bluuebunny/crossref_metadata_2025_split huggingface dataset:
KLF1 Dependent Pathways In Developmental Globin Gene Switching
Authors: Felice Alexander E. 
Published: October 2019

Abstract
Abstract 5162
We provide additional data on members of the family from Malta with Hereditary Persistence of Fetal Hemoglobin (HPFH) due to KLF1 haplo-insufficiency. The data indicated a possible role of additional loci in the pathway of globin gene control. We showed that KLF1 functions as a master regulator of erythropoiesis and developmental globin gene switching (Borg et al., Nature Genetics doi: 10. 1038/ng.630, 2010), at least partly through BCL11A. Given the phenoytpes of the HPFH heterozygotes, the truncating KLF1 p.K288X was best described as a dominant mutation with variable penetrance; most likely due to interplay with other regulatory factors that we have been seeking.
Genome-wide association analysis, in the context of genome-wide expression profiles from cultured Human Erythroid Progenitors (HEPs) of critically informative family members, revealed additional loci of potential interest.
The effect of the Hb F inducer Hydroxyurea on the gamma globin profiles of the KLF1- (p.K288X) HPFH HEPs was enhanced compared to the wild type, and 74 loci were differentially expressed.
It is anticipated that extensive re-sequencing of these new targets may reveal the extent of the molecular pathways under control of KLF1 in erythropoiesis and globin gene switching, and in particular those that may be targeted for therapeutics in patients.

Disclosures:
No relevant conflicts of interest to declare.
Severe Fetal and Neonatal Anemia Due to Heterozygosity for a 198 Kb Deletion Removing the Entire β-Globin Gene Cluster
Authors: Verhovsek Madeleine , Shah Nirmish , Thornburg Courtney , Koenig Sara , Wilcox Ibifiri , Barros Tiago , Steinberg Martin H. , Luo Hong , Chui David H.K. 
Published: October 2019

Abstract
Abstract 5171
Infants who are heterozygous for a number of (γδβ)0-thalassemia deletions are known to present with neonatal hemolytic anemia (Koenig et al, Am J Hematol 84:603, 2009). The breakpoints for most of these large deletions have not been identified. Molecular diagnoses of these deletions therefore can be challenging. We now report an infant girl of Irish/Scottish descent with self-limited fetal and neonatal hemolytic anemia, in whom we have defined the extent of the large deletion removing the entire β-globin gene cluster. At 32-week of gestation, fetal tachycardia prompted percutaneous umbilical cord blood sampling, which revealed a hematocrit of 14%. Umbilical cord transfusion was undertaken followed by Caesarean section delivery. Brisk hemolysis continued in the first few days of life (reticulocyte count of 22 – 24%), with no other causes of hemolysis identified. In the following weeks, the infant was transfused on three occasions. After 2-month of age, she became transfusion-independent with a stable microcytic anemia. The infant's mother also had a history of hemolytic anemia requiring transfusion in the neonatal period, and subsequently became transfusion independent with a microcytic anemia. The mother and several of her family members were extensively investigated, and shown to have a novel (γδβ)0-thalassemia deletion of over 100 kb (Pirastu et al, J Clin Invest 72:602, 1983). Multiplex ligation-dependent probe amplification (MLPA) was carried out in the genomic DNA from the present neonate. The infant was heterozygous for a large deletion spanning at least from 5′ to the HS 5 of the LCR to 3′ of the β-globin gene. Sequential gap-PCR reactions and nucleotide sequencing were done. The deletion was characterized with its 5` breakpoint at nt 5,376,341 and 3` breakpoint at nt 5,178,572 (GenBank NT_009237). The deletion measures 197,770 bp, removing the β-globin LCR, all of the β-like globin genes, and several olfactory receptor genes. A diagnostic gap-PCR test was established for detection of this deletion. This case illustrates the syndrome of neonatal hemolytic anemia caused by large deletions removing the entire β-globin gene cluster. MLPA is a useful tool to screen for these deletions. The pathophysiology of these self-limited and sometimes severe fetal and neonatal hemolytic anemias is presently not understood. We speculate that expression of α-hemoglobin stabilizing protein (AHSP) and/or the proteolytic capacity to degrade excess α-globin chains within erythroid cells might be diminished during fetal and neonatal development, accounting for increased red cell membrane damage and hemolysis in affected fetuses and neonates.

