Distinct Sarcoma Microenvironments Predict Benefit from Addition of Pembrolizumab to Preoperative Radiotherapy and Surgery in SU2C-SARC032

The addition of pembrolizumab to preoperative radiotherapy (RT) improved disease-free survival (DFS) for patients with stage III undifferentiated pleomorphic sarcoma (UPS) and dedifferentiated/pleomorphic liposarcoma (LPS) in the randomized SU2C-SARC032 trial. To precisely identify patients who benefit from pembrolizumab and RT, we performed comprehensive multi-omics profiling of pre- and post-treatment tumor and blood samples, including bulk RNA-seq, flow cytometry, and cytometry by time of flight. Additionally, we built a single-cell RNA-seq atlas spanning 65,786 cells from UPS and LPS to recover single-cell states in bulk tumor samples using digital cytometry. Two opposing tumor microenvironments (TMEs), immune-cold sarcoma ecotype 1 (SE1) and immune-hot sarcoma immune class E (SIC E), benefited from pembrolizumab. Pembrolizumab combined with RT depleted PD-1+ exhausted T cells in SIC E sarcomas and increased effector memory CD4+ T cells in SE1 sarcomas with an overall increase in CD8+ early activated T cells, CD4+ follicular helper T cells, and T cell receptor diversity. Matrix-remodeling stromal and epithelial-like sarcoma cell programs were associated with worse outcomes and diminished with pembrolizumab and RT. Our findings identify different mechanisms of response to pembrolizumab in localized, high-risk UPS/LPS and suggest that sarcoma TME signatures may identify patients most likely to benefit from adding pembrolizumab to preoperative RT.