bluuebunny/crossref_metadata_2025 huggingface dataset:
A Sense Oligonucleotide Specifically Reverses the Prothrombin Depletion and Anticoagulation Caused by ISIS 401025, an Antisense Oligonucleotide Targeting Prothrombin RNA in Mice.
Authors: Zhao Chenguang , Monia Brett P. , Siwkowski Andy , Freier Sue , Witchell Donna , Zhang Hong
Published: October 2019

The ability to safely and effectively neutralize anticoagulant activity is of particular importance in the case of long acting drugs. While antisense oligonucleotides (ASOs) benefit from their slow clearance rate from liver (t1/2 approx 10 days in mice) which permits infrequent dosing, the potential necessity to reverse ASO drug activity presents a challenge. ISIS 401025 is a second generation ASO targeting prothrombin RNA that has previously been shown to reduce thrombin generation and prolong PT and aPTT as a result of depleting prothrombin mRNA levels in mice. To explore the possibility of developing an “antidote” strategy for ASO-mediated anticoagulation, we designed a sense oligonucleotide, ISIS 405277, complementary to the ISIS 401025 sequence and examined its ability to reverse the effects of prior treatment with ISIS 401025 in mice. A PT-INR of 3.0 was established after 3 weeks of treatment with ISIS 401025 at a weekly dose of 60 mg/kg, corresponding to a reduction of prothrombin mRNA transcript levels of 96%. A single injection of the sense oligonucleotide, ISIS 405277, resulted in a dose-dependent reversal of INR to 1.6, 1.1, and 0.9 three days after injection of 30, 60, and 90 mg/kg, respectively. Corresponding prothrombin transcript levels in liver tissue were returned to 18.4%, 27.8%, and 38.9% of normal levels, respectively. Results from a study designed to determine the kinetics of reversal of anticoagulation indicated that a 50% reduction of PT-INR 4.3, established following 3 weeks of treatment with ISIS 401025, required 11 days in the absence of subsequent treatment, while the reduction was achieved in 2.2 days following a single administration of 90 mg/kg of sense oligonucleotide. When mice were pretreated with an ASO targeting prothrombin that was not complementary to the sense oligonucleotide (ISIS 40527), sense oligonucleotide treatment was unable to reverse either target depletion or anticoagulation, indicating a sequence-specific antidote effect. These results demonstrate for the first time that subsequent administration of a sense strand oligonucleotide can neutralize ASO mediated target RNA degradation in animals, and demonstrates its potential utility in reversing ASO-based activity for anticoagulation.
A Role for Mismatch Repair Factors in the FA-BRCA Pathway.
Authors: Williams Stacy A. , Wilson James B. , Thomashevski Andrei , Jones Nigel J , Kupfer Gary
Published: October 2019

Abstract 1095

Poster Board I-117

Introduction

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, congenital abnormalities, and an increased risk for cancer and leukemia. Components of the FA-BRCA pathway are thought to function in the repair of DNA interstrand crosslinks (ICLs). Central to this pathway is the monoubiquitylation and chromatin localization of two FA proteins, FANCD2 and FANCI. Recent reports implicate mismatch repair factors in the repair of ICLs and have shown that FANCJ interacts with the MutLαa complex. Here we show that FANCD2 binds several mismatch repair proteins in vivo and that MSH2 is required for the monoubiquitylation and chromatin localization of both FANCD2 and FANCI.

Methods

Cell lines used: HeLa, human lung carcinoma cell line H1299, FA-A cell line GM6914 and corrected cell line GM6914 + Flag-FANCA, FA-D2 cell line PD20 and corrected cell lines PD20+Flag-FANCD2 and PD20+FANCD2 K561R, human endometrial adenocarcinoma cell line HEC59 (MSH2-deficient) and corrected cell line HEC59+Ch2, and human colon carcinoma cell line HCT116 (MLH1-deficient) and corrected cell line HCT116+Ch3. Cells were treated with the crosslinking agent mitomycin C (MMC). Immunoprecipitation was used to demonstrate the interaction between FANCD2 and MSH2, MLH1, and MSH3. Survival assays were performed by crystal violet staining and extraction. Chromatin loading of FANCD2 and FANCI was determined by cellular fractionation and western blot.

Results

Through chromatographic purification of FANCD2-containing protein complexes, we identified MSH2 and MLH1 as FANCD2-interacting proteins. Immunoprecipitation using HeLa cell extracts confirmed the interaction between FANCD2, MSH2, MSH3, and MLH1 in vivo. These interactions are all induced upon damage with a DNA crosslinking agent and MSH2 specifically interacts only with the monoubiquitylated form of FANCD2. Additionally, the FANCD2-MSH2 interaction requires ATR, but not ATM, BRCA1, MSH3, or ERCC1/XPF. Human cells lacking MSH2 display increased sensitivity to mitomycin C as compared to their corrected counterparts. FANCD2 and FANCI monoubiquitylation is also greatly diminished in these cells, while cells lacking MLH1 show no effect. Cellular fractionation of MSH2-deficient cells shows that FANCD2 and FANCI are not efficiently loaded onto chromatin after treatment with DNA-damaging agents, while MLH1-deficient cells again show no effect. Interestingly, while knockdown of either MSH2 or FANCD2 in H1299 cells results in increased sensitivity to MMC, double knockdown of both proteins corrects this sensitivity on par with controls.

oConclusions

These data suggest that mismatch repair proteins play a key role in the activation of the FA-BRCA pathway, likely through recognition of the DNA lesion. Understanding this role could lead to the development of new therapies for the treatment of patients both with FA and cancer.

