bluuebunny/crossref_metadata_2025 huggingface dataset:
Unrelated Stem Cell Transplantation After Reduced Intensity Conditioning for Patients with Multiple Myeloma Relapsing After Autologous Transplantation A Prospective Multicenter Phase II Study of the Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT).
Authors: Kröger Nicolaus , Shimoni Avichai , Schilling Georgia , Schwerdtfeger Rainer , Bornhäuser Martin , Nagler Arnon , Zander Axel R. , Heinzelmann Marion , Brand Ronald , Gahrton Goesta , Morris Currly , Niederwieser Dietger , de Witte T. M.
Published: October 2019

Abstract 2308

Poster Board II-285

Introduction:

Dose-reduced conditioning followed by allogeneic stem cell transplantation has become a treatment option for patients with multiple myeloma. However, the experience using unrelated donor is limited.

Patients and Methods:

From 2002 to 2007, 49 myeloma patients with relapse to a prior autologous SCT were included in a prospective multicenter trial to determine the efficacy of a reduced melphalan (140 mg/m2)/fludarabine regimen followed by allogeneic SCT from unrelated donors. GvHD prophylaxis consisted of anti-lymphocyte globulin (ATG-Fresenius®), cyclopsorin A and short course of MTX.

Results:

No primary or secondary graft failure was observed and all patients showed leukocyte and platelet engraftment after a median of 15 and 19 days, respectively. Acute graft-versus-host disease (GvHD) grade II to IV occurred in 25% and chronic GvHD in 35% of the patients. Limited GvHD was seen in 29 % and extensive GvHD was seen in 6 % of the patients. Overall response rate at day 100 was 95% including 46% complete remission (CR). Cumulative incidence (CI) of non-relapse mortality at one year was 25% (95% CI: 13-37%) and significantly lower for HLA matched compared to mismatched SCT (10% vs. 53%, p=0.001). During follow-up 22 patients experienced relapse (54 %) resulting in a cumulative incidence of relapse at 1, and 3 years of 27% (95% CI: 14-40%) and 55% (95% CI: 40-70%), respectively. The median time to relapse was 318 days (r: 56 – 861). After a median follow up of 43 months, the estimated 5-year progression-free (PFS) and overall survival (OS) rates were 20% and 26%, respectively and were significantly better for matched in CR at day 100 (41 vs. 7%, p=0.04 and 56 vs. 16%, p=0.02).

Conclusions:

Allogeneic stem cell transplantation from unrelated donors after a reduced intensity regimen is feasible, but an optimal donor selection is mandatory for a low non-relapse mortality. The high relapse incidence remains a major concern should be improved by including posttransplant strategies to upgrade remission status.

Disclosures:

No relevant conflicts of interest to declare.
Chronic Lymphocytic Leukemia with Stereotyped IGHV4-59/IGKV3-20 B Cell Receptors: Another Manifestation of Hepatitis C Virus-Associated B Cell Lymphoproliferation?.
Authors: Kostareli Efterpi , Janus Agnieszka , Gounari Maria , Belessi Chrysoula , Pospisilova Sarka , Oscier David , Foroni Letizia , Francia di Celle Paola , Jurlander Jesper , Rosenquist Richard , Davi Frederic , Thompson Keith , Ghia Paolo , Stamatopoulos Kostas
Published: October 2019

Abstract 2331

Poster Board II-308

The hepatitis C virus (HCV) has been implicated in the development of B-cell lymphoproliferative disorders, including type II mixed cryoglobulinemia (MC-II) and B-cell lymphoma. MC-II is characterized by the presence of monoclonal IgM autoantibodies with rheumatoid factor (RF) activity. The monoclonal IgMs typically form immune complexes by binding polyclonal IgGs that exhibit anti-HCV reactivity. In a series of 6,196 patients affected by chronic lymphocytic leukemia (CLL), we have identified a subset of 12 cases sharing stereotyped mutated IGHV4-59/IGKV3-20 B cell receptors (BCRs) of the MD isotype (subset #13). Comparison of subset #13 heavy chain sequences to a comprehensive dataset of relevant public-database sequences revealed identical gene usage and remarkable junctional homology with the Ig sequence GenBank/U85234, the heavy chain of a RF detected in a healthy donor, as well as the sequence GenBank/AF303916, the clonotypic heavy chain from a CLL case with a history of HCV-associated MC-II. In addition, the light chain IGKV3-20/IGKJ1 stereotyped rearrangements in subset #13 were closely similar if not identical to the rearrangements expressed by clonally expanded IgM+κ+CD27+ B cells in HCV-associated MC-II. For both heavy and light chains, sequence similarities extended beyond junctional regions to shared, “stereotyped” somatic hypermutations across the entire IGHV and IGKV domain, respectively. We established viable and antibody-secreting heterohybridomas from the leukemic cells of a subset #13 case and confirmed the identity of the produced soluble antibody to the IG expressed by the CLL clone. ELISA tests against various antigens revealed that the soluble stereotyped IGHV4-59/IGKV3-20 antibody exhibited RF activity in vitro, while it was not reactive against HCV antigens. In conclusion, the present study for the first time provides evidence for the potential implication of HCV in the pathogenesis of at least a subset of CLL cases with distinctive stereotyped BCRs. The elucidation of the underlying immune mechanisms may pave the way for tailored anti-viral/anti-leukemic therapy for selected cohorts of patients that can be easily identified by molecular techniques during the diagnostic work-up.

