bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of Calcium Sensing Receptor Mediated Hematopoietic Stem Cell Lodgment In the Adult Bone Marrow Niche
Authors: Lam Ben S. , Cunningham Cynthia , Adams Gregor B.
Published: October 2019

Abstract 399

The ability of hematopoietic stem cells (HSCs) to maintain an undifferentiated state and undergo self-renewal is partly regulated by external signals originating from the stem cell niche. One receptor expressed on HSCs that is known to be involved in HSC niche biology is the calcium-sensing receptor (CaR). Our previous study using HSCs obtained from the fetal liver of mice deficient in CaR has shown the crucial role of CaR in HSC lodgment and engraftment in the bone marrow (BM), where CaR-/- HSCs lose their ability to lodge in the endosteal surface of the bone, leading to defective engraftment. To further investigate the mechanism of CaR-mediated lodgment of HSCs, we used a pharmacological approach to activate the receptor and assess the in vitro and in vivo effects. Cinacalcet treatment, which acts as a positive allosteric modulator of CaR to increase the sensitivity of the receptor to activation by extracellular Ca2+, leads to a 3-fold increase in primitive hematopoietic cell activity in vitro as assessed by the cobblestone forming cell assay. The increase in activity in vitro does not appear to be an effect of alterations in cell proliferation, cell survival or the expression of cell adhesion molecules such as VLA-4 or L-selectin. Rather, with CaR stimulation, long-term HSCs have an increased adhesion capability to collagen I, a major ECM molecule present predominantly in the BM endosteal region. We also observed that activation of the CaR following Cinacalcet treatment significantly enhances HSC homing to the BM, lodgment at the endosteal surface and in vivo engraftment capabilities as assessed by a competitive repopulation assay. This enhancement of in vivo activity correlates with increased CXCR4 signaling and migration towards an SDF-1alpha stimulus. Signaling through this receptor is known to be important in cell migration, proliferation, survival, and retention of HSCs in the BM following transplantation. Analysis of the CaR on human cells has demonstrated that there is a distinct population of CaR+ cells present in the CD34+ cell population. The frequency of this population varies between approximately 2% on mobilized peripheral blood CD34+ cells and 6% on BM CD34+ cells. Further analysis is being performed to define the identity of this subpopulation of CaR+CD34+ cells. These mechanisms by which the CaR dictates preferential localization of HSCs in the BM endosteal region may provide additional insights for the fundamental interrelationship between the stem cell niche and stem cell fate. These studies also have implications in the area of clinical stem cell transplantation, where ex vivo modulation of the CaR may be envisioned as a strategy to enhance HSC engraftment in the BM.

Disclosures:

No relevant conflicts of interest to declare.
Circulating T Cells Derived From Patients with Low Risk Myelodysplastic Syndromes Show Altered Chemokine Receptor Expression
Authors: Sand Kristoffer E , Kittang Astrid Olsnes , Bruserud Øystein
Published: October 2019

Abstract 4018

Several T cell abnormalities have been described in myelodysplastic syndromes (MDS), and such abnormalities may become important for identification of patients who will benefit from T cell targeting immunosuppressive treatment. Chemokine receptor repertoires are important in the regulation of both T cell migration and function and may therefore be important in the development of MDS.

Materials and Methods:

The chemokine receptor expression by circulating T cells were investigated by multicolor flow cytometry for patients with newly diagnosed low risk MDS (N=16) and for healthy controls (N=18). CD3+CD8+ and CD3+CD8- cells were examined for expression of CCR2-7, CXCR3-4 and CX3CR1 (only 7 unselected patients examined for CX3CR1 expression). CCR6 and CXCR4 expression was also investigated for specific T cell subsets defined by CD62L and CD45RA expression (naïve, central memory, effector memory and terminal effector). CX3CR1 expression was stratified for CD8+ and CD8- subpopulations according to high, low and negative expression of CCR5.

Results:

Chemokine receptor profiles showed several differences between low risk MDS patients and controls. Total CD8+ T cells from MDS patients showed increased expression of CCR3 (p=0.005) and decreased expression of CCR7 and CCR4 (p=0.023 and p=0.036 respectively), whereas the CD8- T cells from MDS patients showed increased expression of CX3CR1 (p=0.043). In contrast, CCR6 expression was increased only by CD8+ central memory T cells (p=0.044). Finally, CD8+CCR5- and CD8-CCR5high cells from MDS patients showed increased expression of CX3CR1 compared with the controls (p=0.011 and p=0.049).

Discussion:

CCR7 is mainly expressed by central memory and naïve T cells whereas CX3CR1 is especially expressed by cytotoxic effector lymphocytes independent of the lymphocyte subclass (i.e. CD4, CD8, delta/gamma and NK cells). The observed changes are in line with a shift from naïve/central memory to effector/effector memory dominance, especially in the CD8+ population. A similar shift has been described previously using CD45RA and CD62L (Zou et al, Leukemia 2009). Our median values are in line with such a shift. CCR6 expression is associated with IL17 production both for CD8+ and CD4+cells, and increased levels of circulating CD4+ IL17 producing cells in low risk MDS have been described.

Conclusions:

The chemokine receptor profiles of circulating T cells differ between low risk MDS patients and healthy controls, especially for the CD8+ T cell subset. These differences may be important for T cell trafficking and disease development, and they may reflect a shift from naïve to effector/effector memory cell dominance in low risk MDS.

Disclosures:

No relevant conflicts of interest to declare.
Monoclonal Gammopathy of Undetermined Significance and Risk of Infections: A Population-Based Study
Authors: Kristinsson Sigurdur Y. , Min Tang , Pfeiffer Ruth , Björkholm Magnus , Goldin Lynn , Blimark Cecilie , Mellqvist Ulf-Henrik , Wahlin Anders , Turesson Ingemar , Landgren Ola
Published: October 2019

Abstract 4053

Background:

Based on clinical case reports and small hospital-based patient series, monoclonal gammopathy of undetermined significance (MGUS) has been reported to increase morbidity due to bacterial infections; however, no comprehensive evaluation has been conducted.

