bluuebunny/crossref_metadata_2025 huggingface dataset:
Results of a Prospective, Open-Label Trial to Assess the Efficacy, Safety and Immunogenicity of Wilate® in Children Under 6 Years of Age with Inherited Von Willebrand Disease,
Authors: Nowak-Gottl Ulrike , Russo Alexandra , Kruempel Anne , Jansen Martina , Knaub Sigurd
Published: October 2019

Abstract 3330

Introduction:

Wilate® is a new-generation plasma-derived concentrate of von Willebrand factor (VWF) and factor VIII (FVIII) developed for treatment of patients with von Willebrand disease (VWD) and haemophilia A. The objective of this study was to assess efficacy, safety and immunogenicity of Wilate in children below 6 years of age with inherited VWD.

Methods:

The prospective, multi-centre trial included patients with inherited VWD of any type below 6 years of age with insufficient response to DDAVP. Any clinical event requiring VWF/FVIII concentrate treatment within 1 year was treated with Wilate. Efficacy was recorded by using a 4-point VRS and in vivo recovery of FVIII:C and VWF:RCo was analyzed. Immunogenicity was assessed by determination of inhibitors against VWF and FVIII. Safety was measured by monitoring of adverse events. Tolerability was rated as “very good” or “good” for all cases by the investigator.

Results:

Fifteen subjects (6 type 3) were analyzed. Dosing regimens were dependent on specific clinical situations of each subject. The mean incremental in vivo recovery was 1.2 IU/dL per IU/kg for VWF:RCo and 1.6 IU/dL per IU/kg for FVIII:C (n = 7). Forty five bleeding episodes required treatment with Wilate. In all cases the overall haemostatic efficacy ratings by the investigator were “excellent” or “good”. The mean dose was 35.9 IU VWF:RCo/kg. Nine surgical procedures (3 major) were performed in 7 subjects, requiring 2 to 12 infusions/procedure with a mean dose per exposure day of 61.7 VWF:RCo/kg. Haemostatic efficacy for a total of 419 infusions was rated as “excellent” for 362, “good” for 54, “moderate” for 2 (1 missing) infusions. No notable changes in thrombogenicity markers and no thromboembolic events occurred. No inhibitors against VWF or FVIII were detected.

Conclusion:

This study demonstrates the efficacy, safety and tolerability of Wilate® in treatment of VWD in patients below 6 years of age.

Disclosures:

Jansen: Octapharma: Employment. Knaub:Octapharma AG: Employment. Off Label Use: the product, Octanate, is approved in many countries worldwide but is not currently approved in the US.
The Ratio of ADAMTS13 to VWF-Propeptide Can Reflect the Disease Severity and the Extent of Inflammation of the Patients with Severe Sepsis or Septic Shock,
Authors: Nishio Kenji , Fukushima Hidetada , Watanabe Tomoro , Seki Tadahiko , Matsui Hideto , Sugimoto Mitsuhiko , Matsumoto Masanori , Fujimura Yoshihiro , Okuchi Kazuo
Published: October 2019

Abstract 3349

Background:

Sepsis is a critical consequence of self defense to infection, which includes inflammatory and hypercoagulant response. Many stimulatory mediators involved in these responses cause the Weibel-Palade body exocytosis through endothelial activation, leading to surface expression of P-selectin and ultra-large von Willebrand factor (ULVWF), and simultaneously release of VWF-propeptide on a equimolar basis to ULVWF. Where ADAMTS13 is functionally inactivated, ULVWF would not be proteolysed and cause platelet thrombosis in the microcirculation, leading to organ failure. Therefore, the balance of the ULVWF and ADAMTS13 may indicate the prothrombotic state of the patients. However, it is difficult to estimate the amount of ULVWF expressed. It is reasonable to think that plasma VWF propeptide levels will reflect more precisely the extent of expressed ULVWF and P-selectin rather than VWF level. These allow us to speculate that the balance of VWF propeptide and ADAMTS13 can be associated with magnitude of prothrombotic and inflammatory response.

Aim:

To investigate whether the levels of molecules mentioned above are associated with the disease severity of sepsis or inflammation during the clinical course.

Method:

Inclusion criteria are as follows, 1) severe sepsis or septic shock defined by the Society of Critical Care Medicine Consensus Conference Committee 2) Platelets count less than 120,000/μL. SOFA(sequential organ failure assessment)score, APACHEII score, Creatinine, VWF propeptide, VWF, P-selectin, ADAMTS13, TNF-alfa, IL-6 were measured at day 0, 3, 5, 7 (day 0 is the day when the diagnosis as severe sepsis or septic shock was made), and each other's correlation was studied.

Result and Discussion:

VWF propeptide level was increased to 293.8+/−153.8% at day 0, and gradually decreased day by day. VWF was increased to 212.3+/− and slightly increased during the course. P-selectin was not increased during the course. The plasma level of ADAMTS13 at day 0 was decreased to 25+/− 8.7%, and increased during the course in the survivors, however almost not changed in the non-survivors. As we speculated, the value of VWF propeptide/ ADAMTS13 were well correlated to SOFA score, Creatinine, APACHEII, TNF-alfa almost on each study day.

Conclusion:

This result shows that the ratio of VWF propeptide to ADAMTS13 can be used to estimate disease severity of sepsis and magnitude of inflammation. This will improve our understanding of underlying pathophysiology of sepsis.