Disclosures:

No relevant conflicts of interest to declare.
High Resolution Array-CGH Provides New Insights Into the Prognosis of Chronic Lymphocytic Leukemia (CLL): Is 8p Loss Worse Than 17p Loss?.
Authors: Rinaldi Andrea , Mian Michael , Rossi Davide , Forconi Francesco , Deambrogi Clara , Spina Valeria , Cencini Emanuele , Sozzi Elisa , Drandi Daniela , Ladetto Marco , Santachiara Rita , Marasca Roberto , Chigrinova Ekaterina , Cavalli Franco M.D. , Gattei Valter , Zucca Emanuele , Gaidano Gianluca , Kwee Ivo , Bertoni Francesco
Published: October 2019

Abstract 2339

Poster Board II-316

BACKGROUND:

CLL, the most common adult-onset leukemia in the Western world, has a heterogeneous clinical course. Many advances have led to a better understanding of its pathogenesis and to improvements in treatment strategies, but striking solutions are still missing. We conducted a study to evaluate the impact of genomic aberrations on the clinical course.

METHODS:

From January 1980 to May 2008, 395 frozen samples of CLL patients, were prospectively collected in four centers. Extracted DNA was analyzed with Affymetrix Human Mapping 6.0 arrays. Normal matched DNA was analyzed for one fourth of the cases. Correlations between minimal common regions (MCR) and clinical parameters were evaluated with the Fisherôs-exact test and their impact on OS with the log-rank test. A p-value after Bonferroni multiple test correction (MTC) (p-adj.) <0.05 was considered as statistically significant. Up to now 266 samples have been analyzed.

RESULTS:

Analysis of the clinical parameters (CPs) and known risk factors (Rai/Binet, age, doubling time, LDH, beta2, IGVH status, p53 mutations, telomere length, CD38, 11q, 17p) was consistent to previous published series. ZAP70 did not affect the clinical course, likely due inter-laboratories variability. After a median follow up of 53 months, 143/239 (60%) of the patients have started therapy and 63/261 (24%) died. 5-yr OS was 82%. Fisher test between the MCRs and CPs revealed an inverse relation between the presence of trisomy 12 by FISH and del13q14.3, an association between del17p and losses of 8p regions and between CD38 and 12q gain. Before MTC, 46 MCRs had a significant impact on OS and 67. After MTC, 3 regions maintained their role: 8p22 loss (38/248, 15%, p-adj.=0.002, median OS: 26 months vs. 48), 17p13.3-11.2 loss (20/248, 8%, p-adj.=0.001; median OS: 10 months vs. 48). In univariate analysis, the log-rank test among pts with 8p-/17p- (8/248, 3%), 8p- (30/248, 12%), 17p- (12/248, 5%), wild type (198/248, 80%) was statistically significant (p<0.001; see figure). Importantly, none of the analyzed clinical and biological parameters was associated with this aberration.

CONCLUSIONS:

Loss of 8p22 designated a CLL subgroup with a worse outcome among all patients and in the subset with 17p loss. Our data suggested that this aberration might constitute an independent prognostic factor to be evaluated in independent studies. Results, including a Cox regression model, will be presented on all 395 cases.

Disclosures:

No relevant conflicts of interest to declare.
Marrow Stromal Cells Induce TCL1 Expression in Chronic Lymphocytic Leukemia B Cells.
Authors: Sivina Mariela , Hartmann Elena , Keating Michael , Wierda William G , Rosenwald Andreas , Burger Jan A.
Published: October 2019

Abstract 2347

Poster Board II-324

The human T cell leukemia/lymphoma 1 (TCL1) oncogene was initially identified as a target of chromosomal translocations and inversions at the 14q32.1 chromosome breakpoint region in T-cell prolymphocytic leukemia (T-PLL). Increased TCL1 expression is seen in follicular lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, and chronic lymphocytic leukemia (CLL). Transgenic mice over-expressing TCL1 under control of the mu immunoglobulin gene enhancer develop a CD5+ B cell lymphoproliferative disorder that mimics human CLL, indicating that TCL1 plays a central and/or causal role in the pathogenesis of CLL. However, chromosome aberrations that constitutively activate TCL1 have not (yet) been identified in the vast majority of CLL patients, and therefore the oncogenic mechanism(s) of TCL1 activation in CLL remain unclear. There is growing evidence that external signals from the microenvironment control and regulate the survival and proliferation of CLL cells. Marrow stromal cells (MSC) are highly effective in protecting CLL cells from spontaneous and drug-induced apoptosis, and are used as a model system to study the marrow microenvironment. In order to explore the molecular cross talk between CLL cells and MSC, we co-cultured CLL cells with different MSC and analyzed gene expression changes induced by co-cultures with MSC, an approach similar to our recent study with nurselike cells (Blood 113:3050-8, 2009). For this, RNA was extracted from 19-purified CLL cells from 10 different patients (baseline expression, day 0). Also, the same patients' samples were co-cultured on stroma cells (KUSA-H1, NK-Tert) for 2 and 7 days. At these time points, RNA again was isolated after CD19-purification. Then, gene expression was determined using HG U133 plus 2.0 oligonucleotide arrays from Affymetrix. Gene expression changes were analyzed in individual patients' samples, comparing baseline samples' gene expression to samples after 2 and 7 of co-culture on MSC. We observed relatively homogeneous gene expression changes in CLL cells after co-culture with MSC. We found that TCL1 was among the top 5 genes that were most highly up-regulated by MSC, based on at least 3-fold up-regulation in at least 6 of the paired samples. We also found an up-regulation of TCL1 at the protein level when assessed by immunoblotting and flow cytometry in CLL samples after co-culture with MSC. These findings indicate that MSC can induce and regulate TCL1 expression in CLL, suggesting that the microenvironment plays an even greater role in the pathogenesis of this disease than previously recognized.

