PROJECT SUMMARY Molecular Characterization Trial Navigating Extended Specimen Surveys for Therapeutic Selection (ROBIN-NESSTS) Summary: Approximately half of all cancer patients are treated with radiation in the course of their disease. There exists an opportunity and urgent need to apply new biological knowledge in the optimization of radiation treatment and its combination with emerging targeted anti-neoplastic agents. At the core of the CCF/Emory ROBIN U54 Center is a branched Molecular Characterization Trial (MCT) focused on longitudinal collection of biospecimens and multimodal data from patients with muscle invasive bladder cancer (MIBC) and head and neck squamous cell carcinoma (HNSCC) prior to-, during, and after radiotherapy. These molecular characterization cohorts have been designed to incorporate immunomodulatory combination therapies as well as standard-of-care chemoradiotherapy, aiming to address pressing translational questions by integrating these trials throughout the ROBIN Center’s cores and projects. These are immune radiobiology-centered “small N, high-content” study cohorts that will allow us to address critical knowledge gaps related to the biological basis responses to combined modality radiotherapy. The central hypothesis being tested is that the longitudinal collection of clinically annotated biospecimens and multimodal data from patients treated prior to, during, and after radiotherapy from MCT cohorts examining radiation-based immunotherapies and standard of care chemoradiation will provide new insights into the genetic, immunologic, and evolutionary basis of therapeutic response. The ability to interrogate key questions across two distinct cancers will amplify our capacity to identify generalizable molecular mechanisms underlying radiation resistance and immunomodulatory activity. Our Specific Aims are to conduct a specimen collection and characterization trial with 2 cohorts: (1) (MCTa): Adaptive RADiation therapy with concurrent Sacituzumab Govitecan (RAD-SG) for bladder preservation in patients with MIBC and (2) a Phase II trial of IMRT re-irradiation plus concurrent and adjuvant nivolumab. The same sample and data acquisition will be obtained from two parallel cohorts of standard of care cisplatin plus RT treated patients (n=20 for each site). The overarching objective of the high- content study cohorts in this MCT is to rigorously dissect common mechanisms and drivers of efficacy and resistance to the most promising radiation-based combinations in two cancers. We will address knowledge gaps related to the genetic, immunologic, and molecular evolutionary basis of response to immunomodulatory combination therapies by seamlessly integrating multi-modal data from these clinical cohorts across the ROBIN Center’s laboratories, projects, and cores.