bluuebunny/crossref_metadata_2025 huggingface dataset:
Methylcellulose as a Wetting Agent in Blood Clot: In Vivo Studies
Authors: ROSENFELD MORRIS
Published: October 2019

Methylcellulose, injected intravenously in dogs, altered the surface properties of blood plasma, as measured both in the fluid and clotted state. In fluid plasma, it altered the pattern of the surface tension-time curve, instituting the following changes: a reduction in the dynamic value of surface tension, a progressive rise in value contrasted to the normal decline, an elevation of the final static level above the pre-injection value. In clotted plasma, methylcellulose converted the normal water repellent surface to one that was water attracting, or hydrophilic. There was no change of coagulation time in glass, but possibly some prolongation in siliconed tubes. No surface tension or hydrophilic changes were induced by the other colloids tested; namely, dextran, polyvinylpyrrolidone and carboxymethylcellulose. The physical changes induced by methylcellulose are of a type that might favorably influence clot structure and adhesion.
A Consideration of Myeloid-Erythroid Balance in Man
Authors: PATT HARVEY M.
Published: October 2019

The number of myeloid and erythroid cells in marrow, their turnover times and rates of production have been estimated from consideration of mitotic activities and related data. An approximation to the chronology of development in the steady state with constant population is presented and its implications are discussed. Estimates of myeloid and erythroid balance in man are compared with similarly derived values for the dog and rat.
Elucidation of B Cell Antigen Receptor Signaling by Global Phosphoproteomics Reveals Novel Signaling Pathways and Regulatory Mechanisms
Authors: Oellerich Thomas , Corso Jasmin , Engelke Michael , Stanze Daniel , Hsiao He-Hsuan , Wienands Juergen , Urlaub Henning , Serve Hubert
Published: October 2019

Abstract 1056

B cell antigen receptors (BCR) transmit information via posttranslational protein modifications, most importantly protein phosphorylation. BCR signaling is a complex process that regulates differentiation and activation responses of the B-lineage. Dysregulation of BCR signaling can induce malignant transformation that can result in the onset of leukemia or lymphoma. To date our understanding of normal- versus malignant B cell biology points to differences in properties of BCR signaling that may be amenable to selective therapeutic targeting. However, comprehensive knowledge about BCR signaling pathways is still lacking which makes it difficult to understand the molecular events that drive B cell function and malignant transformation. To globally characterize BCR signaling, we have applied quantitative phosphoproteomic techniques that allowed us to identify and quantify about 10000 phosphorylation sites as a function of BCR stimulus, time, and subcellular location. Our phosphoproteome approach uncovered many novel BCR signaling effectors and their site-specific phosphorylation dynamics. The majority of phosphorylation events occured on serine/threonine residues, while only about one percent was detected at tyrosine residues. Interestingly, most of the identified proteins contain multiple phosphorylation sites showing different kinetics upon BCR stimulation, suggesting that they serve as signal integrators. Moreover, our functional analysis revealed that certain phosphorylation sites in adapter proteins are not only relevant for regulating the onset but also the termination of BCR signaling. Such signal termination is at least partly achieved by coupling the phosphotyrosine-dependent formation of BCR signalosomes to the kinetically flexible cytosolic Ca2+ concentration. In summary, our analysis revealed the complex composition of BCR signaling pathways and their regulation, and hence provides a basis for the identification of potential BCR-associated drug targets in the future.

Disclosures:

No relevant conflicts of interest to declare.
Molecular Basis of Phospholipid Synergy in Promoting Blood Coagulation Reactions
Authors: Davis-Harrison Rebecca L , Clay Mary , Rienstra Chad M , Morrissey James H.
Published: October 2019

Abstract 1109

Most steps in the blood coagulation cascade obligatorily take place on membrane surfaces and are dependent on the exposure of phosphatidylserine (PS). Many coagulation proteins bind to PS-containing membrane bilayers in a calcium-dependent manner via gamma-carboxyglutamate-rich (GLA) domains. Furthermore, certain phospholipids, most notably phosphatidylethanolamine (PE), strongly synergize with PS to promote clotting reactions. We have recently been investigating the molecular basis of lipid-Ca2+ and lipid-GLA interactions in blood clotting using solid-state NMR (SSNMR) studies that employ isotopically labeled PS. Our studies have revealed that Ca2+ induces two predominant conformations of the PS headgroup (Boettcher et al., Biochem. 50:2264–73, 2011). We now report the quantification of the intermolecular and intramolecular atomic distances within Ca2+-induced PS nanoclusters using SSNMR. We have also expanded SSNMR studies to phospholipid bilayers composed of trinary mixtures of PS, PC, and PE as a means to further investigate the impact of PE on PS headgroup conformations in an intact lipid bilayer (PE-PS synergy). Initial results suggest that incorporation of PE into the bilayer alters the Ca2+: PS ratio required to fully saturate the L-serine headgroups of PS. Additionally, PE appears to affect the predominant PS headgroup conformations in the presence of Ca2+, and may induce a third L-serine conformation. SSNMR studies utilizing isotopically labeled PE headgroups are currently underway which should provide novel information concerning PE's interactions with PS, Ca2+, and/or GLA domains.

Previous SSNMR studies from our lab have also indicated that a fraction of L-serine headgroups in PS adopt a new conformation when bovine prothrombin fragment 1 is bound to PS/PC bilayers (Tavoosi et al., J. Biol. Chem. 286:23247–53, 2011). These data support the idea that GLA domains contain an L-serine-specific binding site. We have now bound human prothrombin fragment 1 to membranes containing labeled PS headgroups for use in SSNMR studies. In addition, we have also employed surface plasmon resonance (SPR) experiments to investigate the interactions of both human and bovine prothrombin with bilayers containing PS and PE. These data demonstrate that binding affinities and binding footprints are similar for both prothrombin proteins, and that PE enhances binding affinities and increases the number of GLA binding sites for both proteins. Together, results from SSNMR and SPR studies suggest that different GLA domains use similar molecular mechanisms to interact with the membrane surface.

