bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Impact of Aminoacid Substitution at Position 116 Class I HLA, in Unrelated Donor Transplants
Authors: Chiusolo Patrizia , Lamparelli Teresa , Sapienza Giuseppe , Dominietto Alida , Raiola Anna Maria , Di Grazia Carmen , Giammarco Sabrina , Gualandi Francesca , Metafuni Elisabetta , Rossi Monica , Minnella Gessica , Sica Simona , Bacigalupo Andrea , Angelucci Emanuele 
Published: November 2019

Background : The role of aminoacid substitution at position 116 of class I HLA antigens, has been the subject of several contributions, suggesting the possibility of permissive or non permissive mismatches.
Hypothesis. Permissive mismatched at position AA116, yield outcomes comparable to 10/10 matched grafts and superior to AA116 non permissive mismatches.
Patients: We have analyzed 358 unrelated donor transplants (UD) to test this hypothesis. All donors were matched at class II for DRB1 and DQ alleles; 226 were also matched at high resolution for A,B and C alleles; 84 had 1 permissive AA116 mismatch (Pmm), and 48 had 1 non permissive mismatch (NPmm). The 3 groups were comparable for patients age (p=0.5), donors age (p=0.9), diagnosis (p=0.2) and intensity of the conditioning regimen (p=0.4); both NPmm and Pmm had more patients with advanced disease, as compared to matched patients. The stem cell source was peripjheral blood for the large majority of patients.
Results.
The cumulative incidence (CI) of acute GvHD grade II-IV (p=0.01) and III-IV (p=0.001) was greater in patients with 1 allele mismatch, compared to matched patients, irrespective of AA116 permissive substitution. The CI of non relapse mortality (NRM) at 5 years 29%, 35%, 50% respectively in patients grafted from 10/10 matched donors, 1 allele Pmm donors and 1 allele NPmm donors (p=0.005). GvHD with or without infections, as a cause of death was recorded in 19%, 23% and 33% of the three groups respectively (p=0.1). The CI of relapse was respectively 18%, 30%, 20% (p=0.1). The actuarial survival at 5 years was 68% for 10/10 matched patients, 63% for 1 allele Pmm patients and 47% for 1 allele NPmm patients.
Conclusions. We confirm that aminoacid substitution at position 116 of class I HLA antigens, is a risk factor for non relapse mortality and survival. Patients with permissive mismatched AA116 donors have outcome comparable to patients grafted from matched donors.
Figure

Disclosures
Angelucci: Novatis: Honoraria, Other: Chair Steering Committee TELESTO protocol; Celgene: Honoraria, Other: Participation in DMC; BlueBirdBio: Other: Local advisory board; Jazz Pharmaceuticals: Other: Local advisory board; Roche: Other: Local advisory board; Vertex Pharmaceuticals Incorp., and CRISPR Therapeutics: Other: Participation in DMC.
The Neurotransmitter Receptor Gabbr1 Regulates Proliferation and Function of Hematopoietic Stem and Progenitor Cells
Authors: Elujoba-Bridenstine Adedamola , Shao Lijian , Zink Katherine , Sanchez Laura , Cox Brian , Pajcini Kostandin , Tamplin Owen J 
Published: November 2019

