bluuebunny/crossref_metadata_2025 huggingface dataset:
Daunorubicin Induces Procoagulant Activity of Cultured Endothelial Cells through Phosphatidylserine Exposure and Microparticles Release
Authors: Fu Yueyue , Li Huibo , Li Wen , Dong Xiushuai , Hou Jinxiao , Xie Rui , Gao Chunyan , Yang Xue , Zhou Jin , Gilbert Gary E. , Shi Jialan
Published: October 2019

Abstract 5185

Administration of various chemotherapeutic agents is associated with an increased risk of thrombotic events. Although vascular endothelium plays a predominant role in blood coagulation and fibrinolysis, the effect of cytotoxic drugs on the procoagulant activity (PCA) of endothelial cells has not been well evaluated. Our study aims to investigate the possibility that daunorubicin, a chemotherapeutic agent, exerts prothrombotic effect on endothelial cells. We tested the impact of daunorubicin on phosphatidylserine (PS) exposure, endothelial microparticles (EMPs) release and consequent PCA. Human umbilical vein endothelial cells (HUVECs) were treated with daunorubicin (0.1, 0.2, 0.5, 1, 2 μ M) for 24 h. PCA of HUVECs was measured using clotting time and purified coagulation complex assays. Counts and PCA of EMPs were evaluated by flow cytometry and clotting time assay, respectively. Lactadherin was used as a novel probe for detection of PS exposure and EMPs release. We found that daunorubicin dose-dependently increased the PS exposure and consequent PCA of HUVECs. Moreover, daunorubicin treatment also enhanced the release of EMPs which were highly procoagulant. This increment was especially significant at 0.2 μ M of daunorubicin or over. Blockade of PS with lactadherin inhibited over 90% of HUVECs and EMPs PCA. However, anti-TF antibody had no significant inhibition effect. Our results demonstrate that daunorubicin treatment enhanced PCA of HUVECs through PS exposure and shedding of procoagulant EMPs. Lactadherin acts as an efficient anticoagulant in this process.

Disclosures:

No relevant conflicts of interest to declare.
Interleukin-32, α1 Anti-Trypsin (AAT-1) and Graft-Versus-Host-Disease
Authors: Marcondes A. Mario Q. , Tabellini Laura , Hansen John A. , Dinarello Charles A. , Deeg H. Joachim
Published: October 2019

Abstract 522

Graft-versus-host disease (GVHD) is an important complication of allogeneic hematopoietic cell transplantation (HCT). The role of various cell populations, cytokines and chemokines, present pre and post-transplantation, in the development of GVHD has been studied extensively.We investigated the potential role of Interleukin (IL)-32 in alloreactivity and GVHD. IL-32 is the protein product of the NK4 transcript first reported in IL-2 activated T lymphocytes and natural killer cells. IL-32 has pro-inflammatory and pro-apoptotic properties and induces expression of TNF-α in several cell targets. We used one-way mixed lymphocyte cultures (MLC) as a simple in vitro model of GVHD to determine IL-32 expression upon alloactivation. The α and γ isforms of (IL)-32 protein were 2-fold upregulated in allogeneic MLC compared to autologous controls (n=4, p=0.037). Concurrently, the concentrations of TNF-α, IL-6 and IL-8 in the allogeneic MLC supernatants were, as expected, upregulated significantly. This finding led us to evaluate IL-32 expression as a potential marker for GVHD after allogeneic HCT in 45 patients with either active acute GVHD or chronic GVHD, and 16 patients who did not show clinical evidence of GVHD. IL-32 mRNA levels in peripheral blood unsorted white blood cells, correlated with acute GVHD (RT-PCR values expressed as mean +/− SEM of the ratio of expression of IL-32/GUS-B = 1.01, p=0.011, vs control values IL-32/GUS-B = 0.23) but not with chronic GVHD, (IL-32/GUS-B = 0.26, p=0.16) (Figure 1). As the serine protease neutrophil proteinase 3 (PR3) has been shown to serve as activator of IL-32, and to process several inflammatory cytokines, including IL-32, TNF-α and IL-8, we postulated that the addition of the serine protease inhibitor α-1 anti-trypsin (AAT), would interfere with the processing of IL-32 by PR3, and as a result would lead to decreased proliferation of cells in MLC, and reduced cytokine production. To test this hypothesis, MLCs were treated with AAT at concentrations of 1–20 ug/ml and expression of IL-32 and PR3 were determined. In MLCs treated with AAT at the optimal concentration of 5 ug/ml, added to cultures on alternate days for a period of 7 days T lymphocyte proliferation was suppressed when compared to vehicle-treated MLC, (mean CPM=33.000 versus CPM=67.000; p=0.012). Concurrently there was a 2.5 fold decrease in IL-32 and PR3 protein levels (n=4, p=0.023). CONCLUSION: IL-32 is upregulated in patients with acute GVHD. Determination of IL-32 in patients with suspected GVHD may support the diagnosis and the decision to treat.AAT interferred with T cell activation and the release of cytokines, including IL-32, suggesting that administration of AAT may have therapeutic potential in patients with acute GVHD.

Disclosures:

No relevant conflicts of interest to declare.
Genome-Wide Analysis Identifies Regulators of Hematologic Parameters
Authors: Gieger Christian , Soranzo Nicole
Published: October 2019

Abstract SCI-10

Recent genome-wide association scans published by ours and other groups have revealed > 50 genetic loci associated with hematological parameters, including the count and volume of red and white blood cells and platelets. These variants map to known and novel regulators of hematopoiesis, and provide evidence for a high degree of connectivity guiding differentiation of the different blood elements. I will review progress in the field as well as ongoing efforts within the HaemGen consortium, where we have used statistical analyses, animal models and genome-wide functional annotation to address the in-depth genetic exploration of such parameters.

