bluuebunny/crossref_metadata_2025 huggingface dataset:
“Don't Eat me”: CD47 and LFA-1 Modulate CLL Response to Nurselike Cells-Mediated Antibody-Dependent Phagocytosis Induced by Rituximab and Obinutuzumab (GA101)
Authors: Ysebaert Loic , Laprevotte Emilie , Klein Christian , Laurent Guy , Quillet-Mary Anne , Poupot Mary , Fournié Jean-Jacques
Published: October 2019

Abstract 1762

INTRODUCTION AND AIMS: Efficacy of therapeutic monoclonal antibodies depends to a large extent on the induction of effective antibody dependent cell cytotoxicity and phagocytosis (ADCC/ADCP) by CD16A+ immune effector cells. Chao et al. have recently unraveled the role of antibody dependent cell cytotoxicity and phagocytosis (ADCP) in rituximab (RTX)-induced tumor cell clearance using murine models of NHL [Chao M, 2010]. Nurse-like cells (NLC) provide pro-survival signals by direct contact and/or secretion of soluble factors while remaining M2-macrophages by nature [Ysebaert L, 2011]. M2 macrophages rapidly clear multiple opsonized targets [Leidi M, 2009], infiltrate lymph nodes and bone marrow, and are resistant to chemotherapeutic drugs thereby acting as important mediators of anti-CD20 mediated ADCP in CLL. We used an in vitro culture system to assess NLC-mediated ADCP mediated by RTX and obinutuzumab (GA101), a type II, glyco-engineered CD20 antibody with enhanced ADCC/ADCP through improved CD16A binding.

METHODS:

Antibody-mediated B cell depletion was determined by flow cytometry after antibody treatment. PBMCs from CLL patients were cultured for 14 days to allow outgrowth of NLC [Tsukada N, 2002]. Cells were collected and either (i) put in 6-well plates in the same medium without NLC (w/o NLC), or (ii) seeded back onto NLC (CLL+NLC), before anti-CD20 antibodies were added at 10μg/ml for 7d. For CD47 blocking experiments, cells were incubated for 15 min with blocking antibodies before being seeded back onto NLC. An Affymetrix Uplus2.0 chip was used for gene expression profiling (GEP) of 19 NLC and 5 normal CD14+ monocytes samples. For phenotype studies, CLL cells in suspension were thoroughly harvested with medium, and remaining adherent CLL cells were discarded from NLC by vigorous pipetting, before staining with CD47-FITC/LFA-1-PE antibodies.

RESULTS:

CONCLUSIONS:

Our results suggest that NLC may be therapeutically exploited through mediators of ADCP, especially in lymphoid organs. For that purpose, the combination of CD47 and CD20 antibodies may be considered. Due to its efficacy even in LFA-1hi patients, GA101 is considered a better mediator of NLC-ADCP. Exploring the regulation of CD47 in CLL cells is important because it may interfere with strategies targeting surface antigens through FcgammaR-dependent mechanisms.

Disclosures:

Ysebaert: Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.
Centrosomal Disruption As Early Event in Myeloma Development
Authors: Sevcikova Sabina , Kryukov Fedor , Dementyeva Elena , Lenka Kubiczkova , Nemec Pavel , Adam Zdenek , Hajek Roman
Published: October 2019

Abstract 1806

Background:

Multiple myeloma (MM) is a malignancy of differentiated B-lymphocytes characterized by accumulation of clonal plasma cells (PCs) in the bone marrow. Genome of malignant PCs is extremely unstable and characterized by complex combination of structural and numerical abnormalities. The basis of genomic instability underlying MM is still unclear. Centrosome amplification (CA) is present in about a third of MM cases and may represent a mechanism leading to genomic instability in myeloma. We have previously shown that CA is present in pre-plasma cells stages of B-cell development.

Aims:

The objective of our study was to evaluate changes in expression of chosen key genes involved in centrosome duplication process in both B and PCs, including their possible changes during MM progression.

Materials and Methods:

In total, 56 patients were evaluated for this study. Patients' characteristics were as follows: males/females 38/37, median age 69 years (range 43–83 years). Newly diagnosed (27/56) and relapsed (29/56) patients were included in this study; most of them had advanced stage of MM (DS III n=48; ISS II/III n=45). qRT-PCR (51 PCs and 32 B cell samples) was performed on a chosen set of mitotic genes, according to their role in normal centrosome duplication process.

Results:

Expression of genes involving in centrosome duplication process was studied in B cells of MM patients and healthy donors. Group of MM patients showed significant increase in relative quantification coefficient R in the following genes: AURKA, AURKB, CCNB1, CCNB2, HMMR, PLK4, TACC3 and TUBG1. It is notable that TUBG1 was approximately 330-times overexpressed in MM B cells vs healthy donors.

Expression of studied genes was significantly different in B and PCs populations of MM patients: AURKB and TACC3 were upregulated in B cells while CENT2 and TUBG1 were upregulated in PCs.

Analyses of gene expression in newly diagnosed and relapsed patients showed significant changes of the following genes: AURKA and TACC3 were 2-times upregulated in PCs of relapsed patients; no differences were detected in population of B cells.

Conclusion:

Considering revealed changes in B cells of myeloma patients, we suspect early damage in cell-cycle regulating mechanism. This hypothesis correlates with our previous findings of CA in MM B cells. We anticipate that dynamic accumulation of showed changes plays a role in final transformation of malignant PCs. We assume that centrosomal disruption likely contributes to accumulation of genomic abnormalities in tumor cells during MM progression which can be proven in future long-term study.

