bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microfluidic and Flow Cytometric Studies Support a Role for Monocytes and Coated Platelets in the Prothrombotic State in Heparin-Induced Thrombocytopenia (HIT)
Authors: Tutwiler Valerie , Ahn Hyun Sook , Cines Douglas B. , Camire Rodney M. , Poncz Mortimer , McKenzie Steven , Rauova Lubica 
Published: October 2019

Abstract
Abstract 539
HIT is an immune thrombocytopenia associated with a high risk of developing thrombosis. A passive immunization murine model of this disorder has provided important insights into the underlying pathogenesis of this disease, but is limited by its inability to study human cells and limited ability to define the contribution of various hematopoeitic and vascular cells to the prothrombotic state. We used a microfluidic system in conjunction with flow cytometry to further our understanding of the prothrombotic nature of HIT. Platelet adhesion and aggregation was studied in whole blood labeled with Calcein AM, perfused through a microfluidic channel (BioFlux 200 system, Fluxion) coated with von Willebrand factor (vWf) at shear stress of 20 dyne/cm2 at 37°C. A 40–60% increase in platelet adhesion (relative area covered by platelets) with up to a 4 fold increase in average aggregate size was seen in the presence of the pathogenic HIT-like monoclonal antibody (moAb) KKO (50 μg/ml) in conjunction with PF4 (10 μg/ml) when compared to control samples with PF4 only or with PF4 plus a non-pathogenic anti-PF4 moAb RTO (p <0.01). Monocyte-depletion decreased platelet aggregation by 20 – 40% relative to whole blood or after monocyte-repletion (P<0.0001). In HIT, thrombin plays a key role in the formation of platelet aggregates. Addition of thrombin inhibitor PPACK to the whole blood stimulated by KKO and PF4 decreased thrombus formation in the microfluidic chamber by 40% (p<0.001). Coated platelets are prothrombotic and characterized by phosphatidylserine (PS) exposure and binding of FVa and FXa. This activated state requires dual stimulation via thrombin and ITAM receptors. Flow cytometric studies of annexin V and FXa binding showed extensive induction of coated platelets in whole blood by KKO plus PF4 in contrast to PF4 or PF4 plus RTO (annexin V: p<0.0001; Factor Xa p<0.01). These new studies, focused on human blood, support our finding in the passive murine HIT model as to the importance of monocytes to thrombus formation and suggest that the prothrombotic nature of HIT may also be promoted by the generation of coated platelets. Identification of coated platelets may also lead to new diagnostic tests and new therapeutic interventions in HIT.

Disclosures:
No relevant conflicts of interest to declare.
Enrichment of Allogeneic Tumor Antigen-Specific T Cells From Bone Marrow (BM) of Patients Treated with High-Dose Post-Transplant Cyclophoshamide (Cy) – A Novel Approach to Adoptive Immunotherapy
Authors: Noonan Kimberly , Luznik Leo , Borrello Ivan M. 
Published: October 2019

Abstract
Abstract 647

Background:
Donor lymphocyte infusions (DLI) has limited efficacy and is associated with substantial graft vs host disease (GVHD) in treating relapse after allogeneic BMT (alloBMT) in large part due to the absence of significant tumor specificity. Thus, novel strategies are required that increase the tumor specificity with reduced alloreactivity. High-dose Cy post-BMT effectively reduces GVHD through early elimination of allo-reactive T cells and enables immune reconstitution free of ongoing pharmacologic immunosuppression. Since the BM is both the site of disease in most hematologic malignancies and a reservoir of tumor specific T cells, we hypothesized that marrow infiltrating lymphocytes (MILs) collected after alloBMT in patients treated with high-dose Cy could be a source of tumor-specific T cells for adoptive immunotherapy.

Methods:
BM was obtained from patients 2 –12 months after HLA-matched alloBMT on the clinical trial using high-dose post-transplant Cy as a single agent GVHD prophylaxis. The MILs were activated and expanded with CD3/CD28 antibody-coated beads.

