bluuebunny/crossref_metadata_2025 huggingface dataset:
High-Level Expression of Mastermind-Like 2 (MAML2) Contributes to Aberrant Activation of the NOTCH Signaling Pathway In Human Lymphomas
Authors: Köchert Karl , Kreher Stephan , Aster Jon C , Kitagawa Motoo , Jöhrens Korinna , Anagnostopoulos Ioannis , Jundt Franziska , Stein Harald , Janz Martin , Dörken Bernd , Mathas Stephan
Published: October 2019

Abstract 2685

Inappropriate activation of the NOTCH signaling pathway, e.g. by activating mutations, contributes to the pathogenesis of various human malignancies. Here, we demonstrate that aberrant expression of an essential NOTCH co-activator of the Mastermind-like (MAML) family provides an alternative mechanism to activate NOTCH signaling in human lymphoma cells. We detected high-level MAML2 expression in several B cell-derived lymphoma types, including classical Hodgkin lymphoma (cHL) cells, relative to normal B cells. Inhibition of MAML protein activity by a dominant negative form of MAML or by shRNAs targeting MAML2 in cHL cells resulted in down-regulation of the NOTCH target genes HES7 and HEY1, which we identified as overexpressed in cHL cells, and in reduced proliferation. Furthermore, a NOTCH gene-expression signature in cHL cells confirmed their cell-autonomous NOTCH activity. Finally, in line with the essential role of MAML proteins for assembly and activity of the NOTCH transcriptional complex (NTC) we show that MAML-derived small-peptide constructs block NOTCH activity and disrupt NTC formation in vitro. These data strongly suggest direct targeting of the NTC as treatment strategy for NOTCH-dependent malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Validation of Monosomal Karyotype Based Model In the Risk Stratification of Acute Myeloid Leukemia
Authors: Al-Khabori Murtadha K. , Yee Karen , Gupta Vikas , Schimmer Aaron , Schuh Andre , Minden Mark D. , Brandwein Joseph
Published: October 2019

Abstract 2745

Background:

The influence of cytogenetic abnormalities on the prognosis of acute myeloid leukemia (AML) has been well-documented; however, the relative impact of certain miscellaneous abnormalities remains controversial. Recently, monosomal karyotype-based risk stratification has been shown to further discriminate the prognosis within the poor-risk karyotype group (Breems et al. JCO 2008), but this finding requires further validation.

Methods:

We retrospectively reviewed 779 consecutive adult AML patients treated with standard induction chemotherapy, consisting of daunorubicin plus cytarabine (3+7), at our institution from 1998–2008. After excluding patients with favourable risk, normal, missing or failed karyotype, 290 patients remained and were included in the analysis.

Results:

The baseline characteristics of these 290 patients were as follows: median age 59 y (range 18–81), male 181, prior malignancy 110, median white cell count (WBC) 7.6 × 10^9/L (range 0–246). The karyotypic features included single monosomy in 42, 2 or more monosomies in 51, and non-monosomy structural and numerical abnormalities in 197 patients. Of the 290, 116 (40 %) had three or more abnormalities (complex karyotype, CK). A total of 141 patients (49 %) achieved complete remission (CR) with 3+7 induction chemotherapy. Sixty-four patients received allogeneic stem cell transplantation in CR. The median overall survival (OS) for all patients was 12 months (95% CI: 10–14 months). The median OS was 10 (95% CI: 6–18), 7 (95% CI: 6–10) and 14 months (95% CI: 12–16) in the single monosomy, 2+ monosomy and non-monosomy groups, respectively (p < 0.0001 by log-rank test comparing the three groups). Among the patients containing at least one monosomy, the OS was not significantly different between the CK and non-CK groups (p = 0.08 by log rank). Similarly, in the non-monosomy structural abnormality group, the OS was not significantly different between the CK and non-CK groups (p = 0.2).

Conclusions:

Our results provide validation for the monosomal karyotype-based risk stratification for AML, indicating that patients with at least one monosomy have an inferior OS compared to other poor-risk non-monosomy groups. Within each of the monosomy and non-monosomy groups, the presence of a complex karyotype does not significantly influence the OS.

Disclosures:

No relevant conflicts of interest to declare.
IL-9 Production by Regulatory T Cells Recruits Mast Cells That Are Essential for Regulatory T Cell-Induced Immune-Suppression
Authors: Wolf Anna Maria , Wolf Dominik , McKenzie Andrew , Maurer Marcus , Rosenkranz Alexander R , Eller Kathrin
Published: October 2019

Abstract 2782

Tipping the balance between effector and regulatory cell populations is of critical importance in the pathogenesis of various autoimmune disorders. Both, mast cells (MC) and regulatory T cells (Treg) have gained attention as immunosuppressive cell populations. To investigate a possible interaction, we used the Th1- and Th17-dependent model of nephrotoxic serum nephritis (NTS), in which both MC and Treg have been shown to play a protective role.

We recently provided evidence that adoptive transfer of wild-type (wt) Treg into wt recipients almost completely prevents development of NTS. We here show that Treg transfer induces a profound increase of MC in the kidney draining lymph nodes (LN). In contrast, transfer of wt Treg into animals deficient in MC, which are characterized by an exaggerated susceptibility to NTS, do not prevent acute renal inflammation. Blocking the pleiotropic cytokine IL-9, which is known to be critically involved in MC recruitment and proliferation, by means of an antagonizing monoclonal antibody in animals receiving wt Treg abrogated protection from NTS. Moreover, we provide clear evidence that Treg-derived IL-9 is critical for MC recruitment as mediators of their full immune-suppressive potential, as adoptive transfer of IL-9 deficient Treg failed to protect from NTS. In line with our hypothesis, absence of Treg-derived IL-9 does not induce MC accumulation into kidney-draining LN, despite the fact that IL-9 deficiency does not alter the general suppressive activity of Treg, as shown by in vitro testing of their functional capacities. Finally, we observed a significantly decreased expression of the MC chemoattractant Cxcl-1 in the LN of mice receiving IL-9 deficient Treg as compared to mice receiving wt Treg or control CD4+CD25− T cells, which might at least in part explain the deficient MC recruitment under these conditions.

