bluuebunny/crossref_metadata_2025_split huggingface dataset:
Methylation of Specific Microrna Genes In MLL-Rearranged Infant Acute Lymphoblastic Leukemia: - Major Matters at a Micro Scale -
Authors: Stumpel Dominique JPM , Schotte Diana , Lange-Turenhout Ellen AM , Schneider Pauline , Seslija Lidija , Menezes Renee X de , Marquez Victor E , Pieters Rob , Boer Monique L. Den , Stam Ronald 
Published: October 2019

Abstract
Abstract 2483
MLL-rearranged Acute Lymphoblastic Leukemia (ALL) in infants (<1 year) represents one of the most aggressive types of childhood leukemia. In order to develop more suitable treatment strategies, a firm understanding of the biology underlying this disease is of utmost importance. MLL-rearranged ALL displays a unique gene expression profile, partly explained by erroneous histone modifications. We recently showed that t (4;11)-positive infant ALL is also characterized by pronounced promoter CpG hypermethylation. Here we investigated whether this widespread hypermethylation also affected microRNA (miRNA) expression.
We performed CpG methylation analyses at 122 miRNA loci using Differential Methylation Hybridization (DMH), and miRNA expression analyses using quantitative real-time PCR on primary t (4;11)-positive infant ALL samples (n= 22) and normal pediatric bone marrows (n= 7). We identified 11 miRNAs that were markedly down-regulated in t (4;11)-positive infant ALL as a consequence of CpG hypermethylation. Seven of these miRNAs were re-activated after exposure to the de-methylating agent Zebularine. Interestingly, 5 of these miRNAs had already been associated either with the MLL gene or with leukemic MLL fusions. For one of the remaining miRNAs, i.e. miR-152, we demonstrate that high degrees of methylation strongly correlate with a poor clinical outcome. Moreover, we identified MLL and DNA methyltransferase 1 (DNMT1) as potential target genes for miR-152.
Thus, genome-wide DNA methylation in MLL-rearranged infant ALL not only inactivates numerous protein-coding genes, but also affects several miRNA genes. While inhibition of methylation by Zebularine to certain extents re-activates gene expression, re-activation of miRNAs by this agent restores the suppression of associated target genes. As demethylating agents exert their functions by covalently trapping DNMT1 to the DNA, re-activation of miR-152 by Zebularine further supports demethylation by targeting DNMT1 expression.
In summary, our data demonstrates an important role for genome-wide DNA methylation in suppressing miRNA expression and provides additional grounds to initiate efficacy testing of demethylating agents in MLL-rearranged ALL in vivo.

Disclosures:
No relevant conflicts of interest to declare.
Identification and Modeling of TEL-AML1 (ETV6-RUNX1) Molecular Signature In Acute Lymphoblastic Leukemia
Authors: Miles Gregory , Rajagopal Gunaretnam , Strair Roger , Sabaawy Hatem E 
Published: October 2019

Abstract
Abstract 2500
Despite high cure rates for childhood ALL, treatment failure remains a formidable problem. TEL-AML1 (ETV6-RUNX1), the product of t (12;21) translocation, is the most common cytogenetic abnormality in childhood cancer, and is detected in 25–30% of precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL) in children, and in small percentage of adult ALL. Considerable experimental and clinical evidence indicate that the TEL-AML1 fusion is insufficient by itself for leukemic transformation. First, monozygotic twins had the same prenatal TEL-AML1 sequence, but different latency and/or no leukemia development. Second, we have previously established transgenic zebrafish expressing TEL-AML1. These TEL-AML1 transgenic zebrafish have hematopoietic stem cell (HSC) expansion, and develop pre-B ALL at low penetrance, and after prolonged latency. Similarly, transgenic mice expressing TEL-AML1 did not develop pre-B ALL, but showed HSC expansion. Third, retroviral-mediated TEL-AML1 gene transfer into murine HSCs resulted in a preleukemic state, and the incidence of leukemia increased with additional mutation. Here, we report on the identification of a TEL-AML1-specific leukemic signature in ALL cases, and modeling of these mutations in dual and compound transgenic zebrafish. First, we performed meta-analyses of ÓALL compendiumÕ of molecular signatures and expression profiles of 990 ALL cases from six studies, measuring the expression of 14,145 genes in 475 arrays. The normalized data were imported into the Ingenuity Pathway Analysis (IPA) software to identify TEL-AML1-related pathways. We determined a TEL-AML1-specific signature that was organized into modules that are induced or suppressed, based on involvement in several biological pathways comprising the hallmarks of cancer. These analyses identified modules of cell differentiation, cell proliferation, apoptosis, autophagy, cell cycle regulation, and lymphocyte development as the most common modules associated with TEL-AML1. The signature was confirmed using additional microarray analyses, in combination with Q-PCR, and western blotting from the same cases. Similar analyses of marrow cells from transgenic zebrafish with ALL identified 1,128 upregulated and 936 down-regulated genes with 27 genes common with the human TEL-AML1 signature. We next isolated the zebrafish homologues of several induced signature genes to generate dual and compound transgenic zebrafish, and to investigate the effects of their overexpression on TEL-AML1 leukemia development. Analyses of established transgenic fish demonstrate leukemia-enhancing effects of signals within the proliferation and cell cycle modules. These studies provide a model to understand the role of the TEL-AML1 fusion and the secondary mutations required for leukemia development, and might present a rational for leukemia combination therapy.

