bluuebunny/crossref_metadata_2025 huggingface dataset:
A MiR-142-3p/EGR2 Feedback Circuitry In Human CSF-1 Driven Differentiation of Monocytes Into Macrophages
Authors: Lagrange Brice , Droin Nathalie , Aucagne Romain , Paggetti Jérôme , Largeot Anne , Solary Eric , Delva Laurent , Bastie Jean-Noel
Published: October 2019

Abstract 2366

Colony-stimulating factor-1 (CSF-1 or M-CSF) triggers the differentiation of human peripheral blood monocytes into macrophages through and integrated cytokine/transcription factors circuitry. Using microarray profiling to explore the role of microRNAs (miRNAs) in this molecular circuitry, we identified the down-regulation of miR-142-3p in human macrophages obtained from CSF-1-treated monocytes. We show that miR-142-3p is a repressor of the transcription factor EGR2 (Early Growth Response 2) through direct 3'UTR interactions. Interestingly, EGR2 binds the promoter of the pre-miR-142-3p gene to negatively regulate its expression, identifying a self-regulatory feedback loop. Enforced expression of miR-142-3p in primary human monocytes as well as decreased expression of miR-142-3p observed in monocytes from patients with a chronic myelomonocytic leukemia further assess the link between miR-142-3p and EGR2 expression in these cells. A chemical inhibition of the Src kinase family prevents the regulation loop induced by CSF-1. Thus, our study uncovers an EGR2/miR-142-3p circuitry which regulates CSF-1 driven differentiation of human monocytes into macrophages.

Disclosures:

No relevant conflicts of interest to declare.
HSP70 Inhibitor, YK5, Ablates Blast, Progenitor and Stem Cell Populations in Primary Acute Myelogenous Leukemia Cells
Authors: Rico Juan Felipe , Sharma Krishan K. , Kang Yanlong , Taldone Tony , Patel Hardik , Brennan Sarah , Zong Hongliang , Becker Michael W. , Roboz Gail J. , Chiosis Gabriela , Guzman Monica L.
Published: October 2019

Abstract 2493

Prognosis for acute myelogenous leukemia (AML) with traditional chemotherapeutic agents remains poor, most patients relapse and die of their disease. Studies have shown that AML can be originated and maintained by leukemia stem cells (LSCs) and that a high percentage of phenotypically-defined LSCs correlate to especially poor prognosis. Thus, novel therapies that can eliminate LSCs are needed. Upregulation of HSP70 has been described in human cancer cells and is often associated with chemoresistance and poor prognosis. Therefore, HSP70 represents a novel target for cancer therapy. Due to this, we tested a novel inhibitor of tumor-HSP70, YK5, and found that it can induce potent cell death in AML cells including progenitor and stem cell populations with minimal effects in normal hematopoietic cells.

Fifteen primary AML patient samples were treated with YK5 in varying concentrations for 48 hours to determine the LD50 of YK5. The viability of these samples was determined by multiparameter flow cytometry using cell surface markers to distinguish, lymphocytes, blasts, progenitor and stem cell populations in conjunction with Annexin V and 7AAD. Mean LD50 for all samples tested was 4.5μM (CI 95% 2.239–6.853). Three of these samples had very low LD50s below 2.5μM and only one sample demonstrated relative resistance with a LD50 that was greater than 15μM. Notably, progenitor and stem cell populations were also targeted while normal remaining lymphocytes were spared. Furthermore, we found that LSCs in a subset of patient samples treated with YK5 exhibited higher sensitivity to the compound when compared to blast cells. Importantly, treatment of primary AML samples with 5μM YK5 resulted in a 63% decrease in colony forming ability when compared to untreated control (N=5), suggesting that YK5 can impair malignant progenitors. In contrast, normal CD34+ cord blood cells after treatment with 5μM YK5 for 48hrs exhibited a viability of 57% (N=3). Xenotransplant assays are currently ongoing to determine the ability of YK5 to inhibit LSC function. Treatment with YK5 resulted in a decrease in phosphorylation of STAT5, determined by flow cytometry. Taken together, HSP70 inhibition is a promising potential treatment for AML and has the potential to eradicate leukemic stem and progenitor cells while sparing normal hematopoietic cells. Thus, targeting HSP70 in AML appears to hold great promise.

*JFR and KKS contributed equally to this project

Disclosures:

Roboz: EpiCept: Consultancy; ChemGenex: Consultancy; Celgene: Consultancy; Boehringer Ingelheim: Consultancy.
Comparison of Short-Term Outcomes Between CSTIBES02 and ALL Ph08 Trials for Young Patients with Philadephia Chromosome-Positive ALL (Ph+ALL) Differing In Imatinib Dose and Amount of Chemotherapy Before Stem Cell Transplantation
Authors: Ribera Josep-Maria , Garcia Olga , Montesinos Pau , Barrios Manuel , Oriol Albert , Brunet Salut , Gonzalez Jose , Esteve Jordi , Tormo Mar , Potro Eloy Del , Moreno Maria-José , Fernández-Abellán Pascual , Hernández-Rivas Jesú S. M.a , Barba Pere , Sarrá Josep , Abella Eugènia , Bernal Maria-Teresa , Llorente Andreu , Amigo Maria-Luz , Lavilla Esperanza , Bravo Pilar , Sanz Miguel-Angel , Feliu Evarist
Published: October 2019

Abstract 2582

Background and objectives.

Concurrent imatinib and chemotherapy followed by allogeneic stem cell transplantation (SCT) is the most used treatment for young patients with Ph+ALL. However, the amount of chemotherapy before SCT is not well established. The Spanish PETHEMA Group conducted two consecutive protocols (CSTIBES02 -Haematologica 2010; 95: 87–95- and ALL Ph08) differing in the imatinib dose (400 mg/d vs. 600 mg/d) and the amount of chemotherapy (two consolidation cycles vs. one consolidation cycle) before SCT. The short –term outcomes are herein compared.

