Adaptive Immune Gene Signatures Correlate with Response to Flotetuzumab, a CD123 × CD3 Bispecific Dart® Molecule, in Patients with Relapsed/Refractory Acute Myeloid Leukemia

Abstract

Background. The therapeutic approach in patients (pts) with acute myeloid leukemia (AML) has not changed substantially in >30 years. The introduction of new treatment strategies, including immunotherapy, remains a priority. Flotetuzumab, a CD123 × CD3 bispecific DART immunotherapy, is being tested in a phase 1/2 study of relapsed/refractory (R/R) AML. We previously showed that AML pts with an immune-enriched and IFN-γ-dominant tumor microenvironment (TME) experience significantly shorter relapse-free survival, suggesting refractoriness to standard induction chemotherapy (Vadakekolathu J, et al. Blood 2017; 130: 3942A). Herein, we report that an IFN-γ-dominant TME, while predicting resistance to standard therapy, is favoring response of AML to flotetuzumab. Methods. Gene expression was analyzed in 78 bone marrow (BM) samples (36 at baseline, 27 post-cycle 1 and 15 post-cycle 2) from 40 pts with relapsed or refractory AML enrolled in a phase 1/2 clinical trial of flotetuzumab (NCT#02152956). Thirty-six baseline BM samples were included in the analysis, of which 34 from pts who were treated at the target dose of ≥500 ng/kg/day. The NanoString PanCancer IO360™ assay was used to assess the expression of 770 genes, including the levels of 14 immune cell types and of 32 immuno-oncology signatures, and their correlation with response to flotetuzumab. Data are presented as score means per group±SEM and analyzed by unpaired t-test. Results. Gene expression analysis of BM samples at baseline stratifies flotetuzumab-treated pts into 3 clusters within an immunological continuum: immune-depleted, immune-exhausted and immune-enriched (Fig. 1A). Pts with primary-refractory disease (refractory to ≥2 induction attempts, first CR of <6 months, or failure after ≥4 cycles of hypomethylating agents, HMA) showed prevalently an immune-infiltrated TME phenotype, which included higher inflammatory chemokine scores compared with relapse pts (3.27±0.22 vs 2.46±0.07, p=0.026). Within this group, chemotherapy-refractory and HMA-refractory pts further stratify into immune-enriched and immune-exhausted phenotypes, respectively. Specifically, HMA-refractory pts displayed features of immune exhaustion and adaptive immune resistance, including upregulation of TIGIT (5.55±0.34 vs 3.85±0.24, p=0.006), PD-L1 (3.55±0.18 vs 2.4±0.29, p=0.009) and Treg cells (4.87±0.23 vs 3.69±0.19, p=0.0009) together with a trend toward increasingly exhausted CD8+ T cells (CD244, EOMES, LAG3 and PTGER4) compared to chemotherapy-refractory pts (Fig. 1B). Responders to flotetuzumab showed a significantly higher IFN-γ signaling scores at baseline compared to non-responders (3.31±0.32 vs 2.27±0.11, p=0.0005), consistent with the greater frequency of responders in primary refractory pts compared to relapse pts. Accordingly, baseline IFN-γ signaling scores may be predictive of response to flotetuzumab therapy (AUC=0.841; Fig. 1C). Furthermore, comparison of post-cycle 1 BM samples to baseline samples showed treatment with flotetuzumab lead to increased immune cell infiltrate and immune activation scores, as reflected by a higher Tumor Inflammation Signature (Ayers M, et al. J Clin Invest 2017; 127: 2930-40) (6.49±0.20 vs 5.93±0.12, p=0.015) together with enhanced immunoproteasome (5.72±0.07 vs 5.23±0.10, p=0.0002) and IFN-γ signaling (3.38±0.23 vs 2.53±0.14, p=0.0015) scores. Flotetuzumab-induced TME gene activation was therefore reminiscent of an immune-enrichment rather than immune-exhaustion signature. Conclusions. We provide evidence for a range of immune gene expression profiles in AML, with primary refractory pts displaying an enhanced immune infiltration signature compared with relapse pts. Furthermore, an IFN-γ-related gene signature at baseline, a feature of primary refractory pts, was associated with clinical response to flotetuzumab. HMA-refractory pts showed an immune-rich but exhausted phenotype with PD-L1 expression, suggesting these pts may further benefit from flotetuzumab combination therapy with checkpoint inhibition (Rettig M, et al. Blood 2017; 130: 1365). Lastly, treatment with flotetuzumab further shifted the immune signature toward a more immune rich phenotype. The AML TME immune gene expression can influence susceptibility to therapy, with primary refractory AML showing an IFN-γ-dominant signature associated with response to flotetuzumab. Figure Figure. Disclosures Rutella: NanoString Technologies: Research Funding. Church:NanoString Technologies: Employment. Viboch:NanoString Technologies: Employment. Sullivan:NanoString Technologies: Employment. Hood:NanoString Technologies: Employment. Warren:NanoString Technologies: Employment. Cesano:NanoString Technologies: Employment. La Motte-Mohs:MacroGenics: Employment, Equity Ownership. Muth:MacroGenics: Employment. Lelièvre:Servier: Employment. Lowenberg:Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; international Scientific Advisory Board, Institute Gustave Roussy, Paris: Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity's Board of Directors or advisory committees; Editorial Board "European Oncology & Haematology": Membership on an entity's Board of Directors or advisory committees; Editorial Board "The Netherlands Journal of Medicine": Membership on an entity's Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherlands: Membership on an entity's Board of Directors or advisory committees; Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands): Membership on an entity's Board of Directors or advisory committees; Editorial Board "International Journal of Hematology": Membership on an entity's Board of Directors or advisory committees; "Up-to-Date", section editor leukemia: Membership on an entity's Board of Directors or advisory committees; Astex: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees. Davidson-Moncada:MacroGenics: Employment.

