 and hSep15 gradually increased with tumour grade in patients with cancer. We did not find any association between selenoprotein mRNA levels, Se status and selenoprotein genetic polymorphism. Conclusions: This study showed significant down-regulation of hGPx1, hGPx3, hSep15 and hSeP1 mRNA levels in leukocytes of patients with bladder cancer compared to controls. Selenoprotein transcript levels in circulating leukocytes of patients with bladder cancer and controls revealed no potential impact of Se status on selenoprotein expression. Clin Chem Lab Med 2009;47:1125–32.

Genetic polymorphisms in matrix metalloproteinases (MMPs) and tissue inhibitors of MPs (TIMPs), and bladder cancer susceptibility.

To elucidate genetic polymorphisms of the matrix metalloproteinases (MMPs) MMP1 (rs1799750), MMP2 (rs243865), MMP9 (rs3918242), MMP12 (rs2276109) and tissue inhibitors of MMPs (TIMPs) TIMP1 (rs2070584) and TIMP3 (rs9619311) genes that may be involved in susceptibility to bladder cancer (BC).

GSTP1 mRNA expression in human circulating blood leukocytes is associated with GSTP1 genetic polymorphism

OBJECTIVES We explored association between GSTP1 Ile(105)Val (rs1695) polymorphism and GSTP1 mRNA expression in circulating blood leukocytes. DESIGN AND METHODS GSTP1 transcripts level and polymorphism were determined by Real-Time PCR in 51 bladder cancer and 90 healthy men. RESULTS Individuals with at least one GSTP1 Val(105) variant allele possessed higher GSTP1 mRNA level in blood leukocytes compared to GSTP1 Ile(105)Ile carriers. CONCLUSIONS GSTP1 Ile(105)Val gene polymorphism influences its expression in blood, regardless of cancer disease.

Novel Biomarkers in the Diagnosis of Chronic Kidney Disease and the Prediction of Its Outcome.

In its early stages, symptoms of chronic kidney disease (CKD) are usually not apparent. Significant reduction of the kidney function is the first obvious sign of disease. If diagnosed early (stages 1 to 3), the progression of CKD can be altered and complications reduced. In stages 4 and 5 extensive kidney damage is observed, which usually results in end-stage renal failure. Currently, the diagnosis of CKD is made usually on the levels of blood urea and serum creatinine (sCr), however, sCr has been shown to be lacking high predictive value. Due to the development of genomics, epigenetics, transcriptomics, proteomics, and metabolomics, the introduction of novel techniques will allow for the identification of novel biomarkers in renal diseases. This review presents some new possible biomarkers in the diagnosis of CKD and in the prediction of outcome, including asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), uromodulin, kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), miRNA, ncRNA, and lincRNA biomarkers and proteomic and metabolomic biomarkers. Complicated pathomechanisms of CKD development and progression require not a single marker but their combination in order to mirror all types of alterations occurring in the course of this disease. It seems that in the not so distant future, conventional markers may be exchanged for new ones, however, confirmation of their efficacy, sensitivity and specificity as well as the reduction of analysis costs are required.

Comparison of Neodymium-Doped Yttrium Aluminum Garnet Laser Treatment with Cold Knife Endoscopic Incision of Urethral Strictures in Male Patients

OBJECTIVE To assess the effectiveness of visual laser ablation treatment with neodymium-doped yttrium aluminum garnet (Nd:YAG) laser in male patients with urethral strictures and to compare the effects with those obtained in patients treated with Sachses optical urethrotomy. MATERIALS AND METHODS Fifty patients aged 22 to 83 (mean age 61.8) with primary (n = 26, 52%) and recurrent (n = 24, 48%) urethral strictures 0.3 to 2.4 cm long qualified for the study. The patients were randomized into two groups: 30 men treated using visual laser ablation of urethral strictures (VLASU) with Nd:YAGlaser and 20 men treated by correction of urethral strictures using Sachses optical urethrotomy. RESULTS At 12-month follow-up, seven (35%) patients who underwent optical urethrotomy and 21 (70%) in the VLASU group did not require repetition of the procedure. The choice of VLASU as a method of treatment significantly decreased the probability of therapeutic failure and recurrence of urethral strictures (p = 0.02). CONCLUSION VLASU can be used as a method of treatment of this disorder. It is an effective, modern, low-invasive, and repeatable technique and is technically simple and easy to master. It can be used in cases in which introduction of a 22 Char optical urethrotome into the stricture site is impossible, as well as for treatment of multiple strictures during one procedure.

