A 3D tumorwide multi-omic atlas of intratumoral heterogeneity in IDH -mutant glioma.

2067 Background: Isocitrate dehydrogenase mutant (IDHmut) glioma affects over 20,000 adults per year. Treatment failure often results from intratumoral heterogeneity, in which genomic subclones undergo selection for treatment-resistant populations. Understanding this heterogeneity is critical to selecting therapeutic combinations that are efficacious across the entire tumor, but few genomic studies go beyond analyzing a single tumor sample per patient. Methods: 3D whole tumor sampling was used to obtain 324 spatially mapped samples from 32 IDHmut gliomas. Whole exome (n=323), RNA (318), ATAC sequencing (92), and Hi-C (14) were performed. PyClone reconstructed clonal evolution, weighted gene/peak correlation network analyses derived co-expression (RNA) and gene regulatory (ATAC) programs, and comparison of tumor Hi-C results to normal brain identified tumor-specific chromatin loops. Results: Regional tumor content (purity) inversely correlated with distance from tumor centroid ( R = -0.32, p = 2.8×10 -8 ) and inter-sample distance was associated with subclonal ( R = 0.24, p = 8.1x10 -15 ) and transcriptional ( R = 0.12, p = 1.0x10 -5 ) similarity. Beyond IDH1 in the combined 17 astrocytomas and 15 oligodendrogliomas, tumor-wide alterations were recurrently observed in TP53 (altered in 64%; tumor-wide in 58%), ATRX (55%; 39%), TERT promoter (45%; 45%) and FUBP1 (18%; 6%). Despite high cohort prevalence, glioma-associated drivers CIC (altered in 42%), PIK3CA (36%), and ARID1A (18%) were tumor-wide in only one patient each, while MUC4 (33%), NOTCH1 (27%), SETD2 (24%), and PIK3R1 (15%) were never observed in the tumor founding clone. Tumors with CIC alterations harbored a median of 5 unique mutations with a median combined tumor-wide cancer cell fraction of 45%. Hi-C identified 5,579 tumor-specific promoter-enhancer interactions across 4,144 loops. The genes associated with these promoters enriched for tumor-specific RNA programs associated with OPC signature ( p adj = 3x10 -14 ), NPC signature ( p adj = 3.87x10 -14 ), and neuronal mimicry (1.2×10 -13 ). We discovered adversely prognostic gene expression programs in astrocytoma independently validated in chromatin regulatory programs. Conclusions: Tumor-wide sampling of IDHmut glioma revealed evidence of radial tumor growth and 3D patterns of cellular organization, in sharp contrast to intermixed patterns in IDH wildtype glioma. Most inter-patient heterogeneity in driver alterations was subclonal. Our data also revealed regulatory loops that explain tumor cell-specific expression signatures. A significant, adverse prognostic marker in astrocytoma was discovered and cross-validated.