bluuebunny/crossref_metadata_2025 huggingface dataset:
FLT3ITD Expression Confers the Leukemogenic Properties of Growth Factor Independence and Enhanced Self-Renewal to Yolk Sac Progenitors.
Authors: Xiao Nan , Jani Kaushal , Yang Yuxia , Raffel Glen D
Published: October 2019

Abstract 1576

Congenital leukemias are clinically and biologically distinct from those manifesting in later childhood and in adults. Derivation of the leukemic cell of origin from the primitive hematopoietic elements of the yolk sac compartment rather than definitive hematopoietic precursors may explain the unique features of this class of leukemias. We hypothesized that in the cellular context of the yolk sac precursor, common leukemogenic mutations such as the FLT3 receptor internal tandem duplication (FLT3ITD), induce characteristics allowing expansion or propagation of this developmentally self-limited cell population. Yolk sacs were dissected from pregnant C57BL/6 mice at E9.5, disaggregated and then infected with an MSCV-FLT3ITD-GFP or an MSCV-GFP control virus. To assess growth factor independence, infected yolk sac cells were plated in methylcellulose lacking growth factors. Colony numbers were scored after 7–10 days and demonstrated FLT3ITD was capable of establishing growth factor independence in yolk sac precursors, similar to results obtained using transduced fetal liver and adult bone marrow. To determine whether FLT3ITD expression in yolk sac progenitors enhanced self-renewal, the ability to serially replate was tested. Yolk sac cells were infected as above and plated in methylcellulose containing Il-3, Il-6, Scf and Epo. Colonies were scored and the cells were replated every 7–10 days. In contrast to infected fetal liver or adult bone marrow, FLT3ITD-expressing yolk sac cells were capable of replating beyond the 4th round. Analysis of derived FLT3ITD-expressing yolk sac colonies demonstrated a CD41+CD34+c-Kit+ population with an undifferentiated morphology similar to primary yolk sac progenitors. In summary, aberrant expression of the FLT3ITD mutation in yolk sac cells results in the acquisition of growth factor independence and enhanced self-renewal, characteristics essential for leukemogenesis. Although FLT3ITD expression in fetal liver and bone marrow allows growth factor independence, the distinctive ability to promote enhanced self-renewal as well in yolk sac cells suggests the yolk sac is a uniquely vulnerable target for leukemic initiation during fetal development.

Disclosures:

No relevant conflicts of interest to declare.
Role of Cohesin-Resolving Protease, Separase, In Hematopoiesis.
Authors: Nakamura Lanelle V. , Mukherjee Malini , Goodell Margaret A. , Pati Debananda
Published: October 2019

Abstract 1593

Introduction:

Cohesin is an evolutionarily conserved protein complex that forms during the replication of sister chromatids. It is a multi-protein complex that consists of four proteins, Smc1, Smc3, Rad21, and Scc3. Resolution of sister chromatid cohesion at the onset of anaphase depends on Separase, an endopeptidase that separates sister chromatids by cleaving cohesion Rad21. A recent study suggests a new role of Cohesin proteins in gene expression and development with implications in hematopoiesis. Our data indicates that cohesin-resolving protease Separase may play a critical role in hematopoiesis. HYPOTHESIS: We hypothesize that Separase plays a role in hematopoiesis by increasing the quantity of hematopoietic stem cells (HSC). METHODS: Our experimental approach was to isolate murine long-term HSC from WT mice and mice with one mutated copy of Separase (i.e. Separase heterozygotes). In addition, in vivo competitive long term repopulation assays were used assess the function of HSC in Separase heterozyotes. RESULTS: Separase heterozygote have increased HSC numbers (p<0.05) as compared to WT mice. In addition, an improved engraftment in a competitive repopulation assay (p < 0.001) was seen in the Separase heterozyotes. Analysis of the engrafted cells demonstrated no difference between the wild type and Separase heterozygote animals, indicating the increased engraftment may be due to unique features in the primitive hematopoietic stem cells. CONCLUSION: Investigation of the mechanism for improved HSC engraftment in Separase heterozygote mice will significantly contribute to our understanding of marrow engraftment and function. Elucidating the mechanisms of hematopoietic dysregulation will provide insights into the development of life-threatening disorders such as leukemia and, in the setting of bone marrow transplant, engraftment failure.

Disclosures:

No relevant conflicts of interest to declare.
Differentiation of Mature Eosinophils From Human Embryonic and Induced Pluripotent Stem Cells.
Authors: Yang Wenyu , Ma Feng , Matsumoto Kenji , Nishihama Natsumi , Sagara Hiroshi , Ebihara Yasuhiro , Gu Yanzheng , Saito Hirohisa , Zhu Xiaofan , Nakauchi Hiromitsu , Tsuji Kohichiro
Published: October 2019

Abstract 1594

Eosinophils are multifunctional leukocytes implicated in the pathogenesis of numerous inflammatory processes. As the major effectors, eosinophils function in a variety of biological responses, allergic diseases and helminth infections. It is generally accepted human eosinophils develop through a pathway initially sharing common feature with basophils. However, there lacks a clear chart for early development of human eosinophils, such as during embryonic or fetal stages. We recently established an efficient method for producing eosinophils from human embryonic and induced pluripotent stem cells (hESC/iPSCs). By a two-step induction, we first generated multipotential hematopoietic progenitors by co-culturing hESC/iPSCs with mouse AGM-derived stromal cells for 2 weeks. Then, total co-culture cells were transferred into suspension culture favoring eosinophil development with addition of IL-3 and other factors (SCF, IL-6, TPO, Flt-3 ligand) . The maturation of hESC/iPSC -derived eosinophils was shown in a time-dependent manner, first co-expressing eosinophil-and basophil-specific markers [eosinophil peroxidase (EPO), and 2D7, respectively], then the portion of eosinophil markers gradually increased while that of basophil markers decreased (EPO+ cells from 56.4% at day 7 to 94.4% at day 21, while 2D7+ cells from 62.8% to 25.7%, respectively), typically mimicking the development of eosinophils from human adult hematopoietic progenitors. By flowcytometric analysis, an eosinophil-specific surface marker, Siglec-8, was also expressed on these hESC/iPSC-derived eosinophils in a time-dependent manner (from 10.8% at day 7 to 91.3% at day 21), paralleling to those with EPO. The expression of eosinophil-specific granule cationic proteins (EPO, MBP, ECP, EDN) and IL-5 receptor mRNA was also detected by RT-PCR. Furthermore, transmission electron microscopy (TEM) observation confirmed the eosinophil property. Eosinophils derived from hiPSCs hold similar characteristics as those from hESCs. The function of hES/hiPSC-derived eosinophils is being under investigation. Our study provides an experimental model for exploring early genesis of eosinophils, especially in uncovering the mechanisms controlling the development of the initial innate immune system of human being in normal and diseased individuals.

