bluuebunny/crossref_metadata_2025 huggingface dataset:
Adult Langerhans Cells Derive Directly From Embryonic Macrophages.
Authors: Bertrand Julien , Wittamer Valérie , Traver David
Published: October 2019

Abstract 3788

Dendritic cells (DCs) represent the most potent antigen-presenting cells in vertebrates. Upon immune challenge, DCs can uptake antigens and migrate to lymphoid organs to activate naïve T cells. Therefore, understanding their biology is important for designing new treatments. Langerhans cells (LCs) are a subset of DCs residing in the adult epidermis. In adult teleosts, many evidences point to the existence of DCs, but no tools have been generated to enable in vivo imaging, prospective isolation and functional analyses of this subset.

By combining two new transgenic lines, cd45:DsRED and mhc2dab:eGFP, we could isolate mononuclear phagocytes (macrophages/DCs) in the CD45+DAB+ fraction from adult tissues. Zebrafish DC-like cells displayed cytological features that resembled those of mammalian DCs. In the skin, the majority of CD45+DAB+ cells exhibited a typical morphology of LCs. Thus, we can isolate a pure population of LCs from the adult skin.

Most DC subtypes are constantly replenished from hematopoietic stem cell derived progenitors. However, recent studies indicate that LCs self-renew independently from the bone marrow in steady-state conditions, suggesting the existence of a local progenitor in the skin. Recent reports showed that mouse LCs derive from embryonic myeloid precursors that seed the skin before undergoing proliferation. By combining imaging and gene expression analyses, we could show that mhc2dab is first expressed in myeloid cells (CD45+) at 7 days post fertilization (dpf). Accordingly, we can first observe CD45+DAB+ cells at 8 dpf, in the skin of developing embryos, suggesting that LCs derive from early CD45+ cells. In order to unravel their origin, we traced CD45+ cells by using the tamoxifen-inducible cd45:CRE-ERt2 transgenic line, combined to a switch reporter that allows us to permanently label cells. This switch-reporter consists of the b-actin promoter driving the expression of DsRED after a STOP cassette is excised by the CRE recombinase. By exposing double-transgenic embryos to tamoxifen between 20 and 26 hpf, we could induce CRE expression in primitive macrophages only. Several weeks after tamoxifen treatment, we could identify the progeny of primitive macrophages in the skin, showing LC morphology. This strongly suggests that primitive macrophages can persist to adulthood and contribute to the LC network. Additional experiments will be required to prove if they are the only source of LCs in steady-state conditions.

Disclosures:

No relevant conflicts of interest to declare.
First Report and Results From VSCO.NET, the Virtual Society of Clinical Oncology, An Independent Internet-Based Haematology/Oncology Platform.
Authors: Weihrauch Martin R , Brinker Florian , Breuer Jonas
Published: October 2019

Abstract 3832

Introduction.

VSCO.NET, the Virtual Society of Clinical Oncology was founded as an independent internet platform for healthcare professionals working in the field of hematology and oncology. The aim is to bundle all accessible knowledge and experience of the oncology community in one place and to grant an open, free-of-charge source of information.

Methods.

The concept was developed by hematologists/oncologists and was realized together with a team of web programmers. The software was developed in HTML, PHP and JavaScript and runs on a Linux web-server. To enable an internationally accepted platform, VSCO.NET offers its pages in English, Spanish, French and German.

Results.

VSCO.NET went public in December 2009 in a beta-stage with the following functions: 1. a comprehensive chemotherapy order system with over 100 included chemotherapy protocols, 2. staging tools for Hodgkin's and Non-Hodgkin's lymphoma, multiple myeloma, CLL, and CML, 3. texts and tools for oncological emergencies, 4. a knowledge database/file repository for information texts, presentations and pictures, 5. a news channel with oncological abstracts, and 6. a virtual tumor board (discussion forum). Currently, over 400 physicians, mostly hematologists/oncologists, have registered with VSCO.NET. The most frequented tool is ChemoBOS, the chemotherapy order system, which quickly generates chemotherapy orders for patients. Due to its open and free protocol template generator, many physicians have created their own adapted chemotherapy protocols, which can be used by the entire VSCO.NET community. Also, the staging tools were generally well accepted and have a fairly high usage. However, the virtual tumor board as a way to communicate about difficult cases has not been used so far, which could show a reluctance of physicians to discuss their patient cases in an internet-based forum. It should be noted that many physicians registered from developing countries, who cannot afford expensive chemotherapy software and now have the opportunity to improve patient care at their centers.

Conclusions:

As the first internet-based society for hematology/oncology with a wide variety of interactive functions, VSCO.NET shows great potential to internationally connect physicians, expand the accessibility of oncological knowledge and to facilitate the daily clinical routine.

