bluuebunny/crossref_metadata_2025 huggingface dataset:
Drug Metabolites Formed in Vivo Promote the Clearance of Human Platelets by Metabolite-Dependent Antibodies in NOD/SCID Mice
Authors: Bougie Daniel W. , Nayak Dhirendra , Aster Richard H.
Published: October 2019

Abstract 1083

Drug induced immune thrombocytopenia (DITP) is a relatively common and often serious complication of treatment with many drugs. It can be extremely helpful clinically to identify the responsible agent, especially in patients taking multiple drugs. It is sometimes possible to demonstrate a drug-dependent antibody (DDAb) that binds to platelets only when drug is present but results are often negative even in patients with a history that strongly favors DITP. An important reason for this is that a drug metabolite rather than the parent drug can be the sensitizing agent. Because drugs often have many metabolites, most of which are not readily available, identification of the metabolite-specific, DDAbs can be very difficult.

Animal models for studying immune clearance of human cells have not been available because of naturally occurring xenoantibodies. However, recent studies have shown that destruction of human platelets by human antibodies can be studied in the NOD/SCID mouse, which lacks xenoantibodies (Boylan et al 2009). Metabolism of many drugs is similar in mice and humans. We have shown that mice injected with acetaminophen and naproxen produce metabolites that promote in vivo destruction of human platelets by DDAbs specific for metabolites of these drugs (Bougie et al 2010). These findings suggested that the NOD/SCID mouse might be a useful screening tool to identify putative DDAbs specific for metabolites of other drugs in patients suspected of DITP.

To detect metabolite-specific antibodies, mice are transfused with human platelets suspended in test serum. After collecting a baseline blood sample to define the relative number of human platelets in each mouse, the implicated drug is injected (intraperitoneal). Drug metabolites made by the mouse diffuse into the circulation and shorten the survival of human platelets if a metabolite-specific, platelet-reactive antibody is present in the test serum. For a result to be considered “positive” for DDAbs, human platelet survival should be unaltered in mice given test serum alone and normal serum plus drug and should be significantly shortened in mice given test serum plus drug (Figure).

Findings made to date with 94 archived serum samples from DITP suspects exposed to 5 widely used medications are summarized in Table 1. No platelet-reactive antibodies were detected in vitro using unmodified drug. However, 13 patient samples caused accelerated clearance of human platelets when mice were injected with the implicated drug (2 furosemide, 6 clopidogrel, 2 pantoprazole, 3 ibuprofen).

These findings suggest that DITP resulting from sensitization to drug metabolites may be much more common than has been thought and indicate that the NOD/SCID mouse model provides a highly efficient tool to screen candidate sera for metabolite-specific, platelet-reactive antibodies.

Disclosures:

No relevant conflicts of interest to declare.
Thrombin Generation Mediators and Markers in Sepsis Associated Coagulopathy and Their Modulation by Recombinant Thrombomodulin
Authors: Hoppensteadt Debra , Cunanan Josephine , Khan Hussein , Kaul Inder , Osawa Yutaka , Fareed Jawed
Published: October 2019

Abstract 1131

Severe sepsis remains the most common cause of death in critically ill patients and thrombin plays a crucial role in the pathogenesis of sepsis associated disseminated intravascular coagulation (DIC). The purpose of this study was to profile prothrombin fragment (F1.2), thrombin antithrombin complex (TAT) and d-Dimer (DD) throughout the course of hospital stay in subjects identified with sepsis. Plasma samples from seven hundred and fifty eight patients enrolled in the ART-123 study, a phase 2b, international, multicenter, randomized controlled trial, separated into treatment and placebo groups were analyzed for various parameters using ELISA methods. Plasma levels of F1.2, DD, and TAT were recorded at several time points following administration of recombinant thrombomodulin or placebo and the results are tabulated as the median values, in the following tables. All parameters in both treatment groups showed a decrease from the baseline values at day 14. In the group treated with thrombomodulin the median F1.2 levels demonstrated a marked decrease on day 14 exhibiting a 50% decrease, whereas the F1.2 levels fluctuated throughout the study period and showed slight change on day 14, in the placebo group. Although both groups showed a gradual decrease in the DD, the group treated with thrombomodulin demonstrated a stronger decrease over the 14 day period compared to the placebo treated group. While the data was widely scattered, these results show that DIC represents a hypercoagulable state along with other hemostatic abnormalities and the activation of the inflammatory process. Modulation of these activation processes through such targets as DD, F1.2 and TAT may play an important regulatory role in the pathogenesis of sepsis associated coagulopathy. Moreover, this study validates the hypothesis that thrombomodulin down regulates the thrombin generation mediators/markers in sepsis associated DIC.

Median Value For Patients Treated with Placebo in the DIC Study Parameter Baseline Post Day1 Pre Day 3 Post Day 3 Day 7 Day 14 F1.2 (pmol/L) 304.96 304.73 392.90 372.75 328.34 290.19 DD (ng/ml) 4669.5 4825.5 4610 4378.5 3740 3468 TAT (ng/ml) 7.66 6.95 7.28 6.73 6.31 6.04

Median Value For Patients Treated with Recombinant Thrombomodulin in the DIC Study Parameter Baseline Post Day1 Pre Day 3 Post Day 3 Day 7 Day 14 F1.2 (pmol/L0 278.33 240.96 293.42 281.98 228.82 142.59 DD (ng/ml) 5152.50 4465.00 3726.00 3289.00 2785.00 3275.50 TAT (ng/ml) 9.03 6.49 5.99 5.77 5.96 7.53

Disclosures:

