bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical Relevance of Ubiquitin-Proteasome System Profiling in Acute Leukemias.
Authors: Ma Wanlong , Kantarjian Hagop M. , Zhang XI , Wang Xiuqiang , Estrov Zeev , O'Brien Susan , Albitar Maher
Published: October 2019

Abstract 2633

Poster Board II-609

The ubiquitin-proteasome system (UPS) plays a major role in the homeostasis of cellular proteins: chymotrypsin-like (Ch-L), caspase-like (Cas-L), and trypsin-like (Tr-L). The UPS system is involved in most critical cellular processes and its role in oncogenesis is well established. Here we compared UPS protein levels (proteasome and ubiquitin) and proteasome enzymatic activities in peripheral blood plasma from newly diagnosed patients with acute myeloid leukemia (AML; n=147), acute lymphoblastic leukemia (ALL; n=34?), or myelodysplastic syndrome (MDS ; n=27), and 99 apparently healthy control subjects. Proteasome and ubiquitin were measured using immunoassays based on electro-chemiluminescence technology. The proteasome enzymatic activities were measure using a standard enzymatic assay utilizing fluorogenic peptide-AMC substrate. By normalizing these enzymatic activities to the levels of proteasome protein in plasma, we also determined specific proteasome enzyme activities (Ch-L/p, Cas-L/p, and Tr-L, respectively). AML, ALL, and MDS patients all had high levels of proteasome protein, ubiquitin, and enzymatic activities relative to control subjects. However, each specific enzyme activity in general was lower in all 3 disease groups than in control subjects, which suggests that each proteasome has lower enzymatic activity in blast cells but we cannot rule out that the number of proteasomes might be increased. Despite these similarities, AML, ALL, and MDS each exhibited a specific profile of UPS protein and enzymatic activities . Proteasome protein levels and enzymatic activities also correlated with clinical behavior. In AML, proteasome protein level was a strong predictor of survival as a both a continuous (P<0.00001) and a dichotomous (P=0.04) variable, independent of cytogenetics, performance status, and age. In ALL, Ch-L/p showed a significant negative correlation with survival (P=0.0015). In conclusion, these data demonstrate that each disease has a unique UPS profile and confirm that the UPS system plays a major role in the leukemic process. In addition, profiling the UPS in leukemias using plasma provides valuable biomarkers that can be used to help predict clinical behavior and may ultimately help manage disease.

Disclosures:

No relevant conflicts of interest to declare.
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 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 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 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 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 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 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 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 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 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.
Therapy for Chronic ITP in Germany - A Patient Survey.
Authors: Matzdorff Axel C. , Arnold Gabriele , Salama Abdulgabar , Ostermann Helmut , Hummler Simone
Published: October 2019

Abstract 4471

Background

Guidelines recommend glucocorticoids and splenectomy as standard 1st and 2nd line treatments for chronic ITP. We sought to find out how German ITP-patients are treated in respect of these guidelines.

Methods

Members of a patient support association >18 y with self-reported history of chronic ITP (>6 mo) were surveyed. A questionnaire was developed from literature review with clinician and patient input, and administered on-line.

Results

123 questionnaires were evaluated. Age (median 51 years) and gender distribution (38% m, 62% f) are comparable to surveys from other countries. 70% of patients had chronic ITP for more than 5 years and 50% a “usual” platelet count of < 50.000/μl (20% < 30.000/μl). 69% had hematomas or petechiae within the last 12 months, 45% had oropharyngeal bleeds, and 11% had been admitted to a hospital within this year. 88% had received or receive glucocorticoids, 28% were splenectomized. IVIg was given to 55%, rituximab to 22%, anti-D to 11%, cyclosporine to 7%. Complementary and alternative medical treatments had been used by 36%. 38 women were under the age of 50 and 14 (36%) reported that they had been advised not to become pregnant. 23 became pregnant and 10 (44%) required ITP-treatment during their pregnancy.

