Veronika Holubekova

Active cigarette smoking and the risk of breast cancer at the level of N-acetyltransferase 2 (NAT2) gene polymorphisms

The aim of our study was to assess the correlation between the tobacco exposure and NAT2 gene (rs1041983 C/T, rs1801280 T/C, rs1799930 G/A) polymorphisms in association with breast cancer development. We wanted to determine the prognostic clinical importance of these polymorphisms in association with smoking and breast cancer. For the detection of possible association between smoking, NAT2 gene polymorphisms, and the risk of breast cancer, we designed a case-controlled study with 198 patients enrolled, 98 breast cancer patients and 100 healthy controls. Ten milliliters of peripheral blood from the cubital vein was withdrawn from every patient. The HRM (high resolution melting) analysis was used for the detection of three abovementioned NAT2 gene polymorphisms. When comparing a group of women smoking more than 5 cigarettes a day with the patients smoking fewer than 5 cigarettes a day, we found out that if women were the carriers of aberrant AA genotype for rs1799930, the first group of women had higher risk of breast carcinoma than the second group. If patients were the carriers of aberrant TT genotype for rs1041983, for rs1801280CC genotype, and rs1799930AA genotype and they smoked more than 5 cigarettes a day, they had higher risk of malignant breast disease than never-smoking women. Our results confirm the hypothesis that NAT2 gene polymorphisms (rs1041983 C/T, rs1801280 T/C, and rs1799930 G/A) in association with long-period active smoking could be the possible individual risk-predicting factors for breast cancer development in the population of Slovak women.

Determination of malignant potential of cervical intraepithelial neoplasia.

Basic diagnostic procedures in cervical cancer screening are able to set the diagnosis but they do not provide any information about the biological nature and behavior of lesions. The causal link of HPV infection and cervical cancer and discoveries of complex interactions between host and HPV genome opened new possibilities in molecular diagnostics. HPV DNA analysis, determination of viral load, detection of E6 and E7 mRNA transcripts, identifying of methylation profiles, genomic changes, miRNAs, and telomerase activity should be the right choice for exact diagnostics and prediction of behavior of premalignant lesions of the cervix. These findings set a completely new light not only in diagnostic but also in management and treatment of cervical dysplasia and cervical cancer.

Methylation pattern of CDH1 promoter and its association with CDH1 gene expression in cytological cervical specimens

Cervical cancer is the fourth leading cause of cancer mortality in females worldwide. Infection with high-risk human papillomavirus (HPV) is essential but insufficient to cause cervical cancer, and the clearance of HPV infection is mediated by the immune system. The deficit of molecules responsible for adhesion may play a role in the development of cervical cancer. E-cadherin is encoded by the cadherin 1 (CDH1) gene, and is involved in cell adhesion by forming adherens junctions. The aim of present study was to investigate the methylation pattern of the CDH1 promoter and to identify the association between CDH1 promoter hypermethylation, CDH1 gene expression and HPV infection in cervical specimens obtained from 93 patients with low-grade squamous intraepithelial lesions (SILs), high-grade SILs or squamous cell carcinomas, and from 47 patients with normal cervical cytology (HPV-negative). The methylation pattern of the CDH1 promoter was investigated by methylation-specific polymerase chain reaction and quantitative pyrosequencing. CDH1 gene expression was measured by relative quantification. CDH1 methylation was significantly higher in both types of lesions and in cervical cancer than in normal samples, and CDH1 gene expression was significantly reduced during SIL progression (P=0.0162). However, the influence of HPV infection or HPV E6 expression on the methylation pattern of the CDH1 gene or its gene expression levels could not be confirmed. The present results support that the methylation of the CDH1 gene is age-related in patients with cervical lesions (P=0.01085), and therefore, older patients could be more susceptible to cancer than younger patients. The important methylation of the CDH1 promoter occurred near the transcription factor binding sites on nucleotides −13 and +103, which are close to the translational start codon. These results suggest that methylation at these sites may be an important event in the transcriptional regulation of E-cadherin, and in patients harboring these methylated cytosines, this event may facilitate HPV-driven carcinogenesis.

Epigenetic regulation by DNA methylation and miRNA molecules in cancer

Division of Oncology, Biomedical Center Martin, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, SK-03601 Martin, Slovakia Division of Molecular Medicine, Biomedical Center Martin, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, SK-03601 Martin, Slovakia Department of Obstetrics and Gynecology, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin University Hospital, SK-03601 Martin, Slovakia Department of Molecular Biology, Biomedical Center Martin, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, SK-03601 Martin, Slovakia *Author for correspondence: Tel.: +421 43 2633 651; veronika.holubekova@jfmed.uniba.sk

Methylation of CADM1 and MAL together with HPV status in cytological cervical specimens serves an important role in the progression of cervical intraepithelial neoplasia

