bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of KLF4 and KLF12 in γ-Globin Gene Regulation.
Authors: Kalra Inderdeep S , Li Wei , Muralidhar Shalini , Pace Betty
Published: October 2019

Abstract 4075

Poster Board III-1010

Kruppel-like factors (KLFs) are a family of Cys2His2 zinc-finger DNA binding proteins that regulate gene expression through CACCC/GC/GT box binding in gene promoters. The CACCC element is critical for the developmental regulation of the human γ-globin and β-globin genes and studies are being done to ferret out various factors that bind this region and modulate gene activity. We recently identified two Kruppel-like factors, KLF4 and KLF12 whose expression levels decreased based on microarray-based gene profiling, concomitantly with decreased γ-globin expression during erythroid maturation. Decreased expression of both factors was further confirmed using quantitative PCR (qPCR) analysis. KLF4 and KLF12 mRNA levels decreased 56-fold and 16-fold respectively by day 28 compared to levels in day 7 erythroid progenitors. We next determined if KLF4 and KLF12 bind the γ-globin CACC box by electrophoretic mobility shift assay (EMSA) using nuclear proteins extracted from K562 cells and a [γ-32P] labeled γ-CACC probe located between -155 to -132 relative to the γ-globin gene cap site. Three DNA-protein complexes were observed. The specificity of these interactions was confirmed by competition reactions in which preincubation with excess unlabelled γ-CACC oligonucleotide effectively abolished the formation of all DNA/protein complexes; addition of nonspecific oligonucleotide had no effect on binding activity. Addition of polyclonal KLF4 or KLF12 antibodies to the EMSA reaction resulted in a marked decrease in intensity of all DNA-protein complexes suggesting both KLF4 and KLF12 are present. Additional studies were performed to determine the effect of the known fetal hemoglobin inducer hemin on KLF gene expression in K562 cells. Hemin stimulated γ-globin transcription while increasing KLF4 and KLF12 66-fold and 4-fold respectively (p<0.05). Hemin treatment in KU812 erythroleukemia cells which actively transcribe both γ- and β-globin, also produced a 10-fold increase (p<0.05) in KLF4; KLF12 levels were not changed. Our preliminary data suggest these KLFs might play a role in γ-globin regulation. siRNA mediated gene silencing studies are underway to determine if KLF4 and/or KLF12 play a direct role in γ-globin gene regulation. This mechanism could provide important molecular targets for fetal hemoglobin reactivation. This will be highly significant towards developing therapeutic strategies for hemoglobinopathies like sickle cell anemia and β-thalassemia.

Disclosures:

No relevant conflicts of interest to declare.
Outcomes of CAG Regimen for Refractory Biphenotypic Acute Leukemia Patients.
Authors: He Guangsheng , Zhang Xiang , Depei Wu , Sun Aining , Jin Zhengming , Qiu Huiying , Miao Miao , Tang Xiaowen , Fu Chengcheng , Han Yue
Published: October 2019

Abstract 4111

Objective

Biphenotypic acute leukemia (BAL) involves both myeloid and lymphoid cells, and there is lack of uniformity in treatment at present. Optimal approach for therapy of BAL is unknown, and BAL usually becomes refractory to conventional chemotherapy. It was found that CAG regimen [low-dose cytosine arabinoside (Ara-C) plus aclarubicin with concurrent administration of granulocyte colony-stimulating factor (G-CSF)] is effective for both myeloid leukemia and refractory acute lymphoblastic leukemia. To explore the efficiency of CAG regimen for refractory BAL, 12 BAL patients who were failed to daunorubicin/mitoxantrone, cytosine arabinoside, vincristine, prednisone (D/MAOP) regimen, were treated by CAG. 8 were males and 4 was female, with ages ranging from 20 to 43 years (median=34 years). Immunophenotype of B lymphoid lineage/myeloid lineage showed in 8 patients, and T lymphoid lineage/myeloid lineage showed in 4 patients. The patients whose blast cells did not decrease 50% after induction regimen with lymphoid and/or myeloid drugs were defined as refractory cases.

Methods

12 refractory BAL patients were treated by CAG regimen (cytosine arabinoside 10 mg/m2 subcutaneously every 12 hours, day 1-14; aclarubicin 5-7 mg/m2 intravenously daily, day 1-8; and concurrent use of granulocyte colony-stimulating factor 200μg·m-2·d-1 subcutaneously) from November 2002 to April 2009. The efficacy of the regimen was evaluated by response rate, and the side-effects was also measured.

Results

The major chemotherapy toxicity was bone marrow depression, mainly showing as pancytopenia. Median duration of absolute neutrophil count (ANC)<0.50×109/L and of platelet (PLT) count<20×109/L was 13 (range, 1-17) days and 1 day (range, 0-5 days), respectively. Median transfusion of red cells and PLT was 4 (range, 1.5-12) U and 20 (range, 0-40) U, respectively.

Besides bone marrow depression, other side-effects such as infection, bleeding, nausea and vomiting also occurred during therapy. According to chemotherapy toxicity assessment of WHO, there were 7 patients confronting infection, among whom only 2 patient occurred III-‡W infection. No serious nausea vomiting or hepatic function damage happened.

