bluuebunny/crossref_metadata_2025_split huggingface dataset:
Induced Transdifferentiation of Leukemia B-Cells to Macrophages Involves Reconfiguration of the DNA Methylome
Authors: Bueno-Costa Alberto , Piñeyro David , Soler Marta , Javierre Biola Maria , Martínez-Climent José Angel , Esteller Manel 
Published: October 2019

Abstract
The epigenomic changes that occur during the process of cellular differentiation, such as in the blood cell lineage, are currently not well understood, especially at distant regulatory regions such as enhancer sequences. To study the effects of DNA methylation on cellular (de)differentiation, we have used a human B Cell Acute Lymphoblastic Leukemia model of transdifferentiation (BLaER1), which has an estradiol-inducible CEBPA construct that allows the conversion of leukemic B cells to non-tumorigenic macrophage-like cells. By analyzing the DNA methylation landscape of these cells at different time points of transdifferentiation with an Illumina EPIC methylation array, we have found enhancer-associated CpGs that shifts their methylation levels at the end of the transdifferentiation. By merging these results with the data obtained by Genome-wide Chromosome Conformation Capture Capture (Hi-C) in naive B cells and macrophages, we studied the putative interaction between several gene-promoters and our differentially methylated CpGs. We then proceed further to characterize the impact of the observed interactions on gene expression. We have identified the DNA methylation dependent enhancer interactomes of B-cells and macrophages. These target genes are related with vesicle trafficking, endocytosis and immune response. Our data highlight the role of DNA methylation to determine cell identity in the blood cell lineage.

Disclosures
No relevant conflicts of interest to declare.
Autoimmune Hemolytic Anemia and Checkpoint Inhibitors: 68 Cases from the FDA Database and Critical Review
Authors: Tanios Georges , Doley Peter B , Munker Reinhold 
Published: October 2019

Abstract
Immune checkpoint inhibitors (CPI) are widely used in modern oncology and have improved the prognosis of lung cancer, bladder cancer, malignant melanoma and other malignancies. Unlike cytotoxic chemotherapy, drugs like nivolumab, pembrolizumab and ipilimumab are associated with a high frequency of immune-related adverse effects. We recently observed a patient with lung cancer who developed a fulminant warm-antibody autoimmune hemolytic anemia (AIHA) and reviewed the literature and public databases of the Food and Drug Administration (FDA) to help understand the association between CPI use and AIHA. A total of 68 cases of AIHA were identified in the FDA database during the time period 2012-2018; 43 associated with nivolumab, 13 with pembrolizumab, 7 with ipilimumab and 5 with atezolizumab administration (see Table 1). All episodes of AIHA were listed as serious. If the total number of adverse effect cases reported to the FDA is taken as a reference, AIHA is rare, but occurred more frequently with PD-1 or PDL1 targeting agents (0.15 to 0.25%) than with CTLA4-inhibitors (0.06%). The underlying cancer diagnoses corresponded mostly to the approved indications for CPI (32 cases of malignant melanoma, 24 cases of lung cancer). In about a quarter of cases with AIHA other immune related side effects were reported. In addition to our case, the literature review identified 10 similar cases. AIHA can occur earlier and later after the administration of CPI (median of 10 weeks, range 2- 78). Most cases of AIHA responded to steroids, but 2/11 were fatal. In conclusion, we describe AIHA as a rare and serious immune-related side effect of checkpoint inhibitors. Because of its seriousness and the underlying comorbidities, early aggressive management is necessary.

Disclosures
No relevant conflicts of interest to declare.
Development of a Pegylated Dimeric Recombinant Factor VIII-Fc Fusion Protein for the Treatment for Hemophilia a Patients
Authors: Liu Bin , Wang Xiaoshan , Li Xueqin , Yan Haixia , Wang Shuya , Zhu Xi , Wang Pengju , Gao Jie , Wang Yali , Su Hongsheng 
Published: October 2019

