bluuebunny/crossref_metadata_2025 huggingface dataset:
Pediatric Reference Intervals for the DxH 520* Hematology Analyzer
Authors: Smit Astrid C , Seidel Aprille Lynn , Rohrbach Justin , Magari Robert , Lo Karen , Martin Javier , Adeli Khosrow , Higgins Victoria , Shabani-Rad Meer-Taher , Cyfra Maureen , Gwilliam Tracey , Michaud Wendy , Tuson Brian , Schmitt Kristy , Couvrette Nicole , Axelson Sandra , Tejidor Liliana M.
Published: October 2019

Introduction: The DxH 520 is a small hematology analyzer capable of performing CBC and 5-part differential in fresh whole blood samples (venous and capillary) collected in K2EDTA and K3EDTA anticoagulants. Medical care of children and adolescents is significantly dependent on reference intervals to properly interpret laboratory test results. A multi-center study was performed to verify or establish pediatric reference intervals for all parameters (White Blood Cells (WBC), Red Blood Cells (RBC), Hemoglobin (Hgb), Hematocrit (Hct), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Red Cell Distribution Width (RDW), RDW-SD, Platelets (PLT), Mean Platelet Volume (MPV), (Lymphocyte) LY%, LY#, Monocyte% (MO%), MO#, Neutrophil % (NE%), NE#, Eosinophil% (EO%), EO#, Basophil % (BA%), and BA#) with combined genders.

Methods: Whole blood samples from healthy children were tested within 8 hours of collection on the DxH 520 analyzer. Samples generating review flags or suspect messages were excluded from the analyses. Even gender distribution was targeted. A total of 208 specimens were enrolled that included 20 neonates (0 to 30 days), 27 infants (31 days to 2 years), 94 children (3 to 12 years) and 67 adolescents (13 to 21 years). Results were analyzed according to CLSI EP20-A3c guidelines.

Results: Reference intervals for the neonate age group were verified from existing published ranges using the transference method described in the guideline (Table 1). The robust method was used to calculate two sets of reference intervals partitioned by combining data from the infant and children age groups, and from the adolescent age group (Table 2).

Conclusion: Reference Intervals for CBC and differential parameters have been established for pediatric age groups (0 to 30 days, 31days to 12 years, 13 to 21 years) on the DxH 520 Hematology analyzer.

*CE marked. Pending 510(k) clearance by the United States Food and Drug Administration; not yet available for in vitro diagnostic use in the U.S.

Beckman Coulter, the stylized logo, and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries.

Disclosures

Smit: Beckman Coulter: Employment. Seidel:Beckman Coulter: Employment. Rohrbach:Beckman Coulter: Employment. Magari:Beckman Coulter: Employment. Lo:Beckman Coulter: Employment. Martin:Beckman Coulter: Employment. Tejidor:Beckman Coulter: Employment.
Quantitative Proteomic Profiling Identifies Differentially Regulated Pathways in Fanconi Anemia and Aplastic Anemia
Authors: Hou Hui , Lu Qin , Li Dan , Wu Shuiyan , Chu Xinran , Xu Guoqiang , Hu Shaoyan
Published: October 2019

Purpose: Prognosis and therapeutic treatment of Fanconi anemia (FA) and acquired aplastic anemia (AA) are different. However, delayed and incorrect treatments are frequently occurred in FA because its symptoms are very similar to those of AA. This study aimed to elucidate the differences between FA and AA at the molecular level and to identify biomarkers and pathways of FA through quantitative proteomics and bioinformatics analyses.

Methods: Samples were obtained from the bone marrow of patients with FA and AA followed the institutional guideline. Proteins were extracted, digested with Lys-C and trypsin, and chemically labeled with tandem mass tag (TMT) duplex reagents (TMT126 and 127) for mass spectrometric analyses. Proteins with fold changes of > 1.5 or < 0.667 were considered as differentially expressed proteins (DEPs). Gene ontology analysis was performed using the Database for Annotation, Visualization and Integrated Discovery. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted using the ClueGO plug-in in Cytoscape. A DEP-associated protein-protein interaction (PPI) network was constructed using STRING and visualized in Cytoscape. The degree and topological index of these DEPs were calculated using CentiScape. The PPI network was subjected to modular analysis using the Molecular Complex Detection plug-in in Cytoscape.

Results: A total of 114 DEPs, including 71 upregulated and 43 downregulated proteins, were discovered in the FA sample compared with those in the AA sample. The upregulated proteins were enriched in nucleosome assembly, cell or subcellular component movement, glycolysis, and gluconeogenesis pathway. The downregulated proteins were enriched in immune response, negative regulation of apoptosis, proteolysis, phagosome, and pantothenate and CoA biosynthesis. Five important modules were detected from the PPI network of DEPs. Eight hub proteins, a-enolase (ENO1), phosphoglycerate kinase 1 (PGK1), HSP90AA1, HSP90AB1, ACTC1, ACTBL2, EEF1A1, and CFL1, with a high degree of connectivity were obtained.

Conclusions: Quantitative proteomics coupled with bioinformatics analyses are useful for the identification of protein biomarkers and pathways associated with FA and AA. Some critical DEPs, such as ENO1, PGK1, ACTC1, ACTBL2, EEF1A1, and CFL1, likely play important roles in FA and could be used as serologic markers for its early diagnosis and to distinguish FA from AA.