Patients and methods.

CSTIBES02 trial included 30 patients with a median age of 44 yr and ALL Ph08 includes 26 patients (median age 37 yr). The main clinical and biological features of the two series of patients are comparable. The initial induction chemotherapy schedule included VCR, DNR and PDN at the same doses in the two protocols, but in the CSTIBES02 mitoxantrone and ARA-C were administered on day 15 if >5% blast cells were present in bone marrow smear, whereas no change in the chemotherapy schedule was prescribed in the ALL Ph08 trial according to the percentage of blast cells on day 15. The first consolidation cycle included HD MTX, ID ARA-C, MP and teniposide in both protocols. Patients in CR were then submitted to SCT in ALL Ph08 study, whereas those included in CSTIBES02 received a second consolidation cycle before SCT. Imatinib was administered concurrently with chemotherapy from diagnosis (400 mg/d in CSTIBES02 trial and 600 mg/d in ALL Ph08 study).

Results.

The main short-term outcomes are compared in the table

Conclusion.

Despite non-significant improvement has been observed in ALL Ph08 protocol regarding CR attainment, molecular response after induction and consolidation, 2-yr EFS has been improved after the increase of imatinib dose and the decrease of amount of chemotherapy. Both decrease in relapse before SCT and TRM observed in the ALL Ph08 protocol are responsible for the better EFS.

Supported in part by grants RD06/0020/1056 from RTICC and P-EF-11 from FJC

Disclosures:

No relevant conflicts of interest to declare.
Adolescents and Young Adults with Acute Lymphoblastic Leukemia Have Better Outcomes When Treated with Pediatric-Inspired Regimens - Systematic Review and Meta-Analysis of Comparative Trials
Authors: Ram Ron , Wolach Ofir , Vidal Liat , Gafter-Gvili Anat , Shpilberg Ofer , Raanani Pia
Published: October 2019

Abstract 2591

Background:

Survival of adults with acute lymphoblastic leukemia (ALL) is inferior to pediatric patients. Therefore, strategies to improve long-term results in this population are warranted. Objectives: This study aims to evaluate the efficacy and safety of pediatric-inspired regimens given to adolescents and young adults (AYA) with ALL. Methods: Systematic review and meta-analysis of comparative trials of AYA patients with ALL given induction chemotherapy with either pediatric-inspired regimens or conventional adult chemotherapy was conducted. Cochrane Library, MEDLINE and conference proceedings were searched (up-to May, 2011). Outcomes assessed were: all-cause mortality, number of patients achieving complete remission after induction chemotherapy and toxicity of the regimen (evaluated by non-relapse mortality). Relative risks (RR) with 95% confidence intervals (CIs) were estimated and pooled. Results: Our search yielded 11 trials, including 2489 patients. The age of AYA patients ranged between 10 and 57 years. Studies were published between the years 2003 and 2009. AYA patients given pediatric-inspired regimens had a statistically significant lower all cause mortality at 3 years (RR 0.58; 95% CI 0.51–0.79, 7 trials) beyond 3 years (RR 0.58; 95% CI 0.51–0.65, 8 trials) and at the end of study period (RR 0.59; 95% CI 0.52–0.66, 9 trials, Figure). Sensitivity analysis for all cause mortality including studies in which the two cohorts were well compared in either age or disease risk also showed decreased all cause mortality in patients given pediatric-inspired regimen (RR 0.53; 95% CI 0.41–0.70, I2=50%, 4 trials). However, this superiority in survival for pediatric-inspired regimens was not maintained when studies including only patients older than 20 years were analyzed (RR 0.49; 95% CI 0.23–1.06, I2=67%, 2 trials). Complete remission rate after induction chemotherapy was superior in patients given pediatric-inspired regimens when compared to patients given conventional adult chemotherapy (RR 1.05; 95% CI 1.01–1.10, I2=55%, 7 trials) with comparable non-relapse mortality in both groups (RR 0.53; 95% CI 0.19–1.48, I2=56%, 4 trials). Conclusions: For patients up to the age of 20 years, pediatric-inspired regimens are superior to conventional adult chemotherapy in terms of ACM and complete remission rate with similar non relapse mortality. Randomized controlled trials to further investigate the feasibility of this approach in adult ALL patients are warranted.

Disclosures:

No relevant conflicts of interest to declare.
Role of Connective Tissue Growth Factor (CTGF) in Survival and Chemosensitivity of Acute Lymphoblastic Leukemia
Authors: Lu Hongbo , Battula Venkata Lokesh , Shi Yuexi , Lock Richard B , Spong Suzanne , Andreeff Michael , Konopleva Marina
Published: October 2019