Results:
Allo-MILs can be reproducibly expanded (574-fold avg expansion; 14–2000) at all time points tested. The activated allo-MILs are not anergic, exhibit anti-HLA-reactivity against third party allo-antigens but do not respond to recipient allo-antigens. Tumor-specific MILs were significantly expanded as determined by reactivity to HL60/K562 myeloid cell lysates (P<0.0001; pre- vs. post-activation). In contrast, no change was observed in cultures pulsed with irrelevant antigens, thus suggesting the specificity of the response. The ability to expand allogeneic antigen-specific MILs was also confirmed with a 15.5 fold expansion of PR-1 specific (HLA) A*0201-restricted CD8+ T-cells using tetramers.

Conclusions:
We can augment the tumor-specificity of MILs obtained post-transplant with minimal allo-reactivity in patients treated with post-BMT high-dose Cy. This could represent a novel approach to highly tumor-specific DLI. A phase I/II trial is planned to assess the safety and feasibility of administering ex vivo expanded post-transplant, allogeneic MILs to patients with relapsed hematologic malignancies after alloHSCT.

Disclosures:
No relevant conflicts of interest to declare.
Genetic Susceptibility Loci and Risk of Infant Leukemia by Subtype and MLL status: A Children's Oncology Group Report
Authors: Ross Julie A. , Linabery Amy M. , Blommer Crystal , Langer Erica , Spector Logan G. , Hilden Joanne M. , Heerema Nyla A. , Tower Richard L. , Davies Stella M. 
Published: October 2019

Abstract
Abstract 749

Introduction.
Leukemia in infancy is extremely rare and etiologically distinct from leukemia arising in older children. Infant leukemia cases likely arise in utero, typically involve rearrangements of the mixed lineage leukemia (MLL) gene, and have a much poorer survival than older children with leukemia. Since genetic determinants of susceptibility to infant leukemia are likely different from those in older children, we aimed to evaluate genetic variants previously identified by genome wide association studies of childhood acute lymphoblastic leukemia (ALL).

Methods.
We genotyped candidate susceptibility loci in 171 Caucasian infants less than 12 months of age with acute leukemia (including 102 ALL, 67 acute myeloid leukemia (AML), and 2 biphenotypic cases) diagnosed during the period 1996–2006. Controls consisted of 384 healthy Caucasian blood donors. Associations between genetic polymorphisms in IKZF1, ARID5B, and CEBPE and infant leukemia were evaluated using logistic regression and were further stratified by leukemia subtype and presence or absence of MLL gene rearrangements.

Results.
We observed a statistically significant association with two copies of the IKZF1 rs11978267 variant allele in infant leukemia overall (Odds Ratio (OR)=2.3, 95% Confidence Interval (CI)=1.3-4.2), but not with the other susceptibility loci. The association with IKZF1 was concentrated in the AML group, with an increased risk associated with two copies of the variant allele (OR=3.9, 95%CI=1.8-8.4), but not one copy. The association was similarly strong in those with and without a MLL gene rearrangement. In contrast, a strong association with the IKZF1 variant was seen in infants with ALL lacking a MLL rearrangement (OR=5.1, 95%CI=1.8-14.5), but not in those with MLL rearrangements (OR=0.7, 95%CI=0.2-2.2). Moreover, infants with ALL and no MLL rearrangement had a strong association with a genetic variant in the gene, ARID5B (rs10821936, OR=7.2, 95%CI=2.5-20.6), which was also not seen in those with ALL and a MLL rearrangement.

Discussion.
IKZF1 (IKAROS)is expressed in early hematopoiesis, and its role in lymphopoiesis and lymphoid leukemia is well-described. IKAROS is also expressed in precursor myeloid cells, and our report is the first evidence that IKZF1 is important in the etiology of infant myeloid leukemia, irrespective of MLL gene rearrangements. Moreover, our data indicate striking differences in genetic susceptibility to infant ALL with and without rearrangements of the MLL gene. This knowledge could provide important new etiologic insights into this extremely rare but heterogeneous hematopoietic malignancy. Supported by NIH CA079940, T32 CA099936, K05 CA157439 and the Children's Cancer Research Fund, Minneapolis, MN.

