Weinhold, Kent J.

Abstract 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 caused an overall increase in activated CD8 + T cells, CD56 low NK cells, and T cell receptor diversity, while diminishing matrix-remodeling stromal cells and sarcoma cells. 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.

Background Our study was designed to determine the safety, efficacy, and immunological effects of perioperative pembrolizumab in early-stage NSCLC. Methods This is a single-arm phase II study of perioperative pembrolizumab in patients with untreated, clinical stage IB to IIIA NSCLC. Patients received two doses of 200 mg pembrolizumab, surgery, standard adjuvant chemotherapy, followed by four doses adjuvant pembrolizumab. The primary objective of this study was to determine surgical feasibility rate, and secondary objectives are pathological response rate, treatment adverse events, efficacy data, and exploratory analysis of biomarkers. Results 30 patients initiated perioperative pembrolizumab, and 25 completed tumor resection. At median follow-up of 59 months after surgical resection, seven patients had disease progression, while six had died representing. A 5-year progression-free survival (PFS) from time of surgery was 72.0% (56.4%–91.9%) and overall survival (OS) from time of surgery was 75.8% (60.7%–94.7%). Major pathological response (MPR) was found in seven tumors (28%) including two complete responses (4%). Across all treated patients, four receiving neoadjuvant and four receiving adjuvant pembrolizumab experienced treatment-related adverse events of grade 3 or higher with no grade 5 events. Plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) levels increased across our patient cohort over time from baseline until postsurgery and remained elevated at the end of treatment. There was a significant difference between mean plasma PCSK9 levels for patients with MPR versus all other patients on study when checked postoperatively. Conclusions Perioperative pembrolizumab was safe and effective with promising MPR rate, PFS, and OS.

3533 Background: The BACCI trial (NCT02873195) randomized 133 mCRC pts to C+B+A (investigational) or C+B+placebo. Longer progression free survival (PFS) was observed in the investigational vs placebo arm. To investigate T-cell functional heterogeneity, we performed single-cell proteomic analyses on pts with either long or short PFS and evaluated T-cell function as a prognostic and predictive biomarker. Methods: Isolated CD8+ and CD4+ cells were analyzed at baseline and first restaging from pts with long ( > 6 mo, N = 14) or short ( < 2 mo, N = 13) PFS. Single-cell, functional proteomic analyses were conducted using the IsoLight system (Isoplexis), which assessed secretion of 32 cytokines from an average of 786 cells per sample. The cumulative readout was polyfunctional strength index (PSI), a composite score of frequency and magnitude of secreted cytokines. Cytokines are organized into effector (E), stimulatory, chemoattractive (CA), regulatory, and inflammatory (I) clusters. Differential expression analyses were done using Wilcoxon rank-sum tests. Potential predictive effects were assessed using logistic regressions with interaction between treatment and biomarkers. No multiple comparison control was done due to the exploratory nature of analyses. Results: Baseline CD8+ and CD4+ cells were stimulated ex vivo and single-cell cytokine analyses performed. Prognostic analyses indicated that CD8+ cells from pts with long PFS had ~3-fold higher PSI compared to pts with short PFS (p = 0.005). Further analysis identified that the E (p = 0.005) and CA clusters (p = 0.005) most impacted PSI, significant individual cytokines are listed in the table. Baseline CD8+ cell function was not predictive of outcome. Baseline CD4+ cell function was not prognostic or predictive of outcome. Analysis of CD8+ cells after treatment identified that changes in CA cluster was prognostic of long PFS (p = 0.024). Cytokine changes from CD4+ cells were not prognostic; however, early changes in the CD4+ CA cluster were predictive of outcome (p-int = 0.055). Similarly, early changes in the CD8+ I cluster were predictive of outcome (p-int = 0.073). Conclusions: These results reveal pts with long PFS had more functional CD8+ T-cells with a higher PSI than pts with short PFS. Predictive analyses suggested that early changes in the CD4+ CA and CD8+ I clusters were associated with benefit from immunotherapy. These preliminary results highlight the importance of single-cell functional proteomic analyses. [Table: see text]

Abstract Purpose: In this prospective trial, we sought to assess the feasibility of concurrent administration of ipilimumab and radiation as adjuvant, neoadjuvant, or definitive therapy in patients with regionally advanced melanoma. Patients and Methods: Twenty-four patients in two cohorts were enrolled and received ipilimumab at 3 mg/kg every 3 weeks for four doses in conjunction with radiation; median dose was 4,000 cGy (interquartile range, 3,550–4,800 cGy). Patients in cohort 1 were treated adjuvantly; patients in cohort 2 were treated either neoadjuvantly or as definitive therapy. Results: Adverse event profiles were consistent with those previously reported with checkpoint inhibition and radiation. For the neoadjuvant/definitive cohort, the objective response rate was 64% (80% confidence interval, 40%–83%), with 4 of 10 evaluable patients achieving a radiographic complete response. An additional 3 patients in this cohort had a partial response and went on to surgical resection. With 2 years of follow-up, the 6-, 12-, and 24-month relapse-free survival for the adjuvant cohort was 85%, 69%, and 62%, respectively. At 2 years, all patients in the neoadjuvant/definitive cohort and 10/13 patients in the adjuvant cohort were still alive. Correlative studies suggested that response in some patients were associated with specific CD4+ T-cell subsets. Conclusions: Overall, concurrent administration of ipilimumab and radiation was feasible, and resulted in a high response rate, converting some patients with unresectable disease into surgical candidates. Additional studies to investigate the combination of radiation and checkpoint inhibitor therapy are warranted.