bluuebunny/crossref_metadata_2025 huggingface dataset:
Which Hematological Malignancies Are Implicated In Acute Hemolytic Reactions Following Rh (D) Immune Globulin for ITP
Authors: Astacio Hugo , Muller Carmen , Little Gregg , Sinclair Chris , Genereux Maurice
Published: October 2019

Abstract 4670

Background:

Acute Hemolytic Reactions (AHR) and Intravascular Hemolysis (IVH) have been reported rarely with Rh (D) Immune Globulin (WinRho® SDF) administration for normalization of platelet count in ITP. The sporadic and unpredictable occurrence of these events has led to an analysis of our WinRho® SDF safety database to identify populations that may be at higher risk for occurrence of AHR's. A disproportionality analysis revealed that malignancies were disproportionally over represented in cases with AHR compared to Non Hemolytic Reactions and was reported in another abstract.

Aims:

To determine which malignancies were disproportionally overrepresented in patients who developed AHR's.

Methods:

Medical Histories cases with AHR with malignancies were reviewed and classified by type of malignancy. AHR's included both the expected post infusion hemolysis and infrequently reported cases involving clinically compromising hemolysis including IVH.

Hematological malignancies were present in 39/45 of AHR's (86.6 %) and non-hematological malignancies 6 of 45 of AHR (13.3%). 90% of AHR's in patients with Malignancies were adult, 5% children and 5% age unknown. Adults above the age of 65 years accounted for 45% of these cases. CLL was present in 66% of patients with leukemia and Non Hodgkins Lymphoma was present in 92% of patients with Lymphomas. Proliferative disorders were diagnosed in 27% of patients with AHR. Solid tumors were present in 14% of patients with AHR's.

Conclusions:

Hematological malignancies including CLL and NHL are disproportionally over represented in cases presenting with AHR post WinRho® SDF. Alternate therapies to Rh (D) Immune Globulin should be considered in patients with thrombocytopenia and these hematological malignancies due to increased risk of AHR, such as IVH.

Disclosures:

Astacio: Cangene Corporation: Employment. Muller:Cangene Corporation: Employment. Little:Cangene Corporation: Employment. Sinclair:Cangene Corporation: Employment. Genereux:Cangene Corporation: Employment.
Thrombotic Thrombocytopenic Purpura INDUCED PANCREATITIS– A Rare Complication
Authors: Gotlieb Vladimir K. , erma Vaibhav , Jacob Robin , Reddy Padi , Taha Hesham , Ullah Asmat
Published: October 2019

Abstract 4683

Thrombotic Thrombocytopenic Purpura (TTP) is a rare clinical disorder characterized by clinical combination of fever, microangiopathic hemolytic anemia, thrombocytopenia, renal failure and neurological changes.

Pancreatitis may occur as a result of TTP or may rarely trigger TTP. We present a case of acute Pancreatitis as a complication of TTP.

A 29 year-old African-American female with history of alcohol abuse presented with three days of nausea, vomiting, diarrhea, headache and fever. Vitals: BP-120/80mmHg, HR-75/min, Temp-98F, and RR-14/min. Examination was remarkable for lethargy and mild epigastric tenderness. Pertinent laboratory: Hgb-12.4g/dL (Day1) - 8.5g/dL (Day3), reticulocyte count-3.35, Platelets-13,000mm3, BUN/Cr-69/6.3 (mg/dL), LDH-1523U/L, indirect hyperbilrubinemia-3.6mg/dL, Amylase/Lipase-117/87(U/L), fibrinogen-462, D-dimer-15.39, PT/PTT-normal and Haptoglobulin<8mg/dL. Peripheral smear showed 3+ schistocytes. Imaging: CXR, CT Head and CT Abdomen were unremarkable. Work-up for various infections and autoimmune diseases was negative.

TTP was diagnosed and patient was admitted to ICU. Plasmapheresis was initiated with clinical improvement by day 3 with subsequent deterioration by day 7. On day 10, patient developed severe abdominal pain with increase in Amylase (145U/L) and lipase (281U/L). Repeat CT Abdomen showed acute pancreatitis. Day 13 - Rapid worsening in clinical status with death of the patient. Autopsy revealed micothrombi and infarcts in multiple organs including kidney, pancreas, and heart.

Acute Pancreatitis is a rare complication of TTP suggested to occur in 2% of cases of TTP but no case has ever been reported in the literature. There are cases that have been reported where Acute Pancreatitis have triggered TTP-HUS syndrome and chronic relapsing TTP. Idiopathic cases of TTP have a deficiency of ADAMS 13, a metalloprotease enzyme that cleaves von Willebrand factor (vWF) resulting in abnormal vWF multimers causing hyaline thrombi formation with end-organ damage due to ischemia. Pancreatic inflammation in TTP has been regarded as ischemic in nature, due to microthrombi of pancreatic small vessels. It is common to see the inflammation of pancreas in the histological specimens on autopsy, but very rare to develop pancreatitis with clinical symptoms, increase in pancreatic enzymes and CT findings of Pancreatitis. Prognosis of Patients with Acute Pancreatitis in TTP is guarded. Patient should be closely monitored in ICU setting aiming to control TTP by Plasmapheresis and Immunosuppressive therapy (Rituximab) in refractory cases.

Acute Pancreatitis is a rare complication of TTP and carries a worse prognosis.

In patients developing abdominal pain during the course of TTP, Acute Pancreatitis should always be considered in the differential diagnosis.