Disclosures:

No relevant conflicts of interest to declare.
Factor VIIa-CTP, a Novel Long-Acting Coagulation Factor, Displays a Prolonged Hemostatic Effect and Augmented Pharmacokinetics and pharmacodynamics Following IV and SC Administration in Hemophilic Animal Models
Authors: Hart Gili , Hershkovitz Oren , Bar-Ilan Ahuva , Seligsohn Uri , Fima Eyal
Published: October 2019

Abstract 1114

Background:

Prolor Biotech Inc. is a clinical stage public company developing biobetter long acting versions of existing therapeutic proteins utilizing a technology termed CTP. The technology involves fusion of the C terminus peptide of hCG to one or both ends of the target protein. The technology was clinically validated and proven as a safe and efficient way for prolonging the half-lives of several therapeutic proteins while maintaining their biological activity.

Aims:

To determine the pharmacokinetic (PK), pharmacodynamic (PD) and long term hemostatic effects of FVIIa-CTP in murine FVIII−/− mice following IV and SC administrations and to characterize FVIIa-CTP in vitro mechanism of action.

Methods:

FVIIa-CTP was expressed in CHO cells, purified and activated utilizing a purification process involving a CTP specific step. FVIIa-CTP was administered to FVIII−/− mice, and following IV and SC injection the PK and PD profiles were determined. In addition, the long term hemostatic effect was evaluated following bleeding challenge (tail clip assay and in a Tail Vein Transection) as compared to commercial rFVIIa. FVIIa-CTP in-vitro characteristics were also evaluated.

Results:

FVIIa-CTP PK parameters following IV administration, as assessed by a clotting assay, were superior to those of rFVIIa. Its half-life and AUC were 5 and 3.5 fold higher, respectively. A 2 fold improvement in the recovery was observed 15–30 minutes post dosing. Thrombin generation parameters were also superior in these studies. In a Tail Vein Transection study, FVIIa-CTP had a profound effect on survival, which was maintained for more than 24 hours. Reduced duration and intensity of bleeding was also observed in the tail clip study. Following SC administration, FVIIa-CTP's bioavailability, Cmax and half life were superior to commercial rFVIIa in both rats and hemophilic mice models. Correction of thromboelastograply in FVIII −/− mice plasma was maintained longer with FVIIa-CTP than with rFVIIa.

Conclusion:

Attachments of CTP to FVIIa led to a markedly enhanced PK, increased exposure, as reflected by AUC,improved recovery and a prolonged hemostatic effect in hemophilic mice with a comparable specific activity to rFVIIa. In addition, SC administration of FVIIa-CTP resulted in improved bioavailability and exposure was significantly prolonged relative to IV administration. Our data suggest that CTP fused FVIIa has the potential to significantly improve the prophylactic and on demand treatment of hemophilic patients.

Disclosures:

Seligsohn: prolor biotech: Consultancy, clinical advisor Other.
Treatment Irrespective of Symptoms Is a Major Predictive Factor for Thrombus Resolution in Patients with Portal Vein Thrombosis
Authors: Yu Yuan-Bin , Yang Chien-Wen , Liu Chun-Yu , Gau Jyh-Pyng , Hong Ying-Chung , Hsiao Liang-Tsai , Liu Jin-Hwang , Chiou Tzeon-Jye , Hsu Hui-Chi , Tzeng Cheng-Hwai
Published: October 2019

Abstract 1166

Background:

Portal vein thrombosis (PVT) typically presents with abdominal pain, ascites, and splenomegaly, but it is frequently asymptomatic. Current guidelines recommend that anticoagulation be used for symptomatic patients with PVT. However, it remains controversial regarding the benefits of treatments for asymptomatic patients with PVT.

Methods:

We retrospectively enrolled 933 patients with suspicious PVT in the Taipei Veterans General Hospital from January 2002 to December 2011. At total of 93 patients were confirmed using either doppler sonography, computed tomography, or magnetic resonance imaging. Response to treatment was defined as the recanalization or cavernous formation of portal veins. Logistic regression was used to investigate the clinic-laboratory parameters that were predictive for the resolution of PVT.

Results:

Among the 93 patients, the median age was 63 years old (range 1–91), and 61 (66%) of the patients were male. Abdominal pain was the most common symptom, occurring in the 53 patients (57%). Twenty-nine (31%) patients were asymptomatic at the time of diagnosis. For 35 of the treated patients, anticoagulation (71%), anti-platelet agents (20%), and catheter-directed urokinase infusion (26%) were the common modalities. Bleeding was noted in 6 (17%) of the patients, and all of the bleeding incidents were in the gastrointestinal tract. In the multivariate analysis, treatment was the only independent factor for thrombus improvement [odds ratio 8.54, 95% confidence interval 2.61–28.0, P < 0.001]. The results were the same when we analyzed the symptomatic and asymptomatic subgroups. The cumulative probability of improvement was higher among the treated patients compared to untreated patients at 2 years (62.8% vs. 26.2%, P< 0.001), and the benefits of the treatment were evident among the symptomatic and asymptomatic patients.

Conclusions:

Asymptomatic PVT patients may benefit from treatment. Further large-scale or prospective studies are necessary.

