bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sequence-Specific Conversion of βA- to βS-Globin by Small Fragment Homologous Replacement In Hematopoietic Stem/Progenitor Cells.
Authors: Abdolmohammadi Alireza , Maurisse Rosalie , Bedayat Babek , DeSemir David , Laber Damian , Gruenert Dieter C. 
Published: October 2019

Abstract
Abstract 3754

Introduction:
An ultimate goal of gene therapy is the development of effective strategies to correct mutant genomic sequences in pathologic cells. To that end, studies have been undertaken to evaluate the therapeutic potential of an oligo/polynucleotide-based sequence-specific gene modification strategy, small fragment homologous replacement (SFHR) in the correction of the mutation giving rise to sickle cell anemia. Small DNA fragments (SDFs) comprising the sickle cell anemia mutation (an A>T transversion in codon 6) and flanking DNA sequences in the human b-globin gene were introduced into Hematopoietic Stem/Progenitor Cells (HSPCs). The studies presented indicated modification at the level of DNA, RNA, and protein when cells were differentiated into erythrocytes.

Methods:
In this study, SFHR was used to convert A>T in codon 6 of the b-globin gene in CD34+/CD38-/Lin- HSPCs isolated from full term umbilical cord blood as a proof of principle. HSPCs were transfected with a defined number of a 559-bp SDF using the Amaxa electroporation (nucleofection) system. After growing the transfected cells in stem cell media containing EPO for different time intervals up to 32 days, RNA was extracted and DNase I-treated before further analysis. Erythrocytes were also analyzed using antibodies that differentiate between wild-type hemoglobin A (HBA) and sickle cell hemoglobin S (HBS).

Results:
RFLP analysis of a 430-bp PCR product generated from mRNA-derived cDNA with the DdeI enzyme indicated conversion of bA- to bS-globin. Sequencing of the 430-bp amplicon showed the A > T conversion. Analysis of the transfected wild-type HSPC-derived erythrocytes with HBA and HBS specific antibodies demonstrated the presence of a subpopulation of cells expressing HBS. These data are consistent with previous studies showing both conversion of bS- to bA-globin in SC1 cells and bA- to bS-globin in HSPCs after electroporation and microinjection of SDF, respectively.

Conclusion:
These studies represent a critical next step in developing SFHR as a therapy for sickle cell disease, in that they demonstrate long-term SFHR-mediated modification of b-globin following mass transfection by electroporation of HSPCs.

Disclosures:
No relevant conflicts of interest to declare.
Inhibition of BCL11B Expression Leads to Apoptosis of Malignant T Cells but Not CD34+ Cells.
Authors: Huang Xin , Chen Si , Chen Shaohua , Yang Lijian , Shen Qi , Grabarczyk Piotr , Przybylski Grzegorz K. , Schmidt Christian A. , Li Yangqiu 
Published: October 2019

Abstract
Abstract 3755
Altered expression, mutation, disruption, or rearrangement of the B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B gene (BCL11B) has been associated with T-cell malignancies. BCL11B overexpression occurs primarily in T-cell malignancies, suggesting that it may be a target gene for cancer therapy. Our previous study showed that the inhibition of BCL11B expression by siRNA led to apoptosis in human T-cell acute lymphoblastic leukemia cell lines (Jurkat, huT78, and Molt-4), but not in normal, mature T cells. To confirm the potential of BCL11B siRNA as a therapeutic agent and to determine its safety, the present study further analyzed the effects of BCL11B suppression on hemopoietic stem/progenitor cells, CD34+ cells were collected from three samples of umbilical cord blood using magnetic-activated cell sorting. BCL11B expressions in mRNA level of umbilical cord blood CD34+ cells were analyzed by the real-time quantitative PCR with TaqMan technique. For nucleofection of CD34+ cells, BCL11B-siRNA935 and the U-001 program were used of a Nucleofection Device II. Mock-transfected cells nucleofected without siRNA were used as a negative control. To evaluate the role of differentiation and proliferation in CD34+ cells after BCL11B-siRNA935 transfection, erythroid burst-forming units (BFU-E), granulocyte/macrophage colony-forming units (CFU-GM), and megakaryocyte colony-forming units (CFU-Meg) from BCL11B siRNA935-, mock-CD34+ cells were performed using methylcellulose assays. Colonies were assessed 14 days after plating. The BCL11B mRNA expression level in the CD34+ cells was significantly lower than that in Molt-4 cells and in peripheral blood mononuclear cells from eight healthy individuals. There was no significant difference between BCL11B siRNA935-transfected and mock-transfected CD34+ cells with respect to the formation of BFU-E, CFU-GM, or CFU-Meg (P > 0.05). We speculate that the BCL11B gene, because of its very low expression level, may not play a major role in proliferation and differentiation of hematopoietic stem/progenitor cells, but we cannot rule out another anti-apoptotic mechanism. Nevertheless, BCL11B gene silencing alone does not affect the proliferation and differentiation of hematopoietic stem/progenitor cells in vitro. In conclusion, our findings provide evidence for an anti-apoptotic function of BCL11B in T-cell malignancies, but not in hemopoietic stem/progenitor cells, suggesting that BCL11B siRNA is safe and may be considered a new targeted therapeutic strategy for T-cell malignancies.
* National Natural Science Foundation(30771980)& Guangdong Science and Technology Project (2007B030703008) Grants

