bluuebunny/crossref_metadata_2025 huggingface dataset:
Gadd45 Null Mice and Neutrophils/Macrophages Derived From the Bone Marrow of These Mice Exhibit Impaired Innate-Immune/Inflammatory Reponses.
Authors: Salerno Dominic M , Hoffman Barbara , Liebermann Dan A.
Published: October 2019

Abstract 1350

Poster Board I-372

Stress sensor Gadd45 proteins modulate p38-NF-Kb and JNK signaling, which play major roles in leukocyte activation and innate immunity. We have previously documentedthat under conditions of hematological stress, notably acute stimulation with cytokines or inflammation, both gadd45a-deficient and gadd45b-deficient mice exhibited impaired inflammatory responses as indicated by lower percentages of Gr-1-positive cells in the BM and lower numbers of myeloid cells in peritoneal exudates (Gupta et. al Oncogene 25:5539-46, 2006). Recent evidence has implicated Gadd45 proteins in dendritic cell functions that influence effector Th1 responses to inflammation. However, whether gadd45 genes play a role in modulating the myeloid compartment, notably macrophage & granulocyte functions in response to inflammatory stress, remains largely unexplored. To this end, we have employed in vitro & in vivo models of inflammation using BM derived neutrophils and macrophages from WT, gadd45a and gadd45b null mice. The data obtained indicate that chemotaxis and transmigration to various chemo-attractants, including LPS and fMLP, as well as oxidative burst and phagocytic functions were impaired for both neutrophils and macrophages from mice lacking either gadd45a or gadd45b. Furthermore, upon stimulation with LPS, cytokine secretion, notably, but not exclusively IL-12 and TNFa, was significantly reduced in neutrophils and macrophages of gadd45a-/- and gadd455b-/- mice. Western Blot analysis of BM derived neutrophils lacking gadd45a and stimulated with LPS (500ng/mL) exhibited defects in p38 phosphorylation as compared to controls, suggesting a possible mechanism by which the innate response is impaired. P38 phosphorylation in gadd45b null granulocytes stimulated with LPS appeared comparable to what was observed in wt controls. This suggests that gadd45a and gadd45b utilize different signaling pathways to regulate innate-mmune/inflammtory responses. Interestingly, gadd45a, gadd45b & gadd45g null mice injected intraperitoneally with sublethal (25mg/kg body weight) doses of LPS were significantly more susceptible to septic shock compared to wt mice, as indicated by significantly increased morbidity through 5 days post LPS administration. Moreover, 18 hrs. post-injection, the spleens of KO mice were shown to have numerous apoptotic foci in the white pulp, confirmed to be tingible body macrophages ingesting dying cells by IH and IF for macrophage markers. These in vitro and in vivo data suggest a novel role for gadd45 family members in myeloid innate immune responses. Further elucidation of the signaling pathways involved is in progress and is expected to elucidate the molecular basis for the role Gadd45 proteins play in macrophage and granulocyte innate immune functions.

Disclosures

No relevant conflicts of interest to declare.
Nicotinamide Phosphoribosyltransferase (Nampt) Induces NAD+ / SIRT1 Mediated Deacetylation of FOXO3a in Myeloid Cells.
Authors: Lippka Yannick , Thakur Basant Kumar , Skokowa Julia , Welte Karl
Published: October 2019

Abstract 1352

Poster Board I-374

Severe congenital neutropenia (CN) is a heterogeneous disorder of hematopoiesis characterized by a maturation arrest of granulopoiesis at the level of promyelocytes with peripheral blood absolute neutrophil counts below 0.5 × 10/l. G-CSF treatment increases blood neutrophil numbers in more than 90% of individuals with CN. CN is also considered as a pre-leukemic syndrome, since ca. 20% of CN patients develop AML/MDS. Recently we found that NAMPT, a protein involved in biosynthesis of NAD+ was significantly increased in CN patients treated with G-CSF as compared to healthy individuals (Skokowa et al, Nature Medicine, 2009). Increased NAD+ levels correlated with the elevated levels of SIRT1, an enzyme involved in deacetylation of several histone and non -histone proteins (e.g. FOXO3a) by utilising NAD+. In search of the downstream factors regulated by G-CSF/ NAMPT /SIRT1 pathway, we found elevated levels of FOXO3a protein in CN patients treated with G-CSF compared to healthy controls or patients with other types of neutropenia. Therefore, we were interested whether NAMPT/ NAD+ -dependent activated SIRT1 affects FOXO3a levels. Indeed we observed that overexpression of NAMPT leads to the upregulation of FOXO3a protein in 293T cells and endogenous FOXO3a interacts with endogenous SIRT1 in both 293T cells and promyelocytic cell line NB4. The compound FK866 which specifically inhibits NAMPT has recently entered clinical trials as a potential chemotherapeutic agent. As acetylation of FOXO3a is considered to be important for its tumor suppressor function we asked if inhibition of NAMPT using FK866 increases the acetylation of FOXO3a. Indeed we show that acetylation level of FOXO3a is enhanced when 293T and NB4 cells were treated with FK866. Furthermore, increased acetylation of FOXO3a correlates with the decreased interaction between FOXO3a and SIRT1 in 293T cells after treatment with FK866. GADD45, a protein involved in DNA damage repair, is a well known target of FOXO3a. We found that in CD33+ myeloid progenitor cells from G-CSF treated CN patients with high NAMPT / SIRT1 / FOXO3a levels also had low GADD45 mRNA levels. We performed reporter gene assay using luciferase construct containing wild type GADD45 promoter and observed that SIRT1 enhances the FOXO3a mediated downregulation of GADD45 promoter and interestingly NAD+ further augments this effect. Taken together we hypothesized that NAMPT/NAD+ activated SIRT1 mediates deacetylation and activation of FOXO3a protein, leading to downregulation of target genes (e.g. GADD45) with tumor suppression functions which might possibly be involved in the leukemic transformation in CN patients.

