497 Multiomic Analysis of Spinal Ependymomas Following Chemotherapy and MYCN Amplification Reveals Potential Mechanisms of Recurrence

INTRODUCTION: Spinal ependymomas often recur following surgical resection. Grade 3 tumors, particularly those harboring MYCN amplification, have dismal prognoses despite best treatment regimes. METHODS: MYCN amplification was identified with the Oncomine panel and bulk methylation analysis (MYCN). We conducted paired single-nucleus RNA (snRNA) and chromatin accessibility (snATAC) sequencing (10x Genomics) of grade 2 and grade 3 spinal ependymomas (7 patients, 1 with repeat surgery). Additional snRNAseq samples (8 grade 2 and 2 grade 3) were included from NCBI GEO Dataset GSE163686. Canonical cell classes were identified with a combination of cluster- and cell-based strategies. Downstream analysis was conducted using R 4.3 (Seurat, Signac, clusterProfiler, CellChat), and Python 3.11 (scanpy) packages. Significance was assigned at p < 0.05. RESULTS: We identified ependymoma tumor cells distinct from canonical immune cell classes with gene expression analysis and confirmed a distinct copy-number-variation pattern in these cells. In the tumor cells from MYCN samples, we found overexpression of mTORC pathway genes, and increased chromatin accessibility of chromatin modulators and transcription factors. Receptor-ligand analyses revealed unique immunosuppressive VISTA signaling between MYCN tumor cells and tumor-associated macrophages. In a pairwise pre- and post-ICI analysis, we observed an upregulation of the ephrin signaling pathway and post-transcriptional regulation in snRNA and snATAC, respectively. CONCLUSIONS: Recurrent MYCN-amplified spinal ependymomas appear to have upregulation of pathways contributing to cell proliferation and an immune cold microenvironment. Treatment with ICI likely selects cells for that promote cell adhesions and junction stability, contributing to a more aggressive phenotype on recurrence. Further validation is required to identify therapeutic targets for these ependymomas.