bluuebunny/crossref_metadata_2025 huggingface dataset:
Positive Impact of Chronic Graft-Versus Host Disease on Survival in 928 Allogeneic Hematopoietic Stem Cell Transplant Recipients
Authors: Gurman Gunhan , Ataca Pinar , Atilla Erden , Civriz Bozdag Sinem , Toprak Selami Kocak , Yuksel Meltem Kurt , Demirer Taner , Akan Hamdi , Arslan Onder , Topcuoglu Pervin , Beksac Meral , Ilhan Osman , Ozcan Muhit
Published: October 2019

Introduction:

Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is a standard curative treatment option in hematological diseases. The anti-leukemic activity relies not only the effects of conditioning regimen but also the immune mediated graft-versus leukemia (GVL) effect. Donor T cells are responsible for the GVL and causes complications namely acute and chronic graft versus host disease (GVHD). Herein, we aim to present the relation of graft versus host disease with survival and relapse.

Patients and Methods:

We retrospectively evaluated 928 Allo-HSCT between 1989 and 2015 followed in our institution. Chi-square test and student's t test were used in comparison. P <0.05 was considered statistically significant.

Results:

551 patients were male (59%) and 377 patients (41%) were female. The median age of the group was 34 (range 15 -71). Patients received stem cell from related donors more frequently (85%) with HLA full match (66%). Peripheral blood was the source of stem cells in 645 recipient (70%) followed by bone marrow (28%). 75 patients were diagnosed as benign hematological disorders. The most common malign hematological diagnosis was acute myeloid leukemia in 366 patients (39%). Patients received 80% of myeloablative conditioning regimen prior to transplantation. 43% of the patients diagnosed as acute GVHD and 45% had chronic GVHD. 798 achieved engraftment (86%) neutrophil engraftment median of 16 days, platelet engraftment median of 14 days. Relapse was detected in 6% of patients. The overall survival (OS) was 59 months and progression free survival (PFS) was 33 months. Acute GVHD had no impact on OS, PFS, relapse rate. The OS in patients with chronic GVHD was significantly higher than in GVHD negative patients (68.8 vs 56.9, P=0.009). Although chronic GVHD patients had lower risk of relapse and higher PFS, the results were not statistically significant.

Conclusion:

GVHD is a serious complication and important cause of post transplant morbidity. Chronic GVHD is associated with lower risk of relapse in previous studies. In this study, we have concluded the improved OS in patients with chronic GVHD. The immune mediated GVL effect may be the reason for anti-leukemia effect and improved survival.

Disclosures

No relevant conflicts of interest to declare.
The Light Chain of Rat Factor VIII Confers Its Superior Cofactor Activity
Authors: Wang Qizhao , Firrman Jenni , Pokiniewski Katie A , Cao Wenjing , Wei Hongying , Herzog Roland , Xiao Weidong
Published: October 2019

Hemophilia A is caused by genetic defect of human coagulation factor VIII (hFVIII) and patients have to take lifelong replacement therapy to prevent excessive bleedings upon hemostatic challenges. Due to the short half-life of hFVIII, replacement treatment has to be given frequently and inhibitors against infused hFVIII can be developed in about 20-30% of patients. These shortcomings have generated tremendous interest in developing HA gene therapies which is more efficient and long-lasting. However, early preclinical studies have shown FVIII activities were still limited after vector delivery. A Modified hFVIII with higher specific activity and pharmacodynamics properties is highly desirable to overcome the disadvantages of current protein replacement and gene therapy strategies.

In the current study, we successfully constructed a B-domain deleted rat FVIII(rBDDF8) that contained a PACE/furin recognition site (RHQR) within a 14 amino acid linker between A2 and A3 domains. The rBDDF8 displayed significantly higher coagulation activity(~2.5-fold) than hBDDF8 after transfection into HEK 293 cells. In order to explore the mechanism for the observed superior cofactor activity, we constructed heavy chain(rHC) and light chain(rLC) of rFVIII. The rHC and rLC are able to reconstitute 5 times more FVIII activity than their human counter parts. However, when rHC is associated with human FVIII light chain (hLC), the reconstituted FVIII activity is lower that from hHC and hLC, suggesting that high coagulation activity of rFVIII is not mediated by its HC. On the contrary, when FVIII is constituted by hHC with rLC, we found that the activity is increased by 3~5-fold as against hHC and hLC. The hHC antigen level of FVIII reconstituted from hHC and rLC was 1.5-fold higher than that of hHC and hLC, suggesting that higher activity of FVIII with hHC and rLC is not through increased secretion. The specific activity deduced from activity/antigen ratio showed that FVIII with rLC is 3 times higher more than FVIII with hLC. To investigate the potential application of rFVIII in gene therapy, rBDDF8 was delivered in hemophilia A mouse model using AAV8 vectors. The high dose rBDDF8(4*1011 vg/mouse) resulted 2.5U FVIII activity at week 17, which is much higher(about 10-fold) than that of hBDDF8. When the rFVIII was delivered by dual chains strategy, i.e, administering vectors carrying only LC or HC simultaneously, it also showed 2-4 fold increased in FVIII activity. Interestingly, the combination of hHC and rLC also generated similar FVIII activity as rHC and rLC, further proving the rLC is the major contributor to the superior coagulation activity of rFVIII. Our results showed that the rFVIII has higher cofactor activity conferred by its LC. Our results suggest that rFVIII can be further exploited to make an ideal candidate for hemophilia gene therapy using AAV vectors.

