bluuebunny/crossref_metadata_2025 huggingface dataset:
Bioluminescent Monitoring of Microenvironmental Effects on Multiple Myeloma Engraftment In a Humanized Mouse Model
Authors: Wu Christina , Leu Heather , Ma Wenxue , Shih Alice , Carson Dennis , Kulidjian Ana , Jamieson Catriona
Published: October 2019

Abstract 1927

Multiple myeloma (MM) is characterized by clonal proliferation of CD138+ plasma cells in the bone marrow (BM) and remains an incurable disease. Recent identification of a rare population of MM cancer stem cells (MM CSC) is phenotypically similar to memory B cells (CD138- CD34- CD19+) but differs in that they have the self-renewal capacity within the BM and may be responsible for drug resistance. Preclinical testing of novel therapeutic strategies that target MM CSC requires animal models that closely resemble human disease and allow quantitative evaluation of the applied therapy. We have previously reported results on establishing a MM animal model by transplanting MM CSC from autologous mobilized peripheral blood of primary MM patients, transduced with lentiviral luciferase GFP (GLF) and transplanted intrahepatically (IH) into neonatal RAG2/gc double knock-out (RG-KO). Here we evaluate engraftment efficiency in consideration of BM microenvironment by comparing CD45+ human cell engraftment in mice transplanted either IH to neonates or intrafemorally (IF) to gamma-irradiated young adult mice. MM CSC were selected from isolated PBMC after Ficoll gradient centrifugation of fresh BM biopsy from two primary MM patients or from a human MM cell line, H929, followed by immunomagnetic bead depletion of CD34+ and CD138+ cells. The cells were transplanted into RG-KO mice ranging from 53,000 to 10⋀6 cells per mouse either IH or IF. Mice transplanted with GLF-transduced MM CSC were imaged with an in vivo imaging system (IVIS) to detect bioluminescent engraftment. Results showed that bioluminescence signal levels were detected in mice transplanted IF with 53,000 MM CSC per mouse even before 3 weeks by ventral view and as early as 5 weeks by lateral view. To date, tumor growth was only discovered in mice transplanted IH with 2 × 10⋀6 unselected MM PBMC from a fresh BM biopsy as early as 10 week post-transplantation. FACS analysis of these mice demonstrated successful engraftment with the presence of CD45+, CD19+ and CD138+ population in tumor, bone marrow, spleen and liver. In addition, expression of clonal light chain restriction in myeloma cells confirmed myeloma engraftment. Future studies will focus on expression of genes involved in sonic hedgehog pathway as analyzed by PCR to confirm the self-renewal capacity. Moreover, investigations on the effect of B cell-activating factor (BAFF) in BM microenvironment by transplanting MM CSC into BAFFxRG-KO mice are in progress.

Disclosures:

Jamieson: Bristol-Meyers Squibb: Research Funding.
High Mitochondrial Membrane Potential Identifies Patients with Myeloproliferative Neoplasms with a More Aggressive Natural History
Authors: Gardner Jeffrey R , Abdel-Wahab Omar , Frattini Mark , Jurcic Joseph G , Knapp Kristina , Rosenblat Todd L. , Soff Gerald A. , Levine Ross , Heaney Mark
Published: October 2019

Abstract 1992

The myeloproliferative neoplasms (MPN) can have a variable natural history. Polycythemia vera and essential thrombocythemia, in particular, are conditions that can extend over decades, but some patients have clinical progression to myelofibrosis or acute myeloid leukemia. As first articulated by Warburg, cancers are metabolically distinguished from normal tissues by the use of glycolysis under aerobic conditions. To metabolically characterize the blood cells of patients with myeloproliferative neoplasms, we measured the mitochondrial membrane potential using the cyanine dye, JC-1. In examining cells derived from the blood and/or marrow of 159 patients with primary myelofibrosis, polycythemia vera and essential thrombocythemia, we found that the mitochondrial membrane potential (FL2/FL1=electrochemical potential/mitochondrial mass) was elevated compared to the blood cells of normal individuals. Thirty five percent of patients with polycythemia vera and essential thrombocythemia had normal MMP. In contrast, 97% of patients with primary myelofibrosis, post-polycythemia myelofibrosis, post-essential thrombocythemia myelofibrosis and acute myeloid leukemia following an MPN had evidence of cell populations with higher mitochondrial membrane potential. Cells with distinctly higher mitochondrial membrane potential could be indentified in platelets and polymorphonuclear leukocytes; however the MMP of lymphocytes was normal, indicating that the alteration in metabolic state likely occurred in a multipotential myeloid stem cell. Cell populations were confirmed by co-staining with anti-CD19, -CD45, -GlycophorinA and -β3-integrin antibodies. Sequential analysis of patient samples found that the acquisition of higher mitochondrial membrane potential was stable and persistent over 2 years or more of follow up and that elevated membrane potential predisposed patients to disease progression. The balance of patients (65%) with ET had evidence of increased MMP suggesting the possibility of disease in an early state of evolution to a more aggressive condition. The increased MMP did not correlate with the presence of mutation in JAK2. These results indicate that clinically advanced MPN can be characterized by changes in mitochondrial physiology that might be identified non-invasively by flow cytometric staining with JC-1. In addition, the early nature of these changes may help to identify therapeutic targets.

