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.     

Potential use of prostate specific membrane antigen (PSMA) for detecting the tumor neovasculature of brain tumors by PET imaging with <Superscript>89</Superscript>Zr-Df-IAB2M anti-PSMA minibody

Tumor angiogenesis has attracted increasing attention because of its potential as a valuable marker in the differential diagnosis of brain tumors as well as a novel therapeutic target. Prostate-specific membrane antigen (PSMA) is expressed by the neovasculature endothelium of some tumors, with little to no expression by the tumor cells or normal vasculature endothelium. The aim of this study was to investigate the potential of PSMA for the evaluation of the tumor neovasculature of various brain tumors and the possibility of detecting PSMA expression in brain tumors using PET imaging with ⁸⁹Zr-Df-IAB2M (anti-PSMA minibody). Eighty-three tissue specimens including gliomas, metastatic brain tumors, primary central nervous system lymphomas (PCNSL), or radiation necroses were analyzed by immunohistochemical staining with PSMA antibody. ⁸⁹Zr-Df-IAB2M PET scans were performed in three patients with recurrent high-grade gliomas or metastatic brain tumor. PSMA was highly expressed in the vascular endothelium of high-grade glioma and metastatic brain tumor, whereas PSMA was poorly expressed in the vascular endothelium of PCNSL and radiation necrosis. PSMA expression in high-grade gliomas and a metastatic brain tumor was clearly visualized by PET imaging with ⁸⁹Zr-Df-IAB2M. Furthermore, a trend toward a positive correlation between the degree of ⁸⁹Zr-Df-IAB2M uptake and PSMA expression levels in tumor specimens was observed. PET imaging of PSMA using ⁸⁹Zr-Df-IAB2M may have potential value in the differential diagnosis of high-grade glioma from PCNSL or radiation necrosis as well as in the prediction of treatment efficacy and assessment of treatment response to bevacizumab therapy for high-grade glioma.     

Hypermethylation of promoter 5＇CpG island of p16 gene in glioma tissue and plasma

ObjectiveTo explore the clinical significance of methylation status of promoter CpG island of p16 gene in glioma tissue and plasma.MethodsMethylation specific polymerase chain reaction (MSP) was used to determine the methylation status of the promoter for p16 gene within glioma tissue and plasma.Immunohistochemicel method (SP) was used to analyze the expressions of p16 and Ki-67 proteins.ResultsHypermethylation was found in 17/40 (42.5%) of brain gliomas,in comparison with 11/40 (27.5%) plasma specimens (x2 = 1.9780,P = 0.1596).Loss of p16 expression was associated (P = 0.0229) with hypermethylation of CpG island of promoter regions.Hypermethylation of p16 gene CpG island was significantly related to the increase of malignant grade of brain glioma (TissueX2 = 11.4288,P = 0.0007;PlasmaX2 = 8.9439,P = 0.0028).The Ki-67 index increased significantly (P＜0.05) in brain gliomas methylated in contrast to those unmethylated.ConclusionP16 hypermethylation may be one of the major mechanisms of tumorigenesis of gliomas.Methylated tumor-specific DNA may be as a plasma biomarker for prognosis in patients with glioma.     

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。     

Immunohistochemical study for O<Superscript>6</Superscript>-methylguanine-DNA methyltransferase in the non-neoplastic and neoplastic components of gliomas

