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The transcription factor FOXO3 has been associated in different tumor entities with hallmarks of cancer, including metastasis, tumor angiogenesis, maintenance of tumor-initiating stem cells, and drug resistance. In neuroblastoma (NB), we recently demonstrated that nuclear FOXO3 promotes tumor angiogenesis in vivo and chemoresistance in vitro. Hence, inhibiting the transcriptional activity of FOXO3 is a promising therapeutic strategy. However, as no FOXO3 inhibitor is clinically available to date, we used a medium-throughput fluorescence polarization assay (FPA) screening in a drug-repositioning approach to identify compounds that bind to the FOXO3-DNA-binding-domain (DBD). Carbenoxolone (CBX), a glycyrrhetinic acid derivative, was identified as a potential FOXO3-inhibitory compound that binds to the FOXO3-DBD with a binding affinity of 19 µM. Specific interaction of CBX with the FOXO3-DBD was validated by fluorescence-based electrophoretic mobility shift assay (FAM-EMSA). CBX inhibits the transcriptional activity of FOXO3 target genes, as determined by chromatin immunoprecipitation (ChIP), DEPP-, and BIM promoter reporter assays, and real-time RT-PCR analyses. In high-stage NB cells with functional TP53, FOXO3 triggers the expression of SESN3, which increases chemoprotection and cell survival. Importantly, FOXO3 inhibition by CBX treatment at pharmacologically relevant concentrations efficiently repressed FOXO3-mediated SESN3 expression and clonogenic survival and sensitized high-stage NB cells to chemotherapy in a 2D and 3D culture model. Thus, CBX might be a promising novel candidate for the treatment of therapy-resistant high-stage NB and other “FOXO-resistant” cancers.

This work evaluated gene expression differences between a hanging-drop 3D NSCLC model and 2D cell cultures and their in-vivo relevance by comparison to patient-derived data from The Cancer Genome Atlas. Gene expression of 2D and 3D cultures for Colo699 and A549 were assessed using Affymetrix HuGene 1.0 ST gene chips. Biostatistical analyses tested for reproducibility, comparability and significant differences in gene expression profiles between cell lines, experiments and culture methods. The analyses revealed a high interassay correlation within specific culture systems proving a high validity. 979 genes were altered in A549 and 1106 in Colo699 cells due to 3D cultivation. The overlap of changed genes between the cell lines was small (149), but the involved pathways in the reactome and GO- analyses showed a high overlap with DNA methylation, cell cycle, SIRT1, PKN1 pathway, DNA repair and oxidative stress as well known cancer-associated representatives. Additional specific GSEA-analyses revealed changes in immunologic and endothelial cell proliferation pathways, whereas hypoxic, EMT and angiogenic pathways were downregulated. Gene enrichment analyses showed 3D-induced gene up-regulations in the cell lines 38 to be represented in in-vivo samples of NSCLC patients using data of The Cancer Genome Atlas. Thus, our 3D NSCLC model might provide a tool for early drug development and investigation of microenvironment-associated mechanisms. However, this work also highlights the need for further individualization and model adaption to address remaining challenges.

The tumour microenvironment and tumour angiogenesis play a critical role in the development and therapy of many cancers, but in vitro models reflecting these circumstances are rare. In this study, we describe the development of a novel tri-culture model, using non-small cell lung cancer (NSCLC) cell lines (A549 and Colo699) in combination with a fibroblast cell line (SV 80) and two different endothelial cell lines in a hanging drop technology. Endothelial cells aggregated either in small colonies in Colo699 containing microtissues or in tube like structures mainly in the stromal compartment of microtissues containing A549. An up-regulation of hypoxia and vimentin, ASMA and a downregulation of E-cadherin were observed in co- and tri-cultures compared to monocultures. Furthermore, a morphological alteration of A549 tumour cells resembling “signet ring cells” was observed in tri-cultures. The secretion of proangiogenic growth factors like vascular endothelial growth factor (VEGF) was measured in supernatants. Inhibition of these proangiogenic factors by using antiangiogenic drugs (bevacizumab and nindetanib) led to a significant decrease in migration of endothelial cells into microtissues. We demonstrate that our method is a promising tool for the generation of multicellular tumour microtissues and reflects in vivo conditions closer than 2D cell culture.

