bluuebunny/crossref_metadata_2025_split huggingface dataset:
Low Expression of RPS14 Accompanied with p53-Independent Over-Apoptosis in Myelodysplastic Syndromes Patients with 5q- Aberration
Authors: Wu Lingyun , Zhang Yin , He Qi , Zhang Qingxia , Chang Chunkang , Li Xiao 
Published: October 2019

Abstract
Abstract 5046

Purpose
In this study, we focused on evaluating ribosomal protein S14 (RPS14) and p53 expression and their correlation analysis with the purpose of investigating the role of RPS14-MDM2-p53 pathway in hematopoietic cell apoptosis of myelodysplatic syndrome (MDS) patients with 5q- aberration.

Patients and Methods:
Real time quantitative PCR was used to detect the expression levels of RPS14, MDM2 and p53 in 18 MDS patients, among which 7 with isolated del (5q) and 11 del (5q) with other chromosomal abnormalities were included. Besides, alkaline phosphatase-anti-alkaline phosphatase (APAAP) and terminal deoxynucleotidyI transferase dUTP nick end labeling (TUNEL) techniques were carried out simultaneously for each sample to respectively examine the expression of p53 and the apoptosis level of bone marrow (BM) mononuclear cells (MNCs). 10 healthy controls were included in the study.

Results:
under-expression of RPS14 gene was seen in 15/18 patients' (83.3%), of which 6/7 were isolated 5q- and 9/11 were 5q- with other chromosomal abnormalities. RPS14 expression was negatively correlated with the proportion of 5q- clonal cell in BM while it showed a positive correlation with both of the expression of MDM2 and p53. The expression of p53 was not correlated to the high apoptosis level of BM in patients with del (5q). Furthermore, APAAP and TUNEL results demonstrated apoptotic cells in patients BM were p53-negative (see Fig 1). Conclusions: RPS14 gene was under-expressed in MDS patients with 5q- aberration, which did not result in a p53-dependent apoptosis. A p53-independent pathway may contribute to the over apoptosis of 5q- MDS patients.

Disclosures:
No relevant conflicts of interest to declare.
MyD88 Could Promote Multiple Myeloma Cells Growth and Survival
Authors: Xu Yang , Depei Wu , Tian Hong , Wang Rongxian , Yang Zhen 
Published: October 2019

Abstract
Abstract 5086
Toll like receptors (TLRs) are the major agents for innate immunity, which recognize invading microbial products. In previous study, we proved that activated TLR4 and TLR9 could promote MM cells growth and activated NF-κB signaling played a crucial role for ligands of TLR4 and TLR9 via increasing IL-6 autocrine. It was reported that MyD88 could play the key role in TLR signaling pathway and TLR4 and TLR9 shared the same MyD88-dependent pathway. In this study, we observed that expression of MyD88 mRNA in primary MM cells was higher than normal plasma cells and most of MM cell lines had a high MyD88 transcripts. Futher experiments demonstrated that transient over-expression of MyD88 could promote MM primary cells and MM cell lines growth and production of IL-6, IL-1, TNF-α, IFN-γ, VEGF and TGF-β. Moreover, down-regulation of MyD88 by sepecific virus based siRNA plamids in MM cell lines preliminarily resulted in reduced MM cells proliferation and increased MM cells apoptosis. These results highly suggested that MyD88 played a crucial role for TLR ligands to promote MM cells growth and survival, which make it possible to develop TLR system and MyD88 gene as the MM therapy target in the future.

Disclosures:
No relevant conflicts of interest to declare.
Level of Scientific Evidence Underlying Recommendations Arising From the National Comprehensive Cancer Network Clinical Practice Guidelines for Hematologic Malignancies
Authors: Kusuma Benny , Go Ronald S. 
Published: October 2019

Abstract
Abstract 509

Purpose:
The level of scientific evidence on which the National Comprehensive Cancer Network (NCCN) guidelines are based has not been systematically investigated. We describe the distribution of categories of evidence and consensus (EC) among the 10 most common hematologic malignancies with regard to recommendations for staging, initial and salvage therapy, and surveillance.