In summary, our data provide the first evidence that the immunosuppressive effects of adoptively transferred Treg depend on IL-9-mediated recruitment of MC to the kidney draining LN in NTS. This data is in perfect agreement with our previous report showing that CCR7-mediated LN occupancy of Treg is a prerequisite for their immune-suppressive potential and furthers adds a piece of information to the functional understanding of the in vivo anti-inflammatory effects of Treg.

Disclosures:

No relevant conflicts of interest to declare.
IDO Expression In Human Mesenchymal Stromal Cells Mediates T Cell Suppression and Leads to Monocyte Differentiation Into IL-10 Secreting Immunosuppressive CD206+ M2 Macrophages
Authors: Francois Moira , Romieu Raphaëlle , Li Mengyang , Galipeau Jacques
Published: October 2019

Abstract 2784

Clinical trials testing the use of either autologous or allogeneic human bone marrow-derived mesenchymal stromal cells (MSC) as a cell-based pharmaceutical for suppression of autoimmune and alloimmune ailments are underway. Reported results from completed trials vary in effectiveness within and between studies without any clear mechanistic explanation. We propose that these discrepancies may arise from intrinsic variability in the immunosuppressive potential of each MSC donor source. Here, we demonstrate that TNF-a and IFN-g-activated MSC derived from normal adult volunteers suppress T cell proliferation in vitro in a variegated manner, where high IDO producers were more efficient inhibitor of T cell proliferation. We also demonstrate that MSC IDO activity induce the differentiation of monocytes into IL-10 secreting M2 immunosuppressive macrophages (CD14+/CD206+). More precisely, tryptophan metabolite kynurenine, derived from the enzymatic activity of IDO induces the conversion of monocytes into M2 macrophages. Those monocyte-derived M2 in turn suppressed T cell proliferation in an IL-10 dependent and independent manner, thus amplifying the immunosuppressive effect generated by MSC. In summary, the immune plasticity of IFN-g and TNF-α licensed veto MSCs varies amongst donors and defines a central role to inducible IDO activity and its bystander effects on lymphomyeloid immune effectors.

Disclosures:

No relevant conflicts of interest to declare.
Clinical Usefulness and Prognostic Value of Interim PET/CT for the Treatment of Peripheral T Cell Lymphomas.
Authors: Yang Deok-Hwan , Min Jung-Joon , Song Ho-Chun , Jeong Yong Yeon , Bae Soo-Young , Ahn Jae-Sook , Kim Yeo-Kyeoung , Bom Hee-Seung , Chung Ik-Joo , Kim Hyeoung Joon , Lee Je-Jung
Published: October 2019

Abstract 2808

Although interim 18F-fluoro-2-dexoy-D-glucose-positron emission tomography (FDG-PET)/computerized tomography (CT) scan has emerged as a powerful prognostic tool in predicting treatment outcome in Hodgkin's lymphoma (HL) and diffuse large B cell lymphoma (DLBCL), the positive predictive value (PPV) of interim PET/CT scanning has not been determined in patients with peripheral T cell lymphoma (PTCL). The sequential interim PET/CT was prospectively investigated to determine whether it provided additional prognostic information and could be a positive predictable value for the treatment of PTCL.

Patients and Methods:

Fifty-five newly diagnosed patients with PTCL were enrolled from Sep. 2005 to July 2009 at a single institution. The PET/CT analysis was performed at the time of diagnosis and mid-treatment of CHOP/CHOP-like or other chemotherapy (EPOCH and IMEP). The clinical stage and response of the patients were assessed according to revised response criteria for aggressive lymphomas (Cheson, J Clin Oncol, 2007). The positivity of interim PET/CT was determined based on the semi-quantitative assessment of the maximal standardized uptake value (Cut-off SUVmax value of 3.0).

Results:

Median age was 55 years (range: 23–77). 31 patients (56.4%) presented in advanced stages and 13 (23.6%) had bone marrow involvements. The histological subtypes were 40.0% PTCL-unspecified (n=22), 5.1% angioimmunoblastic T cell (n=5), 38.2% nodal or extranodal NK/T cell (n=21), and others. At diagnosis, 24 patients (43.6%) were classified as high-risk by the international prognostic index (IPI) and 22 (40%) were classified as high-risk (more than 2 factors) by the prognostic index for PTCL (PIT). 47 patients could be assessed the interim response and 24 patients (43.6%) remained positive metabolic uptakes in interim PET/CT. The patients with positive interim PET/CT showed a significantly higher relapse rate (75.0%) than those with negative interim PET/CT (43.5%) (P =0.028). After following median 12.7 months, positivity of interim PET/CT was the prognostic factor for both OS and PFS, with a hazard ratio of 4.11 (1.30 – 13.01) and 3.26 (1.19 – 8.96), respectively. Six patients (10.9%) who determined to have positive interim PET/CT were revealed false-positive uptakes after locoregional biopsy (PPV of 0.75).

Conclusions:

Interim PET/CT has a significant predictive value for disease progression and survival of PTCL. The patients with positive interim PET/CT response should be considered an intensive therapeutic plan for overcoming their poor clinical outcome.

Disclosures:

No relevant conflicts of interest to declare.
Contribution of PET/CT In the Design of the Involved Node Radiotherapy (INRT) Concept for Patients with Localized Hodgkin Lymphoma: Preliminary Results on 117 Patients.
Authors: Ribrag Vincent , Edeline Véronique , Celebic Alijosa , Fermé Christophe , Ruelle Claude , Boulet Bérénice , Girinsky Théodore , Auperin Anne
Published: October 2019

Abstract 2817

Background:

The INRT concept, that limit delineates irradiated volume to lymphoma-infiltrated node, requires an extremely precise determination of the number and volume of initially involved nodes. In the study, we tried to assess the additional value of a PET/CT performed before treatment.

