Liver transplanted patients with preoperative autoimmune hepatitis and immunological disorders are at increased risk for Post-Transplant Lymphoproliferative Disease (PTLD).

Long term immunosuppression and therapy of acute rejections result in a 20-120-fold increased risk to develop Non Hodgkin lymphoma (NHL). Since immunosuppressive therapy and immunological disorders are major risk factors for the development of NHL in the non-transplant population we aimed to analyze risk factors for PTLD in our cohort of liver transplanted (LT) patients.We analyzed retrospectively 431 patients liver transplanted between 1998 and 2008.PTLD was diagnosed in eleven of 431 patients (2.6%). PTLD, especially late PTLD, was significantly more frequent in patients who received steroids before LT (Kaplan-Meier: p0.001). Moreover PTLD in immunocompromised patients with preoperative s…

Abstract B072: Phase Ib trial of the RNActive cancer vaccine BI 1361849 (CV9202) and local radiotherapy in patients with stage IV non-small cell lung cancer (NSCLC) with disease control after first-line chemotherapy or during therapy with an epidermal growth factor receptor tyrosine kinase inhibitor: Updated clinical results and immune responses

Abstract Background: Preclinical studies demonstrated that local radiotherapy (RT) acts synergistically with RNActive mRNA vaccines to enhance anti-tumor effects and increase tumor-infiltrating lymphocytes. BI 1361849 is a therapeutic vaccine comprising optimized mRNA constituents encoding six NSCLC-associated antigens. Interim data of a phase Ib study, employing local RT to increase the immune mediated tumor control by BI 1361849, have been previously published (J Clin Oncol 34, 2016, suppl; abstr e20627). Here we report results of immune response analyses as well as updated safety and efficacy data. Methods: Patients (pts) with stage IV NSCLC were enrolled in three cohorts based on histol…

Cylindromatosis (Cyld) gene mutation in T cells promotes the development of an IL-9-dependent allergic phenotype in experimental asthma

Cylindromatosis (CYLD) is a ubiquitously expressed deubiquitinating enzyme which removes activating ubiquitin residues from important signaling molecules of the NF-κB pathway. In CYLDex7/8 transgenic mice, a naturally occurring short isoform (sCYLD) is overexpressed in the absence of full length CYLD, leading to excessive NF-κB activity. Herein, we investigated the impact of the CYLDex7/8 mutation selectively in T cells on the development of experimental allergic airway disease induced by sensitization and challenge with ovalbumin. Compared with their wildtype littermates, mice bearing the T cell-specific mutation (CD4+CYLDex7/8) display stronger eosinophilia and mucus production in the lun…

Prophylactic transfer of CD8-depleted donor lymphocytes after T-cell–depleted reduced-intensity transplantation

AbstractAllogeneic hematopoietic stem cell transplantation (SCT) regimens incorporating the lymphocytotoxic CD52 antibody alemtuzumab demonstrate efficient engraftment and reduced graft-versus-host disease (GVHD). However, these protocols substantially impair posttransplantation antiviral and antitumor immunity. To accelerate immune reconstitution after alemtuzumab-based reduced-intensity SCT, we administered prophylactic CD8-depleted donor lymphocyte infusions (DLIs) starting on days 60 and 120 after transplantation. DLIs were processed in an immunomagnetic good manufacturing practice depletion procedure resulting in a 2.5- to 6-log reduction in CD8 T cells. Of 23 high-risk patients with h…

Strong Expression of Chemokine Receptor CXCR4 by Renal Cell Carcinoma Correlates with Advanced Disease

Diverse chemokines and their receptors have been associated with tumor growth, tumor dissemination, and local immune escape. In different tumor entities, the level of chemokine receptorCXCR4 expression has been linked with tumor progression and decreased survival. The aim of this study was to evaluate the influence ofCXCR4 expression on the progression of human renal cell carcinoma.CXCR4 expression of renal cell carcinoma was assessed by immunohistochemistry in 113 patients. Intensity ofCXCR4 expression was correlated with both tumor and patient characteristics. Human renal cell carcinoma revealed variable intensities ofCXCR4 expression. StrongCXCR4 expression of renal cell carcinoma was si…

29: Rapid expansion of acute myeloid leukemia-reactive cytotoxic T cells from CD8+CD62L+ blood lymphocytes of HLA-matched healthy donors in vitro

297: Establishment of a chimeric NOD-scid/IL2RγcNull transplantation-model to evaluate graft-vs-host and graft-vs-leukemia immune responses of ex vivo modified human T lymphocyte grafts

Quality of Life in NSCLC Survivors - A Multicenter Cross-Sectional Study.

