bluuebunny/crossref_metadata_2025 huggingface dataset:
Controlling Long-Range Genomic Interactions to Reprogram the β-Globin Locus
Authors: Deng Wulan , Rupon Jeremy W , Wang Hongxin , Reik Andreas , Gregory Philip D. , Blobel Gerd A
Published: October 2019

Abstract 280

Distal enhancers physically contact target promoters to confer high level transcription. At the mammalian β-globin loci long-range chromosomal interactions between a distal enhancer, called the locus control region (LCR), and the globin genes are developmentally dynamic such that the LCR contacts the embryonic, fetal and adult globin genes in a stage-appropriate fashion. LCR-globin gene interactions require the nuclear factor Ldb1. Recently, we employed artificial zinc finger (ZF) proteins to target Ldb1 to the endogenous β-globin locus to force an LCR-promoter interaction. This led to substantial activation of β-globin transcription and suggested that forced chromatin looping could be employed as a powerful tool to manipulate gene expression in vivo (Deng et al., Cell 2012). Reactivation of the fetal globin genes in adult erythroid cells has been a long-standing goal in the treatment of patients with sickle cell anemia. Therefore, building on our findings, we investigated whether the developmentally silenced embryonic globin gene βh1 can be re-activated in adult murine erythroblasts by re-directing the LCR away from the adult type globin gene and towards its embryonic counterpart. To this end, Ldb1 was fused to artificial ZF proteins (ZF-Ldb1) designed to bind to the βh1 promoter. ZF-Ldb1 was introduced into definitive erythroid cells in which only the adult but not the embryonic β-like globin gene is expressed. In vivo binding of the ZF-Ldb1 to its intended target was verified by chromatin immunoprecipitation assay. Strikingly, expression of ZF-Ldb1 re-activated βh1 transcription up to approximately ∼15% of total cellular β-globin production. This suggests that forced tethering of a looping factor to a select promoter can be employed to override a pre-existing developmental long-range chromatin interaction to reprogram a developmentally controlled gene locus. We are now in the process of testing whether our approach might be suitable to reactivate the silent fetal globin genes in adult human erythroid cells. These studies are underway and the results will be discussed at the meeting.

Disclosures:

Reik: Sangamo BioSciences, Inc.: Employment. Gregory:Sangamo BioSciences, Inc.: Employment.
Differences in the Angiogenic Response and Subsequent Growth of Plasma Cells From Myeloma and MGUS Patients Xenografted Into Zebrafish Embryos.
Authors: Ma Dongdong , Ramachandran Aravind , Chang Yali , Lin Jianhong , Zhang Weihong , Tai Yu-Tzu , Munshi Nikhil C. , Handin Robert I
Published: October 2019

Abstract 2912

A monoclonal gammopathy of uncertain significance (MGUS) is an obligate precursor to each case of multiple myeloma (MM). MGUS and MM samples share many of the same gene mutations and chromosomal translocations. Despite increasing genetic and genomic information gathered from patient samples, we still do not understand which mutations are responsible for the progression from MGUS to MM. In order to learn more about this progression and the possible involvement of an angiogenic switch as seen in solid tumors, we have developed a zebrafish model to study the angiogenic potential and subsequent growth of CD138+ plasma cells derived from normal human bone marrow, MGUS and myeloma patients. Immunopurified CD138+ cells were labeled with the red fluorescent dye CM DiI, suspended in Matrigelâ and injected into the perivitelline space of Tg(fli1:GFP) zebrafish embryos that had fluorescent vasculature, 48 hpf (hour-post-fertilization). Cell growth and angiogenesis were assessed by conventional and confocal fluorescent microscopy. Inhibitors of myeloma cell growth or tumor-induced angiogenesis were added to embryo water. The injection of up to 2000 plasma cells from normal human bone marrow into zebrafish embryos did not elicit an angiogenic response and the cells did not grow after injection. In contrast, the injection of as few as 100 plasma cells purified from the marrow of patients with multiple myeloma induced a brisk angiogenic response with extension of new vessels ventrally from the sub-intestinal venous system into the site of cell injection. Angiogenesis was accompanied by the growth of the CD138+ cells and, in some cases, movement of cells out of the Matrigelâ plug to distant sites in the developing embryo. MGUS cells induced minimal or no angiogenesis and grew very slowly after injection. The growth of cells from MGUS patients and de novo myeloma patients was inhibited by the addition of the VEGFR2 inhibitor SU5416 to embryo water 24 hpi (hours-post-tumor cell-injection). If the addition of SU5416 was delayed until 48 hpi, when the angiogenic response had been established, there was no effect on cell growth. We also observed that dexamethasone, bortezimib and lenalidomide, singly and in combination, inhibited the growth of MGUS and myeloma cells. These studies document clear biologic differences between normal, MGUS and myeloma plasma cells. In addition, we provide evidence that the zebrafish embryo can be used to study tumor-induced angiogenesis, tumor cell growth and the effects of possible inhibitors. We also provide some new insights into the mechanism underlying the disappointing response of angiogenic inhibitors when given to patients with multiple myeloma.

