bluuebunny/crossref_metadata_2025 huggingface dataset:
The Tak-1 Inhibitor AZ-Tak1 Inhibits XIAP, Activates Caspase-9, and Induces Apoptosis in Mantle Cell Lymphoma.
Authors: Buglio Daniela , Palakurthi Sangeetha , Byth Katharine F. , Younes Anas
Published: October 2019

Abstract 1692

Poster Board I-718

Transforming growth factor-b-activated kinase 1 (TAK1) is a key regulator of NF-kB activation. TAK1 can be activated by a variety of pro-inflammatory cytokines and T and B cell receptors. Recent experiments demonstrated that deletion of TAK1 results in inactivation of both JNK and NF-kB signaling resulting in massive apoptotic death of hematopoietic cells in mice. In this study, we examined the expression pattern of TAK1 and its role as a potential therapeutic target for lymphoma. First, we examined TAK1 expression in a panel of lymphoid cell lines by western blot, and found it to be highly expressed in mantle cell lymphoma cell lines (Mino, SP53, and Jeko-1). These lines expressed relatively low levels of the tumor suppressor protein A20. Mino and SP53 expressed high level of p-p38. Subsequently, we investigated the in vitro activity of the novel TAK1 small molecule inhibitor AZ-Tak1 in these cell lines. AZ-Tak1 is a potent and a relatively selective inhibitor of TAK1 kinase activity, with an IC50 of 0.009 mM. It also inhibits Jak2 but at a much higher concentration (IC50=0.18 mM). AZ-Tak1 treatment decreased the level of p38 and ERK in mantle cell lymphoma cells, and induced apoptosis in a dose and time dependent manner, with an IC50 of 0.1-0.5 mM. Using the annexin-V and PI staining and FACS analysis, After 48 hours of incubation, AZ-Tak1 (0.1 mM) induced apoptosis in 28%, 34% and 86% of Mino, SP53, and Jeko cells, respectively, which was increased to 32%, 42%, and 86% when 0.5 mM concentration was used. Similar activity was also observed when primary mantle cell lymphoma cells were examined. Using pathway-specific protein arrays focusing on apoptosis, kinases, and transcription factors, AZ-Tak1 (0.5 mM) altered the level of several proteins that regulate cell growth and survival, especially members of the inhibitors of apoptosis (IAP) family. Specifically, AZ-Tak1 decreased the level of SMAC/DIABOLO and cytochrome –C in the mitochondria, which was associated with a decrease in the level of the anti-apoptotic protein X-linked IAP (XIAP) and activation of the intrinsic apoptotic pathway as evident by activation of caspase 9, cleavage of caspase 3, and induction of apoptosis. Furthermore, and consistant with its ability to inhibit Jak2 activity, AZ-Tak1 reduced STAT2 and STAT6 levels. AZ-Tak1 demonstrated no significant effect on bcl-2 family members. Finally, co-treatment with HDAC inhibitors demonstrated synergistic effect with low concentrations of AZ-Tak1. Collectively, our data demonstrate that targeting TAK1 by the small molecule inhibitor AZ-Tak1 induces cell death in mantle cell lymphoma by activating the intrinsic apoptosis pathway, suggesting that targeting TAK1 may have a therapeutic value for the treatment of mantle cell lymphoma.

Disclosures

Palakurthi: Astra Zeneca: Employment. Byth:Astra Zeneca : Employment.
Overall Survival (OS) Benefit of Rituximab Based Immunochemotherapy Followed by Post-Induction Treatment in Mantle Cell Lymphoma (MCL): a Retrospective Analysis of 279 Patients Treated by Polish Lymphoma Research Group (PLRG) Centers.
Authors: Jurczak Wojciech , Szymczyk Michal , Giza Agnieszka , Joks Monika , Kisiel Elzbieta , Stella-Holowiecka Beata , Boguradzki Piotr , Wrobel Tomasz , Sikorska Magdalena , Knopinska-Posluszny Wanda , Kalinka-Warzocha Ewa , Walewski Jan , Skotnicki Aleksander B.
Published: October 2019

Abstract 1702

Poster Board I-728

Background

ASCT consolidation in the first remission is the treatment of choice in MCL patients. However lack of adequate response to the first line therapy, elderly age and co-existing co-morbidities makes it feasible for less than a third of patients.

Methods

Retrospective analysis of 279 MCL cases treated at 10 PLRG centers was performed: 52% of them (144 pts) received Rituximab in induction therapy; 35% (97 pts) were subjected to post-induction therapy (ASTC – 16%, radioimmunotherapy consolidation – 13% and Rituximab maintenance – 6%). There were no significant differences in risk factor distribution among analyzed subgroups.

Results

1) At 5 years OS was 40% in the whole group: 77% for those subjected to post-induction therapy vs 25% for those who were not; 5 year PFS is 20%, 48% and 5% respectively (p<0,0001, in both comparisons).

2) Pts with initial good response, whose treatment was NOT continued after induction, had inferior results (5 year OS and PFS – 30 and 10 % respectively) to those subjected to post-induction therapy (p<0,0001).

3) Rituximab included in the first line regimens increased RR, increasing % of pts that could be subjected to post-induction therapy: progression/ death during the first line therapy were 5,5% (8/144 Rituximab treated pts) as compared to 15,5% (21/135 treated without Rituximab).

Conclusions

With all limitations of retrospective analysis, it strongly supports the necessity of Rituximab immunochemotherapy followed by post induction treatment in MCL pts. The role of ASCT is established in younger patients, radioimmunotherapy consolidation or Rituximab maintenance should be considered in elderly and unfit ones.

