bluuebunny/crossref_metadata_2025 huggingface dataset:
Newly Identified Metabolites Connect Colon Cancer to Thrombosis
Authors: Belghasem Mostafa , Roth Daniel Michael , Walker Joshua Aaron , Richards Sean , Spencer Cheryl , Tashijan Joseph Yeghishe , Arinze Nkriuka , Andry Chris , Ravid Katya , Chitalia Vipul
Published: October 2019

Patients with cancer are at 4-to-7-fold higher risk of venous thromboembolism (VTE). The risk of cancer-associated VTE varies with the type of cancer, underscoring the presence of cancer type-specific drivers and a need for appropriate animal models, both of which have been understudied. Here, we generated a colon cancer-specific mouse model and probed for the development of VTE and factors that might induce it. To this end, a group of athymic animals injected with human colon adenocarcinoma cell line (HT-29) underwent inferior vena cava (IVC) ligation, a validated model of venous thrombosis. Compared to corresponding controls (age- and gender-matched), mice on regular chow diet with xenografts exhibited significantly higher IVC clot weights. Plasma analysis of metabolites led us to identify the tryptophan metabolites, Kynurenine and Indoxyl Sulfate, which are known activator of the Aryl Hydrocarbon Receptor (AHR), as significantly elevated in the cancer model (2-3 fold; p<0.05). Plasma from these animals activated the AHR pathway and augmented both tissue factor (TF) and Plasminogen Activator Inhibitor 1 (PAI-1) levels in venous endothelial cells. PAI-1 is a known target of AHR pathway in other cells. Using an AHR inhibitor, we showed that the above effects of the plasma depended on AHR activation. Consistent with these findings, immunostaining of the endothelial layer of IVC in animals with xenografts revealed predominantly nuclear AHR (sign of activation) and clear increases in TF and PAI-1 expression- telltale signs of an activated AHR-TF/PAI-1 axis. As TF augments thrombogenesis and PAI-1 inhibits thrombolysis, these results may explain the enhanced thrombogenicity in this model. While this model represents a novel tool to mechanistically probe mediators of cancer-associated VTE, it links for the first-time cancer-associated VTE to a set of metabolites. Our data raise a possibility of AHR-TF/PAI-1 axis as a mediator of VTE in other tumors, worthy of exploration given its potential as a biomarker and a therapeutic target.

Disclosures

No relevant conflicts of interest to declare.
Rafoxanide, an Organohalogen Drug, Triggers Apoptosis and Cell Cycle Arrest in Multiple Myeloma By Enhancing DNA Damage Responses and Suppressing the p38 MAPK Pathway
Authors: Shi Jumei , Xiao Wenqin , Xu Zhijian , Chang Shuaikang , Gao Lu , Tao Yi , Wang Yingcong , Wu Xiaosong , Zhu Weiliang
Published: October 2019

Rafoxanide is used in veterinary medicine for the treatment of fascioliasis. We previously repositioned the drug as the inhibitor of B-Raf V600E, but its anti-tumor effect in human cancer has never been reported. In this study, we investigated the effects of rafoxanide in multiple myeloma (MM) in vitro and in vivo. We found that rafoxanide inhibited cell proliferation and overcame the protective effect of the bone marrow (BM) microenvironment on MM cells. Rafoxanide induced cell apoptosis by reducing mitochondrial membrane potential (MMP) and regulating the caspase pathway, while having no apparent toxic effect on normal cells. Rafoxanide also inhibited DNA synthesis and caused cell cycle arrest by regulating the cdc25A-degradation pathway. In addition, rafoxanide enhanced the DNA damage response by up-regulating the expression of γ-H2AX. Importantly, as a potent B-Raf V600E inhibitor, rafoxanide could suppress activation of the p38 MAPK pathway. Rafoxanide treatment inhibited tumor growth, with no significant side effects, in an MM mouse xenograft model. Finally, combination of rafoxanide with bortezomib or lenalidomide significantly induced synergistic cytotoxicity in MM cells. Collectively, our results may provide a rationale for use of this drug in MM treatment. This study was supported by grants from the National Natural Science Foundation of China (Nos. 81570190; 81670194 and 81529001).

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
Characterization of a Mouse Model of Hypereosinophilic Syndrome
Authors: Zimmermann Nives , Gibbons William J , Homan Shelli M , Klingler Andrea , Prows Daniel R
Published: October 2019

Introduction: Hypereosinophilic syndrome is characterized by sustained blood and/or tissue hypereosinophilia leading to tissue damage and organ dysfunction. Nearly half the cases are classified as idiopathic, since no known cause is identified and there is no evidence of a clonal disorder. In such cases, nonspecific treatment approaches, including corticosteroids, hydroxyurea and interferon-alpha, are used aimed at mitigating eosinophil-mediated organ damage. However, mortality occurs primarily due to cardiac and thromboembolic complications. Understanding the cause and mechanism of disease would aid in the development of targeted therapies with greater efficacy and fewer side effects. For instance, while the traditional view is that eosinophils are detrimental via toxicity of their granule proteins, recent studies suggest other mechanisms including non-canonical cell death subroutines, such as regulated necrosis, release of pro-thrombotic DNA "traps" and others. Lack of a mouse model hinders testing such hypotheses in vivo. We have developed a mouse model of eosinophil-associated hypereosinophilic syndrome characterized by multi-organ involvement including lethal prothrombotic myocardial damage.