Disclosures:

No relevant conflicts of interest to declare.
Low Molecular Weight Heparin Reduces sVCAM-1 and Lung Congestion In a Murine Model of Sickle Cell Disease
Authors: Guo YiHe H. , Field Joshua , Foster Thomas D. , Scott J. Paul , Wandersee Nancy , Hillery Cheryl A.
Published: October 2019

Abstract 1635

There is evidence for increased levels of procoagulant proteins, thrombin generation and tissue factor activity in individuals with sickle cell disease. Both thrombin and tissue factor also have potent proinflammatory effects that likely further contribute to the vascular dysfunction and organ injury observed in sickle cell disease. In this study, we examine the effect of the LMW heparin, enoxaparin, on markers of vascular injury and inflammation in the Berkeley mouse model of severe sickle cell disease (HbSS mice). HbSS mice or control HbAA mice (that exclusively express normal human HbA) were treated with continuous subcutaneous infusion of enoxaparin (10 mg/kg/day) or saline for 14 days using a surgically implanted Alzet pump (n=12-14 mice per group). Blood was collected at baseline and following 14 days enoxaparin therapy. Anesthetized animals were perfused with PBS at constant physiologic pressure (80 mmg Hg) and lung and liver collected for pathologic analyses. Enoxaparin-treated mice had steady state LMW heparin levels of 0.45 ± 0.17 anti-Xa U/mL compared to <0.1 anti-Xa U/mL in saline treated animals. Enoxaparin reduced d-dimer levels by approximately 35% in both HbSS and HbAA mice compared to saline-treated mice (p=0.03 and p=0.02, respectively), showing a functional reduction in coagulation and fibrinolytic activity. The hemoglobin level did not change and no obvious hemorrhage was noted in enoxaparin-treated mice suggesting no major bleeding complications. sVCAM-1 is a measure of endothelial injury and is increased at baseline in HbSS mice compared to HbAA mice (p<0.001). sVCAM-1 levels were reduced in HbSS mice after 14 days of enoxaparin therapy (1424 ± 83 ng/mL baseline, 1314 ± 85 ng/mL post enoxaparin, p=0.02), but not in saline treated HbSS mice (1462 ± 69 ng/mL baseline, 1392 ± 85 ng/mL post saline, p=0.3). Furthermore, the sVCAM-1 level negatively correlated with the steady state heparin level (r=-0.48, p=0.02) suggesting a modest dose-dependent effect of heparin on sVCAM-1 level. There was no effect of enoxaparin or saline therapy on sVCAM-1 levels in control HbAA mice. Preliminary histological analysis revealed a reduced number of congested pulmonary arteries in enoxaparin-treated HbSS mice compared to saline treated HbSS mice (p=0.01). These data suggest that enoxaparin therapy may improve endothelial injury as measured by sVCAM-1 and pulmonary vascular congestion in a mouse model of severe sickle cell disease. The data also suggest that heparin-based anticoagulant therapy may be a novel therapeutic approach to improve vascular health in individuals who suffer from sickle cell disease.

Disclosures:

Field: Novartis: Honoraria.
Overexpression of IL-3R alpha on CD34+CD38- Stem Cells Defines Leukemia-Initiating Cells In FA AML.
Authors: Du Wei , Sipple Jared , Pang Qishen
Published: October 2019

Abstract 1667

Patients with Fanconi anemia (FA) have high risk of developing acute myeloid leukemia (AML). In this study, we attempted to identify cell surface markers for leukemia-initiating cells (LICs) in FA AML patients. We found that interleukin-3 receptor alpha (IL-3R alpha) is a promising candidate as an LIC-specific antigen for FA AML. While IL-3R alpha expression is undetectable on normal CD34+CD38- hematopoietic stem cells (HSCs), it is overexpressed on CD34+CD38- cells from FA patients with AML. We have examined whether the LIC activity of IL-3R alpha-positive FA AML cells in a “humanized” FA xenotransplant model, in which we separated AML cells into IL-3R alpha-positive and IL-3R alpha-negative CD34 fractions and transplanted them into irradiated recipient mice. In all three FA AML samples, only IL-3R alpha-positive cells showed significant levels of engraftment and developed leukemia in the recipient mice. The FA CD34+IL-3R alpha+ blasts isolated from leukemic mice exhibited hypersensitivity to IL-3 deprivation and JAK2-STAT5 overactivation following IL-3 treatment. Finally, pretreatment of FA CD34+IL-3R alpha+ blasts with the IL-3Ra neutrolizing antibody inhibits IL-3-mediated proliferation and STAT5 activation. These results demonstrate that IL-3Ra is a cell surface marker present on FA AML-LIC and may serve a valuable therapeutic target.