Disclosures

No relevant conflicts of interest to declare.
Small Molecule Bda-366 As a Bcl2-BH4 Antagonist for Multiple Myeloma Therapy
Authors: Deng Jiusheng , Park Dongkyoo , Wang Mengchang , Deng Qiaoya , Matulis Shannon , Boise Lawrence H. , Deng Xingming
Published: October 2019

Multiple myeloma (MM) is the most common hematological malignancy in USA, characterized with excessive abnormal clonal plasma cells in the bone marrow. Despite the great improvement in MM treatment by targeting the normal plasma cell biology of the cells during the last decade, the disease still remains incurable. B-cell lymphoma-2 (Bcl2) protein has four domains: BH1 (Bcl2 homology domain 1), BH2, BH3 and BH4, and plays critical roles in promoting the survival and drug-resistant of MM cells. Unlike the BH1, BH2, and BH3 domains, the BH4 region is responsible for the anti-apoptotic function of Bcl2 protein. Current BH3-mimetic and other Bcl2 inhibitors have been trailed in clinic but showed limited therapeutic efficacy. We recently identified a small molecule BDA-366 as a Bcl2-BH4 domain antagonist with potent anti-MM effect. BDA-366 bound to the BH4 resulted in the decrease of Bcl2 phosphorylation and the exposure of the BH3 domain as detected by anti-Bcl2-BH3 antibodies. Consequently, BDA-366 treatment induced robust apoptotic death on both human MM cell lines (RPMI8226 and U266) and primary MM cells from patients. In NOD-scid/IL2Rγnull (NSG) murine xenograft model, administration of BDA-366 (20mg/kg/day, 5 doses) markedly suppressed the growth of human MM tumor cells in vivo. We propose that BDA-366 as a novel BH4-based anti-MM agent could provide a more efficient pharmacological approach to treat MM.

Disclosures

No relevant conflicts of interest to declare.
Predictive Value of Rothman Index in Patients That Develop Febrile Neutropenia
Authors: Rubio Luis Alberto , Banach David , Lee Alfred I
Published: October 2019

Background: Febrile Neutropenia (FN) in cancer patients is associated with a high mortality rate yet identifying patients with FN at highest risk for short term morbidity and mortality remains challenging. The Rothman Index (RI) is a real-time composite measurement of a patient's condition using 26 clinical variables sensitive to signs of patient clinical decline. RI has been shown to predict intensive care unit (ICU) admission, hospital readmission and short term mortality among inpatients. A prior study from our institution demonstrated that hospitalized patients with hematological malignancies and RI < 60 have a greater likelihood of readmission or discharge to hospice rather than those with higher RI scores. The aim of this study was to explore RI as a predictor of adverse outcomes in patients with hematological malignancies who have FN.

Methods: Chart records of all patients with hematological malignancies and FN who were hospitalized at Yale Cancer Center between February of 2013 and July of 2014 were reviewed. Clinical variables collected included demographics, malignancy type, comorbidities, admission condition and microbiological data related to the FN episode. The primary outcome measure was a composite of in-hospital mortality and discharge to hospice care. Variables associated with the primary outcome in univariate analysis were entered into a multivariate logistic regression model for analysis.

Results: Of 308 patients with hematological malignancies, 180 (58.4%) developed FN during their admission and 38 (12.3%) died during the admission or were discharged to hospice. A total of 85 (27.6%) patients had a RI < 60, 155 (50.3%) had a RI between 60-80, and 71 (23.1%) had a RI > 80. Controlling for malignancy type, major medical comorbidities and microbiologically confirmed bacterial infection, RI < 60 was independently associated with this outcome (Odds ratio 3.02, 95% Confidence Interval 1.25-7.34). Baseline renal disease, pneumonia, respiratory failure and sepsis were also associated with the primary outcome.

Conclusion: RI is an objective measure that can independently predict poor outcomes among inpatients with FN. RI can guide clinicians caring for patients with FN in both prognostication and identifying high risk patients with an RI < 60 who are at higher risk for mortality, discharge to hospice, or high risk of readmission. The utility of this scoring system should be compared to existing risk stratification systems used in Oncology to identify its optimal use in the clinical setting.

Table 1. Factors associated with inpatient mortality or hospice discharge among hematologic malignancy inpatients with febrile neutropenia Variable OR 95% CI for OR p RI < 60 3.02 1.25-7.34 .015 Respiratory Failure 3.51 1.07-11.56 .039 Renal Disease 4.97 1.38-17.9 .014 Sepsis 8.18 1.63-41.2 .011 Pneumonia 8.01 1.42-45.1 .018

Disclosures

No relevant conflicts of interest to declare.
Understanding the Patient's Experience with Acute Myeloid Leukemia: A Patient Interview Study
Authors: Tomaszewski Erin L. , Fickley Catherine , Maddux LeAnne , Krupnick Robert , Bahceci Erkut , Paty Jean , van Nooten Floortje
Published: October 2019

INTRODUCTION: One way to capture the patient reality of living with disease is to talk with patients and listen to them describe their experience. We sought to understand the patient perspective regarding the signs and symptoms of acute myeloid leukemia (AML) and the impact of these on patients, through direct, qualitative patient interviews.

METHODS: We conducted individual interviews with patients with AML (median age=48, 37% male) in Japan and the USA following a semi-structured interview guide, containing both open- and closed- ended questions. These questions aimed to understand the patient's experience both spontaneously and with specific probing. Patients were asked to describe their individual experience of living with AML including signs, symptoms, impacts, and the experience during treatment of the disease.

RESULTS: Forty-one patients were interviewed in the two regions: 23 in US, and 18 in Japan. The sign, symptom, and impact 'concepts' identified were very similar across regions. Forty-nine symptoms were mentioned by US patients; 32 by Japanese patients. The signs/symptoms reported most frequently and as most bothersome included: fatigue, bruising, weakness, dizziness, shortness of breath, and bleeding (Table 1). Twenty-three unique impacts of signs/symptoms were mentioned in the US and 20 in Japan. The most frequent and bothersome impacts reported included: decreased ability to maintain social/familial roles, anxiety, decreased ability to function, depression, fear, remission uncertainty, appetite loss, and low sex drive (Table 1). These concepts were corroborated by two additional sources: interviews with clinicians and published literature.

