Single-Cell Mass Cytometry Identifies Mechanisms of Resistance to Immunotherapy in AML

Background: The mechanisms of resistance to immunotherapies in patients with acute myeloid leukemia (AML) are not well characterized and biomarkers for improved immunotherapeutic strategies are critical. Multiple phase II/III clinical trials combining hypomethylating agents (HMA), azacitidine (AZA) and decitabine, with immune checkpoint inhibitors are now underway for patients with newly diagnosed and relapsed/refractory (R/R) AML and MDS based on data suggesting that 1) hypomethylating agents increase tumor expression of PD-L1 and PD-1 in myeloid malignancies(Yang H et al. Leukemia 2014); 2) blocking the PD-1/PD-L1 signaling axis has profound anti-leukemic response (Zhou et al. Blood 2011); and 3) clinical responses to Azacitidine/PD1 inhibitor (nivolumab) in relapsed AML (Daver et al., Cancer Discovery). Here, we characterize the baseline immune landscape and potential mechanisms of resistance using single-cell mass cytometry (CyTOF) profiling of serially collected samples from R/R AML patients undergoing therapy with AZA and PD-L1 inhibitor avelumab [NCT02953561]. Methods: Bone marrow (BM) and peripheral blood (PB) samples were collected from 9 patients prior to treatment with AZA and avelumab and after cycles 1, 3, and 5 (as available). To interrogate immune profiling in these samples, we have developed and optimized a novel CyTOF antibody panel that includes immunophenotypic markers to distinguish AML stem cells and blasts from adjacent immune cell subsets (T, B, NK cells, and monocytes), as well as known checkpoint ligands and receptors. Data was analyzed using the uniform manifold approximation and projection (UMAP) algorithm (McInnes et al. arXiv 2018) implemented through Cytofkit (Chen et al. PLoS Comput Biol 2016). Results: We first used the multi-parameter immunophenotyping to characterize the immune microenvironment in normal and leukemic BM prior to therapy. At baseline, the composition of AML BM CD4 and CD8 T cells contained a significantly smaller fraction of naive T-cells when compared to healthy bone marrow controls (both p Conclusions: Single-cell characterization of R/R AML reveals that the immune landscape of these patients at baseline is significantly altered when compared to healthy bone marrow. The ratio of CD:/CD8 and composition of residual T cells appear to be the most important predictors of response to HMA/PD-L1 inhibition. Finally, AML cells express a variety of other immune checkpoints, particularly PD-L2 but also OX40 and TIM3, that should be considered for future combination therapy. Download : Download high-res image (283KB) Download : Download full-size image Disclosures Short: AstraZeneca: Consultancy; Amgen: Honoraria; Takeda Oncology: Consultancy, Research Funding. Konopleva: Astra Zeneca: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Genentech: Honoraria, Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Calithera: Research Funding; Agios: Research Funding.