bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytomegalovirus Reduces the Relapse Risk for Acute Leukemia After Transplant: There Is a Virus-Versus-Leukemia Effect.
Authors: Elmaagacli Ahmet H , Lindemann Monika , Opalka Bertram , Koldehoff Michael , Gromke Tania , Ottinger Hellmut , Ross Rudolf S. , Bauer Sebastian , Hilgendorf Inken , Beelen Dietrich W.
Published: October 2019

Abstract 2261

Poster Board II-238

Cytomegalovirus reactivation (HCMV) occurs frequently after hematopoetic stem cell transplantation and is associated with an increased treatment-related mortality. We aimed first to evaluate the cytotoxic effects of CMV on AML in vitro, and further, the clinical outcome of patients with AML with a documented CMV reactivation after transplant.

First, we show that HCMV infects acute leukemia cell line Kasumi-1 (AML) and SD-1 (ALL), inhibits their proliferation and induces apoptosis almost in all cells after 14 days. HCMV further inhibits the proliferation of bone marrow progenitor cells derived from healthy volunteers as demonstrated here by the reduction of the number and size of colony forming units in methylcellulose assays. The induction of apoptosis is unusual because HCMV possesses various strategies to prevent apoptosis in infected cells in order to delay their cell death and maintain its own virus replication. Apoptosis via a caspase-dependent pathway occurred although HCMV induced a significant up-regulation of the anti-apoptotic gene cFLIP and anti-stress gene Gadd45a and simultaneously down-regulation of pro-apoptotic genes p53, Bcl-2, Gadd45γ in Kasumi-1 and SD-1 cells, showing that the anti-apoptotic mechanisms of HCMV to prevent cell death failed. Concordantly, the coapplication of the caspase-specific inhibitor zVAD.fmk to HCMV- infected Kasumi-1 cells inhibited the induction of apoptosis to a great extent.

In the second part of our study we evaluated the clinical outcome of patients with AML after transplant in whom a HCMV reactivation occurred. We evaluated a homogenous group of 140 patients with AML after myeloablative conditioning, non-T cell depleted allogeneic HSCT from a HLA-identical sibling donor, and compared the clinical results to 60 patients with CML after transplant. CMV reactivation was documented by CMV-related matrix protein pp65 antigenemia test and routinely accompanied by a CMV preemptive therapy with ganciclovir.

CMV reactivation in AML patients was associated with reduced risk for relapse (11.6% v 50.5%; p<0.0001) and improved overall survival estimates (73.6% v 42.5%, p=0.039), but increased risk for acute graft-versus-host-disease (GVHD) grade 2-4 (75.1% v 37.5%, p=0.004) and TRM in our analyses, whereas CML-patients had no advantageous effects. Multivariate analyses for risk of relapse confirmed CMV reactivation as an independent factor (RR 0.15, 0.03-0.67;[95% CI], p<0.013) besides chronic GVHD, disease stage, and cytogenetics.

We show here for the first time a strong anti-leukemic effect of HCMV in AML, which improves the outcome of patients after transplant, and might be defined as “virus versus leukemia effect”.

Disclosures:

No relevant conflicts of interest to declare.
Myeloid-Derived Suppressor Cells in Multiple Myeloma.
Authors: Van Valckenborgh Els , Van Ginderachter Jo , Movahedi Kiavash , Menu Eline , Vanderkerken Karin
Published: October 2019

Abstract 2794

Poster Board II-770

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous mix of myeloid cells in different maturation stages generated in the bone marrow. The role of MDSCs in cancer is to suppress T-cell responses, thereby likely regulating tumor progression. In mice, MDSCs are identified by the expression of the surface markers CD11b and Gr-1. Recently, Ly6G+ granulocytic (PMN-MDSC) and Ly6G− monocytic (MO-MDSC) subsets could be distinguished (Movahedi et al. Blood 2008, 111:4233-44). In multiple myeloma patients, the immune function is impaired and this is caused by an immunologically hostile microenvironment and cellular defects, such as decreased numbers of immune cells, and DC or T-cell dysfunction. However, the role of MDSCs in immune suppression in multiple myeloma is not yet described.

In this study, we investigated the immunosuppressive activity and mechanism of MDSC subsets in the syngeneic and immunocompetent 5TMM mouse model (5T2 and 5T33 models). In first instance, CD11b+Ly6G− and CD11b+Ly6G+ lineage-committed myeloid MDSC subsets were detected in 5TMM-diseased bone marrow by flow cytometry. These subsets were purified via MACS from the bone marrow of naïve and 5TMM tumor-bearing mice, and analyzed for T-cell suppressive activity. Hereto, CD8+ TCR-transgenic OT-1 splenocytes were stimulated with ovalbumin protein in the presence of purified MDSC subsets, after which T-cell proliferation was measured via 3H-thymidine incorporation. Both MDSC subsets from 5TMM bone marrow were able to suppress antigen-specific T-cell responses at a higher level compared to purified MDSC subsets from normal bone marrow. On average, Ly6G− MDSCs were more suppressive than Ly6G+ MDSCs. The 5T2MM model has a tumor take of approximately 12 weeks. Three weeks after intravenous inoculation of the tumor cells, the suppressive effect of the myeloid subsets was already observed (while the plasmacytosis in the BM was very low and no detectable serum M spike was observed), indicating that T-cell suppression is an early event in MM development. To unravel the suppressive mechanism of the MDSC subsets, inhibitors were used in ovalbumin-stimulated cocultures. Ly6G− MDSC-mediated suppression was partially reversed by the iNOS inhibitor L-NMMA and the COX-2 inhibitor sc-791, both of which lower the NO concentration in culture. In contrast, superoxide dismutase and especially catalase enhance NO concentrations, resulting in enhanced T-cell suppression. None of these inhibitors had any impact on the Ly6G+ MDSC-mediated suppression.

