bluuebunny/crossref_metadata_2025 huggingface dataset:
Upregulation of CD274 facilitates ex Vivo Expanded Hematopoietic Stem Cells Overcome the MHC Barrier in Allogeneic Transplantation
Authors: Zhang Chengcheng
Published: October 2019

Abstract 2965

Success of allogeneic transplantation is severely hampered by low levels of donor engraftment and high risks of graft-versus-host disease (GVHD). Transplantation of purified allogeneic blood stem cells (HSCs) diminishes the risk of GVHD, but also results in decreased engraftment. Here we show that ex vivo expanded HSCs efficiently overcame the major histocompatibility complex (MHC) barrier and engrafted allogeneic recipient mice. As measured by limiting dilution analysis, there was a 40-fold increase in the allograft ability of the 8-day-cultured HSCs compared to that of the freshly isolated HSCs. We found that both increased numbers of HSCs and cultured-induced elevation of expression of the immune inhibitor on the surface of HSCs contributed to the enhancement. CD274, an immune inhibitor that is expressed on freshly isolated HSCs, was significantly upregulated on cultured HSCs. The use of CD274-deficient HSCs or HSCs treated with a CD274 neutralizing antibody abrogated the ability of cultured but unexpanded HSCs to cross the MHC barrier. To test whether cultured HSCs can be used to cure genetic diseases in allogeneic recipients, we used ex vivo expanded allogeneic HSCs for transplantation and successfully rescued the lethal phenotype of DNA-PK knock-in mice. Our study suggests that HSCs can be manipulated to regulate their immune privilege and this will likely lead to development of new strategies for successful allogeneic transplantation for human patients.

Disclosures:

No relevant conflicts of interest to declare.
90yttrium Ibritumomab Tiuxetan-BEAM Followed by Autologous Stem Cell Transplantation Significantly Improves Overall Survival After Rituximab Containing Induction Therapy in Patients with High-Risk Aggressive B Cell Non-Hodgkin's Lymphoma
Authors: Wondergem Marielle , de Rooij Madelon , Zijlstra Josee , Visser Otto J , Ossenkoppele Gert , Huijgens Peter C.
Published: October 2019

Abstract 3078

Introduction

Adding rituximab to induction therapy has improved overall survival in patients with relapsed or refractory diffuse large B cell lymphoma (DLBCL) after high dose chemotherapy followed by autologous stem cell transplantation (AuSCT) to 50–60%. However, there is still room for improvement. Addition of 90Yttrium ibritumomab tiuxetan prior to the BEAM conditioning regimen seems feasible and results in promising data with respect to disease free and overall survival in high risk DLBCL patients even when treated with rituximab containing induction therapy. At the VU University Medical center, rituximab was added to (re-)induction therapy starting July 2001. From 2006 we started to add 90Yttrium ibritumomab tiuxetan (Zevalin®) to BEAM (Z-BEAM) in high risk DLBCL patients. In this retrospective analysis we compare outcome of Z-BEAM versus BEAM, both followed by AuSCT.

Patients and methods

All high risk DLBCL patients consolidated with high dose (radio-immuno) chemotherapy and AuSCT in CR or PR after rituximab-containing induction therapy were included. High-risk DLBCL was defined as either relapsed or refractory DLBCL or as histological transformation of indolent NHL. AuSCT was preceded by BEAM conditioning and 90Yttrium ibritumomab tiuxetan (0, 4 mCi/kg, max 32 mCi, starting 2006: Z-BEAM group) or by BEAM only (BEAM group). EFS and OS were estimated using the Kaplan-Meier method and compared using the log rank test.

Results

43 patients received Z-BEAM and 42 patients received BEAM conditioning. Median age was 56 and 52 years respectively. No significant differences in disease characteristics were seen. Median follow up (range) was 15 months (6–54) and 39 months (0–112) respectively. Overall survival was significantly better in the Z-BEAM group compared with the BEAM group (p=0.02) with an estimated 2 year overall survival of 90% vs. 65%. (fig 1.) In the first 2 years of follow up 7 patients in the Z-BEAM group relapsed compared to 11 in the BEAM group, this did not reach significance (p=0.09). Median time to recovery of neutrophils and thrombocytes was not significantly different. Moreover, there was no significant difference in TRM (no TRM in the Z-BEAM group versus 2 patients in the BEAM group). Patients who relapsed, in both groups, were able to receive re-induction chemotherapy and, if indicated, allogeneic SCT without being compromised by decreased bone marrow reserve or non haematological toxicities.

Conclusion

Adding 90Yttrium ibritumomab tiuxetan to the BEAM conditioning regimen preceding AuSCT leads to a significant improvement in overall survival in high risk DLBCL patients, even if they have received rituximab during (re)induction.

Disclosures:

No relevant conflicts of interest to declare.
Relapse of POEMS Following Autologous Stem Cell Transplantation: A Single Center Experience
Authors: D'Souza Anita , Buadi Francis K , Lacy Martha Q , Gertz Morie A , Hayman Suzanne R , Kumar Shaji , Dingli David , Zeldenrust Steven R , Dispenzieri Angela
Published: October 2019

Abstract 3101

The POEMS (polyneuropathy, organomegaly, endocrinopathy, M-protein, skin changes) syndrome is a rare paraneoplastic disorder secondary to a clonal plasma cell process. The most experience with successful outcomes in POEMS with disseminated disease has been with high dose chemotherapy followed by autologous stem cell transplantation (ASCT). However, the risk of relapse of POEMS following an ASCT or the outcome of the patients who relapse is not known. We therefore performed a retrospective review of POEMS patients who underwent ASCT at our center and subsequently relapsed or progressed.