Disclosures:

No relevant conflicts of interest to declare.
Prolonged Thrombocytopenia Following Allogeneic Hematopoietic Stem Cell Transplantation: Association with Reduced Ploidy and Immaturation of Megakaryocytes
Authors: Zhang Xiao-hui , Fu Haixia , Xu Lan-Ping , Liu Dai-Hong , Liu Kaiyan , Huang Xiao Jun
Published: October 2019

Abstract 4695

Prolonged thrombocytopenia is a frequent complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT), but its pathogenesis has remained obscure. In present study, by using a flow cytometry technique, we determined the frequency of bone marrow megakaryocytes (MKs) and MKs ploidy distributions in allo-HSCT recipients with or without prolonged thrombocytopenia (n=32 and 27, respectively) and in 13 healthy volunteers. Along with that, the expression of c-Mpl in MKs was measured. The result showed a significant difference in MKs ploidy distributions between the 3 groups. Both the allo-HSCT patients with or without prolonged thrombocytopenia showed a significant shift to low ploidy cells (left shift) with 55.63±18.62% and 44.63±19.38% of their MKs in ploidy classes 2N to 8N, respectively. In contrast, the healthy volunteers had only 25.97±10.70% of their MKs in ploidy classes 2N to 8N. And the patients with prolonged thrombocytopenia showed a marked increase in cells ≤8N, a significant decrease in the 16N and ≥32N cells comparing to patients without prolonged thombocytopenia, which told us there were more less mature MKs in the allo-HSCT recipients with prolonged thrombocytopenia. We concluded that prolonged thrombocytopenia and slow platelet engraftment after allo-HSCT may be related to reduced ploidy and immaturation of MKs.

Disclosures:

No relevant conflicts of interest to declare.
Selectively T Cell Depleted Allografts From HLA-Matched Sibling Donors Followed by Low-Dose Post Transplant Immunosuppression to Limit Disease Relapse In Patients with Hematological Malignancies
Authors: McIver Zachariah A. , Mielke Stephan , Shenoy Aarthi , Fellows Vicki , Stroncek David , Leitman Susan , Childs Richard , Battiwalla Minoo , Koklanaris Eleftheria K. , Haggerty Janice , Savani Bipin N. , Rezvani Katy , Barrett A. John
Published: October 2019

Abstract 4705

We evaluated a photodepletion technique to selectively deplete graft-versus-host disease (GVHD) alloreacting T cells from stem cell transplants. Donor lymphocytes were stimulated with irradiated in-vitro expanded recipient T lymphocytes. Alloactivated T cells preferentially retaining the photosensitizer 4, 5-dibromorhodamine 123 (TH9402) (Kiadis Pharma, NL) were eliminated by light exposure. Twenty-four patients with hematological malignancies (16 high-risk) conditioned with fludarabine, cyclophosphamide and total body, irradiation received a CD34-selected stem cell allograft from an HLA identical sibling and 5×106/kg selectively depleted (SD) donor T cells. Low-dose cyclosporine was used as the only post-transplant immunosuppression. The overall probability of grade III-IV a-GVHD was 13%. Fourteen patients developed chronic GVHD (c-GVHD). Five patients relapsed, 2 of whom remain alive in remission after further treatment. Thirteen patients survive at a median of 22 months. Overall survival and disease free survival probabilities (+/− SEM) were 43 +/− 13% and 35 +/− 13% respectively. The SD technique resulted in a low incidence of relapse (24 +/− 10%), but was complicated by late non-relapse mortality associated with c-GVHD and infection of 50 +/− 14% at 4 years follow up. These results suggest that SD may effectively reduce severe a-GVHD while conserving graft-versus leukemia effects, but the depletion strategy has yet to be optimized.

Disclosures:

Mielke: Kiadis Pharma: Research Funding.
Studies on the Role of TGF-β1/TGF-βR1/Smad4 In the Occurance and Development of Myelocytic Leukemia and Breast Cancer
Authors: Xiaoli Liu , Xia Liping , Zhang Jinfang , Du Qingfeng , Li Rong , Xu Na , Huang Bintao
Published: October 2019

Abstract 4714

Previous studies showed that the function of TGF-β/Smad passway is aberrant in many human cancers, the expresses of TGF-β1, TGF-βR1 and Smad4 proteins were related to the clinicopathologic characteristic of tumors and prognosis. The aim of the present work was to study the role of TGF-β1, TGF-βR1 and Smad4 in the occurance and development of myelocytic leukemia and breast cancer. Firstly, 2 myelocytic leukemia cell lines (KG1, HL60), 2 breast cancer cell lines (SKBR-3, MCF-7) and 293 cells (normal control) were selected. Secondly, we detected the expression levels of mRNA and protein in the 4 cell lines by fluorescence quantitative PCR (qRT-PCR), immunohistochemistry, and Western blotting. qRT-PCR showed that the gene copies of TGF-β1, TGF-βR1 and Smad4 in SKBR-3 cells and KG1 cells, which were poorly differentiated, were lower than that in MCF-7 cells and HL60 cells, which were well differentiated (P<0.01); and the gene copies in 293 cells were higher than all of the four cell lines (P<0.01). Western blotting showed that the protein expression levels of TGF-β1, TGF-βR1 and Smad4 in SKBR-3 and KG1 cells were also lower than in MCF-7 cells and HL60 cells (P<0.01), and higher in 293 cells (P<0.01). The same results could also be abtained by immunohistochemistry. This studies may indicate that TGF-β1, TGF-βR1 and Smad4 act possibly as the role of anti-oncogene in myelocytic leukemia and breast cancer, which also may be associated with the biological behaviors of the development of myelocytic leukemia and breast cancer. TGF-β/Smad passway might be a promising candidate target for the controling progression of malignant tumor.

Disclosures:

No relevant conflicts of interest to declare.
Accuracy and Precision Study: Body Fluid White Blood Cell (WBC) Analysis (Peritoneal, Pleural and Peritoneal Dialysate) Using a Light Scatter Technology (ADVIA®2120/2120i) Versus Hemocytometer Manual Counts
Authors: Castellone Donna D , Peerschke Ellinor I.B. , Francisco Nenita , Canfield William , Kling Gail
Published: October 2019

Abstract 4730

Background:

The ADVIA® 2120/2120i body fluid application is an in vitro diagnostic test for the enumeration of the total nucleated cell (TNC) or white blood cell (WBC) count and RBC (red blood cell) count for pleural, peritoneal and peritoneal dialysis (PD) specimens collected in K2 EDTA. Total nucleated cell (TNC) and RBC counts are used in the laboratory assessment of these fluids. The TNC count is an absolute count of the nucleated blood cells, mesothelial cells, and other non-hematopoietic cells in these fluids.