Disclosures:

Matsumoto: Alexion Pharma: Membership on an entity's Board of Directors or advisory committees. Fujimura:Baxter BioScience: Membership on an entity's Board of Directors or advisory committees; Alexion Pharma: Membership on an entity's Board of Directors or advisory committees.
Inhibition of Platelet Glycoprotein β3 by Chimeric Monoclonal Antibody Prevents Arterial Thrombosis in Beagle Dogs,
Authors: Ji Shundong , Jiang Miao , Dong Ningzheng , Bai Xia , Ruan Changgeng
Published: October 2019

Abstract 3357

In this present study in beagle dogs, we evaluated the antithrombotic efficacy of Pulaimab, the chimeric monoclonal antibodies SZ21-F(ab)2 fragments against platelet glycoprotein (GP) β3, in a modified Folts model. The Folts model is widely accepted to be effective and clinically relevant for testing potential anti-thrombotic agents, in this model the cyclic flow reductions (CFRs) are caused by platelet dependent thrombi that form under high-shear conditions at injured stenosed sites of an artery. Thirty beagle dogs of either sex, weighing 7.5 to 14 kg, were randomly divided into five groups of six (three females and three males), the details were following: negative control group (injected normal saline), positive control group (injected 0.2mg/Kg of Reopro), and three experimental groups [injected 0.2, 0.4, 0.8 mg/Kg of SZ21-F(ab)2, respectively]. A dose range from 0.2 to 0.8 mg/kg of Pulaimab significantly reduced the CFRs by 21–73%, without reduction of platelet numbers and prolongation of the bleeding time. Ex vivo ADP-induced platelet aggregation was equally reduced. The present study demonstrates that the inhibition of platelet GP αIIbβ3function by SZ21-F(ab)2 is a powerful intervention to prevent platelet thrombus formation in injured arteries without thrombocytopenia and prolongation of the bleeding time. We therefore conclude that F(ab)2 fragments of inhibitory anti-GPβ3 antibodies may be useful compounds to prevent thrombosis.

Disclosures:

No relevant conflicts of interest to declare.
Storage-Induced Changes Promote Erythrocyte Recognition by Autoantibodies,
Authors: Dinkla Sip , Novotný Vera M.J. , Joosten Irma , Bosman Giel J.C.G.M.
Published: October 2019

Abstract 3371

Physiological erythrocyte removal is associated with a selective increase in the expression of senescent cell antigens related to band 3, and subsequent autologous antibody binding and phagocytosis. Chronic erythrocyte transfusion often leads to immunization and the formation of alloantibodies and autoantibodies. In order to understand the molecular basis for this phenomenon we investigated whether erythrocyte storage leads to increased expression of non-physiological neoantigens.

Immunoprecipitations were performed with erythrocytes and vesicles from blood bank erythrocyte concentrates of increasing storage periods, using plasma containing erythrocyte autoantibodies from selected patients with autoimmune hemolytic anemia (N=6). Patient plasma autoantibody binding to erythrocytes increased with erythrocyte storage time, while the exact opposite was observed for plasma from healthy volunteers, showing that erythrocyte antigenicity changes during storage. SDS polyacrylamide gel analysis combined with proteomics revealed band 3 to be a dominant target of the autoantibodies. From vesicles that are formed during erythrocyte storage, the autoantibodies precipitated protein complexes distinct from those recognized on erythrocytes, indicating that they have a different immunization potential. Soluble immune mediators including complement factors were present in the patient plasma immunoprecipitates, but not in the control immunoprecipitates.

These findings support the hypothesis that disturbed erythrocyte aging during storage of erythrocyte units contributes to transfusion-induced alloantibody and autoantibody formation.

Disclosures:

No relevant conflicts of interest to declare.
The MEIS1 Interactome in Megakaryocytes Reveals a Role in Cell Cycle Regulation,
Authors: Salunkhe Vishal A , Macaulay Iain , Nuernberg Sylvia , McCarthy Cathal , Ouwehand Willem Hendrik , Fitzgerald Desmond J. , Maguire Patricia
Published: October 2019

Abstract 3380

Haematopoiesis is highly coordinated process of fate determination at branch points that is regulated by transcription factors and their cofactors. Our comprehensive catalogue of transcripts in the eight main mature blood cell elements, including erythroblasts and megakaryocytes (MKs) showed that the transcription factor MEIS1 is uniquely transcribed in MKs and the CD34+ haematopoietic stem cell. Gene silencing studies in mice and zebrafish has shown a pivotal role for MEIS1 in haematopoiesis, megakaryopoiesis and vasculogenesis, although its precise hierarchical position and function remain unknown. To gain further insight in the role of MEIS1 in megakaryopoiesis, we used a proteomics approach to search for its nuclear interaction partners.

Co-immunoprecipitation was used to isolate MEIS1 interacting proteins from the nuclear fraction of the MK cell line, CHRF 288–11 and resulting eluates were subjected to proteomics analysis using one-dimensional electrophoresis and liquid chromatography (LC) coupled to tandem mass spectrometry (MS) or GeLC-MS/MS. In total 70 proteins were identified to co-immunoprecipitate with MEIS1 from 3 replicate MS analyses. These included the previously validated MEIS1 interactors PBX1 and HOXB9, as well as numerous novel interactors such as ARID3B and DHX9.

Network analysis of our MEIS1 interactome dataset revealed a strong association with cell cycle regulation. In fact, we had identified a myriad of cell cycle regulators including CDK1, CDK2, CDK9, CUL3, PCNA, CDC5L, ARID3B and MDC1. These interactions are consistent with recent microarray studies in promyelocytic leukemic cell lines that link MEIS1 with cell cycle entry and its regulation of genes such as CDK2, CDK6, CDKN3, CDC7 and Cyclin D3 among others. To confirm the novel interaction of MK MEIS1 with cell cycle regulators we performed reverse immuno-precipitation/immunoblot analysis in CHRF cells and purified MEIS1 containing multiprotein complexes from L8057 murine megakaryoblastic cells. Using a cell cycle specific PCR array, we demonstrate that MEIS1 overexpression in L8057 cells regulates numerous cell cycle regulatory genes. Preliminary analysis using flow cytometry demonstrated that MEIS1 overexpression resulted in an altered cell cycle progression. Furthermore, genome wide ChIP-Seq analysis in CHRF cells for MEIS1 revealed binding sites in Cyclin D3 and CDK6, two known key regulators of the cell cycle and megakaryopoiesis.

Taken together this study provides evidence linking MEIS1 to the cell cycle control of MKs and will help elucidate the role of MEIS1 in cell cycle progression, megakaryopoiesis and associated disorders.

