bluuebunny/crossref_metadata_2025 huggingface dataset:
A Phase II Study of Risk-Adapted Intravenous Melphalan in Patients with AL Amyloidosis,
Authors: Mollee Peter , Tiley Campbell , Cunningham Ilona , Moore John , Prince Miles , Cannell Paul , Gibbons Steve , Tate Jill , Paul Sanjoy , Gill Devinder S
Published: October 2019

Abstract 3973

Aims:

High-dose IV melphalan is standard autograft (ASCT) conditioning in patients with AL amyloidosis, but careful patient selection is required to avoid high transplant-related mortality rates. In patients ineligible for ASCT, intermediate-dose IV melphalan (IDM) may provide advantages over oral dosing where the already variable intestinal absorption is complicated by gastrointestinal amyloid deposition. We aimed to assess a risk-adapted strategy to IV melphalan dosing in patients with AL amyloidosis.

Methods:

The MM8 study was a prospective clinical trial conducted by the Australasian Leukaemia and Lymphoma Group. Transplant candidates (minimal cardiac disease including BNP<300ng/L, age <65, ECOG<2, <2 organs involved) received melphalan at 140 or 200mg/m2 IV. All other patients received IDM (melphalan 20mg/m2 IV d1 and dexamethasone 40mg PO d1-4 every 4 weeks for 3–6 cycles). Treatment was continued until 2 cycles beyond free light chain normalisation or maximal response, or to a maximum of 6 cycles. IDM was discontinued if there was no haematologic response after 3 cycles.

Results:

21 patients enrolled with median age 61yrs. Organ involvement was cardiac 48%, renal 81%, liver 14%, neurologic 43%. 6, 10 and 5 were low, intermediate and high cardiac biomarker risk, respectively. 7 underwent ASCT and 14 IDM.

The trial closed early due to excessive myelotoxicity in the IDM arm. In this cohort, grade 3/4 neutropenia and thrombocytopenia during the 1st cycle occurred in 54% and 23%, with 15% having neutropenic fever. Grade 3/4 neutropenia and thrombocytopenia during any cycle occurred in 85% and 54% of patients, with 38% having neutropenic fever. First cycle severe neutropenia was not predicted by age, cardiac or renal function but was significantly more common with lower body surface area (p=0.01). 8 IDM patients died before the 6 month response assessment, 2 achieved a 50% reduction in baseline involved FLC and 4 failed to respond. All 7 patients receiving ASCT are alive at a median of 33 months.

Conclusions:

IV melphalan at 20mg/m2 is excessively myelotoxic for patients with AL with no evidence of response benefit to offset this toxicity. BNP<300ng/L may identify patients suitable for ASCT.

This study was supported by the Leukaemia Foundation of Queensland and Amgen Australia.

Disclosures:

Mollee: Amgen Australia: Research Funding; Leukaemia Foundation of Queensland: Research Funding. Cannell:Nereus Pharmaceuticals:.
Clofarabine-Based Chemotherapy for Relapsed/Refractory Adult Acute Lymphoblastic Leukemia and Lymphoblastic Lymphoma. the Spanish Experience
Authors: Barba Pere , Sampol Antonia , Calbacho María , Gonzalez Jose , Serrano Josefina , Martinez-Sanchez Pilar , Fernandez Pascual , Garcia-Boyero Raimundo , Martino Rodrigo , Delgado Pilar , Hernandez-rivas Jose Angel , Del Potro Eloy , Gonzalez-Muñiz Soledad , Gomez-Sanchez MAria Isabel , de la Fuente Adolfo , Bueno Javier , Ribera Josep-Maria
Published: October 2019

Abstract 4246

Background:

Patients with relapsed/refractory acute lymphoblastic leukemia (ALL) have a very poor outcome with limited therapeutic options available. Clofarabine (Clo) is a second generation purine nucleoside analogue increasingly used in these patients, mainly in the pediatric setting.

Aims:

To evaluate the toxicity profile and outcome of Clo-based chemotherapy regimens used in adult ALL in the Spanish PETHEMA centers.

Materials and Methods:

Sixteen of the 22 PETHEMA centers to have used Clo in adult patients (≥16 years) with a definitive diagnosis of ALL (n=25) or lymphoblastic lymphoma (LL) (n=5) participated in the study. Patient-, disease-, toxicity- and outcome-related data were retrospectively collected and analyzed.

Results:

Thirty patients received Clo-based regimens from January 2007 to May 2011. Median age at diagnosis was 33 years (range 16–73). B- and T- lineage diseases were found in 20 (67%) and 10 (33%) patients, respectively. Median number of treatment regimens before Clo was 3 (range 1–7) and median time of follow-up for survivors was 5 months (range 2–25). Clo-based combinations included Clo-cyclofosfamide (Cy) (n=11, 36%), Clo-Cy-etoposide (n=8, 27%) Clo-cytarabine (n=6, 20%) and Clo monotherapy (n=5, 17%) for a median of 1 (range 1–3) cycle. Clo total dose/cycle in most patients was 200mg/m2. Hematological and infectious grade III-IV toxicities were found in 30 (100%) and 20 (67%) patients, while hepatic, gastrointestinal and renal grade III-IV toxicities were less common (17%, 17% and 0%, respectively). Eight (27%) and 2 (7%) patients achieved CR (complete remission) and CRi, respectively (overall CR rate 33%), while 7 patients died before disease evaluation. CRs were observed in B- (n=8, 40%) and T- (n=2, 20%) lineage diseases and in patients with normal and adverse cytogenetics (including the only Ph+ case). Younger patients (median age 25 years vs 36 years, p=0.1) and those who received a previous allogeneic hematopoietic cell transplantation (allo-HCT) (67% vs 29%, p=0.1) showed a non-significant trend for a higher overall CR achievement. Six (20%) patients (5 in CR) underwent allo-HCT after Clo-based treatment. Five (17%) patients were alive at 5 months of Clo treatment for an overall survival probability of 10% (95%CI 4–16). The most frequent death causes were disease progression (n= 16, 64%) and infections (n= 3, 12%).

