bluuebunny/crossref_metadata_2025_split huggingface dataset:
Decreased Expression In NF-κB Essential Modulator Due to a Novel Splice-Site Mutation Causes Ectodermal Dysplasia with Immunodeficiency
Authors: Karakawa Shuhei , Okada Satoshi , Tsumura Miyuki , Mizoguchi Yoko , Ohno Norioki , Yasunaga Shin'ichiro , Ohtsubo Motoaki , Kawai Tomoki , Nishikomori Ryuta , Takihara Yoshihiro , Kobayashi Masao 
Published: October 2019

Abstract
Abstract 1732

<Introduction>
X-linked ectodermal dysplasia with immunodeficiency (XL-EDA-ID) is caused by hypomorphic mutations in the nuclear factor κB (NF-κB) essential modulator (NEMO) gene, also called IKBKG, and these mutations impair but do not abolish NF-κB signaling, resulting in distinct clinical and immunologic phenotypes. We presented the patient with XL-EDA-ID showing a novel splice-site mutation. In this report we precisely study the involvement of this mutation in the molecular pathogenesis of XL-EDA-ID.

<Patient>
The patient is 12 years old Japanese boy. He had conical-shaped teeth and hypodontia. He had suffered from recurrent bacterial infections and these pathogenic bacteria were mostly streptococcus pneumonia. The laboratory examination revealed that the number of white blood cell counts, the classification of lymphocytes, and the serum immunoglobulin levels were within normal range. However the specific antibodies against measles and streptococcus pneumonia were negative in spite of these infections.

<Results>
We identified a novel hemizygous mutation, 769-1 G>C, at the splicing acceptor site of exon 7 in the IKBKG gene. In order to clarify the effect of this mutation on mRNA, we performed a cloning analysis. Although various abnormal spliced NEMO (mutant NEMO) mRNAs were observed, a small amount of wild-type NEMO (WT NEMO) mRNA was also identified. The rate of WT and mutant was variable on the time of blood collection. Further we performed FACS and immunoblot analysis in order to evaluate the effect of this mutation at protein level. Two major bands which were presumed to be derived from WT and one mutant were detected in immunoblots using EBV transfected B cells, however the expression levels of these bands were markedly decreased compared to those of the healthy controls. The NEMO protein expression was confirmed to be decreased in various lineages of leukocytes. We generated WT and two representative mutant NEMO constructs and measured their NF-κB transcriptional activity using reporter assay. One mutant NEMO abolished NF-κB transcriptional activity, the other showed weak activity. However, neither of mutant NENO showed a dominant-negative effect against WT NEMO activity. CD14+ cells from the patient produced a lower level of TNF-α in response to IFN-γ stimulation, on the other hand CD3+ cells produced very little IFN-γ in response to IL-12. CD4+ T-cell proliferation was impaired in response to measles and mumps, but not rubella.

<Conclusion>
The hemizygous 769-1 G>C mutation was shown to cause the decreased expression of WT NEMO protein, resulting in the decrease in NF-κB activation and the development of XL-EDA-ID.

Disclosures:
No relevant conflicts of interest to declare.
Bone Morphogenetic Proteins Inhibit CD40L- and IL-21-Induced Ig Production In Human B Cells: Differential Effects of BMP-6 and BMP-7
Authors: Huse Kanutte , Bakkeb∅ Maren , Forfang Lise , Hilden Vera I. , Smeland Erlend B. , Myklebust June H. 
Published: October 2019

Abstract
Abstract 1745

Introduction:
TGF-β has been shown to play an important role in directing class switch recombination in human B cells. Bone morphogenetic proteins (BMPs) are members of the TGF-β family, and we have previously shown that BMP-6 inhibits proliferation of B cell progenitors as well as mature B cells in humans. However, how various BMP members affect Ig production and B-cell maturation to plasma cells have not been investigated. Here we studied the effects of various BMPs in CD27- naive and CD27+ memory B cells from peripheral blood of healthy human donors.