Disclosures:

No relevant conflicts of interest to declare.
Molecular Heterogeneity as a Basis for Rational Therapeutics in Chronic Lymphocytic Leukemia.
Authors: Herreros Beatriz , Wang Shaomeng , Bischoff James Robert , Carro Ruben , Córdoba Raul , Delgado Julio , García-Marco Jose Antonio , Gómez Natalia , García Juan F. , Tomáas José Francisco , Martínez-López Joaquín , Rodríguez Antonia , Villuendas Raquel , Piris Miguel A.
Published: October 2019

Abstract 2348

Poster Board II-325

Chronic lymphocytic leukemia (CLL), the most frequent form of adult leukemia in Western countries, is characterized by a highly variable clinical course. Data from our group have shown that the gene expression profile can be used to classify CLL patients into different risk groups, according to a biology-based predictive model that includes genes from essential survival pathways, such as the B cell receptor (BCR) and NF-κB pathways. We propose that this molecular heterogeneity may be associated with variable sensitivity to drugs affecting these survival pathways. Using the Connectivity Map bioinformatic tool, we identified several drugs that may indirectly regulate BCR signaling. We decided to explore one inhibitor of calmodulin (Calmidazolium) further. Based on previously available information about specific signaling cascades, we also selected a PIM kinase inhibitor (C6) and a small molecule inhibitor (SMI) of the BCL2 family (TW-37), which targets Bcl-2, Bcl-XL, and, unlike most SMIs, Mcl-1. An automated drug dispensation protocol coupled to a cell viability assay was set up to test these compounds in primary CLL samples. EC50s were calculated at 72 hours of treatment for each sample and drug. EC50s were in the low μM range for Calmidazolium (0.9-6.8 μM) and C6 (1.5-6.5 μM) and in the nM range for TW-37 (90-740 nM). Patients were stratified using IgVH, SHM, ZAP70, PIM1, PIM2, MCL1 expression, and BCR signature expression as markers. Pharmacodynamic markers such as p-AKT were chosen. Associations of EC50s with SHM and ZAP70 clinical parameters were examined. Strikingly, unmutated cases (4/13), characterized by a worse prognosis, were associated with greater sensitivity (i.e., lower values of EC50) to TW-37 (p = 0.017). Interestingly, ZAP70-positive cases (4/10) also showed a tendency towards greater sensitivity to the TW-37 treatment (p = 0.079). However, variability in the response to Calmidazolium and C6 was not statistically significant, given the small size of the series (11 and 5 samples, respectively). In summary, we show that samples from patients with a poor prognosis (unmutated IgHV and ZAP70-positive) are more sensitive to the BCL2 family SMI, supporting the hypothesis that molecular heterogeneity in CLL is associated with variable drug response.

Disclosures:

Wang: Ascenta Therapeutics: Consultancy, Patents & Royalties, Research Funding. Bischoff:Eli Lilly & Co: Consultancy.
Nucleophosmin Is Required for Macrophage Function and Maturation.
Authors: Sportoletti Paolo , Clohessy John G , Riccardi Luisa , Khanna-Gupta Arati , Chen Jian , Berliner Nancy , Pandolfi Pier Paolo
Published: October 2019

Abstract 238

Nucleophosmin (NPM1) is a multifunctional phosphoprotein predominantly localized in the nucleolus that is involved in numerous cellular processes. The NPM1 gene is frequently a target of genetic alteration in hematological tumors and recent published reports have highlighted the importance of NPM1 in malignancies that affect predominantly the myeloid lineage. Complete inactivation of Npm1 in the mouse disrupts primitive hematopoiesis and results in embryonic lethality. Npm1 heterozygosity produces features similar to those of MDS patients, and can progress to overt leukemia. Here we generated a conditional mousemutant to inactivate Npm1 in the myeloid compartment crossing mice carrying a floxed conditional allele of Npm1 (Npm1fl/fl) with the MRP8-Cre transgenic mouse model. Specific deletion of the Npm1 allele in Npm1fl/fl;MRP8-Cre+ mice resulted in complete loss of Npm1 at the protein level in 40% of peritoneal macrophages analyzed by immunofluorescence. In vitro analysis demonstrates that Npm1 deletion altered peritoneal macrophage functions and influenced monocytic differentiation in Npm1fl/fl;MRP8-Cre+ mice. Specifically we demonstrate that (i) superoxide production was significantly attenuated over time in Npm1fl/fl;MRP8-Cre+ compared with Npm1fl/fl;MRP8-Cre- macrophages; (ii) The chemotactic response was decreased by up to 46% ± 9.6 in Npm1fl/fl;MRP8-Cre+ macrophages when compared to controls; and (iii) Phagocytic index of Npm1fl/fl;MRP8-Cre+ macrophages dropped to 40% of that for the control Npm1fl/fl;MRP8-Cre- macrophages. Analysis of monocyte transcriptional regulators revealed a significant reduction of Maf-b and Irf-8 mRNA expression in Npm1 deficient macrophages, demonstrating an altered maturation process in the absence of Npm1. Our analysis demonstrates that Npm1 loss in the myeloid compartment affects macrophage function and profoundly alters gene expression patterns required for proper monocyte/macrophage development and maturation.

Disclosures:

No relevant conflicts of interest to declare.
Selective and Titratable Effects On AML Genome Wide Methylation Patterning, Transcription and Clonogenicity Using Very Low Concentrations of 5-Aza-2'deoxycytidine.
Authors: Heuston Elisabeth , Farrar Jason E. , Triche Timothy , Buckley Jonathan , Sorensen Poul , Reynolds C. Patrick , Reaman Gregory , Yegnasubramanian Vasan , Arceci Robert J.
Published: October 2019

