bluuebunny/crossref_metadata_2025_split huggingface dataset:
ZNF802 (JAZF1), a Possible New Therapeutic Target for Treatment of β-Thalassemia
Authors: Wongborisuth Chokdee , Tangprasittipap Amornrat , Anurathapan Usanarat , Sathirapongsasuti Nuankanya , Sripichai Orapan , Songdej Duantida , Hongeng Suradej 
Published: November 2019

β-Thalassemias are groups of inherited blood disorders caused by reduced or absent production of β-globin chains of adult hemoglobin (HbA; α2β2). The severely affected patients with β-thalassemia require lifelong blood transfusion and chelation therapy. Reactivation of fetal hemoglobin (HbF; α2γ2) could ameliorate the symptoms of patients via increased total hemoglobin production and reduced imbalanced α- to non-α-globin synthesis. Hemoglobin switching during human development requires activities of several transcription factor proteins, including those bind to the globin gene promoters. However, the regulation of fetal to adult hemoglobin switching is not fully understood. Previously, we have carried out the microarray analysis comparing human erythroblasts derived from therapeutic aborted fetal liver and adult peripheral blood. The results showed that ZNF802 mRNA is among the transcripts that up-regulated in adult erythroblast of greater than 2-fold change. ZNF802, also called JAZF1, is a 27 kDa protein containing two zinc finger motifs that acting as transcriptional repressor by binding to the direct repeat (DR) elements on DNA. In this study, quantitative RT-PCR was performed in order to validate the microarray data. We observed 13-fold up-regulation of ZNF802 in adult erythroid cells compared to those cells derived from fetal liver. To further elucidate the role of ZNF802 in reduction of HbF expression in adult erythroids, the lentivirus carrying ZNF802 specific shRNA and non-targeted shRNA were designed and constructed for transduction in erythroblasts. Hematopoietic CD34+ progenitor cells were isolated from β-thalassemia/HbE patients (n=9) and subjected to ex vivo two-phase liquid erythroid culture. The results showed the reduction of greater than 90% of ZNF802 transcript and nearly undetectable level of ZNF802 protein in cells transduced with the ZNF802 shRNA lentivirus. As expected, the level of γ-globin transcript and levels of HbF were increased in ZNF802 knockdown erythroid cells in β-thalassemia/HbE (5-fold changed and HbF 26.9±7.4% vs 18.5±6.8% in non-targeted shRNA (p-value = 0.04), respectively). Interestingly, we found up-regulation of the embryonic ε-globin and ζ-globin transcripts in knockdown ZNF802 cells compared to non-targeted shRNA control (15-fold and 10-fold changed (p-value = 0.01), respectively). To note, the diminishing of ZNF802 effect on the proliferation and differentiation pattern of erythriod cells was not found. Altogether, our findings suggested that ZNF802 may play a role as the transcriptional repressor of γ-globin gene in human adult erythroid cells, thus served as a possible new therapeutic target for treatment of β-thalassemia. The molecular mechanisms underlying the ZNF802 mediated HbF reduction are underway.

Disclosures
No relevant conflicts of interest to declare.
Haploidentical Hematopoietic Stem Cell Transplantation May Improve Prognosis in Non-Infant Children with t(v;11q23)/MLL-Rearranged B-Acute Lymphoblastic Leukemia
Authors: Bai Lu , Cheng Yi-Fei , Lu Ai-Dong , Suo Pan , Wang Yu , Zuo Ying-Xi , Yan Chen-Hua , Wu Jun , Jia Yue-Ping , Sun Yu-Qian , Chen Yu-Hong , Chen Huan , Liu Kai-yan , Han Wei , Xu Lan-Ping , Wang Jing-Bo , Tang Xiang-Feng , Chen Hui-Ren , Zhang Le-Ping , Huang Xiao-Jun 
Published: November 2019

