bluuebunny/crossref_metadata_2025 huggingface dataset:
Low-Dose Uroacitides(CDA-II) Downregulates Telomerase Activity and Inhibits Proliferation in the Telomerase-Expressing Maliganant Cell Lines Through Inactivation of Akt Signaling Pathway
Authors: Huang Jian , Jin Jie
Published: October 2019

Abstract 4920

Uroacitides, another name CDA-II (cell differentiation agent II), is a mixture product and isolated from healthy human urine in China. It is characterized as a novel molecular targeting agent for cancer therapy. Multiple active components, such as peptides (MW 400–2800), organic acids, pigments and phenylacetylglutamine, with different mechanisms of action act concurrently to contribute to the anticancer effect of CDA-II. We have reported that high-dose CDA-II could induce acute myeloid leukemia (AML) cell apoptosis. Here, in the light of the important role of telomerase in malignant transformation. We evaluated the effect of low-dose CDA-II on telomerase activity (TA) and regulation in various malignant cell lines. CDA-II caused a dose-dependent inhibition of TA (up to 80% at a concentration of 0. 5 mg/mL) in Hela (cervix uteri cancer), MCF-7(breast cancer), SW480(colon cancer), RPMI8226(myeloma), HL-60 (AML), Raji (Burkitt lymphoma), L428 (Hodgkin's disease). Low-dose CDA-II did not affect the activity of other DNA polymerases. Inhibition of TA was associated with 60% inhibition of proliferation. The inhibition of proliferation was associated with a decrease in the S phase of the cell cycle and an accumulate of cells in G1 phase. No apoptosis was observed. Inhibition of TA was caused by dephosphorylation of Akt and by early downregulation of hTERT transcription. Other steps of telomerase regulation were not affected by low-dose CDA-II. This study demonstrated an additional cellular target of low-dose CDA-II that causes inhibition of TA and cell proliferation.

Disclosures:

No relevant conflicts of interest to declare.
Mechanism of Gambogic Acid-Induced Apoptosis in DB Cells
Authors: Xu Jingyan , Ouyang Jian , Zhou Min , Zhang QiGuo , Zhou Rong-Fu
Published: October 2019

Abstract 4923

This study was purposed to explore the apoptotic effect of gambogic acid on DB cells and its mechanism. The DB cells were treated with GA at different concentrations(2, 1, 0. 5, 0 ug/ml) in 24 hr. Annexin V-fluorescein-isothiocyanate/propidium iodide was used to detect apop tosis of DB cells. Western blot was used to determine the expressions of P53, Bcl-2 and Bax. The results showed that the Gambogic acid dose-dependently induced the apoptosis of DB cells in 24 hr, downregulated the expression of Bcl-2, upregulated the expressions of Bax, and the expression of P53 did not change. It is concluded that gambogic acid induces the apoptosis of DB cells through Bax upregulation, Bcl-2 downregulation without influence on P53.

Disclosures:

No relevant conflicts of interest to declare.
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 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 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 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 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 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 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 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 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 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.
Clinicopathologic Significance of CD11c, CD16 and FOXP3 Expression in Diffuse Large B-Cell Lymphoma (DLBCL) Patients Receiving Rituximab, Cyclophosphamide, Anthracycline, Vincristine, and Prednisolone (R-CHOP) Combination Chemotherapy
Authors: Oh Sung Yong , Jung Jin Sook , Kim Sung Hyun
Published: October 2019

Abstract 5112

Backgrounds:

The principal objective of this study was to determine whether the expressions of CD11c, CD16 and FOXP3 are predictive of clinical outcomes in diffuse large B-cell lymphoma (DLBCL) patients receiving treatment with rituximab, cyclophosphamide, anthracycline, vincristine, and prednisolone (R-CHOP) combination chemotherapy.

Method:

The study population consisted of 25 patients with DLBCL between January 2006 and December 2007. All the patients had been treated with R-CHOP combination chemotherapy. The R-CHOP regimen was as follows: 375 mg/m2 rituximab, 750 mg/m2 cyclophosphamide, 50 mg/m2 anthracycline, 1. 4 mg/m2vincristine were intravenously administered on day 1, and prednisolone 100 mg was medicated on days 1 to 5. This regimen was repeated every three weeks. The expressions of CD11c, CD16 and FOXP3 of primary tumors were immunohistochemically evaluated.

