bluuebunny/crossref_metadata_2025 huggingface dataset:
PU.1 Is Indispensable for Development of Mature Dendritic Cells and Their Function That Plays a Critical Role in Maintenance of Normal T Cell Pool.
Authors: Iino Tadafumi , Jeong Yong , Mizuno Shin-ichi , Kohno Kentaro , Ito Kyoko , Tenen Daniel G. , Reizis Boris , Akashi Koichi
Published: October 2019

Abstract 1471

Poster Board I-494

PU.1, a hematopoietic transcription factor, is indispensable for development of myelo-lymphoid cells from hematopoietic stem cells (HSCs). PU.1-deficient mice fail to develop common myeloid progenitors (CMPs) or common lymphoid progenitors (CLPs), resulting in complete loss of dendritic cells (DC) in addition to mature myeloid and lymphoid cells. By disrupting PU.1 specifically at the mature DC stage, we here show that PU.1 is necessary for maintenance of mature DC pool and their functions. We crossed PU.1 floxed/floxed mice with a mouse line harboring the Cre transgene driven by the CD11c-BAC. In these mice, PU.1 gene was disrupted in all conventional DCs but not in other hematopoietic cells, including lymphoid cells, myeloid cells and their progenitors. Development of DC precursors such as Lin−c-KitloFLT3+MCSFR+, FLT3+ CLP and FLT3+CMP were not affected. The number of CD11c+B220− DCs, however, significantly reduced in all lymphoid tissues including the thymus, the spleen, the lymph node and the skin, down to <40%, <25%, <10% and <5% as compared with the wild-type control, respectively. Moreover, the number of mature T cells reduced to ∼60% in the spleen as compared to the control. PU.1-deficient DCs displayed impaired functions to induce antigen-driven T cell proliferation, and to produce inflammatory cytokines (TNFa, IL-6, IL-12) in response to Toll like receptor (TLR) stimulation. These results clearly show that PU.1 is required for development of the peripheral DC pool and for maintenance of their immunological functions, which might be required for maintenance of the peripheral T cell pool.

Disclosures:

No relevant conflicts of interest to declare.
Elevated Levels of Tissue Factor Bearing Microparticles in Patients with Antiphospholipid Antibodies: Analysis of Fresh, Unfrozen Samples.
Authors: Khan Mohammad , Cockrell Erin , Espinola Ricardo , Bartholomew John R. , McCrae Keith R.
Published: October 2019

Abstract 148

Introduction:

The antiphospholipid syndrome (APS) is characterized by venous or arterial thrombosis, and/or recurrent fetal loss in the presence of circulating antiphospholipid antibodies (APL). The pathogenesis of this disorder may involve, in part, the activation of vascular endothelial cells, platelets, and monocytes. Upon activation, these cells shed components of their plasma membranes as microparticles (MP), and procoagulant MPs are associated with an increased risk for thromboemboli. The goal of these studies was to characterize, for the first time, the frequency by which increased levels of endothelial cell, platelet and monocyte-derived microparticles are observed in freshly-obtained, unfrozen plasma from patients with APL.

Patients and Methods:

Whole citrated (3.2%) blood samples from patients/healthy donors were centrifuged at 1500xg for 15 min, and the supernatant further centrifuged at 13000xg for 2 min to obtain platelet free plasma (PFP). To detect microparticles, and their origin, PFP was labeled with the following conjugated monoclonal antibodies and analyzed by flow cytometry using an LSR II flow cytometer (BD): Annexin V-APC; CD105-PE and CD144-PE (endothelial cells); CD41-PECy5 (platelets); CD14-PE (monocytes); and anti-tissue factor mAb-Alexa Fluor 647 (a kind gift from Dr. Kenneth Mann, Univ of VT). The binding of control antibodies labeled with identical fluorophores as the monoclonal antibodies was subtracted to adjust for nonspecific binding. We assessed microparticles in 47 patients with APL and 144 healthy controls.

Results

Levels of annexin-V, CD105, CD144, CD41 and tissue factor positive microparticles were significantly higher in patients (47,000±7900, 10,140±2164, 46,690±8783, 127,200±28,050 and 7270±2810 microparticles/ml, respectively) versus controls (17,000±1800, 3,824±374, 17,320±2636, 54,910±7132 and 2,537±747 microparticles/ml, respectively). These differences were statistically significant. In contrast, there was no significant difference in CD14 positive microparticle levels (1035±167 in APL patients versus 1409±465 in controls; p=0.655).

Conclusions:

Our results demonstrate elevation of annexin V+, CD105+, CD144+, CD41+ and tissue factor-bearing microparticles in freshly-obtained, unfrozen plasma obtained from patients with APL. These results suggest that ongoing activation of platelets and endothelial cells occurs in many patients with APL even in the absence of active thrombosis, and may contribute to the prothrombotic phenotype of these patients. Data is currently being analyzed for associations of microparticle levels with APL serologies, as well as the expression of functional microparticle-associated tissue factor activity.

