bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential Expression of Micro RNAs Accompanies Differential Reactivity Via Platelet FcγRIIa in Humans and Transgenic Mice.
Authors: Abraham Shaji , Andre Pierrette , Zhou Yuhang , Edelstein Leonard C. , Shaw Chad , Bray Paul F. , McKenzie Steven E. 
Published: October 2019

Abstract
Abstract 2165
One of the major clinical challenges in heparin-induced thrombocytopenia (HIT) is that antibodies to the heparin/PF4 complex are necessary but not sufficient to cause thrombocytopenia and thrombosis. While contributions of macromolecular antigen structure, antibody recognition and procoagulant activation of leukocytes and endothelial cells are increasingly appreciated, the sine qua non of HIT is platelet activation via FcγRIIa. It has been recognized for over 20 years that there is substantial inter-individual variation in human platelet reactivity to IgG immune complex agonists (such as in HIT) acting via FcγRIIa. In the course of our Platelet RNA And eXpression-1 (PRAX1) study on the genomics of platelet reactivity, we observed differential reactivity among 154 healthy donors of platelets when platelet-rich plasma aggregated in response to the agonist anti-CD9, a well-established model of FcγRIIa-mediated activation relevant to HIT. Importantly, even after accounting for the known sources of variation in reactivity - level of platelet surface FcγRIIa expression and for the H/R131 ligand-binding polymorphism - most of the variation in platelet reactivity remained unexplained. In parallel, we established human FcγRIIa transgenic mouse lines of the C57Bl6 strain and the 129SvJ strain. Unexpectedly, we identified differential reactivity to FcγRIIa-mediated platelet activation between these different mouse strains. The availability of models of variation in FcγRIIa-mediated platelet activation in both humans and mice allowed us a unique opportunity to test the hypothesis that differential expression (DE) of platelet RNA species accounts for differential reactivity. Total RNA was isolated from leukocyte-depleted platelets from each human donor and from each transgenic mouse strain. Using the species-appropriate Nanostring digital quantification assay, microRNAs (miRs) expressed in platelets were identified. 4 DE miRs in humans and 7 in mice were identified with a statistically significant difference in normalized expression, after Benjamini-Hochberg correction for multiple testing. DE was validated by qRT-PCR. Of note, several DE miRs were shared by human and mouse platelets. Work is ongoing as to the identity and functions of the mRNAs targeted by these DE miRs.

Disclosures:
No relevant conflicts of interest to declare.
Influence of Minor Histocompatibility Antigens' Disparities On Graft Versus Host Disease and Overall Survival After Allogeneic Hematopoietic Cell Transplantation From Siblings
Authors: Dzierzak-Mietla Monika , Markiewicz Miroslaw , Siekiera Urszula , Mizia Sylwia , Karolczyk Agnieszka , Koclega Anna , Zielinska Patrycja , Sobczyk-Kruszelnicka Malgorzata , Kyrcz-Krzemien Slawomira 
Published: October 2019

Abstract
Abstract 4172

Introduction:
Minor histocompatibility antigens (MiHA) are acknowledged non-HLA genetic factors which, in case of their incompatibility between the donor and the recipient, may contribute to post-transplant complications and impact the results of transplantation. The aim of this study was to investigate whether immunogenic MiHA disparities in either Graft-versus-Host or Host-versus-Graft direction influence Graft Versus Host Disease (GVHD) and overall survival after allogeneic hematopoietic cell transplantation (allo-HCT) from matched siblings.

Methods:
Alleles encoding 11 MiHAs: HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, HwA-9, HwA-10, UGT2B17 and HY were examined in 62 sibling donor/recipient pairs with use of Dynal AllSet mHA typing kit and PCR-SSP method. Only immunogenic MiHA disparities determined in accordance to dbMinor database, with regard to their GVH or HVG direction, were assessed. Median age of donors and recipients was 35(14–60) and 38(14–59) years, respectively. Median time from diagnosis to allo-HCT was 0.62(0.24–12.91) years. Allo-HCTs were performed between 2000–2008 for following indications: AML, ALL, MDS, CML and NHL. The conditioning treatment before allo-HCT was myeloablative (BuCy, TBI/Cy), reduced toxicity (Treo/Flu) or nonmyeloablative. Median follow-up was 3 (0.04–10) years.