DISCUSSION: The priority concepts expressed by patients were relevant and important to all four AML patient groups in both regions. This data provides valuable insight into patients' experiences with AML and helps to select the appropriate patient-reported outcomes for clinical trials.

Table 1. AML Symptom and Impact Frequencies US Frequency (N=23), n (%) Japan Frequency (N=18), n (%) Symptom Fatigue 22 (95.6) 16 (88.8) Bruising 18 (78.2) 7 (38.8) Weakness 18 (78.2) 15 (65.2) Dizziness 15 (65.2) 12 (52.1) Shortness of Breath 13 (56.5) 15 (65.2) Bleeding 12 (52.1) 13 (72.2) Impact Decreased ability to maintain social/familial roles 21 (91.3) 13 (72.2) Anxiety 20 (86.9) 17 (94.4) Decreased ability to function 20 (86.9) 17 (94.4) Depression 16 (69.5) 7 (38.8) Fear 15 (65.2) 12 (52.1) Remission uncertainty 15 (65.2) 17 (94.4) Appetite loss 14 (60.8) 13 (72.2) Low sex drive 11 (47.8) 7 (38.8)

Disclosures

Tomaszewski: Astellas Pharma, Inc.: Consultancy. Fickley:Astellas Pharma, Inc.: Consultancy. Maddux:Astellas Pharma, Inc.: Consultancy. Krupnick:Astellas Pharma, Inc.: Consultancy. Bahceci:Astellas Pharma Global Development: Employment. Paty:Astellas Pharma, Inc.: Consultancy. van Nooten:Astellas: Employment.
Three Dimensional Speckle Tracking Echocardiography (3D-STE) for Early Detection of Subtle Myocardial Deformation Dysfunction in Thalassemic Patients
Authors: Moustafa Agha Hala Mounir Mohamed , AbdelMassih Antoine , Abd El Rahman Mohamed Youssef , Milanesi, Ornella , Castaldi Biagio , Geranio Giulia , Frizziero Ludovica , Basso Giuseppe , Putti Maria Caterina , Essam Ahmed , Esmail Reem , El-Kamah Ghada , Hamdy Mona , El Baz Heba , El Behary Noha , Hanna Carol , El Tagy Hassan , Atef Mishriky Mary , Mamdouh Mirna , Khalifa Hossam , Adam Soheir
Published: October 2019

Background: Among patients with beta-thalassemia early detection of transfusion-induced myocardial iron loading and its intervention with aggressive chelation therapy may delay or reverse heart failure. Three dimensional speckle tracking echocardiography (3D-STE) is a novel tool that may early detect myocardial affection in these patients.

Methods: Thirty-two thalassemic patients with a mean age of 18.1± 7.03 years and 30 aged matched healthy control subjects have been included in the study. Patients have been recruited from pediatric hematology clinics in both Cairo University, Egypt (n=18) and Padova University, Italy (n=14). 3D-STE was performed to all patients and control subjects in addition to the myocardial relaxometry T2* by cardiac MRI.

Results: The left ventricular ejection fraction (LVEF) derived from 3D echocardiography among the studied thalassemia patients was within normal range 62.5± 5.6%. Compared to the normal subjects, thalassemia patients had a statistical significant reduction of the left ventricular global longitudinal strain (-16.81± 0.93% vs -18.76 ± 1.12 %, p=0.001), the left ventricular global circumferential strain (-10.56 ± 0.61% vs -11.83 ± 0.71 %, p=0.001 ) and the left ventricular global area strain (-20.13 ±1.18% vs -22.48 ±,1.29 %, p=0.001). No statistical significant correlation was found between the severity of myocardial iron overload measured by T2* and the measured left ventricular global strain.

Conclusion: In asymptomatic thalassemia patients with preserved left ventricular global systolic function 3D-STE derived strain can detect early subtle myocardial dysfunction. The observed subtle myocardial deformation dysfunction is not related to the extent of myocardial iron deposition.

Disclosures

No relevant conflicts of interest to declare.
Platelet-Neutrophil Aggregates Promote Pulmonary Arteriole Microembolism in Sickle Cell Disease
Authors: Bennewitz Margaret F. , Vats Ravi , Tutuncuoglu Egemen , Gladwin Mark T. , Sundd Prithu
Published: October 2019

Introduction: Sickle cell disease (SCD) is an autosomal recessive genetic disorder that affects ~100,000 Americans and millions of people worldwide. The acute chest syndrome (ACS), a form of acute lung injury, is a major cause of morbidity among SCD patients. The current treatment for ACS is primarily supportive and the molecular mechanism remains largely unknown. SCD patients hospitalized with vaso-occlusive pain crisis (VOC) often develop ACS within the ensuing days, suggesting a role for pulmonary vaso-occlusion in the onset of ACS. However, the cellular and molecular mechanism and the anatomical site of pulmonary vaso-occlusion are still elusive.

Materials and Methods: Intravenous (IV) bacterial lipopolysaccharide (LPS) was used to induce VOC in SCD mice. Intravital multiphoton excitation (MPE) fluorescence microscopy was used to study the blood cell trafficking within the pulmonary microcirculation of live SCD or control mice. Fluorochrome-conjugated anti-mouse Ly-6G, Ter-119, and CD49b antibodies were administered IV for in vivo staining of circulating neutrophils, red blood cells and platelets, respectively. Cellular trafficking was recorded at baseline and 2 hours after IV challenge with LPS. Image sequences were analyzed to identify vaso-occlusion, which was defined as cellular aggregation and stasis of blood flow within the pulmonary blood vessels.

