bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation and Clinical Application of a New Method for Detecting ADAMTS13 Activity.
Authors: Wang Anyou , Dong Ningzheng , Ma Zhenni , Zhang Jingyu , Su Jian , Ruan Changgeng
Published: October 2019

Abstract 2134

Poster Board II-111

Introduction:

A severe deficiency of ADAMTS13 activity contributes to the pathogenesis of thrombotic thrombocytopenic purpura (TTP). Measuring the activity of ADAMTS13 is helpful for the diagnosis of TTP and the prognostic monitor in TTP patients. Most available assays are cumbersome and costly, and not appropriate for routine laboratories. ADAMTS13 cleaves the von Willebrand factor (VWF) within the domain A2, which locates between the domains A1 and A3. Therefore, specific assays for ADAMTS13 activity could be based on the different structure of VWF before and after the cleavage.

Materials and Methods:

To determine the activity of ADAMTS13, a new simple method has been developed in this study. Firstly, plasma samples were exposed in denaturing condition to allow cleavage of VWF by ADAMTS13. Then, the ADAMTS13 activity was measured with two novel monoclonal antibodies (SZ-129 and SZ-125), which are specifically recognize the VWF A1 and A3 domain, respectively, by using a two-site sandwich ELISA. Comparing with a residual-collagen binding assay (R-CBA), plasma ADAMTS13 activities in 161 samples were assessed, and the inhibitory activities of ADAMTS13 autoantibodies in 24 TTP patients were determined. The relationship of these two assays was analyzed by linear correlation, and the sensitivity and specificity of the new assay was also evaluated.

Results

Our results showed that plasma ADAMTS13 activities determined by the new assay were consistent with those of R-CBA, the squared correlation factor was 0.9183 of the two assays, and the coefficient of variation for the new assay was 6.17%. In 23 idiopathic TTP patients, the inhibitor activities of ADAMTS13 autoantibodies were ranged from 12% to 100%, while no inhibitory activity was detected in one hereditary TTP patient.

Conclusions

This new and simple assay for ADAMTS13 activity could be used routinely in clinic to determine the activity of ADAMTS13.

Disclosures:

No relevant conflicts of interest to declare.
Title:Dose-Adjusted EPOCH Plus Rituximab (DA-EPOCH-R) in Untreated Patients with Poor Risk Large B-Cell Lymphoma. A Phase 2 Study Conducted by the Spanish PETHEMA Group.
Authors: Purroy Noelia , López Andrés , Vallespi Teresa , Gironella Mercedes , Bergua Juan , Sancho Juan Manuel
Published: October 2019

Abstract 2701

Poster Board II-677

Background.

Since the development of CHOP chemotherapy, many efforts have been made to improve first line treatment in aggressive lymphomas, mainly in poor prognosis cases. To assess the efficacy and safety of an infusional DA-EPOCH plus Rituximab (DA-EPOCH-R) regimen in poor prognosis large B-cell lymphomas, the Spanish PETHEMA group conducted a phase 2 study in patients with a higher than 1 aaIPI in this setting.

Methods:

Eligibility criteria included histologically documented DLBCL, grade 3b follicular lymphoma or primary mediastinal B-cell lymphoma, IPI higher than 2 or aaIPI higher than 1 (or equal to 1 plus bulky disease). No restriction was made based on ECOG performance status when they could understand and sign the informed consent. Radiation therapy as consolidation was permitted on bulky masses detected at diagnosis.

Results:

Herein, 71 patients were evaluated for safety and outcome: 32 were male and 39 female. Median age was 60 years (range: 21-77). DLBCL was diagnosed in 57 (80%), PMBCL in 6 (9%), and grade 3b follicular lymphoma 8 (11%). Eighty nine percent had advanced (III-IV) CS, and 58% presented B symptoms. Bulky disease (3 10 cm maximum diameter) was present in 18%. ECOG was higher than 2 in 13 patients (18%). b-2-microglobulin was elevated in 62%, and LDH in 90% of patients. AaIPI was: 1 in 7 patients (10%), 2 in 36 (51%), and 3 in 28 (39%). Four hundred and fifteen cycles were given. A grade 3-4 mucositis occurred in 9 occasions, and neutropenic fever or infections in 66 cycles (16%). Fifty nine patients (83%) achieved a CR or CRu, 6 (8%) a PR and 4 (6%) an inadequate response, there were 2 early deaths: a toxic one, due to septic shock, and one due to non-related cause (traumatism). Twenty six percent of patients who achieved a CR relapsed. Twenty one patients (30%) died so far: 76% due to progressive disease, one patient who had undergone an ASCT after relapse died from secondary MDS. With a median follow-up of 40 months the event-free survival and overall survival is 60% and 61% respectively at 5 years. Disease-free survival is 72% at 5 years for patients who achieved a CR or CRu. No statistically significant difference in overall survival were seen according to aaIPI risk factors.

Conclusion:

DA-EPOCH-R is an immunochemotherapy regimen with a clinically significant activity and a very acceptable toxicity profile in patients with poor prognosis aggressive B-cell lymphomas and could somehow overcome IPI risk factors.

Disclosures:

No relevant conflicts of interest to declare.
Anti-CD20 Antibody GA101 Shows Higher Cytotoxicity but Is Competitively Displaced by Rituximab in Mantle Cell Lymphoma.
Authors: Heinrich Daniel A. , Klein Christian , Decheva Kristina , Weinkauf Marc , Hutter Grit , Zimmermann Yvonne , Weiglein Tobias , Hiddemann Wolfgang , Dreyling Martin H.
Published: October 2019

Abstract 2704

Poster Board II-680

Background:

Mantle cell lymphoma (MCL) is characterized by a poor long-term prognosis with a median survival of 3–5 years. Type I anti-CD20 antibody rituximab has demonstrated a clear anti-proliferative effect in MCL and achieves increased response rates in combination with chemotherapy. GA101, a third-generation IgG1 anti-CD20 antibody displays improved ADCC and superior direct cell death induction by virtue of glycoengineering compared to rituximab and its targeting a type II epitope on CD20, respectively.