The objective was to assess quality of life (QoL) in lung cancer survivors, compare it to the general population, and identify factors associated with global QoL, physical functioning, emotional functioning, fatigue, pain, and dyspnea.Data from NSCLC patients who had survived 1 year or longer after diagnosis were collected cross-sectionally in a multicenter study. QoL was assessed with the European Organisation for the Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire (QLQ)-C30 and the lung cancer module QLQ-LC13 across different clinical subgroups and compared to age- and sex-standardized general population reference values. Multivariable linear regression analyses wer…

Erratum to: Cetuximab-induced skin exanthema: prophylactic and reactive skin therapy are equally effective

Purpose Treatment with cetuximab is accompanied by the development of an acneiform follicular skin exanthema in more than 80 % of patients. Severe exanthema (grade III/IV) develops in about 9–19 % of patients with the necessity of cetuximab dose reduction or cessation. Methods The study presented was a retrospective analysis of 50 gastrointestinal cancer patients treated with cetuximab in combination with either FOLFIRI or FOLFOX. One cohort of 15 patients received an in-house reactive skin protocol upon development of an exanthema. A second cohort of 15 patients received a skin prophylaxis starting with the first dose of cetuximab before clinical signs of toxicity. A third historic group o…

Analysis of the expression of SDF-1 splicing variants in human colorectal cancer and normal mucosa tissues

C-X-C motif chemokine ligand 12 (CXCL12), also termed stromal cell-derived factor-1 (SDF-1) is a small protein 8-14 kDa in length that is expressed as six isoforms, consisting of SDF-1α, SDF-1β, SDF-1γ, SDF-1δ, SDF-1ε and SDF-1θ. All six isoforms are encoded by the single CXCL12 gene on chromosome 10. This gene regulates leukocyte trafficking and is variably expressed in a number of normal and cancer tissues. The potential role of the novel CXCL12 splice variants as components of the CXCR4 axis in cancer development is not fully understood. The present study aimed to analyze the expression profile of the various SDF-1 isoforms and SDF-1 polymorphisms, and the association with the clinicopat…

Rapid identification of minor histocompatibility antigen HA-1 subtypes H and R using fluorescence-labeled oligonucleotides

Acknowledgments: We thank Brigitte Schuch and Karola Schmidt for excellent technical assistance. This work was supported by a grant of the Deutsche Krebshilfe (Nr. 70-2427 and Nr. 70-2428). Abstract: Donor-recipient disparitiy of the minor histocompatibility antigen HA-1 is relevant for the development of graft-versus-host disease after HLA-matched sibling allogeneic bone marrow transplantation in HLA-A*0201-positive individuals. Two different alleles of HA-1 with a single amino acid polymorphism have been identified. Here we describe a time- and cost-efficient method for HA-1 typing of genomic DNA, using site-specific hybridization probes with the LightCycler. This method was compared with…

Xenograft models for undifferentiated pleomorphic sarcoma not otherwise specified are essential for preclinical testing of therapeutic agents

Undifferentiated pleomorphic sarcoma not otherwise specified belongs to the heterogeneous group of soft tissue tumors. It is preferentially located in the upper and lower extremities of the body, and surgical resection remains the only curative treatment. Preclinical animal models are crucial to improve the development of novel chemotherapeutic agents for the treatment of undifferentiated pleomorphic sarcoma. However, this approach has been hampered by the lack of reproducible animal models. The present study established two xenograft animal models generated from stable non-clonal cell cultures, and investigated the difference in chemotherapeutic effects on tumor growth between undifferenti…

Coexpression of receptor-tyrosine-kinases in gastric adenocarcinoma-a rationale for a molecular targeting strategy?

