bluuebunny/crossref_metadata_2025 huggingface dataset:
Divalent Cations Define the Structural Requirements for αIIbβ3 Integrin Activation
Authors: Kamata Tetsuji , Handa Makoto , Kawai Yohko , Ikeda Yasuo , Aiso Sadakazu
Published: October 2019

Abstract 260

Platelet αIIbβ3 integrin undergoes structural rearrangements to increase the affinity for ligands. Two major changes in integrin three-dimensional structure are critical for this activation process. One is the straightening of integrin legs (extension), and the other is the swing out movement of the β3 hybrid domain (swing-out). In this study, we have found that this structural requirement for activation differs depending upon the divalent cations present. We previously reported that the swing-out-defective mutant αIIbD319C/β3V359C did not bind fibrinogen in the presence of Ca2+/Mg2+. However, the same mutant bound fibrinogen in the presence of Mn2+, when activated by monoclonal antibody PT25-2 or by forcing the integrin legs to extend by concomitant αIIbQ595NTT mutation. We engineered another mutation that was designed specifically to prevent the swing-out. Likewise, this β3G327C/β3V419C mutation blocked fibrinogen binding in the presence of Ca2+/Mg2+, but not in the presence of Mn2+. On the other hand, when integrin extension was specifically prevented by αIIbD464C/αIIbS728C mutation, fibrinogen binding was observed neither in the presence of Ca2+/Mg2+ nor in Mn2+. Thus, swing-out is not essential for activation in Mn2+, as long as integrin legs are in extended state, while both swing-out and extension are required in Ca2+/Mg2+. To explore the underlying mechanism originating this difference, three distinct cation-binding sites in the βA domain were mutated. Among the three sites, MIDAS is occupied by Mg2+, while ADMIDAS and LIMBS/SyMBS are occupied by Ca2+ in Ca2+/Mg2+ condition. When amino acid residues composing these sites were mutated to Ala, none of them bound fibrinogen, except for D126A and D127A in the presence of Ca2+/Mg2+. Notably, these mutations restored fibrinogen binding in the swing-out-defective mutant. Amino acid residues D126 and D127 compose ADMIDAS together with S123 and M335. Swing-out of the hybrid domain disrupts M335 from ADMIDAS. This allows Ca2+ to move toward MIDAS, bringing the β1-α1 loop closer to ligand to provide contact site. However, similar rearrangement of the cation/ligand binding sites has been shown to take place in Mn2+ without the swing-out. The results suggest partial disruption of ADMIDAS is the key event for activation in Ca2+/Mg2+, while it is not so in Mn2+. Our results may reconcile the dispute over the apparently contradicting findings in the crystal structure that integrin with a closed head with Mn2+ represents an active conformation as one with an open head with Ca2+/Mg2+.

Disclosures:

Kawai: Daiichi Sankyo: Consultancy; Bayer: Consultancy; Toyama Chemical: Consultancy.
Monocytosis Has Adverse Prognostic Significance and Impacts Survival in Patients with T-Cell Lymphomas.
Authors: Sacchi Stefano , Bari Alessia , Tadmor Tamar , Marcheselli Luigi , Liardo Eliana Valentina , Pozzi Samantha , Luminari Stefano , Baldini Luca , Federico Massimo , Polliack Aaron
Published: October 2019

Abstract 2647

New data concerning the important role of microenvironment on lymphoma growth are emerging, and in recent years surrogate biomarkers have been identified as prognostic factors for survival in non-Hodgkin lymphoma (NHL). Unlike follicular (FL), diffuse B-cell lymphoma (DLBCL) and Hodgkin lymphoma (HL), there is still no specific internationally accepted risk stratification scoring system for peripheral T-cell lymphomas (PTCL), and the International Prognostic Index (IPI) or the Prognostic Index for T-cell lymphomas (PIT) model are been used to identify higher risk cases of PTCL. Here we retrospectively analyzed the relevance of the well recognized prognostic parameters for T-NHL in 172 patients with different types of PTCL. In 94 cases in whom peripheral blood monocyte count (PBMC) at diagnosis was available, we evaluated whether monocytosis (PBMC >800/mm3) could be used as a simple prognostic factor for overall survival (OS) and outcome in PTCL.

For the entire group with a median follow-up of 19 months (range 1–168 months), the 5-years OS was 42%, and the median OS 48 months. Monocytosis was present in 23% of the evaluable cases and patients with high PBMC (>800/mm3) at diagnosis had a worse OS (median 12 months) compared to those with PBMC < 800/mm3.This difference showed strong statistical significance (p=0.003) (Fig 1) and the Hazard ratio (HR) for PBMC >800/mm3, stratified by histopathological subtype, was 2.81. In particular 3-years OS of patients with PBMC >800/mm3 with anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, and peripheral T-cell lymphoma not otherwise specified was 50%, 25%, and 12%, respectively compared to 86%, 65%, and 50% for those patients with PBMC <800/mm3. In univariate analysis age >60y, advanced stage, bone marrow involvement, ECOG PS >1, LDH>UNL, PBMC >800/mm3, hemoglobin <12 gr/dL, albumin <3.5 gr/L were associated with inferior OS. In multivariate analysis, monocytosis alone retained a negative prognostic value even when adjusted for PIT and stratified by histolopathological subtype (HR 2.41, p=0.015).

In this study, as in others on B-NHL and HL, monocytosis had an independent negative impact on survival in patients with T-NHL. This data, which provide convincing support for the use of monocytosis as a simple prognostic parameter, now need to be further validated in a larger cohort of patients with T-NHL.