Abstract 2593

Connective tissue growth factor (CTGF/CCN2) is a member of the CCN family of proteins involved in extracellular matrix production, tumor cell proliferation, adhesion, migration, and metastasis. Recent studies have shown that CTGF expression is elevated in 75% of acute lymphoblastic leukemia (Br J Haematol, 2007; 138(6):740–8), and that increased expression of CTGF is associated with inferior outcome in B-ALL (Blood, 2007; 109(7):3080–3). In this study, we characterized the functional role and downstream signaling pathways of CTGF in ALL cells. First, we utilized lentiviral shRNA to knock-down CTGF in RS4;11 and REH ALL cells expressing high levels of CTGF mRNA (479.3±37.2 and 57.3±5.9 copies per 100 copies of ABL1, respectively). Silencing of CTGF (CTGF-knockdown, CTGF-kd) resulted in significant suppression of leukemia cell growth (57% in RS4;11 and by 70% in REH) compared to control vector. CTGF knockdown moderately reduced adhesion of RS4;11 to fibronectin (27%±0.1%). In the in vitro culture system, CTGF knockdown significantly enhanced growth inhibition and apoptosis induction after 48 hour exposure to chemotherapy agents (annexinV(+): Vincristine 25.8±3.5%, Vincristine/CTGF-kd 42.6±2.8%; Dexamethasone 66.3±1.8%, Dexamethasone/CTGF-kd 99.3±0.6%; Methotrexate, 17.4±0.6%, Methotrexate/CTGF-kd 39.5±3.9). Analysis of signaling pathways showed that CTGF down-regulation inhibits Src phosphorylation at Tyr416. Remarkably, phosphorylation of AKT at Ser473, and of mTOR downstream targets S6 Ribosomal Protein and 4E-BP1 were significantly inhibited in CTGF-knockdown RS4;11 cells, concomitantly with upregulation of expression of AKT targets Bim and p27. No changes in the levels of apoptotic regulators cIAP1 and Bcl-xL were found. This data suggest that CTGF regulates growth and chemosensitivity of ALL cells through Src and AKT/mTOR signaling. We previously reported that an anti-CTGF monoclonal antibody significantly extended median survival of mice implanted with xenografts derived from a primary CTGF expressing ALL sample in NOD/SCID mice. We are now investigating the effects of combining anti-CTGF treatment with cytotoxic chemotherapy in this model. Blocking CTGF signaling may represent a useful adjunct to cytotoxic therapies in acute lymphoblastic leukemia.

Disclosures:

Spong: Fibrogen: Employment, Equity Ownership.
CCR4 Expression in Hodgkin Lymphoma
Authors: Visser Lydia , van West Myrna , Rutgers Bea , Poppema Sibrand , Van Den Berg Anke
Published: October 2019

Abstract 2626

In Hodgkin lymphoma (HL) a minority (<1%) of tumor cells is found in a reactive background. The tumor cells shape their environment by the production of several chemokines and cytokines. Production of the chemokine TARC will lead to the attraction of Th2 and Treg cells specifically to involved lymph nodes. Although Treg and Th2 cells are prominent in the infiltrate the expression of its receptor CCR4 is not very high in lymphocytes surrounding the tumor cells. We hypothesized that this is due to internalization of the receptor. We analyzed in which specific compartment of lymphocytes CCR4 is expressed and if CCR4 expression can be recovered when TARC and MDC levels are low.

We analyzed 11 lymph node suspensions involved with HL and 11 with follicular hyperplasia (RLN) for expression of CD4, CD26, FoxP3 and CCR4 at t=0 and t=24 hours. We tested the effect of TARC and MDC on CCR4 expression and the mechanism of internalization on normal PBMCs.

At t=0 the CD4+ T cell compartment was significantly lower for CD26 in HL (49% vs 68% in RLN, p<0.01) (Table 1). In the comparison between CD26+ and CD26- cells no difference was seen in CCR4 expression in HL but in RLN CCR4 in CD26- cells was significantly lower than in CD26+ cells (p<0.01).Recovery of CCR4 (in average %) after 24 hours was significantly lower in CD26+ cells and FoxP3+ cells in RLN (p<0.01 and p<0.001). Recovery of CCR4 as factor change is significantly higher in HL than in RLN for CCR4 as well as the 3 subpopulations (Table 2). Generally, internalization of CCR4 on lymphocytes is higher with MDC (54 to 90% internalization with 10 and 500ng/ml MDC) than TARC (28 to 48% with 10 and 1000ng/ml TARC) after 2 hours. Internalization occurs through the lipid raft/caveolae dependent pathway as filipin III could inhibit internalization completely and not by clathrin-mediated endocytosis since no effect of inhibition by sucrose was seen. Recovery of CCR4 back on the membrane is fast and by exocytosis since it can be disrupted by exocytosis inhibitor Brefeldin A.Table 1:Percentages subpopulations CD4+ T cells in Hodgkin lymphoma and reactive lymph node by flowcytometry on cell suspensionsHLRLNpCD264968<0.01CCR44137nsFoxP34.41.8nsCCR4/CD26+1925nsCCR4/CD26-2212nsCCR4/FoxP3+5560nsTable 2:Factor of CCR4 recovery after 24 hours in CD4+ subpopulations in Hodgkin lymphoma and reactive lymph node cell suspensionsHLRLNpCCR41.70.9<0.05CCR4 in CD26+1.30.8<0.05CCR4 in CD26-2.91.1<0.05CCR4 in FoxP3+1.60.8<0.05

In conclusion, CD26 expression is significantly lower in HL than RLN. CCR4 expression is equally divided over CD26+ and CD26- cells in HL, but in RLN less is found in CD26-. Recovery of CCR4 expression after culture for 24 hours is lower in RLN than in HL. More CCR4 is recovered in HL especially in the CD26- CD4+ T cell population that is surrounding the tumor cells in HL.

Disclosures:

No relevant conflicts of interest to declare.
Re-Assessment of the Prognostic Value of International Prognostic Index (IPI) and Revised IPI (R-IPI) in Patients with Diffuse Large B-Cell Lymphoma Treated with or without Rituximab in Chinese Populations: A Retrospective Multicenter Study by Shanghai Lymphoma Research Group
Authors: Huang Honghui , Xiao Fei , Chen Fangyuan , Wang Ting , Li Junmin , Wang Jianmin , Cao Junning , Wang Chun , Zou Shanhua
Published: October 2019

Abstract 2649

Background:

The International Prognostic Index (IPI) is a widely accepted prognostic factor system for diffuse large B cell lymphoma (DLBCL) patients treated with chemotherapy. However, the prognostic value of IPI has been a focal point of the debate in the era of immuno-chemotherapy. Recently, the study of British Columbia group suggested that a revised IPI (R-IPI) which redistributed the IPI factors into 3 distinct prognostic groups provided a more clinically useful prediction of outcome for patients with DLBCL. In order to reassess the value of IPI and R-IPI in unselected Chinese population, we conducted this study.