Disclosures

No relevant conflicts of interest to declare.
PET Scan Results of NCCTG N0489: Epratuzumab and Rituximab in Combination with Cyclophosphamide, Doxorubicin, Vincristine and Prednisone Chemotherapy (ER-CHOP) in Patients with Previously Untreated Diffuse Large B-Cell Lymphoma.
Authors: Micallef Ivana N. , Maurer Matthew J. , Witzig Thomas E. , Nikcevich Daniel , Kurtin Paul , Cannon Michael W. , Flynn Patrick J. , Soori Gamini S. , Lowe Val , Wiseman Gregory
Published: October 2019

Abstract 137

Background:

FDG-PET/CT after 2–4 cycles of chemotherapy has been shown to be predictive of outcome in patients with aggressive lymphoma. This multicenter NCCTG phase II study was carried out to assess efficacy of ER-CHOP in newly diagnosed DLBCL; functional response as measured by FDG-PET/CT was done after 2 cycles and after 6 cycles of therapy.

Methods:

Patients received immunochemotherapy on the following schedule: epratuzumab 360 mg/m2, rituximab 375 mg/m2, and standard dose CHOP every 3 weeks for 6 cycles. FDG-PET/CT was done after 2 cycles and again after 6 cycles of therapy. All PET scans were centrally reviewed by an expert in nuclear medicine who was blinded to the outcome of the patients.

Results:

107 patients were accrued from Feb 2006 to Aug 2007. 27 patients were ineligible resulting in 80 eligible patients. Baseline patient characteristics for the eligible patients included median age 60 (range 21–82); 56% were male. 80% had advanced stage; 73% had an elevated LDH. Performance score was 0–1 in 71 pts and 2–3 in 9 pts. IPI was 0–1 in 18 pts (23%), 2 in 22 pts (28%), 3 in 29 pts (36%) and 4–5 in 11 pts (14%). PET scans were available on 76 patients (95%); 3 patients completed treatment but scans were unavailable and 1 died prior to cycle 2 PET. The PET CR rate after 2 cycles was 79%. The PET CR rate after 6 cycles was 90%. Using an intent to treat (ITT) analysis, 67 patients (87%) achieved PET CR of which 61 (79%) also completed all 6 cycles of treatment. Based on interim PET after 2 cycles, the EFS at 24 months (EFS24) was 73% for the PET negative patients vs 60% for the PET positive patients (p=0.25); OS at 24 months (OS24) was 83% vs 73% for the PET negative and positive pts, respectively (p=0.17). In comparison, EFS24 for patients with a negative PET after 6 cycles was 80% vs 57% for those with a positive PET (p=0.11); OS24 was 92% vs 57%, respectively (p=0.01). In intent to treat (ITT) analysis, patients that achieved a functional CR had improved overall survival (OS24 = 84%) and event free survival (EFS24 = 74%) compared to patients that failed to achieve a functional CR (OS24 = 50%, EFS24 = 40%, p=0.01 and p=0.006 respectively).

Conclusions:

ER-CHOP every 21 days × 6 cycles results in a functional CR rate of 87%. Early PET scan during therapy does not significantly predict outcome. Achievement of PET negativity by completion of therapy is associated with a good outcome

Disclosures:

No relevant conflicts of interest to declare.
In-Hospital Mortality and Trends Associated with Splenectomy in Patients with Immune-Mediated Thrombocytopenia (ITP).
Authors: Jaglowski Samantha M. , Byrd John C. , Jones Jeffrey A.
Published: October 2019

Abstract 1398

Poster Board I-420

Background:

Splenectomy remains a standard treatment for ITP patients not responding to medical management, but anecdotal reports suggest that use of the procedure is in decline. We studied patterns of use and outcome of splenectomy performed for ITP at the population level.

Methods:

Using data from the Nationwide Inpatient Sample and ICD-9 diagnosis and procedure codes, we identified 39,543 splenectomies among hospital admissions including a diagnosis of ITP (ICD-9 287.3) from 1993-2005. Admissions were characterized by patient and hospital facility characteristics. Laparascopic procedures were identified by published procedure coding algorithms. Factors influencing in-hospital mortality for 2005 were further evaluated using multivariate logistic regression models.

Results:

Annual estimates for incidence of splenectomy are displayed in Figure 1. Between 1993 and 2005, there was a decrease in the total number of splenectomies performed for ITP, with the most significant drop occurring from 1997 to 2000, concurrent with the FDA approval of rituximab. Over the same period, there has been an increase in the proportion of splenectomies performed laparoscopically from 3.4% to 18.6%. Patient gender, age, presence of comorbid malignancy, and Charlson score were not significantly associated with type of splenectomy procedure. Among facility factors, only hospital teaching status was a statistically significant predictor of laparoscopic splenectomy use, early but not later in the observation period. On an annual basis, in-hospital mortality did not vary significantly over the observation period, with risks ranging from 1.5% (95% CI 0.83-2.86%) in 1993 to 4% (95% CI 2.8%-5.7%) in 1997. Annual mortality risk between open and laparoscopic procedures likewise did not significantly differ. However, over the total 13-year observation period there was a >60% increased risk of death with an open versus laparoscopic procedure (OR 1.669, p<0.0001). In 2005, 2869 splenectomy procedures were performed. Multivariate logistic regression models for in-hospital mortality that year found that presence of a malignancy (OR 9.65, p=0.003) significantly increased mortality risk. Charlson comorbidity approached statistical significance (0 v. ≥1, OR 6.83, p=0.087). Hospital bed-size (OR 0.87, p=0.73), location (rural v. urban, OR 3.80, p=0.127), and teaching status (OR 0.39, p=0.203) were not significantly associated with outcome.

