bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a Stable Thrombotic Core with Limited Access to Plasma Proteins During Thrombus Formation In Vivo
Authors: Stalker Timothy J. , Traxler Elizabeth A. , Diamond Scott L. , Brass Lawrence F.
Published: October 2019

Abstract 2013

Several studies examining thrombus formation in vivo have described the formation of a thrombotic core composed of stably adherent, apparently activated platelets covered by several layers of minimally activated, loosely adherent platelets. Using confocal fluorescence intravital microscopy in mouse cremaster muscle arterioles, we sought to 1) determine whether the stable thrombus core could be defined by the presence of platelet activation markers (e.g. P-selectin); 2) define the spatial and temporal characteristics of the stable thrombus core as it develops; and 3) determine whether the accessibility of plasma proteins to the inner regions of a stable thrombus is limited. We found that P-selectin platelet surface expression is associated with stable platelet incorporation into a growing thrombus following either laser- or wall puncture-induced vascular injury in vivo. The P-selectin positive core originated at the site of vascular injury and subsequently expanded outward into the central region of the developing thrombus with kinetics that were distinct from total platelet accumulation. Further, using two related approaches, we determined that the accessibility of plasma components to the stable thrombus core is limited. In the first approach, fluorescently labeled anti-P-selectin antibody was infused after a stable thrombus had formed (25 minutes after injury), and was found to bind to a monolayer of platelets on the luminal surface of the stable thrombus core, but was unable to penetrate this layer of platelets and bind to platelets in the central region of the thrombus. In the second approach, we directly measured the porosity of thrombi as they evolved by infusing fluorescently labeled dextran to illuminate the plasma in the gaps between platelets. We found that the porosity of the stable thrombus core was significantly decreased as compared to the porosity of the growing platelet mass before the core developed. Taken together, these results demonstrate that a stable core composed of degranulated platelets develops during thrombus formation in vivo with spatial localization and kinetics that are distinct from total platelet accumulation, and that development of this core limits the accessibility of plasma components to the central region of a stable thrombus.

Disclosures:

No relevant conflicts of interest to declare.
The Hepatic Asialoglycoprotein Receptor Regulates Platelet Homeostasis
Authors: Grozovsky Renata , Jansen Gerard , Hoffmeister Karin M.
Published: October 2019

Abstract 2025

The human body produces and removes more than a 100 billion of platelets every day. The mechanisms responsible for platelet homeostasis are subject to speculation since the 1950's. The most popular hypothesis to date has been antibody-mediated clearance, platelet consumption due to massive blood loss and an internal “senescence timer”. We and others have recently demonstrated that sialic acid deficient platelets due to external triggers such as sepsis or chilling are cleared by hepatic asialoglycoprotein receptors (ASGPR) independently of macrophages. Here, we investigated whether loss of sialic acid mediates platelet clearance in vivo. We show that 1) Injection of the specific sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) lengthened the survival of biotinylated platelets by ∼50% (T1/2 of 72h), compared to mock treated (PBS injected) control mice (T1/2 of 49h); 2) Similarly, biotinylated platelet survival in ASGPR-null mice was prolonged by ∼ 50% (T1/2 of 74h) compared to platelet survival in wild type (WT) mice (T1/2 of 48h); 3) ASGPR-null mice have significantly increased platelet counts, compared to WT (p=0.0004) and platelets isolated from ASGPR-null mice are ∼15% smaller than WT (p=0.03); 4) Platelets isolated from ASGPR-null mice showed significant increased in b-galactose exposure (∼50% increase, i.e. decrease of sialic acid), compared to WT, as evidenced by binding of the b-galactose specific lectin (RCA-I). These data show that the ASGPR not only removes desialylated platelets due to sepsis or chilling, but also regulates platelet homeostasis.

Disclosures:

No relevant conflicts of interest to declare.
Dynamic Erythropoietin Receptor Trafficking within Cell Surface, and Intracellular Pools
Authors: Singh Seema , Leu Karen , Mortensen Bruce , Emerson Christine , Pradeep Anamika , Young Peter R. , Schatz Peter J , Green Jennifer M , Wojchowski Don M.
Published: October 2019

Abstract 2046

The erythropoietin receptor mediates pro-erythroblast production; can promote erythrocytosis; and can cytoprotect select non-hematopoietic tissues. For certain key aspects of EPOR biology, knowledge nonetheless remains limited. This includes regulated EPOR trafficking, which we presently investigate using a novel panel of specific rabbit monoclonal anti-EPOR antibodies. In exponentially growing EPO-dependent UT7epo cells, a single high-Mr species was detected at low levels, and EPOR turnover appeared to predominate. The limiting of EPO led to the accumulation of two high Mr EPOR forms, EPOR-68K and EPOR-70K. EPO exposure rapidly converted an indicated cell surface EPOR-70K form to an EPOR-72K tyrosine-phosphorylated species (and to lower Mr fragments). In contrast, EPOR-68K was not rapidly modulated by EPO, and proved to comprise an intracellular, endoglycosidase-sensitive EPOR pool. At the cell surface, EPOR repopulation also was regulated and interestingly was accelerated in response to higher-dose EPO exposure. Constitutive EPOR turnover was observed, but as comparably limited ligand-independent trafficking. EPOR expression in primary erythroid progenitors also was analyzed. Flow cytometry and western blotting each unexpectedly revealed peak EPOR expression at a GPAhigh late erythroblast stage. These studies provide new insight into dynamic, EPO dose-dependent EPOR trafficking, together with an observed sustained expression of the EPOR within maturing primary human erythroblasts.

