bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigation of the Morphology of Recombinant Von Willebrand Factor by Two High-Resolution Microscopic Techniques
Authors: Rottensteiner Hanspeter , Seyfried Birgit K , Friedbacher Gernot , Allmaier Guenter , Ilk Nicola , Sleytr Uwe B , Ehrlich Hartmut J. , Turecek Peter L
Published: October 2019

Abstract 1409

Von Willebrand factor (VWF) is the largest known glycoprotein circulating in human blood plasma and is composed of a series of multimers with molecular weights ranging from 600 to 20,000 kDa or even more. In this study, we investigated the morphology of recombinant VWF (rVWF) and compared it to that of plasma-derived VWF (pdVWF) by tapping mode atomic force microscopy and transmission electron microscopy.

Tapping mode atomic force microscopy showed that both rVWF and pdVWF contain globular and stretched domains. Mean chain lengths of the filaments and diameters of the core globular domains were determined and analyzed on a statistical basis. About 70% of the rVWF and pdVWF molecules were 100–300 nm long. The portion of very long molecules (>300 nm) was only slightly greater in rVWF than in pdVWF (20% versus 18%). The diameters of the globular core structures were in the range of 12 to 30 nm for both types of VWF. Inspection of a purified rVWF dimer revealed a similar range for the globular domain (14-32 nm). Upon exposure to high shear stress, a dramatic conformational change was observed for rVWF, just as has been reported for pdVWF.

Investigation of negatively stained preparations of rVWF by transmission electron microscopy showed a highly organized structure. The heterogeneity in size observed was as expected for a polymeric VWF that consists of a mixture of multimers of varying sizes. The micrographs also revealed the presence of coiled and elongated structures. Analysis of pdVWF led to similar results, which were also in agreement with data from the literature.

The results confirmed that the morphology of rVWF is similar to that observed for VWF purified from human normal plasma.

Disclosures:

Rottensteiner: Baxter Innovations GmbH: Employment. Seyfried:Baxter Innovations GmbH: Employment. Friedbacher:Vienna University of Technology: Consultancy. Allmaier:Vienna University of Technology: Consultancy. Ilk:University of Natural Resources and Applied Life Sciences: Consultancy. Sleytr:University of Natural Resources and Applied Life Sciences: Consultancy. Ehrlich:Baxter Innovations GmbH: Employment. Turecek:Baxter BioScience: Employment.
Recombinant Dimeric IgA Antibodies as Tumor-Specific Agents.
Authors: Dechant Michael , Lohse Stefan , Derer Stefanie , Beyer Thomas , Klausz Katja , Peipp Matthias , Leusen Jeanette H. W. , van de Winkel Jan G. J. , Valerius Thomas
Published: October 2019

Abstract 1488

Dimeric IgA antibodies contribute significantly to the humoral part of the mucosal immune system. However, their potential as immunotherapeutic agent has hardly been explored. Here, we describe the production, purification and functional evaluation of recombinant dimeric IgA, exemplarily directed against the epidermal growth factor receptor (EGF-R). Human J-chain-containing IgA was produced under serum-free conditions by transfecting non-adherent CHO-K1 cells expressing EGF-R specific monomeric IgA with a vector coding for the His-tagged human J-chain. For purification of J-chain-containing dimeric IgA two different affinity (anti-human-kappa and anti-His-tag) and one size exclusion chromatography were combined, resulting in a homogenous preparation of highly pure IgA dimers, as determined by gel electrophoresis under denaturing and native conditions using silver stain, Coomassie blue or Western blots for protein detection, respectively. Functional studies demonstrated dimeric IgA (dIgA) to be at least as effective as monomeric IgA (mIgA) in triggering ADCC of A431 tumor cells by isolated monocytes (EC50 0.35 and 0.30 μg/ml for mIgA and dIgA, 0.23 μg/ml for the corresponding IgG1; maximal killing 51.2 vs 43.3 vs 56.3 %), by isolated PMN (EC50 0.96, 1.20 and 0.8 μg/ml for mIgA, dIgA and IgG1; maximal killing 52.6 vs 50.6 vs 19.9 %) and in human whole blood assays (EC50 0.32 and 0.32 μg/ml for mIgA and dIgA; maximal killing 7.9 vs 12.5 %; IgG1: maximal killing 0.7 %, EC50 not determinable). Importantly, dimeric IgA was more effective in F(ab)-mediated mechanisms: inhibition of binding of FITC-labelled EGF to A431 cells by dimeric IgA was achieved at significantly lower concentrations than by monomeric IgA (EC50 7.1 vs 18.1 μg/ml, respectively). In addition, growth of EGF-R expressing DiFi colon carcinoma cells was inhibited at significantly lower concentrations by dimeric than by monomeric IgA (EC50 1.6 vs 3.7 μg/ml). Both IgA isoforms and their IgG1 variant were similarly effective in triggering apoptosis of DiFi cells, as analyzed by PARP cleavage. Furthermore, only dimeric but not monomeric IgA or IgG was directionally transported by the polymeric immunoglobulin receptor (pIgR) through an epithelial cell monolayer, as analyzed by an experimental transcytosis assay with polarized MDCK cells stably transfected with human pIgR. Together, these studies demonstrate that recombinant dimeric IgA antibodies recruit a distinct repertoire of effector functions compared to monomeric IgA or IgG1 antibodies, trigger important biological functions and constitute an interesting antibody format for future tumor therapies.

