bluuebunny/crossref_metadata_2025 huggingface dataset:
TFPI Antagonist Aptamer ARC19499 Is a Procoagulant In Vitro and In Vivo.
Authors: McGinness Kathleen E. , Waters Emily K. , Genga Ryan M. , Olson Karen A. , Nelson Jennifer A. , Kurz Jeffrey C. , Schaub Robert G.
Published: October 2019

Abstract 1134

Hemophilia is a bleeding disorder that results from deficiencies in coagulation factor VIII (FVIII; hemophilia A) or factor IX (FIX; hemophilia B), primarily impacting the intrinsic coagulation pathway. The extrinsic coagulation pathway remains intact in hemophiliacs and is negatively regulated by tissue factor pathway inhibitor (TFPI). The primary role of TFPI is regulation of the FVIIa:tissue factor complex through inhibition of FVIIa and FXa. Inhibition of TFPI may provide an effective treatment for hemophilia by allowing sufficient thrombin generation via the extrinsic coagulation pathway to bypass the defect in clot propagation caused by deficiency of FVIII or FIX.

ARC19499 is an aptamer that is a potent, specific inhibitor of TFPI. We have employed a variety of in vitro and in vivo methods to demonstrate that ARC19499 mediates a procoagulant effect in hemophilic plasma and in a model of hemophilia A in non-human primates (NHP). Plasma-based experiments that measure thrombin generation over time demonstrate that ARC19499 mediates a procoagulant response in both hemophilia A and hemophilia B plasma, restoring thrombin generation to near normal levels at 10 – 100 nM aptamer. Experiments in TFPI-depleted plasma and in vitro binding experiments demonstrate that this procoagulant effect is dependent on the specific interaction between ARC19499 and TFPI. ARC19499 restores thrombin generation to a level that is equivalent to or better than 14% FVIII replacement at a concentration of 30 nM in hemophilia A plasma and has an additive effect on thrombin generation when used in combination with FVIII. In a NHP model of hemophilia, cynomolgus monkeys acquire a hemophilia A-like state following administration of an anti-FVIII antibody as measured by ex vivo thromboelastography (TEG). TEG coagulation parameters are corrected following administration of ARC19499. In addition, antibody-mediated FVIII depletion moderately prolongs saphenous-vein bleeding time. ARC19499 corrects bleeding times to normal.

These experiments demonstrate that the anti-TFPI aptamer ARC19499 mediates a procoagulant hemostatic effect both in vitro and in vivo in hemophilia model systems that is dependent on specific interaction between the aptamer and TFPI. ARC19499 may provide an effective alternative to replacement factors and bypassing agents for the treatment of hemophilia. Additionally, aptamer therapeutics have the advantage of subcutaneous bioavailability which provides an opportunity for improved treatment regimens in hemophilia.

Disclosures:

McGinness: Archemix Corporation: Employment. Waters:Archemix Corporation: Employment. Genga:Archemix Corporation: Employment. Olson:Archemix Corporation: Employment. Nelson:Archemix Corporation: Employment. Kurz:Archemix Corporation: Employment. Schaub:Archemix Corporation: Employment.
Structural Insights Into How Clotting Proteins with GLA Domains Bind to Membrane Surfaces.
Authors: Davis-Harrison Rebecca L , Tavoosi Narjes , Clay Mary , Boettcher John M , Rienstra Chad M , Morrissey James H.
Published: October 2019

Abstract 1141

Most steps in the blood coagulation cascade obligatorily take place on membrane surfaces and are dependent on the exposure of phosphatidylserine (PS). Many coagulation proteins bind to PS-containing membrane bilayers in a calcium-dependent manner via gamma-carboxyglutamate-rich (GLA) domains. In spite of their importance, a clear picture of how GLA domains bind to the membrane interface has yet to emerge. A further intriguing aspect of the membrane's role in blood coagulation is that certain phospholipids, most notably phosphatidylethanolamine (PE), strongly synergize with PS to promote clotting reactions. The mechanisms of this synergy, and of PE's contribution to GLA domain binding, are poorly understood – although a number of hypotheses have been put forward. We now propose a new hypothesis to explain GLA domain binding to membranes, which we term the ABC (Anything But Choline) hypothesis; it invokes two main types of protein-phospholipid interactions: a single L-serine-specific binding site in each GLA domain; and multiple “phosphate-specific” interactions in which the phosphate groups of non-phosphatidylcholine phospholipids form coordination complexes with the tightly bound calcium ions in GLA domains. We have utilized liposomes and nanoscale phospholipid bilayers (Nanodiscs) in studies employing a series of techniques including solid-state NMR (SSNMR) and surface plasmon resonance (SPR) to address the mechanism of GLA domain-membrane interactions. We provide direct evidence in favor of the ABC hypothesis for GLA domain binding to membrane surfaces. Using SSNMR, we demonstrate that two distinct PS headgroup conformations are induced by binding of calcium ions, and that a third, novel PS headgroup conformation is induced when the prothrombin GLA domain engages the membrane. SPR studies have allowed for the determination of thermodynamic profiles of GLA domains interacting with phospholipid bilayers containing PS and/or PE, providing further insights to the mechanisms of GLA domain-membrane interactions.

