bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Hepcidin on Hypoxia-Induced Erythropoietin Production
Authors: Dallalio Gail A , Means Robert T.
Published: October 2019

Abstract 3219

The anemia of chronic disease (ACD) is the major etiology of the anemia observed in in chronically ill patients. ACD typically manifests itself as a hypoproliferative anemia accompanied by a low serum iron concentration despite adequate reticuloendothelial iron stores. In ACD, a slight shortening of red cell survival creates a demand for a small increase in red cell production by the bone marrow. The marrow cannot respond adequately to this demand due to impaired erythropoiesis and impaired mobilization of reticuloendothelial system iron stores. Increased production of the iron regulatory peptide hepcidin has been proposed as the primary factor resulting in ACD. Hepcidin has also been reported to decrease erythroid colony formation in vitro under conditions of restrictive Epo concentration. A blunted erythropoietin (Epo) response to anemia is a characteristic feature of ACD. If hepcidin is the major factor responsible for ACD, then it should also contribute to the impaired Epo production observed in this syndrome.

The effect of hepcidin on hypoxia-induced Epo production was evaluated in HepG2 cells exposed to 5% oxygen for 24 hr. 24 hr exposure to hepcidin during hypoxia at the concentrations studied (up to 100 ng/mL) had no adverse effect on HepG2 cell viability compared to controls as evaluated by cell number and Trypan blue exclusion or on cellular synthetic function as measured by alpha fetoprotein. Epo production (whether measured by Western blot or by ELISA) was increased by hypoxia; however, this increase was blunted by the addition of hepcidin to the incubation medium. Impairment of hypoxia-induced Epo production by hepcidin showed a dose-response relationship. The addition of iron-replete transferrin to the incubation mixture did not significantly alter hepcidin effects, suggesting that these effects do not primarily reflect changes in iron availability.

In order to evaluate mechanisms by which hepcidin might decrease Epo production, effects of hepcidin 0–100 ng/mL on hypoxia-inducible factor (HIF)-1α protein expression were evaluated in HepG2 cells (HIF expression was normalized to actin expression). The increment in HIF-1α caused by hypoxia was decreased by hepcidin in a dose-dependent manner. The ratio of Epo to HIF was not altered by hepcidin, suggesting that hepcidin effects on HIF may be the mechanism of its effects on Epo production.

In conclusion, hepcidin appears to blunt the increment in Epo production induced by hypoxia in vitro. This mechanism does not appear to be reversible by exposure to increased quantities of transferrin-bound iron. This finding is consistent with a role for hepcidin in the impaired Epo production of ACD independent of its effects on iron flux.

Disclosures:

No relevant conflicts of interest to declare.
Implication of Genetic Variations of Ikzf1, ARIDB5, and CEBPE Genes In the Risk of Childhood Acute Lymphoblastic Leukemia In Korea
Authors: Han Dong Kyun , Kim Hee Nam , Shin Min Ho , Eguchi-Ishimae Minenori , Eguchi Mariko , Ishii Eiichi , Baek Hee Jo , Park Sun Ju , Kim Hyeoung Joon , Hwang Tai Ju , Kook Hoon
Published: October 2019

Abstract 3235

Background:

Recent western studies have showed the implication of the germline genomic variations in IKZF1 gene at 7p12.2, ARIDB5 gene at 10q21.2, and CEBPE gene at 14q11.2 on the risk of childhood acute lymphoblastic leukemia (ALL); the most significant association was observed in the single nucleotide polymorphism (SNP) rs4132601 which located at 3' region of the IKZF1. IKZF1 plays important role in lymphocyte differentiation, proliferation and function, ARIDB5 in embryogenesis and growth regulation, and CEBPE in regulation of myelopoiesis. Genomic variants in these genes are therefore considered to be involved in transcriptional regulation and differentiation of B cell progenitors. However, there have been no reports on the role of germline variations in leukemogenesis of childhood ALL in Asian countries. The aim of this study is to show the impact of these genetic variants on childhood ALL in Korea.

Patients and Methods:

To examine the association between genetic variations (IKZF1 rs4132601, ARIDB5 rs7089424, and CEBPE rs2239633) and the risk of childhood ALL, we here analyzed 228 children with ALL and 508 healthy individuals in Korea.

Results:

In ARIDB5 rs7089424, TG and GG genotypes were significantly associated with a risk for ALL (odds ratio [OR], 1.63; 95% confidential interval [CI], 1.07–2.48; P=0.02 for TG genotype, OR, 2.69; 95% CI, 1.42–5.07; P=0.002 for GG genotype). The allele incidence of ARIDB5 rs7089424 was also significantly associated with a risk for ALL (OR, 1.66; 95% CI, 1.24–2.22; P=0.0006). CEBPE rs2239633 TT genotype showed a significant association with a decreased risk for ALL (OR, 0.54; 95% CI, 0.33–0.90; P=0.02 for TT genotype). The allele incidence of CEBPE rs2239633 was also associated with a decreased risk for ALL (OR, 0.77; 95% CI, 0.61–0.97; P=0.02). There was no significant association between IKZF1 rs4132601 polymorphism and a risk for ALL in this study.

Conclusion:

These results suggest that genomic variations of ARIDB5 and CEBPE may play an important role in the risk for childhood ALL in Korea, compared with findings from western countries showing a significant relation between IKZF1 and childhood ALL. Several factors should be considered to explain a discrepancy between our results and the previous studies, which include different genotype frequencies in polymorphisms and varied susceptibility to ALL in different ethnic groups. Further studies incorporating larger number of cases and analyzing other SNPs or other Asian countries are warranted in childhood ALL.

