bluuebunny/crossref_metadata_2025 huggingface dataset:
Donor-Cell Leukemia After Hematopoietic Stem Cell Transplant: a Retrospective Review of a Single Center's Experience
Authors: Washburn Katie , Selby George B. , Dunn S. Terence , Chakrabarty Jennifer L. Holter
Published: October 2019

Abstract 1997

Donor-cell leukemia (DCL) is a rare complication of allogeneic hematopoietic stem cell transplant (ASCT) with a reported incidence varying from 0.1% to 5.0% of all post-transplantation relapses. However, the incidence is likely to be highly underestimated due to the difficulty in making the diagnosis. In the 1970s and 1980s, diagnosis of DCL relied on cytogenetic and fluorescence in situ hybridization studies in sex-mismatched donor/recipient pairs. Recent advances in molecular-based laboratory methods have considerably improved our ability to identify and confirm DCL in ASCT patients, especially when there is not a sex-mismatch between donor and recipient. Recently, an algorithm has been proposed by Wang et al. 2011 (Am J Clin Pathol 135;525–40) to help make the diagnosis of DCL in ASCT patients who develop overt leukemia or a clonal cytogenetic abnormality using these new advanced techniques. We retrospectively evaluated all patients who underwent ASCT at our center for acute leukemia from 2005 to present. Out of 43 patients, 6 apparent DCLs were documented using short tandem repeat (STR) analysis (12 separate loci examined); each ASCT patient relapsed with the full complement of donor STRs. Our data confirms that the true incidence of DCL is likely underestimated, since within our select population, we had a 14% incidence of DCL. Of note, 1/6 were sex mismatches, 3/6 received cord blood transplants, 4/6 cases were ABO mismatches, and 4/6 cases were AML (Table 1). Our findings concur with sparse available reports in the literature that purport there may be a shorter time to relapse in patients who are undergoing ASCT for malignant reasons; indeed, our average latency period was 14 months as opposed to 24 months found by Wang et al. However, we did not find that a disproportionate number of sex-mismatched donor/recipient pairs developed DCL; in fact, nearly all of our DCLs arose in the setting of a sex-matched pair. The apparent increased incidence in cord blood transplants, sex-mismatched donor/recipient pairs and ABO-mismatched transplants needs to be assessed in a large-scale registry using standard methodologies, which could help identify and define potential causes of DCL.Table 1:Patient Characteristics with Donor-Derived LeukemiaSexAge (years)Leukemia TypeTransplant TypeTime to Relapse (months)Sex MismatchABO MismatchChemotherapy PreparationM22ALLunrelated donor15NoYesalemtuzumabF1AMLrelated sibling14NoNoBu/Cy/Mito1M24AMLunrelated double-cord blood40NoNoTVTG2M20AMLunrelated donor5NoYesCy/TBI3M26AMLunrelated double-cord blood10.5YesYesFlu/Cy/TBI4M26ALLunrelated double-cord blood3.5NoYesFlu/Cy/TBI41. busulfan, cytarabine, mitoxantrone2. topotecan, vinorelbine, thiotepa, gemcitabine3. cyclophosphamide & total body irradiation4. fludarabine, cyclophosphamide & total body irradiation

Disclosures:

No relevant conflicts of interest to declare.
Achievement of Complete Remission After High-Dose Melphalan and Autologous Stem Cell Transplantation Is the Only Important Prognostic Factor in Patients with Multiple Myeloma
Authors: Shin Seunghwan , Kim Jeong-A , Lee Bohee , Min Chang Ki , Kwak Jae-Yong , Yhim Ho-Young
Published: October 2019

Abstract 2018

Background:

High-dose melphalan and autologous transplantation (HDM-AT) is the most effective treatment for survival benefits in multiple myeloma (MM) patients. However, the relationship between the HDM-AT response and long-term outcome is not clear. In this study we examined the relationship.

Method:

We analyzed the clinical data of 137 patients treated with HDM-AT in three institutions between 2000 and 2011. The international Myeloma Working Group Uniform Response Criteria was used to evaluate.

Results:

Out of 137 patients, clinical data of 126 patients were evaluable for treatment response according to the medical records. Their median age was 55 years (range, 36–65 years) and 69 (55%) patients were male. Among the patients who had received induction treatment (IT), 79%(100/126) of the patients were treated with bortezomib-containing regimens. After IT, the response rates were 29% CR, 18% VGPR, 37% PR, 15% SD, and 1% PD. After HDM-AT, response rates were 64% CR, 15% VGPR, 15% PR, 4% SD, and 2% PD. Median follow-up duration was 25 months (range, 5–146 months). 5-year overall survival (OS) and progression-free survival (PFS) were 54% and 43 %, respectively. Multivariate analysis regarding OS showed that CR after HDM-AT compared to ≤CR is an important prognostic factor (5-year OS rate of CR vs ≤CR, 78% vs 29%, respectively, p=0.002). However, CR after HDM-AT is not prognostic factor for PFS.

79 (79/126, 64%) patients showed the CR after HDM-AT. Among the 79 patients, 44 patients did not achieve CR after IT however, turned into CR after HDM-AT; 35 patients already achieved CR after IT. However, there were no significant differences between the two groups of patients (5-year PFS, 44% vs. 45%, P=0.245 and 5-year OS 77% vs. 79%, P=0.690 respectively).

Conclusion:

Achievement CR after HDM-AT is the only important prognostic factor to obtain the survival benefit regardless of response after IT in MM patients.

Disclosures:

No relevant conflicts of interest to declare.
Anticoagulation in Ambulatory Cancer Patients
Authors: Pishko Allyson , Smith Kenneth J. , Ragni Margaret V.
Published: October 2019

Abstract 2071

Background:

Venous thromboembolism (VTE) risk is increased in patients with malignancy, occurring in up to 20%, and is a major cause of cancer mortality. VTE prophylaxis is currently recommended for inpatients with malignancy and in those undergoing surgery (Chest 2008). Despite evidence that low molecular weight heparin (LWMH) has anti-tumor effect and improves short-term survival (Cochrane, 2011), the cost-effectiveness of anticoagulation in ambulatory cancer patients is unknown.

