bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vivo Efficacy of Bcl11a Erythroid-Enhancer Deletion in Humanized Mouse Models of Anemia
Authors: Ryan Thomas M , Fontenard Suean Daimia , Liu Shanrun , Lockhart Jonathan , Berlett Michael 
Published: October 2019

Abstract
Autologous cell therapy holds great promise for the treatment of beta thalassemia major and hemoglobinopathies like sickle cell anemia. Gene editing of a patient's own stem cells to reactivate the silenced gamma globin gene is one approach under active development. Prior to directly testing these new therapies in patients, we can answer some basic questions about their in vivo efficiency and efficacy in humanized mouse models of anemia. These models have their endogenous adult alpha and beta globin genes replaced with human alpha, gamma, and beta globin genes. These mice synthesize high level of human fetal hemoglobin during fetal life and complete their fetal-to-adult hemoglobin switch after birth. Experimental strategies designed to reactivate the silenced fetal gamma globin genes in adult erythroid cells are easily tested in vivo in these humanized hemoglobin switching mouse models. The silenced human fetal gamma globin genes can be activated by mutating the erythroid-specific enhancer of Bcl11a by gene editing. CRISPR sgRNAs, designed to target the +62 kb DNase I hypersensitive site in the second intron of Bcl11a, were microinjected along with Cas9 mRNA, into fertilized mouse embryos collected from humanized hemoglobin (Hb A) mice. The indel mutations that were generated in the founder animals were characterized and bred to homozygosity. The data demonstrates that the sgRNAs tested were successful in creating multiple unique mutations at the erythroid enhancer target sites. These mutations were transmitted through the germline allowing the effect of individual edited alleles to be analyzed. The majority of the mutations showed marginal increases in the number of F-cells over control animals. Significantly, despite having homozygous mutation of the erythroid-enhancer in all cells, fetal hemoglobin expression remains heterocellular. Importantly, the therapeutic efficacy of reactivating fetal hemoglobin with specific Bcl11a erythroid-enhancer mutations for the treatment of beta thalassemia major and sickle cell anemia was directly measured in vivo in these humanized models of disease. The reactivation of gamma globin in these humanized mouse models provides us with an opportunity to further interrogate the Bcl11a enhancer element, identify additional factors involved in hemoglobin switching and elucidate the mechanism driving pancellular vs heterocellular fetal hemoglobin expression.

Disclosures
No relevant conflicts of interest to declare.
Structural and Functional Insight of Sphingosine 1-Phosphate-Mediated Pathogenic Metabolic Reprogramming in Sickle Cell Disease
Authors: Sun Kaiqi , Xia Yang , D'Alessandro Angelo , Ahmed Mostafa H , Zhang Yujin , Song Anren , KO Tzu-Ping , Nemkov Travis , Haines Julie , Wu Hongyu , Adebiyi Morayo G. , Liu Hong , Huang Aji , Peng Zhangzhe , Wen Yuan Edward , Wen Alexander , Berka Vladimir , Bogdanov Mikhail , Abdulmalik Osheiza , Kato Gregory J , Tsai Ah-lim , Idowu Modupe , Juneja Harinder S. , Kellems Rodney E. , Hansen Kirk C , Safo Martin K. 
Published: October 2019

