Supplement 248

36th Seminar of the Austrian Society for Surgical Research Vienna, November 22–24, 2012 Guest Editor: M. Bergmann, Vienna, Austria

“Bench to Bedside” in Surgical Oncology Kindly supported by

Eur Surg Vol. 44 · Supplement Nr. 248 · 2012

Published online: 18 November 2012

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36th Seminar of the Austrian Society for Surgical Research

Circulating Tumor Cells in Ovarian Cancer: a Study of the OVCAD Consortium E. Obermayr1,7, I. Alpers2, D. Pils1, I. Braicu3, T. Van Gorp4,5, S. Mahner6, J. Sehouli3, I. Vergote5, P. Speiser1, D. Cacsire-Castillo Tong1, B. Brandt2, R. Zeillinger1,7 Dept. of Obstetrics and Gynecology, Medical University of Vienna, Austria, 2 Inst. for Tumor Biology, University Medical Center HamburgEppendorf, Hamburg, Germany, 3 Dept. of Gynecology, European Competence Center for Ovarian Cancer, Campus Virchow Klinikum, Charité – Universitätsmedizin Berlin, Germany, 4 Dept. of Obstetrics and Gynaecology, MUMC+, GROW - School for Oncology and Developmental Biology, PO Box 5800, 6202AZ Maastricht, The Netherlands, 5 Div. of Gynaecological Oncology, Department of Obstetrics and Gynaecology, Universitaire Ziekenhuizen Leuven, Katholieke Universiteit Leuven, Belgium, 6 Dept. of Gynecology and Gynecologic Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 7 Ludwig Boltzmann Gesellschaft – Cluster Translational Oncology, Vienna, Austria 1

Aim •  Identification of biomarkers for circulating tumor cells (CTCs) in patients with epithelial ovarian cancer (EOC) •  evaluating the clinical impact of CTCs. Methods  mRNA markers were selected by a comparative microarray analysis of EOC tissues and volunteer blood and validated by RT-qPCR. Blood samples were analyzed for the selected markers and EpCAM by RT-qPCR (EOC FIGO stage II–IV: N = 216; volunteer blood: N = 39). Patients’ samples were collected at initial diagnosis (baseline) and six months after completion of platinumbased chemotherapy (follow-up). All samples were enriched for mononuclear cells (eventually containing CTCs) using a two- layer density gradient centrifugation. Samples were also analyzed with the CellSearchTM CTC Test or by immunocytochemistry with an antibody mixture. Results  11 mRNAs were identified as potential CTC markers. 25% of EOC patient samples were CTC-positive at baseline and 20% at follow-up. Cyclophilin C (PPIC) was the most frequently overexpressed gene (17 % baseline, 14 % follow-up), whereas EpCAM gene expression was only detected in few samples (2 % baseline, 1 % follow-up). The presence of PPIC+ CTCs at follow-up correlated with platinum resistance and PPIC+/CTC was a predictor of poor outcome (DFS: HR = 3.545, CI 1.815-6.926, p<0.001; OS: HR = 2.312, CI 1.117-4.784, p=0.024). Only 3 % of samples were classified CTC-positive with the CellSearchTM CTC Test at baseline, but 27 % were positive when stained with an antibody mixture without prior EpCAM-based enrichment. The nature of CTCs was proven by FISH analysis for amplifications on chromosomes 3 and 8. Conclusion  CTCs in EOC were found to lack EpCAM expression and to be very heterogeneous. The novel mRNA markers for the detection of CTCs allow for further characterization of particularly aggressive EOC. Mere enumeration of CTC should be replaced by molecular characterization. Moreover, the findings potentially contribute to the development of new targeted anti-PPIC therapies and thus, more personalized treatment strategies for EOC patients

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The combined use of known anti-viral reverse transcriptase inhibitors, AZT and DDI, induce anti-cancer effects at low concentrations Thomas Aschacher1, Florian Enzmann1, Sandra Sampl2, Brigitte Wolff1, Klaus Holzmann2,3, Michael Bergmann1,3  epartment of Surgery, Medical University of Vienna, Vienna, D Austria, 2 Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria, 3 Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria 1

A hallmark of tumor cell survival is the maintenance of elongated telomeres. It is known that antiviral reverse transcriptase inhibitors (RTI) such as azidothymidine (AZT) and didanosine (ddI) lead to telomere shortening at high, potentially toxic concentrations. We hypothesized that those drugs might have synergistic effects enabling successful therapy with low, non-toxic concentrations. Biological effects of AZT and ddI were analyzed at concentrations which correspond to minimal plasma levels achieved during human HIV therapy. Long term co-application of low dose AZT and ddI induced a significant shortening of telomeres in the tumor cell lines HCT-116, SkMel-28, MelJuso and Jurkat. Treatment of cells with both RTI, but not with single RTI led to a significant accumulation of γH2AX, to p53 phosphorylation and cell apoptosis in all cell lines. Oral low-dose dual RTI application but not low-dose single RTI application was associated with a significantly reduced tumor growth of HCT-116 cells in mice. This anti-proliferative activity of the combined use of AZT and ddI at low, clinically applicable concentrations warrants clinical testing in human solid cancer. Interestingly, treatment of cells with RTI led to an increase of LINE-1 in tumor cells. The similarity of LINE-1 and telomerase is discussed. Adapted from neoplasma 2012;14(1):44–53

p38MAPK inhibition prevents impairment of kidney function resulting from ischemia/reperfusion injury (IRI) Ashraf Muhammad Imtiaz1, Matthias Ebner1, Christoph Wallner1, Martina Haller1, Stephan Sickinger1, Martin Hermann2, 3, Afschin Soleiman4, Stefanie Vallant1, Christina Steger5, Gerald Brandacher1, Raimund Margreiter1, Jakob Troppmair1 Daniel Swarovski Research Laboratory, Department of Visceral-, Transplant- and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria, 2 Department of Anesthesiology and Critical Care Medicine, Innsbruck Medical University, Innsbruck, Austria, 3 Department of Pediatrics II, Innsbruck Medical University, Innsbruck, Austria, 4 Soleiman Pathologie, Hall, Austria, 5 Department for Pathology, Innsbruck Medical University, Innsbruck, Austria 1

In the course of solid organ transplantation excessive production of reactive oxygen species (ROS) is a major contributor to the development of ischemia/reperfusion injury (IRI). Preventing ROS actions through the use of antioxidants proved unsatisfactory in the clinical setting. In various in vivo (IR) and in vitro (hypoxia/reoxy-

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Supplement 248 genation, HR) models we consistently observed the activation of the stress kinase p38MAPK. Moreover, we obtained evidence linking the activation of p38MAPK to mitochondrial ROS accumulation and cell death. These data suggest that inhibition of p38MAPK could be therapeutically exploited for the prevention of IR-induced organ damage. Reperfusion following kidney clamping or transplantation was marked by a profound increase in the activity of p38MAPK and the putative effector MK2 which was significantly prevented by the p38MAPK inhibitor BIRB-796. p38MAPK inhibition almost completely prevented functional impairment caused by IR during kidney clamping as measured by reduced serum creatinine, urea, cystatin c and NGAL levels. p38MAPK inhibition also protected from oxidative damage and significantly reduced the percentage of tubular epithelial cell apoptosis, suggesting that the protection resulted from decreased redox stress and apoptotic cell death. Most importantly, a significant improvement in the kidney function could be achieved in the kidney transplant model. Inhibiting p38MAPK signaling during IR thus may provide a potent strategy for limiting IRI.

Conclusion:  Our study shows that the methylation marker profile of triple-negative breast cancers differs significantly between responders and non-responders to epirubicin and docetaxel-based neoadjuvant chemotherapy. If validated in an independent patient cohort, the results of our study could lead to further individualization of neoadjuvant chemotherapy in patients with triple-negative breast cancer.

Supported by funds from the FWF and the Jubiliäumsfond OeNB.



DNA methylation analysis and correlation with response to neoadjuvant epirubicin/ docetaxel chemotherapy in triple-negative breast cancer patients Marco Gardini, Walter Pulverer, Núria Llamas, Zsuzsanna Bago-Horvath, Rupert Bartsch, Andreas Weinhäusel, Gerda Egger, Thomas Bachleitner-Hofmann Medical University of Vienna, Department of Surgery Background and Aims:  Neoadjuvant systemic chemotherapy is a cornerstone in the multidisciplinary management of triplenegative breast cancer, i.e., breast cancer that lacks expression of hormone receptors and HER-2/neu. However, individual response to neoadjuvant chemotherapy is widely variable and accurate response prediction tools remain yet to be defined. Aberrant methylation of DNA has been described as a crucial step in breast cancer oncogenesis. Aim of the present study was to assess whether triplenegative breast cancers responding to epirubicin/docetaxel neoadjuvant chemotherapy display a methylation marker profile that is distinct from triple-negative tumors that do not respond to therapy. Patients and methods:  Twenty-four patients with histologically confirmed triple-negative breast cancer who received neoadjuvant chemotherapy with six cycles of epirubicin and docetaxel between January 1999 and December 2008 at the Medical University of Vienna were included into the present study. DNA was extracted from formalin-fixed, paraffin-embedded pre-therapeutic biopsies using laser capture microdissection. The quantity and quality of DNA was assessed by real time PCR assay followed by genome wide methylation analysis (>485,000 CpG sites) using the Infinium HumanMethylation450 BeadChip Kit (Illumina Inc. San Diego, California, U.S.). Results:  There were 11 responders and 13 non-responders in our patient population. DNA of sufficient quality for methylation analysis could be extracted from all samples. Genome-wide methylation analysis showed that the methylation profile of tumors that responded to neoadjuvant chemotherapy was significantly different from tumors that did not respond to neoadjuvant chemotherapy, with approximately 1500 CpG sites being differently methylated at a significance level of p < 0.005.

