bluuebunny/crossref_metadata_2025_split huggingface dataset:
Plerixafor Abrogates the Supportive Function of MSC for Self-Renewal of Human Hematopoietic Stem Cells,
Authors: Ludwig Annette , Saffrich Rainer , Eckstein Volker , Lenze Angela , Diehlmann Anke , Ho Anthony D. , Wuchter Patrick 
Published: October 2019

Abstract
Abstract 3408
The CXCR4-SDF1α axis plays an important role in maintaining the stemness of human hematopoietic stem cells (HSC). In the present study we established a surrogate model for the bone marrow niche by culturing HSC on a feeder-layer of human mesenchymal stromal cells (MSC) and investigated the proliferation and differentiation behaviour upon perturbation by Plerixafor.
HSC (CD34+ cells) were isolated from umbilical cord blood by fluorescent activated cell sorting (FACS). MSC were derived from bone marrow aspirates from healthy voluntary donors. HSC were stained with carboxyfluorescein succinimidyl ester (CFSE) and cultured on MSC feeder-layer for 6 days. For evaluating the influence of the culture medium on MSC cultures, three different media conditions (M1-M3) were used. Medium 1 (M1) contained 2% fetal calf serum (FCS), medium 2 (M2) contained 10% FCS and medium 3 (M3) contained GMP-grade human platelet lysate (hPL). Proliferation of HSC was calculated by analyzing the distribution of the CFSE dye (measured at day 1 and day 6). Plerixafor was added in concentrations of 0.1, 1.0 and 10.0 μM. On day 6, HSCs were harvested and analyzed by flow cytometry for CD34, CD38 and CXCR4 expressions in relationship to the cell division rate.
When co-cultured with MSC, the division kinetics of HSC was increased, while the proportion of CD34+ cells remained significantly higher compared to HSC without MSC. Accordingly, more CD38− HSC were found after 6 days upon co-culture with MSC. All three MSC preparations supported self-renewing proliferation of HSC, whereas MSC M1 induced the strongest effect. This underlines that co-culture with MSC has a significant supportive function for hematopoiesis. The additional exposure to Plerixafor in the co-culture system partially reversed this effect in a dose-dependent manner: division rate of HSC and the proportion of CD34+ and CD38− cells were reduced with higher concentrations of Plerixafor. The reduction of self-renewing proliferation by Plerixafor was not observed in controls consisting of HSC without MSC. Plerixafor also rendered the CXCR4 receptors undetectable on the surface of CD34+ cells for up to 6 days, most probably due to a persisting blockade of the antibody-binding site.
Human HSC co-cultured with MSC showed an increased cell division rate and produced a higher proportion of CD34+/CD38− cells. Different MSC culture media were systematically analysed in this setting and subtle differences in the supportive function could be observed. The addition of Plerixafor neutralized the effects of MSC, leading to an earlier loss of “stemness” and to lineage-commitment of HSC, thus providing evidence for the role of the CXCR4/SDF1α axis in terms of supportive function of MSC for self-renewal of HSC.

Disclosures:
Ho: Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.
Dysregulated Bone Wound Repair and Marrow Functions in Senescence Accelerated Mice (SAMP6),
Authors: Epperly Michael W. , O'Sullivan Regina P. , Cao Shaonan , Dixon Tracy M. , Goff Julie P. , Glowacki Julie , Greenberger Joel S. 
Published: October 2019

Abstract
Abstract 3415

Introduction:
The Senescence Accelerated-Prone mouse (SAMP6) shows normal growth followed by rapid aging, development of osteopenia and shortened lifespan, compared with control R1 mice. The bone defect has been attributed to reduced osteoblast potential of marrow stromal cells. We compared in vivo repair of tibial bone wounds. We compared in vitro hematopoiesis, irradiation sensitivity, stromal cell biology, and osteoblastogenesis with SamP6 and R1 marrow. We tested the hypothesis that SAMP6 mice have poor healing of osseous wounds and that in vitro properties of SAMP6 marrow are abnormal.

Methods:
As a model for spontaneous bone repair, a 2-mm unicortical wound was made in each proximal tibia. At intervals, wound diameter was measured on radiographs. Long term bone marrow cultures (LTBMCs) were established from other SAMP6 and R1 mice to measure hematopoiesis and to establish marrow stromal cell lines (MSCLs) for analysis. MSCL gene expression and sensitivity to irradiation were measured; osteoblastogenic potential was determined by culture in osteoblastogenic medium (1% FBS, 10 nM dexamathasone, 5 mM b-glycerophosphate, 50 mg/mL ascorbate-2-phosphate) and gene expression analysis for osteoblast markers alkaline phosphatase (ALP), PTH-receptor-1 (PTHR1), and Osteocalcin (OC).