Disclosures:

No relevant conflicts of interest to declare.
Exportin 7 (RanBP16) Plays An Essential Role In Terminal Erythroid Differentiation
Authors: Hattangadi Shilpa , Burke Karly , Lodish Harvey
Published: October 2019

Abstract 3877

Members of the nuclear transport receptor family of importins and exportins regulate the passage of proteins between the nucleus and cytoplasm. Although evolutionarily conserved across several species, Exportin 7 (Xpo7 or RanBP16) and its cargo are not well understood. In our study, we find that Xpo7 is highly erythroid-specific, as all other exportins are downregulated during terminal erythroid differentiation, a process including the induction of a highly specialized erythroid expression program, a set number of 3–5 terminal cell divisions, and chromatin condensation and eventual enucleation. Xpo7, in contrast, is highly induced during terminal erythropoiesis. Using retroviral shRNA knockdown of Xpo7 in in vitro fetal liver erythroid cell cultures, we demonstrate that exportin 7 is necessary for normal cellular proliferation and terminal erythroid differentiation, specifically for normal enucleation. Through microarray and biocomputational analysis of mRNA isolated from the knockdown cultures, we have found that the promoters of genes that are dysregulated after Xpo7 knockdown are enriched for binding sites for the activating transcription factor 4 (ATF4). Given that the erythroid phenotype of the ATF4 knockout mouse is very similar to the specific erythroid defects we observe in our in vitro knockdown analysis, our data suggests that either ATF4 or its binding protein may be Xpo7's cargo during terminal erythroid differentiation. Ongoing studies aimed at confirming this mechanism, the interaction between ATF4 and Xpo7, and the role and cargo of Xpo7 in terminal erythroid differentiation, are underway.

Disclosures:

No relevant conflicts of interest to declare.
Expression of the c-Met Oncogene Correlates with Favorable Progression Free Survival In Classical Hodgkin Lymphoma
Authors: Xu Chuanhui , Van Den Berg Anke , Plattel Wouter , Ruther Nele , Xin Huang , Wang Miao , Van Imhoff Gustaaf , Viardot Andreas , Möller Peter , Poppema Sibrand , Visser Lydia
Published: October 2019

Abstract 3880

Background

The HGF/c-Met signaling pathway regulates a variety of biological processes, including proliferation, survival and migration. Aberrant c-Met activation has been implicated in the pathogenesis of many human cancers. Furthermore, c-Met is of prognostic significance in several types of cancer such as diffuse large B cell lymphoma, bladder cancer, breast cancer, colorectal cancer and ovarian cancer. Expression of c-Met in Hodgkin lymphoma (HL) patients, as well as increased levels of HGF in the urine of HL patients has been reported, but no prognostic studies or functional data have been reported.

Methods

We studied the prognostic significance of c-Met expression by immunohistochemistry on paraffin sections of classical HL (cHL) patients from two independent patient cohorts. Functional studies were performed on HL cell line L428 that showed high c-Met levels, constitutive phosphorylated c-Met and no HGF expression. The effect of stimulation by HGF and inhibition by c-Met kinase inhibitor SU11274 was investigated by Western blot, cell proliferation and cell cycle progression analysis.

Results

Expression of c-Met was detected in Hodgkin Reed-Sternberg (HRS) cells in 52% (79/152) of primary cHL tissue samples and expression of its ligand, hepatocyte growth factor (HGF), was detected in HRS cells in 8% (10/120) of the cHL patients. Co-expression of HGF and c-Met in HRS cells was observed in only 3% (4/120) of cHL patients. A variable percentage of infiltrating cells stained positive for HGF, supporting a predominant paracrine activation route. In contrast to its adverse prognostic impact in other cancers, high c-Met expression significantly correlated with favorable 5 year progression free survival in both patient cohorts. To explain these unexpected findings we studied the c-Met/HGF signaling pathway in the L428 cHL cell line. The levels of phosphorylated c-Met, Akt, and Erk1/2 were upregulated upon HGF stimulation and this induction could be blocked by inhibiting c-Met activation with SU11274. Activation with HGF did not effect cell growth, while SU11274 alone suppressed cell growth. SU11274, as well as inhibitors of PI3K, MEK1/2 and Erk1/2 (downstream targets of the HGF/c-Met signaling pathway) induced G2/M cycle arrest.

Conclusion

Expression of c-Met in tumor cells was observed in 52% of cHL patients. In contrast to its prognostic value in other cancers, expression of c-Met in HRS cells of cHL patients correlated with favorable prognosis in two independent cohorts. Although triggering of c-Met in L428 cells with HGF induced activation of its downstream factors, no effect was observed on proliferation. Remarkably, the c-Met inhibitor did suppress cell growth of L428 cells.

Disclosures:

No relevant conflicts of interest to declare.
Hodgkin's Disease and HIV Infection (HD-HIV): Prognostic Factors In 596 Patients (pts) within the European Group for the Study of HIV and Tumours (GECAT)
Authors: Spina Michele , Ribera Josep-Maria , Gabarre Jean , Wyen Christoph , Montoto Silvia , Jaeger Ulrich , Talamini Renato , Re Alessandro , Miralles Pilar , Tirelli Umberto
Published: October 2019

Abstract 3883

Background:

Hodgkin's disease (HD) is the most common non-AIDS defining tumour diagnosed in HIV setting. The introduction of highly active antiretroviral therapy (HAART) has opened a new prospective in the treatment of pts with HD-HIV as the better control of the underlying HIV infection allows the use of more aggressive chemotherapy regimens, including high dose chemotherapy. However, up to now prognostic factors on overall survival (OS) or time to treatment failure (TTF) have not been identified yet.

Methods:

in order to identify prognostic factors, we analyze data on 596 pts with HD-HIV diagnosed and treated in 90 different Institution from 6 European countries from October 1983 to March 2010. All factors were analyzed for OS and TTF.

Results:

86% of pts were male and the median CD4 cell count was 224/mL (range 3–1274); 52% of pts had mixed cellularity subtype, stages III-IV were diagnosed in 72% of cases and 55% of pts had extranodal involvement (bone marrow 35%, spleen 21%, liver 14%). Table 1 summarizes the results of multivariate analysis.

Conclusion:

We identified a new “European Score” (IPS>2 and CD4 cell count <200/mL) for HD-HIV able to predict different outcomes in these patients. This score should be considered for future prospective studies.