Disclosures:
No relevant conflicts of interest to declare.
Complement Enhances Foxp3+ T Regulatory Cell Induction by B Cell Lymphoma
Authors: Veeramani Suresh , Dahle Chris , Weiner George 
Published: October 2019

Abstract
Abstract 5176
The effect of complement on the immune system is complex and includes inhibition of aspects of the immune response. For example, we previously reported that C3b can inhibit the ability of mAb to activate NK cell-mediated antibody-dependent cellular cytotoxicity and that polymorphisms in C1q associated with lower C1q levels correlate with better therapeutic outcome. Polymorphisms in C5 and C9 complement components have also been shown to be associated with lymphoma risk. The current studies were designed to assess whether complement components may exert their inhibitory effects through the immune regulatory network, such as that mediated by T regulatory (Treg) cells.
Peripheral Blood Mononuclear Cells (PBMCs) from healthy donors were co-cultured with Raji B cell lymphoma cells for 48 hours in the presence of unmodified autologous human serum or complement-depleted serum [heat-inactivated (HI) or cobra-venom factor (CVF)-treated serum]. Cells were stained with anti-CD3-APC-Cy7, CD4-PE-Cy7, CD25-PerCP-Cy5.5 and Foxp3-APC and the percent of CD4+ T-cells that were CD25highFoxp3+ (henceforth referred to as Tregs) determined. The ability of cells cultured in this manner to inhibit granzyme B expression in PHA-activated CD8+ autologous T cells was also determined.
Tregs were not induced when PBMCs were cultured without Raji cells regardless of whether the cells were cultured in serum (data not shown). However, PBMCs co-cultured with Raji B lymphoma cells showed significantly higher percentages of Tregs in the presence of unmodified human serum (*p=0.0387) when compared to HI or CVF-treated human serum (**p=0.0370 and 0.0227, respectively) (Fig. 1). Tregs enriched from PBMCs cultured with unmodified human serum and Raji cells also showed an increase in immunosuppressive function, as determined by their inhibitory effect on PHA-induced GrzB expression by autologous CD8+ T cells (*p=0.0029) (Fig. 2).unmodified or complement-inactivated humanunmodified or complement-inactivated human
We conclude that complement enhances generation of Treg cells induced by B lymphoma cells as indicated by both phenotypic and functional studies. Ongoing studies are exploring the role of individual complement components in Treg induction, and the possibility of using complement depletion as a strategy to boost immune responses against lymphoma.

Disclosures:
No relevant conflicts of interest to declare.
Protected by Nature: Effects of Exercise In Non-Severe Haemophilia Patients
Authors: Uijl Ingrid E.M. den , Groen Wim G , van der Net Jan Jaap , Grobbee Diederick E , de Groot Philip G , Fischer Kathelijn 
Published: October 2019

Abstract
Abstract 545

Background:
An increase of clotting factor VIII (FVIII) within 10 minutes after physical exercise has been reported in healthy subjects and patients with von Willebrand disease. The mechanism of FVIII increase through exercise is thought to be similar to that of desmopressin, which increases FVIII. Therefore, moderate and mild haemophilia A patients should also be able to increase their endogenous FVIII levels after exercise. A proof of principle study was performed to test this hypothesis.

Methods:
Changes in coagulation associated with exercise of 15 haemophilia A patients, aged 20–39 years, were measured. Eight had moderate haemophilia (FVIII 1–5 IU/dl), 7 had mild haemophilia with endogenous FVIII activity levels at the lower end of the range (FVIII 6–16 IU/dl) Patients were encouraged to cycle until volitional exhaustion, on an electronically braked cycle ergometer. Blood samples were drawn before and 10 minutes after the incremental exercise test. Blood was frozen and analyzed for FVIII, von Willebrand factor (vWF) and von Willebrand propeptide (vWpr), in one batch to avoid inter-assay variation. In mild haemophilia patients, changes in coagulation profile were compared to DDAVP tolerance tests.

Results:
All 15 patients had an increased FVIII level after exercise. Median relative increase was 2.5 times (interquartile range (IQR)= 1.2–4.0). The relative increase did not vary across severities (median relative increase of FVIII was 2.4 times (IQR= 1.3–4.0) for moderate haemophilia patients compared to median 2.5 times (IQR=1.2-4.0) in patients with mild haemophilia Mann-Whitney: p =0.91). However, the absolute FVIII levels were very different with a median FVIII level of only 9 IU/dl; IQR= 8–11 IU/dl) in patients with moderate haemophilia, compared with a much higher median FVIII level of 21 IU/dl; IQR=19-44 IU/dl) attained in patients with mild haemophilia. vWF and vWFpr also increased independent of severity (p=0.3-0.6) in all patients, median 50%; IQR 30–79% for vWF and median 165%; IQR 130–244% for vWFpr, reflecting acute release of coagulation factors. DDAVP response tests were available only for patients with mild haemophilia. Relative increase after DDAVP administration was median 3.5 times (IQR= 2.5–4.5), similar to increase in FVIII after exercise (Wilcoxon: p=0.14).

