bluuebunny/crossref_metadata_2025 huggingface dataset:
A Cost-Utility and Budget Impact Analysis of Allogeneic Hematopoietic Stem Cell Transplantation for Severe Thalassemic Patients In Thailand.
Authors: Hongeng Suradej , Pakakasama Samart , Sirachainan Nongnuch , Chuansumrit Ampaiwan
Published: October 2019

Abstract 1536

Background:

Hematopoietic stem cell transplantation (HSCT) is the only curative treatment available to severe thalassemic patients. The treatment, however, is very costly, particularly in the context of low and middle income countries, and no studies have been carried out to explore its economic justifiability. This study aimed to estimate the cost-utility of HSCT compared with blood transfusions combined with iron chelating therapy (BT-ICT) for severe thalassemia in Thailand, and to investigate the affordability of HSCT using a budget impact analysis.

Methods:

A Markov model was used to estimate the relevant costs and health outcomes over the patients’ lifetimes taking a societal perspective as recommended by Thailand's health technology assessment guidelines. All future costs and outcomes were discounted at a rate of 3% per annum. Primary outcomes of interest were lifetime costs, quality adjusted life years (QALYs) gained, and the incremental cost-effectiveness ratio (ICER) in Thai baht (THB) per QALY gained.

Results:

Compared to BT-ICT, the incremental cost-effectiveness ratio increased with patient age from 80,700 to 183,000 THB per QALY gained for related HSCT and 209,000 to 953,000 THB per QALY gained for unrelated HSCT among patients aged 1 to 15 years (US$1= 34 THB). The governmental budget impact analysis showed that providing 200 related HSCT to patients aged 1 to 10 years, in accordance with the current infrastructure limitations, would initially require approximately 90 million additional THB per year.

Conclusions:

At a societal willingness to pay of 100,000 THB per QALY gained, related HSCT was likely to be a cost-effective and affordable treatment for young children with severe thalassemia in Thailand.

Disclosures:

No relevant conflicts of interest to declare.
Multiple Apoptotic Defects In Hematopoietic Cellsfrom Mice Lacking Lipocalin 24p3.
Authors: Devireddy L.R. , Liu Zhuoming , Ciocea Alieta , Yang Amy , Wang Zhengqi , Bunting Kevin D. , Green Michael
Published: October 2019

Abstract 1560

The lipocalin mouse 24p3 has been implicated in diverse physiological processes including apoptosis, iron trafficking, development and innate immunity. Studies from our lab as well as others demonstrated the pro-apoptotic activity of 24p3 in a variety of cultured models. However, a general role for the lipocalin 24p3 in the homeostasis of the hematopoietic system has not been tested in vivo. To study the role of 24p3 in apoptosis, we derived 24p3 null mice on 129 SVE and C57BL/6 genetic backgrounds, which enabled us to test, for the first time, the in vivo role of 24p3 without any confounders. We find that homozygous 24p3-/- mice have apoptotic defects in several hematopoietic lineages, including neutrophils, cytokine-dependent mast cells, thymocytes and erythroid cells. The progressive accumulation of lymphoid, myeloid and erythroid cells in 24p3-/- mice demonstrate the importance of 24p3 in the regulation of homeostasis of hematopoietic cells in adults. Thymocytes isolated from 24p3 null mice displayed resistance to apoptosis-induced by Dexamethasone. In addition, Dexamethasone-induced apoptosis is also reduced in 24p3-/- mice. Collectively, these results indicate that 24p3 is an important target gene involved in GC-mediated apoptosis of lymphocytes and other hematopoietic cell types. Finally, our results also indicate that the genetic background of mice can modulate the effects of 24p3 deficiency. In summary, the results presented here, in conjunction with those of previous studies, reveal 24p3 as a regulator of the hematopoietic compartment with important roles in normal physiology and disease progression.

Disclosures:

No relevant conflicts of interest to declare.
IFNy Regulates the Balance Between Monocyte and Neutrophil Production During Immune Activation.
Authors: de Bruin Alexander M. , Touw Ivo , Nolte Martijn A.
Published: October 2019

Abstract 1562

Recruitment of the appropriate cells from the innate immune system to sites of infection is essential for the defense against pathogens. Depending on the type of invading pathogen, infection induces a rapid release from innate immune cells from the bone marrow, followed by an increased production of those myeloid cells that are most suitable to assist the ensuing immune response in the rapid eradication of the pathogen. However, it is thus far poorly understood what the cellular and molecular mechanism is that underlies this change in hematopoiesis.

Using a mouse model with increased numbers of IFNγ-producing T cells in the bone marrow, we have previously found that IFNg plays an important role in the production of myeloid cells and can directly inhibit the formation of eosinophils (De Bruin et al., Blood. 2010 Jun 29). Here we demonstrate that IFNg also skews the balance between monocyte and neutrophil production in the direction of the monocytes. Increased monocyte production was also observed in WT mice infected with LCMV, which was IFNg-dependent, as it was not observed in IFNg-deficient mice. Subsequently, we set out to investigate how IFNγ can modulate the balance between monocyte and neutrophil production. We found that IFNγ reduces the proliferation and differentiation of murine hematopoietic progenitors in vitro in response to G-CSF, while M-CSF responses were not inhibited, but even slightly increased. In addition, IFNγ reduces the capacity of GMPs to form granulocyte colonies in semi-solid cultures and increases their ability to form monocyte/macrophage colonies. On a molecular level, we demonstrate that IFNγ induces a strong upregulation of SOCS3 levels and thereby reduces the phosphorylation of STAT3 in response to G-CSF, thus explaining the reduction in granulocyte formation. The expression levels of M-CSF-R and G-CSF-R by GMPs were not affected by IFNγ, but we found that IFNγ does induce the expression of the monocyte-inducing transcription factors PU.1 and IRF8 in these cells. In conclusion, these data demonstrate that IFNγ can direct myelopoiesis in a lineage-specific manner, thereby modulating the production of the appropriate type of innate immune cells required to combat infection.

