bluuebunny/crossref_metadata_2025 huggingface dataset:
The Study of Thrombosis in JAK2V617F Mutated Myeloproliferative Neoplasms
Authors: Bai Jie , Zhang Yuhui , Wang Yingshao , Teng Guangshuai , Wang Yan , Du Chenxiao , Chen Yafang , Zhang Huiqin , Li Yanqi , Fu Lixia , Zhang Lei , Zhou Yuan
Published: November 2019

Introduction: Thrombosis is the most important risk factor affecting event-free survival and overall survival (OS) of patients with myeloproliferative neoplasms (MPNs). To explore the risk factors of thrombosis in patients with JAK2V617F-mutated MPN, a cohort of 1537 Chinese patients with JAK2V617F-mutated MPN was retrospectively analyzed for clinical characteristics, laboratory characteristics, cytogenetics, thromboembolism, disease progression and OS.

Methods: The qualitative data were compared by the Chi-square test or Fisher's test, and continuous variables were compared with the Mann-Whitney U test or Kruskal-Wallis test. The Kaplan-Meier method and multivariate Cox analysis were used to study the risk factors of thrombosis in patients with JAK2V617F-mutated MPN.

Results: Among the 1537 MPN patients, 931, 468 and 138 had polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), respectively. The median follow-up time was 7 years (range 1-47), and 12.8% of patients (197/1537) died during this period. A total of 16.8% (259/1399) of PV and ET patients had secondary myelofibrosis, and 2.5% (38/1537) of patients developed acute myeloid leukemia (AML). Thrombosis events occurred in 43.9% (675/1537) of patients, among which 91.4%(617/675) were arterial thrombosis and 16.6% (112/675) were venous thrombosis. The number of thrombosis events in PV, ET and PMF patients was 439 (47.2%), 197 (42.1%) and 39 (28.2%), respectively. The multivariate analysis indicated that age≥60 years old (P=0.003, HR=1.76, 95% CI[1.214, 2.552]), HCT≥48% (P=0.022, HR=1.635, 95% CI[1.073, 2.492]), at least one cardiovascular risk factor (P=0.024, HR=1.559, 95% CI[1.061,2.291]), a history of thrombosis (P<0.0001, HR=2.313, 95% CI[1.573,3.401]), and JAK2V617Fallele burden (V617F%) ≥50% (P=0.003, HR=1.804, 95% CI [1.221, 2.665]) are risk factors for thrombosis in JAK2V617F-mutated MPN. According to the results of the multivariate analysis, a risk model of thrombosis was established and comprised low-risk (0 points), intermediate-risk (1 points) and high-risk (≥2 points) groups, among which the incidence of thrombosis was 9.1%, 33.7% and 72.9%, respectively.

Conclusions: The Chinese thrombosis risk model of JAK2V617F-mutated MPN established in this study suggests that elderly patients with a history of thrombosis should reduce V617F%, control HCT to less than 48% and mitigate cardiovascular risk factors, all of which are necessary measures to prevent thrombosis in patients with JAK2V617F-mutated MPN.

Disclosures

No relevant conflicts of interest to declare.
Examining the Role of CD30 in an Anaplastic Large Cell Lymphoma Mouse Model
Authors: Poggio Teresa , Graessel Linda , Kreutmair Stefanie , Miething Cornelius , Pfeifer Dietmar , Andrieux Geoffroy , Boerries Melanie , Follo Marie , Turner Suzanne , Zeiser Robert , Duyster Justus , Illert Anna Lena
Published: November 2019

Anaplastic large cell lymphoma (ALCL) represents a heterogeneous group of T-cell non-Hodgkin lymphoma (NHL) mainly affecting children and young adults. About 70% of systemic ALCLs are associated with a characteristic chromosomal translocation, t(2;5)(p23;35) which fuses the anaplastic lymphoma kinase (ALK) gene on chromosome 2 with the nucleophosmin (NPM) gene on chromosome 5, resulting in the NPM-ALK fusion gene, its over-expression and constitutive kinase activity. Immunophenotypic characterization of human ALCL revealed highly CD30-positive cells of T- or Null-Cell-origin and resulted in promising clinical trials with CD30-coupled antibodies. However, the impact of CD30 on disease development remains unclear and the relationship between NPM-ALK and CD30 has been investigated mostly using cell line models. Syngeneic mouse models of cancer can serve as useful models since the tumors develop in situ where the contribution made by the immune system and the extracellular matrix can be investigated.

Here, we focus on the involvement of CD30 in a retroviral murine bone marrow transplantation model of ALCL. In this model, the BM of Lck-Cre-transgenic mice is infected with a MSCV-Stop-NPM-ALK-IRES-EGFP vector leading to expression of NPM-ALK in early T-cells. With a latency of 3-4 months, mice develop lymphomas and die from neoplastic T-cell-infiltration of BM and lymphatic organs. To investigate the impact of abrogation of CD30 signals on the development of NPM-ALK+ ALCL in our model, CD30 knockout mice were crossed with Lck-Cre mice. Both Lck-Cre NPM-ALK CD30 wt and Lck-Cre NPM-ALK CD30 ko recipients develop a human ALCL-like lymphoma with a pure T-cell phenotype characterized by Thy1.2+ cells infiltrating the thymus, lymph nodes, spleen and BM. First results from Lck-Cre NPM-ALK CD30 ko transplanted mice showed impaired disease induction and prolonged survival compared to CD30 wt animals. Moreover, secondary transplantation of NPM-ALK thymic lymphomas led to distinct deceleration of disease development upon CD30 deletion. Microarray analyses have shed some light on the mechanisms underlying the delayed lymphoma progression of CD30 deleted tumors with an upregulation of inflammatory pathways and proteins that are master players in inflammation and immune responses. Further characterization of the role of CD30-mediated immune response in disease progression using this mouse model and immunocompromised mice is ongoing. An improved understanding of how the immune system affects tumor progression will extend the rationale in translational strategies to use immunotherapies for patients with T-NHLs.

