胶质母细胞瘤MGMT基因启动子甲基化、MGMT蛋白表达和预后相关性分析

目的探讨脑胶质母细胞瘤中O-6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)基因启动子甲基化状态和MGMT蛋白表达水平与临床预后的相关性。方法收集119例人脑胶质母细胞瘤石蜡包埋样本,提取基因组DNA并进行亚硫酸氢盐修饰,用MethyLight法检测MGMT基因启动子甲基化状态,用免疫组织化学染色法检测MGMT蛋白表达水平,对MGMT基因启动子甲基化状态和MGMT表达水平与患者预后行相关性分析。结果在119例胶质母细胞瘤样本中,有42例检测到MGMT基因启动子甲基化,甲基化发生率为35.3%(42/119例),MGMT基因启动子甲基化与无进展生存期(P=0.011)及总体生存期(P=0.036)延长相关。MGMT蛋白表达水平和临床预后无相关性(P0.05),与MGMT基因启动子甲基化状态之间也无相关性(P0.05)。结论 MGMT基因启动子甲基化与胶质母细胞瘤患者预后呈正相关,由免疫组化法测得的MGMT蛋白表达水平和预后及基因启动子甲基化之间无关联性,MGMT基因启动子甲基化状态可以作为评判预后的生物学指标之一。     

恶性脑胶质瘤MGMT基因启动子甲基化状态与患者临床预后的相关性

目的 探讨恶性脑胶质瘤组织O6-甲基鸟嘌呤-DNA-甲基转移酶(MGMT)基因启动子甲基化状态及MGMT蛋白表达与患者生存预后的关系,评价MS-MLPA技术检测MGMT基因启动子甲基化状态对脑胶质瘤化疗的意义. 方法 选择自2006至2010年南方医科大学珠江医院神经外科手术治疗且病理证实为恶性脑胶质瘤(WHOⅢ级、Ⅳ级)的39例患者为研究对象,应用免疫组化染色法检测肿瘤组织MGMT蛋白表达,应用MS-MLPA技术检测MGMT基因启动子甲基化状态,并观察患者化疗后总生存时间. 结果 肿瘤组织中MGMT蛋白表达阳性、弱阳性者与表达阴性者生存时间比较差异有统计学意义(P=0.003),MGMT蛋白表达阳性者比表达阴性者预后更差.肿瘤组织中MGMT基因启动子未甲基化者、低度过甲基化者、中度过甲基化者、高度过甲基化者生存时间两两比较差异均有统计学意义(P＜0.05),MGMT基因启动子过甲基化率越高者预后越好.MGMT蛋白表达与MGMT基因启动子甲基化状态存在统计学相关性(r=0.697,P=0.000),基因甲基化程度越高者蛋白表达越低. 结论 MGMT蛋白表达与MGMT基因启动子甲基化状态均可以作为接受烷化剂化疗的恶性胶质瘤患者生存期的预测指标.MS-MLPA技术是一种可靠的检测MGMT基因启动子甲基化状态的方法.     

胶质瘤MGMT启动子甲基化定量检测的MS-HRM方法建立及评估

目的建立甲基化特异高分辨率溶解曲线(Methylation sensitive high resolution melting curve,MS-HRM)定量检测胶质瘤MGMT基因启动子甲基化的方法,用于指导胶质瘤患者术后化疗及预后判断。方法从标本库随机选取胶质瘤组织37例,提取DNA进行甲基化修饰,应用MS-HRM方法和甲基化特异性PCR(methylation specific PCR,MSP)进行MGMT基因启动子区甲基化检测。结果 MSP方法检测显示部分甲基化标本29例(78.4%),较甲基化(13.5%)和未甲基化(8.1%)差异显著。MS-HRM发现MGMT基因启动子甲基化水平在10%~25%和25%~50%的标本分别有14例(37.8%)和17例(49.5%)。结论成功建立MS-HRM检测胶质瘤MGMT启动子甲基化的方法,该方法特异性高、灵敏度强和可重复性,有望应用于临床胶质瘤MGMT启动子甲基化高通量定量检测,以指导个体化治疗。     

