FORSCHUNGSLABORATORIEN

Transkript

1 FORSCHUNGSLABORATORIEN Universitätsklinik für Frauenheilkunde 173

2 L ABOR- FORSCHUNGS - EINHEITEN DER UNIVERSITÄTS - KLINIK FÜR FR AUENHEILKUNDE Die Labor-Forschungseinheiten der Universitätsklinik für Frauenheilkunde entwickelten sich aus den Labors der ehemaligen I. und II. Universitäts-Frauenklinik und sind seit Mitte der 1990er Jahre in einem Cluster auf Ebene 5Q zusammengefügt. Sie sind dem Vorstand der Klinik, o.univ. Prof. Dr. Peter Husslein direkt unterstellt. Wurde ursprünglich die Routine und Forschung gleichermaßen abgedeckt, so liegt heute der Schwerpunkt auf Grundlagen- und angewandter Forschung. Sechs Arbeitsgruppen, die international in vielen Partnerschaften vernetzt sind, führen eine Vielzahl von Projekten durch, die sich mit speziellen Fragestellungen im Bereich der Geburtshilfe, der Gynäkologie, der gynäkologischen Onkologie und Endokrinologie beschäftigen. 174 Universitätsklinik für Frauenheilkunde

3 Molekulare Onkologie Die Arbeitsgruppe entwickelte sich aus dem Hormonlabor der I. Universitäts-Frauenklinik, wird von ao.univ.prof. Dr. Robert Zeillinger geleitet und ist eng mit dem Ludwig Boltzmann Cluster Translational Oncology verknüpft. Der Forschungsschwerpunkt liegt bei der Erforschung des Ovarialkarzinoms. Weitere Projekte befassen sich mit dem Nachweis und der molekularen Charakterisierung im Blut zirkulierender Tumorzellen und der Früherkennung verschiedener Krebsarten. Reproductive Biology Die Arbeitsgruppe wird von ao.univ.prof. Mag.Dr. Martin Knöfler geleitet. Sie hat die Differenzierung und Entwicklung der humanen Plazenta als Schwerpunkt und beschäftigt sich mit molekularen Mechanismen der Plazentadifferenzierung unter physiologischen und pathologischen Bedingungen, mit plazenta-spezifischen Transkriptionsfaktoren und Signalwegen, die die Invasion des Trophoblasten kontrollieren, sowie der transkriptionellen Kontrolle von Schwangerschaftshormonen. Gynäkologische Endokrinologie Geleitet wird die Arbeitsgruppe von ao.univ.prof. Mag.Dr. Christian Schneeberger. Die Schwerpunkte liegen in der Erforschung von Signaltransduktionswegen, die an der Entstehung der Endometriose beteiligt sind, der Bedeutung von SNPs und mirnas für verschiedene Fragestellungen in der gynäkologischen Endokrinologie und in der Isolierung, Kultivierung und Differenzierung von hämatopoietischen Progenitorzellen aus Nabelschnurblut. Prädiktive Onkologie Die von Univ.Prof. Dr. Christian Singer geleitete Arbeitsgruppe setzt sich schwerpunktmäßig mit dem erblichen Brust- und Eierstockkrebs und der Rolle von BRCA 1 und 2 bei verschiedenen Aspekten von Brustkrebserkrankungen auseinander. Weitere Forschungsprojekte beziehen sich auf die Rolle von Wachstumsfaktoren bei der mitogenen Stimulation und die Ausbreitungsmechanismen von Tumorzellen. Darüber hinaus wird intensiv die Rolle der Estrogenrezeptoramplifikation (ESR1 Amplifikation) beim endokrinresponsiven Mammakarzinom untersucht. Onkogenomics Der Arbeitsgruppe steht ao.univ.prof. Mag.Dr. Martin Schreiber vor. Das Forschungsthema der Arbeitsgruppe ist Brustkrebs, wobei der Schwerpunkt auf genomischen und molekularbiologischen Forschungsansätzen liegt. Eines der Hauptziele der Forschungsarbeiten ist es, molekulare Mechanismen der Invasivität und Metastasierung aufzuklären und Proteine sowie funktionelle RNAs zu identifizieren, die dabei eine Schlüsselrolle spielen. Medical Genetics 2011 wurde Univ.Prof. Dr. Berthold Streubel der Universitätsklinik für Frauenheilkunde zugewiesen und etablierte eine Forschungsgruppe mit Schwerpunkt in der genetischen Ursachenforschung von pränatalen und postnatalen Krankheitsbildern sowie in der Tumorentstehung. Hierbei kommen entsprechend den technologischen Entwicklungen vermehrt Hochdurchsatzgenomanalysen zur Anwendung. Universitätsklinik für Frauenheilkunde 175

4 MOLEKUL ARE ONKOLOGIE From left to right: Top: Andrea Wolf, Stephanie Aust, Eva Obermayr, Dietmar Pils, Eva Schuster, Barbara Holzer, Nina Pecha, Robert Zeillinger; Centre: Anna Bachmayr-Heyda, Magdalena Gamperl, Eva Blümel, Caroline Kreuzinger, Peter Borossay, Grazyna Dudek, Martin Stöger, Dan Cacsire Castillo-Tong; Bottom: Bettina Savarese-Brenner, Simon Deycmar, Nyamdelger Sukhbaatar, Elisabeth Maritschnegg, Katharina Auer, Markus Niebuhr, Agnes Reiner, Paul Speiser PROJEK TE 2014 GANNET53 Companion diagnostics of a drug study in metastatic, platinum-resistant ovarian cancer patients Collaborators: Maritschnegg E, Obermayr E, Zeillinger R, Holzer B, Pils D, Deycmar S, Cacsire-Castillo Tong D The FP7 EU-funded study GANNET53 targets the predominant, aggressive Type II ovarian tumours, which are characterized by resistance to platinum-based chemotherapy. In 176 Universitätsklinik für Frauenheilkunde

5 that course, mutant p53 should be targeted via an innovative Hsp90 (heat shock protein 90) inhibition applying the second-generation inhibitor Ganetespib. After successful completion of Phase I, Phase II will start in March Different aspects are covered in the companion diagnostics, for example a test, for possible prediction of responsiveness to Ganetespib should be established. Therefore ascites will be collected and cultured in the presence of different concentrations, or the absence of Ganetespib. The interaction of p53 with Hsp90 will be determined by the in situ Proximity ligation assay (PLA). A strongly reduced signal or no signal after treatment will be interpreted as in vitro response. Correlations of in vitro response with clinical outcome measures will be performed, allowing evaluation of the assay s predictive value. An example of a PLA performed on ascites cells is shown, where each red dot represents the interaction between a p53 and HSP90 protein. A second part involves monitoring of the patients via analysis of circulating tumour cells (CTCs). Tumour cells will be enriched via the Parsortix microfluidic system and analysed concerning their RNA expression of specific markers, and detected and characterized by immunofluorescence staining. Recent publications showed that the p53 protein, especially in its mutant form, aggregates into prion-like amyloid oligomers and fibrils. These structures might play a role in the development of therapy resistance. Still, these studies lack patient data and the methods used for analysis are limited. We established an ELISA based detection method, where a special coated plate is able to capture protein aggregates. We ve applied the assay to various ovarian cancer cell lines, with different p53 status. As expected, in p53 mutant cell lines, p53 prions were more prevalent than in p53 wild type ones. Cell lines carrying p53 null mutations showed the lowest levels. Another side project is the evaluation of the extracellular HSP90 levels in plasma. Normal cells only secrete ehsp90 under stress, whereas tumour cells show constitutive secretion linked to motility, invasion and metastasis. First tests in a small number of patients showed, that the plasma HSP90 levels were significantly increased not only in late stage, but also early stage ovarian cancer patients, compared to healthy controls. Evaluation of the Parsortix micro-fluidic technology to enrich CTCs Collaborators: Obermayr E, Maritschnegg E, Pecha N, Schuster E, Borrosay P, Holzer B, Wolf A, Speiser P, Zeillinger R We intended to develop a protocol combining the novel micro-fluidic enrichment technology (Parsortix, Angle plc., UK; see figure 1) and RT-qPCR for the molecular analysis of CTCs. Recently we identified CTC-specific mrna markers allowing CTC detection in 29% of breast cancer patients and in 24.5% of ovarian cancer patients at diagnosis. However, the detection of cancer cells was hampered by the large number of contaminating leukocytes. The necessary Figure 1: The Parsortix device for the enrichment of CTCs from blood samples Universitätsklinik für Frauenheilkunde 177

6 use of cut-off values for positivity reduced the specificity of the down-stream PCR assay. By improving the purity of cancer cells and PCR analysis we sought to increase both sensitivity and specificity of the diagnostic procedure. For reducing the blood sample volume (up to 20ml) density gradient centrifugation was used when needed. The samples were passed through the micro-fluidic disposable Parsortix cassette (see figure 2), which captures tumour cells based on their less deformable nature and larger size compared to blood cells. Figure 2: The microscope slide sized microfluidic separation cassette (left) which is inserted into the Parsortix device. When the blood sample is passed through the cassette, large and less deformable cells are captured within the 10µm separation step (right). Lysis of the captured cells was done directly in the cassette and total RNA was extracted. After a cdna pre-amplification step gene expression levels of leukocyte-specific and CTC-related markers were measured using RT-qPCR. The efficiency of the combined protocol was assessed in blood samples taken from patients with primary and recurrent malignant diseases. As a result, 7 out of 13 pre-selected RNA markers were not detectable in blood samples from 11 healthy volunteers. In cancer patients we observed measurable gene expression of at least one out of these 7 RNA markers. 5 out of 5 breast cancer (primary disease: N=1; metastatic: N=4), 9 out of 10 primary and 4 out of 8 relapsed ovarian cancer patients were classified correctly by the test. The most striking finding is the detection rate of 90% for primary ovarian cancer at a specificity of 100%. We conclude that the enrichment of rare cells from blood samples using micro-fluidics results in a highly pure cell population. This enables the application of extremely sensitive methods, like RT-qPCR to specifically detect rare events. By combining the novel micro-fluidic Parsortix technology for cell enrichment with molecular analysis we have taken a major step forward, which allows the implementation of liquid biopsies in cancer detection studies and as a companion diagnostic in clinical trials. Bio Bank of the Division of Gynecology and Gynecological Oncology for Research, Documentation and Analysis, in cooperation with the Center for Medical Statistics, Informatics, and Intelligent Systems. Collaborators: Pecha N, Pieber K, Obermayr E, Schuster E, Borrosay P, Holzer B, Wolf A, Wrba T, Zeillinger R. Tissue (bio) banking is what will take us to the next level of cancer treatment and cure. - Judy Garber, MD, MPH, Professor of Medicine, Harvard Medical School Bio Banks are facilities that are developed to collect, store and distribute samples for further use in basic and translational cancer research. Collection of high-quality bio samples, such as tumor tissue and peripheral blood, is the basis for discovering new biomarkers as well as novel therapy targets. Recent progress in the diagnosis and treatment of cancer can be attributed mainly on translational oncology research. Translational oncology focuses on novel research investigations which bridge the laboratory and clinical settings, including risk assessment, cellular and molecular characterization, prevention, detection, diagnosis and treatment of human cancers with the overall goal of improving the clinical care of oncology patients. Collecting bio samples for research immediately prompts the need for sound administration of the collected and conserved samples, enabling the availability of corresponding clinical data and tracking of the samples. A research database by the name of Tumorbank Molekulare Onkologie, located at the laboratory of the Molecular Oncology Group, Division of Gynecology and Gynecological Oncology at the University Clinic of Obstetrics and Gynecology, has been already implemented in Documentation of all data and setting up queries to enable data management is an ongoing project, with exportable data being available for statistical analysis now. Since December 2014, tissue samples as well as liquid samples (e.g. blood, ascites, saliva) has been collected and stored partly already in tubes with a barcoding system. This allows a unique sample assignment and recording in our Bio Bank database. As mentioned above, identification of the individual sample is crucial for a well-maintained Bio Bank. To fasten this process, the use of barcode scanners is supported by the RDA application. Barcodes can be read into any text field available in electronic case report forms. Hand-written tags, as used at the Molecular Oncology laboratory right now, pose a potential risk for mistakes such as wrong or unreadable labeling. For a fast and definitive identification of samples, barcoded sample tubes with barcode scanners are set up within reach at the laboratory workbenches. For an optimal use of barcoded systems, the import of scanned character strings into the databa- 178 Universitätsklinik für Frauenheilkunde