Disclosures:

No relevant conflicts of interest to declare.
Prevalence and Clinical Effect of TET2 Mutations in Cytogenetically Normal Acute Myeloid Leukemia (CN-AML)
Authors: Markus Birgit , Thol Felicitas , Morgan Michael A , Wagner Katharina , Heuser Michael , Krauter Juergen , Ganser Arnold , Damm Frederik
Published: October 2019

Abstract 1448

Mutations in the TET2 gene were recently identified in a variety of myeloid neoplasms including acute myeloid leukemia (AML). To investigate the frequency and clinical relevance of TET2 mutations in cytogenetically normal AML (CN-AML), the largest cytogenetic subgroup of adult AML, the total coding region of TET2 was analyzed by direct sequencing in 215 adult CN-AML patients younger than 60 years. All patients were uniformly treated on the AML SHG 0295 or AML SHG 0199 treatment protocols, both including double induction and intensive consolidation regimes. To test the prognostic value of TET2 mutations in the context of other prognostic molecular markers, mutational analyses for CEBPA, DNMT3A, FLT3-ITD, IDH1/2, MLL-PTD, ND4, NPM1, NRAS and WT1 and expression analyses for BAALC, ERG, EVI-1, IDH1, MLL5, MN1 and WT1 were performed. At least 1 sequence variation in TET2 was found in 16 out of 215 patients (7.4%). In 3 of these patients the somatic origin of the mutation could not be validated, so these patients were considered wildtype in all subsequent analyses. Mutations were detected throughout the entire gene with the highest frequency found in exon 3 (n=6). Patients harboring TET2 mutations showed higher platelet counts (P=.041), a higher frequency of concomitant NPM1 (11 of 13, P=.047) and DNMT3A (10 of 13, P=.001) mutations and tended to be older (P=.078) when compared to TET2 wildtype patients. The recently reported mutational exclusiveness of IDH1/2 and TET2 was validated in the present cohort (P=.042). No differences for complete remission (CR) rate (P=.76), relapse-free survival (RFS; P=.63) or overall survival (OS; P=.59) was observed based on the TET2 mutation status. When restricting the analyses to CN-AML patients assigned to the European LeukemiaNet (ELN) favorable-risk or intermediate1-risk groups (ELN-favorable risk-group: CN-AML patients with mutated CEBPA and/or mutated NPM1 without FLT3 -ITD, n=96; ELN-intermediate-1-risk group CN-AML with wild-type CEBPA and wild-type NPM1 and/or FLT3-ITD; n=106), the TET2 mutation status again did not impact on CR rate, RFS or OS. We conclude that in younger CN-AML patients, TET2 mutations occur with a lower prevalence compared to other myeloid malignancies such as MDS or CMML, and have no prognostic effect. Therefore, TET2 mutation analysis at present is not required for risk-stratified protocols in AML.

Disclosures:

No relevant conflicts of interest to declare.

Disclosures:

No relevant conflicts of interest to declare.
JAK2 mutations and CRLF2 rearrangements in Down Syndrome-Associated Acute Lymphoblastic Leukemia in Japan
Authors: Hanada Isamu , Terui Kiminori , Toki Tsutomu , Kudo Ko , Sato Tomohiko , Kamio Takuya , Sasaki Shinya , Takahashi Yoshihiro , Hayashi Yasuhide , Sugita Kanji , Kojima Seiji , Koike Kenichi , Kosaka Yoshiyuki , Kobayashi Masao , Ito Etsuro
Published: October 2019

Abstract 1451

Children with Down syndrome (DS) have a 10- to 20-fold increased risk of developing acute lymphoblastic leukemia (ALL). In DS-associated ALL (DS-ALL), the chromosome aberrations which are generally common in childhood ALL, such as hyperdiploidy and t(12;21), are less frequent. Recent studies have shown that activating JAK2 mutations and overexpression of cytokine receptor-like factor 2 (CRLF2) gene are identified in approximately 20% and 50–60% of DS-ALL in Western countries, respectively. Most of the patients with CRLF2 overexpression have been reported to be associated with interstitial deletions of the pseudoautosomal region 1 (PAR1) of the sex chromosomes and the P2RY8-CRLF2 fusion gene. In addition, one report showed that the activating CRLF2 F232C mutation was identified in about 10% of DS-ALL. However, there have been no studies to determine the incidence of these genetic aberrations in Asian patients with DS-ALL. In this study, 23 patients with DS-ALL in Japan were screened for mutations in the pseudokinase domain of the JAK2 gene, the P2RY8-CRLF2 fusion gene, and the CRLF2 F232C mutation by PCR/RT-PCR and direct sequencing. Fourteen patients, whose bone marrow RNAs were available, were also screened for CRLF2 overexpression by real-time quantitative RT-PCR. We identified the JAK2 R683G mutation in 2 patients (9%) and the P2RY8-CRLF2 fusion gene in 4 patients (17%). The CRLF2 F232C mutation was not detected in any patient. CRLF2 overexpression was observed in 2 of 14 patients examined (14%). Although bone marrow RNA was available in only 1 of 4 patients positive for P2RY8-CRLF2, high-level expression of CRLF2 was confirmed in this patient. The other patient with CRLF2 overexpression was negative for P2RY8-CRLF2, indicating the involvement of the other type of CRLF2 rearrangement, IGH@-CRLF2 in this patient. We also performed a preliminary study on JAK1, JAK3, and Interleukin-7 receptor-α (IL7R) mutations, and 14, 11, and 12 patients were screened for mutations in the pseudokinase domain of JAK1, JAK3, and exon 5 and 6 of IL7R, respectively. However, no mutations were identified in any patient. Our results show the lower incidence of CRLF2 rearrangements in DS-ALL patients in Japan than that in Western countries. Gene alterations other than CRLF2 rearrangements may contribute to leukemogenesis in Japanese patients with DS-ALL. To clarify if the incidences of the mutations in JAK1-3, CRLF2, and IL7R are also lower in DS-ALL patients in Japan than those in Western counties, more patients need to be studied.

