bluuebunny/crossref_metadata_2025_split huggingface dataset:
ASXL1 Is a Key Regulator for Erythroid Development and Asxl1 Loss Impairs Erythropoiesis In Vivo
Authors: Yamamoto Shohei , Shi Hui , Chen Shi , Zhang Peng , Zhou Yuan , Xu Mingjiang , Yang Feng-Chun 
Published: October 2019

Abstract
Objective:
Recently, ASXL1 mutations are found in a spectrum of numerous of myeloid malignancies, including myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML). ASXL1 mutation is a poor prognostic markers. Recently, we reported that Asxl1+/- mice developed myelodysplastic syndrome (MDS)-like disease and MDS/MPN. Regardless of the existence of defective erythroid lineage maturation and anima in patients with MDS or MPN, the role of ASXL1 in erythropoiesis remains unveiled.
Methods:
Asxl1+/- mice were utilized for hematopoietic analysis, including peripheral blood counts, blood smear, morphology, flow cytometry and histology. To assess the erythroid phenotypes in vivo, blood smears were subjected to acridine staining for reticulocyte counts. The percentage of reticulocytes and erythroid cell differentiation in peripheral blood and bone marrow were evaluated flow cytometrically following thiazole orange and CD71/Ter119 staining. Colony-forming cells (CFU) and in vitro erythroid differentiation assays were performed with methylcellulose (EPO, SCF) and liquid (EPO, transferrin) cultures of Lin-Kit+ cells of BM, spleen and fetal liver cells. In addition, the nuclear fractions were prepared from cultured erythoblasts of WT and Asxl1+/- mice and subjected to western blotting analyses to determine the histone levels of H3K27me3. RNA-Seq was performed to survey the differentially expressed genes in LK cells of WT and Asxl1+/- mice. Real time PCR was performed to confirm the alteration of erythroid development related gene.
Results:
Adult Asxl1+/- mice were anemic. High frequencies of immature and dysplastic (tear drop) red blood cells were observed in the peripheral blood of Asxl1+/- mice. Flow cytometric analysis revealed an increased percentage of pro-erythroblasts and basophilic erythroblasts populations in peripheral blood and bone marrow cells compared to that of WT controls, suggesting a blockage of erythroid maturation in Asxl1+/- mice. In vitro methylcellulose cultures showed a dramatic decrease of BFU-E and CFU-GEMM in the bone marrow of Asxl1+/- mice compared to WT controls. Western blot analysis showed that Asxl1+/- erythroblast cells exhibited decreased global levels of H3K27me3 and qPCR of Asxl1+/- erythroblast cells revealed altered expression of genes involved in development and homeostasis of erythroid lineage, including Gata1, Ldb1, Cdc42, and Zfpm1.
Conclusion:
Our study demonstrates that loss of Asxl1 results in blockage of erythroid cell maturation, indicating that ASXL1 is a key regulator for erythroid development and Asxl1 loss impairs erythropoiesis in vivo.

Disclosures
No relevant conflicts of interest to declare.
CHD7 Deficiency Inhibits CBFB-MYH11 -Induced Leukemogenesis
Authors: Zhen Tao , Zhao Ling , Hyde R. Katherine , Alemu Lemlem , Liu Pu Paul 
Published: October 2019