7 se has to be set up, too. Due to a planned cooperation of the Molecular Oncology Group with the Central Laboratory of the Vienna General Hospital (AKH), the interface between the RDA and the laboratory information system will be introduced as well. Along with the occurring of -omics platforms, requirement of high quality cancer tissues or peripheral blood for research of genome, transcriptome, proteome and metabolome is increasing rapidly. Therefore, the creation of high quality banks of biological resources at large medical centers or institutes is a fundamental and valuable work. The results of research using tissue samples will not affect therapeutic care of the patient right after donation of the sample, but if the cancer reoccurs in the future, however, new treatment options may be available. Stored tissue material of the cancerous disease at the time of first occurrence will be available. To compare the original tissue with the recurrent disease could help to not only distinguish between true recurrences versus second primary tumor, but help to improve the clinical care of the cancerous disease for two reasons: In comparison to a pathological institute, that often only stores paraffin embedded tissue samples, the Bio Bank of the Molecular Oncology group has been storing fresh frozen tissue samples and liquid biopsies such as blood, saliva and lavage samples, as well. The stored samples have already been tested on. New treatment options as well as novel biomarkers may be available by the time the patient is diagnosed with metastasis. The primary tissue samples stored already at the Bio Bank can then be tested with newly discovered diagnostic and prognostic biomarkers and compared to the recurrent disease. The patient will therefore profit from the scientific advances made by applied research done on already stored sample in the past donated by other individuals and hopefully profit from new advances in translational oncology like new drug targets. Lavage of the uterine cavity as potential tool for the diagnosis of ovarian cancer, its precursor lesions and endometrial cancer. Collaborators: Speiser P, Pecha N, Maritschnegg E, Horvat R, Zeillinger R Epithelial ovarian cancer (EOC) is generally diagnosed at an advanced stage, translating into a poor survival rate. There is increasing evidence that EOC often originates in the fallopian tubes and furthermore, tumours in the ovary may exfoliate cells that are transported into the uterine cavity via the fallopian tube. We have established an approach for lavage of the uterine cavity to detect shed cancer cells. This technique was further optimized, to be performed in an outpatient setting and in a non-invasive manner. Genetic changes were analysed in the lavage specimens to confirm the presence of cancer cells. This principle is applied in different studies. The LUDOC study will give information on the specificity and sensitivity of our approach in detecting EOC and its application in differential diagnosis of suspicious pelvic masses. Six European centres are involved so far. In the corresponding proof of concept study we obtained a lavage of the uterine cavity from 42 patients. Twenty-seven of these had EOCs, six had other malignancies with involvement of gynaecological organs, while nine had benign tumours. These samples, as well as corresponding tumour tissue if available, were examined for the presence of somatic mutations in a panel of genes using massively parallel sequencing. Lavage technique could successfully be applied and sufficient amounts of DNA obtained in all patients. Mutations in the genes PPP2R1A, TP53, CDKN2A, FBXW7, KRAS, PTEN, AKT1, EGFR, FGFR2 were identified in 16/26 (62%) lavage samples of patients with malignant tumors and only in 1/8 (12.5%) of patients with benign tumours (P=0.02, Fisher Exact Probability Test, one-tailed) (manuscript submitted). Figure 1: Laboratory entry form Figure 1: MPS mutation analysis of lavage specimens depicted as percent mutant alleles present. Shown are the results obtained from analysis of patients included in final analysis. Disease type, as well as stage of disease of EOC patients is listed on the y axis. The fraction of mutant alleles varied between 0.95% and 50.72%. Universitätsklinik für Frauenheilkunde 179

8 By adding yet three more studies, using the same molecular detection principle, we further extend the potential application spectrum of the Lavage technique: We hypothesize that even premalignant changes from high risk patients, could be detected applying this procedure, since already the precursor lesions, so called STICs, show a high exfoliation tendency. In the course of a project funded by the Anniversary Fund of the Austrian National Bank (LUSTIC) which started in 2014, we will be able to address this question. Six different European centres have already entered patients in this study. One pilot study is aiming at detecting exfoliated cells from endometrial cancer, particularly of type II EC, in the lavage fluid. The third study tested the feasibility of the procedure, measuring objective parameters like the amount of liquid that can be obtained as well as the condition of the lavage and complications occurring. 87 lavages have been protocoled in Vienna and in four additional cooperating centres in Europe. No adverse event occurred so far (manuscript under preparation). HPV-Papillocheck: Image and PCR based detection of cervical cancer derived CTC s Collaborators: Niebuhr M, Maritschnegg E, Holzer B, Zeillinger R Human papillomavirus (HPV) is involved in about 99% of all cervical carcinoma cases. A possible relapse detection method for HPV caused cervix carcinoma is the analysis of off-scaled circulating tumour cells in peripheral blood. For this purpose CTC detection via immunofluorescence and PCR should be established. For secondary immunofluorescence based image detection a panel of known CTC marker (EpCAM and Cytokeratin) is extended by the HPV oncogene E7 and upregulated cell cycle promotor (CCNE2 and p16). An example of cervical derived CTCs immunofluorescence staining is shown. Here the overlying signals of HPV E7 (yellow), EpCAM (green) and Cytokeratin (blue) verifying the cervical cancer offspring. In order to increase the general specificity and throughput a PCR based CTC detection should be established. Due to its constitutive transcription, the E7 mrna represents a reliable PCR marker. Therefore genotype specific probes targeting HPV E7 should be designed and evaluated. Currently 58 patients are included. Since the clinical relevance and abundance of HPV is mainly based on the genotypes HPV 16 and HPV 18, a reliable genotyping for the evaluation of both methods is needed. Therefore a sub-project started to evaluate the retrospective HPV genotyping method using FFPE starting material. It is planned to include 30 CIN I to CIN III patients in the genotyping evaluation. Correlation of circular RNA abundance with proliferation - exemplified with colorectal and ovarian cancer, idiopathic lung fibrosis, and normal human tissues. Collaborators: Bachmayr-Heyda A, Reiner AT, Auer K, Sukhbaatar N, Aust S, Bachleitner-Hofmann T, Mesteri I, Grunt TW, Zeillinger R, Pils D Circular RNAs are a recently (re-)discovered abundant RNA species with presumed function as mirna sponges, thus part of the competing endogenous RNA network. We analysed the expression of circular and linear RNAs and proliferation in matched normal colon mucosa and tumour tissues. We predicted >1,800 circular RNAs and proved the existence of five randomly chosen examples using RT-qPCR. Interestingly, the ratio of circular to linear RNA isoforms was always lower in tumour compared to normal colon samples and even lower in colorectal cancer cell lines. Furthermore, this ratio correlated negatively with the proliferation index. The correlation of global circular RNA abundance (the circrna index) and proliferation was validated in a non-cancerous proliferative disease, idiopathic pulmonary fibrosis, ovarian cancer cells compared to cultured normal ovarian epithelial cells, and 13 normal human tissues. We are the first to report a global reduction of circular RNA abundance in colorectal cancer cell lines and cancer compared to normal tissues and discovered a negative correlation of global circular RNA abundance and proliferation. This negative correlation seems to be a general principle in human tissues as validated with three different settings. Finally, we present a simple model how circular RNAs could accumulate in non-proliferating cells (Figure 1). Sci Rep Jan 27;5:8057. doi: /srep08057 Figure1(HPV-Papillocheck): Immunofluorescence staining of patients enriched mononuclear blood cell fraction. Clear distinction between cells of hematopoietic offspring and cervical cancer CTCs. Grey DAPI, green EpCAM, red CD45, yellow HPV E7, blue Cytokeratin. Figure 1. Schematic model of circular and linear RNAs in proliferating and nonproliferating cells. 180 Universitätsklinik für Frauenheilkunde

9 Peritoneal Tumor Spread in Serous Ovarian Cancer - Epithelial Mesenchymal Status and Outcome Collaborators: Auer K, Bachmayr-Heyda A, Aust S, Sukhbaatar N, Reiner AT, Grimm Ch, Horvat R, Zeillinger R, Pils D In this study we aimed to analyse the biological mechanisms underlying apparently different modes of peritoneal tumour spread in serous ovarian cancer: miliary (widespread, millet-like lesions) versus non-miliary (bigger, exophytically growing implants). Tumour tissues and ascites from 23 chemotherapy naive patients were analysed by RNA-sequencing and flow cytometry. On the basis of differential gene expression between miliary and non-miliary, gene signatures were developed. A calculated tumour spread factor revealed a significant independent negative impact of miliary spread on overall survival (HR 3.77; CI ; p=0.029) in an independent cohort of 165 serous ovarian cancer patients. Comparing previously published epithelial-mesenchymal transition (EMT) gene signatures, non-miliary spread correlated significantly with a reduced epithelial status. We conclude that serous ovarian cancer is a heterogeneous disease with distinct modes of peritoneal tumour spread, differing not only in clinical appearance, but also in molecular characteristics and outcome. EMT, peritoneal inflammation status, and therapeutic options are discussed and findings summarized in Figure 2. Significance. More than half of serous epithelial ovarian cancer patients present with a newly described type of intraperitoneal tumour spread, associated with differences in the inflammation status, activated oncogenic pathways, Figure 2. Summary of results (flow cytometry and transcriptomics) comparing cancer cells of patients with miliary and non-miliary peritoneal tumour spread. Non-miliary ascites samples showed more CD44+ cells, lower keratin expression, and higher MMP9/12 expression compared to miliary ascites samples. Ascites tumour cells of non-miliary patients showed a substantial reduced epithelial character compared to tumour cells from all other origins and tumour spread types, whereas ascites and solid tumour cells of non-miliary patients a globally increased mesenchymal characteristic revealed compared to ascites and solid tumour cells from miliary patients. lack of EMT, and thus reduced overall survival. Both, the diminished immune reaction and the enhanced epithelial and malignant characteristics of the tumour cells open new avenues for therapeutic options and strategies, like Catumaxomab, already in clinical use. Surface plasmon fluorescence spectroscopy for EV analysis Collaborators: Agnes T. Reiner1,2, Wolfgang Knoll1,2, Jakub Dostalek2, Alain Brisson3, Sai-Kiang Lim4, Robert Zeillinger, Dietmar Pils 1Centre for Biomimetic Sensor Science, School of Materials Science and Engineering, Nanyang Technological University, Singapore, Singapore 2BioSensor Technologies, AIT-Austrian Institute of Technology, Vienna, Austria 3Molecular Imaging and NanoBioTechnology, University of Bordeaux CNRS-UMR-CBMN, Pessac, France 4Institute of Medical Biology, A*STAR, Singapore, Singapore Extracellular vesicles (EVs) are gaining interest in many fields of research, but still methods for their analysis are mostly complex and lack sensitivity or specificity. Recently plasmonic biosensors appeared as EV analysis tools making use of surface plasmon resonance for detection of EVs captured by specific antibodies to the sensor surface. Additionally surface plasmons have the capability of coupling with fluorophores in their proximity and thus enhancing fluorescence emission. This attribute is employed in surface plasmon enhanced fluorescence spectroscopy (SPFS), on basis of which we want to develop a method for EV analysis (setup shown in Figure 3). EVs were purified by filtration and ultracentrifugation from cell culture supernatant of different cell lines and from biological fluids (i.e. ascites and plasma of ovarian cancer patients and plasma of healthy individuals). The vesicles were analysed with flow cytometry in fluorescence mode examining general EV makers and markers for ovarian cancer cell derived EVs. A model system using SPFS for EV analysis was built, where Annexin-V is used as capturing molecule on the sensor surface. With the means of flow cytometry in fluorescence mode EVs purified by ultracentrifugation could be detected and analysed. The general EV markers were, as expected, present on EVs in all samples. The ovarian cancer specific markers were only present on EVs from CaOV3 cell culture supernatant and ascites, but not from plasma of ovarian cancer patients, indicating that the concentration of cancer derived EVs in plasma might be below the detection limit. The SPFS model system could detect standard vesicles down to fm concentrations. Combining the specificity of molecular binding, the sensitivity of SPFS and the simplicity of plasmonic sensors, SPFS can provide a cheap and simple platform for EV analysis for scientific and clinical application. Universitätsklinik für Frauenheilkunde 181

