bluuebunny/crossref_metadata_2025_split huggingface dataset:
Increased Copy Number of Interleukin-6 Receptor Gene Is Associated with Adverse Survival In Multiple Myeloma Patients Treated with Autologous Stem Cell Transplantation
Authors: Kim Seon Young , Min Hyun Jung , Park Hyun Kyung , Oh Bora , Kim Tae Young , Kim Hyun Kyung , Kim Inho , Yoon Sung-Soo , Park Seonyang , Kim Byoung Kook , Lee Dong Soon 
Published: October 2019

Abstract
Abstract 1895

Background:
Interleukin-6 (IL-6) is a potent pleiotropic cytokine that regulates plasma cell (PC) growth via IL-6 receptor (IL-6R). We hypothesized that upregulation of IL-6R in myeloma cells might confer the growth privilege to myeloma cells over bone marrow (BM) hematopoietic cells. We investigated the frequency and prognostic implication of increased copy number of IL-6R gene by fluorescence in situ hybridization (FISH) in patients with newly diagnosed multiple myeloma (MM). To confirm the role of IL-6R, the change of proliferative potentials after blocking of IL-6R by monoclonal antibody was observed in a myeloma cell line.

Methods:
102 patients with newly diagnosed MM were enrolled. FISH study for IL-6R was performed using home-made probe for the locus. The BAC (bacterial artificial chromosome) clone RP11 350G8 (BACPAC Resources, Oakland, CA) was used as the IL-6R gene-positive clone and the FISH probe was labeled by a nick translation reaction. FISH signals were counted among BM plasma cells sorted by immunoglobulin light chain staining (cIg FISH). U266 myeloma cell line was treated with IL-6R monoclonal antibody (MRA, Chugai, Japan) at concentration of 0.06 to 1.25 μM and then cell viability test were performed.

Results:
The amplification of IL-6R was detected in 53/102 patients (52.0%). The 5-year overall survival (OS) rate of patients with IL-6R gene amplification was 41.3% versus 44.8% for those with a normal IL-6R (P = 0.425). In 44 patients treated with high-dose chemotherapy and autologous stem cell transplantation (ASCT), patients with 3.1 or more mean IL-6R gene copy number per PC showed worse 5-year OS compared to those who had less than 2.1 mean copies of IL-6R gene (44.4% versus 78.0%, P = 0.024). In multivariate analysis, the increase of IL-6R copy numbers (mean copy/PC ≥ 3.1) could be considered as an independent prognostic factor for MM patients underwent ASCT (hazard ratio, 30.9; 95% CI, 1.74–548.6; P = 0.020). Treatment of IL-6R monoclonal antibody on the U266 cell line induced marked reduction of cell viability compared with control, ranged from 7.1% to 98.7% according to the increase in treated antibody level.

Conclusions:
The gain of IL-6R gene is frequent in myeloma, showing an association with adverse prognosis in MM patients treated with ASCT. In vitro suppression of cell proliferation by IL-6R blocker suggests the potential role of IL-6R in myeloma cell growth and therapeutic implication of IL-6R blocker in the future.

Disclosures:
No relevant conflicts of interest to declare.
Valuation of MCP-1 and VEGF Chemokines In Serum of Patients with AL Amyloidosis
Authors: Moscetti Alessandro , Saltarelli Francesca , Bianchi Maria Paola , Bruno Guglielmo , Salerno Gerardo , Corsetti Maria Teresa , Cardelli Patrizia , Ferranti Giovanna , La Verde Giacinto 
Published: October 2019