Disclosures:

No relevant conflicts of interest to declare.
MAZ Is a Substrate for the Deubiquitinating Enzyme DUB1
Authors: Kim Yong-Soo , Shin Dong-Mi , Wang Hongsheng , Morse Herbert
Published: October 2019

Abstract 3913

Ubiquitination is a fundamental mechanism of signal transduction that regulates immune responses and many other biological processes. Similar to phosphorylation, ubiquitination is a reversible process that is counter-regulated by ubiquitinating enzymes and deubiquitinating enzymes (DUBs).

DUB1 has been identified as a member of a subfamily of deubiquitinating enzymes that are induced by Interleukin-3 (IL-3) in Ba/F3 cells. Recently, we've known that DUB1 is expressed in some pro-B and pre-B cell lines and is differentially regulated during normal B cell differentiation with highest expression in small pre-B cells. To understand the functional role of DUB1 in early B cell development, we identified a transcription factor, MAZ, as an interacting partner or substrate of DUB1 in the Abelson-transformed 220-8 pro-B cell line by using a retrovirus-based protein-fragment complementation assay (RePCA) screen. MAZ has been identified as a critical regulator of p21 gene induction, which controls cell cycle progression in synovial fibroblast cells.

Immunoprecipitation and immunoblot analysis confirmed that MAZ and DUB1 interact with each other in cells. DUB1 expression significantly increased the steady-state cellular levels of MAZ. Notably, the half-life of MAZ in the pEGFP-DUB1-transfected cells was significantly increased by DUB1 expression, whereas the half-life of MAZ in the mock-transfected cells was less than 1 hr. These data therefore demonstrate that DUB1 specifically stabilizes MAZ in vivo.

We found that the overexpressed MAZ was polyubiquitinated and that the polyubiquitinated MAZ was the substrate for proteasome inhibitor MG132 caused a robust increase of MAZ polyubiquitination. Overexpression of DUB1 in 293T cells caused a decrease of MAZ polyubiquitination in a DUB1 dose-dependent manner. Taken together, these results indicate that DUB1 mediate the ubiquitination-dependent degradation of MAZ.

Other study shows that the DUB1 targets both K-48 and K-63 linked ubiquitination suggesting that it may be involved in both protein degradative and non-degradative functions during early B cell differentiation.

Disclosures:

No relevant conflicts of interest to declare.
Decitabine Is Effective and Safe In Patients with Chronic Myelomonocytic Leukemia
Authors: Jabbour Elias , Kantarjian Hagop M. , Ravandi Farhad , Cornelison A. Megan , Kadia Tapan , Borthakur Gautam , Cortes Jorge E. , Garcia-Manero Guillermo
Published: October 2019

Abstract 4032

Background:

Chronic myelomonocytic leukemia (CMML) is a rare yet indolent disease. The median survival duration in CMML is 12 to 18 months and patients with poor prognostic features do even worse, with median survival time ranging 3 to 6 months. Activity with decitabine in CMML has been previously reported. We sought to analyze the clinical experience of 17 adults with a diagnosis of CMML treated on two decitabine studies.

Methods:

A subset of patients with CMML from a pivotal phase III 3-day dosing and an open-label trial of 5-day dosing were identified and reviewed to determine the overall response rate (ORR, based on IWG 2006 criteria), duration of response, time to response, and overall survival (OS).

Results:

A total of 17 patients with CMML were included in this review. Mean age at diagnosis was 71 years (range, 47 to 81 years) with a mean time from diagnosis of 406.4 days. The majority of CMML patients had de novo (94.1%), good risk cytogenetics (58.8%) with an IPSS classification of Intermediate-1 (64.7%). Baseline mean white blood count (WBC), hemoglobin (HGB), and platelets (plts) were 7.5 × 103/μ L, 14.6 g/dL and 81.9 × 103/μ L, respectively. A larger proportion of CMML patients at baseline were plt and RBC transfusion independent. Objective response rate (ORR) was 41% [17.6% complete response (CR) and 23.5% marrowCR (mCR)]; Hematologic improvement (HI) was observed in 11.7% and stable disease in 29.4% of patients. Median survival was 391 (95% CI 239, 678) days and 2 (11.7%) patients progressed to AML. The adverse event profile was similar to observations in previous trials with myelosuppression and infectious complications.

Conclusions:

This retrospective review of responses in CMML patients supports previous findings of decitabine experience in this population. In this analysis an overall response rate of 41.4% was achieved. Decitabine provided anti-CMML activity with an acceptable safety profile.

Disclosures:

Jabbour: Eisai Inc.: Editorial and statistical support from Eisai Inc., Honoraria. Kantarjian:Novartis: Research Funding; Pfizer: Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Consultancy. Ravandi:Eisai Inc.: Research Funding; Eisai Inc.: Honoraria. Borthakur:Eisai Inc.: Research Funding. Cortes:Novartis: Research Funding; Pfizer: Consultancy, Research Funding; Bristol Myers Squibb: Research Funding.
Interleukin-16 Is An Important Growth-Promoting Factor and a Novel Diagnostic and Therapeutic Target for Multiple Myeloma
Authors: Atanackovic Djordje , Hildebrandt York , Luetkens Tim , Zander Axel R. , Bokemeyer Carsten , Kroeger Nicolaus
Published: October 2019

Abstract 4056

Background:

Multiple myeloma (MM) is a malignancy characterized by the expansion of a plasma cell (PC) clone that localizes to the bone marrow (BM). Myeloma cells and BM stromal cells both produce soluble factors promoting the survival and progression of MM. Interleukin-(IL)-16 is involved in regulating migration and proliferation of normal leukocytes, however, it has been unclear whether IL-16 also plays a role in the pathophysiology of human cancers.