Results: We discovered a spontaneous mouse mutant in our colony with a hypereosinophilic phenotype. Mice develop peripheral blood eosinophilia, infiltration of lungs, spleen and heart by eosinophils, and extensive myocardial damage and remodeling. This ultimately leads to heart failure and premature death, often by 14 weeks of age. Histopathologic assessment of the hearts from affected mice (Fig. 1) revealed a robust inflammatory infiltrate of the ventricular myocardium. Immunohistochemistry demonstrated that this infiltrate is composed primarily of eosinophils (MBP) and B lymphocytes (B220), leading to myocardial damage and replacement fibrosis. In some cases, hearts showed dilatation and thinning of the right ventricular wall, suggestive of an inflammatory dilated cardiomyopathy. An exam of most mice revealed primarily atrial thrombi, which often filled the entire/majority of the chamber. Expression analysis by protein multiplex assay in heart homogenates revealed overexpression of Th1 and Th2 cytokines and chemokines, including IL-4, IL-6, LIF, IL-16, IP-10/Cxcl10, MIG/Cxcl9, Eotaxin-1/Ccl11, RANTES/Ccl5, MIP-1β/Ccl4, MIP-3β/Ccl19, MCP-1/Ccl2, MCP-3/Ccl7, MDC/Ccl22 and TARC/Ccl17.

Conclusion: The pathologies observed in the mutant line are reminiscent of those seen in patients with hypereosinophilia, where cardiac-related morbidities, like congestive heart failure and thrombi, are the most common causes of death. As such, our model provides an opportunity to test mechanistic hypotheses and develop targeted therapies.

Disclosures

No relevant conflicts of interest to declare.
Low-Dose Decitabine Could Improve the Imbalance of M1/M2 Ratio in Immune Thrombocytopenia
Authors: Shao Xia , Li Feng , Cheng Luya , Chen Pu , Min Zhihui , Ji Lili , Wu Boting , Cheng Yunfeng
Published: October 2019

Background

Primary immune thrombocytopenia (ITP) is an acquired autoimmune hemorrhagic disease characterized by increased immune-mediated platelet destruction and insufficient platelet production. As the impaired balance of M1 macrophage and M2 macrophage subsets increases platelet destruction, the M1/M2 ratio has become the focus of attention. The demethylating agent, decitabine (DAC) has been successfully used in the treatment of myelodysplastic syndrome with a moderate response in thrombocytopenia. Interestingly, at low doses, DAC induces megakaryocyte differentiation and maturation in vitro via inducing hypomethylation, thus having the potential to promote platelet release in ITP patients. In addition, several reports suggest that DAC can promote the polarization of macrophages to M2 macrophages. Therefore, we hypothesized that low dose of DAC treatment could lead to shrinkage of platelet destruction in murine model of ITP via promoting the M2 macrophage polarization.

Methods

Platelets isolated from Wistar rats were transferred into CBA/Ht mice via intraperitoneal injection weekly to induce a marine model of primary ITP. Starting on day 8, ITP mice were treated with low-dose DAC (0.25 mg/kg/d, n = 8) or PBS (n = 8) for two consecutive weeks intraperitoneally. Platelet counts were recorded every three days. The spleens were removed at day 10, from which M1 macrophages (F4/80+ CD11c+) and M2 macrophages (F4/80+ CD206+) were identified by flow cytometry analysis and immunofluorescence.

Results

The platelet levels in ITP group began to descend sharply at the second week after transferring platelets when compared with the control group (735.0 ± 25.2 × 109vs. 390.3 ± 70.0 × 109, p < 0.05). Mice in DAC-treated group recovered from thrombocytopenia by day 10 (713.8 ± 29.1 × 109vs. 311.0 ± 4.5 × 109, p < 0.001). In the following days, it still showed significantly higher level of platelet in DAC-treated group than ITP group despite the trend to decline. Comparing with the ITP group, immunofluorescence revealed that spleen samples showed stronger M2 expression pattern in DAC group, and the ratio of M1/M2 declined regardless of the fact that both the percentage of M1 macrophage and M2 macrophage was increased when DAC was applied.

Conclusions

Low dose DAC can promote the polarization of both M1 and M2 macrophage, especially M2 macrophage, thus reversing the unbalanced M1/M2 ratio in ITP group. Further studies are required to investigate the mechanism underlies the specialized polarization of macrophages.

Disclosures

No relevant conflicts of interest to declare.
Corticosteroids Combined with Cyclosporine a Is Superior to Corticosteroids As First-Line Treatment for Autoimmune Hemolytic Anemia after Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Lv Weiran , Qu Hong , Wu Meiqing , Fan Zhiping , Huang Fen , Xu Na , Xuan Li , Lin Ren , Zhao Ke , Sun Jing , Lai Yongrong , Xu Yajing , Liu Qifa
Published: October 2019

We retrospectively investigated the incidence and outcomes of allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients with autoimmune hemolytic anemia (AIHA). A total of 1,377 patients enrolled in this study, and the 3-year cumulative incidence of AIHA was 2.2±0.4%. Twenty-five patients with AIHA received treatment, including 15 patients who received corticosteroids combined with Cyclosporine A(CsA )and 10 who received corticosteroids monotherapy. After four weeks of treatment, the patients treated with corticosteroids combined with CsA had a higher complete response rate than those with corticosteroids monotherapy (66.7% vs. 11.1%; P=0.013). One patient (6.7%) experienced AIHA relapse in the combined group, and 5 (50.0%) relapsed in the monotherapy group (P=0.023). The 5-year cumulative incidence of malignant disease relapse was 5.5±5.1% and 27.4±1.4% (P=0.032), the OS was 86.3 ± 7.4% and 65.2 ± 1.6% (P=0.046), and the transplant-related mortality (TRM) post-transplantation was 9.3±6.4% and 22.8±1.6% (P=0.140), respectively, for patients with AIHA and those without AIHA. Our results indicate that corticosteroids combined with CsA is superior to corticosteroids as first-line treatment for AIHA, and AIHA does not contribute to increased primary disease relapse and TRM.

Disclosures

No relevant conflicts of interest to declare.
A Potential Mechanism for the Antioxidant Compound Kushen Injection Targeting the ROS Signaling Pathway in Acute Myeloid Leukemia
Authors: Jin Yanxia , Yang Qian , Liang Li , Ding Lu , Liang Yuxing , Zhang Dongdong , Yang Tian , Zhao Mingfeng , Zhou Fuling
Published: October 2019

Abstract

It is a challenge for treatment of acute myeloid leukemia (AML) in clinic. The increase in the levels of reactive oxygen species (ROS) in AML patients has been previously described; thus, it is important to regulate ROS levels in AML.