Result:

The mean age of the patients was 58 years (range, 27–77 years), and the pilot study population included 11 (44%) females and 14 (56%) males. The stages of the patients were I, II, III, and IV in 4 (16%), 5 (20%), 7 (28%), and 9 (36%), respectively. 60% of the patients were categorized as the low (44%) or low-intermediate risk group (16%) according to the International Prognostic Index (IPI). After a median follow-up of 57months, 8 patients (32%) developed disease recurrence or death. The estimated 5-year Progression free survival (PFS) and Overall survival (OS) were 65% (Figure 1) and 74% (Figure 2), respectively. 8 (32%), 18 (72%), and 17 (68%) were positive for CD11c, CD16 and FOXP3 expression, respectively. Univariate analysis of CD11c, CD16, FOXP3 for PFS and OS are shown in Figures 3–5. We noted no significant correlation between immunostaining of CD11c, CD16, FOXP3, and PFS. Patients with a CD11c -expressing DLBCL had a significantly higher 5-year OS rate than those without (p=0. 033, Figure 3). We classified patients according to the risk group. Each one counted for 1 point; In CD11c and FOXP3 stain, positive was counted as 0, negative was 1. In CD16, positive was counted as 1, negative was 0- and they were summed. The points were separated into low risk (0–1) and high risk (2–3) groups (Figure 6). We could found significant differences of PFS (p=0. 015) and OS (p=0. 046) between two groups.

Conclusion:

Even though this result was obtained from small number and retrospective data set, we can get a possibility of clinical significance of CD11c, CD16 and FOXP3 in DLBCL patients receiving R-CHOP therapy. Prospective large scale study is needed for confirming these promising our pilot study results.

Disclosures:

No relevant conflicts of interest to declare.
NPM-RAR Enhances NFκB Transcriptional Activity
Authors: Redner Robert L. , Abecassis Irina , Rush Elizabeth A. , Chattopadhyay Anuja
Published: October 2019

Abstract 5117

Acute Promyelocytic Leukemia (APL) is characterized by recurrent translocations of 17q21, which result in expression of fusion proteins of the retinoic acid receptor alpha (RARα). We have been studying the t(5;17)(q35;q21) variant of APL, which fuses the N-terminal 117 amino acids of nucleophosmin (NPM) to the C-terminus of RARα. NPM-RAR is capable of binding to retinoic acid response elements (RAREs), and we have previously shown that NPM-RAR can recruit both corepressor and coactivator complexes to retinoic acid target promoters, to modulate transcription in a ligand-dependent fashion, though with a different sensitivity to ligand than RARα. The N-terminal NPM-sequence of NPM-RAR contains a dimerization domain capable of mediating not only homodimerization of the fusion protein, but also heterodimerization with wild-type NPM, to potentially modulate NPM functionality. NPM serves as a molecular chaperone, and binds to a diverse set of proteins. Amongst these are the p50 and p65 subunits of NFκB. NPM has been shown to serve as a coactivator for the NFκB transcriptional complex. We hypothesized that NPM-RAR might interact with NPM to modulate NFκB dependent transcription. We focused on NFκB regulation of the Superoxide Dismutase 2 (MnSOD, SOD2) promoter, since NPM regulation of NFκB mediated transcription has been most extensively studied in this model system. We found that NPM-RAR enhances both basal and TNFα-induced transcription of an NFκB-reporter construct (containing a minimal MnSOD promoter/enhancer) in HepG2 and Hela cells. This effect was not dependent upon translocation of NFκB to the nucleus, indicating that the positive effect on NFκB was mediated downstream of the canonical activation pathway of NFκB. The MnSOD promoter does not contain a consensus RARE, and this effect was not seen in cells transfected with RARα, suggesting that this effect is not mediated through the DNA binding motifs of the NPM-RAR C-terminal RARα sequences. NPM-RAR does not directly interact with NFκB; preliminary experiments using siRNA to down-regulate NPM indicate that NPM-RAR enhancement of MnSOD transcription is dependent upon the presence of wild-type NPM. This leads us to propose a model whereby NPM-RAR binds to the NPM/NFκB transcriptional complex through interaction with NPM, to enhance NFκB mediated transcription.