10 pulation of resistant cells that are ultimately responsible for relapse and that by targeting this population front-line, we may prolong disease-free survival or even achieve cure. Figure 3. Setup of the model system for EV detection and analysis with SPFS. OCTIPS: Ovarian Cancer Therapy Innovative Models Prolong Survival FP7 EU Project ( ) HEALTH : Translational research on cancers with poor prognosis Coordinator: Cacsire Castillo-Tong D Collaborators: Wolf A, Kreuzinger C, Gamperl M, Reinthaller A and the Onco-Team Website: Concept: About 75% of advanced epithelial ovarian cancer patients respond to first-line surgery and chemotherapy but most relapse and ultimately acquire platinum resistance which soon leads to death. Relapsed high grade serous ovarian cancer is the single main cause of epithelial ovarian cancer. We hypothesize that the primary tumor includes a small po- Objectives: cancer, identifying molecules or pathways responsible for resistance and relapse, and defining new therapeutic targets and strategies. which represent resistant high grade serous ovarian cancer. Results in 2014: was organized together with a project partner Dr. Charles Theillet from INSERM, Institut National de la Santé et de la Recherche Médicale, Montpellier, France. The second periodic reports were submitted to the European Commission. The project entered its third and the last period. cancer cell lines: Definition of the establishment of cell lines In ascites, tumor cells often appeared in form of clusters which were easily separated and purified by filtration. In some primary cultures, tumor cells formed an island-like structure surrounded by fibroblasts (Figure 1I) which mimicked the tumor structure in vivo. The fibroblasts were reduced and finally eliminated by repeating selective trypsinisation, until pure tumor cell culture had been established (Figure 1H). All cell lines have been passaged more than 40 times. After TP53 mutations were determined by the functional yeast assay in the corresponding tumor tissues, the purity of the cell culture was determined using ddpcr. A cell line 182 Universitätsklinik für Frauenheilkunde

11 was defined when 100% cells in the culture were confirmed to harbor the specific TP53 mutation. To ensure the authenticity of the cells, short tandem repeat analysis of 7 markers (TPOX, vwa, CSF180, D16S539, D7S820, D13S317, D5S818, Applied Biosystems Life Technologies) was performed regularly. Morphology of the cells 7 cell lines have been established (examples in figure 1). Two of them were derived from the same patient, one at primary diagnosis and the other at the recurrence. The cell lines presented with irregular sizes and shapes and had a high ratio of nucleus to cytoplasm (Figure 1A). Most of the cell lines grew as monolayers. One cell line grew as a mixture of clusters and adherent islands. These clusters could contain a couple of cells up to several hundred cells (Figure 1B). Figure 2: Scratch Assays from all cell lines at 0h and after 48h (brightfield microscopy images). 0 hour: the scratched area was defined as 100%; 48 hours: proportion of the remaining cell free area was indicated. A: 58%* remaining cell free area was manually calculated because of cell clustering. response to the first line chemotherapy (Table 1). Two patients (12370 and 13363) could not be optimally debulked, having a tumor mass of <5mm and >5mm after surgery, respectively. Although they showed partial response to the first line chemotherapy, they died of disease progression at 3 and 9 months after the last dose, respectively. Mutations in the TP53, KRAS, BRAF and BRCA1/2 genes All patients had a somatic TP53 gene mutation (Table 1). The percentage of the mutant cells was examined to de- Figure 1. Morphology of established cell lines (brightfield microscopy images). A: HE staining of 13914_1; B: 12370; C: 13363; D: 15233; E: 13699; F: 13914_1; G: 14433_1; H: 8587 in pure culture; I: 8587 tumor cell islands surrounded by fibroblasts. Growth and mobility of the cells Split ratios of the cell lines differed from 1:2 to 1:3. Some cell lines had a shorter doubling time of two to three days whereas some needed six to nine days. Scratch assay showed that some cell lines (for example 13699) did not have any mobility (Figure 2D) while other lines had a similar migration rate, filling around 1/3 to 4/5 of the scratched areas (Figure 2A-2C, 2E-2G). One pair of the matched cell lines derived from the same patient (13363 and 15233) had similar high migration ability (Figure 2B and 2C). Patients and tumor characteristics Among the seven established cell lines, the and One cell line 8587 was derived from tumor tissue and all others were established from ascites. The age of the patients at diagnosis ranged from 33 to 67 with a median age of 55. All primary samples set for cell culture were taken before any chemotherapy. All patients received standard treatments and presented with partial or complete clinical Table 1. Clinical data of patients and clinicopathological characteristics of tumors 1 cpr=clinical partial response; 2 ccr= clinical complete remission; 3 Point Mutation at intron 5 (bp 13239) leading to a splice variant (g.13193_13238del, Val172Val), which causes a frameshift and a truncated protein (fsx60); 4 DBD: central DNA-binding core domain; 5 OD: homo-oligomerization domain Universitätsklinik für Frauenheilkunde 183

12 TT = tumor tissue; 0: no expression; 1: weak expression in the minority of the cells; 2: weak expression in the majority of the cells; 3: strong expression in the minority of the cells, 4: strong expression in the majority of the cells. termine the purity of the cell lines. It was confirmed that the TP53 mutations were stable through all passages. Sequencing of the corresponding blood DNA confirmed that none of the cell lines were from patients with a germline TP53 mutation. A non-sense mutation (c.8557a>t, p.lys2853x) in BRCA2 and a 11bp deletion (c.3481_3491del, p.glu1161fsphex3) in BRCA1 were found in the cell lines 14433_1 and 13914_1, respectively, both being germline mutations. The latter was detected from a patient, who had breast cancer previously. No mutations in the KRAS and BRAF genes were detected in the cell lines. Antigen expression of cell lines and the corresponding tumors Antigen expression of the established cell lines was compared with that in the corresponding tumor tissues (Table 2). Table 2. Immunohistochemistry staining in all established cell lines and their corresponding tumor tissues Cytokeratins 8, 18 and 19 and EpCAM were expressed in all tumor cells in tissues as well as in the cell lines (Figure 3A, 3B and 3G, 3H). CA125 was expressed in the majority of the cell lines as well as in tumor tissues (Figure 3M, 3N and 3O, 3P). Most of the patients had very few CD44 and vimentin positive tumor cells in tissues (Figure 3J, 3L and 3D, 3F). In the cell lines, the expression of these two proteins was quite heterogeneous (Figure 3C, 3E and 3I, 3K). In vitro chemosensitivity Five cell lines did not show any remarkable difference in the responsiveness to carboplatin, all being highly sensitive to the drug. One cell line (13914_1) as well as the cell line established from ascites under treatment (15233) were highly resistant (Figure 4). Figure 4: IC 50 +/- SD [µm] values of all cell lines treated with carboplatin. Prospects in 2015: HGSOC will be validated pression in primary and recurrent lines Figure 3: Immunohistochemistry staining examples from cell lines and corresponding tumor tissue (brightfield microscopy images). A,B: Cytokeratin 8,18,19 staining of 13914_1 with corresponding tumor tissue; C,D: Vimentin staining of with corresponding tumor tissue, E,F: Vimentin staining of 8587 with corresponding tumor tissue; G,H: EpCAM staining of 14433_1 with corresponding tumor tissue; I,J: CD44 staining of with corresponding tumor tissue; K,L: CD44 staining of 13914_1 with corresponding tumor tissue; M,N: CA125 staining of with corresponding tumor tissue; O,P: CA125 staining of 13914_1 with corresponding tumor tissue. 184 Universitätsklinik für Frauenheilkunde

13 Post OVCAD: Ovarian Cancer Diagnosis of a silent killer FP6 EU Project ( ); post period: LifeSciHealth: LSH Coordinator: Robert Zeillinger Collaborators: Dan Cacsire Castillo-Tong, Andrea Wolf, Eva Schuster Website: Background: After closing the project successfully in 2010, the consortium decided to continue the cooperation on ovarian cancer research with the materials collected during the project, including samples from blood, ascites, and tumor tissues from 275 ovarian cancer patients with advanced epithelial ovarian cancer. Objectives: gnosis. the patients. and progression of ovarian cancer. Results in 2014: do research work with OVCAD resources and publish the data. In 2014, 5 publications had appeared in scientific journals. have been included into the study. At the end of 2013, all patients have had a clinical follow-up of at least five years. The data were thoroughly updated. Prospects in 2015: produced from the projects, after the updating of the clinical data of patients. Universitätsklinik für Frauenheilkunde 185

