Spatial–Temporal Diversity of Extrachromosomal DNA Shapes Urothelial Carcinoma Evolution and the Tumor Immune Microenvironment

Abstract

Extrachromosomal DNA (ecDNA) presents a promising target for cancer therapy; however, its spatial–temporal diversity and influence on tumor evolution and the immune microenvironment remain largely unclear. We apply computational methods to analyze ecDNA from whole-genome sequencing data of 595 patients with urothelial carcinoma. We demonstrate that ecDNA drives clonal evolution through structural rearrangements during malignant transformation and recurrence of urothelial carcinoma. This supports a model wherein tumors evolve via the selective expansion of ecDNA-bearing cells. Through multiregional sampling of tumors, we demonstrate that ecDNA contributes to the evolution of multifocality and increased intratumoral heterogeneity. ecDNA is present in 36% of urothelial carcinoma tumors and correlates with an immunosuppressive phenotype and poor prognosis. Single-cell RNA sequencing analyses reveal that ecDNA+ malignant cells exhibit diminished expression of MHC class I molecules, enabling them to evade T-cell immunity. Finally, we show that sequencing of urinary sediment–derived DNA has excel-lent specificity in detecting ecDNA.