Methods:

Using an antibody array we screened supernatants of myeloma cell lines for the presence of a variety of cytokines/chemokines. We confirmed IL-16 expression in myeloma cell lines as well as in malignant PC and BM plasma from MM patients applying real-time PCR, western blots, ELISA, and flow cytometry. We applied inhibitory RNA to analyze IL-16 function and we used anti-IL-16 antibodies to evaluate possible therapeutic options for MM.

Results:

We found IL-16 to be strongly overexpressed in the BM of myeloma patients. Myeloma cell lines as well as primary tumor cells from MM patients constitutively expressed IL-16 RNA and protein and spontaneously secreted soluble IL-16. Functional analyses revealed that IL-16 supports the proliferation of myeloma cells. Accordingly, silencing of IL-16 expression had an anti-proliferative effect on the tumor cells. Most importantly, the application of a monoclonal antibody directed against IL-16had a strong growth-inhibiting influence on myeloma cells.

Conclusions:

These findings suggest that cytokine IL-16 is an important growth-promoting factor in MM and might represent a novel diagnostic and therapeutic target for this incurable human malignancy.

Disclosures:

No relevant conflicts of interest to declare.
Weak Sensitivity of Myeloma Cells to CD38 or CD138-ADCC
Authors: Clemenceau Beatrice , Vivien Regine , Descamps Géraldine , Maiga Sophie , Amiot Martine , Vié Henri , Pellat-Deceunynck Catherine
Published: October 2019

Abstract 4072

The development of novel treatments such as those based on human monoclonal antibodies (mAb) are currently being evaluated in preclinical and early clinical studies in cancers. There is convincing evidence supporting a key role for antibody-dependent cell-mediated cytotoxicity (ADCC) in the clinical response of several therapeutic monoclonal antibodies. In myeloma, CD38 and CD138, both highly expressed by all myeloma cells, represent the best targets for ADCC and humanized Abs are currently in development for both antigens. The aim of the present study was to assess the CD38 and CD138-ADCC sensitivity of a large panel of human myeloma cell lines (HMCLs) representative of the disease heterogeneity.

To reproductively assess ADCC, we used mouse FcgRIII expressing human T lymphocytes as cytotoxic effector cells. We measured ADCC sensitivity of 20 HMCLs with two mouse IgG1 anti-CD38 (clones T16 and HIT2), with two mouse IgG1 anti-CD138 (clones BB4 and MI15) and with a mouse IgG2a anti-HLA class-I (clone W6.32) by a standard 51Cr release assays performed at an effector to target ratio of 10:1.

Unexpectedly, despite high level of CD38, only 3 cells lines among 20 were sensitive to anti-CD38/ADCC. No correlation was observed between anti-CD38/ADCC sensibility and the CD38 expression level. No correlation was observed too between sensitivity and representative molecular heterogeneity of HMCLs. The absence anti-CD38/ADCC was not due to intrinsic resistance lysis through ADCC since these cell lines were lysed when using an anti-HLA class-I mAb (or anti-CD20 mAbs for one CD20+ HMCL). Furthermore, absence of ADCC was not due to antibody-induced antigenic modulation. Surprisingly too, none HMCL was lysed by anti-CD138 mAbs despite high expression of CD138. Moreover, primary myeloma cells purified from peripheral blood of a patient were also resistant to both anti-CD38 and anti-CD138 mAbs.

These results show that ADCC activity mediated by a particular monoclonal antibody varies according to the target cell and independently of the antigen expression level, suggesting that the antigen “environment” might affect the interactions between antibody, Fc receptor and effector cells. Further studies will be required to determine the CD38 and CD138 “environment” factors on myeloma cell that can influence ADCC.

Disclosures:

No relevant conflicts of interest to declare.
CREBBP Mutations In Relapsed Acute Lymphoblastic Leukemia
Authors: Mullighan Charles , Zhang Jinghui , Kasper Lawryn H. , Lerach Stephanie , Payne-Turner Debbie , Phillips Letha A , Collins-Underwood J. Racquel , Heatley Susan L. , Ma Jing , Buetow Kenneth , Pui Ching-Hon , Brindle Paul K. , Downing James R.
Published: October 2019