Methods:

We constructed a Markov model to evaluate prophylactic AC in ambulatory cancer patients with no previous VTE, during 4 months of chemotherapy following diagnosis. In the model, AC was enoxaparin. Major and minor bleeding risk, VTE risk, mortality, and other clinical parameters were obtained from the Cochrane 2011 analysis and other published studies. Drug costs were based on average wholesale price. Outcomes included medical costs, effectiveness measured by mortality reduction, quality-adjusted life-years (QALY), and incremental cost-effectiveness ratio (ICER) over a 24-month time horizon.

Results:

Base case results are shown in the Table. Compared with no LMWH, primary prophylaxis with LMWH in ambulatory cancer patients over a 4-month period was associated with a relative mortality risk of 0.92 over 24 months (relative risk reduction of 0.08), at a gain of 0.0484 QALY, for an ICER of $76,922/QALY gained, well within the accepted $100,000/QALY threshold. The results were not sensitive to variation in VTE relative risk (RR) with AC (base case RR 0.55), nor to AC-associated major bleeding (base case RR 1.3) or minor bleeding (base case RR 1.05) risks. Variation of the relative mortality risk with AC to ≥ 0.94, or the cost of AC (Figure) to ≥ $1,429/month (base $1,132), increased the ICER to >$100,000/QALY.

Discussion:

Prophylactic AC appears cost-effective in ambulatory cancer patients during the first four months of chemotherapy. Clinical trials will be needed to confirm the survival benefit of LMWH prophylaxis in ambulatory cancer patients.

Disclosures:

No relevant conflicts of interest to declare.
KLF1 Modulates Delta-Aminolevulinic Acid Dehydratase (ALAD) Gene Transcription in a Context-Specific Manner
Authors: Desgardin Aurelie , Abramova Tatiana , Lin Jenny , Shim Eun-Hee , Cunningham John M
Published: October 2019

Abstract 2094

Krüppel-like factor 1 (KLF1) is a zinc finger-encoding transcription factor that recognizes CACC elements, and is essential for maximal erythroid-specific gene transcription. Several critical mechanisms dependent on KLF1 and required for gene activation have been elucidated, predominantly using the beta-globin locus. KLF1 has been associated with the ordered recruitment of SWI/SNF and RNA polymerase-II complexes, necessary for chromatin remodeling and gene transcription respectively. KLF1 has also been reported to influence erythroid-specific heme biosynthesis. Studies in KLF1-null fetal erythroblasts and a KLF-1 deficient cell line have demonstrated that mRNA levels of the first three enzymes of the biosynthetic pathway are underrepresented. However, although in vitro studies of the rate-limiting enzymes ALAS2 and PBGD suggested a potential regulatory role for KLF1, in vivo studies failed to validate these findings. ALAD is the second enzyme of the pathway. Complete loss of ALAD expression in erythroid cells results in catastrophic events during zebrafish ontogeny. Interestingly, no human erythropoietic defect has been reported as a consequence of aberrant ALAD expression. To extend the analysis of KLF1's regulation of heme biosynthesis, we evaluated KLF1 binding of enzyme regulatory sequences by EMSA and ChIP studies, identifying a KLF1 binding CACC element in the erythroid-specific ALAD promoter. This regulatory element was transactivated specifically by a KLF1 transgene in KLF1-deficient cells. Using a unique 4-OH-Tamoxifen (4-OHT) mediated KLF1-inducible erythroid cell line (K1-ERp), we identified KLF1 as an essential, and early (within 2 hours of induction) activator of transcription of the endogenous ALAD, but not ALAS2 or PBGD genes. Further studies in K1-ERp cells, including DNAseI hypersensitivity and ChIP assays revealed that KLF1 occupancy at the erythroid-specific ALAD promoter triggers a series of molecular events including histone modifications, and enhanced recruitment of the sequence-specific transcription factors, GATA-1, NF-E2 and the TAL-1/SCL multiprotein complex. Importantly, we identified differences in the kinetics of recruitment of the closely related histone acetyltransferases proteins CBP and p300 and the SWI/SNF ATPase Brg1. The latter complex was recruited subsequent to KLF1 binding, although the ALAD promoter was already DNAseI hypersensitive. These results suggest strongly that KLF1 plays a major role in the regulation of heme biosynthesis in erythroid cells. Furthermore, our data challenges a model in which an identical temporal cascade of molecular events are required for transcription at KLF1-dependent promoters.

Disclosures:

No relevant conflicts of interest to declare.
A Robust Ex Vivo culture System for Production of Mature Human Reticulocytes From a Single Donor
Authors: Cogan Nicola , Kupzig Sabine , Parsons Steve F , Anstee David J
Published: October 2019

Abstract 2096

We have formulated a robust culture system simulating human erythropoiesis, producing mature reticulocytes from adult peripheral blood CD34+ progenitor cells in 20 days. Expansion of cell numbers over the course of the culture exceeded 104-fold and enucleation efficiency was up to 90% without the use of feeder layers. Mature reticulocytes and erythrocytes were isolated from culture by passage through a standard leucocyte filter. Scanning and transmission electron microscopy of the reticulocytes revealed morphological features indistinguishable from those of mature reticulocytes isolated from the peripheral blood of normal donors. Post-filtration cells expressed adult haemoglobin and had a capacity to bind and release oxygen comparable to that of adult peripheral blood erythrocytes. Examination of blood group active- and other cell surface antigens revealed a normal glycosylation profile and normal cell surface protein expression. Most notably, the cells did not express cryptantigens T and Tn. LORCA analysis indicated that cultured cells had slightly reduced membrane stability and deformability compared to peripheral blood erythrocytes. Haematology parameters demonstrated a typical mean cell volume of 131fL and mean cell haemoglobin of 37.7pg. This, scalable approach has allowed us to culture cells from a single apheresis cone to produce 1010 reticulocytes, which represents a packed cell volume of 1ml. These results clearly demonstrate the feasibility of producing cultured red cell products suitable for transfusion therapy ex vivo from adult peripheral blood stem cells.

