bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Role of Mel18 Overlaps with BMI1 In the Self-Renewal of Human Hematopoietic Stem Cells.
Authors: Reyal Yasmin , Bonnet Dominique 
Published: October 2019

Abstract
Abstract 2619
Complex mechanisms regulate the ability of hematopoietic stem cells (HSCs) to self-renew, some of which may be exploited by leukemic stem cells. BMI1, a member of the polycomb group (PcG) proteins is known to be a positive regulator of this process, largely by repressing the p16/INK4a locus. However the role of other PcG proteins is unclear. We initially screened HSCs and progenitor populations from umbilical cord blood (CB), for the expression patterns of a number of PcG genes. Levels of expression were heterogeneous, indicating that there may be different roles for different PcG in HSCs versus progenitors. As a starting point we have focused on Mel18 (PCGF2) as it has been suggested in murine hematopoiesis that it acts to counteract BMI1. Lineage negative CB cells were transduced with lentiviral vectors expressing shRNA against Mel18, BMI1 and a control sequence. Specific knockdown by these constructs was confirmed at the RNA level to be at least 80% for both genes and was verified at the protein level by Western blot. Our data indicates that knockdown of Mel18 impairs the proliferation of primitive cord blood cells in both stromal-dependent and -independent culture, in a similar manner to BMI1. Furthermore Mel18 deficiency impedes both primary and secondary colony formation of all myeloid lineages in methylcellulose. These findings have been confirmed in vivo with significant reduction in engraftment of CB lineage negative cells in NOD/SCID mice at twelve weeks. We are investigating whether over expression of Mel18 can rescue BMI1 deficient cells to establish if these homologous genes have redundant functions. The data so far suggests that BMI1 is not unique in its role in HSC self - renewal, and Mel18 may share overlapping functions. This highlights a possible difference between human and murine hematopoiesis. Moreover it is likely that other members of the PcG family are also important in human HSCs. It will be of interest to investigate whether like BMI1, they are also implicated in the maintenance of the leukemic stem cell.

Disclosures:
No relevant conflicts of interest to declare.
Tumor Microenvironment In Pediatric Hodgkin Lymphoma: Clues for Disease Biology
Authors: Henry Meret , Poulik Janet , Buck Steven , Ravindranath Yaddanapudi , Savasan Sureyya 
Published: October 2019

Abstract
Abstract 2681
The non-malignant elements in Hodgkin lymphoma (HL) tissue are very prominent and represent a reactive process either directly against or induced by tumor cells. Thus far, only clinical characteristics have been used in the risk assessment of HL. Biologic markers of disease remain largely unidentified. This study examined cellular immunologic features of the tumor microenvironment in pediatric HL patients seen at a single children's hospital since 1997 for whom flow cytometric evaluation at diagnosis was available. Retrospective chart review was conducted to gather other relevant clinical and laboratory data on the cases. Student t-test, nonparametric Mann-Whitney U and Kruskal-Wallis tests, and Pearson's correlation analysis were used for statistical evaluation. Thirty-one patients, 14 male and 17 female, with a median age of 14 years (range 3–17) were included in the study. Twenty-one cases were nodular sclerosis (NS) [71% female, with a median age of 14 years], 6 mixed cellular (MC) [17% female, with a median age of 11 years] and 4 undetermined subtype. While T cells were predominant in nodular sclerosis (NS), the infiltrate was B cell-rich in mixed cellular (MC) subtype. CD4+ T cells were significantly higher in NS (55 vs. 30.5; p=0.01) and, conversely, CD8+ cells in MC (21.3 vs. 11.3; p=0.02). Consistently, CD4/CD8 ratio was significantly higher in NS (9.6 vs. 1.6; p=0.02). All of the MC tumors were in situ EBV-positive, compared to only 24% of NS tumors (p=0.008). The WBC and absolute lymphocyte count were higher in NS compared with MC, 12×109/L vs. 6.1×109/L and 3.9×109/L vs. 1.5×109/L, respectively. Occurrence of primary continuous complete remission (CCR) without recurrence or refractoriness was significantly higher in MC group (p=0.009). Higher hemoglobin, DR+CD38+ lymphocyte percentage and lower CD4/CD8 ratio were also predictive of primary CCR status (p<0.05) in the entire study group. In the NS group, higher DR+CD38+ lymphocyte percentage was again associated with primary CCR (p=0.004). In this group, there was also an association between primary CCR and higher hemoglobin level (p=0.053). While DR+CD38+ lymphocyte percentage negatively correlated with CD4/CD8 ratio, there was positive correlation with WBC. Finally, the immunophenotype characteristics of the EBV-positive NS cases were intermediate to the MC group and EBV-negative NS cases. Albeit a small series, these results point to striking tumor microenvironment differences between NS and MC subtypes and EBV status-associated patterns. Moreover, DR+CD38+ lymphocyte proportion and CD4/CD8 ratio appear to be associated with the risk status of childhood HL, suggesting their potential role as prognostic biologic factors.