In conclusion, these data reveal the presence of MDSCs as a novel immune suppressive strategy employed by multiple myeloma cells in the bone marrow, already occurring early in the disease process.

Disclosures:

No relevant conflicts of interest to declare.
An Angiogenic Risk Score Improves the Prognostic Information of the International Staging System in Patients with Symptomatic Multiple Myeloma.
Authors: Jakob Christian , Wernecke Theresa , Sterz Jan , Goerke Andrea , Heider Ulrike , Kaiser Martin , Mieth Maren , Ziefle Susanne , Sezer Orhan
Published: October 2019

Abstract 2802

Poster Board II-778

Introduction:

Bone marrow angiogenesis has been shown to correlate with growth, disease progression and survival in multiple myeloma (MM). The angiogenic stimulus in the bone marrow microenvironment is mediated by various angiogenic factors. Serum levels of the angiogenic cytokines basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) can be elevated in MM patients in different disease stages.

Patients and methods:

In the present study we investigated the prognostic value of circulating levels of serum-bFGF, serum-HGF and plasma-VEGF by enzyme-immunoassays in 100 newly diagnosed MM patients, 24 with smoldering MM and 76 patients with symptomatic MM. In addition, the parameters of the recently introduced International Staging System (ISS), beta-2-microglobulin (β2M) and albumin were evaluated in comparison to the angiogenic cytokines in a multivariate prognostic model.

Results:

Circulating levels of serum bFGF, serum HGF and plasma VEGF were significantly elevated in symptomatic versus smoldering MM (P= 0.015, P= 0.005 and P=0.015 respectively). In a univariate survival analysis, all angiogenic factors and a combined angiogenic cytokine score (0-1 versus 2-3 angiogenic factors elevated) were identified as significant prognostic factors for overall survival in the total cohort of MM patients (P= 0.046, P= 0.005 and P< 0.009, respectively) and in symptomatic MM (P= 0.025, P< 0.001 and P= 0.018, respectively). In addition to angiogenic cytokine levels, beta-2-microglobulin (β2M) and albumin as parameters of the ISS were also entered in a multivariate analysis. In this model, bFGF, HGF and VEGF were found to be independent prognostic factors (hazard ratio: 2.5, 3.6 and 2.6, respectively) in symptomatic MM (Table 1). A combined angiogenic risk score (0-1 or 2-3 angiogenic factors elevated) was also a significant prognosticator for overall survival in symptomatic MM (P< 0.001) and clearly separated two subgroups with a good and a worse prognosis within ISS stage III (P= 0.003). In patients with a high angiogenic risk score (2 or 3 factors elevated) there was no significant survival benefit of high-dose therapy, in contrast to patients with a low risk score (0-1 factors elevated).

Conclusions:

Our data show that elevated angiogenic cytokines predict poor prognosis and gives additional prognostic information in comparison to the ISS parameters β2M and albumin in symptomatic MM. Since a variety of drugs with anti-angiogenic properties are currently in use or under investigation in MM, our data support the idea of using angiogenic cytokines as risk factors to identify poor prognosis groups, which may be considered for novel anti-angiogenic treatment strategies.

Disclosures:

No relevant conflicts of interest to declare.
The Krüppel-Like Factor 4 (KLF4) Modulates Survival and Tissue Distribution of Natural Killer Cells.
Authors: Shik Park Chun , Lee Ping-Hsien , Yamada Takeshi , Mamonkin Maksim , Lacorazza H. Daniel
Published: October 2019

Abstract 282

Natural Killer (NK) cells are important mediators of the innate immune system that could be targeted therapeutically to treat hematologic malignancies and to prevent graft-versus-host disease. Hence, a better understanding of NK cell survival and tissue trafficking at steady state is vital to develop cell-based therapies. Genes that control proliferation are often involved in tissue distribution of lymphocytes, such as KLF2 in T cells. KLF4, another member of the Krüppel-like factor family, can activate and repress genes involved in diverse cellular processes. We recently reported that KLF4 is part of a novel inhibitory pathway that prevents proliferation of naïve T cells during homeostasis and retain memory T cells in lymph nodes (Yamada et al., Nature Immunology, 2009). In this work, we studied the role of KLF4 in the development and maintenance of NK cells by deleting Klf4-floxed gene (fl/fl) using the Mx1-Cre system. The percentage of NK1.1+TCR- cells is significantly reduced in peripheral blood of Klf4-deficient (▪/▪) mice (fl/fl: 3.4±1.1 versus ▪/▪: 1.2±0.1, n=9) and also absolute numbers in spleen (▪/▪: 1.1±1.3 ×106, n=6) due to increased percentage of Annexin V positive cells (fl/fl: 9.2±3.2 versus ▪/▪: 22.9±15.5, n=15). The number of CD49d+TCR- cells was also significantly reduced in peripheral blood and spleen of Klf4-deficient mice. In contrast, the number of NK cells in bone marrow and lymph nodes of Klf4-deficient mice was similar to controls. Deletion of Klf4 gene led to reduced numbers of NK1.1+TCR-CD27+CD11b+ and NK1.1+TCR-CD27-CD11b+ cells, which correlated with increasing apoptosis of these subsets. Yet, the percentages of these NK cell subsets were normal in bone marrow ruling out a developmental defect in this tissue. Transplant of wild type and Klf4-deficient bone marrow cells into wild type mice suggested environmental rather than cell intrinsic defects. NK cells (NK1.1+TCR-) isolated from spleen of Klf4-deficient mice showed to be functional in a cytotoxicity assay using a mixture of differentially CFSE-labeled RMA-S (target) and EL4 (control). In summary, KLF4 plays a key role in the maintenance of mature NK cells in peripheral blood and spleen.

