bluuebunny/crossref_metadata_2025 huggingface dataset:
Safe and Efficient Chelation by Deferasirox In Thalassemia Major Patients with Low Liver and Cardiac Iron Concentrations
Authors: Grosse Regine , Janka Gritta , Jarisch Andrea , Nielsen Peter , Yamamura Jin , Fischer Roland
Published: October 2019

Abstract 5168

Chelation treatment of iron overload from chronic blood (RBC) transfusion is still a challenge to both, patients and medical caretakers. Different treatment regimes have been recommended so far, especially for chronically transfused patients with low or even normal liver iron concentration. We report the results from 16 regularly transfused patients with thalassemia major (TM) who were on iron chelation treatment under normal to mild liver iron concentration (LIC). All patients received deferoxamine (DFO) treatment before they changed to deferasirox (DSX) treatment.

16 TM patients (mean age 13.6 y) were treated with DSX (median dose 18 mg/kg/d, range: 7 – 33 mg/kg/d) for 6 to 71 months. Liver iron measurements by biomagnetic susceptometry (BLS) and/or MRI-R2 as well as cardiac MRI-R2* were performed in intervals of 6 to 12 months. The median LIC was 782 μ g/g-liver wet weight (range: 460 μ g – 1122 μ g). Median RBC transfusion rate was 8500 ml/y, equivalent to about 2 erythrocyte concentrates per 3 weeks or a daily iron influx of 16.2 mg/d. For each measurement interval, the ratio of daily iron influx and DSX dose rate was calculated. This represents the equilibrium molar efficacy for iron balance.

In all 16 TM patients no severe side effects were observed and creatinine was in the normal range of < 0.9 mg/d throughout the treatment with DSX. From baseline DFO treatment interval to the endpoint of DSX treatment, liver iron decreased by 124 – 4689 μ g/g-liver (conversion factor of 6 for mg/g-dry-wgt), while serum ferritin decreased by -596 to 8283 μ g/l. For all measurement intervals, molar chelation efficacies between 18 % and 56 % were calculated at equilibrium with a median efficacy of 31 % (interquartile range = 16 %). This agrees with molar efficacies of DSX reported earlier, but for relatively higher LIC and chelation doses (Blood 2005; 106(11):#2690 and Blood 2007; 110(11):#2776). The cardiac R2* (median R2* = 38 s-1) was either below the normal threshold of 50 s-1 (T2* > 20 ms) or decreased by about 24 %/y under DSX treatment. In these few patients at low LIC, this was even higher than recently reported.

Conclusion:

Even in patients with normal to mild LIC iron chelation treatment with DSX is safe, does not result in increased creatinine levels or severe side effects and is as efficient as in patients with higher LIC.

Disclosures:

No relevant conflicts of interest to declare.
Upregulation of Von Willebrand Factor, Von Wilelbrand Factor Propeptide, and ADMATS-13 In End-Stage Renal Disease
Authors: Fareed Jawed , Litinas Evangelos , Hoppensteadt Debra , Thethi Indermohan , Bansal Vinod
Published: October 2019

Abstract 5189

End-Stage Renal Disease (ESRD) is a complex syndrome in which systemic vascular pathophysiologic changes contribute to adverse cardiovascular and cerebrovascular manifestations. Cardiovascular disease alone is present in over 60% of patients with ESRD and contributes heavily to mortality among this population. Von Willebrand Factor (vWF) plays a well known role in platelet aggregation and has been shown to be upregulated in many thromboembolic conditions. However in many conditions, the role of von Willebrand Factor Propeptide (vWFpp), a peptide cleaved prior to assembly of active vWF multimers, and ADAMTS-13, the enzyme responsible for disassembly and deactivation of vWF multimers, have not been examined. The purpose of this study is to better characterize the relationship among vWF, vWFpp, and ADAMTS-13 as it relates to ESRD. Plasma samples from 51 patients with ESRD were collected prior to maintenance hemodialysis. A group of 50 normal individuals, both male and female, was included as control. Concentrations of vWF and vWFpp were quantified using the GTI® vWF & Propeptide Assay. A separate assay was used to measure ADAMTS-13 in these samples (GTI®). This data as well as the ratio of vWF:vWFpp were analyzed using the Mann-Whitney U test (two-tailed with Gaussian approximation). Compared to the controls, there was an upregulation of all three analytes in patients with ESRD. Levels of vWF were increased 1.3 fold, (mean 128 ± 69 U/dL, range 9.2 to 308) compared to control (mean 101 ± 60, range 22 to 332). Similarly, there was a 1.6 fold increase in both vWFpp (mean 148 ± 92, range 8.0 to 361) compared to (mean 92 ± 23, range 60 to 163) and ADAMTS-13 (mean 131 ± 27, range 67 to 150) compared to (mean 84 ± 22, range 36 to 128). However, the ratio of vWFpp:vWF (mean 1.2 ± 0.6, range 0.4 to 3.3) was unchanged compared to control (mean 1.2 ± 0.7, range 0.4 to 3.6). This study shows a previously uncharacterized increase in vWFpp and further validates the upregulation of vWF in ESRD. The normal vWFpp:vWF ratio implies an increased production in vWF in response to endothelial damage without any apparent alteration in vWF degradation or clearance. The proportional increase in ADAMTS-13 activity suggests that the increased vWF is being cleaved into inactive monomers. However, further studies are required to assess the proportion of vWF in each mulitmeric form and better understand the exact interaction among these components in ESRD.

Disclosures:

No relevant conflicts of interest to declare.
Reduced Von Willebrand Factor Secretion Is Associated with Loss of Weibel-Palade Formation
Authors: Castaman Giancarlo , Giacomelli Sofia Helene , Jacobi Paula M. , Obser Tobias , Schneppenheim Reinhard , Haberichter Sandra L
Published: October 2019

Abstract 541

Background.

Von Willebrand Disease (VWD) is caused by mutations in von Willebrand factor (VWF) that have different pathophysiologic effect in causing low plasma VWF levels. Type 1 VWD includes patients with quantitative plasma VWF deficiency with normal VWF structure and function.