Disclosures:

Leu: Affymax, Inc.: Employment. Mortensen:Affymax, Inc.: Employment. Young:Affymax, Inc.: Employment. Schatz:Affymax, Inc.: Employment. Green:Affymax, Inc.: Employment.
Risk Factors for Pulmonary Hypertension In Patients with Thalassemia Intermedia
Authors: Karimi Mehran , Musallam Khaled M , Cappellini Maria Domenica , El-Beshlawy Amal , Belhoul Khawla , Daar Shahina , Saned Mohamed-SalahEldin , Temraz Sally , Taher Ali T
Published: October 2019

Abstract 2069

Introduction:

Mechanisms leading to pulmonary hypertension (PHT) in patients with thalassemia intermedia (TI) are still controversial. Moreover, clinical and laboratory characteristics of patients who eventually develop PHT have not been identified. Our aim was to identify factors that characterize TI patients who develop PHT.

Methods:

Data was retrospectively retrieved from the Thalassemia Intermedia Registry, a database of 584 TI patients currently registered at six comprehensive care centers in Lebanon, Italy, Iran, Egypt, United Arab Emirates, and Oman. Institutional review boards at each center approved the study protocol. Two Groups of patients were identified: Group I, TI patients with documented PHT (n=64; mean age 37.3 ± 10.6; 44% males); and Group II, age- and sex-matched TI patients without PHT (n=64; mean age 37.9 ± 11.4; 44% males). Collected data included demographics, laboratory parameters, disease-complications, and received treatments that may influence PHT development and reflected the period prior to PHT occurrence.

Results:

There were no statistically significant differences in mean platelet counts, total or fetal hemoglobin levels between the two Groups. However, mean serum ferritin level was higher in Group I compared to Group II (1233.2 ± 499.2 vs. 654.7 ± 234.5 ng/ml; P=0.01). Moreover, mean nucleated red blood cell (NRBC) count was higher in Group I compared to Group II (354.2 ± 199.2 vs. 214.7 ± 94.5 ×10⋀6/l; P=0.03). A higher proportion of patients were splenectomized (84.4% vs. 46.9%; P<0.001) or had a previous history of thromboembolic events (40.6% vs. 7.8%; P<0.001) in Group I compared to Group II. Conversely, a higher proportion of patients received transfusion (78.1% vs. 56.2%; P<0.001), iron chelation (62.5% vs. 37.5%; P<0.001), or hydroxycarbamide (34.4% vs. 17.2%; P<0.001) therapy in Group II compared to Group I. There were no statistically significant differences in the proportion of patients with heart failure, prothrombotic mutations, or receiving antiplatelet or anticoagulant therapy between the two Groups. Multivariate logistic regression analysis revealed that patients in Group I are more likely to be splenectomized (OR:4.9, 95%CI:1.9-8.5); transfusion-naive (OR:3.5, 95%CI:2.1-6.25); on no hydroxycarbamide (OR:2.6, 95%CI:1.1-5.25) or iron chelation therapy (OR:2.3, 95%CI:1.2-4.25); and have NRBC count >300 ×10⋀6/l (OR:2.59, 95%CI:1.69-6.05) or a previous history of thromboembolism (OR:3.69, 95%CI:2.38-7.05).

Conclusion:

Splenectomy, previous history of thromboembolism, and a high NRBC count characterize TI patients who develop PHT. Transfusion, iron chelation, and hydroxycarbamide therapy deserve evaluation for a protective role against PHT in TI.

Disclosures:

No relevant conflicts of interest to declare.
A Bispecific Protein Targeting the NKG2D Receptor on Natural Killer Cells: In Vitro and In Vivo activity of ULBP2-CEA
Authors: Madlener Marie , Rothe Achim , Jachimowicz Ron , Reiners Katrin S. , Chatterjee Sampurna , Ullrich Roland , Yazaki Paul , Engert Andreas , von Strandmann Elke Pogge
Published: October 2019

Abstract 2095

Natural killer (NK) cells are effector lymphocytes of the innate immune system. NK cell activity is tightly regulated by inhibitory and activating surface receptors to ensure that NK cells recognize and eliminate target cells, whereas healthy cells escape from the NK cell-mediated immune surveillance. The inability of the immune system to recognize and kill malignant target cells has been partly attributed to the ineffective activation of NK cells. It is well accepted, that the activating NK receptor NKG2D (natural killer group 2 member D) plays an important role in tumor rejection. The Immunoligand ULBP2-CEA is a recombinant bispecific protein designed to enhance NK cell-mediated lysis of CEA-expressing target cells. Consisting of the NKG2D-ligand ULBP2 (UL16 binding protein 2) and an anti-CEA single chain antibody, this construct provides the ability of cross-linking NK cells and CEA-expressing cells for specific tumor targeting. CEA (carcinoembryonic antigen) is frequently overexpressed in colorectal and other carcinomas. The binding and specificity of ULBP2-CEA fusion protein was tested in FACS analysis. Transfected human CEA+ murine colon carcinoma cells (MC38-CEA) were incubated with ULBP2-CEA and recombinant NKG2D receptor (NKG2DR). The protein was able to bind both, tumor cells and recombinant NKG2DR simultaneously. No specific cell binding was detectable in control experiments using fusion constructs with another binding moiety (e.g. ULBP2-BB4) or soluble NKG2DR alone. In cytotoxicity assays ULBP2-CEA enhances the susceptibility to NK cell mediated lysis of CEA-expressing cells. CEA+ human colon carcinoma cells (LS174T) were incubated with NK cells isolated from healthy donors in the presence or absence of ULBP2-CEA. The increase of cell lysis was significant at all effector-target ratios compared to the controls demonstrating specific targeted in vitro efficacy of ULBP2-CEA. In order to investigate anti-tumor activity in vivo we use a syngeneic mouse model. MC38-CEA cells were implanted subcutaneously in CEA transgenic C57BL/6 mice and tumor growth was assessed using in vivo bioluminescence imaging (BLI). First results on the in vivo efficacy of ULBP-CEA and effects on the activation status of splenic and peripheral blood NK and T cells upon treatment will be discussed. This syngeneic mouse model is a useful tool to study efficacy and mode of action of ULBP2-CEA in vivo.

