ALBERT

Description: Key Points L5 is elevated in ischemic stroke patients, and its receptor, LOX-1, plays a critical role in increasing stroke size. L5 induces platelet secretion of Aβ to potentiate platelet activation and aggregation via LOX-1 and IKK2.

Description: Emerging evidence suggests that progression of hematologic malignancies is associated with angiogenesis. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can provide global and functional imaging of tumor angiogenesis. In this study, we performed bone marrow DCE-MRI prospectively at diagnosis and after induction chemotherapy in 78 de novo acute myeloid leukemia (AML) patients and correlated it with treatment outcome. An algorithm to assess bone marrow angiogenesis by measuring the DCE-MRI time-intensity curve pixel by pixel was developed using 3 distinct parameters: peak enhancement ratio (Peak) to indicate tissue blood perfusion; amplitude (Amp) to reflect vascularity; and volume transfer constant (K trans) to indicate vascular permeability. The Peak and Amp decreased significantly at remission status after induction chemotherapy. Patients with higher Peak or Amp at diagnosis had shorter overall survival and disease-free survival than others. Cox multivariate analysis identified higher Peak value (hazard ratio, 9.181; 95% confidence interval, 1.740-48.437; P = .009) as an independent predictor for overall survival in addition to unfavorable karyotype and old age. Our findings provide evidence that increased bone marrow angiogenesis measured by DCE-MRI can predict adverse clinical outcome in AML patients. DCE-MRI may help to select high-risk phenotype AML patients for tailored antiangiogenic therapy and to monitor treatment response.

Description: Abstract 2750 Neutrophil-derived microparticles (MP) have become important mediators of inflammation, coagulation and vascular homeostasis. MP carry surface antigens, proteins and adhesion molecules from their originating cell and can mediate intercellular cross-talk. Human neutrophil contain high levels of the anti-inflammatory protein annexin 1 (AnxA1), which contributes to mechanisms activated in the host to keep under control cell activation and trafficking in the resolution of inflammation. We investigated the role of MP released by all-trans retinoic acid (ATRA)-treated acute promyelocytic leukemic (APL; NB4) cells during the process of granulocytic differentiation. Materials & Methods: We determined the expression of AnxA1 and its receptor (FPRL1) on the NB4 cells and their MP by flowcytometry, real-time PCR and western blotting. The anti-inflammatory effect of AnxA1was determined by the transmigration assay. Results: AnxA1 was constitutively expressed on the surface of ATRA-untreated NB4 cells. ATRA treatment of NB4 cells can significantly enhance their surface expression of Anx-A1 in a time dependent manner, but did not change their mRNA expression or release of free AnxA1 into the conditioning medium. We further determined the amount of MP in the CM by flowcytometry. Significantly higher number of MP was released by the ATRA-NB4 cells, as compared with those by ATRA-untreated NB4 cells (p

Description: Three murine monoclonal antibodies, named 2H9, 1E9 and 1A2, were produced after immunization of BALB/c mice with cells of the SU-DHL-1 cell line from a true histiocytic lymphoma. In frozen sections from various lymphomas, 2H9 and 1A2 selectively stained the cell membranes of neoplastic cells in true histiocytic lymphoma and Hodgkin's disease. Antibody 1E9 stained the nuclear membranes of the tumor cells in true histiocytic lymphoma and malignant histiocytosis. No staining was seen in 56 cases of B and T cell lymphoma. Several tissue culture cell lines, including T cell acute lymphoblastic leukemia and pre-B cell lines, were not stained. With 2H9, however, a positive reaction was noted for two Epstein-Barr virus (EBV)-positive African Burkitt's lymphoma cell lines (Daudi and P3HRI), one human T cell lymphoma/leukemia-virus-positive cell line (HUT 102), and one EBV- transformed normal B lymphoblastoid cell line (RPMI 8057). In normal lymphoid tissues, 2H9 and 1E9 reacted with the nuclear membranes of histiocytes and interdigitating reticulum cells, whereas 1A2 stained only rare cells of an unknown type. All three antibodies failed to react with B or T cells in frozen tissue sections of normal lymphoid tissues. The use of these three antibodies should facilitate the diagnosis of histiocyte and interdigitating reticulum (IR) cell-related neoplasms, namely, true histiocytic lymphoma, malignant histiocytosis, and Hodgkin's disease. True histiocytic lymphoma and Hodgkin's disease exhibit similar reactivities with these three and with two other monoclonal antibodies (HeFi-1 and Tac), suggesting that these two types of lymphoma are related. In contrast, malignant histiocytosis was negative for 2H9, 1A2, Tac, and HeFi-1. The difference in the phenotypic expression of true histiocytic lymphoma and malignant histiocytosis indicates that they are two different disease entities.