Abstract 2385

Poster Board II-362

5-Aza-2'deoxycytidine (5AzadC) has significantly contributed to the treatment of myelodysplatic syndromes (MDS) and acute myeloid leukemia (AML). But while the cytotoxic effects of 5AzadC have been well characterized, its influence on methylation-induced cellular reprogramming remains poorly understood. We have treated several AML cell lines at extremely low concentrations of 5AzadC (0 nM to 1.0 nM) over the course of three days, followed by the determination of genome wide methylation changes, alterations in transcription profiles as well as cell viability, proliferation, apoptosis and changes in clonogenicity. The results demonstrate titratable responses on both genomic methylation and transcriptional patterns as well as a selective effect on clonogenicity compared to cytotoxicity. An alternative chemotherapeutic cytosine analog, cytosine arabinofuranoside (AraC), does not show the same selective depletion of clonogenic cells, suggesting that 5AzadC's effects are likely due to altered epigenetic changes associated with cellular reprogramming rather than a direct cytotoxic effect. We are currently evaluating 5AzadC and AraC effects on this population using immunophenotyping methods as well as xenograft models of tumorigenicity. These findings describe a potential role for very low concentrations of 5AzadC in treating acute myeloid leukemia through a selective affect on genome wide methylation patterns leading to altered transcription that differentially effects the clonogenic, leukemic stem cell compartment.

Disclosures:

No relevant conflicts of interest to declare.
AML Survival and In Vivo Progression Is Dependent On β-Catenin Signaling.
Authors: Siapati Elena K , Papadaki Magda , Kozaou Zoi , Rouka Erasmia , Michali Evridiki , Gogos Dimitrios , Savvidou Ioanna , Kyriakou Despoina S , Anagnostopoulos Nikolaos.I. , Vassilopoulos George
Published: October 2019

Abstract 2398

Poster Board II-375

B-catenin is the central effector molecule of the canonical wnt signaling pathway which governs cell fate and differentiation during embryogenesis as well as self-renewal of hematopoietic stem cells. Deregulation of the pathway has been observed in various malignancies including myeloid leukemias where over-expression of β-catenin is an independent adverse prognostic factor. In the present study we examined the functional outcome of stable β-catenin down-regulation through lentivirus-mediated expression of short hairpin RNA (shRNA). Reduction of the β-catenin levels in AML cell lines and patient samples diminished their in vitro proliferation ability without significantly affecting cell viability. In order to study the role of β-catenin in vivo, we transplanted leukemic cell lines with control or reduced levels of β-catenin in NOD/SCID animals and analyzed the engraftment levels in the bone marrow. We observed that while the immediate homing of the cells was not affected by the β-catenin levels, the bone marrow engraftment was directly dependent on its levels. Subsequent examination of bone marrow sections revealed that the reduced engraftment was partly due to the inability of the cells with lower β-catenin levels to dock to the endosteal niches, a finding that was confirmed in competitive repopulation assays with untransduced cells. When we examined the expression levels of adhesion molecules and integrins in engrafted cells in vivo, we observed a significant down-regulation of CD44 expression, a molecule that participates in the interaction of HSCs with the niche. Gene expression analysis of the components of the wnt signaling pathway showed that the pathway is subject to tight transcriptional regulation with minor expression deviations. We did, however, observe an up-regulation in components that participate in the non-canonical wnt signaling pathways such as the WNT5B ligand. Ongoing experiments in normal cord blood CD34+ cells will determine the in vivo role of β-catenin signaling in normal hematopoietic progenitors. In conclusion, our study showed that β-catenin comprises an integral part in the development and progression of AML in vivo, indicating that manipulation of the wnt pathway may hold a therapeutic potential in the management of AML.

Disclosures:

No relevant conflicts of interest to declare.
Memory Phenotype CD8+T Cells Are Superior to Naive CD8+T Cells in Separating Graft Anti-Tumor Activity From Gvhd After Bone Marrow Transplantation; Application to DLI.
Authors: Dutt Suparna , Baker Jeanette , Kambham Neeraja , Kohrt Holbrook E , Negrin Robert , Strober Samuel
Published: October 2019

Abstract 2452

Poster Board II-429

We compared the graft anti-tumor effect and GVHD activities of naturally occurring naive and memory phenotype subsets of C57BL/6 (H-2b) CD8+ T cells after bone marrow transplantation into BALB/c (H-2d) mice. The tumor used for the study was the BCL1 lymphoma, a spontaneously arising non-transfected cell line. We found that naive CD8+ T cells (CD62LhiCD44lo) express higher levels of gut homing molecules a4b7, CCR9 and CD103 compared to memory subsets comprised of both effector (CD62LloCD44hi) and central memory cells (CD62LhiCD44hi). The proliferation of the naive cells was ten-fold higher than memory phenotype cells against BALB/c stimulators and memory cells secreted significantly more IFNg in the MLR. All BALB/c hosts without tumor cells that were given donor memory CD8+T cells along with T cell depleted bone marrow cells (TCD BM) survived over 100 days of transplantation without significant weight loss compared to TCDBM controls. However, 25% of the mice given naive CD8+ T cells and TCD BM died due to GVHD and had significantly greater weight loss compared to TCD BM controls or to hosts given memory CD8+ T cells and TCD BM. BALB/c hosts given 500 BCL1 and TCD BM all died of lymphoma within 28 days of transplantation. Hosts given BCL1 cells, TCD BM and either memory or naive cells cleared the tumor by day 28 but weight loss and survival was significantly improved in the memory versus naive cell groups despite non significant differences in the GVHD scores in the liver and gut at day 100. Ex vivo imaging of mice receiving BCL1 tumor cells, TCD BM, and CD8+T cells from luciferase transgenic donors showed increased accumulation of naive phenotype CD8+T cells in the liver and the gut compared to memory phenotype cells after 6 days of transplantation. We compared the memory and naive CD8+ T cells in a model of donor lymphocyte infusion (DLI) in which BALB/c mice with progressive growth of BCL1 tumor after TCD BM transplantation were given infusion of the CD8+ T cells. Both naive and memory cells are effective in clearing the tumor, and converting the hosts from mixed to complete chimeras. Differences in GVHD are observed. In conclusion, CD8+ memory phenotype T cells are a desirable subset that mediates potent anti-tumor activity without severe GVHD, and can be used in a DLI model.