Disclosures:

No relevant conflicts of interest to declare.
Thrombocytopenia Induced-Tumor Vascular Leakiness Increases Drug Efficacy
Authors: Demers Mélanie , Ho-Tin-Noé Benoit , Schatzberg Daphne , Yang Janie J , Wagner Denisa D.
Published: October 2019

Abstract 2105

Important research goals in cancer therapy include delivering high doses of drug to tumor sites for maximum treatment efficacy while minimizing side effects to healthy organs. We have recently demonstrated that platelets contribute to tumor vascular homeostasis through prevention of severe tumor hemorrhage. In order to define a new way of increasing drug delivery to the tumor site, we hypothesized that the selective induction of tumor vascular leakiness by thrombocytopenia may facilitate the delivery of chemotherapeutic agents to tumors. Using a mammary carcinoma murine model, we showed that platelet depletion induced hemorrhage specifically at the tumor site and favored the accumulation of fluorescently-labeled microspheres in the tumor. Moreover, the induction of thrombocytopenia in tumor-bearing mice before injection of the chemotherapeutic drug paclitaxel significantly reduced tumor growth compared to mice with normal platelet count that were treated with paclitaxel. Analysis of the tumors ex vivo revealed an increase in tumor apoptosis and a reduction in tumor cell proliferation in thrombocytopenic mice that received therapy. No increased toxicity was seen on other organs and blood cells. Taken together, our results indicate that low platelet count selectively induces leakiness of tumor vessels and allows better access of a chemotherapeutic agent to the tumor, enhancing its tumoricidal effect.

Disclosures:

No relevant conflicts of interest to declare.
Heparin Prevents Histone-Induced Thrombocytopenia and Mortality
Authors: Fuchs Tobias A. , Wagner Denisa D.
Published: October 2019

Abstract 2106

Histones and DNA are released from activated neutrophils to form extracellular traps (NETs) which bind and kill bacteria. Histones contribute to this antimicrobial activity, but they are a double-edged sword because they are a major mediator of death in sepsis. Histones are cytotoxic and intravenous infusion of histones into mice can lead to death. Recently we have shown that NETs provide a scaffold supporting platelet adhesion, activation and thrombus formation. NETs and histones were found in deep vein thrombi. Here we show that platelets in blood are preferentially targeted by histones. Histone binding to platelets induces calcium influx, recruits fibrinogen and causes platelet aggregation which depends on the interaction of histones, fibrinogen and αIIβ3-integrins. Mice challenged intravenously with histones at sublethal concentrations developed a rapid and profound thrombocytopenia reflecting platelet aggregation in vivo. Heparin administration abolished histone interaction with platelets and prevented thrombocytopenia. Heparin also prevented histone induced cytotoxicity towards endothelial cells and rescued mice after a challenge with a lethal dose of histones. The biological function of heparin stored in granules of mast cells is not known. We propose that it may be to counteract histone-mediated thrombosis and injury during innate immune response.

Disclosures:

No relevant conflicts of interest to declare.
Multicenter Retrospective Study on the Development of Peripheral Lymphocytosis During Second-Line Dasatinib Therapy for Chronic Myeloid Leukemia
Authors: Lee Ha Yeon , Lee Su Jin , Ha Boram , Yi Jun Ho , Jung Chul Won , Kim Dae-Young , Lee Je-Hwan , Lee Kyoo Hyung , Sohn Sang Kyun , Kim Sung Hyun , Lee Won-Sik , Park Joon Seong , Yhim Ho-Young , Kwak Jae-Yong , Kim Hyeoung Joon , Kim Yeo-Kyeoung , Kim Inho , Park Seonyang , Kim Dong Hwan (Dennis)
Published: October 2019

Abstract 2275

Background:

Dasatinib is known to induce large granular lymphocyte (LGL) expansion, which correlates with better clinical efficacy. The current retrospective study attempted to investigate the incidence of lymphocytosis following second-line dasatinib therapy in chronic myeloid leukemia (CML) and to analyze the clinical factors predictive of the development of lymphocytosis, as well as association with treatment outcomes.

Method:

Fifty CML patients who failed imatinib treatment and received dasatinib for 3 months or more, were enrolled from 9 centers in the Republic of Korea. The cumulative incidence of lymphocytosis was assessed, and cytogenetic and molecular response, treatment failure, loss of response, progression to advanced disease, and survival were evaluated and analyzed according to the development of lymphocytosis.

Results:

After a median of 17 months of dasatinib therapy, complete cytogenetic (CCR) and major molecular response (MMR) was noted in 23 and 16 patients, respectively. Twenty three patients (46%) developed lymphocytosis following dasatinib therapy (median onset 4 months). No clinical predictive factor for the development of lymphocytosis was found. The cytogenetic response was significantly better in the group that developed lymphocytosis (LC+), as compared to the group without lymphocytosis (LC-); the LC+ group showed a higher complete cytogenetic response (CCyR; 78.3% vs. 29.6%, p=0.001) and major molecular response (MMR; 52.2% vs. 14.8%, p=0.005), in comparison to the LC- group. The development of lymphocytosis after dasatinib was identified as a favorable independent marker for predicting a CCyR (p=0.002) or MMR following dasatinib therapy (p=0.003).

Conclusion:

The present study suggested that 1) lymphocytosis following dasatinib therapy is not rare phenomenon with incidence of 46%; 2) it might be associated with higher response following dasatinib therapy. Further study is necessary to identify which subset of lymphocytes was expanded and to reveal the exact mechanism by which dasatinib induces lymphocyte expansion.

