bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Changes on hematopoietic Stem Cells and mIcroenvIronment of childhood acute Lymphoblastic Leukemia at dIagnosIs and after chemotherapy
Authors: Lee Mee Jeong , Park Chan-Jeoung , Yoon Chan-Hee , Jang Seongsoo , Chi Hyun-Sook , Im Ho Joon , Kim Bo Eun , Seo Jong Jin 
Published: October 2019

Abstract
Abstract 4807

Background:
Hematopoietic microenvironment consists of endosteal and vascular microenvironment. Osteopontin and osteonectic are released by osteoblast which is one of the factors regulating endosteal microenvironment. CXCL12 and CXCR4 are included in the factors regulating vascular microenvironment. Markers indicating the degree of differentiation of hematopoietic stem cells (HSC) include CD133, CD34 and CD117. This study is to evaluate the expression pattern of markers of hematopoietic microenvironment and HSC in childhood acute lymphoblastic leukemia (ALL) at diagnosis and the changes of them after chemotherapy.

Methods:
Between January, 2007 and December, 2009, 32 patients were diagnosed as ALL at Asan Medical Center. Bone marrow (BM) biopsies were obtained at diagnosis, after induction, consolidation, interim maintenance and delayed intensification. There were 22 male and 10 female patients with a mean age of 7.33 years. The diagnoses were a common cell ALL in 29. By immunohistochemistry, we analyzed the expressions of CD133, CD34, CD117, osteopontin, osteonectin, CXCL12, and CXCR4 in these BM biopsy specimens.

Results:
CD133+ cells decreased at diagnosis, and recovered after consolidation. The CD34+ cells decreased after induction, and then gradually increased again. CD117+ cells were fewer at diagnosis, but increased after chemotherapy. The expression of osteopontin was depressed at diagnosis, and gradually recovered. The expression of osteonectin was also suppressed at diagnosis and recovered after delayed intensification. CXCL12 was suppressed at diagnosis, recovered after consolidation and decreased after delayed intensification. CXCR4 was also suppressed at diagnosis, but increased after therapy.

Conclusions:
This study is the first report on the changes of hematopoietic microenvironment and HSC of childhood ALL at diagnosis and after chemotherapy, evaluated by immunohistochemistry. CD133+ cells, CD34+ cells and CD117+ cells increased in the reconstructive phase. CD34 can be a marker reflecting the BM recovery. Proliferation of osteoblasts and stromal cells were active after interim maintenance. The reconstruction of HSC and BM matrix increased after consolidation. The recovery of BM microenvironment was established after consolidation and interim maintenance chemotherapy. These findings may serve as basic data for future studies dealing with the hematopoietic microenvironment and HSC of childhood ALL.

Disclosures:
No relevant conflicts of interest to declare.
The Growth Response of Erythrocyte Production to Increased Dense Condition
Authors: Choi Hye sook , Lee Eun-mi , Ajjappala Brijesh Siddaveerappa , Huh Ji Young , Yoon Hwi-Joong , Baek Eun Jung 
Published: October 2019

Abstract
Abstract 4812
Erythroid precursors proliferate, differentiate, and enucleate in the bone marrow niches called erythroblastic islands in contact with macrophages. However, the cell-to-cell communication between erythroblasts which are undergoing terminal maturation leading to enucleation is not well known. Moreover, the cell-to-cell interaction at a terminal erythroblast maturation stage was hardly detected in conventional suspension culture systems due to cell surface characteristics of electrostatic repulsion.
To elucidate the communication between late maturing erythroblasts, erythroid cells, which were derived from human CD34+ cells, were cultured at conventional or supra-optimal densities. Surprisingly, the final yields of red blood cells (RBCs) were significantly increased in supra-optimal culture density, even in the absence of feeder stromal cells and serum or plasma. Also observed were increases in the rates of cell survival, expansion, and enucleation. Contact between cells observed in these supra-optimal cultures was associated with increases in adhesion-related signaling proteins (150 kDa and 123 kDa), the latter of which was previously unknown to be involved in erythropoiesis. The cultured erythroid cells showed erythropoiesis specific markers such as CD71, GPA, GATA-1 and b-globin.
Therefore, we firstly elucidated that the erythroid cells which were in the final maturation step can communicate each other and affect their survival and maturation including enucleation without the help of macrophages. Also, the increased attachments between erythroid cells evoked adhesion-related signals. We anticipate that the culture of these cells at supra-optimal density may enhance in vitro RBC production for human transfusion.