Figure 1: Overall survival (OS) of 94 patients with PTCL according to peripheral blood monocyte count (PBMC): monocytosis (PBMC >800/mm3) has a negative impact on OS (p=0.003) Figure 1:. Overall survival (OS) of 94 patients with PTCL according to peripheral blood monocyte count (PBMC): monocytosis (PBMC >800/mm3) has a negative impact on OS (p=0.003)

Disclosures:

No relevant conflicts of interest to declare.
“Early Lesion” of Follicular Lymphoma (FL) Precedes Clinically Overt FL: A Retrospective Study of Lymph Nodes (LNs) Obtained At Surgery with LNs Dissection for Preceding Cancer.
Authors: Ota Yasunori , Izutsu Koji , Ota Hikari , Nishida Aya , Ishiwata Kazuya , Tsuji Masanori , Yamamoto Hisashi , Yamamoto Go , Asano-Mori Yuki , Wake Atsushi , Fujii Takeshi , Taniguchi Shuichi
Published: October 2019

Abstract 2716

Follicular lymphoma (FL) is the most common subtype of indolent lymphoma and most patients with FL are diagnosed with advanced stage. “Early lesion” of FL including partial involvement of FL and FL in situ was proposed recently, of which clinical implication remains uncertain. One report suggested that some patients with FL in situ can develop into clinically overt FL afterward (Blood 2011; 118:2976). However, it is unclear whether clinically overt FL is usually preceded by “early lesion”. In order to answer this question, we retrospectively reviewed the archived pathological specimen of lymph nodes (LNs) that had been obtained at surgery with LNs dissection for a preceding other malignancy before the diagnosis of FL in patients with clinically overt FL.

Materials and Methods

Cases for study were diagnosed as FL at Toranomon Hospital between January 2001 and March 2012 and had undergone surgery with LNs dissection for a preceding malignancy. We reexamined the dissected LNs by morphology and immunohistochemistry. The hematoxylin and eosin stained slides were reviewed and immunohistochemical staining was performed using a streptavidin-biotin complex technique. Antibodies included the following: CD5, CD10, CD20, CD21, BCL2, BCL6, MIB1. This study was performed with the approval by the Institutional Review Board.

Results

Among the 167 patients who were diagnosed as FL at Toranomon Hospital during this period, 8 patients (4.8%) had undergone surgery with LNs dissection for a preceding malignancy (Table). Preceding malignancy was gastric carcinoma (n=3), esophageal carcinoma (n=2), and endometrial carcinoma (n=2), and colon cancer (n=1), respectively. Surgery for preceding malignancy had been performed at a median of 18 months (9–186 months) before the diagnosis of FL. No patient had lesions suggestive of lymphoma on preoperative imaging studies or endoscopic examinations. With immunostaining of the archived specimen, FL was found in all of the 8 cases, of which 6 cases were FL in situ and 2 cases were usual FL. The number of resected LN was median 38 (19–153) and the involvement of FL was detected in 60% of the resected LNs. LNs metastasis of carcinoma was found in 4 cases. Median duration from surgery for precedent malignancy to the diagnosis of overt FL was 26 mo(12–186) in patients preceded by the FL in situ and 13 mo (9,17) in patients preceded by clinically latent but pathologically usual FL.

Conclusion

We found that FL is preceded by latent FL that is only detectable by pathology. Remarkably, 6 out of 8 cases were preceded by FL in situ. This retrospective series suggests that “early lesion” of FL is a precursor lesion of clinically overt FL.

Disclosures:

No relevant conflicts of interest to declare.
Activation Of Natural Killer Cells By Soypeptide Lunasin and Cytokine: Implication In Cancer Immunotherapy For Lymphoma
Authors: Tung Chun-Yu , Kyazike Sharifah , Lewis David , Han Ling , Kolb Alexander , Pelloso David , Sinn Anthony , Pollok Karen E. , Srivastava Shivani , Robertson Michael J. , Chang Hua-Chen
Published: October 2019

Introduction

Immunostimulatory cytokines can enhance anti-tumor immunity and are part of the therapeutic armamentarium for cancer treatment. We have previously reported that chemotherapy-treated lymphoma patients have acquired deficiency of Signal Transducer and Activator of Transcription 4 (STAT4), which results in defective IFNg production during clinical immunotherapy. With the goal of further improvement in cytokine-based immunotherapy, we examined the effects of a soybean peptide called lunasin that exhibits immunostimulatory effects on natural killer (NK) cells.

Experimental Design

PBMCs of healthy donors and chemotherapy-treated lymphoma patients were stimulated with or without lunasin in the presence of IL-12 or IL-2. NK activation was evaluated, and its tumoricidal activity was assessed using in vitro and in vivo tumor models. Chromatin immunoprecipitation (ChIP) assay was performed to evaluate the histone modification of gene loci that are regulated by lunasin and cytokine.

Results

Adding lunasin to IL-12- or IL-2-cultuted NK cells demonstrated synergistic effects in the induction of IFNG and genes involved in cytotoxicity. The expression level of CD16 and granzyme B was increased in CD56-bright population of NK cells following stimulation with lunasin and cytokine. The combination of lunasin and cytokines (IL-12 plus IL-2) was capable of restoring IFNg production by NK cells from post-transplant lymphoma patients. In addition, NK cells stimulated with lunasin plus cytokines had higher tumoricidal activity than those stimulated with cytokines alone using in vitro and in vivo tumor models. Moreover, lunasin augmented antibody-dependent cellular cytotoxicity (ADCC) of NK cells against anti-CD20 coated human B-lymphoma cell line. The underlying mechanism responsible for the effects of lunasin on NK cells is likely due to epigenetic modulation on target gene loci.

Conclusion

We have discovered a novel use of lunasin that exerts synergistic effects together with IL-12 or IL-2. This synergism leads to stronger NK-mediated anti-tumor activity, suggesting the potential clinical use of lunasin to augment the therapeutic responses in cytokine-based immunotherapy.

