Abstract
Abstract
Background: The immune status of patients with acute myeloid leukemia (AML) is not fully investigated and predictive biomarkers for immunotherapies are needed. Blockade of PD-1/PD L1 pathways in T cell subsets can exert anti-leukemia responses. We here investigated, at the single T cell level, functionality of bone marrow (BM) T cells in AML at diagnosis and relapse, and in patients responding/not responding to PD1 inhibition.
Methods: Pre-therapy BM aspirates were collected from newly diagnosed and relapsed AML patients and from relapsed patients treated with azacitidine/nivolumab (NCT02397720). CD4 and CD8 T cells were stimulated with anti-CD3/CD28 and loaded into IsoCode chip containing ~12,000 micro-chambers with a 32-plex antibody array. Protein secretion from ~1000 single cells was analyzed by fluorescence ELISA-based assay; polyfunctional profile was analyzed by IsoSpeak (IsoPlexis).
Results: We demonstrate that BM T cells from new AML (n=13) exhibit polyfunctionality and polyfunctional strength index (PSI) indistinguishable from that of normal control lymphocytes. However, newly diagnosed, untreated patients with AML carrying TP53 mutations show a pronounced decrease in PSI (wt p53: 73.5 ± 27.6, vs. mt p53: 6.6 ± 2.8, p=0.013) and polyfunctionality (p = 0.0476). The difference was observed in both CD4 and CD8 T cell subsets, respectively (CD4: 90.5 ± 44.5 vs. 9.0 ± 5.3; CD8: 56.7 ± 35.5 vs. 4.2 ± 2.0). Relapsed AML (n = 13) also showed significantly impaired polyfunctionality (p < 0.05). Patients with relapsed or p53 mutant AML exhibited decrease in protein secretions including Granzyme B, IFN-γ, MIP-1α, MIP-1β, perforin, TNF-α and sCD137 indicating a crucial role of IFN-γ. The pre-treatment PSI of CD4 T cells significantly segregated patients with AML who responded (CR/CRi) to azacitidine and nivolumab from non-responding patients. (p=0.0002). All responding and none of the non-responding patients had PSI>10, Overall survival of patients with </> 10 was significant at p= 0.0018. The markedly enhanced PSI of BM CD4 T cells in the responders was primarily composed of increased granzyme B, IFN-γ, IL-5, TNF-α, IL-8, and IL-10 proteins compared to non-responders.
Conclusions: Single-cell multiplexed proteomics precision profiling identifies a polyfunctional downregulation in BM T cells in patients with relapsed AML compared to newly-diagnosed patients, and an equally pronounced functional defect in newly diagnosed patients with TP53 mutations, while newly diagnosed AML patients with wtTP53 display normal polyfunctionality. The pre-therapy PSI was a strong predictor of response and OS in relapsed patients treated with Nivolumab/azacitidine and could be used to select patients in prospective clinical trials.
Citation Format: Naval Daver, Hagop Kantarjian, Sean Mackay, Brianna Flynn, Sreyashi Basu, Guillermo Garcia-Manero, Mansour Alfayez, Marina Konopleva, Jairo Matthews, Steven Kornblau, Ellias Jabbour, Jing Zhou, Michael Andreeff. Polyfunctionality determined by single-cell proteomics of bone marrow-derived CD4 T cells from patients with acute myeloid leukemia identifies patients responding to anti-PD-1-based therapy and discovers profound T cell defect in mutant TP53 disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-222.