Patients and methods:

Using population-based data from Sweden, we assessed the risks of viral and bacterial infections (reported to the Patient-Registry) in 5,326 MGUS patients diagnosed 1958–2006, compared to 20,161 matched population-based controls. We fit Cox proportional hazard models to estimate hazard ratios (HRs) as measures of risk.

Results:

At 5 years of follow-up MGUS patients had a 2.1-fold (95% confidence interval (CI) 2.1–2.3) increased risk of developing any infection compared to controls; at 10 years of follow-up, the risk was very similar (HR=2.2; 95% CI 2.0–2.3). MGUS patients had a 2.2-fold (95% CI 2.0–2.4) and 2.1-fold (95% CI 2.0–2.3) increased risk of developing bacterial infections at 5 and 10 years, respectively. A significantly increased risk (P<0.05) was found for pneumonia, osteomyelitis, septicemia, pyelonephritis, cellulitis, endocarditis and meningitis. We also assessed the risk of developing viral infections; compared to controls, MGUS patients had a 2.7-fold (95% CI 2.2–3.3) and 2.9-fold (95% CI 2.3–3.7) increased risk at 5 and 10 years, respectively, with a significantly increased risk for influenza and herpes infections (P<0.05). Risk of infections did not differ by MGUS isotype. MGUS patients with an M-protein >2.5 g/dL at diagnosis had higher risks of infections compared to those <0.5 g/dL. However, compared to controls the risk of infections was also significantly increased among MGUS patients with a concentration <0.5 g/dL. MGUS patients with (versus without) infections had no excess risk of developing multiple myeloma or related malignancies.

Summary and Conclusions:

Based on over 5,000 MGUS patients, we found a 2-fold higher risk of developing bacterial and viral infections, compared to controls. The risk was highest among MGUS patients with high M-protein concentrations at diagnosis (>2.5 g/dL); however, the risk was still significantly increased among those with a concentration <0.5 g/dL. Infections in patients with MGUS were not associated with an increased risk of malignant transformation. We recently showed that patients with MGUS have a 3-fold risk of death in bacterial infections. Our findings may have clinical implications for treatment strategies and prophylactic measures, as well as surveillance of MGUS patients.

Disclosures:

No relevant conflicts of interest to declare.
Liver Infiltration In Splenic Marginal Zone Lymphoma: Prevalence and Prognostic Implication
Authors: Domingo-Domenech Eva , Romagosa Vicente , González-Barca Eva , Rodriguez-Aliberas Marta , Oliveira Ana , de la Banda Esmeralda , Mercadal Santiago , Sarra Jose , Petit Jose , Fernandez de Sevilla Alberto
Published: October 2019

Abstract 4137

Introduction:

There are no studies in the literature about the frequency of liver infiltration by splenic marginal zone lymphoma (SMZL). In the last review published by the International Group of SMZL (Matutes E et al, Leukemia 2008;22:487) clinical liver infiltration seems to be infrequent and it is considered, as other organ involvement, to appear during the course of the disease and confers a bad prognosis.

Objective:

To investigate in a selected sample of patients with SMZL the prevalence of liver infiltration, as well as its possible prognostic implication in survival or transformation.

Material and methods:

In the majority of our SMZL patients who underwent a splenectomy between 1995 and 2001, a liver biopsy was realised during the surgical procedure in order to investigate hepatic infiltration by lymphoma.

Results:

Forty-eight patients with SMZL were diagnosed in our center during this period; of them, 19 were splenectomized being liver biopsy performed in 16 (only in those cases that gave informed consent). Splenectomy was performed at diagnosis in 11 patients (time from diagnosis to splenectomy < 3 months). Liver biopsy was positive for SMZL infiltration in 81.3% (13/16) of the cases. Baseline demographic and clinical characteristics of both groups of patients are shown in the following table; there were no significant differences between the groups. We neither find statistical significant differences in overall survival between both groups: mean overall survival for positive and negative liver biopsy patients was 116 ± SD 13 months and 74 ± 29 months, respectively (p=0.69).

Conclusion:

Histological hepatic infiltration is present in almost all SMZL patients when splenectomized. In our experience, this finding has no prognostic implication. Due to the small number of patients of this study, its significance is limited but to the best of our knowledge, it is the first report that investigates the prevalence of liver infiltration in SMZL and its impact in survival. Larger series are necessary to confirm our findings.

Disclosures:

No relevant conflicts of interest to declare.
MicroRNA Signatures In B-Cell Lymphomas
Authors: Di Lisio Lorena , Sanchez-Beato Margarita , Gomez-Lopez Gonzalo , Rodriguez Maria E. E. , Montes-Moreno Santiago , Mollejo Manuela , Menarguez Javier , Martinez Miguel A. , Alves Javier , Pisano David G. , Piris Miguel A. , Martinez Nerea
Published: October 2019

Abstract 4155

Beyond the conventional criteria of lymphoma classification (integrated clinical, morphological, immunophenotypic, and molecular features) additional markers are still required for a more precise differential diagnosis and a better understanding of lymphoma pathogenesis.

MicroRNAs (miRNA) are non-coding small RNAs that play an important role in gene expression regulation, contributing to cell differentiation and tumorigenesis. Specifically, miRNAs have been already described to play a relevant role in B cell differentiation, and in some cases proposed to constitute lymphoma-type specific markers and possible therapeutic targets.

We explore the potential diagnostic application of miRNA expression in a large series of 147 cases including all B-cell non-Hodgkin lymphomas (NHL) major types and appropriate controls. As an example of a practical application, data were also used to identify miRNAs differentially expressed when comparing Burkitt Lymphoma (BL) and Diffuse Large B-Cell Lymphoma (DLBCL) in paraffin-embedded samples.