Disclosures:

No relevant conflicts of interest to declare.
Erythropoietin-Associated CFU-E Expansion: A Novel Mechanism Responsible for Erythroid Recovery Following Sublethal Radiation.
Authors: Peslak Scott , Wenger Jesse , Kingsley Paul , Koniski Anne , Bemis Jeffrey , McGrath Kathleen E. , Palis James
Published: October 2019

Abstract 1483

Poster Board I-506

Although the synthesis of two million red blood cells every second is essential for the maintenance of steady state levels of blood cells as well as our well-being, very little is known about the sensitivity of the erythroid lineage to injury or its kinetics and mechanisms of recovery. A novel multispectral imaging flow cytometry analysis utilizing the AMNIS ImageStream and functional colony assays were utilized to study the response of the erythroid lineage in C57Bl/6 mice following 4 Gray whole body irradiation. BrdU cell cycle analysis and erythropoietin ELISA assays were also utilized to investigate erythroid recovery after radiation injury. Two days post-radiation, all erythroid progenitors and precursors in the marrow were severely depleted. Over the next week, early-stage progenitors (BFU-E) only made a partial recovery. In marked contrast, late-stage progenitors (CFU-E) dramatically expanded in numbers at 5-6 days post-radiation to greater than 200% of normal. These CFU-E subsequently generated a wave of erythroid precursors that culminated in the emergence of circulating reticulocytes beginning at 8-9 days post-radiation. Rapid expansion of the CFU-E compartment was associated with an increase in cell cycling. CFU-E expansion was also temporally associated with an acute 20-fold increase in circulating levels of erythropoietin (EPO). Additionally, exogenous EPO administration at 4 days post-radiation resulted in further expansion of CFU-E 2 days later. Interestingly, erythroid progenitors and precursors were not found in the spleen after radiation, indicating that recovery of the erythroid lineage after direct marrow injury is not associated with extramedullary erythropoiesis. We conclude that erythroid progenitors and precursors, unlike circulating erythrocytes, are exquisitely sensitive to clastogenic insults such as radiation. Furthermore, we conclude that the acute recovery of erythropoiesis following sublethal radiation injury is centered on profound expansion and robust cycling of CFU-E. The temporal association of this CFU-E expansion both with the endogenous upregulation and with the exogenous administration of EPO suggests that CFU-E expansion is due primarily to erythropoietin stimulation. We hypothesize CFU-E expansion in the marrow may represent a novel adaptive mechanism of the erythroid lineage to acute injury. Overall, a more thorough understanding of radiation-induced erythroid injury and recovery will ultimately lead to new treatments to protect and mitigate the hematopoietic system from clastogenic agents such as radiation and chemotherapy.

Disclosures:

No relevant conflicts of interest to declare.
TIMP–1 Plays a Functional Role in CD34. + Hematopoietic Stem and Progenitor Cells.
Authors: Wilk C. Matthias , Czibere Akos G. , Cadeddu Ron-Patrick , Buest Sebastian , Schildberg Frank A. , Lauterbach Marcel A. , Westphal Volker , Hell Stefan W. , Haas Rainer , Bruns Ingmar
Published: October 2019

Abstract 1487

Poster Board I-510

TIMP–1 protein (Tissue Inhibitor of Metalloproteinases) is a recently identified tetraspanin interacting cell surface protein in the immortalized human breast epithelial cell line MCF10A. Tetraspanins like CD63 are proteins that consist of four transmembrane domains and are known to interact with integrins. Integrins play a crucial role in hematopoietic stem cell homing and mobilization. We first screened gene array data sets of CD34+ human hematopoietic stem and progenitor cells (HSPCs) and found TIMP–1 mRNA expression. In this study we show that TIMP–1 co-localizes with the tetraspanin CD63 and Beta-1-Integrin. Furthermore, we found a functional interaction of TIMP-1 with its receptor complex on G-CSF mobilized HSPCs.

All experiments were carried out using highly enriched CD34+ cells. Co-immunoprecipitation shows that TIMP–1 binds to CD63. Using high resolution Stimulated Emission Depletion (STED) microscopy we could confirm co-localization of TIMP-1 and CD63 as well as Beta-1 Integrin and CD63. To further characterize the role of TIMP-1 in the Beta-1-Integrin signaling, we used an antibody specific to the active form of Beta-1- Integrin. Flow cytometric analysis revealed a significantly higher number of active Beta-1-Integrin in TIMP-1 stimulated cells suggesting TIMP-1 to activate the receptor complex on CD34+ cells. For functional analysis of the receptor complex formation, transwell migration assays were performed showing significantly increased migratory capacities of TIMP–1 treated cells. Additionally, TIMP–1 stimulation leads to a significantly increased adhesion rate of CD34+ cells to the fibronectin-coated dish. To assess a potential role of TIMP–1 in apoptosis, CD34+ HSPCs were co-incubated with thapsigargin and TIMP–1 or DMSO as a control. Subsequent flow cytometric analysis of cleaved Caspase-3 revealed a decrease of apoptotic cells in the TIMP-1 treated samples.

In summary, we can show that TIMP-1, CD63 and Beta-1-Integrin form a complex on CD34+ HSPCs. TIMP-1 activates the Beta-1-Integrin signaling in HSPCs and alters the adhesive as well as the migratory behavior of CD34+ HSPCs. Furthermore, TIMP-1 induces an antiapoptotic effect in CD34+ cells. The functional effects of TIMP-1 in HSPCs might be of relevance in clinical hematopoietic stem cell transplantation so that we are currently about to verify these effects in an in vivo model.

Disclosures:

No relevant conflicts of interest to declare.
The Rho GTPase Rac1 Is Not Required for Definitive Hematopoietic Specification in ES-Derived Hematopoiesis.
Authors: Milsom Michael D. , Yabuuchi Akiko , Daley George Q. , Williams David A.
Published: October 2019