Hematopoietic stem and progenitor cells (HSPCs) have multi-lineage potential and can be used in transplants as a curative treatment for various hematopoietic diseases. HSPC function and behavior is tightly regulated by various cell types and factors in the bone marrow niche. One level of regulation comes from the sympathetic nervous system that innervates the niche and releases neurotransmitters to stromal cells. However, the direct regulation of HSPCs via cell surface expression of neural receptors has not been functionally explored. Using imaging mass spectrometry, we detected strong and regionally specific gamma-aminobutyric acid (GABA) neurotransmitter signal in the endosteal region of mouse bone marrow. GABBR1 is known to be expressed on human HSPCs (Steidl et al., Blood 2004), however its function in their regulation has not been investigated. Based on published mouse HSPC single cell RNA-seq data (Nestorowa et al., Blood 2016), we found that a subset of HSPCs expressed the GABA type B receptor subunit 1 (Gabbr1). We confirmed by surface receptor expression that a subset of mouse bone marrow HSPCs express Gabbr1 protein. Using the same single cell RNA-seq data as above, our own gene set enrichment analysis (GSEA) revealed positive correlation of Gabbr1 expression with genes involved in immune system processes, such as response to type I interferons. We generated a CRISPR-Cas9 Gabbr1 mutant mouse model on a C57/BL6 background suitable for hematopoietic studies. Analysis of Gabbr1 mutant bone marrow cells revealed a reduction in the absolute number of Lin-Sca1+cKit+ (LSK) HSPCs, but no change in the number of long-term hematopoietic stem cells (LT-HSCs). With further hematopoietic profiling, we discovered reduced numbers of white blood cells in peripheral blood that was primarily due to fewer B220+ cells. We show that Gabbr1 null HSPCs display reduced proliferative capacity, as well as diminished reconstitution ability when transplanted in a competitive setting. An in vitro differentiation assay revealed the impaired ability of Gabbr1 null HSPCs to produce B cell lineages. We tested our predicted association with type I interferon response by administration of poly(I:C) and found reduced HSPC proliferation in Gabbr1 null mice. Our results may translate well to humans, as a rare human SNP within the GABBR1 locus was found that correlates with altered leukocyte counts (Astle et al., Cell 2016). Our results indicate an important role for Gabbr1 in the regulation of HSPC proliferation and differentiation, highlighting Gabbr1 as an emerging factor in the modulation of HSPC function and behavior.

Disclosures
No relevant conflicts of interest to declare.
Long Range Sequencing Shows Improved Resolution in the Detection of Beta Globin Cluster Variants
Authors: Rangan Aruna , Hein Molly S. , Koganti Tejaswi , Jenkinson William G. , Hilker Christopher A. , Blommel Joseph H. , Porter Tavanna R. , Jen Jin , Hoppman Nicole , He Rong , Viswanatha David S. , Aleff Ross , Hoyer James D. , Wieben Eric , Klee Eric W. , Kipp Benjamin R. , Oliveira Jennifer L. 
Published: November 2019

Introduction: Globin gene sequencing has long been impeded by repetitive regions, homology and structural variation. Currently, long-read sequencing technology has been limited by high cost and error rate. These challenges can be mitigated by sample multiplexing of targeted capture sequencing and the use of consensus reads, respectively. This ongoing study analyzes the utility of long range sequencing technology in identifying historically problematic beta globin cluster mutations.
Materials and methods: Long range sequencing was performed on complex beta globin gene cluster cases utilizing the Sequel platform (Pacific Biosciences, Menlo Park, California, United States). Sample input was 1 microgram of DNA extracted from whole blood. Circular Consensus Sequence (CCS) FASTQ sequences files were mapped to reference genome (GRCh37/hg19) using NGMLR, followed by annotation of variants using a custom bioinformatics workflow. Comparison to multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing, array CGH, short-range NGS (300 bp), hemoglobin protein, and clinical data was performed.
Results: CCS read length varied from 1 to 10 kb, average 3.5. Analysis of the beta globin cluster region showed impressive superiority and comprehensiveness to Sanger, MLPA, array CGH and short range sequencing technology: 1) single nucleotide variants (SNVs) were identified to a sensitivity and specificity of 99.5%; 2) large structural variants (SVs) such as large deletions, duplications, insertions, crossovers and fusions spanning many kilobases were characterized in the heterozygous, homozygous and compound heterozygous state to a precise genomic coordinate (table 1); 3) phasing SNVs identified Hb S haplotypes (Central African region (CAR), Benin, Senegal, Arab-Indian and Cameroon).
Conclusion: 1) Long range sequencing holds significant promise in genotyping hemoglobin disorders. This method improves efficiency with potential for a single test offering with increased resolution, particularly in large deletion analysis. Complex cases greatly benefited from improved range and resolution. 2) The decreased requirement of blood volume for extensive testing is of particular interest for the pediatric population. 3) Genotyping of Hb S patients detected by positive newborn screening will help predict need for prophylaxis or therapy by assessing variant zygosity, phasing and haplotypes. In conclusion, we believe that long range sequencing has immense potential to contribute extensively to thalassemia and hemoglobinopathy diagnostics and is far superior to the currently available technologies for beta globin cluster analysis.

