bluuebunny/crossref_metadata_2025_split huggingface dataset:
LY2784544, a Novel JAK2 Inhibitor, Decreases In Vitro Growth of Hematopoietic Human Progenitors From JAK2 V617F Positive Polycythemia Vera Patients
Authors: Florensa Lourdes , Bellosillo Beatriz , Arenillas Leonor , Ma Liandong , Walgren Richard , Alvarez Alberto , Gonzalez Judit , Vila Rosa M , Longarón Raquel , Besses Carles 
Published: October 2019

Abstract
Abstract 5054

Introduction:
The discovery of JAK2 V617F mutation in patients with myeloproliferative disorders (MPD) has opened new perspectives for the development of targeted therapies. We have studied the efficacy of a novel molecule LY2784544 with JAK2 inhibitory activity in the in vitro growth of myeloid progenitors from JAK2 V617F-positive polycythemia vera (PV) patients.

Objectives:
To investigate the efficacy of LY2784544 in the inhibition of endogenous(e)BFU-E and CFU-GM growth in PV patients.

Methods:
In vitro cultures in semisolid media were performed from peripheral blood mononuclear cells (PBMC) of 6 PV patients who had never received cytoreductive treatment (4 patients with homozygous JAK2 V617F and 2 patients with heterozygous JAK2 V617F). PBMC were suspended in methylcellulose (Methocult. StemCell, Vancouver, Canada) without the addition of EPO and containing 0–30.0 μM LY2784544 drug. Concurrent plates containing EPO were plated as control cultures. The medium was distributed in multidishes and they were incubated at 37° with 5% CO2 and 95% humidity. Hemoglobinized colonies and granulomonocytic colonies were counted on day 14 by standard criteria (BFU-E defined by an aggregate of >50 hemoglobinized cells or three or more erythroid subcolonies and CFU-GM was defined by an aggregate of >50 cells). Each in vitro assay was performed in duplicate. DNA was obtained from peripheral blood granulocytes from each patient to quantify the JAK2 V617F allele burden at the time of culture assay.

Results:
LY2784544, at concentrations ranging from 0.03–30.0 μM, inhibited growth of unselected peripheral blood eBFU-E and CFU-GM from PV patients carrying the JAK2 V617F mutation in a dose-dependent manner, although without achieving complete inhibition of all colonies (fig.1).

Conclusions:
In vitro studies show that LY2784544 decreases the eBFU-E and CFU-GM growth in therapy-naive JAK2 V617F positive PV patients. Our data suggest that LY2784544 may be a candidate for the treatment of MPD carrying the JAK2 V617F mutation.

Disclosures:
Ma: Eli Lilly and Company: Employment. Walgren:Eli Lilly and Company: Employment.
Chronic Myeloprolipherative Neoplasms (MPNs) and Splanchnic Venous Thrombosis(SVT): Incidence and Risk Factors
Authors: Martino Bruno , Alati Caterina , Cufari Patrizia , Vincelli Iolanda , Ronco Francesca , Oriana Vincenzo , Nobile Francesco 
Published: October 2019

Abstract
Abstract 5057
There is evidence in literature of the clinical and pathogenetic role of JAK2 V617F mutation in MPN, while its contribute as an additional thrombotic risk factor in MPN patients is still under discussion. The jak2 mutation has been identified as occult marker in several patients with splanchnic venous thrombosis (SVT); morover, 45% of patients with Budd-Chiari syndrome (BCS) and 34% of patients with portal vein thrombosis (PVT) have associated MPN. On the other hand, the majority of BCS patients without MPN present additional congenital or acquired thrombosis risk factors.
The aim of the present study is to evaluate the incidence of SVT in MPN patients. We also evaluated the presence of other prothrombotic risk factors in MPN patients, in addition to the JAK-2 mutation.
Out of the 460 Ph1 negative MPN patients observed in our center from January 2000 to January 2010 and retrospectively evaluated, 9 patients (7 females and 2 males; 2%) presented SVT.
Six cases had Essential Thrombocythemia (ET), 2 Primary Myelofibrosis (PMF), and 1 Polycitemia Vera (PV). Five of the 6 cases with ET were females. Among the entire population of ET, SVT incidence was 3%. All the 9 patients diagnosed of SVT were JAK2 V617F positive and they were treated with antiaggregant and anticoagulation therapy; 6 received hydrossiurea.
Seven patients had SVT before MPN diagnosis, 2 of them had splenectomy at diagnosis for surgical decision. In these patients developing SVT before MPN diagnosis no other major thrombotic event occurred during follow-up. The remaining 2 MPNs patients presented asymptomatic SVT diagnosed by imaging techniques routinely performed during MPN follow up.
Interestingly, 75% of MPNs patients with SVT demonstrated at least one prothromobtic risk factor, such as factor V Leiden, Protein C deficiency, hyperhomocystinemia and 50% had 2 or more associated defects. MPNs patients without SVT (396) had a lower prevalence of prothrombotic risk factors and developed venous thrombosis in different anatomical sites: in these cases white blood cell count, platelets values and the presence of JAK2 V617F mutation correlate with the development of the thrombotic event.

