Fig. 1. Genomic analysis of urothelial carcinomas with squamous cell differentiation.

a Hematoxylin and Eosin (H&E) staining of a representative mixed histology bladder cancer with discrete and separable regions of urothelial carcinoma, NOS (UC), and squamous differentiation (SqD). In comparison to the urothelial component, the squamous regions were characterized by the presence of tumor cells with keratin formation (block arrow), a microscopic feature unequivocal for squamous differentiation. Scale bar = 2 mm. b Whole exome sequencing of macrodissected UC and SqD regions of 21 mixed histology bladder cancers revealed a significantly higher tumor mutational burden in the SqD versus UC regions (two-sided Wilcoxon paired test, p 0.02). c Oncoprint showing the mutation status of genes commonly mutated in bladder cancer. For each patient, the mutation status in the UC component is shown on the left, and the SqD component on the right. d, e Phylogenetic analysis of two representative urothelial carcinomas with squamous differentiation highlighting that the UC and SqD components were derived from a shared precursor. The number of non-synonymous mutations in each region is shown on top with shared mutations represented in dark green and mutations private to each component in light green. The cancer cell fraction (CCF) of individual mutations is indicated by the degree of blue shading. The UC (orange), SqD (green), and their hypothetical normal cell of origin (gray) are indicated by the different colors. f Average of concordant and discordant mutations between paired UC and SqD samples revealed higher concordance of oncogenic and likely oncogenic mutations (63.2%) versus all nonsynonymous mutations (33.9%).