Disclosures:

No relevant conflicts of interest to declare.
Induction of REMISSION with Clofarabine IN CHILDREN with Relapsed Acute LYMPHOCYTIC Leukemia.
Authors: Moschovi Maria M , Trimis Georgios G , Karamolegou Kaliopi , Tourkantoni Niki , Tzanoudaki Maria , Divane Anna , Botsa Eleni , Bouzios Elias , Koultouki Eleftheria , Tzortzatou-Stathopoulou Fotini F
Published: October 2019

Abstract 4112

New treatment protocols have improved significantly the survival of patients with acute lymphocytic leukemia (ALL). However, there is a risk of relapse, which depends on disease classification and the protocol of chemotherapy administered. In case of relapse, new remission has to be achieved to proceed to bone marrow transplantation (BMT). For this reason, new therapies for remission induction, except standard chemotherapy, are under investigation. Clofarabine, a new class agent, is a nucleoside analogue of purine which inhibits DNA synthesis and repair by blocking ribonucleoside reductase and DNA polymerase. It also disrupts mitochondrial membrane, driving to cell apoptosis. We describe two children with B-cell ALL, in whom clofarabine was administered because of disease resistance. Case 1: Girl, 12 years old, with ALL, presented a second bone marrow (BM) relapse. The first relapse was also in BM and was presented 30 months after the end of primary chemotherapy. Remission was achieved with chemotherapy based on protocol ALL-REZ-BFM 96. In the second relapse, that occurred one year after the end of ALL-REZ-BFM 96, conventional cytogenetics analysis showed an apparently normal female karyotype 46,XX, while molecular cytogenetics analysis (FISH) showed 3 and 4 copies of AML1 gene in 10.7% and 82.4%, of analyzed nuclei respectively. Clofarabine was administered in 52mg/m2/24h for 5 days, every 4-6 weeks. It was well tolerated. Moderate myelotoxicity presented, early after the end of regimen that was uncomplicated. Mild and transient elevation of transaminases was detected in the peripheral blood. Reevaluation after 3 cycles of clofarabine, revealed morphologic and cytogenetic remission. The patient underwent successful heterologous BMT. Case 2: Boy 18 years old with ALL presented a first BM relapse 22 months after the end of initial chemotherapy. Conventional cytogenetics analysis showed an apparently normal male karyotype 46,XY, while molecular cytogenetics analysis (FISH) was negative for clonal rearrangement involving the MLL gene and TEL/AML1, or BCR/ABL hybrid genes. Chemotherapy was administered based on protocol ALL-REZ BFM 96, but remission was not achieved. Clofarabine in 52mg/m2/24h for 5 days was administered. The patient presented mild uncomplicated myelotoxicity. Remission was achieved after the first cycle of clofarabine and the patient underwent successful heterologous BMT.

Conclusion

Clofarabine administration in children with refractory ALL seems to achieve disease remission and is well tolerated. More studies are needed to establish clorafabine as first line therapy in patients with relapsed ALL, prior to bone marrow transplantation.

Disclosures:

No relevant conflicts of interest to declare.
VEGF Autocrine System Is Demonstrated in Some of Acute Promyelocytic Leukemia Cases: Relationship to WBC Count at the Disease Onset.
Authors: Sugao Toshihiko , Oka Yoko , Tashiro Haruko , Shirasaki Ryosuke , Akiyama Nobu , Kawasugi Kazuo , Shirafuji Naoki
Published: October 2019

Abstract 4127

Aims:

In acute promyelocytic leukemia (M3), a prominent production of vascular endothelial growth factor (VEGF) is reported, and the obvious angiogenesis is observed in bone marrow specimens; however, VEGF receptors (VEGFRs) are not expressed in M3 cells in general. We analyzed VEGF systems in M3 blasts.

Materials and Methods:

Bone marrow cells were obtained from informed M3 patients, whose mononuclear cells were prepared with density-sedimentation method. Cells were cultured for one day for the elimination of an adherent cell-fraction. RNA was extracted from the non-adherent mononuclear cell-fraction, and cDNA was synthesized. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the expression of VEGF and VEGFRs. When the expression of VEGFR type-1 and -2 was demonstrated with RT-PCR, cells were analyzed on the protein level with FACS. VEGF-A levels in sera and in the conditioned media from the cultured M3 cells were determined with ELISA kit. When M3 blasts expressed VEGFR type-1, -2, and also secreted VEGF-A, a growth-inhibition by anti-VEGF antibody was assayed in in vitro cultures.

Results and Discussion:

In all 25 cases examined VEGF-A production was observed, in which VEGFR type-3 was not expressed in any cases. VEGFR type-1 and -2 were expressed in 3 cases, in all of which WBC count at the onset of the disease was above 20,000/μl. When VEGF antibody was added to the blast cell-cultures, the cell-growth was inhibited significantly. These observations indicate that VEGF system works on proliferation in a few of M3 cases with hyper-leukocytosis.

Disclosures:

No relevant conflicts of interest to declare.
CD33-Negative Acute Myeloid Leukemia (AML) Has Similar Characteristics to CD33-Positive Disease.
Authors: Arana Yi Cecilia Y. , Tan Wei , Wilding Gregory , Ford Laurie Ann , Sait Sheila N.J. , Block AnneMarie W. , Deeb George , Wallace Paul K. , Starostik Petr , Thompson James E. , Wang Eunice S. , Wetzler Meir
Published: October 2019