Disclosures:

No relevant conflicts of interest to declare.
Shear Induced Reversible Conglomerates of Von Willebrand Factor Networks with Aggregates of Inactivated Platelets - a Fast Switchable Plugging Mechanism
Authors: Reininger Armin J. , Napoleone Marina , Angerer Jennifer , Falah Babak , Schneppenheim Reinhard , Schneider Matthias
Published: October 2019

Abstract 2104

Thrombus growth rate has been reported to be a linear function of very high shear rates beyond 40,000 s-1 (Ku DN, Bioreology. 2007;44(4):273-84), which are thought to occur in severe arterial stenosis. We therefore applied shear rates continuously increasing from 2,000 s-1 to 40,000 s-1 and examined platelet and von Willebrand factor (VWF) interaction in a parallel plate flow chamber under direct visualization. Above a critical threshold of 10,000 to 15,000 s-1 aggregates of inactivated platelets formed when VWF was present in solution and immobilized on the perfused surface (Ruggeri, … Reininger; Blood, 108: 1903–1910, 2006). A new finding was the shear dependent simultaneous formation of VWF strands in and around those platelet aggregates, growing to strands up to several microns thick and up to several tens of microns long, thus forming large VWF networks. Platelets appeared to be enmeshed in the VWF networks but were nevertheless a prerequsite for their formation, mutually fostering each other. Platelet-VWF conglomerates formed at the perfused VWF surface, were constantly rolling in the flow direction, grew in size with increasing shear rate, and were completely reversible below the critical shear rate threshold of 10,000 s-1. VWF networks assembled in whole blood as well as in washed blood cell suspensions reconstituted with VWF. Recombinant full length VWF generated larger VWF networks than a commercial VWF preparation from pooled human plasma. Perfusion of collagen coated surfaces caused assembly of VWF networks anchored to the collagen fibrils and capture of activation-blocked platelets within them at shear rates of 2,000 s-1, leading to residence times of platelet-VWF conglomerates of more than a minute. Newly formed platelet conglomerates could be visualized rolling on the surfaces of already attached conglomerates. When we used a gain of function mutation of VWF, rolling platelet aggregates could be detected at a shear rate as low as 1,500 s-1. We conclude that shear rate activation of VWF leads to reversible platelet-VWF network generation, which may be a crucial mechanism of fast platelet accrual and prolonged arrest times at sites of thrombosis as well as physiological hemostasis.

Disclosures:

No relevant conflicts of interest to declare.
A Retrospective Multi-Center Analysis of Prognostic Factors Determining Outcomes In Patients with Secondary AML
Authors: Mohan Sanjay R. , Maciejewski Jaroslaw P , Tejura Bindu , Erba Harry P , Stone Richard M , Sekeres Mikkael A.
Published: October 2019

Abstract 2139

Introduction:

Secondary AML (sAML), a well recognized poor prognostic factor in AML, is a heterogeneous group of diseases, including both AML evolving from myelodysplastic syndromes (MDS, MDS→AML) and AML following chemotherapy for another malignancy, i.e. treatment-related AML (tAML). These two subtypes of poor risk AML have similar outcomes when treated with the same cytarabine-based chemotherapy regimen, supporting their inclusion into the same clinical category. A combined data set of patients from a single centre and a phase II study was analyzed to determine which patient and disease factors were of prognostic significance in predicting response to treatment. This is a large data set of sAML patients including patients treated with a variety of cytarabine-based induction regimens.

Materials and Methods:

Data from 77 newly diagnosed sAML patients treated with cytarabine-based induction chemotherapy at the Cleveland Clinic between 1997 and 2007 and 88 newly diagnosed sAML patients treated with amonafide + cytarabine in a phase II study were combined. The focus of the analysis was to ascertain the prognostic factors affecting overall survival (OS) from time of diagnosis. Initially, univariate Cox proportional hazard models for each prognostic factor were fitted. Hazard ratios and their associated 95% confidence intervals and Wald test p-values were produced. Prognostic factors were: age at diagnosis (<60 years, ≥60 years), gender (male, female), WBC (<20 x109/L, ≥20 x109/L), peripheral blasts (<20%, ≥20%), bone marrow blasts (<40%, ≥40%), cytogenetics CALGB class (unfavorable, intermediate, favorable, unknown), secondary disorder class (MDS, tAML), prior chemotherapy (yes, no), prior radiotherapy (yes, no). Subsequently a multivariate model was produced using the stepwise selection method and a significance level of 5%, having imputed missing data with mean/median values as appropriate.

Results:

In the combined data set of 165 patients with newly diagnosed sAML, the results for each significant prognostic factor are outlined in the table below.

The final multivariate model (displayed in the table below) determined unfavorable cytogenetics, male gender and advanced age as prognostic factors predictive of worse OS.

