The Correlation Between Malignant Glioma MGMT Gene Promoter Methylation Status, Protein Expression And Patient Clinical Prognosis

Alkylating agent resistance and patient suvivals, in recent years.Methylation specific PCR (MSP) used to be the first chioce for most study to test promoter methylation status. However, this method is doubted because its defects such as lack of quantitative analysis. New developed methods detected promoter can show the methylation state status by quantitative, half quantitative analysis way, having their own advantages and shortages. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) technology develop from the MLPA technology, it test gene promoter status by combine probes hybridization and PCR amplification. Compared with the classical MSP method,MS-MLPA has the advantage that bisulfite conversion can be omitted, different CpG dinucleotides can be analyzed simultaneously, give half quantitative result, The same primer can be use to amplificate all probe low-molecular-weight DNA isolated from paraffin-embedded tissue(p-DNA) can be analyzed as the MLPA probes are small(approximately50-60bp), only a small amount of DNA is required (100ng), and copy number detection can be performed simultaneously, etc. It is a reliable testing method.Our study use MS-MLPA ME011-B1MMR kit(mrc-holland) to test methylation status, the brief protocol:Firstly degeneration reaction transfer a target DNA into single sequences. Then in the hybrid reaction, hybrid sequence in the long and short probe hybrid with the single target sequence. The reaction then is split into two tubes. One tube is processed as a standard MLPA reaction. This reaction provides information on copy number changes. The other tube of the MLPA hybridization reaction is incubated with the methylation-sensitive HhaI endonuclease while simultaneously,the hybridized probes are ligated. Hybrids of (unmethylated) probe oligonucleotides and unmethylated sample DNA are digested by the HhaI enzyme. Digested probes will not be exponentially amplified by PCR and hence will not generate a signal when analyzed by capillary electrophoresis. In contrast, if the sample DNA is methylated, the hemi-methylated probe-sample DNA hybrids are prevented from being digested by HhaI and the ligated probes will generate a signal.ObjectiveTo research the correlation between MGMT gene promoter methylation status, MGMT gene copy number, MGMT protein expression and survival prognosis in patients.To explore the advantages and shortages of MS-MLPA to detect the gene methylation status. To study avariety of problems for IHC method to test the MG-MT protein expression. To discuss postoperative treatment strategies for glioma patients of high activity MGMT.METHODSSpecimens:All glioma Formalin-fixed Paraffin-embedded (FFPE) specimens come from department of pathology,zhujiang hospital, southern medical university. All the Non-neoplastic reference specimens taken from department of neurosurgery, zhujiang hospital, southern medical university.Object of observation:Collection of39cases confirmed by pathology which are gliomas (WHOⅢ classification and WHO IV classification) with a complete follow-up data in the Zhujiang Hospital of south Medical University from2006to2010,including24male and15female, aged from20to68years, mean46.9years old. WHOⅢclassification19cases, WHO IV classification20cases.All patients were gived with routine postoperative radiotherapy and chemotherapy with TMZ after the operation.Meanwhile,6cases of the normal brain tissue were chosen as the control group.Observation Method:tracking the patient’s overall survival (OS). The glioma organization MGMT protein expression was detected by immunohistochemical method, The MGMT gene promoter methylation and genetic copy several state were detected by MS-MLPA method. The patients were divided into MGMT protein expression group, The MGMT gene promoter methylation group and genetic copy several state group. Evaluation the relationship of three group with OS. Observe the relationship of MGMT protein expression and MGMT gene promoter methylation with the age, gender, Pathology classification of the patients.Statistical Methods:The data were analyzed by SPSS13.0statistical software Kaplan-Meier survival curves were used to indicated the relationship of survival time between MGMT protein expression, methylation status, copy number and survival time,respectively. Survival time expressed as mean±standard error.Log-Rank test were used to measured the overall comparison and drawed survival curves. For multiple comparison between groups was proceeded if overall comparison has statistical differences. Spearman correlation coefficient was used to analysis correlation between MGMT protein expression and MGMT promoter methylation. the