Conclusion:

Despite their toxicity, Clo-based chemotherapy regimens may allow CR achievement in heavily pretreated relapsed/refractory ALL and LL patients.

Disclosures:

Barba: Genzyme: Consultancy.
Leukemia Cell-Derived Exosomes Targeted Dendritic Cells Can Induce More Strong Anti-Leukemia Response Than Exosomes Alone
Authors: Sheng Chang , Sun Jing , Wang Chun , Hao Siguo
Published: October 2019

Abstract 4278

Exosomes are the newest family number of ‘bioactive vesicles’ that play important roles in antigen presentation. Recent clinical trials about exosomes have shown minor clinical benefit. However, exosomes derived from leukemia cell lines were less studied, In this study, we investigated the biological properties and anti-leukemia immunity of leukemia cell-derived exosomes (LEX). The results showed that like other tumor cells, similarly, leukemia cells can release exosomes too. Transmission electron microscopy showed that exosomes derived from NB4 cell, a promyloblastic leukemia cell line and K562 cell, a chronic myeloid leukemia cell line were membrane vesicles with diameter about 50–100μ m, and loading their specific moleculars such as RAR-α and ABL and adherent moleculars such as ICAM-1. Similarly, our data also demonstrated the presence of Hsp70 in these two leukemia cell-derived exosomes. The data from two-dimensional electrophoresis of K562 cell-derived exosomes showed that most of molecules expressing on leukemia cells were detected on their exosomes, indicating that LEX load most of proteins expressed on their original leukemia cells. Out data also demonstrated that LEX can induce anti-leukemia CTL response and LEX-targeted DC can induce more efficient anti-leukemia immunity compared with LEX alone. In conclusion, exosome-based therapies could be considered as a promising strategy to prolong disease-free survival in patients with leukemia after consolidation or hematopoietic stem cell transplantation.

Disclosures:

No relevant conflicts of interest to declare.
Anti-Leukemic Effect of Tetra-Arsenic Oxide in Acute Promyelocytic Leukemia Cells in Vitro and In Vivo
Authors: Chong So Young , Jang Moon Ju , Lee Sun Ju , Huh Ji Young , Bae Jeehyeon , Chung Kwang-Hoe , Oh Doyeon , Kwaan Hau C.
Published: October 2019

Abstract 4306

Arsenic trioxide (As2O3; ATO) has been established to be an effective agent for treating acute promyelocytic leukemia (APL). Tetra-arsenic oxide (As4O6; TAO) is a new arsenic compound which has shown anti-proliferative and apoptosis-inducing effects against human leukemic and solid tumor cells, but TAO has never been studied in APL so far. We investigated the effect of TAO in APL cell lines (NB-4), and evaluated their anti-leukemic effect in murine xenograft model. In both the XTT assay and in FACS analysis, TAO inhibited cell proliferation and induced apoptosis in NB4 cell lines as effective as ATO in dose dependent manner. In FACS analysis, TAO induced apoptosis in NB4 cell lines as effective as ATO in a dose dependent manner. However, expression of differentiation (CD11b) was unchanged. For in vivo study, we established a xenograft model in nude mice (BALB/cBy J-nu) using the NB4 cell line. 1 × 107 NB4 cells were inoculated into the flank subcutaneously. When the tumor size reached 1.0 cm3, intra-peritoneal ATO or TAO treatment (5mg/kg or 10mg/kg) was initiated and administration was performed daily. We found that TAO (10mg/kg) significantly inhibited tumor growth of the inoculated NB4 cell as effective as ATO (10mg/kg). These results suggest that TAO may have a potential for the treatment of APL.

Disclosures:

No relevant conflicts of interest to declare.
Variants At 6q21 Associated with Radiation-Induced Second Malignancies After Hodgkin Lymphoma Differentially Regulate PRDM1 Expression Following Radiation Exposure
Authors: Onel Kenan , Best Timothy , Yu Shan
Published: October 2019

Abstract 431

Childhood Hodgkin lymphoma (HL) survivors are particularly susceptible to second malignant neoplasms (SMNs) caused by radiation therapy (RT). To identify genetic risk factors for SMNs, we undertook a genome-wide association study of 96 SMN cases and 86 SMN-free controls. Both cases and control sets consisted of long-term survivors of childhood HL who received antecedent RT. Two variants at chromosome 6q21 intergenic between PRDM1 and ATG5 were associated with SMNs (rs4946728: P = 1.26×10−9, OR = 3.17 [2.16–4.65] and rs1040411: P = 4.68×10−8, OR= 2.41 [1.76–3.32]). These results were replicated in an independent set of 63 SMN cases and 75 SMN free controls (rs4946728: P =. 003, and rs1040411: P =. 04). The risk variants form a haplotype associated with decreased basal expression of PRDM1, as well as impaired induction of the PRDM1 protein by radiation exposure. An analysis of the genomic region spanned by the SMN-associated haplotype revealed four evolutionarily conserved putative enhancers, characterized by both H3K27 acetylation and DNase I hypersensitivity. To determine whether these putative enhancers modulate expression following radiation exposure, we cloned each into the pGL3 vector downstream of a luciferase reporter gene. We then transiently transfected each construct into lymphoblastoid cell lines. Data will be presented showing how carriage of either the risk or protective haplotype alters the activity of these enhancers in response to radiation. Furthermore, ChIP-Seq data indicate that NFkB variably binds each putative enhancer. As NFkB has previously been shown to induce PRDM1 expression and is itself activated by radiation, we treated LCLs with an NFkB inhibitor to determine whether NFkB is involved in induction of PRDM1 following radiation exposure. These data demonstrate that incorporating etiologic exposures, such as radiation, can enhance the discovery disease-associated genetic variation and guide functional follow-up experiments.