新诊断胶质瘤MGMT基因启动子甲基化水平定量分析

目的 探讨新诊断胶质瘤O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）基因启动子甲基化（简称MGMT甲基化）水平的变化。方法 回顾性分析2016年1月至2019年6月郑州大学附属肿瘤医院神经外科收治的225例新诊断胶质瘤的临床资料,定量分析MGMT甲基化。结果 新诊断胶质瘤MGMT甲基化水平为（23.68±14.88）%,阳性率89.33%。≥40岁病人MGMT甲基化水平[（24.89±15.67）%]显著高于<40岁病人[（19.85±11.31）%;P<0.05]。少突胶质细胞瘤MGMT甲基化水平[（30.41±12.99）%]显著高于其他类型的胶质瘤[星形细胞瘤为（20.81±13.53）%、少突星形细胞瘤为（23.00±8.21）%、胶质母细胞瘤为（23.39±17.85）%;P<0.05]。胶质瘤MGMT甲基化水平与病人年龄呈正相关（r=0.135,P<0.05）,而与病人性别和肿瘤级别无明显关系（P>0.05）。结论 新诊断胶质瘤MGMT甲基化水平与病人与年龄正相关,少突胶质细胞瘤MGMT甲基化水平明显高于其他类型胶质瘤     

胶质瘤干细胞对替莫唑胺敏感性的研究

目的 探讨胶质瘤干细胞(GSC)对替莫唑胺(TMZ)的敏感性及耐药机制.方法 新鲜多形性胶质母细胞瘤(GBM)标本培养后获得GSC.免疫荧光技术榆测未分化GSC的CD133及分化生长GSC的GFAP的表达,MTS法检测对替莫唑胺的敏感性,流式细胞技术对CD133阳性细胞的比例进行定量,荧光标记的甲基化特异性PCR分析MGMT启动子区域的甲基化状态,Western blot检测抑癌基因PTEN的表达.结果 (1)5例GBM标本中成功获得GSC,符合肿瘤下细胞定义.(2)5个GSC细胞株多数对TMZ不敏感.其中,T509的半数抑制浓度(IC50)为22.3 μmol/L(敏感),T411的IC50为286.3 μmol/L(中度敏感),其余3个细胞株T402,T405及T509的IC50皆大于1000μmol/L(不敏感).(3)CD133阳性细胞比例大于10%的GSC细胞株对TMZ不敏感.(4)MGMT启动子区域呈去甲基化状态的GSC对TMZ不敏感或仅为中度敏感.(5)5个GSC细胞株中,PTEN表达水平差异大,与GSC对TMZ的敏感性无明显关联.结论 GSC对TMZ普遍耐药,与MGMT启动子区域甲基化状态及CD133阳性细胞有关,而与PTEN蛋白表达水平无明显关联.     

MGMT表达在胶质瘤对烷化剂耐药中的作用

恶性脑胶质瘤是最常见的原发性颅内恶性肿瘤,包括小分子、脂溶性、能穿过血脑屏障的烷化剂的化疗方案,使部分病人的生存期明显延长。烷化剂的化疗效果同MGMT(O6-methylguanine-DNA methyltransferase MGMT)基因启动子甲基化沉默了MGMT表达相关,因此,阻断其修复能力可使烷化剂更有效。其表达可由甲基化特异性PCR(MSP)、免疫组化(IHC)、血清MGMT活性检测等方法直接或间接测定。MGMT基因启动子甲基化水平可作为识别病人对烷化剂敏感性的指标,并以此指导临床治疗。     

MGMT基因对脑胶质瘤患者启动子甲基化变化的价值研究

目的探究MGMT基因表达、启动子甲基化与脑胶质瘤患者预后间的关联性。方法择取我院2012-06-2014-09收治的82例行脑胶质瘤个体化综合治疗的恶性胶质瘤患者为研究对象,依据脑胶质瘤MGMT基因表达与启动子甲基化状况进行分组,术后予以同步放化疗,分析各组患者近期疗效、安全性及无进展生存时间。结果 MGMT蛋白表达与恶性胶质瘤MGMT基因启动子甲基化状态呈负相关(P0.05),MGMT基因启动子甲基化组近期疗效(79.54%vs75.00%)高于MGMT蛋白低表达组(P0.05),MGMT甲基化低表达组1、2、3a生存率(90.62%vs 75.00%;56.25%vs41.66%;31.25%vs 16.66%)明显高于MGMT甲基化高低表达组(P0.05)。结果 MGMT基因表达、启动子甲基化与脑胶质瘤患者预后密切相关,临床上应引起足够重视。     