Conclusions:

In a combined data set of 178 patients with sAML from 22 US centers, cytogenetics, age and gender were identified as prognostic factors of significance in determining the outcome when treating sAML patients with induction chemotherapy. For patients with these risk factors, additional therapeutic strategies, including early stem cell transplantation, should be considered.

Disclosures:

Tejura: Antisoma: Employment. Sekeres: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.
RANKL Expressed by Acute Myeloid Leukemia Cells Impairs NK Cell-Mediated Immune Surveillance
Authors: Schmiedel Benjamin J , Wende Constantin M , Baessler Tina , Scheible Carolin , Wirths Stefan , Azuma Miyuki , Schneider Pascal , Grosse-Hovest Ludger , Kanz Lothar , Salih Helmut R
Published: October 2019

Abstract 2164

NK cells play an important role in tumor immunosurveillance, especially of leukemia. Their reactivity is governed by various activating and inhibitory molecules expressed by their targets including multiple members of the TNF family. The TNF family member Receptor Activator of NF-κB ligand (RANKL) and its receptors RANK and osteoprotegerin (OPG) are key regulators of bone remodelling, but recently have also been shown to influence progression of hematopoetic malignancies. Here we studied the yet unkown role of the RANK/RANKL molecule system in NK cells and their reactivity against acute myeloid leukemia (AML). Primary leukemia cells from AML patients were found to substantially express RANKL mRNA and surface protein in 75% of the investigated cases (n=40). Reverse signaling via surface-expressed RANKL into AML blasts induced the release of soluble factors including the immunoregulatory cytokines TNF and IL-10, which impaired NK cell anti-tumor reactivity. Moreover, we observed upregulation of RANK on NK cells among PBMC of healthy donors upon exposure to IL-10. This was not caused by direct effects on NK cells, but was rather due to yet unidentified factors released by monocytes among the PBMC upon IL-10 exposure and could be prevented by the activating cytokine IL-2. Furthermore, functional experiments with NK cells and RANKL transfectants or RANKL-negative controls revealed that forward signaling into RANK-expressing NK cells by tumor-expressed RANKL also directly impaired NK cytotoxicity and IFN-γ production. In line, blocking RANK-RANKL interaction using anti-RANKL antibodies or RANK-Fc fusion protein increased cytotoxicity and cytokine production of allogenic NK cells in cultures with RANKL-positive primary AML cells. Our data indicate that RANKL expression enables immune evasion of leukemia cells both by directly inhibiting reactivity of RANK-expressing NK cells and by orchestrating a reciprocal interplay between AML cells, monocytes and NK cells resulting in an immunosuppressive cytokine milieu. Thus, therapeutic modulation of the RANK/RANKL system, e.g. with Denosumab/AMG162, which is presently being evaluated for treatment of both non-malignant and malignant osteolysis, holds promise to reinforce NK reactivity against hematopoietic malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Ara-C Treatment of Acute Myeloid Leukemia Does Not Lead to Prolonged Enrichment of Stem Cells or a Cell Cycle Arrest
Authors: Perry Robin L. , Sarry Jean-Emmanuel , Danet-Desnoyers Gwenn-ael , Carroll Martin
Published: October 2019

Abstract 2178

The major therapeutic barrier in acute myeloid leukemia is chemotherapy resistance. Although most patients will respond to treatment with chemotherapy over 50% of responders will relapse and eventually die of disease. Many hypotheses have been proposed to explain chemotherapy resistance but none of these have lead to new therapies or a complete understanding of the molecular mechanisms of AML chemotherapy resistance. In order to develop an improved understanding of chemotherapy resistance in AML, we have developed a chemotherapy model of human AML in NSG mice. Mice are engrafted with primary AML samples from patients seen at diagnosis or relapse of disease or who demonstrated primary chemotherapy resistance. After demonstrating AML engraftment, mice are treated with cytosine arabinoside (Ara-C) given IP daily for 5 days as a single agent at 10mg/kg daily, 30mg/kg daily, and 60 mg/kg daily which correlates with human dosing. In >75% of mice treated with 10 mg/kg of Ara-C there is a cytoreductive effect at 2 weeks post-treatment with relapse at 4 weeks post-treatment. In all mice treated with 30 mg/kg of Ara-C there was a cytoreductive effect at 2 weeks post-treatment with relapse at 4 weeks post-treatment. In all mice treated with 60 mg/kg of Ara-C there was a cytoreductive effect at 2 weeks post-treatment with relapse 4–13 weeks post-treatment when relapse occurred, demonstrating that there is a dose response relationship in the model in terms of nadir leukemic burden and time to peripheral blood relapse. Two weeks after treatment, there was up to a 50-fold decrease in total AML cell burden in the peripheral blood of mice treated with 30 mg/kg and and up to a 70-fold decrease in the peripheral blood of mice treated with 60 mg/kg of Ara-C. We found no enhancement in quiescent or G0 cells after chemotherapy treatment. We did however, in 1/5 samples tested see a change in phenotype after chemotherapy treatment with an increase in the total number of CD34+38+ cells with a concomitant decrease in CD34-38+ cells. In all other samples tested, there was no change in phenotype after chemotherapy treatment. These results are in contrast to recent studies using a 1 gm/kg dose of Ara-C and analysis at Day 3 following a single treatment. These results have implications for understanding the physiologic response to Ara-C at different doses. We are currently analyzing mRNA expression arrays of AML without or with Ara-C treatment to identify novel mechanisms of chemotherapy resistance. Taken together, this model provides a novel approach for development of new therapies in AML.