Disclosures

No relevant conflicts of interest to declare.
Outcome of Lymphocyte Predominant Hodgkin Lymphoma: A Single Institutional Analysis
Authors: Ezekwudo Daniel E , Anusim Nwabundo , Konde Anish S , Gbadamosi Bolanle , Khoury John , Macari David , Zakalik Dana , Blankenship Leann , Gaikazian Susanna , Jaiyesimi Ishmael
Published: October 2019

Background: Nearly 5% of patients diagnosed with Hodgkin lymphoma have the nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), also known as Hodgkin paragranuloma or lymphocytic and histiocytic predominant Hodgkin disease. Given the rarity of this variant of Hodgkin lymphoma, there is no consensus on the standard of care/regimen in this subgroup of patients. Therapeutic approach has spanned from radiotherapy alone, combined chemo-radiotherapy to chemotherapy alone or observation and recently the use of Rituximab anti CD20 monoclonal antibody as these cells expresses CD20 marker. We opt to evaluate the outcome of patients with newly diagnosed NLPHL in our institution. We also reviewed outcome of NLPHL patients treated with various chemotherapy such as ABVD, CVP or RCHOP ± Radiation.

Methods: We retrospectively reviewed medical records of over 300 patients [mean age 45 (13-76)] with Hodgkin Lymphoma at Beaumont Hospital, an academic community center from January 2007 - January 2017. Given the rarity of the disease, a total of 13 patients were found to have pathologically confirmed diagnosis of NLPHL. Data regarding age, sex, stage of disease, treatment and outcome was analyzed.

Results: Total 13 patients with NLPHL with age range from 13 to 80 years were studied. Demographic distribution of studied patients were 8(62%) white, 4 (31%) African Americans and 1 (7.6%) Asian. Out of these, 8 (62%) were males and 5 (38%) females. A total of 9 (69%) patients have stage III disease on diagnosis while 4 (31%) patients had stage II disease on diagnosis. Out of 9 patients with stage III disease, 6 (67%) received chemotherapy alone (ABVD or CVP) whereas 3 (33%) received combined chemotherapy and radiotherapy. One patient in the chemotherapy alone arm, recurred within 24 months of treatment and received local therapy with radiation. Among those with stage II disease on diagnosis, 3 patients received radiation therapy alone while one patient was observed and did not receive any treatment. Overall, out of 13 patients studied, 12 (92%) are still alive and continues to follow with our clinic with no recorded relapse to date. One patient developed astrocytoma grade III and died of intracranial hemorrhage.

CONCLUSION: Although the standard therapeutic management for patients with NLPHL remains controversial. Our data showed that regardless of the regimen selected (chemotherapy alone or combined chemo-Radiotherapy), patients with Lymphocyte predominant Hodgkin Lymphoma treated with ABVD ± Radiation have excellent outcome. Although our study represents a single institutional analysis, it concurs with recent studies that clinical outcome in patients with NLPHL is favorable with chemotherapy.

Disclosures

No relevant conflicts of interest to declare.
Tandem P-Selectin Glycoprotein Ligand Immunoglobulin Prevents Lung Vaso-Occlusion in SCD Mice
Authors: Vats Ravi , Tutuncuoglu Egemen , Tejero Jesus , Shaw Gray , Sundd Prithu
Published: October 2019

Introduction: Sickle cell disease (SCD) is an autosomal-recessive-genetic disorder, which leads to red blood cell sickling, hemolysis and vaso-occlusion. Acute systemic painful vaso-occlusive crisis (VOC) is the predominant pathophysiology requiring emergency medical care by SCD patients. 10-20% of SCD patients hospitalized with VOC tend to develop acute chest syndrome (ACS), a type of lung injury within next few days, suggesting a role for pulmonary vaso-occlusion in ACS. This epidemiology also provides a window for therapeutic intervention provided treatments to prevent vaso-occlusion are available. Earlier, we have shown that VOC involves entrapment of large neutrophil-platelet aggregates in lung arterioles of SCD mice, which is inhibited following intravenous administration of P-selectin function blocking antibody. Recently, a tandem-P-selectin-glycoprotein-ligand-immunoglobulin (TSGL-Ig) with two P-selectin binding sites in tandem, has been shown to prevent P-selectin-dependent liver injury in mice. Here, we test the ability of TSGL-Ig in attenuating P-selectin dependent lung vaso-occlusion in SCD mice.

Materials and Methods: Townes knock-in humanized SS (hα/hα:βS/βS) mice were used as SCD mice. SCD mice were intravenously (IV) challenged with 10 µmole/kg Oxy-hemoglobin (Oxy-Hb) without or with 100 µg of TSGL-Ig to trigger lung vaso-occlusion. Fluorescent anti-mouse mAbs against CD49b and Ly6G, and FITC dextran were IV administered for in vivo staining of platelets, neutrophils, and visualizing lung microvasculature, respectively. Presence or absence of lung vaso-occlusion was assessed using multi-photon excitation enabled quantitative fluorescence intravital lung microscopy (qFILM).

Results and Discussion: IV administration of Oxy-Hb led to occlusion of pulmonary arterioles by large neutrophil-platelet aggregates. Remarkably, IV administration of TSGL-Ig significantly attenuated lung vaso-occlusion in SCD mice by reducing the number as well as size of neutrophil-platelet aggregates in the pulmonary arterioles of SCD mice.

Conclusion: Systemic challenge with Oxy-Hb promotes lung vaso-occlusion in SCD mice. TSGL-Ig significantly reduced oxy-Hb induced lung vaso-occlusion in SCD mice, highlighting the potential of TSGL-Ig to prevent ACS.

Disclosures

No relevant conflicts of interest to declare.
Thalassemia Gene Therapy By In Vivo Transduction of Mobilized Hematopoietic Stem Cells (HSCs) with an Integrating Hybrid Adenovirus Vector System
Authors: Georgakopoulou Afrodite , Wang Hongjie , Kapsali Chrysi , Psatha Nikoleta , Koufali Angeliki , Li Chang , Izsvak Zsuzsanna , Anagnostopoulos Achilles , Papayannopoulou Thalia , Yannaki Evangelia , Lieber Andre
Published: October 2019