Disclosures:

No relevant conflicts of interest to declare.
Mast Cells Reduce Gvhd Severity In Allogeneic Transplantation by Reducing the Proliferation of Conventional T Cells
Authors: Leveson-Gower Dennis B , Sega Emanuela I , Kalesnikoff Janet , Florek Mareike , Galli Stephen J , Negrin Robert
Published: October 2019

Abstract 243

Mast cells are important effector cells in many innate and adaptive immune responses, and can influence the function of several immune cells including granulocytes, monocytes/macrophages, dendritic cells, T cells, B cells, NK cells, and NKT cells. In contrast to their well-know pro-inflammatory roles, in certain models, mast cells have been shown to decrease inflammation and tissue injury. We explored the biological activity of mast cells in a well-established model of mouse graft-versus-host disease (GVHD) by comparing C57BL/6-KitW-sh/W-sh mice (which lack mast cells) to wild-type (WT) C57BL/6 (H-2b) recipients in a major-MHC mismatched model of allogeneic transplantation. After myeloablative irradiation, recipients received 5×106 T-cell depleted bone marrow cells from FVB/N donors (H-2q) on day 0 followed by transfer of 2×106 FVB/N CD4 and CD8 conventional T cells (Tcon) at a 2:1 ratio on day 4. A dramatic decrease in survival was observed in the animals lacking mast cells where 100% of KitW-sh/W-sh recipients died by day 15 following transplantation while over 50% of WT recipients were alive at day 60 (p < 0.0001). The exacerbated GVHD in KitW-sh/W-sh mice correlated with an increase in Tcon proliferation as indicated by bioluminescence imaging (BLI), particularly in lymph node, liver, and gastrointestinal tract tissue sites (p < 0.001). The percentage of Foxp3 positive Treg cells was similar before and after transplantation in KitW-sh/W-sh and WT recipients, and CD4+CD25hi Treg from KitW-sh/W-sh mice were capable of suppressing T cell proliferation in a mixed leukocyte reaction. When KitW-sh/W-sh mice were given 5×106 bone marrow-derived cultured mast cells (BMCMCs) i.p., mast cell re-population of the gut and peritoneal cavity was indicated by both BLI and traditional staining methods. Preliminary experiments with KitW-sh/W-sh recipients engrafted with BMCMCs i.p. 6 weeks before allogenic transplantation demonstrated improved survival in the above GVHD model, but not if the BMCMCs were derived from IL-10-/- C57BL/6 mice. Together, these results support the hypothesis that mast cells can decrease Tcon proliferation and reduce GVHD severity by a mechanism that involves production of IL-10 by mast cells but which is independent of CD4+CD25+ Treg.

Disclosures:

No relevant conflicts of interest to declare.
Profiling of Aberrant DNA Methylation In AML Reveals Subclasses of CpG Islands with Epigenetic or Genetic Association
Authors: Gebhard Claudia , Sadeh Mohammed , Glatz Dagmar , Schwarzfischer Lucia , Spang Rainer , Ehninger Gerhard , Thiede Christian , Andreesen Reinhard , Rehli Michael
Published: October 2019

Abstract 2498

CpG islands show frequent and often disease-specific epigenetic alterations during malignant transformation, however, the underlying mechanisms are poorly understood. We used methyl-CpG immunoprecipitation (MCIp) to generate comparative DNA methylation profiles of 30 patients with acute myeloid leukemia for human CpG islands across the genome. DNA methylation profiles across 23.000 CpG islands revealed highly heterogeneous methylation patterns in AML with over 6000 CpG islands showing aberrant de novo methylation in AML. Based on these profiles we selected a subset of 380 CpG islands (covering 15.000 individual CpGs) for detailed fine-mapping analyses of aberrant DNA methylation in 185 patients with AML (50% normal karyotype). We found that a proportion of patients (5/185) displayed a concerted hypermethylation at almost all studied loci, representing the rare CpG island methylator phenotype (CIMP) in AML. Meta analysis of methylation profiling and published ChIP sequencing data separated CpG islands in two groups. A highly correlated subgroup of CpG island regions was strongly associated with histone H3 lysine 27 trimethylation in human hematopoietic progenitor cells, suggesting that disease-related de novo DNA methylation at these CpG islands is linked with polycomb group protein (PcG)-mediated repression. The group of mainly non-PcG target CpG islands showed heterogeneous methylation patterns across patients and unsupervised hierarchical clustering revealed a correlation of methylation profiles with genetic disease markers, including oncofusion proteins as well as CEBPA- and NPM1-mutations. Our study suggests that both epigenetic as well as genetic aberrations may underlay AML-related changes in CpG island DNA methylation states.

Disclosures:

No relevant conflicts of interest to declare.
Amelioration of Murine Immune Thrombocytopenia by CD44 Antibodies: a Potential Therapy for ITP?
Authors: Crow Andrew R. , Song Seng , Suppa Sara J , Lazarus Alan H
Published: October 2019

Abstract 2528

Intravenous immunoglobulin (IVIg) is used to treat autoimmune diseases such as ITP. IVIg is a limited resource and its dosage and cost are both high. Although considered safe, it will always carry a theoretical risk of transferring infectious disease. Thus it would thus be highly desirable to improve the efficacy of IVIg or develop monoclonal antibodies, capable of mimicking the clinical effects of IVIg. Work by others has successfully improved the efficaciousness of IVIg in murine models of autoimmunity: It has been shown that enriched sialylated IVIg has a therapeutic effect at a 1 log fold lower dosage than IVIg; and a sialylated recombinant Fc fragment of IgG functions successfully at a 2 log fold lower dosage than IVIg.

CD44 is a widely expressed cell surface polymorphic glycoprotein. It is involved in many processes including tumour metastasis and inflammation. CD44 antibodies have been successfully used to treat several murine autoimmune disease models, including inflammatory arthritis and experimental autoimmune encephalomyelitis, although their mechanism of action remains unclear. To explore the potential for monoclonal antibodies as a treatment for ITP as well as further explore their mechanisms of action, we tested 8 monoclonal CD44 antibodies in murine ITP, and found 4 antibodies which could successfully ameliorate ITP; 2 of these antibodies function at a full 3 log fold lower dosage as compared to IVIg. Further characterization of the 2 most successful antibodies (5035-41.1D and KM114) demonstrated that, similar to IVIg, i) the expression of the inhibitory IgG receptor FcγRIIB was required for their ameliorative function and ii) complement-depleted mice also responded to anti-CD44 treatment. Dissimilar to IVIg, the Fc portion of the CD44 antibody was not required: an F(ab')2 fragment of antibody KM114 also significantly ameliorated thrombocytopenia at an equivalent molar concentration as intact KM114. Thus while KM114 functions by an FcγRIIB sensitive mechanism, FcγRIIB is unlikely a direct target of the Fc region of this antibody and likely plays a downstream role in the amelioration of immune thrombocytopenia, similar to IVIg. These data demonstrate that CD44 antibodies can function therapeutically in murine ITP and we speculate that they could potentially provide a very low dose recombinant therapy for the treatment of ITP.