Disclosures:

No relevant conflicts of interest to declare.
Patterns of Monoclonal Immunoglobulins and Serum Free Light Chains Are Significantly Different in African-American Compared to Caucasian MGUS Patients.
Authors: Weiss Brendan , Minter Alex , Laquer Matthew , Howard Robin , Abadie Jude , Ascencao Joao , Schechter Geraldine P. , Kuehl Michael , Landgren Ola
Published: October 2019

Abstract 2838

Poster Board II-814

Background.

Monoclonal gammopathy of undetermined significance (MGUS), the precursor of multiple myeloma (MM), is 2- to 3-fold more common in African-Americans (AA) than Caucasians (CA). Prior studies have reported clinical features of MGUS and MM to be different among AA compared to CA. Recent studies on CA MGUS cases have found serum free kappa and lambda light chain assays (sFLC) to be highly predictive of MM progression. We have conducted the first study designed to evaluate patterns of sFLC markers in AA.

Methods.

All AA MGUS patients seen at Walter Reed Army Medical Center (WR) and the Washington DC Veteran's Affairs Medical Center (VA) 2005-2009 were eligible. WR patients were enrolled through a retrospective chart review as sFLC has been performed routinely on MGUS patients. VA patients were enrolled in a prospective trial and sFLC was performed at WR. All patients were categorized for their risk of progression using the Mayo Clinic model (Rajkumar SV Blood 2005) and compared to the Caucasian Mayo Clinic population by the Fisher's exact test (2-tailed).

Results.

Eighty-six patients were enrolled, the median age was 73 yrs (42-92) and 74% were male. The MGUS isotype was 87% IgG, 10% IgA, 2% IgM and 1% biclonal. Kappa and lambda light chain disorders were present in 61% and 38%, respectively. The median monoclonal Ig concentration was 0.56 gm/dL (trace-2.33), 47% had a concentration '0.5 g/dL, 30% 0.51-1.0, 20% 1-2 g/dL, and 2% 2-3 g/dL. An abnormal sFLC ratio was present in 49% of patients. Based on the Mayo Clinic model, the risk of progression was: low 42% (95%CI 32-52), low-intermediate 49% (39-59), high-intermediate 8% (4-16) and high 1% (0-7). In comparing our data to the Mayo Clinic data, AA have significantly different distributions of MGUS isotype (p =0.001), lower monoclonal Ig concentration (p=0.0005), presence of an abnormal sFLC ratio (p=0.004) and distributions of Mayo Clinic risk groups (p=0.008) (see table).

Conclusions.

The clinical and laboratory features of AA patients with MGUS are distinctly different than in Caucasians, in particular a lower proportion of IgM MGUS, lower monoclonal Ig levels, a higher proportion of abnormal sFLC ratios, and fewer higher-risk patients. IgM MGUS is the precursor to Waldenstrom's macroglobulinemia (WM) and the lower proportion of IgM MGUS in AA is consistent with the very low rate of WM in AA. Non-IgM MGUS should be considered a separate entity and a direct comparison of AA and CA non-IgM MGUS patients using the Mayo Clinic data is planned. The significance of these differences on the risk of malignant progression will require prospective studies in racially diverse populations.

Disclosures:

Weiss: The Binding Site, Inc.: Research Funding.
Matrix Metalloproteinses-13 (Collagenase-3) Secreted by Multiple Myeloma Cells Enhances the Bone Resorptive Activity of Osteoclasts.
Authors: Feng Rentian , Kurihara Noriyoshi , Ma Huihui , Roodman G. David , Lentzsch Suzanne
Published: October 2019

Abstract 2853

Poster Board II-829

Introduction:

Osteolytic bone disease is a frequent complication of multiple myeloma (MM) that is characterized by reduced bone formation and increased bone resorption. MM cells produce several cytokines causing dysregulation of the bone remodeling process that results in excessive bone resorption mediated by activated osteoclasts (OCLs). Matrix metalloproteinases (MMPs) play a critical role in tumor invasion and progression in many solid tumors such as breast cancer but the role of MMPs in MM has have not been well elucidated.

Methods and Results:

We analyzed the MMP expression profiles (MMP1, 2, 3, 7, 8, 12, 13) of MM cells and found very high expression (400-fold higher) of MMP13 compared to other MMPs. Tissue array analysis for MMP-13 expression in MM tumor samples and normal control tissue indicated that MMP13 was only highly expressed in MM tumor samples. ELISA analysis of serum samples revealed a positive correlation of MMP13 expression with MM bone disease. MMP13 was not detectable in the sera of healthy donors, whereas 62% of MM patients expressed MMP13 in their serum samples, among which 87% had extensive bone disease. Co-culture of MM cells with primary bone marrow stromal cells (BMSC) resulted in a significant (P<0.001) increase (up to 14-fold) of MMP13 secretion by MM cells. RT-PCR analysis indicated that the expression of MMP13 gene in MM cells was markedly enhanced by BMSC conditioned medium, suggesting that BMSC-secreted soluble factors, instead of direct cellular interactions, induce MMP13. Functional inactivation of different cytokines with neutralizing antibodies identified IL-6 as a critical cytokine for the induction of MMP13. Electromobility shift assays showed that IL-6 mediated AP-1 activation promotes MMP13 transcription. Importantly, MMP13 significantly enhanced osteoclastic bone resorption on dentin slices induced by RANKL plus M-CSF. However, the strong induction of bone resorption was not derived from increased OCL formation since MMP13 only slightly increased OCL numbers. MMP13 significantly increased the OCL size as well as the nuclear number/OCL which increases the bone resorption activity per osteoclast.

Conclusions:

Our results suggest that MMP13 augments the pathogenic process of osteolytic lesions in MM by increasing the bone resorption capacity/OCL. These results suggest that targeting MMP13 may represent a novel approach to ameliorate osteolytic lesions in MM.