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MAPKAP kinase two over expression influences prognosis in gastrointestinal stromal tumors and associates with copy number variations on chromosome one and expression of p38 MAP kinase and ETV1 Peter Birner, Berthold Streubel, Sebastian F. Schoppmann Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria,

Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract. ETV1 has been proposed to be activated by KIT mutations in GIST, playing a key role in progression of this disease. The aim of the study was to evaluate the clinical role of ETV1 and associated proteins in GIST. Expression levels of ETV1, MAPKAP kinase two (MAPKAPK2), phosphorylated p38 MAP kinase (pp38), phosphorylated MSK1 (pMSK1), phosphorylated RSK1, COP1, and KIT protein were determined immunohistochemically in 139 GISTs. Sequence analysis of KIT, PDGFRA, and MAPKAPK2 and FISHs of ETV1 as well as chromosomes one and seven were done. Prominent ETV1 expression was seen in 50 % of GISTs, but no correlation with clinical outcome was found. Correlation of ETV1 expression and KIT mutation was seen in 60 % of cases. MAPKAPK2 over expression (n = 62/44.6  %) correlated with pp38 expression (P = 0.021, χ(2) test) and alterations of chromosome one (n = 17, P = 0.024, χ(2) test). In one of 20 sequenced cases with high MAKAPK2 expression, a putative damaging MAPKAPK2 gene mutation was found. All relapsing GISTs with very low/low risk according to Fletcher showed high MAPKAPK2 and KIT expression. MAPKAPK2 over expression was an independent prognostic factor for disease-free survival (P = 0.006, Cox regression). Our data show that ETV1 is not universally over expressed in GIST and seems to also be induced by pathways other than KIT mutation. Nevertheless, its clinical relevance is low. Over expression of ETV1 inhibitor MAPKAPK2 is associated with shorter survival in GIST, indicating a clinically relevant role of this gene not reported previously. Patients with low-risk GISTs showing MAPKAPK2 over expression might profit from early adjuvant tyrosine kinase inhibitor therapy. Adapted from clinical cancer research 2012;18(7):1879–87

HSP 27—a novel therapeutic target in melanoma? Stefan Blunder, Coralie Briand, Waltraud Jerney, Hubert Pehamberger, Christoph Hoeller, Nikolaus Schicher Department of Dermatology, Medical University of Vienna, Vienna, Austria

Heat shock proteins (Hsps) are molecular chaperones which are up regulated upon a broad variety of cell stress. Recent studies have shown that the up regulation of HSP27 is common in differ-

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36th Seminar of the Austrian Society for Surgical Research ent cancers and is linked to poor prognosis and metastasis. HSP27 interferes with apoptosis in the intrinsic as well as in the extrinsic pathway. Hence, it can confer resistance to apoptosis elicited through chemotherapeuticdrugs, resulting in decreased chemoand radiosensitivity of cancer.The aim of this study was to identify the relevance of HSP27 in melanoma using a specific antisense oligonucleotide (ASO). Cell proliferation, migration and apoptosis were used as readout-parameters. 518A2 (BRAF V600E), M24 (NRAS Q61R), MelJuso (NRAS Q61L), CHL1 (wt) and 607B (NRAS Q61K) melanoma cells were transfected with ASO or a scrambled control oligonucleotide (SCO) for two consecutive days. HSP27 protein expression status was determined by Western Blotting. MTS assays were used to assess cell death and proliferation; scratch assays were performed to test the impact on migration. Apoptosis was determined by Annexin-FACS staining and Caspase-3 Cleavage activity. The determined IC50 for the ASO was used to assess a potential sensitizing effect of ASO treatment towards chemotherapeutic agents. Treatment of melanoma cells with HSP27 ASO is an effective tool to downregulate HSP27 protein levels at low nanomolar concentrations. Further, ASO treatment clearly reduces cell proliferation and induces cell death through apoptosis, irrespective of the genetic background. In scratch assays, ASO transfection impairs migratory capabilities of melanoma cells. Upon ASO treatment melanoma cells show increased chemosensitivity towards cytotoxic drugs. These data underline the role of HSP27 in melanoma.Further validation in a xenotransplant melanoma mouse model is being performed. Correlation of HSP27 expression levels and disease stage of melanoma will be important to further assess the potential role of HSP27 directed agents in melanoma.

Miniaturised chemical proteomics to profile clinically-relevant kinase inhibitors in tumour needle biopsies Ivo Chamrád2#, Uwe Rix3#, Manuela Gridling1, Katja Parapatics1, André C. Müller1, Alexey Stukalov1, Giulio Superti-Furga1, Eric B. Haura3, Keiryn L. Bennett1 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria, 2 Palacký University, Olomouc, Czech Republic, 3 H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA 1

Background  Specific anticancer agents have been undeniably successful in the treatment of the disease, however, several issues still remain. Even when enormous efforts have been made to design and develop compounds affecting only given molecular nodes in cancer circuits, a strictly selective agent has yet to be discovered. The majority of novel, potential therapeutics display broader inhibitory profiles than originally envisaged. Affecting unexpected targets can lead to unwanted toxicity and cause adverse side effects. Alternatively, incidental drug promiscuity is not necessarily always harmful. Hence, for every drug, determining the specificity profile and delineating a plausible mechanism-of-action should be an integral part of the modern drug discovery pipeline. Chemical proteomics is a compound-centric affinity approach that utilises immobilised drugs to isolate protein interactors from complex protein mixtures. Identification of the specific protein sets is achieved via modern high-end mass spectrometry (MS). Research in our laboratory has previously achieved successful downscaling of the affinity procedure to 500 µg total protein [1]. The study presented here, entailed a further miniaturisation of the protocol to 100 µg—the range of material that can be obtained from a needle biopsy. The possibility to perform systematic chemical proteomics

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with material from core needle biopsies taken from patient tumours would revolutionise the field of personalised medicine. Methods  The broad-spectrum tyrosine kinase inhibitor bosutinib was chemically-modified and coupled to sepharose beads. Cell lysate was generated from the human K562 cell line (derived from chronic myelogenous leukaemia). Drug pulldowns were performed according to Fernbach et al. [1] except that the total quantity of input starting material was lowered to 100 µg protein. Experimental conditions such as protein concentration, bead bed volume, total quantity of drug, drug concentration on the beads, incubation time, agitation speed, elution volume, and quantity of trypsin used for protein digestion were assessed as a multi-dimensional matrix. Samples were analysed by LCMS on hybrid LTQ Orbitrap instruments and based on the number of kinases identified, the experimental conditions were optimised for drug pulldowns from low quantities of protein. Preliminary data  In the first phase of this study, the published protocol [1] was assessed with 100 µg protein input. Approximately 100 protein groups (98 and 120) were unambiguously identified in two replicates, with the target kinases accounted for approximately 20 % of all protein identifications. For specific interaction partners (approximately 30 kinases), many known major targets of bosutinib such as SRC kinases (LYN and YES), Bcr-Abl, CSK, BTK, GAK, EPHB4 and PTK2 were present. Functional annotation analysis of the proteins revealed an over-representation of gene ontology terms associated with kinase activity. These results were highly-encouraging, as lowering the protein input by 5-fold still resulted in the identification of a high proportion of kinases. Only ten fewer kinases were apparent as compared to the data obtained with 500  µg protein input. Phase two of the study involved a systematic assessment of several experimental metrics that can influence the result of the chemical proteomic experiment. To monitor the various permutations of factors investigated in optimising the protocol, normalised sequence abundance factor (NSAF) was utilised to evaluate the abundance of the isolated proteins. Several blocks of experimental factors were designed and evaluated, prior to selecting the ideal conditions to design the next experiments. After several rounds of experimentation, the following conditions were selected: protein concentration (0.5 mg/mL); bead volume (50 µL); total quantity of drug (50 nmol); drug concentration (1  nmol/µL); incubation time (2  h); agitation speed (10 r.p.m.); formic acid elution volume (250 µL); and quantity of trypsin (1.25 µg). To ensure that our optimised approach was not tuned specifically for bosutinib, the established down-scaled methodology will be performed with additional tyrosine kinase inhibitors. Ultimately, the approach will be applied to lysates of core needle biopsies from tumour samples and the protein profile determined from clinically-relevant tyrosine kinase inhibitors. Novel aspect  The optimisation and miniaturisation of a robust method to study the kinase inhibitor profile of human tumour needle biopsies.

References 1. Fernbach N, et al. J Proteome Res. 2009;8:4753–65.

Targeting BAG-1 protein interactions to inhibit tumor growth Marion Enthammer1,2, Martin Deutsch2, María Salomé Gachet3, Stefan Schwaiger3, Hermann Stuppner3, Gerhard Wolber4, Jakob Troppmair2 Institute of Pharmacy, Department of Pharmaceutical Chemistry, Leopold-Franzens-University, Innsbruck, Austria,

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Supplement 248 Daniel Swarovski Research Laboratory (DSL), Department of Visceral, Transplant and Thoracic Surgery, Center of Operative Medicine, Innsbruck Medical University, Innsbruck, Austria, 3 Institute of Pharmacy, Department of Pharmacognosy, LeopoldFranzens-University, Innsbruck, Austria, 4 Institute of Pharmacy, Department of Pharmaceutical Chemistry, Freie Universität Berlin, Berlin, Germany 2

BAG-1 is a multifunctional protein, which regulates cell growth, survival, intracellular signaling, and protein folding. Important for its function is the ability to associate with a variety of different proteins via its C-terminal BAG-domain. Interaction partners include Bcl-2, heat shock protein 70 (Hsp70/Hsc70) and RAF kinases. Recently first evidence has been obtained that disrupting the protein complex BAG-1/Hsc70 and to a lesser degree of BAG-1/RAF by Thioflavin S abolished transformation in vitro. However, Thioflavin S is a mixture of reaction products unsuitable for lead development. In order to identify component(s) responsible for activity a purification protocol was established affording several single, chemically well characterized compounds. Among them the most promising compound (Thio-2) was extensively biologically characterized to confirm effects on growth inhibition, apoptosis induction, proteinprotein interactions, MAPK signaling and most importantly on the transformation by oncogenic RAF, which is found in about 10 % of all tumors. Our work resulted in the establishment of an inhibitor of protein-protein interaction for BAG-1/Hsc70 and possibly BAG-/ RAF, with the ability to suppress signaling via oncogenic RAF and tumor cell growth. Given the frequent involvement of BAG-1 and its protein binding partners in tumorigenesis, targeting these interactions might lead to new therapeutic approaches in cancer therapy. This work was supported by the COMET Center ONCOTYROL, which is funded by the Austrian Federal Ministries BMVIT/BMWFJ (via FFG) and the Tiroler Zukunftsstiftung/Standortagentur Tirol (SAT).

