bluuebunny/crossref_metadata_2025_split huggingface dataset:
TET2 Gene Expression in Bone Marrow Cells in Myelodysplastic Syndromes Patients and the Effect of Silencing TET2 by siRNA On Biological Characteristics of Healthy CD34+ Cells
Authors: Shao Zonghong , Zhang Wei , Fu Rong , Li Lijuan , Wang Huaquan 
Published: October 2019

Abstract
Abstract 4934

Objective
To investigate the expression of TET2 mRNA in bone marrow mononuclear cells(BMMNC), CD3+ T cells and CD34+ cells of patients with myelodysplastic syndrome(MDS)and to study the effect of silencing TET2 by Small interfering RNA (siRNA) on the biological characteristics of CD34+ cells.

Methods
CD3+ T cells and CD34+ cells were sorted by magnetic activated cell-sorting system. The expression of TET2 mRNA was detected by qPCR. The silencing effect of RNA interference (RNAi) on TET2 expression was identified by qPCR and Western blot analysis. The cell cycle kinetics and cell apoptosis were detected by flowcytometry.

Results
The expressions of TET2 mRNA in BMMNC, CD3+ T cells and CD34+ cells were down-regulated in MDS compared with that in controls. The siRNA targeting TET2 suppressed the expression of TET2 in CD34+ cells. Meanwhile, the proliferation activity was significantly enhanced and the apoptosis rate was declined in cells which transfected with TET2 siRNA as compared to those of the cells transfected with scrambled siRNA and control cells.

Conclusions
The mRNA expressions of TET2 in BMMNC, CD3+ T cells and CD34+ cells of MDS patients were decreased. Suppression of TET2 expression renders the CD34+ cells harboring more aggressive phenotype. This preliminary finding suggests that CD34+ cells lowering expression of TET2 may play an oncogenic role on myeloid tumor.

Disclosures:
No relevant conflicts of interest to declare.
Incidence and Outcomes for Low Risk Myelodysplastic Syndrome: A Surveillance, Epidemiology and End Results (SEER) Study
Authors: Zhang Yue , Podoltsev Nikolai A. , Gould Rothberg Bonnie E. , Dhodapkar Madhav V. , Morgensztern Daniel 
Published: October 2019

Abstract
Abstract 4944

Background:
Despite its common occurrence, there are few large population-based studies on low-grade myelodysplastic syndromes (MDS), which became available to United States central cancer registries only in 2001. We evaluated the outcomes for low-grade MDS according to histological subtype and cause of death.

Methods:
The Surveillance, Epidemiology and End Results (SEER) database was searched for patients with refractory anemia (RA), RA with ringed sideroblasts (RARS), refractory cytopenia with multilineage dysplasia (RCMD) and MDS with 5q deletion (5q-), diagnosed between 2001 and 2009 and with complete demographic information. Overall survival (OS) and disease-specific survival (DSS) were calculated from diagnosis to death from any cause and death from MDS or acute leukemia, respectively. Univariate survival was estimated by the Kaplan-Meier method and curves compared by log rank. Univariate and multivariable Hazard ratios were calculated by Cox Proportional Hazards. Incidence rates were calculated from SEERStat 7. 1.

Results:
8606 patients met inclusion criteria, including 3866 (45%) RA, 2639 (31%) RARS, 1498 (17%) RCMD and 603 (7%) 5q-. The percentage of females was highest in 5q- (61%) and lowest in RCMD (36. 5%) (p < 0. 001). Incidence rates per 100, 000 population were 0. 55, 0. 44, 0. 26 and 0. 09 for RA, RARS, RCMD and 5q-, respectively. Deaths from MDS or acute leukemia compared to other causes were 15. 9% vs. 38. 4% in RA, 17. 4% vs. 33. 6% in RARS, 29. 9% vs. 28. 9% in RCMD and 33% vs 24. 8% in 5q-. Both OS and DSS were significantly higher for RA and RARS compared to RCMD and 5q- in both univariate and multivariable analyses (Table).

