bluuebunny/crossref_metadata_2025 huggingface dataset:
Safety of Recombinant Human Factor IX, Nonacog Alfa, for Usual Use In Pediatric Patients: Results From a Prospective Registry of European Hemophilia B Patients.
Authors: Rendo Pablo , Berntorp Erik , Keeling David M. , Makris Michael , Hermans Cedric R. , Mauser-Bunschosten Ereline Pauline , Musso Roberto , Tagliaferri Annarita , Kollmer Carl , Baumann James , Charnigo Robert
Published: October 2019

Abstract 3664

Objective:

To evaluate the long-term safety of nonacog alfa in pediatric hemophilia B patients during usual use.

Design/Methods:

An open-label, multicenter, prospective registry was conducted in 9 European countries to evaluate the safety of nonacog alfa in patients with hemophilia B in a usual-use setting. The registry was initiated in March 2002, with a period of enrollment of 7 years. Patients had received at least 1 dose of the original formulation of BeneFIX or a newer, reformulated version of the product. Of 218 patients enrolled in the registry, 66 (30.3%) were pediatric patients <18 years of age.

Results:

A total of 41 (62.1%) of the pediatric patients completed the registry per protocol. Seven events of special interest (ESIs) occurred in 6 (9%) patients. Allergic reaction was the sole ESI in 2 patients, lack of drug effect/low FIX recovery occurred in 3 patients, and 1 patient had an allergic reaction and developed a factor IX inhibitor. Two (3.0%) patients withdrew from the registry due to an ESI and/or a serious adverse event. Both were patients who developed an allergic reaction and one was the patient who also developed an inhibitor.

Conclusion:

The events reported for pediatric patients participating in this long-term prospective study demonstrated an acceptable safety profile for nonacog alfa in the pediatric population. The events did not differ in type, frequency, or severity from those previously observed in pediatric hemophilia B patients treated with nonacog alfa.

Disclosures:

Rendo: Pfizer: Employment. Keeling:Boehringer-Ingelheim:; Bayer: Membership on an entity's Board of Directors or advisory committees, lecture fee. Kollmer:Pfizer: Employment. Baumann:Pfizer: contract. Charnigo:Pfizer: Employment.
Bleeding and Thrombosis In 90 Iranian Patients with Inherited Afibrinogenemia.
Authors: Lak Manijeh , Toogheh Gholamreza , Keyhani Manoutchehr
Published: October 2019

Abstract 3667

Background:

Afibrinogenemia is a rare coagulation disorder transmitted as an autosomal recessive trait. In Iran, where consanguineous marriages are common coagulation disorders occur approximately ten times more frequently than in western countries. Afibrinogenemia is characterized by abnormal thrombin time, prothrombin time, activated partial thromboplastin time and absent plasma fibrinogen levels. We investigated the clinical manifestations of bleeding and thrombosis in a series of patients with Afibrinogenemia in Iran. To our knowledge this is the largest series ever reported.

Patients & Methods:

All patients in our cohort were diagnosed and registered in the Iranian National Registry of Inherited Bleeding Disorders. Every patient with an inherited blood disorder is reported to this registry because it is the only source of free treatment for bleeding disorders in the country. Afibrinogenemia has been tracked in Iran since 1965. Diagnoses in all patients was confirmed when plasma fibrinogen levels were absent both as functional activity (the Clauss method) and using the antigen level (electroimmunoassay). We retrospectively evaluated data from all 90 Afibrinogenemia patients available in the registry.

Results:

There were 46 males and 44 females in our cohort. Ages ranged from 1 to 71 years. 82% of patients were born from consanguineous marriages with asymptomatic parents. The most frequent symptoms in our patients were umbilical cord bleeding (87%), hematomas (74%) epistaxis (70%) and hemarthrosis (52%). Central nervous system bleeding was relatively rare (2.2%). In women of reproductive age, menorrhagia occurred in 75% of cases. Recurrent spontaneous abortions (more than 2) were seen in 28% of women (range 2 to 6). With prophylactic treatment (cryoprecipitate and limited fibrinogen) two (16.6%) of our patients with recurrent spontaneous abortion out of the 12 treated were able to carry pregnancies to term. A total of 7 patients in our population developed arterial or venous thromboses (3 males and 4 females). Four cases of the thromboses observed occurred after infusion of fibrinogen concentrate. Three episodes of spontaneous thrombosis were observed including a case of mesenteric thrombosis.

Discussion:

Afibrinogenemia is associated with severe and life threatening bleeding symptoms. Treatment can help women afflicted with this disease carry pregnancies to term. As reported elsewhere, thrombotic complications both spontaneous and acquired from treatment were also seen in our population.

Disclosures:

No relevant conflicts of interest to declare.
Evaluation of Clotting Factor Concentrates for Treatment of Thrombotic Thrombocytopenic Purpura.
Authors: Falter Tanja , Qorraj Mirjeta , Steinemann Sarah , Vigh Thomas , Scharrer Inge
Published: October 2019

Abstract 3678

Introduction:

Thrombotic thrombocytopenic purpura (TTP) is characterized by microthrombi, hemolytic anemia as well as thrombocytopenia. These symptoms are caused by a decreased activity of the protease ADAMTS13 which cleaves the von Willebrand Factor (VWF), due to mutation of the ADAMTS13-gene or autoantibodies. At the moment, the only available immediate therapy is plasmapheresis with Fresh Frozen Plasma (FFP) which may induce side effects. Therefore an alternative therapy might be the treatment with clotting factor concentrates.