To overcome the cost and complexity of current thalassemia ex vivogene therapy protocols, we developed a minimally invasive and readily translatable approach for in vivo HSC gene delivery which abrogates the need for HSC leukapheresis, CD34+ cell selection, ex vivo HSC culture, myeloablation and ultimately, transplantation. Our approach involves HSC mobilization with G-CSF/AMD3100 andintravenous injections of a hybrid vector system consisting ofa CD46-targeting, helper-dependent adenoviral vector and the hyperactive Sleeping Beauty transposase (SB100x) that mediates integration of thevector-encoded γ-globin and mgmtP140K genes. Pretreatment with glucocorticoids before virus injectionsis used to blockthe release of pro-inflammatory cytokines andimmunosuppression is applied in order to avoid responses against human g-globin- and MGMT protein-expressing cells. We tested our approach in a mouse model recapitulating the phenotypeof human β-thalassemia intermedia (Hbbth-3/hCD46++ mice). At week 8 post transduction, hCD46+/+/Hbbth-3 mice expressed HbF in 31.2±2.7% of circulating erythrocytes. Due to a significant drop in HbF expression by week 16 (11.9±3.0%), a 4-dose O6BG/BCNU treatment was administeredin order to in vivo select forgene corrected hematopoietic progenitors, thus recovering the HbF expression in76.0±5.7% of the circulating erythrocytes, by week 29 post in vivo transduction. With an average vector copy number of 1.4/cell, the human γ-globin to mouse α-globin expression was ~10% by HPLC and the human γ-globin to mouse β-globin mRNA ratio ~10%, by qRT-PCR. Hematological parameters (RBCs, Ht, Hb, MCV, RDW, Reticulocytes) at week 29 post in vivo transduction, were significantly improved over baseline or were indistinguishable from normal values, suggesting near to complete phenotypic correction. Treated mice showed significant reduction of spleen size, extramedullary erythropoiesis and parenchymal hemosiderosis. After secondary transplantation and without in vivo selection, more than 90% of donor-derived erythrocytes (CD46+) were g-globin-positive, up to 20 weeks post-transplant. Safety was demonstrated by the good tolerability of treatment, the absence of alterations in hematopoiesis, the normal colony-forming potential of bone marrow cells and the random integration pattern of our vector system. Overall, we present a simplified platform for gene therapy of thalassemia, which can serve as a cost-efficient and "portable" approach to make gene therapy accessible even to resource-poor regions where thalassemia major is endemic but only minimally complex strategies could be adopted.

Disclosures

No relevant conflicts of interest to declare.
Human Plasminogen Replacement Therapy Vs. Best Supportive Care in Patients with Congenital Plasminogen Deficiency: A Comparison of Health Care Resource Use over a 48-Week Period
Authors: Dranitsaris George , Ortiz Amparo Santamaria , Dorward Kristine , Kizilocak Hande , Celkan Tiraje
Published: October 2019

Background: Congenital plasminogen deficiency (CPD), is an ultra-rare autosomal recessive disorder that causes severe and debilitating clinical sequelae. Clinical signs are characterized by ligneous conjunctivitis, gingivitis, otitis media, impaired wound healing and even hydrocephalus. Current disease management includes surgery and systemic/topical therapies, but successful clinical responses are few and recurrences are typically rapid. Intravenous (IV) human plasminogen is a new treatment for CPD currently undergoing regulatory review in the United States. A recent clinical trial demonstrated that regular infusions of plasminogen IV (every 2 to 4 days) induced near complete and long term durable responses in affected patients. The current study compared overall health care resource use required to manage clinical manifestations of CPD for patients treated with human plasminogen in the clinical trial relative to a historical control group.

Methods: The registration trial database was used to extract health care resource data in CPD patients (n=14) who received human plasminogen IV therapy throughout a 48-week period. Total health care resource use was compared to a control sample consisting of CPD patients (n=16) who did not receive human plasminogen IV and were managed with only best supportive care (BSC) over a one year period from the date of diagnosis. Health care resource extraction consisted of topical and systemic therapies such as fresh frozen plasma (FFP), clinic visits, surgical interventions and hospital admissions related to CPD.

Results: A total of 16 patients who received BSC in either the United States (n=3), Spain (n=3) and Turkey (n=10) were identified and formed the control group. Over a 12-month assessment period, there were 304 clinic visits and 65 days of hospitalization for CPD related complications. IV FFP was given to 12 of 16 (75%) patients, which translated to 246 individual FFP doses administered (dose range = 5 to 15 mg/kg) over 12 months. Furthermore, there were 54 eye surgeries performed on the 16 control patients to remove ligneous membranes. In contrast, none of the 14 patients treated with plasminogen IV required surgical interventions, hospital admissions or FFP infusions over the 48-week treatment period (p < 0.001). There were also significant reductions in all types of supportive therapy in the human plasminogen treated patients relative to their utilization at baseline (p < 0.05).

Conclusions: Human plasminogen replacement therapy was able to significantly offset health care resource use compared to patients receiving BSC only. Patients in the trial were also able to incorporate IV human plasminogen therapy into their weekly routine, with administrations often performed at home.

Disclosures

Dranitsaris: Prometic Life Sciences Inc.: Consultancy. Dorward:Prometic Life Sciences Inc.: Employment.
Novel Antibodies Against Glycoprotein Ibα Inhibit Pulmonary Metastasis By Affecting Vwf-Gpibα Interaction
Authors: Yingxue Qi , Chen Wenchun , Xu Ke , Wu Fengying , Fan Xuemei , Ren Shengxiang , Liu Junling , Deng Wei , Zhang Jun , Li Renhao , Liu Jianwen , Liang Xin
Published: October 2019

Background:

Platelet glycoprotein Ibα (GPIbα) extracellular domain, which is part of the receptor complex GPIb-IX-V, plays an important role in tumor metastasis. However, the mechanism through which GPIbα participates in the metastatic process remains unclear. In addition, potential bleeding complication remains an obstacle for the clinical use of anti-platelet agents in cancer therapy.