Patients and methods:

Early-stage lymphoma patients entered in the EORTC-GELA-IIL.

Data imaging comprising PET/CT prior to treatment and CT simulation prior to radiotherapy were retrieved using the DICOM/DICOM-RT imaging network connecting all French cancer centers participating in the H10 trial. A lymph node was considered to be involved if it fulfilled 3 requirements: FDG uptake was greater than the background, was clearly identifiable on CT, and either decreased or disappeared after chemotherapy. Involved lymph nodes were delineated on CT first before adding the information provided by PET. Differences between the number of involved lymph nodes and areas were compared before and after PET/CT assessment.

Results:

From March 2007 to February 2010, 117 patients from 18 French Cancer Centers and hospitals were included in the study. PET/CT identified at least one additional FDG-avid lymph node in 82/117 patients (70%; CI 95%: 61–78%). Additional lymph nodes were observed significantly (p = 0.008) more frequently when PET/CT was performed without IV contrast (in 79% of the patients versus 56%). At least one additional lymph node area was detected by PET in 49/117 patients (42%; CI 95%: 33–51%). There was a 15% (CI 95%: 9–22%) increase in the gross target volume (GTV) due to the additional information provided by PET (a 20% increase in 10% of the patients). On the other hand, after chemotherapy, there was a 10% (CI 95%: 5–18%) increase in the clinical target volume (CTV) in the 96 assessable patients (a 20% increase in 10% of the patients).

Conclusions:

PET/CT with IV contrast may be an important imaging procedure for implementing the INRT concept.

Disclosures:

Ribrag: LFB: Honoraria, Research Funding; servier: Research Funding; celgene: Research Funding; pfizer: Honoraria; novartis: Honoraria.
The Pan-Deacetylase Inhibitor Panobinostat Downregulates HIF-1α and VEGF and, Synergizes with Everolimus In Hodgkin Lymphoma Cell Lines
Authors: Lemoine Manuela , Buglio Daniela , Jona Adam , Derenzini Enrico , Medeiros L. Jeffrey , Berry Donald A , Younes Anas
Published: October 2019

Abstract 2851

Deacetylases (DAC) inhibitors are promising new class of anticancer agents. Panobinostat (LBH589) is a pan-DAC inhibitor with evidence of clinical activity in patients with relapsed classical Hodgkin Lymphoma (HL). However the mechanisms of action of this new drug are not completely understood. The aim of this study was to investigate the mechanisms of antiproliferative effect induced by LBH589 in HL-derived cell lines. Experiments were performed in HL derived-cell lines (HDLM-2, KM-H2 and L-428). Cells were treated with 0.01–1μM of LBH589 alone or in combination with 0.01–1μM of everolimus (RAD001), an inhibitor of the mammalian target of rapamycin (mTOR), for 24–72 hours. Growth inhibition and apoptosis were analyzed in response to treatment using MTS assay, Western blotting and flow cytometric analyses. Effect of panobinostat on a panel of 30 cytokines and chemokines was assayed on cells after incubation of 24 hours using a multiplex assay. Panobinostat demonstrated antiproliferative activity in HL cell lines in a dose-and time-dependant manner with an IC50 ranging between 20 and 40nM at 72 hours. The antiproliferative activity was associated with downregulation of the X-linked inhibitor of apoptosis protein (XIAP), activation of caspase 9 and caspase 3, cleavage of Poly ADP Ribosome polymerase (PARP) and induction of apoptosis. In addition, panobinostat downregulated the level of the transcription factor hypoxia-inducible factor 1 alpha (HIF-1α) a major regulator of tumor cell adaptation to hypoxic stress. Furthermore, panobinostat decreased the level of the vascular endothelial growth factor (VEGF), an HIF-1α target gene, in the cell culture supernatants.The combination of panobinostat and everolimus had synergistic antiproliferative activity in the cells lines. This synergy was due to reciprocal inhibition of negative feedback loops induced by mTOR inhibitor and panobinostat therapy. Collectively, our data demonstrate that panobinostat induces apoptosis in HL cell lines by modulating several survival pathways. Furthermore, the observed synergy between panobinostat and everolimus provides rationale for combining these two active agents in a phase I/II clinical trial in patients with relapsed HL.

Disclosures:

No relevant conflicts of interest to declare.
RNA Interference Targeting Thiopurine Methyl Transferase Gene Alters the Cytotoxic Effect of the 6-Mercaptopurine In Human Leukaemia Cells
Authors: Albertioni Freidoun , Fotoohi Alan , Hashemi Jamileh , Yektaei Elham , Larsson Catharina
Published: October 2019

Abstract 2891

Thiopurines; mercaptopurine (6-MP) and 6-thioguanine (6-TG) are important drugs in treatment of paediatric cancer patients. The activity of these drugs depends on the activity of several common enzymes in the metabolism pathways such as thiopurine methyl transferase (TPMT) and guanine monphosphate synthetase (GMPS). In the present study the efficacy of thiopurines was investigated upon inhibition of TPMT and GMPS gene expression by RNA interference (siRNA). Treatment of the MOLT4 human T-cell leukemia cells with TPMT and GMPS siRNA resulted in decreased mRNA expression as determined by Real-Time PCR by 60% and 70% respectively. When reducing TPMT mRNA, the MOLT-4 cells were 70% less sensitive to 6-MP while the sensitivity to 6-TG was unchanged. When down-regulating GMPS using siRNA the sensitivity was unchanged upon treatment with both drugs. A microarray experiment was conducted using wild type on MOLT-4, 6-MP and 6-TG resistant MOLT-4 variants. The aim was to identify affected genes in response to the resistance induction. The mRNA levels of several nucleoside transporter genes were down-regulated in both thiopurine-resistant sub-lines; concentrative nucleoside transporter 3 (CNT3) and equilibrative nucleoside transporter 2 (ENT2). The expression of genes encoding the purine de novo synthesis enzymes was reduced to the same extent in both resistant cell lines as well as the expression of GMPS (>40% in both resistant sub-lines) which indicates defected purine metabolism. The activity of TPMT and other enzymes in metabolism pathway of 6-MP and 6-TG was unchanged in resistant cells. Our results suggest that the contribution of TPMT activity in cytotoxicity of 6-MP is greater than for 6-TG, probably due to inhibition of de novo purine synthesis by methylated nucleotides, whereas TPMT inactivates 6-TG by methylation.