In this study, we found that intracellular ROS levels in AML cells were decreased, the total antioxidant capacity (T-AOC) and glutathione (GSH) contents were increased and xanthine oxidase (XOD) vitality was decreased when treated with the compound kushen injection (CKI). This shows that CKI inhibited the proliferation of AML cell lines and patient cells and enhanced the cytotoxicity of AML cells, which has few toxic effects on haematopoietic stem cells (HSCs) and T cells. At the single-cell level, individual AML cells died gradually by CKI treatment on optofluidic chips. CKI could promote apoptosis and arrest cell cycle at G1/G0 phase in U937 cells.

Furthermore, higher peroxiredoxin-3 (Prdx3) expression levels were found in CKI-treated U937 cells through quantitative proteomics detection. Mechanically, the expression of Prdx3 and peroxiredoxin-2 (Prdx2) was up-regulated in CKI-treated AML cells, while thioredoxin 1 (Trx1) was reduced. Laser confocal microscopy showed that the Prdx2 and Trx1 proteins could be co-localized by CKI treatment.

In vivo, in the CKI-treated group, survival was longer in an AML patient-derived xenograft model in B-NSG mice. The disease was partially alleviated by decreased CD45+ immunophenotyping in peripheral blood and bone marrow smear analysis. After CKI injection, the T-AOC vitality, GSH content and CAT activity increased and the concentration of H2O2decreased in mouse plasma. A bone marrow biopsy and immunohistochemistry analysis showed that Prdx3 and Prdx2 expression was increased, while Trx1 expression was decreased.

In a conclusion, we provided a model for the anti-leukaemic effects of CKI. CKI decreased intracellular ROS levels by up-regulating the expression of Prdx2 in the cytoplasm and Prdx3 in the mitochondria and down-regulating Trx1 expression, which maintained the intracellular REDOX and further inhibited AML cell proliferation.

Therefore, antioxidant CKI is a promising drug for the treatment of AML in the clinic. We aimed to explore the therapeutic efficacy of low toxicity natural antioxidants against AML by targeting ROS pathways and providing new strategies to improve survival in AML patients.

Disclosures

No relevant conflicts of interest to declare.
Trends in Multiple Myeloma Incidence in the United States (US): Demographic and Geographic Considerations and Focus on Young Adults
Authors: Ruan Gordon , Goyal Gaurav , Hashmi Shahrukh K. , Kapoor Prashant , Go Ronald S.
Published: October 2019

Background: The incidence of multiple myeloma (MM) in the US is increasing among non-Hispanic (NH) White males and females as well as NH Black males (Costa LJ, Blood Advances 2017). However, more specific incidence studies accounting for geographic and age subgroups are limited. There is also a growing concern that incidence of many cancers is increasing in the younger population. Utilizing the Surveillance, Epidemiology, and End Results Program (SEER), we investigated the trends in MM incidence in the US according to demographic and geographic subgroups with a focus on the young adults.

Methods: Incidence rates (case/100,0000) were calculated using SEER*Stat and 2000-2015 data from SEER-18 registries age-adjusted to the U.S. 2000 standard population. We calculated the annual percentage change (APC) of incidence rates according to age groups and stratified by sex, race, and registry. Age at diagnosis was analyzed in 10-year intervals starting at 20 years. All analyses used ICD-O-3 codes 9732/3 with microscopically confirmed MM.

Results: There were 69,613 MM patients included in the analysis. The mean age at diagnosis was 68 years and decreased over time from 68.5 years in 2000 to 67.6 years in 2015. Men comprised 55.5% of the population. Patients <50 years constituted 7.9% of the population with 6.6% between 40-49 years. The incidence significantly (all p values <0.01) increased from 2000-2015 for the entire cohort (incidence 6.9-9.5; APC 1.1) and for males (8.6-9.5; 1.1) and females (5.7-6.3; 0.8). The highest increase in incidence among the age groups (Figure) was in 40-49 years (1.9-2.7; 2.1). When analyzing the trends for age groups stratified by sex, women ages 40-49 years had the highest increase in incidence (1.6-2.6; 2.4). The APCs of SEER-18 registries stratified by age and sex are reported in the Table. The highest APCs (>3.0) were observed in the registries of Atlanta, Greater Georgia, Hawaii, and New Jersey. When the SEER-18 registries were further stratified by race, white men ages 40-49 years in Seattle and New Jersey had APCs of 5.5 and 2.9 respectively, while Black women ages 40-49 years in Greater Georgia had an APC of 3.3. Only 3 groups of individuals had a significant decline in the incidence: Asian women ≥70 years in Los Angeles (APC -6.4), White women ≥70 years in Los Angeles (APC -1.9), and men 50-59 years in San Jose (APC -1.5).

Conclusions: The overall incidence of MM is increasing in the US with the highest increment among young adults between the age groups 40-49 years. We observed geographic and demographic differences in the incidence rates, which may reflect genetic and environmental influences. Interestingly, a few geo-demographic subgroups had a decline in the incidence. Further research is needed to determine the reasons behind the observed trends.

Disclosures

Kapoor: Celgene: Research Funding; Takeda: Research Funding.
Efficacy of Early Convert to Dasatinib in Chronic Myeloid Leukemia Patients Who Inresponse or Intolerance to Imatinib
Authors: Liang Liu , Xu Na , Zhou Xuan , JiXian Huang , Chen Chen , Tong Lin , wan-Er Wu , Yin Changxin , Liu Qifa , Xiaoli Liu
Published: October 2019

Blackground:Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm hallmarked by the presence of fusion protein kinase derived from reciprocal translocation between chromosome 9 and 22, breakpoint cluster region (BCR)-Abelsonleukemia virus (ABL). Since the discovery of a tyrosine kinase inhibitor (TKI), CML has become a chronic illness if managed appropriately. The use of the first generation of TKI such as imatinib, benefit for most CML patients, but there are still a significant number of patients resistance to imatinib , need switched to second generation TKIs, such as dasatinib or nilotinib. however the efficiency and safety of CML patients who inresponse or intolerance to imatinib early convert to dasatinib should clinical studies.