Methods:

Using a panel of MCL cell lines (Rec-1, HBL-2, Jeko-1, Granta-519, JVM-2 and Z-138) we determined the effect of GA101 alone as well as in combination with rituximab on cell viability and proliferation. Karpas-422 (Diffuse Large B-Cell Lymphoma) was used as a control cell line. MCL and Karpas-422 cells were treated with GA101 or rituximab at concentrations of 1 – 20μg/ml and rituximab. Cell viability was analyzed by trypan-blue exclusion tests at 0h, 24h, 48h and 72h. The panel of MCL cell lines and Karpas-422 were then treated with GA101 and rituximab each at 1 and 10 μg/ml to determine potential synergism of antibody combinations. Accordingly, a fractional product calculation was performed: synergism > 0,1; antagonism < −0,1. In addition, Western-blot and RNA-array-analyses were performed to elucidate potential intra-cellular downstream pathway mechanisms.

Results:

After mono-exposure with GA101 (1 μg/ml), Granta-519 and Rec-1 showed the highest sensitivity (65–75% cell reduction in Granta-519 and 35–40% in Rec-1). Intermediate results were gained for Z-138, HBL-2, Jeko-1 and JVM-2 and Karpas-422 (15–20%). rituximab mono-exposure at 12,5 μg/ml showed a 25% reduction of cell count in Granta-519, 20% in HBL-2 and < 5% in Rec-1, Jeko-1 and Z-138. Combination experiments suggested the competitive binding of the two antibodies. Thus, GA101 plus rituximab combination experiments resulted in a lower cytotoxicity than GA101 alone, according to fractional product calculations.

Conclusions:

Although GA101 is competitively displaced by rituximab, GA101 demonstrates higher efficacy in MCL cell lines than rituximab, even at a more than 10-fold lower concentration. Currently RNA-array- and Western blot analysis are being performed to identify the critical pathways responsible for the superior cytotoxicity of GA101.

Disclosures:

Klein: Discovery Oncology, Roche Diagnostics GmbH: Employment. Weinkauf:Lilly Deutschland GmbH: Research Funding. Hutter:Lilly Deutschland GmbH: Research Funding. Zimmermann:Lilly Deutschland GmbH: Research Funding. Dreyling:Roche: Honoraria, Research Funding.
GA101-Coated Target Cells Are More Effective Than Rituximab-Coated Target Cells at Activating NK Cells When Complement Is Present.
Authors: Spaunhorst Britnie , Weiner George J
Published: October 2019

Abstract 2707

Poster Board II-683

Rituximab has had a major impact on the treatment of B cell malignancies. The mechanisms responsible for mediating the anti-tumor effects of rituximab are complex. For example, complement can have both positive and negative effects on the ability of rituximab to induce target cell lysis. In particular, we recently reported that rituximab-mediated complement activation results in C3b deposition on the rituximab Fc. C3b then impedes interaction between rituximab and NK cell CD16, thereby limiting NK cell activation and ADCC.

GA101 is a type II anti-CD20 monoclonal antibody that mediates enhanced direct cell death induction. It has significantly reduced CDC activity compared to type I anti-CD20 antibodies such as rituximab. In addition, GA101 was engineered to mediate increased ADCC (Umana et al., ASH 2007). The current studies were designed to assess whether the decreased ability of GA101 to activate complement results in an enhanced ability of GA101 to activate NK cells when complement is present.

Peripheral blood mononuclear cells (PBMCs) were obtained from normal donors and added to Raji cells (Burkitt lymphoma cell line) at a 1:1 ratio. Various concentrations of rituximab or GA101 were added along with media, 20% autologous serum or 20% heat-inactivated autologous serum (heated to 57°C for 30 minutes). Samples were cultured for 20 hours. NK cell (CD3−, CD56+) activation, as determined by phenotypic changes, was evaluated by flow cytometry based on prior studies demonstrating that downmodulation of CD16, and upregulation of CD54 and CD69 are reproducible surrogates for mAb-induced NK activation and ADCC. Raji cells coated with either rituximab or GA101 were able to activate NK cells when cultures were performed in media alone or with heat-inactivated serum (left panel). In contrast, serum blocked the ability of rituximab to activate NK cells, but not the ability of GA101 to activate NK cells (right panel). Similar results were found when upregulation of CD69 or downmodulation of CD16 were evaluated as markers of NK activation and using PBMCs from two other donors.

We conclude that the presence of complement does not limit the ability of GA101-coated target B cells to activate NK cells. This is in contrast to rituximab-coated target B cells which are unable to activate NK cells in the presence of serum. These results suggest that the decreased ability of GA101 to fix complement could, paradoxically, enhance the efficacy of GA101 by resulting in enhanced activation of NK cells and increased ADCC.

Disclosures:

No relevant conflicts of interest to declare.
Induction of Noxa Via Generation of Reactive Oxygen Species Plays a Key Role in Induction of CLL Cell Apoptosis by Two Novel p53-Independent Cytotoxic Agents.
Authors: Steele Andrew , Prentice Archibald G , Chanalaris Anastasios , Hoffbrand A. Victor , Cwynarski Kate , Wickremasinghe Ranmohan
Published: October 2019