Disclosures:

No relevant conflicts of interest to declare.
PIM Kinases Inhibition, a Rational Strategy in Peripheral T-Cell Lymphomas,
Authors: Martin-Sanchez Esperanza , Rodriguez-Pinilla Socorro M. , Sanchez-Beato Margarita , Dominguez-Gonzalez Beatriz , Wozniak Magdalena B. , Mollejo Manuela , Alves Javier , Rodriguez-Peralto Jose Luis , Menarguez Javier , Cigudosa Juan C. , Ortiz-Romero Pablo L. , Garcia Juan F. , Bischoff James R. , Piris Miguel A.
Published: October 2019

Abstract 3494

The search of an efficient therapy for Peripheral T-cell lymphomas (PTCL) patients is still a challenge, in part due to the very little knowledge about the PTCL pathogenesis, and the absence of appropriate models. This heterogeneous group of very aggressive malignancies can not be cured with conventional therapies; therefore, new therapeutic strategies are needed to improve the poor outcome in these patients. PIM family is composed of 3 kinases (PIM1, PIM2 and PIM3) which play an essential role in cell proliferation and survival. They are mainly activated through JAK/STAT signaling pathway, and are frequently altered in human malignancies by amplification, mutation and overexpression. The aim of this study is to determine the efficiency and the mechanism of action of PIM inhibition in PTCL.

Gene expression profiling of twenty two PTCL cases and seven reactive lymph nodes was performed. We observed a strong overexpression of the three PIM family genes in PTCL cases, especially PIM2. In addition, Gene Set Enrichment Analysis identified an overexpression of STAT3 and IL-2 pathways in PTCL cases, probably responsible for the strong expression of PIMs we found. Furthermore, PIM genes expression was confirmed by quantitative RT-PCR in 6 PTCL-derived cell lines compared to normal T cells from healthy donors, highlighting again the relevance of PIM2.

Genetic inhibition was carried out using small interference RNA to specifically abolish the expression of each PIM1, PIM2 and PIM3 in a panel of 6 PTCL cell lines belonging to different PTCL subgroups. Additionally, pharmacological inhibition with one PIM inhibitor (ETP-39010) was performed.

Surprisingly, genetic inhibition of each of the PIM gene alone did not show any cellular effect, neither cell cycle arrest nor apoptosis. But interestingly, we found that specific inhibition of each of the PIM genes caused an increased expression of the other PIM family members, probably leading to a compensatory mechanism among these kinases balancing the lack of one of them, avoiding pro-apoptotic effects and allowing cell survival.

Accordingly, a simultaneous inhibition of PIM1, PIM2 and PIM3 using the pharmacological pan-PIM inhibitor produced a decrease in cell viability and a strong induction of apoptosis in all cell lines, without cell cycle arrest. Several PIM inhibitor biomarkers have been identified at the mRNA level, involving the DNA damage response signaling.

In conclusion, our results indicate that PIM kinases inhibition could be an effective therapeutic approach for PTCL.

Disclosures:

No relevant conflicts of interest to declare.
Gas6 Receptors, Tyro3, Axl and Mer, Differentially Participate in Glycoprotein VI-Mediated Platelet activation
Authors: Zhou Junsong , Wang Lu , Wu Yi
Published: October 2019

Abstract 364

Growth arrest-specific gene 6 (Gas6) has three receptors on platelets - Tyro3, Axl and Mer. It has been shown that these three receptors are equally important in platelet activation (J Clin Invest. 2005;115:237–46), and they synergizes with ADP receptor for platelet activation, but not with collagen receptor (J Thromb Haemost. 2010;8:1797–808). Considering the fact that Tyro3, Axl or Mer plays a distinct role in the function of endothelial cells and leukocytes, we hypothesized that these three receptors play specific role in platelet activation, and examined the role of each receptor in Gycoprotein VI (GPVI)-mediated platelet activation using single receptor knockout mice. In response to 100 ng/ml convulxin or 5 ng/ml poly(PHG), a GPVI-specific synthetic peptide, the aggregation of Axl−/− or Tyro3−/− platelets was almost completely prevented. However, the aggregation of Mer−/− platelets was only delayed, and still could reach the maximum level. In the presence of 100 ng/ml convulxin or 5 ng/ml poly(PHG), the spreading of Axl−/− or Tyro3−/− platelets, but not Mer−/− platelets, on fibrinogen-coated surfaces was markedly inhibited. Moreover, convulxin-stimulated P-selectin expression and JON/A binding were both significantly decreased in Axl−/− and Tyro3−/− platelets, but not in Mer−/− platelets. Upon stimulation with 100 ng/ml convulxin, tyrosine phosphorylation of Syk, linker for activation of T cells (LAT), SLP-76(SH2 domain-containing leukocyte protein of 76) and phospholipase C (PLCγ2) was markedly reduced in the Axl−/− or Tyro3−/− platelets, but not in the Mer−/− platelets. Interestingly, anti-Axl or anti-Tyro3 antibody (20 mg/ml) completely blocked convulxin-stimulated human platelet aggregation, however, the neutralizing anti-Gas6 antibody (60 mg/ml) did not have any effect, suggesting the homodimer formation of Axl or Tyro3 is involved in GPVI-mediated platelet activation. As detected by flow cytometry, there was no difference in the expression of GPVI and integrin aIIbb3 on membrane surface among Tyro-3−/−, Axl−/−, Mer−/− and wild type platelets. In conclusion, Gas6 receptors are not equally important in platelet activation, Axl and Tyro3 play more critical role than Mer in GPVI-mediated platelet activation. The trans-interaction of Axl or Tyro3 mediates cell-cell contact-dependent activation, which facilitates the activation of GPVI in platelets. Thus, Axl and Tyro3 may serve as better targets than Mer for the development of new antiplatelet agents.

