Enhanced MGMT expression contributes to temozolomide resistance in glioma stem-like cells

O6-methylguanine DNA methyltransferase （MGMT） can remove DNA alkylation adducts, thereby repairing damaged DNA and contributing to the drug resistance of gliomas to alkylating agents. In addition, glioma stem-like cells （GSCs） have been demonstrated to be involved in the recurrence and treatment resistance of gliomas. In this study, we aimed to investigate MGMT expression and regulatory mechanisms in GSCs and the association of MGMT with temozolomide （TMZ） sensitivity. GSCs were enriched from one MGMT-positive cell line （SF-767） and 7 MGMT-negative cell lines （U251, SKMG-4, SKMG-1, SF295, U87, MGR1, and MGR2） through serum-free clone culture. GSCs from the U251G, SKMG-4G, SF295G, and SKMG-1G cell lines became MGMT-positive, but those from the U87G, MGR1G, and MGR2G cell lines remained MGMT-negative. However, aJl the GSCs and their parental glJoma cell lines were positive for nuclear factor-KB （NF-KB）. In addition, GSCs were more resistant to TMZ than their parental glioma cell lines （P 〈 0.05）. However, there was no significant difference in the 50% inhibition concentration （ICo） of TMZ between MGMT-positive and MGMT-negatJve GSCs （P 〉 0.05）. When we treated the MGMT-positive GSCs with TMZ plus MG-132 （an NF-KB inhibitor）, the antitumor activity was significantly enhanced compared to that of GSCs treated with TMZ alone （P 〈 0.05）. Furthermore, we found that MGMT expression decreased through the down-regulation of NF-KB expression by MG-132. Our results show that MG-132 may inhibit NF-KB expression and further decrease MGMT expression, resulting in a synergistic effect on MGMT-positive GSCs. These results indicate that enhanced MGMT expression contributes to TMZ resistance in MGMT-positive GSCs.     

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。      

胶质瘤干细胞样细胞中MGMT表达以及与替莫唑胺的耐药关系研究

背景与目的:O6甲基鸟嘌呤DNA甲基转移酶(O6-methylguanine DNA methyltranferase,MGMT)是一种能将鸟嘌呤DNA第六位氧氧原子上的甲基加合物移除和修复损伤DNA的酶,临床上能影响甲基化类化疗药物的疗效。胶质瘤干细胞样细胞被认为是胶质瘤复发的根源之一。本研究旨在探讨MGMT在胶质瘤干细胞样细胞中的表达以及与替莫唑胺耐药的关系。方法:采用悬浮克隆球形成法自胶质瘤细胞株U251、SKMG-4、SF295、SKMG-1、U373、U87、MGR1和MGR2中富集胶质瘤干细胞样细胞。应用免疫荧光技术检测胶质瘤干细胞样细胞相关分子标志;裸鼠移植瘤试验检测胶质瘤干细胞样细胞的成瘤能力。RT-PCR和Western blot检测胶质瘤干细胞样细胞中MGMT的表达;甲基化特异性PCR分析胶质瘤干细胞样细胞MGMT启动子甲基化状况;CCK-8法检测不同浓度替莫唑胺对胶质瘤干细胞样细胞和胶质瘤细胞增殖的作用。结果:分别自8个胶质瘤细胞株中成功富集胶质瘤干细胞样细胞:U251G、SKMG-4G、SF295G、SKMG-1G、U373G、U87G、MGR1G和MGR2G。胶质瘤干细胞样细胞高表达CD133、Nestin和Sox-2等干细胞标志,而且低表达GFAP和TUJ1。胶质瘤干细胞样细胞均能在裸鼠移植成瘤。MGMT在8株胶质瘤细胞及U87G、MGR1G和MGR2G中为阴性,而在U251G、SKMG-4G、SF295G、SKMG-1G和U373G中为阳性表达。替莫唑胺对胶质瘤干细胞样细胞和胶质瘤细胞的抑制作用差异具有显著性。胶质瘤干细胞样细胞与胶质瘤亲代细胞相比更加耐药(P<0.05)。另外,替莫唑胺对MGMT阳性及MGMT阴性胶质瘤干细胞样细胞IC50间的差异无统计学意义(P>0.05)。结论:MGMT阴性表达的胶质瘤细胞经干细胞样培养后,MGMT表达可转为阳性;胶质瘤干细胞样细胞较胶质瘤亲代细胞更耐受替莫唑胺;MGMT的表达与胶质瘤干细胞样细胞对替莫唑胺的耐受之间无明显关联,提示胶质瘤干细胞样细胞对替莫唑胺的耐药可能还有MGMT以外的机制参与。     

