Abstract A38: Whole methylome explorations of paired lung tumor and non-tumor clinical samples
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AbstractBackground: Hypermethylation in specific candidate gene promoters has been found during progressive lung carcinogenesis. To explore common methylation events on a genome-wide scale in lung cancer, we analyzed the methylation profiles of paired NSCLC tumor and far adjacent non-tumor samples using the HELP-microarray assay, which yields information on 1.2 million fragments throughout the genome.Methods: The HELP (HpaII tiny fragment Enriched by Ligation mediated PCR) assay is based on the generation of restriction enzyme libraries generated by methylation sensitive (HpaII) and methylation insensitive (MspI) isoschizomers, in its second generation as a microarray platform. The assay lends itself to low starting amounts of DNA (3 ug) and robust assessment of methylation status by comparing ratios of HpaII- generated-fragments to MspI- generated fragments co-hybridized to a Nimbelgen custom high-density microarrays. The CCGG sites were weighted if neighboring CCGGs were methylated in same direction. Here, 24 pairs of tumor and adjacent non-tumor samples were analyzed using the HELP assay.Summary of results: At p = 5E-6, we identified 26,138 differentially methylated fragments (corresponding to 2 CpG sites each) in tumor versus non-tumor. The overall trend was consistent with genome-wide hypomethylation and locus specific hypermethylation (localized to CG-island containing promoters). We could identify both known and novel regions of the genome as well as specific gene-promoters that are hypermethylated in tumor versus non-tumor.Region # loci # signif loci T Hypomethyl T HypermethylPromoter 151,568 576 69% 31%Gene Body 551,628 9,817 62% 38%Intergenic 540,473 15,745 97% 3%Conclusion: An interrogation of methylation status of 1.2 million loci throughout the genome in paired lung tumor/non-tumor specimens reveals many more differential methylation events in gene bodies and intergenic regions than in promoters. That said, many previously unreported differentially-methylated gene promoters were identified. We are able to discover individual methylation events common across different clinical specimens. Based on a set of priors, we have narrowed down promoter-specific hypermethylation events for further validation using tagged Bisulfite Genomic Sequencing. We are also working on the integration of methylome date with other genome-wide epigenetic and expression data from the same clinical samples.[Funding source: NCI 1RC1 CA145422-01; 1K24-CA139054-01]