Disclosures:

No relevant conflicts of interest to declare.
Influence of Residual Normal Metaphases In Patients with Monosomal Karyotype.
Authors: Xie Bin , Othus Megan , Medeiros Bruno C. , Fang Min , Appelbaum Frederick R. , Estey Elihu H.
Published: October 2019

Abstract 1671

Breems et al.(J Clin Oncol 2008;26:4791-97) observed that a monosomal karyotype(MK: ≥ 3 cells with ≥2 autosomal monosomies or with 1 autosomal monosomy plus a structural abnormality) conveyed a worse prognosis in untreated AML than a complex karyotype (CC ≥ 4 distinct clonal abnormalities),although there was considerable overlap between MK and CC. The SWOG (Medeiros et al. Blood e pub June, 2010) has confirmed these findings and extended them to patients age 60 or above. Previous data (Estey et al. Haematologica 2000;85:246-49) suggested that the presence of residual normal cells improved prognosis in patients with monosomy 7, prompting us to see if the same was true in patients with MK. Our analysis focused on survival and used the same 176 MK patients identified by Mederios et al. from 1,344 patients enrolled in one of 10 successive SWOG clinical trials. The MK patients had a median age of 61 years and 94% had a CC (≥ 3 distinct clonal abnormalities); 68% received induction with standard dose ara-C, 14% with other ara-C doses, and 18% without ara-C. A univariate proportional hazards model indicated that survival improved as the % of normal cells increased and accordingly as the % of abnormal cells (MK and non MK) decreased. Further investigation indicated that this reflected the number of normal, rather than the number of abnormal, cells. Thus having one or more normal cells was associated with a hazard ratio (HR) of 0.95 (p = 0.02), while the presence of 5 or more normal cells decreased the HR to 0.76. In contrast, there was no association between the number of abnormal MK cells and survival (p=0.15). Accounting for age and treatment (as noted above) did not alter these conclusions (HR for having 1 or more normal cells 0.95, p = 0.03).Despite the p-values these findings have limited medical significance for MK patients; since even the aforementioned decrease in HR to 0.76 would result in a median survival of only 5.3 months vs. the median of 4 months for all 176 MK patients. Nonetheless the results indicate that it might be worthwhile to examine the prognostic effect of residual normal metaphases in patients with better prognosis karyotypes.

Disclosures:

No relevant conflicts of interest to declare.
Decreased Expression In NF-κB Essential Modulator Due to a Novel Splice-Site Mutation Causes Ectodermal Dysplasia with Immunodeficiency
Authors: Karakawa Shuhei , Okada Satoshi , Tsumura Miyuki , Mizoguchi Yoko , Ohno Norioki , Yasunaga Shin'ichiro , Ohtsubo Motoaki , Kawai Tomoki , Nishikomori Ryuta , Takihara Yoshihiro , Kobayashi Masao
Published: October 2019

Abstract 1732

X-linked ectodermal dysplasia with immunodeficiency (XL-EDA-ID) is caused by hypomorphic mutations in the nuclear factor κB (NF-κB) essential modulator (NEMO) gene, also called IKBKG, and these mutations impair but do not abolish NF-κB signaling, resulting in distinct clinical and immunologic phenotypes. We presented the patient with XL-EDA-ID showing a novel splice-site mutation. In this report we precisely study the involvement of this mutation in the molecular pathogenesis of XL-EDA-ID.

The patient is 12 years old Japanese boy. He had conical-shaped teeth and hypodontia. He had suffered from recurrent bacterial infections and these pathogenic bacteria were mostly streptococcus pneumonia. The laboratory examination revealed that the number of white blood cell counts, the classification of lymphocytes, and the serum immunoglobulin levels were within normal range. However the specific antibodies against measles and streptococcus pneumonia were negative in spite of these infections.

We identified a novel hemizygous mutation, 769-1 G>C, at the splicing acceptor site of exon 7 in the IKBKG gene. In order to clarify the effect of this mutation on mRNA, we performed a cloning analysis. Although various abnormal spliced NEMO (mutant NEMO) mRNAs were observed, a small amount of wild-type NEMO (WT NEMO) mRNA was also identified. The rate of WT and mutant was variable on the time of blood collection. Further we performed FACS and immunoblot analysis in order to evaluate the effect of this mutation at protein level. Two major bands which were presumed to be derived from WT and one mutant were detected in immunoblots using EBV transfected B cells, however the expression levels of these bands were markedly decreased compared to those of the healthy controls. The NEMO protein expression was confirmed to be decreased in various lineages of leukocytes. We generated WT and two representative mutant NEMO constructs and measured their NF-κB transcriptional activity using reporter assay. One mutant NEMO abolished NF-κB transcriptional activity, the other showed weak activity. However, neither of mutant NENO showed a dominant-negative effect against WT NEMO activity. CD14+ cells from the patient produced a lower level of TNF-α in response to IFN-γ stimulation, on the other hand CD3+ cells produced very little IFN-γ in response to IL-12. CD4+ T-cell proliferation was impaired in response to measles and mumps, but not rubella.

The hemizygous 769-1 G>C mutation was shown to cause the decreased expression of WT NEMO protein, resulting in the decrease in NF-κB activation and the development of XL-EDA-ID.

Disclosures:

No relevant conflicts of interest to declare.
Bone Morphogenetic Proteins Inhibit CD40L- and IL-21-Induced Ig Production In Human B Cells: Differential Effects of BMP-6 and BMP-7
Authors: Huse Kanutte , Bakkeb∅ Maren , Forfang Lise , Hilden Vera I. , Smeland Erlend B. , Myklebust June H.
Published: October 2019

Abstract 1745

Introduction:

TGF-β has been shown to play an important role in directing class switch recombination in human B cells. Bone morphogenetic proteins (BMPs) are members of the TGF-β family, and we have previously shown that BMP-6 inhibits proliferation of B cell progenitors as well as mature B cells in humans. However, how various BMP members affect Ig production and B-cell maturation to plasma cells have not been investigated. Here we studied the effects of various BMPs in CD27- naive and CD27+ memory B cells from peripheral blood of healthy human donors.