Disclosures:

van de Winkel: Genmab: Employment, Membership on an entity's Board of Directors or advisory committees. Valerius:Genmab: Consultancy, Research Funding.
Extended Molecular and Clinical Phenotype of Human G6PC3 Deficiency.
Authors: Boztug Kaan , Rosenberg Philip S. , Böhm Marie , Moulton Thomas , Curtin Julie , Rezaei Nima , Corns John , Innis Jeffrey , Avci Zekai , Tran Hung Chi , Pellier Isabelle , Gatti Simona , Fruge Rachel , Parvaneh Nima , Darbyshire Phil , Buchanan George R. , Alter Blanche P , Boxer Larry , Donadieu Jean , Welte Karl , Klein Christoph
Published: October 2019

Abstract 1495

Severe congenital neutropenia (SCN) is a heterogenous group of disorders characterized by an increased susceptibility to bacterial infections. Recently, our discovery of G6PC3 deficiency in 12 patients with SCN and various developmental features highlighted the role of glucose metabolism in the viability of neutrophil granulocytes. To further delineate the molecular and clinical phenotype of this complex syndrome, we analyzed a second cohort of 23 SCN patients referred to us for molecular analysis of G6PC3 mutations. All patients had at least one syndromic feature in addition to congenital neutropenia such as such as congenital heart defects, urogenital malformations or increased venous marking. Among these 23 patients, we identified 14 patients with biallelic mutations in G6PC3. 10 patients had novel mutations in G6PC3. A comprehensive review of the clinical characteristics of these patients underlined the phenotypic variability of G6PC3 deficiency. In addition to known manifestations including cardiac (14/14) and urogenital malformations such as cryptorchidism (3/14), novel features such as facial dysmorphy (11/14) or malformation of the outer genitalia (4/14) were found. No obvious genotype-phenotype correlations could be established. All patients except one had a good response to treatment with G-CSF characterized by increased peripheral neutrophil counts and decreased frequency and severity of infections.

To assess the risk of leukemogenesis, we performed a meta-analysis comparing the 14 G6PC3-deficient SCN patients combined with the 12 patients in the original publication with a cohort of 374 patients SCN patients bearing mutations in ELANE or HAX1, in which 61 developed MDS or AML. The rate of MDS/AML was found to be significantly lower in G6PC3-deficient patients (p=0.02). Our analysis suggests that the risk of transition to MDS/AML may be lower in G6PC3-deficient SCN compared with other genetically defined SCN subgroups.

Disclosures:

No relevant conflicts of interest to declare.
Patterns of HLA-DR Surface Expression on CD14+ Monocytes During Adverse Events After Hematopoietic Stem Cell Transplantation.
Authors: Döring Michaela , Müller Ingo , Erbacher Annika , Handgretinger Rupert , Hofbeck Michael , Kerst Gunter
Published: October 2019

Abstract 1499

The human leukocyte antigen DR (HLA-DR) surface expression on CD14+ monocytes reflects the degree of activation of these cells. In line with the central role of monocytes and macrophages in the immune system, a decreased HLA DR expression on CD14+ monocytes has been shown to be a hallmark of an altered immune status during the systemic inflammatory response syndrome (SIRS). We therefore hypothesized that HLA-DR expression might likewise be altered after hematopoietic stem cell transplantation (HSCT).

HLA-DR surface expression of CD14+ monocytes was assessed by flow cytometry in 30 pediatric patients (below 15 years, n=23) and young adults (15 years and above, n=7) up to one year after HSCT (7 autologous and 23 allogeneic stem cell transplants). Normal values were derived from a control group of healthy children (n=18) and young adults (n=22).

During the conditioning period, a significant increased HLA-DR expression in pediatric patients and young adults was observed. This increase could not be attributed to the administration of anti-thymocyte globulin (ATG). After HSCT, HLA-DR expression was not altered in general. However, HLA-DR expression decreased significantly up to 5 days before (p<0.05) and during (p<0.01) bacterial infections or sepsis. In contrast, HLA-DR expression levels were increased 7–10 days before and at the time of diagnosis of viral infections. HLA-DR expression was also elevated during acute graft-versus-host disease (GVHD). At the time of hepatic veno-occlusive disease (VOD), HLA-DR expression on CD14+ monocytes was reduced. Neither the administration of granulocyte colony-stimulating factor (G-CSF) nor the occurrence of a relapse was associated with a change of HLA-DR expression.

In conclusion, adverse events after HSCT were associated with altered HLA-DR expression levels. Therefore, HLA-DR expression on CD14+ monocytes appears as a promising parameter which might allow identifying patients at risk after HSCT.

Disclosures:

No relevant conflicts of interest to declare.
Increased Prevalence of Neoplasms In Patients Diagnosed with Common Variable Immunodeficiency.
Authors: Grossman William J , Bonnet Patrick O , Xiong Yan
Published: October 2019

Abstract 1531

Introduction:

Common variable immunodeficiency (CVID) is a heterogenous disorder characterized by hypogammaglobulinemia, poor specific antibody responses, and increased numbers of infections. In addition, patients with CVID have been reported to carry an increased risk of developing other co-morbidities, including autoimmunity and certain types of neoplasms. Data establishing the overall risk and prevalence of these co-morbidities in patients with CVID has been limited due to the small sizes of patient cohorts examined to-date, and the lack of comprehensive primary immunodeficiency registries. The objective of this study was to document the prevalence rates of lymphoma select neoplasms in a large cohort of patients with CVID.

Methods:

The MedStat MarketScan database, which contains approximately 48 million patients, was evaluated for the number of IVIG-treated CVID patients using the 279.06 ICD-9 code for CVID over a 6-year period (2002-2008). Ten-fold the number of randomly selected patients, based on age, gender and length of enrollment, were used for matched control comparisons to our CVID cohort. Each cohort was evaluated for the rate of select neoplasms using ICD-9 codes for malignant lymphatic and hematopoietic tissue neoplasms (ICD-9 codes 200 to 208).

