bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantification of Human Equilibrative Nucleoside Transporter 1 in Blood Leukocytes Using Flow Cytometry
Authors: Davis Bruce H. , Curtis Adam , Booij Birgitte , Sandvold Marit Liland 
Published: October 2019

Abstract
Abstract 4737
We have developed an assay intended to measure the human Equilibrative Nucleoside Transporter 1 (hENT1) level in leukemic blast cells in blood or bone marrow samples from patients with acute myeloid leukemia or myelodysplasia (MDS). This is ultimately intended as companion diagnostic assay for hENT1 to identify those patients with poor treatment prognosis with cytarabine (ara-C) or other cytotoxic therapies that rely on hENT1 transport into abnormal cells. We optimized an intracellular staining assay incorporating a patent pending technique of utilizing fluorescent labeled monoclonal antibodies, internal fluorescent calibration beads and analytical software that allows identification of leukocyte subpopulations using a novel quantitative flow cytometric assay incorporating antibody binding inhibition by the immunizing peptide to demonstrate specific antigen binding by the anti-hENT1 monoclonal antibody. Initial validation studies were performed on blood samples from both healthy adults and some clinical samples, including samples from HIV+ individuals (samples 4A* and 4B*), infection/sepsis patients (samples 9–11**), and MDS as shown in figures. These preliminary studies showed two novel observations. Firstly expression differs in the major leukocyte populations with expression highest in eosinophils (hENT1 index range of 17.36 – 61.52), followed by neutrophilic granulocytes and monocytes and lymphocytes with the lowest level. Secondly while intra-assay imprecision was low with a CV <7%, some day to day variation was seen in healthy donors and greater variation seen with patient samples, thus indicating significant modulation of cellular expression within myeloid and monocytic cells. These preliminary results suggest retroviral therapy may decrease hENT1 expression, infection or its treatment increases hENT1 expression in myeloid cells, and MDS myeloid cells are also altered in hENT1 levels from the healthy state. These findings indicate potential clinical utility for a quantitative assay of hENT1 expression in hematolymphoid cells in a variety of conditions.

Disclosures:
Davis: Clavis Pharma: Consultancy; Trillium Diagnostics, LLC: Equity Ownership. Curtis:Trillium Diagnostics, LLC: Employment. Booij:Clavis Pharma: Employment. Sandvold:Clavis Pharma: Employment.
Tumor Lysis Syndrome As a Major Risk Factor of Posterior Reversible Encephalopathy Syndrome in Patients with Lymphoid Malignancies of Childhood
Authors: Suzuki Daisuke , Kobayashi Ryoji , Iguchi Akihiro , Sano Hirozumi , Yasuda Kazue , Kobayashi Kunihiko 
Published: October 2019

Abstract
Abstract 4754

Background
Posterior reversible encephalopathy syndrome (PRES) is a neurological complication that is caused by various factors such as hypertension, the use of calcineurin inhibitor(cyclosporin or tacrolimus). Its initial symptoms usually are seizure, headache, visual disorder. Although PRES associated with lymphoid malignancies has been reported, it only consits of case reports or small series. Furthermore, its risk factors have never been well-documented.

Patients and methods
A total of 126 pediatric patients with lymphoid malignancies received chemotherapy in our hospital between January 2000 and October 2010. Of these patients 84 were boys and 42 were girls. Age of patients at the onset of their respective diseases was 4 months to 15 years(median: 5y). One hundred and eight patients had acute lymphoblastic leukemia, 15 were Non-Hodgkin lymphoma and 2 were Hodgkin lymphoma.

Result
Of 126 patients, 6 developed PRES. The initial symptoms were seizure, cortical blindness and upper limb paralysis. All the patients had hypertension before developing PRES. In addition, 3 patients had tumor lysis syndrome(TLS). The median time from the initiation of treatment was 10.5 days (range: 6–35 days), Two patients developed epilepsy as a sequela. On statistical analysis, the only risk factors for PRES was the development of TLS.

Conclusion
Although various factors were reported, TLS-related PRES has never been described. Some patients developed PRES in the early period of induction treatment while the others in the late period. The difference between them remains to be seen. However, TLS may cause renal dysfunction in combination with hypertension induced by chemotherapeutic drugs such as prednisolone. Renal dysfunction and following hypertension might have induced PRES. We must pay attention to the state especially of the patients who developed TLS in the course of chemotherapy.