Disclosures:

Roodman: Novartis: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy; Celgene: Consultancy; Acceleron: Consultancy. Lentzsch:Celgene: Consultancy, Speakers Bureau; Pfizer: Consultancy.
Waldenstrom's Macroglobulinemia: A SEER Database Review From 1981-2005.
Authors: Sekhar Jeevan , Morgensztern Daniel , Zhang Qin , Vij Ravi
Published: October 2019

Abstract 2926

Poster Board II-902

Introduction:

Waldenstrom's Macroglobulinemia (WM) is a relatively rare hematopoietic neoplasm, with literature data mostly limited to small series of patients. We conducted a review of the Surveillance Epidemiology and End Results (SEER) database for WM analyzing 2969 cases from 1981-2005.

Results:

The incidence of reported cases of WM was stable during the studied time period at 0.3 per 100,000. The median age at diagnosis was 73 (range 23-98). There were 1661 male cases of WM and 1038 female cases (incidence ratio of 1.6:1). There were 2435 Caucasian cases and 264 non-Caucasian cases (incidence ratio of 9.2:1). Median overall survival for the entire cohort was 65 months (95% CI, 61-69). Five-year overall survival was 52% (95% CI, 50-55%). There was a significant difference in survival for individuals age <60 years versus age ≥60 years with age-adjusted 5 year overall survivals of 78% (95% CI, 44-49%) and 47% (95% CI, 44-49%), p<0.0001, respectively. Gender and race had no impact on overall survival (p=0.23 and 0.13, respectively). There was no improvement in survival over the time period analyzed. The cohort of patients diagnosed in 1981-1995 had a 5-year overall survival of 50% (95% CI, 46-54%), and those diagnosed in 1996-2005 had a 5-year overall survival of 54% (95%CI, 51-57%), p=0.18.

Conclusion:

This survey represents the largest cohort of WM analyzed to date. Age was shown to be the sole predictor of overall survival. Furthermore, these results underscore the little therapeutic progress we have made for this disease considering the lack of improvement in overall survival over a quarter century.

Disclosures:

Vij: Otsuka Pharmaceuticals: Research Funding.
Detection of LMO2 in Normal Human Tissues and Haematopoietic and Non-Haematopoietic Tumours by a Newly Developed Rabbit Monoclonal Antibody.
Authors: Agostinelli Claudio , Paterson Jennifer C , Gupta Rajeev , Righi Simona , Sandri Federica , Piccaluga Pier Paolo , Bacci Francesco , Sabattini Elena , Pileri Stefano A. , Marafioti Teresa
Published: October 2019

Abstract 2942

Poster Board II-918

Introduction:

LMO2 is a transcription factor with a central role in haematopoietic development and angiogenesis. High levels of LMO2 transcript were reported in CD34+ stem cells and strong protein expression was observed in endothelia, germinal centre B cells (GCB) and some B cell lymphomas. Activation of LMO2 has been demonstrated in T-ALL as a consequence of translocations or deletions and its expression in diffuse large B-cell lymphomas is associated with favourable outcome. Furthermore, correlation between LMO2 expression and tumour progression has been reported in prostatic carcinoma.

Aims:

The expression pattern of a newly developed rabbit monoclonal antibody raised against a fixation-resistant epitope of the LMO2 protein was studied in human normal and pathologic samples.

Material and Methods:

Normal haematopoietic and non-haematopoietic tissue sections, tissue micro-arrays including 1452 neoplastic samples, 11 lymphoma-derived cell lines and CD34+/CD38− stem cells, purified from human umbilical cord blood, were investigated by single and double immunostaining.

Results and discussion:

Strong LMO2 expression was found in CD34+/CD38− stem cells and in contrast to previous studies, the anti-LMO2 rabbit monoclonal distinctively labelled normal mantle, monocytoid and splenic marginal zone B cells. A few LMO2+/CD34+ cells were detected in the thymic cortex, most likely representing T cell precursors. Tables 1 and 2 summarise the results obtained in tumours and lymphoma-derived cell lines. Among B cell neoplasms, LMO2 was highly expressed in B-lymphoblastic lymphoma/leukaemia (87%) and heterogeneously in MCL (14%), FL (57%) and BL (37%). In DLBCL, LMO2 positivity was closely related to those cases with a “germinal centre” phenotype. LMO2 nuclear positivity was observed in all cases of LPHL; interestingly Reed-Sternberg cells in CHL showed LMO2 cytoplasmic staining in 10% of cases. The constant expression in T-ALL highlights the pathogenetic role and diagnostic significance of LMO2 in this setting as opposed to PTCLs (usually negative). Detection of LMO2 was observed in the majority of AML and CML cases investigated. Different types of carcinoma expressed LMO2 that was found to be mainly cytoplasmic.

Conclusion:

The newly developed anti-LMO2 rabbit monoclonal antibody represents a powerful reagent for detection of LMO2 protein in routine samples as compared to the previously published monoclonal anti-LMO2. Its frequent expression among haematological malignancies (e.g. T-ALL, B-ALL and AML) indicates LMO2 as putative therapeutic target as evidenced by RNA-silencing studies in experimental models.

Disclosures:

No relevant conflicts of interest to declare.
Minimal Disseminated Disease Is An Independent Poor Prognosis Marker Among High Risk Burkitt's Lymphoma Patients .
Authors: Mussolin Lara , Pillon Marta , Tridello Gloria , Boaro Maria Paola , d'Amore Emanuele S , Nigro Luca Lo , Buffardi Salvatore , Lombardi Alessandra , Sala Alessandra , Favre Claudio , Aricò Maurizio , Rosolen Angelo
Published: October 2019

Abstract 2944

Poster Board II-920

Introduction:

The chromosomal translocation t(8;14)(q24;q32) represents a specific tumor marker in Burkitt's lymphoma (BL). This chromosomal aberration involves the MYC oncogene on chromosome 8 and the immunoglobulin heavy-chain (IgH) locus on chromosome 14. We have previously demonstrated that this genetic abnormality can be used as a marker of Minimal Disseminated Disease (MDD) in BL (Mussolin et al, JCO, 2007). The aim of the study was to assess of the prevalence of MDD in BL at diagnosis in children enrolled in the AIEOP LNH-97 clinical protocol and the evaluation of its impact on prognosis.