Kaul: Asahi Kasei Pharma America Corporation: Employment. Osawa:Asahi Kasei Pharma America Corporation: Employment.
Structural Comparability Between Recombinant FVIII-Fc and Its Isolated FVIII and Fc Constituents
Authors: Kulman John , Tsutakawa Susan , Bou-Assaf George , Berkowitz Steven , Light David R. , Mezo Adam , Wang Deping , Whitlow Marc , Culyba Liz , Tainer John , Peters Robert
Published: October 2019

Abstract 1135

rFVIIIFc is a recombinant fusion protein comprising human B domain-deleted (BDD) coagulation factor VIII covalently attached to the Fc domain of human immunoglobulin G1 (IgG1). rFVIIIFc is currently in a phase 3 clinical study as a novel long-lasting FVIII molecule designed to potentially reduce dosing frequency and provide prolonged protection from bleeds for patients with hemophilia A receiving prophylaxis or on-demand treatment, or undergoing surgery. In this study, we evaluated the structural comparability of BDD rFVIII and rFVIIIFc using small angle X-ray scattering (SAXS) and hydrogen/deuterium exchange (HDX) mass spectrometry. SAXS analysis of BDD rFVIII and rFVIIIFc, performed at the Advanced Light Source Synchrotron, provided the electron pair radius distribution of each molecule and accurate but inherently low resolution structural information. Both BDD rFVIII and rFVIIIFc were monomeric in solution. BDD rFVIII had a radius of gyration (Rg) of 39.7 Å, compared with a Rg of 38 Å calculated from its published atomic coordinates (2R7E.pdb). rFVIIIFc had a larger Rg of 51 Å, which was consistent with a simple computational model of BDD rFVIII tethered to Fc that predicted an Rg of 50 Å. HDX mass spectrometric analysis yielded 94% coverage of the BDD rFVIII component and 87% coverage of the Fc component. Rates of deuterium exchange within peptides derived from each of these components in the context of the rFVIIIFc fusion protein were very similar to those of the isolated BDD rFVIII and Fc constituents. Taken together, the results of SAXS and HDX analyses support the conclusion that fusion of the Fc moiety to BDD rFVIII does not alter the higher order structure of either component.

Disclosures:

Kulman: Biogen Idec: Employment. Bou-Assaf:Biogen Idec: Employment. Berkowitz:Biogen Idec: Employment. Light:biogenidec: Employment. Mezo:Biogen Idec: Employment. Wang:Biogen Idec: Employment. Whitlow:Colabrativ Inc.: Employment; Biogen Idec: Consultancy. Culyba:Biogen Idec: Employment. Peters:Biogen Idec: Employment.
Real Life Efficacy and Safety of Rivaroxaban for Acute VTE Treatment – First Results of the Prospective Noac Registry (NCT01588119)
Authors: Gelbricht Vera , Koehler Christina , Werth Sebastian , Haensel Ulrike , Schreier Thomas , Eulitz Maria , Tittl Luise , Beyer-Westendorf Ines , Beyer-Westendorf Jan
Published: October 2019

Abstract 1159

Background:

In the EINSTEIN study rivaroxaban (RX) has been found to be at least as effective and safe as warfarin in treatment of acute deep vein thrombosis (DVT), which lead to approval of RX in many countries. However, patients in RCT‘s present a selected population treated under a strict protocol and followed for a short period of time. Consequently, efficacy and safety of new oral anticoagulants (NOAC) need to be confirmed in unselected patients in daily care.

Objectives:

To evaluate the efficacy, safety and management issues of rivaroxaban anticoagulation in acute VTE in daily care.

Patients and methods:

A network of 200 physicians from private practice and hospitals enrol patients in the prospective NOAC registry. Inclusion criteria are: 1) indication for NOAC anticoagulation >3 month; 2) age > 18 years; 3) written informed consent; 4) availability for follow-up. No Exclusion criteria apply. In the registry, up to 2000 patients will receive prospective follow up (FU) by phone visits at day 30 day and quarterly thereafter to collect efficacy and safety data.

Results:

Until July 31th 2012, 938 patients were registered. Of these, 105 patients received RX for acute VTE treatment (demographic data in table 1). In our registry, the population receiving acute VTE treatment is older than the EINSTEIN population (62.2 vs. 55.8 years). Most patients are treated for major VTE (proximal deep vein thrombosis (DVT) pulmonary embolism (PE)), but about 20% are treated for isolated distal DVT.

The results of 1-, 3- and 6-months FU are shown in table 2. Until now, no recurrent VTE or VTE-related death occurred. Two patients (1.9%) experienced a major vascular event (acute limb ischemia) at the beginning of NOAC therapy and one patient experienced a minor vascular event (tachyarrhythmia). Bleeding events were frequent (22.3%) but only five patients (4.8%) experienced major bleeding events, one of which was a fatal intracranial bleeding. Three patients (2.9%) died during FU (1 intracranial bleed, 2 of underlying diseases). At 6 month, only eight patients (7.8%) were switched to other anticoagulants and one patient (1.0%) had an unscheduled discontinuation of anticoagulant therapy.

Conclusion:

In unselected patients in daily care, acute VTE treatment with RX is effective and safe with low rates of cardiovascular or bleeding events during the first 180 days of treatment.