B7-Positive and B7-Negative Acute Myeloid Leukemias Display Distinct T Cell Maturation Profiles, Immune Checkpoint Receptor Expression, and European Leukemia Net Risk Profiles

“…The "inflamed/non-inflamed" AML dichotomy might explain why patients with identical AML entities and risk profiles may have different outcomes that deviate from the initial ELN prognostic prediction (42). Recent research has demonstrated that an "inflamed" AML immune profile can predict the resistance to cytotoxic therapy but also the patient's responsiveness to immunotherapies such as ICBs or DART (9,29,43). However, future research is necessary to investigate how B7 immune profiling can complement the predictive ability of the ELN risk classification and guide immunotherapy.…”

Immune landscapes predict chemotherapy resistance and immunotherapy response in acute myeloid leukemia

“…Using large cohorts of subjects, the current study is the first to reveal underlying transcriptomic features that stratify the TME of AML into immune subtypes and may assist therapeutic predictions by defining patients who will potentially derive the greatest benefit from immunotherapies 11,46 . We identified two subtypes of differentially-infiltrated tumors, an observation that was validated in independent childhood and adult AML series, reinforcing the notion that unique molecular features can distinguish AML across age groups 33…”

“…T-cell markers and cytolytic effectors (CD8A, CD8B, GZMB, PRF1), counter-regulatory immune checkpoints and immunotherapy drug targets (IDO1, CTLA4, PD-L1 and BTLA), and molecules involved in antigen processing and presentation (TAP1, TAP2, HLA-A, HLA-B and HLA-C). Conceivably, high T-cell infiltration, MHC expression and PD-L1 levels in the immune-infiltrated AML subtype reflected a pre-existing IFN-γ-driven adaptive immune response which has previously been associated with suppressed anti-tumor immune reactivity 13,26 , but also with immunotherapy responses in patients with solid tumors 9,18,27 and AML 11 . The expression of STAT1, a central component of the IFN-γ signaling pathway and predictor of response to immune checkpoint blockade 28 , was more strongly correlated with the presence of T-cell inhibitory receptors TNFRSF14 (a ligand for the immunoglobulin superfamily members BTLA and CD160), PD-L1, HAVCR2 (Tim-3) and LAG-3, and with IFN-stimulated genes MX1, IFIT1 and IRF1 in the immune-infiltrated relative to the immunedepleted subtype, consistent with their coordinated regulation in an inflamed TME (Fig.…”

“…In solid tumors, six immune subtypes have been described (wound healing, interferon (IFN)-γ-dominant, inflammatory, lymphocyte-depleted, immunologically quiet, and transforming growth factor (TGF)-β-dominant). These are characterized by differences in macrophage or lymphocyte signatures, T helper type (Th)-1 to Th2 cell ratio, extent of intratumoral heterogeneity and neoantigen load, aneuploidy, cell proliferation, expression of immunomodulatory genes, and patient survival 6 .Although immunotherapy may be an attractive modality to exploit in patients with AML 7 , the ability to predict the groups of patients and the forms of leukemia that will respond to immune targeting remains limited [8][9][10][11] . Clinical studies in patients with solid tumors have shown that responses to anti-programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1)-targeted immunotherapy occur most often in individuals with immune-inflamed lesions that are characterized by pre-existing CD8 + T-cell responses, release of proinflammatory and effector cytokines 12-14 , and an augmented T-cell receptor (TCR) clonal diversity pre-treatment 15,16 .…”

“…Although immunotherapy may be an attractive modality to exploit in patients with AML 7 , the ability to predict the groups of patients and the forms of leukemia that will respond to immune targeting remains limited [8][9][10][11] . Clinical studies in patients with solid tumors have shown that responses to anti-programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1)-targeted immunotherapy occur most often in individuals with immune-inflamed lesions that are characterized by pre-existing CD8 + T-cell responses, release of proinflammatory and effector cytokines 12-14 , and an augmented T-cell receptor (TCR) clonal diversity pre-treatment 15,16 .…”