Disclosures:

No relevant conflicts of interest to declare.
Serious Adverse Drug Reactions (sADRS) Associated with Hematopoietic Growth Factors: A Systematic Review From the Southern Network on Adverse Reactions (SONAR) Program
Authors: Norris LeAnn , Qureshi Zaina , Barnato Sara , Lai Stephen , Bennett Charles
Published: October 2019

Abstract 2555

Erythrpoiesis stimulating agents (ESAs) and the granulocyte hematopoietic growth factors granulocyte colony stimulating factor (G-CSF) and pegylated G-CSF revolutionized supportive care approaches to anemia and neutropenia, respectively. Thrombopoietin-receptor agonists improve antibody-mediated thrombocytopenia management. For these drugs, as with all agents, a comprehensive understanding of sADRs is important, however, often difficult to do. We reviewed sADRS described in meta-analyses and web-site materials maintained by regulatory agencies and manufacturers reports (1989 to July 2010; n=2,094). Overall, 28 systematic reviews, six package inserts, three medication guides, four FDA Risk Evaluation and Mitigation Systems (REMS) materials, 16 Summaries of Product Characteristics/Product Assessment Reports were included. For ESAs, common sADRs associated with increased relative risks (RRs) included: mortality among cancer patients with chemotherapy-associated anemia or cancer patients not receiving active treatment (RRs: 1.1–1.2); cerebrovascular and cardiovascular events among chronic kidney disease patients ((RRs: 1.3, 1.9), and venous thromboembolism in the cancer setting (RRs;16-1.7). For G-CSF/pegylated-GCSF, rare instances of myelodysplasia/acute myeloid leukemia (MDS/AML) associated with statistically increased RRs were reported among breast cancer patients receiving chemotherapy in trials reported from one NCI-sponsored clinical trials group, one meta-analysis, and one review of Medicare/SEER databases (RR: 1.9–2.1), although the absolute risks were very small (< 2 per 100,000 treated patients). Other rarely reported sADRs (total N < 20) included splenic rupture following G-CSF stimulation of healthy peripheral blood stem donors. Bone marrow fibrosis and collagen deposition were reported with thrombopoietin receptor agonists, although the implications of this finding is uncertain. Also, instances of VTEs were reported among persons who developed high platelet counts following administration of these thrombopoeitin receptor agonists. SADRs are commonly associated with ESA administration in a range of settings, are uncommon occurrences with G-CSF/pegylated G-CSF, and are not well characterized for thrombopoieitin receptor agonists. Formal pharmacovigilance initiatives are ongoing, including FDA mandated REMS with restricted distribution requirement for ESAs in the cancer setting (APPRISE) and the NEXUS and the PROMACTA CARES programs for thrombopoeitin receptor agonists.

ESAs (Cancer) ESAs (CKD) G-CSF/Pegylated G-CSF VTE Mortality Cerebrovascular and CVD AML/MDS RR 1.57 and 1.67 1.10 to 1.17 1.34 and 1.92 2.14 and 1.92 95% CI 1.31–1.87 and 1.35–2.06 1.02–1.20 and 1.06–1.30 1.03–1.74 and 1.38–2.26 1.12–4.08 and 1.19–3.07 Baseline Risk Absolute Risk Common Common Common Rare (2/100,000)

Disclosures:

Bennett: Pfizer: Consultancy.
Ethnicity Significantly Influences Incidence Patterns and Predicts Mortality: Adult Hodgkin Lymphoma (HL) In the United States (US)
Authors: Evens Andrew M , Shiuan Eileen , Kwon Soyang , Gordon Leo I , Chiu Brian
Published: October 2019

Abstract 2683

Background:

Pediatric HL studies have suggested survival differences based on ethnicity. However, little data is available regarding the impact, if any, of ethnicity on incidence patterns, disease histology, and/or survival among adult HL.

Methods:

We examined data for 13 US SEER areas, several of which contain large Hispanic and Black populations. Case information was obtained from the 11/2009 SEER data submission released April 2010. We analyzed incidence, HL histology, and mortality rates according to ethnicity, age, and gender. We also examined incidence patterns across the past four decades. All analyses used SEER*Stat.

Results:

A total of 16,783 HL cases were diagnosed among residents in the 13 SEER registry areas during 1992–2007, with non-Hispanic Whites contributing the largest number (n=11,890), followed by Hispanics (n=2,190), and Blacks (n=1,724). Consistent with SEER 9 results (1973 data), Whites show a continued bimodal age-incidence curve (6.0/100,000 ages 25–29, 2.5/100,000 ages 50–54, and 4.5/100,000 age 75–79). However, Blacks have a much less apparent bimodal pattern (4.5/100,000 ages 25–29, 2.6/100,000 ages 50–54, and 3.0/100,000 ages 75–79), while Hispanics are distinctly not bimodal with a small increase at 20–24 (2.4/100,000) followed by an exponential-like increase with peak HL incidence at ages 80–84 (7.0/100,000). Moreover, among persons >65 years, HL is currently significantly more common in Hispanics than Whites (4.7-7.0/100,000 vs 3.9–4.5/100,000, respectively, p<0.05). With gender, HL is more common in males than females, regardless of ethnicity. Interestingly, the male excess, however, does not occur until ages 30–34 (all ethnicities). Furthermore, from 1975–2007, HL incidence increased in Black females (annual percent change (APC) = 2.5; p<0.05) and White females (APC = 0.4; p<0.05). According to histology, both nodular sclerosis and mixed cellularity are more common in Whites followed by Blacks and Hispanics, while in persons age 60–84, both histologies are significantly more common in Hispanics compared with Whites and Blacks. Over the past 20 years, mortality has declined within each race by 10.3%–13.7% (p<0.05). However, age-specific ethnic survival disparities are apparent (Figure 1). For ages 65–84, Hispanics have a significantly increased mortality rate compared with Whites/Blacks (p<0.05). Conversely, among ages 20–44, Hispanics have a lower mortality rate versus Whites and Blacks.

Conclusions:

Multiple important epidemiologic and mortality differences are evident across and within ethnicities in adult HL.

*Both sexes (1992-2007). Rates are per 100,000. Mortality source: US Mortality Files, National Center for Health Statistics, CDC. Accessed August 12th, 2010.

Disclosures:

No relevant conflicts of interest to declare.
The Novel Organic Arsenical, Darinaparsin (ZIO-101), Induces Apoptosis through AKT and MEK/ERK Pathways In Hodgkin Lymphoma (HL) and T-Cell Lymphoma (TCL) Cell Lines
Authors: Bhalla Savita , Prachand Sheila , Gordon Leo I , Evens Andrew M
Published: October 2019

Abstract 2840

Background:

The inorganic arsenic, arsenic trioxide (ATO), has a narrow therapeutic index, which has limited its clinical use in most malignancies. Darinaparsin (ZIO-101, S-dimethylarsino-glutathione), synthesized by conjugating dimethylarsenic to glutathione, is a novel organic arsenical that is under investigation as a novel agent for the treatment of cancer. Furthermore, early-phase clinical trials with darinaparsin have demonstrated low toxicity and encouraging clinical efficacy in relapsed/refractory hematologic malignancies.

Methods:

We treated several TCL cell lines (Jurkat, C10MJ, Hut-78, and MT2) and the resistant HL cell line, L428, with increasing concentrations of darinaparsin (0.5-5μM) +/− the MEK inhibitor, U0126, or ERK siRNA (Qiagen HiPerFect transfection). Cell survival and apoptosis were measured by MTT and Annexin-V/propidium iodide staining, respectively. Further, tumor intracellular darinaparsin and ATO concentrations were assessed with mass spectrometry, while transcription pathway intermediates were analyzed by Western blotting.

Results:

Darinaparsin inhibited cell growth and induced apoptosis in all cell lines at 1–3μM. At 2μm (48 hours), darinaparsin induced approximately 80% apoptosis in each of the four TCL lines, while 3μM resulted in 65% apoptosis in L428 cells. By comparison, >10μM of ATO (48 hours) was required to induce 40% apoptosis in TCL and 25% apoptosis in L428. At 1–3μM, darinaparsin induced significant increases in caspase 3 and PARP activation in TCL, while interestingly, minimal caspase or PARP was observed in L428. Notably, in L428 cells at 1 hour, mass spectrometry showed that intracellular accumulation of darinaparsin was >10-fold higher as compared with equivalent ATO concentrations (p<0.01). We also treated L428 cells with U0126 (5μM) or ERK2 siRNA, both combined with darinaparsin. Pre-incubation with U0126 or siRNA knock down of ERK2, followed by treatment with darinaparsin, significantly enhanced darinaparsin-induced apoptosis (p<0.05). To further investigate darinaparsin-induced signaling pathways, we analyzed phospho-AKT (p-AKT), and phospho-ERK (p-ERK) in Jurkat and L428. We found down-regulation of p-AKT in Jurkat as well as L428 cells, while total AKT remained unchanged. Additionally, an increase in p-ERK was observed in L428 cells with 2–3μM darinaparsin, while p-ERK was down-regulated in Jurkat cells.

Conclusions:

Darninaparsin induces significant cell death in HL and TCL cell lines that is mediated through AKT and MEK/ERK-based pathways. Additionally, markedly higher intracellular darinaparsin levels are achieved in lymphoma cells compared with equivalent concentrations of ATO. Continued pre-clinical and clinical trial investigation of darinaparsin in HL and TCL is warranted.

Disclosures:

No relevant conflicts of interest to declare.
Targeting the PI3K/AKT/Survivin Pathway with Ritonavir In Therapy-Resistant Mantle Cell Lymphoma
Authors: Nordgren, Tara M , Hegde Ganapati , Munger Corey , Vose Julie M. , Joshi Shantaram
Published: October 2019

