bluuebunny/crossref_metadata_2025_split huggingface dataset:
Clinicopathological Features and Mutational Analysis of Patients with Aplastic Anemia and Hypoplastic Myelodysplastic Syndrome
Authors: Zhang Xiaohui , List Alan F , Lancet Jeffrey E. , Jinming Song , Moscinski Lynn C. , Sokol Lubomir , Zhang Ling 
Published: October 2019

Abstract

Background: Pancytopenia and bone marrow aplasia/hypoplasia are caused by a heterogeneous group of disorders, most commonly aplastic anemia (AA), hypoplastic myelodysplastic syndrome (MDS), paroxysmal nocturnal hemoglobinuria (PNH), and T-cell large granular lymphocytosis (T-LGL). Clinical and morphological distinction among these entities is often challenging, particularly between AA and hypoplastic MDS. This study is to examine the clinicopathological and genetic features of a group of AA and hypoplastic MDS patients, with or without concurrent T-LGL and/or PNH, in order to better understand and differentiate the two entities.
Methods and Materials: We retrieved 45 cases with cytopenias and hypoplastic bone marrow at Moffitt Cancer Center. Peripheral blood complete blood counts, bone marrow morphological findings, flow cytometric analyses for LGL and PNH, and cytogenetics data were extracted from electronic medical records. Targeted next-generation sequencing (54 myeloid neoplasm related genes) was performed on the bone marrow.
Results: The 45 patients showed peripheral blood cytopenias and bone marrow aplasia or marked hypocellularity. There were 26 cases diagnosed with AA with no morphologic evidence of dysplasia or increased blasts in the bone marrow, and 19 cases diagnosed with hypoplastic MDS based on morphological and cytogenetic criteria. In the meantime, distinct T-LGL population was identified in 2 of 11 cases with AA (18.2%) and 3 of 11 cases with hypoplastic MDS (27.3%); PNH clones were identified in 8 of 17 cases with AA (47%) and 3 of 11 cases with hypoplastic MDS (27.3%). Clonal cytogenetic abnormalities were found in 2 of 25 cases with AA (8%) and 11 of 18 cases with hypoplastic MDS (61.1%). Twelve of 26 cases of AA (46%) showed one or more gene mutations with allele burden ranging from 7% to 52%, and most of these cases (9 of 12; 75%) involved only one gene. In contrast, 15 of 19 cases of hypoplastic MDS (78.9%) had one or more gene mutations with allele burden ranging from 19% to 53%. Seven of the 15 cases (46.7%) had two or more gene mutations. The most common mutated genes in the two groups in this study were ASXL1 and TET2.
Conclusion: Although there are overlapping clinical and morphological features between AA and hypoplastic MDS, differences are present between the two entities including presence of PNH clones, cytogenetic changes, and gene mutation frequencies. These features may help to make differential diagnosis and identify the cases with more progression potential. Clinical outcomes with different treatment and larger scale studies are needed to better characterize and define the two different entities.

Disclosures
No relevant conflicts of interest to declare.
Bortezomib-Induced Apoptosis in Myeloma Cell Via Oxidation of Peroxiredoxin III and Thioredoxin 2
Authors: Mun Yeung-Chul , Ahn Jee-Young , Kang Seung Hye , Yoo Eun-Sun , Lee Kyoung Eun , Nam Eunmi , Lee Soon Nam , Woo Hyun Ae , Rhee Sue Goo , Seong Chu-Myong 
Published: October 2019