Disclosures:

No relevant conflicts of interest to declare.
Gfi1 Is Repressed by p53 and Inhibits DNA Damage-Induced Apoptosis
Authors: Du Pei , Tang Fangqiang , Qiu Yaling , Dong Fan
Published: October 2019

Abstract 1228

Gfi1 is a transcriptional repressor that plays a critical role in hematopoiesis. Gfi1 has also been implicated in lymphomagenesis when aberrantly activated as a result of deregulated expression. It is still poorly understood how Gfi1 expression is regulated and how it acts in the hematopoietic system. We show here that Gfi1 transcription was repressed by the tumor suppressor p53 in hematopoietic cells. Treatment of cells with doxorubicin (Doxo), which induces topoisomerase II-mediated DNA double strand breaks (DSB), led to a steady increase in p53 protein level, which was accompanied by a gradual decline in Gfi1 expression at both protein and mRNA levels. Knockdown of p53 resulted in increased Gfi1 expression and also abolished Doxo-induced Gfi1 downregulation. In contrast, Gfi1 expression was reduced and its downregulation in response to DNA damage was restored in p53-deficient cells transfected with the p53/estrogen receptor ligand binding domain fusion protein (p53ERTAM) in the presence of 4-hydroxytamoxifen (4-OHT). In luciferase reporter assays, Doxo treatment inhibited the activity of a ∼2.4-kb Gfi1 promoter fragment in p53+/+ cells, but not in p53−/− cells. Consistent with this, the wild type p53, but not a DNA binding-defective p53 mutant, repressed the Gfi1 promoter fragment. Chromatin immunoprecipitation (ChIP) assays demonstrated that p53 bound to the proximal region of the Gfi1 promoter. Detailed mapping of the Gfi1 promoter indicated that the core promoter region of Gfi1 spanning −33 to +6 bp is sufficient for p53-mediated repression. This core promoter region contains a putative p53 repressive response element (RRE) and, when the RRE was mutated, p53 failed to bind to and repress the Gfi1 promoter. Significantly, apoptosis induction by Doxo treatment was inhibited upon Gfi1 overexpression, but augmented following Gfi1 knockdown. Together, these data establish for the first time that Gfi1 is repressed by p53 and indicate that Gfi1 acts to protect hematopoietic cells from DNA damage-induced apoptosis. Our findings have important implications for understanding the roles of Gfi-1 in normal hematopoiesis and lymphomagenesis.

Disclosures:

No relevant conflicts of interest to declare.
Origin and Characterization of CD166+ Cells During Human Marrow Ontogeny
Authors: Mokhtari Saloomeh , Colletti Evan , Porada Christopher D , Almeida-Porada Graca
Published: October 2019

Abstract 1252

The development of hematopoiesis during embryonic and fetal life has served as a valuable model for characterizing putative HSC markers, as well as for studying the ontogeny of cells comprising the hematopoietic niche, and defining the nature of the interactions of hematopoietic stem/progenitor cells with these microenvironmental cells that drive HSC maintenance and/or differentiation. Alcam (CD166) has been shown to be expressed during ontogeny by the endothelium within the yolk sac and dorsal aorta, playing a key role in capillary tube formation and differentiation, but the adult aorta is devoid of this molecule. In the adult bone marrow, CD166 has been shown to be expressed by both highly primitive HSC and mesenchymal cells of the microenvironment, and to mediate homophilic adhesion between these two cell types. Here, we investigated the origin of the CD166+ cells during ontogeny and extensively characterized their phenotype as the marrow developed to better understand their function. To this end, we used flow cytometry and confocal microscopy to analyze human bone marrow (BM) during the period of development from 10 to 20 gestational weeks. CD166 expression was not detected on any cell population within the BM until week 18 of gestation, a time point when hematopoiesis is already well established within this tissue, as determined by the presence of CD34+CD45+ cells. CD166+ cells were rare at week 18, localized with other circulating cells in hematopoietic areas/spaces, and co-expressed FLK-1, CD44, N-cadherin, and Stro-1. By 20 weeks of gestation, the CD166+ population had increased significantly in number, and could be separated based on phenotype and spacial localization. CD166+/Stro-1+/CD44+/N-cadherin+/FLK-1+ cells increased approximately 4-fold in number between weeks 18 and 20, and they continued to be localized within the hematopoietic zones. In contrast, the more numerous CD166+/Stro-1+/CD44+/FLK-1- population was confined to the perivascular niche, with some of these cells integrating within the forming/maturing vasculature. In conclusion, we found that CD166 expression defines two distinct populations during development; one that seemingly associates with the perivascular niche, and another that localized with circulating hematopoietic cells. Interestingly, even though some CD166+ cells co-expressed N-cadherin, these cells did not localize to the osteoblastic niche. Studies are currently underway to further characterize these two populations with respect to their ability to support and/or give rise to hematopoiesis.

Disclosures:

No relevant conflicts of interest to declare.
Integrated RNA-Seq-Mirnome Analysis Reveal Interplay of Multiple Cancer Signaling Pathways Involved in B Lymphomagenesis
Authors: Muniandy Parameswary A , Haas Brian , Csizmadia Eva , Purushotham Jyothi , Washington Cyrus , Balter Barbara , Yan Catherine T.
Published: October 2019

Abstract 1294

Conditional inactivation of both the essential non-homologous DNA end-joining repair gene XRCC4 and the tumor suppressor gene p53 in mature B cells in mice frequently leads to mature B cell lymphomas. The majority of B lymphomas recurrently harbor reciprocal IgH/c-Myc translocations, activating c-Myc, a hallmark characteristic of Burkitt's lymphoma. We designed an integrated tumor RNA-Seq transcriptome profiling and miRnome based approach to dissect the interplay of signaling pathways and oncogenic signatures that lead to the development of the tumors in our model. Differential expression analysis of mRNA and 709 miRNAs reveal specific alterations in distinct signaling pathways, including upregulation of both Wnt and PI3K pathway signaling. For example, we found that Wnt10b, the wnt ligand expressed in B cells is upregulated. Correspondingly, frizzled receptors and it's downstream effector disheveled are also upregulated, factors that contribute to beta-catenin stabilization. Our studies also reveal increased expression PIK3CD, a PI3K catalytic isoform expressed in B cells, with corresponding downregulation of miRNAs with seed matches to 3'UTR of PIK3CD in the tumor; a factor that likely contribute to the stabilization of PIK3CD mRNA. Our analyses reveal an interplay of multiple pathway proteins and genes that serve as novel therapeutic targets for human B lymphoma.