Each lymphoma type (BL, CLL, DLBCL, FL, MCL, MZL/MALT, NMZL and SMZL) was compared to the whole series of NHL by Significant Analysis of Microarray (SAM) method. The analysis identified a set of 128 characteristic miRNAs (FDR<0.01 and Fold change >1.5 log2). All lymphoma types were characterized by specific miRNA signatures, reflecting cell of origin and/or discrete oncogene alterations. Of interest is also the comparison with reactive lymphoid tissues, since it revealed a specific B-cell lymphoma miRNA profile, which includes a cluster of downregulated miRNAs, such as let7 family, miR-1 and miR-200 family.

Burkitt Lymphoma was also directly compared to DLBCL, and 43 miRNA selected by SAM analysis were studied in a new series of 28 BL and 43 DLBCL samples using quantitative RT-PCRIn this second step, the differential expression of a set of 19 miRNAs was confirmed between BL and DLBCL. (FDR < 0.05 after t-test (limma)).

These findings expand the potential diagnostic markers in lymphoma diagnosis and provide useful information on lymphoma pathogenesis.

Disclosures:

No relevant conflicts of interest to declare.
Endogenous Oncogenic Nras Mutation Leads to Aberrant GM-CSF Signaling In Granulocytic/Monocytic Precursors In a Murine Model of Chronic Myelomonocytic Leukemia
Authors: Wang Jinyong , Liu Yangang , Li Zeyang , Du Juan , Ryu Myung-Jeom , Taylor Philip , Fleming Mark D. , Young Ken H , Pitot Henry , Zhang Jing
Published: October 2019

Abstract 4180

Oncogenic NRAS mutations are frequently identified in myeloid diseases involving monocyte lineage. However, its role in the genesis of these diseases remains elusive. We report a mouse bone marrow transplantation model harboring an oncogenic G12D mutation in the Nras locus. Approximately 95% of recipient mice develop a myeloproliferative disease resembling the myeloproliferative variant of chronic myelomonocytic leukemia (CMML), with a prolonged latency and acquisition of multiple genetic alterations, including uniparental disomy of oncogenic Nras allele. Based on single-cell profiling of phospho-proteins, a novel population of CMML cells is identified to display aberrant GM-CSF signaling in both the ERK1/2 and Stat5 pathways. This abnormal signaling is acquired during CMML development. Further study suggests that aberrant Ras/ERK signaling leads to expansion of granulocytic/monocytic precursors, which are highly responsive to GM-CSF. Hyperactivation of Stat5 in CMML cells is mainly through expansion of these precursors rather than upregulation of surface expression of GM-CSF receptor. Our results provide insights into the aberrant cytokine signaling in oncogenic Nras-associated myeloid diseases. Our mouse model will serve as a powerful system to identify and validate cooperating mutations of oncogenic Nras in myeloid leukemias as well as assess the therapeutic efficacy of molecular agents in treating these leukemias.

Disclosures:

No relevant conflicts of interest to declare.
A Large, Population Based Study of Sex Differences In the Risk of Recurrent Venous Thromboembolism
Authors: Kondal Dimple , Taglalakis Vicky , Moride Yola , Boivin Jean-François , Kahn Susan
Published: October 2019

Abstract 4204

Background.

Most reports of patient sex differences in the risk of recurrent VTE have been based on small or moderate sized cohorts of selected patients with VTE.

Objectives.

We used the administrative hospital discharge registry of Quebec, Canada to conduct a population-based retrospective cohort study to evaluate the effect of sex on rates of recurrent VTE.

Methods.

Using data from the province-wide hospital discharge database (Med-Echo) which systematically records information on all hospital admissions in the province of Quebec since 1967, we constructed a cohort of all individuals who had a first-time discharge diagnosis of deep vein thrombosis (DVT) or pulmonary embolism (PE) between January 1, 1996 and December 31, 2004 and no prior discharge diagnosis for DVT or PE back to 1983. Subjects were followed from the time of diagnosis until first VTE recurrence, death, or end of study (December 31, 2005). DVT and PE were defined based on the International Classification of Diseases, 9th edition, Clinical Modification.

Results.

The cohort comprised 55,314 patients (43% men and 57% women) with incident VTE. Mean age was 61.9 years (60.4 years (men) vs. 63.1 years (women); p=0.0001), and 33,535 (61%) had DVT alone, 14,574 (26%) had PE alone and 7,205 (13%) had both DVT and PE. During a mean follow-up of 3.9 years, 5,243 (9.5%) of patients developed recurrent VTE, which corresponded to an incidence rate of 2.4 events per 100 patient-years (95% confidence interval (CI), 2.3–2.5 per 100 patient-years). In survival analysis, men had significantly higher rate of recurrence than women (age- and comorbidity-adjusted hazard ratio = 1.14; 95% CI, 1.08–1.21), and at 5 years, the cumulative risk of recurrent VTE was 8.3% among men vs. 7.3% among women (p=0.0001). Mean age at the time of VTE recurrence in men vs. women was 61.1 years and 64.4 years, respectively (p=0.0001).

Conclusion.

Our study of over 55,000 patients hospitalized with VTE is the largest to date to report an effect of patient sex on risk of VTE recurrence, with men having about a 15% higher risk of recurrence than women. This provides further evidence that the difference in risk of VTE recurrence between men and women is significant and if confirmed by ongoing prospective management studies, patient sex should be considered in determining duration of anticoagulation treatment in patients with VTE.

