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。     

非小细胞肺癌组织MGMT基因启动子过甲基化与临床病理特征相关性的研究

目的:探讨非小细胞肺癌(NSCLC)组织中DNA修复酶O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)过甲基化状况与临床病理特征的关系.方法:甲基化特异性PCR(MSP)检测120例病理确诊的非小细胞肺癌组织及癌旁正常肺组织和30例病理确诊为肺炎症组织中MGMT的甲基化状况.结果:120例NSCLC组织中MGMT基因启动子甲基化率(35%)显著高于癌旁正常组织甲基化率(5%,P<0.05),亦显著高于30例正常肺组织中甲基化率(0,P<0.05).NSCLC中MGMT基因启动子过甲基化状态与淋巴结转移、TNM分期、吸烟史、肿瘤分化程度有关(P值均<0.05),与患者年龄、性别、病理组织类型无关(P值均>0.05).结论:在NSCLC中,MGMT基因启动子甲基化可能与肺癌的发生、发展相关.     

MGMT表达指导下的复发恶性胶质瘤挽救性化疗疗效分析（附30例报告）

O6-甲基鸟嘌呤-DNA甲基转移酶（O6-methylguanine-DNA methyltransferase,MGMT）是与恶性胶质瘤对替莫唑胺（temozolomide,TMZ）等烷化剂耐药相关的重要指标。本文总结根据MGMT表达状态选择不同的化疗方案,对复发恶性胶质瘤患者进行挽救性化疗的临床疗效。方法:经手术后病理确诊的复发恶性胶质瘤患者30例,均有可评价病灶。应用免疫组化法检测肿瘤MGMT表达状态,分为阳性组和阴性组。阳性组患者应用非TMZ常规5天方案或非烷化剂药物进行化疗,阴性组患者不限制化疗方案。结果:全组患者客观有效率为20%,中位无进展生存时间为8个月（95%CI:4.3～11.7）,中位生存时间为16个月（95%CI:7.4～24.6）。其中MGMT阳性组16例,阴性组14例。阳性组和阴性组患者的客观有效率分别为18.8%和21.4%,中位无进展生存时间分别为7个月（95%CI:3.1～10.9）和8个月（95%CI:3.9～12.1）,中位生存时间分别为16个月（95%CI:5.4～26.6）和16个月（95%CI:7.3～24.7）,差异均无统计学意义（P>0.05）。结论:复发恶性胶质瘤挽救性化疗具有良好的临床获益,根据肿瘤MGMT表达进行个体化化疗,特别是对于MGMT阳性复发恶性胶质瘤患者,能够避免耐药,得到与MGMT阴性患者相当的临床疗效。      

miR-370调控MGMT增强替莫唑胺对胶质瘤细胞化疗敏感性的实验研究

目的 探讨microRNA-370(miR-370)通过调控O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)增强替莫唑胺(TMZ)对胶质瘤化疗敏感性的机制研究.方法 采用TMZ浓度梯度递增法体外建立U87/TMZR耐药细胞株,分别转染阴性空质粒和miR-370 mimics作为miR-NC组和miR-370 mimics...     

O~6-甲基鸟嘌呤-DNA甲基转移酶在恶性脑胶质瘤组织中的表达及意义

目的探讨O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)在恶性脑胶质瘤组织中的表达及其与预后的关系。方法选取2011年1月至2016年1月间中山市陈星海医院收治的经手术切除的64例恶性脑胶质瘤(Ⅲ、Ⅳ级)患者,采用免疫组化法检测肿瘤组织中MGMT蛋白的表达,评定治疗效果,追踪生存时间(OS),评价MGMT与生存时间之间的关系。结果 MGMT阳性表达率为62.5%,MGMT阴性表达率为37.5%。WHOⅢ级恶性胶质瘤组织中MGMT阳性表达率与Ⅳ级比较,差异无统计学意义(P>0.05);但Ⅳ级恶性胶质瘤MGMT(++)表达率为60.0%,明显高于Ⅲ级的48.3%,差异有统计学意义(P<0.05)。MGMT阴性患者总有效率(ORR)为62.5%,高于阳性患者的17.5%;与MGMT(-)比较,MGMT(+)和MGMT(++)平均OS均降低,差异均有统计学意义(均P<0.05)。MGMT阳性患者最长无进展生存时间(PFS)为32个月,MGMT(-)最长PFS为40个月。结论 MGMT表达与脑胶质瘤恶性程度有关,表达强度越高,恶性程度越高,检测MGMT可一定程度预知脑胶质瘤治疗的有效率及预后。     