Abstract
Backgrounds : Bortezomib (BTZ) is the first generation proteosome inhibitor providing excellent response in newly diagnosed multiple myeloma. BTZ treatment increases oxidative stress in myeloma cells. However the roles of antioxidant enzymes during upregulation of ROS and the mechanisms of resistance from BTZ treatment are unclear. The aims of current study are to elucidate that the changes of redox enzyme could have a important roles of anti-myeloma effect during BTZ treatment.
Methods : MM.1S, MM.1R, and RPMI8226, the human myeloma cell lines, were treated with BTZ to induce apoptosis. 2, 7-dichlrodihydro-fluorescein-diacetate (H2DCF-DA) and MitoSOX Red were used to detect cellular and mitochondrial reactive oxygen species (ROS) respectively. Sulfinic acid, (SO2) form of preoxiredoxin (Prx) was studied by western blot assay using Prx SO2 form-specific antibody. Monomer/dimer assay for subtypes of Prx and thioredoxin (Trx) was performed by western blot using non-reducing gel. To evaluate the effect of down regulation on sulfiredoxin (Srx), myeloma cells were transfected with small interfering RNA (siRNA) followed by Western blot analysis.
Results : Mitochondrial over cytosolic ROS of MM cells was increased significantly after 19 hour of BTZ (2.5 nM). Apoptosis of MM cell after BTZ treatment was increased in concordance with mitochondrial ROS increment of MM cells. N-acetylcystein (NAC) reversed BTZ-induced mitochondrial ROS elevation and apoptosis of MM cells as well. Increased expressions of cleaved caspase-9 and cleaved caspase-3 were also observed during BTZ-induced MM cell apoptosis. Monomer, indicated active and reduced form, of Prx III was decreased and dimer, indicated inactive and oxidized form, of Prx III was increased in MM cells after treatment of BTZ among Prxs. Similarly, monomer of Trx 2, mitochondrial Trx was decreased in MM cells after BTZ treatment. However, increment of cysteine SO2 Prx III was not observed. Meanwhile, Srx, reducing enzyme of SO2 Prx, was induced in MM cells after BTZ treatment. Down regulation of Srx by siRNA did not promote ROS generation or apoptosis in BTZ-treated MM cells, otherwise.
Conclusions : Our results showed inactivation of Prx III by multimer formation as hyperoxidation is found during BTZ-induced mitochondrial ROS generation and apoptosis in MM cells. This Prx III oxidation was due to down regulation of reduced Trx 2 in BTZ-treated MM cells. To design of raising ROS stress and down regulation of anti-oxidants(ie, Trx), as a treatment-strategies may be worthwhile to potentiate BTZ-induced apoptosis in MM cells.

Disclosures
No relevant conflicts of interest to declare.
Reducing on-Target Off-Tumor Effects of CD38-Chimeric Antigen Receptors By Affinity Optimization
Authors: Drent Esther , Themeli Maria , Poels Renée , van de Donk Niels W.C.J. , Lokhorst Henk M. , Mutis Tuna 
Published: October 2019

Abstract
Chimeric Antigen Receptors (CARs) are engineered transmembrane proteins consisting of an antibody (ab)-derived antigen recognition domain linked to intracellular T cell signaling domains. Cytotoxic T cells endowed with tumor-reactive CARs are highly promising tools for immunotherapy of cancer. There are however only a few truly tumor specific molecules that can be targeted by CARs, a drawback for the broad application of CAR T cell therapy. Indeed, when we recently aimed at targeting CD38high multiple myeloma (MM) with T cells transduced with high affinity CD38CARs, we observed that they not only lysed the CD38high MM cells but also CD38+ normal hematopoietic cells, pointing towards potential safety issues of such tumor-associated, but not entirely tumor-specific CARs. Therefore, using CD38 as a model for antigen we now tested whether it would be possible to reduce the on target, off-tumor effects of such CARs by optimizing their target cell affinity. To this end, we generated a new panel of CD38 abs through the "light chain exchange" method, in which heavy chains of two high affinity CD38 abs were combined with 176 different germ line light chains. This approach revealed around 100 new abs, which displayed 10- >1000 fold lower affinity to CD38 as compared to the parental abs. After categorizing them in three classes based on CD38 binding affinity, we used 8 abs from each class to generate 24 different CD38-CAR constructs. Testing the cytotoxic activity of T cells transduced with these CD38-CARs against CD38++ MM cell lines, primary MM cells and CD38+ normal hematopoietic cells in vitro and in vivo demonstrated that CD38-CAR T cells with ca. 1000 fold lower affinity to CD38 could still effectively lyse CD38++ MM cells while there was little or no cytotoxicity against CD38+ healthy hematopoietic cells. The results of this study reveal that it is possible to reduce the on-target off-tumor effects of CARs by optimizing their affinity. Thus, tailored affinity of the ab binding domain may open up new roads for CAR therapy.

