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507 High Dimensional Flow Cytometry Analysis in Newly Diagnosed Acute J Immunother Cancer: first published as 10.1136/jitc-2020-SITC2020.0507 on 9 November 2020. Downloaded from Abstracts LAG-3, and CSF1R at the PM interface were associated with made R code, we performed dimensionality reduction, cluster- worse progression-free survival (PFS), while gene sets associ- ing, and pseudotime analysis. ated with productive T cell immune response were associated Results The IR-score discriminated NR and CR (p = 3e-02, with improved PFS (figure 4). AUC 0.84) after treatment with CD57 and KLRG1 accounting Conclusions In contrast to primary bone osteosarcoma for most of this difference (p = 2e-02, AUC = 0.79). Next ‘immune deserts,’ osteosarcoma PMs represent an ‘immune- we investigated CD8+ T cell populations that best correlated excluded’ TME where immune cells are present but are halted with response to chemotherapy. FlowSOM revealed seven at the PM interface. TILs can produce effector cytokines, sug- major clusters: naive and naive-like, CD28+KLRG1+ acti- gesting their capability of activation and recognition of tumor vated-effector, CD28+KLRG1+PD1+ dysfunctional, PD1 antigens. Our findings suggest cooperative immunosuppressive +CD57+ senescent effector-memory and two clusters of ter- mechanisms in osteosarcoma PMs that prevent TILs from pen- minally differentiated CD45RA+KLRG1+ cells. Since the acti- etrating into the PM interior, including immune checkpoint vation and differentiation states accounted for most of the molecule expression and the presence of immunosuppressive subpopulation variability, we grouped the clusters into resting myeloid cells. We identify cellular and molecular signatures (naive, naive-like), activated (activated-effector, dysfunctional), that are associated with PFS of patients, which could be and terminally differentiated cells (senescent effector-memory, potentially manipulated for successful immunotherapy. terminally differentiated). UMAP, developmental trajectories Ethics Approval This study was approved by Johns Hopkins and differential abundance testing showed increased frequency University’s Ethics Board, approval number FWA00005752. of activated cells at diagnosis (p-adj = 2.9e-05) and of resting cells after treatment (p-adj = 1.3e-02) in CR, while terminally REFERENCES differentiated T cells prevailed in NR (p-adj = 5.3e-08) after 1. Mirabello L, Troisi RJ, Savage SA. Osteosarcoma incidence and survival rates from treatment (figures 1 and 2). 1973 to 2004: Data from the surveillance, epidemiology, and end results pro- gram. Cancer 2009;115(7):1531–43. Conclusions The increased number of functional activated T 2. Tawbi HA, Burgess M, Bolejack V, Van Tine BA, Schuetze SM, Hu J, et al. Pem- cells at diagnosis and the persistence of a naive/naive-like res- brolizumab in advanced soft-tissue sarcoma and bone sarcoma (SARC028): A ervoir at the time of response is a signature associated with multicentre, two-cohort, single-arm, open-label, phase 2 trial. Lancet Oncol achievement of CR. Lack of response (NR) correlates with 2017;18(11):1493–501. 3. Davis KL, Fox E, Merchant MS, Reid JM, Kudgus RA, Liu X, et al. Nivolumab in accumulation of the terminally differentiated and senescent children and young adults with relapsed or refractory solid tumours or lymphoma cells in the bone marrow. These results uncover an inter- (ADVL1412): A multicentre, open-label, single-arm, phase 1–2 trial. Lancet Oncol twined relationship between skewing of T cell differentiation – 2020;21(4):541 50. and clinical response to chemotherapy. The data provide 4. D’Angelo SP, Mahoney MR, Van Tine BA, Atkins J, Milhem MM, Jahagirdar BN, et al. Nivolumab with or without ipilimumab treatment for metastatic sarcoma rationale to either remove senescent or augment activity of (alliance A091401): Two open-label, non-comparative, randomised, phase 2 trials. Lancet Oncol 2018;19(3):416–26. 5. Paoluzzi L, Cacavio A, Ghesani M, Karambelkar A, Rapkiewicz A, Weber J, et al. Response to anti-PD1 therapy with nivolumab in metastatic sarcomas. Clin Sar- coma Res 2016;6:24. http://dx.doi.org/10.1136/jitc-2020-SITC2020.0506 507 HIGH DIMENSIONAL FLOW CYTOMETRY ANALYSIS IN http://jitc.bmj.com/ NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA PREDICTS PATIENTS OUTCOMES 1Francesco Mazziotta*, 1Rupkatha Mukhopadhyay, 2Hanna A Knaus, 1Anish Chowdhury, 1Amanda Blackford, 1Ivana Gojo, 1Leo Luznik. 1Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA; 2Medical University of Vienna, Abstract 507 Figure 1 UMAP embedding of T cells in CR, NR, at Vienna, Austria on October 1, 2021 by guest. Protected copyright. diagnosis (BM_DG) and after chemotherapy (BM_post), HC colored by T Background We have previously characterized phenotypic and cell state (resting, activated, terminal differentiated), overlaid with a contour plot transcriptional profile of CD8+ T cells in acute myeloid leu- kemia (AML) and their differences between responders vs. nonresponders to chemotherapy.1 Goal of ongoing work was to further probe uniqueness of AML in sculpting CD8+ T cell responses and the plasticity of their signatures upon che- motherapy response. Methods We first examined the cumulative expression of mul- tiple inhibitory receptors (IRs) (detected by 2 different panels) on CD8+ T cells and created an IR-score which summarizes the relative amount of PD-1, Tim3, KLRG1, 2B4, CD160, CD57, and BTLA-positive CD8+ T-cells in relation to the well-characterized maturation states of CD8+ T cells. Serial bone marrow samples from 33 newly diagnosed AML patients Abstract 507 Figure 2 Boxplots showing the differential cluster with well-annotated clinical data (21 complete responders abundance and adjusted p-values for CR, NR, at diagnosis (BM_DG) (CR) and 12 nonresponders (NR) to chemotherapy) and 11 and after chemotherapy (BM_post), HC in the three different T cell healthy controls (HC) were analyzed. FInally, using custom states (resting, activated, terminal differentiated) A312 J Immunother Cancer 2020;8(Suppl 3):A1–A559 J Immunother Cancer: first published as 10.1136/jitc-2020-SITC2020.0507 on 9 November 2020. Downloaded from Abstracts naïve/naïve-like T cells as a strategy to reinforce antileukemia immunoreactivity and sensitivity to immune checkpoint blockade. Science 2016 – immunity. Mar 25;351(6280):1463 9. http://dx.doi.org/10.1136/jitc-2020-SITC2020.0508 REFERENCE 1.. Knaus HA, Berglund S, Hackl H, et al. Signatures of CD8+ T cell dysfunction in AML patients and their reversibility with response to chemotherapy. JCI Insight 2018; 3(21). 509 POTENT AND SELECTIVE INHIBITION OF AXL RECEPTOR TYROSINE KINASE FOR THE TREATMENT OF CANCER http://dx.doi.org/10.1136/jitc-2020-SITC2020.0507 Susan Paprcka*, Akshata Udyavar, Subhasree Sridhar, Dillon Miles, Yu Chen, Sean Cho, Corinne Foley, Rebecca Grange, Manmohan Leleti, Sharon Zhao, Lixia Jin, Stephen Young, Jay Powers, Matthew Walters. Arcus Biosciences, Hayward, CA, USA 508 DIFFERENT NEOANTIGEN EXPRESSION PATTERNS IMPACT THE STRENGTH OF ANTI-TUMOR IMMUNE Background AXL receptor tyrosine kinase (AXL) is a trans- RESPONSES membrane protein that is over-expressed in a variety of cancer Kim Nguyen*, Stefani Spranger, Christopher Copeland. Massachusetts Institute of and immune cells. AXL signaling has been implicated in creat- Technology, Cambridge, USA ing an immunosuppressive tumor microenvironment (TME) through both tumor-intrinsic and immunomodulatory mecha- Background Many cancer immunotherapies depend on the nisms1,2,3,4,5 promoting resistance to various therapies.6,7,8,9 ability of cytotoxic CD8+ T cells to recognize neoantigens on Methods Compound inhibition potency against the kinase MHCI complexes to effectively eliminate tumor cells. How- activity of AXL and other kinases was determined by detecting ever, patient response following immunotherapy is highly vari- phosphorylated substrate using homogeneous time-resolved flu- able, with recent work suggesting that neoantigen expression orescence (HTRF). Binding affinity of inhibitor to intracellular patterns can impair patient response. Specifically, it was AXL kinase was determined by monitoring displacement of a observed that the immune response is dampened when neoan- competitive fluorescent tracer using an AXL NanoBRET assay. tigens are expressed only by a subset of tumor cells (heteroge- Recombinant Gas6, cancer cell lines, whole blood or isolated neous expression).1 To study why anti-tumor immunity is cells from healthy donors were used to determine the reduc- reduced in a heterogeneous setting we developed a transplant tion in AXL-mediated signaling in-vitro. PK/PD and anti-tumor murine tumor model engineered to express neoantigens in a effects of selected AXL inhibitors were evaluated in murine heterogeneous pattern or homogenously. models. Methods A curated list of neoantigens with varying predicted Results AXL is highly expressed on a subset of immune cells, MCHI binding affinities was used to established an array of including DC’s, NK cells and M2 macrophages as well as cell lines expressing at one to three neoantigens. The lines fibroblasts, which contribute to a blunted anti-tumor response. were inoculated subcutaneously in immunocompetent mice as Consistent with these observations, AXL is strongly associated mixtures (heterogenous) or as a single line (homogenous) to with increased infiltration of macrophages, exhausted NK and study the resulting immune response. Tumors were harvested T-cells, as well as significantly increased CD73 expression in at days 7, 10 and 14 and flow cytometry analysis was used to multiple
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