bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Recombinant Antibody to EphA3 with Pro-Apoptotic and Enhanced ADCC Activity Against Various Hematologic Malignancies Shows Selective Inhibition of Colony Formation From Long-Term Culture-Initiating Cells (LTC-ICs) In Primary Leukemia Samples
Authors: Palath Varghese , Vekhande Rohini , Baer Mark , Ching Wendy , Tomasevic Nenad , Martinez David , Yi Christina , Bhaskar Swathi , Luehrsen Kenneth , Williams Jason , Leung John , List Alan F. , Zhang Ling , Lackmann Martin , Boyd Andrew , Scott Andrew , Cilloni Daniela , Yarranton Geoffrey , Bebbington Christopher 
Published: October 2019

Abstract
Abstract 2897
EphA3 is a receptor tyrosine kinase expressed at high level on the surface of some solid tumors and certain leukemias. KB004 is a high-affinity non-fucosylated, recombinant antibody developed by KaloBios derived from a mouse monoclonal anti-EphA3 antibody using Antibody Humaneering™ Technology. The antibody binds EphA3 from human and cynomolgus macaque with equivalent affinity (610 pM for human and 570 pM for cynomolgus as determined by surface plasmon resonance (spr) analysis) making the latter a relevant toxicology species. KB004 has a direct pro-apoptotic effect on EphA3-positive cells and a induces potent antibody-dependent cellular cytotoxicity (ADCC) activity mediated by CD16-expressing effector cells. The non-fucosylated antibody has enhanced affinity for both human and cynomolgus CD16a. The affinity of KB004 for cynomolgus CD16a (7.2 nM, determined by spr analysis) was comparable to the affinity for the high-affinity variant of CD16a from humans (6.6 nM), indicating that KB004 has similar potential for ADCC in cynomolgus macaques.
We first demonstrated cell-surface expression of EphA3 by flow cytometry in approximately 50% of primary human leukemia cells. Here we extend the analysis to multiple myeloma (MM) in which 3/5 patient samples show EphA3 expression. EphA3 expression was confirmed in a panel of chronic myeloid leukemia (CML) samples [n=14] and in acute myeloid leukemia (AML) [n=40] in which EphA3+ cells were identified in each French American British (FAB) classification sub-type except M0, for which only two samples were studied. CD123-positive leukemia stem cells (CD34+ CD38− CD123+) also displayed surface expression of EphA3. KB004 induced apoptosis in EphA3+ primary cells from each of the diseases analyzed, including imatinib-sensitive and resistant CML, with no activity in EphA3-negative specimens, including normal CD34+ bone marrow cells. KB004 inhibited myeloid leukemia colony formation (CFU-L) from primary AML samples in methylcellulose colony assays without affecting normal hematopoietic colony recovery and also showed dose-dependent inhibition of long-term culture-initiating cells (LTC-IC) from AML stem cells. EphA3 expression was not detected on primary human CD34+ bone marrow cells from healthy individuals.
These data support the development of an anti-EphA3 antibody for the treatment of hematologic malignancies. The selective activity against leukemic stem cells supports a novel strategy for therapeutic targeting of leukemia-initiating cells.

Disclosures:
No relevant conflicts of interest to declare.
Lenalidomide reexposure After Short Interruption In Del(5q) MDS Patients at Relapse of Transfusion Dependence
Authors: Radkowski Robert Marek , Haase Sabine , Schlegelberger Brigitte , Goehring Gudrun , Banisch Stefanie , Heinsch Michael , Aul Carlo , Giagounidis Aristoteles 
Published: October 2019

Abstract
Abstract 2923

Introduction:
Lenalidomide is effective in myelodysplastic syndroms (MDS) in patients with del(5q) chromosomal abnormality (List et al 2006). However, relapse of transfusion dependence occurs consistently and therapeutic measures at that moment are scarce. We report on our experience to achieve a second remission of transfusion dependence using lenalidomide after a variable period of lenalidomide treatment interruption.

Methods:
5 patients with a del(5q) chromosomal abnormality (low- and intermediate-1-risk MDS according to IPSS) were treated within clinical trials, all transfusion-dependent with a median age of 67 years and with female prevalence. Mean MDS duration before start of lenalidomide therapy was 4.2 years. Patients were treated with doses of oral lenalidomide ranging from 5 mg every other day to 10 mg for 28 days of every 28 days cycle. All patients had a response to the initial lenalidomide treatment and achieved transfusion independence. All 5 patients relapsed and became red cell transfusion dependent, again. Median time to relapse was 24.2 months (13-55 months).The therapy was stopped and after a therapy-free interval of 7.2 months (2-13 months) we resumed lenalidomide therapy. The patients were treated with the initial lenalidomide dose.