Inherited susceptibility to colorectal cancer Philipp Hofer, Andreas Baierl, Florian Frommlet, Erich Doljesi, Judith Karner-Hanusch, Thomas Bachleitner, Michael Bergmann, Anton Stift, Gernot Leeb and Andrea Gsur Institute of Cancer Research, KIMI, MUW Abstract:  Many colorectal cancers (CRCs) develop in genetically susceptible individuals, only a small fraction is attributable to high penetrance mutations, but the vast majority to the co-inheritance of multiple low-penetrance variants. So far 20 common low-penetrance genetic variants for CRC suscepitibility have been identified by genome-wide association studies (GWAS). GWAS have yet not identified all CRC susceptibility variants and there is still missing heritability hidden for this disease. Therefore we are under way to perform a GWAS on 1.000 CRC patients and 1.000 controls from our CRC DNA bank, using a new Affymetrix platform, the Axiom arrays. In our molecular epidemiology colorectal cancer study of Austria (CORSA) more than 6.000 Caucasian participants were recruited since May 2002 within a large province-wide screening project in the province Burgenland and in cooperation with the Department of Surgery, MUW. As member of the COGENT (Colorectal cancer GENeTics) consortium we will pool our GWAS data with already available GWAS data of this international consortium which has access to more than 50.000 CRC cases and 50.000 controls to obtain ample statistical power to reach solid conclusions on genetic susceptibility variants. The objective of this study is to understand the impact of inherited susceptibility in CRC for profiling individual risk and performing early screening and treatment monitoring.

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CpG island methylator phenotype in rectal cancer: a rare event Ruth Exner1, W. Pulverer2, M. Diem3, Brigitte Wolf1, M. Sonntagbauer2, Michael Bergmann1, A. Weinhäusel2, G. Egger3 Department of Surgery, Medical University of Vienna, Vienna, Austria, 2 Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria, 3 Surgical Research Laboratories, Medical University of Vienna, Vienna, Austria, 4 AIT—Austrian Institute of Technology GmbH, Health & Environment Department, Molecular Diagnostics , Austria 1

Background  In patients with rectal cancer, the clinical value and the impact on prognosis of epigenetic changes like the CpG island methylator phenotype (CIMP) are still unknown. A recent study showed worse disease free survival in CIMP positive patients with locally advanced rectal cancer after neoadjuvant radiochemotherapy [1]. Methods  Eighty-eight patients with pT2 and pT3 rectal adenocarcinoma were included in this analysis. CIMP was assessed by methylation specific PCR isolated from FFPE material using CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1 as a marker panel. In these patients, microsatellite instability (MSI) was measured. Results  Seventy-three percent of rectal carcinomas do not show CIMP specific DNA methylation and only three CIMP-high cases were identified within the 88 tumor samples. There was no significant correlation between CIMP and tumor size, affected lymph nodes or grading, but we found a tendency towards better survival (100 vs.76  %) with worse disease free survival (67 vs. 82  %) in the CIMP positive group, but data were not statistically significant due to low patient numbers in this group. Analysis for MMR status did not reveal any microsatellite instability. Conclusion  In rectal cancer, CIMP positivity, defined by methylation of at least 3 out of 5 specific gene promoters, is a rare event. In our patients we found a tendency towards worse disease free survival, but due to its low frequency CIMP might not be useful as a prognostic or predictive marker.

References 1. Jo P, et al. Surgery. 2012;151:564–70. 2. Weisenberger, et al. Nature genetics. 2006;38:787–93.

Identification of a novel PKCβ phosphorylation motif within the PTB domain of p66SHC essential for ROS production and cell death under cellular stress Martina Haller1, Friedrich Fresser2, Daniela Pirkebner1, Muhammad Imtiaz Ashraf1, Martin Hermann3, Michael Leitges4, Marco Giorgio5, Gottfried Baier2, Jakob Troppmair1* Daniel Swarovski Research Laboratory, Department of Visceral-, Transplant- and Thoracic Surgery, Innsbruck Medical University, Austria, 2 Division of Cell Genetics, Medical University Innsbruck, Austria, 3 Division of Human Genetics, Innsbruck Medical University, Austria, 1

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36th Seminar of the Austrian Society for Surgical Research Department for Anesthetics and Intensive Care, Innsbruck Medical University, Austria, 4 Biotechnology Center of Oslo, Oslo, Norway, 5 European Institute of Oncology, Milan, Italy 3

Stressed mitochondria contribute to the development of many diseases through excessive production of reactive oxygen species (ROS). Antioxidants frequently proved inefficient and novel therapeutic options may come from understanding the cytoplasmic-mitochondrial crosstalk that controls mitochondrial ROS production. Under stress p66SHC translocates to the mitochondria, where it oxidizes cytochrome c to yield H2O2, which in turn initiates cell death. PKCβ-mediated phosphorylation of serine (S)36 on p66SHC has been implicated in mitochondrial translocation. However, in our experiments inhibiting PKCβ efficiently prevented ROS production without affecting S36 phosphorylation. Here we identified serine (S)213 within the phosphotyrosine binding (PTB) domain as a p66SHC pro-oxidant and pro-apoptotic function regulating PKCβ phosphorylation site. Consistently, the neutral exchange mutant P66SHCS213A was impaired in stressinduced mitochondrial translocation, which correlated with decreased Pin1 binding. Mutation of a second predicted PKCβ phosphorylation site located in the same domain, threonine (T) 206, had no phenotype. Intriguingly, mutation of both sites to glutamic acid (E) showed a gain-of-function phenotype with significantly increased ROS production and cell death induction. Our data argue for a novel mechanism of PKCβ-dependent p66SHC activation involving a motif surrounding S213. Given p66SHC’s role in causing redox damage preventing its activation may have far-reaching therapeutic implications. Supported by funds from the FWF and the Jubiliäumsfond OeNB.

Essential role for DNA methylation and DNMT1 in NPM-ALK mediated lymphomagenesis Melanie R. Hassler1, Aleksandra Klisaroska, Karoline Kollmann, Irene Steiner, Martin Bilban, Ana-Iris Schiefer, Veronika Sexl, Gerda Egger Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria,

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Epigenetics studies heritable changes in gene expression mediated by chromatin modifications, but not by changes in DNA sequence. An important epigenetic regulatory mechanism is DNA methylation, which occurs at CpG sites in the genome and leads to silencing of genes. In cancer, aberrant DNA methylation at promoter regions and global DNA hypomethylation are observed during disease progression and contribute to the malignant transformation and genomic instability of the cancer cell. In this study, we aimed to investigate the role of DNA methylation for the progression of NPM-ALK positive lymphomas, a class of CD30 + T-cell lymphomas expressing the oncogenic NPM-ALK fusion protein. We analysed tumor specific DNA methylation patterns of NPM-ALK positive patients by using the Illumina Infinium Methylation array and show that chemical inhibition of DNA methyltransferases leads to reduced tumor cell growth in vitro and in vivo. Furthermore, in transgenic mice expressing the oncogenic NPM-ALK fusion protein under the CD4 promoter, which causes rapid tumor formation in the thymus, T-cell specific deletion of the maintenance methyltransferase DNMT1 rescues mice from tumorigenesis. We therefore conclude that altered DNA methylation is essential for ALCL development and progression and might thus be a promising therapeutic target.

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Pretreatment serum C-reactive protein levels predict benefit from multimodality treatment including radical surgery in malignant pleural mesothelioma Mir Alireza Hoda, Bahil Ghanim, Thomas Klikovits, Max-Paul Winter, Madeleine Arns, Peter Schenk, Wolfgang Pohl, Michael Grusch, Martin Filipits, Christoph Zielinski, Balazs Hegedus, Balazs Dome Walter Klepetko, Walter Berger Translational Thoracic Oncology Laboratory, Division of Thoracic Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria,

Objective  To evaluate the prognostic and predictive relevance of pretreatment serum C-reactive protein (CRP) in malignant pleural mesothelioma (MPM) patients. Background  MPM is a rare but aggressive disease with poor treatment outcome. Therapeutic decision is challenging and predictive biomarkers for better treatment stratification are urgently needed. Methods  Clinical data, including survival and pretreatment CRP levels, were retrospectively collected from 115 patients with histologically proven MPM. Patients with any evidence for infectious disease were excluded. The association between CRP levels and survival was analyzed using Cox models adjusted for clinical and pathological factors. Results  Median pretreatment CRP of all patients was 1.19 mg/ dl (range: 0.00–22.62  mg/dl). Patients with elevated CRP levels (≥ 1  mg/dl; n = 62, 53.9  %) had a significantly shorter overall survival compared to those with normal CRP (hazard ratio [HR] 2.81, 95  % confidence interval [CI] 1.82–4.33; p < 0.001). In multivariate survival analyses, elevated CRP was confirmed as an independent prognostic factor in MPM (HR 2.07, 95  % CI 1.23–3.46; p = 0.01). Most interestingly, we observed a significant interaction between CRP and treatment modality (p < 0.001). Among patients with normal CRP levels, radical tumor resection within multimodality therapy was associated with distinctly prolonged overall survival when compared to treatment protocols without surgery (HR 7.26, 95  % CI 3.40–15.49; p < 0.001). In contrast among patients with elevated CRP, no survival benefit was achieved by radical surgery within multimodality approaches (HR 0.911, 95 % CI 0.53–1.58; p = 0.74). Conclusions  Our results suggest that multimodality regimens including radical resection increase survival selectively in MPM patients with normal pretreatment serum CRP levels.

Secretome from mononuclear cells confers immunosuppression in a murine autoimmune myocarditis model Konrad Hoetzenecker, Matthias Zimmermann, Thomas Schweiger, Dagmar Kollmann, M Milder, Balazs Hegedus, Andreas Mitterbauer, Stefan Hacker, Peter Birner, C. Gabriel, Mariann Gyöngyösi, P. Blyszczuk, U. Eriksson, Hendrik Jan Ankersmit Department of Thoracic Surgery, Medical University of Vienna, Vienna, Austria,

Inflammatory dilated cardiomyopathy (iDCM) is a common cause of heart failure in young patients. Experimental autoimmune myocarditis (EAM) is a CD4 + T cell dependent autoimmune model, which mirrors important pathogenic aspects of iDCM. We have recently shown that a high dose application of paracrine factors

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Supplement 248 obtained from mononuclear cells (MNC) modulates the inflammatory response following myocardial ischemia. In this subsequent study, we sought to evaluate possible immunosuppressive features of MNC secretome using the EAM model. Cell culture supernatants derived from murine MNC were injected intraperitoneally after induction of autoimmune myocarditis with a cardiac myosin peptide homologue. The inflammatory response was determined by histopathological evaluations and by ELISA. Impact of MNC secretome on proliferation and cell viability of CD4 + T- cells was measured by flow cytometry, 3[H]-thymidine incorporation and histone release assays. Treatment of EAM mice with a single high dose of MNC secretome resulted in an attenuation of myocardial infiltrate (myocarditis score 2.7 ± 0.4 vs 0.01 ± 0.01; p = 0.0089). We further evaluated the effect of MNC secretome on JURKAT cells and purified human CD4 + T cells. Coincubation of MNC secretome with T-cells led to a caspase-8 dependent induction of apoptosis. Our data give first evidence that secretome obtained from MNC possess immunosuppressive features in an autoimmune myocarditis model. This anti-inflammatory effect of MNC secretome points to a novel and simple potential treatment concept in inflammatory heart diseases.

