bluuebunny/crossref_metadata_2025 huggingface dataset:
A MODEL to STUDY the Function of NPM-MLF1 IN MYELODYSPLASIA.
Authors: Lee Wen-hsin
Published: October 2019

Abstract 1571

Myelodysplastic syndromes (MDS) are bone marrow disorders characterized by ineffective haematopoiesis and peripheral cytopenia(s) with frequent evolution to acute myeloid leukemia (AML). Apoptosis is significantly deregulated in early MDS whereas advanced MDS is characterized by deregulation of DNA damage response. As MDS proceeds to AML, the ratio of apoptosis to proliferation decreases, resulting in clonal outgrowth of abnormal cells.

The t(3;5)(q25;q34) translocation, creating the NPM-MLF1 fusion, has been found as a sole cytogenetic abnormality in MDS. It is recurrent, with poor prognosis but the precise mechanism through which NPM-MLF1 induces malignant transformation remains unknown. We aimed to model this disease in vitro and in vivo by expressing NPM-MFL1 in mouse bone marrow hematopoietic progenitor cells (HPCs) and analyzing any changes in HPC self-renewal and response to DNA damage.

NPM-MLF1 did not impair haematopoiesis in vitro and in vivo. FLT3/ITD was frequently associated with NPM mutant in AML patients; however, NPM-MLF1 did not collaborate with FLT3/ITD in our system. To recapitulate NPM hemizygosity in t(3;5)-MDS patients, we have expressed NPM-MLF1 in HPCs derived from Npm+/− mice. A transient increase in the self-renewal of the NPM-MLF1-expressing Npm+/− HPCs was seen. These cells did not exhibit enhanced proliferation as confirmed by growth curve and analysis of DNA synthesis. Interestingly, unlike control cells, NPM-MLF1-expressing Npm+/− HPCs showed prolonged self-renewal ability in vitro, and an elevated expression of c-Myc, Hoxa9, Hoxa10 and Meis1 genes. In addition to altering HPC self-renewal, NPM-MLF1 was also found to modulate their DNA damage response. This study suggests that the ability of NPM-MLF1 to maintain HPC self-renewal and impaired DNA damage responses may favour the accumulation and outgrowth of the aberrant HPCs, contributing to the abnormal haematopoiesis.

Disclosures:

No relevant conflicts of interest to declare.
Activation of Dormant Hematopoietic Stem Cells In Vivo by the Endotoxin LPS.
Authors: Essers Marieke , Lutz Raphael , Kern Andrea , Wurzer Stephan , Trumpp Andreas
Published: October 2019

Abstract 1613

Maintenance of the blood system is dependent on dormant haematopoietic stem cells (HSCs), which are characterized by pluripotency and life long self-renewal capacity. In order to both maintain a supply of mature blood cells and not exhaust HSCs throughout the lifespan of the organism, most adult HSCs remain deeply quiescent during homeostasis and only a limited number are cycling at any given time. The balance between self-renewal and differentiation of HSCs is controlled by external factors such as chemokines and cytokines, as well as interactions of HSCs with its niche environment.

We have recently shown that the cytokine IFNa very efficiently activates dormant HSCs in vivo. Within hours after treatment of mice with IFNa, HSCs exit G0 and enter an active cell cycle. In general, IFNa is produced in response to viral infections by cells of the immune system, and plays an important role in the antiviral host defense. We now questioned whether endogenous IFNa is also produced in response to other types of bone marrow stress and whether this affects the proliferation rate of HSCs. To monitor IFNa production in the bone marrow in vivo, we have generated MxCre; ROSA-R26-EYFP mice and found that treatment with both the chemotherapeutic agent 5-FU as well as the endotoxin LPS leads to the production of IFNa in the vicinity of HSCs and progenitors. In addition, LPS treatment in vivo induced a strong increase in HSC cycling. Surprisingly, since mice lacking the IFNa receptor (Ifnar-/-) still respond to LPS this effect is independent of IFNAR signaling. LPS binds and signals via the TLR4-CD14 receptor complex and HSCs of mice lacking TLR4 (TLR4-/-) are no longer activated by LPS. Strikingly, LPS induced HSC activation correlated with increased expression of Sca-1, similar to what occurs upon IFNa treatment. Moreover, as for IFNa, the upregulation of Sca-1 is required for LPS induced proliferation, since Sca-1-/- mice fail to respond to LPS stimulation.

In summary, these data suggest that not only virus inducible IFNa, but also infections by gram negative bacteria produced LPS induces cycling of progenitors and otherwise dormant HSCs. Finally, both IFNa and LPS induced activation of HSCs are dependent on the up-regulation of Sca-1, suggesting a more general role for Sca-1 in the activation of dormant stem cells in response to injury signals.

Disclosures:

No relevant conflicts of interest to declare.
Ribosomal RNA Expression In CD34+ Hematopoietic Progenitor Cells Inversely Correlates with Ribosomal DNA Methylation In Myelodysplastic Syndromes.
Authors: Raval Aparna , Pollyea Daniel A , Shridhar Kunju J , Patel Shripa , Greenberg Peter L. , Mitchell Beverly S.
Published: October 2019