Disclosures:

Munshi: Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.
CD82 Regulates STAT5/IL-10 and Supports Survival of Acute Myelogenous Leukemia Cells.
Authors: Nishioka Chie , Ikezoe Takayuki , Takeuchi Asako , Yang Jing , Yokoyama Akihito
Published: October 2019

Abstract 2981

We recently reported that adhesion molecule CD82 is aberrantly expressed in CD34+/CD38− leukemia stem cells (LSCs) (ASH 2010; Abstract 2168.). Here, we report the results of a functional analysis of CD82 in CD34+/CD38− acute myelogenous leukemia (AML) cells. Short hairpin (sh)RNA-mediated downregulation of CD82 resulted in dephosphorylation of STAT5 and a decrease in the level of IL-10. In contrast, forced expression of CD82 in CD34+/CD38+ AML cells by transduction with CD82-expressing lentiviral particles resulted in an increase in the levels of p-STAT5 and IL-10. Chromatin immunoprecipitation (ChIP) assay results indicated that STAT5A binds to the promoter region of the IL-10 gene, while reporter gene assay results indicated stimulation of IL-10 expression at the transcriptional level. These results suggest that CD82 positively regulates the STAT5/IL-10 signaling pathway. Notably, exposure of CD34+/CD38− AML cells to IL-10 stimulated clonogenic growth of these cells. Moreover, shRNA-mediated downregulation of CD82 expression in CD34+/CD38− AML cells impaired AML reconstitution in immunodeficient mice in parallel with downregulation of STAT5. Taken together, our data suggest that the CD82/STAT5/IL-10 signaling pathway is involved in the survival of CD34+/CD38− AML cells and may thus be a promising therapeutic target for eradication of AML LSCs.

Disclosures:

No relevant conflicts of interest to declare.
Outbreak of Nosocomial Respiratory Syncytial Virus Infections in a Hematology and Transplant Unit.
Authors: Lehners Nicola , Schnitzler Paul , Egerer Gerlinde , Dreger Peter , Eisenbach Christoph , Aichinger Elisabeth , Weißbrich Benedikt , Frank Uwe , Heeg Klaus , Ho Anthony D
Published: October 2019

Abstract 3032

Respiratory syncytial virus (RSV) is a pathogen associated with bronchiolitis in infants and small children. However, immunocompromised adults might also be at risk for severe lower respiratory tract involvement induced by RSV. We report the rapid control of an outbreak of nosocomial RSV infections in an institution with special focus on allogeneic and autologous stem cell transplantations.

Between November 2011 and March 2012 56 patients were diagnosed with RSV infection by RT-PCR at our institution. In order to identify possible risk factors for lower respiratory tract involvement in RSV infected patients, clinical course of infection as well as radiological and laboratory findings were evaluated. Characterization of RSV strains in respiratory specimens was achieved by sequence analysis of part of the RSV glycoprotein G gene and phylogenetic comparison of outbreak and community strains was performed.

Out of 56 patients with RSV infection, 39 patients showed signs of lower respiratory tract involvement. 14 fatal outcomes were observed. In most of the lethal cases coinfections with other pathogens such as Aspergillus spp. or Pseudomonas aeruginosa were present. Therefore the exact impact of RSV on the fatal outcome remains difficult, if not impossible, to assess. Age, sex, underlying disease, disease control or transplant status did not differ significantly between severe and non-severe cases. However, hypogammaglobulinemia was associated with a higher risk for fatal infection (p = 0.03) in univariate analysis, whereas therapy with oral ribavirin might show a protective effect (p = 0.02). Prolonged viral shedding was frequently observed (median 22.5 [1 – 143] days), most pronounced in patients folllowing allogeneic transplantation (p = 0.04). The major mode of RSV transmission seemed to be direct patient-to-patient contact as suggested by evaluation of patient movements on the wards. Sequence analysis on respiratory specimens obtained from 46 patients revealed the presence of a particular outbreak strain in 40 patients, characterized by an identical nucleotide sequence of RSV GA2 subtype. Phylogenetic analysis showed low prevalence of the outbreak strain in the community. By implementation of rigorous isolation measures containment of outbreak was achieved.

Highly contagious viral pathogens such as RSV are able to cause rapidly spreading outbreaks among immunocompromised patients. Hypogammaglobulinemia might be a risk factor for severe lower respiratory tract infection. Treatment with oral ribavirin might have a positive effect on outcome. Rigorous isolation measures are essential to the containment of outbreak.

Disclosures:

Off Label Use: ribavirin aerosol is FDA approved for severe cases of RSV in infants and young children.
Effective Prevention of Acute and Chronic Graft-Versus-Host Disease with Anti-Lymphocyte Globulin (ATG) without Increase of Relapse in HLA-Identical Sibling Peripheral Blood Stem Cell Transplantation.
Authors: Kröger Nicolaus , Zabelina Tatjana , Ayuk Francis , Alchalby Haefaa , Wolschke Christine , Stübig Thomas , Klyuchnikov Evgeny , Berger Jürgen , Zander Axel R.
Published: October 2019

Abstract 3055

Acute and chronic graft versus host disease (GvHD) are severe complications after allogeneic stem cell transplantation but occurrence of GvHD is associated with reduced risk of relapse. Therefore, strategies to prevent GvHD without increasing the risk of relapse are urgently needed.

We investigate retrospectively whether anti-lymphocyte globulin (ATG Fresenius®, Fresenius Biotech, Graefelfing, Germany) as part of the conditioning regimen may reduce the risk of GvHD without increasing the risk of relapse after peripheral blood stem cell transplantation from HLA-identical siblings or HLA-compatible relatives. Out of 462 patients who received HLA-identical stem cell transplantation between 1990 and 2011 in our institution, we selected 238 consecutive patients who received allogeneic peripheral blood stem cell grafts after the year 2000. The median age of the patients was 48 years (r.,18–73y) and diagnosis were: AML (n=93), ALL (n=24), CML (n=25), MDS (n=23) or lympho-proliferative disorders (n=73). Patients were classified as good risk (n=95) or bad risk (n=143). 79 patients did receive ATG within the conditioning regimen with a median dose of 30mg/kg (r., 20–90mg/kg) and 159 patients did not receive ATG. In the ATG group there were more HLA mismatch donors (6% vs. 1%, p=0.02), more bad risk patients (70% vs. 50%, p=0.04), more reduced intensity conditioning regimens (65% vs. 34%, p< 0.001) and older patients (median age 50 vs. 46 years, p=0.03). Acute GvHD grade I to IV was less observed in the ATG group (27% vs. 40%, p= 0.04), but the difference in severe GvHD grade III and IV did not show statistical significance (10% vs. 18%, p=0.1). Chronic GvHD was less observed in the ATG group (30% vs. 52%, p=0.002), which was most obvious for extensive chronic GvHD (14% vs. 40%, p<0.001). After a median follow up of 57 months in the non-ATG and 63 months in the ATG group the cumulative incidence of non-relapse mortality at 5 years was 20% for the ATG and 34% for the non-ATG group (p=0.06) and the cumulative incidence of relapse at 5 years was 34% for the ATG group and 29% for the non –ATG group (p=0.2), resulting in a comparable 5 year progression-free (ATG: 46% and non-ATG 37%, p=0.5) and overall survival (ATG: 54% and non-ATG 46%, p=0.4).