Disclosures:

No relevant conflicts of interest to declare.
Molecular Dynamics As a Computational Tool To Analyze Lymphocyte Alloreactivity In Hematopoietic Transplant Recipients
Authors: Guerrero Estefania Garcia , Moreno Irene Diaz , De la rosa Miguel Angel , Pérez Simón Jose Antonio , Quintana Antonio Diaz
Published: October 2019

Introduction

The generation of the immune response requires the recognition of peptides presented by the major histocompatibility complex (MHC) through the T cell receptor (TCR). In the hematopoietic transplantation context, T cells (LT) from the donor recognize foreign MHC or own MHC bound to foreign peptides (pMHC), generating an alloimmune response. Currently, the molecular mechanisms of LT alloimmune activation are unknown. In order to analyze the molecular interactions between peptides, MHC and TCR, we have implemented Molecular Dynamics techniques. We have compared immunologically reactive complexes (HLA-A2/TAX/TCR-A6; HLA-A2/HUD/TCR-A6) to non/weakly reactive complexes (HLA-A2/V7R/TCR-A6; HLA-A2/P6A/TCR-A6; HLA-A2/Y8A/TCR-A6).

Methods

Starting structures of two reactive complexes were downloaded from the PDB database and used to model mutations known to lead to different degrees of immune reactivity. Dynamics simulations were performed and analyzed using the program AMBER version 9. The simulation time was approximately 10 ns. Further analysis was carried out using the script ARO (Díaz-Moreno et al. 2009) in the VMD Tk console.

Results

A total of 17 MD trajectories have been reckoned, to simulate the behavior of isolated components of the different MHC-TCR complexes. Analysis of the fluctuations shows that pMHC binding barely restrains TCR motions, affecting mostly to CDR3 loops. Opposite, pMHC displayed substantial changes in its dynamics upon comparing its free versus ternary form (pMHC-TCR). Furthermore, taking as reference the positions of the MHC´s helices in free binary structures (MHC-peptide) and comparing them to the positions in ternary structures (pMHC-TCR), we can observe that in reactive complexes MHC exhibits a higher variability than in non-reactive complexes, suggesting that the MHC must tighten and acquire a position further away from its position in free binary form.

We also analyzed the position of the peptide in the groove of MHC and we found that at position 5 (aa aromatic) the peptide is diverted although its position in the groove of MHC seems to be unrelated to the reactivity of the interaction TCR-pMHC.

Conclusions

The MHC shows strong changes in its molecular dynamics upon binding TCR, decreasing its mobility. The structure of MHC is slightly perturbed in reactive complexes, but not in non-reactive ones.

Financial support

Spanish MINECO (BFU2009-07190/BMC, BFU2012-31670/BMC) the Andalusian Government (BIO198) and Instituto de Salud Carlos III (PFIS - FI12/00189 and FIS PI11/02366)

Disclosures:

No relevant conflicts of interest to declare.
In Vitro Study Of The Mechanisms Involved In The Bone Marrow Mesenchymal Stromal Cell Modulatory Effect On B Cell Function
Authors: Amati Eliana , Bassi Giulio , Di Trapani Mariano , Liotta Francesco , Annunziato Francesco , Perbellini Omar , Ricciardi Mario , Pizzolo Giovanni , Scupoli Maria Teresa , Krampera Mauro
Published: October 2019

Human bone marrow Mesenchymal Stromal Cells (MSC) are potent modulators of T cell activation and proliferation, mainly through the production of partially defined soluble factors, including the IFN-g-induced tryptophan-degrading enzyme IDO, a key immunosuppressive effector pathway. Actually, MSC may affect the functions of virtually all immune effector cells, including B cells. However, current literature concerning MSC immunomodulatory activity on B cells is still controversial, due to both biological peculiarities of B cells, which do not produce IFN-γ, a key MSC-triggering cytokine, and to different and poorly comparable experimental approaches.

Human purified B cells, either resting or activated for 4 days with a stimulation cocktail (CD40 ligand + enhancer polyhistidine mAb MAB050 + rhIL-2 + mouse F(ab’)2 anti-human IgM/IgA/IgG + CpG oligodeoxynucleotide 2006) were co-cultured with MSC, either at resting conditions or following inflammatory priming (MSC pre-incubation with IFN-γ + TNF-α for 48 hours), or with MSC supernatants. CD27-positive (memory) and CD27-negative (naïve) B cell survival, proliferation, and intracellular activation status (through signaling network analysis by Phosphoflow) were assessed. Our results showed that MSC are normally supportive cells, not intrinsically capable of suppressing B cell proliferation, and require inflammatory priming to acquire B cell inhibitory potential. Inflammatory-primed MSC impair significantly activated B cell growth in a cell contact-independent manner, as supernatant is sufficient to provide maximal inhibition of B cell proliferation. B cell inhibition by MSC is not related to either induction of B cell apoptosis or early signaling events necessary for B cell activation. In addition, IDO pathway triggered in IFN-γ-primed MSC seems to have a role also in B cell inhibition by interfering with the tryptophan metabolism.

Overall, B cell behavior following the interaction with MSC depends on the functional state of both B cells and MSC. The role of IDO in B cell regulation needs further investigation, as it may be relevant to develop new therapeutic approaches in pathological conditions related to B cell hyper-activation.