Objective:

The objective is to demonstrate concordance between the manual (hemocytometer) method versus the light scatter principle used on the ADIVA® 2120i analyzer.

Methods:

The ADVIA® 2120/2120i Body Fluid Application uses the Basophil/Lobularity and RBC/PLT channels to enumerate the WBC and RBC counts. WBC counts are derived from the Basophil/Lobularity channel by a process in which the cytoplasmic membrane is stripped and the suspension of cells is intercepted by light from the laser diode where the low-angle light scatter (2° to 3°) and high-angle light scatter (5° to 15°) signals of each cell are counted. The manual method uses a hemocytometer in which a chamber is charged with fluid and both areas of the chamber are counted and the result averaged. De-identified remnant peritoneal, pleural and peritoneal dialysate samples were processed within 2 hours in the Hematology Laboratory according to their standard operating procedure. Samples with TNC counts that fell in the range of 0.030 to 400 × 103 cells/μ L (30 to 400,000) for each type of fluid were included. Ten consecutive runs were performed for each type of fluid to determine precision.

Results:

The WBC reproducibility study (n=10) demonstrates that within the run, precision of the body fluid counts demonstrates a coefficient of variation (CV) ≤ 10 CV. Regression analysis (n=34) demonstrates an r=.93 using samples across the reportable range.

Conclusion:

These results suggest that the light scatter technology used on the ADVIA® 2120/2120i gives both accurate and precise WBC determinations and compares very well to the manual method for determination of WBC analysis in peritoneal, peritoneal, dialysate, and pleural fluids. Utilizing this method will decrease turn-around time for this process, resulting in a faster time to result and ultimately better patient outcomes.

Disclosures:

Castellone: Siemens Healthcare: Employment. Canfield:Siemens: Employment. Kling:Siemens: Employment.
Hemophilia A: Patient Impact and Economic Burden of the Disease
Authors: Strandberg-Larsen Martin , Gater Adam , Marlow Emma , Thomson Andrew
Published: October 2019

Abstract 4763

Introduction:

Hemophilia A is a rare but serious bleeding disorder caused by a deficiency in blood clotting factor VIII (FVIII). To examine the impact of hemophilia A and replacement FVIII therapies from a humanistic and economic perspective, a targeted literature review was conducted.

Methods:

Searches were conducted in MEDLINE® and the National Health Service Economic Evaluation Database, using combinations of disease, patient-reported outcome and economic-related key words, limited to articles published in English between January 2000 and January 2010 (inclusive). A total of 34 full-text articles (from 653 abstracts retrieved) were selected for detailed consideration.

Results:

Findings revealed increased mortality rates and decreased life expectancy among people with hemophilia A, compared with the general population. This is largely a result of the transmission of blood-borne viruses (e.g. HIV and Hepatitis-C) due to use of plasma-derived FVIII (pdFVIII) concentrates. Improvements in viral attenuation processes plus donor screening practices for pdFVIII products have reduced the risks of viral transmission, but risk of transmission still exists in relation to non-enveloped viruses and other unknown pathogens. Newly developed recombinant FVIII therapies minimizes these risks; however, such therapies are not currently widely available globally. All available FVIII therapies use demanding regimens requiring regular time-consuming injections, which can be detrimental to patients’ health-related quality of life and limit adherence to replacement FVIII therapy. Non-adherence to factor replacement therapy is associated with diminished product efficacy, poorer health outcomes and increasing economic expenditure. Treating hemophilia A is costly, primarily due to the high acquisition cost of replacement FVIII products. However, indirect costs related to patient disability and level of care also contribute to economic burden. Prophylactic treatment initially may appear to be more expensive than on-demand treatment but is associated with greater clinical efficacy and improved long-term outcomes, which may lead to cost savings over the course of a patient's lifetime.

Conclusions:

Hemophilia A is associated with considerable humanistic and economic burden. There remain substantial unmet needs with regard to the safety, convenience, global access to treatment incl. reliable product supply and total care costs associated with factor replacement therapies.

Disclosures:

Strandberg-Larsen: Novo Nordisk A/S: Employment. Gater:Novo Nordisk A/S: Research Funding. Marlow:Novo Nordisk A/S: Research Funding. Thomson:Novo Nordisk A/S: Research Funding.
A Mechanism Based Pharmacokinetic/Pharmacodynamic Model of the Effect of a Prolyl Hydroxylase Inhibitor on Erythropoietic Response In Mice
Authors: Singh Indrajeet , Krzyzanski Wojciech , Singh Pratap
Published: October 2019

Abstract 4769

Background:

The oxygen sensing pathway is one of the major controlling mechanisms of erythropoiesis in humans and other mammals. The transcription factor, Hypoxia-Inducible-Factor (HIF) regulates the local gene expression of the hormone erythropoietin (EPO). Degradation of HIF1α is regulated via oxygen dependent pathway by Prolyl Hydroxylases (PHDs) enzymes and the inhibition of PHD enzyme activity stimulates the erythropoietic response. The purpose of this study was to develop a mechanistic Pharmacokinetic/Pharmacodynamic (PK/PD) model to describe the effect of a Prolyl Hydroxylase Inhibitor (PHI) on the erythropoietic process in mice.