Aim of the study.

We report three different novel type 1 VWF mutations (A1716P, C2190Y and R2663C) which although located in different VWF domains are associated with reduced secretion and lack of formation of Weibel-Palade body-like granules.

Methods.

Transient expression of recombinant mutant full-length VWF in 293 EBNA cells was performed and secretion, collagen binding, and GpIb binding assessed in comparison to wild-type VWF. Furthermore, expression was also examined in HEK293 cells that form Weibel-Palade body (WPB)-like granules when transfected with wt VWF.

Results.

The multimer analysis of plasma VWF was compatible with type 1 VWD. The results of 3 different expression experiments showed a slightly reduced VWF synthesis and drastically impaired secretion into the medium with homozygous expression.

In HEK293 cells, homozygous A1716P and C2190Y VWF variants failed to form WPB-like granules, while R2663C was capable of forming granules, but had fewer cells with granules and more with ER-localized VWF. Heterozygous expression of A1716P and C2160Y VWF variants had a negative impact on wild-type VWF and WPB-like granules were observed in transfected cells.

Conclusions.

Our results demonstrate that homozygous and heterozygous quantitative VWF deficiency caused by missense VWF mutations can be associated with inability to form endothelial Weibel-Palade-like granules and mutations in different VWF domains can affect the formation of these organelles.

Disclosures:

No relevant conflicts of interest to declare.
Race, ABO Blood Type, Gender, Age and Venous Thromboembolism Risk: Not Black and White
Authors: Fang Chunhui , Billett Henny Heisler
Published: October 2019

Abstract 568

Venous thromboembolism (VTE) prevalence has been noted to be different for different racial groups: Non-Hispanic blacks tend to have a higher risk compared with non-Hispanic whites. Different ABO blood types have also been previously shown to be associated with different risk of VTE, with individuals with O blood type carry the lowest VTE risk. Given that blacks have a higher percentage of O blood type, one would expect to see a lower prevalence of VTE. In order to explain this paradoxical finding, we utilized the Clinical Looking Glass (CLG) system to study the relative influence of race, gender, age, and ABO blood type on the VTE risk. A total of 61,077 adult patients admitted to our large diverse urban hospital between 2000 and 2009 who had blood typing performed were included in the study. Four cohorts were established according to ABO blood group and the prevalence of VTE among each cohort was examined. We confirmed the higher prevalence of VTE in blacks (7.05% vs. 6.75%, p<0.001). While it has been previously shown that male gender is associated with higher VTE risk, we found this to be true only for the younger patient population: in adult patients less than 45yrs, male carried a higher prevalence of VTE (5.97% vs. 3.06%, p<0.001); in patients aged between 45 and 64yrs, no gender difference in the prevalence of VTE could be determined (8.40% vs. 8.33%, p=0.86). For patients aged between 65 and 84yrs, male gender was actually associated with lower prevalence of VTE (10.11% vs. 12.07%, p=0.009). No gender difference was seen in patients older than 80 (11.11% vs. 11.99%, p=0.356). When the prevalence of VTE was examined within each blood type, we confirmed that O blood type in general carries a lower VTE risk compared with other non-O blood types (6.9% vs. 8.4%, p<0.001); this held true for both genders (See Figure). To better analyze this complex interaction between race, ABO blood type, gender, age and VTE risk, we used logistic regression analysis. Race appeared to be the strongest determinant (black vs. white OR 1.43; 95% CI: 1.33, 1.52), followed by ABO blood type (Type A 1.2, 95% CI: 1.13–1.3; Type AB 1.2, 95% CI: 1.05, 1.40; Type B 1.33, 95% CI 1.23, 1.44), then gender (male vs. female OR 1.11; 95% CI: 1.04, 1.18) and finally, age (OR 1.03; 95% CI 1.03 – 1.03).). In conclusion, VTE risk for any individual is a composite of multiple variables. Our study suggests that race, gender, ABO blood type and age might allow us, in combination with other known risk factors, to develop a prognostic score for VTE risk stratification for each patient.

Disclosures:

No relevant conflicts of interest to declare.
Transfer of MHC Class I by Coating Platelets Enables Tumor Cells to Evade NK Cell Immune Surveillance by Conferring a “Pseudo Self” Phenotype
Authors: Placke Theresa , Kopp Hans-Georg , Schaller Martin , Jung Gundram , Kanz Lothar , Salih Helmut R.
Published: October 2019

Abstract 583

NK cells are a central component of the cytotoxic lymphocyte compartment capable of lysing tumor cells without prior immune sensitization of the host. The mechanisms leading to activation of NK reactivity are described by the principles of ‘missing-self' and ‘induced-self', which imply that cells with a low or absent expression of MHC class I (‘missing-self') and/or a stress-induced expression of ligands of activating NK receptors like e.g. NKG2D (‘induced-self') are preferentially recognized and eliminated by NK cells. Thus, a balance of various activating and inhibitory signals determines whether NK cell responses are initiated or not. Tumor cells often downregulate expression of MHC class I to evade T cell-mediated immune surveillance, which results in enhanced NK susceptibility. Besides the direct interaction with their target cells, NK activity is further influenced by the reciprocal interplay with various other hematopoietic cells. We and others demonstrated previously that thrombocytopenia inhibits metastasis in murine models, which is reversed by additional depletion of NK cells (e.g., Jin et al., Nature Med. 2006, Palumbo et al., Blood 2005). However, the mechanisms by which platelets impair NK-tumor interaction are largely unclear, especially in humans. Recently we reported that platelets release TGF-β upon interaction with tumor cells causing downregulation of NKG2D on NK cells, which impairs anti-tumor immunity by disturbing the principle of “induced self” (Kopp et al., Cancer Res. 2009). Here we demonstrate that platelets further enable tumor cells to evade NK cell immune surveillance by preventing detection of “missing self”: We found that tumor cells rapidly get coated in the presence of platelets, the latter expressing large amounts of MHC class I on their surface. In case of MHC class I-negative or -low cancer cells, this process results in MHC class I “pseudoexpression” on the tumor cell surface as revealed by flow cytometry, immunofluorescent staining, and electron microscopy. Platelet-derived MHC class I was found to inhibit the reactivity of autologous NK, both upon activation with cytokines and, most importantly, in cultures with platelet-coated tumor cells. Using constitutively MHC class I-negative/low tumor cells we found that blocking MHC class I restored NK cytotoxicity and IFN-γ production against platelet-coated tumor cells, but did not alter NK reactivity against the tumor cells in the absence of coating platelets. Taken together, our data indicate that platelets enable a molecular mimicry of tumor cells, allowing the latter to downregulate MHC class I in order to escape T cell immunity without inducing sufficient NK tumor immune surveillance due to conferred platelet-mediated “pseudo self”.

