bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Human AF9 Homologue in Zebrafish Is Involved in Primitive Hematopoietic Development.
Authors: Germano Giuseppe , Guariento Ilaria , Tiso Natascia , Argenton Francesco , Basso Giuseppe 
Published: October 2019

Abstract
Abstract 3653
Poster Board III-589

Introduction
Human AF9 (alias MLLT3) gene was initially identified as one of the most common translocation partners of MLL associated with acute myeloid leukemia. More recently it has been demonstrated that AF9 can be a positive regulator of early erythroid/megakaryocytic lineage decisions. However, many biological functions of AF9 and its role in leukemia remain unclear. Here, we aimed to identify the zebrafish homologue of human AF9 and to analyze its function using the zebrafish model system.

Methods and Results
A single zebrafish af9 gene was identified by homology searching at zebrafish genome database (www.ncbi.nlm.nih.gov/). The putative af9 protein showed 60% identity and 73% similarity to human counterpart. These results from the perspective of phylogenetic and synteny analyses indicated that zebrafish af9 was the likely homologue of human AF9. We used whole-mount in situ hybridization to analyzed its spatial and temporal expression patterns during embryonic development. Af9 was expressed specifically in the posterior-lateral mesoderm at the 5-somite stage and in the intermediate cell mass (ICM, site of primitive hematopoiesis) between 18 to 24 hours post fertilization (hpf). In order to characterize the role of af9 in early hematopoietic development, we performed over-expression and loss-of-function experiments in zebrafish embryos, and then analyzed various hematopoietic markers by reverse transcriptase quantitative PCR (RT-qPCR). Inducing ectopic af9 expression by mRNA injection we found an increased expression of gata1 and hbae1 (erythroid markers) and reduced expression of runx1, scl, lmo2, c-myb (stem cell/precursor associated markers) as well as pu.1 and l-plastin (myeloid markers) at 22 hpf-old embryos. Conversely af9 loss-of-function obtained using anti-sense morpholinos showed decreased expression of gata1 and hbae1 mRNA, while pu.1 and l-plastin increased their expression in morphant embryos at 22 hpf. The same morphants did not show any appreciable change in stem cell/precursor marker expression.

Conclusion
The finding that gata1 expression is controlled by af9 suggests that this gene might promote the erythroid development in zebrafish primitive hematopoiesis. As the genetic programs of AF9 are evolutionary conserved between human and zebrafish, these findings provide new insights and opening further perspectives in leukemia research and hematopoiesis studies.

Disclosures:
No relevant conflicts of interest to declare.
Modulation of Natural Killer Cells by Therapeutic Antibody Preparations.
Authors: Hokland Marianne , Bohn Anja Bille , Harbo Thomas , Vorup-Jensen Thomas , Petersen Line 
Published: October 2019

Abstract
Abstract 3680
Poster Board III-616
Therapeutic antibody preparations are used in the treatment of several diseases. Thus, human immunoglobulin is administered as high dose intravenous immunoglobulin (IVIg) to patients suffering from a wide range of disorders. Moreover, various monoclonal antibodies (MoAbs) are approved for therapy in cancer and immune diseases. Natural Killer (NK) cells exert important immune effector functions. As these cells express the IgG receptor, CD16 (FcγRIIIa), it is of clinical importance to investigate how these cells react to therapeutic antibody preparations. We found that cytotoxic effector functions of NK cells were significantly inhibited by therapeutic antibody preparations in vitro. Natural cytotoxicity and antibody dependent cell-mediated cytotoxicity (ADCC) were inhibited by treating peripheral blood mononuclear cells (PBMCs) with the polyclonal immunoglobulin preparation Beriglobin, as well as the MoAb preparation Rituximab, prior to standard 51Cr-release assays compared to untreated cells. Moreover, our data showed that freshly isolated NK cells had IgG bound to the cell surface. The level of bound IgG increased following short-time exposure to both therapeutic antibody preparations, and the level of CD16, which is the key receptor for inducing ADCC, decreased. To investigate whether the NK-inhibitory effects of IVIg were demonstrable in vivo too, we analyzed blood from patients diagnosed with chronic inflammatory demyelinating polyneuropathy (CIDP), a disorder in which high-dose IVIg as monotherapy has shown striking effects. Comparing peripheral blood samples obtained from patients before treatment and 3 days following high-dose IVIg using multiparameter flow cytometry, we demonstrated a substantial decrease in the number of CD56+/CD3- NK cells. Moreover, as shown in vitro, the expression of CD16 on the NK cells was markedly reduced following high-dose IVIg treatment due to IgG engaged by the FcγRIIIa receptor (CD16). Finally, we found that NK mediated cytotoxicity decreased significantly when comparing peripheral blood samples from patients before and 3 days following IVIg treatment. In conclusion, our data presented in this study show that high-dose immunoglobulin therapy induces NK inhibitory effects in vitro as well as in vivo and thus may have important consequences for the overall therapeutic efficiency.

