Single-cell functional genomics reveals determinants of sensitivity and resistance to natural killer cells in blood cancers.

Cancer cells can evade natural killer (NK) cell activity, thereby limiting anti-tumor immunity. To reveal genetic determinants of susceptibility to NK cell activity, we examined interacting NK cells and blood cancer cells using single-cell and genome-scale functional genomics screens. Interaction of NK and cancer cells induced distinct activation and type I interferon (IFN) states in both cell types depending on the cancer cell lineage and molecular phenotype, ranging from more sensitive myeloid to less sensitive B-lymphoid cancers. CRISPR screens in cancer cells uncovered genes regulating sensitivity and resistance to NK cell-mediated killing, including adhesion-related glycoproteins, protein fucosylation genes, and transcriptional regulators, in addition to confirming the importance of antigen presentation and death receptor signaling pathways. CRISPR screens with a single-cell transcriptomic readout provided insight into underlying mechanisms, including regulation of IFN-γ signaling in cancer cells and NK cell activation states. Our findings highlight the diversity of mechanisms influencing NK cell susceptibility across different cancers and provide a resource for NK cell-based therapies.

Results of a phase I trial with Haploidentical mbIL-21 ex vivo expanded NK cells for patients with multiply relapsed and refractory AML.

Natural killer (NK)-cells have potent anti-tumor effects, yet it remains unclear if they are effective for patients with relapsed acute myeloid leukemia (AML). In a phase I clinical trial, we treated 12 patients (median age 60 years) with refractory AML (median 5 lines of prior therapy, median bone marrow blast count of 47%) with fludarabine/cytarabine followed by 6 infusions of NK-cells expanded from haploidentical donors using K562 feeder cells expressing membrane-bound IL21 and 4-1BBL. Patients received 106-107/kg/dose. No toxicity or graft-versus-host disease (GVHD) was observed and MTD was not reached. Seven patients (58.3%) responded and achieved a complete remission (CR) with/without count recovery. Median time to best response was 48 days. Five responding patients proceeded to a haploidentical transplant from the same donor. After a median follow-up of 52 months, 1-year overall survival (OS) for the entire group was 41.7%, better for patients who responded with CR/CRi (57.14%), and for patients who responded and underwent transplantation (60%). Persistence and expansion of donor-derived NK-cells were identified in patients' blood, and serum IFNγ levels rose concurrently with NK cell infusions. A higher count-functional inhibitory KIR was associated with higher likelihood of achieving CR/CRi. In conclusion, we observed a significant response to ex vivo expanded NK-cell administration in refractory AML patients without adverse effects.

Cerebral organoids containing an AUTS2 missense variant model microcephaly.

Variants in the AUTS2 gene are associated with a broad spectrum of neurological conditions characterized by intellectual disability, microcephaly, and congenital brain malformations. Here, we use a human cerebral organoid model to investigate the pathophysiology of a heterozygous de novo missense AUTS2 variant identified in a patient with multiple neurological impairments including primary microcephaly and profound intellectual disability. Proband cerebral organoids exhibit reduced growth, deficits in neural progenitor cell (NPC) proliferation and disrupted NPC polarity within ventricular zone-like regions compared to control cerebral organoids. We used CRISPR-Cas9-mediated gene editing to correct this variant and demonstrate rescue of impaired organoid growth and NPC proliferative deficits. Single-cell RNA sequencing revealed a marked reduction of G1/S transition gene expression and alterations in WNT-ß-catenin signalling within proband NPCs, uncovering a novel role for AUTS2 in NPCs during human cortical development. Collectively, these results underscore the value of cerebral organoids to investigate molecular mechanisms underlying AUTS2 syndrome.

Cellular therapy: Adoptive immunotherapy with expanded natural killer cells.

