bluuebunny/crossref_metadata_2025_split huggingface dataset:
5-Aza-2'-Deoxycytidine Has a Biphasic Effect On Ex Vivo Expanded Natural Killer Cells.
Authors: Kopp Lisa M. , Denman Cecele J. , Zhu Shiguo , Lee Dean A. 
Published: October 2019

Abstract
Abstract 2664
Poster Board II-640

Adoptive transfer of activated autologous or allogeneic natural killer (NK) cells has emerged as a safe and potentially efficacious immunotherapy for cancer. Alloreactive HLA haploidentical NK cells in the HSCT setting have been reported to enhance engraftment, reduce GVHD and prevent relapse of leukemia. The main restrictions to developing NK cells for immunotherapy are the limited quantity of cells available for adoptive transfer and the modulation of NK cell receptors and/or NK cell ligands as a recognized mechanism of immune escape in oncogenesis. To address the first hurdle, we have developed an efficient method to expand CD3−CD56+ primary NK cells in vitro using K562 artificial APCs expressing membrane-bound IL-21. Altering the balance of activating and inhibitory signals with immunosensitizing drugs is an attractive approach to addressing the second hurdle of NK cell therapy. DNA methylation inhibitors such as 5-Aza-2'-deoxycytidine and 5-azacytidine are being tested for their activity in AML, and have been shown to sensitize AML blasts to lysis by NK cells. However, there is little information on the direct immunologic effect of these hypomethylating agents on NK cell function, reconstitution, and phenotype.
Using the calcein release assay, we investigated NK cell survival and lytic function against the NK cell-sensitive HLAnull 721.221 cells after exposing expanded NK cells to 5-Aza-2'-deoxycytidine. Human peripheral blood NK cells that had been expanded ex vivo in our laboratory were incubated with a wide range of 5-Aza-2'-deoxycytidine concentrations for 3 days in the presence of 50 IU/mL IL-2. Cell viability was tested every 24 h.
We found that 5-Aza-2'-deoxycytidine at higher concentrations resulted in reduced NK cell viability, but at lower concentrations inhibited NK cell killing. At these lower doses, NK cell inhibition was not associated with loss of viability, resulting in an overall U-shaped dose response curve to 5-Aza-2'-deoxycytidine with maximum inhibition in the 0.3 – 2.5 micromolar range.
This biphasic response to 5-Aza-2'-deoxycytidine matches that previously described for malignant cells in which reduced proliferation at higher concentrations resulted in reduced demethylation inhibition (Qin et al, Clin Cancer Res. 2007 Jul 15;13(14):4225–32). To assess the role of demethylation in NK cells in a more dynamic setting, we are investigating 5-Aza-2'-deoxycytidine effects on NK cell proliferation and phenotype during ex vivo expansion, and assessing NK cell recovery, phenotype, and function in a prospective cross-over trial of patients receiving 5-azacytidine post HSCT.

Disclosures:
No relevant conflicts of interest to declare.
Clinical Scale Generation of Functional Human Natural Killer Cells From Umbilical Cord Blood CD34-Positive Cells for Immunotherapy.
Authors: Spanholtz Jan , Tordoir Marleen , Trilsbeek Carel , Paardekooper Jos , Eissens Diana , van der Meer Arnold , Joosten Irma , Preijers Frank , de Witte T. M. , Schaap Nicolaas , Dolstra Harry 
Published: October 2019

Abstract
Abstract 2666
Poster Board II-642
Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been isolated and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. The use of GBGM culture media in a clinical applicable protocol allows the ex vivo expansion and differentiation of CD34+ cells to more than 4-logs into CD56+CD3- NK cells with very high purity. These ex vivo-generated CD56+ cell products contain NK cell subsets expressing CD94/NKG2A and KIR as well express high levels of activating NKG2D and NCRs. Functional analysis showed that CD56+ cell products containing alloreactive NK cells efficiently kill myeloid leukemia and melanoma tumor cells as well as primary acute myeloid leukemia (AML) cells. We have currently translated the protocol to clinical scale production using a closed cell culture bioprocess. CD34+ selection from frozen UCB samples (n=9) using the CliniMACS device was optimized and the selection resulted in a CD34+ enrichment with a mean number of 2,2 ± 1,6 × 106 cells and a purity of 71 ± 14 % CD34+ cells. Validation experiments using the CliniMACS selected cells showed, that the generation of sufficient numbers of NK cells without contaminating T-cells or B-cells under a closed system process is feasible. The cell cultures using bags show a mean expansion of 1273 ± 506 fold (range 759–1770 fold, which generated 8,6×108 − 1,9×109 NK cells from cord blood-derived CD34+ cells within maximal 6 weeks of culture. The mean purity of the NK cell product was 71 ± 9 % (range 63–80%) of the total cells in the bag cultures. These NK cell products will be used for immunotherapy in elderly AML patients. This study is a phase I dose escalation study in a series of 12 AML patients who have successfully achieved CR (<5% blasts in the bone marrow) after standard intensive chemotherapy.