Conclusion.
Even though SVT has a low incidence in MPNs patients, according to the results of the present retrospective study we suggest the potential benefit of searching for additional prothrombotic risk factors in the whole MPN population in order to prevent and/or properly treat this complication.

Disclosures:
No relevant conflicts of interest to declare.
A Retrospective Epidemiological Analysis of 1572 Cases of Ph1- Myeloprolypherative Neoplasms (MPNs) From 9 Centers In the Latium: Preliminary Results
Authors: Montanaro Marco , Latagliata Roberto , Montefusco Enrico , Cedrone Michele , Villivà Nicoletta , Cotroneo Ettore , Rago Angela , Spirito Francesca , De Muro Marianna , Breccia Massimo , Anaclerico Barbara , Porrini Raffaele , Pessina Gloria , Felici Stefano , Trapè Giulio , Tafuri Agostino , Mazzucconi Maria Gabriella , Cimino Giuseppe , Majolino Ignazio , Avvisati Giuseppe , Lo Coco Francesco , Andriani Alessandro 
Published: October 2019

Abstract
Abstract 5065
The Latium is a region of central Italy that counts approximatively 5.600.000 of residents. We reported herein a preliminary analysis of 1572 patients affected by MPN diagnosed in the last 20 years and treated in 9 of the Centers belonging to our group. Our centers are mainly located in Rome (7 centers) and neighboring districts (2 centers, Latina and Viterbo). The diagnosis was performed according to PVSG criteria until 2001 and then according to the WHO criteria: the majority of the patients underwent bone marrow biopsy. 218 patients were affected by primary myelofibrosis (PMF), 779 by Essential thrombocytemia (ET) and 575 by Policytemia Vera (PV). Epidemiological and clinical findings of all patients at diagnosis as well as thrombotic complications and evolution are reported in the table:ET (779 pts)PV (575 pts)PMF (218 pts)M/F288/491350/225134/84Age (yrs)59 (r: 20-93)60 (r: 19-91)67 (r: 30-86)WBC (x109/L)9192 (+/− 3483 SD)10423 (+/− SD 4307)13313 (+/−5220)Hb (g/dL)13, 9 (+/− 1,8)18,1 (+/− 2,3)11,7 (+/− 2,8)Plts (x 109/L)877 (+/− 353)445 (+/−247)457 (+/− 358)Splenomegaly (%)193579Bone marrow biopsy (performed/total))557/779 (71,5%)254/575 (44,2%)207/218 (95%)JAK-2 V617F mut (%)284/484 (59%)256/369 (69,4%)69/116 (59,5 %)Jak-2 allele burdenPerformed in 188/284 cases 26,49% (SD +/− 24)Performed in 199/256 cases: 59,4 % (SD +/−30,9)Performed in 51/69 cases 57,5% (SD +/− 29,4)Median Follow-up (years)7,157,863,89Thrombosis pre-diagnosis of MPNN. of patients (%)108/779 (13,8%)100/575 (17,4%)29/218 (13,3%)(arterial+venous)74+3476+2424+5Thrombosis post diagnosis of MPNN. of patients (%)71/779 (9,1%)66/575 (11,5%)20/218(9,17%)(arterial+venous)42+2942+2412+8EvolutionMyelofibrosis9/779 (1,1%)24/575 (4,1%)AML9/779 (1,1%)13/575 (2,3%)17/143 (11,8%)

Comments:
In our opinion, this casistic of patients with different types of MPNs reflects clinical presentation and evolution of three variants of the same disease. Many clinical findings in our unselected cohort of patients are similar to those reported in literature; in particular, thrombotic events were seen in about 13 – 17% of patients, without any correlation with Jak-2 status in both pre-diagnosis and follow-up. However, 2 main differences were noted: a lower incidence of JAK-2 V617F mutation among our PV patients and a lower rate of evolution in myelofibrosis and AML among our ET and PV patients. Both these features warrant further insights to be fully elucidated.