Abstract

Hemophilia A is a hereditary bleeding disorder resulting from reduced factor FVIII activity. It occurred in 1/5000 male. Currently, the treatment option is with the factor FVIII replacement therapy. A long-acting recombinant monomeric FVIII-Fc fusion protein product (Eloctate®) has been approved in 2014 by the US FDA, it requires to infuse the drug for every 3 days or twice a week. There is a clinical need to develop longer half-life product to extend the treatment option to once a week infuse for hemophilia A patients. Recently, we have developed a dimeric recombinant factor VIII-Fc (drFVIII-Fc) fusion protein therapeutic candidate, which is entering the clinical development in China. To generate a longer half-life recombinant FVIII product, we have developed a PEGylation method to PEGylated this drFVIII-Fc fusion protein to PEGdrFVIII-Fc. We have analyzed and characterized the fusion protein by various analytic methods, as well as in vivo animal tests. It was shown that PEGdrFVIII-Fc fusion protein has been modified with about five Y type of 40kd PEG; the remaining activity is around 700 IU/mg, and the in vivo tests in cynomolgus monkey demonstrated that the fusion protein has a half-life of about 37 hours. The data also showed that there was no detectable affinity binding activity of vWF to a PEGdrFVIII-Fc fusion protein, as compared with the binding activity of 5.16X10-4M for the molecule of a drFVIII-Fc fusion protein. In conclusion, we are able to generate a PEGylated form of a drFVIII-Fc molecule with the relevant specific activity and has been shown the molecule with the prolonger half-life in the in vivo tests. The further biochemical analysis demonstrated PEGdrFVIII-Fc fusion protein with no detectable vWF binding activity, which might explain why its half-life is longer than vWF's ~15hours half-life in vivo. This molecule is likely to be used as a once-weekly treatment option for hemophilia A patients. Currently, we are in the development stage of an IND filing in China.

Disclosures
No relevant conflicts of interest to declare.
Defective Autophagy Process and Increased ROS Formation in Primary Myelofibrosis
Authors: Shi Guanfang , Kodali Sreenath , Wong Ching , Gotlieb Vladimir K. , Wang Jen-Chin 
Published: October 2019

Abstract
Introduction: We and others have reported markedly increased ROS formation in primary myelofibrosis (MF) including post-ET or PV-MF when compared with essential thrombocytosis (ET), polycythemia vera (PV) and normal controls. A growing body of evidence illustrates the interdependency between the Keap1-Nrf2 pathway and p62-mediated selective autophagy, are playing an important role in major cellular defense mechanisms against oxidative and electrophilic stress ; dysregulation of p62-Keap1-Nrf2 axis has been implicated in tumor development. Therefore, we studied p62 mediated autophagy and Nrf2 pathways in patients with MF ,ET and PV.
M aterials and Methods :33 patients including 15 patients with MF, 7 patients with PV, 11 patients with ET and 10 normal controls were studied. Cellular ROS measurement: Cellular ROS was determined by a dichlorofluorescein (DCF) assay. The oxidation of H2DCFDA was measured by flow cytometry. Real-time PCR assay: Total RNA was extracted from normal control or patient mononuclear cells using RNeasy Kits from Qiagen (Germantown, MD). Real-time PCR was performed using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA) on Bio-Rad iQ5 Multicolor Real-Time PCR Detection System. Western blotting: Total cell lysates were obtained from normal control or patient mononuclear cells. Primary antibodies against Beclin-1, LC3 and p62 were purchased from Cell Signaling Technology (Danvers, MA).
Results: Fig.1a shows significantly elevated ROS in MF (2127±261.6) when compared with PV (1090 ± 163.8) or ET (1031 ± 140.9) or controls (549.1 ± 52.50). Fig.1b. shows that Nrf2 protein levels (ratio to controls) was significantly reduced in MF (41.84 ± 29.33), PV (14.42 ± 2.96), ET (14.05 ± 3.02), than the controls ( 101.3 ± 7.64). Fig.1c shows that the RT-PCR values of p62 was significantly increased in MF (1.34 ± 0.06) and grouped PV (1.21 ± 0.07), ET (1.14 ± 0.08) and MF as MPN (1.27 ± 0.04) than controls (1.00 ± 0.05),( P<0.05); ET or PV were not significantly elevated than controls.
Conclusions: Patients with MF had increased levels of ROS when compared with PV and ET while Nrf2 levels were significantly reduced in all three( ET,PV, MF) which implies defective detoxification process in MPNs. P62, a marker for defective autophagy was significantly increased in MF than controls . This implies that there is a defective autophagy process in MF, contributing further to the formation of P62 and ROS. There is also a defective detoxification mechanism which further leads to more ROS formation and further DNA damage. Further studies are in progress on other autophagy markers which will enlighten more about the pathophysiology of MF.