Disclosures:
Li: National Natural Science Foundation (30771980) and Guangdong Science and Technology Project (2007B030703008, 2009B050700029) Grants: Research Funding.
Impaired Lentiviral Transgene Expression In Vivo Caused by Massive Methylation of SFFV Promoter Sequences.
Authors: Herbst Friederike , Ball Claudia R , Tuorto Francesca , Wang Wei , Kloz Ulrich , Hoeven Frank van der , Lyko Frank , Kalle Christof Von , Glimm Hanno 
Published: October 2019

Abstract
Abstract 3760
Lentiviral vectors (LV) assure stable transgene expression in vivo, allowing to investigate genes and their functions. In recent years, lentiviral gene transfer was considered to facilitate the generation of transgenic mice with a higher yield of transgenic offspring as compared to commonly used DNA microinjection. We applied LV to generate a mouse model transgenic for SETBP1 and eGFP.
Murine zygotes were infected at dE0.5 with lentiviral particles directly injected into the perivitelline space. Specific PCRs for either the SETBP1 transgene or for the WPRE element of the lentiviral construct verified complete lentiviral integration in newborn pups (F0). Lentiviral integration sites were detected using highly sensitive LAM-PCR in 65% of 31 analyzed F0 mice. Germline transmission was shown in a total of 33% vector positive offspring from 5 out of 9 F0 mice. However, no ectopic transcription and overexpression of neither SETBP1 nor eGFP could be detected in transgenic mice. We therefore analyzed the methylation status of the internal SFFV promoter (SFFVp) by bisulfite sequencing. Extensive methylation (around 90%) could be assessed in 18 of 18 analyzed CpGs within the promoter region in F0 animals and in all progeny determined (n=12). We transduced mES cells with LV.SFFV.Setbp1.IRES.eGFP or the corresponding eGFP-expressing control vector to exclude transgene effects on epigenetic silencing of SFFVp sequences in self-inactivating LVs. Differentiation of ES cells infected with the transgene vector and SFFV driven control vector led to a 1.8 – 3.5 fold decrease of eGFP expression. To analyze whether methylation of SFFVp sequences is a common event even in adult tissues, we analyzed the methylation status of peripheral blood in mice transplanted with bone marrow cells transduced with either gammaretroviral vectors (RV) or LV 3 months after transplantation (n=7). Interestingly, SFFVp sequences in peripheral blood of mice transplanted with LV transduced bone marrow were stronger methylated than CpGs of SFFVp in RV transplants.
Our data demonstrate that the commonly used SFFV promotor is highly methylated with remarkable strength and frequency during development in vivo and differentiation in vitro. We conclude that lentiviral vectors using an internal SFFV promoter are not suitable for the generation of transgenic mice or constitutive expression studies in hematopoietic cells.

Disclosures:
No relevant conflicts of interest to declare.
Bone Turnover Markers In Gaucher Disease Patients.
Authors: Giuffrida Gaetano , Cingari Maria Rocca , Parrinello Nunziatina , Romano Alessandra , Triolo Anna , Franceschino Magda , Raimondo Francesco Di 
Published: October 2019

Abstract
Abstract 3780

Background:
Gaucher Disease (GD) is an autosomal recessive hereditary disorder of glycosphingolipid metabolism, characterized by accumulation of glucosylceramide in cells of the reticulo-endothelial system, due to the deficient activity of the lysosomal enzyme glucocerebrosidase. Bone manifestations are frequent in GD; nevertheless the pathophysiology of skeletal involvement is still not well understood.