Disclosures:
No relevant conflicts of interest to declare.
Nuclear Area Predicts Outcome In Peripheral T-Cell Lymphoma, Not Otherwise Specified: Morphometric Image and Immunohistochemical Analyses of 78 Patients
Authors: Niino Daisuke , Kimura Yoshizo , Kiyasu Junichi , Takeuchi Masanori , Yoshino Tadashi , Nakamura Shigeo , Ohshima Koichi 
Published: October 2019

Abstract
Abstract 5081

Background:
Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is an uncommon tumor with unfavorable prognosis. The purpose of this retrospective study was to investigate the cytological features and treatment outcomes in patients with PTCL-NOS and to obtain new information on the therapeutic implications of PTCL-NOS.

Method:
A total of 6 institutions in Japan and Asia submitted clinical and pathologic information on PTCL-NOS diagnosed and treated at their respective centers. Among these cases, 55 (71%) were from Japan, 8 (10%) were from Hong Kong, 8 (10%) were from Thailand, and 7 (9%) were from Korea. From 1992 to 2006, 78 cases of PTCL-NOS were examined for cytological features, focusing on morphometric image analysis. The average nuclear area of lymphoma cells, as well as the median, the minimum, the maximum and the standard deviation, was estimated in 50 cells for each case. In addition, we performed an immunohistochemical study of CCR4 expression in all cases. Univariate analyses were performed using Kaplan-Meier survival estimates, and data were compared using the log-rank test. Cox proportional-hazard regression test was used for multivariate analysis. P-values of less than 0.05 were considered significant.

Results:
Clinical information was available for review in 78 cases. There was a 1.52:1 male/female ratio, and median age was 62 years (range, 3 to 87 years). Sixty-two percent of patients had advanced clinical stages. Lactic dehydrogenase (LDH) was elevated in 53% of cases, and most patients were treated with combination chemotherapy. Median survival was 693 days. On the morphometric image analysis, median nuclear area was 41.7 μ m2 (range, 22 to 119 μ m2). Thirty-three of the cases (42%) showed positive CCR4 immunostaining. Statistical analysis confirmed that nuclear area of lymphoma cells in PTCL-NOS is correlated with overall survival, whereas immunohistochemical expression of CCR4 is not correlated with overall survival or nuclear area of lymphoma cells. Multivariate analysis confirmed that the nuclear area of lymphoma cells in PTCL-NOS is an independent prognostic factor.

Conclusion:
These results indicate that nuclear area of lymphoma cells in PTCL-NOS is an independent prognostic factor that may predict overall survival. Because PTCL-NOS is a heterogeneous disease with regard to histological type and pathological state, nuclear area of lymphoma cells could be used to stratify patients with PTCL-NOS for therapies, although we are continuing to accumulate data.

Disclosures:
No relevant conflicts of interest to declare.
Absence of Mutations of the Histone Methyltransferase Gene EZH2 In Splenic B-Cell Marginal Zone Lymphoma
Authors: Martínez-Avilés Luz , Salido Marta , Bellosillo Beatriz , Adema Vera , Ferrer Ana , Salar Antonio , Garcia Mar , Espinet Blanca , Florensa Lourdes , Besses Carles , Serrano Sergi , Sole Francesc 
Published: October 2019

Abstract
Abstract 5086

Background
Splenic marginal zone lymphoma (SMZL) is a rare low-grade B-cell lymphoproliferative disorder with characteristic clinical, cytological, histological and immunophenotypical features. The most common cytogenetic abnormality, present in 30–40% of the patients is the 7q deletion, that extends from 7q21 to 7q36. This aberration may represent a primary pathogenic event in SMZL. Recently, mutations in the EZH2 gene, located at 7q36.1, have been described in different hematological malignancies including B-cell lymphomas. However, the role of the EZH2 gene in SMZL has to be elucidated.

Aim
To determine the prevalence of EZH2 mutations in a cohort of SMZL patients.

Patients and Methods
Twenty-nine patients with SMZL were screened for mutations in the EZH2 gene. From the whole cohort, 11 patients presented 7q deletion (three of them as a single anomaly), 11 had a normal karyotype and 7 had other cytogenetic aberrations. The mutational analysis of the EZH2 gene was performed by direct sequencing using primers covering the whole exome of the gene. DNA was extracted from CD19 isolated B-cells from peripheral blood or from total lymphocytes if the percentage of pathologic B-cell was higher than 50%.

Results
From the whole cohort of 29 SMZL patients, no pathogenic mutations (frameshift or nonsense mutations) were detected in the EZH2 gene in any of the patients analyzed. Five patients harboured the missense mutation D185H in exon 6, that has been previously described as a single nucleotide polymorphism (SNP).

