bluuebunny/crossref_metadata_2025_split huggingface dataset:
CD19 Fc-Fusion Protein for Detection of Cells Expressing Anti-CD19 Chimeric Antigen Receptors.
Authors: Oliveira Satiro N. De , Wang Jiexin , Hollis Roger P. , Kohn Donald B. 
Published: October 2019

Abstract
Abstract 3756
Chimeric Antigen Receptors (CARs) consist of the antigen-recognition portion of a monoclonal antibody fused to an intracellular signaling domain that is capable of activating T cells. CARs are displayed on the surface of transduced cells, performing non-MHC-restricted antigen recognition and activating intracellular signaling pathways for induction of target cytolysis, cytokine secretion and proliferation. Clinical trials are in progress assessing the use of modified mature T lymphocytes transduced with CARs targeting CD19 antigen to treat B-lineage malignancies. CD19 is an attractive target for immunotherapy because of its consistent and specific expression in most of the stages of maturation and malignancies of B lymphocyte origin, but not on hematopoietic stem cells. Antibodies against the extracellular domain of the CAR molecule (anti-Fab, Fc or idiotype) have been used for detection of CAR expression in research and clinical samples by flow cytometry, but may need development for each construct and present significant background in samples from xenograft models. We developed a specific reagent for the detection of anti-CD19 CAR expressed on cells, a soluble fusion protein consisting of human CD19 extracellular domains and the Fc region of human IgG1 (19sIg). Genes encoding 19sIg fusion proteins were constructed by fusing either Exons 1 to 3 (19sIg1-3) or Exons 1 to 4 (19sIg1-4) of the human CD19 cDNA to a human IgG1Fc fragment. These fusion proteins are intended to work in similar fashion as the MHC Tetramers used for identification of antigen-specific T cells, and may also have other applications in studies of activation of anti-CD19 CAR bearing cells. The 19sIg proteins were produced from 293T cells by stable lentiviral vector transduction and purification from culture medium. ELISA assays using several different monoclonal antibodies to CD19 demonstrated dose-related specific binding by the fusion molecule 19sIg1-4, but no binding to 19sIg1-3. Conjugation of the 19sIg1-4 fusion protein to Alexa Fluor 488 allowed specific and sensitive staining of anti-CD19 CAR-bearing cells for flow cytometry assays. Large-scale production and stable conjugation of this fusion molecule will provide a useful reagent to detect anti-CD19 CAR derived from any monoclonal antibody.

Disclosures:
No relevant conflicts of interest to declare.
Olfactomedin 4, An Indicator of the Existence of Neutrophil Subsets.
Authors: Clemmensen Stine Novrup , Roervig Sara , Wren Jonathan , Illemann Martin , Glenthoj Andreas , Mora-Jensen Helena , Cramer Elisabeth , Larsen Maria Torp , Jacobsen Lars C. , Cowland Jack B , Borregaard Niels 
Published: October 2019

Abstract
Abstract 3775
Olfactomedin 4 (OLFM4) was initially identified as a gene highly induced in myeloid stem cells by G-CSF treatment and independently as a gene highly expressed in colon cancers. OLFM4 was predicted in a bioinformatics analysis as associated with neutrophil specific granules. We analyzed the expression of OLFM4 mRNA in myeloid cells from normal human bone marrow and demonstrated that expression of OLFM4 mRNA is similar to the expression of LCN2 which codes for the specific granule protein NGAL (Figure 1), but distinct from expression of mRNA for myeloperoxidase and gelatinase which are marker proteins for azurophil granules and gelatinase granules, respectively. Subcellular fractionation of peripheral blood neutrophils demonstrated complete co-localization of OLFM4 with NGAL, and stimulation of neutrophils with fMLP or PMA resulted in co-release of NGAL and OLFM4, indirectly proving that OLFM4 is a genuine constituent of neutrophil specific granules.
Figure 1. mRNA expression profiles for OLFM4 and LCN2 in populations enriched in myeloblasts/promyelocytes (MB/PM), myelocytes/metamyelocytes (MY/MM), banded cells/segmented cells (BC) and peripheral blood neutrophils (pb-PMN) normalized to ACTB. Figure 1. mRNA expression profiles for OLFM4 and LCN2 in populations enriched in myeloblasts/promyelocytes (MB/PM), myelocytes/metamyelocytes (MY/MM), banded cells/segmented cells (BC) and peripheral blood neutrophils (pb-PMN) normalized to ACTB.
Interestingly, immunohistochemistry showed OLFM4 expression in only a subset of neutrophils (figure 2). We suspected that this might be dependent on the antibody, but two different commercial antibodies and an in-house antibody raised against a synthetic OLFM4 derived peptide, all polyclonal, showed similar patterns. Flow cytometry confirmed the existence of two populations of neutrophils, one expressing OLFM4 the other not.
Figure 2. Immunohistochemistry of OLFM4 in neutrophils. Figure 2. Immunohistochemistry of OLFM4 in neutrophils.
Immunohistochemistry of bone marrow cells showed that OLFM4 appears in myelocytes and is maintained in the cells during further maturation of the cells to segmented neutrophils. Again, only 30% of the neutrophil precursors from bone marrow stain positive for OLFM4 indicating, that different subsets of human neutrophils may exist.