Disclosures:

No relevant conflicts of interest to declare.
Venous Thromboembolism (VTE) and Survival in a Cancer Chemotherapy Outpatient Clinic: A Retrospective Chart Review Validation of a VTE Predictive Model.
Authors: Kearney Julia C. , Rossi Suzanne , Glinert Keren , Henry David H.
Published: October 2019

Abstract 2503

Poster Board II-480

Because venous thromboembolism (VTE) often occurs as a complication of cancer and chemotherapy, cancer patients have a 47-fold elevation of risk of VTE compared to the general population (Khorana et al., Journal of Thrombosis and Haemostasis 2007). However, until recently there was not a model to accurately predict which cancer patients would experience a VTE during their treatment. Khorana et al. have developed such a model (Khorana et al., Blood 2008). We conducted a retrospective study to test the effectiveness of Khorana and colleagues' clinical model at predicting risk of VTE in patients with metastatic cancer. We collected data from an outpatient oncology clinic for 112 patients with solid tumors or malignant lymphoma who had undergone active chemotherapy in the last two years. The data included the five predictive variables outlined in Khorana et al.'s model: site of cancer (2 points for a very high-risk site, 1 point for a high-risk site); a platelet count of 350 × 109/L or more; hemoglobin less than 100g/L and or/use of erythropoiesis-stimulating agents; leukocyte count more than 11 × 109/L; and body mass index of 35kg/m2 or more (1 point each) (Khorana et al., Blood 2008). All data was collected on the first day of chemotherapy or the most recent labs before the start of chemotherapy. A score of 0 points represented a low risk of VTE, 1-2 points represented an intermediate risk and 3-4 points represented a high risk. There were 20 low risk patients, 63 intermediate risk and 29 high risk. We found that the clinical model accurately predicted which patients would experience a VTE (Table 1). Of the total 112 patients, 23 experienced a VTE. We determined that patients considered high risk were 8-fold more likely to experience a VTE as compared to low risk patients (see Table 1). Kuderer et al. demonstrated that this risk model also predicts overall mortality for patients receiving chemotherapy (Kuderer et al., ASH 2009). The results of our study also showed a strong correlation between overall mortality and a higher risk score. The mortality rate of high risk patients during the trial period was 69.0% compared to 30.0% of low risk patients and 42.9% of intermediate risk patients. Our retrospective review validates Khorana and colleagues' clinical model to predict VTE and survival rates in cancer patients. Our results, combined with data from previous studies, indicate that patients who are at risk for VTE also have a higher mortality rate. With an accurate predictive model, primary prophylaxis treatment for VTE could be considered for high risk patients.

Disclosures:

No relevant conflicts of interest to declare.
Single-Cell STAT5 Signal Transduction Profiling in Normal and Leukemic Stem and Progenitor Cell Populations Reveals Highly Distinct Cytokine Responses.
Authors: Han Lina , Wierenga Albertus T.J. , Rozenveld-Geugien Marjan , van de Lande Kim , Vellenga Edo , Schuringa Jan Jacob
Published: October 2019

Abstract 2510

Poster Board II-487

Signal Transducer and Activator of Transcription 5 (STAT5) plays critical roles in normal and leukemic hematopoiesis. We have observed that introduction of activated STAT5A in human stem/progenitor cells enhances long-term stem cell self-renewal, while lentiviral downmodulation of STAT5 expression in both normal as well as primary leukemic CD34+ cells impairs long-term growth and self-renewal. Many cytokines that act on the immature human stem cell compartment are known to be able to activate STAT5, such as TPO and SCF. Yet, little is known about the specificity and kinetics of STAT5 signaling in response to early-acting and lineage-restricted cytokines in specifically defined stem cell and progenitor compartments, particularly in the case of acute myeloid leukemia. We optimized a multiparametric flow cytometry protocol to analyze STAT5 phosphorylation upon cytokine stimulation in stem and progenitor cell compartments at the single-cell level. In normal cord blood (CB) cells, STAT5 phosphorylation was efficiently induced by TPO, IL-3 and GM-CSF within CD34+CD38− hematopoietic stem cells (HSCs). EPO and SCF-induced STAT5 phosphorylation was largely restricted to the megakaryocyte-erythroid progenitor (MEP) compartment, while G-CSF, as well IL-3 and GM-CSF were most efficient in inducing STAT5 phosphorylation in the myeloid progenitor compartments. Strikingly, mobilized adult peripheral blood (PB) CD34+ cells responded much less efficiently to cytokine-induced STAT5 activation, with the exception of TPO. In leukemic stem and progenitor cells, highly distinct cytokine responses were observed, differing significantly from their normal counterparts. A number of different types of responses were observed, being a) a strong STAT5 activation induced by TPO only; b) a strong STAT5 activation induced by IL3 and GM-CSF, but not TPO; c) STAT5 activation induced by various cytokines; and d) constitutive STAT5 phosphorylation that could not significantly be further induced by cytokines. These responses could not be predicted by the expression level of cytokine receptors. Also, heterogeneity existed in cytokine requirements for long-term expansion of AML CD34+ cells on stroma. In most examined cases multiple cytokines acted in an additive fashion in inducing long-term growth of primary AML CD34+ cells in bone marrow stromal cocultures, and in only 1 out of 10 cases cytokine-independent growth was observed. In none of the cases only IL3 or TPO were sufficient to induced long-term expansion of primary AML CD34+ cells. In conclusion, our study demonstrates variable cytokine responses in STAT5 phosphorylation in both normal and leukemic stem/progenitor cells.