Abstract 2752

Poster Board II-728

Approximately 15% of CLL patients present with a deletion of chromosome 17p, resulting in the loss of the p53 gene. The percentage of 17p-deleted patients can increase to between 30–50% following treatment with cytotoxic agents which induce CLL apoptosis via upregulation of p53. Therefore, identification of novel agents which kill CLL cells independently of p53 is of crucial importance. We have recently shown that two chemically unrelated agents, 2-phenylacetylenesulfonamide (PAS) and the sesquiterpene lactone LC1 kill CLL cells regardless of the functional status of p53. Furthermore, in contrast to the conventional drugs chlorambucil and fludarabine, PAS and LC1 induce apoptosis in the absence of p53 elevation. However, the mechanisms of action by which these agents induce p53-independent apoptosis are unclear. We have previously shown that both PAS and LC1 initiated CLL cell apoptosis within 6-10h with, maximal killing by 48h. Here we show that treatment of CLL cells with either agent results in the generation of reactive oxygen species (ROS), the activating phosphorylation of the pro-apoptotic MAP kinase family member JNK (Fig 1), resulting in turn in upregulation of its downstream target, the transcription factor c-JUN (Fig 2). The BH3-only pro-apoptotic protein Noxa was originally described as a pro-apoptotic target for upregulation by p53. However, both PAS (Figs1 and 2) and LC1 upregulated Noxa in a p53-independent manner. Addition of N-acetylcysteine (NAC), a free radical scavenger, decreased ROS generation by PAS or LC1 and also prevented phosphorylation of JNK and Noxa upregulation (Fig 1) and also the upregulation of c-JUN. NAC also strikingly abrogated apoptosis induction by either agent, as shown by quantitation of cleavage of the caspase 3 substrate poly (ADP ribose) polymerase (PARP; Fig 1). Taken together, the data suggest both PAS and LC1 induce p53-independent apoptosis via upregulation of ROS and the subsequent induction of Noxa. The data are also compatible with a role for JNK and c-JUN in the events leading to Noxa upregulation.

Disclosures:

No relevant conflicts of interest to declare.
KIT Inhibition Affects Heat Shock Protein (HSP) Activity: A Potential Rescue Mechanism towards KIT Tyrosine Kinase Inhibition and a Rationale for Dual KIT/HSP Inhibition.
Authors: Kampa Kerstin M , Grandl Tanja , Mueller Sandra , Normant Emmanuel , Walter Michael , Schuetz Wolfgang , Kanz Lothar , Heinrich Michael C , Schittenhelm Marcus M
Published: October 2019

Abstract 2753

Poster Board II-729

Activating mutations of the KIT class III receptor tyrosine kinases are associated with core binding factor leukemias (CBF AML), systemic mastocytosis (SM), gastrointestinal stromal tumors (GIST), melanomas, seminoma/dysgerminoma and sinonasal natural killer/T-cell non-Hodgkin lymphoma. Despite the encouraging therapeutic potential of KIT-tyrosine kinase inhibitors (TKI), resistance leading to disease progression occurs in many patients, specifically after TKI monotherapy. We hypothesized that resistance to therapy is promoted by activation of alternative signaling pathways which override TKI inhibition.

To explore the downstream signaling pathways of class III receptor tyrosine kinases, we performed unbiased phoshoproteomic analyses of mutant FLT3 or KIT leukemia and mastocytosis cell lines before and after TKI treatment. Tantalizingly, immunoaffinity purification of phosphopeptides followed by tandem mass spectrometry following KIT-inhibition with Imatinib at IC90 (100nM) revealed a significant upregulation of phosphorylation levels of peptides identified as members of the heat shock protein (HSP) family. Of interest, mRNA GeneChip® Array analysis of hematopoietic Ba/F3 cells transfected with either a mutant KIT isoform (D816V) or a mutant FLT3 isoform (ITD) and treated with TKI revealed significant downregulation of HSP family members in the FLT3 model – but stable mRNA levels in the KIT model. Taken together, our phosphoproteome and mRNA data suggest a protective function of HSP in mutant-KIT tumors treated with TKI.

Next we studied the antiproliferative and proapoptotic effects of the HSP90 inhibitor IPI-504, a 17-AGG-derivative, in mutant-KIT cell models. IPI-504 potently inhibited proliferation and induced apoptosis with an IC50 of 0.5 up to 5μM depending on the KIT isoform. Importantly, combination of IPI-504 with TKIs resulted in potentiation of the antiproliferative and proapoptotic effects achieved by either drug alone. Antitumor efficacy in combination therapy was observed even at HSP90 inhibitor concentrations that did not display antitumor activity if administered alone.

In conclusion, our model suggests that inhibition of KIT affects heat shock protein activity serving to stabilize the functionality of targeted autoactivated receptor tyrosine kinases, which provides a potential mechanism for resistance to TKI therapy. Importantly, we provide a rationale to combine TKI with (low-dose) HSP-inhibitors such as IPI-504 to optimize TKI therapy.

Disclosures:

Normant: Infinity: Employment.
Consequences of 4-1BB-4-1BB Ligand Interaction for NK Cell Anti-Tumor Immunity: Opposite Effects of Seemingly Analogue Molecules in Mice and Men.
Authors: Baessler Tina , Charton Jean E , Buechele Corina , Schmiedel Benjamin J , Gruenebach Frank , Krusch Matthias , Wacker Alexander , Kanz Lothar , Salih Helmut R
Published: October 2019