Disclosures:
No relevant conflicts of interest to declare.
Learn While You Wait: An Experience of a Novel, Interactive VHA e-Notebook Education Program for Multiple Myeloma, Final Results
Authors: Yacoub Abdulraheem , Mehta Paulette 
Published: October 2019

Abstract
Abstract 4759
We evaluated a novel e-notebook -based education program for patients with multiple myeloma. The program was developed by the Myeloma Initiative in the Veteran's Administration (MIVA) consortium to educate patients about myeloma while they wait for clinic appointments or procedures. The goals of this study were to assess the feasibility of the program in a busy clinic setting, patients ‘knowledge after the program and patients’ acceptance of the e-notebook format. Study subjects were patients with multiple myeloma being treated at the Central Arkansas Veterans Healthcare System McClellan Hospital. Eligibility criteria were diagnosis of multiple myeloma and ability to follow instructions in the program and complete a paper questionnaire. Study subjects evaluated the module while awaiting their clinic visit and then completed a survey consisting of ten multiple-choice questions designed to measure the goals of the study. The scope of the questions were: patients' primary source of medical education, patients' perceived benefit of the education program in improving their understanding of their disease (three questions), patients' preferences in regard to educational formats (three questions), and multiple myeloma knowledge (three questions).
Fifteen consecutive patients were enrolled in the study. Patient flow was not adversely affected by use of the module in the waiting room and the program was well accepted by the patients. Most patients in our study (93%) considered their primary source of medical information about their disease to be their oncologist, followed by internet resources (20%). Most patients (93%) stated that the education program helped them better understand the information provided by their oncologists and better understand multiple myeloma treatment options. Twelve patients (60%) answered that the program motivated them to learn more about myeloma. Fourteen patients (93%) recommended that this program be expanded to spouses or family members, and twelve patients (80%) advocated creating a video format of the program for repeat viewing. Nine patients (60%) stated they plan to view the content again at a later time. Of all multiple myeloma knowledge questions, 76% were answered correctly and 60% of the patients who answered correctly attributed their answers to information they learned in the education program.
We conclude that this e-notebook -based multiple myeloma education program is well accepted and can be adopted in a busy clinic. It is an effective and motivating tool for patient education in adjunct with physician efforts. It can be a productive utilization of waiting room time and has the potential for further applications.

Disclosures:
No relevant conflicts of interest to declare.
Hypoxia-Inducible Factor 1-Mediated Human GATA1 Induction Promotes Erythroid Differentiation Under Hypoxic Conditions
Authors: Zhang Jun-Wu , Zhang Feng-Lin , Shen Guo-Min , Liu Xiao-Ling , Wang Fang 
Published: October 2019

Abstract
Abstract 4794
The stimulation of red blood cell (RBC) production is one of the systemic adaptions to hypoxia. Hypoxia-inducible factor (HIF) promotes erythropoiesis through coordinated cell type-specific hypoxia responses. Hematopoietic transcription factor GATA1 is essential to normal erythropoiesis and plays a crucial role in erythroid differentiation. In this study, we show that hypoxia-induced GATA1 expression is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased GATA1 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34+ hematopoietic stem/progenitor cells (HPCs) derived from human cord blood. Enforced HIF1Á expression increased GATA1 expression, while HIF1Á knock-down by RNA interference decreased GATA1 expression in K562 cells. We searched the human GATA1 gene sequence on NCBI and identified a putative HRE in the 3'-flanking sequence of the gene. The results from reporter gene and mutation analysis suggested that this element is necessary for hypoxic response. Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo. These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1Á or GATA1. Flow cytometry analysis also indicated that hypoxia or desferrioxamine or CoCl2 induced expression of erythroid surface marker CD71 and CD235a, while expression repression of HIF1Á or GATA1 by RNA interference led to a decreased expression of CD235a. These results suggested that HIF1-mediated GATA1 upregulation promotes erythropoiesis in order to satisfy the needs of an organism under hypoxic conditions.

