PROJECT SUMMARY/ABSTRACT Glioblastoma is one of the most lethal of human cancers, with very few long-term survivors and no definitive cures for this disease. Immunotherapy is an appealing strategy because of the potential ability for immune cells to traffic to and destroy infiltrating tumor cells. Although immunotherapies, such as checkpoint blockade, have revolutionized the treatment of several cancers, its benefit in GBM has been limited to small randomized trials in the neoadjuvant setting, and limited benefit in Phase III trials in the adjuvant setting. Recently, we published a surgical trial of neoadjuvant PD-1 antibody blockade, to address the immunologic effects of this agent in recurrent glioblastoma. While it was a small, randomized clinical trial, the median overall survival of the neoadjuvant treatment cohort was 417 d (13.7 months) from registration date, while that of the adjuvant treatment cohort was 228 d (7.5 months; hazard ratio 0.39). Neoadjuvant PD-1 blockade was associated with upregulation of T cell– and interferon-γ-related gene expression, but downregulation of cell-cycle-related gene expression within the tumor, which was not seen in patients that received adjuvant therapy alone. These findings suggest that the neoadjuvant administration of PD-1 blockade enhances both the local and systemic antitumor immune response and may represent a more efficacious approach to the treatment of this uniformly lethal brain tumor than traditional treatment in the adjuvant setting. While provocative, the benefit was restricted to patients whose T cell infiltration and IFN-γ signature was elevated. Our hypothesis is that the combination of CTLA-4 and PD-1 blockade in the neoadjuvant setting will significantly increase the tumor-specific T cell infiltration, allowing for PD-1 blockade to be more effective. To test this hypothesis, we will utilize our unique access to samples and patient data from an ongoing investigator-initiated clinical trial with PD-1 +/- CTLA-4 antibody blockade in the neoadjuvant setting for patients with recurrent GBM. The Specific Aims of this project are: • Aim #1: To evaluate the immunogenicity, toxicity, and clinical benefit for the combination of neoadjuvant CTLA-4 + PD-1 antibody blockade in recurrent GBM patients. • Aim #2: To evaluate how PD-1 and CTLA-4 mAb blockade independently modify the tumor microenvironment and the expansion of tumor-specific T cells following neoadjuvant checkpoint blockade in recurrent GBM patients. • Aim #3: To develop non-invasive imaging biomarkers of response or adaptive resistance in recurrent GBM patients treated with combinations of neoadjuvant CTLA-4 +/- PD-1 antibody blockade. This project could potentially be transformative, as a better understanding of how neoadjuvant immunotherapy alters immune responses within the tumor could teach us important lessons about the critical requirements for productive anti-tumor responses in glioblastoma and how adaptive resistance occurs...