This retrospective study shows that ATG can prevent severe chronic GvHD without obvious risk of relapse and similar PFS and OS in HLA identical sibling transplantation. A randomized trial including QoL measurement comparing ATG vs. no ATG in HLA identical sibling transplantation is ongoing (registered NCT 00678275).

Disclosures:

Kröger: Fresenius Biotech: Honoraria, Research Funding. Off Label Use: ATG in HLA identical sibling transplantation.
Long-Term Outcomes of Autologous Transplantation in Multiple Myeloma: Prognostic Significance of Complete Response.
Authors: Krejci Marta , Hajek Roman , Adam Zdenek , Pour Ludek , Zahradova Lenka , Pelcova Jana , Mayer Jiri
Published: October 2019

Abstract 3126

Background:

Autologous stem cell transplantation (ASCT) after high-dose melphalan 200mg/m2 has got an important role in the treatment of symptomatic multiple myeloma (MM). The prognostic significance of achieving complete response (CR) after ASCT was cause of great debate for a long time. Some studies have shown the strong prognostic significance of achieving CR in MM, but other studies have failed to correlation between strength of the response and outcome.

Aim:

In this report we describe the long-term outcome of cohort 232 MM patients (pts) after ASCT with aim to establish the actual prognosis for the different response categories and to analyse other factors that might predict for long-term survival.

Methods:

We evaluated 232 pts with newly diagnosed symptomatic MM who received ASCT as a part of the first-line treatment between 1995 and 2005, median follow-up from ASCT was 131 months (range 61–195).

Results:

Following ASCT, overall response rate was 90% (202/232), 23% (52/232) of pts were in complete remission (CR), very good partial response (VGPR) was achieved in 45% of pts (100/232), partial response (PR) in 22% of pts (50/232), minimal response (MR) or stable disease (SD) in 10% of pts (22/232). Median progression-free survival (PFS) and overall survival (OS) from ASCT were 30.8 and 71.9 months, respectively. Progression-free survival at 12 years after ASCT in different response categories was 41% for pts with CR, 11% for pts with VGPR and 10% for pts with PR. Overall survival at 12 years after ASCT was 51% for pts with CR, 22% for pts with VGPR and 20% for pts with PR. The achievement of CR after ASCT was independent factor for long-term survival, significance differences in OS and PFS were found between CR and non-CR groups (P under 0.001 and P under 0.001, respectively).

On multivariate analysis, the other factors associated with significantly better OS were ISS stage under III (P = 0.002), no presence of renal impairment (P = 0.008), age under 60 years (P = 0.001), no presence of deletion 1q21 (P = 0.029) and lenalidomide treatment in the post-transplant relapse (P = 0.002).

Conclusion:

The achievement of complete response after ASCT in multiple myeloma is the most important prognostic factor, even after long-term follow-up. The relapse rate is low in patients who remained in CR after 12 years from ASCT. A long-term complete remission should be a goal of treatment.

Disclosures:

Hajek: Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Honoraria.
Functions and Regulation of Erythrocyte Equlibrative Nucleoside Transporter 1 (ENT1) in Acute Hypoxia Mediated Tissue Injury
Authors: Song Anren , Zhao Cheng , Zhang Yujin , Li Jessica , Blackburn Michael R. , Grenz Almut , Eltzschig Holger , Kellems Rodney E. , Xia Yang
Published: October 2019

Using a nonbiased high throughput metabolomic screen, coupled with genetic and pharmacological approaches, recent studies demonstrated that excessive adenosine signaling through the A2B adenosine receptor triggers sickling by induction of 2,3-bisphosphoglycerate (2,3-BPG), an erythroid specific metabolite that induces O2 release from hemoglobin. Adenosine is a signaling nucleoside that elicits numerous physiological and pathological effects by engaging membrane receptors. Notably, equlibrative nucleoside transporters (ENTs) on erythrocytes have been long speculated to regulate extracellular adenosine concentrations under hypoxic conditions. Thus, we hypothesize that ENT is likely a key molecule responsible for elevated circulating adenosine levels and protects tissues from hypoxia induced injury. To test this hypothesis, we first conducted in vivo Carbon-14 labeled adenosine (C14-Ado) injection and in vitro functional C14-Ado uptake assays. We found that erythrocyte plays a key role in regulation of circulating adenosine. We then conducted western blot analysis to compare expression profiles of ENTs on erythrocyte. We found that ENT1 is the major ENT expressed on both mouse and human erythrocytes. Using genetic approach, we successfully generated an erythrocyte ENT1 knockout mouse model. Using this genetic model and pharmacological approach combined with in vivo C14-Ado injection and in vitro C14-Ado uptake assay, we demonstrated that ENT1 1) is the major adenosine transporter in erythrocyte and 2) erythrocyte is the major cell type involved in regulating circulating adenosine levels through ENT1’s function. Using erythrocyte ENT1 knockout mouse model, we found that, during acute hypoxia treatment, the loss of erythrocyte ENT1 can cause faster increase in circulating adenosine level, subsequently promoting 2,3-BPG production, triggering oxygen release, and protecting acute hypoxia-mediated tissue injury. Mechanistically, we demonstrated that hypoxia regulates ENT1 activity through adenosine-ADORA2B-PKA signaling pathway. Overall, our studies demonstrate that 1) ENT1 is a major adenosine transporter expressed by erythrocytes and erythrocytes are the major cell type responsible for regulating circulating adenosine. 2) Hypoxia regulates ENT1 activity through adenosine-ADORA2B-PKA signaling pathway. 3) Inhibition or deletion of erythrocyte ENT1 results in enhanced adenosine-mediated 2,3-BPG induction and hemoglobin deoxygenation in RBCs when hypoxia is encountered. Thus, our findings suggest that erythrocyte ENT1 and ADORA2B are novel targets to prevent hypoxia-mediated tissue injury.

