CCRI CHILDREN S CANCER RESEARCH INSTITUTE SCIENTIFIC REPORT 2006/2007/2008 ST. ANNA KINDERKREBSFORSCHUNG JAHRESBERICHT 2006/2007/2008

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2 CCRI CHILDREN S CANCER RESEARCH INSTITUTE SCIENTIFIC REPORT 2006/2007/2008 ST. ANNA KINDERKREBSFORSCHUNG JAHRESBERICHT 2006/2007/2008

3 Inhaltsverzeichnis/Table of contents Vorwort/Preface 1 Einleitung/Introduction 3 Tumor Biologie/Tumor Biology 5 Immunologische Diagnostik/Immunological Diagnostics 11 Tumorimmunologie/Tumor Immunology 15 Biologie von Stammzellen/Biology of Stem Cells 19 Transplant-Immunologie/Transplant-Immunology 23 Molekularbiologie/Molecular Biology 27 S 2 IRP Studien und Statistik/Studies and Statistics for Integrated Research and Projects 33 Molekulare Mikrobiologie/Molecular Microbiology 37 Biologie der Leukämien/Biology of Leukemias 43 Leukämiegenetik/Genetics of Leukemia 49 Varia 53 Publikationen/Publications 57 Impressum/Medieninhaber: Published by: St. Anna Kinderkrebsforschung Children s Cancer Research Institute (CCRI) (Leiter/Head: Univ. Prof. Dr. H. Gadner) A-1090 Wien Korrespondenz an: Mag. Marion Zavadil Correspondence to: Tel./Phone: zavadil@ccri.at Koordination/Coordination: Fotos: Univ. Doz. Dr. Heinrich Kovar Mag. Elisabeth Tax Mag. Marion Zavadil Stefan Liewehr, ComCom, St. Anna Kinderkrebsforschung

4 Vorwort Preface 2008 feiert die St. Anna Kinderkrebsforschung ihr 20jähriges Jubiläum. Warum war es eigentlich nötig, 1988 ein Forschungsinstitut aus dem Nichts zu entwickeln? Warum gerade im St. Anna Kinderspital, das von einer Reihe von Forschungslaboratorien umgeben ist? Und warum wurde der Schwerpunkt Kinderkrebs gewählt? Alle drei Fragen wecken Erinnerungen, die es gilt anlässlich dieses runden Geburtstages aufzufrischen. Das St. Anna Kinderspital wurde1837 als erstes Kinderspital in Wien und drittes in Europa gegründet und war auf die kostenlose Versorgung sozial schwacher kranker Kinder ausgerichtet. Der Gründer und seine renommierten Nachfolger haben die Kinderheilkunde in den folgenden Jahren erstmalig in Europa zu einem eigenständigen klinischen Fach erhoben und das Spital war von 1850 bis 1911 Universitätsklinik für Kinderheilkunde. Nach den Wirren der Weltkriege begann ein neuerlicher Aufstieg erst in den 70er Jahren des 20. Jahrhunderts mit der Entscheidung zur Aufnahme und Behandlung krebskranker Kinder, an deren Betreuung kein besonderes Interesse bestand. Um die Voraussetzungen für eine optimale Versorgung dieser Kinder zu schaffen, wurden 1980 mit großem Elan bauliche Erneuerungen eingeleitet. Nach Einführung der Therapie-Optimierungs- Protokolle im Rahmen nationaler und internationaler Kooperationen stiegen die Heilungsraten der vorher zum Großteil als unheilbar betrachteten Krebsformen innerhalb weniger Jahre von ca. 35% auf 70% an. Dies war die Grundlage zur Etablierung eines Dokumentations- und Studienzentrums im St. Anna Kinderspital, um die österreichweiten Fortschritte der Behandlung laufend zu überwachen und weltweit zu vergleichen. Es wurde bald klar, dass weitere Fortschritte nur zu erreichen wären, wenn es gelänge, Einblicke in die Biologie der Krebszellen zu bekommen, um daraus Erkenntnisse für eine individuell angepasste Therapie abzuleiten gelang es, eine Elterninitiative ins Leben zu rufen und die Öffentlichkeit mit dem dringenden Bedürfnis nach einer Kinderkrebsforschung vertraut zu machen. Der Spendenfluss ermöglichte, im bislang leeren Dachboden des St. Anna Kinderspitals nach einjähriger Bauzeit 1988 ein Forschungslaboratorium zu eröffnen, das zunächst mit einer Arbeitsgruppe startete, die sich innerhalb eines Jahres auf fünf Forschungsgruppen vermehrte. Die Schwerpunkte der Forschung konzentrierten sich auf die Verfeinerung der Diagnostik, die angewandte Forschung zur Vorbereitung und Begleitung der Stammzelltransplantation, und die Grundlagenforschung. Dieses Spektrum reflektierte von Anfang an das vordergründige Bemühen, das wissenschaftlich-fachliche know-how im Labor mit den klinischen Bedürfnissen in Einklang zu bringen, heute bezeichnen wir das als translational research. Es zeigte sich, dass die Eingliederung des Forschungsinstitutes in den unmittelbaren Nahbereich der Behandlungsstationen des St. Anna Kinderspitales zur Steigerung des persönlichen Engagement der Mitarbeiter auf beiden Seiten entscheidend beiträgt. In den folgenden Jahren führte die Expansion des Forschungsprogrammes zu einem weiteren Zuwachs auf insgesamt neun Arbeitsgruppen. Entsprechend ist die Zahl der Mitarbeiter kontinuierlich gestiegen. Es gelang, die dafür notwendigen finanziellen Ressourcen durch verstärkte Öffentlichkeitsarbeit und erfolgreiche Einwerbung von Drittmitteln, besonders durch Teilnahme an internationalen Vernetzungen der Europäischen Union, signifikant anzuheben. Um die Qualität der wissenschaftlichen Arbeit zu sichern, wurde ein ständiger Beirat ins Leben gerufen, der seit 1993 regelmäßig Evaluierungen vornimmt. Im Jänner 2009 wird das Forschungsinstitut in ein eigenes Institutsgebäude mit direkter Brückenverbindung zum St. Anna Kinderspital übersiedeln marks the 20 th anniversary of the Children s Cancer Research Institute which certainly brings back a flood of memories. Why was it suddenly so important in 1988 to establish a cancer research institute out of nothing? Why precisely at St. Anna Children s Hospital, which was already surrounded by several research labs at that time? Why focus on children s cancer research? After two decades of CCRI it might be adequate to refresh our memories. The St. Anna Children s Hospital s history goes back a long way: founded in 1837 as the first paediatric hospital in Vienna, and the third oldest in Europe, it focussed on the free medical care of socially deprived children. In the following years, the founder and his renowned successors established, for the first time in history, paediatrics as an independent clinical department in Europe; from 1850 to 1911 the hospital was University Clinic for Paediatrics. Having endured two world wars, its rise began anew in the 1970ies, supported by the decision to admit and treat children with cancer, whose medical care was of little interest at that time. In 1980, essential structural renovations including the construction of a bone marrow transplantation unit were initiated, to offer the best possible health care to these children with cancer. By entering the so-called Treatment Optimising Studies (TOS) within national and international co-operations, the cure rate of certain forms of childhood cancer, considered incurable so far, were improved from 35% to 70% within a few years. As a consequence, a centre for documentation and statistics was established at St. Anna Children s Hospital in order to evaluate the progress of treatment and outcome in Austria and compare it with international studies. Soon, it became clear that further progress could only be achieved by gaining insights into the biology of the cancer cells to provide optimal therapy regimens for each individual. For this reason an appropriate research program had to be developed; however, the hospital did not have sufficient research funds available. In 1985, a parents association was founded, who successfully informed the public about the urgent need for children s cancer research. As a consequence, the association raised enough donations to start the conversion of the then empty loft in the building of St. Anna Children s hospital into laboratory space for the cancer research lab to open in What had started with one working group became a team of 5 working groups of 20 motivated people within one year. The first research activities concentrated on basic research and patient orientated diagnostics, applying research in the context of stem cell transplantation. From the very beginning, the major endeavour has always been devoted to bringing the know-how from the laboratories to the bedside, bringing together the clinicians with the researchers. Today, this is called translational research. One benefit of having a research institute within the walls of the paediatric hospital is the possibility to foster the commitment and dedication of the employees on both sides. In the following years, the CCRI continued to grow until today it is encompassing nine different working groups. Consequently, the number of staff members has constantly been growing as well. It is through increased public relation and successful acquisition of external grants, especially through participation in international networks within the European Union, that the CCRI succeeded in raising and maintaining the adequate financial resources. To guarantee that the high quality of the scientific work will be maintained as well, we have established an international advisory board who has been regularly evaluating all research projects since The next important expansion of the institute will take place in January 2009 when it is moving to its own building while staying connected with the hospital by a bridge. 1

5 Das enge Zusammenwirken von Forschung und klinischer Umsetzung ermöglicht mittlerweile eine Heilungsrate von 80% bei den uns anvertrauten krebskranken Kindern, und so hat unsere 1988 zaghaft formulierte Vision eine erfolgreiche Umsetzung erfahren. Abschließend möchte ich diesen Leistungsbericht nutzen, meinen MitarbeiterInnen, den kooperierenden Institutionen, den verschiedenen Wissenschaftsförderungsfonds, den Mitgliedern des Vorstandes, des Fördervereins St. Anna Kinderkrebsforschung, und der Foundation für ihre uneigennützige und engagierte Unterstützung zu danken, die die 20-jährige Erfolgsgeschichte der St. Anna Kinderkrebsforschung ermöglicht haben. Ein besonderer Dank gilt den unzähligen SpenderInnen, die uns seit vielen Jahren die Treue und damit die Kinderkrebsforschung am Leben halten. Univ. Prof. Dr. Helmut Gadner (Institutsleiter) Within two decades of close collaboration between scientists and clinicians, an 80% cure rate of the paediatric cancer patients entrusted to our care was made possible. Viewed in this light, our vision of 1988 has finally been realised. Concluding, I want to take the opportunity to thank all staff members, all collaborating institutions, all science promotion funds, the board members, the sponsors of St. Anna Kinderkrebsforschung, and the St. Anna foundation for their commitment and support, without which these achievements would not have been possible. I also wish to express my sincere thanks to the countless charitable donors whose unremitting contributions and loyalty have been keeping the Children s Cancer Research Institute alive through all these years. And I do hope that there will be many more years as pleasant and as successful as the years gone by. Univ. Prof. Helmut Gadner (MD) (Director) Einleitung Während der letzten drei Jahre, dem Berichtszeitraum, durchlebte die biomedizinische Forschung weltweit einige revolutionäre Entwicklungen. Vor allem genomische und post-genomische Hochdurchsatzmethoden wurden Standardwerkzeuge für die Analyse von Krebsgenomen. Sie führten auf mehreren Beobachtungsebenen zur Anhäufung enormer Mengen hoch komplexer Daten, deren Interpretation die Anwendung sehr anspruchsvoller bioinformatischer Analysen notwendig macht. Diese Methoden sind aber immer noch sehr teuer, weshalb der Zugang zu ihnen für eine hauptsächlich durch Spenden finanzierte Organisation schwierig ist. Im Berichtszeitraum ist es dem CCRI jedoch gelungen, diesem Problem durch die erfolgreiche Einwerbung von gut dotierten externen Projektmitteln und die Etablierung entsprechender nationaler und internationaler Zusammenarbeiten zu begegnen. Tatsächlich konnte der Anteil extern finanzierter Projektmittel am Forschungsbudget auf 38% gehoben werden, was zu einem guten Teil auf die Teilnahme an nationalen (GEN-AU Programm) und internationalen (6. EU-Forschungsrahmenprogramm) Kooperationsprojekten zurückzuführen ist. Diese Entwicklung wurde von den Mitgliedern einer internationalen Gutachtergruppe während ihres Besuches am CCRI im Winter 2007 sehr positiv vermerkt. Zusätzlich half uns das GEN-AU Mobilitätsprogramm des Bundesministeriums für Wissenschaft und Forschung, junge CCRI Wissenschafter für ein weiterführendes Training in renommierte ausländische Labors zu schicken und in der Folge die Bioinformatik als Unterstützung im eigenen Haus zu etablieren. Die Analyse von Hochdurchsatzgenomischen Daten in mehreren am Institut studierten Erkrankungen und Zelltypen führte zur Identifikation pathogener Mechanismen, die in experimentellen in-vitro Systemen überprüft wurden. Die Anwendung der RNA-Interferenztechnologie spielte bei diesen Validierungen eine zentrale Rolle und wurde zu einem Routinewerkzeug am CCRI. Zusätzlich zur molekulargenetischen Grundlagenforschung, setzte das CCRI seine Arbeiten im Bereich der Transplantations-Immunologie, der Diagnostik und Prognostik fort, wodurch die angewandte Forschung als eine wesentliche Säule des Institutes weiter gestärkt wurde. Klinische Studien erhielten exzellente Unterstützung durch die reorganisierte Abteilung Studies and Statistics als ein weiteres Verbindungsglied zwischen dem Forschungsinstitut und dem St. Anna Kinderspital. Letztlich übernahm das CCRI durch verstärkte Kommunikation mit der Öffentlichkeit auch soziale Verantwortung. Diese erfolgte nicht nur an den jährlichen Tagen der offenen Tür ( Lange Nacht der Kinderkrebsforschung ), sondern auch durch die Leitung eines von der EU finanzierten internationalen Wissenschaftskommunikationsprojektes ( DIRECT ). Dieser Bericht soll die mannigfachen wissenschaftlichen Aktivitäten des CCRI während der letzten drei Jahre dokumentieren. Da das CCRI im Jahr 2008 seinen 20. Geburtstag feiert, ist jedem Einzelbericht eine Rückschau über die wesentlichen Erfolge seit dem Bestehen des Institutes vorangestellt. Der Leser mag sich ein Bild der beachtlichen Fortschritte der Arbeitsgruppen machen, auf die wir als der Kinderkrebsforschung verschriebenes Team sehr stolz sind. Introduction Over the last three years, the period reviewed in this report, worldwide biomedical research has experienced a number of revolutionizing developments. In particular, genomic and post-genomic high-throughput methods have become a standard tool in the analysis of cancer genomes yielding an enormous amount of highly complex data on multiple observation levels, and requiring sophisticated bioinformatic analyses for interpretation. However, these methods are still based on rather expensive technologies and thus, access poses a major problem to a predominantly charity based organization. However, in the reporting period, the CCRI has successfully managed to cope with this problem by obtaining well funded external grants and by building-up adequate national and international collaborations. In fact, it was possible to raise the contribution of external competitive grants to the research budget to 38%, which in part was due to the successful participation in national (GEN-AU program) and international (EC framework program 6) cluster projects. This development was very positively received during a site visit of the CCRI s external review board in winter Further, the GEN-AU mobility program of the Austrian ministry of science was instrumental for the training of young CCRI scientists in expert laboratories outside of Austria. As a result, it was possible to initiate in-house bioinformatic support. Analysis of high through-put genomic data in several diseases and cell types studied at the CCRI led to the identification of pathogenic pathways that were experimentally tested in in-vitro models. RNA interference technology played a major role in this validation process and has now become a routine tool at the CCRI. In addition to basic molecular genetic studies, the CCRI has continued research into transplantation related, diagnostic and prognostic issues further strengthening translational research, a major pillar of the institute. Clinical studies obtained excellent support by the re-organized Studies and Statistics section of the CCRI, a further link to the St. Anna Kinderspital. Finally, the research institute has taken social responsibility by enhancing communication with the lay public not only during the yearly open days ( Lange Nacht der Kinderkrebsforschung ) but also by running an ECfunded international science communication project (DIRECT). This report shall document the thriving scientific activities of the CCRI during the last three years. Since 2008 marks the 20 th anniversary of the CCRI, each group report is preceded by a short paragraph recalling the individual research groups major achievements over the years. The reader may judge the remarkable developments of which we as a team dedicated to childhood cancer research are extremely proud of. Assoc. Prof. Dr. Heinrich Kovar (Wissenschaftlicher Direktor) Assoc. Prof. Heinrich Kovar (Ph.D.) (Scientific Director) 2 3