Disclosures:

Carroll: Sanofi Aventis Corporation: Research Funding; Kyowa Hakko Kirin Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding.
Methotrexate-Asparaginase-Dexamethasone Regimen for Blastic Plasmocytoid Dendritic Cell Neoplasm treatment
Authors: Vaida Ioana , Royer Bruno , Gruson Berengere , Damaj Gandhi , Harrivel Veronique , Marolleau Jean Pierre
Published: October 2019

Abstract 2194

Blastic plasmacytoid dendritic cell neoplasm (BPDCN), previously named CD4 CD56 haematodermic neoplasia is a rare malignancy, associated with poor prognosis; it affects older patients and has a typical clinical pattern, with initially skin involvement and evolution toward leukaemia. To date, best responses for patients younger than 60 yrs are obtained after induction chemotherapy acute lymphoblastic leukaemia-like and allogeneic bone marrow transplantation (Dalle, BJD 2010); for older patients there is no standardized treatment in first line; the CHOP or CHOP-like regimens don't improve the outcome of these patients. We report our experience in 6 patients with BPDCN treated with Methotrexate-Asparaginase (MTX ASP) steroid regimen, active in lymphoid malignancies.

From august 2006 to December 2009, 6 patients (pts) were newly diagnosed BPDCN according to main immunophenotyping features of this entity: CD4+, CD56+, HLA-DR+, CD123+, absence of B-, T- and myeloid associated markers (Tsagarakis, Leukemia Research, 2010); the mean age was 62 yrs (range, 55–74); 83% were males; 100% of patients presented at diagnosis with cutaneous disease and bone marrow involvement; all patients received the MTX-ASP steroid regimen; 4 pts were treated in 1st line and 2 pts in relapse, after CHOP-like regimen. The treatment schema was: Methotrexate 3g/m2 day 1, L-asparaginase 6000UI/m2, on day 2, 4, 6, 8 and Dexamethasone 40 mg day 1 to 4; the treatment was administered monthly till progression.

Results:

Overall complete remission (CR) rate was 83, 3% after 3 cycles: 5 pts were in CR and one patient had a progressive disease (PD). The 5 responders followed the same regimen until progression (total number of cycles for 6 to 11). The mean CR duration was 7, 8 months. One patient underwent allo HSCT after 7 courses and is still alive. The MTX-ASP regimen was well tolerated without severe haematological and non-haematological toxicity; there was no episode of infection and despite high doses of L-Asparaginase, no deep venous thrombosis was seen.

Conclusions:

The BPCDN treatment is not standardised, mainly due to the rarity of this entity; for younger patients best responses are obtained after allogeneic bone marrow transplantation and induction chemotherapy LAL-like; for older patients the CHOP or CHOP-like regimens don't improve the outcome of these patients. In our experience, methotrexate-asparaginase steroid regimen induces a high rate of CR and seems an interesting treatment option especially for older patients. The treatment toxicity is acceptable. We need to standardize our therapeutic approach in this rare malignancy.

Disclosures:

No relevant conflicts of interest to declare.
Jak2 Plays a Critical and Non-Redundant Role In Hematopoiesis throughout Pre- and Post-Natal Life
Authors: Park Sung , Gnanasambandan Kavitha , Sayeski Peter P.
Published: October 2019

Abstract 2238

In early Jak2 murine models, conventional germline knockout of Jak2 tyrosine kinase resulted in embryonic lethality at E12.5 due to severe defects in hematopoiesis. However, what role, if any, that Jak2 plays in late gestational development and post natal life is currently unknown. To achieve some understanding of this, we utilized a conditional knockout approach that allowed for the temporal deletion of Jak2 at various stages of embryonic and postnatal growth. Specifically, Jak2 conditional knockout mice were produced by crossing estrogen receptor inducible Cre mice (RosaCreER) with loxP inserted Jak2 conditional knockout mice (FL/FL). Injection of the bigenic mice with tamoxifen (TM), a synthetic estrogen receptor modulator, accordingly results in a Jak2 null allele. TM was injected into pregnant dams at E12.5 and into postnatal mice at day 4 and day 60. The animals were then euthanized at E16.5 (late gestation), postnatal at day 18 (an active stage of postnatal development) and day 90 (adult stage), respectively. We found that deletion of Jak2 throughout all stages of murine development resulted in marked hypocellular bone marrow, significantly reduced spleen size, an absence of extramedullary hematopoiesis, and profound anemia. Analysis of peripheral blood samples indicated that the Jak2 null mice have significantly decreased hematocrits characterized by increased numbers of abnormal red blood cells that were either immature or lacking hemoglobin. Furthermore, the number of platelets was also significantly reduced in the null mice when compared to aged matched littermate controls. Overall, these results indicate that Jak2 plays a critical and non-redundant role in hematopoiesis throughout all stages of prenatal and postnatal life.

Disclosures:

No relevant conflicts of interest to declare.
The Optimal Timing for Stem Cell Collection After Induction Therapy for Patients with Multiple Myeloma
Authors: Nazha Aziz , Vogl Dan T. , O'Doherty Una , Mangan Patricia , Cunningham Kathleen , Luger Selina , Porter David L. , Schuster Stephen J. , Gardler Marie , Sell Mary , Siegel Don L. , Stadtmauer Edward A
Published: October 2019

Abstract 2252

Introduction:

High dose chemotherapy and stem cell transplant remains an integral part of the therapy for Multiple Myeloma patients under age of 70. The collection of sufficient number of stem cells for one or more transplant is however sometimes a challenge. Moreover, the optimal timing for stem cell collection after induction chemotherapies is controversial. The standard recommendation is for stem cell collection after 4–6 cycles of non-alkylator regimen, however studies to support this practice are limited.