Methods:

PHI was given orally at three single doses of 10, 30 and 100mg/kg to male BALB/C mice (N=3-6, per time point). The blood plasma and kidney tissue samples were collected upto 96hrs. The PD markers were: kidney HIF1α concentration, kidney EPO mRNA expression, EPO plasma concentration and reticulocyte count. A two compartment model with first order absorption was fitted to describe the PK in plasma. Two different dose dependent apparent volume of distribution was assumed for the central (plasma) compartment at lower and higher doses. For the PD model, under quassi steady state assumption the enzymatic hydroxylation leading to degradation of HIF1α by PHDs is assumed to be an irreversible reaction. The drug present in plasma stimulates the removal of PHDs which results in accumulation of HIF1α and further EPO stimulation. This stimulation of kidney EPO mRNA, EPO protein level and reticulocytes were described by a cascade of indirect response models. ADAPT5 was used for all model fittings.

Results:

At higher PHI dose levels, ~30-40 fold increase in HIF1α was observed which caused ~1000 fold increase in EPO mRNA and EPO plasma concentrations within 6–8 hrs whereas the reticulocytes level increased by 3–4 fold within 3–5 days of single dose administration. Model parameters such as PHI elimination rate (kel, 0.43hr-1), the dose dependent apparent volume of distribution (V/F, 2–8L), the drug potency (IC50, 0.68μ M) for the degradation of PHDs; rate of loss (kout, 0.26hr-1) of HIF1α; the depletion rate of EPO mRNA (kd1, 0.48hr-1), plasma EPO (kd2, 0.25hr-1); and half maximal effective concentration (EC50, 7.01 ng/mL) of EPO for reticulocyte stimulation were estimated with high precision levels. Overall, the PK/PD model adaquetly captured the experimental data for all three doses.

Conclusion:

The erythropoietic process follows a cascade of biological events which is well described by the presented PK/PD model. Our model uses the systems biology approach to vertically integrate the intracellular HIF1α and EPO mRNA responses with in vivo markers of erythropoiesis such as EPO plasma levels and reticulocyte counts.

Disclosures:

No relevant conflicts of interest to declare.
Whole Genome Transcriptional Analysis of Cd4+ and Cd8+ T-Lymphocyte Immunomodulated by Mesenchymal Stromal Cells
Authors: Saldanha-araujo Felipe , Panepucci Rodrigo , Araujo Amélia , Covas Dimas , Zago Marco A.
Published: October 2019

Abstract 4777

Introduction.

Recently, several researchers have showed that mesenchymal stromal cells (MSCs) are useful for preventing and treating the graft versus host disease, however the molecular mechanisms involved in immunomodulation of T lymphocytes by MSCs remains to be explored.

Aims.

With that in mind, we performed a whole genome microarray study to explore transcriptional differences in activated TCD4 and TCD8 lymphocytes immunomodulated or not by MSCs.

Materials and Methods.

Peripheral blood CD3+ T lymphocytes from 3 individuals were activated by anti-CD3/CD28 beads and cultured either in the absence or in the presence of MSC (5 to 1 ratio). Following a 5 day period, CD4+ and CD8+ lymphocytes cultivated or not with MSCs were immunomagnetically purified to over 95% purity (flow cytometry) and profiled by whole genome microarrays. Differentially expressed genes were analyzed in search of regulatory networks and levels of selected transcripts were evaluated by RT-PCR.

Results.

T lymphocytes proliferation was inhibited significantly by MSC (BrdU incorporation assay) and induced the expression of classical regulatory T cell genes (IL10, FOXP3, CTLA-4 and GITR). Our microarray analyses revealed that lymphocytes cultured with MSCs displayed several transcriptional modifications related to distinct signaling pathways. Among them, TCR signaling, cell cycle (G1/S progression), NF-kB pathway, mTOR signaling and in enzymes and factors related with chromatin modification and remodeling. An insignificant number of transcript behaved distinctly (eg. with changes in opposite direction) when TCD4+ and TCD8+ lymphocytes where compared.

Conclusion.

Our results clearly show that both CD4+ and CD8+ T lymphocytes are immunomodulated by MSCs by similar mechanisms and this effect occurs by means of distinct molecular mechanisms. Supported by FAPESP, CNPq and FINEP.

Disclosures:

No relevant conflicts of interest to declare.
Human Umbilical Cord Blood Derived IPS Cells as a Source of Hematopoietic Progenitors Cells
Authors: Moraghebi Roksana , Rönn Roger Emanuel , Parker Aaron , Lutz Margaret , Berggren Travis , Woods Niels-Bjarne
Published: October 2019

Abstract 4790

The ability to generate hematopoietic stem cells (HSCs) from an unlimited source of cells, such as from patient derived induced pluripotent stem (iPS) cells, would enable the generation of an unlimited supply of HLA matched transplantable HSCs for therapeutic purposes. Umbilical cord blood is an ideal source of fetal/neonatal cellular material for iPS reprogramming due to the proliferative capacity of the cells as well as the reduced exposure of these cells to environmental factors compare to commonly used skin fibroblasts. In addition, it has been proposed that the cellular starting material imparts an epigenetic memory to the iPS cell line that influences lineage predisposition upon its differentiation. This project seeks to evaluate human umbilical cord blood as a starting cell source for generating iPS cells, with the ultimate goal to generate transplantable HSCs. We have isolated, cultured, and characterized an adherent cell fraction from the hemato-endothelial lineage. These cells were found to have an endothelial progenitor phenotype with CD45neg, CD133neg, VE-Cadherinhi, VEGFR2med, CD31hi, CD34hi cell surface markers as determined by FACS and formed tubules in the matrigel tubular assay. The iPS cell lines were generated using a 5-factor cocktail of inducible lentiviral vectors with an efficiency of 0.02%. The iPS cell lines were then evaluated for blood cell lineage differentiation capacity using our state-of-the-art ES/iPS-to-blood differentiation protocol. From the 5 iPS lines we tested, we saw 30 +/− 20% hematopoietic (CD45+) cells in our differentiation cultures. Moreover, the percentage of hematopoietic progenitors (CD45+ CD34+) of the hematopoietic cell fraction was 19.8+/− 0.6%, and also showed the presence of the more primitive CD45+ CD34+ CD38- progenitor fraction. Clonogenic progenitor cell counts determined by methylcellulose colony assay showed 44 +/− 3 colony forming units per 10,000 plated CD45+ cells. This is in the range of colonies obtained from umbilical cord blood mononuclear cell isolates (mean= 35+/− 2). The efficiency of hematopoietic generation for the lines ranged from 8 to 60% CD45+ suggesting that there are significant differences between the lines in terms of the completeness of reprogramming towards the pluripotent state. Further investigation into the epigenetic status of these lines is being performed. These data demonstrate the utility of human umbilical cord blood derived iPS cells for generating and expanding hematopoietic progenitors. This project advances iPS technologies towards treating life threatening hematological malignancies and diseases.