// Dagmar Quandt 1 , Hans Dieter Zucht 2 , Arno Amann 3 , Anne Wulf-Goldenberg 4 , Carl Borrebaeck 5 , Michael Cannarile 6 , Diether Lambrechts 7 , Herbert Oberacher 8 , James Garrett 9 , Tapan Nayak 10 , Michael Kazinski 11 , Charles Massie 12 , Heidi Schwarzenbach 13 , Michele Maio 14 , Robert Prins 15 , Bjorn Wendik 16 , Richard Hockett 17 , Daniel Enderle 18 , Mikkel Noerholm 18 , Hans Hendriks 19 , Heinz Zwierzina 3 and Barbara Seliger 1 1 Institute of Medical Immunology, Martin Luther University Halle-Wittenberg, Halle, Germany 2 Protagen, Dortmund, Germany 3 Department of Haematology and Oncology, Medical University Innsbruck, Innsbruck, Austria 4 Experimental Pharmacology and Oncology, Berlin-Buch GmbH, Berlin, Germany 5 Create Health Translational Cancer Center, Lund University, Lund, Sweden 6 Roche Pharmaceutical Research and Early Development, Roche Innovation Center, Munich, Germany 7 Laboratory for Translational Genetics, VIB Center for Cancer Biology, Leuven, Belgium 8 Institute of Legal Medicine and Core Facility Metabolomics, Medical University of Innsbruck, Innsbruck, Austria 9 Global Drug Development, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, USA 10 Roche Pharmaceutical Research and Early Development, Roche Innovation Center Basel, Basel, Switzerland 11 Qiagen, Hilden, Germany 12 CRUK Cambridge Institute, Cambridge, UK 13 University Medical Center Hamburg-Eppendorf, Department of Tumor Biology Hamburg, Hamburg, Germany 14 Medical Oncology and Immunotherapy, University Hospital of Siena, Siena, Italy 15 Department of Neurosurgery, David Geffen School of Medicine at UCLA, Los Angeles, California, USA 16 Perkin Elmer, Hamburg, Germany 17 Biodesix, Boulder, Colorado, USA 18 Exosome Diagnostics GmbH, Martinsried, Germany 19 Hendriks Pharmaceutical Consulting, Purmerend, The Netherlands Correspondence to: Barbara Seliger, email: Barbara.Seliger@uk-halle.de Keywords: liquid biopsy, biomarker, tumor, high throughput analysis, immunotherapy Received: February 08, 2017 Accepted: April 04, 2017 Published: April 24, 2017 ABSTRACT During the last decade, novel immunotherapeutic strategies, in particular antibodies directed against immune checkpoint inhibitors, have revolutionized the treatment of different malignancies leading to an improved survival of patients. Identification of immune-related biomarkers for diagnosis, prognosis, monitoring of immune responses and selection of patients for specific cancer immunotherapies is urgently required and therefore areas of intensive research. Easily accessible samples in particular liquid biopsies (body fluids), such as blood, saliva or urine, are preferred for serial tumor biopsies. Although monitoring of immune and tumor responses prior, during and post immunotherapy has led to significant advances of patients’ outcome, valid and stable prognostic biomarkers are still missing. This might be due to the limited capacity of the technologies employed, reproducibility of results as well as assay stability and validation of results. Therefore solid approaches to assess immune regulation and modulation as well as to follow up the nature of the tumor in liquid biopsies are urgently required to discover valuable and relevant biomarkers including sample preparation, timing of the collection and the type of liquid samples. This article summarizes our knowledge of the well-known liquid material in a new context as liquid biopsy and focuses on collection and assay requirements for the analysis and the technical developments that allow the implementation of different high-throughput assays to detect alterations at the genetic and immunologic level, which could be used for monitoring treatment efficiency, acquired therapy resistance mechanisms and the prognostic value of the liquid biopsies.

Purpose Improvements in systemic treatment have led to a prolongation of survival and quality of life in patients with metastatic tumors in recent years. However, despite this improved standard of care, it is expected that the progression-free survival (PFS) for patients with refractory cancers will continue to decline over subsequent therapy lines. In those patients, studies and meta-analyses showed that treatment based on multiplatform molecular profiling (MMP) of tumor tissue may derive a clinical benefit. The aim of this study was to analyze if molecular-based therapy may prolong PFS compared with the PFS of the immediately prior therapy. Methods We pooled clinical data of 140 patients treated within 3 recently conducted pilot studies and included an additional 21 patients who were treated within the ongoing ONCO-T-PROFILE program. The PFS of the molecular-based treatment was compared with the PFS of the previous therapy using Kaplan-Meier curves. Results In heavily pretreated cancer patients, the PFS could be significantly improved using molecular-based treatment options (120.0 vs. 89.5 days). More than 50% of patients showed a clinical benefit from MMP-guided therapy as defined by a PFS ratio of 1.3 or greater. Conclusions We conclude that pretreated cancer patients can benefit from incorporation of molecular profiling, as demonstrated by not only an increase of the PFS ratio but also PFS. Further randomized trials in specific tumor subtypes may help establish specific patient populations who might benefit most from MMP guidance.

Introduction Patients with refractory metastatic cancer have been shown to benefit from molecular profiling of tumor tissue. The ONCO-T-PROFILE project was launched in March 2014 at the Innsbruck Medical University. Within 2 years our project aims to recruit 110 patients with stage IV cancer refractory to standard therapy. Our data presented here are based on an interim-analysis. Methods Tumor tissue specimens were submitted for molecular profiling to the certified laboratory (Caris Life Science, USA). Druggable tumor targets were selected based on biomarker status to agents with potential clinical benefit. Clinical benefit was defined as a PFS ratio (=PFS upon treatment according to the molecular profile/ PFS upon the last prior therapy) ≥ 1.3. Results As of April 2015, tumors from 50 patients have been molecularly profiled and one or more targets were detectable in 48 specimens (98%). So far, 19 (38%) patients have been treated according to their molecular tumor profile. To date, 8 (42%) patients have reached a PFS ratio of ≥ 1.3. Conclusions We could show that molecular profiling is feasible in the clinical routine. A proportion of patients might benefit from an individualized treatment approach based on molecular profiling. As a result, we will proceed to enroll patients in ONCO-T-PROFILE.

Background The focus of the outlined work is the establishment of a three-dimensional lung model for various drug-screening applications. Methods The non-small cell lung cancer (NSCLC) cell line Colo699 was cultivated as monolayer (2D) on plates for 5 days or as microtissues (3D) using a hanging-drop system for 5 and 10 days. Cells and microtissues were treated with afatinib (10–80 µM), cisplatin (100–800 µM) or vinorelbine (25–200 µM) for 24 or 48 hours (h). Cell proliferation and viability were analysed by intra-cellular adenosine triphosphate (ATP) and lactate dehydrogenase release (LDH) assays, annexin V/propidium iodide (PI) staining, and cell cycle determination. Microtissue morphology and size, as well as cell death were evaluated via phase contrast microscopy. Results Our results demonstrate the valid determination of viability and cell death using established assays in the 3D system for drug testing. The comparison of ATP, LDH and cytometry data showed moderate (0.40) to very strong (0.99) correlations. Thereby, we observed partially significant differences in drug efficacy between microtissues and 2D cultures dependent from the applied treatment and read-out method. Altogether, microtissues developed resistance to cisplatin and vinorelbine; but remained more vulnerable to afatinib. These findings were confirmed with microscopy. Conclusion In summary, we established an NSCLC 3D test system with multiple assays compatible for drug-testing applications of substances with different mechanisms of action. In addition, our data support the usage of microtissues as more accurate tools for drug-efficacy testing with the possibility of long-term cultivation and treatment. Electronic supplementary material The online version of this article (doi:10.1007/s00432-016-2198-0) contains supplementary material, which is available to authorized users.