Results:
3 of 5 patients (60%) became transfusion independent, again. The median increase in hemoglobin from baseline to the maximum hemoglobin achieved was 4.4 g/dl. Patients are followed up and all of them are currently in ongoing transfusion independence with a median time of 16.3 months (11, 15, 23 months, respectively). The other two patients stayed transfusion dependent and lenalidomide was interrupted after 3 and 4 months, respectively. Both later progressed to higher MDS subtypes or acute myeloid leukemia. Responses seemed to be independent on age, time to relapse, interval to retreatment or doses of lenalidomide.

Discussion:
In low/int-1 IPSS del(5q) patients relapsing with red cell transfusion dependence during lenalidomide treatment, current algorithms recommend discontinuation of the drug. Our results suggest efficacy of lenalidomide in a significant portion of the patients when they are rechallenged with a standard dose after a therapy-free interval of 2 to 6 months. Second remissions might be as long lasting as, or exceeding, the initial one. We are unable to define predictive factors for second responses. Lack of further response may be an ominous prognostic sign and, in our experience, heralded progression to higher risk MDS subtypes or acute myeloid leukemia after a short time.

Disclosures:
Giagounidis: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.
Comparison of Stromal Cells of Adipose Tissue From Multiple Myeloma Patients and Healthy Donors
Authors: Hébraud Benjamin , Labat Elodie , Espagnolle Nicolas , Corre Jill , Attal Michel , Bourin Philippe 
Published: October 2019

Abstract
Abstract 2979

Introduction:
Multiple myeloma (MM) is a B-cell neoplasia characterized by the accumulation of malignant plasma cells in the bone marrow (BM) patients causing severe bone disease (with osteolytic lesions, pain, pathological fractures) and cytopenias. These lesions are irreversible, even for patients in complete response to treatment. We have proposed to treat those lesions with mesenchymal stem cells (MSC) of the BM using their ability to differentiate into osteoblasts and to support haematopoiesis. But our previous results demonstrated that MM MSC are abnormal. We thus studied cells from adipose tissue (AT) with similar properties than MSC; the Adipose derived Stromal Cells (ASC). The aim of this study is to demonstrate the normality of ASC in MM context for a potential use in autologous stem cell transplantation. Here we propose the first study comparing ASC issue from MM patients and healthy donors (HD).

Patients and Methods:
We studied ASC from 15 patients with newly diagnosed MM and 15 HD between 18 and 65 years. All gave their informed consent. The stromal vascular fraction was isolated from subcutaneous AT by and centrifugation. The ASC were sorted by adhesion to the plastic flask and expanded for 3 passages. We then performed the following assays to compare ASC from MM and HD:

Results:
The cell culture assay of ASC didn't show any difference between MM and HD for all the studied parameters: expansion capacity (HD 470± 45, MM 208±194; p=0.13), cell population doubling (HD 16.0±0.3, MM 15.0±1.3; p=0.17) and progenitor frequency (HD 3.4%±3, MM 3.7%±5.5; p=0.165). ASC phenotype didn't show any significant difference for all the checked markers and confirm their stromal feature: positive for CD90, CD73 and CD105, and negative for CD14 and CD45. The differentiation assay was evaluated for osteogenic, chondrogenic and adipogenic lineages. We did not underline any difference comparing ASC from MM or HD (Table 1). We previously showed that MM MSC secreted increased amount of IL-6, GDF15 and DKK-1 when compared with HD MSC. Interestingly, no difference was observed between MM ASC and HD ASC (Table 2). In the same way, contrary to MM MSC, MM ASC didn't promote the proliferation of MOLP-6 cell line better than HD ASC (proliferation rate: 1.49±0.34 for HD and 1.66±0.80 for MM; p=0.33).

Conclusion:
To our knowledge, this is the first study to compare ASC from MM patient and HD. These preliminary data suggest that ASC from MM patients are normal and could potentially be used in autologous stem cell transplantation for MM patients. We are currently completing this study by performing haematopoiesis support and microarrays assays.