Methods and Results:

To generate antibodies that bind to mouse platelet GPIbα, washed mouse platelet lysate was used as the antigen for rat immunization. Obtained hybridoma clones were screened in ELISA for binding affinity to the GPIb-IX complex. Positive clones were further screened for their abilities to inhibit platelet-cancer cell adhesion. Finally, at static condition, two antibodies, 2B4 and 1D12, had virtually no effect on the activation of integrin αIIbβ3, which is used to indicate platelet activation. Then, we characterized the binding sites of 2B4 and 1D12 by 20 purified recombinant GPIbα fragments binding. Results showed that 2B4 and 1D12 shared the same binding sites with vWF.

To determine whether 2B4 and 1D12 affect vWF binding, we tested the binding by flow cytometry using recombined mouse vWF, and then, we investigated platelet aggregation induced by several agonists, including vWF binding agonist ristocetin. Our data demonstrated clearly that 2B4 and 1D12 could inhibit vWF binding.

To investigate whether the inhibition of vWF-GPIbα interaction was associated with tumor metastasis, we examined the effect of 2B4 and 1D12 in each of the following interactions in vitro: between activated platelets and tumor cells, platelets and endothelial cells. Meanwhile, We further investigated the inhibitory effect of these antibodies in vivo using the experimental metastasis model and the spontaneous metastasis model. Results showed that 2B4 and 1D12 could potently inhibit the adhesion of cancer cells in vitro, and metastasisin vivo.

We next investigated whether 2B4 and 1D12 could affect platelet activation and/or induce thrombocytopenia in vivo. Results showed that the addition of 2B4 or 1D12 to PRP did not induce platelet aggregation and injection of 2B4 or 1D12 Fab at appropriate dose did not affect tail-bleeding time and platelet count.

Based on the above findings, we obtained anti-human platelet GPIbα monoclonal antibody YQ3 using the same approach to explore the role of human GPIbα in cancer metastasis. As expected, YQ3 inhibited lung cancer adhesion and demonstrated similar value in metastasis. More importantly, for all three mAbs in our study, none of their Fabs induced thrombocytopenia.

Conclusion:

Our results therefore supported the hypothesis that GPIbα contributes to tumor metastasis, and suggested potential value of using anti-GPIbα mAb to suppress cancer metastasis.

Disclosures

Li: Neoletix: Consultancy, Equity Ownership.
Regulatory T Cell Selective IL-2-Fc Fusion Proteins for the Treatment of Autoimmune Diseases
Authors: Varma Rajat , Liu Ke , Avery Kendra , Rashid Rumana , Schubbert Suzanne , Leung Irene , Bonzon Christine , Bogaert Liz , Desjarlais John , Bernett Matthew J.
Published: October 2019

Regulatory T cells (Tregs) characterized as CD4 positive cells, expressing CD25 (IL-2Rα) and the forkhead transcription factor FoxP3 maintain immune tolerance in tissues by suppressing the function of both CD4 and CD8 effector T cells. It is well documented that there is a paucity and/or dysfunction of Tregs in most autoimmune diseases and therefore a promising therapeutic approach has been to restore their numbers and function. Treg homeostasis depends on IL-2 derived signals and Tregs are sensitive to IL-2 stimulation in part due to the high abundance of CD25. As a result, encouraging results have been seen in patients with autoimmune diseases who were treated with low doses of IL-2. Single or repeat low doses of IL-2 led to selective expansion of Tregs over effector cells and provided clinical benefit. Such IL-2 based therapies can be further improved by enhancing the selectivity of IL-2 for Tregs and improving the pharmacokinetics of the drug. Here we have developed an IL-2-Fc fusion protein that selectively acts on Tregs in vitro and in vivo. IL-2 mediated signal transduction occurs through the formation of a high affinity tetrameric complex consisting of IL-2, CD25, CD122(IL-2Rβ) and CD132(IL-2Rγ). IL-2-Fc fusion proteins were designed to be monomeric by fusing variant IL-2 molecules to one side of our heterodimeric Fc-region. To achieve Treg selectivity, we designed variants of IL-2 that have weakened interaction with CD122 and/or enhanced interaction with CD25 such that signaling is only observed in CD25 high cells. These IL-2-Fc variants were produced with good yield and purity. We identified novel variants that selectively led to phosphorylation of STAT5 transcription factor in Tregs from human peripheral blood mononuclear cells (PBMCs). These Treg-selective IL-2-Fc variants stimulated Tregs with varying potencies. Further, they selectively enhanced and sustained the proliferation of Tregs in vivo in non-human primates. These promising preclinical results suggest that clinical development of Treg selective IL-2-Fc fusion proteins for autoimmune diseases is warranted.

Disclosures

Varma: Xencor: Employment, Equity Ownership. Liu:Xencor: Employment, Equity Ownership. Avery:Xencor: Employment, Equity Ownership. Rashid:Xencor: Employment, Equity Ownership. Schubbert:Xencor: Employment, Equity Ownership. Leung:Xencor: Employment, Equity Ownership. Bonzon:Xencor: Employment, Equity Ownership. Bogaert:Xencor: Employment, Equity Ownership. Desjarlais:Xencor: Employment, Equity Ownership. Bernett:Xencor: Employment, Equity Ownership.
A Population-Based Study of Prognosis and Survival in Primary Hepatobiliary Lymphoma
Authors: Sindel Ariel , Al-Juhaishi Taha , Sabo Roy , Yazbeck Victor Youssef
Published: October 2019

Background:

Primary Hepatobiliary Lymphoma (PHBL) is a rare disease. In review of the literature, there are only few retrospective studies. On prior studies, the median age at presentation was 62 years and was associated with a median overall survival of 162 months. In this study, we sought to characterize standard prognostic factors and their effects on overall survival utilizing the Surveillance, Epidemiology and End Results (SEER) database.

Methods

The Surveillance, Epidemiology and End Results (SEER) database was used to identify patients diagnosed with PHBL between 1973 and 2014. B-Cell and T-Cell lymphomas of all stages were included. Patient characteristics and demographics are summarized. Overall survival (OS) was estimated using the Kaplan-Meier method for all patients and for sub-groups. Comparisons are made between the survival functions between the levels of these categorical measurements using the log-rank test, where we adjust the significance level to account for multiple comparisons using the Bonferroni correction.