Disclosures:

No relevant conflicts of interest to declare.
Myeloid Lineage-Associated Mutations Are Detected in Blastic Plasmacytoid Dendritic Cell Neoplasm
Authors: Wang Yongbao , Windham Justin , Billouin-Frazier Shere , Jones Dan
Published: October 2019

Abstract 5123

Background/Purpose:

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare but highly aggressive hematologic malignancy that frequently presents with skin, lymph node, and leukemic disease. Since BPDCN can evolve into acute myelomonocytic leukemia (AMML) with other cases associated with monocytosis, a close relationship of BPDCN to the myelomonocytic lineage is suspected. Here we investigate the frequency of myeloid-associated DNA mutations in BPDCN using a targeted exon sequencing panel.

Methods:

Nine cases of BPDCN (5 skin, 1 lymph node, 3 bone marrow clot sections) were studied. All showed blastic cytomorphology; characteristic expression of CD56 and CD123; CD4 and/or TCL1 expression; and absence of definitive lymphoid, myeloid, and monocytic marker expression. AMML recurrence at 18 months, seen in 1 patient, was also assessed. Genomic DNA was extracted from formalin-fixed paraffin-embedded sections and subjected to a 161-amplicon PCR-based DNA sequencing assay that includes the commonly mutated areas of KRAS, NRAS, IDH1, IDH2, TET2, EZH2, JAK2, and ASXL1. Limited multiplex PCR and product harvesting was performed on an Access Array chip (Fluidigm). Ion adaptors were added and DNA sequencing was performed on a PGM sequencer using the 316 chip with 200 base-pair sequencing chemistry (Life Technologies). Sensitivity of mutation detection varied between 5–10%. Sequencing data were analyzed by SeqNext software (JSI MedSystems). Detected mutations were confirmed using conventional Sanger sequencing, with an approximate sensitivity of 15%, or pyrosequencing (5%).

Results:

Mutations in one or more of the tested genes were found in 6 of 9 patients, with unconfirmed mutations in 2 additional cases due to low-level mutations in samples with poor quality DNA. One patient with a typical BPDCN showed an IDH2 mutation (R140Q) with recurrence as AMML showing this mutation as well as a double mutation in KRAS (G12R, Q61R). Other mutations identified included 3 in ASXL1 including W608C, NRAS G12D, EZH2 C-terminal truncation, and 2 TET2indels.

Conclusions:

The presence of myeloid-associated mutations particularly in IDH2, TET2 and ASXL1 in cases of BPDCN is support for a relationship to the myelomonocytic lineage. The presence of shared and new mutations in the AMML recurrence in 1 patient with BPDCN further indicates a pathogenetic relationship between the two entities.

Disclosures:

Wang: Quest Diagnostics: Employment. Windham:Quest Diagnostics: Employment. Billouin-Frazier:Quest Diagnostics: Employment. Jones:Quest Diagnostics: Employment.
Frequency and Distribution of Asymptomatic Brain Lesions in Patients with β-Thalassemia Intermedia
Authors: Karimi Mehran , Haghpanah Sezaneh , Bagheri Mohammad Hadi , Bordbar Mohammad Reza , Pishdad Parisa , Rachmilewitz Eliezer A.
Published: October 2019

Abstract 5141

We aimed to determine the frequency of asymptomatic brain lesions in a group of patients with β-thalassemia intermedia (β-TI) and to evaluate correlation of asymptomatic brain lesions with splenectomy, thrombocytosis, blood transfusions and clinical parameters. Ninety five neurologically intact patients with β-TI were randomly enrolled in this cross-sectional study. Diffusion Weighted Imaging (DWI) brain MRI was performed in every patient to detect cerebral white matter lesions (WML). We found an overall frequency of 15 (15. 8%) for WMLs, 14 (23. 7%) in splenectomized and 1 (2. 8%) in nonsplenectomized patients. The presence of WML was significantly associated with splenectomy (P= 0. 008) and thrombocytosis (P=0. 009). However, after adjustment for splenectomy, thrombocytosis was not significantly associated with the presence of WML (P>0. 05). The number of patients with regular blood transfusions and normal MRI was not significantly higher compared to those with abnormal findings (52. 5% vs 26. 7%; P=0. 092). In untransfused patients, hydroxyurea (HU) administration was associated with a lower incidence of WML (P<0. 001). Although in univariate analysis either splenectomy or thrombocytosis showed significant correlation with the presence of single or multiple WMLs, thrombocytosis by itself did not significantly contribute in developing asymptomatic brain lesions. The lack of significant correlation between lesions and regular blood transfusions could be related to the treatment with HU in untransfused patients, which increased fetal hemoglobin levels and improved the morphology and the pathological indices of the red blood cells. Larger prospective studies are suggested for evaluation of the correlation of these factors with developing asymptomatic brain lesions accurately.

Disclosures:

No relevant conflicts of interest to declare.
Aspirin Induces Platelet Apoptosis
Authors: Zhao Lili , Zhang Weilin , Chen Mengxing , Zhang Jiansheng , Zhang Mingyi , Ruan Changgeng , Dai Kesheng
Published: October 2019

Abstract 5144

Aspirin is widely used in the treatment of a number of clinical conditions, such as fever, pain, strokes, and heart attacks. Although aspirin is being thought of a relatively “safety” medicine, it also has some side effects, particularly the risk of bleeding which may be severe and lead to death. The mechanisms, however, are not totally understood. The effectiveness of aspirin has been attributed to its ability to inhibit prostaglandin production by inhibiting the cyclooxygenase (COX) enzyme. Interestingly, it has been reported recently that aspirin induces apoptosis in many cell types. Platelet apoptosis induced by either physical or chemical compounds or platelet storage occurs widely. We have reported that the glycoprotein Ibα-von Willebrand factor interaction, hyperthermia, and calmodulin antagonists induce platelet apoptosis, suggesting that platelet apoptosis might play important roles in controlling the number and function of circulated platelet under physiological conditions, or in the development of platelet-related hemorrhagic diseases. Thus, the aim of the current study is to explore whether aspirin induces platelet apoptosis. Washed platelets (3×108/ml) were incubated with various concentrations of aspirin (2. 5 mM, 5 mM, 10 mM, 20 mM) at 37 °C for 2 hours. The effect of aspirin on platelet mitochondrial membrane potential (ΔΨm) depolarization and phosphatidylserine (PS) exposure were analyzed by cell-permeable lipophilic cationic dye tetramethylrhodamine ethyl ester (TMRE) and annexin V binding, respectively. The data show that ΔΨms depolarization and PS exposures were dose-dependently induced by aspirin in platelets. In order to further confirm that aspirin incurs platelet apoptosis, caspase-3 activation was measured in platelets incubated with aspirin by detecting 32 kD pro-caspase-3 and 17 kD cleaved caspase-3 fragment, and the result shows that aspirin induced caspase-3 activation. Furthermore, platelet shrinkage appeared in platelets incubated with aspirin (20 mM). Caspase inhibitor z-VAD-fmk inhibited apoptotic platelet shrinkage, and blocked ΔΨm depolarization, but had no effect on PS exposure, suggesting that caspase-3 acts upstream of ΔΨm depolarization, and plays a key role in aspirin-induced platelet apoptosis. Whereas, protein kinase C (PKC), protein kinase B (PKB), p38 mitogen-activated protein kinases (MAPK), protein 53 (p53), reactive oxygen species (ROS), and mitogen-activated protein kinase kinase (MAPKK) were not involved in aspirin-induced platelet apoptosis. In addition, platelets incubated with another COX inhibitor indomethacin did not incur ΔΨm depolarazation and PS exposure in platelets. Taken together, the data indicate that aspirin induces platelet apoptosis via caspase-3 activation, and independent of the COX signaling.

Disclosures:

No relevant conflicts of interest to declare.
Erythropoietin Induced Erythroid Precursor Pool Depletion Causes Erythropoietin Hyporesponsiveness
Authors: Yan Xiaoyu , Ait-Oudhia Sihem , Krzyzanski Wojciech
Published: October 2019

Abstract 5156

Background:

Erythropoietin (EPO) hyporesponsiveness is demonstrated by a persistence of anemia despite high dose of recombinant human erythropoietin (rHuEPO) or requirement of large doses to maintain the target hemoglobin concentration. The purpose of this study is to demonstrate that the bone marrow depletion induced by rHuEPO treatment may another contributing factor to hyporesponsiveness.

