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Inadequate molecular and clinical stratification of the patients with high-risk diffuse large B-cell lymphoma (DLBCL) is a clinical challenge hampering the establishment of personalized therapeutic options. We studied the translational significance of liquid biopsy in a uniformly treated trial cohort. Pretreatment circulating tumor DNA (ctDNA) revealed hidden clinical and biological heterogeneity, and high ctDNA burden determined increased risk of relapse and death independently of conventional risk factors. Genomic dissection of pretreatment ctDNA revealed translationally relevant phenotypic, molecular, and prognostic information that extended beyond diagnostic tissue biopsies. During therapy, chemorefractory lymphomas exhibited diverging ctDNA kinetics, whereas end-of-therapy negativity for minimal residual disease (MRD) characterized cured patients and resolved clinical enigmas, including false residual PET positivity. Furthermore, we discovered fragmentation disparities in the cell-free DNA that characterize lymphoma-derived ctDNA and, as a proof-of-concept for their clinical application, used machine learning to show that end-of-therapy fragmentation patterns predict outcome. Altogether, we have discovered novel molecular determinants in the liquid biopsy that can noninvasively guide treatment decisions.

Antibodies that bind CD47 on tumor cells and prevent interaction with SIRPa on phagocytes are active against multiple cancer types including T-cell lymphoma (TCL). Here we demonstrate that surface CD47 is heterogeneously expressed across primary TCLs, whereas major histocompatibility complex (MHC) class I, which can also suppress phagocytosis, is ubiquitous. Multiple monoclonal antibodies (mAbs) that block CD47-SIRPa interaction promoted phagocytosis of TCL cells, which was enhanced by cotreatment with antibodies targeting MHC class I. Expression levels of surface CD47 and genes that modulate CD47 pyroglutamation did not correlate with the extent of phagocytosis induced by CD47 blockade in TCL lines. In vivo treatment of multiple human TCL patient-derived xenografts or an immunocompetent murine TCL model with a short course of anti-CD47 mAb markedly reduced lymphoma burden and extended survival. Depletion of macrophages reduced efficacy in vivo, whereas depletion of neutrophils had no effect. F(ab')2-only fragments of anti-CD47 antibodies failed to induce phagocytosis by human macrophages, indicating a requirement for Fc-Fc? receptor interactions. In contrast, F(ab')2-only fragments increased phagocytosis by murine macrophages independent of SLAMF7-Mac-1 interaction. Full-length anti-CD47 mAbs also induced phagocytosis by Fc? receptor-deficient murine macrophages. An immunoglobulin G1 anti-CD47 mAb induced phagocytosis and natural killer cell-mediated cytotoxicity of TCL cells that was augmented by cotreatment with mogamulizumab, an anti-CCR4 mAb, or a mAb blocking MHC class I. These studies help explain the disparate activity of monotherapy with agents that block CD47 in murine models compared with patients. They also have direct translational implications for the deployment of anti-CD47 mAbs alone or in combination.

Antibodies that bind CD47 on tumor cells and prevent interaction with SIRPα on phagocytes are active against multiple cancer types including T-cell lymphoma (TCL). Here we demonstrate that surface CD47 is heterogeneously expressed across primary TCLs, whereas major histocompatibility complex (MHC) class I, which can also suppress phagocytosis, is ubiquitous. Multiple monoclonal antibodies (mAbs) that block CD47-SIRPα interaction promoted phagocytosis of TCL cells, which was enhanced by cotreatment with antibodies targeting MHC class I. Expression levels of surface CD47 and genes that modulate CD47 pyroglutamation did not correlate with the extent of phagocytosis induced by CD47 blockade in TCL lines. In vivo treatment of multiple human TCL patient-derived xenografts or an immunocompetent murine TCL model with a short course of anti-CD47 mAb markedly reduced lymphoma burden and extended survival. Depletion of macrophages reduced efficacy in vivo, whereas depletion of neutrophils had no effect. F(ab′)2-only fragments of anti-CD47 antibodies failed to induce phagocytosis by human macrophages, indicating a requirement for Fc-Fcγ receptor interactions. In contrast, F(ab′)2-only fragments increased phagocytosis by murine macrophages independent of SLAMF7-Mac-1 interaction. Full-length anti-CD47 mAbs also induced phagocytosis by Fcγ receptor-deficient murine macrophages. An immunoglobulin G1 anti-CD47 mAb induced phagocytosis and natural killer cell-mediated cytotoxicity of TCL cells that was augmented by cotreatment with mogamulizumab, an anti-CCR4 mAb, or a mAb blocking MHC class I. These studies help explain the disparate activity of monotherapy with agents that block CD47 in murine models compared with patients. They also have direct translational implications for the deployment of anti-CD47 mAbs alone or in combination.

Introduction:Recent analyses of diffuse large B-cell lymphoma (DLBCL) have highlighted the clinical importance of immune tumor microenvironment (iTME), and based on the composition of the iTME, different DLBCL subtypes have been proposed (Kotlov et al. Cancer Discov. 2021, Steen et al. Cancer Cell. 2021). However, studies have mainly focused on the impact of different cell type proportions, whereas the clinical importance of their spatial organization has remained unclear.

