bluuebunny/crossref_metadata_2025 huggingface dataset:
Liver Tropism and Factor VIII Transgene Expression after Intrauterine Chorionic Villus Stem Cell Transplantation
Authors: Miguelino Maricel G. , Kumar Priyadarsini , Pivetti Christopher , Lanford Lee , Powell Jerry S. , Wang Aijun
Published: October 2019

INTRODUCTION: Gestation is characterized by rapid mitosis and histogenesis in an immune naïve environment. Growth and development of vital organs are occurring continuously at different time points throughout pregnancy under the regulation of paracrine signals, transcription and growth factors. Appropriate timing to target the developing organ of interest during in utero stem cell transplantation (IUT) may result in high level engraftment in that organ as well as recognition of foreign cells as self. Hemophilia A, is an X-linked recessive bleeding disorder caused by a mutation of the gene that encodes Factor VIII (FVIII) which is primarily produced by the liver. We hypothesize that introduction of stem cells in utero during hepatogenesis in mice (e9.5-15 days) will result in localization and persistence of stem cells in the liver.

METHODOLOGY: We reported isolation and characterization of early preterm chorionic villus derived mesenchymal stem cells (PMSCs) at GA 9-13 weeks. PMSCs (n =5) were transduced with a lentiviral vector encoding B-domain deleted FVIII, GFP and luciferase reporter genes (pCCLc-MNDU3-eGFP/LUC-PGK-F8-WPRE) at an MOI (Multiplicity of Infection) of 3. Chromogenic assay was performed on cell supernatant of transduced PMSCs 72 hours after transduction. Prior to transplantation, PMSCs were plated at different densities ranging from 250,000 cells to 2 x10^6 cells and subjected to bioluminescence imaging. Intraperitoneal transplantation of PBS (control group) or with 1 x10^6 PMSCs (experimental group) was conducted in fetal mice at e12-14 days using a 33 gauge non-coring needle. Bioluminescence analysis was performed 24 hours after IUT on dams, and 12-15 days after transplantation. Mice were euthanized and liver tissue was harvested from all animals. Expression of FVIII was analyzed by RT-PCR from both groups.

RESULTS: Bioluminescence imaging revealed focal density and transgene expression for transduced cells in vitro, in vivo at 24 hours and in postnatal pups 12-14 days after transplantation. Postmortem bioluminescence analysis of IUT transplanted mice showed positive bioluminescence in the livers of pups injected with transduced PMSCs. RT-PCR analysis of the liver samples using human specific FVIII primers revealed FVIII expression in liver tissues in PMSC treated mice while negative for PBS treated mice.

CONCLUSION: These results demonstrate the functional survival and localization of genetically modified PMSCs in the liver after intrauterine transplantation for up to two weeks. This study provide better understanding of the cell fate of stem cells after IUT and suggest that appropriate timing of stem cell transplantation in utero may lead to immunological tolerance.

Disclosures

Powell: CSL Behring: Employment.
Binding of rhFVIIa (LR769) to the Endothelial Protein C Receptor
Authors: Grandoni Jerry A
Published: October 2019

Congenital Hemophilia A and B treatment may be complicated by the development of inhibitors to the coagulation factors used in replacement therapies. In such cases, factor replacement becomes ineffective and use of a bypassing agent is needed to treat or prevent bleedings. A recombinant form of activated Factor VII has been available for this purpose. In this study, a new second generation recombinant human Factor VIIa (rhFVIIa, LR769) produced by LFB USA was studied. The goal is to provide patients with Hemophilia A and B who develop inhibitors an alternative treatment option. In work presented previously, we demonstrated that LR769 was similar to NovoSevenRT with respect to activation of FX and FIX, tissue factor binding, and inactivation by antithrombin. LR769 bound better to platelets than NovoSevenRT at saturation but had a similar binding constant(1). It is known that FVIIa binds to Endothelial Protein C Receptor (EPCR) with the same affinity as Protein C (2-4). Several studies suggest EPCR is involved in therapeutic mechanism rhFVIIa (5,6,7,8). In addition to its effect on thrombin generation, binding to EPCR may protect against permeability induced by VEGF and LPS (6). In the work presented here, the binding of LR769 to EPCR on the surface of cultured HUVEC cells was determined and compared to NovoSevenRT. A dose-dependent binding was observed at rhFVIIa concentrations expected in patients undergoing treatment with this product. The specificity for EPCR binding was confirmed by competition with Protein C, soluble EPCR, or anti-EPCR which reduced binding to these cells. Addition of soluble tissue factor increased the binding by approximately 20-30%. Overall, the results show that LR769 binds to primary human endothelial cells via EPCR and that tissue factor increases the binding affinity. Binding was found to be higher with LR769 than NovoSevenRT. The significance of the tissue factor effect on rhFVIIa binding with respect to Hemophilic joint arthropathy is discussed. The methods used will be used to further characterize the interactions of rhFVIIa with important components of the vascular system.

1) Grandoni, J.A. et al. (2013) ASH 2013 poster

2) Gosh et al. (2007) JBC 282 11849-11857

3) Lopez-Sagaseta et al. (2007) JTH 5 1817-1824

4) Preston et al. (2006) JBC 281 28850-28857

5) Pavani, G et al. (2014) Blood 7 1157-65

6) Sundarham et al. J. Thromb and Hemost (2014)12:690-700

7) Monroe DM, et al. (1997) Br J Haematol;99:542-7

Disclosures

Grandoni: LFB USA: Employment.
Biological Effects of Different Extracellular Vesicles Population on Reversal of Marrow Cells Radiation Damage
Authors: Wen Sicheng , Cheng Yan , Dooner Mark , Pereira Mandy , Del Tatto Michael , Papa Elaine , Goldberg Laura R. , Aliotta Jason M. , Quesenberry Peter J.
Published: October 2019

We have shown that Human or mouse mesenchymal stem cell (MSC) derived vesicles separated by differential centrifugation (300g, 10kg &100kg) can reverse radiation whole bone marrow (WBM) damage in vitro on the hematopoietic cell line FDC-P1 and in vivo on recovery of stem cells and differentiated blood cells after whole body irradiation. The vesicle separation is a classic method for preparations of exosomes. However, this separation isolates the smaller vesicles, discarding larger vesicles. Biologic healing effects of larger vesicles with or without smaller vesicles have not been investigated. Here, we investigated the healing effects of 3 different preparations of vesicles: 10k pellet (microvesicles), 100-10k pellet (exosomes) & 100k pellet (no 10k spin).