Disclosures
van de Donk: Janssen: Research Funding; Celgene: Research Funding; BMS: Research Funding; Amgen: Research Funding. Lokhorst:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding. Mutis:Celgene: Research Funding; Genmab: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.
High Disease-Free and Overall Survival Rate Following Allogeneic Hematopoietic Stem Cell Transplantation for FLT3-Mutated Acute Myeloid Leukemia Even in Non-Remission Status
Authors: Nishida Aya , Yuasa Mitsuhiro , Kageyama Kosei , Ishiwata Kazuya , Takagi Shinsuke , Yamamoto Hisashi , Asano-Mori Yuki , Yamamoto Go , Uchida Naoyuki , Izutsu Koji , Wake Atsushi , Yoneyama Akiko , Makino Shigeyoshi , Taniguchi Shuichi 
Published: October 2019

Abstract
[Background] FMS like tyrosine kinase 3 (FLT3) mutations occur in about 30% of patients with acute myeloid leukemia (AML). Patients with FLT3-mutated AML have a poor prognosis and are referred for early allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission (CR1). So far, there is still limited data available on allo-HSCT for FLT3-mutated AML in non-remission status.
[Objective and method] To assess the clinical features and outcome of allo-HSCT for FLT3-mutated AML, we retrospectively analyzed patients underwent first allo-HSCT for FLT3-mutated AML excluding acute promyelocytic leukemia (FAB M3) from January 2011 to March 2016.
[Result] During the study period, 332 patients received first allo-HSCT for AML in our institute. One hundred and thirty-eight were tested for the presence of FLT3-mutation and 35 showed positive results and were subjected to the analysis. The median follow-up day of survivors was 602 (101-1867). The median age of the patients was 55 years (range, 21-72). Twenty-one patients had de novo AML, 12 had AML with myelodysplasia related changes, and 2 had therapy related AML. Eighteen had normal karyotype, 4 had complex, and 13 had others. Seven were in remission (5 in CR1, and 2 in CR2), and 28 were in non-remission (8 in primary induction failure, 13 in relapse 1, and 7 in chemo naïve status). Twenty-nine patients used unrelated cord blood, 2 did unrelated bone marrow, and 4 did related peripheral blood stem cell as grafts. All but 1 received myeloablative pretransplant conditionings. At 2 years after transplantation, overall survival (OS), disease free survival (DFS), relapse rate (RR), and non-relapse mortality (NRM) of whole studied population were 65.9%, 50.2%, 28.4%, and 21.4%, respectively. Among those in non-remission before transplantation, OS, DFS, RR, and NRM at 2 years post-transplant were 62.2% (Figure 1A), 49.9%(Figure 1B), 24.3%, and 25.8%, respectively. Only younger age (<55 years) was the factor associated with better OS with statistical significance in multivariate analysis.
[Conclusion] Our data indicated that allo-HSCT could overcome the poor prognosis of FLT3-mutated AML even for those in non-remission status, despite the profound chemo-resistant character of FLT3-mutated AML.
Figure 1A Figure 1A. Figure 1B Figure 1B.

Disclosures
Izutsu: Abbvie: Research Funding; Gilead: Research Funding; Celgene: Research Funding; Janssen Pharmaceutical K.K.: Honoraria; Eisai: Honoraria; Kyowa Hakko Kirin: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria; Mundipharma KK: Research Funding.
Precise and Efficient Crispr/Cas9 Mediated Gene Editing in Long-Term Engrafting Human Hematopoietic Stem/Progenitor Cells
Authors: Heath Jack M , Chalishazar Aditi , Lee Christina S , Selleck William , Cotta-Ramusino Cecilia , Bumcrot David , Gori Jennifer L 
Published: October 2019