Disclosures:

No relevant conflicts of interest to declare.
Differential Expression of Transforming Growth Factor Beta Receptor 2 (TGFβR2) In Diabetic CD34+ Cells: Implications for Vascular Repair
Authors: Bhatwadekar Ashay , Stepps Valerie , Grant Maria B , Bartelmez Stephen H.
Published: October 2019

Abstract 4795

We recently reported that the vascular reparative ability of diabetic CD34+ cells can be restored by transient inhibition of transforming growth factor β1 (TGF-b1) using antisense phosphorodiamidate morpholino oligomers (TGF-β1-PMO) (Bhatwadekar 2010). TGF-β1 mediates its action by binding to TGF-β-R2 receptor homodimers, which then form heterotetrameric complexes with two TGF-βR1 subunits. We investigated whether healthy or diabetic CD34+ cells differentially express TGFβR2 receptor and whether inhibition or knock-out of endogenous TGF-β1 expression would affect the surface expression of TGF-βR2.

TGF-βR2 expression of murine bone marrow (BM) lin- Sca-1+/− cells from TGF-β1 knockout mice or wild type mice was studied using flow cytometry. Unexpectedly, TGF-β-R2 expression was essentially absent in BM lin- Sca-1 +and - cells from TGF-β1 knockout mice compared to wild type cells. Because the absence of endogenous and exogenous TGF-β1 resulted in complete down regulation of TGF-βR2 in primitive murine hematopoietic cells, we explored the relationship of endogenous TGF-β1in human mature and immature CD34+ cells in blood, bone marrow and cord blood.

Human CD34+CD45+ CD38+ or - cells from healthy and diabetic individuals were FACS sorted based on surface expression the analysed for TGF-βR2 expression. At the same time, TGF-βR2 and TGF-β1 mRNA expression was determined for sorted populations using quantitative real time RT-PCR.

A comparison of TGF-βR2 surface expression between mature progenitors (lin- CD34+ CD45+ CD38+) from diabetic and healthy cord blood revealed that TGF-βR2 expression did not differ between these groups. In contrast, TGF-βR2 surface expression in immature cells (CD34+ CD45+ CD38-) was very low in diabetic patients compared to healthy controls (p = 0.001).

Next, we inhibited endogenous TGF-β1 in healthy CD34+ cells which resulted in a significant (p<0.01) reduction in TGF-β-R2 mRNA expression. However, in diabetic CD34 cells, TGF β1-PMO treatment abolished TGFβR2 expression.

Our study suggests that healthy and diabetic CD34+ cells differentially regulate TGF-βR2 expression which may be the result of the elevated intracellular and extracellular levels of TGFb1 in diabetic individuals. A reduction in TGF-βR2 expression in diabetic CD34+ CD38- cells may physiologically enhance proliferation of the primitive CD34+ CD38- cell compartment, an outcome that may be necessary to keep up with increased demand for CD34+ cells in the diabetic individual.

Disclosures:

Stepps: BetaStem Therapuetics: Employment. Bartelmez: BetaStem Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.
Hematopoietic Stem Cell Expansion Leads to Monocytosis In Animal Models of Atherosclerosis
Authors: Akhtari Mani , Murphy Andrew J , Pagler Tamara A , Tall Alan R
Published: October 2019

Abstract 4797

Atherosclerotic cardiovascular disease is associated with leukocytosis. Hypercholesterolemic animal models like apolipoprotein E deficient (Apoe-/-) mice develop leukocytosis and monocytosis. However, the mechanisms linking hypercholesterolemia to leukocytosis are poorly understood. Recent studies in our laboratory have shown increased proliferation of hematopoietic stem and progenitor cells (HSPCs) and leukocytosis in mice deficient in efflux of cholesterol to HDL. Apoe also appears to be highly expressed in HSPCs, suggesting a role in cholesterol efflux. We hypothesized that leukocytosis in Apoe-/- mice might be associated with increased proliferation and expansion of HSPCs and that lowering their cholesterol levels with atorvastatin would suppress this expansion. Wild type (WT), LDL receptor-deficient (Ldlr-/-), and Apoe-/- mice were fed a Western-type diet (WTD) for 0, 10 and 20 weeks (8 mice per group per timepoint). After 10 weeks of WTD, 8 Apoe-/- received WTD with atorvastatin (0.01% wt/wt) for the remainder of the study. On chow diet Apoe-/- mice had monocytosis (+66% vs WT, P<.01), neutrophilia (+90%, P<.0001), and expanded HSPCs (+46% compared to the other groups, P<.05). After 10 weeks of WTD, leukocytosis developed further in Apoe-/- mice, along with increased bone marrow proliferation (+86%, P<.01) and more granulocyte-macrophage (GM)-colony formation (110% more, P<.01). After 20 weeks, all mice had developed monocytosis, neutrophilia, and expansion of the HSPCs compared to their chow starting point. Atorvastain reduced plasma cholesterol by 60% (P<.01) and attenuated monocytosis (30% reduction, P<.05) and neutrophilia (35% reduction, P<.05), but did not reduce the HSPCs levels in Apoe-/- mice. In conclusion, leukocytosis and expansion of HSPCs in Apoe-/- mice is observed on a chow diet and develops earlier than in the other models on the WTD, consistent with a role of APOE in modulating HSPCs cholesterol content. However, dietary hypercholesterolemia eventually led to HSPCs expansion paralleling leukocytosis in all groups, suggesting this is a general response to dietary hypercholesterolemia. Leukocytosis was attenuated with atorvastatin treatment through a mechanism unrelated to the reversal of HSPCs expansion.