Disclosures

No relevant conflicts of interest to declare.
DOWN'S Syndrome Acute Lymphoblastic LEUKEMIA: A HIGHLY Heterogeneous DISEASE DRIVEN by an Aberrant CRLF2/JAK2 Cooperation – A REPORT FROM the Ibfm-STUDY GROUP.
Authors: Hertzberg Libi , Vendramini Elena , Ganmore Ithamar , Cazzaniga Giovanni , Schmitz Maike , Shiloh Ruth , Chalker Jane , Iacobucci Ilaria , Shochat Chen , Zeligson Sharon , Cario Gunnar , Stanulla Martin , Strehl Sabine , Russel Lisa J , Harrison Christine , Bornhauser Beat , Yoda Akinori , Rechavi Gideon , Bercovich Dani , Borkardt Arndt , Kempski Helena , Kronnie Geertruy te , Bourquin Jean-Pierre , Domany Eytan , Izraeli Shai
Published: October 2019

Abstract 11

Children with Down's Syndrome (DS) have an increased risk for acute lymphoblastic leukemia (ALL). We have previously identified somatic mutations in R683 of JAK2 in 20% of DS-ALLs (Bercovich et al, Lancet 2008;372:1484). However the cooperating cytokine receptor and other molecular features have been obscured. Here we report that gene expression analysis reveals that DS-ALL is a highly heterogeneous disease not definable as unique ALL subtype. However, 62% (33 of 53) of the DS-ALL samples analyzed were characterized by aberrant expression of the type I cytokine receptor CRLF2 caused by two types of genomic aberrations. CRLF2 encodes one chain of the heterodimeric receptor of the TSLP cytokine involved in inflammation and lymphoid development and has been recently reported to be involved in translocations to the IgH locus or micro-deletions in 3% of ALLs in children without DS. We have confirmed its cell surface expression on primary DS-ALL cells by Flow cytometry analysis. Consisting with a possible role of CRLF2 as a cytokine receptor, all specimens with the R683 mutated JAK2 had high CRLF2 expression. We further show that CRLF2 and R683 mutated JAK2 cooperate in conferring cytokine independent growth of pro-B cells. Furthermore in 3 of 23 DS-ALL patients with wt. JAK2 we identified a novel activating somatic mutation in CRLF2 replacing the juxta-membrane F232 with Cystein. Mutated CRLF2 but not wild type CRLF2 conferred cytokine independent growth of BaF3 cells associated with phosphorylation of STAT5. Aberrant expression of CRLF2 was also associated with a younger age at diagnosis and worse prognosis. Intriguingly, bioinformatic analysis revealed that gene expression of DS-ALLs is enriched with DNA damage and BCL6 responsive genes suggesting the possibility that DS is associated with B-cell lymphocytic genomic instability leading to the genomic aberrations activating CRLF2. Thus DS confers increased risk for genetically highly diverse ALLs with frequent somatic anomaly in CRLF2, whose higher expression further selects for the activating mutations in itself or in JAK2 (figure). Our data also suggests that the majority of DS children with ALL may benefit from therapy blocking the CRLF2/JAK2 pathway.

Disclosures:

No relevant conflicts of interest to declare.
Population Pharmacokinetics of Cyclophosphamide in Patients with Thalassaemia Major Undergoing HSCT Shows Body Weight, CYP450, GST and ALDH Polymorphisms as Covariates Explaining Inter-Individual Variation.
Authors: Balasubramanian Poonkuzhali , Panetta John Carl , Desire Salamun , Velayudhan Shaji R , Mathews Vikram , George Biju , Viswabandya Auro , Chandy Mammen , Srivastava Alok
Published: October 2019

Abstract 1182

Poster Board I-204

Cyclophosphamide (Cy) in combination with busulfan is an important component of myeloablative conditioning regimen used prior to hematopoietic stem cell transplantation (HSCT) for both malignant and non-malignant conditions. We have previously reported up to 20 fold inter-individual variation in the pharmacokinetics (PK) of Cy in patients with beta thalassaemia undergoing HSCT [Blood (ASH Annual Meeting Abstracts), Nov 2004; 104: 99]. PK parameters of Cy have been shown to be associated with regimen related toxicity and outcome of transplant. To explain the basis of the inter-individual variation in Cy PK, we have developed a population PK model. We analyzed the PK of Cy in consecutive children with beta thalassaemia major who received HSCT from HLA identical matched sibling donor at the Christian Medical College, Vellore from 2001 till 2004. A total of 900 cyclophosphamide concentration measurements from 55 patients were included and correlated with age, sex, body weight and 10 polymorphisms in enzymes involved in the metabolism or biotransformation of Cy namely GST A1, M1, T1, P1, CYP2B6, CYP2C9, CYP2C19 and ALDH genes. Non-linear mixed effects modeling analysis was performed with Monolix (version 2.4, www.monolix.org) to investigate the effect of patient covariates on PK, and to estimate the relative magnitude of inter-individual and inter-occasion variability. A two-compartment pharmacokinetic model was used to describe the data. The pharmacokinetic parameters estimated included elimination rate constant and volume (ke (1/hr), V (L or L/kg)), and the inter-compartmental parameters (k12 and k21 (1/hr)). The distribution of the parameters was assumed log-normal. Body weight was the main covariate which explained the largest portion of the IIV (28% and 20% of V and ke IIV, respectively). In addition, the following genotypes showed differences in the pharmacokinetics: GSTP1*B (1.7X higher ke in MUT versus WT or HET; p<0.05), CYP3A4*1B (2X higher ke in HET versus WT; p<0.05), and ALDH1A1*2 (2X higher ke in HET versus WT; p<0.05). We have developed a population PK model for Cy in thalassaemic children by considering morphological and biological covariates, which explains more than 45% and 22% (V and ke IIV, respectively) of the variation in Cy PK in these patients. This model-based algorithm may be used to design and plan targeted dose therapy in this group of pediatric patients and to predict the risk of toxicity and outcome of HSCT.