Abstract 4129

Several immunophenotypic subgroups within AML are associated with specific disease characteristics, e.g., CD19-positive AML is associated with t(8;21)(q22;q22) and CD56 expression is associated with adverse outcome. CD33 is a sialic acid-binding immunoglobulin-like lectin (Siglec) located on chromosome 19q13.3. It is highly expressed on early multilineage hematopoietic progenitors but absent from the pluripotent hematopoietic stem cells. It contains two tyrosine phosphorylation motifs whose phosphorylation depends on Src and is the target of gemtuzumab ozogamicin. We asked if lack of CD33 expression (<10%) on the surface of AML blasts was associated with different disease characteristics. We identified 15 such newly diagnosed AML cases and compared their characteristics with 45 (1:3 ratio) cases matched by age and French-American-British subtypes (only cases with M0, M1 and M2 were included). The mean white blood cell count at diagnosis (18.3 ×109/L, 41.7×109/L; p=0.149) and percent of secondary AML [either antecedent hematologic disorder (13%, 18%; p=1) or therapy-related (7%, 16%; p=0.6657)] among the CD33-negative group was similar to the CD33-positive cohort. The percent of unknown (21%, 19%), intermediate (64%, 51%) and unfavorable (14%, 30%) karyotype (p=0.5484) and the percent of cases with FLT3 (20%, 18%; p=0.6058) and NPM1 (7%, 22%; p=0.1562) was also similar among the CD33-negative and the CD33-positive cohorts. The patients were treated with available protocols. There was no significant difference between CD33-negative and CD33-positive treatment assignment. A total of 50% of the CD33-negative cases achieved complete remission compared to 48% of the CD33-positive cases. The median overall survival was 17.1 [95% confidence interval (CI) 10.3, 22.5] versus 10.6 (95% CI 7.1, 16.9) months (p=0.7488) for the CD33-negative compared with the CD33-positive cases. Finally, disease-free survival was also similar between the two groups; 11.0 (95% CI 6.2, 22.6) months for the CD33-negative cohort versus 9.5 (95% CI 4.5, 118.2) months for the CD33-positive group (p=0.966). In summary, CD33 expression does not alter disease characteristics and does not appear to affect outcome in AML unless treatment with gemtuzumab ozogamicin is considered.

Disclosures:

No relevant conflicts of interest to declare.
Immunophenotypic Profile of Acute Myeloid Leukemia in Elderly Patients: a Tertiary Center Experience in Brazil.
Authors: Perini Guilherme F , Santos Fabio P S , Seguro Fernanda , Bacal Nydia , Velloso Elvira , Hamerschlak Nelson
Published: October 2019

Abstract 4137

Acute myeloid leukemia (AML) has an extremely poor prognosis in elderly patients. However, AML is considered a heterogeneous disease, and few reports have analyzed the immunophenotypic profile of AML in elderly patients. We thus analyzed the immunophenotypic markers of newly diagnosed AML in elderly patients in order to determine the incidence of anomalous markers and the correlation with cytogenetic findings. We analyzed samples from 127 patients older than 65 years old with newly diagnosed adult AML seen at our institution from January, 2002 until December, 2007. Markers were assessed by flow cytometry, and the cut-off for positivity was 20%. The expression of CD33 and CD13 was positive in 82.7% and 88.2% of the samples, respectively. HLA-DR was found in 73.2% of cases. The most common aberrant lymphoid markers were CD56 (22.2%), CD7 (16.5%) and CD2 (6.3%). The expression of CD4 and CD11b was correlated with an abnormal karyotype (p<0.05). CD36 was significantly more expressed in patients younger than 75 years of age (p=0.005). AML in elderly patients is a biologically heterogeneous disease, and the immunophenotypic profile reflects this. Future studies should analyze the prognostic value of immunophenotypic abnormalities in this population.

Disclosures:

No relevant conflicts of interest to declare.
Hepatotoxicity in Patients Treated with Gemtuzumab Ozogamicin - Specific Targeting of Hepatocytes?
Authors: Maniecki Maciej Bogdan , Hasle Henrik , Bendix Knud , Møller Holger Jon
Published: October 2019

Abstract 4150

The immunotoxin gemtuzumab ozogamicin (Mylotarg‘, GO) comprises a potent cytotoxic agent, calicheamicin, and a recombinant humanized monoclonal antibody directed against CD33. GO is increasingly used for treatment of acute myeloid leukemia (AML) as CD33, a trans-membranous glycoprotein, is highly expressed on most leukemic blast cells, but, in normal cells confined to early multilineage hematopoietic progenitors and myelomonocytic precursors. Adverse effects of GO treatment are primarily related to myelosuppression (infections, fever, bleedings). However, severe sinusoidal obstructive syndrome (SOS) and hepatotoxicity have also been reported. Since the introduction of GO treatment into routine clinical practice, the incidence of SOS has been reported to span from 0% to 33%, but, the direct cause of the liver-associated effects remain unknown. Using immunohistochemistry we show that the CD33 receptor is widely distributed in the liver tissue and highly expressed on hepatocytes making them susceptible for targeted CD33 chemotherapy by GO. Moreover, utilizing CD163 as a specific marker for macrophages, double immunofluorescence staining revealed that Kupffer cells strongly express CD33. Therefore, the reported hepatoxicity associated with GO treatment may be due to accumulation of antibody-toxin conjugates in hepatocytes and Kupffer cells causing calicheamicin-induced damage.

