bluuebunny/crossref_metadata_2025 huggingface dataset:
Formation of FVIIa-Antithrombin Complexes After Administration of rFVIIa to Hemophilia Patients.
Authors: Ezban Mirella , Martin Erika , Barrett John Christian , Kuhn Janice , Nolte Mindy , Pelzer Hermann , Agerso Henrik , Petersen Lars Christian , Hedner Ulla , Carr Marcus E. , Brophy Donald
Published: October 2019

Abstract 1295

Poster Board I-317

Introduction

Recent animal studies suggest that measurable amounts of factor VIIa and antithrombin (AT) complexes are formed and accumulate following rFVIIa administration. The in vivo rate of inhibition has been reported to be faster than the un-stimulated in vitro reaction between AT and free rFVIIa and of the same order of magnitude as the rate determined in the presence of tissue factor. To study the impact of AT inhibition on the elimination of rFVIIa in humans, we measured the pharmacokinetics (PK) of rFVIIa and rFVIIa-AT complex formation in 10 hemophilia A or B patients.

Patients and Methods

The PK of single-dose rFVIIa 90 μg/kg (Novo Nordisk A/S) was evaluated in 10 severe FVIII- or FIX-deficient patients in a non-bleeding state. The plasma concentrations of FVIIa activity (FVII:C), FVII antigen (FVII:Ag), FVIIa-AT, D-dimer and F1+2 fragment were determined immediately before, and at 0.5, 1, 2, 4 and 6 hours following rFVIIa dosing.

Results

Significant amounts of FVIIa–AT complex were formed in vivo after rFVIIa administration, and reached a maximum of 5.4 ± 0.8 nmol/L [mean ±SD] at 2 hours following rFVIIa administration and declined to 4.4 ± 0.9 nmol/L at 6 hours, as compared to 0.1 ± 0.05 nmol/L at baseline. While the FVII:C PK data in this study were consistent with previous data, there was greater total body clearance (Cltot), a larger volume of distribution (Vdss) and a shorter plasma half-life (T1/2) of FVII:C relative to FVII:Ag (Table). No change in D-dimer was observed after the administration of rFVIIa, while a slight increase in F1+2 fragment levels to 258 ± 73 pmol/L was measured 4 hours after rFVIIa dosing, as compared to 141 ± 45 pmol/L at baseline.

Conclusion

A significant divergence between the clearance of rFVIIa, as determined by either FVII:C or FVII:Ag measurements, can be accounted for by AT complex formation. Inhibition by AT appears thus to have a significant impact on the elimination of FVII:C activity from the circulation when rFVIIa is administered at a therapeutic dose. Similar to animal data, the formation of the FVIIa-AT complexes in vivo was faster than anticipated from in vitro studies, indicating that the exposure to the vessel wall stimulates the FVIIa inhibition by AT. Analyses of coagulation parameters did not indicate induction of systemic coagulation.

Disclosures

Ezban: NovoNordisk A/S: Employment. Pelzer:NovoNordisk: Employment. Agerso:NovoNordisk: Employment. Petersen:NovoNordisk: Employment. Hedner:NovoNordisk: Employment. Carr:NovoNordisk: Employment.
Quantitation of Changes in VWF and FVIII Following Elective Orthopedic Surgery in Normal Individuals.
Authors: Kahlon Amrit , Grabell Julie , Tuttle Angie , Engen Dale , Hopman Wilma , Lillicrap David , James Paula
Published: October 2019

Abstract 1296

Poster Board I-318

Background

Patients with inherited bleeding disorders undergoing surgery typically receive therapy to correct the deficient factor to a level of ∼1.0 IU/mL. There is a paucity of well-constructed descriptive data quantifying the changes in coagulation that occur in response to surgical stress in normal individuals, however. Additionally, von Willebrand factor (VWF) and factor VIII (FVIII) are acute phase reactants, and thus, supratherapeutic levels of these proteins that might develop in response to the surgical stress would predispose patients to thrombotic events. Aim: With this in mind, our objective was to quantify the changes in hemostatic variables that occur in response to elective orthopedic surgery in normal individuals.

Methods

Eligible subjects > 18 years of age undergoing total knee or total hip replacement were recruited. Exclusion criteria included a known bleeding disorder, hormone replacement therapy, use of oral contraceptives or pregnancy. Following informed consent, blood samples were drawn at five time points: 1) baseline - in clinic 1-2 weeks pre-operatively (pre-op), 2) pre-op the morning of surgery – same day admitting center, 3) intra-operatively (intra-op) – 30 minutes after the start of surgery, 4) 30 minutes post-operatively (post-op) - recovery room, and 5) post-op day one (POD 1) – on the ward. Samples were analyzed for complete blood counts (CBC), prothrombin time (PT), activated partial thromboplastin time (aPTT), VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), FVIII coagulant activity (FVIII:C) and whole blood thromboelastography (TEG) without tissue factor activation. Repeated measures analysis of variance was used to evaluate trends over time; paired samples t-tests were done to maximize the sample size for pairwise comparisons.

Results

37 subjects were recruited (25 females, 12 males) with a mean age of 64 years (range 33 – 87). Means and ranges for all laboratory parameters are displayed in Table 1 in addition to statistical analysis of the change in all variables over the 5 time points. Statistically significant changes were seen in all variables except PTT and TEG angle. In general, VWF and FVIII indices decreased during surgery (presumably due to consumption) and then increased in the post-operative period (presumably due to a stress response). A great deal of variability was seen in post-operative levels of VWF and FVIII between individuals with some showing borderline low levels and some achieving very high levels.

Conclusion

These data establish a baseline on which to reconsider current strategies for the peri-operative management of patients with inherited bleeding disorders, and provide relevant information when considering the relative risks of bleeding or thrombosis associated with surgery.