Disclosures:

No relevant conflicts of interest to declare.
Yttrium-90-Ibritumomab Tiuxetan and BuCyE High-Dose Chemotherapy Compared with BuCyE Alone As Conditioning Regimen in Patients with B-Cell Non-Hodgkin Lymphoma: IPI Matched Case Control Study At a Single Center,
Authors: Jo Jae-Cheol , Yoon Dok Hyun , Kim Shin , Jang Seongsoo , Park Chan-Jeoung , Chi Hyun Sook , Park Chan-Sik , Huh Jooryung , Lee Sang-wook , Suh Cheolwon
Published: October 2019

Abstract 3703

Background:

Currently, there are accumulating data on the role of radioimmunotherapy in autologous stem cell transplantation (ASCT) with promising results. We compared the efficacy and safety of yttrium-90-ibritumomab tiuxetan (90Y-ibritumomab) combined with intravenous busulfan, cyclophosphamide, and etoposide (BuCyE) with those of BuCyE alone followed by ASCT in patients with relapsed or refractory B-cell non-Hodgkin lymphoma (NHL).

Patients and Methods: From October 2005 to May 2011, 71 NHL patients underwent high-dose chemotherapy with BuCyE (n=52) or 90Y-ibritumomab plus BuCyE (n=19) followed by ASCT at the Asan Medical Center. Of 52 patients receiving BuCyE, we found 19 patients who had B-cell NHL and they comprised control group of BuCyE alone (n=19). Retrospective comparison matched by the same IPI was performed for the efficacy and safety between two groups.

Results:

The patient cohort consisted of 38 individuals (19 males), of median age 52 years (range, 27–65 years). The baseline characteristics were well balanced between the two groups. The median time to platelet engraftment (>20,000/mm3) and the median time to neutrophil engraftment (>500/mm3) did not differ between 90Y-ibritumomab plus BuCyE (12 days [range, 3–103 days] and 10 days [range, 8–12 days], respectively) and BuCyE alone (12 days [range, 8–35 days] and 10 days [range, 9–12 days], respectively). The objective overall response rate was 89.5% (17/19): continued CR, 42.1% (8/19); induced CR, 36.8% (7/19); PR, 10.5% (2/19) in 90Y-ibritumomab plus BuCyE group, and 78.9% (15/19): continued CR, 26.3% (5/19); induced CR, 47.4% (9/19); PR, 5.3% (1/19) in BuCyE group, respectively (p =0.649). Median follow-up duration for survivors was 30 months. Event-free survival tended to be better in 90Y-ibritumomab plus BuCyE group with a median event-free survival duration of 12.5 months in 90Y-ibritumomab plus BuCyE group and 6.2 months in BuCyE group (p =0.236) (figure 1-a).There seemed to be no significant difference in overall survival between the two groups (p =0.767, figure 1-b). The toxicities including the incidence of grade 3 or higher stomatitis, nausea, vomiting, diarrhea, hyperbilirubinemia were similar in the two groups.

Conclusion:

Addition of 90Y-ibritumomab to BuCyE high-dose chemotherapy may potentially benefit patients with relapsed or refractory B-cell NHL at no cost of additional toxicity, which warrants further investigation.

Disclosures:

No relevant conflicts of interest to declare.
Angiogenesis and Tumor Growth Are Increased in Kininogen Deficient Mice
Authors: Zhang Wan-Ming , Hsi Linda , McCrae Keith R.
Published: October 2019

Abstract 371

High molecular weight kininogen (HK) is an abundant plasma protein and member of the contact phase of coagulation that is involved in a number of important biological processes. We have previously reported that kallikrein-cleaved HK (HKa) induces rapid apoptosis of proliferating endothelial cells and inhibits angiogenesis. We have recently produced a kininogen deficient mouse by deleting the murine gene for kininogen that is expressed in liver, mKng1. These mice lack detectable kininogen, despite persistent expression of mRNA from the mKng2 gene, primarily expressed in kidney. To further determine the importance of kininogen in the regulation of angiogenesis, we assessed angiogenesis in mKng1−/− mice. These mice demonstrated increased angiogenesis in subcutaneously-implanted Matrigel plugs containing vascular endothelial cell growth factor (VEGF) or basic fibroblast growth factor (bFGF), compared to wild-type littermates. We also assessed the growth of B16F10 melanoma cells and Lewis lung carcinomas planted subcutaneously in mKng1−/− mice. In both cases, in particular with the B16F10 implants, tumor growth occurred substantially faster in the setting of kininogen deficiency (mean tumor volume of mKng1−/− mice 4820 ± 728 mm3 vs 1990 ± 412 mm3 in wild type mice; P = 0.003). Differences in growth rates were apparent approximately 8 days after tumor implantation, and were associated with increases in tumor microvascular density. Several reports suggest that the urokinase receptor (uPAR), which binds HKa, mediates its antiangiogenic activity. However, using Matrigel plug assays we observed that HKa inhibited angiogenesis equally well in wild type and uPAR−/− mice. These studies demonstrate that kininogen serves as an endogenous regulator of angiogenesis. Current studies are focused on better defining the mechanisms of this activity.

Disclosures:

No relevant conflicts of interest to declare.
Phospho-SFKs (Y416) As a Potential Predictive Marker for Dasatinib Therapy in B-Cell Non-Hodgkin Lymphoma and Chronic Lymphocytic Leukemia,
Authors: Zhao Xiaoxian , Hsi Eric D.
Published: October 2019

Abstract 3727

The Src family of protein tyrosine kinases (SFKs) plays an important role in regulating multiple signaling networks including B-cell receptors (BCR) mediated pathways and abnormal SFK kinase activation promotes B lymphoma cell survival. Dasatinib is an oral BCR/ABL1 and SKF inhibitor useful in the treatment of imatinib-resistant CML and Ph+ALL. Given its broad inhibitory activity, dasatinib may be useful in the treatment of other hematologic malignancies and having a biologic predictor of response would be helpful in rational selection of this targeted therapeutic. We hypothesized this agent could have therapeutic potential against lymphoma patients with p-SFK (Y416) expression.