Disclosures:

No relevant conflicts of interest to declare.
Role of Anticoagulation in Patients with Inferior Vena Cava Filter but with No Risk Factor or History of DVT/PE
Authors: Khalife Mustapha A. , Dabak Vrushali S. , Hammoud Marwa , Arnaout Karim
Published: October 2019

Abstract 4317

Introduction:

Inferior Vena Cava (IVC) filters have been available for almost 40 years but their clinical utility and safety have not been completely evaluated in patients with no previous history of deep vein thrombosis (DVT) or pulmonary embolism (PE). The role of anticoagulation in patients with IVC filter with no history of DVT/PE is questionable. In this study, we try to determine if there is a role or benefit from anticoagulation in patients with an IVC filter placed but without any other risk factor for deep vein thrombosis (DVT) or pulmonary embolism (PE).

Methods:

we retrospectively reviewed the charts of 562 patients who had an IVC filter placed between 2003 and 2005. 442 patients were excluded because they had a history of DVT/PE, or because of a hypercoagulable state (genetic predisposition, prolonged hospitalization/immobilization, surgery, or malignancy). Of the 120 remaining patients included in this study, 6 had their IVC filter removed. And therefore we only analyzed the charts of 114 patients who had a permanent IVC filter placed for prophylactic reasons. Group 1 consisted of 17 patients who received different forms of anticoagulation (subcutaneous heparin, low molecular weight heparin or coumadin). Group 2 consisted of the remaining 97 patients who did not receive any form of anticoagulation.

Results:

2 out of 17 patients in group 1 had a DVT and 14 out of 97 patients in group 2 had a DVT. The incidence of DVT was 11.8% in group 1 versus 14.4% in group 2 (p-value 0.770). The median onset of DVT/PE after IVC filter placement was 31 days. The median time of follow up was 77.33 months.

Conclusion:

Patients who had a permanent prophylactic IVC filter placed but with no history or risk factors for DVT/PE appear to be at an elevated risk for new DVT/PEs. In these patients, the role of anticoagulation is questionable. With a median 6 year follow up, anticoagulation seemed to non significantly lower the risk of DVT/PE. Larger randomized prospective trials are needed to examine the efficacy and duration of anticoagulation in patients with a prophylactic IVC filter placed.

Disclosures:

No relevant conflicts of interest to declare.
Generation of Red Blood Cells From Human Embryonic Stem Cells (ES) with Increased Efficacy
Authors: Srivastava Anand S , Azad Babak Esmaeli , De Necochea Campion Rosalia , Carrier Ewa
Published: October 2019

Abstract 4334

It is estimated that for every unit of donated blood, two units are required in North America. The current rate of blood donation is stagnant while the need increases by 6–8% annually. In order to overcome this difficulty, we have developed an improved method to generate red blood cells from human embryonic stem cells (H9) with increased efficacy. In addition to xeno-free conditions and standard cytokine cocktail used for hematopoietic differentiation of human embryonic stem cells (Carrier et al. J Transl Med. 2009; Vol 7: 27), we have introduced a new method of improved growth and differentiation of human ES cells with hypoxia-induced mesenchymal stem cells, obtained from allogeneic adult bone marrow donors. This technique increased efficacy of red blood cell production by 5–25 fold.

We have developed a bioscaffold–> microsphere-based culture system with highly porous surface allowing culturing of a very large number of embryonic stem cells per one culture condition. This culture system avoids shear forces and damage to the cells, and facilitates removal and recycling of the microspheres. The in vitro obtained human ES-derived red blood cells are enucleated and do not produce tumors (efficacy of enucleation is 65–95%). The laser-based system is utilized to eliminate nucleated cells from the culture.

The problem with hES-derived red blood cells is that they are produced in small numbers and process is very costly. We are developing a 3-phase bioreactor with computerized programming, which will increase every step of the differentiation process and allow recycling of feeder cells and cytokines. In this system we will utilize iron-loaded microspheres coated with hypoxia-processed mesenchymal stem cells as a main culture unit. The in vitro generated human ES-derived red blood cells upscaled in a bioreactor will be used for the off-shelf production of red blood cells for clinical use.

Disclosures:

Srivastava: Giostar: Employment, Equity Ownership. Azad:Dnamicroarray, Inc.: Employment, Equity Ownership. Carrier:Giostar: Consultancy; Samaritan Pharmaceuticals: Consultancy; Entest Biomedical: Consultancy, Equity Ownership; America Stem Cells: Consultancy, Equity Ownership; Millenium: Speakers Bureau; NovaRx: Employment, Equity Ownership.
Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine
Authors: Majumder Rinku , Liang Xiaoe , Quinn-Allen Mary Ann , Kane William H. , Lentz Barry R.
Published: October 2019

Abstract 4344

Localization of blood coagulation enzymes and cofactors to the surface of procoagulant anionic phospholipid membranes is a central paradigm in hemostasis and thrombosis. The prototype for these complexes is the prothrombinase complex that catalyzes the conversion of prothrombin to thrombin. This enzyme complex consists of the enzyme factor Xa, the cofactor factor Va and calcium bound to a phospholipid surface. Formation of the prothrombinase complex results in a greater than 105-fold rate enhancement in the conversion of prothrombin to thrombin. Abnormal regulation of the prothrombinase complex has been demonstrated to be critical in the pathogenesis of thrombotic disorders.