Introduction The interaction between the immune system and malignant diseases is a proven key target for cancer therapy. We describe an innovative 3D cell culture system comprising both immune and cancer cells to evaluate their interaction and immune cell infiltration to provide an innovative in vitro screening of immunomodulatory agents and biomarkers. Methods 3D tumor microtissues were cultivated using a hanging drops system. Human non-small-cell lung cancer cell lines were incubated for 7 days to form microtissues. On day 5, peripheral blood mononuclear cells (PBMC) were added with or without interleukin-2 (IL-2) for 24 or 48 h. Viability of cancer cells and the infiltrating PBMC subpopulations were investigated by flow cytometry. Aggregation of tumor cells and PBMC and the infiltration of the PBMC into the tumor microtissues were analyzed by immunohistochemistry. Quantification of infiltration was measured by applying the TissueFAXS system. Results Immunohistochemistry revealed PBMC infiltration in all cell lines which increased under IL-2 stimulation. Analysis of infiltrating populations showed both lymphocyte subpopulations and monocytes within the tumor microtissues. In all three co-cultures, CD3+CD8+ and CD3+CD8+CD45R0+CD28+ lymphocytes were increased with IL-2, whereas CD3+CD8+CD45R0-CD28+ PBMCs were decreased with and without IL-2 stimulation. Conclusion In summary, we present a novel cell culture system to study the interaction between cancer cells and immune cells in 3-dimensional microtissues. In addition, we report for the first time an in vitro infiltration assay based on 3D microtissues. This model has the potential to provide a tool for ex-vivo drug testing and biomarker screening of immunomodulatory agents.

Because of recent failures of novel drugs in phase II and III trials innovative in vitro models are needed that may predict drug efficacy in patients. Although, numerous modifications of traditional cell culture and animal models were undertaken significant improvements in cell culture- based drug development have not been achieved. Three-dimensional (3D) cell culture may represents an modern alternative to bridge the gap between 2D cell cultures and animal models.

Aviscumine, a recombinant lectin I, has been identified as an immunomodulatory agent within a new class of ribotoxic stress-inducing anticancer substances that have demonstrated efficacy in phase I/II trials. The aim of the present study was to elucidate the presumed effect of aviscumine on enhancing human natural killer (NK) cell antitumor cytotoxicity. To measure the effect of aviscumine on human NK cell cytotoxicity, chromium-51-release assays against K-562 cells were performed with isolated NK cells from the whole blood of 34 healthy volunteers. Two effector-to-target cell ratios (12.5:1 and 25:1) were used by two independent investigators with a focus on the concentration-dependent effect (0.5 vs. 1 ng/ml aviscumine), reproducibility (first vs. second investigator) and the specificity of the effect by comparison to a heat-inactivated aliquot and interleukin 2 (IL-2) stimulation (10 ng/ml). The mediation of the effect via degranulation was demonstrated by flow cytometric analyses of CD107α expression. Statistics were performed with SPSS using Student's t-tests for normally distributed data. Aviscumine induced a significant and reproducible, concentration-dependent increase in NK cell cytotoxicity (n=22; P

This work evaluated gene expression differences between a hanging-drop 3D NSCLC model and 2D cell cultures and their in-vivo relevance by comparison to patient-derived data from The Cancer Genome Atlas. Gene expression of 2D and 3D cultures for Colo699 and A549 were assessed using Affymetrix HuGene 1.0 ST gene chips. Biostatistical analyses tested for reproducibility, comparability and significant differences in gene expression profiles between cell lines, experiments and culture methods. The analyses revealed a high interassay correlation within specific culture systems proving a high validity. 979 genes were altered in A549 and 1106 in Colo699 cells due to 3D cultivation. The overlap of changed genes between the cell lines was small (149), but the involved pathways in the reactome and GO- analyses showed a high overlap with DNA methylation, cell cycle, SIRT1, PKN1 pathway, DNA repair and oxidative stress as well known cancer-associated representatives. Additional specific GSEA-analyses revealed changes in immunologic and endothelial cell proliferation pathways, whereas hypoxic, EMT and angiogenic pathways were downregulated. Gene enrichment analyses showed 3D-induced gene up-regulations in the cell lines 38 to be represented in in-vivo samples of NSCLC patients using data of The Cancer Genome Atlas. Thus, our 3D NSCLC model might provide a tool for early drug development and investigation of microenvironment-associated mechanisms. However, this work also highlights the need for further individualization and model adaption to address remaining challenges.

Insights into tumour biology of breast cancer have led the path towards the introduction of targeted treatment approaches; still, breast cancer-related mortality remains relatively high. Efforts in the field of basic research revealed new druggable targets which now await validation within the context of clinical trials. Therefore, questions concerning the optimal design of future studies are becoming even more pertinent. Aspects such as the ideal end point, availability of predictive markers to identify the optimal cohort for drug testing, or potential mechanisms of resistance need to be resolved. An expert panel representing the academic community, the pharmaceutical industry, as well as European Regulatory Authorities met in Vienna, Austria, in November 2012, in order to discuss breast cancer biology, identification of novel biological targets and optimal drug development with the aim of treatment individualization. This article summarizes statements and perspectives provided by the meeting participants.

In the EU, the centralized procedure (CP) of the EMA is mandatory for marketing authorization for innovative anticancer drugs. However, numerous independent healthcare systems are in operation across the EU, and each HTA body follows its own methodologies and scientific value judgements in the assessment of the added value of an innovative anticancer drug. Initiatives to improve the interface between the different stakeholders are currently on the way, but are unlikely sufficient enough to overcome these fundamental problems.

Cancer proteomics is a rapidly developing field and promises to accelerate the discovery of new diagnostic, prognostic and therapy-related biomarkers. Increasingly, the mechanisms of action of cancer drugs are defined at the molecular level. The possibility of detecting hundreds and thousands of proteins at the same point of time is a tool to define proteins associated with response or resistance to therapy. This strategy has been applied successfully in cell culture models and is implemented as translational research tool in early clinical trials of new anti-cancer agents. This review aims to summarize basic concepts and techniques of proteome analysis, its challenges and limitations, and discusses the opportunities for cancer therapy.