Constitutive p-SFK (Y416) expression (indicating active SFK signaling) was detected in both B-lymphoma cell lines and a subset of primary lymphoma tissues including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Burkitt lymphoma and small lymphocytic leukemia (SLL). Dasatinib induced apoptosis of B-lymphoma Raji cells correlated with high level expression of constitutive p-SFK (Y416) and dasatinib rapidly reduced the global level of tyrosine phosphorylations including p-SFK (Y416) in Raji cells. 19 of 28 lymphoma cases (67.9%) were positive for p-SFK (Y416) by Western blot analysis. Dasatinib displayed in vitro dose-dependent (10–200 nM) killing activity against 17 of those 19 p-SFK (Y416) cases (89.5%). In contrast, only 2 of 9 p-SFK (Y416) negative cases (22.2%) had response to dasatinib exposure. Thus presence of p-SFK (Y416) was associated with in vitro response to dasatinib (p <0.0001). Similar to tested Raji cells, dasatinib induced apoptosis of primary B-cell lymphoma cells was accompanied with de-phosphorylation of p-SFK (Y416) and cleavage of caspase-3. 6 of 9 tested CLL cases were p-SFK (Y416) positive. Dasatinib displayed in vitro killing activities against 5 of 6 positive cases with a range of killing from 12% to 53% (mean 26.5%) of malignant B-cells. Meanwhile, one of three negative cases showed response to dasatinib (17% killing).

We conclude that p-SFK (Y416) may be a useful predictive marker of response to dasatinib. Potential uses include pharmacodynamic monitoring or integral biomarker for selecting appropriate patients with B-cell malignancies for clinical trials.

Disclosures:

No relevant conflicts of interest to declare.
Socs2 Is Dispensable for BCR/ABL1-Induced Chronic Myeloid Leukemia-Like Disease in Mice and for Normal Hematopoietic Stem Cell Function,
Authors: Hansen Nils , Agerstam Helena , Wahlestedt Martin , Ehinger Mats , Johnels Petra , Ishiko Jun , Melo Junia V. , Alexander Warren S , Bryder David , Järås Marcus , Fioretos Thoas
Published: October 2019

Abstract 3743

Background:

The P210 BCR/ABL1 fusion protein initiates signaling through several down stream pathways such as STAT5, PI3K, AKT, RAS, and WNT. However, only few down stream mediators have so far been thoroughly studied in vivo in the context of BCR/ABL1-mediated induction of CML-like disorder in mice deficient for the specific genes. Suppressor of cytokine signaling 2 (SOCS2) is known as a feedback inhibitor of cytokine signaling, a negative regulator of the STAT5 pathway, and is markedly upregulated in primary bone marrow cells from patients with chronic myeloid leukemia (CML). However, it has not been clear whether SOCS2 is involved in BCR/ABL1 induced cell transformation or whether it is important for normal hematopoietic stem cell (HSC) function.

Methods:

To evaluate the stem cell function of Socs2, HSCs from wild type and Socs2 deficient mice were competitively transplanted into lethally irradiated recipients. Blood chimerism and lineage distribution were analyzed by flow cytometry at 4 and 18 weeks after transplantation. To investigate the potential role of Socs2 in CML, c-kit enriched Socs2(−/−) and wild type cells were transduced with a BCR/ABL1 expressing retroviral vector and transplanted into lethally irradiated recipients. The in vivo development of malignant disease was analyzed by peripheral blood cell counts and, upon euthanization, by flow cytometry and histopathology. Socs family gene expression was assessed by Q-RT/PCR and Stat5 phosphorylation by Western blotting on BCR-ABL1 expressing cells.

Results:

Although Socs2 was previously found to be upregulated in long-term repopulating HSCs by gene expression arrays, Socs2 deficient HSCs were indistinguishable from wild type HSCs when challenged in competitive bone marrow transplantation. Furthermore, when expressing BCR/ABL1, both Socs2 deficient and wild type cells induced a CML-like disease with an average survival of three weeks after transplantation. The leukemic mice suffered from elevated white blood cell counts and splenomegaly. To investigate if other Socs family members were upregulated to compensate for the Socs2 deficiency, we compared the expression of the Socs gene family members in Socs2(−/−) with wild type, leukemic cells. The expression levels of all other Socs genes were similar between Socs2(−/−) and Socs2 wt cells, suggesting that compensatory mechanisms of other Socs genes do not account for the lack of Socs2 function. In addition, the finding that the phosphorylation of Stat5 was unaffected in Socs2(−/−) cells indicates that BCR/ABL1-induced Stat5-phosphorylation is insensitive to Socs2 levels.

Conclusions:

Collectively, our results clarify that Socs2 is dispensable for normal HSC function and for BCR/ABL1-induced CML-like disease.

Disclosures:

No relevant conflicts of interest to declare.
B Cell Receptor Stimulation of CLL Cells Leads to Upregulation of IRF4 Proteinexpression Influenced by SNP Expression,
Authors: Patz Michaela , Linde Nina-Felizitas , Frenzel Lukas P. , Pallasch Christian P. , Brinker Reinhild , Claasen Julia , Hallek Michael , Wendtner Clemens
Published: October 2019

Abstract 3886

Introduction:

As previously published by Di Bernado et al. (2008) the single nucleotide polymorphism (SNP) RS872071 located in the 3'UTR of IRF4 (Interleukin Regulatory Factor 4) has influence on the risk for developing chronic lymphocytic leukemia (CLL). IRF4 is a key player in the development of B lymphocytes and multiple myelomas. The SNP is either expressed as Adenine (A) or Guanine (G). Expression of G/G increases the risk to develop CLL. MicroRNAs (miRNAs) adhere posttranscriptional to the 3'untranslated region (UTR) of mRNAs and can influence the expression of mRNA and proteins. The SNP lies within the 3'UTR of IRF4 and offers some putative binding sites for miRNAs. The aim of this project is to proof that this SNP has influence on the binding behavior of miRNAs, which are influencing the IRF4 expression.