Abstract 2855

Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma accounting for about 6% of non-Hodgkin's lymphoma cases in the US. While multiple therapy regimens are available to treat MCL, patients ultimately relapse within 3–4 years from therapy-resistant MCL, making MCL carry the worst prognosis of all non-Hodgkin's B cell lymphomas. To improve therapies for patients with MCL, we must understand the biological causes for relapse and therapy-resistance in MCL and develop mechanisms to target the unique properties in relapsing MCL. Recently, we have isolated and produced therapy-resistant MCL cell lines by inoculating NOD-SCID mice with a human MCL cell line, Granta-519 (GP), and subsequently treating with CHOP chemotherapy regimen plus bortezomib. When mice relapsed following therapy, tumor cells were isolated from the kidneys and livers, and cultured to produce stable cell lines, named GRK and GRL, respectively. GRK and GRL cells exhibited increased proliferative and therapy-resistant properties in vitro, and GRL showed increased aggressiveness in vivo also, as compared with GP. Using quantitative PCR (qRT-PCR) to assess the activation of pathways relating to lymphoma progression, the PI3K/Akt/survivin pathway was found to be differentially activated in GRL and GRK compared to GP, including a 6.5 fold increase in survivin in GRL. Therefore, we inhibited survivin in GRL using the FDA-approved protease inhibitor ritonavir (Abbott Laboratories), as recent studies suggest this compound is able to downregulate survivin in lymphoblastoid B cells (Dewan, et al. Int J Cancer 2009; 124[3]: 622–629). When GRL cells were incubated with ritonavir plus vincristine or doxorubicin, MTT assays showed significant inhibition of cell proliferation/survival (p < 0.05) compared to the effects of ritonavir alone. Also, flow cytometric analysis of Annexin V demonstrated dose-dependent induction of apoptosis upon ritonavir treatment in GRL. In addition, qRT-PCR analysis demonstrated a decrease in mRNA expression of the pro-survival genes cyclin D2, Rel A, survivin, and BCL2 in GP and GRL cells after ritonavir treatment. Together, these studies demonstrate the potentials of utilizing ritonavir in a combined treatment regimen designed to target therapy-resistant MCL. As ritonavir is already an FDA-approved drug, these studies hold promise for expediting future in vivo and clinical studies that may eliminate therapy-resistant cells responsible for relapse in MCL patients.(This research was supported by the Lymphoma Research Foundation, New York, NY).

Disclosures:

No relevant conflicts of interest to declare.
Monosomal Karyotype In Primary Myelofibrosis Is Prognostically Worse Than Otherwise Unfavorable Karyotype
Authors: Vaidya Rakhee , Caramazza Domenica , Begna Kebede , Gangat Naseema , Van Dyke Daniel L. , Hanson Curtis A. , Pardanani Animesh , Tefferi Ayalew
Published: October 2019

Abstract 3069

Background:

Monosomal karyotype (MK) is defined as the presence of two or more distinct autosomal chromosome monosomies or a single autosomal monosomy associated with at least one structural abnormality (Breems DA et al. J Clin Oncol 2008; 26: 4791). In acute myeloid leukemia (AML), MK has been shown to be prognostically worse than complex or other unfavorable karyotype (Breems DA et al. J Clin Oncol 2008; 26: 4791). In primary myelofibrosis (PMF), complex karyotype or isolated trisomy 8 predicts inferior survival (Hussein K et al. Blood 2010; 115: 496).

Objective:

To determine if MK in PMF is prognostically distinct from previously defined poor cytogenetic risk categories including complex karyotype and isolated trisomy 8.

Methods:

The Mayo Clinic database for PMF was used to identify consecutive patients with unfavorable karyotype including complex karyotype and sole trisomy 8. WHO criteria were used for PMF diagnosis and leukemic transformation (Vardiman JW et al. Blood 2009; 114: 937).

Results:

Among 793 PMF patients with cytogenetic information at the time of their first time referral to the Mayo Clinic, 452 displayed a normal karyotype and 341 (43%) an abnormal karyotype. Of the latter, 41 (12%) displayed complex karyotype and 21 (6%) sole trisomy 8. Among the 41 patients with complex karyotype, 17 (42%) met the criteria for MK and 24 (58%) displayed complex karyotype without monosomies.

Overall survival was significantly inferior in patients with MK compared to those with either complex karyotype without monosomies (p=0.02; HR 2.3, 95% CI 1.1–4.8) or trisomy 8 (p=0.02; HR 2.4, 95% CI 1.2–5.1) (Fig. 1). Prognosis among all three groups was significantly worse than patients with normal karyotype (Fig. 1).

Leukemia-free survival was also significantly inferior in patients with MK compared to those with either complex karyotype without monosomies (p=0.02; HR 6.9, 95% CI 1.3–37.3) or trisomy 8 (p=0.02; HR 14.8, 95% CI 1.7–130.8) (Fig. 2). LFS in patients with normal karyotype was similar to those with either complex karyotype without monosomies (p=0.31) or trisomy 8 (p=0.86) (Figure 2).

Conclusions:

Monosomal karyotype in PMF is distinctly associated with extremely poor overall and leukemia-free survivals that are significantly worse than those seen in PMF patients with other unfavorable karyotype including complex karyotype without monosomies and sole trisomy 8 abnormalities.

Disclosures:

No relevant conflicts of interest to declare.
Down-Regulation of Signal Transducer and Activator of Transcription 3 Enhances Acute Myeloid Leukemia-Derived Dendritic Cell Function
Authors: Brady Michael T , Miller Austin , Sait Sheila , Ford Laurie A , Hans Minderman , Wang Eunice S. , Lee Kelvin P , Baumann Heinz , Wetzler Meir
Published: October 2019