Disclosures:
No relevant conflicts of interest to declare.
Comparison of IV Busulfan and Fludarabine Vs Oral Busulfan Plus Cyclophosphamide As Myeloablative Conditioning Regimens for Acute Leukemias and Myelodisplastic Syndromes
Authors: Jaimovich Gregorio , Requejo Alejandro , Milovic Vera , Escobar Nicolas Fernandez , Drelichman Guillermo , Real Juan , Brioschi Sandra , Feldman Leonardo Julio 
Published: October 2019

Abstract
Abstract 4575

Introduction:
The Busulfan Cyclophosphamide (Bu-Cy) combination has been broadly used as a myeloablative conditioning regimen for allogeneic hematopoietic cell transplantation for patients (pts) affected by myeloid malignancies. The Busulfan Fludarabine (Bu-Flu), a newly introduced mieloablative conditioning regimen association showed, in several reports, less regimen related toxicities. We compared in a retrospective study the effectiveness and toxicity of both regimens.

Patients and methods:
From 12/2002 to 12/2010, 59 pts. affected by AML, CML, MDS and ALL were included in the study. Forty two pts. received Bu-Cy (oral Bu 1 mg/kg, every 6 hours × 16doses days -8 to -5 followed by Cy 60 mg/kg per day, days -4 and -3) and 17 pts. received Bu-Flu (Bu IV 3.2 mg/kg, once daily for four days and Flu 40 mg/kg /daily for four days, days – 6 to -3). There were no statistical significant differences between both groups comparing age, diagnosis, disease status and CD34+ cells infused. Regarding the cell source there were more patients in the Bu-Flu group that received peripheral blood stem cells and cord blood and more pts in the Bu-Cy were infused with bone marrow. Another imbalance was the use of more unrelated donors in the Bu-Flu arm. GVHD prophylaxis was with cyclosporine A or tacrolimus and metotrexate. Protective isolation and antibiotic prophylaxis was used in both groups.

Results:
Considering hematopoietic recovery, there were no differences in days to reach 500 granulocytes and 20.000 platelets. Transfusion requirements were without statistical significant differences. The following observations were statistically significant (p<0.001) favoring Bu-Flu: Pts receiving Bu-Flu required less hospital stay days (27,8 vs 35,6) and less days on antibiotics (8,5 vs 16,5). Acute GVHD incidence grade II to IV was 23,5% in the Bu-Flu arm vs 50% in the Bu-Cy group. Early TRM was 5,9% with Bu-Flu vs 16,6% with Bu-Cy. Overall survival and relapse rate at 1 year of follow up was 70.58% vs 50% and 23.5 % vs 30.9% for Bu-Flu and Bu-Cy respectively.

Conclusions:
In our experience Bu-Flu is associated with lower toxicity, neutropenia duration, and severe acute GVHD. The overall survival and relapse rate are also superior. More pts should be included in a prospective study to confirm these observations.

Disclosures:
No relevant conflicts of interest to declare.
Risk Factors of HHV6 Reactivation and Relationship Between Syndrome of Inappropriate Antidiuretic Hormone Secretion After Stem Cell Transplantation in Pediatric Patients
Authors: Kobayashi Ryoji , Toriumi Naohisa , Yoshida Makoto , Iguchi Akihiro , Sarashina Takeo , Suzuki Daisuke , Sano Hirozumi , Ogata Masao 
Published: October 2019

