bluuebunny/crossref_metadata_2025 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 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 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 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 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 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 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 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 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 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.
Harnessing Invariant Natural Killer T Cells for Cancer Therapy
Authors: Dhodapkar Madhav V.
Published: October 2019

Abstract SCI-20

Natural killer T (NKT) cells are distinct innate lymphocytes that recognize lipid ligands in the context of nonclassical MHC molecules called CD1d. NKT cells have been broadly classified into two major types based on their T-cell receptors (TCRs). One group, termed type I invariant, or iNKT cells, bears a noncanonical conserved TCR (Va24/Vb11 in humans, and Va14 in mice), while others, termed type II NKT cells, express diverse TCRs. Recent studies have identified several naturally occurring lipid ligands recognized by these cells. iNKT cells are efficiently activated by α-galactosylceramide (α-GalCer), a lipid ligand first isolated from a marine sponge. Activation of iNKT cells in vivo leads to activation of NK cells, as well as downstream activation of adaptive immunity, leading to strong antitumor effects in mice. Several hematologic malignancies express CD1d and are attractive targets of NKT-targeted therapeutic approaches. Progression from premalignant gammopathy to clinical myeloma is associated with loss of the interferon-γ-secreting effector function of NKT cells. Adoptive transfer of α-GalCer-loaded dendritic cells (DCs) led to activation and expansion of circulating NKT cells in vivo in patients with advanced cancer. However, the functional properties of NKT cells were found to be impaired. Lenalidomide leads to enhanced activation and cytokine secretion by NKT cells in a ligand-dependent manner in culture. This led us to treat patients with early multiple myeloma with three cycles of low-dose lenalidomide and α-GalCer-loaded DCs. Preliminary studies from this trial show that this combination leads to activation of diverse innate cells, including NKT and NK cells, and upregulation of NKG2D and CD56. Activation of these innate lymphocytes also led to an increase in serum-soluble interleukin-2 receptor and induction of eosinophilia. Together, these data demonstrate the capacity of this combination to recruit several innate lymphocytes and mediate antitumor effects. In contrast to conventional MHC I/II restricted T cells, understanding the diversity and functional properties of lipid-reactive T cells is still in its infancy. The biology of NKT cells may have an impact, due to the cells' immune-regulatory properties, in diverse inflammatory states, including cancer, atherosclerosis, and allergy. This research was supported in part by funds from the Translational Research Program of the Leukemia and Lymphoma Society.

Disclosures:

Dhodapkar: KHK: Research Funding; Celgene: Research Funding. Off Label Use: Lenalidomide in asymptomatic myeloma.
The Role of Iron in Toxicity of Stored Red Blood Cell Units
Authors: Hod Eldad A. , Brittenham Gary M. , Spitalnik Steven L.
Published: October 2019

Abstract SCI-46

Accumulating evidence suggests that transfusion of red blood cells (RBCs) stored for longer durations is associated with increased rates of infection, morbidity, and mortality in hospitalized patients. During refrigerated storage in vitro, RBCs undergo cumulative biochemical and biomechanical changes that reduce their survival in vivo. After transfusion, storage-damaged RBCs are rapidly cleared from the circulation by reticuloendothelial macrophages, usually within the first hour. The associated hemoglobin iron is then rapidly catabolized and returned to the plasma at a pace that can exceed the rate of uptake by the physiologic iron transporter, transferrin, thereby producing plasma non-transferrin-bound iron. Our overarching hypothesis is that the rapid clearance of storage-damaged RBCs is responsible for some of the adverse effects of transfusion, specifically by delivering a large bolus of toxic hemoglobin-derived iron to macrophages. In particular, plasma non-transferrin-bound iron can produce oxidative damage to lipids, proteins, and DNA, thereby activating endothelial cells and platelets to increase thrombotic risk. Furthermore, increased macrophage iron and plasma non-transferrin-bound iron can enhance infectious risk by providing sources of iron to microbial pathogens both within macrophages and within the systemic circulation. Our studies in healthy human volunteers demonstrate that transfusions of “older” RBCs (stored for 40–42 days), but not “fresh” RBCs (stored for less than seven days), significantly increase circulating non-transferrin-bound iron levels in vivo (1) and enhance bacterial growth in vitro in posttransfusion serum samples. Animal studies (2) confirm these findings and provide additional evidence that transfusion exacerbates infection in vivo with Salmonella typhimurium, Escherichia coli, and Staphylococcus aureus. In addition, murine studies suggest that rapid transfusion of the equivalent of two units of older, stored RBCs produces a proinflammatory cytokine response, which was not seen after slow transfusion of one unit of older, stored RBCs to healthy human volunteers. Studies in dogs are currently under way to test whether the RBC transfusion rate and dose affects the proinflammatory response. In conclusion, transfusion of older, stored RBCs results in delivery of a potentially toxic dose of iron to macrophages that both increases macrophage intracellular iron and produces plasma non-transferrin-bound iron. This iron toxicity may increase infectious and thrombotic risks and potentially exacerbate an existing proinflammatory response.