Objective: To evaluate the efficiency and safety of CML patients who inresponse or intolerance to imatinib early convert to dasatinib.

Methods:We retrospectively analyzed 70 CML patients who inresponse or intolerance to imatinib in Southern Hospital from March 2014 to April 2016;real-time quantitative PCR method was used to detect BCR-ABL1 fusion gene Sanger sequencer.

Results: 37 CML patients BCR/ABL (IS)>10% treated by imatinib for 3 to 6 months,

had early conversion to dasatinib. Among them, 31 cases had low response to imatinib (sokal score was middle and high risk) and 6 cases intolerance to imatinib. 24 cases (64.9%) reached BCR/ABL (IS) < 10% after convert to dasatinib for 3 months, and 6 cases (40.5%) reached MMR after convert to dasatinib for 6 months.15 cases (40.5%) reached MMR in 12 months after conversion to dasatinib.33 patients (treated with imatinib for 3 to 6 months) did not undergo early transformation. Among them, 31 patients had poor response to imatinib and 2 patients intolerance to imatinib ,these patients continued imatinib or add imatinib dose to 600mg or 800mg qd. 13 patients (51%) did not reach the BCR/ABL (IS) < 10% for next 3 months, only 12 cases (36.3%) achieved MMR after 12 months follow-up, and 4 patients changed to dasatinib or nilatinib becase detected mutation during follow-up.The most common hematological adverse effects during dasatinib treatment were thrombocytopenia, neutropenia, anemia, and non-hematological adverse effects such as edema, pleural effusion, alopecia, gastrointestinal reactions and rare pulmonary hypertension. AEs can be alleviated by reducing or suspending dasatinib use.

Conclusion: The cml patients who inresponse or intolerance to imatinib early convert to dasatinib will gain an earlier and greater benefit, and adverse effects of dasatinib are controllable.

Key words:Chronic myeloid leukemia, Imatinib, Dasatinib, Early conversion

Disclosures

No relevant conflicts of interest to declare.
AYA ALL: Real World Experience from a Tertiary Care Center in North India
Authors: Das Satyaranjan , Yanamandra Uday , Moslem Mir , Kapoor Rajan , Pramanik Suman , Kumar Rajiv , Khurana Harshit , Sharma Sanjeevan , Verma Tarun , Jasjit Singh , Sharma Ajay , Nair Velu
Published: October 2019

Introduction: The outcomes of Adolescent and young adult (AYA) have been studied in various hemato-oncology subsets. Patients of AYA-ALL have improved outcomes by paediatric-inspired or fully paediatric protocols. The outcomes published from trials are different from the actual practise, that too in the real-world settings with resource constraints.

Objective: To study the characteristics and outcomes of AYA-ALL from real world settings.

Methodology: It is a retrospective observational study wherein the data of all patients of AYA-ALL managed at a tertiary care center in North India over the last 14 years (2004-2018) were analyzed. All case records of the AYA-ALL were perused, digitalized and their survival statistics derived.

Results:Amongst a total of 611ALL case records, 116 (18.98%) were AYA-ALL. AYA-ALL cases with complete data (n-116) were analysed for overall survival. The mean age of the patients was 26.65 ± 6.61 years (range 16-39) (Fig. A). Males constituted 32.7% (n-78) and females, 67.2% (n-38) of AYA-ALL. On risk stratification, 67 (57.7%), 26 (22.4%), and 23 (19.8%) patients were classified as standard, intermediate and high risk. Phenotypically, 78 patients were B-ALL, 23 were T-ALL and rest were MPAL.

Of these 61 patients (52.5%) received adult ALL (GMALL protocol), 26 patients (22.3%) received paediatric BFM protocol, 19 patients (16.3%) received Hyper-CVAD, 8 patients (6.8%) MCP841 protocol and 2 received modified drugs not adhering to any fixed protocol. Week 4 Bone marrow evaluation was in CR in 91.4% of patients. L-Asparaginase was given in only 74.5% of the patients. An interruption in the therapy of more than 2 weeks for various reasons was present in 35% of the patients. Prophylactic cranial irradiation was given in 42.4% of the patients and high dose methotrexate was given in 25.2% of the patients. Relapse was seen in 27.7% of the patients. CNS disease was present in 9.4% of the patients. Only 8% of the patients were subjected to transplant.

The cumulative overall survival at 1y (1y-OS) was 86.98% with 3y and 5y OS being 67.9% and 55.6% (Fig. B). The survivalsbased on the type of protocol was statistically different with the best survival with GMALL protocol and the least survival with Hyper-CVAD therapy (log rank p-0.0011) (Fig.C). The survival was also significantly different between different risk groups (p-0.003) (Fig.D). The survival was not statistically different between males and females (p-0.5), B-ALL vs T-ALL (p-0.6) (Fig. E, F).

Conclusion: We have demonstrated in this study the improved outcomes of AYA-ALL who usually present in late chronic phase in resource constraint settings. Contrary to the belief, adult GMALL protocol had better survival vis-à-vis pediatric BFM protocol in this cohort of AYA-ALL.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
Zinc Finger Protein X-Linked Exhibits Pro-Leukemia Activity Via WNT3 in Chronic Myeloid Leukemia Stem/Progenitor Cells
Authors: Zhang Xiuyan , Wang Yu , Xiao Lun , Zhou Haixia , Zhu Li , Zhao Yun
Published: October 2019

Zinc Finger protein, X-linked (ZFX) is a transcriptional regulator, which controls the self-renewal of both embryonic and hematopoietic stem cells and participates in pathogenesis of various cancers. Zfx deficiency impairs Notch intracellular domain (NotchIC) induced acute T-cell leukemia (T-ALL) or MLL-AF9 induced acute myeloid leukemia (AML) in mice models. However, the function of ZFX in chronic myeloid leukemia (CML) stem/progenitor cells has not been elucidated yet.