人脑胶质瘤组织MGMT和EGFR及Ki-67表达临床意义分析

目的:探讨人脑胶质瘤组织中O6-甲基鸟嘌呤DNA甲基转移酶(MGMT)、表皮生长因子受体(EGFR)和细胞增殖相关核蛋白Ki-67的表达及其相关性。方法:选取64例脑胶质瘤组织标本,采用免疫组织化学染色法检测MGMT、EGFR及Ki-67的表达。结果:MGMT、EGFR、Ki-67总体阳性率分别为76.6%、59.4%和56.3%。在Ⅰ、Ⅱ、Ⅲ、Ⅳ级胶质瘤中,MGMT阳性表达率分别为0、58.8%、70.0%和100.0%;EGFR阳性表达率分别0、41.2%、60.0%和76.0%;Ki-67阳性表达率分别为0、23.5%、65.0%和76.0%。MGMT、EGFR和Ki-67的阳性表达率在脑胶质瘤不同病理分级间差异有统计学意义,P值分别为0.001、0.043和0.002;且三者的表达强度与胶质瘤病理分级呈正相关性,r值分别为0.315、0.273和0.400,P值分别为0.011、0.029和0.001。3种检测指标的阳性表达率与发病年龄及性别均无明显的相关性,差异无统计学意义,P>0.05。分层分析发现,Ki-67的表达与MGMT和EGFR的表达有相关性,r分别为0.296和0.466,P值分别为0.018和0.000。结论:人脑胶质瘤中MGMT、EGFR及Ki-67的表达与病理分级均有相关性,测定三者的表达水平可评价胶质瘤细胞的恶性程度,对胶质瘤预后判断可能有指导作用。     

IDH1基因突变在脑胶质瘤中的研究进展

脑胶质瘤是中枢神经系统较常见的肿瘤之一, 尽管目前诊疗技术得到长足的发展, 但疗效仍然不尽如人意。随着分子病理学的发展, 人们越来越关注脑胶质瘤中一些分子水平的异常在诊断和治疗疾病中的意义。近年来研究发现, 脑胶质瘤患者存在高频率的异柠檬酸脱氢酶-1(isocitrate dehydrogenases-1, IDH1)基因突变现象, 而且该突变与脑胶质瘤的诊断分型和临床预后有明确的关系, 对其深入研究, 有望找到脑胶质瘤治疗的新靶点, 对改善当前脑胶质瘤治疗现状有着深远的意义。      

O~6-甲基鸟嘌呤-DNA-甲基转移酶与肿瘤预见性化疗

亚硝脲类药物造成细胞DNA烷化损伤,杀伤肿瘤细胞.O~6-甲基鸟嘌呤-DNA-甲基转移酶(MGMT)可以修复这种损伤,使细胞产生抗药性.我们以中国人肿瘤细胞株和临床肿瘤标本为材料,分析了MGMT酶活,应用ACNU和BCNU进行肿瘤预见性化疗的基础研究.主要结果如下:1 分析了20株中国人肿瘤细胞(包括肝癌、肺癌、鼻咽癌、脑瘤、胃癌、恶性黑色素瘤、直肠癌、舌癌、乳腺癌、食管癌和宫颈癌等)MGMT酶活性及对亚硝脲药物的敏感性.发现MGMT酶活性高的细胞对亚硝尿有抗性,MGMT酶活低的细胞对亚硝脲药物敏感.     

胶质瘤患者外周血 DNA 修复基因甲基化与蛋白表达的相关性

目的：探讨胶质瘤患者O6-甲基鸟嘌呤-DNA-甲基转移酶（ O6-Methylguanine -DNA methyltransferase,MGMT）及人类错配修复基因 MSH2（Human mutS homolog2,hMSH2）蛋白表达与外周血相应基因启动子甲基化的相关性。方法分别采用免疫组化法及甲基化特异性PCR （ MSP ）检测275例胶质瘤患者肿瘤组织MGMT、hMSH2蛋白的表达及外周血中这两个基因启动子甲基化情况。结果脑胶质瘤患者肿瘤组织MGMT和hMSH2蛋白阴性表达率分别为47．2％和62．5％;基因启动子区甲基化阳性率分别为41．8％和22．4％。统计学分析显示外周血MGMT基因启动子甲基化与肿瘤组织蛋白阴性表达相关（P＜0．05）。 hMSH2基因启动子甲基化与肿瘤组织hMSH2蛋白表达不具有相关性（P＞0．05）。结论 MGMT基因甲基化是脑胶质瘤发生过程中常见的分子事件,可能与胶质瘤的发生有关;而hMSH2基因启动子甲基化可能并不是胶质瘤hMSH2蛋白失活的主要原因,可能存在其他重要因素影响其表达。     