Disclosures

No relevant conflicts of interest to declare.
The Flt3 ITD Accelerates An Already Established Myeloid Leukemia and Alters Chemotherapy Response In Vitro and In Vivo in a p53 Dependent Manner.
Authors: Pardee Timothy , Zuber Johannes , Lowe Scott
Published: October 2019

Abstract 1719

Poster Board I-745

Acute myeloid leukemia (AML) is an aggressive disease with heterogeneous genetics and variable prognosis. The presence of an internal tandem duplication within the FLT3 gene (Flt3 ITD) is a marker for poor prognosis and has been linked to anthracycline resistance in cell lines and primary patient samples in vitro. The effect of this mutation on response to chemotherapy in vivo has not been examined and its effect on response to cytarabine is not known. In this study we use a genetically defined mouse model of AML to examine the effects of the Flt3 ITD on response to cytarabine and the anthracycline doxorubicin in vitro and in vivo. In vitro the Flt3 ITD conferred resistance to doxorubicin and the combination of doxorubicin and cytarabine but sensitivity to cytarabine alone in comparison to the identical leukemia without the Flt3 ITD. In vivo the presence of the Flt3 ITD provided an advantage in leukemic engraftment and accelerated disease onset. This advantage could be partially reversed by treatment of the animals with cytarabine but not by treatment with doxorubicin. Surprisingly, in vivo the Flt3 ITD conferred a marked increase in sensitivity to cytarabine when compared to the parental leukemia without this mutation. In contrast to the parental leukemia, the addition of doxorubicin to cytarabine provided no advantage over cytarabine alone. When the DNA damage response was assessed the presence of the Flt3 ITD resulted in an increase in the levels of p53 following treatment with either doxorubicin or cytarabine. Induction of the p53 target genes p21 and MDM2 was also increased. Surprisingly, the Flt3 ITD had no effect on disease onset or chemotherapy response in vitro or in vivo in the setting of p53 null AML. These data when taken together demonstrate that the Flt3 ITD confers a mixed sensitivity and resistance to standard chemotherapy and provides an engraftment advantage in a manner that depends on an intact p53 allele. This may at least in part explain the rarity of dual p53 null and Flt3 ITD positive AML. Furthermore, these data suggest that patients with Fl3 ITD positive AML may benefit more from treatment with high dose Ara-C then with combinations containing an anthracycline.

Disclosures

No relevant conflicts of interest to declare.
Valproic Acid Selectively Inhibits STAT3 Phosphorylation.
Authors: Zhu Shiguo , Denman Cecele J , Lee Dean A.
Published: October 2019

Abstract 1720

Poster Board I-746

Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor (1), was found to enhance NK cell mediated antitumor activity by upregulating the surface expression of NKG2D ligands MICA and MICB on hepatoma cells (2). In contrast, VPA was found to impair NK cell antitumor activity by downregulating the surface expression of NKp30 and NKp46 on NK cells by blocking NFkB activation (3). Recently, we also found that VPA efficiently upregulates the surface expression of MICA, MICB, and ULBP family ligands on human osteosarcoma and neuroblastoma cells, but downregulates NKG2D surface expression on human NK cells. Similarly, the HDAC inhibitor MS275 efficiently upregulates expression of MICA, MICB, and ULBP family ligands on human osteosarcoma and neuroblastoma cells, but surprisingly, MS275 significantly upregulates NKG2D expression on NK cells. In addition, we found that VPA inhibited the cell growth of osteosarcoma cell lines more readily than MS275. To investigate the mechanism of these differing effects of VPA and MS275, we compared the effect of these HDAC inhibitors on Signal Transducer and Activator of Transcription (STAT) signaling, which are important signaling pathways in NK cells and many cancers. Here we show that VPA is a potent inhibitor of STAT3 phosphorylation at the tyrosine 705 residue in NK cells, whereas MS275 has no effect on STAT3 phosphorylation (Figure A). Moreover, this inhibition is highly specific, as VPA has no effect on STAT1 and STAT5 phosphorylation (Figure B). To further confirm VPA-mediated STAT3 inhibition, we detected STAT3 expression and the degree of tyrosine phosphorylation affected by VPA in a panel human osteosarcoma cells. We found that constitutive STAT3 phosphorylation is present in osteosarcoma SaOS2 and LM7 cells, but not in OS187 cells, which corresponded to the stronger growth inhibition caused by VPA in SaOS2 and LM7 cells compared to OS187 cells. These results are for the first to show that VPA, a HDAC inhibitor, is also a selective STAT3 inhibitor. VPA can upregulate NKG2D ligand expression on the tumor cell surface by HDAC inhibition, but may downregulate NKG2D expression on the NK cell surface by STAT3 inactivation. The antitumor activity of VPA may be via a mechanism of both HDAC inhibition and STAT3 inactivation. The knowledge of potent STAT3 inhibition by VPA is important for identifying potential new clinical applications for this drug.

Disclosures

No relevant conflicts of interest to declare.
Akt Inhibitors Induce Apoptosis in Chronic Lymphocytic Leukemia Cells.
Authors: de Frias Mercè , Iglesias-Serret Daniel , Cosialls Ana M , Coll-Mulet Llorenç , Santidrián Antonio F , González-Gironès Diana M , de la Banda Esmeralda , de Sevilla Alberto Fernandez , Pons Gabriel , Gil Joan
Published: October 2019