Although the expression O⁶-methylguanine-DNA methyltransferase (MGMT) is an important hallmark for decision of nitrosourea chemotherapy for glioma patients, no immunohistochemical method for analysis of MGMT has been standardized yet. Gliomas usually contain non-neoplastic cells even deep in the tumor. It is not known which of these components expresses MGMT. To clarify this point, we investigated MGMT expression in the non-neoplastic cells in autopsy and surgical specimens by immuno-histochemistry. High grade gliomas were also studied to find a cut-off point for treatment decision. MGMT immunohistochemistry in the normal brain or brain with non-neoplastic disease revealed nuclear staining in some endothelial cells, inflammatory cells, ependymal cells, astrocytes and oligodendroglias. Some cells were double stained with CD68 (macrophages or microglias). The neurons were consistently MGMT-negative. High grade gliomas always contained an MGMT-positive non-neoplastic component. Although the endothelial cells were easily distinguished from the neoplastic cells, other cells were often mistaken for tumor cells. The population of MGMT-positive non-neoplastic cells was usually less than 10%. We set a cut off-point at 10% between the positive and negative groups because the statistical difference in the overall survival was most distinct at this value. In 51 high grade glioma patients, who received both radiotherapy and chemotherapy with nimustine (ACNU), the median overall survival of the MGMT-negative group (23months) was significantly longer than that of the MGMT-positive group (14months) (P<0.009). Multivariate analysis revealed that the negative MGMT expression was a significant prognostic variable next to the degree of surgical removal for the overall survival. In the MGMT-positive group, addition of platinum-based chemotherapy did not improve the survival.     

A combination of IFN-β and temozolomide in human glioma xenograft models: implication of p53-mediated MGMT downregulation

Purpose Methylation of the O⁶-methyguanine-DNA methyltransferase (MGMT) gene promoter in gliomas has been reported to be a useful predictor of the responsiveness to temozolomide (TMZ). In our previous experiments, we observed that IFN-β sensitized TMZ-resistant glioma cells with the unmethylated MGMT promoter and that the mechanism of action was possibly due to attenuation of MGMT expression via induction of TP53. In this study, (1) we explored the synergistic effect of IFN-β and TMZ in the animal model, and (2) clarified the role of IFN-β induced TP53 in the human MGMT promoter. Methods (1) Nude mice with either subcutaneous T98 (TMZ-resistant) or U251SP (TMZ-sensitive) tumor were treated with IFN-β/TMZ for 5 consecutive days. (2) The MGMT promoter activity was assayed by a luciferase reporter system in Saos2 (p53-null) cells transduced with a p53-adenoviral vector, and T98 glioma cells treated with IFN-β. Results (1) A combination of IFN-β/TMZ had significant synergistic antitumor activity on the growth of both T98 and U251SP tumors. (2) MGMT promoter activity was suppressed by either adenovirally transduced p53 or IFN-β. Conclusions It would be appealing to consider a prospective clinical trial in which genetic markers are used for personalized drug selection, eliciting other forms of treatment or inhibition of MGMT for those with MGMT expression. In this context, IFN-β inactivates MGMT via p53 gene induction and enhances the therapeutic efficacy to TMZ.     

Deregulation of the p14ARF/Mdm2/p53 pathway and G1/S transition in two glioblastoma sets

Sixty-one glioblastomas have been studied, subdivided into the categories of classic glioblastomas (GBM) and glioblastomas with astrocytic (GBA) and oligodendroglial (GBO) differentiated areas. On surgical samples, TP53, Mdm2, CDKN2A/p16–p14 alterations were studied by molecular biology techniques and by immunohistochemistry. It has been found that Mdm2 amplification was more frequent in GBM than in GBA and GBO, that p14^ARF was inactivated in a high percentage of cases in the three tumor categories. Both these and other alterations did not reach a statistical significance, with the exception of CDKN2A/p16 homozygous deletion which showed the highest frequency in GBO. The latter finding could be in line with the observation that CDKN2A/p16 inactivation is a step in the molecular pathway to tumor progression in oligodendrogliomas. TP53 mutations and Mdm2 amplifications were mutually exclusive, whereas TP53 mutations and CDKN2A/p14 inactivation coexisted in 5 cases. The alterations of the p53/Mdm2/p14^ARF pathway occurred in 73% of cases and in 80% of cases if CDKN2A homozygous deletions were associated. All glioblastomas with gemistocytic areas showed p14^ARF inactivation. Immunohistochemistry showed higher percentages of positivity in comparison with molecular genetics, but with similar variations.     