Abstract 3140

Immunotherapies, including stimulating patients' immune system with acute myeloid leukemia (AML)-derived dendritic cell (DC) vaccines, are being sought for the treatment of AML. However, these vaccines have proven to be less successful than anticipated. We have previously shown that STAT3 is constitutively activated in the blasts of approximately half of AML patients and to correlate with poor prognosis. STAT3 regulates a variety of cellular events and has been shown to play a role in immunosuppression by inhibiting DC differentiation, resulting in induction of T cell tolerance rather than T cell activation. We hypothesize that AML-DC with constitutively activated STAT3 are impaired to fully differentiate and to efficiently stimulate T cells. The current study investigated the correlation between STAT3 activity, DC maturation and the ability to stimulate allogeneic T cells in primary AML-derived DCs. AML blasts differentiated to DCs regardless of the level of activated STAT3, however AML-DC with high levels of activated STAT3 elicited less T cell response than AML-DC with low levels of activated STAT3. Knock-down of STAT3 protein with inhibitory shRNAmirs in 4/4 patient AML-DCs increased stimulation of allogeneic T cells compared to control DCs without affecting the immunophenotype or endocytotic activity. Treatment of AML-DC with the STAT3 inhibitors AG490, arsenic trioxide (ATO), JSI-124 and NSC-74859 during early differentiation did not significantly enhance T cell stimulation. Treatment with ATO, but not the remaining inhibitors, during the last 24 hours of maturation led to more mature phenotype and enhanced T cell stimulation in 2/2 AML cell lines and 8/9 patient samples (p=0.0001) while having minimal effect on normal cord blood-derived DC. ATO-treated AML-DCs had an increased expression of the co-stimulatory markers CD80 and CD86, the mature DC marker CD83 and increased HLA-DR expression indicating more mature DCs. Four of six ATO-treated AML-DCs secreted higher amounts of the T cell stimulating cytokine interleukin (IL)-12p40 (average increase 2.5 fold); exogenous administration of IL-12 recapitulated ATO's effect. These data demonstrate that AML-DCs have improved immunogenicity after reducing STAT3 protein levels during differentiation. The observation that treatment with ATO is superior to the more targeted JAK/STAT inhibitors suggests that DC maturation is regulated by other pathways in addition to STAT3. We propose to use ATO-treated AML-DC vaccines in future clinical trials. ATO treatment would not only lead to more immunogenic vaccines, but would also increase the number of mature AML-DCs that could be differentiated from each patient, solving two of the major obstacles for the use of AML-DC vaccines.

Disclosures:

No relevant conflicts of interest to declare.
Dominant Negative Effect of Palmitoylation-Deficient NRAS In Suppression of BCR/ABL Leukemogenesis
Authors: Cuiffo Benjamin , Ren Ruibao
Published: October 2019

Abstract 3157

RAS mutations are found in ∼30% of all human cancers, with NRAS being the RAS isoform found most frequently mutated in hematological malignancies including acute myelogenous leukemia (AML) and chronic myelomonocytic leukemia (CMML). We have previously shown that expression of oncogenic NRAS efficiently induces a CMML- or AML-like disease in mice. Like all RAS proteins, NRAS must undergo a series of post-translational modifications for differential targeting to distinct cellular membrane microdomains. We have previously found that palmitoylation, in addition to prenylation, is an essential process for NRAS leukemogenesis. The results suggest that targeting RAS palmitoylation may be an effective therapy for hematological malignancies as well as other NRAS related cancers.

In addition to the direct activation by mutations, RAS can also be functionally activated by other oncogenic mutations, including activated protein tyrosine kinases such as BCR/ABL. While significant advances have been made in the treatment of chronic myelogenous leukemia (CML) by targeting the kinase activity of BCR/ABL, these treatments do not constitute a cure, as BCR/ABL-positive cells persist and eventually develop mutations that cause resistance to the kinase inhibitors. In addition, although advances have also been achieved in the treatment of BCR/ABL+ B-acute lymphoblastic lymphoma (B-ALL) through the use of combination chemotherapy along with the ABL kinase inhibitor imatinib, BCR/ABL+ B-ALL continues to pose a huge challenge; they are highly refractory to imatinib, and are still the least treatable subtype of ALL. The identification of molecular targets that impede the progression of BCR/ABL+ leukemias not relying on targeting the kinase activity of BCR/ABL is likely beneficial. Previous studies have shown that RAS is a critical downstream transducer of the oncogenic signaling by BCR/ABL. We have shown that palmitoylation-deficient oncogenic NRAS is mislocalized away from the plasma membrane yet still capable of binding GTP in cells. This finding suggests that palmitoylation-deficient, activated RAS may have a dominant negative effect in RAS signaling. As a proof-of-concept study for treating cancers driven by RAS regulators through blocking RAS palmitoylation, we evaluated the effect of blocking RAS palmitoylation in BCR/ABL leukemogenesis. We found that expression of palmitoylation-deficient oncogenic NRAS or palmitoylation-deficient wild type NRAS significantly impeded progression of BCR/ABL induced B-ALL- and CML-like diseases in mice. The palmitoylation-deficient NRAS suppresses the activation of downstream effectors of RAS, possibly by sequestrating RAS effectors away from the plasma membrane. The results suggest that targeting RAS palmitoylation may also constitute an effective therapy in cancers driven by oncogenes upstream of RAS.

Disclosures:

No relevant conflicts of interest to declare.
Prevalence of Thrombophilia In Patients with Sickle Cell Disease and Osteonecrosis
Authors: Al-Khabori Murtadha K. , Pathare Anil , Al Farsi Khalil , Al Huneini Mohammed , Alkindi Salam
Published: October 2019

Abstract 3184

Background:

Osteonecrosis (ON) of the femoral and humeral heads is frequently seen in patients with sickle cell disease (SCD). Earlier studies reported a high prevalence of thrombophilia in patients with ON.

Aims:

To study the prevalence of thrombophilia in patients of SCD with ON.

Methods:

Case records of SCD patients with ON were retrospectively reviewed for protein S, protein C, and anti-thrombin deficiency, along with activated protein C resistance (APCR).