Disclosures:

No relevant conflicts of interest to declare.
Microenvironmental Factors and The Role Of Tumor Necrosis Factor Receptor Type 1 (TNFR-1) In Chronic Lymphocytic Leukemia
Authors: Duerr Claudia , Schulz Angela , Ohl Sibylle , Zenz Thorsten , Stilgenbauer Stephan , Lichter Peter , Seiffert Martina
Published: October 2019

B-cell Chronic Lymphocytic Leukemia (CLL) represents the most common leukemia among adults in the western world and remains still incurable. CLL is characterized by the accumulation of malignant B-cells in the peripheral blood and the lymphoid organs due to apoptosis resistance. Removing the cells from their physiological microenvironment spontaneously drives them into apoptosis in vitro unless they are supported by bystander cells (e.g. stromal cells or accessory leukocytes) or soluble factors such as cytokines produced by these cells.

In order to identify differences in the biology of healthy B-cells and the leukemic clones we performed comparative microarray analyses of both cell types before and after cultivation in high cell density that allows cell-cell interactions of CLL cells with accessory leukocytes. These studies revealed the establishment of an inflammatory microenvironment in CLL characterized by the transcriptional upregulation of several cytokines. Antibody array analyses of culture supernatants and blood serum samples confirmed the upregulation of these proteins and their relevance for CLL. The 11 most deregulated cytokines were quantified in the serum of 250 CLL patients of the German CLL8 study cohort and 50 age- and sex-matched controls in order to identify novel predictive or prognostic markers. Bioinformatical analyses of the data are currently ongoing.

Among other proteins, we found Tumor Necrosis Factor Receptor Type 1 (TNFR-1) to be significantly upregulated in the malignant B-cells under survival-maintaining cultivation as well as in CLL serum, but not in healthy controls (Figure 1). To exploit the difference in TNFR-1 biology, we treated CLL cells with TNF-α in combination with wogonin which is known to block the survival supporting pathway propagated by TNFR-1 upon TNF-α binding. This combinational treatment strategy effectively triggered

Disclosures:

No relevant conflicts of interest to declare.
Self-Complementary AAV Vectors Delivered Intramuscularly Do Not Break Tolerance In a Partial Knockout Model Of Hemophilia B
Authors: Rogers Geoffrey L. , Herzog Roland W
Published: October 2019