Disclosures:

No relevant conflicts of interest to declare.
RASGRP1:APTX Gene Expression Ratio Enriches for Responders to the Combination of Tipifarnib and Etoposide in Elderly Newly Diagnosed AML.
Authors: Raponi Mitch , Vener Tatiana , Derecho Carlo , Greer Jacqueline , Malek Sami , Palma John , Karp Judith E
Published: October 2019

Abstract 4154

A 2-gene ratio (RASGRP1:APTX) has previously been shown to predict response to the single agent farnesyltransferase inhibitor, tipifarnib, in patients with both newly diagnosed or relapsed and refractory acute myeloid leukemia (AML) (Raponi et al. Blood 2008, 111:2589). Here we investigated the utility of the 2-gene ratio to predict response to the combination of tipifarnib and etoposide (VP16) in a phase 1 study (Karp et al. Blood 2009, 113:4841) and continuing phase 2 study of elderly patients with newly diagnosed AML. A Taqman quantitative RT-PCR assay was performed on leukemic blasts from a total of 81 patients who were evaluable for response. A receiver operator characteristic analysis indicated that the predictive value of the 2-gene ratio was 76% for predicting overall response (CR+PR) in the Phase 1 study (51 patients). When using an optimized cutoff the overall response rate doubled from 25% to 50% with a negative predictive value of 88% (p = 0.008). These data indicate that the RASGRP1:APTX gene ratio has utility in predicting response to the combination of tipifarnib and etoposide in elderly newly diagnosed AML. This assay could find broader utility in enriching for responders to tipifarnib combination therapy.

Disclosures:

Malek: Cephalon: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Affymetrix: Research Funding.
Increase of Sensitivity to Chemotherapy and NK-Mediated Cytotoxicity of Leukemic Stem Cell by a Farnesyltransferase Inhibitor.
Authors: She Miaorong , Li Jinggao , Chen Xilin , Niu Xingqing , Guo Kunyuan , Du Xin , Zhou Maohua , He Yanjie , Le Yi
Published: October 2019

Abstract 4161

Acute myeloid leukemia (AML) is a serious and often lethal hematopoietic malignant disease arising from stem cells. Leukemic stem cells (LSCs) play the central role in the relapse and refractory of AML and highlight the critical need for the new therapeutic strategies to directly target the LSC population. We previous found that LSC were resistant to chemotherapy and NK-mediated cytotoxicity, which were resulted from the apoptosis defect and NKG2D ligands change. Our previous data also showed that Manumycin induced apoptosis in leukemia cells through mitochondria pathway. Here, we demonstrated that manumycin increased the sensitivity of LSC to chemotherapy and NK cell-mediated cytotoxicity. Manumycin enhanced sensitivity to chemotherapy in LSCs via induced apoptosis. Analysis of signaling pathways revealed that manumycin enhanced mitoxantrone activation of caspase 3 and significantly decreasing Bcl2 protein. These finding indicated that manumycin sensitized mitoxantrone to chemotherapy via down-regulating Bcl2. Importantly, manumycin enhanced the sensitivities of LSCs to NK cells cytotoxicity through up-regulating the expressions of NKG2D ligands MICA/B and ULBP3. Thus, Manumycin present a promising novel therapeutic approach for AML therapy especially when resistant to chemotherapy and NK cell immunotherapy.

Disclosures:

No relevant conflicts of interest to declare.
A Concept for Long-Term Anticoagulation Based On the Individual Risk of Lethal Pulmonary Embolism (PE) and Lethal Bleeding Using Oral Anticoagulants.
Authors: Zotz Rainer B. , Sucker Christoph , Gerhardt Andrea
Published: October 2019

Abstract 4174

Objectives

Indications for an indefinite oral anticoagulation are a matter of debate in patients with a first unprovoked proximal DVT.

Methods

An individual risk-benefit analysis is made using published prospective studies by determining the patient-specific lethal risk of bleeding under oral anticoagulation compared with the estimation of lethal PE-risk by type of initial thrombosis (spontaneous vs. secondarily caused, with or without PE).

Results

According to this risk-benefit analysis, long-term oral anticoagulation is indicated to prevent lethal PE in all patients with low risk of bleeding (1% per year, 0.1 % lethal bleeding per year) in the risk group with lethal PE > 0.2 % per year. This risk group includes patients with idiopathic proximal thrombosis and PE in the initial event (also without thrombophilic risk factors) and patients with an idiopathic initial event without PE, who have relevant thrombophilic risk factors with a relative risk ≥ 2, such as antithrombin deficiency, homozygous factor V Leiden or a combined heterozygous factor V Leiden and prothrombin G20210A mutation. In case of a higher bleeding tendency (0.3% lethal bleeding per year in a patient group with 1-2 bleeding risk factors like age >65 or diabetes) other risk-benefit estimations are present.

Conclusions

Our individual risk stratification is in contrast to current therapy recommendations, which generally consider long-term oral anticoagulation for patients with an idiopathic initial proximal DVT with a low bleeding risk, but do not specify these in individual cases.

Disclosures:

No relevant conflicts of interest to declare.
Thrombin Generation Assessment in Platelet Rich Plasma Offers Additional Criteria for Low Molecular Weight Heparin Antithrombotic Fingerprint Characterization.
Authors: Gerotziafas Grigorios T. , Vasso Galea , Walenga Jeanine M. , Kalodiki Evi , Chaari Mourad , Mohamed Hatmi , Fareed Jawed , Ismail Elalamy
Published: October 2019

Abstract 4177

Introduction

National and international organizations recognize that low molecular weight heparins (LMWHs) are distinct entities and that they should not be used interchangeably in clinical practice. Physicochemical properties, such as molecular weight anti-Xa/anti-IIa ratios, are used by health authorities to establish LMWHs differentiation. LMWHs are multitargeted drugs in which small differences in physicochemical properties may lead to significantly different inhibition of blood coagulation. In the present in vitro study we have investigated if thrombin generation assay, reflecting the overall coagulation process, is a suitable tool for the LMWH variability evalution.