Conclusion

Glucocorticoids are the most common therapy for chronic ITP but complementary and alternative treatments already come second and less than 1/3 of the patients are splenectomized. This and the frequent use of complementary medicines suggests dissatisfaction with conventional therapeutic approaches. Many patients receive off-label therapies (rituximab, anti-D, cyclosporine are not licensed for ITP in Germany). There is a major need for adequate counseling and care for pregnant ITP-patients.

Disclosures:

Matzdorff: GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AMGEN: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Baxter: Consultancy, Honoraria. Off Label Use: Rituximab for chronic ITP Complementary medicines for ITP. Hummler:GlaxoSmithKline: Employment.
Short-Term Inhibition of p53 Combined with Keratinocyte Growth Factor Improves Thymic Epithelial Cell Recovery and Enhances T-Cell Reconstitution After Murine Bone Marrow Transplantation.
Authors: Kelly Ryan , Goren Emily , Taylor Patricia , Scott Hamish S , Komarova Elena , Gudkov Andrei , Hollander Georg A. , Blazar Bruce R.
Published: October 2019

Abstract 4485

Myeloablative conditioning prior to bone marrow transplantation (BMT) results in thymic epithelial cell (TEC) injury, T cell immune deficiency, and susceptibility to opportunistic infections. Conditioning regimen-induced TEC damage directly contributes to slow thymopoietic recovery following BMT. Keratinocyte growth factor (KGF) is a TEC mitogen that stimulates proliferation and, when given prior to conditioning, reduces TEC injury. Some TEC subsets are refractory to KGF and functional T-cell responses are not fully restored in KGF-treated BMT recipients. Therefore, we investigated whether the addition of a pharmacological inhibitor, PFT-β to transiently inhibit p53 during radiotherapy could spare TEC from radiation-induced damage in congenic and allogeneic BMT. The combined pre-BMT administration of KGF plus PFT-β additively restored numbers of cortical and medullary TEC and improved thymic function post-BMT, resulting in higher numbers of donor-derived, naïve peripheral CD4+ and CD8+ T-cells. Improved T-cell reconstitution correlated with enhanced recovery of fibroblastic reticular cells in the lymph node stromal compartment, and a superior immune response against L. monocytogenes infection. Thus, transient p53-inhibition combined with KGF represents a novel and potentially translatable approach to promote rapid and durable thymic and peripheral T-cell recovery following BMT.

Disclosures:

No relevant conflicts of interest to declare.
Synthetic Retinoid Am80 Ameliorates Chronic Graft-Versus-Host Disease.
Authors: Nishimori Hisakazu , Sugiyama Haruko , Kobayashi Koichiro , Yamasuji Yoshiko , Kondo Eisei , Shinagawa Katsuji , Tanimoto Mitsune , Takeuchi Kengo , Maeda Yoshinobu
Published: October 2019