Safety of aviscumine by subcutaneous route was assessed in patients with advanced cancer refractory to chemotherapy. Patients with progressive disease received escalating doses twice weekly Treatment of the accrued 26 patients (10 colorectal cancer (CRC), 6 soft tissue sarcoma (STS), 5 melanoma (MM), 5 others) was well tolerated without substance-related grade 3 or 4 toxicities. Grade 1/2 toxicities were predominantly injection site reactions. Aviscumine lacked dose-limiting toxicity (DLT) up to a maximal dose of 10 ng/kg. An increase of interleukin-1 beta and interferon-gamma from baseline was seen in the patient's plasma between the 1st and 11th injection. Highest release of both cytokines was in the dose range of 45.9 ng/kg. Interferon-gamma was not detected after doses higher than 6 ng/kg. Eight patients (5 CRC, 1 MM, 1 STS, 1 RCC) had disease stabilisation for 79-250 days (median122 days) associated with an increase of interleukin (IL)-1 beta and interferon (IFN)-gamma. Aviscumine was well tolerated and appeared to possess clinical activity at a biologically active dose between 4 and 6 ng/kg. (C) 2008 Elsevier Ltd. All rights reserved.

PURPOSE: To throw light on some of the issues in the choice of therapy for patients with metastatic colorectal cancer (mCRC) and to provide consensus recommendations on which combination(s) and sequence(s) of systemic therapy to be used in different treatment situations. MATERIAL AND METHODS: An expert panel comprising clinicians from Austria, Belgium, the Czech Republic, Germany, Hungary, Italy, Romania, Slovakia, Slovenia, Spain and the UK with expertise in medical oncology, clinical oncology, surgery and dermatology, and specialist knowledge of the treatment of patients with CRC was convened by the Central European Cooperative Oncology Group (CECOG) in Vienna in 2007. Members were then asked to agree on a consensus statement following a period of discussion at the end of a series of presentations. RESULTS AND CONCLUSIONS: The consensus recommendations for the state-of-the-art treatment of colon cancer arrived at by an "expert panel" of clinicians were that: oral 5-fluorouracil (5-FU) prodrugs or protracted intravenous infusion of the antimetabolite are preferable to bolus administration; all active drugs (fluoropyrimidines, irinotecan, oxaliplatin, bevacizumab, cetuximab) should be used during strategic patient management; after neoadjuvant treatment and consultation of an interdisciplinary team, surgery should be considered for metastatic disease wherever possible and "fit" elderly patients should not be denied the same treatment as younger patients.

This study aimed to characterize the antitumor activity of 5-Chloro-N-{2-[2-(4-chloro-phenyl)-3-methyl-butoxy]-5-trifluoromethyl-phenyl}-2-hydroxy-benzamide (CTFB), a novel anticancer agent, in head and neck cancer cell lines, FaDu, SCC-25 and cisplatin-resistant CAL-27. CTFB was generated as a result of an extensive medicinal chemistry effort on a lead compound series discovered in a high-throughput screen for inducers of apoptosis. All cell lines showed significant growth delay in response to CTFB treatment at a concentration of 1 μmol/L with 17.16 ± 2.08%, 10.92 ± 1.22%, and 27.03 ± 1.86% of cells surviving at 120 h in FaDu, CAL-27, and SCC-25, respectively. To define proteins involved in the mechanism of action of CTFB, we determined differences in the proteome profile of cell lines before and after treatment with CTFB using two-dimensional difference gel electrophoresis followed by computational image analysis and mass spectrometry. Eight proteins were found to be regulated by CTFB in all cell lines. All these proteins are involved in cytoskeleton formation and function and/or in cell cycle regulation. We showed that CTFB-induced cell growth delay was accompanied by cell cycle arrest at the G0-G1 phase that was associated with the up-regulation of p21/WAF1 and p27/Kip1 expression and the down-regulation of cyclin D1. Furthermore, we showed that activity of CTFB depended on the down-regulation of nuclear factor-κB (NF-κB) and NF-κB p65 phosphorylated at Ser536. The level of proteasome activity correlated with the response to CTFB treatment, and the down-regulation of NF-κB is accompanied by enhanced proteasome activity in all investigated head and neck cancer cell lines. In this report, we show that CTFB reveals multiple effects that lead to delayed cell growth. Our data suggest that this compound should be studied further in the treatment of head and neck cancer. [Mol Cancer Ther 2007;6(6):1898–908]

Epidermal growth factor (EGF) receptor (EGFR) signal transduction is organized by scaffold and adaptor proteins, which have specific subcellular distribution. On a way from the plasma membrane to the lysosome EGFRs are still in their active state and can signal from distinct subcellular locations. To identify organelle-specific targets of EGF receptor signaling on endosomes a combination of subcellular fractionation, two-dimensional DIGE, fluorescence labeling of phosphoproteins, and MALDI-TOF/TOF mass spectrometry was applied. All together 23 EGF-regulated (phospho)proteins were identified as being differentially associated with endosomal fractions by functional organelle proteomics; among them were proteins known to be involved in endosomal trafficking and cytoskeleton rearrangement (Alix, myosin-9, myosin regulatory light chain, Trap1, moesin, cytokeratin 8, septins 2 and 11, and CapZbeta). Interestingly R-Ras, a small GTPase of the Ras family that regulates cell survival and integrin activity, was associated with endosomes in a ligand-dependent manner. EGF-dependent association of R-Ras with late endosomes was confirmed by confocal laser scanning immunofluorescence microscopy and Western blotting of endosomal fractions. EGFR tyrosine kinase inhibitor gefitinib was used to confirm EGF-dependent regulation of all identified proteins. EGF-dependent association of signaling molecules, such as R-Ras, with late endosomes suggests signaling specification through intracellular organelles.