Abstract 1731

Poster Board I-757

Phosphatidylinositol-3-kinase (PI3K)/Akt pathway has been described to be critical in the survival of chronic lymphocytic leukemia (CLL) cells. Here, we have analyzed the effect of two selective chemical inhibitors of Akt (Akti-1/2 and A-443654) in the survival of CLL cells. We studied by cytometric analysis the cytotoxic effects of Akt inhibitors on peripheral B and T lymphocytes from patients with CLL and from healthy donors. Both inhibitors induced apoptosis in CLL cells in a dose-dependent manner. Moreover, B cells from CLL samples were more sensitive to Akt inhibitors than T cells from CLL samples, and B or T cells from healthy donors. Survival factors for CLL cells, such as IL-4 and SDF-1a, were not able to block the apoptosis induced by both Akt inhibitors. We studied the changes induced by Akti-1/2 and A-443654 at mRNA level by performing reverse transcriptase multiplex ligation–dependent probe amplification (RT-MLPA). Akti-1/2 did not induce any change in the mRNA expression profile of genes involved in apoptosis, while A-443654 induced some changes, including an increase in NOXA and PUMA mRNA levels, suggesting the existence of additional targets for A-443654. We also studied the changes induced by both Akt inhibitors in some BCL-2 protein family members on CLL cells by Western blot. Both inhibitors induced an increase in PUMA and NOXA protein levels, and a decrease in MCL-1 protein level. Moreover, Akti-1/2 and A-443654 induced apoptosis irrespective of TP53 status. These results demonstrate that Akt inhibitors induce apoptosis of CLL cells and might be a new therapeutic option for the treatment of CLL.

Disclosures

No relevant conflicts of interest to declare.
Molecular and Biological Characteristics of Acquired Vorinostat-Resistant Cutaneous T-Cell Lymphoma Cells.
Authors: Zhang Chunlei , Zhang Xiang , Li Baoqiang , Duvic Madeleine
Published: October 2019

Abstract 1732

Poster Board I-758

Vorinostat (suberoylanilide hydroxamic acid, SAHA), a pan-histone deacetylase (HDAC) inhibitor, has an overall response rate of 24-30% in two phase II studies of patients with cutaneous T cell lymphoma (CTCL). Since a considerable proportion of CTCL patients did not reach a partial response and loss of response could occur after only a few months, development of resistance became an important clinical problem. Although we have shown that constitutive activation of STAT signaling may be involved in resistance to vorinostat, the other mechanisms of resistance to vorinostat in CTCL are largely unknown. To further investigate mechanisms of vorinostat resistance, we have generated a vorinostat-resistant CTCL cell line (HH/VOR) from a parent vorinostat-sensitive HH CTCL cell line by long term exposure to stepwise increasing concentrations of vorinostat. The HH parental cells were cultured in increasing concentrations of vorinostat starting at 10 nM. Viable cells were then passaged into a higher concentration of vorinostat in 10 nM increments until a concentration of 1 μM vorinostat was reached. The HH/VOR cells were then maintained in complete RPMI1640 medium containing 1 μM vorinostat. We studied the molecular and biological characteristics of the vorinostat-resistant CTCL cells. Compared with the parental vorinostat-sensitive HH cells, the vorinostat-resistant HH/VOR cells were highly resistant to vorinostat-mediated growth inhibition and apoptosis. Of interest, the HH/VOR also exhibited cross resistance to another pan-HDAC inhibitor panobinostat and a class I HDAC inhibitor SNDX-275. The vorinostat-resistant HH/VOR cells had reduced accumulation of acetylated histones (H3 & H4) and decreased expression of class I and class II HDACs (1-11), and no expression of multi-drug resistant (MDR) efflux transporters. Moreover, the vorinostat-resistant HH/VOR cells had elevated DNA binding of NF-κB and increased expression of phospho-NF-κB p65 and phospho-STAT-1 compared with the parent sensitive HH cells. Co-treatment with the RXR-selective retinoid bexarotene selectively enhanced vorinostat-induced apoptosis in the vorinostat-sensitive HH and -resistant HH/VOR CTCL cell lines as well as in patients' Sézary cells compared to normal CD4+ T-cells. Taken together, our findings provide further evidence for the potential of vorinostat to cause acquisition of HDAC inhibitor resistance in CTCL. This acquired HDAC inhibitor resistance neither correlates with over-expression of HDACs nor with expression of MDR but correlates with abnormal activation of the NF-κB and STAT-1. Bexarotene may override this acquired resistance of CTCL cells to vorinostat and other HDAC inhibitors.

Disclosures

Zhang: Merck: Research Funding. Duvic:Merck: Honoraria, Research Funding, Speakers Bureau.
A Meta-Analysis of Randomized Controlled Trials of Aspirin in Primary Prevention of Cardiovascular Disease.
Authors: Raju Nina C , Sobieraj-Teague Magda , Eikelboom John W
Published: October 2019

Abstract 174

Primary prevention with aspirin reduces the risk of non-fatal cardiovascular events but has not been demonstrated to reduce mortality. We performed an updated meta-analysis of randomised controlled trials of aspirin in primary prevention to obtain best estimates of the benefits and harm of aspirin compared with no aspirin with a focus on mortality. Eligible articles were identified by computerized search of MEDLINE, EMBASE, Cochrane library and CINAHL databases, review of bibliographies of relevant publications and a related article search using PubMed. The outcomes of interest included all cause mortality, cardiovascular mortality, the composite of myocardial infarction, stroke or death, and bleeding. 2 reviewers independently extracted study information and data. Data were pooled from individual trials using the DerSimonian-Laird random-effects model and results are presented as relative risk (RR) and 95% confidence intervals (CI). 8 studies comprising a total of 96,726 subjects were included. Aspirin reduced all-cause mortality (RR 0.94; 95%CI 0.88–1.00), the composite of myocardial infarction, stroke or cardiovascular death (RR 0.87; 95%CI 0.82–0.93), and myocardial infarction (RR 0.8; 95%CI 0.66–0.98) but did not significantly reduce cardiovascular mortality (RR 0.94; 95%CI 0.82–1.08) or stroke (RR 0.93; 95%CI 0.81–1.07). Aspirin increased the risk of major bleeding (RR; 1.69 95%CI 1.38–2.08), gastrointestinal bleeding (RR 1.38; 95%CI 1.16–1.65) and hemorrhagic stroke (RR 1.36; 95%CI 1.01–1.84). There was no interaction between subjects with or without diabetes for the outcomes of all cause mortality, cardiovascular mortality, the composite of myocardial infarction, stroke or death. Aspirin therapy in subjects with no prior history of cardiovascular disease reduces the risk of cardiovascular events, myocardial infarction and overall mortality. These benefits are achieved at the expense of increased bleeding.