Material and Method:

We conducted a retrospective analysis of 366 patients who were diagnosed with multiple myeloma and mobilized at the Hospital of University of Pennsylvania between January 2002 and December 2008. Patients who did not meet the initial inclusion criteria were those who had induction regimens containing an alkalytor agent or whose regimens were not well documented and were excluded from futher analysis (85). Every 4 cycles of any non-alkalytor agent was considered to be one treatment session for the purpose of this analysis. 245 patients received 1 or 2 treatment sessions and 36 received > 2. All patients were mobilized with either Cyclophosphamide/G-CSF (CY/G-CSF), Plerixafor/G-CSF (AMD/G-CSF), or G-CSF alone.

Result:

The mean number of collected CD 34+ cells (CD 34+) was 9.22 × 106 CD34+/Kg in the patients who received 1 or 2 sessions and 6.87 × 1106 CD34+/Kg in the patients who received > 2 sessions (P= 0.005). The number of the patients who collected > 6 × 106 CD34+/Kg was 63%(153/246), 53%(19/36) respectively, (p= 0.005). The patients who mobilized with either CY/G-CSF or AMD/G-CSF collected higher number of CD34+ than the patients mobilized with G-CSF alone in both groups. (Table 1, 2.)

The mean number of collected stem cells was 7.14 × 106 CD34+/Kg in the patients who received more than 2 sessions of different regimens and 6.26 × 106 CD34+/Kg in the patients who received > 2 sessions of the same regimen.

Conclusion:

The patients who mobilized after fewer than 8 cycles of non-alkylator agents (2 sessions) collected a higher number of CD 34+ than those with greater than 8 cycles. CY/G-CSF or AMD/G-CSF are similar and superior to G-CSF alone in the more heavily treated patients. The patients who received multiple sessions of the same regimen have similar outcome compared to those who received multiple different regimens suggesting that the duration of the treatment may impact stem cell collection more than the content of the regimen. Prospective studies in this regards are warranted.

Disclosures:

No relevant conflicts of interest to declare.
Adoptive Transfer of EBNA1-Specific T-Cells as a Treatment of Epstein-Barr-Virus Reactivation Following Allogeneic Stem Cell Transplantation
Authors: Icheva Vanya , Opherk Kathrin , Bethge Wolfgang A. , Kayser Simone , Greil Johann , Handgretinger Rupert , Lang Peter , Feuchtinger Tobias F.
Published: October 2019

Abstract 2342

Reactivation of Epstein-Barr-Virus (EBV) after allogeneic stem cell transplantation (SCT) is responsible for significant morbidity and mortality. EBV is also assotiated with the development of some malingancies, such as Burkitt-Lymphoma or nasopharyngeal carcinoma (NPC). In particular, the EBV-induced lymphoproliferative disorder (PTLD) is a rare but severe condition after SCT. PTLD is often associated with insufficient immune responses against EBV in transplant recipients. There is no effective antiviral drug treatment against EBV by now. Given that T-cell immunity is crucial for protection against infection or reactivation of EBV, cellular immunotherapy is a promising therapeutic option. The Epstein-Barr-Virus Nuclear Antigen 1 (EBNA-1) has been shown to contain immunodominant T-cell epitopes with T-cell responses in the majority of the healthy population. Here we report adoptive EBNA-1-specific T-cell transfer in seven pediatric and adult patients with chemorefractory EBV-reactivation after allogenic SCT. Four patients had PTLD and one had metastatic relapse of a NPC. EBNA-1-specific T-cells were isolated from the SCT-donor by using an IFNγ-capture technique. These small T-cell populations were immediately infused to the patient without in vitro expansion steps. The adoptive T-cell transfer contained both, CD4+ T-helper cells and CD8+ cytotoxic T-cells. The patients with a mean age of 20 years were treated with antigen specific T-cells from haploidentical, matched unrelated or matched sibling donor SCT between day 72 and 410 post SCT. The T-cell dose varied from 150–7750 T-cells/ kg. No acute toxicity was observed. In vivo T-cell responses before adoptive T-cell transfer were absent and were detectable in all of the patients within the first weeks after adoptive transfer, associated with a partial clinical and/or virological response to the adoptive T-cell transfer. In three of the patients a second specific T-cell administration was needed to achieve an improvement of the EBV-related condition. PTLD or EBV-infection was not a cause of death in any of the other six patients. In conclusion we could show that adoptive T-cell-immunotherapy is safe, feasible and a promising therapeutic option in patients with EBV- infection and/or PTLD, having the advantage of not being immunosuppressive compared to chemotherapy against PTLD. Infusion of small IFNγ producing EBNA-1-specific T-cell populations resulted in an in vivo expansion of specific T-cells. Emergence of in vivo T-cell responses was closely associated with a clearance or reduction of the viral load.

Disclosures:

No relevant conflicts of interest to declare.
Older Patients with AML Undergoing Matched Related Allogeneic Transplantation Have Significantly Older Donors but Equivalent Survival as Patients Receiving Unrelated Donor Transplants
Authors: Hill Brian T. , Rybicki Lisa , Bolwell Brian J. , Dean Robert , Pohlman Brad , Tench Shawnda , Sobecks Ronald , Andresen Steven , Copelan Edward A. , Kalaycio Matt
Published: October 2019

Abstract 2356

Introduction:

Allogeneic stem cell transplantation (SCT) is commonly offered to patients with acute myeloid leukemia (AML) over the age of 55 with good performance status. For this patient population, potential sibling donors are necessarily older and have more comorbidities than siblings of younger patients. It is not known whether the advanced donor age of older patients outweighs the beneficial effect of having a sibling donor. To address this question, we retrospectively analyzed data from 62 consecutive patients who received allogeneic SCT at the Cleveland Clinic from 1990–2009.