Abstract 1494

Poster Board I-517

Rac1 is a Rho GTPase involved in integrating signaling pathways that regulate numerous cellular processes including adhesion, migration, proliferation and HSC engraftment. Homozygous deletion of Rac1 is lethal in the murine embryo prior to E9.5 and Rac1−/− embryos demonstrate defective gastrulation associated with reduced epiblast adhesion and motility. We have recently demonstrated using lineage-specific conditional deletion that Rac1 insufficiency results in severely impaired hematopoiesis in the embryonic sites of hematopoiesis (AGM, aortic clusters and fetal liver) in the setting of normal hematopoietic development in the yolk sac (YS) and reduced HSC and progenitors in the fetal circulation. This data appears to support the controversial hypothesis that YS derived HSC seed embryonic sites, but an alternative explanation is that Rac1 is essential for some aspect of the induction of intraembryonic hematopoiesis in situ. Another possibility is that Vav1-Cre-mediated excision of Rac1 occurs prior to the onset of hematopoiesis in the embryo proper but not early enough to affect yolk sac hematopoiesis. To test whether Rac1 insufficiency perturbs the normal early differentiation of hematopoietic cells in vitro, we used a lentivirus expressing a Rac1-specific shRNA to knock down expression in an ES line previously characterized to have good hemogenic potential. We observed that the de novo knockdown of Rac1 expression appeared to have no impact upon derivation of hematopoietic progenitors. To demonstrate that this was not the result of inefficient knockdown of Rac1, we derived Rac1−/− ES lines from blastomeres resulting from the mating of Rac1+/− mice. Rac1−/− ES lines were produced in normal Mendelian ratios (4 Rac1+/+: 9 Rac1+/−: 3 Rac1−/−) and did not demonstrate any evidence of abnormal expansion on murine embryonic fibroblasts. In order to assess the impact of Rac1 deficiency on the hemogenic potential of ES cells, standard in vitro differentiation via embryoid body formation was utilized. Neither Rac1 haploinsufficiency nor complete absence of Rac1 had any impact on the production of CD41+/c-Kit+ hematopoietic progenitors within embryoid bodies (Table 1). Furthermore, colony forming assays demonstrated that Rac1 insufficiency did not alter the relative frequency of hematopoietic progenitor compartments (Table 2). We conclude that in the absence of a requirement for vascular migration of HSC, Rac1 is not required for the specification of definitive hematopoiesis. These data, together with our previously published in vivo data continue to support the hypothesis that HSC migration from the YS to the embryo may be required for development of hematopoiesis in the embryo proper.

Disclosures:

No relevant conflicts of interest to declare.
The California Sickle Cell Database.
Authors: Wun Ted , Brunson Ann , Brown Monica
Published: October 2019

Abstract 1530

Poster Board I-553

INTRODUCTION

The Cooperative Study of Sickle Cell Disease (CSSCD) was a landmark prospective observational study that helped to define the natural history of sickle cell disease. Although this study provided (and is still yielding) a wealth of robust data, there are still gaps in knowledge on long term complications, especially in adults. In addition, the CSSCD was conducted by investigators at specialized centers, and it is possible findings might be different in a population based cohort. Therefore, we wished to use available administrative datasets to study Californians with sickle cell disease (SCD) and create a California Sickle Cell Database (CSCD).

METHODS

We conducted a retrospective study with which to determine complications and health care utilization amongst patients with sickle cell disease. The California Office of Statewide Health Planning and Development (OSHPD) Hospital Discharge Dataset (HDD) collects data on all hospital discharges in California (exclusive of federal facilities) since 1991. Since 2005, all non-federal emergency department (ED) visits have also been reported to the Emergency Department Utilization (EDU) system of OSHPD. Data elements include basic demographics and diagnoses. Patients can be tracked using social security numbers; therefore, longitudinal data on individual patient hospitalizations and ED utilization can be generated. The ICD-9 codes used were 282.6X.

RESULTS

There were 11,351 individual SCD patients identified in the OSHPD HDD (1991-2007) and EDU (2005-2007) data systems. Analysis to date has yielded some basic demographic data on utilization. Females comprised 59.3% of the cohort. As anticipated most patients (81.9%) were non-Hispanic Black. However, 7.9% were reported as non-Hispanic White and 6% as Hispanic. The majority was less than 60 years of age; however, 744 were between 60-69 years and 406 were 70-79 years of age. There were 124,455 admissions of patients with SCD from 1991 to 2007 (mean ± SD =7321 ± 473 per year). 2135 ± 168 patients per year accounted for these hospitalizations with mean hospitalizations per patient of 3.4 with SD's from 3.6 to 4.6. In the three years 2005-2007 inclusive a mean of 1,871 ± 127 patients a year utilized the ED. The mean number of ED encounters per year per patient was 5 with SD's of 13 to 22.

CONCLUSIONS

Limitations include those inherent with any retrospective study dependent on administrative data, including inaccurate coding. In addition, true prevalence could be underestimated due to the acquisition bias of the requirement for hospitalization and/or ED utilization to be counted. Longitudinal data may be affected by migration to other states. Nonetheless, the large number of identified cases in the CSCD will allow determination of rates for many complications of interest that commonly require hospitalization and/or ED utilization, and provide important information on the individual and societal burden of disease.

Disclosures

No relevant conflicts of interest to declare.
Chronic Kidney Disease and Mild Elevations in Pulmonary Arterial Systolic Pressure Are Associated with Increased Mortality in Adult Patients with Sickle Cell Disease.
Authors: Lorch Daniel , Spevack Daniel , Little Jane
Published: October 2019

Abstract 1539

Poster Board I-562

Background

Pulmonary arterial hypertension (PAH) has recently been shown to be an important predictor of prognosis in sickle cell disease (SCD). We studied this association in a large population of adults with SCD.

