Expression Of Promoter Methylation Of MGMT In Adenocarcinoma Of The Esophagogastric Junction

Objective: Adenocarcinoma of the esophagogastric junction(AEG)is one of the most common cancer in China,It's morbidity rate and mortality rate are higher than others.The incidence rate and mortality rate in several counties of Hebei province is significant,there are 1/3 patients in advanced stages when they were diagnosed,It's important to explore early diagnosis and treatment of adenocarcinoma of the esophagogastric junction.In recent years,epigenetics has become a hot research direction in the development and progression of tumors,DNA methylation is an important epigenetic change in many tumourigenesis.However few studies have been conducted on the correlation between DNA methylation and AEG.In this study we used methylation specific polymerase chain reaction(MSP)to detect MGMT promoter hypermethylation status in AEG and paired surgical marginal AEG tissues,and immunohistochemical(IHC)was used to investigate the expression of MGMT protein in these tissues.By analyzing the methylation status of MGMT and its protein expression in AEG and adjacent normal tissues,to clarify the association between promoter methylation of MGMT with clinical features and survival time,and whether or not it could be a biomarker for early diagnosis and predicting the prognosis in AEG.Method: 107 cases who underwent surgical resection at thoracic surgery of Fourth Hospital of Hebei Medical University between January 2011 to December 2011 were obtaind,all of the specimens had been confirmed adenocarcinoma with pathologically.Only patients have no blood relationship with each other,without preoperative radio or chemotherapy and without clinical evidence for distant metastasis were selected.Someone who had serious complications or got mental illness,etc had been excluded.There comprised 74 male and 33 female,and aged between 50 to 75,the average age is 63.40±6.64 years.Tumor tissues were taken from non central necrotic areas,the adjacent normal tissues were taken from at least 5cm distant from the tumor.There was no sitatistically significant difference in gender,age,et al between the two groups.107 cases of AEG and its adjacent normal paraffin-embedded surgical tissue specimens were sliced at a depth of 5?m.Genomic DNA was extracted form these spcimens,modificate them by sodium sulfite,PCR was used to amplificate the modificated DNA.At last the methylation status of MGMT promoter was detected by methylation specific polymerase chain reaction(MSP)method(Fig.1).Immunohistochemistry staining method was used to detect protein expression in AEG and paired surgical marginal normal AEG tissues(Fig.1,Fig.2).Through reviewing the patients' operation cases,recorded their gender,age,pathology,tumor size,number of positive lymph nodes,etc,then the patients were followed up with a history of chemotherapy,survival time,cause of death,etc.Data were obtained by telephone,letters,and many kinds of ways.All statistical analysis was performed with SPSS 21.0.P<0.05 was considred statistically significant.Chi-square test and Wilcoxon signed-rank test were used to compare the differents,Speraman correlation analysis was used to find the correlation.Kaplan-Meier method was used to calculate the median survivl time.Cox analysis was used to find the risk factors of 5 years survival time.Results:(1)The promoter methylation rate of MGMT in the tumor tissues was significant higher than that in the adjacent normal tissues(45.8% vs.16.8%,Z=-4.258,P<0.01),the differences were significant.The MGMT expression rate in the tumor tissues was significant lower than that in the adjacent normal tissues(50.5% vs.81.3%,Z=-4.533,P<0.01),the differences were significant.(2)In the tumor tissues 15/49(30.6%)tumors with MGMT expression,showd promoter methylation of MGMT,there were 39/58(67.2%)MGMT expression,showd promoter unmethylation of MGMT.In the adjacent normal tissues 5/18(27.8%)tumors with MGMT expression,showd promoter methylation of MGMT,there were 82/89(92.1%)MGMT expression,showed promoter unmethylation of MGMT.There were significant correlation between promoter methylation of MGMT and MGMT expression in two groups by Spearman correlation(r=-0.365,r=-0.618).(3)There were no correlation between MGMT promoter methylation and its expression with age,gender,residual carcinoma,tumor size(P>0.05).In contrast,promoter methylation of MGMT and its expression correlated with lymph node metastasis and pTNM stage(P<0.05).(4)The median survivl time of patients with AEG was 35 months in our study,the median survival time of patients with methylation of MGMT promoter(26months,95%CI:19.544~32.456)was shorter than patients without methylation(44 months,95%CI:38.117~49.883)(P<0.05)(Table 4,Fig.4).Similarly,the median survival time of patients without MGMT protein expression(26months,95%CI:20.672 ~31.328)was shorter than patients with its protein expression(46months,95%CI:37.356~50.644)(P<0.05)(Table 4,Fig.5).(5)The Cox multivariate regression analysis indicated that gender,age,residual carcinoma,tumor size,lymph node metastasis,didn't affect patients 5 years survival time.In contrast,MGMT promoter methylation(P=0.027),MGMT protein expression(P=0.031),chemotherapy(P=0.031)and p TNM stage(P=0.040)were significant influence factors for 5 years survival time.Conclusions:1 There were relationship between promoter methylation of MGMT and MGMT expression in AEG.2 The promoter methylation of MGMT and its expression were associated with lymph node metastasis and pTNM stage.3 The promoter methylation of MGMT,MGMT protein expression,chemotherapy and pTNM stage were significant influence factors for 5 years survival time.The promoter methylation of MGMT and MGMT protein were used to predict prognosis.