Results and Discussion: Preliminary data using MPE imaging in transgenic SCD mice revealed that vaso-occlusion was absent at baseline in unchallenged SCD mice and the cellular trafficking within the pulmonary microcirculation was comparable in SCD and control mice. Doses of IV LPS (0.01-5 mg/kg of body weight), which were innocuous to control mice were found to be lethal to SCD mice. Remarkably, MPE imaging of the lung microcirculation revealed that IV LPS led to microembolism of the pre-capillary pulmonary arterioles by platelet-neutrophil aggregates in SCD but not control mice. The microembolism involved either entrapment of circulating platelet-neutrophil aggregates or in situ aggregation through sequential steps of neutrophil arrest on the arteriolar endothelium, followed by platelet nucleation on arrested neutrophils and microthrombus formation.

Conclusions: Initial findings demonstrate that pulmonary vaso-occlusion in SCD mice involves microembolism of the pre-capillary pulmonary arterioles by platelet-neutrophil aggregates. Future studies will determine the molecular interactions responsible for pulmonary arteriolar microembolism.

Acknowledgments: This study is supported by the 11SDG7340005 from the American Heart Association (P.S.) and the VMI startup account (P.S.). M.F.B is supported by the NIH NHLBI VMI T32 training grant T32HL110849.

Disclosures

No relevant conflicts of interest to declare.
The Heme Scavenger Hemopexin Reverts Heme-Driven Pro-Inflammatory Phenotypic Switching of Macrophages in Sickle Cell Disease
Authors: Vinchi Francesca , Costa da Silva Milene , Ingoglia Giada , Petrillo Sara , Brinkman Nathan , Zuercher Adrian , Cerwenka Adelheid , Tolosano Emanuela , Muckenthaler Martina U.
Published: October 2019

Hemolytic diseases, such as sickle cell anemia and thalassemia, are characterized by enhanced release of hemoglobin and heme into the circulation, heme-iron loading of reticulo-endothelial system macrophages as well as chronic inflammation. Here we demonstrate that in addition to activating the vascular endothelium, hemoglobin and heme alter the macrophage phenotype in sickle cell disease.

We show that exposure of cultured macrophages to hemolytic aged red blood cells, heme or iron causes their functional phenotypic change towards a pro-inflammatory phenotype, with increased expression levels of inflammatory markers such as IL-6 (P<0.001), TNFα (P<0.0001), IL-1β (P<0.001) MHCII (P<0.001), CD86 (P<0.05) and CD14 (P<0.0001) and decreased expression levels of anti-inflammatory markers such as IL-10 (P<0.0001), CD206 (P<0.0001), Arginase-1 and Ym1. Interestingly, hemolysis and macrophage heme/iron accumulation in a mouse model of sickle cell disease triggers similar pro-inflammatory phenotypic alterations in hepatic macrophages, increasing the expression levels of IL-6 (P<0.0001), MHCII (P<0.05), CD86 (P<0.0001) and iNOS (P<0.01). On the mechanistic level, heme-induced pro-inflammatory phenotype switching of macrophages critically depends on ROS production and activation of the TLR4 signaling pathway.

We further demonstrate that the heme scavenger hemopexin protects reticulo-endothelial macrophages from heme overload in heme-loaded Hx-null mice (P<0.05) and reduces production of cytokines (IL-6, TNFα: P<0.01) and reactive oxygen species (P<0.001). Importantly, in sickle mice the administration of human exogenous hemopexin attenuates the inflammatory phenotype of macrophages, by decreasing the expression of IL-6 (P<0.05), MHCII (P<0.01), CD86 (P<0.001) and iNOS (P<0.05).

Taken together, our data suggest that therapeutical administration of hemopexin is beneficial to counteract heme-driven macrophage-mediated inflammation in sickle cell disease.

Disclosures

Brinkman: CSL Behring: Employment. Zuercher:CSL Behring: Employment.
Neutrophil Transmigration into the Joint of RA-Induced Mouse Is Markedly Blocked By EC-18, Via STAT3 Signaling
Authors: Shin Jae Min , Kim Young-Jun , Yoon Sun Young , Kim Myung-Hwan , Suh Cheolwon , Sohn Ki-Young , Kim Heung-Jae , Han Yong-Hae , Chong Saeho , Kim Jae Wha
Published: October 2019

Neutrophil trafficking during the inflammatory response is effectively regulated by the enhanced IL-6 expression which orchestrates chemokine production and leukocyte apoptosis. Inappropriate regulation of leukocyte trafficking can lead to impaired neutrophil clearance and increased tissue damage from the accumulation of neutrophil-secreted proteases and reactive oxygen species at the site of inflammation. In collagen-induced arthritis (CIA) mouse model, arthritic symptoms were recapitulated with an increase of IL-6 level in the synovium, which was recuperated by the treatment of EC-18 to the level comparable with commercial therapeutics (such as Remicade or Methotrexate). When the tissues were stained with neutrophil-specific antibodies, EC-18 significantly reduced the infiltration of neutrophils into the synovium. EC-18, a monoacetyl-diglyceride (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol), has been isolated from the antlers of Sika deer (Cervus nippon Temminck), which were known to have immunosuppressive and anti-arthritic activities. In this study, we have discovered that EC-18 regulates the activity of signal transducer and activator of transcription 3 (STAT3), which is a master regulator of IL-6 expression. EC-18 caused the decrease of IL-6 production in a macrophage cell line, RAW264.7, and RA-fibroblast-like synoviocyte (RA-FLS) via the regulation of STAT3 signaling without affecting NF-κB signaling, which is also a well-known regulator of IL-6 expression. The expression of RANKL, a key factor for osteoclast differentiation and activation, was also regulated by STAT3, and decreased by EC-18 treatment. In fact, EC-18 inhibited osteoclastogenesis of hematopoietic stem cells. Therefore, blocking STAT3 activity by EC-18 treatment was able to inhibit the inflammatory cytokine loop triggering RANKL expression, thereby inhibiting joint destruction. Cardinal features of rheumatoid arthritis (RA) are chronic synovial inflammation and joint damage caused by the activity of cytokines and proteins expressed from cells in synovial fluid. Interleukin (IL)-6, a multifunctional pro-inflammatory cytokine, plays a critical role in the pathogenesis of joint and systemic inflammation in RA. Therefore, EC-18 could be utilized as a potential therapeutic agent for the treatment of sustained inflammation and joint destruction.