Aim:
to investigate the role of the bone turnover biochemical markers in GD. In particular we evaluated markers involved in bone formation: Osteoprotegerin (OPG), carboxyterminal propeptide of type I procollagen (PICP), osteocalcin (OST) and in bone resorption: receptor activator of nuclear factor (NF)-kb ligand (RANKL), tartrate resistant acid phosphatase (TRAP5b).

Methods:
Bone turnover biochemical markers were evaluated on sera obtained from 5 GD patients and 5 healthy subjects matched for age and sex, by Elisa commercial available kits according to the manufacturers‘ instructions.

Results:
In GD patients we observed an increase of RANKL and a significant reduction of OPG, with a consequent significantly lower OPG/RANKL ratio. Despite of higher levels of TRAP5b and lowest levels of PICP in GD compared to healthy subjects the differences did not achieve statistical significance, likely due to the small number of subjects involved in the study. No differences we observed in serum levels of OST compared with controls. Data (means ± DS) are reported in Table 1.

Conclusions:
Our data confirm the presence of unbalanced bone turnover in GD patients, characterized by an altered modulation of the OPG/RANKL system (due mainly to reduced expression of OPG, besides increased production of RANKL), an increased resorption phase (TRAP5b) and a decreased neoformation phase (PICP), all of them contributing to skeletal alterations characteristics of these patients.

Disclosures:
No relevant conflicts of interest to declare.
California Emergency Department Utilization in Patients with Sickle Cell Disease: Sociodemographic Predictors.
Authors: Wolfson Julie , Schrager Sheree M , Khanna Rachna , Coates Thomas D. , Kipke Michele 
Published: October 2019

Abstract
Abstract 3820

Background:
While patients with sickle cell disease (SCD) visit emergency departments (ED) in rates leading to a significant health system burden, limited comprehensive evaluations of utilization patterns have been published using data connecting visits to patients across facilities.

Purpose:
This study aims to examine sociodemographic predictors of ED utilization in SCD.

Methods:
This retrospective cohort study employed 2007 data from the California Office of Statewide Health Planning and Development (OSHPD). Data included all inpatient and ED discharges from all non-federal California hospitals; identifiers connected each visit to an individual patient, across all facilities in the state. Primary predictors of interest included age, insurance status and distance to the nearest source of comprehensive SCD care. Additional influential variables included disease severity, urbanicity, gender, race, and ethnicity. Geographic Information Systems software and social networking methods were used to derive a measure of distance from patient residence to the nearest self-identified SCD comprehensive care provider. Multivariate regression techniques evaluated sociodemographic predictors of utilization while also adjusting for confounding variables.

Results:
In 2007, 2,920 California SCD patients made 16,364 ED visits. Adults ≥ 21 had higher ED visit rates than children and were more likely to both be in the highest tier of users and visit multiple facilities. Patients living further from the nearest source of a self-identified provider of comprehensive SCD care had higher ED visit rates with a lower likelihood of inpatient admission from the ED. Publicly insured patients had higher ED visit rates and were more likely to be in the highest tier of users than were the privately insured or uninsured.

Conclusions:
Age ≥ 21, distance from comprehensive SCD care, and insurance status are significant predictors of ED utilization in SCD. As a routine source of care decreases ED utilization, these findings prompt concern that these factors act as barriers to accessing comprehensive SCD care.

Disclosures:
No relevant conflicts of interest to declare.
Impact of Health Care Funding on overall Survival of Patients with Multiple Myeloma and Monoclonal Gammopathy of Uncertain Significance.
Authors: Devarakonda Srinivas S , Nair Binu S. , Gonzales Ryan A. , Shi Runhua , Koshy Nebu , Burton Gary , Glass Jonathan , Turturro Francesco 
Published: October 2019

Abstract
Abstract 3821

Introduction:
The impact of socioeconomic status on patients with cancer has been previously reported. Health care funding improves access to care and thus outcome. There is little information on the impact of health care funding on hematological malignancies including multiple myeloma.

Objectives:
To analyze the effect of health care funding on the survival of patients with multiple myeloma (MM) and monoclonal gammopathy of uncertain significance (MGUS) in North Louisiana. A retrospective study of all patients diagnosed with multiple myeloma/MGUS and treated at Louisiana State University Health Science Center in Shreveport, LA between the years 1997 and 2010 was completed.