Abstract
Inversion of chromosome 16 is a consistent finding in patients with acute myeloid leukemia subtype M4 with eosinophilia (AML M4Eo), which generates a CBFB-MYH11 fusion gene. Mechanistic studies showed that the interaction between CBFbeta-SMMHC (encoded by CBFB-MYH11) and RUNX1 plays a critical role in the leukemogenesis of this kind of leukemia. Recently, it was shown that chromodomain-helicase-DNA binding protein 7 (CHD7) interacts with RUNX1 and suppresses RUNX1-induced expansion of hematopoietic stem and progenitor cells. These results suggest that CHD7 also plays a role in CBFB-MYH11 induced leukemogenesis. To test this hypothesis, we crossed Cre-based conditional Chd7 knockout mice (Chd7f/f) with Cre-based conditional Cbfb-MYH11 knockin mice (Cbfb+/56M) to generate Chd7f/f, Mx1-Cre, Cbfb+/56M mice, which express Cbfbeta-SMMHC but not CHD7 after poly I:C treatment to induce Cre expression. Three weeks after poly I:C treatment, the Lin- and the Lin-/Sca1-/c-Kit+ (LK) progenitor cell populations were significantly lower in Chd7f/f, Mx1-Cre, Cbfb+/56M mice than that in Mx1-Cre, Cbfb+/56M mice. In addition, there were fewer BrdU+ cells in the Lin- and LK populations in the Chd7f/f, Mx1-Cre, Cbfb+/56M mice. Importantly, Chd7f/f, Mx1-Cre, Cbfb+/56M mice developed leukemia with a much longer latency than the Mx1-Cre, Cbfb+/56M mice. Secondary transplantation assay demostrated that spleen cells isolated from Chd7f/f, Mx1-Cre, Cbfb+/56M and Mx1-Cre, Cbfb+/56M leukemic mice were transplantable, but recipients that received spleen cells from Chd7f/f, Mx1-Cre, Cbfb+/56M leukemic mice had a longer latency than those who received cells from Mx1-Cre, Cbfb+/56M mice. The above results suggest that CHD7 deficiency slows leukemia initiation and development by inhibiting the proliferation ability of the Lin- and LK populations. Further mechanistic studies showed that CHD7 interacted with RUNX1 but not CBFbeta-SMMHC by co-Immunoprecipitation and immunofluorescence staining, suggesting that CHD7 is a partner of the RUNX1-CBFbeta-SMMHC transcription complex. In vitro luciferase assay showed that CHD7 enhanced RUNX1 and CBFbeta-SMMHC's transcriptional activity on M-CSFR, a RUNX1 target gene. These data indicate that CHD7 is important for Cbfb-MYH11 induced leukemogenesis by facilitating RUNX1 regulation of transcription and cellular proliferation.

Disclosures
No relevant conflicts of interest to declare.
Ten Year Single-Center Experience in Fertility Preservation of 459 Patients Suffering from Acute Leukaemia
Authors: Noetzli Jasmine , Voruz Sophie , Wunder Dorothea , Primi Marie-Pierre , Crosazzo Laura , Gavillet Mathilde , Spertini Olivier , Blum Sabine 
Published: October 2019

Abstract
Patients with acute leukaemias suffer from a life threatening disease that has to be treated immediately. Fertility preservation is recommended in these patients, but quite often not possible.
Methods
This study is a retrospective analysis of 459 patients suffering from acute leukaemia, 221 with acute myeloid leukaemia (AML) or high risk MDS, 238 with acute lymphoblastic leukaemia (ALL) diagnosed between 2002 and 2012 in the University clinics Lausanne, Switzerland. Patients were aged between 0 and 93 years. Of course, not all of these patients were send to the preservation laboratry because of their age or their performance status.
Results
Of 221 AML cases, 113 were male and 108 female. 46 male patients were between 14 and 60 years of age, of these, 14 were sent for cryopreservation. 12 sperm samples could be frozen, 2 were sample failures due to azoospermia. Of the frozen samples, 7 were destroyed (6 because of death of the patient, 1 according to wish of patient), 4 are still frozen without tempting procreation and 1 was used for in vitro fertilization (IVF) resulting in a healthy child in 2010. 32 female patients were between 17 and 45. No sample of ovocytes was taken, 1 sample of ovarian tissue could be frozen, that was afterwards destroyed because of death of the patient.
Of 238 ALL cases, 123 were male and 115 female. 55 male patients were between 14 and 60 years of age, of these 19 were sent for cryopreservation. 16 sperm samples could be frozen, 2 were sample failures due to azoospermia, 1 because of ejaculation failure. Of the frozen samples, 11 were destroyed (8 because of death of the patient, 2 due to missing payment and 1 according to the wish of the patient). 5 samples are still frozen, 1 was used for a IVF resulting in a healthy child in 2009. 12 female patients were between 17-45. No sample taking was tempted.
Discussion
In a 10-year period in our institution, of 459 patients suffering from acute leukaemia, 145 of these within the age range defined by our institution to qualifying to cryoconservation. Only 29 samples were taken (20%), non of these in paediatric patients. These samples were leading to 2 IVFs. Of note, one male patient whose samples were taken and resulted in one of the IVFs was suffering from ALL and had an allogenous stem cell transplantation with full conditioning. This patient fathered after the IVF two other healthy children without assisted fertilization. The other patient whose sperm sample was taken for a IVF suffered from AML, he too fathered a child spontaneously after the IVF.
The data show that only 20% of eligible acute leukaemia patients had the opportunity to freeze sperm or ovocytes samples. Certainly, this was not possible for a lot of patients due to performance status, but more effort has to be done to improve sampling in these patients.

