SARC028 Samples Reveal an Interplay between TGF-β, IFN Signaling, and Low HLA Class I Expression as Contributors to Ewing Sarcoma Checkpoint Blockade Resistance.

Purpose: Ewing sarcoma, in contrast to some adult sarcoma subtypes, generally does not respond to single-agent immunotherapy targeting PD1. The features of Ewing sarcoma that preclude the effectiveness of immunotherapy remain largely unknown. To address this question, we utilized biopsies from patients with Ewing sarcoma obtained before and after pembrolizumab (anti-PD1) therapy from the phase II clinical trial SARC028 to interrogate the Ewing tumor microenvironment and features associated with resistance to checkpoint inhibition.

Experimental design: We utilized multiplexed immunofluorescence, spatial proteomics, and spatial transcriptomics to analyze paired pretreatment and 8-week posttreatment biopsy specimens from patients with Ewing sarcoma enrolled in SARC028.

Results: Pembrolizumab therapy did not alter the quantity of immune cell infiltration in Ewing tumor biopsies. Analysis of tumor-associated protein markers revealed increased immunoregulatory markers after pembrolizumab. Spatial transcriptomics identified 10 cellular neighborhoods (CN) across patients consisting of specific cell subsets. CN10 was consistently observed across patients with a poor response. This CN was enriched for a tumor subpopulation with a high TGF-β response, low IFN response, and low HLA class I expression. IFN response, HLA class I expression, and overall immune infiltration were correlated.

Conclusions: Analyses of paired Ewing sarcoma tumor samples from SARC028 reveal an immunosuppressive triad, the disruption of which should be pursued to improve antitumor immunity. This work highlights the unique insight that can be gained from the analysis of paired patient Ewing sarcoma tumor biopsy samples from clinical trials.