Disclosures:

No relevant conflicts of interest to declare.
Natural Course in Childhood Chronic Immune Thrombocytopenic Purpura (ITP) and Their Relation to CD4+CD25+FoxP3+ Regulatory T Cells.
Authors: Kim Ji Yoon , Choi Yong-Mook , Lee Kun Soo
Published: October 2019

Abstract 1318

Poster Board I-341

Purpose

The proposal of the present study was to determine the frequency of CD4+CD25+FoxP3+ regulatory T cells (Treg) in the peripheral blood and to assess the serum levels of IL-2, IL-10 and TGF-β1 in childhood chronic ITP with thrombocytopenia and spontaneous remission. In anticipation of the findings would elucidate the possible role in order to predict spontaneous recovery and pathogenesis of childhood chronic ITP.

Patients and Methods

Eleven children with chronic ITP (7 thrombocytopenic and 4 recovered cases, mean age 8.8 years; range, 1.7∼14.9 years) were enrolled. Five healthy children and eight healthy adults were included in this study as controls. The frequency of Treg was evaluated in peripheral blood by flow cytometry. The serum levels of IL-2, IL-10 and TGF-β1 were measured by ELISA technique.

Results

During this study, it was observed that four patients (36%) achieved spontaneous remission within 2.8 years (mean year; range, 1.0∼4.4 years). The frequency of Treg was significantly decreased in the patients with thrombocytopenia as compared to spontaneously recovered patients and controls. A significant positive correlation was found between the frequency of Treg and the platelet count in children. The serum level of IL-10 was higher in the patients recovered spontaneously comparing to the patients with thrombocytopenia, but the difference was not significant statistically. The serum level of IL-2 was not able to detect as too low to be analyzed. TGF-β1 showed no significant differences in this study.

Conclusion

These data suggest that the decreased frequency of Treg would contribute to breakdown of self-tolerance as well as form a base for future specific immunomodulatory therapies and have prognostic significances toward the natural course and long-term outcome of childhood chronic ITP.

Disclosures

No relevant conflicts of interest to declare.
Neutrophil Extracellular Traps Induce Platelet Adhesion and Thrombus Formation.
Authors: Fuchs Tobias , Brill Alexander , Dürschmied Daniel , Schatzberg Daphne , Hartwig John H. , Myers Daniel , Wakefield Thomas W. , Wagner Denisa D.
Published: October 2019

Abstract 1345

Introduction

Thrombus stability is provided by very large polymers adhering to platelets and anchoring the thrombus to the vessel wall. The best described polymers are fibrin and von Willebrand Factor (VWF). Activated neutrophils and other leukocytes can form an extracellular fibrous network which is composed of DNA, histones, and granular proteins. These neutrophil extracellular traps (NETs) are present in various inflammatory diseases. In deep vein thrombosis (DVT) inflammation closely cooperates with thrombosis. Here we examine whether NETs provide a new means to support the adhesion and recruitment of platelets and whether NETs are present in DVT. Methods and Results: To study the interaction of platelets with NETs, we isolated human neutrophils, induced NET formation and perfused over the NETs human platelets in plasma or whole blood anticoagulated with the thrombin inhibitor PPACK. Microscopic analysis revealed that under flow platelets adhere avidly to NETs. Perfusion of whole blood at physiological shear resulted in formation of thrombi on NETs in a time dependent manner. Addition of DNase1 degraded NETs and removed all platelets and thrombi demonstrating their adhesion to NETs. Thrombus formation on NETs was absent if blood was supplemented with EDTA indicating the requirement for divalent cations. Perfusion of NETs with heparinized blood dismantled NETs and prevented thrombus formation. Incubation of NETs with heparin alone released histones from NETs, indicating that heparin destroys the chromatin backbone of NETs. Furthermore, immunocytochemistry revealed that NETs were able to bind platelet adhesion molecules VWF and fibronectin from human plasma. Immunohistochemical analysis of a baboon deep vein thrombus showed abundant extracellular chromatin which co-localized with fibronectin and VWF. Conclusions: We show that extracellular traps are able to promote thrombosis in vitro and are abundant in vivo in DVT. We propose that extracellular chromatin provides a new type of scaffold that promotes platelet adhesion, activation, and aggregation and may be important for thrombus initiation or stability.

Disclosures

No relevant conflicts of interest to declare.
The Second Signal in Antigen-Presenting Cells: Complementary JAK-1 Directs Pro- and Anti-Inflammatory Cytokine Production Induced by CD40L.
Authors: Conzelmann Michael , Wagner Andreas , Hildebrandt Anke , Rodionova Elena , Hess Michael , Zota Annika , Giese Thomas , Falk Christine , Breit Stephen , Hecker Markus , Ho Anthony D. , Dreger Peter , Luft Thomas
Published: October 2019

Abstract 1348

Poster Board I-371

Aberrant expression of CD40 ligand (CD40L) represents a strong endogenous danger signal associated with chronic inflammatory disease. However, CD40 activation alone is unable to induce interleukin (IL)-12p70 secretion, whereas IL-6, IL-12p40, TNF-alpha and IL-10 are produced. We demonstrate that cytokine-induced Janus kinase (JAK)-1 is unique in mediating a complementary pro-inflammatory signal for the production of IL-12p70 whilst at the same time inhibiting IL-10 secretion in human APCs.

CD40- and JAK/STAT signals recruit complementary pathways: CD40 signals involve RelA and cRel for the induction of IL-12p70 and RelA for IL-10. In contrast, RelB inhibited IL-12p70 production. JAK1 complemented CD40-induced NF-kB signals by phosphorylating STAT-1 and inducing IRF-1 and IRF-8 expression resulting in IL-12p70 production. JAK-1-dependent inhibition of IL-10 transcription was mediated either by STAT-1 directly binding to the IL-10 promotor or by IRF-8 that in a complex with PU.1 (Spi-1) binds to inhibitory IFN-gamma-activated sequence (GAS)-sites of the IL-10 promotor. Our results demonstrate that specific inhibition of the “second signal” JAK1 will shift the balance of CD40-induced cytokine panels from IL-12p70 towards IL-10 in human APCs.

