julilili42/papers huggingface dataset:
Elimination Of Minimal Residual Disease (MRD) In AML Patients With a Novel Fc-Optimized FLT3 Antibody (4G8-SDIEM)
2013-11-15

Abstract Chemotherapeutic treatment induces a morphological complete remission (CR) in the majority of leukemia patients, but minimal residual disease (MRD) remains detectable by sensitive methods and constitutes the basis for relapse. Immunotherapeutic strategies are rarely sufficient to eliminate large tumor burden, but can successfully eliminate MRD. This is exemplified by the efficacy of donor lymphocyte infusion (DLI) and allogeneic stem cell transplantation (SCT) in leukemia. Here we report the first clinical experience with a novel Fc-optimized antibody directed to the receptor tyrosine kinase FLT3 expressed on the surface of leukemia cells. This antibody may potently eliminate MRD after conventional therapy and achieve this goal with less toxicity (i.e. GvHD) than DLI and SCT. After in vitro characterization of the antibody, which revealed its markedly increased ability to mediate antibody dependent cytotoxicity (ADCC), in particular of NK cells, against leukemic cells (Hofmann et al, Leukemia 2012), the antibody (4G8SDIEM) was produced in a university owned unit and applied on a compassionate need basis. Upon application in AML patients with active disease, potent NK cell activation and anti-leukemia activity were observed, but effects were rather short lived, most likely due to unfavorable NK:leukemia cell ratios in the bone marrow. Consecutively we applied 4G8SDIEM to AML patients in morphological CR after SCT which displayed increasing MRD levels (mutNPM1) indicative of imminent relapse and for which no other treatment options were available. In 3 of the 4 patients treated so far, a single application of 4G8SDIEM (in 2 cases followed by treatment with one dose of DLI) resulted in rapid elimination of MRD. The non-responsive patient displayed high titers of preexistent anti-Fab antibodies capable of binding 4G8SDIEM, which provides a potential explanation for the lacking response. One patient that had received subsequent DLI died of complications due to GvHD after reaching and maintaining a molecular CR. The other two patients (1 with, 1 without DLI) remained in molecular CR for at present 17 and 12 months after 4G8-SDIEM application without further specific treatment. No stem cell toxicity was observed. Our results imply that 4G8SDIEM is capable to reduce or eliminate MRD after conventional therapy. Based on these observations, a phase I/II trial enrolling AML patients in CR with detectable MRD is in preparation. Disclosures: Hofmann: Synimmune, GmbH, Tuebingen, Germany: Employment. Grosse-Hovest:Synimmune, GmbH, Tuebingen, Germany: Employment. Aulwurm:Synimmune, GmbH, Tuebingen, Germany: Employment. Jung:Synimmune GmbH, Tuebingen, Germany: Equity Ownership, Honoraria.
Platelets Impair NK Cell Immunosurveillance Of Metastasizing Tumor Cells By Altering Surface Expression and Shedding Of Ligands For The Activating Immunoreceptor NKG2D
2013-11-15

Abstract NK cells play an important role in the immunosurveillance of tumor cells. The mechanisms leading to NK cell activation are described by the ‘missing-self’ and “induced-self’ hypotheses, implying that cells with low or absent expression of MHC class I and stress-induced expression of ligands for activating receptors like e.g. NKG2D (NKG2DL) are preferentially recognized and eliminated by NK cells. Besides the direct interaction with their target cells, NK activity is further influenced by various other hematopoietic cells. In mouse models, thrombocytopenia impairs metastasis, and this is reversed by additional depletion of NK cells. However, the knowledge regarding the molecular mechanisms by which platelets influence NK cells is still fragmentary. We recently reported that release of TGF-β by platelets upon their interaction with (metastasizing) tumor cells downmodulates NKG2D on NK cells (Kopp et al., Cancer Res. 2009; Placke et al., J Innate Immun. 2011). Moreover, platelets transfer “healthy” MHC class I to the tumor cell surface. Thus, platelets may facilitate metastasis by interfering with both, “induced and missing self’ NK cell recognition. Here we provide evidence for a yet unknown mechanism by which platelets further impair NKG2D-mediated immunosurveillance. Tumor cells were incubated with platelets from healthy donors resulting in coating of tumor cells and activation of the platelets, or treated with platelet-derived soluble factors (releasate) obtained either by tumor cell-induced platelet activation (TCIPA) or the platelet agonist thrombin. Presence of platelet derived factors derived either from coating of tumor cells or contained in platelet releasate substantially reduced NKG2DL surface expression on tumor cells. This was paralleled by enhanced levels of soluble NKG2DL in culture supernatants, indicating that platelet-derived factors mediate NKG2DL shedding from the tumor cell surface. Diminished NKG2DL surface expression resulted in decreased NKG2D-dependent cytotoxicity of NK cells as revealed by blocking experiments using NKG2D antibody and NKG2DL-specific F(ab)2 fragments targeting the specific modulated NKG2DL. Our data thus identify induction of NKG2DL shedding as novel mechanism by which interaction of platelets with metastasizing tumor cells impairs NK cell immunosurveillance. Disclosures: No relevant conflicts of interest to declare.
Leukocyte Adhesion and Activation in Atherothrombosis
2013-11-15