Methods and Results

215 patients with sickle cell syndromes in whom echoes had been performed, 113 as outpatients and 102 as inpatients, were identified from our database. Clinical characteristics were analyzed, and survival data were extracted from a national mortality registry. At the time of examination, subjects were 39 ± 14 years old, with hematocrits (Hcts) of 23.6 ± 4.4. 78 out of 213 patients who had undergone echoes had PAH (PASP ≥ 40 mmHg) on echocardiogram. Those patients were older (41 ± 15 vs 37 ± 13 years, p=0.03), had higher creatinine values (1.3 vs 0.9 mg/dL, p=0.01) higher serum alkaline phosphatase levels (172.2 ± 23.2 vs 104.5 ± 5.5 U/L, p=.0058), and were more often taking anti-hypertensive medications (38 vs 17%, p<0.001). During a median follow-up of 7.8 years [interquartile range (IR); 3.9, 10.0] there were 30 deaths. In a multivariate Cox proportional hazards model, PAH (hazard ratio (HR) 11.3; 95% CI 2.7, 47.9), chronic kidney disease (CKD) (HR 17.0; 95% CI 3.7, 79.1, defined as an estimated glomerular filtration rate, eGFR, of <60 cc/min/1.73 m2) and hyperbilirubinemia (HR 13.1; 95% CI 2.0, 83.9) independently predicted mortality after adjustment for inpatient/outpatient status, age, sex, blood counts, measurement of chamber size and ventricular function on echocardiogram, oxygen saturation, history of transfusion and background medical therapy. The association of PAH and mortality was not modified by inpatient/outpatient status (p=0.6).

Conclusion

In a community-based academic center, we found that mild elevation in PASP on echocardiogram, whenever obtained (see figure), depressions in eGFR (see figure), and hyperbilirubinemia were highly predictive for mortality in sickle cell disease. It is unclear whether these relationships are causal or an epiphenomenon of severe underlying disease. Hemolysis (lower Hcts, higher Lactate dehydrogenase levels), implicated in other studies, was not prominent in the PAH cohort.

Disclosures

No relevant conflicts of interest to declare.
Increased Efflux of Oxidized Glutathione (GSSG) Causes Glutathione Depletion in Sickle Erythrocytes.
Authors: Nur Erfan , de Waart Rudi , Verwijs Mirjam , Otten Hans-Martin , Brandjes Dees P. , Elferink Ronald P.J. Oude , Biemond Bart J.
Published: October 2019

Abstract 1542

Poster Board I-565

Introduction

Sickle cell disease (SCD) is characterized by increased oxidative stress playing an important role in the pathophysiology of hemolysis, vascular occlusion and organ damage in sickle cell patients. Sickle erythrocytes are both an important source and target of reactive oxygen species (ROS). Levels of both total and reduced form of glutathione (GSH), a major intracellular anti-oxidant, have been demonstrated to be decreased in sickle erythrocytes, despite the increased de novo synthesis of GSH in these cells. The mechanism leading to this depletion of intracellular glutathione in sickle erythrocytes is not known yet. After reaction with ROS, GSH is oxidized into its oxidized form (GSSG) and can be transported actively out of the erythrocyte. We questioned whether, during episodes of increased oxidative stress, GSSG efflux in sickle erythrocytes is higher than in normal erythrocytes.

Materials and methods

Erythrocytes of 10 homozygous sickle cell patients and 9 race-matched healthy controls were stimulated with 2,3-dimethoxy-l,4-naphthoquinone (DMNQ), which induces intracellular ROS generation, and hydrogen peroxide (H2O2) to stimulate GSH consumption. Intra- and extracellular levels of GSH and GSSG were measured at baseline and after 210 minutes of DMNQ and H2O2 stimulation.

Results

While both intra- and extracellular GSSG levels (μM) at baseline were comparable in sickle and control erythrocytes (14.5(11.5–22.7) vs. 14.3(11.6–16.3) and 0.05(0.00–0.19) vs. 0.07(0.00–0.20) respectively), GSSG levels were significantly higher in sickle erythrocytes after 210 minutes DMNQ stimulation (intracellular: 74.4(52.9–93.1) vs. 45.3(40.8–66.7),P=0.005; extracellular: 23.3(18.2–37.3) vs. 13.2(11.1–14.6),P=0.001) which suggests an increased generation of GSSG intracellularly and a resulting elevated efflux to the extracellular environment. These observations were confirmed with H2O2 stimulation of erythrocytes, showing that, while comparable at baseline, the GSSG levels were higher in sickle erythrocytes after 210 minutes stimulation (intracellular: 26.1(22.8–30.1) vs. 17.5(14.2–20.1),P=0.043; extracellular: 6.9(2.3–16.6) vs.1.2(0.6–1.6),P=0.008). In contrast to the control erythrocytes, where intracellular GSH levels remained unchanged, GSH levels decreased significantly in sickle erythrocytes during DMNQ stimulation, suggesting a limited anti-oxidative reserve capacity in SCD.

Conclusion

GSSG efflux in sickle erythrocytes is increased and results in net loss of intracellular glutathione, rendering sickle erythrocytes more susceptible to oxidative damage. The higher rate of GSH consumption during an episode of oxidative exacerbation in sickle erythrocytes suggests a reduced anti-oxidative reserve capacity in SCD.

Disclosures

No relevant conflicts of interest to declare.
Association of Janus Kinase 2(JAK2) A830G Polymorphism with Acute Leukemia Susceptibility.
Authors: Chen Bao-An , Zhong Yue-Jiao , Feng Ji-feng , Li Yu-feng , Ding Jia-Hua , Gao Chong , Cheng Jian , Xia Guo-Hua , Sun Ning , Chen Ning-Na , Zhang Xiao-ping , Xu Pei-pei , Pei Xiao-Ping , Qian Jun , Cheng Lu , Lu Zu-hong
Published: October 2019

Abstract 1573

Poster Board I-599

Background

Aberrant activation of the The Janus kinase(JAK)/signal transducer and activator of transcription (STAT) pathway may predispose to leukemia cells due to deregulation of proliferation, differentiation or apoptosis. One of the members of this family, JAK2, plays a very important role in metabolizing carcinogens and medications. In this study, we aimed to determine whether any association exists between genetic polymorphism in JAK2 A830G and individual susceptibility to acute leukemia.

