bluuebunny/crossref_metadata_2025_split huggingface dataset:
Outcome of Elderly Patients with Acute Myeloid Leukemia (AML) Post Hypomethylating Agent (HMA) Failure.
Authors: Takahashi Koichi , Kantarjian Hagop M. , Ghanem Hady , Ravandi Farhad , Cortes Jorge E. , Kadia Tapan M. , O'Brien Susan , Pierce Sherry A. , Jain Nitin , Garcia-Manero Guillermo , Jabbour Elias J. 
Published: October 2019

Abstract
Abstract 2627

Background and Aims:
HMA such as 5-azacitadine or decitabine have been increasingly used as induction therapy in elderly patients with AML. The prognosis of such patients who failed initial HMA and the impact of subsequent chemotherapy on outcome are unknown. Therefore the aims of the current analysis are 1) to evaluate the prognosis of elderly patients with AML post HMA failure and 2) to evaluate the efficacy of using intensive cytarabine-based salvage therapy vs. low intensity investigational therapy.

Methods:
We analyzed 54 patients older than 60 years old with AML who received induction treatment with HMA using either 5-azacitadine (n=17; 31%) or decitabine (n=37; 69%) under various clinical trials.

Result:
Median age of the group was 68 years (range; 60–84); 39% patients were female. Forty three patients (77%) had performance status (PS) ≤1 and 11 patients had PS of 2 (23%). Mean initial white blood cell count was 10.9 ± 3.2 (x103/μl), hemoglobin 9.9 ± 0.2 (g/dl), platelet count 77 ± 9 (x103/μl), and bone marrow blast count 44 ± 2.9 (%). Cytogenetic category was classified as intermediate in 30 (55%) patients and poor in 24 (45%) based on the MRC criteria. Six patients (11%) carried FLT3-ITD mutation and 1 carried FLT3 D835 mutation. Complete remission (CR) was achieved in 24 patients (44%) with median numbers of cycles required to achieve CR being 3 (range; 1–6). All patients received further salvage therapies, at the time of failure, with a median of 2.5 (range, 2–5) salvage regimens. As part of the salvage regimen, high-dose cytarabine-based regimen (HDAC) was given to 32 patients (59%). Objective response rate to HDAC was 53%, with 18 pts achieving CR. Median number of courses given was 2 (range, 1–7) with a median duration of response of 3 months. No induction death was reported. Stem cell transplant was performed in total of 9 patients (17%) as a consolidation post HDAC (n=4) or as salvage therapy (n=5). With a median follow-up of 5 months post HMA failure, 5 (9%) patients remained alive receiving therapy. The median overall survival (OS) was 5.4 months (range, 2.3–8.5). The 1-year overall survival rate was 26%. There was no difference in OS between patients who received HDAC versus those who received investigational agents (p=0.25).

Conclusion:
The outcome of untreated elderly patients with AML who failed HMA is poor with a median survival of 5 months. HDAC based regimen, although active with low treatment related morbidity, yielded no survival improvement compared to investigational agents. Such patients should benefit from more innovative approaches.

Disclosures:
Ravandi: Celgene: Honoraria, Research Funding; Eisai: Honoraria, Research Funding.
Early Response to Methotrexate, Vincristine and Procarbazine Chemotherapy-Adapted Strategy for Primary CNS Lymphoma: Omitted Radiotherapy for Patients Achieving Complete Response in Interim Analysis.
Authors: Kim Yu Ri , Kim Se Hoon , Chang Jong Hee , Suh Chang-Ok , Kim Soo Jeong , Kim yun Deok , Hwang Doh Yu , Jang Ji Eun , Hyun Shin Young , Cheong June-Won , Min Yoo-Hong , Kim Jin Seok 
Published: October 2019

