bluuebunny/crossref_metadata_2025 huggingface dataset:
Congenital Factor VII Deficiency and Surgery In a Single Center Experience.
Authors: Park Young sil , Park Ji Kyoung
Published: October 2019

Abstract 3658

Congenital factor VII (FVII) deficiency is rare hemorrhagic disorder, and, in affected patients, surgery is likely to cause excessive bleeding. Recombinant form of activated factor VII (Novoseven® from Novo Nordisk, Bagsvaerd, Denmark), that was developed as a second-generation bypassing agent, is used in management of bleeds in patients with congenital FVII deficiency recently. Doses lower than those recommended for patients with hemophilia are usually effective in patients with FVII deficiency.

We review the results of 6 surgical procedures in 4 patients with congenital FVII deficiency in East-West Neo Medical Center. Between January 2008 and February 2010, 6 surgeries for patients with congenital FVII deficiency were performed. We administerated rFVIIa pre- and postoperatively. Plasma FVII:C levels were measured in baseline, before and after the intravenous administration of routine dose FVIIa.

Between January 2008 and February 2010, 6 surgeries for 4 patients with congenital FVII deficiency were performed successfully without complications in our center. The median level of FVII:C was 2% (range 2–7%). The median age of patients was 29.5 years (range, 7–37). Types of surgery were 4 orthopedic surgeries, 1 tonsillectomy, and 1 dental extraction. The median duration of hospitalization was 14 days (range 6–15). rFVIIa was used at all surgery except dental extraction. FVIIa was administrated every 4 hours with the first dose being administered 1 hour preoperatively at least 48 h with dose 15–30 μμ¤/kg. After 48 h, the frequency was reduced according to the patient's clinical status. Plasma FVII:C levels were measured before testing and then again after the intravenous administration of routine dose FVIIa. FVII:C levels increased to about 200 %. Dental extraction was performed with only fibrinolytic agents without any replacement. There was no bleeding and thrombogenic complication.

rFVIIa was well-tolerated, maintained effective haemostasis with good clinical outcome. Patients with congenital FVII deficiency requiring surgery can be treated by the use of rFVIIa and antifibrinolytic agents efficiently and safely.

Disclosures:

No relevant conflicts of interest to declare.
Non-Invasive Management of Alloimmune Thrombocytopenia: Prediction of the Fetal Status.
Authors: Bertrand Gerald , Drame Moustapha , Martageix Corinne , Kaplan-Gouet Cecile
Published: October 2019

Abstract 3690

Fetal/neonatal alloimmune thrombocytopenia (F/NAIT) is the most common cause of severe thrombocytopenia in the fetus and the newborn in maternity wards. To counter the bleeding consequences of severe fetal thrombocytopenia, antenatal therapies have been implemented for subsequent pregnancies with incompatible fetuses. Predictive parameters for fetal severe thrombocytopenia are important for the development of non-invasive strategy and tailored intervention. We report here data concerning 67 HPA-1bb women with 81 managed pregnancies with IVIG. In 51% of the cases, the diagnosis of F/NAIT was made during the first gestation, following an intracranial haemorrhage (ICH) in 8 cases (12%). Severe thrombocytopenia was recorded for 88% of the newborn. Analysis of the index cases did not show any correlation between the severity of the disease and the maternal genetic background (ABO group and HLA-DRB3 allele). Subsequent pregnancies were managed and therapy effectiveness was evaluated. The highest mean newborn plt count was observed for a combination of intravenous immunoglobulin (IVIG) and corticoids (135.109/L; 54 newborn), in comparison with IVIG alone (89.109/L; 27 newborn). No ICH was recorded in these 2 groups. The maternal anti HPA-1a antibody concentration measured before any treatment and before 28 weeks of gestation was predictive of the fetal status: a high antibody concentration (≥28 International Units/mL) was correlated with a severe thrombocytopenia of the fetus (p=0.0016). Follow-up of the antibody concentrations during 34 pregnancies with antenatal management allowed demonstrating for the first time that the areas under curves (AUC) weighted by the weeks of gestation were a predictive parameter of therapy failure. The weighted AUC was significantly higher for women who delivered severely thrombocytopenic newborn than newborn with platelet count above 50.109/L (p=0.0107). To conclude, this large retrospective survey gives new insights on maternal predictive parameters for fetal status and therapy effectiveness allowing non-invasive strategies.