Abstract 1682

Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells characterized by ineffective hematopoiesis and, in the high risk setting, by progression to acute myelogenous leukemia (AML). The DNA hypomethylating agents, 5-azacytidine (5AC) and 5-aza-2’-deoxyazacytidine (DAC), are effective treatments for patients with MDS, increasing time to progression to AML and improving overall response rates and survival. Genome-wide DNA hypermethylation in MDS has been documented. However, promoter methylation signatures in MDS samples have not correlated with the clinical response to epigenetic drug therapies. Recently, attention has been drawn to the potential etiologic role of reduced expression of specific ribosomal proteins in del (5q) MDS, as well as in other bone marrow failure states. We therefore asked whether CD34+ cells from the bone marrow of MDS patients had reduced expression of ribosomal RNA (rRNA) and whether this correlates with hypermethylation of ribosomal DNA (rDNA). Using quantitative SYBR-Green RT-PCR, we found that rRNA expression was significantly reduced in CD34+ hematopoietic progenitor cells from 15/29 MDS patients as compared to normal controls (p value <0.01). We then studied quantitative rDNA methylation using pyrosequencing in DNA from CD34+ cells isolated from 5 normal controls and 6 MDS samples. Two of six MDS samples had reduced rRNA expression and increased DNA methylation, not only within the upstream core element but also in the intergenic rDNA sequences. For each of the CpGs evaluable within these highly CpG rich regions, the extent of DNA methylation in patients with reduced rRNA expression were increased by more than 2-fold compared to that in normal samples. This DNA hypermethylation was not observed in rDNA from 4/6 MDS samples where rRNA expression was not reduced in relation to controls. In addition, cells treated with DAC demonstrated a two-fold induction in rRNA gene expression and an up to four-fold reduction in rDNA promoter methylation as compared to controls, suggesting that promoter methylation may have a direct effect on rRNA expression. We are currently examining MDS patient samples before and after DAC treatment for levels of rDNA methylation and expression. It is possible that the methylation status at the rDNA locus may serve as a better predictor of response to hypomethylating agents than genome-wide methylation status. Regulation of rDNA gene expression is complex, but may be a fundamental underlying mechanism of bone marrow failure in a subgroup of MDS patients and perhaps in other bone marrow failure states.

Disclosures:

No relevant conflicts of interest to declare.
Clinical, Biological Profile and Outcome of Biphenotypic Acute Leukemia: a Case Series.
Authors: Zhang Yanming , Depei Wu , Sun Aining , Qiu Huiying , He Guangsheng , Jin Zhengming , Tang Xiaowen , Miao Miao , Fu Chengcheng , Han Yue , Zhu Mingqing , Xue Yongquan , Chen Zixing , Ruan Changgeng
Published: October 2019

Abstract 1683

Biphenotypic acute leukemia (BAL) is a very rare type of acute leukemia, which presents a high heterogeneity and poor prognosis. We identified 51 cases (3.0%) BAL from 1693 newly diagnosed acute leukemia patients according to the EGIL scoring system between January 2003 and July 2009. The biological features, treatment and outcome of 39 evaluable BAL patients were analyzed retrospectively. There were 23 (59.0%) cases of myeloid and B-lymphoid (M/B) phenotype, 14 (35.9%) cases of myeloid and T-lymphoid (M/T) phenotype, one case (2.6%) of trilineage phenotype or B-lymphoid and T-lymphoid phenotype respectively. The high expressions of CD34 (84.6%) and HLA-DR (54.5%) on the blast cells of BAL support the notion that BAL probably arises from hemopoietic stem/progenitor cells. It seemed that CD7-positive patients had poorer median survivals comparing with those CD7-negative patients, however, there was no statistical difference (P=0.076). Abnormal karyotypes were detected in 75.7% of 37 BAL patients with valid analysis and displayed a high heterogeneity, which were associated with structural rearrangement and numerical abnormalities including t(8;21) (16.2%), t(9;22) (13.5%), structural rearrangement of 11 chromosome (16.2%), 11q23 (5.4%), complex karyotype (21.6%) and other abnormal karyotypes (10.8%). Combined regimens for both AML and ALL, ALL-type regimens appeared a better complete remission (CR) rate than AML-type regimens (71.4% vs. 63.6% vs. 33.3%). The median survival for overall survival (OS) and disease-free survival (DFS) in our series was 14 and 12 months, the probability of OS and DFS at 2 years was 26.0% and 18.5%, respectively. No statistical differences were observed in CR rate, OS and DFS between M/B and M/T cases. It showed that BAL patients with complex karyotype or rearrangement of 11 chromosome had a significantly worse survival in contrast to normal karyotype, t(8;21) and t(9;22) group (P=0.001). Although BAL with t(8;21) seemed to be appeared a better survival than normal karyotype and t(9;22) group, there were no statistical significance (P=0.436). Our data indicate that combined-type regimens or ALL-based protocols are more effective and complex karyotype, rearrangement of 11 chromosome have the unfavorable prognosis for BAL.

Disclosures:

No relevant conflicts of interest to declare.
Is There a Role for Autologous Stem Cell Transplantation (Auto SCT) for Patients with Acute Myelogenous Leukaemia? A Retrospective Analysis.
Authors: Novitzky Nicolas , Thomas Valda , Toit Cecile du , McDonald Andrew
Published: October 2019

Abstract 1689

Introduction:

To better counsel our patients on the role of auto SCT for patients with AML we studied consecutive individuals in CR 1 who had tissue compatible siblings and underwent allogeneic (allo) SCT with subjects who had no HLA donor and actually underwent auto SCT.

Methods:

Patients were in CR 1 following induction combinations containing 7 days of cyatarabine and etoposide with 3 days daunorubicin followed by similar consolidation therapy. The choice for the type of graft was based on availability of HLA identical siblings. Allogeneic donors underwent PBPC mobilisation with filgrastim and for GvHD prophylaxis grafts were exposed ex vivo to alemtuzumab 1mg/1010 mononuclear cells. Patients were prescribed cyclosporin until day 90 post transplant. Individuals lacking a donor underwent PBPC mobilization with etoposide 2 gr/m2 and harvested PBPC were cryopreserved. Patients received similar myeloablative conditioning followed by infusion of the grafts. Patients were stratified by clinical and laboratory factors as well as cytogenetic risk. The end points were TRM, DFS and OS.