Disclosures:

No relevant conflicts of interest to declare.
Mastl Kinase Gene Disruption Causes Embryonic Lethality.
Authors: Johnson Helen Jan , Davizon Pavel D , Lopez Jose A. , Gilligan Diana M.
Published: October 2019

Abstract 1567

Microtubule-associated serine/threonine like kinase (MASTL kinase) was originally identified by genetic linkage as the probable mutant gene responsible for the platelet phenotype in a family with autosomal-dominant thrombocytopenia. MASTL kinase is the mammalian ortholog of Drosophila Greatwall kinase, which is required for cell cycle regulation. Affected members of the thrombocytopenic family carried a mutant MASTL kinase allele with a single nucleotide change that converted a glutamic acid codon to one specifying aspartic acid at amino acid 167 of the protein; no unaffected members of the family carried the mutant allele. Affected family members had a 60% reduction in the numbers of circulating platelets and megakaryocytes that were less mature and less polyploid than normal megakaryocytes. We have sought to determine how MASTL kinase affects platelet production and circulation by generating a strain of mice deficient in MASTL kinase. For this, we obtained embryonic stem (ES) cells containing a gene trap mutation within the murine Mastl gene from the Mutant Mouse Regional Resource Center of the National Center for Research Resources (MMRRC, University of California, San Diego). The mutation was generated by an insertional disruption, and the resulting mutant allele carries a beta–galactosidase/neomycin fusion expression cassette within the fourth intron of the Mastl kinase gene. Chimeric animals were bred and their offspring were genotyped by Southern blotting, confirming germ-line transmission. F1 heterozygote offspring were produced and appear developmentally normal and healthy, with platelet counts indistinguishable from those of their wild-type littermates. We have yet to observe a single weaned animal that is homozygous for the mutant allele in the F1 mice, indicating that MASTL kinase deficiency results in embryonic lethality. We observed decidual swellings at 13.5 days post coitus (dpc), which contained no traces of either embryos or yolk sac membranes. While this has made it impossible to genotype these malformed embryos, it does indicate that the embryos are viable to 3.5–5.5 dpc and are able to implant within the uterine horn. This unexpected phenotype demonstrates a significant and previously unknown role for Mastl kinase in embryonic development. We hypothesize that Mastl kinase may be involved in regulating cell differentiation during embryogenesis. In terms of role of MASTL kinase in familial thrombocytopenia, the mutant animals have allowed us to conclude that the thrombocytopenic syndrome is very unlikely to be the result of haploinsufficiency of MASTL kinase because heterozygous animals have normal platelet counts. Because of the dominant nature of the syndrome, this suggests that the mechanism involves either gain-of-function of the mutant allele or a dominant-negative inhibition of MASTL function specific to the megakaryocyte, two possibilities we are examining.

Disclosures:

No relevant conflicts of interest to declare.
Heat Shock Protein 47: A New Platelet Collagen Receptor
Authors: Kaiser William J , Sage Tanya , Sasikumar Parvathy , Kemp Frederick , Pugh Nicholas , Dobson Laura , Holbrook Lisa , Gibbins Jonathan M.
Published: October 2019

Abstract 158

We recently reported that Heat shock protein 47 (Hsp47), a well established collagen binding protein, was present on the surface of human platelets. In the current study we demonstrate that anti-Hsp47 antibodies and a small molecule inhibitor of Hsp47 selectively prevent aggregation of platelets induced with collagen fibrils. Inhibition of Hsp47 was also found to reduce the size of thrombi produced upon perfusion of blood under arterial flow conditions over collagen in vitro. Inclusion of Integrilin, to prevent platelet-platelet interactions via fibrinogen, indicated that the reduced size of thrombi was due to fewer direct platelet-collagen interactions. Collagen-induced tyrosine phosphorylation of Syk, an early marker of signalling from the collagen receptor GPVI was unaffected by the Hsp47 inhibitor, suggesting that any contribution to platelet function by Hsp47 may be independent of GPVI-proximal signalling. Surface plasmon resonance analysis of recombinant his-tagged human Hsp47 revealed its ability to interact directly with collagen fibrils, an interaction that was disrupted by the small molecule inhibitor of Hsp47.

Mouse platelets were also found to contain Hsp47, and aggregation induced with collagen was reduced by inhibitors of Hsp47. Infusion of Hsp47 inhibitor into mice increased tail bleeding times and volumes of blood lost, even in the absence of anticoagulant. This effect was likely due to inhibition of platelet function, since the thrombi formed in cremaster muscle arterioles using an intravital laser-injury model of thrombosis were smaller following administration of the Hsp47 inhibitor.

Together these data indicate Hsp47 to function on the platelet surface as a collagen receptor that potentially represents a new target for antithrombotic therapy.

Disclosures:

No relevant conflicts of interest to declare.
Bmi1 Confers Stress Resistance to Self-Renewing Hematopoietic Stem Cells.
Authors: Nakamura Shunsuke , Iwama Atsushi
Published: October 2019