Abstract 4487

Chronic graft-versus-host disease (cGVHD) remains a major cause of late death and morbidity after allogeneic hematopoietic cell transplantation, and the treatment of cGVHD remains challenging. All-trans retinoic acid (ATRA), a potent vitamin A derivative, can regulate immune responses. Synthetic retinoid Am80, which shows biological activity approximately 10 times more potent than that of ATRA by binding to RARα and RARβ, but not RARγ, also reduces the severity and progression of immune disease models, including those for contact dermatitis, collagen-induced arthritis, allergic encephalomyelitis, and atherosclerosis. We hypothesized that the administration of ATRA or Am80 could modulate cGVHD. BALB/c (H-2d) mice were subjected to sublethal irradiation and injected with 8 × 106 T-cell-depleted bone marrow cells and 8 × 106 spleen cells from major histocompatibility complex-matched, multiple minor histocompatibility antigen-mismatched B10.D2 (H-2d) mice. Then, the mice were given oral ATRA (200 μg/body), Am80 (1.0 mg/kg body weight), or vehicle daily, beginning from day 0. ATRA slightly decreased the clinical cGVHD score, whereas Am80 significantly reduced the score (Table). When administered daily, Am80 decreased the clinical score beginning from day 21, as in a treatment setting (data not shown). Histopathological examination of the skin showed significantly reduced GVHD pathology in recipients of Am80 (Table). Flow cytometry analysis of the peripheral draining lymph nodes on day 16 showed significant reductions in IFN-γ+, IL-17+, and Foxp3+ cells in Am80-treated recipients compared to controls (Table), whereas no reduction in IL-13+ cells was observed. Cytometric bead arrays and enzyme-linked immunosorbent assays (ELISA) revealed decreased levels of the cytokines IFN-γ, IL-17, TNF-α, and IL-6 in the supernatant of peripheral lymph nodes from Am80-administered recipients (data not shown). Real-time RT-PCR of ears from these recipients on day 21 showed that the administration of Am80 reduced the expression of Foxp3 and TGF-β (Table and data not shown). Therefore, the administration of retinoids ameliorates cGVHD by reducing Th1 and Th17 inflammatory cytokines and the fibrosis factor TGF-β. Thus, treatment with retinoids may be effective for prophylaxis and treatment of cGVHD.

Disclosures:

No relevant conflicts of interest to declare.
Vav3 and Rac2 Activation Are Required for B Progenitor Transformation Induced by p190-BCR-ABL.
Authors: Chang Kyung-Hee , Sanchez-Aguilera Abel , Sengupta Amitava , Arnett Jorden , Dunn Susan , Williams David A , Cancelas Jose
Published: October 2019

Abstract 449

The Philadelphia chromosome-positive (Ph+) hematologic malignancies are characterized by the reciprocal translocation between the BCR serine/threonine kinase and the ABL tyrosine kinase genes. Fifty percent of adult B-cell Ph+ acute lymphoblastic leukemia (B-ALL) and most cases of pediatric Ph+ B-ALL express the p190 BCR-ABL isoform, which causes abnormal expansion of lymphoid progenitors and a poor prognosis despite the administration of tyrosine kinase inhibitors. A major signaling pathway activated by BCR-ABL is the Rac/Rho GTPase signaling pathway. We had previously demonstrated that p210-BCR-ABL activates Rac GTPases and the deficiency of Rac2, or even more, the deficiency of both Rac1 and Rac2, impairs myeloid leukemogenesis induced by p210 BCR-ABL in vitro and in vivo (Thomas EK et al., Cancer Cell 2007). The p190-BCR-ABL isoform also activates Rac GTPases and the deficiency of Rac2 induces resistance to develop proB-ALL (Sanchez-Aguilera et al., ASH 2008). Since p190-BCR-ABL is devoid of guanine nucleotide exchange factor (GEF) activity through the absence of the BCR Dbl-homology (DH) domain, it probably requires the recruitment of intermediate GEFs to activate Rac GTPases. The Vav family of GEF has been shown to bind the SH3/SH2 domains of ABL and become activated. Three isoforms of Vav (Vav1, Vav2 and Vav3) are expressed in hematopoietic cells. Several studies have indicated that Vav3 has a role distinct from Vav1 or Vav2. Here, we hypothesized that the hematopoietic specific Rho GTPase, Rac2, may mediate key signals in the transformation of B-cell progenitors upon Vav-mediated activation. We have found that both Vav1 and Vav3 are overexpressed and hyperphosphorylated in p190 BCR-ABL expressing human B-ALL cells as well as in BCR-ABL transduced murine proB cells. While the combined deficiency of Vav1 and Vav2 does not impair leukemogenesis in vivo, Vav3 deficiency reverses p190-BCR-ABL induced transformation phenotypes. Specifically, Vav3 deficiency in p190-BCR-ABL expressing proB-ALL cells associated with a significant reduction of Rho and Rac activation (75% and 56%), apoptosis induction (4-fold) and impaired ex-vivo expansion (33% reduction). In addition, p190-BCR-ABL-induced adhesion-to-fibronectin defect and hypermigratory response to CXCL12 were corrected by deletion of Vav3 (2.1-fold increase and 60% decrease, respectively), suggesting that Vav3 deficiency reverses the transformation-associated phenotype associated to BCR-ABL expression. Collectively, our work identifies a critical role of Rac2 and its activator Vav3 for p190 BCR-ABL induced B-cell progenitor leukemic transformation and may define a novel therapeutic target for this poor prognosis disease.