14 REPRODUCTIVE BIOLOGY UNIT P E R S O N A L S T A N D W I S S E N S C H A F T L I C H E M I T A R B E I T E R / I N N E N Mag. Dr. Martin Knöfler Ao. Univ. Prof. Mag. Dr. Jürgen Pollheimer Ass.Prof. DIin (FH) Valerie Fock Dissertantin Mag. Philipp Velicky Dissertant Mag.a Gerlinde Otti Dissertantin MSc. Kerstin Plessl Dissertantin MSc. Leila Saleh BMA (Klinik) Peter Haslinger BMA (MUW, 100%) Mag. a Sandra Haider BMA (MUW, 75%), Dissertantin Mag. a Gudrun Meinhardt BMA (MUW (100%), Dissertantin D I P L O M S T U D I E R E N D E Sophie Herzer Stephanie K. Milla Stefanie Kaltenberger Humanmedizin Humanmedizin Biomedizinische Wissenschaften Kurzbeschreibung Während der Schwangerschaft adhäriert die Plazenta an der mütterlichen Dezidua und bildet somit den extravillösen Trophoblasten (EVT). Dieser trophoblastäre Subtyp invadiert die Dezidua sowie Spiralarteriolen, um eine adäquate Nährstoffversorgung und die immunologische Akzeptanz in einem allogenen Umfeld zu gewährleisten. Die EVT Differenzierung ähnelt teilweise der Krebszellentstehung, unterscheidet sich aber durch strikte Kontrollmechanismen, deren Ursprung bisher noch nicht vollständig geklärt ist. In diesem Zusammenhang konnten wir bereits zeigen, dass invasive Trophoblasten ein bemerkenswertes Repertoire an ERBB Rezeptoren exprimieren (Figur 1). Interessanterweise, finden wir ausschließlich ERBB2 und ERBB3 an der Oberfläche dieses trophoblastären Zelltypus. Frau Mag. a Valerie Fock konnte in einer aktuellen Studie zeigen, dass beide Rezeptoren vom Wachstumsfaktor Neuregulin 1 (NRG1) stimuliert werden können und dadurch die Apoptose in invasiven Trophoblasten unterdrücken. Darüber hinaus konnten wir erstmals zeigen, dass NRG1 von dezidualen Stromazellen gebildet und sezerniert wird. In einer weiteren, bereits in Begutachtung befindlichen Studie assoziierten Frau Mag. a Fock und Plessl die Expression von EGFR und ERBB4 mit der proliferativen Kapazität von Trophoblasten. Beide ERBB Rezeptoren regen, nach Stimulierung mit heparin-binding epidermal growth factor (HB-EGF) und epidermal growth factor (EGF), die Zellteilung in primären Trophoblasten an. In Zuge einer Zusammenarbeit mit Frau Dr. Sabine Dekan (Institut für Pathologie, MUW) konnten wir des Weiteren zeigen, dass das hyperproliferative Krankheitsbild der kompletten Blasenmole mit einem starken Anstieg der EGFRpositiven Trophoblastsubtypen einhergeht. Ä R Z T L I C H E M I T A R B E I T E R / I N N E N Dr. Harald Zeisler Dr. Katharina Worda Dr. Christof Worda Ao. Univ. Prof. Priv. Doz Priv. Doz Z U S Ä T Z L I C H E M I T A R B E I T E R / I N N E N Jakob Knöfler Laborassistenz (Drittmittel, 25%) WISSENSCHAFTLICHE PROJEKTE LIGAND-INDEPENDENT CONTROL OF EXTRAVILL- OUS TROPHOBLAST DIFFERENTIATION Im Rahmen des vom FWF geförderten Projektes (P25187) studieren wir die Rolle der ERBB (EGFR) Rezeptoren in der invasiven Differenzierung des humanen Trophoblasten. M I T A R B E I T E R I N N E N : Fock V, Plessl K, Haider S, Knöfler M und Pollheimer J. Abb. 2. Reziproke Expression von verschiedenen Rezeptor-Tyrosinkinasen (RTK) in primären nicht invasiven (CTB) und invasiven (EVT) Trophoblasten. Die heat map zeigt die RTK mrna expression von verschiedenen CTB und EVT Isolaten. Jede Reihe repräsentiert ein Probenset. Der Farbcode zeigt den n-fachen Unterschied im Vergleich zum Median. (rot, überexprimiert; blau, reprimiert; grau, unverändert). 186 Universitätsklinik für Frauenheilkunde

15 Fock V, Plessl K, Fuchs R, Dekan S, Milla S, Haider S, Fiala C, Knöfler M, and Pollheimer J. Trophoblast subtype-specific EGFR/ERBB4 expression correlates with cell cycle progression and hyperplasia in complete hydatidiform moles. 2014, Revision underway for Human Reproduction Kaltenberger S, Meinhardt G, Fiala C and Pollheimer J. ERBB2 gene amplification is associated with the invasive differentiation program in human trophoblasts. 2014, manuscript in preparation Fock V, Plessl K, Otti G, Fiala C, Knöfler M and Pollheimer J. Neuregulin-1 suppresses apoptosis in trophoblasts via the activation of the oncogenic unit ERBB2/ERBB , manuscript in preparation THE ROLE OF ADAM12 IN NORMAL AND PATHOLOGICAL PREGNANCIES In diesem von der Nationalbank (P17147) geförderten Projekt untersuchen wir die Funktion der Protease ADAM-12 im humanen extravillösen Trophoblasten. M I T A R B E I T E R I N N E N : Fock V, Biadasiewicz K, Knöfler M und Pollheimer J Kurzbeschreibung: ADAM-12 wird in zwei verschiedenen Varianten, durch alternatives Splicing, gebildet. ADAM-12L, die längere Form wird an der Zellmembran exprimiert und die kürzere Variante, ADAM-12S wird als solubles Protein sekretiert. Obwohl sehr wenig über die biologische Bedeutung dieser Protease bekannt ist gibt es Hinweise, dass ADAM-12L in der Krebsentstehung und Metastasierung eine Rolle spielt. In einer erst kürzlich publizierten Arbeit konnten wir zeigen, dass uteroplazentares ADAM-12 ausschließlich vom Trophoblasten gebildet wird. Des Weiteren konnten wir ADAM-12 als einen positiven Regulator der Trophoblasteninvasion identifizieren. Hierbei ist es uns gelungen die ADAM-12 mediierte Aktivierung des pro-migratorischen Moleküls Integrin beta 1 nachzuweisen. Darüber hinaus postulieren wir, dass ADAM-12 eine wichtige Rolle in der Degradation von IGFBP3 einnimmt und somit eine tragende Rolle in der Entstehung von Schwangerschaftserkrankungen spielen könnte. Biadasiewicz K, Fock V, Dekan S, Proestling K, Velicky P, Haider S, Knöfler M., Fröhlich C, Pollheimer J. Extravillous trophoblast-associated ADAM12 exerts pro-invasive properties. Biol Reprod Pollheimer J, Fock V, Knöfler M. Review: The ADAM metalloproteinases - Novel regulators of trophoblast invasion? Placenta IDENTIFICATION OF FACTORS CONTROLLING DIFFE- RENTIATION IN HUMAN DECIDUAL STROMAL CELLS OF EARLY PREGNANCY M I T A R B E I T E R I N N E N : Otti G, Saleh L, Pollheimer J und Knöfler M. Kurzbeschreibung Dezidualisierung, der Umbau des uterinen Endometriums in das Endometrium der Schwangerschaft, ist ein kontrollierter zellulärer Prozess, der im Wesentlichen durch camp und Progesteron gesteuert wird. Da die Versorgung mit gesunden endometrialen Zellen extrem limitiert ist, haben wir ein Modellsystem mit dezidualen Stromazellen, die von Plazenten isoliert werden, aufgebaut. Bislang konnten wir zeigen, dass diese Zellen analog zu jenen, die aus dem normalen Endometrium isoliert wurden, reagieren (Saleh et al., 2011). Im Rahmen ihrer Dissertation konnte nun G. Otti zeigen, dass Notch signalling eine wichtige Rolle bei der Funktion und Differenzierung dieser Zellen spielt, beispielsweise die Expression von dezidua-spezifischen Hormonen wie Prolaktin steuert. Diese Daten wurden heuer im internationalen Top-Journal Plos One publiziert (Otti et al., 2014) Des Weiteren wurde eine entsprechende Arbeit zur Thematik kürzlich publiziert (Saleh et al. 2011). Diese Studie beschreibt die Etablierung eines primären Zellsystems zur Simulation der Dezidualisierung. In diesen Modellsystemen untersuchte G. Otti die Funktion des kanonischen Notch signalling. Sie konnte zeigen, dass der Rezeptor Notch2 sowie die Liganden Jag1, Dll1 und Dll4 in dezidualen Stromazellen vorhanden sind und deren Expression von camp/ Porgesteron abhängt. Ein kanonischer Notch Reporter wird während der in vitro Differenzierung angeschalten, sodass die Vermutung nahe liegt, dass der Notch Signalweg eine kritische Rolle in der humanen Dezidualisierung spielt. Weitere funktionelle Studien zu diesem Thema sind zurzeit in Arbeit. Saleh L, Otti G, Fiala C, Pollheimer J, and Knöfler M Evaluation of human first trimester decidual and telomerase-transformed endometrial stromal cells as model systems of in vitro decidualization. Reprod Biol Endocrinol. 2011; Dec 7;9:155 Otti G, Saleh L, Fiala C, Pollheimer J, and Knöfler M Notch signalling controls differentiation of human decidual stromal cells. PLoS One. 2014; Nov 14;9(11) CRITICAL SIGNALLING PATHWAYS REGULATING TRO- PHOBLAST INVASION OF THE HUMAN PLACENTA Im Rahmen des vom FWF geförderten Projektes (P22587) studieren wir die Rolle des konservierten Signalweges Notch sowie anderer Signalketten in der invasiven Differenzierung des humanen Trophoblasten. Universitätsklinik für Frauenheilkunde 187

16 M I T A R B E I T E R I N N E N : Haider S, Velicky P, Otti GR, Pollheimer J, Knöfler M Notch signalling ist generell für Aufrechterhaltung von Stammzellen, aber auch für Homöostase und Differenzierung von Geweben verantwortlich. Um Notch Signalling auszulösen bedarf es eines direkten Zell-Zell Kontakts zwischen Notch Rezeptoren und den ebenfalls Membrangebundenen Notch Liganden. Jüngere Befunde aus der Literatur weisen weiters darauf hin, dass die Expression von Notch Rezeptoren und deren Liganden in präeklamptischen Plazenten verändert sein könnten. Die Funktion des Pathways bei humaner Plazentaentwicklung und Differenzierung ist jedoch gänzlich unbekannt. Im Rahmen ihrer Dissertation untersucht S. Haider die Produktion von Notch Signalkomponenten und die Funktion des Notch Signalweges in verschiedenen Trophoblastmodellsystemen. Bislang konnte sie zeigen, dass die Inhibition des Notch Signallings zu vermehrter Invasion und Proliferation des Trophoblasten führt (Haider et al., 2014). Weiters lassen sich Trophoblast Subtyp-spezifische Expressionen bestimmter Notch Rezeptoren feststellen. Neben der Funktion in der Trophoblastenproliferation dürften die Notch Liganden auch parakrine Effekte auf deziduale Zellen ausüben. Im Rahmen seiner Dissertationen untersucht weiters Philipp Velicky die Funktion des Notch-abhängigen Transkriptionsfaktors RBPJkappa (Velicky et al. 2014), während eine andere Dissertantin des Labors, Gerlinde Otti, Notch Signaltransduktion in der Dezidua studiert (siehe oben). Haider S, Meinhardt G, Velicky P, Otti GR, Whitley G, Fiala C, Pollheimer J, Knöfler M. Notch signaling plays a critical role in motility and differentiation of human first-trimester cytotrophoblasts. Endocrinology Jan;155(1): Function of the Wingless ligand Wnt5A in placental differentiation and senescence In diesem von der Herzfelderschen Familienstiftung unterstützen Projekt (AP00574OFF) untersuchen wir die Funktion des Wnt-Liganden Wnt5A während der Differenzierung und Seneszenz der Plazenta MitarbeiterInnen: Meinhardt G, Knöfler M Die Wingless (Wnt)- Signalkaskade ist ein hochkonservierter Signalweg, der in Embryogenese und Onkogenese eine wichtige Rolle spielt und Zellfunktionen wie Wachstum, Proliferation, Überleben und Zelldifferenzierung steuert. Der Wnt Ligand Wnt5A wurde kürzlich als Regulator des Wachstums und der Alterung von Ovarialkarzinoma-Zellen identifiziert und könnte diese Prozesse auch in anderen reproduktiven Zelltypen steuern. Tatsächlich konnten wir und andere Wnt5A sowohl in der Plazenta als auch im schwangeren Endometrium, der Dezidua, nachweisen. Die Funktion des Liganden in diesem biologischen System ist jedoch noch unbekannt. Wir studieren daher mittels verschiedener molekularbiologischer Techniken und Trophoblastzell-Modellsystemen die Rolle von Wnt5A in der Plazentafunktion und-differenzierung. Unsere Experimente zeigten, dass der Ligand in zahlreichen Zelltypen (mesenchymalen, leukozytären und trophoblastären Zellen) an der feto-maternalen Grenzfläche nachweisbar ist und hier eine wichtige Rolle zu spielen scheint. In plazentären villösen Gewebskulturen konnten wir zeigen, dass Wnt5A einerseits die Zellproliferation der epithelialen villösen als auch der Zellsäulentrophoblasten in vitro fördert, andererseits apoptotische Prozesse unterdrücken kann und somit eine wichtige Rolle in der Gewebshomeostase spielt. Des Weiteren konnten wir eine Aktivierung der MAPK(ERK) Signalkaskade durch den Liganden nachweisen. Velicky P, Haider S, Otti GR, Fiala C, Pollheimer J, Knöfler M Notch-dependent RBPJ inhibits proliferation of human cytotrophoblasts and their differentiation into extravillous trophoblasts. Mol Hum Reprod Aug;20(8): Universitätsklinik für Frauenheilkunde