Conclusions:

While the overall mortality risk from splenectomy in ITP is low, it is influenced by the presence of malignancy and other comorbid conditions. Further studies designed to evaluate newer medical management strategies (e.g. rituximab, thrombopoeitin mimetics, etc.) versus surgical intervention in these higher-risk populations are warranted.

Disclosures:

No relevant conflicts of interest to declare.
A Multifaceted Strategy to Reduce Inappropriate Use of Fresh Frozen Plasma Transfusions in the Intensive Care Unit.
Authors: Arnold Donald M. , Whittingham Heather , Lauzier Francois , Clarke France , McDonald Ellen , Tkaczyk Andrea , Greiter Angela , Waugh Lily , Crowther Mark A. , Cook Deborah
Published: October 2019

Abstract 1412

Poster Board I-435

Introduction:

The overuse of fresh frozen plasma (FFP) transfusions has been well documented, especially among critically ill patients. In a mixed medical surgical intensive care unit (ICU), we documented that 43% of FFP transfusions were given for indications other than those proposed in published guidelines (Lauzier 2007).

Methods:

We developed a 3-Phase multifaceted behavior-change strategy to curtail inappropriate FFP transfusions, documenting all patients who had FFP, excluding plasmapheresis. Phase I was a 3-month baseline assessment period with no intervention, in which the FFP transfusion orders prescribed at the discretion of the ICU team were recorded. Phase II was a 3-month intervention targeted to all ICU clinicians, comprised of education on the appropriate use of FFP transfusions, audit and feedback of performance indicators, and a pre-order FFP Request Form to specify the indication and the pre-transfusion INR. Phase III was a 9-month assessment period incorporating only the FFP Request Form. At the end of the study, the indications for all transfusions were adjudicated independently in triplicate by 2 ICU clinicians and 1 hematologist, to determine whether each FFP transfusion was a) consistent with published guidelines, b) inconsistent with guidelines but appropriate for the ICU context, or c) inconsistent and inappropriate. Discrepancies were resolved in all cases. FFP orders were not withheld if FFP Request Forms were not completed.

Results:

Chance-corrected agreement (which considers clustered transfusions within patients) between ICU reviewers on whether transfusions were consistent or appropriate versus inconsistent and inappropriate was high (phi = 0.80). During Phase I (3 months), 66 FFP transfusions were administered (n= 26 patients), of which 30 were for bleeding. During Phase II (3 months), 24 transfusions were administered (n = 11 patients), of which 11 were for bleeding. During Phase III (7 months of data), 96 transfusions were given (n= 41 patients), of which 57 were for bleeding. Rates of FFP transfusions per month for all indications and for bleeding indications were 22 and 10, respectfully in Phase I; 8 and 4 in Phase II; and 14 and 8 in Phase III. A FFP Request form accompanied 39 (40.6%) of 96 FFP transfusions in Phase III.

Conclusions:

A multifaceted behavior-change strategy appears to be an effective method of changing inappropriate FFP transfusion practices; however satisfactory uptake of a pre-transfusion FFP Request Form requires consistent reminders. We recommend that transfusion guidelines are improved to explicitly incorporate FFP transfusion criteria appropriate for the ICU setting.

Disclosures:

No relevant conflicts of interest to declare.
Prognostic Significance of Circulating Epstein-Barr Virus (EBV) DNA in Non-Hodgkin's Lymphoma.
Authors: Kang Myoung Joo , Cho Yoo Jin , Ahn Heui June , Kim Shin , Lee Dae Ho , Kim Sang We , Chi Hyun-Sook , Suh Cheolwon
Published: October 2019

Abstract 1936

Poster Board I-959

Introduction:

Epstein-Barr virus (EBV) is well-known as an etiologic agent of lymphoproliferative disorders. Almost all types of sporadic non-Hodgkin's lymphoma (NHL) of both B-cell and T-cell origins have been demonstrated to have EBV-infected tumor cells. However, the prognostic significance of EBV viral load has not been established yet. Methods: To monitor quantitative EBV DNA load, real time quantitative PCR of EBV BZLF1 DNA was done using RealArt® EBV LC PCR reagents (Artus) from whole blood samples drawn from newly diagnosed NHL patients at the time of diagnosis and during the follow-up. Results: Between July 2006 and March 2009, a total of 233 patients (including 120 [51.5%] diffuse large B-cell lymphoma [DLBCL]; 18 [7.7%] natural killer (NK)/T-cell lymphoma; and 11 [4.7%] peripheral T-cell lymphoma, unspecified) were reviewed and analyzed. Among them, 33 (14%) patients, had a detectable EBV DNA (positive group) while 200 (85%) patients did not (negative group). Those in positive group had a more advanced disease and a higher LDH level than those in negative group (P=0.003 and 0.007, respectively). With a median follow up of 11.3 months (range 1.2-34.9), 14 (42.4%) of the 33 patients in positive group had a progressive or relapsed disease while 30 (15%) of the 200 patients in negative group did (P<0.001). One-year overall survival (OS) rate was 72.5% in positive group while that was 82.9% in negative group (log-rank P=0.001). Interestingly, conversion to negative EBV DNA during the follow-up was associated with a lower probability of progressive or relapsed disease (P=0.004). NK/T-cell lymphoma patients having an EBV DNA survived shorter than those having not. However, in DLBCL patients, there was no difference in survival between those having an EBV DNA and those not. Conclusions: The presence of EBV DNA at diagnosis and the negative conversion of EBV DNA during the follow-up might have prognostic significance in patients with NHL.