Disclosures:

Cancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.
Improving Tumor Immunotherapy by Using Distinct Subpopulations of the γδT Cell Repertoire with High Anti-Tumor Reactivity.
Authors: van Dorp Suzanne , Hol Samantha , Marcu-Malina Victoria , Thuss Nicole , Lokhorst Henk , Kuball Juergen
Published: October 2019

Abstract 4504

Introduction

Currently innate immune cells such as γ9δ2T cells are explored in tumor immunotherapy e.g. by adoptive transfer of in vitro expanded bulk γ9δ2T cells.

Objective

We speculated that γ9δ2T cells are highly variable in function and specificity due to differences in γ9δ2TCR, NKG2D and KIR expression and that efficacy of adoptively transferred γ9δ2T cells can be increased by transfer of defined subpopulations or clones rather than bulk γ9δ2T cells.

Methods

A variety of γ9δ2T cell clones, derived from a healthy donor, were tested for expression levels of γ9δ2TCR, NKG2D and KIRs by flow cytometry analysis. The sequence of the γ9δ2TCR of different clones was further analyzed. Reactivity of γ9δ2T cell clones to a panel of tumor and normal cell lines was tested and these functional analyses were correlated to receptor expression levels and compared with bulk γ9δ2T cells of the same donor.

Results

Functional analyses revealed a high interclonal variability in recognizing leukemia or solid tumor cell lines. Consequently, γ9δ2T cell clones with high anti-tumor reactivity were superior in killing tumor cells when compared to bulk γ9δ2T cells. Different variable regions of γ9δ2-chains and different expression levels of NKG2D and KIRs were detected in multiple clones. No correlations could be found between TCR, NKG2D, and KIR expression on γ9δ2T cell clones and their response to different tumor cell lines when clones expressed different γ9δ2TCRs. However, analysis of γ9δ2T-cell clones with identical γ9δ2TCRs revealed that a clone with higher reactivity against cancer cells expresses higher amounts of NKG2D and lower inhibitory KIRs when compared to a clone with lower reactivity.

Conclusion

γ9δ2TCR, NKG2D and KIR expression in γ9δ2T cells is highly variable and cannot be directly correlated to an effective anti-tumor response. Only, when T-cells express one defined γ9δ2TCR, a modulating activity of NKG2D and KIRs can be observed. Thus, we conclude that anti-tumor reactivity is fine tuned by all three receptors and thereby we speculate that the γ9δ2TCR defines tumor-specificity and activity is further modulated by NKG2D and KIRs. These results support the application of distinct subpopulations or of genetically engineered γδT cells with defined receptors rather than bulk γ9δ2T cells in the context of anti-tumor immunotherapies.

Disclosures:

No relevant conflicts of interest to declare.
Microvesicle Mediated Genetic Phenotype Modulation.
Authors: Aliotta Jason M , Pereira Mandy , Dooner Mark , Dooner Gerri , Ramratnam Bharat , Lee David , Johnson Kevin , Hixson Douglas , DePaz Nicole , Berz David , Josic Djuro , Brilliant Kate , Quesenberry Peter J.
Published: October 2019

Abstract 4509

Objective

We have previously reported that lung-derived microvesicles (MVs) can enter target marrow cells, resulting in increased levels of lung-specific mRNAs (Stem Cells 25:2245, 2007). Marrow cells which have been exposed to MVs also show increased production of pulmonary epithelial cells after transplantation into irradiated mice. The present studies have addressed the universality of the mRNA modulation and the underlying mechanisms.