Abstract
Abstract 2719
The aim of this study was to evaluate treatment outcomes and prognostic factors of high-dose MVP (methotrexate, vincristine and procarbazine) chemotherapy and response adapted intensification therapy in primary central nervous system lymphoma (PCNSL). We evaluated the expression of Epstein-Barr virus (EBV)-encoded RNA in tissue and pretreatment EBV-DNA in whole blood. Forty patients with PCNSL who treated with high-dose MVP chemotherapy were retrospectively reviewed. Additional radiotherapy or autologous stem cell transplantation were performed for the patients who could not achieve complete response (CR) at interim response (Figure 1). The median age was 55 years. CR rate was 50.0% and objective response rate was 80.0%. Two-year overall survival (OS) was 59.8% and 2-year progression free survival (PFS) was 47.1%. Grade 3 or 4 neutropenia was seen in 19 of 40 (47.5%) patients and thrombocytopenia was developed in 13 (32.5%) patients. Treatment-related mortality was occurred in 4 patients. Delayed neurotoxicity was occurred in 4 patients. Poor performance status, elevated cerebrospinal fluid protein level and whole blood EBV-DNA positivity showed inferior OS (P=0.036, P=0.022, P=0.013, respectively, Figure 2). We detected whole blood EBV-DNA in 10 (29.4%) of 34 patients. It was related with poor OS and PFS in high risk patients according to International Extranodal Lymphoma Study Group scores (P=0.015, P=0.027, respectively). This study suggests that high-dose MVP chemotherapy without intrathecal chemotherapy and response adapted strategy may be an effective regimen for the newly diagnosed PCNSL. Whole blood EBV-DNA positivity could predict the inferior outcome in high-risk patients.
Figure 1: Treatment scheme HD-MVP high dose methotrexate, vincristine and procarbazine, CR complete response, CRu unconfirmed complete response, PR partial response, SD stable disease, PD progressive disease, Tx treatment, NE not evaluated, ASCT autologous hematopoietic stem cell transplantation, RT radiotherapy Figure 1:. Treatment scheme HD-MVP high dose methotrexate, vincristine and procarbazine, CR complete response, CRu unconfirmed complete response, PR partial response, SD stable disease, PD progressive disease, Tx treatment, NE not evaluated, ASCT autologous hematopoietic stem cell transplantation, RT radiotherapy
Figure 2: Overall survival and progression free survival according to EBV-DNA status in high risk patients. EBV Epstein-Barr virus Figure 2:. Overall survival and progression free survival according to EBV-DNA status in high risk patients. EBV Epstein-Barr virus

Disclosures:
No relevant conflicts of interest to declare.
Inherited Biallelic Loss-Of-Function Mutations In CSF3R Define a Novel Type Of Severe Congenital Neutropenia With Full Myeloid Cell Maturation and Refractoriness To RhG-CSF
Authors: Triot Alexa , Järvinen Päivi M , Arostegui Juan I. , Racek Tomas , Puchalka Jacek , Díaz Jose Luis Dapena , Kohistani Naschla , Gertz E. Michael , Schäffer Alejandro A. , de Heredia Rubio Cristina Díaz , Ozdemir Mehmet A. , Patiroglu Turkan , Karakukcu Musa , de Toledo Codina José Sanchez , Yagüe Jordi , Unal Ekrem , Klein Christoph 
Published: October 2019

Abstract
Severe congenital neutropenia (SCN) is a heterogeneous group of disorders characterized by defective production and viability of neutrophil granulocytes and predisposition to life-threatening bacterial infections. Currently, OMIM lists five defined monogenic SCN: SCN1 ELANE, SCN2 GFI1, SCN3 HAX1, SCN4 G6PC3. Here, we describe a novel SCN subtype (SCN6) caused by recessively-inherited loss-of-function mutations in the gene encoding the granulocyte colony-stimulating receptor (CSF3R). We have identified four affected children in two distinct families. Family A had a homozygous missense mutation in close proximity of the highly conserved WSXWS motif (c.922T, p.Arg308Cys) and family B had two compound heterozygous small deletions provoking frameshift mutations (p.Gly316fs and p.Gly415fs). Mutated G-CSFR p.Arg308Cys protein was characterized by perturbed N-glycosylation and aberrant localization to cell surface. G-CSF induced phosphorylation of STAT3 and STAT5 was greatly diminished. In contrast to other SCN subtypes, all patients had morphological evidence of full myeloid cell maturation in bone marrow. However, none of the patients responded to granulocyte colony-stimulating growth factor (GCSF) treatment in vivo, confirming aberrant GCSF-receptor dependent signaling. Our studies highlight the genetic and morphological variability of SCN and provide evidence both for functional importance and redundancy of G-CSFR-mediated signaling in human granulopoiesis.