Disclosures:
No relevant conflicts of interest to declare.
MEK Inhibition Modulates the Growth of Nf1 mutant Hematopoietic Cells and Induces Clinical Improvement in a Murine Model of Juvenile Myelomonocytic Leukemia (JMML)
Authors: Chang Tiffany Y. , Krisman Kimberly , Harding-Theobald Emily Rose , Braun Benjamin S. , Shannon Kevin M. 
Published: October 2019

Abstract
Abstract 797FN2
Neurofibromatosis type 1 (NF1) is a dominant genetic disorder that is caused by germ line mutations in the NF1 tumor suppressor gene. Children with NF1 have a 200- to 500-fold increase in the risk of developing malignant myeloid disorders, particularly JMML, an aggressive myeloproliferative neoplasm (MPN) characterized by over-production of myeloid lineage cells that show extensive tissue infiltration, anemia, and thrombocytopenia. The only curative therapy for JMML is hematopoietic stem cell transplantation, and 50% of children relapse after HSCT and die of recurrent JMML or of transformation to acute myeloid leukemia (AML). Somatic inactivation of Nf1 in hematopoietic cells causes a progressive MPN in Mx1-Cre, Nf1flox/flox mice that closely models JMML. We administered the MEK inhibitor PD0325901 (901) to Mx1-Cre, Nf1flox/flox mice with MPN to investigate the therapeutic effects of inhibiting Raf/MEK/ERK signaling in vivo and to interrogate the underlying mechanism of any observed response. Mx1-Cre, Nf1flox/flox mice (n = 35) and WT littermates (n = 38) were randomized to receive 901 (5 mg/kg/day) or control vehicle at 6 months of age. Treatment was continued for 10 weeks or until the mice became moribund. Mx1-Cre, Nf1flox/flox mice that received 901 demonstrated a rapid reduction in leukocyte counts and enhanced erythropoiesis. In addition, spleen size was markedly reduced with correction of aberrant proliferation and differentiation of progenitor cells. Flow cytometric analysis showed that 901 treatment restored a normal pattern of erythroid differentiation and greatly reduced splenic hematopoiesis. Remarkably, genetic studies revealed persistence of Nf1-deficient hematopoietic cells despite dramatic improvement in hematologic abnormalities. Based on these data, we conclude that treatment with 901 rebalances the proliferation and differentiation of Nf1 mutant cells in vivo rather than eliminating them. These preclinical data and a recent study in Kras mutant mice with MPN (Lyubynska N, et al. Science Trans Med 2011; 3: 76ra27) support clinical trials of MEK inhibitors in JMML and chronic myelomonocytic leukemia and suggest differentiation of malignant cell as a potential mechanism of response to this class of targeted agents.

Disclosures:
No relevant conflicts of interest to declare.
Inotuzumab Ozogamycin (I0), a CD22 Monoclonal Antibody Conjugated to Calecheamicin, Is Active in Refractory-Relapse Acute Lymphocytic Leukemia (R-R ALL)
Authors: o'Brien Susan , Thomas Deborah A. , Ohanian Maro , Jorgensen Jeffrey L. , Kebriaei Partow , Jabbour Elias , Rytting Michael E. , York Sergernne , Ravandi Farhad , Kwari Monica , Faderl Stefan , Rios Mary Beth , Cortes Jorge E. , Fayad Luis , Tarnai Robert , Wang Sa A. , Champlin Richard , Advani Anjali S. , Kantarjian Hagop M. 
Published: October 2019