Results:

The median presentation age for both transplant groups was 35 (14-60) years. Of the 112 consecutive patients achieving remission 37 had HLA identical siblings, but 3 relapsed and donors became unavailable in 2. Thus, autologous or allogeneic grafts were actually transplanted to 43 and 32 patients respectively. There was no significant difference in the presentation clinical features, laboratory parameters, marrow morphology or proportion of low and intermediate cytogenetic risk for both transplant options. Treatment mortality as well as relapse rate was similar (14 and 15%; 39 and 27%, respectively). At a median of 1609 and 1819 post transplant days, 56% and 63% in each group survive. In univariate analysis performance status, cytogenetic risk, morphological features of dysplasia, blast count and LDH were significant factors for survival. While for the entire group there was no difference in survival between both modalities, all patients with unfavourable cytogenetics receiving an autologous graft died of disease recurrence (3 year survival 35% vs 0%; p= 0.05).

Conclusions:

We conclude that patients with AML who have low or intermediate cytogenetic risk undergoing myeloablative conditioning followed by autologous or allogeneic T-cell depleted stem cell transplantation appeared to have similar outcome. However, those with unfavourable karyotype are unlikely to be cured with autologous grafts and are candidates for experimental modalities.

Disclosures:

No relevant conflicts of interest to declare.
A Proteomic Platform for EBV and KSHV Serological Screening
Authors: Zheng Dasheng , Wang Leyao , Chiou C.-J. , Liao Gangling , Zhu Heng , Hayward Gary S , Ambinder Richard F. , Hayward S.D.
Published: October 2019

Abstract 1747

Individuals with HIV infection have an increased susceptibility to EBV- and KSHV-associated malignancies. Evaluating a possible role for specific humoral responses to individual viral proteins in the control of viral load and prevention of EBV or KSHV related disease development in this population has been hampered by the limited number of viral antigens currently used for screening serum samples. We have developed a protein array platform that can be utilized for EBV and KSHV serological screening. The array consists of EBV and KSHV proteins printed onto glass slides. All eighty four EBV open reading frames and eighty six KSHV open reading frames were cloned into bacterial vectors and the inserts validated by DNA sequencing in both directions. Authenticated inserts were transferred to a yeast vector that expresses proteins as N-terminal GST-fusions. Seventy nine EBV proteins and eighty one KSHV proteins were successfully purified from yeast. First generation EBV/KSHV protein arrays have been printed with these proteins plus a variety of control proteins that include GST, and human IgG, IgM and IgA. Initial serological assays compared IgG antibody responses in HIV positive individuals, cancer patients and healthy adults. The HIV positive group (i) had a broader EBV antigenic response than the cancer patients or healthy normal's and (ii) recognized a wider range of EBV antigens compared to KSHV. This may reflect more extensive exposure to replicating EBV versus KSHV in these patients. In addition to the previously described dominant antigenic responses to EBV and KSHV, the assays identified multiple additional immunogenic viral proteins. The ability to compare serological responses to the complete repertoire of individual EBV and KSHV encoded proteins should provide new insight into B cell mediated immune regulation of these viruses.

Disclosures:

No relevant conflicts of interest to declare.
Induction of Autophagic Cell Death Circumvents Azacitidine-Resistance In Myelodysplastic Syndrome-Derived Cell Lines
Authors: Cluzeau Thomas , Robert Guillaume , Karsenti Jean-Michel , Luciano Frederic , Puissant Alexandre , Raynaud S. Dominique , Mannone Lionel , Cassuto Jill-Patrice , Mounier Nicolas , Auberger Patrick
Published: October 2019

Abstract 1817

Azacitidine (AZA) is the first line treatment for IPSS (index prognostic scoring system) high-risk myelodysplastic syndrome (HR-MDS). To date, only Khan et al. (Exp Hematol. 2008) and Hollenbach et al. (PLoS One. 2009) have reported apoptosis as a mechanism of AZA effect on MDS cell lines. Nevertheless, approximately 40% of patients treated with AZA are refractory to this molecule. To investigate the possible mechanisms of AZA resistance in MDS cells, we developped AZA-resistant cell clones (AZA-R) from the well-characterized MDS cell line SKM1. The bulk resistant SKM1 cell line (AZA-R) was obtained following long time exposure of cells to iterative and increasing doses of AZA ranging from 0.1 to 8mM. We first showed that AZA triggered loss of cell metabolism in SKM1 parental cells but not in their AZA-resistant counterpart at a maximally effective dose of 1mM. AZA-mediated loss of cell metabolism accounted mainly for induction of apoptosis as judged by both an increase in caspase 9 and 3 activities triggered by this compound and by a significant protection in the presence of the pan-caspase inhibitor Z-VAD-fmk in SKM1 parental cells. Conversely, no or very few activation of caspases and apoptosis were detected in AZA-R cells strongly suggesting that apoptosis is impaired in AZA-R SKM1 cells. Finally, unlike in SKM1 cells, AZA failed to induce mitochondrial membrane permeabilization in AZA-R SKM1 cells. Importantly, basal autophagy was increased in AZA-R versus AZA-S cells as shown by LC3-I cleavage into LC3-II, p62/SQSTM1 protein expression, cathepsin B activation, mTOR and S6 ribosomal protein dephosphorylation and finally electronic microscopy experiments. In addition, Acadesine, an adenosine derivative and AMPK agonist which targets autophagy was capable to circumvent AZA resistance in both AZA-R SKM1 cells and in medullary cells from five MDS patients resistant to AZA after 6 cycles of Azacitidine. In conclusion, targeting autophagy appears as an attractive therapeutical strategy to circumvent AZA resistance in both established MDS cell lines and cells from MDS patients. Therefore, drugs capable of inducing autophagy or autophagic cell death, such as Acadesine (Robert et al., PLoS One. 2009) which is currently in phase II clinical trials for the treatment of Chronic Lymphoblastic Leukemia could be also beneficial for HR-MDS patients resistant to AZA.