Disclosures:

No relevant conflicts of interest to declare.
Alterations In Wnt Signalling In the Megakaryocytic Lineage Leads to Bone Marrow Failure and Myelofibrosis
Authors: Calaminus Simon D , Inman Gareth , Ghaevert Cedric , Sansom Owen , Watson Steve P , Holyoake Tessa L. , Machesky Laura
Published: October 2019

Abstract 628

Introduction:

Wnt signalling is fundamental in controlling stem cell self-renewal, cell proliferation and development in multicellular organisms. Stabilization of beta catenin or loss of the scaffold protein adenomatis polyposis coli (APC) causes aberrant activation of wnt signalling and often leads to cancer. Mutations to wnt pathway members in haematopoietic stem cells leads to haematopoietic failure and rapid lethality. In this study, we demonstrate that aberrant wnt signalling in the megakaryocyte lineage underlies myelofibrosis.

Methods:

We created a series of mice with altered wnt pathway signalling in their megakaryocytic lineage using PF4-Cre (platelet factor 4 cre) as follows: Ctnnb1fx(ex3)/wt_ Expresses stabilized active beta catenin henceforth termed PF4bcat+; APCfx/fx_ Loss of APC stabilises beta catenin termed PF4APC-KO; Ctnnb1fx/fx b-catenin knockout termed PF4bcat-KO.

Results:

By day 40, PF4bcat+ and PF4APC-KO mice are severely underweight, anaemic (wt 6.8+0.2×106/ml v. PF4bcat+ 4.2+0.3×106/ml, PF4APC-KO 5.5+0.5×106/ml), and have a significant reduction in platelet number (wt 1033+37×106/ml v. PF4bcat+ 717+57×106/ml, PF4APC-KO 747+68×106/ml). Furthermore PF4bcat+ and PF4APC-KO mice develop bone marrow fibrosis and consistently die within 50 days of birth. Both populations of mice have splenic extramedullary haemopoiesis with hyperplasia of splenic megakaryocytes, leading to a dramatic increase in spleen to body size ratio. In addition, both PF4bcat+ and PF4APC-KO mice have increased peripheral blood levels of active TGFb, providing a likely molecular basis of the induction in bone marrow fibrosis. PF4APC-KO and PF4bcat+ mutant mice show a dramatic (>10-fold) increase in platelet b-catenin protein levels over wt samples. By comparison, human myelofibrosis patients (n=16) show a 2.7+0.6-fold increase in platelet b-catenin expression over controls. Moreover, overexpression of b-catenin within human patient samples is correlated with a worsening Primary Myelofibrosis prognostic score (Blood, 2009 vol 113 p. 2895), with those patients with a low score (n=7) having a 1.3+/−0.37-fold increase over control, intermediate score (n=4) 4.52+/−1.23-fold, and high score (n=1) 4.56-fold. In contrast PF4bcat-KO mice show no changes in whole blood counts, weight, or evidence of splenic extramedullary haematopoiesis, indicating that b-catenin removal does not adversely affect megakaryocyte development or function.

Conclusions:

Stabilisation of b-catenin within mouse megakaryocytes leads to a myelodysplastic disorder and myelofibrosis. This finding demonstrates a defined role for aberrant activation of the wnt signalling pathway and marks the wnt pathway and the megakaryocyte lineage as important potential drug targets for the treatment of myelodysplastic disorders.

Disclosures:

No relevant conflicts of interest to declare.
Gemcitabine, Busulfan and Melphalan (GemBuMel) Is a New High-Dose Chemotherapy (HDC) Regimen with High Activity In Refractory Hodgkin's Lymphoma (HL) Patients Receiving An Autologous Stem-Cell Transplant (ASCT): A Contemporaneous Comparison with BEAM and Busulfan/Melphalan (BuMel)
Authors: Nieto Yago , Anderlini Paolo , Popat Uday , Liu Ping , Andersson Borje S , Shpall Elizabeth J. , Kebriaei Partow , Hosing Chitra , Alousi Amin M , Qazilbash Muzaffar H. , Ciurea Stefan O. , Parmar Simrit , Champlin Richard E. , Jones Roy
Published: October 2019

Abstract 690

Background:

BEAM is considered standard HDC for primary refractory or relapsed HL. However, pts with refractory HL have <30% chance of long-term event-free survival (EFS), underscoring the need for more active HDC regimens.

Methods:

We developed a new HDC regimen of gemcitabine (Gem), busulfan (Bu) and melphalan (Mel) (GemBuMel) exploiting their synergy. Bu was given as 4 daily doses on days −8 to −5 targeting an AUC of 4,000/d. Mel was given at 60 mg/m2/d on d-3 and d-2. Gem was infused over 3 hours at a fixed dose rate of 10 mg/m2/min (total dose 1875 mg/m2) on days −8 and −3 immediately preceding Bu and Mel, respectively. We compared the subset of refractory HL pts enrolled in this trial with all other refractory HL pts treated at MDACC with HDC during the same time period, who were eligible for the GemBuMel trial but either received BEAM off protocol or were enrolled in a separate trial of BuMel. All of these pts met ≥1 of the following criteria of refractory disease: primary induction failure (PIF) (defined as less than PR to 1st line chemotherapy), CR1 <6 mo, >1 relapse or progressive disease (PD) at HDC. Pts with relapsed but not refractory HL were not included in this analysis.