Description: Three murine monoclonal antibodies, named 2H9, 1E9 and 1A2, were produced after immunization of BALB/c mice with cells of the SU-DHL-1 cell line from a true histiocytic lymphoma. In frozen sections from various lymphomas, 2H9 and 1A2 selectively stained the cell membranes of neoplastic cells in true histiocytic lymphoma and Hodgkin's disease. Antibody 1E9 stained the nuclear membranes of the tumor cells in true histiocytic lymphoma and malignant histiocytosis. No staining was seen in 56 cases of B and T cell lymphoma. Several tissue culture cell lines, including T cell acute lymphoblastic leukemia and pre-B cell lines, were not stained. With 2H9, however, a positive reaction was noted for two Epstein-Barr virus (EBV)-positive African Burkitt's lymphoma cell lines (Daudi and P3HRI), one human T cell lymphoma/leukemia-virus-positive cell line (HUT 102), and one EBV- transformed normal B lymphoblastoid cell line (RPMI 8057). In normal lymphoid tissues, 2H9 and 1E9 reacted with the nuclear membranes of histiocytes and interdigitating reticulum cells, whereas 1A2 stained only rare cells of an unknown type. All three antibodies failed to react with B or T cells in frozen tissue sections of normal lymphoid tissues. The use of these three antibodies should facilitate the diagnosis of histiocyte and interdigitating reticulum (IR) cell-related neoplasms, namely, true histiocytic lymphoma, malignant histiocytosis, and Hodgkin's disease. True histiocytic lymphoma and Hodgkin's disease exhibit similar reactivities with these three and with two other monoclonal antibodies (HeFi-1 and Tac), suggesting that these two types of lymphoma are related. In contrast, malignant histiocytosis was negative for 2H9, 1A2, Tac, and HeFi-1. The difference in the phenotypic expression of true histiocytic lymphoma and malignant histiocytosis indicates that they are two different disease entities.

Description: Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), accounting for roughly 30% of newly diagnosed cases in the United States (US). DLBCL is a heterogeneous lymphoma, including the activated B cell-like (ABC) and germinal center B cell-like (GCB) subtypes, which have different gene expression profiles, oncogenic aberrations, and clinical outcomes (Alizadeh, Nature 2000; Staudt, Adv Immunol 2005). ABC-DLBCL is characterized by chronic active B-cell receptor (BCR) signaling (Davis, Nature 2010), which is required for cell survival. Thus, the BCR signaling pathway is an attractive therapeutic target in this type of B-cell malignancy. Bruton's tyrosine kinase (BTK), which plays a pivotal role in BCR signaling, is covalently bound with high affinity by ibrutinib, a first-in-class BTK inhibitor approved in the US for mantle cell lymphoma and chronic lymphocytic leukemia (CLL) patients (pts) who have received at least one prior treatment, CLL with del17p, and WaldenstršmÕs macroglobulinemia. A recent phase 2 clinical trial of single-agent ibrutinib in DLBCL pts revealed an overall response rate of 40% for ABC-DLBCL (Wilson, Nat. Med 2015); however, responses to single kinase-targeted cancer therapies are often limited by the cellÕs ability to bypass the target via alternative pathways or acquired mutations in the target or its pathway (Nardi, Curr Opin Hematol 2004; Gazdar, Oncogene 2009). The serine/threonine-protein kinase PIM1 is one of several genes exhibiting differential expression in ibrutinib-resistant ABC-DLBCL cells compared with wild-type (WT) cells. We identified and report herein the role of PIM1 in ABC-DLBCL ibrutinib-resistant cells. Methods: PIM1 gene expression was analyzed by RT-qPCR. In vitro, cell viability was assessed in the human ABC-DLBCL cell line HBL-1 after treatment with ibrutinib and/or a pan-PIM inhibitor for 3 days, and the effect on colony formation was determined 7 days post-treatment. PIM1 mutational analysis was performed with clinical tumor biopsy samples from 2 studies, PCYC-04753 (NCT00849654) and PCYC-1106-CA (NCT01325701). PIM1 protein stability was analyzed by treating cells with cycloheximide and examining protein levels at different time points up to 8 hours. Results: Gene expression profiling of ibrutinib-resistant ABC-DLBCL cells revealed an upregulation of PIM1 (15-fold increase compared with WT cells) as well as PIM2 and PIM3. We also found that, compared with single-drug treatment, in vitro cell growth could be synergistically suppressed with a combination of ibrutinib and a pan-PIM inhibitor. This effect was observed in both WT (combination index (C.I.) = 0.25; synergy score = 3.18) and ibrutinib-resistant HBL-1 cells (C.I. = 0.18; synergy score = 4.98). In HBL-1 cells, this drug combination reduced colony formation and suppressed tumor growth in a xenograft model (Figure 1). In 48 DLBCL patient samples with available genomic profiling, PIM1 mutations appeared more frequently in pts diagnosed with ABC-DLBCL compared with GCB-DLBCL (5 out of 6 DLBCL pts with PIM1 mutations were ABC-subtype). 4 of these 5 pts exhibited a poor clinical response to ibrutinib, ie, 80% of ABC-DLBCL pts with PIM1 mutations had progressive disease, compared with only 13 of 26 (ie, 50%) ABC-DLBCL pts without PIM1 mutations. Subsequent characterization of the mutant PIM1 proteins (L2V, P81S, and S97N) confirmed that they were more stable than WT PIM1, suggesting increased protein levels by 2 potential mechanisms (WT PIM1 gene up-regulation or increased mutant PIM1 protein half-life). The impact of these mutations on PIM1 function and ibrutinib sensitivity is under investigation. Conclusions: Ibrutinib-resistant ABC-DLBCL cells have increased PIM1 expression, and synergistic growth suppression was observed when ibrutinib was combined with a pan-PIM inhibitor. PIM1 mutations identified in ABC-DLBCL pts with poor responses to ibrutinib contributed to increased PIM1 protein stability. A better understanding of the role of PIM1 in ibrutinib-resistant ABC-DLBCL tumors could provide a rationale for the design of combination therapies. Figure 1. Combination of ibrutinib and a pan-PIM inhibitor in the HBL-1 xenograft model. Ibrutinib and PIM inhibitor treatment suppressed tumor growth by 62% compared with the vehicle-treated group (*p 〈 0.01, repeated measures MANOVA adjusted univariate F-test). Figure 1. Combination of ibrutinib and a pan-PIM inhibitor in the HBL-1 xenograft model. Ibrutinib and PIM inhibitor treatment suppressed tumor growth by 62% compared with the vehicle-treated group (*p 〈 0.01, repeated measures MANOVA adjusted univariate F-test). Disclosures Kuo: Pharmacyclics LLC, an AbbVie Company: Employment. Hsieh:pharmacyclics LLC, an AbbVie Company: Employment. Schweighofer:Pharmacyclics LLC, an AbbVie Company: Employment. Cheung:Pharmacyclics LLC, an AbbVie Company: Employment. Wu:Pharmacyclics LLC, an AbbVie Company: Employment. Apatira:Pharmacyclics LLC, an AbbVie Company: Employment. Sirisawad:Pharmacyclics LLC, an AbbVie Company: Employment. Eckert:Pharmacyclics LLC, an AbbVie Company: Employment. Liang:Pharmacyclics LLC, an AbbVie Company: Employment. Hsu:Pharmacyclics LLC, an AbbVie Company: Employment. Chang:Pharmacyclics LLC, an AbbVie Company: Employment.

