Vice Chair for Faculty Affairs Baylor College of Medicine
Introduction: Urothelial carcinoma in situ (CIS) is a high-grade intraepithelial cancer whose natural history is associated with ahigh probability of progression. It can be broadly categorized by RNA expression into luminal and basal-likesubtypes, however the molecular drivers unique to CIS as compared to adjacent papillary tumor or normalurothelium are underexplored. Comprehensive genomic analysis of CIS and comparisons to papillary andprogressed tumors may help define progression pathways and BCG treatment response/resistance mechanisms. Todetermine feasibility and proof of concept, we performed molecular characterization of CIS, paired papillary tumorsand normal urothelium Methods: TUR and matched bladder biopsies were obtained from 32 samples in 19 patients. A total of 10 CIS, 9 papillarytumor and 8 normal samples underwent whole transcriptome RNA sequencing. Wilcoxon test was used to filterdifferentially expressed genes. TCGA single sample classifier was used to assign molecular subtypes. Whole exomesequencing (WES) was performed for 19 patients with matched CIS and papillary tumor samples (nCIS=34 andntumor=33). Using multiplex IF and IHC analyses, 24 samples from 15 patients were analyzed for the presence ofcytotoxic T-cells (CTLs), T-helper cells, regulatory T-cells (Tregs), B-cells, M1 and M2 macrophages and PD-1 andPD-L1-expressing cells Results: M:F ratio was 15:4 and the CIS TCGA subtypes were 3 basal, 2 luminal, 5 luminal infiltrative, 7 luminal papillary,and 1 neuronal. We identified a 46-gene signature of differentially expressed genes in CIS samples that includedknown druggable targets: MTOR, TYK2, AXIN1, CPT1B, GAK and PIEZO1 and selectively downregulated BRD2 andNDUFB2 (p < 0.05). We assessed the robustness of these markers in an independent dataset from Dyrskjøt et al(GSE3167)and a reduced 36-gene signature accurately identified the CIS samples (AUC=0.90). We validatedselective overexpression of MTOR, GUSBP11, KMT2D, URB1 and under expression of HMGB1, RPS7, NDUFB4,BRD2, HNRNPK, CANX and HSP90B1in CIS samples. In the WES cohort, KDM6A was the most frequently mutatedgene across CIS and papillary tumor samples. When analyzing CIS samples alone, CCDC138 was the mostcommonly mutated gene. We observed clonality between CIS lesions and matched papillary tumors. We observedfewer immune cells within the tumorareas compared to stromal regions and significantly higher fractions of T-helper cells and B-cells in all stromal areas combined compared to areas of epithelial origin. Additionally, there wasa significantly larger fraction of PD1-expressing cells in CIS regions compared to papillary regions (p=0.031) Conclusions: We identified CIS lesions having a unique molecular signature and potentially actionable mutations. KDM6A andCCDC138 were commonly mutated, and levels of T-helper and B-cells were highest in stromal regions while CISharbored more PD1-expressing cells. SOURCE OF Funding: None