AIM: To define the (co-)expression pattern of target receptor-tyrosine-kinases (RTK) in human gastric adenocarcinoma. METHODS: The (co-)expression pattern of VEGFR1-3, PDGFRα/β and EGFR1 was analyzed by RT-PCR in 51 human gastric adenocarcinomas. In addition, IHC staining was applied for confirmation of expression and analysis of RTK localisation. RESULTS: The majority of samples revealed a VEGFR1 (98%), VEGFR2 (80%), VEGFR3 (67%), PDGFRα (82%) and PDGFRβ (82%) expression, whereas only 62% exhibited an EGFR1 expression. 78% of cancers expressed at least four out of six RTKs. While VEGFR1-3 and PDGFRα revealed a predominantly cytoplasmatic staining in tumor cells, accompanied by an additiona…

Structure-Based Discovery of Small Molecules Binding to RNA

Ribonucleic acids (RNAs) constitute attractive drug targets. The wealth of structural information about RNAs is steadily increasing making it possible to use this information for the design of new ligands. Two methods that make heavy use of structural knowledge for ligand discovery are molecular docking and fragment screening. In molecular docking the structure of the binding site is used as a template for the design of new ligands using computational methods whereas in fragment screening biophysical methods are used for the detection of weak binding ligands which are subsequently elaborated into tighter binding molecules. In this chapter, we give an overview of both methods in the context …

Rapid Expansion of Acute Myeloid Leukemia-Reactive Cytotoxic T Cells from CD8+CD62L+ Blood Lymphocytes of HLA-Matched Healthy Donors In Vitro

Abstract Allogeneic cytotoxic T-lymphocyte (CTL) therapy in acute myeloid leukemia (AML) is hampered by the poor efficiency of growing leukemia-reactive CTLs from healthy donors in vitro. We established an allogeneic mini-mixed lymphocyte-leukemia culture (MLLC) approach by stimulating comparably small numbers (104/well) of CD8+ T cells isolated from healthy donors against irradiated primary AML blasts in 96-well plates. Prior to use, CD8+ T cells were immunomagnetically separated into a CD62L(high)+ subset enriched for naive precursors and central memory cells as well as a CD62L(low)+/negative subset containing effector memory cells. The culture medium contained IL-7, IL-12, and IL-15. Aft…

Dissemination of hepatocellular carcinoma is mediated via chemokine receptor CXCR4

In different tumour entities, expression of the chemokine receptor 4 (CXCR4) has been linked to tumour dissemination and poor prognosis. Therefore, we evaluated, if the expression of CXCR4 exerts similar effects in human hepatocellular carcinoma (HCC). Expression analysis and functional assays were performed in vitro to elucidate the impact of CXCL12 on human hepatoma cells lines. In addition, expression of CXCR4 was evaluated in 39 patients with HCC semiquantitatively and correlated with both, tumour and patients characteristics. Human HCC and hepatoma cell lines displayed variable intensities of CXCR4 expression. Loss of p53 function did not impact on CXCR4 expression. Exposure to CXCL12 …

Campath-1H-Based Reduced-Intensity Conditioning Followed by Allogeneic Blood Stem-Cell Transplantation and Preemptive CD8-Depleted Donor Lymphocyte Infusions.

Abstract Recent reports have demonstrated the feasibility of using the anti-CD52 antibody Campath-1H for in vivo T-cell depletion (TCD) in the context of a fludarabine/melphalan-based reduced-intensity conditioning regimen (Kottaridis et al. Blood 2000, 96:2419). Major disadvantage of this protocol is the severe immunosuppression which results in an increased rate of opportunistic infections and disease relapses. Donor lymphocyte infusions (DLI) are frequently used to overcome this limitation. The application of DLI, however, is associated with a profound risk of GvHD. Depletion of CD8+ lymphocytes from either the allotransplant or from DLI has proven feasible to reduce the incidence of GvH…

Acute myeloid leukemia (AML)-reactive cytotoxic T lymphocyte clones rapidly expanded from CD8(+) CD62L((high)+) T cells of healthy donors prevent AML engraftment in NOD/SCID IL2Rgamma(null) mice.