Method

We carried out a case-control study using a China sample set with 243 acute leukemia cases and 282 controls matched by age, ethnicity. 68 of the acute leukemia patients were diagnosed with acute lymphoblastic leukemia (ALL) and 175 patients with acute myeloid leukemia (AML). Genomic DNA was isolated from peripheral blood and genomic DNA samples were assayed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) in the A830G on JAK2 gene. The data were analyzed statistically employing chi-square and logistic regression analyses.

Result

The frequencies of G/G genotype (wild type) were 4%, 8% and 31.4% in ALL, AML and control groups, respectively. The frequencies of polymorphic G/A genotype (heterozygous variant) were found to be 69% in ALL patients, 71% in AML patients and 48.6% in controls. The A/A genotype (homozygous variant) were existed to be 69% in ALL patients, 20% in AML patients and 20% in controls. Logistic regression analyses showed a significant correlation between the JAK2 A830G polymorphism (G/G) and acute leukemia patients (OR = 1.309, 95% CI =0.839-2.043, p<0.001).

Conclusion

Our findings indicate that G/G genotype may be an important genetic determinant for acute leukemias. According to our knowledge, this is the first report of an association between acute leukemia cases and the JAK2 A830G polymorphism.

Disclosures

No relevant conflicts of interest to declare.
Molecular Characterization of a Novel 3' Mutation in RUNX1 in a New Pedigree with Familial Platelet Disorder.
Authors: Churpek Jane E , Garcia Jacqueline S , Madzo Jozef , Jackson Sarah , Onel Kenan , Godley Lucy A
Published: October 2019

Abstract 1626

Poster Board I-652

Introduction

Familial Platelet Disorder with Propensity to Acute Myelogenous Leukemia (FPD/AML; OMIM 601399) is a rare disorder with an autosomal dominant inheritance pattern characterized by varying degrees of thrombocytopenia, clinical bleeding due to platelet dysfunction, and an increased risk of developing myeloid malignancy. To date, twenty-six families with this disorder have been described and all carry germline RUNX1 mutations as the causative genetic abnormality. The spectrum of RUNX1 mutations includes point mutations within the RUNT domain and frameshift and termination mutations throughout the body of the gene. Here we report identification of a new family with FPD with a novel nonsense mutation resulting in premature protein termination at amino acid 388.

Patients and Methods

Our four generation pedigree includes a mother (II:4) diagnosed with dysplasia and normal karyotype acute myeloid leukemia now in remission after a matched sibling allogeneic stem cell transplant, and her daughter (III:2) with thrombocytopenia since childhood, excessive bleeding with childbirth, and 5q- syndrome diagnosed at 37 years old. Genomic DNA was obtained from all available family members, and RUNX1 cDNA (transcription variants a through c) was sequenced. In addition, RUNX1 cDNA was analyzed for second mutations in bone marrow samples from both patients at the time of diagnosis of their initial bone marrow malignancy.

Results

RUNX1 sequencing of germline DNA revealed heterozygosity for a novel nonsense mutation in exon 8 (c.1163C>A), which is predicted to result in premature protein truncation (p.Ser388X). Full sequencing of RUNX1 cDNA from II:4's AML does not show any secondary mutations. Our current efforts include full sequencing of RUNX1 cDNA from III:2's bone marrow malignancy as well as functional studies of the truncated protein.

Conclusions

We have identified a novel 3' RUNX1 mutation within exon 8, which is predicted to result in premature protein truncation at amino acid 388. To date, this is the most distal mutation identified in an FPD/AML pedigree. The identification of this mutation suggests that the last 100 amino acids, which are known to contain the RUNX1 inhibition domain, contribute an essential function. Further characterization of this RUNX1 mutation and its encoded truncated protein may yield insight into RUNX1's role in leukemogenesis in FPD and de novo AML.

Disclosures

No relevant conflicts of interest to declare.
An in Vitro Model of Differentiation of Memory B Cells Into Plasmablasts and Plasma Cells Including Detailed Phenotypic and Molecular Characterization.
Authors: Jourdan Michel , Caraux Anouk , De Vos John , Fiol Geneviève , Larroque Marion , Cognot Chantal , Bret Caroline , Duperray Christophe , Hose Dirk , Klein Bernard
Published: October 2019

Abstract 1636

Poster Board I-662

Human plasma cells (PCs) and their precursors play an essential role in humoral immune response, but are rare and difficult to harvest. We report here i) the generation of human syndecan-1+ and immunoglobulin secreting PCs starting from memory B cells (MBCs) in a 3-step- and 10-day (D) culture, including a 6-fold cell amplification. ii) We report the detailed phenotypic and Affymetrix gene expression profiles of these in vitro PCs as well as of intermediate cells - activated B cells (actBCs) and plasmablasts (PBs) - compared to MBCs and bone marrow PCs, which is accessible through an open web ATLAS (http://amazonia.transcriptome.eu/). iii) We show this B cell to PC differentiation to involve IRF4 and AICDA expression in D4 actBCs, decrease of PAX5 and BCL6 expressions and increase in PRDM1 and XBP1 expressions in D7 PBs and D10 PCs. It involves downregulation of genes controlled by Pax5, induction of genes controlled by Blimp-1 and XBP1 (unfold protein response). iv) The phenotype of D10 PCs resembles that of peripheral blood PCs detected after immunization of healthy donors. This in vitro model will facilitate further studies in PC biology. It will likewise be helpful to study plasma-cell dyscrasias, including Multiple Myeloma.