Conclusions:
The survival for 5q- was similar to RCMD and significantly inferior to RA and RARS. This surprisingly shorter survival for 5q-, particularly with the highest incidence of death from MDS or acute leukemia has not been previously reported. Since 5q- is the least common subset of MDS and studies usually have a small number of patients, it is possible that a larger database such as SEER, which includes 21% of all cases diagnosed in US, may have unveiled a more accurate prognosis for this MDS subset. Although we cannot rule out the presence of additional genetic abnormalities to the 5q- subgroup, the poor outcomes observed in our study warrant further evaluation.

Disclosures:
Dhodapkar: Celgene: Research Funding; KHK: Research Funding.
Inhibition or Enhancement of Proliferation of Multiple Myeloma Cell Lines by CD137 Ligand Signaling?
Authors: Fu Chengcheng , Liu Hui , Wang Juan , Depei Wu 
Published: October 2019

Abstract
Abstract 4998
Multiple myeloma is an plasma cell malignancy. Despite new adjuvant biotherapeutics (thalidomide-like immunomodulatory drugs and proteasome inhibitors) have improved prognosis and survival, MM is still incurable. So unraveling the intricate pathobiology and exploitation of novel therapy are needed. CD137 and its ligand are members of the Tumor Necrosis Factor (TNF) receptor and TNF superfamilies, respectively. Since CD137 ligand cross-linking enhances proliferation and survival of healthy B cells we wonder that it would also act as a growth stimulus for B cell cancers.
From the study, the constitutive expression of CD137 ligand on U266, RPMI 8226, LP1, MY5 and OPM2 MM cell lines were detected as 89. 2%, 90. 9%, 57. 9%, 77. 9% and 78. 7% by Flow cytometry. Then U266 cell and RPMI 8226 cell were selected to explore the biological effect of CD137L signaling and its underlying mechanism. Resultly, in vitro study showed the proliferation and survival of U266 is enhanced by stimulating- CD137L mAb (1F1) as 4. 2*10e5 cells/ml(1F1 group) compared to 3. 5*10e5 cells/ml(control group) by cell counting at incubation for 48h. On the other wise, RPMI 8226 was not susceptible to 1F1 and was inhibited proliferation and induced cell death, by cell counting as 4. 9*10e5 cells/ml(1F1 group) compared to 5. 4*10e5 cells/ml (control group) at 48h and by CFSE as 277 compared to 197 (X-Mean of FCM) at 48h assay demonstrated. The cell cycle analysis showed that CD137 ligand induces proliferation and increases in the number of cells in the S phase from 36. 1% to 42. 5% of U266, while, showing the opposite from 50% to41% of RPMI 8226. Intracellular cytokine staining showed that treatment of cells with 1F1 increased the production of IL-6 of two cell lines, 3. 8% to 63. 9% of U266 and 90pg/ml to 250pg/ml of RPMI 8226. Finally, the two cells were treated with bortezomib and the growth of cells is analyzed by WST assay. It demonstrated that bortezomib can inhibit the function of 1F1 at U266 cells but have synergistic effection of 1F1 at RPMI 8226 cells. Inhibition rate of bortezomib were 22%, 51% and 58% of U266 and 58%, 80% and 77% of RPMI 8226 at 24h, 48h and 72h. As the inhibition rate of the two cells treated with bortezomib and 1F1 were 22. 9%, 46. 7% and 58% of U266 and 56%. 85% and 79. 7% of RPMI 8226.
It is at first sight surprisingly that CD137 ligand acts in concert with 1F1 having two opposite effection in different MM cell lines. Even so, we think it needed to explain some mechanism regarding different pass ways in different cell lines(the experiment under way).