Description: Abstract 866 Background: Stem cells are retained in the bone marrow via the trophic effects of the binding of chemokine stromal cell-derived factor-1α (SDF-1α) to its receptor, CXC chemokine receptor 4 (CXCR4). TG-0054 inhibits SDF-1α/CXCR4 binding and therefore mobilizes stem cells into peripheral blood. Animal studies in mice showed rapid and effective mobilization of CD34+ hematopoietic stem cells (HSCs) and CD133+ endothelial progenitor cells (EPCs) into peripheral blood after TG-0054 administration. A Phase I study was conducted in healthy volunteers to assess safety, tolerability, pharmacokinetics (PK) and stem cell mobilization of TG-0054. Materials and Methods: This is a phase I, randomized, double-blind, placebo-controlled, single ascending dose study. In each cohort, 2 volunteers received placebo and 6 received 0.10, 0.14, 0.28, 0.56, 1.12, 2.24, 3.14, or 4.40 mg/kg of TG-0054 (dose was calculated based on TG-0054 free base) via 15 minutes single IV infusion. All subjects underwent PK sampling at pre-dose, 5 and 15 minutes during infusion, and at 1, 2.5, 5, 10, 30 minutes and 1, 2, 4, 6, 9, 12, 24, 36 hours after infusion. The pharmacodynamics (PD) sampling time points were at pre-dose, 1, 2, 4, 6, 9, 24, and 36 hours after infusion. General tolerability, adverse events (AEs), electrocardiogram (ECG), vital signs and laboratory tests were recorded. Results: In this study, the maximum tolerated dose (MTD) was not reached in TG-0054 doses up to 4.40 mg/kg in healthy volunteers. Dose escalation was stopped due to plateau of mobilized CD34+ and CD133+ cell numbers. TG-0054 was well tolerated up to 4.40 mg/kg. The majority of AEs were mild in severity (53 out of 55 events), and all AEs resolved by the end of the study without medical treatment. The number of subjects reporting the most common AEs included: abdominal pain (7/64, 11%), diarrhea/loose stools (5/64, 8%), dizziness (3/64, 5%), nausea (3/64, 5%), and diaphoresis (3/64, 5%). No significant abnormalities were noted in vital signs, ECG, holter monitoring, telemetry, pulse oximetry, physical examination, or laboratory tests. The area under the plasma concentration vs. time curve (AUC0-t) and maximum plasma concentration (Cmax) showed dose proportionality over the dose range studied. The mean of terminal elimination half-life (t1/2) was approximately 2.5 to 5 hrs. Single-dose administration of 1.12 - 4.40 mg/kg of TG-0054 significantly increased CD34+ cell counts in peripheral blood. At peak time, TG-0054 caused a 3 - 14 fold increase in circulating CD34+ cells from baseline. The mean CD34+ cell counts at peak time were 27.1 ± 9.3 cells/μL (1.12 mg/kg TG-0054), 35.9 ± 27.3 cells/μL (2.24 mg/kg), 32.5 ± 27.7 cells/μL (3.14 mg/kg), and 29.2 ± 12.9 cells/μL (4.40 mg/kg). The increase in circulating CD34+ cell counts was evident within 2 hours of TG-0054 administration, peaked at 4 - 6 hours after TG-0054 administration, followed by a gradual decline to baseline at 24 hours post-dosing. Similarly, increases in WBC and CD133+ cell counts were observed in all subjects. No AEs were deemed to be associated with WBC increases. Conclusion: TG-0054 exhibited a favorable safety and PK profile in healthy subjects in this Phase I study. PD analysis also displayed potent mobilization of CD34+ HSCs and CD133+ EPCs from TG-0054 dose levels of 1.12 - 4.40 mg/kg. These results support subsequent clinical investigations. Disclosures: Chung: TaiGen Biotechnology, Inc.: Employment. Chang:TaiGen Biotechnology, Inc.: Employment. Huang:TaiGen Biotechnology, Inc.: Employment. Tsai:TaiGen Biotechnology, Inc.: Employment. Hsu:TaiGen Biotechnology, Inc.: Employment. King:TaiGen Biotechnology, Inc.: Employment. Yuan:TaiGen Biotechnology, Inc.: Employment. Yen:TaiGen