Results:

We analyzed 115 pts treated in one of the following three cohorts: 1) GemBuMel (N=51) since 01/07, median follow-up: 10 (2-43) mo; 2) BEAM (N=26) since 01/07, median f/u: 13 (3-56) mo; 3) BuMel (N=38) since 04/05, median f/u: 36 (17-56) mo. The GemBuMel cohort had significantly higher % PIF, median # prior relapses, % PET + tumors at HDC and % PD at HDC, with all other demographic and clinical features comparable (Table 1).

There were no treatment-related deaths in any cohort. GemBuMel pts had improved EFS (Fig. 1) and OS (Fig. 2). GemBuMel was superior in patients with either PET- or PET+ tumors at HDC (Table 2). Cox proportional hazards regression models identified the use of a regimen other than GemBuMel (HR 2.38, P=0.02 for EFS; HR 8.25, P=0.009 for OS), >1 prior relapse (HR 2.91, P=0.006 for EFS) and B symptoms (HR 6.57, P=0.009 for OS) as independent adverse outcome predictors.

Conclusions:

Despite its worse prognostic features, the cohort of refractory HL pts treated with GemBuMel showed superior outcome to contemporaneous patients receiving BEAM or BuMel. A randomized trial of GemBuMel v BEAM is warranted.

Disclosures:

Off Label Use: Off-label use of gemcitabine for Hodgkin's lymphoma. Popat:Otsuka: Research Funding. Andersson:Otsuka: Consultancy. Champlin:Otsuka: Research Funding.
Analysis of TIF1gamma Conditional Knockout Establishes a Requirement for the Differentiation of Multiple Hematopoietic Lineages
Authors: Bai Xiaoying , Trowbridge Jennifer , Lee Joseph , Orkin Stuart H. , Zon Leonard I.
Published: October 2019

Abstract 744

Vertebrate hematopoiesis is regulated by cell-specific transcription factors that couple to RNA polymerase-associated basal machinery. Mutation of the chromatin factor TIF1gamma (TIF1g) gene in the zebrafish moonshine mutant causes a profound decrease in expression of most erythroid genes. Our previous work in the zebrafish system established an essential role of TIF1g in transcription elongation by coupling the SCL transcription factor complex to the transcription elongation machinery, and erythroid-specific transcription is paused in moonshine mutant (Bai et al., Cell 2010). Here we examined the role of TIF1g in murine hematopoiesis by studying conditional knockout (KO) models. Deletion of TIF1g was either induced in adult mice by Mx-Cre or during mouse development by vav-Cre. Both Cre systems induce excision at the HSC level and in all hematopoietic lineages. We observed the same trend of multi-lineage defects in both Cre lines, including decreased erythropoiesis in the bone marrow, loss of mature B cells and expansion of granulocytes. Bone marrow analysis of TIF1g-deleted mice revealed a block in erythroid differentiation starting as early as the BFU-E stage, consistent with the erythroid defect in the zebrafish moonshine mutant, confirming an evolutionarily conserved role for TIF1g in vertebrate erythropoiesis. A subset of the vav-Cre induced KO mice developed a MPD (myeloproliferative disease)-like phenotype at 1–3 months after birth, including a dramatic increase of dysplastic granulocytes in the peripheral blood and massive extramedullary hematopoiesis in spleen and liver, suggesting a requirement for TIF1g in myeloid differentiation and proliferation. Consistent with the KO mouse phenotype, we observed an increase of definitive myelopoiesis in the zebrafish TIF1g mutant. In addition, mature B cells were absent in KO mice, and this loss was observed as early as the pre-B stage, suggesting a defect occurring at the pro-B to pre-B transition. At the progenitor level, we observed an increase of GMP, MPP, and HSCs in bone marrow, suggesting that TIF1g may be required to control proliferation at the stem/progenitor cell stage or in the committed myeloid lineage. Our study reveals an essential function of TIF1g in regulating the differentiation and proliferation of multiple hematopoietic lineages.

Reference

Bai X, Kim J, Yang Z, Jurynec M, Akie T, et al. (2010) TIF1gamma controls erythroid cell fate by regulating transcription elongation. Cell. 142:133-43.

Disclosures:

Zon: FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Mir-15a/16-1 Cluster Is Frequently Deleted In Primary Hodgkin Lymphoma and Modulates Multiple Survival Pathways Including AP-1
Authors: Chapuy Bjoern , Green Michael R , Lu Jun , Monti Stefano , Hao Yansheng , Yeda Kelly T , Rodig Scott J , Currie Treeve , Takeyama Kunihiko , Golub Todd R , Kutok Jeffery , Shipp Margaret A.
Published: October 2019

Abstract 746

Classical Hodgkin Lymphoma (cHL) Reed-Sternberg (RS) cells have crippling mutations of their rearranged Ig genes and lack B-cell receptor-mediated survival signals. As a consequence, cHLs rely on alternative survival and proliferation pathways including AP-1. Although cHL RS cells express high levels of the AP-1 components, cJun and JunB, and depend on AP-1-mediated proliferation signals, the mechanism of constitutive AP-1 activation in cHL remains undefined. In a screen for genetic abnormalities in cHL, we integrated microRNA (miR) expression profiles with high resolution copy number data and identified single copy loss of the miR-15a/16-1 locus (chromosome 13q14) and decreased miR-15a/16 expression in 75% of examined HL cell lines. We isolated primary Hodgkin RS cells using laser-capture microdissection and confirmed frequent single copy loss of the miR-15a/16-1 locus by quantitative PCR and associated decreased miR-15a/16 abundance by RT-PCR in 10/23 (43%) of primary tumors. Using retroviral vectors containing miR-15a and miR-16, we reconstituted miR-15a/16 expression in HL cell lines with single copy loss of chromosome 13q14. In these HL lines, miR-15a/16 reconstitution was associated with cell cycle arrest, significantly decreased cellular proliferation and induction of apoptosis (88% apoptotic cells at 48 hours). In addition, miR-15a/16 reconstitution resulted in significant downregulation of multiple survival pathway components and known miR-15a/16 targets including panAKT, BCL2 and cyclin D1. Using a stand-alone miRanda algorithm with direct sequence entry, we also identified a candidate miR-15a/16 binding site in the AP-1 component, cJun. In multiple HL cell lines, miR-15a/16 reconstitution decreased the expression of cJun and additional known AP-1 targets such as galectin-1. Therefore, miR-15a/16-1 deletion is a frequent genetic abnormality in HL cell lines and primary tumors, a candidate mechanism of AP-1 activation in HL and a modulator of multiple critical survival pathways in this disease.