Disclosures:

No relevant conflicts of interest to declare.
Functional Evaluation of HbF-Associated Region of BCL11A Locus
Authors: Bauer Daniel E. , Xu Jian , Fujiwara Yuko , Stamatoyannopoulos John A. , Orkin Stuart H.
Published: October 2019

Abstract 2148

The severity of the major β-globin disorders, sickle cell disease and β-thalassemia, is modified by the residual level of fetal hemoglobin (HbF, α2γ2). Reactivation of HbF by derepression of γ-globin gene expression constitutes a major therapeutic goal, though an incomplete understanding of globin gene regulation has limited such efforts. The postnatal level of HbF is a highly heritable quantitative trait. Genome-wide association (GWA) studies have identified several HbF-associated loci, including the BCL11A gene. BCL11A has been demonstrated to be a bona fide repressor of γ-globin expression in mouse and man. However, the mechanisms by which common genetic variation modulate BCL11A expression remain poorly understood. Numerous GWA studies, conducted in both healthy individuals and those with hemoglobinopathies, and including populations of European, Asian, and African ancestries, have consistently found that HbF-associated variants cluster within a 14-kb region of the second intron of BCL11A. At least twenty HbF-associated SNPs have been identified within this region, with various of these genetic markers considered as “tag SNPs” most strongly associated with HbF levels depending on the particular GWA study. Based on patterns of linkage disequilibrium among SNPs, several common haplotypes have been identified. The tag SNP-dense intronic region, more than 55 kb downstream of the BCL11A transcription start site, demonstrates evolutionary sequence conservation. Here we show that this region of BCL11A is decorated by epigenetic features in human erythroid precursors indicative of distal regulatory potential. Specifically, this intronic region is marked by the presence of the histone modifications H3K4me1 and H3K27ac and the absence of H3K4me3, indicating an enhancer signature. The erythroid transcription factors GATA1 and SCL/TAL1 co-occupy discrete sequences within this region, with the binding peaks of these master erythroid regulators overlapping those of the histone marks. Furthermore, this tag SNP-dense intronic region of BCL11A displays CpG hypomethylation and exhibits DNase I hypersensitive sites restricted to the erythroid lineage but not other lineages that express BCL11A. Moreover, in in vivo transient transgenic assays, this region displays erythroid-restricted enhancer activity. A minimal 1.8-kb sequence possessing erythroid enhancer function has been mapped. This region includes GATA1 and SCL/TAL1 binding sites. We hypothesize that common polymorphisms are in linkage disequilibrium with functional variants that modulate erythroid enhancer activity. These studies further delineate pathways critical for hemoglobin switching and serve as an example of how common genetic variation may influence traits by affecting non-coding regulatory elements.

Disclosures:

No relevant conflicts of interest to declare.
Relationship Between Pulmonary Artery Pressure and Spleen Volume in Non-Splenectomized Hemoglobin E/β-Thalassemia Patients
Authors: Atichartakarn Vichai , Chuncharunee Suporn , Yamwong Sukit , Yingchoncharoen Teerapat , Jongjirasiri Sutipong , Archararit Napaporn , Udomsubpayakul Umaporn , Aryurachai Katcharin
Published: October 2019

Abstract 2156

Introduction:

Clinically significant pulmonary arterial hypertension (PAH) has been reported in splenectomized hemoglobin E/β-Thalassemia patients. Like primary PAH, the underlying basis is hypercoagulability related thrombotic pulmonary arteriopathy, which results in an increased pulmonary vascular resistance. In the non-splenectomized counterparts with no clear evidence of hypercoagulability only mild PAH is found. To evaluate role of splenomegaly and the accompanying increased blood volume in the pathogenesis of PAH in this group of patients, we studied the relationship between spleen volume and PA pressure.

Patients and Methods:

Nineteen clinically stable hemoglobin E/β-Thalassemia outpatients, on no medication aside from folic acid and who received no blood transfusion in the preceding 4 weeks were first analyzed in this ongoing study. All gave written informed consent, and study protocol was approved by the institution ethics committee on studies in humans (ID 07–53-01). PA pressure (PAP) was estimated by a single echocardiologist (T.Y.) using Philips iE33 echocardiographic system with a S5 probe (Philips Medical Systems, Andover, MA). Spleen volume was determined by CT scan with non-contrast, 320-slice, 3 mm axial cut (Aquilion one, Toshiba, Japan). Statistical analysis of clinical and laboratory data was done by SPSS version 18 (SPSS Inc, Chicago, Ill), considering a P value <0.05 as statistically significant. Results: Patients' characteristics are shown. Results are expressed as mean ± SD or median (range) where appropriate. All had normal left ventricular systolic ejection fraction and no valvular heart disease. There was good correlation (r=0.609, P=0.006) between spleen volume and mean PAP.

Conclusions:

Non-splenectomized patients with hemoglobin E/β-Thalassemia have elevated PAP in proportion to spleen size, likely secondary to elevated blood volume. This situation is distinct from the pathologic PAH that results from increased pulmonary vascular resistance in splenectomized patients.

Disclosures:

No relevant conflicts of interest to declare.
Onset of Cardiac Iron Loading in a Large and Homogeneous Cohort of Thalassemia Major Paediatric Patients
Authors: Pepe Alessia , Meloni Antonella , Favilli Brunella , Capra Marcello , D'Ascola Domenico Giuseppe , Palazzi Giovanni , Quarta Antonella , Casini Tommaso , Putti Maria Caterina , Cianciulli Paolo , Franceschi Lucia De , Positano Vincenzo , De Marchi Daniele , Lombardi Massimo , Filosa Aldo
Published: October 2019

Abstract 2157

Introduction:

Magnetic Resonance Imaging (MRI) by the T2* technique allows highly reproducible and non invasive quantifications of myocardial iron burden and it is the gold standard for quantifying biventricular function parameters. It is important to determine the appropriate age to start MRI screening, because its high cost. Few data are available in the literature and they are contrasting. So the aim of this study was to address this issue in our paediatric patients with thalassemia major (TM).