Disclosures

No relevant conflicts of interest to declare.
Use of Fluid and Oxygen Therapy in Normoaxaemic Paediatric Patients Presenting with Acute Sickle Vaso-Occlusive Episodes Requiring Hospital Admission and Analysis of Clinical Outcome
Authors: Butt Zain , Chakravorty Subarna
Published: November 2019

Introduction: There is limited consensus on the optimal regime and benefit of fluid and oxygen therapy when treating vaso-occlusive episodes (VOEs) in children with sickle cell disease (SCD). The purpose of this study was to investigate the current prevalence of intravenous fluid and oxygen therapy when treating VOEs in children with SCD in a large tertiary hospital in London, UK and analyse the clinical outcome of these patients, in order to design a future study to determine optimum use of fluid and oxygen therapy in children presenting with VOE.

Methods: We studied a random selection of 100 vaso-occlusive events in paediatric sickle cell patients resulting in hospital admission between 2008-2019. All patients received assessment and initial treatment according to established hospital guidelines at the emergency department (ED) and were subsequently admitted to an inpatient ward. Baseline clinical and laboratory information, alongside data on the admission, duration, treatment and clinical outcome of each VOE were recorded. Data were compared between therapy and non-therapy groups.

Results: Out of 100 VOE events, 46 were given fluid therapy and 24 were given oxygen therapy. All cases of VOE on fluid therapy were treated with IV dextrose saline (0.5% dextrose and 0.45% NaCl), and the average rate of fluid given was 71.4 ml/kg/24hour (±20.7). No intravenous bolus of normal saline was administered. Fluid therapy was associated with a higher reduction in pain score points at discharge (5.4±2.3 vs 4.8±2.6). However, it was also associated with more chest x-rays (CXR) positive for consolidation (20% vs 7%) and more acute chest syndrome (ACS) diagnosis (9% vs 2%). The mean oxygen saturation in air of patients given oxygen therapy in ED was 96.6% (±4.8) and those that were not given oxygen therapy in ED was 98.2% (±1.6). Oxygen therapy was associated a higher reduction in pain score points at discharge (6.3±2.5 vs 4.7±2.4). However, in the oxygen therapy group, more CXRs were undertaken (71% vs 30%), more CXRs were positive for consolidation (42% vs 4%) and there was more (ACS) diagnoses (21% vs 0%).

Conclusion: These data reveal that about half of children presenting with VOEs received intravenous fluid therapy and about a quarter received oxygen therapy for the treatment of VOEs in this single-centre study. These therapies were associated with positive and negative impacts on clinical outcome. This highlights the need for randomised-controlled trials to evaluate the optimal regime for such therapies.

Disclosures

No relevant conflicts of interest to declare.
FLT3ITD Target Enhancers Are Primed but Inaccessible in Fetal Hematopoietic Progenitors
Authors: Li Yanan , Patel Riddhi M , Casey Emily , Magee Jeffrey A.
Published: November 2019

The FLT3 Internal Tandem Duplication (FLT3ITD) is common somatic mutation in acute myeloid leukemia (AML). We have previously shown that FLT3ITD fails to induce changes in HSC self-renewal, myelopoiesis and leukemogenesis during fetal stages of life. FLT3ITD signal transduction pathways are hyperactivated in fetal progenitors, but FLT3ITD target genes are not. This suggests that postnatal-specific transcription factors may be required to help induce FLT3ITD target gene expression. Alternatively, repressive histone modifications may impose a barrier to FLT3ITD target gene activation in fetal HPCs that is relaxed during postnatal development. To resolve these possibilities, we used ATAC-seq, as well as H3K4me1, H3K27ac and H3K27me3 ChIP-seq, to identify cis-elements that putatively control FLT3ITD target gene expression in fetal and adult hematopoietic progenitor cells (HPCs). We identified many enhancer elements (ATAC-seq peaks with H3K4me1 and H3K27ac) that exhibited increased chromatin accessibility and activity in FLT3ITD adult HPCs relative to wild type adult HPCs. These elements were enriched near FLT3ITD target genes. HOMER analysis showed enrichment for STAT5, ETS, RUNX1 and IRF binding motifs within the FLT3ITD target enhancers, but motifs for temporally dynamic transcription factors were not identified. We cloned a subset of the enhancers and confirmed that they could synergize with their promoter to activate a luciferase reporter. For representative enhancers, STAT5 binding sites were required to activate the enhancer - as anticipated - and RUNX1 repressed enhancer activity. We tested whether accessibility or priming changed between fetal and adult stages of HPC development. FLT3ITD-dependent changes in chromatin accessibility were not observed in fetal HPCs, though the enhancers were primed early in development as evidenced by the presence of H3K4me1. Repressive H3K27me3 were not present at FLT3ITD target enhancers in either or adult HPCs. The data show that FLT3ITD target enhancers are demarcated early in hematopoietic development, long before they become responsive to FLT3ITD signaling. Repressive marks do not appear to create an epigenetic barrier to enhancer activation in the fetal stage. Instead, age-specific transcription factors are likely required to pioneer enhancer elements so that they can respond to STAT5 and other FLT3ITD effectors.

Disclosures

No relevant conflicts of interest to declare.
TRAF1-ALK Fusion Predicts Poor Prognosis for ALK Positive Anaplastic Large Cell Lymphoma Patients with Chemotherapy and ALK Inhibitor
Authors: Huang Ling , Jiang Xinmiao , Guo Hanguo , Sichu Liu , Wei Xiaojuan , Feili Chen , Liang Zhanli , Li Wenyu
Published: November 2019

Background: Relapsed/refractory anaplastic lymphoma kinase (ALK) positive anaplastic large cell lymphomas (ALCLs) respond to ALK inhibitors, but resistance, which bear a poor prognosis. No biomarkers were found to predict long duration of response to ALK inhibitors. The ALK gene was first identified as the fusion partner of the nucleophosmin (NPM1) gene in the recurrent t(2;5)(p23;q35) found in a subset of ALCL. However, several distinct ALK fusions which result in highly different characteristics have also been described in lymphomas.