Conclusion:
Although normal levels were not achieved, the increase of FVIII activity during exercise may reduce bleeding risk and therefore help promote participation in sports and other strenuous activities for moderate and mild haemophilia A patients with modest endogenous FVIII activities who might be considered at high bleeding risk.

Disclosures:
No relevant conflicts of interest to declare.
Generation of HIV Resistant and Functional Macrophages From Hematopoietic Stem Cell Derived Induced Pluripotent Stem Cells
Authors: Kambal Amal , Mitchell Gaela , Cary Whitney , Gruenloh William , Jung Yunjoon , Nacey Catherine , Kalomoiris Stefanos , Lindsey Matt , Bauer Gerhard , Nolta Jan A. , Anderson Joseph S. 
Published: October 2019

Abstract
Abstract 562
Induced pluripotent stem cells (iPSCs) have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from adult individuals. By developing iPSCs to treat HIV, there is the potential for generating a continuous supply of therapeutic cells for transplantation into HIV infected patients. In this study, we have utilized human hematopoietic stem cells (HSCs) to generate anti-HIV gene expressing iPSCs for HIV gene therapy. HSCs were de-differentiated into continuously growing iPSC lines with four reprogramming factors and a combination anti-HIV lentiviral vector containing a CCR5 shRNA and a human/rhesus chimeric TRIM5α gene. Upon directed differentiation of the anti-HIV iPSCs towards the hematopoietic lineage, a robust quantity (>35%) of colony forming CD133+ HSCs were obtained. These cells were further differentiated into functional end-stage macrophages which displayed a normal phenotypic profile. Upon viral challenge, the anti-HIV iPSC derived macrophages exhibited strong protection (>3 logs) from HIV-1 infection. Here we demonstrate the ability of iPSCs to develop into HIV-1 resistant immune cells and highlight the potential use of iPSCs for HIV gene and cellular therapies.

Disclosures:
No relevant conflicts of interest to declare.
The Activated K-RAS Protein Accelerates Human Derived-MLL/AF4 Induced Leukemo-Lymphomogenicity In Transgenic Mice Model
Authors: Tamai Hayato , Miyake Koichi , Takatori Miyuki , Miyake Noriko , Yamaguchi Hiroki , Dan Kazuo , Shimada Takashi , Inokuchi Koiti 
Published: October 2019

Abstract
Abstract 61
Acute lymphoblastic leukemia (ALL) with MLL/AF4 fusion is a common infant leukemia associated with a poor prognosis. Research into the molecular mechanisms underlying ALL with MLL/AF4 has been hampered by the absence of a good animal model: there is a large discrepancy between the time to disease onset in infants and the long latency seen in previously established murine models (human derived-AF4 knock-in and inverter model). We speculated that the absence of associated K-RAS mutations could explain the long latency seen in murine models of ALL with MLL/AF4. To establish a model combining MLL/AF4 fusion and activated K-RAS, we first established MLL/AF4+ transgenic (Tg) mice through overexpression of human MLL/AF4 fusion protein. These MLL/AF4+ Tg mice exhibited splenomegaly and lung tumors at 8–12 months of age (median, 12 months), and histopathological analysis showed disruption of the spleen structure and infiltration of the liver, lung and spleen by lymphoma cells consisted of pro B-cells (CD45R/B220+CD43+). Western blot analysis showed expression of both MLL/AF4 and HoxA9 was upregulated in all of the tissues tested. Infiltration of tumor cells into the peripheral blood was observed after a long latency period in MLL/AF4+ Tg mice (median, 14 months), at which time abnormal lymphocytes accounted for 7.2±1.2% (n=5) of white blood cells. FACS analysis showed these abnormal lymphocytes to be CD45R/B220+CD43+CD19+ cells, which is the same phenotype as the lymphoma cells. To develop a more aggressive oncogenic model mouse, we generated MLL/AF4+K-RAS mutation+ Tg mice by crossing MLL/AF4+ Tg mice and K-RAS mutation (G12D)+ Tg mice. The resultant mice developed lymphoma significantly earlier (median, 6 months) than MLL/AF4+ Tg mice (median LFS: 5.5 months vs. 12 months, P=0.001). Infiltration of tumor cells into the peripheral blood was observed after a short latency period in MLL/AF4+K-RAS mutation+ Tg mice (median age, 6 months), at which time leukemia cells accounted for 3.2±0.9% of WBCs, suggesting the collaborative acceleration of leukemo-lymphomogenicity through overexpression of MLL/AF4 and mutant K-RAS. We anticipate that the MLL/AF4+K-RAS mutation+ Tg mouse model will be useful for studying the molecular mechanisms of MLL/AF4 leukemogenesis and for developing new therapies against MLL-related malignancies.