Methods:

We studied retrospectively 72 patients (47 males, 4.2–17.9 years old, mean age 13.03 ± 3.70 years), enrolled in the MIOT (Myocardial Iron Overload in Thalassemia) network. Myocardial iron overload was measured by T2* multislice multiecho technique. Biventricular function parameters were quantitatively evaluated by cine images.

Results:

The global heart T2* value was 29.7 ± 11.2 ms (range 6.2 – 48.0 ms). No significant correlation was found between global heart T2* value and age (see figure). The global heart T2* value did not show significant differences according to the sex (male 30.2 ± 11.0 ms versus female 28.7 ± 11.8 ms, P=0.568). Sixteen patients (22%) showed an abnormal global heart T2* value (<20 ms) and none of them was under 8 years of age. Global heart T2* value was negatively correlated with mean serum ferritin levels. Odds Ratio for high serum ferritin levels (≥ 1500 ng/ml) was 8.4 (1.01–69.37, OR 95%CI) for abnormal global heart T2* values (< 20 ms). The global heart T2* value did not show a significant difference with respect to the chelation therapy (P=0.322). No significant correlations were found between the global heart T2* values and the bi-atrial areas or the LV and RV morphological and functional parameters. Eight patients showed a left ventricular (LV) ejection fraction (EF) < 57% and none of them was under 7 years of age. Two patients showed a right ventricular (RV) EF < 52% and none of them was under 14 years of age.

Conclusion:

The MRI screening for both cardiac iron overload and function assessment can be started for TM patients at the age of 7 years. At this age not sedation is generally needed. If the availability of cardiac MRI is low, the serum ferritin levels could be used as a discriminating factor.

Disclosures:

No relevant conflicts of interest to declare.
Leukocyte Integrin Activation and Alterations in Parathyroid Hormone Levels Following G-CSF Administration
Authors: Donahue Robert E. , Tuschong Laura , Bauer Thomas R. , Hickstein Dennis D. , Yau Yu Ying , Leitman Susan F.
Published: October 2019

Abstract 2164

Following administration of granulocyte colony-stimulating factor (G-CSF) there is a marked but transient drop in circulating neutrophils. To determine the contribution of integrin activation to these events, a monoclonal antibody (mAb 24) that recognizes the activated α-subunit cation binding domain of the integrin family of receptors was used to evaluate human leukocytes following G-CSF administration. A single 480 mcg dose of human recombinant G-CSF (Neupogen, Amgen) was administered in the upper arm of six healthy human subjects (3 male, 3 female). EDTA and heparinized blood samples were collected prior to and at 15, 30, 60, 90, 120 and 240 minutes after injection. Mean absolute neutrophil count decreased from 2,557 ± 654/μl at baseline to 227 ± 193 (p<0.001) at 30 and 45 minutes. Flow cytometric analysis revealed a dramatic increase in binding of mAb24, which recognizes the occupied divalent cation binding site of the activated integrin α subunit, to neutrophils but not other circulating nucleated cells within 15 to 30 minutes after G-CSF administration, concurrent with a dramatic decrease in circulating neutrophil numbers. This activation, like the drop in neutrophil count, was transient, with a return to baseline status by 60 minutes. Intact parathyroid hormone (iPTH) levels were also evaluated. Within 30–90 minutes following G-CSF injection there was a significant drop in iPTH from a baseline of 52.5 ± 31.1 to a nadir value of 30.6 ± 20.9 pg/ml (p=0.01). A rebound in iPTH levels to 63.4 ± 27.0 pg/ml (p<0.001) occurred at 90 to 240 minutes. There were no significant changes in ionized calcium or magnesium levels. These results demonstrate that the integrin on circulating neutrophils is transiently activated following G-CSF administration and that this activation may mediate the transient neutropenia seen in this setting. In addition, alterations in PTH levels occur following G-CSF administration. As it is known that iPTH influences the hematopoietic stem cell (HSC) niche, fluctuations in iPTH levels following G-CSF administration may play a role in HSC expansion.

Disclosures:

No relevant conflicts of interest to declare.
Non-AIDS-Defining Hematologic Malignancies in HIV-Infected Patients: A Nationwide Epidemiologic Study in Japan
Authors: Hagiwara Shotaro , Yotsumoto Mihoko , Okada Seiji
Published: October 2019

Abstract 2183

Background.

The prevalence of HIV infection is estimated to be less than 0.1% in Japan; however, the number of newly reported HIV-infected persons has gradually increased, and the estimated number of HIV-infected patients now ranges from 6300 to 10000. Since the introduction of highly active antiretroviral therapy (HAART), the morbidity and incidence of AIDS-defining cancers has decreased. However, the incidence of non-AIDS-defining cancers has increased in Japan. To elucidate the incidence and clinical outcome of non-AIDS-defining hematologic malignancies, excluding non-Hodgkin's lymphomas, in HIV-infected patients, we conducted a nationwide epidemiologic study.

Methods.

We surveyed 429 regional AIDS centers and 497 educational hospitals certified by the Japanese Society of Hematology. Epidemiologic data for HIV was sourced from the Joint United Nations program on HIV/AIDS.

Results.