Ki-67及TopoisomeraseⅡ在脑胶质母细胞瘤组织中的表达及其生物学意义

目的 探讨人胶质母细胞瘤中Ki-67及TopoisomeraseⅡ(TopoⅡ)之间的相关性及其与胶质母细胞瘤病人预后的关系.方法 收集O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)低表达的胶质母细胞瘤标本60例,应用免疫组化法检测Ki-67和TopoⅡ的表达,研究其相关性.结果 在MGMT低表达的胶质母细胞瘤中,Ki-67与TopoⅡ表达呈正相关(γ=0.83,P＜0.05).对本组标本来源的60例胶质母细胞瘤病人随访12-23个月,其中死亡38例,肿瘤复发46例.结论 Ki-67和TopoⅡ在胶质母细胞瘤中的表达强度相对一致;其表达能客观反映肿瘤细胞的增殖活性和恶性程度,且可能影响胶质母细胞瘤病人的预后.     

Li-Fraumeni综合征

目的总结1例以胶质母细胞瘤为临床表现的Li-Fraumeni综合征患者的临床病理学特点。方法与结果女性患者,33岁,以无明显诱因的左侧肢体抽搐发病,头部MRI显示右侧额顶叶交界区占位性病变,增强扫描病灶呈"花环"样强化征象,行右侧顶叶占位性病变切除术。术中切开右侧顶叶皮质2.50 cm,其下即见肿瘤组织,呈椭圆形,大小约5 cm×4 cm×3 cm,质地柔软,紫红色,边界清晰,血供丰富,分块全切除肿瘤。组织学形态,胶质细胞异型性增生明显,部分区域呈梭形伴大量瘤巨细胞;免疫组织化学染色,肿瘤细胞胞质表达胶质纤维酸性蛋白、胞核弥漫性表达P53蛋白,Ki-67抗原标记指数为25%;网织纤维染色呈阴性;分子病理学检查未检测到异柠檬酸脱氢酶1/2(IDH1/2)基因外显子4突变、端粒酶逆转录酶(TERT)基因启动子突变和O6-甲基鸟嘌呤DNA甲基转移酶(MGMT)基因甲基化,荧光原位杂交未检测到染色体1p/19q共缺失。病理诊断为(右侧顶叶)胶质母细胞瘤,IDH-野生型(WHOⅣ级)。患者共住院15 d,出院后随访至2016年,死亡。详细追问家族史得知患者家系有肿瘤病史:其姊罹患右侧乳腺癌,其父因胃癌死亡,祖母因乳腺癌死亡,叔父因肺癌死亡,伯父因造血系统肿瘤死亡。进一步对患者胶质母细胞瘤标本和右侧乳腺浸润性导管癌标本行TP53基因检测,均未检测到突变;对冻存的血液标本行全外显子测序,TP53基因存在c.1009CT(p.Arg337Cys)杂合致病性突变,为错义突变。最终诊断为Li-Fraumeni综合征,该家系诊断为Li-Fraumeni综合征家系。结论 Li-Fraumeni综合征是遗传性肿瘤综合征,呈常染色体显性遗传,以乳腺癌、骨与软组织肉瘤、中枢神经系统肿瘤和肾上腺皮质肿瘤等高肿瘤发病风险为特征,TP53基因是最常见的Li-Fraumeni综合征相关致病基因。     

胶质母细胞瘤中COL9A1与MGMT的表达相关

目的分析胶质母细胞瘤中O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）的相关基因。方法从NCBI基因芯片数据库GDS1962中选取78例胶质母细胞瘤样本的基因表达数据,通过Pearson积差相关分析法统计与MGMT表达高度相关的基因,然后通过实时定量逆转录多聚酶链反应（qRT-PCR）在18例胶质母细胞瘤临床样本中检验这些基因与MGMT的表达相关性。结果基因芯片分析表明,COL9A1（IX型胶原α1链）等基因与MGMT表达显著负相关（r=-0.589,P=0.000）,对18例临床样本中检测到的COL9A1和MGMT的相对表达数据的统计分析进一步证实了这些相关性。结论 COL9A1可以作为胶质母细胞瘤MGMT分子治疗的重要候选靶点,增强患者对化疗的敏感性。     