Disclosures:
No relevant conflicts of interest to declare.
Comparative Analysis on Cellular Components of Bone Marrow Microenvironment in Normal and Aberrant Hematopoiesis
Authors: Lee Young-Ho , Kwon Young-hee , Hwang Kyoujung , Jun Hyunju , Park Byungbae , Park Chan-Jeoung , Yoon Chan-Hee , Jang Seongsoo , Chi Hyun-Sook , Kim Dae-Young , Lee Jung-Hee , Lee Je-Hwan , Lee Kyoo-Hyung 
Published: October 2019

Abstract
Abstract 4808

Background:
It is now evident that hematopoietic stem cells (HSCs) reside preferentially at the endosteal region within the bone marrow (BM) where bone-lining osteoblasts are a key cellular component of the HSC niche that directly regulates HSC fate. We investigated the microenvironmental differences including osteoblastic activities and HSC components in myeloproliferative (chronic myeloid leukemia, CML) and hypogenerative disease (aplastic anemia, AA) as well as normal control (NC).

Methods:
The immunohistochemistry for osteonectin, osteocalcin, stromal cell derived factor (SDF, CXCL12), T cell, T helper/inducer cell, T suppressor/cytotoxic cell, hematopoietic stem/progenitor (CD34, CD117) and megakaryocytes was performed on BM biopsy specimens from 10 AA patients, 10 CML patients and 10 NC (lymphoma without BM involvement). The positive cells for immunohistochemical stainings except osteocalcin on each slide were calculated on 10 high power fields (HPF, ×400), and then corrected by the cellularity. The positive cells for osteocalcin were counted on the peritrabecular line on each slide, and then corrected by the mean length measured.

Results:
The CD34+ cells (p=0.012) and megakaryocytes (p<0.0001) were significantly lower in AA than in NC, but CD117+ cells was comparable in AA, CML, and control samples. The osteonectin+ cells (p=0.0003) were lower in CML than in AA and NC, however the osteocalcin+ cells showed wide variation (0-903/2035um) and no significant difference. The SDF+ cells (p<0.0001) was significantly higher in AA and very lower in CML, compared with NC. The counts for T cell and T cell subsets were significantly lower in CML than in NC, and higher in AA than in NC (p<0.0001).

Conclusions:
Cellular components of BM microenvironment in 2 hematologic diseases representative of myeloproliferation (CML) and hyporegeneration (AA) respectively are quite different. Further studies would be required to explore the role of these components for hematopoiesis and the rationale for therapeutic application.

Disclosures:
No relevant conflicts of interest to declare.
Interleukin-1β Promotes the Expression of CD34 and Granulocyte Colony-Stimulating Factor-Receptor in Adult Dermal Fibroblasts
Authors: Tashiro Haruko , Shirasaki Ryosuke , Oka Yoko , Yamamoto Tadashi , Akiyama Nobu , Kawasugi Kazuo , Shirafuji Naoki 
Published: October 2019

Abstract
Abstract 4815

Background and Aims:
We reported that acute myelogenous leukemia blasts and chronic myelogenous leukemia cells converted to stromal myofibroblasts to create an environment for the proliferation of leukemic cells in vitro and also in a non-obese diabetes/ severe combined immunodeficiency (NOD/SCID) murine bone-marrow in vivo. In normal hematopoiesis, hematopoietic stem cell (HSC) and stromal immature mesenchymal stem cell (MSC) are speculated to have a cross-talk, and some reports indicate that the HSC generates MSC, and also a specific fraction of MSC shares similar molecular expressions to that of HSC. We made a hypothesis that HSC might be generated from MSC. To make clear this issue, expression cloning was performed to isolate a molecule that stimulated bone-marrow stromal myofibroblasts to express hematopoietic stem cell marker, CD34. And, we also observed the effect of the isolated molecule to an adult human dermal fibroblast (HDF).

Materials and Methods:
cDNA-expression library was constructed using PHA-P-stimulated normal human blood lymphocytes, and the prepared plasmids were transfected to COS7 cells. After 3 days of culture, supernatants were added to the normal human bone-marrow-derived myofibroblasts (final 10%), and cells were further cultured for one week. RNA was extracted from the cultured myofibroblasts, and cDNA was synthesized. Positive clones were selected on CD34-expression with reverse transcription-polymerase chain reaction, and a single clone was isolated. The purified protein from the isolated single clone was added to HDF-culture, and the morphological changes and the expression of specific hematopoiesis-related proteins were analyzed.