We obtained data from 511 institutes (55.4%). From 1991 to 2010, 47 cases of non-AIDS-defining hematologic malignancies were found—19 cases of Hodgkin's lymphoma (HD), 13 of AML, 7 of ALL, 4 of CML, 2 of multiple myeloma, 1 of CLL, and 1 of MDS-RAEB1. The mean age was 49.3 years (21–70 years), and 93.6% patients were male. The mean CD4-positive cell count was 303.7/μL (1–1371/μL). The mean duration from the diagnosis of HIV infection to hematologic malignancy was 45.0 months (0–204 months). Before diagnosis, 68.1% patients had been treated with HAART and 51.1% patients had AIDS. Sixteen of 19 HD patients were treated with ABVD or radiation therapy. Of these, 80% patients achieved complete remission, with a 2-year overall survival of 62.3%. Twelve of AML and 6 of ALL patients were treated with standard induction chemotherapy. Eight of twelve patients (66.7%) with AML and five of six patients (83.3%) achieved complete remission. The median survival for both AML and ALL patients was 16 months, with 42.8% ALL and 30.8% AML patients alive after 4 years. Comparison of the last 2 decades (1991–2000 and 2001–2010) showed a 4.7-fold increase in the incidence of hematologic malignancies (11.5 vs. 54.6/100000 person-years).

Conclusion.

A nationwide epidemiologic study revealed that HIV-infected patients are at high risk of hematologic malignancies, and that the incidence of hematologic malignancies has increased in the past decade. The prognosis was equivalent to that of HIV-negative patients, and standard chemotherapy may be a feasible treatment option for HIV-infected patients with hematologic malignancies.

Disclosures:

No relevant conflicts of interest to declare.
In Vitro Generation of “Long-Lived” Human Plasma Cells
Authors: Stephenson Sophie , Cocco Mario , deTute Ruth , Rawstron Andy , Doody Gina , Tooze Reuben
Published: October 2019

Abstract 2187

Plasma cells, the terminal effectors of the B-cell lineage include both short- and long-lived cells. The latter persist for extended periods in the absence of cell division, supported in niche environments. No model system has successfully recapitulated the function of the niche to allow the in vitro generation of long-lived plasma cells. This limits investigation into the factors controlling and targeting plasma cell populations. Here we describe the generation of mature human plasma cells with extended lifespan from peripheral blood B-cells. Cell division accompanies phenotypic maturation between plasmablasts and plasma cells. These cells then persist in the absence of cell division, remaining functional and viable in extended culture, currently limited solely by elective termination. Extended survival is accompanied by maturation to a phenotype consistent with human bone marrow plasma cells. By establishing a set of conditions sufficient to allow the development and persistence of mature human plasma cells in vitro, we recapitulate the essential function of the plasma cell niche. We definitively link phenotypic maturation to lifespan and provide the first platform with which to explore and manipulate the full trajectory of human plasma cell differentiation.

Disclosures:

No relevant conflicts of interest to declare.
Pathological Shear Regulates ADAM10 Activity on Circulating Platelets
Authors: Gardiner Elizabeth E , Qiao Jianlin , Tan Cheewee , Arthur Jane Frances , Al-Tamimi Mohammad , Ludwig Andreas , Ward Christopher M , Andrews Robert K
Published: October 2019

Abstract 2194

Metalloproteinase-mediated ectodomain shedding of platelet-specific receptors for collagen (GPVI) and von Willebrand factor (GPIbα of the GPIb-IX-V complex) is triggered by either ligand-induced platelet activation-dependent pathways, or by activation-independent pathways mediated by Factor Xa or induced by the thiol-modifying agent, N-ethylmaleimide. We recently reported that shed soluble GPVI (sGPVI) was elevated in plasma of 159 ischaemic stroke patients compared with 159 community-based controls (P=0.0168), and in 29 patients with disseminated intravascular coagulation compared with healthy donors (n=25, P=0.002), consistent with a pathophysiological role for GPVI shedding from human platelets. Our new studies now show that transient exposure of human platelets to arterial or pathological shear rates of 3000–10,000 s−1 for 1–5 min ex vivo in a cone-plate viscometer, in the absence of GPVI ligand or platelet activation, activated sheddases producing a 2- to 3-fold increase in plasma sGPVI and a corresponding loss of surface GPVI from sheared platelets. Shear-induced GPVI shedding was blocked by GM6001 or GI254023, a selective inhibitor of ADAM10. In contrast to shear-induced platelet aggregation, shedding was unaffected by inhibitors of aggregation (VWF-blocking anti-GPIbα mAb, AK2, or the αIIbβ3 antagonist, RGD peptide) or by the absence of VWF in a patient with von Willebrand's disease Type III (VWF antigen levels <1%). Further, shedding of GPVI increased for up to 10 min after cessation of a short exposure of platelets to shear even when signalling, secretion and aggregation was blocked by inhibiting intracellular kinases (PP2, piceatannol), thromboxane generation (aspirin), ADP (apyrase) and calcium flux (BAPTA). Together, the combined results provide the first evidence that receptor sheddase activity can be regulated by hydrodynamic shear stress independent of cellular activation. This may represent a novel protective mechanism for down-regulating platelet reactivity as a response to pathological shear.

Disclosures:

No relevant conflicts of interest to declare.
Trypsin Causes Platelet Activation Independently of Known Protease-Activated Receptors
Authors: Mao Yingying , Kunapuli Satya P.
Published: October 2019

Abstract 2197

In platelets, Thrombin is the major agonist for PAR1 and PAR4, although PARs can be activated by other proteases. The physiological agonist of PAR3 has not been identified to date. To identify a physiological agonist of PAR3, we used PAR4 null murine platelets, which were known to express only PAR3. In this study, we tested several proteases and found that trypsin, but not heat-inactivated trypsin, activated PAR4 null murine platelets. Even at high concentrations, trypsin caused shape change without increasing intracellular calcium levels in PAR4 null murine platelets. Consistent with this result, Gq inhibitor YM-254890 or calcium chelator BAPTA-AM had no effect on trypsin-induced shape change. However, trypsin-induced platelet shape change was completely abolished by ROCK inhibitor Y27632 or H1152, suggesting that trypsin-induced PAR4 null murine platelet shape change is mediated by G12/13, but not Gq pathway. Furthermore, trypsin caused phosphorylation of myosin light chain (Thr18), but not Akt or Erk. To investigate whether Trypsin activated PAR3, we desensitized PAR4 in both WT and PAR3 null murine platelets. Surprisingly, trypsin caused similar shape change in PAR4-desensitized PAR3 null murine platelets as in PAR4 null murine platelets, indicating that trypsin did not activate PAR3 to cause shape change. More interestingly, a Src family kinase (SFK) inhibitor PP2 abolished trypsin-induced, but not AYPGKF-induced, shape change. Hence, trypsin activated a novel signaling pathway through RhoA/p160ROCK and was regulated by SFKs. In conclusion, our study demonstrates a novel protease signaling pathway in platelets that is independent of PARs. This protease-induced novel signaling pathway regulates platelet shape change through SFKs and p160ROCK.