Abstract 1607

The polycomb group (PcG) proteins form chromatin-modifying complexes that implement transcriptional silencing. There are two major PcG complexes, polycomb repressive complex (PRC) 1 and PRC2. PRC1 ubiquitylates histone H2A at lysine 119 and PRC2 trimethylates lysine 27 of histone H3. Among PcG proteins, Bmi1, a core component of PRC1, plays an essential role in the self-renewal and maintenance of various kinds of stem cells including hematopoietic stem cells (HSCs), neural stem cells, and leukemic stem cells. We previously reported that forced expression of Bmi1 using a Bmi1 retrovirus promotes symmetrical cell division of HSCs, resulting in a marked ex vivo expansion of multipotent progenitors and an enhancement of repopulating capacity of HSCs in vivo. However, the impact of overexpression of Bmi1 in HSCs remains to be precisely addressed. To this end, we generated a mouse line where Bmi1 can be conditionally overexpressed under the control of the Rosa promoter in a tissue-specific fashion by the Cre-LoxP system. We crossed the mice to Tie2-Cre mice (Tie2-Cre; Rosa-Bmi1fl/+) and induced overexpression of Bmi1 in hematopoietic cells in vivo. Real-time PCR analysis demonstrated that expression of Bmi1 inpurified bone marrow (BM) c-Kit+Sca-1+Lineage-marker- (KSL) cells was 6-fold higher in Tie2-Cre; Rosa-Bmi1fl/+ mice than control Tie2-Cre mice mice. Overexpression of Bmi1 did not significantly affect steady state hematopoiesis. The number of HSCs and progenitors (multipotent progenitors, CMPs, GMP, MEPs, and CLPs) and lineage composition (myeloid cells, B cells, and T cells) in BM of Tie2-Cre; Rosa-Bmi1fl/+ mice was not significantly changed compared to those in control mice. We then performed serial transplantation assay. The repopulating capacity of Tie2-Cre; Rosa-Bmi1fl/+ BM cells was comparable to those of the control cells in primary recipients. However, Tie2-Cre; Rosa-Bmi1fl/+ cells retained higher repopulating capacity during serial transplantation compared to those of the control cells. Moreover, ex vivo culture of Tie2-Cre; Rosa-Bmi1fl/+ HSCs for 10 days contained approximately 2-fold more high proliferative potential colony-forming cells (HPP-CFCs; colony diameter>1mm) and colony-forming unit-neutrophil/macrophage/erythroblast/megakaryocyte (CFU-nmEM) which retain multi-lineage differentiation potential along myeloid lineage than the control. Of note, cells in ex vivo culture of Tie2-Cre; Rosa-Bmi1fl/+ HSCs exhibited significantly augmented repopulating capacity in recipient mice and we are now engaged in competitive repopulation unit (CRU) assays to determine the net HSC expansion by overexpression of Bmi1 during ex vivo culture. These results indicate that overexpression of Bmi1 confers stress resistance to HSCs during ex vivo culture and serial transplantation. Our findings also provide Bmi1 as a potential target for efficient manipulation of HSCs ex vivo.

Disclosures:

No relevant conflicts of interest to declare.
Filamins Regulate Differentiation of Megakaryocytes and Platelets From Cultured Embryonic Stem (ES) Cells.
Authors: Kanaji Taisuke , Okamura Takashi , Newman Peter J.
Published: October 2019

Abstract 1618

Filamins (Flns) A and B are major non-muscle actin binding proteins that play important roles in cross-linking cortical actin filaments into three-dimensional networks. In addition to their role as cytoskeletal scaffolding molecules, filamins are also known to bind more than 30 other proteins, regulating their subcellular location and coordinating their ability to signal. The role of filamins in hematopoietic stem cell differentiation, however, remains unclear, in part because gene-targeted mice lacking filamins die early on in embryonic development. To investigate the role of filamins A and B in the differentiation of embryonic stem cells (ESCs) along the megakaryocyte/platelet axis, we designed shRNA-containing vectors that targeted both FlnA and B under the control of either the CMV immediate-early promoter (CMV-FlnABLow), or an endogenous Rosa26 promoter (Rosa26-FlnABLow). Compared with wild-type ESCs, FlnABLow ESCs formed small, tightly packed undifferentiated colonies that expressed high levels of the ESC transcription factor, Nanog, and low levels of ERK activity – all indicators of an undifferentiated state. Embryoid prepared from FlnABLow ESCs, were allowed to differentiate, and examined for markers of mesoderm differentiation (Flk-1) and megakaryocyte differentiation (CD41). Whereas Day 6 EB-derived FlnABnormal wild-type cells were 8% Flk-1 positive and 13% CD41 positive, Day 6 FlnABLow cells were 9% Flk-1 positive and only 4% CD41 positive, consistent with the notion that loss of Fln A and B results in a delay of mesoderm to hematopoietic differentiation. To evaluate the effect of Fln knockdown on the ability of the CD41-positive cells to further differentiate into megakaryocytes, form proplatelet extensions, and produce platelets, CD41 positive cells isolated from day 8 EBs were cultured in the presence of a thrombopoietin (TPO)-producing TERT stromal cell line. We found that FlnABLow CD41-positive cells formed far fewer and smaller megakaryocytes compared with their FlnABnormal wild-type counterparts. Proplatelets derived from FlnABLow cells exhibited an abnormal, enlarged morphology with swellings and thick shafts that released platelets prematurely, yielding platelets that were nearly twice the size of those derived from FlnABnormal cells. Taken together, we conclude that not only do filamins function prominently in hematopoietic cell differentiation, they also play an important role in platelet production, likely via their ability to by recruit and organize the necessary signaling molecules near the inner face of the plasma membrane.

Disclosures:

Newman: New York Blood Center: Membership on an entity's Board of Directors or advisory committees; Children's Hospital of Boston: Membership on an entity's Board of Directors or advisory committees.
Sickle-Cell Disease and Malaria: Evaluation of Seasonal Intermittent Preventive Treatment with Sulfadoxine-Pyrimethamine in Senegalese patients—a Randomized Placebo-Controlled Trial
Authors: Diop Saliou , Seck Moussa , Gueye Youssou Bamar , Soudre Fabienne , Fall Awa Oumar Touré , Sall Abibatou , Thiam Doudou
Published: October 2019