Acknowledgment:

This study was supported by grants NT11154, NS10207, MSM0021622434

Disclosures:

No relevant conflicts of interest to declare.
Thalidomide As Maintenance Therapy in Multiple Myeloma (MM) Improves Progression Free Survival (PFS) and Overall Survival (OS): A Meta-Analysis
Authors: Nooka Ajay K. , Behera Madhusmita , Boise Lawrence H. , Watson Melanie , Kaufman Jonathan L. , Lonial Sagar
Published: October 2019

Abstract 1855

Background:

Conflicting data regarding the survival benefit of thalidomide maintenance exist in the literature. Recent phase 3 study MMRC.M10 has demonstrated significant toxicity with thalidomide maintenance in combination with steroids. The role of thalidomide maintenance in prolonging PFS and OS is less clear. We conducted a meta-analysis of phase III randomized control trials (RCT) that evaluated thalidomide as maintenance therapy in transplant-eligible patients.

Methods:

We conducted an electronic literature search of public databases (MEDLINE, EMBASE, Cochrane library) and manual search of conference proceedings of ASH and ASCO. There were seven phase III RCTs that reported the maintenance therapy of thalidomide and survival in MM (Total Therapy 2, IFM 99-02, ALLG trial, HOVON 50, MRC IX trial, NCIC CTG MY.10). One study (Abdelkefi et al., 2008) was retracted and hence not included in the analysis. A formal meta-analysis was conducted using Comprehensive Meta Analysis software (Version 2.0). The outcome data were pooled and reported as hazard ratio (HR). The primary outcome of interest was progression free survival (PFS) and overall survival (OS).

Results:

A total of 3194 patients (maintenance arm/control arm- 1479/1715, median age 58 years) were evaluated. The 3 year-OS ( HR 0.80, 95% CI 0.70 to 0.917, P=0.001) and 3 year-PFS (HR 0.62, 95% CI 0.55 to 0.69, P=0.000) were superior with maintenance therapy. ‘Steroid and thalidomide’ maintenance therapy was associated with significant OS and PFS improvement (OS HR 0.65, 95% CI 0.47 to 0.89); PFS (HR 0.54, 95% CI 0.44 to 0.67). Overall toxicities with thalidomide maintenance were peripheral neuropathy (PN) 2.184 (95% CI 1.523–3.133) and thromboembolic events (TEE) 1.632 (95% CI 1.076–2.475). Toxicities were increased with thalidomide and steroid maintenance - PN 28.78 (95% CI 1.676–494.13); TEE 2.896 (95% CI 0.76–11.06).

Conclusions:

Thalidomide as a single agent or in combination with steroids as maintenance therapy improves progression free survival and overall survival. However, high toxicity with steroid combination was observed.

Disclosures:

Kaufman: Millenium; Onyx; Novartis; Keryx: Consultancy; Merck, Celgene: Research Funding. Lonial:Millennium: Consultancy; Novartis: Consultancy; Celgene: Consultancy; BMS: Consultancy; Onyx: Consultancy; Merck: Consultancy.
Distinct Role of Antigen-Specific Tc1 and Tc17 Cells in Tumor Eradication In Vivo
Authors: Yu Yu , Cho Hyun Il , Wang Dapeng , Kaosaard Kane , Anasetti Claudio , Celis Esteban , Yu Xue-Zhong
Published: October 2019

Abstract 1910

Background:

Adoptive cell transfer (ACT) of tumor-reactive T cells is one of the most promising approaches for the treatment of established melanoma. Recently, limited studies provide some evidence that Th/Tc17 cells may also have potent anti-tumor activities, but the conclusion is far from reach. Methods: Human gp10025-specific Tc1 or Tc17 cells were generated from pmel-1 transgeneic mice and used as cell source for ACT. Luciferase-transduced B16 melanoma was intravenously injected into C57BL/6 mice to establish lung-metastasis. After 7 days, tumor-bearing mice were lethally irradiated and transferred gp-10025 specific Tc1 or Tc17 cells in the combination of syngeneic bone marrow. Survival of those tumor-bearing mice was monitored daily, and tumor growth was monitored weekly using in vivo bioluminescent imaging (BLI). Donor T-cell expansion and cytokine secretion from the spleen and lung of tumor bearing mice were analyzed using flow cytometry and ELISPOT assays. To evaluate the role of IFNγ in anti-tumor immunity, we used a B16 melanoma cell line that was transduced with a plasmid encoding a dominant-negative IFNγ receptor (B16-IFNγRDN), and IFNγR knockout mice as tumor-bearers. Results: As expected, irradiation and transfer of syngeneic bone marrow had little or no effect on established melanoma. Adoptive transfer of tumor-specific Tc17 cells significantly suppressed the tumor growth, whereas Tc1 cells induced long-term regression of established melanoma. After ACT, Tc1 cells maintained their phenotype to produce IFNγ. However, Tc17 cells largely preserved their ability to produce IL-17, but a subset of them secreted IFNγ, indicating the plasticity of Tc17 cells in vivo. Mechanistically, Tc1 cells executed their anti-tumor immunity primarily through the direct effect of IFNγ on melanoma cells because Tc1 cells had essentially no effect on B16-IFNγRDN tumor. However, Tc1 cells had a similar therapeutic effect on IFNγR knockout as wild type mice, indicating that IFNγ signaling in host cells was not critical. In contrast, despite the fact that Tc17 cells also secreted IFNγ, Tc17-mediated anti-tumor immunity was independent of the effect through IFNγ. Ironically, IFNγ was inhibitory to Tc17-mediated anti-tumor activity. Conclusions: Taken together, these studies demonstrate that both Tc1 and Tc17 cells can mediate effective anti-tumor immunity, but Tc1 is superior to Tc17. These findings also demonstrated for the distinct effect mechanisms of antigen-specific Tc1 and Tc17 cells in anti-tumor response, and direct IFNγ signaling on tumor cells is a key effect to eradicate established tumors mediated by Tc1 cells.