The mitogen activated protein kinase pathway regulates the expression of p18INK4c and p21Waf1/Cip1 cyclin-dependent kinase inhibitors through the activating protein-1 transcription factor Ahmad Jalili1, Christine Wagner1, Kirsten D. Mertz1, Mikhail Pashenkov2, Georg Stingl1, Sridhar Ramaswamy3, Stephan N. Wagner1 DIAID, Department of Dermatology, Medical University of Vienna, Vienna, Austria, 2 Laboratory of Clinical Immunology, National Research Center Institute of Immunology, Moscow, Russia, 3 Massachusetts General Hospital Cancer Center, Boston 1

Background  Gain-of-function mutations in upstream members of the MAPK pathway, BRAF and NRAS are among the most common alterations in human melanoma. Single agent BRAF or MEK inhibitors alone only show a very short-lasting or no meaningful clinical activity in human melanoma, respectively. Objectives  MAPK activity is context-dependent as the result of a dynamic and complex regulatory network of diverse protein kinases. We therefore hypothesized that downstream effectors of MAPK pathway would be better suited for therapeutic intervention. However, these downstream effectors are largely undefined in melanoma. Results  Here, we show that BRAFV600E can induce the AP-1 transcription factor. In human melanoma cell lines interference with AP-1 activity by over expressing dominant negative AP-1 or c-Jun (the main AP-1 member) siRNAs resulted in G1 cell cycle arrest, reduced cell proliferation and induced apoptotic cell death both in vitro and in vivo. Induction of G1 cell cycle arrest upon AP-1 deactivation was mediated through combined induction of p18INK4c and p21Waf1/Cip1 cyclindependent kinase (CDK) inhibitors. AP-1 repressed p18INK4c expression through direct binding to its promoter and the p21Waf1/Cip1 indirectly through its upstream negative regulator Tbx2 but not p53 or c-Myc. Combined silencing of p18INK4c and p21Waf1/ Cip1 was able to completely rescue the human melanoma cell death upon AP-1 deactivation. Interestingly, simultaneous pharmacological inhibition of p18INK4c and p21Waf1/Cip1 targets CDK2 and CDK4, in combination with

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or without MAPK inhibitors resulted in decreased human melanoma cell viability in vitro and in vivo. Conclusions  Our results demonstrate how MAPK activity can be linked to cell cycle control in a specific cellular context (BRAF activating mutation) in human melanoma and identify novel therapeutic strategies.

Epidermal growth-factor: induced transcript isoform variation drives mammary cell migration Wolfgang J. Köstler1, Amit Zeisel, Cindy Körner, Jonathan M. Tsai, Jasmine Jacob-Hirsch, Nir Ben-Chetrit, Kirti Sharma, Hadas Cohen-Dvashi, Assif Yitzhaky, Eric Lader, Ulrich Tschulena, Gideon Rechavi, Eytan Domany, Stefan Wiemann, Yosef Yarden Clinical Division of Oncology, Department of Medicine 1, Medical University of Vienna, Vienna, Austria,

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Signal-induced transcript isoform variation (TIV) includes alternative promoter usage, as well as alternative splicing and alternative polyadenylation of mRNA. To assess the phenotypic relevance of signal-induced TIV, we employed exon arrays and breast epithelial cells, which migrate in response to the epidermal growth factor (EGF). We show that EGF rapidly—within 1 h—induces widespread and non-monotonous TIV in a significant fraction of the transcriptome. Importantly, TIV characterizes many genes that are not differentially expressed upon stimulus, and similar EGF-dependent changes occur across a panel of breast cell lines. A functional screen, which utilized isoform-specific siRNA oligonucleotides, indicated that several isoforms play essential, non-redundant roles in EGF-induced mammary cell migration. Taken together, our findings highlight the importance of TIV in the rapid evolvement of a phenotypic response to extracellular signals.

Einfluss des NO-DONORS S-NO-HSA während globaler und lokaler Ischämie am Rattenherzen Elda Dzilic, Maximilian Kreibich, David Santer, Joanna Krynicka, Felix Nagel, Seth Hallström, Bruno Podesser, Karola Trescher Ludwig Boltzmann Cluster für Kardiovaskuläre Forschung, Wien, Österreich, 

Grundlagen  Wir haben die neue HTK-N Kardioplegie mit der klinisch etablierten Vorgängerlösung HTK am akut geschädigten Rattenherzen verglichen. Anschließend wurde der Einfluss des NODonors S-NO-HSA während globaler Kardioplegie und lokaler invivo Ischämie evaluiert. Methodik  Nach 60‚ LAD Ligatur und 120‘ Reperfusion in-vivo wurden die Herzen von SD-Ratten entnommen und am Erythrozyten perfundierten isolierten Herzapparat während 45‚ präischämischer Messung, 60‘ globaler durch HTK [Gruppe-1: n = 8], HTK-N [Gruppe-2: n = 8] oder S-NO-HSA angereicherter HTK-N Lösung (Gruppe-3: n = 8; Gruppe-4: n = 8] geschützten Ischämie und 45 ‚postischämischer Messungen evaluiert. Gruppe-4 erhielt während der in-vivo Ischämie i.v. S-NO-HSA für 75‘ (0,27 μmol/ kg/h). Infarktgröße (IS) und area at risk (AAR) wurden durch Färbungen kontrolliert. Ergebnisse  Die Färbung zeigte gleiche IS (Kontrolle: 39 ± 3 % vs. Therapie: 38 ± 2 % n.s.) bei signifikant verkleinerter AAR (Kontrolle:

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36th Seminar of the Austrian Society for Surgical Research 79 ± 7 % vs. Therapie: 55 ± 3 % p ≤ 0,05). Das Recovery in den durch S-NO-HSA angereicherten HTK-N Gruppen war signifikant besser: External Heart Work (Gruppe-1: 74 ± 3 % vs. Gruppe-2: 84 ± 3 % n.s.; Gruppe-1 vs. Gruppe-3: 97 ± 4  % p ≤ 0,05; Gruppe-1 vs. Gruppe-4: 105 ± 7 % p ≤ 0,01), LVPsys (Gruppe-1: 96 ± 1 % vs. Gruppe-2: 94 ± 1 % n.s.; Gruppe-1 vs. Gruppe-3: 101 ± 1 % n.s.; Gruppe-1 vs. Gruppe-4: 114 ± 5  % p ≤ 0,01), Koronarfluss (Gruppe-1: 79 ± 5  % vs. Gruppe-2: 95 ± 3 % n.s.; Gruppe-1 vs. Gruppe-3: 118 ± 6 % p ≤ 0,01; Gruppe-1 vs. Gruppe-4: 112 ± 3 % p ≤ 0,01). Bei gleicher MVO2 (Gruppe-1: 76 ± 4 % vs. Gruppe-2: 99 ± 2 % n.s.; Gruppe-1 vs. Gruppe-3: 102 ± 4 % p ≤ 0,05; Gruppe-1 vs. Gruppe-4: 112 ± 8  % p ≤ 0,05) verbesserte S-NO-HSA das MDO2 signifikant (Gruppe-1: 82 ± 1  % vs. Gruppe-2: 96 ± 5  % p ≤ 0,05; Gruppe-1 vs. Gruppe-3: 105 ± 5  % p ≤ 0,01; Gruppe-1 vs. Gruppe-4: 112 ± 9 % p ≤ 0,01) in den Blutgasanalysen. Schlussfolgerungen  HTK-N zeigt ein überlegenes postischämisches Recovery. Zusätzliche Verbesserung konnte durch S-NOHSA Gabe zur Kardioplegie oder in-vivo beobachtet werden. Hierfür könnte ein verbesserter Endothelschutz verantwortlich sein. Wir glauben, dass S-NO-HSA eine interessante Therapieoption für Patienten mit ACS und PTCA oder Bypasschirurgie sein kann.

Self-optimization of an influenza virus vector leads to the stabilization of transgene expression Irina Kuznetsova1, Tobias Arnold1, Anna-Polina Shurygina3, Markus Wolschek1, 4, Oleg Kiselev3, Michael Bergmann1, 2*, Andrej Egorov3  epartment of Surgery, Medical University of Vienna, Vienna, D Austria, 2 Comprehensive Cancer Centre, Medical University of Vienna, Vienna, Austria, 3 Research Institute of Influenza, Department of Molecular Virology, Russian Academy of Medical Sciences, St. Petersburg, Russia, 4 Avir Green Hills Biotechnology AG, Vienna, Austria 1

The development of chimeric viral vectors enables alternative vaccine strategies, new diagnostic tools in virology, and the concept of armed virotherapy to combat cancer. The use of influenza A virus vectors for this purpose has been hampered by the instable nature of the virus, leading to the loss of the transgene during the production and application of vaccine viruses. Here, we used the influenza virus’ inherent property of self-optimization to select a vector that is capable of growing and stably expressing transgenes in mouse melanoma cells (B16f1). This was achieved by the insertion of a green fluorescence protein (GFP) sequence into the open reading frame (ORF) of partially truncated viral non-structural protein one (NS1) followed by the selection of bright fluorescent plaques during multiple passages of the virus on B16f1 cells. Although adaptation was associated with the appearance of four coding mutations in several viral genes, the stability of the transgene expression was primarily dependent on a single mutation Q20R in the nuclear export protein (NEP). Adaptation was correlated with a decreased cellular innate immune response of the infected cells that was reflected in the diminished production of interleukin-6 (IL-6). Importantly, in being adapted to B16f1 murine melanoma cells, the selected vector gained stability in other cell types and in vivo in mouse lungs. Moreover, when the GFP-reporter insert was exchanged to another foreign sequence, such as human interleukin-2 (IL-2), the stable high expression of the transgene was retained. Using the IL-2-expressing influenza NS-116/A virus vector for oncolytic tumor therapy led to complete remission in 25 % of B16f1 tumor-bearing mice.