Disclosures:

No relevant conflicts of interest to declare.
TIMP-1 Deficiency Subverts Cell Cycle Dynamics in Long-Term HSCs.
Authors: Rossi Lara , Goodell Margaret
Published: October 2019

Abstract 2514

Poster Board II-491

In addition to the consolidated role in extracellular matrix remodeling, the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) has been suggested to be involved in the regulation of numerous biological functions, including cell proliferation and survival. We therefore hypothesized that TIMP-1 might be involved in the homeostatic regulation of hematopoietic stem cells (HSCs), whose biological behavior is the synthesis of both microenvironmental and intrinsic cues. We found that TIMP-1−/− mice have decreased HSC numbers and, consistent with this finding, TIMP-1−/− HSCs display reduced capability of long-term repopulation. Interestingly, the cell cycle distribution of TIMP-1−/− LT-HSCs is profoundly distorted, with a consistent proportion of the stem cell pool arrested in the G1 phase, suggesting that TIMP-1 is intrinsically involved in the regulation of the HSC proliferation dynamics. Indeed, HSCs exhibit a higher proliferation rate, leading to an increased formation of CFU-C in vitro and spleen colonies (CFU-S) after transplant. Of note, TIMP-1−/− HSCs present decreased levels of CD44 glycoprotein, whose expression has been proven to be controlled by p53, the master regulator of the G1/S transition. Our findings establish TIMP-1 role in HSC function, suggesting a novel mechanism presiding over stem cell quiescence and potentially involved in the development of hematological diseases.

Disclosures:

No relevant conflicts of interest to declare.
Genome Expression Profiling of the First Hematopoietic Progenitors of the Mouse Embryo.
Authors: Baron Margaret H. , Isern Joan , Fraser Stuart T. , He Zhiyong , Tuck David , Schulz Vincent , Gallagher Patrick G.
Published: October 2019

Abstract 2536

Poster Board II-513

The transcriptional networks that regulate lineage commitment and expansion of the earliest hematopoietic progenitors in the mammalian embryo have not been well studied, due to a lack of methods for isolating these cells. We have begun to address this problem by purifying the first hematopoietic-committed progenitors in the mouse embryo based on expression of a human ε-globin::H2B-EGFP transgene that is expressed exclusively within the primitive erythroid (EryP) lineage, as early as embryonic day (E) 7.5. EryP are the first lineage-specific cell type to form in the embryo. They arise in large numbers from yolk sac-derived progenitors at the end of gastrulation, enter the circulation as nucleated cells soon thereafter, and continue to mature in a stepwise, synchronous fashion until they enucleate. The early and lineage specific expression of the GFP reporter allowed us to isolate not only circulating EryP (E9.5-E11.5) but also a population from dispersed E7.5-8.5 embryos that is enriched in EryP progenitors. Genome expression profiling allowed us to define the transcriptome from each stage of development and revealed highly dynamic changes during the progression from progenitor to maturing erythroblast. Hierarchical clustering analysis was used to organize genes on the basis of overall similarity in expression patterns; six major cluster patterns were identified. Genes within these clusters comprised distinct functional classes. For example, particularly prominent increases in expression were detected for genes involved in ribosome biogenesis, translation, chromosome condensation, and autophagy. Genes that were downregulated included those involved in DNA replication, cell cycle, and nucleolar and organelle biogenesis. We have focused on the emergence of EryP in the yolk sac. Expression of Gata2 is high in the progenitor population at E7.5 and decreases dramatically by E8.5. In contrast, Gata1, Scl, and Eklf are all upregulated during maturation of EryP progenitors, suggesting that these transcription factors have distinct functions during primitive erythropoiesis. Consistent with expression of the GFP reporter as early as E7.5, we find that endogenous mouse embryonic globin genes are also expressed at this stage. Therefore, globin gene expression is an early feature of EryP development. Analysis of promoters of differentially expressed genes allowed us to identify candidate transcriptional regulators, some of which have not previously been implicated in erythroid development. This is the first lineage specific transcription profiling of a differentiating hematopoietic cell type in the early mouse embryo. While we have focused on the development of EryP, insights from this study should have broader relevance to the definitive erythroid lineage.

Disclosures:

No relevant conflicts of interest to declare.
AMPK Regulates Nanog Gene Expression Via p53 in Mouse Embryonic Stem Cells.
Authors: Chae Hee-Don , Broxmeyer Hal E.
Published: October 2019

Abstract 2537

Poster Board II-514

High glucose level is usually required for embryonic stem cell (ESC) culture but culture medium without glucose is a rather favorable condition to enhance differentiation of ESCs. However, the exact molecular relationship of how glucose metabolism affects ESC pluripotency remains unclear. AMP-activated protein kinase (AMPK), a key regulator for controlling the metabolism of glucose and fatty acids, is activated in response to a high intracellular AMP to ATP ratio (ATP-exhausting stress). We hypothesized that AMPK activity is associated with maintenance of ESC pluripotency and tested this hypothesis utilizing aminoimidazole carboxamide ribonucleotide (AICAR) and Compound C, respectively as an activator and a selective inhibitor of AMPK. Here, we demonstrate that in the mouse ESC line, R1, in the presence of LIF, AICAR significantly reduced protein expression of Nanog and SSEA-1 (Nanog: 365+/−21 vs. 270+/−25; SSEA-1: 13195+/−707 vs. 7572+/−507, mean fluorescence intensity (MFI), +/-SD, control vs. AICAR (0.5 mM, 24h treatment), n=3 expts, p<0.01). In contrast, Compound C enhanced protein expression of SSEA-1 and Nanog (Nanog: 365+/−21 vs. 555+/−35; SSEA-1: 13195+/−707 vs. 15864+/−894, mean fluorescence intensity (MFI), +/-SD, control vs. AICAR (0.005 mM, 24h treatment), n=3 expts, p<0.02). This indicates that AMPK has a positive effect on protein expression of Nanog and SSEA-1. We extended our study to investigate whether increased Nanog protein expression level via AMPK activity resulted from enhanced stabilization of Nanog protein by measuring the half-life of nanog protein following treatment of mouse ESCs with cycloheximide, a protein synthesis inhibitor. Nanog protein stability was not affected by AMPK activity. Next, we examined whether AMPK activator or inhibitor could change Nanog and Oct-4 mRNA levels. AICAR reduced Nanog mRNA expression by 40%, but did not influence Oct-4 mRNA expression. AMPK induces cell cycle arrest or apoptosis under ATP-exhausting stresses by phosphorylating and activating the p53 tumor suppressor, which could repress Nanog gene expression. Activated AMPK induced phosphorylation of p53 at serine 15 and 392 without disturbing LIF induced STAT3 signaling in this mouse ESC line (Fig. 1). Thus, we postulate that activated AMPK might inhibit Nanog gene activity by phosphorylation of p53. Taken together, our data suggest that AMPK activity in response to metabolic ATP and AMP levels may play a primary role in controlling ESC pluripotency through p53-mediated regulation of Nanog gene expression.

