Modeling of bladder cancer evolution from field effects by multi-platform spatial mapping on the whole-organ scale

Introduction: Understanding the mechanisms driving evolution of the mucosal field effects to invasive cancer is not possible unless they are analyzed in the context of their geographic distribution in the entire organ. Bladder cancer is an ideal model disease for such studies as it effects an anatomically simple organ permitting the multi-platform analysis of the affected mucosa on the scale of whole organ. Methods: We performed comprehensive multi-platform analyses on nine cystectomies with invasive bladder cancer comprising of 433 mucosal tissue samples analyzed by bulk whole-exome and mRNA sequencing, genome-wide copy number variation and methylation profiling complemented with proteomics, metabolomic and single cell sequencing spatial mapping. The data from multi-platform profiling were geographically annotated to microscopically normal urothelium (NU) and low-grade intraurothelial neoplasia (LGIN; n=243), high-grade intraurothelial neoplasia (HGIN; n=90), and invasive urothelial carcinoma (UC; n=100). Results: We identified ~16000 non-silent mutations per cystectomy. In two maps the hypermutator phenotypes with ~48000 and ~57000 mutations were detected. The mutational analysis identified three types of mutations based on the variant allele frequency and geographic distribution referred to as alpha, beta, and ϒ. Time modeling by a parsimonious time-continuous Markov model incorporating cell migration (immigration) and growth (branching) revealed that bladder carcinogenesis takes approximately three decades and can be divided into dormant and progressive phases. Low selection alpha mutations were private and most frequent. They continuously developed over 30 years primarily in the dormant phase of carcinogenesis. beta mutations clonally expanded regionally and signified the advent of progressive phase of carcinogenesis which lasted five years. ϒ mutations were the ultimate drivers of the progressive phase and emerged 2-3 years before the final progression to invasive bladder cancer. The mutational landscape developed on the background of severely dysregulated urothelial differentiation and increased immune infiltration with T-cell exhaustion. The proteomic and metabolomic changes involved a wide-spread disorganization of glycolipid energy metabolism with downregulation of mitochondrial oxidative phosphorylation. Conclusion: Dysregulated mitochondrial energy metabolism converging on anerobic glycolysis and oxidative phosphorylation with Warburg phenotype emerged as the leading mechanism driving the progression of mucosal field effects to invasive cancer.