Disclosures:

No relevant conflicts of interest to declare.
Increased INR>3.0 In Patients on Warfarin Therapy Is Associated with Acute Changes In the Serum Creatinine and Increased Mortality Rate
Authors: Brodsky Sergey , Hebert Lee , Wu Haifeng , Liu James , Ware Kyle , Brodsky Polina , Chen Robert , Von isger Jon
Published: October 2019

Abstract 819

Recently we reported that an excessive anticoagulation with warfarin (INR>3.0) can result in acute kidney injury (AKI). Morphologic findings included glomerular hemorrhage and renal tubular obstruction by red blood cell (RBC) casts. The clinical outcome in these patients was unfavorable; more than half of them did not recover from acute kidney injury even after normalization of INR. Later we analyzed serum creatinine (SC) and INR in patients with chronic kidney disease (CKD) on warfarin therapy. We found that 46% of patients had increase in SC levels >0.3 mg/dl associated with INR>3.0. SC remained elevated above baseline after the first episode of abnormal INR. The slope of the following SC increase was higher after this abnormal INR episode. We called this condition warfarin related nephropathy (WRN). The current study is based on medical records of 4059 consecutive patients who were on warfarin therapy at the Ohio State Medical Center for a 5-year period. Of these, 838 (21%) experienced an increase in SC>0.3 mg/dl within 1 week after INR>3.0 (WRN group). The remaining 3221 patients (79%) were designated no-WRN. The WRN group had a 5-year mortality rate of 42%, as compared to 27% for the no-WRN group (p<.001). The highest risk of death in the WRN cohort occurred within the 1st month after INR>3.0 (hazard ratio =2.15). For both WRN and no-WRN groups, the 5-year mortality rate was consistently higher in those with CKD compared to those with no-CKD (50.8% vs. 37.0% for the WRN cohort; 39.7% vs. 24.5% for the no-WRN cohort; p<.0001). Compared to no-WRN patients, WRN patients tended to be older (63.7±14.7 years vs. 61.7±15.6 years, p=.025), diabetic (47% vs 37%, p<.0001), hypertensive (82% vs 72%, p<.001) and had a history of heart failure (62% vs 42%, p<.001). Preliminary models indicate that WRN still is a significant predictor of death even after adjusting for these factors. We conclude that WRN is associated with increased mortality rate in the elderly, the diabetic, and those with CKD and cardiovascular diseases. The possible pathophysiologic mechanisms may be glomerular hematuria and formation of occlusive RBC casts. Physicians, involved in the clinical care of patients on systemic anticoagulation therapy, should be aware of this serious renal complication of warfarin overdose and carefully monitor the kidney function and coagulation parameters in these patients

Disclosures:

No relevant conflicts of interest to declare.
Evolution of the Donor T Cell Repertoire In Allogeneic Stem Cell Transplant Recipients In the Second Decade After Transplantation
Authors: Le Robert Q. , Melenhorst J. Joseph , Hill Brenna , Memon Sarfraz , Battiwalla Minoo , Savani Bipin N. , Shenoy Aarthi , Hensel Nancy F. , Koklanaris Eleftheria K. , Keyvanfar Keyvan , Hakim Frances T. , Douek Daniel C. , Barrett A. John
Published: October 2019

Abstract 831

After allogeneic stem cell transplantation (SCT), donor T lymphocyte immune function is slowly re-established in the recipient through reconstruction of the donor's post-thymic T cell repertoire and from T cell neogenesis in the thymus. Although long-term survivors from SCT appear healthy, their immune repertoire and differences from that of their donors have not been characterized. We studied 38 healthy patients surviving more than 10 years from a myeloablative SCT for hematological malignancy (median follow-up 12 years, range 10–16 years). T cell and natural killer (NK) cell repertoires in these patients were compared with cells from their stem cell donors cryopreserved at time of transplant and from the same donors at 10 year after SCT. The median age of both recipients and their sibling donors at time of transplant was identical (36 years). Patients received cyclosporine GVHD prophylaxis and delayed add-back of donor lymphocytes 30–90 days post transplant. Only one patient was on continued immunosuppressive treatment at the time of study. Compared with the donor pre-transplant counts there was no significant difference in the absolute lymphocyte, neutrophil, monocyte, CD4+ and CD8+ T cell, NK cell, and B cell subset counts. However, compared to their donors, recipients had a) significantly fewer naïve CD4+ and CD8+ T cells; b) lower T cell receptor excision circles levels; c) fewer CD4+ central memory T cells; d) more effector CD8+ T cells; e) and more FOXP3+ regulatory T cells. These data suggest that the patient had a persistent deficiency on T cell neogenesis. Molecular examination of the T cell receptor Vbeta (TCRBV) repertoire by spectratype analysis showed that there was no significant difference in total complexity score, defined as the sum of the number of discrete peaks for each Vbeta subfamily, between the patients and their donors. TCRBV subfamily spectratyping profiles of patients and donors, however, had diverged, with both gains and losses of peaks identifiable in both patient and donor. In conclusion, patients surviving 10 or more years after allogeneic SCT still show a T cell repertoire that reflects expansion of the donor-derived post thymic T cell compartment, with a limited contribution by new T cell generation and persistently increased Tregs. It therefore appears that a diverse TCRBV repertoire predominantly derived from the memory T cell pool is compatible with good health.