Results:

A total of 1751 patients were included. The mean age was 64 years (2-97), with the majority of patients being male (n=1081, 62%) and Caucasian (n=1278, 73%). B-cell Non-Hodgkin's Lymphoma was the predominant lymphoma (n=1297, 75%), followed by Not Otherwise Specified (n=338, 19%), and T-cell NHL and Hodgkin's Lymphoma were nearly identical at n=54 and 50 respectively. Staging at presentation varied with the majority being 1 and 4 (n=703, 40% and n=698, 40%), this was followed by stage 2 (n=196, 11%). The median overall survival time was 16 months (95% CI; 12, 23). By diagnoses, the median survival time (Figure 1) for B-Cell NHL was 23 months (95% CI; 17, 33) and was significantly longer survival than patients with T-Cell NHL (1 month, 95% CI; 0, 8, p=0.0014) and NOS (9 months, 95% CI; 6, 12, p=0.0076) but was not significantly different than patients with Hodgkin's Lymphoma (12 months, 95% CI; 1, 71 p=0.15). By staging, patients with stage 1 (38 months, 95% CI; 24, 52) did not have a statistically significant difference in survival in comparison to stage 2 (15 months, 95% CI; 9, 48, p=0.85) or 3 (10 months, 95% CI; 2, 34, p=0.0173). Results were not statistically significant regarding gender nor ethnicity. On a multivariant analysis, surgery, chemotherapy, or radiation were all associated with better survival when adjusted for patient age, sex, race, disease type and cancer stage. However, there did not appear to be a statistical significance when compared to each other.

Conclusion

PHBL is a rare lymphoma affecting mostly elderly, Caucasian males. B-Cell NHL subtype and stage 1 were associated with a more favorable prognosis. However, compared to DLBCL, PHBL seem to have a poorer outcome. While treatment alone conferred a longer survival, the type of treatment option did not have an overall change to prognosis.

Disclosures

No relevant conflicts of interest to declare.
Successful Treatment with Intrathecal Tocilizumab(IL-6 antibody) for 2 Cases of Graft-Versus Host Disease of the Central Nervous System after a Second Allogeneic HSCT from Haploidentical Donor
Authors: Gui Ruirui , Zhao Juanjuan , Zu Yingling , Han Lu , Zhang Yanli , Yu Fengkuan , Li Zhen , Zhao Huifang , Fang Baijun , Lin Quande , Gao Quanli , Song Yongping , Zhou Jian , Liu Delong
Published: October 2019

Graft-versus-host disease (GVHD) of central nervous system (CNS) is a rare complication after allogeneic hematopoietic cell transplantation (allo-HSCT), which is easily misdiagnosed and has limited treatment and poor efficacy. In this study, we reported two children(1 case of severe aplastic anemia and 1 case of acute myeloid leukemia) who were subjected to the secondary haploid hematopoietic stem cell transplantation of another door after the first implantation failure. Two patients developed epileptic seizures during hematopoietic recovery. Peripheral blood and cerebrospinal fluid cytokines were detected and diagnosed as acute GVHD in the central nervous system. The cytokine elevation is mainly IL-6(IL-6 level in peripheral blood was 2276.25 pg/ml, and in cerebrospinal fluid was 2353.89pg/ml of case 1). We administered multiply consecutive plasmapheresis, intravenous infusion of tocilizumab combined with intrathecal injection of tocilizumab and dexamethasone. The epileptic symptoms of 2 cases are effectively controlled after the first dose. The IL-6 levels in peripheral blood and cerebrospinal fluid of both patients decreased significantly after 4(IL-6 level in peripheral blood was 6.74 pg/ml, and in cerebrospinal fluid was 195.72pg/ml of case 1) and 5 doses, respectively. Central nervous system aGVHD is prone to hematopoietic recovery after secondary transplantation; intrathecal injection of tocilizumab is safe and effective in the treatment of central nervous system aGVHD with elevated IL-6 mainly, which is worth further exploring.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
B-Cell Mediated Humoral Responses Are Impaired in Sepsis Survivors
Authors: Rana Minakshi , Sherry Barbara , Diamond Betty
Published: October 2019

Sepsis survivors exhibit a sustained compromise in innate immunity, and high mortality rates due to increased vulnerability to infections. Previously we demonstrated in a cecal ligation and puncture (CLP) model, that sepsis survivors had a reduced TNFα response to LPS-challenge in vivo, which is accompanied by an increase in CD4+ChAT+ T cells. These data together with clinical observations of low endogenous levels of IgG in sepsis survivors led us to hypothesize that T-cell dependent (T-D) B cell responses might also be altered after CLP. In order to assess alterations in the adaptive humoral response in sepsis survivors, we immunized C57BL/6J sham surgery and CLP mice with a single intraperitoneal (I.P.) injection of NP (29)-KLH (T-D antigen) in alum 4 weeks post-CLP (or sham surgery). We observed sepsis survivors showed reduced serum NP-specific IgG and IgG1 antibodies compared to sham controls. Furthermore, to confirm whether this reduced immune response is mediated through a T-D or T cell-independent (T-I) type II mechanism, we used other carrier molecules CGG (T-D) and Ficoll (T-I), respectively. To evaluate time dependent kinetics, we immunized mice with single I.P. injection of either NP (20)-CGG or NP (55)-Ficoll. We analyzed both low-affinity and high-affinity anti-NP antibodies through NP (23)- and NP (7)- BSA ELISAs, respectively. We found a significant reduction in anti-NP IgG and IgG1 antibodies in CLP mice post-NP-CGG immunization (p<0.01 and p<0.001, respectively) while the response was not diminished after NP-Ficoll immunization at day 7; suggesting that compromised antibody response is mediated through a T-cell dependent pathway. Flow cytometry data also revealed that the splenic frequency of NP-specific CD138+ intracellular IgG1 plasma cells was decreased in CLP mice immunized with NP-CGG as compared to sham mice at day 7, 14 and 21 (p<0.001, p<0.05 and p<0.05, respectively). Since follicular dendritic cells (FDCs) play a key role in B-cell activation and affinity maturation of antibodies, we performed immunofluorescence staining of FDCs in spleens from sham surgery and CLP mice. Interestingly, we found a decreased number of FDC clusters in CLP mice compared to sham controls. In summary, our study is clinically relevant as it may explain the hypogammaglobulinemia seen in sepsis surviving patients.

