Daniel H. Hovelson, Aaron M. Udager, Andrew S. McDaniel, Petros Grivas, Phillip Palmbos, Shuzo Tamura, Lorena Lazo de la Vega, Ganesh Palapattu, Brendan Veeneman, Layla El-Sawy, Seth E. Sadis, Todd M. Morgan, Jeffrey S. Montgomery, Alon Z. Weizer, Kathleen C. Day, Nouri Neamati, Monica Liebert, Evan T. Keller, Mark L. Day, Rohit Mehra, Scott A. Tomlins

Integrated molecular profiling has identified intrinsic expression-based bladder cancer molecular subtypes. Despite frequent histological diversity, robustness of subtypes in paired conventional (urothelial) and squamous components of the same bladder tumor has not been reported. To assess the impact of histological heterogeneity on expression-based bladder cancer subtypes. We performed clinically applicable, targeted DNA and/or RNA sequencing (multiplexed DNA and RNA sequencing [mxDNAseq and mxRNAseq, respectively]) on 112 formalin-fixed paraffin-embedded (FFPE) bladder cancer samples, including 12 cases with paired urothelial/squamous components and 21 bladder cancer cell lines. Unsupervised hierarchical and consensus clustering of target gene expression enabled derivation of basal/luminal molecular subtyping. Across 21 bladder cancer cell lines, our custom mxRNAseq panel was highly concordant with whole transcriptome sequencing, and assessed targets robustly determined expression-based basal/luminal subtypes from The Cancer Genome Atlas data (in silico) and internally sequenced FFPE tissues. Frequent deleterious TP53 (56%) and activating hotspot PIK3CA (30%) somatic mutations were seen across 69 high-quality tissue samples. Potentially targetable focal ERBB2 (6%) or EGFR (6%) amplifications were also identified, and a novel subgene copy-number detection approach is described. Combined DNA/RNA analysis showed that focally amplified samples exhibit outlier EGFR and ERBB2 expression distinct from subtype-intrinsic profiles. Critically, paired urothelial and squamous components showed divergent basal/luminal status in three of 12 cases (25%), despite identical putatively clonal prioritized somatic genomic alterations. Limitations include lack of profiled paired normal tissues for formal somatic alteration determination, and the need for formal analytical and clinical validation. Our results support the feasibility of clinically relevant integrative bladder cancer profiling and challenge the intrinsic nature of expression subtypes in histologically diverse bladder cancers. A targeted RNA sequencing assay is capable of assessing gene expression-based subtypes in individual components of clinical bladder cancer tissue specimens. Different histological components of the same tumor may yield divergent expression profiles, suggesting that expression-based subtypes should be interpreted with caution in heterogeneous cancers.