Disclosures:

No relevant conflicts of interest to declare.
The PIM Kinase Inhibitor K00135 Sensitizes Primary Acute Lymphoblastic Leukaemia (ALL) Cells to DNA-Damaging Agents
Authors: Da Costa David , Weston Victoria J , Knapp Stefan , Taylor A. Malcolm R , Kearns Pamela R. , Stankovic Tatjana
Published: October 2019

Abstract 3238

Relapsed ALL is the most common cause of death in children, with only 30% being cured despite intensified treatment and stem cell transplantation. We previously showed that a subset of paediatric ALL tumours have a defect in the apoptotic response to DNA damage in vitro which correlates with a poor clinical response in vivo. Proviral integration site for Moloney murine leukaemia virus (PIM) proteins are a family of serine/threonine kinases which mediate prosurvival signals in response to cytokines, growth factors, hypoxia and DNA damage. PIM kinases have been implicated in several haematological malignancies such as chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), acute myeloid leukaemia (AML), and T-ALL. However, the role of PIM kinases and their contribution to treatment resistance in a large cohort of primary paediatric ALL has not been comprehensively addressed. To address the frequency of PIM overexpression in ALL we used quantitative real-time PCR to quantify the levels of PIM-1 and PIM-2 in an unselected cohort of 86 primary ALL tumours compared to the PIM-expressing lymphoid cell line K562. We observed high PIM-1 levels in only 2/86 (2.3%) ALLs, whereas PIM-2 was highly expressed in 20/86 (23%) primary tumours. PIM-2 overexpression was detected in tumours with a range of chromosome translocations or hyperdiploidy. In representative samples, the level of PIM-2 protein expression correlated with abundance of mRNA transcript. We then investigated the role of PIM overexpression on apoptosis using the highly specific PIM inhibitor, imidazo[1,2-b]pyridazine K00135, to sensitize tumour cells with high PIM expression to ionising radiation (IR)-induced apoptosis. Pretreatment with K00135 led to sensitization of representative primary tumour cells with high PIM-2 expression to 5Grays IR at an half maximal lethal concentration (LC50) of 10μM, and killing was mediated by caspase-dependant apoptosis. Furthermore, consistent with the role of PIM-2 in the regulation of c-Myc protein stability (Zhang Y, Wang Z, Li X, Magnuson NS. 2008; Oncogene; 27;4890-4819), K00135-induced killing coincided with the downregulation of c-Myc protein. We are currently addressing the impact of PIM inhibition in an ALL xenograft model. Constitutive expression of PIM-2 may be an underlying mechanism of treatment resistance in a subset of paediatric ALL. Pharmacological inhibition of PIM-2 may prove to be a useful therapeutic option for poorly responding ALL in children in the future.

Disclosures:

No relevant conflicts of interest to declare.
Thirty Percent Long Term Survival In Children with Acute Leukemia and Complications Requiring Mechanical Ventilation
Authors: Steinbach Daniel , Wilhelm Bernhard , Kiermaier Hans-Rudolf , Creutzig Ursula , Schrappe Martin , Zimmermann Martin , Debatin Klaus-Michael , Gruhn Bernd , von Stackelberg Arend , Jürgens Heribert , Strahm Brigitte , Reinhardt Dirk , Möricke Anja
Published: October 2019

Abstract 3243

Objective:

Previous reports indicated that short term prognosis for patients with malignant diseases and serious adverse events requiring mechanical ventilation (SAEV) is not dismal any more. The purpose of this study was to determine whether patients who survive such complications can also achieve long term cure from leukemia. It might influence end-of-live decisions on the intensive care unit if patients with an SEAV only survive intensive care to succumb to relapse.

Patients and Methods:

We report the outcome of children with SAEV treated in the multicenter studies ALL-BFM 95 and AML-BFM 98. Data from 1182 patients with acute lymphoblastic leukemia (ALL) and 332 patients with acute myeloid leukemia (AML) were analyzed. 88 patients (51 ALL; 37 AML) developed an SAEV.

Results:

The prognosis was almost identical in ALL and AML (50% survival of SAEV; 30% overall survival after 5 years). This was independent from the time between diagnosis of leukemia and SAEV. Even children who required hemodialysis (n=14) or cardiac resuscitation (n=16) achieved 20% long term survival but no patient survived (n=16) who fulfilled more than 3 out of 6 identified risk factors: age≥10 years, high risk leukemia, C-reactive Protein≥150mg/l, administration of inotropic infusion, cardiac resuscitation, and hemodialysis.

Conclusions:

To our knowledge, this is the first report about cure rates in a malignant disease after successful treatment of SAEV. Our results show that intensive care medicine contributes to short and long term survival of children with leukemia. Sixty percent of all children with acute leukemia who survive an SAEV achieve long term cure. However, we could also identify risk features that still indicate a devastating prognosis.

Disclosures:

No relevant conflicts of interest to declare.
Anti-HLA-DR Monoclonal Antibody Evokes a Novel Non-Apoptotic Cell Death Pathway In Acute Lymphoblastic Leukemia
Authors: Naidoo Khimara , Alduaij Waleed , Honeychurch Jamie , Cheadle Eleanor , Alexander Seema , Byers Richard , Saha Vaskar , Illidge Tim
Published: October 2019

Abstract 3245

Whilst modern treatment approaches cure a high number of patients with acute lymphoblastic leukemia (ALL), little progress has been made in the treatment of refractory and relapsed ALL and new treatment approaches are needed. We recently demonstrated that anti-HLA-DR class II monoclonal antibody (mAb) L243 induces a novel non-apoptotic mode of cell death in B-cell lymphoma lines, defined by homotypic adhesion (HA), actin reorganisation and lysosomal activity (Ivanov et al. J Clin Invest, 2009). Here, we extend these important observations and examine whether this novel form of mAb induced cell death occurs in pre-B ALL cell lines.