Disclosures:

Cluzeau: Celgene: Consultancy. Raynaud:Celgene: Consultancy.
Hedgehog Pathway Inhibition Is a Novel Therapeutic Approach in Lymphomas and Its Efficacy is Inversely Related to PI3 Kinase Inhibition
Authors: Walsh Katherine J. , Fan Siyao , Patel Amee , Jacobs Cassandra L , Smith Jason , Liu Qingquan , Dave Sandeep
Published: October 2019

Abstract 1848

Background:

The hedgehog signaling pathway has been shown to be important in the development of malignancies, however, the role of this pathway in lymphomas is uncertain. GDC-0449 is a novel small molecule inhibitor that has been shown to block hedgehog activity by inhibiting SMO. The purpose of this study was to determine a potential role for hedgehog inhibition in the treatment strategies for lymphomas. We further sought to understand the determinants of sensitivity and potential resistance to therapeutic inhibition of this pathway.

Methods and Results:

We tested the small molecule inhibitor GDC-0449 in cell lines that span the spectrum of lymphoma including Burkitt lymphoma (N=4), ABC DLBCL (N=3), GCB DLBCL (N=7), mantle cell lymphoma (N=2), multiple myeloma (N=2), lymphoid leukemia (N=2), and T cell leukemia (N=2). Using the MTT assay, we identified the IC50 of drug response in these cell lines. We performed gene expression profiling in these cell lines and applied the Cox proportional hazards model using IC50 as the survival variable to identify genes that were associated with either sensitivity or resistance to the drug.

We found that the drug was lethal in these cell lines at physiologically achievable concentrations with IC50s ranging from 5 to 22μM. We applied hierarchical clustering to the genes associated with sensitivity and resistance and found that the method distinguished two groups of samples that differed in their average IC50s by over 2-fold (8μM vs 17μM). There was a suggestion of association with lymphoma cell type with Burkitt lymphoma lines being the dominant cell type in the resistant group and DLBCL being the dominant cell type in the sensitive group.

Resistance to hedgehog inhibition was found to be highly associated with the phosphatidylinositol 3′-kinase (PI3K) signaling pathway, suggesting that the hedgehog pathway and PI3K pathways act through independent, complementary mechanisms to promote tumor proliferation. In order to test the hypothesis that PI3K signaling is independent of hedgehog signaling we tested the lethality of PI3K inhibition using a dual PI3K/mTOR inhibitor (BEZ235) in the same cell lines.

We found an inverse relationship between the hedgehog inhibitor and PI3K inhibitor, i.e. the cell lines that were relatively sensitive to hedgehog inhibition (mean IC50 of 8μm) were resistant to PI3K inhibition(mean IC50 of 0.69μm) and the cell lines that were relatively resistant to hedgehog inhibition (17μm) were relatively sensitive to PI3K inhibition (0.32μm).

Conclusion:

Hedgehog pathway inhibition is a promising therapeutic approach in a carefully defined subset of patients with lymphoma. Hedgehog and PI3K inhibitors appear to act by distinct mechanisms and may be complementary in the treatment of lymphomas.

Disclosures:

No relevant conflicts of interest to declare.
Prediction of Relapse and Impact of Immunomodulation Based On Quantitative RT-PCR of Insertion/Deletion Polymorphism for Chimerism Analysis in Acute Leukemia
Authors: Jacque Nathalie , Nguyen Stephanie , Golmard Jean Louis , Garnier Alice , Uzunov Madalina , Leblond Veronique , Vernant Jean Paul , Bories Dominique , Dhedin Nathalie
Published: October 2019

Abstract 1966

Detection of increasing mixed chimerism (IMC) using standard PCR is correlated with relapse after allogeneic stem cell transplantation (allo-SCT) for acute lymphoblastic and myeloid leukemias (AL); however, it is not known whether using a very sensitive method such as quantitative real time PCR of insertion/deletion polymorphism (indel-qPCR) would be of benefit. Here, we studied whether this technique is useful for predicting relapse after transplant for AL, and whether an immunomodulation (IM) based on the detection of IMC would improve outcome. Eighty-nine transplants were included, with a median follow-up of 30 months for the survivors (range 4–86). The 3-year overall survival (OS) was 57.4% (95% CI: 48.1–66.7%). Twenty-one patients relapsed, leading to a 3-year incidence of relapse of 28.9%. An IMC was defined by an increasing of patient's DNA rate of 1 log when it was under 0.1% and an increasing of at least 0.1% beyond this threshold. Among the 57 transplants presented IMC, 21 relapsed, whereas none of the 32 patients with stable or decreasing chimerism relapsed. By multivariate analysis, the detection of IMC and the disease status at transplant were strongly associated with relapse (p=0.0002; HR: 27.6, 95%CI: 3–246 and p=0.0037; HR: 3.4 95%CI: 1.4–8.2, respectively). After the detection of one IMC (n=57), an IM was performed in 27 patients, consisted in withdrawal of immunosuppressive drugs for all, and followed by donor lymphocyte infusion (DLI) in 2. For these 27 patients, the 1 and 3-year relapse rate was 15.7%, versus 57.6% in the 30 other patients (p=0.0007). After IM, 2 patients developed acute graft versus host disease (GVHD) (grade 2 and grade 4) and died of infection. Both of them had received DLI. Four patients presented extensive chronic GVHD and two of them died. Altogether, the 3-year overall survival was 68.3% after IM versus 42.6% without any immunological intervention (p=0.02). These results suggest that chimerism analysis using indel-qPCR is a useful tool to early detect and prevent relapse after allo-SCT for AL.

Disclosures:

No relevant conflicts of interest to declare.
Autologous Stem Cell Transplantation with Yttriumm-90-Ibritumomab Tiuxetan (Zevalin) Plus BEAM Conditioning in Patients with Refractory Non-Hodgkin Diffuse Large B-Cell Lymphoma: Results of a Prospective, Multicenter, Phase II Clinical Trial
Authors: Briones Javier , Novelli Silvana , Marco Jose Antonio Garcia , Tomas Jose Francisco , Bernal Teresa , Grande Carlos , Canales Miguel , Torres Antonio , Moraleda José M. , Panizo Carlos , Palmero Fernanda , Jarque Isidro , Hernandez Miguel , Barca Eva Gonzalez , Lopez Dulce , Caballero Dolores
Published: October 2019

Abstract 1978

Background:

Autologous stem cell transplantation (ASCT) is the treatment of choice for patients with relapsed diffuse large B-cell lymphoma (DLBCL). However, in the rituximab era, patients with persistent disease receiving ASCT have a very poor outcome. Recent studies including radioimmunotherapy as part of the conditioning treatment suggest an improved outcome for this group of patients.