Results:

814 CVID patients with at least 18 months of data were identified in the database, with 61% being female and 39% being male. The average age for females and males was 45 and 37 years, respectively (p<.0001). The CVID cohort carried a significantly higher prevalence of having a diagnosis code for a malignant neoplasm of the lymphatic and hematopoietic tissue than the control cohort [10.1% vs. 0.6%, respectively (p<.0001)]. Most of these neoplasm diagnoses occurred after the diagnosis of their CVID; however, 26.8% of neoplasm diagnoses occurred before the diagnosis of CVID. Additionally, the rates for each of the select neoplasms examined showed a significantly higher rate in the CVID cohort than in the control cohort.

Conclusions:

This study showed that CIVD patients have a higher prevalence of being co-diagnosed with certain neoplasms than matched controls. These data also suggest that CVID patients should not only be evaluated longitudinally for the development of neoplasms, but that certain cancer patients may also need to be considered and evaluated for underlying immune deficiencies such as CVID.

Disclosures:

Grossman: Baxter BioScience: Employment. Bonnet:Baxter BioScience: Employment. Xiong:BioScience: Employment.
Resource Use and Expenditures Among Adult Sickle Cell Patients with and without Depression
Authors: Kamble Shital , Reed Shelby D , Flahiff Charlene , Adam Soheir , DeCastro Laura M
Published: October 2019

Abstract 1534

Objective:

Depression is commonly associated with less favorable medical outcomes among patients with sickle cell disease (SCD), yet little is known about its associated impact on medical resource use and expenditures. In this study, we descriptively compared inpatient stays, number of hospital admissions, emergency department (ED) visits, outpatient visits, and expenditures for SCD patients with and without depression.

Methods:

150 adult SCD patients were prospectively enrolled in a cross-sectional cohort study in 2009 to evaluate the prevalence of depression and its association with quality of life, disease severity scores measuring end organ damage, and health care utilization. Detailed cost accounting and administrative physician billing records from the Duke University Health System were obtained for all enrolled patients and used to generate estimates of medical care utilization and costs. Indexing on the enrollment date, we included data representing one year prior to and six months following study entry. Based on the Beck Depression Inventory (BDI) scores, we categorized SCD patients into those with depression (BDI score ≥14) and those without depression (BDI score <14). Given the skewed distributions of resource use and expenditures, we used generalized linear regression models (GLM) with negative binomial distributions and log links to compare inpatient stays, number of hospital admissions, ED visits, and outpatient visits and GLMs with gamma distributions and log links to compare costs associated with each resource use category between patients with and without depression.

Results:

Data from 142 patients analyzed, 81 females and 61 males with a mean age of 34.2 years. We identified 50 patients with depression and 92 patients without depression. At study entry, females represented 72% of SCD patients with depression and 49% of SCD patients without depression (P< 0.01). Median age was 32.5 years among patients with depression and 29.5 years among those without depression (P= 0.22). Hospital admissions, ED visits, and outpatient visits were generally similar between patients with and without depression one year prior to and six months after study entry. Patients with depression spent more days in the hospital during both time periods. Total inpatient expenditures, including physician fees paid, were higher for patients with depression than those without depression for both time periods (Table).

Conclusion:

SCD patients with depression incur higher expenditures and longer stays than SCD patients without depression. Efforts should be made for early diagnosis and active therapeutic intervention for depression among SCD patients, to decrease health care utilization and cost.

Disclosures:

No relevant conflicts of interest to declare.
GSK3β Signaling Regulates the Motility of Hematopoietic Progenitors Via Prune.
Authors: Lapid Kfir , D‘Uva Gabriele , Kalinkovich Alexander , Ludin Aya , Golan Karin , Itkin Tomer , Zollo Massimo , Lapidot Tsvee
Published: October 2019

Abstract 1553

One of the hallmarks of hematopoietic stem and progenitor cells (HSPC) is their motility. In steady state, HSPC are mostly retained in the bone marrow (BM), allowing ongoing hematopoiesis, concomitantly with slow release to the circulation as part of homeostasis and host defense mechanisms. While stress-induced recruitment and clinical mobilization processes are extensively studied, steady state egress mechanisms are poorly understood. In this study, we demonstrate that inhibition of Glycogen Synthase Kinase 3b (GSK3β) directly or via upstream Insulin-like Growth Factor-1 (IGF-1) signaling limited murine HSPC egress to the circulation. Indeed, inhibition of GSK3β resulted in reduced HSPC migration capacity towards a gradient of the chemokine stromal derived factor-1 (SDF-1, also termed CXCL12) in vitro and was found to reduce HSPC mobilization by IGF-1 receptor antagonist treatment. Interestingly, GSK3β signaling also regulated SDF-1 transcription by BM stromal cells in vitro and in vivo, probably as part of HSPC maintenance, since murine CXCR4 signaling is essential for hematopoietic stem cell quiescence. We revealed that the involvement of GSK3β in directional HSPC motility is mediated by the downstream phosphodiesterase Prune. Prune, which is over-expressed in several human cancers, was recently found to localize in focal adhesion sites, promoting the motility of malignant cells. Herein, we show that Prune is also expressed in normal leukocytes, including HSPC. Accordingly, inhibition of Prune resulted in reduced SDF-1 induced migration of murine HSPC in vitro as well as reduced steady state egress in vivo. Prune activity was also shown to regulate the actin cytoskeleton by contributing to its polymerization. In general, highly regulated actin turnover is essential for spontaneous and directional motility mechanisms. Altogether, we present GSK3β and Prune as novel players in physiological HSPC motility, dictating an active rather than passive nature for steady state egress from the BM reservoir to the blood circulation as part of homeostasis.

