bluuebunny/crossref_metadata_2025_split huggingface dataset:
Small Leucine Rich Proteoglycans as Novel Tumor Markers In Chronic Lymphocytic Leukemia
Authors: Mikaelsson Eva , Jeddi-Tehrani Mahmood , Ã–Sterborg Anders , Shokri Fazel , Rabbani Hodjattallah , Mellstedt Håkan 
Published: October 2019

Abstract
Abstract 694

Background:
Small leucine rich proteoglycans (SLRPs) are a family of glycosylated proteins normally expressed in the extracelluar matrix (ECM) of collagen rich tissues. The biological role of the SLRPs is multifactoral, but mostly these proteins are secreted and bind to membrane receptors or ECM proteins affecting cell proliferation and cell migration. Some SLRPs (eg. Decorin, Lumican) have been reported to be expressed in cancer, but the expression as well as the glycosylation pattern differ. Microarray studies have revealed that the SLRP family member fibromodulin (FMOD) gene is overexpressed in chronic lymphocytic leukemia (CLL). We later reported the specific expression of FMOD in CLL and mantle cell lymphoma (MCL). FMOD is located on chromosome 1q32 adjacent to two other members of the SLRP family, proline/arginine-rich end leucine-rich repeat protein (PRELP) and opticin (OPTC).

Aims:
To analyse 1) the expression of PRELP and OPTC in CLL and other hematological malignancies. 2) the glycosylation pattern of PRELP and OPTC, and cellular localization of OPTC in CLL cells.

Methods:
PRELP: Gene expression was tested by RT-PCR and realtime-PCR. Protein expression was tested by western blot. Chemical deglycosylation was performed to characterize the glycosylation pattern of the PRELP protein. OPTC: Cell fractionation was performed using four different methods. Protein expression (molecular weight and cellular location) was tested by western blot. Chemical deglycosylation was performed to characterize the glycosylation pattern of the OPTC protein.

Results:
PRELP was expressed at the gene level (RT-PCR) in all CLL patients tested (n=30) and in 3/5 patients with mantle cell lymphoma, but not in normal leukocytes (n=10) or in other hematological malignancies (7 different types, n=35). The PRELP protein was detected in all CLL samples tested but not in leukocytes of healthy donors (western blot). Molecular analysis of the CLL PRELP protein revealed a unique unglycosylated 38 kDa core protein, with an intact signal peptide. This protein was not detected in serum that, in combination with the uncleaved signal peptide, suggests cellular retention. A “normal” OPTC protein (50 kDa) was detected in cell lysates of both CLL tumor cells (n=30) and normal leukocytes (n=10). However, the CLL cells also expressed a 37 kDa OPTC that was not detected in healthy controls. This 37 kDa OPTC was detected in all CLL samples and molecular analysis revealed a unique unglycosylated core protein that was located in the cell nucleus and endoplasmatic reticulum of the CLL cells.

Conclusion:
The unique expression of the SLRPs PRELP and OPTC in CLL (in addition to the previously reported FMOD) is unexpected and merits further studies since the specific expression of three closely related SLRP genes may indicate a role in the pathobiology of the disease.

Disclosures:
No relevant conflicts of interest to declare.
A Predictive Model for the Development of High-Responding Inhibitor In Children with Severe and Moderate Hemophilia A- Results of a Multicenter Cohort Study
Authors: Holzhauer Susanne , Bidlingmaier Christoph , Ettingshausen Carmen Escuriola , Halimeh Susan , Iorio Alfonso , Kenet Gili , Pautard Brigitte , Nowak-Gottl Ulrike 
Published: October 2019

Abstract
Abstract 710

Background:
Pediatric cohort studies in previously untreated [PUPs] children with hemophilia A [HA] identified potential risk factors for clinical meaningful inhibitor development [IH], among them HA severity < 1%, high risk gene mutations [HRS], high dose FVIII replacement therapy [HD] and use of recombinant FVIII concentrates [rFVIII]. The aim of the present study was to develop a simple score to rate inhibitor development in HA children.