Disclosures:
No relevant conflicts of interest to declare.
Ex Vivo Expanded Megakaryocyte Progenitors Generated From Human Mobilized Peripheral Blood CD34+ Stem Cells Can Be Cryopreserved and Transplanted Into NSG Mice to Generate Functional Platelets
Authors: Karsunky Holger , Tressler Robert J. , Chananukul Joy 
Published: October 2019

Abstract
Abstract 482
Thrombocytopenia is a common side effect of high-dose chemotherapy that can compromise cancer treatment by requiring treatment delay and/or dose reduction for the patient. Platelet transfusion is typically given to prevent severe hemorrhage. However, several factors including acquisition, banking, and associated risks of bacterial infections and alloimmunization are hampering reliance on platelet transfusion. Growth factors are also used to stimulate proliferation and differentiation of megakaryocytes to increase platelet production, but in severely myelosuppressed patients these have only had modest benefit. The limitations of these two modalities for the treatment of chemotherapy-induced thrombocytopenia indicates that additional treatment approaches are needed. We have developed a novel approach to reconstitute megakaryocytes and platelets in thrombocytopenic patients which is presented here. We have identified a scalable culture system using serum-free medium and a defined cytokine cocktail free of animal products to expand CD34+ hematopoietic stem cells from G-CSF mobilized peripheral blood donors in vitro and direct their development to the megakaryocyte lineage to yield committed human megakaryocyte progenitors (MKPs). These MKPs can be readily cryopreserved while retaining their capacity to generate CFU-MK and platelets in vitro. When infused into NSG mice, ex vivo expanded MKP generate clinically relevant platelet levels of platelets in blood within a few days with sustained platelet levels for several weeks. The platelets generated from MKP in vivo are also functional as assessed by CD62P expression in responses to ADP stimulation in vitro. Our results present a compelling approach for the development of off-the-shelf storable MKPs for the treatment of thrombocytopenia.

Disclosures:
Karsunky: Cellerant Therapeutics Inc.: Employment, Patents & Royalties. Tressler:Cellerant Therapeutics, Inc.: Employment, Equity Ownership. Chananukul:Cellerant Therapeutics Inc.: Employment, Patents & Royalties.
A Sensitive and Quantitative Polymerase Chain Reaction-Based Non-Homologous End Joining in Vitro Assay for Hematopoietic Stem Cells
Authors: Shao Lijian , Feng Wei , Zhou Daohong 
Published: October 2019

Abstract
Abstract 4825
Hematopoietic stem cells (HSCs) are longevity cells that are responsible for sustaining hematopoietic homeostasis and regeneration after injury for the entire lifespan of an organism. Maintenance of genomic stability is crucial for the preservation of HSCs, which depends on their efficient repair of DNA damage, particularly DNA double strand breaks (DSBs). Because of the paucity of HSCs and lack of sensitive assays, directly measuring the ability of HSCs to repair DSBs has been difficult. Therefore, we developed a sensitive and quantitative in vitro non-homologous end joining (NHEJ) assay using the Bgl II-digested linear pDsRed2ER plasmids and real-time polymerase chain reaction (qPCR) technique. This assay can sensitively detect DSB repair via NHEJ in less than 0.25 μg nuclear proteins or nuclear extracts from about 1000 human BM CD34+ hematopoietic cells. Using this assay, we confirmed that human bone marrow HSCs (CD34+CD38− cells) are less proficient in the repair of DSBs by NHEJ than HPCs (CD34+CD38+ cells). In contrast, mouse quiescent HSCs (Pyronin-Ylow LSK+ cells) repaired the damage more efficiently than cycling HPCs (LSK− cells). The difference in the abilities of human and mouse HSCs and HPCs to repair DSBs through NHEJ is likely attributed to their differential expression of key NHEJ DNA damage repair genes such as ATM and ligase 4. These findings suggest that the qPCR-based plasmid NHEJ assay can be used to sensitively measure the ability of HSCs to repair DSBs. This assay can also be applied to other rare tissue stem cells as well.