17 GYNÄKOLOGISCHE ENDOKRINOLOGIE expressed in endometrium of patients with versus without endometriosis. With this study we aim to search for differences in the plasma of secreted mirnas of patients with versus without endometriosis. Additionally, we plan to evaluate the correlation of these differentially secreted mirnas with the disease. This shall allow assessing the potential use of mirnas as monitoring or prognostic markers for endometriosis. Leiter/Verantwortliche des Projekts: I. Yotova, P. Pateisky, Kooperation mit Gynäkologie W I S S E N S C H A F T L I C H E M I T A R B E I T E R I N N E N Christian Schneeberger ao. Univ.-Prof. Mag. Dr. Andrea Kolbus Univ.-Doz. Dipl. Biol. Dr. Mario Mairhofer Mag. Dr. Detlev Pietrowski Dipl. Biol. Dr. Iveta Yotova Dr. Marco Medjimorec BSc Marion Martins BSc Gerald Hofstätter BSc Daniel Faust BSc Barbara Widmar Ladislaus Szabo AR Ä R T Z L I C H E M I T A R B E I T E R I N N E N Walter Tschugguel ao. Univ.-Prof. Dr. Petra Pateisky Dr. Aulona Gaba Dr. PROJEKTE 2014 Circulating mirnas as potential biomarkers for Endometriosis Endometriosis is a disease which is affecting up to 10% of women in their reproductive age. The symptoms are very heterogeneous and may range from dysmenorrhea and dyspareunia to infertility. Up to now the gold standard of diagnosis is still laparoscopic evaluation of the pelvis. Therefore, the search for new and less invasive diagnostic and/or monitoring markers is an interesting research field. MicroRNAs are a class of short, non-coding RNAs known to regulate gene expression post- transcriptional. There exists evidence that those factors are differentially EU-Projekt HYPERLAB Das von der EU im 7. Rahmenprogramm geförderte Forschungsprojekt HYPERLAB (High Yield and Performance Stem Cell Lab) wurde im September 2009 gestartet. Der Schwerpunkt dieses Projektes liegt auf der Optimierung von gegenwärtigen Stammzell-Kultivierungsmethoden. Innovative neue Techniken (Pipet Robots, PipeBased Bioreactors) werden an die besonderen Anforderungen der Stammzell-Kultivierung adaptiert und sollen so den Arbeitsaufwand für die Kultivierung reduzieren, sowie ein High-Throughput-Screening von unterschiedlichen Medien und Wachstumsfaktoren ermöglichen. An der Universitätsklinik für Frauenheilkunde werden in Zusammenarbeit mit den Projektpartnern Protokolle zur Expansion und Differenzierung von CD34-positiven hämatopoetischen Stammzellen aus Nabelschnurblut etabliert und optimiert. Leiter/Verantwortliche des Projekts: A. Kolbus, M. Mairhofer E-rare Projekt EMINA-2 Das E-rare Projekt EMINA-2 (Analysis of VPS13A function in ips-derived erythroid cells) wurde im Februar 2013 gestartet und widmet sich der Aufklärung der molekularen Mechanismen der seltenen Erkrankung Neuroacanthozythose. PatientInnen mit dieser Erkrankung, welche durch Defekte/ Mutationen im VPS13A Gen ausgelöst wird, zeigen neben Huntington s Disease-ähnlichen neuro-degenerativen Symptomen typisch sternförmig deformierte Erythrozyten. Die Funktion des VPS13A Proteins in humanen Zellen ist noch völlig unklar und soll im Rahmen des internationalen Kooperationsprojektes mit Partnern aus Deutschland, Holland und Israel untersucht werden. Als Modellsystem wird mit patientenspezifischen induzierten pluripotenten Stammzellen gearbeitet, welche in-vitro in erythroide Zellen differenziert werden. Als weiteres Modellsystem stehen primäre erythroide Zellen aus Nabelschnurblut zur Verfügung. Weitere Informationen zum Projekt sind auf der Projekthomepage unter tu-dresden.de/emina2/ zu finden. Leiter/Verantwortlicher des Projekts: M. Mairhofer Universitätsklinik für Frauenheilkunde 189

18 EUROSTARS-Projekt MUSST (Multi-step Streptamer systems for clinical stem cell research) Im Projekt MUSST wird zusammen mit Projektpartnern aus Deutschland ein neuartiges System zur Anreicherung/ Reinigung von hämatopoietischen Stammzellen aus Nabelschnurblut entwickelt, welches eine mehrstufige Anreicherung erlaubt, die in derzeit verwendeten Selektionssystemen nicht möglich ist. Die neuartigen Affinitätsreagenzien werden von Firma IBA entwickelt, und Projektpartner IBA stellt das Know-how im Mikrofluidikbereich zur Verfügung. Die Hauptaufgabe unserer Arbeitsgruppe ist die Evaluierung von neuen Reagenzien und Methoden mit Stammzellen aus der Nabelschnur sowie der Vergleich zum derzeitigen state-of-the-art im Bereich der Zellselektion. Leiter/Verantwortlicher des Projekts: M. Mairhofer Mathematische Modellierung des Einflusses von Genvarianten im VEGF-VEGF Rezeptorsystem bei OHSS Patienten In Vorarbeiten haben wir zeigen können, dass das Auftreten des ovariellen Überstimulations-syndroms (OHSS) mit genetischen Unterschieden im VEGF-VEGF-Rezeptorsystem zusammenhängt. Diese Unterschiede lassen sich auch auf Proteinebene im Serum von Patienten nachweisen (siehe Projekt Nr. 4; Nouri et al (2014), Pietrowski et al (2012) Im Rahmen dieses Nachfolgeprojekts soll untersucht werden, ob sich ein mathematisches Modell für einen direkten Genvarianten-Proteinexpressions-Zusammenhang in dieser Patientengruppe darstellen lässt. Damit würde erstmalig für OHSS Patienten der Einfluss einzelner Genvarianten auf die Proteinexpression im VEGF-VEGF-Rezeptorsystem berechenbar sein und könnte damit auch als Prognosefaktor in der IVF-Therapie eingesetzt werden. Leiter/Verantwortliche des Projekts: D. Pietrowski, M. Schreiber (Kooperation mit AG Schreiber) Effect of testosterone on Human Uterine Vein Endothelial Cells (HUVEC) Androgens are the most abundant sex steroids in men, as well as in women after menopause and are also used pharmacologically. While the effects of estrogens on vasculature are studied extensively, the mechanisms implicated in the androgen regulation of endothelial cell behavior are far from being thoroughly understood. A growing body of evidence suggests that androgens affect the behavior of endothelial cells, modulating proliferation, migration and angiogenesis. The results of different studies remain contradictory with regard to the involvement of Androgen Receptor in Testosterone mediated signaling. The effects of androgens on vasculature seem to be tissue and vessel caliber dependent. Our project aims to investigate whether the effects of Testosterone on endothelial cells from macrovasculature (HUVEC) are different then on microvasculature and whether migration and proliferation are mediated by the Androgen Receptor. Leiter/Verantwortliche des Projekts: A. Gaba Analyse des Auftauverfahrens von Ovargewebe im Rahmen des Ovarian Tissue Banking Die Durchführung einer Chemotherapie ist in der Regel gonadotoxisch. Im Rahmen des Ovarian Tissue Bankings (OTB) wird daher insbesondere jüngeren Patientinnen, angeboten, ein Teil ihres Ovargewebes als Fertilitätsreserve, einfrieren zu lassen. Es werden hierbei Kryoprotokolle genutzt, die im wesentlichen DMSO als Kryoprotektant enthalten. Die zurzeit erfolgreich genutzten Auftauprotokolle hingegen enthalten Sucrose als wesentliches Medium. Schon aus physikalischen Gründen ist davon auszugehen, dass der Wechsel von DMSO haltigem Einfriermedium hin zu einem Sucrose haltigem Auftaumedium für die Zellen im Ovargewebe einen zusätzlichen osmotischen Stress beinhaltet, der zu einem deutlich erhöhten Anteil an apoptotischen und/oder nekrotischen Zellen führen kann. Im Rahmen dieses Projekts soll daher untersucht werden, inwieweit es möglich ist optimierte Bedingungen zu schaffen, um den Stress für die Zellen und damit auch die Erfolgsrate einer Retransplantation zu verbessern. Modellhaft werden die experimentellen Arbeiten an einer Granulosa Zelllinie durchgeführt. Für dieses Projekt ist bereits ein Ethikvotum vorhanden. Leiter/Verantwortliche(r) des Projekts: D. Pietrowski, M. Kokotsaki, Ch. Schneeberger ONCOGENOMICS W I S S E N S C H A F T L I C H E M I T A R B E I T E R I N N E N Martin Schreiber, ao.univ.-prof. Mag. Dr. M.Sc. Andrea Friesenhengst, B.Sc. Tamara Pribitzer-Winner, B.Sc. Wissenschaftliche Projekte (Auszug) Assoziation des G473A Polymorphismus und der Expression von Lysyl Oxidase mit dem Brustkrebsrisiko und der Prognose Die Lysyl Oxidase (LOX) ist ein extrazelluläres Enzym mit einer essentiellen Rolle in der kovalenten Vernetzung von extrazellulären Matrixproteinen sowie wahrscheinlich noch weiteren Funktionen. Das sekretierte LOX-Proenzym wird proteolytisch zum fertigen, funktionellen Enzym prozessiert. Die LOX-Expression kann im Tumor sowohl erhöht als auch vermindert sein und ist sowohl mit der Tumorsuppres- 190 Universitätsklinik für Frauenheilkunde