Materials and methods

LMWHs (bemiparin, enoxaparin, nadroparin, dalteparin and tinzaparin) and unfractionated heparin (UFH) were added in vitro in normal citrated platelet rich plasma from 10 healthy individuals, at clinically relevant concentrations (ranging from 0.2 to 1 anti-Xa IU/ml). Thrombin generation was studied with Thrombogram-Thrombinoscope® assay using diluted thromboplastin (Innovin®, 1/1000 final dilution, Siemens France). The concentrations doubling the lag-time, the time to Peak, the IC50 for Peak, the endogenous thrombin potential (ETP) and the mean rate index of propagation phase (MRI) were determined for each compound.

Results

LMWHs compared on their anti-Xa activity basis showed variable inhibitory effect on thrombin generation. At equivalent anti-Xa concentrations tinzaparin was significantly more potent than the other LMWHs, being almost similar to UFH profile. Enoxaparin, nadroparin and dalteparin showed a similar inhibitory activity. Bemiparin had the lesser pronounced inhibitory effect on thrombin generation. The impact of anti-Xa and anti-IIa activities on each phase of thrombin generation process is different. Thrombogram chronometric parameters are mainly influenced by the anti-IIa activity. Similarly, ETP inhibition depends more on anti-IIa rather than anti-Xa activity. The MRI of the propagation phase is more sensitive to the anti-Xa activity of LMWHs.

Conclusion

Thrombin generation assessment in platelet rich plasma allows to evaluate the anticoagulant fingerprint of LMWHs and to differentiate them on global and functional criteria. Each LMWH has a particular inhibitory impact on each phase of thrombin generation process. These functional criteria need to be standardized and probably required for a better characterization of LMWHs heterogeneity by health authorities. They could be used also to evaluate bioequivalence of generic and original LMWHs.

Disclosures:

No relevant conflicts of interest to declare.
Higher Prevalence of Heparin-Induced Thrombocytopenia Antibodies in Asian Indian Population: Is This Due to Contaminated Heparin?
Authors: Kannan Meganathan , Ahmad Firdaus , Saxena Renu , Fareed Jawed
Published: October 2019

Abstract 4182

Heparin treatment may result in an adverse effect because of the formation of antibodies against heparin-platelet factor 4 (H-PF4) complexes, which in turn leads to platelet/endothelial cell activation followed by heparin-induced thrombocytopenia (HIT). Several recent publications have reported a higher incidence of H-PF4 antibodies in patients treated with contaminated heparin. The main contaminant in recalled heparin was identified to be oversulfated chondroitin sulfate (OSCS), the same compound known to have caused several deaths due to generation of bradykinin, a potent vasoactive mediator, and generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. It is known that the quality-control measures in the European community and the United States are stringent; such control measures are not enforced in other countries. In our experience, that was performed prior to the global recall of contaminated heparins and the implementation of new analytical procedures, 11 of 47 cardiovascular surgery patients who have undergone heparin therapy were H-PF4 antibody positive by serotonin release assay. Of the 11 patients who showed positive H-PF4 antibody, only 5 of whom had thrombocytopenia and the remaining 6 patients did not show thrombocytopenia or any adverse effects. Even though a greater prevalence of H-PF4 antibody has also been reported with the bovine heparin preparations, the increase in prevalence of these antibodies may also be due to the quality of heparin used. Our subsequent study in another fifty cardiovascular surgery patients, who underwent heparin therapy, did not show any positive H-PF4 antibody. This demonstrates that the quality of heparin used may play a role in the generation of H-PF4 antibodies. Published reports suggest that the hypersulfated glycosaminoglycans such as the pentosan polysulfate also produces a much higher incidence of H-PF4 antibodies. Several other contaminants, such as the oversulfated derivatives of dermatan sulfate, heparin sulfate and heparin, may also be present in substandard heparin preparations. Thus, the higher generation of H-PF4 antibodies in these patients observed may be due to the heparin used; and heparin needs to be re-evaluated and subject to quality-control measures when a batch of heparin shows higher prevalence of H-PF4 antibodies. Also, to confirm true HIT in heparin treated patients, it is important to identify the functional forms of these H-PF4 antibodies.

Disclosures:

No relevant conflicts of interest to declare.
Effect of the Erythrocyte Apheresis On Immunological Parameters in Sickle Cell Anemia Patients in Crisis Period.
Authors: Kilinc Yurdanur , Tekinturhan Ferda , Guvenc Birol , Burgut Refik , Sasmaz Ilgen , Antmen Bulent
Published: October 2019

Abstract 4191

PURPOSE:

Erythrocyte apheresis is an effective therapy in the acute and chronic treatment of sickle cell anemia (SCA). The main goal of erythrocyte apheresis is to improve blood perfusion through reducing Hemoglobin S (HbS) to < %30 and keeping hemoglobin (Hb) at desired level. The aim of this study was to evaluate the effect of the erythrocyte apheresis on immunological parameters in SCA patients with crisis.

METHODS

Between February 2009 and June 2009, 37 patients (20 female / 17 male) received 44 apheresis treatments at Hemapheresis Unit of Cukurova University School of Medicine, Adana, Turkey. The median age was 22 (Range, 4-56) years and the mean body weight was 51.9±18.6 kilograms. The apheresis procedures were carried out using a Cobe Spectra Cell Separator. A mean of 1.77 body volume calculated red blood cell volumes were exchanged with a mean duration of 125.9±34.9 minutes (Table 1). Sickling negative and leukoreduced packed red cells were used for apheresis treatments. Hemoglobin electrophoreses, complete blood counts, immunoglobins, specific surface markers for T, B and natural killer (NK) cells were performed before and after each procedure. While three patients had a SCA-induced functional asplenia, none of the patients was HIV-seropositive.