Abstract Platelets interact with a variety of cells, including leukocytes and progenitor cells. Upon activation, platelets release chemokines such as CXCL12/SDF-1 that modulate important functional aspects of vascular inflammation and atherogenesis. Platelet-derived SDF-1 and CyPA regulate chemotaxis, migration, and cell activation in the microenvironment of accumulating platelets. Previously we have shown that platelets are the major source of CXCL12/SDF-1. Platelet-derived SDF-1 provides a strong signal for recruitment of circulating progenitor cells and leukocytes towards vascular and myocardial injury. Patients with severe coronary artery disease have enhanced levels of plasma and platelet-associated SDF-1 that correlates with an enhanced number of circulating progenitor cells and the development of cardiovascular diseases. In patients with both acute myocardial infarction platelet-SDF-1 and an increase in circulating platelet/progenitor cell aggregates, the combination is associated with myocardial recovery and improved prognosis, indicating that platelet-SDF-1 is critical for repair and regeneration of vascular or myocardial injury. Administration of a stable recombinant SDF-1 fusion protein reduces infarct size and preserves myocardial infarction in mice. We showed that platelet-derived SDF-1 is an important survival factor both for platelets (in an autocrine manner) and for surrounding cells. SDF-1 released from activated platelets binds to its receptor CXCR4 and induces internalization of the SDF-1/CXCR4 complex. SDF-1-ligation of CXCR4 augments surface expression of CXCR7; a process coupled via ERK1/2 and cyclophilin A. SDF-1α caused downstream phosphorylation of Erk1/2 and cyclophilin A (CyPA). NIM-811 a CyPA-PPIase-activity inhibitor significantly abolished SDF-1α-driven CXCR7 surface exposure. Moreover, Cypa-/-murine platelets failed to show SDF-1α/CXCR4-mediated CXCR7 translocation. SDF-1α-induced ubiquitination of CXCR7 was essential for its surface translocation and was dependent on Erk1/2 and CyPA-PPIase activity, inhibited by U0126 and NIM-811, respectively. In contrast to wild-type, Cypa-/- murine platelets failed to exhibit a dynamic change in CXCR7 ubiquitination status upon SDF-1α exposure and thus failed in subsequent CXCR7 externalization. SDF-1/CXR4-driven CXCR7 translocation inhibits activation-induced apoptosis of platelets and prolongs platelet survival in vitro and in vivo. Similarly, platelet SDF-1 interacts with monocytes, reduces apoptosis and promotes differentiation of monocytes into macrophages. Inhibition of platelet release and adhesion to endothelial cells reduces monocyte migration and differentiation into macrophages in vitro and attenuates vascular inflammation and atheroprogression in apoE-deficient mice. In conclusion, platelets and platelet interaction with leukocytes are critical in regulation of vascular inflammation and atheroprogression and may offer promising targets for modulating inflammation in disease states. Disclosures: No relevant conflicts of interest to declare.
Characterization Of The HLA Class II Ligandome In Acute Myeloid Leukemia (AML) Reveals Novel Candidates For Peptide-Based Immunotherapy
2013-11-15