Disclosures

No relevant conflicts of interest to declare.
Rapamycin and IL-2 Prevents Lethal Acute Graft-Versus Host Disease by Expansion of Donor Type CD4+CD25+Foxp3+ Regulatory T Cells.
Authors: Shin Ho-Jin , Baker Jeanette , Leveson-Gower Dennis , Sega Emanuela I , Negrin Robert
Published: October 2019

Abstract 1334

Poster Board I-356

Previous work has demonstrated that both rapamycin (RAPA) and IL-2 enhance CD4+CD25+Foxp3+ regulatory T cells (Treg) proliferation and function. We investigated whether the combination of RAPA and IL-2 administered in vivo could reduce acute graft-versus-host disease (aGVHD) induction and increase survival after bone marrow transplantation (BMT) by induction of Treg. T cell depleted bone marrow (TCD-BM) cells from wild type C57BL/6 mice were injected after lethal irradiation with (800 cGy) into Balb/c recipients on day 0. To induce aGVHD and evaluate the proliferation of donor T cells by in vivo bioluminescence imaging, 1 × 106 conventional CD4+ and CD8+ T cells (Tcon) from luciferase-expressing (luc+) transgenic C57BL/6 were injected intravenously on the same day. RAPA was administered intraperitoneally (dose of 0.5 mg/kg/day) for 14 days and IL-2 (dose of 5 × 104 IU) for 3 days (twice a day). RAPA plus IL-2 significantly reduced the expansion of luc+ Tcon more than RAPA (P=0.04) or IL-2 alone (P=0.002) or no treatment (P = 0.01). Weight loss and aGVHD score were significantly reduced in the mice which were injected with the combination of RAPA and IL-2 compared with those animals injected with RAPA (P = 0.03) or IL-2 alone (P = 0.05). The percentage of donor type CD4+CD25+ cells was increased and CD4+CD25− T cells were reduced from thymic and extrathymic tissues after treatment with RAPA plus IL-2 on day 7 after BMT. The combination of RAPA and IL-2 resulted in a 2.4 fold (mesenteric LN), 2.7 fold (peripheral LN), 4.2 fold (spleen) and 2.1 fold (thymus) increase in the percentage of donor type CD4+CD25+Foxp3+ Treg. In animals receiving the combination of RAPA and IL-2, the percentage of CD4+CD25+Foxp3+ T cells increased from 8.98% (RAPA alone) to 24.2% (RAPA plus IL-2) at 7 days after BMT, representing a 2.7 fold expansion. The cell numbers of CD4+ and CD8+ T cells and CD4+CD25− T cells were reduced and CD4+CD25+Foxp3+ Treg were expanded in the thymus and extrathymic tissues after administration RAPA and IL-2. To study the origin of the expanded Treg in RAPA plus IL-2, BALB/c recipient mice were injected with purified donor CD4+CD25highFoxP3+ Treg and CD4+CD25− and CD8+CD25− (both Foxp3−) Tcon after lethal irradiation followed by RAPA plus IL-2 for 7 days. The combination of RAPA and IL-2 increased the expansion of donor type CD4+CD25+Foxp3+ Treg, but did not result in an increase in the conversion of Foxp3+ Treg from donor CD25− Tcon. We also evaluated whether Foxp3+ non-Treg were present in the expansion of Treg by RAPA plus IL-2 by evaluating interferon-γ- and IL-2-production which was minimally detected in the Foxp3+ cells. In conclusion, the combination of RAPA and IL-2 after BMT synergistically promoted the expansion of donor CD4+CD25+Foxp3+ Treg resulting in prevention of lethal aGVHD.

Disclosures

No relevant conflicts of interest to declare.
In-Hospital Outcomes Among Sickle Cell Patients with Acute Chest Syndrome: Results From a National Database.
Authors: Kamble Shital , Reed Shelby D , Grussemeyer Chelsea , Telen Marilyn J.
Published: October 2019

Abstract 1367

Poster Board I-389

Objective:

Acute chest syndrome is the leading cause of death and the second most common cause of hospitalization in patients with sickle cell disease (SCD), yet there are no recent estimates of its associated impact on medical resource use and costs. In this study, we compared inpatient mortality, length of stay (LOS) and total costs among hospital discharges for SCD patients with acute chest syndrome (ACS), SCD patients with crisis, and SCD with neither ACS nor crisis (controls).

Methods:

Using the 2006 Nationwide Inpatient Sample (NIS) data of the Healthcare Cost and Utilization Project, we developed three groups of discharges with SCD (Clinical Classification Code 61): (1) ACS with or without crisis on the basis of ICD-9-CM codes 517.3 (ACS) or 486 (pneumonia), (2) sickle cell crisis without ACS, using ICD-9-CM codes 282.42, 282.62, 282.64 or 282.69; and (3) SCD patients without a diagnosis of ACS or sickle cell crisis. We excluded cases representing elective admissions, patients discharged to short-term hospitals, born in the hospital, and women giving birth. We used generalized linear regression models to compare LOS (negative binomial distribution, log link) and inpatient costs (gamma distribution, log link) between study groups while controlling for gender, age, payer, 29 comorbidities, and hospital characteristics, including rural vs. urban location, teaching status, bed size, ownership type, and hospital region. We did not perform adjusted comparisons for mortality due to small numbers.