Disclosures

No relevant conflicts of interest to declare.
Erythropoietin Treatment Is Associated with Augmented Immune Response to the Influenza Vaccine in Hematologic Patients.
Authors: Oster Howard S , Prutchi-Sagiv Sara , Halutz Ora , Golishevski Nataliya , Binyamin Moshe , Orkin Ruth , Hoffman Michael , Neumann Drorit , Mittelman Moshe
Published: October 2019

Abstract 1637

Poster Board I-663

Introduction

Recombinant human erythropoietin (EPO) has been effective in treating various types of anemia. EPO receptors on non-erythroid cells suggest that EPO may also have non-erythroid effects. Indeed, we have previously shown that EPO has anti-neoplastic immunomodulating effects in both patients and mice (Mittelman PNAS 2001; Mittelman Eur J Haematol 2004). EPO effects were demonstrated in both the cellular and humoral immune systems (Katz Acta Haematol 2005; Katz Eur J Immunol 2007; Prutchi-Sagiv Br J Haematol 2006; Prutchi-Sagiv Exp Hematol 2008; Lifshitz Mol Immunol 2009). Hassan et al. (Ren Fail 2003) have reported that patients with renal failure treated with EPO had a higher antibody (Ab) titer in response to the Hepatitis B vaccine. We therefore hypothesized that EPO plays a general role in augmenting the immune response. To test this hypothesis we examined the effect of EPO on the antibody response to the seasonal influenza (flu) vaccine.

Methods

Three groups, all receiving the flu vaccine, were tested: A) Healthy controls (HC), B) Hematologic patients not receiving EPO (H-NoEPO), C) Hematologic patients on EPO for anemia (H-EPO). Mean ages of the participants [95% CI] were 59.5 [56.5-62.4], 59.6 [50.9-68.2], and 73.9 [68.8-79.0] respectively. Flu vaccine and EPO were ordered by the attending physicians only, irrespective of the study. Blood samples were drawn prior to and 3 weeks, 7 weeks and 4 months after the flu vaccination. Anti-flu Ab titer was measured using the complement fixation test. The results are presented as the number (percentage) of tested individuals who responded with at least two- or four-fold increase in anti-flu titers, compared to their baseline. P value represents comparison of H-EPO to H-NoEPO.

Results

Of the HC individuals, 90% doubled (at least) and 58% quadrupled their anti-flu Ab titers. Of the H-NoEPO patients only 56% doubled and 19% quadrupled their titers. However, 72% of the H-EPO patients doubled and 56% quadrupled their titers, a response approaching that of healthy controls, despite their being significantly older.

Conclusions

1) Hematologic patients respond poorly to the flu vaccine, compared to healthy subjects. 2) EPO treatment is associated with an improved immune response to the flu vaccine in hematologic patients. This suggests that EPO may serve as an immune augmenting agent for the flu vaccine and possibly other vaccines as well.

Disclosures

Off Label Use: Non erythroid effects: immune, anti-cancer (all under investigation). Prutchi-Sagiv:BioGAL- Start up (inactive): Equity Ownership. Mittelman:BioGAL- Start up (inactive): Equity Ownership, Patents & Royalties.
Predictive role of Early Interim FDG-PET in Hodgkin Lymphoma.
Authors: Stefoni Vittorio , Broccoli Alessandro , Alinari Lapo , Ambrosini Valentina , Derenzini Enrico , Fanti Stefano , Fina Mariapaola , Gandolfi Letizia , Marchi Enrica , Pellegrini Cinzia , Quirini Federica , Zinzani Pier Luigi , Baccarani Michele
Published: October 2019

Abstract 1659

Poster Board I-685

Background

Hodgkin lymphoma (HL) is a curable malignancy with a long-term survival of around 80%. FDG-PET is a noninvasive imaging modality widely used in lymphoma patients. Early PET assessment of response to therapy is a routine part of management in HL patients, and an independent, strong predictor of progression-free survival.

Patients and Methods

178 patients, with a diagnosis of HL, underwent to an early PET evaluation during their course of chemotherapy and were considered eligible for the study. 85 patients (48%) were male and 93 (52%) female; the median age at diagnosis was 33 (13-78) years. 6 patients (3%) had stage I disease; 106 patients (60%) stage II; 34 (19%) stage III and 32 (18%) stage IV (bone marrow involvement in 5 cases).

B-symptoms were detected in 81 patients (46%). A mediastinal bulk was detected in 54 cases (30%). The majority of patients (173, 97%) underwent to ABVD as first line therapy; 5 received BEACOPP chemotherapy (3%). Early PET evaluation was performed after the second course of therapy. Results were classified into complete response (CR), partial response (PR), stable disease (SD) or progressive disease (PD) according to International Workshop standardized response criteria. PET scan was performed again at the end of the first-line treatment. 44 patients have been addressed to a second-line therapy, in presence of PR, PD or relapsing disease; in particular, 39 patients received an autologous stem-cells transplantation (ASCT), and 3 an allogeneic bone marrow transplantation (ABMT).

Results

At a median follow up of 41,85 (5,23-141,77) months, 152 patients are alive and in CR; 7 in PR; 3 alive with SD and 7 present a PD. 9 patients have died. 150 patients presented with a negative PET after 2 cycles, and 28 with a positive one (26 in PR, 1 with SD and 1 with PD). More specifically, of the 178 initial patients, 150 (84%) had a negative early PET and 28 (16%) a positive early PET. Of those with a negative PET, 135 (90%) experienced a continuous CR, while among those with a positive early PET, none obtained at least a stable CR. Of this unfavourable group of patients, 9 (32%) reached, and still maintain, a CR after ASCT.

Conclusions

Our experience indeed confirms the highly predictive value of a negative early PET during the therapy for HL. Moreover we may suggest the potential role of ASCT in inducing a CR in around one-third of those unfavorable patients with a positive early interim PET.