Methods:

We used flow immunomagnetic beads and flow cytometry cell sorting to purify CD19+CD27- naive and CD19+CD27+ memory B cells from peripheral blood of healthy donors, and investigated BMP induced effects on in vitro proliferation, cell death and Ig production as measured by thymidine incorporation assay, propidium iodide staining and ELISA, respectively. To separate direct inhibition of plasma cell differentiation from indirect effects via suppression of proliferation and induction of apoptosis, CFSE tracking of cell division was combined with immunophenotyping of cultured cells to identify CD27+CD38+ plasma cells. Expression of transcription factors and AICDA were measured by quantitative real-time PCR.

Results:

BMP-2, -4, -6 and -7 specifically inhibited CD40L- and IL-21-induced production of IgM, IgG and IgA. BMP-6 was the most potent inhibitor, reducing the Ig production by 70% in memory B cells and more than 50% in naive B cells. By investigating the mechanisms for reduced Ig production, we found a striking difference between the structurally similar BMP-6 and BMP-7. BMP-6 directly inhibited differentiation to CD27+CD38+ plasma cells, whereas BMP-7 only had minor effects on differentiation. Instead, BMP-7 mainly affected Ig production indirectly by inducing apoptosis. Furthermore, we explored BMP-6-induced signaling and gene regulation in more detail in memory B cells. BMP-6 up-regulated Id1, Id2 and Id3 gene expression in CD40L- and IL-21-stimulated cells (6.1-fold, 1.7-fold and 4.0-fold induction after 48 hours culture with BMP-6, respectively). In contrast, BMP-6 potently inhibited CD40L- and IL-21-induced upregulation of the transcription factor XBP-1, necessary for the late stages of plasmacytic differentiation. Expression of transcription factors regulating earlier stages (IRF4, PRDM1) was not affected, indicating that BMP-6 only modulates late events of plasma cell development.

Conclusion:

These results show that BMPs potently suppress Ig production in mature human B cells by inhibiting differentiation or by inducing cell death.

Disclosures:

No relevant conflicts of interest to declare.
BACOPP-D - An Etoposide-Free Dose-Escalated Polychemotherapy Regimen In Advanced Hodgkin‘s Lymphoma
Authors: Naumann Ralph , Kluge Diana , Haenel Annette , Wetzko Katrin , Friedel Nadja , Schmidt Helmuth , Dawel Monic , Beuthien-Baumann Bettina , Schwanebeck Uta , Ordemann Rainer , Haenel Mathias , Ehninger Gerhard
Published: October 2019

Abstract 1754

Introduction:

The development of the escalated BEACOPP regimen let to an improved outcome in patients with advanced Hodgkin‘s lymphoma (HD9 study of the GHSG). However, the application of high dose etoposide (cumulative 4,8 g/m2 per 8 cycles) seems to be associated with an increased incidence of secondary MDS and AML, respectively. Therefore, the aim of our multicenter pilot study was to evaluate the efficacy and toxicity of the etoposide-free as well as dose-intensified BACOPP-D protocol.

Methods:

From May 2000 until August 2008 a total of 139 untreated patients with Hodgkin‘s lymphoma (HL) stage IIB, III, and IV were treated with BACOPP-D which included cyclophosphamide 1250 mg/m2 (d1), adriamycin 25 mg/m2 (d1+2), dacarbazine 250 mg/m2 (d1-3), procarbazine 100 mg/m2 (d1-7), prednisolone 40 mg/m2 (d1-14), bleomycin 10 mg/m2 (d8) and vincristine 1,4 mg/m2 (maximum 2 mg, d8) at three-weekly intervals with granulocyte colony-stimulating factor (G-CSF). A consolidating involved field radiation (30 Gy) was performed only in patients who achieved less than CR following chemotherapy. Post-treatment follow-up included PET imaging.

Results:

All patients (median age 34 years, range 16–65; 86 male, 53 female) are assessable for toxicity and treatment outcome. We analyzed the acute toxicity for 1060 cycles of BACOPP-D. CTC grade III/IV haematological toxicities per patient were observed as follows: leukopenia 92%, anemia 40%, and thrombocytopenia 35%. CTC grade III/IV non-haematological side effects included documented infection (8%) and lung toxicity (one patient). Consolidation radiotherapy was given in 73 patients (52,5%). A total of 125 patients (89,9%) achieved complete remission, 9 patients (6,5%) achieved partial remission, five patients (3,6%) had progressive disease. At a median observation time of 46 months (5-109 months), 9 patients (6,5%) have relapsed, and 11 deaths were documented (4 HL-specific and 4 treatment related deaths, 1 death due to ruptured Meckel diverticulum with peritonitis, one 65 year-old woman died in CR following myocardial infarction and 1 death due to secondary malignancy). Only two patients developed a second neoplasia (hypopharyngeal carcinoma in an alcoholic; melanoma). The overall survival and progression free survival rates at 46 months were 89,7% and 85,9%, respectively. Discussion: BACOPP-D regimen appears as a feasible and effective treatment which induced a complete morphologic remission in a high proportion of patients with advanced HL. The treatment was associated with moderate acute toxicity. No secondary AML or MDS occurred so far.

