Early response to radiotherapy and immunotherapy in rectal cancer: an integrated molecular, cellular, and spatial approach - Project Abstract With increased incidence in young populations and ~65% three-year survival of high-risk locally advanced rec- tal cancer, innovative approaches to improve outcomes is imperative. Neoadjuvant therapy with radiation (RT) and chemotherapy (CT) is now the standard curative treatment, but there is demand for non-operative man- agement (NOM) if the disease can be cured with local and systemic therapy. Thus, innovations could be trans- formative with the aim to improve durable complete responses by personalizing therapy, which includes how to deliver RT, CT, and the incorporation of novel agents such as immunotherapy. Cancer Immunotherapy has had little impact on colorectal cancer outside of mismatch repair deficient tumors. The αCD40 agonist antibody is an emerging class of immunotherapy and sotigalimab has shown promise in phase I and multiple ongoing phase II trials. The CD40 receptor is important in both innate and adaptive immune responses and a greater therapeutic effect can be achieved combining αCD40 with RT in animal models. We hypothesize that short course RT (SCRT) and CT when combined with αCD40 in human tumors can result in a greater antitumor im- mune response, reduce risk of metastatic progression, and extend survival in a poorly immunogenic malig- nancy like rectal cancer. We developed the INNATE trial, a phase II randomized trial of neoadjuvant SCRT fol- lowed by CT with or without the addition of sotigalimab for locally advanced rectal cancer. This trial design has allowed us to collect fresh pre- and post-SCRT biopsy tissue, which we have obtained from 21 of 30 patients enrolled to date. In this proposal, we focus on the central hypothesis that an integrated molecular, cellular, and spatial assessment of treatment response dynamics in the tumor microenvironment early after SCRT can re- veal insights into the immunobiological responses, which can inform mechanisms of efficacy and therapeutic selection. We will perform 1) molecular and cellular single cell (sc) RNAseq with proteomic and immune reper- toire analysis, 2) molecular, cellular, and spatial multiplex immunofluorescence, and 3) cellular and spatial quantitative deep learning based histopathologic analysis to achieve our aims. These three technologies will enable us to investigate early changes across RT and αCD40 treated groups. Then we will aim to identify ther- apeutic opportunities for the combination of SCRT with immune active agents though an integrated RT re- sponse assessment. Lastly, we will establish innate and adaptive immunologic signaling events triggered by SCRT in combination with αCD40 immunotherapy and factors that enhance or hinder efficacy. We will use models to start validating key findings and aim to propose future therapeutic directions to build off these efforts and ultimately improve outcomes. Specifically, regarding our patient population, we expect this proposal will lead to evidenced based trials for most patients with locally advanced rectal cancer who strive to achieve cure without a morbid surgery.
