Multiomics profiling of urothelial carcinoma in situ reveals CIS-specific gene signature and immune characteristics

Abstract

Urothelial carcinoma in situ (CIS) is an aggressive phenotype of non-muscle-invasive bladder cancer. Molecular features unique to CIS compared to high-grade papillary tumors are underexplored. RNA sequencing of CIS, papillary tumors, and normal urothelium showed lower immune marker expression in CIS compared to papillary tumors. We identified a 46-gene expression signature in CIS samples including selectively upregulated known druggable targets MTOR, TYK2, AXIN1, CPT1B, GAK, and PIEZO1 and selectively downregulated BRD2 and NDUFB2. High expression of selected genes was significantly associated with CIS in an independent dataset. Mutation analysis of matched CIS and papillary tumors revealed shared mutations between samples across time points and mutational heterogeneity. CCDC138 was the most frequently mutated gene in CIS. The immunological landscape showed higher levels of PD-1-positive cells in CIS lesions compared to papillary tumors. We identified CIS lesions to have distinct characteristics compared to papillary tumors potentially contributing to the aggressive phenotype.

Keywords: Cancer; Immunology; Omics.

Graphical abstract

Figure 1

Morphologic spectrum and subtype heterogeneity of urothelial carcinoma in situ (CIS) (A) Full thickness, partially detached CIS with prominent nuclear pleomorphism and hyperchromasia, architectural disorder, and lack of polarity (10x); (B) prominent denudation and discohesion of CIS with a single layer of tumor attached to the basement membrane. Note the prominent vasculature in the lamina propria (block arrows); (C) High-grade UC involving von Brunn’s nests, which may mimic invasion (4x). The surface tumor is both papillary (shown) and flat (not shown); (D) variable tree showing site, gender, and subtypes for 19 high-quality samples.

Figure 2

CIS gene signature and pathway analysis (A) Gene signature categorizing samples into luminal, ECM and smooth muscle, EMT and claudin, basal, squamous, Immune, neuronal differentiation, CIS surrounding, and Sonic hedgehog markers. (B) Composite signature scores shown as boxplots for CIS and tumor samples (Wilcoxon rank-sum test derived p-value shown) (C) Differentially expressed hallmark pathways specific to CIS tumors. Upregulated and downregulated pathways are shown in red and blue, respectively.

Figure 3

CIS gene signature in CIS and papillary tumors with validation at individual gene level (A) CIS-specific gene signature composed of 46 genes (up- and downregulated) were identified. Unsupervised clustering shows clear segregation of CIS samples from other samples. (B) AUC curve showing performance of CIS-specific gene signature average score in predicting CIS samples from an independent cohort (GSE3167). (C) volcano plot showing fold change and p value for individual gene validation from the signature in GSE3167 dataset. Genes passing both Log2 fold change cutoff of 0.5 and Wilcoxon rank-sum test p value cutoff of 0.05 are shown as red dots.

Figure 4

Mutational landscape of CIS and papillary tumor samples (moderate and high impact SNVs and indels) Columns correspond to unique samples. Groups of columns correspond to all analyzed samples from a single patient. Top: the number of the four mutation types in every sample. Middle: oncoplot showing the four mutation types in selected genes, sorted by mutation frequency across patients. Frequencies of patients with mutations in the given gene are indicated to the left. Bars to the right indicate the numbers of the different mutation types within each gene across the samples in the cohort. Bottom: clinical and sample information. BCG outcome: high-grade disease within two years after BCG treatment or progression to muscle-invasive bladder cancer (MIBC) progression to MIBC. BCG Timing: sample timing in relation to BCG treatment.

Figure 5

Immunofluorescence and immunohistochemistry workflow and immune cell characterization (A) Workflow of the mIF and IHC methods. Multiple visits from the same patients are marked with different letters, and numbers following letters mark samples collected from the same clinical visit. Sections stained with different mIF panels or antibodies are shown in the order it was applied. Examples of analyzed regions of interest (ROIs) are shown. (B) From top bar to bottom bar: percentage of immune cells in ROIs. Immune cell proportions in regions where all stainings were available. Heatmap indicating the levels of immune cells and markers in different ROIs/patients. Bottom annotation includes sample information with patient of origin, sample types, and ROIs as well as clinical information with prior BCG treatment, post-BCG outcome (CR: complete response at 6 months. Failure: any persistent high-grade cancer), and progression status (T-stage progression). (C) Comparisons of the levels of immune cell types and markers in different ROIs. All stromal areas have been combined to one ROI. Adjusted p values are indicated when below-significance level of 0.05 (Wilcoxon rank-sum test).