Disclosures

No relevant conflicts of interest to declare.
Personalized Prophylaxis in Hemophilia a: A Pediatric Center Experience in Mexico
Authors: Aquino Efrain
Published: October 2019

Summary. Prophylaxis is the current standard of care for Hemophilia patients. However, the regimen should be individualized as much as possible based on age, bleeding phenotype and physical activity. PK based regimens have been proposed as an effective way to achieve more successful outcomes. According to 2016 WFH data, Mexico has 1.425 IU/capita mean use, which barely overcomes the limit of 1IU/ minimum target to achieve survival of the hemophilia population (WFH Report on the Annual Global Survey). Objectives. To examine a Mexican Center experience obtaining limited sampling PK profiles in pediatric patients. To determine the difference in frequency dosification in standard prophylaxis vs PK guided. Methods. Limited sampling was done in 14 pediatric patients with Hemophilia A without inhibitors, moderate to severe, in prophylaxis aged 2 to 14 years old using population PK tool myPKFiT. PK profiles based on the same data points plus patient characteristics were generated by a Bayesian algorithm with myPKFiT. Results. The mean age was 5.5 years (range 1-14 years) with a mean standard dose of 35 IU/k 2 times a week. The PK based mean dose was 27IU/kg 4 times a week. The mean half-life was 9.5 hours (range 6.8-11.6 hours. Conclusions. Shorter half life should be expected in pediatric patients, and be taken into account with other variables to achieve a personalized prophylaxis. Tailored prophylaxis facilitate patient education and adherence. Pediatric PK suggest shorter intervals or higher dosing are needed to achieve a desired target level. Use of myPKFiT and clinical, radiological and adherence scores to improve outcomes, in particular in the pediatric population. Integrating PK data into routine hemophilia care could provide promising results in developing countries.

Disclosures

No relevant conflicts of interest to declare.
Knockout Tumor Microenvironment HO-1 Neutralizes Myeloid-Derived Suppressor Cells and Enhances the Antitumor Effect of PD-1 Inhibition in Murine Models of Acute Myeloid Leukemia
Authors: Zhou Zhen , Fang Qin , Ma Dan , Wei Danna , Hu Xiuying , Liao Yudan , Wang Jishi
Published: October 2019

Background/Aims: Myeloid-derived suppressor cells (MDSCs) has been shown to be involved in tumor immune escape mechanisms and B cell-specific immunity in Acute myeloid leukemia (AML) patients. In our previous study shown that HO-1 protein have immunomodulatory activity including targeting of immune suppressor cells in the tumor microenvironment. Thus, we decided to assess whether HO-1 could enhance anti-PD-1 treatment and investigate those alterations in the immunosuppressive tumor microenvironment that contribute to the combined antitumor activity.

Method: We utilized C57/BL6 (HO-1 knockout) mouse were radiation with 6.5GY once in a row to repress residual immunity. Malignant tumor HL-60 cells respectively (1×107 cells) per animal were injected subcutaneously into the right abdomen. The xenograft mouse models of AML were euthanised on the 14th day after treatment with a PD-1 inhibitor once a day (20 mg/kg). The tumor volumes were measured and calculated by ruler. Survival curve of individual groups was evaluated from the first day of treatment until death using Kaplan-Meier curves.

Results: HO-1 gene knockout enhanced the antitumor effect of PD-1 inhibition in xenograft mouse models of AML by reducing tumor growth and increasing survival. HO-1 gene knockout inhibited the immunosuppressive function of both polymorphonuclear (PMN)- and monocytic-myeloid derived suppressor cell (M-MDSC) populations. Analysis of MDSC response to HO-1 gene knockout revealed significantly reduced arginase-1, iNOS, and COX-2 levels, suggesting potential mechanisms for the altered function. We also observed significant alterations in cytokine/chemokine release in vivo with a shift toward a tumor-suppressive microenvironment.

Conclusions: Our results demonstrate that HO-1 gene knockout enhances the antitumor effect of PD-1 targeting through functional inhibition of MDSCs and a transition away from an immune-suppressive tumor microenvironment.

[Key words] HO-1 gene knockout; PD-1 inhibitor; tumor microenvironment; MDSCs; AML

Disclosures

No relevant conflicts of interest to declare.
Does Neutrophil to Lymphocyte Ratio (NLR) Predict the Prognosis of Diffuse Large B-Cell Lymphoma?
Authors: Nakaya Aya , Fujita Shinya , Satake Atsushi , Nakanishi Takahisa , Azuma Yoshiko , Tsubokura Yukie , Saito Ryo , Konishi Akiko , Hotta Masaaki , Yoshimura Hideaki , Ishii Kazuyoshi , Ito Tomoki , Nomura Shosaku
Published: October 2019

Background: The international prognostic index(IPI) was widely used to predict aggressive lymphoma patients' outcome and to choose the best therapeutic treatment. However, this scale is unsatisfactory in identifying patients who would receive best benefit from rituximab containing regimens. Recently, NLR has been recognized as a poor prognostic indicator in various solid tumors. Here we quantify the prognostic impact of NLR in de novo DLBCL patients. However, various studies of the usefulness of the NLR have used different cut-off values, and the methods of selecting these NLR cut-offs were unclear. Therefore, we verify the adaptive cut-off value.