Abstract
Abstract 4580
Many reports were seen about reactivation of human herpes virus 6 (HHV6) after stem cell transplantation (SCT) in adult patients, and this reactivation sometimes induce severe condition of patients. However, few reports were seen about pediatric patients. Therefore, we examined HHV 6 reactivation after stem cell transplantation in patients with children, retrospectively. The cases were 80 patients, 48 male, 32 female, and the median age was 6 years old (range 0–20 years old). Transplantations were 23 related bone marrow transplantations (BMT) or peripheral blood stem cell transplantations (PBSCT), 18 unrelated BMT, one related cord blood transplantation (CBT), 31 unrelated CBT, and seven autologous BMT or PBSCT. We analyzed HHV6 DNA samples of serum with these patients before SCT, 20 days and 40 days after SCT using PCR method. In addition, we analyzed relationship between HHV6 reactivation and syndrome of inappropriate antidiuretic hormone secretion (SIADH).
In samples of 20 days after SCT, 35.0% of samples were positive for HHV6 DNA. On the other hand, 2.5% and 5.0% were positive before SCT and 40 days after SCT, respectively. From 24 out of 28 samples, over 10E3 of HHV6 DNA were detected in positive samples. Factors associated with HHV6 reactivation were CBT, unrelated donor, malignant diseases, use of total body irradiation as conditioning, cyclosporine and methyl prednisolone as GVHD prophylaxis, acute GVHD (>grade 2), chronic GVHD and use of steroid using univariate analysis. Moreover, CBT was an only risk factor of HHV6 reactivation using multivariate analysis.
In 14 patients with SIADH, 78.6% of patients had HHV6 reactivation. On the other hand, 25.8% of patients had HHV6 reactivation in 66 patients without SIADH. This result was statistically significant (p<0.001).
From this analysis, we can understand HHV6 reactivation was seen in many patients with children. In addition, we thought about the possibility of SIADH is one symptom of the encephalopathy by the HHV6 reactivation.

Disclosures:
No relevant conflicts of interest to declare.
The Biological Role of BAG3 in Human Chronic Lymphatic Leukemia
Authors: Zhu Huayuan , Liu Peng , Li Jianyong 
Published: October 2019

Abstract
Abstract 4589

Objective:
BAG3, a member of BAG family, is shown to sustain cell survival and underlies resistance to chemotherapy in human cancer cells, through down-modulation of apoptosis. It can also enhance cell adhesion and migration to promote tumoral invasion. However, the role of BAG3 in human chronic lymphatic leukemia (CLL) remains unclear. Our study aims to discover the roles of BAG3 in the apoptosis and migration of primary CLL cells.

Method:
In our study, Peripheral blood mononuclear cells were freshly isolated from 18 previously untreated patients. The primary CLL cells were cultured for 6 hours, following, three siRNA (siRNA-1, siRNA-2 and siRNA-3) targeting with different regions of BAG3 mRNA and one non-targeting siRNA were transfected into CLL cells by using Lipofectamine 2000. 6 samples were collected for checking the transfection efficiency by real-time PCR and western blot after 24 or 48 hours. The other 6 samples were exposured with or without fludarabine 12 hours later after tansfection; with another 24 or 48 hours, both cells were harvested, stained with annexin V/PI,and then apoptosis were analyzed using a FACSCalibur flow cytometer.on the other hand, transwell assay were performed in another 6 samples 12 hours after tansfection. to analysis cell migration, the cells were collected and counted 24 hours later.

Result:
By using the siRNA technique, we successfully down-regulated the expression of BAG3 by 3 different siRNAs. BAG3 mRNA was decreased □‘6 folds (p < 0.05). In order to analyze the effect of BAG3 on CLL cell apoptosis, we used flow cytometry to measure cell apoptosis. Here, the apoptosis rates of CLL cells were increased □‘2 folds after 24 hours transfection by siRNA-2 and siRNA-3 (p < 0.05), and □‘2.5 folds after 48 hours transfection by BAG3 siRNA-1, siRNA-2 and siRNA-3 (p < 0.05). These indicate that BAG3 makes an anti-apoptotic, and knocking-down of it increases the cell apoptosis. To assess the role of BAG3 in the migration of CLL, we used transwell assay to measure cell migration. We found that the migration of CLL cells was decreased 28% by BAG3 siRNA-1 treatment (p < 0.05), 47% by siRNA-2 (p < 0.01) and 50% by siRNA-3 (p < 0.01), respectively. These data showed that BAG3 enhances the CLL migration, and down-regulation of it can inhibit cell migration. Furthermore, fludarabine and BAG3 siRNA were co-treated with CLL cells, to check whether knocking-down of BAG3 can reverse the CLL cells’ resistance against fludarabine. However, the apoptosis rates are not different between co-treated cells and fludarabine-treated cells, which indicated knocking-down of BAG3 may not reverse the fludarabine resistance, though it can promote cell apoptosis and inhibit cell migration.
Conclusion: Based on these observations, we conclude that BAG3 may be a potential therapeutic target of human CLL, and inhibition of BAG3 expression can induce cell apoptosis and inhibit cell migration.