Disclosures:

No relevant conflicts of interest to declare.
Loss of Foxo3a In FA Mice Leads to Hematopoietic Stem Cell Exhaustion That Can Be Attenuated by Natural Anti-Oxidant Quercetin.
Authors: Li Jie , Sipple Jared , Pang Qishen
Published: October 2019

Abstract 1166

Fanconi anemia (FA) is a genetic disorder characterized by genomic instability, bone marrow (BM) failure and predisposition to cancer. However, FA mouse models do not show spontaneous genetic instability. Previous study shows that FOXO3a is associated with the FA pathway through oxidative stress-specific interaction with FANCD2. To address the consequence of loss of FOXO3a function in FA hematopoiesis, we generated Foxo3a-/-Fancd2-/- and Foxo3a-/-Fancc-/- double-knockout (DKO) mice by crossing Foxo3a+/− with Fancd2+/− or Fancc+/−; mice. Reactive oxygen species are increased in low-density BM (LDBM) cells isolated from DKO mice compared to those from single KO (SKO) or wt mice. Analysis of hematologic parameters shows significantly increased number of nucleate cells and high ratio of eosinophils in peripheral blood of DKO mice. CFU assay shows more progenitor cells in peripheral blood isolated from DKO mice. Moreover, BM progenitor cells from DKO mice exhibit lower adhesion but higher migration activity, compared to those from wt or SKO mice. Consistent with this, Cdc42 pull-down assay shows lower Cdc42 activity in DKO LDBM cells than in wt or SKO cells, indicating that decreased Cdc42 may contribute to the observed aberrant adhesion and migration activities. DKO mice show significant decrease in primitive progenitor (Lin-Sca-1+c-kit+; LSK) cells, increase in BrdU+ and G1-phase LSK cells, and impaired repopulating capacity after competitive BM transplantation, which can be attenuated by the anti-oxidant Quercetin. Taken together, loss of Foxo3a in FA mice results in FA-like syndrome, which may be resulted from increased reactive oxygen species accumulation.

Disclosures:

No relevant conflicts of interest to declare.
Disturbed NK Cell Compartment In Human CML and Bcr-Abl Positive Mice.
Authors: Chen Christiane I-U , Koschmieder Steffen , Kamp Linda , Altvater Bianca , Pscherer Sibylle , Maecker Holden , Berdel Wolfgang E. , Juergens Heribert , Lee Peter P , Rossig Claudia
Published: October 2019

Abstract 1207

Targeted pharmacologic therapy with tyrosine kinase inhibitors (TKIs) has become the first-line treatment for patients with CML. However, BCR-ABL+ stem cells resist elimination by continuous TKI treatment in most patients. By contrast, graft-versus-leukemia effects during allogeneic hematopoietic stem cell transplantation can eradicate the disease, suggesting an important role of the immune system in this disease. Besides cytotoxic T cells, natural killer (NK) cells may be involved in immune control of CML. Here, we explored numbers and functionality of NK cells in CML patients and in a transgenic inducible BCR-ABL mouse model.

In 18 patients with newly diagnosed chronic-phase CML, the relative proportions of peripheral blood CD56+CD3- NK cells within the lymphocyte gate were significantly reduced compared to age-matched healthy controls (7.0 ± 5.8% versus 13.1 ± 5.1, p=0.005) and did not recover to normal levels during imatinib-induced remission (9.2 ± 5.9%, p=0.024, follow-up 10–59 months). Functional experiments showed reduced in vitro expansion of CML NK cells at diagnosis in response to stimulation with 4-1BBL/mbIL-15 transduced K562 cells (23.5 ± 14.46 fold vs 41.2 ± 7.2 fold, p=0.013) and under imatinib treatment (31.5 ± 10.5 fold, p=0.03), and a reduced degranulation response to K562 target cells by CD107 upregulation (2.8 ± 2.7% at diagnosis and 9.0 ± 13.2% under treatment, vs. 19.1 ± 8.0% in controls, p=0.003 and p=0.045, respectively).

To investigate, whether the defective NK-cell compartment in CML is a consequence of the characteristic BCR-ABL-induced myeloproliferation, we addressed the quantity and functionality of NK cells in a double transgenic mouse model of human CML. Consistent with the results in human CML, the relative proportions of NK1.1+ NK cells among total splenic lymphocytes were significantly reduced in BCR-ABL induced mice (6.4 ± 3.5% vs. 14.7 ± 1.8%, p=0.005). Moreover, compared to NK cells isolated and expanded from BCR-ABL-non-induced control mice, the degranulation response of splenic NK cells from BCR-ABL+ mice to YAC-1/NIH-3T3 cells was significantly decreased (25.7 ± 1.6% vs 42.4 ± 5.6%, p=0.002), and analogous results were obtained with NK cells expanded from bone marrow of these mice (7.7 ± 4.9% vs. 25.0 ± 7.1%, p=0.033).

These results suggest both quantitative and qualitative defects within the NK cell compartment in CML. Further work will aim at identifying the underlying mechanisms of the NK cell deficiency in CML, and the development of strategies to utilize NK cells for immunotherapy of CML.