Results:

A total of sixty-three patients were identified, 35 of whom were males, with a median age of 21 years (range 15to 46). Median haemoglobin, total white cell count and platelet counts were 10 g/dL (range 7.7to 13.3), 7.5 ×109/L (range 3.4 to 16.7) and 302 ×109/L (range 72 to 1101) respectively. Twenty-eight patients were on hydroxyurea. Thrombophilia testing showed that 29% (95% confidence interval: 17–40), 47% (95% CI: 29–64) and 79% (95% CI: 65–93) of the patients had low levels of functional (<60 unit/dl), total antigenic (<70 unit/dl) and free antigenic (<70 unit/dl) protein S respectively, while 21% (95% CI: 10–31) and 67% (95% CI: 46–87) had low levels of functional (<70 unit/dl) and antigenic (<70 unit/dl) protein C respectively. In addition, 14% (95% CI: 5–23) and 22% (95% CI: 0–56) of the patients had low levels of functional (<80 unit/dl) and antigenic (<80 unit/dl) anti-thrombin levels respectively. Only 2% (95% CI: 0–5) of these patients had an abnormally low APCR (APCR ratio ≤2.3).

Summary/Conclusions:

Patients with SCD and ON have a high prevalence of thrombophilia. These results indicate that a prospective study with more detailed thrombophilia work-up, along with confirmatory DNA analysis, as well as a study of the role of prophylactic anticoagulation in such patients is highly warranted.

Disclosures:

Pathare: Sultan Qaboos University: Employment, Research Funding. Alkindi:Sultan Qaboos University: Employment, Research Funding.
Detection of Lupus Anticoagulant In Women with Obstetric complications
Authors: Avigliano Andrea Laura , Galli Juan Carlos , Grand Beatriz
Published: October 2019

Abstract 3185

Introduction:

Lupus anticoagulant (LA) is one of the laboratory criteria for the diagnosis of antiphospholipid syndrome. To detect the presence of LA, ISTH recommends performing two assays based on different principles. Our LA profile includes dilute Russell viper venom time (dRVVT) and two different activated partial thromboplastin time (aPTT) reagents.

The aim of the study was to evaluate sensitivity and specificity of aPTT and dRVVT in women with a history of pre-eclampsia, intra-uterine growth restriction, early recurrent abortion, late fetal loss and placental abruption collectively termed “placenta mediated complications”.

Materials and Methods:

We studied a total of 247 patients and 287 samples in a 10 months period. Based on consensus criteria, samples were retested within a period of three months. 62/247 plasmas samples were collected during pregnancy.

Laboratory studies: Screening tests: aPTT was performed using PTT-LA (Stago) and an aPTT home made reagent with diluted cephalin. dRVVT was performed using Russel viper venom RVV (Stago) and diluted cephalin. Negative control plasma was prepared according to ISTH LA recommendations for the mixing tests. Confirmatory tests: We performed a home made reagent for platelet neutralization procedure (PNP)-aPTT and dRVVT; Rosner Index and % of Correction as criteria for mixing and confirmatory tests interpretation.

Statistical evaluation was performed by ROC method using EP Evaluator 9.9 software.

Results:

172/287 samples (59.9%) were negative, while 115/287 samples (40.1%) were positive for LA. 44/115 samples (38.26%) were positive for both tests (aPTT and dRVVT), 60/115 samples (52.17 %) were positive only for dRVVT and 11/115 samples (9.57%) were positive only for aPTT.

LA was confirmed in 9/62 pregnant women samples (14.5 %). 7 (11.3%) had abnormal dRVVT, the remaining 2 samples were positive for both tests.

Conclusions:

Our results demonstrated: 1- High prevalence of dRVVT positive plasmas 104/115 (90.43%); 2- Despite showing a low performance regarding sensitivity when compared to dRVVT, both aPTT tests were able to detect LA in 47.83% of the positive studies. 3- According to our experience, considering this initial cohort of selected patients, the use of dRVVT as the first and aPTT as the second screening test may help improve the performance for the diagnosis of LA.

Disclosures:

No relevant conflicts of interest to declare.
The Attitude of Canadian University Students towards a Behaviour-Based Blood Donor Health Assessment Questionnaire
Authors: Go Stephanie Lynn , Lam Cindy Tze-Yung , Lin Yahui Tammy , Wong Deborah Joanne , Lazo-Langner Alejandro , Chin-Yee Ian H.
Published: October 2019

Abstract 3342

Background:

As part of Canadian Blood Services policy (CBS), men who have sex with men (MSM) are indefinitely deferred from donating blood due to an increased risk for HIV infection. This policy has generated controversy, especially amongst student populations, due its perceived discrimination against homosexual men.

Objectives:

1) To determine the acceptability of a behaviour-based donor health questionnaire amongst Canadian university students and assess its suitability as an alternative to the current MSM policy. 2) To determine the perception of blood safety associated with specific risk behaviours.

Methods:

We conducted a survey amongst students at the University of Western Ontario in London, Ontario, Canada. A questionnaire was designed that included questions found on the current CBS donor health survey, and behaviour-based questions derived from studies assessing high risk factors for HIV. For each question, participants were asked to rate its acceptability and its perceived effect on blood safety using 7-point Likert scales. They were also asked whether the question would deter them from future donations. Data was analyzed using non-parametric tests.

Results:

741/3500 (21.2%) students participated in the study. Students rated both current and behavioural questions as equally acceptable and the majority (89-97%) would not be deterred from donating blood in the future by any question. The response scores for both sets of questions were significantly correlated. Questions on donor's sexual practices were rated less acceptable compared to those on high risk behaviours. Sexually transmitted infections, injection drug use and sex for money were considered high risk behaviours for blood safety (69-95% unsafe). In contrast, sexual practices (MSM, anal intercourse, condom use, knowledge of partner's sexual background) were rated less important for blood safety (30 to 62% unsafe). We found an inverse correlation between the perception of safety and the acceptability of questions (Table). 24.4% of students rated both questions on MSM status and a behaviour-based alternative as equally unacceptable.

Discussion:

A behaviour-based screening modification is unlikely to change opinions or satisfy those who object the current MSM policy. Acceptability of these questions might be related to a poor understanding of the effect of sexual practices on blood supply safety.

Disclosures:

No relevant conflicts of interest to declare.