In the present study, qRT-PCR analysis showed that ZFX expression was significantly higher in CD34+ cells from CML patients in chronic phase (CP) (n=8, 3.1-fold, P=0.0052) and patients in blast crisis (BC) (n=8, 18.6-fold, P=0.0050) compared with that in healthy donors (n=4). Two independent shRNA sequences against ZFX were delivered in CD34+ cells with lentiviral vector. The silence of ZFX had a stronger inhibitory effect on colony-forming cell (CFC) ability of CML CD34+ cells (75±5%) than that of healthy donor CD34+ cells (44±5%). Furthermore, ZFX silencing augmented Imatinib Mesylate (IM) sensitivity of CML CD34+ cells, especially in IM-resistant samples; due to the fact that ZFX silencing increased apoptosis induced by IM. To obtain the molecular insights of how ZFX acts, we generated transcriptome data comparing ZFX silenced CML CD34+ cells with control cells. qRT-PCR data validated that ZFX silencing caused a significantly declined expression of WNT3 in K562, MEG-01 and CML CD34+ cells (n=5). In addition, ZFX silencing decreased WNT3 protein expression as well. Interestingly, WNT3 had significantly higher expression in CD34+ cells from patients in CP (n=10, 5-fold, P=0.0006) compared with that in healthy donors (n=8). Silence of WNT3 inhibited the growth of K562 cells and enhanced IM sensitivity of these cells as well. Overexpression of WNT3 restored the growth inhibition and IM hypersensitivity upon ZFX silencing. Chromatin immunoprecipitation (ChIP) analysis revealed that ZFX was able to bind with WNT3 promoter, and luciferase assay showed that ZFX silencing significantly decreased the activity of WNT3 promoter. Finally, we also found that the expressions of c-Myc and cyclin D1 were reduced by ZFX silencing or WNT3 silencing, suggesting decreased WNT/Catenin signaling.

Taken together, we have demonstrated that ZFX is aberrantly expressed in CML stem/progenitor cells, and it modulates the growth and IM response of CML stem/progenitor cells via wnt/Catenin pathway indicating ZFX is a new regulator of CML stem/progenitor cells, which deepens the understanding of CML pathology and potentially provides clues for novel therapies against this disease.

Disclosures

No relevant conflicts of interest to declare.
Children with Inflammatory Bowel Disease Exhibit Insensitivity to Tissue Factor Pathway Inhibitor
Authors: Schlagenhauf Axel , Haidl Harald , Sina Pohl , Jörg Jahnel , Muntean Wolfgang , Siegfried Gallistl
Published: October 2019

Background: Patients with inflammatory bowel diseases (IBD) exhibit chronic inflammation of the digestive tract associated with a prothrombotic shift in the plasmatic coagulation system as well as heightened platelet reactivity and preactivation. Recently, microbial and platelet-derived inorganic polyphosphate has been shown to inhibit tissue factor pathway inhibitor (TFPI), thus, influencing the hemostatic balance during inflammation. We hypothesized that polyphosphate plays a role in the pathophysiology of inflammatory bowel disease resulting in refractoriness to TFPI activity.

Aims: We aimed to determine the hemostatic sensitivity to exogenous TFPI in IBD patients and healthy controls.

Methods: Plasma from pediatric patients with active Crohn's disease (CD) (N=10) or ulcerative colitis (UC) (N=10) and age-matched healthy controls (N=20) was spiked with recombinant TFPI (150 ng/ml). Thrombin generation with/without exogenous TFPI was performed using Calibrated Automated Thrombography. Differences in lag time of thrombin generation with/without TFPI were calculated (∆lag time).

Results: Without addition of exogenous TFPI, IBD patients exhibited a shorter lag time than controls (IBD: 2.65±0.41 min; Controls: 3.72±0.48 min, P<0.001) Addition of exogenous TFPI prolonged the lag time significantly in healthy controls (∆lag time= 4.69±0.61 min; P<0.001), while the lag time was just slightly prolonged in IBD patients (CD: ∆lag time=0.92±0.37 min; P<0.001; UC: ∆lag time=0.34±27 min; P<0.05).

Conclusion: Plasma samples from pediatric IBD patients exhibit refractoriness towards the anticoagulant activity of exogenous TFPI. This hyposensitivity potentially extends to the action of endogenous TFPI which adds to the prothrombotic phenotype associated with IBD. Further studies are needed to determine potential associations with disease activity and the susceptibility to develop thrombosis.

Disclosures

No relevant conflicts of interest to declare.
Changing the Paradigm in Hemophilia Care: Extended Half-Life Products (rFVIIIFc and rFIXFc)
Authors: Aledort Louis M. , Jain Nisha , Li Nanxin , Krishnan Sangeeta , Hagberg Benjamin , Su Jun
Published: October 2019

Background:

Prophylactic treatment with factor products has demonstrated superior outcomes and care compared to on-demand treatment in hemophilia patients, including patients with pre-existing joint diseases. However, 40% to 50% of severe or moderate hemophilia A and B patients are still treated on-demand in the US. Recombinant factor VIII and IX Fc fusion proteins (rFVIIIFc and rFIXFc) were the first extended half-life (EHL) products approved in the US (in 2014) with the promise of optimizing prophylactic regimens by achieving low bleeding rates and improving joint health with fewer injections overall.

Objective:

This is the first real-world study to assess the impact of EHL rFVIIIFc and rFIXFc on overall prophylaxis rates, particularly to understand how patients have transitioned from on-demand treatment.