Disclosures:

No relevant conflicts of interest to declare.
Growth Factor Independence 1 b (Gfi1b) as a New Regulator of Hematopoietic Stem Cell Fate
Authors: Khandanpour Cyrus , Vassen Lothar , Gaudreau Marie-Claude , Kosan Christian , Moroy Tarik
Published: October 2019

Abstract 837

Donor matched transplantation of bone marrow or hematopoietic stem cells (HSCs) are widely used to treat hematological malignancies, but are associated with high mortality. Methods for expansion of HSC numbers and their mobilization into the bloodstream of a donor could significantly improve therapy. We show here that the zinc finger transcriptional repressor Gfi1b is highly expressed in hematopoietic stem cells (defined as CD 150+, CD 48-, Lin-, Sca1+ and c-kit+) cells and is down-regulated more than 10 fold upon differentiation into multipotential progenitors (defined as CD 150+ or CD150-, CD 48+, Lin-, Sca1+ and c-kit+). Constitutive germline deletion of Gfi1b is lethal at midgestation due to impaired development of erythrocytes and megakaryocytes. We have therefore developed a conditional knock-out of Gfi1b to study its role specifically in the adult hematopoietic system. Deletion of Gfi1b leads to a 30-fold increase of HSC numbers in bone marrow and around a100 fold increase in spleen and peripheral blood. This was due to a higher rate of HSCs undergoing cell cycling. Concomitantly, the number of quiescent HSCs was reduced 5–6 times.

We then performed an gene expression array of wt and Gfi1b deficient HSCs and observed that loss of Gfi1b leads to an altered RNA expression of integrins and adhesion molecules, for instance CXCR4, VCAM-1 and Tenascin C, which usually retain HSCs in a dormant state in the endosteal niche. These changes were also confirmed on protein level. Finally, we could observe a higher levels of Reactive Oxygen Species (ROS) in the Gfi1b deficient HSCs compared to wt HSCs. We verified whether elevated level of ROS are causative for the expansion of HSCs and noticed that application of N-Acetyl-Cystein, which counteracts the effects of ROS, limits significantly the expansion of HSCs, underscoring the important role of ROS in the expansion of Gfi1b deficient HSCs. Despite markedly increased proliferation, Gfi1b-/- HSCs can reconstitute lymphoid and myeloid lineages to the same extent as wt HSCs when transplanted in competition with wt HSCs. Furthermore, Gfi1b deficient HSCs also feature an expansion after transplantation and expand 5–10 fold more than wt HSC when transplanted initially in equal numbers with wt HSCs. It is possible that lower expression of CXCR4, VCAM-1 and other surface proteins leads to release and egression of Gfi1b deficient HSCs from the hypoxic endosteal stem cell niche and exposes the HSCs to more oxygen which in turn increases ROS levels. Elevated ROS could promote entry of Gfi1b-/- HSCs into cell cycle. In conclusion Gfi1b regulates HSC dormancy, pool size and potentially also the egress and mobilization of HSCs and might offer a new therapeutic approach to improve human HSC transplantation.

Disclosures:

No relevant conflicts of interest to declare.
14-3-3 Regulates the Lnk/JAK2 Pathway In Hematopoietic Stem and Progenitor Cells
Authors: Balcerek Joanna , Jiang Jing , Bersenev Alexey , Song Yiwen , Wu Chao , Tong Wei
Published: October 2019

Abstract 86

Hematopoietic stem and progenitor cell (HSPC) homeostasis is regulated by intricate signaling networks. The tyrosine kinase JAK2 plays an essential role in cytokine signaling during hematopoiesis and its dysregulation can lead to hematologic malignancies. Recently activating mutations in JAK2 were found in a large fraction of patients with myeloproliferative neoplasms (MPNs). We previously demonstrated that lymphocyte adaptor protein (Lnk) binds JAK2 and attenuates its activity, thereby limiting HSPC expansion (Bersenev et al., JCI, 2008;118:2832-2844). We further showed that loss of Lnk accelerates and exacerbates oncogenic JAK2-induced MPNs in mice (Bersenev et al., JCI, 2010;120:2058-2069). Lnk directly inhibits oncogenic JAK2 as well as acting through JAK2- independent pathways to constrain MPN development. Consistently, aged Lnk–/– mice spontaneously developed a CML-like MPN (Bersenev et al., JCI, 2010;120:2058-2069). More importantly, loss-of-function mutations in Lnk are found in human MPN patients (Oh, et. al., Blood, 2010, in press). This work suggests that Lnk plays a pivotal role in regulating both normal and malignant HSPC expansion. However, it remains to be determined how Lnk attenuates JAK2 activity since Lnk itself does not possess any enzymatic activity. Therefore, we began exploring Lnk regulatory mechanisms by identifying novel Lnk partners through protein purification and mass spectrometric analysis. This resulted in the identification of a number of novel Lnk binding proteins, which include the 14-3-3 proteins as the most robust interactors. 14-3-3s are a group of scaffold proteins that regulate many disease-relevant gene products and play important functions in many aspects of cellular processes. We found that Lnk is phosphorylated at two serine residues, which serve as the critical binding sites for 14-3-3. 14-3-3 abrogates the Lnk-JAK2 interaction thereby alleviating Lnk inhibitory function in both JAK2 signaling and cell growth. Furthermore, 14-3-3 binding is necessary and sufficient to maintain Lnk in an inactive state. We also investigated the signals that regulate Lnk phosphorylation, revealing Lnk as a signaling nodal point that integrates multiple signaling pathways in controlling HSPC homeostasis. The physiological significance of the Lnk-14-3-3 interaction in HSPC development will be discussed. Thus, our data implicate previously unappreciated serine phosphorylation events in Lnk-dependent hematopoietic function and regulation of JAK2. In addition, identification of novel signaling molecules that influence hematopoiesis might facilitate stem cell therapies and provide novel therapeutic targets for the treatment of MPNs.