Disclosures:
No relevant conflicts of interest to declare.
Activation of K-Ras in Arf-Null Mice Is Not Sufficient for Malignant Transformation of Post-Germinal Center B-Cells
Authors: Mullins Chelsea D. , Su Mack Y. , Hucthagowder Vishwanathan , Chu Liang , Lu Lan , Kulkarni Shashikant , Vij Ravi , Tomasson Michael H. 
Published: October 2019

Abstract
Abstract 5016
Activating mutations in Ras (N- and K-) are the most common mutations in multiple myeloma (MM), and are associated with advanced clinical stage and poor outcomes. As previously reported, we re-sequenced coding regions of highly expressed cytokine signaling candidate genes from MM patient samples and found the most common mutations to affect Ras oncogenes (N and K-). To test the role of Ras activation in MM pathogenesis directly, we generated mice harboring a mutant K-ras allele, Lox-stop-lox(LSL)- K-ras G12D (K-ras+/G12D) with Cγ1-Cre knock-in mice (Cγ1+/Cre), activated specifically in germinal center (GC) cells. K-ras+/+/Cγ1+/Cre mice served as negative controls. We found 58% of unstimulated K-ras+/G12D/Cγ1+/Cre mice developed fatal double-positive CD4/8 T-cell lymphomas (n=12) and 42% developed lung adenocarcinomas (n=12), phenotypes that have previously been reported for K-ras+/G12D mice. Tumor cells demonstrated successful recombination of the K-Ras locus. We showed recombination in splenic germinal center cells (B220+, GL7+ and IgG1+), as well as in post-germinal center cells (B220-, CD138+). No evidence of plasma cell proliferation was seen by serum ELISA, SPEP, flow cytometry or histological examination. We confirmed these results by crossing the K-ras+/G12D mice to a second strain, AID-Cre, which expresses the recombinase earlier and more specifically in the GC. We found 100% (n=20) of K-ras+/G12D/AID-Cre-YFP mice possessed visible, small benign papillomas on the ventral neck, as early as 3 weeks of age. To stimulate malignant transformation, vitamin D deficient chow and/or sub-lethal radiation was given to K-ras+/G12D/AID-Cre-YFP and negative control, K-ras+/G12D mice. Dual treatment increased the size and number of papillomas, giving a median survival of 26 weeks, compared to 52 weeks with control. However, no evidence of MM was detected by serum ELISA, SPEP, flow cytometry or histological examination. Finally, we generated AID-Cre-K-ras+/G12D triple transgenic mice in a tumor prone Arf-null background. After 13 wks, we found sarcomas in 66% (n=3) of K-ras+/G12D/AID-Cre-YFP/Arf−/− mice. Additionally, 100% (n=3) of these mice had rapid growing benign papillomas and only a minimal increase in the plasma cell compartment, compared to K-ras+/G12D/AID-Cre/Arf +/− control. Together, these data demonstrate that Ras activation is not sufficient to transform primary germinal center or plasma cells, even in an Arf-null context, and suggests that specific myeloma events, yet to be identified, are required for malignant transformation.

Disclosures:
No relevant conflicts of interest to declare.
Identification of the Black Tea Polyphenol Theaflavin-3, 3'-Digallate From Screen of Natural Product Inducers of Endoplasmic Reticulum Stress
Authors: Doudican Nicole A , Wen Shih Ya , Mazumder Amitabha , Orlow Seth J 
Published: October 2019

Abstract
Abstract 5021

Background:
A distinguishing characteristic of myeloma plasma cells is the large quantity of paraprotein that they synthesize and secrete, rendering them especially sensitive to the effects of endoplasmic reticulum (ER) stress. Consistent with this notion, the proteasome inhibitor bortezomib disrupts protein equilibrium in the ER by preventing misfolded proteins from being properly degraded. Given the clinically validated importance of targeting ER stress mediated pathways in the treatment of multiple myeloma (MM), we sought to identify natural products that modulate pathways known to be an effective therapeutic target for MM for potential use to inhibit progression of asymptomatic MM to symptomatic MM without the limiting side effects of current targeted therapies.