Meningeal hemangiopericytomas: A clinicopathological study with emphasis on MGMT (O6‐methylguanine‐DNA methyltransferase) promoter methylation status

Meningeal hemangiopericytomas (HPCs) are aggressive dural‐based tumors, for which no prognostic or predictive marker has been identified. Gross total resection is treatment of choice, but not easily achieved; hence, alkylating agents like temozolomide (TMZ) are now being tried. O⁶‐methylguanine‐DNA methyltransferase (MGMT) promoter methylation has proven prognostic and predictive value in glioblastomas. This study evaluates MGMT promoter methylation in meningeal HPCs to determine its role in HPC oncogenesis and its association with patient outcome. Meningeal HPCs diagnosed between 2002 and 2011 were retrieved and clinicopathological features reviewed. MGMT promoter methylation status was assessed by methylation‐specific polymerase chain reaction (MSP) and immunohistochemistry (IHC) for MGMT protein. HPCs accounted for 1.1% of all CNS tumors. Forty cases were analyzed; the majority were adults (mean age = 41.4 years). Seventy percent were primary and 30% were recurrent tumors; 60% were grade II and 40% were grade III. MGMT promoter methylation was identified in 45% of cases, including Grade II (54.2%) and Grade III (31.3%) (P = 0.203). Promoter methylation was significantly (P = 0.035) more frequent in primary (57.1%) than in recurrent (16.7%) tumors. No correlation was noted between MGMT promoter methylation by MSP and MGMT protein expression by IHC, or with progression‐free survival. Thus, a significant proportion of HPCs demonstrate MGMT promoter methylation, suggesting possible susceptibility to TMZ. As promoter methylation is more frequent in primary tumors, TMZ may serve as a therapeutic option in residual primary tumors. Epigenetic inactivation of MGMT in HPCs necessitates the assessment of prognostic and predictive value of MGMT promoter methylation in HPCs in larger clinical trials.     

O6 -methylguanine DNA methyltransferase gene promoter methylation in high-grade gliomas: a review of current status

Assessment of promoter methylation of the O 6 -methylguanine DNA methyltransferase (MGMT) gene has recently gained importance in molecular profiling of high-grade gliomas. It has emerged not only as an important prognostic marker but also as a predictive marker for response to temozolomide in patients with newly diagnosed glioblastoma. Further, recent studies indicate that MGMT promoter methylation has strong prognostic relevance even in anaplastic (grade III) gliomas, irrespective of therapy (chemotherapy or radiotherapy). This article provides an overview of its use as a predictive and prognostic biomarker, as well as the methods employed for its assessment and use in therapeutic decision making.     

人脑胶质瘤IDH1突变状态与MGMT启动子甲基化、P53和TERT突变相关性

目的 探讨脑胶质瘤异柠檬脱氢酶1（IDH1）突变与O6-甲基鸟嘌呤-DNA甲基转移酶 （MGMT）启动子甲基化状态、P53和端粒酶逆转录酶（TERT）突变之间的相关性。方法 收集2016年6月至2017年9月手术切除并经病理诊断为胶质瘤标本72例（WHO Ⅱ级14例,Ⅲ级19例,Ⅳ级39例）。采用PCR荧光探针法检测MGMT基因启动子甲基化状态,毛细管电泳法检测基因IDH1、P53、TERT突变情况;采用列联系数分析IDH1突变与MGMT基因启动子甲基化、P53、TERT突变状态的相关性。采用多因素Logistic回归分析检验IDH1突变的相关因素。结果 72例中,IDH1突变率为29.2%,MGMT启动子甲基化率为47.2%,P53突变率为41.4%,TERT突变率为50%。相关性分析发现IDH1突变与MGMT启动子甲基化（列联系数=0.44;P<0.001）、P53突变（列联系数=0.32;P<0.05）均有显著相关性,但与TERT启动子突变无明显相关性（P>0.05）。IDH1野生型胶质瘤中MGMT启动子甲基化与TERT启动子基因突变具有相关性（列联系数=0.28,P<0.05）。IDH1突变型胶质瘤中MGMT启动子甲基化与P53基因突变具有相关性（列联系数=0.27,P<0.05）。多因素Logistic回归分析,结果显示病人年龄、MGMT启动子甲基化是IDH1突变独立相关因素（P<0.05）。结论 胶质瘤IDH1突变可能与MGMT启动子甲基化、P53和TERT突变之间存在复杂的相互调节作用。     

