bluuebunny/crossref_metadata_2025_split huggingface dataset:
PU.1 Relieves Its GATA-1-Mediated Repression near Cebpa and Cbfb During Transdifferentiation of Murine Erythroleukemia - Tool of Inducing Leukemic Blasts to Differentiate.
Authors: Burda Pavel , Curik Nikola , Kokavec Juraj , Mikulenkova Dana , Skoultchi Arthur , Zavadil Jiri , Stopka Tomas 
Published: October 2019

Abstract
Abstract 547
Transcription factors GATA-1 and PU.1 interact on DNA to block transcriptional programs of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells that co-express GATA-1 and PU.1 are blocked at the blast stage but respond to down-regulation of PU.1 or up-regulation of GATA-1 by inducing terminal erythroid differentiation. To test whether GATA-1 blocks PU.1 in MEL cells we have conditionally activated a transgenic PU.1 protein fused with the estrogen receptor ligand-binding domain (PUER), resulting in activation of a myeloid transcriptional program. Gene expression arrays identified components of the PU.1-dependent transcriptome negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein (C/EBP) alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) that encode two key hematopoietic transcription factors. Inhibition of GATA-1 by siRNA resulted in derepression of PU.1 target genes. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb co-occupied by PU.1 and GATA-1 in the leukemic blasts. Substantial derepression of Cebpa and Cbfb is achieved in MEL cells by either activation of PU.1 or knockdown of GATA-1. Furthermore, transcriptional regulation of these loci by manipulating the levels of PU.1 and GATA-1 involves quantitative increases in a transcriptionally active chromatin mark: acetylation of histone H3K9. Collectively, we demonstrate that either activation of PU.1 or inhibition of GATA-1 efficiently reverse the transcriptional block imposed by GATA-1 and lead to activation of a myeloid transcriptional program directed by PU.1.
The mechanism of PU.1 and GATA-1 in leukemic state and upon leukemia differentiation involves the following putative steps: at myeloid genes such as Cebpa, PU.1 binds directly to DNA but is repressed by GATA1 that binds directly to PU.1 molecules on DNA. Activation of PU.1-ER and stable levels of GATA-1 create excess of availabel PU.1, which is not paired by availabel GATA-1 on DNA, allowing thus gene activation. Similarly, on erythroid genes such as Nfe2, GATA1 is bound to DNA, but is repressed by PU.1 that binds to this GATA-1 molecule. Activation of GATA1-ER creates an excess of availabel GATA-1 which is not paired on DNA by availabel PU.1, also allowing gene activation. Our mechanistic study implicates that transcription factor manipulation, such as inhibition of GATA-1 or activation of PU.1 in erythroleukemias, may represent an efficient tool of inducing leukemic blasts to differentiate. (Grants NR9021-4, 10310-3, 2B06077)

Disclosures:
No relevant conflicts of interest to declare.
Surface IgM of Chronic Lymphocytic Leukemia Cells Displays Unusual Glycans Indicative of Antigen Engagement In Vivo.
Authors: Packham Graham , Krysov Serge , Mockridge Christopher Ian , Potter Kathy N , Stevenson Freda K 
Published: October 2019

Abstract
Abstract 55
Several lines of evidence support the idea that surface immunoglobulin M (sIgM) plays a key role in determining the clinical behavior of chronic lymphocytic leukemia (CLL). For example, the presence of somatic mutations in immunoglobulin variable region genes is a strong prognostic marker with unmutated CLL (U-CLL) associated with a poor outcome relative to mutated CLL (M-CLL). U-CLL also generally express higher levels of sIgM and retain the ability to signal via this receptor. In this study, we used surface biotinylation to analyse sIgM in CLL and discovered that it exists in two forms with differing mobility on SDS-PAGE. Treatment with glycosidases revealed that these forms were due to different N-glycosylation patterns in the μ constant region. One form is similar to that of normal B cells in bearing mature complex glycans common to most cell surface glycoproteins. The other is an immature mannosylated form more characteristic of endoplasmic reticulum (ER)-located μ chains. CLL cells expressed variable proportions of the immature mannosylated form and quantitative analysis demonstrated that, on average, the proportion of mannosylated sIgM was approximately 2-fold higher (p=0.006) in U-CLL compared to M-CLL. Although normal B cells isolated from blood expressed only the mature form of sIgM, in vitro treatment with anti-μ resulted in upregulation of the immature form, suggesting that glycan modification is a consequence of antigen exposure. Consistent with this, in vitro incubation of CLL cells was associated with increased expression of the mature form of sIgM. Phosphotyrosine analysis demonstrated that both forms of sIgM were able to signal following sIgM engagement in vitro. Taken together, these findings support the concept that CLL cells are continuously exposed to antigen in vivo. This process leads to a change in the N-glycosylation pattern of the re-expressed sIgM so that a mannosylated form predominates, especially in U-CLL. Strikingly, expression of mannosylated sIgM is also characteristic of follicular lymphoma, where it is constitutively displayed via N-glycosylation sites in the Ig variable region (Radcliffe et al. J Biol Chem. 2007; 282, 7405-15). Persistent mannosylation of sIgM appears to be a feature common to several B-cell malignancies, suggesting a role in pathogenesis.

Disclosures:
No relevant conflicts of interest to declare.
Accuracy of Reporting of Venous Thromboembolism in Hospitalized Patients.
Authors: Perez Alejandro , Hubman Margie S. , Martin Craig , Michaels Jeannie , Bartholomew John R. 
Published: October 2019

Abstract
Abstract 557

Introduction:
Venous thromboembolism (VTE), which includes deep vein thrombosis (DVT) and pulmonary embolism (PE), is an important clinical event that needs to be accurately reported. The Agency for Healthcare Research and Quality (AHRQ) has categorized VTE as a reportable patient safety indicator (PSI) and has distributed a software package for recording events such as VTE based upon diagnosis codes. At our institution, there was concern that VTEs identified by this software method may be over estimating the actual number.

Objective:
To verify the cases of VTE as identified by AHRQ methodology through a review of the medical record for each event reported.

Methods:
Through a review of the electronic medical record (EMR), the events recorded as a VTE by PSI software over a 7 month period from November 2008 to May 2009 were further characterized into categories of acute, remote or age indeterminate DVTs and were distinguished from events that were either isolated superficial vein thrombosis (SVT) or that were not VTE at all. Discharge summaries, hospital notes, operative reports and imaging reports were used to verify identified cases of VTE. VTE occurrences were grouped by the month of discharge of the patient. The PSI software utilized diagnosis codes that have been abstracted for a patient's hospitalization based upon physician documentation.

Results:
437 events were recorded as VTE over a 7 month period. 59 cases (13.5%) were incorrectly coded as VTE. 35 cases (8.0%) were superficial vein thrombosis mistakenly classified as DVT. 12 cases (2.7%) had remote or age indeterminate DVTs. 12 cases (2.7%) cases had no VTE and were considered further abstraction errors. After interim reporting of the first 4 months of VTE data, vascular laboratory study reports were amended so that superficial vein thrombosis would no longer be identified by this terminology and would subsequently be classified as superficial vein thrombophlebitis. The last three months of data collection had 9 cases (4.7%) incorrectly identified as DVTs that were actually SVTs versus 26 cases (10.6%) prior to implementation of the nomenclature change.

Conclusion:
Hospitals must be aware of reporting practices in their own institution as national protocols may not completely depict individual hospital occurrences. Careful attention to the manner in which the medical record reflects events can make substantial difference in accounting of VTE occurrence. Because of the lack of specificity of current diagnosis coding guidelines, an appropriate medical term was not sufficient for medical record abstractors to categorize an event correctly in this study. As hospitals become monitored for quality of care with these methodologies, the accuracy of reporting becomes paramount.

Disclosures:
No relevant conflicts of interest to declare.
Regulation of ABCB1 (p-glycoprotein) by the FOXO1 Transcription Factor in Acute Myeloid Leukemia.
Authors: Seedhouse Claire , Ahluwalia Sophie , Mills Ken I , Grundy Martin , Shang Shili , Burnett Alan K. , Russell Nigel H. , Pallis Monica 
Published: October 2019

Abstract
Abstract 589
FOXO1 mediates a cellular stress response by inhibiting cell growth and up-regulating expression of molecules involved in apoptosis and antioxidant defences. The membrane pump p-glycoprotein (pgp, expressed by the ABCB1 gene) is responsible for the efflux of chemotherapeutic drugs and the redistribution of components of the ceramide signalling pathway. In AML, pgp over-expression is associated with failure to respond to remission-induction chemotherapy, but attempts to modulate pgp function have enjoyed little success and new approaches are needed. We studied transcriptional control of the ABCB1 gene in AML cells. The Pgp protein was measured using MRK-16; ABCB1 mRNA was measured using realtime PCR in patients entered into UK AML 14 and AML 15 clinical trials. 141 of these trial samples were also studied using the Affymetrix U133A (22,283 probesets) or the U133Plus2 (∼54,000 probesets) gene chips to establish correlations between pgp and factors which might influence expression. FOXO1 was one of the most closely correlated genes with pgp (P<0.001). FOXO3 and 4 did not correlate. To confirm these results FOXO1 mRNA levels were measured by real time PCR in a separate cohort of 178 trial samples: FOXO1 and ABCB1 were also strongly correlated in this cohort (P<0.001). Protein levels of FOXO1 were measured by flow cytometry, and corresponded to mRNA levels in patient samples. Using the TF1 cell line, which normally expresses high levels of FOXO1 and ABCB1 we studied the regulation of pgp by FOXO1 using siRNA. FOXO1 siRNA was found to decrease both FOXO1 and ABCB1 mRNA expression by 70% and 60% respectively. High pgp expression rarely occurs in a sample with a FLT3-ITD mutation. In this context we established that the presence of a FLT3-ITD in AML samples is associated with low FOXO1 expression (P<0.001, median FOXO1/B2M ratio = 0.51 in wildtype samples (n=268) and 0.18 in ITD samples (n=101)) and that siRNA to FLT3 in MV4.11 (FLT3-ITD) cells upregulates FOXO1 more than 2-fold. As the ABCB1 promoter is known to have a FOXO1 binding site, we conclude that FOXO1 is likely to be a major regulator of ABCB1 expression in AML and may therefore be a therapeutic target to modulate drug resistance. We further conclude that FOXO1 may be at least partially responsible for the distinction between two major poor risk phenotypes in AML–namely FLT3-ITD (causing low FOXO1, predictive for relapse) and pgp overexpression (induced by high FOXO1, predictive for chemoresistant disease).

Disclosures:
No relevant conflicts of interest to declare.
Supportive Care Only Versus Allogeneic Hematopoietic Cell Transplantation in Patients ≥ 60 Years with De Novo High-Risk MDS.
Authors: Platzbecker Uwe , Finke Jürgen , Trenschel Rudolf , Kerstin Schaefer-Eckart , Bornhauser Martin , Kobbe Guido , Scott Bart L , Ehninger Gerhard , Beelen Dietrich , Deeg H. J. , Germing Ulrich 
Published: October 2019

Abstract
Abstract 596
Myelodysplastic syndromes (MDS) occur primarily in older patients, and allogeneic hematopoietic cell transplantation (HCT) only infrequently is considered a therapeutic option. Until recently best supportive care (BSC), only including transfusion support, has been the standard of care for the majority of these patients (pts). Novel treatment modalities and innovations in transplant protocols have changed that approach and have led to increased frequency of allogeneic HCT in pts more than 60 or 65 years of age. However, no randomized trial comparing outcomes with BSC and allogeneic HCT has been conducted.
Therefore, we performed a matched pair analysis including 126 patients 60-77 (median 65) years old with de novo high-risk MDS by FAB classification (RAEB n=88, RAEB-t n=20, CMML n=18) who underwent allogeneic HCT at a median of 8.6 months from initial MDS diagnosis. Cytogenetics were availabel in 108 pts (good: n=63, intermediate: n=18, poor: n=27), allowing for IPSS scoring in 107 pts: 28 were INT-1, 45 INT-2, and 34 HIGH risk. Before HCT most pts had progressed to more advanced disease (RAEB n=45, RAEB-t n=10, CMML n=10, AML n=61). At the time of HCT the median blast count in the marrow was 12%. While 51 pts had received supportive care only prior to HCT the remaining had undergone chemotherapy of various intensities including methyltransferase-inhibitors (n=10). Patients were prepared for HCT with one of several reduced intensity (RIC, n=79) or more conventional intensity (CI, n=47) conditioning regimens and transplanted from related (n=50) or unrelated (n=76) donors. The outcome after HCT was compared to outcome with BSC only in a matched pair group from the Düsseldorf registry. Matching criteria were age, gender, marrow blast count, FAB and IPSS category. One prerequisite was that controls were still under supportive care at the time of HCT of the corresponding matched patient.
With a median follow-up of 60 months from MDS diagnosis the 5-year overall survival (OS) rate was 45% for the HCT and 25% for the BSC cohort (p=0.008). These retrospective data suggest, therefore, that allogeneic HCT from related or unrelated donors, using various conditioning approaches, compared to BSC only, offers a meaningful survival advantage and the potential of cure for patients with high-risk MDS, even in the 7th decade of life. There is a need for prospective clinical trials in order to determine the place of allogeneic HCT within the growing therapeutic opportunities for pts with MDS.