Disclosures:

No relevant conflicts of interest to declare.
Fc-Engineered RANK-Fc Fusion Proteins for Neutralization of Soluble RANKL and Induction of Antibody-Dependent Cellular Cytotoxicity (ADCC) Against Multiple Myeloma
Authors: Schmiedel Benjamin J , Scheible Carolin , Baessler Tina , Wende Constantin M , Wirths Stefan , Azuma Miyuki , Schneider Pascal , Grosse-Hovest Ludger , Kanz Lothar , Salih Helmut R
Published: October 2019

Abstract 3039

Bone resorption is commonly associated with aging, but also with certain cancers. Recent studies identified Receptor Activator of NF-κB (RANK) ligand (RANKL) and its receptors RANK and osteoprotegerin as key regulators of bone remodelling. Multiple myeloma (MM) disrupts the balance within this molecule system towards osteoclastogenesis and bone destruction. Neutralization of RANKL by the monoclonal antibody Denosumab (AMG162) is presently being evaluated for treatment of both non-malignant and malignant osteolysis. We found, in line with previously published data, that primary MM cells (9 of 10) express substantial levels of RANKL at the cell surface and that MM cells directly release RANKL in soluble form (sRANKL). Next we evaluated the possibility to combine neutralization of sRANKL with targeting of MM cells for antibody-dependent cellular cytotoxicity (ADCC) of NK cells utilizing RANK-Ig fusion proteins with modified Fc portions. Compared to wildtype RANK-Fc, our mutants (S239D/I332E and E233P/L234V/L235A/DG236/A327G/A330S) displayed highly enhanced (RANK-Fc-ADCC) and abrogated (RANK-Fc-KO) affinity, respectively, to the NK cell FcγRIIIa, but comparable capacity to neutralize RANKL in binding competition and osteoclast formation assays. Analyses with RANKL transfectants and RANKL-negative controls confirmed the high and lacking potential of the RANK-Fc-ADCC and the RANK-Fc-KO to induce NK ADCC, respectively, and ascertained that the RANK-Fc-ADCC specifically induced NK cell lysis of RANKL-expressing but not RANKL-negative target cells. Most notably, in cultures of NK cells with RANKL-expressing primary MM cells RANK-Fc-ADCC potently enhanced NK cell degranulation, cytokine release and MM cells lysis due to enhanced NK reactivity. Thus, our Fc-engineered RANK-Fc-ADCC fusion protein may both neutralize detrimental effects of sRANKL and enhance NK anti-tumor reactivity by targeting RANKL-expressing malignant cells thereby constituting an attractive immunotherapeutic means for treatment of MM.

Disclosures:

No relevant conflicts of interest to declare.
In Vitro and In Vivo Efficacy of CD38 Directed Therapy with Daratumumab In the Treatment of Multiple Myeloma
Authors: Groen Richard W , van der Veer Michael , Hofhuis Frans M , van Kessel Berris , de Weers Michel , Parren Paul W.H.I. , Lokhorst Henk M , Mutis Tuna , Martens Anton
Published: October 2019

Abstract 3058

Daratumumab (DARA) is a fully human antibody against CD38, a membrane-associated antigen, which is considered to be a highly relevant target for the treatment of hematological malignancies such as multiple myeloma (MM) and chronic lymphocytic leukemia (CLL). DARA has the ability to effectively kill target cells by CDC, ADCC and by induction of apoptosis. While CD38 is frequently overexpressed on MM cells, it has a variable degree of expression on the malignant CLL cells. To gain detailed insight into the potential therapeutic power of DARA in tumors with different CD38 expression levels, we now investigated the correlation between the level of CD38 expression and DARA-induced kill by CDC and ADCC. To this end, we first used the human MM cell lines L363 and UM9, expressing low and intermediate CD38 levels, respectively. While both cell lines could be lysed via ADCC, they were not susceptible to killing via CDC. To delineate a possible correlation between CD38 expression and induction of CDC, we lentivirally transduced the CD38 gene into L363 and UM9 cells to generate cell lines ranging in CD38 expression from 50,000 to 800,000 molecules per cell. Hereby we covered the range of CD38 expression found in CLL and MM patients. The L363 and UM9 cell lines were also transduced with the luciferase-GFP marker genes to enable their in vivo quantitative monitoring by bioluminescence imaging (BLI). In CDC assays, up to 95% lysis of CD38-transduced L363 and UM9 cells was achieved and showed an excellent correlation with the level of CD38 expression (r2=0.90). In ADCC assays, however, killing already reached maximum levels at DARA concentrations as low as 1 ng/ml at the lowest CD38 expression levels tested. These in vitro assays indicated that cell killing via CDC is enhanced with increasing CD38 expression levels, while CDC resistant, low CD38-expressing tumors could be targeted by DARA via ADCC. To explore this in an in vivo setting, we used a newly developed MM model in Rag2−/−gc−/− mice and we inoculated the mice with nontransduced, CD38low, UM9 cells, which can be lysed by ADCC but not by CDC. Treatment of the mice with DARA one day after tumor inoculation completely prevented the outgrowth of these UM9 cells; treatment of established UM9 tumours at week 3 resulted in a significant delay in tumor growth. These results confirmed that the CDC resistant UM9 could be readily targeted by DARA in vivo, possibly via ADCC mediated by monocytes in this model. In vivo experiments with variants that express high levels of CD38 are in progress. Taken together, these in vitro and in vivo data underscore the potency of DARA as a novel antibody for CD38-targeted therapy in hematological malignancies, and warrant further exploration in clinical trials.