Table 1: Maternal anti HPA-1a alloantibody concentrations and fetal platelet counts: statistical analysis. Predictive parameter Platelet count (×109/L) p-value Se (%) Sp (%) PPV (%) NPV (%) <50 ≥50 Number of mothers with antibody concentration (<28wg) <28 IU/mL 3 Fetuses 10 0.0016* 81.2 91.7 92.3 79.9 ≥28 IU/mL 13 2 Number of pregnancies with weighted AUC <24 IU/mL/wg 5 Newborn 18 0.0153* 64.3 82.6 69.2 79.2 ≥24 IU/mL/wg 9 5 * P<0.05. AUC: area under curve; IU: international units; Se: sensitivity, Sp: specificity; PPV: positive-predictive value; NPV: negative-predictive value. Wg: weeks of gestation.

Disclosures:

No relevant conflicts of interest to declare.
Strong CD28 Costimulation Suppresses Induction of Regulatory T Cells From Naïve Precursors through Lck Signaling.
Authors: Semple Kenrick , Nguyen Antony , Yu Yu , Anasetti Claudio , Yu Xue-Zhong
Published: October 2019

Abstract 3728

CD28 costimulation is required for the generation of naturally-derived regulatory T cells (nTregs) in the thymus through Lck-signaling. However, it is not clear how CD28 costimulation regulates the generation of induced Tregs (iTregs) from naïve CD4 T-cell precursors in the periphery. To address this question, we induced iTregs (CD25+Foxp3+) from naïve CD4 T cells (CD25−Foxp3−) by TCR-stimulation with additional TGFβ in vitro, and found that the generation of iTregs was inversely related to the level of CD28 costimulation independently of IL-2. By using a series of transgenic mice on CD28-deficient background that bears WT CD28 or mutated CD28 in its cytosolic tail incapable of binding to Lck, PI3K or Itk, we found that CD28-mediated Lck-signaling plays an essential role in the suppression of iTreg generation under strong CD28 costimulation. Furthermore, we demonstrate that T cells with the CD28 receptor incapable of activating Lck were prone to iTreg induction in vivo, which contributed to their reduced ability to cause graft-versus-host disease. These findings reveal a novel mechanistic insight into how CD28 costimulation negatively regulates the generation of iTregs, and provide the rationale for promoting T-cell immunity or tolerance by regulating Tregs through targeting CD28-signaling.

Disclosures:

No relevant conflicts of interest to declare.
High Frequency of Alloantigen-Specific Regulatory T-Cells In Human Blood Allow Rapid Ex Vivo Expansion for Adoptive Therapy.
Authors: Veerapathran Anandharaman , Pidala Joseph , Beato Francisca , Yu Xue-Zhong , Anasetti Claudio
Published: October 2019

Abstract 3747

Evidence from animal studies has demonstrated that allogeneic regulatory T (Treg) cells can be used as effective therapeutic tools in the prevention of allograft rejection and graft-vs-host disease (GVHD). However, translating Treg-based therapies from animal models to human clinical trials requires methods for the isolation and expansion of functional Treg. Loss of function has been a concern for Treg expanded broadly with anti-CD3 and CD28 antibody-coated beads. Based on preclinical data in rodents, allospecific Tregs are anticipated to be more potent and exert more selective immunoregulation than polyclonal Treg in the prevention or treatment of GVHD. Currently, no effective approach has been established for selective expansion of human allospecific Treg ex vivo. In this study, we show the selective direct and indirect recognition of alloantigen by specific human CD4+CD25+CD127− Tregs. CD4+CD25+CD127− Treg were freshly isolated from normal human blood, labeled with CFSE and stimulated by MHC (HLA)-mismatched APC's, in the presence of IL-2 and IL-15 to amplify the proliferative responses, and sirolimus to suppress other T cells. The precursor frequency of the antigen specific Tregs was calculated by Limiting Dilution Analysis and found to be 1/49 -1/138 among all Tregs. The expanded antigen specific Tregs with low CFSE content were in general greater than 90% by days 12–15 in culture. CFSE-low Tregs were sorted by flow cytometry and exhibited complete suppression against CD25-negative CD4 T cell responses up to ratios of 1:100, in contrast CFSE-high Tregs were not suppressive. Ex vivo expanded antigen specific Tregs maintained high Foxp3, retained lymphoid homing receptor CD62L and chemokine receptor CCR7 expression, suggesting that they are functional and able to migrate to lymphoid tissue in vivo. Among the APC's tested, DC was found to be better stimulus with potent suppressive potential when compared to T-cell stimulated PBMC's. Specific indirect allorecognition was elicited when Tregs were stimulated with autologous APC's pulsed with allogeneic cell lysate in the presence of sirolimus, IL-15 and IL-2. Precursor frequency of antigen specific Tregs with indirect allorecognition was 100 fold lower than the precursor frequency of Tregs with direct allospecificity. Highly suppressive antigen specific Tregs show a duplication time of approximately 24 hours in the presence alloantigen, cytokines and sirolimus, and can be expanded in vitro by 400 fold in a 12 day period. This magnitude of expansion predicts the feasibility of conducting translational clinical trials. Our data may provide a platform for the selective expansion of Tregs against minor histocompatibility antigens to prevent GVHD while sparing graft-vs.-leukemia effects.