Background: B-acute lymphoblastic leukemia (B-ALL) with t(v;11q23)/MLL-rearrangement (MLL-r) in children (1 year or older) is rare, and its outcome and optimal treatment options remain controversial. This study aimed to analyze the clinical characteristics and outcomes of these patients, and to explore the role of allogeneic hematopoietic stem cell transplantation (allo-HSCT), especially haploidentical HSCT (haplo-HSCT) in the treatment of these patients.
Methods: At the time of the last follow-up (July 1, 2019), we retrospectively analyzed clinical data of 42 non-infant children with t(v;11q23)/MLL-r B-ALL. Comparison of outcomes was made between patients received allo-HSCT in the first complete remission (CR1) and chemotherapy only.
Results: The median follow-up was 41 (1-106) months. The median age at diagnosis was 4.5(1-14) years and the median leukocyte count was 56.0 (2.2-735.2)×109/L. One was excluded for death during induction. For the remaining 41 patients, the complete remission rate after induction therapy was 40/41 (97.6%), the estimated 4-year probabilities of overall survival (OS), event-free survival (EFS) and cumulative incidence of relapse (CIR) were 59.7%, 51.6% and 43.4%, respectively. 19 patients received allo-HSCT in CR1 (HSCT in CR1 cohort), notably, 18/19 cases in this cohort received haplo-HSCT, and the remaining 22 patients continued the consolidation therapy (Non-HSCT in CR1 cohort). The estimated 4-year probabilities of OS, EFS and CIR in the HSCT in CR1 cohort were 86.6%, 89.2% and 5.3%, respectively. Meanwhile, the estimated 4-year probabilities of OS, EFS and CIR in the Non-HSCT in CR1 cohort were 37.5%, 19.9% and 75.6%, respectively. They were considered to be statistically significant. Of the 17 patients who relapsed during consolidation chemotherapy, 9 patients who underwent chemotherapy only (Non-HSCT after relapse cohort) all died within 44 months. For the remaining 8 patients who chose allo-HSCT (HSCT in CR2 cohort) when they achieved the second complete remission (CR2), the estimated 4-year probability of OS was 47.6% (P=0.002). Multivariate analysis showed that HSCT in CR1 was the only independent protective factor for OS, EFS and CIR, and age at diagnosis (≥10 years) was an independent risk factor of OS.
Conclusions: Allo-HSCT (especially haplo-HSCT) in CR1 may reduce the risk of relapse and improve prognosis in non-infant children with MLL-r B-ALL. In addition, allo-HSCT also seemed to be an effective approach to improve the prognosis of relapsed patients. Thus, haplo-HSCT could be an alternative approach for non-infant children with MLL-r B-ALL.

Disclosures
No relevant conflicts of interest to declare.
Genetic Variants of ATM & Non-Homologous End Joining Pathway Genes in Acute Myeloid Leukemia: A South India Case-Control Study
Authors: Vuree Sugunakar , Cingeetham Anuradha , Nageswara Rao Dunna , Gorre Manjula , Sinha Sudha , Digumarthi Raghunadharao , Satti Vishnupriya , Gundeti Sadashivudu 
Published: November 2019