Disclosures:

No relevant conflicts of interest to declare.
Impact of Bendamustine Pretreatment on Stem Cell Mobilization and Autologous Stem Cell Transplantation in Patients with Multiple Myeloma
Authors: Pönisch Wolfram , Wiesler Julia , Wagner Ina , Leiblein Sabine , Edel Elvira , Al-Ali Haifa K. , Vucinic Vladan , Hegenbart Ute , Heiß Christiane , Niederwieser Dietger , Goldschmidt Hartmut
Published: October 2019

Abstract 1933

Introduction:

Bendamustine is a bifunctional alkylating agent with low toxicity that produces both single- and double-strand breaks in DNA, and shows only partial cross resistance with other alkylating drugs. Treatment of patients with newly diagnosed multiple myeloma using Bendamustine and Prednisone in comparison to Melphalan and Prednisone results in superior complete response rate and prolonged time to treatment failure (Poenisch et al, Res Clin Oncol 132: 205–212;2006). So far, however, reliable information on stem cell toxicity and mobilization of stem cells for autologous stem cell transplantation (SCT) after Bendamustine therapy is missing.

Material and Methods: A retrospective analysis of peripheral blood stem cell mobilization and autologous SCT was performed in 63 patients with multiple myeloma who had received Bendamustine pretreatment at the university Hospitals Leipzig and Heidelberg over a period of sixteen years. Patients had a median age of 59 (range, 31–72) years. The cumulative dosis of Bendamustine per patient ranged between 120 and 2400mg/qm and was administered during a median of three (range 1–10) cycles. The mobilization regimen consisted of Cyclophosphamide 4g/qm (n=41) or 7g/qm (n=4) and G-CSF (2×5ug/kg). Alternative regimens such as CAD, CED, TCED and others were also used in the remaining patients. Apheresis was started as soon as peripheral blood CD34+ counts exceeded 10×106/l with a harvest target of 4×106 CD34+/kg using 4 times the blood volume. The minimal accepted target was 2×106 CD34+/kg.

Results:

Stem cell mobilization and harvest was successful in 60 of the 63 patients (95 %). In 19 of 60 patients (32 %) a single apharesis was sufficient to reach the target. The median number of aphareses was two (range 1–7) and the median CD34+ cell-count/kg was 5.9 (range 1.7–20.4) x106. Information on autologous SCT is available from all 60 patients with successful harvest. Engraftment was successful in 59 of 60 patients. The median time to leucocytes count > l ×109/l was reached after 12 days and the time to untransfused platelet count of >50×109/l was 14 days. 54 patients (90%) responded after the autologous SCT with 6 CR, 4 nCR, 12 VGPR, and 32 PR. The event free survival at 36 months was 31 % and overall survival was 68 %.

In conclusion, the stem cell mobilization and autologous SCT is feasible in multiple myeloma patients who have received Bendamustine pretreatment.

Disclosures:

Pönisch: Mundipharma: Honoraria, Research Funding. Niederwieser:Mundipharma: Research Funding.
Cytomegalovirus (CMV) gB3 Genotype Is Associated with Acute Gvhd and CMV Disease in Allogeneic Hematopoietic Stem Cell Transplant Recipients (HSCT)
Authors: Dieamant Débora de Campos , Bonon Sandra Helena Alves , Aranha Francisco J P , Duarte Gislaine O. , Fernandes Virginio C.O. , Souza Carmino Antonio De , Costa Sandra Cecília Botelho , Vigorito Afonso Celso
Published: October 2019

Abstract 1958

Based on sequence variation in the UL55 gene that encodes glycoprotein B (gB), human cytomegalovirus (CMV) can be classified into four gB genotypes. Previous studies have suggested an association between CMV gB genotype and clinical outcome in patients who underwent an allogeneic hematopoietic stem cell transplant (HSCT). Objectives: The goals of this study were: identify patients with active infection caused by CMV in recipients of HSCT; determine the prevalence of CMV genotypes in the study group; correlate genotype with the CMV disease, acute GVHD and overall survival. Study design: The diagnosis of active CMV infection after allogeneic HSCT was detected by Antigenemia (AGM) and/or Nested-PCR (N-PCR). Positive samples from patients with active CMV infection were submitted to genotyping using the N-PCR to amplify a region of UL55, followed by restriction analysis based on HinfI and RsaI digestion. Real-time PCR (qPCR) was used to determine the viral load during active CMV infection and antiviral treatment. Results: Were evaluated 63 allogeneic HSCT recipients, 49/63 patients (78%) presented active CMV infection detected by AGM and/or N-PCR, in a median time of 38 days after the transplant. The distribution of CMV gB genotypes in these 49 patients with active CMV infection was as follow: gB1, 19/49 (38.8%); gB2, 17/49 (34.7%); gB3, 3/49 (6.1%); gB4, 7/49 (14.3%) and three patients (6.1%) had mixed infection with gB1+gB3, gB1+gB4 and gB2+gB4. Acute GVHD grade II-IV occurred in 17/49 (34.7%) patients: 8/19 (gB1-42%), 1/17 (gB2 - 5.9%), 4/4 (gB3 - 100%) and 4/9 (gB4 - 44.4%). The distribution of the frequency of acute GVHD grade II-IV between the genotypes was statistically different (p=0.008). CMV disease occurred in 3/49 (6.1%) patients, characterized for gastrointestinal disease and these three patients had infection with CMV gB3 genotype. This genotype of CMV was also associated with higher viral load during antiviral treatment and worse survival. Conclusions: This study demonstrated that the frequency of active CMV infection in HSCT population was high (78%). The most prevalent genotype in patients with active CMV infection was gB1 and gB3 genotype was associated with acute GVHD grade II-IV, CMV gastrointestinal disease, higher viral load during antiviral treatment and worse survival.