Methods:

A multicenter retrospective analysis of DLBCL patients treated with CHOP-like chemotherapy alone or plus rituximab was performed by Shanghai Lymphoma Research Group. In total, 438 patients of newly diagnosed DLBCL treated at 6 participated hospitals were included during the period of 1997–2008. The prognostic value of IPI and R-IPI at diagnosis with regards to overall survival (OS) and progression-free survival (PFS) was evaluated.

Results:

The median age at diagnosis was 50 years (range, 18–83 years), and the median follow-up was 34 months (range, 3–145 months). Among them, 241 patients received CHOP-like regimen, whereas 197 had rituximab (R)-CHOP-like regimen. While IPI remained predictive in CHOP-like group, it could not distinguish between each prognostic category in the R-CHOP-like group (Fig.1). Redistribution of the IPI factors into a R-IPI identified three distinct prognostic groups with significantly different outcomes both in the patients treated with and without rituximab. In R-CHOP-like arm, these three risk groups had distinctly different rates of 3-year progression-free survival rates of 96%, 84.3% and 67.5% (P<0.001), respectively, and 3-year overall survival rates of 96%, 87.6% and 71.1% (P<0.001), respectively (Fig.2).

Conclusions:

Our study underscores the power of R-IPI as a simplified and more clinically relevant predictor of the disease outcomes than the standard IPI in Chinese DLBCL populations in the rituximab era, and it deserves a further study in larger population-based prospective study.

Disclosures:

No relevant conflicts of interest to declare.
Central Nervous System (CNS) Relapse In a Population of 143 Patients (Pts) with Mantle Cell Lymphoma (MCL) From Two Centers
Authors: Conconi Annarita , Franceschetti Silvia , Lobetti-Bodoni Chiara , Stathis Anastasios , Bruna Riccardo , Riccomagno Paola , Bertoni Francesco , Ghielmini Michele , Cavalli Franco , Gaidano Gianluca , Zucca Emanuele
Published: October 2019

Abstract 2660

Inconsistent information concerning the pattern of CNS relapse have been reported in MCL pts. We retrospectively analyzed the clinical variables at diagnosis and outcome, with special reference to CNS relapse, in a population of consecutive pts with confirmed diagnosis of MCL from two hematology centers.

Among 2426 non Hodgkin's lymphoma pts treated from 1979 to 2011, 142 cases (44 female, 98 male) of MCL were selected. Median age at diagnosis was 68 years (17–94 years); 116 pts (82%) had stage III-IV, 89 (67% of the 132 cases in whom the data was available) had intermediate-high/high International Prognostic Index (IPI) risk. Extranodal disease was reported in 127 pts (89%), serum LDH was elevated in 45 pts (40% of 113 tested pts). Information concerning first line treatment was available in 139 pts. Fourteen pts (10%) did not receive active treatment at diagnosis, in 7 (5%) of these, systemic treatment followed the initial expectant strategy. Four pts (3%) only received radiotherapy and/or surgery. One hundred twenty two pts (88%) were treated with chemotherapy, 46 pts (33%) had rituximab, alone (6 pts, 4%) or in combination with chemotherapy. Eighteen pts (13%) received chemotherapy regimens including drugs crossing the blood-brain barrier or prophylactic intrathecal chemotherapy, 10 pts (7%) had autologous stem cell transplantation.

After median follow-up of 8 years, CNS relapse was observed in 11 cases (7.7%; 95%CI:4–13%). CNS disease occurred at a median of 13.8 months (range:3.7–95 months) from diagnosis. Cumulative risk of CNS relapse raises until 10 years, being 7.2% (95%CI:4–14%) at 3 years, 10.6% (95%CI:6–19%) at 5 years and 13.6% (95%CI:7–25%) at 10 years. Elevated serum LDH at diagnosis was significantly associated with higher risk of CNS relapse at univariate analysis (P=0.006). Actuarial risk of CNS relapse was significantly shorter in pts with higher risk according to IPI (P=0.018). Median survival after CNS relapse was 6.3 months (range: 1.5–77 months). CNS relapse had a dismal impact on survival (P=0.04). No specific treatment approach at diagnosis, including autologous stem cell transplantation, intrathecal chemotherapy, high dose cytarabine or rituximab, alone or in combination with chemotherapy, significantly reduced the risk of CNS relapse.

CNS relapse is one of the most challenging events in the management of MCL. Our data confirmed the adverse clinical outcome of MCL after CNS relapse. Better definition of clinico-pathological profile at diagnosis suggesting higher risk of CNS relapse could select pts candidate to prophylactic approach addressed to prevent CNS disease.

Disclosures:

No relevant conflicts of interest to declare.
Mean Platelet Volume and Integrin Alleles Strongly Correlate with Increased Levels of Integrins αIIbβ3 and α2β1: Clinical Implications for Platelet Reactivity in Acute Coronary Syndome
Authors: Kunicki Thomas J , Williams Shirley A , Nugent Diane J. , Yeager Mark J
Published: October 2019

Abstract 32

Background

- Differences in the level of integrins expressed on the surface membranes of platelets can have a significant effect on platelet reactivity and adverse outcomes in a variety of thrombotic and hematologic dyscrasias. Among normal individuals, the basal level of platelet integrin aIIbb3 varies at least two-fold, while that of the integrin a2b1 exhibits a greater range of variation. The total number of integrin molecules and other receptors on platelets is influenced to a large extent by the total surface area of plasma membrane and consequently the mean platelet volume (MPV), which itself has a very strong genetic component. We sought to determine the relative contribution of mean platelet volume (MPV) and integrin gene alleles to expression levels of these integrins in patients with acute coronary syndrome (ACS) and normal subjects.