Leukocyte migration to sites of inflammation is regulated by several endothelial adhesion molecules. Vascular adhesion protein-1 (VAP-1) is unique among the homing-associated molecules as it is both an enzyme that oxidizes primary amines and an adhesin. Although granulocytes can bind to endothelium via a VAP-1–dependent manner, the counter-receptor(s) on this leukocyte population is(are) not known. Here we used a phage display approach and identified Siglec-9 as a candidate ligand on granulocytes. The binding between Siglec-9 and VAP-1 was confirmed by in vitro and ex vivo adhesion assays. The interaction sites between VAP-1 and Siglec-9 were identified by molecular modeling and confirmed by further binding assays with mutated proteins. Although the binding takes place in the enzymatic groove of VAP-1, it is only partially dependent on the enzymatic activity of VAP-1. In positron emission tomography, the 68Gallium-labeled peptide of Siglec-9 specifically detected VAP-1 in vasculature at sites of inflammation and cancer. Thus, the peptide binding to the enzymatic groove of VAP-1 can be used for imaging conditions, such as inflammation and cancer.

Leukocyte migration to sites of inflammation is regulated by several endothelial adhesion molecules. Vascular adhesion protein-1 (VAP-1) is unique among the homing-associated molecules as it is both an enzyme that oxidizes primary amines and an adhesin. Although granulocytes can bind to endothelium via a VAP-1–dependent manner, the counter-receptor(s) on this leukocyte population is(are) not known. Here we used a phage display approach and identified Siglec-9 as a candidate ligand on granulocytes. The binding between Siglec-9 and VAP-1 was confirmed by in vitro and ex vivo adhesion assays. The interaction sites between VAP-1 and Siglec-9 were identified by molecular modeling and confirmed by further binding assays with mutated proteins. Although the binding takes place in the enzymatic groove of VAP-1, it is only partially dependent on the enzymatic activity of VAP-1. In positron emission tomography, the 68Gallium-labeled peptide of Siglec-9 specifically detected VAP-1 in vasculature at sites of inflammation and cancer. Thus, the peptide binding to the enzymatic groove of VAP-1 can be used for imaging conditions, such as inflammation and cancer.

Inadequate molecular and clinical stratification of the patientswith high-risk diffuse large B-cell lymphoma (DLBCL) is a clinical challenge hampering the establishment of personalizedtherapeutic options. We studiedthe translational significance of liquid biopsy in a uniformly treated trial cohort.Pretreatment circulating tumor DNA (ctDNA)revealed hiddenclinical and biological heterogeneity, and high ctDNA burdendeterminedincreasedrisk of relapse and deathindependentlyof conventional risk factors. Genomic dissection of pretreatment ctDNA revealed translationally relevantphenotypic, molecular,and prognostic information that extended beyond diagnostic tissue biopsies.During therapy, chemorefractory lymphomas exhibited diverging ctDNA kinetics, whereas end-of-therapy negativity for minimal residual diseasecharacterized cured patientsand resolvedclinical enigmas, including false residual PET positivity.Furthermore, we discovered fragmentation disparities in the cell-free DNA that characterize lymphoma-derived ctDNA and,as a proof-of-conceptfor their clinical application,utilized machinelearningto show that end-of-therapy fragmentation patterns predict outcome. Altogether, wehave discovered novel molecular determinantsin the liquid biopsythatcan non-invasively guide treatment decisions.

Background:Tumor infiltrating immune cells can modulate cancer progression and are attractive therapeutic targets. We have previously identified a tumor microenvironment (TME) immune cell signature in diffuse large B-cell lymphoma (DLBCL), which contains genes for cytolytic factors, immune checkpoint molecules, T-cells and macrophages, and separates the patients into immune cell high and low or T-cell inflamed and non-inflamed subgroups (Autio et al., 2020). As macrophages and T-cells are key components of the TME, our aim was to characterize their phenotypes and relationships in the tumor tissue and associate the findings to clinical outcome in patients with DLBCL.

Leppa: Novartis: Consultancy; Takeda: Consultancy; Celgene: Research Funding; Bayer: Research Funding; Incyte: Consultancy; Roche: Consultancy, Research Funding.

Leppa: Roche: Honoraria, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees.

Jørgensen: Roche: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees. Jerkeman:Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Roche: Honoraria, Research Funding. Holte:Novartis: Honoraria, Other: Advisory board. Leppa:Celgene: Consultancy; Bayer: Research Funding; Roche: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding.