Expression of HLA-DR was determined using flow cytometry in a panel of ALL cell lines (REH, SupB15 and SD1). HLA-DR was expressed at high levels on each of the cell lines. The ability of L243 to induce HA and cell death (Annexin V/PI positivity) was assessed using microscopy and flow cytometry respectively. L243 was able to evoke both strong HA and cell death in all of the ALL cell lines (e.g. in SupB15 cells 46±1.7% death versus 7±0.5% in controls, p<0.001 by Student's t-test). Inhibitors of actin polymerization (cytochalasin D, latrunculin B) were used to assess the role of actin in cell death and HA induced by L243. These inhibitors of actin polymerization inhibited both HA and cell death elicited by L243 (e.g. in SupB15 cells 24±0.5% death versus 10.3±0.8% with Latrunculin B, p<0.001), demonstrating the dependence of HA and cell death on actin reorganisation. The importance of cell to cell contact in this form of antibody induced cell death was confirmed by the addition of low-melting point agarose which physically blocked cell to cell contact and markedly attenuated cell death induced by L243. In contrast using the pan-caspase inhibitor QVD OPH had no effect on cell death induced by L243, indicating that this mode of death is non-apoptotic.

These findings demonstrate that anti-HLA DR mAb L243 induces a novel model of cell death in ALL cell lines that is independent of caspase activation and dependent on actin reorganization. This data suggests that this novel mAb induced death pathway is independent of apoptosis and potentially exploitable in the clinic in leukemias resistant to chemotherapy apoptosis induction.

Disclosures:

No relevant conflicts of interest to declare.
Comparision of Dual PI-3K/mTOR Inhibitors with mTOR Inhibitors Using a Pre-Clinical Model of Acute Lymphoblastic Leukemia
Authors: Wong Jacky , Welschinger Robert , Baraz Rana , Weiss Jocelyn , Bradstock Ken , Bendall Linda J.
Published: October 2019

Abstract 3258

ALL cells are highly dependent on bone marrow stromal support for in vitro proliferation and survival. The major regulators of patient-derived ALL cell growth and survival convey their proliferative and survival signals through the phosphoinositide 3-kinase (PI-3K) pathway. It has been recently demonstrated that signalling through PI-3K and AKT is the most important pathway for the proliferative responses of ALL cells to CXCL12, the chemokine predominantly responsible for stromal dependent growth of ALL cells. In addition, inhibition of the mTOR signalling molecule downstream of PI3K with RAD001 has been shown to inhibit proliferation and induce cell death. Although PI-3K and mTOR have similar and overlapping functions, mTOR can be activated independently of PI-3K, and proliferation and survival can be stimulated by PI-3K in an mTOR independent manner. Therefore combining PI-3K and mTOR inhibition is likely to be advantageous over inhibition of either kinase alone, suggesting disruption of PI-3K/AKT/mTOR signalling will provide a new approach for the treatment of ALL. We investigated the dual kinase inhibitors BEZ235 and BGT226. Here, we demonstrate that PI-3K and mTOR inhibition with the dual kinase inhibitor BEZ235 significantly inhibits ALL proliferation in vitro, with IC50 values in the range of 7–20nM, indicating a 3 log greater potency in comparison to the mTOR inhibitor RAD001. The ability to induce cell death differed between the dual mTOR and PI-3K inhibitors, with BGT226 potently inducing cell death at 1.6μM, but more than 16μM of BEZ235 was required to kill ALL cells, with a combination of autophagy and apoptosis being detected. While cell death was induced with higher concentrations of BEZ235 than needed to inhibit proliferation, clonogenic assays revealed a major decrease in the survival capacity of cells exposed to the agent. We also demonstrate the activity of these dual kinase inhibitors in a NOD/SCID xenograft model of human ALL with significantly prolonged survival of mice. The potential synergy of dual kinase inhibitors with conventional chemotherapy drugs and in mTOR inhibitor resistant cases remains to be studied. Dual kinase inhibitors may offer an improved therapeutic index through reduced toxicity over mTOR inhibitors, and potentially reduce the risk of development of resistance to kinase inhibition.

Disclosures:

No relevant conflicts of interest to declare.
Platelet Inhibition by Adjunctive Cilostazol Versus High Maintenance-Dose Clopidogrel In Patients with Acute Myocardial Infarction According to Cytochrome P450 2C19 Genotype
Authors: Kim In-Suk , Jeong Young-Hoon , Kim Arum , Lee Gyeong-Won
Published: October 2019

Abstract 3322

Objectives

The aim of this study was to assess the degree of platelet inhibition by adjunctive cilostazol in patients with acute myocardial infarction (AMI) according to CYP2C19 genotype.

Background

Although adjunctive cilostazol intensifies platelet inhibition in AMI patients, it is not established whether this regimen can overcome the loss-of-function effect of CYP2C19 variants.

Methods

We randomly assigned 126 AMI patients with available CYP2C19 genotyping to receive adjunctive cilostazol (triple group; n = 64) or high maintenance-dose (MD) clopidogrel of 150-mg/day (high-MD group; n = 62). Using conventional aggregometry and VerifyNow, platelet reactivity was measured at pre-discharge and 30-day follow-up. Primary endpoint was change in maximal platelet aggregation (Aggmax). High post-treatment platelet reactivity (HPPR) was defined as 5 μmol/l ADP-induced Aggmax > 50%.

Results

In non-carriers, the two groups did not differ with respect to changes in platelet measures, and could achieve fewer rates of HPPR at 30-day follow-up (< 5%). In carriers, changes in 5 and 20 μmol/l ADP-induced Aggmax were significantly higher in the triple (n = 39) versus high-MD group (n = 38) (21.8 ± 13.9% vs. 9.0 ± 13.3%, p < 0.001, and 24.2 ± 17.2% vs. 7.7 ± 15.5%, p < 0.001, respectively). Likewise, changes in late platelet aggregation and P2Y12 reaction unit were consistently greater in the triple vs. high-MD group. Fewer patients in the triple group met the criteria of HPPR at 30-day follow-up compared with the high-MD group (2.6% vs. 21.1%, p = 0.014).