Methods/Results

Co-culture of heart, brain, liver, and lung tissue across from murine marrow, but separated by a 0.4 micron cell-impermeable membrane, show tissue specific elevations of mRNA. MVs were found to contain lung-specific mRNA and 200 microRNAs. Proteomic studies of MVs showed up to 75 individual proteins, some of which are known to be associated with MV biogenesis and trafficking. Studies using rat/mouse hybrid cultures demonstrated that the target cell induced lung-specific mRNA elevations were mediated by transcriptional mechanisms. In these experiments, rat lung was co-cultured across from murine marrow cells and RT-PCR was performed using rat or mouse-specific primers for surfactant B. High levels of rat-specific surfactant B were seen in the co-cultured marrow cells indicating that transcription had been induced in the target cells. These conclusions were supported by additional studies employing the transcription factor inhibitors actinomycin-D and alpha-amantin. RNase treatment of conditioned media prior to marrow cell co-culture suggested that transfer of RNA may be involved in these mRNA elevations. However, our transcriptional studies indicate that we are not observing a simple transfer of MV lung-specific mRNA. One possible mechanism may be transfer of microRNA with epigenetic changes resulting in lung-specific mRNA production.

Conclusion

In summary, these observations suggest the existence of unique pathways for information transfer and cell phenotype determination. MV transfer could represent an underlying mechanism for much of the previous reported stem cell plasticity in different tissues.

Disclosures:

No relevant conflicts of interest to declare.
Human Granulocytic Anaplasmosis: a Case Report and Review of Literature.
Authors: Ouyang Jian , Zhang Qiguo , Xu Jingyan , Chen Bing , Zhou Rong-Fu
Published: October 2019

Abstract 4518

Purpose

To discuss the clinical and laboratory features, diagnosis and treatment of human granulocytic anaplasmosis.

Methods

We present the clinical and laboratory features, diagnosis and treatment of a patient with human granulocytic anaplasmosis.

Case presentation

The patient with human granulocytic anaplasmosis presented with fever,cough?Adiarrhea?Amyalgia?Afacies typhosa?Arelative infrequent pulse and swelling of lymph nodes. Laboratory examination showed the patient had leukopenia?Athrombocytopenia?Aproteinuria?Aliver injury?Ablood clotting abnormal?AEBV-DNA positive. We also found the patient's ferritin?Acreatase?Aamylase and lipase increased. In the patient's bone marrow, plasma cells were increased, hemophagocyte and intragranulocytic inclusions were found. The patient did not respond to the treatment of imipenem, cefepime hydrochloride and teicoplanin. But he was treated successfully with moxifloxacin.

Conclusion

Patient with human granulocytic anaplasmosis can present leukopenia, thrombocytopenia, blood clotting abnormal and plasma cells increased in bone marrow. It's quite necessary to make differential diagnosis with some blood diseases. The patient can be accompanied with EBV infection and hemophagocytic syndrome. The patient can be cured by antibiotics-quinolones.

Disclosures:

No relevant conflicts of interest to declare.
Two-Dimensional Speckle Strain for Evaluation of Left Ventricular Systolic Deformation in Children with Thalassemia Major.
Authors: Abd Rab-Rassol Tolba Osama , El-Shanshory Mohamed Ramadan , Hagag Adel Abd El-Halum
Published: October 2019

Abstract 4520

Objective

In β-thalassemia major, Identifying early myocardial insult is important since significant cardiac involvement may predate a decrease in left ventricular systolic functions. The aim of this study is to clarify the value of 2 D-strain (2DS) in early detection of segmental and global left ventricular systolic dysfunction in thalassemic children.