Methods:
NCCN uses a system of guideline development distinct from other major professional organizations. The NCCN definitions for EC are as follows: category I, high level of evidence with uniform consensus; category IIA, lower level of evidence with uniform consensus; category IIB, lower level of evidence without a uniform consensus but with no major disagreement; and category III, any level of evidence but with major disagreement.

Results:
Of the 1160 recommendations found in the 10 guidelines, the proportions of category I, IIA, IIB, and III EC were 3%, 93%, 4%, and 0%, respectively. Recommendations with category I were found in acute myeloid leukemia (4%), multiple myeloma (7%), Hodgkin's lymphoma (1%), diffuse large B-cell lymphoma (4%), follicular lymphoma (11%). Chronic lymphocytic leukemia/small lymphocytic lymphoma, mantle cell lymphoma, marginal zone lymphoma, AIDS-related B-cell lymphoma, and Burkitt lymphoma did not have any category I recommendations. Three percent of all therapeutic recommendations were category I. Guideline with the highest proportion of category I therapeutic recommendations was for diffuse large B-cell lymphoma (46%). No category I recommendations were found on staging or surveillance.

Conclusion:
Recommendations issued in the NCCN guidelines are largely developed from lower levels of evidence but with uniform expert opinion. This underscores the urgent need and available opportunities to expand evidence base in oncology.

Disclosures:
No relevant conflicts of interest to declare.
Potentiation of bortezomib's Anti-Myeloma Effects in Combination with Anti-DR5 Agonistic Antibody: The Role of ER Stress in DR5 Editing and DR-Mediated Apoptotic Signaling
Authors: Miki Hirokazu , Abe Masahiro , Kagawa Kumiko , Oda Asuka , Amou Hiroe , Ikegame Akishige , Watanabe Keiichiro , Hiasa Masahiro , Harada Takeshi , Fujii Shiro , Nakamura Shingen , Nakano Ayako , Takeuchi Kyoko , Ozaki Shuji , Shiotsu Yukimasa , Ri Masaki , Iida Shinsuke , Matsumoto Toshio 
Published: October 2019

Abstract
Abstract 5104
Bortezomib (BTZ) is widely used in the treatment of myeloma (MM) with marked response rates in both relapsed/refractory as well as newly diagnosed MM. However, significant numbers of patients still remain outside benefit of the BTZ treatment; and various combinatory treatments with BTZ have been implemented to improve BTZ's anti-MM effects. On the other hand, immunotherapies seem attractive for yet incurable malignancies by chemotherapeutic agents such as MM and their clinical application has been studied. One such approach is a TNF-related apoptosis-inducing ligand (TRAIL)-mediated immunotherapy. In the present study, we therefore explored the role of BTZ on TRAIL receptor editing and its downstream signaling with special reference to endoplasmic reticulum (ER) stress and the cytotoxic effects of BTZ and anti-TRAIL receptor agonistic antibody in combination on MM cells. Most MM cells expressed DR4 but weakly DR5, while normal peripheral blood mononuclear cells expressed neither DR4 nor DR5. BTZ at 10 nM markedly up-regulated the surface levels of DR5 and its mRNA expression but not those of DR4 in MM cell lines and primary MM cells. Furthermore, BTZ decreased the levels of c-FLIP, an inhibitor of DISC, along with activation of caspase-8 and caspase-3, suggesting potentiation of the DR-mediated extrinsic apoptotic pathway. Consistently, BTZ and anti-DR5 agonistic antibody cooperatively enhanced the cytotoxicity against MM cells. BTZ induced phosphorylation of eIF2alpha, ATF4 and CHOP, along with disappearance of anti-apoptotic proteins including Mcl-1 in MM cells, suggesting the enhancement of ER stress and subsequent suppression of protein translation by BTZ. However, such induction of ER stress by BTZ was not observed in BTZ-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ, with a point mutation in BTZ-binding proteasome beta5 subunit (Ri et al. Leukemia 2010). In KMS-11/BTZ and OPM-2/BTZ, surface protein as well as mRNA levels of DR5 were not up-regulated by BTZ, suggesting a critical role of ER stress in up-regulation of DR5 expression by BTZ. Because DR5 expression has been shown to be transcriptionally up-regulated by CHOP, the up-regulation of DR5 mRNA and protein in MM cells by BTZ is suggested to be at least in part due to CHOP induced by BTZ-mediated ER stress. Although BTZ exerts its anti-MM effects through induction of ER stress, the present study demonstrates that induction of ER stress by BTZ is also able to sensitize MM cells to TRAIL-mediated immunotherapy. Therefore, the combination of BTZ and TRAIL-mediated immunotherapy is warranted for further study.