Disclosures

No relevant conflicts of interest to declare.
Multiple Oral Therapeutics Suppress Repressors (LSD-1, HDACs, and BCL11A) of Gamma Globin Gene Expression
Authors: Dai Yan , Faller Douglas V , Sangerman Jose I , Fucharoen Suthat , Perrine Susan P
Published: October 2019

Induction of gamma globin expression to certain target levels reduces clinical severity in sickle cell disease, globin imbalance, and anemia in the beta thalassemias. However, achieving high level expression in diverse patients with variable baseline HbF levels is clinically challenging. To determine whether different therapeutics have complimentary molecular actions that could be combined for higher efficacy, we evaluated a panel of oral fetal globin-inducing therapeutics in clinical use or trials for effects on transcriptional suppressors of HBG, including components of the NuRD complex, LSD1 and HDACs 1-3, and the downstream repressor BCL11A, in erythroid progenitors cultured from sickle cell and beta thalassemia patients. Using chromatin immunoprecipitation assays, immunoblot, and qRT-PCR, we investigated two pan-HDAC inhibitors, MS-275 and SB939, a short chain fatty acid derivative without global histone deacetylation effects, sodium dimethylbutyrate (ST20), and PB-04, a PK-enhancing drug identified through high throughput screening (HTS), which has a long-standing benign clinical safety profile. These therapeutics induce fetal globin expression by 1.5 to 12-fold in different individuals’ progentiors over untreated controls and are active in 60-90% of patients’ progenitors. BCL11A mRNA and protein were suppressed by 3-7 fold and 5-10 fold, respectively, over untreated cells from the same subject by all agents tested. PB-04 treatment decreased LSD1 mRNA by 5-fold. MS-275 and ST20 suppressed KLF-1 mRNA by 3- and 2.5-fold respectively. ChIP analyses demonstrated that PB-04 reduced LSD1 and HDAC-3 occupancy on the gamma globin gene promoter by 4-fold and 3-fold, respectively, coincident with gamma globin induction; ST20 decreased HDAC-2 and LSD1 binding by 6- and 2-fold respectively. Further, histone marks associated with gene activation were enriched at the gamma-globin promoter by treatment with PB-04, including acetylated Histone H3K9 (AcH3K9), the target of HDAC3, and dimethylated H3K4 (H3K4me2), a target of LSD1. Collectively, these results identify oral clinical stage therapeutics which disrupt two major mechanisms of transcriptional repression. The findings provide a basis for targeting therapies in individuals and combining pharmacologic agents which induce HbF expression through complimentary molecular mechanisms to provide high level induction.

Disclosures

Faller: Phoenicia BioSciences: Employment, Membership on an entity's Board of Directors or advisory committees. Perrine:Phoenicia BioSciences: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Multi-Center Study of DEP Regimen As a Salvage Therapy for Adult Refractory Hemophagocytic Lymphohistiocytosis
Authors: Wang Yini , Wang Zhao , Huang Wenqiu , Hu Liangding , Cen Xinan , Li Lihong , Wang Jijun , Shen Jianliang , Wei Na
Published: October 2019

Purpose: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome. Current standard therapy increased the complete remission (CR) rate to more than 50%. Even so, about 30% patients fail the standard treatment. This study aimed to investigate the efficacy of liposomal doxorubicin together with etoposide, and high dose methylprednisolone (DEP) as a salvage therapy for adult refractory HLH.

Methods: Refractory HLH was defined as patient who did not achieve at least partial remission (PR) 2 weeks after initial standard HLH therapy. The DEP regimen included liposomal doxorubicin 25mg/m2 d1, etoposide 100mg/m2 Qw and methylprednisolone 15mg/kg d1-3, 2mg/kg d4-6, 1mg/kg d7-10,0.75mg/kg d11-14. We estimated the curative effect after 2 weeks therapy and 4 weeks therapy.

Results: We observed 63 refractory HLH patients from Jun 2013 to Jun 2014, including 42 males and 21 females. All the data were gathered from Beijing Friendship Hospital, 307 Hospital of PLA, Peking University People’s Hospital, Peking University Third Hospital, Beijing Chao-Yang Hospital, and Navy General Hospital. The median age was 34 years old (Range 18-78). Seventeen patients (27.0%) achieved CR and 31 patients (49.2%) achieved PR. The overall response reached 76.2% (48/63). The underlying disease of HLH were diagnosed within 2 to 8 weeks after HLH diagnosis, including 29 cases of lymphoma associated HLH, 22 cases of EBV associated HLH and 4 cases of familial HLH. There were still 8 cases had unknown underlying disease. The patients who had no response to DEP were died within 2 to 4 weeks after salvage therapy. Twenty-nine of the 48 patients who achieved PR or CR survived to undergo subsequent chemotherapy, allogenic hematopoietic stem cell transplantation (allo-HCT) or splenectomy. There were significant improvements on leukocytes, ferritin and soluble CD25 (sCD25) after 2 weeks therapy. Thrombocytes and glutamic-pyruvic transaminase (GPT) had improvements after 4 weeks therapy.

