Identification of regions correlating MGMT promoter methylation and gene expression in glioblastomas.

The O(6)-methylguanine-DNA methyltransferase gene (MGMT) is methylated in several cancers, including gliomas. However, the functional role of cysteine-phosphate-guanine (CpG) island (CGI) methylation in MGMT silencing is still controversial. The aim of this study was to investigate whether MGMT CGI methylation correlates inversely with RNA expression of MGMT in glioblastomas and to determine the CpG region whose methylation best reflects the level of expression. The methylation level of CpG sites that are potentially related to expression was investigated in 54 glioblastomas by pyrosequencing, a highly quantitative method, and analyzed with respect to their MGMT mRNA expression status. Three groups of patients were identified according to the methylation pattern of all 52 analyzed CpG sites. Overall, an 85% rate of concordance was observed between methylation and expression (p < 0.0001). When analyzing each CpG separately, six CpG sites were highly correlated with expression (p < 0.0001), and two CpG regions could be used as surrogate markers for RNA expression in 81.5% of the patients. This study indicates that there is good statistical agreement between MGMT methylation and expression, and that some CpG regions better reflect MGMT expression than do others. However, if transcriptional repression is the key mechanism in explaining the higher chemosensitivity of MGMT-methylated tumors, a substantial rate of discordance should lead clinicians to be cautious when deciding on a therapeutic strategy based on MGMT methylation status alone.

[1]  H. Heinzl,et al.  Anti‐O6‐Methylguanine‐Methyltransferase (MGMT) Immunohistochemistry in Glioblastoma Multiforme: Observer Variability and Lack of Association with Patient Survival Impede Its Use as Clinical Biomarker * , 2008, Brain pathology.

[2]  Elizabeth Eisenhauer,et al.  Nomograms for predicting survival of patients with newly diagnosed glioblastoma: prognostic factor analysis of EORTC and NCIC trial 26981-22981/CE.3. , 2008, The Lancet. Oncology.

[3]  H. Wheeler,et al.  Variation of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation in serial samples in glioblastoma , 2008, Journal of Neuro-Oncology.

[4]  G. Reifenberger,et al.  Intratumoral homogeneity of MGMT promoter hypermethylation as demonstrated in serial stereotactic specimens from anaplastic astrocytomas and glioblastomas , 2007, International journal of cancer.

[5]  T. Siegal,et al.  Novel mechanism whereby nuclear factor kappaB mediates DNA damage repair through regulation of O(6)-methylguanine-DNA-methyltransferase. , 2007, Cancer research.

[6]  Ivo G Gut,et al.  DNA methylation analysis by pyrosequencing , 2007, Nature Protocols.

[7]  P. Wesseling,et al.  MS-MLPA: an attractive alternative laboratory assay for robust, reliable, and semiquantitative detection of MGMT promoter hypermethylation in gliomas , 2007, Laboratory Investigation.

[8]  K. Hoang-Xuan,et al.  MGMT prognostic impact on glioblastoma is dependent on therapeutic modalities , 2007, Journal of Neuro-Oncology.

[9]  K. Hoang-Xuan,et al.  MGMT methylation: A marker of response to temozolomide in low‐grade gliomas , 2006, Annals of neurology.

[10]  R. McLendon,et al.  Quantitative analysis of O6-alkylguanine-DNA alkyltransferase in malignant glioma , 2006, Molecular Cancer Therapeutics.

[11]  M. Sanson,et al.  Genetic alterations associated with acquired temozolomide resistance in SNB-19, a human glioma cell line , 2006, Molecular Cancer Therapeutics.

[12]  A. Verma.  MGMT Gene Silencing and Benefit From Temozolomide in Glioblastoma , 2006 .

[13]  I. Ferrer,et al.  Prognostic Significance of O6-Methylguanine-DNA Methyltransferase Determined by Promoter Hypermethylation and Immunohistochemical Expression in Anaplastic Gliomas , 2005, Clinical Cancer Research.

[14]  T. Mukai,et al.  The essential role of histone H3 Lys9 di-methylation and MeCP2 binding in MGMT silencing with poor DNA methylation of the promoter CpG island. , 2005, Journal of biochemistry.

[15]  T. Ushijima,et al.  Detection and interpretation of altered methylation patterns in cancer cells , 2005, Nature Reviews Cancer.

[16]  M. Wolter,et al.  Frequent promoter hypermethylation and low expression of the MGMT gene in oligodendroglial tumors , 2005, International journal of cancer.

[17]  I. Gut,et al.  De novo quantitative bisulfite sequencing using the pyrosequencing technology. , 2004, Analytical biochemistry.

[18]  B. Bataille,et al.  Correlation of Clinical Features and Methylation Status of MGMT Gene Promoter in Glioblastomas , 2004, Journal of Neuro-Oncology.

[19]  Luca Regli,et al.  Clinical Trial Substantiates the Predictive Value of O-6-Methylguanine-DNA Methyltransferase Promoter Methylation in Glioblastoma Patients Treated with Temozolomide , 2004, Clinical Cancer Research.

[20]  K. Hoang-Xuan,et al.  Primary brain tumours in adults , 2003, The Lancet.

[21]  M. Turker.  Gene silencing in mammalian cells and the spread of DNA methylation , 2002, Oncogene.

[22]  Susan J Clark,et al.  DNA methylation and gene silencing in cancer: which is the guilty party? , 2002, Oncogene.

[23]  Scar,et al.  Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. , 2000, The New England journal of medicine.

[24]  J. Herman,et al.  Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. , 1999, Cancer research.

[25]  C. Ramana,et al.  Activation of human O6-methylguanine-DNA methyltransferase gene by glucocorticoid hormone , 1999, Oncogene.

[26]  T. Brent,et al.  Methylation hot spots in the 5' flanking region denote silencing of the O6-methylguanine-DNA methyltransferase gene. , 1997, Cancer research.

[27]  J. Costello,et al.  Methylation of discrete regions of the O6-methylguanine DNA methyltransferase (MGMT) CpG island is associated with heterochromatinization of the MGMT transcription start site and silencing of the gene , 1997, Molecular and cellular biology.

[28]  Min Wu,et al.  Mutations of O6‐methylguanine‐DNA methyltransferase gene in esophageal cancer tissues from Northern China , 1997, International journal of cancer.

[29]  R. Pieper,et al.  Understanding and manipulating O6-methylguanine-DNA methyltransferase expression. , 1997, Pharmacology & therapeutics.

[30]  J. Costello,et al.  Methylation of CpG Island Transcription Factor Binding Sites Is Unnecessary for Aberrant Silencing of the Human MGMT Gene* , 1996, The Journal of Biological Chemistry.

[31]  J. Costello,et al.  Graded methylation in the promoter and body of the O6-methylguanine DNA methyltransferase (MGMT) gene correlates with MGMT expression in human glioma cells. , 1994, The Journal of biological chemistry.

[32]  P. Potter,et al.  Characterization of the promoter region of the human O6-methylguanine-DNA methyltransferase gene. , 1991, Nucleic acids research.

[33]  J. Costello,et al.  Methylation-related chromatin structure is associated with exclusion of transcription factors and suppressed expression of O 6 -methylguanine-DNA methyltransferase , 1994 .