Conclusions

Among AMI patients with CYP2C19 variants, adjunctive cilostazol enhances platelet inhibition and reduces the rate of HPPR, as compared with high-MD clopidogrel. (Adjunctive Cilostazol Versus High-MD ClopidogrEL in Patients With Acute Myocardial Infarction According to CYP2C19 genotype [ACCEL-AMI-CYP2C19]; NCT00915733).

Disclosures:

No relevant conflicts of interest to declare.
Inhibition of Thrombin Generation: An Additional Biological Criterion for the Evaluation of the Anticoagulant Mechanism of Action of Generic LMWHs
Authors: Gerotziafas Grigoris T , Sassi Mouna , Galea Vassiliki , Fareed Jawed , Walenga Jeanine M. , Elalamy Ismail
Published: October 2019

Abstract 3334

Background.

All low molecular weight heparins (LMWHs) exert their antithrombotic effect primarily via the binding of pentasaccharide domain on antithrombin but vary in physicochemical properties, anti-Xa/anti-IIa ratios, and their effect on global coagulation tests such as the inhibition of thrombin generation (TG). Definition of criteria for biological similarity between generic LMWHs and the original product is a challenging issue.

Aim of the study.

We evaluated the capacity of Lovenox® and seven generic enoxaparins to inhibit TG in platelet poor and platelet rich plasma (PPP and PRP).

Materials and Methods.

Citrated PPP and PRP from 15 healthy volunteers was spiked with increasing concentrations (2-20 μg/ml) of Lovenox®, Novex®, Enoxa®, Dilutol®, Versa®, Cutenox®, Loparin®, Fibrinox®. Platelet count in PRP was adjusted to 200 /L. The anti-Xa and anti-IIa activities in PPP were measured on STAR analyser using chomogenic assays from Diagnositca Stago. Thrombin generation in fresh PPP and PRP was triggered with PPP-reagent® and 1/200 diluted Innovin® (from Siemens) respectively and assessed with Calibrated Automated Thrombogram® (Stago, France). Mean Rate Index (MRI) of the propagation phase of TG and endogenous thrombin potential (ETP) were analysed. The enoxaparin concentrations which reduced 50% of theTG parameters (IC50) were calculated.

Results.

All studied compounds showed the same anti-Xa activity/mg and anti-Xa/anti-IIa ratio as the comparator Lovenox®. The IC50 for MRI and ETP in PPP was not significantly different between Lovenox® and each one of the generic enoxaparins. In PRP the IC50 of Lovenox® for MRI and ETP was significantly lower compared to the generic enoxaparins. The IC5O of Fibrinox®, Dilutol®, Cutenox® and Versa® was 1.5- to 2-fold higher compared to that of Lovenox® (Table 1).

Conclusion.

Lovenox® and seven generic enoxaparins were the same in terms of anti-Xa activity/mg and anti-Xa/anti-IIa ratio. They exerted similar inhibitory effect on thrombin generation in PPP. In contrast, in the presence of platelets, the sameness and even the biological similarity of the studied compounds are not evident. The reasons for these diversities need to be elucidated. The results of the present study introduce the inhibition thrombin generation in platelet rich plasma as an additional biological criterion for the comparison of generic LMWHs to the innovator product.

Disclosures:

No relevant conflicts of interest to declare.
Platelet-Targeted Pro-Urokinase as a Novel Thromboprophylaxis Fibrinolytic Strategy
Authors: Fuentes Rudy , Kowalska M. Anna , Tliba Samira , Bougie Daniel W. , Aster Richard H. , Cines Douglas B. , Muzykantov Vladimir R. , Poncz Mortimer , Zaitsev Sergei
Published: October 2019

Abstract 3339

Use of plasminogen activators (PAs) is restricted to life-threatening thrombotic conditions because high concentrations are required to diffuse into clots, overcome PA inhibitors and compensate for rapid clearance, thereby predisposing to bleeding. We hypothesized that targeting pro-PAs to platelets would circumvent these obstacles and preferentially lyse nascent, pathological clots that are actively recruiting platelets, while sparing preformed hemostatic clots. To test this concept, we expressed a chimeric protein (anti-PLT scFv/uPA-T) composed of an N-terminal scFv generated from a monoclonal antibody MoAb 312.8 directed to human αIIb integrin chain linked via a serine-rich linker peptide to human low molecular weight thrombin activatable pro-urokinase (uPA-T) wherein the plasmin cleavage site was replaced with a thrombin cleavage site, which we reasoned would be activated preferentially at sites of active clot propagation. Anti-PLT scFv/uPA-T expressed in Drosophila S2 insect cells bound specifically to human platelets and to transgenic mouse platelets that contain only human αIIb/mouse β3 on their cell surface (HαIIb+ Tg), but not to wild type mouse platelets. The anti-PLT scFv/uPA-T bound specifically to immobilized human αIIbβ3 with a Kd of ∼80 nM. The anti-PLT scFv/uPA-T did not interfere with either ADP-induced platelet aggregation or activation as demonstrated by expression of P-selectin by flow cytometry, and retained its zymogenic properties until activated specifically by thrombin. The fibrinolytic activity of anti-PLT scFv/uPA-T and uPA-T were compared using the FeCl3 carotid artery injury model in HαIIb+ Tg mice. The mice were protected from forming occlusive thrombi for at least 10 hrs post injection of anti-PLT scFv/uPA-T whereas even five-fold higher concentrations of uPA-T were effective for only 2–5 min. These studies support a novel approach for prophylactic targeting drug delivery by combining a pro-drug that requires activation by thrombin with platelet delivery to sites of incipient thrombotic vascular occlusion.