Methods:
We used flow immunomagnetic beads and flow cytometry cell sorting to purify CD19+CD27- naive and CD19+CD27+ memory B cells from peripheral blood of healthy donors, and investigated BMP induced effects on in vitro proliferation, cell death and Ig production as measured by thymidine incorporation assay, propidium iodide staining and ELISA, respectively. To separate direct inhibition of plasma cell differentiation from indirect effects via suppression of proliferation and induction of apoptosis, CFSE tracking of cell division was combined with immunophenotyping of cultured cells to identify CD27+CD38+ plasma cells. Expression of transcription factors and AICDA were measured by quantitative real-time PCR.

Results:
BMP-2, -4, -6 and -7 specifically inhibited CD40L- and IL-21-induced production of IgM, IgG and IgA. BMP-6 was the most potent inhibitor, reducing the Ig production by 70% in memory B cells and more than 50% in naive B cells. By investigating the mechanisms for reduced Ig production, we found a striking difference between the structurally similar BMP-6 and BMP-7. BMP-6 directly inhibited differentiation to CD27+CD38+ plasma cells, whereas BMP-7 only had minor effects on differentiation. Instead, BMP-7 mainly affected Ig production indirectly by inducing apoptosis. Furthermore, we explored BMP-6-induced signaling and gene regulation in more detail in memory B cells. BMP-6 up-regulated Id1, Id2 and Id3 gene expression in CD40L- and IL-21-stimulated cells (6.1-fold, 1.7-fold and 4.0-fold induction after 48 hours culture with BMP-6, respectively). In contrast, BMP-6 potently inhibited CD40L- and IL-21-induced upregulation of the transcription factor XBP-1, necessary for the late stages of plasmacytic differentiation. Expression of transcription factors regulating earlier stages (IRF4, PRDM1) was not affected, indicating that BMP-6 only modulates late events of plasma cell development.

Conclusion:
These results show that BMPs potently suppress Ig production in mature human B cells by inhibiting differentiation or by inducing cell death.

Disclosures:
No relevant conflicts of interest to declare.
BACOPP-D - An Etoposide-Free Dose-Escalated Polychemotherapy Regimen In Advanced Hodgkin‘s Lymphoma
Authors: Naumann Ralph , Kluge Diana , Haenel Annette , Wetzko Katrin , Friedel Nadja , Schmidt Helmuth , Dawel Monic , Beuthien-Baumann Bettina , Schwanebeck Uta , Ordemann Rainer , Haenel Mathias , Ehninger Gerhard 
Published: October 2019

Abstract
Abstract 1754

Introduction:
The development of the escalated BEACOPP regimen let to an improved outcome in patients with advanced Hodgkin‘s lymphoma (HD9 study of the GHSG). However, the application of high dose etoposide (cumulative 4,8 g/m2 per 8 cycles) seems to be associated with an increased incidence of secondary MDS and AML, respectively. Therefore, the aim of our multicenter pilot study was to evaluate the efficacy and toxicity of the etoposide-free as well as dose-intensified BACOPP-D protocol.

Methods:
From May 2000 until August 2008 a total of 139 untreated patients with Hodgkin‘s lymphoma (HL) stage IIB, III, and IV were treated with BACOPP-D which included cyclophosphamide 1250 mg/m2 (d1), adriamycin 25 mg/m2 (d1+2), dacarbazine 250 mg/m2 (d1-3), procarbazine 100 mg/m2 (d1-7), prednisolone 40 mg/m2 (d1-14), bleomycin 10 mg/m2 (d8) and vincristine 1,4 mg/m2 (maximum 2 mg, d8) at three-weekly intervals with granulocyte colony-stimulating factor (G-CSF). A consolidating involved field radiation (30 Gy) was performed only in patients who achieved less than CR following chemotherapy. Post-treatment follow-up included PET imaging.

