Methylation Status Of GSTP1 Research Articles

Methylation of Glutathione-S-Transferase P1 Promotor in Egyptian Females with Breast Cancer

Background:Breast cancer (BC) is the second most common cancer after the lung cancer worldwide and number one killing cancer in Egyptian females . It is a multifactorial disease driven by different environmental, hormonal, genetic and epigenetic factors. Epigenetic alterations have been studied in cancer breast. Role of GSTP1 promotor methylation in breast cancer has been studied in different ethnic groups. Objectives:Current study aimed at studying the methylation status of the promotor region of glutathione-S-transferase P1 in breast ductal carcinoma of a cohort group of Egyptian females and its correlations with histopathological and prognostic parameters. Methods:Control group included 15 fresh normal breast tissues taken from BC female patients after leaving a clearly defined safety margin and a Patient group included confirmed 35 fresh breast ductal carcinoma tissue biopsies taken from female patients postoperatively. To all patients clinical examination, radiological examination (plain X-ray chest and or CT scan, ultrasonography of abdomen and pelvis were done), in addition to histopathological examination, typing, grading and staging of tumour, hormonal receptors status and molecular typing of breast mass. GSTP1 methylation status was evaluated using methyl specific polymerase chain reaction. Results:Statistical significant increase was found in methylation status of GSTP1 promotor gene in BC cases than that in control group, (60% of patients samples had methylated GSTP1 promotor vs only 6.7% of controls) (p= >0.001). No association was found between GSTP1 promotor methylation status and the poor prognostic factors neither with hormonal profile nor molecular type. However, GSTP1 promotor methylation were two times higher in postmenopausal than premenopausal cases and three times higher in late grade (III). Also GSTP1 promotor methylation was 2.4 times higher in Her2 positive cases than either ER or PR positive cases. Conclusion: Glutathione-S-Transferase P1 Promotor methylation plays a role in breast cancer development.

The methylation status of GSTP1, APC, and RASSF1 genes in human prostate cancer samples: Comparative analysis of diagnostic informativeness of MS-HRM and hybridization on the Illumina Infinium HumanMethylation450 BeadChip

There is a clear need in molecular markers of prostate cancer (PC) that would specify stratification of patients into risk categories and serve as additional parameters to clinical prognostic factors. Altered DNA methylation status is one of molecular processes that occur during PC development. The gene methylation status may be evaluated in laboratory diagnostics by a PCR approach known as means of methylation-sensitive high resolution melting (MS-HRM) curve analysis. This method is particularly promising due to minimal amounts of DNA required for analysis (10 ng). To date, numerous data based on the use of Infinium HumanMethylation450 BeadChip (HM450) technology have been accumulated on DNA methylation in PC samples. However, it remains unclear whether the MS-HRM results are consistent with chip hybridization data. The aim of this study was to perform the comparative analysis of the diagnostic informativeness of MS-HRM and HM450 chip hybridization methods in determination of the methylation status of three genes GSTP1, APC, and RASSF1 in samples obtained from PC patients. The study has shown that using the MS-HRM method it is possible to discriminate PC tumor samples from samples of histologically unaltered prostate tissue by analyzing the methylation status of each gene. Analysis of chip hybridization data confirmed the results obtained by MS-HRM. Differences between tumor tissue and histologically intact tissue obtained from each PC patient in the methylation levels of GSTP1, APC, and RASSF1, determined by MS-HRM or chip hybridization were consistent with each other. Thus, the assessment of the methylation level of gene GSTP1, APC, and RASSF1 using MS-HRM can become the basis for the development of a laboratory test, the specifying diagnosis of prostate cancer.

A methylation-specific dot blot assay for improving specificity and sensitivity of methylation-specific PCR on DNA methylation analysis.