Abstract
Sphingosine 1-phosphate (S1P) is a bioactive signaling lipid highly enriched in mature erythrocytes. Previous study has revealed that levels of S1P are significantly elevated in patients and mice with Sickle Cell Disease (SCD), a devastating and highly prevalent genetic hemolytic disorder that causes life-threatening hemolysis, tissue damage, and organ dysfunction with very limited treatment. Moreover, the activity of S1P generating enzyme-Sphingosine Kinase 1 (SphK1) is increased in human and mouse SCD erythrocytes, and inhibition of SphK1 decreased erythrocyte sickling. However, the structural and functional basis for the pathogenic nature of S1P in SCD remains obscure. Here, we report that increased erythrocyte S1P promotes pathogenic metabolic reprogramming coupled to increased channeling of glucose to glycolysis rather than through the pentose phosphate pathway (PPP). Suppressed PPP causes compromised glutathione homeostasis and increased oxidative stress, while enhanced glycolysis induces production of 2,3-bisphosphoglycerate (2,3-BPG) and thus increasing deoxygenated sickle Hb (deoxyHbS), deoxyHbS polymerization, sickling, hemolysis and disease progression. S1P functioning intracellularly binds to deoxyHbS, facilitates deoxyHbS anchoring to the membrane, induces release of membrane-bound glycolytic enzymes and in turn switches glucose flux towards glycolysis relative to the PPP. Extending from SCD, we unexpectedly found that S1P and 2,3-BPG work synergistically to decrease both HbA and HbS oxygen binding affinity. The crystal structure of HbA complexed with S1P alone or in combination with 2,3-BPG at 1.9 Å resolution revealed the overall architecture and unique features of S1P-2,3-BPG-deoxyHbA complex. In the presence of 2,3-BPG, S1P binds to the surface of 2,3-BPG-deoxyHbA and causes additional conformation changes to the T-state Hb. Phosphate moiety of the surface bound S1P engages in a highly positive region close to a1-heme while its aliphatic chain snakes along a shallow cavity making hydrophobic interactions in the "switch region", as well as with b2-heme like a molecular "sticky tape" with the last 3-4 carbon atoms sticking out into bulk solvent. Altogether, our findings provide functional and structural bases underlying pathogenic consequences of elevated S1P in SCD and its potential role in normal erythrocyte physiology.

Disclosures
Kato: Mast Therapeutics: Consultancy; Bayer: Research Funding.
VWF Multimer Quantitation for Assessment of Cardiac Lesion Severity and Bleeding Risk
Authors: Bryan Justin , Austin Christopher , Thomas Colleen , Safford Robert E. , Chen Dong , Blackshear Joseph 
Published: October 2019

Abstract
Introduction: Recent reports have utilized von Willebrand factor (VWF) multimerquantitation in the assessment of native cardiac lesions and prosthetic valve dysfunction. However there is no standardized method for quantitation. We compared 3 methods of assessment which utilized a normal plasma control.
Methods: We analyzed 476 samples and their control plasma from 395 patients including normal subjects, patients with aortic stenosis, hypertrophic cardiomyopathy, mitral or aortic regurgitation, normal and abnormal left cardiac valve prostheses, and left ventricular assist device therapy (LVAD). VWF multimers were assessed as normal or abnormal, or as normalized (patient / normal plasma) VWF multimerratios of gel bands >15/2-15 (NMR 15) or gel bands > 10/2-10 (NMR 10). We tested whether one technique was more strongly associated with hemodynamic severity of the cardiac lesion, with a history of acquired bleeding, and compared the results to a separate test of VWF function, platelet function analyzer 15 00 (PFA).
Results: Two hundred seventeen patient samples reflected normal or mildly abnormal hemodynamics, 111 moderate, and 113 severely abnormal hemodynamics. Abnormal multimers were present in only 19% of samples with normal or mildly abnormal hemodynamics compared to 79% abnormal in the presence of moderate to severe hemodynamic abnormality. The distinction between moderate and severe hemodynamic abnormality was less striking, 65% versus 88% (figure, left). Quantitative NMR values declined with increasing cardiac lesion severity (figure, middle). The two quantitative multimermethods showed similar relationships to lesion severity by ANOVA, r=0.40 (NMR 15) versus r=0.36 (NMR 10), and by Spearman correlation, r=0.68 for NMR 15, and r=0.66 for NMR 10, and PFA results were comparable (all p< 0.001). Bleeding rates were associated with NMR 15, 10 and PFA (Mann-Whitney p<0.001, figure, right).
Conclusion: The dichotomous normal / abnormal VWF multimer method appears to distinguish ≥ moderate hemodynamic disruption, whereas there is a continuous relationship between hemodynamic severity and quantitative multimer methods, which may be clinically useful in circumstances in which clinical estimation of lesion severity is challenging, such as with dysfunctional prosthetic valves. Progressive loss of VWF multimersappears to be associated with bleeding, suggesting that quantitative measures may provide incremental value over the dichotomous normal / abnormal designation.
Figure Figure.