Abstract 413

Relapsed acute lymphoblastic leukemia (ALL) is a leading cause of death due to disease in young people, but the biologic determinants of treatment failure remain poorly understood. To identify novel sequence mutations contributing to relapsed in ALL, we resequenced 300 genes in matched diagnosis and relapse samples from 23 patients with ALL. The cohort included B-progenitor ALL with high hyperdiploidy (N=3), TCF3-PBX1 (N=1), ETV6-RUNX1 (N=3), rearrangement of MLL (N=3), BCR-ABL1 (N=3), and low hyperdiploid, pseudodiploid, or miscellaneous karyotypes (N=10). This identified 52 somatic non-synonymous mutations in 32 genes, many of which were novel, including mutations in the transcriptional coactivators CREBBP and NCOR1, the transcription factors ERG, SPI1, TCF4 and TCF7L2, components of the Ras signalling pathway, histone genes, genes involved in histone modification (CREBBP and CTCF), and genes previously shown to be targets of recurring DNA copy number alteration in ALL. Analysis of an extended cohort of 63 diagnosis-relapse cases and 200 acute leukaemia cases that did not relapse found that 19% of relapse cases had sequence or deletion mutations of CREBBP, which encodes the transcriptional coactivator and histone acetyltransferase (HAT) CREB-binding protein (CBP). The mutations were either present at diagnosis, acquired at relapse, or duplicated to homozygosity at the time of relapse. Moreover, several mutations acquired at relapse were detected in subclones at diagnosis, suggesting that the mutations confer a selective advantage and promote resistance to therapy. The mutations either resulted in truncated alleles or deleterious substitutions in highly conserved residues of the HAT domain. To examine the functional consequences of the mutations, we introduced wild type or mutant Crebbp alleles into Cbp/Ep300flox/flox murine embryonic fibroblasts, (dKO MEFs), and examined histone acetylation, expression of CREBBP target genes, and cellular proliferation. The HAT domain mutations resulted in impaired acetylation of the key Crebbp substrate, H3K18, and resulted in impaired transcriptional regulation of multiple CREBBP targets and pathways, including cAMP, dsRNA and dexamethasone responsive genes. The latter observation suggests that CREBBP mutations may directly result in resistance to corticosteroid therapy, which is a hallmark of high risk ALL. Together, these data these results extend the landscape of genetic alterations in leukemia, and identify mutations targeting transcriptional and epigenetic regulation as a mechanism of resistance in ALL.

Disclosures:

Pui: EUSA Pharma: Honoraria; Enzon: Honoraria; Sanofi-Aventis: Honoraria.
Immunomodulation of NK Cells through 4-1BB (CD137) to Improve the Anti-Lymphoma Activity of Rituximab: Antibody-Based Anti-Lymphoma Synergy
Authors: Kohrt Holbrook E , Houot Roch , Goldstein Matthew J , Weiskopf Kipp , Chao Mark , Chen Lieping , Tedder Thomas , Levy Ron
Published: October 2019

Abstract 422

Background:

Antibody-dependent cell-mediated cytotoxicity (ADCC), largely mediated by natural killer (NK) cells, is thought to play an important role in the efficacy of rituximab, an anti-CD20 monoclonal antibody (mAb) used to treat patients with B cell lymphomas. CD137 is a costimulatory molecule expressed on a variety of immune cells following activation, including NK cells. We hypothesized that the anti-lymphoma activity of rituximab could be enhanced by stimulation of NK cells with an anti-CD137 agonistic mAb.

Methods:

Rituximab induced upregulation of CD137 on NK cells was assessed using lymphoma cell lines and primary lymphoma patient samples. In-vitro NK cell degranulation and cytotoxicity were assessed by CD107a mobilization and chromium release. A murine lymphoma tumor model targeted by mouse anti-mouse CD20 mAb was used to assess in-vivo synergy of anti-CD20 and anti-CD137 mAbs. Mechanism of synergy was explored by T cell, NK cell, and macrophage depletion in the immune competent mouse model. A xenotransplant model in SCID mice with disseminated, luciferase-labeled lymphoma was used to demonstrate efficacy of anti-CD137 mAb and rituximab, and sufficiency of an innate immune response.

Results:

NK cells in human primary lymphoma samples do not express CD137 at baseline, however these cells highly upregulate CD137 when encountering rituximab-coated tumor B cells. Rituximab-induced NK cell degranulation and cytotoxicity as measured by CD107a mobilization (p=.006) and chromium release (p=.01) are enhanced by anti-CD137 agonistic mAb. In a murine lymphoma model, anti-CD137 mAb significantly enhances anti-tumor activity of anti-CD20 mAb leading to complete tumor resolution (p<.001) and prolonged survival (p=.048). Sequential administration of anti-CD20 mAb followed by anti-CD137 mAb (p=.027) is required for the synergistic effect. In-vivo administration of anti-CD20 mAb induces upregulation of CD137 on mouse NK cells (p=0.001), allowing subsequent targeting with anti-CD137 mAb. NK cell depletion completely abrogates the therapeutic effect of anti-CD20 plus anti-CD137 mAb combination (p<.001). In a xenotransplant disseminated lymphoma model (Figure 1A), rituximab plus anti-CD137 mAb provided superior reduction in tumor burden, as quantified by bioluminescence (p=.001; Figure 1B), and prolonged overall survival (p=.013; Figure 1C).

Conclusions:

Our results demonstrate the synergy of anti-CD137 mAb and rituximab by stimulation of rituximab-activated NK cells with anti-CD137 mAb to enhance ADCC. These results support a novel, sequential antibody approach against B cell malignancies by targeting first the tumor and then the host immune system.

Disclosures:

No relevant conflicts of interest to declare.
Chronic IFNγ Production Induces Anemia by Reducing Erythrocyte Lifespan and Inhibiting Erythropoiesis through An IRF-1/PU.1-Axis
Authors: Libregts Sten F. W. M. , Gutierrez Laura , de Bruin Alexander M. , Wensveen Felix M. , Papadopoulos Petros , van Ijcken Wilfred , Ozgur Zeliha , Philipsen Sjaak , Nolte Martijn A.
Published: October 2019

Abstract 4234

Anemia of chronic disease (ACD) is a complication accompanying many inflammatory diseases. The pro-inflammatory cytokine IFNγ has been implicated in this form of anemia, but the underlying mechanism remains unclear. Using a novel model for ACD, we demonstrate that IFNγ reduces both the lifespan and formation of red blood cells. Molecular analysis revealed that IFNγ induces expression of the transcription factors IRF-1 and PU.1 in both murine and human erythroid precursors. We found that IRF-1 binds to the promoter of PU.1 and induces its expression, leading to inhibition of erythropoiesis. Notably, downregulation of either IRF-1 or PU.1 expression is sufficient to overcome IFNγ-induced inhibition of erythropoiesis. PU.1 is a key transcription factor in hematopoietic lineage determination and its expression is sufficient to block erythroid differentiation and enhance myeloid differentiation. We therefore postulate that expression of IFNγ during infection can briefly shift the balance of erythroid towards myeloid differentiation, which will benefit the anti-microbial response. However, continuation of this feedback mechanism will hamper erythroid development and subsequently lead to anemia.