Constituents of platelet membranes regulate the activity of the prothrombinase complex. We demonstrate that membranes containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) bind factor Va with high affinity (Kd∼10 nM) in the absence of phosphatidylserine (PS). These membranes support formation of a 60–70% functional prothrombinase complex at saturating factor Va concentrations. While reduced interfacial packing does contribute to factor Va binding in the absence of PS, it does not correlate with the enhanced activity of the Xa-Va complex assembled on PE-containing membranes. Instead, specific protein-PE interactions appear to contribute to the effects of PE. In support of this, soluble C6PE binds to recombinant factor Va2 (Kd ∼6.5 mM) and to factor Xa (Kd ∼ 91 mM). C6PE and C6PS binding sites in factor Xa are specific, distinct and linked, as binding of one lipid enhances the binding and activity effects of the other. C6PE triggers assembly (Kdapp ∼40nM) of a partially active prothrombinase complex between factor Xa and factor Va2, compared to Kdapp for C6PS ∼2nM. We report here seven significant new observations:PE effects kcat more than KM. A kcat effect strongly implies an enzyme or cofactor conformational change, while a KM effect could be due to conformational changes or to altered binding of enzyme-cofactor and substrate to the membrane.PE promotes binding of factor Va to membranes.PE promotes assembly of a prothrombinase complex due its effects on factors Va and Xa independent of the presence of a membrane and thus not due to its effects on membrane packing.30% PE does not produce a measurable effect on factor Xa membrane binding.C6PE and C6PS bind synergistically to sites on factor Xa to alter its activity.C6PE binds to factor Va with greater affinity than does C6PS, but binding of these lipids to sites on this protein is not synergistic.Both PS and PE promote prothrombinase activity, but the magnitudes of these effects do not correlate with their effects on membrane interfacial packing.

These findings provide new insights into the possible synergistic roles of platelet PE and PS in regulating thrombin formation, particularly when exposed membrane PS may be limiting.

Disclosures:

No relevant conflicts of interest to declare.
Clinical and Hematologic Manifestations in Patients with Diamond-Blackfan Anemia
Authors: Kim Soon Ki , Kang Hyoung Jin , Ahn Hyo Seop , Park Kyung Duk , Shin Hee Young , Kim Chul Soo
Published: October 2019

Abstract 4378

Background

Diamond-Blackfan anemia (DBA), characterized by impaired red cell production, is a rare condition that usually occurs symptomatic in early infancy. The purpose of this study was to assess the nationwide experiences of DBA that were encountered over a period of 20 years as a part of further molecular analyses.

Methods

Medical records of 56 patients diagnosed to have DBA were reviewed retrospectively from Nov. 1984 to July 31, 2010. Fifteen clinics including 13 university hospitals have been participated.

Results

Male to female ratio was 1.67:1 (males 35 cases, females 21). The median age of diagnosis was 4 months (range birth to 52 months of age), with the diagnosis of 74.1% before 1 year of age. From 2000 to 2009, 33 cases with DBA were registered, which showed the incidence of 6.6 cases per million. Excluding growth retardation, 21 cases (38.2%) showed congenital defects: thumb deformities (8 cases), ptosis (5), coarctation of aorta (5), ventricular septal defect (4), strabismus (3), cleft palate (2), mitral valve prolapsed (2). The mean hemoglobin (Hb) concentration was 5.1±1.9 g/dL, and median, 5.2 g/dL. The mean MCV was 93.4±11.6 fL, mean HbF 7.7±11.3%, mean reticulocytes 19,700/mm3 and mean adenosine deaminase 21.3. The mean cellularity of bone marrow was 75%, with the ME ratio of 20.4:1. Among 47 patients who had been treated with steroids, 23 patients (48.9%) did not need further steroids after remission. The mean duration of steroid use was 7 years and 2 months (maximum 22 years). Among 28 cases who received steroid, 9 patients had been controlled with the oral steroid of 5 mg or less per day. Eight patients with DBA that received hematopoietic transplantation have survived. Cancers developed in 2 cases (3.6%).

Conclusion

The incidence of DBA is similar with the data already published, but male predilection in our study. Although all patients responded to initial treatment with steroids, about a half needed further steroids after remission. It is necessary to collect the further data including management pathways from nationwide DBA registries, with the molecular analyses.

Disclosures:

No relevant conflicts of interest to declare.
Survival Of AML Patients Relapsing After Allogeneic Stem Cell Transplantation: A Center For International Blood and Marrow Transplant Research Study
Authors: Bejanyan Nelli , Weisdorf Daniel J. , Wang Hailin , Logan Brent R. , Devine Steven M. , de Lima Marcos , Bunjes Donald W. , Zhang Mei-Jie
Published: October 2019

Despite the curative potential of allogeneic hematopoietic cell transplantation (alloHCT), a large number of patients with acute myeloid leukemia (AML) experience leukemia relapse after HCT. Although subsequent remissions can be achieved with salvage therapy, they are usually short lived. We evaluated survival of AML patients relapsing after alloHCT to identify prognostic factors for survival after post-transplant relapse. 1788 pediatric and adult AML CR1 & CR2 patients relapsed post-transplant (1990-2010). The median age at HCT was 30 years (yr; range, <1-76). De novo leukemia occurred in 81% and intermediate risk cytogenetics in 45%. 1374 patients (77%) underwent myeloablative (MA) and 936 patients (52%) related donor HCT. Active GVHD prior to relapse occurred in 727 subjects (41%). Median time from transplant to relapse was 7 months (mo; range, 1-177). At relapse, 357 patients (20%) received supportive care, whereas 1231 patients (69%) received intensive therapy, including chemotherapy (CT) alone (n=660), donor lymphocyte infusion (DLI)±CT (n=202; DLI+CT=51) and 2nd alloHCT±CT and/or DLI (n=369; 49% 2nd alloHCT were myeloablative). Among evaluable patients (n=846) who received intensive therapy, CR rate was 29% (n=246) [15.6% for CT alone (n=443), 37.4% for DLI±CT (n=139) and 47.3% for 2nd alloHCT±CT±DLI (n=264)]. After a median follow up of 39 mo (range, <1-193) after relapse, 229 patients (13%) survived with 5-yr overall survival of 3% (95% CI 2-5%) for patients relapsing within 6mo, 11% (95% CI 8-14%; p<0.0001 comparing to <6 mo) for 6mo-2yr, 21% (95% CI 14-28%; p<0.0001 comparing to <6 mo) for 2-3yr and 31% (95% CI 23-39%; p<0.0001 comparing to <6 mo) for ≥3yr groups. Persisting AML (n=1271, 71%) was the most common cause of death. In multivariable analysis, longer time from alloHCT to relapse (RR 0.55 6mo-2yr, 0.39 2-3yr and 0.28 ≥3yr; p<0.0001) and reduced intensity conditioning (RIC) (RR=0.77; 95% CI 0.66-0.88, p=0.0002) were associated with significantly lower mortality. In contrast, age over 40 yr (RR=1.42, 95% CI 1.24-1.64; p<0.0001), active GVHD at relapse (RR=1.25, 95% CI 1.13-1.39; p<0.0001), adverse cytogenetics (RR=1.37, 95% CI 1.09-1.71; p=0.0062), mismatched URD (RR=1.61, 95% CI 1.22-2.13; p=0.0008) or cord blood (RR=1.23, 95% CI 1.06-1.42; p=0.0078) HCT had inferior survival. We confirm poor survival of AML patients relapsing after alloHCT. Yet patients with relapse beyond 6 mo have better survival and may benefit from intensive therapy, DLI or 2nd alloHCT.