Disclosures:
No relevant conflicts of interest to declare.
Here Is the Sub-Title:Apoptosis on Multiple Myeloma U266 Cell by Phosphorylation c-Jun of Brucine
Authors: Ma Yanping , Li Zhihua , Wang Yihua , Feng Jing 
Published: October 2019

Abstract
Abstract 5112

Objective:
To investigate the Mechanism of the apoptotic effect of brucine on human multiple myeloma.

Method:
U266 cells (5×104) were plated in the presence or absence of the brucine (0, 0.05, 0.1, 0.2, 0.4 mg/ml) in 96 well culture plates for 24–72 h. The anti-proliferative response of brucine was assessed by MTT assay. The analysis of cell cycle of U266 cell with or without brucine was mesured by flow cytometry. The expression change of c-Jun after joining brucine, brucine and JNK specific inhibitor SP600125 was detected using RT-PCR.

Results
The apoptotic effect of brucine show a dose and time dependent manner. Cell cycle analysis using flow cytometry revealed accumulation of cells at sub-G0/G1 phase. The apoptosis rate separately were (4.137±0.01)%, (10.55±0.03)%, (12.31±0.04)%, (27.67±0.08)%, (29.67±0.09)% (p<0.01). Detecting c-Jun gene expression respectively after joining brucine, brucine and JNK specific inhibitor SP600125 by RT-PCR. The gray scale values were (0.7961±0.007),(0.4683±0.003).

Conclusions
Within the 0.4mg/ml concentration of brucine can induce apoptosis in U266 cells. Brucine by JNK signaling pathway through phosphorylation of c-Jun induced apoptosis in U266 cells.

Disclosures:
No relevant conflicts of interest to declare.
Role of NKG2D in Cytokine-Induced Killer (CIK) Cells Against Multiple Myeloma Cells
Authors: Lu Xu-zhang , Chen Bao-An , Ma Lin-di , Cai Xiao-hui , Zhou Min 
Published: October 2019

Abstract
Abstract 5119
Cytokine-induced killer (CIK) cells are T lymphocytes enriched in CD3+CD56+ cells, which can be easily and rapidly expanded in vitro from human peripheral blood, bone marrow or cord blood mononuclear cells with the sequential addition of interferon (IFN)-{gamma}, OKT-3 and high doses of interleukin (IL)-2. The cytokine-induced killer (CIK) cells have been reported to have potent cytotoxicity against a variety of tumor cells including multiple myleoma (MM) cells. The mechanism of CIK cell recognizing MM cells remains unknow. Recent studies indicated that ligation of NKG2D on immunological cells directiy induce cytotoxicity. We suspect whether NKG2D receptor induction on CIK cells by cytokines is responsible for the killing of MM cells by CIK. We expended CIK cells from healthy controlswith interferon (IFN)-γ, CD3 monoclonal antibodies (mAb) and IL-2, and checked expression of NK cell receptors on CIK cells by flow cytometry. We found higher expression of NKG2D receptor and lower other NK receptors, such as CD158a,CD158b and NCRs on expanded CIK. These CIK cells showed higer cytotoxicity to multiple myleoma cell line U266 expressing NKG2D ligands. Interestingly, when cocultured with U266 cells, only NKG2D expressing CIK cells released IFN-γ detected by flow cytometry. We next analyzed NKG2D ligands expression on primary plasma cells in 22 MM patients by flow cytometry, the primary plasma cells in 16/22 (72.7%) MM patients expressed different levels of ULBPs or MICA/B on the cell surface. CIK cells showed higher cytotoxicity (12.5%) to NKG2D ligands expressing primary plasma cells compared to those did not express NKG2D ligands. The killing of CIK against MM cells were partially blocked by treatment of CIK with anti-NKG2D antibody. We conclude that NKG2D-NKG2D ligangd interaction may be one of the mechanisms by which CIK cells kill MM cells.