Disclosures:

No relevant conflicts of interest to declare.
Prophylaxis of Acute Gvhd Using Stromal Cell Therapy: Preliminary Results After Administration of Single or Multiple Doses of Multistem® in a Phase I Trial
Authors: Maziarz Richard T. , deVos Timothy , Bachier Carlos , Goldstein Steven C. , Leis Jose F. , Perry Robert , Deans Robert , Hof Wouter J Van't , Lazarus Hillard M.
Published: October 2019

Abstract 1987

Background.

MultiStem®, a multipotent adult progenitor cells (MAPC) collection, is an immune-modulatory, bone marrow-derived adult adherent allogeneic stromal cell product manufactured to large scale, assuring consistency of universal donor cell product during clinical evaluation. Pre-clinical studies showed safety of IV infusion and survival benefit in a haploidentical transplantation rat model of acute GVHD (Kovacsovics 2008, 2009).

Methods.

An open label Phase I clinical dose escalation study is being conducted with the primary goal to assess safety of MAPC as an adjunct treatment for human subjects undergoing myeloablative allogeneic HSCT for hematologic malignancies. Patients have been enrolled for MultiStem® administration as a single dose or in multiple weekly doses. Infusional toxicity and RRTs are assessed for 30 days following the last study drug dose administration. Secondary endpoints include incidence of acute GVHD, infection and survival through day 100. Dose escalation is guided by the Continual Reassessment Method (CRM).

Results.

In the single dose arm, 18 patients with AML (9), MDS (4), CML (3) and ALL (2) from 5 clinical centers were administered MultiStem® IV at 1, 5, or 10 million cells per kg at day 2 after allogeneic HSCT. There was no observed infusional toxicity. Two patients experienced Bearman RRTs (Gr 3 mucositis; Gr 3 renal & pulmonary failure) deemed unrelated to study product. Engraftment occurred in all 18 patients. The median time to neutrophil engraftment for related (n=7) and unrelated (n=11) HSCT was 15 days (range, 12–16 days) and 14 days (range, 11–25 days), respectively. The 100-day cumulative incidence of Grade II-IV and III-IV GVHD was 28% and 6% (n=18). In the highest MultiStem® dose group (n=9) there was no grade III-IV GVHD, and only one case of grade II GVHD, which subsequently resolved with treatment. 17/18 patients have been enrolled in the multidose arm. Results of safety endpoints and interim results of all secondary endpoints of repeated MultiStem® administrations following HSCT will be presented.

Conclusion.

Single dose administration of MultiStem® is well-tolerated, without observation of infusional toxicity or graft failure. Together with the observed low incidence of severe aGVHD, these findings are supportive of the concept that stromal stem cell therapy is safe and may be harnessed as an efficacious therapeutic option for acute GVHD prophylaxis following HSCT.

Disclosures:

Maziarz: Athersys, Inc: Patents & Royalties, Research Funding. Perry:Athersys, Inc.: Employment. Deans:Athersys, Inc: Employment. Van't Hof:Athersys, Inc.: Employment.
The Impact of Anti-HLA Antibodies on Allogeneic Hematopoietic Stem Cell Transplantation Outcomes After Reduced-Intensity Conditioning Regimens
Authors: Detrait Marie Y. , Dubois Valerie , Sobh Mohamad , Morisset Stephane , Tedone Nathalie , Nicolini Franck E. , Labussiere Helene , Gillis Lilia , Barraco Fiorenza , Cannas Giovanna , Ducastelle Sophie , Fattoum Jihane , Thomas Xavier , Chelghoum Youcef , Michallet Mauricette
Published: October 2019

Abstract 2038

Introduction:

Anti-HLA antibodies are associated with several complications in solid organ transplantations but their impact after allogeneic hematopoietic stem cell transplantation (HSCT) is not very well defined yet.

Patients and methods:

To evaluate the relevance of anti-HLA antibodies, we have retrospectively analyzed 107 peripheral blood allogeneic HSCT after reduced-intensity conditioning (RIC) regimen between 2005 and 2010. Acute myeloid leukaemia (n=52,48.5%) and multiple myeloma (n=18,17%) were the most common diagnosis in the cohort. The detection of anti-HLA antibodies was systematically performed in all patients before transplantation.

Results:

We found in 24 patients (22%) the presence of anti-HLA antibodies. There was no association between the presence of anti-HLA antibodies and engraftment, incidence of relapse or acute GvHD. The presence of anti-HLA antibodies was associated with worse survival in univariate analysis (HR 2.04; [95%CI,1.21–3.44], p=0.0056) and in multivariate analysis (HR 2.63; [95%CI, 1.32–5.25], p=0.006). The 3-year probability of OS was 34% (95%CI, 24–49) without anti-HLA antibodies and 16% (95%CI, 6–41) in their presence (Figure 1). The other significant factors on survival were the disease status at transplantation, the age, minor and major ABO incompatibilities between donor and recipient, the sex-mismatching and the CMV status. Moreover, the positive anti-HLA antibodies group showed a trend of higher TRM in univariate analysis (p=0.07) and in multivariate analysis (HR= 1.9; [95%CI, 0.94 – 3.9], p=0.076). The TRM at 3 years after transplantation were 31% (95%CI, 26–37) without anti-HLA antibodies and 46% (95%CI, 36–57) in their presence (figure 2). The causes of the pejorative effect of the anti-HLA antibodies were not defined yet but we observed microangiopathy associated with vascular endothelium damage which could be related to the presence of anti-HLA antibodies.

Conclusion:

Our study suggests that anti-HLA antibodies should be tested and considered as an important prognostic factor for transplant outcomes after RIC HSCT.

