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

Abstract

PurposeEwing 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. Experimental DesignWe 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. ResultsPembrolizumab 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-{beta} response, low interferon (IFN) response, and low HLA class I expression. IFN response, HLA class I expression, and overall immune infiltration were correlated. ConclusionsAnalyses of the tumor microenvironment from Ewing sarcoma biopsies 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. Statement of translational relevanceSingle agent checkpoint inhibitors have demonstrated limited clinical benefit in bone sarcomas, including Ewing sarcoma (EwS). While in vitro and limited patient tumor analyses have suggested mechanisms of immunotherapy resistance in EwS, tumor biopsies following checkpoint inhibitor administration have not been examined. SARC028 was a phase II study investigating blockade of programmed cell death-1 (PD-1) in patients with relapsed/refractory sarcomas, including EwS. Tumor biopsies were collected prior to and 8 weeks after anti-PD1 therapy. Here, we leverage a unique opportunity to understand EwS immunotherapy resistance through analysis of paired tumor samples from SARC028. Using a multi-omics approach, we identify features of EwS that contribute to primary resistance to anti-PD1 including TGF-{beta}, reduced interferon signaling and low HLA class I expression. This study provides an improved understanding of tumor characteristics driving poor response to immunotherapy in relapsed EwS and will guide the development of future trials to modulate this immunosuppressive axis.