6 TUMOR BIOLOGIE TUMOR BIOLOGY Gruppenleiter (Group Leader): Univ. Doz. Dr. Peter F. AMBROS (Ph.D.) Wiss. Mitarbeiterinnen (Staff Scientists): Dr. Ingeborg M. AMBROS (M.D.) Dr. Cornelia STOCK (Ph.D.) (bis/until 07/07) Dr. Eva BOZSAKY (Ph.D.) Technische Mitarbeiterinnen (Technicians): Andrea LUEGMAYR Bettina BRUNNER Doktorandin (Ph.D. Students) Agata KOWALSKA (bis/until 03/08) Diplomandinnen (Diploma Students) Heide-Maria BINDER (seit/since 03/07) Magdalena PABINGER (seit/since 08/07) Klin. Mitarbeiterin (Clinical Collaborator): Univ. Doz. Dr. Ruth LADENSTEIN (M.D.) St. Anna Kinderspital Univ. Doz. Dr. Peter F. AMBROS Geschichtlicher Überblick Ein wesentliches Ziel unserer Arbeitsgruppe ist und war es, Marker zu identifizieren, die es erlauben, die Behandlung auf die Eigenschaften des jeweiligen Tumors abzustimmen, um ein Zuviel- oder Zuwenig an Therapie zu verhindern. Dies wurde in weltweiter Kooperation durch Analyse genetischer, im Tumor auffindbarer Marker, die bestimmten klinischen Krankheitsverläufen zugrunde liegen, ermöglicht. Im Fall des Neuroblastoms, dem häufigsten Tumor des frühen Kindesalters, konnte durch die Untersuchung einer großen Zahl von Tumoren das genetische Profil von benignen und aggressiven Formen aufgeklärt werden. Damit bestand und besteht nun die Möglichkeit, aggressive von nicht aggressiven Tumoren abzugrenzen und die Therapie entsprechend zu steuern. Der Einsatz einer auch auf biologischen/ genetischen Kriterien basierenden Therapieentscheidung war in österreichischen und später auch in europäischen Studien sehr erfolgreich und konnte die Mortalitätsrate bei einzelnen Tumorstadien drastisch senken. Grundlagen dieser neuen Stratifizierungskriterien waren eine vereinheitlichte Nomenklatur und standard operating procedures, die von uns initiiert wurden und heute die Basis einer weltweiten Regelung darstellen. Unsere Gruppe konnte auch in der automatisierten Darstellung und Quantifizierung von Tumorzellen im peripheren Blut, Knochenmark und Aphereseprodukten einen innovativen Ansatz verfolgen. Durch die kombinierte Darstellung von immunologischen und genetischen Markern konnte eine bis dahin nicht erreichte Sensitivität und Spezifität des Nachweises erreicht werden. Dieses Verfahren ist seit über 10 Jahren im klinischen Einsatz und hat entscheidende neue Informationen über die klinische Relevanz von hämatogenen Metastasen bei Diagnose geliefert und sich als nicht invasives Verfahren zum Nachweis des Therapieerfolges bewährt. In den letzten Jahren haben wir uns neben diagnostischen/ prognostischen Aspekten auch möglichen therapeutischen Ansätzen, nämlich der Induktion der Alterung von Tumorzellen, zugewandt. Wir haben zunächst für das Neuroblastom ein Modellsystem zur Tumorzellalterung entwickelt. Mit diesem in vitro System lassen sich hochaggressive Tumorzellen in alternde, nicht mehr aggressive Zellen verwandeln, die möglicherweise das Immunsystem stimulieren können. Historical survey One of our group s essential objectives has always been to identify markers which enable treatment of patients exactly according to the tumor s biological characteristics in order to avoid over- or under-treatment. This aim has been achieved by worldwide co-operations through the analyses of genetic patterns which underlie the clinical course of the respective disease. In the case of neuroblastoma, the most frequently occurring tumor in childhood, the investigation of a large number of tumors has led to the discovery of the genetic profiles of benign as well as malignant forms of neuroblastoma. As a consequence, we are now able to distinguish between aggressive and non-aggressive tumors and patients can be treated accordingly. As several Austrian (and, later, also European) Studies showed, the survival rate of patients with certain stages of tumor increased considerably as soon as clinicians started to base their therapy decisions on biological/genetic data. The basis for these new criteria is a uniform nomenclature and standard operating procedures which were initiated by us and which today are the basis for worldwide regulations. Our group has also been very successful in pursuing an innovative approach within the area of automatic identification and quantification of tumor cells in the peripheral blood, bone marrow and apheresis products: the combined visualisation of immunological and genetic markers leads to an unparalleled sensitivity and specificity of results. This procedure has been used for more than 10 years now and has yielded new information on the clinical relevance of hematogenetic metastases at diagnosis and has proven to be a noninvasive method to assess the success of therapy. In recent years, besides pursuing diagnostic/prognostic aspects, we have also devoted ourselves to possible therapeutic approaches, in particular to the induction of tumor senescence. We have in fact developed a model for senescence for neuroblastoma: in this in vitro model highly aggressive cells are turned into senescent, non-aggressive cells which can possibly stimulate the immune system. 4 5

7 Biologic pathways and genetic features in neuroblastic tumours. Tumour cell ploidy (grey columns) can be used to subdivide neuroblastoma tumours into two broad groups (separated by the long punctuated line). Although the ploidy subgroups roughly correspond to the biologic subgroups (aggressive neuroblastomas marked by a red background either with MNA in dark red and separated by a short punctuated line from neuroblastomas without MNA versus less aggressive behaving neuroblastomas indicated by a green background), they do not totally match. Although aggressive near-triploid neuroblastomas (in red below the long punctuated line) have been observed, it is less clear if benign diploid neuroblastomas without any structural aberrations (in green above the long punctuated line) occur. Benign clinical behaviour refers either to spontaneous regression/maturation without any therapy or with surgery only (no cytotoxic therapy). Gruppenbeschreibung Genetik des Neuroblastoms: In den letzten Jahren kam es, dank der Aktivitäten Internationaler Komitees (SIOPEN, SIOP Europe Neuroblastoma und INRG, International Risk Grouping), zu bedeutenden Weiterentwicklungen und Veränderungen, sowohl in der Stadieneinteilung von Neuroblastompatienten als auch in der Therapiestratifizierung. Unsere Gruppe konnte in folgenden Bereichen zu dieser Entwicklung beitragen: i) Einführung einer neuen multi-genomischen Technik in die Diagnostik, ii) retrospektive Analyse der prognostischen Bedeutung von segmentalen Chromosomenveränderungen in LNESG I (Localized Neuroblastoma European Study) Tumoren und iii) Etablierung technischer Standards, stratifizierender Biomarker und einer eindeutigen Terminologie. Ad. i) Die Behandlungsstratifizierung von Neuroblastompatienten basiert gegenwärtig bereits auf Veränderungen des Tumorgenoms (v.a. des MYCN Onkogens). In der Zukunft wird jedoch ein umfangreiches Panel an DNA Markern (pan-/multigenomische Techniken) für Algorithmen zur Risikoeinschätzung verwendet werden [1-3]. Diese Entwicklungen erfordern eine Umstellung der molekular-genetischen Tumoraufarbeitung innerhalb der Europäischen Neuroblastomstudien, die herkömmliche Analysen nur einzelner genetischer Marker mittels Interphase FISH oder PCR ablösen wird. Da die Neuroblastomforschung der letzten Jahre die prognostische Bedeutung von segmentalen Chromosomenaberrationen zeigen konnte, war es die Aufgabe unserer Gruppe, eine Technik einzuführen, die es ermöglicht, eine Vielzahl chromosomaler Regionen, die alle bekannten segmentalen Aberrationen beinhalten, in einem Experiment zu erfassen. Wir adaptierten, gemeinsam mit MRC Holland, eine PCR Technik für das Neuroblastom, die sog. MLPA (multiplex ligation-dependent probe amplification) [4]. Diese zeichnet sich durch Robustheit und gute Anwendbarkeit in den meisten molekularbiologischen Laboratorien aus und ermöglicht nun eine multigenomische Tumoruntersuchung aller in Europäischen Studien gemeldeter Neuroblastompatienten. Ad. ii) Die Anwendung neuer genomischer Techniken ermöglichte nun auch die pan-/multigenomische Aufarbeitung von Tumoren der ersten SIOPEN Studie (LNESG I) und damit die Aufklärung der prognostischen Bedeutung von segmentalen Chromosomenveränderungen in lokalisierten, nicht MYCN amplifizierten Neuroblastomen nach ausschließlich chirurgischer Behandlung unabhängig von einem möglichen Tumorrest (Manuskript in Vorbereitung). Ad. iii) In Zusammenarbeit mit der SIOPEN Biologiegruppe haben wir für die diagnostische Aufarbeitung von Neuroblastomen technische Standards, eindeutige Biomarker und eine einheitliche Terminologie für die verschiedenen genomischen Veränderungen in Abhängigkeit von der Untersuchungsmethode entwickelt. Diese Standards erreichten nun auch weltweite Akzeptanz und bilden die Basis der Empfehlungen der INRG Biologie Gruppe (Ambros, BJC, eingereicht). Group description Neuroblastoma genetics: Neuroblastoma research of the last years has led to profound changes in staging and treatment planning based on the efforts of International Committees of clinicians, biologists and other disciplines (SIOPEN Committees; INRG, International Neuroblastoma Risk Grouping, Committees). The contribution of our group concerns: i) the implementation of a new multi-genomic technique into the diagnostic work-up, ii) the retrospective analysis of the prognostic meaning of segmental chromosome aberrations in LNESG I (Localized Neuroblastoma European Study) tumours, and iii) the development of technical standards, decisive biomarkers and a common language. Ad. i) Presently, treatment stratification of neuroblastoma patients is highly dependent on tumour cell genomic features (especially status of the MYCN oncogene), and future risk assignment algorithms will use a comprehensive panel of DNA-based biomarkers (genome-wide techniques). These developments require a fundamental change of the molecular diagnostic work-up within European Neuroblastoma Studies which will supersede the analysis of only individual genetic markers by interphase FISH or PCR techniques in the past. Since the prognostic meaning of segmental chromosome aberrations in neuroblastic tumours has emerged in the last years [1-3], we have been committed to introduce strategies to visualize all common segmental aberrations in a single experiment. From the different techniques presently known, we have focused on the PCR based multiplex ligationdependent probe amplification (MLPA) technique which is robust and can be applied in virtually every molecular biology laboratory [4]. Consequently, tumours enrolled in current European Neuroblastoma studies are generally analyzed by MLPA. Ad. ii) Pan-/multi-genomic techniques applied in retrospect for LNESG I tumours helped to clarify the prognostic impact of segmental chromosome aberrations in localized, not MYCN amplified, neuroblastomas treated by surgery alone (manuscript in prep.). Ad. iii) As a general achievement of our group to better define the diagnostic work-up of neuroblastomas and other tumours we, in collaboration with the SIOPEN Biology Group, developed technical standards and definitions of decisive biomarkers which are now accepted by the worldwide neuroblastoma community and build the basis of the INRG Biology recommendations (Ambros, BJC, submitted). [3] Schleiermacher G., Michon J., Huon I., d Enghien C.D., Klijanienko J., Brisse H., RibeiroA., Mosseri V., Rubie H., Munzer C., Thomas C., Valteau-Couanet D., Auvrignon A., Plantaz D, Delattre O., Couturier J., Sociéte Francaise des Cancers de l Enfant (SFCE). Chromosomal CGH identifies patients with a higher risk of relapse in neuroblastoma without MYCN amplification. Br J Cancer, 2007 jul 16;97(2): Epub 2007 Jun19. [4] Schouten J.P., McElgunn C.J., Waaijer R., Zwijnenburg D., Diepvens F., Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res Jun 15;30(12):e57. 6 [1] Mosse Y.P., Diskin S.J., Wasserman N., Rinaldi K., Attiyeh E.F., Cole K., Jagannathan J., Bhambhani K., Winter C., Maris J.M. Neuroblastomas have distinct genomic DNA profiles that predict clinical phenotype and regional gene expression. Genes Chromosomes Cancer Oct;46(10): [2] Vandesompele J., Baudis M., De Preter K., Van Roy N.,Ambros P., Bown N., Brinkschmidt C., Christiansen H., Combaret V., Lastowska M., Nicholson J., O'Meara A., Plantaz D., Stallings R., Brichard B., Van den Broecke C., De Bie S., De Paepe A., Laureys G., Speleman F. Unequivocal delineation of clinicogenetic subgroups and development of a new model for improved outcome prediction in neuroblastoma. J Clin Oncol Apr 1;23(10):