Disclosures
Blackshear: Baxalta, Inc: Consultancy, Research Funding.
Incidence and Risk Factors of Venous Thromboembolism in Patients with Multiple Myeloma Receiving Immunomodulatory Agents
Authors: Kimpton Miriam , Buyting Ryan , Corsi Daniel J , Rupani Natasha , Carrier Marc , McCurdy Arleigh 
Published: October 2019

Abstract
Multiple myeloma (MM) is an incurable plasma cell disorder representing 10% of all hematologic malignancies. Cancer is a known risk factor for venous thromboembolism (VTE). Patients with MM are at a particularly high risk of developing VTE owing to patient characteristics (e.g. previous history of VTE), disease characteristics, and treatment characteristics including use of the immunomodulatory agents (IMIDs). Unfortunately, standard criteria to identify the patients most at risk for developing VTE in MM while receiving IMIDs are unknown. We sought to assess the incidence of VTE and its associated risk factors in MM patients receiving IMID therapy.
A retrospective cohort study including 1680 consecutive patients with multiple myeloma treated at our centre between January 01, 1995 and January 26, 2016 was conducted. The annual incidence of VTE on immunomodulatory agents including thalidomide, lenalidomide, and pomalidomide was derived. Univariate incidence ratio analyses of VTE for different risk factors was performed including: previous history of VTE, concomitant use of dexamethasone, and ≥/< 6 months after IMID initiation.
A total of 309 MM patients treated with an immunomodulatory agent were identified. Nineteen patients were excluded (incomplete data, lost to follow). Of the remaining 290 patients, the mean age was 67.9 and 42.4% were female. Twenty-seven VTE events were recorded. The overall risk ratio was 6.1 for the development of VTE. Patients with a personal history of VTE had an increased risk of suffering a VTE while on IMID therapy (IRR 5.4; CI, 1.9-13.6). The time from the initiation of the IMID therapy (less than 6 months) also increased the risk of developing a VTE (IRR 51.7; CI,19.4-160.1). The concomitant use of dexamethasone was not associated with a statistically significant increased risk (IRR 1.7; CI, 0.3-69.5). Incidence risk ratios for these risk factors are depicted in Table 1.
Our results suggest that a personal history of VTE and the time from the initiation of the IMID (less than 6 months) are associated with an increased risk of VTE in MM patients receiving IMID therapy. This may be helpful in determining which multiple myeloma patients treated with an IMID agent warrant more aggressive thromboprophylaxis. Further prospective studies are needed to determine the optimal agent, intensity, and duration of thromboprophylaxis in patients with MM on IMID therapy.

Disclosures
McCurdy: Celgene: Honoraria.
Myelodysplastic/Myeloproliferative Neoplasms Unclassified (MDS/MPN-U) Overlap: Can We Alter the Natural History?
Authors: Hendrix Kaitlin , Alali Najla , Padron Eric , Sallman David , List Alan F. , Lancet Jeffrey E. , Komrokji Rami S. 
Published: October 2019

Abstract
Introduction:
The 2008 World Health Organization classification recognized a unique overlap category that combines features of myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN). The least well characterized of the 4 overlap diseases is a rare entity known as MDS/MPN Unclassifiable (MDS/MPN-U), comprising <5% of myeloid disorders. The outcome of this subtype has been reported to be poor. The response to and impact of hypomethylating agents (HMA) on outcome is not well studied.
Methods:
We identified MDS/MPN-U patients within the Moffitt MDS database, baseline characteristics were reviewed, and responses to HMA utilizing IWG 2006 response criteria were assessed. The Kaplan-Meier method was applied to estimate overall survival (OS).
Results:
Among 127 patients with MDS/MPN-U, 62 patients received HMA treatment. There was no difference in baseline characteristics among those who received HMA or not, except that those treated with HMA were less likely to have low risk disease by IPSS, MD Anderson model or very low risk by revised IPSS (R-IPSS). Among 16 patients who had NextGen sequencing data, the 5 most common mutations were ASXL-1 (56%), TET2 (31%), JAK2 (33%), SRSF2 (25%), as well as SETBP1, DNMT3A, and EZH2(19%). The best overall response to HMA (hematological improvement [HI] or better) according to the International Working Group (IWG) 2006 criteria was 26%. Only 11 patients (low risk) underwent allogeneic stem cell transplant (allo-SCT) with no improvement or decrease in OS.
The median OS was 33 months (mo). Among patients with IPSS low/int-1 risk disease, the median OS was 39 and 33 mo respectively for those treated with HMA vs. HMA untreated. (p=0.50). For Int-2/high risk IPSS, the median OS was 11 and 5 mo for those who received HMA and those who did not. (p=0.02). Based on R-IPSS the median OS for very low/low risk was 47 and 39 mo respectively for those treated with HMA and without (p=0.96), for intermediate risk, the median OS was 39 and 25 mo respectively (p 0.75), and for high and very high risk the median OS was 13 and 5 mo respectively for those treated with HMA and those who did not. (p < 0.005). The median OS for patients with HI+, stable disease or progressive disease was 68, 59, and 36 mo respectively (p=0.08).
Conclusions:
Treatment with HMA is associated with modest improvement in OS among higher risk MDS/MPN-U, reinforcing the unmet need to improve outcomes in this group. Targeted therapy may prove to be of benefit as allo-SCT does not appear to be beneficial for patients even with low risk disease. Furthermore, these data demonstrate the need for prospective analysis of the outcomes of allo-SCT in patients with MDS/MPN-U to determine potential benefit to high risk patients.