Disclosures:
No relevant conflicts of interest to declare.
A Novel PCR-Based Gene-Specific Chromatin Condensation Assay
Authors: Vatolin Sergei , Shahper Khan Nazeer , Reu Frederic J. 
Published: October 2019

Abstract
Abstract 2508
We developed a method for efficient, quantitative, and site specific characterization of chromatin activity in myeloma cell populations. This method is based on modified salt extraction of chromatin directly from intact cells and PCR amplification of site of interest. The whole procedure (cell collection, chromatin isolation, PCR, and data analysis) takes from 4 to 6 hours and requires only 100–200 cells per probe. The general idea behind this procedure is that most active chromatin is sensitive to mild denaturing conditions which make its DNA accessible for primers, whereas inactive chromatin remains “closed”. No antibodies, fixation, viral or plasmid-based constructs, or nuclease treatments are involved. The method corroborated findings on ChIP and pyrosequencing of bisulfite modified DNA for CDKN2A (p16) regulatory regions. After optimization with decitabine it correctly identified other epigenetically active agents in a small scale screen. In summary, we have developed a method for detection of gene specific chromatin changes that requires less than 1% of cells needed for other epigenetic assays, is cheaper, faster, and detects various changes of chromatin. We expect it will revolutionize drug screening for epigenetic reactivators and silencers, epigenetic profiling, and elucidation of chromatin modifying pathways in myeloma and other cancers.

Disclosures:
No relevant conflicts of interest to declare.
Differentiating Human ES and Patient Derived IPS Cells to Hematopoietic Stem/Progenitor Cells.
Authors: Rönn Roger Emanuel , Moraghebi Roksana , Parker Aaron , Lutz Margaret , Berggren Travis , Woods Niels-Bjarne 
Published: October 2019

Abstract
Abstract 2628
The ability to generate hematopoietic stem cells (HSC's) from patient derived induced pluripotent stem (iPS) cells, would enable the generation of an unlimited supply of HLA matched transplantable cells for the treatment of both hematological disorders and malignancies. The goal of this project is to find the molecular determinants of human ES and iPS cell differentiation to transplantable HSC's. We have developed an advanced ES/iPS-to-blood differentiation protocol that mimics embryonic development towards the hematopoietic lineage. Current progress has resulted in up to 84% of human CD45+ hematopoietic cells (mean 41 ± 16%, n = 7) and with significant numbers of hematopoietic progenitor cells (CD45+CD34+, 29 ± 4%, n = 6) as reported by FACS analysis. Moreover, the numbers of hematopoietic progenitor cells generated (mean 20 ± 1 for a keratinocyte derived iPS (kiPS)), as measured by colony forming unit assays were comparable to numbers obtained from fresh whole blood umbilical cord mononuclear cell isolates (mean 35 ± 2) on a per 10.000 CD45+ input cells. We now plan to overexpress key factors predicted to support the generation and maturation of bona fide HSC's with repopulating ability. For this we have generated a tet-regulatable lentiviral based gene expression system to, out of a pool of over 30 genes, express up to 5 cDNA's of interest simultaneously. By clonal selection of lenti-transduced ES and iPS cells we have generated novel pluripotent lines that carry the tet-transactivator gene (m2rtTA), capable of efficient differentiation into blood. FACS analysis of these lines show blood generation of 30.7% and 11.8% of cells positive for CD45 for the human ES line Hues3 and the kiPS line respectively, with progenitor levels (CD45+CD34+) at 8.8% and 20.2%. These values are similar in efficiency to that of untransduced sister lines from which they were derived. These lines strongly express genes of interest following lenti-viral transduction and the addition of Doxycyclin (as reported by GFP). It is our goal to show successful reconstitution with in vitro generated HSC's, and ultimately pave the way for therapeutically relevant HSC's for treating hematological malignancies.

Disclosures:
No relevant conflicts of interest to declare.
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
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
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
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
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
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
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.