Abstract 1633

Malaria is a real public health problem in Africa, more than 300 million new cases and approximately two million deaths arise every year. Sickle-cell disease (SCD) patients are at high risk of developing malaria which is a major contributor to morbidity and mortality in this disease. In Senegal, malaria transmission is high during rainy season, between July and October, and it was noted that sickle-cell crisis are frequent during this period. Then we carried out a double-blind randomized controlled trial to compare the impact of monthly sulfadoxine-pyrimethamine (SP) during the high-transmission season versus placebo on malaria incidence and morbidity of sickle-cell anemia. Sixty (60) SCD patients were randomized either to receive three intermittent preventive treatment (ITP) with SP or placebo using the random permutation table with nine elements. The drug was administrated as follows: sulfadoxine 25 mg/kg and pyrimethamine 1.25 mg/kg and this treatment was given once during the following months: September, October, and November. Overall four episodes of malaria disease were diagnosed, all these cases in the placebo arm. Thus, overall prevalence was 6.6% and there was no other case of malaria in the SP arm during the study period. Parasitological diagnosis confirmed the presence of Plasmodium falciparum in all four cases. No patient death was encountered during the study. SP treatment was well tolerated as only one patient (1.6%) in the SP arm reported pruritis. A significant reduction of patients’ complaints (p= 0.002) and blood requirements (p = 0.001) was noted in the SP group; whereas, no impact was observed on vaso-occlusive crisis and hospitalization occurrence. Malaria prophylaxis by monthly intake of SP during the transmission period of the parasite reduced the prevalence of malaria and was safe in SCD patients leaving in malaria endemic area.

Disclosures:

No relevant conflicts of interest to declare.
Allogeneic Hematopoietic Stem Cell Transplantation Overcomes the Adverse Prognostic Impact of CD20 Expression In Both Adult and Pediatric Acute Lymphoblastic Leukemia.
Authors: Bachanova Veronika , Sandhu Karamjeet , Weisdorf Daniel J. , Verneris Michael , Burke Michael J , Cao Qing
Published: October 2019

Abstract 1681

The heterogeneous expression of CD20 antigen on leukemic blasts may harbor prognostic value and therapeutic potential. CD20 positivity (CD20+) has been recently associated with disease recurrence and shorter overall survival in adults with pre-B acute lymphoblastic leukemia (ALL). The influence of CD20 expression on outcomes following allogeneic donor hematopoietic stem cell transplantation (HCT) is not known.

Results:

We analyzed CD20 expression on pre-B ALL marrow blasts at diagnosis in 157 consecutive patients who underwent allogeneic HCT in first or second complete remission (CR) from 1999–2010. Thirty-two patients with no available flow cytometry data were excluded. Out of 125 evaluable patients, 52 (42%) were < 20 years of age; 73 (58%) were ≥20. All patients had high risk ALL and were in CR at HCT (57% in CR1, 43% in CR2). A majority of patients were Ph+ (60%) and received myeloablative conditioning (84%). Grafts were from sibling (36%), unrelated umbilical cord blood (UCB) (59%) and matched adult URD (5%). CD20+, defined as >20% expression on marrow blasts, was observed in 58 (46%) patients. 55% of children and 39% of adults were CD20+ and expression was similar in CR1 & CR2. Gender, donor source (sibling vs UCB), patient CMV serostatus, cytogenetics (Ph+ vs other), and conditioning (myeloablative vs reduced intensity) was similar in CD20+ and CD20- groups. Disease-free survival (DFS) at 3 years was 48% (95% CI 39–57%) for all patients; 42% (95% CI 30–54%) for CD20- patients, and 55% (95% CI 40–67%) for CD20+ patients (p=0.14). CD20+ expression did not significantly impact relapse rate or DFS in adults (Table) while in patients <20 years CD20+ expression was associated with a slightly favorable relapse rate. Similar overall survival (OS) and DFS was seen in both age groups, independent of CD20 expression. Treatment-related mortality was unaffected at 18% (95% CI 9–27%) and 19% (95% CI 9–29%) in CD20+ and CD20- cohorts, respectively (p= 1.00). In adjusted multivariate regression, the CD20+ group had a slightly but not significantly lower risk of relapse (RR 0.54 [0.27-1.09]; p=0.09), yet similar OS (RR 0.66 [0.38-1.14], p=0.13) and DFS (RR 0.67 [0.39-1.14], p=0.14) compared to the CD20- group.

Conclusion:

Pre-B ALL with a CD20+ immunophenotype reportedly confers higher risk of disease recurrence and unfavorable OS with conventional chemotherapy. Our data demonstrates that the poor prognosis associated with CD20+ ALL blasts is overcome by allogeneic HCT. In addition, post-HCT survival is promising in both adults and children, independent of CD20 expression. CD20 targeting monoclonal antibodies may further improve these outcomes.

Disclosures:

No relevant conflicts of interest to declare.
CD70 Expression on HTLV-1 Infected T Cells of Carriers and ATL Patients and Its Clinical Significance
Authors: Masamoto Izumi , Horai Sawako , Kozako Tomohiro , Yoshimitsu Makoto , Niimoto Junko , White Yohann , Uozumi Kimiharu , Suzuki Shinsuke , Arima Naomichi
Published: October 2019

Abstract 1730

Human T-lymphotropic virus type-1(HTLV-1) is the causative agent of adult T cell leukemia/lymphoma (ATL). HTLV-1 infected T cell growth or leukemogenesis in ATL is controlled by various host immune surveillance systems. Among them, CD70 on HTLV-1 infected T cells coupled with CD27 on virus specific cytotoxic T cells has been suggested to play an important role in ATL leukemogenesis. The CD70 molecule is the only known ligand for CD27, a member of the tumor necrosis factor (TNF) receptor superfamily 7. This negative immunoregulatory pathway downregulates cytotoxic T lymphocyte activity against CD70-expressing virus infected cells. In the present study, we examined CD70 expression on primary lymphocytes of HTLV-1 carriers and ATL patients, its relationship with HTLV-1 Tax protein expression, and the effect on CTL induction. CD70 expression was higher on peripheral blood mononuclear cells (PBMCs) of HTLV-1 infected carriers compared with healthy donors (p = 0.021, n = 21, Mann-Whitney U test), and higher in ATL patients compared to carriers (p = 0.045, n = 38, Mann-Whitney U test). CD70 expression may be observed in CD4 T cells, as well as B cells, but not in CD8 T cells or monocytes. CD70 expression in CD4 T cells is related to HTLV-1 infection, because of increased detection of HTLV-1 Tax protein during over night culture of CD70-expressing cells. Experiments using an ATL cell line, in which Tax expression is inducible by doxycycline stimulation, demonstrated enhanced CD70 expression when Tax protein was induced in HTLV-1 infected cells. Anti-CD70 antibody enhanced CD107a mobilization, a marker of recent cytotoxic degranulation, in HTLV-1 Tax specific CTLs in PBMCs from asymptomatic carriers in vitro, suggesting that the CD70/CD27 pathway plays an important role in the immune response to HTLV-1 infection in carriers, as well as ATL patients.