Methods and Results

- Monoclonal antibodies AP2 and 8C12 (or 12F1) were used in flow cytometry to measure platelet aIIbb3 and a2b1, respectively, in whole blood from 341 patients with ACS and 128 normal subjects. Comparisons were made with MPV, platelet count (PC) and in the normal subjects ITGA2 rs1126643 and ITGB3 rs5918 alleles. In both ACS patients and controls, there was a strong direct correlation between MPV and aIIbb3 level (p < 0.001). In control subjects, MPV and aIIbb3 level did not correlate with ITGB3 rs5918 alleles. For a2b1, MPV contributed modestly in either patients or controls, while ITGA2 rs1126643 alleles exerted the greatest effect. Moreover, we observed an inverse correlation between MPV and the rs1126643 minor allele.

Conclusions

- Our results indicate that MPV is the major effector of platelet membrane levels of aIIbb3, in both ACS patients and normal subjects, while ITGA2 rs1126643 contributes most strongly to a2b1 levels. Hyperactivity of larger platelets results from increased levels of key platelet receptors, notably integrin aIIbb3, in support of the contention that MPV is an accurate marker of risk for adverse outcome in ACS. This is consistent with published findings that MPV, the most common metric of platelet size, correlates very well with platelet reactivity, such that larger platelets have greater prothombotic potential, and elevated MPV is associated with increased platelet aggregation, thromboxane synthesis, b-thromboglobulin release, and expression of adhesion molecules. The most intriguing finding of our study is the inverse association of the ITGA2 rs1126643 minor allele T with MPV. The rs1126643 minor allele likely exerts this effect via the documented influence of a2b1 on megakaryocyte (MK) maturation.

Disclosures:

No relevant conflicts of interest to declare.
Immature T-Cell Populations in Lymph Nodes of Castleman Disease and Angioimmunoblastic T-Cell Lymphoma Suggest Alternate Sites of T-Cell Development,
Authors: Ohgami Robert S. , Chun Shuchun , Ohgami Jane , Zehnder James L. , Van de Rijn Matthew , Arber Daniel A. , Natkunam Yasodha , Warnke Roger
Published: October 2019

Abstract 3238

A central dogma of human immunology is that proliferation of immature T-cells, and their development after release from the bone marrow, occurs in the central thymus. Recently, we identified several patients with aberrant polyclonal immature TdT+ precursor T-cell populations in extra-thymic lymphoid tissues. Here we demonstrate that immature precursor T-cell populations, with a cortical thymocyte phenotype, in fact, are expanded in extra-thymic lymphoid tissues of patients with Castleman disease (P < 0.001; n = 29), and angioimmunoblastic T-cell lymphoma (P = < 0.001; n =31) and increased in cases of Castleman disease in association with follicular dendritic cell sarcoma (Figures 1 and 2). Analysis of the proliferation marker, MiB-1, and the morphologic presence of mitoses reveal that these populations are undergoing extra-thymic proliferation and expansion, arguing against simple release from the central thymus and sequestration in these extra-thymic organs. Finally, these populations of immature T-cells are not associated with a particular anatomic site (i.e. neck or mediastinum). These findings challenge the dogma that proliferation of immature human T-cell populations occurs nearly exclusively in the central thymus and demonstrates that stimulation and significant proliferation of extra-thymic immature T-cells does, and can occur in a subset of patients.

Disclosures:

No relevant conflicts of interest to declare.
An Important Role for Rac1 in Glycoprotein Ib-IX Induced Platelet Activation,
Authors: Delaney Michael Keegan
Published: October 2019

Abstract 3250

The platelet receptor for von Willebrand factor, the glycoprotein Ib-IX (GPIb-IX) complex, not only plays a critical role in mediating platelet adhesion at sites of vascular injury, but also transmits signals leading to platelet activation. GPIb-IX mediated activation of the ligand binding function of integrin αIIbβ3 is required for stable platelet adhesion under high shear rate conditions. Previous studies have suggested that ligand occupancy of GPIb-IX leads to activation of the Src family kinase Lyn, phosphoinositide 3-kinase (PI3K), Akt, cGMP-dependent protein kinase, and the MAPK and ITAM signaling pathways. However, it remains unclear how the PI3K/Akt pathway is activated during GPIb-IX dependent signal transduction. Using platelet-specific conditional Rac1−/− mice and the Rac1 specific inhibitor NSC23766 (NSC), we examined the role of Rac1 in GPIb-IX induced platelet activation. Platelet-specific conditional Rac1−/− mice were generated by crossing Pf4-Cre transgenic mice with mice containing the Rac1 conditional allele. Rac1−/− mouse platelets had a defect in VWF/botrocetin induced aggregation, TXA2 synthesis, and ATP secretion. Rac1−/− mouse platelets were also defective in stable adhesion to VWF under shear stress and in spreading on immobilized VWF, as compared to WT. The defects observed in Rac1−/− mouse platelets were recapitulated in human platelets treated with the Rac1 inhibitor NSC23766. Importantly, we show that ligand binding to GPIb-IX induced Rac1 activation, which was abolished in Lyn−/− mouse platelets. We also show that Rac1−/− (or NSC-treated) platelets have a defect in GPIb-IX induced Akt and P38 MAPK activation. Altogether, this study reveals that Rac1 plays an important role in GPIb-IX dependent platelet activation and stable adhesion to VWF under shear stress. Rac1 is activated downstream of Lyn and is important for GPIb-IX and Lyn-dependent activation of Akt and MAPK signaling pathways.