Disclosures:
No relevant conflicts of interest to declare.
Real-World Analysis of Venous Thromboembolism Rates and Thromboprophylaxis Prescribing In US Patients with Cancer.
Authors: Amin Alpesh N , Lin Jay , Thompson Stephen , Wiederkehr Daniel 
Published: October 2019

Abstract
Abstract 3802

Background:
Deep-vein thrombosis (DVT) is a frequent complication of cancer and treatment of cancer. Among clinical guidelines, there is broad consensus regarding the importance of thromboprophylaxis in hospitalized cancer patients, including prolonged prophylaxis in high-risk patients. The objective of this analysis was to assess the real-world use of prophylaxis for DVT, as well as symptomatic rates of DVT and pulmonary embolism (PE) in cancer patients, both during hospitalization and after hospital discharge.

Methods:
Data were extracted from the US Premier Perspective(tm)-i3 Pharma Informatics linked database for non-surgical cancer patients who were admitted to hospital from January 2005 to November 2007. Included patients were aged ≥ 18 years and had ≥ 6 months' continuous plan enrollment. Patients were excluded if they were discharged to an acute-care facility, had length of hospital stay ≤ 0 or > 30 days, had missing/unknown gender or age data, or if they were diagnosed with atrial fibrillation during index hospitalization. Clinical rates of DVT/PE, as well as thromboprophylaxis status and duration were evaluated for during hospitalization and post-discharge.

Results:
Of the 3,759 cancer patients analyzed, 51.9% received inpatient pharmacological and/or mechanical prophylaxis, and 2.8% received outpatient pharmacological prophylaxis in the 14 days following discharge. During index hospitalization 40.0% of patients received mechanical prophylaxis and 21.1% pharmacological prophylaxis, with 9.3% of patients receiving a combination of mechanical and pharmacological prophylaxis. Mean (± standard deviation) duration of prophylaxis was 1.3 (± 2.3) days for inpatients and 0.7 (± 4.5) days post-discharge among all cancer patients. Symptomatic DVT/PE occurred in 2.4% of patients during hospitalization. In the 30 days following hospital discharge, 1.7% of patients was either rehospitalized for DVT/PE or treated for DVT/PE in the outpatient setting.

Conclusions:
This real-world analysis demonstrates considerable symptomatic rates of DVT/PE in hospitalized cancer patients, with approximately half of patients not receiving any thromboprophylaxis. The risk of DVT/PE persisted into the outpatient setting, but few patients received anticoagulants post-discharge. Further efforts are needed to ensure that patients with cancer receive appropriate thromboprophylaxis and that it is of adequate duration to reduce the large burden of DVT/PE.
Acknowledgment: This study was funded by sanofi-aventis U.S., Inc. The authors received editorial/writing support in the preparation of this abstract provided by Tessa Hartog, PhD of Excerpta Medica, funded by sanofi-aventis U.S., Inc.

Disclosures:
Amin: sanofi-aventis US Inc.: Honoraria, Speakers Bureau. Lin:sanofi-aventis US Inc.: Employment, Research Funding. Thompson:sanofi-aventis US Inc.: Employment. Wiederkehr:sanofi-aventis US Inc.: Research Funding.
Publication Patterns of Cancer Cost-Effectiveness Studies Presented at the American Society of Hematology: Timeliness of Publication, Quality and Possible Bias.
Authors: Mozessohn Lee , Chan Kelvin KW , Cheung Matthew C 
Published: October 2019

Abstract
Abstract 3816

Background:
The timely publication of cancer cost-effectiveness analyses (CEA) is essential to inform decisions regarding new drug adoption by relevant policy makers and stakeholders. This study examined the publication pattern and quality of CEA presented in the annual meetings of the American Society of Hematology (ASH).