Disclosures:

No relevant conflicts of interest to declare.
G0/G1 Switch Gene 2 (G0S2) Is a Positive Regulator of Quiescence in Hematopoietic Stem Cells and Naïve T Cells.
Authors: Yamada Takeshi , Park Chun Shik , Bae Leon , Lacorazza H. Daniel
Published: October 2019

Abstract 2538

Poster Board II-515

The G0/G1 switch gene 2 (G0S2) was identified in blood mononuclear cells activated with mitogens. G0S2 is a small basic protein located in ER/Golgi in mouse fibroblast. Even though G0S2 is upregulated in inflammatory processes (i.e. rheumatoid arthritis, psoriasis, endometriosis) and by all-trans-retinoic acid treatment in acute promyelocytic leukemia cells, its role in the proliferation of hematopoietic stem cells (HSC) and T lymphocytes is largely unknown. Similarly to HSC, a delicate balance between quiescence and homeostatic proliferation maintains the T cell pool over time. An emerging paradigm suggests that cellular quiescence is an actively regulated state. G0S2 expression in bone marrow (BM) cells is downregulated following cytoablation, suggesting a role in the maintenance of stem cell quiescence. Gain-of function experiments, using retroviral gene transfer and BM transplantation, showed that G0S2 increased number of Lin- Sca-1+ c-kit+ cells in BM by inhibiting their proliferation at steady state. In addition, ectopic G0s2 expression inhibits homeostatic expansion of CD11b+ Gr-1+ and CD3+ cells. Competitive BM transplantation showed that cells overexpressing G0S2 are outcompeted by wild type BM cells due to lower proliferative potential, based on in vivo BrdU incorporation experiments. Inhibition of T cell proliferation by ectopic G0S2 expansion is consistent with suppression of G0S2 transcription in naïve CD8+ T cells upon T-cell receptor activation via MAPK and calcium-calmodulin pathways, suggesting that G0S2 also maintains quiescence of naïve T cells. G0S2 expression is silenced in myeloid and lymphoid leukemic cell lines by gene methylation, supporting a leukemogenic role. Collectively, our study uncovered an important role of G0S2 in normal and malignant proliferation of HSC and T cells.

Disclosures:

No relevant conflicts of interest to declare.
Large Scale Generation of Functional Megakaryocytes From Human Embryonic Stem Cells (hESCs) Under Stromal-Free Conditions.
Authors: Li Feng , Lu Shi-Jiang , Feng Qiang , Lanza Robert
Published: October 2019

Abstract 2540

Poster Board II-517

Platelets collected from donors have very limited shelf life and are increasingly needed for transfusions. In contrast to donor dependent cord blood or bone marrow CD34+ stem cells, hESCs are a promising alternative source for continuous in vitro production of platelets under controlled conditions. Current procedures for in vitro generation of megakaryocytes/platelets from hESCs are not efficient and require undefined animal stromal cells. We have developed a novel system to generate megakaryocytes (MKs) from human ES cells under serum and stromal-free conditions. In the current system, hESCs are directed towards megakaryocytes through distinct steps including embryoid body formation and hemangioblast development (Lu et al, Nature Methods, 4:501–509, 2007). A transient bi-potential cell population expressing both CD41a and CD235a markers has been identified at the end of hemangioblast culture. These cells are capable of generating both MKs and erythroid cells as demonstrated by FACS sorting and CFU assays. TPO, SCF and IL-11 are used to further direct MK differentiation of hemangioblasts derived from human ES cells in suspension culture. Currently, up to 2.5×107 MKs (CD41a+) can be generated from 1×106 hESCs, which is approximately 10 times more efficient than recently reported methods (Takayama et al Blood, 111(11):5298–5306, 2008). Without further purification, >90% of live cells from the suspension cultures are CD41a+ and the majority of these cells are also CD42b+ (>70%). These in vitro derived MK cells have morphological characteristics of mature, polyploid MKs as shown by Giemsa staining and immunofluorescent staining of vWF in cytoplasmic granules. Importantly, proplatelet forming cells are constantly observed at the late stage of MK culture indicating that MKs generated in this system are able to undergo terminal differentiation under feeder-free conditions. Platelet-like particles are also detected in culture media by FACS. When plated on OP9 cells, these MKs generate functional platelets that are responsive to thrombin stimulation. In summary, we have established a novel system for the generation of platelet-producing MKs from human ES cells that is suitable for scale up and future preclinical and clinical studies.