Frequent epigenetic inactivation of RASSF1A and BLU genes located within the critical 3p21.3 region in gliomas

RASSF1A is a major tumor suppressor gene located at 3p21.3. We investigated the role of aberrant promoter region hypermethylation of RASSF1A in a large series of adult gliomas. RASSF1A was frequently methylated in both primary tumors (36/63; 57%) and tumor cell lines (7/7; 100%). Hypermethylation of RASSF1A in glioma cell lines correlated with loss of expression and treatment with a demethylating agent-reactivated RASSF1A gene expression. Furthermore, re-expression of RASSF1A suppressed the growth of glioma cell line H4 in vitro. Next, we investigated whether other members of the RASSF gene family were also inactivated by methylation. NORE1B and RASSF3 were not methylated in gliomas, while NORE1A and RASSF5/AD037 demonstrated methylation in glioma cell lines but not in primary tumors. We then investigated the methylation status of three other candidate 3p21.3 tumor suppressor genes. CACNA2D2 and SEMA3B were not frequently methylated, but the BLU gene located just centromeric to RASSF1 was frequently methylated in glioma cell lines (7/7) and in 80% (35/44) of glioma tumors. In these tumor cell lines, BLU expression was restored after treatment with a demethylating agent. Loss of BLU gene expression in glioma tumors correlated with BLU methylation. There was no association between RASSF1A and BLU methylation. RASSF1A methylation increased with tumor grade, while BLU methylation was seen at similar frequencies in all grades. Our data implicate RASSF1A and BLU promoter methylation in the pathogenesis of adult gliomas, while other RASSF family members and CACNA2D2 and SEMA3B appear to have only minor roles. In addition, RASSF1A and BLU methylation appear to be independent and specific events and not due to region-wide changes in DNA methylation.     

p14ARF在宫颈癌中的表达及其与p53 表达相关性的研究

 目的　探讨宫颈癌组织p14^ARF蛋白的表达及其与p53 表达的相关性。方法　应用免疫组化方法检测p14^ARF 、p53 基因在41 例宫颈癌及20 例正常宫颈组织中的表达。结果　p14^ARF在正常宫颈组织中不表达,41 例宫颈癌中35 例表达阳性,占85. 4 %。病理分级为G1 、G2 级的宫颈癌的p14^ARF阳性表达率为68. 4 % ,G3 级为100 % ,两者比较,有显著性差异( P < 0. 05) 。宫颈癌组织中p53 蛋白表达阳、阴性者中p14ARF蛋白表达阳性率分别为75. 0 %(12/ 16) 和92. 0 %(23/ 25) ,两者比较,无显著性差异,p14^ARFF与p53 表达不相关。结论　p14^ARF在宫颈癌中高表达有一定的诊断和估测预后价值,可能是宫颈癌新的肿瘤标志。     

RASSF1A基因在胶质瘤组织中甲基化研究及临床意义

目的：探讨胶质瘤组织及脑正常组织中抑癌基因RASSF1A启动子区甲基化程度及与临床特征的关系。方法：采用甲基化特异性聚合酶链反应（MS-PCR）方法检测46例脑胶质瘤（其中星形细胞瘤19例，室管膜瘤16例，胶质母细胞瘤11例）及6例脑正常组织中RASSF1A基因启动子区甲基化状态。并对RASSF1A基因启动子区甲基化发生情况与临床各因素之间的关系进行分析。结果：（1）46例脑胶质瘤组织DNA标本中RASSF1A基因启动子区甲基化发生率为65．2％（30／46）；6例脑正常组织中RASSF1A基因未发生甲基化；RASSF1A在胶质瘤和脑正常组织之间发生甲基化率比较差异有显著性（P=0.017）。在46例脑胶质瘤中RASSF1A有30例发生甲基化，其中低级别组14例（14／26），高级别组16例（16／20），两组之间甲基化率比较无明显差异（P〉0．05）。其中星形细胞瘤、室管膜瘤、胶质母细胞瘤中甲基化发生率分别为63．2％（12／19）、68、8％（11／16）、63．6％（7／11），各组之间甲基化发生率比较均无统计学意义（P=0．211）。（2）RASSFIA基因启动子区甲基化程度与脑胶质瘤病理分型、肿瘤大小之间无显著相关性。结论：RASSF1A在胶质瘤中有甲基化发生，在脑正常组织中未发生甲基化，检测胶质瘤中RASSF1A基因启动子区甲基化情况对临床判断肿瘤发生发展有指导意义。     