Abstract
Abstract 1922
AL amyloidosis is a pathology characterised by the deposition of fibrillary aggregates of immunoglobuline light chains with β-sheet conformation. The light chains are synthetized by neoplastic plasma cell and fibrils deposition can infiltrate tissues leading to multi systemic organ damage. To evaluate if vascular modifications are involved in AL amyloidosis, inflammatory activity of cytokines as MCP-1 and VEGF was investigated. MCP-1 is a chemokine that activates mononuclear phagocytes by promoting leukocyte-endothelium binding and migration to sites of inflammation, while VEGF is an endothelial cell mitogen and permeability factor that is potently angiogenic in bone marrow of AL amyloidosis patients. Aim of this study is to evaluate serum cytokines MCP-1 and VEGF levels in patients with systemic or localized AL amyloidosis at presentation to find out potential differences useful to define a characteristic inflammatory pattern. Blood samples were collected from 8 patients with systemic amyloidosis and from 4 patients with localized amyloidosis and analyzed for serum MCP-1 and VEGF levels. Mann-Whitney test and Spearman correlation were used to compare results. MCP-1 level was significantly higher in the serum of patients with systemic disease (350.52±58.70 pg/ml) compared to the group of patients with localized amyloidosis (147.82±26.03; p=0.004); VEGF was also significantly increased in systemic disease group (p= 0.028). In addition, a positive correlation between MCP-1 and VEGF (r2= 0.755; p=0.031) has been found in the group of patients with systemic amyloidosis. Results seems to suggest a difference in serum cytokine MCP-1 and VEGF levels between AL systemic and localized amyloidosis. In systemic amyloidosis the neoplastic plasma cells interact with bone marrow microenvironment resulting in VEGF release leading to a new angiogenesis also supported by an inflammatory cells increase. The MCP-1 activates and promotes leukocyte-endothelium binding increasing the inflammatory process. The high correlation between MCP-1 and VEGF suggests a positive relationship between a new angiogenesis and a migration of inflammatory cells in the bone marrow stroma. On the basis of our results, MCP-1 and VEGF chemokines can be used to evaluate the inflammatory process in patient with systemic or localized AL amyloidosis.

Disclosures:
No relevant conflicts of interest to declare.
Long-Term Maintenance with Lenalidomide Improves Progression Free Survival In Myeloma Patients with High-Risk Cytogenetics: An IFM Study
Authors: Avet-Loiseau Hervé , Caillot Denis , Marit Gerald , Lauwers-Cances Valerie , Roussel Murielle , Facon Thierry , Hulin Cyrille , Lamy Thierry , Pegourie Brigitte , Maisonneuve Herve , Garderet Laurent , Moreau Philippe , Stoppa Anne-Marie , Dib Mamoun , Jaubert Jerome , Casassus Philippe , Mathiot Claire , Harousseau Jean-Luc , Moreau Philippe , Attal Michel 
Published: October 2019

Abstract
Abstract 1944
Cytogenetic abnormalities have been associated with specific outcome in myeloma, as in many other hematologic malignancies. Among the large spectrum of chromosomal changes observed in myeloma, at least two of them are clearly associated with a specific poor outcome, both in terms of PFS and OS, i.e., t(4;14) and del(17p). Recent data suggested that bortezomib can at least partially overcome the prognostic value of t(4;14). However, t(4;14) remains a prognostic factor in the IFM series. Deletion 17p is rare (< 10%), but is associated with a very poor outcome, whatever the treatment proposed to these patients. In 2005, the IFM designed a clinical trial aiming to test the role of lenalidomide maintenance until relapse in patients under 65 years of age treated with a VAD or bortezomib/dexamethasone induction, followed by high-dose melphalan. Six hundred and fourteen patients have been enrolled, and the clinical results of this trial are presented in another abstract. Chromosomal data focused on t(4;14) and del(17p) were available for 488 of those patients, as analyzed at diagnosis by FISH on sorted plasma cells. The t(4;14) was observed in 13.3% of the patients and del(17p) (defined by presence in at least 60% of the plasma cells) was present in 6.6% of them, incidences that are in agreement with previous reports. The median PFS (calculated from the date of randomization) for patients with t(4;14) were 15 months and 27 months for patients in the placebo and lenalidomide arms, respectively. The median for patients with del(17p) were 14 months and 29 months for patients in the placebo and lenalidomide arms, respectively. The comparison of PFS in the lenalidomide arm for patients with t(4;14) or del(17p), as compared with those lacking the chromosomal abnormalities shows a significant difference, in favor of the “no abnormality” group. In conclusion, lenalidomide maintenance very significantly improves the PFS of patients with high-risk cytogenetics, albeit these chromosomal changes retain their prognostic value.

Disclosures:
Facon: celgene: Consultancy, Research Funding; johnson and johnson: Consultancy. Attal:celgene: Consultancy, Research Funding; johnson and johnson: Consultancy, Research Funding.
Development of a Stable Thrombotic Core with Limited Access to Plasma Proteins During Thrombus Formation In Vivo
Authors: Stalker Timothy J. , Traxler Elizabeth A. , Diamond Scott L. , Brass Lawrence F. 
Published: October 2019