Disclosures:

No relevant conflicts of interest to declare.
Free Thiol Groups In Von Willebrand Factor (VWF) Are Required For Its Proper Function Under Physiological Flow Conditions
Authors: Solecka Barbara , Fuchs Birte , Weise Christoph , Kannicht Christoph
Published: October 2019

Background

The multimeric plasma glycoprotein von Willebrand factor (VWF) is exceptionally rich in cysteine, and its structure is largely determined by inter- and intramolecular disulfide bonding. Additionally, VWF was shown to contain unpaired cysteine residues potentially affecting protein function. The significance of free thiols on the surface of plasmatic VWF has been confirmed previously with respect to platelet binding under pathologically high shear stress. Furthermore their potential involvement in functional VWF self-association occurring at elevated shear stress has been suggested.

Aims

Objective of the present study was to investigate whether free thiol groups of plasma VWF contribute to the physiological VWF function under high physiological arterial shear stress conditions. Furthermore, we aimed to elucidate possible underlying mechanisms involved in this regulation.

Methods

Free and accessible thiol groups of plasma-derived VWF were blocked with N-ethylmaleimide (NEM). Derivatization was followed by detailed structural and functional examination including multimer analysis (MMA) and Fourier transform infrared spectroscopy (FTIR). Functional differences between the NEM-derivatized sample and the control sample were detected using an in vitro flow chamber system with respect to VWF-mediated platelet adhesion to collagen. Interactions with collagen type III and platelet glycoprotein (GP)Ib receptor were investigated using surface plasmon resonance (SPR). Identification of accessible cysteine residues was accomplished using biotin-linked maleimide (MPB) followed by analysis of multimer and domain incorporation as well as mass spectrometry.

Results

Blocking free thiol groups provoked substantial loss of VWF activity with respect to platelet recruitment to collagen type III under flow. The lowered platelet adhesion to collagen type III was shown to be a combined effect of inhibition of (i) the initial VWF binding to collagen type III as well as (ii) VWF-platelet GPIb interaction. Free thiol groups were accessible for derivatization solely on the surface of coiled multimers. Domain incorporation studies revealed a high level of derivatization in VWF N- and C-terminus. This was in accordance with the mass spectrometric analysis, where 19 MPB-derivatized peptides, predominantly located at the N- and C-terminus, could be identified.

Conclusion

Blocking free thiol groups in VWF significantly impaired mediation of platelet adhesion under physiological shear stress conditions. This result suggests an essential functional role of free thiol groups in VWF regarding binding to subendothelial matrix as well as platelet recruitment.

Disclosures:

Solecka: Octapharma AG: Employment. Fuchs:Octapharma AG: Employment. Kannicht:Octapharma AG: Employment.
Recombinant Human Thrombopoietin Promotes The Recovery Of Megakaryocyte Lineage In Patients With Severe Aplastic Anemia Receiving Immunosuppressive Therapy
Authors: Shao Zonghong , Dong Qi'e , Fu Rong
Published: October 2019

Objective

To assess the effectiveness of recombinant human thrombopoietin (rhTPO) in severe aplastic anemia (SAA) patients receiving immunosuppressive therapy (IST).

Methods

Eighty SAA patients receiving IST during a period from January 2007 to December 2011 were included in this retrospective analysis. Thirty-two subjects also received rhTPO treatment (15,000 U, three times a week, subcutaneously). The remaining 48 patients did not receive rhTPO treatment. The choice of using (or not using) rhTPO was based on physician discretion, and more importantly, patient will (partly based on financial capability to afford the medication). rhTPO was discontinued when platelet count returned to normal range. Hematologic response, bone marrow recovery, transfusion interval (platelet or red-cells), and the time to transfusion-free status were compared.

Result

At 6th months after the treatment, hematologic response along at least one lineage was achieved in 65.6% of the subjects receiving rhTPO vs. 41.7% in those who did not receive rhTPO (p=0.04). Response rate of megakaryocyte and erythroid lineage at 3rd months was also higher in subjects receiving rhTPO than in those who did not (43.8% vs. 10.4%, p=0.001; 50.0% vs. 20.8%, p=0.006). The mean number of megakaryocyte per bone marrow slide was higher in subjects receiving rhTPO than those who did not (9.7±3.1 vs. 2.6±4.2, p=0.002) after three months. The percentage of nucleated erythroid cells in bone marrow was also higher in subjects receiving rhTPO after three months (22.2±13.2% vs. 13.6±13.9% in those who did not receive rhTPO; p=0.007). The percentage of reticulocytes in peripheral blood was higher in subjects receiving rhTPO (1.9±1.4% vs. 0.7±0.4% in those who did not receive rhTPO; p=0.001) after three months. Myeloid percentage in bone marrow did not differ at any time points (3, 6, or 9 months). The need for platelet transfusion was lower in subjects receiving rhTPO (transfusion interval: 13.8±14.3 vs. 6.9±5.2 and 26.3±28.9 vs. 15.7±13.1 days in those who did not receive rhTPO during the first three and six months, respectively; P=0.004, P=0.03). The need for red cell transfusion was also lower in subjects receiving rhTPO (interval: 32.5±22.0 vs. 11.9±7.2 days and 50.4±27.9 vs. 23.9±20.1 days during the first three and six months, respectively; p=0.001 P=0.009). Time to independence from platelet transfusion was significantly shorter in subjects receiving rhTPO (99.9±49.9 vs. 156.3±14.5 days in those who did not receive rhTPO; p=0.01).