In conclusion, we here provide evidence for both a cellular and molecular mechanism by which IFNγ modulates erythropoiesis and how this contributes to the development of ACD.

Disclosures:

No relevant conflicts of interest to declare.
Role for BH3-Only Protein NOXA In Growth-Factor Deprivation and Early Erythropoiesis
Authors: Geest Christian R. , Wensveen Felix M. , Libregts Sten F.W.M. , de Bruin Alex M. , Derks Ingrid A.M. , Ekert Paul G. , Labi Verena , Villunger Andreas , Nolte Martijn A. , Eldering Eric
Published: October 2019

Abstract 4235

Red blood cell production is a strictly regulated process and homeostatic maintenance of the erythropoietic system requires equilibrium between the rate of erythroid cell production and red blood cell destruction. Hematopoietic cytokines play a crucial role in regulating expansion, differentiation and survival of erythrocyte progenitors. Shortage of growth factors triggers the mitochondrial apoptosis pathway, which is critically dependent on Bcl-2 family members. However, the contribution of this mechanism in the regulation of erythropoiesis remains ill-defined. This prompted us to screen for candidate genes involved in this process in erythroid progenitors. We found that the expression of Noxa, a pro-apoptotic Bcl-2 family member, is upregulated during erythroid differentiation and following cytokine-withdrawal in erythroid progenitor cells. Knockdown or deletion of Noxa in IL-3 dependent human and murine erythroid progenitor cell lines increased Mcl-1 levels, which correlated with markedly decreased apoptosis following cytokine withdrawal. Importantly, Noxa ablation in mice increased extra-medullary erythropoiesis, resulting in enhanced numbers of early splenic erythroblasts and circulating reticulocytes. Noxa-deficient hematopoietic progenitors were more resistant to apoptosis induced by growth factor deprivation and displayed increased colony-forming potential. In addition, combined loss of Noxa and Bim resulted in enhanced resistance of erythroid progenitors to cytokine withdrawal compared to WT or single Bim knockouts, suggesting a non-redundant role for Noxa and Bim in regulating survival of erythroid progenitors in response to cytokine deprivation. Finally, in a model of acute haemolytic anaemia, deletion of Noxa enhanced subsequent hematocrit recovery. Together, these findings identify a non-redundant role for BH3-only protein Noxa in the regulation of erythroblast survival during early erythropoiesis. Therefore, Noxa may be a novel component to control red blood cell numbers and modulation of this pathway could be envisaged in therapeutic options for treatment of anaemia.

Disclosures:

No relevant conflicts of interest to declare.
Studies on Heme Oxygenase 1 During Erythroid Differentiation
Authors: Santos Daniel Garcia , Eisenberg Jesse , Schranzhofer Matthias , Ponka Prem
Published: October 2019

Abstract 4254

Heme is indispensable for the function of all aerobic cells as a prosthetic group of innumerable proteins. However, “free heme” (uncommitted) can initiate the formation of free radicals and cause lipid peroxidation, which can lead to cellular damage and tissue injury. Therefore, the rate of heme biosynthesis and catabolism must be well balanced by tight control mechanisms. The highest amounts of organismal heme (75-80%) are present in circulating red blood cells (RBC), whose precursors synthesize heme with rates that are at least one order of magnitude higher (on the per cell basis) than those in the liver – the second most active heme producer in the body. The degradation of heme is exclusively carried out by heme oxygenases 1 and 2 (HO1 and HO2), which catalyze the rate-limiting step in the oxidative degradation of heme. Although the heme-inducible HO isoform, HO1, has been extensively studied in hepatocytes and many other non-erythroid cells, virtually nothing is known about the expression of HO1 in developing RBC. Similarly, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using both a murine erythroleukemia cell line (MEL) and primary erythroid cells isolated from mouse fetal livers, we have demonstrated that during erythroid differentiation HO1 is up-regulated at both mRNA and protein levels. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase. These data suggest that in developing RBC, in addition to the continuous assembly of heme with globin chains, there is an increase in levels of uncommitted heme, which upregulates HO1 expression. Additionally, we have shown that down-regulation of HO1 via siRNA increased hemoglobinization in differentiating MEL cells. In contrast, induction of HO1 expression by NaAsO2 reduced the hemoglobinization of MEL cells. This effect could be reversed to control levels by the addition of HO1 inhibitor tin-protophorphyrin (SnPP). These results show that in differentiating erythroid cells the balance between levels of heme and HO1 have to be tightly regulated to maintain hemoglobinization at appropriate levels. Our results lead us to propose that disturbances in HO1 expression could play a role in some pathophysiological conditions such as thalassemias.