Results:
Immunogenic MiHA mismatches were observed in 42 (68%) donor-recipient pairs: GVH- or HVG- directed in 18 pairs each and bidirectional in 6 pairs. Acute GVHD was observed in 27 patients, in 24 of whom it was severe (grade III or IV) and it was influenced by GVH-directed disparities of MiHA encoded by Y-chromosome (p=0.037), as shown in Fig. 1. Chronic GVHD was diagnosed in 25 patients, in 12 of them extensive, and it's incidence was influenced by the same kind of MiHA disparities (p=0.017), as shown in Fig. 2. Analysis of overall survival showed unfavorable impact of GVH-directed disparities of Y-chromosome encoded MiHAs (p=0.011), as presented in Fig. 3.

Conclusions:
Mismatches of Y-chromosome encoded MiHAs significantly impact the occurrence of severe acute and extensive chronic GVHD and decrease overall survival.

Disclosures:
No relevant conflicts of interest to declare.
Sequential Measurement of Bone Mineral Density Identifies Ongoing Recovery of Detrimental Bone Mass in Very Long Term Adult Survivors of Childhood Cancer
Authors: Klap Bibi C. , Winkel Mariël L. Te , van Waas Marjolein , Neggers Sebastiaan J.C.M.M , Boot Annemieke M. , Pieters Rob , van den Heuvel-Eibrink Marry M. 
Published: October 2019

Abstract
Abstract 4296

Background
Aim of this study was to investigate status and longitudinal evolvement of BMD in long-term adult survivors.

Patients and methods
A single-center cohort of 410 adult long-term survivors (171 females) was included in this retrospective study. Age at diagnosis was 6.6 years (0.0–16.8 yrs), and at follow up 24.5 years (18.0–49.3 yrs). Median follow-up time was 15.2 years (5.1–39.8 yrs).
BMD of the lumbar spine (BMDLS) and total body (BMDTB) were measured. Results were compared with healthy age matched controls.

Results
Overall BMDTB and BMDLS were low in long-term survivors (median standard deviation score (SDS) −0.60, P < 0.001 and median SDS −0.40, P < 0.001 respectively). In ALL survivors both BMDTB (median SDS −0.65, P < 0.001) and BMDLS (median SDS −0.48, P < 0.001) were low. Backwards elimination of the multivariate linear regression analysis showed that both cranial irradiated and/or total body irradiated survivors were at significant risk for low BMDTB. BM(A)DLS was not different between survivors and controls and no risk factors were identified for low BM(A)DLS. In general, a slight but significant improvement of BMDTB and BMDLS was found in our sequential measurement. This BMD improvement was not found for ALL survivors alone.

Conclusions
Our study shows low BMD in childhood cancer survivors treated with cranial or total body irradiation. However, sequential DXA analysis shows ongoing recovery of bone mass, which suggests a potential for reaching peak bone mass later in life.

Disclosures:
No relevant conflicts of interest to declare.
Monitoring the Antileukemic Immune Response with an Active Specific Immunization Strategy
Authors: Yang Ting , Chen Zhizhe , Kolb Hans-Jochem , Buhmann Raymund 
Published: October 2019

Abstract
Abstract 4354

Background:
Donor lymphocyte transfusion (DLT) may induce the graft-versus-leukemia (GVL) effect for patients with AML relapsed after transplant. However, the limited overall efficacy of DLT in clinical practice emphasizes the importance of identifying a specific subgroup of patients who might benefit from this treatment approach.

Objective:
To monitor the cellular immune response after DLT, we developed an active specific immunization strategy using in vitro generated AML-trained T cells to induce a highly specific antileukemic T-cell response and thus established a novel nonradioactive assay system.

Methods:
The myeloid blasts derived from five patients with AML relapsed post allogeneic hematopoietic stem cell transplantation (allo-HSCT) were first labeled with CFDA (5,6-carboxyfluorescein diacetate succinimidyl ester). To analyze the growth inhibitory potential of the donor T cells trained by AML cells, the myeloid blasts were induced to proliferate by means of a cytokine cocktail. The T cell mediated growth inhibitory potential was detected after 5 days by flow cytometry and correlated with [3H]-thymidine uptake.

Results:
Here, we applied a CFDA dye to track the proliferation and expansion of AML blasts in response to the cytokine cocktail in vitro. AML-trained T cells, expressed high levels of the activation markers CD25 and CD69, and were generated to recognize the leukemic progenitor cells and inhibit cytokine-induced leukemic cell proliferation, which is an active specific immunization strategy circumventing the identification of leukemia-associated antigens. The capability of proliferation inhibition of AML-trained T cells evaluated with the nonradioactive, CFDA-based assay provided comparable results with the classic [3H]-thymidine assay.

Conclusion:
The active specific immunization strategy was realized to monitor the antileukemic immune response measured with radioactive and nonradioactive assay system.