Disclosures:

Werth: Bayer Healthcare: Honoraria. Beyer-Westendorf:Bayer Healthcare: Bayer provided a grant to support the NOAC registry in part Other, Honoraria; Boehringer Ingelheim: Boehringer provided a grant to support the NOAC registry in part, Boehringer provided a grant to support the NOAC registry in part Other, Honoraria; Bristol Myers Squibb: Honoraria; Pfizer: Honoraria.
The Balance of Positive and Negative Effects of TGF-Beta Signaling Regulates the Hemangioblast Development in Human Pluripotent Stem Cells
Authors: Bai Hao , Xie Yin-Liang , Gao Yong-Xing , Cheng Tao , Wang Zack Z
Published: October 2019

Abstract 1220

Derived from mesoderm precursors, hemangioblasts are bipotential common progenitors of hematopoietic cells and endothelial cells. The regulatory events controlling human hemangioblast development are largely unknown. Our previous study demonstrated that CD34 progenitors from human embryonic stem cells (hESCs) contain a population of cells that give rise to hematopoietic cells and endothelial cells. In this study, we established a serum-free and feeder-free system to investigate the signals that direct differentiation of human pluripotent stem cells (hPSCs), including hESCs and induced-pluripotent stem cells (hiPSCs). The hemangioblast development in hPSCs was established in three major steps: i) the formation of uniformed embryoid bodies (EBs) in hanging-drops, ii) the development of multipotential mesoderm precursors in cultivation of EBs, and iii) the generation of CD34+CD31+VE-cadherin+ progenitors, which have hemangioblast potential to give rise to both hematopoietic and endothelial cells (via hemogenic endothelium). The hESC-derived hematopoietic cells expressed CD41, CD61, CD117, CD71, and CD235a. The endothelial cells were CD31+ and VE-cadherin+ cells. We found that inhibition of Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) by Y27632 was critical for hanging-drop EB formation. Transforming growth factor beta (TGF-beta) is a key regulator of hematopoiesis with potent inhibitory effects on progenitor and stem cell proliferation. The expression of TGF-beta was increased during hPSC differentiation. Inhibition of TGF-beta signaling by the TGF-beta inhibitor, SB431542, significantly decreased the efficiency of CD34+ progenitors, suggesting a crucial role of TGF-beta signaling in hPSC differentiation. To further investigate when TGF-beta signaling affects hPSC differentiation to CD34+ progenitors, we added TGF-beta inhibitor at different stages of hPSC differentiation. We found that TGF-beta signaling is required for the early mesoderm induction. Inhibition of TGFb signaling before hemangioblast commitment during PSC differentiation significantly decreases the frequency of hemangioblasts (CD34+/CD31+/VE-cadherin+ cells) and the expression of mesoderm markers, such as Brachyury, Tie1, EOMES, and MIXL1. On the other hand, inhibition of TGF-beta signaling after mesoderm induction increases the number of CD34+/CD31+/VE-cadherin+ cells. EdU cell proliferation assays indicated that the proliferation of CD34+ progenitors were not affected by SB431542, suggesting that TGF-beta signaling suppresses hemangioblast generation. Our study demonstrated that TGF-beta signaling has the double-edged effect on hPSC differentiation to hemangioblasts.

Disclosures:

No relevant conflicts of interest to declare.
Development of a Multi-Step Leukemogenesis Model of MLL-Rearranged Leukemia Using Humanized Mice
Authors: Moriya Kunihiko
Published: October 2019

Abstract 1311

Mixed-lineage-leukemia (MLL) fusion oncogenes are involved in de novo and secondary therapy-related acute leukemia in humans. To understand the pathogenesis of MLL-rearranged leukemia, several murine models for this disease have been established. However, the leukemia derived from murine hematopoietic stem cells (HSCs) may not be fully comparable with human leukemia. In vivo studies, in which primary human leukemia cells were transplanted into immunodeficient mice, provided us significant advances to understand the pathogenesis of human leukemia. However, these models are not suitable for studying physiological leukemogenesis because the initiation and progression processes are hard to be evaluated. Therefore, a new in vivo experimental model for analyzing leukemogenesis derived from primary human HSCs and by genetic hits is required.

Here we developed a humanized murine model for human leukemia by transplanting human cord blood-derived CD34-positive HSCs transduced with an MLL-AF10 oncogene and second hits into a supra-immunodeficient mouse strain, NOD/Shi-scid, IL-2Rγ−/− (NOG) mice.

Injection of the MLL-AF10-transduced HSCs into the liver of NOG mice enhanced multilineage hematopoiesis, however, MLL-AF10 alone could not induce leukemic transformation. Accumulating evidence points to a multistep pathogenesis for leukemia development and progression. In the multi-genetic step models of leukemogenesis, particularly in the two-hit model of leukemia, the initial genetic hit often leads to abnormal cell differentiation (Type II mutation), while subsequent mutations may activate specific signaling pathways that are involved in cell growth, such as the Ras/MAP kinase pathway (Type I mutation). Because active mutations in Ras genes are often found in MLL-related leukemia, we next transduced constitutively active form of K-Ras mutant along with the MLL-AF10 oncogene. Enforced expression of the mutant K-Ras alone was insufficient to induce leukemia. Eight weeks after transplantation, all of the recipient mice transduced both MLL-AF10 and mutant K-Ras developed acute monoblastic leukemia (the M5 phenotype in French-American-British classification). We could also observe organ infiltration in liver and spleen in vivo that mimicked clinical symptoms observed in human leukemia.

We thus successfully established a murine model for human MLL-rearranged leukemia derived from human HSCs. The present model is a useful experimental platform for the multi-step leukemogenesis of human leukemia and for new therapeutic approaches that should be evaluated in vivo.