Adoptive immunotherapy with natural killer cells was pioneered 30 years ago in human clinical trials with the development of cytokine-induced killer cells-unfractionated peripheral blood mononuclear cell (PBMC) populations activated overnight with IL-2. Higher doses were subsequently made possible through the advent of steady-state apheresis, allowing the collection of PBMC numbers equivalent to an entire adult blood volume, and increased purity made feasible through magnetic CD3-depletion and/or CD56-selection methods. Still, these approaches rarely achieved clinical dosing above a single infusion of 108 NK cells/kg, except with substantial donor-recipient size mismatch (eg, parents donating cells to children). To address this shortcoming, leukemia cell lines with NK cell-like function or ex vivo expansion approaches centered on the homeostatic cytokine IL-15 were developed. Here, we describe the development of an ex vivo expansion system based on a feeder cell expressing membrane-bound IL-21 that enables log-phase growth of primary NK cells for many weeks without inducing senescence, and describe the biology, correlative science, and translation to clinical trials for patients with leukemia, brain tumors, and solid tumors.

The Future of Natural Killer Cell Immunotherapy for B Cell Non-Hodgkin Lymphoma (B Cell NHL).

OPINION STATEMENT: Natural killer (NK) cells have played a critical-if largely unrecognized or ignored-role in the treatment of B cell non-Hodgkin lymphoma (NHL) since the introduction of CD20-directed immunotherapy with rituximab as a cornerstone of therapy over 25 years ago. Engagement with NK cells leading to lysis of NHL targets through antibody-dependent cellular cytotoxicity (ADCC) is a critical component of rituximab's mechanism of action. Despite this important role, the only aspect of B cell NHL therapy that has been adopted as standard therapy that even indirectly augments or restores NK cell function is the introduction of obinutuzumab, a CD20 antibody with enhanced ability to engage with NK cells. However, over the last 5 years, adoptive immunotherapy with effector lymphocytes of B cell NHL has experienced tremendous growth, with five different CAR T cell products now licensed by the FDA, four of which target CD19 and have approved indications for some subtype of B cell NHL-axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, and tisagenlecleucel. These T cell-based immunotherapies essentially mimic the recognition, activation pathway, and cytotoxic machinery of a CD19 antibody engaging NK cells and lymphoma targets. Despite their efficacy, these T cell-based immunotherapies have been difficult to implement because they require 4-6 weeks of manufacture, are costly, and have significant toxicities. This renewed interest in the potential of cellular immunity-and the manufacturing, supply chain, and administration logistics that have been addressed with these new agents-have ignited a new wave of enthusiasm for NK cell-directed therapies in NHL. With high safety profiles and proven anti-lymphoma efficacy, one or more new NK cell-directed modalities are certain to be introduced into the standard toolbox of NHL therapy within the next few years, be it function-enhancing cytokine muteins, multi-domain NK cell engagers, or adoptive therapy with expanded or genetically modified NK cells.

Adoptive immunotherapy with double-bright (CD56bright /CD16bright ) expanded natural killer cells in patients with relapsed or refractory acute myeloid leukaemia: a proof-of-concept study.

Patients with acute myeloid leukaemia (AML) have a five-year survival rate of 28·7%. Natural killer (NK)-cell have anti-leukaemic activity. Here, we report on a series of 13 patients with high-risk R/R AML, treated with repeated infusions of double-bright (CD56bright /CD16bright ) expanded NK cells at an academic centre in Brazil. NK cells from HLA-haploidentical donors were expanded using K562 feeder cells, modified to express membrane-bound interleukin-21. Patients received FLAG, after which cryopreserved NK cells were thawed and infused thrice weekly for six infusions in three dose cohorts (106 -107 cells/kg/infusion). Primary objectives were safety and feasibility. Secondary endpoints included overall response (OR) and complete response (CR) rates at 28-30 days after the first infusion. Patients received a median of five prior lines of therapy, seven with intermediate or adverse cytogenetics, three with concurrent central nervous system (CNS) leukaemia, and one with concurrent CNS mycetoma. No dose-limiting toxicities, infusion-related fever, or cytokine release syndrome were observed. An OR of 78·6% and CR of 50·0% were observed, including responses in three patients with CNS disease and clearance of a CNS mycetoma. Multiple infusions of expanded, cryopreserved NK cells were safely administered after intensive chemotherapy in high-risk patients with R/R AML and demonstrated encouraging outcomes.