agreements of MGMT protein expression with the patient’s age, gender, and the pathology classification were measure by χ2test, respectively. Promoter methylation status was measured in the same way. Difference between groups have statistical significance if P<0.05Experimental procedures:Staining the FFPE specimens of glioma with i mmunohistochemistry, coloration with DAB way, with the known MGMT positive glioma organization as the positive control, negative control was set through replacing the primary antibody by PBS liquid. To determine positive results:MGMT positioning in the nucleus or cytoplasm, show brown. MGMT expression is divided into three ranks:no MGMT positive expression and MGMT-Positive cells<10%were regard as negative(-);10%<MGMT-Positive cells<30%as Moderate positive(+);>30%Positive cells as strongly Positive(++).MS-MLPA method was employed to analyse MGMT promoter methylation of FFPE tissues. Six non-neoplastic human brain FFPE samples were chosen as reference, and setting a no DNA Control reaction in which replacing sample DNA with Distilled water. Firstly, extraction genomic DNA in FFPE organization and testing the concentration (50ng/ul is needed) with kit of QIAAMP DNA FFPE TISSUE. Then MS-MLPA was proceeding According the sequence of DNA degeneration, hybridization, ligation reaction, PCR amplification and electrophoresis separation. Electrophoresis data was analysis by corresponding software, Then the dosage quotient of copy number and the methylation rate can be calculated. MGMT Copy number status is divided into three ranks:dosage quotient between0.8-1.2regard as normal, dosage quotient<0.8as copy number loss; dosage quotient>1.2as copy number gain. MGMT promoter methylation status is divided into four ranks:methylation ratio<0.25, regard as unmethylation;0.25<ratio<0.5,as mild hypermethylation;0.5<ratio<0.75, as moderate hypermethylation; ratio>0.75,. as extensive hypermethylation.RESULTS1.MGMT protein and patient OS(overall survival):(1). If the specimen is divided into of MGMT-,MGMT+and MGMT++three groups according to MGMT expression, there’s statistical difference of survival time between the group of MGMT-and MGMT++(P=0.001).however, Survival time in other groups have no significant difference.(2). If the specimen is divided into of MGMT-, MGMT+/++two groups according to MGMT expression, there’s statistical difference of survival time between the group of MGMT-and MGMT+/++(P=0.003)2. MGMT gene promoter methylation status and patient OS:there’s Significant statistical difference of survival time between the group of unmethylation, mild hypermethylation, moderate hypermethylation and extensive hypermethylation (P<0.001)。MGMT gene copy number status and patient OS:there’s no statistical difference of survival time between the group of copy number loss, normal and copy number gain (P=0.938)。3. There’s no statistical difference of MGMT protein expression according the grouping way of mail and female,≥50years old and<50years old, WHO grade III and grade Ⅳ.And, There’s no statistical difference of MGMT promoter methylation status according the grouping way of mail and female,≥50years old and<50years old, WHO grade III and grade IV.4. There’s Statistical correlation between MGMT protein expression and gene promoter methylation status. Spearman rank correlation coefficient is0.697(P<0.001)CONCLUSIONS1. MS-MLPA is a reliable method to detected MGMT gene promoter methylation status. Compared with the classical MSP method, MS-MLPA have Obvious advantage.2. There’s Significant correlation between MGMT gene promoter methylation status and OS of malignant glioma patient. There’s no correlation of MGMT gene promoter methylation status with age, gender and Pathology classification of malignant glioma patient. There’s no correlation between MGMT copy number status and OS of malignant glioma patient.3. There’s correlation between MGMT protein expression and OS of malignant glioma patient, Patient whose tumor tissue express MGMT protein has more poor prognosis than ones who’s negative in MGMT protein. The method of IHC needs to be improved on the aspect of cut-off value in group of MGMT-、MGMT+and MGMT++. There’s no correlation of MGMT protein expression with age, gender and Pathology classification of malignant glioma patient.4. There’s statistical correlation between MGMT protein expression and gene promoter methylation status. Both can be regard as Prognostic indicator of OS about patients with malignant glioma Accepted Alkylating agent chemotherapy, but detecting of MGMT promoter status should be the first choice in assessment of MGMT activity.5. Effect of malignant glioma treatment is still not ideal at present, researching in many aspects Should be strengthen such as reducing Cancer drug-resistant.