Disclosures:

No relevant conflicts of interest to declare.
Transfusion Transmission of GB Virus Type C (HGV) In a Cohort of HIV Infected Patients
Authors: Vahidnia Farnaz , Petersen Maya , Rutherford George , Busch Michael , Assmann Susan , Stapleton Jack , Custer Brian
Published: October 2019

Abstract 3341

Background:

GB virus type C (GBV-C) infection is common with 3–5% rates of viremia in blood donors and a much higher prevalence in HIV infected patients. GBV-C is transmitted by sexual or blood exposure. Although infection may persist, most immune competent individuals clear viremia within 2 years. Few data describe the clinical course of acute GBV-C infection following transfusion in HIV infected patients. We estimated risk of GBV-C RNA acquisition following transfusion.

Methods:

We used a limited access database from the National Heart Lung and Blood Institute from the Viral Activation Transfusion Study (VATS). A RCT of leukoreduced (LR) vs. non-LR transfusion, VATS collected blood samples from U.S. HIV infected transfusion naïve patients. GBV-C RNA in pre- and post transfusion samples was tested after completion of VATS. GBV-C RNA acquisition up to 120 days post-transfusion was examined in 294 patients who were GBV-C RNA and antibody negative before transfusion. Discrete hazard of GBV-C RNA acquisition as a function of cumulative units transfused was estimated using pooled logistic regression.

Results:

GBV-C RNA was detected in 22 (7.5%) of 294 subjects within 120 days following first transfusion. Viremia was detected within the first 30 days in 12 (4.1%) subjects and between 31 and 120 days in 10 (3.4%) subjects. Median (IQR) follow-up duration and total blood units transfused for subjects who acquired GBV-C RNA or stayed negative were: 88.5 (80-108) and 77.5 (32-100) days; and 4 (2-7) and 4 (2-6) units, respectively (Table 1). Discrete hazard of GBV-C RNA acquisition increased with each additional unit transfused (OR=1.09, 95% CI=1.06, 1.11) (Table 2). In pooled logistic regression models, lower baseline HIV viral load and use of antiretroviral therapy (ART) predicted subsequent GBV-C RNA acquisition after controlling for units of blood transfused. Leukoreduction status was not associated with GBV-C transmission.

Conclusion:

Blood transfusion is associated with significant risk of GBV-C acquisition in HIV infected patients. Establishing evidence for transfusion transmission of GBV-C will allow additional studies on the impact of acute acquisition on the course of HIV infection in co-infected patients.

Disclosures:

No relevant conflicts of interest to declare.
Staphylococcus Aureus Detection In Platelets Using Short Synthetic Peptides Derived From Phage-Displayed Random Peptide Libraries Coupled with Quantum-Dot Nanocrystals
Authors: Rao Shilpakala Sainath , Ketha Krishna Mohan V. , Atreya Chintamani D
Published: October 2019

Abstract 3359

Introduction:

Bacterial sepsis in recipients of transfusion products due to contamination of blood components is an infrequent but serious and potentially a fatal complication. Unlike red cells, platelet concentrates (PC) are stored for up to 5 days at room temperature after collection, which is conducive for bacterial growth. In 42% of the sepsis cases following PCs, the most common bacterial contaminant is Staphylococcus spp. Timely detection of these contaminated units is a public health priority. Although a multitude of bacterial diagnostic tests are available for PCs, still there is a need for developing more sensitive, specific and rapid detection assays. The primary objective of this study is to develop a simple and highly sensitive procedure that facilitates rapid and enhanced detection of bacteria using Staphylococcus aureus as a model for PCs.

Methods:

A commercially available bacteriophage-displayed random 12-mer peptide library was screened against S. aureus. The screening and subsequent sequencing of the phage DNA identified a phage clone containing a coding sequence for 12-amino acid peptide that selectively binds to S. aureus. We assessed the binding specificity and detection sensitivity of the peptide to a panel of six bacteria spiked in platelets (including S. aureus) by dot blot, ELISA and fluorometry. The peptides were further labeled with streptavidin-conjugated fluorescent Nanocrystal quantum-dots (Q-dots) to enhance the sensitivity of the assay.

Results:

The membrane-based dot blot method revealed that the peptide binds specifically to S. aureus. ELISA and fluorometry analysis of spiked platelet samples revealed that the peptide binds to S. aureus at 103 CFU/ml concentrations. By using the peptide-Nanocrystal Q-dot combination (peptide probes), the detection sensitivity was further enhanced. The peptide binding remained relatively constant between pH 5 to 7.5.

Conclusion:

This model system demonstrates that peptides derived from random phage library in combination with Nanocrystal Q-dots facilitate high sensitivity detection of S. aureus. In general, this study provides a proof-of-concept that ‘peptide probes' selected against specific bacteria are useful in detecting and monitoring them in various settings relevant to blood safety and transfusion medicine.