Results:
There was a faster rate of closure of the tibial wounds in R1 than in SAMP6 mice; at day 21, wounds in R1 mice were 47% the size of those in SAMP6 mice (p = 0.032). For the first 24 weeks, SAMP6 LTBMCs had significantly greater hematopoiesis than R1, shown by more cobblestone islands (sites of stem cell activity) and by more multilineage colonies from nonadherent cells transferred to semisolid medium. After 24 weeks, however, hematopoiesis ceased in SAMP6 cultures but continued in the SAMR1 cultures for 40 weeks. There was constitutive upregulation of TGF-b, an inhibitor of hematopoiesis, in SAMP6 MSCLs, compared with R1 cells. SAMP6 MSCLs were more sensitive to radiation (Do of 1.5 ± 0.1 Gy), compared with R1 (Do of 4.0 ± 0.4 Gy; p = 0.0080). There was unexpected constitutive expression of ALP, PTHR1, and OC in SAMP6 MSCLs, compared with R1. In osteoblastogenic medium, there was greater expression of osteoblast marker genes in the SAMP6 line compared to R1.

Conclusion:
SAMP6 showed delayed bone wound repair. Marrow from SAMP6 mice showed shortened in vitro hematopoiesis and greater radiation sensitivity, indicative of greater oxidative stress, but the unexpected finding of constitutive expression of osteoblast genes suggest that in vivo factors are more important than innate cellular defects in marrow to account for impaired bone healing.

Acknowledgments:
This project was supported by NIAID U191A168021-06 and NIA R21AG034254.

Disclosures:
No relevant conflicts of interest to declare.
A Phase II Study of Risk-Adapted Intravenous Melphalan in Patients with AL Amyloidosis,
Authors: Mollee Peter , Tiley Campbell , Cunningham Ilona , Moore John , Prince Miles , Cannell Paul , Gibbons Steve , Tate Jill , Paul Sanjoy , Gill Devinder S 
Published: October 2019

Abstract
Abstract 3973

Aims:
High-dose IV melphalan is standard autograft (ASCT) conditioning in patients with AL amyloidosis, but careful patient selection is required to avoid high transplant-related mortality rates. In patients ineligible for ASCT, intermediate-dose IV melphalan (IDM) may provide advantages over oral dosing where the already variable intestinal absorption is complicated by gastrointestinal amyloid deposition. We aimed to assess a risk-adapted strategy to IV melphalan dosing in patients with AL amyloidosis.

Methods:
The MM8 study was a prospective clinical trial conducted by the Australasian Leukaemia and Lymphoma Group. Transplant candidates (minimal cardiac disease including BNP<300ng/L, age <65, ECOG<2, <2 organs involved) received melphalan at 140 or 200mg/m2 IV. All other patients received IDM (melphalan 20mg/m2 IV d1 and dexamethasone 40mg PO d1-4 every 4 weeks for 3–6 cycles). Treatment was continued until 2 cycles beyond free light chain normalisation or maximal response, or to a maximum of 6 cycles. IDM was discontinued if there was no haematologic response after 3 cycles.

Results:
21 patients enrolled with median age 61yrs. Organ involvement was cardiac 48%, renal 81%, liver 14%, neurologic 43%. 6, 10 and 5 were low, intermediate and high cardiac biomarker risk, respectively. 7 underwent ASCT and 14 IDM.
The trial closed early due to excessive myelotoxicity in the IDM arm. In this cohort, grade 3/4 neutropenia and thrombocytopenia during the 1st cycle occurred in 54% and 23%, with 15% having neutropenic fever. Grade 3/4 neutropenia and thrombocytopenia during any cycle occurred in 85% and 54% of patients, with 38% having neutropenic fever. First cycle severe neutropenia was not predicted by age, cardiac or renal function but was significantly more common with lower body surface area (p=0.01). 8 IDM patients died before the 6 month response assessment, 2 achieved a 50% reduction in baseline involved FLC and 4 failed to respond. All 7 patients receiving ASCT are alive at a median of 33 months.

Conclusions:
IV melphalan at 20mg/m2 is excessively myelotoxic for patients with AL with no evidence of response benefit to offset this toxicity. BNP<300ng/L may identify patients suitable for ASCT.
This study was supported by the Leukaemia Foundation of Queensland and Amgen Australia.