Disclosures:

No relevant conflicts of interest to declare.
Interleukin-9 Promotes Survival of Diffuse Large B Cell Lymphoma Cells Through Upregulation of P21cip1
Authors: Lv Xiao , Wang Xin , Feng Lili , Ge Xueling , Fang Xiaosheng , Sui Xiaohui , Liu Xin
Published: October 2019

Abstract 1544

Introduction:

Diffuse large-B-cell lymphoma (DLBCL) is known as an aggressive malignancy arising from B lymphocytes. Despite its greatly improved prognosis as a result of chemotherapy and immunotherapy with monoclonal antibodies, the exact molecular etiology of DLBCL is not well understood. Interleukin-9 (IL-9) is initially described as a growth factor secreted by activated Th2 cells. Various observations have demonstrated its diverse actions in immune disorders. Recent years, the determination of its growth-proliferative and anti-apoptotic activities on multiple transformed cells implies a potential role of this cytokine in tumorigenesis but there are still no reports about its oncogenic activities in DLBCL. Our study is aimed to test the expression of IL-9 and its receptor (IL-9R) in DLBCL patients and illustrate its pathogenic effect on DLBCL cell lines in vitro.

Methods:

Blood samples and araffin-embedded tissues from twenty DLBCL patients were collected prior to therapeutic interventions. Serums from healthy volunteers served as normal control. IL-9 levels in sera were quantified using human ELISA kits. The expression of IL-9R protein in DLBCL tissues and lymphoma cell lines (LY1, LY8, SP53, Mino and Jurket) was determined by immunohistochemical staining and western-blot, respectively. IL-9R genes were knocked down in DLBCL cell lines LY1 and LY8 by lentivirus-mediated gene silencing (interference sequence 5'- GCTCGTGCCATCTGACAATTT -3'). LY1, LY8 and the stable transfected cells were treated with IL-9 alone and in synergy with rituximab (10ug/ml).

Disclosures:

No relevant conflicts of interest to declare.
Chronic Myeloid Leukemia Philadelphia Chromosome Positive (CML Ph+) with Imatinib (IM) Intolerance or Resistant Disease. Response to Thalidomide (TALI) Treatment. Preliminary Report of a Serial of Cases with Encouraging Results
Authors: Hurtado Rafael , Viveros Jose Pablo Vargas , Silvia Carrillo Muñoz , Cruz-Velazquez Judith , Candelaria Myrna , Cervera Eduardo
Published: October 2019

Abstract 1689

The increase dose of Imatinib, the shift to a Tyrosine Kinase (TKI) inhibitor of second generation or hematopoietic stem cell transplantation are treatment options when there is loss or lack of response to IM in CML; However, in patients without access to these therapeutic alternatives, the use of alkylating drugs or investigational agents is indicated. NF Kappa B pathway is constitutively activated in CML patients therefore the combination of IM with a NF Kappa B inhibitor (as thalidomide) contributes a new very interesting field of research. We treated 21 patients (11 female) with CML Ph +− resistant (n=13) or intolerant (n=8) to IM, mean +− SD age was 44 + 11 years. At diagnosis 16 (79%) were classified as high risk, according with Eutos scale. Mean +− SD time of IM treatment before adding thalidomide (100mg daily) was 47.8 +− 31 months. At the time of this preliminary analysis, mean follow up since thalidomide addition was 5 (1–18 months) and only 13 patients are evaluable for response (Global response rate was 76%): Six had hematological response & Four achieved complete cytogenetic response (CCgR). The first case was a 53 year old female, who started treatment with IM in accelerated phase, was intolerant to IM and neither achieve CCgR with IM, but reached it after 6 months of adding thalidomide and remains with this treatment after 18 months. The second case was a 38 year old female, who achieved CCgR after 6 months within IM treatment, lasted 53 months, and finally thalidomide was added after losing CCgR; she achieved a 2nd CCgR after 7 months with this combination, and continues with thalidomide at 11 months. The third one is a 44 year old female, who only achieved partial CCgR (pCGR), and after indicating thalidomide on accelerated phase, she had CCgR after 4 months of this drug and remains at 6 months of treatment. Finally, the fourth case is a 26 year old female that received thalidomide because of a suboptimal response to IM; she achieved CCgR after 2 months within thalidomide. Eight patients are not evaluable for response yet, but are ongoing and all will be detailed at ASH meeting. Toxicity has been acceptable, mainly grade 1 neurotoxicity & none severe adverse events have been documented. This is the first communication of the use of Thalidomide with Imatinib with promising results which may represent an alternative therapeutic in CML with antiproliferative enhancer and/or synergistic effect in patients with resistance to IM without opportunity of access to other treatments. This cases report open a future very interesting field of research in intolerance and resistance CML.