Disclosures:

No relevant conflicts of interest to declare.
Activation of T- and NK- Cells through CD19xCD3 and CD19xCD16A Bispecific TandAb Antibodies in Patients with B-Cell Non-Hodgkin's Lymphoma.
Authors: Poertner Lisa , Uhrberg Markus , Schoenberg Kathrin , Bruennert Daniela , Reusch Uwe , Little Melvin , Haas Rainer , Neumann Frank
Published: October 2019

Abstract 1950

Poster Board I-973

Purpose:

Bispecific, tetravalent.antibodies (TandAbs) directed against the B-cell-surface marker CD19 and activating receptors on T or NK cells (CD19xCD3 or CD19xCD16b) have shown promising effects in vitro and in preclinical studies. The present study examines their effects on the cytotoxic responses of viable T and NK cells isolated from patients with B-cell Non Hodgkin Lymphoma (NHL).

Methods:

Peripheral Blood MNCs were obtained from 30 patients with B-NHL (High grade=6, low grade=24) after successful prior therapy and from 17 healthy donors (HD) and were enriched for NK cells by immunomagnetic separation with CD56-microbeads, the other one was enriched for T cells. After overnight stimulation (RPMI-1640 with 10% FCS, 1% Pen/Strep and human recombinant IL-2 (Novartis) (NK: 1000U/ml; T: 100U/ml) cells were stained with CD107a, a surface marker indicating cytotoxicity. Cells were exposed to K562 alone, to Raji cells alone and finally to the TandAbs CD19xCD3 (T cells) or CD19xCD16A (NK cells) (conc. 1μg/ml). Using FACS analysis we determined the percent fraction of activated CD3 or CD56 cells being positive for CD107a.

Results:

To look for functional NK cells and T cells of HD and NHL patients first we exposed the cells to the K562 cell line. Both groups, HD and NHL patients expressed CD107a similarly (NK: 27.7±12.0% vs. 28.5±14.6%; T: 9.75±7.1% vs. 8.1±5.7%), indicating functional NK and T cells. To observe if NK or T cells would upregulate CD107a expression when exposed to lymphoma cells alone we performed experiments with Raji cells and measured the CD107a response. NK cells of both, patients and HD responded with similar levels of CD107a expression. T-cells of patients showed more cytotoxic activity towards Raji cells than observed in HD (3.1±8.4% vs. 0.1±0.2%). Next we added the CD19xCD3 or the CD19xCD16 TandAb to T/NK cells in the presence of Raji cells. In the patients′ NK-cell-population the fraction of activated cells increased from 10.7% (Raji cells alone) to 30.7±19.2% when TandAb was added. The mean reaction in the group of HD was increased from 9.2% up to 27.7%. CD107a expression of T cells from NHL patients increased from 3.1% when exposed to Raji alone up to 21.3±14.3% when TandAb was added which was comparable to the results of HD (19.6%±11.4%).

Conclusion:

Patients with NHL have functional intact effector cells which show cytotoxic activity in response to TandAbs in presence of lymphoma cells similar to that of HD. These TandAb antibodies are therefore suitable for patients with B-cell NHL as potential maintenance therapy.

Disclosures:

Reusch: Affimed: Employment, Patents & Royalties. Little:Affimed: Employment, Equity Ownership, Patents & Royalties.
Unraveling STAT Signaling in NPM/ALK-Induced Lymphoma: STAT3 but Not STAT5 Is Required for Lymphomagenesis in a Bone Marrow Transplantation Model Using Conditional Knockout Mice.
Authors: Dechow Tobias , Amon Elisa , Miething Cornelius , Grundler Rebekka , Schatz Nicole , Kremer Marcus , Reindl Wolfgang , Ringshausen Ingo , Peschel Christian , Duyster Justus
Published: October 2019

Abstract 1969

Poster Board I-992

Introduction:

NPM/ALK is a constitutively activated, oncogenic kinase leading to the development of anaplastic large cell lymphoma in humans. Multiple pathways have been implicated in NPM/ALK-dependent signaling including STAT3 and STAT5. In a transgenic mouse model it could be demonstrated that STAT3 is critical for NPM/ALK-driven lymphomagenesis. However, deletion of STAT3 did not lead to prolonged survival of the mice. In addition, the role of STAT5 in NPM/ALK-positive lymphoma in vivo is largely unclear.

Methods:

We used the established murine bone marrow (BM) transplantation model using BM derived from STAT3 and STAT5 conditional knock-out (fl/fl) mice. Precisely, we harvested BM from STAT3fl/fl and STAT5fl/fl mice and expressed NPM/ALK-Cre/EGFP in the BM cells by using retroviral gene transfer. BM cells were transplanted in lethally irradiated wild-type mice. Expression of NPM/ALK and deletion of STAT3 and STAT5 was determined in BM cells.

Results:

BM from STAT3fl/fl and STAT5fl/fl mice was successfully harvested, retrovirally infected, and transplanted in recipient mice. Analysis of BM cells revealed expression of NPM/ALK and deletion of STAT3 or STAT5 in NPM/ALK-Cre/EGFP-infected STAT3fl/fl and STAT5fl/fl BM respectively. Within the STAT3 group, control mice transplanted with NPM/ALK-EGFP-infected STAT3fl/fl BM died after 2-3 weeks from a NPM/ALK-positive lymphoma. An additional control group transplanted with NPM/ALK-Cre/EGFP-transduced wild-type BM also died from a NPM/ALK lymphoma but with an increased latency. Notably, mice transplanted with NPM/ALK-Cre/EGFP-infected STAT3fl/fl (STAT3-deleted) BM showed a significantly prolonged survival compared to either control groups including 5 mice that survived at least 300 days after transplantation. Interestingly, only about 60% of these mice died from an NPM/ALK-positive high-grade lymphoma or myeloma-like disease, whereas the remaining animals died from other (irradiation-induced) diseases. In contrast, 150 days post transplantation STAT5 deletion in NPM/ALK expressing BM cells did not increase survival of the transplanted mice compared to the control group with wild-type BM using NPM/ALK-Cre/EGFP retrovirus.