Disclosures

No relevant conflicts of interest to declare.
Control of Neutrophil Endothelial Transmigration By EC-18 in Chemotherapy Induced Neutropenia
Authors: Kim Jae Wha , Kim Yong-Jae , Kim Young-Jun , Yoon Sun Young , Jeong Jinseon , Ko Young-Eun , Shin Su-Hyun , Yoo Nina , Kim Myung-Hwan , Suh Cheolwon , Sohn Ki-Young , Kim Heung-Jae , Han Yong-Hae , Chong Saeho
Published: October 2019

Chemotherapy-induced neutropenia (CIN) is a common dose-limiting side-effect that often compromises chemotherapy resulting in dose reduction and/or delayed treatment. Chemotherapy-induced myeloid suppression is a prevalent mechanism to describe CIN. However, diapedesis of neutrophils from the blood is also a possible mechanism of CIN. Cytokines, chemokines, and damage- (or danger-) associated molecular pattern molecules (DAMPs) are produced from the damaged tissues by chemotherapy, which accelerates neutrophil transmigration. EC-18 is a synthetic monoacetyldiglyceride (MW=635) currently under development as an oral agent to treat and potentially prevent CIN. Here, we showed that neutrophil transmigration is induced in CIN model, and the efficacy and potential mechanism of EC-18 have been evaluated in mice and various cell models. Gemcitabine (50mg/Kg), cyclophosphamide (100mg/Kg) or tamoxifen (50mg/Kg) was administered daily for 3 days in mice (C57BL/6), followed by oral administrations of EC-18 (50mg/Kg) for 21 days. Blood neutrophils were analyzed by complete blood count (CBC) and fluorescence-activated cell sorting (FACS). All three chemotherapeutic agents significantly reduced absolute neutrophil count (ANC) in the blood; 40.4% decrease in gemcitabine, 19.8% in cyclophosphamide, and 21.8% in tamoxifen, which were recovered by the co-administration of EC-18. Microarray analysis of the lymph nodes from gemcitabine-treated mice revealed that the expression of DAMP molecules, S100A8 and S100A9, was induced by gemcitabine and decreased by the co-treatment of EC-18. Using immune cell lines, the expression of neutrophil transmigratory molecules, such as C3 anaphylatoxin, IL-6 and IL-8, was increased by gemcitabine treatment, and EC-18 blocked the expression of those cytokines in dose-dependent manners. Western blot analysis showed that EC-18 regulated STAT3 and STAT6 signaling pathways to modulate the expression of DAMPs, C3, IL-6, and IL-8. The decrease of C3 and IL-6 levels by EC-18 treatment was also verified in PBMCs from normal adults. In a pilot clinical study (WJON 2015 accepted, ASCO e20697), orally administrated EC-18 (1,000mg/day) alleviated neutropenic phenotypes in patients with pancreatic cancer who had been treated with gemcitabine (1,000mg/m2) and erlotinib (100mg daily). As a supportive oncology agent, EC-18 can maximize the antitumor efficacy of myelo-suppressive chemotherapeutic agents by enabling the treatment to continue or even higher doses to be administered.

Disclosures

No relevant conflicts of interest to declare.
Engineered Donor Marrow Macrophages Phagocytose Cancer Cells and Aggressively Shrink Solid Tumor Xenografts Compared to Tumor Associated Macrophages
Authors: Alvey Cory M , Spinler Kyle , Tewari Manu , Discher Dennis
Published: October 2019

Cell-based immunotherapies such as those based on engineered T-cells appear safe and often effective against liquid tumors. In solid tumors, macrophages are typically abundant, but the density of tumor associated macrophages (TAMs) correlates with poor clinical outcomes as they promote tumor growth and immunosuppression. In our studies, less differentiated donor marrow phagocytes are engineered in order to target tumors and selectively phagocytose cancer cells. Xenograft tumors were first made on the flanks of NSG mice using a tdTomato human lung carcinoma cell line (A549). Systemic injections of anti-human IgG (anti-hum) with large tumors (~70 mm2) showed no effect on tumor growth. However, systemic injection of bone marrow from donor NSG mice together with biweekly anti-hum treatments effectively stopped growth of the solid tumors. Replacing anti-hum with a non-specific antibody had no effect on tumor growth. Based on tdTomato signal intensity within macrophages isolated from tumors, 5-10-fold more donor macrophages are phagocytic compared to resident TAMs (2-3% are phagocytic). Since cancer cells express on their surface 'self' markers that limit the phagocytosis of these cells, we inhibited the 'self' receptors on the injected donor phagocytes prior to systemic injection of the donor marrow. This combination of 'self'-receptor inhibition with anti-hum causes a rapid decrease in tumor burden, shrinking tumors by ~40% in just 10 days compared to a similar growth of untreated tumors in the same time period. The anti-hum injection was again necessary as injection of a non-specific antibody failed to affect tumor growth. Tumor analysis showed that >65% of macrophages that were 'self'-receptor inhibited had phagocytosed the tdTomato A549 cells, which is ~20-fold greater than resident macrophages. Importantly, these cell therapy treatments appear safe with no significant decreases in hematocrit or platelets, which is unlike the anemia that has been reported upon systemic injection of 'self' inhibitors. Our results thus suggest that therapies based on engineered macrophages can be safe and effective against solid tumors if three requirements are met: a phagocytic phenotype, target opsonization, and inhibition of 'self' signaling. Future experiments will involve characterizing macrophage phenotypes of donor versus resident macrophages as well as efforts to maintain the necessary phagocytic phenotype.