Methods:

40 plasma samples were tested, consisting of FFP and solvent/detergent treated plasma, four batches of each blood group; VWF/VIII concentrates (Wilate®; Wilfactin®; Haemate®P; Immunate®; Kogenate®, Beriate®). In all samples ADAMTS13 activity, antigen and autoantibodies against ADAMTS13 were investigated. Additionally, the samples were tested for the presence of ultralarge VWF multimers. The BCS method according to Böhm, two ELISAs (Technozym®ADAMTS13 and Actifluor™ADAMTS13) and the electrophoresis on a SDS gel were used.

Results:

ADAMTS13 activity was found in all VWF)VIII concentrates, which are produced from human plasma, but only with a very low activity (Wilate® 5.6%; Wilfactin® 2.8%; Haemate®P 13%; Immunate® 3.7%). ADAMTS13 activity was not detectable in the factor VIII concentrates (Kogenate® <2%; Beriate® <2%). Usual FFP and solvent/detergent treated plasma samples, contained much higher ADAMTS13 activity and antigen values than concentrates (FFP 78.6%, solvent/detergent treated plasma 77.6%). However a difference of activity between individual blood groups was evident in FFP samples (blood group A 69.4%; B 64.9%; AB 98.1%; 0 82.0%). Ultralarge VWF multimers could be demonstrated in VWF containing concentrates, less in VIII concentrate from human plasma and not in FFP and solvent/detergent treated plasma samples. As expected recombinant-produced VIII concentrate contained no traces of ultralarge VWF. In all analyzed samples antibodies were negative.

Conclusion:

For therapy of TTP clotting factor concentrates cannot be used as an alternative to the usual FFP and solvent/detergent treated plasma, because their ADAMTS13 activity values are too low.

In addition, the VWF/VIII concentrates contain higher quantities of ultralarge VWF multimers, which are contraindicated for TTP patients. The differences of ADAMTS13 activity in FFP samples varies between the individual blood groups and batches. Solvent/detergent treated plasma shows less variation in ADAMTS13 activity due to the manufacturing process involving plasma pooling.

Disclosures:

No relevant conflicts of interest to declare.
Potential Identification of High Risk Populations for Acute Hemolytic Reactions Following Rh0 (D) Immune Globulin.
Authors: Ramachandran Paraneedharan , Li Daniel , Muller Carmen , Little Gregg , Sinclair Chris , Genereux Maurice
Published: October 2019

Abstract 3685

Acute Hemolytic Reactions (AHR) post Rh(D) Immune Globulin (Rh IgIV; WinRho® SDF, Cangene Corporation, Winnipeg, MB, Canada) have been reported since its approval for ITP in 1995. Cangene evaluated all post-marketing safety reports to ascertain whether certain populations may be at risk of AHR occurrence. AHR's were defined as one or more of the following: hemoglobin decrease, haptoglobin decrease or elevation of bilirubin occurring within days of Rh IgIV administration. Cases of Intravascular Hemolysis (IVH) were defined as AHR reports with demonstrated plasma hemoglobinemia and/or hemoglobinuria.

Purpose:

Identify populations that may be at higher risk of developing Acute Hemolysis when treated with Rh IgIV for ITP.

Methods:

Adverse Events (AE) were categorized as Acute Hemolytic Reactions (AHR) and Non Hemolytic Reactions (NHR) cases and both were evaluated retrospectively to identify if co-morbid conditions or other risk factors were implicated. Analyses were performed looking at: gender, age group, Rh IgIV dose, co-morbidities grouped by system organ class.

Results:

Between 1995 and March 2010 there were 684 AE reported, AHR were 312/684 (46%) and NHR were 372/684 (54%). Of the 312 AHR reports, IVH was identified in 70 (22%) of cases.

Conclusion:

91% of fatal outcome after AHR were found in patients with significant co-morbid conditions that contributed to death. Elderly patients (above 65 yrs) with following co morbid conditions appear to be more susceptible to AHR including definitive IVH and its complications. The following factors have been reported in the majority of Acute Haemolytic Reactions (AHR):

This review implies that choosing the right patient, the right dose and close monitoring of higher risk patients will improve the safety outcomes among ITP patients treated with Rh IgIV.

Disclosures:

Ramachandran: Cangene Corporation: Employment. Li:Cangene Corporation: Employment. Muller:Cangene Corporation: Employment. Little:Cangene Corporation: Employment. Sinclair:Cangene Corporation: Employment. Genereux:Cangene Corporation: Employment.
Pre-Stem Cell Transplant Induction Therapy Does Not Affect Post-Transplant Survival In Light Chain (AL) Amyloidosis
Authors: Madan Sumit , Kumar Shaji , Lacy Martha , Dispenzieri Angela , Buadi Francis , Hayman Suzanne R. , Dingli David , Rajkumar S. Vincent , Gertz Morie
Published: October 2019

Abstract 370

Background:

Light chain (AL) amyloidosis is characterized by the deposition of amyloid derived from immunoglobulin light chains in various organs. Autologous peripheral blood stem cell transplantation (SCT) is a commonly used and effective treatment for AL. For patients (pts) undergoing this procedure, a proportion of pts receive treatment similar to induction therapy employed in pts with myeloma undergoing SCT. However, the significance of induction therapy and its impact on the overall survival (OS) in AL is unknown. We conducted this study to ascertain the role of pre-SCT therapy on the post-transplant outcomes in AL.

Patients and methods: 435 pts with AL who underwent SCT at our institution between March 1996 and May 2010 form the study group. Groups were compared using Fisher's exact test or t-test, and survival was calculated using Kaplan Meier method. Survival curves were compared using log rank test.