Disclosures:
No relevant conflicts of interest to declare.
Role Of Histone Deacetylase 6 (HDAC6) In The Modulation Of STAT Pathways and Immune Function Of APCs
Authors: Lienlaf Maritza , Perez-Villarroel Patricio , Cheng Fengdong , Lee Calvin K. , Canales Jorge , Knox Tessa , Marante Danay , Yoder Sean J , Woan Karrune V. , Pinilla-Ibarz Javier , Seto Edward , Sotomayor Eduardo M. , Villagra Alejandro 
Published: October 2019

Abstract
Histone deacetylases (HDACs), originally discovered as histone modifiers are now proposed as important regulators of non-chromatin related processes, including the regulation of cellular pathways involved in the production of anti- and pro-inflammatory cytokines and the subsequent function of antigen-presenting cells (APCs). We have recently identified HDAC6 as a positive regulatory factor in the production of IL-10. However, the participation of this HDAC in other immune related cellular processes remains unknown. In this work we present evidence of the important role of HDAC6 in the modulation of the JAK/STAT pathway through the IL-6 regulation.
We generated knockdown cell lines of HDAC6 (HDAC6KD) and non-target (NT) cells as a control in RAW264.7 murine macrophages using lentiviral shRNA. Two HADC6KD and two NT cell lines were treated with LPS or were left untreated and then analyzed by microarray. In HDAC6KD cells we found 1542 genes were down-regulated and 775 up-regulated in HDAC6KD cells. Their ontology distribution revealed significant changes in immune-related and apoptosis/cell cycle control genes. Importantly, we observed that most STAT3 and SP1 target genes were down regulated in HDAC6KD cells, suggesting the participation of HDAC6 in the regulation of these two transcription factors. Further analysis evidenced that the phosphorylation of STAT3 and the acetylation of Sp1 were diminished in HDAC6KD cells when compared against control cells. Chromatin immuneprecipitacion (CHIP) assays indicate that this particular effect of abrogation of HDAC6 involved histone modifications at the IL-6 promoter level, and more importantly, the recruitment of STAT3 and Sp1 to the IL-6 promoter was abrogated. Then, we analyzed the relevance of these findings by studying the tolerogenic JAK/STAT signaling pathway, which is known to be activated by IL-6 and critical in the final outcome of APCs in response to stimuli. Our observations included a complete abrogation in the phosphorylation of JAK2 and STAT3 proteins in HDAC6KD cells in response to LPS, which was reverted when these cells were treated with exogenous IL-6.
Our final results demonstrate a critical role of HDAC6 in the modulation of IL-6 and the potential role of HDAC6 in the regulation of the JAK/STAT3 pathway. In addition HDAC6 is a regulator of SP1 and STAT3 target genes. These findings provide insight into the molecular mechanisms controlling the immunogenicity of APCs, supporting the use of HDAC6 inhibitors to enhance immune activation, and positioning HDAC6 as a potential therapeutic target.

Disclosures:
No relevant conflicts of interest to declare.
Complement Component 3 (C3) Is Transcriptionally Regulated By TWIST1
Authors: Cho Min Soon , Rupaimoole Rajesha , Sood Anil , Afshar-Kharghan Vahid 
Published: October 2019

Abstract
C3, a central component of the complement system, is a plasma protein that is synthesized in the liver. We have found that malignant epithelial cells are also able to synthesize and secrete C3. The regulation of expression of the C3 gene in malignant cells is not well understood. We found that C3 is highly expressed in various cancer cell lines (Cancer Cell Line Encyclopedia: CCLE) and cancerous human patient tissues (The Cancer Genome Atlas: TCGA). We then explored which transcription factors were responsible for regulating C3 gene expression in ovarian cancer cells. To determine C3 transcription factors, we performed a gene promoter analysis, which identified a TWIST1-binding consensus motif on the C3 gene promoter. The TWIST proteins (TWIST1 and TWIST2) are well known transcription factors are associated with more advanced, invasive and metastatic lesions. There is significant evidence suggesting that TWIST1 promotes tumor progression.
To investigate whether TWIST1 is a transcription factor of C3 in human ovarian cancer cells, we performed a chromatin immunoprecipitation (ChIP) analysis and confirmed TWIST1 binding at the C3 promoter (Figure 1A,1B and 1c). The C3 promoter binding affinity is 5.83-fold higher in Twist antibody used pull down compared to the IgG control (n=3, p=0.001, t-test, Figure 1C). To investigate the functional effect of TWIST1 binding to C3 promoter, we performed a Luciferase reporter gene analysis, showing that a mutant TWIST1 binding site on the C3 promoter decreases luciferase reporter gene activity (control vs. C3 vs. mC3=1 vs. 3.3 vs.1.8, n=3, p=0.01, t-test, Figure 1D and 1E).
Figure 1 C3 is a target gene of TWIST1. Figure 1. C3 is a target gene of TWIST1.
Next, we manipulated expression of the Twist1 gene in ovarian cancer cells and monitored its effect on C3 gene expression. Small interfering RNAs (siRNA) against Twist1 drastically reduced C3 expression (n=3, p=0.01, t-test, Figure 1F); on the other hand, transducing these cells with lentivirus containing Twist1 increased C3 expression (n=3, p=0.01, t-test, Figure 1G). Therefore, we conclude that there is a positive correlation between expression of C3 and TWIST1 in ovarian cancer cells.
Taken together, these data provide evidence that a novel transcription factor of C3, TWIST1, upregulates C3 expression in malignant cancer cells, leading to an increase in cell proliferative potential.