Disclosures

No relevant conflicts of interest to declare.
Efficacy and Safety of Liposomal Cytarabine as Intrathecal Prophylaxis in Patients with Diffuse Large B Cell Lymphoma at High Risk of CNS Involvement: A Multicentric Study Including 80 Patients in Spain.
Authors: de la Fuente Adolfo , Salar Antonio , Panizo Carlos , Navarro Belen , Olave Teresa , Peñarrubia María Jesus , Herrero JoaquÃn , Tomas Jose Francisco , Canales Miguel , González-Barca Eva
Published: October 2019

Abstract 1663

Poster Board I-689

Introduction

Lymphomatous meningitis (LM) in patients with Diffuse Large B Cell Lymphoma (DLBCL) is usually an early complication and with poor prognosis. Risk factors have been previously identified for this complication. DepoCyte is an extended released liposomal cytarabine formulation (LC) which has demonstrated better efficacy compared to standard cytarabine for the treatment of LM in one randomized clinical trial.

Purpose and Methods

A retrospective study was carried out in 24 Spanish sites including patients diagnosed of DLBCL and at risk of LM – defined by the presence of at least one of the following: retroperitoneal mass ≥ 10 cm; Waldeyer ring, sinus, vertebral or bone, and testicular involvement; LDH more than twice the upper normal limit; bone marrow involvement > 30% and serology VIH+. All patients had received LC as IT prophylaxis for LM in the period April 2005 – June 2009. Main endpoints were effectiveness (leptomeningeal involvement rate) and safety of the IT prophylaxis.

Results

Data from 80 patients were analyzed. Baseline characteristics were: Mean age 55 ± 16 years (range: 18-80 years). Males 74%; Ann Arbor stage IV 54%. All patients received alkylating based regimens, being R-CHOP as the most frequent one (88%). LC was administered as intrathecal prophylactic treatment for LM in all patients.

64 patients completed the IT prophylaxis treatment with a mean of 2,8 ± 0,83 administrations and a median follow up of 17 months (range: 3-40 months) (8 patient still on treatment and 8 patients died before finish prophylaxis). Just one patient (1,6%) had leptomeningeal spread: 76 years old man with primary testicular DLBCL, treated with R-CHOP regime, who did not reach complete response and died after 8 months of follow up due to respiratory failure.

Twenty four patients out of eighty (30%) showed adverse events, being headache the most frequent side effect (27%), and 14% grade IV. Headache was reversible in all cases. Chemical arachnoiditis prophylaxis was given to 70 patients.

Conclusions

This retrospective study has shown that in DLBCL patients and high risk of LM, prophylaxis with LC was feasible and well tolerated. With a median follow up of 17 months (range: 3-40 months) the incidence of LM was 1,6 %. Prospective, randomized comparative studies versus conventional prophylaxis regimes are needed.

Disclosures

Off Label Use: DepoCyte is an extended released liposomal cytarabine formulation (LC) which has demonstrated better efficacy compared to standard cytarabine for the treatment of LM in one randomized clinical trial. Prphylaxis effectiveness..
A Phase II Study of Temsirolimus (CCI-779) in Combination with Rituximab in Patients with Relapsed or Refractory Mantle Cell Lymphoma.
Authors: Ansell Stephen M , Tang Hui , Kurtin Paul , Koenig Patricia , Inwards David J , Shah Keith , Witzig Thomas E
Published: October 2019

Abstract 1665

Poster Board I-691

Mantle cell lymphoma (MCL) is an aggressive, incurable B-cell malignancy that represents approximately 6% of lymphoma cases. Patients with MCL who relapse after conventional therapy or stem cell transplantation have a poor prognosis and are candidates for treatment with novel agents. The characteristic feature of MCL is overexpression of cyclin D1. Cyclin D1 is under the control of the phosphatidylinositol-3 kinase signal-transduction pathway and is downstream of the mammalian target of rapamycin kinase (mTOR). We have previously shown that temsirolimus (CCI-779), a dihydroester of the selective mTOR inhibitor rapamycin, is an active antitumor agent in MCL. In previous phase 2 trials of temsirolimus as a single agent, we observed a 40% overall response rate (ORR) with a median duration of response (DR) of 6 months in patients with relapsed or refractory disease (J Clin Oncol 23:5347-56, 2005; Cancer 113:508-514, 2008). Due to the fact that rituximab has improved the response rate and overall outcome of lymphoma patients when combined with chemotherapy, we tested the efficacy and toxicity of temsirolimus in combination with rituximab in patients with MCL.

Eligible patients with confirmed relapsed or refractory MCL based on morphologic and phenotypic features received temsirolimus 25 mg intravenously every week and four weekly doses of rituximab with the first cycle and then one dose of rituximab every other cycle. Patients with a tumor response after six cycles were eligible to continue treatment for a total of 12 cycles or two cycles after complete remission, and were then observed without maintenance.

Seventy-one patients were enrolled between May 2005 and March 2009. The median age was 67 years (range, 44 to 86 years), with 51 males and 20 females enrolled. Patients had received a median of two prior therapies (range, 1-9), 31% had received a prior stem cell transplant and 28% were rituximab refractory. The ORR was 48% (34/71 patients) with 20% (14/71) complete responses and 28% (20/71) partial responses. The median DR was 9.5 months (95% CI, 4.5 to 11.3 months). The median DR in the rituximab sensitive patients was 9.5 months (CI: 4.5-11.6) and 7.15 months (CI: 4.1-11.3) in the rituximab refractory patients (p= 0.73). While the combination was generally well tolerated, 12 patients experienced grade 4 toxicity and 2 patients died while on therapy – both from progressive disease. Hematologic toxicities were the most common, with 5 patients having grade 4 thrombocytopenia and 3 having grade 4 neutropenia.

This study demonstrates that temsirolimus in combination with rituximab is well tolerated and has substantial antitumor activity in relapsed MCL. Further comparative studies of this combination in MCL are therefore warranted.