Disclosures:
No relevant conflicts of interest to declare.
Tyrosine 201 Is Critical for Transformation Mediated by JAK2V617F
Authors: Yan Dongqing , Mohi M. Golam 
Published: October 2019

Abstract
Abstract 626
The JAK2V617F mutation is associated with most cases of Ph-negative myeloproliferative neoplasms (MPNs). In normal hematopoietic cells, JAK2 becomes activated upon cytokine stimulation. The JAK2V617F protein is a constitutively activated tyrosine kinase that leads to activation of several intracellular signaling pathways and transformation of hematopoietic cells to growth factor-independence. Previous studies have shown several tyrosine residues within JAK2 are phosphorylated upon growth factor stimulation. However, the role of these tyrosine residues in signaling and transformation mediated by JAK2V617F remains unclear. Tyrosine 201 lies within a YXX(L/V) motif, which is a potential binding site for Src homology 2 (SH2) domain containing protein(s). Here, we sought to determine the role of tyrosine 201 in JAK2V617F-induced hematopoietic transformation by introducing a tyrosine-to-phenylalanine point mutation (Y201F) at this site. We observed that Y201F mutation significantly inhibited the cytokine-independent cell growth and induced apoptosis in Ba/F3-EpoR cells expressing JAK2V617F. The Y201F mutation also resulted in significant inhibition of JAK2V617F-mediated transformation of hematopoietic cell lines and bone marrow progenitors in vitro. Biochemical analyzes revealed that Y201F mutation almost completely inhibited the phosphorylation/activation of JAK2V617F. We found that Shp2 binds to the Y201 site of JAK2 and mediates the interaction between JAK2V617F and EpoR. In addition, we observed constitutive phosphorylation of Erk1/2 and p70S6 kinase mediated by JAK2V617F was inhibited by the Y201F mutation. Treatment of Ba/F3-EpoR-JAK2V617F cells with JAK inhibitor I or mTOR inhibitor rapamycin inhibited the phosphorylation of p70S6 kinase. Moreover, inhibition of p70S6 kinase by rapamycin treatment (0.5-1 nM) significantly impaired the proliferation and clonogenic growth of Ba/F3-EpoR-JAK2V617F cells. Together, these results suggest that p70S6 kinase is a downstream target of JAK2V617F, and mTOR inhibitor could be useful in treating JAK2V617F-evoked MPN. Finally, using a bone marrow transduction/transplantation approach, we found that Y201 plays an important role in efficient induction of MPN mediated by JAK2V617F.

Disclosures:
No relevant conflicts of interest to declare.
Autologous (auto) Versus Allogeneic (allo) Hematopoietic Cell Transplantation (HCT) for T-NHL: A CIBMTR Analysis
Authors: Smith Sonali , Burns Linda J , van Besien Koen , LeRademacher Jennifer , He Wensheng , Laport Ginna G. , Montoto Silvia , Maloney David G , Hari Parameswaran 
Published: October 2019