Disclosures:

Taglalakis: Pfizer: Research Funding; Sanofi Aventis: Honoraria. Kahn:Sigvaris: Research Funding; sanofi-aventis: Advisory Board, Research Funding; Boehringer Ingelheim:.
MASL1 Induces Erythroid Differentiation In Human Erythropoietin-Dependent CD34+ Cells Involving Raf/MEK/ERK Signaling Pathway
Authors: Kumkhaek Chutima , Aerbajinai Wulin , Liu Wenli , Zhu Jianqiong , Uchida Naoya , Hsieh Matthew M , Tisdale John F , Rodgers Griffin P.
Published: October 2019

Abstract 4228

Human erythropoiesis is a dynamic complex multistep process that involves differentiation of early erythroid progenitors to enucleated red blood cells. Importantly, erythroid differentiation involves lineage-specific activation and restriction of gene expression. However, the mechanisms that play a role in erythropoiesis still remain incompletely understood. We previously demonstrated that erythropoietin-stimulated clone-1 (EP1), which is selectively expressed in normal human erythroid lineage cells shares 99.5% identity with the malignant fibrous histiocytoma amplified sequence 1(MFHAS1 or MASL1). MASL1 is an important oncogene that is highly expressed in malignant fibrous histiocytomas (MFH). MASL1 protein has several domains; ras, three leucine zipper, ATP/GTP-binding site and leucine-rich tandem repeat motif which are important structural or functional elements for interactions among proteins related to the cell cycle. However, the function of the MASL1 gene in erythropoiesis has not been studied. We hypothesized that MASL1 gene plays a role in the erythroid differentiation, and used a human liquid erythroid culture system to explore this concept. MASL1 mRNA and protein expression levels were significantly increased during the erythroid differentiation of CD34+ cells following EPO treatment. Conversely, small interfering RNA-mediated knock down of MASL1 in CD34+ cells resulted in a reduction of a double positive of transferrin receptor and glycophorin A (GPA) (14.1 ± 4.7%) when compared with mock (77.9 ± 4.4%) or control vector-transfected CD34+ cells (76.7 ± 8.8%) at day 14. May-Grunwald-Giemsa staining confirmed a phenotype of MASL1 knockdown CD34+ cells that most of cells were pro- or basophilic erythroblasts. Western blotting also showed a significant decrease in hemoglobin protein levels in MASL1 knockdown CD34+ cells. In addition, MASL1-knockdown in CD34+ cells demonstrated an interruption of Raf/MEK/ERK signaling pathway. Inhibition assay of SH3 domain of Son of Sevenless (SOS) which is an upstream adapter protein in EPO-induced erythroid differentiation, confirmed that a suppression of MASL1 reduced the phosphorylation of Raf/MEK/ERK kinases and delays erythroid differentiation of Epo-induced CD34+ cells. Taken together, our study provides novel insights into MASL1-regulated erythropoiesis through the Raf/MEK/ERK signaling pathway.

Disclosures:

No relevant conflicts of interest to declare.
A Simple Regimen to Correct Vitamin D Deficiency In Transfusion-Dependent Thalassemia with High-Dose Ergocalciferol
Authors: Lal Ashutosh , Micaily Ida , Foote Drucilla , Fung Ellen
Published: October 2019

Abstract 4261

Osteoporosis in thalassemia is extremely common and preventive approaches are required to avoid serious complications in adults. Vitamin D deficiency, which contributes to suboptimal bone mineralization, is frequently observed in thalassemia despite routine prescription for supplementation with 400–800 IU vitamin D per day. Screening for vitamin D status was conducted in 71 patients with thalassemia, of which 52 were transfusion-dependent and 19 were transfusion-independent (including 11 patients with hemoglobin H or H Constant Spring disease). Baseline assessment of plasma 25-OH vitamin D revealed 57 (80.3%) patients were either deficient (<20 ng/mL, 47.9%) or insufficient (20-30 ng/mL, 32.4%). Significantly more patients in the transfusion-independent group were deficient in vitamin D compared with transfusion-dependent group (78.9% versus 36.5%, p=0.003). The mean parathyroid hormone level in patients with 25-OH D <20 ng/mL was 35.4 ± 19.6 pg/mL compared to 28.2 ± 17.1 pg/mL in those with ≥20 ng/mL (p=NS). We studied the efficacy of supervised adminstration of high-dose (50,000 IU) oral ergocalciferol every 3 weeks during transfusion visits in 20 patients with transfusion-dependent thalassemia. The mean 25-OH D level increased from 14.0 to 22.9 ng/mL (p<0.001) over a four-month period. A single dose of ergocalciferol given every 3 weeks increased the plasma 25-OH D level by 2.2 ± 2.3 ng/mL. No patient developed toxic 25-OH D level (>80 ng/mL) during the course of the study. These results show that vitamin D deficiency remains widespread despite daily low-dose supplementation. Non-transfused patients are at even greater risk of vitamin D deficiency, which may reflect less attention to nutrition in this group compared to the transfusion-dependent patients. We have found that supervised high-dose oral ergocalciferol supplementation is simple, safe, non-invasive and predictable method to improve vitamin D status in thalassemia.

Disclosures:

No relevant conflicts of interest to declare.
Myocardial Fibrosis by Delayed Enhancement Cardiovascular Magnetic Resonance and HCV Infection In Thalassemia Major Patients
Authors: Pepe Alessia , Meloni Antonella , Borsellino Zelia , Dell'Amico Maria Chiara , Borgna-Pignatti Caterina , Maggio Aurelio , Positano Vincenzo , Cracolici Eliana , Lombardi Massimo , Capra Marcello
Published: October 2019

Abstract 4265

Introduction.

Delayed enhancement (DE) cardiac magnetic resonance (CMR) technique with intravenous administration of gadolinium (Gd) chelates contrast agents is the only validated non-invasive approach for detecting myocardial fibrosis (Mahrholdt H et al, Eur Heart J 2005). This technique has been confirmed safe in patients with hemoglobinopathies (Meloni A et al, Haematologica 2009). In thalassemia major (TM), myocardial fibrosis has been detected using the DE technique and a positive correlation with anti-HCV antibodies has been described (Pepe A et al, Heart 2009). However, HCV-induced cardiomyopathy is still controversial (Matsumori A et al. J Card Fail 2006). The aim of our study was to verify a possible correlation between myocardial fibrosis detected by DE CMR and chronic HCV infection in a large retrospective cohort of TM patients.