Methods: We retrospectively reviewed seven relapsed/refractory ALK positive ALCLs who received ALK inhibitors (six with Crizotinib and one with Alectinib) at Guangdong Provincial People's Hospital from June 2007 through March 2019. We did next generation sequencing (NGS) with paraffin-embedded tissue for two patients who quickly developed resistance.

Results: Of the seven patients, four were male and three were female, with a median age of 18 years (range 15-51) old. The median line of therapies was four (range 3-7) and that of ALK inhibitor usage was three (range 2-5). The overall response rate was 7 of 7 (100%) and the median overall survival of 21.2 months (8.5-86 months). Three patients obtained complete response (CR) on Crizotinib and then received autologous stem cell transplantation, and are still CR. One patient obtained CR, but died of serious infection five months after allogeneic stem cell transplantation. One patient is in CR under continuous crizotinib administration. One patient received Crizotinib obtained CR, but three months later got progression disease, the NGS showed TNF receptor-associated factor 1 gene (TRAF1) exon 6-ALK exon 20 fusion junction. The last patient was CR on alectinib, but quickly developed resistance, with a progression free survival of 1 month, the NGS indicated TRAF1 exon 7-ALK exon 20 fusion.

Conclusions: ALK inhibitors improved survival of relapsed/refractory ALK positive ALCLs. TRAF1-ALK fusion may predict poor clinical outcome to chemotherapy and ALK inhibitors. This ALK fusion may reflect a trend to aggressive behaviour in lymphomas.

Disclosures

Li: Guangdong Province Hospital: Employment.
What Can We Expect after Performing Allogeneic Stem Cell Transplantation for Adult T Lymphoma in Patients from Romania, Europe?
Authors: Tanase Alina Daniela Daniela , Craciun Oana Gabriela , Lipan Lavinia , Ranete Adela , Stefan Diana Laura , Varady Zsofia , Bumbea Horia , Colita Andrei , Hermine Olivier , Pasca Segiu , Teodorescu Patric , Tomuleasa Ciprian
Published: November 2019

Introduction: Adult T lymphoma (ATL) is a rare and aggressive disease shown to be consistently caused by the human T lymphoma virus I (HTLV-I). It generally occurs in Japan and Africa, with a low incidence in Europe. Romania is an exception to this rule, presenting an endemic representation of ATL.

Aim: The aim of this study was to present our experience with allogeneic stem cell transplantation (alloSCT) for ATL.

Methods: The current study was a retrospective analysis of the ATL cases that received alloSCT in the Fundeni Clinical Institute. The patients were included between January 2016 to April 2019.

Results: A total of 7 patients were included in the present study, out of which 2 (28%) presented the acute form, while the rest presented the lymphomatous form. Four (57%) patients presented extranodal involvement, with 3 of those (75%), showing skin involvement. Of note, all patients presenting skin involvement also had bone marrow involvement. Thus, skin involvement was associated with a more advanced disease. Graft versus host disease (GvHD) was present in 4 (57%) patients, two of which were acute GvHD and two, chronic GvHD. Interestingly, the two patients that relapsed presented either acute or chronic GvHD, showing that the graft versus lymphoma (GVL) effect that can be observed in other lymphoma types is not observed in this cohort. Another noticeable observation is the fact that 4 out of the 6 patients tested for HTLV-I at 6 months post alloSCT presented a positive HTLV-I viral load. This event occurred, although, at 30 days, all patients presented 100% donor chimerism and the donor samples were negative for HTLV-I. This shows a possible imperfect jump of the HTLV-I from the recipient to the donor T lymphocytes or the possibility of a reinfection. Three (43%) patients presented a response less than complete remission (CR) after the first line of therapy, two of which progressed to death. The two deaths occurred at 14 and 15 months from the diagnosis, with the other follow-up periods in the cohort being higher than 26 months with the exception of one patient that had a follow-up of 6.8 moths from diagnosis. These two aforementioned patients were also the only ones to relapse thus far.

Conclusion: For ATL, alloSCT represents a viable and potentially curative approach when used in chemosensitive disease. This being said, there are clear differences from other lymphomas, ATL having to be considered separately.

Disclosures

Tanase: Pfizer: Membership on an entity's Board of Directors or advisory committees; Bristol Myers: Consultancy. Hermine:Novartis: Research Funding; Celgene: Research Funding; AB Science: Consultancy, Equity Ownership, Honoraria, Research Funding.
The Effect of Pyruvate Kinase M2: Inhibitor(shikonin) on the Function of Mdc in Severe Aplastic Anemia Mouse Model
Authors: Zheng Mengying , Liu Chunyan , Shao Yuanyuan , Fu Rong , Wang Huaquan , Wang Ting , Qi Weiwei , Shao Zonghong
Published: November 2019

Severe aplastic anemia (SAA) is a rare disease characterized by severe pancytopenia and bone marrow failure. Over-activated myeloid dendritic cells (mDCs) play an important role in the pathogenesis of SAA. In recent years, the role of pyruvate kinase M2 (PKM2) in DC function and autoimmune response has been gradually recognized. In this study, an immune attack-mediated AA mouse model was constructed by total body irradiation and lymphocyte infusion. The AA model mice showed pancytopenia, decreased ratio of CD4+/CD8+ cells, increased expression of cytotoxic molecules perforin and granzyme in CD8+ cells, increased levels of co-stimulatory molecules CD80 and CD86 in DCs and inadequate Treg number. In-vitro animal experiments confirmed the activation of PKM2 in mDCs, which promoted glycolytic metabolism. High levels of PKM2 in mice contributed to the poor survival rate. Additionally, intervention treatment with shikonin or cyclosporin A in the AA mouse model reduced the expression not only of co-stimulatory molecules CD80 and CD86 in mDCs but also of cytotoxic molecules in CD8+ cells. In conclusion, we confirmed the activation of PKM2 in mDCs in AA mouse models. PKM2 is involved in mDC activation and proliferation, which might contribute to activating the downstream cytotoxic T cells (CTLs). PKM2 is a possible novel target in SAA treatment.