Disclosures

Witzig: Wyeth: Patents & Royalties.
Final Report of a Phase-II Study of Rituximab Plus ABVD for Patients with Newly Diagnosed Advanced Stage Classical Hodgkin Lymphoma.: Results of Long Follow up and Comparison to Institutional Historical Data.
Authors: Copeland Amanda R , Cao Yumei , Fanale Michelle , Fayad Luis , McLaughlin Peter , Pro Barbara , Hagemeister Fredrick , Romaguera Jorge , Samaniego Felipe , Rodriguez Alma , Younes Anas
Published: October 2019

Abstract 1680

Poster Board I-706

We previously reported (Wedgwood et al, ASH, 2007) improvement in event free survival (EFS) using Rituximab+ABVD (RABVD) in newly diagnosed patients with advanced stage classical Hodgkin lymphoma (HL) when compared to the previously published historical data from the International Prognostic Score (IPS) project using standard ABVD (Hasenclever et. Al, NEJM, 1998). The purpose of this report is to provide a final update on the EFS with long term follow up of patients who received RABVD. In addition, because of the potential difference in the EFS reported by the IPS project and a single institution results, we provide a new analysis comparing our RABVD data to that of historical data from our institution. After IRB approval, we conducted a retrospective data analysis on patients with advanced HL treated at our center with ABVD from February 1996 to May 2006. Information was collected for the IPS factors: age, stage, gender, hemoglobin, white blood cell count, albumin, and lymphocyte count. 104 consecutive patients who met the eligibility criteria for the RABVD study were identified, and were evaluable for response with at least 2 year follow up. Median age was 35 years old, 48 were female and 56 male. 23 (22%) had stage II disease, 12 of these with bulky disease, 45 (43%) stage III and 36 (35%) with stage IV. 65 (63%) had IPS 0-2 and 39 (37%) had IPS >2. The institutional 5-year EFS for ABVD patients regardless of IPS was 66%. For those with IPS 0-2 the institutional EFS was 71% compared with 74% reported by the IPS project. The institutional 5 year EFS for patients treated with ABVD with IPS >2 was 55%, compared with 55% reported by the IPS project. With a median of 5 year follow up (6-94 months) the projected EFS for RABVD is 87% which is significantly better than institutional results with ABVD (p=0.0036). Improvement in EFS was observed with RABVD in patients with IPS 0-2 (EFS 89% vs. 71%, p=0.0248); and those with IPS >2 (80% vs. 55%; p=0.0532). In conclusion, this long term follow up data confirms the superiority of RABVD to ABVD in patients with advanced stage classical HL in all risk groups. This data serves as the rationale for a multicenter randomized trial comparing ABVD with RABVD in newly diagnosed patients with classical HL with stage III and IV and IPS >2. The study is currently enrolling patients.

Disclosures

Off Label Use: Rituximab in Hodgkin lymphoma. Younes:Genentech: Honoraria, Research Funding.
Incidence and Prevalence of Patients with Myelodysplastic Syndromes (MDS) in Düsseldorf 1996-2005.
Authors: Neukirchen Judith , Schoonen W. Marieke , Aul Carlo , Haas Rainer , Gattermann Norbert , Germing Ulrich
Published: October 2019

Abstract 1774

Poster Board I-800

Background

Population-based data on myelodysplastic syndromes (MDS) are scarce. Since its inception in 1982, the Düsseldorf MDS Registry has captured 3598 patients with MDS, of whom 21 % live in the town district of Düsseldorf. Between 1996-2005 we are confident that all MDS patients residing in the town district of Düsseldorf were entered and followed in the registry through regular follow-ups. As yearly age- and sex-specific population counts are also available for Düsseldorf, the Registry provides a unique opportunity to estimate MDS disease frequency. Here we aim to quantify the incidence and prevalence of MDS disease subtypes.

Methods

Patients were identified from the MDS Registry. Age- and sex-specific yearly population counts were obtained from the Statistical Office of North-Rhine Westphalia. The number of residents in Düsseldorf minimally increased during the study period from 1996 to 2005 (mean: 571,000 residents). The number of patients with a first-time MDS diagnosis in a given calendar year (incident cases) was divided by the total Düsseldorf population in that year to estimate incidence. Prevalence was estimated by dividing the number of existing plus incident MDS patients in a given year by the total population of Düsseldorf that year. Incidence (per 100,000 person-years (PY) and prevalence per 100,000 persons are presented with corresponding 95% confidence intervals (CI) calculated using the delta and Wilson methods, respectively. Low-risk MDS was defined as WHO subtypes RA, RCMD or MDS with del(5q). High-risk included subtypes RAEB-I and RAEB-II.

Results

344 MDS patients were included in our analyses (279 incident patients). Incidence and prevalence of low-risk MDS was 2.87 (95%CI 2.46–3.35) per 100,000PY and 12.4 (95%CI 11.5–13.4) per 100,000 persons, respectively, with no difference between men and women (Table). High-risk disease was less common. The incidence of high-risk disease in men appeared to be higher (1.22 (95%CI 0.87–1.72) per 100,000PY) than in women (0.63 (95%CI 0.40–0.99) per 100,000PY). Prevalence of high-risk disease among men was statistically significantly higher in men compared to women (2.93 (95%CI 2.35 – 3.65) per 100,000 persons) and 1.56 (95%CI 1.17 – 2.08) per 100,000 persons), respectively (Table).

Conclusion

In this population-based study we found that MDS more common in men than in women. The majority of MDS cases had low-risk disease. Incidence and prevalence of high risk disease (but not low risk) is higher in men compared to women.

Financial disclosures: This study was supported by Amgen Inc.

Disclosures

Neukirchen: Amgen Inc.: Research Funding. Schoonen:Amgen Inc.: Research Funding. Aul:Amgen Inc.: Research Funding. Haas:Amgen Inc.: Research Funding. Gattermann:Amgen Inc., Celgene, Novartis: Honoraria, Research Funding. Germing:Amgen Inc., Celgene, Novartis: Honoraria, Research Funding.