Methods:
Electronic medical records from our hospital and Social Security Death Index were used to identify patients and to define patient characteristics, demographic factors, medical funding source and date of death. Medical funding was defined as being funded (Medicaid, Medicare or private insurance) or non-funded (free care or self pay). Descriptive statistics, Product-Limit methods were used to estimate survival and Log rank test was used to compare the survival difference for each factor.

Result:
257 patients were reviewed, of which 208 had multiple myeloma and 49 had MGUS. Median age of patients with MGUS and MM was 59 years and 60 years respectively (range 30–93).There were 92 (37%) Caucasians and 165 (63%) African Americans, 114 (44%) male and 143 (56%) female patients, and 177 (69%) were funded and 80 (31%) were non-funded. Of the patients with MM, 49 (23.5%) were stage 1, 23 (11%) were stage 2, 95 (45.6%) were stage 3A and 41 (19.7%) were stage 3B disease. The 5-year survival for MM was 60% and 42% for funded and non-funded patients respectively (p=0.03). The 5-year survival for MGUS was 95% and 62% for funded and non-funded patients respectively (p=0.012). Funded patients with MM had an overall survival of 6.2 years while it was 3.8 years for the non-funded (p=0.012). The survival difference relative to race and sex was not statistically significant.

Conclusion:
Our analysis demonstrates that patients with multiple myeloma and MGUS with funding have statistically significant increased overall survival compared to patients with no funding. Presumably patients with funding have better access to medical care, which would allow for earlier diagnosis, more effective therapy of smaller tumor burden and access to all therapeutic options. Lead time bias could contribute to some of the improved survival but is doubtful. To our knowledge, this is the first study to explore the impact of health funding on the survival of patients with multiple myeloma and MGUS, however validation with a larger prospective study is needed.

Disclosures:
No relevant conflicts of interest to declare.
Evaluation of the Diagnostic Utility of Cerebral Spinal Fluid (CSF) Flow Cytometry (FC) In Detection of Central Nervous System (CNS) Involvement by Hematological Malignancy.
Authors: Muslimani Alaa , Kizilbash Sani , Jaiyesimi Ishmael , Nadeau Laura , Zakalik Dana , Margolis Jeffery , Huang James 
Published: October 2019

Abstract
Abstract 3837

Introduction:
Flow cytometry (FC) is more sensitive but more expensive than cytomorphology in detection central nervous system (CNS) involvement by hematological malignancy. Previous studies have shown that cerebral spinal fluid (CSF) pleocytosis is absent in about one third of patients with positive FC findings. FC is often performed to investigate CNS involvement on all CSF specimens regardless of cell counts or morphology, which may lead over utilization of FC. Here we investigate the diagnostic value of FC evaluation of CSF specimens with low white blood cell (WBC) count.

Methods:
After IRB approval, we retrospectively reviewed the FC results of CSF samples studied for CNS involvement by hematological malignancy between Jul 2000 and Jun 2010 at William Beaumont Hospital, MI. The FC results were correlated with CSF WBC count, cytomorphology, and documented leptomeningeal symptoms at the time of the sample obtained.

Results:
Of 421 cases included in this study, 73 were acute leukemia and 348 were lymphoma. The mean age was 59 (range from 9–90). 9.5% (40/421) of patients had leptomeningeal symptoms. 13.3% (56/421) had atypical CSF WBC cytomorphology. 11% (46/421) had positive FC. Patients with either atypical cytomorphology or leptomengeal symptoms were more likely have positive FC (p<0.001). Of the 46 patients with positive FC, 32.6% (15/46) of patients did not have CSF leptomengeal symptoms and 19.5% (9/46) had neither CSF pleocytosis (<10/μ l) nor atypical cytomorphology. We did not detect lymphoma or leukemia cells by FC in CSF samples with WBC <3/μ l, which represented 74.7% (314/421) of the specimens studied.

Conclusion:
In this study, we found that FC is more sensitive and specific than leptomeningeal symptoms or cytomorphology in detection of CNS involvement by hematological malignancy in both acute leukemia and lymphoma. However, its sensitivity is dependent on numbers of cells potentially available for analysis. Flow cytometry may not be indicated when CSF WBC count is extremely low (<3/μ l) which is invariably predictive of negative results.