Disclosures:
Mollee: Amgen Australia: Research Funding; Leukaemia Foundation of Queensland: Research Funding. Cannell:Nereus Pharmaceuticals:.
Clofarabine-Based Chemotherapy for Relapsed/Refractory Adult Acute Lymphoblastic Leukemia and Lymphoblastic Lymphoma. the Spanish Experience
Authors: Barba Pere , Sampol Antonia , Calbacho María , Gonzalez Jose , Serrano Josefina , Martinez-Sanchez Pilar , Fernandez Pascual , Garcia-Boyero Raimundo , Martino Rodrigo , Delgado Pilar , Hernandez-rivas Jose Angel , Del Potro Eloy , Gonzalez-Muñiz Soledad , Gomez-Sanchez MAria Isabel , de la Fuente Adolfo , Bueno Javier , Ribera Josep-Maria 
Published: October 2019

Abstract
Abstract 4246

Background:
Patients with relapsed/refractory acute lymphoblastic leukemia (ALL) have a very poor outcome with limited therapeutic options available. Clofarabine (Clo) is a second generation purine nucleoside analogue increasingly used in these patients, mainly in the pediatric setting.

Aims:
To evaluate the toxicity profile and outcome of Clo-based chemotherapy regimens used in adult ALL in the Spanish PETHEMA centers.

Materials and Methods:
Sixteen of the 22 PETHEMA centers to have used Clo in adult patients (≥16 years) with a definitive diagnosis of ALL (n=25) or lymphoblastic lymphoma (LL) (n=5) participated in the study. Patient-, disease-, toxicity- and outcome-related data were retrospectively collected and analyzed.

Results:
Thirty patients received Clo-based regimens from January 2007 to May 2011. Median age at diagnosis was 33 years (range 16–73). B- and T- lineage diseases were found in 20 (67%) and 10 (33%) patients, respectively. Median number of treatment regimens before Clo was 3 (range 1–7) and median time of follow-up for survivors was 5 months (range 2–25). Clo-based combinations included Clo-cyclofosfamide (Cy) (n=11, 36%), Clo-Cy-etoposide (n=8, 27%) Clo-cytarabine (n=6, 20%) and Clo monotherapy (n=5, 17%) for a median of 1 (range 1–3) cycle. Clo total dose/cycle in most patients was 200mg/m2. Hematological and infectious grade III-IV toxicities were found in 30 (100%) and 20 (67%) patients, while hepatic, gastrointestinal and renal grade III-IV toxicities were less common (17%, 17% and 0%, respectively). Eight (27%) and 2 (7%) patients achieved CR (complete remission) and CRi, respectively (overall CR rate 33%), while 7 patients died before disease evaluation. CRs were observed in B- (n=8, 40%) and T- (n=2, 20%) lineage diseases and in patients with normal and adverse cytogenetics (including the only Ph+ case). Younger patients (median age 25 years vs 36 years, p=0.1) and those who received a previous allogeneic hematopoietic cell transplantation (allo-HCT) (67% vs 29%, p=0.1) showed a non-significant trend for a higher overall CR achievement. Six (20%) patients (5 in CR) underwent allo-HCT after Clo-based treatment. Five (17%) patients were alive at 5 months of Clo treatment for an overall survival probability of 10% (95%CI 4–16). The most frequent death causes were disease progression (n= 16, 64%) and infections (n= 3, 12%).

Conclusion:
Despite their toxicity, Clo-based chemotherapy regimens may allow CR achievement in heavily pretreated relapsed/refractory ALL and LL patients.

Disclosures:
Barba: Genzyme: Consultancy.
Leukemia Cell-Derived Exosomes Targeted Dendritic Cells Can Induce More Strong Anti-Leukemia Response Than Exosomes Alone
Authors: Sheng Chang , Sun Jing , Wang Chun , Hao Siguo 
Published: October 2019

Abstract
Abstract 4278
Exosomes are the newest family number of ‘bioactive vesicles’ that play important roles in antigen presentation. Recent clinical trials about exosomes have shown minor clinical benefit. However, exosomes derived from leukemia cell lines were less studied, In this study, we investigated the biological properties and anti-leukemia immunity of leukemia cell-derived exosomes (LEX). The results showed that like other tumor cells, similarly, leukemia cells can release exosomes too. Transmission electron microscopy showed that exosomes derived from NB4 cell, a promyloblastic leukemia cell line and K562 cell, a chronic myeloid leukemia cell line were membrane vesicles with diameter about 50–100μ m, and loading their specific moleculars such as RAR-α and ABL and adherent moleculars such as ICAM-1. Similarly, our data also demonstrated the presence of Hsp70 in these two leukemia cell-derived exosomes. The data from two-dimensional electrophoresis of K562 cell-derived exosomes showed that most of molecules expressing on leukemia cells were detected on their exosomes, indicating that LEX load most of proteins expressed on their original leukemia cells. Out data also demonstrated that LEX can induce anti-leukemia CTL response and LEX-targeted DC can induce more efficient anti-leukemia immunity compared with LEX alone. In conclusion, exosome-based therapies could be considered as a promising strategy to prolong disease-free survival in patients with leukemia after consolidation or hematopoietic stem cell transplantation.