Disclosures

No relevant conflicts of interest to declare.
BTK Inhibitors Induce ABC-DLBCL Cell Apoptosis By Inhibiting CYLD Phosphorylation
Authors: Xu Xin
Published: November 2019

BTK inhibitors induce ABC-DLBCL cell apoptosis by inhibiting CYLD phosphorylation

Xin Xu1, Zhigang Zhu1, Feng Liu1 , Weijie Zhong1,Huabao Xiong2, Tiefeng Feng3, and Qingshan Li3

1Geriatrics Department, GuangZhou First People's Hospital, GuangDong, 510180, People's Republic of China.

2Precision Immunology Institute, Mount Sinai School of Medicine, New York, NY 10029, U.S.A.

3Hemotology Department, GuangZhou First People's Hospital, GuangDong, 510180, People's Republic of China.

Activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL) is a common aggressive diffuse large B cell lymphoma (DLBCL). Even though CD20 antibody (Rituximab) combination therapy improved the total prognosis of DLBCL, there are still many relapsed and refractory ABC-DLBCL cases. The new treatment options are urgently needed.

Pathogenesis of ABC-DLBCL is different from germinal center B cell-like diffuse large B cell lymphoma (GCB-DLBCL), which is related to activation of B cell antigen receptor(BCR) signaling. The Bruton tyrosine kinase(BTK) plays a central role in BCR signaling. Inhibition of BTK function will affect BCR signaling activity, which is involved in maintaining the malignant phenotype in B-cell lymphoma. Now in clinic, BTK inhibitors like Ibrutinib or Acalabrutinib are used extensively in chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), mantle cell lymphoma(MCL) as first line therapy or second line therapy. But in DLBCL its role is unclear now.Some DLBCL clinic trials got the better PFS and OS in BTK inhibitor combination therapy group(combined with Rituximab or RCHOP) but some got the negative results. Whether BTK inhibitors can definitely improve ABC-DLBCL survival and the mechanism how BTK inhibitors inducing cell death are needed further investigations.

CYLD (cylindromatosis) is a tumor suppressor, its activity is inhibited by phosphorylation, which is related to apoptosis regulation pathway NF-kB in many kinds of tumors.In this study, we found that CYLD to be constitutively phosphorylated in ABC-DLBCL cell line models (HBL-1 and OCI-Ly10) as well as primary samples from patients with non-GCB DLBCL. We subsequently examined the effect of BTK inhibitors Ibrutinib (PCI-32765) and Acalabrutinib (ACP-196) on CYLD phosphorylation.

We found that BTK inhibitors Ibrutinib and Acalabrutinib both induced ABC-DLBCL apoptosis by down-regulating CYLD phosphorylation in vivo and in vitro, even in the Rituximab resistant ABC-DLBCL cell line.Combination of BTK inhibitors and Rituximab would enhance this effect. Knockdown of CYLD in ABC-DLBCL cells reduced the level of cell death induced by BTK inhibitors, which showed that this cell death induced by BTK inhibitors was CYLD dependent.

Through this study, we conclude that BTK inhibitors induce CYLD inactivation by regulating CYLD phosphorylation is clinical beneficial in ABC-DLBCL therapy, especially the relapsed or refractory ABC-DLBCL cases after Rituximab therapy.

Disclosures

No relevant conflicts of interest to declare.
Endocytosis of the Thrombopoietin Receptor Mpl Regulates Both Megakaryocyte and Erythroid Maturation in Mice
Authors: Eaton Nathan , Lee-Sundlov Melissa M. , Dlugi Theresa A. , Wieser Jon , Ramsey Haley E. , Sola-Visner Martha , Hoffmeister Karin M. , Falet Hervé
Published: November 2019

The thrombopoietin receptor Mpl plays a critical role in thrombopoiesis, as it is the primary signaling constituent in megakaryocyte (MK) differentiation from hematopoietic stem and progenitor cells (HSPCs) and maintains circulating blood thrombopoietin levels via clathrin-mediated endocytosis. Thus, thrombopoiesis is tightly controlled by Mpl cell-surface expression on HSPCs, MKs, and platelets. Previous work by our group has shown that Dnm2fl/flPF4-Cre (Dnm2Plt-/-) mice, lacking the large endocytic GTPase dynamin 2 (DNM2) specifically within MKs and platelets, develop a variety of clinical phenotypes that closely resemble myelofibrosis, such as bone marrow fibrosis, marked HSPC expansion, MK hyperplasia, extramedullary hematopoiesis, and severe splenomegaly (Bender, Giannini, et al. Blood. 2015;125(6):1014-1024). Dnm2Plt-/- mice also displayed elevated plasma TPO levels and constitutive JAK2 activation in platelets, due to defective Mpl endocytosis. Here, the role of Mpl endocytosis in the maintenance of normal hematopoiesis was assessed by generating Dnm2Plt-/- mice in the Mpl-/- background. At 3 weeks of age, Mpl-/- Dnm2Plt-/- mice displayed significantly reduced HSPCs, a near complete depletion of bone marrow MKs, similar to Mpl-/- mice, indicating that Mpl is the primary receptor contributing to the aberrant hyperproliferative phenotype of Dnm2Plt-/- mice. However, Mpl-/- Dnm2Plt-/- mice also showed severe anemia, defects in erythroblast maturation, grossly elevated plasma erythropoietin (EPO) levels, and splenomegaly, resulting in early fatality by 3 to 4 weeks of age, suggesting that Mpl contributes to normal erythropoiesis in young mice. Mpl-/- and Mpl+/- Dnm2Plt-/- mice displayed reduced erythroblast development at 3 weeks of age, which returned to normal with adulthood. Taken together, the data shows that DNM2-dependent Mpl endocytosis is required for steady-state hematopoiesis and provides novel insights into a developmentally-controlled role for Mpl in normal erythropoiesis.