Abstract
Abstract 2013
Several studies examining thrombus formation in vivo have described the formation of a thrombotic core composed of stably adherent, apparently activated platelets covered by several layers of minimally activated, loosely adherent platelets. Using confocal fluorescence intravital microscopy in mouse cremaster muscle arterioles, we sought to 1) determine whether the stable thrombus core could be defined by the presence of platelet activation markers (e.g. P-selectin); 2) define the spatial and temporal characteristics of the stable thrombus core as it develops; and 3) determine whether the accessibility of plasma proteins to the inner regions of a stable thrombus is limited. We found that P-selectin platelet surface expression is associated with stable platelet incorporation into a growing thrombus following either laser- or wall puncture-induced vascular injury in vivo. The P-selectin positive core originated at the site of vascular injury and subsequently expanded outward into the central region of the developing thrombus with kinetics that were distinct from total platelet accumulation. Further, using two related approaches, we determined that the accessibility of plasma components to the stable thrombus core is limited. In the first approach, fluorescently labeled anti-P-selectin antibody was infused after a stable thrombus had formed (25 minutes after injury), and was found to bind to a monolayer of platelets on the luminal surface of the stable thrombus core, but was unable to penetrate this layer of platelets and bind to platelets in the central region of the thrombus. In the second approach, we directly measured the porosity of thrombi as they evolved by infusing fluorescently labeled dextran to illuminate the plasma in the gaps between platelets. We found that the porosity of the stable thrombus core was significantly decreased as compared to the porosity of the growing platelet mass before the core developed. Taken together, these results demonstrate that a stable core composed of degranulated platelets develops during thrombus formation in vivo with spatial localization and kinetics that are distinct from total platelet accumulation, and that development of this core limits the accessibility of plasma components to the central region of a stable thrombus.

Disclosures:
No relevant conflicts of interest to declare.
The Hepatic Asialoglycoprotein Receptor Regulates Platelet Homeostasis
Authors: Grozovsky Renata , Jansen Gerard , Hoffmeister Karin M. 
Published: October 2019

Abstract
Abstract 2025
The human body produces and removes more than a 100 billion of platelets every day. The mechanisms responsible for platelet homeostasis are subject to speculation since the 1950's. The most popular hypothesis to date has been antibody-mediated clearance, platelet consumption due to massive blood loss and an internal “senescence timer”. We and others have recently demonstrated that sialic acid deficient platelets due to external triggers such as sepsis or chilling are cleared by hepatic asialoglycoprotein receptors (ASGPR) independently of macrophages. Here, we investigated whether loss of sialic acid mediates platelet clearance in vivo. We show that 1) Injection of the specific sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) lengthened the survival of biotinylated platelets by ∼50% (T1/2 of 72h), compared to mock treated (PBS injected) control mice (T1/2 of 49h); 2) Similarly, biotinylated platelet survival in ASGPR-null mice was prolonged by ∼ 50% (T1/2 of 74h) compared to platelet survival in wild type (WT) mice (T1/2 of 48h); 3) ASGPR-null mice have significantly increased platelet counts, compared to WT (p=0.0004) and platelets isolated from ASGPR-null mice are ∼15% smaller than WT (p=0.03); 4) Platelets isolated from ASGPR-null mice showed significant increased in b-galactose exposure (∼50% increase, i.e. decrease of sialic acid), compared to WT, as evidenced by binding of the b-galactose specific lectin (RCA-I). These data show that the ASGPR not only removes desialylated platelets due to sepsis or chilling, but also regulates platelet homeostasis.

Disclosures:
No relevant conflicts of interest to declare.
Dynamic Erythropoietin Receptor Trafficking within Cell Surface, and Intracellular Pools
Authors: Singh Seema , Leu Karen , Mortensen Bruce , Emerson Christine , Pradeep Anamika , Young Peter R. , Schatz Peter J , Green Jennifer M , Wojchowski Don M. 
Published: October 2019

Abstract
Abstract 2046
The erythropoietin receptor mediates pro-erythroblast production; can promote erythrocytosis; and can cytoprotect select non-hematopoietic tissues. For certain key aspects of EPOR biology, knowledge nonetheless remains limited. This includes regulated EPOR trafficking, which we presently investigate using a novel panel of specific rabbit monoclonal anti-EPOR antibodies. In exponentially growing EPO-dependent UT7epo cells, a single high-Mr species was detected at low levels, and EPOR turnover appeared to predominate. The limiting of EPO led to the accumulation of two high Mr EPOR forms, EPOR-68K and EPOR-70K. EPO exposure rapidly converted an indicated cell surface EPOR-70K form to an EPOR-72K tyrosine-phosphorylated species (and to lower Mr fragments). In contrast, EPOR-68K was not rapidly modulated by EPO, and proved to comprise an intracellular, endoglycosidase-sensitive EPOR pool. At the cell surface, EPOR repopulation also was regulated and interestingly was accelerated in response to higher-dose EPO exposure. Constitutive EPOR turnover was observed, but as comparably limited ligand-independent trafficking. EPOR expression in primary erythroid progenitors also was analyzed. Flow cytometry and western blotting each unexpectedly revealed peak EPOR expression at a GPAhigh late erythroblast stage. These studies provide new insight into dynamic, EPO dose-dependent EPOR trafficking, together with an observed sustained expression of the EPOR within maturing primary human erythroblasts.