Methods.

We analysed 434 TM patients (233 males, mean age 31±9 years) consecutively enrolled in the MIOT (Myocardial Iron Overload in Thalassemia) network (Ramazzotti A et al, JMRI 2009). DE images were acquired from 10 to 18 minutes, after intravenous administration of Gadobutrol. DE was evaluated visually using a two-point scale (absent or present) and enhancement was considered present whenever it was visualized in two different views (Pepe A et al, Heart 2009). HCV-RNA tests were sensitive to detect more than 50 copies/ml.

Results.

Figure 1 represents the flow chart of the test results in the 434 TM patients. Ninety out of 434 TM patients (21%) were found to have myocardial fibrosis by DE CMR technique. Chronic hepatitis C (CHC) group was composed by HCV-RNA positive patients plus the patients with a previous diagnosis of CHC successfully treated by alpha-interferon for obtaining a sustained virological response (SVR). There was a significant correlation between the presence of myocardial fibrosis and CHC (P=0.026).

Conclusions.

Our finding supports the hypothesis that HCV infection can be involved in the pathogenesis of myocardial fibrosis in the multitransfused TM patients, who could therefore benefit from therapeutic interventions directed towards the eradication of HCV.

Disclosures:

No relevant conflicts of interest to declare.
Nuclear Factor of Activated T-Cells (NFAT1): Novel Target In T-Cell Acute Lymphocytic Leukemia (T-ALL)
Authors: Potoczak Alexander Douglas , Lesniewski Mathew , Havernik Peter , Pillai Esha , Weitzel R. Patrick , Greco Nicholas J , Forster Margaret , Laughlin Mary J.
Published: October 2019

Abstract 4327

Background:

The well-characterized human T-cell transcription factor NFAT1 has been reported to regulate cell cycle regulatory proteins including Cyclins A, D, and p21. This group has recently identified microRNA-184 to regulate NFAT1 protein translation in human umbilical cord blood CD4 T-cells. We sought to determine if NFAT1 protein expression is dysregulated in T-ALL, and if so, its contribution, if any to T-ALL pathogenesis.

Materials & Method:

MOLT-4 cells were maintained according to ATCC guidelines. Normal adult and umbilical cord blood (UCB) T-cells were selected by AutoMACS (Miltenyi Biotec) and and maintained per standard protocols. Purity was confirmed in excess of 90% by flow cytometry. MOLT-4 cells were transfected with a 10 pmol:3.33 pmol ratio of anti-miR-184 miRNA inhibitor (Ambion) to siGLO non-specific RISC-free control (Dharmacon) in complete RPMI alongside corresponding controls via lipofectamine RNAi max (Invitrogen) overnight per manufacturers’ instructions. Protein was isolated 48 hours after transfection and quantified by standard protein assay (BioRad). 10 μ g of each fraction was analyzed by 10% SDS-PAGE followed by Western Blotting with anti-NFAT1 (BD Biosciences) and γ-tubulin (Santa Cruz Biotechnology) antibodies. Bands were visualized using Supersignal West Pico chemiluminescent substrate (Pierce).

Result:

The relative levels of both NFAT1 protein and mir-184 were first compared to normal adult and UCB CD4+ T-cells. As miR-184 and NFAT1 expression levels can change over the developmental life of CD4+ T-cells, a comparison of MOLT-4 cells was made to normal UCB T-cell controls with similar surface phenotypes (CD4+/8+). NFAT1 protein expression was found to be significantly decreased in the MOLT-4 cells by Western Blot. Further, comparisons of miR-184 expression in UCB double positive T-cells to MOLT-4 by quantitative reverse transcription real-time PCR (qPCR) demonstrated a 56-fold higher expression of mir-184 in MOLT-4 compared to controls. Next, in order to test the ability of miR-184 to regulate NFAT1 levels in the MOLT-4 T-ALL cell line, lipofectamine-mediated transfections of antisense miR-184 were performed. Western Blot analysis showed the expected modest increase in the NFAT1 protein expression in MOLT-4 cells transfected with anti-miR-184.

Discussion:

NFAT1 protein levels are notably reduced in T-ALL cell lines and may be attributable in part to 56-fold higher expression of miR-184. Knockdown of miR-184 renders normalization of NFAT1 protein levels. Ongoing studies are designed to determine cell cycle kinetics and p21 expression in miR-184 transfected MOLT-4 cells. Dysregulation of NFAT1 may contribute to T-ALL pathogenesis, and miR-184 regulation of NFAT1 may be a target for therapeutic studies.

Disclosures:

No relevant conflicts of interest to declare.
Successful Treatment of Acute Promyelocytic Leukemia (APL) In a Patient with Severe Cardiac Disease
Authors: Ayalew Getinet D. , Rotkowitz Michael I. , Mittal Juhi , Sidhu Gurinder , Braverman Albert S. , Khillan Ratesh
Published: October 2019

Abstract 4369

Background:

The standard and often curative regimen for APL includes cardiotoxic daunorubicin and All-Trans Retinoic Acid (ATRA). Arsenic trioxide (ATO), like ATRA, induces leukemic blast differentiation and is active in APL.

