ALBERT

Description: Key Points Genetically heterogeneous subclones with varying leukemia-initiating potential exist in neonatal transient abnormal myelopoiesis. This novel xenograft model of transient abnormal myelopoiesis may provide unique insight into the evolutionary process of leukemia.

Description: Abstract 2631 Little is known about microRNA function in hematopoietic stem and progenitor cells (HSPC). Using a lentivector genetic reporter strategy to functionally detect miRNA activity in hematopoietic cells at single cell resolution, we identified several miRNAs which were specifically expressed in mouse and human HSC and early progenitors, defined according to cell surface phenotype and functional repopulation assays. One of these HSPC-specific miRNAs, miR-126, was further studied. We generated a stable miR-126 knockdown (kd) or forced its expression (“knock-in”, ki) in mouse HSPC using lentiviral vectors. Kd or ki cells were competitively transplanted with congenic, control vector-transduced cells, and hematopoietic chimerism was followed for 〉1 year in both primary and secondary recipients. miR-126 kd HSPC displayed enhanced myeloid and/or lymphoid contribution during the early phases of reconstitution, while they subsequently contributed similarly as the control cells. When this steady state bone marrow (BM) was transplanted into secondary recipients, we noted an even more pronounced over-contribution of miR-126 kd cells to hematopoiesis. In the long run, however, some secondary mice showed signs of exhaustion of miR-126 kd cells. These data suggest that miR-126 kd enhances hematopoiesis, likely at the stem/early progenitor level and in particular under stress conditions. On the other hand, forced expression of miR-126 (ki) resulted in an early competitive disadvantage in vivo, with progressively decreasing contribution to all hematopoietic lineages, paralleled by a nearly complete depletion of Kit+Sca+Lin- (KSL) miR-126 ki cells in the BM at 6 weeks after transplant. At 3 weeks post-transplant, when miR-126 ki KSL cells could still be detected, we found an increased proliferative index in these cells as judged by EdU incorporation in vivo, paralleled by a higher hematopoietic output respect to control cells at week 2–4 after transplant. These data suggest that miR-126 ki might favor HSC commitment at the cost of self-renewal. This phenotype was specific for miR-126 and not due to vector toxicity, as we demonstrate stable, long term overexpression of several control miRNAs in vivo. Moreover, miR-126 ki cells showed normal clonogenic activity in vitro. We then optimized a protocol to stably knock down miR-126 in human cord blood (huCB) HSPC, and validated this approach by demonstrating upregulation of previously described miR-126 targets including the beta subunit of phosphoinositide-3-kinase. Manipulation of miR-126 activity changed cell growth and differentiation of huCB, and we show altered activation of key signal transduction pathways upon miR-126 kd. Identification of additional miR-126 targets is ongoing using unbiased proteomic and transcriptomic approaches. In summary, these data suggest that a narrow range of miR-126 activity is required for robust and sustained HSC function, and that its manipulation may provide novel insights into stem cell biology. Disclosures: No relevant conflicts of interest to declare.

Description: To establish a more appropriate animal recipient for xenotransplantation, NOD/SCID/γcnull mice double homozygous for the severe combined immunodeficiency (SCID) mutation and interleukin-2Rγ (IL-2Rγ) allelic mutation (γcnull) were generated by 8 backcross matings of C57BL/6J-γcnull mice and NOD/Shi-scidmice. When human CD34+ cells from umbilical cord blood were transplanted into this strain, the engraftment rate in the peripheral circulation, spleen, and bone marrow were significantly higher than that in NOD/Shi-scid mice treated with anti-asialo GM1 antibody or in the β2-microglobulin–deficient NOD/LtSz-scid (NOD/SCID/β2mnull) mice, which were as completely defective in NK cell activity as NOD/SCID/γcnull mice. The same high engraftment rate of human mature cells was observed in ascites when peripheral blood mononuclear cells were intraperitoneally transferred. In addition to the high engraftment rate, multilineage cell differentiation was also observed. Further, even 1 × 102 CD34+ cells could grow and differentiate in this strain. These results suggest that NOD/SCID/γcnull mice were superior animal recipients for xenotransplantation and were especially valuable for human stem cell assay. To elucidate the mechanisms involved in the superior engraftment rate in NOD/SCID/γcnull mice, cytokine production of spleen cells stimulated with Listeria monocytogenesantigens was compared among these 3 strains of mice. The interferon-γ production from dendritic cells from the NOD/SCID/γcnull mouse spleen was significantly suppressed in comparison with findings in 2 other strains of mice. It is suggested that multiple immunological dysfunctions, including cytokine production capability, in addition to functional incompetence of T, B, and NK cells, may lead to the high engraftment levels of xenograft in NOD/SCID/γcnull mice.