Methods and results: To identify miRNAs binding to this region we cloned the neighboring region of the SNP into a luciferase expressing vector and generated the SNP by mutagenesis. In luciferase assays 15 miRNAs were checked for differences in binding affinity dependent on SNP expression. Three of them showed significant SNP dependent binding behavior. In all cases expression of SNP G leads to reduced luciferase expression, indicating suppression of IRF4. To elucidate the consequences on SNP expression on cellular level in CLL we collected DNA derived from B cells of CLL patients (n=104) and sequenced the SNP region. All together our pool of patients expressed 25% A/A, 31% A/G and 44% G/G. MRNA and protein expression of IRF4 was considered SNP-dependently and compared to healthy B cells. On mRNA and protein level CLL cells have a significant higher IRF4 expression compared to healthy B cells. SNP-dependent comparison between CLL cells on mRNA-level shows a tendency of less IRF4 expression in B cells of patients expressing the G/G SNP compared to patients expressing A/A SNP. However on protein level this tendency was not detected. As IRF4 is known as a key regulator for extracellular stimuli, we focused on SNP dependent IRF4 regulation after different stimuli. Whereas CD40 and IL-4 stimulation did not show SNP dependent regulation, stimulation of the B cell receptor (BCR) leads to a higher IRF4 induction in patients carrying A/A (n=6) compared to patients carrying G/G (n=5) and A/G (n=8) (p<0.05).

Conclusion:

For the first time connections between the SNP RS872071 and molecular mechanisms explaining his influence on the pathogenesis on CLL were drawn. Dependent on expressed SNP miRNAs bind with different affinities to the 3'UTR. In further experiments the connection between IgM stimulation and miRNA expression has to be tightened.

Disclosures:

No relevant conflicts of interest to declare.
Characterization of Clonotypic B Cell Progenitors in Bone Marrow of Multiple Myeloma Patients,
Authors: Boucher Kelly , Parquet Nancy , Shain Kenneth H. , Baz Rachid , Alsina Melissa , Koomen John , Durand Ashley , Anasetti Claudio , Dalton William , Perez Lia
Published: October 2019

Abstract 4002

The lack of specific molecules to define malignant B progenitor cells in multiple myeloma (MM) has hampered the evaluation of minimal residual disease (MRD). We have identified a bone marrow (BM) CD138- subset that co-express CD19+ with identical κ or λ light chain (LC) restriction as the abnormal plasma cell (PC), as previously shown by others. The majority of LC restricted (LCR) B progenitors are CD19+/CD34- and <0.5% of whole BM (WBM) cells exhibit an immature phenotype: CD19+/CD34+ with aberrant CD27 expression. Immature B cell precursors are undetectable in peripheral blood (PB). LCR CD138-/CD19+ cells represent only 0.72± 0.5% of WBM in newly diagnosed patients (n=23) and do not increase (0.47± 0.51%) in patients with relapsed disease (n=21). The κ/λ LC ratio is 1.46±0.6 regardless of disease stage suggesting that conventional LC ratios for PCs (> 4 or <0.5) may not apply in B progenitors. LCR B progenitors (CD19+/34+ or CD19+/34-) are CD117+, Notch+ and Survivin+ as MM patient's hematopoietic stem cells (HSC). ALDH enzymatic activity is 3.1% (0.1-–7.26%) in LCR B cells. Flow sorted CD138+ did not grow in a colony formation assay (methylcellulose with PHA-LCM), whereas CD19+/CD34- or CD19+/CD34+ grew colonies with efficiency of 1:25,000 or 1:10000 respectively. Cells harvested from colonies have a lympho-plasmacytoid appearance and LCR B progenitors differentiated into CD138+ PC (80±5%) compared to HSC (10±5%). Colony efficiency was optimized (3 fold) using conditioned medium (CM) from HS5-stroma. Isolated CD138-/CD19+ cells were relatively bortezomib and melphalan resistant compared to CD138+ PC. We hypothesize that CD138-/CD19+/CD34+ cells contains earlier progenitor B cells that differentiate into the malignant PC. Surrogate assays for stem cell activity and xenotransplant models should determine cancer stem cell activity of immature B cell precursors. Research studies of MM putative progenitor cells will allow developing novel treatments to eradicate potential MM MRD reservoir.

Disclosures:

Baz: Millenium: Research Funding, Speakers Bureau; Celgene: Research Funding.
The Effect of KIRs Expression Profile in Donor/Recipient Pairs in HLA-Identical Sibling Hematopoietic Stem Cell Transplantation,
Authors: Wei Xiaoai , Tang XiaoWen , Feng Yufeng , Zhu Ziling , He Jun , Xu Yang , Sun Aining , Depei Wu
Published: October 2019

Abstract 4017

Objective:

We investigated the distribution characteristics of KIRs expression profile in donor/recipient pairs with acute leukemia (AL) receiving HLA-identical sibling hematopoietic stem cell transplantation (sib-HSCT). We further explored the effect of KIRs expression profile in donor/recipient pairs on clinical outcome, including dynamics of donor T cell and NK cell engraftment.

Methods:

The genotypes of donor/recipient KIRs were determinated by polymerase chain reaction- sequence specific primer (PCR-SSP) for 80 pairs of donor/recipient receiving HLA-identical sibling hematopoietic stem cell transplantation. The multiple short tandem repeat (STR) PCR was used to evaluate the status of engraftment of donor T cells and NK cells at +14 days, 21 days, 28 days, 60 days and 90 days after transplantation in 24 cases.

Results: 1.

In 80 pairs of donor/recipient: (i) the KIRs were completely identical in 57.5% of donor/recipient pairs; (ii) the donors' KIRs contained the recipients' in 13.75% pairs; (iii) the recipients' KIRs contained the donors' in 17.5% of pairs; (iv) the KIRs were completely different in 11.25% pairs. The graft versus host (GVH) direction KIR-matched group was 75%. The percentage of group donor B/X and group donor A/A was 50%, respectively. 2. Comparing the patients from GVH direction KIR-matched and mismatched group, the incidence of acute (a) GVHD was 60% and 30%, respectively (P =0.0222), and 2-year OS was 62.96% and 94.12%, respectively (P =0.0492). Particularly, grade III-IV aGVHD rate of KIR-matched group was higher than that of non-KIR matched group(15% vs 0%). 3. Donor B/X group had a higher 2-year OS and 2-year relapse-free survival (RFS) compared with donor A/A group (89.23% vs 49.57%, P =0.0159, and 90% vs 59.71%, P =0.0239, respectively). Patients with three or less aKIRs had a lower 2-year OS (58.9% vs 92.44%, P =0.0338) and a lower RFS (65.14% vs 92.59%, P =0.0398), compared with patients with more aKIR. 4. Sequential monitoring of chimerism status of donor NK-cells in 24 cases revealed that on day+14, the percentage of full donor NK cells chimerism was higher in non-KIR matched patients than that of KIR matched patients (85.7% vs 52.9%, P =0.0456).