Conclusions
In conclusion, the EZH2 gene is not mutated in our series of SMZL patients suggesting that this gene is not involved in the pathogeny of this entity.

Acknowledgments:
Fellowship FI2008 (AGAUR) to LMA, This work was supported (in part) by grants from Instituto de Salud Carlos III FEDER; Red Temática de Investigación Cooperativa en Cáncer (RTICC, FEDER): RD06/0020/0031 and RD07/0020/2004; Ministerio de Sanidad y Consumo (Spain): PI07/0586.

Disclosures:
No relevant conflicts of interest to declare.
Activation of Raf-1 through the Depletion of RKIP by Bcr-Abl Promotes CML Cell Proliferation
Authors: Nakamura Satoki , Takemura Tomonari , Nagata Yasuyuki , Yokota Daisuke , Hirano Isao , Ono Takaaki , Shigeno Kazuyuki , Fujisawa Shinya , Ohnishi Kazunori 
Published: October 2019

Abstract
Abstract 5105

[Background and Aims]
RKIP (Raf kinase inhibitor protein) regulates cell proliferation, apoptosis, and cell cycle through the inhibition of Raf-1, and it has been reported that the suppression of the RKIP expression promotes the proliferation of tumor cells. However, its function is little known in CML. We found that the expression of RKIP gene and protein was suppressed in CML cells. In this study, we have investigated the expression and the function of the RKIP in CML cell proliferation. [Methods] The cells used in this study were human CML cell lines, K562, Meg01, and SHG3 cells. Primary CML cells (ALDHhicells) were obtained from the bone marrow of CML (CP) patients (n=12). Human normal ALDHhi cells were isolated from bone marrow of healthy volunteers after obtaining informed consents. For analysis of RKIP mRNA expression, quantitative RT-PCR was performed in all cell lines treated with Abl kinase inhibitors (STI571, AMN107, and BMS354825). For proliferation analysis and the levels of Erk1/2 phosphorylation in CML cells, MTT assays, western blot and cell cycle analysis were performed in all cell lines transfected with Bcr-Abl, RKIP siRNA, or RKIP cDNA respectively. For colony analysis, the colonies of CFU-GEMM, CFU-GM, and BFU-E were counted in CML stem/progenitor cells transfected with RKIP siRNA or treated with Abl kinase inhibitors. [Results] In CML cell lines treated with Abl kinase inhibitors or transfection with Bcr-Abl siRNA, the expressions of RKIP mRNA and protein were significantly increased more than untreated cells. Moreover, in CML cells transfected with the RKIP siRNA, the cell proliferation inhibition by treatment of the Abl kinase inhibitors was weakened compared to RKIP siRNA-untransfected cells, and the phosphorylation levels of Erk1/2 and Raf-1 were markedly increased compared to theRKIP siRNA-untransfected cells. On the other hands, the overexpression of RKIP induced G1 cell cycle arrest through p27 and p21 accumulation, decreased the phosphorylation levels of ERK1/2 and Raf-1, and inhibited the proliferation in CML cells. In CML stem/progenitor cells obtained from patients with CML, the expression of RKIP mRNA was suppressed in 10/10 (100 %) of CML. The transfection with Bcr-Abl siRNA or treatment with Abl kinase inhibitors increased the expression of RKIP mRNA and protein, and the overexpression of RKIP decreased the counts of CFU-GEMM, CFU-GM and BFU-E. [Conclusion] Our results demonstrated that the Bcr-Abl suppressed the expression of RKIP, the depletion of RKIP induced Raf-1 activation, and induced the proliferation of CML cells through ERK1/2 phosphorylation. Moreover, in CML, the induction of RKIP expression inhibited the proliferation of CML stem/progenitor cells.

Disclosures:
No relevant conflicts of interest to declare.
CDP7657, and Other Anti-CD40L Antibody Formats Lacking a Functional Fc Region, Do Not Cause Platelet Activation In Vitro
Authors: Amirkhosravi Ali , Meyer Todd , Harari Olivier , Burkly Linda , Su Lihe , Hsu Yen-Ming , Bourne Timothy , Wakefield Ian , Peters Christopher , Shock Anthony , Francis John 
Published: October 2019

Abstract
Abstract 5128

Objectives:
Hu5c8, a whole IgG1 anti-CD40L antibody, is known to cause thrombotic side effects in patients with Systemic Lupus Erythematosus (SLE). Anti-CD40L-mediated activation of platelets in vitro was shown to be FcγRIIa-dependent1, suggesting that the adverse effects of hu5c8 could be due to this mechanism, and that anti-CD40L antibodies lacking a functional Fc region would not activate platelets. CDP7657 is a monovalent Fab' PEG anti-CD40L antibody being developed for the treatment of SLE. The aim of the present work was to study a range of anti-CD40L antibody formats in isolated platelet assays and to evaluate the role of the Fc region and of valency.