Disclosures:

No relevant conflicts of interest to declare.
The Association of a Single Nucleotide Polymorphism in the Chemokine CXCL10 Gene with Transplant Outcomes After HLA-Matched Unrelated Bone Marrow Transplantation for Low Risk Hematologic Malignancies
Authors: Nakata Katsuya , Espinoza Luis J. , Onizuka Makoto , Fukuda Takahiro , Kawase Takakazu , Morishita Eriko , Kodera Yoshihisa , Morishima Yasuo , Ohtake Shigeki , Nakao Shinji , Takami Akiyoshi
Published: October 2019

Abstract 2048

CXCL10, also known as interferon gamma inducible protein 10, is a chemokine secreted by several cell types including monocytes, endothelial cells and fibroblasts that plays important roles in the stimulation and migration of natural killer cells and T cells. The single nucleotide polymorphism (SNP) rs3921 located in the 3'-untranslated region of the CXCL10 gene has been reportedly associated with acute GVHD and with invasive aspergillosis after allogenic stem-cell transplantation. In this study, we analyzed the impact of rs3921 polymorphism on transplant outcomes in patients undergoing unrelated HLA-matched myeloablative bone marrow transplantation (BMT) through the Japan Marrow Donation Program. The CXCL10 genotypes were retrospectively analyzed in a cohort of 652 pairs of patients with hematologic malignancies and their unrelated donors. The genotype frequencies of GG, CG and CC were 1%, 13% and 86% in recipients and 1%, 12% and 87% in donors (P=0.62). The recipient GG or CG genotype was associated with significantly better overall survival (OS; P=0.02; Fig1). No difference was noted in transplant-related mortality, disease relapse or graft-versus-host disease in relation to the recipients' polymorphism. The multivariate analysis showed that in patients with low risk disease, the recipient GG or GC genotype remained statistically significant for better OS (hazard ratio [HR] 0.55; 95% confidence interval [CI], 0.30 to 1.01; P=0.05). The donor CXCL10 genotypes did not significantly influence the transplant outcomes. These results suggest an association of the recipient GG or GC genotypes with better transplant outcomes after unrelated HLA-matched BMT. These findings could therefore be useful in creating therapeutic strategies such as risk-adapted management of transplanted patients.

Disclosures:

No relevant conflicts of interest to declare.
Population-Based Health Care Cost Estimates Related to Non-Hodgkin Lymphoma (NHL)
Authors: Mittmann Nicole , Cheung Matthew , Isogai Pierre K , Saskin Refik , Liu Ning , Hoch Jeffrey S , Leighl Natasha , Trudeau Maureen E , Evans William K , Dainty Katie N , Earle Craig C.
Published: October 2019

Abstract 2068

Background:

There is limited information on the cost of non-Hodgkin lymphoma (NHL) management. This study provides population-based estimates of the costs for individuals with NHL from a Canadian perspective using provincial administrative databases.

Methods:

All individuals residing in Ontario with a diagnosis of first incident NHL from the Ontario Cancer Registry (2005–2009) were matched to non-NHL controls. Matching was based on age (same birth year), geography, income quintile, and resource utilization bands (2 years prior to cancer diagnosis). Each NHL case was matched to a maximum 5 controls. NHL cases from the Ontario Cancer registry were linked with their unique and encrypted health card number to provincial health claims databases (Ontario Ministry of Health and Long-Term Care and Cancer Care Ontario). Resources for this analysis included physician visits, hospitalizations, emergency room visits, medications, home care and same day surgeries. Unit costs (in 2009 Canadian dollars) were applied to resources. All costs were inflated to 2009. Costs were presented as the mean annual cost for all patients and by clinical stage.

Results:

There were 13,336 NHL cases matched to 65,668 controls. The NHL and control groups were demographically similar. For both groups, the median (25th and 75th percentile) age was 68 (56 to 77) years and 55% were male. Geographically, 86% of both groups were from urban areas. Total and disaggregated mean annual costs for NHL and controls are presented in the table. The mean cost difference between the NHL and control groups represents the mean cost attributable to NHL. Inpatient hospitalization and medication costs attributable to NHL represented 51% and 30% respectively, of the total mean annual cost attributable to NHL. Clinical stage at NHL diagnosis was available for a subset of the NHL cases (37%). Total mean annual cost by stage is also presented in the table. For the entire cohort, mean annual cost attributable to NHL was $16,778. In comparison, mean annual cost attributable to NHL by stage at diagnosis ranged from $9,575 (stage I) to $26,099 (stage IV).

Conclusion:

This study provides total and stage-specific cost estimates for NHL where attributable costs were 3 to 7 fold higher than those for non-NHL controls and increased by stage. Cost drivers included medications and inpatient care. Further work will focus on costing other resources including radiation, other chemotherapies and chronic care.

Disclosures:

No relevant conflicts of interest to declare.
Toll-Like Receptor 4 Mediates Heme Induced Acute Lung Injury: Preclinical Study of Resatorvid in Sickle Cell Disease
Authors: Ghosh Samit , Adisa Olufolake , Yang Yu , Tan Fang , Ofori-Acquah Solomon F
Published: October 2019