Abstract 279

In mouse models, stimulation of the TNF receptor family member 4-1BB/CD137 on cytotoxic lymphocytes by agonistic antibodies potently stimulated anti-tumor immunity, which has led to the development of humanized agonistic 4-1BB mAb that are presently being evaluated in clinical phase I/II studies (www.clinicaltrials.gov, keyword: CD137). However, seemingly analogue immunoregulatory molecules may mediate different effects in mice and men, and in humans yet nothing is known regarding the role of 4-1BB in NK cell reactivity. NK cells play an important role in tumor immunosurveillance, and largely contribute to the success of therapeutic strategies like (haploidentical) stem cell transplantation (SCT) or application of monoclonal antibodies like Rituximab by mediating graft versus leukemia activity and performing antibody-dependent cellular cytotoxicity (ADCC). Here we analyzed the expression of 4-1BB in human NK cells and compared the effect of 4-1BB signaling on effector functions of NK cells in mice and men. While being absent in resting state, both human and mouse NK cells acquire expression of 4-1BB at similar levels with comparable kinetics upon activation. Next we cultured human and mouse NK cells with target cells transfected with human and mouse 4-1BB ligand (4-1BBL) and analyzed the effect of 4-1BB on NK cell effector functions. 4-1BB – 4-1BBL interaction enhanced effector functions of mouse NK cells, while degranulation, cytotoxicity and cytokine production of human NK cells were substantially impaired (all p<0.01, Student's T-test). In line, employing primary leukemia cells from patients with AML (n= 65) and CLL (n=49), which were found to express 4-1BBL in 35% and 100%, respectively, we found that blocking 4-1BB-4-1BBL interaction markedly enhanced granule mobilization, cytotoxicity and interferon-g production of allogenic and autologous human NK cells in response to leukemia cells. In CLL, this inhibitory effect of 4-1BB-4-1BBL interaction on NK cell reactivity was observed both with regard to direct and Rituximab-induced cytotoxicity and cytokine production. Together, our data implicate that one should exercise caution in applying anti-4-1BB therapeutically, underline the necessity of detailed analyses regarding the function of seemingly analogue immunoregulatory molecules in mice compared to men and demonstrate that blocking 4-1BBL-4-1BB interaction may serve to enhance NK cell reactivity in therapeutic settings like allogenic SCT and antibody-treatment of malignancies in humans.

Disclosures:

No relevant conflicts of interest to declare.
Nuclear Expression of the Sox 11 Transcription Factor in Mantle Cell Lymphoma, and Cytoplasmic Expression in Follicular Lymphoma and Multiple Myeloma: Pathogenetic Implications.
Authors: Williams Michael E , Rutherford Sarah , Tang Yunjia , Cousar John
Published: October 2019

Abstract 2925

Poster Board II-901

Mantle cell lymphoma (MCL) is characterized by nuclear cyclin D1 expression resulting from the t(11;14)(q13;q32). As cyclin D1 overexpression alone is insufficient for B-cell transformation, we have investigated other potentially contributing mutations. Sox11 is a member of a large family of transcription factors containing a DNA-binding high-mobility group (HMG) domain and shares homology with Sox4, which is involved in lymphopoiesis. Recent reports have identified Sox11 expression in the majority of MCL, suggesting it may contribute to pathogenesis (Ek et al, Blood 2008;111:800; Wang et al, Br J Haematol 2008;143:248).

Methods:

Patients with MCL diagnosed at the Univ. of Virginia from 1997-2008 and for whom nodal, marrow or spleen tissue blocks were available were identified from the Pathology database. Follicular lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), hairy cell leukemia (HCL) and multiple myeloma (MM) were also analyzed. Formalin-fixed, paraffin-embedded samples were stained by immunohistochemistry for cyclinD1, Sox11 (rabbit a/Sox11, 1:100, Sigma) and Ki-67. Nuclear and/or cytoplasmic expression was determined by two of us (YT, JC).

Results:

All MCL samples revealed nuclear cyclin D1 expression. Nuclear but not cytoplasmic Sox11 expression was identified in all 24 non-blastoid and in 5/6 blastoid samples; 1 blastoid sample showed only cytoplasmic Sox11 staining (Table). Four of 5 HCL expressed cyclin D1 but were negative for Sox11; 4/5 MM also were cyclin D1-positive, with 3 positive for cytoplasmic Sox11 including 2 of the cyclin D1-positive cases. All 5 FL showed cytoplasmic Sox11 positivity, whereas all MZL and SLL were negative for cytoplasmic or nuclear staining.

Conclusions:

Nuclear Sox11 expression was uniformly identified in MCL, with the exception of cytoplasmic expression in a single blastoid case. No nuclear Sox11 was present in HCL or MM despite the expression of cyclin D1, although 3 MM showed cytoplasmic Sox11 staining. These findings suggest that nuclear Sox11 overexpression is not a direct result of dysregulated cyclin D1 signaling but instead occurs by alternative mechanisms. The significance of cytoplasmic staining remains uncertain. A potential pathogenetic role for Sox11 and possibly other Sox family transcription factors warrants further investigation in MCL and other lymphoproliferative neoplasms for diagnostic use and therapeutic targeting.

Disclosures:

No relevant conflicts of interest to declare.
Incidence and Management of Isolated Subsegmental Pulmonary Embolism.
Authors: Shen Yi-Hao , Wells Phil , Dennie Carole , Carrier Marc
Published: October 2019

Abstract 2989

Poster Board II-965

Introduction:

The use of computed tomographic pulmonary angiography (CTPA) in patients with suspected pulmonary embolism (PE) has improved the visualization of subsegmental pulmonary arteries. However, the clinical significance of subsegmental PE is unclear. In the PIOPED Study, PE limited to subsegmental pulmonary arteries was most prevalent among patients with low-probability ventilation/perfusion (V/Q) scans. Patients with non-diagnostic (low or intermediate) V/Q scans can be safely managed without anticoagulation. The incidence and clinical management of subsegmental PE remains uncertain.

Objective:

To evaluate the incidence and clinical management of subsegmental PE in patients with suspected acute PE.

Methods:

This is a retrospective cohort study of consecutive patients with suspected acute PE undergoing CTPA at the Ottawa Hospital from Jan 1, 2007 to Dec 31, 2008. Subsegmental PE was defined as one or more pulmonary artery filling defects located in the subsegmental level, with no filling defects visualized at more proximal pulmonary artery levels. All patients were followed for a minimum of 6 months after the index PE.

Results:

A total of 78 (10.4%, 95% CI: 8.4 to 12.8%) cases of subsegmental PE were identified out of 748 cases of PE diagnosed by CTPA. Forty-three (77%) of these had a single isolated subsegmental filling defect. Data could be extracted in 56 (72%) of the 78 cases. Among these 56 patients, 18 (32%) had unprovoked PE. Further investigations were performed in 50 patients (Ultrasonography (U/S) of legs (n=38), U/S and V/Q scan (n=11) or V/Q scan alone (n=1)). Deep vein thrombosis was detected in 12 patients and two patients had a high probability V/Q scan. Forty-eight (86%) patients were anticoagulated. All patients with concurrent DVT or high probability V/Q scan were started on anticoagulation therapy. Two patients experienced a major bleeding episode after anticoagulation. Out of the 8 patients left untreated, there were no recurrent VTE.