Disclosures:

No relevant conflicts of interest to declare.
Illuminating Clonal Dynamics of a Leukemia Cell Line Using Viral Barcoding and Illumina Deep Sequencing Technology
Authors: Porter Shaina , Highnam Gareth , Mittelman David , Porteus Matthew
Published: October 2019

Abstract 1410

Leukemias are generally clonal in origin, but develop significant heterogeneity as they expand and acquire genetic and epigenetic alterations. Detecting this heterogeneity and tracking the dynamics and evolution of clones within a population are important for our understanding of these cancers and the phenomena of drug resistance and relapse. Attempts to track the contributions of individual cells to the clonality of large populations, however, have been constrained by limitations in sensitivity and complexity. We have created an efficient and high throughput method to overcome these limitations by harnessing the power of viral marking and next generation sequencing technology, allowing us to track the clonal contributions of many thousands of cells with minimal perturbations to the population as a whole. K562s are a common patient-derived CML line featuring rapidly proliferating, highly aneuploid cells. We stably and heritably marked individual cells via the lentiviral-mediated insertion of one of ∼14,000 random, non-coding, 20 basepair DNA “barcodes.” Using Illumina next-generation sequencing we determined the relative frequency of every barcode, thus directly measuring the contribution of each clone to the overall population over 90 population doublings. We anticipated that the the K562 cell line, while very highly aneuploid, would have stable clonal dynamics because it has been grown in culture for years, thus allowing potentially dominant clones to reach equilibrium. We found, however, that K562 cells continue to undergo dramatic changes in clonal representation. As expected, in analysis of our clonally marked population, there was a broad distribution of clones. After culturing under ideal conditions for 90 population doublings, we found that a single clone now represented 9% of the population and that less than 100 clones now represented more than 50% of the population, a massive clonal skewing after just a brief period of time. Cell lines are often assumed to be a homogenous group of identical clones, a seemingly reasonable assumption considering the years of passaging/subculturing. However, our results show that ongoing genomic and/or epigenomic instability leads to proliferative instability, resulting in unexpected clonal dynamics and rapid clonal evolution. These results have profound implications for the experimental use of cell lines, as well as broad applications for clonal analysis of leukemias, other malignancies, and stem cells.

Disclosures:

No relevant conflicts of interest to declare.
Type of Bleeding Among Patients with Nonvalvular Atrial Fibrillation in a Large Managed Care Population
Authors: Deitelzweig Steven , Pinsky Brett , Buysman Erin , Lacey Michael , Makenbaeva Dinara , Wiederkehr Daniel , Graham John
Published: October 2019

Abstract 2073

Background:

Risk of bleeding is an important consideration among patients with nonvalvular atrial fibrillation (NVAF), and different anatomical sites may carry greater risk for bleeding.

Objective:

To describe the incidence of critical anatomical site bleeding events among patients with NVAF in the pre-NOAC (novel oral anticoagulation) era.

Methods:

Administrative claims data were used for this retrospective study. Adults with healthcare claims related to atrial fibrillation (ICD-9-CM 427.31) between Jan 2005 and Jun 2009 but no evidence of valvular disease were included. Patients were followed until the earliest of death, disenrollment from the health plan, or 30 Jun 2010. Bleeding events in the follow-up period were considered major if they were associated with any of the following: inpatient care, blood transfusion, decreased hemoglobin or hematocrit, physician guided medical or surgical treatment, intracranial bleed, or death. Anatomical bleeding sites were identified based on diagnosis codes.

Results:

The mean (SD) age of the study sample (N=48,260) was 67 (13) years and 62.2% of the patients were male. Mean (SD) follow-up duration was 802 (540) days (median 673 days). Over 105,419 patient-years, the critical site with the highest rate of major bleeding events was the gastrointestinal tract, followed by intracranial bleeding (TABLE).

Conclusions:

Gastrointestinal, intracranial, and pericardial bleedings were the most frequently observed major bleedings in the pre-NOAC era. Patients with NVAF have a high rate of major gastrointestinal bleedings.

Disclosures:

Deitelzweig: Bristol-Myers Squibb/OptumInsight: Research Funding, Speakers Bureau. Pinsky:OptumInsight: Employment. Buysman:OptumInsight: Employment. Lacey:OptumInsight: Employment. Makenbaeva:Bristol-Myers Squibb: Employment, Equity Ownership. Wiederkehr:Pfizer: Employment, Equity Ownership. Graham:Bristol-Myers Squibb: Employment, Equity Ownership.
Hedgehog Ligands and Receptors Are Deregulated in Myelodysplastic Syndromes
Authors: Xavier Juliana M. , Dias Amanda I , Latuf-Filho Paulo , Traina Fabiola , Vassallo José , Saad Sara T Olalla
Published: October 2019

Abstract 3817

Background:

The Hedgehog pathway has an important role in self-renew of normal and leukemic stem cells and is upregulated in myeloid leukemias, however there are no studies regarding the Hedgehog pathway in myelodysplastic syndromes (MDS). Hedgehog ligands (Sonic hedgehog [SHh], Indian hedgehog [IHh], and Desert hedgehog [DHh]) are produced by stromal cells and bind to the receptor Patched (PTCH). This binding causes activation of Smoothened (SMO) receptor, resulting in downstream transcription of target genes.

Aim:

To evaluate Hedgehod pathway components in MDS.

Methods:

Bone marrow (BM) samples were collected from 39 MDS (25 low risk, 14 high risk-WHO 2008) patients and 26 healthy donors (HD). Relative expressions of PTCH and SMO were obtained by Real Time PCR. For immunohistochemistry, BM biopsies were collected from 21 MDS (17 low risk and 4 high risk) patients and 7 megaloblastic anemia (MA) were used as control. The BM sections were stained with antibodies for DHh, SHh and c-Kit and the percentage of stained nucleated cells was based on an average of 4 high-powered fields.

