SARC028 samples reveal an interplay between TGFβ, interferon signaling and low HLA class I expression as contributors to Ewing sarcoma checkpoint blockade resistance.

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 pre- and post-pembrolizumab (anti-PD1) therapy from the phase 2 clinical trial SARC028 to interrogate the Ewing tumor microenvironment and features associated with resistance to checkpoint inhibition. We utilize multiplexed immunofluorescence, spatial proteomics and spatial transcriptomics to analyze paired pre- and 8 weeks post- treatment biopsy specimens from patients with Ewing sarcoma enrolled on SARC028. 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 ten cellular neighborhoods (CN) across patients consisting of specific cell subsets. CN10 was consistently observed across patients with poor response. This cellular neighborhood was enriched for a tumor subpopulation with high TGF-β response, low interferon (IFN) response, and low HLA class I expression. IFN response, HLA class I expression, and overall immune infiltration were correlated. Analyses of paired Ewing sarcoma tumor samples from SARC028 reveals 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.