Methods:

Healthy Wistar rats were given single dose (SD) or multiple doses (MD) of rHuEPO. In MD study, animals were challenged with thrice-weekly (100 IU/kg) over two weeks and the pharamcodynamic biomarkers included reticulocyte (RET) and red blood cells (RBC) counts, and hemoglobin (HGB) concentrations. In SD study, in addition to the biomarkers in the peripheral blood, the erythropoietic responses in bone marrow and spleen were also quantified using a flow cytometric immunophenotyping technique. The total marrow and spleen cellularity was quantitatively assessed using flow cytometry. A mathematical approach involving measuring RET age distribution was developed to evaluate the RET loss due to neocytolysis. The pharmacokinetics of rHuEPO was assessed by measuring serum concentrations over time using ELISA.

Result:

In MD study, a slow and oscillatory decline of RET response was observed before the administration of the last dose on day 11. The RET kept decreasing below the baseline and reached a nadir on day 22, followed by a slow return to the baseline on day 28. These observations demonstrated the tolerance and rebound phenomena. In SD study, the RET decreased below the baseline after day 6. A depletion of the bone marrow erythroid precursor cells was also observed. Meanwhile, neocytolysis of RET was only observed from day 3–5 after rHuEPO injection. These results suggested that the reduced levels of RET after day 6 resulted from the depletion in the erythroid precursor cells in bone marrow induced by rHuEPO treatment.

Conclusion:

The EPO hyporesponsiveness is of clinical importance because it is associated with an increased risk of death or cardiovascular events. The findings in this study demonstrate that EPO-induced erythroid precursor depletion may contribute to the rHuEPO hyporesponsiveness. When the erythroid precursor cells are depleted, an increase of rHuEPO dose would not show much benefit, due to the smaller number of cells present to initiate the erythropoiesis. These findings provide additional justification for reducing the doses of erythropoietin stimulating agents in anemic patients demonstrating hyporesponsiveness.

Disclosures:

No relevant conflicts of interest to declare.
Favorable and Sustained Response of Primary Autoimmune Hemolytic Anemia to Rituximab
Authors: Al-Momen Abdul Kareem M , Aleem Aamer , Gahtani Farjah Al , Hasanato Rana , Anjum Farhan Ali , Blecana Sri-Halima , AlSaleh Khalid
Published: October 2019

Abstract 5160

Background:

Primary autoimmune hemolytic anemia (PAIHA) is a rare autoimmune disorder that males and females of all age groups, but more frequent in young adults. Most patients respond initially to steroids and intravenous immunoglobulin's followed by Splenectomy for non-responders. Around one third of the patients do not respond to any standard treatment and another one third show only transient response and therefore labeled as refractory PAIHA. In this refractory group, various immune modulators and suppressants are used with variable response. These include azathioprine, vincristine, cyclophosphamide and mycophenolate.

The Aim:

The aim of our study is to explore the safety and efficacy of Rituximab in the treatment of refractory, PAIHA.

Patients and methods:

Thirteen patients (seven males and six females, aged 33. 7 +/− 15 years) with refractory PAIHA (Hb 73. 1 +/− 13. 9 g/l) were given Rituximab 375 mg/m2 intravenously weekly for four weeks, then monthly for four months.

Results:

All patients showed a rapid and favorable response with significant rise in Hb level to 128 +/− 10. 8 g/l within four months. Rituximab was tolerated very well and the response was sustained in all patients during the follow up to 25. 6 +/− 21. 8 months

Conclusion:

Rituximab is probably highly effective in the treatment of refractory PAIHA with sustainable response. This has been shown in refractory autoimmune hemolytic anemia in adults and children as well as in chronic lymphocytic leukemia associated autoimmune hemolytic anemia (1–3)

Disclosures:

Off Label Use: Rituximab was used in Primary Autoimmune Hemolytic Anemia compassionately.
Hereditary Spherocytosis: How the New Information Provided by the Routine Hematology Analysers May Help in Its Differential Diagnosis or Flagging
Authors: Barella Susanna , Simon-Lopez Ramon , Gaetano Nicola Di , Galanello Renzo
Published: October 2019

Abstract 5163

Introduction:

Hereditary Spherocytosis (HS) is one of the most common inherited hemolytic anemias. Many of them are autosomal dominant, being about 25% of the cases transmitted recessively.