Disclosures:
Leu: Affymax, Inc.: Employment. Mortensen:Affymax, Inc.: Employment. Young:Affymax, Inc.: Employment. Schatz:Affymax, Inc.: Employment. Green:Affymax, Inc.: Employment.
Risk Factors for Pulmonary Hypertension In Patients with Thalassemia Intermedia
Authors: Karimi Mehran , Musallam Khaled M , Cappellini Maria Domenica , El-Beshlawy Amal , Belhoul Khawla , Daar Shahina , Saned Mohamed-SalahEldin , Temraz Sally , Taher Ali T 
Published: October 2019

Abstract
Abstract 2069

Introduction:
Mechanisms leading to pulmonary hypertension (PHT) in patients with thalassemia intermedia (TI) are still controversial. Moreover, clinical and laboratory characteristics of patients who eventually develop PHT have not been identified. Our aim was to identify factors that characterize TI patients who develop PHT.

Methods:
Data was retrospectively retrieved from the Thalassemia Intermedia Registry, a database of 584 TI patients currently registered at six comprehensive care centers in Lebanon, Italy, Iran, Egypt, United Arab Emirates, and Oman. Institutional review boards at each center approved the study protocol. Two Groups of patients were identified: Group I, TI patients with documented PHT (n=64; mean age 37.3 ± 10.6; 44% males); and Group II, age- and sex-matched TI patients without PHT (n=64; mean age 37.9 ± 11.4; 44% males). Collected data included demographics, laboratory parameters, disease-complications, and received treatments that may influence PHT development and reflected the period prior to PHT occurrence.

Results:
There were no statistically significant differences in mean platelet counts, total or fetal hemoglobin levels between the two Groups. However, mean serum ferritin level was higher in Group I compared to Group II (1233.2 ± 499.2 vs. 654.7 ± 234.5 ng/ml; P=0.01). Moreover, mean nucleated red blood cell (NRBC) count was higher in Group I compared to Group II (354.2 ± 199.2 vs. 214.7 ± 94.5 ×10⋀6/l; P=0.03). A higher proportion of patients were splenectomized (84.4% vs. 46.9%; P<0.001) or had a previous history of thromboembolic events (40.6% vs. 7.8%; P<0.001) in Group I compared to Group II. Conversely, a higher proportion of patients received transfusion (78.1% vs. 56.2%; P<0.001), iron chelation (62.5% vs. 37.5%; P<0.001), or hydroxycarbamide (34.4% vs. 17.2%; P<0.001) therapy in Group II compared to Group I. There were no statistically significant differences in the proportion of patients with heart failure, prothrombotic mutations, or receiving antiplatelet or anticoagulant therapy between the two Groups. Multivariate logistic regression analysis revealed that patients in Group I are more likely to be splenectomized (OR:4.9, 95%CI:1.9-8.5); transfusion-naive (OR:3.5, 95%CI:2.1-6.25); on no hydroxycarbamide (OR:2.6, 95%CI:1.1-5.25) or iron chelation therapy (OR:2.3, 95%CI:1.2-4.25); and have NRBC count >300 ×10⋀6/l (OR:2.59, 95%CI:1.69-6.05) or a previous history of thromboembolism (OR:3.69, 95%CI:2.38-7.05).

Conclusion:
Splenectomy, previous history of thromboembolism, and a high NRBC count characterize TI patients who develop PHT. Transfusion, iron chelation, and hydroxycarbamide therapy deserve evaluation for a protective role against PHT in TI.

Disclosures:
No relevant conflicts of interest to declare.
A Bispecific Protein Targeting the NKG2D Receptor on Natural Killer Cells: In Vitro and In Vivo activity of ULBP2-CEA
Authors: Madlener Marie , Rothe Achim , Jachimowicz Ron , Reiners Katrin S. , Chatterjee Sampurna , Ullrich Roland , Yazaki Paul , Engert Andreas , von Strandmann Elke Pogge 
Published: October 2019