Disclosures
No relevant conflicts of interest to declare.
First-in-Man CD123-Specific Chimeric Antigen Receptor-Modified T Cells for the Treatment of Refractory Acute Myeloid Leukemia
Authors: Luo Yi , Chang Lung-Ji , Hu Yongxian , Dong Lujia , Wei Guoqing , Huang He 
Published: October 2019

Abstract
Introduction: Adoptive immunotherapy using T-cells endowed with chimeric antigen receptors (CARs) has emerged as a promising new approach to treating CD19+ acute lymphoblastic leukemia (ALL). However,treatments for relapse/refractory acute myeloid leukemia (AML) have remained largely unchanged for nearly 50 years, and some AML patients have very poor prognosis. The interleukin-3 receptor alpha chain (CD123) has been identified as a potential immunotherapeutic target due to its overexpression in AML cells compared with normal hematopoietic stem cells. Antibodies targeting CD123 for the treatment of AML have demonstrated promising anti-leukemic activity in murine models but showed limited efficacy in clinical trials, suggesting that alternative and more potent therapies targeting CD123 are required.
Methods: We have generated a 4th generation, apoptosis-inducible lentiviral CAR targeting CD123: CD123-scFv/CD28/CD137/CD27/CD3ζ-iCasp9 (4SCAR123), and demonstrated its high AML killing and AP1903-inducible apoptosis functions in ex vivo analyses. In a pilot trial of 4SCAR123, we enrolled a 47-year-old male patient with AML-M2 (FLT3/ITD+). The patient underwent allogeneic hematopoietic stem cell transplantation and relapsed. After 3 chemotherapies combined with sorafenib, his AML cells kept at 59% in bone marrow. He received CTX 250mg/kg/day for 3 days as conditioning regimen followed by 1.8x106/kg 4SCAR123 T cell infusion. Serum cytokine levels were measured by flow cytometric bead assay.
Results: At day 1 after 4SCAR123 T infusion, the patient experienced rigorous chills and fevers, low blood pressure and hypoxemia. We detected elevated serum cytokine levels including interleukin-6 (2,500pg/ml) and tumor necrosis factor-α (33.8 pg/ml) at day 8, and the patient suffered from severe cytokine release syndrome (CRS) on day 4, which was controlled by one dose of Tocilizumab. BM examination detected a decrease of blasts from 59% to 45% 20 days after CAR-T therapy.
Conclusion: Here we report a first-in-man pilot safety study of CD123 CAR-T therapy for AML patients. The 4SCAR123 exhibited potent cytotoxicity against AML in vitro, and in this pilot trial, the patient developed a rapid response consistent with CRS and achieved partial remission within 20 days. Importantly, although CD123 is universally expressed in myeloid and endothelial cells, we did not observe overt off-target cytotoxicity from the 4SCAR123 T cells, except for a controllable CRS. Our pilot study warrants further exploration of CD123 CAR for the management of refractory AML.

Disclosures
Dong: America Yuva Biomed: Consultancy.
Induction of NK Cell Reactivity Against Myeloid Leukemia By a Novel Fc-Optimized CD133 Antibody
Authors: Rothfelder Kathrin , Koerner Samuel , Andre Maya , Leibold Julia , Kousis Philaretos , Bühring Hans-Jörg , Haen Sebastian P. , Kuebler Ayline , Kanz Lothar , Grosse-Hovest Ludger , Jung Gundram , Salih Helmut R 
Published: October 2019