Conclusion

rhTPO improves hematologic response and promotes bone marrow recovery in SAA patients receiving IST.

Disclosures:

No relevant conflicts of interest to declare.
A NUP98-HOXD13 Leukemic Fusion Gene Leads To Aberrant Actin Localization In Dysplastic Megakaryocytes
Authors: Caudell David L. , Okyere Benjamin , Cawley Jacob , Puthiyaveetil Abdul Gafoor A. , Heid Bettina
Published: October 2019

Myelodysplastic syndrome (MDS) is a hematopoietic malignancy characterized by peripheral cytopenias due to bone marrow (BM) failure. Megakarypoiesis, megakaryocyte (MK) production, and platelet release are impaired in in some cases of MDS. Patients often have fewer, but larger circulating platelets, which have abnormal demarcation membrane systems (DMS); the DMS, which determines the number and size of platelets released, is dependent on actin formation. However, the precise role of actin during megakaryopoiesis is poorly understood. Transgenic mice that express the fusion gene NUP98-HOXD13 (NHD13) is a model for MDS and have dysplastic MKs in BM, and macro platelets in circulation. We hypothesized that expression of NHD13 disrupts actin localization during megakaryopoiesis resulting in reduced platelet release and macro platelet formation. To test the hypothesis, BM from wild type (WT) and NHD13 mice were flushed and cultured in media supplemented with Thrombopoietin for 5 days. Following in vitro propagation, MKs were harvested over a discontinuous gradient for downstream experiments. Sternums were also fixed in paraformaldehyde, stained with hematoxylin and eosin, and evaluated by light microscopy to analyze MK morphology in vivo. NHD13 BM contained many dysplastic MKs. Harvested MKs and BM cores from one femur were processed and analyzed by transmission electron microscopy (TEM) and the ultrastructural properties of the DMS detailed. TEM of MKs showed NHD13 leads to formation of an irregular DMS along with abnormal distribution of unusually large granules in MK cytoplasm. Cultured MKs were also cytospun onto glass slides, labeled with fluorescent-tagged F-actin and Myosin IIa and the cytoskeleton visualized by confocal microscopy. WT MKs in vitro had two phenotypes: (1) MKs with myosin and actin evenly dispersed in the cytoplasm and (2) MK with actin predominantly in the periphery of the cytoplasm. In contrast, transgenic MKs displayed only the former phenotype suggesting that actin localization is impaired in NHD13 MKs. Finally, MKs were stimulated with estrogen and adhered to fibrinogen matrices to determine their proplatelet formation functionality. Our results showed impaired proplatelets formation in NHD13 MKs. These data suggest that expression of NHD13 leads to aberrant actin localization leading to dysplastic MK differentiation and macro platelet release. Understanding molecular mechanisms of abnormal megakaryopoiesis in MDS is important as many MDS patients die of hemorrhagic complications. Further studies using this model system will provide a platform for translational research and should reveal potential therapeutic targets in MDS, leading to improved patient care/survival.

Disclosures:

No relevant conflicts of interest to declare.
Expression Pattern Of Mirnas Is Not Predictive For Poor Prognosis In BCR-ABL1-Like Childhood Acute Lymphoblastic Leukemia
Authors: Akbari Moqadam Farhad , Lange-Turenhout Ellen , Veer Arian van der , Marchante João R.M. , Pieters Rob , Den Boer Monique L.
Published: October 2019

Childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a heterogeneous disease in which the 5-years event-free survival rates are currently above 80%. We recently identified a poor prognostic set of patients within unclassified BCP-ALL patients. These patients have a high risk of relapse and a gene expression profile similar to BCR-ABL1-positive ALL cases. This so-called BCR-ABL1-like (or Ph-like) group of patients showed similar high frequencies of deletion in B-cell development genes (den Boer et al, Lancet Oncology, 2009 and Mullighan et al. New Eng J Med, 2009). Here, we investigate the miRNA expression pattern in BCR-ABL1-like ALL. Further, we addressed whether altered expression levels of discriminative miRNAs of BCR-ABL1-like ALL has prognostic importance. MiRNA expression levels of leukemic cells of 91 newly identified children with BCP-ALL: i.e. 15 BCR-ABL1-like, 14 BCR-ABL1-positive, 9 TCF3-rearranged, 14 hyperdiploid (>50 chromosomes), and 15 B-other patients (negative for BCR-ABL1-like ALL and other known genetic lesions) were measured by Taqman® Array Human miRNA cards (TLDA cards, Applied Biosystems). Using the R statistics program (R-2.15, release June 2012), the Limma package was applied to compare the expression levels of miRNAs between the groups. Each genetic subtype was compared to the remaining cases to identify subtype-specific miRNAs.

Our data revealed that children with ETV6-RUNX1-positive, hyperdiploid, TCF3-rearranged and MLL-rearranged ALL demonstrate a subtype-specific miRNA signature. In contrast, BCR-ABL1-positive and BCR-ABL1-like cases showed a more variable miRNA expression pattern, resulting in 2 clusters of patients. The majority of the children with BCR-ABL1-like ALL (11 out of 15) showed a miRNA expression pattern different from that of BCR-ABL1-negative genetic subtypes of pediatric BCP-ALL and cluster with 43% of BCR-ABL1-positive patients (6 out of 14, cluster-I). The remaining 8 BCR-ABL1-positive and 4 BCR-ABL1-like cases showed more heterogeneous expression patterns (cluster-II). The prognosis of patients in both clusters, however, did not differ. Similarly, there was no significant difference in IKZF1, PAX5, JAK2 or CDKN2A/B status between these two clusters. Top-10 most differentially expressed miRNAs of the children with BCR-ABL1-like ALL were miR-324-5p, miR-345, miR-190, miR-130a, miR-545, miR-152, miR-103, miR-191, miR-197 and miR-101. None of these discriminative miRNAs was predictive for clinical outcome of BCR-ABL1-like patients. In conclusion, our data suggest that a miRNA signature is not suitable to dissect good and poor prognostic BCR-ABL1-like cases.