Results:

Hedeghog receptors PTCH and SMO were overexpressed in MDS BM cells compared to normal BM as follows; PTCH: [median (min-max)] healthy donors= 2.23 (0.42–9.22); low risk= 6.09 (0.41–25.28); high risk= 3.97 (0.42– 21.01); HD vs low risk and HD vs high risk p= 0.02; SMO: healthy donors= 2.33 (0.00–16.11); low risk= 14.79 (1.89– 41.93); high risk= 34.44 (8.04– 164.28)]; HD vs low risk and HD vs high risk p<0.001. Immunohistochemistry assays showed a higher expression of Hedgehog ligands in MDS bone marrow compared to control. For DHh, the number of stained cells in low risk MDS were higher than in MA [MA = 102(84 –183), low risk= 196(69 – 317) high risk 159(131 – 236)]; MA vs low risk p=0.018 and MA vs high risk p= 0.11. Furthermore, SHh staining was 2 and 2.5 fold higher in low and high risk MDS, respectively, compared to MA [MA= 47(18 – 74), low risk 98.25(53 – 129), high risk 116.25 (93 – 125)] MA vs low risk p= 0.001, MA vs high risk p=0.01. Similar to SHh, c-Kit staining was higher in MDS cases as follows: MA= 95.3(61 – 114), low risk= 106(57–136), high risk= 95.3(61 – 114). Interestingly, a high correlation was found between c-kit and DHh (p=0.002, r=0.63) or c-kit and SHh (p=0.001, r=0.7).

Conclusion:

To our knowledge, this is the first study of the Hedgehog pathway in MDS. We observed overexpression of Hedgehog receptors and ligands. Furthermore, there is a correlation between these ligands and a marker for hematopoietic stem/progenitors cells. According to these data, we propose that deregulation of Hedgehog in MDS may occur in abnormal progenitors found in MDS bone marrow.

Disclosures:

No relevant conflicts of interest to declare.
Thrombin Generation Capacity of Prothrombin Complex Concentrate in an in Vitro Dilutional Model
Authors: Spronk Henri M.H. , Rossaint Rolf , Yvonne Henskens M.C. , van Oerle Rene , Cate Hugo Ten , Grottke Oliver
Published: October 2019

Abstract 4380

Background:

There is a growing use of prothrombin complex concentrates (PCCs) for the treatment of trauma-induced coagulopathy, which is addressed to their propensity to increase thrombin generation. Despite considerable differences in composition of commercially available PCCs, there is lack of data investigating the procoagulant capacity of different PCCs.

Methods:

The vitamin K-dependent coagulation factors, heparin, and antithrombin were assessed in five commercially available PCCs. The procoagulant potential of the PCCs was assessed in plasma and whole blood from 4 healthy donors by means of classical coagulation assays, thrombin generation assay and thromboelastometry. In order to reflect coagulopathy, whole blood was diluted with 20, 40, 60, and 80% Ringer's lactate solution.

Results:

The five different PCCs were characterised by comparable levels of factors II, VII, IX and X (all around 20–30 IU/mL), whereas the heparin (0 to 17.6 IU/mL) and antithrombin (0.06 to 1.29 IU/mL) levels were remarkably different between manufactures. In vitro dilution of blood induced a prolongation of the PT and aPTT, and attenuation of thrombin generation and ExTem induced thromboelastometry. Overall, non- or low-heparin containing PCCs restored the in vitro dilutional coagulopathy, whereas PCCs containing heparin has an anticoagulant effect. The thrombin generation assay showed to be the most sensitive method for assessment of PCC effects.

Conclusions:

This study shows that most available PCCs are not balanced regarding their pro-and anticoagulants. The effect of measured differences in thrombin generation among different PCCs require further investigations to elaborate the clinical meaning in the treatment of trauma induced coagulopathy.

Disclosures:

No relevant conflicts of interest to declare.
An Intact Immunoglobulin IgAλ Multiple Myeloma Patient Exhibiting Light Chain Escape
Authors: Kraj Maria , Kruk Barbara , Warzocha Krzysztof , Szczepinski Andrzej , Endean Kelly , Harding Stephen
Published: October 2019

Abstract 5008

A 48 year old man was referred to the Institute of Hematology and Transfusion Medicine, Warsaw, Poland in April 2008 with anemia (Hemoglobin; 10. 4 g/dl) and mild renal impairment (eGFR; 75. 4 mL/min/1. 73m2). An initial diagnostic monoclonal protein screen (serum protein electrophoresis (SPE), serum immunofixation electrophoresis (IFE) and serum free light chain (FLC) analysis) revealed an IgAλ monoclonal protein (0. 8g/dL) with monoclonal serum FLC and an abnormal serum FLC κ/λ ratio (0. 0001; RI, 0. 26–1. 65). A bone marrow biopsy at that time confirmed 60% involvement of monoclonal λ - restricted plasma cells; a bone survey did not detect any osteolysis. The patient was diagnosed with multiple myeloma (MM) (ISS stage I, Durie and Salmon stage IA) and was initially treated with 6 cycles of vincristin, doxorubicin and dexamethasone (VAD). The patient responded well to the induction treatment and subsequently underwent a successful autologous stem cell transplantation (ASCT).

The patient was monitored for 3 years subsequent to the ASCT with both serum and urine electrophoresis, serum FLC analysis (Freelite) and heavy chain/light chain (HLC) immunoassays (Hevylite). Sixteen months following the ASCT the dFLC (involved λ FLC– uninvolved κ FLC) concentration began to increase, the FLC κ/λ ratio became abnormal with a trace of λ Bence Jones protein (BJP) detected by urine IFE. However, both SPE and IFE were normal and the HLC ratio (IgAλ/IgAκ) was within the normal range. During the next 9 months the dFLC continued to increase and a λ BJP could now be clearly detected on the urine IFE. 27 months following the ASCT the patient sustained a pathological fracture of the tibiae and was referred to our centre 4 months later. At this point, the dFLC concentration was highly elevated (3168 mg/L) with a λ BJP detectable by both serum and urine IFE. However, there was no detectable monoclonal intact immunoglobulin by serum IFE or HLC analysis, indicating disease relapse by a separate FLC clone; referred to as light chain escape (LCE). A bone marrow biopsy revealed 15% involvement of λ restricted plasma cells; this time a bone survey identified osteolysis. The patient was diagnosed with progression of multiple myeloma and received 6 cycles of bortezomib, cyclophosphamide and dexamethasone (VCD regimen). He responded well to treatment and 3 years following the ASCT achieved a CR as indicated by a normalized κ/λ FLC ratio, negative immunofixation with 1–3% bone marrow plasma cells. The patient is now well and able to continue with normal life.