Disclosures:

No relevant conflicts of interest to declare.
Preclinical Testing of a Novel Axl-Kinase Inhibitor in Chronic Lymphocytic Leukemia
Authors: Göbel Maria , Möllmann Michael , Görgens Andre , Dührsen Ulrich , Hüttmann Andreas , Giebel Bernd , Unger Anke , Choidas Axel , Dürig Jan
Published: October 2019

Abstract 1799

The receptor tyrosine kinase Axl belongs to the TAM (Tyro-3, Axl and Mer) family and is involved in the progression of several human malignancies including chronic lymphocytic leukemia (CLL), where it is has been found to be overexpressed in comparison to normal B-cells. An increasing body of evidence suggests that Axl acts as an oncogene which increases the survival, proliferation, metastatic potential and chemotherapy resistance of tumor cells. Hence, it has been recently identified as a potential therapeutic target in a wide range of tumor entities with deregulated Axl expression including prostate cancer, glioma, lung cancer and CLL. Here, we investigated two different Axl inhibitors for their potential to inhibit the migratory capacity and survival of leukemic cells in preclinical CLL models.

In vitro studies: Freshly isolated PBMC (>90% CD5+CD19+) from CLL patients were incubated in serum free medium for 48h containing concentrations series of 2 different Axl inhibitors: BMS777607, a previously published inhibitor of the MET kinase family, and LDC2636, a novel inhibitor of the TAM receptor tyrosine kinase (RTK) family with high affinity to Axl. Viability of CLL cells was assessed by trypan blue staining and flow cytometry employing annexin V staining. Since a polarized phenotype is required for migration, cell polarization was analyzed by time-lapse video-microscopy. We detected cytotoxic effects in a patient dependent manner that were more prevalent in LDC2636 as compared to BMS777607 treated cells (LD50= 1.4 μM vs. 5.2 μM, p<0.004, n=5). Cell polarization of the remaining viable cells was significantly reduced in a dose dependent fashion in comparison to vehicle only controls (LDC2636 IC50 = 7.2 μM, p<0.00001; BMS777607: IC50=6.2μM; p=0.0004). Of note, both Axl inhibitors exhibited significantly weaker effects on both, the viability and cell polarization of normal PBMC over the whole concentration range tested (p<0.05, n=5).

In vivo studies: To verify our hypothesis that reduced cell polarization results in decreased homing of leukemic cells in vivo we employed a recently developed adoptive transfer model of CLL. In this model NOD/SCID/gcnull(NSG) mice were pre-treated with a single intraperitoneal bolus of LDC2636 or BMS777607 (20 mg/kg) and subsequently transplanted with primary CLL cells. Both Axl inhibitors significantly reduced the homing capacity of CLL cells to the bone marrow of NSG mice by 43% and 59%, respectively, compared to vehicle treated controls (LDC2636: p=0.046, BMS777607 p=0.0077; n=3).

These data demonstrate that Axl inhibitors exert potent in vitro and in vivo activity against human CLL cells, which is caused at least in part by the suppression of CLL homing to their supportive stromal niches.

Disclosures:

No relevant conflicts of interest to declare.
Myeloid Impairment Contributes to Immunoparesis in Multiple Myeloma but Not MGUS
Authors: Romano Alessandra , Parrinello Nunziatina , Vetro Calogero , La Cava Piera , Chiarenza Annalisa , Conticello Concetta , Palumbo Giuseppe A , Di Raimondo Francesco
Published: October 2019

Abstract 1831

Introduction

In Multiple Myeloma (MM), but not in the monoclonal gammopathy of unknown significance (MGUS), the immune function is impaired as consequence of an immunologically hostile microenvironment and cellular defects, including reduction of immuno-surveillance and T-cell immunoparesis.

We conducted an study focused on the myeloid compartment in MM, and its role in the progression from MGUS to MM.

Methods

Between January 2009 and April 2011 peripheral blood obtained from 60 consecutive newly diagnosed MM and 70 MGUS plus 30 healthy subjects was studied for evaluation of myeloid subpopulations and lymphoid paresis.

Myeloid dysfunction was evaluated as percentage and absolute count of circulating myeloid suppressor cells (MDSC) in peripheral blood assessed by flow cytometry as follows: im-MDSC (CD34+/CD11b+/CD13+/CD14-/ HLA-DR-/CD45+), neutrophilic-like N-MDSC (CD11b+/CD13+/CD15+/CD14-/HLA-DR-/Lin-) and monocytic-like mo-MDSC (CD14+/HLA-DRlow/-).

Myeloid function was evaluated by phagocytic activity using a commercially available kit (Phagotest R).

Further, we investigated whether MM-neutrophils were able to induce anergy in T-cells. Neutrophils isolated from healthy donor (N=6), MGUS (N=3) or MM (N=6) peripheral blood (PB) were co-cultured with T-lymphocytes obtained from healthy donors. Expression of markers of activation in response to stimulation with PHA-P for 2 hours was assessed by flow cytometry as antigen density expressed as normalized mean of fluorescence intensity (N-MFI) of CD71 at 48 hours.

Results

The capability of phagocytosis of in neutrophils and monocytes from MM patients at diagnosis was significantly reduced compared to healthy subjects (p<0.001) and MGUS (p<0.0001). While the mature suppressive N-MDSC subset was not increased in MGUS and MM patients, the mo-MDSC subpopulation showed an increasing trend from healthy donors through MM (p=0.06) and the im-MDSC subset was significantly higher in MM vs healthy (p=0.002) and MGUS (p=0.001).

After PHA-P stimulation, expression of CD71 (a marker of activation) in normal T-lymphocytes was increased (2954 ± 240.6 arbitrary units, au), and it was reduced (751.3 ± 30.48 au, p=0.0001) when co-coltured with MM-neutrophils, while no differences were evident in co-colture with MGUS- (2783 ± 206.1 au, p=0.61) or healthy donors-neutrophils (2588 ± 135.4, p=0.38).

Conclusion

Taken together, our findings suggest that in MM but not in MGUS there is a myeloid cell dysfunction that is correlated to impairment of T- cell arm. These alterations may have a role in the development of MM.