Methods:

We have evaluated, in a prospective phase 2 study, the safety and efficacy of yttriumm-90-ibritumomab tiuxetan (Zevalin) combined with standard BEAM in refractory DLBCL patients. Thirty patients with induction failure (primary refractory; n=18) or refractory to salvage immunochemotherapy at relapse (n=12) were included in the study. Patients' response was evaluated with PET-CT according to 2007 Cheson's revised response criteria.

Results:

Patients with a median age of 53 years (range, 25–67) received Zevalin at a fixed dose of 0.4mCi/kg (maximum dose 32 mCi) 14 days prior to standard BEAM. Histology included de novo DLBCL (22) and transformed DLBCL (8). Patients received a median of 3 (range, 2–6) prior therapies before ASCT. All patients had active disease at the time of ASCT, with 25 patients considered to be chemorefractory to the last treatment. Median CD34+ cell dose infused was 3.9 × 106/kg (range, 2–18.3). All patients engrafted. Median time to neutrophil recovery (>500/μl) was 11 days (9–21), and to platelet recovery (>20.000/μl) was 13 days (11–35). Overall response at day +100 was 70% (95% CI, 53.6–86.4) with 60% (95% CI, 42.5–77.5) complete responses. Eleven patients have died. One due to a cerebral hemorrhage before ASCT and 1 due to sepsis immediately post-transplant. Seven patients died of disease progression, and 2 patients died due to late complications: bacterial sepsis and secondary acute leukemia. One patient developed a myelodysplastic syndrome (refractory anemia with excess blasts-2) 33 months after TASP, while being in CR of its lymphoma. After a median follow-up of 22.7 months for alive patients (range, 12.2–39.0), 2-year overall and progression-free survival is 65% (95% CI, 47.8–82.8) and 63% (95% CI, 45.5–80.4), respectively.

Conclusion:

ASCT with conditioning including Zevalin radioimmunotheray plus BEAM is safe, and results in a very high response rate with promising survival in this very poor prognosis group of refractory DLBCL patients.

Disclosures:

No relevant conflicts of interest to declare.
Glycine Supplementation – A Novel Therapeutic Strategy for Congenital Sideroblastic Anemia.
Authors: Berman Jason N , Fernandez-Murray Pedro , Nasrallah Gheyath , Dufay Noelia , Fernandez Conrad V , Jarrar Ameer , Coombs Andrew J , McMaster Christopher
Published: October 2019

Abstract 2087

Congenital sideroblastic anemias (CSA) are inherited diseases, characterized by ineffective haematopoiesis, typically severe microcytic anemia and bone marrow sideroblasts representing excess iron deposition in the mitochondria of the erythroid precursors. More than 40% of CSA cases are attributed to mutations in the X-linked gene ALAS2. ALAS2 encodes the mitochondrial enzyme aminolevulinic acid synthase-2, which utilizes glycine to form 5-aminolevulinic acid (5-ALA), a crucial precursor in heme synthesis. Another gene, SLC25A38, has recently been implicated in the abnormal heme development noted in CSA. The function of the SLC25A38 protein product is uncertain, although it is thought to be an erythroid specific mitochondrial carrier family protein, transporting glycine across mitochondrial membranes. We employed yeast and zebrafish model systems in parallel to evaluate the absence of SLC25A38 or ALAS2 on heme synthesis in vivo and identify potential therapeutic strategies. HEM1 (ALAS2 homologue) mutant yeast were completely unable to make heme, whereas heme synthesis was significantly reduced in YDL119c (SLC25A38 homologue) mutant yeast. To monitor heme synthesis, we utilized a beta-galactosidase reporter linked to Pcyc1, which is only active following binding of the yeast Hap1 transcription activator in the presence of heme. Both HEM1 and YDL119c mutant yeast showed no beta-galactosidase activity, however activity in the YDL119c mutant was returned to 30% with the addition of 5-ALA and to 40% following treatment with glycine. Microarray studies of untreated and glycine treated YDL119c mutant yeast revealed increased expression of genes required to synthesize vitamin B6, a cofactor for the Hem1 enzyme in yeast and humans. Morpholino (MO)-mediated knockdown of the zebrafish homologues of SLC25A38 (slc25a38a and slc25a38b) or alas2 correlated with decreased hemoglobin levels by o-dianisidine staining and increased embryonic malformation and mortality. 5-ALA treatment either by addition to the egg water or by injection into the yolk failed to restore hemoglobinization in alas2 morphant embryos. By contrast, the addition of glycine to the egg water resulted in upregulation of hemoglobin to near normal levels in the majority of slc25a38a/b double morphant embryos. Our study demonstrates conserved heme synthesis pathways through evolution across species and further supports the contention that SLC25A38 functions as a glycine transporter. Most significantly, glycine supplementation emerged as an effective therapeutic strategy to restore heme synthesis in CSA caused by SLC25A38 deficiency, providing the rationale to support use of glycine in a clinical trial that is under development for these patients.

Disclosures:

McMaster: DeNovaMed: Equity Ownership.
Differential Targeting of Signaling Pathways to Selectively Expand Mouse Regulatory T Cells While Inhibiting Conventional T Cells.
Authors: Satake Atsushi , Schmidt Amanda M , Archambault Angela , Wu Gregory F , Kambayashi Taku
Published: October 2019

Abstract 2158

Regulatory T cells (Tregs) are suppressive T cells with therapeutic potential for ameliorating T cell-mediated diseases. Thus, there has been great interest in revealing the mechanisms by which Tregs proliferate. Recently, we reported that TCR signaling is partially dispensable for Treg proliferation in vivo when exogenous IL-2 is administered. Based on this data, we hypothesized that when given in conjunction with IL-2, pharmacological inhibition of TCR signaling might allow Tregs to expand while simultaneously inhibiting conventional T cell (Tconv) proliferation.