In this case study the increase in the dFLC levels was the first indication of disease progression and highlights the importance of monitoring intact immunoglobulin MM patients with serum FLC immunoassays for early detection of LCE.

Disclosures:

Endean: The Binding Site Group Ltd: Employment. Harding:Binding Site: Employment.
Thrombelastographic Demonstration of Nitrite Inhibition of Platelet Function
Authors: Park Ji-Won , Piknova Barbora , Nghiem Khanh , Lozier Jay N. , Schechter Alan N.
Published: October 2019

Abstract 5148

We have recently shown that nitrite ions in blood will inhibit platelet aggregation and activation as measured by aggregometry and flow cytometric analysis of P-selectin under partially deoxygenated conditions. We now show that these phenomena can be measured by thrombelastographic analysis, an alternative method which supplies additional parameters of clotting, using whole blood from healthy donors. Nitric oxide (NO) can be generated by endogenous nitric oxide synthase (NOS) as well as by a serial reduction pathway in which nitrate and nitrite are converted to NO via several enzymatic and nonenzymatic mechanisms. We have previously shown that nitrite anions at 0. 1μM inhibit platelet activation and aggregation in the presence of red blood cells through their reduction to NO and this effect was enhanced by deoxygenation. In the present study, we examined the influence of nitrite on the clotting properties of blood to investigate how nitrite may affect overall clotting processes by modulating platelet function. We measured major clotting parameters such as reaction time (R, minute of initial fibrin formation), angle (velocity of clot growth), and maximum amplitude (MA, clot strength) using thrombelastograph hemostasis analyzer (TEG5000®) in whole blood diluted with plasma to yield 20% Hct. Both the NO donor (DEANONOate) and nitrite showed inhibitory effects on clotting parameters resulting in delayed R, decreased angle, and reduced MA in a dose dependent manner. However, the sources of NO did not change any TEG parameters in plasma. These results suggest that this new clinical method may be used to follow NO inhibition of platelet-induced blood clotting and that the physiological effect of factors which determine NO bioavailability, such as endogenous levels of blood and tissue nitrite, could be used as biomarkers for predicting hemostatic potential.

Disclosures:

Schechter: NIH: Employment, co-inventor of NIH patent on use of nitrite in disease, co-inventor of NIH patent on use of nitrite in disease Patents & Royalties.
Novel Method to Study Mouse Bone Marrow Endothelial Cells in Vivo and in Vitro
Authors: Feng Yuxin , Liu Ming , Guo Fukun , Liu Wei , Sampson Leesa , You Li-Ru , Kuan Chia-Yi , Zheng Yi
Published: October 2019

Abstract 617

Self-renewal, differentiation, and proliferation of hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) are maintained in a complex microenvironment of the adult bone marrow (BM). BM endothelial cells (ECs) have been proposed to be a key component of HSC and LSC niche. However, in contrast to the well-developed culture system of human ECs, current work of murine BM endothelial cells is hindered by a lack of mouse bone marrow endothelial cell primary culture and suitable assay methods to clearly define murine BMEC functionality in vivo and in vitro, which limits genetic and mechanistic studies by using mouse models. To establish an in vivo approach to study the EC function in adult mice, a strain of Tie2-CreER transgenic mice was generated to allow conditional and inducible manipulation of BMECs by Cre recombinase expression under the Tie2 promoter. In vivo lineage tracing was achieved in a Tie2-CreER/TD-tomato or -EGFP reporter mouse strain. Upon a four day Tamoxifen injection regimen, TD-tomato or EGFP reporter was readily visualized in bone marrow vasculature that colocalizes with CD31+ ECs as determined by immunostaining. FACS analysis of Tie2-CreER/EGFP reporter mice showed that the EGFP+ cells in the BM were exclusively in the CD45- VEGFR2+ and CD31+ cell fraction, with no EGFP+ cells being detectable in the CD45+ hematopoietic lineages or osteoblast/stroma cell fractions, suggesting that the Tie2-driven CreER expression is limited to the endothelial lineage in the adult BM. Next, we developed an in vitro method to culture and assay the mouse BMECs functionally. An in vitro selection process allowed us to establish a primary BM cell culture condition that permitted functional expansion and maintenance of mouse BMECs in long-term tissue culture, yielding homogenous CD45- cells expressing endothelial markers CD31, CD34 and VEGFR2. These cells formed capillary-like structures in 2-demensional and 3-demensional tubes/capillaries, and showed TD-tomato reporter color when derived from the Tamoxifen induced Tie2-CreER/TD-tomato mouse BM. They showed expected adhesion and migration activities and morphology of ECs. Lineage chasing assays using isolated CD45+ and CD45- BM cells from the Tie2-CreER/Td-tomato mice demonstrated that the BMECs in our culture system, bearing the Tie2-promoter driven TD-tomato color and CD31+ marker, were exclusively derived from CD45- non-hematopoietic lineage. Taken together, we have established a faithful assay method for studying murine BM EC functions in vivo and in vitro, allowing the tracking and genetic manipulation of adult BM ECs in mice and in culture. The method can be useful for delineating molecular and cellular mechanisms of BMEC regulation and EC-mediated BM niche function, and may have value in testing anti-angiogenic activities of anticancer drugs in animal models.