Disclosures:

No relevant conflicts of interest to declare.
Synergy Between Kit Ligand (KL) and IL-4 In Mast Cells Is Mediated by Cross-Receptor Interactions In Lipid Rafts.
Authors: Mani Maheswaran , Venkatasubrahmanyam Shivkumar , Yang Yujun , Krampf Mark , Huang Jing , Butte Atul , Jahn Thomas , Weinberg Kenneth I.
Published: October 2019

Abstract 1564

Studies of erythroid and thymic differentiation have shown that cross-receptor interactions between the stem cell factor receptor Kit, a receptor tyrosine kinase (RTK), and tissue-restricted Type I cytokine receptors (EpoR and IL-7R, respectively) are necessary for normal development of each lineage. To determine whether these Kit-Type I cytokine receptor interactions are a ubiquitous phenomena in Kit (+) hematopoietic cells, we studied murine bone marrow derived mast cells (BMMC), which express Kit and IL-4R, and are responsive to both cognate ligands. Both KL and IL-4 were individually mitogenic; combinations of KL and IL-4 synergistically promoted BMMC proliferation, even at suboptimal concentrations of each ligand (Synergy Index 2.7 for SCF 20ng/ml + IL-4 5ng/ml). Similar to the results seen previously with Kit and EpoR or IL-7R, activation of Kit by KL resulted in cross-receptor tyrosine phosphorylation of the IL-4Rα and γc subunits of IL-4R, even in the absence of their cognate ligand, IL-4. Each subunit of the IL-4R was independently phosphorylated by activated Kit, in the absence of Jak3. Furthermore, in the malignant mast cell line HMC-1, inhibition of oncogenic Kit by imatinib also reversed constitutive phosphorylation of IL-4R. Previous studies have shown that STAT 1α, STAT 5A, and STAT 5B, but not Stat6, are bound to and directly phosphorylated by activated Kit. In contrast, STAT6 is activated by engagement of IL-4R by its cognate ligand. Cross-receptor phosphorylation of IL-4R by activated Kit in BMMC induced STAT6 phosphorylation, with the same apparent pI as after activation of IL-4R by cognate ligand. Subcellular fractionation showed that activated Kit, γc, Jak3, and STATs were localized in lipid raft fractions upon KL stimulation. Inhibition of lipid raft formation by MβCD resulted in loss of both cross-receptor tyrosine phosphorylation of IL-4R by Kit and synergistic proliferation, but not proliferation induced by each cognate ligand. Gene expression analyses of KL stimulated BMMC from wt and IL-4Rα/γc deficient mice demonstrated that over 30% of the Kit gene signature was dependent on the presence of the IL-4R. Together, the data indicate that the synergy of KL and IL-4 in BMMC is mediated by cross-receptor interactions between Kit and IL-4R in raft membrane microdomains. The Kit-IL-4R interaction is the third cross-receptor interaction described between the Kit RTK and a tissue-restricted Type I cytokine receptor. Besides RBC, thymocytes, and mast cells, such cross-receptor interactions are likely to be a general mechanism for synergistic and tissue-specific pleiotropic signaling by Kit in hematopoiesis and possibly other cell types, e.g., various Kit (+) stem cell populations and cancers.

Disclosures:

No relevant conflicts of interest to declare.
Treatment with mTOR Inhibitor, Everolimus (RAD001) Overcomes Resistance to Imatinib In Ph-Leukemia Quiescent Cells.
Authors: Kuwatsuka Yachiyo , Minami Yosuke , Minami Miho , Tanizaki Ryohei , Kiyoi Hitoshi , Naoe Tomoki
Published: October 2019

Abstract 1579

Recent studies suggest that leukemia stem cells (LSCs) are responsible for relapse of leukemia and eradication of LSCs should be necessary for the cure. In order to examine mechanisms of drug resistance in Ph+ leukemia to imatinib (IM) due to stem cell properties and to seek strategies to overcome the resistance, we've previously established in vivo-murine and ex vivo-culture models using murine hematopoietic pluripotent progenitors transduced with BCR-ABL (Minami, et al., PNAS, 2008). Furthermore, Ph+ leukemia (including T315I-, F311I-mutated CML-BC, or Y253H-mutated Ph-ALL) patient cells were serially xenotransplanted into immunodeficient NOD/SCID/IL2rγnull (NOG) mice. Engrafted bone marrow and spleen cells were almost identical to the original leukemia cells as to phenotypes including karyotypes and distribution of primitive populations. Spleen cells from leukemic NOG mice were treated ex vivo with IM and growth factors, and cell viablility (PI/AnnexinV-staining) was compared between treated and non-treated cells. After treatment with IM, significantly more residual cells were observed in the CD34+/38- population compared to the CD34+/38+ or CD34-/38+ populations. Phosphorylation of BCR-ABL and CrkL was completely inhibited in all populations with IM treatment. Regarding cell cycle states, a higher percentage of quiescent slow-cycling (Hoechst 33342low/Pyronin Ylow) cells was observed in the CD34+/38- population relative to the other populations. Recently, aberrant activation of mTOR signaling has also been reported to be involved in LSCs. Leukemic spleen cells in longer co-culturing with stromal cells were treated with IM and mTOR inhibitor, everolimus (Eve, RAD001). While slow-cycling CD34high+ cells were insensitive to IM, combination treatment with IM and Eve induced significant cell death also in the quiescent population. In vivo-treatment with Eve decreased tumor cells in leukemic NOD mice inoculated with the leukemic NOG cells under the sub-lethally irradiated condition. Treatment with Eve also reduced long-term colony formation of Ph+ leukemia patient samples including Y253H-mutated Ph-ALL with less toxicity to normal cord blood cells. These results imply that treatment with Eve is promising for overcoming the resistance to IM due to quiescent property in Ph+ LSCs.