Disclosures:

No relevant conflicts of interest to declare.
Aplidin® (Plitidepsin) Activity In Peripheral T-Cell Lymphoma (PTCL): Final Results
Authors: Fermé Christophe , Mateos Maria Victoria , Szyldergemajn Sergio , Corrado Claudia S. , Zucca Emanuele , Extremera Sonia , Gianni Alessandro M. , Vandermeeren Andrea , Ribrag Vincent
Published: October 2019

Abstract 1767

Background:

Peripheral T-cell lymphomas (PTCL) are a heterogeneous group of NHL, representing 10–15% of all newly diagnosed cases; their prognosis is poor and optimal therapy is yet to be defined. Most patients (pts) relapse after standard first-line chemotherapy and ultimately die from their disease. Though pralatrexate has been recently FDA-approved for this indication, novel active agents are still needed. From December 2004 to June 2008, 46 adult pts with aggressive non-Hodgkin lymphoma (NHL) were treated in a phase II study aimed at evaluating the activity of plitidepsin. An interim analysis showed responses restricted to pts with PTCL (4/17 vs. 0/29 in pts with B-cell NHL; Fisher's test: p=0.01). Thus, PTCL cohort was expanded. Herein, final results are presented.

Patients and methods:

As of June 2010, 32 pts were treated with plitidepsin 3.2 mg/m2 i.v. infusion over 1-h on days 1, 8 and 15 q4wk. Twenty-nine pts are evaluable for efficacy. Median number of previous regimens was 2 (range, 1–6), including prior stem cell transplantation in 9 pts (28%). Histology: PTCL, NOS 16, anaplastic large-cell 4, angioimmunoblastic 9 and NK/T nasal type 3. Twenty-three pts were male, median age was 57 y (30-80) and ECOG: 0 (14 pts), 1 (12 pts) and 2 (6 pts).

Results:

Two CR and 4 PR were observed, for a 20% objective response rate (95% CI, 8%-39%). Median duration of response was 2.2 months (range, 1 – 28). Median progression-free survival was 1.6 months (95% CI, 1 – 3) and median overall survival was 10.2 months (95% CI, 4 – 24). Toxicity, particularly haematological, was mild. Two patients developed G3, and 2 G4 neutropenia, and 1 pt G3, and 2 G4 thrombocytopenia. Transient and reversible G3 ALT or AST elevations occurred in 7 and 4 pts, respectively, with no patients experiencing G4 events. Clinical toxicities were mainly G1-2 muscular weakness and myalgia in 14% of pts, and G1-2 fatigue, nausea and vomiting in 21%, 28% and 17% of patients, respectively.

Conclusions:

Plitidepsin has activity, with an acceptable safety profile in non-cutaneous relapsed/refractory PTCL patients. Remarkably, lack of hematologic toxicity makes plitidepsin an ideal agent either for treating patients with poor bone marrow reserve or combining it with other active agents. A combination of plitidepsin with gemcitabine is currently being explored in this patient population.

Disclosures:

Szyldergemajn: PharmaMar SAU: Employment. Corrado:PharmaMar SAU: Employment. Extremera:PharmaMar SAU: Employment. Vandermeeren:PharmaMar SAU: Employment.
Low Absolute Lymphocyte Count and Addition of Rituximab Confer High Risk for Interstitial Pneumonia In Patients with Diffuse Large B Cell Lymphoma
Authors: Yu Yuan-Bin , Huang Yu-Chung , Liu Chia-Jen , Pai Jih-tung , Lu Hsueh-Ju , Liu Chun-Yu , Hong Ying-Chung , Hsiao Liang-Tsai , Gau Jyh-Pyng , Hsu Hui-Chi , Chiou Tzeon-Jye , Liu Jin-Hwang , Tzeng Cheng-Hwai
Published: October 2019

Abstract 1805

Purpose:

Several studies reported pulmonary toxicities in patients with diffuse large B cell lymphoma (DLBCL) receiving rituximab and chemotherapy. This retrospective study aimed to determine the risk factors and clinical characteristics of interstitial pneumonia in patients with DLBCL.

Methods:

From January 2000 to May 2009, 529 consecutive patients with DLBCL receiving first-line COP- or CHOP-based chemotherapy with or without rituximab in Taipei Veterans General Hospital were enrolled. Interstitial pneumonia (IP) was defined as diffuse pulmonary interstitial infiltrates found on computed tomography scan as well as respiratory symptoms. Patient characteristics and outcome parameters were retrieved via medical chart review.

Results:

IP was observed in 26 patients (4.9%) and 6 of them were confirmed asPneumocystis jirovecii pneumonia. The median number of chemotherapy course to IP was 4 cycles (range, 1–7). By multivariate logistic regression, absolute lymphocyte count (ALC) less than 1×109/L before treatment (odds ratio [OR] 2.75, 95% confidence interval [CI] 1.23–6.19) and addition of rituximab to chemotherapy (OR 4.56, 95% CI 1.68–12.39) were identified as independent risk factors for IP. In the rituximab-treated patients, low ALC at baseline further increased the risk for IP.

Conclusions:

Incidence of IP is increased in patients with DLBCL receiving rituximab-containing chemotherapy. Specific subgroup with lymphopenia at diagnosis should receive more attention in detecting this pulmonary complication.

Disclosures:

No relevant conflicts of interest to declare.
AKN-028, a FLT-3 Kinase Inhibitor In Preclinical Development, Induces Significant Gene Regulation That Differs From PKC-412
Authors: Parrow Vendela C , Eriksson Anna , Göransson Hanna , Rickardson Linda , Lehmann Fredrik , Lenhammar Lena , Larsson Rolf , Hoglund Martin
Published: October 2019

Abstract 1837

AKN-028 (formerly BVT-II, abstract presented at EHA 2008, Eriksson A et al) is a tyrosine kinase inhibitor originally identified in a screen targeting FLT-3. In vitro testing by fluorometric microculture cytotoxicity assay (FMCA, Lindhagen E. et al, Nat Protoc 2008; 3:1364-9) on primary tumor cells from 29 patients with different haematological malignancies showed that the cytotoxic activity was most pronounced in acute myeloic leukaemia (AML). In vivo, efficacy was demonstrated in a hollow fiber mouse model using MV4-11 cells and primary tumor cells from AML patients.