Disclosures:
No relevant conflicts of interest to declare.
Targeting Multiple Myeloma with Human Ig Light Chain Specific Cytotoxic T Lymphocytes
Authors: Weng Jinsheng , Cha Soung-Chul , Matsueda Satoko , Neelapu Sattva , Kwak Larry W. 
Published: October 2019

Abstract
Abstract 3018
The variable regions of Ig expressed by malignant B cells can serve as a tumor specific antigen. Clinical trials of idiotype (Id) vaccines have demonstrated humoral responses and prolonged remission duration in a recent phase III trial of follicular lymphoma patients in the first remission (Schuster et al, J Clin Oncol 27: 793S, 2009). However, the potentially immunogenic epitopes derived from Ig that stimulate CD8+ T cell immunity have been incompletely characterized. Here, we identified nine out of 14 candidate peptides derived from the Ig L-chain variable region of the human U266 myeloma line, which generated cytotoxic T lymphocytes (CTLs) from 53 HLA A2+ normal donors. These CTLs lines, as well as CTLs line isolated from myeloma patients by stimulation with autologous L-chain Id peptides, specifically produced IFN-γ in response to peptide-pulsed T2 cells and lysed U266 and autologous myeloma cell targets, respectively, but not normal blood B cells. Lysis was HLA class I-dependent, suggesting that primary myeloma cells express Id peptides on the cell surface in combination with HLA molecules. Nine CD8+ Id peptide-specific T-cell clones exhibited the effector memory phenotype and the ability of these T cell clones to eliminate U266 tumor in immune deficient mice is being tested. Finally, sequence analysis revealed shared T-cell epitopes in both framework and CDR regions of the U266 L-chain. CTLs generated against a shared U266 epitope lysed patient-derived myeloma cells expressing the shared sequence, suggesting a strategy to overcome the limitation of patient-specific Id vaccine manufacture. Our data identified novel immunogenic Id L-chain T-cell determinants and suggests that, unlike previously described Ig heavy chains, these sequences harbor common T-cell epitopes that may provide the rationale for shared Id vaccines.

Disclosures:
No relevant conflicts of interest to declare.
Statin Therapy and Levels of Thrombosis Risk Factors In a Healthy Population: the Multi-Ethnic Study of Atherosclerosis
Authors: Adams Nathan B. , Lutsey Pamela , Folsom Aaron , Herrington David , Sibley Christopher T , Zakai Neil , Ades Steven , Burke Gregory , Cushman Mary 
Published: October 2019

Abstract
Abstract 3178

Background:
HMG CoA reductase inhibitors (statins) were recently reported to reduce the rate of venous thromboembolism (VTE) in healthy people. Statins reduce levels of inflammation biomarkers, however the mechanism for a reduction in VTE risk is unknown. We studied cross-sectional associations of statin use with hemostatic factors related to venous thrombosis risk in a large cohort of healthy people.

Methods:
Cross-sectional analyses were performed in the Multi-Ethnic Study of Atherosclerosis (MESA), a cohort study of 6814 healthy men and women age 45–84, free of clinical cardiovascular disease at baseline; 1001 were using statins. Twenty three warfarin users were excluded. Age, race, and sex-adjusted mean hemostatic factor levels were compared between statin users and nonusers, and multivariable linear regression models were used to assess associations of statin use with hemostasis factors, adjusted for age, race/ethnicity, education, income, hormone replacement therapy (in women) and major cardiovascular risk factors.

Results:
The table shows that those using statins had significantly lower levels of D-dimer, C-reactive protein and factor VIII than non-users. Homocysteine and von Willebrand factor did not differ by statin use. Specific adjustment for LDL and triglycerides did not attenuate the observed differences in these factors by statin use.

Conclusions:
Findings of lower D-dimer, factor VIII and C-reactive protein levels with statin use suggest mechanisms whereby statins might lower VTE risk. These associations were not explained by lipid levels on treatment. A prospective study linking these biochemical differences to VTE outcomes is warranted.

Disclosures:
No relevant conflicts of interest to declare.
Decreased Activity of ADAMTS13 In Patients with Deep Vein Thrombosis Associated with Inflammatory Markers
Authors: Mazetto Bruna M , de Andrade Orsi Fernanda Loureiro , Barnabé Aline , Matos Marinez Farana , de Paula Erich , Morelli Vânia Maris , Annichino-Bizzacchi Joyce Maria 
Published: October 2019

Abstract
Abstract 3181

Introduction:
Elevated levels of inflammatory markers and clotting factors have been related to the pathogenesis of DVT. Particularly, the balance between VWF and ADAMTS 13 activity has been previously described in patients with thrombosis. VWF levels are also described to be elevated during inflammatory processes and therefore could play a role linking the activation of inflammatory and coagulation systems in patients with DVT.