Description: Interaction between killer immunoglobulin-like receptors (KIRs) and HLA class I ligands influence development of natural killer cell repertoire and response to allogeneic tissue. Recently several reports suggests donor/recipient KIR-ligand imcompatibility have great influence for outcome of allogeneic Hematopoietic Stem Cell transplantation (SCT). We examined recovery of the NK cell receptor after pediatric SCT to elucidate emergence of NKR repertoire and frequency after SCT is depend on whether donor or recipient type and effect of HLA disparity. [Patients and method] Nine patients receiving allogeneic SCT were examined. Expressions and frequency of KIR on NK cells were determined by flowcytometry using anti CD158a/h, 158b/j, 158e, 158i, 159a, 94, 56,and 3 antibodies. Donor and recipient KIR repertoire frequency prior to SCT were also examined. In cord blood transplantation, donor KIR repertoire was checked using residual thawed and infused cord blood. [Result] Among 9 patients, PBSCT were performed in 3 patients, CBT in 3, unrelated BMT in 3. HLA matched SCT was all 3 cases of unrelated BMT. KIR expression frequency was various in each patient. But in early phase, relatively both high percentage and absolute number of each KIR positive repertoire were observed, and then gradually decreased toward to 1 year after SCT. NK cell receptor repertoire reconstitution was generally well and rapid except for one patient who was treated with steroid pulse therapy for severe GVHD. KIR repertoire expression pattern at 1 year after SCT was similar to donor type compare to that of pre-SCT recipient. Donor/recipient HLA mismatch did not have obvious effect for NKR repertoire reconstitution. [Conclusion] NKR repertoire reconstitution after pediatric SCT depends on donor cell type.