Prognostic Value of MGMT Promoter Methylation and TP53 Mutation in Glioblastomas Depends on IDH1 Mutation

Several molecular markers have been proposed as predictors of outcome in patients with glioblastomas.We investigated the prognostic significance of O6-methylguanine-DNA methyltransferase (MGMT) promotermethylation and TP53 mutation status dependent on isocitrate dehydrogenase 1 (IDH1) mutation in glioblastomapatients. A cohort of 78 patients with histologically confirmed glioblastomas treated with radiation therapy andchemotherapy were reviewed retrospectively. We evaluated the prognostic value of MGMT promoter methylationand TP53 mutation status with regard to progression-free survival (PFS) and overall survival (OS). It was revealedthat mutations in IDH1, promoter methylation of MGMT, TP53 mutation, age, Karnofsky performance status(KFS), and extension of resection were independent prognostic factors. In patients with an IDH1 mutation, thosewith an MGMT methylation were associated with longer PFS (p=0.016) and OS (p=0.013). Nevertheless, thepresence of TP53 mutation could stratify the PFS and OS of patients with IDH1 wild type (p=0.003 and 0.029respectively, log-rank). The MGMT promoter methylation and TP53 mutation were associated with a favorableoutcome of patients with and without mutant IDH1, respectively. The results indicate that glioblastomas withMGMT methylation or TP53 mutations have improved survival that may be influenced by IDH1 mutation status.     

MGMT基因甲基化检测在神经胶质瘤治疗中的应用

摘 要：神经胶质瘤是一种常见的、复杂的恶性肿瘤,现有的治疗方法仍不能明显提高患者的生存期。近年来,越来越多的研究表明MGMT启动子甲基化状态与神经胶质瘤诊断、治疗和预后密切相关。编码修复O6-甲基鸟嘌呤的MGMT可以帮助肿瘤细胞逃避烷化剂的细胞毒作用,但MGMT启动子常会因为发生甲基化降低基因的表达,最终阻碍DNA的修复。研究已证实MGMT基因甲基化是一种可靠的胶质瘤预测标志物,且MGMT基因甲基化的胶质瘤患者对化、放疗更敏感,生存期更长。根据患者的MGMT 基因甲基化状态,制定及实施不同的治疗方案,实现个体化治疗或许可以提高化、放疗疗效和改善预后。因此,本文对MGMT基因甲基化检测在神经胶质瘤治疗中的作用以及目前常用的MGMT基因启动子甲基化检测方法的研究进展进行了综述。     

Immunohistochemical Expression of PD-L1 and IDH1 with Detection of MGMT Promoter Methylation in Astrocytoma

Objective: Programmed death ligand 1 (PD-L1) expression was suggested as a poor prognostic predictor for glioblastoma. While isocitrate dehydrogenase (IDH) has been linked to enhanced overall survival in glioma cells. In glioblastoma patients receiving treatment with alkylating drugs, the methylguanine-DNA methyltransferase (MGMT) promoter’s methylation status has been discovered as a potent and distinct predictor of good survival. In this study, we aimed to investigate the expression rate of PD-L1, IDH1, and MGMT methylation in patients with different grades of  astrocytoma. Methods: The present retrospective study retrieved the data and archived paraffin blocks of 60 cases of astrocytoma. Immunohistochemical evaluation was done to assess the expressions of PD-L1 and IDH1, Methylation-specific-PCR was used to investigate the MGMT promoter. Results: This study included astrocytoma grade II 18% (11/60), grade III 22% (13/60), grade IV 60% (36 cases). PD-L1 expression was detected in 82% of all studied cases (49/60) while IDH1 mutant astrocytoma were 73% (44/60) & methylation was reported in 58.3% (35 cases). High grade astrocytoma showed highrer expression of PD-L1 & IDH1 but with insignificant correlation (p=0.989). Conclusion: There is a relatively high expression of PD-L1 and IDH1 in patients with astrocytoma. More than half of the patients presented with MGMT promoter methylation. Further studies with larger sample size are required to investigate the association between these biomarkers and characteristics of patients with astrocytoma.     

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。     

Correlation of Clinical Features and Methylation Status of MGMT Gene Promoter in Glioblastomas

In an effort to extend the potential relationship between the methylation status of MGMT promoter and response to CENU therapy, we examined the methylation status of MGMT promoter in 44 patients with glioblastomas. Tumor specimens were obtained during surgery before adjuvant treatment, frozen and stored at -80 degrees C until for DNA extraction process. DNA methylation patterns in the CpG island of the MGMT gene were determined in every tumor by methylation specific PCR (MSP). These results were then related to overall survival and response to alkylating agents using statistical analysis. Methylation of the MGMT promoter was detected in 68% of tumors, and 96.7% of methylated tumors exhibited also an unmethylated status. There was no relationship between the methylation status of the MGMT promoter and overall survival and response to alkylating agents. Our observations do not lead us to consider promoter methylation of MGMT gene as a prognostic factor of responsiveness to alkylating agents in glioblastomas.     