Conclusion:

Isolated subsegmental PE represents approximately 10% of all acute PE diagnosed by CTPA. Patients with subsegmental PE diagnosed on CTPA are more commonly receiving anticoagulation than not. Further studies are needed to establish the risk benefit ratio of anticoagulation therapy in patients with subsegmental PE diagnosed on CTPA.

Disclosures:

No relevant conflicts of interest to declare.
Revised Model for Platelet Adhesion to Collagen.
Authors: Stefanini Lucia , Stolla Moritz , Maloney Sean F , Ouellette Timothy Daniel , Roden Claire , Diamond Scott L. , Bergmeier Wolfgang
Published: October 2019

Abstract 2999

Poster Board II-968

The Gi-coupled ADP receptor, P2Y12, is the target of clopidogrel bisulfate (Plavix), currently the most successful anti-platelet strategy used in the clinic. In a recent study, we have shown that the Ca2+-sensing nucleotide exchange factor, CalDAG-GEFI, and P2Y12 represent the major signaling pathways leading to Rap1 and integrin activation in platelets (Cifuni et al., 2008, Blood). In the present study, we have further evaluated the importance of CalDAG-GEFI signaling and Rap1 activation for various aspects of platelet activation, and we have compared thrombus formation of CalDAG-GEFI−/− and WT/clopidogrel platelets under static and flow conditions in vitro. Our studies establish a revised model for platelet activation by collagen. In platelets activated with threshold concentrations of GPVI agonists, CalDAG-GEFI serves as a highly sensitive response element to Ca2+ that allows for the rapid activation of Rap1. CalDAG-GEFI-mediated Rap1 activation triggers a first wave of integrin activation and ERK (MAPK) signaling, followed by TxA2 release. TxA2 provides crucial feedback for the activation of PKC and granule/ADP release. ADP in turn triggers the second, P2Y12-dependent wave of Rap1-mediated signaling events, leading to the sustained activation of integrins and further release of TxA2. Higher concentrations of GPVI agonists lead to the concomitant activation of CalDAG-GEFI and PKC, facilitating platelet aggregation independent of feedback by endogenous TxA2. Under physiological flow conditions, CalDAG-GEFI-dependent platelet activation (clopidogrel-treated WT platelets) allowed for the formation of small but unstable thrombi, which rapidly disintegrated at high shear rates. In contrast, CalDAG-GEFI−/− platelets (P2Y12-dependent platelet activation) in anticoagulated blood firmly adhered to the thrombogenic surface but failed to form thrombi, even at high concentrations of collagen. Addition of exogenous TxA2 to anticoagulated CalDAG-GEFI−/− blood did not restore thrombus formation under flow. However, small thrombi were observed with non-anticoagulated CalDAG-GEFI−/− blood perfused at venous but not arterial shear rates, suggesting that a) locally generated thrombin facilitates the recruitment of free flowing CalDAG-GEFI−/− platelets to already adherent platelets, and b) the slow kinetics of P2Y12-dependent Rap1 activation only supports thrombin-induced platelet-platelet cohesion at low shear conditions. In conclusion, our studies demonstrate that CalDAG-GEFI/Rap1 signaling plays a critical role for the first wave of integrin activation and TxA2 generation important for platelet adhesion to a thrombogenic surface. Signaling by P2Y12/Rap1 is essential for sustained platelet activation/thrombus stabilization and partially compensates for CalDAG-GEFI/Rap1-mediated platelet adhesion under low flow conditions.

Disclosures:

No relevant conflicts of interest to declare.
Inhibition of Calmodulin Triggers Apoptosis Events in Human Platelets.
Authors: Wang Zhicheng , Li Suping , Liu Guanglei , Shi Quanwei , Yan Rong , Ruan Changgeng , Dai Kesheng
Published: October 2019

Abstract 3003

Poster Board II-980

Calmodulin (CaM) is a calcium-sensing protein ubiquitously expressed in every eukaryotic cell type regulating biological processes such as cell proliferation, vesicular fusion, fertilization and apoptosis. CaM antagonists induce apoptosis in various tumor models and inhibit tumor cell invasion and metastasis, thus some of which have been extensively used as anti-cancer agents. Tamoxifen (TMX), a potent antagonist of CaM, has been in the center of management of hormone-sensitive breast cancer, and also represents the best example of chemo-prevention to reduce the incidence of invasive breast cancer. Furthermore, TMX is potentially useful in treatment of other kinds of cancer. However, TMX has some severe side effects, one of which is thrombocytopenia. Up to now, the pathogenesis of thrombocytopenia still remains unclear. In platelets, CaM has been found to bind directly to cytoplasmic domains of several platelet receptors. Incubation of platelets with CaM antagonists impairs the receptors-related platelet function. However, it is still unclear whether CaM antagonists, especially TMX, induce platelet apoptosis. Here, we show that CaM antagonists TMX and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7) dose-dependently induce apoptotic events in human platelets, including depolarization of mitochondrial inner transmembrane potential, caspase-3 activation, gelsolin cleavage and phosphatidylserine (PS) exposure. CaM antagonist did not incur platelet activation as detected by P-selectin surface expression and PAC-1 binding. However, ADP- and botrocetin-induced platelet aggregation and platelet adhesion and spreading on von Willebrand factor surface were significantly reduced in platelets pre-treated with CaM antagonist. Therefore, these findings indicate that CaM antagonists induce platelet apoptosis, which suggests a possible pathogenesis of thrombocytopenia in some patients treated with CaM antagonist drugs, and also may present as a novel mechanism for platelet clearance and dysfunction in vivo or in vitro. The elevation of the cytosolic Ca2+ level may involve in the regulation of CaM antagonist-induced platelet apoptosis.