Developing a methylation-specific dot blot assay (MSP-DB) to increase the sensitivity and specificity of simultaneous methylation analysis of multiple genes is the goal of the present study to evaluate the methylation status of GSTP1 and RASSF1A from prostate cancer in Vietnamese males. The methylation of GSTP1 and RASSF1A was investigated by using the MSP in 50 prostate cancer and 17 benign prostate hyperplasia specimens. The MSP-DB assay that uses a single or multiple probes specifically detected the methylation status of a particular gene or of the two genes GSTP1 and RASSF1A at the same time in a series of samples. The sensitivity and specificity of the MSP-DB were compared to those of the MSP. The probes specifically hybridized with the methylated targets only in the MSP-DB, which allowed detecting GSTP1 and RASSF1A methylation in 23 of 50 and 14 of 50 patients with prostate cancer and in 2 of 17 and 4 of 17 patients with benign prostate hyperplasia. MSP-DB following the MSP assay improved the sensitivity of detection to more than 0.01 % methylated status of a given high CpG-rich region. One methylated MSP product corresponding to the GSTP1, lack of methylated cytosine, was clearly detected on gel electrophoresis but barely visible on MSP-DB. Thus, the MSP-DB is suitable to eliminate the risk of false-positive results. The MSP-DB dispels the weakness of MSP and increases the sensitivity to simultaneous methylation analysis of multiple genes. The MSP-DB is advantageous for the promotion of DNA methylation markers in progressing quickly toward clinical application.

Promoter hypermethylation of the tumor-suppressor genes RASSF1A, GSTP1 and CDH1 in endometrial cancer

Endometrial cancer is a common gynecological malignancy with a good prognosis in early stages of the disease. The CpG island in the promoter region of tumor-suppressor genes are frequently methylated in various types of human cancers. In the present study, we examined the methylation status of the GSTP1, CDH1 and RASSF1A genes in endometrioid endometrial cancer (EEC), endometrial complex hyperplasia (EHP) and healthy endometrium with the aim to identify correlations between promoter hypermethylation, disease risk and clinicopathological parameters. A nested two-stage methylation-specific PCR (MSP) was performed to analyze the promoter CpG methylation status of GSTP1, CDH1 and RASSF1A genes in the population studied. A total of 92 subjects were initially included in the study of which 41 EEC, 19 EHP and 20 controls were processed for final analyses. A significant difference was found between the study groups and the presence of promoter CpG hypermethylation status in the GSTP1 (p<0.05) and RASSF1A (p<0.0001) genes. RASSF1A, GSTP1 and CDH1 gene promoter methylation was present in 85.4, 68.3 and 31.4% of EEC samples when compared to that in the controls with 30.0, 35.0 and 20.0%, respectively. CpG methylation of all three investigated tumor-suppressor genes was found in 12.2% of EEC patients, in 4.2% of EHP patients and in 3.7% of the controls, respectively. Positive findings for the promoter methylation of two investigated genes were found in 48.7% of EEC patients, 26.0% of EHP patients and in 18.5% of the controls. With regard to histopathological variables and CpG methylation, we found significant correlations between the RASSF1A and GSTP1 genes and higher tumor grade, deeper myometrial invasion and positive metastatic involvement of pelvic lymph nodes. No associations were noted between promoter hypermethylation of the CDH1 gene and biological features of the endometrial cancer cases. The results indicate that aberrant CpG methylation of the promoter region in the GSTP1 and RASSF1A tumor-suppressor genes is an important event in carcinogenesis of endometrial cancer and may have an impact on tumor aggressiveness. Finally, the present study suggests that epigenetic alterations may be of diagnostic value for the better clinical management of premalignant endometrial lesions.

Clinical Utility of an Epigenetic Assay to Detect Occult Prostate Cancer in Histopathologically Negative Biopsies: Results of the MATLOC Study

Concern about possible false-negative prostate biopsy histopathology findings often leads to rebiopsy. A quantitative methylation specific polymerase chain reaction assay panel, including GSTP1, APC and RASSF1, could increase the sensitivity of detecting cancer over that of pathological review alone, leading to a high negative predictive value and a decrease in unnecessary repeat biopsies. The MATLOC study blindly tested archived prostate biopsy needle core tissue samples of 498 subjects from the United Kingdom and Belgium with histopathologically negative prostate biopsies, followed by positive (cases) or negative (controls) repeat biopsy within 30 months. Clinical performance of the epigenetic marker panel, emphasizing negative predictive value, was assessed and cross-validated. Multivariate logistic regression was used to evaluate all risk factors. The epigenetic assay performed on the first negative biopsies of this retrospective review cohort resulted in a negative predictive value of 90% (95% CI 87-93). In a multivariate model correcting for patient age, prostate specific antigen, digital rectal examination and first biopsy histopathological characteristics the epigenetic assay was a significant independent predictor of patient outcome (OR 3.17, 95% CI 1.81-5.53). A multiplex quantitative methylation specific polymerase chain reaction assay determining the methylation status of GSTP1, APC and RASSF1 was strongly associated with repeat biopsy outcome up to 30 months after initial negative biopsy in men with suspicion of prostate cancer. Adding this epigenetic assay could improve the prostate cancer diagnostic process and decrease unnecessary repeat biopsies.