Biotechnology, Inc.: Employment. Chen:TaiGen Biotechnology, Inc.: Employment. Lu:TaiGen Biotechnology, Inc.: Employment. Hsu:TaiGen Biotechnology, Inc.: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 4613 Circadian rhythms regulate various functions of human body and disruption of circadian rhythm has been associated with cancer development and tumor progression. Circadian clock genes use transcriptional-translational feedback loops to control circadian rhythms. Many transcriptional regulators are histone acetyltransferases (HAT) or histone deacetylases (HDAC). As clock function and integration of inputs rely on transcriptional regulation, it is possible that chromatin is remodeled during circadian cycles and in response to signals that regulate the clock. SIRT1 (sirtuin 1) is a HDAC that has recently been identified as a crucial modulator of the circadian clock machinery. To date, at least 7 SIRT genes (SIRT1–7) have been identified. In our previous report we have demonstrated the daily expression patterns of PER1, PER2, PER3, CRY1, CRY2, and CKIe in peripheral blood (PB) of healthy individuals were abolished in chronic myeloid leukemia (CML) patients and partial recoveries of daily patterns were observed in CML patients with complete cytogenetic response (CCyR) and major molecular response (MMR) post-imatinib treatment [J Biol Rhythms 2011]. In this study we further investigated the expression profiles of the 7 SIRT genes (SIRT1–7) in PB total leukocytes from 49 CML and 22 healthy volunteers. Collection of PB was carried out at four time points: 2000 h, 0200 h, 0800 h, and 1400 h, respectively. In PB total leukocytes of healthy individuals, the daily pattern of SIRT1 (p 〈 0.01) and SIRT5 (p 〈 0.05) expression level peaked at 0200 h, and SIRT2 (p 〈 0.01) peaked at 0800 h. Daily pattern expression of these 3 genes was abolished in newly diagnosed pre-imatinib mesylate treated and blast crisis-phase CML patients. Partial daily patterns of gene expression recoveries were observed in CML patients with CCyR and MMR. In some serial monitored individual patients, the recoveries of oscillations of SIRT1, 2, and 5 genes expression accompanied with the disappearance of BCR-ABL transcripts were also noted. The expression of SIRT3, 6, and 7 did not show a time-dependent variation among the healthy and CML patients. SIRT4 expression was undetectable both in the healthy and CML patients. Updated in vitro study results of the regulation of SIRT1, 2, and 5 genes on circadian clock genes expression will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

Description: INTRODUCTION: Vaso-occlusion causes tissue ischemia and severe pain in patients with sickle cell disease (SCD). The liver is frequently involved in human SCD complications and elevated serum levels of hepatic transaminases often accompany vasoocclusive pain. It is unknown whether in sickle cell disease, the liver sustains oxidative damage after hypoxia-reoxygenation and if so, how long does it take to recover. Both mitochondrial and cytosolic aconitases are potential targets of oxidants in cells due to the oxidant-mediated loss of iron from the [4Fe-4S] cluster. Oxidant damage can occur from the reactive oxygen species generated by dysfunctional mitochondria dysfunction following ischemia-reperfusion and also from peroxidases later released during the inflammatory reaction to tissue injury. A mouse model of SCD can be used to investigate the cellular mechanisms and temporal sequence of vaso-occlusive tissue damage. OBJECTIVES: After experimental vaso-occlusion by exposing sickle cell mice to hypoxia-reoxygenation, we will determine whether hepatocellular injury shows temporal correlation with a marker of oxidative damage (diminished aconitase activity) and a marker of inflammation (increased myeloperoxidase concentration). No changes are predicted to occur in littermate control mice whose erythrocytes do not sickle with hypoxic challenge. METHODS: Mice expressing exclusively human sickle cell hemoglobin (“Berkeley sickle mice”) and non-sickling littermate controls (“hemizygotes”) were exposed to 10% oxygen for 2 hours, and then restored to normoxia. At 6, 18, 48, or 72 hrs after hypoxia, animals were euthanized to harvest blood and liver tissue. Normoxic control mice of both types had blood and liver harvested without hypoxic exposure. Additional sickle cell mice received intraperitoneal injection of nitrite (2.4 millimole/g body weight) or saline control at the end of 2 hours of hypoxia, then had blood and liver harvested 18 hours after they were restored to normoxia. Serum alanine aminotransferase (ALT) level was assayed as a quantitative measure of hepatocellular injury. Aconitase and myeloperoxidase (MPO) results were compared by t-test. RESULTS: Serum ALT level rose 3-fold in sickle mice by 18 hr after hypoxia, and then declined by 48 and 72 hrs after hypoxia. Hemizygotes showed no change in ALT after hypoxia. Liver homogenate aconitase activity was significantly lower in sickle mice than in hemizygotes at the first time point measured, 6 hrs after hypoxia (p=0.003, n=4 per group), suggesting that oxidative damage to the enzyme had occurred early after hypoxia. MPO concentration in livers harvested 48 hr after hypoxia-reoxygenation was 3-fold elevated in sickle mice vs. hemizygotes (p=0.006, n=4 per group), but not at baseline or 72 hr after hypoxia-reoxygenation. Nitrite injection was associated with complete abrogation of rise in ALT in sickle mice challenged with hypoxia-reoxygenation, but was not associated with any differences in aconitase activity or MPO concentration. CONCLUSION: Serum ALT, liver aconitase activity, and liver MPO concentration had different temporal patterns of changes in sickle mice after hypoxia-reoxygenation challenge as an experimental model of vaso-occlusive tissue injury. Oxidative stress appears to be present within hours after hypoxia, followed by tissue injury (serum ALT rise), which is then followed by inflammation may take 48 hr after experimental vasoocclusion. Although the nitrite is not sufficient as antioxidant to protect against reactive oxygen species and inflammatory leukocytes, nitrite may be protective against tissue injury at the mitochondrial level. This experimental model system may be well-suited for pre-clinical testing of therapy for sickle cell vaso-occlusion.

Description: Introduction: CAR T therapy has improved overall survival for patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) who otherwise have poor outcomes with traditional chemoimmunotherapy. Cytokine release syndrome (CRS) and neurotoxicity are well known side effect from CAR T therapy. Cytopenia has been reported as well. However, to our knowledge, transfusion requirements during the first 30 days post-CAR T treatment have not been reported. Here we report the cytopenia characteristics and transfusion requirements in 15 patients who received commercial Axicabtagene ciloleucel (Yescarta) CAR-T cell therapy. Methods: We retrospectively reviewed all DLBCL patients who received Yescarta between July 2018 through May 2019 at Weill Cornell Medical Center. All patients received cyclophosphamide (500mg/m2) and fludarabine (30mg/m2) lymphodepleting regimen from days -5 to -3 before the CAR T cell infusion. Cytopenia, granulocyte-colony stimulating factor (G-CSF) use, packed red blood cell (RBC) and platelet transfusion requirements during 30 days after CAR T infusion were examined. Cytopenia was defined according to CTCAE criteria. Grade 3 cytopenia was white blood cell (WBC)