Disclosures:

Koschmieder: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees.
Combination of Mycophenolate Mofetil with Cyclosporine A and Methotrexate as Gvhd Prophylaxis In Unrelated Donor Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Zhu Xiaoli , Lai Xiaoyu , Luo Yi , Shi Jimin , Tan Yamin , Huang He
Published: October 2019

Abstract 1280

Graft-versus-host disease (GVHD) is the dominant complication limiting the efficacy and safety of hematopoietic stem cell transplantation (HSCT), especially from unrelated donors. The most widely used regimen of prophylaxis is the combination of cyclosporine A (CSA)and a short-course of methotrexate (MTX). But the patients receiving grafts from matched unrelated donors still have a 50–80% risk of clinical significant GVHD. Thus, more effective prophylaxis is needed. In this study, we try to evaluate the efficacy and safety of the new GVHD prophylaxis regimen combining mycophenolate mofetil (MMF) with CSA and MTX in unrelated donor allogeneic stem cell transplantation. Between November 1998 and June 2008, 153 patients with a variety of hematologic malignancies were enrolled. There were 102 males and 51 females, median age 26 (range, 8–52) years. Diagnoses were acute myeloid leukemia (n=54), acute lymphoblastic leukemia (n=47),myelodysplastic syndrome (n=8), multiple myeloma (n=1) and chronic myeloid leukemia (n=53). One hundred and eight patients had HLA compatible unrelated donors, 37 patients had single locus-mismatched unrelated donors and 8 patients had 2 loci-mismatched unrelated donors. GVHD prophylaxis consisted of CSA,MTX and MMF. CSA was started on day -7 with the initial dosage of 2.5mg/kg daily, and the dose was adjusted to maintain a whole blood steady-state level of 200–400ng/ml. The dose was tapered during the second to third month post-transplant depending on GVHD status. MTX was given at a dosage of 10mg on days+1, +3 and +6. MMF was administered at an oral dose of 500mg daily from day 0 to day +100. The cumulative incidence of grades II-IV aGVHD and grades III-IV aGVHD for all patients were 43.9% and 17.9% respectively for entire group. The cumulative incidence of grades II-IV aGVHD for patients who had HLA-matched donors and mismatched donors were 40.2% and 51.9% respectively (P=0.079). The cumulative incidence of grades III-IV aGVHD for patients who had HLA-matched donors and mismatched donors were 14.0% and 26.9% respectively (P=0.026). The cumulative incidence of cGVHD was 41.4%, with 19.3% extensive cGVHD. At 3 years, the incidence of relapse for entire group was 24.0%. The cumulative incidence of transplant-related mortality at 100 days and 3 years were 12.5% and 34.9%, respectively. The probabilities of overall survival at 2 years and 5 years were 59.2% and 50.2%, respectively. The probabilities of disease-free survival at 2 years and 5 years were 56.3% and 46.7%, respectively. From these results, we conclude that the combination of MMF with CSA and MTX is an effective prophylaxis regimen for acute and chronic GVHD in unrelated donor allogeneic stem cell transplantation without increasing the risk of relapse.

Disclosures:

No relevant conflicts of interest to declare.
Long Term Study of the Impact of Quantitative Molecular Monitoring of Bcr-Abl Transcripts on the Risk of Relapse of CML After Allogeneic HSCT.
Authors: Arpinati Mario , Amabile Marilina , Bochicchio maria Teresa , Poerio Angela , Bandini Giuseppe , Bonifazi Francesca , Stanzani Marta , Castagnetti Fausto , Rosti Gianantonio , Martinelli Giovanni , Baccarani Michele
Published: October 2019

Abstract 1287

Background:

Monitoring of minimal residual disease through RT-PCR analysis of bcr-abl transcripts allows early detection of CML relapse after allogeneic HSCT. However, the introduction of more sensitive techniques, such as quantitative PCR, may result in decreased specificity, leading to false positive results.

Patients and methods:

In this study we reviewed the results of molecular analysis of bcr-abl transcripts in all patients with p210+ CML who underwent allogeneic HSCT from 1983 through 2007. Q-PCR analysis was started in 2002. Out of 189 patients, 87 patients had available Q-PCR data; of these, 63 patients with at least three separate Q-PCR data were included in the study. Median time to the 1st Q-PCR analysis was 2196 days (35-7823). Median age was 36 years (13-56), 62%/38% received transplant from a related/unrelated donor, 62% with BM. 32% were in accelerated phase (AP). Patients with at least one positive Q-PCR value (measured as a ratio of bcr-abl to abl of > 0.001) were classified as Major Molecular Remission (MMR) patients. Each event was defined as one or more consecutive positive results.

Results:

60/63 patients are alive after a median follow up of 3693 days (898-9405). 6 have relapsed 2142 (1419-3746) days after transplant. 52 (83%) patients had at least one positive result (28 with a value of >0.01, 24 with a value of <0.01), whereas 11 (17%) had persistent undetectable transcripts. In MMR patients, 94 events occurred. 29 patients had only one event, while 6 had >3 events. In 10 patients, the event occurred within 1 year after transplant, whereas in 28 it occurred after >5 years. 6/52 MMR patients relapsed, as compared to 0/11 with persistent undetectable transcripts (p=0.19). Relapse did not correlate with the Q-PCR value, the number of events or the time to the event. Finally, of 46 MMR patients who did not relapse, 35 had undetectable transcripts at last follow up. Positive Q-PCR had low specificity (19%) and positive predictive value (12%) in predicting relapse after allogeneic HSC transplantation.

Conclusion:

Our data confirm that the detection of low levels of bcr-abl transcripts (as based on Q-PCR) has a poor accuracy in predicting relapse, and it should not be considered as the sole indication to start treatment. It appears that fluctuation of bcr-abl transcript levels is common as late as > 10 yrs after transplant, possibly suggesting the long term persistence of CML stem cells.

