Maria Constantinou

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.

Familial distal monosomy 3p26.3-pter with trisomy 4q32.2-qter, presenting with progressive ataxia, intellectual disability, and dysmorphic features.

We present a boy diagnosed with partial 3p monosomy and partial 4q trisomy. The patient was 9 years of age with intellectual disability, dysmorphic features, and ataxia. A family history and medical evaluation showed that the father manifested similar facial dysmorphic features, intellectual disability, quadriparesis, and progressive cerebrospinal ataxia. The chromosomal aberration found in the proband was inherited from his father who was found to have a balanced reciprocal translocation of chromosomes 3p and 4q, which was in turn inherited from the paternal grandfather. The final cytogenetic diagnosis according to microarray was 46,XY,der(3)t(3;4)(p26.1;q32.2)arr 3p26.1(39,066–5,363,502)x1,4q32.2q35.2(162,555,236–191,173,881)x3. We describe the cytogenetic investigations that led to the identification of the breakpoints. In addition, we present an overview of the clinical features found in patients with partial 3p monosomies and partial 4q trisomies as reported in the literature.

Concomitance of oncogenic HPV types, CHEK2 gene mutations, and CYP1B1 gene polymorphism as an increased risk factor for malignancy

Introduction Urinary bladder carcinoma ranks the fourth position in malignancy incidence rates in men (6.1%) and the 17th position in women (1.6%). In general, neoplastic diseases should be approached from two perspectives: prevention with implementation of prophylactic measures and early diagnostics. Prophylactics is possible in the preclinical phase of neoplasm, being both justified and plausible in patients from high–risk groups. Thus, it is particularly important to select such groups, not only by referring to environmental carcinogenic factors (occupational and extra–occupational) but also from genetic predisposition, which may be conductive for neoplasm formation. The mutations / polymorphisms of CHEK2 and CYP1B1 genes predispose to neoplasm via multiorgan mechanisms, while the human papilloma virus (HPV) may participate in the neoplastic transformation as an environmental factor. Material and methods 131 patients with diagnosed urinary bladder cancer were qualified to the study. Mutations/polymorphisms of CHEK2 (IVS2 + 1G > A gene, 1100delC, del5395, I157T) and CYP1B1– 355T/T were identified by the PCR in DNA isolated directly from the tumor and from peripheral blood. The ELISA test was used for the studies of 37 HPV genotypes in DNA, isolated tumour tissue. Results 11 mutations of CHEK2 gene were found, 355T/T polymorphism if CYP1B1 gene occurred in 18 patients (12.9%). Oncogenic HPV was found in 36 (29.3%), out of 123 examined patients. Conclusions The concomitance of CHEK2 gene mutations or 355T/T polymorphism of CYP1B1 gene and the presence of oncogenic HPV types statistically significantly correlates with histological malignancy grades of urinary bladder carcinoma.

Genomic imbalance in endometrial hyperplasia and cancer

Endometrial cancer belongs to the most frequently diagnosed malignant neoplasms of female genital organs, and its incidence is steadily growing. For the timebeing, no chromosomal aberrations have been determined unequivocally, which would be specific for the particular stages of endometrial hyperplasia and neoplastic transformation development. The goal of the undertaken studies was an identification of the earliest and specific genetic changes, which could be attributed to an increased risk of neoplastic transformation in a group of patients with endometrial hyperplasia plus the characteristics of genetic changes associated with the mature form of neoplasm. The study involved forty-four (44) patients, including five (5) histopathologically unconfirmed hyperplasia, twenty-six (26) with histopathologically confirmed endometrial hyperplasia and thirteen (13) with diagnosed endometrial cancer. The applied aCGH (array Comparative Genomic Hybridisation) method enabled selection of a few chromosomal regions which indicated a higher incidence of chromosomal rearrangements than in the control group. The study included also an evaluation of the frequency of mutations of the genes specific for neoplastic transformation development, the genes at chromosomal loci, which most frequently presented with genomic imbalance. In cases without hyperplasia, changes were diagnosed, described as CNVs (Copy Number Variations), which occurred with varying prevalence in the genome of the population of healthy subjects. Significant genomic imbalance was identified in 26 (100%) patients with diagnosed hyperplasia and in 11 (84.6%) of the patients with diagnosed endometrial cancer. Also other, till now unreported changes were found, localised at characteristic regions of the genome.

The prevalence of CHEK2 and CYP1B1 mutations/polymorphisms in urinary bladder cancer

This work was supported by the State Committee for Scientific Research (KBN Poland) grant No. N401 197 32/4212.

Artificial neural network in predicting bladder cancer recurrence

The more we learn about human genetics and human variation, the more apparent it becomes that our individual make-up has a noticeble impact on the effectiveness of medications. Urinary bladder cancer is the sixth leading cause of mortality due to malignant neoplasm among Polish man in Lodz region. Many genetic and epigenetic alterations have been identified that contribute directly to the development of bladder tumors. The aim of the project was the creation of the individual risk calculator of bladder cancer recurrence using available clinical data and the results of the long term genetic research.