Disclosures:

Naoe: Kyowa-Kirin: Research Funding; Novartis: Research Funding; Janssen: Research Funding.
Inhibition of Plasminogen Activator Inhibitor (PAI)-1 Corrects Diabetic CD34+ Dysfunction.
Authors: Hazra Sugata , Jarajapu Yagna P.R. , Liu Li , Caballero Sergio , Stepps Valerie , Bhatwadekar Ashay , Boulton Michael E. , Higgins Paul J. , Bartelmez Stephen H. , Grant Maria B.
Published: October 2019

Abstract 1601

Objective:

The dysfunction of human diabetic CD34+ endothelial progenitor cells limits their utility in autologous cell therapy for vascular complications. Previously, we showed that transient inhibition of transforming growth factor-beta 1 (TGF-β1) enhances vascular reparative function of human CD34+ cells isolated from diabetics (Bhatwadekar et al, 2010). Expression of PAI-1, the major gene product of TGF-β1 activation, is increased by high glucose and insulin exposure in endothelial cells and PAI-1 has been shown to be increased in the serum of diabetics. We asked whether the beneficial effects of TGF-β1 blockade on CD34+ cells function were mediated by inhibition of PAI-1 and whether blocking of PAI-1 could correct diabetes associated dysfunction of these cells.

Research Design and Methods:

Plasma determinations of PAI-1 and TGF-β1 (both measured by ELISA) were compared in type 2 (n=17) and type 1 (n=7) diabetic patients. CD34+ cells from these individuals were isolated and analyzed for cell survival (in the presence and absence of growth factors), cell proliferation, cell cycle analysis and migration. The effect of TGF-β1 phosphorodiamidate morpholino oligomers (PMO) treatment on PAI-1 level was determined in CD34+ cells. In CD34+ cells, PAI-1 was blocked using either lentivirus expressing PAI-1 shRNA or PAI-1 siRNA. In vivo homing ability of PAI-1 inhibited CD34+ cells was assessed using an ocular model of ischemia/reperfusion (I/R) Injury.

Results:

Plasma PAI-1 level was increased in type 2 diabetic patients compared to type 1 (p<0.05) and directly correlated with TGF-β1 plasma levels (r= 0.44). TGF-β1 PMO treatment resulted in a reduction of PAI-1 mRNA expression (p=0.0018 in diabetic, p=0.05 in non-diabetic). PAI-1 blockade promoted EPC proliferation in vitro and bypassed the inhibitory effect of TGF-β1 on cell survival (p<0.001) even in the absence of growth factors. PAI-1 blockade enhanced the migration of these cells in response to SDF-1α in (p<0.01) compared to cells treated with scrambled siRNA and improved the in vivo re-endothelialization by CD34+ cells in the I/R model.

Conclusions:

Our results suggest that the cytostatic activity of TGF-β1 in CD34+ cells is mediated largely through PAI-1. Blocking PAI-1 corrects multiple defects in CD34+ cells from type 2 diabetic patients. This approach may offer a promising therapeutic strategy for restoring vascular reparative function in diabetic cells and facilitate their use in autologous cell therapy.

Disclosures:

No relevant conflicts of interest to declare.
Characterization of HbF Decline In Compound Heterozygotes for HbS and Deletional Hereditary Persistence of Fetal Hemoglobin
Authors: Ngo Duyen A. , Aygun Banu , Akinsheye Idowu , Steinberg Martin H. , Hankins Jane S , Chui David H.K.
Published: October 2019

Abstract 1626

In gene deletion hereditary persistence of fetal hemoglobin (HPFH) affecting people of African descent, more than 80 kb of DNA is deleted including the δ- and β-globin genes and downstream sequences. Compound heterozygotes for sickle hemoglobin (HbS) and HPFH have high fetal hemoglobin (HbF) levels and have few if any sickle cell disease-related complications. Among patients with sickle cell disease who were referred for hemoglobin diagnostic testing, 28 compound heterozygotes for HbS and HPFH (6 HPFH type 1 and 22 HPFH type 2) were identified. The diagnosis of HbS and HPFH 1 and 2 were confirmed by DNA-based specific gap-PCR tests. We characterized factors associated with HbF and its changes over time to better understand the modulation of HbF in this population. Laboratory information were obtained through chart review and/or extracted from information provided at the time of hemoglobin diagnostic testing. Data regarding age at which HbF was measured allowed us to study the change in HbF over time. For comparison, we analyzed data collected through the Cooperative Study of Sickle Cell Disease (CSSCD) containing over 3000 HbF measurements at different ages in HbS homozygotes (HbSS) or HbS-β0 thalassemia patients. Statistical analyses were performed using Stata/SE11.1 software. In 28 HbS-HPFH cases, 62 HbF values at different ages ranging from the newborn period to age 20 years were available for analysis. In these patients, HbF was 50% to 90% during infancy and declined steeply within the first few years of life, stabilizing between ages 3 to 5 years. Mean HbF at age 5 or older was 31 ± 2%. Mean hemoglobin concentration was 13 ± 1g/dL and mean MCV was 75 ± 6 fL. Age was log-transformed for all analyses. In univariate and multivariate regression analyses, HbF was significantly associated with age, hemoglobin concentration, and MCV (P<0.001). There was no relationship with gender or genotype of HPFH. There was a strong inverse correlation between HbF and age (r=-0.9, P<0.001). Likewise, in patients with HbSS or HbS-β0 thalassemia from the CSSCD database, HbF was significantly associated with age, hemoglobin concentration, and MCV. In the CSSCD population alone, HbF was also associated with female gender. Patients with compound heterozygosity for HbS and HPFH have a logarithmic age related decline in HbF with marked decline during the first few years of life and plateauing at a HbF of approximately 30% in adulthood. Although these patients maintained a higher HbF after early childhood, their HbF is subjected to a similar age-related decline as seen in patients with other genotypes of sickle cell disease. Future studies aimed at understanding age related regulation and distribution of HbF in these and other patients may offer insight into mechanisms that can halt decline of HbF and ameliorate sickle cell disease.