Disclosures:

de Weers: Genmab bv: Employment. Parren:Genmab: Employment.
NIK Is Involved In the Activation of the Classical and Alternative NF-κB Pathways In Diffuse Large B Cell Lymphoma
Authors: Odqvist Lina , Sánchez-Beato Margarita , Montes-Moreno Santiago , Young Ken H , Acquadro Francesco , Cigudosa Juan , Martinez-Climent Jose A , Sáez Anabel , Sánchez Lydia , Piris Miguel A.
Published: October 2019

Abstract 3099

Deregulated NF-κB activity plays a role in the lymphoma pathogenesis, and has been proposed to constitute a cardinal feature of some subtypes of diffuse large B cell lymphoma (DLBCL). The NF-κB-Inducing Kinase (NIK) is essential for the activation of the alternative NF-κB pathway by inducing the phosphorylation of the NF-κB member p100, which leads to its processing to p52 and its subsequent nuclear translocation. A role for NIK in the classical NF-κB pathway as well has been shown, suggesting NIK as an attractive therapeutic target in lymphomas. Here, we study the frequency and extent of alternative and classical NF-κB activation in diffuse large B cell lymphoma, and the implication of NIK in both pathways. The activation of the classical and alternative NF-κB pathways was present in 28 and 34% of DLBCL cases respectively, as assessed by nuclear expression of p50 (classical pathway) and p52 (alternative pathway) by immunohistochemistry in a series of 301 samples. Activation of both NF-κB pathways was observed in germinal centre B-cell like (GC) and activated B-cell like (ABC) subtypes, with a slight predominance, although not significant, in ABC subtype. In contrast, the levels of p52 and p50 were significantly higher in ABC-DLBCL cell lines than those of GC subtype. The activation of both pathways was mostly overlapped and there was a strong positive correlation between nuclear p52 and p50 (p<0.001). Eighteen % of the cases expressed both p50 and p52 while only 8 and 16% expressed exclusively p50 or p52, respectively. Activation of the alternative NF-κB pathway was strongly associated with Epstein-Barr virus (EBV), since 93% of EBV+ cases expressed nuclear p52 (p<0.001). In our study, no TRAF3 deletions were detected in a panel of 25 DLBCL samples, although absence of TRAF3 was observed in one DLBCL cell line. Since NIK acts as a bottleneck in the activation of the alternative pathway but has also been described to play a role in the classical pathway, we wanted to analyze the effect of the knockdown of NIK on both pathways. Using small interference RNA in two lymphoma cell lines, we observed that the silencing of NIK had an effect on both pathways, decreasing the processing of p100 as well as p105. Taken together, our results show that the activation of NF-κB distinguishes a subset of DLBCL cases, comprising both ABC and GC subtypes, suggest a frequent overlap between the classical and alternative NF-κB pathway in DLBCL, and identify a possible role for NIK in the activation of both pathways.

Disclosures:

No relevant conflicts of interest to declare.
Dysregulation of Micrornas Involved In HTLV-Associated Adult T-Cell Lymphoma
Authors: Beltran Brady , Lin Jianhong , Sokol Lubomir , Zhang Ling , Lwin Tint , Cultrera Jennifer , Quinones Pilar , Morales Domingo , Tannenbaum Victoria , Moscinski Lynn C. , Sotomayor Eduardo M. , Tao Jianguo
Published: October 2019

Abstract 3122

Adult T-cell leukemia/lymphoma (ATL) is an aggressive, fatal malignancy of CD4+ T cells that is etiologically associated with infection by human T-cell leukemia virus-type. ATL has been classified into four main subtypes, indolent smoldering, chronic forms, acute and lymphomatous forms. Most individuals in the chronic stage of ATL eventually undergo progression to a highly aggressive acute stage. Compelling evidence supports a pivotal role for altered tumor suppressor genes involved in leukemogenesis and lymphomagenesis of ATL as well as progression of the disease from the chronic/smoldering to the acute/lymphomatous type. Further investigations are needed as to how the alteration of cell cycle regulatory genes are involved in the aberrant growth of HTLV-infected T cells. The emerging role of microRNAs in tumor development and viral infection has prompted investigations on the role of microRNA in ATL pathogenesis and progression. We therefore performed microRNA microarrays on 16 lymphomatous tissues of ATL patients and normal CD4 lymphocytes of 5 healthy donors. After normalization, we identified a set of aberrantly expressed miRNAs in ATL lymphoma samples when compared with control CD4 lymphocytes including 49 upregulated microRNAs and 89 downregulated microRNAs. MiR-150, miR-24, miR-221, miR-222, miR-142-5p, miR-92b, miR-93, miR-181a, miR-103, miR-107 stands out as highly elevated miRNAs in ATL samples while miR-30b, miR-34a, miR-20b, miR-196b, miR-361-5p, miR-520b/e are found downregulated in ATLL samples. Using gain-and-loss experimentations, we demonstrated miR-181a overexpression decreases, whereas miR-181a inhibition increases a proapoptotic protein, Bim, levels by directly targeting Bim. Furthermore computational analysis, and previous studies revealed that that miR-214, miR-22, miR-93 increased AKT activity by repressing PTEN expression, and miR-221/222 and miR-93 promoted cell proliferation through inhibition of CDKN1B (p27). Overall, this study demonstrated a distinct set of miRNAs that are known to be critical in cellular transformation, representing a novel molecular mechanism and potential therapeutic targets in ATL lymphoma.