Disclosures:

No relevant conflicts of interest to declare.
Higher Doses of Prophylactic Anticoagulation Is More Effective in Preventing Venous Thromboembolism in Morbidly Obese Inpatients.
Authors: Wang Tzu-Fei , Milligan Paul , Wong Catherine A , Thoelke Mark S , Deal Eli N , Gage Brian F
Published: October 2019

Abstract 2268

Background:

Obesity increases the risk for venous thromboembolism (VTE), and the optimal dose of prophylactic anticoagulation to prevent VTE in morbidly obese inpatients is unknown.

Objectives:

To quantify the efficacy and safety of higher doses of prophylactic heparin or enoxaparin for VTE prevention in morbidly obese inpatients with a body mass index (BMI) ≥ 40 kg/m2and/or weight >100 kg within the BJC HealthCare system.

Patients/Methods:

We analyzed 10,239 inpatients with weight > 100 kg and/or morbid obesity (BMI ≥ 40 kg/m2) discharged from the three major hospitals in the BJC HealthCare system from January 1st, 2010 through February 29th, 2012. We used logistic regression to calculate odds ratios (OR) and 95% confidence intervals (CI) and Wald's chi-square to compare proportions. The primary efficacy outcome was an ICD-9 diagnosis code for VTE. The primary safety outcome was an ICD-9 diagnosis codes for bleeding.

Results:

On-treatment analysis showed higher doses of prophylactic anticoagulation (heparin 7500 units three times daily instead of 5000 units two or three times daily or enoxaparin 40 mg twice daily instead of 40 mg once daily) halved the risk (OR 0.52, 95% CI 0.27–1.00; p = 0.05) of symptomatic VTE in the 3928 morbidly obese inpatients (weight > 100kg and BMI ≥ 40 kg/m2). The rate of VTE was 1.48% (35/2369) in these morbidly obese inpatients who received standard prophylactic doses of unfractionated heparin or enoxaparin, compared to 0.77% (12/1559) in those who received higher doses, with an absolute risk reduction of 0.71% and a number needed to treat (NNT) of approximately 140. Intention-to-treat analysis failed to show reduction in VTE rate due to low (< 40%) physician compliance in prescribing the higher doses. Increased doses of prophylactic anticoagulation were not associated with bleeding (OR 0.84, 95% CI 0.66–1.07, p = 0.15).

Conclusions:

Higher doses of prophylactic anticoagulation nearly halve the rate of VTE in morbidly obese inpatients (with BMI ≥ 40 kg/m2 and weight > 100 kg), but do not increase bleeding. Given their safety and ease of implementation, higher doses of prophylactic heparin or enoxaparin should be considered in morbidly obese inpatients. Clinicians' compliance and awareness need to be improved to prevent VTE in this vulnerable population.

Disclosures:

No relevant conflicts of interest to declare.
The Role of Interim-PET and Final-PET in the Outcome of Peripheral T-Cell Lymphoma (PTCL) Treated At the Diagnosis with CHOP.
Authors: Pellegrini Cinzia , Casadei Beatrice , Derenzini Enrico , Broccoli Alessandro , Stefoni Vittorio , Gandolfi Letizia , Quirini Federica , Argnani Lisa , Zinzani Pier Luigi
Published: October 2019

Abstract 2721

Role of interim- and final-PET in peripheral T-cell lymphoma (PTCL) is quite unknown. To determine predictive value of PET on overall survival (OS), we evaluated interim-PET (i-PET) and final-PET (f-PET) in PTCL patients treated in first-line with 6 CHOP-21 courses. From September 2003 to July 2010 we diagnosed and treated in our institution 34 advanced stage PTCL patients (15 females and 19 males). The median age at diagnosis was 46 years (range, 21–81 years); 9 patients were in stage III, and 25 in stage IV. According to the histologic subtype there were 11 PTCL-nos, 6 AILT, 9 ALCL Alk+, 6 ALCL Alk-, and 2 NK/T nasal type patients. Four patients had bulky disease; eight patients had bone marrow involvement, 15 patients had 1 extranodal involvement and 10 had more than 2 extranodal sites. All patients underwent initial staging PET/CT; i-PET was performed after 3 cycles of CHOP-21 and the median time from the end of third course to i-PET was 14 days (range, 7– 18 days). f-PET scans were performed 35 days (range, 30– 45 days) after the end of therapy.

The table summarizes the correspondence between i-PET and f-PET results:

N=34 f-PET negative, n (%) f-PET positive, n (%) i-PET negative 27 19 (70.4) 8 (29.6) i-PET positive 7 1 (14.2) 6 (85.8)

With a median follow-up of 71 months (range, 5.8–120.9 months), 17/19 (89.5%) patients with i-PET negative are in continuous CR (CCR) and only 1/7 (14.2%) patient with i-PET positive is still in CCR. Figures show the overall survival (OS) according to response at i-PET and f-PET. In figure 1a we observe OS plotted according to i-PET results: 78.6% for negative patients (solid line) and 21.4% for positive patients (dashed line) at 88.7 months (p=0.02); in figure 1b we observe OS plotted according to f-PET results: 93.7% for negative patients (solid line) and 21.4% for positive patients (dashed line) (p<0.0001).

In conclusion, our results demonstrate that positive i-PET is not predictive of a worse outcome in PTCL. On the contrary, the f-PET seems to represent a significant step forward in the prediction of survival for these patients. Larger and prospective studies and harmonization of PET reading criteria are needed.