Conclusion: DEP regimen appears to be an effective salvage protocol for refractory HLH. Prospective randomized controlled trials of DEP regimen will further estimated the curative effect and define optimal dosing levels and schedules.

Disclosures

No relevant conflicts of interest to declare.
A New Prognostic Index for Multiple Myeloma Patients in the Era of Novel Agents
Authors: Cho Hyungwoo , Suh Cheolwon , Yoon Dok Hyun , Park Ji Hyun , Kim Shin , Lee Sang-wook , Park Chan-Sik , Huh Jooryung , Lee Kyoungmin , Kang Eun Hee , Park Jung Sun , Park Chan-Jeoung , Kim Jin Seok , Kim Kihyun
Published: October 2019

Introduction

Various prognostic factors for multiple myeloma (MM) have been identified, and combinations of these factors were suggested for staging classification of MM patients. The international staging system (ISS) is a widely accepted prognostic system for MM patients. However, since ISS was established using data collected from 1981 through 2002, only a small portion of patients who received the novel agents (e.g., thalidomide, bortezomib, and lenalidomide) were included. Therefore its validity is being challenged in the era of novel agents. We aimed to develop an alternative prognostic index for MM patients based on easily obtained laboratory measures in the era of novel agents.

Patients and methods

Data were collected from February 2001 through June 2012 at Asan Medical Center. 470 patients were newly diagnosed as symptomatic MM and 156 of them were treated with novel agents including thalidomide, lenalidomide and bortezomib. We analyzed these 156 patients who received novel agent therapy at least once, using prospectively collected data. A new prognostic model was developed based on significant potential prognostic factors in univariate and multivariate analysis, which was validated in external validation data set from two tertiary hospitals in Korea.

Results

With a median follow-up duration of 27.9 months (range, 0.2-122 months), the median overall survival in the whole population was 28.7 months. The median overall survival (OS) of patients according to ISS stage 1, 2 and 3 were 122.2, 43.9 and 30.0 months, respectively. There were no significant differences in median OS between ISS stage 1 and 2 patients and 2 and 3 patients (Figure 1). Furthermore, ISS was not statistically significant in the multivariate analysis. We incorporated serum beta2-microglobulin (Sβ2M), serum albumin and serum lactate dehydrogenase (LDH) as independent risk factors to develop a new prognostic model. Using these three factors, we stratified the patients into three groups; Group 1, Sβ2M < 5.5 mg/L plus serum albumin >= 3.5 g/dL plus serum LDH normal; Group 2, neither group 1 nor 3; Group 3, Sβ2M >= 5.5 mg/L plus serum albumin < 3.5 g/dL plus serum LDH > normal. We found that there were significant differences in median OS among these three groups (122.2, 41.6 and 5.6 months for group 1, 2 and 3, respectively) (Figure 2). This new prognostic index was further validated in the external cohorts (Median OS was 75.2, 36.3 and 23.1 months for group 1, 2 and 3, respectively) (Figure 3).

Conclusion

Our study suggests that this new prognostic index based on easily obtainable variables (Sβ2M, serum albumin, serum LDH) might be a simple and useful tool to predict prognosis for patients with MM in the era of novel agents, which warrants further validation.

Disclosures

No relevant conflicts of interest to declare.
DOCK2 Interacts with FLT3 and Modulates the Survival of FLT3-Expressing Leukemia Cells
Authors: Wu Min , Hamaker Max , Li Li , Small Donald , Duffield Amy S.
Published: October 2019

The FMS-like tyrosine kinase-3 (FLT3) receptor gene is the most commonly mutated gene in acute myeloid leukemia (AML), and patients carrying FLT3/ITD mutations have a poor prognosis. Despite continuing progress in the development of more effective FLT3 inhibitors, long-term success in inhibition of FLT3 activity in AML patients is still elusive. In order to achieve a better understanding of FLT3 biology and more effective strategies for the inhibition of FLT3 activity, a screen was performed on leukemia cell lines to search for FLT3-interacting proteins. One of the proteins identified in the screen was dedicator of cytokinesis 2 (DOCK2).

The DOCK family of proteins acts as guanine nucleotide exchange factors (GEFs) for Rho family of GTPases, which includes Rac GTPases. DOCK2 expression is limited to hematopoietic cells, and is known to regulate several crucial processes, including lymphocyte migration, activation and differentiation of T cells, and cell-cell adhesion and bone marrow homing of various immune cells.

We first verified that DOCK2 is expressed in primary AML samples from patients, and co-immunoprecipitation experiments showed that DOCK2 interacts with both wild-type FLT3 and FTL3/ITD in these cells. Co-immunoprecipitation experiments using leukemia cell lines demonstrated that DOCK2 interacts with FLT3, FLT3/ITD, FLT3/D835Y and FLT3/D835H, and that it predominantly interacts with the unphosphorylated form of FLT3. Knock-down of DOCK2 by shRNA did not significantly affect the growth of cell lines that lack expression of FLT3, but greatly reduced growth of cell lines expressing amplified wild type FLT3 (Sem K2), FLT3/D835H (HB11;19) and FLT3/ITD (MV4;11). Accordingly, colony formation assays revealed that cell lines with elevated expression of wild type or mutant FLT3 produced fewer, smaller and more compact colonies when the expression of DOCK2 was decreased, while colonies from cell lines lacking FLT3 expression showed no significant difference in response to the knock-down of DOCK2. Furthermore, an Annexin V binding assay indicated that reduction in DOCK2 expression level greatly sensitized cells with elevated FLT3 activity (MV4;11 and Sem K2) to cytarabine, resulting in increased apoptosis, but no significant sensitization was observed in cell lines that lack FLT3 expression.