8 Das MYCN Onkogen: Da die Amplifikation des MYCN Onkogens unabhängig vom Tumorstadium und anderen Parametern zu einem aggressiven Tumorwachstum führt, haben wir uns mit dieser Aberration intensiver auseinander gesetzt. In einer vorangegangenen Arbeit konnten wir zeigen, dass dieses Onkogen aus dem Zellkern ausgestoßen werden kann, ein Prozess, der schließlich zu Tumorzellrevertanz und Seneszenz führt [5]. In der Folge konnten wir zeigen, dass dieses Phänomen durch z.b. Hydroxyurea deutlich verstärkt und beschleunigt werden kann. Diese Resultate stimulierten weiterführende Untersuchungen, wie z.b. über die Ursachen der MYCN Ausschleusung, Veränderungen des Expressionsmusters, Methylierungsstatus und mögliche Auswirkungen auf nicht seneszente Tumorzellen und Immunzellen. Chromosomale Information spiegelt die Sequenzinformation wider: In diesem Projekt verfolgten wir die Arbeitshypothese, dass sich der im Genom ungleichmäßig verteilte GC Gehalt auf chromosomaler Ebene widerspiegelt. Wir konnten tatsächlich zeigen, dass diese Annahme zutrifft. Diese Entdeckung erlaubt nun eine hochauflösende Zuordnung von zytogenetischen Informationen zu molekularen Daten und ermöglicht eine neue Nomenklatur auf Basis der heute gängigen Basenpaareinteilung (Kowalska, 2007, Chromosome Res) (Kowalska, 2008, Cytogenet Genome Res). EBV: Wir setzten unsere Untersuchungen über das EBV Genom mit Hilfe einer modernen DNA Spreitungstechnik fort und konnten das erste Mal episomale und chromosomal integrierte virale Genkopien im Fluoreszenzmikroskop visualisieren (Reisinger, 2006, Int J Cancer). The MYCN oncogene: As MYCN amplification ultimately leads to an aggressive tumour growth independent of the tumour s stage, we focused on the expression pattern/changes of this gene and frequently co-amplified genes on chromosome 2p in amplified and non-amplified tumours and cell lines (not described in further detail) and especially pursued the observation of MYCN expulsion. In a preceding work we could show that this oncogene can spontaneously be eliminated from the nucleus, thus leading to tumour cell revertance and cell senescence [5]. To find out whether this mechanism can in principle be used as therapeutic strategy, we used different drugs to accelerate this mechanism. We could show that hydroxyurea is capable of inducing tumour cell revertance and senescence in MYCN amplified neuroblastoma cell lines (Narath et al. 2007). These data indicate a possible therapeutic application and stimulated a number of ongoing research projects covering the causes of the MYCN expulsion, changes in the expression pattern, changes in the methylation status and possible effects of the senescent cells on non-senescent tumour cells and immune cells. Chromosomal information linking sequence information: To fill the gap between the molecular and the visible world and thus to facilitate the correlation of cytogenetic and molecular studies we took advantage of the non random distribution of the GC content within the genome. We could show that differences in the GC distribution are mirrored on the chromosomal level thus enabling translation of every position on the chromosomal level into sequence information and vice versa, leading to a so far unrivalled resolution of cytogenetic data (Kowalska, 2007, Chromosome Res) (Kowalska, 2008, Cytogenet Genome Res). EBV research: Lastly, we could continue our long standing tradition in EBV research by visualizing for the first time episomal and chromosomally integrated viral copies in human tumour cells by a fibre FISH technique (Reisinger, 2006, Int J Cancer). This study will facilitate further insights into the role of integrated viral copies in the tumour genesis. Induction of senescence in MYCN amplified neuroblastoma cell lines by hydroxyurea (In cooperation with P. Boukamp) Recently, it was shown that MYCN amplified cells spontaneously expulse extrachromosomally amplified gene copies by micronuclei formation. Furthermore, it was shown that these cells lose their malignant phenotype and start to age. We tested whether it is possible to encourage neuroblastoma tumor cells to enter the senescence pathway by low concentrations of the micronucleiinducing drug hydroxyurea (HU). We studied the effect of HU on 12 neuroblastoma cell lines with extra- or intrachromosomally amplified MYCN copies and without amplification. Two extrachromosomally amplified neuroblastoma cell lines (with double minutes) were investigated in detail. Already after 3 weeks of HU treatment, the BrdU uptake dropped to 25% of the starting cells. After 4 weeks, enlarged and flattened cells (F-cells) and increased granularity in the majority of cells were observed. A drastic reduction of the MYCN copy number-down to one copy per cellassociated with CD44 and MHCI upregulation in up to 100% of the HU treated neuroblastoma cells was found after 5-8 weeks. Telomere length was reduced to half the length within 8 weeks of HU treatment, and telomerase activity was not detectable at this time, while being strongly expressed at the beginning. All these features and the expression of senescence-associatedbeta-galactosidase (SA-beta-GAL) in up to 100% of the cells support the hypothesis that these cells entered the senescence pathway. Thus, low-dose HU is a potent senescence elicitor for tumor cells with gene amplification, possibly representing an attractive additional strategy for treatment of this subset of tumors. Sequence based high resolution chromosomal CGH (In cooperation with Q.R. Chen, T. Lörch, J. Khan) We aimed to directly align a chromosomal CGH (ccgh) pattern with the gene mapping data by taking advantage of the clustering of the GGCC motif at certain positions in the human genome. The alignment of chromosomal with sequence data was achieved by superimposition of (i) the fluorescence intensity of the sequence specific fluorochrome, Chromomycin A3 (CMA3), (ii) the ccgh fluorescence intensity profile of individual chromosomes and (iii) the GGCC density profile extracted from the Ensembl genome sequence database. The superimposition of these three pieces of information allowed us to precisely localize regions of amplification in the neuroblastoma cell line STA-NB-15. Two prominent ccgh peaks were noted, one at 2p24.3, the position 15.4 mega base (Mb), and the other at 2p23.2, Mb. FISH and high resolution array CGH (acgh) experiments disclosed an amplification of MYCN (16 Mb) and ALK ( Mb), thus confirming the ccgh data. The combined visualization of sequence information and ccgh data drastically improves the resolution of the method to less than 2 Mb. Genes proximal and distal to MYCN are highly expressed in human neuroblastoma as visualized by comparative expressed sequence hybridization MYCN amplification is associated with poor prognosis in neuroblastoma disease. To improve our understanding of the influence of the MYCN amplicon and its corresponding expression, we investigated the 2p expression pattern of MYCN amplified (n = 13) and nonamplified (n = 4) cell lines and corresponding primary tumors (n = 3) using the comparative expressed sequence hybridization technique. All but one MYCN amplified cell line displayed overexpression at 2p. Expression peaks were observed frequently at 2pter and less frequently at 2p24 (MYCN locus), 2p , and/or 2p23.1. Importantly, cell lines and two corresponding primary tumors displayed expression peaks at similar loci. No significant 2p24 expression level was observed for those cell lines displaying a low amplification rate (n = 3) by comparative genomic hybridization. Only the cell lines with an enhanced peak at 2p displayed coamplification of the ALK gene (2p23.2), reported to be associated with unfavorable prognosis. Finally, two of four cell lines without MYCN amplification, both derived from patients with poor outcome, also showed an expression peak at 2p23.2. These data indicate that, besides MYCN, other genes proximal and distal to MYCN are highly expressed in neuroblastoma. The prognostic significance of expression peaks at 2p , independent of MYCN and ALK status, remains to be investigated. Visualization of episomal and integrated Epstein-Barr virus DNA by fiber fluorescence in situ hybridization For many Epstein-Barr virus (EBV)-associated malignancies, it is still a matter of controversy whether infected cells harbor episomal or chromosomally integrated EBV genomes or both. It is well established that the expression of EBV genes per se carries oncogenic potential, but the discrimination between episomal and integrated forms is of great relevance because integration events can contribute to the oncogenic properties of EBV, whereas host cells that exclusively harbor viral episomes may not carry the risks mediated by chromosomal integration. This notion prompted us to establish a reliable technique that not only allows to unequivocally discriminate episomal from integrated EBV DNA, but also provides detailed insights into the genomic organization of the virus. Here, we show that dynamic molecular combing of host cell DNA combined with fluorescence in situ hybridization (FISH) using EBV-specific DNA probes facilitate unambiguous discrimination of episomal from integrated viral DNA. Furthermore, the detection of highly elongated internal repeat 1 (IR1) sequences provides evidence that this method permits detection of major genomic alterations within the EBV genome. Thus, fiber FISH may also provide valuable insights into the genomic organization of viral genomes other than EBV. FISH on DNA-fibers from lymphoma cell line Daudi. Visualization of an intact nondisrupted EBV episome (red color indicates W fragment, yellow corresponds to the two telomeric sequences, the rest of the genome is green). Diagnostic Spin-offs Between January 2006 and September 2008 we performed FISH analyses on 195 neuroblastoma tumor samples to identify the status of the MYCN amplification, presence of a deletion of 1p36 and the presence of a gain on 17q. Further, FISH analyses on 14 Ewing tumor samples concentrating on the presence of the Ewing tumor typical genetic rearrangement of the EWS gene, including the cryptic EWS/ERG translocation. Furthermore we analyzed the presence of the ETV6-NTRK3 gene to verify the diagnosis of congenital fibrosarcoma and rearrangements of the FKHR gene typical of alveolar rhabdomyosarcomas in 25 cases. Presence of integrated/episomal EBV copies was analyzed in 37 cases. In addition, 616 immunological analyses of bone marrow and peripheral blood samples from neuroblastoma, rhabdomyosarcomas and Ewing tumor patients using the Automatic Immunofluorescence Plus FISH (AIPF) method (RCDetect, MetaCyte, Meta- Systems, Germany) were performed. In approximately one third of the samples, FISH analyses of questionable immunological positive cells were performed additionally to verify the exact nature of the immunologically positive cells. Furthermore, we controlled the presence of tumor contamination in apheretic products from 54 neuroblastoma patients. [5] Ambros I.M., Rumpler S., Luegmayr A., et al. Neuroblastoma cells can actively eliminate supernumerary MYCN gene copies by micronucleus formation--sign of tumour cell revertance? EurJCancer 1997;33(12):