Over the past 12 years, 58 patients with POEMS underwent autologous stem cell transplantation at the Mayo Clinic, Rochester between Mar 18, 1999 and Jun 22, 2011. With a median follow up of 40 months (1–147 months), 12 patients have relapsed. The median time to relapse was 41 months. The 1, 2, and 5 year PFS rates were 98%, 94%, and 75%, respectively (Figure 1), and the 1, 2, and 5 year OS rates were 98%, 98%, and 94%.

Of the 12 patients with documented relapse, 7 had baseline PET/CT scans all of which had FDG avid lesions. By 1 year, the FDG avidity had not resolved in 4 of these patients who then went on to either develop additional lesions or increasing SUV of existing lesions. Of the 12 patients with documented relapse, 8 had baseline VEGF measurements, and all but 1 had normalization by 1 year. The first sign of relapse was a clinical deterioration in 2, a rising M-protein in 3, and a rising plasma VEGF level in 5 patients. All of these patients had new or worsening of existing lesions on PET/CT scan (n= 9) or x-ray imaging (n= 3). The symptoms of clinical relapse included pain at the site of the lytic lesion and generalized pain with swelling. Of the patients who relapsed, 5 were treated with radiation therapy, 4 received lenalidomide or thalidomide, 2 had alkylator based therapy and 1 had bortezomib based therapy. One patient was treated with surgical resection of his hepatic plasmacytoma and 2 patients have been observed without needing definitive treatment as of yet. One patient also received anti-VEGF treatment with bevacizumab unsuccessfully after failing cyclophosphamide therapy.

In this large cohort of POEMS patients treated with high dose chemotherapy and autologous stem cell transplantation in a single center, a relapse rate of 21% was noted. Not every patient who relapsed has been deemed to need further treatment, and most patients could be salvaged with chemotherapy or radiation. Only one patient died from relapsed POEMS following stem cell transplantation.

Disclosures:

No relevant conflicts of interest to declare.
Multicentre Retrospective Analysis of Rituximab Use in the Treatment of Steroid Resistant Autoimmune Haemolytic Anaemia in Ireland,
Authors: Maung Su Wai , O'Keeffe Denis , Leahy Maeve , O'Leary Hilary M , Khan Irfan , Cahill Mary , Galligan Oonagh , Murphy Philip , Krawczyk Janusz , McPherson Suzanne , Jackson J F , Ryan Mary , Hennessy Brian , Burke Aine C , Enright Helen , McHugh Johnny
Published: October 2019

Abstract 3171

Autoimmune haemolytic anaemia (AIHA) is characterised by severe haemolysis due to development of auto antibodies directed against patient's own red cells. Rituximab is a chimeric monoclonal antibody that specifically depletes B cells by targeting CD20 on both immature and mature B lymphocytes We evaluated the use of Rituximab in patients with AIHA who are refractory to conventional Immunosupression in 5 centres in ireland.

Methods:

24 cases of AIHA from five centres in the Republic of Ireland were identified who were treated with Rituximab at the standard dose of 375mg/m2 weekly for four weeks. All patients had received prior therapy with steroids and 50% of patients received two or more courses of Immunosupression. One patient had splenectomy prior to Rituximab. Five patients had an underlying lymphoproliferative disorder and two had a myeloproliferative neoplasm. Response was assessed at 1,3,6,12,36, 48 and 60 months. Medium duration of follow up was 34 months. Response was defined as normalisation of Haemoglobin concentration and haemolytic parameters reticulocyte count, bilirubin and lactate dehydrogenase (LDH).

Overall response to Rituximab was 83.3% at 28 days post treatment. Four patients responded to Rituximab in combination with other Immunosupression such as Azathioprine or low dose steroids. Medium response duration was 11.5 months. Sustained response at 1 year, 2 years and 4 years post treatment were 60%, 55% and 30% respectively. Rituximab was well tolerated without significant complications in the majority of patients. One patient developed neutropenic sepsis. One patient received second course of Rituximab after relapse within a year and had sustained response of over two years after the second course.

Conclusion:

Rituximab is a safe and effective treatment for patients with AIHA who are resistant to steroids.The majority of patients will initially respond but a significant number will gradually relapse over time. Re-treatment with rituximab maybe considered on relapse.

Disclosures:

No relevant conflicts of interest to declare.
The Presence of An Internal Tandem Duplicate of FlT3 Receptor (ITD) Is An Unfavorable Prognostic Factor on the Outcome of Adult Patients with Acute Myeloid Leukemia (AML) Autografted in First Remission (CR1) Only in the Absence of An NPM1 Mutation,
Authors: Gorin Norbert C. , Labopin Myriam , Meloni Giovanna , Pigneux Arnaud , Esteve Jordi , Sanz Miguel A. , Bosi Alberto , Arcese William , Milone Giuseppe , Polge Emmanuelle , Mohty Mohamad
Published: October 2019

Abstract 4139

The prognosis of patients with AML harboring the FlT3 ITD is known to be poor in those patients treated with chemotherapy alone. However, it remains uncertain whether this abnormality has also an impact on outcome following high dose intensification and autologous stem cell transplantation (ASCT).

We addressed this question using the EBMT data base: we initially reported on a total of 134 patients in 2010 (Blood 2010, 3356a). Following further queries, we have now extended our study to a total of 357 patients with AML autografted in CR1 between January 2000 to December 2009, and in whom the information on the presence or the absence of FlT3ITD was available. 258 (136 with a normal karyotype) patients were ITD negative and 99 (63 with a normal karyotype) were ITD positive. 96% of the patients received peripheral blood as a source of stem cells. The median follow up was 30 m. (range, 1–118).