Disclosures:

No relevant conflicts of interest to declare.
NK-Cell Acquisition of Chemokine Receptors From Engineered Antigen Presenting Cells
Authors: Somanchi Srinivas S. , Gururajan Anitha , Cooper Laurence J.N. , Lee Dean A.
Published: October 2019

Abstract 1744

Natural Killer (NK) cells are able to extract fragments of cell membrane from antigen presenting cells through the immunological synapse, functionally incorporating the membrane receptors contained therein by a process called trogocytosis. Recently it was demonstrated that NK cells can acquire functional CCR7 from dendritic cells through this process and migrate in response to chemokines (CCL19 and CCL21). We investigated whether this process could be used to transiently modify NK cells ex vivo without genetic intervention.

In previous work, we developed a K562-based artificial antigen presenting cell (aAPC) expressing membrane-bound IL-21 (K562-cl9-mIL21) which enables robust NK-cell expansion, and determined that NK cells did not express detectable CCR7 during this process. To investigate trogocytosis in this system, we genetically modified K562-cl9-mIL21 to express membrane-bound CCR7 (K562 cl9 mIL21CCR7) using the Sleeping Beauty transposon/transposase system. After 24 hours of co-culture the NK cells cultured with K562-cl9-mIL21CCR7 demonstrated marked surface expression of CCR7 compared to NK cells cultured on K562-cl9-mIL21 (Figure 1a). In kinetic experiments using three independent donors, CCR7 peak uptake occured at 24 hours (Figure 1b), followed by a decline that corresponded with the loss of aAPCs in the cultures due to lysis by NK cells. This demonstrates that NK cells can be transiently modified during in vitro expansion to bear receptors that are otherwise not a normal part of their transcriptional repertoire. We are currently establishing the functionality of trogocytosed receptors in vitro and in vivo, and investigating the kinetics of CCR7 persistence after removal of the aAPCs. However, even transient expression as demonstrated might be sufficient for bestowing novel NK-cell migration ability in vivo in response to chemokine signaling, giving the engineered NK cells ability to reach desired tissue targets.

Disclosures:

Lee: Altor BioScience Corp: Research Funding; Celgene: Research Funding.
Phosphatase RNA-Interference Lethality Screening with Suberoylanilide Hyrdoxamic Acid In Myeloid Cell Lines
Authors: Bogenberger James , Kassner Michelle , Chow Donald , Yin Holly , Tibes Raoul
Published: October 2019

Abstract 1851

Background:

In previous work we performed RNAi sensitizer screening of epigenetic modulation with 5-Azacytidine and the human kinome. Few kinase hits were identified. Therefore we asked if phosphatases might be involved in modulating epigenetic therapies. Thus in order to identify potentiators of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) we performed high-throughput RNA-interference (RNAi) lethality screening using the myeloid cell lines HEL, THP-1 and TF-1 in combination with SAHA.

Methods:

A small-interfering RNA (siRNA) library targeting 206 phosphatases with 4 sequences per target gene was used for this study. siRNA were transfected in a single-siRNA-per-well format in 384-well plates using commercial lipid-based transfection reagents. Non-silencing siRNA were included as negative controls to access non-specific siRNA toxicity and universal lethal siRNA were included as positive controls to indirectly assess functional transfection efficiency. After a 96 hour incubation period, cell viability/proliferation was measured with Cell Titer Glo. For analysis of screen data, log2 values of (SAHA + siRNA treatment/siRNA treatment only) were calculated. Potentiation was defined as two standard deviations from the median log2 ratio of all siRNA (excluding positive controls) on a plate-by-plate basis. Hits were defined as ≥ 2/4 siRNA sequences meeting potentiation criteria.

Results:

Transfection efficiency was 95, 90 and 70% for HEL, THP-1 and TF-1, respectively. All three cell lines were similarly sensitive to SAHA with IC50 values of 0.3, 0.4 and 0.6 μM for HEL, THP-1 and TF-1, respectively. Resulting SAHA concentrations for screens were between the IC5 to IC30. For THP-1, 5 phosphatases qualified according to the hit selection criteria applied indicating strong potentiation to SAHA. Of these 5 hits, 3 did not exhibit strong reductions in viability with siRNA alone, suggesting a SAHA-dependent relationship. For HEL and TF-1, although no phosphatases met the strict selection cutoff, there were several phosphatases exhibiting potentiation, suggesting biological relevance. The primary screening data and preliminary analysis will be validated with rigorous secondary experimentation and top hits for all cell lines will be investigated and advanced.

Conclusions:

While there are numerous kinase inhibitors in the clinic and in preclinical development, phosphatases are much less explored as therapeutic targets. Validated potentiating targets from this RNAi screen can facilitate the development of rational SAHA combination therapies.

Disclosures:

No relevant conflicts of interest to declare.
CD81-Dependent Trafficking of CD19: Restoration of CD19 Surface Expression in Human B Cells Harboring A CD81 Mutation
Authors: Levy Shoshana , Kuo Chiung-Chi , Sagi Yael , Chen Homer , Kela-Madar Neta , van Zelm Menno , van Dongen Jacques J.M.
Published: October 2019

Abstract 1049

Introduction:

A 6-year-old girl, who was diagnosed with a primary antibody deficiency, had B cells lacking surface CD19. However, both her CD19 alleles were normal and the impairment was actually caused by a homozygous exon splice site mutation in CD81 (1). The patient's B cells also lacked surface CD81 and produced an immature glycosylated CD19 protein that was retained intracellularly. Interestingly, this human deficiency differed from that of CD81 knockout mice as the latter still express a low level of CD19 on their B cells.