Disclosures:
No relevant conflicts of interest to declare.
Anti-Leukemic Effect of Tetra-Arsenic Oxide in Acute Promyelocytic Leukemia Cells in Vitro and In Vivo
Authors: Chong So Young , Jang Moon Ju , Lee Sun Ju , Huh Ji Young , Bae Jeehyeon , Chung Kwang-Hoe , Oh Doyeon , Kwaan Hau C. 
Published: October 2019

Abstract
Abstract 4306
Arsenic trioxide (As2O3; ATO) has been established to be an effective agent for treating acute promyelocytic leukemia (APL). Tetra-arsenic oxide (As4O6; TAO) is a new arsenic compound which has shown anti-proliferative and apoptosis-inducing effects against human leukemic and solid tumor cells, but TAO has never been studied in APL so far. We investigated the effect of TAO in APL cell lines (NB-4), and evaluated their anti-leukemic effect in murine xenograft model. In both the XTT assay and in FACS analysis, TAO inhibited cell proliferation and induced apoptosis in NB4 cell lines as effective as ATO in dose dependent manner. In FACS analysis, TAO induced apoptosis in NB4 cell lines as effective as ATO in a dose dependent manner. However, expression of differentiation (CD11b) was unchanged. For in vivo study, we established a xenograft model in nude mice (BALB/cBy J-nu) using the NB4 cell line. 1 × 107 NB4 cells were inoculated into the flank subcutaneously. When the tumor size reached 1.0 cm3, intra-peritoneal ATO or TAO treatment (5mg/kg or 10mg/kg) was initiated and administration was performed daily. We found that TAO (10mg/kg) significantly inhibited tumor growth of the inoculated NB4 cell as effective as ATO (10mg/kg). These results suggest that TAO may have a potential for the treatment of APL.

Disclosures:
No relevant conflicts of interest to declare.
Variants At 6q21 Associated with Radiation-Induced Second Malignancies After Hodgkin Lymphoma Differentially Regulate PRDM1 Expression Following Radiation Exposure
Authors: Onel Kenan , Best Timothy , Yu Shan 
Published: October 2019

Abstract
Abstract 431
Childhood Hodgkin lymphoma (HL) survivors are particularly susceptible to second malignant neoplasms (SMNs) caused by radiation therapy (RT). To identify genetic risk factors for SMNs, we undertook a genome-wide association study of 96 SMN cases and 86 SMN-free controls. Both cases and control sets consisted of long-term survivors of childhood HL who received antecedent RT. Two variants at chromosome 6q21 intergenic between PRDM1 and ATG5 were associated with SMNs (rs4946728: P = 1.26×10−9, OR = 3.17 [2.16–4.65] and rs1040411: P = 4.68×10−8, OR= 2.41 [1.76–3.32]). These results were replicated in an independent set of 63 SMN cases and 75 SMN free controls (rs4946728: P =. 003, and rs1040411: P =. 04). The risk variants form a haplotype associated with decreased basal expression of PRDM1, as well as impaired induction of the PRDM1 protein by radiation exposure. An analysis of the genomic region spanned by the SMN-associated haplotype revealed four evolutionarily conserved putative enhancers, characterized by both H3K27 acetylation and DNase I hypersensitivity. To determine whether these putative enhancers modulate expression following radiation exposure, we cloned each into the pGL3 vector downstream of a luciferase reporter gene. We then transiently transfected each construct into lymphoblastoid cell lines. Data will be presented showing how carriage of either the risk or protective haplotype alters the activity of these enhancers in response to radiation. Furthermore, ChIP-Seq data indicate that NFkB variably binds each putative enhancer. As NFkB has previously been shown to induce PRDM1 expression and is itself activated by radiation, we treated LCLs with an NFkB inhibitor to determine whether NFkB is involved in induction of PRDM1 following radiation exposure. These data demonstrate that incorporating etiologic exposures, such as radiation, can enhance the discovery disease-associated genetic variation and guide functional follow-up experiments.