Disclosures:

Rosti: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.
The Cutoff Leukemic Cell Burden to Influence Outcomes of Allogeneic Stem Cell Transplantation for Acute Myelogenous Leukemia In Hematological Complete Remission.
Authors: Lee Sung-Eun , Kim Hee-Je , Min Woo-Sung , Yahng Seung-Ah , Cho Byung-Sik , Eom Ki-Seong , Kim Yoo-Jin , Min Chang-Ki , Lee Seok , Cho Seok-Goo , Kim Dong-Wook , Lee Jong-Wook , Park Chong-Won
Published: October 2019

Abstract 1298

Background:

Pre-transplant disease status is considered a critical prognostic factor of patients undergoing allogeneic stem cell transplantation (SCT) for AML. The previous studies about the impact of leukemic cell burden on transplant outcomes were based on hematological complete remission (CR), which requires <5% blasts in a bone marrow aspirate. This study was performed to evaluate 2 questions: (1) Can we discriminate the impact of leukemic cell burden from CR status according to current hematological CR definition? (2) Is it possible that the poor outcomes by leukemic cell burden are overcome by the graft-versus-host disease (GVHD)-related graft-versus-leukemic (GVL) effect?

Methods:

We retrospectively analyzed 363 patients with de novo AML who underwent allogeneic SCT between January 2002 and December 2008. There were 183 men and 180 women with a median age of 37 years (range 15–68).

Results:

To compare outcomes according to blast counts in patients who were in CR at bone marrow aspirate just before allogeneic SCT, we divided the patients into subgroups which had >2 - <3% blasts (n = 17), >3 - <5% blasts (n = 8) by plotting a receiver operating characteristic (ROC) curve. At a median follow-up of 37.1 months (range: 0.2–90.1) for survivors, there were significant differences in overall survival (OS) (68.3 vs 37.5, P= 0.075), event-free survival (EFS) (68.3 vs 18.8, P= 0.022), relapse (14.1 vs 64.3, P= 0.063) between two groups. In Multivariate analysis for the patients in CR status just before allogeneic SCT, <3% blasts remained a significant favorable independent variable influencing EFS after adjusting for significant pre-transplant variables in compare with the group which had >3 - <5% blasts [HR (95% CI) 4.21 (1.46-12.1), P=0.008] and the development of chronic GVHD was associated with lower relapse [HR (95% CI) 2.93 (1.7-5.06), P=<0.000], which was translated into improved OS [HR (95% CI) 2.0 (1.28-3.16), P=0.003], EFS [HR (95% CI) 2.24 (1.46-3.43), P=<0.001].

Conclusion:

These results indicate that pre-transplant bone marrow status is an important prognostic factor for patients with AML, and even among the patients in CR, strictly more than 3% blasts is suggested as a poor prognostic factor. Therefore, the impact of leukemic cell burden on outcomes of allogeneic SCT for AML should be considered to make more defined SCT plan. Further studies for treatment strategies to enhance the GVL effect according to leukemic cell burden before transplant are needed.

Disclosures:

No relevant conflicts of interest to declare.
Poor FEV1 or DLco on Pre-Transplant PFT Does Not Compromise Outcomes In Patients Undergoing a Reduced-Intensity Allogeneic Progenitor Cell Transplant.
Authors: Sharma Manish , Saliba Rima M. , Qazilbash Muzaffar H. , Okoroji Grace-Julia , Popat Uday , de Lima Marcos , Champlin Richard , Hosing Chitra
Published: October 2019

Abstract 1322

Background:

Pre-transplant work-up prior to an allogeneic stem cell transplant (allo-SCT) generally includes pulmonary functions tests (PFT) with assessment of FEV1 and DLco. It has been previously published that patients with a DLco or FEV1 less than 60% of predicted have higher non-relapse mortality (NRM) when undergoing a myeloablative allo-SCT. However, the PFT criteria for selecting candidates for a reduced intensity (RI) allogeneic transplant allo-SCT remains poorly investigated. We performed a retrospective analysis to assess the impact of low FEV1 or DLco on NRM and overall survival (OS) in patients undergoing a RI allo-SCT.

Methods:

All patients who underwent a RI allo-SCT for hematological malignancies at MD Anderson Cancer Center from Jan 2000 to April 2009 were included. Using Cox's proportional hazards regression, we compared the rate of NRM and OS between patients who had a pre-transplant FEV1 or DLco > 50% (control group) prior to a RI allo-SCT and those with FEV1 and DLco < 50% (study group). The cumulative incidence of NRM was estimated considering disease progression as a competing risk. Actuarial OS was estimated by the Kaplan-Meier method.

Results:

Patient characteristics are presented in Table 1. There were no statistically significant differences between the study and control groups except for age and sex. A significantly higher proportion of patients were > 50 years in the control group (65%) compared with the low PFT group (45%, P=0.02) and there were more males in the control group (P=.001). The median FEV1 and DLco in the study group was 55% (28-111) and 47% (33-98) of predicted, respectively. On univariate analysis abnormal PFTs did not impact OS at 30 months (HR 1.1, 95% CI 0.7–1.8, P=0.7) or NRM at 1 year (HR 1.3, (95% CI 0.6–2.7, P=0.4). Active disease at SCT, and a diagnosis of CLL, NHL or HL were associated with significantly worse OS and NRM, whereas age >50 years was only associated with worse OS. Low PFT had no significant impact on OS and NRM when evaluated separately in patients older or younger than 50 years old.

Conclusion:

Our experience suggests that a low (<50% of predicted) FEV1 or a DLco on pre-transplant evaluation does not adversely impact NRM and OS after RI allo-SCT. These findings need to be validated in a multivariate analysis.