9 IMMUNOLOGISCHE DIAGNOSTIK IMMUNOLOGICAL DIAGNOSTICS Gruppenleiter (Group Leader): Wiss. Mitarbeiterin (Staff Scientist): Technische Mitarbeiterin (Technician): Univ. Doz. Dr. Michael N. DWORZAK (M.D., Assoc. Professor of Pediatrics) Dr. Zvenyslava HUSAK (Ph.D.) Angela SCHUMICH Univ. Doz. Dr. Michael DWORZAK Geschichtlicher Überblick Seit der Gründung der Arbeitsgruppe im Jahr 1993 liegt unser wissenschaftlicher Fokus auf der Charakterisierung der normalen bzw. malignen Hämatopoese, und inkludiert damit insbesondere Leukämien und Lymphome, welche etwa 50% aller Krebserkrankungen des Kindes- und Jugendalters umfassen. Unser Ziel ist es, durchflußzytometrische Methoden zu entwickeln, welche klinisch für Diagnosestellung, Risikostratifizierung und Therapieplanung einsetzbar sind. Seit den Anfängen hat sich die verwendete Technologie von 2-Farben Anwendungen zur nunmehrigen 8-Farben Routine weiterentwickelt, wobei vieles in Kooperation mit der Arbeitsgruppe Stammzellbiologie erfolgte. Unsere wissenschaftlichen Leistungen wurden bislang dreier nationaler Preise für Wert befunden, sowie in fünf Projektansuchen extern finanziell unterstützt. Basierend auf Untersuchungen des Expressionsprofils von CD99 in der frühen Blutzellentwicklung (publiziert 1993) konnten wir in den Folgejahren eine sensitive und kostensparende durchflusszytometrische Methode für die Detektion minimaler Resterkrankung ( MRD ) bei Leukämie entwickeln, welche sich für Prognosestellung und Therapiesteuerung eignet. Unsere erste nationale, multizentrische Studie mit dieser Methode wurde 2002 in Leukämie-Spezialistenkreisen mit Interesse aufgenommen. Dies führte zur Gründung mehrerer international kooperativer Projekte mit unserer Teilnahme, im Rahmen derer bislang etwa 1500 Patienten mit Leukämie (ALL und AML) untersucht wurden. Die Ergebnisse dieser Kooperationen auf der Basis unserer durchflußzytometrischen MRD-Bestimmungsmethode führten nicht nur zu zahlreichen Publikationen, sondern auch dazu, dass diese Diagnostik im nächsten internationalen AIEOP-BFM-ALL Behandlungsprotokoll als Bestandteil der Therapieplanung herangezogen werden wird. In der Zukunft hoffen wir, die Anwendung unserer Methode international weiter verbreiten, sowie technische Weiterentwicklungen im Sinne der Automatisierung vorantreiben zu können. Darüber hinaus haben wir 2007 ein neues kollaboratives Projekt mit dem Ziel begonnen, eine geeignete durchflußzytometrische Methode für den Nachweis aktivierter intrazellulärer Signalwege zu entwickeln. Diese Methode könnte dazu beitragen, Krebsbehandlungen mit Signalweg-Inhibitoren, einer neuen Klasse von Krebsmedikamenten, zielgerichteter und damit in Zukunft erfolgversprechender zu machen. Historical survey Founded in 1993, the Immunological Diagnostics research group has its scientific focus on the characterization of normal and malignant hematopoiesis. This includes specifically the pediatric leukemias and lymphomas which make up for approximately 50% of all cancers in childhood and adolescence. Our goal is the development and evaluation of flow cytometric methodology which can be exploited clinically for diagnostics, risk stratification and treatment tailoring to individual needs. Technically we developed from two-color applications to nowadays 8-color routine, much in co-operation with the research group Biology of Stem Cells. We were awarded three national prizes for outstanding achievements in bio-medical research, as well as endorsed five external grants. Based on observations on CD99 expression profiles in early hematopoiesis which were published in 1993, we developed a sensitive and cost-effective flow cytometric method for the detection of minimal residual disease in leukemia, designed for prognostication and treatment selection. A first nation-wide multi-center study with this methodology was well received by the scientific community in These achievements led to the initiation and fruitful conduct of still ongoing internationally collaborative projects, both in ALL and AML. In these co-operations approximately 1500 patients with leukemia have been investigated up-to-now using and validating our methodology. Apart from numerous publications, our data have led to the integration of flow cytometric MRD-assessment into the up-coming international AIEOP-BFM-ALL therapy protocol aiming at randomized treatment reduction in patients with very rapid leukemia regression kinetics. Future aims are the further dissemination and automation of the MRD-detection assay. Secondly, in 2007 we started a collaborative project aiming at the development and validation of new methodology for activated signal pattern profiling in leukemia. This approach aims at treatment individualization and tailoring towards optimum efficacy of the new anti-cancer drugs which block intra-cellular signal transduction

10 Gruppenbeschreibung Die Entwicklung und Validierung neuer diagnostischer Methoden basierend auf Durchflußzytometrie ist das Hauptziel unserer Forschungsgruppe. Dabei konzentrieren wir uns vor allem auf die pädiatrischen Leukämien sowie Lymphome, die gemeinsam etwa 50% aller Krebserkrankungen im Kindes- und Jugendalter ausmachen. Zusammenfassend sind die Themen unserer Arbeit einerseits die krankheitsassoziierten Besonderheiten in der Proteinexpression und die sich daraus ergebenden Möglichkeiten für Feindiagnostik, Risikostratifizierung und Prognoseevaluation, sowie für die individualisierte Behandlungsplanung (siehe FLOWMRD und SIGNALFLOW ). Andererseits beschäftigen wir uns auch mit leukämiebiologischen Fragestellungen mit dem Ziel der Entwicklung neuer Therapiemöglichkeiten (siehe CD99 ). FlowMRD Acute leukemias and lymphomas have become curable diseases. However, 10-35% of children with these diseases still relapse. To optimize and economize treatment further, therapy must be tailored upon individual factors of relapse risk. Of these, one of the most relevant is response to treatment which can be estimated with high accuracy by assessing minimal residual disease (MRD) using flow cytometry (FLOW). Since the year 2000 we have been conducting an internationally collaborative four-center study of FLOW-MRD in pediatric ALL (Berlin, Monza, Padova, and Vienna M.N. Dworzak, coordinator). We aimed at determining algorithms for risk assessment in a large group of patients treated with the AIEOP-BFM-ALL 2000 protocol in Italy, Germany and Austria. This study launched as a prospective validation approach to our previous retrospective experience with FLOW-MRD in study ALL-BFM 95 [1]. The current protocol is going to close recruitment by end of 2008 in Austria approximately 500 patients have been recruited up to now. In this concerted action we improved FLOW-MRD methodology. An extensive standardization program was installed, and subse- Group description The development and evaluation of new diagnostic approaches based on flow cytometric immunophenotyping is the main goal of our group. In this respect, we focus on the pediatric leukemias and lymphomas which make up for approximately 50% of cases of cancer in childhood and adolescence. Topics of our work are investigations into disease-associated peculiarities of protein expression which could in the future be exploited clinically for elaborate diagnostics, risk stratification and treatment tailoring to individual needs (see topic FLOWMRD and SIGNAL- FLOW ), and development of new therapeutic approaches (see topic CD99 ). Representative example of the analysis and gating strategy in flow cytometric MRD assessment in childhood ALL (leukemic cells = red). quent blinded inter-laboratory comparison tests proved that MRDevaluation by FLOW in ALL can be reliably standardized for multi-centric assessment in large trials (Dworzak, 2008, Clin Cytometry). A further aim of the project was assessing the overtime variation of individual leukemic phenotypes. We found that immunophenotypic modulation is induced in ALL by steroids during induction therapy [2]. This could be reproduced in vitro and appeared to reflect sensitivity to chemotherapy (Gaipa, 2008, Clin Cytometry). However, modulation of antigen expression was found to revert frequently to initial patterns after stop of steroids (Dworzak et al., manuscript in preparation). Notably, we found that CD20 expression increases during induction which translates into an acquired state of higher sensitivity to rituximab-immunotherapy (Dworzak, 2008, Blood, in press). CD20 up-regulation was frequent in high-risk patients, patients with high MRD at endinduction, and patients who suffered later from relapse, but not in TEL/AML1 cases. Based on the collaborative outcome-related FLOW-MRD results, the clinical authorities have decided to integrate FLOW-MRD assessment for treatment stratification (towards reduction of anthracyclines in induction) in the upcoming AIEOP-BFM ALL 2008 protocol. In addition, dissemination of methodology and know-how has been actively pursued: both the ALL-IC-BFM (largest international pediatric ALL-treatment collaboration) and the Moscow-Berlin group (spanning most Russian centers) have called our group into their advisory board for the planning and conduct of FLOW-MRD-based treatment stratification in their upcoming BFM-backbone therapy protocols. Hence, laboratory staff of 13 foreign institutions has been trained in the CCRI in immunodiagnostics and MRD-detection during the observation period. Smaller-sized studies have focussed on FLOW-MRD in AML [3], in lymphomas (ongoing), as well as on other aspects of immunophenotyping in hematological malignancies (Attarbaschi, 2006, CCR) (Attarbaschi, 2007a, Leukemia) (Minkov, 2007, Ped Blood Cancer). [1] Dworzak M.N., Fröschl G., Printz D., Mann G., Pötschger U., Mühlegger N., Fritsch G., and Gadner H. (2002) Prognostic significance and modalities of flow cytometric minimal residual disease detection in childhood acute lymphoblastic leukemia. Blood 99(6): [2] Gaipa G., Basso G., Maglia O., Leoni V., FainiA., Cazzaniga G., Bugarin C., Veltroni M., Michelotto B., Ratei R., Coliva T., Valsecchi M.G., Biondi A., Dworzak M.N. on behalf of the I-BFM-ALL-FCM-MRD-Study Group (2005) Drug-induced immu nophenotypic modulation in childhood ALL: implications for minimal residual disease detection. Leukemia 19:49-56 [3] Langebrake C., Creutzig U., Dworzak M., Hrusak O., Mejstrikova E., Griesinger F., Zimmermann M. and Reinhardt D. (2006) Residual disease monitoring in childhood acute myeloid leukemia by multiparameter flow cytometry: The MRD-AML-BFM Study Group. Journal of Clinical Oncology, 24(22): Signalflow Treatment of acute myeloid leukemia is about to change as a result of the development of new targeted agents, which are in many cases small molecules engineered to block intracellular signalling pathways. The interaction of such drugs with their targets either inhibits pathways essential for cell proliferation or activates pathways that culminate in apoptosis. The full potential of signal inhibition therapy will ultimately depend on the definition and reliable assessment of markers, which can indicate responsiveness to these compounds. This project has been started in 2007 and is conducted in collaboration with the group of Prof. V. Sexl, Institute of Pharmacology, University of Vienna (mouse model work). It has gained external support from the WWTF for a period of four years. The project aims at determining the intracellular signalling status of pediatric AML cases in order to identify new therapeutic options. The interplay of genetic mutations with the effects of coexistent external stimuli from cytokines determines the activation pattern of intracellular signalling networks. Leukemic cells have been shown to exhibit frequently aberrant constitutive activation of several key signalling molecules [1]. Combinations of key signalling activity states may differ between leukemic cells from different patients, thus creating situations where certain patterns of activation in the signalling network may correlate with specific leukemia subtypes, genetic lesions, and/or therapy response to signal interceptors. Moreover it is accepted that in order to eradicate AML, the so called leukemic stem cells which propagate leukemic repopulation need to be targeted. Their signalling profile may, however, differ from that of the more differentiated bulk of leukemic cells. Flow cytometry is specifically suited to investigate the profiles of various subpopulations of cells simultaneously, and it is a rapid and high through-put technology, allowing analysis of several qualities on the single-cell level. Using murine leukemia models and mice transplanted with human AML cells we plan to establish a flow cytometric assay for comprehensive activated signal pattern profiling in childhood leukemia with particular consideration of leukemic stem cells. This assay will be validated on clinical samples from pediatric AML patients from the AML-BFM study centers in Hanover (Prof. D. Reinhardt) and Vienna. The availability of the flow cytometric assay will allow us to characterize the effects of signal interceptors on signalling pathways and cellular outcome in leukemic blasts (induction of apoptosis/cell cycle arrest). We hope eventually to be able to link certain patterns of signalling pathways with potential treatment options. [1] Irish JM, Hovland R, Krutzik PO, et al. Single Cell Profiling of Potentiated Phospho- Protein Networks in Cancer Cells. Cell 2004;118: CD99 CD99 is a human 32 kd transmembrane-sialoglycoprotein expressed on many hematopoietic and non-hematopoietic cell types where it has been implicated in survival and adhesion processes. Malignancies such as Ewing tumor (ET), acute lymphoblastic as well as myeloid leukemia, and lymphoblastic lymphoma were found to be strongly CD99 positive. Experiments with antibody-mediated ligation of CD99 in T cells and ET cell lines showed that such treatment specifically induced apoptosis. This was then successfully engaged as an anti-tumor and anti-metastatic strategy in human ET mouse models. This observation pushed CD99 forward as a potential clinical target in diseases with significant levels of CD99 expression. Our previous investigations showed that CD99 is very strongly expressed by the most immature BCP stages in bone marrow (BM), and that this maturation-linked expression pattern is conserved despite malignant transformation in B cell precursors acute lymphoblastic leukemia (BCP-ALL) [1, 2]. It allowed us to speculate that CD99 may have a pivotal role also in early B-lymphopoiesis. Based on FWF grant P18196-B05 we studied functional effects of CD99 engagement in normal and leukemic BCPs. An association of CD99 expression levels with the cytogenetic background of pediatric BCP-ALLs was found. Among three investigated subgroups, hyperdiploid cases and those with a TEL/AML1 translocation showed high CD99 levels as opposed to cases with normal karyotype. Specifically in TEL/AML1-positive leukemias CD99 modulation by antibodies moderately induced apoptosis, which was however, prevented by stroma-cell contact. Likewise, very immature normal BCP were more sensitive to CD99-mediated apoptosis induction even when grown on mesenchymal stroma cells as compared to later maturational stages. We observed a gross reduction of the main CD99 mrna form during normal BCP maturation that was accompanied by down-regulation of CD99 on the cell surface. CD99-modulation did not affect STAT3 and 5, ERK1/2, or Akt signaling, but led to a specific up-regulation of hsp70 in leukemic cells, both in the cytoplasma as well as on the cell surface. Based on these data, we suggest that clinical targeting of CD99 will have detrimental effects on normal B-lymphopoiesis but affects leukemic BCP cells rather moderately and only in cases with TEL/AML1 translocation. However, CD99- mediated hsp70-induction may offer new perspectives towards induction of NK-cell mediated tumor control. A manuscript on the finding in the study is currently in preparation and our add-on project on this topic has recently gained funding by the ÖNB (Jubiläumsprojekt 13081). [1] Dworzak M.N., Fritsch G., Buchinger P., Fleischer C., Printz D., Zellner A., Schöllham mera., Steiner G., Ambros P.F., Gadner H. (1994) Flow cytometric assessment of human MIC2 expression in bone marrow, thymus, and peripheral blood. Blood 83(2): [2] Dworzak M.N., Fritsch G., Fleischer C., Printz D., Fröschl G., Buchinger P., Mann G., and Gadner H. (1999) CD99 (MIC2) expression in paediatric B-lineage leukaemia/ lymphoma reflects maturation-associated patterns of normal B-lymphopoiesis. British Journal of Haematology 105:

11 TUMORIMMUNOLOGIE TUMOR IMMUNOLOGY Gruppenleiter (Group Leader): Dr. Thomas FELZMANN (M.D.) (MBA) Wiss. Mitarbeiter (Staff Scientists): Dr. Souyet CHANG-RODRIGUEZ (until 06/07) Dr. Alexander DOHNAL (Ph.D.) DoktorandInnen (Ph.D. Students): Mag. Mareike LINDBAUER Mag. Romana LUGER Mag. Michael TRAXLMAYR (until 06/08) DiplomandInnen (M.Sc. Students): Friedrich ERHART (since 09/06) Petra PAUL (until 06/06) Cornelia SCHUH (since 07/08) Sneha VALOOKARAN (since 07/08) Technische Mitarbeiter (Technicians): Christina EICHSTILL Mag. Petra GRUBER (since 05/08) DI Barbara LEITHNER (since 02/08) DI Sidrah UL-HAQ (since 02/06) Dagmar WAGNER (until 12/07) Doris WIMMER (until 04/08) Clinical Trial Management: Heike HÜGEL (MBA) (06/06 12/07) Dr. Thomas FELZMANN Geschichtlicher Überblick Die immunologische Forschung im CCRI wurde von Wolfgang Holter im Jahr 1993 etabliert. Zunächst standen Fragen der Transplantationsimmunologie im Vordergrund, welche später von Andreas Heitger weiter geführt wurden. Im Jahr 1995 begann Thomas Felzmann mit dem Aufbau einer tumorimmunologischen Arbeitsgruppe. Diese beschäftigte sich zunächst in verschiedenen Modellsystemen mit der Frage der Auslösung einer effizienten Immunreaktion gegen Krebszellen. Dabei konnte eine neuartige Methode der Krebsimmuntherapie entwickelt werden. Diese wurde erstmals im Jahr 2000 im Rahmen einer klinischen Pilotstudie im St. Anna Kinderspital bei Patienten eingesetzt. In den folgenden Jahren wurden in vier Pilotstudien mehr als 70 Patienten mit dem Krebsimpfstoff behandelt. Für die Technologie erhielt das CCRI im Jahr 2003 Patentrechte in allen wichtigen Industrieländern. Zur weiteren Entwicklung des Krebsimpfstoffs wurde im Jahr 2003 die Firma Trimed Biotech GmbH als 100% Tochter des CCRI gegründet. Gemeinde Wien, Republik Österreich und EU unterstützten die Entwicklung des Krebsimpfstoffs mit großzügigen Förderungen. Als Gesellschafter zog sich das CCRI im Jahr 2006 aus der Trimed zurück. Die österreichische Pharmafirma AOP Orphan Pharmaceuticals AG übernahm die Mehrheit an Trimed und finanziert seither die klinische Entwicklung des Krebsimpfstoffs. Im Bereich der Grundlagenforschung beschäftigt sich das Labor für Tumorimmunologie mit der molekularen und zellulären Biologie der Steuerung von Immunreaktionen gegen Krebszellen. Ziel dieser Forschungsarbeiten ist es, die Interaktion von Immunsystem mit Tumorgewebe besser zu verstehen und daraus weitere Verbesserungen der Krebsimmuntherapie zu entwickeln. Das Labor für Tumorimmunologie und Trimed arbeiten bis heute sehr eng zusammen. Historical survey Immunological research was established at the CCRI in 1993 by Wolfgang Holter. The initial focus was on questions concerning transplantation immunology, which are now pursued by Andreas Heitger. In 1995 Thomas Felzmann started the Laboratory of Tumour Immunology. Initially, this laboratory used various model systems in order to address the initiation of effective immune responses against cancer cells. This led to a novel method of cancer immune therapy, which was first applied to a patient in the St. Anna Children s Hospital in 2000 in the context of a clinical pilot trial. In the subsequent years, more than 70 patients were treated with the cancer vaccine in four clinical pilot trials. The CCRI was granted intellectual property rights in 2003 in all important industrial nations. For the clinical development of the cancer vaccine the CCRI founded Trimed Biotech GmbH as a 100% subsidiary in The City of Vienna, the Republic of Austria, and the EU generously supported the further development of the cancer vaccine. In 2006 the CCRI exited as a shareholder of Trimed. The Austrian pharmaceutical company AOP Orphan Pharmaceuticals AG assumed majority ownership of Trimed and is now financing the clinical development of the cancer vaccine. In basic research, the Laboratory of Tumour Immunology investigates the molecular and cellular biology of the regulation of immune responses against cancer cells. The aim of this research is an improved understanding of the interactions between immune system and tumour tissue and the translation into improved concepts of cancer immune therapy. The Laboratory of Tumour Immunology and Trimed are closely collaborating to this day

12 Gruppenbeschreibung Das Ziel des Labors für Tumorimmunologie (LTI) ist es, das Verständnis der Biologie der Interaktionen von Immunsystem mit Tumorgewebe von der Grundlagenforschung in die klinische Anwendungen zu übersetzen. Dendritische Zellen (DC) sind die zentralen Koordinatoren der Immunität im Allgemeinen und der Antitumorimmunität im Speziellen. Wenn DCs Gefahrensignale, wie etwa einen Befall mit Mikroorganismen entdecken, nehmen sie einen T Zell-stimulatorischen Phänotyp an und initiieren Immunantworten. Dieser stimulatorische Phänotyp ist allerdings transient, da die DCs weiter in einen Phänotyp differenzieren, der durch negativ-regulatorische Rückkopplungsschleifen gekennzeichnet ist, die eine Immunreaktion daran hindern sollen, außer Kontrolle zu geraten. Es ist eine allgemein anerkannte Tatsache, dass Tumore Antigene exprimieren, die das Ziel einer Immunreaktion sein können. Auf Basis dieser Annahmen entwickelte das LTI ein Forschungsprogramm, das auf zwei Säulen ruht, (i) der Untersuchung der Steuerung von Immunreaktion in Modellsystemen, und (ii) die Beeinflussung von DCs zur Auslösung von Antitumorimmunreaktionen in Krebspatienten. Zum besseren Verständnis der Biologie von DCs während ihrer Differenzierung in der Folge des Wahrnehmens eines Gefahrensignals in einen immunstimulatorischen und danach in einen immunsuppressiven Status ermittelten wir das Genexpressionsprofil von DCs. In diesen Studien konnten wir feststellen, dass DCs während der Differenzierung ein bestimmtes Potenzial zur Flexibilität bewahren, welche unter geeigneten Umständen eine Umkehrung des Differenzierungsprozesses erlauben (Abstrakt siehe unten). Ein anderer Aspekt unserer Studien war die Identifizierung von T Zellen mit dem γδ T Zell Rezeptor als wichtige Effektorzellen in der Suppression der Funktion von T Helfer Zellen und zytotoxischen T Zellen (Abstrakt siehe unten). Das erweiterte Verständnis der Differenzierungskinetik von DCs wurde auch im Design unseres DC Krebsimpfstoffs angewendet. Wir optimierten die Herstellung unseres DC Krebsimpfstoffs in der Weise, dass eine Interaktion von DCs mit T Zellen während des immunstimulatorischen Fensters kurz nach dem Einwirken eines Gefahrensignals ermöglicht wird (Abstrakt siehe unten). Dieser neuartige DC Krebsimpfstoff wurde in einer Serie von vier klinischen Pilotstudien zur Behandlung von pädiatrischen Krebserkrankungen (Abstrakt siehe unten), Nierenkrebs, Prostatakrebs und Knochentumoren erprobt. In diesen Studien erhielten mehr als 70 Patienten den DC Krebsimpfstoff. Die Sicherheit und Durchführbarkeit dieser Behandlung konnte dabei zweifelsfrei dokumentiert werden. Derzeit führen wir eine randomisierte multizentrische Phase II Studie durch, die darauf ausgelegt ist, erste Wirksamkeitsdaten für den DC Krebsimpfstoff zu liefern. Group description The Laboratory of Tumour Immunology (LTI) aims at translating the biological understanding of the interactions between the immune system and a tumour tissue from basic research into clinical application. Dendritic cells (DC) are the central coordinators of immunity in general and of anti-tumour immunity in particular. When sensing danger, such as microbial invasion, DCs assume a T cell stimulatory phenotype and initiate immune responses. This T cell stimulatory status is transient, however, as the DC continues its differentiation into a final status that is dominated by negative regulatory feedback loops that are instrumental in keeping an immune response from running out of control. It is a well-established fact that tumours express antigens that may be the targets of an immune response. Based on these assumptions the LTI has built a research programme that rests on two pillars, (i) addressing issues of immune regulation in model system, and (ii) aiming at modulating DCs in order to trigger antitumour immunity in cancer patients. In order to better understand the biology of DCs during their differentiation upon encountering of a danger signal into an immune stimulatory and subsequently into an immune suppressive status, we assessed the gene expression profile of DCs. In these studies we could demonstrate that DCs retain some developmental flexibility during this differentiation allowing its reversal under certain circumstances (Abstract see below). Another aspect of these studies was the identification of T cells carrying the γδ T cell receptor as important effector cells in the suppression of the function of T helper cells and cytotoxic T cells (Abstract see below). The novel understanding of a DC s differentiation kinetic was applied to the design of a DC cancer vaccine. We optimised the manufacturing of our DC cancer vaccine in such a way that the interaction of the DCs with T cells may take place during the immune stimulatory time window early after exposure to a danger signal (Abstract see below). Using this novel DC cancer vaccine design we conducted a series of four clinical phase I pilot trials for the treatment of paediatric neoplasias (Abstract see below), kidney cancer, prostate cancer, and bone tumours. In these trials more than 70 patients received our DC cancer vaccine. This allowed us to firmly establish safety and feasibility of this treatment approach. Currently we conduct a randomised multi-centre phase II trial with the goal of delivering first efficacy data for DC cancer vaccine treatment. CD40L maintains type 1 immunity in TLR4-activated dendritic cells beyond cytokine exhaustion (In cooperation with P. Paul, D. Fuchs) Pro-inflammation triggered by microbial lipopolysaccharide (LPS) through Toll-like receptor (TLR) 4 in the presence of interferon (IFN) -γ induces cytokine secretion in dendritic cells (DCs) tightly regulated by a defined differentiation programme. This DC differentiation is characterised by a dynamic immune activating but also tolerance inducing phenotype associated with irreversible down-modulation of cytokines. CD40L on activated T cells further modifies DC differentiation. Using DNA micro arrays we showed down-regulated mrna levels of TLR signalling molecules while CD40/CD40L signalling molecules were up-regulated at a time when LPS/IFN-γ activated DCs had ceased expressing cytokines. Accordingly, we demonstrated that CD40/CD40L but not TLR4 or TLR3 signalling mediated by LPS or poly (cytidylic-inosinic) acid (poly I:C) and dsrna re-established the capacity to secret interleukin (IL)-12 in LPS/IFN-γ activated DCs, which have exhausted their potential for cytokine secretion. This resulting TH1 polarising DC phenotype which lacked accompanying secretion of the crucial immune suppressive IL-10 - enhanced activation of cytotoxic T lymphocytes (CTLs). We therefore conclude that immune modulation is restricted to a secondary T-cell mediated stimulus at an exhausted DC state, which prevents an immune tolerant DC phenotype. These findings impact on the rational design of TLR activated DC-based cancer vaccines for the induction of antitumoral CTL responses. (Manuscript submitted to J Cell Mol Med) Immune-suppressive Vγ9Vδ2 T cells as a novel regulatory mechanism modulated by IL-12 secreting dendritic cells (In cooperation with D. Wesch, D. Kabelitz) Maturation of DCs by exposure to LPS and IFN-γ enables them to secret IL-12 resulting in TH1 polarisation and the support of cytotoxic T lymphocytes. We analysed T cell subsets in co-cultures of LPS/IFN-γ stimulated IL-12 secreting human DCs charged with ovalbumin as a model antigen and autologous peripheral blood lymphocytes. We observed strong proliferation of Vδ2 T cells, the predominant γδ T cell subpopulation in human peripheral blood, which mostly co-express Vγ9. This proliferative response was inhibited in cultures supplemented with IL-12 neutralising mabs. Activation of Vδ2 T cells with isopentenyl pyrophosphate (IPP), as well as increasing the number of Vδ2 T cells in the co-culture, was associated with decreased proliferation of CD4+ and CD8+ T cells, suggesting an immune-suppressive role for Vδ2 T cells. In further experiments CD4+ T cells were activated with tetanus toxoid, heat-killed Mycobacterium tuberculosis, Daudi tumour cells, or staphylococcal enterotoxin superantigens in the presence of non-dc APCs. As in the co-culture experiments with ovalbumin charged DCs, CD4+ T cell proliferation was inhibited by the simultaneous activation of Vγ9 T cells confirming the immune suppressive potential of Vγ9 T cells also in the absence of LPS/IFN-γ stimulated DCs. Together these results demonstrate that LPS/IFN-γ stimulated DCs induce proliferation of Vδ2 T cells in an IL-12 dependent manner and that this γδ T cell subset has immune suppressive characteristics. The expansion of regulatory Vδ2 T cells therefore might represent a regulatory feedback mechanism of LPS/IFN-γ matured DCs. (Manuscript submitted to J Immunol) Comparative Evaluation of Techniques for the Manufacturing of Dendritic Cell-Based Cancer Vaccines (In cooperation with S. Graffi, V. Witt) Manufacturing procedures for cellular therapies are continuously improved with particular emphasis on product safety. We previously developed a DC cancer vaccine technology platform that uses clinical grade LPS and IFN-γ for the maturation of monocyte derived DCs. DCs are frozen after 6 hours exposure at a semi mature stage (smdcs) retaining the capacity to secret IL-12 and thus support cytolytic T cell responses, which is lost at full maturation. We compared closed systems for monocyte enrichment from leukocyte apheresis products from healthy individuals using plastic adherence, CD14 selection, or CD2/19 depletion with magnetic beads, or counter flow centrifugation (elutriation) using a clinical grade in comparison to a research grade culture medium for the following DC generation. We found that elutriation was superior compared to the other methods showing 36±4% recovery, which was approximately 5 fold higher as the most frequently used adherence protocol (8±1%), and a very good purity (92±5%) of smdcs. Immune phenotype and IL-12 secretion (adherence: 1,4±0,4; selection: 2,0±0,6; depletion: 1±0,5; elutriation: 3,6±1,5 ng/ml) as well as the potency of all DCs to stimulate T-cells in an allogeneic mixed leukocyte reaction did not show statistically significant differences. Research grade and clinical grade DC culture media were equally potent and freezing did not impair the functions of smdcs. Finally, we assessed the functional capacity of DC cancer vaccines manufactured for three patients using this optimised procedure thereby demonstrating the feasibility of manufacturing DC cancer vaccines that secret IL-12 (9,4±6,4 ng/ml). We conclude that significant steps were taken here towards clinical grade DC cancer vaccine manufacturing. Phase I study of tumour Agloaded IL-12 secreting semimature DC for the treatment of paediatric cancer (In cooperation with V. Witt, W. Holter) Cancer vaccines employing DCs in their capacity as APC have been tolerated well and have shown some efficacy in clinical studies. IL-12, a cytokine critical for type 1 T-helper (TH1) lymphocyte and cytotoxic T-lymphocyte (CTL) differentiation, when released from a DC-based cancer vaccine, may support the generation of a cellular T-cell response. We applied tumour cell lysate plus keyhole limpet haemocyanin (KLH)-loaded and 48 hours LPS plus IFN-γ stimulated fully mature DCs, which do not release IL-12, subcutaneously to eight patients, and maximally 6 hours stimulated semi-mature (sm) DCs, which are potent producers of IL-12, subcutaneously (n=6) or intranodally (n=8) as a cancer vaccine to patients suffering from advanced solid paediatric malignancies. No serious adverse events were observed following application of IL-12 releasing smdc. Following immunisation the majority of patients responded positively to 16 17