Disclosures

Sola-Visner: Sysmex America, Inc.: Other: Laboratory equipment on loan, Research Funding.
Adiponectin Inhibits the Differentiation and Maturation of Osteoclasts Via mTOR Pathway in Multiple Myeloma
Authors: Fu Rong , Zhaoyun Liu , Liu Hui , Li Yanqi , Wang Yangyang , Xing Rui , Mi Fu , Shao Zonghong , Song Jia , Xiang Chenhuan
Published: November 2019

Background: To investigate the correlation of adipose cytokines (visfatin, leptin, and adiponectin) with multiple myeloma bone disease (MBD) and explore the effects and mechanism of adiponectin on the differentiation and maturation of osteoclasts in multiple myeloma (MM).

Methods: The levels of visfatin, leptin and adiponectin were measured .Their relationships between the index of myeloma tumor load and bone disease were analyzed. RT-PCR was used to detect the expressions of RANKL, OSCAR, TRAP and Cathepsin K genes. Flow cytometry (FCM) was used to detect the expressions of AdipoR1 and phosphorylation of mTOR pathway related proteins mTOR and 4EBP1.

Results :There were no significant linear correlation between leptin, visfatin and the indexes of myeloma tumor load and bone disease. Serum adiponectin levels were significantly higher in newly diagnosed multiple myeloma patients and negatively linear correlated with β2-microglobulin.There is a positive linear correlation relationship between the adiponectin and osteocalcin. The number of maturation osteoclasts in adiponectin group was less than the control group. Adiponectin also inhabits the mRNA level of osteoclast related factor- RANKL, OSCAR, TRAP and Cathepsin K. Flow cytometry showed that adiponectin increased the expression of AdipoR1 on the surface of osteoclast precursor((26.21±4.27)% vs (29.86±6.23)%, p<0.05) and reduced the expression of p-mTOR ((7.89±1.00)% vs (5.91±1.26)%, p<0.05) and p-4EBP1 ((26.78±5.00)% vs (22.49±4.24)%, p<0.05).

Conclusion: Adiponectin inhibits the differentiation and maturation of osteoclasts via increasing the expression of AdipoR1 and reducing the phosphorylation levels of mTOR and 4EBP1 proteins in MM patients.

Disclosures

No relevant conflicts of interest to declare.
The Lost or Absence of MRD<0.1% at Any Time after 2 Cycles of Consolidation Chemotherapy in CBFB-MYH11-Positive Acute Myeloid Leukemia Indicates Poor Prognosis
Authors: Duan Wenbing , Jia Jinsong , Wang Jing , Liu Xiaohong , Gong Lizhong , Jiang Qian , Zhao Ting , Wang Yu , Zhang Xiaohui , Xu Lanping , Liu Yanrong , Qin Yazhen , Huang Xiaojun , Jiang Hao
Published: November 2019

Objective: To investigate the long-term prognostic factors of CBFB-MYH11-positive acute myeloid leukemia in low、 intermediate risk patients.

Methods:58 CBFB-MYH11-positive patients were analyzed retrospectively in 1525 acute myeloid leukemia patients who were diagnosed in Peking University Institute of Hematology from October 2013 to August 2018, and the clinical feature、induction remission rate and survival were compared.

Results: 58 (8%) patients with CBFB-MYH11-positive were found in 1525 newly diagnosed actue myeloid leukemia, 41 males and 17 females were included. The median age was 38(17-66) years , and median follow-up time was 29.8(4.8-74.4)months, 25(43.1%) patients received allogenic hematopoietic stem cell transplantation, in which there were 10 patients with c-kit mutation. According to the NCCN guideline, there were 40 patients in low risk group and 18 patients in intermediate risk group (16 patients with c-kit mutation). The rate of complete remission (CR) in the first induction chemotherapy was 98.3%(57/58), the cumulative remission rate of two cycles was 100%, the relapse rate was 25.9%(15/58), and the mortality rate was 19.0% (11 ≤ 58). After 1 and 2 cycles of consolidation, the level of CBFB-MYH11/ABL decreased to less than 0.18% and 0.094% respectively, which were related to the low recurrence rate. Among the 33 patients who did not undergo allogenic hematopoietic stem cell transplantation after two courses of consolidation, the recurrence rate of patients with CBFB-MYH11/ABL level > 0.1% (n =21) was significantly higher than that of patients with < 0.1% (n = 12) (61.9% VS 0%, P =0. 001), and the relapse-free survival(RFS) (3-year RFS rate 37.6% VS 100%, P =0.005 ) 、event-free survival (EFS)(3-year EFS rate 33. 3% VS 100%, P = 0.004) were significantly decreased. The RFS and EFS were lower than those of patients received allogenic hematopoietic stem cell transplantation (n=25) (3-year RFS rate 31.4% VS 84.6%, P = 0.000; 3-year EFS rate 31.4% VS 76.0%, P =0.004).

Conclusions: To the CBFB-MYH11-positive acute myeloid leukemia patients, if the level of CBFB-MYH11/ABL gene presented more than 0.1% at any time after 2 cycles of consolidation, poor prognosis was related to, and allogenic hematopoietic stem cell transplantation could improve the survival.