Disclosures:
No relevant conflicts of interest to declare.
Molecular Response <1% BCR-ABL IS at 12 Months Is Associated with Improved Overall and Progression-Free Survival. the Randomized German CML-Study IV
Authors: Müller Martin C. , Hochhaus Andreas , Lauseker Michael , Jung-Munkwitz Susanne , Pfirrmann Markus , Leitner Armin , Krause Stefan W. , Pletsch Nadine , Hanfstein Benjamin , Schnittger Susanne , Gratwohl Alois , Hasford Jörg , Saussele Susanne , Hehlmann Rüdiger ,   for The German CML-Study Group
Published: October 2019

Abstract
Abstract 669

Introduction:
The prognostic relevance of major molecular remission (MMR, <0.1% BCR-ABL according International Scale, IS) for survival has remained uncertain. Gold standard for the evaluation of treatment response is the achievement of complete cytogenetic remission in spite of its limited sensitivity and the requirement of bone marrow puncture. The standardization of PCR methods and the introduction of conversion factors to account for differences among European laboratories, has resulted in a uniform reporting system allowing comparable BCR-ABL expression levels derived from peripheral blood samples. We sought to evaluate an association of the degree of molecular response and survival.

Patients and Methods:
We have analyzed 848 patients within the CML-Study IV (randomized comparison of imatinib 800 mg vs 400 mg vs 400 mg + IFN). BCR-ABL (IS) was determined by quantitative RT-PCR. Patients with atypical BCR-ABL transcripts were excluded from the analysis. Median observation time was 40 months (minimum 12). Landmark analyses have been performed at 12 months for overall and progression-free survival using 3 groups of response (<0.1%, 0.1%-1%, >1% BCR-ABL IS).

Results:
341 patients achieved a BCR-ABL expression <0.1% (MMR), 240 patients between 0.1% and 1% and 267 patients >1% by 12 months. Independent of treatment approach, the groups of patients achieving MMR and 0.1%-<1% at 12 months showed significantly higher progression free survival (PFS) (p=0.0023; 99% [95% CI: 97–100%] vs 97% [95% CI: 94–99%] vs 94% [95% CI: 90–97%] at 3 years) and better overall survival (p=0.0011; 99% [95% CI: 97–100%] vs 98% [95% CI: 95–100%] vs 93% [95% CI: 90–96%] at 3 years) compared to the group with >1% BCR-ABL by 12 months (Figure).

Conclusion:
Faster and deeper response to imatinib-based treatment by 12 months revealed to be associated with improved PFS and overall survival. The critical cutoff level seems to be 1% BCR-ABL IS which has been shown to closely correlate with complete cytogenetic remission.

Disclosures:
Müller: Novartis: Honoraria, Research Funding. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. German CML-Study Group:Deutsche Krebshilfe: Research Funding; Novartis: Research Funding; Roche: Research Funding; BMBF: Research Funding; Essex: Research Funding.
Methylation Profiling of Mediastinal Gray Zone Lymphoma Reveals a Distinctive Signature with Elements Shared by Classical Hodgkin's Lymphoma and Mediastinal Large B-Cell Lymphoma
Authors: Eberle Franziska C. , Rodriguez-Canales Jaime , Wei Lai , Hanson Jeffrey C. , Killian J. Keith , Sun Hong-Wei , Adams Lisa G. , Hewitt Stephen M. , Wilson Wyndham H. , Meltzer Paul S. , Staudt Louis M. , Emmert-Buck Michael R. , Pittaluga Stefania , Jaffe Elaine S. 
Published: October 2019

Abstract
Abstract 747

Background:
Mediastinal gray zone lymphoma (MGZL) is a newly recognized entity demonstrating transitional morphologic and immunophenotypic features between classical Hodgkin's lymphoma, nodular sclerosis subtype (CHLNS) and primary mediastinal large B-cell lymphoma (PMLBCL). CHLNS and PMLBCL differ in morphology, immunophenotype, and therapeutic consequences. MGZLs present a challenge both to the pathologist and clinician, as the criteria to distinguish MGZL from CHLNS and PMLBCL are still imprecise, and the optimal treatment approach is as yet undetermined.

Purpose of Study:
Epigenetic changes have been implicated in the loss of the B-cell program in CHL, and might provide a basis for the immunophenotypic alterations seen in MGZL. Thus, we performed a large scale DNA methylation array of MGZL, CHLNS, and PMLBCL as well as diffuse large B-cell lymphoma (DLBCL) to investigate the biological underpinnings of MGZL and how it corresponds to the two related entities CHLNS and PMLBCL and the less related entity DLBCL. Microdissection of tumor cells was performed to identify changes in the tumor cell population, and allow comparison with the background inflammatory and stromal milieu.

