bluuebunny/crossref_metadata_2025_split huggingface dataset:
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
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
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
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
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
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
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
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
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.