Abstract 2113

Sickle cell disease (SCD) is characterized by multiple exacerbating events that cause intravascular hemolysis. Heme released into the circulation is scavenged by multiple plasma proteins and delivered to the liver for degradation. Our recent data indicate that this process is impaired in SCD resulting in excess protein-free plasma heme (PFPH) that triggers a lethal form of acute lung injury (ALI) in mice. In this study, we tested the hypothesis that toll-like receptor 4 (TLR4) mediates heme-induced ALI. Wild-type and two TLR4 mutant strains (B6.B10ScN-Tlr4lps-del/JthJ and C3H/HeJ) were intravenously injected with a dose range of ferric heme (0–210 micromoles/kg) and respiratory function monitored using a pulse oximeter. Excess PFPH was associated with reductions in oxygen saturation (SpO2) and breath rate in the wild-type mice but not in the TLR4 variants. Lungs of heme-treated wild-type mice were congested, edematous, hemorrhagic, and had thickened alveolar walls, while no histological abnormalities were found in the TLR4 variants. All heme-treated wild-type mice succumbed within 2 hours, while all TLR4 variants survived. Transgenic mice expressing exclusively human sickle hemoglobin (SS) were intravenously injected with a small molecule TLR4 inhibitor (resatorvid/TAK-242), or a lipid vehicle prior to induction of lung injury with heme (35 micromoles/kg). TAK-242 preserved lung function in the majority of SS mice that failed to scavenge excess PFPH, while both SpO2 and breath rate deteriorated in vehicle treated mice. The unique response to heme by TAK-242 and vehicle-treated SS mice was supported by histological analysis and survival (TAK-242; 76.9% vehicle; 23.5%, n=13–17; log-rank survival test, p<0.01). We provide the first evidence that the interaction between heme and TLR4 can be pathological, specifically causing a lethal form of ALI. Our data on TAK-242, a phase II drug, offers an attractive option to explore TLR4 inhibition as a novel therapeutic strategy to limit progression of acute chest syndrome.

Disclosures:

Ofori-Acquah: Emory University: Patents & Royalties.
Heme Induced Organ Failure in Sickle Cell Disease Is Spatiotemporally Determined by the Amount of Heme Released Into the Bloodstream
Authors: Ghosh Samit , Adisa Olufolake , Aryee Doreen , Tan Fang , Ofori-Acquah Solomon F
Published: October 2019

Abstract 2135

Circulating heme is scavenged by multiple plasma proteins and delivered to the liver for degradation. We have recently demonstrated that heme scavenging is variably impaired in transgenic mice with sickle cell disease (SCD). In this study, we tested the hypothesis that excess protein-free plasma heme (PFPH) in the blood circulation, and excess scavenged protein-bound plasma heme (PBPH) destined for degradation cause different types of organ damage in SCD. Transgenic mice expressing exclusively human sickle hemoglobin (SS) were intravenously injected with a dose of heme (25 micromoles/kg) to elevate PBPH only, or with a dose (70 micromoles/kg) sufficient to raise PFPH. In agreement with our previous findings, PFPH was associated with severe lung injury and 100% lethality within 2 hours. This phenomenon occurred independent of any liver involvement. Modest elevation of circulating heme sufficient to raise PBPH only, increased alanine aminotransferase and aspartate aminotranferase 3- to 4-fold (P<0.001) within 12 hours, caused 55% lethality by 72 hours, with severe liver damage but no lung involvement. There was no effect on lung and liver function in control AA and AS mice administered the same doses of heme given to the SS mice. Recombinant human hemopexin rescued SS mice from heme induced lung damage and sudden death, however, the same group of mice developed severe liver damage and succumbed 24–48 hours later. Finally, in a cohort of 27 patients with SCD, a sudden drop in hemoglobin of 0.5 to 4.0 g/dl was associated with a marked increase in ALT 3–7 days later (p=0.027). Collectively, these results show for the first time that acute elevation of circulating heme causes two distinct organ pathologies with different mortality profiles in SCD. Therapeutic strategies based solely on enhanced scavenging of excess heme may provide short-term pulmonary benefits with deleterious long-term effects on liver function.

Disclosures:

Ofori-Acquah: Emory University: Patents & Royalties.
Quaternary Organization of GPIb-IX Complex and Insights Into Bernard-Soulier Syndrome Revealed by the Crystal Structures of GPIbβ Ectodomain and a GPIbβ/GPIX Chimera
Authors: McEwan Paul A. , Yang Wenjun , Carr Katherine H. , Mo Xi , Zheng Xiaofeng , Li Renhao , Emsley Jonas
Published: October 2019

Abstract 2195

Platelet glycoprotein (GP)Ib-IX receptor complex contains three subunits, GPIbα, GPIbβ and GPIX, which assemble with a ratio of 1:2:1. Dysfunction in surface expression of the complex leads to Bernard-Soulier syndrome (BSS). We have crystallized the GPIbβ ectodomain (GPIbβE) and determined the structure to reveal a single leucine-rich repeat with N- and C-terminal disulfide bonded capping regions. The central region of the structure can be divided into concave parallel β-sheet and convex loops. The crystal structure of a GPIbβE/GPIXE chimera that contains three non-continguous convex loops of GPIX and retains a GPIbβ-binding site of GPIX (Mo et al. J. Thromb. Haemost. 7:1533–40, 2009) was also determined. The chimera, but not GPIbβE, forms a homotetramer in the crystal, revealing a quaternary interface between GPIbβ and GPIX ectodomains. Central to this interface is residue Tyr106 from GPIbβ that inserts into a shallow and largely hydrophobic pocket generated by two convex loops from GPIX. Mutagenesis studies confirmed this interface as a valid representation of interactions between GPIbβ and GPIX in the full-length complex. Eight GPIbβ missense mutations identified from BSS patients were examined in transiently transfected Chinese hamster ovary cells for changes to the GPIb-IX complex surface expression. Six of the eight mutations lead to secretion defect and/or misfolding of GPIbβE. In contrast, the other two mutations, A108P and P74R, were found to maintain normal secretion and folding of GPIbβE but were unable to support GPIX surface expression. The close structural proximity of these mutations to Tyr106 and the GPIbβE interface with GPIX indicates that residues Ala108 and Pro74 in GPIbβ are located at the GPIbβE/GPIXE interfaces. Based on the tetrameric arrangement of the chimera structure, we propose a structural model for the GPIb-IX complex that embodies its organizing principles and helps to provide mechanistic insights on its assembly, function and regulation.