Disclosures:
No relevant conflicts of interest to declare.
Role of Anticoagulation in Patients with Inferior Vena Cava Filter but with No Risk Factor or History of DVT/PE
Authors: Khalife Mustapha A. , Dabak Vrushali S. , Hammoud Marwa , Arnaout Karim 
Published: October 2019

Abstract
Abstract 4317

Introduction:
Inferior Vena Cava (IVC) filters have been available for almost 40 years but their clinical utility and safety have not been completely evaluated in patients with no previous history of deep vein thrombosis (DVT) or pulmonary embolism (PE). The role of anticoagulation in patients with IVC filter with no history of DVT/PE is questionable. In this study, we try to determine if there is a role or benefit from anticoagulation in patients with an IVC filter placed but without any other risk factor for deep vein thrombosis (DVT) or pulmonary embolism (PE).

Methods:
we retrospectively reviewed the charts of 562 patients who had an IVC filter placed between 2003 and 2005. 442 patients were excluded because they had a history of DVT/PE, or because of a hypercoagulable state (genetic predisposition, prolonged hospitalization/immobilization, surgery, or malignancy). Of the 120 remaining patients included in this study, 6 had their IVC filter removed. And therefore we only analyzed the charts of 114 patients who had a permanent IVC filter placed for prophylactic reasons. Group 1 consisted of 17 patients who received different forms of anticoagulation (subcutaneous heparin, low molecular weight heparin or coumadin). Group 2 consisted of the remaining 97 patients who did not receive any form of anticoagulation.

Results:
2 out of 17 patients in group 1 had a DVT and 14 out of 97 patients in group 2 had a DVT. The incidence of DVT was 11.8% in group 1 versus 14.4% in group 2 (p-value 0.770). The median onset of DVT/PE after IVC filter placement was 31 days. The median time of follow up was 77.33 months.

Conclusion:
Patients who had a permanent prophylactic IVC filter placed but with no history or risk factors for DVT/PE appear to be at an elevated risk for new DVT/PEs. In these patients, the role of anticoagulation is questionable. With a median 6 year follow up, anticoagulation seemed to non significantly lower the risk of DVT/PE. Larger randomized prospective trials are needed to examine the efficacy and duration of anticoagulation in patients with a prophylactic IVC filter placed.

Disclosures:
No relevant conflicts of interest to declare.
Generation of Red Blood Cells From Human Embryonic Stem Cells (ES) with Increased Efficacy
Authors: Srivastava Anand S , Azad Babak Esmaeli , De Necochea Campion Rosalia , Carrier Ewa 
Published: October 2019

Abstract
Abstract 4334
It is estimated that for every unit of donated blood, two units are required in North America. The current rate of blood donation is stagnant while the need increases by 6–8% annually. In order to overcome this difficulty, we have developed an improved method to generate red blood cells from human embryonic stem cells (H9) with increased efficacy. In addition to xeno-free conditions and standard cytokine cocktail used for hematopoietic differentiation of human embryonic stem cells (Carrier et al. J Transl Med. 2009; Vol 7: 27), we have introduced a new method of improved growth and differentiation of human ES cells with hypoxia-induced mesenchymal stem cells, obtained from allogeneic adult bone marrow donors. This technique increased efficacy of red blood cell production by 5–25 fold.
We have developed a bioscaffold–> microsphere-based culture system with highly porous surface allowing culturing of a very large number of embryonic stem cells per one culture condition. This culture system avoids shear forces and damage to the cells, and facilitates removal and recycling of the microspheres. The in vitro obtained human ES-derived red blood cells are enucleated and do not produce tumors (efficacy of enucleation is 65–95%). The laser-based system is utilized to eliminate nucleated cells from the culture.
The problem with hES-derived red blood cells is that they are produced in small numbers and process is very costly. We are developing a 3-phase bioreactor with computerized programming, which will increase every step of the differentiation process and allow recycling of feeder cells and cytokines. In this system we will utilize iron-loaded microspheres coated with hypoxia-processed mesenchymal stem cells as a main culture unit. The in vitro generated human ES-derived red blood cells upscaled in a bioreactor will be used for the off-shelf production of red blood cells for clinical use.

Disclosures:
Srivastava: Giostar: Employment, Equity Ownership. Azad:Dnamicroarray, Inc.: Employment, Equity Ownership. Carrier:Giostar: Consultancy; Samaritan Pharmaceuticals: Consultancy; Entest Biomedical: Consultancy, Equity Ownership; America Stem Cells: Consultancy, Equity Ownership; Millenium: Speakers Bureau; NovaRx: Employment, Equity Ownership.