Methods: We retrospectively analyzed 543 patients with de novo DLBCL who diagnosed at Kansai Medical University Hospital and Kansai Medical University Medical Center from January 2003 to December 2017. The prognostic value of NLR at diagnosis was assessed. We put the cut-off of NLR; 3, 4, 5, 6, and evaluate which the most predictive cut-off value is.

Results: The median age was 69(20-95) years old, and male was 59%. The Ann Arbor stage III and IV was 60%. The proportion of patients with IPI, Low, Low-intermediate, High-intermediate, High was 36%, 19%, 21%, 24%, respectively. The optimal cutoff for NLR was 6. NLR(6) was associated with overall survival(OS)(HR 1.76, 95%CI: 1.23-2.51, p= 0.002) and progression free survival(PFS) (HR 2.66, 95%CI: 1.65-4.28, p<0.001), either. Multivariate analysis identified NLR(6) remained as a significant factor affecting PFS (hazard ratio: 1.53, 95%CI: 1.02-2.29, p=0.041).

Conclusion: Our result revealed that the optimal cut-off for NLR was 6. NLR predicted both OS and PFS of the patients with de novo DLBCL. NLR is a simple and useful scale and it might be a useful marker for prediction of patients' survival or disease progression in patients with de novo DLBCL.

Disclosures

Ito: Mundipharma: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis Pharma: Honoraria; Takeda: Honoraria; Pfizer: Honoraria.
Sumoylation of Pyruvate Kinase M2 Inhibits Myeloid Differentiation in Hematopoietic Cells
Authors: Xia Li , Wang Xin-Ran , Wei Ran , Yan Jin-Song , Chen Guo-Qiang , Lu Ying
Published: October 2019

The pyruvate kinase (PK) is a rate-limiting glycolytic enzyme catalyzing the dephosphorylation of phosphoenolpyruvate to pyruvate. M2 form of PK (PKM2) is expressed during embryogenesis and is the predominant form in tumors of different types. In contrast to the essential role of PKM2 in solid tumors, much less is known about the effects of PKM2 in hematopoietic cells and the development of leukemia. Here we found that PKM2 is modified by small ubiquitin-like modifier 1(SUMO1), which can be reduced by a SUMO1-specific protease SENP1 in hematopoietic cells. SUMOylation induced nuclear localization and conformation change from tetramer to dimer of PKM2. Importantly, SUMOylation of PKM2 is prevalent in a variety of leukemic cell lines as well as primary samples from patients with hematologic malignancies. In consistency, predominant nuclear localization and dimeric forms of PKM2 in leukemic cells were observed. Using in vitro SUMOylation reaction-coupled liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), we identified K270 lysine residue of PKM2 as the SUMOylation target. Replacement of endogenous PKM2 with mutant PKM2K270 showed a significant shift of PKM2 from tetramer to dimer. To investigate the potential leukemogenic effect of PKM2 SUMOylation, murine hematopoietic progenitor 32D clone 3 (32Dcl3) transfectants expressing wild type(WT) or mutant PKM2K270 were generated and G-CSF-induced differentiation was evaluated by morphology appearance and expression of myeloid associated surface markers CD11b and Gr-1. The results showed that expression of WT PKM2 but not mutant PKM2K270 significantly blocked myeloid differentiation. Further investigations revealed that SUMO1 modification of PKM2 at K270 is essential in mediating the interaction between PKM2 and Runt-related transcription factor 1(RUNX1), a master transcriptional factor implicated in the differentiation of hematopoietic cells. This interaction led to a downregulation of RUNX1 during G-CSF-induced myeloid differentiation of 32D cells, which could be abrogated by expression of mutant PKM2K270. Collectively, these data indicated that SUMOylated PKM2 blocks myeloid differentiation through suppressing RUNX1. These findings reveal a novel nonmetabolic function of PKM2 in modulating myeloid differentiation and highlight the critical role of SUMOylation in leukemogenesis.

Disclosures

No relevant conflicts of interest to declare.
Minit-Livin Conjugated to Targeted Nanoparticles Inhibits Tumorigenicity
Authors: Abd Elrahman Ihab , Nassar Taher , Khairi Noha , Perlman Riki , Benita Simon , Ben-Yehuda Dina
Published: October 2019

We found that Livin, an Inhibitor of Apoptosis Protein, is specifically cleaved to produce a truncated Livin protein (tLivin) and demonstrated the paradoxical pro-apoptotic activity of tLivin. mini-tLivin (mtLivin) is a 70 aa derivative of tLivin that remarkably, exhibits a pro-apoptotic activity as potent as tLivin. Our findings regarding tLivin and mtLivin suggest unique therapeutic avenue against cancer. To improve the delivery and efficacy of mtLivin for treatment we conjugated mtLivin-to nanoparticles (NPs) of poly (lactide-co-glycolide) (PLGA) and targeted the nano-delivery system to diffuse large B-cell lymphoma (DLBCL) using CD40L. Delivery of mtLivin with nanoparticles should enhance stability, provide prolonged bioavailability, intracellular uptake and efficacy.

We generated bifunctional mtLivin-CD40L-NPs to target mtLivin-NPs to DLBCL cells. Various cell lines were used to evaluate the biological activity of free and conjugated (targeted or not) mtLivin including 293T, 721.221 B cells, L428 Hodgkin's lymphoma, OCI-LY19 DLBCL cells and OCI-LY19- GLuc cells that constitutively express a gaussia luciferase reporter.

Targeted, bifunctional mtLivin-CD40L-NPs elicited significant cell death of DLBCL cells while monofunctional mtLivin-NPs had a lower effect. mtLivin-NPs may induce cell death of some cell lines without targeting (e.g. 293T) yet targeting is required to induce marked cell death of DLBCL cells.

To best mimic lymphoma disease in humans, a disseminated lymphoma model was used to evaluate the antitumor effect of targeted mtLivin treatment. NOD/SCID mice were injected with OCI-LY19- GLuc cells or into the tail vein. Gaussia luciferase reporter allows IVIS imaging to monitor tumor size and location in mice. Mice were treated approximately once a week with various mtLivin-NPs formulations. Luciferase signal was observed as early as day 2 after tumor cells injection.