Disclosures:

Tall: CSL: Consultancy; Merck: Consultancy; Roche: Consultancy; Arisaph: Consultancy.
Evaluation of Possible Risk Factors for Avascular Necrosis of the Femoral head in patients with Sickle Cell Disease
Authors: Al-Momen Abdul Kareem , Owaidah Tarek , Naqshabandi Nizar , Hasanato Rana , Cana Sri-Halima , Qayum Abdul , Zamzami Marwan , Aleem Aamer
Published: October 2019

Abstract 4819

Sickle cell disease is one of the most common causes of avascular necrosis (AVN) of the femoral head. many previous studies have investigated the risk factors for AVN with conflicting conclusions. We hereby report the results of a total of 44 adult patients (26 males and 18 females, age 18–56 years) with severe sickle cell disease seen in our hematology clinic who were investigated for the presence of AVN. Out of these patents 24 (55%) had AVN of the femoral head (6 bilateral and 18 unilateral). All of these patients had further investigations including methelene tetrahydrofolate reductase (MTHFR) gene mutation by molecular technique, along with Hb F level, Hb A2 level and MCV. Six of the 24 AVN patients (24%) were positive for either heterozygous or homozygous for C677T variant compared with four out of the 20 who did not have AVN (the difference is not statistically significant). HbF level was statistically higher in patients with AVN (P- value 0.03). In conclusion, we confirm here that patients with sickle cell disease and AVN have high prevalence of MTHFR mutation and higher level of Hb F, both of which which could be contributing factors for AVN in patients with Sickle Cell Disease.

Disclosures:

No relevant conflicts of interest to declare.
Prevalence of Sickle Cell Anemia In Eastern India
Authors: Chakraborty Abhijit , Basak Jayasri , Majumdar Deboshree , Mukhopadhyay Soma , Chakraborty Sagnik , Mukhopadhyay Ashis
Published: October 2019

Abstract 4822

Background:

Sickle cell disease is an inherited disorder of hemoglobin synthesis. This is due to replacement of Valine for Glutamic Acid in position six of the Beta globin chain of hemoglobin. This genetic alteration yields unstable RBC which lasts for 10–20 days. In stressful conditions the cells become sickle shaped and get lysed. There are about 20 million people with sickle cell disease in India. During January 2009- May2010 camps were held in various parts of West Bengal, Jharkhand, Chattisgarh. Along with various mutations of thalassemia, we also observed sickle cell anemia among them. This triggered our interest to study the spectrum of the sickle mutation co-inheritant with different mutations such as Homozygous Sickle Cell, Sickle Cell-Beta0 Thalassemia, Sickle Cell-Beta+ Thalassemia, Severe β+ thalassemia genes, Moderate β+ thalassemia genes, Mild β+ thalassemia genes Sickle cell-HbE Thalassaemia, Sickle cell-HPFH Thalassaemia, in said part of India. Since Indian patients with SS disease had higher hemoglobin, red cell counts and higher HbF levels and lower HbA2, MCHC, MCV, and reticulocyte counts, hence a high hemoglobin is a risk factor for painful crises and may also be a risk factor for avascular necrosis of the femoral head, proliferative sickle retinopathy, and acute chest syndrome.

Methods:

We have screened 332 individuals in eastern part of India during the period January 2009- May 2010. 3ml of peripheral blood was collected in EDTA vial from each individual. NESTROFT (Naked Eye Single Tube Red Cell Osmotic Fragility Test) was performed on spot. Then Complete Blood Count was done within 24 hours of collection. HPLC (High Performance Liquid Chromatography) was performed to identify the samples for confirmation. In our observation in case of sickle cell anaemia HbF (Fetal haemoglobin), Hb (haemoglobin), MCV (mean corpuscular volume) values ranges between 0–10 %, ≤7-10g/dl, 65–90fl respectively. ARMS (Amplification Refractory Mutation System) PCR (polymerase chain reaction) was done to confirm the mutation.

Result:

Conclusion:

Of the total samples collected in the camps held at various places of Jharkand, Chattisgarh & West Bengal 87 of them was carriers of sickle cell anemia. There was 7 homozygous (SS), 14 sickle beta, 12 double heterozygous for HPFH (High Persistance of Fetal Hemoglobin) & sickle cell anemia. In conclusion, the manifestations of sickle cell disease are influenced by a variety of other genetic and environmental factors. The occurrence of the disease against different genetic and environmental backgrounds provides experimental models that contribute to understanding the variability in clinical and hematological expression of the disease.

Disclosures:

No relevant conflicts of interest to declare.
Gain of the Centromeric Region of Chromosome 8, a New Germline Alteration In Juvenile Myelomonocytic Leukemia
Authors: Steinemann Doris , Ripperger Tim , Tauscher Marcel , Praulich Inka , Göhring Gudrun , Pabst Brigitte , Teigler-Schlegel Andrea , Schlegelberger Brigitte , Niemeyer Charlotte M , Flotho Christian
Published: October 2019