Immunotherapies for pediatric cancer: current landscape and future perspectives.

The advent of immunotherapy has revolutionized how we manage and treat cancer. While the majority of immunotherapy-related studies performed to date have focused on adult malignancies, a handful of these therapies have also recently found success within the pediatric space. In this review, we examine the immunotherapeutic agents that have achieved the approval of the US Food and Drug Administration for treating childhood cancers, highlighting their development, mechanisms of action, and the lessons learned from the seminal clinical trials that ultimately led to their approval. We also shine a spotlight on several emerging immunotherapeutic modalities that we believe are poised to have a positive impact on the treatment of pediatric malignancies in the near future.

Intravenous Bone Marrow Mononuclear Cells for Acute Ischemic Stroke: Safety, Feasibility, and Effect Size from a Phase I Clinical Trial.

Cellular therapy is a promising investigational modality to enhance poststroke recovery. We conducted a single-arm, phase I clinical trial to determine the safety and feasibility of intravenous (IV) administration of autologous bone marrow mononuclear cells (MNCs) after acute ischemic stroke (AIS). Patients with moderate severity of AIS underwent bone marrow harvest followed by IV reinfusion of MNCs within 24-72 hours of onset. A target dose of 10 million cells per kilogram was chosen based on preclinical data. Patients were followed up daily during hospitalization and at 1, 3, 6, 12, and 24 months for incidence of adverse events using laboratory, clinical (12 months), and radiological (24 months) parameters. The trial was powered to detect severe adverse events (SAEs) with incidences of at least 10% and planned to enroll 30 patients. Primary outcomes were study-related SAEs and the proportion of patients successfully completing study intervention. A propensity score-based matched control group was used for the estimation of effect size (ES) for day-90 modified Rankin score (mRS). There were no study-related SAEs and, based on a futility analysis, enrolment was stopped after 25 patients. All patients successfully completed study intervention and most received the target dose. Secondary analysis estimated the ES to be a reduction of 1 point (95% confidence interval: 0.33-1.67) in median day-90 mRS for treated patients as compared with the matched control group. Bone marrow harvest and infusion of MNCs is safe and feasible in patients with AIS. The estimated ES is helpful in designing future randomized controlled trials. Stem Cells 2019;37:1481-1491.

Evaluation of serum-free media formulations in feeder cell-stimulated expansion of natural killer cells.

BACKGROUND: Optimal expansion of therapeutic natural killer (NK) cell products has required media supplementation with human or fetal bovine serum, which raises safety and regulatory concerns for clinical manufacturing. Serum-free media (SFM) have been optimized for T-cell expansion, but few SFM systems have been developed for NK cells. Here, we compare six commercial clinical-grade SFM with our standard fetal bovine serum-containing medium for their ability to support NK cell expansion and function. METHODS: Human peripheral blood NK cells were expanded in selected media by recursive weekly stimulation with K562-based feeder cells expressing membrane-bound interleukin-21 and CD137L. Expansion was the primary readout, and the best-performing SFM was then compared with standard medium for cytotoxicity, phenotype, degranulation and cytokine secretion. Multiple lots were compared for consistency, and media was analyzed throughout for nutrient consumption and metabolic byproducts. RESULTS: TexMACS, OpTmizer, SCGM, ABS-001 and StemXVivo demonstrated equal or inferior NK cell expansion kinetics compared with standard medium, but expansion was markedly superior with AIM V + 5% Immune Cell Serum Replacement (ICSR; mean 5448 vs. 2621-fold expansion in 14 days). Surprisingly, NK cells expanded in AIM V + ICSR also showed increased cytotoxicity, tumor necrosis factor α secretion and DNAM-1, NKG2D, NKp30, FasL, granzyme B and perforin expression. Lot-to-lot variability was minimal. Glucose and glutamine consumption were inversely related to lactate and ammonia production. DISCUSSION: The AIM V + ICSR SFM system supports excellent ex vivo expansion of clinical-grade NK cells with the phenotype and function needed for adoptive immunotherapy.