Disclosures:
Spanholtz: Glycostem Therapeutics: Employment. Tordoir:Glycostem Therapeutics: Employment.
Outcome of Second Line Therapy for Pediatric Patients with Hodgkin Lymphoma Who Relapse Following ABVD Based Therapy.
Authors: Belgaumi Asim , Al-Kofide Amani A , Jamil-Malik Rubina , Joseph Nicey , Khafaga Yasser , Sabbah Rajeh 
Published: October 2019

Abstract
Abstract 2691
Poster Board II-667

Background
Treatment outcomes for patients with newly diagnosed Hodgkin lymphoma have improved significantly. This, however, means that the number of patients relapsing following modern therapy is small and the information regarding their clinical characteristics and treatment outcome is limited.

Methods
Clinical information for patients with HL treated on an ABVD protocol were retrospectively collected. For those patients who suffered relapse or progression, data relating to this event, the treatment received and the outcome were collected and reviewed.

Results
Between May 1990 and December 2006 230 children (<14 years) were treated for HL at our institution. 30 patients suffered treatment failure; 21 relapses and 9 progressions (PD). The median time to failure was 1.1years (0.05–7.29 yrs); 0.58 years for patients who progressed and 1.4 years for relapsing patients. 16 patients maintained their stage at relapse, 9 relapsed at a lower stage and 5 at a higher stage. 24 patients were subsequently treated with chemotherapy alone, one with XRT alone and 4 received CMT. One patient with progressive disease did not receive any salvage therapy. Chemotherapy protocols used included APPE (n=13), COPP (n=6), ESHAP (n=2), MOPP (n=2), COPPABV (n=2), MOPPABV (n=1) and ABVD/COPP (n=1). One patient with a nodular lymphocyte predominance HL progressed as a DLBCL and was treated on a NHL protocol. 10 patients subsequently underwent SCT (9 autologous and one allogeneic). 19/29 patients achieved CR post 2nd line therapy, while 5 each had PR and PD. OS at 6 years is 65.7%. Outcome for patients with PD was significantly worse than for those who relapsed (47.6% v. 73.2%; p=0.048). There was no difference in outcome for those patients who underwent SCT compared with those who did not (63% v. 66.4%; p=NS).

Conclusion
A majority of pediatric patients with HL who relapse following ABVD therapy can be salvaged. Outcome for patients following chemotherapy alone was no different from those who underwent SCT; therefore the necessity for SCT needs to be evaluated based on risk stratification.

Disclosures:
No relevant conflicts of interest to declare.
Epigenetic Inactivation of DBC1 in Acute Myeloid Leukaemia.
Authors: Zhao Chen , Dai Aili , Chen Ling , Sun Xiaoping , Han Xin , Yin C. Cameron , You M. James 
Published: October 2019

Abstract
Abstract 4429
DNA hypermethylation has important implications in the tumorigenesis and prognosis in acute myeloid leukemia (AML). To identify relevant methylated genes in AML, we have compared several expression and methylation profilings. With expression analysis, we identified that TRPC6, DBC1, DCC and SOX9 have decreased expression levels in the most analyzed AML cell lines. Among these candidates, DBC1 (deleted bladder cancer 1), a putative tumor suppressor, drew our attention because it is frequently methylated not only in hematological malignancies, including diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, and acute lymphoblastic leukemia, but also in epithelial cancers. DBC1 may play an important role in the regulation of cell growth and programmed cell death. But the mechanisms of transcriptional control and function role in the hematological malignancies, especially on acute myeloid leukemia, are not well known. In this study, we analyzed the DBC1 expression pattern in 9 AML cell lines with RT-PCR analysis. DBC1 mRNA expression was observed in normal bone-marrow but diminished expression in all of 9 AML cell lines. DBC1 methylation was frequently observed in AML cells (9 of 9, 100%) and inversely correlated with DBC1 mRNA expression in a COBRA analysis (Combined Bisulfite Restriction Analysis). We also detected a frequent methylation of DBC1 in primary AML patient samples (9 of 9, 100%). These findings indicate that DBC1 is frequently silenced by hypermethylation in AML. We are in the process of investigation the functional role of DBC1 in the pathogenesis. In addition, diagnostic and prognostic values of DBC1 in AML are being pursued.* Chen Zhao and Aili Dai contributed equally to the presented work.