Purpose of the study:
Deregulated DNA repair is one of the hallmarks of cancers including Acute Myeloid Leukemia (AML), as it results in genomic instability. ATM gene functions as a sensor, activates cascade of events leading to stimulation of multiple DNA damage- responsive signaling pathways. Principal DNA repair mechanism activated in the hematopoietic stem cells is the Non Homologous End Joining (NHEJ) pathway. However, this pathway was shown to be error prone. Functional SNPs in the genes involved in DNA repair might influence the gene expression leading to altered DNA repair which might confer the risk to AML.
Materials & Methods:
This hospital-based case-control study included 225 AML patients and 326 cancer-free controls from South Indian population. Six polymorphisms of XRCC5, XRCC6, XRCC7 and ATM were genotyped using polymerase chain reaction (PCR)-Restriction Fragment Length Polymorphism (PCR- RFLP) method. Statistical analyses were performed by using SPSS (version20v) and SNPSTAT online tool. Protein-Protein Interaction (PPI) analysis was also done to see the relationship between these genes.
Results:
We found that there was an elevated risk of AML associated with the XRCC5 VNTR 0R repeat and A allele of 2408G>A polymorphism (p-0.04 and p<0.0001 respectively), the frequencies of G allele (p-<0.0001) of XRCC6 -1310C>G and T allele (p-0.003) of ATM -5144A>T polymorphisms were also significantly increased in AML cases. Further, analyses of the variant genotypes with epidemiological and clinical variables revealed a significant association of the risk genotypes with development and progression of AML.
Conclusion: The XRCC5 0R repeat, 2408G>A, XRCC6 -1310 C>G and ATM- 5144A>T polymorphisms, but not XRCC6 -61C>G and XRCC7 6721G>T polymorphisms, play an important role in the pathogenesis of AML.
Figure

Disclosures
No relevant conflicts of interest to declare.
Revealing Homologous Clonality By Synchronous Trisomy 8 in Langerhans CELL Histiocytosis and ACUTE Myeloid Leukemia
Authors: Wang Xinkai , Wang Zhao 
Published: November 2019

Purpose Langerhans cell histiocytosis (LCH) is a disorder in which excess immature Langerhans cells build up in the body. Increasing reports have revealed LCH may share a common origin with myeloid-derived malignancies. Here we report a case of a 45-year-old male, diagnosed as synchronous LCH and acute myeloid leukemia (AML). Relevant tests have been performed to investigate the clonal relationship between LCH and AML. Methods The diagnosis of LCH and AML were based on skin and bone marrow biopsy respectively in Beijing Friendship Hospital, Capital Medical University. No cytotoxic drugs were used before the two diseases were diagnosed. Immunophenotyping was run by immunochemistry or flow cytometry. Karyotype analysis was carried out with G-banding, and the findings were confirmed by fluorescence in situ hybridization (FISH). Mutational analysis of BRAF, KRAS and NRAS genes were performed by ARMS-PCR. Additional gene screening was detected by next-generation sequencing (NGS). Results The case expressed CD1a, S-100, Langerin on the Langerhans cells and CD13, CD34, CD38, CD117, MPO on the leukemic blasts. No other intermingling markers was found on the LCH and AML except CD33 and CD68. Cytogenetic studies showed a karyotype with 47, XY, +8[10], and a confirmatory test revealed the existence of trisomy 8 in both skin and bone marrow lesions by FISH. Cell rate of trisomy 8 on skin, bone marrow and oral mucosa were 9%, 49% and 1%. Mutation analysis revealed KRAS c.436G>A:p.A146T (exon4) in AML and LCH cells, but BRAF c.1799T>A:p.V600E (exon15) only in LCH. Conclusion These findings suggest that LCH cells are derived from myeloid precursors in this case, and BRAF mutation may play an important role in pathogenesis of LCH.

Disclosures
No relevant conflicts of interest to declare.
Defective Autophagy in MPN Were Predominantly Detected in Megakaryocytes in Philadelphia Negative (Ph -) Myeloproliferative Neoplasm
Authors: Wang Jen-Chin , Shi Guanfang , Josan Karan , Ramachandran Preethi , Gotlieb Vladimir , Joseph Gardith , Nasr Sherif A. 
Published: November 2019