Disclosures:

No relevant conflicts of interest to declare.
Low Absolute Lymphocyte Count Predicts Chemotherapy Response and Survival In Peripheral T-Cell Lymphoma, NOS
Authors: Kim Yu Ri , Kim yun Deok , Kim Jin Seok , Cheong June-Won , Kim soo Jeong , Min Yoo-Hong
Published: October 2019

Abstract 3135

Peripheral T cell lymphoma, not otherwise specified (PTCL, NOS) is heterogenous groups of aggressive T-cell lymphoma and treatment outcome is dismal. Lymphopenia is an independent prognostic factor for survival for B-cell lymphoma. The ALC at diagnosis on survival in T-cell lymphoma has not been studied. Thus, we studied the role of ALC at diagnosis on clinical outcome in patients with PTCL, NOS. Between 2001 and 2009, 32 patients with PTCL, NOS reviewed for the study. Median patient age was 57 (range 34–78) years. Median ALC at the time of diagnosis was 1.54 (range 0.41–12.64×109/L). Patients were divided two groups according to ALC count 1.0 ×109/L. Ten patients (31%) had lower ALC at diagnosis. Median follow up duration was 299 days (range 11–2164 days). Overall response rate was 61.5% (16 of 26 patients) and complete response (CR) rate was 42% (11 of 26 patients). Only two patients reached CR in low ALC group.There was no significant difference in overall response rate because of small number of patients. Superior overall survival was observed with an ALC 1.0 × 109/L (N = 22) versus an ALC < 1.0 × 109/L (N=10) (median OS: not reached vs 242 days, OS rates at 5 years, 57% vs 0%, p =0.016, respectively). Multivariate analysis demonstrated ALC to be an independent prognostic indicator for OS (Hazard Ratio 3.5, 95% confidence intervals 1.2–10.2; p<0.019) when compared to the International prognostic index (IPI) and Prognostic Index for PTCLU (PIT). This study suggested that low ALC is an independent prognostic factor for survival in patients with PTCL, NOS.

Disclosures:

No relevant conflicts of interest to declare.
Multiple Mechanisms of Notch-Mediated B-ALL Growth Arrest Via HES1 and PLK1: KIF11, CyclinB and p53
Authors: Kannan Sankaranarayanan , Fang Wendy , Zweidler-McKay Patrick
Published: October 2019

Abstract 3137

Notch signaling inhibits B-ALL growth and survival, while it promotes T-ALL, revealing contrasting cell-specific consequences of Notch signaling in ALL subtypes. We previously reported that HES1 (Hairy and Enhancer of split1) is a Notch pathway member which is sufficient to reproduce these effects in B-ALL.

We have found distinct HES1 protein complexes in B-ALL versus T-ALL, with significantly higher molecular weight complexes found in B-ALL. Through characterization of these complexes, multiple novel HES1 interacting proteins have been identified which regulate HES1 function as well as provide growth inhibiting and pro-apoptotic effects. We previously reported the novel role of HES1:PARP1 complexes in inducing B-ALL apoptosis. This study reports Polo-like kinase1 (PLK1) and Kinesin Family member 11 (KIF11, also known as Eg5) as novel HES1 interacting proteins in B-ALL. PLK1 is highly expressed in B cell leukemias and drives multiple proliferative pathways including activation of KIF11, induction of cyclinB and degradation of p53. KIF11 is also highly expressed in B-ALL and promotes cell cycle progression through enhanced spindle formation, while cyclinB promotes accelerated mitosis.

Through HES1 immunoprecipitation and MALDI-TOF peptide analysis, we identified KIF11 as a potential HES1 interacting protein. Low flow size exclusion chromatography revealed that HES1 and KIF11 co-migrate in high molecular weight complexes. Reciprocal immunoprecipitation (IP) with HES1 and KIF11 antibodies reveal a strong protein-protein interaction in B-ALL cells. PLK1 was also identified in these complexes, revealing a novel interaction of HES1, KIF11 and PLK1. Importantly, induction of Notch signaling via HES1 in B-ALL decreases both PLK1 and KIF11 levels, associated with G1 cell cycle arrest. Furthermore, HES1 expression decreased cyclinB protein levels, but not cyclin A/E, consistent with inhibition of PLK1. Finally, in the same model we observe increased p53 and p21WAF1 protein levels, also contributing to growth arrest.

These data reveal that multiple mechanisms of Notch/HES1-mediated growth arrest may occur via PLK1 in B-ALL. The novel interaction of HES1 with PLK1 and KIF11 and resulting effects on KIF11, cyclinB and p53/p21 pathways may represent important mechanisms of Notch-mediated growth inhibition in B-ALL.

Disclosures:

No relevant conflicts of interest to declare.
A Critical Role of YB-1 In NPMc-Induced Acute Myeloid Leukemia
Authors: Ogawara Yoko , Katsumoto Takuo , Uchiumi Takeshi , Kohno Kimitoshi , Kitabayashi Issay
Published: October 2019

Abstract 3149

Frameshift mutations in Nucleophosmin gene (NPM) are the most frequent abnormality in acute myeloid leukemia (AML), found in approximately 30% of all cases and 50% of patients with normal karyotype (NK) AML. NPM mutations result in an aberrant cytoplasmic localization of NPM protein (NPMc) through a loss of nucleolar localization signal accompanied by acquisition of new nuclear export signal. NPM mutations are heterozygous, so the other wild-type allele is consistently retained. NPMc binds to wild-type NPM through oligomerization domain and impairs its activity by delocalizing to the cytoplasm. It was reported that the NPM-null mice are early embryonic lethal and defective in primary hematopoiesis, suggesting important roles of NPM in early hematopoiesis. However, the molecular mechanism by which NPMc exerts its leukemogenic potential has never been established.