Disclosures:
No relevant conflicts of interest to declare.
Inhibition of NOTCH Activity by GLIOTOXIN Induces Apoptosis in CLL Cells and Overcomes the Supportive Effect of Primary Bone Marrow Stromal Cells
Authors: Hubmann Rainer , Hilgarth Martin , Schnabl Susanne , Ponath Elena , Demirtas Dita , Reiter Marlies , Zielinski Christoph , Jäger Ulrich , Shehata Medhat 
Published: October 2019

Abstract
Abstract 4609
Active NOTCH signaling is observed in an increasing number of human neoplasias including chronic lymphocytic leukemia (CLL) and represents a potential therapeutic target. We have recently reported that the γ-secretase inhibitor DAPT may not be effective in all cases of CLL (Hubmann et al; BJH 2010). The aim of this study was to evaluate the therapeutic value of a newly identified NOTCH transactivation inhibitor (gliotoxin) and to compare its efficiency with the highly selective γ-secretase inhibitor (GSI) DAPT in CLL cells.
Electrophoretic mobility shift assays (EMSA) showed that gliotoxin completely blocked the formation of DNA-bound NOTCH2 in CLL cells independent of their sensitivity to DAPT. The inhibition of NOTCH2 signaling by gliotoxin was associated with down regulation of its target gene CD23 and induction of apoptosis. The IC50 of Gliotoxin was variable between patients (n=20) and ranged between 50–100 nM irrespective to their sensitivity to GSI. Short term (4 hours) exposure of CLL cells to gliotoxin revealed that gliotoxin modulates the mRNA expression of several NOTCH-related genes including JAG1, PRKCD (PKC-δ), and NR4A1. We (Shehata et al; BLOOD 2010) and others have reported on the supportive effect of primary bone marrow stromal cells (BMSC) for CLL cells. Therefore, we tested whether gliotoxin may overcome this supportive effect. FACS analysis and MTT assays showed that gliotoxin abolished the supportive effect of BMSC under co-culture conditions. The effect of gliotoxin was dose dependent and selectively induced apoptosis in CLL cells accompanied by down regulation of NOTCH2 and CD23 transcription. Western blotting analysis demonstrated that gliotoxin also decreased the phosphorylation of Akt-pSer473 suggesting a link to PI3-K signaling. NOTCH1 has been recently shown to be affected by “gain of function” mutations in a subset of CLL patients. We observed that co-culture of CLL cells with BMSC was associated with increased NOTCH1 mRNA expression which could be decreased upon exposure to gliotoxin. In addition, gliotoxin inhibited DNA-bound NOTCH1 complexes in the T-ALL cell line SupT1 which is known to express high NOTCH1 activity. This indicates that gliotoxin may target both NOTCH1 and NOTCH2 isoforms.
In conclusion, the data show that gliotoxin effectively targets NOTCH activity in CLL cells in a mechanism which is independent of γ-secretase. Thus, gliotoxin might have a beneficial effect in a wider range of CLL patients and warrants further evaluation.

Disclosures:
No relevant conflicts of interest to declare.
β-Arrestin-1 Regulates Epigenetics of Whole Genome in Chronic Myeloid Leukemia Cells
Authors: Zou Lin , Li Kang , Zhou Xiaoyan , Bao Liming 
Published: October 2019