Disclosures:
No relevant conflicts of interest to declare.
Histone Deacetylase 6 (HDAC6) Influences T-Cell Activation and Survival: Implications For Cancer Immunotherapy
Authors: Laino Andressa Sodre , Woods David M , Cheng Fengdong , Wang Hongwei , Sotomayor Eduardo M. 
Published: October 2019

Abstract
The role of histone deacetylases (HDACs) as epigenetic regulators of immune function is becoming increasingly clear. Recently, the role of specific HDACs in orchestrating T-cell maturation, survival and function has begun to emerge, giving rationale to selective therapy to direct immune responses in different disease settings, including cancer. In particular, HDAC6 has recently been characterized as a negative regulator of regulatory T-cell suppressive activity (de Zoeten, Molecular and Cellular Biology, 2011). Here we report an expanded, novel role of HDAC6 in regulating T-cell survival and activation. First, the relative expression of the eleven classic HDACs was evaluated in resting and activated T-cells from mouse and human samples. It was found that the majority of HDACs decrease in expression following activation, including HDAC6. Next, in a HDAC6KO mouse model, it was found that T-cells lacking HDAC6 had skewed survival when compared to wild-type murine T-cells. This difference seems to be the result of an increased CD4+ T-cells population in the lymph nodes, with a concomitant decrease in viable CD8+ T-cells. To determine whether this population skewing was the consequence of defects in HDAC6KO mice T-cell development, wild-type murine T-cells were treated with an isotype-selective HDAC6 inhibitor. The results seen in HDAC6KO T-cells were recapitulated when wild-type T-cells were activated and treated with HDAC6 specific inhibitors, indicating a role of HDAC6 outside of thymic development in promoting CD4+ T-cell survival at the expense of CD8+ T-cells. Interestingly, it was found that activated CD4+ T-cells displayed decreased expression of the apoptosis signaling receptor FAS after HDAC6 inhibition while no differences were observed in CD8+ T-cells under the same conditions. In addition to these results implicating HDAC6 in regulating T-cell survival, expression of surface markers was altered in both CD8+ and CD4+ T-cells, including enhanced expression of the activation molecule CD69 in stimulated T-cells treated with an isotype-selective HDAC6 inhibitor. Finally, in vivo studies in tumor-bearing HDAC6KO mice revealed a significantly delayed in tumor progression. Similar results were observed in lymphoma-bearing mice treated with HDAC6 specific inhibitors. Taken together, this data shows that HDACs are dynamic in expression with regards to T-cell activation state. More specifically, we have unveiled hereto-unexplored roles of HDAC6 in regulating T-cell survival and function, pointing at this specific HDAC as an appealing target to harness T-cell immunity in hematologic malignancies.

Disclosures:
No relevant conflicts of interest to declare.
The Role Of Intraplatelet Reactive Oxygen Species In The Regulation Of Platelet Glycoprotein Ibα Ectodomain Shedding
Authors: Zhang Pingping , Du Juan , Zhao Lili , Wang Xiujuan , Zhang Yiwen , Yan Rong , Dai Jin , Liu Guanglei , Zhang Feng , Dai Kesheng 
Published: October 2019

Abstract

Background
Glycoprotein (GP) Ibα contains binding sites for von Willebrand factor (VWF), α-thrombin, P-selectin, and Mac-1 at the extracellular N-terminal 282 residues. Particularly, the interaction of GPIbα with VWF exposed at the injured vessel wall initiates platelet adhesion, and simultaneously triggers intracellular signaling leading to integrin activation and platelet thrombus formation. Therefore, GPIbα ectodomain shedding, which down-regulates the surface expression of the functional receptor and results in the generation of glycocalicin (GC), a soluble N-terminal fragment of GPIbα, has important implications for thrombosis and hemostasis. Stimulations of platelet with either physiological or chemical compounds result in GPIbα ectodomain shedding in vitro and in vivo. The generation of ROS has been observed in platelets stimulated with physiological or chemical compounds, such as collagen, thrombin, and thromboxane A2 analog U46619. In this paper, we plan to clarify the relationship between physiological stimulation-induced ROS generation and GPIbα shedding and the regulatory mechanism of ADAM17-mediated GPIbα shedding.