Methods:

We used an EBV-transformed B cell line from this patient to better understand i) the difference between the human and mouse CD81 deficiencies and ii) how CD81 controls the trafficking of CD19 to the cell surface. We reasoned that the truncated human CD81 mutant (CD81mut) protein might be expressed intracellularly. Indeed, whereas most anti-CD81 mAbs did not recognize CD81mut, we identified one that bound the mutated form and used it in this study. We also expressed the human CD81mut in a CD81-deficient mouse B cell line to determine if it could negatively regulate CD19 surface expression.

Results:

We show that the CD81mut protein is indeed expressed intracellularly in the patient's EBV-transformed B cells. We then used a proximity ligation assay to demonstrate that the truncated CD81mut protein interacts intracellularly with CD19. However, this interaction with the CD81mut protein abrogated carbohydrate maturation and the trafficking of CD19 to cell surface. We therefore expressed the CD81mut in CD81KO mouse B cells, which still express low levels of surface CD19, and found that it did not exert a dominant negative effect on CD19 surface expression. Finally, we used this reconstitution system to identify specific CD81 domains that restored carbohydrate maturation and cell surface expression of the CD19 molecule in the patient's B cells.

Conclusion:

This specific case of antibody deficiency was manifested because of lack of surface expression of CD19, an important B cell signaling molecule. However, the maturation of CD19 and its trafficking to the cell surface require the presence of specific domains of the tetraspanin CD81 molecule.

Disclosures:

No relevant conflicts of interest to declare.
Differential Regulation of Myeloid Leukemias by the Bone Marrow Microenvironment
Authors: Krause Daniela S. , Fulzele Keertik , Catic Andre , Hurley Michael , Lezeau Sanon , Attar Eyal C. , Wu Joy Y. , Lin Herbert Yih-Fuu , Divieti-Pajevic Paola , Hasserjian Robert , Schipani Ernestina , Van Etten Richard A. , Scadden David T.
Published: October 2019

Abstract 1245

Normal hematopoiesis is influenced by constituents of the bone marrow microenvironment (BMM) and is regulated by parathyroid hormone (PTH) via its actions on osteoblasts, osteoclasts and bone remodeling. The role of the niche in the biology of leukemic stem cells (LSC), frequently not eliminated by current therapies, however, is unclear. Targeting of the LSC niche is a novel concept aimed at eradication of LSC, the origin of relapse and progression of myeloid leukemias.

Using genetic and pharmacological murine models of BCR-ABL1+ chronic myelogenous leukemia (CML) and MLL-AF9+ acute myeloid leukemia (AML), we show that PTH treatment increased transforming growth factor β1 (TGFβ1) in the BMM and attenuated CML-like myeloproliferative neoplasia (MPN) without affecting engraftment, while leukemic cell-specific knockdown of the receptor for TGFβ1 (TGFβRI) exacerbated the disease. CML-like MPN is also exacerbated by treatment of mice, which have constitutively active receptor for PTH and PTH related protein, by osteoprotegerin, which inhibits osteoclasts and bone remodeling. In contrast, AML was accelerated by the PTH-modified BMM, which was reversed by overexpressing TGFbetaRI. PTH treatment reduced CML LSC frequency and cycling and, combined with the tyrosine kinase inhibitor imatinib, significantly prolonged survival of mice with CML-like MPN versus imatinib alone. PTH-treatment also reduced BCR-ABL1 transcript levels in a xenotransplantation model of human CML.

In summary, our results suggest that LSC niches in acute and chronic myeloid neoplasms are distinct and differentially modulated by PTH via TGFβ1. Combinations of TKIs with BMM-modulating agents may be a therapeutic strategy for LSC reduction and cure of CML.

Disclosures:

Lin: Ferrumax Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties.
Independent Validation of Chronic Graft-Versus-Host Disease (GVHD) Grading System Proposed by the NIH Consensus Criteria in Terms of Prognostic Stratification of the Patients Developed Chronic Gvhd
Authors: Moon Joon Ho , Sohn Sang Kyun , Uhm Jieun , Gupta Vikas , Lipton Jeffrey H. , Messner Hans A. , Kuruvilla John , Kim Dennis Dong Hwan
Published: October 2019

Abstract 1946

Background:

A new grading system for graft-versus-host disease (GVHD) severity was established by the National Institute of Health (NIH)-consensus criteria (NCC) which was introduced since 2007. However, prognostic implications of the NCC grading system on GVHD-specific survival (GSS) are still on debate.

Methods:

A total of 425 consecutive patients who survived beyond 100 days following allogeneic hematopoietic stem cell transplantation (HSCT) at the Princess Margaret Hospital from Jan 1996 to Oct 2007 were reviewed retrospectively and reclassified using the NCC. The GVHD-specific survival was compared according to the severity at onset or at peak of cGVHD by the NCC.

Results:

Out of 425 patients, 346 patients (81.4%) were diagnosed to have chronic GVHD (cGVHD) by the Revised Seattle criteria (RSC). Among them, 317 patients were reclassified to have classic cGVHD (n=144, 41.6%) and overlap syndrome (n=173, 50.0%) by the NCC. Twenty nine patients (8.4%) were reclassified as late-onset/recurrent acute GVHD. Out of 317 patients diagnosed to have a classic chronic GVHD and overlap syndrome, the severity scores at the onset of cGVHD were as follows: mild (n=137, 43.2%), moderate (n=134, 42.3%) and severe (n=46, 14.5%), while more moderate and severe cGVHD was noted when severity score was assessed at the peak time of cGVHD as mild (41, 12.9%), moderate (n=176, 55.5%) and severe (n=100, 31.6%). With median follow-up duration of 34 months (range 4–93 months), 5 yrs GSS rate was significantly worse in the group with severe grade of cGVHD at onset and at peak: 50.9±7.8% in severe vs. 89.7±3.2% in moderate vs. 93.5±2.4% in mild (at onset, p<0.001) and 69.1±5.2% in severe vs. 93.2±2.1% in moderate vs. 97.3±2.7% in mild (at peak, p<0.001). No differences in GSS were noted between moderate and mild grades of cGVHD at onset and at peak. Multivariate analysis confirmed severe grade of cGVHD at onset (HR 5.18, 95% CI=1.97–13.6) and at peak (HR 3.79, 95% CI=1.79–8.07) by NCC is associated with poor survival.