Methods:

Inclusion criteria were: age ≥ 55 years, diagnosis of AML, and history of myeloablative or nonmyeloablative allogeneic SCT from related or unrelated adult donors. 31 patients underwent transplantation from matched related donors (30 siblings, 1 cousin). 31 patients received unrelated donor transplants. The median age was 59 for patients with related and unrelated donors (P=0.89). There was an equal percentage of males (64.5%) in both groups (P=1.0). There was an equivalent distribution hematopoietic cell transplant comorbidity index scores of low, intermediate and high for patients with related and unrelated donors (38.7%, 37.1% and 24.2% vs. 38.7%, 32.3%, and 29.0% P = 0.65). 45.2% of patients with related donors underwent myeloablative conditioning vs. 54.8% for patients with unrelated donors (P =0.61).

Results:

Sibling donors were significantly older than unrelated donors [median 59 years (range 41–75) vs. 36 (range 24–58), P < 0.001]. The incidence of acute and chronic graft-versus-host disease (GVHD) was similar in both groups. At five years, the cumulative incidence of relapse (43.2% vs. 38.7%, P = 0.88), non-relapse mortality (48.1% vs. 48.8%, P =0.91) and overall survival (17.2% vs. 24.1%, P =0.88) were similar for recipients of matched related and unrelated donor transplants. Older donor age was not predictive of death in univariate or multivariate analysis. Kaplan-Meier estimates of overall survival for recipients of related and unrelated donor transplants are shown.

Conclusion:

Patients with AML 55 years or older who underwent related donor transplantation had significantly older donors but equivalent survival when compared to patient who underwent unrelated donor transplantation. Advanced donor age should not be a contraindication to allogeneic SCT for older patients with AML.

Disclosures:

No relevant conflicts of interest to declare.
Methylation of Specific Microrna Genes In MLL-Rearranged Infant Acute Lymphoblastic Leukemia: - Major Matters at a Micro Scale -
Authors: Stumpel Dominique JPM , Schotte Diana , Lange-Turenhout Ellen AM , Schneider Pauline , Seslija Lidija , Menezes Renee X de , Marquez Victor E , Pieters Rob , Boer Monique L. Den , Stam Ronald
Published: October 2019

Abstract 2483

MLL-rearranged Acute Lymphoblastic Leukemia (ALL) in infants (<1 year) represents one of the most aggressive types of childhood leukemia. In order to develop more suitable treatment strategies, a firm understanding of the biology underlying this disease is of utmost importance. MLL-rearranged ALL displays a unique gene expression profile, partly explained by erroneous histone modifications. We recently showed that t (4;11)-positive infant ALL is also characterized by pronounced promoter CpG hypermethylation. Here we investigated whether this widespread hypermethylation also affected microRNA (miRNA) expression.

We performed CpG methylation analyses at 122 miRNA loci using Differential Methylation Hybridization (DMH), and miRNA expression analyses using quantitative real-time PCR on primary t (4;11)-positive infant ALL samples (n= 22) and normal pediatric bone marrows (n= 7). We identified 11 miRNAs that were markedly down-regulated in t (4;11)-positive infant ALL as a consequence of CpG hypermethylation. Seven of these miRNAs were re-activated after exposure to the de-methylating agent Zebularine. Interestingly, 5 of these miRNAs had already been associated either with the MLL gene or with leukemic MLL fusions. For one of the remaining miRNAs, i.e. miR-152, we demonstrate that high degrees of methylation strongly correlate with a poor clinical outcome. Moreover, we identified MLL and DNA methyltransferase 1 (DNMT1) as potential target genes for miR-152.

Thus, genome-wide DNA methylation in MLL-rearranged infant ALL not only inactivates numerous protein-coding genes, but also affects several miRNA genes. While inhibition of methylation by Zebularine to certain extents re-activates gene expression, re-activation of miRNAs by this agent restores the suppression of associated target genes. As demethylating agents exert their functions by covalently trapping DNMT1 to the DNA, re-activation of miR-152 by Zebularine further supports demethylation by targeting DNMT1 expression.

In summary, our data demonstrates an important role for genome-wide DNA methylation in suppressing miRNA expression and provides additional grounds to initiate efficacy testing of demethylating agents in MLL-rearranged ALL in vivo.

Disclosures:

No relevant conflicts of interest to declare.
Identification and Modeling of TEL-AML1 (ETV6-RUNX1) Molecular Signature In Acute Lymphoblastic Leukemia
Authors: Miles Gregory , Rajagopal Gunaretnam , Strair Roger , Sabaawy Hatem E
Published: October 2019