Methods:

A retrospective analysis was conducted on aggregate, de-identified United States Specialty Pharmacy Provider (SPP) records from Jan 2014 to June 2018 (data pull on June 14th, 2018). Adult hemophilia patients (age 18 and above) were included for an analysis of treatment regimens prior to and after switching to EHL rFVIIIFc and rFIXFc (on-demand or prophylaxis). The results were descriptively compared to those who switch to the other EHL PEGylated rFVIII and rFIX albumin Fusion Protein (rFIX-FP) and conventional factors.

Results:

A total of 304 hemophilia A patients who switched to rFVIIIFc and 133 hemophilia B patients who switched to rFIXFc were included in this study. For the hemophilia A and B patients, the median age was 31 and 32 years and median weight was 82 and 79 kg. The proportion of patients who switched from on-demand treatment with conventional factors to prophylaxis treatment with EHL rFVIIIFc and rFIXFc among all patients who switched to these products increased over time since the FDA approval (3 time points: 2014-2015, 2016-2017, and 2018: rFVIIIFc: 13%, 20%, and 21%; rFIXFc: 25%, 29%, and 57%). Such proportions were higher compared to those who switched from on-demand with conventional factors to prophylaxis with the other EHLs (2016-2017, 2018: PEGylated rFVIII: 16%, 18%; rFIX-FP: 18%, 13%, see comparison in the Figure) and with conventional factors (2014-2015, 2016-2017, and 2018: conventional rFVIII: 10%, 7%, and 9%; conventional rFIX: sample size was too small and not suitable for analysis).

Discussion:

rFVIIIFc and rFIXFc have transformed the paradigm of hemophilia care by shifting on demand patients to prophylaxis significantly faster than conventional factors and any other EHLs to date. This shift is significant as it is helping more hemophilia patients achieve optimal protection through low bleeding rates and improved joint health over the long-term.

Figure. Figure.

Disclosures

Jain: Bioverativ: Employment. Li:Bioverativ: Employment. Krishnan:Bioverativ: Employment. Hagberg:Bioverativ: Employment. Su:Bioverativ: Employment.
Impact of Non-CMV Specific Intravenous Immunoglobulin on Intravenous Ganciclovir Effectiveness
Authors: Drokov Mikhail Yu. , Tikhomirov Dmitry S. , Kuzmina Larisa , Tupoleva Tatiana A. , Vasilyeva Vera , Popova Natalia , Mikhaltsova Ekaterina , Koroleva Olga , Dubnyak Daria , Sidorova Anna , Usikova Ekterina , Konova Zoya , Dmitrova Anna , Gaponova Tatiana V. , Parovichnikova Elena , Savchenko Valery G.
Published: October 2019

Introduction Cytomegalovirus (CMV) reactivation is one of the main problems in allogenic hematopoietic stem cell transplantation (allo-HSCT). Standard of care for CMV-disease is still ganciclovir at 10 mg/kg b.i.d. Use of non-CMV specific intravenous immunoglobulin (IVIG) is still controversial and mostly determined by internal care protocols in every transplant center. Here we report data about impact of non-CMV specific IVIG during ganciclovir treatment in allo-HSCT CMV-patients.

Patients and methods Thirty two patients with hematological malignancies transplanted in NRCH were included in this study. Detailed patients characteristics are listed in Table 1. All patients were CMV-positive by PCR (Amplisens EBV/ CMV /HHV6-screen-FL", InterLabService, Russia) and required to start therapy with ganciclovir 10 mg/kg when was included to this study. Patients in IVIG group also received non-CMV specific IVIG during ganciclovir treatment with a median total dose 20 g (7.5-90 g.). Kaplan-Meier estimator was used to determine probability of achievement of CMV-negativity from time of ganciclovir was started to CMV-negativity (by PCR) or last contact. The logrank test is used to compare differences between two groups. Fisher's exact test was used for 2×2 tables. P-value less than 0.05 was considered statistically significant

Results Influence of IVIG in allo-HSCT patients on probability of achievement of CMV-negativity (by PCR) are shown in the Figure 1. As we can see there is no significant differences on the achievement of CMV-negativity (by PCR) in patients with/without IVIG during ganciclovir therapy.

Conclusion The use of non-CMV specific immunoglobulins after allo-HSCT still lies in the plane of faith and the possibilities of the transplantation center than any evidence base. Using IVIG in patients after allo-HSCT is still not a routine in terms of CMV-reactivation/disease therapy and mostly based on an "experience" of every transplant center. According to our data there is no difference between IVIG/no-IVIG groups during ganciclovir treatment so use of IVIG, for our opinion, does not really make sense for CMV-disease treatment. Anyway a large randomized trial is required for determine indications for therapy with IVIG.

Disclosures

No relevant conflicts of interest to declare.
Exome Sequencing of Extreme Phenotypes Suggests Novel Candidate Genes As Modifiers of Leg Ulcer in Sickle Cell Anemia
Authors: de Carvalho Siqueira Gabriela Queila , Ananina Galina , Borges Murilo Guimarães , de Souza Bruno Batista , Cendes Iscia Teresinha Lopes , Costa Fernando F. , de Melo Mônica Barbosa
Published: October 2019