We previously reported that autophagy was defective in classical Philadelphia negative (Ph -) Myeloproliferative Neoplasm (MPN) by demonstrating increased p62, decreased Beclin-1 by RT-PCR and Western Blot (WB) methods and decreased LC3-II by WB (ASH annual meeting poster 2018) in peripheral blood mononuclear cells. Now, we further performed immunohistochemical staining of these autophagy markers on the bone marrow biopsy specimens. Methods: Formalin-fixed and paraffin-embedded bone marrow samples were stained with primary antibodies against Beclin-1 (mouse monoclonal, Millipore), LC3B (rabbit monoclonal, Cell Signaling Technology), and p62 (mouse monoclonal, Cell Signaling Technology). The tissue sections were analyzed using VENTANA BenchMark Ultra System (Ventana Medical Systems, Inc.) according to the manufacturer's protocol. Patients who were studied included 12 essential thrombocythemia (ET), 10 polycythemia (PV), and 5 myelofibrosis (MF) (including 1 post-ET MF and 1 post-PV MF). Scoring was given based on the degree of cytoplasmic staining. Score 1 included weak cytoplasmic stain, score 2 included moderate cytoplasmic stain and score 3 was given for strong cytoplasmic staining . Results: 1) As shown in Fig1, the predominant cells which stained positive including p62, Beclin-1 , or LC3 B were mostly found on the megakaryoctes, although very few of other cell types were found to have positive staining as well 2) As shown in Table 1, the immunostaining results were in general correlated to the RT-PCR , or western blot findings that a defective autophagy was demonstrated in MPN patients with combined finding of a stronger staining in p62,and weak staining in Beclin-1 and LC3B. Conclusion: We have demonstrated defective autophagy in these Ph (-) MPN, by immunostaining of the bone marrow specimens, and demonstrating positivity for defective autophagy predominantly in megakaryocytes. Since megakaryocytes are the most important cells involved in the pathogenesis of MPN, we propose that defective autophagy in megakaryocytes play an important role in the pathogenesis of these diseases.

Disclosures
Wang: Incyt: Research Funding.
Modeling of Immune Reconstitution Post CD34 Selected Stem Cell Transplantation in Pediatric Patients with Severe Combined Immune Deficiency
Authors: Diana Jean-Sebastien , Bouazza Naim , Couzin Chloé , Castelle Martin , Magnani Alessandra , Magrin Elisa , Joseph Laure , Delville Marianne , Lefrere Francois , Treluyer jean-Marc , Picard Capucine , Moshous Despina , André Isabelle , Blanche Stephane , Neven Benedicte , Cavazzana Marina 
Published: November 2019

Severe combined immunodeficiencies (SCID) are a heterogeneous group of inherited disorders characterized by a profound reduction or alteration of T lymphocyte function. They arise from a variety of molecular defects which affect T lymphocytes development and function. The number of infections prior hematopietic stem cells tansplantaton (HSCT), genotype, and the type of donor are described as prognostic factors for stem cell transplants.
In this retrospective study, we included 30 pediatric patients suffering from SCID who underwent to CD34+-selected grafts between January 2008 to December 2017 in our center. Diagnosis of reticular dysgenesis, ADA deficiency, or leaky SCIDs and intra thymic deficiency were excluded. A mechanistic mathematical model of all available data was performed and provided a dynamic appreciation of immune reconstitution, while removing bias. T-cell populations were maintained through proliferation and loss model and thymic output have been integrated to the production function. This joint modeling approach aimed to predict rate and extent of T cell immune reconstitution over time (mainly CD3+ T cells, CD3+CD4+ helper T cells, and the CD3+CD4+ CD45RA+ cells).
With a median follow-up time of 97.28 months [range 0.85; 131.54], there were 345 points of T cell phenotyping concentrations in total with a median of 12 samples per patient (range, 0 -35 samples) taken post-transplantation. In this data-set, 13 % of the patients (n= 4) died from infections. Time to reach half of the maximal T cell concentrations was estimated to 3.4 months, 95%CI [2.5 - 4.6]. In covariate analysis, genetic diagnosis (p= 0.0047) and conditioning regimen (p= 0.01) were found to be significant pre transplant covariates which impacted the trajectory of T cell concentration with time.
This modeling approach appeared to be the best method to learn about the dynamic T cell reconstitution after transplant in patients suffering from SCID. In the context of new cell therapy approach for T cell depletion and in vitro thymic maturation, this mechanistic joint model can be used for the design and analysis of incoming clinical trials.
Figure