Conclusions:

Donor KIR genotype appears to have a direct impact on aGVHD, OS and RFS. Therefore, donor KIR genotype should be evaluated as an outcome predictor of the HLA-identical sib-HSCT.

Disclosures:

No relevant conflicts of interest to declare.
Prevention of Graft-Versus-Host Disease by HY-Specific Induced Tregs Through Activation-Dependent Manner,
Authors: Li Jun , Semple Kenrick , Koasaard Kane , Haarberg Kelley M.K. , Chen Fang-ping , Anasetti Claudio , Yu Xue-Zhong
Published: October 2019

Abstract 4023

Naturally occurring regulatory T cells (nTregs) may prevent graft-versus-host disease (GVHD) while preserving graft-versus-leukemia (GVL) activity. However, clinical application of nTregs has been severely hampered by their scarce availability and non-selective suppression. To overcome these limitations, we took the alternative approach to induce antigen-specific Tregs (iTregs) and tested their efficacy and selectivity in the prevention of GVHD in a pre-clinical model of bone marrow transplantation (BMT). We selected HY as target antigen, because it is a naturally processed and ubiquitously expressed minor histocompatibility antigen (miHAg) with a proven role in GVHD and GVL effect. To generate HY-specific iTregs, naïve CD4+CD25− cells were isolated from MHC II-restricted, HY-specific transgenic and Foxp3/GFP knock-in mice, and were stimulated with HY peptide and APC, in the presence of TGFb and retinoic acid. Marrow plus polyclonal CD4 T effector cells (Teffs) from B6 donor splenocytes induced GVHD when transplanted into lethally irradiated (B6 x bm12)F1 recipients, and adoptive transfer of HY-specific iTregs (CD4+CD25+GFP+) significantly decreased GVHD mortality in male (HY+) but not female (HY−) recipients. On a per cell basis, HY-specific iTregs were significantly more potent than polyclonal Tregs in the prevention of GVHD. These data indicate that HY-specific iTregs were effective in controlling GVHD in an activation-dependent manner. Because polyclonal Teffs cells recognize bm12 alloantigen whereas iTregs recognize HY miHAg, these data suggest that Tregs may control GVHD through a linked-suppression on Teffs in vivo. Mechanistically, by measuring iTregs and Teffs in spleen and liver of the recipients, we found that HY-specific iTregs expanded extensively and significantly suppressed expansion and infiltration of Teffs in male but not female recipients. We finally generated alloreactive iTregs from polyclonal CD4 precursors, and found that these iTregs highly suppressed the Teffs alloresponse in vitro and in vivo. These results show that Ag-specific iTregs are promising cell therapy for effective GVHD prevention in human allogeneic hematopoietic cell transplantation.

Disclosures:

No relevant conflicts of interest to declare.
IL-15 Administration Increases Graft Versus Leukemia Activity in Recipients of Haploidentical Hematopoietic Stem Cell Transplantation,
Authors: Sauter Christopher , Biswas Chandra , Bailey Cavan , Panis Michelle , Budak-Alpdogan Tulin , Alpdogan Onder
Published: October 2019

Abstract 4027

The success of haploidentical (HI) hematopoietic stem cell transplantation (HSCT), suggests that graft-versus-leukemia (GVL) effect might have a substantial role in this transplant modality. Rigorous T-cell depletion (TCD) of the graft decreases the occurrence of graft-versus-host disease (GVHD) in HI-HSCT, however this results in immunodeficiency and high disease relapse rate, especially in patients with resistant or residual leukemia. Therefore, enhancing GVL activity of HSCT without increasing GVHD is crucial for improving the outcome of haploidentical transplant. Post-transplant IL-15 administration is shown to enhance immune reconstitution, particularly donor-derived NK and CD8+ T cell populations in murine models. We evaluated the efficacy of IL-15 for enhancing GVL effect in recipients of HI-HSCT.

For developing clinically relevant haploidentical transplant models, different hybrid mice with B6 background that share the same haplotype (H2Kb) are used for our murine haploindentical transplant experiments. Lethally irradiated B6D2F1/J (H2Kb/d) mice are transplanted with B6CBAF1/J (H2Kb/k) TCD bone marrow (BM) and T cells at varying doses. Some animals were also given P815 tumor cells on the day of transplant. Administration of IL-15 significantly increased the numbers of CD8+ T and NK cells in the spleen and BM in the T cell depleted model at post-transplant day 28. Infusion of very low dose haploidentical T cells (1×104) with TCD-BM resulted in a conflicting effect on immune reconstitution, i.e. increased T cell numbers, and decreased NK cell population. Post-transplant IL-15 administration also changed this immune reconstitution pattern and significantly increased both T and NK cell numbers in recipients of HI-HSCT. In P815 challenged mice that were transplanted with very low dose T cell added TCD-BM, IL-15 administration significantly increased anti-tumor activity of the graft and improved survival (Figure 1) without increasing GVHD. This effect was observed when IL-15 administration was given at a later time point rather than immediately following transplantation, possibly allowing for more donor cell engraftment and T cell proliferation to take place. IL-15 administration without T cell infusion did not result in any survival improvement. We conclude that in our experimental HI transplant models, IL-15 administration augments anti-tumor effect of the HI-HSCT without increasing GVHD risk, and this effect requires presence of donor derived T cells.