Disclosures:

No relevant conflicts of interest to declare.
CBL Mutations In Pediatric Acute Myelogenous Leukemia Are a Rare Event, a Report From the Children's Oncology Group.
Authors: Kutny Matthew A. , Miller Kristen , Alonzo Todd A. , Gerbing Robert B. , Ho Phoenix A. , Gamis Alan S. , Raimondi Susana C. , Hirsch Betsy A. , Meshinchi Soheil
Published: October 2019

Abstract 1659

The CBL gene encodes a ubiquitin ligase that targets multiple activated tyrosine kinases for degradation. Analysis of several series of patients with myeloid malignancies demonstrated mutations in the RING finger domain and linker domains encoded by exons 8 and 9 of the CBL gene. Loh, et al (Blood, 114:1859-63, 2009) analyzed 159 samples from pediatric patients with juvenile myelomonocytic leukemia (JMML) for mutations in those exons. They found CBL mutations in 27 (17%) of the samples. We sought to determine the prevalence of mutations in CBL exons 8 and 9 in pediatric patients with de novo acute myeloid leukemia (AML). We analyzed 290 samples from pediatric patients treated on the Children's Oncology Group study AAML03P1. DNA was isolated from diagnostic bone marrow samples. Exons 8 and 9 of the CBL gene were amplified by PCR and then sequenced for mutational analysis. Five of 290 (1.7%) patients had CBL exon 8 or 9 mutations (Table 1). Patient 1 had a heterozygous splice site mutation (1096 –1G>T). Patient 2 had a homozygous missense mutation (C384S). Splice-site mutations and mutations of the C384 residue were also seen in JMML. Two patients had synonymous heterozygous mutations (P433P and I429I), and 3 patients had heterozygous insertions and deletions. Patient 3 had a 121-bp deletion that included 66 base pairs at the 3’ end of exon 8 and the first 55 base pairs of intron 8. Loh, et al (Blood, 2009) also found a similar 99-bp deletion in JMML. Patient 4 had a 3-bp duplication in exon 9 (1382-1383ins TGA) with TGA repeated 7 times; TGA is repeated 6 times in wild-type cells. Patient 5 had a complex insertion and deletion: 73 base pairs (31 base pairs in intron 7 and 42 base pairs at the 5’ end of exon 8) were deleted, and 26 base pairs were inserted at the site of the deletion. We examined the clinical features and other genetic abnormalities among these 5 patients with CBL mutations. All of the patients had an abnormal karyotype, including monosomy 7, t(8;21), t(9;11), or inv(16). We tested FLT3 status (internal tandem duplications and point mutations), CEBPA, and NPM1. Patient 5 had a FLT3 point mutation. We conclude that CBL mutations in the RING finger and linker domains are rare in pediatric de novo AML compared to their prevalence in JMML.

Disclosures:

No relevant conflicts of interest to declare.
Activation of a Novel Palmitoyltransferase through a t(6;14) Translocation In T/Myeloid Biphenotypic Leukemia and Subsets of Acute Myeloid Leukemia.
Authors: Ning Yi , Yu Lei , Reader Jocelyn , Chen Ching , Zhao Xianfeng Frank , Ha Jung Sook , Lee Charles , York Teresa , Gojo Ivana , Baer Maria R
Published: October 2019

Abstract 1663

The current World Health Organization (WHO) classification of hematologic malignancies includes a number of entities defined by specific genetic abnormalities. Most known abnormalities result from chromosomal translocations and mutations involving genes encoding transcription factors or tyrosine kinases. We identified the activation of a novel palmitoyltransferase, ZDHHC14, through a chromosomal translocation, t(6;14)(q25;q32). This translocation was initially documented by three independent groups in two children and two adult patients with T/myeloid biphenotypic leukemia, a poorly differentiated form of acute leukemia with a single population of leukemic blasts co-expressing antigens of both T-lymphoid and myeloid lineages. Recently, we detected the same translocation in two additional patients with T/myeloid biphenotypic leukemia. Molecular characterization of the translocation breakpoints revealed the juxtaposition of a regulatory region downstream of BCL11B (at chromosome region 14q32) upstream of an uncharacterized gene, ZDHHC14 (at chromosome region 6q25), leading to transcriptional activation of the ZDHHC14. Notably, this regulatory region from 14q32 has been found to be responsible for transcriptional activation of a homeobox gene, TLX3, as a result of a cryptic translocation t(5;14)(q35;q32) in T-cell acute lymphoblastic leukemia. Our subsequent survey of ZDHHC14 protein levels in blasts from patients with various subtypes of acute myeloid leukemia (AML) showed that the protein was not detectable in AML patients with t(8;21), t(15;17), or inv(16), but was detected in AML patients with FLT3 mutations or with complex karyotypes, indicating a possible association of increased expression of this protein with unfavorable prognostic features of the disease. We also demonstrated that overexpression of ZDHHC14 could inhibit TPA-induced cellular differentiation of K562 cells. Interestingly, the Drosophila homolog of ZDHHC14 has been found to be a palmitoyltransferase regulating Fat signaling. Fat is a Drosophila tumor suppressor gene, encoding a large cadherin that regulates growth and a form of tissue organization. While the function of ZDHHC14 and the role of protein palmitoylation in mammalian hematopoiesis remain unknown, recent studies in neuronal development have demonstrated that palmitoylation regulates diverse aspects of protein trafficking. Our findings suggest that palmitoyltransferase plays a previously unappreciated role in hematopoiesis and that increased expression of ZDHHC14 may be associated with a primitive status of leukocytes. Further characterization of ZDHHC14 may broaden our knowledge of leukemogenesis, contribute to risk stratification of acute leukemia, and provide a novel therapeutic target.