Disclosures
Komrokji: Novartis: Consultancy, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.
Dual Deficiency of mDia1 and Mir-146a in an Age-Related Inflammatory Bone Marrow Microenvironment Induces Ineffective Erythropoiesis in Del(5q) MDS
Authors: Mei Yang , Basiorka Ashley , Zhao Baobing , Yang Jing , List Alan F , Ji Peng 
Published: October 2019

Abstract
Myelodysplastic syndromes (MDS) are a group of age-related clonal hematologic diseases characterized by anemia, neutropenia, and thromobocytopenia. The mechanisms of anemia in MDS are unclear, which is partially due to the heterogeneity of MDS involving many cytogenetic and molecular abnormalities. Using a mouse genetic approach, here we show that dual deficiency of mDia1 and miR-146a, two genes located at chromosome 5q that is commonly deleted in MDS, causes an aged-related anemia and ineffective erythropoiesis that closely mimics human MDS. Bone marrow erythropoiesis at various stages was dramatically affected in mDia1/miR-146a double knockout mice, which induced a massive splenomegaly with potent extramedullary erythropoiesis. Old (> 1 year of age), but not young (2-4 months of age), wild type recipient mice that were transplanted with bone marrow cells from mDia1/miR-146a double knockout mice, exhibited severe anemia and rapid lethality, which indicates that the aged microenvironment is important for the development of ineffective erythropoiesis. Consistent with the roles of mDia1 and miR-146a in the innate immune response, the serum levels of TNFα and IL-6 were significantly elevated in mDia1/miR-146a double knockout mice. Pathogen-associated molecular pattern proteins (PAMPs), or damage-associated molecular pattern proteins (DAMPs), whose levels increase in aged microenvironment, both induced TNFα and IL-6 upregulation in mDia1/miR-146a double knockout granulocytes and T cells. Mechanistically, we demonstrated that the anemia and ineffective erythropoiesis was independent of hepcidin expression in mDia1/miR-146a double knockout mice. Instead, pathologic levels of TNFα and IL-6 inhibit erythroid colony formation and differentially affect terminal erythropoiesis through reactive oxygen species-induced caspase-3 activation and cell apoptosis. Our study highlights the dual roles of age-related microenvironment and cytogenetic abnormalities in the pathogenesis of ineffective erythropoiesis in MDS.

Disclosures
No relevant conflicts of interest to declare.
BKV Disease during the First 100 Days after Allogeneic Hematopoietic Stem Cell Transplantation - Results of a Screening Program and Retrospective Analysis
Authors: Posdzich Philip , Herling Marco , Leitzke Silke , Hamacher Laura , Di Christanziano Veronica , Hoeller Konstantin , Kaiser Rolf , Chemnitz Jens , Scheid Christof , Holtick Udo 
Published: October 2019