Disclosures
Wang: INCYTE: Other: clinical trial.
Human Double-Negative Regulatory T Cells Modulate Effector Functions of Conventional T Cells By Selectively Blocking mTOR Signaling
Authors: Haug Tabea , Aigner Michael , Bruns Heiko , Peuser Moritz , Gebhardt Dorothea , Berger Jana , Koppitz Florentine , Mackensen Andreas , Voelkl Simon 
Published: October 2019

Abstract
Regulatory T cells play an important role in the maintenance of immune tolerance to self-antigens and are involved in modulating immune responses to promote resolution of inflammation. The population of TCRαβ+ CD4-/CD8- (double-negative, DN) T cells has attracted growing attention as a result of their potent immune regulatory function. In murine models, DN T cells were able to prevent rejection of allogeneic and xenogeneic organ grafts by effectively suppressing reactive T cells. In addition, DN T cells possess the capacity to resolve various inflammatory conditions, including graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation. Notably, first clinical studies in patients after stem cell transplantation demonstrated an inverse correlation between the frequency of circulating DN T cells and the severity of acute GvHD, suggesting a therapeutic potential of human DN T cells. To gain a better understanding of the molecular mechanism of suppression, we investigated whether human DN T cells modulate distinct signaling processes in conventional T cells. We found that DN T cells selectively block mTOR signaling but not activation of mitogen-activated protein kinases. Enforced activation of the mTOR pathway by a chemical activator rendered conventional T cells unsusceptible to DN T cell-mediated suppression, confirming the critical function of mTOR signaling. Given that mTOR is a major regulator of cellular metabolism, we further determined the impact of DN T cells on the metabolic framework of conventional T cells. Of interest, DN T cells diminished upregulation of the glycolytic machinery and glucose uptake in conventional T cells, whereas fatty acid uptake was not modified. Next, we investigated the fate and function of effector cells after DN T cell co-culture. Of importance, DN T cells suppressed proliferation but also altered expression of differentiation markers, transcription factors and homing receptors. Further analyses demonstrated that CD4+ T cells failed to produce effector cytokines IL-17 and IFN-γ after coculture with DN T cells, whereas IL-2 secretion was amplified. The selective modification was induced by a direct cell-cell contact dependent mechanism between CD4 and DN T cells and not as a consequence of competition for nutrients or growth factors. Together, our findings expand the understanding of DN T cell functionality and support that human DN T cells represent an interesting opportunity to limit and modulate T-cell reactivity.

Disclosures
No relevant conflicts of interest to declare.
Pegaspargase with Concurrent Radiotherapy in a Once-Every-Three-Weeks Schedule in Patients with Newly Diagnosed, Stage IE to IIE, Nasal, Extranodal NK/T-Cell Lymphoma: A Phase II Study
Authors: Wang Hua , Luo Wei , Lu Yue 
Published: October 2019

Abstract
Introduction: Extranodal natural killer/T-cell lymphoma (ENKTL) is an aggressive form of non-Hodgkin's lymphoma,which is relatively more common in Asia and Latin America.Radiation therapy(RT) is widely administered for patients with localized nasal disease. However, local and systemic failures are observed frequently in patients who receive RT alone.Therefore, chemotherapy is needed in combination with RT to reduce the risk of recurrence. Concurrent chemoradiotherapy (CCRT) is expected to improve both local and systemic disease control and has been established as a standard therapy for several types of solid tumors. Therefore,we conducted a phase II study of pegaspargase-based CCRT to explore a more effective treatment for localized nasal natural killer (NK)/T-cell lymphoma.
Methods: Enrolment began in May 2016 for a phase II single-center clinical trial. Chemotherapy and RT were simultaneously started within 7 days after registration. The drug administration schedule were as follows: day1,deep intramuscular injection of 2000 unit/m2 pegaspargase at three different sites. Chemotherapy was planned to repeat every 3 weeks. Pegaspargase were administered only if the plasma fibrinogen level was greater than 0.75g/L. Six courses of chemotherapy were planned.All patients received three-dimensional (3D) conformal radiation therapy by using 4 or 6MV photons generated from a linear accelerator. The involved-field radiation (IFRT) dose was 50 Gy, which was given as 2.0 grays (Gy) per daily fraction with 5 weeks.
Results: Until May 2, 2018, we have completed the enrolment of 12 patients with newly-diagnosed untreated ENKTL. The main clinical characteristics of the 12 patients are presented in Table 1. All patients (n=12) have begun treatment and are evaluable for response. Of the 12 patients, complete remission (CR) rates is 100% to concurrent chemoradiotherapy alone. None of patients has experienced disease progression until now. Toxicities are recorded as the number of patients experiencing a certain adverse event.The most common grade 1-2 non-haematological (non-heme) adverse effects (AEs) are fatigue (n=2), nausea (n=1),oral mucositis (n=1) and increased transaminases (n=1).Eight (n=8) patients were confirmed to have decreased fibrinogen. Most of these were grade 1/2 and no bleeding or other adverse events were observed.There was no grade 3/4 non-heme toxicities during treatment. Grade 1-2 hematological (heme) AEs included neutropenia (n=6), anemia (n=5) and thrombocytopenia(n=2).Grade3-4 haematological AEs was anemia (n=1). No patients interrupted treatment for severe adverse events and no treatment-related deaths occurred.
Conclusions: Preliminary data indicate that pegaspargase with concurrent radiotherapy in newly diagnosed ENKTL patients was efficacious and well-tolerated, while the long-term outcome is required to be followed up.