Disclosures:

No relevant conflicts of interest to declare.
New and Highly Efficient Therapy for Treatment NPM-ALK Associated Lymphomas
Authors: Kenner Lukas , Laimer Daniela , Dolznig Helmut , Vesely Paul , Kollmann Karoline , Schiefer Ana-Iris Iris , Sexl Veronika , Staber Philipp B , Hoefler Gerald , Penninger Josef , Inghirami Giorgio , Ulrich Jaeger
Published: October 2019

Abstract 1659

Anaplastic Large Cell Lymphoma (ALCL) is a non-Hodgkin lymphoma of children and young adults with poor survival rates. ALCLs often occur with a chromosomal translocation that results in expression of the NPM-ALK oncoprotein. The molecular mechanism of NPM-ALK resulting in lymphoma growth are not fully understood. In a murine NPM-ALK lymphoma-model with T-cell specific deletion of cJun and JunB we could drastically delay lymphoma development and metastases. We identified PDGFRB as a direct transcriptional target of cJun and JunB. Blockage of the PDGFRβ activity significantly prolonged survival of NPM-ALK transgenic mice and severely reduced the growth of xenografted NPM-ALK tumors. In addition, we demonstrate that PDGFRβ is upregulated in human ALCL tumor specimens. Inhibition of the PDGFRβ in a terminal stage patient with NPM-ALK expressing ALCL which was refractory to any conventional therapy resulted in full remission within 10 days of treatment. We herwith certify the PDGFRB as a novel cJun/JunB target essential for ALCL development. Our data for the first time unravel a highly effective targeted therapy with the outlook to make ALCL a curable disease.

Disclosures:

Off Label Use: Imatinib for treatment of ALCL.
Plasma Membrane Loss in Maturing Human Reticulocytes Occurs Through Endocytosis of Large Glycophorin A- Containing Vacuoles Which Fuse with Autophagosomes Before Exocytosis
Authors: Griffiths Rebecca E , Kupzig Sabine , Cogan Nicola , Betin Virginie , Mankelow Tosti J , Lane Jon , Parsons Steve F , Anstee David J.
Published: October 2019

Abstract 177

The erythrocyte is one of the best characterized human cells. However, studies of the process whereby human reticulocytes mature to erythrocytes have been hampered by the difficulty of obtaining sufficient numbers of cells for analysis. We have developed an in vitro culture system permitting the production of millilitre quantities of functional mature human adult reticulocytes from peripheral blood CD34+ cells. Analysis of reticulocytes obtained from culture using confocal microscopy on permeabilized cells reveals that the final stage of reticulocyte maturation occurs by a previously undescribed mechanism in which large Glycophorin A-containing vacuoles forming at the cytosolic face of the plasma membrane are internalised and fuse with autophagosomes (defined by reactivity with anti-LC3) before expulsion from the cell. Our results constitute the first demonstration of linkage between plasma membrane remodelling and autophagy, and suggest reticulocyte plasma membrane remodelling occurs in two stages. Early maturation is characterized by the selective elimination of unwanted plasma membrane proteins (CD71, CD98, β1 integrin) through the endosome-exosome pathway. In contrast, final maturation is characterized by the generation and expulsion of large Glycophorin A-decorated vacuoles of autophagic origin, thereby simultaneously permitting optimisation of membrane:skeleton interactions to yield the more stable and deformable membrane of mature erythrocytes and eliminating unwanted residual organelles.

Disclosures:

No relevant conflicts of interest to declare.
Agelastatin A (AgA), a Marine Sponge Derived Alkaloid, Inhibits Wnt/Beta-Catenin Signaling and Selectively Induces Apoptosis in Chronic Lymphocytic Leukemia Independently of p53
Authors: Choi Michael Y. , Cardenas Johanna Melo , Lu Desheng , Yu Jian , Stout E. Paige , Wu Raymond P. , Horton John M , Gomez Allison , Diaz-Perez Julio A. , Carson Dennis A. , Molinski Tadeusz F. , Kipps Thomas J. , Castro Januario E.
Published: October 2019

Abstract 1786

The Wnt/beta-catenin pathway is highly active in chronic lymphocytic leukemia (CLL) and confers an anti-apoptotic effect in vitro and possibly also in vivo. As such, inhibition of this pathway represents a potential therapeutic target.

Here we report preclinical studies using agelastatin A (AgA), a naturally occurring alkaloid extracted from the marine sponges Agelas dendromorpha and Cymbastella sp. We tested AgA using SW480 cells transfected with a beta-catenin dependent reporter gene expression system. Using this assay, we found that AgA is a potent Wnt signaling inhibitor with IC50 of 12nM. AgA also inhibits the expression of reporter genes in HEK293 cells cotransfected with either Wnt1 or mutated (dominant-active) beta-catenin, suggesting that its mechanism of action is independent of the beta-catenin degradation complex. Moreover, real time-PCR results show that Lef1, a classic Wnt/beta-catenin target gene highly expressed in CLL, is down-regulated in primary CLL cells incubated with AgA at nanomolar concentrations.

AgA, but not structurally related compounds agelastatin C and agelastatin D, selectively induces apoptosis in CLL B-cells (mean IC50 = 56nM) compared with peripheral blood mononuclear cells from healthy volunteers (mean IC50 = 250nM). Interestingly, AgA induces apoptosis at nanomolar concentrations even in samples from CLL patients who had no clinical response to fludarabine, samples with 17p deletion or TP53 gene mutations, and samples in which p53 deficiency was determined by in vitro chemoresistance and absence of inducible p21, CD95, and DR5 after irradiation (Figure 1). In addition, AgA induces apoptosis on CLL cells that are maintained in co-culture conditions with stromal cells, which typically increases leukemia cell viability. This suggests that AgA is capable of disrupting pro-survival signals derived from the microenvironment, including Wnt mediated activation.