Methods:
Isolated human platelets from healthy donors were used in the platelet aggregometry and serotonin release assay systems. Antibodies were added either alone, or pre-complexed with soluble CD40L trimer (sCD40L) at optimal stoichiometry. Antibody IV.3 was used to block FcγRIIa. The antibodies and antibody fragments tested included hu5c8 in IgG1, aglycosyl IgG, Fab′, & (Fab′)2 formats, CDP7657, and the CDP7657 variable regions in di-Fab' PEG, tri-Fab' PEG and aglycosyl formats.

Results:
Hu5c8 in complex with sCD40L caused strong aggregation (in 2–3 minutes) and >85% serotonin release, both alone and in complex with sCD40L. This activation was completely abrogated by blocking FcγRIIa. In contrast, platelet activation did not occur with CDP7657 or other antibody formats, in any assay, regardless of the valency or presence of sCD40L.

Conclusions:
The data support the hypothesis that platelet activation by anti-CD40L antibodies is dependent on the presence of an active Fc moiety, via interactions with FcγRIIa, and indicate that CDP7657 has the potential to deliver the therapeutic effect of anti-CD40L blockade in SLE without thrombotic toxicity.
References:(1) Langer F et al. 2005; Thromb Haemost 93: 1137.

Disclosures:
Amirkhosravi: UCB: Research Funding.
Inadequately Regulated Adrenal Steroids through HPA Axis In Chronic Idiopathic Thrombocytopenic Purpura
Authors: Oh Doyeun , Ha Young Wan , Chong So Young , Bang Soo-Mee , Choi Man Ho , Chung Bong Chul 
Published: October 2019

Abstract
Abstract 5130
Idiopathic thrombocytopenic purpura (ITP) is an immune disorder along with the condition of having a low platelet count of multiple etiologies. Endogenous steroids such as corticosteroids have been known to have potent immunomodulatory effect, and also altered expression of steroid metabolism have been reported on the immune related diseases. The level of endogenous steroids are closely related with the level of cytokines (IL-6, IL-1, TNF-α) through HPA axis. Therefore, the eluciadaton of the relationship between steroid hormones and immune cytokines through hypothalamic-pituitary-adrenal (HPA) axis may help to identify biochemical roles of steroid hormones in ITP. The metabolites of steroid hormones were assayed in the plasma obtained from 54 ITP patients and 44 controls using the validated GC-MS based signatures of 70 endogenous steroids including 22 androgens, 18 estrogens, 15 corticoids, 13 progestin, 2 sterols. We also measured the plasma concentration of three HPA axis-stimulating cytokines (IL-6, IL-1, TNF-α) and adrenocorticotropic hormone (ACTH) in chronic ITP patients and healthy subjects using Luminex xMAP technology. Patients were classified to active and remission state depending on the platelet count (active, less than 50,000/mm3; remission, over 100,000/mm3). The active ITP patients showed significant lower plasma levels of adrenal steroids such as pregnenolone (1.49 ± 0.71 ng/mL vs 3.07 ± 1.30 ng/mL, p = 2.3E–08), dehydroepiandrosterone (2.98 ± 1.57 ng/mL vs 4.82 ± 1.67 ng/mL, p = 7.1E-05) and cortisol (73.8 ± 11.3 ng/mL vs 112.7 ± 30.1 ng/mL, p = 2.6E–10) compared with healthy subjects, while the levels of adrenal steroids in remission ITP patients (pregnenolone, 1.87 ± 0.76 ng/mL; dehydroepiandrosterone, 3.70 ± 1.95 ng/mL, cortisol, 83.2 ± 16.2 ng/mL) were not recovered to normal levels. The cortisol/ACTH (ng/mL vs pg/mL) ratio representing pituitary-adrenal axis was not significantly different between patients (4.63 ± 0.72) and healthy subjects (5.17 ± 0.67). However, there was a positive correlation between ACTH and IL-6 in healthy subjects (R = 0.672, p = 0.0024), while no correlation in ITP patients. In conclusion, the low levels of endogenous adrenal steroids resulted from dysregulated HPA axis may have a role in the pathogenesis of ITP.

Disclosures:
No relevant conflicts of interest to declare.