Methods:
ASH abstracts from 1997 to 2007 were reviewed. Abstracts with a malignant focus and that reported primary outcomes of incremental cost per life-year-gained or quality-adjusted-life-year (QALY) were included. Data including ICER (adjusted to USD) and author affiliation to the pharmaceutical industry was extracted. Quality indicators associated with well-performed CEAs were derived from the literature (Weinstein MC et al., JAMA 1996;276:1253-1258) and used to determine abstract quality. A search for subsequent publication of the abstract findings was conducted using Medline. The primary outcome of time-to-publication was determined using Kaplan-Meier statistics. Predictor variables were tested using the log-rank statistic.

Results:
29 abstracts met inclusion criteria. Only 13 were published (overall rate of 44.8%). The actuarial 1 and 3 year publication rates were 24.1% and 41.4% respectively (see figure). All but 1 abstract presented at ASH had an ICER less than $100 000/QALY (median $33 000/QALY) with 55.2% disclosing author affiliation to the pharmaceutical industry. Specific to abstracts published after 2001, when ASH instituted a policy of disclosure reporting, the proportion of abstracts reporting pharmaceutical affiliation was 72.7%. No variable or quality indicator predicted for time-to-publication though there was a trend for abstracts reporting dominant ICERs to yield more timely publications (p = 0.075). In terms of quality indicators, the reporting of a societal perspective, lifetime horizon, or a sensitivity analysis was noted in only 37.9%, 24.1% and 62.1% of abstracts, respectively.

Conclusions:
The publication rate of CEA abstracts from ASH was low and not timely for open discussion among stakeholders. Although there was no direct evidence of bias in abstracts selected for presentation, most reported highly favorable ICERs and consistent author affiliation with the pharmaceutical industry. Overall, the quality of abstracts appeared suboptimal.

Disclosures:
No relevant conflicts of interest to declare.
Toll-Like Receptors 2 and 4 Mediate the Capacity of Human Bone Marrow-Derived Mesenchymal Stem Cells to Support the Proliferation and Differentiation of CD34+ Cells In a Non-Synergistic Way.
Authors: Wang Xingbing , Cheng Qiansong , Wang Jian , Xia Liang , Zhang Xuhan , Ding Kaiyang , Sun Zimin 
Published: October 2019

Abstract
Abstract 3850
Human bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent nonhematopoietic progenitor cells, which can differentiate into osteoblasts, adipocytes, chondrocytes and other tissues. The most important function of BM-MSCs is to support hematopoiesis. Toll-like receptors (TLRs) are a conserved family of receptors that can be activated by both pathogen components and mammalian endogenous molecules such as heat-shock proteins and extracellular matrix breakdown products. In the past a few year, several studies reported that TLRs are expressed in hematopoietic and non-hematopoietic to modulate their biological functions. We hypothesized that MSCs are equipped with TLRs that enable them to dynamically change hematopoiesis-related cytokines expression pattern and level by sensing correspondent agonists, thus efficiently supporting hematopoiesis.
In this study, BM-MSCs were analyzed for mRNA expression of TLR 1–9 by reverse transcription-polymerase chain reaction. TLR 1–6, but not TLR 7–9 were expressed by MSCs. The expression of TLR2 and TLR4 was also confirmed by flow cytometic assay. We further explored the role of TLR2 and TLR4 in mediating the capacity of MSC to support the proliferation and differentiation of CD34+ cells. The pre-stimulation with TLR2 agonists (Pam3Cys) or TLR4 agonists (LPS) enable MSCs to enhance CD34+ cells proliferation and promote CD34+ cells differentiation towards the myeloid lineage (CD33+, CD11b+), as well as granulocyte colony formation by those cells. The production of interleukin 8 (IL-8), IL-11, stem cell factor (SCF), granulocyte colony-stimulating factor (CSF), macrophage CSF and granulocyte-macrophage CSF were also increased by stimulated MSCs. Interestingly, although Pam3Cys and LPS displayed different inductive magnitudes, they have no synergistic effect on MSCs. We hypothesized there may be some antagonistic effect between TLR2 and TLR4 intracellular signal conductive pathway, or they can downregulate the expressive level of the TLRs on MSCs.
Together, our findings suggest that TLR2 and TLR4 signalings may indirectly regulate hematopoiesis by modulating MSCs' functions. The increased haemopoietic proliferation and myeloid lineage differentiation could be mediated, at least in part, by augmented hematopoiesis-related cytokine production.