The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

Disclosures:

No relevant conflicts of interest to declare.
Involvement of Death-Associated Protein Kinases In DNA Damage Responses of B-ALL Cells.
Authors: Humbert Magali , Medova Michaela , Geering Barbara , Blank-Liss Wieslawa , Simon Hans-Uwe , Fey Martin F. , Zimmer Yitzhak , Tschan Mario P.
Published: October 2019

Abstract 3369

Intact DNA damage response pathways are important for genomic fidelity of cells in order to avoid tumor formation. On the other hand, inhibition of DNA repair provides an important mechanism to enhance the therapeutic efficacy of DNA damaging agents such as gamma-irradiation. Thus, it is important to identify novel players in DNA damage response that might represent novel targets for combination therapies. Death-associated protein kinases (DAPK) are serine/threonine kinases believed to be involved in cell death and autophagy mechanisms, whereby particularly the role of DAPK1 has previously been investigated. The DAPK family is composed of five members: DAPK1, DAPK2 (or DRP-1), DAPK3 (or ZIP kinase), DRAK1 and DRAK2. DAPK1 and DAPK2 share 80% homology in the catalytic domain. Generally, the role of DAPK in DNA damage responses is not well studied. To analyze the role of DAPK1 and DAPK2 in response to gamma-irradiation, we used p53 wild-type REH B-cell acute lymphoblastic leukemia (B-ALL) cells as a model. In response to irradiation, DAPK1 protein expression increased paralleled by an increased of total p53, phospho-Ser20-p53 and p21WAF1/CIP1. DAPK2 expression, however, did not increase. Since upregulation of p21WAF1/CIP1, a classical p53 target in response to DNA damage leads to cell cycle arrest, we asked whether knocking down DAPK1 or DAPK2 might affect the cell cycle. Interestingly, knocking down DAPK2 but not DAPK1 led to a significant increase of S-phase cells upon irradiation. Moreover, knocking down DAPK2 attenuated the induction of DAPK1 upon irradiation indicating a DAPK2-DAPK1 cascade in DNA damage responses. Next, given the significant role of p21WAF1/CIP1 and p53 in DNA damage responses, we tested if DAPK2 might directly participate in a novel signaling pathway by interacting with these proteins. Indeed, pull down assays revealed that p21WAF1/CIP1 and p53 are novel DAPK2 interacting proteins. Clearly, further experiments are needed to define the DAPK2-DAPK1-p53- p21WAF1/CIP1 network in DNA repair pathways. In conclusion, we identified a novel role for DAPK1 and DAPK2 in DNA damage responses of B-ALL cells and propose a novel DAPK2/DAPK1/p53/ p21WAF1/CIP1 DNA damage regulatory pathway.

Disclosures:

No relevant conflicts of interest to declare.
Treatment of AML Relapse After Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) Using Low-Dose Azacitidine (AZA).
Authors: Chiattone Alexandre , Saliba Rima M , Andersson Borje S. , Giralt Sergio , Sharma Manish R , Rondon Gabriela , Kebriaei Partow , Hosing Chitra , Popat Uday , Parmar Simrit , Alatrash Gheath , Bashir Qaiser , Chen Julianne J , Yuen Carrie Hai-Lai , Chiattone Viviane C. , Champlin Richard E. , De Lima Marcos
Published: October 2019

Abstract 3445

Background:

Relapsing AML/MDS after HSCT has a dismal prognosis, with few patients achieving long-term control of the malignancy. AZA is a hypomethylating agent that is moderately active against AML/MDS, and may have beneficial immunomodulatory effects after HSCT. We have shown that a significant minority of patients with recurrent disease respond to this drug. Here, we present long-term follow-up after salvage treatment regimens that included AZA, to treat AML/MDS that recurred after HSCT.

Patients and Methods:

Twenty-three patients received low-dose AZA for recurrence. Decision to use AZA was based on clinical assessment of slow progression of disease and relatively slower disease ‘tempo' and relatively small AML bulk. AZA cohort preparative regimens for 1st HSCT were myeloablative in 12 cases, and of reduced intensity in 11 cases. AZA was used prior to or without a 2nd HSCT (n=17), or after a 2nd HSCT (n=6). Outcomes were compared to controls (n=18) that relapsed ≥ 8 months after HSCT, and did not receive AZA (8 months representing the median disease free survival (DFS) for AZA-treated patients). The control group included all patients that relapsed ≥ 8 months after allogeneic HSCT using myeloablative busulfan 130 mg/m2 and fludarabine 40 mg/m2 for 4 days. AZA was studied as a time dependent variable. AZA and controls had similar baseline characteristics as described in the Table, although median DFS after the first HSCT was 8 (range: 2–51) and 17 (range: 7–59) months, favoring the control group (p=0.08). AZA was administered outpatient, with good tolerance. Fatigue and nausea were commonly observed toxicities. Doses were 8 mg/m2 (n=1), 16 mg/m2 (n=3), 24 mg/m2 (n=10), 32 mg/m2 (n=5), 40 mg/m2 (n=2), and 75 mg/m2 (n=2), administered subcutaneously for 5 days, in 28–32-day cycles.

Results:

Median number of cycles was 4 (range, 1–44). With a median follow-up of 18 months for AZA and control patients, median survival after relapse was 17 versus 6 months, respectively for AZA and control patients. 11 (48%) AZA patients are alive, while 2 (11%) control patients are alive. Two-year overall survival (OS) for AZA and control groups was 40% and 10%, respectively. AZA and controls had similar baseline characteristics as described in the Table.

Conclusion:

Low-dose AZA was a well tolerated outpatient treatment that may improve survival after AML/MDS recurrence in selected cases. Major determinants of survival in this setting, however, were remission duration after HSCT, and use of a 2nd HSCT.

Disclosures:

No relevant conflicts of interest to declare.
Transplantation for Immunoglobulin Light Chain Amyloidosis. A Statistical Analysis of Factors Predicitng Outcome In Over 400 Patients
Authors: Gertz Morie Abraham A. , Lacy Martha , Dispenzieri Angela , Kumar Shaji , Buadi Francis , Dingli David , Ansell Stephen M. , Gastineau Dennis , Inwards David James , Johnston Patrick B , Litzow Mark R. , Micallef Ivana , Porrata Luis , Leung Nelson , Wolf Robert , Hogan William J , Hayman Suzanne R.
Published: October 2019

Abstract 3557

Introduction:

Autologous stem cell transplantation for patients with amyloidosis results in high reported hematologic and organ response rates compared with conventional chemotherapy.