Using mutant mice with defective TCR-mediated PLCγ activation, we found that the activation of PLCγ is dispensable for IL-2-mediated Treg proliferation. In contrast, costimulation-derived mTOR signaling was required for IL-2-induced Treg proliferation. We next used Cyclosporine A (CSA; calcineurin inhibitor) and rapamycin (mTOR inhibitor) to differentially target these signaling pathways. Consistent with our hypothesis, while both CSA and rapamycin suppressed antigen-specific Tconv proliferation, only CSA permitted IL-2-induced Treg expansion in vitro and in vivo. Rapamycin, however, did increase the overall Treg:Tconv ratio due to its negative effects on Tconv survival.

Given that CSA inhibited antigen-specific Tconv proliferation while maintaining IL-2-induced Treg expansion, we hypothesized that the combination of CSA and IL-2 would be beneficial for attenuating T cell-mediated disease. Indeed, CSA synergized with IL-2 in protection against experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Surprisingly, however, the administration of CSA blocked whereas rapamycin augmented the beneficial effect of IL-2 in graft-versus-host disease (GVHD). These differences potentially results from the overt TCR stimulation that Tregs would receive in the allogeneic (GVHD) vs. syngeneic (EAE) environment. Moreover, inducible Treg (iTreg) generation from allogeneic MHC-stimulated naïve Tconvs contributes greatly to the Treg pool during GVHD. This was consistent with our data showing that rapamycin promotes iTreg generation and allows TCR-enhanced Treg proliferation, whereas CSA inhibited both of these processes. Thus, depending on the disease setting, the signaling pathways contributing to expansion of the Treg pool need to be carefully considered and specifically targeted to increase the Treg:Tconv ratio in treatment of T cell-mediated disorders.

Disclosures:

No relevant conflicts of interest to declare.
Dysregulation of Inflammatory and Hemostatic Markers in Sepsis Associated Disseminated Intravascular Coagulation.
Authors: Fareed Jawed , Hoppensteadt Debra , Cunanan Josephine , Mosier Michael , Osawa Yutaka , Kaul Inder
Published: October 2019

Abstract 2223

Disseminated intravascular coagulation (DIC) represents a complex pathophysiologic syndrome where marked alterations in the hemostatic system are manifested. As a result several inflammatory mediators are up regulated through multiple mechanisms. The up regulation of inflammatory mediators such as anaphylatoxin C5a (C5a), procalcitonin (PCT), interleukin 6 (IL-6), interleukin 10 (IL-10), myeloperoxidase (MPO), C reactive protein (CRP), and circulating levels of hemostatic markers including protein C inhibitor (PCI), plasminogen activator inhibitor 1 (PAI-1), and protein C (Pr C) were evaluated in 758 subjects enrolled in a randomized, double-blind, placebo-controlled, Phase-2B study evaluating the safety and efficacy of recombinant thrombomodulin (ART-123) in subjects with sepsis and suspected DIC. Thirty healthy male and female volunteers served as the control group. Commercially available ELISA methods were used to measure the various mediators. Marked deviations in the circulating levels of these markers, as compared to controls, were noted as shown in the following table. Compared with controls, subjects in DIC showed an increase in the circulating levels of most inflammatory markers. The levels of PCT, IL-6 and CRP, where considerably higher in the DIC subjects whereas PCI, Pr C and AT exhibited slight decreases. Wide individual variations were present. The PAI-1 levels were also increased in the DIC subjects. These results are tabulated below. These results clearly indicate that inflammation and impairment of fibrinolysis play a key role in the pathogenesis of DIC

Parameter Nomal (NHP Mean+SEM) DIC (Baseline Mean+SEM) % Change Protein C (% Ag) 82.5 ± 13.6 47.6 ± 23.7 −42.2% Functional Protein C (%) 83.4 ± 13.2 46.2 ± 29.8 −44.6% PCI (% Inhibition) 130.0 ± 24.6 79.4 ± 105.5 −38.9% PAI-1 (ng/ml) 35.4 ± 10.8 140.6 ± 165.6 297.1% CRP (ug/ml) 2.6 ± 0.4 48.0 ± 14.2 1736.9% C5a (ng/ml) 9.2 ± 3.2 17.2 ± 13.3 85.1% IL-6 (pg/ml) 9.3 ± 3.7 620.3 ± 1883.4 6583.9% IL-10 (pg/ml) 13.9 ± 13.1 130.2 ± 118.6 836.1% MPO (ng/ml) 16.0 ± 4.2 108.1 ± 68.6 574.6% PCT (ng/ml) 0.2 ± 0.13 21.9 ± 43.3 14514.5%

Disclosures:

Osawa: Asahi Kasei Pharma America Corporation: Employment. Kaul:Asahi Kasei Pharma America Corporation: Employment.
Protein Z Deficiency in Patients with Bleeding History. Is It a Coagulopathy?.
Authors: Bergmann Frauke , Czwalinna Andreas , Groening Arndt
Published: October 2019

Abstract 2227

Introduction:

Protein Z (PZ) is a vitamin K-dependent protein. In hemostasis PZ has two functions: together with Factor Xa it forms an inhibiting complex with the PZ- dependent protease-inhibitor and it enhances binding of Thrombin to phospholipid surfaces. Therefore, low PZ levels may induce thrombosis or bleeding.

The latter has been questioned by Vasse 2008 and in 1995 Kemkes-Matthes reported in 58% of patients (pts) with a bleeding history PZ deficiency as the only abnormality.

Aim of the study:

To evaluate if low PZ levels are associated with a bleeding tendency, we reviewed data of pts referred to or laboratory. PZ determination is part of our diagnostic work up, including whole blood count, PT, PTT, VWF/FVIII-complex, FXIII and platelet function studies.