Disclosures:

No relevant conflicts of interest to declare.
In Vitro Deprivation of CD8+CD57+t Cells Promotes the Malignant Growth of Bone Marrow Stem/Progenitor Cells in Patients with Myelodysplastic Syndrome.
Authors: Zhang Qianqiao , Li Xiao , Zhang Zheng , Xu Feng , He Qi , Wu Lingyun , Chang Chunkang
Published: October 2019

Abstract 1764

Poster Board I-790

Objective

To investigate the effect of the activated CD8+T (CD8+CD57+) lymphocytes on colony formation (especially malignant colony formation) during myelodysplastic syndrome (MDS) in vitro.

Methods

Bone marrow mononuclear cells (BMNC) from a total of 54 MDS patients were subjected to magnetic-activated cell sorting (MACS) to separate CD8+CD57+T lymphocytes. BMNC were cultured without CD8+CD57+T cells or cocultured with 4 times of CD8+CD57+T cells to study the correlation between the stem/progenitor cell colony formation and the polarization of T cells towards Tc1 lymphocytes. G-band karyotyping was used to identify abnormal karyotypes in MDS patients. The fluorescence in situ hybridization (FISH) method was used to detect stem cells carrying abnormal karyotypes, and the proportions of abnormal cells among BMNC were calculated before and after T cell deprivation. The impact of effector CD8+T cells on the malignant growth of BMNC was especially investigated.

Results

(a) After deprivation of CD8+CD57+T cells, BMNC from 28 MDS patients formed colonies in the culture media. The average number of granulocyte and monocyte colony forming unit (CFU-GM) was 42.9/4×105 cells, and the average number of erythroid colony forming unit (CFU-E) was 11.36/4×105 cells, which were significantly lower than those in the normal control group after deprivation of CD8+CD57+T cells (CFU-GM 83.4/4×105 cells, CFU-E 32.8/4×105 cells; P=0.00). After add-back of CD8+CD57+T cells (4 times), none of the BMNC cultures from any of the 54 MDS patients formed colonies in vitro. (b) In 28 MDS patients whose BMNC formed colonies after T cell deprivation, the bone marrow Tc1/Tc2 ratio was positively correlated with the number of CFU-GM (r=0.463, P=0.01). (c) In 15 MDS patients who had abnormal karyotypes, deprivation of CD8+CD57+T cells significantly increased the proportion of abnormal cells from 43.79% to 56.26% in BMNC culture (P=0.00).

Conclusion

The effector CD8+T lymphocytes inhibit bone marrow hematopoiesis in MDS patients, and the target cells were mainly the cells with a malignant phenotype and abnormal karyotypes.

Disclosures

No relevant conflicts of interest to declare.
Yttrium 90 Plus High Dose BEAM Conditioning with Autologous Stem Cell Transplantation (ASCT); Effects of Prior Rituximab and Outcome of Poor Risk Non Hodgkin Lymphoma (NHL).
Authors: Krishnan Amrita , Raubitschek Andrew , Nademanee Auayporn P. , Kogut N. , Popplewell Leslie , Tsai Ni-Chun , Palmer Joycelynne , Simpson Jennifer , Federico Laura , Yamauchi Dave , Forman Stephen J.
Published: October 2019

Abstract 2323

Poster Board II-300

Introduction:

High dose chemotherapy with ASCT can increase overall survival (OS) in patients with poor risk or relapsed Non- Hodgkin lymphoma (NHL). Novel conditioning regimens combining radioimmunotherapy (RIT) with high dose chemotherapy have demonstrated favorable toxicity profiles and potentially improved response rates. Herein we report on 84 patients who underwent conditioning with Y90 Ibritumomab plus high dose BEAM (BCNU VP16 AraC Melphalan) and the effects of pretreatment rituximab levels on relapse. On day-23 and day-16 of conditioning pts had a blood draw for rituximab levels. If levels were &10ug/ml, pts received rituximab 250mg/m2. This was followed by pretherapy imaging with Indium 111 on day -21 and Y90 treatment on day -14. High dose BEAM chemotherapy started on day-7and all pts received rituximab 250mg/m2 at day+8.

Results:

The median age at ASCT was 58.5 years (range 19-78). Histologies included DLC n=36, Mantle Cell n=27, Follicular n=11, Transformed NHL n=10. Disease status at ASCT was 1st or 2nd CR n=35, 1stPR n=13, 1st or 2nd rel n=18, induction failure (IF) n=18. Median length of f/u for living pts was 34 months. All patients engrafted at a median of 11 days (anc>500) Transplant related mortality was 2.3 %. Two year OS/PFS for the entire cohort was 85.62% (77-91) and 66% (58-73). There was no significant difference in either DFS or OS based on histology (figure 1)Rituximab levels were available in a subset of 18 pts. and of note there was a trend towards higher relapse rates in patients with higher day-23 rituximab levels (35% vs. 0) (see figure 2).

Conclusions.

Y90 BEAM conditioning is well tolerated. Short term outcome is similar among different histologic types of NHL. The true effects of rituximab levels may not yet be fully appreciated due to small patient numbers. However, the trend towards higher relapse rates in the cohort with high circulating pre RIT rituximab levels suggests that titration of rituximab dosing pre RIT may be another strategy in which to improve transplant outcomes by optimizing targeting of the RIT.