Disclosures:
No relevant conflicts of interest to declare.
Increased Body Mass Index Correlates with Higher Risk of Disease Relapse and Differentiation Syndrome in Patients with Acute Promyelocytic Leukemia Treated with Aida Protocols
Authors: Breccia Massimo , Mazzarella Luca , Loglisci Giuseppina , Bagnardi Vincenzo , Disalvatore Davide , Latagliata Roberto , Testi Anna Maria , Cimino Giuseppe , Avvisati Giuseppe , Petti Maria Concetta , Pelicci Pier Guiseppe , Foá Robin , Lo Coco Francesco 
Published: October 2019

Abstract
Abstract 4866
The association between obesity and outcome of acute promyelocytic leukemia (APL) patients is still not defined. We investigated whether increased body mass index (BMI) might have a role in the outcome of APL patients. The study population includes 144 patients consecutively diagnosed as having APL between January 1993 and December 2005 at the “Sapienza” University of Rome and treated with AIDA or AIDA2000 schedule. Diagnosis was confirmed for all patients at molecular level. Patient demographic information, weight and height, treatments received, events and survival outcome data were collected. In particular the diagnosis of definitively present retinoic differentiation syndrome (DS) was clinically established by the presence of at least three of the following signs weight gain, respiratory distress, unexplained fever, interstitial pulmonary infiltrates, pleural or pericardial effusions, according to Frankel et al. There were 66 males and 78 females, median age 39.3 years. Seventy-five patients were aged more than 40 years at diagnosis. Twenty-four patients out of 144 were classified as having variant type according to FAB classification and 83 patients have BCR1 type of transcript. BMI was defined as the individual's body weight divided by the square of him or her height: we stratified 4 categories of patients, according to height and weight at the time of diagnosis: underweight (BMI< 18.5), normal weight (BMI 18.5-<24.9), overweight (BMI 25->29.9) and obese (BMI ≥30). Fifty-seven (39.6%) and 87 (60.4%) patients were underweight/normal (BMI<25) and overweight/obese (BMI≥25), respectively. Increased BMI was associated with older age (p=0.0002) and male gender (p=0.004). Twenty-one patients (14.5%) experienced DS during induction therapy according to reported criteria: we evaluated in multivariable logistic regression model clinico-pathological features at baseline possibly associated to an increased risk of DS and we identified as the significant factor only BMI (p=0.014, OR 7.2, 95% CI 1.5–34.9). After a median follow-up time of 6 years, the estimated cumulative incidence of relapse (CIR) at 5 years was 31.6% (95% CI: 22.7%,43.8%) in overweight/obese and 11.2% (95% CI: 5.3%,23.8%) in underweight/normal weight patients (p=0.029). Multivariable analysis showed that BMI was an independent predictor of relapse (HR = 2.45, 95% CI 1.00, 5.99, in overweight/obese vs under/normal weight patients, p=0.049). In conclusion, increased BMI at diagnosis is associated to higher risk of RAS and relapse in APL.

Disclosures:
Breccia: novartis: Honoraria; Bristol Myers Squibb: Honoraria.
HSP90 Chaperone – An Important Player in CML: Study At RNA and Protein Level
Authors: Zackova Marketa , Lopotova Tereza , Nadvornikova Sylvie , Klamova Hana , Moravcova Jana 
Published: October 2019

Abstract
Abstract 4878
Chronic myeloid leukemia (CML) is the myeloproliferative disease characterised by presence of BCR-ABL tyrosine kinase, which has been proved to play the basic role in CML ontogenesis. BCR-ABL as well as other kinases active during disease progression (e.g. Src kinase) belong to HSP90 client proteins. It is known, that HSP90 protein is overexpressed in cancer cells and leukemias and is related to therapy resistance in CML cells (Gorre et.al. Blood 2002;100:3041-3044).
HSP90 exists in two isoforms: alpha - inducible and beta – constitutively expressed. Taherian et.al. (Biochemistry and Cell Biology, 2008, 86:(1) 37–45) demonstrated that Hsp90α and Hsp90β exhibit similar interactions with cochaperones, but significantly different substrate specificity under stress conditions. Those results reveal both functional similarities and key functional differences between the individual members of this protein family.
In our previous study we found high protein expression of total HSP90 in leukocytes of CML patients in advanced disease states and in patients with poor responses (Zackova et.al. EHA 2011). The HSP90 seems to be an indicator of disease deterioration in patients with chronic myeloid leukemia.
In the current study, we extended our field of interest on HSP90 isoforms alpha and beta on mRNA level. We aimed to find out possible correlation between protein and mRNA expression levels, as well as between mRNA levels and response to the therapy. We wondered to know whether the monitoring of total HSP90 or its isoforms in CML can early predict the disease deterioration.
We tested HSP90 alpha and HSP90 beta mRNA expression levels in patients with various response of CML by real-time RT-PCR method. The mRNA profiles showed high similarity with the data obtained from previous western blot analyses. The analyses showed that high HSP90 levels are associated with poor response to therapy and in advanced disease phases. These levels are probably represented by HSP90-beta isoform (constitutively expressed), which is expressed in higher levels comparing to HSP90 alpha in all samples tested. Expression of HSP90 alpha isoform is much lower while its mRNA expression level highly increases only in blast crisis.
Studying both isoformes separately could distinguish various mechanisms in disease progression.
The results of this and previous studies suggest HSP90 (on protein and mRNA levels) is an important molecule for studying of prognosis in CML patients. Thus HSP90 appears to be a candidate for novel marker of CML progression.