Disclosures:

No relevant conflicts of interest to declare.
Oxidant Pathway Functional Polymorphisms Influence the Risk of Myeloid Leukemia/Transient Myeloproliferative Disorder In Children with Down Syndrome.
Authors: Pope Jennifer M. , Kim Mi-Ok , Alonzo Todd A. , Gerbing Robert B. , Rayburg Melissa , Radloff Gretchen , Gamis Alan S. , Davies Stella M. , Perentesis John
Published: October 2019

Abstract 1680

Individuals with Down syndrome have a 2% cumulative risk for the development of leukemia by the time they reach 30 years of age. In addition, as many as 5–10% of infants with Down syndrome may develop a transient myeloproliferative disorder (TMD). The molecular basis for the increased risk for hematologic disorders and other congenital developmental abnormalities with DS remains unknown. Additional genetic material from the critical Down syndrome locus at 21q22 includes the Cu/Zn superoxide dismutase gene and other genes contributing to high oxidative stress and increased endogenous DNA damage observed in cells from patients with Down syndrome. We hypothesized that functional polymorphic variants in enzymes associated with detoxification of oxidants and repair of oxidant associated DNA damage would be associated with an increased risk for the development of myeloid leukemia (ML) and TMD in children with Down syndrome. We studied the role of functional polymorphisms in the oxidant metabolizing genes, Paraoxonase 1 (PON1) and NADH/NADPH oxidase p22 phox (P22_PHOX) in the potential genetic etiology of ML/TMD in children with Down syndrome. Genotyping was conducted in 192 patients with Down Syndrome ML or TMD, and 251 healthy blood donor controls. All genotype frequencies in patients and control populations were consistent with those expected from Hardy-Weinberg equilibrium. We found that the variant PON1 Gln192Arg allele, associated with reduced free radical metabolism, occurred at a higher frequency in the Down syndrome ML/TMD population compared to healthy controls (OR, 2.71; 95% CI, 1.50–4.97; p=0.0003). Consistent with our hypothesis of increased oxidant sensitivity in these patients, we also observed decreased frequency of the protective variant P22_PHOX His72Tyr allele, purportedly associated with reduced generation of reactive oxygen species, in the Down syndrome ML/TMD population compared to healthy controls (OR, 0.48; 95% CI, 0.23–0.95; p=0.025). Notably, the variant PON1 Gln192Arg allele has also been associated with increased risk for non-Hodgkin's lymphoma in adults (Kerridge et al. Br J Haematol, 2002). Conclusion: These preliminary studies suggest that functional polymorphic variants in the oxidant metabolizing enzymes PON1 and P22_PHOX may influence risk for development of ML/TMD in children with Down syndrome. Studies to confirm these observations in a separate cohort of patients are in progress.

Disclosures:

No relevant conflicts of interest to declare.
Levels of Mir-29b or Mir-101 Do Not Predict Response In Patients (pts) with Acute Myelogenous Leukemia (AML) Treated with the Combination of 5-Azacytidine, Valproic Acid and ATRA
Authors: Yang Hui , Fang Zhihong , Wei Yue , Hu Yumin , Calin George , Kantarjian Hagop M. , Garcia-Manero Guillermo
Published: October 2019

Abstract 1701

Introduction:

MicroRNA (miRNAs) are noncoding RNAs of 19 to 25 nucleotides in length that regulate gene expression by inducing translational inhibition and cleavage of their target mRNAs through base pairing to partially or fully complementary sites. Altered miRNA expression has been associated to various types of cancers, including leukemia. Recently it has been reported that pretreatment expression levels of miR-29b are associated with clinical response in pts with AML treated with a 10-day schedule of decitabine (PNAS 2010; 107:7473-78). These findings are significant as detection of miR-29b, if validated, could be used to select therapy in pts with AML.

Methods:

To follow on these observations, we have analyzed miR-29b and miR-101 expression levels in a cohort of patients with AML or high-risk myelodysplastic syndrome (MDS) previously treated in I/II study of the combination of 5-azacitidine (5-AZA), valproic acid (VPA) and ATRA (Blood 2007; 110:2302-08). We analyzed 45 pts, including 28 previously untreated patients. The characteristics of these pts have been previously reported (above): 8 (29%) pts achieved a complete remission and 1 (4%) partial remission. Total cellular RNA (extracted from unselected peripheral blood mononuclear cells collected before the first cycle of treatment) was used for reverse transcription reactions using TaqMan microRNA reverse transcription kit (Applied Biosystems). For real - time PCR, miR-29b and miR-101 TaqMan - microRNA assays were purchased from Applied Biosystems and analyzed with TaqMan Universal PCR Master Mix (Applied Biosystems) using an Applied Biosystems Prism 7500 Sequencing detection system. The U6 small nuclear RNA was used as internal control.

Results:

Expression levels of miR-29b and miR-101 from 45 patients were compared to 6 healthy donors. Expression levels of miR-29b was down-regulated in patients (P=0.001) (Fig. A). There were no difference in terms of miR-101 expression between patients and health donors. We then compared the miR-29b and miR-101 expression levels between patients who responded to the combination therapy versus non-responders: no differences were observed for both miR-29b (P=0.56) and miR-101 expression (Fig. B).