Abstract
NK cells largely contribute to the success of monoclonal antibody (mAb) application in cancer due to their ability to mediate antibody-dependent cellular cytotoxicity (ADCC), a feature considered critical for therapeutic success. Up to now, no immunotherapeutic antibodies are available for the treatment of myeloid leukemias. Recently, we reported on the development of mAb targeting CD133, which is expressed on a wide variety of tumor cells (Koerner et al., Blood 2014 124:2309). Here we extend our analyses and provide further data on the preclinical characterization of an Fc-engineered CD133 mAb for the treatment of myeloid leukemia. Compared to two other anti-human CD133 mAb (clones AC133 and W6B3), which both bound to primary AML and CML cells in 15/25 and 7/10 cases, respectively, clone 293C3 recognized the leukemic cells in 22/25 AML cases and 7/10 CML cases. Based on these results, clone 293C3 was chosen to generate chimeric mAb with either a wildtype Fc part (293C3-WT) or a variant containing amino acid exchanges (S239D/I332E) to enhance affinity to the activating Fc receptor CD16 on NK cells (293C3-SDIE). Treatment with 293C3-SDIE resulted in significantly enhanced activation, degranulation and lysis of primary CD133-positive AML cells by NK cells in allogeneic and autologous experimental ex vivo settings as compared to its wildtype counterpart. Considering the expression of CD133 on healthy hematopoietic progenitor cells, we further performed colony forming unit assays with healthy bone marrow (BM) cells. In line with the observed lower expression levels of CD133 on healthy compared to malignant hematopoietic cells no relevant toxicity of 293C3-SDIE at the level of committed hematopoietic progenitor cells was observed. Moreover, 293C3-SDIE did not induce lysis of of healthy BM cells by allogeneic or autologous NK cells. In a NOD.Cg-Prkdcscid IL2rgtmWjl/Sz (NSG) xenotransplantation model, induction of ADCC by treatment with 293C3-SDIE resulted in the elimination of patient AML cells by NK cells from a matched human donor. Thus, 293C3-SDIE constitutes an attractive immunotherapeutic compound, in particular for the elimination of minimal residual disease in CD133 bearing leukemia in the context of allogenic SCT.

Disclosures
No relevant conflicts of interest to declare.
Infiltration of Effector Regulatory T Cells Is Associated with Poor Prognosis in Diffuse Large B-Cell Lymphoma, Not Otherwise Specified
Authors: Shoko Nakayama , Motomu Tsuji , Taiji Yokote , Toshikazu Akioka , Takuji Miyoshi , Nobuya Hiraoka , Yuji Hirata , Kazuki Iwaki , Ayami Takayama , Uta Nishiwaki , Yuki Masuda , Jun Hatooka , Toshiaki Hanafusa , Hiroyoshi Nishikawa 
Published: October 2019

Abstract
(Background) Regulatory T cells (Tregs) are the focus of interest as one of the tumor microenvironmental factors because of their critical role in the modification of immune response, such as suppression of tumor-associated antigen-reactive lymphocytes. Tregs-mediated immunosuppression contributes to the progression of tumors and poor prognosis of patients. Forkhead box P3 (FOXP3) had been regarded as a specific marker of Tregs, but recently, FOXP3-positive cells were proved to be heterogeneous in phenotype and function. FOXP3-positive cells include effector Tregs (eTregs), naïve Tregs, and non-Tregs. Of them, eTregs have a substantial immunosuppressive effect. So far, no study has investigated the relationship between infiltration of eTregs and prognosis of patients with lymphoma.
(Aim) The present study aimed to evaluate the relationship between infiltration of eTregs doubly positive for FOXP3/cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and prognosis of patients with diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS).
(Methods) Seventy-four DLBCL, NOS specimens were doubly immunostained with anti-FOXP3 and anti-CTLA-4 antibodies. The relationship between the degree of infiltration of only FOXP3-positive cells or FOXP3/CTLA-4 double-positive eTregs and the prognosis of DLBCL, NOS patients was analyzed using Cox regression analysis. (Results) Presence of numerous FOXP3-positive cells (>52x102/cm2) was significantly predictive of better prognosis than that of fewer FOXP3-positive cells (<52x102/cm2) in terms of overall survival (OS) (p =0.0481) (Figure 1). In contrast, presence of many eTregs (>18/cm2) was significantly predictive of worse prognosis than that of fewer eTregs (<18/cm2) in terms of OS and progression-free survival (p =0.0342 and p =0.0269, respectively) (Figure 2). This was independent of the international prognostic index.
(Conclusions) Higher infiltration of only FOXP3-positive cells showed better prognosis of DLBCL, NOS patients, consistent with existing reports. In contrast, higher infiltration of eTregs showed worse prognosis of DLBCL, NOS. This suggests that infiltrating eTregs play an important role in DLBCL, NOS progression. Recently, immunotherapies targeting eTregs-depletion or functional alteration of eTregs-are being developed and are being tried in some solid tumors. These immunotherapies could also become a novel strategy of treatment for DLBCL, NOS patients with higher eTregs infiltration.
Figure 1. Figure 1. Figure 2. Figure 2.