Immunohistochemistry for anti-CD20, a human B-cell marker, showed infiltration of OCI-ly19-Gluc cells in spleen and brain close to tumor cells injection (Day 2). With time, lymphoma infiltrations were detected in brain, bone marrow, lungs, spleen and liver. All control mice exhibited paralysis and did not survive beyond 28 days. In contrast, 37.5% of animals receiving mtLivin-NPs and 71.4 % of animals receiving mtLivin-CD40L-NPs achieved complete pathological tumor response and survived significantly longer. All treatments in this study were well-tolerated.

In terms of mechanism, we show high caspase-3 activity in tumors from mice that were treated with mtLivin-CD40L-NPs demonstrating the ability of the nanoparticles to target the tumors and to induce apoptotic tumor cell death. The resistance of tumor cells to drug-induced apoptosis is a major cause of cancer treatment failure. We designed mtLitvin targeted nanoparticles constituting novel personalized therapeutic approach for resistant cancer.

Disclosures

No relevant conflicts of interest to declare.
Donor Lymphocyte Infusion Prevented Late Post-Transplant Rejection in 30 Patients with Thalassemia Major
Authors: Liao Jianyun , Liu Xiaoting , He Yuelin , Peng Zhiyong , Liu Huaying , Li Chunfu
Published: October 2019

OBJECTIVE:To investigate the role of donor lymphocyte infusions (DLI) in preventing post-transplant graft rejection (GR) in hematopoietic stem cell transplantation (HSCT) for patients with β-thalassemia major (TM);

METHODS: A total of 50 patients with TM who had mixed chimerism (MC) after HSCT from June 2011 to June 2016 was divided into 4 groups. The group 1 included the patients with grade I MC ratio of 90%-95% (n=20) but not DLI; Group 2 patients had same grade I MC ratio as Group 1 and received DLI (n=11); Group 3 had grade II MC ratio of 75%-90% (n=13); Group 4 had grade III MC of <75% (n=6). Group 1 received gradually reduced immunosuppressive agents; Groups 2 to 4 received DLIs of gradually increasing dose after reduction of immunosuppressive agents;

RESULTS: (1) Full donor chimerism (FDC) conversion rates in the groups 1 to 4 were 100%, 100%, 85%, and 66.7%, respectively; The incidence of graft versus host disease (GVHD) in groups 1 to 4 was 15%, 27.3%, 46.2%, and 33.3%, respectively; (2) There was no significant differeces as comparing incidence of GVHD in group 1 and in group 2 (15% vs. 27.3%, P = 0.638); (3) A significant difference was found in comparing patients receiving initial DLI at ≤180 and > 180 days and the incidence of GVHD was 50% vs 10% (P=0.049); (4) Comparing the accumulated doses of DLI >1×107/kg with ≤1×107/kg, the incidence of GVHD was 50% vs. 27.3% (P=0.142);

CONCLUSION: 1. The optimal choice for post-transplant MC grade 1 patient is only observation and decreasing the immunosuppressive drugs; 2. For grade 2-3 patients with high risk of rejection, the fractional increasing dose DLI can effectively reverse the unstable chimera to FDC; 3. Post-DLI GVHD was associated with DLI timing, and the incidence of GVHD was higher in patients who received DLI at ≤180 days after transplantation.

Disclosures

No relevant conflicts of interest to declare.
Sf3b1 Regulation of Jak/Stat Signaling Is Essential for Hematopoietic Stem Cell Formation
Authors: Bowman Teresa V. , Cameron Rosannah C. , Potts Kathryn S , McKinstry Mia , Gupta Varun , Bai Xiaoying
Published: October 2019

Hematopoietic stem cells (HSCs) maintain the hematopoietic system throughout the lifetime of an organism. During embryonic development, HSCs emerge through an endothelial-to-hematopoietic transition (EHT) from specialized hemogenic endothelial (HE) cells in the dorsal aorta. HSC fate specification depends on gene expression, which is the culmination of coordinated transcription, RNA splicing, and translation. Although transcriptional regulation of HSC fate choice is well studied, the regulatory role of RNA splicing in this process is poorly understood. Using zebrafish loss-of-function mutants for the spliceosomal component splicing factor 3b, subunit 1 (sf3b1), we identified that impaired splicing hindered HSC production. Surprisingly, we found that this constitutive splicing factor selectively regulates the fate of hemogenic endothelium while leaving the identity of closely-related non-hemogenic endothelium unperturbed. To identify Sf3b1-regulated transcripts important in EHT, we performed RNA-sequencing on purified kdrl:gfp+ endothelial cells from sf3b1 mutant and wild-type siblings at 24 hpf. Approximately 900 genes were mis-spliced, 144 of which were differentially expressed. Ingenuity Pathway Analysis identified Janus Kinase (Jak)/Signaling Transducer and Activator of Transcription (Stat) signaling, in particular Stat3, as one of the top perturbed pathways in the mis-spliced gene set. Stat3 is a transcription factor activated in response to several cytokine and inflammatory signals. To determine if altered splicing of stat3 was critical for HSC formation, we injected antisense splice-blocking morpholinos (MO) targeting the Sf3b1-sensitive stat3 exon19 into wild-type and sf3b1 heterozygous embryos, which normally generate equivalent levels of HSCs. We observed an impairment of HSC production in stat3 morpholino-injected sf3b1 heterozygotes, but not wild-type siblings, indicating a synthetic lethal interaction between sf3b1 and stat3. We also found that overexpression of a constitutively active form of Stat3 significantly suppressed the HSC defects in sf3b1 homozygous mutants. Together, these data indicate that Sf3b1-mediated splicing regulation of the Jak/Stat pathway is critical for HSC emergence.

Disclosures

No relevant conflicts of interest to declare.