Disclosures:
No relevant conflicts of interest to declare.
Presence of a Common Haplotype in the Turkish VWD Patients.
Authors: Berber Ergul , Dagdemir Aslihan , Pehlevan Funda , Akin Mehmet , Kavakli Kaan , Caglayan Hande S 
Published: October 2019

Abstract
Abstract 4435
Von Willebrand Factor (VWF) is the cruical molecule in the initiation of hemostatic response to vascular injury. Quantitative or qualitative defects in VWF are associated with Type 1/Type 3 or Type 2 von Willebrand disease (VWD), respectively. VWF is encoded by the VWF gene composed of 52 exons and located on chromosome 12. Nonfunctional copy of the VWF gene containing a region from exon 22 to exon 34 is located on chromosome 22. Plasma VWF levels show a five-fold range within a normal population and genetic varaiablity at the VWF locus accounts for 30% of this variance. VWF gene exhibits a high variablity in its sequence. More than 150 polymorphisms and more than 200 different mutations have been reported in VWF database. Most of the polymorphisms and mutations are detecetd in the region that is duplicated in the pseudogene. Genetic recombination between the gene and its pseudogene may account for this high variability in the VWF gene. Highly polymorphic structure of the VWF gene suggests that haplotypic interactions may account for the variable expressivity of the gene. In this study we report presence of a common haplotype structure in exon 28 of the VWF gene in the Turkish VWD patients. Patient population includes Type1, Type 3 and Type 2 VWD patients. DNA sequencing analysis of 32 VWD patients revealed presence of a novel c.4453G>C change in exon 28 of the 23 patients. c.4453G>C change results in the p.1485V>L substitution. Individuals having p.1485V>L substitution was also carrier for the previously identified c.4500G>A (p.1500A), 4503C>T (p.1501F), c.4508T>C (p.1503L>P), and c. 4517C>T (p.1506S>L) changes in exon 28. c.4500G>A, 4503C>T changes were reported as polymorphisms and c.4508T>C and c. 4517C>T changes were reported as mutations identified in VWD Type 2A patients in VWF database. To determine whether these alterations represent a haplotype, we are currently analyzing exon 28 of the parents of the carrier patients. In 8 of the 11 patient families analyzed, patients inherited the haplotype from the parents. These preliminary data suggests presence of a common haplotype for the Turkish VWD patients. Future studies with the rest of the patient families and normal population will determine whether this haplotype is a benign haplotype or associated with VWD.

Disclosures:
No relevant conflicts of interest to declare.
New Fibrinogen Substitution (gSer313Arg) with Hypodysfibrinogenemia and the Use of Thromboelastography in Diagnosis of Hypo/Dysfibrinogenemia.
Authors: Chitlur Meera B. , Davis Ryan L , Oliver Jocelyn , Brennen Stephen O 
Published: October 2019

Abstract
Abstract 4437

Introduction
Fibrinogen, also known as factor I, is an integral component of both the coagulation and fibrinolytic pathways. The functions of this 340 kDa glycoprotein include fibrin clot formation, non-substrate thrombin binding and platelet aggregation. It is encoded by 3 genes, FGA, FGB and FGG, clustered on chromosome 4q. Disorders of fibrinogen are extremely rare, and may be qualitative or quantitative, resulting from defects in synthesis, processing, polymerization, crosslinking or clot lysis. Clinical manifestations vary from life threatening bleeding disorders to thromboembolism. We report here the identification of a novel g313Ser®Arg substitution in the gD domain that affects both expression level and fibrinogen function in a 3 year old African American child and her mother with a history of epistaxis and menorrhagia respectively.