Results and Discussion:
Isolated single clone was human interleukin 1β (IL-1β). When the purified IL-1β protein was added to the bone-marrow-derived myofibroblast cultures, cell growth was increased, and up-regulation of the expression of several hematopoietic specific proteins, including cytokine receptors and transcription factor SCL, was observed. Based on these observations, we determined the effect of IL-1β to HDF. When HDFs were cultured with human IL-1β for 3 weeks, the expression of granulocyte colony-stimulating factor (G-CSF)-receptor, and SCL was increased. When these IL-1β-stimulated cells were cultured in a non-coated dish, cells were floating, and budding of the cells was also observed. When HDF were cultured with IL-1β for 3 weeks, and then G-CSF and erythropoietin were added to the cultures, expression of transcription factor GATA-1 and CEBPA was significantly increased after one week. These observations indicate that IL-1β can stimulate to induce HDF toward hematopoietic cells. Now we determine the precise actions of human IL-1β to HDF using NOD/SCID transplantation model in vivo.

Disclosures:
No relevant conflicts of interest to declare.
To Use CD64 Expression Level on Neutrophils As An Infection Marker in Children 10 Years Old or younger Cut-off Values Higher Than That in Adults Must Be Established
Authors: Matsushita Shoko , Inagaki Yoshiko , Nakatsuji Yukie , Endo Mikiya , Ito Shigeki 
Published: October 2019

Abstract
Abstract 4930

Objectives:
To develop the use of CD64 expression as an early diagnostic of adult collagen disease, we elucidated normal levels in neonates, infants and children.

Methods:
Blood samples were collected from a total of 62 symptom- and infection-free subjects recruited at our outpatient department: neonates (n=80), infants (n=41), 1- to 5-year-old children (n=77), 6- to 10-year-old children (n=58), children 11 years old or older and adults (n=95). CD64 expression per neutrophil was quantitatively determined by flow cytometry using a BD FACS Calibur HG flow cytometer (Becton Dicknson, BD) and BD CellQuest Pro analysis software (BD). Fluorescence was measured and converted to number of CD64 molecules per cell using a phycoerythrin (PE) fluorescence kit (Quantibrite PE; BD Biosciences) according to the manufacturer's instructions. This study was approved by the ethics committee of Iwate Medical University.

Results:
CD64 expression level in neonates (median, 1981 molecules per neutrophil; range, 908–9844), infants (1666.5, 572–12941), and 1- to 5-year-old children (1778, 545–11283) was significantly higher than that in children 11 years old or older and adults (1280, 533–11205) (p<0.01 or <0.05). There were no differences in expression between 6- and 10-year-old children (1529, 439–9214) and children 11 years old or older and adults.

Conclusion:
Our results suggest that the normal value for adults can not be applied for neonates, infants, and 1- to 5-year-old children. To use CD64 expression level on neutrophils as an infection marker in neonates, infants and 1- to 5-year-old children cut-off values higher than that in adults must be established.

Disclosures:
No relevant conflicts of interest to declare.
Immunologic Effects of An Oral BRAF Inhibitor in a BRAF Wild-Type Murine Model
Authors: Vosganian Gregory S. , Bos Rinke , Sherman Linda 
Published: October 2019

Abstract
Abstract 4935

Introduction:
Metastatic melanoma represents a clinical challenge as aggressive therapy often results in unacceptable toxicity and less intensive therapy results in suboptimal clinical outcomes. PLX-4032 is an orally available small molecule which targets constitutively activated BRAFV600E in melanoma cells. BRAF is an important regulator of cell growth, proliferation and migration. We examined the effect of PLX-4032 on the immune system in two BRAF wild type murine systems: the first to determine the effect of PLX-4032 on cytokine production and the second to determine any functional immune effect of PLX-4032 in an insulinoma model.

Methods:
Effect of PLX-4032 on cytokine production: B10.D2 BRAF WT mice were injected with CD8 lymphocytes expressing a TCR specific for HA518-526 peptide (clone 1) and CD4 lymphocytes expressing a TCR specific for HA110-119 peptide (SFE) on Day 0 and concurrently simulated with appropriate peptide, incomplete Freund's adjuvant, and poly IC. Mice received PLX-4032 200mg/kg or placebo daily for 5 days. On day 6, splenic lymphocytes were harvested and flow cytometry performed to determine CD4 and CD8 IL2, IFN gamma, and TNF alpha production. Effect of PLX-4032 on functional immune response: B10.D2 BRAF WT rat insulin promoter (RIP)-Tag2-hemagglutinin mice bearing insulinoma were injected with clone 1 and SFE lymphocytes on Day 0 and received concurrent stimulation as above. Mice received PLX-4032 200mg/kg or placebo daily for 30 days and serum glucose levels were monitored weekly.