RESULTS

The erythrocyte apheresis resulted in a decrease on white blood cell (WBC) counts as expected. Average pre and post apheresis WBC counts were 12883±5775/mm3 and 8506±3331/mm3 (p<0.001) respectively. Accordingly, a decrease in post procedure lymphocyte, monocyte and granulocyte counts was observed. Because small amounts of patient's plasma were removed with red blood cells during apheresis procedures, serum levels of immuglobulins were also decreased. CD3, CD4 and CD8 for T cells, CD11b for monocytes, CD20 for B cells, CD56 as a NK marker and CD45 for leukocytes were analyzed and all of surface markers showed an increase after erythrocyte apheresis. The average pre apheresis HbS level was 78.90±19.20%, whereas the mean post apheresis HbS level was found to be 23.85±13.27% (p<0.001) (Table 2).

CONCLUSIONS

The erythrocyte apheresis is an effective and rapid procedure to reduce HbS concentration without increasing blood viscosity. Apheresis treatments have also been found to be beneficial in decreasing the leukocyte counts and serum immunoglobulins levels in the blood. Flow cytometric analyses revealed that T, B and NK cells were increased after apheresis treatment. Interestingly, the change in CD4/CD8 ratio was not statistically significant (p>0.05). This shows that vascular wall integrity is maintained and apheresis can be safely performed in SCA patients. In our study, blood perfusion was restored and blood chemistry was improved to optimal levels after erythrocyte apheresis. As a result, crisis periods were shortened.

Disclosures:

No relevant conflicts of interest to declare.
Molecular Basis for A Thrombophilic Patient's Combined Deficiencies in Proteins C and S.
Authors: Smith Tyler W , Carter Isis S. R , Carter Cedric John , MacGillivray Ross T.A
Published: October 2019

Abstract 4198

Background/objectives

Proteins C and S (PC and PS) are vitamin K-dependent plasma proteins with anticoagulant properties. Protein S functions as a non-enzymatic cofactor for activated protein C (APC). APC proteolytically degrades coagulation factors Va and VIIIa, thereby diminishing the activities of the prothrombinase and tenase complexes, respectively. The human PC gene (PROC) is located on the long arm of chromosome 2 (2q13-q14) and contains 9 exons which code for 461 amino acid residues. The human PS (PROS1) gene resides on chromosome 3 (3p11.1-q11.2) and contains 15 exons coding for 636 amino acid residues. Hereditary PS and PC deficiencies are both autosomal dominant disorders in which patients have diminished functional levels of the respective protein (usually ∼50% relative to normal controls). Clinically, this results in increased propensity toward thromboembolic disease, also known as thrombophilia. In this study, we describe a unique thrombophilic patient who has combined deficiencies in both proteins C and S. The objective of the study was to elucidate the precise genetic defect(s) causing these deficiencies.

Methods

Following purification of the patient's DNA from peripheral blood leukocytes, PCR amplifications were performed using oligonucleotide primers flanking all exons of the PROS1 and PROC genes. In addition, the 400bp region upstream of the first exon of PROS1 (corresponding to the promoter region) was also amplified by PCR. The PCR products were purified and their DNA sequences determined in both forward and reverse directions using the dye-terminator method. The resultant nucleotide sequences were compared with the PROS1 and PROC reference sequences [web address].

Results

The patient was found to be heterozygous for a novel missense PROC gene mutation in exon 8, which codes in part for the proteolytic domain of protein C. The resulting ValàGly substitution of residue 221 is in close proximity to the catalytic triad, which could abrogate its enzymatic activity. Although no mutations were present in any of the patient's PROS1 exons, there was a novel heterozygous CàG nucleotide substitution in the PROS1 promoter region. This substitution is present within an Sp1 transcription factor binding site that is highly conserved among mammals. Therefore, this mutation could significantly diminish expression of the otherwise normal PROS1 gene, leading to the observed decreased protein S level. Both mutations are unreported in the literature and are not listed in the PROC and PROS1 mutation databases.

Conclusions

We have successfully identified two novel genetic defects leading to deficiencies of the anticoagulant proteins C and S in a patient with thrombophilia. Further studies are underway to confirm the suggested biochemical effects of the mutation on protein C and protein S gene expression.

Disclosures:

No relevant conflicts of interest to declare.
Antithymocyte Rabbit Globulin Is Safe and Effective in Children with Severe Aplastic Anemia Report Polish Pediatric Hematology Group (Warszawa, Krakow, Wroclaw, Bydgoszcz, Poznan, Lodz, Bialystok, Szczecin, Gdansk).
Authors: Pawelec Katarzyna , Matysiak Michal , Salamonowicz Malgorzata , Balwierz Walentyna , Zaleska-Czepko Ewa , Chybicka Alicja , Szmyd Krzysztof , Sonta-Jakimczyk Danuta , Bubala Halina , Wysocki Mariusz , Kurylak Andrzej , Wachowiak Jacek , Grund Grzegorz , Mlynarski Wojciech , Bulas Monika , Krawczuk-Rybak Maryna , Leszczynska Elzbieta , Urasinski Tomasz , Peregud-Pogorzelski Jaroslaw , Kaczorowska Barbara , Balcerska Anna
Published: October 2019

Abstract 4220

Bone marrow transplantation (BMT) is a therapy of choice in children with severe aplastic anemia (SAA), but it is possible only in about 15-20% patients who have family donors, so the majority of them received the immunosuppressive therapy (IST).

We want to present the results of IST obtained in 116 children treated between 1993-2008 years at 11 pediatric hematology centers of the Polish Pediatric Hematology Group (PPHG),, 55 patients (22 girls and 33 boys aged 0,6-17,5 years) received antithymocyte rabbit globulin (ATG) as a first course of therapy. ATG was administered according to PPHG and Working Party SAA Group of EBMT protocol: ATG 3.75mg/kg iv for 5 days, cyclosporin A (CSA) 5mg/kg orally from day 1 to day 180 and granulocyte-stimulating factor during deep neutropenia.