Disclosures:
No relevant conflicts of interest to declare.
Rapid Response of Biallelic BRAF V600E Hairy Cell Leukaemia to Low Dose Vemurafenib
Authors: Sims Hannah , Bloxham David , Creasey Hannah , Bradford Elaine , Liu Hongxiang , Wright Penny , Bench Anthony , Hopper Melanie A , van't Veer Mars , Crawley Charles , Scott Mike , Follows George 
Published: October 2019

Abstract
Abstract 4890
Following the recent report that identified the V600E BRAF mutation in Hairy Cell Leukaemia (HCL) (Tiacci et al NEJM 2011), we confirmed this finding in 48/48 patients diagnosed with HCL in the Cambridge Haematology Laboratory (Boyd et al BJHaem 2011). One of these patients was found to have a bialleleic V600E BRAF mutation on a bone marrow biopsy taken in 2008. Retrospective molecular analysis of diagnostic material from 1996 confirmed this patient's disease was heterozygous for BRAF V600E at presentation. He initially enjoyed a long period of disease stability following splenectomy and cladribine in 1996/1997. He was retreated with 6 cycles of cladribine in 2008 but within 3 years he relapsed again and was treated with 6 cycles of pentostatin and rituximab completing in 2011. He achieved a partial bone marrow remission with recovery of normal peripheral blood counts, but his disease progressed within 6 months. He became profoundly cytopenic with circulating hairy cells and was platelet/red cell transfusion dependent. He was intolerant of interferon-alpha.
Following the recent report of the successful treatment of a single patient with a BRAF inhibitor (Dietrich et al NEJM 2012), our patient was treated with vemurafenib at a continuous dose of 240mg BD. Pretreatment Day -7 bloods confirmed total white cell count 20.0 × 109/L, neutrophils 0.8 × 109/l, haemoglobin 9.5 g/dl (supported) and platelets 11 × 109/l (supported). In the 3 months prior to starting vemurafenib, he had received 14 units of red cells, 9 transfusions of platelets and on-going G-CSF therapy. Baseline MRI of pelvis and femora revealed diffusely abnormal homogenous bone marrow signal with increased T2 fat suppressed (T2fs) signal and decreased T1 signal
Treatment was tolerated with no discernable side effects to date and renal and liver function remained normal on therapy. Flow cytometry quantification confirmed a rapid reduction in peripheral blood hairy cells from pre treatment 19 x109/l, to 3 x109/l by day 7 and 0.002 x109/l on day 36. The final platelet transfusion was on day 15 and platelet counts increased to 37 × 109/l by day 29 and 94 × 109/l by day 36. The last red cell transfusion was on Day 22 and on day 36 the patient's haemoglobin was 9.4 g/dl. G-CSF was stopped on day 28. The bone marrow remained inaspirable on day 16, but a trephine biopsy revealed early response with some regeneration of normal haematopoiesis. Bone marrow from Day 36 confirmed ongoing haematopoietic recovery but hairy cells persisted. MRI on day 32 showed development of patchy marrow signal throughout consistent with multifocal recovery of haematopoietic marrow. The patient continues on vemurafenib 240mg BD and follow-up data will be presented.

Disclosures:
Off Label Use: Oral BRAF inhibitor.
Outcome of Young Children with Non-Hodgkin's Lymphoma
Authors: Rodriguez Laura , Zolaly Mohammed , Weitzman Sheila , Naqvi Ahmed , Punnet Angela , Abla Oussama 
Published: October 2019

Abstract
Abstract 5106

Background:
The incidence and biology of non-Hodgkin's lymphoma (NHL) vary according to age, and the outcome differs amongst children being less favorable in infants and adolescents.

Objectives:
To analyze the outcome and toxicity patterns of young children with NHL comparing patients aged < 10 years to those 10–18 years of age.

Methods:
A retrospective review of children ≤ 18 years of age diagnosed with NHL over a period of 13 years. Patients were treated according to different protocols and treatment subgroups included: Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), lymphoblastic lymphoma (LL), anaplastic large cell lymphoma (ALCL), and peripheral T-cell lymphoma (PTCL). Post-organ transplant lymphoproliferative disorder (PTLD) and immunodeficient patients were excluded.