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Evaluation of trastuzumab mediated antibody dependent cellular cytotoxicity in HER2/neu positive breast cancer patient using a rapid FACS-based analysis Ljubomir Petricevic1, J. Längle1, Josef Singer2, Günther Steger3, Rupert Bartsch3, Erika Jensen-Jarolim2, Michael Bergmann1 Department of Surgery, Medical University of Vienna, Vienna, Austria, 2 Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria, 3 Department of Oncology, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria 1

Background  Monoclonal antibodies (mAb) such as trastuzumab are valuable addition to HER2/neu breast cancer therapy. The majority of patients treated with trastuzumab exhibit only shortlived partial responses. Currently, there are no clinically proven predictors for drug response to trastuzumab. Antibody-dependent cell cytotoxicity (ADCC) is considered the major mechanisms of mAb, which could be deteriorated by the systemic immunosuppression of cancer patients and therefore be the reason for its poor response. Methods  We investigated whether breast cancer patients reveal decreased ADCC/ADCP activity in comparison to healthy probands, correlated systemic immunosuppressive markers described in cancer patients with ADCC/ADCP activity and furthermore with treatment response. Measurement of ADCC/ADCP activity was done using a three-colour flow cytometric assay. Immunosuppressive markers were identified as T-Regulatory cells (CD4+ CD25+ Foxp3+), Fc-gamma receptor I (CD64+) and III (CD16+) on monocytes (CD14+) and natural killer cells (CD56+) as well as LAMP1 (CD107a+) on NK cells. Treatment response was determined according to RECIST (Response Evaluation Criteria in Solid Tumors) criteria. Results  ADCC activity of patients is lower in comparison to the activity of the healthy probands with high statistical significance. However, absolute numbers of CD14+CD16+, CD56+CD64+ and CD56+CD107a+ cells in the patient group were higher in contrast to healthy probands, whereas the subpopulation CD56+CD14+ appeared to be lower. Moreover, high number of CD14+CD16+, CD56+CD64+ and CD56+CD107a+ cells revealed to have a negative correlation with ADCC activity. Follow-up of the patient will be discussed.

Novel tumor suppressor genes in malignant pleural mesothelioma Viktoria Laszlo, Chrisitine Pirker, Mir Alireza Hoda, Tamas Garay, Karin Schelch, Michael Grusch, Balazs Dome, Ghanim Bahil, Thomas Klikovits, Anita Rozsas, Walter Klepetko, Walter Berger, Balazs Hegedus Division of Thoracic Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria,

Malignant pleural mesothelioma (MPM) is a devastating malignancy characterized by invasive growth, rapid recurrence and therapy resistance. There is an urgent need to identify the molecular mechanisms that lead to this aggressive phenotype of MPM in order to identify potential diagnostic and prognostic markers as well as to find potential therapeutic targets. Accordingly, we performed genome wide array comparative genomic hybridization and

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Supplement 248 gene expression array analysis of international MPM cell lines and recently established long-term primary mesothelioma cultures. We found downregulation of expression or homozygous or heterozygous loss of two tumor suppressor genes that have not been previously described in MPM. Following validation of the loss or decrease at the protein level we performed in vitro functional assays of cell proliferation, cell survival and migration to demonstrate the biological significance of the decreased function. The clinical significance of the findings is currently being explored using a large cohort of clinically annotated specimens with detailed clinical follow-up.

Identification of a chemokine profile during progression of primary human melanoma Ana Soler-Cardona, Andreas Bracher, Robert Loewe Skin and Endothelium Research Division, Department of Dermatology, Medical University Vienna

Metastasis in melanoma requires a complex series of steps covering interconnected processes such as angiogenesis, lymphangiogenesis, tumor and host immunology or tumor cell migration. Crucial for its understanding is the study of molecules linking these processes. Chemokines are important mediators in nearly all the above mentioned steps. In a xenotransplantation melanoma mouse model, the chemokine profile of primary tumors and lymph node metastasis was compared to controls. Expression of twelve chemokines in mRNA level proved to be differently regulated. These chemokines were subsequently analyzed in FFPE samples of human stage T1 to T4 melanomas using RT-PCR. Interestingly, a chemokine pattern primarily reflecting differences in biological behavior and not only the different tumor stages could be identified. From the upregulated chemokines we focused in those involved in the metastatic process through cell migration and angiogenesis/lymphangiogenesis. An increased mRNA level could be identified even in metastasizing T1 melanomas. To proof the biological relevance in vivo, overexpression was carried out in cell lines with diverse metastatic behaviors including two primarly isolated from the same melanoma tumor patient and studied in a xenotransplantation SCID mouse model.

Complement binding in chemotherapeutics-induced cell death Ying Yu Liang, Anna Michlmayr, Tobias Arnold, Desiree Rainprecht, Rudolf Oehler Department of Surgery, Medical University of Vienna, Vienna, Austria,

The complement system is a matter of controversy in the area of cancer research. Apparently, a beneficial or deleterious effect strongly depends on the circumstances under which complement is analyzed. Implications for a potential contribution of complement components to the action of chemotherapeutic agents derive from studies in the area of autoimmunity demonstrating improved phagocytosis of dying cells opsonized with complement. In the present study, we wanted to investigate whether complement binding to cells treated with chemotherapeutic drugs is a general effect. Therefore, Jurkat cells were treated with drugs having different mechanisms of action (oxaliplatin, irinotecan, docetaxel, etoposide and 5-FU) and deposition of complement components C1q and C3d was analyzed by flow cytometry. Additionally, phagocytosis of

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chemotherapeutics-treated Jurkat cells by peripheral blood mononuclear cells (PBMCs) in the presence of active human serum was assessed. These experiments demonstrated that C1q as well as C3d deposition occurred regardless of which chemotherapeutic agent was used, but to a varying extent. The binding of these complement components was restricted to a population of secondary necrotic cells. Interestingly, oxaliplatin and irinotecan showed significantly different percentages of C1q positive secondary necrotic cells despite similar levels of loss of cell membrane integrity. This difference was also reflected by a differential phagocytosis of tumor cells treated with the two chemotherapeutic agents. Our observation that the extent of complement opsonization of dying cells differs between chemotherapeutic drugs, might has implications for the success of cancer therapy. Further comprehensive studies are needed to address this interesting topic in more detail.

Hypoxic signaling leads to deregulation of the AKT/mTOR pathway in prostate cancer stem cells Maximilian Marhold, Erwin Tomasich, Michael Krainer, Peter Horak Department for Internal Medicine I, Medical University of Vienna, Vienna, Austria

Prostate cancer (PCa) is the most widespread malignant disease amongst men in developed countries. Tumor-initiating subpopulations of carcinoma cells, also known as cancer stem cells (CSCs), were identified and characterized in PCa. We hypothesized that the PI3K/AKT/mTOR pathway, which is known to be frequently altered within PCa cells, and one of its main components, the mammalian target of rapamycin (mTOR), play a role in PCa stem cell maintenance. We isolated CSC like subpopulations from the androgen independent human prostate cancer cell line DU145 using fluorescence activated cell sorting (FACS) according to their expression of the stem cell markers CD44 and CD49f. Further, we sorted the murine prostate cancer cell line TRAMP-C1 based on the expression of the murine stem cell markers Sca-1 and CD49f. We used sphere formation assays to confirm the stem and progenitor cell properties of the sorted subpopulations. We observe lower mTOR and higher AKT activity in prostate cancer stem cells isolated from DU145 and TRAMP-C1 cell lines compared to non-CSCs in vitro. Hypoxia is known to regulate CSC maintenance, thus we evaluated the hypoxic signaling in prostate CSCs. Hypoxic treatment leads to a more pronounced decrease in mTOR activity and an increase in AKT activity in CSC like subpopulations. Normoxic and hypoxic CSCs display higher levels of HIF1 in comparison to the non-CSC subpopulation. HIF-mediated negative regulation of mTOR activity and consequent deregulation of the S6K/IRS/PI3K negative feedback loop might offer an explanation for these effects. Indeed, HIF targets involved in negative regulation of mTOR, such as REDD1 and REDD2 are upregulated in prostate CSCs. Further, IRS phosphorylation is lost in CSCs, leading to activation of the PI3K/AKT pathway. Flow cytometric measurements of the AKT and S6 phosphorylation status in prostate tumors isolated from TRAMP mice confirm our in vitro findings, hence showing elevated AKT and decreased S6 phosphorylation within the CSC subpopulations. In addition, we evaluated the effects of mTOR inhibitors rapamycin and everolimus as well as of the PI3K/mTOR inhibitor NVPBEZ235 under normoxic (20 % O2) and hypoxic (3 % O2) conditions in the CSC and non-CSC cell populations. Our results suggest that CSC like prostate cancer cells are more resistant to mTOR inhibitors when compared to their non-CSC counterparts. Most interestingly,

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36th Seminar of the Austrian Society for Surgical Research while the non-CSC population has a higher sensitivity to mTOR inhibitors under hypoxic conditions, the viability of CSCs remains unaffected. Small molecule inhibitors of multiple kinases along the PI3K/AKT pathway, such as NVP-BEZ235, are successful in eradicating the CSC-like cell populations in hypoxia as well as normoxia. Conclusively, we define a deregulation of mTOR signaling in prostate cancer stem cells, leading to increased AKT activity and survival. In view of our data, we suggest that prostate cancer stem cells might be resistant to mTOR inhibitors and that targeting multiple kinases along the PI3K/AKT/mTOR axis would be more effective in this setting. Our findings could be helpful to assess the impact of mTOR targeted therapy in prostate cancer, especially in light of ongoing clinical trials of mTOR inhibitors such as everolimus and temsirolimus in castration resistant prostate cancer.