Disclosures:

No relevant conflicts of interest to declare.
Influenza Virus-Containing Immune Complexes Activate Platelets Through FcγRIIa Resulting In Bioactive Lipid Synthesis and Microparticle Release
Authors: Boilard Eric , Pare Guillaume , Nathalie Cloutier , Dubuc Isabelle , Rousseau Matthieu , Flamand Louis
Published: October 2019

After red blood cells, platelets represent the most abundant cell lineage in blood, where they sentinel the vasculature and play crucial functions in haemostasis and the prevention of bleedings. Platelets also express a vast array of immune mediators and receptors, suggesting that they can also be regarded as tiny immune cells capable of the recognition of pathogens. Indeed, platelets express functional Toll-like receptors and are thought to actively participate to innate immunity. During severe cases of H1N1 influenza A virus infection, circulating platelets display markers of activation. The specific platelet activation triggers during Influenza infection remain however unknown. In this study, we incubated human platelets with H1N1 influenza A virus (IAV) and monitored platelet activation. We found that IAV is a highly potent inducer of de novo lipid mediators production and a trigger for the release of microparticles from platelets. This activation process takes place independently of the Toll-like receptor 4 and requires the presence of serum, pointing to the contribution of soluble factor(s) present in blood. We observed that the virus scaffolds with immunoglobulin G to form large immune-complexes (∼1micron in size) that activate platelets through the engagement of FcgRIIA. Accordingly, the serum of naïve mice housed in a pathogen-free facility is ineffective at initiating human platelet activation when incubated in the presence of IAV. Intrigued by the fact that all the tested human sera were capable of forming immune complexes with IAV, we hypothesized that significant antibody cross-reactivity between different influenza viruses was sufficient to promote the formation of virus-containing immune complexes. Using in vivo approaches, we found that the antibodies from H3N2 influenza virus-immunized mice generate immune complexes when put in presence of H1N1, activating human platelets and transgenic mouse platelets that express FcgRIIA. Taken together, our observations demonstrate that beyond their activities in haemostasis and innate immunity, platelets can also play a role during the active stage of adaptive immune responses against pathogens through FcγRIIa activation.

Disclosures:

No relevant conflicts of interest to declare.
Impaired Hemostasis Leads To Alveolar Hemorrhages and Increased Mortality In Influenza A Infection
Authors: Antoniak Silvio , Tatsumi Kohei , Milner Justin , Beck Melinda , Mackman Nigel
Published: October 2019

Objective

Viral infections lead to activation of coagulation which enhances inflammatory responses. For instance, influenza A is associated with activation of coagulation and the increased risk for thrombotic events, such as myocardial infarction. Protease-activated receptor 4 (PAR-4) is the main functional receptor on mouse platelets. Here, we investigated the role of tissue factor (TF) and platelets in influenza A infection in mice.

Methods

We used male LowTF mice, which express 1% of normal TF levels, PAR-4 deficient mice and respective control mice. All mice were infected intranasal with influenza A H1N1/PR8. Changes in body weights and survival were analyzed up to 14 days after infection. In addition, bronchoalveolar lavage (BAL) was collected and analyzed 7 days after virus challenge.

Results

LowTF mice exhibited increased loss of body weights compared to littermate control mice between day 4 and 6 after infection (P<0.05). At day 7, BAL of LowTF had increased levels of free hemoglobin compared to infected control mice (2.60±0.39 g/dL vs. 0.05±0.02 g/dL, P<0.05). In addition, LowTF lungs exhibited increased protein levels in the BAL compared to controls (1.52±0.18 mg/mL vs. 0.70±0.26 mg/mL, P<0.05). Lung histology revealed that LowTF mice had extensive hemorrhages. The impaired hemostasis resulted in increased mortality in LowTF mice compared to control mice (90% vs 30%, P<0.004, N=9-10, Figure). Similarly, mice with defective thrombin signaling in platelets (PAR-4-/-) exhibited visible lung hemorrhages with increased hemoglobin levels in the BAL (P<0.05) at day 7 and increased mortality compared to controls (57% vs 10%, P<0.03, N=9-14, Figure).

Conclusions

TF mediated activation of coagulation and PAR-4 dependent platelet activation are essential for pulmonary hemostasis during influenza A infection.

Disclosures:

No relevant conflicts of interest to declare.
Inhibition Of Anti-b2GPI-Dependent Thrombus Enhancement In The Laser-Induced Thrombosis Model In Mice
Authors: Kolyada Alexey , Porter Andrew , Beglova Natalia
Published: October 2019

B2GPI is the major antigen for pathogenic antibodies in antiphospholipid syndrome (APS). The presence of anti-b2GPI antibodies correlates with increased risk for thrombosis and pregnancy loss in patients with APS. In animal models of thrombosis, anti-b2GPI antibodies cause the increase in thrombus size after injury to the vessel wall. We engineered a molecule, A1-A1, that prevents the binding of b2GPI to anionic phospholipids and ApoER2, and studied how it inhibits prothrombotic properties of anti-b2GPI antibodies in vivo using the laser-induced arterial thrombosis model. Lupus-prone (NZWxBXSB)F1 male mice express chronic autoimmune anti-b2GPI antibodies. In (NZWxBXSB)F1 mice, infusion of A1-A1 at a dose of 4 ug/g of body weight caused a decrease in thrombus size of more than 85% as compared to thrombus size in the absence of A1-A1. Similarly, A1-A1 inhibits prothrombotic properties of b2GPI/anti-b2GPI antibody complexes in wild type mice following acute infusion with anti-b2GPI positive APS IgG. The thrombus reduction was due to the binding of A1-A1 to b2GPI, because infusion of LA6, a structural homologue of A1 that does not bind b2GPI, did not affect thrombus size. We did not observe thrombus inhibition by A1-A1 in the absence of anti-b2GPI antibodies, indicating that A1-A1 does not affect normal thrombus formation in the absence of anti-b2GPI antibodies.