Results:
All patients (median age 34 years, range 16–65; 86 male, 53 female) are assessable for toxicity and treatment outcome. We analyzed the acute toxicity for 1060 cycles of BACOPP-D. CTC grade III/IV haematological toxicities per patient were observed as follows: leukopenia 92%, anemia 40%, and thrombocytopenia 35%. CTC grade III/IV non-haematological side effects included documented infection (8%) and lung toxicity (one patient). Consolidation radiotherapy was given in 73 patients (52,5%). A total of 125 patients (89,9%) achieved complete remission, 9 patients (6,5%) achieved partial remission, five patients (3,6%) had progressive disease. At a median observation time of 46 months (5-109 months), 9 patients (6,5%) have relapsed, and 11 deaths were documented (4 HL-specific and 4 treatment related deaths, 1 death due to ruptured Meckel diverticulum with peritonitis, one 65 year-old woman died in CR following myocardial infarction and 1 death due to secondary malignancy). Only two patients developed a second neoplasia (hypopharyngeal carcinoma in an alcoholic; melanoma). The overall survival and progression free survival rates at 46 months were 89,7% and 85,9%, respectively. Discussion: BACOPP-D regimen appears as a feasible and effective treatment which induced a complete morphologic remission in a high proportion of patients with advanced HL. The treatment was associated with moderate acute toxicity. No secondary AML or MDS occurred so far.

Disclosures:
No relevant conflicts of interest to declare.
Aplidin® (Plitidepsin) Activity In Peripheral T-Cell Lymphoma (PTCL): Final Results
Authors: Fermé Christophe , Mateos Maria Victoria , Szyldergemajn Sergio , Corrado Claudia S. , Zucca Emanuele , Extremera Sonia , Gianni Alessandro M. , Vandermeeren Andrea , Ribrag Vincent 
Published: October 2019

Abstract
Abstract 1767

Background:
Peripheral T-cell lymphomas (PTCL) are a heterogeneous group of NHL, representing 10–15% of all newly diagnosed cases; their prognosis is poor and optimal therapy is yet to be defined. Most patients (pts) relapse after standard first-line chemotherapy and ultimately die from their disease. Though pralatrexate has been recently FDA-approved for this indication, novel active agents are still needed. From December 2004 to June 2008, 46 adult pts with aggressive non-Hodgkin lymphoma (NHL) were treated in a phase II study aimed at evaluating the activity of plitidepsin. An interim analysis showed responses restricted to pts with PTCL (4/17 vs. 0/29 in pts with B-cell NHL; Fisher's test: p=0.01). Thus, PTCL cohort was expanded. Herein, final results are presented.

Patients and methods:
As of June 2010, 32 pts were treated with plitidepsin 3.2 mg/m2 i.v. infusion over 1-h on days 1, 8 and 15 q4wk. Twenty-nine pts are evaluable for efficacy. Median number of previous regimens was 2 (range, 1–6), including prior stem cell transplantation in 9 pts (28%). Histology: PTCL, NOS 16, anaplastic large-cell 4, angioimmunoblastic 9 and NK/T nasal type 3. Twenty-three pts were male, median age was 57 y (30-80) and ECOG: 0 (14 pts), 1 (12 pts) and 2 (6 pts).

Results:
Two CR and 4 PR were observed, for a 20% objective response rate (95% CI, 8%-39%). Median duration of response was 2.2 months (range, 1 – 28). Median progression-free survival was 1.6 months (95% CI, 1 – 3) and median overall survival was 10.2 months (95% CI, 4 – 24). Toxicity, particularly haematological, was mild. Two patients developed G3, and 2 G4 neutropenia, and 1 pt G3, and 2 G4 thrombocytopenia. Transient and reversible G3 ALT or AST elevations occurred in 7 and 4 pts, respectively, with no patients experiencing G4 events. Clinical toxicities were mainly G1-2 muscular weakness and myalgia in 14% of pts, and G1-2 fatigue, nausea and vomiting in 21%, 28% and 17% of patients, respectively.