Disclosures:

No relevant conflicts of interest to declare.
Thrombin Modulates PAR1-PAR4 Heterodimers
Authors: de la Fuente Maria , Arachiche Amal , Nieman Marvin T.
Published: October 2019

Abstract 2203

Thrombin is a potent platelet agonist. Thrombin activates platelets and other cells of the cardiovascular system by cleaving its receptors, protease activated receptor 1 (PAR1), PAR4 or both. PARs are G-protein coupled receptors that activate cellular signaling through Gq and G12/13. There is structural evidence that GPCRs, as a class, function as dimers and that dimerization can alter signaling specificity. Our previous studies have determined that PAR4 forms homodimers and have mapped the homodimer interface to transmembrane helix 4 (TM4). We have also shown that coexpression of PAR1 with PAR4 lowers the threshold for PAR4 activation by thrombin ∼10-fold. The purpose of the current study is to examine the physical interaction between PAR1 and PAR4 and how these interactions influence PAR1's ability to enhance PAR4 activation. The PAR1-PAR4 heterodimers were examined by bioluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementation (BiFC). Similar to our previous studies with PAR4 homodimers, PAR1 homodimers were constitutive and did not require receptor activation. In contrast, PAR1-PAR4 heterodimers were not detected under basal conditions. However, when the cells were stimulated with 10 nM thrombin, we were able to detect a strong interaction between PAR1 and PAR4. We next examined if PAR1-PAR4 heterodimers would be induced by stimulating PAR1 or PAR4 individually with their agonist peptides TFLLRN (100 μM) or AYPGKF (500 μM), respectively. The agonist peptides were unable to induce heterodimers when added to the cells individually or simultaneously. These data demonstrate that PAR1 and PAR4 require allosteric changes induced by receptor cleavage by thrombin to mediate heterodimer formation. To examine this further, we removed 37 amino acids from the C-terminus of PAR1, which disrupts the eighth helix. The truncated PAR1 was able to form constitutive heterodimers with PAR4 and these heterodimers were unaffected by thrombin. These data suggest that PAR1 is the allosteric modulator of the PAR1-PAR4 heterodimers. Finally, the stability of the constitutive PAR1 and PAR4 homodimers was unchanged in response to thrombin or the agonist peptides. Taken together, these data suggest that PAR1 and PAR4 have a dynamic interaction depending on the context of their expression. Since PAR1 is an attractive antiplatelet target, the molecular interactions of this receptor on the cells surface must be taken into account when developing and characterizing these antagonists.

Disclosures:

No relevant conflicts of interest to declare.
Novel Polyphosphate-Binding Proteins in Human Plasma and Platelet Releasates
Authors: Smith Stephanie A. , Collins Julie N. , Choi Sharon H. , Morrissey James H.
Published: October 2019

Abstract 2270

Introduction:

Inorganic polyphosphates (polyP) are negatively charged, linear phosphate polymers found throughout biology. PolyP is secreted in substantial amounts by activated human platelets and it also accumulates in many infectious microorganisms. We have shown that polyP is a potent hemostatic regulator, acting at multiple points in the clotting cascade; in particular, polyP triggers the contact pathway, enhances FXI activation by thrombin, accelerates factor V activation, and modulates fibrin clot structure. Since there may be other, as-yet undiscovered (patho)physiologic roles for polyP, we undertook a study to identify the major polyP-binding proteins in human plasma and in releasates from activated human platelets.

Methods:

PolyP was end-labeled by biotinylation, then bound to streptavidin coated onto magnetic beads (Dynabeads), polystyrene microtiter plates, or surface plasmon resonance sensorchips. PolyP-Dynabeads were exposed to plasma or to releasates from platelets activated by TRAP; the beads were then washed repeatedly, and polyP-binding proteins were eluted with 1 M LiCl. Eluted proteins were separated by 2-D gel electrophoresis and were identified by mass spectrometry. Some additional proteins known or suspected to bind to polyP were also analyzed in parallel. Binding affinities for selected proteins were quantified by microtiter plate assays or by surface plasmon resonance approaches.

Results:

Candidate poly-P binding proteins identified in plasma pull-downs included parvulin, factor H, complement C1q, complement C1q tumor necrosis factor-related protein, C4 binding protein, and apolipoprotein E. Candidate poly-P binding proteins identified in pull-downs of platelet-releasate included platelet factor 4, multimerin, thrombospondin, and cyclophilin. High affinity binding to polyP was also quantified for thrombin, FXI, FXIa, kallikrien, histones and the isolated polyphosphate-binding domain of E. coli exopolyphosphatase.

Conclusions:

PolyP binds with high affinity to certain proteins that participate in the coagulation cascade as well as several additional proteins. Some of these are involved in reactions known to be modulated by polyP, but several have yet to be evaluated. Further investigation is indicated into potential roles for polyP in modulation of the complement cascade, prolyl isomerase activity, angiogenesis, and platelet responses.

Disclosures:

Smith: University of Illinois: Patents & Royalties. Choi:University of Illinois: Patents & Royalties. Morrissey:University of Illinois: Patents & Royalties.
Generic Low Molecular Weight Heparins May Not Have the Same Safety –Efficacy Profile As the Branded Low Molecular Weight Heparins in Acute Coronary Syndrome
Authors: Hoppensteadt Debra , Jeske Walter , Walenga Jeanine M. , Lewis Bruce E , Fareed Jawed
Published: October 2019