Patients and Methods:

We established a simplified long-distance PCR (LD-PCR) assay which can amplify up to 15-20 Kb DNA sequence making it possible to detect the t(8;14) at the genomic level with the sensitivity of 10-4. The assay was based on 4 separate PCR reactions in which one primer complementary to the first exon of the MYC gene is used with one of four primers for the IgH locus (1 for the JH region and 1 for each of the 3 constant regions).

Results:

LD-PCR was applied to prospectively study 124 BL biopsies and detected a specific PCR product in 88 of them (71%). Of the 88 positive BL patients we studied both the tumor and the bone marrow (BM) at diagnosis in 76: BM was positive by LD-PCR in 25 patients (33%), whereas only 10 (13%) were positive at the standard morphological and/or immunophenotypical analyses. Most of the MDD positive patients (88%) belonged to the R4 Risk Group according to BFM definition (stage III or stage IV according to St. Jude staging classification and LDH≥1000 U/l). The 3-year progression-free survival (PFS) was 68% (SE 10%) in MDD positive R4 patients compared with 96% (SE 4%) in MDD negative R4 patients (p= 0.02), whereas there was no difference in PFS between children with morphological involvement of BM at diagnosis versus those who had negative BM (PFS=62.5% (SE 17%) vs. PFS= 87% (SE 6%), respectively, p= 0.09). By multivariate analysis (including MDD, gender, LDH, CNS involvement) MDD was predictive of higher risk of failure (Hazard Ratio: 8.4 , p= 0.04).

Conclusions:

We demonstrated that MDD identifies a poor prognosis subgroup among high risk Burkitt's lymphoma patients. We suggest that a more effective risk-adapted therapy, possibly including anti-CD20 monoclonal antibody, should be considered in these patients.

Disclosures:

No relevant conflicts of interest to declare.
Aberrant DNA Methylation and Transcriptional Silencing of DLC-1 in Waldenstrom's Macroglobulinemia.
Authors: Xu Lian , Ciccarelli Bryan , Hatjiharissi Evdoxia , Yang Guang , Zhou Yangsheng , Hunter Zachary , Sun Jenny , Gong Ping , Liu Xia , Tseng Hsiuyi , Ioakimidis Thea , Manning Robert , Hanzis Christina , Cao Yang , Lewicki Megan , Patterson Christopher J , Treon Steven
Published: October 2019

Abstract 2954

Poster Board II-930

The deleted in liver cancer-1 (DLC-1) gene encodes a Rho GTPase activating protein (RhoGAP) with potential tumor-suppressor activity. Hypermethylation in the DLC-1 promoter is an aberrant epigenetic modification associated with transcriptional silencing of DLC-1 in various types of human cancers. To explore the epigenetic alteration of DLC-1 in Waldenström's macroglobulinemia (WM), we investigated the methylation status of the DLC-1 promoter and its correlation with DLC-1 mRNA expression in cell lines and primary WM patient samples. Initial analysis using methylation-specific PCR (MSP) showed that DLC-1 promoter was completely methylated in WM-WSU and partially methylated in BCWM.1. Using quantitative RT-PCR, DLC-1 mRNA expression was detectable in BCWM.1, but not WM-WSU. These results suggested a correlation between the DLC-1 methylation status and mRNA expression. Similarly, among multiple myeloma (MM) cell lines, we showed that RPMI and U266 exhibited complete methylation, whereas INA6 showed partial methylation, and no methylation was detectable in MM1S and MM1R cells. Similar in WM cells, DLC-1 methylation status was highly correlated with the mRNA expression in these MM cell lines. Of 37 WM patient samples examined, 24 (65%) exhibited methylation in the DLC-1 promoter. In contrast, no methylation of DLC-1 was observed in 4 healthy volunteers. The methylation status was further confirmed using bisulfite DNA sequencing in a subset of WM patients. Quantitative RT-PCR analysis showed that DLC-1 mRNA expression was significantly lower in WM patients compared to healthy volunteers (p=0.001). Treatment with demethylation agents azacytidine or 5-aza-deoxycytidine resulted in significant reactivation of DLC-1 transcription in the WM cell lines WM-WSU and BCWM.1. In addition, a synergistic induction of DLC-1 transcription was observed in the presence of azacytidine and the HDAC inhibitor Vorinostat in BCWM.1 and primary WM patient cells. Moreover, functional studies showed that overexpression of DLC-1 induced cell growth arrest and apoptosis in BCWM.1. DLC-1 methylation status was also correlated with serum sCD27 levels in WM patients (p=0.004), which is secreted by WM cells, serves as a marker of disease burden and facilitates CD40L directed paracrine stimulation by mast cells. Taken together, these results suggest that the down-regulation of DLC-1 via aberrant DNA methylation plays a role in the pathogenesis of WM, and represents a novel therapeutic target in the treatment of WM.

Disclosures:

No relevant conflicts of interest to declare.
Post-Thrombotic Syndrome (PTS) in Children: A Systematic Review of Prevalence, Validity of Outcome Measures, and Prognostic Factors.
Authors: Goldenberg Neil A , Kahn Susan , Crowther Mark A. , Kenet Gili , Nowak-Gottl Ulrike , Manco-Johnson Marilyn J.
Published: October 2019

Abstract 2978

Poster Board II-949

BACKGROUND:

Post-thrombotic syndrome (PTS) is a condition of chronic venous insufficiency following deep venous thrombosis (DVT) that affects both adults and children. Nevertheless, investigation of PTS in children has lagged behind that in adults.