Disclosures:

No relevant conflicts of interest to declare.
Tregs In Graft-Versus-Host Disease: A Task Force In Trouble?.
Authors: Ukena Sya N. , Grosse Jens , Buchholz Stefanie , Stadler Michael , Ganser Arnold , Franzke Anke
Published: October 2019

Abstract 3748

Graft-versus-host disease (GvHD) remains the major clinical complication in hematopoietic stem cell transplantation (SCT) resulting in severe morbidity and significant mortality. This alloreactive immune response is mainly induced by donor T cells transplanted with the graft. Regulatory T cells (Tregs) play an essential role in the induction and maintenance of peripheral tolerance. In addition, data from murine models have shown that Tregs can prevent GvHD while preserving the graft-versus-leukemia effect. In order to functionally and dynamically characterize human Tregs after allogeneic SCT, we analyzed CD4+CD25highCD127dim T cells isolated from the peripheral blood of more than 80 patients with hematological malignancies every 30 days over half a year following SCT.

Patients were divided into the following clinical groups: (A) no signs of acute or chronic GvHD, (B) acute GvHD, (C) chronic GvHD and (D) acute GvHD passed into chronic GvHD. Human peripheral blood lymphocytes were separated by Ficoll gradient and CD4+CD14−CD25highCD127dim T cells were isolated by MoFlow cell sorting. Isolated RNA was pooled and microarray analysis was performed by using Affymetrix HG_U133_Plus2.0 Arrays. Results were verified by using quantitative realtime RT-PCR. Additionally, Tregs were phenotypically analyzed by FACS.

We monitored a continous but slower recovery of Tregs in GvHD within the first 6 months following PBSCT. Manifestation of acute and chronic GvHD correlated with significantly reduced frequencies of peripheral Tregs in the first month after PBSCT compared to patients without GvHD. Microarray data revealed a high stability of the Treg transcriptome in the first half year representing the most sensitive time window for tolerance induction. Moreover, comparison of the Treg gene expression profiles from patients with and without GvHD point to a reduced suppressive function of Tregs with diminished migration capacity to the target organs likely contributing to the development of GvHD.

Our findings corroborate the impact of human Tregs in the pathophysiology of GvHD and identify novel targets for the manipulation of Tregs to optimize strategies for prophylaxis and treatment of life-threatening GvHD.

Disclosures:

No relevant conflicts of interest to declare.
Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells.
Authors: Marin Virna , Pizzitola Irene , Biondi Andrea , Biagi Ettore , Pule Martin
Published: October 2019

Abstract 3753

Immunotherapy with TCR or chimeric receptor-genetically modified T cells may result in toxicity related to direct target effects, unanticipated off-target effects or lymphoproliferation due to insertional mutagenesis. Therefore it is required to include a suicide gene in the viral vector. We compared different suicide genes in vitro in Epstein Barr Virus-specific cytotoxic T cells (EBV-CTL). Herpes Simplex Virus Thymidine Kinase (HSV-tk), human inducible Caspase 9 (iCasp9), human CD20 and mutant human thymidylate kinase (mTMPK) genes were cloned in frame with truncated CD34 (dCD34, marker gene), separated by the 2A peptide in a SFG-based vector. We previously reported with the iCasp9-coding construct, a lower transduction efficiency and instable expression of the dCD34 marker gene. We therefore codon-optimized the iCasp9 sequence (iCasp9opt) and repeated the cloning in frame with 2A-dCD34 in the SFG vector. EBV-CTLs could be similarly and efficiently transduced with iCasp9opt-2A-dCD34, HSV-TK-2A-dCD34, mTMPK-2A-dCD34 and CD20-2A-dCD34 (mean % CD34+, 80%, n=5), similarly to the control vector containing dCD34 alone. Expression of the marker gene was stable up to 3 weeks. Expression of the suicide genes was not associated with alterations in the expansion rate, immunophenotype and capacity to kill autologous lymphoblastoid cell lines. Transduced and CD34-selected EBV-CTLs have been tested for their sensibility to the corresponding activator in vitro by evaluating residual CD34+ cells. iCasp9opt- transduced cells were rapidly killed with high efficiency by CID, (mean survival, 11% after 24 hours, and 5% after 7 days; n=7). Gancyclovir treated HSV-tk expressing cells showed similar levels of efficacy only after 3 days and CD20 and mTMPK-transduced cells showed only minimal killing at all time points (mean survival after 7 days,84% and 32% respectively).The same results were obtained by analyzing apoptosis induction through Annexin-7AAD staining. In fact, after 24 hours of incubation with CID, nearly 100% iCasp9opt+ cells were apoptotic, whereas a significant lower % of apoptotic cells was observed with the other suicide genes. Altogether our results suggest that the faster activity of iCasp9 might be advantageous in case of occurring severe toxicity, and, together with its lack of immunogenicity and the absence of side-effects of CID, support the clinical applicability of iCap9-based suicide strategy.