These findings demonstrate that DOCK2 interacts with FLT3 in leukemia cell lines, and suggest that this interaction has important roles in regulating the survival of leukemia cells with elevated FLT3 activity, both alone and in combination with conventional anti-leukemic agents. Therefore, DOCK2 is a potential therapeutic target for AML treatment, and better understanding of the interaction between DOCK2 and FLT3 may contribute to the development of novel strategies to effectively inhibit FLT3 activity in AML patients.

Disclosures

No relevant conflicts of interest to declare.
Frequency and Prognosis of JAK2 V617F, Calr, MPL and ASXL1 Mutations in Primary Myelofibrosis
Authors: Sorigue Marc , Cabezón Marta , Garcia Olga , Velez Patricia , Marce Silvia , Boqué Concha , Xicoy Blanca , Fernandez Cristalina , Xandri Marisol , Casanova Enric , Gallardo David , Milla Fuensanta , Ribera Josep-Maria , Feliu Evarist , Zamora Lurdes
Published: October 2019

INTRODUCTION: Several mutations have been described in patients with BCR-ABL1 –negative chronic myeloproliferative neoplasms, including primary myelofibrosis (PMF). The most frequent mutation is JAK2 V617F, followed by calreticulin exon 9 (CALR), MPL exon 10 and ASXL1 exon 12. Currently, less than 10% of patients lack molecular marker. CALR and ASXL1 mutations have been consistently found to have favorable and unfavorable prognostic implications, respectively.

The aim of this study was to describe the frequency and prognostic impact of JAK2 V617F, CALR, MPL and ASXL1 mutations in patients diagnosed with PMF in 6 Spanish hospitals.

METHODS: To detect the presence of JAK2 V617F mutation, an allele-specific PCR using TaqMan probes was used. Screening for insertions and deletions in CALR gene was performed with 6-FAM labeled primers spanning exon 9 and CALR mutations were described by Sanger sequencing. Sanger sequencing was also used to detect MPL exon 10 and ASXL1 exon 12 mutations.

RESULTS: Sixty-eight patients were included in the study. All of them were screened for JAK2 and CALR mutations. Forty-five of them (66%) were positive for the JAK2 V617F mutation, while 11/68 (16%) were positive for CALR mutations. Of the 11 CALR mutations, 10 were JAK2 wild-type. MPL exon 10 mutation analysis was only performed in JAK2 wild-type patients and was positive in 4/23 patients (17%), and all of them were CALR wild-type. At the time of submission, ASXL1 exon 12 has been assessed in 18 patients (analysis in the rest of them is currently ongoing). ASXL1 mutations have been found in 3/18 (17%) patients, two of them also with a CALR mutation and the other one with the JAK2 V617F mutation. All three cases were indel mutations. Overall, no mutation was detected in 9/68 (13%) patients. JAK2 V617F, CALR and MPL mutations had no prognostic impact on overall survival. The effect of ASXL1 mutation on prognosis (with and without CALR mutation) will be assessed once all samples have been sequenced.

CONCLUSION: JAK2 V617F, CALR and MPL mutations were found in our series of PMF patients in the same proportion found in larger series. ASXL1 has so far been found in a smaller percentage but the entire series of patients will need to be sequenced before reaching definitive conclusions. Studying these genes, only 13% of patients with PMF did not have a clonal marker. None of the studied mutations had prognostic significance.

ACKNOWLEDGMENTS: The authors would like to thank Diana Dominguez for her excellent technical assistance and to the grant 2014 SGR225 (GRE), Generalitat de Catalunya.

Disclosures

No relevant conflicts of interest to declare.
Calreticulin Gene Mutations in Suspicion of Myeloproliferative Neoplasms: A Single Institution Experience
Authors: Ben Lassoued Amin , Beaufils Nathalie , Bonnefoy Caroline , Gabert Jean
Published: October 2019

Recently, Klampfl et al. and Nangalia et al. reported recurrent somatic mutations in the calreticulin (CALR) gene in patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 and MPL. All CALR mutations described to date are either deletions or insertions, and occur in exon 9 and result in a frameshift leading to a loss of KDEL sequence (endoplasmic reticulum retention motif) and multiple calcium biding sites with a new basic (instead of acidic) C terminal region.

The aim of this work is to know how far the CALR mutations can fill the molecular diagnostic gap for myeloproliferative neoplasms (MPNs) left by JAK2 and MPL mutations.

The study was approved by the local ethics committee.

Our study population consists of 155 patients with platelet counts > 500 G/L sampled between 2012 and 2013 for suspicion of MPNs. Only JAK2 V617F negative patients were included.

Analyses were performed using genomic DNA isolated from peripheral blood. Patients were screened for CALR mutations using a High Resolution Melting (HRM) and then the precise mutations were characterized by Sanger sequencing which is a new approach for this test.

A total of 21 (13.5%) patients with CALR mutations were detected with 8 distinct variants.

Among CALR mutations, type 1 (52 basepair deletion, c.1092_1143del) and type 2 (5 basepair insertion, c.1154_1155insTTGTC) were the most common (respectively 7 (33%) and 7 (33%) cases). Three patients had 2 concurrent mutations (insertion+substitution / Deletion+substitution / deletion +insertion). We found a deletion that has not been reported so far to our knowledge (c.1125_1147del). Sequencing is ongoing for 4 patients.

Compared with negative patients, CALR-mutated patients demonstrated markedly higher platelet counts (median count: 820 vs 543 G/L).

Disclosures

No relevant conflicts of interest to declare.
Human BDH2 As a Predictor for Leukemia Progression in Myelodysplastic Syndrome
Authors: Yang Wen-Chi , Lin Sheng-Fung
Published: October 2019

Objective: Myelodysplastic syndrome (MDS) is a disorder of hematopoietic stem cells. In MDS patients, leukemia progression is associated with iron overload. Hydroxybutyrate dehydrogenase type 2 (BDH2) catalyzes the production of 2,5-dihydroxybenzoic acid (DHBA), an iron-binding component.