Self-complementary adeno-associated viral (scAAV) vectors have become a popular tool for AAV gene therapies due to their increased transgene expression relative to single-stranded (ss)AAV vectors, at least for models in which the transgene can fit within these restrictive constructs. The most recent clinical trial for hemophilia B used a scAAV8 vector. However, recent reports by our lab and others have suggested that these vectors are also more immunogenic than their single-stranded brethren. We previously showed that, during hepatic gene transfer, the innate immune response is greater with a scAAV vector. Moreover, Wu et al. demonstrated an enhanced T and B cell response to a secreted form of HIV’s gag protein when it was delivered via scAAV. While this was beneficial for their vaccine model, it could potentially be problematic for therapeutic gene transfer applications. To investigate whether this phenomenon could deleteriously impact gene therapy for hemophilia B, we used a partial knockout model of hemophilia B. This mouse model has the murine factor IX gene knocked out, and then a truncated form of human factor IX (hF.IX) is inserted. Specifically, the mice we used have a late stop codon (LS) at amino acid 338; while hF.IX can be detected in the liver, this mutated protein is not released into circulation (crm-). During muscle gene transfer with AAV2, LS mice have an interesting response: while they form antibodies to hF.IX, there is no CD8+ T cell infiltrate detected in the transduced tissue. We then set out to determine whether this partial tolerance could be disrupted using a scAAV vector. Using AAV1, which is a more relevant serotype for muscle gene therapy, we injected single-stranded and self-complementary vectors (1011 vg/mouse) and measured the resulting immune responses. Surprisingly, unlike with AAV2, the LS mice completely tolerated AAV1-mediated intramuscular delivery of hF.IX. They did not make detectable anti-hF.IX IgG1, and their plasma had no Bethesda activity through 4 weeks post-injection. In contrast, hF.IX null mice (HBKO) made 10708±3869 ng anti-hF.IX IgG1 and developed inhibitory activity of 22.5±26 Bethesda units 4 weeks after delivery of a scAAV vector. Consequently, LS mice had circulating hF.IX levels of 148.9±18.64 (ssAAV1) and 60.12±8.71 ng/mL (scAAV1), while hF.IX was undetectable in the plasma of HBKO mice at this time point. The CD8 response was similarly lacking in LS mice, with splenic responses to hF.IX detected by ELISPOT reduced by ∼2-10 fold relative to HBKO mice. Infiltrating CD8 cells were detected in the muscle of HBKO but not LS mice. In conclusion, our data suggest that the underlying mutation within the patient is a more important risk factor for immune responses to the transgene than the molecular form of the AAV genome.

Disclosures:

Herzog: Genzyme: AAV technology, AAV technology Patents & Royalties.
In Vivo Targeting Of Acute Myeloid Leukemia Using CpG-Stat3 siRNA Results In T Cell-Dependent Tumor Eradication
Authors: Hossain Dewan Md Sakib , Santos Cedric Dos , Zhang Qifang , Kozlowska Anna , Liu Hongjun , Gao Chan , Moreira Dayson , Swiderski Piotr , Jozwiak Agnieszka , Kline Justin , Forman Stephen J. , Bhatia Ravi , Kuo Ya-Huei , Kortylewski Marcin
Published: October 2019

Signal Transducer and Activator of Transcription-3 (STAT3) is an oncogene and immune checkpoint commonly activated in cancer cells and in tumor-associated immune cells. As a focal point of downstream signaling from numerous cytokines and growth factors present in the tumor microenvironment, STAT3 is an attractive but also elusive pharmacological target. We previously developed an immunostimulatory strategy based on targeted Stat3 gene silencing in Toll-like Receptor (TLR9)-positive hematopoietic cells using CpG-siRNA conjugates. Here, we assessed therapeutic effect of systemic STAT3 blocking/TLR9 triggering in disseminated acute myeloid leukemia (AML). We used a model of mouse Cbfb/MYH11/Mpl-induced leukemia, which mimics human inv(16) AML. Our results demonstrate that intravenously delivered CpG-Stat3 siRNA, but not control oligonucleotides, can eradicate established AML and impair leukemia-initiating potential. These antitumor effects require host’s effector T cells but not TLR9-positive antigen-presenting cells. Instead, CpG-Stat3 siRNA has direct immunogenic effect on AML cells in vivo leading to up regulation of MHC class II, co-stimulatory molecules and proinflammatory mediators, such as IL-12 and IFNγ, while down regulating expression of co-inhibitory PD-L1 molecule. Systemic injections of CpG-Stat3 siRNA generate potent tumor antigen specific immune responses, increase the ratio of tumor-infiltrating CD8+ T cells to T regulatory cells (Tregs) in various organs and result in CD8+ T cell-dependent regression of leukemia. Targeted STAT3 inhibition/TLR9 triggering also enhances immunogenicity of primary patients’ AML cells which gain ability to stimulate T cell proliferation. Our findings underscore the potential of using targeted STAT3 inhibition/TLR9 triggering to break tumor tolerance and induce potent immunity against AML and potentially other TLR9-positive blood cancers.