Abstract 2500

Despite high cure rates for childhood ALL, treatment failure remains a formidable problem. TEL-AML1 (ETV6-RUNX1), the product of t (12;21) translocation, is the most common cytogenetic abnormality in childhood cancer, and is detected in 25–30% of precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL) in children, and in small percentage of adult ALL. Considerable experimental and clinical evidence indicate that the TEL-AML1 fusion is insufficient by itself for leukemic transformation. First, monozygotic twins had the same prenatal TEL-AML1 sequence, but different latency and/or no leukemia development. Second, we have previously established transgenic zebrafish expressing TEL-AML1. These TEL-AML1 transgenic zebrafish have hematopoietic stem cell (HSC) expansion, and develop pre-B ALL at low penetrance, and after prolonged latency. Similarly, transgenic mice expressing TEL-AML1 did not develop pre-B ALL, but showed HSC expansion. Third, retroviral-mediated TEL-AML1 gene transfer into murine HSCs resulted in a preleukemic state, and the incidence of leukemia increased with additional mutation. Here, we report on the identification of a TEL-AML1-specific leukemic signature in ALL cases, and modeling of these mutations in dual and compound transgenic zebrafish. First, we performed meta-analyses of ÓALL compendiumÕ of molecular signatures and expression profiles of 990 ALL cases from six studies, measuring the expression of 14,145 genes in 475 arrays. The normalized data were imported into the Ingenuity Pathway Analysis (IPA) software to identify TEL-AML1-related pathways. We determined a TEL-AML1-specific signature that was organized into modules that are induced or suppressed, based on involvement in several biological pathways comprising the hallmarks of cancer. These analyses identified modules of cell differentiation, cell proliferation, apoptosis, autophagy, cell cycle regulation, and lymphocyte development as the most common modules associated with TEL-AML1. The signature was confirmed using additional microarray analyses, in combination with Q-PCR, and western blotting from the same cases. Similar analyses of marrow cells from transgenic zebrafish with ALL identified 1,128 upregulated and 936 down-regulated genes with 27 genes common with the human TEL-AML1 signature. We next isolated the zebrafish homologues of several induced signature genes to generate dual and compound transgenic zebrafish, and to investigate the effects of their overexpression on TEL-AML1 leukemia development. Analyses of established transgenic fish demonstrate leukemia-enhancing effects of signals within the proliferation and cell cycle modules. These studies provide a model to understand the role of the TEL-AML1 fusion and the secondary mutations required for leukemia development, and might present a rational for leukemia combination therapy.

Disclosures:

No relevant conflicts of interest to declare.
A Novel PCR-Based Gene-Specific Chromatin Condensation Assay
Authors: Vatolin Sergei , Shahper Khan Nazeer , Reu Frederic J.
Published: October 2019

Abstract 2508

We developed a method for efficient, quantitative, and site specific characterization of chromatin activity in myeloma cell populations. This method is based on modified salt extraction of chromatin directly from intact cells and PCR amplification of site of interest. The whole procedure (cell collection, chromatin isolation, PCR, and data analysis) takes from 4 to 6 hours and requires only 100–200 cells per probe. The general idea behind this procedure is that most active chromatin is sensitive to mild denaturing conditions which make its DNA accessible for primers, whereas inactive chromatin remains “closed”. No antibodies, fixation, viral or plasmid-based constructs, or nuclease treatments are involved. The method corroborated findings on ChIP and pyrosequencing of bisulfite modified DNA for CDKN2A (p16) regulatory regions. After optimization with decitabine it correctly identified other epigenetically active agents in a small scale screen. In summary, we have developed a method for detection of gene specific chromatin changes that requires less than 1% of cells needed for other epigenetic assays, is cheaper, faster, and detects various changes of chromatin. We expect it will revolutionize drug screening for epigenetic reactivators and silencers, epigenetic profiling, and elucidation of chromatin modifying pathways in myeloma and other cancers.

Disclosures:

No relevant conflicts of interest to declare.
Differentiating Human ES and Patient Derived IPS Cells to Hematopoietic Stem/Progenitor Cells.
Authors: Rönn Roger Emanuel , Moraghebi Roksana , Parker Aaron , Lutz Margaret , Berggren Travis , Woods Niels-Bjarne
Published: October 2019

Abstract 2628

The ability to generate hematopoietic stem cells (HSC's) from patient derived induced pluripotent stem (iPS) cells, would enable the generation of an unlimited supply of HLA matched transplantable cells for the treatment of both hematological disorders and malignancies. The goal of this project is to find the molecular determinants of human ES and iPS cell differentiation to transplantable HSC's. We have developed an advanced ES/iPS-to-blood differentiation protocol that mimics embryonic development towards the hematopoietic lineage. Current progress has resulted in up to 84% of human CD45+ hematopoietic cells (mean 41 ± 16%, n = 7) and with significant numbers of hematopoietic progenitor cells (CD45+CD34+, 29 ± 4%, n = 6) as reported by FACS analysis. Moreover, the numbers of hematopoietic progenitor cells generated (mean 20 ± 1 for a keratinocyte derived iPS (kiPS)), as measured by colony forming unit assays were comparable to numbers obtained from fresh whole blood umbilical cord mononuclear cell isolates (mean 35 ± 2) on a per 10.000 CD45+ input cells. We now plan to overexpress key factors predicted to support the generation and maturation of bona fide HSC's with repopulating ability. For this we have generated a tet-regulatable lentiviral based gene expression system to, out of a pool of over 30 genes, express up to 5 cDNA's of interest simultaneously. By clonal selection of lenti-transduced ES and iPS cells we have generated novel pluripotent lines that carry the tet-transactivator gene (m2rtTA), capable of efficient differentiation into blood. FACS analysis of these lines show blood generation of 30.7% and 11.8% of cells positive for CD45 for the human ES line Hues3 and the kiPS line respectively, with progenitor levels (CD45+CD34+) at 8.8% and 20.2%. These values are similar in efficiency to that of untransduced sister lines from which they were derived. These lines strongly express genes of interest following lenti-viral transduction and the addition of Doxycyclin (as reported by GFP). It is our goal to show successful reconstitution with in vitro generated HSC's, and ultimately pave the way for therapeutically relevant HSC's for treating hematological malignancies.

Disclosures:

No relevant conflicts of interest to declare.