Disclosures:
Wang: National Natural Science Foundation (30700329): Research Funding; Anhui Provincial Outstanding Young Investigator Program (08040106810): Research Funding; Fund of Anhui Provincial “115” Industrial Innovation Program: Research Funding.
MT1-MMP Plays a Critical Role In the Modulation of Hematopoiesis.
Authors: Nishida Chiemi , Heissig Beate , Tashiro Yoshihiko , Seiki Motoharu , Nakauchi Hiromitsu , Hattori Koichi 
Published: October 2019

Abstract
Abstract 3851
Specific niches, in which hematopoietic stem cells (HSCs) reside control the balance between HSC quiescence and self-renewal, yet little is known about the extrinsic signals provided by the niche and how these niche signals regulate such a balance. In the recent study, activation of the fibrinolytic pathway via matrix metalloproteinases (MMPs) including MMP-9 resulted in the release of kit ligand (KitL) in the BM niche. Membrane type 1-MMP (MT1-MMP) can activate MMP-9 in the process of mutual activation of MMP. It has already known that BM myeloablation with irradiation or anti-cancer drug induces MT1-MMP expression, but the role of MT1-MMP in hematopoiesis is not well-understood. We examined MT1-MMP deficient mice (MT1-MMP-/-) 12 days after birth. MT1-MMP-/- were suffering from pancytopenia, and are reduced numbers of bone marrow mononuclear cells (BMMCs), splenocytes and number of hematopoietic progenitor cells in the BM although the number of HSCs in BM showed no significant difference. BM cytospins from MT1-MMP-/- mice showed mild erythropoietic disturbance and severer impairment of myelopoiesis. Interestingly, a powerful hematopoietic factor, Kit-ligand (KitL) levels were significantly lower in MT1-MMP-/- mice than wild type. MT1-MMP knockdown by shRNA or/and siRNA impaired KitL expression and secretion in transfected stroma cells compared to Mock controls, demonstrating that reduced KitL plasma levels were due to impaired release/production and not due to reduced numbers of stromal cells in MT1-MMP-/-. Similarly, impaired proliferation and differentiation of MT1-MMP-/- BMMCs in vitro could be restored by exogenous sKitL. Others and we reported that BM ablation induces the production of stromal cell-derived factor-1 (SDF-1; CXCL12), which plays a key role in stem cell homing and B-cell lymphopoiesis. Reduced SDF-1 expression was observed in BMMCs of MT1-MMP-/- mice and genetic knockdown of MT1-MMP resulted in lower SDF-1 expression both on a transcriptional and protein level. BMMCs of MT1-MMP-/- showed a decrease in the percentage of mature B cells compare to controls. Knocking down of MT1-MMP in stromal cell reduced the number of adherent hematopoietic cells, but addition of rec. SDF-1 could reverse the phenotype. These results suggested stromal-derived MT1-MMP was functionally important to maintain HSC function in long-term cultures of WT HSCs. Thus, MT1-MMP is a critical modulator of hematopoiesis, as it alters the growth factor output of niche cells.

Disclosures:
No relevant conflicts of interest to declare.
Role of Polycomb Repressive Complex 1 (PRC1) In Runx1 Mediated Gene Regulation
Authors: Yu Ming , Mazor Tali , Huang Hui , Huang Emily , Kathrein Katie , Woo Andrew , Zon Leonard I. , Fraenkel Ernest , Cantor Alan B 
Published: October 2019