Disclosures

No relevant conflicts of interest to declare.
Serum Citrulline As a Biomarker for Blood Stream Infections in Pediatric Hematopoietic Stem Cell Transplant Patients
Authors: Singla Shyamli , Wong Justin , Jiang Shuang , Hussain Sarah , Coughlin Laura , Zhan Xiaowei , Koh Andrew Y.
Published: November 2019

Background: Invasive microbial infections remain a major cause of morbidity and mortality in hematopoietic stem cell transplant (HSCT) patients. Microbes residing in the GI tract are believed to be the source of mucosal barrier injury-associated blood stream infections (mBSI) in HSCT and cancer patients. Three host factors are critical for preventing microbial translocation from the gut: neutrophils, intact gut barrier function, and gut microbiota homeostasis. Currently there are no reliable biomarkers that can identify patients who will develop mBSIs.

Objectives/Hypothesis: Since all HSCT patients develop severe neutropenia (ANC<100), we hypothesized that HSCT patients who develop mBSI will have more impaired gut barrier function and a greater imbalance in gut microbiota populations (as evidenced by depletion of beneficial obligate anaerobe commensals) compared to counterparts who do not develop mBSI.

Methods: We designed a prospective, non-randomized, case control pilot study for HSCT patients < 20 years of age. Serum citrulline, which has been used as a surrogate to measure overall gut function, was obtained 1-2 times weekly. Bacterial genomic DNA was isolated from stool samples collected every 7 days. Amplicons (16S rRNA V4 variable region) were generated and sequenced using the Illumina MiSeq platform. Microbial taxonomic assignments were determined using QIIME software and further characterized into relative abundances of obligate (beneficial commensal) versus facultative (pathogenic) anaerobes. Clinical characteristics were recorded.

Results: Of the 78 HSCT patients enrolled on the study, 27% (21 patients) developed BSI. Of the patients with confirmed blood stream infections, 16 patients were categorized as mBSI, and ultimately 11 patients were included in our analysis. 48 patients did not develop a BSI and had evaluable citrulline levels and constituted our control group. Patients with mBSI had a more significant decline in citrulline levels during the period from the start of conditioning to the development of mBSIs (linear mixed effect model, p = 0.048, likelihood ratio test) compared to counterparts without mBSI. We found no significant association between levels of facultative (pathogenic) anaerobes and the development of mBSI in this cohort.

Conclusions: Decreased citrulline levels, which suggest impaired gut epithelial function, are associated with the development of severe, invasive bacterial infections. These data suggest that measuring citrulline may have utility as a biomarker for predicting mBSI in HSCT patients.

Disclosures

Koh: Merck: Consultancy.
Hematopoietic Cell Cluster Identifier (HCCI): A Knowledgebase Computational Tool to Facilitate the Characterization of Hematopoietic Cell Populations in Single Cell RNA-Sequencing
Authors: Kumar Gaurav , Ertel Adam , Naranjo Nicole , Shahid Shuvanon , Languino Lucia , Fortina Paolo
Published: November 2019

Single cell transcriptional profiling is critical to interrogate cell types, states and functionality in a complex tissue or disease. Despite rapid advancements in single cell RNA sequencing (scRNA-seq) analysis, major challenge remains in identification of distinct cell types based on specific molecular signatures. Few databases are available to facilitate cell-type characterization, however, their broad all-cells encompassing structure is deemed often inept. At present, manual curation is the only option. To address this, we developed a knowledgebase computational tool, HCCI, that identifies and characterizes up to 28 different hematopoietic cell types in a scRNA-seq dataset. Utilizing the knowledgebase comprised of ~1500 marker genes obtained from data mining, HCCI first uses a voting algorithm to search and match genes from a given cluster to a single or more possible cell type. Next, it normalizes score of each cluster-cell type pair to provide a percentage denoting a cluster to a specific cell-type. To demonstrate this, we utilized a dataset of Peripheral Blood Mononuclear Cells (PBMC) available from 10X Genomics. As the cell populations are already known, this was an ideal dataset to evaluate the performance of HCCI. As expected, unsupervised clustering and UMAP algorithms identified 9 different clusters of cells conforming to the existing knowledge of this dataset. Differentially expressed genes from each cluster were input into HCCI, and the following unique clusters were identified: naïve CD4 (cluster 1), M0 macrophage (cluster 2), CD8 T-cells (cluster 3), naïve B-cell (cluster 4), cytotoxic T-cell (cluster 5), M1 macrophage (cluster 6), activated NK cell (cluster 7), monocyte (cluster 8) and plasmacytoid dendritic cell (cluster 9). Here using HCCI, an improvement in classification was observed for clusters 2,3,6,8 and 9 which were originally classified only in major blood cell types. Classification of clusters 1,4,5 and 7 remained unchanged. The classification by HCCI was observed to be much precise when compared to the manual curation performed originally. To our knowledge, HCCI is the only available tool for characterization of hematopoietic cells from scRNA-seq data.

Disclosures

No relevant conflicts of interest to declare.
Cell-Intrinsic Inflammation Drives Progression from Myelodysplastic Syndromes to Leukemia
Authors: Barreyro Laura , Sampson Avery , Hueneman Kathleen , Choi Kwangmin , Christie Susanne , Wang Dehua , Huang Gang , Starczynowski Daniel T.
Published: November 2019