Remission of the disease was assessed on days 112, 180 and 360 from start of therapy on the basis of blood and bone marrow examination. The results on 112 and 180 were similar. The remission was achieved in 28 of 55 children (51%), complete remission (CR) in 5 children (9%), and partial remission (PR) in 23 children (42%). On day 360 remission was obtained in 30 of 55 patients (42 %), CR in 10 children (18%) and PR in 20 children (36%) There was no response to treatment (NR) in 25 patients (45%).

In the group of 55 patients, 15 died (27%). 5 of them died early (day 52 and day 72 of therapy) due to septicemia and central nervous system (CNS) hemorrhage. The other 10 died late (day 99 and day 360 of therapy) due to CNS hemorrhage. Observation time of patients ranged from 1 to 14 years. During this time we noted four relapse. PNH was observed in one patient. The probability of 14 year survival in our group of patients treated initially with rabbit ATG is 72,7%. The results in the last follow up (Jun 2009) are better then earlier. We observed CR in 23/55 children (42%), PR in 10/55 (18%), NR 18/55 (33%). o information about 4 patients.

We conclude that initial treatment with rabbit ATG is safe and effective in children. Further studies are needed, to assess long-term effectiveness of rabbit ATG in IST.

Disclosures:

No relevant conflicts of interest to declare.
A Randomized Trial Evaluating a New Powered Bone Marrow Aspirate and Core Biopsy System Compared to Traditional Manual Devices.
Authors: Berenson James R. , Donahue David J. , Yellin Ori , Upadhyaya Gargi , Nassir Youram , Swift Regina A.
Published: October 2019

Abstract 4234

Traditional bone marrow procedures using a manual device hand-inserted by the clinician may be associated with pain, bleeding and inability to obtain adequate specimens leading to multiple attempts and a prolonged time to complete the procedure. These procedures take from approximately 7 minutes to more than 30 minutes to collect adequate aspirate and core biopsy samples. Notably, it has been shown that the duration of needle insertion is the sole predictor of patient pain during the procedure. In an attempt to overcome these shortcomings of the traditional technique, the OnControl bone marrow aspiration and biopsy system has recently been developed. It is an FDA-cleared device, comprised of the driver, a metal needle set and a connector. The driver is a small battery-powered device for insertion of a single lumen needle into the intraosseous space of the adult iliac crest. This device allows for a minimally-invasive procedure that in a 2007 study showed a substantially faster time for needle insertion into the medullary space of the iliac crest with a mean insertion time of 5 seconds. In an interim analysis of a single-arm validation study for the OnControl system, 36 patient procedures were completed with a mean user satisfaction score of 4.6 (on a scale of 0-5, with 5 indicating high satisfaction), a biopsy core acquisition success rate of 92%, a mean length of biopsy core of 1.22 cm, and a mean time from needle insertion to core extraction of 2 minutes and 9 seconds. Thus, we are currently enrolling patients on a multicenter, prospective, randomized controlled trial in order to directly compare results of using this new powered core biopsy needle to traditional manually inserted needles in terms of patient level of pain, time from needle insertion to removal, insertion success rate, core biopsy capture rate, quality of yield of this device, rate of complications, and operator satisfaction with the device. Patients requiring conscious sedation/narcotics for pain relief or extra bone marrow for research purposes/additional tests beyond that required for routine clinical evaluation will be excluded. Block randomization with blocks of size 4 will be used to prevent unequal treatment-group sizes. Each patient is being randomized to undergo a bone marrow procedure with the standard manual device or the powered device. Patient pain is being assessed using the Visual Analog Scale of 0 to 10; with 0 = no pain and 10 = extreme pain. The pain level will be assessed immediately following needle removal and at days 2 and 8 following the procedure. We plan to enroll 102 patients in this study and expect to have all patients enrolled in the next 3 months. Updated data as part of an interim analysis of this randomized trial will be presented at the time of the ASH meeting.

Disclosures:

Berenson: Vidacare, Inc.: Research Funding.
Harvesting of Peripheral Blood Stem Cells Using Plerixafor (MOZOBIL) and Granulocyte-Colony Stimulating Factor in Patients Who Had Never Undergone Stem Cell Collection.
Authors: Teoh Gerrard , Goh Keith
Published: October 2019

Abstract 4236

Introduction

The collection of peripheral blood stem cells (PBSC) can be tedious and expensive. Moreover, PBSC yields can be severely compromised in patients who previously received chemotherapy. The use of plerixafor (MOZOBIL) for PBSC mobilization has been shown to significantly improve CD34 stem cell yields in patients whom PBSC collections have been adequate. However, this requires the patient to undergo further PBSC harvesting sessions; adding to the patient's inconvenience, exposure to adverse events and costs. We therefore investigated whether using plerixafor at the outset of PBSC mobilization would improve patient acceptability; reduce PBSC collection days and accordingly the patient's costs.

Patients and Methods

Three adult patients who had never undergone PBSC harvesting were mobilized using subcutaneous (SC) granulocyte-colony stimulating factor (G-CSF) 10 μg/kg daily starting 4 days prior to PBSC harvesting; plus SC plerixafor 240 μg/kg 10 hours prior to PBSC harvesting. Both G-CSF and plerixafor were continued for as long as was required for PBSC collections. Two patients had multiple myeloma (MM) and were both in stringent complete remission (sCR). The third patient suffered from adult-onset spinomuscular atrophy (SMA) and stem cells were required for possible re-infusion therapy. Importantly, the bone marrows of all 3 patients were assessed to be normal prior to PBSC mobilization and collection. The target PBSC CD34 yield was 2 million CD34 cells/kg per PBSC collection.