Frequent but borderline methylation of<Emphasis Type="Italic"> p16</Emphasis><Superscript>INK4a</Superscript> and<Emphasis Type="Italic"> TIMP3</Emphasis> in medulloblastoma and sPNET revealed by quantitative analyses

Certain risk groups among tumors of the central nervous system (CNS) in children take an almost inevitably fatal course. The elucidation of molecular mechanisms offers hope for improved therapy. Aberrant methylation is common in malignant brain tumors of childhood and may have implications for stratification and therapy. Methylation of p16 ^INK4A, p14 ^ARF, TIMP3, CDH1, p15 ^INK4B and DAPK1 in medulloblastoma (MB) and ependymoma has been discussed controversially in the literature. DUTT1 and SOCS1 have not previously been analyzed. We examined methylation in MB, sPNET and ependymoma using methylation-specific PCR (MSP), quantitative Combined Bisulfite Restriction Analysis (COBRA) and direct and clone sequencing of bisulfite PCR products. We detected methylation of p16 ^INK4A (17/43), p14 ^ARF (11/42) and TIMP3 (9/44) in MB and others by MSP. CDH1 was not only methylated in MB (31/41), but also in normal controls. Evaluation of MSP results by quantitative COBRA and sequencing yielded methylation between the detection limits of COBRA (1%) and MSP (0.1%). Only p16 ^INK4A and TIMP3 were methylated consistently in medulloblastomas (p16 ^INK4A 14%, TIMP3 11%) and p16 ^INK4A also in anaplastic ependymomas (1/4 tumors). Methylation ranged from 1–5%. Evaluation of methylation using MSP has thus to be supplemented by quantitative methods. Our analyses raise the issue of the functional significance of low level methylation, which may disturb the delicate growth factor equilibrium within the cell. Therapeutic and diagnostic implications urge into depth analyses of methylation as a mechanism, which might fill some of the gaps of our understanding of brain tumor origin.     

人脑胶质组织中SV40大T抗原表达及p53形成特异性复合物

目的 探讨SV40早期区域基因编码产物大T抗原（Tag）表达及与抑癌蛋白p53的相互作用在人脑胶质瘤发生发展中的意义。方法 采用免疫组织化学方法检测89例人脑胶质瘤组织和11例正常人脑组织中Tag的表达,并对Tag表达阳性肿瘤组织进行免疫共沉淀和Western blot表达阳性33例（阳性率37．1％）,Tag表达水平与胶质瘤病理分级呈显著正相关（pearson列联系数＝0．72,P＜0．01）;33例Tag表达阳性瘤组织中均发现Tag表达全部阴性。结果 SV40感染与人脑胶质瘤病因学密切相关,Tag可能是SV40在胶质瘤发生发展中起作用的重要因素;Tag可能是SV40在胶质瘤发生发展中起作用的重要因素：Tag与p53可形成特异性复合物,Tag-p53特异性复合物的形成导致p53失活,可能是人脑胶质癌发生发展的一个重要机理。     

p14ARF expression in invasive breast cancers and ductal carcinoma in situ – relationships to p53 and Hdm2

Introduction  

p14^ARF stabilises nuclear p53, with a variable expression of p14^ARF mRNA in breast cancers. In vitro, nuclear p14^ARF binds Hdm2 to block Hdm2-dependent nucleocytoplasmic shuttling of p53, which is required before cytoplasmic degradation of p53. p14^ARF is negatively regulated by p53 and through p53-independent pathways. No studies have yet examined levels of p14^ARF protein expression in breast cancer and their relationship to Hdm2/p53 immunoreactivity or subcellular localisation. Previously, immunohistochemical expression of cytoplasmic p14^ARF, p53 and Hdm2 has been described. HER-2 (c-erbB2/neu) predicts prognosis and interacts with the p14^ARF/Hdm2 pathway to inactivate p14^ARF and to influence Hdm2 activity and localisation. This study examined p14^ARF and p53/Hdm2 expression and subcellular localisation by using immunohistochemistry in a series of invasive ductal breast cancers (IDCs) with concomitant ductal carcinoma in situ (DCIS), to evaluate whether findings in vitro were related to clinicopathological parameters such as HER-2 and their effect on patient outcome.     

