title: Extent and patterns of MGMT promoter methylation in glioblastoma- and respective glioblastoma-derived spheres.
abstract: OBJECTIVE
  Quantitative methylation-specific tests suggest that not all cells in a glioblastoma with detectable promoter methylation of the O6-methylguanine DNA methyltransferase (MGMT) gene carry a methylated MGMT allele. This observation may indicate cell subpopulations with distinct MGMT status, raising the question of the clinically relevant cutoff of MGMT methylation therapy. Epigenetic silencing of the MGMT gene by promoter methylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma.
  
  
  METHODS
  Ten paired samples of glioblastoma and respective glioblastoma-derived spheres (GS), cultured under stem cell conditions, were analyzed for the degree and pattern of MGMT promoter methylation by methylation-specific clone sequencing, MGMT gene dosage, chromatin status, and respective effects on MGMT expression and MGMT activity.
  
  
  RESULTS
  In glioblastoma, MGMT-methylated alleles ranged from 10% to 90%. In contrast, methylated alleles were highly enriched (100% of clones) in respective GS, even when 2 MGMT alleles were present, with 1 exception (<50%). The CpG methylation patterns were characteristic for each glioblastoma exhibiting 25% to 90% methylated CpGs of 28 sites interrogated. Furthermore, MGMT promoter methylation was associated with a nonpermissive chromatin status in accordance with very low MGMT transcript levels and undetectable MGMT activity.
  
  
  CONCLUSIONS
  In MGMT-methylated glioblastoma, MGMT promoter methylation is highly enriched in GS that supposedly comprise glioma-initiating cells. Thus, even a low percentage of MGMT methylation measured in a glioblastoma sample may be relevant and predict benefit from an alkylating agent therapy.
authors.affiliation: Laboratory of Brain Tumor Biology, Department of Clinical Neurosciences, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland.
authors.forename: Davide
authors.initials: D
authors.lastname: Sciuscio
authors.forename: Annie-Claire
authors.initials: AC
authors.lastname: Diserens
authors.forename: Kristof
authors.initials: K
authors.lastname: van Dommelen
authors.forename: Danielle
authors.initials: D
authors.lastname: Martinet
authors.forename: Greg
authors.initials: G
authors.lastname: Jones
authors.forename: Robert-Charles
authors.initials: RC
authors.lastname: Janzer
authors.forename: Claudio
authors.initials: C
authors.lastname: Pollo
authors.forename: Marie-France
authors.initials: MF
authors.lastname: Hamou
authors.forename: Bernd
authors.initials: B
authors.lastname: Kaina
authors.forename: Roger
authors.initials: R
authors.lastname: Stupp
authors.forename: Marc
authors.initials: M
authors.lastname: Levivier
authors.forename: Monika E
authors.initials: ME
authors.lastname: Hegi
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001932:Brain Neoplasms / Q000235:genetics*; D002843:Chromatin / Q000648:ultrastructure; D019175:DNA Methylation; D005260:Female; D005787:Gene Frequency; D005909:Glioblastoma / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D002843:Chromatin; D019853:O(6)-Methylguanine-DNA Methyltransferase