Results

As can be seen, the CD34 yields (expressed as x million CD34 cells/kg) exceeded expectations:

All patients tolerated the procedure very well and were discharged within 24 hrs of completing the final PBSC collection. The only adverse event was grade 1 restless legs syndrome in Patient #1 following the first injection of plerixafor. It was improved with oral diazepam and did not recur. Both patients with MM reported a sense of “rejuvenation” 2-3 weeks after PBSC collection. All patients were pleased with the relatively complication-free procedure.

Conclusions

Upfront collection of PBSCs using G-CSF/plerixafor is both highly effective and safe. There is great patient acceptability as well as much fewer PBSC collection days and an accompanying reduction in procedure costs. The added advantage of using the SPECTRA Optia cell separator is worth further investigations.

Disclosures:

Off Label Use: Plerixafor (MOZOBIL) - Use in patients who had never undergone stem cell collection..
A High Resolution Comparative Genomic Hybridization Array of Adult T-Cell Leukemia-Lymphoma in Individuals of African Descent.
Authors: de Lima Rosangela Gomes , Xu Suying , Hedges Dale , Brites Carlos , Barber Glen N. , Fan Yao-Shan , Ramos Juan Carlos
Published: October 2019

Abstract 4241

Adult T-cell leukaemia/lymphoma (ATLL) is a highly aggressive malignancy with a poor prognosis caused by the human T-cell lymphotropic virus type-1 (HTLV-I). While no specific chromosome or genetic abnormalities have been clearly proven to contribute to the pathogenesis of ATLL, recent comparative genomic hybridization (CGH) studies have demonstrated frequent genetic lesions (gains and losses) involving specific chromosomal regions containing a number of genes of potential cancer relevance. In this study, using a high resolution Agilent platform, we performed a comprehensive CGH analysis of 60 ATLL DNA specimens obtained from individuals of African descent from the United States, the Caribbean, and Brazil. Our preliminary results demonstrate the presence of some alterations already reported by others, such as gains in chromosomes 1q22, 4p16, 7p22, 8q24, 9q33-34, 14q31-q32, 16q16, 18q23, 19p13 and deletions at 6q14, 9p21 and q21 and 14q11. Additionally, we have observed several genetic alterations not previously reported in a significant percentage of our ATLL tumors, such as specific gains in regions of chromosomes 1q, 2q, 5p, and 17p and q, and deletions in 13q and 15q. Most of these regions harbor potential cancer related genes but some contain genes of unknown function. Our preliminary results confirm there are common genetic alterations between ATLL patients of Japanese and African origin, but also distinct genetic lesions between both ethnic groups perhaps contributing to the pathogenesis of ATLL. A more comprehensive analysis including validated findings will be presented at the meeting.

Disclosures:

No relevant conflicts of interest to declare.
A Novel Recurrent Chromosomal Inversion, Inversion(9)(p21q34), in Ph-Positive Leukemia Patients.
Authors: Pan Jinlan , Zhang Jun , Xue Yongquan , Wu Yafang , Shen Juan , Wang Yong , Liu Dandan , Chen Suning
Published: October 2019

Abstract 4242

A pericentric inversion(9)(p21q34) was identified in five Ph-positive leukemia patients including four patients with chronic myeloid leukemia(CML) in chronic phase(CP) and one patient with acute myeloid leukemia (AML) since 1998. Among them, two were males, three wrer females. The median age is 31 years (range 21-52 years). Conventional karyotypic analysis with R-banding technique showed that the inv(9)(p21q34) always occurred in the der(9)t(9;22)(q34;q11) and was accompanied by the der(22)t(9;22)(q34;q11) in all metaphases analyzed in four patients with CML-CP at diagnosis. One patient with AML presented three clones: one with normal karyotype, one with sole t(9;22)(q34;q11), one with inv(9)(p21q34) involving the der(9)t(9;22) and additional t(8;12)(q12;p11). FISH using LSI BCR/ABL dual-color, dual fusion probe, chromosome painting(CP) with the paint probes for chromosome 9p and 9q and RT-PCR using the primers of the BCR/ABL fusion genes were performed in four of them. FISH showed the coexistence of clone with sole t(9;22) and another with inv(9)(p11q34) and t(9;22) in four patients in whom, one patient also showed a deletion of partial sequence from BCR on der(9)t(9;22)inv(9)(p21q34). RT-PCR revealed a b3a2 transcript for BCR/ABL fusion gene transcript. FISH proved that the inv(9)(p21q34) disrupted the ABL/BCR fusion gene at the molecular level. However, it does not appear to have any biological significance because BCR/ABL fusion gene is thought to be involved in the pathogenesis of CML, while ABL/BCR fusion gene is merely mechanical consequence of the t(9;22) translocation. As far as we know, inv(9)(p21q34) has not been reperted previously. Thus, it should be regarded as a novel rare but recurrent secondary chromosomal anomaly. In this series, one lost to follow-up, one patient transformed into B cell acute lymphocytic leukemia, who and other two patients survivals of 28 days, 13 and 34 months, respectively. Only one remains alive. Their dismal outcome probably suggests inv(9)(p21q34) having unfavourable impact on prognosis. At present, no firm conclusion can be drawn from this study. More patients with this anomaly need to be investigated to elucidate its prognostic significance.

Disclosures:

No relevant conflicts of interest to declare.