Disclosures
Jen: Celgene: Employment.
Hematopoietic Stem Cells Increase Quiescence during Aging
Authors: Kovtonyuk Larisa V. , Ashcroft Peter , Spaltro Gianluca , Tata Nageswara Rao , Skoda Radek C. , Bonhoeffer Sebastian , Manz Markus G. 
Published: November 2019

Introduction: Definitive hematopoietic stem cells (HSCs) sustain blood production from fetal development throughout life. In mice, most of steady state, young adult HSCs are in the G0 phase of cell cycle (quiescence), and are estimated to divide roughly once a month. Daily hematopoietic production is thus mainly sustained by highly proliferative downstream hematopoietic progenitor cells (HPCs). Aged haematopoiesis was demonstrated to be distinct from young haematopoiesis in various aspects such as i) a shift from lymphopoiesis to myelopoiesis, ii) functional decline of HSCs (self-renewal, homing), and iii) HSCs pool expansion. While several studies attempted to address whether changes in HSCs turnover during aging can account for the distinct aging associated phenotype and function, it remained to be determined whether aged HSCs overall cycle more or less frequently than young HSCs.
Methods: To construct data-based, quantitative models, we measured turnover rates and compartment sizes of populations of HSCs, HSPCs and granulopoiesis/granulocytes, i.e. a post-mitotic mature hematopoietic linage with a short half-life. We examined four age groups: 3 week, 2 month, 1 year and 2 year old mice. Mice in each group were i.p. injected every 4 hours with 1 mcg EdU up to a maximum time of 48 hours. HSC, HSPC and granulopoiesis/granoulocyte compartment sizes and snapshot cell-cycle analysis was performed by FACS at multiple sampling points in BM and peripheral blood (PB), respectively. Based on this data, we built a mathematical model of HSC turn-over and HSPC differentiation during ageing. Moreover, we evaluated HSC cycling by CFSE dilution in steady-state transplantation experiments (as described before; Takizawa et al., J Exp Med 2011).
Results: In line with previous reports, the HSCs compartment size gradually increased with age from 3wk old mice to 2 year old mice. In sharp contrast, cycling activity of HSCs as determined by EdU incorporation decreased gradually and significantly with increasing age. This was driven by decreased activation from the quiescent state, while the time that actively cycling HSCs require to progress through cell-division remains constant with age. Multipotent Progenitor (MPP) cycling showed a non-significant trend towards slower turn-over. These results were confirmed by complementary CFSE-dilution experiments. Mathematical modeling of HSC proliferation and differentiation revealed a higher probability of self-renewing divisions in 3 week old mice as compared to 2 month, 1 and 2 year old mice, with the latter both having nearly equal chances of self-renewing versus differentiating divisions.
Conclusions: Our data clarifies the long-standing question, how the HSC pool increases with age. Instead of an increase in active cycling, an increase in HSC quiescence is responsible for the increased size of the HSCs pool in aging.

Disclosures
No relevant conflicts of interest to declare.
Fetal Hemoglobin Promotes P. Falciparum Growth in Sickle Cell Disease Erythrocytes
Authors: Archer Natasha M. , Duraisingh Manoj 
Published: November 2019