Results:
From 01/1995 to 12/2008, 173 children with NHL were reviewed. Of these, 81 pts (47%) were <10 years of age, with BL (29 pts), DLBCL (5 pts), PTCL (6 pts), LL (27 pts), and ALCL (14 pts). The 10-year overall survival (OS) was 93. 6+2. 8% in patients aged < 10 years compared to 77. 3+7. 2 in patients 10–18 years of age (P=0. 02). The 10-year event-free survival (EFS) was 89. 4+3. 5% in patients aged < 10 years compared to 70+9. 3% in patients 10–18 years of age (P=0. 03). BL was the most common subtype in patients <10 years, with a 10-year EFS of 96. 4+3. 5% versus 82. 6+7. 9% in patients 10–18 years of age (P=0. 10). The most common subtype in patients 10–18 years of age was ALCL (36%), the 10-year EFS was 78. 5+8. 3 versus 92. 9+6. 9 in patients < 10 years (P=0. 29). Children 10–18 years of age were more likely to develop neurotoxicity (9. 7% vs 0%), gastrointestinal toxicity (30% vs 12%), and treatment-related mortality (4. 3% vs 2. 4%).

Conclusions:
Children < 10 years with NHL have better outcomes and less treatment-related toxicity than older children.

Disclosures:
No relevant conflicts of interest to declare.
Antibodies Specific for MHC Class II Antigens Cause TRALI Directly in a Two-Event in Vivo Model
Authors: Kelher Marguerite R , Silliman Christopher C. 
Published: October 2019

Abstract
Abstract 845
Transfusion-related acute lung injury (TRALI) is the most common life threatening adverse event related to the transfusion of blood products. Antibodies to HLA class II antigens have been implicated in clinical TRALI, and in situ animal modeling has demonstrated that antibodies to HLA class II antigens bind to Human monocytes that express the cognate antigen resulting in the release of soluble mediators, which induce neutrophil (PMN)-mediated ALI in isolated rat lungs. This mechanism of TRALI is complex, used Human reagents in rodent lungs, and must meet a number of constraints. We hypothesize that the initial clinical event(s) predisposing to TRALI must 1) change the antigen surface expression of PMNs so that they express HLA class II antigens and 2) activate the pulmonary endothelium so that the subsequent transfusion of an antibody specific to the expressed surface antigen bind and activate the PMNs resulting in endothelial damage, capillary leak, and TRALI.

Methods:
Sprague Dawley rats underwent three different first events: 1) saline given intraperitoneally (IP) followed by a 2–24 hour waiting period, 2) endotoxin (LPS) [2 mg/kg] given IP with a 2 hour waiting period, or 3) interferon-γ(INF-γ) [3 μg/kg] + M-CSF [20 μg/kg] given IP with a 24 hour waiting period along with tumor necrosis factor-α (TNF-α) [65 ng/kg] given intravenously (IV) at 22 hours, following INF-γ/M-CSF injection, with a 2 hour incubation period. A femoral cut down was made, the vessels cannulated, and either blood was removed to assess rat MHC Class II (OX6) surface antigen expression on PMNs with blockade of the Fc receptors to obviate non-specific binding plus isotypic controls or OX6 (150 μg/kg) antibodies were infused as a second event followed by Evans blue dye (EBD) [30 mg/kg] IV injection, at 6 hours blood was obtained, and a bronchoalveolar lavage (BAL) performed to quantify capillary leak/ALI.

Results:
both INF-γ/M-CSF/TNF-α and LPS increased OX6 surface expression vs. buffer-treated control (Table 1). Both TNFa and LPS cause activation of the pulmonary endothelium resulting in PMN sequestration (data not shown). In rats that received either INF-γ/M-CSF/TNF-α or LPS, the infusion of OX6 antibodies caused significant lung leak of EBD (Table 2). In conclusion, if the initial insult(s) in TRALI induces surface expression of class II antigens on the PMNs and activated the pulmonary endothelium, then infusion of class II antibodies caused ALI. Such a pathogenesis may be appropriate for clinical TRALI and provides a much simpler explanation for class II antibody-mediated ALI.

Disclosures:
Silliman: Pall Corporation: Honoraria, Research Funding.
Identification of a Two-Step Mechanism Responsible for Antibody-Mediated Transfusion Related Acute Lung Injury (TRALI)
Authors: McKenzie Christopher G.J. , Kim Michael , Singh Tarandeep , Semple John W. 
Published: October 2019