Abstract CD4+ T cells are crucial for the induction and maintenance of cytotoxic T cell responses, but can also mediate direct tumor rejection. The therapeutic efficacy of peptide-based cancer vaccines may thus be improved by including HLA class II epitopes to stimulate T helper cells. In contrast to HLA class I ligands, only a small number of class II ligands of TAA has been described so far. We recently reported on the overexpression of HLA class II in AML cells as compared to autologous monocytes and granulocytes as well as on the first HLA class I leukemia associated antigens identified directly on the cell surface of primary AML cells (Stickel et. al. abstract in Blood 2012). In this study we characterized the HLA class II ligandome in AML to identify additional ligands for a peptide-based immunotherapy approach. HLA class II ligands from primary AML cells as well as bone marrow and peripheral blood mononuclear cell (BMNCs/PBMCs) of healthy donors were analyzed using the approach of direct isolation and identification of naturally presented HLA peptides by affinity chromatography and mass spectrometry (LC-MS/MS). LC-MS/MS peptide analysis provided qualitative and semi-quantitative information regarding the composition of the respective ligandomes. Comparative analysis of malignant and benign samples served to identify ligandome-derived tumor associated antigens (LiTAAs) and to select peptide vaccine candidates. Most abundantly detected peptides were functionally characterized with regard to their ability to induce a specific CD4+ T-cell response in healthy donors and in tumor patients using ELISpot. Samples from 10 AML patients (5 FLT3-ITD mutated) and 18 healthy donors were analyzed. We identified more than 2,100 AML-derived HLA class II ligands representing &gt;1,000 different source proteins, of which 315 were exclusively represented in AML, but not in healthy PBMC/BMNC. Data mining for broadly represented LiTAAs pinpointed 26 HLA class II ligands from 8 source proteins that were presented exclusively on more than 40% of all analyzed AML samples as most promising targets. Amongst them were already described TAAs (e.g., RAB5A) as well as several so far understated proteins (e.g. calsyntenin 1, glycophorin A, mannose-binding lectin 2). Subset analysis revealed 58 LiTAAs presented exclusively on FLT3-ITD mutated AML cells. Additional screening for HLA class II ligands from described leukemia associated antigens showed positive results for NPM1 (1 peptide sequence) and MPO (13 peptide sequences). Peptides from calsyntenin 1 and RAB5A were able to elicit CD4+-T-cell response in 25% of tested AML patients (n=16). Thus, our study identified, for the first time, HLA class II tumor associated antigens directly obtained from the HLA ligandomes of AML patients and thereby represents a further step to our goal of developing a multipeptide vaccine for immunotherapy of AML. Disclosures: No relevant conflicts of interest to declare.
Immunotherapy With RNA Vaccination Coding For TAA In Renal Cell Carcinoma: Update On Immunological Responses and Correlation With Survival
2013-11-15

Abstract Introduction Recently Walter et al. (Nature med 2012) demonstrated that induction of T cell responses to tumor-associated antigens (TAA) after peptide vaccination (IMA901) is associated with significantly prolonged overall survival in patients with advanced renal cell carcinoma (RCC). Between August 2003 and November 2005 we performed a phase I/II non-randomized study to analyze the feasibility and safety of an mRNA-based vaccination as well as immunological and clinical responses in patients with stage IV RCC (Rittig et al., Mol Ther. 2011). Here we provide an update on survival and correlative analyses of immunological responses and survival. Methods: In vitro transcribed naked mRNA coding for the TAAs mucin 1, carcinoembryonic antigen (CEA), human epidermal growth factor receptor (EGFR) 2, telomerase, survivin, and melanoma-associated antigen 1 was administered to a total of 30 patients. In the first 14 patients (cohort A), vaccinations were applied on days 0, 14, 28, and 42 (20 μg of each antigen). In the consecutive 16 patients (cohort B), an intensified protocol consisting of injections at days 0-3, 7-10, 28, and 42 with 50 μg/antigen was used. After the respective induction periods, patients in both cohorts received monthly vaccinations until tumor progression according to RECIST. Results: In October 2010 monitoring had revealed a survival in cohort A ranging from 3 to 85 months with 4/14 patients (29%) being alive, while survival in cohort B ranged from 2 to 71 months with 4/16 (25%) patients being alive. Follow up was re-assessed in March 2013. In cohort A, 3 patients were still alive (21%) with survival ranging from 105 to 115 months. Two of these surviving patients had intermediate and one had favourable risk according to Motzer score at enrolment. These respective patients had received between 13 and 32 doses of RNA. In cohort B, three patients (19%) were found to be alive, of which two had favourable and one had intermediate risk according to Motzer score at enrolment. Survival in cohort B ranged from 95 to 97 months, and between 10 and 22 doses of the vaccine had been applied to the patients. Impressively and similar to the findings of Walter et al. with the IMA901 peptide vaccine, all six patients that currently are alive displayed immunological responses to the applied antigens in cytotoxic T cell or ELISpot assays, while vice versa no patient without detectable immune response had survived more than 33 months. Conclusion: The current survival update revealed long term survival (&gt;8 years) in about 20% of patients in this small cohort suffering from stage IV RCC. Notably, in line with data obtained after peptide vaccination, prolonged survival after treatment with our RNA vaccine also showed a clear correlation with in vitro detectable immunological responses to the applied TAA. Disclosures: No relevant conflicts of interest to declare.
Induction Of Apoptosis By The Dual PI3K/Mtor Inhibitor NVP-BEZ235 Is Impaired By a Profound G1/G0 Arrest In Acute Leukemia Which Can Be Overcome By Combination Approaches
2013-11-15