In conclusion, AgA has potent activity against CLL cells in vitro. Our studies show that AgA inhibits Wnt/beta-catenin signaling at nanomolar concentrations, induces apoptosis in CLL cells independently of p53, and is able to disrupt pro-survival signaling derived from stromal cell support. AgA warrants further studies and clinical development for the treatment of CLL and potentially other malignancies associated with Wnt/beta-catenin signaling.

Disclosures:

No relevant conflicts of interest to declare.
Outcomes of Myeloma Patients on Hemodialysis in the Era of Novel Therapies: An Analysis of the US Renal Data System
Authors: Weiss Brendan M , Hurst Frank , Agodoa Lawrence , Abbott Kevin C
Published: October 2019

Abstract 1803

Background.

Historically, severe renal failure requiring hemodialysis (HD) complicates multiple myeloma (MM) in 2–13% of patients. Recent epidemiologic studies show marked improvements in the survival of MM in the era of autologous transplantation and novel agents (post-1995). It is not clear how these therapies, which are feasible on HD, have affected the outcome of MM patients on HD. We have performed the first study of outcomes of MM patients on HD treated during the era of novel therapies. Methods. Patients who were initiated on dialysis whose primary cause of ESRD was listed as MM between 1989–2008 were included using the US Renal Data System (USRDS) database, which incorporates extensive baseline and follow-up demographic and clinical data on all patients accessing the Medicare ESRD program in the United States. The patients were divided into three time periods based on the introduction of novel therapies: 1989–1994, 1995–2000 and 2001–2008. Survival was calculated using dates of dialysis initiation, death, or end of study period and compared using Cox regression analysis. Results. The characteristics of the patients are shown in the table. After 1995 MM patients initiated on HD were older, had significantly more co-morbidities, poorer functional status and a lower albumin. The patients in 1989–1994 had markedly lower rates of co-morbidities than the general USRDS population. The median survival was 1.77, 0.74 and 0.69 years (p<0.0001) in 198994, 1995–2000, and 2001–2008, respectively. The rates of survival at 1 and 5 years were significantly lower in the two latter time periods. Adjusting for age, hemoglobin, albumin, and co-morbidities, the latter time periods were associated with a HR for death of 3.21 [95% CI 2.18–4.73, p<0.001] and 3.22 [2.18-4.74, p <0.001)], respectively. Conclusions. Patients with MM initiated on HD in the era of novel therapies have significantly more co-morbidities and worse survival. Given the effectiveness of novel therapies, it is likely that MM patients requiring HD in the era of novel therapies represent a population with aggressive, treatment refractory disease for whom alternative approaches are needed. An analysis of therapies delivered to this population is planned.

Disclosures:

No relevant conflicts of interest to declare.
CD34+/CD38− Acute Myelogenous Leukemia Cells Aberrantly Express Aurora Kinase A
Authors: Yang Jing , Ikezoe Takayuki , Furihata Mutsuo , Nishioka Chie , Yokoyama Akihito
Published: October 2019

Abstract 1886

Aurora kinase A (AURKA) plays a pivotal role in the mitotic processes during cell division. We previously showed that AURKA was aberrantly expressed in hematological malignant cells including those from acute myelogenous leukemia (AML) and acute lymphoblastic leukemia, compared with CD34+ hematopoietic stem/progenitor cells isolated from healthy volunteers. This study found that freshly isolated CD34+/CD38− cells from individuals with AML (n=12) in which leukemia stem cells were supposed to be enriched expressed a greater amount of AURKA than their CD34+/CD38+ counterparts, as measured by real time RT-PCR. Blockade of AURKA by the specific inhibitor MLN8237 significantly inhibited proliferation and induced apoptosis of CD34+/CD38− AML cells, as assessed by the clonogenic assay and detection of the cleaved form of poly (ADP-ribose) polymerase by Western blot analysis, respectively. In addition, exposure of CD34+/CD38− AML cells to MLN8237 down-regulated levels of Bcl-2 family proteins and increased Bax/Bcl-2 expression ratio. Moreover, inhibition of AURKA by MLN8237 (30mg/kg for 14 consecutive days) impaired engraftment of CD34+/CD38− AML cells in severely immunocompromised mice (n=5, 49±32% in control vs 18±16% in MLN8237 treated mice, P<0.05), and prolonged their overall survival (P<0.05), compared with vehicle treated mice. Taken together, AURKA may be a promising molecular target to eliminate chemotherapy-resistant CD34+/CD38− AML cells.

Disclosures:

No relevant conflicts of interest to declare.
Janus Kinase-2 Inhibition Induces Durable Tolerance to Alloantigen by Dendritic Cell-Stimulated Human T Cells, Yet Preserves Immunity to Recall Antigen
Authors: Betts Brian C. , Abdel-Wahab Omar , Curran Shane A , St. Angelo Erin T , Koppikar Priya , Heller Glenn , Levine Ross L , Young James W
Published: October 2019