Results:

We identified 2,971 discharges with ACS, 13,593 with sickle cell crisis, and 4,040 with neither condition coded. Median age was 25 for ACS, 27 for sickle cell crisis, and 29 for sickle cell ‘controls’. Females represented 50.8% of discharges with ACS, 56.1% of discharges with sickle cell crisis, and 55.8% of sickle cell controls. Inpatient mortality was low across all three groups (Table). After adjustment for patient and hospital characteristics, discharges with ACS incurred hospital stays that were 24% longer (95%CI: 1.20-1.28, P<.0001) than those with crisis and 68% longer (95%CI: 1.62-1.74, P>.0001) than SCD controls. Similarly, inpatient costs among ACS discharges were 45% higher (95%CI: 1.40-1.49) than discharges with sickle cell crisis and 62% higher (95%CI: 1.56-1.68,P<.0001) than for SCD controls. Discharges for patients with sickle cell crisis were 35% longer (95%CI: 1.31-1.39, P<.0001) and 12% more costly (95%CI: 1.09-1.15, P<.0001) than for sickle cell controls.

Conclusion:

SCD patients admitted to the hospital with ACS incur significantly higher costs and longer stays than SCD patients with crisis or neither of these conditions.

Disclosures:

No relevant conflicts of interest to declare.
Cost-Effectiveness of Ferric Carboxymaltose Supplementation in Chemotherapy-Induced Anemia Patients Treated with Erythropoiesis-Stimulating Agents.
Authors: Szucs Thomas D. , Blank Patricia R. , Schwenkglenks Matthias
Published: October 2019

Abstract 1372

Poster Board I-394

Background:

Erythropoiesis-stimulating agents (ESA) are widely used in the treatment of chemotherapy-induced anemia (CIA). ESA prescription has come under pressure in this indication due to safety concerns and because of their high acquisition price. Recent studies suggest that the efficiency of ESA can be maximized by optimising iron stores and iron bioavailability. However, little is known about the economic implications of adding intravenous ferric carboxymaltose (iv FCM; an innovative iv iron formulation allowing the administration of high iron dosages in a short time) to ESA treatment.

Aims:

This study aimed at estimating the incremental cost-effectiveness of adding iv FCM to ESA treatment in patients with CIA, in the Swiss health care setting.

Methods:

Using a Swiss third party perspective, incremental cost effectiveness of iv FCM use was expressed as cost per additional CIA patient responding to anemia treatment. Response rates and medical resource use parameters were retrieved as weighted averages from 4 randomised controlled clinical trials of solid tumour patients, which used other iv iron preparations. An assumption of equal clinical effectiveness was made. Mean treatment duration was 11.9 weeks. Unit costs were based on average ex-factory prices for ESAs. The European target ex-factory price for 100 mg iv FCM was €25.

Results:

Per-patient costs were determined to be €4'943 for ESA treatment alone and €5'227 for ESA plus iv FCM. The addition of iv iron to ESA increased the response rate from 55% to 74%. Hence, the incremental cost-effectiveness (cost per additional patient responding to treatment) for iv FCM was estimated at €1′455.

Summary/conclusions:

The addition of iv FCM to ESA treatment appears to be economically viable in patients with CIA, on the basis of a marked increase in response rate due to iv iron use. Additional economic analyses are certainly warranted, as soon as appropriate clinical data become available. Cost savings associated with reduced administration time are not covered by this analysis.

Disclosures:

Schwenkglenks: Amgen: Honoraria, Research Funding.
Peripheral Blood Stem Cell (PBSC) Harvest Is More Expensive Following Bortezomib-Based Therapy in Multiple Myeloma (MM), Related to Increase Number of Days of Collect.
Authors: Barthelemy Matthieu , Boulanger Florence , Debarri Houria , Pascal Laurent , Samarcq Pierre , Cossement Colette , Jouet Jean-Pierre , Facon Thierry , Yakoub-Agha Ibrahim , Leleu Xavier
Published: October 2019

Abstract 1373

Poster Board I-395

Background:

High-dose therapy is the front line treatment of reference in young patients with Multiple Myeloma (MM). Although induction therapy remains a matter of controversy, bortezomib-based therapy is considered more and more as a standard of care. Prior to autologous stem cell transplantation (ASCT), patients undergo PBSC collect, usually starting after cycle 2 to 4 of the induction treatment. Currently, patients receive one transplant at front line, but most of the patients will benefit throughout the MM disease history of a second or a third ASCT procedure. We have noticed that the number of days of collection vary from patient-to-patient following bortezomib-based induction therapy. This increase in collection procedures might increase the cost of PBSC harvest with more patients discomfort and staff unavailability. We have therefore further studied the quality, yields and days of collection in myeloma patients following bortezomib-based therapy as compared to other regimens-based treatment courses.

Material and Method:

We retrospectively studied 70 patients with myeloma that underwent PBSC harvestsafter mobilization with GCSF following debulking with bortezomib-based therapy (58 days of procedures – 26 patients) versus other agents-based therapy [65 days of procedure – 44 patients; VAD vincristine, adriamycin, dexamethasone).

Results:

-Yields. CD34 Mobilization is lower following bortezomib-based therapy as compared to other regimen-based therapy. Similarly, more days of collection are also needed to collect the requested yield of PBSC.

-Engrafment. No significant differences regarding engraftment was noticed among the 2 groups studied. The days to neutrophil and platelet counts recovery, the number of days with fever and the number of red cell and platelet transfusions were not significantly different between the 2 groups, mobilization following bortezomib-based therapy versus other regimen-based therapy, respectively.

Conclusion:

In our series, bortezomib-based induction regimen does not increase the number of PBSC harvest failure and the quality of engraftment was identical to other regimen-based induction treatment. However, CD34 mobilization was lower following bortezomib-based therapy, which explains lower daily harvest counts and therefore an increase number of days of collection in bortezomib-based treated patients. Therefore, PBSC harvest procedure following bortezomib-based therapy significantly increases the cost of PBSC collection. These results need to be confirmed in larger studies. New agents in use for mobilization might be considered for future PBSC collection in bortezomib-based treated patients.