The culmination of over a century’s work to understand the role of the immune system in tumor control has led to the recent advances in cancer immunotherapies that have resulted in durable clinical responses in patients with a variety of malignancies. Cancer immunotherapies are rapidly changing traditional treatment paradigms and expanding the therapeutic landscape for cancer patients. However, despite the current success of these therapies, not all patients respond to immunotherapy and even those that do often experience toxicities. Thus, there is a growing need to identify predictive and prognostic biomarkers that enhance our understanding of the mechanisms underlying the complex interactions between the immune system and cancer. Therefore, the Society for Immunotherapy of Cancer (SITC) reconvened an Immune Biomarkers Task Force to review state of the art technologies, identify current hurdlers, and make recommendations for the field. As a product of this task force, Working Group 2 (WG2), consisting of international experts from academia and industry, assembled to identify and discuss promising technologies for biomarker discovery and validation. Thus, this WG2 consensus paper will focus on the current status of emerging biomarkers for immune checkpoint blockade therapy and discuss novel technologies as well as high dimensional data analysis platforms that will be pivotal for future biomarker research. In addition, this paper will include a brief overview of the current challenges with recommendations for future biomarker discovery.

The incidence of cancer will increase within the next 5 years up to 15 million patients per year worldwide with a further increasing tendency. Therefore, there is a requirement for more emphasis on cancer prevention, research and effective anticancer drugs including a rapid licensing and market availability for the patients. In the EU, the centralized procedure (CP) of the European Medicine Agency (EMA) is mandatory for marketing authorization for anticancer drugs. A CP will result in one marketing authorization for all Member States granted by the European Commission. At variance, numerous independent healthcare sys-tems are in operation across the EU, and each Health Technology Assessment (HTA) body follows its own methodologies and scientific value judgements in the assessment of the ad-ditional clinical benefit of a new anticancer drug. Payer organisations in the various member states consider these assessments to a varying degree as input in their pricing and reim-bursement negotiations. At the same time international reference pricing and parallel trade have an inherent tendency to establish rather uniform price levels across member states. Consequently, drug access for patients differs considerably within the EU. Initiatives to im-prove the interface between the different stakeholders are currently on the way, but are unlikely sufficient to overcome these fundamental problems.

Rituximab (RTX), a chimeric human anti-CD20 monoclonal antibody, is currently employed in the treatment of malignant non-Hodgkin's lymphoma (NHL) either alone or in combination with other cytotoxic approaches. The present study examines the effects of ionizing radiation in combination with RTX on proliferation and apoptosis development in B-lymphoma RL and Raji cells. RTX was used at a concentration of 10 microg/mL 24 hours prior to irradiation at a single dose of 9 Gy. CD20 expression, cell viability, apoptosis, mitochondrial membrane potential and apoptosis-related proteins were evaluated in the treated B cells. The constitutive level of CD20 expression in RL and Raji lymphoma cells did not play an essential role in RTX-induced cell growth delay. Both lymphoma cells showed similar inhibition of cell proliferation without apoptosis development in response to RTX treatment. Exposure to ionizing radiation induced cell growth delay and apoptosis in RL cells, whereas Raji cells showed moderate radio-resistance and activation of cell growth at 24 hours after irradiation, which was accompanied by increased radiation-triggered CD20 expression. The simultaneous exposure of lymphoma cells to ionizing radiation and RTX abrogated radioresistance of Raji cells and significantly enhanced cell growth delay and apoptosis in RL cells. X-linked inhibitor of apoptosis protein (XIAP) and the inducible form of heat shock protein 70 (Hsp70) were positively modulated by RTX in combination with ionizing radiation in order to induce apoptosis. Furthermore, it was demonstrated that mitochondrial membrane potential dissipation is not an essential component to induce apoptosis-inducing factor (AIF) maturation and apoptosis. Our results show that RTX-triggered enhancement of radiation-induced apoptosis and cell growth delay is achieved by modulation of proteins involved in programmed cell death.

The epidermal growth factor receptor (EGFR) is overexpressed in the majority of colorectal carcinomas and represents a target for therapeutic interventions with signal transduction inhibitors. We investigated the ability of CI-1033 to induce apoptosis and inhibition of proliferation in the colorectal cancer cell lines DiFi and Caco-2, which both express high levels of EGFR. While in Caco-2 cells CI-1033 treatment at a concentration 0.1 microM for 72 hours demonstrated only antiproliferative (53.7 +/- 4.3%) but no pro-apoptotic effects, treatment of DiFi cells resulted in a reduced proliferation rate (31.4 +/- 3.1%) and in apoptosis (44.2 +/- 8.9%). In order to define proteins involved in the regulation of apoptosis, we aimed to determine differences in the proteome profile of both cell lines before and after treatment with CI-1033. Cellular proteins were analyzed by 2-D gel electrophoresis followed by computational image analysis and mass spectrometry. Our data show that DiFi cells differ from Caco-2 cells in nine significantly upregulated proteins, and their potential role in apoptosis is described. We demonstrate that induction of apoptosis was triggered via caspase-independent pathways. Overexpression of leukocyte elastase inhibitor (LEI) and translocation of cathepsin D to the cytosol accompanied by upregulation of other defined proteins resulted in Bax-independent AIF translocation from mitochondria into the nucleus and apoptosis. Definition of these proteins can pave the way for functional studies and contribute to a better understanding of the effects of CI-1033 and the pathways of caspase-independent cell death.

Biomarkers that predict response to therapy with inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase remain largely uncharacterized. In order to define proteins involved in potential resistance mechanisms, we examined the effect of gefitinib (ZD1839, Iressa) in the EGFR-positive colon cancer cell lines Caco-2, DiFi, HRT-18 and HT-29. None of them exhibited an activating mutation in exons 19 or 21 of EGFR. Proteome profiling with two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometry revealed 12 proteins differentially expressed in responsive and non-responsive cells. These proteins are involved in metabolic pathways, partially relevant in malignant growth and four of them are known to interact with the EGFR signalling pathway. Ubiquitin carboxyl-terminated hydrolase isozyme L1 (UCH-L1) and galectin-3 are overexpressed in the responsive cell line Caco-2, whereas fatty acid-binding protein (E-FABP) and heat shock protein (hsp) 27 are expressed more in the resistant cell lines HRT-18 and HT-29 suggesting a role in non-responsiveness of cells to gefitinib.