Abstract 4830

Juvenile myelomonocytic leukemia (JMML) is an aggressive childhood myeloproliferative disorder characterized by the clonal hyperproliferation of myelomonocytic cells. The pathogenesis of JMML involves disruption of signal transduction through the RAS pathway, which may be an early event during leukemogenesis. Additional genetic lesions may be necessary for full malignant transformation. We applied microarray BAC/PAC- and high resolution 244k oligo-arrayCGH to bone marrow samples from 21 JMML patients in order to identify subtle genomic alterations. In 8 of13 JMML patients with normal karyotype and 2 of 8 patients with monosomy 7, additional copy number alterations were identified. A recurrent deletion of around 1.4 Mb at 17q11.2 targeting the NF1 gene was identified in 2 patients with clinical diagnosis of neurofibromatosis due to germline mutations and LOH in the tumor as described previously. In addition to the monosomy 7, one patient (D600) with a somatic mutation in PTPN11 showed an additional marker chromosome. The origin of this marker chromosome was resolved by arrayCGH. The BAC/PAC array indicated a gain of the centromeric region: arr8p12-q11.21(37,204,000-49,686,000)×3. Breakpoints were refined to 8p12-q11.21(36,673,794- 50,142,678) by high resolution oligo-aCGH. Interestingly, in patient D703 with a heterozygous CBL germline mutation and homozygosity in hematopoietic cells, we detected a nearly identical gain arr8p12-q11.21(40,076,917-49,622,319)×3 not accompanied by -7 and with slightly different breakpoints. The gain was confirmed by means of FISH using the centromere-specific probe for chromosome 8 CEP8 showing three signals. This patient was also found to have a small additional marker chromosome. In order to rule out that the marker chromosome was inherited, we performed chromosome analyses in the parents of patient D703. Both had a normal karyotype and did not carry an extra copy of the region 8p12-q11.21. However, FISH on buccal epithelial mucosa cells of the patient D703 revealed a mosaic constellation with 34% cells positive for trisomy 8. Likewise, in the second patient, the dup 8p12-q11.21 (D600) was constitutional as 38% of fibroblasts showed 3 CEP8 FISH signals. There has been a longstanding discussion as to whether mosaic trisomy 8 may be constitutional. Our findings provide evidence that at least partial trisomy 8 may indeed be present as a germline mutation. Future work is needed to determine how constitutional partial trisomy 8 might contribute to leukemogenesis in JMML with different mutational subtypes.

Disclosures:

No relevant conflicts of interest to declare.
Concordance of CNS Leukemia Detection Between Morphology and Flow Cytometry Prior to Stem Cell Transplantation In Patients with MDS, AML, ALL and CMML
Authors: Smith Kelly M. , Cherian Sindhu , Wood Brent , Sandmaier Brenda M. , Pagel John M. , Estey Elihu H.
Published: October 2019

Abstract 4842

Accurate diagnosis of CNS involvement is essential for optimizing treatment and maximizing outcomes in patients with leukemia prior to hematopoietic stem cell transplantation (HCT). The most common methods of detecting CNS involvement include cytomorphic analysis and multiparametric flow cytometry (MFC). While cytomorphic analysis still has diagnostic and prognostic importance, MFC may be more than twice as sensitive as standard cytology analyses, raising the question of whether MFC might be able to detect CNS disease in cases where cytology did not.1 We retrospectively evaluated cerebral spinal fluid (CSF) results among 109 continuous patients with MDS, AML, ALL and CMML who underwent a reduced-intensity HCT at the Fred Hutchinson Cancer Research Center between May 2006 and May 2010. Ninety-seven of these patients had a pre-HCT lumbar puncture on arrival to evaluate for CNS disease. Thirty-five patients had CSF sent for morphology alone, 2 patients had CSF sent for MFC alone and 60 patients had CSF sent for both morphology and MFC. Of the 60 patients with both MFC and morphology results, CSF notable for blasts by concordant morphology and MFC was detected in 6 patients. These patients received treatment with intrathecal chemotherapy with or without craniospinal radiation. One patient had a single atypical cell with blast-like morphology seen by cytology with negative MFC. Fifty-three patients had both negative CSF by morphology and MFC. This data suggests that there is high concordance between morphology and MFC, however, it is difficult to determine the concordance with much confidence due to the small number of patients with CNS disease. Given the infrequency of CNS disease in this patient population, more patients will need to be studied in order to fully assess the value of MFC in addition to cytomorphic analysis. Because it is also difficult to be sure that our patient population was representative, a prospective large study, for example, in consecutive patients presenting for treatment of AML, might be worthwhile.

1J.E.C. Bromberg, MD, D. A. Breems, MD, PhD, J. Kraan, BSc, G. Bikker, B. van der Holt, MSc, P. Sillevis Smitt, MD, PhD, M. J. van den Bent, MD, M. van't Veer, MD, PhD and J. W. Gratama, MD, PhD. CSF flow cytometry greatly improves diagnostic accuracy in CNS hematologic malignancies. Neurology 2007;68:1674-1679.

Disclosures:

No relevant conflicts of interest to declare.
Identification of Novel Peptide Ligands Targeting α4β1 Integrins on Patient Lymphoma Cells
Authors: Tseng Harry , Xiao Wenwu , Huang Wenzhe , Wun Ted , Lam Kit
Published: October 2019

Abstract 4854

Peptides are promising cancer-targeting agents—they are small, chemically stable, easy to synthesize and can be readily conjugated to toxins, radionucleotides, or drugs. The “one-bead one-compound” (OBOC) combinatorial libraries developed in the Lam lab consist of millions of beads, each displaying one unique peptide entity. Through high throughput screening peptide combinatorial libraries against cancer cell lines, peptides can be selected and engineered to recognize some certain receptors on the cell surface. From our previous screen, we observed that the LLP2A peptide sequence bound to the α4β1 integrin on the cell surface of a variety of malignant lymphoid cell lines. We hypothesize that more specific ligands against the α4β1 integrin on human patient lymphoma cells could be identified through screening focused LLP2A libraries. Two OBOC-focused combinatorial libraries based on the LLP2A peptide sequence were designed, synthesized and screened against human patient lymphoma cells to search for ligands with higher binding affinity. We selected the candidate peptide sequences that displayed a higher strength for the patient lymphoma cells using the whole cell binding assay, an in vitro approach for quantitating the strength of peptide interaction with cell surface molecules on the cancer cells. We are currently analyzing the data to determine the specific sequence requirements for higher affinity binding. Further assessment of the affinity and kinetics of integrin-peptide binding will be performed using flow cytometry.