Material and Methods:

Over a 1 year period we investigated PZ level in 173 pts. PZ concentration was determined by ELISA (Asserachrom Protein Z; Diagnostica Stago), a sandwich immunoassay using a mouse monoclonal antibody against PZ, normal range 1600 – 3300 μg/l, mean 2600 μg/l published by Miletich. Our normal range obtained by 62 donors 990–2490, mean 1740μg/l (+/− 2SD). PZ <1000μg/l is considered to be abnormal.

Results:

In 75/173 (43%) pts with a bleeding history (e.g. postoperative hemorrhage, epistaxis, menorrhagia) no coagulation abnormality was detected. In 41/98 (42%) low PZ level was the only abnormality (mean PZ 642, range 195–994). 57/98 (58%) were diagnosed with vWD n=17; platelet function disorders n=10 or ASA/drugs causing platelet dysfunction n=20, others n=10. In 21/57 (37%) low PZ level was detected additionally (mean PZ 699μg/l; range 219–949) and in the remaining 36/57 (mean PZ 1689; range 1036–2842).

Conclusion:

Surprisingly, in 24% of pts with a bleeding history the only abnormality was PZ deficiency. Therefore, we consider PZ determination a useful parameter in patients with a bleeding history after ruling out more common disorders. Low PZ levels may cause a coagulopathy in some pts.

Disclosures:

No relevant conflicts of interest to declare.
Risk Factors for Inpatient Acute Venous Thromboembolism (VTE) Despite Thromboprophylaxis.
Authors: Wang Tzu-Fei , Wong Catherine A , Milligan Paul , Thoelke Mark S , Woeltje Keith F , Gage Brian F
Published: October 2019

Abstract 2248

Background:

Venous thromboembolism (VTE) is the most common preventable cause of morbidity and mortality in the hospital. Adequate thromboprophylaxis has reduced the rate of hospital-acquired VTE by 50–70%; however, some inpatients still develop VTE despite thromboprophylaxis. Predictors associated with thromboprophylaxis failure are undefined.

Objectives:

We aimed to identify risk factors for VTE despite use of inpatient thromboprophylaxis.

Patients/Methods:

We used a case-control study to determine independent predictors of inpatient VTE. We matched 94 cases discharged from the BJC HealthCare system from January 1st, 2010 to May 31st, 2011who developed in-hospital VTE despite thromboprophylaxis (including either pharmacological or mechanical prophylaxis or both) to 272 controls without VTE. Matching was done by hospital, age, and date of discharge. Two thirds of the sample was used as derivation model, while one third was used for validation. We used multivariate conditional logistic regression on the derivation sample to develop a VTE prediction model and validated the model. We repeated conditional logistic regression on the pooled data.

Results:

Age and sex were well-matched. Mean age was 62.8 years-old in the VTE group and 63.2 years-old in the no-VTE group. Forty-three percent were female in the VTE group, while 49% were female in the no-VTE group. Using conditional logistic regression model in the pooled data, we identified five independent risk factors for in-hospital VTE: hospitalization for cranial surgery (Odds ratio [OR] 16.1, 95% confidence interval [CI] 3.2–80.4, p<0.001), hospitalization in a critical care unit (OR 3.0, 95% CI 1.5–5.9, p=0.002), admission leukocyte count > 13 × 103/mm3(OR 2.7, 95% CI 1.4–5.1, p=0.003), presence of an indwelling central venous catheter (OR 2.5, 95% CI 1.3–4.7, p=0.007), and admission from a long-term care facility (OR 2.1, 95% CI 1.0–4.2, p=0.04).

Conclusions:

In the hospital setting, we derived and validated five risk factors associated with the development of VTE despite thromboprophylaxis. Clinicians should be vigilant for VTE in these patients and ensure that they have aggressive VTE prophylaxis.

Disclosures:

No relevant conflicts of interest to declare.
Obesity Is a Risk Factor for Recurrent Venous Thrombosis in Young and Middle-Aged Women. Results From Tehs-Follow up, a Nested Cohort Study On 1394 Women with VTE.
Authors: Ljungqvist Maria , Sonnevi Kristina , Bergendal Annica , Holmstrom Margareta , Kieler Helle , Larfars Gerd
Published: October 2019

Abstract 2250

Introduction:

Venous thromboembolism (VTE) is a multifactorial disease with a high risk of recurrence. The risk of recurrence is highest during the first year with an incidence of 10–15%, thereafter recurrence occurs in 3 to 5 % per year. The risk of recurrence is associated with age, gender and whether the first VTE was provoked by a transient risk factor or not. Obesity increases the risk of a first event of VTE but its role for a recurrent event is unclear.

Methods:

We performed a nested cohort study and included 1394 women 18 to 64 years of age with a previous first episode of deep vein thrombosis (DVT) located in the leg or pelvis or with a pulmonary embolism (PE). All women had taken part in TEHS, a population based case-control study on risk factors for VTE. Information on risk factors was obtained by interviews and DNA-analyses immediately after the VTE and information of recurrent VTE was obtained from a follow-up questionnaire or from data recorded in the Swedish Patient Register. Only women who were not on continues anticoagulant treatment were included for assessment of recurrent VTE. Women with a BMI ≥ 30 were considered obese. Risks of recurrence were calculated in Cox regression models and are presented as hazard ratios (HRs) with 95% confidence intervals.

Results:

A total of 964 women (mean age 46 ± 13 years) with a median follow-up of 76 months accepted participation in the nested cohort study. The recurrence rate was 10.3% and 221 women (23%) were obese when diagnosed with their first VTE. At follow up 240 women (25%) were obese. The recurrence rate was higher in obese than in non-obese women (15.8% vs. 8.6%, p=0.002). The HR for recurrence was 1.9 (p=0.02, 95% CI 1.3–2.9) for obese women compared to non-obese. The HR was unchanged in a multivariate analysis, adjusting for age, index VTE (provoked or unprovoked by cast/surgery or hormonal treatment) and presence of factor V Leiden or prothrombin gene mutation.

