ABSTRACT Molecular Characterization Trial The Molecular Characterization Trial (MCT) is a clinical platform to enable tissue acquisition to perform cutting edge laboratory and imaging research to advance the field of radiation biology. The MCT overarching hypothesis is that the contribution of radiotherapy (RT) to the outcome of cancer depends on the balance of the crosstalk among the irradiated tumor and normal tissue, the host microbiome, and the host’s immune system. Rectal cancer is an ideal model to test this hypothesis since imaging and specimens, from patients undergoing a standard short course radiotherapy (SCRT) treatment, are accessible and available at pre- and post RT treatment as well as at time of surgery. As such the RT effects can be analyzed longitudinally in each single tissue and then be integrated globally into a model to correlate biological and imaging data with clinical outcome. Rectal cancer is also a research priority because of its disparity in racial incidence and outcome. The ROBIN P.I.s have assembled an international team of established investigators, to conduct the same trial concurrently, at seven centers, to accelerate accruals and discovery. As such, the MCT represents the ROBIN foundation on which both Project 1 and Project 2 rely to answer fundamental questions regarding RT biology. Novel bioinformatic approaches will be applied to integrate clinical and biological data with imaging data, to enhance the potential for discovery. The initial MCT will create a supporting structure for future trials, iteratively testing new interventions informed by the results of the associated scientific projects. Project 1 will focus on the molecular analyses of tumor and patient’s matched non-tumor tissue with the goals of defining the local immune response to RT and the genomic changes induced by RT and elucidate how the treatment affects the pathways involved in cell fate decisions, and its effects on the local colonic mucosal microbiome. Project 2 will use blood specimens and lymph nodes, collected inside or outside the radiation field, to quantify RT-induced oxidative stress and the biological outcome of it on each immune cell subpopulation. Pathways associated with cellular stress responses, cell death, and immunological fitness will also be evaluated. Additionally, stool samples collected before and after RT will be analyzed to qualitatively and quantitatively map changes in the microbiome. Both projects will address these pivotal questions using state-of-the-art, genomic and proteomic approaches integrated with the patients’ clinical data and multi-modal imaging from an orthogonal radiomic study. After standardization and upload, all laboratory, clinical and imaging data will be harmonized and cured by the Data Sharing and Integrative Analysis Core (DSIA). Data will then be analyzed using novel integrative bioinformatics approaches to identify previously undetectable patterns related to radio-responsiveness and assess t...