Disclosures:

No relevant conflicts of interest to declare.
Direct Association of PAR4 with P2Y12 Is Required for PAR4-Induced Recruitment of Arrestin-2: Structural and Functional Analysis of a GPCR Heterodimer,
Authors: Khan Aasma , Li Dongjun , Ibrahim Salam , Smyth Emer , Woulfe Donna
Published: October 2019

Abstract 3253

We have previously shown that the PAR4 thrombin receptor and the P2Y12 receptor for ADP co-immunoprecipitate from thrombin-stimulated platelets, but not unstimulated platelets. Here we show that PAR4 and P2Y12 directly interact when expressed in HEK293T cells using Bioluminescence Resonance Energy Transfer (BRET) studies, identify a region in the fourth transmembrane (TM4) domain of PAR4 required for its interaction with P2Y12 and, using a mutant variant of PAR4 in the TM4 domain, demonstrate that PAR4 interaction with P2Y12 is required for arrestin recruitment to PAR4. Luciferase-tagged P2Y12 and YFP-tagged PAR4 were expressed in HEK293T cells and demonstrate specific, saturable interaction by BRET analysis of dimer-dependent light emission at 530 nM. PAR4 and P2Y12 can be co-immunoprecipitated from PAR4-stimulated platelets or HEK293T cells stably expressing both receptors, but the receptors fail to co-precipitate from platelets or HEK293T cells pre-treated with apyrase or P2Y12 antagonist. The PAR1 thrombin receptor and the DP1 prostanoid receptor fail to interact with P2Y12 when evaluated by saturation BRET in HEK293T cells expressing the receptors or by co-immunoprecipitation studies in human platelets. Untagged PAR4 was able to compete with YFP-tagged PAR4 for interaction with luciferase-tagged P2Y12, whereas PAR1 was not. Based upon sequence comparison of PAR1 and PAR4 in the transmembrane domains, the most frequently identified sites of G protein-coupled receptor (GPCR) associations, we have identified 3 amino acids at the base TM4 of PAR4 (LGL194-196) that differ significantly from the SFT(220–222) expressed at the aligned sequence of TM4 in PAR1. Mutation of residues LGL194-196 of PAR4 to SFT results in a mutant variant of PAR4 (PAR4-TM4m) that, when expressed in HEK293T cells, supports only 8.8% of the interaction with P2Y12 compared to the native PAR4 according to saturation BRET (average of n=3). However, homo-dimerization of PAR4 does not require this determinant, as luciferase-tagged PAR4-TM4m mutant supports saturation BRET with YFP-tagged PAR4. The PAR4-TM4m variant expressed in HEK293T cells also fails to co-immunoprecipitate with P2Y12 and with arrestin-2, whereas non-mutant PAR4 supports both of these interactions. This is the first demonstration that direct heterodimeric interaction of two GPCRs expressed in platelets is required to recruit the molecular scaffold arrestin-2. Based upon our previous results demonstrating that arrestin-2 is required to recruit PI3K to PAR4 and for sustained, maximal activation of Akt, we hypothesize that the co-recruitment of P2Y12 and arrestin-2 to activated PAR4 in platelets is required for sustaining PI3K-dependent responses to both Rap1 and Akt activation with potential consequences for maintaining platelet plug stability.

Disclosures:

No relevant conflicts of interest to declare.
Tissue Factor Is Internalized by Platelets Through Activation of RhoA and PI3-Kinase Promoting Reversible Cytoskeletal Assembly,
Authors: Escolar Gines , Lopez-Vilchez Irene , Galan Ana M , Roque Merce , White James G , Diaz-Ricart Maribel
Published: October 2019

Abstract 3264

TF associated with platelets may play a critical role in the development of ischemic complications at sites of vascular damage. Platelets can internalize and store tissue factor-rich microvesicles from human origin (hTF), a process that can be potentiated by serotonin (5-HT) (Galan et al, Thromb Haemost. 102:511, 2009). There is limited knowledge on the mechanisms involved in the uptake, redistribution and functional expression of TF by platelets. We have investigated the mechanisms implied in the uptake and traffic of hTF with especial attention to CD36 and the 5HT transporter receptor (SERT), but also to the involvement of PI3-kinase, small GTPases (RhoA) and modifications of cytoskeletal organization during vesicular trafficking. Isolated platelets were exposed to hTF for up to 10 min. Cytoskeletal assembly and activation of RhoA were analyzed by electrophoresis and ELISA, respectively. Aggregometry and ultrastructural microscopy were used to assess platelet activation and hTF uptake. Protein arrays were used to evaluate changes in signaling molecules induced by hTF. In addition, inhibitory strategies to block GPIIbIIIa (Reopro), the scavenger receptor GPIV (anti-CD36), the SERT (S-Citalopram, SCit), and PI3-kinase (Wortmannin, Wo) were applied to platelets prior exposure to hTF. Internalization of hTF resulted in reversible actin polymerization and association of contractile proteins (alpha-actinin, actin binding protein and myosin) to the cytoskeleton, being maximal at 1 min. Maximal activation of RhoA occurred at 1 min, decreasing gradually afterwards, while activation of PI3-kinase occurred later (at 5 min). Screening of signaling proteins revealed activation of Akt, ERK1/2, and SNAP-25, among others. All inhibitory strategies fully prevented platelet aggregation to hTF. Uptake of hTF was slightly prevented by Reopro, while both anti-CD36 and Wo partially inhibited hTF uptake and, interestingly, SCit was the most efficient. We conclude that uptake of hTF by platelets promotes activation of specific signaling molecules with reversible cytoskeletal assembly and membrane fusion. While it may be difficult to design specific strategies to prevent the activation of small GTPases, cytoskeletal arrangements or membrane fusion specifically for platelets, our results indicate that the CD36 scavenger receptor and the 5-HT transporter (SERT) may become realistic therapeutic targets to prevent or reduce the association of TF to platelets and hence its possible prothrombotic action.

