Analysis of gliomas DNA methylation: Assessment of pre-analytical variables

Abstract

ABSTRACTPrecision oncology is driven by molecular biomarkers. For glioblastoma multiforme (GBM), the most common malignant adult primary brain tumor, O6-methylguanine-DNA methyltransferase (MGMT) gene DNA promoter methylation is an important prognostic and treatment clinical biomarker. Time consuming pre-analytical steps such as biospecimen storage before fixing, sampling, and processing are major sources of errors and batch effects, that are further confounded by intra-tumor heterogeneity ofMGMTpromoter methylation. To assess the effect of pre-analytical variables on GBM DNA methylation, tissue storage/sampling (CryoGrid), sample preparation multi-sonicator (PIXUL) and 5-methylcytosine (5mC) DNA immunoprecipitation (Matrix MeDIP-qPCR/seq) platforms were used.MGMTpromoter CpG methylation was examined in 173 surgical samples from 90 individuals, 50 of these were used for intra-tumor heterogeneity studies.MGMTpromoter methylation levels in paired frozen and formalin fixed paraffin embedded (FFPE) samples were very close, confirming suitability of FFPE forMGMTpromoter methylation analysis in clinical settings. Matrix MeDIP-qPCR yielded similar results to methylation specific PCR (MS-PCR). Warm ex-vivo ischemia (37°C up to 4hrs) and 3 cycles of repeated sample thawing and freezing did not alter 5mC levels atMGMTpromoter, exon and upstream enhancer regions, demonstrating the resistance of DNA methylation to the most common variations in sample processing conditions that might be encountered in research and clinical settings. 20-30% of specimens exhibited intratumor heterogeneity in theMGMTDNA promoter methylation. Collectively these data demonstrate that variations in sample fixation, ischemia duration and temperature, and DNA methylation assay technique do not have significant impact on assessment ofMGMTpromoter methylation status. However, intratumor methylation heterogeneity underscores the need for histologic verification and value of multiple biopsies at different GBM geographic tumor sites in assessment ofMGMTpromoter methylation. Matrix-MeDIP-seq analysis revealed thatMGMTpromoter methylation status clustered with other differentially methylated genomic loci (e.g. HOXA and lncRNAs), that are likewise resilient to variation in above post-resection pre-analytical conditions. TheseMGMT-associated global DNA methylation patterns offer new opportunities to validate more granular data-based epigenetic GBM clinical biomarkers where the CryoGrid-PIXUL-Matrix toolbox could prove to be useful.