Disclosures:

No relevant conflicts of interest to declare.
Heparin-Induced Thrombocytopenia: A Rapid, Reliable and Practical, Functional Flow-Cytometric Assay
Authors: Deutsch Varda , Cipok Michal , Kay Sigi , Levy Yvette , On Shoshana Bar , Kirgner Ilya , Naparstek Elizabeth , Tomer Aaron
Published: October 2019

Background

Heparin-induced thrombocytopenia (HIT) is a serious complication of heparin treatment, associated with morbidity and mortality. HIT is characterized by thrombocytopenia and thrombotic complications secondary to the formation of antibodies (Abs) against heparin-platelet-factor 4 (PF4) complexes. The pathologic mechanism involves the binding of the heparin-immune-complex to the platelet-Fc-receptor, resulting in platelet activation, aggregation, and rapid elimination. The diagnosis of HIT requires laboratory confirmation. Common laboratory testing is based on immune detection of antibodies directed against the PF4/heparin complex (ID-H/PF4-PaGIA or ELISA). However, these assays suffer from methodological limitations, especially low specificity, as compared to the platelet functional assays. The “gold standard” functional test for detecting of platelet-activating antibodies is the radioactive [14C] serotonin-release assay (14C-SRA) (Sheridan D, et al, Blood. 1986;67:27-30, Kelton JG, et al.,Blood.1988;72:925-30). However, the assay includes the use of a radiolabeled biological probe and requires considerable expertise to obtain reliable results. Consequently, its use is limited to research laboratories.

Aim

To overcome the methodological limitations associated with current assays, we modified a functional flow-cytometry assay (FCA), which exhibits high sensitivity and specificity (Tomer, A. Br J Haematol, 1997;98: 648-656 , Tomer, A., et al, Am J Hematol, 1999;61: 53-61). This assay, similar in concept to the 14C-SRA, determines the capacity of the patient's serum to activate platelets in the presence of heparin, using a fluorescent probe.

Methods

Consecutive samples from 254 patients clinically suspected for HIT were tested. The FCA assay was compared with the standard ID-H/PF4-PaGIA antigenic assay (DiaMed, Switzerland) with two dilutions to assess specificity (Nellen, V., et al.,Haematologica, 2012;97: 89-97).

Results

Of the total 254 samples tested, 48 (19%) were positive by PaGIA, compared to 13 (5.1%) positive by the functional FCA (Table 1). The number of PaGIA positive samples was reduced to 24 (9.4%) by 1:16 dilution, and to 14 (5.5%) by 1:32 dilution. All FCA positive samples were positive at all PaGIA dilutions (relative sensitivity 93%). Thirty PaGIA negative samples were all negative by the FCA (relative specificity 100%).

Conclusion

The results suggest that the functional FCA is a practical, sensitive, and highly specific test for the reliable diagnosis of HIT.

Disclosures:

No relevant conflicts of interest to declare.
Four-Factor Prothrombin Complex Concentrate (4-PCC) Effectively Reverses Edoxaban Induced Bleeding In a Rabbit Model Of Acute Injury
Authors: Herzog Eva , Kaspereit Franz , Krege Wilfried , Pragst Ingo , Morishima Yoshiyuki , Schulte Stefan , Dickneite Gerhard
Published: October 2019

This study was designed to determine whether the four-factor Prothrombin Complex Concentrate (4-PCC) Beriplex® P/N (Kcentra™) can effectively reverse bleeding following edoxaban administration in a rabbit kidney injury model.

The oral direct and selective factor Xa inhibitor edoxaban (Daiichi Sankyo) is currently available in Japan for the prophylaxis of venous thromboembolism (VTE) in patients undergoing major orthopedic surgery and is undergoing investigation in phase III trials for the prevention of stroke in patients with atrial fibrillation and the treatment and secondary prevetion of VTE. The primary complication of any available anticoagulant therapy is the risk of bleeding. Rapid reversal of anticoagulation may be necessary in patients requiring emergency treatment due to uncontrolled bleeding. PCCs are frequently used to reverse the effect of vitamin K antagonists such as warfarin and have also been suggested to be potentially effective in reversing the effects of the new oral anticoagulants.

The present study was therefore designed to further investigate the ability of 4-PCC to reverse the anticoagulant action of edoxaban using an in vivo rabbit model of acute bleeding.

Animals were treated with a high intravenous bolus dose of edoxaban (1200 µg/kg) leading to increased and prolonged bleeding following standardized kidney injury compared to vehicle administration. Parallel monitoring of biomarkers of haemostasis showed that edoxaban treatment resulted in the prolongation of prothrombin time (PT), activated partial thromboplastin time, and whole blood clotting time (WBCT), and affected the thrombin generation parameters thrombin peak, endogenous thrombin potential (ETP), lagtime and time to peak. Subsequent administration of the 4-PCC (25-75 IU/kg) resulted in a dose-dependent reversal of edoxaban induced bleeding as indicated by reduced time to haemostasis and total blood loss. Both parameters achieved statistical significance compared to placebo at the 4-PCC dose of 50 IU/kg under fully blinded study conditions. The biomarkers correlating best with 4-PCC mediated edoxaban anticoagulation reversal included PT, WBCT and ETP.

In summary, 4-PCC treatment effectively decreased edoxaban induced hemorrhage in an animal model of acute bleeding at clinically relevant dose levels.