Methods:
Using decreased protein processing in the secretory pathway as a measurable hallmark of ER stress, our screen employed the naturally secreted Gaussia luciferase (Gluc) as a reporter that can be easily monitored through extracellular release of luciferase activity in real time. KMS11 and ARP-1 MM cells expressing Gluc were exposed to compounds in our natural products library in order to identify those which potentially induce ER stress as measured by inhibition of Gluc secretion. The growth inhibitory activity of theaflavin-3, 3'–digallate (TF3) was further characterized by MTS assay. Mechanistic studies of ER stress related pathways including the unfolded protein response (UPR) and apoptotic cascades were analyzed by standard Western blotting techniques.

Results:
Our screen identified the black tea polyphenol TF3 as a significant inhibitor of GLUC secretion in ARP-1 and KMS-11 cells. TF3 at 0. 5 μM inhibits GLUC secretion by 73 and 68% in ARP-1 and KMS-11 cells, respectively. This inhibition observed is on par with that observed for bortezomib and tunacamycin (a well known inducer of ER stress). TF-3 effectively inhibits cellular proliferation and induces apoptosis in a panel of MM cell lines at physiologically achievable concentrations. Apoptotic induction is at least partially mediated by ER stress mediated pathways as upregulation of the protein chaperone HSP90 and phosphorylation of eIF2-a, a key mediator of the UPR pathway, occurs prior to caspase and PARP cleavage.

Conclusions:
Our results suggest that TF-3 inhibits protein secretion and MM cell growth through promotion of ER stress generating mechanisms. Based upon these promising results, further mechanistic evaluation and characterization of this safe, natural product as a prophylactic agent in the treatment of asymptomatic conditions like monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) is warranted.

Disclosures:
No relevant conflicts of interest to declare.
Proteasome Inhibitors Do Not Inhibit the Serine Protease HtrA2/Omi
Authors: Senn Joseph , Csizmadia Vilmos , Hales Paul , Dick Larry , Kadambi Vivek J. 
Published: October 2019

Abstract
Abstract 5023
Based on unprecedented efficacy, the proteasome inhibitor (PI) bortezomib has become the cornerstone of multiple myeloma treatment. Nevertheless, in a subset of patients bortezomib causes painful peripheral neuropathy and this side effect can limit its potential benefit for those patients. Although the mechanism of bortezomib associated neuropathy is unknown, we have previously suggested that it is related to the mechanism of action (Csizmadia at. al. Vet Pathol 2010; 47:358–367.). Recently Arastu-Kapur et al. (Clin Cancer Res 2011;17:2734–2743.) have reported that the serine protease HtrA2/Omi was inhibited by bortezomib (a peptide boronate proteasome inhibitor) and not by carfilzomib (an epoxyketone proteasome inhibitor). Further, since HtrA2/Omi is involved in neuronal survival (Martins et al. Mol Cell Biol 2004; 24: 9848–9862.) they suggested that this off target inhibition by bortezomib could be the mechanism underlying bortezomib associated peripheral neuropathy. To confirm and extend these published results, we investigated the effects of these two PIs on HtrA2/Omi activity in recombinant enzyme assays, in SH-SY5Y neuroblastoma-, and wild type and HtrA2/Omi double negative mouse embryonic fibroblast cells (MEF). In contrast to the results of Arastu-Kapur et al., our results clearly demonstrated that neither bortezomib nor carfilzomib inhibits HtrA2/Omi in recombinant enzyme assays at concentrations up to 100μM. As a positive control we used Ucf-101 an HtrA2/Omi specific inhibitor (Cilenti et al. J Biol Chem 2003; 278:11489–11494.) which in our assay behaved in a manner consistent with the published literature. Similarly, in MEF cells, only Ucf-101 prevented the degradation of validated HtrA2/Omi substrates eIF4G1 and UCH-L1, while neither bortezomib nor carfilzomib prevented the degradation of these two substrates. In conclusion, we have assessed the protease activity of HtrA2/Omi both in vitro with purified enzyme and in cultured cells and we find that neither PI inhibits this protease. Therefore we think it is unlikely that PI associated peripheral neuropathy is caused by off target inhibition of HtrA2/Omi. Further research is needed to understand the side effects of PIs.