OBJECTIVE:

This systematic review was undertaken to summarize evidence from the pediatric literature on the prevalence of PTS, the validity of proposed measures of PTS, and prognostic factors for PTS in children.

METHODS:

A comprehensive Medline search was performed employing the following terms: “[pediatric OR children] AND [post-thrombotic syndrome OR post-phlebitic syndrome]”. Single case reports, narrative reviews, and commentaries were excluded.

RESULTS:

Seven case series and cross-sectional studies, nine registries and cohort studies, and one uncontrolled clinical trial were identified that met inclusion criteria; these studies reported on a total of 1316 children with DVT (Table 1). The prevalence of PTS differed substantially between observational and non-observational studies: 15% of 1042 children versus 46% of 274 children, respectively. No pediatric studies have evaluated quality of life (QOL) associated with PTS. Two reports from a single-institutional cohort have identified elevated levels of factor VIII and D-dimer and non-use of thrombolytic therapies as potentially prognostic of PTS in children, particularly among those with veno-occlusive thrombi; these are the only reports employing a PTS outcome measure that has been validated in children (the Manco-Johnson instrument).

CONCLUSIONS:

Overall, high-quality evidence on pediatric PTS is lacking. Collaborative prospective cohort studies and trials that use validated measures of pediatric PTS are needed to assess the incidence of, prognostic factors for, and QOL impact of PTS in children.

Disclosures:

No relevant conflicts of interest to declare.
The Epidemiology of Venous Thromboembolism in the Elderly: a Population-Based Study.
Authors: Tagalakis Vicky , Kahn Susan
Published: October 2019

Abstract 2980

Poster Board II-954

Background.

There are few studies that examine incidence trends of venous thromboembolism (VTE) among the elderly, and moreover data on changes in the prevalence over time of VTE risk factors such as hospitalization are limited.

Objectives.

Using the Province of Québec's administrative health claims (“RAMQ”) and hospitalization (“MED ECHO”) databases, we determined among individuals 65 years of age and older the trend in annual VTE incidence over a ten-year period, and examined the prevalence of hospital vs. community acquired VTE over time.

Methods.

Using RAMQ medical service and prescription claims data, we identified a cohort of elderly persons with incident VTE between 1983 and 1994. All individuals 65 years of age and older between January 1, 1994 and December 31, 2004 with at least one medical service (i.e. physician) claim linked to a VTE ICD-9 code in conjunction with a prescription claim for an anticoagulant in the subsequent 60 days, and who had no prior VTE-coded claim between 1983 and 1994 were included in the cohort. The first (incident) VTE-coded claim (index claim) during the period between January 1, 1994 and December 31, 2004 defined entry into the cohort. Using Québec population census data, we determined annual VTE incidence estimates, adjusting for the population's age and sex distribution as per 1999 census data. An index VTE event that occurred during a hospitalization and up to 90 days following hospital discharge was defined as hospital-acquired VTE, otherwise it was considered to be community-acquired.

Results.

A total of 27 758 persons were included in our cohort. The age and sex adjusted annual VTE incidence among individuals 65 years of age and older was 2.1, 2.7, and 2.8 per 1000 population in 1994, 1999 and 2004, respectively (p trend < 0.001 (across years)). The incidence rates increased with age and were slightly higher in men than women. Overall, 35% of VTE events were hospital-acquired (n=9598) and 65% occurred in the community (n=18160). There was a significant trend over time of an increase in the proportion of VTE associated with hospitalization with 32% of VTE being hospital acquired in 1994 and 39% in 2004.

Conclusion.

In a general population of individuals 65 years of age and older, VTE is a common problem, and its overall annual incidence has increased over time. Our results suggest that hospital admission and recent hospitalization are increasingly important risk factors for VTE occurrence in this population, and that efforts are needed to improve and optimize VTE preventative strategies in the hospitalized elderly.

Disclosures:

No relevant conflicts of interest to declare.
Risk Stratification of Recurrent Venous Thromboembolism.
Authors: Zotz Rainer B. , Gerhardt Andrea
Published: October 2019

Abstract 2981

Poster Board II-956

Background:

A better risk stratification for recurrent venous thromboembolism (VTE) in patients with a first episode of idiopathic venous thromboembolism (VTE) is urgently needed.

Methods:

Retrospective study covering more than 20 years after a first venous thromboembolic event in a group of 1,440 patients with VTE.

Results:

In the subgroup of patients (n=515) with a first spontaneous VTE, the yearly incidence of a recurrent spontaneous VTE was 8% for the time period 0-2 years and 4-6% in the following 8 years, after a first VTE, triggered by a transient risk factor (oral contraceptives, surgery or immobilization, pregnancy), the yearly incidence of a recurrent spontaneous VTE was 2% (first 2 years) and 1.3-3% (following 8 years). The hazard ratio for recurrent spontaneous VTE in patients with a first spontaneous VTE for specific predictors were as follows: prothrombin mutation heterozygous 1.2 (95% CI 0.9-1.7), FVL heterozygous 1.3 (95% CI 0.92-1.8), male sex 1.9 (95% CI 1.4-2.7), D-Dimer 2.3 (0.9-6.4), protein C (<60% activity) 2.6 (95% CI 1.2-5.7), FVL homozygous 3.0 (1.3-7.7), AT (<60% activity) 3.0 (95% CI 0.96-9.6). In Conclusion, in patients with a first spontaneous VTE the yearly recurrence rate of 5% is more than doubled in the presence of relevant thrombophilic risk factors supporting the need of long-term oral anticoagulant therapy after a first idiopathic VTE. In contrast to current ACCP recommendations, thrombophilic risk factors are of clinical relevance.