Subjects and Methods

This study included two age-matched groups:Group 1 (n=30) thalassemic children and Group 2 (n=10) healthy control children. They undergo conventional echo-Doppler measures of left ventricular (LV) systolic and diastolic functions. The pulsed tissue Doppler of lateral mitral annulus was recorded. Longitudinal 2D strain in 18 left ventricular segments using 2 dimensional (2DS) speckle-tracking imaging from apical 4, 2, and 3-chamber views, using a Vivid 7 ultrasound system. The images were analyzed using Echopac soft ware.

Results

There was no significant difference among thalassemic patients and controls in LV fraction shortening, peak systolic and diastolic tissue velocities of lateral mitral annulus. But by the 2 D strain study there were a significant multi-segmental affection of the LV mainly the posterior, anterior walls with predilection mainly to the apical segments, also there was a significant affection of the peak global LV systolic strain with a median value (-17.7 versus -20.75; P = 0.008.). There was no significant correlation between all segmental or global LV systolic strain and the serum ferretin.

Conclusion

2DS is a sensitive and accurate tool for early detection of segmental and global cardiac insult in thalassemic children.

Disclosures:

No relevant conflicts of interest to declare.
Clinical Usefulness and Safety of Peripherally-Inserted Central Catethers (PICC) in Hematological Patients.
Authors: Morano Giacomo Salvatore , Mercanti Caterina , Federico Vincenzo , Matturro Angela , Micozzi Alessandra , Latagliata Roberto , Breccia Massimo , Girmenia Corrado , Brunetti Gregorio Antonio , Berneschi Paola , Giampaoletti Massimo , Foà Robin , Alimena Giuliana
Published: October 2019

Abstract 4545

The use of Peripherally-Inserted Central Catheters (PICC) as an alternative to Central Venous Catethers (CVC) is becoming very frequent in different setting of patients. To highlight the role of PICC also in patients with haematological malignancies, we revised our single Institute experience from 11/2008 to 7/2009. On the whole, 33 PICCs (BARD Groshong 4 Fr) were inserted in 32 patients [M/F 11/21, median age 59.9 years, Interquartile Range (IR) 47.1 – 74.7] for a total number of 1979 days. Twelve patients had Acute Myelogenous Leukemia (AML), 3 Acute Lymphoblastic Leukemia (ALL), 6 Non-Hodgkin Lymphoma (NHL), 5 Hodgkin Lymphoma (HD), 6 Myelodysplastic/Myeloproliferative Disorders (MDS/MPD); as to disease phase, 6 patients were at onset, 10 in complete response (3 before consolidation therapy and 7 before autologous peripheral stem-cell transplantation), 5 at disease relapse, 7 in chronic phase with transfusional requirement and 4 in advanced phase. PICC was successfully inserted in all cases with US-guide (in 21 cases via basilica vein, in 11 via brachial vein and in 1 via cephalic vein). At insertion, platelets count was < 50 × 109/l in 17/33 cases (51.5%) while WBC count was < 1.0 × 109/l in 6/33 cases (18.1%). An accidental PICC extraction occurred after 13 days; in addition, there were 2/33 (6.0%) (0,03/1000 gg) thrombophlebitic complications after 15 and 21 days respectively and 6/33 (18.1%)(0.10/1000 gg) infective complications [4 sepsis catheter-related from Staphylococci (3) or Acromobacter (1) and 2 local flogistic infiltration]. On the whole, 17/33 PICCs (51.5%) were removed after a median period of 39 days (IR 17 – 64); the reasons for removal were completion of treatment in 4 patients, death unrelated to the PICC in 6 and catheter-related complications in 7 (5 for infection, 1 for thrombosis and 1 for accidental extraction). The remaining 16/33 PICCs (48.5%) are still in use after a median period of 73 days (IR 30 – 93). In conclusion, PICC seems to be a useful, safe and promising alternative to conventional CVC for many haematological malignancies in a wide spectrum of clinical settings, ranging from intensive chemotherapy (including autotransplant procedure) to chronic management and very advanced phases requiring palliative approach.

Disclosures:

No relevant conflicts of interest to declare.