Disclosures
Cavazzana: Smartimmune: Other: Founder of Smartimmune.
PEDF Can Rescue the Inhibition of Injured Endothelial Cells on Hematopoietic Stem Cell Expansion Ex Vivo
Authors: Zeng Lingyu , Lu Wenyi , Ding Lan , Ju Wen , Qiao Jianlin , Xu Kailin 
Published: November 2019

Introduction: Endothelial cells (ECs) provide a fertile niche for hematopoietic stem cell (HSC) maintenance, differentiation, and migration.Several studies have indicated that bone marrow (BM) vascular niche was impaired after HSC transplantation and severely inhibited hematopoietic reconstruction. Pigment epithelium-derived factor (PEDF) is an important potential cytoprotection and therapeutic agent for injured cells. The direct role of the injured endothelial cells on hematopoietic stem cells and whether PEDF has protective effect in this system remain unknown. This study aims to observe the influence of enjured ECs on HSCs and to explore the role of PEDF in endothelial-HSC coculture system.
Methods: Injury of Endothelial cells by two important preparative regimenconditioning radiation and Busulfan respectively was evaluated with CCK8 assay. The expression of endothelial tight junctions(TJs),adherent junctions related molecules and endothelial to Mesenchymal Transition molecules such as ZO-1, Occludin,VE-cadherin, ICAM, α-SMA, CD31 and VCAM were detected by RT-qPCR, flow cytometry, immunofluorescence and western blot. The effects of injured endothelial cells on HSC self-renewal, differentiation, cell cycle and apoptosis were evaluated by flow cytometry, photography, viable cell count and clone formation assay. Hematopoiesis regulation factors SCF, IL-6, TGF-β and TNF-α were detected by ELISA. The protective effect of PEDF was also explored.
Results: Both radiation and Busulfan could decrease cell viability of endothelial cells. The expression level of ZO-1, Occludin, VE-cadherin, ICAM, CD31 and VCAM were decreased and α-SMA was increased when EC exposed to radiation or Busulfan suggesting endothelial activation, impaired EC permeability and endothelial to Mesenchymal Transition after EC injured. Compared with normal endothelial cells and hematopoietic stem cell co-culture group, the HSC% of injured endothelial cells and hematopoietic stem cells co-cultured group were significantly decreased, the cell colony formation ability was decreased, the proportion of mature cells increased, and the damage of endothelial cells could not maintain the characteristics of HSC, weakened the self-renewal and multidirectional differentiation potential of HSC and promoted the maturation of HSC. After the administration of PEDF, endothelial to Mesenchymal Transition of EC was suppressed and the EC permeability was improved. Most importantly, the proportion of HSC was significantly increased, and the proportion of mature cells decreased in the coculture system.
Conclusion: Injured endothelial cells can inhibit proliferation of hematopoietic stem cells, self-renewal and promote HSC differentiation. PEDF could ameliorate endothelial injury and promote HSC expansion by suppressing endothelial-mesenchymal transition and protecting TJs and AJs.

Disclosures
No relevant conflicts of interest to declare.
A New Step in Understanding of Fanconi Patients Peripheral Stem Cell Harvesting, a Bridge to Gene Therapy
Authors: Diana Jean-Sebastien , Manceau Sandra , Leblanc Thierry , Couzin Chloé , Magnani Alessandra , Magrin Elisa , Bendavid Matthieux , Joseph Laure , Delville Marianne , Blanche Stephane , Soulier Jean , Cavazzana Marina , Lefrere Francois 
Published: November 2019

