Abstract 738: Single-cell trajectories of metastatic urothelial cancer and individual patterns of resistance to immune checkpoint inhibitors

Abstract Background. Immune checkpoint inhibitors (ICI) improve survival in patients with metastatic urothelial cancers (mUC) but most patients experience disease progression. The mechanisms driving this progression are mostly unknown. We longitudinally assessed mUC microenvironment at the single-cell level in ICI-treated patients to explore the determinants of the response and adaptive resistance mechanisms on therapy. Methods. The prospective MATCH-R study (NCT02517892) included patients with mUC treated with single-agent PD-(L)1 inhibitors. Biopsies of metastatic sites were performed at baseline, on therapy and at progression for single-nuclei RNA sequencing (10X genomics, 5’ v1.1). After stringent quality control and cell type annotation, we compared the proportions and transcriptomic signatures of tumor and immune cell subsets between responders and non-responders, and explored their evolution during treatment. Pseudobulk analyses were used to classify whole samples according to the consensus molecular classification (Kamoun et al. 2020). Results. We enrolled 32 patients among whom eight (25%) achieved objective response (OR). All patients underwent biopsies at baseline, 6 (19%) on therapy, 17 (53%) at progression. Tumor cells showed high transcriptomic heterogeneity within the consensus groups, with the coexistence of basal and luminal cell states within the same samples. The proportion of basal cells at baseline, but not the basal sample classification, was associated with an increased OR (p=0.02). Immune features associated with resistance to ICI at baseline involved both myeloid and lymphoid compartments : within myeloid cells, an abundance of pro-tumoral HES1 macrophages and higher expression of immunosuppressive genes CD163, MS4A4A and STAB1 ; within T cells, a higher proportion of exhausted CD8+ lymphocytes, and higher expression of immune checkpoint genes including PDCD1. Conversely, the baseline abundance of CD4+ naive T helpers was associated with improved OR. Longitudinal sampling showed potential adaptive resistance mechanisms at progression, including downregulation of HLA genes and IFN signaling in tumor cells ; shift from M1 to M2 macrophage polarization within myeloid cells; increased expression of immune checkpoints, downregulation of type-I interferon induced genes and cytotoxicity markers such as GZMA in T cells. Individual patient analyses have shown that these mechanisms may coexist with discrete patterns. Conclusions. We identified potential mechanisms of resistance to ICI in patients with mUC related to both tumor and immune compartments, including specific T cell and macrophage subpopulations. Longitudinal assessments in patients show individual molecular patterns related to progression that may allow for dynamic and tailored therapeutic strategies. Citation Format: Ronan Flippot, Amélie Roehrig, Julien Vibert, Nicolas Stransky, Luc Cabel, Kevin Mulder, Benjamin Besse, Claudio Nicotra, Maud Ngo Camus, Christophe Massard, Etienne Rouleau, Gerome Jules-Clement, Alicia Tran-Dien, Lambros Tselikas, Fabrice Andre, Jean-Yves Scoazec, Céline Vallot, Maud Kamal, Eric Letouze, Yohann Loriot. Single-cell trajectories of metastatic urothelial cancer and individual patterns of resistance to immune checkpoint inhibitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 738.