Conclusion:

The present study has successfully validated the grading system of NCC. The NCC could predict long-term outcome in the patients with cGVHD and could potentially be used to develop risk-stratified approaches to treatment based on NCC grading. The group with severe grade cGVHD presented inferior long-term outcomes than those with mild/moderate grade cGVHD.

GVHD-specific survival according to chronic GVHD grading system

A. GSS according to the Revised Seattle Criteria, B. GSS according to the NIH Global score at onset, C. GSS according to the NIH Global score at peak

Disclosures:

No relevant conflicts of interest to declare.
The Outcome of Hematopoietic Cell Transplantation (HCT) for Myelodysplastic Syndrome (MDS) in Adults ≥65 Years of Age: First Report of the Coverage with Evidence Development (CED) in Medicare Beneficiaries
Authors: Atallah Ehab , Pedersen Tanya L. , Warlick Erica D. , Dircks Anne , Weisdorf Daniel , Horowitz Mary M. , Rizzo J. Douglas
Published: October 2019

Abstract 1983

Despite recent advances in treatment for MDS, allogeneic HCT remains the only curative therapy. Historically, patients (pts) ≥65 years with Medicare did not have coverage for HCT. On August 4th 2010, the Centers for Medicare and Medicaid services (CMS) approved coverage for HCT for MDS through coverage with evidence development (CED). A Center for International Bone Marrow Transplant Research (CIBMTR) study comparing outcomes of pts 55–64 vs. 65 and older was approved. Since the study initiation the number of HCT performed in pts≥65 years old has nearly doubled from a pre-study average of 75/year to 154 in 2011. As of May 2012, 220 pts have been enrolled; 120 have ≥100 days of follow-up. The median age at HCT was 67 (range 65–74); only 24% were >70 years old and 20% (n=23) had therapy related MDS. At the time of HCT, 45% had refractory anemia with excess blasts −1/2 and 32% had refractory anemia ± ringed sideroblasts/refractory cytopenia with multilineage dysplasia. The modified International Prognostic Scoring System (IPSS) score was low, intermediate-1 (INT-1), INT-2 and high risk in 17%, 36%, 32% and 5% of pts, respectively. Donors were an HLA identical sibling, unrelated adult donor (URD) or cord blood (UCB) in 25%, 73% and 2% respectively. Of the 88 patients who received an URD HCT, 70 (80%) were HLA 8/8 allele matched. Twenty five pts (21%) (95% CI, 14–29%) died before 100 days. Causes of death were graft rejection/failure (1), infection (5), interstitial pneumonitis (1), GVHD (3), relapse (6), organ failure (8) and unknown (1). The probability of death at 100 days after HCT in all patients' ages 55–64 transplanted for MDS in 2010–2011 reported to the CIBMTR is 17%. (95% CI, 15–20%).

Conclusion:

In pts ≥65, HCT for MDS results in similar early mortality compared to pts 55–64 years old. This study will be expanded to include 700 pts age 65 and older to facilitate analysis of effect of IPSS, cytogenetics and conditioning regimen on the critical HCT outcomes for MDS of survival and MDS-free survival. Importantly, the increased use of HCT after approval of the CED suggests that insurance and Medicare coverage is a significant barrier limiting access to this potentially curative treatment in older patients with MDS.

Disclosures:

No relevant conflicts of interest to declare.
Long-Term Remission After Hematopoietic Stem Cell Transplantation for Refractory Cutaneous T Cell Lymphoma
Authors: Galvin John P. , Monreal Joanne , Guitart Joan , Rosen Steve T. , Kuzel Timothy , Mehta Jayesh
Published: October 2019

Abstract 2028

Purpose:

Current treatment of advanced stage cutaneous T cell lymphoma (CTCL) includes a number of chemotherapy agents which provide good response rates, but with often short duration. Hematopoietic cell transplantation (HSCT) has the potential to offer a durable remission due to graft-versus-lymphoma effect. We conducted a retrospective study to report the outcome of HSCT in patients with refractory CTCL.

Methods:

From Feburary 1997 through May 2012, twenty-one patients (22–65 yo) with advanced, refractory CTCL underwent HSCT from HLA-identical sibling (n=13), unrelated donor (n=5), umbilical cord (n=1), syngeneic (n=1) and autolougous (n=1) sources. There were 7 tumor stage MF, 6 Sézary syndrome, 2 erythrodermic MF, 1 folliculotropic MF, 1 aggressive CD8+ T cell lymphoma (Berti's type), 2 γ-δ TCL, 1 α-β TCL and 1 CD30-negative large cell peripheral TCL. The intensity of the conditioning regimen was conventional in 10 and reduced in 11. At the time of HSCT, the median disease duration was 5.4 years (1–17 y). Most patients were heavily pretreated, having failed a median number of four (1–9) lines of therapy. The stage of the disease at the time of HSCT was IIB (n=9), III (n=2), IVA (n=9), and IVB (n=1).

