Fig. 5: Subclonal genomic heterogeneity from extrachromosomal DNA double-minutes.

Spatial transcriptomic analysis of a Glioblastoma A1 (left panels) and b Glioblastoma A5 (right panels) reveals spatially distinct subclonal genomic heterogeneity, demonstrating loss of heterozygosity in regions that also show overexpression of ecDNA-amplified genes, including EGFR. As discussed in the text, Glioblastoma A1 showed LOH of chromosome 17 within high EGFR-expressing cluster 9. For each vertical panel: i Schematic of EGFR+ amplified ecDNA fragments based on exome copy number and breakpoint analysis. Glioblastoma A2 shows two different EGFR+ ecDNA variants. ii–iii Log2 fold change and B-allele frequency plots from exome sequencing CNV and LOH analysis. iv–v Exome analysis coverage of ecDNA, indicating breakpoints and mapped regions. vi SCT-normalized spatial expression of genes within ecDNA regions. Violin plot shows the expression of normalized coverage of genes encompassing DM ecDNA (excluding EGFR) for high EGFR and low EGFR spots (low EGFR < = 5 counts). vii. Spatially mapped average expression for gene expression marker of hypoxia, proliferative cells, macrophages, and cancer stem cells. viii. tLOH analysis showing spatially distinct clusters with LOH. ix–x Spot-level B-allele frequency analysis indicates clonal LOH of chromosome 17 in recurrent Glioblastoma A1 and LOH on chromosome 19 in areas with high EGFR expression (high EGFR > 8 counts). Bars and points are color-coded by state: blue for heterozygous, gold for LOH, and gray for undefined.