Abstract Introduction Constitutive phosphorylation of AKT is frequently found in acute leukemia. In a proportion of patients, activation of the PI3K/AKT pathway can be linked to gain-of-function tyrosine kinase mutations. We have previously shown that TKI only incompletely inactivate AKT signaling. PI3K/AKT pathway inhibitors are attractive for targeting this pathway. However, most inhibitors lead to G1/G0 arrest. Likely for this reason, response rates to PI3K inhibitor treatment were moderate in various neoplasms. We here demonstrate that the dual PI3K-MTORC1/2 inhibitor NVP-BEZ235, which is currently under clinical investigation in relapsed acute leukemia, mediates a profound G1/G0 arrest in cells harboring leukemia-driving FLT3 ITD or BCR-ABL1 mutations, impairing induction of apoptosis. Furthermore, combination with TKI or chemotherapy overcomes cellular resistance. Methods Proliferation and apoptosis assays were performed in leukemia cell lines, an isogenic Ba/F3 cell model harboring mutant-KIT, FLT3 or ABL1 isoforms and primary leukemic cells as well as healthy donor cells. Immunoblots were performed to study effects on AKT signaling pathways including downstream effectors of autophagy (p-ULK1), proliferation, cell cycle (p70S6Kinases, cyclinD, pRB) and apoptosis (caspases). Isobolograms were created to compute combination indices. Results NVP-BEZ235 profoundly inhibited phosphorylation of AKT and p70S6K, leading to a potent antiproliferative effect in the low nanomolar range in most cell lines as well as patient blasts. However, no meaningful induction of apoptosis was observed. Instead, a sustained G1/G0 cell cycle arrest with dephosphorylation of RB and upregulation of cyclinD, induction of protective autophagy pathways (via ULK1) and absence of cleaved caspase 3 was detected. An isogenic Ba/F3 model confirmed the low proapoptotic efficacy especially for FLT3 ITD and BCR-ABL1. In an attempt to overcome NVP-BEZ235 induced cell cycle arrest, we co-treated leukemic cells with specific TK inhibitors or chemotherapeutics such as daunorubicin in a fixed dose-dilution ratio. Combination indices revealed additive to superadditive proapoptotic effects for all tested combinations. Conclusion We provide evidence that the antileukemic activity of NVP-BEZ235 in acute leukemia with mutations in tyrosine kinases is abrogated due to induction of profound G1/G0 cell cycle arrest which can be overcome by combination with TKI or chemotherapy. Disclosures: No relevant conflicts of interest to declare.
Heatshockproteins-60 and 90 Are Overexpressed In Leukemia Stem Cells and Stabilize Oncogene Signaling
2013-11-15