Cervical cancer (CC) is the second most common type of cancer affecting the female population. The development of CC takes several years, and involves a precancerous stage known as cervical intraepithelial neoplasia (CIN). A key factor in the development of disease is the human papillomavirus (HPV) infection, which initiates carcinogenesis. Furthermore, CC is also impacted by epigenetic changes such as DNA methylation, which causes activation or exclusion of certain genes, and the hypermethylation of cytosines in promoters, thereby switching off previously active genes. The majority of DNA methylation events occur at cytosine-guanine nucleotides, which in the human genome are known as CpG islands. The aim of the present study was to investigate the methylation levels in intronic sequences of the two tumor suppressor genes cell adhesion molecule 1 (CADM1) and T-lymphocyte maturation associated protein (MAL) using cytological samples and to identify potential biomarkers involved in CIN by pyrosequencing. DNA was isolated from cervical smears from patients with CINs, with healthy patients serving as a control group. Samples were converted by treatment with sodium bisulfite and subsequent pyrosequencing to detect the methylation status of the selected genes. The presence of HPV DNA infection analyzed by the polymerase chain reaction, was detected in each sample. Of the total number of samples (n=91), the present study confirmed the presence of one or two high-risk subtypes of HPV in 39 cases (42.85%) and HPV infection was significantly associated with CIN2+ lesions. For the two genes (MAL and CADM1) the present study confirmed that the median methylation was significantly higher in HPV positive patients [P=0.0097, 95% confidence interval (CI): (−0.030, −0.003)/P=0.0024, 95% CI: (−0.06, −0.01)] when compared with patients negative for HPV DNA infection, and the average methylation was demonstrated to be increased with the degree of cervical lesion. The present study used logistical regression to model the dependence between the case/control statuses (control group vs. Dg. 1–4). The area under the curve values for MAL were: 84% for cervical inflammation, 71% for CIN1, 73.4% for CIN2+ and 77% for squamous cell carcinoma (SCC); and for CADM1 were: 88.6% for cervical inflammation, 68% for CIN1, 80% for CIN2+ and 89% for SCC. The present study confirmed that there were statistically significant differences between the methylation levels of individual CpGs and significantly higher median methylation in patients positive for HPV16/18. CADM1 exhibited higher levels of methylation in almost every study group when compared with MAL during the transition of CIN and appeared to be a promising biomarker for future study.

Methylation status of KLF4 and HS3ST2 genes as predictors of endometrial cancer and hyperplastic endometrial lesions

Endometrial carcinoma is one of the most common tumours in developed countries. In addition to the active role of genetic factors, epigenetic changes also have an important effect. The present study analysed the methylation status of kruppel like factor 4 (KLF4) and heparan sulfate-glucosamine 3-sulfotransferase 2 (HS3ST2) genes in three endometrial tissue types for carcinoma prediction. The sample comprised 91 women with histologically-confirmed endometrial carcinoma (64.16±9.64 years old), 36 women with hyperplasia (53.39±9.64 years old) and 45 with no signs or symptoms of malignancy (48.53±11.11 years old). The CpG dinucleotide methylation levels were examined by quantitative pyrosequencing, and the discrimination accuracy of the model was calculated using the Random Forest classification algorithm of the area under the ROC curve (AUC). The mean values of KLF4 and HS3ST2 methylation indices were 23.83±11.39 and 8.52±2.57 in the control samples; 30.40±8.52 and 33.76±20.66 in hyperplasia and 34.72±10.79 and 34.49±18.39 in the cancerous tissues. Multinomial logistic regression indicated that the HS3ST2 CpG1 methylation status is a predictor of hyperplasia (P<0.05) and that the KLF4 CpG2 dinucleotide can predict carcinoma formation (P<0.001). The AUC value of 0.95 indicates high discrimination accuracy of the CpG nucleotides methylation status model between the controls and the two other diagnoses. The results of the present study establish the likelihood that aberrations in KLF4 and HS3ST2 gene methylation levels are important in the development of endometrial hyperplasia and carcinoma, with hyperplasia an intermediate step between healthy and tumour tissues.

miRNA in a multiomic context for diagnosis, treatment monitoring and personalized management of metastatic breast cancer

Metastatic breast cancer is characterized by aggressive spreading to distant organs. Despite huge multilevel research, there are still several important challenges that have to be clarified in the management of this disease. Therefore, recent investigations have implemented a modern, multiomic approach with the aim of identifying specific biomarkers for not only early detection but also to predict treatment responses and metastatic spread. Specific attention is paid to short miRNAs, which regulate gene expression at the post-transcriptional level. Aberrant miRNA expression could initiate cancer development, cell proliferation, invasion, migration, metastatic spread or drug resistance. An miRNA signature is, therefore, believed to be a promising biomarker and prediction tool that could be utilized in all phases of carcinogenesis. This article offers comprehensive information about miRNA profiles useful for diagnostic and treatment purposes that may sufficiently advance breast cancer management and improve individual outcomes in the near future.