Degree and organization of immune cells including B-cell populations within the active border of human colorectal cancer liver metastases: novel prognostic marker for clinical outcome? Anastasia Meshcheryakova1*, Dietmar Tamandl4*, Martin Svoboda1, Erika Bajna1, Judith Stift2, Milena Sachsenmaier4, Thomas Grünberger4, Erika Jensen-Jarolim1, 3, Michael Bergmann4, Diana Mechtcheriakova1 Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria, 2 Institute of Pathology, Medical University of Vienna, Vienna, Austria, 3 Messerli Research Institute of the Medical University of Vienna, Veterinary University of Vienna and University of Vienna, Vienna, Austria, 4 Department of Surgery, Medical University of Vienna, Vienna, Austria 1

Solid tumors represent an extreme example of tissue where immune cells, either resident or infiltrating, might get aberrantly activated and affect the clinical course of disease. We established an algorithm which allows to assess a link between the patient-specific immunological capacity and the clinical outcome for patients with colorectal cancer liver metastases (CRCLM). The microscopybased TissueFAXS system was used for automated scanning of the large-scale tissues sections, followed by identification of single cells in the complex tissue environment and quantification of positively stained immune cell subtypes using image analysis softwares. Tissue sections were analysed for CD45 to detect all classes of infiltrating immune cells; CD20, AID, CD138, and IgM to characterize the B cell populations; and CD68 to detect macrophages. For specimen’s comparison, we applied a uniform strategy by defining subregions at tumor–liver border and within liver tissue. Results indicate massive infiltration of CD45+ cells in the form of diffuse aggregates or GC-like ectopic follicles confined to the active tumor–liver border of the metastases. We observed two different types of follicles: fully developed CD20+/AID+ surrounded by CD138+ plasma cells and those composed from IgM+/AID- B-cell subset. Of importance, the recurrence free survival was found to be significantly better in patients with high CD45+ and high CD20+ content. The findings emphasize the impact of the B-cell driven local immune response on the CRCLM disease progression. In summary, the established algorithm allows to estimate the prognostic power of infiltrating immune cells within complex malignant tissue as new biomarker for clinical outcome. Supported by The Austrian Science Fund FWF P23228-B19, P22441–P13

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Chemotherapy of breast cancer patients induces a shift in the glycosylation pattern of plasma complement components C3 and C4 Christine Schalko1, Anna Michlmayr1, Rupert Bartsch2, Michael Bergmann1, Thomas Bachleitner-Hofmann1, Rudolf Oehler1 Department of Surgery, Medical University of Vienna, Vienna, Austria, 2 Department of Internal Medicine-I, Medical University of Vienna, Vienna, Austria 1

Using 2D gel electrophoresis, we recently demonstrated that a combination therapy of mamma-carcinoma patients with epirubicin and docetaxel leads to changes of the plasma proteome. After the initial chemotherapeutic dose, changes in plasma levels of specific isoforms of C3 and C4 were observed which correlated with the final response to chemotherapy. Because many C3 and C4 isoforms showed the same molecular weight but different pI we analysed in the present study whether the observed changes are related to glycosylation. Complement components were analysed in human blood plasma by 1D-WB and 2D-WB analysis. Glycosylation was analysed using specific dyes. Moreover we performed in vitro activation experiments. We could show that the C3 alpha chain is present in a glycosylated as well as in a non-glycosylated form. In contrast, all C4 alpha isoforms were positively stained for glycosylation. Variations in staining intensities suggested that the degree of glycosylation differed between the isoforms. Comparing the glycosylation pattern before and after the initial dose of chemotherapy revealed that the treatment induced a shift in the glycosylation pattern. This shift was not observed after in vitro activation of the complement system. Our data clearly show that complement components C3 and C4 can be glycosylated in vivo. However, the reason of the effect of chemotherapy on this glycosylation remains unclear.

Mass spectrometry for medicine and oncology: status quo, applications and challenges Peter Pichler, Thomas Köcher, Karl Mechtler Protein Chemistry and Mass Spectrometry, Research Institute of Molecular Pathology (IMP), Vienna, Austria

Initially being a technique within the field of experimental physics, mass spectrometry (MS) has meanwhile become a part of the protein researcher’s toolbox. The invention of soft ionization techniques such as ESI (electrospray ionization, Fenn J, Nobel Prize in Chemistry 2002) turned the tide, paving the way for applications in molecular biology and, ultimately, medicine. Cancer may be regarded as a disease of aberrant cellular information processing based on genetic and epigenetic changes. Although DNA sequencing and the detection of mutations can provide insight into the “cancer cell wiring plan” (Hanahan D et al. Cell 2011), an analysis of proteins and their post-translational modifications may permit a more direct view of signaling pathways and protein expression: Quantitative MS can be used to detect alterations in protein expression patterns that may uncover candidates for potential biomarkers. The technique can also be used to analyze phosphorylation of signaling proteins and the activity of kinases that “drive” cancer cell behavior (Kubota K et al, Nature Biotechnology 2009). Moreover, proteomics can characterize post-translational modifications of histone tails that constitute the basis of cancer epigenetics. We here present data, including

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Supplement 248 our own work, on the current status quo of the performance of MS, illustrating that MS is getting closer to potential applicability in the clinic, and we discuss limitations and challenges. We suggest that, together with other data, the application of MS in oncology settings can provide information that may be made available to clinicians in terms of a “wiring diagram” of a patient’s cancer, thus establishing a basis for “personalized” treatment. In this way, clinical MS will not only contribute to improvements in prognostic accuracy, but may also help the guidance of treatment with drugs targeting the signaling pathways that “drive” the specific cancer while potentially exerting less side effects on other tissue.

Simvastatin influence on NADPH oxidase four concentration in human AAA wall tissue Aleksandra Piechota-Polanczyk1, Svitlana Demyanets2, Martina Mittlboeck3, Christoph Domenig4, Christoph Neumayer4, Johann Wojta2, Josif Nanobachvili4, Igor Huk4, Markus Klinger4  epartment of Cardiovascular Physiology, Chair of Experimental and D Clinical Physiology, Medical University of Lodz, Łódz´ , Poland, 2 Department of Internal Medicine II, Division of Cardiology, Medical University of Vienna, Vienna, Austria, 3 Center for Medical Statistics, Informatics, and Intelligent Systems, Medical University of Vienna, Vienna, Austria, 4 Department of Surgery, Division of Vascular Surgery, Medical University of Vienna, Vienna, Austria 1

Background  A number of experimental studies have suggested an association of oxidative stress and abdominal aortic aneurysm (AAA). Statins, HMG-CoA reductase inhibitors, have been reported to suppress the progression of AAA. However, the effects of statins on antioxidant mechanisms are poorly understood. Our previous research indicated that simvastatin decreases free radical formation and improves antioxidative parameters of human AAA tissue. However, the role of NADPH oxidase four (NOX4), a free radical generator in endothelial and smooth muscle cells, in AAA remains to be evaluated. Methods  AAA wall tissue of 50 patients (statin patients, n = 30; non-statin patients, n = 15) subjected to elective open AAA repair was analyzed prospectively. The patients were matched by age-, sex-, and AAA diameter. The blood concentration of total cholesterol, LDL and HDL was measured. The tissue NOX4 mRNA expression and protein concentration was determined by real time RT-PCR and Western blot, respectively. Results  Simvastatin treated patients had significantly lower concentration of total cholesterol (191.07 ± 40.19  mg/dl vs. 234.4 ± 40.99  mg/dl, p = 0.002) and LDL (112.92 ± 38.91  mg/dl vs. 151.6 ± 36.59 mg/dl, p = 0.003) when compared to the control. However, there was no significant difference in HDL concentration between examined groups (50.53 ± 11.77 vs. 48.13 ± 9.12, p = 0.45). Furthermore, simvastatin had no influence on mRNA and protein concentration of NOX4 (7.83 ± 6.63 vs. 9.09 ± 7.01 and 0.98 ± 0.34 vs. 1.16 ± 0.5, p = 0.311, p = 0.229, respectively). Conclusions  Simvastatin treatment to patients undergoing AAA open repair improved lipid profile by decreasing total cholesterol and LDL concentration. However, simvastatin treatment has no influence on tissue NOX4 gene and protein expression.

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A role of AXL in EMT and hepatocellular carcinoma progression Patrick Reichl, Franziska van Zijl, Heidemarie Huber, Markus Grubinger, Wolfgang Mikulits Institute of Cancer Research, Department of Medicine I, Medical University of Vienna,Vienna, Austria,

Metastasis is the leading cause of cancer mortality and represents a multi-step process including tumor cell invasion. In hepatocellular carcinoma (HCC), intrahepatic metastasis frequently correlates with a preceding epithelial to mesenchymal transition (EMT) of malignant hepatocytes. EMT is therefore considered as a pivotal event in HCC progression. Several mechanisms have been elaborated to be essentially involved in hepatocellular EMT such as the collaboration of Ras signaling with transforming growth factor-β. Yet, the diversity of signaling cascades that govern EMT in HCC progression is still poorly understood. We established a unique cellular EMT model of human HCC which particularly allows to identify novel molecular mechanisms of HCC cell dissemination. Expression profiling of epithelial versus mesenchymal HCC cells revealed that the receptor tyrosine kinase AXL is a major regulator of EMT and HCC progression. AXL is expressed at the cell surface of mesenchymal HCC cells, whereas epithelial cells are devoid of AXL expression. Negative interference with AXL expression showed abrogation of migratory abilities in HCC cells that have undergone EMT. These in vitro data could be verified by immunohistochemical evaluation of AXL expression in a large number of HCC patient samples. High AXL expression could be correlated with (i) advanced stages of liver cancer, (ii) elevated vessel invasion of HCC cells, (iii) higher risk of tumor recurrence after liver transplantation, and (iv) a significantly lower survival rate within 5 years after tumor resection. In conclusion, our data suggest that Axl might represent a promising target in HCC invasion and intrahepatic metastasis.

Erythropoietin-receptor (EPOR) expression and the tumor specific effect of EPO treatment in lung cancer Anita Rozsas, Balazs Hegedus, Livia Rojko, Istvan Kenessey, Judit Berta, Magdolna Keszthelyi, Jozsef Tovari, Mir Alireza Hoda, Walter Berger, Michael Grusch, Walter Klepetko, Balazs Dome Translational Thoracic Oncology Laboratory, Department of Thoracic Surgery, Medical University of Vienna, Vienna, Austria,

Recombinant human erythropoietin (rHuEPO) is part of the standard care for lung cancer patients with anaemia but the clinical findings on the tumor-related effects are inconsistent. Hypoxic tumors are less sensitive to a number of anticancer therapies and hypoxia itself promotes progression. Nevertheless, the receptor of EPO (EPOR) can also be expressed on lung cancer cells that may support their survival and growth. Accordingly, we determined the expression of EPOR in lung cancer tissue samples and cells and investigated the effect of rHuEPO treatment in vitro and in vivo using xenografts of human lung cancer cells. First, we analyzed bronchoscopy samples of 99 and 27 patients with NSCLC and SCLC, respectively. The expression of EPOR was measured in the tumor and adjacent non-tumorous samples of each patient by real-time PCR. Next, we measured the expression of EPOR in lung cancer cell cultures. Finally, we characterized the proliferation of rHuEPO treated lung cancer cell cultures and the

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36th Seminar of the Austrian Society for Surgical Research growth and vascularization of subcutaneously growing human NSCLC treated with rHuEPO. The expression of EPOR was significantly higher in the NSCLC bronchoscopy samples when compared to SCLC. Nevertheless, we found no significant association between EPOR expression levels and survival. Although a number of lung cancer cell lines expressed EPOR, EPO treatment had no effect on their in vitro proliferation. Importantly, rHuEPO treatment of NSCLC xenografts resulted in increased proliferation of endothelial cells in the tumor and decreased tumor size. Our study suggests that rHuEPO can support the proliferation of intratumoral endothelial cells. This, in turn, can result in better perfusion of tumor tissue, decrease tumor hypoxia and improve the therapeutic efficacy of anti-cancer treatment. However, further studies are needed to establish the effect of EPO treatment on the clinical outcome of lung cancer.