Fc-engineered anti-CD33 monoclonal antibody potentiates cytotoxicity of membrane-bound interleukin-21 expanded natural killer cells in acute myeloid leukemia.

BACKGROUND: Qualitative and quantitative defects in natural killer (NK) cells have been noted in patients with acute myeloid leukemia (AML), providing rationale for infusion of donor-derived NK cells. We previously showed that decitabine enhances expression of NKG2D ligands in AML with additive cytotoxicity when NK cells and Fc (fragment crystallizable region)-engineered CD33 monoclonal antibody (CD33mAb) was used. We conducted a phase 1 study evaluating decitabine and haploidentical NK cells in relapsed AML. Using patient samples from this study, we evaluated whether ex vivo donor-derived expanded NK cells with or without CD33mAb was effective in decitabine-treated AML. METHODS: Bone marrow aspirates were collected from patients at pre- and post-NK cell infusion. NK cells from healthy donors were expanded for 14 days using irradiated K562 feeder cells displaying membrane-bound IL-21 (mbIL-21). Patient samples were used to test in vitro activity of mbIL-21 NK cells ± CD33m Ab-dependent cellular cytotoxicity (ADCC) and AML patient derived xenograft (PDX) mice were developed to test in vivo activity. RESULTS: Upon incubation with primary AML blasts, mbIL-21 NK cells showed variable donor-dependent intra-cellular interferon-γ production, which increased with CD33mAb-coated AML. ADCC assays revealed mbIL-21 NK cells effectively lysed primary AML blasts with higher activity on CD33mAb-coated AML. Importantly, CD33mAb-dependent enhanced cytotoxicity by mbIL-21 NK cells was maintained in AML cells from patients even 24 days post-decitabine treatment. In vivo infusion of mbIL-21 NK cells in AML PDX mice, treated with CD33mAb, reduced the tumor burden. DISCUSSION: These data show the therapeutic utility of mbIL-21 NK cells that can be further potentiated by addition of CD33mAb in AML.

Natural Killer Cell Immunotherapy for Osteosarcoma.

Natural killer (NK) cells are lymphocytes of the innate immune system that have the ability to recognize malignant cells through balanced recognition of cell-surface indicators of stress and danger. Once activated through such recognition, NK cells release cytokines and induce target cell lysis through multiple mechanisms. NK cells are increasingly recognized for their role in controlling tumor progression and metastasis and as important mediators of immunotherapeutic modalities such as cytokines, antibodies, immunomodulating drugs, and stem cell transplantation. Recent advances in manipulating NK cell number, function, and genetic modification have caused renewed interest in their potential for adoptive immunotherapies, which are actively being tested in clinical trials. Here, we summarize the evidence for NK cell recognition of osteosarcoma, discuss immune therapies that are directly or indirectly dependent on NK cell function, and describe potential approaches for manipulating NK cell number and function to enhance therapy against osteosarcoma.

Education-dependent activation of glycolysis promotes the cytolytic potency of licensed human natural killer cells.

BACKGROUND: The mechanism by which natural killer (NK) cell education results in licensed NK cells with heightened effector function against missing self-targets is not known. OBJECTIVE: We sought to identify potential mechanisms of enhanced function in licensed human NK cells. METHODS: We used expanded human NK cells from killer immunoglobulin-like receptor (KIR)/HLA-genotyped donors sorted for single-KIR+ cells to generate pure populations of licensed and unlicensed NK cells. We performed proteomic and gene expression analysis of these cells before and after receptor cross-linking and performed functional and metabolic analysis before and after interference with selected metabolic pathways. We verified key findings using freshly isolated and sorted NK cells from peripheral blood. RESULTS: We confirmed that licensed human NK cells are greater in number in peripheral blood and proliferate more in vitro than unlicensed NK cells. Using high-throughput protein analysis, we found that unstimulated licensed NK cells have increased expression of the glycolytic enzyme pyruvate kinase muscle isozyme M2 and after KIR cross-linking have increased phosphorylation of the metabolic modulators p38-α and 5' adenosine monophosphate-activated protein kinase α. After cytokine expansion and activation, unlicensed NK cells depended solely on mitochondrial respiration for cytolytic function, whereas licensed NK cells demonstrated metabolic reprogramming toward glycolysis and mitochondrial-dependent glutaminolysis, leading to accumulation of glycolytic metabolites and depletion of glutamate. As such, blocking both glycolysis and mitochondrial-dependent respiration was required to suppress the cytotoxicity of licensed NK cells. CONCLUSIONS: Collectively, our data support an arming model of education in which enhanced glycolysis in licensed NK cells supports proliferative and cytotoxic capacity.