Oligodendrogliomas lacking O6-methylguanine-DNA-methyltransferase expression

O6-Methylguanine-DNA-Methyltransferase (MGMT) is a DNA repair protein considered to be a chemosensitivity predictor. We evaluated the immunohistochemical MGMT expression in 28 consecutive oligodendroglial tumors (21 oligodendrogliomas, 5 mixed oligoastrocytomas, and 2 glioblastomas with prominent oligodendroglial features; 13 treated with CCNU) and compared it with that of 13 glioblastomas. Twenty-six (93%) oligodendroglial tumors were MGMT-negative, 2 (7%) were MGMT-positive. Twelve (92%) patients treated with CCNU had MGMT-negative lesions and their median survival was 73 months; 1 patient had an MGMT-positive oligodendroglioma and is alive at 28 months. Three (23%) glioblastomas were MGMT-negative and 10 (77%) MGMT-positive. The lower MGMT expression in oligodendroglial tumors compared to glioblastomas (P < 0.05), which have different chemosensitivity, suggests a possible role of MGMT in the determination of chemoresistance. Nevertheless, the heterogeneous outcome of our MGMT-negative oligodendroglial tumors treated with CCNU, indicates that MGMT expression alone is insufficient to predict the response to alkylating drugs, presumably because of the numerous mechanisms involved.     

MGMT modulates glioblastoma angiogenesis and response to the tyrosine kinase inhibitor sunitinib

Angiogenesis inhibitors, such as sunitinib, represent a promising strategy to improve glioblastoma (GBM) tumor response. In this study, we used the O⁶-methylguanine methyltransferase (MGMT)-negative GBM cell line U87MG stably transfected with MGMT (U87/MGMT) to assess whether MGMT expression affects the response to sunitinib. We showed that the addition of sunitinib to standard therapy (temozolomide [TMZ] and radiation therapy [RT]) significantly improved the response of MGMT-positive but not of MGMT-negative cells. Gene expression profiling revealed alterations in the angiogenic profile, as well as differential expression of several receptor tyrosine kinases targeted by sunitinib. MGMT-positive cells displayed higher levels of vascular endothelial growth factor receptor 1 (VEGFR-1) compared with U87/EV cells, whereas they displayed decreased levels of VEGFR-2. Depleting MGMT using O⁶-benzylguanine suggested that the expression of these receptors was directly related to the MGMT status. Also, we showed that MGMT expression was associated with a dramatic increase in the soluble VEGFR-1/VEGFA ratio, thereby suggesting a decrease in bioactive VEGFA and a shift towards an antiangiogenic profile. The reduced angiogenic potential of MGMT-positive cells is supported by: (i) the decreased ability of their secreted factors to induce endothelial tube formation in vitro and (ii) their low tumorigenicity in vivo compared with the MGMT-negative cells. Our study is the first to show a direct link between MGMT expression and decreased angiogenicity and tumorigenicity of GBM cells and suggests the combination of sunitinib and standard therapy as an alternative strategy for GBM patients with MGMT-positive tumors.     

Oligodendrogliomas Lacking O6-Methylguanine-DNA-Methyltransferase Expression

Abstract

O⁶-Methylguanine-DNA-Methyltransferase (MGMT) is a DNA repair protein considered to be a chemosensitivity predictor. We evaluated the immunohistochemical MGMT expression in 28 consecutive oligodendroglial tumors (21 oligodendrogliomas, 5 mixed oligoastrocytomas, and 2 glioblastomas with prominent oligodendroglial features; 13 treated with CCNU) and compared it with that of 13 glioblastomas. Twenty-six (93%) oligodendroglial tumors were MGMT-negative, 2 (7%) were MGMT-positive. Twelve (92%) patients treated with CCNU had MGMTnegative lesions and their median survival was 73 months; 1 patient had an MGMTpositive oligodendroglioma and is alive at 28 months. Three (23%) glioblastomas were MGMT-negative and 10 (77%) MGMT-positive. The lower MGMT expression in oligodendroglial tumors compared to glioblastomas (P <0.05), which have different chemosensitivity, suggests a possible role of MGMT in the determination of chemoresistance. Nevertheless, the heterogeneous outcome of our MGMT-negative oligodendroglial tumors treated with CCNU, indicates that MGMT expression alone is insufficient to predict the response to alkylating drugs, presumably because of the numerous mechanisms involved.     