抑癌基因TCF21启动子甲基化在肾癌诊断及预后中的意义

目的 探讨TCF21启动子甲基化在肾癌诊断与预后中的意义。方法 使用焦磷酸测序仪对样本的TCF21基因启动子甲基化水平进行定量检测,并进行统计学分析。结果 肾癌组织及尿液中TCF21基因甲基化水平明显升高（P=0.000,P=0.000）。肾癌组织TCF21基因甲基化水平与年龄（P=0.003）、吸烟史（P=0.018）、Fuhrman分级（P=0.046）及临床分期（P=0.048）有关, TCF 2 1 甲基化水平与年龄（ r=0 . 4 0 3 、P=0 . 0 0 2 ） 、吸烟史（r=0.321、P=0.017）和Fuhrman分级（r=0271、P=0.045）呈正相关。肾癌尿液中TCF21甲基化水平与肿瘤大小（P=0.000）、Fuhrman分级（P=0.013）及临床分期（P=0.020）有关,TCF21甲基化水平与肿瘤大小（r=0.662、P=0.000）、Fuhrman分级（r=0.662、P=0.010）和临床分期（r=0.662、P=0.017）呈正相关。TCF21高甲基化组（>35.42%）与TCF21低甲基化组（≤35.42%）患者术后生存率和总生存率差异有统计学意义（P=0.000,P=0.000）。肾癌组织中TCF21甲基化水平诊断肾癌的ROC曲线下面积高于尿液样本,差异具有统计学意义（P=0.004）。结论 TCF21与肾细胞癌的发生、发展及预后有关。联合检测肾细胞癌组织标本和尿液标本可作为诊断肾癌及判断预后等生物学行为的重要指标。     

食管鳞状细胞癌中MGMT和p16基因启动子甲基化关系分析

背景与目的:探讨食管鳞状细胞癌中06-甲基鸟嘌呤-DNA甲基转移酶(O6-methylguanine DNA methyltransferase,MGMT)基因和p16基因启动子的甲基化情况,了解这两个基因启动子甲基化与食管癌发生的关系. 材料与方法:采用甲基化特异性PCR(MSP)方法分别对44例食管鳞状细胞癌组织进行MGMT基因和p16基因的甲基化检测,并对其甲基化频率分别进行差异性检验和关联性分析.结果:在44例食管鳞癌组织中,有18例(40.9%)出现MGMT基因启动子甲基化,23例(52.3%)出现p16基因启动子甲基化,7例(14.9%)两基因均出现甲基化,10例(22.7%)两基因均未出现甲基化.MGMT和p16基因启动子甲基化率差异无统计学意义(P＞0.05).MGMT和p16基因启动子甲基化无明显相关关系(P＞0.05).结论:MGMT和p16基因启动子甲基化均可能与食管鳞状细胞癌发生、发展过程密切相关,但二者是相互独立进行的.     

IDH1 Overexpression Induced Chemotherapy Resistance and IDH1 Mutation Enhanced Chemotherapy Sensitivity in Glioma Cells in Vitro and in Vivo

Isocitrate dehydrogenase (IDH) is of great importance in cell metabolism and energy conversion. IDH mutationin glioma cells is reported to be associated with an increased overall survival. However, effects biological behaviorof therapy of gliomas are unclear. Here, we investigated the influence of wild-type and mutated IDH genes onglioma cell biological behavior and response to chemotherapy. Relevant mechanisms were further explored.We designed our study on the background of the IDHR132H mutation. Stable cell lines were constructed bytransfection. The CCK-8 method was used to assess cell proliferation, flow cytometry for the cell cycle andcell apoptosis, and the transwell method for cell invasion. Nude mouse models were employed to determinetumorigenesis and sensitivity to chemotherapy. Western blotting was used to detect relevant protein expressionlevels. We found that overexpression of wild IDH1 gene did not cause changes in the cell cycle, apoptosis andinvasion ability. However, it resulted in chemotherapy resistance to a high dose of temozolomide (TMZ) in vivoand in vitro. The IDH1 mutation caused cell cycle arrest in G1 stage and a reduction of proliferation and invasionability, while raising sensitivity to chemotherapy. This may provide an explanation for the better prognosis ofIDH1 mutated glioma patients and the relative worse prognosis of their wild-type IDH1 counterparts. We alsoexpect IDH1 mutations may be optimized as new targets to improve the prognosis of glioma patients.