Disclosures:

No relevant conflicts of interest to declare.
Sialic Acid Deficiency Impairs GPVI-Mediated Platelet Activation in ST3Gal-IV−/− Mice.
Authors: Rumjantseva Viktoria , Sørensen Anne Louise , Hoffmeister Karin M , Falet Hervé
Published: October 2019

Abstract 3005

Poster Board II-972

Lack of terminal sialic acid residues on platelet surface glycoproteins results in rapid platelet clearance. Using null mice for the ST3Gal-IV sialyltransferase gene (ST3Gal-IV−/− mice), we have recently identified galactose residues on the N-terminus of the platelet Von Willebrand Factor receptor GPIbαa as a major counter receptor for the lectin domain of the asialoglycoprotein receptor on both hepatocytes and liver Kupffer cells (Sørensen et al., Blood 2009). ST3Gal-IV−/− mice have increased tail bleeding time. However, the role of terminal sialic acid residues on platelet activation is unclear. We investigated here whether loss of sialylation affects platelet activation mediated through the collagen receptor GPVI or by thrombin. Platelets were isolated from ST3Gal-IV−/− and ST3Gal-IV+/+ mouse littermates, stimulated with collagen-related peptide (CRP), convulxin (CVX), or thrombin, and platelet activation was evaluated by flow cytometry using P-selectin expression, as a marker for αa-granule secretion, and fibrinogen binding, as a marker for integrin αaIIbβ3 activation. Stimulation of ST3Gal-IV−/− platelets with CRP and CVX revealed a profound activation defect, compared to ST3Gal-IV+/+ platelets. The defect was not due to loss of surface receptor expression since ST3Gal-IV−/− and ST3Gal-IV+/+ platelets had comparable GPVI expression, as evidenced by flow cytometry. By contrast, activation of ST3Gal-IV−/− platelets with thrombin was normal. The data show that terminal sialic acid residues on GPVI are required for maximal platelet activation by CRP and CVX.

Disclosures:

No relevant conflicts of interest to declare.
BMP Signaling Is Crucial for Regulation Vascular Progenitor Development in Human Embryonic Stem Cells.
Authors: Wang Zack Z. , Bai Hao , Gao YongXing , Arzigian Melanie , Wojchowski Don M. , Wu Wen-shu
Published: October 2019

Abstract 3037

Poster Board II-1013

The generation of vascular cells from pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells, may facilitate tissue transplantation, reperfusion of ischemic tissues, and treatment of pathologies in which endothelial cell dysfunction exists. The signals that direct pluripotent stem cell differentiation into lineage-specific cells remain largely unknown. To identify vascular progenitor cells during hESC differentiation, we characterized various subpopulations that may differentiate into endothelial cells (ECs) and smooth muscle cells (SMCs). In our newly established serum-free medium, hESCs sequentially differentiated into CD34+CD31-, CD34+CD31+, and then CD34-CD31+ cells. Real-time PCR analysis indicated that EC- and SMC-specific genes, including VEGFR2, Tie2, VE-Cad, vWF, SMA, SM22-alpha, calponin, and caldesmon, were expressed significantly high in CD34+CD31+ cells. Furthermore, the CD34+CD31+ cells that were cultured in EC or SMC growth medium had high differentiation potential to become ECs and SMCs. These data suggested that CD34+CD31+ cells contain vascular progenitor cells. By treating hESCs with various factors at different time points, we found that BMP4 was critical to initiate hESC differentiation into CD34+CD31+ progenitor cells, whereas VEGF and FGF2 facilitated CD34+CD31+ cell development in the later stage. Conversely, TGFbeta promoted CD34+CD31- cells that were unable to give rise to ECs and SMCs. In addition, TGFbeta blocked the development of CD34+CD31+ cells induced by BMP4, suggesting that TGFbeta signaling negatively regulates the development of CD34+CD31+ cells during hESC differentiation. DNA microarray analysis indicated that a number of mesodermal genes were increased in BMP4-induced CD34+ cells, and a number of ectodermal genes were increased in TGFbeta-induced CD34+ cells. Therefore, the roles of BMP and TGFbeta signals in lineage differentiation may be inversely reflected in mesoderm and ectoderm. In addition, several pluripotent genes were expressed in TGFbeta-induced CD34+ cells, suggesting that TGFbeta signaling affects maintenance of stem cell pluripotency. The BMP-Smad inhibitor, dorsomorphin, inhibited phosphorylation of Smad1/5/8, and blocked hESC differentiation to CD34+CD31+ progenitor cells, suggesting that BMP Smad-dependent signaling is critical for CD34+CD31+ vascular progenitor development. Our findings provide new insight into how pluripotent hESCs differentiate to generate vascular cells.

Disclosures

No relevant conflicts of interest to declare.
High Concentrations of Thrombospondin-1 Suppress Shear Stress Induced Cleavage of Von Willebrand Factor by ADAMTS13.
Authors: Zhou Wenhua , Tsai Han-Mou
Published: October 2019