Abstract
Abstract 875

Background:
Patients (pts) with R-R ALL have a poor prognosis. CD22 is highly expressed in ALL. IO is active in CD22-positive lymphomas. Methods: Pts with R-R ALL received IO 1.8 mg/m2 IV over 1hr every 3 weeks. First 3 pts received 1.3 mg/m2 in course 1. Children were eligible for study after 10 adults were treated; first 3 children received 1.3 mg/m2. Study started June 2010. Results: 49 pts were treated. Median age 36 yrs (range 6–80); 12 pts ≥ 60 yrs; Ph+ in 7, t(4:11) in 5, other abnormalities in 25. CD22 expressed in > 50% blasts in all pts, > 90% in 28. Median number courses so far 2 (1–5). 13 pts received IO as salvage 1 (S1), 24 as salvage 2 (S2), 12 as ≥ salvage 3 (S3). 7 pts had allo SCT before IO. Overall, 9/49 pts (18%) achieved CR, 5 had marrow CR (10%), 14 had CRp (29%), 21 were resistant, 2 died within 4 wks. Overall, CR+mCR+CRp (OR) was 57%: S1 9/13 (54%); S2 11/24 (46%); S3 8/12 (67%). A lower response rate was noted in Ph-positive ALL (3 of 7 = 43%) and in t (4;11) (1 of 5 = 20%) otherwise there were no clear correlations between OR and pts/ALL characteristics or with CD22 expression ≥ vs. < 90%. In 24 pts with evaluable pharmacokinetics studies: 3-hr I0 level > 100 ng/ml associated with OR in 8/9 vs. 5/15 if I0 level < 100 ng/ml. Median survival 5.1 months: S1 not reached (65% at 8 mos), S2 4.1 mos, ≥ S3 6.7 mos. Median response duration, CR+mCR+CRp (OR), not reached (estimated 70% at 8 mos). Median survival in 28 responders 7.9 mos. Fever and mild hypotension occurred in most pts on days 1–2 and resolved with support. Liver function abnormalities (LFA) were noted as IO related in 32 (67%), severe in 4 (13%). 2 pts had liver biopsies showing periportal fibrosis (after 1 and 4 courses). 20 pts were able to proceed to allo SCT (19 in remission, 1 with active ALL); 5 pts post-allo SCT developed veno-occlusive disease, 2 receiving second allo SCT, 4 receiving thio-TEPA + clofarabine as part of their preparative regimen. Conclusions: IO is highly active in R-R ALL. LFAs occur and are reversible. Study of IO weekly schedule is ongoing. IO and chemotherapy combinations should be pursued.

Disclosures:
Cortes: Pfizer: Consultancy, Research Funding. Kantarjian:Novartis: Consultancy, Research Funding; Pfizer: Research Funding; BMS: Research Funding.
Iron Containing Compound Stimulates Expression of Pulmonary Hypertension Promoting Factor PlGF
Authors: Wang Xunde , Kato Gregory J. , Mendelsohn Laurel 
Published: October 2019