Grants: SAF2009-10365, Red HERACLES RD06/0009/1003

Disclosures:

No relevant conflicts of interest to declare.
Activated Protein C Inhibits High Mobility Group Box 1 Signaling in Endothelial Cells,
Authors: Bae Jong-Sup , Rezaie Alireza R.
Published: October 2019

Abstract 3274

A pathogenic role for high mobility group box 1 (HMGB1) protein has been postulated in severe sepsis. Activated protein C (APC) is the only FDA-approved drug for severe sepsis, however, its effect on HMGB1 signaling has never been investigated. Here, we monitored the effect of APC on the LPS-mediated release of HMGB1 and the HMGB1-mediated modulation of pro-inflammatory signaling responses in human umbilical vein endothelial cells. APC potently inhibited the LPS-mediated release of HMGB1 and down-regulated the cell surface expression of VCAM-1, ICAM-1 and E-selectin as well as the adhesion of the monocytic cell line, THP-1, to HMGB1-activated endothelial cells. HMGB1 up-regulated pro-inflammatory responses by interacting with three pathogen-related pattern recognition receptors: toll-like receptors 2, 4 and the receptor for advanced glycation end products. APC not only inhibited the LPS-mediated HMGB1 release, but also down-regulated the cell surface expression of all three HMGB1 receptors on endothelial cells. The protective effects of APC were mediated through both EPCR and PAR-1, as evidenced by the function-blocking antibodies to either receptor abrogating the signaling function of APC. Interestingly, a thrombin derivative containing the Gla-domain of APC recapitulated all protective effects of APC with a 20–50-fold higher efficacy. These results suggest that the EPCR- and PAR-1-dependent protective effects of APC in severe sepsis may partially be mediated through the inhibition of HMGB1 signaling and that the chimeric thrombin mutant has potential therapeutic utility for severe sepsis.

Disclosures:

No relevant conflicts of interest to declare.
Predictors of Acquired Von Willebrand Syndrome in Patients with Aortic Stenosis,
Authors: Chen Dong , Thomas Colleen S , Blackshear Joseph L
Published: October 2019

Abstract 3318

Background:

Acquired von Willebrand syndrome (AVWS) due to loss of high molecular weight multimers (HMWM), is a frequent cause of bleeding in aortic stenosis (AS), and is reversed by valve replacement. The goal of this study was to explore predictors of AVWS in patients with AS.

Methods:

A total of 91 patients (59 men, median age 79 years) with AS (n=64) or heart valve replacement (n=27) were included. We recorded peripheral blood count, vital signs, mean gradient (MG), peak velocity (peak vel), aortic valve time velocity integral (AV-TVI), aortic valve area (AVA) and valve area index (AVAi), time velocity integral ratio (TVI ratio), VWF antigen (VWF:Ag), VWF activity by latex immunoturbidity assay (VWF:Lx), VWF multimer analysis, closure times of platelet function analyzer ADP and epinephrine cartridges (PFA-CADP and –CEPI). HMWM losses were graded as normal, mild or severe when compared to HMWM analyses of pooled plasma samples from normal donors. Potential predictors of HMWM loss were explored using logistic regression analysis.

Results:

Of the 91 patients, 42 (46%) had loss of HMWM (23 mild and 19 severe). In single variable logistic regression analysis, AVWS was associated with a low AVA (<1.0 cm2; OR=5.08, P<0.001), and low AVAi (<0.60 cm2/m2; OR=5.54, P<0.001), low TVI ratio (<0.25; OR=10.80, P<0.001), prolonged PFA-CADP (≥121 sec; OR=6.76, P<0.001), and low VWF:Lx/Ag (≤0.8; OR=8.06, P=0.008). Moderate peak vel (3–4 m/sec) and high peak vel (>4 m/sec) were associated with loss of HMWM (OR[vs. <3 m/sec]=5.80 and 37.70 respectively, P<0.001) as were moderate MG (25–40 mmHg) and high MG (>40 mmHg), (OR [vs. <25mmHg]=4.50 and 32.50 respectively, P<0.001). In exploratory multivariable analysis, mean gradient remained one of the strongest predictor of loss of HMWM. The estimated sensitivity of MG (≥25mmHg) in diagnosing AVWS was 83% (35/42, 95% CI: 69%-93%) and estimated specificity was 71% (35/49, 95% CI: 57%-83%). Considering the very strong correlation with mean gradient, we also considered the diagnostic utility of peak vel. Peak vel≥ 3 m/sec had a sensitivity of 89% (38/42, 95% CI: 77%-97%) and a specificity of 59% (29/49, 95% CI: 44%-73%) in detecting loss of HMWM.

Conclusion:

Our data suggest that mean gradient (>25 mmHg) and peak vel (≥ 3 m/sec), both derived from the Doppler aortic valve velocity envelope, are accurate predictors for AS-AVWS, and should be incorporated into algorithmic approaches to laboratory screening for AVWS.

Disclosures:

No relevant conflicts of interest to declare.
Rofecoxib Does Not Appear to Increase the Risk of Venous Thromboembolism: A Systematic Review of the Literature,
Authors: Goy Jennifer K , Paikin Jeremy , Crowther Mark A.
Published: October 2019

Abstract 3334

Rofecoxib, a selective cyclo-oxygenase-2 (COX-2) inhibitor, has been associated with increased arterial thrombosis. It is unknown whether it is associated with venous thromboembolism (VTE). We investigated, using a systematic review of the literature, the association between rofecoxib and venous thrombosis. A search strategy was developed with the assistance of a research librarian and was implemented in 2 electronic databases. We identified all English language studies in which rofecoxib was compared with placebo, irrespective of the primary outcome of the study. Only placebo controlled trials were included in the review as NSAIDs (the comparator in many rofecoxib studies) may have anti-thrombotic effects. We also limited studies to those with 3 or months of follow-up since we presume that the effects of rofecoxib on the coagulation system would take time to develop. Two reviewers assessed study eligibility, with disagreements resolved by consensus. Data was extracted from each of study in a standardized manner using RefMan software. Quality of the studies was assessed according to pre-established criteria: (1) randomization process (2) blinding, and (3) documentation of losses to follow up. Pooled incidence rates were calculated from the reported number of VTEs and total person years at risk. Confidence intervals and risk difference were calculated using a Poisson distribution. The search strategy identified 1339 papers. After review, a total of 15 studies in 14 populations met inclusion criteria. The majority of trials were short in duration (∼12 weeks). All studies met at least two of the three quality criteria. In 15160 (9217 person years follow up) patients allocated to rofecoxib there were 8 VTEs reported, compared with 9 VTEs in 13147 (9092 person years) patients allocated to placebo (relative risk 0.87, 95% CI 0.29–2.56, p = NS). The estimated incidence of VTE was 86.8 per 100,000 (95% CI 37.5 –171.2) person years with rofecoxib, and 99.1 per 100,000 person years with placebo (95%CI 45.3 – 188). This difference is statistically insignificant (p=0.78). Our findings are limited by the relatively small number of events, although the contributing sample size 28307 subjects (18309 person years) is reasonable. We are also unsure that all venous events were captured, although the fact that many of the contributing studies were performed in support of regulatory filings of rofecoxib suggests that events were unlikely to be missed. All included trials had processes for adjudication of VTE. Keeping these limitations in mind, our findings do suggest that there is no increase in the risk of VTE with rofecoxib use.