Introduction: We have demonstrated that sickle hemoglobin (HbS) polymerization in low oxygen (O2) is the main driver of sickle cell trait (AS) resistance to P.falciparum malaria. This suggests that homozygous sickle cell disease (SS) individuals should have even greater resistance to malaria. Instead SS individuals infected by P. falciparum often have increased malaria morbidity and mortality compared to individuals with normal hemoglobin (AA) or AS. The reasons for this paradox are poorly understood. We propose that fetal hemoglobin (HbF) inhibits polymerization thereby allowing parasite proliferation. To test this hypothesis the following experiments were performed.
Methods: AA and SS erythrocytes were collected within two weeks of the assay date. P. falciparum 3D7 parasites were used for growth assays. Growth assays were performed at 1, 3, 5, 7.5, 10 and 16% O2 using synchronized schizonts obtained by magnet purification via the MACS system. Staging was performed via light microscopy analysis of May-Grünwald-Giemsa-stained cytospins. Parasite Multiplication Rate (PMR) was determined via flow cytometry using parasitemia at 64 and 16 hours post infection (hpi) as previously described.
Results: In contrast to AS erythrocytes, SS RBCs contain varying amounts of HbF that differ by cell and by individual. In hypoxic SS RBCs with low (3.8%) HbF, P. falciparum parasites remain immature (early trophozoites) in 1, 3, and 5% O2 (Fig. 1) at 36 hpi, when they should have matured into schizonts. However, in SS RBCs with high (22.6%) HbF, 12, 16 and 27% of parasites matured into schizonts in 1, 3 and 5% O2 respectively compared to 0, 0, and 1% in the SS RBCs with 3.8% HbF. Preliminary data also demonstrate that the parasite multiplication rate (PMR), a surrogate of proliferation, in SS RBCs improves with increasing O2 and HbF. Using SS/Hereditary Persistence of Fetal Hemoglobin RBCs with 42% HbF, proliferation in 5% O2 or higher is exponential with a PMR of 2.6 or greater (Fig. 2). In RBCs isolated from SS patients treated with hydroxyurea with HbF of 26.4 or 21%, PMR is 2 or more in 7.5% O2 or greater.
Conclusion: These data demonstrate that HbF promotes P. falciparum growth in SS erythrocytes. In addition, the data initiates a resolution of the malaria paradox. HbS in both hypoxic AS and SS surely inhibits P. falciparum growth due to HbS polymerization at low O2. But high levels of anti-sickling HbF observed in erythrocytes from many individuals with SS reverses this inhibition despite the increased content of HbS in SS individuals. Even small increases in P. falciparum proliferation within the broadly compromised SS host may enhance the malaria associated morbidity and mortality seen in this population. These data further suggest the importance of anti-malarial prophylaxis in patients with SS especially those treated with hydroxyurea or anti-sickling agents.

Disclosures
No relevant conflicts of interest to declare.
Outcome of Patients with Newly-Diagnosed Therapy-Related AML (t-AML) and AML with Myelodysplasia-Related Changes (AML-MRC) Treated with Liposomal Daunorubicin and Cytarabine (CPX-351) at an Academic Community-Based Health System
Authors: Khoury John , Anusim Nwabundo , Macari David , Duenn Angela , Jaiyesimi Ishmael 
Published: November 2019

Method: A query was generated to retrieve all cases treated with CPX-351 at Beaumont Health System. SPSS software was used to perform descriptive statistics.
Results: We found 11 cases treated with CPX-351 from 2017 to 2019. Six patients were male and 5 females. The median age at diagnosis was 67. Eight patients were Caucasian, two African American and one Asian. The median body mass index (BMI) was 27.1 and median body surface area (BSA) was ! treated with CPX-351 after a diagnosis of AML-MRC and 4 patients (36.3%) with t-AML. The median hospital stay was 35 days. The median units of red blood cells transfusion during hospital stay were 9 units and the median number of transfused platelet units was 6. Only 2 patients (18.1%) achieved complete response (CR) and 2 patients (18.1%) achieved complete response with incomplete blood count recovery (CRi). Three patients developed allergic reactions presented as a rash, 2 patients had significant bleeding and all 11 patients developed neutropenic fever. Three patients died during induction therapy and 2 patients underwent allogeneic hematopoietic stem cell transplant. Six patients (54.5%) died within 90 days of starting induction therapy.
Conclusion: t-AML and AML-MRC remain to have dismal prognosis. Our study highlights the high early mortality rate associated with induction therapy with CPX-351. The most common cause of death was refractory or relapsed leukemia. The high incidence of neutropenic fever was also more prevalent to what has been reported.

