PDTB-14. INVESTIGATING HISTONE H3 POST-TRANSLATIONAL MODIFICATIONS USING PARAFFIN-EMBEDDED PEDIATRIC GLIOMA TISSUE SAMPLES

Mutant Histone H3 protein is increasingly thought to play a role in pediatric gliomagenesis. Chromatin immunoprecipitation sequencing (ChIP-Seq) is a powerful method for studying the epigenetic effects of histone protein modifications. However, analysis of rare or surgically-inaccessible tumors, such as pediatric diffuse intrinsic pontine glioma (DIPG), is often precluded by the relative paucity of fresh tissue available, while tumor primary cell lines may not accurately reflect the tumor epigenome. ChIP-Seq of formalin-fixed, paraffin-embedded (FFPE) tumor samples, derived via tumor biopsy or autopsy, could therefore provide valuable insight to tumor biology in lieu of fresh tissue. Here we present, to our best knowledge, the first successful ChIP-Seq of FFPE pediatric brain tumor tissue, as an alternative to fresh tissue analysis. Archival FFPE pediatric glioma tissue (n=8) was used to evaluate Histone H3K27M mutation status via Sanger Sequencing and genomic deposition of H3K27M, H3K4me3 and H3K27ac via ChIP-Seq. Four 20-micron FFPE sections were deparaffinized then rehydrated via ethanol incubation at decreasing concentrations. Extracted chromatins were sonicated to produce DNA fragments for subsequent ChIP-Seq analyses. ChIP-Seq libraries were prepared using the KAPA HTP Library Preparation Kit and sequenced on the Illumina NextSeq 500 Sequencing System. Successful extraction of chromatins was achieved from H3K27 wild-type FFPE Juvenille Pilocytic Astrocytoma tissue (n=1). ChIP-Seq revealed genomic enrichment of H3K4me3 and H3K27ac, consistent with known patterns of gene expression regulation in pediatric glioma. Analysis of H3K27M DIPG specimens is currently underway (n=7) for comparison to ChIP-Seq results from DIPG primary cell lines (n=2). We present the technical feasibility of FFPE pediatric glioma ChIP-Seq. Epigenetic analysis of FFPE tumor tissue, which may be more accessible than fresh tumor samples, could provide new insight into tumor biology and identify novel therapeutic targets for improved patient outcomes in rare but highly aggressive tumors, such as DIPG.