Myelodysplastic Syndromes (MDS) often progress to Acute Myeloid leukemia (AML) typically following acquisition of mutations in RUNX1, NRAS/KRAS or AXSL1. Inflammatory processes associated with autoimmune diseases or natural aging are thought to contribute to the development of MDS and progression to AML. Moreover, dysregulation of innate immune signaling is broadly observed in hematopoietic stem and progenitor cells (HSPC) from MDS patients. However, how dysregulation of innate immune signaling and/or inflammatory signals might lead to malignant transformation and overt AML has not been fully elucidated. We have developed a mouse model of cell-intrinsic inflammation-driven progression from MDS to AML that will allow us to determine the mechanisms of oncogenic transformation. MicroRNA miR-146a is a 5q gene that is frequently deleted in MDS and AML and its deletion promotes dysregulation of innate immune signaling and a systemic inflammatory state. Deletion of miR-146a results in myeloproliferation and marrow failure in mice associated with attrition of HSPC, but alone is insufficient to induce overt AML. Using a genetically engineered approach, we show that deficiency of miR-146a (miR-146a-/-) combined with a C-terminal truncation of the RUNX1 (S291fsX300) protein causes anemia, thrombocytopenia and ineffective erythropoiesis mimicking human MDS in younger mice, but then the mice progresses to a fatal AML. The inflammatory environment induced by miR-146a deletion was exacerbated in mice with miR-146a-/-expressing RUNX1-S291fsX300 (RUNX1-mut) and correlated with the increased incidence and aggressiveness of hematologic malignancies. In particular, the levels of IFNγ were increased in plasma in mice with miR-146a-/-expressing RUNX1-mut in comparison to mice reconstituted with miR-146a-/-or RUNX1-mut-expressing BM cells. As previously reported, miR-146a-/-cells exhausted during serial transplantation, however RUNX1-mut rescued the hematopoietic repopulation deficiency of miR-146a-/-HSPC, suggesting that the RUNX1 mutation promotes malignant transformation to AML under inflammatory conditions. To establish whether cell-intrinsic immune signaling is required for RUNX1-mut;miR-146a-/- leukemic cells, we evaluated an inhibitor (NSC697923) that targets TRAF6, a key target of miR-146a in MDS/AML, via inhibition of its co-factor UBE2N. Treatment with NSC697923 or a RUNX1 inhibitor (Ro 5-3335) abolished colony formation by RUNX1-mut;miR-146a-/- leukemic cells, indicating that cell-intrinsic innate immune signaling is not only required for initiating AML, but is also critical for sustaining the leukemic phenotype. These findings describe the first evidence that dysregulation of innate immune signaling and/or inflammatory signals contribute to the progression and maintenance of AML.

Disclosures

Starczynowski: Kurome Therapeutics: Consultancy.
Rezafungin Prevention of Pneumocystis Pneumonia and Pneumocystis reactivation Using Different Doses and Durations of Prophylaxis in a Mouse Model
Authors: Cushion Melanie T , Ashbaugh Alan , Ong Voon
Published: November 2019

Rezafungin (RZF) is a novel echinocandin in development for prevention of invasive fungal disease caused by Candida, Aspergillus, and Pneumocystis species in patients undergoing blood and marrow transplantation. RZF has a favorable safety and tolerability profile, a low risk of drug-drug interactions and, with its stability, pharmacokinetics that allow for once-weekly dosing with broad distribution to the lung and other fungal target organs.

We previously demonstrated RZF efficacy in preventing Pneumocystis infection in an immunosuppressed mouse model. The present study addressed whether Pneumocystis infection in a similar immunosuppressed mouse model could re-activate after 2 to 8 wks of prophylactic therapy using different dosing regimens of RZF.

C3H/HeN mice were infected with P. murina intranasally at 2 x 106/50 µl after immunosuppression with dexamethasone. Mice were administered vehicle as a negative control (C/S), trimethoprim/sulfamethoxazole (T/S) as a positive control, caspofungin (CASPO) as a comparator echinocandin or RZF intraperitoneally at time of challenge. Study drug administration was stopped at 2, 4, 6, and 8 weeks, at which time mice were immunosuppressed for an additional 6 weeks to allow any residual P. murina to re-activate. Mice were then euthanized and lungs were processed for analysis of nuclei and asci.

All RZF dose regimens at all timepoints significantly reduced both nuclei and asci burdens versus the C/S group (Figure 1). After 4 weeks of RZF prophylaxis (plus 6 wks additional immunosuppression [week 10 timepoint]; Figure 1), both RZF 20 mg/kg groups (3x/wk and 1x/wk) had prevented P. murina organisms from activating an infection. After 6 and 8 weeks of RZF prophylaxis (week 12 and 14 timepoints; Figure 1), no re-activation of infection was present in any of the study groups. After 2 and 4 weeks of prophylaxis (week 8 and 10 timepoints), there was a significant reduction of nuclei and asci counts in all groups of RZF versus CASPO. After 2, 4, and 6 weeks of RZF prophylaxis, there was a significant reduction of nuclei and asci counts between all groups of RZF versus T/S 1x/week. There was a significant benefit in survival between the RZF group at 20 mg/kg/3x/week versus CASPO at week 14 (Figure 2).

In this study, prophylaxis with RZF for as little as 4 weeks prevented P. murina organisms from developing infection after cessation of therapy and showed more efficacy than CASPO. These results in the mouse model of Pneumocystis pneumonia provide evidence that RZF can prevent Pneumocystis reactivation and that such regimens hold promise for prophylaxis against Pneumocystis in at-risk patients undergoing blood and marrow transplantation.

Disclosures

Cushion: Cidara Therapeutics: Consultancy, Research Funding.
Maintenance Sorafenib Is Superior to Prophylactic DLI in Improving the Prognosis of FLT3-ITD-Positive AML after Allogeneic HSCT
Authors: Shi Jimin , Cao Liqin , Luo Yi , Zhao Yanmin , Tan Yamin , Yu Jian , Lai Xiaoyu , Zhu Yuanyuan , Hu Yongxian , He Jingsong , Sun Jie , Zheng Weiyan , Wei Guoqing , Huang He
Published: November 2019