Disclosures:

No relevant conflicts of interest to declare.
Germinal Center Specific Activation of K-Ras, Common In Multiple Myeloma, Is Selected Against and Is Not Sufficient to Initiate Plasma Cell Transformation In Mice
Authors: Mullins Chelsea D. , Tomasson Michael , Lu Lan , Su Mack Y. , Hutchagowder Vishwanathan , Vij Ravi , Kulkarni Shashikant , Chu Liang
Published: October 2019

Abstract 137

Activating mutations in Ras (N- and K-) are the most common mutations known in multiple myeloma (MM), and are associated with advanced clinical stage and poor outcomes. We re-sequenced coding regions of highly expressed cytokine signaling candidate genes from MM patient samples and found rare novel mutations. Still, the most common recurrent somatic mutations we found affected N and K-Ras. Previous studies of MM patient samples suggest that Ras mutations may be late/progression events, but the role of Ras activation in MM pathogenesis has not been tested directly. Toward this end, we generated double-transgenic mice by crossing Lox-stop-lox(LSL)- K-ras G12D knock-in mice (K-ras+/G12D) with Cγ1-Cre knock-in mice (Cγ1+/Cre) expressing the Cre recominase specifically in germinal center (GC) B-cells. K-ras+/+/Cγ1+/Cre mice served as negative controls. We found 58% of unstimulated K-ras+/G12D/Cγ1+/Cre mice developed fatal double-positive CD4/8 T-cell lymphomas (n=12) and 42% developed lung adenocarcinomas (n=12). The median survival of these mice with lymphomas was 19 weeks, compared to the 300 day end point of unstimulated K-ras+/+/Cγ1+/Cre control mice. Tumor cells from these mice demonstrated successful recomination of the K-Ras locus. In contrast, no evidence of multiple myeloma or monoclonal gammopathy was found by serum ELISA, SPEP, flow cytometry or histologic examination. We isolated B-cell subsets from K-ras+/G12D/Cγ1+/Cre mice and documented successful recombination in splenic germinal center cells (B220+, GL-7+) but not in bone marrow plasma cells. Together, these data suggest that Ras activation by “leaky” off-target Cre expression was sufficient to initiate tumorigenesis in two non-B-cell compartments, but that activation of the K-rasG12D allele in germinal center B-cells was associated instead with clearance of recombined cells. We conclude that activating Ras mutations are disease progression events in MM and that earlier mutations are required before primary plasma cells are transformed. This novel mouse model will be useful for identifying and testing early genetic events that drive MM development in cooperation with Ras.

Disclosures:

No relevant conflicts of interest to declare.
Type 3 Von Willebrand Disease with Alloantibodies and Its Challenging Management During Episode of Bleeding
Authors: Verma Anupam , Yaish Hassan , Rodgers George M.
Published: October 2019

Abstract 1405

Type 3 Von Willebrand Disease (VWD) is the most severe and least common of the three VWD types. The incidence varies by ethnicity ranging from 0.1–5.3 cases/million. 8.5% of patients develop alloantibodies to Von Willebrand Factor (VWF), becoming non-responsive to treatment or prone to life threatening reactions. Predisposing factors include extent of previous exposure to VWF and mutations causing a null-allele gene. Variability of the clinical course is attributed to different antigenic specificities and potency of the antibodies. Management of these patients during events of bleeding is challenging. We report 2 cases of girls with type 3 VWD. The first was diagnosed at birth and had her initial VWF transfusion as a neonate. The second was diagnosed at 4 years of age. Both were frequently treated with VWF and developed alloantibodies. The clinical course, severity and circumstances leading to the diagnosis were different in both cases. Life threatening anaphylactic reactions like hypotension, bronchospasm and tongue edema was noticed in the first. The second showed poor factor recovery and response to treatment with urticaria. She continued to tolerate therapy but failed tolerance induction. Peak antibody level was 84 BU compared to 3 BU in the first. Novel VWF gene mutations were detected in both. Recently the first patient was managed for profuse bleeding post extensive dental procedure. Recombinant Factor VIII infusion was administered every 3 hours with monitoring of aPTT and Factor VIII levels. Based on peak and post 3 hour levels of Factor VIII, we concluded the half life of Factor VIII could be as short as 2 hours in patients with type 3 VWD. Post infusion peak level might approach 200% in some instances. Due to persistent bleeding despite adequate correction of Factor VIII and aPTT, platelet transfusion was cautiously administered which proved to be not only effective in controlling bleed but was also safe and not associated with any anaphylactic reaction. In conclusion, patients with type 3 VWD and alloantibodies can be successfully managed with aggressive frequent therapy with recombinant Factor VIII infusions. Platelet transfusions during episodes of severe bleeding can also be safe and effective.Administration of Factor VIII by continuous infusion could be an option if high post infusion peaks are to be avoided.

Disclosures:

No relevant conflicts of interest to declare.
Tacrolimus Induced Refractory Immune Thrombocytopenia.
Authors: Alkhateeb Hassan , Manchanda Naveen , Mehta Rakesh P
Published: October 2019

