TBIO-05. GENOME-SCALE NUCLEOTIDE-SPECIFIC CHARACTERIZATION OF 5-HYDROXYMETHYLCYTOSINE IN PEDIATRIC CENTRAL NERVOUS SYSTEM TUMORS

Though aberrant cytosine modifications are prevalent in cancer, nucleotide-specific 5-hydroxymethylcytosine (5hmC) modifications remain understudied, including in pediatric CNS tumors. Brain 5-hydroxymethylation is linked with development and differentiation. We measured genome-scale nucleotide-specific 5hmC in patients with diagnoses of glioma, ependymoma, and embryonal tumors under age 18 (n=36), and in non-tumor pediatric brain tissues (n=3). DNA was processed with tandem oxidative (OxBS) and bisulfite (BS) treatments followed by hybridization to the Illumina Methylation EPIC Array that interrogates over 860,000 CpG sites. We used the OxyBS R package to determine levels of 5hmC and 5mC. Mean 5hmC levels were lower in tumors (gliomas 4.1%, ependymomas 3.9%, and embryonal tumors 3.4%) compared to nontumor tissues (5.3%). We subset to the CpGs with the 5% highest 5hmC content for downstream analyses (37,173 CpGs). These sites were enriched among regulatory elements, including TFBS (Odds Ratio 1.14 p-value 3.57E-20) and super-enhancers (OR 1.93, p-value 1.14E-126). Linear mixed-effects models adjusted for age, sex, and cell type proportions tested the CpG-specific differences in 5hmC between tumor and nontumor samples, as well as between tumor subtypes. 5hmC levels were depleted across tumors compared with nontumor brain tissues, including at CpG islands. Model-based clustering (RPMM) results indicated that patients with low 5hmC patterns have poorer overall survival and increased risk of recurrence. Our results indicate that 5hmC localizes to sites in the DNA critical to gene regulation and is associated with patient outcomes. This study offers an opportunity to potentially contribute to classification markers for childhood brain tumors.