Disclosures:

No relevant conflicts of interest to declare.
GLI2 Transcription Factor Modulates CD40 Ligand Expression In Bone Marrow Stromal Cells
Authors: Neil Matthew S. , Dennison Joel R. , Fernandez-Zapico Martin E. , Elsawa Sherine F.
Published: October 2019

The interaction between tumor cells and their surrounding microenvironment is essential for the growth and persistence of cancer cells within the host. Cytokines play a key role in mediating this crosstalk between malignant cells and the tumor microenvironment. Although this interaction is clearly established, many of the molecular mechanisms mediating these signaling events remain elusive. In previous work, we have shown that the cytokine CCL5 can regulate the expression and activity of the transcription factor GLI2 in stromal cells. GLI2 is an effector of hedgehog (HH) signaling; however, our studies reveal a role for GLI2 independent of HH. GLI2, in turn, increases the expression and secretion of IL-6 in the tumor microenvironment, highlighting a novel role for GLI2 in directly modulating cytokine genes. We therefore screened for additional cytokines regulated by this axis in HS-5 bone marrow stromal cells using expression arrays. We found that GLI2 can modulate the expression of several cytokines including CD40 ligand (CD40L), CCL2, CCL7 and CXCL11. CD40L is an immunomodulatory and proinflammatory molecule that has been shown to play a role in multiple hematologic malignancies including chronic lymphocytic leukemia (CLL), Waldenström macroglobulinemia (WM) and acute myeloid leukemia (AML) pathogenesis. We first characterized the regulation of CD40L, because of the importance of CD40L in malignant cell biology. Gene expression results were validated by RT-PCR in two additional bone marrow stromal cell lines Saka and L88 where overexpression of GLI2 resulted in increased CD40L expression. Furthermore, we detected increased levels of soluble CD40L (sCD40L) in L88 and Saka cells overexpressing GLI2. Conversely, knockdown of GLI2 resulted in reduced expression and secretion of this cytokine. Biochemical mapping of the domains critical for this GLI2 regulatory mechanism reveals that N-terminal transcriptional domain is dispensable, as a mutant form of GLI2 lacking this domain (delta N-GLI2) increases CD40L gene expression by RT-PCR, protein expression by western blot and sCD40L secretion by ELISA, beyond that of the full-length GLI2 levels. Bioinformatics analysis of the CD40L promoter indicates the presence of 3 candidate binding sites for GLI proteins, suggesting the modulation of CD40L by GLI2 is mediated by direct interaction with its promoter. Ongoing experiments are aimed at determining the mechanism by which GLI2 regulates CD40L expression (either by direct binding to the CD40L promoter or through another indirect pathway). Together, our data identify a novel mechanism controlling CD40L in stromal cells in the bone marrow microenvironment. Because of the importance of CD40L in malignant cell biology, understanding the mechanism of its regulation is of great importance for the development of therapies aimed at targeting the malignant bone marrow tumor microenvironment.

Disclosures:

No relevant conflicts of interest to declare.
The Classical and Alternative NF-κB Activity Supports Cell Survival and Confers Chemo-Sensitivity Of Chronic Lymphocytic Leukemia Cells
Authors: Feng Guo , Xu Jingjing , Sun Aining , Chen Zi-Xing
Published: October 2019