Disclosures:
No relevant conflicts of interest to declare.
A Phase I/II Study of Bortezomib in Combination with Doxorubicin and Intermediate-Dose Dexamethasone (iPAD therapy) for Relapsed or Refractory Multiple Myeloma
Authors: Takamatsu Yasushi , Sunami Kazutaka , Muta Tsuyoshi , Tsukada Junichi , Miyamoto Toshihiro , Eto Tetsuya , Higuchi Masakazu , Hata Hiroyuki , Suzumiya Junji , Tamura Kazuo 
Published: October 2019

Abstract
Abstract 5137

[Background]
Bortezomib and doxorubicin have synergistic activity against myeloma cells. However, the combination of bortezomib and cytotoxic agents has not long been permitted in Japan because the safety of the combination therapy has not been determined in Japanese patients. We, therefore, decided to undergo a phase I/II study of bortezomib, doxorubicin and intermediate-dose dexamethasone (iPAD) combination therapy in Japanese patients with relapsed or refractory myeloma.

[Methods]
In a phase I study, the maximally tolerated dose of bortezomib was determined when given in combination with a fixed dose of doxorubicin and dexamethasone. Bortezomib was administered iv on days 1, 4, 8 and 11 at a dose of 1.0 mg/m2 in cohort 1 and 1.3 mg/m2 in cohort 2. Doxorubicin 9 mg/m2 was given iv on days 1–4, and dexamethasone 20 mg po on days 1–2, 4–5, 8–9 and 11–12. After a recommended dose of bortezomib was determined, a phase II study was undertaken to examine overall response rate, survival time and toxicity profiles.

[Results]
The dose-limiting toxicity (DLT), defined as grade 4 hematological toxicity lasting more than 5 days and/or grade 3 or higher non-hematological toxicity, was observed in 2 of 6 patients in cohort 1, indicating this dose was considered tolerable. In cohort 2, 3 of 5 patients developed DLTs including grade 4 thrombocytopenia lasting more than 5 days, grade 3 elevated hepatic transaminase levels and grade 3 ileus in one of each patient, indicating this dose was intolerable. Thus, 1.0 mg/m2 of bortezomib is a recommended dose in iPAD therapy. In a phase II study, 27 patients, aged 40 to 78 (median 63), were enrolled to receive iPAD therapy by using 1.0 mg/m2 dose of bortezomib. Overall response (CR+PR) was achieved in 89% (95% CI, 76–100%), and complete response (CR+nCR) in 30% (95% CI, 11–48%). The median overall survival time was not reached and progression-free survival time was 12.3 months (95% CI, 6.1–18.5 months). One patient died of pneumonia. Grade 3/4 thrombocytopenia, neutropenia and anemia developed in 67%, 41% and 19%, respectively, and febrile neutropenia in 11%. Sensory neuropathy was observed in 78% with grade 3/4 in 22% of the patients. Grade 3/4 hypokalemia developed in 15%, hyponatremia in 11%, and fatigue, nausea and diarrhea in 4%.