Disclosures:
No relevant conflicts of interest to declare.
Establishment of Bioluminescence Imaging Based Leukemia In Vivo Model for Preclinical Testing
Authors: Lee Myoung Woo , Kim Hye Jin , Kim Dae Seong , Son Meong Hi , Lee Soo Hyun , Jung Hye Lim , Yoo Keon Hee , Sung Ki Woong , Koo Hong Hoe 
Published: October 2019

Abstract
Abstract 4879

Background.
A hematological malignant animal model is an essential tool for evaluating efficacy of anti-cancer drugs and elucidating underlying mechanism of leukemogenesis. Intraperitoneal (IP) and intravenous (IV) xenograft of acute lymphoblastic leukemia (ALL) cells have limited capacity as in vivo anti-cancer drug screening system.

Purpose.
In this study, we aimed to establish an ALL animal model using NOD/SCID mouse and evaluate efficiency and sensitivity of the model as a preclinical drug screening system.

Materials and Methods.
Firefly luciferase (fLuc)-gene introduced ALL (ALL/fLuc) cell line and patient-originated ALL cells were transplanted into a tibia of NOD/SCID mouse. We conducted a comparative analysis of intra-bone marrow (IBMT) transplanted leukemia model with IP and IV transplantation of leukemic cells.

Results.
IBMT of ALL/fLuc cells effectively established a bioluminescent leukemia NOD/SCID mouse model. Upon comparison of IBMT model with IP and IV transplantation models, infusing identical number of ALL/fLuc cells into NOD/SCID mice resulted in IBMT model with evaluable bioluminescent signal, but not in IP and IV models. In IBMT model, bioluminescent signals of ALL/fLuc cells emitted from peripheral blood, tibia and infiltrated organs indicated that leukemia model was established. The changes in these signals' strength reflected dose-dependent cytotoxic effects of vincristine, which allowed leukemia model with evaluable bioluminescent signal to be utilized as a preclinical drug screening system. IBMT leukemia model was also established using primary ALL cells that can provide additional insights for the development of leukemia therapeutics.

Conclusion.
IBMT of ALL/fLuc cells enables development of leukemia mouse model with the greater bioluminescent sensitivity than IP and IV in NOD/SCID to evaluate candidate for development of anti-cancer drug.

Disclosures:
No relevant conflicts of interest to declare.
MYL9 Is Prerequisite for the CD3 Expression in Cytoplasm Membrane of T Lymphocytes
Authors: Aoki Takumi , Miyazaki Koji , Higashihara Masaaki 
Published: October 2019

Abstract
Abstract 4925

[Introduction]
Normal T lymphocytes express CD3 protein, major component of T-cell receptor, in the cytoplasm membrane. However, CD3 expression is suppressed in malignant T cells. The mechanism of suppression of CD3 expression remains unknown. We previously reported the cloning of three isoforms of non-muscle myosin light chain, including MYL9 (transcript variant 1). MYL9 mRNA was expressed in monocyte-macrophage cell lines, but not in lymphoid cell lines (J. Smooth Muscle Res.44: 29–40, 2008). We report here some evidence, suggesting the prerequisite role of MYL9 (transcript variant 1) in the process of CD3 expression in cytoplasm membrane.

[Materials and Methods]
Leukemic cell lines were cultured in RPMI1640 plus 10% FCS. mRNA contents were measured with RT-PCR. CD3 expression was measured using FACScan FITC-labeled or PE-labeled second antibody. Whole CD3 contents were measured using IntraPrep™ (Beckman Coulter). Transfection of MYL9(transcript variant 1)gene into Jurkat cell (wild-type) was performed using Trans Fasc Reagent (Promega). In the process of screening the change the percentage of surface marker, we found that some clones showed the CD3 positive, and Jurkat (clone I), which expresses CD3 (85∼90%) in the cytoplasm membrane, was selected. We also used Jurkat, Clone E6-1(TIB-152)(ATCC®), strong positive in CD3 (90∼95%) in cytoplasm membrane.