Disclosures:

Crowther: Various: Consultancy.
Cerebral Artery Velocity in Patients with β-Thalassemia Intermedia Compared to β-Thalassemia Major Using Transcranial Doppler Ultrasonography,
Authors: Karimi Mehran , Haghpanah Sezaneh , Shariat Abdolhamid , Ashjazadeh Nahid , Nazeri Masoume , Emami Sajad , Foroughi Amin Abolhasani , Petramfar Peyman , Yaghoubi Ehsan , Lak Manijeh
Published: October 2019

Abstract 3335

Introduction:

Patients with β-thalassemia, especially β-thalassemia intermedia (β-TI), are prone to hypercoagulable state and subsequently cerebral thromboembolic events. In this study we aimed to determine cerebral artery velocity as a determinant of stroke risk in patients with β-TI compared to β-thalassemia major (β-TM) and healthy subjects. Method: Sixty patients with β-TM and 64 β-TI were randomly selected in this cross sectional study. Sixty healthy adults comprised as a control group. The Time average Maximum Mean Velocity (TAMMV) was measured in large cerebral arteries using Transcranial Doppler (TCD) Ultrasonography. The TAMMVs in different arteries were compared between the patients with β-TI, β-TM and controls. Results: TAMMV in all arteries was significantly higher in β-TI patients than β-TM patients and controls (P< 0.001). Also β-TM patients had significantly higher TAMMVs than controls in all arteries except for the left and right posterior cerebral arteries (P< 0.001). In univariate analysis we found that the lack of transfusion, thrombocytosis, splenectomy, higher platelet counts and lower age could contribute to increase TAMMV (P< 0.05). However, in the multiple linear regression models the only important factors influencing TAMMV were the lack of transfusion in all evaluated arteries following with age only in some investigated arteries. Conclusion: We showed that patients with β-TI has a higher TAMMV in comparison with the healthy subjects as well as with the β-TM patients probably due to the lack of transfusion and chronic anemia. In addition, β-TI patients are more prone to thromboembolic events and subsequently cerebral infarction due to hypercoagulable state. We recommend MRI screening in β-TI patients for detection of silent lesions, in the presence of asymptomatic lesions, TCD would be beneficial. In patients with high cerebral artery velocity and silent lesion further evaluation, close observation and prophylactic treatment including regular blood transfusion and antiplatelet therapy should be considered. However further prospective studies are suggested to confirm the correlation between the stroke risk and high cerebral velocity as well as the protective effect of regular transfusion in β-TI patients.

Disclosures:

No relevant conflicts of interest to declare.
Progestin-Only Contraceptives and the Risk of Venous Thromboembolism: Systematic Review and Meta-Analysis,
Authors: Mantha Simon , Raghavan Vidya , Karp Rebecca , Terrin Norma , Bauer Kenneth A. , Zwicker Jeffrey
Published: October 2019

Abstract 3344

Background:

Combined oral contraceptives (COC) containing estrogens and progestins are associated with a 2 to 4-fold increased risk of venous thromboembolic events. Progestin-only contraceptives are commonly prescribed to women with a higher risk for thrombosis, although a lower thrombotic risk than COC has not been clearly established. We performed a systematic review and meta-analysis of published studies to estimate the risk ratio of venous thromboembolism (VTE) in women taking progesterone for the purpose of contraception.

Materials and methods:

We performed a literature search of cohort, case-control and randomized studies including women treated with progestin-only contraception compared with a no-hormone control group. All administration routes (oral, injectable and intra-uterine) were considered. The primary endpoint was the corrected risk ratio of VTE analyzed using a random effects model.

Results:

1150 references were retrieved; 7 studies were retained for analysis. The summary estimate of the adjusted risk ratio of VTE for women taking a progestin for contraception versus no hormone was 1.02 (95% CI=0.76–1.37, p=0.89). Heterogeneity between studies was minimal (Q-value=7.1, I2=15.49 and p=0.31). An analysis combining the unadjusted odds ratios of VTE gave a similar result (OR=1.24, 95% CI=0.93–1.66, p=0.14). Analysis by subgroup suggests a higher risk of thrombotic events for individuals using an injectable progestin, with an estimated risk ratio of VTE equal to 2.67 (95% CI=1.29–5.53, p=0.008), compared to 0.87 (95% CI=0.53–1.42, p=0.58) for oral administration.

Conclusions:

Progestin-only oral formulations used for contraception do not appear to increase the risk of venous thromboembolic disease, although injectable administration was associated with an increased incidence of VTE relative to women not taking hormones that is comparable to the risk associated with COC.

Estimated risk ratio of VTE for progestin versus no hormone, using adjusted values; A, all routes of administration for all studies; B, injectable progestins only, for studies where those results were reported.

This publication was supported by Grant Number UL1 RR025752 from the National Center for Research Resources. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NCRR.

Disclosures:

No relevant conflicts of interest to declare.