[Conclusion]
The iPAD therapy is feasible and effective to treat relapsed or refractory myeloma in Japanese population.
(This clinical study was registered in University hospital Medical Information Network (UMIN) (registration number; UMIN000001210))

Disclosures:
No relevant conflicts of interest to declare.
MSCs From Patients with Myelofibrosis Display a Fibrotic Phenotype
Authors: Schneider Rebekka K , Leisten Isabelle , Ziegler Susanne , Schumacher Anne , Brümmendorf Tim H , Jost Edgar , Ziegler Patrick 
Published: October 2019

Abstract
Abstract 5160
Myeloproliferative neoplasms (MPNs) are characterized by the excessive production of terminally differentiated nonlymphoid cells or platelets in the bone marrow. They represent a heterogenous group of clonal hematologic malignancies and are classified into chronic myeloid leukemia (CML), essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) and other rarer diseases.
MPNs are often associated with extramedullary hematopoiesis and progressive hepatosplenomegaly due to displacement of hematopoietic progenitor cells from the BM to spleen and liver. Progenitor mobilization follows the enhanced deposition of extracellular matrix proteins, which can be found in the bone marrow of MPN patients.
Nonhematopoietic bone marrow stromal cells (BMSCs) and their precursors (mesenchymal stem cells) are believed to be conditioned by abundantly released growth factors from the pathological hematopoietic clone in MPNs and in turn contribute to a modified niche environment which participates in the maintenance of the malignant clone and in disease progression.
We therefore aimed at the comparative analysis of BMSCs from MPN patients (displaying myelofibrosis 0–1) and healthy donors as well as at their matrix remodelling capacity.
BMSCs from PMF patients were obtained by trephine biopsies and isolated by plastic adherence in IMDM, 20% FCS and cortisone. BMSCs from PMF patients fulfilled MSC criteria according to common consensus (Dominici et al., 2007). To compare their potential to support hemato-lymphopoiesis, we analyzed their hemato-lymphotropic cytokine transcription by RT-PCR. MSCs from MPN patients and healthy donors expressed gene transcripts for M-CSF, SDF-1, LIF, FLT3L, Oct-4, SCF, IL-6 and IL-7, suggesting no significant difference in cytokine production. However, when activated through contact with collagen I/III and embedded in three-dimensional scaffolds, MSCs from MPN-patients extensively remodelled the collagenous matrix compared to healthy donors. Under osteogenic and undifferentiated culture conditions, the extracellular matrix (ECM) production by MPN-MSC was strongly enhanced compared to MSCs from healthy controls as shown by RT-PCR and immunohistochemistry for collagen I, IV, fibronectin, laminin and osteopontin. Furthermore, MSCs from MPN patients significantly contracted and densified the collagenous matrix and the ECM deposition by MSCs from MPN patients was highly comparable to the ECM changes observed in corresponding bone punches. We therefore hypothesize that MSCs from MPN patients are primed to produce enhanced ECM proteins, whereas their capacity to support hematopoiesis seems to be unchanged.

Disclosures:
No relevant conflicts of interest to declare.
The Prevalence of HIV-1 DNA in AIDS-Related Lymphoma throughout the Epidemic in the US
Authors: Huysentruyt Leanne C , Kaplan Lawrence D , McGrath Michael S. 
Published: October 2019

Abstract
Abstract 5205
Kaposi sarcoma (KS) and non-Hodgkin lymphoma (NHL) are the two most common AIDS-defining cancers and significantly contribute to HIV-related deaths. While highly active antiretroviral therapy (HAART) has produce a sharp decline in the incidence of KS and AIDS-related NHL (ARL), HIV-infected patients are still at a high risk for developing these malignancies. Recent studies evaluating HIV-1 DNA in 91 AIDS-related KS cases demonstrated a significant decrease in the prevalence of HIV-1 DNA positive KS cases post-HAART (after 1996; 16.7%) as compared to pre-HAART (before 1996; 45%) corresponding to the decrease in incidence of this cancer since the introduction of HAART. Additionally HIV was more prevalent in sites of visceral KS (51.9%) as compared to skin KS (30.7%). We have recently evaluated the HIV sequence evolution in the context of metastatic ARL and identified lymphoma-specific HIV compartmentalized within tumor sites that was genetically distinct from HIV in non-tumor sites suggesting a specific form of HIV arises within individuals who develop ARL. The goal of this research was to determine the prevalence and quantity of HIV DNA in 119 ARL biopsies representing the entire span of the HIV epidemic (1982–2005). Whole genomic amplification was performed on DNA extracted from three 10um paraffin embedded tissue sections and the presence of HIV-1 DNA was determined by quantitative amplification of the HIV gag gene. Of the 119 cases, 45% contained detectable levels of HIV DNA. The HIV antigen was predominantly localized to macrophages. A subset of the ARL cases in this study contained approximately 1 HIV copy per cell which is extremely high considering previously reported data describing HIV-infected lymph nodes contained approximately 1 HIV copy per 1000 cells. Similar to what was observed for KS, the prevalence of HIV-positive ARLs has decreased in the post-HAART era (39%) when compared to pre-HAART (54%). While the prevalence of HIV-1 DNA positive ARLs declined in the post-HAART era, it was not to the same extent as what was observed for KS. Thus, our data is consistent with a decrease in the incidence of both tumor types in the post-HAART era. The higher prevalence of HIV-1 DNA in visceral sites of KS and lymphoma specific-HIV sequences in sites of metastatic lymphoma suggests that HIV, especially HIV infected macrophages, may play a role in the pathogenesis of KS and ARL disease progression.