Telomere maintenance mechanism in colorectal cancer Sandra Sampl, Stefan Stättner, Brigitte Marian, Klaus Holzmann Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria, 

Tumors can overcome cellular senescence, one of the main barriers to tumor development and immortality by telomere maintenance mechanism (TMM). Most of colorectal carcinomas (CRC) use telomerase activity (TA) as TMM. Mechanisms of remaining CRC tumors may utilize alternative lengthening of telomeres (ALT) or a not defined telomere maintenance mechanism (NDTMM). Both TA and expression of telomeric repeat containing RNA (TERRA) were recently identified to correlate with tumor grade. This led us to evaluate possible correlations of TMM and TERRA levels to clinical data. Tumor and matched adjacent non-tumor tissues from CRC patients (N = 68) and cell lines (N = 7) were assessed. TMM were evaluated by measuring TA and by detection of ALT with c-circles. Mean telomere length (TL) and expression were determined by qPCR as relative quantity (RQ). RQ values were analyzed for significant differences between groups and were assessed after log-transformation with correlation calculation. TL of tumors was comparable with TL of non-tumor tissues. TA was detected in 90  % tumors and 36  % non-tumors. Seven (10  %) tumor cases showed a TA below the detection limit and similar to median activity found in non-tumors. ALT was detected in 4 tumors, one case with and three cases without TA, demonstrating prevalence of ALT in TA negative cases. NDTMM was identified in four tumors. TA was detected in all cell lines. TERRA levels showed down regulation in 84  % of tumors compared to non-tumor tissues, a moderate negative correlation with TA (Pearson r = − 0.47, p < 0.001) and were higher in tumors with ALT, supporting the inhibitory function of TERRA on TA as recently described in vitro. No correlation with tumor grade/stage and survival was observed. In conclusion, we identified all combinations of TMM in our CRC patient series. Our preliminary data suggest that TERRA expression correlates with TMM and thus can be considered promising candidate as marker.

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Leoligin, the major lignan of edelweiss, influences serum cholesterol levels in ApoE/mice Bernhard Scharinger, Adrian Türkcan, Barbara Messner, David Bernhard Cardiac Surgery—Research Laboratories, Department of Surgery, Medical University of Vienna, Vienna, Austria,

Cardiovascular diseases (CVD) are still the number one cause of death in the world. As cholesterol imbalance (increased low density lipoprotein—reduced high density lipoprotein) is a major driving force underlying atherosclerosis initiation and progression, the search for new lipid lowering agents is still a highly relevant task to improve the treatment of dyslipidemia. Systemically applied, Leoligin reduces serum cholesterol levels (SCL) in a short treatment mouse model. The present study is designed to investigate the systemic effect of Leoligin in a long treatment mouse model. Leoligin was administered orally to 40 ApoE/mice over 17 weeks at three different concentrations (1 µM, 10 µM, 50 µM) and blood samples were taken at 0, 5, 10 and 17 weeks. Additionally two intraperitoneal glucose tolerance tests (IPGTT) were performed after 3 and 17 weeks. After sacrificing the mice, organ and blood samples were taken. Analyses of the blood samples were performed and statistically evaluated. Analyses of blood samples gave that Leoligin significantly lowered SCL (e.g. control: 287.2 mg/ml versus Leoligin 1 µM: 236.6 mg/ ml; p-value: 0.018) as well as low density lipoprotein cholesterol (control: 201.3  mg/ml versus Leoligin 1  µM: 170  mg/ml; p-value: 0.016) at 5 weeks, but not at the endpoint. A significant gain in body weight was observed in the control group compared to the Leoligin treated mice as well. Further the IPGTT after 3 weeks showed lowered postprandial serum glucose levels after intraperitoneal injection of glucose in the Leoligin treated groups. Leoligin could be a novel substance to avoid and prevent the impacts of dyslipidemia. The change in blood lipid profiles, glucose sensitivity and body weight indicate that it has a broad effect on metabolism and may therefore reduce the risk of CVD. However the detailed biochemical interaction of Leoligin with metabolism, proteins, and cells is still unclear and needs further studies.

Monocyte subsets in colorectal cancer Dominic Schauer, Patrick Starlinger, Christian Reiter, Nikolaus Jahn, Philipp Zajc, Lorand Pop, Elisabeth Buchberger, Thomas Bachleitner-Hofmann, Michael Bergmann, Anton Stift, Thomas Gruenberger, Christine Brostjan Medical University of Vienna, Vienna, Austria,

Based on the differential expression of CD14 and CD16 monocytes can be divided into three populations with distinct biological functions. We have compared the prevalence of these monocyte subsets in the blood of colorectal cancer patients and in healthy individuals. The so-called “intermediate monocytes” were found to exhibit a significant marker potential, in particular for the diagnosis of early stages of disease. Furthermore, this monocyte population increased during chemotherapy and showed a highly significant correlation with treatment response. During in vitro experiments the intermediate monocytes were found to preferentially migrate in response to tumor-derived stimuli. When this monocyte subset was isolated from patient blood, we found high levels of HLA-DR, CCR5 and TNFα expression, indicative of a potent pro-inflammatory phenotype. The expression profile and migratory behavior of interme-

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Supplement 248 diate monocytes was not altered by chemotherapy. In conclusion, the population of intermediate monocytes is increased in colorectal cancer patients; it exhibits pro-inflammatory properties and has diagnostic and predictive marker potential.

Podoplanin expressing cancer associated fibroblasts are associated with unfavourable prognosis in adenocarcinoma of the esophagus

and 14/116 (10.4 %) of ACs showed ALK amplifications. Concomitant EML4 amplifications were present in 27/28 cases with ALK amplifications. Three cases (2 SCC, one with additional ALK&EML4 amplification and 1 AC) showed EML4 translocations not involving ALK. None of the tumors with ALK amplification showed ALK protein expression, and no correlation with clinical parameters, survival or pSTAT3 expression was observed. Conclusions  While ALK translocations are not present in esophageal cancer, ALK amplifications are common events with comparable rates in SCC and AC. Since ALK amplified breast cancer cells were shown to respond to ALK inhibitors, ALK amplified esophageal cancers might be considered as possible candidates for therapies targeting ALK.

Sebastian F. Schoppmann1, Peter Birner2  epartment of Surgery, Medical University of Vienna, Vienna, D Austria, 2 Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria 1

Over expression of the podoplanin in cancer associated fibroblasts (CAFs) was recently shown to be associated with tumor progression, metastasis and poor prognosis in lung and breast cancer. In the present study we investigate the role of podoplanin expressing CAFs in esophageal adenocarcinoma (AC), its precursor lesions and metastases. Podoplanin was investigated immunohistochemically in 200 formalin-fixed, paraffin embedded specimens of invasive esophageal ACs, their corresponding metastases and 35 precursor lesions. Podoplanin expressing CAFs (CAF+) were observed in 22  % of patients with invasive AC, but not in precursor lesions. CAF+ correlated with tumor stage (p = 0.004), lymphovascular tumor invasion (p = 0.018) and lymph node metastasis (p = 0.0016). Patients with CAF+ had a significant shorter disease free and overall survival (p < 0.05, Cox regression). Podoplanin expressing CAFs were only rarely observed in lymph node and distant metastases, as well as in local recurrences of ACs. Podoplanin expression in AC tumor cells was seen in only four cases. Podoplanin expressing CAFs are evident In around 20  % of patients with esophageal AC, defining a high risk subgroup, and might represent new therapeutical targets.

ALK gene status and protein expression esophageal cancer Sebastian F. Schoppmann1, Berthold Streubel2, Peter Birner2 Department of Surgery, Medical University of Vienna, Vienna, Austria, 2 Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria 1

Introduction  Translocations of ALK to various fusions partners and formation of oncogenic fusions proteins have been demonstrated in a variety of human malignancies. These fusion-proteins are potential pharmaceutically targets. Aim of this study was to investigate ALK gene status in a large cohort of squamous cell carcinoma (SCC) and adenocarcinoma (AC) of the esophagus. Methods  One hundred seventeen SCCs and 136 ACs were included into this study. ALK and EML-4 gene status were evaluated by fluorescence in situ hybridization (FISH) using a triple color break apart single fusion probe. ALK and EML4 protein expression was determined by immunohistochemistry. Data on expression of ALK downstream effector tyrosine -705 phosphorylated STAT3 (pSTAT3) was available from a previous study. Results  FISH was performed successfully in 251 cases, All cases were negative for ALK translocations, while 14/135 (12.1 %) of SCCs

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Early pulmonary spreading of primary colorectal carcinoma is associated with carbonic anhydrase IX expression and tobacco smoking Thomas Schweiger1, 2, Dagmar Kollmann3, Christoph Nikolowsky1, Denise Traxler-Weidenauer1, Emanuella Guenova4, György Lang1, Peter Birner5, Walter Klepetko1, Hendrik Jan Ankersmit1, 2, Konrad Hoetzenecker1, 2 Department of Thoracic Surgery, Medical University of Vienna, Vienna, Austria, 2 Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration, Medical University of Vienna, Vienna, Austria, 3 Department of Pathophysiology, Medical University of Vienna, Vienna, Austria, 4 Harvard Skin Disease Research Center, Harvard Medical School, Boston, MA, USA, 5 Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria 1

Introduction  Pulmonary metastasectomy is nowadays a common practice in thoracic surgery. However, the selection of patients, which will benefit from a surgical resection is still challenging. Carbonic anhydrase IX (CA9), originally described as a marker for tumor hypoxia, has been described as associated with poor clinical outcome in various malignancies. Data on CA9 expression in pulmonary metastases and its potential role as a predictive value are lacking. Also tobacco smoking has been associated with rapid pulmonary spread and upstream signaling in the CA9 pathway. We hypothesized that CA9 expression affects pulmonary metastasis and might be influenced by smoking. Methods  The impact of nicotine exposure on phosphorylation of STAT3, HIF-1α and CA9 expression was assessed in HT29 cells. Furthermore, 32 patients, who suffered from primary colorectal carcinoma (CRC) and underwent curative pulmonary metastasectomy at the Department of Thoracic Surgery, MUV, were included in this study. We determined the expression of CA9 in pulmonary metastases and corresponding primaries by immunohistochemistry. The expression level was correlated with clinical parameters and patients’ smoking habits. Results  Nicotine treated HT29 cells showed an induction of CA9 in vitro. This induction was accompanied with STAT3 phosphorylation and was independent of HIF-1α. In tissue specimens, CA9 expression was evident in 100  % from the primary CRC and 84.6 % of paired pulmonary metastases. High expression of CA9 in resected pulmonary metastases and corresponding primary tumors correlated with early pulmonary metastasis (p = 0.011 and p = 0.032, respectively) and established clinical risk factors. CA9 expression was also increased in former smokers.