Disclosures:

No relevant conflicts of interest to declare.
Analysis of Rare deletional Thalassemia Using Custom CGH Array DNA Chip
Authors: Pissard Serge , Ceccaldi Raphael , Faubert-Laugé Emmanuelle , Velayoudane Valerie , Delattre Valerie , Lecerf Laure
Published: October 2019

Abstract 4278

Deletions are the main etiology for alpha thalassemia and account for 9 to 10 % of beta thalassemia. They are currently detected using “in house” methods as QMPSF or the use of commercially available assays as MLPA. The accurate determination of the breakpoint could be of importance for news and rare events to have a proper characterization of the deletion and to understand the consequences of the event. However, the imprecision due to the relative small numbers of markers in these methods, the distance to be covered and the high percentage of repeated sequences located into the intergenic regions, makes the breakpoint determination difficult. To overcome the limitation of the number of markers we have setup a custom “Comparative Genomic Hybridization” assay to study the beta and the alpha globin cluster. Based upon the Agilent multiple arrays system, we have designed an array covering chromosome 11 and chromosome 16, containing 15 000 probes with an average spacing of 150 bp on both clusters. This CGH DNA chip was used to successfully clone the breakpoint of several rare and new deletions removing from 10 kb to up to 1 megabase of DNA sequence on chromosome 11 and chromosome 16. The results show the efficiency of such an approach and the multiple array format allows a good cost value by comparison to step by step cloning. In 50 % from more than 100 beta locus deletional events the 5′ breakpoint lies between the Aγ globin and the delta globin gene indicating the location of hotspots of recombination herein. Recent findings have shown that intergenic sequences in β-globin cluster and probably in α-globin cluster may be involved in the global regulation of the globin genes expression. Interestingly, a non coding small RNA was recently discovered in the same region and supposed to be involved in fetal hemoglobin expression. Such multiple arrays CGH chips provide an easy and efficient tool to define critical intergenic regions involved in the global regulation of the globin genes. Such hypothesis is currently under study.

Disclosures:

No relevant conflicts of interest to declare.
RASGRP1/APTX Ratio Is a Strong Biomarker of Clinical Response and Survival In AML Patients Treated with Tipifarnib: A Phase I-II Preliminary Results
Authors: Martinelli Giovanni , Ottaviani Emanuela , Paolini Stefania , Papayannidis Cristina , Curti Antonio , Parisi Sarah , Iacobucci Ilaria , Cilloni Daniela , Arruga Francesca , Saglio Giuseppe , Russo Domenico , Visani Giuseppe , Zaccaria Alfonso , Rondoni Michela , Bocchia Monica , Candoni Anna , Fanin Renato , Gherlinzoni Filippo , Baccarani Michele
Published: October 2019

Abstract 4359

We conducted a phase I-II study aiming to assess efficacy and toxicity of tipifarnib-bortezomib treatment in elderly AML patients. RASGRP1/APTX genetic ratio, which is associated with treatment response in patients treated with tipifarnib alone, was tested.

Eighty patients were enrolled with secondary-AML: 14 had high risk cytogenetics; 42 were previously untreated. Seventy-five patients were treated.

Nine patients achieved complete remission (CR), 1 patient obtained a partial response (PR) and in 2 cases an hematological improvement (HI) was documented for an overall response rate (ORR) of 19%. Median time to response was 112 days, corresponding to 4 cycles (range 2–14). Marrow response (CR+PR) was significantly associated with overall survival (OS) (p<0.0001). RASGRP1/APTX was evaluated before treatment initiation on bone marrow (BM), peripheral blood (PB) or both. The median RASGRP/APTX value on BM was 15.3 (15-19.8) in responder patients and 2.2 (0.5-25.9) in non responders, respectively (p=0.00006). Its median value on PB was 31.6 (19.3-35.5) in responders and 6.4 (0.5-27.1) in non responders, respectively (p=0.00001). Interestingly, no marrow responses were recorded in patients with marrow RASGRP1/APTX ratio <8, while the response rate was 43% [how many were CR?] in patients with RASGRP1/APTX >8 (p<0.0001). Finally, RASGRP1/APTX levels significantly correlated with OS (p=0.001) with a median OS of 490 days and 162 days in patients with RASGRP1/APTX >8 and <8 respectively.

We conclude that the clinical efficacy of the combination of tipifarnib and bortezomib was evident. We confirmed that the RASGPR1/APTX BM or PB level is an effective predictor of response and survival and our now studying the response of such patients to tipifarnib, alone.

Supported by: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione – Università 2007 – 2009.

Disclosures:

Martinelli: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy. Baccarani:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria.
Primary Clinical Research of FA Regimen as Consolidation Therapy for Patients with Acute Myeloid Leukemia
Authors: Lu Huina , Qin Wei , Liang Aibin
Published: October 2019

Abstract 4380

In our study, 28 patients with AML treated in our department between 2004 and 2009 under complete remission, median age of 54 years (range 23–77) were consolidated with FA regimen which included fludarabine 30mg/m2 (days 1–3) and Ara-C 2g/m2 (days 1–3). Ara-C were administered as a continuous sequential infusion 4 hours later when the application of fludarabine had been over. Another 14 AML patients with matched conditions treated with high dose Ara-C (HDAra-C, 3g/m2, q12h×3d) were designated as controls. Event-free survival of both 2 groups of these patients were analysed.

Overall, 14 patients (50%) achieved continuous complete remission for more than 2 years. There is no statistically significant differences between FA group and HDAra-C group (P=0.214>0.05) in CCR. Patients aged >60 years also had no statistically significant differences compared to the patients aged <60 years in CCR. Seven patients relapsed after this treatment in a median survival of 11 months. Two of the relapsed patients had karyotype abnormalities and all of them had a markedly elevated WBC count at diagnosis.