MGMT在脑胶质瘤组织中的表达及其与患者生存期的关系

背景与目的:目前的研究已经证实DNA修复酶——6-氧-甲基鸟嘌呤DNA甲基转移酶(O6-methylguanine-DNAmethyltransferase,MGMT)在脑胶质瘤组织中的表达与肿瘤的耐药性有一定的关系,并且能够影响肿瘤的化疗效果。本研究通过分析MGMT在脑胶质瘤组织中的表达及其与患者生存期的关系,为基于耐药机制上的脑胶质瘤分子分类提供参考资料。方法:用组织芯片技术和免疫组织化学方法检测311例脑胶质瘤石蜡标本中MGMT的表达情况,并对所有患者进行手术后的5年随访。结果:MGMT表达阳性者126例,占40.51%(126/311)。其中,星形细胞瘤中阳性率为50.41%(61/121),少枝胶质细胞瘤中为25.71%(18/70),少枝星形细胞瘤中为28.13%(18/64),胶质母细胞瘤中为51.79%(29/56);在Ⅰ～Ⅱ级胶质瘤中,MGMT表达的阳性率为36.56%(68/186),而在Ⅲ～Ⅳ级胶质瘤中为46.40%(58/125),经χ2检验分析,两者之间有显著性差异(P<0.001);将MGMT的表达与患者生存期的关系绘制成Kaplan-Meier生存曲线,并进行log-rank分析,MGMT表达阳性者与阴性者之间的差异有显著性(P<0.05)。结论:MGMT在脑胶质瘤的异常表达与肿瘤的组织类型、病理级别有关,MGMT表达阳性患者的生存期明显低于表达阴性的患者。     

替莫唑胺联合干扰素α/β治疗胶质瘤移植瘤的实验研究

背景与目的:6-氧甲基鸟嘌呤-DNA甲基转移酶(06-methylguanine-DNA-methyl transferase,MGMT)是肿瘤对甲基化类药物耐药的重要原因之一.替莫唑胺(temozolomide,TMZ)常规方案对MGMT阳性胶质瘤的化疗效果不理想.本实验在动物体内观察干扰素α/β(interfero...     

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.     

Management and survival rates in patients with glioma in China (2004–2010): a retrospective study from a single-institution

To analyze the clinical characteristics and prognostic factors in patients with glioma in an academic institute in China. From October 2004 to August 2010, total 1,285 patients were diagnosed as glioma at the Glioma Center of Beijing Tiantan Hospital. Clinical and molecular pathology features and survival rates were analyzed. The median overall survival (OS) times were 78.1, 37.6 and 14.4 months for low-grade glioma (WHO grade II), anaplastic glioma (WHO grade III) and glioblastoma (WHO grade IV), respectively. In patients with low-grade glioma, age, preoperative Karnofsky performance scale (KPS), pathological type, radiotherapy, O⁶-methylguanine-DNA methyltransferase (MGMT) expression and Ki-67 expression, were significantly associated with OS in multivariate analyses; and preoperative KPS and radiotherapy were significantly associated with progression-free survival (PFS). For anaplastic gliomas, age, preoperative KPS, pathological type, extent of resection, radiotherapy, p53 expression and phosphatase and tensin homolog (PTEN) expression were associated with OS. For glioblastomas, age, preoperative KPS, pathology type, extent of resection, radiotherapy and chemotherapy were associated with OS; and age, gender, preoperative KPS, extent of resection, radiotherapy and chemotherapy were associated with PFS. This is the largest survey for glioma management in China to date. We found significant differences in age, presenting symptoms and the expression of p53, MGMT, PTEN, and Ki-67 among patients with different types of glioma. Age, preoperative KPS, tumor grades, radiotherapy, chemotherapy and Ki-67 expression were significantly associated with clinical prognosis.     