Disclosures

No relevant conflicts of interest to declare.
TET2 Regulates CD8+ T Cell Responses to Acute and Chronic Viral Infection
Authors: Carty Shannon A. , Gohil Mercy , Stelekati Erietta , Banks Lauren B. , Wherry E. John , Koretzky Gary A. , Jordan Martha S
Published: October 2019

DNA methylation is one of the major epigenetic mechanisms that controls cellular differentiation. The ten-eleven translocation (TET) family of methylcytosine dioxygenases mediates active DNA demethylation through the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and subsequent intermediates. Here we demonstrate that TET2 regulates CD8+ T cell differentiation in vivo following acute and chronic viral infection. At steady-state, mice with a T-cell specific deletion of TET2 have intact thymic and peripheral T cell populations. Following acute viral infection with LCMV-Armstrong, TET2 loss enhances LCMV-specific CD8+ T cell memory differentiation in a cell-intrinsic manner without disrupting antigen-specific cell expansion or cytokine production. However, TET2-deficient memory CD8+ T cells exhibit altered recall responses with blunted re-expansion, retained expression of phenotypic memory markers and restricted re-expression of activation markers. During chronic viral infection with LCMV-clone 13, TET2 controls CD8+ T cell expansion and alters differentiation. Importantly, though mice with T-cell specific loss of TET2 developed similar levels of CD8+ T cell exhaustion as wild-type mice, TET2 loss specifically reduced PD-1 expression suggesting that TET2 may direct DNA demethylation of the PD-1 locus. Together, our data indicate that TET2 is an important regulator of CD8+ T cells following both acute and chronic viral infections and suggest targeting epigenetic regulators have potential for enhancing antiviral immunity.

Disclosures

No relevant conflicts of interest to declare.
Non-Proteolytic Regulation of the Multimerization of Von Willebrand Factor By ADAMTS13 Based on a Novel Cysteine Thiol Redundancy
Authors: Rottensteiner Hanspeter , Skalicky Susanna , Seyfried Birgit , Kaufmann Stefan , Fiedler Christian , Hasslacher Meinhard , Dong Jing-fei , Zheng X. Long , Plaimauer Barbara , Scheiflinger Friedrich
Published: October 2019

The platelet dependent-clotting process is a highly complex pathway that needs to be tightly controlled by a variety of soluble and cellular components. Failure to regulate the activity of multimeric von Willebrand factor (VWF) by ADAMTS13 for instance causes thrombotic thrombocytopenia purpura (TTP). Regulation is achieved through the established function of ADAMTS13 as a VWF-cleaving protease; however, it has been reported that ADAMTS13 may additionally curtail VWF multimer fibrillation by forming disulfide bridges with VWF. Since the latter mechanism remained ill-defined, we aimed to directly visualize disulfide bond formation between ADAMTS13 and VWF using multimer gel electrophoresis. Covalent interaction between ADAMTS13 and VWF was time-, concentration-, temperature-, and shear-stress dependent. The covalent linkage was independent of the proteolytic activity of ADAMTS13 and could be assigned to a C-terminal fragment comprising TSP1 domains 5 to 8 plus the CUB domains. This interaction was blocked by thiol-reactive agents, indicating that association was accomplished by a thiol/disulfide exchange reaction. Furthermore, a polyclonal anti-ADAMTS13 antibody that inhibited the protease in a FRETS-VWF73 activity assay also inhibited the formation of VWF-ADAMTS13 adducts.

Using a coupled reversed phase HPLC-ESI-QTOF-MS system, several partially reduced free thiols were identified in ADAMTS13, with cysteines 1254 and 1275 being the most prominent. Together with the observation that a C1275S point mutation retained the ability to form covalent linkages with VWF, this result indicated that ADAMTS13 has disulfide plasticity. The same conditions that led to VWF complex formation also caused a hitherto undetected homo-oligomerization of ADAMTS13. An even more pronounced oligomerization was noted in the presence of thiol-sensitive agents such as glutathione.

We propose a dynamic network of redundant, partially free thiols in ADAMTS13 that undergo intra- and inter-molecular redox reactions capable not only of depolymerizing VWF, but also to prevent further VWF multimerization by facilitating the aggregation of ADAMTS13 around the monomers.

Disclosures

Rottensteiner: Baxalta Innovations GmbH: Employment. Skalicky:Baxalta Innovations GmbH: Employment. Seyfried:Baxalta Innovations GmbH: Employment. Kaufmann:Baxalta Innovations GmbH: Employment. Fiedler:Baxalta Innovations GmbH: Employment. Hasslacher:Baxalta Innovations GmbH: Employment. Plaimauer:Baxalta Innovations GmbH: Employment. Scheiflinger:Baxalta Innovations GmbH: Employment.
Widespread and Dynamic Translational Control of Red Blood Cell Development
Authors: Hu Wenqian , Zhang Xu , Alvarez-Dominguez Juan
Published: October 2019

Cell development requires tight yet dynamic control of protein production. Here, we use murine erythropoiesis as a model to study translational regulatory dynamics during mammalian cell differentiation. We uncover pervasive translational control of protein synthesis, including widespread alternative translation initiation and termination, stoichiometric synthesis, and dynamic use of upstream reading frames. In addition, we identify hundreds of mRNAs with dynamic translation efficiencies during erythropoiesis. Interestingly, these mRNAs have significantly enriched binding motifs of many mRNA-binding proteins that are expressed with high specificity in hematopoietic cells, suggesting that those mRNA-binding proteins are important players in the translational regulatory programs in erythropoiesis. A major such program involves enhanced decoding of specific mRNAs depleted in terminally differentiating/enucleating cells with decreasing transcriptional capacity. We functionally characterized one such protein, Rbm38, which was previously identified as a splicing factor in terminal erythropoiesis, can enhance the translation of a subset of those mRNAs by interacting with the general translation initiation factor eIF4G in erythroid cells. Altogether, our findings reveal widespread and dynamic translational control in supporting specialized mammalian cell formation.