Disclosures:

Off Label Use: yttrium 90 in combination with high dose BEAM chemotherapy.
In Vitro Activity of the Type II Anti-CD20 Antibody GA101 in Refractory, Genetic High-Risk CLL.
Authors: Zenz Thorsten , Volden Matthias , Mast Theresa , Sarno Antonio , Winkler Dirk , Schnaiter Andrea , Bühler Andreas , Klein Christian , Umana Pablo , Döhner Hartmut , Stilgenbauer Stephan
Published: October 2019

Abstract 2379

Poster Board II-356

Introduction:

Anti-CD20 (i.e. rituximab) based immuno-chemotherapies are now considered standard of care in CLL. However, addition of rituximab does not appear to decisively alter the dismal clinical outcome for 17p- / TP53 mutated CLL. Relatively little is known about activity of different anti-CD20 antibodies in genetic subgroups of CLL.

Methods:

In an effort to assess the activity of the next generation mAb GA101 in vitro, we studied B cell depletion / apoptosis in a set of CLL patients. CLL samples were genetically characterized with respect to genetics (genomic aberrations, TP53 mutation, IGHV mutation status), as well as clinical course and immunophenotype. To study the effect on CLL cells, we assessed GA101, Rituximab and Alemtuzumab by in vitro treatment after ficoll separation with FACS (7-AAD) for viability. In addition, in order to maintain ADCC and to mirror the in vivo situation, we studied the effect of GA101 in a whole blood culture (n=10).

Results:

With increasing concentrations (0.01-100mg/ml) of GA101 we observed significant cell death as measured by a B cell depletion assay (whole blood) after GA101 exposure (n=10). After 3 hours the maximum effect was observed at a concentration of 10mg/ml with a reduction of CLL cells to 43% of untreated cells.

The results after 8h were similar, again showing the profound effect in GA101 treated samples (mean B-cell depletion to 34.6% remaining cells). Interestingly, further increasing the dose to 100mg/ml did not increase the B-cell depletion by the antibody, but appeared to decrease the ability to deplete CLL cells (Fig. 1).

In contrast treatment with control antibodies (isotype control, rituximab, alemtuzumab) did not lead to similar B-cell depletion (101%, 69%, and 73% of control cells at 3h). We were particularly interested in any potential effect in cases with TP53 mutation. While the sample number is still low (TP53 mutation / 17p deletion n=3), currently we have no indication of differential response in different genetic subgroups.

Conclusion:

Compared to the currently most widely used mAb in CLL (Rituximab), GA101 appears more potent at equivalent concentration in depleting CLL cells. Further in vitro studies are currently ongoing to assess this antibody in particular in genetic high-risk, refractory CLL.

Disclosures:

Zenz: Roche: Honoraria. Klein:Roche: Employment, Equity Ownership, Patents & Royalties. Umana:Roche: Employment, Equity Ownership, Patents & Royalties. Döhner:Roche: Research Funding. Stilgenbauer:Roche, Bayer Schering Pharma: Honoraria, Research Funding.
Human Cytomegalovirus Inhibited Megakaryocytic Differentiation, Maturation and Induced Apoptosis in Vitro.
Authors: Chen Jian Liang , Chan Godfrey ChiFung , Ye Jie Yu , He Zheng Xian , Wang Qing Wen , Pan Si Nian , Li Xiao Feng , Deng Rui Xia , Yang Mo
Published: October 2019

Abstract 2413

Poster Board II-390

Human cytomegalovirus (HCMV) can cause life-threatening infection in immunocomprimised individuals, such as patients undergoing intensive chemotherapy or bone marrow transplantation. Thrombocytopenia is one of the manifestations in active HCMV infection, which may be a consequence of viral suppression on megakaryopoiesis. The exact underline mechanisms remain uncertain. Our previous studies suggested that HCMV directly infects megakaryocytic progenitors and inhibits their proliferation. Colony-formation of HCMV-infected CFU-MK decreased in a dose-dependent manner (Blood, 2003, abstract). In present study, we explored the mechanisms further by using a phorbol 12-myristate 13-acetate (PMA) stimulating polyploidization to mimic the late stage of megakaryocytic differentiation and maturation in vitro. After co-culture of a megakaryocytic cell line CHRF-288-11 with HCMV AD169 experimental strain from day 0 to day 3 (multiple of infection, MOI=1), the polyploidization of megakaryocyte was determined by DNA content analysis using flow cytometry. Compared with negative control, the proportion of polyploidy (ploidy N ≥ 8) megakaryocytes decreased by 52%, 32% and 16% in HCMV-infected cell at day 3, day 6 and day 9 respectively. As a specific receptor for megakaryopoietic differentiation, the c-Mpl protein (TPO receptor) was also examined in CHRF-288-11 cell line. The proportion of c-Mpl positive cells showed a 23% decrease in HCMV-infected group in compared to the mock infection control (using ultralviolet treated HCMV) at day 5. In addition, apoptotic signals from megakaryocytic surface, cytoplasma and mitochondria were detected in HCMV-infected cells by flow cytometry with Annexin V, Caspase-3 and JC-1 assay. Compared to mock infection control at day 5, annexin-V positive cells population increased by 57%; active caspase-3 signal increased by 125% in viable cell population; and cell population with damaged mitochondial membrane showed a 5-times increase. In conclusion, our data demonstrated that: (1) HCMV inhibited megakaryocytic differentiation and maturation at late stage; (2) HCMV reduced c-Mpl positive cell population; (3) HCMV induced megakaryocytic apoptosis through intrinsic apoptotic pathway as shown by the functional alteration of mitochodial membrane, activation of caspase-3 and structural damage of outer cellular membrane. HCMV-induced thrombocytopenia is the consequence of multiple processes involving inhibition of megakaryocytic proliferation, differentiation, maturation and also increased megakaryocytic apoptosis.