Constitutive classical NF-κB activation protects chronic lymphocytic leukemia (CLL) B-cells from spontaneous cell death and plays a critical role in the chemo-resistance. RelB, representing the alternative NF-κB activity, functions specifically in lymphoid organogenesis and B-cell maturation. RelB indeed plays a tumor-supportive role and confer radiation-resistance in solid tumors. However, the involvement of RelB in CLL is unrevealed. Here, we analyzed the NF-κB activation in CLL bone marrow (BM). Both the RelA and RelB activity could be detected in CLL B-cells from BM, in spite of inevitable variability. High RelB activity, concomitant with high RelA activity, was linked to an unfavorable prognosis of CLL. CLL-derived BM stromal cells (BMSCs) were functionally similar to disease-free BMSCs in their capability to support CLL B-cells survival. The migration and adhesion ability of CLL B-cells was not affected by the RelB activity. High RelB activity, together with the RelA activity, maintained the basal survival of cells. The induction of RelA and RelB expression in the nuclear was responsible for the better survival of CLL B-cells supported by BMSCs. In addition, the presence of high RelB activity in CLL B-cells was correlated with the sensitivity to proteasome inhibitor but not fludarabine. Taken together, we provided evidences that not only RelA but also RelB, subunits of NF-κB family, plays an important role in the cellular behavior of CLL cells from BM. The strength of RelB activity influenced the prognosis of CLL patients.

Disclosures:

No relevant conflicts of interest to declare.
A6 Peptide Is Selectively Cytotoxic For Chronic Lymphocytic Leukemia Cells
Authors: Zhang Suping , Lai Hsien , Liu Grace , Rassenti Laura , Choi Michael Y. , Howell Stephen B , Finlayson Malcolm , Kipps Thomas J.
Published: October 2019

Chronic lymphocytic leukemia (CLL) cells express high levels of CD44, a cell-surface glycoprotein receptor for hyaluronic acid (HA). We found that a mAb specific for CD44 was directly cytotoxic for leukemia B cells, but had little effect on normal B cells. Moreover, this anti-CD44 mAb could induce CLL cells that expressed the zeta-associated protein of 70 kDa (ZAP-70) to undergo caspase-dependent apoptosis, independent of complement or cytotoxic effector cells (Proc Natl Acad Sci, USA 2013, PMID: 23530247). The cytotoxic effect of this mAb was not mitigated when the CLL cells were co-cultured with mesenchymal stromal cells (MSCs) or hyaluronic acid or when they were stimulated via ligation of the B-cell receptor with anti-µ.

A6 (Angstrom Pharmaceuticals) is an 8-amino acid peptide that has marked homology with a linear sequence of CD44. A6 can bind CD44 within a region of the ligand-binding domain, leading to inhibition of the migration and metastatic potential of CD44-expressing cancer cells in vitro and in vivo (Mol Cancer Ther, 2011 PMID: 21885863).

We evaluated the cytotoxic activity of A6 against primary leukemia cells of patients with CLL (n = 22). We found that A6 peptide also was directly cytotoxic for CLL cells isolated from different patients in a dose-dependent manner at concentrations that may be achieved in vivo. The A6 peptide appeared less cytotoxic for CLL cells than the intact anti-CD44 mAb, but still had greater direct cytotoxicity for CLL cells that expressed ZAP-70 than for CLL cells that were ZAP-70 negative. Furthermore, the A6 peptide had negligible effect on the viability of lymphocytes isolated from the blood of healthy donors (n = 3). Because clinical studies have found the A6 peptide to be well-tolerated and without dose-limiting toxicity in patients with solid tumors who have been treated to date (N = 40), a clinical study is planned to evaluate the safety and activity of the A6 peptide in the treatment of patients with CLL.

Disclosures:

Howell: Angstrom Phamaceuticals: Membership on an entity’s Board of Directors or advisory committees. Finlayson:Angstrom Phamaceuticals: Employment.
Tocilizumab Overcomes Chemo-Resistance Of CLL Cells
Authors: Liu Fengting , Jia Li , Wang Ping , Farren Tim , Agrawal Samir G
Published: October 2019