Three different fractions (10k pellet, 100-10k pellet &100k pellet) of murine/human MSC or murine WBM derived vesicles were isolated by differential centrifugation. FDC-P1 cells were exposed at 500cGy irradiation and cultured with or without the addition of 3 different preparations of murine WBM or human MSC-derived vesicles for 7 days. A significant increase in cell proliferation was observed after 3 different vesicle fractions treatments. However, exosomes were clearly inferior, microvesicles and the combined exosomes and microvesicles population were superior. The capacity of human MSC-derived vesicles of different fractions on reversal of murine bone marrow damage was also evaluated in vivo. Again, the combined fractions shown a significant increase donor chimerism in bone marrow at 6 month post-transplant with 5x's the level of engraftment compared to irradiation control. The 10k and 100-10k fractions showed intermediate healing. Exposure to vesicles in irradiated FDC-P1 cells downregulated phosphorylated H2AX, the DNA damage marker and decreased apoptosis marker, PARP cleavage part. We further investigated the effect of murine marrow MSC-derived vesicles on the gene expression in peripheral blood cells from radiation damaged mice. 28 genes were altered by irradiation, 22 showed partial or complete reversal of these alterations. The characterization of three different fractions of vesicles derived from murine bone marrow cells and human MSC were evaluated by miRNA realtime PCR array (total 750 miRNA). Several MSC-EV associated miRNA responsible for the reversal of radiation damage were evaluated. Our preliminary data shown that miR-221, miR654-3p and miR210-5p could partially reverse radiation damage in FDC-P1 cells

There are different biological effects in different extracellular vesicles populations. For reversal of radiation toxicity the most effective vesicle population would include both smaller (exosomes) and larger vesicles (microvesicles).

Acknowedgements: This work was support by the NIH grant 5UH2TR00080

Disclosures

No relevant conflicts of interest to declare.
Whole Exome Sequencing Defines the Genetic Differences Between Pediatric and Adult Follicular Lymphoma
Authors: Ondrejka Sarah Lynn , Jaffe Elaine S , Gong Shunyou , Rajagopalan Deepthi , Love Cassandra , Dave Sandeep
Published: October 2019

Follicular lymphoma (FL) is the most common low grade lymphoma in adults, but uncommon in children. While the two diseases are similar in morphology and immunophenotype, pediatric-type FLs manifest higher grade histology with most cases being localized and curable with standard therapy. Pediatric-type FLs are thought arise from germinal center B cells and typically demonstrate higher expression of the germinal center B cell markers BCL6 and CD10.

While the genetic basis of adult FL has been studied previously, the genetic basis of pediatric-type FL remains largely unknown. In this study, we sought to define the gene coding mutations occurring in pediatric FL and compared them to those of 48 adult FLs, as well as other adult lymphomas derived from germinal center B cells including 88 GCB DLBCLs and 67 cases of BL that we, and others, have published previously.

We obtained a total of 32 pediatric-type FLs along with paired normal tissue. The median age wa 8 years with 62% male patients. We performed whole-exome sequencing for these cases using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes (CCDS database), as well as ~700 human miRNAs from miRBase (v13). In all, we generated over 6 GB of sequencing data using high throughput sequencing on the Illumina platform.

Using bioinformatics approaches that we have described previously, we identified 26 candidate cancer genes that were recurrently somatically mutated in pediatric FL. We found that the genes mutated in pediatric FLs were from three predominant ontologies/pathways. These included DNA damage repair (40%, ATM, RB1), chromatin modification (34%, SETD2, SMARCA4 and TET2) and alternative splicing (12%, SF3B1). We also noted IRF4 translocations and TNFRSF14 mutations in 10% of the cases as has been described previously.

We further compared pediatric FLs to other germinal center B cell derived lymphomas and, in particular, FLs occurring in adults. We found that the mutations that characterize adult FLs including MLL2, CREBBP, and GNA13 were absent in pediatric FLs. There was only one pediatric FL case with an EZH2 mutation, which otherwise occur commonly in germinal center B cell derived lymphomas.

Thus, pediatric FLs are genetically unlike both adult FLs and other germinal center B cell derived lymphomas. Our data provide new clues to the distinctive genetic basis of these tumors and their clinical behavior.

Disclosures

No relevant conflicts of interest to declare.
The Homeobox Gene VENTX Collaborates with AML1-ETO in Inducing an Acute Leukemia in Mice
Authors: Gentner Eva , Vegi Naidu , Mulaw Medhanie A. , Quintanilla-Fend Leticia , Döhner Hartmut , Döhner Konstanze , Buske Christian , Rawat Vijay P.S. , Feuring-Buske Michaela
Published: October 2019