Disclosures:

No relevant conflicts of interest to declare.
The Population Based Incidence of Myelodysplastic Syndrome (MDS): Utility of Multiple Data Sources and Follow-up.
Authors: Kumar Rajat , Musuka Charles , Khair Humaira , Seftel Matthew D. , Szwajcer David , Clague Cheryl , Noonan Gail , Demers Alain
Published: October 2019

Abstract 245

Background:

The incidence of MDS in Canada is not known. Diagnosis of MDS is often challenging as dysplastic features on bone marrow may be non-specific, requiring exclusion of other disorders. The province of Manitoba, with a population of 1.2 million, has a cancer registry which has included patients with MDS since 2001. In this province, hematology diagnostic services are centralized at two teaching hospitals and the few bone marrows performed outside are reviewed centrally. This provided us with the opportunity to use registry data and bone marrow records to determine the incidence of MDS. We hypothesized that for an accurate estimate, a proportion of MDS cases would require follow-up data.

Methods:

Retrospective study to examine all cases of MDS, which included chronic myelomonocytic leukemia (CMML) diagnosed in Manitoba. All adult Manitobans diagnosed with MDS and CMML (excluding RAEB-T), from 01/2006 to 12/2007 were identified from the cancer registry using ICD-O-3 topography code C42.1 and morphology codes 9980/3, 9982/3, 9983/3, 9985/3, 9986/3, 9987/3, 9989/3 and 9945/3. Bone marrow records for the same period were reviewed to identify all cases that had features of MDS. The clinical charts of all these patients were reviewed centrally to exclude those whose clinical course or repeat investigations suggested an alternative diagnosis.

Results:

A total of 80 patients with newly diagnosed MDS were identified. The age adjusted incidence of MDS was 3.26/100,000. Incidence was higher in men (4.05/100,000) as against women (2.57/100,000). Incidence varied significantly with age at diagnosis: <49yr: 0.12; 50–59yr: 2.24; 60–69yr: 10.63; 70–79yr: 20.41 and >80yr: 21.93. Eleven cases (13.75%) were not known to the cancer registry but were detected on reviewing the bone marrow data. From the registry, nine cases (11.25%) were excluded as the chart review and follow-up revealed alternative diagnoses.

Conclusions:

The incidence of MDS for Manitoba is similar to published rates in Europe and the USA. This may be an underestimation of the actual incidence, as elderly patients may not undergo bone marrow examination if the therapeutic intervention is only supportive. Cancer registries that include MDS based on one-time bone marrow reports should include a review process of confirming or excluding the diagnosis of MDS based on follow up investigations and course of illness.

Disclosures:

No relevant conflicts of interest to declare.
Rapamycin-Induced Autophagy Generates Anti-Apoptotic Th1/Tc1 Cells That Mediate Increased Xenogeneic Gvhd.
Authors: Amarnath Shoba , Costanzo Carliann M , Foley Jason , Mariotti Jacopo , Konecki Daniel M , Gangopadhyay ANu , Eckhaus Michael , Wong Susan , Levine Bruce , June Carl H. , Fowler Daniel H.
Published: October 2019

Abstract 2456

Poster Board II-433

Murine T cells exposed to rapamycin maintain flexibility towards Th1/Tc1 differentiation; the degree to which rapamycin might inhibit human Th1/Tc1 differentiation has not been fully evaluated. In the presence of rapamycin, T cell co-stimulation and polarization with IL-12 or IFN-α permitted human CD4+ and CD8+ T cell differentiation towards a Th1/Tc1 phenotype: by intracellular flow, median percentage expression of Foxp3, IFN-γ, and T-bet was 4%, 20%, and 72%, respectively. Phospho-flow cytometry revealed that such Th1/Tc1 cells expressed activated STAT1 and STAT4 in spite of mTOR blockade; STAT activation was abrogated by PI3 kinase inhibition. Rapamycin-resistant human Th1/Tc1 cells (Th1/Tc1.R cells): (1) had increased expression of the autophagy-related gene LC3BII by gene array and protein analysis; (2) preferentially expressed anti-apoptotic bcl-2 family members (reduced Bax, Bak; increased phospho-Bad); (3) maintained mitochondrial membrane potentials; and (4) had reduced apoptosis relative to control Th1/Tc1 cells not generated in rapamycin (p=0.04). The anti-apoptotic phenotype of Th1/Tc1.R cells was abrogated by co-incubation with the autophagy inhibitor, 3-methyl adenine. The in vivo effect of the Th1/Tc1.R cells was evaluated using two xenogeneic GVHD (x-GVHD) models. First, in an LPS-induced x-GVHD model, Th1/Tc1.R cells resulted in lethality in 75% recipients; soluble TNF-α receptor therapy with etanercept reduced the frequency of lethality to 15%. Second, using a non-LPS natural history model of x-GVHD, recipients of Th1/Tc1.R cells (relative to recipients of control Th1/Tc1 cells) had increased human T cell engraftment (day 30 post-BMT, p=0.001), increased human T cell cytokine levels, increased human T cell expression of the cytotoxic degranulation molecule CD107 (p=0.05), and increased human T cell infiltration of skin, gut, and liver. In this model, lethality due to x-GVHD was also increased in Th1/Tc1.R cell recipients (lethality increased from 20% to 70%, p=0.04). We conclude that rapamycin therefore does not impair human T cell capacity for type I differentiation. Rather, by promoting autophagy rapamycin permits stable expression of T-bet and generates an anti-apoptotic Th1/Tc1 effector phenotype, thereby yielding increased x-GVHD.

Disclosures:

No relevant conflicts of interest to declare.
Cost-Utility Analysis of Von Willebrand Disease Screening in Adolescents with Menorrhagia.
Authors: Sidonio Robert F. , Smith Kenneth J , Ragni Margaret V.
Published: October 2019

Abstract 2476

Poster Board II-453

Introduction:

It is unknown whether it is cost effective to screen adolescents with menorrhagia for von Willebrand disease (VWD) prior to starting oral contraceptive pills (OCPs). Because OCPs can mask a diagnosis of VWD, a woman is at risk for potential future traumatic or surgical bleeding, once OCPs are stopped. We, therefore, designed a decision analysis model to evaluate the cost utility of VWD testing in adolescents with menorrhagia.