Methods: The bone marrow (BM) of 187 MDS patients, 119 de novo acute myeloid leukemia (AML) patients, and 43 lymphoma patients (stage I-III) with normal BM were analyzed. BDH2mRNA expressions from BM and BDH2-KD THP1 were analyzed using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Cell cycle and cell maturation were determined by flowcytometry and surface markers in RNA interference–mediated-knockdown of BDH2 (BDH2-KD) THP1, an acute leukemia cell line. Immunohistochemistry was performed to detect BDH2 protein expression in the bone marrow.

Results: Elevated BDH2mRNA expression was observed in bone marrow in MDS patients (P=0.009) and was related to ferritin levels (P=0.049). The higher BDH2 expression (15%) group showed a greater risk for leukemia progression than the lower expression group (3.18%) (P=0.017). Leukemia-free-survival was longer in patients with low BDH2mRNA expression (P=0.028) and patients with ferritin level <1000 ng/mL. Additionally, cell cycle analysis, surface markers, and special stain studies indicated that BDH2-KD induced differentiation and decreased the growth rate of THP1 cells, which was associated with the retardation of cell cycle.

Conclusions: We identified new molecular mechanisms of BDH2. BDH2 is involved in cell cycle arrest and inhibition of differentiation in malignant cells. Further, high BDH2 expression in MDS patients suggest that it could act as a poor prognostic factor.

Disclosures

No relevant conflicts of interest to declare.
Evaluation of Azacitidine Clinical Efficacy in Patients with Myelodysplasia Syndrome Dudina G.a., Golenkov a.K., Kataeva E.V., Mitina T.a. Moscow Regional Clinical Research Institute
Authors: Golenkov Anatoly
Published: October 2019

Introduction: The anti-tumor effect of azacitidine in patients with myelodysplasia syndrome is based on cytotoxicity against pathologically changed hemopoetic cells of bone marrow and DNA hypomethylation.

Objective: evaluation of clinical efficacy of azacitidine in patients with myeloddysplasia syndrome.

Materials and methods: we have monitored 7 patients with myelodysplasia syndrome and refractory anaemia with excessive blast count, type II. IPSS with a high risk of acute leucosis development was found in 4 patients; intermediate risk was reported in 3 patients. Mean age – 62 years. Chromosomal aberrations (multiple clone transformations) were found in 4 patients during cytogenetic analysis. Normal karyotype was reported in 3 patients. Treatment regimen: azacitidine 75 mg/m² daily s/c injections, Days 1 - 7. Totally, 3 – 7 induction cycles were conducted, median (Me) – 4 cycles. Further maintenance treatment with 6-8 weeks interval was conducted in 5 patients. The mean index of haemoglobin level decrease was used to evaluate the hemopoetic ability in non-selective group; it was calculated as the ratio of the haemoglobin level at the time of discharge minus haemoglobin level at the time of the next hospitalization to the duration of the interval between treatment courses. The number of blood transfusions during the interval between treatment courses was constant: 2-3 transfusions of RBCs.

Results: 4 azacitidine treatment courses resulted in decrease of blast cells number in bone marrow from 16.4 ± 0.2 % to 5.4% ± 0.7 % (р<0.05). Mean index of haemoglobin level decrease after the first hospitalization was 1.9 g/l per day and 0.3 g/l per day after the 4-th treatment course (р<0.05) The following results were reported using standard Cheson criteria: full response – 1 patient, hematologic improvement – 2 patients, bone marrow remission – 3 patients. Mean duration of response 8.5 months. Treatment complications: pleural empyema, abscess of left lung lower lobe after the 2-nd treatment cycle – 1 patient; broncho-pulmonary aspergillosis after 3-rd treatment cycle – 1 patient. In other patients drug toxicity that could possibly affect the interval between courses or decrease the drug dose was not reported. Delayed treatment results: 100% of patients survived the 12 – months period.

Conclusions: in patients with myelodysplasia syndrome and refractory anaemia with excessive blast count, type II treatment with azacitidine (median treatment course duration – 4 months) resulted in full response in 14% of cases (1 patient), bone marrow remission was reported in 43% of cases (3 patients). Use of original method of haemoglobin level decrease index determination enables evaluation of the cumulative azacitidine dose effect on hemopoesis recovery after 4 treatment cycles.

Disclosures

No relevant conflicts of interest to declare.
The Role BAFF and BAFF Receptors in Macrophage Induced Drug Resistance of Multiple Myeloma
Authors: Chen Jing , He Donghua , Guo Xing , Yang Li , Li Yi , Chen Qingxiao , He Jingsong , Yang Yang , Zhang Enfan , Wu WenJun , Han Xiaoyan , Zheng Gaofeng , Zhao Yi , Huang He , Cai Zhen
Published: October 2019

Background: B-cell–activating factor (BAFF) is a member of TNF family that critical for maintenance of B-cell development and homeostasis. BAFF also modulates the proliferation, survival and drug resistance of multiple meyloma (MM) cells. BAFF can be secreted by neutrophils, monocytes,dentritic cells and macrophages. Our previous study showed that, macrophages protect MM cells from drug-induced apoptosis by a cell-to-cell interaction between MM cells and macrophages. We supposed that the interaction of BAFF and BAFF receptors plays a role in macrophages induced MM resistance.

Methods: First, we detected the expression levels of BAFF and its three receptors in MM cells were detected by semiquantitative real time-polymerase chain reaction (qPCR) and flow-cytometry. The concentration of BAFF in the culture supernatants of MM cell lines was detected by ELISA. Second, we collect peripheral blood monocytes form healthy donors. Monocytes were induced into macrophages by culturing with M-CSF for 7 days.BAFF expression level in macrophages was detected by qPCR, flow-cytometry and ELISA. Then MM cells were sole-cultured or coculture with macrophages for the indicated time (usually 24 h), after that bortezomib was added to the culture system. Cell viability and apoptosis of MM cells were verified by MTT and flow cytometry.At last,the recombinant human BAFF were added to MM cells, MTT and flow cytometry were used for detection of cell viability and apoptosis of MM cells.