Conclusion:

Specific deletion of STAT3 but not STAT5 in NPM/ALK-expressing cells markedly attenuates disease progression. The described transplantation model using BM from conditional knock-out mice and an Cre-containing expression vector is extremely helpful to specifically study signaling in oncogene-driven hematological malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Abnormal Distribution of Erythroid Progenitors Populations in Mice Lacking p45 NF-E2.
Authors: Gasiorek Jadwiga , Chevillard Gregory , Nouhi Zaynab , Blank Volker
Published: October 2019

Abstract 1988

Poster Board I-1010

The NF-E2 transcription factor is a heterodimer composed of a large hematopoietic-specific subunit called p45 and widely expressed 18 to 20-kDa small Maf subunits. In MEL (mouse erythroleukemia) cells, a model of erythroid differentiatin, the absence of p45 is inhibiting chemically induced differentiation, including induction of globin genes. In vivo, p45 knockout mice were reported to show splenomegaly, severe thrompocytopenia and mild erythroid abnormalities. Most of the mice die shortly after birth due to haemorrhages. The animals that survive display increased bone, especially in bony sites of hematopoiesis. We confirmed that femurs of p45 deficient mice are filled with bone, thus limiting the space for cells. Hence, we observed a decrease in the number of hematopoietic cells in the bone marrow of 3 months old mice. In order to analyze erythroid progenitor populations we performed flow cytometry using the markers Ter119 and CD71. We found that p45 deficient mice have an increased proportion of early erythroid progenitors (proerythroblasts) and a decreased proportion of late stage differentiated red blood cells (orthochromatic erythroblasts and reticulocytes) in the spleen, when compared to wild-type mice. We showed that the liver of p45 knockout adult mice is also becoming a site of red blood cell production. The use of secondary sites, such as the spleen and liver, suggests stress erythropoiesis, likely compensating for the decreased production of red blood cells in bone marrow. In accordance with those observations, we observed about 2 fold increased levels of erythropoietin in the serum of p45 knockout mice.Overall, our data suggest that p45 NF-E2 is required for proper functioning of the erythroid compartment in vivo.

Disclosures:

No relevant conflicts of interest to declare.
Investigation of the Role of Heme Oxygenase 1 During Erythroid Differentiation.
Authors: Santos Daniel Garcia dos , Eisenberg Jesse , Schranzhofer Matthias , Chies Jose Artur Bogo , Ponka Prem
Published: October 2019

Abstract 1997

Poster Board I-1019

Heme is a complex of iron with protoporphyrin IX that is essential for the function of all aerobic cells. However, if left unguarded, non-protein-bound heme promotes free radical formation, resulting in cell damage and tissue injury. The highest amounts of organismal heme (75-80%) are present in circulating red blood cells (RBC) whose precursors synthesize heme with rates that are at least 1 order of magnitude higher than those in the liver (on the per cell basis), which is the second most active heme producer in the body. The only physiological mechanism of heme degradation is by heme oxygenases (HO1 and HO2) that catalyze the rate-limiting step in the oxidative degradation of heme and are, therefore, involved in the control of cellular heme levels. Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Although the heme-inducible HO isoform, HO1, has been extensively studied in hepatocytes and many other non-erythroid cells, virtually nothing is known about the expression of HO1 in developing RBC. Similarly, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. In this study we have shown that HO1 protein is expressed in uninduced murine erythroleukemic (MEL) cells and that its levels, somewhat surprisingly, do not decrease during DMSO-induced erythroid differentiation. Moreover, we demonstrated that heme significantly induces HO1 in both uninduced and induced MEL cells. Additionally, we investigated the effect of sodium arsenite (NaAsO2), HO1 inducer, on heme and iron metabolism in MEL cells induced to erythroid differentiation. MEL cells treated with NaAsO2 displayed a significant reduction in globin expression and increased ferritin levels. Moreover, NaAsO2treatment decreased levels of transferrin receptor in cell membranes. These effects triggered by NaAsO2 could be prevented by the addiction of tin-protophorphyrin (SnPP), HO1 activity inhibitor. Using a siRNA specifically targeting HO1, we observed an increase in globin expression together with a small decrease in the expressin of ferritin in DMSO-induced MEL cells. These results suggest that an as yet unknown mechanism exists to protect heme against endogenous HO1 action during erythroid differentiation. In summary, our results showing that NaAsO2-induced HO1 in erythroid cells cause a defect in erythroid differentiation suggest that HO1 could play a role in some pathophysiological conditions such as thalassemias.

Disclosures:

No relevant conflicts of interest to declare.
Myeloid Colony-Stimulating Factors Do Not Increase the Risk of Mortality or Disease Activity Measures in Patients with Acute Myeloid Leukemia: Systematic Review and Meta-Analysis.
Authors: Gurion Ronit , Belnik-Plitman Yulia , Gafter-Gvili Anat , Paul Mical , Vidal Liat , Ben-Bassat Isaac , Shpilberg Ofer , Raanani Pia
Published: October 2019

Abstract 2045

Poster Board II-22

Myeloid colony-stimulating factors (M-CSFs) are recommended as primary prophylaxis for the prevention of febrile neutropenia (FN) in patients who are at high risk for developing it. Since acute myeloid leukemia (AML) cells express functional growth factor receptors on their surface, concerns have been raised regarding the effect of M-CSFs on these cells. We assessed the safety of M-CSFs in AML patients. Systematic review and meta-analysis of all randomized controlled trials that compared the addition of M-CSFs during and following chemotherapy to chemotherapy alone in patients with AML was conducted. Trials evaluating the role of M-CSFs administered for the purpose of stem cell collection and/or priming e.g., before and/ or only for the duration of chemotherapy, were excluded. Both patients with and without neutropenia and/ or fever on admission were included. Two reviewers appraised the quality of trials and extracted data independently. The primary outcome was all-cause mortality at defined points in time. Secondary outcomes included complete remission (CR), disease free survival (DFS), relapse and infection rates. Relative risks (RR) with 95% confidence intervals (CIs) were estimated and pooled. The search yielded 17 trials including 4800 patients (range 53-803 per trial). 14 trials included patients undergoing induction chemotherapy, two trials included patients undergoing consolidation and one trial included patients undergoing salvage chemotherapy. The addition of M-CSFs to chemotherapy yielded no difference in all-cause mortality at 30 days, 1 year and at the end of follow-up [RR 1.00 (95% CI.0.82-1.22), RR 1.07 (95% CI 0.97-1.19) and RR 1.02 (95% CI 0.98-1.05), respectively] (Fig.1). There was no difference with regard to CR [RR 1.03 (95% CI 0.98-1.07)], relapse [RR 0.99 (95% CI 0.91-1.08)] and DFS [HR 1.00 (95% CI 0.90-1.13)]. M-CSFs did not decrease the occurrence of bacteremias, RR 0.96 (95% CI 0.82-1.12) or invasive fungal infections, RR 1.40 (95% CI 0.90-2.19). The addition of M-CSFs to chemotherapy does not affect all-cause mortality and leukemia associated clinical end points, e.g. CR, relapse rate and DFS. Hence, the use of M-CSFs for prevention of FN in patients with AML is safe. However, since prevention of neutropenia does not result in improved survival, they should not be used on a routine basis. Their use in the individual patient can be considered according to the clinical situation.

Fig. 1: All Cause Mortality Fig. 1:. All Cause Mortality

Disclosures:

No relevant conflicts of interest to declare.
Lack of Inhibition of AKT Predicts Clinical Resistance to Therapy with Imatinib + Reinduction Chemotherapy in Relapsed/Refractory c-Kit+ Acute Myeloid Leukemia.
Authors: Brandwein Joseph , Hedley David , Chow Sue , Schimmer Aaron D. , Yee Karen WL , Schuh Andre , Gupta Vikas , Minden Mark D.
Published: October 2019

Abstract 2073

Poster Board II-50

Patients age 18-65 with acute myeloid leukemia (AML) who had persistent leukemia after 3+7 induction with cytarabine and daunorubicin, or relapsed within 24 months of achieving CR, and whose blasts were c-kit (CD117) positive, were enrolled in a phase I/II clinical trial with imatinib on Days 1-10 combined with mitoxantrone 10 mg/m2/day IV Days 4-8, etoposide 100 mg/m2/day IV Days 4-8 and cytarabine 1.5 g/m2 IV q12h × 4 doses on Days 9-10. Imatinib was escalated through 3 dose levels (200, 300, 400 mg daily) in successive 6 patient cohorts. The combination was well-tolerated up to 400 mg/day imatinib, and an additional 15 patients were enrolled at this dose level. Of the 21 patients treated at the 400 mg imatinib dose, 13 (62 %) achieved CR, 7 (33 %) were non-responders, and one died during reinduction. The CR rate was 82% (9/11) among patients with standard risk AML karyotpye, compared to 33% (3/9) for patients with adverse risk karyotype. The CR rate for primary 3+7 non-responders was 6/14 (43%) vs. 7/7 (100%) for relapsed patients. For 14 patients on the Phase II portion of the study, AML blasts from peripheral blood were assayed for pAKT and pERK by flow cytometry, after stimulation with stem cell factor, on Days 1 and 4, pre- and 1 and 2 hours post-imatinib dosing on each day. A heterogeneous response pattern was seen, with some samples exhibiting marked inhibition of pAKT and/or pERK after imatinib therapy, while others demonstrated little change in levels. Of 8 patients achieving CR with re-induction, 6 demonstrated marked (>50%) pAKT inhibition with imatinib therapy, 1 showed no inhibition, while 1 showed intermediate levels of inhibition. In contrast, all 6 non-responders to re-induction demonstrated a lack of pAKT inhibition with imatinib therapy (p<0.01). Of 5 patients with adverse risk karyotype, 4 demonstrated lack of pAKT inhibition with imatinib therapy and none of these responded to re-induction; the remaining patient whose blasts demonstrated pAKT sensitivity to imatinib did achieve CR with re-induction. In contrast pERK inhibition did not correlate with response to therapy. These results indicate that lack of pAKT inhibition in vivo correlates with resistance to re-induction therapy using this regimen. Further studies using agents that are able to inhibit AKT more effectively are warranted to try to overcome drug resistance in this setting. The treatment regimen appears active in relapsed patients, but this requires confirmation in a larger series.

Disclosures:

Off Label Use: Imatinib for c-kit positive AML.
The Epigenetic Therapies Azacitidine and Sodium Valproate Augment Immune Responses to the MAGE Cancer Testis Antigen in Acute Myeloid Leukemia and Myeloma.
Authors: Goodyear Oliver Charles , Agathanggelou Angelo , Ryan Gordon , Novitsky-Basso Igor , Stankovic Tatjana , Moss Paul , Craddock Charles F.
Published: October 2019