Disclosures:

No relevant conflicts of interest to declare.
Impact of Reduced Dose Intensity for Elderly Patients with Diffuse Large B-CELL Lymphoma Receiving R-CHOP Every 14 or 21 Days: An Analysis of the LNH03–6B GELA Study.
Authors: Delarue Richard , Tilly Herve , Salles Gilles , Thieblemont Catherine , Mounier Nicolas , Molina Thierry Jo , Bologna Serge , Haioun Corinne , Bosly Andre
Published: October 2019

Abstract 2723

Introduction:

Elderly patients with diffuse large B-cell lymphoma (DLBCL) frequently receive reduced dose-intensity (DI) chemotherapy (Lyman GH et al. J Clin Oncol 2004) but impact on outcome in the era of classical or dose-dense immunochemotherapy is unknown. Final results of the LNH03–6B did not show any difference in PFS (HR: 0.99 [95%CI: 0.78–1.26]; p=0.90) and OS (HR: 0.96 [95%CI: 0.73–1.26]; p=0.75) between R-CHOP14 and R-CHOP21 (Delarue et al. ASCO 2012).

Methods:

Patients between 60 and 80 years old with DLBCL and aaIPI≥1 were eligible. They were randomized between R-CHOP14 and R-CHOP21 for 8 cycles. Dose-intensity (DI) was calculated for all patients for cyclophosphamide (CPM), doxorubicin (DOX) and rituximab (RTX) and patients were separated into 4 quartiles for each of these drugs. Outcome (PFS and OS) according to final DI was evaluated for all patients and in R-CHOP14 and R-CHOP21 arms.

Results:

602 pts were randomized, 600 were evaluable, 304 with R-CHOP14 and 296 with R-CHOP21. Median age was 70 years. Patient characteristics were similar in both arms. The percentage of cycles administered with G-CSF was 89% in R-CHOP14 and 66% in R-CHOP21. Median interval between 2 cycles was 14 d [9–94] in R-CHOP14 arm and 21 d [15–66] in R-CHOP21 arm. Median dose-intensity was 88% in R-CHOP14 and 97% in R-CHOP21 for CPM, and 88% in R-CHOP14 and 96% for R-CHOP21 for DOX. There was no difference of median dose-intensity for CPM and DOX according to G-CSF use at C1 in R-CHOP14 arm. In the R-CHOP14 arm, the increase of DI at the end of treatment, calculated according to 3-week interval as a reference, was 133% for CPM and DOX. When separating patients in 4 quartiles according to final dose intensity, there was no impact for CPM, DOX and RTX for the entire cohort in term of PFS and OS. Moreover, decreased DI for CPM, DOX and RTX did not impact negatively PFS and OS in patients randomized in the R-CHOP14 arm. On the other hand, for patients included in the R-CHOP21 arm, PFS was negatively impacted by lower DI of CPM (p=0.02), DOX (p=0.007) and RTX (p=0.006). OS was also negatively impacted by lower DI of CPM (p=0.0002), DOX (p<0.00001) and RTX (p<0.0001). In the R-CHOP21 arm, the negative impact was pronouncedly marked when comparing patients in the first and the second quartiles with those in the fourth quartile.

Conclusion:

While low DI has a major impact on PFS and OS in patients who receive conventional dose immunochemotherapy, consequences for patients receiving dose-dense immunochemotherapy seem less pronounced. As a consequence, immunochemotherapy could be safely decreased for elderly patients who receive R-CHOP14 and experienced adverse events.

Disclosures:

No relevant conflicts of interest to declare.
Bendamustin-Rituximab Induction Followed by Observation or Rituximab Maintenance for Newly Diagnosed Patients with Waldenström's Macroglobulinemia: Results From a Prospective, Randomized, Multicenter Study (StiL NHL 7–2008 –MAINTAIN-; ClinicalTrials.gov Identifier: NCT00877214).
Authors: Rummel Mathias J. , Lerchenmüller Christian , Greil Richard , Görner Martin , Hensel Manfred , Engel Erik , Jaeger Ulrich , Breuer Friedhelm , Hertenstein Bernd , Prummer Otto , Buske Christian , Barth Juergen , Burchardt Alexander C. , Brugger Wolfram
Published: October 2019

Abstract 2739

Background:

Bendamustine-rituximab (B-R) has demonstrated substantial efficacy in the primary treatment of indolent lymphomas. A multicenter prospective randomized trial was initiated to investigate the impact of adding rituximab maintenance following B-R first-line induction. The trial included patients (pts) with Waldenström's Macroglobulinemia (WM), marginal zone, small lymphocytic and mantle cell lymphomas. Here we present first and preliminary results for pts with WM.

Methods:

Treatment consisted of a maximum of 6 cycles of B-R (bendamustine 90 mg/m2, rituximab 375 mg/m2) administered every 28 days plus 2 cycles of rituximab every 4 weeks. Responding pts (≥ PR) were eligible for further treatment and were randomized to observation or 2 years of rituximab maintenance every two months. The primary endpoint is PFS.

Results:

From April 2009 to July 2012, 57 centers included a total of 162 pts with newly diagnosed WM with a median age of 67 years (31% < 60 years, 69% > 60 years). At baseline/inclusion/screening, the median values for b2-microglobulin (b2M), hemoglobin and IgM were 3.3 mg/L, 10.1 g/dL and 2110 mg/dL (max. 13400 mg/dL), respectively. To date (Aug 2012) 116 pts are evaluable for response (43 women [37%] and 73 men [63%]). 100 pts have responded to B-R leading to an overall response rate (ORR) of 86%. At the time of response evaluation, the median Hb was 12.6 g/dl and the median IgM was 380 mg/dl.

90 pts have undergone randomization to date, 43 to observation and 47 to maintenance. Randomization is ongoing. No results can be reported from that ongoing part of the trial. No uncommon toxicities were observed during B-R induction.

Conclusion:

Initial results of our trial confirm that for patients with Waldenström's macroglobulinemia, treatment with B-R is an efficacious treatment with a manageable safety profile. The role of rituximab maintenance in this disease is under investigation.