Abstract
Abstract 846
Transfusion-related acute lung injury (TRALI) is one of the leading causes of transfusion fatalities, and most TRALI reactions are thought to be caused by donor antibodies. It is currently thought that the donor antibodies activate pulmonary neutrophils to produce reactive oxygen species that damage lung tissue. There have been several animal models of TRALI developed including, for example, ex vivo lung models demonstrating the importance of human anti-neutrophil antibodies in TRALI, and in vivo models showing how biological response modifiers can induce recipient lung damage. An in vivo murine model of antibody-mediated TRALI was developed in 2006, and has also shown several similarities with human TRALI induction (Looney MR et al., J Clin Invest 116: 1615, 2006). Specifically, a monoclonal anti-mouse MHC class I antibody (34-1-2s) causes significant increases in excess lung water, lung vascular permeability and mortality within 2 hours after administration. These adverse reactions were found to be due to the antibody's ability to activate pulmonary neutrophils to produce reactive oxygen species (ROS) in an Fc receptor (FcR)-dependent manner. In contrast, however, it was recently shown that 34-1-2s induces pulmonary damage by activating macrophages to generate ROS in a complement (C5a)-dependent process (Strait RT J et al., Exp Med 208: 2525, 2011). In order to better understand this apparent controversy, we attempted to determine the nature of how 34-1-2s mediates its lung damaging properties. 34-1-2s was digested with pepsin or papain to produce F(ab')2 or Fc fragments respectively, and the fragments were tested for their ability to mediate TRALI reactions. In control mice, when intact 34-1-2s antibody was intravenously injected into either CB.17 mice with severe combined immunodeficiency or C5 deficient DBA/2 mice, increased shock, serum MIP-2 (murine equivalent to human IL-8) levels, pulmonary neutrophil accumulation, pulmonary edema and mortality all occurred within 2 hours. In contrast, however, injection with 34-1-2s F(ab')2 fragments was only able to generate MIP-2 production and pulmonary neutrophil accumulation; no lung damage or mortality occurred. Injection of 34-1-2s Fc fragments either alone or together with equal molar concentrations of F(ab')2 fragments failed to induce any lung damage or mortality. These results suggest that 34-1-2s recognition of it's cognate MHC class I antigen may be a priming reaction that stimulates MIP-2 and chemotaxis of neutrophils to the lungs, whereas the Fc portion of the intact molecule is responsible for the second step of exacerbating TRALI symptoms in a complement independent manner.

Disclosures:
No relevant conflicts of interest to declare.
Intracellular Retention, Enhanced Clearance, and Defective FVIII Binding Are Common Features of Von Willebrand Factor D'-D3 Domain Mutations in Patients with Von Willebrand Disease Type 1 From the European Mcmdm-1VWD Study
Authors: Schneppenheim Reinhard , Budde Ulrich , Batlle Javier , Castaman Giancarlo , Eikenboom Jeroen C. J. , Federici Augusto B. , Goudemand Jenny , Obser Tobias , Oyen Florian , Rodeghiero Francesco , Schneppenheim Sonja , Peake Ian R. , Goodeve Anne C. 
Published: October 2019

Abstract
Abstract 99

Background:
Von Willebrand disease (VWD) type 1 is characterized by a partial reduction of structurally and functionally normal VWF with normal VWF multimers. As part of a large European study (Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease (MCMDM-1VWD) patients previously diagnosed with VWD type 1 were studied systematically to assess the phenotypic and genotypic spectrum.

Objective:
To confirm the pathogenicity of VWF gene mutations and to elucidate the molecular mechanisms of VWD type 1.

Patients and methods:
VWD type 1 patients were recruited by twelve expert centers in nine European countries. VWF genotyping was performed in all index cases (IC). The eight mutations studied here are located in the VWF D'-D3 domain and corresponded to 57 patients from 19 families. They were reproduced by recombinant expression with subsequent phenotypic characterization, two of them in cis and one in trans with a second mutation.

Results and Discussion:
Intracellular VWF:Ag of all mutants was normal or near normal suggesting normal expression levels. However, seven mutations (p.M771I, p.I1094T, p.C1130R, p.C1130G, p.C1130F, p.W1144G and p.Y1146C) caused intracellular retention and impaired VWF secretion. In addition, we observed a major loss of high molecular weight multimers as in type 2A and a novel finding of a severe VWF:FVIII binding defect in most of the homozygously expressed mutants. Additional mutations either in cis or in trans had no modifying effect. The recombinant VWD type 1 Vicenza mutation p.R1205H with or without the allelic variant p.M740I seen in three Italian IC was secreted normally and had normal function leaving enhanced clearance of mutant VWF as the only pathomechanism. In conclusion, the majority of mutations in the D3 domain impair VWF multimerization, cause intracellular retention and correlate with defective FVIII binding. An elevated ratio of VWF propeptide to VWF:Ag suggests enhanced VWF clearance as an important pathomechanism of most mutations and particularly of p.R1205H.

Disclosures:
No relevant conflicts of interest to declare.