19 sion als auch mit der verstärkten Ausbildung von Metastasen assoziiert. Der G473A Polymorphismus (rs ) führt zu einer Arg158Gln Aminosäure-Substitution im LOX- Propeptid, welche die tumor-suprimierende Wirkung von LOX beeinträchtigt und in einer chinesischen Studienpopulation mit erhöhtem Brustkrebsrisiko assoziiert ist. In unserer erstmals mit europäischen Frauen durchgeführten Studie konnten wir zeigen, dass das A-Allel dieses Polymorphismus mit einem höheren Manifestationsalter, schlechterer Prognose hinsichtlich krankheitsfreier und metastasenfreier Überlebensrate, jedoch nicht mit einem erhöhten Brustkrebsrisiko assoziiert ist. Die LOX mrna-expression war in Tumoren von Patientinnen von über 55 Jahren, von postmenopausalen Patientinnen, in Östrogen-Rezeptor (ER) positiven Tumoren und in p53-negativen Tumoren signifikant erhöht. Der G473A Genotyp hatte jedoch keinen signifikanten Einfluss auf die LOX Expression, weder in Tumorgewebe noch in Brustkrebszelllinien. Eine hohe LOX- Expression war mit einer schlechten Prognose bei Patientinnen mit ER-negativen Tumoren, aber nicht bei solchen mit ER-positiven Tumoren assoziiert. Bei multivariablen Analysen zeigte sich die LOX-Expression als unabhängiger prognostischer Parameter, der G473A Genotyp hingegen nicht. Eine kleine Subgruppe von Östrogenrezeptor (ER)- negativen Patientinnen mit erhöhter LOX-Expression wies eine sehr ungünstige Prognose hinsichtlich des krankheitsfreien (p=0,001) und Metastasen-freien (p=0,0003) Überlebens auf und stellt somit ein vielversprechendes Kollektiv für zukünftige, individualisierte Ansätze der Brustkrebsdiagnose und therapie dar wir, dass eine zytoplasmatische Überexpression von ILEI mit der Metastasierung und mit einer schlechteren Prognose bei Brustkrebspatientinnen assoziiert ist. Entscheidend für den biologischen Effekt auf den Krankheitsverlauf war dabei die genaue subzelluläre Lokalisation von ILEI. Diese Entdeckungen konnten wir nun in einem fast doppelt so großen Patientinnenkollektiv bestätigen. Unsere Kooperationspartner entdeckten zusätzlich, dass die Lokalisation von ILEI massgeblich durch extrazelluläre Proteasen, insbesondere Plasminogen und den Rezeptor für Plasminogen-Aktivator vom urokinase-typ (upar) beeinflusst wird. Imunhistochemische Analysen unseres Patientinnenkollektivs bestätigten dass die Lokalisation von ILEI deutlich mit der Expression von upar in Tumorzellen korreliert, und dass die hohe prognostische Aussagekraft der ILEI-Lokalisation durch gleichzeitige Auswertung der upar Expression und Berücksichtigung beider Marker in Überlebensanalysen weiter verbessert werden kann (Abbildung 2). Abbildung 1: Kaplan-Meier Analysen des krankheitsfreien und des Metastasen-freien Überlebens von Patientinnen mit hoher vs. niedriger LOX- Expression sowie positivem und negativem Östrogenrezeptor (ER) Status. ILEI (Interleukin-like epithelial-to-mesenchymal transition inducer) und das plasminogen upar (urokinase plasminogen activator receptor) System als prognostische Marker des Mammakarzinoms In einer früheren Studie konnten wir in einer Kooperation mit dem IMP Wien zeigen, dass das Protein interleukin-like EMT inducer (ILEI) notwendig und hinreichend für die epithelial-mesenchymale Transition, Tumorbildung und Metastasierung normaler Epithelzellen ist. Außerdem fanden Abbildung 2: Kaplan-Meier Analysen des Metastasen-freien Überlebens basierend auf immunhistochemischen Färbungen von ILEI (oben), upar (Mitte), oder einer Kombination aus ILEI und upar (unten). Universitätsklinik für Frauenheilkunde 191

20 Assoziation des TT Genotyps des rs10046 Polymorphismus der Aromatase (CYP19) mit einem verstärkt prämenopausalen Auftreten von Brustkrebs Das CYP19 Gen kodiert für Aromatase, ein Enzym das die Produktion von Östrogenen aus Androgenen katalysiert. Der rs10046 single nucleotide polymorphism (SNP) befindet sich in der 3 untranslatierten Region des CYP19 Gens. Wir untersuchten in diesem Projekt den Einfluss dieses SNP auf das Brustkrebsrisiko und das Erkrankungsalter, sowie die Assoziation mit klinischen und histopathologischen Charakteristika des Mammakarzinoms. Wir fanden heraus dass ein signifikant erhöhter Teil der Patientinnen mit dem TT Genotyp des rs10046 SNPs bereits in einem jungen Alter von unter 50 Jahren an Brustkrebs erkrankten (41.8% der Patientinnen mit dem TT Genotyp, aber nur 26.6% der Trägerinnen eines oder zweier C-Allele; p = 0.018, Chi-Quadrat Test; Abbildung 3A). Im Gegensatz dazu war keiner der drei rs10046 Genotypen signifikant mit einem erhöhten Brustkrebsrisiko, oder mit weiteren klinischen Parametern assoziiert. Beim Vergleich der Kurven der kumulativen Brustkrebsinzidenz zeigten sich charakteristische Unterschiede zwischen dem TT Genotyp und den beiden anderen Genotypen. Die Kurve der TT Patientinnen zeigte zunächst einen deutlich steileren Anstieg bei einem Alter zwischen 40 und 50 Jahren, wies dann aber einen auffälligen Knick und eine Plateauphase bei einem Alter von Jahren auf, welches in etwa dem typischen Alter der Menopause entspricht (Abbildung 3B). In postemenopausalen Patientinnen waren die drei Genotypen wieder im Gleichklang, sodass es in der Gesamtinzidenz keine signifikanten Unterschiede gab (Abbildung 3B). Abbildung 3: Einfluss des CYP19 rs10046 SNP auf das Erkrankungsalter beim Brustkrebs. (A) Anteil der Patientinnen, die bereits in einem Alter unter 50 Jahren erkrankten. (B) Kurven der kumulativen Brustkrebsinzidenz nach Erkrankungsalter der drei Genotypen. MEDICAL GENETICS L E I T E R : Univ.Prof.Dr. Berthold Streubel Wissenschaftliche Projekte (Auszug) (16 Publikationen 2014) Perinatal Global and single gene DNA methylation in umbilical cord blood cells after elective caesarean: a pilot study. OBJECTIVE: To evaluate global and single gene methylation patterns as a sign for epigenetic modulation of the immune system in infants born by elective cesarean section (CS) and vaginal delivery (VD). STUDY DESIGN: For this prospective pilot study a two-step approach was chosen. Initially 41 newborn infants comprising 23 delivered by VD and 18 delivered by elective CS were included. Global DNA methylation of umbilical cord blood was determined. In a second step, methylation status of 96 single genes linked to T cell activation, cytokine production, inflammatory response, and stem cell transcription was evaluated in 48 newborn infants, 20 delivered by VD and 28 delivered by CS. RESULTS: Global methylation did not differ significantly between CS and VD (p=0.732). The methylation status was low (threshold: 3%) for the majority of single genes (n=92) in both groups. FOXP3, CD7, ELA2, and IRF1 showed hypermethylation in both groups. In the CS group, ELA2 (p<0.001) and IRF1(p =0.017) showed significantly higher methylation compared to the VD group. CONCLUSION: We found no difference in global methylation between newborn infants in the VD group compared to the elective CS group. Methylation of single genes was significantly higher in newborn infants delivered by elective CS. Further research is needed to determine the significance of these findings. Tumor FOLFOX4 Plus Cetuximab for Patients With Previously Untreated Metastatic Colorectal Cancer According to Tumor RAS and BRAF Mutation Status: Updated Analysis of the CECOG/CORE Study. 192 Universitätsklinik für Frauenheilkunde

21 BACKGROUND: This updated analysis of the CECOG/CORE study investigated the association between clinical outcome and RAS and BRAF mutations in metastatic colorectal cancer (mcrc) patients treated with FOLFOX4 plus cetuximab. PATIENTS AND METHODS: Available DNA samples from CECOG/CORE study patients with KRAS exon 2 wild type (wt) (at codons 12 and 13) tumors were screened for mutations at other loci in the KRAS and NRAS (RAS) coding regions by Sanger sequencing, and for BRAF codon 600 mutations by Sanger sequencing and pyrosequencing. Clinical outcome was compared among different mutation subgroups. RESULTS: Of 152 KRAS wt mcrc patients, 148 were evaluable for RAS and BRAF mutation status. Eleven RAS mutations were detected in 10 patients tumors (7%). BRAF mutations were detected in 14 patients tumors (9%). RAS and BRAF tumor mutations were mutually exclusive. Compared with patients with RAS wt/braf wt tumors (n = 124; median overall survival, 28.5 months), those with RAS mutations (n = 10; median, 16.3 months; hazard ratio, 0.43; 95% confidence interval, ; P =.020) or BRAF mutations (n = 14; median, 11.7 months; hazard ratio, 0.23; 95% confidence interval, ; P <.0001) had worse overall survival, which remained significant (P <.04) when adjusting for differences in baseline characteristics among the mutation subgroups. CONCLUSION: These findings support those from recent studies that RAS and BRAF mutations are associated with poor outcome in patients receiving an epidermal growth factor receptortargeted monoclonal antibody in combination with oxaliplatin-based chemotherapy. Furthermore, mutation testing should not only include RAS codons 12 and 13 but should also be extended to the entire coding regions. PR ÄDIKTIVE ONKOLOGIE L E I T U N G : Univ. Prof. Dr. Christian SINGER, MPH W I S S E N S C H A F T L I C H E M I T A R B E I T E R I N N E N : Marie-Theres Kastner (Biomedizinische Analytikerin) Daniela Muhr (Biomedizinische Analytikerin) MMag. Christine Rappaport-Fürhauser (Dissertantin) Sigrid Weingartshofer, MSc (Biomedizinische Analytikerin) B A C H E L O R S T U D E N T I N N E N : Jennifer Jankovich Veronika Weiler WISSENSCHAFTLICHE PROJEKTE Prognostic and predictive value of ESR1 Amplification in postmenopausal receptor-positive women with early breast cancer treated with TAM vs sequential TAM and AI Singer, Holst, Rudas, Filipits, Stahl, Weingartshofer Several independent studies have now also confirmed the existence of ESR1 amplifications in breast cancer, and the presence of intratumoral ESR1 gene copy number alterations has been suggested to harbour relevant prognostic information. Recently, ESR1 amplification has also been suggested to predict responsiveness to endocrine therapy although the data are somewhat contradictory. We have therefore performed an ESR1 gene amplification in breast cancer retrospective biomarker trial in women with endocrine-responsive early breast cancer who had been randomized into the tamoxifen-only arm of the prospectively designed endocrine ABCSG-06 trial and in whom FFPE tumor samples were available. When ESR1 gene amplification status was evaluated by FISH analysis, we found copy number gains and amplifications in a significant fraction of tumor specimen and detected a strong and independent association with the clinical outcome. At a median follow-up of 10 years, women with intratumoral ESR1 copy number gains had a significantly longer distant recurrence-free survival and breast cancer-specific survival when compared to women with normal ESR1 copy numbers. We therefore concluded that the ESR1 gene copy status is an independent and powerful predictor for long-term distant recurrencefree and breast cancer-specific survival in postmenopausal women with endocrine-responsive early-stage breast cancer. In the present research initiative we propose to confirm the prognostic role of ESR1 in postmenopausal breast cancer patients, which we have previously demonstrated in the preliminary experiments described above, and to evaluate the predictive role of ESR1 in tamoxifen and AI-treated postmenopausal women. To this end, we will utilize an archived subset of breast cancer samples from more than 3200 postmenopausal women who have been randomized Universitätsklinik für Frauenheilkunde 193