MMP, VEGF and TIMP as prognostic factors in recurring bladder cancer

OBJECTIVES To investigate the clinical correlates and prognostic utility of MMP, VEGF and TIMP genes expression in bladder cancer (BCa) recurrence. METHODS Expression of MMP1, MMP2, MMP9, VEGFA and TIMP1, TIMP3 was analyzed by qRT-PCR using SYBR Green in peripheral blood leukocytes (PBLs) of BCa patients at two time points (diagnosis (n=40), and first recurrence (n=40)) and an age-matched group of healthy controls (n=100). Plasma concentrations of MMP1 (pro- and active forms) were measured using ELISA in BCa patients. RESULTS The expression of MMP1 mRNA was significantly lower in BCa patients with first recurrence compared to control (p=0.019). Expression of other genes did not differ significantly between the groups. MMP9 gene expression was associated with differentiation grade (p=0.043), with the highest expression in poorly differentiated tumors (G3) and was higher in smokers than in non-smokers (p=0.039) in BCa patients at diagnosis. The results at two time points showed that MMP9 and VEGFA genes expression was increased in patients with moderately differentiated BCa (p=0.029), and advanced pathologic stage (p=0.048), respectively. Moreover, gene expression of TIMP1 was increased for G3 (p=0.043), and was decreased for early recurrence (p=0.003). CONCLUSIONS Our study suggests that the expression of MMP9 in PBLs of BCa patients at diagnosis is associated with the differentiation grade of the BCa, and smoking status. Genes expression of MMP9, VEGFA and TIMP1 in PBLs may play a pivotal role in regulation of progression of BCa. Additionally, TIMP1 gene expression may be important factor for early recurrence of BCa.

MMP7 and MMP8 genetic polymorphisms in bladder cancer patients

Introduction Breakdown of the extracellular matrix by matrix metalloproteinases (MMPs), as we know, is one of mechanisms involved and required in tumor invasion. MMP7 is a negative prognostic factor of various malignances, while MMP8 exhibits an inhibitory effect on tumorigenesis and metastasis. We evaluated the potential association of functional polymorphisms in the promoter of the MMP7 (rs11568818) and MMP8 (rs11225395) genes and bladder cancer (BCa) risk. Materials and methods The study included 241 BCa cases and 199 healthy population controls that were collected at the First Department of Urology, Medical University (Łódź, Poland) and at the Nofer Institute of Occupational Medicine (Łódź, Poland). Genomic DNA samples were isolated from venous blood and genetic polymorphisms were analyzed by real–time polymerase chain reaction using TaqMan fluorescent probes. Associations between genotype and allele status were estimated by logistic regression models adjusted for classic risk factors (e.g. age, gender and cigarette smoking). Results MMP7 and MMP8 genotypes were distributed similarly in BCa patients and in controls and at least one variant allele was not associated with BCa cancer risk (OR, 0.91; 95% CI, 0.60–1.39; p = 0.662 for MMP7 and OR, 0.96; 95% CI, 0.63–1.46; p = 0.836 for MMP8). We observed higher prevalence of MMP7 GG genotypes among BCa patients than in controls (OR, 1.54; 95% CI, 0.93–2.55; p = 0.093). Additionally, genetic polymorphisms in the MMP7 and MMP8 were not associated with the tumor grade or stage. Conclusions Our results suggest that genetic variations in two genes encoding members of the MMP7 and MMP8 are not associated with a risk of BCa in the Caucasian population.

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.