Conclusion:

our results fail to detect a relationship between miR-29b levels and response to 5-azacitidine based therapy. This could be explained by the fact that: 1) in our study, we used 5-azacitidine in combination versus decitabine; 2) source of RNA (peripheral blood versus bone marrow); And 3) relatively small sample size. Despite these limitations, these results indicate that larger studies need to be performed to confirm the role of miRNAs in response to hypomethylating agent based therapy.

Disclosures:

No relevant conflicts of interest to declare.
Phase II Study of Intrathecal Long Acting Liposomal Cytarabine (Depocyte®) In the Prophylaxis of Lymphomatous Meningitis In HIV-Related Non-Hodgkin's Lymphoma
Authors: Spina Michele , Martellotta Ferdinando , Berretta Massimiliano , Zanet Ernesto , Lleshi Arben , Canzonieri Vincenzo , Bulian Pietro , Bibas Michele , Antinori Andrea , Tirelli Umberto
Published: October 2019

Abstract 1750

Background:

Around 5% of patients with aggressive non-Hodgkin's lymphoma (NHL) develop central nervous system (CNS) progression or relapse during the course of their disease. Patients with human immunodeficiency (HIV)-related NHL often develop CNS progression despite the use of adequate prophylaxis. Liposomal cytarabine has show a significant activity in lymphomatous meningitis but there are limited data in the prophylactic setting.

Methods:

Since May 2006, we are running a prospective phase II study of intrathecal liposomal cytarabine (Depocyte®) at the dose of 50 mg in 40 patients with HIV-NHL with the aim to evaluate the feasibility and activity of this drug in the prevention of lymphomatous meningitis.

Results:

Thirty-five patients were males and the median age was 44 years (range 18–69 yrs). As far as the histological subtype of NHL, 47% of patients had a diffuse large B-cell (DLBC) NHL and 40% Burkitt NHL. Stage III-IV was diagnosed in 80% of patients and 68% of DLBC were age-adjusted IPI 2 or more. An extranodal involvement was diagnosed in 70% of patients (gastrointestinal 30%, bone 27%, spleen 10%, liver 22%, bone marrow 17%). Liposomal cytarabine was well tolerated with headache grade I to III being the most frequent side effect in only 37% of patients. Less common toxicity (all grade I) included cortical changes (5%), fever (3%), vomiting (3%), hypertension (3%), chills (3%). With a median follow up of 12.4 months only one patient (3%) with Burkitt lymphoma developed a combined systemic and meningeal relapse. Moreover, in our experience previously the present study, we used methotrexate as practical use in 426 HIV-NHL with a meningeal progression or relapse of 14% (p=0.09). The use of a liposomal formulation allowed to significantly reduce the number of lumbar injections in comparison to the standard schedules (approximately of 50%) with an improvement of quality of life of patients and with a reduction of professional exposure risk for health care staff.

Discussion:

In this first prospective study on prophylaxis of lymphomatous meningitis in HIV-NHL reported in the literature, liposomal cytarabine seems safe and active and it reduces of approximately 50% the number of lumbar punctures and exposure risk for health staff as well.

Disclosures:

No relevant conflicts of interest to declare.
Prognostic Significance of Interim 18f-FDG PET/CT for the Treatment of Diffuse Large B-Cell Lymphoma In the Post-Rituximab Era.
Authors: Yang Deok-Hwan , Min Jung-Joon , Song Ho-Chun , Jeong Yong Yeon , Chung Woong-Ki , Bae Soo-Young , Ahn Jae-Sook , Kim Yeo-Kyeoung , Bom Hee-Seung , Chung Ik-Joo , Kim Hyeoung Joon , Lee Je-Jung
Published: October 2019

Abstract 1799

18F-fluoro-2-dexoy-D-glucose-positron emission tomography (FDG-PET)/computerized tomography (CT) has been used for staging and monitoring responses to treatment in patients with diffuse large B cell lymphoma (DLBCL). The sequential interim PET/CT was prospectively investigated to determine whether it provided additional prognostic information and could be a positive predictable value within patients with the same international prognostic index (IPI) after the use of rituximab in DLBCL.

Patients and methods:

One hundred and sixty-one patients with newly diagnosed DLBCL were enrolled between August 2004 and December 2009 at a single institution. The assessment of the PET/CT was performed at the time of diagnosis and mid-treatment of R-CHOP chemotherapy. The clinical stage and response of the patients were assessed according to revised response criteria for aggressive lymphomas (Cheson, J Clin Oncol, 2007). The positivity of interim PET/CT was determined based on the semi-quantitative assessment of the maximal standardized uptake value (SUVmax cut-off value of 3.0).

Results:

Sixty-seven patients (41.6%) presented in advanced stage disease and 27 (16.8%) had bulky lesions. At diagnosis, 53 patients (32.9%) were classified as high/high-intermediate risk by the IPI and two patients could not check the interim response due to treatment-related mortality (TRM). Forty-three patients (26.7%) continued to have positive metabolic uptakes with a significantly high relapse rate (62.8%) compared to the patients with a negative interim PET/CT (12.1%) (P<0.01). After a median follow-up of 30.8 months, the positivity of interim PET/CT was found to be a prognostic factor for both OS and PFS, with a hazard ratio of 4.07 (2.62 – 6.32) and 5.46 (3.49 – 8.52), respectively. In the low-risk IPI group, the 3-year OS and PFS rate was significantly different in the patients with positive (53.3 and 52.5%) and negative (93.8 and 88.3%) interim PET/CT, respectively (P<0.01). These significant prognostic differences of interim PET/CT responses were consistent with the results of the patients with high-risk IPI group (P< 0.01).

Conclusions:

Interim PET/CT scanning had a significant predictive value for disease progression and survival of DLBCL in post-rituximab treatment; it might be the single most important determinant of clinical outcome in patients with the same IPI risk.

Disclosures:

No relevant conflicts of interest to declare.