DNA Methylation ofGSTP1in Human Prostate Tissues: Pyrosequencing Analysis

PurposeDNA methylation is an important epigenetic mechanism of gene regulation and plays essential roles in tumor initiation and progression. Differences in methylation patterns between neoplastic and normal cells can be used to detect the presence of cancer. The aim of the present study was to evaluate the usefulness of glutathione-S-transferase-Pi (GSTP1) hypermethylation in discriminating between normal and prostate cancer (PCa) cells and in predicting tumor characteristics by use of quantitative pyrosequencing analysis.Materials and MethodsA total of 100 human prostate tissues obtained from our institute were used in this study: 45 for benign prostatic hyperplasia (BPH) and 55 for PCa. The methylation level of GSTP1 was examined by a quantitative pyrosequencing analysis. The associations between GSTP1 methylation level and clinico-pathological parameter were also compared.ResultsThe level of GSTP1 methylation was significantly higher in PCa samples than in BPH samples (56.7±32.7% vs. 1.6±2.2%, p<0.001). The sensitivity and specificity of GSTP1 methylation status in discriminating between PCa and BPH reached 85.5% and 100%, respectively. Even after stratification by stage, Gleason score, and prostate-specific antigen (PSA) level, similar results were obtained. A positive correlation between GSTP1 methylation level and serum PSA level was observed (r=0.303, p=0.002). There were no associations between GSTP1 methylation level and age, Gleason score, and staging.ConclusionsOur study demonstrates that GSTP1 methylation is associated with the presence of PCa and PSA levels. This methylation marker is a potentially useful indicator for the detection and monitoring of PCa.

Abstract B62: Epigenetic DNA methylation changes may underlie racial differences and susceptibility to prostate cancer.

Abstract Prostate cancer (PCa) is a common malignancy and a leading cause of cancer deaths among men in the United States. African American (AA) men have both a higher incidence and significantly higher mortality rates than Caucasian (Cau) men. Abundant evidence has accumulated to suggest that epigenetic DNA methylation changes may appear earlier during PCa development than genetic changes, as well as more commonly and consistently. Most studies have emphasized DNA hypermethylation as an important mechanism for inactivation of key regulatory genes in prostate cancers. Thus methylated genes can serve as biomarkers for the detection of cancer from clinical specimens such as tissue biopsies or body fluids. We used pyrosequencing to quantitatively measure methylation status of GSTP1, AR, RAR 2, SPARC, TIMP3 and NKX2-5 in prostate tissue specimen from AA and Cau men. Statistical analysis showed significantly higher methylation in the prostate cancer tissue samples in comparison with matched normal samples. Overall, we observed significant differences (p &amp;lt; 0.05) in the methylation level for all genes (except GSTP1) in the AA samples in comparison to the Cau samples. Furthermore, regression analysis revealed significantly higher methylation for NKX2-5 (p = 0.008) and TIMP3 (p = 0.039) in normal prostate tissue samples from AA in comparison with Cau, and a statistically significant association of methylation with age for NKX2-5 (p = 0.03) in AA in comparison with Cau. In conclusion, we observed higher prevalence of DNA methylation changes in prostate tissue samples from AA in comparison to Cau men. The DNA methylation differences in AA in comparison to Cau men may potentially contribute to racial disparity associated with prostate cancer. Gene specific DNA methylation changes could potentially lead to the identification of “ethnic sensitive” biomarker for PCa detection. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):B62.

Identification of Differentially Methylated Genes in Normal Prostate Tissues from African American and Caucasian Men

Aberrant DNA methylation changes are common somatic alterations in prostate carcinogenesis. We examined the methylation status of six genes in prostate tissue specimens from African American (AA) and Caucasian (Cau) males. We used pyrosequencing to quantitatively measure the methylation status of GSTP1, AR, RARbeta2, SPARC, TIMP3, and NKX2-5. Real-time PCR was used to determine gene expression, and gene reactivation was analyzed by 5-aza-2'-deoxycytidine and/or trichostatin A treatment. Statistical analysis showed significantly higher methylation in the prostate cancer tissue samples in comparison with matched normal samples for GSTP1 (P = 0.0001 for AA; P = 0.0008 for Cau), RARbeta2 (P < 0.001 for AA and Cau), SPARC (P < 0.0001 for AA and Cau), TIMP3 (P < 0.0001 for AA and Cau), and NKX2-5 (P < 0.0001 for AA; P = 0.003 for Cau). Overall, we observed significant differences (P < 0.05) in the methylation level for all genes, except GSTP1, in the AA samples in comparison with the Cau samples. Furthermore, regression analysis revealed significantly higher methylation for NKX2-5 (P = 0.008) and TIMP3 (P = 0.039) in normal prostate tissue samples from AA in comparison with Cau, and a statistically significant association of methylation with age for NKX2-5 (P = 0.03) after adjusting for race. Our findings show higher methylation of several genes in prostate tissue samples from AA in comparison with Cau and may potentially contribute to the racial differences that are observed in prostate cancer pathogenesis.

Cell‐free DNA and RNA in Plasma as a New Molecular Marker for Prostate and Breast Cancer

In this study, we examined several molecular markers in prostate and breast cancer patients and in normal individuals. The markers tested were: variations in the quantity of plasma DNA, glutathione-S-transferase P1 gene (GSTP1), Ras association domain family 1A (RASSF1A), and ataxia telangiectasia mutated (ATM) methylation status in plasma, carcinoembryonic antigen (CEA) and prostate-specific membrane antigen (PSMA) mRNA in peripheral blood mononuclear cells (PBMC) and plasma samples from prostate cancer patients. DNA quantification in plasma was performed using real-time PCR (RT-PCR). We assessed the methylation status of GSTP1 in plasma DNA using methylation-specific PCR (MSP) assay, while the methylation status of RASSF1A and ATM genes was examined by the MethyLight technology. RT-PCR analysis was used for the detection of mRNA, PSMA, and CEA. In 58.3% of newly diagnosed prostate cancer patients and 26.7% of prostate cancer patients under therapy, plasma DNA levels were increased. Additionally, 48.5% of breast cancer patients showed plasma DNA levels above the cutoff limit. GSTP1 Promotor hypermethylation was detectable in 75% of plasma samples obtained from patients with newly diagnosed prostate cancer and in 36.8% of patients under therapy, whereas 26% and 14% of the breast cancer patients tested were positive for RASSF1A and ATM methylation, respectively. The combination of DNA load and promotor methylation status identified 88% of prostate cancer patients and 54% of breast cancer patients. This study shows that free-circulating DNA can be detected in cancer patients compared with disease-free individuals, and suggests a new, noninvasive approach for early detection of cancer.

Methylation-specific PCR for DNA-based detection of occult tumor cells in lymph nodes of prostate cancer patients.

To assess the feasibility of methylation-specific PCR (MSP) for the detection of promoter hypermethylation of the detoxifying glutathione-S-transferase P1 gene (GSTP1) to detect occult prostate cancer cells in lymph nodes (LNs). Paraffin-embedded pelvic LNs from 20 patients with pT2pN0M0R0 prostate cancer who developed PSA relapse were assessed by MSP. In 18 of the patients, samples of the primary tumor were obtained for MSP. In 19 patients, bone marrow (BM) aspirates were analyzed preoperatively for disseminated tumor cells by immunocytochemistry (mAb A45-B/B3). In 16 patients, biopsies of the anastomotic region were performed following PSA relapse. As a negative control GSTP1 methylation status was also assessed in LNs from 9 patients for whom an autopsy was performed for non-cancer-related causes. All primary tumors displayed GSTP1 hypermethylation (HM). Preoperative BM assessment showed disseminated tumor cells in 8/20 cases (40%). In 4 patients, biopsies of the vesico-urethral anastomosis showed local tumor recurrence. The LNs in the cancer patients showed GSTP1 HM in 18/20 cases (90%) versus 1/9 patients (11.1%) in the non-cancer cohort (p<0.0001). GSTP1 HM appears to be well suited for molecular staging of prostate cancer and accurately detects disseminated tumor cells in LNs, which was seen in 90% of the patients with PSA relapse. However, the limited number of patients and the finding of benign and malignant prostatic tissue at the vesico-urethral anastomosis as a putative local source of PSA recurrence does not allow us to draw conclusions on the prognostic significance of our findings, yet.