13 KLH in a delayed-type hypersensitivity (DTH) test. In addition, three of six intranodally treated patients responded to the tumour antigen in the DTH test. We conclude that treatment with a DC cancer vaccine enabled to release the immune regulatory cytokine IL-12 is safe and feasible and has the potential to induce a cellular immune response in paediatric cancer patients. BIOLOGIE VON STAMMZELLEN BIOLOGY OF STEM CELLS Gruppenleiter (Group Leader): Doz. Dr. Gerhard FRITSCH (Ph.D., Assoc. Professor) Wiss. Mitarbeiter (Staff Scientists): Dr. Roswitha FRIEDL (Ph.D., 02/06-04/08) Dr. Rene GEYEREGGER (Ph.D., since 08/08) Mag. Claus SCHERMANN (until 01/06) Techn. Mitarbeiter (Technicians): Ing. Dieter PRINTZ Daniela SCHARNER Julia STEMBERGER Dijana TRBOJEVIC Elke ZIPPERER Klin. Mitarbeiter (Clinical Collaborators): Dr. Susanne MATTHES-MARTIN (M.D.) Dr. Volker WITT (M.D.) Doz. Dr. Gerhard FRITSCH Geschichtlicher Überblick Nach Gründung des Forschungsinstituts vor 20 Jahren wurde das Stammzell-Labor als letztes der ursprünglich 5 Forschungseinheiten Ende 1989 bezogen. Erstes Ziel war die Analyse von Stammzellen, die wir bereits nach einem Jahr aus Patientenblut isolieren und erstmals mikroskopisch sichtbar machen konnten. Untersuchungen von Knochenmark, Blut und Nabelschnurblut zeigten, dass diese Materialien nicht nur verschieden viele, sondern vor allem jeweils typische Stammzell-Subtypen enthielten [1-3]. Eine Studie belegte, dass sich Stammzellen aus mobilisiertem Blut besonders gut für die Transplantation eigneten [4]. Die durchflusszytometrische Zellanalyse wurde optimiert, sodass nach 6 Jahren die 4-Farben FACS Routine zur Verfügung stand, um neben den Stammzellen auch alle anderen Leukozytensubtypen quantitativ zu messen und die Ergebnisse der Klinik zur Verfügung stellen zu können. Aus Experimenten zur Anreicherung und Reinigung von Stammzellen wurde 1996 die positive Stammzell-Selektion, die erstmals eine Transplantation über HLA-Barrieren hinweg erlaubte [5]. Mit Beginn der klinischen Stammzellsammlung übernahm das Labor auch die Kryokonservierung von autologen Stammzellen und von Spenderzellen für die allogene Zellspende. In enger Zusammenarbeit mit Ärzten und Wissenschaftlern wurden viele Methoden in der klinischen Anwendung optimiert, wie das Monitoring der hämatopoetischen Regeneration [6-7], die standardisierte Messung von Stammzellen [8-9], die Steuerung des Chimärismus im transplantierten Patienten [10-12], oder die Optimierung der Sammeleffizienz bei der Zellapherese [13] (Witt, 2007, J Clin Apher). Aktuelle Aktivitäten konzentrieren sich auf den Nachweis virusspezifischer Immunzellen (Schermann, 2008, Cytotherapy, in press) und auf neue Methoden der adoptiven Immuntherapie, um lebensbedrohliche virale Infekte im Patienten behandeln zu können. Alle Arbeiten des klinischen Labors laufen seit der Betriebsbewilligung des Bundesministeriums (2004) und der Zertifizierung durch Jacie (2005) unter GMP Bedingungen im Reinraum ab. [1] Fritsch et al., Exp Hematol 1991 [2] Fritsch et al., Blood 1993 [3] Fritsch et al., Bone Marrow Transplant 1996 [4] Fritsch et al., Blood 1994 [5] Fritsch et al., Onkologie 2000 [6] Fritsch et al., Cytotherapy 1999 [7] Pichler et al., Cytometry 2002 Historical survey After foundation of the Research Institute 20 years ago, the Stem Cell Laboratory was the last of the 5 laboratories to commence work. Our primary goal was the analysis of stem cells. After one year, we managed to isolate blood stem cells from a mobilized patient for the first microscopical observation. Comparative analyses of bone marrow, blood and cord blood revealed differences in both stem cell number and composition of subtypes [1-3]. A study suggested that, for transplantation, mobilized peripheral blood stem cells were superior over those of bone marrow [4]. Within 6 years, we optimized the flow cytometric measurements and established a 4-color analysis as routine tool to provide information to clinicians about stem cells as well as all other blood leukocytes. Experiments to enrich stem cells resulted in the first stem cell selection in 1996 and enabled transplantation across HLA barriers [5]. When clinical cell collection became available at the St. Anna Children s Hospital, we were in charge of cryopreservation and storage of both allogeneic and autologous blood components. In collaboration with clinicians and scientists, a variety of clinical applications were optimized, like the analysis of hematopoietic regeneration [6-7], the standardization of cell quantification [8-9], the monitoring of donor-recipient chimerism [9-12], or the optimization of the collection efficacy during cell apheresis [13] (Witt, 2007, J Clin Apher). Recent activities are directed towards virus-specific immune cells (Schermann, 2008, Cytotherapy, in press) as well as new approaches to treat lifethreatening viral infections by means of adoptive immune therapy. We obtained the operating approval from the Bundesministerium für Gesundheit und Frauen in 2004, as well as JACIE accreditation for cell processing in 2005, and all work of the clinical laboratory is since performed under GMP conditions. [8] Witt et al., Infus Ther Transfus Med 2001 [9] Gutensohn et al., Infusionstherapie und Transfusionsmedizin 1996 [10] Matthes-Martin et al., Leukemia, 2003 [11] Dubovsky et al., Leukemia 1999 [12] Lion et al., Leukemia 2001 [13] Felzmann et al., Cytotherapy