Case Report:

A 58 year old African American man had a metallic aortic valve replacement 6 years prior to presentation, for which he was on warfarin, coronary artery disease, a left ventricular ejection fraction of 20%, and an implanted cardioverter-defibrillator. He presented with dyspnea, fever, scattered rales and ecchymoses and petichiae. His white blood count was 38,000/mcl, with 51% promyelocytes and 8% neutrophiles, Hb 9.7 g/dl, and platelets of 12,000/mcl. Fibrinogen was 172 mg/dl, aPTT 28 seconds, and international normalized ratio 2.3; lactate dehydrogenase was 1593 U/dl. The bone marrow (BM) was replaced by promyelocytes, karytotype revealed t (15;17) (q22;q12), and fluorescent in situ hybridization (FISH) showed the promyelocytic leukemia – retinoic acid receptor fusion product (PML-RARA). The diagnosis was high risk APL. Warfarin was suspended, and treatment was initiated with ATRA 45 mg/M2. On day 7, he developed shortness of breath; cough and chest radiograph illustrated marked interstitial edema as well as bilateral pleural effusion. These features of the differentiation syndrome resolved rapidly after transfer to the Medical Intensive Care Unit and dexamethasone initiation. On day 10, daily ATO at 0.15 mg/kg IV was initiated. His QTC interval ranged from 430–600 ms, showed interactions with 5-HT inhibitors and antifungal drugs, and stabilized after discontinuation of these drugs. By day 23, his white blood cell counts had normalized, and BM aspiration showed normal morphology. ATO was discontinued, platelet count rose to 50,000/mcl by day 28, when warfarin was resumed. Post-induction the patient received 4 cycles of ATO 5 days per week for 4 weeks, each followed by 4 weeks off treatment and ATRA 45 mg/m2 daily for 2 weeks every 4 weeks for a total of 7 cycles. Cytogenic remission was confirmed by FISH analysis of BM on days 23 and 113, and subsequently by quantitative RT-polymerase chain reaction on peripheral blood specimens for one year. By these criteria he remains in complete remission one year after presentation.

Conclusion:

With careful cardiac monitoring, ATRA-ATO induced a durable complete remission of APL in a patient with several major cardiac contra-indications to anthracyclines. Despite the patient's metallic aortic valve, he tolerated suspension of anti-coagulant therapy well.

Disclosures:

No relevant conflicts of interest to declare.
Effect of Heparin:PMX60056 Complexes on Platelet Aggregation Induced by ADP or HIT Antibody
Authors: Jeske Walter , McAllister R. Eric , Walenga Jeanine M. , Paulus Michelle , Fareed Jawed
Published: October 2019

Abstract 4386

Introduction:

A neutralization of anticoagulant activity occurs when heparin binds to a variety of positively charged substances such as protamine and platelet factor 4 (PF4). PMX60056 (PolyMedix, Radnor, PA) is a novel compound that is being developed as a heparin antagonist. Since heparin-PF4 complexes are antigenic, with antibodies against this complex activating platelets to trigger thrombin generation, thrombus formation and associated morbidities, it is of interest to determine whether heparin:PMX60056 complexes affect platelet function. This study compares the effect of PMX60056 and protamine, alone and complexed to heparin, on platelet function as assessed by platelet aggregometry.

Materials and Method:

Whole blood, collected from 10 healthy individuals, was anticoagulated with 3.2% sodium citrate and centrifuged to make platelet rich plasma (PRP). PRP was supplemented with 10 μ g/ml heparin (~1.5 IU/ml), 10 μ g/ml heparin antagonist (protamine or PMX 60056) or a complex of 10 μ g/ml heparin and 10 μ g/ml heparin antagonist. Platelet aggregation was stimulated by the addition of ADP (5 or 10 μ M final concentration) or serum from a patient with heparin-induced thrombocytopenia.

Result:

ADP-induced platelet aggregation was not affected by the addition of heparin, protamine, PMX 60056, or complexes of heparin with heparin antagonist. In the HIT system, heparin + HIT serum led to a significant increase in platelet aggregation vs. saline (46.7 ± 3.2 % vs. 8.2 ± 2.9%). HIT serum + heparin antagonist did not induce platelet aggregation (PMX60056: 10.2 ± 4.4%; protamine: 11.6 ± 3.5%). The aggregation responses to HIT serum + heparin (46.7 ± 3.2%), HIT serum + heparin:PMX60056 (43.6 ± 5.8%) and HIT serum + heparin:protamine (47.8 ± 3.8%) were not significantly different.

Conclusion:

When mixed at equigravimetric amounts, protamine and PMX60056 do not prevent formation of immune complexes consisting of HIT antibody and heparin which lead to platelet activation. Previous data from human trials has suggested that smaller amounts of PMX60056 (less than equigravimetric) may effectively neutralize heparin. Thus, it is speculated that smaller heparin:PMX60056 complexes may induce less antibody formation than larger heparin:protamine complexes. Validation of this hypothesis in animal models or clinical studies is warranted.

Disclosures:

McAllister: PolyMedix, Inc.: Employment.
Comparison of Incidence and Investigation of In-Hospital Pulmonary Embolism In Patients Receiving Dalteparin Prophylaxis Following Elective Knee Replacement at Two Hospitals
Authors: Linkins Lori Ann , Warne Connor , Crowther Mark A.
Published: October 2019

Abstract 4388

Background:

The incidence of in-hospital pulmonary embolism (PE)in patients who received dalteparin prophylaxis following elective total knee replacement (TKR) at a tertiary care centre was reported to be surprisingly high at 4.6% (Kerr et al, Thromb Haemost 2010, 103:123-128).

Objective:

To compare the incidence and investigation of in-hospital PE between two centres that used dalteparin prophylaxis following elective TKR.

Method:

Charts of consecutive patients who underwent elective TKR and received dalteparin prophylaxis between January and June 2008 were retrospectively reviewed. A standardized case report form was used to abstract data on patient age, gender, type of surgery, risk factors for venous thromboembolism (VTE)(i.e. active cancer, prior history of VTE, hormonal therapy), results of imaging studies performed for suspected VTE, and in-hospital death. Electronic records were reviewed for 90 days after the date of surgery to capture subsequent studies for VTE performed at the same institution. Because the original study identified postoperative day 3 (POD#3) as the day that the majority of investigations were ordered for PE, a subgroup of 100 patients were randomly selected at each site, and data were abstracted on vital statistics which may have raised suspicion of PE: requirement for at least 2 litres of oxygen to keep oxygen saturation ≥ 92% or heart rate > 100 beats per minute or systolic blood pressure < 100 mmHg (These values were selected based on data from the original study).