Abstract
Transplantation of gene-modified autologous hematopoietic stem/progenitor cells (HSPCs) is an effective treatment for several hematologic diseases. However, a number of blood disorders may not be amenable to gene augmentation-based therapeutics. Targeted genome editing in human HSPCs could provide a therapeutic approach for these otherwise untreatable diseases. Here we demonstrate that CRISPR/Cas9 ribonucleoprotein (RNP) edits target genes in human HSPCs with high efficiency and precision. Human adult and umbilical cord blood (CB) CD34+ cells from 20 donors were electroporated with S. pyogenes or S. aureus Cas9 RNP targeting HBB, AAVS1, or CXCR4. Sequence analysis demonstrated up to 80% editing in CB CD34+ cells (mean±s.d: 61%±9%) and up to 57% in adult CD34+ cells (39%±13%). Delivery of Cas9 RNP and a single-stranded oligodeoxynucleotide donor (ssODN) led to up to 12% ssODN-mediated homology directed repair (HDR) and also led to a 20% increase in total gene editing (HDR+NHEJ)(RNP: 48%±15%; RNP+ssODN: 69%±8%). Both Cas9 RNP gene-edited CD34+ cells and donor-matched untreated control CD34+ cells reconstituted human hematopoiesis in primary and secondary recipient immunodeficient mice, with ~85% human CD45+ cell peripheral blood reconstitution 4 months after primary transplantation. Human T and B lymphoid, erythroid, and myeloid cells were detected in the spleen, thymus, and bone marrow with 20% CD34+ cell engraftment in the marrow of mice transplanted with RNP gene-edited or control CD34+ cells. The level of targeted gene editing in human erythroid, myeloid, and CD34+ cells that were recovered and enriched from the hematopoietic organs of primary recipients (~50%) was similar to the level of gene editing detected in the pre-infusion product (~60%). In summary, these results indicate that Cas9 gene-edited human HSPCs retain long-term engraftment potential and support multilineage blood reconstitution in vivo, thus supporting further investigation of CRISPR/Cas9 mediated gene-edited hematopoietic stem/progenitor cell therapies.

Disclosures
Heath: Editas Medicine: Employment. Chalishazar:Editas Medicine: Employment. Lee:Editas Medicine: Employment. Selleck:Editas Medicine: Employment. Cotta-Ramusino:Editas Medicine: Employment. Bumcrot:Editas Medicine: Employment. Gori:Editas Medicine: Employment.
Strategies for Selection of AAV Vectors for Administration to Liver: Studies in Nonhuman Primates
Authors: Wang Lili , Wilson James M , Calcedo Roberto , Bell Peter , He Zhenning , White John , Hewitt Matthew , Makaron Leah , Kattenhorn Lisa , Wadsworth Sam , Goode Tamara 
Published: October 2019

Abstract
Vectors based on adeno-associated virus (AAV) have demonstrated early promise in clinical trials, including published reports in patients with hemophilia B where therapeutic levels of factor IX have been achieved using AAV serotype 8, a member of the clade E family. With the success of these hemophilia B clinical trials utilizing AAV8, numerous alternative AAV capsid types that target the liver have begun to enter clinical testing across multiple disease indications. Specific properties of the various AAV capsids should be taken into account such as overall efficiency of hepatocyte gene transfer, differential gene transfer across the porto-central axis within the liver, durability of gene expression and the potential for vector re-administration. In this study, we evaluated these aspects of AAV-directed liver gene transfer in rhesus macaques across various capsid serotypes. Animals were injected with vectors expressing the secreted reporter gene rhesus bhCG and produced using different capsids [AAV5, AAV3b and two clade E vectors (AAVrh10 and AAV8)]. Nonhuman primates (NHPs) injected with clade E vectors expressing bhCG were administered 3 months later with AAV5 or AAV3b vectors expressing rhesus derived AFP. The key findings were: 1) in naïve animals, clade E vectors demonstrated the highest levels of periportal gene transfer with AAV5 vectors having the lowest levels of periportal gene transfer; 2) AAVrh10 and AAV5 elicited higher levels of neutralizing antibodies (NAb) than AAV8 and AAV3b; 3) significant animal-to-animal variation in transgene expression was noted with AAV3b in seronegative animals; and 4) within the short time frame tested NAb elicited from AAVrh10 appears to have inhibited subsequent in vivo transduction with the serologically distinct AAV3b serotype; prior exposure to AAV8 did not interfere with AAV3b transduction. These studies highlight the influence that capsids can play in efficiency and immunogenicity of AAV vectors for liver gene therapy.

Disclosures
Wilson: Dimension Therapeutics: Consultancy, Equity Ownership, Patents & Royalties, Research Funding; Solid Gene Therapy: Consultancy, Membership on an entity's Board of Directors or advisory committees; REGENXBIO: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Kattenhorn:Dimension Therapeutics: Employment. Wadsworth:Dimension Therapeutics: Employment, Equity Ownership.
Mutation of a GATA1 Binding Site in ALAS2 Intron 1 Arrests Murine Erythroid Development In Vivo
Authors: Zhang Yingchi , Zhang Jingliao , An Wenbin , Wan Yang , Gao Jie , Shi Lihong , Cheng Tao , Zhu Xiaofan 
Published: October 2019