Disclosures:

Leleu: Janssen Cilag: Research Funding.
Outcome of Fungal Infection in Patients Undergoing Myelosuppressive Chemotherapy in a Tertiary Care Centre During 12 Years (1995-2006).
Authors: von Lilienfeld-Toal Marie , Glasmacher Axel , Marklein Günter , Brossart Peter , Hahn-Ast Corinna
Published: October 2019

Abstract 1384

Poster Board I-406

Invasive fungal infections (IFI) contribute significantly to mortality and morbidity in patients receiving myelosuppressive chemotherapy for hematological malignancies. The present study evaluates the incidence of IFI, the overall survival (OS), the infection-related mortality and changes in treatment of IFI in our department from 1995 until 2006. Data of all consecutive chemotherapy courses were retrospectively collected with a standard questionnaire. EORTC/MSG criteria for IFI were applied with a modification: A positive PCR-result for Aspergillus spp. in bronchoalveolar lavage was also defined as probable IFI. In total, 1693 courses of 592 patients were evaluated. Sixty-three percent were given to treat acute myeloid leukemia, the rest for acute lymphoblastic leukemia or aggressive lymphoma. IFI were observed in 139/592 patients (23%, 95% confidence interval (CI) 20-27%), and in 149/1693 (8.8%, 95%CI 8-10%) courses. IFI-related mortality was 57% in 1995-2001 and 29% in 2002-2006, p<0.001. Accordingly, median OS in patients with IFI increased in the later years: 54 days (95%CI 26–82 days) in 1995-2001 versus 229 days (95% CI 35–423 days) in 2002-2006, p=0.001, figure 1. By multivariate analysis, factors predictive for better OS were controlled disease after chemotherapy (hazard ratio (HR) 0.226, p<0.001), possible IFI (in contrast to proven/probable IFI, HR 0.511, p=0.002), age < 60 years (HR 0.611, p=0.015), and use of novel antifungals (HR 0.493, p=0.002). In conclusion, IFI-related mortality decreased and OS in patients with IFI increased significantly in recent years compared to 1995-2001. In our cohort improved OS was associated with controlled underlying disease, certainty of IFI diagnosis (possible), younger age, and the use of novel antifungal agents.

Figure 1 p=0.001 Figure 1. p=0.001

Time period ········ 2002-2006 —— 1995-2001 Number at risk 2002-2006 78 28 18 9 6 2 1995-2001 61 8 6 5 5 5

Disclosures:

von Lilienfeld-Toal: MSD: Honoraria, Research Funding. Glasmacher: Celgene: Employment, Equity Ownership. Hahn-Ast: MSD: Research Funding.
Development of Personal Health Records (PHR) for Patiets with Malignancies Using Interactive Internet Service: Experience at National Center for Hematology in Moscow.
Authors: Shklovskiy-Kordi Nikita E , Zingerman Boris V , Varticovski Lyuba , Kremenetskya Alexandra , Vorobjov Andrei
Published: October 2019

Abstract 1407

Poster Board I-429

Purpose.

To compare the requirements of physicians and patients for the Internet interactive service which allows patients to manage their own medical records and communicate with physician via the Internet.

Background.

USA federal rule defining “the Meaningful Use of Electronic Health Records” is similar to that of the National Standard of Russian Federation “The Electronic Case History (EHR)”, operating since 2008. This National Standard was developed based on experience of EHR system at the National Center for Hematology in Moscow (NCH). In 2009, we started the Personal Health Records service (PHR service) that allows patients to manage their own medical records and have internet-based communication with physicians. Simple interface for patients which blocks the full capacity of the PHR service is similar to that of EHR system of NCH. It permits integrated data presentations on a uniform axis of time and access to additional information (reported to ASH in 2001). The PHR service raises question of “meaningful use” requirements not only for EHR provider organization, but for the service users - patients and doctors.

Methods.

Using questionnaires and interviews, we compared expectations and acceptance of the PHR service by doctors and their patients.

Results and Discussion.

Preliminary results indicate that doctors are more likely to use the PHR service than the System of HER. Although the entire format of PHR service is familiar to physicians at NCH, they mostly use its information capabilities (viewing the results of the analysis, making appointments for research and planning patient's visits). The patients use PHR service with great enthusiasm (increasing with younger age and higher level of education). The complexity of integration interfaces, which we leave for the patients in the second term, gives them more inspiration than that the physicians. However, few patients take seriously the responsibilities that exist in relation to the accurate maintenance of their records.

Conclusion.

PHR can be widely used if integration of sources for medical information and unification format can simplify the “manual” work of PHR management. Key Words: Telemedicine, PHR, EHR

Disclosures:

No relevant conflicts of interest to declare.
Deletion of the Protein Tyrosine Phosphatase Gene PTPN2 in T-Cell Acute Lymphoblastic Leukemia.
Authors: Kleppe Maria , Lahortiga Idoya , El Chaar Tiama , De Keersmaecker Kim , Mentens Nicole , Graux Carlos , Van Roosbroeck Katrien , Ferrando Adolfo A. , Langerak Anton W , Meijerink Jules P.P. , Sigaux Francois , Haferlach Torsten , Wlodarska Iwona , Vandenberghe Peter , Soulier Jean , Cools Jan
Published: October 2019

Abstract 141

Introduction:

T-cell lymphoblastic leukemia (T-ALL) arises from clonal expansion of a lymphoid progenitor that has undergone stepwise alteration at distinct stages of differentiation. It is suggested that a set of cooperative mutations that affect different pathways are required before thymocytes become fully malignant. Despite major improvements in our understanding of the molecular genetics of T-ALL, the underlying mechanisms that lead to the abnormal proliferation and enhanced survival of the leukemic cells remain largely unknown.

Results:

Array CGH analysis revealed an acquired homozygous microdeletion at chromosome 18p11 in 6 % of T-ALL cases. The deleted region was only 125 kb in size and restricted to the PTPN2 (protein tyrosine phosphatase, non-receptor type 2) locus. PTPN2 encodes an intracellular non-transmembrane tyrosine-specific phosphatase that functions as a negative regulator of a variety of signaling proteins including several members of the janus kinase (JAK) and of signal transducer and activator of transcription (STAT) families, growth factor receptors and SRC family kinases. Homozygous deletion of PTPN2 was specifically found in cases with aberrant expression of the TLX1 transcription factor, with two cases also harboring the NUP214-ABL1 fusion. Analysis of additional TLX1 positive cases by quantitative PCR identified loss of one copy of PTPN2 in 5 out of 20 cases. No mutations were detected in the coding region of PTPN2. To determine the effect of loss of PTPN2 in T-cells, we downregulated the expression of PTPN2 using RNAi technology. siRNA mediated knock-down of PTPN2 affected activation of JAK1 associated cytokine receptors implicated in T-cell development. Ligand stimulation of IL7 and interferon gamma receptor resulted in an augmented and prolonged phosphorylation of JAK1 as well as downstream targets STAT1 and STAT5 in T-ALL cell lines with knock-down of PTPN2. In addition, knock- down of Ptpn2 sensitized the pro B-cell line Ba/F3 to transformation by wild type JAK1 confirming a clear relationship between loss of PTPN2 and JAK1 activation. Knock-down of PTPN2 expression also provided a proliferative advantage and reduced sensitivity to kinase inhibitors in lymphoblastic leukemia cell lines HSB-2 and ALL-SIL.

Conclusion:

In conclusion, our data provide genetic and functional evidence for a tumor suppressor role of PTPN2 in T-ALL and warrant testing of JAK inhibitors for the treatment of this specific subset of T-ALLs as well as further analysis of a potential negative impact of loss of PTPN2 on responsiveness to anti-cancer treatments.

Disclosures:

Ferrando: Merck, Pfizer: Research Funding.
The Humanized Multiple Myeloma Mouse Model: Opportunities for Studying the Pathogenesis of MM in Its Natural Environment.
Authors: Groen Richard W.J. , Hofhuis Frans M. , Prins Henk-Jan , Rozemuller Henk , van Kessel Berris , de Weers Michel , Lokhorst Henk , Martens Anton C.M.
Published: October 2019

Abstract 1847

Poster Board I-873

Multiple myeloma (MM), one of the most common hematological malignancies in adults, is a neoplasm of terminally differentiated B cells, i.e. plasma cells. The transition of a plasma cell to a fully transformed, aggressive myeloma is a multistep process, which requires the acquisition of mutations in multiple genes. Most of this evolution takes place in the bone marrow (BM). Studying the pathogenesis of MM is seriously hampered by the lack of appropriate conditions for the engraftment of patient derived MM cells (pMM) which, unlike MM cell lines, strongly depend on a human microenvironment to engraft, survive and expand, indicating that the interaction of MM cells with the cellular and extracellular components of the human BM microenvironment plays a crucial role in the growth behavior of MM cells. To date only few animal models are described that facilitate the outgrowth of pMM, however, they depend on the co-implantation of human fetal tissues or prior implantation of rabbit bone. Moreover, these do not represent the human adult BM microenvironment in which MM cells normally exist (e.g. in the SCID-Rab model and in the NOD-SCID mouse model) or there are ethical concerns (e.g. the SCID-Hu model).

Here we report the development of a unique mouse model to study the pathobiology of MM by implementing a technology for creating a natural human bone environment in the immune deficient RAG2−/−γc−/− mouse. To this end we combined a procedure to culture-expand human BM-derived mesenchymal stromal cells (MSC) that were seeded on biphasic calcium phosphate (BCP) particles and subsequently implanted subcutaneously in RAG2−/−γc−/− mice. Within 6 to 8 weeks this leads to the formation of so-called ossicles that contain substantial amounts of human bone, while the remaining open structures are filled with hematopoietic cells (of mouse origin) at various stages of erythroid, megakaryocytic and myeloid differentiation, creating an environment that strongly resembles the human bone marrow. A stricking finding was that this humanized environment in the mouse proved to be capable to not only support human GFP-luciferase gene-marked MM cell lines but also pMM cells. Intravenous or intracardial injection of MM cell lines or pMM cells showed homing to, engraftment and outgrowth of tumor cells in connection with the human bone layer in the ossicles. Hence, this novel humanized mouse model will provide the first opportunity to investigate patient derived MM plasma cells in their natural environment which may lead to better insights in the pathogenesis of this disease and serve as a model for preclinical testing of new therapeutic approaches for the treatment of Multiple Myeloma patients.

Disclosures:

de Weers: Genmab: Employment.
Stem Cell Transplant in Multiple Myeloma: Impact of Response Failure in the Era of Novel Therapies.
Authors: Gertz Morie A , Kumar Shaji , Lacy Martha Q. , Dispenzieri Angela , Dingli David , Hayman Suzanne R. , Buadi Francis , Hogan William
Published: October 2019

Abstract 1878

Poster Board I-900

Autologous stem cell transplant as a platform for multiple myeloma treatment is the standard of care for patients who can safely withstand the procedure. Before novel agents were introduced, one-third to one-half of patients did not achieve partial response at transplant. Previously published medical literature has showed that in this past era, absence of initial response to induction therapy had no impact on progression-free survival and overall survival after high-dose therapy. Lack of response to initial induction did not preclude a good response after stem cell transplant. With the introduction of novel agents—immunomodulatory drugs and proteasome inhibitors—response rates with initial therapy are now between 70% and 100%. This retrospective study analyzes progression-free survival and overall survival in patients who do not have a partial response after induction therapy with a regimen that contains a novel agent. Unlike patients in reports published previously—before novel agents—patients who do not achieve partial remission have a significantly shorter overall survival from transplant (74.0 vs 43.5 months) and a shorter progression-free survival (22.6 vs 13.1 months; P<.001). Absence of a response to induction therapy with a novel agent predicts a poorer outcome after high-dose therapy.{abstabft}.b CR+VGPR for plateau, P<.001 compared with other 3 categories.

Failure to respond to novel-agent induction leads to shorter posttransplant progression-free survival (PFS).

Failure to respond to novel-agent induction leads to shorter posttransplant overall survivalDisclosures: Gertz: celgene: Honoraria; millenium: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Kumar: celgene: Honoraria; millenium: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Lacy: celgene: Honoraria; millenium: Honoraria.TableMultivariable Analysis of Posttransplant Progression-Free SurvivalVariableP ValuePlateau vs relapsed-refractory.003Albumin.86Sex.94b2-Microglobulin.89Bone marrow plasma cells.18Age.75Abnormal cytogenetics.002CTX mobilization.51Labeling index.002
Effect of Phlebotomy Therapy On Hemoglobin Concentration and Tricuspid Regurgitation Velocity in Chuvash Polycythemia.
Authors: Miasnikova Galina Y , Niu Xiaomei , Nouraie Mehdi , Sergueeva Adelina I , Okhotin Daniel J , Ammosova Tatiana , Nekhai Sergei , Aliyu Zakari , Sable Craig , Dham Niti , Gordeuk Victor R.
Published: October 2019

Abstract 1897

Poster Board I-920

Background:

Chuvash polycythemia is caused by homozygosity for the VHL598C>T mutation, which leads to up-regulation of HIF-1a and HIF-2a in normoxia. As the result, circulating concentrations of erythropoietin are elevated. Chuvash polycythemia patients suffer from cardiovascular abnormalities that include pulmonary arterial hypertension, thrombosis and stroke. Phlebotomy is a common therapy for patients to decrease symptoms such as plethora and headache. However, the outcomes of phlebotomy have not been assessed for these patients. The objective of this analysis is to evaluate the effect of phlebotomy on hemoglobin concentration, serum concentrations of ferritin and erythropoietin, and echocardiographically-determined tricuspid regurgitation velocity, which reflects systolic pulmonary artery pressure.

Methods:

One hundred twenty patients homozygous for VHL598C>T and 38 controls of comparable age and gender from Chuvash Republic of the Russian Federation were studied. Clinical and demographic characteristics were determined and echocardiography was performed. Serum ferritin and erythropoietin concentrations were measured by ELISA.

Results:

The median (interquartile) age for Chuvash polycythemia cases was 36 (22–48) years. They included 68 females (56%). Chuvash polycythemia patients had higher serum erythropoietin concentration (medians of 46 versus 8 mIU/ml, P = 0.0001) and lower serum ferritin concentration (medians of 12 versus 48 ng/ml, P = 0.0001) compared to controls. Tricuspid regurgitation velocity was higher in cases than controls (medians of 2.5 vs. 2.3 m/sec, P = 0.007). Among the cases, 87 (71%) had a history of phlebotomy and 54 of these had phlebotomy within the last year. Phlebotomy was associated with higher erythropoietin concentration (P = 0.033) and lower ferritin concentration (P = 0.024) but no significant difference in hemoglobin concentration (P = 0.9) (Table 1). After adjusting for the effect of age, phlebotomy was associated with significantly higher odds of tricuspid regurgitation velocity ≥2.5 (m/sec) (odds ratio: 3.3; 95% CI: 1.1–9.9).

Conclusion:

Patients with Chuvash polycythemia tend to mobilize iron stores and increase erythropoietin production to maintain a constant, elevated hemoglobin concentration despite phlebotomy therapy. In this process, estimated pulmonary systolic blood pressure appears to increase. Therefore, phlebotomy therapy might be a risk factor for pulmonary hypertension in the context of Chuvash polycythemia.

Disclosures:

No relevant conflicts of interest to declare.
JAK2V617F Mutation Selectively Exerts the STAT3 Pathway for Enhancing a Neutrophil Activation Marker.
Authors: Oku Seido , Takenaka Katsuto , Shide Kotaro , Kumano Takashi , Yoshikane Kikushige , Kuriyama Takuro , Urata Shingo , Iwamoto Chika , Yamauchi Takuji , Shimoda Haruko , Miyamoto Toshihiro , Shimoda Kazuya , Akashi Koichi
Published: October 2019

Abstract 1901

Poster Board I-924

Leukocyte alkaline phosphatase (LAP) is considered as a neutrophil activation marker. The level of LAP is quantitated as the LAP score. It is well known that patients with chronic myelogenous leukemia (CML) usually have low LAP scores, whereas those with BCR-ABL negative chronic myeloproliferative disorders (MPD) have elevated LAP scores. In CML patients, the premature release of granulocytes from the bone marrow into the peripheral blood is considered as the cause of low LAP scores. However, the reason for elevated LAP scores in BCR-ABL negative MPD patients has been unclear. An acquired JAK2V617F mutation is observed in most patients with BCR-ABL negative MPD. It has been shown that the JAK2V617F mutation induces constitutive activation of its downstream signaling pathways such as STAT3/STAT5, Ras/MAPK and PI3K pathways. We speculated that an elevated LAP score might be due to the activation of Jak2 downstream pathways through the JAK2V617F mutation.

We analyzed LAP expression in BCR-ABL negative MPD patients. JAK2V617F homozygous patients had higher LAP expression than JAK2V617F heterozygous or negative patients. AG490, the Jak2 inhibitor, was shown to significantly decrease the LAP expression in neutrophils of JAK2V617F positive patients.

The myeloid cell line NB4 was transfected with the JAK2V617F mutation and a wild-type Jak2 using lentivirus vectors. It was observed that the JAK2V617F mutation, but not wild-type Jak2, enhanced cell proliferation. Then the LAP expression in NB4 cells was evaluated after these cells were differentiated by all-trans retinoic acid and granulocyte colony-stimulating factor. It was observed that the JAK2V617F mutation, but not wild-type Jak2, increased LAP expression. Next, we examined which of the Jak2 downstream pathways played a major role in increasing LAP expression and prompting cell proliferation. By using MEK1/2 inhibitor U0126, PI3K inhibitor LY294002, STAT3 siRNA and STAT5 siRNA, we demonstrated that the JAK2V617F mutation primarily used the STAT3 pathway to increase LAP expression. On the other hand, the JAK2V617F mutation used the STAT5, the Ras/MAPK and the PI3K pathways, but not the STAT3 pathway, to prompt proliferation of NB4 cells.

In conclusion, we obtained direct evidence that the JAK2V617F mutation induced elevation of LAP scores via the STAT3 pathway, and prompted proliferation of NB4 cells via the STAT5, the Ras/MAPK and the PI3K pathways. Our findings showed the possibility that the JAK2V617F mutation might use specific downstream pathways depending on various phenotypic manifestations of BCR-ABL negative MPD.

Disclosures:

No relevant conflicts of interest to declare.
p53-Inducible Micrornas 192 and 215 Regulate p53 Expression and IGF1 Axis in Multiple Myeloma.
Authors: Pichiorri Flavia , Suh Sung-Suk , De Luca Luciana , Taccioli Cristian , Benson Don , Hofmeister Craig , Aqeilan Rami , Croce Carlo M.
Published: October 2019

Abstract 1973

Poster Board I-996

Multiple myeloma (MM) is an incurable neoplastic disease of B cell origin, affecting 50,000 patients in the United States, occurring in approximately 16,000 new individuals each year. In MM, mutation or deletion of p53 is rarely detected at diagnosis, although both become more frequent in advanced disease. Therapeutic activation of p53 might therefore be particularly suitable for the treatment of MM. The discovery of microRNAs (miRNAs) has revealed a new level of regulation of protein expression. We have shown that an altered miRNA expression profile plays a role in the malignant transformation of plasma cells (PCs); we recently published the first comprehensive global microRNA expression profiles of MM, monoclonal gammopathy of undetermined significance (MGUS) and contrasted these expression patterns with that of normal plasma cells (PCs). Now we have used nutlin-3a, a recently developed small-molecule activator of p53, that efficiently disrupts the p53-MDM2 interaction, to profile a p53 miRNA activation pathway in the MM1s cell line. Results of the analysis have shown differential expression of 46 individual miRNAs after p53 activation, 14 showing up-regulation and 32 showing down-regulation. We confirmed our data by qRT-PCR in p53 wild type and mutated MM cell lines and in primary MM cells. Our results confirm published results; after p53 stabilization we found up-regulation of the miR-34a, but we also detected strong up-regulation of several other miRNAs. In fact we found that p53 induces two additional, mutually related clusters of miRNAs, leading to the up-regulation of miR-192, miR-194, and miR-215. The same miRNAs were detected at high levels in normal plasma cells (n.3) but were severely reduced in many untreated MM samples (n.22). Here we characterize the promoter and the p53 activation site for miR-192-194 cluster on chromosome 11(q13.1) but we have also confirmed the previous published p53 consensus site for miR-215-194 cluster on chromosome 1(q41) in multiple myeloma cells. Furthermore we found that miR-192 and its cousin miR-215 but not miR-194 can each contribute to enhanced CDKN1A/p21 levels, colony suppression, cell cycle arrest in multiple myeloma cells acting directly on p53 regulation. On the other hand, we define that IGF-1 and IGFR are direct targets of miR-192-215. In fact the re-expression of these miRNAs in multiple myeloma cells blocks their migration through both endothelial barriers and associated bone marrow stroma. Our data suggest that downstream signaling in the p53 pathway is functional in MM cells and able to induce miRNAs that play a pivotal role in cell cycle progression, mobility and invasive properties of these cells. These studies provide the basis for the development of new miRNA-targeted therapies for MM.

Disclosures:

No relevant conflicts of interest to declare.
Compound Deficiency of Nrf2 and Selenoproteins in Hematopoietic Cells Leads to Severe Defects in Erythroid and Lymphoid Lineage Development.
Authors: Kawatani Yukie , Suzuki Takafumi , Shimizu Ritsuko , Kelly Vincent , Yamamoto Masayuki
Published: October 2019

Abstract 1993

Poster Board I-1015

Precise regulation of reactive oxygen species (ROS) level is essential for normal cellular homeostasis. Defect of ROS scavenging system results in excess accumulation of ROS in cells, leading to the cellular aging and senescence. Nrf2 (NF-E2-related factor 2) is a basic leucine zipper-type transcription factor, which regulates expression of a set of genes related to the ROS scavenging and detoxification. While accumulating lines of evidence support the significance of Nrf2 for the cellular protection in many types of cells, Nrf2-deficient mice live out their natural life span under the homeostatic condition, and show no significant hematological abnormality except for anemia with aging. We previously found that the Nrf2 activity rose in the macrophages and liver cells derived from conditional knockout (CKO) mice of the selenocystein tRNA (Trsp) gene. The Trsp gene is essential for production of selenoproteins, including glutathione peroxidase and thioredoxin reductase. Thus, there are two prevalent ROS scavenging systems based on different molecular mechanisms; Nrf2 and selenoproteins seemingly act compensatory and cooperatively for maintenance of hematopoietic homeostasis. To explore the roles Nrf2 and selenoproteins play in hematopoietic cells, we conducted conditional knockout of the Trsp gene by crossing Trsp-floxed mice with the interferon-inducible Mx1-Cre transgenic mice in combination with Nrf2 gene knockout (KO) in germline. The Trsp-CKO mice in the background of Nrf2 wild type (Trsp single KO mice) showed thymic atrophy with massive reduction of CD4/CD8 double-positive cells in thymus. This T-lymphocytic phenotype was not changed in the Trsp-CKO mice under the circumstance of Nrf2-null condition (Trsp:Nrf2 double KO mice), suggesting that selenoproteins were prerequisite for the T-cell development. The Trsp single KO mice suffered from mild anemia with increased number of erythroid progenitors in bone marrow and spleen, and importantly this erythroid phenotype was dramatically exacerbated in the Trsp:Nrf2 double KO mice. A large number of poikilocytes and teardrop-shaped cells were observed in the blood smear of the Trsp:Nrf2 double KO mice, whereas these deformed red blood cells were vaguely found in the Trsp single KO mice. Intracellular ROS level was significantly elevated in erythroid progenitors recovered from the Trsp:Nrf2 double KO mice, although that of the Trsp single KO mice were maintained within normal level, indicating that Nrf2 compensates for the defect of ROS scavenging function caused by the loss of selenoproteins in erythroid cells. These results thus demonstrate for the first time that the ROS scavenging conducted by Nrf2 and selenoproteins is essential for the homeostatic maintenance in erythroid and lymphoid cells in a lineage-specific manner.

Disclosures:

No relevant conflicts of interest to declare.
Both Nramp1 and Dmt1 Are Necessary for Efficient Macrophage Iron Recycling.
Authors: Soe-Lin Shan , Apte Sameer S , Mikhael Marc R , Kayembe Lidia , Nie Guangjun , Ponka Prem
Published: October 2019

Abstract 1994

Poster Board I-1016

Divalent metal transporter 1 (DMT1) and natural resistance-associated macrophage protein 1 (Nramp1) are iron transporters that localize, respectively, to the early and late endosomal compartments. DMT1 is ubiquitously expressed, while Nramp1 is found only within macrophages and neutrophils. Our previous studies have identified a role for Nramp1 during macrophage erythrophagocytosis; however, little is known about the function of DMT1 during this process. Wild-type RAW264.7 macrophages (Nramp1-/-), and those stably transfected with Nramp1 (Nramp1+/+) were treated with either DMT1-siRNA, or with ebselen, a selective inhibitor of DMT1. While macrophages lacking either functional DMT1 or Nramp1 experienced a moderate reduction in iron recycling efficiency, the ability of macrophages lacking both functional DMT1 and Nramp1 to recycle hemoglobin-derived iron was severely compromised. Compared to macrophages singly deficient in either DMT1 or Nramp1 transport ability, macrophages where DMT1 and Nramp1 were both compromised exhibited an abrogated increase in labile iron pool content, released less iron, and experienced diminished upregulation of ferroportin and heme-oxygenase 1 levels following erythrophagocytosis. These results suggest that while the loss of either Nramp1 or DMT1 transport ability results in minor impairment following erythrophagocytosis, the simultaneous loss of both Nramp1 and DMT1 iron transport activity is detrimental to the iron recycling capacity of the macrophage.

Disclosures:

No relevant conflicts of interest to declare.
Deciphering Mitochondrial Iron Metabolism Using a Knockout Mouse.
Authors: Huang Michael , Becker Erika , Whitnall Megan , Rahmanto Yohan Suryo , Ponka Prem , Richardson Des R.
Published: October 2019

Abstract 1995

Poster Board I-1017

We utilized the muscle creatine kinase conditional frataxin knockout mouse to elucidate how frataxin-deficiency alters iron metabolism. This is of significance since frataxin-deficiency leads to the neuro- and cardio-degenerative disease, Friedreich's ataxia. Using cardiac tissues, we demonstrate that frataxin-deficiency leads to down-regulation of key molecules involved in three mitochondrial utilization pathways: iron-sulfur cluster (ISC) synthesis (iron-sulfur cluster scaffold protein1/2 and the cysteine desulferase, Nfs1); mitochondrial-iron storage (mitochondrial ferritin); and heme synthesis (5-aminolevulinate dehydratase, coproporphyrinogen oxidase, hydroxymethylbilane synthase, uroporphyrinogen III synthase and ferrochelatase). This marked decrease in mitochondrial-iron utilization and resultant reduced release of heme and ISC from the mitochondrion could contribute to the excess mitochondrial-iron observed. Indeed, this effect is compounded by increased iron availability for mitochondrial uptake through: (1) transferrin receptor1 up-regulation that increases iron uptake from transferrin; (2) decreased ferroportin1 expression, limiting iron export; (3) increased expression of the heme catabolism enzyme, heme oxygenase1, and down-regulation of ferritin-H and —L, both of which likely lead to increased “free iron” for mitochondrial uptake; and (4) increased expression of the mammalian exocyst protein, Sec15l1, and the mitochondrial-iron importer, mitoferrin-2 (Mfrn2), that facilitate cellular iron uptake and mitochondrial-iron influx, respectively. This study enables construction of a model explaining the cytosolic iron-deficiency and mitochondrial-iron-loading in the absence of frataxin that is important for understanding the pathogenesis of Friedreich's ataxia.

Disclosures:

No relevant conflicts of interest to declare.