22 into the ABCSG 8 trial and who have either received 5 years of tamoxifen or 2 years tamoxifen followed by 3 years of the aromatase inhibitor anastrozole. We will then extend our research objective to premenopausal women and investigate the prognostic and predictive utility of ESR1 in tumor samples from the ABCSG 12 trial. In this prospectively designed phase III trial, more than 1800 premenopausal women with endocrine-responsive early breast cancer have received either tamoxifen or the aromatase inhibitor anastrozole in combination with GnRH for 3 years, which provides us with a unique possibility to validate our initial findings in tamoxifen- and AI-treated premenopausal women. ABCSG 12 is the only prospectively randomized clinical trial worldwide that has directly compared tamoxifen with an AI in this patient setting. While the previous research strategies comprise retrospective biomarker analyses rom prospective trials, we will also measure ESR1 copy numbers in a predefined, prospective manner in a neo-adjuvant trial in which postmenopausal women with luminal A tumors receive 6 months of an AI prior to curative surgery. The efficacy of the studied AI letrozole will be measured by the rate of pcr or near pcr and will be correlated with the ESR1 amplification status in order to investigate whether a gene dose effect exists also for AI treatment. We hypothesize that the analysis of ESR1 copy number alterations allows to predict response to endocrine treatment (tamoxifen and AI) in pre- and in postmenopausal women with endocrine-responsive early breast cancer. We hypothesize that the analysis of ESR1 copy number alterations has a prognostic role in premenopausal women with endocrine-responsive early breast cancer We hypothesize that the analysis of ESR1 copy number alterations has a role in predicting near or complete pathological response in postmenopausal women with endocrine responsive, luminal A tumors that receive preoperative endocrine therapy with an AI for 6 months. To evaluate the predictive role of ESR1 copy number alterations in pre- and postmenopausal women with endocrine responsive early breast cancer who receive endocrine treatment in a prospective-retrospective study design. To evaluate the prognostic role of ESR1 copy number alterations in premenopausal women with endocrine-responsive early breast cancer who receive either tamoxifen or AI in combination with GnRH for 3 years in a prospectiveretrospective study design. To evaluate the utility of ESR1 copy number alterations in predicting near or complete pathological response in postmenopausal women with endocrine responsive, luminal A tumors that receive preoperative endocrine therapy with an AI for 6 months. To establish a standardized protocol for the treatment and analysis of ESR1 FISH, and to develop and cross-validate an analysis algorithm ESR1 gene amplification in breast cancer. Figure 1. FISH analysis on FFPE samples of invasive breast carcinomas. (left side) ESR1 gene amplification as indicated by multiple green ESR1 specific signals in each nucleus. The centromere 6 is red, ESR1 gene is green labeled, 100x. ER-expression performed by immunohistochemistry. (right side) Transmitted-light microscopic image (4x) of an immunohistochemical stained cryo section (4µm). ER-protein is colored brown in the nucleus. 194 Universitätsklinik für Frauenheilkunde

23 Methyl-binding domain protein-based DNA isolation from human blood serum combines DNA analyses and serum-autoantibody testing Wielscher, Pulverer, Peham, Hofner, Rappaport-Fürhauser, Singer, Jungbauer, Nähammer, Weinhäusel Circulating cell free DNA in serum as well as serum-autoantibodies and the serum proteome have great potential to contribute to early cancer diagnostics via noninvasive blood tests. However, most DNA preparation protocols destroy the protein fraction and therefore do not allow subsequent protein analyses. In this study a novel approach based on methyl binding domain protein (MBD) is described to overcome the technical difficulties of combining DNA and protein analysis out of one single serum sample. Serum or plasma samples from 98 control individuals and 54 breast cancer patients were evaluated upon silica membrane- or MBD affinity-based DNA isolation via qpcr targeting potential DNA methylation markers as well as by protein-microarrays for tumor-autoantibody testing. In control individuals, an average DNA level of 22.8 ± 25.7 ng/ml was detected applying the silica membrane based protocol and 8.5 ± 7.5 ng/ml using the MBD-approach, both values strongly dependent on the serum sample preparation methods used. In contrast to malignant and benign tumor serum samples, cell free DNA concentrations were significantly elevated in sera of metastasizing breast cancer patients. Technical evaluation revealed that serum upon MBD-based DNA isolation is suitable for protein-array analyses when data are consistent to untreated serum samples. MBD affinity purification allows DNA isolations under native conditions retaining the protein function, thus for example enabling combined analyses of DNA methylation and autoantigene-profiles from the same serum sample and thereby improving minimal invasive diagnostics. Figure 2. Increased level of cell free DNA in serum of Breast cancer patients with metastasizing disease. Amount of cell free DNA isolated from 1 ml serum of breast cancer patients with metastasizing tumors and control individuals obtained from AKH (source 3). An increased serum DNA amount was detected in sera from metastasizing tumors with both isolation strategies. (A), silica based isolation protocol (P = , Wilcoxon test); (B) MBD loaded bead based purification (P = , Wilcoxon test). Figure 2. Increased level of cell free DNA in serum of Breast cancer patients with metastasizing disease. Amount of cell free DNA isolated from 1 ml serum of breast cancer patients with metastasizing tumors and control individuals obtained from AKH (source 3). An increased serum DNA amount was detected in sera from metastasizing tumors with both isolation strategies. (A), silica based isolation protocol (P = , Wilcoxon test); (B) MBD loaded bead based purification (P = , Wilcoxon test). Figure 3. Quality of cell free serum DNA. (A) reflects the amplification success for each sample using MBD or silica membrane based serum DNA. A maximum of 12 fragments per sample was possible and in sum 34 samples per isolation approach were analyzed. (B) shows the amplification success of each fragment getting amplified across all analyzed samples. Both plots (A, B) are based on the analysis of two multiplex PCRs performed on serum or plasma DNA isolates (source 1, 2). Figure 1. Extraction of cell free DNA from control individuals. Box plot of DNA amount isolated from 1 ml serum or plasma from (1) Austrian Institute of Technology (n = 12), (2) Austrian Red Cross (n = 24) and (3) AKH (n = 24). Serum DNA levels were dependent on serum source. Using the silica-based extraction protocol, mean amounts of DNA could be isolated ranging from (1), 11.9 ± 10.9ng/ml (mean ± SD) and (3), 12.2 ± 9.7 ng/ml to (2), 39.7 ± 32.8 ng/ml. By contrast using the MBD-based protocol, serum DNA concentrations of (1) 2.5 ± 1.9 ng/ml, (2) 11.5 ± 7.3 ng/ml and (3) 2.4 ± 1 ng/ml were observed. Figure 4. Autoantibody tests of MBD processed serum. Pearson correlation plots upon X-values of autoantibody protein micro arrays analyzing serum and plasma samples with and without MBD treatment. All samples originate from source 1 (AIT). Comparison of plasma and serum samples were performed on samples obtained from one single blood withdrawal. The cor-value states the Pearson s correlation. Universitätsklinik für Frauenheilkunde 195

24 Antiestrogenic effects of the natural fetal estrogen estetrol (E4) in women with estrogen-receptor-positive early breast cancer Singer, Bennink, Natter, Steurer, Moinfar, Rudas, Visser, Appels, Kubista Estetrol (E4) is a fetal estrogen which exerts estrogenic effects on vaginal epithelium, hot flushes and bone, but has estrogen-antagonistic effects on breast cancer cell lines in vitro and in the rat DMBA model. Therefore E4 may be suitable for Hormone Replacement Therapy (HRT) in women with breast cancer including women treated with aromatase inhibitors. We have investigated the effect of 14 days pre-operative treatment with 20 mg E4 per day on proliferation, apoptosis, ER-receptors and sex steroid levels in a prospective, randomised, double-blind, placebo-controlled, neo-adjuvant study in 15 pre- and 15 postmenopausal women with estrogen-receptor positive early breast cancer. Estetrol induced a significant increase of SHBG, a significant decrease of FSH in postmenopausal women and no increase of gonadotrophins in premenopausal women. Estetrol had no effect on Ki67 expression and on apoptosisrelated Bax and Bcl-2, but the apoptosis index in tumor tissue increased significantly. Systemic IGF-1 levels decreased significantly. Surprisingly the intratumoral epithelial ER-alpha expression decreased significantly, whereas the ER-beta expression showed a trend to increase. Fig. 2. Representative immunohistochemistry photomicrographs depicting ER staining before (a) and after (b) 2 weeks of E4 treatment, and ER staining before (c) and after (d) 2 weeks of E4 treatment. This data show that E4 has estrogenic endocrine effects. The data support the hypothesis that E4, may be suitable and safe for HRT in women with spontaneous or induced menopausal symptoms, since apoptosis increases, IGF-1 decreases and no unfavourable effects are observed on Ki67, Bax and Bcl-2. The decrease of ER-alpha and the increase of ER-beta suggest a mechanism of action, explaining why the natural fetal estrogen E4 has estrogenantagonistic effects on breast cancer tissue. Carcinogenesis vol.35 no.11 pp , 2014 doi: /carcin/bgu144 Advance Access publication July 5, 2014 Fig. 1. Ki67 expression (%) in breast tumor tissue before (day 0) and after 14 days of oral treatment with 20 mg E4 or placebo per day. Fig. 3. Relative changes (%) in SHBG (a), FSH (b), bioavailable testosterone (c) and IGF-1 (d) serum concentrations after 14 days of treatment with E4 or placebo in preand post-menopausal BC patients. 196 Universitätsklinik für Frauenheilkunde

25 Serum-Autoantibody testing for early diagnosis of Breast Cancer Singer, Weinhäusel (AIT), Zeillinger, Pecha, Koch Early detection of breast cancer will be the main focus of this projectproposal, although prognosis and prediction of clinical outcomes might be a potential task using our immunological approach. Early detection reduces the suffering and cost to society associated with the disease. A sensitive assay to identify biomarkers that can accurately diagnose the onset of breast cancer using non-invasively collected clinical specimens is ideal for early detection. The earlier and more accurate the diagnostic biomarker can predict disease onset, the more valuable it becomes. Autoantibodies and abnormal tumor-specific DNA methylation found in cell-free serum DNA may provide the best opportunity for constructing multiplexed tests, which will be sufficiently specific and sensitive for early detection of breast cancer. Although we have identified DNA-methylation based classifiers in primary tumors and tested those along with published breast cancer methylation markers for detection of abnormal tumor-specific DNA methylation in serum, we could not distinguish females with benign and malignant breast tumors (unpublished results). Although PCR testing is relatively simple and robust, we have found that a simpler assay might be advantageous. This is especially true for presymptomatic screening or improving diagnostic testing of females with nodular (including benign and malignant) breasts. In these settings, a minimal invasive serum test would be of great value, especially when a breast tissue biopsy for histopathological assessment of nodules could be avoided. Therefore we aim to develop a technically simple and cheap immunologic serum test for minimal invasive diagnostics, and expect that technologies developed for breast cancer detection will be useful for other types of cancer. 5) a peptide-based proto-type assay will be developed, transferred to the clinical research laboratory and validated on a retrospective study-cohort (n=1200). All these techniques and methods are already established and will be combined for the most comprehensive approach for autoantibody-based serum diagnostics. In this cooperational project AIT has successfully conducted immune-profiling of cases and controls using AITs 16k protein microarray for distinguishing malignant, benign and healthy controls from each other. The significant proteins were then used for a tiling-design of a peptide array. Together with peptides deduced from antigenic protein classifiers for colon-, lung, and prostate cancer, together with peptides presenting the top 500 known mutations in Our innovative approach will 1) provide a protein-microarray which enables highly reproducible identification of marker-candidates, 2) integrate already defined candidate markers derived from macro-array- and SEREX- pre-screenings, and 3) evaluate peptides deduced form marker-candidates. In addition we will 4) apply phage-display-technology for selection of seroreactive peptides from controls and clinico-pathologically different patient-groups defined during clinical examinations. Phage-display will elucidate antigenic peptides in a very efficient manner without the need of tumor derived cdna libraries. Both the microarray-derived and the phage display derived antigenic-peptides panels of candidate markers will be chemically synthesized and used for confirmation and elucidation of a classifier using state-of the art statistical bioinformatics. Thus Universitätsklinik für Frauenheilkunde 197