Kinase inhibitors interacting with the ATP-binding pocket usually targets more than one kinase. PKC-412, presently in phase III trials in AML, is a staurosporine analogue targeting many kinases. In this study we show the kinase inhibition profile of AKN-028 when characterised on a broad panel of 320 different kinases. AKN-028 is relatively specific compared to the staurosporine analogues. To further analyse the difference in mechanism of action between the multi-targeting inhibitor PKC-412 and the more selective AKN-028, we have performed global gene expression analysis by Affymetrix arrays using two different leukaemia cell-lines, HL-60 and MV4-11 (expressing FLT3-ITD), and tumour cells from a patient with FLT3-ITDpos AML. The cells were treated with 10 μM of either compound or vehicle control for 6 h. Principal component (PC) analysis was used to visualise the global gene expression pattern, see fig 1. Searching for differential expression of mRNA levels showed that treatment with AKN-028 resulted in significantly altered gene expression in all three cell types tested, compared to vehicle control treated cells. 430 mRNAs were down-regulated, and 280 were up-regulated. By contrast, treatment with PKC-412 caused very few significant alterations of mRNA levels when compared to vehicle treated cells. Further analysis of gene expression patterns to elucidate the mechanism of action of AKN-028 is ongoing. In conclusion, even though AKN-028 is a relatively selective kinase inhibitor targeting FLT-3, it has more profound effects altering gene expression both in cultured AML cell-lines and a primary AML tumor sample than the multikinase inhibitor PKC-412.

Disclosures:

Parrow: Akinion Pharmaceuticals: Employment, Equity Ownership. Lehmann:Akinion Pharmaceuticals: Consultancy. Larsson:Akinion Pharmaceuticals: Consultancy.
GlycoVariant Anti-CD37 Small Modular Immuno-Pharmaceutical Exhibits Superior Natural Killer Cell Mediated Cytotoxicity Against Chronic Lymphocytic Leukemia Cells at Low Concentrations and Low Antigen Density
Authors: Rafiq Sarwish , Cheney Carolyn M , Lozanski Gerard , Lapalombella Rosa , Mo Xiaokui , Siadak Anthony , Jarjoura David , Byrd John C. , Muthusamy Natarajan
Published: October 2019

Abstract 1847

CD37 is a tetraspanin transmembrane family protein that is strongly expressed on the surface of mature human B cells and transformed mature B cell lymphoma and leukemia cells, including CLL cells. It is absent or minimally expressed on normal T cells, NK cells, monocytes, and granulocytes. Predominant expression of CD37 on CLL cells makes it an ideal candidate to target with potential agents for treatment of CLL. TRU-016, a small modular immunopharmaceutical protein (SMIP) that specifically binds to an extracellular region of CD37, is presently in clinical trials in CLL patients. TRU-016 includes humanized immunoglobulin variable regions (scFv) fused to a human IgG1 Fc region. We have previously reported that SMIP-016, the chimeric version of the humanized TRU-016, induced apoptosis in CLL B cells in the presence of goat anti-human Fc antibody cross-linker through a novel, caspase-independent pathway. Furthermore, SMIP-016 showed potent in vivo activity in a SCID xenograft mouse model. Aside from direct cytotoxicity, SMIP-016 mediates antibody-dependent cellular cytotoxicity (ADCC) by NK cells both in vitro and in vivo. In an attempt to enhance its ADCC function, a new variant of SMIP-016, SMIP-016GV, was designed with a modification of the glycosylation of the Fc portion of the molecule. SMIP-016GV exhibits enhanced binding to both low- and high-affinity molecular variants of human CD16 (FcγRIII) and augments ADCC potency when compared to SMIP-016. In this study, we compared SMIP-016GV and SMIP-016 in direct cytotoxicity and ADCC against CLL B cells. While SMIP-016 and SMIP-016GV mediated comparable direct cytotoxicity at 48 hrs in the presence of goat anti-human Fc crosslinker, the SMIP-016GV resulted in 2 to 4 fold increase in NK cell mediated ADCC function at all effector to target ratios tested. This increased ADCC with SMIP-016GV was observed using NK cell effectors derived from both normal as well as CLL-affected individuals. In addition, this enhanced cytotoxicity was sustained at concentrations of SMIP-016GV as low at 5E-6 μg/ml. These low concentrations of SMIP-016GV were also able to mediate superior ADCC in 697 cells expressing as few as 10,000 molecules of surface CD37 antigen. Furthermore, NK cells stimulated with the glycovariant were potently activated and released 3 to 4 fold more IFNγ compared to SMIP-016. Ongoing studies are aimed at defining other effector cells which may interact with SMIP-016GV via different Fcγ Receptors. Collectively, these results suggest potential use of the SMIP-016GV with enhanced ADCC function as an alternate for TRU-016 in B cell malignancies including CLL therapy.

Disclosures:

Siadak: Trubion Pharmaceuticals: Employment.
TRAIL Preferentially Affects Cell Cycle-Arrested Tumor Cells Including Stem- and Progenitor Cells From Patients with Acute Lymphoblastic Leukemia
Authors: Ehrhardt Harald , Alves Catarina Castro , Wachter Franziska , Jeremias Irmela
Published: October 2019

Abstract 1879

Leukemic stem- and progenitor cells exhibit low cycling activity which might represent a major cause for their increased treatment resistance. TRAIL (TNF-related apoptosis inducing ligand) is a novel putative anticancer drug currently in phase I and II clinical testing. We recently showed that TRAIL is able to address stem- and progenitor cells from patients with acute lymphoblastic leukemia (ALL) in xenotransplantation assays (Alves et al., Blood 2012,119,4224). As stem- and progenitor cells are often non-cycling, we asked here, whether TRAIL is able to address resting leukemia cells.