We assessed the tolerability, safety, pharmacokinetics and dose-limiting toxicity (DLT) of the recombinant humanized IgG4 anti-vascular endothelial growth factor (VEGF) monoclonal antibody, HuMV833, in patients with advanced cancer. Cohorts of patients with progressive solid tumours received escalating doses of HuMV833 as a 1-h intravenous (I.V.) infusion on days 1, 15, 22, and 29. Twenty patients (median Eastern Cooperative Oncology Group (ECOG) score 1) were accrued. HuMV833 infusions were well tolerated and there were no grade III or IV toxicities definitely related to the antibody. Grade I or II toxicities probably related to the antibody included fatigue, dyspnoea and rash. There were two episodes of asymptomatic hypocalcaemia, one at grade III and one grade IV, which were recorded in early follow-up. There were eight grade I episodes of asymptomatic elevation of activated partial thromboplastin time (APTT) and two grade III events; one in a patient receiving 1 mg/kg and the other receiving extended doses of 10 mg/kg. Pharmacokinetic analysis revealed a non-linear kinetic and an elimination half-life of between 8.2 (0.3 mg/kg) and 18.7 (10 mg/kg) days. One patient with ovarian cancer experienced a partial response (PR) of 9 months duration and eight had disease stabilisation (SD) including one patient with colorectal carcinoma whose disease was stable for 14 months. In 13 of the 14 samples taken from 12 patients, the plasma concentration of hepatocyte growth factor (HGF) was reduced 24 h after drug administration. HuMV833 is safe and lacked DLT at doses up to 10 mg/kg on this schedule. Multiple doses were well tolerated, despite occasional asymptomatic elevations in APTT. By combining pharmacokinetic, pharmacodynamic and toxicity data, we can identify doses of 1 and 3mg/kg for further investigation. HuMV833 appears to possess some clinical activity.

Einleitung Aufgrund des besseren Verstandnisses molekularer Mecha-nismen, die der Kanzerogenese zugrunde liegen, hat sich in der klinischen Entwicklung neuer Krebstherapeutika in den letzten Jahren ein grundsatzlicher Wandel ergeben. Bei der Prufung neuer Substanzen in praklinischen und klinischen Studien steht daher heute nicht mehr (nur) die Histopatho-logie eines Tumors, sondern vielmehr die von den transfor-mierten Zellen exprimierten Zielstrukturen («targets») im Mittelpunkt. Einige zielgerichtete Wirkstoffe (so genannte «targeted thera-pies») sind inzwischen zugelassen und werden bereits zur Behandlung diverser solider und hamatologischer Neoplasien eingesetzt. Die Tatsache, dass sich mehrere der neuen Sub-stanzen in fruhen Phase-I/II-Studien als sehr vielverspre-chend erwiesen haben, in spateren Phase-III-Studien aber teilweise enttauschende Ergebnisse erzielt wurden, zeigt, dass die klinische Entwicklung neuer zielgerichteter Therapeutika nicht unproblematisch ist. Es ist inzwischen klar geworden, dass die Prinzipien, die der Entwicklung konventioneller Zytostatika zugrunde liegen, nicht ohne weiteres auf die Ent-wicklung der neuen Substanzen ubertragen werden konnen [1, 2]. Vielmehr muss der Entwicklungsprozess individuell auf die einzelne Substanz sowie deren Wirkmechanismus und biologischen Effekte zugeschnitten werden. Auch ist zu beachten, dass molekulare Zielstrukturen einer Tumorzelle nicht statisch sind, sondern im Verlauf der Tumorerkrankung einer Dynamik unterliegen konnen. Da die zielgerichteten Wirkstoffe teuer sind, ist es zudem vordringlich, die Therapie zu individualisieren, d.h. ihren Einsatz auf Patienten zu begrenzen, deren Tumoren das entsprechende Target expri-mieren und bei denen daher ein Ansprechen auf die jeweilige Substanz angenommen werden kann. Der translationalen Forschung, mit deren Hilfe die Target-Inhibition durch neue Wirkstoffe gemessen und Biomarker als Pradiktoren fur ein Ansprechen identifiziert werden sollen, wird daher zukunftig ein hoher Stellenwert zukommen.

The present study examines the effects of ionizing radiation in combination with rituximab (RTX), a chimeric human anti-CD20 monoclonal antibody, on proliferation, cell cycle distribution and apoptosis in B-lymphoma RL and Raji cells. Exposure to ionizing radiation (9 Gy) induced cell growth delay and apoptosis in RL cells, whereas Raji cells showed moderate radio-resistance. The simultaneous exposure of lymphoma cells to ionizing radiation and RTX (10 microg/mL) markedly enhanced apoptosis and cell growth delay in RL and Raji cells. Cooperative antiproliferative and apoptotic effects of RTX and radiation were achieved through the inhibition of c-myc and bcl-XL expression. Furthermore, RTX-modulated expression of cell cycle regulating proteins, such as p53, p21/WAF1, p27/KIP1, contributed to the development of radiation-induced cell killing and growth arrest. Each NHL cell line that underwent apoptosis induced by combination treatment revealed enhanced caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage as compared to only irradiated cells. These findings show that rituximab synergistically enhances radiation-induced apoptosis and cell growth delay through the expression of proteins involved in the programmed cell death and cell cycle regulation pathways.

At American Society of Clinical Oncology’s 2013 meeting, the clinical development of innovative drugs targeting the immune system and progress in biomarker research represented some of the highlights. During recent years, immunomodulatory agents have become a very active field in clinical cancer research. After the successful development of the anti-cytotoxic T-lymphocyte antigen 4 (CTLA4) antibody ipilimumab in melanoma, the “programmed cell death protein 1” pathway is expected to become a cornerstone in several tumor types. One key challenge for anticancer treatment, in general, is the definition of biomarkers and the further individualization of therapy. Progress in molecular phenotyping and in serum proteomics as predictive markers was reported.