Disclosures:

No relevant conflicts of interest to declare.
Essential Role of the Transcription Factor ZBP-89 in Lymphopoiesis
Authors: Woo Andrew J. , Huang Hui , Piers Taylor , Cantor Alan B
Published: October 2019

Abstract 277

We previously identified the Krüppel-type zinc finger transcription factor ZBP-89 (also called zfp148) as a novel GATA1 associated protein in erythroid and megakaryocytic cells. ZBP-89 also associates with other GATA family members such as GATA2 and GATA3, Friend of GATA (FOG) cofactors, and RUNX1. It is ubiquitously expressed, but has high-level expression in a subset of tissues including thymus, spleen, bone marrow, lung and brain. Our prior studies using morpholino knockdown in zebrafish, in vitro differentiation and chimeric mouse analysis of ZBP-89 genetrap embryonic stem cells, and lentiviral shRNA knock down in primary human CD34+ cells demonstrated a functional role for ZBP-89 in erythroid and megakaryocyte maturation. In this study, we generated conditional knock out mice to further examine the requirements for ZBP-89 in vivo. On a mixed strain background, full ZBP-89 knock out mice were born at close to Mendelian ratios. However, the ZBP-89−/− mice were severely runted and had increased mortality over the first 30 days of life. Surviving pups had significant growth failure, but eventually matched their wild type and heterozygous littermates by about 6 weeks of age. Analysis of erythroid maturation in bone marrow and spleen using flow cytometry for CD71 and Ter119 demonstrated impaired erythroid maturation in a ZBP-89 allele-dose dependent manner. On a pure C57BL/6 genetic background, nearly 100% of the ZBP-89−/− mice died within the first 10 days of life from unclear causes. ZBP-89fl/fl, Mx1-Cre mice developed lymphopenia and platelet abnormalities following activation of Cre by polyI-polyC injection. The lymphopenia was due to reduction in both B and T cells. Further delineation of the T-cell defect using ZBP-89fl/fl, Lck-Cre mice demonstrated impaired maturation of double negative (CD4−CD8−) T cells at the DN3 (CD25+ CD44−) to DN4 (CD25−CD44−) stages in thymi from 5–6 week old mice. These findings indicate that ZBP-89 plays functional roles in multiple hematopoietic lineages. Moreover, they identify ZBP-89 as a novel transcriptional regulator of lymphocyte development. We speculate that this latter role involves its known interactions with GATA3, FOG-1, and/or RUNX1, which are all similarly involved in lymphopoiesis. We also show that the ZBP-89 family member ZBP-99 is highly expressed in thymus and other hematopoietic tissues, and may therefore play partially overlapping roles with ZBP-89.

Disclosures:

No relevant conflicts of interest to declare.
CTCF Mediated Enhancer and Promoter Interaction Regulates Differential Expression of TAL1 Oncogene in Normal and Malignant Hematopoiesis
Authors: Patel Bhavita , Deng Changwang , Litt Michael , Riberio Mariana St. Just , Cui Kairong , Kang Yuanyuan , Qiu Yi , Zhao Keji , Huang Suming
Published: October 2019

Abstract 281

The key hematopoietic transcription factor TAL1/SCL is essential for the specification of hematopoietic stem cells (HSCs) and differentiation along myeloid and erythroid lineages. However, ectopic activation of TAL1 gene is the most frequent gain-of-function mutation associated with childhood T-cell acute lymphoblastic leukemia (T-ALL) in approximately 60% of patients. To understand underlying epigenetic mechanisms of TAL1 activation in normal and in malignant hematopoiesis, we performed ChIP, ChIP-seq, and chromatin conformation capture assays to investigate chromatin structure profile which correlates with transcriptional activation, at the TAL1 locus comparing erythroid progenitors, K562 and CD36+ cells, with T-ALL cells, Jurkat and Rex cells. We found distinct epigenetic landscape and chromatin organization across the TAL1 locus in these two different types of hematopoietic cells. Although H3K4me3 is enriched at the TAL1 promoter in both erythroid progenitors and in T-ALL cells, the erythroid specific +51 enhancer is marked by active histone modifications only in erythroid progenitors, but not in T-ALL cells. Furthermore, we demonstrated that +51 enhancer interacts with the TAL1 promoter 1a via a long range chromatin loop in vivo in erythroid CD36+ and K562 cells. The recruitment of hSET1 HMT complex facilitates this enhancer/promoter chromatin interaction and depletion of hSET1 leads to loss of H3K4 methylation, enhancer/promoter interaction, RNA PolII loading, and TAL1 transcription at both the TAL1 promoter 1a and the +51 enhancer. In addition, loss of SET1 in CD34+ HSCs exhibited a significant reduction in the formation of CFU-E and BFU-E colonies. In contrast, neither H3K4 methylation nor enhancer/promoter interaction was detected at the +51 enhancer in T-ALL cells. Finally, we investigated the role of insulator protein CTCF in the regulation of TAL1 expression in normal and in malignant cells. We found that regardless of similar CTCF binding patterns at the TAL1 locus in erythroid progenitors and T-ALL cells, CTCF elements flanking the TAL1 locus mediate a long-range loop that encloses TAL1 promoter and enhancer in the same chromatin domain in erythroid progenitors, whereas in T-ALL Jurkat cells the CTCF site located between the promoter 1a and the +51 enhancer forms a repressive loop that expels the +51 enhancer from the same domain with the promoter 1a. Overall, our study demonstrates that a novel long range intrachromosomal interaction between the TAL1 promoter 1a and the +51 enhancer regulated and organized by hSET1 histone methyltransferase and insulator protein CTCF controls TAL1 promoter activity in normal versus malignant hematopoiesis.

Disclosures:

No relevant conflicts of interest to declare.