Methods
Washed platelets (3×108/ml) were pre-incubated with or without N-acetylcysteine (NAC) (10 mM), dithiothreitol (DTT) (3 mM), or vehicle control (DMSO) at RT for 15 minutes, and then were incubated with A23187 (5 μM), thrombin (1 U/ml), collagen (5 ug/ml), or dibucaine (1 mM) at 37 °C (or at RT for dibucaine) for different time. Western blot and flow cytometry was used to evaluate the GPIbα ectodomain shedding. ROS levels in platelets were examined using ROS assay kit according to the manufacture’s instruction.

Results and conclusions
Collagen, thrombin, and calcium ionophore A23187 induced ROS generation, and simultaneously incurred GPIbα ectodomain shedding. ROS scavengers NAC and DTT abolished not only collagen, thrombin, and A23187 induced ROS production, but also GPIbα ectodomain shedding. Interestingly, a recognized calpain activator, dibucaine, induced both ROS production and GPIbα shedding, which were also obviously reduced by NAC and DTT. Furthermore, calpain inhibitors calpain inhibitor I and carbobenzoxy-valinyl-phenylalaninal (MDL), obviously reduced dibucaine-induced ROS production, and inhibited ROS generations induced by A23187 and thrombin. These data indicate that ROS, regulated by calpain, plays a key role in collagen, thrombin, and A23187 induced GPIbα ectodomain shedding. These findings will help to understand the negative-regulatory mechanisms of platelet function, and may suggest a novel strategy to design new class of anti-platelet drug.

Disclosures:
No relevant conflicts of interest to declare.
Cooperative Binding Of Anti-Factor VIII Inhibitors and Induced Conformational Change Detected By Hydrogen-Deuterium Exchange Mass Spectrometry
Authors: Meeks Shannon L. , Sevy Alexander M. , Healey John F. , Deng Wei , Spiegel P. Clint , Li Renhao 
Published: October 2019

Abstract
The development of anti-factor VIII (fVIII) antibodies (inhibitors) is a significant complication in the management of patients with hemophilia A leading to significant increases in morbidity and treatment cost. Using a panel of anti-fVIII monoclonal antibodies to different epitopes on fVIII, we recently have shown that epitope specificity, inhibitor kinetics, and time to maximum inhibition are more important than inhibitor titer in predicting response to fVIII and the combination of fVIII and recombinant factor VIIa. Thus the ability to quickly map the epitope spectrum of patient plasma using a clinically feasible assay may fundamentally change how clinicians approach the treatment of high-titer inhibitor patients. To this end, we have characterized the binding epitopes of 4 monoclonal antibodies (MAbs) targeted against fVIII C2 domain by hydrogen-deuterium exchange coupled with liquid chromatography-mass spectrometry (HDX-MS). MAbs included both classical (inhibiting binding of fVIII to von Willebrand factor and phospholipid) and non-classical inhibitors (inhibiting activation of fVIII), which target separate regions of fVIII C2 domain and have distinct inhibitory mechanisms. HDX-MS analysis showed clear differences in binding patterns between classical and non-classical inhibitors, centering on the protruding hydrophobic loop at Met2199. The binding epitopes of classical and non-classical inhibitors mapped by HDX-MS agree well with previously reported ones characterized by structural studies and mutagenesis analysis. Classical and non-classical inhibitors could be distinguished by a limited subset of C2-derived peptides, simplifying analysis significantly. In addition, HDX-MS was able to detect subtle differences in binding patterns of various classical inhibitors, based on the HDX protection pattern around the hydrophobic loop at Leu2251. Interestingly, two MAbs, G99 and 3E6, exhibited an observable shift in HDX protection when bound to C2 as a ternary complex, as opposed to when bound individually, thus providing evidence for cooperative binding of these two MAbs (Figure 1). In summary, our results demonstrate the effectiveness and robustness of the HDX-MS method in the rapid epitope mapping of fVIII inhibitors. This method can be expanded to map epitopes of inhibitors against other domains of fVIII, potentially leading to a more personalized treatment of hemophilia A patients.Figure 1Figure 1.

Disclosures:
No relevant conflicts of interest to declare.