Disclosures:

No relevant conflicts of interest to declare.
Long-Term Outcomes of Renal Function in Patients with TTP Associated with Severe ADAMTS13 Deficiency
Authors: Reese Jessica A , Deford Cassandra C , Al-Nouri Zayd , Terrell Deirdra , Vesely Sara K. , Strebel Johanna A. Kremer Hovinga , Lammle Bernhard , George James N.
Published: October 2019

Abstract 2215

Introduction:

Among survivors of TTP associated with severe ADAMTS13 deficiency, long-term outcomes other than relapse are not well documented. The recent report of persistent minor neurocognitive abnormalities attributed to subcortical microvascular thrombosis (Transfusion 2009;49:1092) suggested that other organs may also be affected by subclinical abnormalities. Therefore we documented renal function in patients in the Oklahoma TTP-HUS Registry who survived acute episodes of TTP associated with severe ADAMTS13 deficiency (activity <10%).

Methods:

ADAMTS13 activity has been measured in 299 of 321 (93%) patients, 1995–2010. Glomerular filtration rate (GFR) was calculated by the Chronic Kidney Disease-Epidemiology Collaboration (CKD-EPI) equation (Ann Int Med 2009;150:604). Urine protein was measured by dipstick. Patient GFRs were compared to GFRs for the US population using data from the National Health and Nutrition Examination Survey (NHANES), 1998–2006, that had been analyzed in the CKD-EPI study.

Results:

68 of 299 (23%) patients from the Oklahoma TTP-HUS Registry had severe ADAMTS13 deficiency. Fifty-five of the 68 (81%) patients with severe ADAMTS13 deficiency survived their initial episode of TTP. Among the 55 survivors, 48 (87%) have had ≥1 year of follow-up (median, 6.4 years; range, 1.0 – 14.9 years). The 7 patients whose follow-up was less than 1 year included 3 patients whose initial episode was within the past year; 2 with systemic lupus erythematosus who died within one year; 1 who relapsed and died at one year; 1 lost to follow-up. Sixteen (33%) of the 48 patients had 1–4 relapses. None of the 48 patients developed end stage renal disease and none developed severe chronic kidney disease (GFR <30 ml/min/1.73m2). Three of the 48 patients had chronic kidney disease with moderately decreased GFR (GFR 30–59 ml/min/1.73m2); two of these patients had hypertension and one had hypertension and diabetes prior to their initial episodes of TTP. The median GFR was 97 ml/min/1.73m2 (range, 30 – 139 ml/min/1.73m2). The GFR for the ADAMTS13 deficient TTP patients was not different from the GFR of the U.S. population (p=0.289) (Figure 1). Urine protein was measured on 34 patients (median follow-up, 6.7 years; range, 1.2–14.9 years): 32 (94%) were negative; 2 (6%) were trace, equivalent to <30 μg albumin/mg creatinine.

Conclusion:

Renal function of TTP patients with severe ADAMTS13 deficiency who had been followed for a median of 6.4 years following recovery from their initial episode was not different from the US population.

Disclosures:

No relevant conflicts of interest to declare.
ISIS-FXIRx, a Potent Antisense Inhibitor of Factor XI, Suppresses FXI Activity and Elevates Aptt without Increased Risk of Bleeding in Cynomolgus Monkeys Following 13 Weeks of Treatment
Authors: Younis Husam , Crosby Jeff R. , Zhao Chenguang , MacLeod Robert , Monia Brett P. , Henry Scott
Published: October 2019

Abstract 2255

Complications associated with increased bleeding risk are the main limitations with current anticoagulant therapy. Inhibition of factor XI (FXI), a component of the intrinsic coagulation pathway, has received considerable interest because of the potential to produce sufficient anticoagulant activity without bleeding. The objective of these studies were to characterize the efficacy and safety of ISIS-FXrx and to assess the bleeding risk associated with FXI depletion under experimental surgical settings in cynomolgus monkeys. ISIS-FXIRx was selected from a series of 2nd-generation (2'-O-methoxyethyl) antisense oligonucleotides (ASOs) targeted to FXI which were evaluated for both tolerability and efficacy in cynomolgus monkeys (25 mg/kg twice per week for 13 weeks, SC). ISIS-FXIRx emerged as the most efficacious among the FXI ASOs evaluated, producing marked reductions of FXI RNA in liver, and FXI protein and activity levelsin plasma with a good tolerability profile. A more extensive dose-response evaluation of ISIS-FXrx (4,8,12 and 40 mg/kg/wk, SC) was conducted over a 13-week treatment period in cynomolgus monkeys to further assess tolerability and activity. ISIS-FXIRx produced a dose-dependent reduction in plasma FXI activity (>80% at 4 weeks of treatment at 40 mg/kg) with a concomitant increase in aPTT (33%). No effects on PT, platelets and no evidence of bleeding were observed after 13 weeks of treatment with ISIS-FXrx. ASO treatment was well tolerated at all doses tested and only produced changes typical for this chemical class of ASOs (e.g., basophilic granules in multiple tissues due do drug accumulation). ISIS-FXIRx (20 mg/kg, SC for 6 weeks) was also evaluated for bleeding risk in two experimental surgical models in cynomolgus monkeys; a tail amputation model and gum laceration model. Enoxaparin (2 mg/kg), known to increase the risk of bleeding, produced a statistically significant (p<0.05) increase in bleeding time and blood volume loss compared to saline control animals in both the tail amputation and the gum laceration models. Animals treated with ISIS-FXIRx displayed no increase in bleeding parameters compared to control animals. These results further support the conclusion that targeting FXI is an effective and safe strategy for development of novel antithrombotic agents with minimal increase in bleeding risk. ISIS-FXIRx is currently under evaluation in Phase 1 studies.