Disclosures

No relevant conflicts of interest to declare.
DYRK2 Inhibits the Self-Renewal of Leukemic Stem Cells in Chronic Myeloid Leukemia By Inducing Degradation of c-Myc Downstream of the Reprogramming Factor KLF4
Authors: Park Chun Shik , Shen Ye , Suppipat Koramit , Lewis Andrew , Tomolonis Julie , Puppi Monica , Mistretta toni-Ann , Ma Leyuan , Green Michael R , Lacorazza Daniel
Published: October 2019

Chronic myeloid leukemia (CML) is a blood cancer originated by expression of BCR-ABL, a constitutively activated kinase product of the chromosomal translocation t(9;22), in hematopoietic stem cells (HSC). Although tyrosine kinase inhibitors (TKI) can efficiently induce molecular remission in CML patients, drug discontinuation often leads to relapse caused by reactivation of leukemic stem cells (LSC) spared from TKI therapy via BCR-ABL-independent mechanisms of self-renewal and survival. Thus, there is a need for alternative drugs for relapse patients to prevent expansion of BCR-ABL-positive LSC during discontinuation of chemotherapy or emergence of chemoresistance. We found that somatic deletion of the reprogramming factor Krüppel-like factor 4 (KLF4) in BCR-ABL(p210)-induced CML severely impaired disease maintenance. This inability to sustain CML in the absence of KLF4 was caused by a progressive attrition of LSCs in bone marrow and the spleen and impaired ability of LSCs to recapitulate leukemia in secondary recipients. Analyses of global gene expression and genome-wide binding of KLF4 revealed that the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2) is repressed by KLF4 in CML LSCs. Immunoblots revealed elevated levels of DYRK2 protein that were associated with a reduction of c-Myc protein and increased levels of p53 (S46) phosphorylation and PARP cleavage in KLF4-deficient LSCs purified from the bone marrow of CML mice. Genomic silencing of KLF4 in the murine CML cell line 32D-BCR-ABL resulted in increased levels of DYRK2 and phosphorylated c-Myc (S62) leading to diminished levels of c-Myc protein, which was reverted by treatment with a proteasome inhibitor, suggesting that KLF4 prevents c-Myc degradation triggered by DYRK2-mediated priming phosphorylation. Consistent with an inhibitory role in leukemia, DYRK2 levels are significantly reduced both in CD34+CD38+ and CD34+CD38− cells from CML patients compared to normal stem/progenitor cells. Aiming at pharmacological activation of DYRK2 to abrogate self-renewal and survival of CML cells, we treated CML cells with vitamin K3 that inhibits Siah2, an ubiquitin E3 ligase involved in Dyrk2 proteolysis. Vitamin K3, and not Vitamin K1 and K2, induces dose-dependent cytotoxicity in a panel of human-derived CML cell lines by stabilizing Dyrk2 protein and consequently promoting c-Myc degradation. Interestingly, combination of vitamin K3 with Imatinib exhibit additive effect inducing cytotoxicity in CML cells. Collectively, the identification of Dyrk2 as a critical mediator of LSC downfall is a novel paradigm poised to support the development of LSC-specific therapy to induce treatment-free remission in conjunction with Imitinib in CML patients.

Disclosures

No relevant conflicts of interest to declare.
Individual Variation in Hemostatic Alterations Caused By Tyrosine Kinase Inhibitors - a Way to Improve Personalized Cancer Therapy?
Authors: Lotfi Kourosh , Deb Suryyani , Sjöström Clara , Tharmakulanathan Anjana , Boknäs Niklas , Ramström Sofia
Published: October 2019

Introduction

During the last two decades, Bcr-Abl tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myelogenous leukemia (CML), and are now considered standard treatment for this disease. However, TKIs can induce serious hemostatic side effects including cardiovascular disease and bleeding disorders. Blood platelet aggregation and formation of pro-coagulant platelets are important to allow a well-balanced hemostatic response. Therefore, a detailed understanding of what effect different TKIs exert on platelets and hemostasis could help to understand if there are differences of importance to minimize the risk of bleeding complications in treated patients.

Aim

To investigate how TKIs used in CML (imatinib, dasatinib, nilotinib, bosutinib, and ponatinib) affect platelet activation and hemostasis.

Materials and Methods

We have developed a multi-parameter six color flow cytometry protocol to study different aspects of platelet function upon activation, e.g. formation of aggregatory (PAC-1-positive) and pro-coagulant (phosphatidylserine-exposing) platelets, exocytosis of alpha- and lysosomal granules and mitochondrial membrane potential.This protocol was performed in presence or absence of TKIs in blood from normal donors and in treated patients. Whole blood aggregometry (Multiplate®), thrombin generation in platelet-rich plasma and in vitro thrombus formation by free oscillation rheometry (ReoRox G2) was further evaluated in some situations.

Results

At clinically relevant concentrations, dasatinib significantly decreased the formation of procoagulant platelets. Ponatinib induced a slight decrease in formation of procoagulant platelets, whereas bosutinib and nilotinib showed opposite tendencies (n=7). Dasatinib also decreased platelet aggregation (n=4-6) and in vitro thrombus formation (n=3). Thrombin generation was not significantly affected by therapeutic levels of TKIs, whereas higher doses of dasatinib, bosutinib, ponatinib and imatinib significantly changed one or several of the thrombin generation parameters (n=7-8). Interestingly, large differences in response to the drugs were observed among the healthy donors, especially for dasatinib and bosutinib. Major inter-individual variations were also observed in dasatinib-treated patients.

Conclusions

Different TKIs show varying potency to affect platelet-based hemostasis. In addition, we found large inter-individual variations in how some drugs affected platelet function. Therefore, we suggest that development of a clinically useful protocol for platelet function testing could help to identify patients more susceptible to adverse drug reactions. Such a protocol could potentially help clinicians to gain insight into the risk of side effects, which could help to choose the most suitable drug for each individual patient.