Patients and methods:
184 consecutively ascertained PUPs [enrollment between 1982 and 2008] with HA [severity < 5%] followed over a period of 200 exposure days were randomly selected into two groups [G1; G2] of 92 patients each. Three variables [HRS: 2 points], [HD: median single dose > 30 IU/kgbw within 8 to 12 weeks after therapy start] and [rFVIII] 1 point each, were incorporated into a risk assessment model, which was i) evaluated in patient group G1 [pilot cohort: patients not included in the CANAL study] and ii) validated in the second group [G2: confirmation cohort]. The risk discrimination cut-off was calculated using two independent statistical methods [ROC-analysis: MedCalc 11.0.0; CART regression tree: CART 6.0]. In addition, model specific cumulative IH-free survival [Cox regression analysis: hazard ratio (HR) and 95% confidence interval (CI)], negative predictive values [NPV], sensitivity and specificity were calculated. In Cox regression analysis adjustment was performed for i) age at first symptomatic bleed [proxy for early start of prophylaxis], ii) severity of factor VIII-deficiency, and iii) for treatment periods [1982-2008: divided into 3 to 5 year intervals].

Results:
The risk discrimination cut-off in the pilot cohort G1 was calculated as “2”. Model sensitivity was 78.8 % and specificity was 49.8% respectively. In the confirmation cohort G2 seventeen children with HA developed a high responding IH [18.5%]: 16 out of 52 in children with a risk score > 2 [30.8%] and one out of 40 with a risk score < 2 [2.5%: p<0.001]. Compared to children with a risk score < 2 the cumulative IH-free survival in HA patients with a risk score > 2 was significantly reduced with an adjusted HR [CI] of 11.2 [1.5-83.5]. The corresponding NPV to rule out a high responding IH development in the confirmation cohort was 92% [80.5-98.5].

Conclusion:
The model is able to rule out the development of high responding IH in white children with HA < 5% with good discriminative ability. Thus the model can be instrumental to individualize treatment strategies in HA patients. The score needs to be validated in other populations to test its generalizability.

Disclosures:
No relevant conflicts of interest to declare.
Rational Design and Characterization of Platelet Factor 4 Antagonists
Authors: Sachais Bruce S , Rux Ann H , Hinds Jillian L , Rux John J 
Published: October 2019

Abstract
Abstract 724

Background:
Heparin-induced thrombocytopenia and thrombosis (HITT) is a serious complication of heparin therapy, placing patients at increased risk for thrombosis and death. The chemokine platelet factor 4 (PF4) is central to the pathogenesis of HITT. Tetrameric PF4 forms ultralarge complexes (ULC) with heparin, which, when bound to specific antibodies, lead to thrombocytopenia and thrombosis. We have previously shown that the tetrameric form of PF4 is required for ULC formation. Since a large number of hospitalized patients are exposed to heparin, HITT is a major iatrogenic cause of morbidity and mortality in this patient population. There are currently no specific therapies for the treatment of HITT.

Objectives:
Identify small molecule antagonists of PF4 tetramerization to serve as reagents for the mechanistic study of HITT as well as lead compounds for development of novel HITT therapies.

Approach:
Using the crystal structure of PF4, we identified a potential antagonist binding site near residues glutamic acid 28 (E28) and lysine 50 (K50). These residues form salt bridges between two PF4 dimers which are required for tetramer formation. We then performed an in silico screen of 1.1×10^6 lead-like small molecules using the program DOCK to identify compounds likely to bind to this site. A subset of these compounds were obtained and tested for their ability to inhibit PF4 tetramerization and PF4:heparin ULC formation in vitro.

Results:
In silico screening identified 109 compounds with DOCK scores (lower scores predict higher binding affinity) which were greater than 10 standard deviations below the mean (see Figure). We have obtained 12 of these compounds for in vitro testing. The ability of antagonists to inhibit PF4 tetramerization was measured as previously described by quantitation of silver stained SDS-PAGE gels of cross-linked PF4 in the absence and presence of putative antagonists. 5 of the compounds showed inhibition of tetramerization in a dose dependent manner, with 2 of these compounds (PA31 and PA35) demonstrating micromolar potency (IC50 of tetramerization = 500uM and 600uM respectively). The ability of these antagonists to inhibit ULC formation between PF4 and heparin was measured by both gel filtration and ELISA as previously described. PA31 and PA35 both inhibited ULC formation with potencies similar to that for inhibition of tetramerization.