Abstract
Congenital sideroblastic anemia (CSA) comprises a heterogeneous group of genetic disorders characterized by decreased heme synthesis and the presence of bone marrow (BM) ring sideroblasts. The most common form of CSA is X-linked sideroblastic anemia (XLSA, OMIM#300751), which is caused by mutations in the gene encoding 5-aminolevulinic acid synthase 2 (ALAS2). However, to date the experimentally confirmed ALAS2 mutations mostly occurred at the exons of ALAS2 and are missense mutations.
In our study, we identified a non-coding GATA1 binding site mutation in ALAS2 intron 1 in a large XLSA pedigree. We addressed the functions of the GATA1 binding site in ALAS2 intron 1 in vivoby generating mice lacking this GATA site and its flanking sequence (13bp) and found that this deletion led to an embryonic lethal phenotype due to severe anemia, indicating that this fragment is indispensable during erythroid development. Next we demonstrated that GATA1 activated ALAS2 transcription by forming an enhancer loop with TAL1, LDB1 and Pol II through GATA binding sites in the promoter and in introns 1 and 8. Finally, we discovered that int-1-GATA site deletion completely abolished ALAS2 transcription due to disruption of the enhancer loop and a simultaneous lack of Pol II enrichment at the intron 1 enhancer region and promoter.
Our findings first revealed the essential roles of GATA1 binding site in ALAS2 intron 1 in erythroid development in vivo and uncovered the mechanisms how GATA1 regulated the expression of ALAS2. We believe that these findings will be of wide general interest and will provide valuable information for the clinical diagnosis of XLSA patients with unknown mutations.

Disclosures
No relevant conflicts of interest to declare.
Corticosteroids Impair Granulocyte Transfusion Therapy By Targeting NET Formation and Neutrophil Antifungal Functions Via ROS/Dectin1 Pathways
Authors: Kalleda Natarajaswamy , Amich Jorge , Poreddy Spoorthi , Arslan Berkan , Friedrich Mike , Mokhtari Zeinab , Ottmüller Katja , Jordán-Garrote Ana-Laura , Einsele Hermann , Brock Matthias , G Heinze Katrin , Beilhack Andreas 
Published: October 2019

Abstract
Introduction:
Invasive pulmonary Aspergillus fumigatus infections cause high morbidity and mortality in neutropenic patients. Granulocyte transfusions have been tested as an alternative therapy for the management of high-risk neutropenic patients with invasive A. fumigatus infections. To increase the granulocyte yield for transfusion, donors are treated with corticosteroids. Yet, the efficacy of granulocyte transfusion and functional defense mechanisms of granulocytes collected from corticosteroid treated donors remain largely elusive. Therefore, we investigated the efficacy of granulocyte transfusion and functional defense mechanisms of corticosteroid treated granulocytes using mouse models.
Methods:
To determine the effects of corticosteroids on granulocytes to control A. fumigatus infections, we performed granulocyte adoptive cell transfers using in vivo mouse models, in vitro human and mouse granulocyte and A. fumigatus functional co-culture experiments in combination with flow cytometry, cytokine analysis, fluorescence and electron microscopy.
Results:
Transfusion of granulocytes from corticosteroid treated mice did not protect cyclophosphamide immunosuppressed mice against lethal A. fumigatus infection in contrast to granulocytes from untreated mice. Upon infection increased levels of inflammatory cytokines helped to recruit granulocytes to the lungs without any recruitment defects in corticosteroid treated and infected mice or in cyclophosphamide immunosuppressed and infected mice that had received the granulocytes from corticosteroid treated mice. However, corticosteroid treated human or mouse neutrophils failed to form neutrophil extracellular traps (NETs) under in vitro and in vivo conditions. Furthermore, corticosteroid treated granulocytes exhibited impaired ROS production against A. fumigatus. Notably, corticosteroids impaired the β-glucan receptor Dectin-1 (CLEC7A) on mouse and human granulocytes to efficiently recognize and phagocytize A. fumigatus, which markedly impaired fungal killing.
Conclusions:
We conclude that corticosteroid treatment of granulocyte donors for increasing neutrophil yields or patients with ongoing corticosteroid treatment could result in deleterious effects on granulocyte antifungal functions, thereby limiting the benefit of granulocyte transfusion therapies against invasive fungal infections.

Disclosures
Einsele: Celgene: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Speakers Bureau.