The Interplay between Mutagenesis and Extrachromosomal DNA Shapes Urothelial Cancer Evolution

Advanced urothelial cancer is a frequently lethal disease characterized by marked genetic heterogeneity. In this study, we investigate the evolution of the genomic signatures caused by endogenous and external mutagenic stimuli and their interplay with complex structural variants. We superimposed mutational signatures and phylogenetic analyses of matched serial tumors from patients with urothelial cancer to define the evolutionary patterns of these processes. We show that APOBEC3-induced mutations are clonal and early, whereas mutational bursts comprising hundreds of late subclonal mutations are induced by chemotherapy. Using a novel genome graph computational paradigm, we observed frequent circular high copy-number amplicons characteristic of extrachromosomal DNA (ecDNA) involving double-minutes, breakage-fusion-bridge, and tyfonas events. We characterized the distinct temporal patterns of APOBEC3 mutations and chemotherapy-induced mutations within ecDNA, gaining new insights into the timing of these events relative to ecDNA biogenesis. Finally, we discovered that most CCND1 amplifications in urothelial cancer arise within circular ecDNA amplicons. These CCND1 ecDNA amplification events persisted and increased in complexity incorporating additional DNA segments potentially contributing selective fitness advantage to the evolution of treatment resistance. Our findings define fundamental mechanisms driving urothelial cancer evolution and have therapeutic implications for treating this disease. ### Competing Interest Statement B.M. Faltas: Consulting or Advisory Role: QED therapeutics, Boston Gene, Astrin Biosciences Merck, Immunomedics/Gilead, QED therapeutics, Guardant, Janssen. Patent Royalties: Immunomedics/Gilead. Research support: Eli Lilly. Honoraria: Urotoday. Grants and research support: NIH, DoD-CDMRP, Starr Cancer Consortium, P-1000 Consortium. O. Elemento: Stock and Other Ownership Interests: Freenome, OneThree Biotech, Owkin, Volastra Therapeutics. Personal fees: Pionyr Immunotherapeutics, Champions Oncology. S.T. Tagawa: Consulting or Advisory Role: 4D Pharma, Abbvie, AIkido Pharma, Amgen, Astellas Pharma, Bayer, Blue Earth Diagnostics, Clarity Pharmaceuticals, Clovis Oncology, Convergent Therapeutics, Dendreon, Endocyte, Genentech, Genomic Health, Gilead Sciences, Immunomedics, Janssen, Karyopharm Therapeutics, Medivation, Myovant Sciences, Novartis, Pfizer, POINT Biopharma, QED Therapeutics, Sanofi, Seagen, Telix Pharmaceuticals, Tolmar. Research Funding: Abbvie (Inst), Amgen (Inst), Astellas Pharma (Inst), AstraZeneca (Inst), AVEO (Inst), Bayer (Inst), Boehringer Ingelheim (Inst), Bristol-Myers Squibb (Inst), Clovis Oncology (Inst), Dendreon (Inst), Endocyte (Inst), Exelixis (Inst), Genentech (Inst), Immunomedics (Inst), Inovio Pharmaceuticals (Inst), Janssen (Inst), Karyopharm Therapeutics (Inst), Lilly (Inst), Medivation (Inst), Merck (Inst), Millennium (Inst), Newlink Genetics (Inst), Novartis (Inst), POINT Biopharma (Inst), Progenics (Inst), Rexahn Pharmaceuticals (Inst), Sanofi (Inst), Stem CentRx (Inst). Patents, Royalties, Other Intellectual Property: Patent Royalty from Immunomedics / Gilead. Travel, Accommodations, Expenses: Amgen, Immunomedics, Sanofi. Uncompensated Relationships: ATLAB Pharma, Phosplatin Therapeutics. D.M. Nanus: Consulting or Advisory Role: AstraZeneca. Research Funding: AstraZeneca (Inst), Boehringer Ingelheim (Inst), Clovis Oncology (Inst), Exelixis (Inst), Immumedics (Inst), Janssen (Inst), Novartis (Inst), Pfizer (Inst), Zenith Epigenetics (Inst). J.T. Nauseef: Consulting or Advisory Role: AIQ Solutions. Travel, Accommodations, Expenses: Digital Science Press. C.N. Sternberg: Consulting or Advisory Role: Astellas Pharma, AstraZeneca, Bayer, Bristol-Myers Squibb/Medarex, Foundation Medicine, Genzyme, Immunomedics, IMPAC Medical Systems, Incyte, Medscape, Merck, MSD, Pfizer, Roche, UroToday. A.M. Molina: Consulting or Advisory Role: Eisai, Exelixis, Janssen. J. M. Mosquera: Research Funding: Personal Genome Diagnostics. Travel, Accommodations, Expenses: Personal Genome Diagnostics. D. Scherr: Research Funding: Urogen Pharma (Inst), Cepheid (Inst), Anchiano (Inst), CryoLife (Inst). D.D. Nguyen, W.F. Hooper, T.R. Chu, H. Geiger, J.M. Shelton, M. Shah, Z.R. Goldstein, L. Winterkorn, M. Sigouros, J. Minohar, J. Moyer, D. Wilkes, R.R. Singh, W. Liu, A. Sboner, G. Inghirami, and N. Robine declare no competing interests.