Background: Despite resulting from homozygosity of a single mutation at position 6 of the beta-globin locus, the clinical aspects of sickle cell anemia (SCA) are very heterogeneous. Leg ulcer is one of the many resulting complications, exerting a quite negative impact in the quality of life of patients and is related to the severity of the disease. The pathogenesis of such complication is complex and still not well explained. Objective: Seeking for novel genes associated with leg ulcer in SCA, we proposed exome sequencing of extreme phenotypes in a sample of the Brazilian population. Methods: Our sample consisted of 40 unrelated patients with SCA from the Hematology and Hemotherapy Center of the University of Campinas (Unicamp), Campinas, SP, Brazil. The cohort was composed by 20 patients who didn't have leg ulcer, with ages varying from 40 to 61 years (it is unusual the appearance of this disorder at such age) and 20 patients with chronic leg ulcer. DNA was collected from peripheral blood leukocytes and submitted to exome sequencing. Capture and enrichment were performed with the Nextera Rapid Capture Exome kit (Illumina) and samples were loaded in the Illumina HiSeq2500. The bioinformatics process was based on the GATK Pipeline and manual filters in vcftools and excel. Briefly, we prioritized the variants according to some conditions, like score quality, depth, Hardy-Weinberg equilibrium and minor allele frequency greater than 0.1. Subsequently, through PLINK, we selected the variants with p value less than 0.05 from Fisher association test. The annotation was made through wAnnovar. We excluded the synonymous variants, remaining with 244 variants at this point. Finally, the deleterious variants were selected, in addition to those not classified by SIFT, PolyPhen2 and FATHMM. Twenty-one variants remained, all of them in genes beyond the usual set of a priori biological candidate genes for this phenotype. We picked up 6 variants with the lowest p values: rs4857302 (CRYBG3), rs3782489 (KRT77), rs11800462 (TNFRSF25), rs13428956 (FOXD4L1), rs201853154 (UBTFL1) and rs11454536 (VWDE). We suggest that these variants and their genes could potentially be related to the development of leg ulcer in SCA. However, they should all be validated in other populations for confirmation.

Disclosures

No relevant conflicts of interest to declare.
Identification of Syndecan-1 As a Key Dependency of Myeloid Leukemia Growth and Dissemination
Authors: Spinler Kyle , Reya Tannishtha , Bajaj Jeevisha , Ito Takahiro , Zimdahl Bryan , Ahmadi Armin , Koechlein Claire , Lytle Nikki , Kwon Hyog Young , Anower-E-Khuda Ferdous , Kritzik Marcie , Karlseder Jan , Park Pyong Woo , Esko Jeffrey
Published: October 2019

Intratumoral heterogeneity is a common feature of many myeloid leukemias and a significant reason for treatment failure and relapse. Thus identifying the cells responsible for residual disease and leukemia re-growth is critical to better understand how they are regulated. Here we show that a knock-in reporter mouse for the stem cell gene Musashi 2 (Msi2) allows identification of therapy resistant leukemia propagating cells in aggressive myeloid malignancies, and provides a new strategy for defining their core dependencies. Specifically, we carried out a high throughput screen using Msi2 reporter blast crisis chronic myeloid leukemia (bcCML) and identified syndecan-1 (Sdc1), a cell surface proteoglycan, as preferentially expressed in therapy resistant bcCML cells, and critical for bcCML function. Specifically, in Sdc1-/- mice, Sdc1 loss led to a defect in bcCML growth and propagation in vitro and in vivo, and markedly improved survival. Further, live imaging revealed that Sdc1 loss had a striking impact on the spatiotemporal dynamics of leukemia cells, impairing their localization, migration and systemic dissemination. Mechanistically, distinct elements of Sdc1 contributed to leukemia growth and dissemination, with the core protein alone being able to rescue the growth defect, but the heparin sulfate chains that mediate matrix attachment being needed for migration. These data present a new platform for delineating the biological underpinnings of leukemia stem cell function, and identify Sdc1 as a central regulator of leukemia stem cell growth and dissemination.

Disclosures

No relevant conflicts of interest to declare.
Relationship of Markers of Inflammation, Infection and Endothelial Function to Mortality and Severity of Coagulopathy in Patients with Sepsis-Associated DIC
Authors: Hoppensteadt Debra , Walborn Amanda , Rondina Matthew T. , Fareed Jawed
Published: October 2019

Background: Sepsis-associated disseminated intravascular coagulation (DIC) is a complex clinical scenario involving derangement of many processes, including hemostasis. Assessment of markers of these other aspects of disease, including inflammation, immunity, endothelial function, and endogenous anticoagulants may provide insight into DIC pathophysiology and lead to improved methods for assessment of patient condition and response to treatment. This study was designed to measure biomarkers representative of multiple systems involved in DIC development in a cohort of patients with sepsis and DIC and determine the association of these biomarkers with disease severity and mortality.

Materials and Methods: Citrated plasma samples were prospectively collected from 102 patients with sepsis and suspected DIC at ICU admission (day 0) and again serially on ICU days 4 and 8 under an IRB approved protocol. DIC score was determined using the ISTH scoring algorithm (e.g. platelet count, PT/INR, fibrinogen, and D-Dimer). Plasma from healthy individuals was purchased from a commercial laboratory (George King Biomedical, Overland, KS). . CD 40 Ligand (CD40L), Plasminogen inhibitor 1 ( PAI-1), nucleosomes, Procalcitonun (PCT), Microparicle Tissue Factor (MP-TF), and Prothrombin Fragment 1.2 (F1.2) were measured using commercially available ELISA kits. Protein C activity was measured using a clot-based assay. Interleukin 6 (IL-6), Interleukin 8 (IL-8), Interleukin 10 (IL-10) , Tumor Necrosis Factor Alpha (TNFα) , Monocyte Chemoattractant protein 1 (MCP-1) were measured using a biochip technology.

Results: Significant differences in levels of Protein C (p=0.009), PCT (p=0.0005), IL-6 (p=0.019), IL-8 (p= 0.015), and PAI-1 (p=0.015) were observed between survivors and non-survivors (Table 1). Significant variation of Protein C (p=0.002), nucleosomes (p=0.05), PCT (P<0.001), IL-6 (p=0.001), IL-8 (p=0.003), IL-10 (p=0.011), TNFα (p=0.021), and MCP-1 (p=0.21), were observed based on severity of DIC score.

Conclusion: Markers from multiple systems perturbed in DIC were associated with mortality suggesting that while these systems may not be routinely evaluated in the course of patient care, dysfunction of these system contribute significantly to mortality. In addition, numerous inflammatory cytokines showed an association with DIC score. This study suggests that the measurement of additional markers in sepsis-associated DIC may be of value in the prediction of mortality and may be helpful in guiding treatment for these patients.