长周期替莫唑胺治疗脑胶质瘤:附32例报告

背景与目的 :替莫唑胺(temozolomide,TMZ)国内外多推荐为胶质瘤的一线化疗药物,化疗周期通常为六周期。长周期(超过六周期)TMZ治疗胶质瘤国外已有多篇文献报道,但中国脑胶质瘤患者这方面信息较少。本文总结我们近年来长周期TMZ治疗32例胶质瘤的临床经验,重点探讨其安全性。方法:32例高级别胶质瘤(high-grade gliomas,HGGs)或低级别胶质瘤(low-grade gliomas,LGGs)采用了TMZ长周期治疗。TMZ化疗方案的选择基于肿瘤组织DNA甲基转移酶(O6-methylguanine-DNA methyltransferase,MGMT)的免疫组化检测结果,用甲基化特异PCR(MSP-PCR)检测了其中6例的MGMT启动子甲基化程度。MGMT阴性表达(±或-)者接受TMZ标准化疗(200mg/(m2.d),d1-5,四周方案),MGMT阳性表达(+或++)者接受TMZ剂量密度方案[75mg/(m2.d),d1-21,4周方案],或顺铂(cisplatin,DDP)联合TMZ化疗方案[DDP 75mg/(m2.d),d1-2;TMZ 200mg/(m2.d),d2-6,四周方案]。结果:32例患者共接受318周期TMZ方案化疗。患者化疗周期数为7~24(中位周期数为9.4)。最常见的严重毒性反应是Ⅲ度淋巴细胞减少症与白细胞减少症,发生率均为9.4%(3/32)。最常见的轻至中度毒性反应依次为疲乏(86.9%)、中性粒细胞减少症(46.9%)、脱发(46.9%)、血小板减少症(40.6%)、便秘(41.2%)及淋巴细胞减少症(25.0%)等。中位无进展生存(progress free survival,PFS)为28.6月。6月PFS、12月PFS分别为100%与71%。32例患者中,15例肿瘤完全切除,至今无病生存。依据意向性治疗原则(intention-to-treatprinciple,ITT),1例(3.1%)取得完全缓解(complete response,CR),14例(43.8%)微效(minor response,MR),2例(6.2%)稳定(stable disease,SD)。总反应率(overall response rate,ORR)为81.5%(95%CI,50%~96%),疾病控制率(disease control rate,DCR)为89.2%(95%CI,64%~98%)。结论:长周期TMZ化疗治疗胶质瘤是安全的。长周期TMZ化疗具有较高的反应率(ORR)与无进展生存(PFS)。     

Quantitative analysis of O<Superscript>6</Superscript>-methylguanine DNA methyltransferase (MGMT) promoter methylation in patients with low-grade gliomas

Methylation of the MGMT promoter is supposed to be a predictive and prognostic factor in glioblastoma. Whether MGMT promoter methylation correlates with tumor response to temozolomide in low-grade gliomas is less clear. Therefore, we analyzed MGMT promoter methylation by a quantitative methylation-specific PCR in 22 patients with histologically verified low-grade gliomas (WHO grade II) who were treated with temozolomide (TMZ) for tumor progression. Objective tumor response, toxicity, and LOH of microsatellite markers on chromosomes 1p and 19q were analyzed. Histological classification revealed ten oligodendrogliomas, seven oligoastrocytomas, and five astrocytomas. All patients were treated with TMZ 200 mg/m² on days 1–5 in a 4 week cycle. The median progression-free survival was 32 months. Combined LOH 1p and 19q was found in 14 patients; one patient had LOH 1p alone and one patient LOH 19q alone. The LOH status could not be determined in two patients and was normal in the remaining four. LOH 1p and/or 19q correlated with longer time to progression but not with radiological response to TMZ. MGMT promoter methylation was detectable in 20 patients by conventional PCR and quantitative analysis revealed the methylation status was between 12 and 100%. The volumetric response to chemotherapy analyzed by MRI and time to progression correlated with the level of MGMT promoter methylation. Therefore, our retrospective case series suggests that quantitative methylation-specific PCR of the MGMT promoter predicts radiological response to chemotherapy with TMZ in WHO grade II gliomas.     