Abstract
Abstract 3863
The transcription factor Runx1 is required for the generation of all definitive hematopoietic stem cells (HSCs), and for normal megakaryocyte, lymphocyte and granulocyte terminal maturation. Runx1 and its cofactor CBF-β are also the most common targets of chromosomal translocations in human leukemias. Somatic and germline point mutations in Runx1 occur in myelodysplastic syndrome and undifferentiated leukemias, and are associated with a poor prognosis. Despite the key roles that Runx1 plays in normal and malignant hematopoiesis, its transcriptional mechanisms remain incompletely understood. In this study, we purified Runx1 containing multiprotein complexes from megakaryocytic cells and identified several associated chromatin-remodeling complexes, including Polycomb Repressive Complex 1 (PRC1), NuRD, SWI/SNF and MLL/TrxG. Interactions were validated by independent biochemical assays and demonstrate a direct interaction between Runx1 and the PRC1 component Bmi1. ChIP-seq studies identified a large overlap between Runx1/CBF-β and Ring1b (another PRC1 core component) occupied sites, with 45% of the peaks at these genes < 200 bp from each other. ShRNA mediated gene knockdown of CBF-β shows differential gene expression of many of the co-occupied genes. Among the direct CBF-β/Ring1b co-occupied targets are other key hematopoietic transcription factors including FOG-1, SCL and Lyl1, and a number of cell adhesion related genes. ShRNA knockdown of Ring1b impairs megakaryocyte endomitosis, partially phenocopying Runx1 deficient megakaryocytes. Morpholino mediated knockdown of Ring1b or Bmi1 in zebrafish embryos reduces the number of phenotypic definitive HSCs, also partially phenocopying Runx1 morphants. We also show that Runx1/CBF-β interact with Ring1b in the human T cell line Jurkat, and that Ring1b occupies Runx1/CBF-β bound sites of key direct target genes in primary murine thymocytes, including CD4, TCRβ, and Th-POK. Surprisingly, we did not find enrichment for histone 2A monoubiquitination at most of the megakaryocytic and T-lymphocyte co-occupied sites examined, suggesting that PRC1 acts through alternate mechanisms at these genes. Collectively, these data provide evidence for a broad role of PRC1 in Runx1 mediated gene regulation.

Disclosures:
Zon: FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Cantor:Amgen, Inc: Consultancy.
Temporal and Sequential Expression of EBF1 and PAX5 Restricts the Non-B Cell Fate In Early Lymphopoiesis
Authors: Zandi Sasan , Zetterblad Jenny , Tsapogas Panagiotis , Månsson Robert , Sigvardsson Mikael 
Published: October 2019

Abstract
Abstract 3867
The generation of B cells from multipotent hematopoietic stem cells involves activation of B cell specific and repression of alternative lineage programs. This process to a large extent is under the control of transcription factors such as EBF1, E2A, IKAROS and PAX5. Inactivation of PAX5 and EBF1, by point mutations or deletions can be found in 30% of the acute lymphocytic leukemia (ALL) cases, probably due to a disturbance of differentiation in early B cell development. Conditional deletion of PAX5 and EBF1 leads to a complete block in B cell production and subsequent accumulation of early immature B cell progenitors. Although the roles of these transcription factors have been established in B cell development, the presence of a feedback loop between PAX5 and EBF1 as well as lack of well defined early B cell developmental events and cell populations, lead to some discrepancies in the current literature regarding the role of these factors in restricting non-B cell lineages and the establishment of B cell fate. By using a combination of RAG1/EBF1 reporter mice and newly identified surface markers i.e Ly6D, we have shown the exact point of myeloid (M), natural killer (NK) and T cells restriction within conventional common lymphoid progenitors (CLP) population. The Lin-IL7r+flt3+kitloSca1lo(CLP)Ly6D-λ5- population retains the residue of myeloid potential together with dendritic (D), Nk, T and B potentials. Upon expression of Ly6D, these cells lose the residual myeloid, NK and D potentials, and eventually the expression of λ5, associated with loss of T cell potential, marks the B-cell committed cells. Based on this new model of lymphoid development we have revisited the functional roles of EBF1 and PAX5 in B-cell commitment. This work suggests that EBF1 restricts the myeloid, NK and D potentials in the transition from CLPLy6D- to CLPLy6D+ stage while the expression of PAX5 in the transition from CLPLy6D+λ5- to CLPLy6D+λ5+ restricts the T cell potential by possibly by counteracting Notch1 signaling in the bone marrow.
Considering the high prevalence of PAX5 and EBF1 mutations in ALL and diffuse large B cell lymphoma (DLBL) cases and the fact that inactivation of PAX5 and EBF1 leads to accumulation of B cell progenitors in mice models, understanding the temporal and spatial expression of EBF1 and PAX5 in regard to restriction of non-B cell fates in early stages of lymphoid development will allow us to identify the leukemia-initiating cells and the mechanisms of leukemogenesis in these diseases.

Disclosures:
No relevant conflicts of interest to declare.