Defining the impact of intra-tumoral morphologic, immune and mutational heterogeneity in urothelial carcinoma Bladder cancer is the ninth most common cancer worldwide and the fourth most common cancer in men. Despite intensive multi-modality therapy, approximately 50% of patients with muscle-invasive disease develop distant metastases and historically such patients had little hope of long-term survival. The development of immune checkpoint inhibitors is the most significant therapeutic advance in bladder cancer in three decades and the development of these agents have provided renewed hope to many patients with previously incurable metastatic disease. Anti-PD1/PD-L1 antibodies can induce durable complete responses in patients with metastatic bladder cancer with several immune checkpoint inhibitors are now FDA-approved for this indication. However, the majority of patients with metastatic urothelial cancers do not benefit from immune checkpoint blockade and some patients who initially respond later develop acquired resistance. The biologic basis for innate and acquired resistance to immune checkpoint blockade in urothelial cancer remains poorly defined. Urothelial cancers display a wide spectrum of variant morphologies that often co-exist within individual tumors. We have shown that this morphologic heterogeneity is often associated with intra-tumoral mutational heterogeneity. The current proposal is based upon preliminary data indicating that morphologic heterogeneity in bladder cancer is associated with genomic and immune heterogeneity and is predictive of a worse response to atezolizumab (an anti-PD-L1 inhibitor). Three aims are proposed. In Aim 1, we will perform integrated histologic, genomic and immune analyses of paired, macro-dissected, morphologically distinct areas from morphologically heterogeneous tumors from patients treated with immune checkpoint blockade to define the prevalence and extent of intratumoral genetic and immune heterogeneity. In Aim 2, these tissue profiling studies will be integrated with detailed clinical and patients response data to define the role of pre-existent histologic, genomic and immune heterogeneity in determining response to systemic immunotherapy. Finally, in Aim 3, we will study tumors collected at the time of disease progression in patients treated with immune checkpoint inhibitors to determine whether pre-existent drug resistant clones were present in morphologically heterogeneous primary tumors and that these less immunogenic cancer cells are a basis for drug resistance and disease progression in patients with morphologically heterogeneous tumors. The long-term translational objective will be to use the biologic insights gained to develop improved biomarkers of immunotherapy sensitivity and resistance, and to develop rational immune-based combination strategies that prevent or delay the emergence of drug resistant clones.