Disclosures:

No relevant conflicts of interest to declare.
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 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 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 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 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 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 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 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 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 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.
A Functional Analysis of Variants Associated with Therapy-Induced Second Malignancies After Hodgkin Lymphoma Identified by a Genome-Wide Scan
Authors: Best Timothy , Skol Andrew D , Gamazon Eric , Onel Kenan
Published: October 2019

Abstract 3892

Survivors of Hodgkin lymphoma (HL) are susceptible to radiation-induced second malignant neoplasms (SMNs). In a genome-wide association study (GWAS) of patients treated for HL who did or did not develop SMNs, we identified and validated two SMN-associated single nucleotide polymorphisms (SNPs) at 6q21, intergenic between PRDM1 and ATG5 [rs4946728: P = 1.04×10-9, OR = 3.21 (95% CI = 2.37–6.42), and rs1040411: P = 4.24×10-8, OR = 2.43 (95% CI = 1.76–3.34)]. Recently, it was demonstrated that disease-associated SNPs are more likely to be expression quantitative trait loci (eQTLs), SNPs that regulate gene expression, than are randomly chosen SNPs matched for their population allele frequencies. Indeed, we found that the 1000 SNPs most associated with SMNs are significantly enriched for eQTLs (P = 0.01). Exploring the processes regulated by SMN-associated SNPs can inform the mechanism by which SMNs result in patients treated with radiation therapy. As an initial investigation of the effect of these SNPs on gene expression, we studied the effect of the validated 6q21 haplotype (comprised of rs4946728 and rs1040411) on global gene expression in HapMap lymphoblastoid cell lines (LCLs). Gene set enrichment analysis of genes differentially expressed (log2>0.05) between cell lines carrying either the risk or protective haplotype revealed that carriage of the risk-associated haplotype was associated with increased expression of transmembrane proteins (enrichment P = 2.1×10-13) and immune response proteins (enrichment P = 1.2×10-6). Because the 6q21 haplotype is in close physical proximity to ATG5 and PRDM1, we investigated its functional consequence on expression of these genes. We discovered the risk-associated haplotype was significantly associated with lower levels of PRDM1 mRNA (P = 0.04) but not ATG5 mRNA. As exposure to radiation is the primary etiologic factor for SMNs, we assessed the effect of the risk haplotype on protein levels of PRDM1 and ATG5 in six LCLs (three with the risk haplotype and three with the protective haplotype) following 10Gy of gamma irradiation (IR). PRDM1 protein levels were significantly lower in LCLs carrying the risk-associated haplotype in the absence of IR. In all lines, PRDM1 levels increased following radiation exposure, but this effect was significantly attenuated in presence of the risk haplotype. In sum, these data suggest that SNPs associated with SMNs following HL are enriched for SNPs that regulate gene expression. We demonstrate that the validated risk alleles at 6q21 are associated with differences in PRDM1 mRNA and protein levels and response to radiation. These observations suggest a model in which PRDM1 may be a key regulator of the radiation-response that protects against the emergence of SMNs.