奥沙利铂甘露醇联合替尼泊苷治疗O~6-甲基鸟嘌呤-DNA甲基转移酶阳性的恶性胶质瘤:附48例经验

背景与目的:O6-甲基鸟嘌呤-DNA甲基转移酶(O6-methylguanine-DNA methyltransferase,MGMT)阳性胶质瘤易对甲基化类药物耐药。本研究应用奥沙利铂(oxaliplatin)甘露醇与替尼泊苷(teniposide,VM-26)联合方案治疗恶性脑胶质瘤患者,观察其对恶性脑胶质瘤的近期临床治疗效果,评价其不良反应。方法:2009年12月至2011年8月,我科收治48例经病理活检确诊的恶性脑胶质瘤患者(WHOⅢ级24例,WHOⅣ级24例)。基于肿瘤组织MGMT的免疫组织化学检测结果选择化疗方案。MGMT阳性(+或++)者接受VM-26与oxaliplatin甘露醇联合方案化疗。方案为VM-26,80~100 mg/(m2.d),d1～3,4周重复一次;oxaliplatin甘露醇,130 mg/(m2.d),d1,4周重复一次。按RECIST(Response Evaluation Criteria in Solid Tumours,RECIST)疗效评价标准评价疗效。按美国国立癌症研究所(National Cancer Institute,NCI)评价标准评价不良反应。结果:48例患者资料完整,均可行近期疗效评价。上述病例共行160周期化疗。化疗周期数为2至8(中位周期数为3.3)。依据意向性治疗原则(intention-to-treat principle,ITT),无明确可评价病灶9例(18.8%),完全缓解(complete response,CR)3例(6.3%),部分缓解(partial response,PR)11例(23.0%),稳定(stable disease,SD)12例(25%),进展(progressivedisease,PD)13例(27.1%)。客观有效率(CR+PR)为47.9%(95%CI,37%~80%),疾病控制率(CR+PR+SD)为72.9%(95%CI,50%~90%)。最常见的严重毒性反应是Ⅲ度或Ⅳ度中性粒细胞减少症,发生率为10.6%(17/160)。最常见的轻至中度毒性反应依次为脱发(68.8%)、中性粒细胞减少症(58.8%)、疲乏(48%)、便秘(41.2%)以及恶心、抑郁与呕吐(均为32%)。结论:基于肿瘤组织MGMT检测结果,VM-26与oxaliplatin甘露醇联合方案治疗恶性脑胶质瘤是安全的,可取得较为满意的总反应率、疾病控制率。     

MGMT不同表达水平与替莫唑胺对胃肠胰神经内分泌肿瘤疗效的相关性

目的:通过检测肿瘤组织中O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）的表达,探讨其与胃肠胰神经内分泌瘤以替莫唑胺为基础化疗患者预后的相关性。方法:收集2017年至2019年在我院接受替莫唑胺联合替吉奥化疗的15例晚期胃肠胰神经内分泌瘤患者的肿瘤组织,采用免疫组织化学法检测肿瘤组织中MGMT蛋白表达情况,根据表达情况分为MGMT阴性组和MGMT阳性组,并对患者长期随访,进行无进展生存时间和药物安全性的评定,对MGMT表达水平与替莫唑胺治疗效果行相关性分析。结果:15例患者肿瘤组织MGMT表达阴性者为8例（53.3%）,MGMT表达阳性者为7例（46.7%）;MGMT阴性组患者化疗6个疗程后,客观缓解率（ORR）为35.7%(3/8),明显高于MGMT阳性组的0(0/7);MGMT表达阳性组患者的中位无进展生存时间（mPFS）为6个月,而MGMT表达阴性组患者的mPFS目前无法得出,但明显长于MGMT阳性组,两组间差异具有统计学意义（P=0.000 2）。化疗不良反应均为2级以下的骨髓抑制和消化道反应。结论:基于替莫唑胺化疗方案的疗效与MGMT在肿瘤组织中的表达状态相关,MGMT表达与否可以用作胃肠胰神经内分泌瘤患者对替莫唑胺治疗反应的生物学指标。     

Differential expression of miR200a-3p and miR21 in grade II–III and grade IV gliomas: Evidence that miR200a-3p is regulated by O6-methylguanine methyltransferase and promotes temozolomide responsiveness