Conclusions:
We have identified two small molecule PF4 antagonists. These compounds will be useful in future mechanistic studies of HITT. They also may serve as lead drug discovery compounds for chemical modification to create PF4 antagonists with increased affinity and potency. Such compounds may be tested as novel therapeutics for the treatment and/or prevention of HITT.

Disclosures:
Rux: In sillico Molecular, LLC: Employment, Owner.
Assessment of Residual Bulky Tumor Using FDG-PET In Patients with Advanced-Stage Hodgkin Lymphoma After Completion of Chemotherapy: Final Report of the GHSG HD15 Trial
Authors: Engert Andreas , Kobe Carsten , Markova Jana , Haverkamp Heinz , Borchmann Peter , Hitz Felicitas , Zijlstra Josee , Eich Hans , Mueller Rolf-Peter , Dietlein Markus , Diehl Volker 
Published: October 2019

Abstract
Abstract 764

Introduction
The role of additional radiotherapy after chemotherapy for advanced-stage Hodgkin lymphoma is unclear. The German Hodgkin Study Group (GHSG) thus performed the HD15 trial in which advanced-stage Hodgkin lymphoma patients having residual disease after 6–8 cycles of BEACOPP were evaluated by 18F-fluorodesoxyglucose positron emission tomography (PET) following chemotherapy.

Methods
Entry criteria for the PET question in HD15 were partial remission (PR) after the end of chemotherapy with at least one involved nodal site measuring more than 2.5 cm in diameter by computed tomography (CT). Exclusion criteria included diabetes, elevated blood sugar levels and skeletal involvement with risk of instability. Calculations were restricted to those cases with either progressive disease (PD) or relapse within 12 months after PET or at least 12 months of follow-up. A total of 2,137 patients with de novo HL were included in HD15 of whom 728 had a tumor bulk ≥ 2.5 cm after BEACOPP chemotherapy and were qualified for the PET question. An expert panel performed the assessment of response and PET. Only PET-positive patients were scheduled for radiotherapy of residual disease. The negative prognostic value (NPV) of PET was defined as the proportion of PET-negative patients without progression, relapse or radiotherapy despite being PET-negative within 12 months.

Results
The full analysis set included 728 patients of whom 699 had at least 12 months of follow-up. Median age was 30 years, 57% were males and 66% had NS histology. Of the 728 qualified patients with residual disease ≥ 2.5 cm after BEACOPP, 74.2% were PET-negative and 25.8% PET-positive. In the PET-negative group, a total of 28 patients relapsed or had radiotherapy despite being PET-negative (8 patients including 1 relapsing patient) resulting in a negative prognostic value of 94.6% (95% CI 92.7% to 96.6%). With a median follow-up of 38 months, the time-to-progression after PET at 3 years was 92.1% for PET-negative patients counting radiotherapy as failure and 86.1% for PET-positive patients (95%-CI for difference -11.9% to -0.1%). Overall, only 11% of patients had additional radiotherapy as compared to 71% after BEACOPPescalated in our prior HD9 trial. In addition, there was no difference in PFS or overall survival as compared to our earlier trials in advanced-stage HL.

Discussion
The NPV of PET of 0.95 suggests that indeed only patients with residual disease after chemotherapy who are PET-positive need additional radiotherapy. PET-negative patients at least after BEACOPP can be spared from additional radiotherapy.

Disclosures:
No relevant conflicts of interest to declare.
A Novel Pro-Survival Function of Cyclin-D1 Underlies Its Oncogenic Role and Potential as a Therapeutic Target In Mantle Cell Lymphoma
Authors: Beltran Elena , Fresquet Vicente , Martinez-Useros Javier , Richter-Larrea Jose A. , Sagardoy Ainara , Sesma Izaskun , Almada Luciana L. , Montes-Moreno Santiago , Gesk Stefan , Siebert Reiner , Calasanz MaJose , Prosper Felipe , Piris Miguel A. , Fernandez-Zapico Martin E. , Martinez-Climent Jose A 
Published: October 2019

