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Intratumoral heterogeneity (ITH) is associated with clinical challenges such as possible differences in response to treatment and difficulties in classifying the tumor. Previously, ITH has been described in bladder cancer using detailed genetic analyses. However, in this disease, it is not known to what extent ITH actually occurs, or if it involves molecular subtyping, when assessment is achieved by immunohistochemistry (IHC) on the protein level using tissue microarrays (TMAs), the method most widely applied when analyzing large sample numbers. We aimed to investigate ITH by IHC in bladder cancer TMAs. Staining for eleven immunohistochemical markers (CK5, Cyclin D1, E-Cadherin, EGFR, FGFR, GATA3, HER2, p16, p63, P-Cadherin and RB1) was performed, and differences in staining patterns were assessed both within 1981 individual tissue-cores and by comparing two cores from the same tumor in 948 cases according to our pre-specified criteria. Presence of ITH was associated with clinicopathological data such as stage, grade, molecular subtype and survival. Intracore ITH in one or several markers was associated with grade 3, stage T1 and the genomically unstable molecular subtype. ITH in three or more markers was found in 5% between cores (intercore heterogeneity) and in 2% within cores (intracore heterogeneity). No association with survival was found for any of the ITH groups. We observed ITH in a small proportion of the tumors, suggesting that ITH has only a limited impact on TMA bladder cancer studies.

We recently defined molecular subtypes of urothelial carcinomas according to whole genome gene expression. Herein we describe molecular pathologic characterization of the subtypes using 20 genes and IHC of 237 tumors. In addition to differences in expression levels, the subtypes show important differences in stratification of protein expression. The selected genes included biological features central to bladder cancer biology, eg, cell cycle activity, cellular architecture, cell-cell interactions, and key receptor tyrosine kinases. We show that the urobasal (Uro) A subtype shares features with normal urothelium such as keratin 5 (KRT5), P-cadherin (P-Cad), and epidermal growth factor receptor (EGFR) expression confined to basal cells, and cell cycle activity (CCNB1) restricted to the tumor-stroma interface. In contrast, the squamous cell cancer–like (SCCL) subtype uniformly expresses KRT5, P-Cad, EGFR, KRT14, and cell cycle genes throughout the tumor parenchyma. The genomically unstable subtype shows proliferation throughout the tumor parenchyma and high ERBB2 and E-Cad expression but absence of KRT5, P-Cad, and EGFR expression. UroB tumors demonstrate features shared by both UroA and SCCL subtypes. A major transition in tumor progression seems to be loss of dependency of stromal interaction for proliferation. We present a simple IHC/histology-based classifier that is easy to implement as a standard pathologic evaluation to differentiate the three major subtypes: urobasal, genomically unstable, and SCCL. These three major subtypes exhibit important prognostic differences.

We assessed DNA methylation and copy number status of 27,000 CpGs in 149 urothelial carcinomas and integrated the findings with gene expression and mutation data. Methylation was associated with gene expression for 1,332 CpGs, of which 26% showed positive correlation with expression, i.e., high methylation and high gene expression levels. These positively correlated CpGs were part of specific transcription factor binding sites, such as sites for MYC and CREBP1, or located in gene bodies. Furthermore, we found genes with copy number gains, low expression and high methylation levels, revealing an association between methylation and copy number levels. This phenomenon was typically observed for developmental genes, such as HOXgenes, and tumor suppressor genes. In contrast, we also identified genes with copy number gains, high expression and low methylation levels. This was for instance observed for some keratin genes. Tumor cases could be grouped into four subgroups, termed epitypes, by their DNA methylation profiles. One epitype was influenced by the presence of infiltrating immune cells, two epitypes were mainly composed of non-muscle invasive tumors, and the remaining epitype of muscle invasive tumors. The polycomb complex protein EZH2that blocks differentiation in embryonic stem cells showed increased expression both at the mRNA and protein levels in the muscle invasive epitype, together with methylation of polycomb target genes and HOXgenes. Our data highlights HOXgene silencing and EZH2expression as mechanisms to promote a more undifferentiated and aggressive state in UC.