Abstract 3063

Poster Board II-1039

ADAMTS13 is a circulating metalloprotease that cleaves and down regulates the activity of von Willebrand factor (VWF) in the circulation. Deficiency of ADAMTS13 causes thrombotic thrombocytopenic purpura (TTP). VWF is critical for supporting platelet adhesion and aggregation at sites of microvascular injury. Nevertheless, the factors that protect VWF from cleavage by ADAMTS13 remain unknown. Thrombospondin-1 (Thbs-1), an adhesive protein that exhibit binding to multiple proteins such as collagen, fibronectin and fibrin, promotes thrombus adherence in animal models of vascular injury. To investigate the role of Thbs-1 in the regulation of VWF cleavage by ADAMTS13, we analyzed the cleavage of VWF flowing through a capillary tube. In the presence of physiological concentrations of ADAMTS13, VWF fragments were generated in a shear stress dependent manner. The cleavage of VWF in the capillary tube device was inhibited by Thbs-1 in a concentration dependent manner. At physiological concentrations (30 ng/mL) of Thbs-1, the cleavage of VWF was decreased insignificantly to 85% of control. The cleavage was decreased to 50% at 100 ng/mL Thbs-1, and to less than 10% at 1000 ng/mL Thbs-1 (P<0.0001). The suppressive effect of Thbs-1 was also observed with a truncated variant of ADAMTS13 that lacks the distal six thrombospondin type 1 repeats and the CUB domains, suggesting that the C-terminal part of ADAMTS13 is not necessary for the suppressive effect of Thbs-1. Furthermore, the presence of platelets did not negate the suppressive effect of Thbs-1 on VWF cleavage. Thbs-1 did not suppress the cleavage of FRET-VWF73 or guanidine hydrochloride treated VWF. It also did not affect the cleavage of VWF multimers induced by vortexing in the presence of ADAMTS13 and platelets. In summary, the suppressive effect of Thbs-1 on VWF cleavage is shear stress dependent. Thbs-1 is not expected to play a major role in regulating the size of circulating VWF multimers. Nevertheless, high concentrations of Thbs-1 released from platelets may protect sheared VWF from cleavage by ADAMTS13. This protective effect of Thbs-1 on VWF proteolysis may help stabilize the hemostatic plugs at sites of microvascular injury.

Disclosures

No relevant conflicts of interest to declare.
Aberrant Expression of Mir-29b, Mir-181a and Mir-181b in T-Cell Acute Lymphoblastic Leukemia.
Authors: Gimenes-Teixeira Hamilton L. , Zanette Dalila L. , dos Santos Guilherme Augusto S. , Scheucher Priscila S. , Dalmazzo Leandro F. , Araújo Amélia G. , Falcao Roberto P. , Júnior Wilson A. Silva , Rego Eduardo M
Published: October 2019

Abstract 3065

Poster Board III-2

Deregulations of miRNA expression and function in B-cell acute lymphoblastic leukemia (B-ALL) have been associated with specific recurrent citogenetic abnormalities and clinical outcomes. In contrast, there is few data about miRNAs in T-cell acute lymphoblastic leukemia (T-ALL). We have determined the miRNA expression profile of 48 T-ALL patients' blasts and compared with normal mature T cells. We used the Taqman MicroRNA Assay Human Panel to screen 164 known mature miRNA sequences. Normal CD3+ cells were isolated from peripheral blood of four healthy subjects by immunomagnetic labeling. Total RNA was pooled and reverse transcribed with specific looped RT primers, and expression was evaluated by quantitative real-time PCR (RQ-PCR). Reactions were performed in duplicates and samples with a coefficient of variation greater than 5% were excluded. Furthermore, we considered as differentially expressed those miRNAs with fold change values higher than 10 or lower than 0.1. With this strategy we identified four miRNAs that were hyper-expressed (miR-181a, miR-181b, miR-213 and miR-29b) and three hypo-expressed (miR-150, miR-95, miR-338) in the leukemic pool. In order to confirm our findings, we then performed the analysis of miR-181a, miR-181b and miR-29b expression on 52 individual samples (48 T-ALL and 4 normal T cell samples) using RQ-PCR. Forty-five (93.7%) and 46 (95.8%) of the T-ALL samples presented expression levels of miR-29b and of miRs 181a/181b higher than the maximum detected in the normal samples. The analysis of the predicted targets for these three miRNAs was performed using miRNApath. MAPK signaling was the pathway with the highest number of target genes with 60 genes, of which MAP4K4, FOS, RAP1B, AKT3 and NLK were commonly targeted by all three miRNAs. As deregulation of the MAPK pathway in T-ALL has been previously described, we hypothesized that the hyper-expression of miR-29b, miR-181a and miR181b may be associated with this aberrant MAPK signaling.

Disclosures

No relevant conflicts of interest to declare.
Trends in Incidence and Survival of Myeloid Malignancies Since 1980, in the Côte D'or Department, Burgundy, France.
Authors: Maynadié Marc , Manivet Ines , Mounier Morgane , Mugneret Francine , Bailly François , Caillot Denis , Petrella Tony , Flesch Michel , Carli Paule Marie , Girodon François
Published: October 2019

Abstract 3122

Poster Board III-59

Objective

The Registry of Haematological Malignancies (HM) has been established on January 1st, 1980 in the department of Côte d'Or (pop 500 000 inhabitants). It was the first specialized registry in haematology in the world. During the course of 25 years (1980-2004), 5026 cases of HM were recorded including 1553 Myeloid malignancies (MM) in which entities not initially considered as malignant were taken in account such as Myelodysplastic syndrome (MDS) and some Myeloproliferative neoplasms (MPN). This allow us to present trends in incidence and survival of myeloid malignancies by entities since 1980.

Method

MM diagnosed in the population between 01/01/1980 and 31/12/2004 were registered. They were coded according to ICD-O-3 and following the principles of the 2001 WHO classification i.e. new threshold of definition of acute myeloid leukaemia (AML). World population standardized incidence rate were calculated by sex, age, by urban /rural repartition and by period of time. Five-year, 10-y, 15-y and 20-y relative survival was calculated using Estève's method and based on vital status updated at the end of 2007.

Results

Incidence rate for whole MM were 3.73/100 000/y in men and 2.74 in women. They were 2.82 for AML, 3.73 in MPN and 2.83 in MDS. Sex ratio was 1.5/1 as a whole and urban/ratio was always close to 2, data being statistically significant for numerous entities. Incidence rates increase with age until 75y-o for AML and 80y-o for MPN and then decreased except for MDS in which incidence continue to increase. Along the period, only incidence of MDS increases significantly with an annual medium rate of 3% (2.7 in men and 3.3 in women). In MPN, a significant increase was observed only for women (2%). Within AML, surprisingly a significant increase was found for AML with recurrent cytogenetic abnormality in men (8.03%). 15-years relative survival was the best for MPN (46%), being significantly better in women (59% vs 31%), in which Essential Thrombocythemia was the best (74%). On the contrary, the worst 15-y survival was for was for MDS (9%). Within AML, the best survival was found for AML recurrent cytogenetic abnormalities (15-y: 54%) compare to other categories (4-8%). Survival has not increased along the period, has increased significantly in MPN. In AML, 10-y survival has increased until 1999 to reach 39% but has decreased for the 2000-2004 period (8-y: 11%).

