Epco-21. Single-Nuclei Analysis Identifies Novel Neoplastic Idh Mutant Astrocytoma Proliferative and Stemness Phenotypes Driving Poor Patient Outcomes

BACKGROUND Gliomas carry a high disease burden, particularly the aggressive IDH-wildtype subtype. However, some IDH-mutant gliomas can transition from a less aggressive G-CIMP-High (GCH) state to a more malignant G-CIMP-Low (GCL) phenotype, mimicking IDH-wildtype tumors. Understanding the cellular and molecular mechanisms behind this progression is vital for developing effective therapies. HYPOTHESIS We propose that single-nuclei resolution analysis can reveal distinct neoplastic cell subtypes driving the GCH-to-GCL transition through activation of stemness- and proliferation-related gene programs. METHODS We analyzed 22 glioma specimens from 15 patients (including matched primary and recurrent tumors) using single-nuclei RNA- and ATAC-sequencing. After quality control and neoplastic cell identification via somatic CNVs, we applied non-negative matrix factorization (NMF) to identify cell subclusters based on gene expression patterns. We further characterized these clusters using gene set enrichment, protein-protein interaction networks, developmental trajectory analysis, and integration with external RNA-seq datasets. Chromatin accessibility was assessed via snATAC-seq to identify regulatory elements and transcription factor activity. RESULTS We identified six major neoplastic cell subtypes: Astrocytic-Support, Pro-neural Housekeepers, Proteo-synthetic Cells, Mitotic Proliferators, Neural Guidance & Growth Cells, and Mesenchymal-mutant Cells. Each showed distinct gene expression signatures, pathway enrichments, and survival associations. Two subtypes—Mitotic Proliferators and Mesenchymal-mutant Cells—were linked to high-grade tumors and poor patient outcomes. Chromatin accessibility patterns corroborated transcriptional findings, highlighting differential TF regulation in aggressive subtypes. CONCLUSION This study reveals two aggressive GCL-related neoplastic cell subtypes with distinct stem-like and proliferative features. These findings provide insight into glioma progression mechanisms and uncover potential molecular targets for therapeutic intervention in IDH-mutant gliomas.