Disclosures:

No relevant conflicts of interest to declare.
LPS Mediates the Activation of Platelet by Toll-Like Receptor 4.
Authors: Ma Liping , Wei Jing , Chang Jian-Xing , Zhang Cheng , Pei Zhi-Xin , Yang Qiu-Hong
Published: October 2019

Abstract 3001

Poster Board II-978

Lipopolysaccsharide (LPS) is a principal outer membrane component of gram-negative bacteria. It initiates an inflammatory response to infection by activating Toll-like receptor-4 (TLR4) on various tissues or cells in host. Platelet contributes to the inflammation process through respond to invading pathogens, membrane adhesion molecule (CD62P, P-selectin) and CD40L on platelet are the indexes to determine platelet activation. The present experiment was designed to investigate the expression of Toll-like receptor 4 (TLR4) on platelet and to determine whether platelet TLR4 involves in platelet activation induced by Lipopolysaccsharide (LPS). Human platelet-rich plasma (PRP) and platelet suspension obtained from 15 healthy people were pretreated with a concentration of 0.2μg/ml of LPS in the presence or absence of thrombin (1 U/ml) for 1 hour. The expressions of TLR4,CD62P and CD40L on platelets were detected through flow cytometry, and platelet TLR4 was further determined by performing western blot analysis. The results show that the percentage of TLR4-positive platelet induced by thrombin was increased by 32.34% compared with the resting platelets (25.44%, P<0.01). TLR4 expression on platelets treated with LPS was remarkedly elevated in the presence or absent of thrombin. However, the expression level was much higher in presence of both than thrombin alone( 39.16%,P<0.01). Moreover, the similar results were found in Western blot analysis. Synchronously, the expressions of CD62P and CD40L on resting platelets were 6.39% and 2.45%, they were also markedly increased when treated with thrombin(42.68% and 14.80%) and LPS respectively, and the increase of CD62P was more significant when stimulated with both of LPS and thrombin(63.03%). Although anti-TLR4 antibody inhibited significantly the increases of TLR4, CD62P and CD40L on platelets induced by LPS, it didn't effect their increases induced by thrombin. The experiment results support the evidence that functional TLR4 can be expressed on human platelet. It may involve in platelet activation as an important mediator of LPS- induced CD62P and CD40L expressions on platelets.

Disclosures:

No relevant conflicts of interest to declare.
SHIP-1 Associates with Tyrosine Phosphorylated Protein Kinase C-Delta at 155 Residue.
Authors: Chari Ramya , Bhavanasi Dheeraj , Daniel James , Kunapuli Satya P.
Published: October 2019

Abstract 3004

Poster Board II-981

Protein Kinase C-delta (PKCδ) is a novel PKC isoform that differentially regulates platelet dense granule secretion. PKCδ positively regulates Protease activated receptor (PAR)-mediated dense granule secretion, whereas it negatively regulates glycoproteinVI (GPVI)-mediated dense granule secretion in platelets. PKCδ, a serine/threonine kinase is phosphorylated on its tyrosine residues. There are nine potential tyrosine phosphorylation sites in the regulatory domain of PKCδ. Phosphorylation at different tyrosine residues regulates its substrate specificity. We have previously shown that the association of PKCδ with Lyn and SHIP-1 negatively regulates GPVI-mediated dense granule secretion. However, the event leading to the association between PKCδ and SHIP-1 is not known. We hypothesize that the differential tyrosine phosphorylation of PKCδ downstream of PARs or GPVI receptors result in the preferential association with SHIP-1. In the current study, we show that PKCδ is phosphorylated at tyrosine residues Y332, Y523, Y525 and Y565 upon PAR or GPVI stimulation. Y311 residue is predominantly phosphorylated upon stimulation of PARs, whereas Y155 residue is preferentially phosphorylated upon GPVI stimulation. PAR-mediated Y311 phosphorylation peaks at later timepoint, whereas GPVI-mediated Y155 phosphorylation peaks at an early timepoint. correlating with dense granule secretion. Furthermore, we show that agarose-conjugated Y155 phosphorylated PKCδ peptide associates with SHIP-1 upon GPVI stimulation, and not PARs. These data suggest that the phosphorylation of PKCδ at distinct tyrosine residues differentially regulate its association with SHIP-1. Therefore, we conclude that the GPVI-mediated phosphorylation of PKCδ at 155 is required for its association with SHIP-1.

This study is supported by pre-doctoral fellowship to Ramya Chari from American Heart Association, Pennsylvania-Delaware affiliate.

Disclosures:

No relevant conflicts of interest to declare.
Preliminary Identification of ATP Compartment Associated Proteins On Erythrocyte Membrane.
Authors: Chu Haiyan , Hoffman Joseph F. , Low Philip S.
Published: October 2019

Abstract 3019

Poster Board II-995

A compartment/pool of ATP that is generated by glycolytic enzymes has been reported to exist on the erythrocyte membrane where it channels ATP directly to the Na+/K+-ATPase (Proverbio F and Hoffman JF. J. Gen. Physiol. 1977, 69:605-632. Mercer RW and Dunham PB. J. Gen. Physiol. 1981, 78:547-567). In order to identify the protein components enclosing this compartment of ATP, a photoactivatable probe, 8-azido-[αa-32P]ATP, was loaded into porous erythrocytes under conditions that fill the compartment with ATP, and the radiolabeled ATP was induced to label proximal proteins by illumination with UV light. Analysis of radiolabeled bands reveals that spectrin, adducin, protein 4.1, and actin constitute major components of the compartment. To further verify the involvement of these proteins in the ATP compartment, antibodies against the aforementioned proteins were pre-incubated with porous erythrocytes before attempting to load the compartment with ATP. Analysis of the efficiency of ATP loading in the presence of these blocking antibodies reveals that antibody binding prevents normal filling of the ATP pool. These findings confirm Hoffman's earlier hypothesis that a membrane-bound compartment of ATP exists. The data also suggest that the location of the compartment might reside at the junctional complex, and that the complex of enzymes that fill the ATP pool is different from the complex organized around band 3.