Abstract 1903

Janus kinase 2 (JAK2) conveys signals from receptor binding by several inflammatory cytokines, including IL-6, IL-12, and IL-23, via phosphorylation of signal transducer and activator of transcription 3 (STAT3). These cytokines promote the development and expansion of T helper 1 (Th1) cells, which use IL-12, and Th17 cells, which use IL-6 and IL-23. Th1 and Th17 cells can in turn induce alloreactive end organ damage in GvHD. JAK2 is therefore a principle gatekeeper of alloreactivity and inflammation, and it represents an attractive target to control GvHD. We demonstrate that TG101348, a synthetic small molecule, highly selective JAK2 inhibitor, when present during initial and secondary encounters between human T cells and allogeneic monocyte-derived dendritic cells (moDCs), induces durable, profound, and specific T cell tolerance on reexposure to the same alloantigens. TG101348 ablates IL-6/JAK2-mediated phosphorylation of STAT3 in T cells (24% v 0%, P<0.05, n=5) but has no off-target effects on IL-2 or IL-15/JAK3/pSTAT5-dependent signaling, which would interfere with Tregs and other effector T cell responses. JAK2 inhibition preserves Treg numbers and thereby enhances the ratio of CD4+ Tregs to CD8+CD25+ effector T cells in favor of Tregs (Treg:Effector 1:2 vs 1:1, P<0.05, n=5). JAK2 inhibition also reduces the production of IL-6 (255 vs 157 pg/ml, P<0.05, n=5) and TNF-alpha (50 vs 15 pg/ml, P<0.05, n=4) in allogeneic MLRs, impairing the activation of central and effector memory T cells as well as the expansion of responder Th1 (24% vs 14%, P<0.05, n=4) and Th17 (2% vs 1%, P<0.05, n=4). T cells, stimulated by moDCs in the presence of TG101348, demonstrate a marked reduction in proliferative capacity upon reexposure to fresh allogeneic moDCs from the original stimulating party, even in the absence of the JAK2 inhibitor during the secondary MLR (P<0.05, n=4). Responses to stimulation de novo by influenza matrix peptide (fluMP), a pathogenic nominal antigen, remain intact (P=NS, n=4). Conversely, TG101348 also inhibits secondary MLR responses by T cells, which have already been sensitized to alloantigens in a primary MLR without the JAK2 inhibitor (P<0.05, n=4). The durable tolerance against full HLA disparities in each of these settings provides preclinical data in vitro that JAK2 represents a relevant biologic target for preventing or treating graft-versus-host disease (GvHD), or even solid organ allograft rejection, without broader immune impairment.

Disclosures:

No relevant conflicts of interest to declare.
Effect of Plerixafor on Graft Lymphocyte Subsets in NHL Patients Mobilizing Poorly with Chemotherapy Plus G-CSF
Authors: Jantunen Esa , Varmavuo Ville , Kuittinen Taru , Nousiainen Tapio , Mäntymaa Pentti
Published: October 2019

Abstract 1929

A combination of chemotherapy plus G-CSF (chemomobilization) is commonly used to mobilize CD34+ cells to circulation. Mobilization of CD34+ cells is poor or suboptimal in 20–30 % of patients. Plerixafor, a CXCR4 antagonist, increases the mobilization of CD34+ cells and may also have effect on graft composition subsequently collected. There are no data on lymphocyte subsets in the grafts collected after chemomobilization plus pre-emptively given plerixafor.

We have analyzed lymphocyte subsets (CD3, CD4, CD8, NK cells, CD19) in grafts collected on the next morning after plerixafor injection in 13 chemomobilized patients with non-Hodgkin lymphoma. As controls we had the first collections from 13 NHL patients mobilized with chemotherapy plus G-CSF and with yield of 2–6 × 106/kg CD34+ cells with 1–2 aphaereses.

The median CD34+ content of the analyzed grafts was 1.45 × 106/kg in the plerixafor group compared to 1.8 × 106/kg in the controls (p=n.s.). The number of T-cell subsets and NK cells were significantly higher in plerixafor mobilized grafts (Table 1). CD19+ B cells were infrequent in both groups.Table 1.Lymphocyte subsets of the grafts.Stem cell collection with plerixafor, median (range)Stem cell collection without plerixafor, median (range)Significance pGraft volume (ml)100 (43–190)80 (45–140)0.280Graft sample preservation time (days)299 (31–450)291 (103–397)0.898CD34+ cell content (x 106/ kg) after 7-AAD1.45 (0.40–4.40)1.80 (0.31–4.74)0.858CD3+ cell content (x 106/kg)75.3 (14.6–327.3)21.3 (9.1–159.4)0.004CD4+ cell content (x 106/kg)32.7 (10.6–132.8)12.4 (6.9–51.5)0.002CD8+ cell content (x 106/kg)33.4 (4.2–200.5)8.8 (2.2–125.0)0.006CD19+ cell content (x 106/kg)0 (0–0)0 (0–0)NANK cell content (x 106/kg)5.1 (0.2–30.40)1.5 (0.3–8.0)0.045CD4+/CD8+ cell ratio0.98 (0.34–3.04)1.41 (0.28–5.06)0.2287-AAD, 7-Aminoactinomycin D; NK, natural killer.

All except one patient has received high-dose therapy with blood stem cell support. The median CD34+ cell dose was 3.1 × 106/kg in plerixafor treated group and 3.3 × 106/kg in the control group, respectively. Time to neutrophil engraftment was comparable between the groups. There were two patients in the plerixafor group with late platelet engraftment (1 and 6 months).

Addition of plerixafor to chemomobilization in poor mobilizers results in increased content of T lymphocytes and NK cells in the graft but do not appear to mobilize B lymphocytes. Whether higher T cell and NK cell content are associated with more rapid immune reconstitution and survival should be evaluated in larger patient series with longer follow-up.

Disclosures:

Jantunen: Genzyme: Honoraria. Kuittinen:Roche: Consultancy.
A Phase 1 Open Label, Dose Escalation Study of Nilotinib in Steroid Dependent/Refractory Chronic Graft-Versus-Host Disease
Authors: Chen George L. , Carpenter Paul A. , Broady Raewyn , Gregory Tara B. , Arai Sally , Johnston Laura , Flowers Mary E.D. , Beumer Jan H. , Mendolsohn Jodie , Zhou Xin , Perloff Spenser H. , Otani Joanne M , Miklos David B
Published: October 2019

Abstract 1986

Imatinib is clinically active in cGvHD. Nilotinib, a tyrosine kinase inhibitor, targets the same receptors as imatinib but with different affinities. These targets and their IC50s are: DDR1 3.7 nM, DDR2 5.2 nM, ABL 25 nM, PDGFRA 53 nM, KIT 158 nM, and NQO2 1800 nM. We hypothesized that nilotinib is safe, tolerable, and clinically efficacious in cGvHD. We present preliminary safety and pharmacokinetic data from a phase 1 trial of nilotinib for steroid dependent/refractory cGvHD.