Abstract
Abstract 689

T-cell lymphomas (T-NHL) are aggressive, heterogeneous malignancies, marked by chemoresistance with fewer than one-third of patients achieving disease control with non-transplant therapies. There are no large comparative studies of autoHCT and alloHCT in T-NHL. We analyzed post-transplant outcomes of 241 recipients (≤ 60 yrs) of autoHCT (n=115) and alloHCT (n=126, 76 matched siblings) for T-NHL between 1996–2006 reported to the CIBMTR. Median age was 43 yrs for autoHCT and 38 yrs for alloHCT. In the alloHCT cohort: 59% received myeloablative conditioning, 60% had matched sibling donors. Histology was heterogeneous (Table 1).
Median time to transplant after diagnosis was similar in both groups (10m vs. 11m). AutoHCT, compared to alloHCT, was used more in ALCL (53% vs. 40%, p=0.04) and in those with less advanced and more sensitive disease: first complete remission (CR1) (35% vs. 14%, p=0.001), chemosensitive disease (86% vs. 60%, p<0.0001), and ≤ 2 lines prior therapy (65% vs. 44%, p<0.001). Median follow-up was 71m for autoHCT and 49m for alloHCT. Treatment related mortality (TRM) at 100 days in the autoHCT and alloHCT cohorts was 2% and 17%, respectively. For those beyond CR1, overall survival (OS) at 1 yr was 62% vs. 52% and at 3-yrs 53% vs. 41% for the autoHCT and alloHCT cohorts, respectively (Table 2). Relapsing T-NHL was the cause of death in 73% of autoHCT and 44% of alloHCT pts.
In multivariate analysis, alloHCT and more pretransplant chemotherapy regimens were associated with a higher risk of TRM. Relapse risk was higher with chemotherapy resistant disease and those not in CR. PFS and OS were similar after autoHCT or alloHCT with a greater number of prior chemotherapy regimens and chemotherapy resistance impacting survival outcomes adversely. Among 101 peripheral T cell lymphoma (PTCL) pts, the risk of relapse was significantly lower with alloHCT (RR 0.5, 95% CI 0.26–0.97) but countered by increased TRM for alloHCT (RR 3.03, 95% CI 1.02 –8.96). PFS and OS after autoHCT or alloHCT for PTCL were similar and adversely affected by pretransplant chemotherapy resistance. Among alloHCT recipients, there were no differences in outcomes based on donor source, conditioning regimen type or intensity.
In summary, pts undergoing autoHCT for T cell NHL appear to be selected for less advanced disease and greater chemosensitivity, making direct outcome comparisons with alloHCT difficult. AlloHCT is an effective strategy for high risk patients, albeit with significant TRM. Higher numbers of chemotherapy regimens prior to transplant adversely impacted both TRM and survival, suggesting that HCT should be considered earlier in the disease course.

Disclosures:
No relevant conflicts of interest to declare.
Trends In Mortality Rates and Age of Death In Sickle Cell Disease (SCD): 1979–2005
Authors: Lanzkron Sophie , Haywood Carlton 
Published: October 2019

Abstract
Abstract 736
While improvements in care have resulted in significant decreases in mortality for children with SCD, it is unclear if similar decreases in mortality rates have occurred in adults with the disease. The purpose of this research is to describe mortality rates and trends in age of death for those with SCD over the last 3 decades. We used the National Center for Health Statistics Multiple Cause of Death (MCOD) files to examine age at death and calculate mortality rates from 1979–2005. ICD9 and ICD10 codes for SCD were used as appropriate to identify sickle cell related deaths. Mortality rates were calculated as deaths per 100,000 African American population. The number of African Americans each year was determined using available census data. Trends in mortality rates were examined using negative binomial regression and age of death was examined using t-tests and linear regression. After excluding certificates with codes for sickle trait and those with multiple sickle codes we identified 16,654 sickle-related deaths. The age range was 0 to 107 years. Mean age of death was significantly different for men (33.4, 95% CI [33.0, 33.7]) and women (36.9, 95% CI [36.5, 37.4]). SCD was the most common listed underlying cause of death (COD) at 62.8%. Infection was the second most common COD (5.9%). Controlling for sex and the presence of infection as COD, the mean age of death increased significantly by 0.08 years (p<0.001) each year over the time period studied, with men on average dying 4.3 years earlier than women (p<0.001). The mean age of death in 2005 was 43 yrs for women and 37 yrs for men. Those with COD of pulmonary hypertension, stroke and renal disease had a significantly older age of death than those without those diagnoses, while having infection as the underlying COD was associated with a younger age at death. The overall mortality rate increased by 0.7% (p<0.001) each year over the time period studied. Mortality rates for adults and children over time are shown in Figure 1. The adult (>19 yrs) mortality rate increased by 1% (p<0.001) each year over the time period studied. The pediatric mortality rate decreased by 3% (p<0.001) each year over the time period studied. When controlling for the pediatric mortality rate the adult mortality rate increased by 1.6% (p<0.001) each year. This data confirms prior studies showing a significant decrease in mortality for children with sickle cell disease over the last 30 yrs. The mortality rate for the adult population appears to be steadily increasing over the same time period. It seems unlikely that this is due merely to an influx of younger patients surviving to adulthood. Further investigation as to the cause of the increasing mortality rate in adults is needed.

Disclosures:
No relevant conflicts of interest to declare.