Study On Aberrant Promoter Methylation Of Tumor Genes With Clinical Prognosis In Gliomas

Glioma is the most common malignant tumor of central nervous system, how to understand the mechanism of tumorigenesis and look for new strategy to treat with gliomas, to improve patients’ survival and quality of life, it’s always the challenge for neurosurgeons. Epigenetics refers to stable alterations in gene expression with no underlying modifications in the genetic sequence, DNA methylation is central to the aberrant epigenetics of cancer, which is also happened in gliomas. Cancer cells often have both a loss of global methylation and a gain of methylation at the promoters of selected CpG islands, induced to tumorigenesis because of the activation of oncogenic and/or inactivation of tumor suppressor pathways. On this study, we intend to find out the association with aberrant promoter methylation of tumor genes and clinical prognosis in gliomas, and look for a suitable assay to evaluating the DNA methylation status, to provide theoretical basis for gliomas’early diagnosis, recurrence monitoring, prognosis evaluation, individualization treatment options and molecular biological therapies.Part Ⅰ MethyLight assay for aberrant promoter methylation of MGMT in gliomas and comparative analysis with the nested MSP assayObjective:Epigenetic silencing of the MGMT gene has been shown to correlate with improved survival of glioma patients treated with alkylating agent therapy, to compare the feasibility and reliability of direct, real-time PCR assay with the clinically validated nested, gel-based MSP assay with known predictive value in the clinical setting in the assessment of MGMT methylation status.Methods:54Fresh frozen glioma tissue samples and2traumatic brain samples were obtained after informed consent from adult patients. Genomic DNA was extracted using modified alkaline lysis method and bisulfite modification was performed on certain concentration of genomic DNA using CpGenomeTM DNA Modification Kit. MGMT methylation status was measured with both direct real-time PCR assay and the clinically validated nested, gel-based MSP assay, and Cohen’s kappa coefficient was used to compare the feasibility and reliability of direct, real-time PCR assay with the clinically validated nested, gel-based MSP assay. Results:The reproducibility between independent replicates, in agreement with the characteristics of the reliability of real-time PCR, Pearson correlation at0.908. There is a good concordance between the two tests (48/56,85.7%). Comparison with the clinically validated nested, gel-based assay shows, sensitivity and specificity were84%(21/25) and87%(27/31), respectively, and the positive and negative predictive values were84%(21/25) and87%(27/31)Conclusion:A sensitive and specific direct, real-time MSP assay has been developed to reliably detect the methylation status of the MGMT gene promoter, The results of the methylation status of this test are in good concordance with results obtained with the nested, gel-based MSP assay.Part Ⅱ the Correlation between Aberrant Promoter Methylation of MGMT and Immunohistochemical Expression with Clinical Prognosis in GBMObjective:Promoter methylation of MGMT is associated with improved treatment outcome for newly diagnosed glioblastoma treated with standard chemoradiation, however, the correlation between the methylation status of MGMT promoter and MGMT protein expression levels in GBM and patients’survival is still controversial. To determine the methylation status of the MGMT promoter and the MGMT protein expression in gliomas, and to analyze the correlation between MGMT methylation status and patients’ outcome.Methods:119formalin-fixed and paraffin-embedded GBM tissue samples and10traumatic brain samples were obtained after informed consent from adult patients. Genomic DNA was extracted using modified alkaline lysis method and MGMT methylation status was measured with MethyLight assay after bisulfite treatment. The protein expression levels of MGMT was determined by immunohistochemical staining. The correlation between MGMT methylation status and MGMT expression levels was measured with Chi-square test, and the correlation between patients’ outcome and MGMT methylation status or MGMT expression levels was determined by log-rank tests.Results:The MGMT promoter was found to be methylated in35.3%patients (42/119). A significant correlation was observed between MGMT promoter methylation and patients’ survival (PFS and OS). No correlation was found bteween MGMT promoter methylation and MGMT expression determined by immunohistochemical staining (P>0.05), or MGMT expression and patients’survival (P>0.05)Conclusions:our results support the hypothesis that MGMT promoter methylation status may be more suitable as a predictive biomarker than the MGMT expression in the treatment of patients with GBM.Part Ⅲ the Correlation between Aberrant Promoter Methylation of Stem Cell Marker CD133and Immunohistochemical Expression with Clinical Prognosis in gliomasObjective:CD133has played a pivotal role in the identification and isolation of brain tumor stem cells, but the correlation between the methylation status of CD133promoter and CD133protein expression levels in glomas and patients’ survival is still controversial. To determine the methylation status of the regions1-3of the CD133promoter and the CD133protein expression in gliomas, and to analyze the correlation between CD133methylation status, expression level and patients’ outcome.Methods:170formalin-fixed and paraffin-embedded glioma tissue samples and3traumatic brain samples were obtained after informed consent from adult patients. Genomic DNA was extracted using modified alkaline lysis method and bisulfite modification was performed on certain concentration of genomic DNA using EZ DNA methylation kit. CD133methylation status was measured with the clinically validated nested, gel-based MSP assay. The protein expression levels of CD133was determined by immunohistochemical staining. The correlation between CD133methylation status and CD133expression levels was measured with Weighted kappa, and the correlation between patients’ outcome and CD133methylation status or CD133expression levels was determined by log-rank tests.Results:We detected five CD133promoter methylation patterns in170glioma samples:methylation only (M+, U-)36cases, high methylation and low unmethylation (M+, Ul)23cases, both methylation and unmethylation equally (M+, U+)40cases, low methylation and high unmethylation (Ml, U+)7cases, and unmethylation only (M-, U+)64cases. By multivariate survival analysis, we found CD133promoter unmethylation status was significant (P<0.01) prognostic factors for adverse progression-free survival and overall survival independent of tumor grade, extent of resection, or patient age. CD133immunostaining showed considerable variability among tumors. While, there was lack of correlation between CD133protein expression determined by immunohistochemical staining and patient’s survival (P>0.05). Furthermore, no correlation between CD133protein expression and CD133promoter methylation status was observed (Kw=-0.165)Conclusions:CD133promoter methylation status in glioma is closely correlated with patient survival, there was lack of correlation between CD133protein expression determined by immunohistochemical staining and patient’s survival or CD133promoter methylation status, indicating that CD133promoter methylaiton pattern might be a promising tool for diagnostic purposes.