Disclosures:

No relevant conflicts of interest to declare.
Influenza A H1N1 Pandemic Infection In Onco-Hematologic Patients
Authors: Pierrotti Ligia Camera , Bellesso Marcelo , Garcia Cilmara Polido , Pereira Juliana , Atta José Antonio , Hajjar Ludhmila Abrahão , Hoff Paulo Marcelo Gehm , Chamone Dalton FA , Abdala Edson
Published: October 2019

Abstract 4869

Background:

Epidemiological, virological and clinical data about H1N1 pandemic infection in immunocompromised patients are needed.

Objectives:

To describe a series of onco-hematologic patients with H1N1 pandemic infection who required hospitalization.

Method:

Review from institutional database and electronic patients charts. Specific diagnosis defined as a positive nasal swab PCR. Flu syndrome defined as fever, cough and/or sore throat; severe respiratory disease as fever higher than 38°C, cough and dyspnea.

Results:

From July 24th to September 30th 2009, 114 patients were investigated; 18 (15.8%) was confirmed and 9 were onco-hematologic patients. Hematological diseases were: chronic myeloid leukemia (3), lymphoma (3), amiloidosis (2) and myelofibrosis (1) – 2 with previous TCTH (for amiloidosis and myelofibrosis). Clinical manifestations in table 1. One patient died (11.1%). Two patients had received chemotherapy in the last 4 weeks. Radiological findings were mainly interstitial (4) and interstitial-alveolar (1). Lymphocytes less than 1000 cel/mm3 were detected in 3 patients; the patient who died had 1000 lymphocytes/mm3. Three patients required ICU; 1 of 2 who required mechanical ventilation died. Time between symptoms onset and start of oseltamivir in table 2; mean therapy length was 6.1 days (3-10 days); 3 days in the case of obit. Prolonged viral shedding was investigated in 3 patients - 1 negative (5 days after first oseltamivir dose) and 2 positive (5 and 7 days after).

Conclusion:

We did not include outpatients because in our country most of the time PCR was available only for those requiring hospitalization, despite of clinical suspicion and oseltamivir prescription. An important challenge recognized was atypical clinical presentation including acute respiratory distress without fever, leading us to consider that we probably lost opportunity to make diagnosis in some other cases. For both reasons, we were not able to know the real impact of H1N1 pandemic infection among onco-hematological patients. We recognize however that, among our hospitalized patients, mortality rate was low as reported recently in hematological cancer patients.

Disclosures:

No relevant conflicts of interest to declare.
Granulocyte Colony-Stimulating Factor Induces High Expression of HLA-G In CD3+CD4+ T Lymphocytes and CD3+CD8+ T Lymphocytes
Authors: Liu Qifa , Chen Yinkui , Wu Xiuli , Du Xuan , Li Rui , Fan Zhiping , Xuan Li , Sun Jing
Published: October 2019

Abstract 4870

Background and objectives:

Human leukocyte antigen –G (HLA–G) is a nonclassic HLA class I molecule and has the immunosuppressive activity. We investigate the effects of Granulocyte colony-stimulating factor (G-CSF) on the expression and secretion level of HLA-G in peripheral blood and bone marrow cells, and attempt to explain the mechanism of the low incidence of graft-versus-host disease (GVHD) in G-CSF-primed bone marrow (G-BM) or peripheral blood stem cells (G-PBSC) transplantation.

Methods:

Bone marrow and peripheral blood cells of 10 donors were collected pre-mobilization and at day 5 after G-CSF mobilization. Flow cytometry was used to detect the expression of membrane-bound HLA-G (mHLA-G) of CD3+CD4+ T lymphocytes, CD3+CD8+ T lymphocytes, CD19+ B lymphocytes, CD56+ NK cell, CD14+ monocytes and CD33+ granulocytes. The levels of soluble HLA-G (sHLA-G) and cytokine were determined by enzyme-linked immunosorbent assay (ELISA). Bone marrow cells and peripheral blood cells of pre-mobilization were incubated for 24h, which are divided into 5 groups: (a) blank group, (b) with G-CSF, (c) with G-CSF and blocking antibody of IL-10, (d) with G-CSF and blocking antibody of IFN-γ, (e) with G-CSF, blocking antibody of IL-10 and blocking antibody of IFN-γ.

Results:

The levels of mHLA-G of CD3+CD4+ lymphocytes, CD3+CD8+ lymphocytes and the levels of sHLA-G at day 5 after G-CSF mobilization groups were significantly higher than the pre-mobilization groups both in bone marrow and peripheral blood cells, especially in CD3+CD8+ lymphocytes. The levels of mHLA-G of CD19+ B lymphocytes, CD56+ NK cells, CD14+ monocytes and CD33+ granulocytes had no significant difference in the two groups. The levels of IL-10 or IFN-γ in plasma of bone marrow or peripheral blood at day 5 after G-CSF mobilization groups were much higher than the pre-mobilization groups. The levels of mHLA-G of CD3+CD4+ lymphocytes, CD3+CD8+ lymphocytes and the levels of sHLA-G in bone marrow cells or peripheral blood cells incubating with G-CSF for 24h were significantly higher than the groups without incubating with G-CSF. And the blocking antibody of IL-10 or the blocking antibody of IFN-γ could not reduce the expression levels of mHLA-G and sHLA-G in vitro.

Conclusion:

G-CSF can act on cells directly to increase the expression levels of HLA-G in CD3+CD4+ T lymphocytes and CD3+CD8+ T lymphocytes. It might be assciated with the low incidence of GVHD in G-BM transplantation or G-PBSC transplantation.

Supported by National Natural Science Foundation of China (30971300), Science and Technology Planning Project of Guangdong Province of China (2009A030200007) and China Postdoctoral Science Foundation (200902332, 20080440776).

Disclosures:

No relevant conflicts of interest to declare.