Objective Current in vitro techniques for isolating leukemia-reactive cytotoxic T lymphocytes (CTLs) from healthy donors are of relatively low efficiency and yield responder populations with unknown biological significance. This study aimed at the development of a more reliable approach, allowing generation and expansion of acute myeloid leukemia (AML)-reactive CTLs using primary in vitro stimulation. Materials and Methods We established allogeneic mini-mixed lymphocyte-leukemia cultures (mini-MLLCs) by stimulating donor CD8 + T cells with human leukocyte antigen (HLA) class I–matched AML blasts in microtiter plates. Before culture, CD8 + T cells were separated into CD62L (high)+ and CD62L …

Characterization of Renal-Cell Carcinoma (RCC)-Reactive Cytotoxic T-Lymphocyte Responses Generated from Peripheral Blood of HLA-Matched Sibling and Unrelated Healthy Individuals in Vitro.

Abstract Although current allo-transplantation therapy can induce considerable graft-versus-tumor (GvT) effects in RCC patients, it is also accompanied by severe, even life-threatening side effects, mainly due to graft-versus-host disease (GvHD). Efforts aiming to improve the specificity and efficiency of allogeneic cell therapy in this disease (e.g. specific donor lymphocyte infusion or vaccination) will certainly benefit from the identification of potential anti-tumor effector mechanisms and their corresponding target structures. We recently demonstrated that RCC-reactive cytotoxic T-lymphocyte (CTL) clones can be isolated from peripheral blood of healthy donors matched with the RCC stimu…

Rapid identification and sorting of viable virus-reactive CD4+ and CD8+ T cells based on antigen-triggered CD137 expression

Abstract Current methods for the detection and isolation of antigen-specific CD4 + and CD8 + T cells require the availability of peptide/MHC multimers or are restricted to cells that produce cytokines after antigen contact. Here we show that de novo cell surface expression of the TNF-receptor family member CD137 (4-1BB) identifies recently activated, but not resting, human CD4 + and CD8 + memory T cells. Maximum CD137 expression level is uniformly observed in both T-cell subsets at 24h after stimulation with antigen. In experiments with CMV and EBV-reactive T cells, we confirmed the specificity of CD137 expression by co-staining with peptide/HLA tetramers. Substantial proportions of CD137 +…

Superior antitumor in vitro responses of allogeneic matched sibling compared with autologous patient CD8+ T cells.

AbstractAllogeneic cell therapy as a means to break immunotolerance to solid tumors is increasingly used for cancer treatment. To investigate cellular alloimmune responses in a human tumor model, primary cultures were established from renal cell carcinoma (RCC) tissues of 56 patients. In three patients with stable RCC line and human leukocyte antigen (HLA)-identical sibling donor available, allogeneic and autologous RCC reactivities were compared using mixed lymphocyte/tumor cell cultures (MLTC). Responding lymphocytes were exclusively CD8+ T cells, whereas CD4+ T cells or natural killer cells were never observed. Sibling MLTC populations showed higher proliferative and cytolytic antitumor …

Chemokine receptor CCR7 enhances intrahepatic and lymphatic dissemination of human hepatocellular cancer.

Despite many pathophysiological analyses, the process of tumor dissemination of hepatocellular carcinoma (HCC) remains vague. In diverse tumor entities, expression of the chemokine receptor, CCR7, has been linked to tumor dissemination and poor prognosis. Therefore, we evaluated, whether CCR7 exerts similar effects in human HCC. CCR7 expression analysis was performed in vitro on human hepatoma cell lines (Huh7, Hep3B, wt HepG2, p53 dominant negative transfected HepG2). In addition, CCR7 expression was evaluated in 39 patients with hepatocellular cancer and correlated with both, tumor and patients characteristics. Human hepatocellular carcinoma samples and hepatoma cell lines displayed varia…

Cetuximab-induced skin exanthema: prophylactic and reactive skin therapy are equally effective

Purpose Treatment with cetuximab is accompanied by the development of an acneiform follicular skin exanthema in more than 80 % of patients. Severe exanthema (grade III/IV) develops in about 9–19 % of patients with the necessity of cetuximab dose reduction or cessation.

186: Superior antitumor in vitro responses of allogeneic matched sibling compared to autologous patient CD8+ T cells

Allogeneic cell therapy as a means to break immunotolerance to solid tumors is increasingly used for cancer treatment. To investigate cellular alloimmune responses in a human tumor model, primary cultures were established from renal cell carcinoma (RCC) tissues of 56patients. In three patients with stable RCC line and human leukocyte antigen (HLA)-identical sibling donor available, allogeneic and autologous RCC reactivities were compared using mixed lymphocyte/ tumor cell cultures (MLTC). Responding lymphocytes were exclusively CD8 + T cells, whereas CD4 + T cells or natural killer cells were never observed. Sibling MLTC populations showed higher proliferative and cytolytic antitumor respon…

Targeting the activation-induced antigen CD137 can selectively deplete alloreactive T cells from antileukemic and antitumor donor T-cell lines.