Abstract
Abstract 900
We have previously reported elevated plasma levels of placental growth factor (PlGF) and endothelin-1 (ET-1) in patients with sickle cell disease with high estimated pulmonary artery systolic pressure, a marker of pulmonary hypertension, and gene transfer experiments in mice document that PlGF stimulates ET-1 expression and pulmonary hypertension. Among other markers, PlGF in SCD subjects is correlated with markers of excessive iron burden, including serum ferritin and transferrin, markers that have been previously associated with pulmonary hypertension in SCD in many studies. We therefore hypothesized that iron stimulates expression of PlGF. Because tissue postnatal expression of PlGF in vivo is restricted primarily to early erythroid cells, we chose K562 erythroleukemia cells as a cell culture model. We find that heme-bound iron (hemin) induces PlGF mRNA robustly in a dose-dependent and time-dependent fashion. The PlGF transcript rises robustly within hours of hemin stimulation, and reaches levels of 40 to 80-fold over baseline in a real time PCR assay. Heme analog compound, mesoporphyrin without iron fails to induce PlGF, but as a control induces hemeoxygenase-1 mRNA. Iron plus mesoporphyrin induces PlGF strongly, indicating a critical requirement for iron in inducing PlGF transcript. In promoter-reporter luciferase constructs transfected into K562 cells, hemin induces the human PlGF promoter significantly, and a minimal promoter construct contains binding sites for erythroid Kruppel-like factor (KLF-1). KLF-1 mRNA levels rise in hemin-stimulated K562 cells in parallel with PlGF, and in peripheral blood mRNA from SCD subjects and healthy controls, the level of KLF-1 transcript correlates closely with PlGF transcript (r=0.82, p<0.0001). Additional studies of KLF-1 are under way.
Our results indicate for the first time a specific mechanistic pathway induced by iron that is linked in humans with SCD and in mice to vasculopathy and pulmonary hypertension. Rather a simple epiphenomenon of frequent transfusion or source of nonspecific oxidative stress, iron may trigger a KLF-1/PlGF/ET-1 linear mechanistic pathway to stimulate pulmonary hypertension. This suggests that PlGF levels are linked to the iron overload in patients with SCD. Further details in this pathway are under investigation.

Disclosures:
No relevant conflicts of interest to declare.
The Notch Signaling Pathway as a Suppressor of Myeloid Transformation
Authors: Aifantis Iannis 
Published: October 2019

Abstract
Abstract SCI-13
Notch signaling is a central regulator of differentiation in a variety of organisms and tissue types. Its activity is controlled by the multi-subunit γ-secretase complex (γSE) complex. Although Notch signaling can play both oncogenic and tumor suppressor roles in solid tumors, in the hematopoietic system, it is exclusively oncogenic, notably in T cell acute lymphoblastic leukemia (T-ALL), a disease characterized by Notch1 activating mutations. We identified somatic inactivating Notch pathway mutations in a fraction of chronic myelomonocytic leukemia (CMML) patients. Inactivation of Notch signaling in mouse hematopoietic stem cells (HSC) results in an aberrant accumulation of granulocyte/monocyte progenitors (GMP), extramedullary hematopoieisis and the induction of CMML-like disease. Transcriptome analysis reveals that Notch signaling regulates an extensive myelomonocytic-specific gene signature, through the direct suppression of gene transcription by the Notch target Hes1. These studies identify a novel role for Notch signaling during early hematopoietic stem cell differentiation and suggest that the Notch pathway can play both tumor-promoting and suppressive roles within the same tissue. These observations also suggest that Notch activity is not simply a promoter of the T cell lineage in the thymus but that Notch signaling thresholds could regulate commitment and/or survival of distinct hematopoietic lineages in the bone marrow. To address these issues in vivo, we have generated Notch receptor lineage tracing and activity reporter genetic tools. Analysis of these animal models identified unique novel functions for the Notch pathway during early bone marrow hematopoiesis.

Disclosures:
No relevant conflicts of interest to declare.
Role of Notch Signaling in Hematopoietic Stem Cell Function
Authors: Espinosa Lluis , Bigas Anna 
Published: October 2019