Fanconi anemia (FA) is an inherited disorder, clinically characterized by congenital abnormalities, a fatal progressive bone marrow failure (BMF), and a predisposition to develop malignancies. Gene therapy by infusion of FA-corrected autologous hematopoietic stem cells (HSCs) may offer a potential alternative cure and to get around the problems of the Hematopoietic stem cell transplantation toxicity or the donor restriction. For gene therapy, an adequate number of HSC collected is a key point to a successful engraftment. However, the HSC collection in FA patients implies particular challenges because of their reduced BM stem cells numbers and implies a theorical risk of an inner depletion in stem cell reserve following collection.The main objective of this pilot study was to evaluate the feasibility and the safety of co-administration of G-CSF and plerixafor in patients with FA for the mobilization and collection of peripheral HSC for potential use in a GT trial.
We present the results of this open-label phase I/II trial (N°EUDRACT 2014-005264-14) from 4 selected FANCA mutated patients (FA-A) with a weight >10 Kg and an age between 2 to 18 years old. A systematic combination of G-CSF (12μg/kg twice a day) plus plerixafor (Mozobil® 0.240 mg/kg/d ) was used to maximise the CD34+ cells mobilization. CD34+ cells and white blood cells (WBC) blood counts were monitored tightly along the mobilization protocol. No short-term adverse events linked to the mobilization and the collection procedures were observed. The combination of G-CSF and Plerixafor allowed crossing the PB mobilization threshold (≥5 CD34+cells/μL) for 2 patients. Interestingly, CD34+cells were mobilized quickly but transitionally after plerixafor injection. One patient mobilization had more than 100 CD34+cells μ/L with a early peak 2h after injection. The peak disappeared 11 hours after injection. We adapted the time of collection to the C34+ cells mobilization. No CD34+ blood cell rebound was observed after the apheresis was stopped.
Our new datas suggest that mobilization of FA patients with G-CSF and plerixafor is safe. However, the age of the patient, a potential cytopenia or the lack of bone marrow progenitor cell may heavely compromise the collection. Nevertheless, the datas show a stable cytopenia despite the stimulation and collection of stem cells during the following months. This study underlines that a very cautious collection of stem cell in the Fanconi anemia to consider gene therapy is a necessity. These results also confirm that the kinetic of CD34+ cells mobilization is one of the key point to a successful stem cell harvesting for gene therapy trial.

Disclosures
Cavazzana: Smartimmune: Other: Founder of Smartimmune.
CD Maps - Dynamic Profiling of CD1 to CD100 Surface Expression on Human Leukocyte and Lymphocyte Subsets
Authors: Fišer Karel , Kalina Tomas , Perez-Andres Martin , Kužílková Daniela , Engel Pablo , Cuenca Marta , Álvarez Elena Blanco , Bartol Sophinus J.W. , van Zelm Menno C. 
Published: November 2019

CD molecules are surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication and sensing the microenvironment. These molecules are essential markers for the identification and isolation of leukocytes and lymphocyte subsets and their malignant counterparts.
Here, we present the results of the first phase of the CD Maps study, mapping the expression of CD1-100 (n=110) on 47 immune cell subsets from blood, thymus and tonsil using an 8-color standardized EuroFlow approach and quantification of expression.
The resulting dataset included median antibody binding capacities (ABC) and percentage of positivity for all markers on all subsets and was developed into an interactive CD Maps web resource. Using the resource, we examined differentially expressed proteins between granulocyte, monocyte and dendritic cell subsets, and profiled dynamic expression of markers during thymocyte differentiation, T-cell maturation, and between functionally distinct B-cell subset clusters.
The CD Maps resource will serve as a benchmark of antibody reactivities ensuring improved reproducibility of flow cytometry-based research. Moreover, it will provide a full picture of the surfaceome of human immune cells and serves as a useful platform to increase our understanding of leukocyte biology, as well as, to facilitate the identification of new biomarkers and therapeutic targets of immunological and hematological diseases.
CD Maps project is supported with reagents from BD Biosciences, BioLegend and Exbio. TK is supported by Ministry of Health Czech Republic grant 15-26588A and LO1604. KF is supported by Ministry of Health Czech Republic grant NV18-08-00385.

Disclosures
No relevant conflicts of interest to declare.