Disclosures:

No relevant conflicts of interest to declare.
Influence of Genetic Polymorphisms in CYP2C8 and ABCB1 on the Mobilization Ability and Toxicity of Paclitaxel,
Authors: Lozano María L. , Niño Elkin A. , Anton Ana Isabel , Padilla Jose , Rivera Jose , Cerezo Juan José , Castilla-Llorente Cristina , Garcia Vicente
Published: October 2019

Abstract 4050

Introduction.

We and others have described the efficacy of paclitaxel-based chemotherapy in mobilizing large amounts of hematopoietic progenitors (HP) both in patients with solid tumors,(Bone Marrow Transplant 2000;25:231–5), and with hematological malignancies (Haematologica 2008; 93:161–3). Like most drugs, the taxanes are not controlled by the actions of one gene, but believed to be dependent on several polymorphic proteins. Single nucleotide polymorphisms (SNPs) in the ABCB1 gene, which encodes the transport protein P-glycoprotein, or in in metabolic enzymes, such as CYP2C8, have been suggested to influence the pharmacokinetics and clinical response to paclitaxel. Aim. To retrospectively evaluate the effects of five known allelic variants in the CYP2C8, and ABCB1genes on the mobilization ability and toxicity of the anticancer agent paclitaxel. Patients and Methods. 107 patients with hematological malignancies (43 lymphoma, 41 myeloma, 23 acute leukemia) received paclitaxel 170 mg/m2 i.v. by continuous infusion for 24 hours (day 1) followed by 8 mg/kg s.c G-CSF daily until the last apheresis. 77% received this treatment after failure of mobilization with G-CSF, and the rest as first line therapy because of risk factors for failure to achieve successful mobilization. The genetic variants (ABCB1 rs1045642 A >G, ABCB1 rs2032582 C>A, ABCB1 rs2032582 C>T, CYP2C8 rs10509681 C>T, and CYP2C8 rs11572080 A>G), were genotyped by allelic discrimination polymerase chain reaction (PCR) assays using TaqMan®Genotyping Assays (Applied Biosystems). The effect of genotypes on mobilization efficacy and on maximal hematological toxicity (according to the NCI version 3) was retrospectively assessed. Results. Allelic frequencies for rs1045642 A>G, rs2032582 C>A, rs2032582 C>T, rs10509681 C>T, and rs11572080 A>G variants, were 0.46, 0.40, 0.8, 0.13, and 0.13, respectively. Successful mobilization (>2 106/kg CD34+ cells) was achieved in 57% of patients. No reproducible significant associations between genotype and outcome (evaluated as number of CD34+ cells/kg in the first and total apheresis, number of collections performed, and on the mobilization success [p>0.05]), nor on myeloid or platelet toxicity (p>0.05) were found for any of the SNPs analyzed. Discussion. This study on a well-defined patient population suggests that the presently evaluated variant alleles in CYP2C8 and ABCB1 genes do not explain the substantial interindividual variability in the outcome or hematological toxicity of the mobilization schedule using paclitaxel and G-CSF. Funding. This study was supported in part by a research grants 04515/GERM/06; RECAVA RD06/0014/0039, and FIS 10/02594.

Disclosures:

No relevant conflicts of interest to declare.
High Level Secretion of HLA-G5 Might Inhibit the Incidence of Gvhd,
Authors: Wu Xiuli , Chen Yinkui , Liu Qifa , Xuan Li , Ling Yiwen , Zhang Yu , Fan Zhiping
Published: October 2019

Abstract 4068

Human leucocyte antigen G (HLA-G) is a nonclassical HLA class I molecule, the tolerogenic role of HLA-G is highly supported in pregnancy immunization, tumor immune escape and organ transplant. HLA-G molecules also act as an inhibitor of the T-lymphocytes, NK cells and antigen presenting cells. HLA-G can be expressed as 7 different isoforms including 4 membrane-bound (HLA-G1 to -G4) and 3 soluble (HLA-G5 to-G7) proteins. The soluble HLA-G5 isoform encoded by intron-4 retaining spliced transcript has been previously detected in vivo in sera and grafts from solid transplanted patients who had significantly better graft acceptance. In the present study, we analyzed the expression levels of HLA-G in patients after hematopoietic stem cell transplantation (HSCT) and investigated the relationship between HLA-G and GVHD. The levels of soluble HLA-G (sHLA-G) in the plasma of peripheral blood from 90 patients underwent HSCT were determined by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used to detect the expression of membrane bound HLA-G (mHLA-G). The levels of mHLA-G of patients at pre-HSCT, +15d post-HSCT and +30d post-HSCT were similar with the normal individuals. High levels of sHLA-G in the plasma of recipients were found after allogeneic HSCT (allo-HSCT). The soluble HLA-G5 and HLA-G6 levels in the plasma of recipients at +15d post-HSCT and +30d post-HSCT were significantly higher than that at pre-HSCT, but the soluble HLA-G7 levels in the plasma of recipients at +15d post-HSCT or +30d post-HSCT were similar with that at pre-HSCT. In contrast, the levels of sHLA-G in the plasma of recipients had no significant change after autologous HSCT (auto-HSCT). High level secretion of HLA-G5 after allo-HSCT (the levels of sHLA-G increased more than 1.0 fold compared to the pre-HSCT group) significantly reduced the incidence of acute GVHD and chronic GVHD; High level secretion of HLA-G6 after allo-HSCT also significantly reduced the incidence of chronic GVHD, but had no influence to the incidence of acute GVHD; High level secretion of HLA-G7 after allo-HSCT had no influence to the incidence of acute GVHD or chronic GVHD. The increased levels of HLA-G5 after allo-HSCT which compared with the pre-HSCT group had a significant negative correlation with the severity of GVHD. In conclusion, the high level secretion of HLA-G5 might inhibit the incidence of acute and chronic GVHD. Detected the secretion level of HLA-G5 after HSCT might benefit to predict and evaluate the GVHD.

Disclosures:

Liu: National Natural Science Foundation of China (No.30971300), Science and Technology Planning Project of Guangdong Province of China (No.2009A030200007): Research Funding.