Patients:

Four-hundred and thirty-four patients have undergone transplantation between March 8, 1996, and April 13, 2010 Table 1. Clinical parameters seen in patients with amyloid are given in Table 2. The overall day-100 mortality seen in this group of patients is 44 of 434 patients (10%).

Results:

The most important determinant of outcome is stage defined by BNP and troponin levels. Figure 1 demonstrates the survival for patients for all 3 cardiac stages. Troponin <.035; NT-proBNP <332. Stage 1 both low, Stage 3 both elevated. The survival of all patients based on whether they achieved a complete response, a partial response (>50%), or no response was analyzed. Median survival has not been reached for the complete response group, is 107 months for the partial response group, and is 32 months for the no response group (p<0.0001). Figure 2, divides the patients at the median dFLC level of 13.5 mg/dL demonstrating that for patients with a higher level of free light chain, the median survival is 87.6 months and has not been reached for those with a lower level of free light chain. A proportional hazards model for variables that impact on survival was done in all patients, and the only relevant predictor of outcome was stage (p<0.0001). When the same analysis was performed using a landmark of 6 months, the only predictor of outcome was stage (p=0.0005). When best response was incorporated into the landmark model, it was the strongest predictor of survival. The amyloid stage predicted survival (p= 0.0005), and the best response to therapy (p <0.0001). When the same analysis was performed in the entire group of 434, stage and best response each remained significant (p<0.0001).

Discussion:

There is a high response rate associated with high-dose therapy that was not observed in the melphalan and prednisone era. For eligible patients who can be transplanted safely, high-dose melphalan is an effective therapy for many patients with amyloidosis. Best response and stage at diagnosis are the best predictors of overall survival.

Disclosures:

Lacy: Celgene: Research Funding. Dispenzieri:Celgene: Honoraria, Research Funding; Binding Site: Honoraria. Kumar:Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.
Reapplication of High-Dose Chemotherapy with Melphalan Followed by Autologous Hematopoietic Stem Cell Transplantation as Salvage Therapy for Patients with Relapsed Multiple Myeloma
Authors: Sellner Leopold , Teodorov Sonja , Heiss Christiane , Benner Axel , Egerer Gerlinde , Dreger Peter , Ho Anthony D. , Goldschmidt Hartmut , Neben Kai
Published: October 2019

Abstract 3568

Introduction:

High dose chemotherapy with melphalan followed by autologous peripheral blood stem cell transplantation (PBSCT) is a standard treatment regimen for young patients with multiple myeloma. However, there are few studies, mainly with low patient counts, testing the benefit of the reapplication of high dose chemo therapy in relapsed or refractory myeloma.

Methods:

Here we retrospectively analyzed 178 patients (56% male, 44% female, median age 60 years) with relapsed or refractory myeloma who were treated by reapplying high dose chemotherapy with melphalan followed by autologous PBSCT in our institution over the last 18 years. The median follow up of this study was 54 months.

Result:

Median progression free survival (PFS) and overall survival (OS) after relapse autologous transplantation were 16 and 35 months, respectively. 66% of the patients received newer antimyeloma agents for reinduction therapy (39% thalidomide, 6% bortezomib, 21% lenalidomide). In univariate analysis, time between first transplantation and progression of disease had a significant impact on PFS and OS (p=0.001 and p<0.001). Estimated hazard ratio (HR) for prolongation of time to progression (TTP) after first transplantation of one year for PFS and OS are 0.85 and 0.73, respectively. The effect of TTP after first PBSCT on PFS and OS with respect to different clinically relevant cutoff values is illustrated in Table 1.

Conclusion:

Reapplication of high dose chemotherapy can be an effective treatment option for relapsed or refractory myeloma, in particular in patients with a time to progression after first autologous transplantation of more than one year.

Disclosures:

No relevant conflicts of interest to declare.
Active Chromatin Structure near MYB Occupancy at the Mir-155 Host Gene Promoter Coincides with Increased Mir-155 and MYB Levels In Chronic Lymphocytic Leukemia
Authors: Stopka Tomas , Vargova Karin , Curik Nikola , Burda Pavel , Basova Petra , Pospisil Vit , Kulvait Vojtech , Savvulidi Filipp , Necas Emanuel , Berkova Adela , Obrtlikova Petra , Karban Josef , Trneny Marek , Zavadil Jiri
Published: October 2019

Abstract 3589

Elevated levels of microRNA miR-155 represent a candidate pathogenic factor in chronic B-lymphocytic leukemia (B-CLL). In our study, B-CLL patients (N=71, F/M 31/40, median age 65) were subject to molecular analyses that found significant overexpression of MYB and miR-155 in B-CLL and physical association of MYB with the promoter of MIR155 host gene (MIR155HG, also known as B-cell integration cluster). In CD19+ B-cells derived from healthy control individuals (N=13), MYB and miR-155 expression was significantly lower. Next, we found that MYB positively regulates MIR155HG transcription in reporter assays. The endogenous chromatin structure of the MIR155HG promoter in B-CLL cells is characterized by spreading of active chromatin mark, histone H3K9 acetylation. Gene expression arrays on B-CLL patients (N=11) identified a set of predicted miR-155 target genes (N=94) that are downregulated. Their expression pattern displayed significant negative correlation with pri-miR-155 levels. Similarly, a number of MYB target genes were found deregulated (N=99) in B-CLL. Gene annotation of differentially expressed miR-155 and MYB targets in B-CLL revealed their biological functions as regulators of apoptosis (BCL2, API5), proliferation (CCND1, CCND2) and mediators of B-cell function (CD200, F11R). In addition, expression patterns of miR-155, MYB and their targets are currently being compared in a larger patient group with prognostic hallmarks of CLL (ZAP70, CD38, del(17)(p13.1), and IgVH). Our data collectively support novel candidate mechanism in B-CLL that includes MYB directly stimulating MIR155HG promoter coincident with its epigenetic dysregulation. (Grant # IGA 10310-3, MSMT 2B06077, 0021620806, LC06044, SVV-2010-254260507).