Disclosures:

Herzog: CSL Behring GmbH: Employment. Kaspereit:CSL Behring GmbH: Employment. Krege:CSL Behring GmbH: Employment. Pragst:CSL Behring GmbH: Employment. Morishima:Daiichi Sankyo: Employment. Schulte:CSL Behring GmbH: Employment. Dickneite:CSL Behring GmbH: Employment.
The Transfer Of Dabigatran and Rivaroxaban Across a Dually Perfused Isolated Human Placental Cotyledon – Implications For Therapy In Pregnancy
Authors: Bapat Priya , Kedar Reuven , Melamed Nir , Matlow Jeremy , Lubetsky Angelika , Aleksa Katarina , Berger Howard , Koren Gideon
Published: October 2019

Background

Anticoagulant therapy is often required in cases of high-risk pregnancy for the prophylaxis of venous thromboembolism following surgery, atrial fibrillation, and congestive heart failure, and for the prevention of pregnancy loss in thrombophilic women. During pregnancy, the concentrations of many blood-clotting factors rise, thereby increasing the need for anticoagulants that are safe to use throughout gestation. Dabigatran (Pradaxa®) and rivaroxaban (Xarelto®) are newer generation oral anticoagulants that are increasingly being prescribed to women of reproductive age for the treatment of thromboembolic disorders. Dabigatran acts by directly inhibiting thrombin, and rivaroxaban acts as a direct factor Xa inhibitor. However, the information regarding fetal safety and placental transfer of these drugs is currently lacking. If there is limited transfer of either drug across the placenta, then it may not increase the risk of bleeding in the fetus. The objective of this study was to determine the transplacental kinetics of dabigatran and rivaroxaban.

Methods

Placentae were obtained with informed consent after elective caesarean section of healthy term pregnancies in Toronto, Ontario. The transplacental transfer of dabigatran and rivaroxaban were separately assessed using ex vivo dual perfusion of an isolated human placental cotyledon. Dabigatran, at a concentration of 35 ng/ml, was added to the maternal circulation at the start of the experimental phase. Maternal and fetal samples were taken throughout the pre-experimental (1 h) and experimental (3 h) phases for measurement of dabigatran and markers of placental viability. Separate placenta perfusions with rivaroxaban were conducted at an initial maternal concentration of 250 ng/ml. The perfused drug was measured in maternal and fetal samples using liquid chromatography-mass spectrometry (LC/MS).

Results

There was slow transfer of dabigatran from the maternal to fetal circulation. The fetal-to-maternal (F:M) concentration ratio was 0.33 ± 0.13 after 3 hours (n=3). In contrast, the transfer of rivaroxaban from maternal to fetal circulation was much more rapid, as characterized by a F:M ratio of 0.72 ± 0.12 at 3 hour (n=4), suggesting rapid equilibrium between maternal and fetal circulations. Placental viability markers for all perfusions were within normal ranges.

Conclusions

This is the first direct evidence of the transfer of dabigatran and rivaroxaban across the human placenta from the mother to fetus. It suggests less fetal exposure to dabigatran.

Disclosures:

No relevant conflicts of interest to declare.
Viral Infection Sentinel Rig-I Maintains Hematopoietic Stem Cell Function Through Regulating DNA-Damage Response
Authors: Zhang Wu , Ding Meng-Lei , Li Xian-Yang , Guo He-Zhou , Zhang Hong-Xin , Chen Sai-Juan , Zhu Jiang
Published: October 2019

Throughout life hematopoietic stem cells (HSCs) have to cope with various kinds of insults from inflammation to DNA damage constantly to maintain the integrity of stemness. It is possible that certain core factors are commonly implicated in the maintenance of HSC pool and function under discrete physiological and pathological conditions. However, the underlying mechanisms remain largely unexplored. Previous works have demonstrated that retinoic acid inducible gene I (Rig-I) plays an essential role in recognizing viral RNA and activating type I IFN transcription, but whether Rig-I is involved in the core program governing HSCs’ behaviors is unclear. Here, we report that in the steady status Rig-I deficiency significantly increased HSC number by dysregulating the cell-cycling status of HSCs in mice. However, HSCs in Rig-I-/- mice were actually more sensitive to genotoxic treatments such as irradiation as compared to wild type HSCs, causing more Rig-I-/- mice to die of hematopoietic exhaustion. In accordance, HSC transplantation assays showed a significant impact of Rig-I loss on the hematopoietic regeneration capacity. Mechanistically, we found that Rig-I represented a pivotal component of the molecular pathways that mediate DNA-damage response and the repair of DNA lesions. Taken together, these data indicate a crucial role of innate immunity-regulatory factor Rig-I in the maintenance of HSCs.

Disclosures:

No relevant conflicts of interest to declare.
Germline Events In GFI1 In Myelodysplastic Syndrome
Authors: Thota Swapna , McMahon Sarah , Makishima Hideki , Przychodzen Bartlomiej P , Guinta Kathryn M , Dienes Brittney , Hosono Naoko , Sekeres Mikkael A. , Maciejewski Jaroslaw P.
Published: October 2019

Germ line mutations in growth factor independent-1 (GFI1) have been described in a small subset of patients (pts) with severe congenital neutropenia. Subsequently, the GFI136N polymorphism, present in 3-5% of controls, has been found overrepresented (11%) in pts with primary (p) acute myeloid leukemia (AML), conveying 1.6 fold risk of development of AML. Mutant GFI136S and N variants lack affinity to HOXA9 (overrepresented in corresponding AML cases), shows increased proliferative potential in vitro and accelerates RAS-driven myeloproliferative neoplasm (MPN) disease in mice.