Background: Compared with traditional chemotherapy, allogeneic hematopoietic stem cell transplantation (allo-HSCT) can improve the prognosis of patients with FLT3-ITD positivity. Relapse after transplantation is still an important factor affecting survival. Prophylactic donor lymphocyte infusion (DLI) and maintenance sorafenib are two methods for preventing relapse after allo-HSCT in FLT3-ITD-positive acute myeloid leukemia (AML) patients, and their roles in these patients after allo-HSCT need to be further studied. Methods: From January 2014 to December 2018, 69 FLT3-ITD-positive patients who received allo-HSCT at our center were included in this retrospective study. All patients were divided into four groups according to the different treatments received: group A (23 patients who received maintenance sorafenib), group B (12 patients who received prophylactic DLI), group C (33 patients who received neither prophylactic DLI nor maintenance sorafenib), and group D (1 patient who received a combination of prophylactic DLI with maintenance sorafenib), because there was only one patient in group D, it was not included in the analysis. Results: For all patients, the 2-year estimated overall survival (OS) and leukemia-free survival (LFS) were 79.9% and 78.3%, respectively, and the 2-year cumulative incidence of relapse (CIR) was 17.2%. The 2-year estimated OS, LFS and CIR in group A, group B and group C were 95.7%vs75%vs66.8%, 95.7%vs74.1%vs70.2% and 4.3%vs6.7%vs26.1%, respectively. Group A was the best one in OS, LFS and CIR. Between the group A and group C, there were significant difference in OS(P=0.03) and LFS(P=0.04), but not in CIR(P=0.06).The OS in group A was significantly higher than group B(P=0.047), but the difference was not statistically in LFS and CIR (P=0.14 and P=0.21). Conclusion: Maintenance sorafenib after allo-HSCT was associated with improved outcomes for FLT3-ITD positive AML patients and was superior to prophylactic DLI in promoting FLT3-ITD-positive patients obtain a better survival after allo-HSCT.

Figure

Disclosures

No relevant conflicts of interest to declare.
HIF-1a Regulating the Chemoresistance By Upregulating the Expression of xCT and GCLM in the Diffuse Large B Cell Lymphoma
Authors: Wei Yongqiang , Zeng Hong , Wei Xiaolei , Huang Weimin , Song Jialin , Zheng Jingxia , Feng Ru
Published: November 2019

Background

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-hodgkin lymphoma with great heterogeneity in clinical behavior and response to treatment. Although the addition of rituximab to CHOP regimen has significantly improved the survival of DLBCL, 1/3 will be eventually relapse and progression. HIF1a has been reported to be related with the drug resistance in DLBCL, but the mechanism remains unknown.

Methods

Two DLBCL cell lines (Riva and SuDHL2) were treated with doxorubicin and HIF-1a inhibitors digoxin, YC-1. The proliferation of these cell lines (Riva and SuDHL2) were measured by MTT and apoptosis were detected by FCM after staining with Annexin V/SYTOX Green. Expression of IF-1α, PHD, xCT, GCLM and apoptosis-related proteins was detected by Western Blot.

Results

With the increase concentration of doxorubicin treatment, the proliferation of Riva and SuDHL2 cell lines could be gradually inhibited. xCT and GCLM expression were upregulated after treated with doxorubicin and can be reversed by N-acetylcysteine. HIF-1a inhibitors digoxin and YC-1 could inhibited the expression of xCT and GCLM, proliferation and apoptosis induced by doxorubicin. Furthermore, we treated shHIF-1a RNA to significantly reduce the expression of HIF-1a, and the decrease of HIF-1a expression enhanced the proliferation inhibition and apoptosis of lymphoma cells by Dox. Downregulation the expression of HIF-1a by shRNA enhanced the doxorubicin induced apoptosis and inhibited the proliferation in DLBCL cell lines.

Conclusions

Taken together, our results showed that HIF-1a could regulate the expression of xCT and GCLM and further mediate drug resistance in the diffuse large B cell lymphoma.

Disclosures

No relevant conflicts of interest to declare.
Curcumin Enhances the Sensitivity of Bortezomib By Inhibiting NF-Κb in ABC Diffuse Large B-Cell Lymphoma Cells
Authors: Wei Xiaolei , Song Jialin , Wei Yongqiang , Zeng Hong , Huang Weimin , Zheng Jingxia , Feng Ru
Published: November 2019

Background

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-hodgkin lymphoma with great heterogeneity in clinical behavior and response to treatment. According to gene expression profile, DLBCL can be divided into at least two subtypes: germinal center B cell type (GCB) and activated B cell type (ABC). ABC-type DLBCL is commonly activated by the NF-κB pathway, but the proteasome inhibitor bortezomib does not significantly improve the prognosis of ABC-DLBCL. Curcumin inhibit the proliferation of various tumor cells by inhibiting the activation of NF-κB. Our purpose was to evaluate whether curcumin could enhance the ensitivity of Bortezomib in ABC-DLBCL.

Methods

MTT assay was used to evaluate the proliferation of 2 ABC-DLBCL cell lines by treated with curcumin and bortezomib. Apoptosis were detected by FCM after staining with Annexin V/SYTOX Green.Western Blot was used to evaluated the expression of PARP, NF-κB, I κBα/p-IκBα and caspase-3 in ABC-DLBCL cells treated with curcumin and bortezomib.

Results

Both curcumin and bortezomib could inhibit the proliferation and induce apoptosis in ABC-DLBCL cell lines. Curcumin decreased the expression of p-IκBα, NF-κB/p65 and increased the expression of Cleaved PARP in ABC-DLBCL cell lines. Caspase Inhibitor Z-VAD could reverse the curcumin induced proliferation inhibition and apoptosis by decreasing the cleaved PARP expression. The combination of curcumin and bortezomib could further enhance the proliferation inhibition and apoptosis in ABC-DLBCL cell lines by inhibiting NF-κB.

Conclusions

Curcumin induced proliferation inhibition and apoptosis by inhibiting NF-κB in ABC-DLBCL. It may sensitize ABC-DLBCL cell lines to the cytotoxic effects of bortezomib by inhibiting NF-κB.

Disclosures

No relevant conflicts of interest to declare.