Conclusions:
Plitidepsin has activity, with an acceptable safety profile in non-cutaneous relapsed/refractory PTCL patients. Remarkably, lack of hematologic toxicity makes plitidepsin an ideal agent either for treating patients with poor bone marrow reserve or combining it with other active agents. A combination of plitidepsin with gemcitabine is currently being explored in this patient population.

Disclosures:
Szyldergemajn: PharmaMar SAU: Employment. Corrado:PharmaMar SAU: Employment. Extremera:PharmaMar SAU: Employment. Vandermeeren:PharmaMar SAU: Employment.
Low Absolute Lymphocyte Count and Addition of Rituximab Confer High Risk for Interstitial Pneumonia In Patients with Diffuse Large B Cell Lymphoma
Authors: Yu Yuan-Bin , Huang Yu-Chung , Liu Chia-Jen , Pai Jih-tung , Lu Hsueh-Ju , Liu Chun-Yu , Hong Ying-Chung , Hsiao Liang-Tsai , Gau Jyh-Pyng , Hsu Hui-Chi , Chiou Tzeon-Jye , Liu Jin-Hwang , Tzeng Cheng-Hwai 
Published: October 2019

Abstract
Abstract 1805

Purpose:
Several studies reported pulmonary toxicities in patients with diffuse large B cell lymphoma (DLBCL) receiving rituximab and chemotherapy. This retrospective study aimed to determine the risk factors and clinical characteristics of interstitial pneumonia in patients with DLBCL.

Methods:
From January 2000 to May 2009, 529 consecutive patients with DLBCL receiving first-line COP- or CHOP-based chemotherapy with or without rituximab in Taipei Veterans General Hospital were enrolled. Interstitial pneumonia (IP) was defined as diffuse pulmonary interstitial infiltrates found on computed tomography scan as well as respiratory symptoms. Patient characteristics and outcome parameters were retrieved via medical chart review.

Results:
IP was observed in 26 patients (4.9%) and 6 of them were confirmed asPneumocystis jirovecii pneumonia. The median number of chemotherapy course to IP was 4 cycles (range, 1–7). By multivariate logistic regression, absolute lymphocyte count (ALC) less than 1×109/L before treatment (odds ratio [OR] 2.75, 95% confidence interval [CI] 1.23–6.19) and addition of rituximab to chemotherapy (OR 4.56, 95% CI 1.68–12.39) were identified as independent risk factors for IP. In the rituximab-treated patients, low ALC at baseline further increased the risk for IP.

Conclusions:
Incidence of IP is increased in patients with DLBCL receiving rituximab-containing chemotherapy. Specific subgroup with lymphopenia at diagnosis should receive more attention in detecting this pulmonary complication.

Disclosures:
No relevant conflicts of interest to declare.
AKN-028, a FLT-3 Kinase Inhibitor In Preclinical Development, Induces Significant Gene Regulation That Differs From PKC-412
Authors: Parrow Vendela C , Eriksson Anna , Göransson Hanna , Rickardson Linda , Lehmann Fredrik , Lenhammar Lena , Larsson Rolf , Hoglund Martin 
Published: October 2019

