SUMMARY

The non-Mendelian inheritance of extrachromosomal DNA (ecDNA) exacerbates tumor genomic heterogeneity and evolution. Due to short-read sequencing’s reconstruction deficiencies, ecDNA abundance in clinical tumor tissues requires further estimation, and the mechanisms driving tumor evolution remain underexplored. Here, we perform long-read whole-genome sequencing on primary and paired metastatic tumor tissues from 12 patients with high-grade serous ovarian cancer (HGSOC) and 6 normal tubal tissues, constructing a comprehensive ecDNA profile. In HGSOC, ecDNA exhibits significantly greater genomic instability and lower methylation than chromosomal DNA. Beyond oncogene and immunomodulatory gene amplification, ecDNA amplifies extensive enhancers to promote gene expression via mechanisms including enhancer hijacking and mobile enhancers. Notably, we observe activation of transposable elements on hypomethylated ecDNA, which is strongly correlated with epithelial-mesenchymal transition gene expression and poor clinical outcomes. These findings reveal ecDNA as a multifunctional driver of genomic variations and tumor progression in HGSOC, highlighting its therapeutic potential.