Disclosures:

No relevant conflicts of interest to declare.
The Oncogenic Mir-17-92 MicroRNA Cluster Is Inhibited by EGR2 During Macrophage Differentiation Via JARID1b-Mediated Histone 3 Lysine 4 Demethylation
Authors: Pospisil Vit , Vargova Karin , Kokavec Juraj , Rybarova Jana , Savvulidi Filipp , Necas Emanuel , Laslo Peter , Zavadil Jiri , Stopka Tomas
Published: October 2019

Abstract 390

The miR-17-92 cluster (Oncomir1) encodes seven related microRNAs associated with cell proliferation, apoptosis and development and is overexpressed in number of malignancies including myeloid leukemias. The miR-17-92 cluster is highly expressed in myeloid progenitors, while it becomes downregulated upon the onset of macrophage differentiation. Conversely, sustained expression of miR-17-92 is associated with a differentiation blockade (Fontana 2007). Here we report a novel mechanism of the regulation of miR-17-92 cluster within differentiating myeloid progenitors. During macrophage differentiation, the myeloid transcription factor PU.1 transcriptionally induces the secondary determinant Early growth factor 2 (Egr2). Subsequently, Egr2 binds to the miR-17-92 cluster promoter and recruits histone demethylase Jarid1b resulting in histone H3 lysine K4 (H3K4) demethylation within the CpG island upstream miR-17-92 cluster leading to the repression of miR-17-92 expression. Reporter assays using deletion constructs of the miR-17-92 promoter region revealed that Egr2 is required for targeting the Jarid1b onto critical minimal region within CpG island of upstream miR-17-92 locus and its decreased H3K4 methylation. In addition, functional assays identified the 3'UTR of Egr2 as the target of multiple miRNAs of the miR-17-92 cluster, indicating existence of a mutual regulation between miR-17-92 cluster and Egr2, putatively involved in macrophage differentiation characterized by a bistable state, where Egr2 negatively regulates miR-17-92 cluster in differentiating cells and, in turn, miR-17-92 cluster negatively regulates Egr2 in highly proliferating progenitor cells to achieve homeostatic regulation. Ectopic expression of miR-17-92 cluster within myeloid progenitors blocked macrophage differentiation indicating that leukemogenesis may involve miR-17-92 mediated differentiation blockade. To determine if the newly identified negative regulatory mechanism of miR-17-92 is involved in leukemogenesis, we tested peripheral blood mononuclear cells isolated from acute myeloid leukemia (AML) patients (N=27). 14 of 27 AML patients exhibited significantly downregulated transcriptional factors PU.1 and Egr2 and high levels of the miR-17-92 cluster expression. Ectopic expression of Egr2 within AML blast cells (N=2) led to decreased levels of miR-17-92 and restored the expression of p21 and BIM, two established miR-17-92 targets downregulated in AML. We conclude that PU.1 represses miR-17-92 cluster during macrophage differentiation by Egr2 mediated recruitment of the Jarid1b demethylase and that dysregulation in the miR-17-92 repression mechanism may contribute to the pathogenesis of AML. (Grants # IGA 10310-3, MSMT 2B06077, 0021620806, LC06044, SVV-2010-254260507).

Disclosures:

No relevant conflicts of interest to declare.
CXCR7 Regulates Retention of Splenic Marginal Zone B Cells and Availability of SDF-1/CXCL12
Authors: Wang Hongsheng , Shin Dong Mi , Qi Chenfeng , Morse Herbert
Published: October 2019

Abstract 3902

CXCR7 is a newly discovered receptor for the chemokines I-TAC/CXCL11 and SDF-1/CXCL12. Overexpression of CXCR7 in certain tumors has been associated with increased activities of adhesion, invasion and survival. CXCR7 has thus been investigated as a potential chemotherapeutic target in the treatment of metastatic cancers. Our analyses of murine B cell lymphomas revealed that marginal zone B (MZB) cell lymphomas expressed higher levels of CXCR7 than other types of lymphomas. This prompted us to investigate the expression and function of CXCR7 in normal B cells. In this report, we demonstrate that normal MZB cells expressed the highest level of CXCR7 among all B cell subsets. This pattern of expression was consistent with gene profiling studies using cDNA microarrays. Injection of mice with CCX754 or CCX771, a specific blocker of CXCR7, resulted in a significant reduction of MZB cells in the spleen. Immunohistological analyses revealed disrupted integrity and reduced size of the MZ in spleens of CCX754-treated mice. In addition, CCX754 significantly blocked internalization of CXCR7 resulting in an increase of CXCR7 expression on MZB cells but not follicular B cells. This indicates that CXCR7 constantly removes its ligands from the extracellular environment. Taking together, our data suggest that CXCR7 controls CXCL12 availability influencing MZB cell retention in the spleen.

Disclosures:

No relevant conflicts of interest to declare.