Conclusion

Analysis of the largest epidemiologic database on HM allow to produce information such as a significant increase of incidence of MDS in which survival remained very poor in both sex, a good survival of MPN compared to other categories and a worrying decreased of survival of AML in the more recent period.

Disclosures

No relevant conflicts of interest to declare.
Parvovirus B19 Is Inactivated by Pasteurization, a Dedicated Virus Inactivation Step in the Manufacturing Process of Plasma-Derived Products.
Authors: Groener Albrecht , Nowak Thomas , Schäfer Wolfram
Published: October 2019

Abstract 3152

Poster Board III-89

Human parvovirus B19 (B19V), a small non-enveloped virus, is the causative agent of the childhood disease erythema infectiosum (fifth disease) but causes, especially when the disease occurs during adulthood, a number of clinical symptoms as arthralgia, arthritis, (transient) aplastic crisis, anaemia, and hydrops fetalis (during pregnancy). B19V is normally spread via the respiratory route, however, parenteral transmission can occur through blood or blood components and plasma-derived products. In order to demonstrate the safety of plasma-derived products regarding B19V, virus validation studies are performed, using usually animal parvoviruses as models for B19V which indicate that this virus is highly resistant to commonly used inactivation methods as heat. Employing a cell culture infectivity assay for B19V, established at CSL Behring's virology laboratory, a considerable higher sensitivity of B19V to pasteurization (heat treatment in aqueous solution at 60°C for 10 hours) could be demonstrated for B19V in contrast to the animal parvovirus CPV (canine parvovirus). B19V was not only very sensitive to heat in unstabilised product intermediates but also heat sensitive when studying intermediates of various plasma products containing high concentrations of the stabilizers sucrose and glycine. Although these protein stabilizers had also a stabilizing effect on B19V this virus was nevertheless considerably more sensitive to pasteurization than CPV. As shown below, pasteurization results in an effective inactivation of B19V in a wide range of plasma-derived products.

Product Mean Virus Reduction Factor [log10] due to Pasteurization B19V CPV VWF / FVIII (Humate-P) ≥3.9 1.1 FVIII (Beriate P) a ≥3.8 0.7 Fibrinogen (RiaSTAP) b ≥4.5 1.6 PCC (Beriplex P/N) a 3.5 0.5 C1-INH (Berinert P) a 3.9 1.4 Human Thrombin a 3.5 0.5 FXIII (Fibrogammin P) a ≥4.0 1.0 scIG (Vivaglobin) ≥5.0c 2.3 Human albumin (different products) ≥4.3 1.6 a product not licensed in USA b Pasteurization time 20 hours c studied in a porcine immunoglobulin intermediate to avoid neutralization by human IgG

Based on these experimental data on inactivation of B19V by pasteurization and a plasma pool for fractionation not exceeding 104 IU B19V DNA/ml, the virus safety of plasma-derived products regarding B19V can be assessed more correctly demonstrating an appropriate margin of safety.

Disclosures

Groener: CSL Behring: Employment. Nowak:CSL Behring: Employment. Schäfer:CSL Behring: Employment.
Viability and Engraftment of Hematopoietic Progenitor Cells (HPC) After Long Term Cryopreservation: Effect of Diagnosis and % Dimethylsulfoxide (DMSO) Concentration.
Authors: Veeraputhiran Muthu Kumaran , Theus John , Pesek Gina , Barlogie Bart , Cottler-Fox Michele H.
Published: October 2019

Abstract 3161

Poster Board III-98

Introduction

There are few reports of HPC viability after more than 9 y of storage. It has been suggested that HPC from myeloma patients (pts) may survive storage less well than those from other diseases. The appropriate concentration of DMSO for long term storage is unclear. Our program has collected and stored HPC since November 1989, changing from 5% DMSO to 10% DMSO in August 2000. We present a retrospective analysis of viability by diagnosis and DMSO concentration in pts who underwent autologous transplant using HPC after long-term storage (up to 17.8 y).

Materials and Methods

HPC were harvested and preserved using a controlled rate freezer and 5% or 10% DMSO, then stored in liquid nitrogen. Viability was tested by flow cytometry using unwashed samples and looking at propidium iodide or 7-AAD uptake within the CD34+ population. Data from 242 samples were analyzed (224 myeloma pts and 18 other diagnoses): 100 consecutively thawed samples with storage time <1 y (all 10% DMSO), 100 consecutive samples stored from 5-9 y ( 7 samples, 10% and 93 samples, 5% DMSO), and all samples stored and used for transplant after >9y (42 samples, 5% DMSO). No statistically significant difference was seen in viability between <1y and 5-9y, so these groups were combined and compared to >9y. White blood cell (WBC) and platelet engraftment were compared for the 2 groups using an unpaired t test.

Results

Mean viability for the 0-9y and >9 y groups were 91.62% and 92.79% respectively, with no statistically significant difference in viability between groups (p=0.28). Using a 2-tailed t test, no difference was seen in viability based on diagnosis (p=0.19) or between 5% and 10% DMSO (p= 0.12). No difference was seen in WBC engraftment (p=0.28) or platelet engraftment between groups (p=0.57).

Discussion

HPC collections remained viable after 17.8 years, the longest time reported for use after storage to date. No difference in viability was seen with 5% or 10% DMSO, or between myeloma and other diseases. Storage time did not affect engraftment.

Disclosures

No relevant conflicts of interest to declare.