Results:

The protocol at both hospitals dictated that the first dose of dalteparin (5,000 units subcut) was to be given on POD#1. At Hospital A only, 54 patients (12%) received dalteparin 2,500 units subcut 6 hours after surgery (personal practice of one surgeon).

None of the in-hospital PEs were fatal. The incidence of VTE (non-fatal and fatal) after discharge is less reliable because patients may have presented for medical attention at centres other than the hospital where they underwent surgery or died at home.

In the POD#3 substudy, the proportion of patients investigated for in-hospital PE was 11% and 3%, at Hospital A and Hospital B, respectively and the proportion of patients investigated for PE who were diagnosed with PE was 54% (6/11) and 0, respectively. The proportion of patients with at least one vital statistic which may have raised suspicion of PE on POD#3(hypoxia, tachycardia, hypotension) was similar at the two sites (45% vs 43%).

Conclusion:

The higher incidence of in-hospital PE in one of two hospitals using the same method of thromboprophylaxis following elective TKR was likely the result of a more aggressive approach to investigation. The clinical importance of detecting these emboli is uncertain.

Disclosures:

Crowther: Pfizer: Consultancy; Leo Pharma: Research Funding; Boehringer Ingelheim: Research Funding.
Effect of Very Dose Warfarin therapy Upon the Markers of Activcation of Clotting Following Major Joint Replacement Surgery
Authors: Bern Murray M. , Adcock Dorothy , Wheaton Diane , McCree Paula , Deeran Elisabeth
Published: October 2019

Abstract 4395

Prophylaxis against post-operative deep vein thrombosis using low dose warfarin is appealing as it is inexpensive and with low risk of hemorrhage. Investigators have examined this issue with variable outcomes. Critical to our thinking is that the drug must be started prior to the elective surgical intervention in order to be an effective antithrombotic agent, while not causing potential hemorrhagic anticoagulation. This abstract describes the effect of such a regimen on the generation of markers of coagulation activation, comparing fixed very low dose warfarin (1 mg daily) to variable dose warfarin among patients having elective replacement of hip or knee surgery. The fixed low dose warfarin is begun 7 days prior to surgery and continued 28 days post operatively. Variable dose warfarin is begun at 5 mg the night prior to surgery and is continued for 28 days post operatively with the target INR 2.0–2.5, adjusted twice per week. For this study the markers of coagulation activation are prothrombin fragment F1+2 and thrombin-anti-thrombin complex (T-AT). They are measured prior to start of warfarin therapy (baseline), on the morning of surgery (OR Day), and on postoperative days 3 and 28. PIVKA II is measured at the same time points as a measure of warfarin activity other than the INR. For this study 10 patients for each group are taken from a larger group of patients participating in a randomized prospective study of this regimen now in progress. Both studies are IRB approved. Table 1 demonstrates the results.

As expected the PIVKA II is increased over normal for both groups by OR Day and on post-operative days 3 and 28. Greater increases in PIVKA II, as expected, occur among patients receiving variable dose warfarin, as this group received higher doses. The T-AT is elevated by post-operative day 3 and is returned to normal on post-operative day 28 in both groups. At each time point the T-AT values are equal for the two study groups. The F1+2 values are modestly increased for both groups on post-operative day 3, with better suppression of F1+2 generation by the variable dose regimen on day 28. In conclusion, the fixed low dose warfarin regimen is as effective as the variable dose warfarin as measured by the generation of T-AT, and is possibly somewhat less effective as determined by the generation of F1+2. The clinical significance of this difference is unknown, especially given the suppression of the T-AT. None of these patients suffered postoperative thromboembolic disease.

Disclosures:

Adcock: Esoterix Laboratory Services, Inc: Employment.
Evidence of Common Mechanisms Underlying the Survival Advantage of the PIG-A mutant and 13q- Hematopoietic Stem Cell Clones In Patients with Immune-Mediated Bone Marrow Failure
Authors: Hosokawa Kohei , Katagiri Takamasa , Sugimori Naomi , Qi Zhirong , Espinoza Luis J. , Sanada Masashi , Ogawa Seishi , Nakao Shinji
Published: October 2019

Abstract 4429

Background:

Numerical karyotypic abnormalities of undetermined significance are occasionally detected in patients with bone marrow failure (BMF), including aplastic anemia (AA) and low-risk myelodysplastic syndrome (MDS). BMF patients with 13q deletions (13q-) are likely to respond to immunosuppressive therapy (IST) without a propensity to develop into leukemia (Ishiyama K et al, Br J Haematol; 117: 747. 2002), and they possess small populations of glycosylphosphatidyl-inositol anchored protein (GPI-AP)- deficient granulocytes and erythrocytes more frequently than patients with normal karyotypes%, 15 with AA and 8 with MDS) of 1705 patients. The proportion of 13q- clones in all metaphase cells ranged from 5% to 100% with a median of 27.5%. Fourteen of the 23 (61%) patients showed 13q- alone and 9 (39%) showed other karyotypic abnormalities in addition to 13q-. GPI-AP- cells that accounted for 0.009% to 6.7% (median 0.148%) of granulocytes were detected in all (100%) of the 14 patients with 13q- alone, while the prevalence of increased GPI-AP- cells in patients with 13q- plus other abnormalities and those with a normal karyotype was 44% and 43%, respectively. A FISH analysis of PB granulocytes in individual patients revealed apparently lower percentages of GPI-AP- granulocytes than those of 13q- granulocytes, indicating that the GPI-AP- granulocytes were derived from non 13q- HSCs. Ten patients with 13q- alone were treated with IST (ATG+cyclosporine in 4 and cyclosporine alone in 6) and all of them achieved either a partial remission or a complete remission. The SNP array analysis of 5 patients revealed common deletion of a 15 Mb (13.3 to 14.3) region containing the RB gene. Wild-type and PIG-A mutant K562 cells were incubated in the presence of TGF-beta, which can inhibit leukemic cell proliferation by down-regulating phosphorylated RB protein (pRB), to determine whether the RB gene loss in 13q- HSCs has any connection with a proliferative advantage of PIG-A mutant HSCs. PIG-A mutant K562 cells were less sensitive to TGF-beta inhibition of proliferation and down-regulation of pRB levels than wild-type K562 cells.