Phase 1 clinical trial using mbIL21 ex vivo-expanded donor-derived NK cells after haploidentical transplantation.

Relapse has emerged as the most important cause of treatment failure after allogeneic hematopoietic stem cell transplantation (HSCT). To test the hypothesis that natural killer (NK) cells can decrease the risk of leukemia relapse, we initiated a phase 1 dose-escalation study of membrane-bound interleukin 21 (mbIL21) expanded donor NK cells infused before and after haploidentical HSCT for high-risk myeloid malignancies. The goals were to determine the safety, feasibility, and maximum tolerated dose. Patients received a melphalan-based reduced-intensity conditioning regimen and posttransplant cyclophosphamide-based graft-versus-host disease (GVHD) prophylaxis. NK cells were infused on days -2, +7, and +28 posttransplant. All NK expansions achieved the required cell number, and 11 of 13 patients enrolled received all 3 planned NK-cell doses (1 × 105/kg to 1 × 108/kg per dose). No infusional reactions or dose-limiting toxicities occurred. All patients engrafted with donor cells. Seven patients (54%) developed grade 1-2 acute GVHD (aGVHD), none developed grade 3-4 aGVHD or chronic GVHD, and a low incidence of viral complications was observed. One patient died of nonrelapse mortality; 1 patient relapsed. All others were alive and in remission at last follow-up (median, 14.7 months). NK-cell reconstitution was quantitatively, phenotypically, and functionally superior compared with a similar group of patients not receiving NK cells. In conclusion, this trial demonstrated production feasibility and safety of infusing high doses of ex vivo-expanded NK cells after haploidentical HSCT without adverse effects, increased GVHD, or higher mortality, and was associated with significantly improved NK-cell number and function, lower viral infections, and low relapse rate posttransplant.

Correction to: Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI.

There was a typo in author Andrew Wahba's name in the initial online publication. The original article has been corrected.

Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI.

PURPOSE: Medulloblastoma (MB) is the most common malignant brain tumor in children. Recent studies have shown the ability of natural killer (NK) cells to lyse MB cell lines in vitro, but in vivo successes remain elusive and the efficacy and fate of NK cells in vivo remain unknown. METHODS: To address these questions, we injected MB cells into the cerebellum of immunodeficient mice and examined tumor growth at various days after tumor establishment via bioluminescence imaging. NK cells were labeled with a fluorine-19 (19F) MRI probe and subsequently injected either intratumorally or contralaterally to the tumor in the cerebellum and effect on tumor growth was monitored. RESULTS: The 19F probe efficiently labeled the NK cells and exhibited little cytotoxicity. Fluorine-19 MRI confirmed the successful and accurate delivery of the labeled NK cells to the cerebellum of the mice. Administration of 19F-labeled NK cells suppressed MB growth, with the same efficacy as unlabeled cells. Immunohistochemistry confirmed the presence of NK cells within the tumor, which was associated with induction of apoptosis in tumor cells. NK cell migration to the tumor from a distal location as well as activation of apoptosis was also demonstrated by immunohstochemistry. CONCLUSIONS: Our results show that NK cells present a novel opportunity for new strategies in MB treatment. Further, 19F-labeled NK cells can suppress MB growth while enabling 19F MRI to provide imaging feedback that can facilitate study and optimization of therapeutic paradigms.