Results:
Effect of PLX-4032 on cytokine production: We noted no significant difference in the absolute number of splenic lymphocytes recovered in the two groups. There was no significant difference in the percent of CD8+ clone 1 cells producing TNF alpha, IL2 or IFN gamma. There was similarly no significant difference in the percent of CD4+/SFE lymphocytes recovered between the treatment and control groups, nor was there a significant difference in the percent of cells producing IFN gamma. Effect of PLX-4032 on functional immune response: Treatment with PLX-4032 resulted in no significant inhibition of the immune response of transferred lymphocytes against insulinoma. Mice in both groups developed significantly elevated serum glucose levels at the same time and remained diabetic for a similar duration.

Discussion:
Our data demonstrate that PLX-4032 does not appear to exert a quantitative or functional effect on the immune system in BRAF WT mice. Melanoma therapies often include medications which augment patient immune function or specifically target immune regulation; these data suggest that PLX-4032 does not inhibit normal immune function and therefore may have a role in combination with immunologically directed therapy, possibly resulting in synergistic anti-tumor effect by targeting multiple pathways involved in tumor survival.

Disclosures:
No relevant conflicts of interest to declare.
Lymphoproliferative Disorders (LPD) in Individuals ≥80 Years of Age–Incidence, Response, and Survival
Authors: France Fallon O , Chikkappa G. , Pasquale Donald 
Published: October 2019

Abstract
Abstract 4945

Introduction
Clinical trials describing LPD therapy and outcomes overwhelmingly exclude individuals ≥80 years of age. Thus, ability to deliver LPD therapy to this age group is not defined, and tolerance to therapy and response rates and survival data are not available.

Methods
We retrospectively identified all individuals diagnosed with any LPD at ≥80 years of age at our institution between 1997 and 2010. Data included age at diagnosis, diagnosis, therapy for LPD, comorbidities (disease count), survival from date of diagnosis of LPD, chemotherapy dose intensity (percent of 100% dose based on standard regimen dosing), hospitalization for any reason during chemotherapy, and response. Survival was analyzed by Kaplan-Meier analysis and data sets were compared using Student's t-test or Chi-square as appropriate.

Results
Of a total of 518 individuals diagnosed with any LPD, we identified 33(6.4%) who were diagnosed ≥80 years of age. All were male consistent with our veteran population. Data is illustrated in tables 1 and 2. Fifteen (15) had Large B-cell lymphoma, 4 CLL/SLL, 2 follicular, 2 mantle zone, 1 marginal zone, 2 lymphoplasmacytic, 3 T-cell. Two (2) could not be classified despite AFIP review. Twenty-two (22) individuals were treated with 73 cycles of chemotherapy (25 CHOP, 30 R-CHOP, 3 CVP, 11 R-CVP, 4 PO cyclophosphamide). One (1) individual with LBCL had no evidence of disease following excisional biopsy and declined further therapy, and 1 received radiotherapy. A total of 27 (82%) patients died during observation. Deaths were predominantly due to LPD. Of those not treated, 5 of 9 had low-grade LPD and were among longer-term survivors. Median survival, illustrated in the figure, was 18.8 months and was not different between treated and untreated group. This compares with 72.7 months predicted survival for an average 84-year old (WWW.SSA.GOV).

Discussion
LPD is common in individuals ≥80 years of age with large B-cell lymphoma being most common in our population. Despite multiple co-morbidities, the data show that reasonable dose intensity combination chemotherapy may be delivered to treat octogenarians, however hospitalization was required for 1/3 patients during chemotherapy. While survival was not different between treated and untreated groups, the individuals who were treated likely had more severe or advanced stage LPD. Prospective studies with sufficient number of individuals within each diagnostic category should be done to clarify benefits of chemotherapy in ≥80 year old patients with LPD.

Disclosures:
No relevant conflicts of interest to declare.