Abstract
Abstract 769
Despite the many and diverse therapeutic approaches used to treat patients with mantle cell lymphoma (MCL), it remains an incurable disease. Recently, attention has turned into novel therapies targeting MCL-specific oncogenic pathways important for the growth and maintenance of the transformed phenotype. The chromosomal translocation t(11;14)(q13;q32) leading to cyclin-D1 over-expression is the hallmark of MCL. Constitute cyclin-D1 activation in B-lymphocytes maintains retinoblastoma protein in a phosphorylated state and promotes cell cycling, thus initiating the tumorigenesis process. Cyclin-D1 has been postulated as a putative target for therapeutic intervention, however its evaluation has been hampered by the incomplete understanding of the mechanism underlying this cyclin oncogenic function and by the lack of valid MCL models. To investigate these issues, we developed a combined cellular-genomics screening whereby responses to known cytotoxic compounds targeting cancer-related molecular pathways were correlated with genomic, gene expression and proteomic profiles of human MCL cells. Results showed that cyclin-D1 silencing had minimal antitumoral effects but significantly increased the therapeutic efficacy of several compounds, especially the BH3 mimetics that inhibited anti-apoptotic protein BCL-2. To further evaluate this finding we generated a MCL mouse model by transducing a tetracycline-regulatable cyclin-D1-expressing vector in murine pro-B cells, which allowed modulating cyclin-D1 expression levels. These mice generated lymphomas recapitulating most of the cellular, histopathological and molecular features of human MCL. Similar to the previous in vitro findings, cyclin-D1 inhibition in this model did not induce lymphoma regression, but sensitized cells to apoptosis. Analysis of the mechanisms underlying this therapeutic synergy identified a novel role for cyclin-D1 as a pro-survival molecule. Specifically, cyclin-D1 sequestrated the pro-apoptotic effector protein BAX in MCL cells, thereby favoring BCL2 anti-apoptotic function. Accordingly, therapeutic cyclin-D1 inactivation released BAX, thus sensitizing cells to apoptosis and inducing lymphoma regression. Interestingly, pharmacological blockade in vivo of cyclin-D1 with Roscovitine synergistically cooperated with the BH3 mimetic ABT-737 to effectively inhibit MCL tumor growth. In summary, our study reveals a novel role for cyclin-D1 in deregulating apoptosis in MCL cells and highlights the potential benefit of cyclin-D1 targeting, thus providing the rationale for the clinical evaluation of drugs targeting cell proliferation and survival pathways in MCL.

Disclosures:
Siebert: Abbott: Honoraria.
Up-Regulation of Anti-Inflammatory, STAT3-Activating Hepatocyte Growth Factor and Interleukin-11 In Polycythemia Vera Is Independent of JAK2V617F and Contributes to the Growth of Clonal Erythroblasts
Authors: Boissinot Marjorie , Cleyrat Cedric , Vilaine Mathias , Jacques Yannick , Corre Isabelle , Hermouet Sylvie 
Published: October 2019

Abstract
Abstract 796

Context:
The V617F activating mutation of JAK2, JAK2V617F, characterizes Polycythemia vera (PV). However, expression of JAK2V617F does not always result in clone expansion, implying that additional events are required for the growth of JAK2-mutated progenitors and development of the PV phenotype. In the present study, we provide evidence that anti-inflammatory cytokines are required for the growth of JAK2V617F-mutated erythroid progenitors.

Methods:
We searched for cytokines over-expressed in PV using cytokine antibody arrays and ELISAs for analyses of serum and bone marrow (BM) plasma, and quantitative RT-PCRs for analyses of cells purified from PV patients and controls. Transient transfection of JAK2V617F and invalidation of JAK2V617F with siRNA were used to study the effect of JAK2V617F on cytokine expression.