[Results]
1) CD3 were negative in Jurkat cell (wild-type), and highly positive in both clone I and clone E6-1. 2) Whole CD3 (cytosole and cytoplasm membrane) were positive in these three cells, suggesting that transportation of CD3 protein from cytosole to cytoplasm membrane is suppressed in Jurkat cell (wild-type). 3) Long term culture (i.e. 30∼60 days) induced, although weakly, both MYL9 mRNA and CD3 of cytoplasm membrane in Jurkat cell (wild-type). 4) Clone E6-1 shows positive in MYL9 mRNA. 5) In Jurkat cell (wild-type), PMA induced CD3 expression in cytoplasm membrane, but not MYL9 mRNA. Pre-treatment with staurosporine showed partial inhibitory effect on PMA-induced CD3 expression. 6) Neither concanavalin A nor A23187 induced CD3 expression in both clone I and Jurkat cell (wild-type).

[Discussion]
These results suggest that MYL9 is prerequisite for the CD3 expression in cytoplasm membrane of normal T lymphocytes. However, in T-cell malignant cells, MYL9 gene is suppressed by unknown mechanism. Our data show that CD3 expression in cytoplasm membrane may be regulated by MYL9-dependent and MYL9-independent fashions.

Disclosures:
No relevant conflicts of interest to declare.
The Mortality of Vancomycin-Resistant Enterococci Bloodstream Infections (VRE BSI)
Authors: Choi Philip Young-Ill , Straube Belinda , Cook Christine , Van Der Weyden Carrie , Ramanathan Sundra , Taylor Peter , Roncolato Fernando , Chong Beng 
Published: October 2019

Abstract
Abstract 4928

Background
VRE are nosocomial pathogens with resistance to most commonly used antimicrobial agents. VRE BSI occurs in as few as 4% of patients colonised with VRE[i]. A review of factors contributing to the development of VRE BSI was performed to improve patient safety at St George Hospital.
Alarmingly, there were 9 new cases of VRE BSI detected amongst haematology inpatients in early 2010, as compared with only 2 in the previous 6 months. In high risk populations, the rate of VRE BSI amongst patients colonised with VRE can be as high as 29%[ii].
VRE BSI is associated with an increased length of hospital stay from 10.5 to 46 days[iii][iv]and an estimated increased cost of $27,190 per patient[v]. Annual net savings of $100,000-150,000 can be achieved by hospitals detecting 6–9 cases of VRE BSI per year by utilising enhanced infection control strategies[vi].

Methods
Admission details for haematology in-patients and their microbiology results between 1/6/2009-30/11/2010 were reviewed. A subsequent case-controlled analysis was performed matching for patient age, disease and disease stage.
Interventions to reduce the rate of VRE transmission were introduced in July 2010: improved hand hygiene education, additional staffing allocations, additional cleaning services, antimicrobial stewardship, improved patient education, increased staff awareness, monthly census screening of all patients on the 4 East Oncology/Haematology ward for VRE and contact tracing measures.

Results
471 patients were admitted a total of 943 times. VRE was isolated in 61 patients. Average length of stay was significantly longer in patients with VRE than for patients without VRE [16.3±3.0 vs 8.4±1.0 days, p=0.01].
16 patients had VRE BSI. After a median follow-up of 9.8 months, eight of these patients have died (50% mortality), mostly due to progressive disease.
45 patients had non-BSI VRE and were followed up for a median 8.4 months: 24 died (53% mortality), also mostly due to progressive disease.
Case-controlled Analysis
An age, case and stage of disease matched analysis compared 14 patients with VRE and 14 with no evidence of VRE. Median follow-up from presentation with disease was 16.3 months and from VRE detection was 8 months. Mortality rate was 64% vs 29%. Seven out of nine deaths in the VRE cohort were due to progressive disease.

Conclusions
The clinical significance of VRE BSI over other modes of detection remains uncertain for individual patients, however high rates may reflect uncontrolled VRE transmission. Case controlled analysis demonstrates an associated additional mortality risk for VRE positive patients.

Disclosures:
No relevant conflicts of interest to declare.