Disclosures:

No relevant conflicts of interest to declare.
Circulating t(14;18)+ Cells As Predictive Markers Of Follicular Lymphoma Development
Authors: Roulland Sandrine , Morgado Ester , Kelly Rachel , Sungalee Jocelyne Stephanie , Colombat Philippe , Solal-Celigny Philippe , Salles Gilles Andre , Vineis Paolo , Nadel Bertrand
Published: October 2019

Background

The (14;18) translocation constitutes both a genetic hallmark and critical early event in the natural history of Follicular Lymphoma (FL). However, t(14;18) is also detectable in the blood of otherwise healthy persons, and the relationship with progression to disease remains unclear. A key gap in our understanding is whether such t(14;18)+ cells in healthy individuals constitute committed FL precursors, and if high t(14;18) frequencies in blood represent a suitable predictive biomarker of FL development.

Methods

Among 520,000 healthy participants enrolled in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort, we identified 100 subjects who developed FL 2-161 months after enrolment. Blinded Prediagnostic blood from these and 218 controls were screened for t(14;18) by sensitive PCR-based assays. Results were subsequently validated on an independent cohort (65 cases, 128 controls). Clonal relationships between t(14;18) cells and FL biopsy were also assessed by molecular backtracking of paired prediagnostic/tumor samples.

Results

Clonal analysis of t(14;18) junctions in paired prediagnostic blood vs. tumor demonstrated that progression to FL occurred from t(14;18)+ committed precursors. Furthermore, we show that the prevalence of t(14;18) was significantly higher in individuals that subsequently develop FL compared to the controls (56% vs. 28.9%, p<10-3). Using a ROC curve analysis, we estimated that an optimal frequency value for predicting FL development was 10-4. Findings from the discovery cohort were fully validated in an independent cohort and when combined, we confirmed the optimal frequency value of 10-4. Overall, the combined analysis reports a 23-fold higher risk of subsequent FL associated with the 10-4 threshold (OR: 23.17; 95% CI, 9.98-67.31; P=7.7x10-9). Remarkably, stratified analyses on time to diagnosis revealed that risk estimates remained high and significant up to 15 years before diagnosis.

Conclusion

We demonstrated that high t(14;18) frequencies in blood from healthy individuals discriminates commitment to FL development up to 15 years before diagnosis and define the first predictive biomarker for FL. Providing they represent bona fide precursors, this molecular blood-based biomarker could therefore be of great value in identifying groups of at-risk individuals and their detailed geno-phenotypic characterization should provide important insights into the factors and kinetics of events driving early FL progression.

Disclosures:

No relevant conflicts of interest to declare.
Impact Of Age On Toxicity Associated With Chemotherapy For Acute Lymphoblastic Leukaemia (ALL): Results From The UK Prospective Study UKALL2003
Authors: Hough Rachael E. , Rowntree Clare , Wade Rachel , Goulden Nicholas , Mitchell Chris , Vora Ajay J.
Published: October 2019

Despite the substantial improvements made in the outcomes of paediatric ALL, with ‘cure' rates now in excess of 90%, survival in teenage and young adult (TYA) patients has remained inferior. The reasons for this are likely multifactorial, including tumour biology, toxicity, compliance, access to clinical trials and protocol (adult or paediatric) used. We report the toxicity profiles observed in children, teenagers and young adults treated on the UK intensive, minimal residual disease (MRD) directed ALL protocol, UKALL2003.

Of a total of 3126 patients treated, 1520 patients were under 5 years old, 767 were aged 5-9 years, 610 aged 10-15 years and 229 aged 16-24 years, with a median overall follow-up of 4 year and 10 months. The risk of serious adverse events (SAEs) was higher in patients older than 10 years (56% in 10-15 year olds, 53% in 16-24 year olds) compared to those aged 9 or younger (30% in under 5 years and 31% in 5-9 years)(p<0.0001), with no difference in the those aged 16-24 compared to younger teenagers (p=0.5). The incidence (per number of patients in each group) and distribution of toxicities according to age group is summarised in the table.Table 1Age in years<55-910-1516-24AllTotal number of patients1520767610229 NB: 56 pts≥20 years3126Infection n (%)328 (21.6%)130 (17.0%)145 (23.8%)72 (31.4%)675 (21.6%)Asaparaginase n (%)57 (3.8%)57 (7.4%)64 (10.5%)31 (13.5%)209 (6.7%)Methotrexate n (%)100 (6.6%)74 (9.6%)123 (20.2%)33 (14.4%)330 (10.6%)Steroid n (%)54 (3.6%)37 (4.8%)141 (23.1%)52 (22.7%)284 (9.0%)Vincristine n (%)34 (2.2%)11 (1.4%)22 (3.6%)7 (3.0%)74 (2.4%)Other SAEs94 (6.2%)42 (5.5%)90 (14.8%)25 (10.9%)251 (8.0%)

The incidence of certain toxicities including viral infection (5.3%), asparaginase hypersensitivity (1.9%) and vincristine neurotoxicity (2.1%) appeared equivalent across all age groups. Avacular necrosis was seen predominantly in adolescents (83% of 147 events in 10-19 year olds) and was rare in those younger than 10 years (n=18) or older than 20 years (n=7). Asparaginase thrombotic events increased in frequency with increasing age (1.5% in under 5 years, 3.3% in 5-9 years, 4.4% in 10-15 years and 8.3% in 16-24 year olds)(p<0.0001). All other toxicities were more frequently observed in over 10 year olds compared to patients aged 9 or younger, with no difference between 16-24 year olds and 10-15 year olds.

