bluuebunny/crossref_metadata_2025_split huggingface dataset:
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
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
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
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
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
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
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
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
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.