Disclosures:
No relevant conflicts of interest to declare.
Hemophagocytic Lymphohistiocytosis Is Caused by Engulfment of Hematopoietic Stem Cells by Macrophages Through Stem Cell-Specific Suppression of CD47 Under Influence of Cytokine Deregulation
Authors: Kuriyama Takuro , Takenaka Katsuto , Kohno Kentaro , Yoshimoto Goichi , Kishimoto Junji , Abe Yasunobu , Miyamoto Toshihiro , Akashi Koichi 
Published: October 2019

Abstract
Abstract 522
Hemophagocytic lymphohistiocytosis (HLH) is a syndrome characterized by hemophagocytosis in the bone marrow with systemic inflammatory reactions. We have reported that the inflammatory cytokinemia including interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) might play a key role in activating macrophages to induce HLH (Akashi et al. Br J Haematol, 1994). Recent studies have shown that the engulfment of blood cells by macrophages is regulated in part by the interaction of CD47 and its ligand, signal regulatory protein alpha (SIRPA). Blocking the CD47 and SIRPA interaction by using anti-CD47 monoclonal antibodies can induce engulfment of blood cells, suggesting that the SIRPA signaling provide “don't eat me signals” to prevent macrophages from digesting self blood cells. These data led us to hypothesize that HLH is caused by the disruption of self-recognition by macrophages through impairment of the CD47-SIRPA system. To test this hypothesis, we evaluated the expression level of CD47 in bone marrow cells in 24 patients with HLH. Interestingly, the expression of CD47 was significantly downregulated (by ∼2-fold reduction in average at the protein level) in the CD34+CD38− hematopoietic stem cell (HSC) fraction in HLH patients, whereas that of the CD34+CD38+ progenitor and other mature blood cell fractions was unchanged. We then purified the CD34+CD38− HSCs and CD34+CD38+ progenitor cells from HLH patients and normal controls, co-cultured them with human macrophages in the presence of IFN-gamma and lipopolysaccharide, and evaluated the percentage of phagocyting macrophages. The numbers of phagocyting macrophages were significantly higher in cultures of CD34+CD38− HSCs from HLH patients, as compared to those in cultures of normal HSCs and of progenitor populations, suggesting that the expression level of CD47 inversely correlated with the efficiency of hemophagocytosis. Furthermore, normal HSCs but not CD34+CD38+ progenitors or other mature blood cells down-regulated CD47 in vitro in response to IFN-gamma, TNF-alpha, and IL-6, suggesting that these cytokines plays a critical role in down-regulation of CD47 especially in HSCs. In contrast, the expression level of SIRPA did not differ in myeloid cells between HLH patients and normal controls, and mutations of SIRPA were not found in HLH patients. Thus, in HLH, inflammatory cytokines down-regulate CD47 selectively in HSCs, resulting in the engulfment of HSCs by macrophages. Our data strongly suggest that the cytokine deregulation can lead to HLH in human, through disruption of self-recognition guided by the CD47-SIRPA system at the HSC stage.

Disclosures:
No relevant conflicts of interest to declare.