Introduction:

Urothelial carcinoma in situ (UCIS) of the bladder is a high-grade intraepithelial cancer with who’s natural history is associated with a high probability of disease progression. It can be broadly categorized by RNA expression into luminal and basal-like subtypes as described by Kim et al (PMID:30851984), however the molecular drivers unique to CIS as compared to adjacent papillary tumor or normal urothelium are underexplored in part due to challenges obtaining fresh CIS samples and small biopsies in FFPE sections. The aim of the present study was to explore the molecular landscape of UCIS, paired papillary tumors and normal urothelium by transcriptome profiling using RNAseq.

Methods:

Transurethral resection and matched bladder biopsies were obtained from 32 samples in 19 patients. After path QC and RNA extraction, 10 CIS, 9 papillary tumor and 8 normal urothelial samples underwent whole transcriptome RNA sequencing. Strand-specific, poly-A+ RNA-seq libraries for sequencing on the Illumina platform were prepared as previously described (PMID: 25360585). ANOVA and Wilcoxon test were used statistically to filter differentially expressed genes. TCGA single patient classifier was used to assign molecular subtypes. A statistical significance of p-value < 0.05 was used for potential gene candidtates of CIS and a validating independent dataset from  Dyrskjøt et al (GSE31679) comprised of 5 CIS and 55 other tumor samples were used to validate those potential CIS genes and their expressions from their surgical specimens of this study.

Results:

Male-to-female gender ratio was 15:4 and the TCGA classification showed 3 Basal, 2 luminal, 5 luminal infiltrative, 7 luminal papillary, and 1 neuronal subtypes (Fig. 1). Overall, the CIS samples with lower expression of immune markers. A 46 gene signature of differentially expressed genes in CIS samples were identified including known druggable targets incuding selectively upregulated MTOR, TYK2, AXIN1, CPT1B, GAK and PIEZO1 and selectively downregulated BRD2 and NDUFB2 (p<0.05). To assess the robustness of these markers in an independent dataset from Dyrskjøt et al (GSE31679)  comprised of 5 CIS and 55 other tumor samples, selectively overexpressed MTOR, GUSBP11, KMT2D, URB1 were validated  in CIS samples.

Conclusion:

This study identified CIS lesions having a unique molecular signature that may contribute to an aggressive feature of progressive disease phenotype of the bladder cancer.

Funding: This study is supported by the Grant of National Institutes of Health and and Albert Institute