Disclosures:

Younis: Isis Pharmaceuticals, Inc.: Employment. Crosby:Isis Pharmaceuticals: Employment. Zhao:Isis Pharmaceuticals: Employment. MacLeod:Isis Pharmaceuticals: Employment. Monia:Isis Pharmaceuticals: Employment. Henry:Isis Pharmaceuticals, Inc.: Employment.
Detection of Continuous Flow Left Ventricular Assist Device -Associated Acquired Von Willebrand Factor (VWF) Abnormality by An Automated Immunoturbidimetric VWF Activity Assay
Authors: He Rong , Crow Sheri , Joyce Lyle D. , Milano Carmelo , Heit John A. , Chen Dong
Published: October 2019

Abstract 2273

Background:

Continuous flow left ventricular assist device (CF-LVAD) recipients have high tendency of post-surgical gastrointestinal (GI) bleeding. We previously described the loss of high molecular weight VWF multimers (HMWM), due to either an accelerated clearance or enhanced cleavage of the HMWM, in most CF-LVAD recipients (Ann Thorac Surg. 90:1263–9). However, besides the tedious plasma VWF multimer analysis, neither VWF ristocetin cofactor nor collagen binding activity assay provides sufficient sensitivity for detecting such an acquired VWF abnormality (AVWA). In this study, we examined a new automated latex particle-enhanced immunoturbidimetric VWF activity assay (VWF:Lx) for its ability of detecting CF-LVAD-AVWA. We also analyzed the VWF pro-peptide (VWF:pp) to explored the potential mechanism of CF-LVAD-AVWA.

Design:

As part of an on-going prospective multicenter study, pre- and post-CF-LVAD implantation (7, 30 days and 5–7 months) blood samples were collected from 15 LVAD recipients (median age 52 years; 10 male and 5 female; 2008∼2009). Plasma VWF antigen (VWF:Ag), VWF:Lx activity, VWF:pp/Ag ratios were measured; and plasma VWF multimer analyses were performed on all available plasma samples. Standard statistical analyses were employed.

Result:

Loss of VWF HMWM was observed in 3 patients prior to CF-LVAD implantation and virtually all patients after surgery. VWF:Ag, VWF:Lx or VWF:pp/Ag ratios of the pre- and post-implantation samples were not significantly different (P> 0.1). However, VWF:Lx/Ag ratios of the post-implantation samples were significantly decreased (P<0.05). At a cut off of 0.8, the VWF:Lx/Ag ratio has a 90% sensitivity and specificity for detecting AVWA.

Conclusions:

VWF:Lx/Ag ratio has excellent sensitivity and specificity in detecting CF-LVAD-AVWA, and its impact on predicting post-surgical bleeding tendency in CF-LVAD recipients is being investigated. The distinct CF-LVAD-AVWA is most likely caused by enhanced cleavage, rather than clearance, of the HMWM.

Disclosures:

No relevant conflicts of interest to declare.
Is Deep-Vein Thrombosis Always the Origin of Pulmonary Embolism? Imaging of Veins with a Total-Body Direct Thrombus MRI Technique
Authors: van Langevelde Kirsten , Srámek Alexandr , Vincken Patrice W.J. , van Rooden Jan-Kees , Rosendaal Frits R. , Cannegieter Suzanne C.
Published: October 2019

Abstract 2297

Introduction

Pulmonary embolism (PE) is thought to originate from embolisation of a deep-vein thrombosis (DVT), resulting in two clinical manifestations of one disease: venous thrombosis. However, in up to 50% of patients with PE, no DVT is found with ultrasound. A new technique in diagnosing DVT is ‘MR Direct Thrombus Imaging’ (MRDTI), using methaemoglobin in the thrombus as endogenous contrast. We performed total body MRIs to visualize thrombi from calf veins to subclavian veins, to assess the origin of PE.

Methods

Consecutive patients with a first PE diagnosed by Multi Detector CT scan are included. All patients undergo a 1.5 T MRDTI scan. In addition, blood is sampled for determination of genetic risk factors: the Factor V Leiden (FVL) and prothrombin G20210A (FII) mutations.

Results

99 patients have been enrolled in the study. In 44/99 (44%) a thrombus was identified on MR images. Most thrombi originated from the left side of the body (27/44). The upper leg was involved most often (22/44), with thrombi extending into the calf in 16 of these patients. Other thrombi were present in the pelvic veins (4/44), and deep calf veins alone (12/44). 6 patients had merely thrombophlebitis. The FVL mutation was present in 14% (95% CI 6–27) of patients with a thrombus on MRI, and in 7% (95% CI 3–18) of patients without a thrombus. For FII, the prevalence in the group with a DVT on MRI was 2% (95% CI 0.4–12) and without a DVT 4% (95% CI 1–13).

Conclusion

In less than half of PE patients a thrombus can be found with total body MRI. When thrombi were present, they were mainly found in the upper leg, extending into the calf veins. Interestingly, 27% of PEs with a thrombus on MRI was related to isolated calf vein thrombosis and 14% to thrombophlebitis. In PE patients with no thrombus on MRI, a local origin of PE, a thrombus from cardiac origin or complete embolisation needs to be considered.