26 cancer (derived from the COSMIC database), a high density array was presenting different peptides was designed. This array was then probed with breast-cancer, benign breast-tumor and healthy control serum-samples (n=72). The 179k peptide array data enabled correct classification of all the three different patient groups with high accuracy (see ROC curves depicted below). Then serum from initial diagnostics of patients representing cases and hospital control were collected and provided to partners at AIT for testing the candidate peptides to generate a targeted assay using Luminex bead array technology. These assays, when providing high accuracy will be provided to MUW for cross-validation early in Evaluation and fine mapping of FISH-detected lowlevel ESR1 amplification using SNP-GeneChip analysis to reveal amplicon breakpoints in fresh frozen breast cancer tissue specimens Weingartshofer, Holst, Ambros (CCRI), Ambros (CCRI), Rudas, Kriegner (AIT), Weinhäusel (AIT), Singer Breast cancer is still the most common cancer in women worldwide. The rapid development in the field of diagnostic and therapeutic possibilities regarding to the underlying causes and mechanisms of the disease there are many questions still unresolved. It is known that estrogen positive breast cancer is associated with estrogen receptor overexpression, which is linked to estrogen receptor 1 (ESR1) amplification. In a previous study, it was particularly striking that women who received tamoxifen monotherapy, in terms of breast cancer recurrence and 5-year survival had a better prognosis than women who had no ESR1 amplification. Therefore, it was of great interest in this work, to elucidate the underlying mechanism of amplification, which is responsible for the copy number increase. In addition, we wanted to ascertain if the start and end points (break points) of the additional copy of the gene happen randomly or are localized to specific sites on the gene. For the study we used 25 fresh frozen breast carcinoma specimens of women with early breast cancer who had been naïve to any therapies, neither chemotherapy nor endocrine therapy to exclude any negative influences or other potential effects regarding the sample quality as a result of treatment. Additionally, a neuroblastoma control cell line (SHEP cells) was cultured and used for further specific investigations. For the detection of ESR1 amplifications a fluorescence in situ hybridization (FISH) protocol was developed with and without RNase pretreatment and the amplified regions on correlating frozen sections then were marked. FISH analysis samples with cell nuclei showing tight as well as confluent signal clusters and distinguishable signals, have been included into the study. An average ratio 2 was rated as amplification, and 1.3 as gain. Cases showing defragmented DNA or normal signals were deleted. Six tumors, which were gained and one amplified sample, could be selected for the following comparative genomic hybridization (CGH) array (Figure 1). To avoid dilution effects performing SNP-GeneChip analysis, marked areas were lasered out by laser microdissection (Figure 2), DNA then was isolated therefrom and subsequently a SNP GeneChip analysis was performed to locate the exact breakpoints of an additional copy within and around the ESR1 gene. To investigate the potential involvement of intrachromosomal low copy repeats (LCRs) in recurrent chromosomal translocation formation, we performed computational Basic Local Alignment Search Tool (BLAST) analysis that can potentially mediate chromosomal translocations (Figure 3). BLAST analysis was accomplished within ESR1 gene regions using the long and a short isoform of ESR1. For this, we downloaded the genomic sequence of the ESR1 ±10 kb from UCSC and generated a BLAST database with 17,311 fragments. These fragments then were blasted against whole ESR1 gene sequence including 10 kb upstream and downstream sequences with selected criteria including E- value 1E-10, homology 95 % and hit length of 100 % and resulted in 508 hits. We also wanted to know how much of these 508 hits are within a repeat region identified by RepeatMasker and found 10 (0.058 %) hits. It was also of great interest to identify potential in amplification involved repetitive sequences in ESR1 flanking regions. Here, genomic sequence of ESR1 ± 200 kb was downloaded from UCSC followed by BLAST analysis and homology mapping. Several candidates such as AluYa5 and L1MB8 that were adjacent and showed homologous sequences could be identified in ESR1 flanking regions. But very exceptional was the highest level of sequence homology of all ESR1 flanking RepeatMasker sequences we could observe in AluYb8 with only two bases mismatches over 312 bp. This was unique in this area. To confirm the exact location of the suspected break points in this range, the results were compared with the data of SNP-GeneChip analysis and further a PCR analysis was carried out. In laser micro dissected samples we could not identify break points within or in expected flanking regions of ESR1. However, in the neuroblastoma cell line we found a small and partial gain (Figure 4). Based on the data of the amplified region identified with array CGH it was of great interest to know if starting and endpoint of the gained region is correlating to AluYb8. There were identified homologies but none, which really correlated to the gained region. Results showed that there are missing fragments in a size of 300 kb on the left and right side from ESR1. Nevertheless, we could identify the MLT1C, a retrotransposon, within the gained region, but none homologous element confining this area. Beside FISH analysis, microdissection and further SNP array we wanted to investigate, if the recombination of amplified samples is localized to the assumed homologous sequences beside the ESR1 gene. For this, the Alu element 198 Universitätsklinik für Frauenheilkunde

27 AluYb8, where the highest level of sequence homology of all ESR1 flanking RepeatMasker sequences was shown, was used for primer design. The primers were designed rather PCR analysis was so performed that if a recombination would take place in AuYb8 area, a corresponding amplification product would be produced. Primer C was corresponding to the 3 ESR1 sequences directed to the right side and primer B was corresponding to 5 ESR1 sequence with the direction to the left side. So if here would be held recombination from our DNA samples (non-laser and laser microdissected), we would have an amplification product. However, no PCR amplification products could be detected using this kind of analysis. Although within the cell line a gain could be found by CGH-array, we could not visualize amplification by RNAse FISH analysis. With this result the limitation of FISH analysis could be demonstrated. Sequences may were directly adjacent and consequently no additional FISH signal could be identified because the hybridized sequences by FISH would appear together as one signal. That means that the FISH analysis could not detect additional gene signals, suggesting that the additional ESR1 sequences were not translocated, but probably lie directly behind in the original locus. Summing up, ESR1 amplification, its underlying mechanisms and its role in therapy and prognosis as potential biomarker is still an exciting topic and has to be resolved. Figure 2. Selected cryo sample before and after the laser microdissection. Light microscopy image (4x) of a histological cryo section (7µm) of a breast tumor before and after microdissection. The red outlined tumor regions in this picture (left) are isolated after microdissection (right). Figure 4. ESR1 homology regions overview. H = homology (black) e.g. H1 upstream + H1 downstream or H8 upstream + H8 downstream are homologous. These data were performed by using BLAST analysis; U = upstream (blue), e.g. U4 upstream against downstream analysis (second attempt) for long isoform (above ESR1 isoforms). U = upstream (blue) e.g. U4 = d2, d4, d13. There is a minimum homology from fragment U4 with fragment d2, d4 and d13 downstream for short isoform (below ESR1 isoforms). ESR1 (blue lines): Long ESR1 isoform including the five short isoforms is shown. Figure 1. Selected tumor samples with ESR1 copy number increase. RNase FISH was performed for laser microdissection. Images A to F show RNase FISH with low-level copy number increase (gain). (ESR1 signal is green, Centromere 6 is red, 100x) Figure 4: Neuroblastoma cell line with partial gain in ESR1 gene. Universitätsklinik für Frauenheilkunde 199

28 The role of G-CSF and GM-CSF as biomarker in breast cancer Singer, Salama, Jankovich, Weiler, Weingartshofer Among women in the western industrial countries, breast cancer is the most common type of cancer. Biomarkers are used to obtain more detailed information concerning diagnosis and prognosis or in order to improve treatment. In this respect, G-CSF becomes more and more interesting, as it was found that there are differences regarding the serum levels of breast cancer patients compared to healthy women. In several studies, positive effects of G- CSF in terms of stimulating growth and development of the tumor have been demonstrated. To prevent severe forms of neutropenia caused by chemotherapy, prophylactic G- CSF is used. Therefore it is vital to evaluate the influence of this growth factor on the progression of breast cancer. As higher concentrations of G-CSF are expected in advanced stages, the serum levels of breast cancer patients with and without lymph node involvement were compared to a control group of women without breast cancer. The granulocyte macrophage colony-stimulating factor (GM-CSF) and its function as biomarker in breast cancer was also determined. Until today there are only two biomarkers which are used for the interpretation of the status of breast cancer upa and PAI-1. For this reason it is important to find other biomarkers which support cancer diagnosis. GM-CSF was found to change its concentration in patients with colorectal cancer. Because of this fact, GM- CSF serum concentrations maybe also changes in patients with breast cancer. This growth factor is used combined to chemotherapies because of its ability to raise the cell population of granulocytes and macrophages. Due to the fact that GM-CSF has a very low serum concentration it is necessary to compare the characteristics between the multiplex systems and the common ELISA if detecting cytokines. Also a trouble shooting is a central theme in this study because it helps to prevent problems when using this kind of assay. In this study we compared patient groups of a total of 149. The first group consisted of healthy women with changes in their breastparenchym. The remaining groups were sera of women with breast cancer (with and without involvement of lymph nodes). G-CSF and GM-CSF serum concentrations were detected by using the Bio-Plex multiplex system (Bio Rad/Luminex). This assay is based on the binding between the analyte and the specific coated bead, which is detected by flow cytometry. There was no significant elevation in the serum concentrations of G-CSF of breast carcinoma patients compared to women without breast cancer (p=0.74). Moreover, the serum levels in patients with lymph node involvement in comparison to women without lymph node metastasis showed no significant difference (p=0.421). The same applies to subgroups of other clinical relevant parameters like the ER- or PR-status (p=0.494; p=0.428), the HER2-status (p=0.755), the tumor grading (p= 0.282) and the menopausal status (p=0.89). However, controversial results have been shown by other studies. The statistical analysis in the serum concentrations of GM- CSF has represented that there is a significant difference (p=0.002) between healthy women and breast cancer patients (Figure 1). All other clinical relevant parameters (age, menopause status, hormones) had no significant difference (Figure 2). This study was able to show that the Bio-Plex multiplex System (Luminex) is an appropriate method to detect the GM- CSF serum concentrations. Furthermore it was possible to confirm the literarily known significant difference between healthy persons and breast cancer patients. Further research is needed to evaluate the true value of G-CSF as prognostic biomarker and its influence on tumor development. Figure 2. Significant difference (p=0.002) in GM-CSF serum concentrations (Obs Conc) between healthy women and women with Mamma carcinomas using the Mann-Whitney-U-Test. Figure 3. No correlation between age and GM-CSF serum concentration. 200 Universitätsklinik für Frauenheilkunde