Disclosures
No relevant conflicts of interest to declare.
Imatinib (IMA) Low Dose Reduction Program (LDR) in Chronic Myeloid Leukemia Ph+ (CML Ph+) in a Single Reference Institution in Mexico City. Ongoing Preliminary Results
Authors: Hurtado Monroy Rafael , Vargas-Viveros Pablo , Silvia Carrillo Munoz 
Published: November 2019

Imatinib (IMA) total stop therapy has interesting worldwide results in selected patients (pts) with CML Ph+ who have achieved 4 to 5 log reduction of BCR/ABL copies, however there is no information of LDR.
The observation that IMA intolerance induces a dose reduction in our cases and, the fact that during the follow-up of their RT-PCR for BCR/ABL remains in > 4 log reduction after two years, was the rationale for an open prospective LDR program with the main objective to optimizing CML Ph+ treatment due to economical limitations and drug availability in Mexico. Herein we report our ongoing preliminary results:
A total of 73 of 152 (48%) pts was eligible for LDR after achieving Major Molecular Response (MMR) who reduced of > 4 log in at least two RT-PCR determinations in one or two years. The molecular response was assessed by peripheral blood real-time polymerase chain reaction at selected laboratory, able to express the results according to the International Scale (Hughes T, et al. Blood 2006;108:28-37). LDR included reduction of IMA to 25% of the 400mg for the chronic phase and 33% for the accelerated phase or those who reduced from 800 to 600 and even 400mg.
Among 73 pts, 11 (15%, historic group) were intolerant to IMA and their median dose reduction was 50% (Min-Max; 25-100%) with a median follow-up of 28 months (12-48) with only one relapse after 48 months of dose reduction. The LDR included 62 of 152 (40%) pts with a median age of 37 years (18-74), the gender was, female/male (n-30/32), the median time (MT) duration of CML was 8 years (4-18 years), the MT of reduction was 16 months (12-30). In 52 of 62 pts (83%) the proportion of LDR was 25% and the others in the range of 800 to 400mg. Molecular relapse was seen in 4 cases (6.4%) of the 25% reduction group and in 2 cases of the 50% (600-to 800mg) that represented 1.2%.
The LDR program is an interesting alternative for optimizing CML Ph+ IMA treatment with range from one to two and a half years in MMR with a 7.2% global relapse rate which is lower than the total interruption of TKIs in previous literature reports; however, the inclusion of more pts with follow-up as well, is mandatory to validate our program and the economic impact.

Disclosures
No relevant conflicts of interest to declare.
Novel Thermostable Inhibitor of Contact Activation Tica Effectively Blocks Contact Activation in Low Tissue Factor Thrombin Generation
Authors: Van De Berg Tom , Suylen Dennis P.L. , Thomassen M.G.L. Christella D. , van Oerle Rene , Spronk Henri M.H. , Apitz Rafael , Hemker H. Coenraad , Hackeng Tilman M. 
Published: November 2019

Background: Thrombin generation and other clotting assays suffer from a wide variation of pre-analytical variables. One of those pre-analytical variables is contact activation through blood withdrawal methods, different syringes, differences in blood coagulation tubes, blood transport and sample handling. It has been shown that the addition of contact activation inhibitors in low tissue factor activated thrombin generation leads to a correction of the, in these circumstances significant, increase in thrombin generation due to contact activation. We compare the novel 'thermostable inhibitor of contact activation' (TICA) to the current standard 'corn trypsin inhibitor' (CTI).
Aim: Comparing the effectiveness of novel contact activation inhibitor TICA to the current standard CTI in low tissue factor-induced thrombin generation and recalcification in sodium citrate anticoagulated platelet poor plasma (PPP) and platelet rich plasma (PRP).
Methods: We compared TICA, Corn trypsin inhibitor and plasma without contact activation inhibitors in low tissue factor PPP thrombin generation and in PRP recalcification thrombin generation, the latter the most sensitive condition for contact activation. In addition, we compared low tissue factor activated thrombin generation in plasma from severe hemophilia A patients with and without TICA during and after blood drawing. Thermostability - as a measure of shelf life - was measured and compared to CTI.
Results: TICA is able to fully block contact activation in PRP recalcification experiments and is comparable to CTI in doing so. TICA significantly lowers low tissue factor induced thrombin generation by blocking contact activation. Pre-loading vacuum blood collection tubes with contact activation inhibitors is superior in inhibiting contact activation compared to addition of the inhibitor during the thrombin generation assay itself. TICA did not alter coagulation activity when added to FXIIa deficient plasma in thrombin generation. In contrast to CTI TICA is heat stable which will be of benefit to shelf life of pre-loaded blood drawing tubes.
Conclusion: TICA is able to fully block contact activation and has several advantages over CTI.

Disclosures
No relevant conflicts of interest to declare.