Supported by Netherlands Heart Foundation grant number 2009B050

Disclosures:

No relevant conflicts of interest to declare.
Venous Thromboembolism (VTE) in Patients with Hematologic and Non-Hematologic Malignancies: Incidence and Risk Factors for Recurrent VTE
Authors: Vadhan-Raj Saroj , Zhou Xiao , Shah Jatin J , Benjamin Robert S , Gladish Gregory
Published: October 2019

Abstract 2303

The incidence of VTE and risk for recurrence is known to be higher in patients (pt) with malignancy than in other patients. However, the exact incidence and risk factors predictive of recurrent VTE in patients with hematologic malignancies (Hem) and solid tumors (ST) are not well defined. A retrospective study was conducted to evaluate the incidence of VTE and the recurrent events during one year period at MD Anderson Cancer Center. The medical records of all patients with VTE confirmed by the radiologic studies in 2006 were reviewed. The data were collected for the incidence and type of VTE, the recurrent events during a one year follow-up from the time of primary event, and the risk factors for recurrent events, including, the pt demographics, diagnosis, prior history of VTE, transfusions, use of erythropoiesis-stimulating agents, and the laboratory parameters at the time of the index VTE event. Cox proportional hazard models were established to determine the independent predictive factors for recurrent VTE. There were 24,806 unique patients (each patient counted once) in active treatment at the Cancer Center between January 2006 and December 2006. Of the 980 pts diagnosed with VTE (480 DVT, 477 PE, and 23 DVT/PE) during this period, there were 770 ST, 208 Hem, and 2 benign conditions. The incidence of VTE was higher in Hem pts than in ST pts [208/3603 (6%) vs. 770/20212 (4%), p<0.0001]. Among Hem pts, the incidence was significantly higher in myeloma as compared to lymphoma and leukemia (9%, 6%, and 4%, respectively, p<0.0001). The proportion of VTE pts with PE was significantly higher among ST pts compared with Hem pts (55% vs 37%, p<0.0001). The incidence of recurrent VTE, as defined by any new event or progression of the index event, was 14% (140/978 pts) during one year follow-up period, and it was not different for Hem (16%) vs. ST (14%). Among Hem pts, the recurrence was higher for myeloma (19%) than lymphoma (16%) and leukemia (13%). Majority of the recurrent events (100/140, 71%) were seen during the initial 3 month period from the index event. The independent risk factors for recurrent VTE during 3 months, 6 months and 1 year were summarized in the following table:3 months6 months1 yearRisk factorsHazard ratio (95% CI)PHazard ratio (95% CI)PHazard ratio (95%CI)PPE vs. non-PE1.86 (1.20–2.88)0.0051.67 (1.12–2.42)0.0061.74 (1.20–2.51)0.003Age (<60 vs. ≥60 years)2.05 (1.34–3.15)0.0011.55 (1.08–2.23)0.0171.62 (1.14–2.32)0.008Men vs. women1.70 (1.10–2.63)0.0181.44 (0.994–2.07)0.054PE, pulmonary embolism; CI, confidence interval.

Conclusions:

The incidence of VTE is higher in Hem pts, especially in myeloma. Younger age (<60 years) and PE are independent risk factors predictive of recurrence during 3 month, 6 month and 1 year period.

Disclosures:

No relevant conflicts of interest to declare.
Thrombosomes Show Dose-Dependent Increase in Thrombus Formation in a Model of Deep Arterial Injury
Authors: Joshi Nikhil Vilas , Raftis Jennifer , Lucking AJ , Tandon Narendra , Fitzpatrick M , Feuerstein Giora , Newby David E
Published: October 2019

Abstract 2319

Introduction

Thrombosomes are novel lyophilized human platelet derivatives that retain a number of key platelet structural and functional properties. Building on preliminary in vitro studies, we here sought to investigate whether Thrombosomes, suspended in platelet free plasma (PFP), would enhance and be incorporated in thrombus generated under flow conditions within a validated and well-characterised model of deep arterial injury.

Methods

PFP was obtained by centrifuging citrated whole blood from six healthy non-smoking volunteers and filtering with a 0.22μm filter. Thrombosomes were suspended in 60 mL of PFP to generate final concentrations of 20 and 200 × 106Thrombosomes /mL. Immediately prior to use, 1.2 mL of 1M CaCl2 was added to permit fibrin generation. Thrombus formation was assessed using the Badimon Chamber at low (212 s−1) and high (1690 s−1) shear rates with porcine aortic tunica media as the thrombogenic substrate. Total thrombus area and platelet-rich area were measured histomorphometrically using conventional and immunohistochemical staining respectively. Fluorescent labeled Thrombosomes were added to the extracorporeal circuit in the Badimon chamber to study the ex vivo thrombus generation in the whole blood. Electron microscopy of Thrombosomes and platelets was undertaken.

Results

Thrombosomes contributed towards thrombus formation in whole human blood as evidenced by incorporation of fluorescent-labeled Thrombosomes into the thrombus. Immunohistochemical staining of the glycoprotein IIb/IIIa receptor confirmed incorporation of Thrombosomes into the thrombus. In the high shear chamber, mean thrombus area increased in a dose-dependent manner following the addition of Thrombosomes (704 μm2 [95% CI, 224 – 1184 μm2], 1511 μm2 [95% CI, 687 – 2336 μm2] and 2378 μm2 [95% CI, 1567 – 3189 μm2] for PFP and Thrombosomes at concentrations of 20 and 200 × 106/mL respectively [P= 0.003]). In the low shear chamber, total thrombus area for the PFP was 4962 μm2 (95% CI, 2489 – 7434 μm2). The addition of Thrombosomes at concentrations of 20 and 200 × 106/mL led to a numerical increase in mean thrombus area to 6170 μm2 (95% CI, 3944 – 8397 μm2) and 7504 μm2 (95% CI, 3864 – 11144 μm2) respectively, although this was not statistically significant (P= 0.2969).

Conclusions

Thrombosomes suspended in PFP and exposed to injured arterial tissue under physiologically relevant flow conditions are incorporated into thrombus and enhance thrombus formation in a dose dependent manner. These findings act as impetus to undertake clinical studies of this rehydrated, lyophilized infusible hemostatic platelet product.

Disclosures:

Fitzpatrick: Cellphire Inc.: Employment, Equity Ownership. Feuerstein:Cellphire: Consultancy. Newby:Cellphire: Research Funding.