Conclusion:

The markedly high prevalence of increased GPI-AP- cells and good response to IST indicate that the presence of 13q- clones is highly associated with the immune pathophysiology of BMF. Decreased sensitivity to inhibitory signals by TGF-beta due to RB gene loss in 13q- HSCs and the loss of GPI-AP-type TGF-beta receptors in PIG-A mutant HSCs may therefore be a common mechanism that confers a survival advantage to these mutant HSCs over normal HSCs in patients with immune-mediated BMF.

Disclosures:

No relevant conflicts of interest to declare.
Detection and Analysis of Autoantigens on the Membrane of Bone Marrow Cells In the Patients with BMMNC- Coomb's Test(+) Hemocytopenia
Authors: Fu Rong , Liu Hui , Shao Zonghong , Wang Jun , Li Lijuan
Published: October 2019

Abstract 4434

Objective

To investigate the autoantigens on the membrane of bone marrow cells in the patients with bone marrow mononuclear cell Coomb's(BMMNC-Coomb's) test(+) Immuno-related pancytopenia(IRP),for further purification and cloning.

Methods

20 newly diagnosed IRP patients, 10 AA patients, 10 MDS patients and 10 healthy donors as controls were enrolled in this study.The level of autoantibody-IgG in bone marrow was measured by Western blot; The autoantigens targeted by autoantibody IgG in IRP were identified from membrane protein extracts by SDS-PAGE and Western blot.

Results

The level of autoantibody IgG in bone marrow of untreated IRP patients was 75% (15/20), which was significantly higher than that of AA or MDS patients (0%) and normal controls(10%)(P<0.01). Autoantibody IgG in IRP could react with several autoantigens with approximate MWs of 25–30kDa, 60–65 kDa, 73 kDa and 83 kDa. Positive rates of individual autoantigens were 35%,50%,20% and 10%,respectively.

Conclusion

Autoantibody IgG reacted with membrane antigens could be found in bone marrow in IRP patients. The molecular weights of the autoantigens were mainly 25–30kDa, 60–65 kDa, 73 kDa and 83 kDa. The result showed that autoantibody IgG played an important role in IRP.

Disclosures:

No relevant conflicts of interest to declare.
Successful Mobilization of Peripheral Blood Stem Cells After Intensive Bendamustine Pre-Treatment In Patients with Multiple Myeloma
Authors: Pönisch Wolfram , Wiesler Julia , Leiblein Sabine , Edel Elvira , Al-Ali Haifa K. , Basara Nadezda , Hegenbart Ute , Heiß Christiane , Niederwieser Dietger W , Goldschmidt Hartmut
Published: October 2019

Abstract 4439

Introduction

The alkylating agent bendamustine has structural similarities to both alkylating agents and purine analogs, and is effective in the treatment of patients with multiple myeloma. So far, no data are available on stem cell toxicity or on stem cell mobilization. Since autologous stem cell transplantation is an established treatment for multiple myeloma after primary treatment, we were interested in analysing the experience of stem cell mobilization after bendamustine treatment.

Material and Methods

A retrospective analysis over a period of fifteen years was carried out in 56 (34 male and 22 female) patients with multiple myeloma after bendamustine pretreatment at the university hospitals Leipzig and Heidelberg. Patients had a median age of 58 (range 31–72) years. The median number of cycles was 3 (range 1–10) and the cumulative bendamustine dose ranged from 120 to 2400 mg/qm. The mobilization regimen in 37 cases was either cyclophosphamide 4 g/qm (n=33) or 7 g/qm (n=4) followed by G-CSF (2×5 ug/kg s.c.). Alternative regimens such as CAD, CED, TCED and others were used for mobilization in the remaining 19 patients. Apheresis was started as soon as peripheral blood CD34+ counts exceeded 10×106/l with a harvest target of 4×106 CD34+/kg using 4 times the blood volume. The minimal accepted target was 2×106 CD34+/kg.

Results

Stem cell harvest was successful in 54 of the 56 patients. In one patient the peripheral blood CD34+ cell count failed to reach 10 × 106/l and no apheresis was performed. In one further patient a rapid decrease in peripheral blood CD34+ counts resulted in insufficient recovery of stem cells in the apheresis product. In 18 out of 54 patients (33%) the target was reached with a single apharesis. The median number of aphareses in the 54 patients was 2 (range 1–7) and the median CD34+ cell-count obtained was 5.5 (range 1.7–20.4) × 106/kg. Engraftment was successful in 52/53 patients receiving a stem cell transplant. One patient was successfully harvested and did not receive the transplant yet.

Conclusion

From this retrospective analysis we conclude that mobilization of PBSC is possible after intensive bendamustine pretreatment.

Disclosures:

Niederwieser: Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau. Goldschmidt:Celgene: Membership on an entity's Board of Directors or advisory committees; Ortho Biotech: Membership on an entity's Board of Directors or advisory committees; Ortho Biotech: Research Funding; Celgene: Research Funding; Chugai Pharma: Research Funding; Amgen: Research Funding.