Disclosures

No relevant conflicts of interest to declare.
Role of Spleen Tyrosine Kinase Signaling in Early B Cell Acute Lymphoblastic Leukemia.
Authors: Perova Tatiana , Nutter Lauryl , Matei Irina , Grandal Ildiko , Pine Polly , Hitoshi Yasumichi , Hitzler Johann K. , Guidos Cynthia J , Danska Jayne S
Published: October 2019

Abstract 3092

Poster Board III-29

Introduction

Early B cell acute lymphoblastic leukemia (B-ALL) is the most common type of childhood malignancy, characterized by abnormal accumulation and proliferation of progenitor-B or precursor-B (pre-B) cells. Current challenges associated with B-ALL treatment include fatal relapses, treatment-related toxicities and long-term morbidities underscoring a need to develop new targeted therapies aimed at eradicating leukemia cells and their stem cells. To achieve this, a better understanding of molecular mechanisms involved in leukemia initiation and progression is required. Our laboratory developed p53-/- PrkdcSCID/SCID double mutant (DM) strain as a mouse model of early B-ALL. We showed that DM leukemias progress through discrete developmental stages of leukemogenesis despite the absence of a pre-B cell receptor (pre-BCR), a crucial checkpoint in B cell development. Spleen tyrosine kinase (SYK), a key proximal component of pre-BCR signaling, was activated in the DM leukemias despite the absence of pre-BCR and was required for their survival. Approximately 70% of pediatric pre-B-ALLs also do not express pre-BCR, which lead us to investigate SYK signaling in human pre-B-ALL and to test potential therapeutic application of SYK inhibition in these leukemias.

Patients and Methods

We examined 22 viably frozen primary pediatric pre-B-ALL bone marrow samples to test their responses to SYK inhibition in vitro and in vivo and have investigated the molecular basis for aberrant SYK-mediated signaling in B-ALL.

Results

Western blot analyses revealed that SYK and BLNK, a dominant target of SYK, were expressed in pre-B-ALL patient samples. The majority of human pre-B ALL samples tested (14/22) displayed significantly attenuated proliferation in the presence of SYK inhibitors suggesting that SYK is necessary for their survival and/or proliferation. Treatment with SYK inhibitor R406 prevented phosphorylation of downstream SYK targets including BLNK and PLC-γ2. We are continuing to study the effects of SYK inhibition using phospho-flow cytometry and genome wide expression arrays. Preliminary data will also be presented on therapeutic efficacy of an orally bioavailable form of R406-mediated SYK inhibition in vivo by xenotransplantation of human leukemias into immuno-deficient mice.

Conclusions

Understanding the molecular mechanisms of pre-BCR-independent SYK activation involved in proliferation and survival of leukemic blasts may provide a rational basis for development of effective treatment for ALL. Specifically, targeted therapeutic inhibition of SYK signaling may be effective B-ALL treatment that may improve outcomes of current treatment regiments with minimal additional treatment-related toxicity.

Disclosures

Pine: Rigel Pharmaceuticals: Employment, Equity Ownership. Hitoshi:Rigel Pharmaceuticals: Employment, Equity Ownership.
Warfarin Use May Be Associated with Increase Prevalence of Valvular Calcification.
Authors: Sekhri Arunabh , Lerner Robert G. , Aronow Wilbert S , Palaniswamy Chandrasekar , Ahn Chul , Singh Tarunjit , Sandhu Rasham , McClung John A
Published: October 2019

Abstract 3137

Poster Board III-74

Background

Warfarin affects the synthesis and function of the matrix Gla protein, a vitamin K–dependent protein, which is a potent inhibitor of tissue calcification. We aimed to study the association of use of warfarin with calcification of the mitral valve, mitral annulus, and aortic valve.

Methods

We reviewed 1155 patient's charts with a diagnosis of nonvalvular atrial fibrillation. Based on the chart review, patients were grouped into the warfarin group and the no warfarin group. The following parameters were recorded in both groups: age, ethnicity, glomerular filtration rate, calcium-phosphate product, lipid profile, coronary artery disease, diabetes, hypertension and intake of medications including aspirin, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, statins, and beta blockers. All the echocardiograms were analyzed by two cardiologists blinded to the clinical details of subjects but alerted to evaluate for the presence of mitral valve calcium (MVC), mitral annular calcium (MAC), aortic valve calcium (AVC), or any of the three. Stepwise logistic regression analysis was used to identify significant independent risk factors associated with any calcification using twenty five variables.

Results

Out of 1155 patient records reviewed, 725(63%) were in the warfarin group and 430(37%) were in the no warfarin group. MVC, MAC, or AVC was present in 473 of 725 patients (65%) on warfarin versus 225 of 430 patients (52%) not on warfarin (p<0.0001). Stepwise logistic regression analysis showed significant independent risk factors for any valve calcification were use of warfarin (Odds ratio 1.972, p-value< 0.001), age (Odds ratio 1.078 per one year increase, p-value <0.001), dyslipidemia (Odds ratio 0.235,p-value <0.001), calcium-phosphate product (Odds ratio 1.015, p-value 0.024), and coronary artery disease ( Odds ratio 13.563, p-value <0.0001).

Conclusion

Warfarin intake is associated with a significantly higher prevalence of MVC, MAC or AVC. Further studies are needed to confirm this association and the duration of warfarin intake for development of calcification. A pilot study has been started to study the effect of oral vitamin K on valvular calcification.

Disclosures

Off Label Use: Oral Vitamin K in preventing calcification.