Description: A single-center trial of CD19 directed, lentiviral transduced chimeric antigen receptor (CAR) T cells (CTL019) for relapsed and refractory (r/r) B-ALL pediatric patients showed rates of CR 〉90% with prolonged CAR T cell persistence/CR without further therapy in the majority of patients infused (Maude NEJM 2014). We report here the feasibility, safety and efficacy of the first multicenter global pivotal registration CAR T cell trial. Features of this trial include: i) the first trial in which industry-manufactured cells were provided to all patients; ii) enrollment across 25 centers in the US, EU, Canada, Australia, and Japan; iii) successful transfer and manufacturing of cells in a global supply chain; and iv) successful implementation of cytokine release syndrome (CRS) management across a global trial. All patients had CD19 positive B-ALL with morphologic marrow tumor involvement at registration (〉5% blasts), and were either primary refractory; chemo-refractory after first relapse, relapsed after second line therapy; or ineligible for allogeneic SCT. CTL019 was manufactured from patient PBMC under GMP conditions in the US, at a centralized "sponsor-owned" manufacturing facility, and supplied to all sites. The primary endpoint of overall remission rate (CR+CRi) within 3 months and secondary endpoints (EFS, DOR, OS and safety) were assessed by an independent review committee. Based on preliminary data as of March 2016, 57 patients were enrolled. There were 3 manufacturing failures (5%), 5 patients were not infused due to death or adverse events (9%), and 15 patients were pending infusion at the data cut off. Following fludarabine/cyclophosphamide lymphodepleting chemotherapy in the majority of the patients, 34 patients (median age 11 [3-23], 50% with prior HSCT) were infused with a single dose of CTL019 at a median dose of 2.9 x106 transduced CTL019 cells/kg (0.2 to 4). Among 29 patients reaching D28 prior to the data cutoff, 83% (24/29) achieved CR or CRi by local investigator assessment, all of which were MRD-negative. Two early deaths occurred prior to initial disease assessment, one due to disease progression and one due to intracranial hemorrhage. Two patients did not respond. One patient was in CR by BM at D28, but CSF was not assessed, therefore this patient was classified as "incomplete" assessment. Safety was managed by a protocol-specified CRS algorithm with no cases of refractory CRS. Using the Penn CRS grading scale, 82% of patients experienced CRS, with 7 grade 3 (21%) and 8 grade 4 (24%) events. 44% patients with CRS required anti-cytokine therapy; all received tocilizumab with or without other anti-cytokine therapy, with complete resolution of CRS. Besides CRS, the most common grade 3 and 4 non-hematologic AEs were febrile neutropenia (29%), increased bilirubin (21%), increased AST (21%), and hypotension (21%). 21% of patients experienced grade 3 or 4 neuropsychiatric events including confusion, delirium, encephalopathy, agitation and seizure; no cerebral edema was reported. CTL019 in vivo cellular kinetics by qPCR demonstrated transgene persistence in blood in responding patients at and beyond 6 months. Overall exposure (AUC 0-28d) and maximal expansion (Cmax) of CTL019 DNA measured by qPCR was higher in responding compared with non-responding patients. In summary, this pivotal global study in pediatric and young adult patients with r/r B-ALL receiving CTL019, confirms a high level of efficacy and a similar safety profile to that shown in the prior single center experience. Safety was effectively and reproducibly managed by appropriately trained investigators. The study has completed accrual. At the meeting, updated data from a planned formal interim analysis including safety, efficacy (primary and selected secondary endpoints), cellular kinetics, and impact of anti-cytokine therapy will be presented for more than 50 patients infused at 25 global sites. Disclosures Grupp: Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy. Laetsch:Novartis: Consultancy; Loxo Oncology: Consultancy. Bittencourt:Seattle Genetics: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Educational Grant. Maude:Novartis: Consultancy. Myers:Novartis Pharmaceuticals: Consultancy. Rives:Novartis: Consultancy; Jazz Pharma: Consultancy. Nemecek:Medac, GmbH: Research Funding; Novartis: Consultancy; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees. Schlis:Novartis: Honoraria. Martin:Jazz Pharmaceuticals: Other: One time discussion panel; Novartis: Other: Support of clinical trials. Bader:Medac: Consultancy, Research Funding; Riemser: Research Funding; Neovii Biotech: Research Funding; Servier: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Peters:Novartis: Consultancy; Jazz: Speakers Bureau; Amgen: Consultancy; Pfizer: Consultancy; Medac: Consultancy. Biondi:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Cellgene: Other: Advisory Board; BMS: Membership on an entity's Board of Directors or advisory committees. Baruchel:Servier: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Jazz: Consultancy; Baxalta: Research Funding. June:University of Pennsylvania: Patents & Royalties; Johnson & Johnson: Research Funding; Celldex: Consultancy, Equity Ownership; Pfizer: Honoraria; Immune Design: Consultancy, Equity Ownership; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Tmunity: Equity Ownership, Other: Founder, stockholder . Sen:Novartis: Employment. Zhang:Novartis: Employment. Thudium:Novartis: Employment. Wood:Novartis Pharmaceuticals: Employment, Other: Stock. Taran:Novartis: Employment. Pulsipher:Chimerix: Consultancy; Jazz Pharmaceutical: Consultancy; Novartis: Consultancy, Other: Study Steering Committee; Medac: Other: Housing support for conference.