Whole exome next generation (WE NGS) technology facilitates comprehensive screens for the presence of both somatic and germ line genetic alteration. In this study, we used NGS to search for germline variants of the GFI1 gene. We screened 140 pts (mean age 66.8 years, range 44-85) with MDS and related disorders (MDS/MPN and secondary (s) AML) for the presence of GFI1 variants. We found non-synonymous variants in 11 cases (8%), including the previously described pathogenic p.S36N (n=8), p.P107A (n=2), or p.L400F (n=1), while the corresponding frequencies for these alterations were .04 and .001, .002 in the general population. This frequency appears comparable or higher to those previously reported for pAML, but our screen of the TCGA AML cohort, perhaps due to very low coverage for this gene, did not reveal any GFI1 polymorphisms. We next focused on the clinical features of altered GFI1 carriers. A significant proportion of GFI1 cases were younger (age<60 years) 45% (5/11) compared to 19% (p=.05) for wild type (WT) and predominantly male (82% vs. 52%, p=.02). MDS and sAML and MDS/MPN were present in 4, 3 and 4 pts, respectively. Normal cytogenetics at presentation was present in 64% vs. 43% (p=0.22). The availability of the somatic mutational profile allowed us to investigate whether specific genes are more commonly mutated in GFI1 variant cases. Among the 100 most commonly mutated genes, somatic PTPN11 (18% vs. 2%, p=.09), ASXL1 (18% vs. 8% p=0.2), SF3B1 (18% vs. 9%, p=0.3) were the most frequently encountered. Response rate to therapy with hypomethylating agents amongst carriers of GFI1 variants was 50% compared to 35% among WT (p=.32). The mean overall survival of the GFI1mutants was also higher compared to WT (40 vs. 33.6m).

In sum, our results demonstrate that potentially pathogenic GFI1 mutations are present in increased frequency in younger pts with MDS and thus may constitute a new predisposing factor for MDS and related myeloid neoplasms.

Disclosures:

Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.
In Vivo RNAi Screening Identifies HDAC4 As a Mediator Of Chemoresistance In Acute Myeloid Leukemia
Authors: Lettermann Stephanie , Agelopoulos Konstantin , Rohde Christian , Lindtner Beate , Marek Linda , Hempel Georg , Stelljes Matthias , Berdel Wolfgang E. , Kurz Thomas , Müller-Tidow Carsten
Published: October 2019

The majority of AML patients still relapse, despite intensive chemotherapy. A subset of more chemoresistant leukemia cells might be responsible for this phenomenon. These relapse-initiating cells might differ epigenetically from the more chemosensitive leukemia bulk. Since interaction of leukemic cells with the niche might contribute to chemoresistance, we decided to perform an in vivo RNAi screen for modulators of chemoresistance. We constructed a focussed shRNA library with ∼400 constructs that target 142 genes involved in stem cell functions, leukemogenesis and epigenetic regulation. Myc/Bcl2 induced murine leukemias were retrovirally transduced and injected into sublethally irradiated syngeneic mice. Mice (n=24) were either treated with Cytarabine, Azacytidine, Decitabine, or vehicle. Upon signs of leukemia, DNA was extracted and the shRNA pool was PCR-amplified from bone marrow, spleen and liver of each individual mouse. Overall, 61 libraries were sequenced with more than 500-fold coverage of each construct. Suppression of HDAC4, Pik3r1, Atr, Ccna2 and HDAC1 significantly enhanced sensitivity towards Cytarabine treatment. Histone deacetylase 4 (HDAC4) was identified as a top candidate since it enhanced the efficacy of all three drugs. It was depleted more than sixteen-fold upon Cytarabine treatment. HDAC4 suppression by the specific shRNAs was confirmed by real-time RT-PCR and western blot analysis. In a competitive re-transplantation assay in vivo, HDAC4 suppression inhibited leukemic growth. Further, we analyzed inhibition of HDAC4 by LMK235, a novel and specific inhibitor of HDAC4 and 5 in human HL60 leukemia cells. Synergistic inhibition was demonstrated for LMK235 and Cytarabine in proliferation assays and in colony formation assays. These findings demonstrate that in vivo RNAi screening for therapeutic efficacy is feasible. HDAC4 might be an important target to enhance efficacy of anti-leukemic drugs.

Disclosures:

No relevant conflicts of interest to declare.
Prevention Of Minimal Residual Disease In Ph+ ALL
Authors: Appelmann Iris , DeStanchina Elisa , Carbonetti Gregory , Chen Chong , Lowe Scott W. , Sherr Charles J.
Published: October 2019

Aggressive Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) that genetically and phenotypically mimics the human disease can be induced by the introduction of cultured p185 (BCR-ABL)-expressing Arf-null pre/pro-B cells into healthy, unconditioned syngeneic mice. Only 20 polyclonal donor cells are sufficient to induce lethal ALL within 30 days of their IV administration, indicating that BCR-ABL expression and Arf inactivation are sufficient to guarantee leukemogenesis in healthy recipient animals. Leukemic mice enter transient remission in response to treatment with potent second generation tyrosine kinase inhibitors (TKI) such as dasatinib (SprycelTM). However, like human patients with Ph+ ALL, the continuously treated animals ultimately relapse with the emergence of leukemic clones containing clinically relevant BCR-ABL mutations, the nature of which depends upon the intensity of TKI treatment. Premature withdrawal of dasatinib when animals are in remission results in re-emergence of leukemia; surprisingly, leukemic B cells recovered from these animals lack BCR-ABL mutations and remain sensitive to dasatinib ex vivo. Hence, minimal residual disease depends upon salutary signaling within the hematopoietic microenvironment. In agreement, the response to TKI therapy can be significantly improved by abrogating cytokine signaling through a knockdown of the common gamma chain of the cytokine receptor. Administration of the Janus kinase (JAK) inhibitor ruxolitinib (Jakafi™) mimics this response. Although ruxolitinib demonstrated no anti-leukemic activity of its own, the overall survival of leukemic mice inoculated with 200,000 p185+ Arf-/- pre/pro-B cells was significantly extended after administration of a targeted combination therapy of ruxolitinib and dasatinib in comparison with mice treated with dasatinib alone. Addition of dexamethasone further reduced the leukemic burden, prevented CNS relapse, and led to prolonged survival. This implicates prevention of minimal residual disease and relapse by a non-toxic combination of targeted treatments. These studies have provided a rationale for a Phase I/II clinical trial employing these agents, particularly in older patients who are ineligible for bone marrow transplantation or do not tolerate cytotoxic chemotherapy.