Methylation,expression, and mutation analysis of the cell cycle control genes in human brain tumors

Methylation status of the p15(INK4B), p16(INK4A), p14(ARF) and retinoblastoma (RB) genes was studied using methylation specific polymerase chain reaction (MSP) in 85 human brain tumors of various subtypes and four normal brain samples. These genes play an important role in the control of the cell cycle. Twenty-four out of 85 cases (28%) had at least one of these genes methylated. The frequency of p14(ARF) methylation was 15 out of 85 (18%) cases, and the expression of p14(ARF) in methylated gliomas was significantly lower than in unmethylated gliomas. The incidence of methylation of p15(INK4B), p16(INK4A) and RB gene was 4%, 7%, and 4%, respectively. Samples with p14(ARF) methylation did not have p16(INK4A) methylation even though both genes physically overlap. None of the target genes was methylated in the normal brain samples. In addition, the p53 gene was mutated in 19 out of 85 (22%) samples as determined by single strand conformation polymorphism (SSCP) analysis and DNA sequencing. Thirty out of 85 (35%) brain tumors had either a p53 mutation or methylation of p14(ARF). Also, the p14(ARF) expression in p53 wild-type gliomas was lower than levels in p53 mutated gliomas. This finding is consistent with wild-type p53 being able to autoregulate its levels by down-regulating expression of p14(ARF). In summary, inactivation of the apoptosis pathway that included the p14(ARF) and p53 genes by hypermethylation and mutation, respectively, occurred frequently in human brain tumors. Down-regulation of p14(ARF) in gliomas was associated with hypermethylation of its promoter and the presence of a wild-type p53 in these samples.     

p14ARF induces apoptosis via an entirely caspase‐3‐dependent mitochondrial amplification loop

The p14^ARF tumor suppressor triggers cell death or cell cycle arrest upon oncogenic stress. In MCF‐7 breast carcinoma cells, expression of the tumor suppressor gene p14^ARF fails to trigger apoptosis but induces an arrest in the G1 and, to a lesser extent, in the G2 phase in the cell division cycle. Here, inhibition of cell cycle arrest resulted in apoptosis induction in caspase‐3 proficient MCF‐7 cells upon expression of p14^ARF. This occurred in the absence of S‐phase progression or mitotic entry. In contrast, syngeneic, caspase‐3‐deficient MCF‐7 cells remained entirely resistant to p14^ARF‐induced apoptosis. Thus, cell cycle checkpoint abrogation overcomes resistance to p14^ARF‐induced cell death and promotes cell death via a caspase‐3‐dependent pathway. Cell death coincided with dissipation of the mitochondrial membrane potential, release of cytochrome c, and was inhibitable by pan‐caspase inhibitors and the caspase‐3/7 inhibitor zDEVD‐fmk. Of note, mitochondrial events of apoptosis execution depended entirely on caspase‐3 proficiency indicating that caspase‐3 either acts “up‐stream” of the mitochondria in a “non‐canonical” pathway or mediates a mitochondrial feedback loop to amplify the apoptotic caspase signal in p14^ARF‐induced stress signaling.     

Correlation of <Emphasis Type="SmallCaps">l</Emphasis>-methyl-<Superscript>11</Superscript>C-methionine (MET) uptake with <Emphasis Type="SmallCaps">l</Emphasis>-type amino acid transporter 1 in human gliomas