Expression of carcinoma, apoptosis, and cell-death-related genes are determinants for sensitivity of pediatric cancer cell lines to lysis by natural killer cells.

Natural killer (NK) cells have potential utility in pediatric cancer immunotherapy for their ability to lyse diverse tumor targets, lack of dependence on mutation-associated tumor antigens, and for their relative safety demonstrated so far in clinical trials. Here, we evaluate the cytotoxic potential of expanded NK cells against a well-characterized panel of pediatric cancer cell lines representing Ewing sarcoma, rhabdomyosarcoma, neuroblastoma, lymphoma, leukemia, and brain tumors. We correlate their sensitivity NK cell lysis with tumor phenotypic, transcriptomic, and genetic determinants, and correlate known immunogenetic determinants with donor NK cell potency. Although ligand expression on cell lines stratified according to hematologic versus nonhematologic cancer types, the sensitivity to NK cell lysis varied widely and did not correlate with cancer type, expression of individual activating or inhibitory ligands, gene-expression clusters of NK cell ligands, disease status (newly diagnosed or relapsed), or MYCN amplification. Rather, sensitivity to NK cell-mediated lysis was associated with a novel 96-gene cluster of predominantly carcinoma-, apoptosis-, and cell death-related pathways, and with functional p53 status. NK cell potency was strongly associated with activating KIR gene content, but not with KIR/KIR-ligand mismatch. This study suggests that adoptive immunotherapy with expanded NK cells has the potential for a wide range of pediatric cancers, identifies potential biomarkers of efficacy and response, and establishes a foundation for using this cell line panel for the preclinical evaluation of immunotherapies.

Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells.

Adoptive immunotherapy retargeting T cells to CD19 via a chimeric antigen receptor (CAR) is an investigational treatment capable of inducing complete tumor regression of B-cell malignancies when there is sustained survival of infused cells. T-memory stem cells (TSCM) retain superior potential for long-lived persistence, but challenges exist in manufacturing this T-cell subset because they are rare among circulating lymphocytes. We report a clinically relevant approach to generating CAR+ T cells with preserved TSCM potential using the Sleeping Beauty platform. Because IL-15 is fundamental to T-cell memory, we incorporated its costimulatory properties by coexpressing CAR with a membrane-bound chimeric IL-15 (mbIL15). The mbIL15-CAR T cells signaled through signal transducer and activator of transcription 5 to yield improved T-cell persistence independent of CAR signaling, without apparent autonomous growth or transformation, and achieved potent rejection of CD19+ leukemia. Long-lived T cells were CD45ROnegCCR7+CD95+, phenotypically most similar to TSCM, and possessed a memory-like transcriptional profile. Overall, these results demonstrate that CAR+ T cells can develop long-term persistence with a memory stem-cell phenotype sustained by signaling through mbIL15. This observation warrants evaluation in clinical trials.

Recurrent Stimulation of Natural Killer Cell Clones with K562 Expressing Membrane-Bound Interleukin-21 Affects Their Phenotype, Interferon-γ Production, and Lifespan.

A pattern of natural killer cell (NK cell) heterogeneity determines proliferative and functional responses to activating stimuli in individuals. Obtaining the progeny of a single cell by cloning the original population is one of the ways to study NK cell heterogeneity. In this work, we sorted single cells into a plate and stimulated them via interleukin (IL)-2 and gene-modified K562 feeder cells that expressed membrane-bound IL-21 (K562-mbIL21), which led to a generation of phenotypically confirmed and functionally active NK cell clones. Next, we applied two models of clone cultivation, which differently affected their phenotype, lifespan, and functional activity. The first model, which included weekly restimulation of clones with K562-mbIL21 and IL-2, resulted in the generation of relatively short-lived (5⁻7 weeks) clones of highly activated NK cells. Levels of human leukocyte antigen class II molecule-DR isotype (HLA-DR) expression in the expanded NK cells correlated strongly with interferon-γ (IFN-γ) production. The second model, in which NK cells were restimulated weekly with IL-2 alone and once on the sixth week with K562-mbIL21 and IL-2, produced long-lived clones (8⁻14 weeks) that expanded up to 107 cells with a lower ability to produce IFN-γ. Our method is applicable for studying variability in phenotype, proliferative, and functional activity of certain NK cell progeny in response to the stimulation, which may help in selecting NK cells best suited for clinical use.