Abstract
BK virus (BKV) is a human polyomavirus often acquired in childhood which can reactivate after allogeneic hematopoietic stem cell transplantation (HSCT). BKV reactivation may present indifferent ways from asymptomatic viruria to hemorrhagic cystitis or nephritis.
Methods
We evaluated 377 patients (159 male, 218 female, median age 52 years) who were transplanted at the University of Cologne between 2008 and 2013 to assess incidence and risk factors for BKV disease after allogeneic HSCT. All 377 patients were screened routinely for BKV in urine and serum. We defined BKV disease as hemorrhagic cystitis (HC) with BK virus >100.000/ml in urine, hematuria (2+ until 4+ in dipstick test) and negative urine microbiology. To find risk factors for BKV disease, the impact of conditioning, CMV status, graft-versus-host disease (GvHD), underlying disease, donor mismatch and stem cell source was analyzed.
Results
According to our definition, BKV cystitis occurred in 37 of 377 patients (9,8%) in the first 100 days after HSCT. In 14 of these 37 patients (37,8%) BKV was also detected in serum. BKV was detected in the urine of 194 patients (51,4%) at any time during the screening program, but only 19,1% developed hemorrhagic cystitis. Sixty-two percent of patients suffering from BKV disease were female, 37,8% male. BKV disease was diagnosed in 13,3% of patients not in complete remission as opposed to 5,4 % of patients in complete remission prior to conditioning (p = 0,010). BKV disease was less common in matched related or unrelated (7,7%) as compared to mismatched or haplo-identical donors (16,7%; p=0,012). Acute GvHD grade II to IV was described in 122 of 377 patients (32,36%). Of them, 13,9% suffered from BKV cystitis in comparison to 7,84% with no or grade I acute GvHD, which did not reach statistical significance. We could not detect any correlation between BKV disease and administration of cyclophosphamide, total body irradiation or anti-thymocyte globuline. There was no association with the graft source. BKV disease was no predictor for non-relapse mortality, overall and relapse-free survival.
Conclusion
BKV disease is a common complication after allogeneic HSCT. The virus can be found in the urine of about half of our transplanted patients, but only a small proportion developed an HC. State of remission before conditioning and donor mismatch are associated with the development of BKV disease, which had no impact on survival in our cohort.

Disclosures
Scheid: Novartis: Other: funding outside this work; Celgene: Other: funding outside this work; Janssen: Other: funding outside this work.
Optimal Dose of Antithymocyte Globulin in Conditioning Regimens for Unmanipulated Haploidentical Haematopoietic Stem Cell Transplantation: Long-Term Outcomes of a Prospective Randomised Trial
Authors: Chang Yingjun , Wang Yu , Mo Xiao-Dong , Zhang Xiao-Hui , Xu Lan-Ping , Yan Chen-Hua , Chen Huan , Chen Yu-Hong , Chen Yao , Han Wei , Wang Feng-Rong , Wang Jing-Zhi , Liu Kai-Yan , Huang Xiao-Jun 
Published: October 2019

Abstract
Background: Antithymocyte globulin (ATG) is an important component of conditioning regimens to prevent severe GVHD in patients undergoing unmanipulated haploidentical stem cell transplantation (haplo-SCT). However, the optimal dose of ATG is unknown.
Methods: In this open-label extension of a prospective, randomised trial, we compared the long-term outcomes of two ATG doses used in myeloablative conditioning before unmanipulated haplo-HSCT. Patients were randomly assigned (1:1) to 10 mg/kg (ATG-10) or 6 mg/kg (ATG-6) ATG. Patients and individuals assessing outcomes were masked to treatment allocation. Analysis of disease-free survival (DFS), GVHD-free/relapse-free survival (GRFS), relapse, non-relapse mortality (NRM), and chronic GVHD (cGVHD) included the entire population. Late effects were assessed in disease-free patients who had survived for at least 6 months and had received regular follow-up evaluations.
Results: Between December 2010 and May 2012, 224 patients were recruited. The median follow-up period was 1614 days (28-1929 days). The 5-year cumulative incidence was comparable between the ATG-6 and ATG-10 groups for relapse (12·8% vs. 13·4%, p=0·832) and NRM (11·6% vs. 17·0%, p=0·263). The 5-year probability of DFS was comparable between groups (75·6% vs. 69·6%, p=0·283). The 5-year cumulative incidence was higher with ATG-6 for cGVHD (75·0% vs. 56·3%, p=0·007; moderate-to-severe cGVHD: 56·3% vs. 30·4%, p<0·0001) and late effects (71·2% vs. 56·9%, p=0·043). The 5-year probability of GRFS was higher with ATG-10 (41·0% vs. 26·8%, p=0·008). In the multivariate analysis, ATG-10 was associated with lower cGVHD risk and improved GRFS.
Conclusions: ATG 10 mg/kg may be the optimal dose for conditioning regimens before unmanipulated haplo-SCT.

Disclosures
No relevant conflicts of interest to declare.