Disclosures:
No relevant conflicts of interest to declare.
Escalated BEACOPP Versus ABVD-Like Chemotherapy for Hodgkin Lymphoma Patients: a Cochrane Review.
Authors: Brillant Corinne , Bauer Kathrin , Herbst Christine , Monsef Ina , Skoetz Nicole , Engert Andreas 
Published: October 2019

Abstract
Abstract 3705
Poster Board III-641

Introduction
The main challenge in treating Hodgkin lymphoma (HL) is to find the optimal treatment with the best efficacy and least toxicity. So far there are two different international standards for the treatment of intermediate and advanced stage HL: chemotherapy with ABVD regime and chemotherapy with escalated BEACOPP regime. This review aims to clarify advantages and disadvantages of both treatments.

Methods
MEDLINE, CENTRAL and EMBASE were systematically searched for randomised controlled trials from 1985 to 2008. Trials comparing treatment with at least 2 cycles escalated BEACOPP versus chemotherapy with at least 4 cycles of ABVD within patients in intermediate or advanced stages HL were included. Trials without any published results will be excluded from the meta-analysis. Trial selection, quality assessment and data extraction were done independently by two review authors. Time-to-event outcome were analyzed with hazard ratios (HR) and 95% confidence interval (CI) in a fixed effects model.

Results
A total of 683 references were screened. Four eligible trials with 1970 patients were identified and included in the main analysis: the HD9 and HD14 trials from Germany, the HD2000 and GSM-HD trials from Italy. The escalated BEACOPP regime was more toxic during treatment than ABVD. Progression free survival (PFS) was statistically significantly longer for escalated BEACOPP: HR was 0.53 (95% CI [0.42, 0.67], I2=0%). In the main analysis of overall survival (OS), escalated BEACOPP was statistically significantly better than ABVD: HR was 0.70 (95% CI [0.50, 0.98], I2=0%). However, this result is not robust. In the OS-analysis restricted to advanced stage trials, the HR was 0.93 (95% CI [0.52, 1.04], I2=0%). Due to the high weight of the HD9 trial (57%), the OS-results are strongly influenced by this trial. In a sensitivity OS analysis without the HD9 trial the HR was 0.83 (95% CI [0.50, 1.38]). Only one trial (HD9) had a follow-up of over 5 years; the median of the other trials was approx. 3 years.

Conclusions
This meta-analysis showed a better PFS and suggests a benefit in OS for escalated BEACOPP in comparison to ABVD-like regimen. However, many trials had a short follow-up. Longer follow-up and the inclusion of the ongoing EORTC 20012 trial should allow a more definitive answer concerning OS.

Disclosures:
No relevant conflicts of interest to declare.
RAD001 Exerts Anti-Tumor Activity in Waldenstrom Macroglobulinemia.
Authors: Vesole Steven , Jia Xiaoying , Roccaro Aldo M. , Sacco Antonio , Ngo Hai T. , Azab Feda , Azab Abdel Kareem , Melhem Molly R , Runnels Judith M. , Quang Phong , Anderson Kenneth C. , Ghobrial Irene M. 
Published: October 2019

Abstract
Abstract 3732
Poster Board III-668

Background
Waldenstrom Macroglobulinema (WM) is an incurable B-cell disorder characterized by the presence of IgM monoclonal gammopathy and bone marrow infiltration of lymphoplasmacytic cells. Apart from promising advances in therapeutic strategy, response rates and treatment-free survival remain inconsistent across prescribed regimens. As we have previously shown, the PI3K/Akt pathway actively mediates tumor growth, survival, migration, and cell cycle within primary WM cells. An active target of this pathway includes the downstream mammalian target of rapamycin (mTOR). By inhibiting the mTOR pathway with RAD001 (Afinitor™, Novartis Pharmaceuticals), we hoped to constructively regulate aforementioned cellular function, and also understand from a preclinical perspective its specificity of action in our microenvironmental models, both alone and the presence of monoclonal antibody against CD20 and proteasome inhibitor.