Disclosures
No relevant conflicts of interest to declare.
Venous Thromboembolic Disease and Bone Marrow Transplant: A Remarkable Cause of Mortality and Morbidity — Potentially Preventable
Authors: Rivas Luque Marta , Morente Constantin Estefanía , Romero Garcia Pablo , Garcia-Ruiz Maria Almudena , Jurado Manuel 
Published: October 2019

Abstract
Thrombotic events are frequent in patients undergoing HSCT, being an important cause of morbidity and mortality. The highest incidence occurs three months after the transplant. There are several risk factors, which add to those already known for VTE: neoplasia, central venous catheters, immobilization, chemotherapy, infections, GVHD. In the series described, the frequencies are variable, between 0.5 and 23.5%, with an overall incidence of 5%. In patients with GVHD, this incidence increases, with up to 35% of events.
METHODS
A retrospective observational study that includes patients transplanted in our Unit between 2014 and 2017 has been conducted, with the objective of analyzing the incidence of VTE peri-TPH. Likewise, we have analyzed if it is associated to catheter, presence of CVRF, if there was a known medical history of thrombophilia, number of platelets at time of thrombosis, the heparin used and whether anticoagulation was maintained indefinitely or not.
RESULTS
Out of the 235 patients included in our series, 130 underwent an autologous transplant and 105 an allogeneic transplant. 18 thrombotic events occurred (9 men and 9 women, aged between 18 and 65 years), which means 7.5% (14 occurred between days 0-100, 12 in patients undergoing autologous hematopoietic stem cell transplantation). Three of them had thromboembolism and the rest deep vein thrombosis, 4 of which with catheter. The platelet count at the time of the event ranges from 21 to 409,000 / mm3. Regarding the heparin used, 2 were treated with Tinzaparin and the rest with Bemiparin. Only 1 of the patients presented prior VTE. Among the patients, there were some with CVRF and others without relevant medical history. Just in one patient, a family thrombophilia study had been performed prior to his hematological diagnosis, resulting in a deficit of protein S. In 8 of the patients, anticoagulation was maintained indefinitely with LMWH in the post-transplant period.
CONCLUSIONS
Our incidence approaches the literature, albeit the series of published cases are heterogeneous and with variable differences. Although the incidence of thrombosis in these patients is a cause of marked morbidity and mortality, the risk of bleeding also increases, therefore routine prophylaxis is not recommended in all patients. We must undergo an exhaustive analysis of the data to identify individually which patients may be candidates for prophylaxis, with the aim of reducing the incidence without raising the hemorrhagic risk of our patients.

Disclosures
No relevant conflicts of interest to declare.
Very High Efficacy of Bendamustine, Gemcytabine and Dexamethasone (BGD) in Relapsed/Refractory Classical Hodgkin Lymphoma
Authors: Swoboda Ryszard , Najda Jacek , Dulik Katarzyna , Kwiatkowska-Pamula Anna , Twardosz Maja , Giebel Sebastian 
Published: October 2019