Abstract 2086

Poster Board II-63

The mechanism by which epigenetic therapies exert an anti-tumor effect in Acute Myeloid Leukemia (AML) and myeloma is unknown. We have studied immune responses to cancer testis antigens (CTA) a family of immunodominant tumor-associated antigens, in 12 patients with AML treated with the DNA methyltransferase inhibitor 5-Azacitidine (AZA) and the histone deacetylase inhibitor sodium valproate (VPA). The frequency of CTA-specific CD8+ T cells was studied in 12 patients with high risk AML prior to treatment with AZA (75 mg/m2 × 7 days per 28 day cycle) and sodium valproate (1-2.5 g daily administered continuously as tolerated) on a Phase II clinical trial. CTA specific CTLs were detectable in 4 of 12 patients with relapsed AML undergoing treatment with AZA and VPA. In one patient CTLs specific to the peptide MAGE-A2157-166 were detectable prior to commencement of trial therapy at a frequency of 0.001%.and persisted throughout treatment. In three patients CTLs were only detectable after commencement of AZA therapy. A polyclonal T cell line was subsequently generated from one patient and tetramer staining of the line revealed the presence of 1% MAGE-A2157-166-specific T cells and this could be enriched using anti-PE magnetic beads (Miltenyi Biotec). Two patients in whom AZA induced a CTL response to MAGE achieved a partial response to trial therapy by Cheson criteria. Treatment with AZA resulted in up to 100fold increased levels of expression of MAGEA4, MAGEA1, HAGE, SSX1, NY ESO1, MAGEC1, LAGE and XAGE mRNA in AML cell lines: HL60, KG1a, U937 and NB4 and multiple myeloma cell lines: U266 and JJN3. We next studied whether prior treatment with AZA augmented recognition of hematopoietic tumors by MAGE specific T cell clones. Prior treatment with AZA increased recognition of two myeloma cell lines (JJN3 and U266) by a MAGEA1 specific T cell clone. This is the first demonstration that AZA induce a CTL response to MAGE in patients with AML. Taken together our data are consistent with the possibility that epigenetic manipulation of cancer testis antigen expression and induction of a tumor specific CTL response may contribute to the clinical activity of AZA in hematologic malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Inactivation of Human Leukocytes in Platelet Products After Pathogen Reduction Technology Treatment in the Presence of Platelet Additive Solution.
Authors: Fast Loren D. , Marschner Susanne , DiLeone Gilbert , Goodrich Raymond
Published: October 2019

Abstract 2116

Poster Board II-93

During the transfusion of blood or blood products, a recipient can receive a large number of allogeneic leukocytes. This can lead to leukocyte-mediated adverse reactions in the recipient and include donor anti-recipient responses such as the life-threatening transfusion-associated graft versus host disease (TA-GVHD) and cytokine production; or recipient anti-donor responses that are induced by direct presentation of foreign antigen by donor leukocytes or indirectly after processing of the donor cells by recipient antigen-presenting cells. To avoid or minimize leukocyte mediated reactions, the leukocytes present in blood products are inactivated or depleted prior to administration. Nucleic acid targeted pathogen reduction processes (PRT) are well suited for leukocyte inactivation. The Mirasol® PRT System uses riboflavin (Vitamin B2) and ultraviolet (UV) light to reduce the active pathogen load and inactivate residual leukocytes in blood products used for transfusion. To make the PRT System more widely applicable, the effect of treating leukocytes in the presence of platelet additive solution (PAS) was tested. Human peripheral blood mononuclear cells (PBMNC) were purified by Ficoll-Hypaque discontinuous centrifugation and placed in 350 ml of storage solution consisting of 65% PAS (SSP+) and 35% plasma. An untreated control sample was removed before addition of 35 ml of riboflavin (500 μM) and exposure to UV light (9.1 J/ml). PBMNC were recovered after treatment and tested for their ability to proliferate in response to polyclonal stimulators such as phytohemagglutinin, and anti-CD3/CD28 or to allogeneic stimulator cells in a mixed lymphocyte culture (MLC). Treatment was found to inhibit proliferation as well as T cell activation as measured by the upregulation of CD69 expression when incubated with phorbol 12-myristate 13-acetate. Treated PBMNC were unable to produce inflammatory or TH1/TH2 cytokines when stimulated with lipopolysaccharide for 24 hours or anti-CD3/CD28 for 72 hours. Levels of cytokines that are released in the absence of activation, such as IL-6, IL-8 and IL1β, were reduced below levels of detection of the assay after PRT-treatment. Quantitation of the degree of inactivation using limiting dilution assays showed that 5.2 log inactivation could be achieved at the specified energy doses. These treatment conditions resulted in acceptable platelet cell quality over 8 days in storage. In summary, PRT treatment was able to functionally inactivate leukocytes in the presence of PAS to the levels seen with gamma-irradiation without adversely affecting the quality of the platelets.

Disclosures:

Fast: CaridianBCT Biotechnologies: Research Funding. Marschner:CaridianBCT Biotechnologies: Employment. Goodrich:CaridianBCT Biotechnologies: Employment.
Activated Platelet-Derived Antimicrobial Peptides Exhibit Virucidal Properties against Vaccinia Virus in Plasma.
Authors: Ketha Krishna Mohan V. , Rao Shilpakala Sainath , Atreya Chintamani D
Published: October 2019

Abstract 2118

Poster Board II-95

Introduction:

Contamination of blood and blood components by bacteria, virus, fungi and parasites is a major safety risk in transfusion medicine. While there has been a tremendous success in inactivating virus contamination in blood products through UV-irradiation, new and novel proof-of-concepts for microbial reduction that enhance risk to benefit ratio of the treated products are still a public health need in transfusion medicine. In the present study, we tested four novel synthetic antimicrobial peptides originating from thrombin-induced human platelet-derived antimicrobial proteins named PD1-PD4 against an enveloped virus, Vaccinia Virus (VV) spiked in plasma as a model system. We have recently shown these peptides to be useful in reducing bacterial burden in plasma and platelets. These short synthetic peptides are human platelet-derived and known to cause no immune response in humans and non-hemolytic in nature as reported by others.

Methods:

The PD1-PD4 peptides were tested on plasma samples spiked with 10-fold dilutions of the wild-type lab strain of Vaccinia Virus (WR strain). Each spiked sample was pre-incubated individually with a peptide (PD1-PD4) for 1 hour at 37°C. Spiked sample without any peptide was included as control. A cell culture-based standard plaque reduction assay method was utilized to monitor the virucidal effectiveness of the peptides. Minimal inhibitory concentration of the peptides was also estimated by testing the peptides at doubling dilutions of 100 μg/ml, 50 μg/ml, 25 μg/ml and 12.5 μg/ml concentrations.