14 Gruppenbeschreibung Bei der klinischen Routinediagnostik lag der Arbeitsschwerpunkt weiterhin beim Analysieren der verschiedenen Leukozyten Subpopulationen, vornehmlich der allogen transplantieren Patienten, sowie beim Sortieren der Zellen für deren anschließende Genotypbestimmung mittels PCR oder FISH. Im Rahmen der Qualitätskontrolle beteiligte sich unser Labor regelmäßig an Rundversuchen von INSTAND (Quantifizierung CD34 positiver Zellen) und von ÖQUASTA (Immunstatus sowie Quantifizierung von CD34+ Zellen und von Restleukozyten). Derzeit laufen Messungen mit dem Ziel der Etablierung einer standardisierten 6-Farben CD34 Messung. Eine Diplomarbeit verglich verschiedene Methoden der Detektion CMV-spezifischer T Lymphozyten und etablierte eine durchflusszytometrische Multiparameter- Analyse auf Basis HLA-spezifischer Tetramere. Bezüglich Adenoviren wurde eine Messmethode in die Routine übernommen, mit der die Zahl der ADV-spezifischen T Zellen bestimmt wird. Zudem wurden erste Untersuchungen unternommen, die auf eine adaptive Immuntherapie bei ADV Infektionen abzielen. Seit 2004 ist unsere Sort-Routine auf die 8-9 Farben-Technik umgestellt, sodass aus 2 Zellpräparationen mindestens 8 sortierte Zellpopulationen für weitere Untersuchungen zur Verfügung gestellt werden können. Seit 2006 sind nach Erwerb eines LSR II auch FACS Analysen mit >9 Farben möglich. Die Zahl der durchflußzytometrischen Anwendungen (inklusive Core Facility) lag weiter auf hohem Niveau und bewegte sich im Berichtszeitraum zwischen und Zellsorts und bis zu Zell- Analysen. Im Bereich der klinischen Zellmanipulation (GMP Labor) wurde die bislang bei HLA Unverträglichkeit zwischen Spender und Empfänger durchgeführte CD34 Positivselektion zur Gänze durch die CD3/CD19 Depletion ersetzt. Im Falle einer ABO-Unverträglichkeit zwischen Spender und Empfänger wurde das Plasma vor Infusion der Zellen depletiert. Im Bedarfsfall wurden vor Infusion aufgetauter Zellen das DMSO ausgewaschen, oder zu große Volumina eingeengt. Zudem war das Labor weiterhin zuständig für die Kryokonservierung, die Lagerlogistik und das Auftauen (meist autologer) Zellprodukte. Unsere Betriebsbewilligung gemäß 63 Arzneimittelgesetz vom Bundesministerium für Gesundheit und Frauen wurde 2007 nach einem Audit der AGES wiedererteilt. Ein weiteres Audit Anfang 2008, gemeinsam mit der Station für Allogene Stammzelltransplantation und der Apherese Gruppe des St. Anna Kinderspitals, führte zur Re-Zertifizierung durch JACIE. Group description Regarding the routine work of the clinical laboratory, the main focus has again been on flow cytometric analysis of leukocyte subtypes as well as on cell sorting for subsequent PCR- or FISHbased determination of donor/recipient genotype. Quality Control (QC) was addressed by regular participation in QC trials organized by ÖQUASTA and INSTAND, for both the flow cytometric analysis of the immune status as well as the enumeration of CD34+ cells and of residual leukocytes in blood and blood components. We are presently validating a new 6-color approach to establish a standardized CD34 analysis. In a diploma thesis, different methods were compared to detect CMV-specific T lymphocytes, and a sensitive, tetramer-based flow cytometric multi parameter analysis was established. In terms of Adenovirus, a method was worked out to quantify the amount of ADV-specific T cells. Recent trials aim at new technologies to provide adaptive immune therapies to ADV-infected patients. Since 2004, we run an extended sorting routine using 8 to 9 colors. From 2 cell preparations, we routinely provide 8 purified leukocyte subpopulations for further studies. Also FACS analyses of >9 colors became possible since a LSR II was purchased in Including the Core Facility, the number of flow cytometric applications remained on a high level, ranging from 2,000 to 2,800 cell sorts and up to 45,000 cell analyses. In the field of clinical cell manipulation (GMP Laboratory), we switched from CD34 positive selection to CD3/19 depletion (for HLA-mismatched transplantation). In case of ABO mismatch between donor and recipient, plasma was depleted prior to infusion. If necessary, DMSO was removed from thawed cell products, or too large volumes were narrowed down. In addition, the laboratory was responsible for cryopreservation, storage and reinfusion of (mainly autologous) cell components. The operating approval from the Bundesministerium für Gesundheit und Frauen was renewed in 2007 after a re-audit by AGES. Another audit early in 2008 confirmed our JACIE accreditation obtained together with the units for allogeneic transplantation and cell collection. Detection of HCMV-specific T-lymphocytes in human blood: Comparison of two methods (In cooperation with G. Fischer, V. Witt, M. Kurz) Human cytomegalovirus (HCMV) infection remains a major cause of morbidity and mortality in immunocompromised patients undergoing allogeneic stem cell transplantation (SCT). In case of HCMV reactivation, the well defined detection of virus-specific effector cells in patients might positively impact antiviral treatment. We examined blood samples from healthy volunteers serologically typed for HCMV-IgG. Based on multicolor flow cytometry analysis, we addressed HCMV-specific CD8+ effector T lymphocytes using HCMV-specific tetramers for the respective MHC class I type. As a second approach, we employed the Cytokine Secretion Assay (CSA), which allows the indirect detection of target-specific CD4+ and CD8+ T cells via their IFN-γ secretion upon HCMV-pp65 in vitro stimulation. We hypothesized to detect HCMV-specific lymphocytes in >50% of healthy Caucasians that are IgG-seropositive for HCMV. In terms of specificity, both assays showed comparably good results (specificity of 100%, CI>95%). Regarding sensitivity, both assays met the zero hypothesis. However, with 45/52 (86.5%) the tetramer technology was superior over the CSA which detected 34/52 (65.4%) based on CD8+ T cells, and 41/52 (78.8%) based on both CD4+ and CD8+ T cells. A good correlation was observed between both assays, although the tetramers address only CD8+ HCMV-specific T cells, whereas IFN-γ secretion is detected on all T cell types. Disadvantages of the CSA are the time-consuming stimulation, the extensive cell washing steps, and the fact that the target cells are detected indirectly. The analysis with tetramers is rapid and reliable, but their general use is hampered because restricted to few HLA types. Quality Control (QC) Internal QC mechanisms are in place and employed regularly according to own standard operating procedures (SOPs) and to JACIE guidelines. In addition, we participate in the regular Austrian and German quality control trials organized by Öquasta and Instand, regarding the quantitative flow cytometric analysis of 1) subtypes of blood leukocytes, 2) peripheral blood CD34+ cells, 3) residual leukocytes in blood products (plasma, platelets) Spin offs During our 14 year experience in flow cytometric analysis, we have been continuously increasing the number of applications and optimizing the different techniques. This applies also for manipulation of cells in the context of bone marrow transplantation. Our laboratory offers services as summarized: Diagnostics The following technologies are routinely used, but are also provided as a service for other internal and external groups: - Flow cytometric quantification (4-to 9-color, single-platform) of CD34+ cells, of T lymphocytes (for DLI), and of residual leukocytes, erythrocytes or platelets in blood components; - Flow cytometric monitoring of engraftment (4-color, dualplatform); - Flow cytometric enumeration of leukocyte subtypes (4- to 9- color, single- or dual-platform), in blood, bone marrow, and apheresis products; - Flow cytometric (up to 9-color) cell sorting of cells from different sources like blood, bone marrow, or cell culture. Cell manipulations All cell manipulations are performed under GMP conditions and include QC measurements like flow cytometric analysis and sterility testing: - CD3/CD19 depletion prior to bone marrow transplantation in case of defined donor/recipient HLA mismatches; - Plasma depletion from bone marrow or apheresis products prior to infusion of components obtained from AB0 incompa tible donors; - Cryopreservation and storage logistics of autologous or allogeneic blood products (stem cells, DLI); - In vitro generation of dentritic cells presenting pp65-derived MHC1 epitopes, to generate anti-cmv-specific immunity

15 TRANSPLANT-IMMUNOLOGIE TRANSPLANT-IMMUNOLOGY Gruppenleiter (Group Leader): Univ.Doz.Dr. Andreas HEITGER (M.D., Assoc. Professor) Postdoktoranden (Postdoct.Res.Fellows): Dr. Ursula HAINZ, (Ph.D.) (bis/until 12/06) Dr. Birgit JÜRGENS (Ph.D.) (seit/since 12/08) Doktorandinnen (Ph.D. Students): MMag. Birgit JÜRGENS (M.S.) (03/05 11/08) Mag. Edda VEITH (M.S.) (seit/since 08/07) Diplomstudentin (Diploma student): Linda KREMPL (12/07 11/08) Univ.Doz.Dr. Andreas HEITGER Geschichtlicher Überblick Die Arbeitsgruppe Transplantationsimmunologie hat sich im Jahre 2001 unter der Leitung von Univ. Doz. Dr. Andreas Heitger formiert. Das Ziel dieser Arbeitsgruppe ist es, die immunologischen Vorgänge im Rahmen einer hämatopoietischen Stammzelltransplantation (HSZT) besser zu verstehen und darauf aufbauend therapeutische Strategien zu entwickeln. Nachdem bei einer HSZT auch das Spender-Immunsystem mittransplantiert wird, ist das Kernproblem die möglichst rasche Entwicklung von immunologischer Toleranz zwischen Empfängerorganismus und transplantiertem Spenderimmunsystem ohne medikamentöse Immunsuppression. Störungen der Entwicklung von Toleranz und Immunkompetenz sind wesentlich an den teils schweren bis lebensbedrohlichen transplantassoziierten Komplikationen beteiligt. Wir haben uns in unseren Forschungsbemühungen auf den Mechanismus des gesteigerten Tryptophanmetabolismus, der durch die enzymatische Aktivität der Indoleamine 2,3-Dioxygenase (IDO) vermittelt wird, als immunologischen Mechanismus der Toleranzinduktion konzentriert. Die IDO Aktivität spielt nach jüngsten Erkenntnissen als potenter Mechanismus der immunologischen Toleranzentwicklung in vielen Gebieten der humanen Biologie, etwa bei Schwangerschaft, Autoimmunerkrankungen, der immunologischen Tumorkontrolle und bei Organ- und Stammzelltransplantationen, eine entscheidende Rolle. Seit 2003 testen wir das sind neben dem Gruppenleiter 2 DoktorandInnen und ein/e DiplomandIn in durch den Fonds zur Förderung der wissenschaftlichen Forschung (FWF) unterstützten Projekten die Möglichkeit, Spender-abgeleitete Immunzellen außerhalb des Körpers, also in vitro, gegen den Empfängerorganismus zu tolerisieren, ohne deren immunologische Kompetenz gegen pathologische Krankheitserreger zu beeinträchtigen. Diese tolerisierten Zellen sollen dem Empfänger rücktransferiert werden, mit dem Ziel seine Immunkompetenz zu stärken ohne eine pathologische Spender-gegen-Empfänger Reaktion auszulösen. Unsere bisherigen Untersuchungen in dieser Richtung sind vielversprechend. Nach Aufklärung und Optimierung der genauen molekularen Mechanismen streben wir in den nächsten Jahren an, diesen Ansatz soweit weiterzuentwickeln, dass er schließlich therapeutisch eingesetzt werden kann. Unser ultimatives Ziel ist es durch dieses Verfahren die belastenden transplant-assoziierten immunologischen Komplikation, die Abwehrschwäche und pathologische Abwehrreaktionen, zu vermeiden und die HSZT zu einem sicheren, breit anwendbaren Therapieverfahren zu machen. Historical survey The working group Transplant-Immunology was formed in 2001 under the leadership of Associate Professor of Pediatrics, Andreas Heitger. The scope of this group has been to better understand the immunologic processes involved in hematopoietic stem cell transplantation (HSCT) and to develop strategies for therapeutic intervention. Because HSCT involves transplantation of the donor immune system, the core problem of HSCT is the generation of immunologic tolerance between the donor-derived immune cells and the host organism without using immunosuppressive drugs. Disturbances of the development of tolerance and of regenerating immunocompetence continue to significantly contribute to the still unacceptably high incidence of transplantassociated morbidity and mortality. In our research we have focussed on the tolerogenic potential of an augmented tryptophan metabolism, which is largely governed by the enzymatic activity of indoleamine 2,3-dioxygenase (IDO). IDO activity and the resulting tryptophan depletion and accumulation of tryptophan catabolites are rather novel and potent factors of tolerance development in human biology, e.g. in pregnancy, autoimmune diseases, immunologic tumor control and organ- and stem cell transplantation. Since 2003, we this is besides the group leader 2 PhD students and 1 diploma student have been testing the possibility to tolerize donor-derived T cells against the host organism ex vivo, following the hypothesis that giving these cells back to the host of HSCT will improve the host s immunocompetence without aggravating the risk of a graft-versus-host reaction. Our thus far performed investigations are promising. In the coming years, having found support from the Austrian Science Foundation, we will enlighten and optimize in detail the mechanisms IDO-mediated tolerizing effects to subsequently be able to transfer this approach into the clinic. Our ultimate goal is to help to prevent the severe immunologic transplant-associated complications and thus make HSCT a safe and widely applicable mode of curative treatment