Disclosures:

No relevant conflicts of interest to declare.
Loss of the Cell Cycle Regulator p26INCA1 Induces Exhaustion of Leukemic Stem Cells
Authors: Tschanter Petra , Baeumer Nicole , Lohmeyer Lisa , Berkenfeld Frank , Tickenbrock Lara , Diederichs Sven , Stehling Martin , Köhler Gabriele , Serve Hubert , Berdel Wolfgang E. , Koschmieder Steffen , Müller-Tidow Carsten
Published: October 2019

Abstract 96

Acute myeloid leukemia is a clonal disease characterized by a malignant proliferation and accumulation of myeloid progenitor cells. Current therapeutic strategies are often not able to eradicate the leukemic cells. Malignancy is associated with deregulation of cell cycle check- points and the deregulation of checkpoints is associated with altered stem cell properties. A better understanding of malignant stem cells and their cell cycle regulation might help to develop new therapies. Recently, we identified p26INCA1 as a novel cell cycle regulator. GST pulldown assays revealed binding of INCA1 predominantly to CDK2- specific Cyclins and we demonstrated an inhibitory effect of INCA1 on CDK2/ CyclinA complexes in kinase activity assays. The loss of INCA1 and its inhibitory effect on the cell cycle regulation led to an increased cell cycling and consequently to an enlarged stem cell pool in vivo. Upon cytotoxic stress, the loss of Inca1 enhanced cell cycling and bone marrow exhaustion. To analyze a potential role of INCA1 in leukemogenesis we retrovirally transduced wildtype and Inca1−/− bone marrow cells with AML1-ETO9a (A1E9a) and transplanted these cells into wildtype recipients. Most of the wildtype cell- transplanted recipients died due to AML. In contrast, only one of the Inca1−/− cell- transplanted mice developed AML. Engraftment was higher upon transplantion of Inca1−/− cells but engraftment was not sustained. To consider the repopulation capacity of the leukemic cells we performed transplantation of primary leukemic cells into secondary recipients. A1E9a induced leukemia in Inca1 wildtype cells was transplantable and lethal. However Inca1−/− leukemic cells were severely impaired in leukemia development in secondary recipients. Colony forming units and replating capacity were significantly reduced in A1E9a Inca1−/− bone marrow cells although these cells showed increased CDK2 activity. Exhaustion of leukemic cells in the absence of Inca1 was confirmed by cloning efficiency assays. Further analyses were performed with c-myc induced leukemias. Interestingly, Inca1 deletion precluded the development of leukemias in secondary recipients. Taken together, these findings identify an important role for p26INCA1 in the maintenance of leukemia and potentially the self-renewal capacity of leukemic stem cells.

Disclosures:

No relevant conflicts of interest to declare.
Genomics in 2K10: Fulfilling the Promise of a Sequenced Human Genome
Authors: Green Eric D.
Published: October 2019

Abstract SCI-16

The Human Genome Project's completion of the human genome sequence in 2003 was a landmark scientific achievement of historic significance. It also signified a critical transition for the field of genomics, as the new foundation of genomic knowledge started to be used in powerful ways by researchers and clinicians to tackle increasingly complex problems in biomedicine. To exploit the opportunities provided by the human genome sequence and to ensure the productive growth of genomics as one of the most vital biomedical disciplines of the 21st century, the National Human Genome Research Institute (NHGRI) is pursuing a broad vision for genomics research beyond the Human Genome Project. This vision includes facilitating and supporting the highest-priority research areas that interconnect genomics to biology, to health, and to society.Current efforts in genomics research are focused on using genomic data, technologies, and insights to acquire a deeper understanding of biology and to uncover the genetic basis of human disease. Some of the most profound advances are being catalyzed by revolutionary new DNA sequencing technologies; these methods are already producing prodigious amounts of DNA sequence data, including from large numbers of individual patients. Such a capability, coupled with better associations between genetic diseases and specific regions of the human genome, are accelerating our understanding of the genetic basis for complex genetic disorders and for drug response. Together, these developments will usher in the era of genomic medicine.

Disclosures:

No relevant conflicts of interest to declare.
DNMT3A and IDH1/2 Mutations Are Stable During the Progression of Acute Myeloid Leukemia
Authors: Kihara Rika , Hoshino Hideaki , Kiyoi Hitoshi , Naoe Tomoki
Published: October 2019

Abstract 1466

Several genetic alterations, which are involved in the pathophysiology of acute myeloid leukemia (AML), have been identified. Recently some mutations in genes affecting DNA and histone methylation have been reported. Their stabilities during the disease progression are not fully clarified. We analyzed mutations of FLT3, NRAS, NPM1, TP53, CEBPA, IDH1/2 and DNMT3A genes in paired initial diagnosis and the first relapse samples from 44 adult AML patients. Informed consent was obtained from all patients, and approval was obtained from the ethics committee of Nagoya university school of medicine. At diagnosis, we identified FLT3 mutation in 12 patients, NPM1 mutation in 17, N-RAS mutation in 7, TP53 mutation in 2, CEBPA mutation in 7, IDH1/2 mutations in 10 and DNMT3A mutation in 6. At relapse, the acquisition and loss of FLT3 mutation were observed in 4 and 1 patients, respectively. Those of N-RAS mutation were in 2 and 3 patients, respectively, and those of TP53 mutation were in each one. No patient acquired NPM1 mutation, but one patient lost it at relapse. And no patient acquired CEBPA mutation, but 2 patients lost it at relapse. One patient acquired IDH1/2 mutation, but no patient lost them at relapse. The acquisition and loss of DNMT3A mutation was not observed. Notably, one patient harboring IDH1 and FLT3 mutations at the diagnosis lost FLT3 mutation, but not IDH1 mutation at relapse; one patient harboring IDH1, DNMT3A and NRAS mutations at the diagnosis lost NRAS mutation, but not IDH1 and DNMT3A mutations at relapse and one patient harboring IDH2 and CEBPA mutations at the diagnosis lost CEBPA mutation, but not IDH2 mutation at relapse. These results indicated that Class 2 mutation, which is more stable than Class 1 mutations, may be lost during the progression of AML. However, IDH1/2 and DNMT3A mutations were stable during the disease course of AML suggesting aberrations of the DNA methylation is an early event in the leukemogenesis.Type of changeFLT3 (n = 44)NRAS (n = 44)NPM1 (n = 44)TP53 (n = 27)CEBPA (n = 34)IDH1/2 (n = 44)DNMT3A (n = 44)Gain at relapse4301010Loss at relapse1311200Total no. of changes(%)5(11)6(14)1(2.2)2(7.4)2(5.9)1(2.2)0(0)