Objective:
To evaluate the activity of ADAMTS13 and VWF in patients with DVT and its association with inflammatory markers.

Patients and Methods:
Fifty-five patients with DVT, 6 months to five years after the acute episode, attended at the Hematology Center of UNIFESP were included in this study and 121 normal subjects were selected as controls. The activity of ADAMTS 13 was performed by binding of residual VWF to collagen; VWF antigen, IL-6, IL-8, F1+2 and MCP1 was determined by ELISA and D-dimer and CRP was performed by turbidimetry. Continuous variables were analyzed by Mann- Whitney test, categorical variables by the chi-square test and Spearman rank test was used for correlation analysis.

Results:
Thirty-tree patients had DVT caused by transient risk factor, especially the use of oral contraceptives, and 22 patients had spontaneous DVT. No patient had renal, hepatic or malignant disease. The median ADAMTS 13 activity was not statistically different between patients (median= 65.8%) and controls (median=78.5%, p= 0.3043). The plasma levels of VWF antigen, IL-6 and CRP were higher in patients than in controls (p= 0.008, p= 0.0003 and p= 0.008, respectively). The levels of IL-8 and MCP1 did not differ betwee n patients and controls (p= 0.65 and p= 0.07, respectively). The levels of VWF correlated significantly with CRP (r= 0.28, p= 0.005) and F1+2 (r= 0.3, p= 0,02) in patients. There was an inverse correlation between ADAMTS13 activity and F1+2 concentrations (r= -0,33, p= 0.01).

Conclusion:
This study suggests that, even after the acute episode of DVT, a chronic inflammatory state would contribute to increasing levels of VWF in patients' plasma and hypercoagulability. The downregulation of ADAMTS13 activity may also contribute to the activation of the coagulation. However the role of ADAMTS13 in the interaction between inflammatory and coagulation systems in patients with thrombosis is yet to be determined. These factors together may contribute to recurrent DVT, which affects over 25% of patients within 10 years from the first episode.

Disclosures:
No relevant conflicts of interest to declare.
Pseudoexpression of Glucocorticoid-Induced TNF-Related (GITR) Ligand Upon Coating of Cancer Cells by Platelets Impairs NK Cell Anti-Tumor Reactivity
Authors: Placke Theresa , Kanz Lothar , Salih Helmut R. , Kopp Hans-Georg 
Published: October 2019

Abstract
Abstract 3193
NK cells as part of the innate immune system substantially contribute to cancer immune surveillance. They prevent tumor progression and metastasis due to their ability to mediate cellular cytotoxicity and to produce cytokines like IFN-γ, which, among others, stimulates subsequent adaptive immune responses. NK reactivity results from an integrative response emerging upon recognition of multiple ligands for activating and inhibitory NK cell receptors including various members of the TNFR family. Apart from the direct interaction with their target cells, NK cell activity is further influenced by the reciprocal interplay with various other hematopoietic cells like e.g. dendritic cells. Metastatic tumor spread in experimental animals is dramatically reduced in thrombopenic mice. Additional depletion of NK cells reverses this effect, indicating that platelets may impair NK anti-tumor reactivity. However, the underlying mechanisms have not been fully elucidated, especially in humans. Recently, we demonstrated that NK anti-tumor immunity is impaired by platelet-derived TGF-β, which is released upon interaction of platelets with tumor cells (Kopp et al., Cancer Res. 2009). Here we report that the ligand for the TNFR family member GITR (GITRL) is upregulated on megakaryocytes during maturation resulting in substantial GITRL expression by platelets. Since we recently identified GITR as inhibitory NK receptor involved in tumor immune escape (e.g., Baltz et al., Blood 2008, Baessler et al., Cancer Res. 2009) we investigated how platelet-derived GITRL influences platelet function and NK immune surveillance. Signaling via GITRL into platelets upon interaction with NK-expressed GITR or recombinant GITR-Ig fusion protein did not alter platelet activation as revealed by analysis of the activation marker CD62P and release of TGF-β. Interestingly, we found that GITRL-negative tumor cells rapidly get coated by platelets, which confers a seemingly GITRL-positive phenotype. “GITRL pseudoexpression” on tumor cells caused a substantial reduction of NK cell cytotoxicity and cytokine production. This reduced NK reactivity was not due to induction of apoptosis via GITR and could be restored by addition of a blocking GITR antibody. Thus, coating of tumor cells by platelets inhibits NK reactivity, which is in part mediated by platelet-derived GITRL. Our data provide a functional basis for the previously observed finding that platelets increase metastasis i.e. by enabling evasion of tumor cells from NK-mediated immune surveillance.