Disclosures:
Senn: Millennium Pharmaceuticals, Inc.: Employment. Csizmadia:Millennium Pharmaceuticals, Inc.: Employment. Hales:Millennium Pharmaceuticals, Inc.: Employment. Dick:Millennium Pharmaceuticals, Inc.: Employment. Kadambi:Millennium Pharmaceuticals, Inc.: Employment.
Clinical Profile of Multiple Myeloma in Asia - an Asian Myeloma Network (AMN) Study
Authors: Lee Jae Hoon , Kim Kihyun , Kim Jin Seok , Min Chang Ki , Yoon Sung-Soo , Shimizu Kazuyuki , Chou Takaaki , Kosugi Hiroshi , Suzuki Kenshi , Hata Hiroyuki , Chen Wenming , Hou Jian , Lu Jin , Huang Shang-Yi , Chng Wee-Joo , Tan Daryl , Teoh Gerrard , Chim James Chor Sang , Siritanaratkul Noppadol , Nawarawong Weerasak , Durie Brian GM 
Published: October 2019

Abstract
Abstract 5035

Background:
The incidence of multiple myeloma (MM) is known to be variable according to ethnicity. However the difference of clinical characteristics between ethnic groups is not well-defined. In Asian countries the incidence of MM has been lower compared with Western countries. However, there are growing evidences that MM is increasing very rapidly in this region. Until now, only few data of Asian patients has been reported. Asian myeloma network (AMN) decided to analyze the first multinational project to explore clinical characteristics of Asian MM patients and clinical practice performed in Asian countries.

Methods:
Data were collected from 22 centers from 7 countries and regions were collected retrospectively. Clinical characteristics of 3377 symptomatic MM patients at diagnosis were described. Overall survival (OS) and prognostic factors were analyzed for 3324 patients who have survival data.

Results:
Clinical and genetic characteristics were summarized at table 1. Median OS was 47 months (95% CI 48. 0–60. 0). Patients who were diagnosed before 2000 were shorter survival. Transplantation was performed to 607 patients with better survival (84 vs 40 months, p<0. 001). First line treatment of 2906 evaluable patients was analyzed. Overall response rate was 71% including VGPR or better 31%. Novel drug-based regimens including bortezomib, thalidomide or lenalidomide were used for 32. 5% of all 2906 patients without difference in response rate or OS.

Conclusions:
We successfully described clinical characteristics of Asian MM patients and this project will be the base for future studies or clinical trials for Asian MM patients. Updated analyses and comparison with Western data will be presented. AMN, supported by the International Myeloma Foundation (IMF) IMWG initiative.

Disclosures:
No relevant conflicts of interest to declare.
Multiple Myeloma Care in the Community Setting by Utilizing Progressive Clinical Care Pathways
Authors: Dhillon Vikram , Martincic Danko , Lee Christopher M , Njuguna Ndegwa , Schmick David , Nagle Annette K , Garman Rachel L , Kaya Hakan 
Published: October 2019

Abstract
Abstract 5053
Multiple myeloma (MM) is the second most common hematologic malignancy in the United States. There are over 20, 000 newly diagnosed MM cases every year and despite the recent advances in care of MM patients, more than 10, 000 MM patients still die from their disease. After experiencing only modest improvements in the care of patients for over 30 years, treatment of MM has dramatically improved over the last decade due to the advent of multiple novel agents. Significant variations in practice patterns among medical oncologists have been observed. In addition to problems with quality of care, health care costs for MM patients can vary dramatically because physicians often treat patients with the same condition differently in choice of drugs. Many times significant variety is present even among oncologists who work in the same practice. Cancer Care Northwest (CCNW) is the largest cancer group in the Inland Northwest with over 20 board certified cancer physicians. In addition to having a well-established myeloma program, CCNW also offers autologous stem cell transplantation to the patients in the community setting (approximately 25 transplants/year). CCNW implemented “Multiple Myeloma Clinical Care Pathways” in 2008. These pathways have been regularly updated according to the newest data presented at the national meetings, recent publications, and new FDA approvals. Over the last 4 years more than 100 unique MM patients have been treated on clinical care pathways. At the meeting, we will present our data including patient characteristics and outcomes. We will also show how our center's multiple myeloma clinical care pathways have evolved over the years. Treating multiple myeloma according to clinical care pathways standardizes treatment and decreases variation.