Abstract
Abstract 2095
Natural killer (NK) cells are effector lymphocytes of the innate immune system. NK cell activity is tightly regulated by inhibitory and activating surface receptors to ensure that NK cells recognize and eliminate target cells, whereas healthy cells escape from the NK cell-mediated immune surveillance. The inability of the immune system to recognize and kill malignant target cells has been partly attributed to the ineffective activation of NK cells. It is well accepted, that the activating NK receptor NKG2D (natural killer group 2 member D) plays an important role in tumor rejection. The Immunoligand ULBP2-CEA is a recombinant bispecific protein designed to enhance NK cell-mediated lysis of CEA-expressing target cells. Consisting of the NKG2D-ligand ULBP2 (UL16 binding protein 2) and an anti-CEA single chain antibody, this construct provides the ability of cross-linking NK cells and CEA-expressing cells for specific tumor targeting. CEA (carcinoembryonic antigen) is frequently overexpressed in colorectal and other carcinomas. The binding and specificity of ULBP2-CEA fusion protein was tested in FACS analysis. Transfected human CEA+ murine colon carcinoma cells (MC38-CEA) were incubated with ULBP2-CEA and recombinant NKG2D receptor (NKG2DR). The protein was able to bind both, tumor cells and recombinant NKG2DR simultaneously. No specific cell binding was detectable in control experiments using fusion constructs with another binding moiety (e.g. ULBP2-BB4) or soluble NKG2DR alone. In cytotoxicity assays ULBP2-CEA enhances the susceptibility to NK cell mediated lysis of CEA-expressing cells. CEA+ human colon carcinoma cells (LS174T) were incubated with NK cells isolated from healthy donors in the presence or absence of ULBP2-CEA. The increase of cell lysis was significant at all effector-target ratios compared to the controls demonstrating specific targeted in vitro efficacy of ULBP2-CEA. In order to investigate anti-tumor activity in vivo we use a syngeneic mouse model. MC38-CEA cells were implanted subcutaneously in CEA transgenic C57BL/6 mice and tumor growth was assessed using in vivo bioluminescence imaging (BLI). First results on the in vivo efficacy of ULBP-CEA and effects on the activation status of splenic and peripheral blood NK and T cells upon treatment will be discussed. This syngeneic mouse model is a useful tool to study efficacy and mode of action of ULBP2-CEA in vivo.

Disclosures:
No relevant conflicts of interest to declare.
Shear Induced Reversible Conglomerates of Von Willebrand Factor Networks with Aggregates of Inactivated Platelets - a Fast Switchable Plugging Mechanism
Authors: Reininger Armin J. , Napoleone Marina , Angerer Jennifer , Falah Babak , Schneppenheim Reinhard , Schneider Matthias 
Published: October 2019

Abstract
Abstract 2104
Thrombus growth rate has been reported to be a linear function of very high shear rates beyond 40,000 s-1 (Ku DN, Bioreology. 2007;44(4):273-84), which are thought to occur in severe arterial stenosis. We therefore applied shear rates continuously increasing from 2,000 s-1 to 40,000 s-1 and examined platelet and von Willebrand factor (VWF) interaction in a parallel plate flow chamber under direct visualization. Above a critical threshold of 10,000 to 15,000 s-1 aggregates of inactivated platelets formed when VWF was present in solution and immobilized on the perfused surface (Ruggeri, … Reininger; Blood, 108: 1903–1910, 2006). A new finding was the shear dependent simultaneous formation of VWF strands in and around those platelet aggregates, growing to strands up to several microns thick and up to several tens of microns long, thus forming large VWF networks. Platelets appeared to be enmeshed in the VWF networks but were nevertheless a prerequsite for their formation, mutually fostering each other. Platelet-VWF conglomerates formed at the perfused VWF surface, were constantly rolling in the flow direction, grew in size with increasing shear rate, and were completely reversible below the critical shear rate threshold of 10,000 s-1. VWF networks assembled in whole blood as well as in washed blood cell suspensions reconstituted with VWF. Recombinant full length VWF generated larger VWF networks than a commercial VWF preparation from pooled human plasma. Perfusion of collagen coated surfaces caused assembly of VWF networks anchored to the collagen fibrils and capture of activation-blocked platelets within them at shear rates of 2,000 s-1, leading to residence times of platelet-VWF conglomerates of more than a minute. Newly formed platelet conglomerates could be visualized rolling on the surfaces of already attached conglomerates. When we used a gain of function mutation of VWF, rolling platelet aggregates could be detected at a shear rate as low as 1,500 s-1. We conclude that shear rate activation of VWF leads to reversible platelet-VWF network generation, which may be a crucial mechanism of fast platelet accrual and prolonged arrest times at sites of thrombosis as well as physiological hemostasis.

Disclosures:
No relevant conflicts of interest to declare.