Here we show that ectopic expression of NPMc, but not wild type (WT) NPM, in mouse bone marrow (BM) cells enhanced their colony formation activity in methylcellulose media. Increased expression of HoxA7, 9 and 10 genes were observed in cells expressing NPMc but not in those expressing WT NPM. It has been reported that the expression levels of HOXA genes are upregulated in various types of AML including NPMc+ AML. Since overexpression of HoxA9 immortalizes hematopoietic progenitor cells, our findings suggest that up-regulation of HoxA genes are involved in NPMc-mediated leukemogenesis.

To clarify roles of NPMc in leukemogenesis, we purified the NPM protein complex and identified Y box-binding protein 1 (YB-1) as a binding partner for NPM. YB-1 belongs to the cold shock family and functions in gene transcription and RNA processing. YB-1 strongly bound to WT NPM but not to NPMc. In addition, interaction between YB-1 and NPM was impaired in the presence of NPMc. YB-1-deficient mice were embryonic lethal and their fetal liver were small. YB-1-deficient yolk sac cells showed decreased colony-forming activity, and decreased number of hematopoietic cells were observed when AGM region of YB-1-deficeint embryo were cultured on OP9 cells. Furthermore, expression of Hoxa9 was decreased in fetal liver cells derived from YB-1 knockout mice. To investigate the roles of YB-1 in NPMc-associated leukemogenesis, WT and YB-1-null E14.5 fetal liver cells were infected with retrovirus expressing NPMc. Analyses of colony-forming activity and mRNA expression showed that YB-1 was essential for NPMc-induced increases in colony formation activity as well as in expression of HoxA genes. However, YB-1 was not necessary for colony formation activity induced by other AML-associated fusion genes, such as AML1-MTG8 and MLL-AF10. These data indicate that YB-1 is specifically required for NPMc-induced leukemogenic transformation of hematopoietic cells.

Disclosures:

No relevant conflicts of interest to declare.
An In Vivo role of Runx1 Subnuclear Targeting In Regulating the Gene Expression Program of Definitive Hematopoiesis
Authors: Dowdy Christopher R , Xie Ronglin , Frederick Dana , Zaidi Sayyed K. , Jones Stephen N. , Lian Jane B. , van Wijnen Andre J. , Stein Janet L. , Stein Gary S.
Published: October 2019

Abstract 3151

Runx1, a master regulator of hematopoiesis, is critical for the emergence of the hematopoietic stem cell (HSC), as observed by gene ablation or replacement studies where either the entire gene is disrupted or replaced with specific mutations that abolish DNA binding activity. Like many phenotypic transcription factors, endogenous Runx1 localizes to distinct foci within the nucleus through a unique subnuclear targeting signal. Clinically relevant is the observation that many Runx1 translocations that are associated with acute myeloid leukemias (e.g., AML1/ETO) retain Runx1 DNA binding activity but exhibit modified localization due to the loss of subnuclear targeting signal and transactivation domains. We hypothesized that altered subnuclear routing of translocation-encoded leukemic proteins plays a key role and provides a common mechanism in the onset and progression of human leukemias. We created a knock-in mouse with a C-terminal truncation by introducing a single nucleic acid substitution (Runx1 Q307X) in the native Runx1 locus. This mutation models genetic lesions observed in patients with leukemia and myeloproliferative disorders. The Runx1 Q307X homozygous mouse exhibited mid-gestation lethality at 12.5dpc due to central nervous system hemorrhage and a complete lack of HSC function. Importantly, Runx1 Q307X, which retains DNA binding activity, failed to activate target genes, resulting in deregulation of various hematopoietic markers. This phenocopy of the complete Runx1 ablation models highlights the critical importance of subnuclear targeting and transactivation activity for Runx1 function during development. We next examined specific contributions of subnuclear targeting in vivo to this phenotype by creating another knock-in mouse model replacing endogenous Runx1 with a mutant (Runx1 HTY350-352AAA). This mutation specifically abrogates subnuclear localization of the protein without altering other known functions. Embryos homozygous for Runx1 HTY350-352AAA bypass the mid-gestation lethality observed with the other Runx1 mutants and survive till birth. Thus, the precise subnuclear targeting of Runx1 does not appear to be essential for emergence of the HSC and the start of definitive hematopoiesis. However, expression of Runx1 target genes that mediate hematopoiesis is modified in mice with this mutant. These findings together suggest a development independent role for Runx1 subnuclear targeting in controlling the hematopoietic gene program that is often modified in human leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Safety and Clinical Utility of Four Clinical Decision Rules In the Diagnostic Management of Acute Pulmonary Embolism – the Prometheus Diagnosis Study
Authors: Mos Inge CM , Douma Renée A , Erkens Petra MG , Nizet Tessa AC , Durian Marc F , van Houten Anja A , Hovens Marcel M , Hofstee Herman MA , Klok Frederikus A , Cate Hugo Ten , Ullmann Erik F , Büller Harry R , Kamphuisen Pieter W , Huisman Menno V
Published: October 2019

Abstract 3186

Background

Several clinical decision rules (CDRs) are available for the exclusion of acute pulmonary embolism (PE). This prospective multi-center study compared the safety and clinical utility of four CDRs (Wells rule, revised Geneva score, simplified Wells rule and simplified revised Geneva score) in excluding PE in combination with D-dimer testing.

Methods

Clinical probability of patients with suspected acute PE was assessed using a computerized based “black box”, which calculated all CDRs and indicated the next diagnostic step. A “PE unlikely” result according to all CDRs in combination with a normal D-dimer result excluded PE, while patients with “PE likely” according to at least one of the CDRs or an abnormal D-dimer result underwent CT-scanning. Patients in whom PE was excluded were followed for three months.