We used cell lines and primary tumor cells from children with ALL which were amplified in severely immuno-compromised mice (NSG mice). Cell cycle arrest was induced (i) by addition of conventional cytotoxic drugs which are known to act as cytostatic drugs such as doxorubicine; (ii) by biochemical inhibitors known to induce cell cycle arrest at different defined points of the cell cycle such as mimosine; (iii) by molecular approaches and knockdown of cyclinB arresting cell cycle in G2 or knockdown of cyclinE arresting cell cycle in G1.

Unexpectedly, TRAIL-induced apoptosis was enhanced, whenever cell cycle was arrested. Cell cycle arrest sensitized towards TRAIL-induced apoptosis independently from the point or phase of cell cycle which was arrested (G0, G1 or G2) and independently from the agent used to arrest the cell cycle. Similarly, knockdown of cyclinB or cyclinE both clearly sensitized cell line cells towards TRAIL-induced apoptosis.

Cytotoxic drugs and cell cycle inhibitors might arrest the cell cycle by activation of p53. Accordingly, when caffeine was added which inhibited p53 activity and drug-induced cell cycle arrest, sensitization towards TRAIL-induced apoptosis was blocked.

We have recently established a novel method which enables performing knockdown experiments in tumor cells derived from ALL patients (Höfig et al., Cell Comm. Signal. 2012,10,8). Using this method and most important for clinical translation, we could show that knockdown of either cyclinB or cyclinE clearly sensitized patient-derived ALL cells towards TRAIL-induced apoptosis.

Taken together and in contrast to most conventional cytotoxic drugs, TRAIL exerts anti-tumor activity preferentially against tumor cells in cell cycle arrest and less against actively cycling tumor cells. This special feature of TRAIL might explain its anti-tumor activity against stem- and progenitor cells in patients with ALL. Thus, TRAIL might represent an interesting drug to treat disease stages with accumulation of stem- and progenitor cells and static tumor disease, e.g., during minimal residual disease.

Disclosures:

No relevant conflicts of interest to declare.
CXCR4 Inhibitors Selectively Eliminate CXCR4 Expressing Human Acute Myeloid Leukemia Cells in NOG Mouse Model
Authors: Zhang Yanyan , Patel Satyananda , Wittner Monika , De Botton Stephane , Solary Eric , Vainchenker William , Coppo Paul , Louache Fawzia
Published: October 2019

Abstract 1881

The chemokine receptor CXCR4 favors the interaction of acute myeloid leukemia (AML) cells with their niche but the extent to which it participates to pathogenesis is unclear. Here we show that CXCR4 expression at the surface of leukemic cells allowed distinguishing CXCR4high (25/47; 53%) from CXCR4neg/low (22/47, 47%) AML patients. Leukemic engraftment in NOD/Shi-scid/IL-2Rnull (NOG) mice was observed for both the CXCR4high and CXCR4neg/low groups. When high levels of CXCR4 are expressed at the surface of AML cells, blocking the receptor function with small molecule inhibitors could promote leukemic cell death and reduce NOG leukemia-initiating cells (LICs). Conversely, these drugs had no efficacy when AML cells do not express CXCR4 or when they do not respond to CXCL12. Mechanisms of this anti-leukemic effect included interference with the retention of LICs with their supportive bone marrow microenvironment niches, as indicated by a mobilization of LICs in response to drugs, and increased apoptosis of leukemic cells in vitro and in vivo. CXCR4 expression level on AML blast cells and their migratory response to CXCL12 are therefore predictive of the response to the inhibitors and could be used as biomarkers to select patients that could potentially benefit from the drugs.

Disclosures:

No relevant conflicts of interest to declare.
Double Haploidentical Hematopoietic Stem Cell Transplantation (HSCT) Enhances Anti-Tumor Activity After Transplant
Authors: Budak-Alpdogan Tulin , Bailey Cavan P , Panis Michelle , Sauter Christopher , Agrawal Vikas , Biswas Chandra , Flomenberg Neal , Alpdogan Onder
Published: October 2019

Abstract 1897

We have previously shown that haploidentical (HI) HSCT with low dose donor T-cell infusion provides a survival advantage in tumor bearing mice when compared to parent F1 or MHC-matched transplant models. We suggest that MHC difference in HI-HSCT generates early T-cell clonal activation against the unshared MHC haplotype, which eliminates residual tumor cells that express the unshared MHC haplotype. However, alteration in MHC antigen expression is a significant contributor to tumor escape from graft-versus-tumor (GVT) activity. Recent haploidentical transplant data revealed that uniparental disomy, the loss of the HLA haplotype, is a clinically relevant mechanism of tumor escape that leads to post-transplant leukemia relapse. Murine renal cell carcinoma, RENCA-TGL, cell line normally expresses only H2Kd as a MHC molecule. Therefore, in our haploidentical transplant model, T cell clonal activity is usually restricted against H2Kd molecule only. For circumventing the single haplotype expression of tumor model, we transfected this cell line with a H2Kb expressing vector, pAcGFP-NeoR-H2Kb, and generated stable clones with G418 selection. The clone that has more than 95% H2Kb expression used for in vivo experiments. Both tumor cell lines, i.e. parental and transfected clone, had similar in vivo tumor growth acceptance and growth rate. We then used two different haploidentical donors that were targeting different MHC haplotypes. Lethally irradiated B6D2F1 (H2Kb/d) mice were transplanted with T cell depleted bone marrow (TCD-BM) from either B6C3F1 (H-2Kb/k) (single haplo-1; SH1), or C3D2F1 (H2Kk/d) (single haplo 2; SH2) or both donor mice with low-dose (1×105) T-cells. In some experiments, animals were also injected either H2Kd or H2Kb/d expressing RENCA-TGL cells for the evaluation of GVT activity.