Abstract 2308

Low molecular weight heparins such as enoxaparin and dalteparin are widely used for the management of Acute Coronary Syndrome (ACS). Recently, several generic versions of enoxaparin and dalteparin have been approved in various countries for all of the branded product's indications. However, no data on their clinical equivalence in ACS is available. Since generic versions of enoxaparin and dalteparin are manufactured by different processes and may use starting material from different sources, these drugs may differ in their pharmacological profile in simulated ACS settings. To compare the pharmacodynamic effects, a branded version of enoxaparin was compared with three generic versions in a primate model at a dosage of 1 mg/kg IV. Such pharmacokinetic parameters as TFPI release, TAFI modulation, vWF release, and TF mediated platelet activation were measured. Anticoagulant effects of these drugs were also measured after IV administration using iSTAT ACT and aPTT. Simulated catheter related thrombosis studies were carried out to differentiate each agent in contact, intrinsic and extrinsic clotting systems. The generic versions of enoxaparin namely, Cutenox (Gland Pharma), Fibrinox (Sandoz AG) and Versa (EuroPharma) exhibited product based pharmacodynamic differences compared with the branded product and the results are provided in the following table. Each of the generic products exhibited its own specific pharmacologic profile despite comparable molecular weight distributions and anti-Xa potencies. Significant differences were noted in the anticoagulant effects of each of these agents as compared with the branded products. Some differences were also noted in HIT antibody mediated aggregation studies. The differences between the branded and generic versions of LMWHs may be due to the higher dosages used and the IV administration which leads to higher circulating levels of these agents. These observations suggest the need for additional animal studies and clinical trials to validate the use of generic versions of LMWHs in such critical indications as ACS and related syndromes.

Disclosures:

No relevant conflicts of interest to declare.
Infusion of Recombinant Full-Length ADAMTS13 and Carboxyl-Terminal Truncated Variant Alters Composition of Ferric Chloride-Induced Arterial Thrombi in Adamts13−/− mice
Authors: Skipwith Christopher G. , Xiao Juan (Jenny) , Weisel John W. , Zheng X. Long
Published: October 2019

Abstract 2313

Proteolytic cleavage of ultra large von Willebrand factor (ULVWF) released from endothelial cells by ADAMTS13 metalloprotease is critical for maintaining normal hemostasis. However, the effect of infusing ADAMTS13 on thrombus composition remains poorly understood. In this study, we determined the morphology and composition of thrombi formed in carotid arteries after topical application of FeCl3 in Adamts13−/− mice receiving PBS, recombinant human full-length ADAMTS13 (FL) and carboxyl-terminal truncated variant after spacer domain (S), using scanning electron microscopy and quantitative image analysis. We showed that in Adamts13−/− mice 5 min after FeCl3 injury, formed arterial thrombi were comprised ∼39% platelets, ∼26% red blood cells, and ∼35% fibrin. The arterial thrombi in these mice were structurally deformed. An infusion of recombinant FL (final concentration of 10 nM) significantly reduced the accumulation of platelets (∼18%) but increased the fibrin network (57%) without affecting the composition of red blood cells (∼25%) in the arterial thrombi formed at the same time point after FeCl3 injury. Similar effects on the morphology and composition of FeCl3-induced arterial thrombi were observed after infusion of recombinant S (10 nM) into Adamts13−/− mice. Kinetic analysis showed that there was a decrease in platelet accumulation over the time of 30 min during thrombus formation with a slight increase in accumulation of red blood cells and formation of fibrin in Adamts13−/− mice. But, the infusion of recombinant FL and S into Adamts13−/− mice restored the kinetics of platelet/red blood cell accumulation and fibrin formation to those observed in wild-type mice. Our findings, revealing the apparent difference in thrombus composition, provide novel insight into the mechanism of ADAMTS13 function in vivo, which may shed more light on the pathogenesis of thrombotic thrombocytopenic purpura and other arterial thrombotic disorders associated with deficiency of plasma ADAMTS13 activity.

Disclosures:

No relevant conflicts of interest to declare.
A Novel Nr4a1GFP Reporter System Reveals That Nr4a1 Expression Identifies Long-Term Hematopoietic Stem Cells
Authors: Land Ruben , Barlowe Trevor , Manjunath Shwetha , Eiger Sophie , Gross Matthew , Garrett Russell , Cunningham Nicole , Emerson Stephen G. , Punt Jenni A.
Published: October 2019

Abstract 2339

Recent studies have highlighted the importance of the NR4A nuclear receptor family (Nur77 (Nr4a1), Nurr1 (Nr4a3), Nor1 (Nr4a2)) in the regulation of hematopoiesis. In murine models, NR4A gene deficiencies lead to aberrant proliferation of hematopoietic stem cells, and can lead to acute myeloid leukemia (AML). NR4A gene deficiencies also appear to be a feature in human AML cells. In order to better understand the pattern of expression and function of NR4A family members during normal hematopoiesis, we have developed a novel reporter mouse where the Nr4a1 promoter drives GFP expression (Nr4a1GFP). Our analyses reveal a hierarchy in Nr4a1 expression among bone marrow hematopoietic stem cells: long-term (LT) HSC's (CD150+CD48-LSKs) express the highest levels of Nr4a1GFP, more mature HSC's and multilineage progenitor populations (CD150+CD48+ and CD150-CD48+ LSKs) express intermediate levels, and common myeloid progenitors (CMLs, defined as Lin-c-kit+sca-1-) express no Nr4a1GFP. Interestingly, circulating LSK's in the spleen express Nr4a1GFP at higher levels than their bone marrow counterparts. In support of data suggesting that Nr4a family members regulate quiescence, we find that 1) all hematopoietic stem cells that remain in the bone marrow after acute (36h) 5-FU treatment express Nr4a1GFP, 2) Nr4a1GFP expression decreases among circulating splenic LSKs 48 hours after treatment with PolyI:C, and 3) Nr4a1GFP expression increases markedly when stem cells are cultured in vitro under conditions that promote quiescence. We will use this novel system to more directly address the role of Nr4a1 expression in hematopoiesis by evaluating the cell cycle status and defining the reconstitution potential of HSC's on the basis of their Nr4a1GFP expression.

Disclosures:

No relevant conflicts of interest to declare.