Disclosures:

Trneny: ROCHE: Honoraria, Research Funding.
High IGSF4 expression In Pediatric Acute Monoblastic Leukemia with t(9;11)
Authors: Coenen Eva A , Kuipers Jenny , Balgobind Brian V , Stary Jan , Baruchel Andre , Haas Valerie de , Bont Evelina S. De , Reinhardt Dirk , Kaspers Gertjan J. , Cloos Jacqueline , Oorschot Astrid Danen-van , Boer Monique L. Den , Marschalek Rolf , Meyer Claus , Pieters Rob , Zwaan Christian M. , Heuvel-Eibrink Marry van den
Published: October 2019

Abstract 3614

Pediatric MLL-rearranged acute monoblastic leukemia with t(9;11)(p22;q23) has a good outcome as compared to other MLL-rearranged AML. The biological background for this difference is unknown. Therefore, we compared gene expression profiles (GEP) of -t(9;11)(p22;q23) patients with other MLL-rearranged AML patients to identify differentially expressed genes.

We performed GEP (Affymetrix HG U133 plus 2.0) in 245 pediatric AML patients (237 de novo and 8 secondary AML patients) and used RT-qPCR and Western Blot to validate expression. Methylation specific PCR was used to investigate epigenetic regulation. We tested the effect of the demethylating agent decitabine and the effect of knock-down by siRNA on both proliferation and drug sensitivity in AML cell lines.

IGSF4, a cell-cell adhesion molecule, was highly expressed in AML-t(9;11). Expression within AML-t(9;11) was 18.5 times higher in FAB-M5 versus other FAB-types (p=0.013). RT-qPCR and Western Blot confirmed this. Methylation status investigation showed that high IGSF4 expressing AML-t(9;11) patients with FAB-M5 have no promoter hypermethylation, whereas all other cases do. This was also seen in cell lines. Cell line incubation with decitabine resulted in promoter demethylation and increased expression of IGSF4. Downregulation of IGSF4 by siRNA did not affect proliferation nor drug sensitivity in suspension culture.

In conclusion, we identified IGSF4 overexpression to be discriminative for AML-t(9;11) with FAB-M5, regulated partially by promoter methylation.

Disclosures:

No relevant conflicts of interest to declare.
Gene Expression-Based Chemical Genomics Identifies Valproic Acid to Revert the Oncogenic Effect of GATA1s In Down Syndrome Megakaryoblastic Leukemia.
Authors: Heitmann Kirsten , Li Zhe , Klusmann Jan-Henning , Thakur Basant Kumar , Schöning Jennifer , Bourquin Jean-Pierre , Orkin Stuart H. , Reinhardt Dirk
Published: October 2019

Abstract 3646

Children with Down syndrome (DS) are at high risk to develop acute megakaryoblastic leukemia (DS-AMKL) and the antecedent transient leukemia (DS-TL). Acquired mutations in the hematopoietic transcription factor GATA1, leading to expression of a shorter GATA1 variant (referred to as GATA1s) truncated at its N-terminus, are consistently present in the affected cells of children with DS-AMKL and DS-TL. Mechanistically, we recently found that in fetal megakaryocytic progenitor cells, GATA1 coordinates proliferation and differentiation by repressing E2F target genes through a direct interaction with E2F activators. Failure of this GATA1-E2F interaction in mutated GATA1s likely converges with overactive IGF signaling to promote cellular transformation.

The treatment of DS-AMKL is hampered by their sensitivity against current cytostatic agents, resulting in treatment-related mortality as the main cause of death. To develop novel targeted and less toxic treatment options for DS-AMKL and DS-TL, we conducted a gene expression-based chemical genomic screen. We connected a DS-AMKL gene expression signature (compared to non-DS-AMKL, i.e. GATA1s vs. GATA1) to a reference collection of gene-expression profiles from cultured human cells treated with bioactive small molecules (Connectivity Map). We discovered the histone deacetylase (HDAC) inhibitor valproic acid (VPA) reverses the DS-AMKL gene expression program.

Cell viability assays, cell cycle analyses, growths curves and colony-forming assays revealed exceptional sensitivity of DS-AMKL cell lines (CMK, CMY; IC50 1mM) and primary DS-AMKL and DS-TL blasts to VPA treatment compared to control cell lines K562 (IC50 4.75mM), M07 (IC50 6.75mM) and CD34+ hematopoietic stem and progenitor cells (IC50 4.75mM). VPA induces apoptosis (26.8% 7-AAD-)Annexin V+ and 38.8% 7-AAD+ CMK cells after 48h at 2mM VPA) and cell cycle arrest (50% reduction of CMK cells in S-phase at 2mM) via activation of the cell cycle inhibitor p21 and the proapoptotic genes BAX and BAK. Gene expression profiles indicated that VPA interferes with the oncogenic effects of GATA1s by globally repressing the deregulated E2F targets. The effects of VPA on leukemic growth in DS-AMKL could be attributed to its HDAC inhibitory function, as the global HDAC inhibitors SAHA and TSA induced a similar response.

Thus, by using a gene expression-based chemical genomic approach, we identified VPA as an efficient and well-tolerated treatment option for DS-AMKL and DS-TL by targeting GATA1s-mediated deregulation of the E2F transcription network.

Disclosures:

No relevant conflicts of interest to declare.