Result:

The median (range) age of pts at the time of SCT was 57.4 years (26-75); 260 (60%) were males. The median (range) duration from AL diagnosis to SCT was 4 mos (1-87), and 284 (65%) pts were alive at the time of analysis. Melphalan 200/m2 (271 pts) or Mel/TBI (17 pts) was used for conditioning in 66% pts, whereas the remaining one-third had reduced doses of melphalan (100-160 mg/m2). The median OS for pts that received pre-SCT therapy (N=286) compared with those who did not (N=149) was 94.7 and 96.5 months, respectively (P=0.6) (Fig 1). Among the group of pts who underwent pre-SCT therapy and were evaluable for response, the median OS for those with a hematologic response (N=42) and no hematologic response (N=42) was 82.1 and 51 months (P=0.2), respectively (Fig 2).

Conclusion:

Our study demonstrates no difference in the OS of AL patients who received a pre-SCT therapy compared with those who received a SCT after their diagnosis of AL. In patients receiving pre-SCT therapy, there was a trend towards reduced OS among those with no hematologic response to pre-transplant therapy, but this difference was not significant.

Disclosure:

Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding. Dispenzieri:Celgene: Honoraria, Research Funding; Binding Site: Honoraria.
Efficient and Selective Prevention of Graft-Versus-Host Disease by Ag-Specific Induced Regulatory T Cells In Mice.
Authors: Semple Kenrick , Yu Yu , Wang Dapeng , Anasetti Claudio , Yu Xue-Zhong
Published: October 2019

Abstract 3745

Naturally occurring regulatory T cells (nTregs) suppress the development of graft-versus-host disease (GVHD). The non-selective suppression against tumor associated antigens in some models severely dampened our enthusiasm for the application of nTregs in the control of GVHD after allogeneic hematopoietic cell transplantation (HCT). In this study, we used an alternative strategy to generate antigen-specific, induced Tregs (iTregs), and tested their potential in the prevention of GVHD in murine model of myeloablative BMT. CD4+CD25+Foxp3+ iTregs generated from OT-II TCR transgenic mice specific for OVA target antigen efficiently prevented GVHD induced by polyclonal T effector cells (Teffs) in allogeneic recipients that express OVA protein but not in those that do not express OVA. The efficacy of these antigen-specific iTregs was significantly higher than polyclonal iTregs in preventing GVHD. As controls, OT-II CD4+Foxp3− cells had no effect on GVHD development in OVA− recipients and exacerbated GVHD in OVA+ recipients when transplanted together with polyclonal Teffs. Mechanistically, OT-II iTregs expanded extensively, and significantly suppressed expansion and infiltration of Teffs in OVA+ recipients. In sharp contrast, OT-II iTregs failed to expand and had no effect on Teffs in OVA− recipients. These results reveal the therapeutic potential of TGFβ-induced, antigen-specific iTregs to prevent GVHD efficiently and selectively.

Disclosures:

No relevant conflicts of interest to declare.
Potentially Fatal Neutropenia Associated with the Use of Cocaine Adulterated with Levamisole.
Authors: Shah Surbi , Goswami Umesh , Rachel Koreth , Williams David N , Mathew Josy
Published: October 2019

Abstract 3790

Background:

Cases of neutropenia associated with the use of cocaine adulterated with levamisole are being increasingly reported and have become a serious public health hazard. As reported by the US Drug Enforcement Administration (DEA) up to 69% of total cocaine seized in the US is adulterated with this veterinary antihelminthic known to cause agranulocytosis in certain susceptible populations. The rationale for selecting levamisole as an adulterant is unclear, but is likely related to the fact that it shares some physico-chemical properties with cocaine and potentiates the activity of dopaminergic (D2) and NMDA receptors in brain thus enhancing its effects.

Methods:

A case review was performed of adult patients who presented with neutropenia after the use of levamisole adulterated cocaine. Their laboratory results and treatment outcomes were studied to establish an expected course of the illness.

Results:

Over a one year period, 5 patients (4 female, 1 male) with age range of 45–60 years, presented with neutropenia in the setting of cocaine use. 4 were positive for levimasole in urine (detected by the use of gas chromatography/mass spectrometry analysis within 4–6 hours of presentation). Bone marrow biopsies were performed in 4 cases showed granulocytic hypoplasia with myeloid maturation arrest, lymphocytic hyperplasia and polytypic plasma cells. Antigranulocyte antibodies (AGA) were positive in 3 of the 4 patients tested. One each had positive serology for serum fluorescent antinuclear antibody (FANA), rheumatoid factor (RF) and antineutrophil cytoplasmic antibody (ANCA). Four of the five patients had hepatitis C infection. One patient had marginal zone B cell lymphoma and another was diagnosed with HIV at her first presentation. The median duration of neutropenia was 5–7 days. 2 patients died as a result of their various co-morbidities. Discussion: Use of cocaine adulterated with levamisole can result in profound neutropenia which could be fatal despite aggressive medical therapy. In patients presenting with neutropenia and a history of substance abuse, a high index of suspicion for cocaine adulterated with levamisole is needed and can be confirmed with rapid testing for both substances in patients' urine. Further diagnostic studies including a bone marrow biopsy with characteristic findings as described above and serologic testing for AGA can aid in confirming the diagnosis and ruling out other common causes of neutropenia. The treatment is usually supportive and based on the Infectious Diseases Society of America (IDSA) guidelines for managing neutropenic patients. The role of public health advisories in providing awareness among patients and care givers cannot be overstated, which can lead to early detection and reduced mortality.

Disclosures:

No relevant conflicts of interest to declare.
Safety of Outpatient Treatment In Acute Pulmonary Embolism.
Authors: Erkens Petra MG , Gandara Esteban , Wells Phil , Shen Alex Yi-Hao , Bose Gauruv , Gal Gregoire Le , Rodger Marc A. , Prins Martin H , Carrier Marc
Published: October 2019

Abstract 3796

Introduction:

Systematic reviews have shown that subcutaneous low molecular weight heparins (LMWH) are at least as safe and effective in the treatment of venous thromboembolism (VTE) as intravenous unfractionated heparin (UFH). LMWH allows patients with VTE to be treated as outpatients. However, patients with pulmonary embolism (PE) are still systematically admitted to the hospital for a few days to avoid potential complications. Physicians are reluctant to discharge patients due to insufficient data supporting the safety of outpatient management of PE. This study evaluates the safety of outpatient treatment of acute PE at the Ottawa Hospital.

Methods:

This is a retrospective cohort study of consecutive patients presenting at the Ottawa Hospital with acute PE diagnosed between January 1, 2007 and December 31, 2008. PE was defined as an arterial filling defect on CTPA or a high probability V/Q scan. Patients diagnosed with PE during hospitalization, patients with chronic PE and patients in whom anticoagulation treatment was not initiated (e.g. palliative care patients, small clinical non-significant PE) were excluded from the analyses. Patients were managed as outpatients if they were hemodynamically stable, did not require supplemental oxygenation and did not have contraindications to low molecular weight heparin therapy.

Results:

In this cohort of 473 patients with acute PE, 260 (55.0%) were treated as outpatients and 213 (45.0%) were admitted to the hospital. The majority of the patients were admitted because of severe comorbidities (45.5%) or hypoxia (22.1%).No outpatient died of fatal PE during the 3 month follow-up period. At the end of follow-up, the overall mortality was 5.0% (95% CI: 2.7 to 8.4%). The rates of recurrent venous thromboembolism (VTE) in the outpatient group were 0.4% (95% CI: 0.0 to 2.1%) and 3.8% (95% CI: 1.9 to 7.0%) within 14 days and 3 months, respectively. The rates of major bleeding episodes were 0% (95% CI: 0 to 1.4%) and 1.5% (95% CI: 0.4 to 3.9%) within 14 days and 3 months, respectively. Four (1.5%) outpatients were admitted to hospital within the first 14 days because of progressive shortness of breath and pain, a pre-syncopal episode or heparin induced thrombocytopenia.

Conclusion:

A majority of patients with acute PE can be managed as outpatients with a low risk of mortality, recurrent VTE and major bleeding episodes. Outpatient treatment in PE is feasible and safe in uncomplicated patients.

Disclosures:

Rodger: Pfizer: Research Funding; Leo Pharma: Research Funding; Sanofi Aventis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Canadian Institutes of Health Research: Research Funding; Heart and Stroke Foundation: Research Funding.
ESAS (Edmonton Symptom Assessment System) as An Effective Outpatient Serial-Longitudinal Symptom Assessment Tool In Patients with Sickle Cell Disease.
Authors: Lopez Gabriel , Liles Darla K , Knupp Charles L
Published: October 2019

Abstract 3798

Purpose:

Our goal is to implement an ongoing symptom assessment strategy using a validated palliative care tool in the outpatient management of adult patients with sickle cell disease at a comprehensive sickle cell center.

Methods:

Sickle cell disease is a chronic lifetime illness with intermittent acute exacerbations to daily symptoms. The extension of palliative care symptom management strategies to patients with sickle cell disease has been proposed. Symptom assessment tools such as the ESAS (Edmonton Symptom Assessment System), validated in the palliative care population, may have a role in sickle cell disease management. Ten symptoms are assessed, with the average 24 hour symptom score reported on a 0 to 10 numerical scale, 10 being the worst possible. In our study, 75 adult patients with sickle cell disease presenting for their outpatient visit at a comprehensive sickle cell center were enrolled; they were asked to complete the ESAS and a survey regarding their opinion of the ESAS at each clinic visit.

Results:

Study patients were 100% African American, 56% (42/75) female, ages 19–67, with disease types 71% (53/75) HgbSS, 19% (14/75) HgbSC, and 11% (8/75) HgbS/beta thal. At least 49% (37/75) of patients had one follow up visit after enrollment, for a total of 112 clinic visits. On the initial visit, 92% (65/71) agreed or strongly agreed with the statement “the ESAS is easy to complete”; 97% (71/73) reported instructions as clear; 96% (72/75) reported no words were difficult to understand; 83% (59/71) were satisfied or very satisfied with the ESAS as a way to report symptoms; 93% (68/73) would recommend the ESAS to other patients. 62% (45/73) of patients reported the ESAS helped them remember symptoms they were experiencing; 34% (25/73) were encouraged to discuss medical issues more openly with their health care provider. In a comparison of visit 1 (V1) and 2 (V2) in the 37 patient subset with a single follow up visit, 94% (33/35) on V1 and 94% (33/35) on V2 recommended the ESAS to other patients; 78% (28/36) on V1 and 86% (31/36) on V2 were satisfied or very satisfied with the ESAS. Including all 75 patients on their initial visit, the most intense symptoms (≥ 4 on ESAS scale) recorded were pain 4.6 (± 3.1) and sleep 4.1 (±3). In the 37 patient subset with follow up, V1 pain 4.7 (± 2.7) and V1 sleep 3.5 (± 2.5); V2 pain 4.4 (± 3.2) and V2 sleep 4.3 (± 2.6).

Conclusion:

The ESAS is an acceptable, brief, easy to understand symptom assessment tool which can be integrated into an outpatient sickle cell clinic as part of a serial-longitudinal symptom management strategy. Future studies should include measuring the impact of symptom-directed interventions on self-reported symptoms.

Disclosures:

No relevant conflicts of interest to declare.
Small Molecule Inhibitor of ITK Disrupts TCR Signaling and Demonstrates Anti-Tumor Efficacy
Authors: Faris Mary , Malyankar Uriel M , Zeng Qingping , Flynn Gary A , Feuer Gerold , Kertesz Nathalie L , Vuga Danka , Lee Sandra , Rosario Darlene , Bot Adrian
Published: October 2019

Abstract 3932

ITK (Interluekin-2 Inducible Tyrosine Kinase) is a member of the TEC family of intracellular protein tyrosine kinases. ITK is highly expressed in T cells and NK cells, with expression detected in mast cells. ITK plays a key role in several aspects of T cell biology, including T cell development, differentiation, migration, proliferation and activation. The function of ITK in immunity and allergy is well documented. T cells from ITK knock out mice show several developmental and functional defects, including defective signal transduction, altered CD4+ to CD8+ T cells ratios, reduced Th2 lineage differentiation, diminished IL4 and IL2 production and reduced T cell proliferation. Importantly ITK deficient mice fail to mount an immune response to infection and show reduced allergic asthma reactions.

In contrast to its well described role in immune function, ITK's function in cancer biology is still emerging. Recent studies had reported enhanced ITK expression and activation of the ITK pathway in several types of leukemias and lymphomas. In addition, the dependence of T cell malignancies on an ITK-regulated pathway, namely the IL2/IL2R (CD25) pathway, has also been observed. Taken together, this information indicates that ITK is a therapeutic target, with applicability in leukemias and lymphomas.

MannKind scientists have developed a series of selective small molecule ITK inhibitors, including the orally available tool compound described within, and evaluated their activity in enzyme, cell-based and in vivo studies. In cellular assays, the compounds showed significant inhibition of the T cell-receptor mediated activation of the ITK pathways and related downstream cytokine production. In addition to inhibiting the phosphorylation of ITK and its downstream mediator, PLCg, our tool compounds inhibited the production of IL2 and expression of CD25 in a dose dependent manner. Importantly, our compound regulated the in vitro growth of tumor T cells but not that of unrelated control cells. In vivo studies revealed that the tool compounds inhibited the growth and progression of patient derived ATL tumors in a xenograft pre-clinical model, and prolonged the survival of treated mice in a dose dependent manner, in addition to regulating cytokine production in vivo. In summary, our team has identified ITK selective compounds with demonstrated on-target and anti-tumor activity in vitro and preclinical T cell tumor models, and validated this pathway relative to T cell malignancies. This effort provides a platform for further compound optimization and evaluation for hematologic malignancies.

Disclosures:

Faris: MannKind Corp: Employment. Malyankar:MannKind Corp: Employment. Zeng:MannKind Corp: Employment. Kertesz:Mannkind Corporation: Employment, Equity Ownership. Vuga:MannKind Corp.: Employment. Rosario:MannKind Corp: Employment. Bot:MannKind Corp: Employment.
The Biocompatibility and Security of Magnetic Nanoparticles Fe3O4-DNR Used In Hematologic Malignancies Therapy
Authors: Chen Bao-An , Wu Wei-wei , Cheng Jian , Wang Jun , Gao Feng , Xu Wen-lin , Gao Chong , Ding Jia-hua , Sun Yun-yu , Song Hui-hui , Bao Wen , Liu Li-jie , Xia Guo-hua , Wang Xue-mei
Published: October 2019

Abstract 3970

The objective of this paper is to study the biocompatibility and security of self-prepared magnetic nanoparticles Fe3O4 (Fe3O4-MNPs) loaded with daunorubicin (DNR), which has the potential application in hematologic malignancies therapy. Hemolysis test was carried out to estimate it s blood toxicity; Fe3O4-MNPs loaded with DNR were intra peritoneally injected into mouse to calculate the LD50; micronucleus (MN) assay was reckoned to identify its genotoxicity; acute toxicity testing was evaluated its influence to mouse hepatic and renal functions. The result of hemolysis rate (HR) of Fe3O4-MNPs loaded with DNR was 2.908%, far less than 5%. Therefore, we concluded that self-prepated Fe3O4-MNPs loaded with DNR nanoparticles had no hemolytic reaction, and they consistent with the requirement of hemolytic test of biomaterials. The LD50 of Fe3O4-MNPs loaded with DNR nanoparticles to the mice was 1009.71mg/kg (relative content of DNR was 10mg/kg) and the 95% confidence interval was 769.11∼1262.40mg/kg, it had no significant difference compared with LD50 of using DNR only, which data was 8.51mg/kg and the 95% confidence interval was 6.48∼10.37mg/kg, and it had wide safety value circumscription. In micronucleus assay, compared Fe3O4-MNPs loaded with DNR nanoparticles experimental groups with negative control group, we found that the result had no significant difference (P > 0. 05) in micronucleus formation rate, while compared experimental groups with positive control group, the result had significant difference (P < 0. 05). The result indicated that Fe3O4 magnetic nanoparticles had no cacogenesis and mutagenesis. Acute toxicity testing showed that mice body weigh of control group, Fe3O4-MNPs loaded with DNR nanoparticles experimental group and isodose DNR group had no significant difference in 24h, 48h, and 72h after intra peritoneally injection; they had normal activity, eating and evacuation; toxic reactions such as instability of gait, convulsion, paralysis and respiratory depression were not been found; the alanine transarninase (ALT), blood urea nitrogen (BUN), and creatinine clearance rate (CCr) of Fe3O4-MNPs loaded with DNR nanoparticles experimental group was 66.0±28.55u/L, 9.06±1.05mmol/L, and 18.03±1.84umol/L, respectively, which had no significant difference compared with control group and isodose DNR group. From the results of our experiment, we could consider that self-prepared Fe3O4-MNPs loaded with DNR nanoparticles is a kind of high biocompatibility and security materials and perhaps is suitable for further application in hematologic malignancies therapy.

Disclosures:

No relevant conflicts of interest to declare.
Curcumin Reverses Resistance to Histone Deacetylase Inhibitors In Cutaneous T-Cell Lymphoma Cells: a Potential Role for NF-κB Signaling
Authors: Zhang Chunlei , Zhang Xiang , Duvic Madeleine
Published: October 2019

Abstract 3973

Histone deacetylase inhibitors (HDACi), including vorinostat (SAHA), depsipeptide (FK228), panobinostat (LBH589), belinostat (PXD101), and entinostat (SNDX275), show in-vitro and clinical activity against cutaneous T-cell lymphoma (CTCL) cell lines and patients' skin lesions [Zhang & Duvic, Expert Rev Dermatol 5: 393–401, 2010]. Vorinostat and depsipeptide were recently approved [Duvic et al, Blood 109: 31-9, 2007; Olsen et al, J Clin Oncol 25: 3109-15, 2007; Piekarz et al, J Clin Oncol 27: 5410-7, 2009], at response rates of 29% and 42%, respectively, but development of resistance remains an important clinical problem. Because we have shown that curcumin, the active ingredient of turmeric, exhibits anti-cancer activity through selective induction of tumor T-cell apoptosis and inhibition of NF-κB signaling in CTCL [Zhang et al, J Invest Dermatol 130: 2110-9, 2010], we now investigated whether curcumin combined with HDACi has synergistic anti-tumor effects in CTCL. HDACi-resistant MJ, HDACi-sensitive HH and HDACi cross-resistant HH/VOR CTCL cells were treated with HDACi (panobinostat, vorinostat, or enlinostat) plus or minus curcumin for up to 48 hrs. Cell viability was examined by the MTS assay and apoptosis by FACS analysis of annexin V/PI binding populations and/or cell cycle distribution. The NF-κB signaling pathway was analyzed by electrophoretic mobility gel shift assay and Western blotting. In MJ and HH cell lines, 5 nM panobinostat induced 1.4% and 11.4% apoptosis and 10 μM curcumin induced 24.5% and 29% apoptosis compared to vehicle controls. Panobinostat combined with curcumin induced 46.9% and 83.4% apoptosis in MJ and HH cell lines, respectively. Of interest, the HDACi cross-resistant HH/VOR CTCL cells were sensitive to curcumin alone and curcumin further enhanced panobinostat-induced apoptosis by 30% in the HH/VOR CTCL cells. Moreover, panobinostat combined with curcumin synergistically suppressed the DNA binding of NF-κB and decreased protein expression of the NF-κB activator RANK and NF-κBp65. Synergism was associated with down-regulation of NF-κB-regulated anti-apoptotic proteins (bcl-2, bcl-xL, and survivin), anti-proliferative proteins (c-myc and cyclooxygenase-2), and pro-invasive protein matrix metalloproteinase-9. Similar synergism was also seen when vorinostat or entinostat was combined with curcumin. These results suggest that HDACi could be combined with curcumin to enhance apoptosis of malignant T-cells through inhibition of NF-κB signaling in CTCL. Curcumin alone and in combination with HDACi may be an attractive strategy for the treatment of HDACi-refractory CTCL patients.

Disclosures:

Zhang: Novartis: Research Funding. Duvic:Novartis: Research Funding.
Indirubin-3'-Monoxime Induces Apoptosis and Autophagy In Acute Lymphoblastic Leukemia Cells and Chronic Myelogenous Leukemia Cells, Exhibits Limited Cytotoxicity In CD34+ Hematopoietic Stem Cells, Lymphocytes and Granulocytes
Authors: Lee Ming-Yang , Liu Yi-Wen , Chen Ming-Ho , Chuang Jing-Jing
Published: October 2019

Abstract 3984

The rates of treatment-related mortality (TRM) and relapse are unacceptably high in adults undergoing antileukemia treatments for acute lymphoblastic leukemia (ALL). So far has no better therapy with low side effects to improve long-term survival in these patients. Indirubin, a Chinese translational anti-chronic myelogenous leukemia (CML) agent, is able to induce cellular apoptosis. However, until now the functional action of IO on ALL remains still unknown. Therefore, here we investigated and compared the cytotoxic efficacy and action of indirubin-3'-monoxime (IO) in JM1 (ALL cell) and K562 (CML cell). ALL and CML cells were treated with a series of IO dose for 24 and 48h, and cell survival was determined by WST-1 assay. ALL and CML cells were shown to be similar susceptible to IO cytotoxicity. In order to clear in which way of cell death induced by IO, we performed analyses for apoptosis, necrosis and autophagy, respectively. After IO treatment, both ALL and CML cells were arrested in the G2/M cell cycle phase. In addition, an increase of sub-G1 proportion was caused. We found also increasing of caspase-3 activation and formation of cleaved PARP in a dose dependent manner. These were associated with the form of apoptosis. However, the caspase inhibitor Z-VAD-FMK only could partially prevent cell death in ALL and CML cells. When further analyzing the necrotic phenomenon through measuring LDH release, the result clearly showed that LDH release was not remarkable after cell treatment with high dose of IO. Besides, we observed surprisingly in Western blot the increasing expression of microtubule-associated protein light chain 3-II (LC3-II), which generally correlates to formation of autophagosomes. Because better antileukemic drug should not induce toxicity in normal blood cells as much as possible, so the cytotoxic effect of IO in CD34+ hematopoietic stem cells, lymphocytes and granulocytes was analyzed. Excitingly, results showed that IO could not affect cell viability of granulocytes, and IO cytotoxicity in lymphocytes was only marginal. If CD34+ hematopoietic stem cells were treated with IO, the rate of cell survival and their ability of differentiation were almost identical in contrast with non-treated control. Apparently, these data indicated that IO possesses the capability to induce apoptosis and autophagy in both CML and ALL cells. The most important is that the IO hardly influences the cell survival and the differentiation of CD34+ hematopoietic stem cells, the cytotoxic effect in granulocytes and lymphocytes is only limited. In conclusion, IO can be considered as a potential agent for clinical anti- ALL treatment.

Disclosures:

No relevant conflicts of interest to declare.
VEGFA Controls Haematopoietic Stem Cell Specification In a Dose-Dependent, Isoform-Specific Manner
Authors: Leung Amy , Ciau-Uitz Aldo , Vyas Paresh , Patient Roger , Porcher Catherine
Published: October 2019

Abstract 406

VEGFA signalling is critical for endothelial and haematopoietic development during embryogenesis. Perturbation of the VEGFA pathway results in defective haemopoietic stem cell (HSC) specification and vascular/arterial formation. Because of the intimate relationship between arterial and blood development, the contribution of VEGFA to HSC specification is unclear. Characterising the exact function of VEGFA is of primary interest for understanding mechanisms regulating haematopoietic specification. Here, we show that morpholino-induced knock-down of the co-repressor and oncoprotein ETO2 in Xenopus embryos leads to a failure of HSC specification via a VEGFA–dependent mechanism, independently from dorsal aorta (DA) formation and arterial specification. Knock-down of ETO2 represses Vegfa levels in somitic tissues along the migratory pathway from the dorsal lateral plate to the midline. This, in turn, leads to absence of Notch1 expression in the hemogenic endothelim of the DA and failure to activate the HSC transcriptional programme. DA formation and arterial specification occur normally, thus uncoupling VEGFA requirements for DA versus HSC formation. Then, we examined expression of the 3 VEGFA isoforms that differ in their physical and biological properties. In Eto2 morphant embryos, there was reduction (∼50%) in mRNA expression of the medium and long Vegfa variants in the somites. Importantly, Vegfa hypomorph embryos phenocopy the Eto2 morphant phenotype; this further demonstrates that reduction in VEGFA levels is sufficient for failure of HSC specification. Furthermore, restoration of medium Vegfa isoform (Vegfa170) levels alone in either Eto2 morphants or Vegfa hypomorph embryos was sufficient to re-establish the HSC programme. In summary, this is the first report of a dose-dependent, isoform-specific function of VEGFA at the onset of the HSC programme, independent from its functions in vessel formation and specification of the DA. We propose that signalling from VEGFA170 in somites instructs the HSC programme of the DA/HSC progenitors as they migrate towards the midline, in a paracrine manner. This VEGFA170 signal is critically required later in the haemogenic endothelium of the newly formed DA to initiate Notch1 expression and trigger the haematopoietic transcriptional programme. This work also reveals for the first time that ETO2 controls the onset of the HSC programme in a non cell-autonomous manner. This work furthers our understanding of the complex interplay between the cell populations controlling key signaling events leading to HSC specification. Manipulation of these signaling cascades may allow us to better develop strategies to produce and expand HSCs for therapeutic and regenerative purposes.

Disclosures:

No relevant conflicts of interest to declare.
High Bcl-2/Mcl-1 Gene Expression Ratio Identifies Molecular Sub-Groups of Multiple Myeloma Patients Extremely Sensitive to ABT-737
Authors: Bodet Linda , Pellat-Deceunynck Catherine , Gomez-Bougie Patricia , Dousset Christelle , Touzeau Cyrille , Le Gouill Steven , Avet-Loiseau Hervé , Bataille Regis , Amiot Martine
Published: October 2019

Abstract 4061

Multiple myeloma (MM) is a plasma cell malignancy presently incurable with heterogeneity in molecular abnormalities and treatment response. While the importance of Mcl-1 in maintaining survival of Human myeloma cell lines (HMCLs) has been well documented we recently identified a sub-group of HMCLs that highly expressed Bcl-2. This finding prompted us to investigate the sensitivity of HMCLs to ABT-737, a cell-permeant compound that binds with high affinity to Bcl-2 and antagonizes its anti-apoptotic function. The effect of ABT-737 on myeloma cell viability was assessed by APO2.7-PE staining on a large panel of HMCLs (n=26) representative of Multiple Myeloma molecular heterogeneity. We found 7 HMCLs highly sensitive to ABT-737 with an EC50 ranging from 7±0.4nM to 150±7.5nM. Myeloma cell death induced by ABT-737 occurred as early as 4 hours after treatment and led to Bax and Bak activation. While all these sensitive cell lines expressed a high level of Bcl-2, only Bcl-2/Mcl-1 gene expression ratio allowed the discrimination of highly ABT-737 sensitive from resistant HMCLs. High sensitivity (EC 50 <150 nM) was correlated to a ratio > 0.4. This result is in agreement with the high affinity of ABT-737 for Bcl-2 in contrast to Mcl-1. Induction of apoptosis may be due to the displacement of Bim, Puma or Bik from their endogenous complexes with Bcl-2, as demonstrated by immunoprecipitation assays. Interestingly, we demonstrated that all sensitive HMCLs harbored a t(11;14) while 77% (7 out of 9) of t(11;14) HMCLs are sensitive to ABT-737. Then, interrogation of the public expression database of MM patients showed that the Bcl-2/Mcl-1 ratio of both t(11;14) (median 0.29, range 0.05–0.71) and hyperdiploid (median 0.24, range 0.04–0.79) patients was significantly higher to all other groups of patients (p<.001). In order to fairly identify MM patients expressing high Bcl-2/Mcl-1 ratio we set up a flow cytometry assay. Preliminary results confirmed that primary myeloma cells with a high Bcl-2/Mcl-1 ratio are also highly sensitive to ABT-737. Altogether, the very high sensitivity of Bcl-2 high Mcl-1weak HMCLs and primary myeloma cells to ABT-737 (similar to the one of CLL) supports the idea of a clinical trial using Bcl-2 antagonists in a selected subset of t(11;14) and hyperdiploid Multiple Myeloma patients.

Disclosures:

Avet-Loiseau: Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees.
A New Xenograft Model for Studying Multiple Myeloma and Drug Response
Authors: Deutsch Varda , Farnoushi Yona , Cipok Michal , Kay Sigi , Naparstek Elizabeth , Goldstein Ronald
Published: October 2019

Abstract 4073

While new treatment options are available, multiple myeloma (MM) still remains an incurable malignancy of plasma cells with a grim prognosis. Practical in vivo models to study human MM may enable a better understanding of the biology of the disease, and better optimization of therapeutic strategies. The best current xenograft model, the immune-deficient NOD/SCID mice, recapitulates MM in vivo, however, the price is very costly and maintenance complex, with >1 month required to establish engraftment. Our goal was to develop a user friendly rapid alternative xenograft system for the preclinical assessment of MM growth and therapy. We recently described this new in-vivo system for studying human leukemia in the pre-immune turkey embryo 1,2. These embryos are inexpensive, require no maintenance, and are easily manipulated experimentally. Described here are the first attempts at application of this novel system to study MM and test therapies. Cell lines ARH-77 and CAG line and fresh patient cells (5 × 106/embryo) were injected IV into turkey egg chorioallantoic membrane veins on embryonic day E11. Engraftment of human cells in hematopoietic organs, bone marrow (BM) and liver was detected 7 days later (E18) by RTPCR, immunohistochemistry and flow cytometry and by circulating free light chain (6-25 mg/L) in the peripheral blood of 100% of the injected cell lines and 50% of patients myelomas. Treatment with Velcade (Bortezomib) or Revlimid IV on E13 (48 hours after MM cell injection), at drug levels that were precalibrated to be non-toxic to the developing embryonic BM, dramatically reduced engraftment, demonstrating the utility of this new model for testing drug activity in vivo. ARH-77 cells, detected by flow cytometry of the embryonic BM cells with anti-human CD19, CD38 and CD138, were inhibited from 8.5% engraftment to 0.72% after a single Velcade treatment, with an 18 fold decrease compared to untreated embryos in the ratio of human to avian cells in BM tissue. determined by Q-RT-PCR analysis of human alpha satellite and avian GAPDH DNA normalized per cell. Very similar results were obtained with Revlimid. The results presented suggest that with further work the turkey embryo model may provide an affordable, rapid and practical xenograft system in vivo for studying the biology of MM, for affordably testing MM therapies, as well for developing a new method for individualized patient screening for response or resistance to particular therapeutic agents.

1. Taizi M, Deutsch VR, Leitner A, Ohana A, Goldstein RS. A novel and rapid in vivo system for testing therapeutics on human leukemias. Exp Hematol. 2006;34:1698-1708.

2. Grinberg I, Reis A, Ohana A, et al. Engraftment of human blood malignancies to the turkey embryo: a robust new in vivo model. Leuk Res. 2009;33:1417-1426.

Disclosures:

No relevant conflicts of interest to declare.
A Novel Anilinoquinazoline Derivative Inhibits Growth of High-Risk Myeloma Cells In Vivo and Regulates Differentiation of Osteoclasts
Authors: Ozaki Yoshie , Naito Yuhei , Yanagawa Hiroshi , Suzuki Yuya , Du Wenlin , Yamada Taketo , Matsuo Koichi , Doi Nobuhide , Iijima Shiro , Matsushita Maiko , Tahara Kayoko , Hattori Yutaka
Published: October 2019

Abstract 4076

Purpose:

Despite recent advances in the use of newly developed drugs, high-risk multiple myeloma (MM) patients harboring del13q, t(4;14) or del17p revealed significantly shorter survival. To overcome the limitation, we have screened forty synthetic anilinoquinazoline (AQ) derivatives, and found a novel compound, Q15, which significantly inhibited the growth of MM cell lines with high-risk chromosomal abnormalities. The purpose of this study is to examine anti-tumor and anti-osteoclastogenic activities of Q15 and to clarify the possibility of development of new drug effective for high-risk MM and bone diseases.

Methods and Results:

Forty AQ derivatives were synthesized and screened for anti-proliferative effect on KMS34 cells. Q15 strongly inhibited growth of t(4;14)-positive KMS34 cells and induced apoptosis in much lower concentration (IC50=78nM) compared with gefitinib (IC50=2500nM), a representative AQ. Q15 also inhibited growth of other MM cell lines harboring high-risk chromosomal abnormalities. It was also found that Q15 did not inhibit intracellular tyrosine phosphorylation induced by EGF, FGF-2, HGF and IL-6, suggesting that Q15 showed anti-tumor activity in a different mechanism from that of gefitinib. In vivo anti-myeloma activity was evaluated by intraperitoneal injection of Q15 into KMS34-bearing lcr/SCID mice. Twenty mg/kg Q15 significantly delayed the tumor growth in these mice. Histopathological examinations revealed apoptosis of MM cells in Q15-treated mice. Growth of colony-forming cells was not suppressed by much higher concentrations (25μ M) of Q15 than IC50, suggesting low hematopoietic toxicity of Q15. In pharmacokinetic study using high-performance liquid chromatography (HPLC), the plasma concentration of Q15 in mice reached a maximum (Cmax=4.5μ M) at 1.5hr after injection, and its half life (T1/2) was 4.5hr. In addition, anti-osteoclastogenic activity was also examined by adding Q15 to M-CSF/RANK ligand-induced osteoclastogenic culture of bone marrow mononuclear cells from C57BL/6JJcl mice. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts was reduced in the presence of Q15.

Conclusion:

Q15, a novel AQ derivative, has anti-MM activity in vivo and is a potentially safe and effective drug for high-risk MM with bone lesions.

Disclosures:

No relevant conflicts of interest to declare.