Disclosures:

Li: Stem Cell & Regenerative Medicine International: Employment. Lu:Stem Cell & Regenerative Medicine International: Employment. Feng:Stem Cell & Regenerative Medicine International: Employment. Lanza:Stem Cell & Regenerative Medicine International/Advanced Cell Technology, Inc: Employment.
The Hematopoietic Stem Cell (HSC) Compartment in Tristetraprolin (TTP) Knock-out Mice Is Abnormal Phenotypically but Normal Functionally.
Authors: Kaplan Ian M , Morisot Sebastien , Heiser Diane , Cheng Wen-Chih , LaDana Christina , Civin Curt I.
Published: October 2019

Abstract 2541

Poster Board II-518

The RNA binding protein TTP binds to AU-rich elements (AREs) located in the 3′UTR of mRNAs and dramatically reduces their half-life through exosome-mediated RNA degradation. TTP provides a conserved mechanism for the temporal and fine-tuning regulation of gene expression. We found that the Lin−Kit+Sca+ (KSL) population, a phenotypic marker combination that enriches for HSCs, in adult (6–8 week old) C57Bl/6 marrow expressed TTP mRNA at a level 25-fold higher than whole bone marrow mononucuclear cells. Furthermore, adult TTP KO mice had twice the number of KSL cells per mouse compared to WT littermates, with no difference in bone marrow cellularity. Thus, we reasoned that TTP might represent an as yet unexplored avenue for HSC regulation. The TTP KO mouse has been described previously* and displays myeloid hyperplasia, cachexia and arthritis. Consistent with the reported phenotype there was a 2-fold increase in the number of phenotypic granulocyte-monocyte progenitors (GMPs) and normal numbers of both common myeloid progenitors (CMPs) and megakaryocyte-erythroid progenitors (MEPs) in KO marrow. Within the KSL population of KO marrow, we observed an increase in the Flt3−CD34− fraction, which has been shown to be highly enriched for long-term HSCs (LT-HSCs). However, when we measured the frequency of functional long-term engrafting cells by limiting dilution competitive repopulation transplantation assays, there was no difference between WT and KO marrow. We also found no difference in the in vivo responses of WT and KO HSCs in chimeric transplant recipients to bi-monthly 5-FU injections for 18 weeks. We tested the hypothesis that the increase in the number of KSL cells is due to a lack of regulation of the Sca1 mRNA in KO marrow. In an RNA half-life assay in NIH3T3 cells, TTP had no effect on the half-life of a reporter message carrying the Sca1 3′UTR; whereas the TNFa 3′UTR, a known TTP target, did cause the half-life of the reporter message to decrease in the presence of TTP in our assay. Interestingly, the Lin−CD150+CD48−Kit+Sca+ labeling scheme gave a much more accurate representation of the relative frequency of HSCs in KO vs WT marrow. In studying HSC biology, the ability to prospectively identify alterations in the HSC compartment of genetically and pharmacologically manipulated marrow is of utmost importance. These studies demonstrate that in genetically manipulated marrow, the phenotypic and functional phenotypes are not always in accordance.

*Taylor GA and Blackshear PJ et al. Immunity (4) 1996

Disclosures:

No relevant conflicts of interest to declare.
The HDAC Inhibitors Trichostatin A (TSA) and Suberoylanilide Hydroxamic Acid (SAHA) Exhibit Multiple Modalities of Benefit for the Vascular Pathology of Sickle Disease.
Authors: Hebbel Robert P , Vercellotti Gregory M. , Pace Betty , Solovey Anna , Kollander Rahn , Abanonu Chine , Nguyen Julia , Vineyard Julie V. , Belcher John D , Abdulla Fuad , Osifuye Shade , Eaton John W. , Kelm Robert , Slungaard Arne
Published: October 2019

Abstract 2586

Poster Board II-562

The chronic, inflammatory vasculopathy of sickle cell anemia accounts for much of the non-infectious morbidity and mortality of this disease. The pathogenesis of vasculopathy involves multiple sub-biologies (sickling, hemolysis, inflammation, adhesion, coagulation, NO deficiency, stasis, and reperfusion injury). Moreover, these various sub-biologies overlap and transactivate each other. So a single modality approach would very likely fail to be effective. For this reason, effective preventative and interventional therapy for sickle vasculopathy will very likely require true multi-modality therapy, preferably with as few drugs as possible. To this end, we have evaluated the histone deacetylase (HDAC) inhibitors TSA and SAHA for beneficial vascular effects in sickle transgenic mouse models. We used the hBERK1 mouse, which displays abnormal endothelial activation even at ambient air; and we used the NY1DD mouse which only acquires significant endothelial activation upon exposure to hypoxia/reoxygenation. Both drugs significantly inhibited the abnormal endothelial expression of tissue factor, predicting beneficial effects for sickle coagulopathy. Both drugs significantly inhibited abnormal endothelial expression of VCAM1, a molecule involved in both adhesion and inflammation sub-biologies. Both drugs worked in both mouse models. For these benefits, both drugs were effective when given as preventative therapy (NY1DD model) or as longer term therapy (hBERK1 model), although previously-established endothelial activation (e.g., the hBERK1 model) required long term (3 weeks) therapy to observe the most significant benefits. TSA markedly inhibited the vascular stasis in the dorsal skinfold chamber model of hBERK1 mice that is seen after their exposure to H/R; SAHA did so in S+SAntilles mice after H/R; our previous studies have implicated VCAM1 and abnormal cell/cell interaction as the etiology of stasis in this model. Both drugs induced gamma globin and levels of HbF in K562 cells in vitro. Both drugs exhibited activity as iron chelators, as detected by absorption spectra in vitro. Since both drugs are hydroxamic acids, they may promote NO formation by the same mechanism that hydroxyurea is said to work, although we did not test this. We suggest that SAHA, which is already approved for other human clinical use, should be considered for testing in a pilot study to determine drug tolerance and in vivo efficacy in sickle subjects.

Disclosures:

No relevant conflicts of interest to declare.