Methods.

All subjects had extensive steroid dependent/refractory cGVHD treated previously with ≥ 2 agents. Previous treatment with imatinib was allowed. Steroid refractory was defined as progressive cGVHD despite prednisone ≥ 0.5 mg/kg/d for ≥ 1 month; steroid dependence was cGVHD requiring prednisone ≥0.25 mg/kg/d for ≥ 3 months. This was a dose escalation trial with 4 sequential dose levels: 200 mg daily, 200 mg twice daily, 400 mg daily, and 400 mg twice daily followed by a dose extension phase at the maximum tolerated dose (MTD). Enrollment followed a standard 3+3 design. Steroids and two other immunosuppressants were allowed. Steroids were tapered as tolerated and other immunosuppressant dosing remained constant. Safety was determined by the observation of adverse events graded according to CTCAE v. 4.0 criteria. Trough plasma nilotinib concentrations were determined by HPLC before the daytime day 8 dose.

Results.

Median age was 49 years (range 23–75). Mean time to study enrollment from transplant was 3.3 years (range 1.7–7.8) and from cGVHD diagnosis 2.9 years (range 0.4–6.6). Sixteen subjects have been enrolled and 14 are evaluable. The median follow-up is 4.5 months (range 0.1–8.0). The MTD was reached at 200 mg daily. The maximum administered dose (MAD) was 200 mg twice daily. The dose limiting toxicities (DLTs) were grade 3 Pneumocystis pneumonia and grade 3 asymptomatic lipase elevation. Grade 2–3 adverse events possibly/probably attributed to nilotinib are shown below; no grade 4–5 adverse events attributed to nilotinib have occurred to date. Median trough plasma concentration of nilotinib 1 week after drug initiation at the MTD was 997 nM (range 661–1380, n=3) and at the MAD was 3110 nM (range 1210–4200, n=3). Trough nilotinib concentrations were higher at the MAD and were associated with DLT occurrence.

Conclusions.

200 mg daily is the MTD in the cGvHD population. Safety and clinical effect at the MTD is being evaluated in an ongoing 35 subject extension study. The MTD results in a trough plasma concentration that may be adequate to inhibit the nilotinib receptor targets except for NQO2. Confirmatory pharmacodynamic studies are planned.

Disclosures:

Off Label Use: Nilotinib for treatment of chronic graft versus host disease.
Modification of An Interactive Surface in the C1 Domain Reduces Immune Responses to FVIII in a Mouse Model of Hemophilia A
Authors: Wroblewska Aleksandra , van Haren Simon D , Herczenik Eszter , Rusiecka Aleksandra , Kaijen Paul H , ten Brinke Anja , Mertens Koen , Voorberg Jan
Published: October 2019

Abstract 199FN2

The X-chromosome-linked bleeding disorder hemophilia A is caused by the absence or dysfunction of blood coagulation factor VIII (FVIII) which can be corrected by regular intravenous administration of FVIII. Development of antibodies directed against FVIII (referred to as FVIII inhibitors) is common complication in hemophilia care. The formation of FVIII-neutralizing antibodies in hemophilia A patients is initiated by the endocytosis of FVIII by professional antigen-presenting cells. Endocytosis of FVIII by human monocyte-derived dendritic cells can be significantly blocked by monoclonal antibody KM33, directed towards the C1 domain of FVIII. We have previously shown that C1 domain residues 2092 and 2093 are required for KM33 binding to FVIII (Meems et al., 2009). However, replacement of both these residues by Ala did not completely abolish KM33 binding. We created an additional C1 domain variant which included an arginine at position 2090. A FVIII variant in which three residues, 2090, 2092 and 2093 were substituted by alanine, showed only minimal binding to KM33. Functional analysis revealed that this variant designated FVIII-2090/2092/2093 retains pro-coagulant activity as measured by a chromogenic assay. FVIII-2090/2092/2093 displayed a strongly reduced internalization by human monocyte-derived dendritic cells and macrophages, as well as murine bone marrow-derived dendritic cells. These results emphasize a role for C1 domain residues 2090, 2092 and 2093 in FVIII endocytosis by antigen-presenting cells. We subsequently investigated the in vivo ability of this variant to induce inhibitors in FVIII−/− mice (E17KO). We show that E17KO mice treated with the FVIII variant have significantly lower anti-FVIII antibody titers when compared to mice treated with wild type FVIII. In accordance with these findings reduced numbers of FVIII-specific antibody-secreting cells were detected in the spleen of mice treated with FVIII-2090/2092/2093. Also, FVIII-specific CD4+ T cell responses of splenocytes derived from FVIII-2090/2092/2093 infused mice were greatly reduced when compared to that of splenocytes derived from wild type FVIII infused mice. These findings show that alanine substitutions at positions 2090, 2092 and 2093 result in a FVIII molecule that is significantly less immunogenic when compared to wild type FVIII. Collectively, our data suggest that FVIII variants displaying a reduced uptake by antigen-presenting cells show a reduced immunogenicity in vivo. Based on our findings we hypothesize that FVIII variants displaying a reduced uptake by antigen-presenting cells provide a novel therapeutic approach to reduce inhibitor development in patients with hemophilia A.

Disclosures:

No relevant conflicts of interest to declare.
A Retrospective Observational Study of Annual Healthcare Costs for Patients with Forms of Myeloproliferative Neoplasms (MPN)
Authors: Price Gregory L , Pohl Gerhardt M , Xie Jin , Walgren Richard A
Published: October 2019

Abstract 2060

Background:

Three subtypes of MPN (myelofibrosis (MF), polycythemia vera (PV) and essential thrombocythemia (ET)) are characterized by activation of JAK2 signaling, with the majority having the JAK2 V617F mutation. Median survival in these subtypes ranges from months to years for MF (Gangat JCO 2011) and up to a decade or more for some patients with PV and ET. Some symptomatic treatment options exist, but with the exception of hematopoietic stem cell transplant, none are curative. Little is known about healthcare costs associated with these diseases.

