bluuebunny/crossref_metadata_2025_split huggingface dataset:
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
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
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
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
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
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
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
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
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.