Abstract LB468: Histological subtypes of metastatic bladder cancer have distinct tumor microenvironments and high intratumoral heterogeneity

Abstract Background: Metastatic bladder cancer (BLCA) is an aggressive disease complicated by the emergence of variant histological subtypes and variable responses to treatment. Understanding the heterogeneity that exists both between these histological subtypes and within individual BLCA tumors is imperative in advancing precision medicine for BLCA. Approach: The University of Washington/Fred Hutchinson Cancer Center metastatic BLCA rapid autopsy program is a first-of-its-kind program collecting both primary and metastatic tumor samples from advanced BLCA patients, with a focus on aggressive variant subtype tumors. To explore intra-tumoral heterogeneity of both tumor and stromal compartments, single-nucleus RNA sequencing, in combination with matching bulk DNA and RNA sequencing, was performed on 15 tumors from 9 patients. The resulting dataset totaled 39,888 cells across a variety of histological subtypes: conventional urothelial carcinoma (UC), UC with squamous differentiation (UCSD), and plasmacytoid UC (PUC). Results: Our unique snRNAseq dataset, the first in either metastatic or variant subtype BLCA, was used to dissect genomic and transcriptomic heterogeneity within BLCA tumors. On a genomic level, we observe subclonal copy number variations (CNVs) in BLCA driver genes in almost all (8/9) patients. Furthermore, we observe varying levels of intra-tumoral transcriptomic heterogeneity, where metastatic survival time is positively associated with tumor heterogeneity. Despite this heterogeneity, particular pathways define the epithelial transcriptomics of variant histological subtypes, including the upregulation of cell cycle and DNA repair upregulated in PUC tumors, which is validated in both bulk RNA sequencing and immunohistochemistry of a larger UW/FH BLCA rapid autopsy cohort. Investigation of the tumor microenvironment revealed that PUC tumors may be more immune-hot than UC, with increased B- and T-cell infiltration, while UCSD tumors have immunosuppressive microenvironments enriched in tissue-associated macrophages. Interestingly, these differences across subtypes are supported by changes in inter-cellular signaling between epithelial and fibroblast cells and the immune cells. Impact: These findings lay the groundwork for development of precision medicines for histological subtypes in BLCA by revealing heterogeneity and immune microenvironments that underlie disease aggressiveness and therapeutic resistance. Citation Format: Samantha L. Schuster, Pushpa Itagi, Sonali Arora, Thomas Persse, Patricia C. Galipeau, John K. Lee, Robert B. Montgomery, Jonathan L. Wright, Petros Grivas, Funda Vakar-Lopez, Andrew C. Hsieh, Hung-Ming Lam, Gavin Ha. Histological subtypes of metastatic bladder cancer have distinct tumor microenvironments and high intratumoral heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB468.