Introduction

Introduction

Outcomes for patients with aggressive T-cell lymphomas (TCLs) remain dismal. Monoclonal antibodies (mAbs) that inhibit engagement of the “don't eat me” signal CD47 with SIRPα on myeloid cells may induce phagocytosis and have exhibited promise in multiple cancers. We dissected the role of CD47 blockade in opsonization and phagocytosis-induction through Fc-Fc gamma receptor (FcγR) interactions. CD47 was expressed at higher levels on 22 TCL cell lines by flow cytometry compared to quiescent T cells from 10 healthy donors (mean fluorescent intensity, 176.9 versus 69.85; Welch t-test p=0.002). We determined tumor cell-specific protein expression of CD47 by calculation of H scores in a tissue microarray (TMA) of 68 primary TCLs. The common subtypes (PTCL-NOS (n=16), AITL (n=19), and ALCL (n=12)) each had heterogeneous CD47 expression that was not associated with progression-free or overall survival. We utilized B6H12, an anti-CD47 mAb to assess apoptosis, antibody-dependent cell mediated-cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) of TCL cells (HuT-78, HH, Myla, SU-PM2, KIJK, MAC-2A, SMZ1 and K-299) and no increase was observed except for ADCC by murine FcγRIII-expressing engineered Jurkat cells. Upon addition of B6H12 or SRF231, a fully human IgG4 mAb against CD47 that is currently in a Phase I clinical trial (NCT03512340), phagocytosis of CFSE+TCL cells with human monocyte-derived macrophages (hMDMs) and murine bone-marrow derived macrophages (mBMDMs) was increased. In contrast, induction of phagocytosis of resting T cells from healthy donors was tested using B6H12 and no increase was observed (22.7 and 19.5%). A flow cytometry-based receptor occupancy assay demonstrated a linear correlation between receptor binding and phagocytosis induction in Jurkat T-cell lymphoblastic leukemia cells. hMDMs failed to induce phagocytosis of TCL cells with F(ab')2portion of B6H12 suggesting complete dependence on Fc receptors. In contrast, mBMDM-mediated phagocytosis of TCL cells with the F(ab')2portion of B6H12 (mean, 21.5%) was increased compared to negative control but reduced compared to full length (mean, 33.4%), suggesting both Fc-dependent and Fc-independent mechanisms. mBMDMs from FcγR knockout mice demonstrated a statistically significant decrease in phagocytosis (compared to wild-type mBMDMs) of Jurkat, HuT-78, MAC2A and K-299 cells with B6H12, confirming a role for Fc-FcγR engagement.Phagocytosis of tumor cells during CD47 blockade may depend on the ability of SLAMF7 to interact with the integrin Mac-1 (Chen J, Nature 2017). Phagocytosis assays of TCL cells with mBMDMs from Mac-1 knockout mice with full length and F(ab')2B6H12 demonstrated a reduction in phagocytosis using Mac-1 knockout macrophages (compared to wild-type) only with full-length B6H12. Thus, the Mac-1 contribution to phagocytosis induced by B6H12 in this context is Fc-dependent. We tested SRF231 in vivousing patient-derived xenograft (PDX) models of systemic hepatosplenic (HSTL), transformed cutaneous, and NKTCL. After 10 days, SRF231-treated mice had remarkable reductions in tumor burden compared to untreated mice, across multiple compartments. SRF231 treatment resulted in prolonged survival despite a short course of therapy (Figure 1). Bone marrow from xenografted mice, collected at day 11 post-treatment with SRF231, had significantly increased cytokines known to promote monocyte recruitment. Ex-vivophagocytosis assays using mBMDMs and hMDMs, enhanced engulfment of HSTL, T-PLL and anaplastic large cell-lymphoma PDX harvested cells upon exposure to SRF231. Finally, we observed that in our HSTL PDX model, depletion of macrophages with clodronate completely abrogated the antitumor efficacy of SRF231 while depletion of neutrophils with anti-Ly6G Ab had no effect, highlighting macrophages as the essential effectors of phagocytosis in this context. In summary, monoclonal antibodies that disrupt CD47-SIRPα have striking in vitroand in vivopreclinical efficacy but require interactions with human FcγR on macrophages to maximize efficacy.

Peluso: Surface Oncology: Employment, Equity Ownership. O'Connor:Surface Oncology: Employment, Equity Ownership. Paterson:Surface Oncology: Employment, Equity Ownership. Holland:Surface Oncology: Employment, Equity Ownership. Weinstock:Astra Zeneca, JAX, Samumed, Regeneron, Sun Pharma, Prescient: Patents & Royalties; Travera: Equity Ownership; Novartis: Consultancy, Research Funding; Novartis, Astra Zeneca, Abbvie, Aileron, Surface Oncology, Daiichi Sankyo: Research Funding; Genentech/Roche, Monsanto: Consultancy; Novartis, Dragonfly, Travera, DxTerity, Travera: Consultancy.

Introduction:Despite better therapeutic options and improved survival of diffuse large B-cell lymphoma (DLBCL), 30-40% of the patients still relapse and have dismal prognosis. Recently, the impact of genomic aberrations, allowing lymphoma cells to escape immune recognition on DLBCL pathogenesis has been recognized. However, whether immune related signatures could be used as determinants for treatment outcome has not been rigorously evaluated. Here, our aim was to elucidate the immunologic characteristics of the tumor microenvironment, and associate the findings with outcome in patients with high-risk DLBCL.

Leppä: Amgen: Research Funding; CTI Life Sciences: Honoraria; Janssen: Research Funding; Takeda: Honoraria, Other: Travel expenses; Roche: Honoraria, Other: Travel Expenses, Research Funding; Mundipharma: Research Funding; Bayer: Honoraria, Research Funding.

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