AbstractIn HLA-incompatible hematopoietic stem cell transplantation, alloreactive donor T cells recognizing recipient mismatch HLA cause severe graft-versus-host disease (GVHD). Strategies allowing the selective depletion of alloreactive T cells as well as the enhancement of graft-versus-malignancy immunity would be beneficial. We generated donor CD8 T-cell lines in vitro using allogeneic recipient cells mismatched at a single HLA class I allele or haplotype as stimulators. Recipient cells were obtained from acute myeloid leukemias, renal-cell carcinomas, and CD40L-induced B lymphoblasts. Resulting alloreactive T cells were activated by incubating day 21 T-cell cultures with HLA-mismatch tr…

Preemptive Transfer of GMP-Grade CD8-Depleted Donor Lymphocytes after T-Cell Depleted Reduced-Intensity Transplantation.

Abstract The combination of fludarabin (150mg/m2) / melphalan (140mg/m2) based reduced-intensity allo-transplantation (RIT) with in vivo T-cell depletion (TCD) by the anti-CD52 antibody alemtuzumab (100mg) has demonstrated efficient engraftment and reduced graft-versus-host disease (GVHD) (Kottaridis et al., Blood 2000). However, the slow lymphocyte recovery following this protocol is associated with reduced anti-infectious and antitumor immunity. In an attempt to improve immune-reconstitution after TCD / RIT, we investigated the early preemptive use of CD8-depleted donor lymphocyte infusions (DLIs). For CD8 depletion of donor leukaphereses, a new depletion protocol using clinical grade CD8…

PDGFRa/β expression correlates with the metastatic behavior of human colorectal cancer: A possible rationale for a molecular targeting strategy

As new multi-target tyrosine kinase inhibitors are emerging in the therapy of various malignancies, our aim was to define the co-expression pattern of receptor-tyrosine-kinase platelet-derived growth factor receptors alpha and beta (PDGFRalpha/beta) in human colorectal cancer. The co-expression pattern of PDGFRalpha/beta was analyzed by RT-PCR in 99 histologically confirmed human colorectal carcinomas and five colorectal cancer cell lines. In addition, immunohistochemical (IHC) staining was applied for confirmation of expression and analysis of receptor tyrosine kinase (RTK) localisation. The colorectal cancer cell lines that were analysed revealed varying expression intensities of PDGFRalp…

Proteasome-inhibited dendritic cells demonstrate improved presentation of exogenous synthetic and natural HLA-class I peptide epitopes.

The design and successful clinical implementation of cancer vaccines targeting the induction of T-cell mediated immunity is a rapidly evolving field that is hampered by an empirical selection of antigen and adjuvant. In particular, vaccines using defined tumor-associated peptide epitopes elicit only a restricted T-cell repertoire in a minority of patients. In this regard, vaccines comprising the whole spectrum of antigens presented by individual autologous tumors would be advantageous. In an in vitro model, we evaluated the capacity of naturally processed Epstein-Barr virus-transformed B-lymphoblastoid-cell line (LCL)-derived peptides to activate virus-specific CD8+ T cells of seropositive …

Psychological distress in lung cancer survivors at least 1 year after diagnosis-Results of a German multicenter cross-sectional study.

Strong expression of chemokine receptor CXCR4 by pancreatic cancer correlates with advanced disease

Certain chemokines have been proposed to distinctly contribute to tumor growth, dissemination and local immune escape. Expression of the chemokine receptor CXCR4 has been linked to tumor progression in diverse tumor entities. The aim of this study was to evaluate if the expression of CXCR4 influences progression of human pancreatic cancer. CXCR4 expression of pancreatic cancer was retrospectively assessed by immunohistochemistry in 103 patients with pancreatic cancer. Intensity of CXCR4 expression was correlated with both tumor and patient characteristics. Human pancreatic cancer revealed variable intensities of CXCR4 expression. Strong CXCR4 expression was significantly associated with adv…