During the last decade, numerous new agents were introduced into the therapy of metastatic colorectal cancer. Treatment duration and maintenance therapy, however, still remain a controversial issue. More and more results from clinical trials become available that assess the role of maintenance chemotherapy in combination with the angiogenesis inhibitor bevacizumab and epidermal growth factor inhibitors. Taken together, these data suggest that some form of maintenance therapy leads to a better patient outcome and is thus appropriate. Our challenge remains the need to identify and predict the required therapeutic intensity whether by biomarkers or clinical parameters. As also side effects may increase due to drug combinations, quality of life issues need to be carefully considered for each individual patient.

Aviscumine is an Escherichia coli-derived recombinant type II ribosome-inactivating protein with potent antitumor activity in vitro and in vivo. It is the recombinant counterpart of natural mistletoe lectin-I. The current study was performed to determine the safety profile, dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of the intravenous (i.v.) administration of aviscumine in cancer patients. Translational research included the evaluation of pharmacokinetics and monitoring of plasma cytokine and anti-aviscumine antibody induction after administration of the drug.Aviscumine was given twice weekly as a 1 h central i.v. infusion in patients with advanced, refractory progressive, solid malignant tumors who had not been previously exposed to natural mistletoe preparations. They had histologically or cytologically verified disease, wereor =18 years old, had an Eastern Cooperative Oncology Group performance statusor =2 and adequate bone marrow, liver and renal function. DLT was defined as any non-hematological grade 3-4 toxicity (National Cancer Institute Common Toxicity Criteria version 2.0), neutrophil count500/microl foror =7 days, febrile neutropenia or thrombocytopenia grade 4. The MTD was defined as the dose at which20% of patients experienced DLT during the first treatment cycle. The Continual Reassessment Method was used to determine the number of patients required per dose level.Forty-one fully eligible patients (19 male, 22 female) with a median age of 56 years (range 37-74) were enrolled. Colorectal, ovarian, renal cell and breast cancer were the most common tumor types. Dose levels of aviscumine ranged from 10 to 6400 ng/kg. The median number of cycles was two (range one to eight). Common clinical toxicities in cycle 1 were fatigue, fever, nausea, vomiting and allergic reactions. Fatigue grade 3 was dose limiting in one of six patients at 4000 ng/kg and reversible grade 3 liver toxicity (elevation in alkaline phosphatase, transaminases and/or gamma-glutamyltransferase) occurred in one of 10 patients at 4800 ng/kg and in two of five patients at 6400 ng/kg. The best response (RECIST criteria) was stable disease in 11 patients, lasting for two to eight cycles. The pharmacokinetic evaluation revealed a short alpha half-life of 13 min and linear kinetics on dose levelsor =1600 ng/kg. Aviscumine stimulated the immune system with a release of cytokines such as interleukin (IL)-1beta, IL-6 and interferon-gamma, and induced immunoglobulin (Ig) G- and/or IgM-anti-aviscumine antibodies of uncertain clinical relevance.The recommended dose for further clinical trials is 5600 ng/kg twice weekly. Based on the short half-life of the recombinant protein observed in this trial, the exploration of prolonged infusion schedules of aviscumine is warranted.

The epithelial-to-mesenchymal transition (EMT) is highly involved in the development of metastases. EMT transforms epithelial carcinoma cells into mesenchymal-like cells, characterized by increased cell migration and invasiveness. Transforming growth factor β (TGFβ) appears to be crucial in this process. Metformin and salinomycin have demonstrated an EMT inhibitory effect. The current experiments indicate that these substances specifically inhibit TGFβ-induced EMT in non-small cell lung cancer (NSCLC) cell lines. The NSCLC cell lines A549 and HCC4006 were stimulated with TGFβ for 48 h to induce EMT. Metformin or salinomycin was added simultaneously with TGFβ to inhibit TGFβ-induced EMT. Western blot analyses of E-cadherin and vimentin were performed to detect changes in EMT marker expression, and a wound healing assay was conducted to determine the potential effects on cell migration. The effects of the two drugs on cell viability were also investigated using MTS tetrazolium dye assays. The results revealed that cells undergoing EMT by application of TGFβ exhibited a downregulation of E-cadherin and an upregulation of vimentin protein expression on western blot analyses, and an increased capacity for cell migration. Simultaneous application of TGFβ and metformin specifically inhibited EMT and increased E-cadherin expression. At the higher dose tested, salinomycin also inhibited EMT, despite an increase in vimentin expression in the two cell lines. Furthermore, metformin and salinomycin, at the two concentrations tested, inhibited cell migration. These findings demonstrate that metformin and salinomycin are able to block EMT and inhibit EMT-induced cell migration. Thus, these two substances are novel EMT inhibiting drugs that have the potential to specifically control EMT and metastatic spread in NSCLC.

Early drug development at EORTC has always been subject to structural changes to adapt to the rapid changes that occur in oncological drug development. The expertise of early drug developers has always been cross-fertilised with disease-/tumour-oriented groups and also backwards to the laboratory research groups. This results in the establishment of a solid and dedicated network of medical oncologists with focused expertise in cancer drug development. The EORTC Data Center is fully equipped with all expertise to support clinical research activities and includes regulatory, safety, and quality assurance desks. The EORTC New Drug development Programme (NDDP) provides methodological expertise to early clinical trials and coordinates phase I and phase II studies addressing various approaches. Through NDDP, the early clinical groups and the disease-/tumour-oriented groups have created specific networks to address early drug development in specific tumour types. This results in very efficient networks which have the resources and the patients to address and conduct challenging clinical trials in a standardised fashion ensuring the highest standards in cancer treatment.