Results
Plasma samples showed low Clauss fibrinogen concentrations (0.5 and 0.4 g/L) and extended thrombin clotting times (40 and 39 s) for both the proband and her mother (normal 1.5-4.0 g/L and 18-26 s respectively). Estimation of physical fibrinogen concentrations by HPLC quantitation of fibrinopeptides gave values of 1.2 and 1.5 g/L respectively (normal 1.5-4.0 g/L). While these latter values clearly indicate hypofibrinogenemia, the discrepancy between the activity based and physical concentrations suggest a concomitant functional defect. Thromboelastography (TEG) performed on the child showed a low angle and reduced maximum amplitude indicating a platelet or fibrinogen dysfunction or deficiency. A TEG® platelet mapping‘ study confirmed a significant impairment of fibrinogen function. Examination of purified fibrinogen by SDS-PAGE showed a normal pattern of Aa, Bb and g chains, with an additional minor band in the g region showing increased mobility. Electrospray ionisation mass spectrometry of separated chains again confirmed an abnormality in the g chain with measured masses of 48,399 and 48,400 Da from two separate measurements compared to control values of 48,394 Da (SEM 0.6, n=3). DNA sequencing of FGG revealed a novel heterozygous mutation of AGT to CGT at codon 339 (c.1015A>C). The g313Ser®Arg substitution has not been previously reported and we have named the new variant fibrinogen Detroit II.

Conclusions
The majority of variant chains are not secreted resulting in hypofibrinogenemia. However, a minor population of dysfunctional variant g chains (∼15 % of total g chains) was expressed in plasma fibrinogen causing hypodysfibrinogenemia. TEG was a useful assay for the diagnosis of the hypo/dysfibrinogenemia and should be considered in the evaluation of patients with bleeding symptoms.

Disclosures:
No relevant conflicts of interest to declare.
The Clinical Development of the First Recombinant FVIII From a Human Cell Line.
Authors: Knaub Sigurd , Zozulya Nadezhda 
Published: October 2019

Abstract
Abstract 4439

Objective
In order to provide a new recombinant FVIII to the hemophilia community with potentially improved features including improved VWF binding and reduced immunogenic potential, a recombinant FVIII produced in a human cell line was developed (human-cl rhFVIII). The requirements for registration of a new recombinant FVIII in Europe are specified in a guideline. In addition, any global development plans need to be discussed with the US FDA.

Methods
The clinical development plan of human-cl rhFVIII follows the European guideline and the requirements as discussed with the FDA.
Here we introduce the first recently started clinical trial with human-cl rhFVIII. It is a prospective, randomized, open, cross-over phase II pharmacokinetic and efficacy/safety trial in 20 patients with severe hemophilia A conducted at one center in Russia. In this trial the pharmacokinetic properties of human-cl rhFVIII will be compared with an established rFVIII product. After a wash-out phase of at least 72 hours patients will be randomly assigned to one of the two products and receive a single dose of 50 IU of FVIII/kg B.W.. Thereafter, blood will be collected at pre-specified time points over 48 hours, followed by a wash-out phase after which the second product will be administered. Subsequently, patients will be prophylactically treated for 6 months with human-cl rhFVIII to document the efficacy and safety of the product. At the end of the study, a second PK assessment with human-cl rh FVIII will be conducted. FVIII:C will be measured with validated one-stage and chromogenic assays.
An “Independent Data Monitoring Committee“ will supervise the safety aspects of the trials and will perform an independent adjudication of the hemostatic efficacy assessment.

Results
Seven patients have been enrolled and followed for 2 – 50 exposure days on prophylactic treatment. The data of the first 7 patients indicate similar PK characteristics for human-cl rhFVIII compared to the established rFVIII. Human-cl rhFVIII was well tolerated and no safety issue has been reported yet. There were two breakthrough bleeds in target joints in two patients that were controlled with one dose of human-cl rhFVIII each. Further patients have been enrolled and updated information will be presented accordingly.

Conclusion
The novel human-cl rhFVIII has been successfully introduced to patients and appears to have comparable PK characteristics to a well-established marketed rFVIII and seems to be safe and well tolerated in this limited patient group. A first clinical study with human-cl rhFVIII in the US is planned to be initiated soon.