Disclosures
Shoko: Osaka Community Foundation: Research Funding.
Palbociclib (PD-0332991), a Potent Selective CDK4/6 Inhibitor, Overcomes Therapy-Resistant and Sensitizes Diffuse Large B-Cell Lymphoma Pre-Clinical Models to Chemo-Reagents
Authors: Gu Juan J , Zhang Qunling , Mavis Cory , Czuczman Myron S. , Hernandez-Ilizaliturri Francisco J. 
Published: October 2019

Abstract
Background: Results from the Collaborative trial in relapsed aggressive lymphoma (CORAL) study highlighted the poor clinical outcome of relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients previously treated with rituximab-based therapy. To better understand the molecular mechanisms underlining the acquired resistance to rituximab, we generated and characterized several rituximab-resistant DLBCL cell lines (RRCLs). We found that RRCL exhibited a unique cell cycle distribution (accumulation in S-phase) and an increase in cyclin D Kinase-4 (CDk4) mRNA/protein levels. In our current work, we evaluated Palbociclib, a novel orally bioavailable CDK4/6 inhibitor recently approved by the Food and Drug Administration for the treatment of breast cancer in rituximab-sensitive and -resistant lymphoma pre-clinical models.
Methods: A panel of rituximab-sensitive cell lines (RSCL) or RRCL were exposed to escalating dosages of PD0332991 (0-80µM) for 24-72 hrs. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 values were calculated using the GraphPad Prism6 software. Subsequently RSCL or RRCL were exposed to PD0332991 or vehicle control and doxorubicin (0.5, 1, 2µM), dexamethasone (1µM), ibrutinib (1µM), bortezomib (10-20nM) or carfilzomib (10nM) for 48 hours. Changes in cell viability were determined by the Presto Blue assay. Coefficient of synergy was calculated using the CalcuSyn software.
Results: Palbociclib induced dose- and time-dependent decrease in cell viability in both RSCL and RRCL. Of interest, palbociclib was more potent in RRCL than RSCL. The IC50 values were lower in RRCL Raji-4RH and RL-4RH (12.12µM and 13.43µM) than in RSCL Raji and RL cells (14.7µM and 19.99µM). In vitro exposure of lymphoma cells to palbociclib resulted in G1 cell cycle arrested and it was more evident in RRCL than in RSCL. Synergistic activity was observed by combining palbociclib with doxorubicin, dexamethasone and proteasome inhibitors (bortezomib and carfilzomib) in vitro. Our data suggests that, palbociclib is active against in rituximab-sensitive or resistant pre-clinical models. In addition, CDK4/6 inhibition resulted in synergistic effects with several chemotherapeutic agents. A better understanding in the molecular events triggered by palbociclib is necessary to better develop optimal combination strategies using this novel agent in future clinical trials. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund).

Disclosures
Czuczman: Celgene: Employment; MorphoSys: Consultancy; Immunogen: Other: Advisory board; Boehringer-Ingelheim: Other: Advisory Board.
The Quality, Content, Accuracy and Readability of Information about Sickle Cell Anemia on the Internet
Authors: Breakey Vicky R. , Harris Lauren , Davis G Omar , Agarwal Arnav , Ouellette Carley , Akinnawo Elizabeth M , Stinson Jennifer N 
Published: October 2019

Abstract
Objective: A comprehensive website review was conducted to assess the quality, content and readability of online information for teens with sickle cell anemia (SCA).
Methods. Key words relevant to SCA were searched across the five most commonly used search engine domains. Websites that contained information about the diagnosis and management of SCA were reviewed. Quality of the information was appraised using the validated DISCERN tool. Two physicians rated website content completeness and accuracy independently. Readability of the sites was documented using SMOG scores and the Flesch Reading ease scoring system.
Results. Search results yielded more than 600 sites of which 25 websites met the criteria for DISCERN quality review. The majority of sites targeted parents and only 5/25 (20%) were specific to teens with SCA. The overall quality of the website information was "fair", with the average DISCERN quality rating score being 50.1 (± 9.3, range 31.0-67.5). Only 12/25(48%)of the websites had DISCERN scores above 50 (mean 57.37 + 4.93, range 52.17-67.50). The average completeness score of the sites was 20 out of 29 (±5; range 12-27) and accuracy was consistently rated 4/4, indicating high accuracy with moderate completeness. The average SMOG score was 12.44 (±2.01; range 10.21-16.08), and the mean Flesch Reading Ease score was 46.45 (±13.22; range 17.50-66.10) indicating that the material was written well above the acceptable level for patient education materials.
Conclusion. Given the paucity of high quality Internet health information at an appropriate reading level for teens with SCA, there is a critical need for the development of Internet programs to meet their unique self-management needs.

Disclosures
No relevant conflicts of interest to declare.