Methods:

A 20-year Markov decision analytic model was constructed to evaluate the cost-utility of two strategies: testing or not testing for VWD. The hypothetical patient is a 15-year-old female presenting with menorrhagia and a decision regarding VWD testing is made. The model includes probabilities of remaining well, suffering an acute menorrhagia bleeding event, surgical complications, OCP complications or dying. Probabilities, costs and utilities were estimated from published literature. The prevalence of type 1 VWD in adolescent females with menorrhagia was estimated to be 13%. We assumed all patients were treated with OCPs regardless of whether hemostatic testing was performed. All patients were assumed to have mild type I VWD, the most common subtype, and all were responsive to intravenous DDAVP. Probabilities of surgical complications in patients with and without VWD were based on adenotonsillectomy hemorrhage rates in children. The probability of acute menorrhagia bleeding necessitating hospitalization or red blood cell transfusion was estimated to be 1% in an adolescent with VWD and 0% in an adolescent with normal hemostatic testing. The sole complication from OCPs incorporated into this decision analytic model was acute deep vein thrombosis.

Results:

The cost of testing adolescents with menorrhagia for VWD was $1,638, vs. $1,251 for not testing for VWD. The effectiveness of not testing in quality-adjusted life-year (QALY) gained (14.237 QALY) was similar to the VWD testing strategy (14.246 QALY). Compared to not testing for VWD, screening for VWD had an incremental cost-effectiveness ratio (ICER) per QALY of $45,061/QALY, a value typically considered economically reasonable. In one-way sensitivity analysis, the outcome and ICER were most sensitive to the probabilities of an acute bleeding event, surgical complication, and the prevalence of VWD. Monte Carlo probabilistic sensitivity analysis, substituting input distributions rather than a single point estimate for the probabilities, costs and utilities, was also performed. Under this assumption, testing for VWD is a cost-effective strategy approximately 60% of the time when the cost-effectiveness threshold is $100,000/QALY.

Conclusions:

It is cost effective to perform VWD testing in adolescents presenting with menorrhagia prior to initiating OCPs. This is related to avoiding cost from postoperative and other bleeding when the diagnosis is masked by OCPs.

Disclosures:

No relevant conflicts of interest to declare.
Disparities of Common Hematological Malignancies in Asian Indian and Pakistani Population Living in the USA: A SEER Database Based Analysis From 1988 to 2006.
Authors: Hossain Akm , Rahman Rubayat , Abraham Jame
Published: October 2019

Abstract 2478

Poster Board II-455

Background:

It is estimated that Asian Indian and Pakistani (AIP) comprises about 1.5% of the total US population and is rapidly growing. Hematological malignancies account for about 15% of all the cancers diagnosed in this group. The aim of this study is to analyze epidemiologic and survival parameters of common hematological malignancies in AIP living in the USA and compare it to the US White (USW) population for the first time.

Methods:

The SEER (Surveillance Epidemiology and End results) database has been reporting cancer incidence since 1973. AIP with cancer diagnosis was reported separately under race and ethnicity in SEER database from 1988. Using SEER database 1988-2006, appropriate frequency and survival sessions (Kaplan-Meier survival method) were performed and epidemiologic characteristics for common hematological malignancies were compared between AIP and USW.

Results:

Conclusions:

Our analysis shows that AIP are diagnosed with common hematological malignancies at a younger age than USW. The distribution of these malignancies according to gender follows other major groups but there is no statistical difference in age at diagnosis, stages, tumor biology, treatment methodologies or survival between male and female AIP. Noticeable better 1-5 years survival could be attributed to difference in tumor biology, cytogenetics, emigrational factors, higher socioeconomic status, better access to health care, educational background with awareness and environmental factors. Future studies are warranted to examine the behavior and biology of tumor in this population.

Disclosures:

No relevant conflicts of interest to declare.
National Burden of Emergency Department Care for Sickle Disease: Impact of Age, Insurance Status, Income, Hospital Type and Location On Subsequent Hospital Admission.
Authors: Goel Ruchika , Viswanathan Prabhu , Krishnamurti Lakshmanan
Published: October 2019

Abstract 2488

Poster Board II-465

BACKGROUND.

Limited data exist about the national burden of emergency department (ED) care for patients with sickle cell disease (SCD). The proportion of patients being seen in the ED who require hospitalization and factors contributing to the above are poorly understood. We present here analysis of such potential factors from the largest nationally representative ED visit data till date.

METHODS.

The Nationwide Emergency Department Sample (NEDS) is one of a family of databases and software tools developed as part of the Healthcare Cost and Utilization Project (HCUP), a Federal-State-Industry partnership sponsored by the Agency for Healthcare Research and Quality. The NEDS was constructed using records from both the HCUP State Emergency Department Databases and the State Inpatient Databases. The NEDS is the largest all-payer ED database and contains almost 26 million records for ED visits for over 950 hospitals and approximates a 20-percent stratified sample of U.S. hospital-based ED's thus generating weighted estimates of over 120 million ED visits.

The following ICD-9-CM diagnosis codes were used to define SCD related hospitalizations: 28241, 28242, 2825, 28260, 28261, 28262, 28263, 28264, 28268, and 28269 and only ‘primary discharge diagnosis' patients were considered. We analyzed the NEDS data for the year 2006.

RESULTS.

Approximately three-quarters of the total in-patient admissions for SCD in 2006 came through the emergency department. The total number of patients presenting to the ED with SCD as primary diagnosis was 166,043. Of these visits, 68,420 (41.2%) resulted in admission to the hospital.

There was a statistically significant difference (p<0.001) in the proportion of ER visits resulting in hospitalization for the following factors (each studied independently):

CONCLUSION.