Abstract
Abstract 1837
AKN-028 (formerly BVT-II, abstract presented at EHA 2008, Eriksson A et al) is a tyrosine kinase inhibitor originally identified in a screen targeting FLT-3. In vitro testing by fluorometric microculture cytotoxicity assay (FMCA, Lindhagen E. et al, Nat Protoc 2008; 3:1364-9) on primary tumor cells from 29 patients with different haematological malignancies showed that the cytotoxic activity was most pronounced in acute myeloic leukaemia (AML). In vivo, efficacy was demonstrated in a hollow fiber mouse model using MV4-11 cells and primary tumor cells from AML patients.
Kinase inhibitors interacting with the ATP-binding pocket usually targets more than one kinase. PKC-412, presently in phase III trials in AML, is a staurosporine analogue targeting many kinases. In this study we show the kinase inhibition profile of AKN-028 when characterised on a broad panel of 320 different kinases. AKN-028 is relatively specific compared to the staurosporine analogues. To further analyse the difference in mechanism of action between the multi-targeting inhibitor PKC-412 and the more selective AKN-028, we have performed global gene expression analysis by Affymetrix arrays using two different leukaemia cell-lines, HL-60 and MV4-11 (expressing FLT3-ITD), and tumour cells from a patient with FLT3-ITDpos AML. The cells were treated with 10 μM of either compound or vehicle control for 6 h. Principal component (PC) analysis was used to visualise the global gene expression pattern, see fig 1. Searching for differential expression of mRNA levels showed that treatment with AKN-028 resulted in significantly altered gene expression in all three cell types tested, compared to vehicle control treated cells. 430 mRNAs were down-regulated, and 280 were up-regulated. By contrast, treatment with PKC-412 caused very few significant alterations of mRNA levels when compared to vehicle treated cells. Further analysis of gene expression patterns to elucidate the mechanism of action of AKN-028 is ongoing. In conclusion, even though AKN-028 is a relatively selective kinase inhibitor targeting FLT-3, it has more profound effects altering gene expression both in cultured AML cell-lines and a primary AML tumor sample than the multikinase inhibitor PKC-412.

Disclosures:
Parrow: Akinion Pharmaceuticals: Employment, Equity Ownership. Lehmann:Akinion Pharmaceuticals: Consultancy. Larsson:Akinion Pharmaceuticals: Consultancy.
GlycoVariant Anti-CD37 Small Modular Immuno-Pharmaceutical Exhibits Superior Natural Killer Cell Mediated Cytotoxicity Against Chronic Lymphocytic Leukemia Cells at Low Concentrations and Low Antigen Density
Authors: Rafiq Sarwish , Cheney Carolyn M , Lozanski Gerard , Lapalombella Rosa , Mo Xiaokui , Siadak Anthony , Jarjoura David , Byrd John C. , Muthusamy Natarajan 
Published: October 2019

Abstract
Abstract 1847
CD37 is a tetraspanin transmembrane family protein that is strongly expressed on the surface of mature human B cells and transformed mature B cell lymphoma and leukemia cells, including CLL cells. It is absent or minimally expressed on normal T cells, NK cells, monocytes, and granulocytes. Predominant expression of CD37 on CLL cells makes it an ideal candidate to target with potential agents for treatment of CLL. TRU-016, a small modular immunopharmaceutical protein (SMIP) that specifically binds to an extracellular region of CD37, is presently in clinical trials in CLL patients. TRU-016 includes humanized immunoglobulin variable regions (scFv) fused to a human IgG1 Fc region. We have previously reported that SMIP-016, the chimeric version of the humanized TRU-016, induced apoptosis in CLL B cells in the presence of goat anti-human Fc antibody cross-linker through a novel, caspase-independent pathway. Furthermore, SMIP-016 showed potent in vivo activity in a SCID xenograft mouse model. Aside from direct cytotoxicity, SMIP-016 mediates antibody-dependent cellular cytotoxicity (ADCC) by NK cells both in vitro and in vivo. In an attempt to enhance its ADCC function, a new variant of SMIP-016, SMIP-016GV, was designed with a modification of the glycosylation of the Fc portion of the molecule. SMIP-016GV exhibits enhanced binding to both low- and high-affinity molecular variants of human CD16 (FcγRIII) and augments ADCC potency when compared to SMIP-016. In this study, we compared SMIP-016GV and SMIP-016 in direct cytotoxicity and ADCC against CLL B cells. While SMIP-016 and SMIP-016GV mediated comparable direct cytotoxicity at 48 hrs in the presence of goat anti-human Fc crosslinker, the SMIP-016GV resulted in 2 to 4 fold increase in NK cell mediated ADCC function at all effector to target ratios tested. This increased ADCC with SMIP-016GV was observed using NK cell effectors derived from both normal as well as CLL-affected individuals. In addition, this enhanced cytotoxicity was sustained at concentrations of SMIP-016GV as low at 5E-6 μg/ml. These low concentrations of SMIP-016GV were also able to mediate superior ADCC in 697 cells expressing as few as 10,000 molecules of surface CD37 antigen. Furthermore, NK cells stimulated with the glycovariant were potently activated and released 3 to 4 fold more IFNγ compared to SMIP-016. Ongoing studies are aimed at defining other effector cells which may interact with SMIP-016GV via different Fcγ Receptors. Collectively, these results suggest potential use of the SMIP-016GV with enhanced ADCC function as an alternate for TRU-016 in B cell malignancies including CLL therapy.

