Abstract

<div>Abstract<p><b>Purpose:</b> Quantitative methylation-specific tests suggest that not all cells in a glioblastoma with detectable promoter methylation of the O6-methylguanine DNA methyltransferase (<i>MGMT</i>) gene carry a methylated <i>MGMT</i> allele. This observation may indicate cell subpopulations with distinct <i>MGMT</i> status, raising the question of the clinically relevant cutoff of <i>MGMT</i> methylation therapy. Epigenetic silencing of the <i>MGMT</i> 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.</p><p><b>Experimental Design:</b> Ten paired samples of glioblastoma and respective glioblastoma-derived spheres (GS), cultured under stem cell conditions, were analyzed for the degree and pattern of <i>MGMT</i> promoter methylation by methylation-specific clone sequencing, <i>MGMT</i> gene dosage, chromatin status, and respective effects on <i>MGMT</i> expression and MGMT activity.</p><p><b>Results:</b> In glioblastoma, <i>MGMT</i>-methylated alleles ranged from 10% to 90%. In contrast, methylated alleles were highly enriched (100% of clones) in respective GS, even when 2 <i>MGMT</i> 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, <i>MGMT</i> promoter methylation was associated with a nonpermissive chromatin status in accordance with very low <i>MGMT</i> transcript levels and undetectable MGMT activity.</p><p><b>Conclusions:</b> In <i>MGMT</i>-methylated glioblastoma, <i>MGMT</i> promoter methylation is highly enriched in GS that supposedly comprise glioma-initiating cells. Thus, even a low percentage of <i>MGMT</i> methylation measured in a glioblastoma sample may be relevant and predict benefit from an alkylating agent therapy. <i>Clin Cancer Res; 17(2); 255–66. ©2010 AACR</i>.</p></div>

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