Bone marrow (BM), spleens and thymi were harvested from recipients of single and double HI-HSCT at day 35 and showed similar cellularities. Interestingly, spleen and bone marrow had similar chimerism from both donors in DH-HSCT. There were no early transplant mortality, graft failure, weight loss and GVHD scoring difference among the double or single-haploidentical transplant recipients. In two other sets of experiments, we followed the tumor growth and the survival of tumor bearing mice after transplant. The recipients of DH-HSCT showed a better survival and GVT activity than the recipients of SH-HSCT in RENCA-TGL (H2Kb/d) bearing tumor model. These observations confirmed that MHC targeting plays a prominent role in tumor surveillance, and immune targeting the unshared MHC haplotype with haploidentical transplant induce remarkable survival advantage. Double HI-HSCT provides an unique anti-tumor activity that continues to exert GVT effect, even in case of MHC haplotype loss.

Disclosures:

No relevant conflicts of interest to declare.
Post-Transplant Pulmonary Function Abnormalities Normalize in Survivors by the Second Decade After Allogeneic Stem Cell Transplantation
Authors: Jain Natasha A , Koklanaris Eleftheria K. , Chawla Kamna , Pophali Priyanka A , Klotz Jeffrey K. , Hourigan Christopher S. , Savani Bipin N. , Ito Sawa , Barrett A. John , Battiwalla Minoo
Published: October 2019

Abstract 1967

Following allogeneic stem cell transplant (SCT), late-onset severe pulmonary complications occur in more than 10% of patients and confer an increased risk of mortality in the first few years post transplant. Whether pulmonary repair can occur subsequently is not well characterized. We prospectively collected pulmonary data on 138 (60 female, 78 male) recipients of allogeneic SCT from HLA identical siblings surviving > 5 years. The median age at transplant was 36 years. Indications for transplant were CML (63), AML (31), MDS (19), ALL (12) and others (14). 126 patients received ≥1200 cGy TBI-based conditioning with or without lung shielding, 3 received 400 cGy TBI-based conditioning and 9 patients received no irradiation. The graft was ex vivo T cell depleted in 131 recipients except six who received non-myeloablative SCT. Of the survivors, 26 had no cGvHD, 70 had cGvHD lasting less than 3 years and 42 had prolonged cGvHD requiring systemic immunosuppression for more than 3 years. Of the 17 patients who died beyond 5 years, 4 died of pulmonary causes, including two with lung cancer. Pre-transplant abnormalities (< 80% predicted) in PFTs were found in 15.9% of subjects in the following declining frequency: FVC%, VC, TLC, FEV1, DLCO_Adj (adjusted for hemoglobin and alveolar ventilation) Hb/VA and FEV1/FVC. Baseline (BL) PFTs served as the reference for all subsequent measurements at 5, 10 and 15 years for each survivor. The only parameter showing a clinically significant decline post transplant was DLCO_Adj at the 5-year mark, with subsequent normalization [paired t-test]. The median DLCO_Adj at 5, 10 and 15 years were 88%(p<0.001), 98%(p=NS) and 100%(p=NS) of BL, respectively. Multivariable modeling (forward-stepwise multiple regression) identified the presence of chronic GVHD (p=0.017) and abnormal BL DLCO_Adj (P=0.023) as the only factors associated with the decline in DLCO_Adj at 5 years but excluded smoking, conditioning intensity, TBI dose to the lungs and demographic variables. In conclusion, poor pulmonary function as measured by DLCO_Adj within 5 years of SCT can normalize by 10 years. Chronic GVHD and pretransplant DLCO_Adj are important contributory factors.

Disclosures:

No relevant conflicts of interest to declare.
Are Disparities in Acute Myelogenous Leukemia Due to Poor Prognostic Factors At Diagnosis? A National Study
Authors: Patel Manali I. , Ma Yifei , Mitchell Beverly S. , Rhoads Kim
Published: October 2019

Abstract 2061

Background:

Disparities in Acute myelogenous leukemia (AML) have been described but not studied extensively in adults. Younger age at diagnosis and cytogenetic profiles of t(8;21) and acute promyelocytic leukemia (APL) subtypes of AML have been associated with improved survival from AML but no studies to date have evaluated these factors by race. The purpose of the current study is to evaluate differences by race/ethnicity in age and select cytogenetic factors at diagnosis. The hypothesis is that disparities for minorities will be explained by older age at diagnosis and lower rates of select favorable cytogenetics.

Methods:

Patients with AML were identified in the Surveillance Epidemiology End Results (SEER) database during the years 1999–2008. Kaplan-Meier survival curves were generated to predict survival by race/ethnicity, stratified by age. Multivariable Cox proportional hazard models estimated mortality by race with adjustment for age, gender, year of diagnosis, t(8;21) and APL subtypes.

Results:

A total of 27,252 patients were included in the study. Blacks and Hispanics were diagnosed at younger ages (<61 years) and had higher rates of t(8;21) and APL compared with non-Hispanic Whites (NHW). The overall KM curve demonstrates that NHWs had a worse survival compared to other races/ethnicities. However when KM curves were stratified by age, blacks and Hispanics had worse survival in every age category below age 61. In multivariable models adjusted for age, black patients had an increased risk of death compared to NHWs (HR 1.10 95% CI (1.04–1.16). In multivariable models adjusted for t(8;21) and APL subtypes, there was no attenuation for the disparity. For APL subtype, in particular, there was an increased disparity for blacks (HR 1.11 95% CI (1.05–1.17) and Hispanics (HR 1.05 95% CI (1.01–1.11) compared to NHWs.

Conclusions:

Despite younger age and higher prevalence of favorable cytogenetics, blacks have worse mortality from AML compared to other racial/ethnic groups. Blacks and Hispanics with the APL subtype have worse mortality compared to other racial/ethnic groups. Future studies should investigate other reasons for disparities among black and Hispanics with acute myelogenous leukemia.

Disclosures:

No relevant conflicts of interest to declare.