Abstract
Abstract 4636
Chronic myeloid leukemia (CML) is a clonal myeloproliferative expansion of transformed, primitive hematopoietic progenitor cells, which is an incidence of 1 to 2 cases per 100,000 people per year and accounts for 15 percent of adult leukemias. Although the-philadelphia (Ph) chromosome and the BCR-ABL fusion gene are known as the featured molecular characteristic, the pathogenetic mechanism of CML is still not fully clear. In recent years, epigenetics, the heritable changes of gene expression without change in DNA sequence, has rapid development in the area of regulating gene transcription and expression. The epigenetic modification includes the types of DNA methylation, histone acetylation, protein phosphorylation, ubiquitination, chromatin remodeling, and so on. β-arrestin1, one key member of arrestin family that are multifunctional scaffold proteins, was reported to regulate histone acetylation and gene transcription in GPCR signaling. However, little is known about the role of β-arrestin1 in mediating the whole genome epigenetics, especially in CML. Here, we firstly knocked down β-arrestin1 expression in K562 cells, the typical cell lines for CML, and then measured and analyzed the alterations of whole genome epigenetics by combinedly using chromatin immunoprecipitation(ChIP)-on-chip and methylation chip. The data from ChIP-on-chip showed that β-arrestin-1 mediated 7498 genes histone H4 acetylation, mainly afffected the genes of cell part, cell vability and prolieration when β-arrestin1 was knocked down in CML cells. Clustering analysis showed that these affected genes of histone H4 acetylation were involved in MAPK signaling. Moreover, the data from methylation chip indicated that the methylative level of the whole genome were decreased, and genome hypermethylated was more obvious than hypomethylated, when β-arrestin1 knockout. Further analysis found that the differences of CpG island number primarily located in 500–1000bp promoter region. And methylative modifications of influenced CpG island number easily occurred in chromosome 1, 4, 13, 14, 19 and X, compared with other chromosomes. Most interestingly, the distinct changes of acetylation and methylation for some cell part genes (ZNF259, IL23A, COPS6 and COASY) were simultaneously regulated by β-arrestin1 with GO analysis, which could shift the epigenetic alterations by β-arrestin1 in CML cells. These common genes both involved in these two kinds of epigenetics, and function analysis are further study. Taken together, our study shed new light on β-arrestin1 in regulating epigenetics of whole genome in chronic myeloid leukemia cells.

Disclosures:
No relevant conflicts of interest to declare.
Rituximab Has Similar Efficacy in Treatment of Acute Refractory or Chronic Relapsing Idiopathic Thrombotic Thrombocytopenic Purpura (TTP) and Refractory Secondary TTP: A Systematic Review
Authors: Tun Nay Min , Villani Gina M 
Published: October 2019

Abstract
Abstract 4673

Background:
Thrombotic thrombocytopenic purpura (TTP) is a potentially life-threatening disorder. Secondary TTP was known to be more resistant to plasma exchange and have higher mortality. Limited clinical data suggest that rituximab may be effective in the treatment of acute refractory or chronic relapsing nonfamilial idiopathic TTP (ITTP) as well as refractory secondary TTP (STTP). We performed a systematic review to evaluate the comparative efficacy of rituximab in ITTP and STTP.

Method:
An electronic literature search of Medline, CINAHL and the Cochrane library databases using the terms “rituximab” and “thrombotic” yielded 179 articles from March 2002 to June 2011. Studies were included if they met the following criteria: TTP in a first episode or subsequent relapse and TTP refractory to plasma exchange. Exclusion criteria were prophylactic administration of rituximab to patients in remission and familial TTP. Secondary TTP cases were author-defined. Case series without extractable individual data were excluded. Two-Tailed Fisher Exact Test was used to investigate the difference in complete remission rates between ITTP and STTP. Independent-Samples Mann-Whitney U Test was applied to detect the difference in platelet recovery days.

Result:
20 case series and 33 case reports including a small prospective study were eligible. Individual data of 139 patients from Europe, Asia, Australia, South and North America were analyzed. Overall, median age was 40 years (range: 1–72). Male constituted 30% and female 70%. Complete remission rate was 88% (90 out of 102) and 84% (31 out of 37) for ITTP and STTP respectively. There was no statistically significant difference in complete remission rates between the two groups (Two-Tailed Fisher Exact Test p value = 0.569). Median follow-up time was 12 months. Median platelet recovery (more than 150×109/L) from the first dose of rituximab was 8 days (range: 2–74) and 19 days (range: 7–90) for ITTP and STTP respectively. The distribution of platelet recovery days was the same between ITTP and STTP (Independent-Samples Mann-Whitney U Test p value = 0.132).

Conclusion:
Rituximab has the same efficacy in both STTP and ITTP in terms of response rate and platelet recovery days. It appears to be effective and well-tolerated. We recommend the use of rituximab as a salvage therapeutic modality in refractory acute or chronic relapsing nonfamilial idiopathic as well as secondary TTP.

Disclosures:
No relevant conflicts of interest to declare.