Abstract
Abstract SCI-15
The Notch pathway controls the generation of different cell types in most tissues including blood, and dysregulation of this pathway is strongly associated with oncogenic processes. In many systems, Notch is also required for the maintenance of the stem cell populations. However, in the adult hematopoietic system this link between Notch and stemness has not been established. Instead, work of several groups, including ours, has clearly demonstrated that Notch has a prominent role in the generation of hematopoietic stem cells (HSC) during embryonic development. Although the first wave of blood cells appears in the mouse embryo around day 7.5 of development and is independent of Notch function, embryonic HSC are formed around day 10 of development from endothelial-like progenitors that reside in the embryonic aorta surrounded by the gonad and mesonephros, also called AGM region. By analyzing different Notch pathway mutant mouse embryos, we have demonstrated the involvement of the Jagged1-Notch1-GATA2 axis in this event. However, the formal demonstration that Notch regulates the GATA2 gene during HSC generation is still lacking. We have now found that GATA2 is a direct Notch target in vivo during embryonic HSC generation. However, whereas Notch positively activates GATA2 transcription in the HSC precursors, it simultaneously activates hes1 transcription, which acts a repressor of the same GATA2 gene. This finding directly implicates hes1 in the regulation of HSC development although further studies using loss-of-function mutant embryos are still needed. Altogether, our results indicate that both Notch and hes1 are required to finely regulate the levels, distribution, and likely the timing of GATA2 expression through an incoherent feed-forward loop. In parallel, we have identified other downstream targets of Notch in the AGM region by ChIP-on-chip and expression microarray analysis that we are currently characterizing.

Disclosures:
No relevant conflicts of interest to declare.
Clinical Insights Derived From B-Cell Immunodeficiency Disorders
Authors: Rawlings David 
Published: October 2019

Abstract
Abstract SCI-19
Over the last four decades, the genetic defects leading to more than 140 distinct inherited immune disorders have been discovered. A subset of these diseases primarily impact B cell development, homeostasis, and/or activation. This talk will provide an overview of these B cell disorders. It will highlight new information regarding human immune function that has been gained from studying these disorders including: the role of key receptors or signaling effectors in immune development or activation, how such changes may impact pathogen specific versus autoimmune responses, and how targeting these pathways may provide a means to modulate other more common human immune diseases. Finally, we will provide a brief overview of efforts to correct one of these disorders in patients using genetic therapies.

Disclosures:
No relevant conflicts of interest to declare.
p53-Mediated Stress and Tissue-Dependent Cell Fate Decisions; Implications for p53 Targeting
Authors: Lozano Guillermina (Gigi) 
Published: October 2019

Abstract
Abstract SCI-3
The activity of the p53 tumor suppressor is tightly regulated by Mdm2, an E3 ubiquitin ligase that targets p53 for degradation. Loss of Mdm2 in embryos or in individual tissues in vivo results in cell lethal events that are p53-dependent. A hypomorphic allele of p53, p53515C (encoding the p53R172P protein), rescues the embryonic lethality of Mdm2–/– mice. Mdm2–/– p53515C/515C mice, however, die by postnatal day 13 due to hematopoietic failure. In these mice, hematopoiesis is normal during embryogenesis. After birth, these mice had elevated reactive oxygen species (ROS), which activated p53R172P. p53R172P, in turn, induced ROS, creating a loop that caused cell death in the hematopoietic compartment. This phenotype was partially rescued with antioxidant treatment. The cell cycle inhibitor, p16, was also stabilized due to ROS, and its loss increased cell cycling and partially rescued hematopoiesis and survival. Thus, Mdm2 is required to control ROS-induced p53 levels for sustainable hematopoiesis.

Disclosures:
No relevant conflicts of interest to declare.
Platelet Cytokines and Vascular Diseases
Authors: Weber Christian 
Published: October 2019

Abstract
Abstract SCI-36
During the past decade it has become increasingly clear that platelets exert important functions in the context of inflammation, beyond their role in hemostasis. Platelets may adhere to intact endothelial cells and promote local vascular inflammation by recruiting leukocytes via direct interactions or by secreting inflammatory mediators such as cytokines and chemokines. Accordingly, platelet-derived chemokines play a crucial role in directing leukocytes to sites of vascular injury or dysfunction, thereby contributing to neointimal hyperplasia or atherosclerosis. In addition, platelet-derived cytokines can shape the local inflammatory environment. In this overview, I will discuss the function of platelets as immune cells that potentiate vascular inflammation with a special focus on platelet-derived chemokines: their effects and interactions and their potential quality as targets for the treatment and/or prevention of cardiovascular disease.

Disclosures:
Weber: Carolus Therapeutics: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.