Disclosures:
No relevant conflicts of interest to declare.
Novel Evidence That the Small Chemokine Macrophage Migration Inhibitory Factor (MIF) Is Highly Secreted by Human Rhabdomyosarcomas, Activates Both SDF-1–binding Receptors, CXCR4 and CXCR7, and Unexpectedly Inhibits Recruitment of Stromal Cells to the Growing Tumor.
Authors: Tarnowski Maciej , Liu Rui , Tarnowska Joanna , Ratajczak Janina , Mitchell Robert , Ratajczak Mariusz Z , Kucia Magdalena 
Published: October 2019

Abstract
Abstract 3849
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of adolescents and children and frequently infiltrates the bone marrow (BM) to the degree that it mimics acute lymphoblastic leukemia. Here we show that human RMS cells highly express and secrete the small chemokine, macrophage migration inhibitory factor (MIF). MIF overexpression has been observed in several tumors and is implicated in oncogenic transformation and tumor progression. MIF activates CXCR2 and CD74 receptors, and surprisingly, as recently reported, may also bind to the stromal-derived factor-1 (SDF-1)–binding receptor CXCR4 (Nat Med 2007;13:587). Here we report that RMS-secreted MIF i) induces phosphorylation of MAPKp42/44 and AKT, ii) stimulates RMS cell adhesion, and iii) enhances tumor vascularization. Furthermore, since RMS cells employed in our studies do not express CXCR2 and CD74 receptors, the biological effects of MIF on RMS cells depend on its interaction with CXCR4. Moreover, as we report here for the first time, receptor internalization/binding studies reveal that MIF may also engage another SDF-1–binding receptor (CXCR7) as well. Since bone marrow fibroblasts highly express the MIF-binding receptor CXCR2, we became interested in the potential role of the MIF-mediated interaction between RMS and stromal cells and, to our surprise, observed that RMS-secreted MIF decreases recruitment of stromal fibroblasts to expanding sarcomas. In support of this finding, downregulation of MIF in RMS cells inoculated into immunodeficient mice lead to formation of larger tumors that displayed higher stromal-cell support. Based on these observations, we postulate that MIF is an important autocrine/paracrine factor that stimulates both CXCR4 and CXCR7 receptors to enhance the adhesiveness of RMS cells. We also envision that MIF in those situations when it is locally secreted by a growing tumor, may desensitize CXCR4 and CXCR7 receptors expressed on the tumor cells and thus prevents their responsiveness to SDF-1 secreted at potential future sites of metastasis and thereby prevents egress of cancer cells into the circulation. On the other hand, despite its obvious pro-angiopoietic effects, MIF inhibits recruitment of stromal cells to the growing tumor. This MIF-mediated impaired recruitment of stromal elements significantly slows down as evidenced by our in vivo xenograft studies tumor growth/expansion. Based on this, we suggest that therapeutic inhibition of MIF in expanding solid tumors may accelerate both metastasis and tumor growth. Thus, the potential application of MIF inhibitors for treatment of MIF-secreting tumors should be reconsidered.

Disclosures:
No relevant conflicts of interest to declare.
C-Myb Controls Hematopoietic Stem Cell Quiescence and Differentiation Via Regulation of p57Kip2
Authors: Cooke Michael P , Sutton Susan E , Parker Albert E 
Published: October 2019

Abstract
Abstract 3878
A seminal feature of long-term hematopoietic stem cells (HSC) is quiescence. We recently described a mutation of the transcription factor c-Myb, M303V that leads to thrombocytosis and a ten-fold increase HSC number. Here we report that increased HSC number in c-MybM303V mice results from increased cycling of long-term and short-term HSC. Analysis of cell cycle genes revealed a decrease in the cell cycle inhibitor p57kip2 (Cdkn1c), a gene expressed in long-term but not short-term HSC. Mechanistic studies reveal that c-Myb binds and activates the p57 promoter and this capacity is diminished by the c-MybM303V mutation. Restoration of p57 in c-MybM303V HSC prevents thrombocytosis and shRNA mediated reduction of p57 in HSC followed by transplantation leads to enhanced numbers of HSC. These data highlight c-Myb and p57 as key regulators of HSC quiescence and differentiation.

Disclosures:
Cooke: Novartis: Employment. Sutton:Novartis: Employment. Parker:Novartis: Employment.