Homeobox genes are key factors in the development of acute leukemias. So far, little is known about the role of non-clustered homeobox genes such as VENTX. The Vent-like homeobox gene VENTX is a member of the Vent gene family in mammals and is the mammalian homolog of the Xenopus xvent gene. Our group has previously shown that VENTX promotes myeloid differentiation when overexpressed in normal CD34+ human stem - and progenitor cells and shows high and aberrant expression in human acute myeloid leukemia (AML) characterized by the AML1-ETO (AE) fusion (Rawat et al., PNAS 2010). To prove the functional relevance of aberrant VENTX expression in this AML genotype, we set up in vitro assays and a murine bone marrow transplantation model, mimicking high expression of VENTX by retrovirally engineered expression of the gene in murine bone marrow progenitor cells. In the CFU-S assay coexpression of both genes significantly increased the frequency of spleen colonies 14-fold and 7-fold compared to VENTX and AE, respectively (p<0.02), and more than 7-fold compared to the control (p<0.001). Most importantly and in contrast to AE alone, coexpression of AE and VENTX induced an acute leukemia after a median latency of 11.3 months post transplant. All leukemias evaluated were rapidly transplantable with a median latency until leukemia induced death of 40 days. The median blast percentage in primary recipient mice was 66% (range 35-100%, n=24). Of note, massive expansion of the red pulpa was documented with the appearance of erythroblasts in the spleen. Furthermore, blasts in the bone marrow expressed CD19, partly in combination with Sca1 and Gr1, pointing to an aberrant expression of this antigen in the AE/VENTX leukemias. Ex vivo, leukemic cells grew permanently generating AE/VENTX positive cell lines. RNA-Seq analyses from CD34+ cord blood transduced with VENTX documented 279 differentially expressed genes compared to the GFP control, hitting 7 pathways (Hematopoietic cell lineage, Cytokine-cytokine receptor pathway, Jak-STAT signaling pathway, Porphyrin and Chlorophyll Metabolism, Asthma, Thyroid cancer and Endocytosis) in the KEGG-Analysis and showed a downregulation of genes necessary for terminal erythroid differentiation such as the EPO-receptor and GATA1. Taken together, these data characterize VENTX as an important contributing factor to AE positive leukemias. Importantly, coexpression of AE and VENTX allows the generation of a murine AE model, which in contrast to all other published mouse models mimics aberrant expression of CD19 in human AE positive AML, a hallmark of this AML genotype.

Disclosures

Buske: CELLTRION, Inc.: Consultancy, Honoraria.
Lmo2's Oncogenic Function in T-Cell Leukemia Requires Ldb1
Authors: Li Liqi , Layer Justin H. , Warzecha Claude , Tripathi Rati , Love Paul , Dave Utpal P.
Published: October 2019

LIM domain Only-2 (LMO2) is one of the most frequently deregulated oncogenes in T-cell acute lymphoblastic leukemia (T-ALL). LMO2 encodes a small protein with 2 LIM domains that is part of a large multiprotein complex in hematopoietic stem and progenitor cells, where it is required for HSC specification and maintenance. Many of LMO2's protein partners in HSPCs are expressed in T-ALL implying that protein complexes similar to those nucleated by LMO2 in HSPCs also play a role in leukemia. In this study, we analyzed a critical component of the LMO2 associated complex, LIM domain binding1 (LDB1). LDB1 appears to be an obligate partner of LMO2 in HSPCs but it is not required for T-cell development from committed progenitors. LDB1 is concordantly expressed with LMO2 in human T-ALL although its expression is more widespread than LMO2. To further define Ldb1's role in leukemia, we induced its conditional knockout in CD2-Lmo2 transgenic mice. CD2-Lmo2 transgenic mice develop T-ALL with high penetrance and closely model the human disease. We discovered that Lmo2-induced T-ALL was markedly attenuated in penetrance and latency by Ldb1 deletion. Since Lmo2 induces a distinct differentiation arrest in T-cell progenitors prior to leukemic transformation, we analyzed the differentiation of T-cell progenitors in CD2-Lmo2 transgenic/floxed-Ldb1/Lck-Cre mice and in non-Lmo2 transgenics: floxed-Ldb1/Lck-Cre mice. Ldb1 deletion by Lck-Cre was efficient in double negative and double positive T-cell progenitors. In striking contrast, Ldb1 deletion could not be induced in CD2-Lmo2 transgenic T-cell progenitors. Consistent with this finding, T-ALLs that developed in CD2-Lmo2/floxed-Ldb1/Lck-Cre mice had incomplete deletion of Ldb1. These results imply that Ldb1 is a required factor for Lmo2 to induce T-ALL. Lastly, gene expression analysis of Lmo2-induced T-ALLs and ChIP-exonuclease analysis of Ldb1 occupancy in T-ALL suggested that the Lmo2/Ldb1 complex enforced a gene signature similar to that seen in HSPCs and in Early T-cell Precursor ALL. In conclusion, Ldb1 is a required partner for Lmo2 to induce T-ALL. Additionally, the HSPC function of Lmo2/Ldb1 complexes may be recapitulated in T-cell progenitors prior to T-ALL.

Disclosures

No relevant conflicts of interest to declare.
Lymphocyte-to-Monocyte Ratio at Diagnosis and Survival in De Novo Double/Triple Hit Diffuse Large B-Cell Lymphoma
Authors: Porrata Luis , Ristow Kay , McPhail Ellen D. , Macon William , Maurer Matthew J , Nowakowski Grzegorz S. , Ansell Stephen , Witzig Thomas E. , Markovic Svetomir , Habermann Thomas M.
Published: October 2019