Results: Two receptors of BAFF, transmembrane activator and CAML interactor (TACI) and B-cell maturation antigen (BCMA), are highly expressed in various MM cell lines evidenced by real-time PCR and flow cytometry. The expression of BAFF in PBMC-induced macrophages are heterogeneous. We verify macrophage-mediated MM drug resistance by directly coculturing MM cells (ARP-1, 8226 and OPM2) with PBMC-derived macrophages from healthy donors. Functional studies show that recombinant human BAFF rescues RPMI8226 myeloma cells from dexamethasone-induced apoptosis.

Conclusions: Our data showed that macrophage might induce drug resistance of MM cells by the interaction of BAFF and BAFF receptors. Further study will focused on the mechanism of interaction between BAFF and BAFF receptors.

Disclosures

No relevant conflicts of interest to declare.
Antitumor Activity of a Novel Proteasome Inhibitor BSc2118 Against Multiple Myeloma
Authors: Zang Meirong , Liu Lanting , Li Xin , Li Wei , Kuckelkorn Ulrike , Zhou Wen , Qiu Lugui , Jin Fengyan
Published: October 2019

Background: Although the first generation of proteasome inhibitor bortezomib is very effective, the development of resistance limits it long-term utility. In this study, we explored the efficiency and molecular mechanisms of the novel, irreversible proteasome inhibitor BSc2118, particularly, the reversal of bortezomib resistance.

Materials and Methods: Human MM cell lines (MM.1S, MM.1R, RPMI-8226, U266, NCI-H929) were treated with BSc2118 at various concentrations for 48h, and assessment for cell viability by CCK-8 assay. MM.1S and MM.1R cells were treated with BSc2118 for 24 hours, and cell cycle and apoptosis analysis were conducted by flow cytometry. Associated molecules were detected by qRT-PCR and western blot. Chymotrypsin-like proteasome activity assay was performed by using the 20S proteasome assay kit. Ubiquitinated proteins were isolated and determined with ubiquitin enrichment kit.

Results: Our results revealed that treatment of MM cell lines with BSc2118 inhibits the chymotrypsin-like proteasome activity and induces accumulation of ubiquitinated proteins. BSc2118 inhibits MM cell growth and induces MM apoptosis via induction of G2/M phase arrest, activation of cleaved caspase-3, caspase-8 and caspase-9 and PARP, increasing p53, p21 and E2F1, and inhibition of autophagy in MM.1S, MM.1R and RPMI-8226 cell lines. In addition, BSc2118 dramatically inhibits cytokines mRNA, such as IL-6, VEGF and bFGF in both myeloma cells line and primary bone marrow stromal cells from myeloma patients. More importantly, BSc2118 could overcome bortezomib resistance in vitro by using primary CD138 positive plasma cells from bortezomib-resistant myeloma patients and bortezomib resistance cell line ANBL-6 (ANBL-6.BR), most likely as the consequence of inhibition of autophagy flux which is responsible for bortezomib resistance.

Conclusion: Our study revealed BSc2118, a novel irreversible proteasome inhibitor, exerts anti-MM effect, mainly through activation of caspase pathway and inhibition of basal autophagy. It is of great importance that BSc2118 could overcome bortezomib resistance via inhibition of autophagy flux. A head to head of BSc2118 versus Bortezomib is performing in human plasmacytoma xenograft tumor model to evaluate drug safety, anti-tumor efficiency, in particular, reversal of bortezomib resistance. Our preclinical study supports clinical evaluation of BSc2118, particularly, overcomes bortezomib resistance, as a potential MM therapy.

Disclosures

No relevant conflicts of interest to declare.
Effectiveness of Lenalidomide and Dexamethasone for the Treatment of Extramedullary Plasmacytoma in Patients with Multiple Myeloma: Report of Two Cases
Authors: Masárová Katarína , Stefanikova Zdenka , Mistrik Martin , Batorova Angelika
Published: October 2019

Extramedullary plasmacytoma (EMP) can occur at the time of primary diagnosis of multiple myeloma (MM), during the disease, or as solitary plasmacytoma without bone marrow involvement. The presence of EMP at any time in the course of disease is associated with significantly shorter overall survival and progression-free survival. We report a series of two patients with EMP who were successfully treated with lenalidomide and dexamethasone.

The first patient was diagnosed in 2006 with EMP in the spine without presence of MM at the time of diagnosis. Reconstruction surgery followed by high dose dexamethasone treatment led to stabilization of patient’s condition. Patient received zoledronic acid for prevention of skeletal fractures. In 2011 the patient was diagnosed with MM and two new EMP lesions in the spine were discovered. The patient received bortezomib and achieved a partial remission. In 2012 a significant growth of EMP in retroperitoneum was observed. Tumor was surgically removed, plasmacytoma was confirmed histologically. Patient was treated with lenalidomide and dexamethasone with subsequent autologous peripheral blood stem cell transplantation (APBSCT) and achieved complete remission.

Patient Two had advanced stage of MM at the time of diagnosis, without presence of EMP. She was treated with induction therapy followed by APBSCT. EMP in the area of clivus, diagnosed three years after initial diagnosis of MM was successfully managed with conventional chemotherapy and radiotherapy. In 2010 another EMP in the left sphenoid sinus progressed during relapse of MM. Treatment with lenalidomide and dexametazone led to remission of MM and disparition of EMP. Both patients are in complete remission at the time of submitting this abstract.

Our clinical experience strongly suggests that lenalidomide with dexamethasone is an effective agent for treating EMP in patients with MM. The treatment was well tolerated with manageable side effects.

Disclosures

No relevant conflicts of interest to declare.