Table Frequencies of gain and loss of gene mutations at relapse in AML

Disclosures:

Naoe: Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.
WT1 Gene Expressions Predicting Posttransplant Outcome
Authors: Kim Heeje , Choi Hee-Baeg , Sohn Hyun-Jeong , Lee Jong-Wook , Min Woo-Sung , Kim Tai-Gyu
Published: October 2019

Abstract 1499

Patients with AML show over expression of Wilms' tumor gene 1 (WT1). WT1 is a valuable prognostic parameter for minimal residual disease (MRD). Quantitation of this gene transcript by real-time quantitative PCR (RQ-PCR) may be useful for predicting prognosis in AML. We prospectively investigated the prognostic potential of the leukemia-associated antigen, WT1. We simultaneously determined the expression of the gene in bone marrow (BM) from 160 adult patients with newly diagnosed AML and healthy normal controls using RQ-PCR. The molecular profiles were then evaluated with multiple parameters. We evaluated whether the expression level of the gene in the patients affected complete remission (CR) and survival rates before and after hematopoietic stem cell transplant (HSCT). The median follow-up period for all patients who were event-free survivors was 30 months (range: 2–56). The Kaplan-Meier estimated overall survival (OS) and event-free survival (EFS) rates in the whole population of evaluable patients were 52% (95% CI, 31%–41%) and 50% (95% CI, 31%–43%), respectively. The median expression levels of the gene in normal control and patients were 7.4 (range 0.74–30.3), 58.6 (range 0.9–1950.5), respectively. Of note, WT1 levels of expression at initial diagnosis revealed influential to the EFS after HSCT (P =0.041). Interestingly, there were no correlated results with CR, OS, and EFS rates for treated AML, specifically before HSCT. This finding may suggest that initial expressions of WT1 possibly in association with the leukemic stem cells remained post-transplant can be the critical target of future immunotherapeutic trial.

Disclosures:

No relevant conflicts of interest to declare.
AZD1208, a Novel, Potent and Selective Pan PIM Kinase Inhibitor, Demonstrates Efficacy in Models of Acute Myeloid Leukemia
Authors: Keeton Erika , Palakurthi Sangeetha , Alimzhanov Marat , Grondine Michael , Chen Yuching , Brown Jeffrey , McEachern Kristen , Cao Yichen , Chinnappan Dharmaraj , Shen Minhui , Dakin Les , Zheng Xiao , Lamb Michelle , Wu Allan , Chen Huawei , Lyne Paul , Huszar Dennis
Published: October 2019

Abstract 1540

The PIM serine/threonine kinase family is composed of three highly homologous members; PIM1, PIM2 and PIM3, identified by the ability of the prototype member PIM1 to drive lymphomagenesis in mice. Upregulation of PIM1 and PIM2 is observed in leukemias and lymphomas, including AML, NHL and CLL, highlighting the potential of these kinases as therapeutic targets in these indications. PIMs are downstream effectors of many cytokine and growth factor signaling pathways and are direct transcriptional targets of STAT transcription factors activated by these pathways. PIMs can phosphorylate multiple substrates to mediate cell proliferation and survival. Here we describe the function of AZD1208, an orally available, potent and highly selective PIM inhibitor that effectively inhibits all three isoforms. AZD1208 inhibits the growth of several AML cell lines and sensitivity correlates with the level of PIM1 expression, STAT5 activation and presence of protein tyrosine kinase mutation. AZD1208 causes cell cycle arrest and apoptosis in MOLM-16 cells in culture. This is accompanied by a dose-dependent reduction in pBAD serine 112 and other substrates, as well as an increase in cleaved caspase 3. AZD1208 suppresses the growth of MOLM-16 and KG-1a xenograft tumors in vivo in a dose proportional manner. In addition, AZD1208 leads to potent inhibition of colony growth of primary AML cells from bone marrow aspirates and downregulates phosphorylation of PIM targets. These results underscore the therapeutic potential of PIM kinase inhibition by AZD1208 for the treatment of AML.

Disclosures:

Keeton: AstraZeneca: Employment, Equity Ownership. Palakurthi:AstraZeneca: Employment, Equity Ownership. Alimzhanov:AstraZeneca: Employment, Equity Ownership. Grondine:AstraZeneca: Employment, Equity Ownership. Chen:AstraZeneca: Employment, Equity Ownership. Brown:AstraZeneca: Employment, Equity Ownership. McEachern:AstraZeneca: Employment, Equity Ownership. Cao:AstraZeneca: Employment, Equity Ownership. Chinnappan:AstraZeneca: Employment, Equity Ownership. Shen:AstraZeneca: Employment, Equity Ownership. Dakin:AstraZeneca: Employment, Equity Ownership. Zheng:AstraZeneca: Employment, Equity Ownership. Lamb:AstraZeneca: Employment, Equity Ownership. Wu:AstraZeneca: Employment, Equity Ownership. Chen:AstraZenenca: Employment, Equity Ownership. Lyne:AstraZeneca: Employment, Equity Ownership. Huszar:AstraZeneca: Employment, Equity Ownership.