Disclosures

No relevant conflicts of interest to declare.
Role of Point-of-Care Device in Improving Sickle Cell Newborn Screening Feasibility in Haiti
Authors: Alvarez Ofelia A. , Hustace Tally , Voltaire Mimose , Rizil Rodrigueson , Liberus Ulrick , Saint Fleur Rony
Published: October 2019

Introduction: Until 2017, hemoglobinopathy newborn screening (NBS) was not offered in Haiti, a country with about 243,000 births per year. Sickle SCAN is a rapid point-of-care (POC) with lateral flow immunoassay technology, but its role in NBS is unknown. Recently, over 100 dried blood samples from newborns were tested [Nguyen-Khoa T, Ann Biol Clin (Paris), 2018] in France with accurate results. Objective: This work had the objective to be proof-of-concept that a hospital-based NBS program is feasible in Haiti and to examine the role of Sickle SCAN in NBS in a larger sample size. Methods: The Ethics Committee at the Universitaire Justinien Hospital (HUJ) and the Institutional Review Board at the University of Miami approved this study. We formed a Haitian team composed of a pediatrician on staff (RSF), two dedicated NBS nurses, a program coordinator (MV), two community health workers, a laboratory technician (RR), and a data manager (UL). We acquired isoelectric focusing equipment (IEF) and performed NBS from dried blood samples only for the first three months while the technician gained proficiency. Thereafter, we have performed dual screening method with the POC device Sickle SCAN and IEF. Confirmatory testing was obtained with both methods. Data were entered into RED Cap. Results: Beginning in August 2017 until present (June 2018), we have screened 1,800 newborns, of which 10.5% have sickle cell trait, 3.3% have hemoglobin C trait, 0.78% have hemoglobin SS, 0.27% have hemoglobin SC and one child has been confirmed to have sickle beta thalassemia plus. Currently, there are 15 children followed at HUJ, for a SCD incidence of 0.83% (8 SS, 6 SC and 1 S-beta thalassemia+). Before the POC screening was implemented two infants have already died when the mothers were contacted (one who screened positive for FS and another for FC). The POC allows for immediate referral and penicillin prophylaxis for at-risk children until the cases are confirmed. Systemic barriers encountered are the electrical outages and intense heat which impacted IEF performance, material procurement (laboratory materials, NBS materials, oral penicillin) which are dependent of shipments from outside of Haiti.

Conclusions: A hospital-based NBS Program is feasible. SCD is highly prevalent with an incidence of 0.83% among newborns in the population studied. Based on this incidence, we estimate that every year around 2,000 children will be born with sickle cell disease in Haiti. The point-of-care device enhanced the screening program by obtaining immediate screening results and maximizing family notification for confirmatory testing.

Disclosures

No relevant conflicts of interest to declare.
Expression Status of Prohibitin Gene Predicts the Prognosis of Acute Myeloid Leukemia with Normal Cytogenetics
Authors: Shin Myung Geun , Lee Jun Hyung , Kim Hye Ran , Na Bo Ram , Lee Young Eun , Choi Hyun Jung , Kim Soo Hyun , Shin Jong Hee , Suh Soon Pal
Published: October 2019

Introduction: Cytogenetically normal acute myeloid leukemia (CN-AML) accounts for 40 to 50 percent of all AML and shows significant heterogeneity in clinical outcomes. Despite the advancements in the molecular pathophysiology of CN-AML, clinical outcome of this leukemia remains to be elucidated. Prohibitin (PHB) were typically known to have the potential act as a tumor suppressor, anti-proliferative regulator of cell-cycle in normal cells. However, our previous study discovered that high expression of PHB in AML cells was involved in escaping apoptosis process. In this study, we investigated the clinical significance of expression levels of PHB1 and PHB2 genes in patients with CN-AML.

Methods: We developed quantitative reverse transcription PCR (qRT-PCR) detection system for mRNA expression status for PHB1 and PHB2.The expression levels was analyzed in 69 CN-AML patients. Total RNA was extracted from bone marrow samples at the diagnosis using InvitrogenRNAqueous Total RNA Isolation Kit (Thermo Fisher Scientific, USA). The concentration of isolated total RNA was measured by Micro UV-VIS spectrophotometer (Malcom, Japan). Quantification of mRNA of PHBs were performed in CFX96 Touch Real-Time PCR system (BIO-RAD, USA) using our laboratory-developed, customized primers and probes.The gene expression levels werenormalized compared to those of ABL1 gene.Survival analysis was performed compared with other prognostic factors: age, NPM1 mutation, and FLT3-ITD. FLT3-ITD was tested by PCR & fragment analysis (LeukoStrat FLT3 Mutation kit; Invivoscribe, USA) and NPM1 mutation was tested using qRT-PCR kit (ipsogen NPM1 MutaQuant; Qiagen, Germany). All statistical computations and graphical process were performed using R version 3.5.1. A P value ≤ 0.05 (for 95% confidence interval) was considered statistically significant.

Results: The median age of enrolled patients was 68 (ranged 23-85) at the diagnosis. The ratio of male and female was 1:0.86 (37:32). There was no significant difference in age, sex, WBC count in peripheral blood, blast percentage of peripheral blood and bone marrow, the achievement rate of complete remission, and the enforcement of stem cell transplant according to expression status of PHB1 and PHB2. Using 0.61 as cutoff, PHB1 high-expressed group showed superior overall survival (OS) (47.7% vs 15.1%; P = 0.014) and low hazard ratio (0.43 [95% CI: 0.215-0.861]; P = 0.017) compared to PHB1 low-expressed group. In PHB2, using a cutoff 2.39, high-expressed group showed as inferior OS (17.5% vs 40.0%; P = 0.16) and high hazard ratio (1.93 [95% CI:0.739-5.042]; P = 0.18) compared to low expressed group.

Conclusions: PHB1 expression status was associated with favorable clinical outcome. In contrast, those of PHB2 was related with poor prognosis in CN-AML.

Disclosures

No relevant conflicts of interest to declare.