The impact of age on SAEs associated with intensive ALL chemotherapy varies according to specific toxicities. In general, toxicity is higher in those over 10 years compared to younger patients, with no excess toxicity in those aged 16-24 compared to 10-15 years. However, specific toxicities may increase with increasing age (thrombosis), be restricted to adolescence (AVN) or be unrelated to age (vincristine neurotoxicity, asparaginase hypersensitivity).

Disclosures:

No relevant conflicts of interest to declare.
Unique and Potent Tumor Specific Antibodies in Graft Versus Leukemia Responses
Authors: Gillissen Marijn A. , Yasuda Etsuko , Levie Sophie , Bakker Arjen Q , Kedde Martijn , Claassen Yvonne B , Huisman Cynthia , Spits Hergen , Beaumont Tim , Hazenberg Mette D.
Published: October 2019

Introduction

Acute myeloid leukemia (AML) is a high-risk malignancy with a poor prognosis. Allogeneic hematopoietic stem cell transplantation (HSCT) can be curative if it induces a potent graft versus leukemia (GvL) response. GvL responses and graft versus host disease (GvHD) are typically considered T cell mediated, because T cell depletion from hematopoietic stem cell grafts reduces the risk of GvHD at the cost of leukemia relapse. In addition, depletion of B-lymphocytes with rituximab has led to amelioration of GvHD in a number of studies suggesting that B cells are also important in the pathophysiology of GvHD and, in analogy, in GvL responses. However, the characteristics of the antibodies produced by these leukemic specific B cells have not yet been studied.

Methods

We selected three patients with high-risk myelomonocytic leukemia who remained disease free years after allogeneic HSCT, from whom we established clonal human B cell lines, using a unique and innovative technology that was developed in our laboratory (Kwakkenbos ea, Nat Med 2010). These B cell lines, that concomitantly express immunoglobulin on their membranes and secrete antibodies, were used to select antibodies specific for cell surface antigens on AML cell lines with similar morphologic and immunophenotypic characteristics as the patients’ leukemic blasts, using a FACS based assay.

Results

From each patient, several AML specific B cell clones were retrieved. Their antibodies recognized surface antigens on primary AML blasts derived from multiple patients and on AML cell lines, but not on healthy bone marrow, peripheral blood mononuclear cells or tissues such as liver, skin and colon. The majority of the antibodies were of the IgG3 isotype. Approximately 40% of the AML-specific antibodies induced direct death of cultured AML cell lines and of primary AML blasts. The cell death pathway induced by these cytotoxic antibodies was oncotic rather than apoptotic.

Conclusion

Our data demonstrate that high-risk AML patients with a potent GvL response mount robust antibody responses against surface antigens that are specifically expressed on tumor cells and binding of these antibodies induces direct cell death. The targets recognized by the recovered antibodies are also expressed on leukemic blast from other patients suggesting evidence for a common immune mechanism responsible for AML clearance. Our findings suggest that antibody responses are important in GvL and open up new ventures for specific antibody treatment of AML patients.

Disclosures

No relevant conflicts of interest to declare.
A Prospective Randomized Comparison of Cyclophosphamide Versus Fludarabine in Addition of Antithymocyte Globuline for Allogeneic Hematopoietic Cell Transplantation in Patients with Adult Severe Aplastic Anemia; Interim Analysis
Authors: Kim Hawk , Lee Won-Sik , Kim Yeo-Kyeoung , Joo Young-Don , Park Jinny , Jung Chul Won , Jo Jae-Cheol , Lee Jung-Hee , Lee Je-Hwan , Kim Dae-Young , Lee Kyoo-Hyung
Published: October 2019

Our previous study showed that a less toxic regimen comprising reduced cyclophosphamide (Cy), fludarabine and anti-thymocyte globulin (ATG) (Cy-Flu-ATG), was less toxic for allogeneic hematopoietic cell transplantation (alloHCT) compared with standard Cy-ATG in patients with adult severe aplastic anemia (AA). We postulated that replacing Cy with Flu (Flu-ATG) would be more beneficial. Therefore we performed a randomized phase III study to compare the regimen-related toxicities (RRTs) of two different conditioning regimens: Cy-ATG vs. Flu-ATG. We present the interim alaysis. Patients in the Cy-ATG arm received Cy at 200 mg/kg. Those in the Flu-ATG arm received fludarabine (Flu) at 180 mg/m2. A total of 36 patients (21 in the Cy-ATG and 15 in the Flu-ATG) were enrolled. The basic patientsÕ characteristics were similar between both arms except for donor type and HLA-matching. There were more unrelated donor (38.1% vs. 73.3%; p=0.037) and HLA mis-matching (0% vs. 40%; p=0.001) in Flu-ATG arm. All predefined RRTs were similar between Cy-ATG and Flu-ATG (33.3% vs. 33.3%; p=1.000). There was no primary engraftment failure in both arms and only one patients in Cy-ATG died of treatment-related hepatic toxicity before engraftment. Also there were no differences between Cy-ATG and Flu-ATG arms in terms of secondary engraftment failure (20% vs. 20%; p=1.000), hepatic sinusoidal obstruction syndrome (0% vs. 0%; p=1.000), hemorrhagic cystitis (4.8% vs. 0%; p=1.000), pulmonary complications (12.5% vs. 16.7%; p=1.000). The incidence of acute graft-versus-host disease (GvHD) (14.3% vs. 20.0%; p=0.677) and chronic GvHD (11.8% vs. 7.7% ; p=1.000) were also similar. The 3-year survival rate did not differ (77.3% vs. 77.0%; p=0.995; Figure 1). Flu-ATG can be

Figure 1 promising in terms of RRT without increasing engraftment failure in Flu-ATG arm when considering more unrelated and HLA-mismaching patients were enrolled. We will continue this phase III trial. Figure 1. promising in terms of RRT without increasing engraftment failure in Flu-ATG arm when considering more unrelated and HLA-mismaching patients were enrolled. We will continue this phase III trial.

Disclosures

No relevant conflicts of interest to declare.