Patients who were ITD positive had higher white cell counts at diagnosis (54 vs. 12.5 109/L; p<0.0001) and had a higher proportion of slow remitters (needing more than one induction course to achieve CR) (22% vs. 11%; p=0.04). Interestingly, patients who were ITD positive were more frequently found to be NPM1 mutated (59% vs. 40%; p=0.03).

In the univariate analysis, Leukemia-Free Survival (LFS) at 3 years was significantly lower in patients who were ITD positive (34±5% vs. 52±4%; p=0.001) and the relapse incidence was higher (58±5% vs. 42±4%; p=0.002). The Non-Relapse Mortality (NRM) rate was similar (8±3% vs. 6±1%; p= 0.7).

By multivariate analysis, the only unfavorable prognostic factors for outcome were failure to achieve CR with more than one induction course (slow remitters), and the presence of an ITD, which were associated with higher relapse incidence (p= 0.006; p=0.03) and lower LFS (p=0.05; p=0.005).

In this series, it was interesting to observe that the only group with a significantly lower LFS in univariate analysis, was the group of patients who were FlT3ITD positive without NPM1 mutation (23±11% vs. 56±6% for FLT3ITD negative/NPM non-mutated, 46±8% for FLT3ITD negative/NPM mutated and 48±13% for FLT3ITD positive /NPM mutated; p=0.03).

We conclude that in the context of autografting for AML in CR1, the presence of a FLT3ITD is an unfavorable prognostic factor only in less than 50% of the patients corresponding to those with the absence of a NPM1 mutation. This study helps to better define the population of patients with AML that would benefit most from intensification with autologous stem cell transplantation.

Disclosures:

No relevant conflicts of interest to declare.
Health Care Resource Utilization and Cost in Patients with Chronic Myeloid Leukemia in a Privately Insured Patient Population in the United States,
Authors: Iyer Shrividya , Trask Peter C , Siu Gordon , Mardekian Jack
Published: October 2019

Abstract 4220

Objective:

To estimate health care resource use and related costs in patients with chronic myeloid leukemia (CML).

Methods:

A retrospective cohort analysis was conducted using the Thomson Reuters MarketScan Commercial Claims and Encounters and Medicare Supplemental databases, which is composed of medical and pharmacy claims for approximately 43 million beneficiaries. Cases with at least 2 medical claims associated with a diagnosis of CML (ICD-9-CM code: 205.1) between Jan 1, 2002 and Dec 31, 2009 were extracted from the database. Index date was defined as the date of the first diagnosis of CML. A minimum of six months pre-index and 12 months post index enrollment was required. Disease and non-disease related utilization and costs were estimated. Resource utilization was calculated from index date to last available claims data point and then annualized per patient.

Results:

A total of 2583 patients were identified with an average follow up of 2.7 years. The mean age of the cohort was 59 years, and 45% were female. Proportions of patients having at least one inpatient, outpatient, and ER CML related visit were found to be 32.4%, 94.9%, and 15.1%, respectively. The average number of visits (standard deviation [SD]) per patient year was found to be 1.3 (1.4) and 1.6 (2.4) for inpatient and ER visits, respectively, among patients who had at least one visit. Average number (SD) of outpatient and office visits per patient year was found to be 40.6 (34.5) and 15.3 (11.6), respectively. Average number of prescriptions filled for CML was 3.3 per patient year. Disease-related health care costs ($23,166) constituted 36% of the total health care costs ($64,441) per patient year. Inpatient ($24,462 ± 77,429), outpatient ($24,391 ± 48,439), and prescription drug costs ($15,588 ± 18,327) accounted for 38%, 38%, and 24% of the total health care costs, respectively. CML drug costs accounted for 73% of the prescription drug costs.

Conclusion:

Cost burden of chronic myeloid leukemia are substantial. Effective disease management could help reduce resource utilization and cost while improving overall disease outcomes.

Disclosures:

Iyer: Pfizer: Employment. Trask:Pfizer Inc (at time of work completion): Employment; Sanofi: Employment. Mardekian:Pfizer Inc: Employment, Equity Ownership.
Nelarabine Is Safe and Effective In Adult Relapsed or Refractory T Cell Acute Lymphoblastic Leukemia (T-ALL) and Lymphoblastic Lymphoma (T-LBL): The Bologna Experience
Authors: Papayannidis Cristina , Iacobucci Ilaria , Abbenante Mariachiara , Lonetti Annalisa , Guadagnuolo Viviana , Ferrari Anna , Ottaviani Emanuela , Testoni Nicoletta , Baldazzi Carmen , Curti Antonio , Parisi Sarah , Clissa Cristina , Baccarani Michele , Martinelli Giovanni
Published: October 2019

Abstract 4250

Background.

Nelarabine (N) is approved for the treatment of T-ALL and T-LBL that have not responded to or has relapsed after treatment with at least 2 chemotherapy regimens.

Aim. To evaluate safety profile and efficacy of N as savage therapy in 16 adult relapsed or refractory T-ALL or T-LBL.

Methods.

After obtaining an informed consent, 16 patients (median age 33 years, range 19–45, M/F= 13/3) affected by T-ALL (n=10) and T-LBL (n=6) received savage therapy with N (median cycle=1, range 1–3), administered at standard adult dosage (1500 mg/sqm on days 1, 3 and 5, every 21). Four patients were primary resistant to induction treatment, 7 patients were relapsed after two previous chemotherapy regimens (including allogeneic BMT in 4 cases and autologous SCT in 1 case); the remaining 6 patients had a molecular relapsed disease (MRD positive). Molecular characterization was performed, including NOTCH and WT-1 genes mutational status. GEP analysis, according to Ferrando A. stratification (Cancer Cell 2002), is still ongoing.

Results.

Currently, 12 out of 16 patients are evaluable, due to a too short follow up in the other 4 cases. Seven out of 12 patients obtained a complete remission (CR) (5 T-ALL 2 T-LBL);a partial remission (PR) was documented in 2 cases, with an overall response rate (ORR) of 75%. Median duration of CR was 10 weeks (range 2.8–54+). Among these, 2 out of 4 patients in molecular relapse reached a molecular CR and underwent an allogenic BMT (currently in CR after a median follow up of 12 months). Extra- hematological toxicity, not clearly related to the drug, occurred in 3 cases, determining, a complete and irreversible paraplegia, a condition of mental confusion, and a peripheral neuropathy, respectively.

Conclusions.

N showed a strong efficacy also in cases with low levels of residual disease, in addition to a good safety profile. Neurological toxicity needs to be strictly monitored.

Acknowledgments.

European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, PRIN 2009, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione – Università 2007 – 2009.

Disclosures:

Baccarani: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Martinelli:Pfizer: Consultancy; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.
Lycorine Modulates the Expression of p21 Via a p53-Independent Pathway in HL-60 Cells
Authors: Liu Jing , Wang Shu-Ling , Fang Lin , Ye Mao , Sun Zhi-Wei , Li Lv , Wang Zi , Yan Hui-Wen , Luo Sai-Qun , Chen Hui-Yong , An Xiuli , Hu Wei-Xin
Published: October 2019

Abstract 4297

Leukemia is one of the most life-threatening cancers today, and acute promyelogenous leukemia is a common type of leukemia. We have previously shown that lycorine, a natural alkaloid extract from Amaryllidaceae, exhibited anti-leukemia effects in vitro and in vivo. Lycorine treatment of HL-60 cell arrested cell cycle at G2/M phase and induced apoptosis. In the present study, we sought to explore the molecular mechanisms for the anti-leukemia action of lycorine. Gene chip analysis revealed that lycorine treatment of HL-60 cells induced more than 9 fold increase of p21, a cyclin-dependent kinase inhibitor, whose expression is mainly regulated by p53. Since HL-60 cells are p53 null, the above findings suggest that lycorine activates p21 expression through p53-independent pathway. To further explore the alternative pathways for the activation of p21 induced by lycorine, we examined the effect of lycorine on the expression of Rb, pRb, E2F, c-Myc and HDACs which have shown to regulate p21 expression. We show that expression of pRb (ser780) and c-Myc was down-regulated, Rb and E2F were up-regulated, while the expression of HDAC1 and HDAC3 was not changed. Together these findings suggest that lycorine exerts its anti-leukemia effect by activating p21 expression via pRb/E2F and c-Myc pathways.

Disclosures:

No relevant conflicts of interest to declare.
Artificial Antigen Presenting Cells Expand NY-ESO-1 Antigen-Specific CD8+ T Cells From Patients with Melanoma
Authors: Maus Marcela V. , Riviere Isabelle , Borquez-Ojeda Oriana , Wang Xiuyan , Yuan Jianda , Merghoub Taha , Plotkin Jason , O’Reilly Richard J. , Wolchok Jedd D , Sadelain Michel
Published: October 2019

Abstract 4309

The NY-ESO-1 antigen is an attractive prototype cancer-testis (CT) antigen because it is expressed in a wide variety of tumors, including myeloma, melanoma, and synovial cell sarcoma, among others, but has restricted expression in normal tissues (testis and placenta). Based on our previous experience with 3T3-based artificial antigen presenting cells (AAPCs) in expanding influenza and CMV-specific T cells, we hypothesized that AAPCs expressing the NY-ESO-1 antigen and optimal costimulatory molecules would efficiently expand NY-ESO-1 specific T cells for potential use as adoptive immunotherapy. Accordingly, AAPCs were transduced to express the common MHC allele HLA-A*0201, beta2-microglobulin, and the full-length NY-ESO-1 protein, together with the T cell costimulatory ligands CD58, CD54, and CD80. These A2/NY-ESO-1 AAPCs expanded NY-ESO-1 specific T cells from normal donors after prolonged culture (4 weeks). In patients with melanoma who had detectable NY-ESO-1 antibodies, AAPC stimulation of PBMC expanded NY-ESO-1 antigen-specific CD8+ T cells approximately 3–4 logs in 3 weeks. These expanded T cells were functional in that they lysed peptide-pulsed targets (T2 cells) and native melanoma tumor targets (SK Mel 37). Expanded CD8+ T cells also produced interferon-gamma, MIP-1 beta, TNF-alpha and CD107a (but not IL-2) in response to antigen, indicating a polyfunctional response. We are currently performing scale-up experiments and pre-clinical validations of these AAPCs to embark on a clinical trial of adoptive cell therapy of NY-ESO1-specific T cells in patients with NY-ESO1-expressing tumors.

Disclosures:

No relevant conflicts of interest to declare.
Clinical Grade Generation of T-Helper-1-Driven Polyclonal T-Cell Populations for Adoptive Immunotherapy Against Tumor Associated Antigens
Authors: Kayser Simone , Boß Cristina , Icheva Vanya , Stevanovic Stefan , Lang Peter , Handgretinger Rupert , Feuchtinger Tobias F.
Published: October 2019

Abstract 4314

Adoptive T cell therapy has been shown an option to treat patients with malignancies. In contrast to vaccinations, T cells for adoptive T-cell therapy are generated ex vivo to be re-infused into the recipient. This enables treatment of immunocompromized hosts and use of allogeneic T cells to exploit graft versus tumor effects. Adoptive T-cell therapy involving CD4+ T-helper cells (Th cells), intends to induce sustained T-cell responses in vivo. The Th1 cytokine interferon-gamma (IFN-γ) has not only an effect in orchestrating cytotoxic T-cell reponses, IFN-γ by itself has antitumor effects. Transferring T cells in a lymphopenic host furthermore eliminates regulatory T cells (Tregs) and offers access to homeostatic cytokines. The aim of our study was the translation of preclinical data into a GMP conform clinical scale protocol to generate specific T cells for adoptive T-cell therapy against tumor associated antigens. Large scale generations of NY-ESO-1 specific T cells was performed according to current GMP regulations in a GMP facility. In brief, peripheral blood mononuclear cells from healthy donors were primed with an overlapping NY-ESO-1 15-mer peptide mix. The priming was done in the presence of IL-7 and IL-2. T cells were enriched using IFN-γ capture technique and expanded for two weeks in autologous culture conditions with IL-7, IL-15 and IL-2. T-cell specificity, function and proliferation capacity was analyzed by flow cytometry. The T-cell products showed high numbers of specifically IFN-γ+, TNF-alpha+ T cells. Tolerance inducing cytokines like IL-10 were absent. Enrichment of Tregs was excluded. Both, CD4+ and CD8+ T cells with an effector memory phenotype proliferated in response to NY-ESO-1. CD107a assays demonstrated cytotoxic capacities of T cells. The T-cell product did not include alloreactive T cells. In summary GMP-conform generation of NY-ESO-1 specific T cells was established. Although tumor associated antigens are potential self antigens, it is possible to induce a functional Th1 response in peripheral blood T cells from healthy donors. Adoptive T-cell therapy against tumor associated antigens could have implications for multiple tumor entities in autologous as well as allogeneic treatment approaches.

Disclosures:

No relevant conflicts of interest to declare.
Which Patients Would Be Willing to Change Anticoagulation From Vitamin-K Antagonists to a New Oral Anticoagulant?
Authors: Harenberg Job , Marx Svetlana , Abou-Ayash Nadja , Kremer Christophe , Hoeing Vera , Schirk Katrin , Weiss Christel , Froelich Lutz
Published: October 2019

Abstract 4318

New oral anticoagulants have generated promising data on the prophylaxis of systemic and non-systemic embolism in patients with atrial fibrillation and treatment of acute venous thromboembolism and prolonged prophylaxis of recurrent events. For patients on chronic treatment with vitamin-K antagonists (VKA) we analysed the motivation and willingness to change the anticoagulation from VKA to new oral anticoagulants.

Patients (n=110) on stable treatment with VKA for at least 3 months (indication for anticoagulation: atrial fibrillation or VTE) completed a validated personality inventory (Freiburger Persönlichkeitsinventar FPI-R), and a self-developed questionnaire on general attitudes regarding anticoagulant therapy (Q1). Patients were divided in two groups according to the reply to the question weather they were willing to switch to a new oral anticoagulant. Out of these sets of questions 7 questions were identified by means of a logistic regression analysis for the willingness to change anticoagulation with VKA to a new oral anticoagulant. The same patients completed this shortened questionnaire (Q2) (n=85) thereafter.

Logistic regression analysis defined the 7 items of the FPI and Q1 questionaires as relevant for willingness of patients to change the medication. The probability to change medication was 98% using the 7 questions (Q2) compared to the 2 comlete questionnaires. The items were: extraversion – introversion scale on the FPI-R consisting of 14 questions, and from Q1: hope for a better quality of life with a new anticoagulant, no scepticism for new drugs, wish of a lack of routine monitoring for dose adjustment, relevance of the practitioners opinion, thoughts in the past of alternatives for anticoagulation, and difficulty to adjust the prothrombin time. Using Q2 85% of patients confirmed to be willing to change the anticoagulant drug compared to Q1 (chi square test p<0.0001).

Seven questions were identified and confirmed to identify patients for their willingness to change anticoagulation from VKA to a new oral anticoagulant.

Disclosures:

No relevant conflicts of interest to declare.
Cross Reactivity of Natural Hirudin Compared to the Recombinant Hirudins with Sheep Anti Hirudin Antibody
Authors: Paramatmuni Vijaya L , Hoppensteadt Debra , Setty Saayi , Litinas Evangelos , Sadeghi Nasir , Fareed Jawed
Published: October 2019

Abstract 4321

Recombinant versions of hirudin such as lepirudin (Refludan®) and desirudin (Iprivask®) are currently used as parenteral anticoagulants for various indications. The recombinant hirudin preparations differ from the natural hirudin in lacking a sulfate group on tyrosine at the 63rd position and a single amino acid substitution. It is hypothesized that these minor differences in the natural and recombinant versions of hirudins contribute to a differential immunogenic behavior. The purpose of this investigation was to compare the relative immunoreactivity of the three forms of hirudin to a sheep antin–hirudin antibody. Antibodies (anti-n-hirudin) were generated in sheep treated with natural hirudin isolated from the medicinal leech (Hirudo medicinalis ). SDS-PAGE immunoblot and Western transfer were conducted using the native hirudin and two recombinant hirudins against the sheep anti-n-hirudin IgG antibodies. Cross-reactivity was quantified by measuring total optical density of the bands using a UVP densitometric scanning system and chemiluminescence detection, by comparing the protein-antibody complex band density from the western blot showed a comparable behavior of the Desirudin and Lepirudin in contrast to the normal or native hirudin which exhibited a much higher band density (2-fold increase). Moreover, the native hirudin exhibited specific bands representing mono, di- and tetra-meric forms, whereas the two recombinant forms exhibited primarily monomeric and dimeric forms. Interestingly several other preclinical versions of recombinant hirudins exhibited fifferent immunoblotting patterns. Although, the structural differences and the molecular weight represent relatively minor variations in the natural and recombinant hirudins, these studies strongly suggest a differential behavior of natural and recombinant hirudins in terms of their cross-reactivity with anti-n-hirudin antibodies.

Disclosures:

No relevant conflicts of interest to declare.
Comparison of Prothrombin Time INR Derived From Point-of-Care and Laboraotry Test According to Fibrinogen Level
Authors: Kim Sue Jung , Kim Juwon , Song Jaewoo
Published: October 2019

Abstract 4325

Coumarin-derived anticoagulants such as warfarin inhibit the vitamin K expoxide reductase (VKORC1) and block vitamin K cycle rendering coagulation factors II, VII, IX, X left in functionally null state (PIVKAs). Because of the narrow therapeutic range of warfarin, prothrombin time standardized into international normalized ratio (INR) should be frequently measured to prevent unwanted bleeding or recurring thrombosis. Point of care (POC) tests for patient self monitoring of anticoagulation was developed with the intention that more frequent monitoring of would enhance the compliance of the patients and prevent overdose. The accordance between results derived from these POCs tests and clinical laboratory tests has been repeatedly reported and the patients self monitoring strategy is now generally accepted as a reliable way for maintaining safer anticoagulation therapy. However, we pointed out that the POC test, especially CoaguChek (Roche) wide in use domestically, can show different correlation pattern with laboratory tests according to fibrinogen level, because the assay principle of the POC test is basically measuring thrombin activity. We found negative bias of POC INR compared with laboratory test from specimen with fibrinogen level lower than 130 mg/dl while positive biases were observed from that with fibrinogen level higher than 130 mg/dl. The difference in INR range from −0.7 to 1.7 and the maximum differences in both directions were observed in specimen with INR above 3.0, that is exceeding therapeutic range. These results suggest that patient selection according to fibrinogen status should precede the implementation of patient self monitoring and periodic laboratory tests and comparison with POC results should be continued as before the initiation of self monitoring.

Disclosures:

No relevant conflicts of interest to declare.
Reversion of the Experimental Hemodilutional Coagulopathy Induced by Crystalloids and Colloids Using Different Coagulation Factor Concentrates
Authors: Caballo Carolina , Galan Ana M , Diaz-Ricart Maribel , Lopez-Vilchez Irene , Lozano Miguel , Cid Joan , Pino Marcos , Beltran Joan , Basora Misericordia , Pereira Arturo , Escolar Gines
Published: October 2019

Abstract 4349

BACKGROUND:

Massive bleeding and subsequent coagulopathy are responsible for 35% of deaths in trauma patients. Hemodilution during resuscitation may worsen the coagulopathy and perpetuate bleeding.

STUDY DESIGN AND METHODS:

Blood samples from healthy donors were diluted (30–60%) using crystalloids (saline, Ringer’s lactate, Plasmalyte™) or colloids (6%hydroxyethylstarch (HES130/0.4), 5% human albumin, and gelatin). The impact of hemodilution on platelet adhesion, thrombin generation (TG), and clot viscoelastic properties by thromboelastometry (TEM) was analyzed. Effects of fibrinogen (Fbn), prothrombin complex concentrates (PCCs), rFVIIa, or cryoprecipates (cryo) on hemodilution were also assessed.

RESULTS:

Hemodilution caused a significant decrease in platelet interaction that was not improved by the addition of any of the plasma derivatives. A decrease in TG and important alterations of TEM were also observed. HES130/0.4 was the expander with the most deleterious action. TG was significantly enhanced by PCCs and their combination with Fbn whereas rFVIIa only slightly accelerated it. Fbn restored the alterations of TEM caused by hemodilution including those more deeply altered by HES 130/0.4. The combination of Fbn with PCC or rFVIIa did not have an additional effect in TEM. Cryo significantly improved the alterations caused by hemodilution on TG and TEM parameters. Effects of cryo on TG disappeared after ultracentrifugation, suggesting that contaminating microvesicular material could account for this effect.

CONCLUSION:

Hemostatic alterations caused by hemodilution are multifactorial and affect both blood cells and coagulation. In our in vitro approach, HES 130/0.4 seemed to exert a more deleterious effect on hemostasis. None of the concentrates improved platelet-mediated hemostasis, although they always showed variable beneficial effects on coagulation parameters. Our data indicate that PCC, rFVIIa and cryo enhance or accelerate thrombin generation. Fbn concentrates could be useful to preserve blood clotting abilities during fluid resuscitation of critically ill patients without exposing them to enhanced thrombin generation.

Grants: PET(2008_0231), FIS(CP04-00112, PS09/00664), SAF2009-10365, RD06/0009

Disclosures:

No relevant conflicts of interest to declare.
2D-DIGE As a Tool to Analyze Lot-to-Lot Consistency of Complex Therapeutic Products Such As BAX 855, a PEGylated Recombinant FVIII
Authors: Rottensteiner Hanspeter , Monetti Christina , Gritsch Herbert , Weber Alfred , Ehrlich Hartmut J. , Scheiflinger Friedrich , Turecek Peter L
Published: October 2019

Abstract 4360

2-Dimensional difference gel electrophoresis (2-D DIGE) is a method that circumvents the gel-to-gel variability inherent in conventional 2-dimensional gel electrophoresis (2-DE). We developed a 2-D DIGE protocol for recombinant factor VIII (rFVIII), a therapeutic protein used for the treatment of hemophilia A. The FVIII heterodimer is composed of heterogenous, strongly glycosylated heavy and light chains that are held together by a divalent cation. 2-DE of rFVIII led to a separation of the various fragments and their identity could be determined by Western blot. A comparison of two rFVIII batches by 2-D DIGE revealed their identical composition, whereas an rFVIII variant lacking its central B domain was congruent with the smallest heavy and light chain fragments of rFVIII only. A simpler pattern was obtained upon removal of the terminal sialic acids of rFVIII’s glycans due to a better focusing in the first dimension. 2-D DIGE was also well suited to structurally evaluate BAX 855, a PEGylated longer-acting variant of recombinant FVIII. 2-D DIGE thus proved an excellent and straightforward method for structural analysis of rFVIII. Our data suggest that the method could serve as a tool to characterize and control quality of very complex pharmaceutically active ingredients such as PEGylated proteins.

Disclosures:

Rottensteiner: Baxter Innovations GmbH: Employment. Monetti:Baxter Innovations GmbH: Employment. Gritsch:Baxter Innovations GmbH: Employment. Weber:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment.
The Somatic Mutation of PIG-A Gene and the Expressions of EGR-1 and WT1 Genes in Bone Marrow Cells of the Patients with Paroxysmal Nocturnal Hemoglobinuria
Authors: Fu Rong , Li Liyan , Shao Zonghong , Wang Jun , Li Lijuan , Liu Hui
Published: October 2019

Abstract 4379

Objective

To investigate the somatic mutations of PIG-A gene and the expressions of EGR-1 and WT1 genes in the bone marrow cells of the patients with Paroxysmal nocturnal hemoglobinuria, then explore the characteristics of PIG-A gene mutation in Chinese patients with PNH and the possible mechanism of PNH clonal expansion related to EGR-1 and WT1 genes.

Methods

The quantities of CD55− cells and CD59− cells in both peripheral blood and bone marrow were examined with flow cytometric assay(FCM), and then sorting CD59− cells from bone marrow mononuclears (BMMNCs). The mutant segments of PIG-A gene were identified by DNA sequencing; The expressions of EGR-1 and WT1 mRNA in CD59− and CD59+ BMMNC were measured with semiquantitive RT-PCR.

Results

Nine in sixteen patients with PNH and one in four patients with AA-PNH were measured for their PIG-A gene mutation. Five single base substitution were found, including A→G, G→T, A→T, T→G and G→C, resulting in one consense and seven missense mutations. Each of 3 PNH patients had more than two point mutations of PIG-A gene. Insert or deletion mutation of PIG-A gene were not found. The relative mRNA expressions of EGR-1and WT1 genes in CD59− BMMNC, CD59+ BMMNC of PNH or AA-PNH patient and BMMNC of normal controls were (1.00±0.11), (0.86±0.14), (0.85±0.06) and (1.06±0.16), (0.90±0.14), (0.88±0.06), the expression in CD59− BMMNC was significantly higher than that in CD59+ BMMNC or normal controls (p<0.05). There was no significant difference between the expression in CD59+ BMMNC and normal controls(p>0.05). The relative mRNA expressions of EGR-1 and WT1 in BMMNC of PNH and AA-PNH cases were positively correlated with the proportion of CD59− cells(coefficient correlation was 0.509 and 0.481 respectively, p<0.05), but not related to the proportion of CD59+ cells.

Conclusions

The PIG-A gene mutation in some Chinese patients mainly manifested as single base substitution, occurred at random sites and without hot spot, the overexpression of EGR-1 and WT1 genes in PNH clone could promote the abnormal clone expansion.

Disclosures:

No relevant conflicts of interest to declare.
Preliminary Study of the Autoantigens on the Membrane of Erythropoietic Cells of the Patients with BMMNC- Coomb’s Test(+) Hemocytopenia
Authors: Fu Rong , Liu Hui , Shao Zonghong , Wang Jun , Li Lijuan
Published: October 2019

Abstract 4382

Objective

To observe the relationship between EPO receptor(EPOR) and autoantibodies-IgG/IgM(auto-Ab) on the membrane of erythropoietic cells of the patients with IRP and then explore the probable autoantigens of auto-Ab in IRP.

Methods

46 newly diagnosed IRP patients(15 with auto-Ab on erythropoietic cells and 31 without auto-Ab on erythropoietic cells) and 18 healthy controls were enrolled in this study. EPOR expression on their nuclear erythrocytes were tested with FCM to observe the relationship between EPOR and auto-Ab; EPOR mRNA were tested by RT-PCR; Stat5 and P-Stat5 proteins in nucleared erythrocytes were measured by Western blot; EPOR expression on the nucleared erythrocytes membrane were tested again after stripping autoantibodies with glycine buffer.

Results

(1)EPOR of auto-Ab(+) arm(1.59±0.87)% was significantly lower than that of auto-Ab (−) arm(4.58±4.09)%(P<0.01)and the latter was significantly higher than that of normal controls(2.27±1.76)%(P<0.05); GEPOR of IRP patients was inversely correlated with their auto-Ab (r=−0.543,P=0.000).(2) EPOR mRNA of auto-Ab(+) arm(0.685±0.136)was significiantly higher than that of auto-Ab (−) arm(0.554±0.116)(P<0.01)and normal controls(0.580±0.119)(P<0.05);(3) Protein Stat5 of auto-Ab(+) arm(1.45±0.94) was significantly higher than that of normal controls(0.54±0.36)(P<0.05). While P-Stat5 of auto-Ab(+) arm(0.42±0.18) was significantly lower than that of normal controls(0.85±0.38)(P<0.05). (4) EPOR expression increased significantly after auto-Ab stripped from nucleared erythrocytes with glycine buffer.

