Abstract 5120: The novel claudin-low molecular subtype of high-grade urothelial bladder cancer is highly immunogenic yet immunosuppressed
Authors: Vincent Benjamin G. , Kim William , Kardos Jordan , Chai Shengjie , Parker Joel , Mose Lisle , Selitsky Sara , Iglesia Michael , Milowsky Matthew 
Published: July 2016

Abstract
Rationale: High-grade urothelial carcinoma of the bladder is a heterogeneous disease, with molecular subtypes characterized by distinct tumor biologies and prognoses. Our group and others have described the basal and luminal subtypes, and we now report on the discovery of the claudin-low subtype, all of which resemble analogous subtypes in breast cancer.
Methods: We analyzed mRNA sequencing and whole exome sequencing data for The Cancer Genome Atlas (TCGA) bladder tumors and tumors collected at UNC. We performed unsupervised hierarchical clustering on relative gene expression values with significance testing using SigClust to identify the claudin-low subtype. Basal, luminal, and claudin-low bladder tumors were compared for enrichment of genetic features (single nucleotide and copy number variation), gene set expression, immune gene signature expression, T cell receptor (TCR) and B cell receptor (BCR) gene segment expression, and number of predicted MHC Class I and Class II neoantigens. We further report on the first use of our VDJician software to reconstruct full-length rearranged BCR sequences from short-read RNA sequencing data in bladder cancer.
Results: Claudin-low bladder tumors were defined by low expression of tight junction claudin-proteins, high expression of epithelial-to-mesenchymal transition genes and immune gene signatures, and were associated with reduced overall survival compared to luminal tumors. A minimal gene set classifier to identify claudin-low tumors showed some but not complete overlap with an optimal classifier derived in breast cancer. Claudin-low bladder tumors were enriched for multiple genetic features: increased rates of RB1, EP300, and NCOR1 mutations, increased frequency of EGFR amplification, decreased rates of FGFR3, ELF3, and KDM6A mutations, and decreased frequency of PPARG amplification. Claudin-low tumors showed the highest expression of immune gene signatures (including an immunosuppression signature), however in contrast to basal tumors, increased immune gene signature expression was not associated with prolonged survival. Claudin-low tumors also showed the highest overall expression of rearranged BCR sequences but lowest BCR repertoire diversity. Predicted neoantigen burden did not vary significantly by subtype, however broad cytokine and chemokine expression levels were elevated in claudin-low tumors, potentially related to low PPARG activity driving increased NFKB activity.
Conclusions: Claudin-low bladder cancer is a novel molecular subtype with distinct molecular and immunologic features and prognostic significance. Given the presence of dense immune infiltrates, BCR repertoire characteristics consistent with an antigen-driven response, and high expression of immunosuppression genes, claudin-low bladder tumors may be primed to respond to immune checkpoint inhibitor therapy.
Citation Format: Benjamin G. Vincent, William Kim, Jordan Kardos, Shengjie Chai, Joel Parker, Lisle Mose, Sara Selitsky, Michael Iglesia, Matthew Milowsky. The novel claudin-low molecular subtype of high-grade urothelial bladder cancer is highly immunogenic yet immunosuppressed. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5120.