Methylation status of the MGMT gene promoter fails to predict the clinical outcome of glioblastoma patients treated with ACNU plus cisplatin

We analyzed the methylation status of the O6‐methylguanine‐DNA methyltransferase (MGMT) promoter using a methylation‐specific polymerase chain reaction (MSP) in glioblastoma patients treated with 1‐(4‐amino‐2‐methyl‐5‐pyrimidinyl)methyl‐3‐(2‐chloroethyl)‐3‐nitrosourea (ACNU) plus cisplatin followed by radiation therapy. Forty‐eight patients with interpretable MSP results were included in this study. The MGMT promoter was methylated in 26 patients (54.2%, methylated group) and unmethylated in 22 patients (45.8%, unmethylated group). Comparison of clinical outcomes between the two groups revealed that the methylation status of the MGMT gene promoter was not a prognostic factor for overall survival (P = 0.516) or a predictive factor for radiological response to ACNU plus cisplatin treatment (P = 0.529). The most noteworthy explanation for the result is that the synergistic antitumor effects of ACNU and cisplatin resulting from inactivation of MGMT by cisplatin in MGMT active tumors offset the drug resistance.     

Immunohistochemistry for O6-methylguanin-DNA methyltransferase in glioblastomas defined by WHO2016: Correlation with promoter methylation status and patients’ progression-free survival with the cut-off value determined by ROC analysis

Although promoter methylation status is known to correlate with response to alkylating agents, immunohistochemistry (IHC) is the only available method for MGMT status in many institutions. However, the clinical utility of MGMT IHC is controversial. A hundred and twenty four cases of primary glioblastoma diagnosed by morphology-based criteria over 2 decades were re-appraised based on WHO 2016 classification. Tumor MGMT status was evaluated by IHC and methylation-specific PCR (MSP) to see if any correlation between the results of the 2 methods. The association with patients’ prognoses was also investigated. Among 124 cases, 116 were confirmed to be glioblastoma by definition of WHO2016, and median overall survival (OS) of glioblastoma, IDH-wildtype and epithelioid glioblastoma were 18 and 27 months, respectively. MGMT promoter methylation status significantly correlated with progression-free survival (PFS) (p = 0.014) but not with OS (p = 0.364) in patients with glioblastoma by the integrated diagnosis. With the cut-off value of 24.5% of positive cell ratio as determined by receiver operating characteristic (ROC) analysis, there was a good correlation between the results of IHC and MSP (p = 0.08 × 10⁻²⁴). Accordingly, there was a marginal association between the results of IHC and patients’ PFS (p = 0.063), but not OS (p = 0.563). When the patients were divided into pre and post temozolomide era, the association of MGMT promoter methylation status with PFS was only noted in the patients of temozolomide era (pre, p = 0.432; post, p = 0.015).     

Radiation‐induced glioblastoma in a medulloblastoma patient: A case report with molecular features

We report a case of glioblastoma (GBM) occurring 8 years after radiation therapy for a medulloblastoma. A 15‐year‐old boy underwent surgery and radiotherapy for a medulloblastoma and 8 years later he developed a second tumor at the same site. The second lesion showed different histological and molecular features, was diagnosed as a glioblastoma and fulfilled the criteria of radiation‐induced neoplasm. Mutational analysis of the p53 gene showed a C to G transition at codon 176 in tumor DNA. LOH was detected at 17p and 19q. The tumor also showed O6‐methylguanine‐DNA methyl‐transferase (MGMT) promoter methylation and no amplification of EGF receptor. In conclusion, the radiation‐induced MGMT hyper‐methylation and p53 mutations may have a role in the development of a subgroup of radio‐induced glioma (RIG), suggesting that these molecular alterations directly cooperate in the genesis of the post‐irradiation GBM. Moreover RIGs seem to be a heterogeneous group of tumors that may resemble either primary or secondary GBM.