Abstract Introduction Activating mutations of the KIT class III receptor tyrosine kinase (TK) are associated with the pathophysiology of acute leukemia, especially core binding factor leukemia (CBFL), and systemic mastocytosis (SM). Despite considerable antiproliferative and proapoptotic activity of several KIT TK inhibitors in vitro, clinical efficacy in AML and SM is generally moderate. We hypothesized that resistance to therapy is promoted by activation of alternative signaling pathways. Previously we reported that KIT TK inhibition results in significantly increased phosphorylation of heat shock protein (HSP) family members and that KIT is a client protein of phosphorylated (p)HSP90 putatively stabilizing KIT protein function in the presence of KIT TK-inhibitors (Kampa-Schittenhelm et al., ASH annual meeting 2010). This prompted us to further test the HSP inhibitor IPI-504 in KIT dependent CBFL, including the leukemic stem cell fraction. Methods Protein expression levels of (p)HSPs in leukemic blasts of high-risk and CBFL patients were studied by flow cytometry focusing on the CD34+/CD38- leukemia stem/progenitor cell fraction. Cellular proliferation and induction of apoptosis in leukemia cells treated with the HSP90 inhibitor IPI-504 was determined by XTT- and annexin V-based assays. Results (p)HSP90/60 levels were preferentially upregulated in CBFL associating with KIT dysregulation. Consequently, HSP90 inhibition with IPI-504 potently degraded KIT expression causing a direct antiproliferative and antiapoptotic effect in CBFL in in vitro and ex vivo models. Efficacy of IPI-504 was potentiated when combined with TK inhibitors. Importantly, high expression of (p)HSP90 and HSP60 was particularly observed in the CD34+/CD38- putative leukemia stem cell fraction arguing for a function as protection mechanism to conserve (leukemic) progenitor cell function upon cell stress such as antileukemic treatment. Conclusion HSPs are upregulated in CBFL, and IPI-504 induces antiproliferative and proapoptotic effects in primary leukemia samples. Importantly, in particular the putative malignant progenitor cell pool in KIT-associated acute leukemia expresses high levels of (p)HSP90, identifying HSP90 inhibition as an attractive novel strategy to overcome the therapy-refractory behavior of malignant stem/progenitor cells. Our results provide a rationale for the evaluation of HSP90 inhibitors such as IPI-504 in CBFL. Disclosures: No relevant conflicts of interest to declare.
Performance on the Frontal Assessment Battery is sensitive to frontal lobe damage in stroke patients
2013-11-16

Abstract Background The Frontal Assessment Battery ( FAB ) is a brief battery of six neuropsychological tasks designed to assess frontal lobe function at bedside [Neurology 55:1621-1626, 2000]. The six FAB tasks explore cognitive and behavioral domains that are thought to be under the control of the frontal lobes, most notably conceptualization and abstract reasoning, lexical verbal fluency and mental flexibility, motor programming and executive control of action, self-regulation and resistance to interference, inhibitory control, and environmental autonomy. Methods We examined the sensitivity of performance on the FAB to frontal lobe damage in right-hemisphere-damaged first-ever stroke patients based on voxel-based lesion-behavior mapping. Results Voxel-based lesion-behavior mapping of FAB performance revealed that the integrity of the right anterior insula (BA13) is crucial for the FAB global composite score, for the FAB conceptualization score, as well as for the FAB inhibitory control score. Furthermore, the FAB conceptualization and mental flexibility scores were sensitive to damage of the right middle frontal gyrus (MFG; BA9). Finally, the FAB inhibitory control score was sensitive to damage of the right inferior frontal gyrus (IFG; BA44/45). Conclusions These findings indicate that several FAB scores (including composite and item scores) provide valid measures of right hemispheric lateral frontal lobe dysfunction, specifically of focal lesions near the anterior insula, in the MFG and in the IFG.
Lipopolysaccharide induces cell volume increase and migration of dendritic cells
2013-11-16

ABSTRACT Migration of dendritic cells (DCs) plays an important role in T‐cell‐mediated adaptive immune responses. Lipopolysaccharide (LPS) sensed by Toll‐like receptor 4 (TLR4) serves as a signal for DC migration. We analyzed LPS‐induced DC volume changes preceding the directed movement towards chemoattractants. Treatment with LPS resulted in rapid, prolonged cell swelling in wild‐type (WT), but not in TLR4 −/− bone marrow‐derived (BM) DCs indicating that TLR4 signaling is essential for LPS‐induced swelling. As a consequence, LPS‐treatment enhanced the migratory activity along a chemokine (CCL21)‐gradient in WT, but not in TLR4‐deficient BMDCs suggesting that the LPS/TLR4‐induced swelling response facilitates DC migration. Moreover, the role of calcium‐activated potassium channels (K Ca 3.1) as putative regulators of immune cell volume regulation and migration was analyzed in LPS‐challenged BMDCs. We found that the LPS‐induced swelling of K Ca 3.1‐deficient DCs was impaired when compared to WT DCs. Accordingly, the LPS‐induced increase in [Ca 2+ ] i detected in WT DCs was reduced in K Ca 3.1‐deficient DCs. Finally, directed migration of LPS‐challenged K Ca 3.1‐deficient DCs was low compared to WT DCs indicating that activation of K Ca 3.1 is involved in LPS‐induced DC migration. These findings suggest that both TLR4 and K Ca 3.1 contribute to the migration of LPS‐activated DCs as an important feature of the adaptive immune response.