Disclosures:
Knaub: Octapharma AG: Employment. Zozulya:Octapharma AG: Membership on an entity's Board of Directors or advisory committees.
Recombinant Factor VIIa in the Management of Bleeding Associated with Congenital Factor V Deficiency.
Authors: Dixon Natalia , Smith Anita , Sabio Hernan 
Published: October 2019

Abstract
Abstract 4444

Background
Congenital factor V deficiency is one of the rare bleeding disorders and is identified in approximately 1/1,000,000 persons. Current management relies on the use of plasma preparations to replace factor V. The clinical variability and limited therapeutic options contribute to make congenital factor V deficiency a challenging disorder.

Case report
An 18 years old male was diagnosed with severe congenital factor V deficiency (1% activity) after prolonged bleeding following trauma. He reported a past history consistent with hemarthroses and episodes of soft-tissue bleeding. After receiving a cumulative total of 67 units of plasma during his life he developed respiratory symptoms including dyspnea and chest pain. These symptoms worsened during subsequent infusions. The patient developed persistent bleeding associated with onychocryptosis and a periungual infection which was not responsive to local hemostatic measures. Because of his intolerance of plasma, he was treated with recombinant factor VIIa (rVIIa) at a dose of 50 micrograms/kg resulting in effective hemostasis. Subsequently he underwent a Syme amputation with permanent nail removal and ablation with resection of the tuft of the distal phalanx. During the perioperative period the patient received infusions of rVIIa (50 micrograms/kg) resulting in excellent hemostasis. The patient also experienced a very good response to rVIIa infusions at the same dose for the treatment of an expanding soft-tissue hematoma following accidental trauma to the posterior cervical area. The patient has tolerated the rVIIa infusions without difficulty.

Discussion
We report the successful use of rVIIa to control soft-tissue bleeding and to provide hemostasis during a surgical procedure in a patient with congenital factor V deficiency. Limited previous experience with the use of rVIIa in the management of congenital factor V deficiency includes its use for dental procedures as reported by Altisent (Haemophilia 6:408, 2000) and for synovectomy as reported by Gonzalez-Boullosa (Haemophilia 11:167, 2005). Although a mechanism of action has not been identified, it can be speculated that residual platelet factor V may become more available through rVIIa leading to increased thrombin generation.

Conclusion
rVIIa infusions resulted in improved hemostasis in a patient with severe congenital factor V deficiency.

Disclosures:
Off Label Use: recombinant factor VIIa used to control bleeding in congenital factor V deficiency.
Cytokine Production Profile of CD4+ T Cells From Patients with Immune Thrombocytopenia.
Authors: Zou Shanhua , Li Feng , Wang Weiguang , Cheng Yunfeng 
Published: October 2019

Abstract
Abstract 4460

Introduction
To balance self-tolerance and immunity, the immune system depends on both activation mechanisms and down regulatory mechanisms. CD4+ T cells are important for the regulation of immune responses and they exert their effects through the secretion of cytokines. Immune thrombocytopenia(ITP) is a common hematologic disorder manifested by immune-mediated platelet destruction. ITP in adults often has a persistent course and frequently requires treatment to prevent bleeding. The mechanism of ITP has historically been attributed to platelet autoantibody production and the resultant platelet destruction. However, more recent evidence suggests that cell immunity pathogenesis plays an important role in ITP.

Methods
Real-time PCR assays was used for the quantification of mRNA of cytokines IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, IL-23, IL-17, IFN-γ, TNF-α and TGF-β in CD4+ T cells from adult ITP patients and healthy subjects. The mRNA of T-bet, GATA-3, RORγt, and FOXP3 were also analyzed using Real-time PCR. The plasma concentrations of IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, IL-23, IFN-γ, TNF and IL-17 were determined by enzyme-linked immunosorbent assay at the same time.

Results
The plasma concentrations of IL-2, IL-17 and IFN-γ in CD4+T cells were increased, while levels of IL-4, IL-6, IL-10 and TGF-β were decreased, comparing adult patients with ITP with healthy subjects. Patients with ITP presented significant increased mRNA expression levels on IL-2,IL-17,T-bet and RORγt.

Conclusions
In adult ITP patients, the expression levels of IL-2 and IL-17 were up-regulated, expression levels of TGF-β and IL-10 were down-regulated, suggesting that ITP is a Th1 and Th17 predominant disease although the precise mechanisms await further study.

Disclosures:
No relevant conflicts of interest to declare.