Abstract 1426

Tacrolimus, a calcineurin inhibitor, has become a standard immunosuppressive therapy in solid organ transplantation. It is known to cause a thrombotic microangiopathy, and has been implicated to cause immune thrombocytopenia (IT) in children. Our institution has recognized a delayed onset IT in adult liver transplant patients that appears associated with chronic therapy with Tacrolimus. Between 2007–2009, 4 patients (3 females and one male) between the ages of 28–71 years who were status post orthotopic liver transplant and were on Tacrolimus developed a refractory IT. The onset of the IT was 3–12 years after starting on the Tacrolimus. One of the patients had a history of an autoimmune hemolytic anemia, which occurred one month after transplantation and resolved with IVIG therapy. Two of the patients were diagnosed with Hepatitis C reactivation after transplantation, but were not on therapy before the onset of IT. At presentation, the platelet counts were between 2,000 – 4,000/μL. IT was diagnosed after ruling out other causes of thrombocytopenia. Bone marrow biopsies were done in all four patients, and were consistent with increased platelet destruction. The thrombocytopenia was refractory to conventional IT treatment with steroids and IVIG. Other therapies included Rituximab (3 patients), Vincristine (2 patients), and splenectomy (1 patient), but none of these significantly improved the platelet count. However, switching the Tacrolimus to Cyclosporine in all the patients led to a rise in platelet count within 6 days to two months. These patients have been observed for over 1 year without a recurrence of their IT. One patient requires chronic therapy with Romiplostim, while being treated with Ribavirin and Interferon for his Hepatitis C reactivation. His platelet count has not fallen below 100k/μL. CONCLUSION: Tacrolimus may cause a refractory IT that occurs several years after its initiation as an immunosuppressant. When this situation occurs, discontinuing the Tacrolimus and replacing the immunosuppression with Cyclosporine is the most effective therapeutic approach.

Disclosures:

No relevant conflicts of interest to declare.
Incidence of Isolated Heparin-Induced Thrombocytopenia and the Risk of Developing Subsequent Thrombosis Using the New IgG Specific Anti-PF4/Heparin ELISA
Authors: Radaideh Sofyan M. , Frenkel Eugene P. , Sarode Ravindra , Shen Yu-Min
Published: October 2019

Abstract 1448

BACKGROUND:

The new IgG specific ELISA for anti-platelet factor 4 (PF4)/heparin antibody is recently introduced for the diagnosis of heparin-induced thrombocytopenia (HIT). It is as sensitive but more specific than the poly-specific IgG, M, A ELISA. About 50% of patients testing positive for the anti-PF4/heparin antibody have thrombocytopenia alone as initial manifestation of HIT without thrombosis (isolated HIT). These patients are thought to be at high risk (≂f30%) for developing thrombosis within next 28 days. The incidence of isolated HIT using IgG-specific ELISA is unknown.

OBJECTIVES:

Evaluate the incidence of isolated HIT using the IgG specific anti-PF4/heparin ELISA and the risk of developing subsequent thrombosis in these patients.

PATIENTS AND METHODS:

We performed a retrospective observational study of patients being tested for the anti-PF4/heparin antibody at our institution from December 2008 to May 2010 using the IgG-specific anti-PF4/heparin ELISA with a two-step assay using high dose heparin in the second step to demonstrate heparin specificity. A positive test was defined as optical density (OD) >0.4 with >50% inhibition.

RESULTS:

319 patients were tested during the study period. 23 (7.2%) patients were diagnosed with HIT based on clinical findings (4T score) and ELISA. 16/23 (70%) had thrombosis at diagnosis whereas 7/ 23 (30%) had isolated HIT. All 7 patients with isolated HIT had follow up data for at least 3 months after diagnosis. Only 2/7 were treated with direct thrombin inhibitors (DTI) and 4/7 were treated with warfarin for at least 1 month. While only 3 of the 7 isolated HIT patients had compression ultrasonography to rule out occult lower extremity thrombus, none of these 7 patients (including 4 with an OD of >1.0) developed symptoms or signs of thrombosis in the subsequent 3 months after the diagnosis of isolated HIT.

CONCLUSION:

The incidence of isolate HIT in this study (7.2%) is significantly lower than previously reported by others and our own historical patients (57.4%, Altuntus et al, Euro J of Haematology 2008) using the IgG, M, A poly -specific anti-PF4/heparin ELISA (z value 2.092, p=0.036). It is possible that many patients previously thought to have HIT by the poly-specific anti-PF4/heparin ELISA assay were false positive. Using the IgG-specific anti-PF4/heparin ELISA not only improved the specificity of the ELISA assay, it also reduced the number of patients with isolated HIT significantly who also seem to have lower risk of developing subsequent thrombosis.

Disclosures:

No relevant conflicts of interest to declare.
Blockade of T Cell Anergy by Tyrosine Kinase Inhibitor, Sorafenib May Induce Exacerbation of Gvhd In Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Yokoyama Hisayuki , Harigae Hideo , Childs Richard
Published: October 2019

Abstract 1458

Combining molecular targeted drugs with allogeneic hematopoietic stem cell transplantation could be an attractive approach to treat diseases associated with a high risk of relapse after allogeneic transplantation. However, influences of molecular targeted drugs on immune function during the period around allogeneic transplantation remain obscure. In our experiment, tyrosine kinase inhibitor, sorafenib exacerbates Graft-versus-Host disease in mouse model when given following allogeneic hematopoietc stem cell transplantation (Blood in press). To explore the mechanisms of this phenomenon, we examined in vitro effects of sorafenib on proliferation of T cells. 3H uptake study showed that the addition of sorafenib did not alter mixed lymphocyte reaction, but when stimulated with anti-CD3 antibody (OKT3) alone, T cell proliferation was enhanced significantly in the presence of sorafenib (median Stimulation index: 4.6, range). This effect was disappeared when stimulated with OKT3 and anti-CD28 antibody. We hypothesize that this stimulatory effect of sorafenib would result from inhibition of T cell anergy. To test this hypothesis, expression of anergy related genes (DGKa, Itch, Egr2, GRAIL) in T cell was examined by RQ-PCR. After stimulation of OKT3 alone, expressions of these genes were increased but in the presence of sorafenib, GRAIL expression was decreased, while expression of DGKa, Itch and Egr2 was not changed. This result may indicate that sorafenib exacerbates GVHD presumably through inhibition of T cell anergy.