Disclosures:
No relevant conflicts of interest to declare.
The Transient Receptor-Potential Channel 6 Plays a Critical Role In Platelet Function
Authors: Murad John P , Ting Harold J , Khasawneh Fadi T. 
Published: October 2019

Abstract
Abstract 3201
In spite of the fact that changes in the intracellular levels of calcium (e.g., calcium entry (CE)) is a central step in platelet activation, the underlying mechanism of this CE is still ill defined. Previous studies have demonstrated that of the transient receptor potential channel (TRPC) family, TRPC1 and, to a higher extent, TRPC6 are expressed in blood platelets, whereas TRPC4 is found in megakaryocytes (at the mRNA level). Furthermore, recent studies have shown that TRPC1 deficient platelets exhibited no defects in platelet function, and unaltered calcium homeostasis. Based on these considerations, we hypothesized that TRPC6 plays an essential role in receptor-operated calcium entry and platelet function. Our preliminary studies reveal that platelets deficient in TRPC6 exhibited a defect in platelet aggregation mediated by the thromboxane receptor (TPR), at least, at low agonist levels (i.e., low levels of receptor activation). Moreover, TRPC6 deficient mice also exhibited a significantly prolonged bleeding time, and were protected against thrombosis development, using a FeCl3 carotid-artery injury model. Together, these findings are consistent with the role of TPRs in hemostasis and the genesis of thrombosis. In future studies, we will investigate the role of TRPC6 in: 1. calcium homeostasis; 2. separate platelet functional responses such as dense granule secretion and shape change; and 3. in platelet activation via separate platelet G-protein coupled receptors such as PAR4 and P2Y1. These studies may define a new therapeutic target for managing multiple thrombosis-based disorders.

Disclosures:
No relevant conflicts of interest to declare.
Mutation In Erythroid Specific Transcription Factor KLF1 Causes Hereditary Spherocytosis In the Nan (Neonatal Anemia) Hemolytic Anemia Mouse Model
Authors: White Robert A , Heruth Daniel P. , Hawkins Troy , Logsdon Derek , Gibson Margaret , Sokolovsky Inna , Nsumu Ndona , Major Stephanie , Fegley Barbara , Woods Gerald , Lewing Karen , Neville Kathleen , Cornetta Kenneth , Peterson Kenneth 
Published: October 2019

Abstract
Abstract 3217
The zinc finger protein Erythroid Krüuppel-like factor (EKLF, KLF1) regulates definitive erythropoiesis and terminal differentiation of red blood cells. KLF1 facilitates transcription through high affinity binding to CACCC elements within its erythroid-specific target genes which include genes encoding erythrocyte membrane skeleton (EMS) proteins. Deficiencies of EMS proteins lead to the hemolytic anemia Hereditary Spherocytosis (HS). We have identified a new HS gene by studying the hemolytic anemia mouse mutant Nan (Neonatal Anemia). Here we report that a mutation, E339D, in the second zinc finger domain of KLF1 is responsible for HS in Nan mice. The causative nature of the E339D mutation was verified with an allelic test cross between Nan/+ and heterozygous Klf1+/− knockout mice. Homology modeling predicted Nan KLF1 binds CACCC elements more tightly, suggesting that Nan KLF1 is a competitive inhibitor of wild type KLF1. Competitive inhibition may help explain the apparent disconnect between the finding that Nan/+ heterozygous mice are anemic, whereas Klf1+/− heterozygous mice are normal and haplo-sufficient. This is the first direct association of a KLF1mutation with a disease in adult mammals. After examining a small population of HS patients, we also discovered one HS patient with a KLF1 mutation, which resulted in a significant amino acid substitution (T251I) in the activator/repressor domain, 28 amino acid residues upstream of the first zinc finger domain. This HS subject had no known mutations in the exons or intron/exon boundaries of EMS genes (SPTA1, SPTB, ANK1, SLC4A1) which comprise 85% of HS mutations in humans. The lack of a known genetic mutation in EMS genes leaves this patient's KLF1 mutation as the leading candidate defect. The identification of the gene causing the Nan mutation is significant because the Nan mutant has allowed discovery of a new HS gene which may also cause this disease in humans. In addition, the putative dominant/negative competitive inhibition of the Nan mutation makes the Nan mouse an excellent model system to study the function of KLF1.

Disclosures:
No relevant conflicts of interest to declare.