Gene Expression Profile of the Clinically Aggressive Micropapillary Variant of Bladder Cancer

Abstract

Background: Progression of conventional urothelial carcinoma of the bladder to a tumor with unique microscopic features referred to as micropapillary carcinoma is coupled with aggressive clinical behavior signified by a high propensity for metastasis to regional lymph nodes and distant organs resulting in shorter survival.

Objective: To analyze the expression profile of micropapillary cancer and define its molecular features relevant to clinical behavior.

Design, setting, and participants: We retrospectively identified 43 patients with micropapillary bladder cancers and a reference set of 89 patients with conventional urothelial carcinomas and performed whole-genome expression messenger RNA profiling.

Outcome measurements and statistical analysis: The tumors were segregated into distinct groups according to hierarchical clustering analyses. They were also classified according to luminal, p53-like, and basal categories using a previously described algorithm. We applied Ingenuity Pathway Analysis software (Qiagen, Redwood City, CA, USA) and gene set enrichment analysis for pathway analyses. Cox proportional hazards models and Kaplan-Meier methods were used to assess the relationship between survival and molecular subtypes. The expression profile of micropapillary cancer was validated for selected markers by immunohistochemistry on parallel tissue microarrays.

Results and limitations: We show that the striking features of micropapillary cancer are downregulation of miR-296 and activation of chromatin-remodeling complex RUVBL1. In contrast to conventional urothelial carcinomas that based on their expression can be equally divided into luminal and basal subtypes, micropapillary cancer is almost exclusively luminal, displaying enrichment of active peroxisome proliferator-activated receptor γ and suppression of p63 target genes. As with conventional luminal urothelial carcinomas, a subset of micropapillary cancers exhibit activation of wild-type p53 downstream genes and represent the most aggressive molecular subtype of the disease with the shortest survival. The involvement of miR-296 and RUVBL1 in the development of micropapillary bladder cancer was identified by the analyses of correlative associations of genome expression profiles and requires mechanistic validation.

Conclusions: Micropapillary cancer evolves through the luminal pathway and is characterized by the activation of miR-296 and RUVBL1 target genes.

Patient summary: Our observations have important implications for prognosis and for possible future development of more effective therapies for micropapillary bladder cancer.

Keywords: Expression profile; Micropapillary bladder cancer; Molecular signature; Prognosis.

Figure 1. Whole genome mRNA expression profiling of micropapillary and conventional bladder cancer

(A) The top 50 upregulated and top 50 downregulated genes in 43 cases of micropapillary cancer compared to 89 cases of conventional urothelial carcinoma. (B) Hierarchical cluster analysis of the cohort shown in A using the top 10 upregulated and the top 10 downregulated genes identified in micropapillary cancer. (C) The distribution of samples in clusters A and B identified by hierarchical clustering analysis. (D) Expression of the top 10 canonical pathways enriched in micropapillary cancers compared with expression in conventional bladder cancers. (E) Kaplan-Meier analysis of survival in micropapillary and conventional bladder cancers. (F) Kaplan-Meier analysis of survival in clusters A and B. P, one-tailed Fisher’s exact p-value; r, ratio of expression values. Con UC, conventional urothelial carcinoma; MP UC, micropapillary urothelial carcinoma.

Figure 2. Luminal and basal molecular subtypes in conventional and micropapillary bladder cancers

(A) The expression of luminal, p53, and basal markers in molecular subtypes of conventional and micropapillary bladder cancers. (B) The distribution of molecular subtypes in conventional and micropapillary bladder cancers. (C) Kaplan-Meier plots of molecular subtypes of conventional and micropapillary bladder cancers. (D) The immunohistochemical expression of signature luminal and basal markers in representative luminal and basal cases of conventional bladder cancer as well as representative luminal micropapillary cancer.

Figure 3. Expression pattern of signature transcriptional regulators in conventional and micropapillary bladder cancers

(A) The top 10 upstream regulators enriched in micropapillary cancer. (B) Expression patterns of PPARγ and p63 target genes in molecular subtypes of conventional and micropapillary bladder cancer. (C) PPARγ expression signatures of micropapillary and conventional basal bladder cancers compared by GSEA. (D) p63 expression signatures of micropapillary and conventional basal of bladder cancers compared by GSEA. P, one-tailed Fisher’s exact p-value; r, ratio of expression values.

Figure 4. Enrichment of canonical pathways and upstream regulators in micropapillary cancers as compared with conventional luminal urothelial carcinomas

(A) The top 10 cannoical pathways enriched in micropapillary cancer. (B) The top 10 upstream regulators enriched in micropapillary cancer. (C) Expression pattern of miR-296 and RUVBL1 target genes in molecular subtypes of conventional and micropapillary subtypes of bladder cancer. (D) Expression levels of miR-296 in conventional and micropapillary bladder cancers. (E) GSEA of RUVBL1 expression signature by comparing micropapillary with conventional luminal subtype of bladder cancer. (F) Expression levels of RUVBL1 in conventional and micropapillary bladder cancers. P, one-tailed Fisher’s exact p-value; r, ratio of expression values.