Targeted DNA and RNA Sequencing of Paired Urothelial and Squamous Bladder Cancers Reveals Discordant Genomic and Transcriptomic Events and Unique Therapeutic Implications

摘要

BackgroundIntegrated 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. ObjectiveTo assess the impact of histological heterogeneity on expression-based bladder cancer subtypes. Design, setting, and participantsWe 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. Outcome measurements and statistical analysisUnsupervised hierarchical and consensus clustering of target gene expression enabled derivation of basal/luminal molecular subtyping. Results and limitationAcross 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 deleteriousTP53(56%) and activating hotspotPIK3CA(30%) somatic mutations were seen across 69 high-quality tissue samples. Potentially targetable focalERBB2(6%) orEGFR(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 outlierEGFRandERBB2expression 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. ConclusionsOur results support the feasibility of clinically relevant integrative bladder cancer profiling and challenge the intrinsic nature of expression subtypes in histologically diverse bladder cancers. Patient summaryA 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.