Disclosures:
Kaya: Millennium, Celgene, Onyx, Novartis: Honoraria, Speakers Bureau.
Mir-16 and 21 Deregulation Are Linked to Anti-Apoptotic Bcl-2 Gene Expression in Polycythemia Vera
Authors: de Souza Nunes Natalia , Tognon-Ribeiro Raquel , Moura Livia G , Kashima Simone , Covas Dimas T , Souto Elizabeth xisto , Zanichelli Maria Aparecida , Simoes Belinda Pinto , de Souza Ana Maria , de Castro Fabiola Attie 
Published: October 2019

Abstract
Abstract 5057

Background:
Polycythaemia vera (PV) is a clonal disorder characterized by an accumulation of normal red and white cells, platelets, and their progenitors in absence of a definable stimulus. Despite the advances in PV diagnosis its physiopathology remains not well elucidated. Apoptosis deregulation seems to contribute to disease establishment. MicroRNAs are small noncoding RNAs, post transcriptional repressors of genes by deadenilation, which play relevant roles in regulation of apoptotic machinery. Here we investigate the relation of apoptosis deregulation and apoptomirs expression in PV patients.

Aims:
To quantify the apoptomirs −16, −21, −15a, −26a, 130b, 29c and let-7d expression in peripheral leukocytes and CD34+ hematopoietic stem cells (HSC) in Polycythemia Vera (PV) patients and to correlate with targets genes expression data.

Subjects and Methods:
30 PV patients, 13 men and 17 women with a mean age (ma) of 64. 7y. 30 healthy subjects, 13 males and 17 females, ma=60. 2y and 23 bone marrow donors, 14 males and 9 females, ma=32. 6y. Peripheral leukocytes were obtained by Haes-Steril method and CD34+ hematopoietic stem cells were enriched by using the magnetically activated cell sorting, total RNA was extracted according to Trizol® method and High Capacity® Kit was used to synthesize cDNA. The microRNAs and apoptosis-related genes expression quantification was performed by real time PCR. Results were given as 2-ΔΔCt and statistical analyses were carried out by Mann-Whitney and Spearman tests. Western Blot was applied to detect Bcl-2 protein expression.

Results:
miR-16 and miR21 levels were increased in PV leukocytes (median= 3. 45 and 6. 97 respectively)) and CD34+ HSC (median= 3. 86 and 4. 07 respectively) in comparison to controls (control leukocytes median= 0. 483 and 0. 989 respectively) (CD34+ HSC median= 0. 978 and 1. 15, respectively) (p= 0. 0001 and 0. 0001 in leukocytes) and (p= 0. 0001 and 0. 0005 in CD34+ HSC, respectively). The Bcl-2 gene expression was decreased in PV leukocytes (m= 0.18) when compared to controls (m= 1. 03; p= 0. 019). In addition we found in CD34+ HSC negative correlation between miR-21 and anti-apoptotic gene Bcl-2 (r= −0. 417; p= 0. 033) and between miR16 and anti-apoptotic gene Bcl-2(r= −0. 384; p=0. 046). Bcl-2 western blot showed half expression in PV leukocytes when compared to controls.

Conclusions:
The results indicate the apoptomirs expression is linked to apoptosis deregulation in Polycythemia Vera physiopathology.

Disclosures:
No relevant conflicts of interest to declare.