IL-6 in the cancer microenvironment plays a crucial role in the constitutive activation of STAT3, which contributes to cancer cell progression. IL-6 binds to a heterodimer of membrane-bound receptors, IL-6R (CD126) and gp130, that triggers phosphorylation of JAK2 and downstream STAT3. The phosphorylated STAT3 acts as a transcription factor, binding target DNA and promoting tumorigenesis, cell proliferation and survival. IL-6 signaling pathways have been extensively studied as therapeutic targets in both cancer and autoimmune conditions. The anti-IL6R antibody Tocilizumab is a humanized anti CD126 antibody, approved by the FDA (Food and Drug Administration) for treatment of rheumatoid arthritis, that can block IL-6 classic and trans-signalling pathways. We investigated IL-6R expression in CLL cells and analysed the correlation of IL-6R expression with chemo-sensitization and the potential of targeting IL-6R to overcome drug resistance in vitro.

41 CLL patients were studied. Membrane-bound and soluble IL-6 receptors, CD126 and gp130, were measured by flow cytometry or ELISA. Constitutive activation of STAT3 and the expression of Bcl-2 family proteins were detected by western blotting. Cell cycle distribution was assessed with Ki67 and cell viability indicated by propidium iodide (PI) negative staining.

CLL cells were found to express variable levels of membrane-bound receptors of IL-6, CD126 (average 22%, range 3-60%) and gp130 (average 12%, range 3-25%), which was significantly higher than normal B-cells (CD126 average 1.7%, range 0-6.3%; gp130 average 1.3%, rage 0-7.9%). Although in vitro Chlorambucil-induced cell death was variable among the cases, there was a strong correlation with CD126 expression (Figure 1): higher CD126 expression was associated with increased resistance to Chlorambucil.

Treatment of CLL cells with Tocilizumab led to inhibition of constitutive STAT3 activation decreased Mcl-1, Bcl-xl expression and cells exited from G0 phase of the cell cycle. Tocilizumab did not induce cell death in CLL cells. Combined treatment with both Chlorambucil and Tocilizumab significantly increased cell death (p < 0.01). Interestingly, higher CD126 expression was highly significantly correlated with the increased cell death seen with combined Chlorambucil and Tocilizumab (p < 0.0001, Figure 2)

These results suggest that inhibiting IL6 signal pathways via IL-6R blockade with Tocilizumab is a new approach in CLL, leading to increased chemo-sensitisation to standard chemotherapeutic agents. Although CLL cells with higher CD126 expression exhibited more drug resistance in vitro, they also showed better response to Tocilizumab. Targeting the combination of Tocilizumab with anti-cancer therapy to CLL patients with high IL-6R expression might achieve clinical therapeutic efficacy.

Disclosures:

No relevant conflicts of interest to declare.
Arsenic Trioxide Induces Apoptosis In Chronic Lymphocytic Leukemic Cells Through Down-Regulation Of Survivin Via The p53-Dependent Signaling Pathway
Authors: Zhang Xiaohui , Lv Meng , Jiang Qian , Zhu Honghu , Qin Yazhen , Huang Xiaojun
Published: October 2019

Arsenic trioxide induces cell apoptosis in a variety of tumor cells. However, the molecular mechanism underlying the killing effects of arsenic trioxide on tumor cells is not fully understood. Here, we demonstrate a signal pathway of arsenic trioxide treatment that leads to apoptosis in a B-cell chronic lymphocytic leukemia (WSU-CLL) cell line. Arsenic trioxide treatment significantly inhibited proliferation and induced apoptosis of WSU-CLL cells in a time- and dose-dependent manner. This anti-tumor effect of arsenic trioxide on WSU-CLL cells was mediated through the down-regulation of survivin and up-regulation of p53. Knock-down of intracellular survivin with siRNA potentiated the inhibitory effect of arsenic trioxide on proliferation in WSU-CLL cells. Also, knock-down of p53 by a specific siRNA prevented the down-regulation of survivin by arsenic trioxide and reduced the cytotoxic effect of arsenic trioxide to WSU-CLL cells. These results indicate that arsenic trioxide may be used to as novel therapeutic agent for chronic lymphocytic leukemia, which down-regulates survivin expression via a p53-dependent signaling pathway.

Disclosures:

No relevant conflicts of interest to declare.