Disclosures:

No relevant conflicts of interest to declare.
Memory T Cells Masquerading as Naïve Cells: Implications on Adoptive T Cell Immunotherapy.
Authors: Cieri Nicoletta , Provasi Elena , Camisa Barbara , Magnani Zulma , Bondanza Attilio , Bonini Chiara
Published: October 2019

Abstract 1471

Differentiation stage of adoptively transferred T cells has a critical impact on the success of immune-gene therapy. Ex vivo T cell manipulation often induces their terminal differentiation, resulting in poor persistence and activity of transferred cells. We previously showed that costimulation and culture with γ-chain cytokines generates gene modified T cells with a functional central memory (TCM) phenotype superior to effector/effector memory (TEM) counterparts for expansion potential and antitumor activity. Here we investigated the consequence of initial targeting of selected T cell subpopulations. We activated and efficiently transduced FACS-sorted T Naïve (TN), TCM and TEM cells by viral vectors. In contrast to TCM and TEM, TN had a greater expansion potential and more sustained expression of IL7-Rα. Strikingly, manipulation of TN resulted in a predominant population of post-mitotic lymphocytes expressing the CD45RA+CD62L+CCR7+ naïve phenotype. These cells expressed markers common to early differentiated cells (CD27, CD28, CD127 and Bcl2) and markers proper of memory lymphocytes (CD45RO, CD122 and CXCR3). Post-mitotic TN produced lower levels of IFNg and Granzyme A, expressed higher levels of c-Kit and CXCR4, and lower levels of HLA-DR, CCR5 and PD1 than memory counterparts. To verify their self-renewal and differentiation potential upon antigen stimulations, TN, TCM and TEM were transduced to express a WT1-specific TCR. Upon multiple stimulations TN expanded at higher numbers and were unique in the ability to generate a mixed population of CD127+/CD127- lymphocytes. When infused in immunodeficient mice, transduced TN proved higher engraftment and persistence potential than memory counterparts, reconstituted a mixed CD45RA±CD62L± phenotype and were significantly higher xenoreactive. These results suggest that gene transfer into TN lymphocytes might increase the efficacy of cancer immunotherapy.

Disclosures:

Bonini: MolMed: Consultancy.
Investigating the Experiences and Educational Needs of Adolescents with Hemophilia Prior to Transition of Care: A Website Development Project.
Authors: Sterling Lynn , Nyhof-Young Joyce , Blanchette Victor S. , Breakey Vicky R.
Published: October 2019

Abstract 1526

Background:

Adolescents with hemophilia (AWH) have heightened educational needs as they become more independent and transition from pediatric to adult care. Poor compliance during adolescence may lead to suboptimal management and its long-term sequelae. An innovative website is being developed to meet these educational needs and foster the acquisition of self-management skills.

Purpose:

To examine the educational needs of AWH treated at The Hospital for Sick Children and explore how a web-based transition program can best support them.

Methods:

Qualitative, semi-structured interviews with a purposive sample of AWH addressed subjects’ experiences with hemophilia, views about transition, internet use, and thoughts about an educational website. The interviews were audio-recorded and transcribed verbatim. Themes were identified through constant comparative analysis by three independent reviewers.

Results:

Eleven of twelve subjects that were approached consented to be interviewed and data saturation was achieved in the areas of interest. Important themes surrounding the hemophilia experience included personal identity and perception of their condition, challenges, raising awareness, and sources of support. Some participants had concerns about transition to adult care but many also recognized its benefits. There was a broad spectrum of perceived preparedness for transition. All participants felt comfortable using the Internet and overall, felt a website would help them to learn about haemophilia and explain it to others. Social networking with others with haemophilia was deemed desirable by many.

Conclusions:

AWH are interested in a hemophilia website and have identified a variety of features which they believe may help to support them during transition of care and beyond.

Disclosures:

Breakey: Baxter Bioscience Canada: Fellowship salary provided through unrestricted educational grant.
Changing Age Distribution of Deep Venous Thrombosis In the United States
Authors: Viswanathan Prabhu , Krishnamurti Lakshmanan
Published: October 2019

Abstract 1539

Background:

Deep vein thrombosis (DVT) and pulmonary embolism (PE) are the major cardiovascular killers in the hospital setting. While there is increasing understanding of the public health importance of DVT there are limited data on the trends in the distribution of DVT in different age groups.

Aim:

The aim of this study was to study national trends in age distribution of DVT in patients over the age of 45 years in the last decade.

Methods:

We used the National Inpatient Sample (NIS) to analyze the epidemiology of hospitalizations in various age groups from 1997 – 2007. The NIS is a part of the Healthcare Cost and Utilization Project (HCUP) sponsored by the Agency for Health Care Research and Quality (AHRQ) and contains information from a 20% stratified sample of hospitals extrapolated to show the entire national utilization in United States. For our study we selected the following ICD-9 codes to represent DVT (ICD-9 codes 451.11, 451.19, 451.2, 451.81, 451.9, 453.40, 453.41, 453.42, 453.8, 453.9). For each hospitalization, NIS allows a total of up to 15 diagnostic entries. The entry in the diagnostic (DX) field 1 is called the Principal Diagnosis (PD) is the principal reason for admission and the entries between DX fields 1 and DX field 15 are called all-listed diagnoses and include the principal diagnosis plus additional conditions that coexist at the time of admission, or that develop during the stay. We derived at Secondary diagnoses (SD) by excluding PD of DVT from the list of AD. U.S. Population Census Bureau was used to calculate the change in population. Predictive Analytics Software, IBM PASW Statistics 18.0 data analysis tool were used for data mining and statistical analysis and least square regression analysis was used to calculate ‘p’ value.