Disclosures

No relevant conflicts of interest to declare.
The Gene RUNX1 and Its Possible Relation with the Alteration of Granulocytes Cells and with the Progression of Chronic Myeloid Leukemia.
Authors: Mascarenhas Cintia C , Cunha Anderson Ferreira , Brugnerotto Ana Flavia , Gambero Sheley , Costa Fernando Ferreira , Pagnano Katia Borgia Barbosa , Souza Carmino Antonio De
Published: October 2019

Abstract 2215

Poster Board II-192

The exact action mechanism of the protein BCR-ABL is unknown, studies in vitro and in animal models showed that the activity of protein tyrosine kinase (TK), by itself, is sufficient to cause the development of CML. Thus, the progression of the disease to accelerated phase or blast crisis may be associated with genomic instability. The use of methods to detect the difference in gene expression between CML and normal granulocytes could bring new insights in the understanding of these complex mechanisms, highlighting new therapeutic targets for the CML treatment. Evaluation of the differential gene expression between CML and normal granulocytes using the Subtractive Suppression Hybridization (SSH) and investigation of the involvement of a set of genes in modulating the development of CML. The SSH libraries were constructed using a pool of granulocytes RNA's extracted from CML patients and from healthy blood donors. Real-Time (RT-PCR) was used to validate the results and evaluation of gene expression in granulocytes and in mononuclear cells in the pool of patients and controls used for the construction of libraries. The expression of the RUNX1 gene, whose expression was the most differential between the libraries, was evaluated in mononuclear cells of patients with CML using RT-PCR. The comparison between granulocytes of CML patients and control granulocytes showed 39 genes exclusively expressed in CML and 169 genes in controls. For validation, we evaluated the expression of genes RUNX1, KPNA6, TOB1, SEPT5, CDC42SE, ITCH, MIER and GP1BA by RT-PCR in the pool of patients and controls used for the construction of libraries. Among these genes, the RUNX1 gene showed substantial difference, and was also studied in mononuclear cells from 20 patients with CML in different stages of the disease and using different types of treatment with TK inhibitors. The expression of this gene was highly altered in patients in advanced phase of disease when compared with patients who are responding to treatment. The genes identified in this study may be related to the development and progression of CML. Among them, the RUNX1 gene was shown to be one of the most promising, since the occurrence of chromosomal translocations in this gene has been associated with different types of acute leukemia – however its association with CML is not yet clear. The results showed a relationship between the expression of this gene and progression of the disease, beyond the identification of several genes that may lead to a better understanding of CML and help in identifying new therapeutic targets. This work was supported by FAPESP.

Disclosures:

Off Label Use: Development of TKI in the treatment of CML.
Treatment of Severe Acyclovir-Resistant Herpes Simplex Virus Infection with Continuous Infusion of High Dose Acyclovir Following Hematopoietic Cell Transplantation.
Authors: Kim Janet H. , Schaenman Joanna , Ho Dora Y. , Brown Janice (Wes) M.
Published: October 2019

Abstract 2224

Poster Board II-201

Although acyclovir (ACV)-resistant herpes simplex virus (HSV) occurs in healthy patients, higher rates have been reported in 7-27% of immunocompromised patients, particularly in the setting of acyclovir prophylaxis. Infection due to ACV-resistant HSV (ACV-R HSV) is frequently severe and poses challenges to treatment, particularly following hematopoietic cell transplantation (HCT). Unfortunately, currently available alternative therapies including foscarnet, cidofovir, and ganciclovir are frequently limited by their toxicity profile. We present a series of five HCT patients from 1997-2008 with severe infections due to ACV-R HSV who were successfully treated with continuous infusion of high dose acyclovir. A Pubmed search of published literature from 1948-2009 confirmed this as the first report of ACV-R HSV in HCT patients treated with this regimen.

The characteristics of the patients are summarized in the table. Patients ranged in age from 24 to 55 and had undergone an allogeneic HCT following a myeloablative preparative regimen. All grafts were composed of peripheral blood hematopoietic cells. Underlying diseases included acute myelogenous leukemia, myelodysplastic syndrome, and essential thrombocytosis. Four of the five patients were receiving treatment for GVHD of the skin (grades 2-4), with regimens that included varying doses of prednisone. Infection was due to HSV-1 in three patients and HSV-2 in two. In vitro testing revealed that high median inhibitory concentrations were necessary to inhibit viral replication by 50% (MIC50) for all five isolates consistent with resistance to acyclovir, ganciclovir, and foscarnet. Failed HSV treatment regimens included standard, intermittent doses of acyclovir (5-10 mg/kg IV every eight hours), foscarnet, cidofovir, and/or famciclovir. Adverse reactions to these agents included severe renal insufficiency, hyponatremia, and neutropenia. The doses of ACV administered via continuous infusion ranged from 30 to 50 mg/kg per day, and time to resolution of lesions ranged from 1 week to 2 months. No patients experienced toxicity associated with continuous infusion of high dose acyclovir and therapy could be continued as an outpatient when appropriate.

Conclusions:

Continuous infusion of high dose acyclovir for acyclovir resistant HSV is an attractive and more tolerable alternative for patients failing standard dose intermittent infusion acyclovir or intolerant of alternative standard therapies such as foscarnet, cidofovir, or ganciclovir. At this time it is not clear how the altered pharmacokinetics that results from continuous infusion of high doses of acyclovir overcomes viral resistance. We are currently investigating the mechanisms by which the sustained levels of ACV provide this therapeutic advantage.

Disclosures:

No relevant conflicts of interest to declare.
Different Clinical Results with the Use of Different Dosing Schedules of Azacitidine in Patients with Myelodysplastic Syndrome Managed in Community-Based Practice: Effectiveness and Safety Data From the Spanish Azacitidine Compassionate Use Registry.
Authors: Garcia Regina , de Miguel Dunia , Bailen Alicia , MD José Ramón González, , Sanz Guillermo , Falantes Jose F , Andreu Rafael , Tormo Mar , Duarte Rafael F , Figueredo Antonio
Published: October 2019

Abstract 2773

Poster Board II-749

Background:

Azacitidine (AZA), a hypomethylating agent recently approved in Europe for the treatment of myelodysplastic syndrome (MDS), prolongs the median survival time in patients enrolled in clinical trials (Fenaux et al 2009). AZA was available for clinical trial or compassionate use in Spain before receiving its marketing authorization from the Spanish Medicines Agency in May 2009. The dosing of AZA in community-based hematology clinics could differ from that approved by the health authorities.

Material and Methods:

We present the preliminary analysis of the clinical results of data from a longitudinal, multicenter Spanish patient registry, which retrospectively collected data from community-based hematology clinics on the disease course and management of patients with MDS treated under compassionate use conditions with AZA, in whom the dosing schedule administered was documented. AZA was administered to the patients in three different dosing schedules over 28-day cycles; Group A: days 1, 2, 3, 4 and 5 / Group B: days 1, 2, 3, 4, 5, 8 and 9 / Group C: days 1, 2, 3, 4, 5, 6, 7. Treatment assignation was based on patient status and the feasibility of weekend drug administration. As of August 1, 2009, data from 147 patients with MDS diagnosed according to the WHO criteria had been collected.

Results:

We evaluated 144 patients in whom the dosing schedule was documented at the time of this analysis. At baseline, the patient characteristics were comparable, except for ECOG performance status, with a higher prevalence of an ECOG ≥ 2 in the day 1-7 dosing schedule group (Group C) (Table 1). Group A accounted for 36% of the patients, group B for 32%, and group C for 32%. The mean initial dose of AZA was comparable between groups. Despite the different dosing schedules, the median number of cycles administered was similar between groups. The overall treatment response rates (International Working Group 2006 criteria) varied according to dosing schedule: 58% in group A, 65% in group B and 74% group C (Table 1). AZA was generally well tolerated, but the adverse events profile differed according to dosing schedule group (Table 1)

Conclusion:

Our results demonstrate that different treatment schemes are feasible for patients with MDS treated with AZA in the community-based setting, but they have different effectiveness and the safety profiles. These data show a better efficacy/safety profile for the dosing schedule approved by the EMEA. Further analyses are awaited.

Disclosures:

No relevant conflicts of interest to declare.
P52 Activation in Monomorphic B-Cell Post-Transplant Lymphoproliferative Disorders without BAFF-R Expression.
Authors: Proust Alexis , Rince Patricia , Creidy Rita , Lazure Thierry , Fabre Monique , Joab Irène , Guettier Catherine , Raphael Martine
Published: October 2019

Abstract 2932

Poster Board II-908

Post-transplant lymphoproliferative disorders (PTLD) are one of the worse prognostic complications after solid organ or bone marrow stem cells transplantation. Most of them are associated to EBV known to activate NF-kB pathway especially by constitutive p65 expression. BAFF cytokine, a B cell-activating factor belonging to the tumour necrosis factor (TNF) family, have been described to modulate cell growth and survival in non Hodgkin lymphomas. However, little is known on the association between EBV, BAFF/BAFF-R signalling and canonical and non canonical NF-kB pathways expression in PTLD. Thus, we intend to study the role of EBV, NF-κB and BAFF/BAFF-R expression in PTLD.Our study has been investigated in two different contexts of diffuse large B-cell lymphoma (DLBCL). 20 cases of DLCBL resulting from immunocompetent patients (DLBCL/IC) and 13 from post-transplant recipients (DLBCL/PTLD) were compared. Indeed, all cases were characterized by histology and immunohistochemistry (IHC) was used to detect B-cell markers and to identify their germinal center (GC) or non GC (NGC) origin. EBV was detected by in situ Hybridisation (ISH) using EBER probe. Latent proteins LMP1 and EBNA2 as well as replicative protein ZEBRA were also detected by IHC. In addition, p50 and p52 proteins expression was carried out to study NF-kB pathway activation.

We showed in DLBCL/IC, regardless of the ontogenic profile GC/NGC, that BAFF-R is expressed in 40% of cases, while in DLBCL/PTLD NGC pattern, BAFF-R is expressed in only one case out of 13 (7,7%) (p<0,05). Moreover, there was no significant difference in p50 expression between the categories of DLBCL studied. In contrast, we have shown a significant expression of p52 in DLBCL/PTLD (8 out of 13 cases) compared to DLBCL/IC (4 out of 20 cases) (p<0.005). In DLBCL/PTLD, there was no expression of BAFF-R ; this pattern could be related to the presence of EBV and LMP1 since p52 expression is mostly observed in EBV+ DLBCL/PTLD (5 out of 7 p52+ cases).

In conclusion, the activation profile of DLBCL/PTLD is mostly not associated with BAFF/BAFF-R expression while NF-kB2 pathway is activated.

Disclosures:

No relevant conflicts of interest to declare.
ALL-Associated JAK1 Mutations Confer Hypersensitivity to the Anti-Proliferative Effect of Type I Interferon.
Authors: Hornakova Tekla , Chiaretti Sabina , Lemaire Muriel , Foà Robin , Tartaglia Marco , Renauld Jean-Christophe , Knoops Laurent
Published: October 2019

Abstract 3066

Poster Board III-3

Recently, we and others reported activating mutations in JAK1 in acute lymphoblastic leukemia (ALL). These mutations are relatively common in adult patients with T cell ALL. JAK1 is a tyrosine kinase that associates to different cytokine receptors to mediate signal transduction. The associations of the mutant JAK1 with receptors like IL-2R or IL-9R are necessary to promote tumorigenicity by inducing constitutive signaling via the activation of the receptor complex. Because JAK1 mutations confer poor prognosis to the patients, there is a need for new therapies that could specifically target the leukemic blast.

Starting from patient samples, we show here that JAK1-mutant ALL blasts are characterized by a type-I interferon (IFN) transcriptional signature. This signature was recapitulated in vitro by the expression of JAK1 mutants in BW5147 and BaF3 hematopoietic cell lines. Binding of JAK1 to the IFN receptor was essential since mutations in the FERM domain abrogated this effect. Beside the constitutive activation of the type I IFN signaling cascade, JAK1 mutations also strongly potentiated the response to IFN in vitro. Typically, the proliferation of cell lines expressing JAK1A634D was abrogated by type I IFNs. Interestingly, we found that different JAK1 mutations differentially potentiate responses to type I IFNs or to IL-9, another cytokine using JAK1 to mediate its effects. This suggests that the type of mutation influences the specificity of the effect on distinct cytokine receptor signaling. Finally, we also showed in an in vivo leukemia model that cells expressing JAK1A634D are hypersensitive to the anti-proliferative and anti-tumorigenic effect of type I IFN, suggesting that type I IFNs should be considered as a potential therapy for ALL with JAK1 activating mutations.

Disclosures

No relevant conflicts of interest to declare.
CD44-Induced Differentiation of Acute Monoblastic Leukemia Cells Is Mediated by Prolonged Activation of ERK1/2 and by Cytokines.
Authors: Bourcier Sébastien , Sansonetti Arnaud , Durand Laetitia , Chomienne Christine , Robert-Lézènès Jacqueline , PhD Florence Smadja-Joffe Sr.,
Published: October 2019

Abstract 3103

Poster Board III-40

We have previously reported that specific monoclonal antibodies (mAbs) to the cell adhesion molecule CD44, trigger monocytic differentiation of blasts from patients with acute monoblastic leukemia (1) and that cytokines contribute this differentiation (2). To further analyze the mechanisms mediating CD44-induced monocytic differentiation of leukemic cells, we have used as a model the THP-1 leukemia cell line which differentiation has been induced by the Hermes-3 anti-CD44 mAb. Monocytic differentiation has been checked at day 3 by measuring, using flow cytometry, the increase in the expression levels of CD11b (myeloid-specific) and CD14 (monocyte-specific) differentiation antigens. Control THP-1 cells (untreated or treated with isotypic IgG) display only a little CD11b and do not express CD14.

First, using western blot analysis, we show that CD44-ligation with Hermes-3 mAb rapidly triggers ERK1/2 MAP kinase phosphorylation, that persists for about 6-8 hours. This prolonged ERK1/2 phosphorylation is essential for CD44-induced differentiation, since its delayed inhibition (using the specific chemical inhibitor U0126) fully abrogates the differentiation process. Similar results have been obtained in Hermes-3 - treated primary blasts from patients with acute monoblastic leukemia. Interestingly, ERK1/2 signalling mediates normal monocytic differentiation induced by cytokines (3). Accordingly, our present results suggest that CD44-ligation might reactivate this physiological differentiation pathway persisting in monoblastic leukemia cells (THP-1 cells and primary leukemic monoblasts).

Second, using real-time RQ-PCR and ELISA assays, we show that ERK1/2 activation leads to synthesis and secretion of a burst of cytokines, including IL-1b, IL-6, IL-8, GM-CSF and TNF-alpha. Among these cytokines, TNF-alpha- and IL-6, acting through an autocrine-paracrine pathway, play an important part in the CD44-induced differentiation process, since their functional inhibition with specific mAbs dramatically decreases the extent of this differentiation. TNF-alpha has a critical role in the increase of CD11b expression level. In addition, it cooperates with IL-6 for inducing CD14 expression. However, TNF-alpha- and IL-6 inhibition does not totally abrogate CD44-induced differentiation. This suggests that another unidentified cytokine is perhaps involved in the differentiation process. Alternatively, CD44-activated ERK1/2 pathway may directly reprogram the expression of monocytic differentiation genes. Interestingly, addition of exogenous IL-6 enhanced the level of CD14, suggesting that IL-6 and CD44-ligation may cooperate for differentiation therapy of acute monoblastic leukemia.

1. Charrad et al. Nat. Med 5:669,1999 2. Delaunay et al. Leukemia 22:873, 2008

Disclosures

No relevant conflicts of interest to declare.
Prevention of Venous Thromboembolism (VTE) Associated with Pregnancy in Women with a Past History of VTE.
Authors: Rozanski Camilla , Lazo-Langner Alejandro , Kovacs Michael J.
Published: October 2019

Abstract 3132

Poster Board III-69

Women with a past history of venous thrombosis are at higher risk for venous thromboembolism (VTE) during and after pregnancy. The highest risk period is the first four weeks post partum. For a woman whose previous event was secondary to a major transient risk factor the antepartum risk of recurrent VTE is low whereas for women whose previous event was idiopathic the antepartum risk is higher. In our institution, for women whose previous event was secondary to a major transient risk factor, standard treatment is to follow closely with no thromboprophylaxis antepartum and then receive either prophylactic low molecular weight heparin (LMWH) or warfarin for six weeks postpartum. For women whose previous event was idiopathic or who were on warfarin at the time of becoming pregnant they receive prophylactic LMWH antepartum with a scheduled delivery and are put back on warfarin if they have long term anticoagulation needs or LMWH for six weeks if they don't have longterm anticoagulation needs. We report the outcome for our patients from 1997 to 2008. All patients were followed for the duration of pregnancy and for 6 weeks postpartum for pregnancy outcome, recurrent VTE, and major bleeding in the thrombosis clinic of London Health Sciences Centre. There were a total of 90 women; 30 women (mean age 30.6 years) with a history of previous secondary thrombosis with 37 pregnancies and 60 women (mean age 29.5 years) with past idiopathic thrombosis with 99 pregnancies. For the secondary group there was 1 episode (2.70%; 95% CI 0.48-13.82) of antepartum recurrent VTE whereas for the idiopathic group there were 3 episodes (3.03%; 95% CI 1.04-8.53). There was no statistical difference between groups (p=1.0). There were no episodes of postpartum VTE recurrence or major hemorrhage for either group. For the secondary group there was 1 fetal loss at 23 weeks (2.7%; 95% CI 0.48-13.82) whereas for the idiopathic group there were 6 fetal losses at 8, 10, 10, 11, 22, 37 weeks gestation (6.06%; 95% CI 2.81-12.60). There was no statistically significant difference between groups (p=0.77). This retrospective review suggests that for pregnant women with a past history of VTE, a strategy of no antepartum prophylaxis for previous secondary thrombosis and antepartum prophylactic LMWH for previous idiopathic thrombosis as well as prophylactic LMWH or warfarin postpartum is efficacious and safe.

Disclosures

Off Label Use: low molecular weight heparin for prevention of VTE in pregnancy.
Identification of a Functional Interferon-Gamma Response Element in the Fibrinogen FGG Gene.
Authors: Rein Chantelle M. , Farrell David H.
Published: October 2019

Abstract 3174

Poster Board III-114

Fibrinogen is the zymogen precursor to the main structural protein in blood coagulation, and is proteolytically activated during coagulation to form fibrin clots. Fibrinogen is an acute phase protein that is produced by the liver, and elevated fibrinogen levels are a known risk factor for cardiovascular disease, including heart attack and stroke. Fibrinogen is synthesized from three separate genes, FGA, FGB, and FGG encoding the Aαa, Bβ, and γ chains, respectively. Several inflammatory cytokines, particularly interleukin-6 (IL-6), are known to up-regulate fibrinogen synthesis, while interleukin-1β, tumor necrosis factor-αa, and transforming growth factor-β are known to antagonize IL-6 stimulation of fibrinogen synthesis. Here we report that interferon-γ (IFN-γ), a cytokine that is highly expressed in atherosclerotic lesions, decreases the production of fibrinogen by two-fold over a 24 hour period in HepG2 hepatocellular carcinoma cells, both at the protein and mRNA level. In addition, IFN-γ antagonizes IL-6 up-regulation of fibrinogen production. Since IFN-γ is known to signal via the IFN-γ receptor through a STAT1-dependent pathway, we investigated the mechanism by which IFN-γ inhibits fibrinogen production. We identify a previously unknown IFN-γ activation sequence (GAS), AGGAGCTTACATAAAGGGACAA, within the promoter of the human γ chain gene FGG. This sequence is homologous to the known GAS consensus sequence TTNCNNNAA. Nuclear extracts from IFN-γ-stimulated HepG2 cells, but not from unstimulated HepG2 cells, form a complex with oligonucleotides containing this GAS sequence, as demonstrated by electrophoretic mobility shift assays. The formation of this complex is blocked by unlabeled competing oligonucleotides and by an antibody to phosphorylated STAT1. The involvement of pSTAT1 in this complex is consistent with the known role of pSTAT1 downstream of IFN-γ receptor signaling. In addition, IFN-γ stimulates STAT1 phosphorylation in a dose-dependent manner in HepG2 cells. Furthermore, using an FGG promoter construct fused to a luciferase reporter gene in transiently transfected HepG2 cells, we show that IFN-γ inhibits luciferase expression in a dose-dependent manner. Together, these data demonstrate that IFN-γ down-regulation of fibrinogen production occurs through a pSTAT1-mediated pathway. Since IFN-γ is known to demonstrate both pro and anti-atherogenic actions, this down-regulation of fibrinogen production may serve an anti-atherogenic function in vivo.

Disclosures

No relevant conflicts of interest to declare.
Predictors of Long-Term Cytogenetic Response Following Dasatinib Therapy of Patients with Chronic-Phase Chronic Myeloid Leukemia (CML-CP).
Authors: Jabbour Elias , Bahceci Erkut , Zhu Chao , Lambert Alexandre , Cortes Jorge
Published: October 2019

Abstract 3296

Poster Board III-184

BACKGROUND

Achievement of major and complete cytogenetic response on imatinib in newly diagnosed CML-CP has been associated with excellent survival. Dasatinib is a potent oral tyrosine kinase inhibitor with marked activity and good tolerability in patients with any-phase CML who have failed prior therapy, including imatinib.

AIM

To investigate factors predictive of cytogenetic response and survival outcomes for patients who receive dasatinib after imatinib failure.

METHODS

Collated patient data from trials of dasatinib (START-C, START-R, and CA180-034) in patients with CML-CP were analyzed. All patients had failed prior treatment with imatinib as a result of resistance or intolerance. Patients with highly imatinib-insensitive BCR-ABL mutations (L248V, G250E, Q252H/R, Y253H/F, E255K/V, T315I/D, F317L, and H369P/R) were ineligible for START-R. Across the three trials, patients were administered dasatinib 50 (n=167) or 70 (n=655) mg twice daily, or 100 (n=165) or 140 (n=163) mg once daily. Analysis of efficacy parameters was performed on all patients receiving at least one dose of dasatinib (n=1150). A multivariate logistic regression model was applied to evaluate patient treatment characteristics as predictive factors for cytogenetic response.

RESULTS

After a median follow-up of 26 months, the rate of major cytogenetic response (MCyR) was 62% and of complete cytogenetic response (CCyR) was 51%. By multivariate analysis, the following were selected as independent favorable prognostic factors for achievement of MCyR: younger age, lower percentage of Ph+ cells, absence of the T315I mutation, prior MCyR with imatinib, imatinib intolerance (vs. resistance), no prior stem cell transplantation (SCT), shorter time from CML diagnosis to dasatinib therapy (Table 1). The same baseline factors independently predicted CCyR.

CONCLUSION

This study confirms the clinical efficacy of dasatinib among patients with CML-CP who have failed prior therapy. Furthermore, we identified baseline factors associated with improved response to dasatinib. Work to determine additional predictive factors for survival outcome with dasatinib is ongoing. The subgroups of patients who may not respond optimally to dasatinib are small, and a modification to their treatment regimen may be called for.

Disclosures

Jabbour: Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau. Bahceci:Bristol-Myers Squibb: Employment. Zhu:Bristol-Myers Squibb: Employment. Lambert:Bristol-Myers Squibb: Employment. Cortes:Bristol-Myers Squibb: Research Funding.
Prevalence of Coronary Heart Disease in Patients with Hemophilia: a Nationwide Study in Brazil.
Authors: Bley Claudia , Santos Fabio P S , Pesaro Antonio E P , Helman Ricardo , Oliveira Sergio A , Pfeferman Elcio , Serrano Carlos V , Saraiva Ana S L , Amico Elbio D , Hamerschlak Nelson
Published: October 2019

Abstract 3495

Poster Board III-432

Background

It is well known that patients with hemophilia (both A and B) have a lower incidence of coronary artery disease (CAD). However, patients with hemophilia may develop atherosclerosis, and with increases in life expectancy it is possible that the incidence of CAD may increase in these patients. There are no studies analyzing the prevalence of CAD in patients with hemophilia and possible risk factors.

Aims

To determine the prevalence of CAD in patients with hemophilia treated in Brazil.

Methods

All patients with hemophilia in Brazil receive therapy with Factor VIII or Factor IX at government-designed hemophilia centers. We thus contacted all centers in Brazil by e-mail and information from databases concerning health information for all patients was reviewed by the responsible physician at each center. A questionnaire about risk factors for CAD and history of coronary events was sent to patients that had a positive history of CAD.

Results

There are 6,881 patients registered with a diagnosis of hemophilia A and 1,291 patients with a diagnosis of hemophilia B in Brazil. Fifty-six percent of hemophilia centers answered the query, and information on 71.7% of patients with hemophilia A (4,934 patients) and 61.4% of patients with hemophilia B (792 patients) was reviewed. There were only 3 patients (0.05%) with a positive history of CAD. Their clinical characteristics are summarized in Table 1. During surgical treatment for CAD, replacement with Factor VIII and Factor IX was done by continuous infusion to increase Factor activity to 80-100%. Antiplatelet therapy was used in all patients at some point, and one patient (#1) developed hemarthrosis after two months of ASA. Patient #3 did not start antiplatelet therapy after CABG, and developed thrombosis of the graft. ASA and clopidogrel were started after percutaneous coronary angioplasty.

Conclusion

We found a very low incidence of CAD in hemophiliac patients, even after doing a nationwide survey that included more than 5,000 patients. No information about risk factors can be gleaned due to the small number of patients. Surgical treatment after increasing factor activity appears to be safe in such patients. Despite a theoretical risk for increased bleeding, therapy with antiplatelet agents appears to be necessary in hemophiliac patients with CAD.

Disclosures:

No relevant conflicts of interest to declare.
Long-Term Effects of In Vivo Administration of An Anti-VLA-4 Antibody (natalizumab) on Mobilization of Committed and Primitive Hematopoietic Progenitor Cells In Humans
Authors: Domenech Jorge , Goustille Julien , Pallix Maud , Bekhechi Zakia , Ducrocq Elfi , Degenne Michel , Herault Olivier , Thibault Gilles , Guennoc Anne-Marie , Binet Christian
Published: October 2019

Abstract 2264

The alpha4 beta1 integrin (VLA-4) exert a critical role on hematopoiesis by confining hematopoietic stem cells (HSC) and progenitor cells (HPC) within the niche. Previous preclinical studies have pointed out this role showing that HPCs can be mobilized by in vivo administration of a blocking anti-VLA-4 antibody (Ab) (Papayannopoulou et al, 1993). Very recently, two papers (Bonig et al, 2008; Zohren et al, 2008) have shown that such Ab exhibits a similar effect in humans for one month after treatment by natalizumab. In the present study, we have investigated long-term hematopoietic effects (up to 23 months) of repeated infusions of natalizumab in patients treated for multiple sclerosis (n=22). Seven patients have been explored sequentially (every month for one year) and 15 have been studied punctually (6 before and 9 after one year of treatment). We found that peripheral blood leukocytosis was consistently increased for one year in relation to lymphocytes, particularly to B lineage (by 3-fold). In parallel, an increase of circulating HPCs (CD34+ cells and total CFU) was observed and appeared more pronounced with levels above baseline values in all the patients studied (by 6-fold) while no increase of circulating mesenchymal stromal cells (CFU-F) was found. The increase was noted for both lymphoid (T and B lineages) and myeloid (granulo-monocyte, erythroid, and megakaryocyte) committed progenitor cells but also for primitive HPC (CD34+CD38- cells and CAFC). This effect was still found at long-term (up to two years of treatment) for both committed and primitive HPC. In conclusion, the HPC mobilizing effect of chronic administration of anti-VLA-4 Ab in humans involves all types of HPCs (lymphoid and myeloid, committed and primitive ones) and is not exhausted with time.

Disclosures:

No relevant conflicts of interest to declare.