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36th Seminar of the Austrian Society for Surgical Research Conclusions  This study provides first evidence of CA9 expression in pulmonary metastases of CRC and suggests a role of CA9 as a prognostic marker. Moreover, our in vitro and in vivo data indicate an association between tobacco smoking and CA9 expression, which might contribute to the worse prognosis of former smokers with malignant disease.

KRAS status and outcome of liver resection after neoadjuvant chemotherapy including bevacizumab Stefan Stremitzer, Judith Stift, Birgit Gruenberger, Dietmar Tamandl, Thomas Aschacher, Brigitte Wolf, Friedrich Wrba, Thomas Gruenberger Department of Surgery, Medical University of Vienna, Vienna, Austria,

Background  The prognostic value of KRAS mutation in patients with colorectal cancer liver metastases (CLM) receiving neoadjuvant chemotherapy including Bevacizumab prior to liver resection is unclear. Methods  KRAS and BRAF status of resected CLM from prospectively studied patients were assessed. Mutations were correlated to recurrence-free and overall survival. Only patients with remaining vital tumor cells in the resected specimens and those without progression were analyzed (patients with progressive disease were excluded from resection). Results  A total of 60 patients were enrolled: 50 of 60 (25  %) patients showed KRAS mutation (mt), whereas BRAF mutation was not detected in any patient. Radiological response to neoadjuvant chemotherapy including Bevacizumab assessed according to RECIST revealed partial response (PR) in 52 (87  %) and stable disease (SD) in eight patients (13 %). Partial responses in KRAS mt patients were equally frequent as in KRAS wild-type (wt) patients (12 of 15 patients versus 40 of 45 patients, P = 0.400). KRAS mutation had a negative prognostic effect on recurrence-free survival (HR 2.48, 95 % CI 1.26–4.89, P = 0.009) and overall survival (HR 3.51, 95 % CI 1.30–9.45, P = 0.013). Conclusions  This study revealed further evidence of the prognostic importance of KRAS status regarding RFS and OS. Neoadjuvant chemotherapy including Bevacizumab demonstrated a response irrespective of KRAS status in this selected CLM patient group.

Interleukin-24 and toll-like-receptor three activation synergize to induce apoptosis in tumor cells Rene Weiss1, Monika Sachet1, Thomas Aschacher1, Henning Walczak3, Balazs Hegedus1, 2, Michael Bergmann1, 2  epartment of Surgery, Medical University of Vienna, Vienna, D Austria, 2 Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria, 3 Department of Tumor Immunology, Department of Medicine, Imperial College of London, Hammersmith Hospital, London, UK 1

Despite the fact that the physiological function of interleukin-24 (IL-24) remains unknown, its pro-apoptotic activity when expressed in adenovirus background is well documented, rendering IL-24 an attractive agent for anti-cancer virotherapy. Interestingly, when used as a single agent, IL-24 only induces apoptosis in

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certain tumor cell lines. Here we analyzed the oncolytic properties of an IL-24-expressing influenza A virus. The analysis of the tumorablative potential of this chimeric RNA virus revealed that it induced a toll-like-receptor three (TLR3)-dependent apoptosis. Apoptosis was dependent on downregulation of th e cellular FLICE-inhibitory protein (cFLIP), resulting in successive activation of caspases-8 and 3. Importantly, we found stimulation of TLR3 to be sufficient to promote IL-24 induced apoptotic cell death. We conclude that the influenza A virus is an excellently suited vector to support the proapoptotic effect of IL-24 in cancer cells.

Clinical trials for p53 marker validation Brigitte Wolf, Sonja Kappel, Daniela Kandioler Research Laboratories, Department of Surgery, Medical University of Vienna, Vienna, Austria,

The moderate efficacy of cancer therapy is still the major challenge in surgical oncology. The use of genetic markers has been suggested to improve treatment efficacy. However, a clear algorithm for the clinical evaluation of a potential marker is currently lacking. Based on the well-established phase I-III clinical trials we present an algorithm, which we propose for reliable clinical evaluation of genetic markers like p53. Phase I marker studies aim to demonstrate the robustness, specificity and prevalence of the potential new marker and the formulation of a marker hypothesis. Phase II marker studies focus on the marker test; concerning reproducibility, sensitivity and specificity of the test, and the result interpretation. Phase I and phase II marker studies may be performed retrospectively using adequately collected samples. Phase III marker trials aim to confirm the clinical relevance of the marker providing a high level of evidence (level I). The latter trials have to be prospective, randomised, controlled investigations taking the qualified trial design into particular consideration. The clinical utility of a marker depends on its ability to guide three therapeutic decisions: “Who to treat”, “How to treat” and “How much to treat”. These questions have to be answered by different marker types –prognostic, predictive and pharmacodynamic- implicating that the projected phase III trial endpoints have to be different. As a marker, p53 has passed phase I and II. Currently phase III trials evaluating p53 are missing probably because it is not clear whether p53 should be evaluated as prognostic or predictive marker. Adapted from Eur. J. Human Genetics 2012;20(s1):191

The influence of local cooling and local anaesthesia on capsaicin induced pain and epidermal nerve fibre density reduction Markus Zadrazil, Gabor Geza Kovacs, Gisela Scharbert, Michael Schemper, Erich Knolle  epartment of Anaesthesia, General Intensive Care and Pain D Management, Medical University of Vienna, Vienna, Austria,

Background  High-dose capsaicin is approved for topical treatment of peripheral neuropathic pain, e.g. cancer associated poly-neuropathic pain, scar-pain and pain from compression of peripheral nerves. The long-term pain relief following capsaicin treatment is associated with a reversible reduction of epidermal nerve fibres (ENF) [1]. As a severe side effect high-dose capsaicin provokes a strong application related pain because it activates the

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Supplement 248 TRPV1-channel, a receptor-ion channel complex that responds to noxious temperature (> 43  °C), pH and specific exogenous and endogenous stimuli [2]. With this study we aimed to test if pretreatment with EMLA (eutectic mixture of local anaesthetics) or cooling down the skin by 5–10 °C prevents the application related pain without compromising the reduction of ENF density. Methods  Four quarters of a capsaicin 8  % patch were applied for 60  min to both thighs of 12 healthy volunteers. According to randomization one thigh was cooled with cool-packs during application. The application sites on each thigh were pre-treated with EMLA or placebo 60  min in advance. At distinct time points the application related pain was ascertained for each site on a visual analogue scale (VAS). One week later skin biopsies were taken from each site to determine ENF density based on diagnostic guidelines [3]. Results  The application pain experienced in the cooled sites was VAS 1.3 ± 1.4 and significantly lower by more than 30  % than the pain in the non-cooled sites VAS 7.5 ± 1.9, whereas pretreatment with EMLA did not influence the application pain. In all treated areas ENF density was significantly reduced by > 60  % following capsaicin exposure with no influence of EMLA-pretreatment. ENF density reduction was slightly lower in the cooled areas. Conclusions  Local cooling markedly prevents the application site pain caused by topical high-dose capsaicin, whereas topical anaesthesia is much less effective and unnecessary. Capsaicin seems to sensitize the TRPV1-ion channel to act at a lower temperature than 43 °C.

References 1. Kennedy W R, et al. J Pain. 2010. 2. Anand P, et al. Br J Anaesth. 2011. 3. Lauria G, et al. Eur J Neurol. 2010.

³Division of Cardiac Surgery, Department of Surgery, Medical University of Graz, Graz, Austria, 4 Clinical Institute of Pathology, Medical University of Graz, Graz, Austria, 5 Section for Surgical Research, Division of Transplant Surgery, Department of Surgery, Medical University of Graz, Graz, Austria

Cardiac diseases are the leading cause of mortality throughout the world. Fibrosis is an important structural substrate leading to various cardiovascular pathologies. The quantity and the quality of fibrosis strongly correlate with the appearance of rhythm disorders. Especially micro fibrosis could not be efficiently detected up to now, thus limiting the accuracy of catheter ablation. The present study aimed to detect micro fibrosis via cardiac near field measurement—for the first time in isolated perfused Langendorff rat hearts. Micro-conduction mapping methods as well as physiological measurements were performed on native (n = 4) and on cyclosporine treated (n = 6) rat hearts. The main objective was to show if a possible correlation between the morphology of electrograms and the amount of underlying fibrosis induced by cyclosporine treatment, could be estimated. In 50 % of the cyclosporine treated rats, areas of minimal fibrosis were histopathologically confirmed. As expected this did not significantly affect the physiological cardiac performance. Nevertheless these minor structural changes could be detected through micro—conduction mapping using a new developed sensor. Significant differences were found. Following biophysical parameters: e (t)(p = 0.029), d e (t)/dt(p = 0.002) were decreased, while the fractionation index was higher in the cyclosporine treated group. Although these findings need to be confirmed in future studies, they strongly imply the possibility to detect micro fibrosis and thereby offer a reasonable potential to be transferred into a clinical application.

Micro conduction measurements of cardiac fibrosis in isolated rat hearts R. Grgic¹, R. Arnold², A. Wasler³, M. Aßlaber4, M. Schwarz5 ¹Division of Transplant Surgery, Department of Surgery, Medical University of Graz, Graz, Austria, ²Institute of Biophysics, Medical University of Graz, Graz, Austria,

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