Abstract
Introduction. Although Hodgkin lymphoma (HL) is one of the most curable malignancies, 10-30% of patients experience resistance or relapse following initial response. Treatment options for relapsed/refractory (R/R) disease include usually multi-agent chemotherapy followed by autologous hematopoietic stem cell transplantation (autoHSCT). Although many chemotherapy-based regimens are used as salvage, the optimal one has not been defined so far. The goal of this study was to evaluate efficacy and safety of bendamustine in combination with gemcitabine and dexamethasone (BGD) in R/R HL.
Patients and Methods. This was a retrospective, single center study, including 30 consecutive R/R HL patients treated between 2016 and 2018. Median age was 33 (19-82) years. Ten patients (33%) had been pre-treated with at least 2 lines of chemotherapy; 11 patients (37%) with radiotherapy, 6 patients (20%) with autoHSCT while 4 patients (13%) with allogeneic (allo)HSCT. Six patients (20%) were resistant to previous salvage treatment.
BGD therapy consisted of bendamustine 90mg/m2 on days 1 and 2, gemcitabine 800 mg/m2 on days 1 and 4, dexamethasone 40 mg on days 1-4. Best response after at least 2 courses of therapy was the primary study end-point.
Results. Median number of BGD cycles was 4 (2-7). Nineteen (63%) patients achieved complete remission (CR) confirmed by FDG-PET, while 6 patients (20%) achieved partial response (PR), accounting for 83% overall response (OR) rate. Stable disease was reported in 3 patients and progressive disease - in 2 cases. Many patients achieved CR (33%) or PR (33%) after 2 cycles of BGD. The OR rate was 85% for patients in whom BGD was administered as first salvage and 80% when used in more advanced stages of the disease. Fourteen patients proceeded to autoHSCT while 4 patients were subsequently treated with alloHSCT.
Six patients experienced infections of the upper respiratory tract while two patients had allergic skin reactions. No organ toxicity was reported. One patient required transfusion of red blood cells while none was treated with platelet transfusions.
Conclusion. BGD is a highly effective, well-tolerated salvage regimen for patients with R/R HL. The protocol may be used as a bridge to autoHSCT or alloHSCT. Further, prospective studies are warranted to define its role in future treatment algorithms.

Disclosures
No relevant conflicts of interest to declare.
Hedgehog Pathway Inhibitor for Treatment of Steroid-Refractory Sclerodermatous Chronic Graft-Versus-Host Disease (GVHD)
Authors: Gupta Ridhi , Albabtain Abdulwahab , Arai Sally , Kwong Bernice , Weng Wen-Kai 
Published: October 2019

Abstract
Background: Sclerodermatous chronic GVHD (scGVHD) is a unique form of cutaneous GVHD that predominantly involves the skin, subcutaneous tissue, fascia and joints. Steroid-refractory scGVHD is especially challenging to manage since it is usually a progressive condition with devastating effects on the quality of life of affected patients. Inhibition of hedgehog signaling has been shown to be effective in the treatment of murine scGVHD. Here, we report a retrospective review of patients with scGVHD who were treated with hedgehog pathway inhibitor, Vismodegib, at Stanford University.
Results: A total of n=8 patients with steroid-refractory scGVHD were treated with Vismodegib 150 mg daily as an off-label use since 2016. The male:female ratio is 5:3 and median age was 49 (range 39-62) at time of start of Vismodegib. The median time from transplant to diagnosis of scGVHD was 26.9 months (range 8.9-37.5) and the median time from diagnosis of scGVHD to Vismodegib treatment was 14.4 months (range 2.7-45.2). All patients had GVHD involvement of other organ besides scGVHD and have received multiple lines of therapy prior to Vismodegib.
Hair thinning/alopecia and muscle cramps were noted in 100% of the patients. 75% (6/8) had dysgeusia, 88% (7/8) had fatigue (gr 2) and 1 patient had an asymptomatic elevated lipase level. All patients required dose reduction or drug holiday due to side effects (mainly muscle cramps). Four patients (50%) stopped the drug due to side effects (2/4) or lack of response (2/4). All Vismodegib-related side effects resolved after discontinuation of the drug. The median duration of Vismodegib treatment was 6.1 months (range 2.3-17.1) for the entire group. One patient received multiple courses of Vismodegib with benefit noted with each course.
Five patients (63%) had partial response including 1 patient responded to multiple courses of Vismodegib and 1 patient had subjective symptom improvement. The median time to clinical response was 103 days (range 24-145) and the duration of response was 7.8 months (range 1.6-13.4). Three patients had experienced prolonged clinical response off the Vismodegib. At the last follow-up visit, all patients were alive and 4 patients continued to take Vismodegib including 1 patient who had been tapered off systemic steroid.
Conclusion: Steroid-refractory scGVHD is an extremely difficult disease to manage with a high rate of morbidity. Our experience with hedgehog pathway inhibitor, Vismodegib, is very encouraging. Additional study to evaluate the optimal dosing, side effects and its efficacy is under way.

Disclosures
No relevant conflicts of interest to declare.