In previous studies we have shown that the expression of the transformingHa-ras oncogene in NIH 3T3 fibroblasts stimulates cellular calcium entry, which triggers oscillatory calcium induced calcium release from internal stores. The intracellular calcium oscillations lead to cytoskeletal remodeling by actin stress fiber depolymerization and activation of the Na+/H+ exchanger thus mediating cell swelling and intracellular alkalosis, both important mitogenic signals. This is evidenced by abrogation of Ha-ras induced growth factor independent cell proliferation by interference with any of these events, i.e. by inhibition of cellular calcium entry or inhibition of the Na+/H+ exchanger.As shown in this study, simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key enzyme for cholesterol biosynthesis, is able to prevent these events following the expression of the transforming Ha-ras oncogene. We show, that simvastatin inhibits farnesylation dependent membrane translocation of a CAAX motive bearing yellow fluorescent protein and suppresses Ha-ras stimulated cellular calcium influx, which can be identified as capacitative calcium entry. In addition simvastatin is able to block regulatory volume decrease channels and to suppress the cytoskeletal remodeling, intracellular alkalinization, increase in cell volume and growth factor independent cell proliferation induced by the oncogene.Thus simvastatin is able to prevent crucial cellular events following expression of the transforming Ha-ras oncogene.

The gene of the leukocyte-specific transcript (LST1) is encoded within the TNF region of the human MHC. The LST1 gene is constitutively expressed in leukocytes and dendritic cells, and it is characterized by extensive alternative splicing. We identified 7 different LST1 splice variants in PBMC; thus, 14 LST1 splice variants (LST1/A-LST1/N) have been detected in various cell types. These isoforms code for transmembrane as well as soluble LST1 proteins characterized by two alternative open reading frames at their 3′ end. We demonstrate the presence of the transmembrane variant LST1/C on the cell surface of the monocytic cell lines U937 and THP1. Recombinant expression of LST1/C permitted its profound inhibitory effect on lymphocyte proliferation to be observed. In contrast, the alternative transmembrane variant LST1/A, the extracellular domain of which shows no amino acid sequence homology to LST1/C exerted a weaker but similar inhibitory effect on PBMC. These data demonstrate the protein expression of LST1 on the cell surface of mononuclear cells, and they show an inhibitory effect on lymphocyte proliferation of two LST1 proteins although they have only a very short amino acid homology.

Myelodysplastic syndromes (MDS) caused by a clonal hematopoietic stem cell disorder progress to either overt leukemia or cytopenia, which leads to lethal infection or bleeding. Although several clinical trials have attempted to reverse cytopenia by using hematopoietic growth factors (HGF), success has been limited due in part to a limited understanding of the role of HGF in MDS progression. The FLT3 ligand, which binds to and activates the FLT3 receptor, does not have a stimulatory effect on hematopoietic cells, but can synergize with other HGF to support the expansion of both immature and committed progenitors. Using ELISA technology we measured endogenous serum levels in 93 patients with MDS: 29 RA, 1 RARS, 31 RAEB, 23 RAEBt, 9 CMML. 48.3% of RA patients' sera had significantly elevated FLT3 ligand levels ranging from 404 to 5735 pg/ml, whereas none of the RAEB, RAEBt, or CMML patients sera had levels different from controls. No significant correlation was found between FLT3 ligand levels and peripheral blood counts, bone marrow cellularity, age, cytogenetic abnormalities, or survival. Our data suggest that FLT3 ligand levels can be upregulated early in the course of MDS, which may represent an appropriate response to a decreased number of normal progenitors, or alternatively a dysregulated HGF system.

Adjuvant treatment of stage II colon cancer remains an issue of controversy. Though there is evidence indicating that adjuvant therapy is associated with a limited survival benefit, large clinical trials published so far did not provide enough evidence to regard adjuvant therapy of stage II colon cancer as standard of care. The ultimate clinical decision should be based on the presence of high-risk prognostic features, the evidence supporting treatment, the anticipated side effects and morbidity of treatment as well as patient preferences. Considering all these circumstances, regimen containing oxaliplatin together with 5-FU/leukovorin may be regarded as standard therapy. Stage II rectal cancer represents an excellent example for the need of close collaboration between both surgeons and medical oncologists together with radiation oncologists. The combination of radiotherapy and chemotherapy given in the neoadjuvant or adjuvant setting can be regarded as standard therapy. The potential benefit of the addition of oxaliplatin is currently under investigation. The goal of ongoing studies is to investigate whether combination of chemotherapy with therapeutic antibodies such as cetuximab or bevacizumab will become standard of care in the adjuvant setting.

1. Stimulation of chemotaxis of human polymorphonuclear leucocytes (PMNs) with the chemoattractive peptide fMLP (N-formyl-Met-Leu-Phe) is paralleled by profound morphological and metabolic alterations like changes of intracellular pH (pHi) and cell shape. The present study was performed to investigate the interrelation of cell volume (CV) regulatory ion transport, pHi and migration of fMLP stimulated PMNs. 2. Addition of fMLP to PMNs stimulated directed migration in Boyden chamber assays and was accompanied by rapid initial intracellular acidification and cell swelling. 3. Inhibition of the Na+/H+ exchanger suppressed fMLP stimulated cell migration, accelerated the intracellular acidification and inhibited the fMLP-induced cell swelling. 4. Step omission of extracellular Na+ caused intracellular acidification, which was accelerated by subsequent addition of gastric H+/K+ ATPase inhibitor SCH 28080, or by omission of extracellular K+ ions. In addition Na+ removal caused cell swelling, which was further enhanced by fMLP. 5. H+/K+ATPase inhibitors omeprazole and SCH 28080 inhibited stimulated migration and blunted the fMLP-induced increase in CV. 6. Increasing extracellular osmolarity by addition of mannitol to the extracellular solution caused cell shrinkage followed by regulatory volume increase, partially due to activation of the Na+/H+ exchanger. In fMLP-stimulated cells the CV increase was counteracted by simultaneous addition of mannitol. Under these conditions the fMLP stimulated migration was inhibited. 7. The antibacterial activity of PMNs was not modified by Hoe 694 or omeprazole. 8. Western analysis with a monoclonal anti gastric H+/K+ ATPase beta-subunit antibody detected a glycosylated 35 kD core protein in lysates of mouse and human gastric mucosa as well as in human PMNs. 9. The results indicate that fMLP leads to cell swelling of PMNs due to activation of the Na+/H+ exchanger and a K+-dependent H+-extruding mechanism, presumably an H+/K+ ATPase. Inhibition of these ion transporters suppresses the increase in CV and precludes PMNs from stimulated migration.