Abstract PR16: Whole-genome analysis of the epigenetic mark 5-hydroxymethylcytosine reveals differential profiles in low- , intermediate- , and high-risk neuroblastomas

Background: Neuroblastoma is characterized by a paucity of somatic mutations, suggesting epigenetic modifications likely play a key role in regulating gene expression driving tumor phenotype. 5-Methylcytosine is an epigenetic DNA modification that decreases transcription and silences several neuroblastoma tumor suppressors. New technology allows evaluation of genome-wide 5-hydroxymethylcytosine (5hmC), a marker of activated transcription. We hypothesize that linking whole-genome 5hmC profiling with tumor transcriptomes will identify epigenetically regulated oncogenic drivers of neuroblastoma phenotype, and may ultimately lead to the discovery of new therapeutic targets. Methods: 5hmC was quantified by nano-hmC-Seal-Seq using 100ng of DNA from locally banked diagnostic neuroblastoma tumors. 50bp sequencing reads underwent quality control with Trimmomatic and were aligned to hg19 with Bowtie2. Homer software quantified peaks by genomic feature. Raw fragment counts were called using featureCounts and normalized with DeSeq2, which also identified genes with differential 5hmC. Gene Ontology (GO) pathway analysis of differential 5hmC genes was performed with the goseq package in R. 5hmC patterns were compared to gene expression in the E-MTAB-1781 cohort from ArrayExpress. Results: 53 patients (27 low- , 12 intermediate- , and 14 high-risk) were evaluated. Low-risk tumors had increased 5hmC peaks per sample compared to high-risk tumors (140,062 vs. 79,727, p=0.014). Peaks were predominantly intronic in all risk groups. Global 5hmC profiles distinguished four groups of patients, low- , intermediate- , MYCN-amplified high-risk, and MYCN-nonamplified high-risk. After controlling for sex, batch, and MYCN-amplification status, 578 genes had differential 5hmC peaks between low- and high-risk tumors (padjusted