Conclusion

The auto-Ab of some IRP patients might block or competitively inhibit the EPOR on the membrane of erythropoietic cells. EPOR might be one of autoantigens in IRP.

Disclosures:

No relevant conflicts of interest to declare.
Off-the-Shelf Cord Blood Mesenchymal Stem Cell Bank
Authors: Montemurro, Tiziana , Lavazza Cristiana , Andriolo Gabriella , Viganò Mariele , Baluce Barbara , Rebulla Paolo , Giordano Rosaria , Lazzari Lorenza
Published: October 2019

Abstract 4403

Our hospital-based GMP facility, named Cell Factory, built in Milano (Italy) and the first one authorized by the National Drug Agency (AIFA) in 2007, has pioneered innovative manufacturing technologies in order to supply therapeutic clinical development programs.

By our group and by others, cord blood (CB) mesenchymal stem cells (MSC) have been already tested both in vitro and in extensive pre-clinical disease models, demonstrating that CBMSC are safe and effective. Therefore, CBMSC are the best candidate product for several clinical trials including neurologic and renal repair (1–3).

In this context, our group developed the first off-the-shelf (OTS) bank where CBMSC are expanded and banked in GMP conditions, and now they are available as “a drug”, to be distributed for patient doses, much like an off-the-shelf pharmaceutical product. From informed consent CB donations, CBMSC were grown and expanded in CellSTACK kit that includes devices for a closed-system cell culture (Macopharma) with EMEA-grade Australian FBS and they were manufactured through a multi-step controlled and well-validated process. Cell Factory established this innovative culture approach through sufficient passages to produce hundreds of thousands of potential doses from an individual donor. After culture, our quality control approach has been designed to ensure that controls are implemented and completed satisfactorily during manufacturing operations, and that developed procedures and specifications are appropriate and followed, including those performed by contract testing laboratories. In fact, at each step and at the end of the production process before cryopreservation, CBMSC were analyzed and qualified according to pre-established criteria (cell count, viability, extensive immunophenotype, CFU-F, karyotype, CGHArray, telomerase activity, sterility, mycoplasma and endotoxin assays) to ensure that a consistent, well characterized product candidate is produced. The same CBMSC batches can be provided for pre-clinical testing and for further validations to prepare the investigational medicinal product dossier (IMPD) for each clinical trial approvals.

Now these GMP batches of CBMSC are cryopreserved and available in our OTS CBMSC bank in different cell doses to comply any kind of medical needs. Products are provided under specific contract conditions developed with the clinical centers. (Contact: cellfactorypoliclinico@gmail.com)

Disclosures:

No relevant conflicts of interest to declare.
K562-R As a Model to Study Jak2 in a Non Bcr-Abl Addicted Cell Line
Authors: Perazzona Bastianella , Lin Yu-Hsi , Arlinghaus Ralph B.
Published: October 2019

Abstract 4412

Chronic myeloid leukemia (CML) is a hematological disease caused by the fusion protein Bcr-Abl tyrosine kinase. Development of the tyrosine kinase inhibitor Imatinib Mesylate (IM) has significantly improved the long-term survival of early stage CML patients. However, occurrence of drug resistance, permanence of residual disease and recurrence of active leukemia if IM is discontinued, remain problems awaiting solution. Therefore, new therapeutic strategies aimed at targeting alternative signaling pathways or CML progenitor cells that survive IM treatment are needed. We have previously shown that Janus kinase 2 (Jak2) is activated in Bcr-Abl+ cells. We have demonstrated that reduction of Jak2 activity by the Jak2-specific inhibitor TG101209 (TG) or by genetic knock down (Jak2 shRNA and siRNA) in Bcr-Abl+ cell lines, IM-resistant cells and CML blast crisis cell lines resulted in reduced levels of phosphorylation of Tyr177 and of total Bcr-Abl protein. Jak2 inhibition results in diminished activation of the Ras, PI-3 kinase pathways and reduced levels of pTyrSTAT5 (Samanta et al., Leukemia 2011). During these studies we observed that K562 cells and IM-resistant cell line K562-R had different susceptibility to the effect of TG, with K562-R showing increase sensitivity to lower concentrations of TG resulting in faster destabilization of the Bcr-Abl protein. Based on these observations, we hypothesized that increased sensitivity of the K562-R cells was due to the different state of activation of Jak2. In addition, based on recent studies by (Dawson et al., Nat 2009) and by (Rinaldi et al., Blood 2010) we also hypothesized that different levels of Jak2 activation may influence the localization of Jak2 in the cell. We used cell fractionation and western blotting analysis to show that in K562-R cells, active Jak2 is mostly localized in the nucleus with a minor pool found in the cytoplasm. In K562 cells, active Jak2 is equally distributed in both cytoplasmic and nuclear compartment. In addition, immuno-fluorescence confocal analysis of total Jak2 distribution in K562 shows a very organized localization of Jak2 at one pole of the cells but this organization is lost in K562-R and total Jak2 appears uniformly distributed within the cell. K562-R cells were isolated as a Bcr-Abl independent IM resistant cell line that expressed high levels of activated Lyn kinase (Donato et al., Blood 2003). We used K562-R as a model to study the role of Jak2 in a non Bcr-Abl addicted cell line. Since we have previously published that Jak2 up-regulates Lyn kinase activity (Samanta et al., Oncogene 2009), we propose that the higher activation of Jak2 in K562-R is the main driver of oncogenicity and IM resistance and that this cell line may be used to model the role of Jak2 in a cell that is not Bcr-Abl “addicted.”

Disclosures:

No relevant conflicts of interest to declare.