Results

807 consecutive patients were included and PE prevalence was 23%. The number of patients categorized as “PE unlikely” ranged from 62% (simplified Wells rule) to 72% (Wells rule). Combined with a normal D-dimer level, the CDRs excluded PE in 22–24% of patients. The total failure rates of the CDR-D-dimer combinations were similar (1 failure, 0.5– 0.6%, upper 95% CI 2.9– 3.1%). Despite 30% of the patients had discordant CDR outcomes, PE was missed in none of the patients with discordant CDRs and a normal D-dimer result.

Conclusions

All four CDRs show similar safety and clinical utility for exclusion of acute PE in combination with a normal D-dimer level. With this prospective validation, the more straightforward simplified scores are ready for use in clinical practice.

Disclosures:

No relevant conflicts of interest to declare.
Ser559 of Glycoprotein Ibα Is Phosphorylated by Protein Kinase A Which Plays Key Roles In Regulating Von Willebrand Factor Binding, Surface Expression and Signaling of Glycoprotein Ib-IX
Authors: Weilin Zhang , Guanglei Liu , Yanhong Yuan , Lili Zhao , Rong Yan , Yi Liao , Ruan Changgeng , Kesheng Dai
Published: October 2019

Abstract 3192

The interaction of glycoprotein (GP) Iba with von Willebrand factor (VWF) is regulated by the associations of 14-3-3ζ and filamin A with the cytoplasmic domain of GPIba. We reported recently that phosphorylation at Ser559 of GPIba is important for 14-3-3ζ binding, however, the protein kinase responsible for Ser559 phosphorylation remains unknown. Here we show that a peptide corresponding to the Arg557-Pro561 region of GPIba and purified GPIba could be phosphorylated at Ser559 by the catalytic subunit of protein kinase A (PKA). Ser559 phosphorylation was enhanced by PKA activators and reduced by PKA inhibitors in platelets and Chinese hamster ovary (CHO) cells expressing GPIb-IX. Furthermore, PKA inhibitor enhanced, however, reduced VWF binding to two kinds of CHO cells expressing GPIb-IX mutant replacing Ser559 of GPIba (S559A) or Ser166 of GPIbβ (S166A) with alanine, respectively. GPIb-IX association with filamin A and membrane expression were enhanced in S559A cells, whereas reduced in S166A cells. The peptide disrupting the interaction of 14-3-3ζ with Ser559 of GPIba inhibited platelet spreading on VWF matrix and GPIb-IX-VWF interaction-induced association of Src with GPIb-IX. These data indicate that Ser559 of GPIba is phosphorylated by PKA which plays key roles in regulating VWF binding, surface expression, and signaling of GPIb-IX.

Disclosures:

No relevant conflicts of interest to declare.
Hepcidin Levels and Erythropoietin Alfa Resistance In Patients with Chemotherapy Induced Anemia (CIA)
Authors: Schipperus M. , Rijnbeek B. , Reddy M. , Li W. , Vercammen E. , Cornfeld M.
Published: October 2019

Abstract 3216

Introduction:

Hepcidin is a key regulator of iron metabolism and is a central mediator of anemia associated with inflammation. Hepcidin has previously been associated with resistance to ESAs in renal anemia and may also be a potential mediator of ESA resistance in chemotherapy-induced anemia (CIA). We therefore assessed hepcidin in a cohort of patients with CIA who received erythropoietin alfa (EPREX, Janssen-Cilag, Ltd.) as part of a post-approval safety study.

Patients and Methods:

All subjects were anemic (defined as baseline Hb ≤11 g/dL) and almost all patients had one of the following malignancies: breast, ovarian, gastric and pancreatic cancer, myeloma. Patients were receiving standard doses of EPREX for management of CIA. Patients with anemia secondary to iron deficiency (defined as transferrin saturation ≤ 20%) were excluded from study enrollment. Serum erythropoietin was not assessed as a condition of eligibility. Hemoglobin (Hb) response was defined as an increase of ≥ 1 g/dL through week 4. Baseline serum hepcidin levels were measured retrospectively using a hepcidin C-ELISA. The relationships between hepcidin, Hb, iron status (serum iron, ferritin, transferrin saturation), C-Reactive Protein (CRP) (a marker of inflammation and surrogate for IL-6 activity) and Hb response were evaluated.

Results:

Among 51 anemic patients with a median age of 56 (range 25–72), 42 were Hb responders. None of the patients studied received blood transfusions during the first 4 weeks of treatment. No significant difference in baseline hepcidin or CRP levels was found between the Hb responders and non-responders; hepcidin 129.43 ng/mL (9.5-722.4) versus 210.7 (9.9-399.6) and CRP 47.1 nmol/L (0-428.6) versus 46.0 (2.1-1145.7) respectively. All subjects who had a decrease in Hb through week 4 were tumor non-responders. Baseline hepcidin levels were inversely correlated with baseline Hb (p = 0.04) but did not predict for Hb response. Neither did baseline CRP nor iron status. Adjusting hepcidin for iron status did not improve predictive accuracy. No significant correlation was found between CRP change at week 4 and Hb response. A correlation was found between baseline hepcidin and week 4 CRP (p = 0.0004) but not with baseline CRP levels.

Conclusions:

Baseline hepcidin was not predictive of Hb response to ESA therapy. Hepcidin change may be a better predictor and warrants further evaluation. Lack of information on baseline erythropoietin levels is a significant limitation of this study. Correlation with additional markers (e.g., soluble transferrin receptor, inflammatory markers like IL-6) will also be important to fully determine the role of hepcidin as a predictor for EPO resistance in settings of chronic inflammation.

Disclosures:

Rijnbeek: Johnson & Johnson: Employment. Reddy:Johnson & Johnson: Employment. Li:Johnson & Johnson: Employment. Vercammen:Johnson & Johnson: Employment. Cornfeld:Johnson & Johnson: Employment.