Results:
Principal component analysis (PCA) demonstrated that MGZLs have a distinct epigenetic profile intermediate between CHLNS and PMLBCL but clearly different from that of DLBCL. Analysis of common hypo- and hypermethylated CpG targets in MGZL, CHLNS, PMLBCL and DLBCL was performed. MGZL showed great overlap with CHLNS (49 common targets) and PMLBCL (50 common targets). In contrast, MGZL shared only two common targets with DLBCL. However, using the epigenetic profiles we were able to establish class prediction models that could distinguish between MGZL, CHLNS and PMLBCL with a final combined prediction of 100%. Pyrosequencing for selected CpG sites from different genes were performed and confirmed the high accuracy of methylation array results.

Conclusions:
MGZLs share several clinical and pathological features with CHLNS and PMLBCL. Our findings further underscore the close biological relationship between MGZL, CHLNS and PMLBCL, and ready distinction from DLBCL. However, MGZL has a distinct epigenetic identity that shares elements of both parent disorders. As the first biological study on MGZL, our results provide novel insights into MGZL pathogenesis, and its relationship to CHLNS and PMLBCL.

Disclosures:
Emmert-Buck: NIH: Michael Emmert-Buck is an inventor on all NIH patents covering laser capture microdissection technology and receives royalty payments through the NIH technology transfer program., Patents & Royalties.
Reinforcement of Cancer Immunotherapy by Adoptive Transfer of Cblb-Deficient Cytotoxic T Lymphocytes Combined with a Dendritic Cell Vaccine
Authors: Lutz-Nicoladoni Christina , Stoizner Patrizia , Pircher Magdalena , Wallner Stephanie , Wolf Anna Maria , Gastl Guenther , Penninger Josef , Baier Gottfried , Wolf Dominik 
Published: October 2019

Abstract
Abstract 957

Introduction:
Various approaches to induce immunological rejection of tumors including transfer of autologous tumor infiltrating lymhocytes (TIL) after ex vivo clonal expansion or application of ex vivo transduced antigen specific T cell (TCR) transgenic T cells have been elaborated. In general, adoptive T cell transfer (ATC) has been combined with lympho-depleting agents (e.g. cyclophosphamide). However, the therapeutic efficacy of these cancer immunotherapy approaches is limited due to insufficient in vivo activation, expansion and survival of transferred effector immune cells, which is mainly due to suppressive mileu signals and immune evasion mechanisms induced by TGF-β. The E3 ubiquitin ligase Cbl-b is a key regulator of T cell activation and is assumed to confer TGF-β resistance. Thus we performed a proof-of-concept study evaluating Cbl-b targeting as “intracellular adjuvant” strategy to improve ATC for cancer immunotherapy.

Material and Methods:
We first tested the in vitro sensitivity of CTL towards TGF-β mediated immuno-suppressive cues and then in vivo evaluated the anti-tumor reactivity of cblb-deficient cytotoxic T lymphocytes (CTL) in murine tumor models alone or in combination with a dendritic cell (DC) vaccine.

Results:
Cblb-deficient CTL are hyper-responsive to TCR/CD28-stimulation in vitro and protected from the negative cues induced by TGF-β as determined by quantification fo IFN-g secretion and quantification of their proliferative capacity. Unexpectedly, adoptive transfer of polyclonal, non TCR-transgenic cblb-deficient CD8+ CTL, however, is not sufficient to reject B16ova or EG7 tumors in vivo, which is in clear contrast to previous reports using lymphopenic animals receiving adoptively transferred TCR-transgenic T cells. Thus, we next evaluated in vivo re-activation of adoptively transferred cblb-deficient T cells by a DC vaccine (i.e. SIINFEKL-pulsed DC). In strict contrast to ATC monotherapy, this approach now markedly delays tumor outgrowth and significantly increase survival rates, which is paralleled by an increased CTL infiltration rate to the tumor site and an enrichment of ova-specific and IFN-g-secreting CTL in the draining lymph nodes. Moreover, compared to wild-type CTL, cblb-deficient mice vaccinated with the DC vaccine show an increased cytolytic activity in vivo.

Conclusions:
In summary, we provide experimental evidence that genetic inactivation of cblb in polyclonal, non-TCR transgenic adoptively transferred CTL might serve as a novel “adjuvant approach”, suitable to augment the effectiveness of anti-cancer immunotherapies using ATC in immune-competent recipients.

Disclosures:
No relevant conflicts of interest to declare.