Venous Thromboembolism in Pediatric Hematopoietic Cell Transplant: A Multicenter Cohort Study.

Hematopoietic cell transplant (HCT) is associated with a proinflammatory, procoagulant environment that places recipients at increased risk of venous thromboembolism (VTE). Although the incidence of VTE in adult HCT recipients has been extensively studied, similar data for children are lacking. We conducted a multicenter retrospective study to analyze the prevalence of VTE and associated risk factors in a large cohort of patients who underwent HCT at tertiary care US children's hospitals. The Pediatric Health Information System database, a large administrative database that contains clinical and resource utilization data from 49 freestanding children's hospitals in the United States, was used to extract data. International Classification of Diseases, Ninth Revision, Clinical Modification codes were used to identify HCT recipients, VTE events, post-HCT complications, and associated risk factors up to 1 year post-transplant. Data on patients who received HCT from January 2010 through September 2014 were collected. A total of 4158 unique patients mean ± standard deviation age at transplant admit, 8.8 ± 6.5 years; range, birth to 33.4 years) were identified. After HCT 290 subjects (6.9%) developed VTE. VTE prevalence was greater in patients aged ≥ 13 versus <13 years (8.54% versus 6.33%; P = .01) and in recipients of allogeneic versus autologous grafts (7.7% versus 5%; P ≤ .01). VTE was associated with prolonged median duration of hospitalization (81 versus 54 days; P ≤.01) and increased 1-year mortality (13.9% versus 5.9%; P ≤ .01). Infections and presence of any graft-versus-host disease (GVHD) were significantly associated with VTE occurrence in recipients of allogenic grafts. Prevalence of VTE in patients who underwent HCT at pediatric tertiary care hospitals is about 7%. Age ≥ 13 years and allogeneic grafts were significant pre-HCT VTE risk factors, with GVHD and infections seen more frequently in patients with VTE.

Ex vivo-expanded NK cells from blood and ascites of ovarian cancer patients are cytotoxic against autologous primary ovarian cancer cells.

Ovarian cancer (OC) is the leading cause of gynecological cancer-related death in North America. Most ovarian cancer patients (OCPs) experience disease recurrence after first-line surgery and chemotherapy; thus, there is a need for novel second-line treatments to improve the prognosis of OC. Although peripheral blood-derived NK cells are known for their ability to spontaneously lyse tumour cells without prior sensitization, ascites-derived NK cells (ascites-NK cells) isolated from OCPs exhibit inhibitory phenotypes, impaired cytotoxicity and may play a pro-tumourigenic role in cancer progression. Therefore, it is of interest to improve the cytotoxic effector function of impaired OCP ascites-NK cells at the tumour environment. We investigated the efficacy of using an artificial APC-based ex vivo expansion technique to generate cytotoxic, expanded NK cells from previously impaired OCP ascites-NK cells, for use in an autologous model of NK cell immunotherapy. We are the first to obtain a log-scale expansion of OCP ascites-NK cells that upregulate the surface expression of activating receptors NKG2D, NKp30, NKp44, produce robust amounts of anti-tumour cytokines in the presence of OC cells and mediate direct tumour cytotoxicity against ascites-derived, primary OC cells obtained from autologous patients. Our findings demonstrate that it is possible to generate cytotoxic OCP ascites-NK cells from previously impaired OCP ascites-NK cells, which presents a promising immunotherapeutic target for the second-line treatment of OC. Future work should focus on evaluating the in vivo efficacy of autologous NK cell immunotherapy through the intraperitoneal delivery of NK cell expansion factors to a preclinical xenograft mouse model of human OC.