Result:

From 1997 – 2007,

Conclusion:

These data describe for the first time, trends in the relative contribution of different age groups to the burden of DVT among hospitalized patients in the United States. The population between age group 45–64 years is growing symmetrically as a proportion of the total population, total hospitalized population and total hospitalized population with DVT. On the other hand the population 65–84 years of age is decreasing as a proportion of the total hospitalized population with DVT to an even greater degree than as a proportion of the total population and the total hospitalized population. The group over 85 years remains a disproportionately large contributor to the population with DVT. These data suggest the need for greater study of the distribution of DVT, the underlying causes and potential strategies to prevent DVT in different age groups

Disclosures:

No relevant conflicts of interest to declare.
Lack of the BH3-Only Proteins Bim, Bmf and Puma In Haematopoietic Stem and Progenitor Cells Facilitates Early Reconstitution and Long Term Haematopoiesis.
Authors: Erlacher Miriam , Bertele Daniela , Woess Claudia , Niemeyer Charlotte Marie , Villunger Andreas , Labi Verena
Published: October 2019

Abstract 1542

During haematopoietic stem cell transplantation (HSCT) the transplanted haematopoietic stem and progenitor cells (HSPC) suffer a transient deprivation of survival signals (i.e. cytokines or adhesion molecules) normally provided by the stem cell niche. Therefore, cell loss may occur prior to successful engraftment and thus restrict haematopoietic reconstitution.

Apoptosis induced in response to lack of cytokines or contact to the extracellular matrix is regulated by members of the Bcl-2 family. Bcl-2 family members can be divided into the pro-apoptotic ‘BH3-only’ proteins and anti-apoptotic Bcl-2 like proteins, the two subclasses antagonizing each other in their function. Using different gene-modified mouse models lacking the ‘BH3-only’ proteins Bim, Bmf or Puma, all implicated in leukocyte homeostasis, we aimed to delineate which one of these Bcl-2 family proteins is critically involved in limiting successful reconstitution of the haematopoietic system.

Our results demonstrate that HSPC lacking Bim show accelerated reconstitution of lethally irradiated recipient mice. Moreover, competitive reconstitution experiments reveal that wild type haematopoiesis is completely displaced in wt:bim-/- and strongly suppressed in wt:bmf-/- and wt:puma-/- bone marrow chimeras. The effects of Bim are comparable to those observed upon induced overexpression of Bcl-2 and cannot be enhanced further by additional loss of Puma or Bmf, identifying Bim as the major BH3-only protein limiting cell survival during haematopoiesis. Since both lymphopoiesis and myelopoiesis are similarly affected, a direct role of Bim on early progenitor or even stem cells can be assumed. However, wild type lymphocytes are additionally outcompeted by bim-/- and bcl-2 tg lymphocytes during differentiation, since the balance between Bim and Bcl-2 is critically involved in many selection processes occurring during a lymphocyte's life (i.e. negative selection in the thymus).

In summary, inhibition of apoptosis in HSPC by transiently interfering with Bim-function may be a promising strategy to increase the efficacy of HSCT and reduce transplantation-related morbidity.

Disclosures:

No relevant conflicts of interest to declare.
TIMP-1 Deficiency Subverts Cell Cycle Dynamics In HSCs.
Authors: Rossi Lara , Goodell Margaret A.
Published: October 2019

Abstract 1545

Although TIMP-1 was initially described as a mere inhibitor of Metalloproteinases (MMPs), recent findings have offered a different perspective on its biological role, contributing to unveil its multifaceted nature. In addition to inhibiting MMP activity, TIMP-1 has been proven to play MMP-independent, cytokine-like activities and to be involved in the regulation of numerous biological functions, including cell proliferation and survival. We therefore hypothesized that TIMP-1 might be involved in the homeostatic regulation of hematopoietic stem cells (HSCs), whose biological behavior is the synthesis of both microenvironmental and intrinsic cues. Bone marrow hematopoietic stem cells (HSCs) were isolated from TIMP-1-/- mice based on the phenotype Side Population c-Kit+Lin- Sca-1+ (SPKLS). In vitro cultural assays as well as in vivo transplantation assays were employed to investigate how TIMP-1 obliteration affects murine hematopoiesis. Cell-cycle dynamics in KO SPKLS HSCs were characterized by Pyronin Y/Hoechst staining, Ki-67 staining, as well as evaluation of RNA expression of cell cycle inhibitors, such as p53, p57, and p21.

We found that TIMP-1-/- mice have decreased HSC numbers and, consistent with this finding, TIMP-1-/- HSCs display reduced capability of long-term repopulation. Interestingly, the cell cycle distribution of TIMP-1-/- LT-HSCs is profoundly distorted, with a consistent proportion of the stem cell pool arrested in the G1 phase, suggesting that TIMP-1 is intrinsically involved in the regulation of the HSC proliferation dynamics. Of note, TIMP-1-/- HSCs present decreased levels of CD44 glycoprotein, whose expression has been proven to be controlled by p53, the master regulator of the G1/S transition. Interestingly, p53 RNA levels are indeed increased in TIMP-1-/- SPKLS HSCs compared to controls. Likewise, the expression level of other cell-cycle inhibitors, such as p57 and p21, were found to be higher in KO SPKLS HSCs, indicating a disregulation of cell-cycle dynamics.Our study highlights a novel biological role of TIMP-1 in the regulation of the HSC compartment and suggest a novel mechanism presiding over stem cell quiescence in the framework of the BM milieu.

Disclosures:

No relevant conflicts of interest to declare.