Methods
WM cell lines (BCWM1) and IgM secreting cell lines MEC1 and RL were used. Bone marrow stromal cells (BMSC) were obtained from patients with WM. Peripheral blood mononuclear cells were harvested from healthy donors. Agents tested included RAD001, Rituximab and Bortezomib. Cytotoxicity, DNA synthesis, and cell cycle were measured using MTT assay, [3H]-thymidine uptake, and flow cytometry/PI staining, respectively. Antibody-dependent cellular cytoxicity (ADCC), transwell, and Matrigel assays were preformed to measure cell lysis, SDF-1 chemotaxis-induced migration, and angiogenesis, respectively. Adhesion to fibronectin has been evaluated with WM cells and BMSC in the presence of RAD001, with and without Bortezomib.

Results
RAD001 induced cytotoxicity and inhibition of DNA synthesis with an IC50 of 1-10 nM in BCWM1 cells at 48 hours. Similar effects were seen in IgM cell lines with an effective dose of 0.1-1 nM. In contrast, at IC50 treatment level donor cells displayed no significant cytotoxicity (>85% survival). Cell cycle analysis showed corresponding G1 arrest, and angiogenesis was also inhibited. RAD001, Bortezomib, and Rituxan combinations showed synergistic cytotoxicity, which was attenuated when WM cells were co-cultured with BMSC. Migration of BCWM1 and adhesion of BCWM1 to BMSC was reduced under presence of RAD001.

Conclusions
These functional assays therefore show that RAD001 has significant and synergistic antitumor activity in WM which will inform the design of future clinical trials.

Disclosures:
Ghobrial: Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.
Vorinostat Induced Cellular Stress Disrupts the Balance Between p38 MAPK and Erk Pathways Leading to Apoptosis in WM Cells.
Authors: Sun Jenny , Tseng Hsiuyi , Xu Lian , Hunter Zachary , Ciccarelli Bryan , Fulciniti Mariateresa , Yang Guang , Gong Ping , Zhou Yangsheng , Liu Xia , Munshi Nikhil C. , Patterson Christopher J , Treon Steven 
Published: October 2019

Abstract
Abstract 3740
Poster Board III-676
Despite advances, Waldenstrom's Macroglobulinemia (WM) remains incurable, and novel agents are urgently needed. Histone deacetylase (HDACs) are involved in transcription regulation and signal transduction of cells through a genome wide alteration in histone modification and other proteins, leading to significant increase in cellular stress in tumor cells. We therefore examined the activity of Vorinostat, a histone deacetylase inhibitor (HDAC-I), and dissected its pro-apoptotic molecular pathways in WM cells. Vorinostat exhibited dose dependent killing of both primary, bone marrow derived WM cells, as well as BCWM.1 WM cells with an LD50 of 3.5 to 5uM using Annexin V and PI staining.
Vorinostat induced apoptosis in WM cells through activation of specific caspases at different time points. Caspase 3, 6, 8, and 9 were activated after 24 hours of Vorinostat. Even though caspase 7 is downstream of caspase 3, we observed that caspase 7 was activated earlier, starting at 6 hours. We therefore hypothesized that the regulators of caspase 7 may be affected by Vorinostat at an earlier time point. Further investigation confirmed that there was significant down-regulation of inhibitor of apoptosis (IAP) family members, including c-IAP1, c-IAP2, XIAP and Livin after 12 hours of treatment with Vorinostat, and may elude to greater sensitivity for IAPs in modulating Caspase 7 versus Caspase 3 in WM cells.
We also studied the stress pathways including Erk, JNK and P38 pathways in Vorinostat treated WM cells. Activated p38, phospho-p38, was upregulated starting at 12 hours, while phopho-Erk abruptly decreased following 24 hours of treatment with Vorinostat. There was minimal change in the activity of JNK pathway following Vorinostat treatment. The activation of the p38 pathway coincided with a reduction in c-IAP1, c-IAP2, XIAP and Livin following treatment of WM cells with Vorinostat for 12 hours.
Taken together, these studies support that stress induced apoptosis in WM cells is mediated through disruption in the balanced activity between the Erk and p38 MAPK pathways. Vorinostat induced cellular stress results in the activation of p38 MAPK pathway and a reduction of the IAP family members, leading to early activation of caspase 7. While the inhibition of Erk pathway by Vorinostat results in delayed activation of caspase 3, 6, 8, and 9 at 24 hours, the collective signaling strength of p38 activation as well as inhibition of Erk likely determines the apoptotic fate WM cells upon Vorinostat treatment.