Disclosures

No relevant conflicts of interest to declare.
CML Treatment in Dutch Hospitals with Low Patient Volumes Is Associated with a Substandard Quality of Molecular Response Monitoring
Authors: Geelen Inge G.P. , Thielen Noortje , Janssen Jeroen J.W.M. , Roosma Tanja , Visser Otto , Cornelissen Jan J. , Westerweel Peter E.
Published: October 2019

Introduction

Chronic myeloid leukemia (CML) is a relatively rare condition with approximately 170 new patients per year in the Netherlands. Patients at risk of progression to advanced stage disease need to be identified based on their molecular BCR-ABL1response, for which detailed guidelines are available. We evaluated the quality of molecular monitoring in Dutch clinical practice.

Methods

Chronic phase CML patients with a minimum of one year available follow-up were abstracted from a real-world population based registry of patients newly diagnosed between January 2008 and April 2013 in the Netherlands (PHAROS-CML). Hospitals were categorized into three groups based on the average number of CML-treatment initiations per year: low (<1/year), intermediate (1-2/year) and high volume (>2/year). The minimum standard of care was defined as molecular monitoring of response at least three times in the first year of treatment.

Results

In 253 eligible patients, molecular response assessment within the first year after diagnosis was performed a minimum of three times in 78%, twice in 15%, once in 4% and not performed in 3% of patients. Treatment for these patients was initiated in 54 different hospitals, corresponding to an average just below 1 new CML patient per year per hospital. In univariate logistic regression analysis treatment in an academic hospital, clinical trial inclusion, hospital volume and first line second generation TKI (2GTKI) treatment proved to be significant predictors of the minimum standard of care. Only the latter two remained significant predictors in multivariate analysis.

Treatment was initiated for 153 patients (61%) in high volume hospitals (n=16), 66 patients (26%) in intermediate volume hospitals (n=18) and 34 patients (13%) in low volume hospitals (n=20). In the high volume hospitals, 90% of patients received the minimum standard of care, compared to 67% in the intermediate (p<.001) and 50% in the low volume hospitals (p<.001). Moreover, monitoring in imatinib treated patients was poorer than in patients initially treated with 2GTKI (74% vs 94%, p=.002).

Conclusion

Approximately 40% of newly diagnosed CML patients in the Netherlands are treated within centres that see less than two new patients per year. Such low treatment volumes are associated with a substandard quality of molecular response monitoring. Surprisingly, patients treated with imatinib as first line treatment are monitored less stringent, even though the rate of treatment failure is known to be higher than 2GTKI treated patients. Our observations support a potential patient benefit of centralization of CML treatment.

Disclosures

Janssen: Novartis: Research Funding; ARIAD: Consultancy; BMS: Honoraria; Pfizer: Honoraria.
Assessment of Molecular Response in CML Patients with Atypical BCR-ABL Tanscripts. Recommendations By the EUTOS Collaboration
Authors: Ernst Thomas , Schäfer Vivien , White Helen E , Saussele Susanne , Lange Thoralf , Gerrard Gareth , Cross Nicholas CP , Hochhaus Andreas
Published: October 2019

Approximately 2‐3% of CML patients (pts) have variant BCR‐ABL fusions transcripts that cannot be monitored by RQ‐PCR using standard methodologies. Most rare variants are accounted for by seven variant fusions (e1a2/3, b2/3a3, e6a2, e8a2, e19a2). Within the European Treatment and Outcome Study (EUTOS) we sought to establish and validate robust RQ-PCR methods for each of these abnormalities. RNA was extracted from leukocytes obtained from 20 mL peripheral blood. Samples were received either locally or by mail and spent between 1 to 3 days in transit. Multiplex reverse transcription polymerase chain reaction (RT-PCR) experiments were performed to identify BCR-ABL1 transcripts (Cross et al., Leukemia 1994). In all cases, gel electrophoresis showed an unusual band. PCR products were then sequenced to characterize BCR-ABL1 rearrangements. Monitoring of residual disease was carried out by RQ-PCR with specific forward and/or reverse primers and probes using a serial dilution of individual BCR-ABL1 or GUSB plasmid DNA calibrators. GUSB transcripts were quantified as internal control and results were expressed as the BCR-ABL1/GUSB ratio with no conversion according to the international scale (IS) as it is recommended for only typical major (M) BCR-ABL1 and cannot be applied to other transcripts in CML. 43 pts from 8 prospective studies and pts outside of studies were investigated. A total of 435 samples (1-72 per patient, median 8) were analyzed. Pts expressed 6 different atypical BCR-ABL1 transcripts (e19a2, n=18; e1a2, n=10; b2a3, n=5; b3a3, n=5; b2a3&b3a3, n=2, e8a2, n=3). At the start of the observation period, ratios BCR-ABL1/GUSB ranged between 0.01% and 179.1% (median 13.75%). Follow-up results were determined in comparison to the starting level and expressed as relative log reduction. 9 pts were in deep molecular remission considering a minimum GUSB transcript level of 20,000/reaction. During the observation period, six pts relapsed with a significant increase of BCR-ABL1/GUSB ratios. We conclude that very few pts are diagnosed with unusual BCR-ABL1 transcripts, but their characterization is important for proper assessment of treatment response in affected patients, and to avoid false negative assessment of residual disease status. Based on the individual PCR strategies, results cannot be expressed with precision on the International Scale and thus the common molecular milestones and triggers for treatment discontinuation are difficult to apply. We suggest that residual disease in these cases should be assessed primarily by considering trends in BCR-ABL/reference gene ratios over time.

Disclosures

Saussele: NOVARTIS PHARMA: Consultancy, Honoraria; BMS: Honoraria. Cross:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau.