胶质瘤组织中DNA修复基因MGMT启动子区过甲基化研究

背景与目的：O^6-甲基鸟嘌呤-DNA甲基转移酶（O^6-methylguanine-DNA methyltransferase,MGMT）是肿瘤细胞产生亚硝脲药物抗药性的分子基础，启动子区过甲基化而导致MGMT基因的转灵失活，影响MGMT蛋白表达，本研究探索了胶质瘤组织中MGMT基因启动子区过甲基化状态及其与MGMT蛋白表达的关系。方法：采用甲基化特异性聚集合酶链反应分析胶质瘤组织中MGMT基因启动子区过甲基化状态，同时采用免疫组织化学法分析胶质瘤组织中MGMT蛋白表达情况。结果：在27例胶质瘤患者的肿瘤组织标本中，18例MGMT蛋白表达呈阳性的胶质瘤组织中7例MGMT基因启动甲基化，阳性率为38.9%；9例MGMT蛋白表达呈阴性的胶质瘤组织中7例MGMT基因启动子甲基化、阳性率为77.8%（P〈0.05）。结论：MGMT基因启动子区的甲基化状态与MGMT蛋白的表达相关，MGMT基因启动子过甲基化，MGMT蛋白表达较低；MGMT基因启动子去甲基化，MGMT蛋白表达较高。     

MGMT基因启动子甲基化检测在脑胶质瘤化疗中的意义

背景与目的：如何预测和克服肿瘤细胞对化疗药物的耐药性,实施个体化治疗是肿瘤化疗急需解决的问题。与基因启动子甲基化密切相关的DNA损伤修复基因O^6-甲基鸟嘌呤-DNA甲基转移酶（O^6-methylguanine-DNA methyhransferase,MGMT）表观沉默与肿瘤对烷化剂药物化疗敏感性密切相关。本研究探讨检测MGMT基因启动子CpG岛甲基化在判断脑胶质瘤患者预后及预测肿瘤对烷化剂药物耐药性中的意义。方法：甲基化特异性PCR（MSP）法检测脑胶质瘤组织及肿瘤细胞株MGMT基因启动子甲基化状态,蛋白印迹和免疫组化法测定蛋白表达。MTF法检测肿瘤细胞株对烷化剂药物敏感性,将患者随访资料针对MGMT甲基化状态绘制Kaplan-Meier生存曲线,并进行log—rank检验分析。结果：39例脑胶质瘤患者组织MGMT基因启动子甲基化发生率为46．2％,蛋白表达阳性率为61．5％,且肿瘤组织中MGMT基因甲基化状态与蛋白表达显著相关（P〈0．05）：6例正常组织均未检测出基因甲基化。MGMT基因过甲基化的脑胶质瘤SHG44细胞株用5-Aza-CdR处理后完全脱甲基化．MGMT蛋白恢复了表达,同时细胞株对烷化剂药物敏感性也发生逆转．由敏感转变为耐受。在采用手术、放疗和烷化剂尼莫司汀化疗等综合治疗的39例脑胶质瘤患者中,MGMT基因甲基化的患者生存率显著高于MGMT基因未甲基化患者（P〈0．05）。结论：MGMT基因甲基化状态与蛋白表达及肿瘤细胞对烷化剂药物敏感性密切相关,有可能替代MGMT蛋白检测成为判断脑胶质瘤患者预后和预测肿瘤对烷化剂化疗耐药性的标志分子。     

脑胶质瘤O6-甲基鸟嘌呤-DNA甲基转移酶启动区甲基化及其表达异质性的研究

目的研究脑胶质瘤组织不同部位O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）启动区甲基化状态及其表达的差异性。方法在54例脑胶质瘤组织的中心和周边部位分别获取标本,共获得92份标本,采用巢式甲基化特异性PCR（MSP）进行MGMT启动区甲基化状态的检测,同时采用免疫组织化学方法进行MGMT蛋白表达的检测。结果 38例胶质瘤中获得了肿瘤两个不同部位MG-MT启动区甲基化的状态,其中24例胶质瘤MGMT启动区甲基化的状态是一致的,占总数的63.2%（24/38）。在29例胶质瘤患者中获得了肿瘤两个不同部位MGMT蛋白表达的情况,其中10例胶质瘤MGMT蛋白表达是一致的,占总数的34.5%（10/29）。两总体一致性概率间差值的95%置信区间为（5.6%和51.8%）,两者的差异有统计学意义。巢式MSP和免疫组化都获得检测结果的标本有77份,在61份MGMT启动区甲基化标本中,25份MGMT蛋白表达阴性,14份蛋白表达可疑阳性,18份蛋白表达阳性,4份蛋白表达强阳性。在16份MGMT启动区非甲基化标本中,2份MGMT蛋白表达阴性,2份蛋白表达可疑阳性,9份蛋白表达阳性,3份蛋白表达强阳性,MGMT启动区甲基化状态与蛋白表达呈负相关（r=-0.318,P=0.005）。结论脑胶质瘤MGMT蛋白表达在同一肿瘤的不同部位存在明显的异质性。虽然脑胶质瘤MGMT启动区甲基化的状态在同一肿瘤的不同部位存在一定的异质性,但是大多数脑胶质瘤MGMT启动区甲基化的状态在同一肿瘤的不同部位是一致的。脑胶质瘤MGMT启动区甲基化状态的一致性优于蛋白表达的一致性。脑胶质瘤MGMT启动区甲基化,MGMT蛋白表达较少,MGMT启动区非甲基化,MGMT蛋白表达较多。     

脑脊液MGMT启动子甲基化在胶质瘤诊断中的应用

目的 比较胶质瘤患者脑脊液与外周血中MGMT（O6-甲基鸟嘌呤-DNA-甲基转移酶）启动子甲基化在胶质瘤初期诊断中的敏感度和特异性。方法 收集283例胶质瘤患者肿瘤组织及其对应的术前外周血和脑脊液,10例颅脑外伤患者挫伤的脑组织及其对应的术前外周血、脑脊液。MGMT启动子甲基化状态通过甲基化特异性PCR（methylation-specific PCR, MSP）确定,比较脑肿瘤组织,外周血和脑脊液中MGMT启动子甲基化率。结果 不同级别胶质瘤组织MGMT启动子甲基化阳性率分别为68.13%（WHO Ⅱ）、65.69% （WHO Ⅲ）以及 67.78%（WHO Ⅳ）,三者之间差异无统计学意义（P>0.05）。胶质瘤瘤体组织、对应的外周血和脑脊液启动子甲基化率为分别为67.14%、44.87%和66.08%,阴性对照组中检测结果均为0。MGMT启动子甲基化阳性瘤体组织,对应的脑脊液中MGMT启动子甲基化敏感度为79.66%（141/177）,较对应的外周血58.82%（110/187）高（P<0.05）。结论 术前患者肿瘤组织、脑脊液和外周血中MGMT启动子甲基化率与肿瘤级别无关。与外周血相比,脑脊液中的 MGMT 启动子甲基化特异性和敏感度更高。     

人原发胶质母细胞瘤组织中NF-κB、TP53及MGMT甲基化与MGMT蛋白表达的相关性研究

背景与目的：胶质瘤化疗敏感性与一些分子表达相关。本研究检测和分析原发性胶质母细胞瘤（glioblastoma multifolille,GBM）患者肿瘤组织中核因子κB（NF-κB）、突变型P53（TP53）及O^6-甲基鸟嘌呤-DNA甲基转移酶｜（O^6-methylguanine—DNA methyltransferase,MGMT）基因启动子区甲基化与MGMT蛋白表达的相关性．以探讨调控MGMT蛋白表达的作用机制。方法：采用甲基化特异性PCR方法（methylation specific PCR,MSP）检测我院收治的120例原发性GBM患者肿瘤组织标本MGMT基因启动子区甲基化：采用免疫组化方法检测NF—κB、TP53、MGMT蛋白表达情况。采用SPSS17．0软件及Spearman相关系数分析方法进行统计学分析。结果：免疫组化表明NF—κB、TP53表达与MGMT表达呈正相关（r=0．244,r=0．271,P均〈0．05）,NF—κB与TP53表达亦呈正相关r=-0．329,P〈0．05）。MSP结果显示MGMT基因启动子区甲基化率与MGMT蛋白表达强度无相关性。结论：转录因子NF—κB与TP53对原发性GBM肿瘤组织中MGMT蛋白表达可能存在正调控作用,而MGMT基因启动子区甲基化率与MGMT蛋白表达强度无相关性。     

胶质瘤MGMT启动子甲基化及其临床意义

目的 分析O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）基因启动子甲基化状态及其与胶质瘤临床病理特征、预后的关系。方法 收集2012年1月至2013年6月间行手术治疗的70例胶质瘤组织和14例非肿瘤患者正常脑组织，采用甲基化特异性 PCR法（MSP）检测MGMT甲基化水平，分析其与胶质瘤临床病理特征的关系。比较不同MGMT甲基化状态的高、低级别胶质瘤患者的总生存（OS），Cox比例风险回归模型分析影响低级别胶质瘤患者的OS的因素。结果 70例胶质瘤患者中48例（68.6％）MGMT基因启动子甲基化，而正常脑组织标本中仅2例（14.3％）MGMT甲基化，差异有统计学意义（P＜0.05）。MGMT甲基化与年龄、性别、肿瘤类型、KPS评分、p53和Ki-67表达无关（P＞0.05）；与病理分级有关（P＜0.05）。低级别脑胶质瘤患者中，MGMT甲基化患者中位OS为30个月，明显长于非甲基化者的11个月，差异具有统计学意义（P＜0.05）。单因素分析显示WHO病理分级、烷化剂化疗、MGMT甲基化与低级别脑胶质瘤患者OS有关（P＜0.05）。多因素分析WHOⅡ级、未接受烷化剂化疗、MGMT非甲基化是影响低级别胶质瘤患者OS的独立危险因素（P＜0.05）。结论 MGMT甲基化与胶质瘤的发生、发展有关，在判断胶质瘤恶性度、评估预后及指导临床治疗方面具有一定的价值。     

NSCLC患者血浆p16和MGMT基因甲基化检测及临床意义

目的：检测非小细胞肺癌（non—small cell lung cancer，NSCLC）患者外周血血浆中p16基因、O^6-甲基乌嘌呤-DNA甲基转移酶（O^6-methylguanine—DNA methyhransferase，MGMT）基因启动子的甲基化状态，探讨p16、MGMT基因启动子的异常甲基化在NSCLC筛查及早期诊断中的意义。方法：利用巢式甲基化特异性聚合酶链反应法检测NSCLC患者外周血血浆p16、MGMT基因启动子的甲基化状态。结果：65例NSCLC血浆样品中分别发现19例（29．23％）p16基因启动子异常甲基化和16例（24．62％）MGMT基因启动子异常甲基化，45例正常对照血浆组未检测到p16、MGMT基因启动子的异常甲基化（P〈0．05），血浆中两基因甲基化检出率与NSCLC的分型及临床分期无明显相关性（P〉0．05）。结论：利用巢式甲基化特异性PCR法检测外周血血浆中p16、MGMT基因启动子的甲基化，可为NSCLC的筛查、早期诊断及预后判断提供有价值的信息。     

DNA修复基因MGMT启动子区过甲基化与食管鳞状细胞癌

目的：O^6－甲基鸟嘌呤DNA甲基转移酶（MGMT）可以转移DNA加合物O^6－甲基鸟嘌呤中的甲基,从而修复DNA损伤,许多肿瘤中发现MGMT基因启动子过甲基化导致该基因失活,我们研究了MGMT基因启动子甲基化状态与食管癌的关系。方法：采用甲基化特异性聚合酶链反应及测序方法分析食管癌。癌旁组织和正常食管上皮中MGMT启动子甲基化状态。结果：在检测的199例食管癌组织中,46例（38．7％）有MGMT基因启动子过甲基化,相应癌旁组织22例中也有5例（22．7％）出现MGMT基因甲基化,而21例正常食管上皮均无此种改变。结论：MGMT基因启动子过甲基化是食管癌中常见的分子事件,可能发生在癌过程的早期阶段。     

MGMT启动子甲基化对神经胶质瘤患者治疗及预后的临床意义

目前以替莫唑胺（temozolomide,TMZ）为基础的化疗已成为神经胶质瘤术后辅助治疗的标准方案,然而TMZ对部分患者疗效欠佳。DNA修复蛋白O6-甲基鸟嘌呤-DNA甲基转移酶（O6-methylguanine-DNA methyltransferase,MGMT）启动子甲基化是胶质瘤患者的重要分子标志物,与胶质瘤预后及烷基化药物如TMZ的耐药有关。在新诊断的胶质母细胞瘤中,MGMT启动子甲基化已成为独立预后指标。MGMT启动子甲基化是抑制MGMT蛋白表达的关键机制,可抑制DNA修复,增加TMZ化疗敏感性。本文综述了MGMT启动子甲基化与神经胶质瘤患者预后、疗效的最新数据及临床试验,对MGMT启动子甲基化在临床中的应用进行总结,以期为神经胶质瘤患者的个体化治疗提供参考。      

食管胃交界部腺癌中MGMT基因启动子区甲基化状态对预后的影响北大核心CSCD

目的检测食管胃交界部腺癌组织中O6-甲基鸟嘌呤-DNA甲基转移酶(O^6-methylguanineDNA methyltransferase,MGMT)基因启动子区甲基化状态以及蛋白表达情况,评价其与临床参数及预后的关系。方法选取107例食管胃交界部腺癌患者,应用甲基化特异性聚合酶链反应(methylmion specific PCR,MSP)检测MGMT基因甲基化情况,免疫组织化学法检测MGMT蛋白表达情况,分析两者与临床特征及生存时间的关系。结果癌组织中MGMT基因启动子区甲基化率显著高于癌旁正常组织;癌组织中MGMT蛋白阳性率显著低于癌旁正常组织。Spearman等级相关分析表明MGMT基因启动子区甲基化与MGMT蛋白表达呈负相关。MGMT基因启动子甲基化及蛋白表达与淋巴结转移、pTNM分期之间具有相关性。多因素Cox回归分析,MGMT基因启动子区甲基化、MGMT蛋白表达及pTNM分期是影响患者生存的独立预后因素。结论 MGMT基因启动子甲基化、MGMT蛋白表达与pTNM分期是影响食管胃交界部腺癌患者预后的独立影响因素。     

Aberrantly Methylated MGMT, hMLH1 and hMSH2 in Tumor and Serum DNA of Gliomas Patients

OBJECTIVE This study is to investigate the prevalence of promoter CpG island methylation of O6-methylguananine-DNA methyltransferase (MGMT), mismatch repair genes (hMLH1 and hMSH2) in both tumor and serum samples of gliomas.METHODS Methylation-specific PCR (MSP) was employed to detect promoter CpG island methylation of the MGMT, hMLH1 and hMSH2 genes in 39 samples taken from surgery and 32 samples of pretreatment serum all from the patients with gliomas.RESULTS Promoter CpG island methylation of MGMT, hMLH1 and hMSH2 was detected and the results were 46.2%, 10.3% and 20.5%, respectively in tumor DNA of the cases with gliomas,and 40.6%, 9.4% and 18.8%, respectively in serum DNA of the cases. The methylation pattern in primary tumor and serum was found to be concordant in matched tissue and serum samples of 21 patients. In the cases with positive result of methylation for MGMT, hMLH1 and hMSH2 in tumor tissues, the results of detection for those in the paired serum sample were 77.8% (7/9),66.7% (2/3) and 75.0 % (3/4), respectively. False positive results were not obtained in any of the patients who did not exhibit methylation. No association was found between the promoter methylation of MGMT, hMLH1, and hMSH2 genes in primary gliomas and gender, age, localization, grade of malignant or tumor stage.CONCLUSION Promoter CpG island methylation is a frequent event in gliomagenesis. Methylation analysis appears to be a promising predictive factor of the prognosis for the glioma patients treated with alkylating drugs and a noninvasive tumor marker in serum DNA.     

Prognostic significance of O6-methylguanine-DNA methyltransferase determined by promoter hypermethylation and immunohistochemical expression in anaplastic gliomas.

PURPOSE: Anaplastic gliomas constitute a heterogeneous group of tumors with different therapeutic responses to adjuvant chemotherapy with alkylating agents. O6-Methylguanine-DNA methyltransferase (MGMT), a DNA repair protein, is one of the implicated factors in glioma chemoresistance.The prognostic value of MGMT remains controversial due in part to the fact that previous published studies included heterogeneous groups of patients with different tumor grades. The aim of this study was to evaluate the prognostic significance of MGMT in patients with anaplastic glioma. EXPERIMENTAL DESIGN: Ninety-three patients with anaplastic glioma were analyzed for MGMT protein expression by immunohistochemistry. In addition, for those patients from whom a good yield of DNA was obtained (n = 40), MGMT promoter methylation profile was analyzed by methylation-specific PCR. MGMT prognostic significance was evaluated together with other well-known prognostic factors. RESULTS: Fifty-one tumors (54.8%) showed nuclear staining of MGMT. There was a trend towards longer overall survival for those patients with negative MGMT immunostaining (hazard ratio, 1.66; P = 0.066). In a secondary analysis including those patients who actually received chemotherapy (n = 72), the absence of MGMT expression was independently associated with better survival (hazard ratio, 2.12; P = 0.027). MGMT promoter methylation was observed in 50% of the analyzed tumors. No statistical correlation between MGMT expression and MGMT promoter hypermethylation was observed. CONCLUSIONS: Unlike previous studies, we did not find a correlation between MGMT promoter methylation and survival. However, we observed a correlation between MGMT protein expression and survival in those patients who received chemotherapy thus suggesting that the absence of MGMT expression is a positive predictive marker in patients with anaplastic glioma.     

TMS1/ASC基因CpG岛甲基化与膀胱移行细胞癌发生的关系

目的检测抑癌基因TMS1/ASC启动子区5′CpG岛甲基化状态及其在膀胱移行细胞癌（BTCC）中mRNA和蛋白表达水平。方法应用MSP技术检测膀胱移行细胞癌中TMS1/ASC基因启动子区甲基化状态,RT-PCR和Western blot法分别检测其mRNA和蛋白表达水平。结果TMS1/ASC基因在正常膀胱组织中未发生甲基化,而在癌组织中甲基化频率为46.9%（15/32）,并且随着肿瘤分级、分期的增加,其甲基化水平逐渐升高（χ^2=23.106,P〈0.05）。在15例启动子异常甲基化的BTCC标本中,14例同时伴有TMS1/ASC基因表达缺失或下调,两者存在明显的相关性（γ=0.5842,P〈0.05）。TMS1/ASC mRNA和蛋白表达在正常膀胱组织和BTCC组织中分别为81.3%（26/32）、18.8%（6/32）（P〈0.01）,不同病理分级、临床分期间差异有统计学意义（P〈0.05）。结论TMS1/ASC基因启动子区异常甲基化可能导致该基因转录表达失活,使其mRNA和蛋白表达减少,甚至缺失,这可能是膀胱癌发生、发展的原因之一。     

Intratumoral homogeneity of MGMT promoter hypermethylation as demonstrated in serial stereotactic specimens from anaplastic astrocytomas and glioblastomas

Hypermethylation of the DNA repair gene O(6)-methyl-guanine DNA methyltransferase (MGMT) has been linked to prolonged survival in glioblastoma patients treated with alkylating agents. It was aimed to analyze prospectively whether the MGMT status of malignant gliomas could be determined from small-sized stereotactic biopsies (maximum volume: 1 mm(3)). Special attention was directed towards the intratumoral distribution of the MGMT promoter methylation, the MGMT protein expression and potential correlations between both. Twenty-five adult patients were included (20 patients with primary World Health Organisation (WHO) Grade III or IV malignant gliomas, 5 patients with secondary malignant gliomas). About 2-4 biopsy specimens per tumor were collected from different sites within the tumor. Promoter methylation of the MGMT gene was assessed by methylation-specific PCR (MSP) and sodium bisulfite sequencing in each of the collected specimens (overall number of specimens: 69). Both methods were validated for application in small-sized tissue samples (1 mm(3)). The MGMT protein expression was analyzed by immunohistochemistry. The overall MGMT promoter methylation rate was 30% in the de novo group and 80% in the tumor progression group. The success rates of MSP and sequencing were 100% and 80%, respectively. Sequence analysis and MSP exhibited 100% concordant findings. No differences in MGMT promoter methylation were detected between the different samples of each individual tumor in 24 of 25 patients. One false negative result was obtained due to the contamination of the biopsy specimen by necrotic tissue. Tissue samples taken from different sites of each individual tumor (13 tumors investigated) exhibited equal or highly similar MGMT protein expression. No correlation between MGMT protein expression and MGMT promoter methylation was observed. The MGMT promoter methylation status of malignant gliomas can be reliably determined from small-sized stereotactic biopsies. The methylation profile, as defined by MSP and sodium bisulfite sequencing, constitutes a homogeneous marker throughout malignant gliomas. The lack of correlation between MGMT status and MGMT protein expression needs further evaluation.     

子宫内膜息肉及子宫内膜癌组织中雌激素受体β的甲基化与表达研究

目的检测正常子宫内膜、子宫内膜息肉及子宫内膜癌中雌激素受体β（estrogenreceptor—β,ER—β）基因启动子区CpG岛的甲基化状态及ER—β蛋白的表达,同时分析ER—β启动子甲基化与ER—β的表达间的关系,以探讨子宫内膜息肉的发病机制。方法采用免疫组化ELIVISON二步法检测40例正常子宫内膜、40例子宫内膜息肉及26例子宫内膜癌组织中ER—β蛋白的表达,同时运用甲基化特异性PCR（MSP）检测上述组织中ER—β的甲基化情况。结果正常子宫内膜中ER—β的表达较子宫内膜息肉及子宫内膜癌显著增高（均P〈0．01）,子宫内膜息肉与子宫内膜癌则无显著差异（P〉0．05）。正常子宫内膜及子宫内膜息肉组织中ER-β基因甲基化率较子宫内膜癌显著降低（均P〈0．05）。正常子宫内膜、子宫内膜息肉及子宫内膜癌组织中ER—β的阳性表达与ER—β基因启动子甲基化状态均呈负相关。结论子宫内膜组织中ER-β的基因甲基化可能与子宫内膜息肉乃至子宫内膜癌的发生和复发密切相关。     

肝细胞癌中APC基因启动子甲基化及蛋白表达的研究

目的：探讨肝细胞癌中APC基因启动子甲基化状态及蛋白表达的临床意义。方法：应用甲基化特异性聚合酶链反应（MSP）和免疫组织化学方法检测61例HCC及相应的癌旁肝组织中APC基因启动子甲基化状态和蛋白表达水平,分析甲基化与临床资料及蛋白表达的关系。结果：癌组织及癌旁组织中APC基因启动子甲基化阳性率分别为26.23%和11.47%（P〈0.05）。癌与癌旁组织APC蛋白表达无统计学差异（P〉0.05）。APC基因启动子甲基化与临床分期、门脉癌栓、术后复发、肝外转移、肿瘤大小、肿瘤分化、肿瘤个数及血清AFP值无关。APC基因启动子甲基化与蛋白表达无相关性。结论：APC启动子区甲基化可能参与了肝癌的发生,但在HCC的发展中的作用仍需进一步研究。