Objective:

To compare annual medical and pharmacy costs in three subtypes of MPN patients (MF, PV, ET) to matched non-cancer controls using a large US commercial claims database.

Methods:

Data were drawn from the Thomson Reuters MarketScan database, which contains claims for insured employees, retirees and dependents from approximately 100 US payers. Index date was the first claim with an MPN diagnosis code in the time period 2005 to 2008. Eligible patients had one year continuous enrollment post-index with no evidence of non-AML secondary malignancy. Controls had one year of continuous enrollment after their match date and were matched to patients in a ratio of 5:1 based on gender, year of birth, geographic region and insurance type (Comprehensive, Health Maintenance Organization, Preferred Provider Organization, etc.). Controls were excluded if they had a service claim with a diagnosis for cancer or a pharmacy claim for chemotherapy. Costs were based on total gross payments to the provider, which equals the amount eligible for payment under the medical plan terms after applying rules for discounts, but before applying coordination of benefits, copayments and deductibles. Total cost was the sum of MPN and non-MPN related medical costs which included inpatient, outpatient and emergency room service and pharmacy. Pharmacy included injectable and non-injectable chemotherapy, supportive care and other prescription drug costs.

Results:

A total of 25,145 MPN patients (MF: 509, PV: 16,165, ET: 8,471) were included and assigned matching controls. The mean cost (standard deviation) for cases and controls are shown in the table.

Medical and Pharmacy Costs:

Conclusions:

Annual medical and pharmaceutical costs for patients with MPNs are 2 to 6 times those of matched non-cancer patients and represent medical management challenges and payer burden. Outpatient visits accounted for about half of the total healthcare costs, followed by inpatient visits, pharmaceutical costs and ER visits.

Disclosures:

Price: Elil Lilly and Company: Employment; Eli Lilly and Company: Equity Ownership. Pohl:Eli Lilly and Company: Employment; Eli Lilly and Company: Equity Ownership. Xie:Eli Lilly and Company: Employment; Eli Lilly and Company: Equity Ownership. Walgren:Eli Lilly and Company: Employment; Eli Lilly and Company: Equity Ownership.
Comparing Patterns for Transitioning the Care of Young Adults with Sickle Cell Disease Versus Hemophilia: The Toronto Experience
Authors: Simpson Ewurabena , Ward Richard , Kirby Melanie , Odame Isaac
Published: October 2019

Abstract 2072

Background:

The Hospital for Sick Children (HSC) in Toronto, Canada cares for more than 700 children with sickle cell disease (SCD), which is the largest Canadian population of children with SCD. Since 2009, the SCD Program at HSC has partnered with adult hematologists within the Red Blood Cell Disorders program at Toronto General Hospital (TGH) to develop a coordinated strategy for transitioning the care of young adults with SCD. Hemophilia is a chronic hematological condition which, like SCD, has a spectrum of disease severity that requires multidisciplinary follow up. At HSC, we care for nearly 200 patients with hemophilia A and B and have a long-established partnership with adult hematologists, which has led to an effective transfer of patients with hemophilia into adult care. In Ontario, adult health providers are remunerated according to a fee-for-service billing schedule. In contrast, pediatric subspecialists are mainly salaried under an alternate funding plan. Until 2010, adult hematologists who provided medical care for individuals with hemophilia received a significantly higher pay scale than those who cared for individuals with SCD. This was changed in July 2010 so that adult hematologists receive commensurate remuneration for services rendered for both hemophilia- and SCD-related medical care.

Objectives:

1. To compare the patterns for transitioning patients of varying disease severity within the pediatric and adult SCD and hemophilia programs in Toronto, Ontario.

2. To identify barriers and enablers that have influenced the transition of young adults with SCD within a universal health care system.

Methods:

Data for active, transitioned and inactive patients in the HSC and TGH clinical programs are maintained in a database at HSC. These patient numbers were gathered according to sickle cell genotype. Similar data were available for hemophilia patients who were transitioned from HSC to adult care. Chi-square analyses were used to compare the proportions of patients in the sickle cell and hemophilia programs that were transitioned between 2009 and 2011.

Results:

Conclusion:

The HSC-TGH- partnership has significantly reduced the number of youth with SCD who continue to be followed at HSC or are lost to follow up. However, a significant number of young adults within the HSC SCD program still need to be transitioned to adult care. For the sustainable expansion of this transitional care strategy, health policymakers must collaborate with tertiary and community level health care providers to develop a coordinated and distributed strategy for the effective delivery of comprehensive health care services for young adults with SCD.

Disclosures:

No relevant conflicts of interest to declare.
A High Throughput Screening System for the Identification of Novel Genes in the Regulation of Mammalian Erythropoiesis
Authors: Ji Peng , Wong Piu , Lodish Harvey F.
Published: October 2019