Disclosures:

No relevant conflicts of interest to declare.
Clinical Factors Predictive Of Myelofibrotic Evolutions In Patients With Essential Thrombocythemia and Polycythemia Vera
Authors: Tiribelli Mario , De Marchi Federico , Barraco Daniela , Marin Luciana , Codarin Erika , Buttignol Silvia , Damiani Daniela , Fanin Renato
Published: October 2019

Introduction

Evolution to myelofibrosis (MF) represents a relatively rare but always severe event in patients with essential thrombocythemia (ET) and polycythemia vera (PV). Few reports have focused on the clinical and biological features at diagnosis of ET and PV that correlate with progression to MF.

Aims and Methods

We retrospectively studied a series of patients with post-ET and post-PV MF and compared with a group of ET and PV patients with a long follow-up without myelofibrotic evolution, with the aim to identify prognostic factors for MF. Forty-three patients with post-ET (n=29) and post-PV (n=14) MF followed at our institution were compared with 125 ET and 75 PV patients with at least 9 years of follow-up without evolution. Diagnosis of ET and PV was confirmed according to WHO criteria (including JAK2 analysis, performed since 2006 and study), evolution to MF was defined according to IWG-MRT proposed criteria. The following parameters, available for all patients at diagnosis of ET or ET, were taken into consideration to find prognostic risk factors for myelofibrosis: age, platelet (PLT) count, hemoglobin (Hb) and hematocrit (Hct) levels, white blood cell (WBC) count. Statistical analyses were conducted using Student t test.

Results

Median time from diagnosis of ET/PV and progression to MF was 156 months (range: 29-314). Comparing baseline characteristics of patients who evolved to MF and those who did not, we did not found any significant correlation. Mean data at diagnosis for patients with (n = 43) or without (n=200) subsequent evolution to MF were as follow: age 52.1 vs 53.1 years (p=0.79), Hb 15.4 vs 15.7 g/dl (p=0.59), Hct 47.2 vs 47.1% (p=0.67), WBC 9.8 vs 9.1 x 109/l (p=0.11), PLT 713 vs 689 x 109/l (p=0.87). Also when considering only the 29 post-ET MF and the 125 ET patients, there was no clinical feature present at diagnosis that could foresee a future myelofibrotic evolution. Conversely, in the 14 post-PV MF and 75 PV patients, progression to MF was predicted only by higher WBC count (11.4 vs 9.3 x 109/l, p=0.046), while no correlation was found with age, Hb, Hct or PLT [Table 1].

Conclusions

Concordant with some previous reports, our data suggest a possible role of leucocytosis as an adverse risk factor for progression to MF in patients with PV, though not in ET. Other clinical characteristics present at diagnosis, such as advanced age, anemia or polycythemia and thrombocytosis do not seem to be associated with higher risk of fibrotic evolution in patients with myeloproliferative neoplasms.

Disclosures:

No relevant conflicts of interest to declare.
Phase 1b Study Of Otlertuzumab (TRU-016), An Anti-CD37 ADAPTIR™ Protein, In Combination With Rituximab In Patients With Previously Untreated Chronic Lymphocytic Leukemia (CLL)
Authors: Maddocks Kami , Pagel John M. , O'Brien Susan , Byrd John C. , Stromatt Scott , Awan Farrukh T
Published: October 2019

Background

CD37 is a tetraspanin protein expressed on the surface of normal and transformed B-cells across a wide range of maturational stages and demonstrates death signaling via SHP1. Otlertuzumab is a CD37-specific therapeutic protein built on the ADAPTIR™(modular protein technology) platform that has shown significantly greater direct killing of CLL cells than rituximab and higher levels of Fc-mediated cellular cytotoxicity of CLL cells than either alemtuzumab or rituximab in pre-clinical models. This phase 1b trial was conducted to evaluate the safety and efficacy of otlertuzumab in combination with rituximab in patients with previously untreated CLL.

Methods

Patients with untreated CLL that required treatment, had adequate organ function, ECOG ≤2, and absolute neutrophil count ≥800/μL were eligible. Patients were ineligible for chemotherapy as first-line therapy due to patient age, comorbidity, or patient refusal of chemotherapy Patients received otlertuzumab (20 mg/kg) weekly by IV infusion for two 28-day cycles then once a month for 4 months. Rituximab (375 mg/m2 the first dose then 500 mg/m2) was administered after otlertuzumab by IV infusion on the same schedule. Safety was evaluated using CTCAE and IWCLL 2008 Grading scale for Hematologic Toxicity in CLL Studies. Response was determined using the 1996 NCI and the 2008 IWCLL Criteria.

Results

24 patients have been treated. Patient characteristics and adverse events are shown in the table. The majority of the patients have not completed 6 cycles of therapy. The preliminary response by investigator assessment using NCI criteria at the last assessment is an ORR 88% (21/24); 2 patients have had PD. Six patients have had CT scans and bone marrow assessments 3 months after their last dose of study drug; by IWCLL criteria 1 has a CR, 4 have a PR and 1 has SD. The patient with a CR was MRD negative by five color flow cytometry of bone marrow aspirate. One patient discontinued therapy for a systemic inflammatory response. Severe (Grade 3 or 4) neutropenia was reported in 13% of patients; the incidence of severe infections was low (8%). Serious adverse events were reported for 4 patients: pneumonia, systemic inflammatory response, and deep vein thrombosis in 1 patient; and lymph node pain, worsening sinusitis, and fever in 1 patient each.

Conclusions

The preliminary response rate with otlertuzumab in combination with rituximab is promising. Follow-up is ongoing and the IWCLL response rate will be presented.