Glioblastoma multiforme (GBM) is the most common primary brain tumor and is among the deadliest of human cancers. Dysregulation of microRNAs (miRNAs) expression is an important step in tumor progression as miRNAs can act as tumor suppressors or oncogenes and may affect cell sensitivity to chemotherapy. Whereas the oncogenic miR21 has been shown to be overexpressed in gliomas, the expression and function of the tumor-supressor miR200a in GBMs remains unknown. In this study, we show that miR21 is upregulated in grade IV (GBMs) vs. grade II–III (LGs) gliomas, confirming that miR21 expression level is correlated with tumor grade, and that it may be considered as a marker of tumor progression. Conversely, miR200a is demonstrated for the first time to be downregulated in GBMs compared with LGs, and overexpression of miR200a in GBM cells is shown to promote TMZ-sensitivity. Interestingly, miR200a but not miR21 expression level is significantly higher in TMZ-responsive vs. -unresponsive tumoral glial cells in primary culture. Furthermore, miR200a appears negatively correlated with the expression of the DNA repair enzyme O⁶-methylguanine methyltransferase (MGMT), and the inhibition of MGMT activity results in an increase of miR200a expression in GBM cells. Taken together, these data strongly suggest that miR200a is likely to act as a crucial antitumoral factor regarding glioma progression. Interplay between miR200a and MGMT should be considered as potential mechanism involved in therapeutic response.     

人脑胶质瘤中CD147的表达及其意义

背景与目的：以往研究发现CD147与某些恶性肿瘤存在相关性。本研究探讨CD147在人脑胶质瘤中的表达及其与病理分级的相关性。方法：收集61例星形细胞肿瘤及18例瘤旁脑组织标本．制作组织芯片。进行免疫组织化学染色,用半定量逆转录聚合酶链反应法（RT-PCR）进行CD147mRNA检测。结果：（1）胶质瘤组织中CD147蛋白的阳性表达率为74．3％．正常脑组织中仅血管内皮细胞浆有阳性着色。（2）Ⅲ、Ⅳ级星形细胞肿瘤瘤细胞胞浆CD147着色显著强于Ⅰ、Ⅱ级低级别星形细胞肿瘤。（3）CD147mRNA在胶质瘤组织中阳性率为83．3％,明显高于正常脑组织中的阳性率25％（P〈0．05）。CD147mRNA在星形细胞肿瘤组织中的表达量为0．274±0．087,明显高于正常组织0．081±0．043,两者差异具有显著性（P〈0．05）。结论：CD147的表达可能在人脑胶质瘤的发生发展中起重要作用．     

O<Superscript>6</Superscript>-methylguanine DNA methyltransferase status determined by promoter methylation and immunohistochemistry in gliosarcoma and their clinical implications

O⁶-methylguanine-DNA methyltransferase (MGMT) is known as a DNA repair protein, and loss of function in MGMT is related to an increase in survival in patients with malignant gliomas treated with alkylating agents. In the present study, we determined the status of MGMT using methylation-specific polymerase chain reaction (PCR) and immunohistochemistry on paraffin-embedded specimens in 12 human gliosarcomas, and these results were then related to overall survival (OS) and response to alkylating agents. The MGMT promoter was methylated in six patients. Immunostaining of MGMT was positive in 58.3% of patients. MGMT methylation status was correlated with immunostaining results in five patients (41.7%). The median OS and progression-free survival (PFS) of the whole population were 13.4 months [95% confidence interval (CI), 12.3–14.5 months] and 8.3 months (95% CI, 7.4–9.2 months), respectively. In patients with methylated MGMT promoter, median OS was 15.0 months, compared with 11.3 months in the unmethylated group. Median PFS of gliosarcoma patients was 10.3 months for the methylated group, whereas it was 7.3 months for the unmethylated group. On multivariate analysis, patients with methylated MGMT promoter had better prognosis than patients with unmethylated MGMT promoter with respect to OS and PFS (P = 0.045 and 0.034, respectively). However, there was no statistical significance between MGMT protein expression and survival. The results show that a significant fraction of gliosarcomas have MGMT promoter methylation and protein expression, and suggest that patient survival is associated with MGMT methylation status.     