Abstract 2093

Mammalian erythropoiesis is globally regulated by erythropoietin (Epo). Epo binds to its receptor on the cell surface of erythroid precursors. This induces a series of downstream pathways that promote cell differentiation and inhibit apoptosis. A recent genome wide transcriptional profile study demonstrated that over 600 genes are up-regulated during erythropoiesis. Surprisingly, the roles that most of the identified genes play in erythroid cells are still unknown. Understanding the functions of these unknown genes in the erythroid cells is necessary to elucidate the pathogenesis of red cell disorders such as congenital dyserythropoietic anemias, fanconi anemia, aplastic anemia, megaloblastic anemia, as well as leukemia and myelodysplastic syndromes with leukemic or dysplastic erythroid features. The goal of our study is to identify novel genes involved in different stages of erythropoiesis. To achieve this goal, we developed a high-throughput flow cytometry based assay that simultaneously detects erythroid cell differentiation and enucleation. In this assay, mouse fetal liver erythroblasts were purified and infected with lentiviruses containing a mammalian shRNA knockdown library obtained from the Broad Institute. The infected cells were cultured in a 96-well plate. Over the following two days the unaffected cells fully differentiate with approximately 60% of the cells enucleated. However, those cells in which shRNAs have knocked down genes critical for erythropoiesis are expected to show alterations in differentiation and/or enucleation. The system was validated using lentiviruses expressing shRNAs against Gata1 and mDia2, known proteins that are involved in the early and late stages of erythropoiesis, which showed inhibitions of differentiation and enucleation, respectively. We have pre-screened the most up-regulated 100 genes that play unknown functions in the erythroid cells. Future studies will be focused on the identified novel genes on their functions in erythroid lineage commitment, Epo mediated signaling pathways, hemoglobin enrichment, chromatin condensation, and enucleation.

Disclosures:

No relevant conflicts of interest to declare.
HPFH From Klf1 Haploinsufficiency Combined to Transcriptional Heterozygous β-Thalassemia
Authors: Moi Paolo , Marini Giuseppina M. , Asunis Isadora , Porcu Loredana , Cabriolu Annalisa , Satta Stefania , Perseu Luciana , Maccioni Liliana , Cao Antonio , Galanello Renzo
Published: October 2019

Abstract 2151

KLF1 mutations are emerging as a frequent cause of hereditary persistence of hemoglobin F (HPFH). Although it has been initially proposed that KLF1 haplo-insufficiency could determine KLF1 related HPFH, following studies have shown that isolated mono allelic Klf1 defects do not or only occasionally raise HbF and that biallelic KLF1 mutations are needed for HPFH to fully develop. Here we present a different mechanism of HPFH in a Sardinian family with a rare genetic combination in which HPFH with HbF levels up to 20% is caused by Klf1 haploinsufficiency combined to a β-thalassemia transcriptional defect from a −87C to G mutation in the proximal CACCC box, the canonical Klf1 binding site. Other known determinants of HPFH in the Myb, γ-globin or Bcl11 gene or variation in the expression of Bcl11 could not account for the increased HbF expression in the compound heterozygotes compared to the simple KLF1 or βThal-87 heterozygotes siblings. Hence, in this family delayed hemoglobin switching and HPFH are not dependent upon the absolute reduction in the free Klf1 concentration, as suggested by current models, but from the reduction in the amount of CACCC bound Klf1. This observation suggests that CACCC bound Klf1 is the primary anchor site for the LCR and that Klf1 mediated bridging between LCR and β-globin promoter is the most important determinant for hemoglobin switching in humans.

Disclosures:

No relevant conflicts of interest to declare.
Only Dendritic Cells, but Not Lymphocytes, Acquired Tolerogenicity by PUVA-Treatment by Upregulation of Indoleamine 2,3-Dioxygenase (IDO)
Authors: Maeba Hideaki , Kuroda Rie , Niida Yo , Mase Shintaro , Araki Raita , Fujiki Toshihiro , Yachie Akihiro , Koizumi Shoichi , Nishimura Ryosei
Published: October 2019

Abstract 2176

Extracorporeal photopheresis (ECP) is a leukapheresis-based immunomodulatory therapy by exposing isolated white blood cells to photoactive 8-MOP (psoralen) and UVA radiation (PUVA) for refractory acute and chronic graft-versus-host disease (GVHD). Although the effects of PUVA treatment on immune system remain poorly characterized, we have reported that bone marrow-derived dendritic cells (BM-DCs), which are generated from bone marrow cells plus GM-CSF, acquired tolerogenicity by PUVA-treatment in mice. In mixed lymphocyte reaction (MLR) assays, strong tolerogenicity was observed when adding PUVA-DCs generated from the same strain of stimulator cells, or responder cells, or even from third-party (Figure 1) strain into MLR mixture. That is, the PUVA-treated DCs have tolerogenic function in a MHC-independent manner (52nd ASH), in part, due to the reduced expression levels of CD80 and CD86, both of which are costimulatory molecules for T cell activation. To clarify the mechanisms of how PUVA-treated DCs induce tolerogenicity in further detail, we performed MLR with the addition of neutralizing antibodies against IL-10 or TGF-β1 or both, which have immunosuppressive effects. Neutralization of immunosuppressive cytokines had no effects on MLR. We then hypothesized that cell-to-cell contact between PUVA-DCs and alloreactive T-cells was needed to mediate the regulatory effect. To this end, we performed MLR using transwell to prevent cell-to cell contact. MLR was not suppressed when transwell was used, suggesting that PUVA-DCs dominantly regulates the alloreaction in a cell contact-dependent manner. Next we compared the expression of the indoleamine 2,3-dioxygenase (IDO), which induces T-cell anergy by tryptophan depletion and by the production of metabolic byproducts collectively known as kynurenines, by real-time PCR between PUVA-DCs and BM-DCs. An increased IDO gene transcription level was observed in PUVA-DCs about 5 times more than in BM-DCs (p<0.01) as shown in Figure 2. This suggested that the PUVA-treated DCs induced tolerogenic function by depleting tryptophan in the immediate environment. The question we have to consider next is whether tolerogenic capacity is acquired only in DCs or other lymphocytes subpopulations, because recent rodent studies have shown that the transfer splenocytes treated with PUVA prevent GVHD. To test this, PUVA-treated CD4, CD8, and B220 cells from the same strain of stimulator cells were added into the MLR mixture. MLR was suppressed only when PUVA-DCs were added. In conclusion, PUVA-DCs, but not other lymphocytes subpopulations, acquired regulatory function in a MHC-independent manner by upregulation of IDO. Infusion of PUVA-treated DCs could have a great potential to treat lethal acute GVHD in clinical settings.

Disclosures:

No relevant conflicts of interest to declare.