Description: We have reported a NOD/SCID/γcnull (NOG) mice model, which enable efficient engraftment of human hematopoietic stem cells and their multi-lineage differentiation including T cells. Using this model, we investigated whether various subpopulations of T cells were generated in this unique murine microenvironment. Freshly collected cord blood was depleted of phagocytes with Silica ® followed by CD34 positive selection using auto MACS ®. The purity of CD34 positive cells always exceeded 98%. These cells were transplanted into irradiated NOG mice intravenously. About 3 months after the transplantation, human T cells in peripheral blood, bone marrow and spleen were analyzed by flow cytometry. As we have reported previously, more than half of the human cells seen in the spleen were human CD3+ T cells and as many as 30% of them expressed CD4 and CD25 without activation markers such as CD69. To examine if these CD4+ CD25+ cells have regulatory activity, CD4+ CD25− cells were stimulated with anti-human CD3 antibody along with irradiated autologous antigen presenting cells in the presence of limiting dose of CD4+ CD25+ cells. The inhibition of proliferation by CD4+ CD25+ cells was analyzed by 3H-thymidine uptake. CD4+ CD25+ cells successfully suppressed the CD4+ CD25− T cell proliferation and RT-PCR analysis revealed the expression of Foxp3, a marker for regulatory T cells, specifically in the CD4+ CD25+ cell population. These results suggest that regulatory T cells can develop from hematopoietic stem cells in our NOG mice model. As human T cells appear first in the thymus of NOG mice, these regulatory T cells are considered to arise in the murine thymus. Our model provides a new and versatile tool to investigate development and function of human regulatory T cells, which are often difficult to study because of complicated history of infection or genetic differences among individuals.

Description: Abstract 3248 Background and Purpose: Acute lymphoblastic leukemia (ALL) is the most common type of childhood hematologic malignancy. Although improvements in treatment regimen have raised the 5-year survival rate as high as 80% for pediatric ALL patients, a minority of patients with various risk factors, including central nervous system (CNS) infiltration continue to have poor prognosis. Recently, bone marrow (BM) microenvironments which support leukemic stem cells have become noticed as an important element which can influence treatment response and relapse of the disease. Although leukemic cells appear to be completely eradicated through treatment, they are thought to survive within bone marrow and/or extramedullary microenvironments, such as CNS, causing disease recurrence. However, little is known about the CNS microenvironment for leukemic cells because of the lack of appropriate animal model. Even though several investigators have tried to establish a CNS infiltrated model of leukemia, major limitation with these studies are the use of leukemic cell lines and the preconditioning of recipient mice, which did not represent CNS leukemia observed in patients. Here we report the establishment of a novel xenograft model for primary human ALL using NOD/SCID/γc null (NOG) mouse. Without irradiation, this model recapitulates CNS as well as extramedullary leukemic infiltration (hereby referred to as the h-leukemic NOG model). Result: Primary bone marrow samples were collected from 9 children with ALL at the time of diagnosis with informed consent. The leukemic cells (1×106cells) were injected into the tail veins of non-irradiated 8- to 10-week old NOG mice. Primary samples from 8 out of 9 patients were successfully engrafted. Engrafted leukemic cells could be serially transplanted into secondary, tertiary and quaternary recipients. Morphological and FACS analyses revealed as high as 95% BM chimerism and showed that blast phenotypes were conserved through serial transplantations. Of note, extramedullary organs including the CNS, liver, spleen, and kidneys showed the leukemic invasion consistent with those of the donor ALL patients. Liver pathology in the h-leukemic NOG model is identical to that seen in the ALL patients. We also showed the existence of a functional niche in the liver mediated by SDF-1/CXCR4 axis. In terms of the CNS involvement, we observed the progressive infiltration of leukemic cells into the Virchow-Robin space that is consistent with the pathology of human ALL patients. Using this model, we examined the mechanism of dissemination and harboring of leukemic cells in the CNS niche. Discussion: NOG mice model for engraftment of human leukemic cells provides useful insights into the biology of ALL and allows us to answer various questions concerning the mechanism of extramedullary invasion and expansion. We have reported that NOG mice have significantly better human hematopoietic cell engraftment in the BM and extramedullary organs than other immunodeficient mice (Hiramatsu H. Blood. 2003), and is capable of supporting the growth of human neoplastic cells (Kato M. Nature. 2009). Here we report that this non-preconditioned mouse xenograft model reproduces leukemic extramedullary involvement, including the CNS, in sustaining leukemic cells. This approach provides a more sophisticated and physiological model suitable for the evaluation of molecular interactions between patient leukemic cells and host niche. Our h-leukemic NOG model will provide a powerful tool to analyze the CNS niche that harbors leukemia initiating cells. Moreover, this model would be a useful platform for developing novel anti-leukemic therapies that target CNS extramedullary niche. Disclosures: No relevant conflicts of interest to declare.