Disclosures:
No relevant conflicts of interest to declare.
Complete Responses with Denileukin Diftitox in Cutaneous T-Cell Lymphoma Studies.
Authors: Foss Francine , Duvic Madeleine , Olsen Elise A. , Kozlovski Anna 
Published: October 2019

Abstract
Abstract 3745
Poster Board III-681
Cutaneous T-cell lymphoma (CTCL) is a disorder of CD4+ helper T-cells with manifestations in the skin, nodes, and, in advanced stages, blood and visceral sites. For many patients, the clinical course of the disease is chronic and progressive, despite multiple therapeutic interventions. To date, are few controlled clinical trials which demonstrate durable complete responses (CR) in advanced stage patients. Denileukin diftitox (DD) is a recombinant fusion protein targeting interleukin-2 receptor which has demonstrated efficacy in early and advanced stage CTCL with response rates of 30% and 44% in two clinical trials of CTCL pts with Stage Ib-IVA relapsed and refractory CTCL or Stage I-III disease, ≤3 prior therapies respectively. In these trials, patients (pts) were randomized to receive DD at a dose of either 9 or 18 ug/kg/d daily x 5 every 21 days. Another rollover trial enrolled pts who had progressed after prior response with DD (N=29) or were CD25- (N=36); all pts in the rollover trial were treated with DD at 18ug/kg dose. Of 263 intent-to-treat pts in these trials, 227 had CD25+ skin infiltrates by immunohistochemistry (IHC) confirmed by a reference pathologist and 36 were CD25-. Overall, 24 (9.1%) pts attained durable complete response (CR). The median age of the responders was 59, and 12 CR pts were over age 60. Of the CR group, 15 pts had early stage (I-IIa) disease and 9 had advanced stage (IIb-IV) CTCL. The mean prior therapies was 3.6 with 37% of patients having received >3 therapies. Of the 24 CR pts, 21 were CD25+ and 3 were CD25-. Two CR were pts who had previously responded to DD in an earlier clinical trial. Of all pts receiving 9 ug/kg dose (N=80), there were 6 (7.5%) CR; for all pts receiving 18 ug/kg dose (N=183), there were 18 (9.8%) CR. There was no significant difference frequency of CR between the 9 and 18 ug/kg groups (P=0.56) or between the CD25+ (N=118) and CD25- (N=36) pts treated at 18 ug/kg dose (P=0.64). CR rate was similar between the early and advanced stage patients (10.7% vs 8.9% respectively). The median time to response was 53.5 vs 41days for CD25+ pts treated in the 9 and 18 ug/kg groups respectively, and 43 days for the CD25- group. The response durations ranged from 57 days to >1325 days in the CD25+ and 190-400 days in the CD25- groups. Of the 24 CR, 7 have progressed as of the time of the analysis and 17 remain in CR. The overall median PFS at the time of analysis has not been reached (range 169-1388+ days). Of the 24 CR, 3 pts had hypersensitivity reactions and 3 had capillary leak syndrome associated with DD treatment. In summary, these studies demonstrate clinical benefit of DD with CR in both early and advanced CTCL at both 9 and 18 ug/kg doses and with durable responses in a small number of pts whose malignant lymphocytes were CD25- by ICH.

Disclosures:
Foss: Eisai : Speakers Bureau. Olsen:Eisai: Research Funding. Kozlovski:Eisai Pharm: Employment.
Irgm1 Is a Negative Regulator of Interferon-Gamma Signaling in Hematopoietic Stem Cells.
Authors: King Katherine Y , Baldridge Megan T , Weksberg David C , Goodell Margaret A 
Published: October 2019