The main toxicity was myelosuppression. WBC count and platelet count reached the lowest (about 0.8×10^9/L and 2.0×10^9/L) 6–13 days after the begining of the regimen. Median recovery time for granulocyte (≥1.0×10^9/L) and platelet (≥20×10^9/L) was 12 and 13 days, respectively. Fever(>38°C) caused by granulopenia occurred in 21 patients, recovered to normal for a median time of 5 days (range, 2–23). Heart, liver and enteric toxicity were negligible. There were no significant differences in hematologic toxicity or nonhematologic toxicity between the FA and HDAra-C group. The average hospitalization was about 20 days.

The FA is effective and safe as consolidation regimen in AML with acceptable toxicity and is not associated with an increased incidence of infections compared to conventional HDAra-C regimen.

Disclosures:

No relevant conflicts of interest to declare.
Homoharringtonine Affects the Expression of PI3K and p-AKT of NB4, SHI-1 Leukemia Cells
Authors: Yang Mingzhen , Zhang Xiaoyu , Huang Zhenqi , Liu Qinhua , Wang Lin , Li Qingsheng
Published: October 2019

Abstract 4381

Background:

Homoharringtonine (HHT) was efficient in therapying patients with acute myeloid leukemia (AML) in China, but little is known about the mechanism of its action. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling axis plays a central role in cell proliferation, growth, and survival under physiological conditions and many human cancers, including acute myelogenous leukemia (AML). We try to explore the effect of HHT on PI3K/Akt pathway in AML cells, thus supplying theoretical basis for wider use of HHT.

Method:

The NB4 and SHI-1 cells were cultured in 20% FCS RPMI-1640 with different concentration of HHT, cell proliferation was detected with MTT, apoptosis was measured by FCM, the protein of PI3K and p-Akt were determined by Western blot.

Result:

5ug/L HHT suppressed NB4 and SHI-1 cells proliferation and induced apoptosis after culture 24hr, 100ug/L HHT suppressed 71.29% NB4 and 64.83% SHI-1 cells proliferation respectively. Apoptosis increased obviously with the increasing HHT concentration and the culture time, the leukemia cell apoptosis was significant at 500ug/L HHT, about 41.84% NB4 cells and 46.88% SHI-1 cells were apoptosis when the HHT concentration was 100ug/L. The protein expression of PI3K, and p-Akt gradually declined with HHT concentration increasing, when 500ug/L HHT co-cultured with leukemia cells for 24 hours, The protein expression of PI3K and p-Akt were lowest. The p-Akt of NB4 and SHI-1 cells decreased 28.4% and 34.5% respectively at 5ug/L HHT for 48hr, the PI3K of NB4 and SHI-1 cells decreased 31.56% and 37.38% respectively at 10ug/L HHT for 48hr.

Conclusion:

HHT could inhibit NB4, SHI-1 cells proliferation and induce leukemia cells apoptosis, and could down-regulate the expression of PI3K and p-Akt significantly, this might be the one of mechanisms that HHT induce NB4 and SHI-1 cells apoptosis, we presume that HHT inhibit proliferation of acute myelogenous leukemia cells through effect of PI3K/Akt signaling pathways.

Disclosures:

No relevant conflicts of interest to declare.
Phenotyped Blood Reduces Alloimmunization Rates Among Cancer Patients
Authors: Novaretti Marcia Cristina Zago , Miyaji Sandra Cristina , Ito Gláucia Munemasa , Dinardo Carla Luana , Chamone Dalton FA
Published: October 2019

Abstract 4405

Background:

Alloimmunization against RBC antigens is a complication of blood transfusion/pregnancy that may cause hemolytic transfusion reactions and can difficult compatible blood availability in future transfusions. Frequency of transfusion, as well as patient's age, type of illness and treatment may influence the development of alloantibodies. Alloimmunization rates of 3.4% in hospitalized patients have been reported. The main purpose of this study is to compare the frequency of alloimmunization to red blood cell antigens in cancer patients that were transfused with phenotyped blood with those who were not.

Method:

Transfusion medical records of 669 oncologic patients were analyzed. Patients were evaluated by cancer type, number of transfusions, presence of alloantibodies and its clinical significance. Antibody screening and identification to red cell antigens were performed using gel-test (Diamed S/A, Lagoa Santa Brazil). All hematological patients were phenotyped for Rh (C, c, E, e, Cw) and K1 antigens using gel-test (Diamed S/A). Patients with hematological diseases were exclusively transfused with RBC units also matched for Rh/K1 antigens. Patients with other types of cancer were transfused with non-phenotyped RBC units. Patients that were previously immunized were excluded from the study.

Result:

Of 2,781 transfusions in 669 oncology patients, 1,026 were performed using phenotyped matched RBC units for Rh and K1 antigens. Patients with hematological diseases were more transfused when compared to patients with other types of cancer (p<0.001). However, alloimmunization rate found in patients that were transfused with phenotyped RBC units was statistically significant lower (1.026/1,000 transfusions) when compared to those one non-phenotyped blood (7.98/1,000 transfusions), (p=0.015). Moreover, the relative risk of a cancer patient to develop RBC alloantibodies when transfused with non-phenotyped Rh and Kell RBC units is 2.519 (IC 95%=2.181-2.908).

Conclusion: The rate of alloimmunization was statistically higher in patients that were transfused non-phenotyped RBC, even though this group was less transfused. We conclude that phenotyping RBC for Rh and K1 antigens reduces alloimmunization among cancer patients and prevents future complications when transfusing them.

Disclosures:

No relevant conflicts of interest to declare.