Disclosures:
Siadak: Trubion Pharmaceuticals: Employment.
TRAIL Preferentially Affects Cell Cycle-Arrested Tumor Cells Including Stem- and Progenitor Cells From Patients with Acute Lymphoblastic Leukemia
Authors: Ehrhardt Harald , Alves Catarina Castro , Wachter Franziska , Jeremias Irmela 
Published: October 2019

Abstract
Abstract 1879
Leukemic stem- and progenitor cells exhibit low cycling activity which might represent a major cause for their increased treatment resistance. TRAIL (TNF-related apoptosis inducing ligand) is a novel putative anticancer drug currently in phase I and II clinical testing. We recently showed that TRAIL is able to address stem- and progenitor cells from patients with acute lymphoblastic leukemia (ALL) in xenotransplantation assays (Alves et al., Blood 2012,119,4224). As stem- and progenitor cells are often non-cycling, we asked here, whether TRAIL is able to address resting leukemia cells.
We used cell lines and primary tumor cells from children with ALL which were amplified in severely immuno-compromised mice (NSG mice). Cell cycle arrest was induced (i) by addition of conventional cytotoxic drugs which are known to act as cytostatic drugs such as doxorubicine; (ii) by biochemical inhibitors known to induce cell cycle arrest at different defined points of the cell cycle such as mimosine; (iii) by molecular approaches and knockdown of cyclinB arresting cell cycle in G2 or knockdown of cyclinE arresting cell cycle in G1.
Unexpectedly, TRAIL-induced apoptosis was enhanced, whenever cell cycle was arrested. Cell cycle arrest sensitized towards TRAIL-induced apoptosis independently from the point or phase of cell cycle which was arrested (G0, G1 or G2) and independently from the agent used to arrest the cell cycle. Similarly, knockdown of cyclinB or cyclinE both clearly sensitized cell line cells towards TRAIL-induced apoptosis.
Cytotoxic drugs and cell cycle inhibitors might arrest the cell cycle by activation of p53. Accordingly, when caffeine was added which inhibited p53 activity and drug-induced cell cycle arrest, sensitization towards TRAIL-induced apoptosis was blocked.
We have recently established a novel method which enables performing knockdown experiments in tumor cells derived from ALL patients (Höfig et al., Cell Comm. Signal. 2012,10,8). Using this method and most important for clinical translation, we could show that knockdown of either cyclinB or cyclinE clearly sensitized patient-derived ALL cells towards TRAIL-induced apoptosis.
Taken together and in contrast to most conventional cytotoxic drugs, TRAIL exerts anti-tumor activity preferentially against tumor cells in cell cycle arrest and less against actively cycling tumor cells. This special feature of TRAIL might explain its anti-tumor activity against stem- and progenitor cells in patients with ALL. Thus, TRAIL might represent an interesting drug to treat disease stages with accumulation of stem- and progenitor cells and static tumor disease, e.g., during minimal residual disease.

Disclosures:
No relevant conflicts of interest to declare.