Diagnostic of HS:

Classic testing for HS includes: Hematologic testing of red blood cell indices (RDW, MCHC, Reticulocyte count), peripheral blood smear (presence of spherocytes), osmotic fragility and Eosin-5-maleimide binding to band 3 and Rh-related proteins forms that may be used as screening tests for hereditary spherocytosis.

Objective:

Recently have been developed new parameters and information in the new automated hematology analyzer called DxH8008™ from Beckman Coulter as @MSCV (@Mean Sphered Cell Volume), @RSF, @MAF, @ LHD%. All this parameters may be used to create flagging for laboratory use only (LUO) or Research use only (RUO). The purpose of this study is to investigate the possible use or utility of this new information for the screening/flagging of Hereditary Spherocytosis. There are previous studies showing the possible benefit of using MCV minus @MSCV for the detection/flagging of cases with spherocytes.

Patient and Methods:

We have collected 28 patients with Hereditary Spherocytosis. All of them were confirmed by red cell morphology, osmotic fragility and Eosin-5-maleimide binding to band 3 and Rh-related proteins forms.

Results:

Using ROC analysis, the best parameters differentiating the Hereditary Spherocytosis from the normals were: RET% (AUC 0. 996), MCV - @MSCV (AUC 0. 996), @MSCV (AUC 0. 969), RDW(AUC 0. 892), MCHC (AUC 0. 860), HGB (AUC 0. 787).

Using ROC analysis, the best parameters differentiating the Hereditary Spherocytosis from other anemias (excluding normals)were: MCV - @MSCV (AUC 0. 991), MCHC (AUC 0. 987), RET% (AUC 0. 857).

Disclosures:

Simon-Lopez: Beckman Coulter: @LHD, @MAF, @RSF, @LHD, @MAF, @RSF Patents & Royalties, Employment. Di Gaetano:Instrumentation Laboratory spa: Work for a distributor of Beckman Coulter Instruments in Italy Other. Galanello:Novartis: Research Funding, Speakers Bureau; Apopharma: Research Funding, Speakers Bureau; Ferrokin: Research Funding.
Iron Chelation Therapy and Mobilization of Hematopoietic Peripheral Progenitors in Patients with β-Thalassemia Major
Authors: Forni Gian Luca , Podestà Marina , Musso Marco , Piaggio Giovanna , Musallam Khaled M , Balocco Manuela , Pozzi Sarah , Frassoni Francesco
Published: October 2019

Abstract 5178

Several reports established an association between iron chelation therapy with deferasirox and hematopoietic improvement in patients with myelodysplastic syndromes. Data in β-thalassemia major (TM) patients is absent. We evaluated levels (frequency and aboslute number) of several hematopoietic peripheral progenitors (HPP: Colony Forming Unit-Granulocyte/Macrophage [CFU-GM], Erythroid Burst-Forming Unit [BFU-E], Colony Forming Unit-Granulocyte, Erythrocyte, Macrophage, Megakaryocyte [CFU-GEMM], and Long Term Culture-Initiating Cells [LTC-IC]) in 26 TM patients (median age 28. 5 years, 42. 3% males) and 12 age-matched controls. All TM patients had to be using the same iron chelator for at least 6 months with >80% compliance. The levels of all HPP were significantly higher in TM patients than controls, and varied between splenectomized and nonsplenectomized patients (lower CFU-GM and BFU-E, and higher LTC-IC in splenectomized patients). Patients using deferasirox (n=9) showed significantly higher levels of BFU-E compared with both deferoxamine (n=10) and deferiprone (n=7) treated patients (p<0. 01). After adjusting for age, sex, splenectomy status, serum ferritin changes, the association between higher BFU-E levels and deferasirox compared with deferoxamine or deferiprone therapy remained statistically significant (R2=0. 698, p=0. 006). Mobilization of BFU-E in TM patients receiving regular iron chelation therapy depends on the type of chelator used, independent of iron status.

Disclosures:

Forni: Novartis Pharmaceuticals: Research Funding; Ferrokin: Research Funding. Musallam:Novartis Pharmaceuticals: Honoraria.