Abstract
Abstract 382
Hematopoietic stem cells (HSCs) are a self-renewing population of bone marrow cells that give rise to all of the cellular elements of the blood and retain enormous proliferative potential in vivo. We have a growing understanding that the controls on HSC proliferation are tied in part to regulation by the immune system—specifically, that HSC proliferation and mobilization can be stimulated by the immune cytokines interferon-alpha and interferon-gamma (IFNg). Our previous work has demonstrated that HSC quiescence and function are aberrant in mice lacking the immunity-related GTPase Irgm1 (also Lrg47). Indeed, the bone marrow of Irgm1-deficient animals at baseline mimics the bone marrow of wild type animals that have been stimulated with IFNg. We hypothesized that the HSC defects in Irgm1-deficient animals are due to overabundant IFNg signaling, and that Irgm1 normally serves to dampen the stimulatory effects of IFNg on HSCs. To test this hypothesis, we used RNA expression profiling to compare gene expression in wild type versus Irgm1-deficient mice. We found that interferon-dependent signaling is globally upregulated in the HSCs of Irgm1-deficient mice. Next we generated Irgm1-/-IFNgR1-/- and Irgm1-/-Stat1-/- double knock out animals. In contrast to the phenotype of Irgm1 single knock out mutants, the hyperproliferation and self-renewal defects in HSCs were both rescued in the double knock out animals, indicating that IFNg signaling is required for manifestation of the Irgm1-deficient phenotype. Futhermore, we found that Irgm1 is expressed in HSCs in a Stat1- and IFNgR-dependent fashion, suggesting that it forms a negative feedback loop for IFNg signaling in the HSC population. Collectively, our results indicate that Irgm1 is a powerful negative regulator of IFNg-dependent stimulation in HSCs. These findings demonstrate that IFNg provides a significant stimulus for HSC proliferation even in the absence of infection, and that IFNg-dependent signaling must be tightly regulated to preserve HSC self-renewal capacity. This study provides evidence that the Irgm1 protein can serve as a link between immunity and regulation of hematopoiesis at the level of the stem cell. We speculate that utilization of Irgm1 for its immune functions may detract from its ability to regulate HSC self-renewal capacity, thus ultimately contributing to myelosuppression and increased risk of death from chronic infections such as tuberculosis.

Disclosures:
No relevant conflicts of interest to declare.
Has the Prognostic of Primary Plasma Cell Leukemia Improved with New drugs?
Authors: Chaoui Driss , Leleu Xavier , Roussel Murielle , Royer Bruno , Rubio Marie-therese , Ducastelle Sophie , Merabet Fatiha , Garderet Laurent , Kolb Brigitte , Debarri Houria , Arkam Yazid , Harousseau Jean-Luc , Moreau Philippe 
Published: October 2019

Abstract
Abstract 3869
Poster Board III-805

Introduction
The outcome of patients with plasma-cell leukemia (PCL) is poor. Avet-Loiseau reported on behalf the IFM, our first experience in PCL patients and showed that the median overall survival (OS) was 8 months (Avet-Loiseau, Blood, 2001). Since 1999, novel agents such as Thalidomide, Bortezomib (Velcade) or Lenalidomide (Revlimid) have been widely used in the treatment of multiple myeloma, both at the time of relapse or part of upfront therapy.

Patients and methods
In this retrospective analysis, we have looked at the outcome of PCL patients treated within the IFM since 1999 in order to study the impact of novel agents on survival.

Results
31 cases, 20 males, 11 females, median age 55 years (34-78) were analyzed. Twenty one patients less than 65 years received high-dose therapy as part of frontline treatment : 19 autologous haematopoietic stem cell transplantation (HSCT) and 5 allogeneic transplantation. Novel agents were used part of induction therapy in 6 cases, at the time of relapse for 9 patients, for both induction and relapse in 16 cases. Thirteen patients received 1 novel agent, 11 received 2 and 7 patients received the 3 novel agents. The median number of lines of therapy was 2 (1 to 4). Bortezomib was used as up front treatment in 15 patients and at relapse for 9 patients. Overall response rate according the IMWG criterias was 70% (17/24) including 11 CR or VGPR (45%). PAD (Bortezomib, Adriamycin and Dexamethasone) and VTD (Bortezomib, Thalidomide, Dexamethasone) regimens provided the best response rates. Lenalidomide was used in 13 patients mostly at relapse. A response was obtained in 53% of patients including 2CR and 2 VGPR (30%). Nineteen patients were treated with Thalidomide-based regimens. Overall response rate was 52% (10/19) including 2 CR and 6 VGPR (31%).
Overall, for the whole group of patients, the median progression-free survival was 8 months (0-26) and the median OS was 15 months (6-108). When comparing this survival with that described in our previous experience reported before 1999, we clearly showed that the use of novel agents improved the survival of patients with PCL.

Conclusion
In this retrospective study, novel agents improved the prognosis of P-PCL. Prospective IFM phase II studies are ongoing to confirm these results.

Disclosures:
No relevant conflicts of interest to declare.