SCD is responsible for a significant burden of ED care in the US. A number of factors including of patient age, insurance status, income, hospital type and location may impact the likelihood of subsequent admission to the hospital. High quality care of uncomplicated pain crises in an ED or day hospital is believed to result in a decrease of avoidable hospitalizations. However, the multiplicity of factors independently associated with likelihood of subsequent hospitalization as demonstrated by these data suggest the need for caution in using the proportion of ED visits resulting in hospitalizations as a surrogate marker of quality of care.

Disclosures:

No relevant conflicts of interest to declare.
Ly6C+ Inflammatory Monocytes Are Extrahepatic Precursors of Hepatic Stellate Cells in Liver Injury.
Authors: Masuya Masahiro , Araki Hiroto , Ohishi Kohshi , Katayama Naoyuki
Published: October 2019

Abstract 2518

Poster Board II-495

Objective:

It has been known that hepatic stellate cells (HpSCs) play an important role in the development of liver fibrosis. We previously reported that the HpSCs are hematopoietic origin in carbon tetrachloride (CCl4)-induced liver injury. However, the differentiation pathway from hematopoietic stem cells to HpSCs remains largely unknown. In this study, we tried to elucidate the ability of terminally differentiated cells to transdifferentiate into HpSCs.

Methods:

Male enhanced green fluorescent protein (EGFP)-transgenic mice 10–12 weeks old were used as bone marrow and spleen donors. Bone marrow mononuclear cells were incubated with purified anti-CD45R/B220, anti-CD4, anti-CD8, anti-Ly6G, anti-TER119, anti-c-kit, anti-Sca-1, anti-Thy-1.2, anti-IL-7 receptor α chain, and immunomagnetic beads. The resulting lineage negative cells were stained with anti-Ly6C, anti-c-kit, and anti-CD45.2. CD45.2+Ly6Clowc-kit+ cells (monocytic precursors), CD45.2+Ly6Chighc-kit− cells (mature monocytes), FSClowSSChigh CD45.2+ cells (eosinophils) were enriched by FACSAria. Ly6G+ cells (neutrophils), CD45R/B220+ cells (B cells) and CD3ε+ cells (T cells) were isolated from bone marrow or spleen using magnetic cell sorter device. We transferred these isolated cells into CCl4-injured mice twice a week for two weeks, and analyzed the presence of EGFP+ HpSCs in the injured liver using immunofluorescence and laser confocal scanning microscopy.

Results:

Total number of transfused monocytic precursors, mature monocytes, eosinophils, neutrophils, B cells, and T cells per mouse was 1.1 × 106, 6.4 × 106, 4.8 × 106, 22.0 × 106, 29.0 × 106, and 12.5 × 106, respectively. Intravenous infusion of each type of cell showed their ability to localize to the injured liver. In the liver of mice, which were given eosinophils, neutrophils, B cells, and T cells, EGFP+ cells were all positive for CD45. However, in the liver of mice, which received monocyte precursors and mature monocytes, 20% and 39% of EGFP+ cells were negative for CD45. Vimentin and ADAMTS13 are identified in HpSCs but not hematopoietic cells. About 18% of EGFP+ cells in the liver of mice, which received monocytic precursors, were positive for both antigens. In the liver of mice, which received mature monocytes, 35% and 27% of EGFP+ cells were positive for vimentin and ADAMTS13, respectively.

Conclusion:

Both Ly6Clowc-kit+ monocytic precursors and Ly6Chighc-kit− mature monocytes can give rise to HpSCs in CCl4-injured liver.

Disclosures:

No relevant conflicts of interest to declare.
TET Family Oncogene Fus Is Essential for the Maintenance of Self-Renewing Hematopoietic Stem Cells.
Authors: Sugawara Takeaki , Oguro Hideyuki , Iwama Atsushi
Published: October 2019

Abstract 2529

Poster Board II-506

A Proto-oncogene FUS (fusion derived from malignant liposarcoma), also known as TLS (translocated in liposarcoma), was originally identified in chromosomal translocation of human soft tissue sarcoma. FUS is also known to be fused with an ETS family transcription factor ERG in human myeloid leukemia with t(16;21) which is associated with poor prognosis. Based on its protein structure, DNA- and RNA-binding activity and involvement in many human cancers as the fusion with various transcription factors, FUS is now grouped with EWS and TAFII68 into TET (FET) oncogene family. Multiple functions have been postulated for FUS, including non-coding-RNA-mediated transcriptional repression, posttranscriptional RNA processing and the maintenance of genomic integrity. Fus-deficient (Fus−/−) mice showed a non-cell-autonomous defect in B lymphocyte development, defective B cell activation and increased sensitivity to radiation in previous studies. However, its physiological function in hematopoiesis remains unknown. In this study we performed detailed analyses of Fus−/− hematopoietic stem cells (HSCs). Fus−/− fetal livers at embryonic day 14.5 exhibited a mild reduction in numbers of hematopoietic stem and progenitor cells compared with the wild type. Disruption of Fus, however, did not grossly affect proliferation or differentiation of hematopoietic progenitors. Of note, Fus−/− HSCs had significantly reduced repopulating activity of hematopoiesis in competitive repopulation assays, and did not repopulate hematopoiesis at all in tertiary recipients. Moreover, Fus−/− HSCs were highly sensitive to radiation both in vitro and in vivo and showed a drastic reduction in numbers in recipient mice after sublethal irradiation. All these findings implicate Fus in the maintenance and radioprotection of HSCs. Studies of chromosome stability, telomere length, apoptosis and levels of reactive oxigen species (ROS) appeared not accountable for the apparent defect of Fus−/− HSCs. However, gene expression profiling identified changes in expression of several genes in Fus−/− HSCs, and dysregulated expression of some of these genes might be responsible for the defective function of Fus−/− HSCs.

Disclosures:

No relevant conflicts of interest to declare.