Results:

For the 21 patients evaluated there was a median follow-up of 62 months (3–186 mo). 11 patients had relapse post-transplant: 5 patients had an early relapse (<100 days) and 6 patients had a late relapse (>100 days). Relapsed disease appeared to be of a lower grade clinically than at pre-transplantation and responded to therapies they had previously failed. 1 early relapse died of disease (SS, auto-SCT) and 1 late relapse died of disease (γ-δ TCL, RIC allo-SCT). Of the nine remaining relapsed patients, 1 has died of a cerebral hemorrhage. (CD30-neg large cell peripheral TCL, alloSCT) and 1 of GVHD (γ-δ TCL, RIC allo-SCT). Of the 7 remaining relapsed patients, 4 went into CR with reduction of immunosuppression. 14 patients developed acute GVHD (fatal in 3), and 10 developed chronic GVHD. Of the surviving patients (n=15) all are in CR. Overall survival was 71%, with a median OS of 27.1 months (range, 0.5–185.9). Progression free survival (PFS) was 43% with a median PFS of 4.1 months (range, 0.5–80).

Conclusions:

Our results suggest that HSCT can induce durable clinical remissions in patients with refractory CTCL through graft-versus-lymphoma effect.

Disclosures:

No relevant conflicts of interest to declare.
Interleukin-6, Hepcidin, and Other Biomarkers in Anemia of Chronic Disease (ACD) and Chemotherapy-Induced Anemia (CIA): Potential Therapeutic Targets.
Authors: Vadhan-Raj Saroj , Zhou Xiao , Bueso-Ramos Carlos E. , Patel Shreyaskumar , Benjamin Robert S , Ngyuen Martin
Published: October 2019

Abstract 2086

Background:

Anemia in patients with malignancies can be multifactorial including anemia of chronic disease (ACD), also known as anemia of inflammation (AI), and chemotherapy (CT)-induced anemia (CIA) from myelosuppression. Although, exact mechanism for ACD is not known, induction of hepcidin, a key iron-regulatory hormone, by Interleukin (IL)-6 and other pro-inflammatory cytokines with resulting hypoferremia and limitation of iron supply to the bone marrow appear to be major contributors to pathogenesis of anemia. Hepcidin reduces iron levels by inducing degradation of the cellular iron exporter, ferroportin. The objective of this study was to examine the levels of various cytokines/regulators that may play role in ACD.

Methods:

Chemo-naïve patients with sarcoma scheduled to initiate first-line doxorubicin-based chemotherapy had blood samples drawn at baseline, and following chemotherapy (post cycles1, 3 and 6) for analysis of pro-inflammatory cytokines/other biomarkers of anemia. Serum samples were analyzed for IL-1β, IL-6, TNF-α, Hepcidin, hemojuvelin, ferroportin, soluble transferrin receptor (sTFR), and C-reactive protein (CRP) using ELISA techniques (R&D Diagnostics, Uscn Life Science Inc, or Abnova). Correlations between these biomarkers and Hgb levels at baseline and during the study period were calculated by linear regression analysis (SAS 9.2).

Results:

Of the 49 patients enrolled on to the clinical trial, there were 26 (53%) women and 23 (47%) men, with median age 45 years (range 19–65 years). Twenty-five percent of the patients had Hgb less than 12g/dL (range, 8.9–15.9 g/dL) prior to CT. At baseline, 50% of the pts had hypoferremia with low serum iron and transferrin saturation <20%. Baseline serum levels of IL-6 (r= −0.73, p<0.0001), hepcidin (r= −0.46, p=0.005), CRP (r= −0.46, p=0.003), sTFR (r= −0.32, p=0.064) inversely correlated with hemoglobin levels prior to CT, supporting their role in ACD. During CT (median 4, range; 1–6 cycles), Hgb declined in all pts with 55% requiring PRBC transfusions (77% of pts starting with baseline Hgb < 12 g/dL vs 47% of pts with baseline Hgb > 12 g/dL). Interestingly, as shown below, Hepcidin, IL-6, and sTFR all significantly negatively correlated with Hgb levels during CT.

No significant correlation was found for IL-1β, TNF-α, ferroportin, or hemojuvelin levels with Hgb.

Conclusions:

IL-6 and Hepcidin pathway appears to play an important role in anemia in cancer patients before and during CT. Treatment with novel agents targeting this pathway may provide effective strategies for prevention and treatment of ACD and CIA.

Disclosures:

Vadhan-Raj: JNJ: Research Funding.
Prevalence and Risk Factors of Left Ventricular Noncompaction in Patients with β-Thalassemia.
Authors: Piga Antonio , Longo Filomena , Musallam Khaled M , Veltri Andrea , Davicino Lia , Bonamini Rodolfo
Published: October 2019

Abstract 2127

Left ventricular noncompaction (LVNC) is a rare cardiomyopathy with potentially serious outcomes. It results in multiple and excessive trabeculations, deep intertrabecular recesses, and a thickened ventricular myocardium with two distinct layers, compacted and noncompacted. The condition is most commonly congenital although acquired forms have been described. A recent report of LVNC detected in a β-thalassemia twin suggested an association with cardiac siderosis. In a cross-sectional study of 135 transfusion-dependent β-thalassemia patients (130 major and 5 intermedia, mean age 29.6 ± 7.7 year, 49.6% males) presenting for cardiac iron assessment by magnetic resonance imaging (MRI), we evaluated the prevalence and risk factors for LVNC. None of the patients had neuromuscular or congenital heart disease. Eighteen patients (13.3%, 95% CI: 8.6–20.1) fulfilled the pre-assigned strict criteria for LVNC on cardiac MRI. There were no statistically significant differences between patients with and without LVNC in respect to: demographics; hemoglobin levels; splenectomy status; systemic, hepatic and cardiac iron overload indices; hepatic disease and infection studies; or iron chelator type. Patients with LVNC were more likely to have heart failure (adjusted OR: 1.77, 95% CI: 0.29 to 10.89); although with high uncertainty. Patients with β-thalassemia have a higher prevalence of LVNC than normal individuals. As this finding could not be explained by conventional risk factors in this patient population, further investigating of the underlying mechanisms of LVNC is warranted. This remains crucial for an entity with adverse cardiac outcomes, especially in a β-thalassemia patients where cardiac disease remains a primary cause of mortality.

Disclosures:

No relevant conflicts of interest to declare.