Results:

Our analysis revealed that peptides PD3 and PD4 were potent against vaccinia virus resulting in reduction of viral titers in the plasma. PD3 peptide demonstrated the highest virucidal activity by bringing about a 2-log reduction of VV titers. PD4 peptide treatment resulted in a 1-1.5 log reduction in viral titers. The minimal inhibitory concentration analysis revealed that at 50 μg/ml concentration both the PD3 and PD4 were able to bring about a log reduction in viral titers.

Conclusion:

The present study reports a novel antiviral agent for reducing vaccinia virus contamination in plasma. Safety profiles of these peptides as reported by others in conjunction with our current studies, provide a new proof-of-concept that could be useful as safer and simpler alternatives to the viral reduction agents in transfusion medicine.

The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

Disclosures:

No relevant conflicts of interest to declare.
Mechanisms of Human Neutrophil Elastase-Catalyzed Inactivation of the Factor VIII(a).
Authors: Nishiya Katsumi , Nogami Keiji , Matsumoto Tomoko , Ogiwara Kenichi , Takeyama Masahiro , Shima Midori
Published: October 2019

Abstract 2125

Poster Board II-103

Inflammation and coagulation are linked in a variety ways. Since both neutrophil elastase and cathepsin G bind to activated platelets, they can be localized on the platelet membrane providing negatively-charged phospholipid, that is essential for assembly of tenase complex. Although it has been reported that cathepsin G provides some procoagulant effect by activating factor (F)VIII, effect of elastase on FVIII is poorly understood. We now examine the effect of human neutrophil elastase (HNE) on FVIII(a). FVIII activity (used 100 nM) was rapidly decreased and was undetectable within 10 min after the addition of HNE (0.5 nM) in a one-stage clotting assay. This time-dependent inactivation was little or mildly affected (∼35% inhibition at 10 μg/ml) in the presence of phospholipid or von Willebrand factor, respectively. Rate constant of FVIII inactivation by HNE possessed ∼15-fold greater than that by APC/protein S (PS). SDS-PAGE analysis revealed that HNE proteolyzed the 90∼210-kDa heavy chain of FVIII to 47- and 37-kDa terminal products, via some proteolyses within the B domain. Western blot and N-terminal sequence analyses showed that these fragments derived from the heavy chain were identified as the A11-357 and A2375-708, respectively, by cleavages at Val357 and Val374 in A1 and Val708 in A2. The cleavages at Val374 and Val708 by HNE were faster than that at Val357. The 80-kDa light chain was proteolyzed to 75-kDa fragment by cleavage at Val1670, quite different with the pattern of FVIII cleavage by known serine proteases. Of note, HNE-catalyzed inactivation of FVIIIa was much slower (by ∼25-fold) than that of FVIII, supporting that HNE preferably attacked to FVIII. This discrepancy was attributed to much slower cleavage at Val708 in FVIIIa. Rate of inactivation of FVIIIa by HNE was ∼30-, ∼8-, and ∼3-fold lower than those by plasmin, APC/PS, and FXa, respectively. SDS-PAGE using FVIII(a) fragments revealed that each cleavage at Val357 and Val708 was regulated by the presence of the light chain and heavy chain (intact A1-A2), respectively. These results demonstrate that the importance of cleavage at Val708, constitutes a FIXa-interactive site in tenase complex, for the mechanism of HNE-catalyzed FVIII inactivation. Furthermore, this cleavage appears to be selectively modulated by the A1-A2 domain that might interact with HNE.

Disclosures:

No relevant conflicts of interest to declare.
Tissue Factor Is Frequently Expressed in Multiple Myeloma Cells.
Authors: Gupta Sameer , Hayes Tanisha R , Liu Yuchuan , Hurford Matthew T , Solomides Charalambos , Bromberg Michael
Published: October 2019

Abstract 2132

Poster Board II-109

Tissue factor (TF) is a 47 kDa transmembrane glycoprotein that initiates blood coagulation when complexed with factor VIIa. TF is constitutively expressed in a variety of tumor cells and has been shown to have a role in cellular signaling, angiogenesis and solid tumor progression. However, the role of TF in the hematologic malignancies is poorly defined. Multiple myeloma (MM) is associated with an increased risk of venous thromboembolic disease. However, whether increased TF expression contributes to the hypercoagulable state associated with MM remains controversial. In this study, we determined the expression of TF on archived bone marrow biopsies and plasmacytomas, and human MM cell lines. Immunohistochemical staining of TF was carried out on paraffin-embedded specimens from eighteen advanced stage MM patients. Staining for TF expression was scored as 0 (null), 1+ (weak), 2+ (moderate) and 3+ (strong). TF expression for the MM cell lines (U266B1, MM1.RL and MM1.S) was carried out by semi-quantitative real time RT-PCR. TF mRNA levels were normalized to 18S ribosomal mRNA levels. The TF: 18S ratios were then compared to that of a low TF expressing human breast cancer cell line cell line, MCF-7. Paraprotein distribution in the MM patient specimens was: IgG kappa (7), IgG lambda (5), IgA kappa (3), lambda light chain (2) kappa light chain (1). Overall, TF expression was observed in 10/18 (56%) of the patient specimens. Six specimens stained 1+, and two each stained 2+ and 3+. Staining was mainly cytoplasmic and did not correlate with the type of secreted paraprotein. TF expression (relative to MCF-7) was 17.3, 1.97 and 0.77 for the U266B1, MM1.RL and MM1.S cell lines, respectively. Results from these studies suggest that TF is frequently expressed in MM cells and might contribute to the hypercoagulability associated with this disease. In addition, TF may play a role in MM cell progression.

Disclosures:

No relevant conflicts of interest to declare.