Conclusion:

Obesity is a strong independent risk factor for recurrent VTE in women. Obese women with a first VTE might benefit from long time treatment with anticoagulants and consultation on weight reduction.

Disclosures:

No relevant conflicts of interest to declare.
Fondaparinux Versus Argatroban and Danaparoid for the Treatment of Suspected or Confirmed Heparin-Induced Thrombocytopenia: A Propensity Score Analysis.
Authors: Kang Matthew , Alahmadi Majed , Sawh Sonja , Kovacs Michael J. , Lazo-Langner Alejandro
Published: October 2019

Abstract 2262

Background:

Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy leading to platelet activation, thrombocytopenia and potentially thrombosis. Upon suspicion, current guidelines recommend heparin cessation and switching to a non-heparin anticoagulant such as argatroban or danaparoid. Fondaparinux could be potentially effective but information supporting its use is limited.

Methods:

We retrospectively evaluated 239 patients admitted to London Health Sciences Centre from January 10, 2004 to June 21, 2011 receiving a non-heparin anticoagulant (133 fondaparinux, 59 danaparoid, 47 argatroban) for suspected or confirmed HIT. Using logistic regression a propensity score was constructed based on age, gender, creatinine, 4T scores and comorbidities (assessed by the Charlson comorbidity index modified by Quan) and used to match 133 patients to 60 controls. Efficacy outcome was thrombosis or thrombosis-related death. Safety outcome was major bleeding. Group comparisons were done using χ2, Fisher's exact or Mann-Whitney tests. Survival analysis was conducted using the Kaplan-Meier method. Analyses were conducted in SPSS 20.0 and R 2.12.

Results:

Population characteristics are shown in Table 1. Well-balanced groups were obtained after matching. There were no differences in the proportion of thrombotic or bleeding events between control and fondaparinux groups (Table 2). Subgroup analyses comparing fondaparinux versus argatroban or danaparoid and unmatched analyses showed similar results. Sensitivity analysis stratified by propensity score quintiles showed no differences in either outcome. Survival analysis showed no differences in thrombosis or bleeding (Log-rank p = 0.415 and 0.779, respectively; Figure 1). Subgroup survival analysis for the 3 drugs independently found no difference in thrombotic events (Log-rank p=0.582) and a trend towards increased major bleeding in patients on argatroban (Log-rank p=0.068). In the fondaparinux group 60% of patients received prophylactic doses.

Conclusion:

Fondaparinux has similar effectiveness and safety as argatroban and danaparoid in patients with suspected HIT. Prophylactic fondaparinux doses seem to be effective if no indication for full anticoagulation exists.

Disclosures:

Off Label Use: Fondaparinux as a treatment option in heparin-induced thrombocytopenia. Lazo-Langner:Pfizer: Honoraria; LeoPharma: Honoraria.
The Characterization of Microparticle Phenotypes in Stored Red Blood Cell Units.
Authors: Tran Edward D , Wu Xiaoping , Blacken Grady R. , Lopez Jose A. , Dong Jing-fei , Fu Xiaoyun , Watkins Timothy R.
Published: October 2019

Abstract 2282

The transfusion of red blood cells (RBC) is associated with adverse outcomes in critically ill patients. One hypothesis explaining this observation involves the accumulation of potentially inflammatory/prothrombotic microparticles (MPs), released from cellular membranes during unit processing and storage. Despite their implication, the exact nature of MPs within RBC units remains incompletely defined. Here, we characterized the MP phenotypes present within stored RBC units and quantified the various populations. We directly sampled 11 O-positive non-leukoreduced RBC unit bags and isolated MPs by sequential centrifugation. We used a two-step process to detect MPs by flow cytometry. First, we used commercially available Megamix beads to define MPs according to their size during flow cytometric analysis and Trucount beads to quantify MP counts/mL (reported as median [IQR]). MPs ≤1 μm in size were subsequently classified into the following populations: total annexin V-positive, tissue factor-positive (CD142), and those staining for RBC- (intra/extracellular domain-specific glycophorin A, and band-3), leukocyte- (CD45), platelet- (CD42b), and endothelial-specific (CD144) markers. The total concentration of MPs was 1.52×109 MPs/mL [1.39×109]; however, only 4.3% were Annexin V-positive (5.16×107 MPs/mL [7.27×107]), and 11.7% were tissue factor-positive (4.24×107 MPs/mL [1.11×108]). Likewise, MPs with leukocyte-, platelet-, and endothelial-specific markers accounted for only 0.05% (4.44×105 MPs/mL [7.83×105]), 0.17% (1.99×106 MPs/mL [3.11×106]), and 0.06% (7.58×105 MPs/mL [8.04×105]), respectively (Figure 1). The amount of RBC-derived MPs varied depending on the antibody used. While only 4.2% of MPs (3.40×107 MPs/mL [2.91×107]) stained positive for the extracellular domain of glycophorin A, 41.3% (8.20×108 MPs/mL [4.06×108]) and 20.6% MPs (3.03×108 MPs/mL [3.53×108]) stained positive for the cytoplasmic domain of glycophorin A and band-3 respectively (Figure 1). The concentration of MPs positive for the cytoplasmic domain of glycophorin A was significantly greater their extracellular domain-specific counterparts (p<0.01). The results indicate that MPs in stored RBC units are primarily derived from RBCs, but also from other blood cells and vascular endothelial cells. A strong stain for the cytoplasmic domains of two RBC markers (cytoplasmic domain-specific glycophorin A and band 3) suggest the presence of a unique inside-out MP phenotype, potentially derived by RBC membrane “flipping” during microvesiculation. Ongoing studies are required to confirm these results and focus on 1) the mechanism of MP formation and (patho)physiological significance of inside-out RBC MPs and 2), the association between these MPs and the adverse effects seen in patients receiving RBC transfusions.

Disclosures:

No relevant conflicts of interest to declare.