高级别胶质瘤手术联合术后辅助放化疗的疗效分析（附56例）

目的:探讨高级别胶质瘤的手术和术后放化疗的治疗效果。方法:回顾性分析56例高级别胶质瘤患者的临床资料,均采用手术、术后同步放化疗及6个周期以上的替莫唑胺化疗,对其治疗效果和不良反应进行评价分析。结果:56例患者手术全切除10例（17.9%）,次全切除35例（62.5%）,部分切除11例（19.6%）。分子病理提示IDH1132H突变19例;MGMT基因启动子甲基化20例。随访过程中13例未见明显复发;35例复发;1例出现脊髓的多发转移灶;7例出现放射性脑损伤,其中3例再次手术减压,4例保守治疗。12个月、24个月和36个月总生存率分别为66.1%、50.0%和32.1%;无进展生存率分别为57.1%、39.3%和25.0%。结论:高级别胶质瘤预后不佳,术后残留、复发、转移和放射性损伤是治疗的重点和难点;MGMT基因启动子甲基化患者总体生存率和无进展生存率高于MGMT启动子未甲基化患者。     

Temozolomide-mediated radiation enhancement in glioblastoma: a report on underlying mechanisms.

PURPOSE: In this study, we investigated the mechanisms by which temozolomide enhances radiation response in glioblastoma cells. EXPERIMENTAL DESIGN: Using a panel of four primary human glioblastoma cell lines with heterogeneous O(6)-methylguanine-DNA methyltransferase (MGMT) protein expression, normal human astrocytes, and U87 xenografts, we investigated (a) the relationship of MGMT status with efficacy of temozolomide-based chemoradiation using a panel of in vitro and in vivo assays; (b) underlying mechanisms by which temozolomide enhances radiation effect in glioblastoma cells; and (c) strategies to overcome resistance to radiation + temozolomide. RESULTS: Temozolomide enhances radiation response most effectively in glioblastomas without detectable MGMT expression. On concurrent radiation + temozolomide administration in MGMT-negative glioblastomas, there seems to be decreased double-strand DNA (dsDNA) repair capacity and enhanced dsDNA damage compared either with radiation alone or with sequentially administered temozolomide. Our data suggest that O(6)-benzylguanine can enhance the antitumor effects of concurrent radiation + temozolomide in MGMT-positive cells by enhancing apoptosis and the degree of dsDNA damage. O(6)-Benzylguanine was most effective when administered concurrently with radiation + temozolomide and had less of an effect when administered with temozolomide in the absence of radiation or when administered sequentially with radiation. Our in vivo data using U87 xenografts confirmed our in vitro findings. CONCLUSIONS: The present study shows that temozolomide enhances radiation response most effectively in MGMT-negative glioblastomas by increasing the degree of radiation-induced double-strand DNA damage. In MGMT-positive glioblastomas, depletion of MGMT by the addition of O(6)-benzylguanine significantly enhances the antitumor effect of concurrent radiation + temozolomide. These are among the first data showing mechanisms of synergy between radiation and temozolomide and the effect of MGMT.     

Temozolomide preferentially depletes cancer stem cells in glioblastoma

The prognosis of patients suffering from glioblastoma (GBM) is dismal despite multimodal therapy. Although chemotherapy with temozolomide may contain tumor growth for some months, invariable tumor recurrence suggests that cancer stem cells (CSC) maintaining these tumors persist. We have therefore investigated the effect of temozolomide on CD133(+) and CD133(-) GBM CSC lines. Although differentiated tumor cells constituting the bulk of all tumor cells were resistant to the cytotoxic effects of the substance, temozolomide induced a dose- and time-dependent decline of the stem cell subpopulation. Incubation with sublethal concentrations of temozolomide for 2 days completely depleted clonogenic tumor cells in vitro and substantially reduced tumorigenicity in vivo. In O(6)-methylguanine-DNA-methyltransferase (MGMT)-expressing CSC lines, this effect occurred at 10-fold higher doses compared with MGMT-negative CSC lines. Thus, temozolomide concentrations that are reached in patients were only sufficient to completely eliminate CSC in vitro from MGMT-negative but not from MGMT-positive tumors. Accordingly, our data strongly suggest that optimized temozolomide-based chemotherapeutic protocols might substantially improve the elimination of GBM stem cells and consequently prolong the survival of patients.