Disclosures:

Lowe: Blueprint Medicines: Consultancy; Constellation Pharmaceuticals: Consultancy; Mirimus Inc.: Consultancy.
Treatment With G-CSF Reduces Acute Myeloid Leukemia (AML) Blasts Viability In Presence Of Bone Marrow Stroma
Authors: Nomdedeu Meritxell , Pratcorona Marta , Díaz-Beyá Marina , Calvo Xavier , Lara-Castillo Mari Carmen , Esteban Daniel , Rozman Maria , Costa Dolors , Esteve Jordi , Risueño Ruth M.
Published: October 2019

Background

The simultaneous administration of G-CSF and chemotherapy as a priming strategy has resulted in a clinical benefit in determined subsets of patients diagnosed with acute myeloid leukemia (AML) (Löwenberg et al, NEJM 2003; Pabst T, et al, Blood 2012). However, the mechanism responsible for this anti-leukemic effect is not fully characterized. We hypothesize that the clinical benefit may occur at least partially by the effect of G-CSF on leukemic stem cells (LSC).

Objective

The main goal of this project was to determine the effect of G-CSF on primary AML samples in vitro, especially on LSCs.

Methods and patients

Peripheral blood mononuclear cells (PBMC) from 10 AML patients were treated with G-CSF at increasing doses, alone or in co-culture with HS-5 stroma cells. Cell viability (7-AAD -eBioscience- cell death exclusion and volumetric cell counting) and surface phenotype was determined by flow cytometry (FACSVerse, BD) 72 hours after treatment. Data were analyzed using the FlowJo (Trastar) software. For clonogenicity assays, AML primary samples were treated for 18 hours with G-CSF at increasing concentrations and cultured in H4034 Optimum MethoCult (StemCell Technologies) for 14 days. Colonies were counted based on cellularity and morphology criteria.

Results

G-CSF treatment showed no effect on cell viability of the bulk leukemic population or on the CD34 + immature subpopulation. A dose-dependent increase in CXCR4 surface expression was observed, reaching a 1.4-fold of change at the highest concentration of G-CSF (100 μg/mL). In contrast, treatment of leukemia cells with G-CSF in the presence of stroma cells reduced the overall cell viability. Thus, a 32% decrease of cell viability was measured at the highest concentration used (p = 0.0006), while no significant changes in the frequency of each leukemic subpopulations were observed. Clonogenic capacity was significantly reduced in a dose-dependent manner upon treatment with G-CSF, achieving a 41% reduction at the highest G-CSF concentration (100 μg/mL).

Conclusions

G-CSF reduces the viability of leukemic cells when these cells are in co-culture with the HS-5 stroma cell line, suggesting that the presence of stroma cells is required for the cytotoxical effect of G-CSF on the blast population. Interestingly, G-CSF treatment decreased the clonogenic capacity of AML samples, therefore suggesting that G-CSF exerts its effect at least partially on LSCs. Our findings support the design of studies to explore new strategies of chemotherapy priming in AML patients.

Disclosures:

No relevant conflicts of interest to declare.
Clinical Efficacy and Safety Of Dasatinib For Newly Diagnosed Chronic Myeloid Leukemia In Chronic Phase In Japan
Authors: Yamaguchi Kohei , Murai Kazunori , Ito Shigeki , Akagi Tomoaki , Ogawa Kazuei , Meguro Kuniaki , Inomata Mitsue , Watanabe Reiko , Miyagishima Takuto , Wakasa Kentaro , Shindo Motohiro , Tsukushi Yasuhiko , Kubo Kohmei , Ishida Yoji
Published: October 2019

Background

Dasatinib is a second-generation BCR-ABL inhibitor that has a 325-fold higher potency than imatinib and a 16-fold higher potency than nilotinib in vitro. The previous report from the global DASISION trial showed dasatinib resulted in significantly higher and faster rates of complete cytogenetic response (CCyR) and major molecular response (MMR) compared with imatinib. We conducted a phase II study to evaluate the efficacy and safety of dasatinib in patients with newly diagnosed chronic-phase chronic myeloid leukemia (CML-CP) in Japan.

Methods

Eighty newly diagnosed CML-CP patients were include in this study. Patients received dasatinib 100mg once daily. Treatment was continued until disease progression or unacceptable toxicity. Primary end point was the rate of major molecular response (MMR) by 12 months. MMR defined as a BCR-ABL transcript level of 0.1% or lower on the International scale by means of a real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in peripheral blood. Secondary end points were the rate of complete cytogenetic response (CCyR) by 12 months, the rate of MR4.5 (either (i) detectable disease with <0.0032% BCR-ABL1 IS or (ii) undetectable disease in cDNA with >32,000 ABL1 transcripts in the same volume of cDNA used to test for BCR-ABL1) by 12 months and adverse events of dasatinib (UMIN #000006358).

Results

Eighty newly diagnosed CML-CP patients were included in this study. All except one patient administered dasatinib 100 mg once daily. One patient was withdrawal before administration of dasatinib. So far, there were 71 patients with 6 months follow-up and 51 patients with 12 months follow-up. The estimated MMR rates were 69.5 % (95%CI, 58.7-80.3 %) by 6 months and 82.7% (95%CI, 73.0-92.4 %) by 12 months. The estimated MR4.5 rates were 27.1 % (95%CI, 16.7-37.5 %) by 6 months and 48.9% (95%CI, 36.0-61.7 %) by 12 months. Only 6 patients were withdrawal because of adverse event (5 patients) and ineffectiveness (1 patient).

Conclusion

Dasatinib treatment results in higher rates of molecular responses in newly diagnosed CML-CP patients in Japan. Dasatinib as the first-line agent might be acceptable for CML-CP patients because of better clinical efficacy and less toxicity.

Disclosures:

No relevant conflicts of interest to declare.