Disclosures:

O'Brien: Emergent Product Development: Research Funding. Stromatt:Emergent Biosolutions: Employment. Awan:Lymphoma Research Foundation - Research Funding: Research Funding, Speakers Bureau; Spectrum Pharmaceuticals, Inc. - Speakers bureau: Research Funding, Speakers Bureau.
Microrna-150 Inhibits Tumor Invasion and Metastasis By Targeting The Chemokine Receptor CCR6 In Advanced Cutaneous T-Cell Lymphoma
Authors: Ito Mitsugu , Teshima Kazuaki , Ikeda Sho , Watanabe Atsushi , Nara Miho , Kitadate Akihiro , Yamashita Junsuke , Oshima Koichi , Sawada Kenichi , Tagawa Hiroyuki
Published: October 2019

MicroRNA (miRNA)s are a class of small regulatory RNA molecules that play important roles in tumor development by pairing with the 3' untranslated region (UTR) of target messenger RNAs to repress their productive translation. Thus although the genes responsible for T-cell lymphomas remain largely unknown, in some lymphoma subtypes, miRNA dysregulation may contribute to tumorigenesis. For example, altered expression of various oncRogenic or tumor suppressive miRNAs has been identified in lymphomas/leukemias and solid tumors We show here that a tumor suppressive miRNA, miR-150, is significantly downregulated in advanced cutaneous T-cell lymphoma (CTCL), and that the downregulation is strongly associated with tumor invasion and metastasis. Inoculation of cutaneous T-cell lymphoma cell lines into NOD/Shi-scid IL-2γnul mice led to CTCL cell migration to multiple organs, but this invasion/metastasis was substantially reduced in miR-150-transfected CTCL cells due to direct downregulation of CCR6, a specific receptor for the chemokine CCL20. MiR-150 thus appears to negatively regulate the interaction between CCL20 and CCR6 in CTCL cells, thereby inhibiting autocrine CTCL cell metastasis. CD4+ T helper cells are divided into TH1, TH2, TH17, and TH22 subsets. Among these, the TH22 subset produces only IL-22, while TH17 cells produce both IL-17 and IL-22. Normally, IL-22 activates CCL20 transcription by binding to the IL-22RA1/IL-10RB receptor, which is not expressed in lymphoid organs or lymphocytes. In the present study, we further found that both IL-22 and its receptor subunit, IL-22RA1, are aberrantly overexpressed in advanced CTCL, but IL-17 is not expressed, suggesting CTCL is derived from the TH22 subset. In the presence of continuous upregulation of CCR6 accompanied by downregulation of miR-150, IL-22 activation could lead to continuous CCL20-CCR6 interaction in CTCL cells and, in turn, autocrine metastasis to distal organs. This is the first report demonstrating an invasion/metastasis mechanism in advanced CTCL.

Disclosures:

No relevant conflicts of interest to declare.
EZH2 Inhibitor DZNep Induces Apoptosis In Adult T-Cell Leukemia/Lymphoma Cells By BCL2 Suppression Via Regulation Of Mir-181a
Authors: Daisuke Sasaki , Hiroo Hasegawa , Hiroaki Taniguchi , Imaizumi Yoshitaka , Naoki Uno , Yoshitomo Morinaga , Miyazaki Yasushi , Katsunori Yanagihara
Published: October 2019

Background

Adult T-cell leukemia/lymphoma (ATL) is a mature T-cell neoplasm originating from human T-cell leukemia virus type-1 (HTLV-1) infected cells. Its clinical behavior differs among patients and is sub-classified into 4 sub-types: smoldering and chronic as indolent subtypes, and acute and lymphoma as aggressive subtypes. Prognosis for patients with the aggressive subtypes is very poor with standard chemotherapy, thus a new therapeutic approach is urgently needed. Previously, we reported that EZH2, a part of the PRC2 complex, not only methylates histone, but also serves as a recruitment platform for DNA methyltransferases that methylate the promoter regions of target genes (tumor suppressor gene and miRNAs) and is overexpressed in ATL cells. We found that ATL cell lines were sensitive to DZNep (3-deazaneplanosin A), which has been shown to deplete EZH2 expression, and their proliferation was attenuated. In the present study, we clarified which target genes and miRNAs are involved in DZNep-induced ATL cell death.

Results

We performed microarray analyses using the ATL cell lines KK1, SO4, LMY1, and KOB to compare their gene expression profiles between cells treated and untreated with DZNep. The results showed that BCL2 transcripts in ATL cell lines were commonly suppressed by DZNep. In accordance with those findings, quantitative PCR analysis revealed that BCL2 transcripts were suppressed in DZNep-treated as compared to untreated ATL cell lines. We also confirmed that EZH2 and BCL2 protein expressions were clearly suppressed in the ATL cell lines by DZNep. These findings indicated that DZNep suppresses BCL2 expression at the transcriptional level in ATL cells. In addition, we performed another set of microarray analyses for miRNA expression profiles using primary ATL cells obtained from ATL patients and CD4 positive T-cells from healthy volunteers. The ATL cells showed decreased expression levels of several miRNAs, such as miR-101, miR-126, and miR-181a. Importantly, miR-181a has been shown to be regulated by EZH2, while miR-181a is a candidate negative regulator of BCL2 expression. Quantification of miR-181a transcripts to determine whether miR-181a is induced by DZNep showed that miR-181a was up-regulated in ATL cells by DZNep. These results strongly support the notion that miR-181a is suppressed by EZH2 and that BCL2 expression is regulated by miR-181a in ATL cells. Together, our findings indicate a unique apoptotic pathway of BCL2 suppression via miR181a and that the EZH2 inhibitor DZNep may become a novel therapeutic agent for ATL.

Disclosures:

No relevant conflicts of interest to declare.