Results:
We found that PV patients over-expressed anti-inflammatory hepatocyte growth factor (HGF) and interleukin-11 (IL-11), BM stromal cells (BMSC) and erythroblasts being the main producers. HGF induced the production of IL-11 and both cytokines activate the STAT3 pathway. We used neutralizing antibodies specific for IL-11 or c-MET, the HGF receptor, to block autocrine/paracrine cytokine stimulation of erythroblasts in in vitro cultures. The growth of JAK2V617F-mutated PV erythroblasts and HEL cells was inhibited by at least 40%, indicating that JAK2V617F-mutated cells depend on HGF and IL-11 for their growth. Consistent with activation of HGF/IL-11 pathways, mRNA levels of gp130 (the receptor chain responsible for signalling common to cytokines of the IL-11/IL-6 family) and STAT3 were significantly elevated in PV erythroblasts. Moreover, mRNA levels of c-MET, HGF and IL-11 were correlated, a logical finding as c-MET, HGF and IL-11 act in cascade. No correlation was found with JAK2V617F mRNA levels. To the opposite, we provide evidence that the up-regulation of HGF and IL-11 in PV is not a consequence of JAK2V617F: transient expression of JAK2V617F in BaF-3/Epo-R cells and invalidation of JAK2V617F in HEL cells using anti-JAK2 siRNA had no effect on HGF and IL-11 expression.

Conclusion:
Anti-inflammatory, STAT3-activating HGF and IL-11 are up-regulated in PV independently of JAK2V617F. Both cytokines contribute to the proliferation of JAK2V617F-mutated erythroblasts, suggesting that drugs blocking the HGF/IL-11 pathways, such as c-MET antagonists, could be of interest as an additional therapeutic option in PV.
Marjorie Boissinot and Cedric Cleyrat contibuted equally.

Disclosures:
No relevant conflicts of interest to declare.
The Role of Nucleophosmin In Hematopoietic Stem Cells and the Pathogenesis of Myelodysplastic Syndrome
Authors: Ito Keisuke , Sportoletti Paolo , Clohessy John G , Silvia Grisendi , Pandolfi Pier Paolo 
Published: October 2019

Abstract
Abstract 95
Myelodysplastic syndrome (MDS) is an incurable stem cell disorder characterized by ineffective hematopoiesis and an increased risk of leukemia transformation. Nucleophosmin (NPM) is directly implicated in primitive hematopoiesis, the pathogenesis of hematopoietic malignancies and more recently of MDS. However, little is known regarding the molecular role and function of NPM in MDS pathogenesis and in stem cell biology. Here we present data demonstrating that NPM plays a critical role in the maintenance of hematopoietic stem cells (HSCs) and the transformation of MDS into leukemia.
NPM is located on chromosome 5q and is frequently lost in therapy-related and de novo MDS. We have previously shown that Npm1 acts as a haploinsufficient tumor suppressor in the hematopoietic compartment and Npm1+/− mice develop a hematologic syndrome with features of human MDS, including increased susceptibility to leukemogenesis. As HSCs have been demonstrated to be the target of the primary neoplastic event in MDS, a functional analysis of the HSC compartment is essential to understand the molecular mechanisms in MDS pathogenesis. However, the role of NPM in adult hematopoiesis remains largely unknown as Npm1-deficiency leads to embryonic lethality.
To investigate NPM function in adult hematopoiesis, we have generated conditional knockout mice of Npm1, using the Cre-loxP system. Analysis of Npm1 conditional mutants crossed with Mx1-Cre transgenic mice reveals that Npm1 plays a crucial role in adult hematopoiesis and ablation of Npm1 in adult HSCs leads to aberrant cycling and followed by apoptosis. Analysis of cell cycle status revealed that HSCs are impaired in their ability to maintain quiescence after Npm1-deletion and are rapidly depleted in vivo as well as in vitro. Competitive reconstitution assay revealed that Npm1 acts cell-autonomously to maintain HSCs. Conditional inactivation of Npm1 leads to an MDS phenotype including a profoundly impaired ability to differentiate into cells of the erythroid lineage, megakaryocyte dyspoiesis and centrosome amplification. Furthermore, Npm1 loss evokes a p53-dependent response and Npm1-deleted HSCs undergo apoptosis in vivo and in vitro. Strikingly, transfer of the Npm1 mutation into a p53-null background rescued the apoptosis of Npm1-ablated HSCs and resulted in accelerated transformation to an aggressive and lethal form of acute myeloid leukemia. Our findings highlight the crucial role of NPM in stem cell biology and identify a new mechanism by which MDS can progress to leukemia. This has important therapeutic implications for de novo MDS as well as therapy-related MDS, which is known to rapidly evolve to leukemia with frequent loss or mutation of TRP53.

Disclosures:
No relevant conflicts of interest to declare.