l-Type amino acid transporter 1 (LAT1) is a neutral amino acid transport system and is a major route for the transport of large neutral amino acids, including methionine, through the plasma membrane. LAT1 requires the heavy chain of 4F2 cell surface antigen (4F2hc) for its functional expression. Positron emission tomography (PET) with l-[methyl-¹¹C] methionine (MET) provides information about amino acid metabolism in brain tumors. We conducted a clinicopathologic study to elucidate the correlation of LAT1 and 4F2hc expression with MET uptake in patients with newly diagnosed human gliomas. Thirty-three newly diagnosed glioma patients were enrolled in this study. Uptake of MET in the tumor was evaluated with the maximum standardized uptake value (SUVmax). Expression of the LAT1, 4F2hc, and CD34, and Ki-67 labeling index of the tumor were analyzed by immunohistochemical staining, and the correlation with the SUVmax in the tumors was examined. Expression of LAT1 and 4F2hc was higher in high-grade gliomas than in low-grade gliomas. The grade of LAT1 immunostaining increased with glioma grade. LAT1 was mainly expressed in the tumor cytoplasm and vascular endothelium and 4F2hc was mainly expressed in the tumor cytoplasm and plasma membrane. Expression of LAT1 but not 4F2hc was significantly correlated with MET SUVmax. Expression of LAT1 in the tumor vascular endothelium is significantly correlated with CD34 positive microvessel density. In conclusion, MET SUVmax correlates with LAT1 expression in the tumor in newly diagnosed gliomas. MET transport may be increased by an increased number of microvessels combined with a higher density or activity of LAT1 in the tumor endothelial cells in high-grade gliomas. Use of MET-PET as a molecular target combined with anti-angiogenesis in glioma therapy should be addressed in future studies.     

胶质瘤患者血浆与肿瘤组织P16基因甲基化的检测及意义

Objective:To explore the clinical significance of methylation status of promoter CpG island of p16 gene in glioma tissue and plasma.Methods:Methylation specific polymerase chain reaction (MSP) was used to determine the methylation status of the promoter for p16 gene within glioma tissue and plasma.Immunohistochemicel method (SP) was used to analyze the expressions of p16 and Ki-67 proteins.Results:Hypermethylation was found in 17/40 (42.5%) of brain gliomas,in comparison with 11/40 (27.5%) plasma specimens (x2 = 1.9780,P = 0.1596).Loss of p16 expression was associated (P = 0.0229) with hypermethylation of CpG island of promoter regions.Hypermethylation of p16 gene CpG island was significantly related to the increase of malignant grade of brain glioma (Tissue:X2 = 11.4288,P = 0.0007;Plasma:X2 = 8.9439,P = 0.0028).The Ki-67 index increased significantly (P＜0.05) in brain gliomas methylated in contrast to those unmethylated.Conclusion:P16 hypermethylation may be one of the major mechanisms of tumorigenesis of gliomas.Methylated tumor-specific DNA may be as a plasma biomarker for prognosis in patients with glioma.     

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。     

Promoter hypermethylation of multiple genes in astrocytic gliomas

Promoter hypermethylation represents a primary mechanism in the inactivation of tumor suppressor genes during tumorigenesis. To determine the frequency and timing of hypermethylation during carcinogenesis of astrocytic tumors, we analysed promoter methylation status of ten tumor-associated genes (MGMT, GSTP1, DAPK, p14ARF, THBS1, TIMP-3, p73, p16INK4A, RB1 and TP53) in a series of 88 astrocytic gliomas, including 24 diffuse astrocytomas; 21 anaplastic astrocytomas, and 43 glioblastomas (33 primary and 10 secondary), as well as two non-neoplastic brain samples, by methylation-specific PCR. Aberrant CpG island methylation was detected in all ten genes analysed, and all but one sample displayed anomalies in at least one gene. The methylation index (number methylated genes/total genes analysed) was 0.3, 0.38, 0.33 and 0.29 for diffuse astrocytomas, anaplastic astrocytomas and secondary and primary glioblastomas, respectively. Some differences may be established regarding the methylation profiles of specific genes and tumor types: MGMT, THBS1, TIMP-3, and p16INK4A appear hypermethylated in low-grade tumors (at least in 45% of cases), whereas GSTP1, DAPK, and p14ARF are mostly changed in 15-50% of the higher grade forms versus <10% in low-grade tumors. Some variation also exists regarding the methylation values for p73 and RB1 (10-40% of cases) among all groups. TP53 presented hypermethylation rates <10% in all tumor subtypes. Our findings thus suggest that methylation represents a common mechanism that contributes to inactivating cancer-related genes in astrocytic neoplasms. This epigenetic change is, in general, an early event in the development of astrocytic neoplasms but this gene silencing mechanism may also appear as a late event involving some loci.