The lymphocyte-to-monocyte ratio at diagnosis (LMR-D) has been reported to be a prognostic factor for clinical outcomes in both T-cell and B-cell lymphomas. However, there are no reports testing the prognostic ability of LMR-D in patients diagnosed with de novo double/triple hit diffuse large B cell lymphoma (DLBCL). Thus, we set out to investigate if the LMR-D is a prognostic factor for survival in de novo double/triple hit lymphomas. From 10/5/1998 until 1/16/2015, thirty-four patients with de novo double/triple hit DLBCL were identified for this study. Double and triple hit were defined by interphase FISH evaluating three fusion signals to identify the BCL2 translocation and IGK/MYC D-FISH probe to identify whether the partner is IG or non-IG. Interphase fluorescence in situ hybridization (FISH) was performed on paraffin sections using probes that included 8q24 (5'MYC, 3'MYC); t (2;8), IGK/MYC; t(8,14), MYC/IGH; t(8;22), MYC/IGL; 3q27 (3'BCL6, 5'BCL'6); and 18q21 (3'BCL2, 5'BCL2). The cohort included 14 females and 20 males. The median follow-up for the cohort was 9.0 months (range: 0.4-72.6 months). Using the median for the LMR-D as the cut-off value, patients with a LMR-D ≥ 1.2 experienced superior overall survival (OS) [Hazard ratio (HR) of 0.127, 95% confidence interval (CI) of 0.019-0.530, p < 0.004] and progression-free survival (PFS) [HR of 0.107, 95 CI of 0.024-0.335, p < 0.0001]. The median follow up for OS for patients with a LMR-D ≥ 1.2 was not reached with a 5-year OS rate of 82% (95% CI of 49%-95%) compared with a median follow-up of 10 months for patients with a LMR-D < 1.2 with a 0% 5 year OS rate, p < 0.003 (Figure 1A). The median PFS for patients with a LMR-D ≥ 2 was not reached with a 5 years PFS of 74% (95%CI, of 43%-91%) compared with a median follow-up of 4.7 months for patients with a LMR-D < 1.2 and 0% 5 year PFS rate, p < 0.0001 (Figure 1B). After adjusting for the International Prognostic Index, multivariate analysis showed that the LMR-D remained an independent prognostic factor for OS [HR = 0.180, 95% CI of 0.254-0.784, p < 0.02] and for PFS [HR of 0.127, 95%CI of 0.029-0.409, p < 0.0003]. In spite of the small cohort of de novo double/triple hit DLBCL, the LMR-D was identified as a prognostic factor for clinical outcomes for this specific set of aggressive lymphomas. Further studies are warranted to confirm our findings. Table.LMR-D ≥ 1.2, N = 18Events = 2LMR-D < 1.2, N = 16Events = 8P < 0.003LMR-D ≥ 1.2, N = 18Events = 3LMR-D < 1.2, N = 16Events = 14P < 0.0001Figure 1AB

Figure 1. Figure 1.

Disclosures

Maurer: Kite Pharma: Research Funding. Ansell:Bristol-Myers Squibb: Research Funding; Celldex: Research Funding. Off Label Use: New agent in a combination regimen..
Racial Disparities in Incidence and Survival for Patients with Multiple Myeloma in the United States
Authors: Radhakrishnan Lakshmi , Lonial Sagar , Nooka Ajay K.
Published: October 2019

Background: Over the past two decades the incidence of myeloma has been gradually increasing in the United States. The incidence rates are higher in men than women, and higher in blacks than whites. Similar to the differential incidence, overall survival rate between blacks and whites are also dissimilar; a difference that is not completely explained and may be attributable to genetic variations between the two groups.

Methods: Using data from 18 SEER registries, we examined differences in incidence, mortality and survival from 1973-2012 for 89,867 myeloma patients (68,701 white, 16,364 black and 4,802 others) by race, gender and age-stratification. ICD-O-3 and morphologic (9732/3) codes were used to identify cases. Age-adjusted incidence and mortality rates, regression analysis and survival curves were calculated by race. Statistics were computed using the National Cancer Institute SEER*Stat software, version 8.2.0. and SAS software, version 9.4 (SAS Institute Inc, Cary, NC).

Results: Median age at diagnosis was 70 years (range 20-100 years) for the overall population (blacks: 66 years; whites: 71 years, and others: 69 years (P<0.01). The age-adjusted incidence rates per 100,000 populations were: blacks- 11.9 (95% CI 11.6, 12.1, P-value<0.05); whites- 5.1 (95% CI 5.0, 5.2) and others- 3.7 (95% CI 3.6, 3.9). The incidence rates were higher for black males, 14.2 (95% CI 14, 14.7) followed by black females, 10.3 (95% CI 10, 10.6, P-value<0.05). Using white male as reference, incidence rate ratios for black males and females are 2.20 (95% CI 2.12, 2.28) and 1.60 (95% CI 1.54, 1.65) respectively. The 2-year relative survival rates (RSR) for the study population were: whites- 60.4%, blacks: 64.1% and others: 68.4%, respectively. The 5-year RSR by race and gender are included in figure 1. On the survival analysis, black race is an independent factor to have improved survival (HR=0.884, P-value<0.0001). This was corroborated on regression analysis, showing decreased hazard ratios for blacks (HR 0.856 95% CI 0.834, 0.878) compared to whites (P-value<0.001).

Conclusions: Black patients are diagnosed younger than whites and other races. The IRRs for black males are twice compared to white males. While the 5-year RSRs are improving for all races and genders, black race is an independent factor to have improved survival. Further population-based studies focused on the exploring the underlying biological mechanisms of disease may explain the earlier presentation with disease and better overall survival among black patients.

Figure 1. Figure 1.

Disclosures

Lonial: Novartis: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding. Nooka:Onyx Pharmaceuticals: Consultancy; Spectrum Pharmaceuticals: Consultancy.
Predictors of Increase in Emergency Room (ER) Visits and Hospitalizations in Patients with Chronic Lymphocytic Leukemia (CLL) Treated with Chemotherapy
Authors: Feinberg Bruce , Schenkel Brad , McBride Ali , Ellis Lorie , Bhor Menaka , Radtchenko Janna , Lal Lincy S
Published: October 2019

Background: Acute care interventions negatively impact patients' quality of life and represent one of the major cost drivers in oncology care. Understanding the predictors for increased healthcare resource utilization is essential for better management of patients with CLL. This study evaluated predictors for ER visits and hospitalizations in patients with CLL treated with chemotherapy.

Methods: Using claims data (MORE2 Registry®), patients receiving chemotherapy for CLL were retrospectively identified by ICD-9 codes (204.1X) during a 48-month period (August 2009-2013). Patients with secondary malignancies, pregnancy codes, and age <18 were excluded. Univariate regression analysis was conducted to determine covariates associated with an increase in ER visits and hospitalizations. Results for significant variables were reported as odds ratio/p-value. Odds ratio >1 indicates increase in chance of event.

Results: A total of 2,013 patients with CLL were identified. Median age at diagnosis was 72 years, 61% were male, 34% were treated in the relapsed setting, 42% had ER visits, and 39% were hospitalized throughout their treatment history. Significant increases in ER visits and hospitalizations were associated with the following variables (OR/p-value): age (ER: 1.015/.001, hospitalization: 1.001/.038), Charlson comorbidity index (ER: 1.253/.013, hospitalization: 1.038/.018), supportive care use (ER: 2.087/<.001, hospitalization: 2.429/<.001), number of CLL related adverse events (ER: 12.311/<.001, hospitalization: 29.467/<.001), chemotherapy duration (ER: 1.001/<.001, hospitalization: 1.001/<.001), bendamustine use (ER: 1.404/.001, hospitalization: 1.381/.002), fludarabine use (ER: 1.405/<.001, hospitalization: 1.395/.001), Northeast region of the United States (ER: 1.293/.008, hospitalization: 1.321/.005), and treatment following relapse (ER: 3.413/<.001, hospitalization: 4.018/<.001). Significant covariates associated with a decrease in hospitalizations or ER visits were not found.

Conclusions: This retrospective study demonstrated that patient demographic and clinical characteristics as well as chemotherapy choice were associated with ER visits and hospitalizations in patients with CLL. ER visits and hospitalizations have similar drivers. These data warrant consideration of age and comorbidity-adjusted treatment choice in CLL patients who need active treatment with antineoplastics.

Disclosures

Feinberg: Janssen Scientific Affairs, LLC: Consultancy. Schenkel:Janssen Scientific Affairs, LLC: Employment. McBride:Janssen Scientific Affairs, LLC: Consultancy. Ellis:Janssen Scientific Affairs, LLC: Employment. Bhor:Janssen Scientific Affairs, LLC: Consultancy. Radtchenko:Janssen Scientific Affairs, LLC: Consultancy. Lal:Janssen Scientific Affairs, LLC: Consultancy.
The Dysfunction of Platelets in Paroxysmal Nocturnal Hemoglobinuria
Authors: Fu Rong , Meng Yinping , Wang Yihao , Liu Hui , Liu Yi , Li Lijuan , Ding Shaoxue , Ruan Erbao , Wang Huaquan , Wang Guojin , Song Jia , Shao Zonghong
Published: October 2019

Introduction Thrombosis is one of the most dangerous complications in PNH, which can cause high mortality. However, its underlying mechanism remains unclear. To explore the mechanism of thrombosis in PNH, the function of platelets and complements were investigated in our study.

Material and Methods Serum level of terminal complement complex (sC5b-9) was detected by ELISA. The quantities of C5b-9,CD61 and CD62p on the membrane of platelets were detected by flow cytometry. Clinical tests were collected, including D-Dimer and platelet aggregation rates induced by adenosine diphosphate (ADP) and arachidonic acid (ARA).

Results The serum sC5b-9 level was significantly lower in the PNH/PNH-AA group than that in the control group (p<0.01). The deposition of C5b-9 on CD59- platelets (17.53%±6.27%) was higher than those on CD59+ platelets in PNH/PNH-AA patients(11.33%±5.03%) and normal controls(10.88%±3.58%) (p<0.01). D-dimer was significantly higher in PNH/PNH-AA patients (485ng/dL) than that in normal controls (311ng/dL)(p<0.05),especially in patients with LDH>1000U/L(789ng/dL). CD61 and CD62p expression on CD59+ platelets in PNH patients (76.87%,39.99%) were significantly lower than those in normal controls(98.41%,64.21%)(p<0.05,p<0.01).CD62p expression on CD59- platelets (40.07%) was significantly lower than normal controls(p<0.01). Platelet aggregation stimulated by agonists ADP (37.54±24.25)% and ARA (24.60%) were lower in the PNH/PNH-AA group than that in the control group(59.20±23.30)%, (14.54%) (p < 0.05). Interestingly,CD61 expression on CD59+ platelets in PNH patients was higher in the patients with higher type II PNH clone.

Conclusions The function of platelets was inversely inhibited,especially CD59+ platelets even if hypercoagulation continuously exists in PNH/PNH-AA.

Disclosures

No relevant conflicts of interest to declare.
Significant Improvement of Survival By T2* Cardiovascular Magnetic Resonance in Thalassemia Major
Authors: Pepe Alessia , Meloni Antonella , Del Vecchio Giovanni Carlo , Romeo Maria Antonietta , Gamberini Maria Rita , Bonetti Federico , Neri Maria Giovanna , Chiodi Elisabetta , Positano Vincenzo , Maggio Aurelio , Borgna-Pignatti Caterina
Published: October 2019

Background: In 2004 seven Italian centers reported survival data for patients with thalassemia major (TM) and showed that heart disease due to iron overload was the most common cause of death (Borgna et al Haematologica 2004). In the same years the accurate and noninvasive assessment of cardiac siderosis was made possible in Italy by the introduction of the T2* cardiovascular magnetic resonance (CMR).

Purpose: We aimed to evaluate if the deployment of T2* CMR had an impact on the mortality rate.

Methods: Four centers contributed to the present study, updating the data of the enrolled patients until August 31, 2010. For the patients who died, the date of the death represented the end of the study. 577 patients (264 females and 313 males) were included.

Results: One-hundred and fifty-nine (27.6%) patients died, 124 of whom (77.9%) died before the year 2000.

The Table shows the comparison between dead patients and survivors. Dead patients were significantly younger and they were more frequently males. Dead patients started chelation therapy significantly later. HIV, arrhythmias and heart failure were significantly more frequent in dead patients. According to the Cox model, the following variables were identified as significant univariate prognosticators for the death: male sex (HR=1.87, 95%CI=1.34-2.60, P<0.0001), HIV (HR=2.55, 95%CI=1.25-5.20, P=0.010) and heart failure (HR=8.86, 95%CI=6.37-12.31, P<0.0001).

MRI was not performed in 406 patients (70.4%) and no patient had been scanned before his/her death. Among the survivors, MRI was not performed in the 59% of the cases (P<0.0001). The absence of an MRI scan was a significant univariate prognosticator for death (HR=43.25, 95%CI=11.32-165.33, P<0.0001).

The study was restricted to the patients dead after 2004 (19/159=12%) or followed until August 2010 (N=357). In this subgroup of 376 patients, MRI was not performed in the 52.4% of the survivors and in all dead patients (P<0.0001). The absence of a MRI exam was reconfirmed as a strong predictive factor for death (HR=49.37, 95%CI=1.08-2263.24, P=0.046). The Kaplan-Meier curve is showed in Figure 1. The log-rank test revealed a significant difference in the curves (P<0.0001).

Conclusions: Our data suggests that the use of T2* CMR, that enables individually tailored chelation regimes reducing the likelihood of developing decompensated cardiac failure, allowed the reduction of cardiac mortality in chronically transfused TM patients.

Table 1. Table 1. Figure 1. Figure 1.

Disclosures

Pepe: Novartis: Speakers Bureau; Chiesi: Speakers Bureau; ApoPharma Inc: Speakers Bureau.
Antiplatelet Glycoprotein Antibodies Profile in Adult Patients with Primary Immune Thrombocytopenia and the Comparison with Regards to the Treatment Response to Corticosteroids
Authors: Atmakusuma Tubagus Djumhana , Sukrisman Lugyanti , Hardi Fransiska
Published: October 2019

Background: Platelet destruction in immune thrombocytopenia (ITP) is mediated by autoantibodies against platelet antigens. Anti-GPIIb/IIIa and anti-GPIb/IX antibodies are two major antibodies spesific for platelets and megakaryocytes. The diversity of clinical characteristics and therapeutic responses have been thought to be influenced by heterogeneity of spesific glycoprotein complexes. However, there has been no published data regarding either antiplatelet antibodies profile or response rates to corticosteroid, in adult patients with primary ITP.

Objectives: To determine antiplatelet glycoprotein antibodies profile in adult patients with primary ITP and to compare the response rate to corticosteroid referring to the profile.

Methods: This study is a cross sectional study in adult patients with primary ITP who visited Hematology Clinics in Dr Cipto Mangunkusumo Hospital, Jakarta, Indonesia, in a period of March to October 2013. The tests to determine the auto antibodies against the platelet GPIIb/IIIa and GPIb/IX surface antigens were performed by a direct MAIPA technique.

Results: A total of 40 patients who were diagnosed as primary ITP have been enrolled into the study. The subjects of the study were divided in two groups, 10 subjects with newly diagnosed ITP, and 30 subjects with persistent or chronic ITP. Most of the subjects were female (82.5 %) with the median age was 24,5 (17-55) years old.. Seventy (??) subjects had ITP Bleeding Score (IBLS) 2.. In a persistent/chronic ITP group (n= 30), the proportion of anti-GPIIb/IIIa was 20 / 30 (66,67 %) with the median optical density (OD) was 0,461 (0,093-2,116) and the proportion of anti-GPIb/IX was 25/30 (83.33%) with the median OD was 0,507 (0,190-1,924). Meanwhile, in a newly diagnosed ITP group (n=10) the proportion of anti-GPIIb/IIIa was 7/10 (70%) with the mean OD was 0,802 ± 0,71 and the proportion of anti-GPIb/IX was 8/10 (80%) with the mean OD 0,82 ± 0,57. The response rates to corticosteroid were as follows: 42.5 % of the subjects achieved complete response, 45 % achieved response, and 12.5 % achieved no response. Comparison between the subjects with and without anti-GPIIb/IIIa and anti-GPIb/IX antibodies showed similarity response rates to prednisone.

Conclusion: Sincemost of patients with primary ITP had anti GPIIb/IIIa and anti GPIb/IX antibodies, those antibodies may support a diagnosis of primary ITP. Since no differences in terms of the response rates to corticosteroid between subjects with and without anti-GPIIb/IIIa and anti-GPIb/IX antibodies, those antibodies can not be used as a predictor of the treatment

Disclosures

Sukrisman: Division of Hematology Medical Oncology: Other: Colleague.
Clinical Validation of a Radiolabeled PF-4 Staph-a Binding Assay for Confirming Heparin Induced Thrombocytopenia
Authors: Takala Hemasri , Davis John A. , Schwartz Kenneth A.
Published: October 2019

Introduction:Confirmatory laboratory assays for heparin induced thrombocytopenia (HIT) can broadly be classified as functional which have high specificity and rely on activation of platelets by the platelet factor 4 (PF4)-heparin-IgG immune complex or as immune based assays that are relatively more sensitive. At the time when the clinician is evaluating heparin as a cause of thrombocytopenia the 4 "T" criteria are helpful. However, an increase in the patient's platelet count after the heparin has been stopped is critical for confirmation of the diagnosis.

Methods: We developed a variation of a previously described technique (Newman Thromb Haemost 1998;80:292) and used clinical criteria as the standard for comparison to evaluate the assay. Radiolabeled 125-I-PF4 is incorporated into the immune complex of PF-4-heparin-immunoglobulin and the amount of radiolabeled immune complex is measured after binding to staphylococcal A protein sepharose (Staph-A). The hospitalized subjects medical record was reviewed to: measure a 4 "T" score, to determine if the patient's platelet count increased after heparin was stopped and to exclude other plausible causes of thrombocytopenia.

Aim: The assay relies on the binding of the heparin immune complexes to Staph A. Staph A preferentially binds to larger as compared to smaller immune complexes and the larger complexes produce a greater degree of F(c) mediated platelet activation when compared with the smaller complexes. This suggests the hypothesis that a Staph A based assay will have have better specificity and sensitivity than the currently used methodologies.

Results: 28 patient samples were evaluated. True positives were observed in 4 hospital patients and 4 known positives. 19 were true negatives and included 7 from hospital patients and 12 from thrombocytopenic patients who were not treated with heparin. 1 sample was negative in our assay, and was judged as false negative. Concordance between the radiolabeled PF-4 assay and the commercial PF-4 assay was observed in all the 28 patients.

Conclusions: To date when judged using clinical criteria, the radiolabeled PF-4 assay correctly distinguished true positives and true negatives in 27 of the 28 samples.

Disclosures

No relevant conflicts of interest to declare.
Effect of Carica Papaya leaf Extract on Platelet Count in Chronic Immune Thrombocytopenic Purpura: A Case Series
Authors: Hampilos Katherine E. , Corn Joshua , Hodsdon Wendy , Anderes Elise N. , Roop Ryan P. , Wagner Peter , Troy Lynn R.
Published: October 2019

Background:

The leaves of Carica papaya have been used to treat thrombocytopenia in the critical phase of Dengue fever in areas where the virus is endemic. This case series describes the use of C. papaya leaf liquid extract (CPLE) as an adjunctive therapy for four patients receiving standard-of-care treatment for chronic immune thrombocytopenic purpura (ITP).

Case 1:

A 67 yo M diagnosed with ITP in 1999 presented on 8/17/2013 with bruising and a platelet count of 5 x 103/ml. No response was seen to prednisone 125 mg QD. He subsequently received a course of IV Ig and IV methylprednisolone sodium succinate, but platelet count remained under 5 x 103/ml on 9/13/13. He was prescribed CPLE 3,000 mg QD on 9/30/13. Platelets increased to 148 x 103/ml by 10/8/13 and remained elevated above 250 x 103/ml for five months.

Case 2:

A 21 yo F diagnosed with ITP in 2011 underwent splenectomy on 11/7/13. Despite this intervention, platelet count decreased to 95 x 103/ml on 4/22/14. She was prescribed CPLE 3,000 mg QD as a monotherapy and by 5/8/14, platelet count increased to 156 x 103/ml. CPLE was discontinued and platelet count remained in normal range at 146 x 103/ml on 5/15/14.

Case 3:

A 56 yo M diagnosed with ITP in 2011 was referred for splenectomy on 9/19/12 after his platelet count dropped below 40 x 103/ml despite corticosteroid therapy. Prior to surgery, on 9/30/12, he initiated CPLE 3,000 mg QD in addition to prednisone 10 mg QD. On 11/7/12, pre-surgical labs revealed a platelet count of 115 x 103/ml and splenectomy was cancelled due to adequate platelet count. Platelets remained elevated over threshold for surgery for over four months.

Case 4:

A 60 yo F diagnosed with ITP in 1992 was referred for splenectomy and refused the procedure on 6/5/13. The patient's platelet count was 86 x 103/ml while on prednisone 10 mg QD and she was prescribed CPLE 3,000 mg QD. Her platelet count increased to 109 x 103/ml on 5/12/13 but subsequently dropped to 98 x 103/ml on 5/20/13. CPLE was discontinued at this time due to lack of beneficial action on platelet count.

Conclusion:

CPLE may prove beneficial in the management of refractory ITP for patients interested in alternative therapy before progressing to second-line treatments. Based on the association observed in these cases, a larger clinical trial is warranted to evaluate CPLE as an adjunctive therapy in chronic ITP.

Disclosures

No relevant conflicts of interest to declare.
Cyclic Thrombocytopenia: A Case Report
Authors: Fontenele Leila P. S. , Santucci Rodrigo , Centrone Renato , Aranha Milton A. F. , Bellesso Marcelo , Dias Daniela Ferreira
Published: October 2019

Cyclic thrombocytopenia (CTP) is a very rare disorder characterized by periodic fluctuations in platelet counts, typically resulting in episodes of thrombocytopenia alternating with normal platelet counts. Most cases are idiopathic while some are associated with a primary hematologic disease. Frequently CTP patients are misdiagnosed as idiopathic thrombocytopenic purpura (ITP), but usually they do not respond to standard ITP treatment.

A 49 year-old male presented a history of one year of symptomatic thrombocytopenia (<10x10(9)). He had been treated initially with corticosteroids without a consistent response. Later, he was subjected to splenectomy. After splenectomy platelet counts was checked weekly. On the first week the cont was 1.000x10(9)/L falling on the third week to 27x10(9)/L. As the patient had no bleeding we decided for no treatment, and platelet counts had been fluctuating from 800x10(9)/L to 20x10(9)/L about every four weeks, for three months of monitoring. No treatment was further required.

When evaluating patients with suspected ITP, CTP should always be included in the differential diagnosis, especially if they are not responding to standard ITP treatment.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Identification of Novel Mutations with Molecular Modelling of Missense Mutations of Congenital Afibrinogenemia Patients in Pakistan
Authors: Khan Tehmina nafees sonia , Naz Arshi , Biswas Arjit , Goodeve Anne , Ahmed Nisar , Ahmed Shariq , Shamsi Tahir , Oldenburg Johannes
Published: October 2019

Background:

Congenital afibrinogenemia (OMIM #202400) is a rare coagulation disorder which was first described in 1920. It is transmitted as an autosomal recessive trait characterized by absent levels of fibrinogen (factor I) in plasma. Consanguinity in Pakistan and its neighboring countries has resulted in higher cases of congenital fibrinogen deficiency in their respective populations.

Aims: This study focuses on the detection of mutations in the fibrinogen genes by DNA sequencing and molecular modeling of missense mutations in all three genes (Fibrinogen gene alpha (FGA), beta (FGB) and gamma (FGG) in Pakistani patients.

Methods:

This descriptive and cross sectional study was conducted in Karachi and Lahore and fully complied with the Declaration of Helsinki. Patients with fibrinogen deficiency (tested by Fibrinogen functional assay from Laboratoire Stago, Asnieres, France) were screened for mutations in the Fibrinogen gene alpha (FGA), beta (FGB) and gamma (FGG) genes by direct sequencing. Molecular modeling was performed to predict the putative structure functional impact of the missense mutations identified in this study

Results:

Ten patients had FGA mutations, four have FGB mutations and one mutation was detected in FGG. Thirteen of these mutations were novel. The missense mutations are predicted to result in loss of stability since they (a) break ordered regions (b) cause clashes in the hydrophobic core of the protein.

Conclusion:

Congenital afibrinogenemia is a rapid growing problem in countries such as Pakistan where consanguinity is frequently practiced. This study illustrates the fact that mutations in FGA are relatively more common in our population than those in FGB where as FGG mutations appear rarer.

Disclosures

Oldenburg: SOBI: Consultancy.
Molecular Characterization of F8 Secreting Cell
Authors: El-Maarri Osman , Jamil Muhammad Ahmer , Pezeshkpoor Behnaz , Nüsgen Nicole , Hofmann Andrea , Hoffmann Per , Heilmann Stefanie , Nöthen Markus M , Frohlich Holger , Oldenburg Johannes
Published: October 2019

Different endothelial cells share common features but also acquire organ specificity: for example the liver sinusoidal endothelial cells (LSECs) are major site of F8 secretion as compared to other endothelial cells. To decipher this specificity in LSECs and to understand the molecular mechanism of F8 synthesis and secretion, we have compared genome wide expression and epigenetic data of fetal LSECs (N=3) and non-liver endothelial cells (HCMEC, HPAEC, HPMEC and HUVEC; N=3), hepatocytes (N=3) and adult total blood (N=3). At a false discovery rate of <5% we found 4134 differentially methylated CpG sites and 663 differentially expressed probes (corresponding to 623 genes/loci) between LSECs and other endothelial cells. The loci were found by ingenuity pathway analysis (IPA) to be enriched in EIF2, eIF4, p7056K and mTOR signaling pathways together with LXR/RXR activation pathway. On the other hand, only 21 different loci and 152 CpGs were found to be statistically different (at <5% FDR) between hepatocytes and LSECs. According to IPA these were found enriched in interferon signaling, activation of IRF by cytosolic pattern recognition receptors and role of pattern recognition receptor in recognition of bacteria and viruses. Additionally, gene ontology analysis showed enrichment in GO-terms such as regulation of cellular metabolic process for LSECs vs. endothelial cells and blood vessel development, cell adhesion and regulation of cell migration for LSECs vs. hepatocytes. Moreover, in each of the GO sub categories of general coagulation (GO:0007596) and transcription factors (GO:003700) a small number of loci successfully differentially identify the LSECs from other endothelial cells. The above described results show specific molecular signature that characterize the F8 secreting cells and highlight specific cellular pathways associated with this process.

Disclosures

Oldenburg: SOBI: Consultancy.
Factor VII Deficiency in the Negev - a 10 Years' Experience of One Tertiary Center
Authors: Fruchtman Yariv , Ben harosh Miri , Kapelushnik Joseph , Mazar Julia , Kenet Gili , Rosenberg Nurit
Published: October 2019

Inherited factor VII (FVII) deficiency is the most common among the autosomal recessive rare bleeding disorders, with an estimated prevalence of 1:300,000 in European countries. Affected individuals display a wide range of clinical phenotypes, ranging from mild non spontaneous bleeding to life threatening (i.e. central nervous system[CNS] bleeding, gastrointestinal [GI] bleeding or haemarthrosis), whereas up to one-third of individuals with a FVII deficiency are asymptomatic and are mainly diagnosed during family studies or after screening for surgery. Unfortunately, the residual activity of FVII does not predict the individual propensity to bleed, and even in individuals with the same mutation, differences in clotting phenotypes can be seen.

As our tertiary center serves a unique population in the Negev, we aimed at studying the prevalence and phenotype of FVII deficiency within the last decade.

Methods: We searched all electronic records for the last 10 years depicting rare bleeding disorders by ICD 9 code - 2863 and compared them to the hematologic record of factor VII deficiency depicted in our lab - 50% or less activity. Patients with any record of genetic diagnosis, were compared with clinical findings.

Results: The population in the Negev is estimated as 700000 people

Most of them are Jewish and 150000 of them are Arab-Bedouins. We found 800 records of rare bleeding disorders (ICD 9-2863), Including 200 with FVII deficiency - 100/200 had FVII levels below 50%.

Most (90%) of cases were of Jewish origin (mostly oriental Jews) and only 10% were Arab- Bedouins. Forty patients were asymptomatic with 50-30% FVII activity and 20 patients with 30-10% FVII activity were either asymptomatic or presented with mild bleeding diathesis. Out of 23 cases with lower than 10% FVII activity, 7 were symptomatic and suffered severe life threatening bleedings (2 infant died of perinatal ICH. Five families (3 Bedouin and 2 oriental Jews) were identified with severe FVII deficiencies. The 4 Bedouin patients were identified to be homozygous to unique mutation. Interestingly, most medical records depicted FVII deficiency were of women studies due to fertility problems.

Conclusions: The prevalence of FVII deficiency depicted in the Negev is much higher in comparison to literature reports (200/700000) Severe FVII deficiency was found in 23: 700000, consistent with 1: 30000 prevalence. As patients are highly variable, in order to "tailor" treatments according to disease severity, new directions should be pursued to identify those with the most severe phenotypes.

Disclosures

Kenet: Bayer, Novo Nordisk: Other: Advisory Boards, Speakers Bureau; Opko Biologics: Consultancy, Other: Advisory Boards; BPL; Baxelta: Research Funding; Pfizer: Honoraria.