16 Gruppenbeschreibung Für die Arbeitsgruppe Transplantationsimmunologie steht die Erarbeitung von Strategien zur Generation von allo-antigen spezifisch toleranten T-Zell Populationen im Mittelpunkt des wissenschaftlichen Interesses. Allo-antigen spezifisch tolerant bedeutet, dass T-Zellen gegen Fremdantigene, z.b. eines Organspenders, keine Immunreaktion im Sinne einer Abstoßungsreaktion auslösen, jedoch gegen Pathogene der Umwelt (z.b. Viren, humanpathogene Pilze, etc) normal funktionell aktiv sind. Allo-antigen spezifisch tolerante T-Zellen eigenen sich hypothetisch optimal für eine adoptive Zelltherapie nach hämatopoietischer Stammzelltransplantation (HSZT), indem sie, ohne das Risiko einer Spender-gegen-Empfänger Krankheit (Graft-versushost-disease, GvHD) zu erhöhen, die immunologische Kompetenz des Knochenmarkempfängers verbessern, bis die Integrität des Immunsystems durch körpereigene Regeneration wieder hergestellt ist. Der derzeitige Fokus unserer Forschungsaktivität ist die Toleranzinduktion über eine gesteigerte Metabolisierung der Aminosäure Tryptophan [1], (Hainz, 2007, Transpl Int). Der Metabolismus von Tryptophan wird wesentlich durch die Aktivität des Enzyms Indoleamine 2,3-dioxygenase (IDO) bestimmt. Einem neu entwickelten Konzept zufolge führt die Immunstimulation durch antigenpräsentierende Zellen (APZ), die einen gesteigerten Tryptophanabbau, i.e. eine erhöhte IDO Aktivität, aufweisen, an der interzellulären Schaltstelle zwischen den stimulierenden APZ und T-Zellen zu einem Tryptophanmangel und Anhäufung von Tryptophanabbauprodukten (z.b. Kynurenine) und dadurch zu einer Tolerisierung von T-Lymphozyten [2]. Generell beruht die Toleranzentwicklung nach HSZT dem derzeitigen Verständnis nach auf (i) der Depletion antigen-reaktiver Zellen und (ii) der Induktion von immunregulierenden Mechanismen [3]. Wir konnten zeigen, dass humane IDOkompetente dendritische Zellen (DZ) (i) besonders in Anwesenheit exogener Kynurenine zu einer selektiven Apoptose allo-antigen aktivierter T-Zellen führen und (ii) in allo-antigen aktivierten T-Zellen immunregulatorische Funktionen anregen (Jürgens et al., Manuskript in Vorbereitung). Wir haben weiters damit begonnen, diese in vitro vielversprechenden Ergebnisse in einem Mausmodell in vivo zu testen. Analog zu dem Ansatz bei Menschen erhalten Mäuse eine HSZT, und simultan einen Transfer von den hypothetisch tolerisierten Spender T-Zellen und werden in Hinblick auf ihre Immunrekonstitution und GvHD überwacht. Unsere weitere Arbeit im humanen System wird sich darauf konzentrieren, die Toleranzinduktion über IDO auf fein-molekularer und Signaltransduktionsebene besser zu verstehen und zu optimieren, mit dem schlussendlichen Ziel, dieses Verfahren klinisch einsetzen zu können. Eine weitere Aktivität unserer Gruppe ist die Identifizierung von individuellen Risikofaktoren für eine GvHD und/oder infektionsbedingte transplant-assoziierte Morbidität. Hierzu haben wir vergangenes Jahr eine Untersuchung begonnen, die den Einfluss einer Defizienz des Mannose-bindenden Lektins (MBL) auf die post-transplant bedingte Anfälligkeit für schwere Infektionen aufklären soll. Das MBL als ein Komplementfaktor ist an der Früherkennung von invasiv wachsenden Bakterien und Pilzen beteiligt. Die in der Regel nicht krankheitsrelevante, in der Normalbevölkerung häufige (bis 20%) Defizienz des MBL spielt möglicherweise unter den immunologisch extremen Bedingungen einer HSZT eine signifikante infektionsbegünstigende Rolle. Group description The core goal of the transplantation immunology is to improve the immune competence after hematopoietic stem cell transplantation (HSCT). Specifically we attempt to generate donor-derived T cells which are tolerant against the recipient but retain full immunological activity against environmental pathogens. These T-cell populations are hypothetically suitable for adoptive cell transfer procedures to improve post-transplantation immunity without bearing the risk of inducing graft-versus-host disease (GVHD), and thus to bridge the hazardous immediate post-hsct period. In the past years we continued to focus our approach of inducing T-cell tolerance via an accelerated tryptophan catabolism [1], (Hainz, 2007, Transpl Int). Tryptophan catabolism in mammals is largely governed by the enzymatic activity of indoleamine 2,3- dioxygenase (IDO). As to the current understanding, T-cell stimulation by IDO competent antigen-presenting cells (APC), will result in a tolerogenic response because of tryptophan depletion and accumulation of tryptophan metabolites (kynurenines) at the intercellular interface of APCs and responding T cells [2]. Immunologic tolerance, i.e. an acceptable down-regulation of an immune response to an antigenic challenge for the benefit of the host, is a complementary interplay between (i) depletion of antigen-reactive T cells and (ii) regulation of T cells responses towards the target antigen [3]. In the past 3 years we were indeed able to demonstrate that IDO competent human dendritic cells (DCs) (i) promoted apoptotic decline of and (ii) induced regulatory activity in allo-reactive T cells (Jürgens et al., manuscript in preparation). This finding constitutes a promising and advanced step towards our ultimate goal of achieving allo-antigen-specific tolerance. In a further experimental approach we began to test the above mentioned hypothesis in a murine model to elaborate on whether these encouraging in vitro results hold true in vivo. Mice will receive HSCT and adoptive therapy of donor-derived hypothetically tolerized T cells and will be monitored for GvHD and for the reconstitution of immunocompetence towards environmental pathogens. Additionally, our continuing studies in human DCs will strive to elucidate IDO-mediated tolerogenesis on a fine molecular level, including effects on signal transduction pathways, with the ultimate goal of improving our understanding and thereby optimizing IDO-mediated tolerance induction in allogeneic T cells. In a further approach we currently explore the significance of a deficiency of mannose-binding lectin (MBL) in HSCT. MBL is involved in the early recognition of invasive bacterial or fungal infection. In collaboration with the transplant unit this study will test whether under the immunologically extreme conditions of HSCT an otherwise inapparent MBL deficiency is significant in facilitating infection-related morbidity in HSCT recipients. [1] Hainz U., Obexer P., Winkler C., Sedlmayr P., Takikawa O., Greinix H., Lawitschka A., Potschger U., Fuchs D., Ladisch S., Heitger A. (2005) Monocyte-mediated T-cell suppression and augmented monocyte tryptophan catabolism after human hematopoietic stem-cell transplantation. Blood 105, [2] Mellor A.L. & Munn D.H., Nat Rev Immunol, 2004, 4: [3] Lechler R.I., Garden O.A., Turka L.A. Nat Rev Immunol, 2003; 3: In our research we test the hypothesis, that allo-antigen specific tolerance can be achieved by induction of indoleamine 2,3-dioxygenase (IDO) in human dendritic cells (DCs) (by lipopolysaccharide and interferon g), and that these DCs, by supporting intercellular tryptophan depletion and tryptophan metabolite (kynurenine) accumulation, induce both, depletion and regulation of allo-reactive T cells. Non-alloreactive T cells will be suitable for adoptive cell transfer strategies to support immunocompetence in HSCT recipients. Allo-antigen-specific tolerance induced by human dendritic cells expressing the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase The immunomodulatory role of tryptophan metabolism by IDO has recently attracted specific interest for its proposed role in tolerance induction. Here, we studied the immunomodulatory effect of human IDO competent DCs on allogeneic T cell responses in vitro. Our significant findings were: (1) IDO competence (IDO protein expression and IDO metabolic activity) in human monocyte derived DCs when using LPS as a maturation stimulus (i) required the presence of IFNγ and (ii) was restricted to a mature DC state. (2) At a high DC/T cell ratio (1:1) IDO competent DCs had a significantly reduced capacity to stimulate allogeneic T cells. The addition of the IDO inhibitor methyl-thiohydantointryptophan (MTH-trp) to cultures of IDO competent DCs resulted in enhanced T cell proliferation, indicating an IDO-dependent effect. competent DCs displayed a regulatory phenotype, (CD4 + CD25 high FoxP3 + CTLA-4high). In fact, these T cells were able to potently suppress the proliferative response of naïve CD4 + T cells to an anti-cd3 mediated stimulation. CD4 + CD25 + T cells recovered from IDO neg cultures failed to suppress. A blockade of IDO by MTH-trp resulted in a reduction of the suppressive capacity of CD4 + CD25 + cells. Together, these findings strongly support the tolerogenic potential of human IDO competent DCs, which apparently have the potential to generate regulatory T cells. In our continuing studies we will investigate further aspects of IDO competence in human DCs upon co-culture with allogeneic T cells: (1) apoptotic decline potentially segregating in allo-activated cells (2) potential antigen specificity of T regulatory cells generated by IDO competent DC (3) examine the cytokine profile and its potential manipulation in IDO competent DC and in the T cells recovered from co-cultures with IDO competent DC to optimize their tolerogenic effect (4) possibilities to up-scale the amount of T cells recovered from co-cultures with IDO competent DC to a level at which adoptive transfer strategies will become possible. (3) When cultures were repeatedly supplied with fresh cell culture medium (to prevent the otherwise rapid exhaustion of cultures) the CD4 + T cells recovered from a co-culture with IDO Supported by FWF Grant P B

17 Employing indoleamine 2,3-dioxygenase (IDO) activity for the ex vivo generation of allo-antigen specific tolerized T cells to be used for adoptive transfer in murine hematopoietic stem cell transplantation Based upon the promising findings made in the human in vitro studies this project addresses our continuing interest in the generation of T cells specifically tolerized to allo-antigens, by IDO competent DCs (see abstract 1). We propose to explore tolerogenesis by recipient-derived IDO competent DCs towards donorderived allogeneic T cells in an in vivo fully HLA disparate murine model of hematopoietic stem cell transplantation. C57BL/6 recipients receive lethal irradiation and are supplemented with bone marrow cells obtained from Balb/c donors. In this approach we will test the hypothesis that donor T cells after an ex vivo exposure to recipient IDO competent DCs can be safely transferred to HSCT recipients, i.e. they effectively transfer T cell immunity while not inducing GVHD. As a first step, in vitro experiments will have to confirm the essential findings of our in vitro human findings, namely (1) similarly effective induction of IDO competence in murine DCs, (2) similar immunomodulatory activity of IDO competent DCs upon co-cultured allogeneic donor derived T cells. A successful completion of this study will then set the basis to move forward towards adopting this approach for clinical trials in humans and thus to contribute to the efforts to make HSCT in humans a safe and broadly applicable procedure. Supported by FWF Grant B13 The role of genetic variants of mannose binding lectin (MBL) for the susceptibility to severe infection in patients undergoing HSCT This study addresses the issue of a link between a genetic alteration of mannose binding lectin (MBL) and a predisposition to severe infection in pediatric HSCT. It is performed in close collaboration with the clinical unit of HSCT and the Department of Pediatrics, General Hospital of Vienna. MBL, being part of the innate immune system, recognizes and binds to a broad spectrum of pathogens and activates the lectin pathway of the complement cascade to initiate immune responses. The MBL gene is subject to substantial genetic polymorphisms leading to either complete or partial MBL deficiency in ~ 30% of the Caucasian population. The clinical significance of MBL deficiency, particularly under circumstances of pharmacological immunosuppression, is unclear. By a combined examination of (i) MBL gene expression in recipients and donors before HSCT and (ii) monitoring MBL serum levels during the course of immune recovery following HSCT and (iii) recording the clinical status and course of patients after HSCT we will generate solid knowledge, whether MBL in fact does represent a risk factor for HSCT recipients in the pediatric population. This study will involve ~100 pediatric patients undergoing allogeneic HSCT in the St. Anna s transplantation unit (2 years term). Ultimately, the study will clarify the so far controversial issue, whether (1) the measurement of MBL either on the genetic or on the serum level is a useful parameter to screen for patients at risk for TRM and (2) in patients with MBL deficiency the therapeutic substitution with recombinant MBL protein (which is available but costly) is justified. Supported by ÖNB Grant MOLEKULARBIOLOGIE MOLECULAR BIOLOGY Gruppenleiter (Group Leader): Wiss. Mitarbeiter (Staff Scientist): Postdoktoranden (Postdoct.Res.Fellows): Geschichtlicher Überblick Die Abteilung Molekularbiologie ist neben der Zytogenetik eine Keimzelle des Forschungsinstitutes. Nach dem Aufbau der für die Anwendung modernster molekulargenetischer und biochemischer Methoden notwendigen Infrastruktur widmeten wir uns schon sehr früh der Erforschung von Knochentumoren des Kindes- und Jugendalters, insbesondere dem Ewing Sarkom. Diese sehr bösartige Erkrankung stellte vor 20 Jahren ein schwieriges differentialdiagnostisches Problem dar. Zudem wusste man über die Herkunft und Mechanismen dieser Erkrankung fast gar nichts. Durch die Aufdekkung und Charakterisierung des ersten molekular definierten Merkmals (MIC2, heute bekannt als CD99), und die Mitwirkung an der Entdeckung der dem Ewing Sarkom zu Grunde liegenden Mutation, einer Chromosomenumlagerung, die zu einer neuartigen Genfusion führt (EWS-FLI1), erlangte das Labor internationale Bekanntheit. Beide Merkmale gelten heute weltweit als Standarddiagnosekriterien für die Erkrankung. Unter unserer Führung wurde darüber hinaus die Nutzbarkeit des molekularen Nachweises der EWS-FLI1 Genfusion für die Verlaufskontrolle der Erkrankung (minimale Resterkrankung) einer multizentrischen, internationalen Überprüfung unterzogen, deren Ergebnisse nun vorliegen. Über diesen deskriptiven Aspekt hinaus war es immer Ziel dieserarbeitsgruppe, ein funktionelles Verständnis der spezifischen Tumorentwicklung und des klinischen Krankheitsverlaufes zu erhalten. Maßgebliche Erfolge konnten dabei in der Beurteilung von wichtigen generellen Wachstumskontrollmechanismen erzielt werden. Dabei richtete sich unseraugenmerk vor allem auf die zentralen Regulatoren p53 und Rb. Wir erkannten den Verlust des INK4A Gens, welcher diese beiden Regulationsmechanismen miteinander verbindet, als die bisher häufigste sekundäre molekulargenetische Veränderung beim Ewing Sarkom. Unsere neuesten Ergebnisse definieren zudem einen völlig neuen Mechanismus der p53 Inaktivierung, dem möglicherweise eine über das Ewing Sarkom weit hinaus gehende Bedeutung zukommt. Hier gelang es uns erstmals komplexe funktionelle Zusammenhänge innerhalb der Ewing Tumorzelle zu definieren und damit mögliche neue therapeutische Ansatzpunkte aufzudecken. Das Molekularbiologielabor sieht seit jeher seineaufgabe auch darin, Wissen und Erfahrung an junge Mitarbeiter weiterzugeben, wissenschaftliches Verständnis bei den klinischen Kollegen zu fördern, und Zugang zu den modernsten molekularen Technologien zu ermöglichen. Durch die Übersiedlung in ein neues Institutsgebäude, in dem sich mehrere Arbeitsgruppen ein gemeinsames Labor teilen, soll diese fruchtbare Interaktion noch weiter gestärkt werden. Doz. Dr. Heinrich KOVAR (Ph.D., Assoc. Professor) Dr. Dave ARYEE (Ph.D.) Dr. Jozef BAN (Ph.D.) Dr. Idriss BENNANI-BAITI (Ph.D.) Dr. Oskar SMRZKA (MD) (seit/since 07/06) Dr. Max KAUER (Ph.D.) (seit/since 05/2007) DoktorandInnen (Ph.D. Students): Mag. Ruth JOAS (bis/until 12/06) D.I. Michael KREPPEL (M.Sc.) (bis/until 12/06) Mag. Radostina Bachmaier (M.Sc.) (bis/until 06/07) Mag. Lucia RIEDMANN (M.Sc.) (seit/since 05/08) Mag. Raphaela SCHWENTNER (M.Sc.) (seit/since 07/08) Diplomand (M.Sc. Student): Stefan NIEDAN (seit/since 03/08) Technische Mitarbeiterinnen (Technicians): Ing. Gunhild JUG Ing. Karin MÜHLBACHER Freiwillige Mitarbeiterin (Volunteer): DDr. Caroline HUTTER (M.D., Ph.D.) Historical survey Doz. Dr. Heinrich KOVAR The Molecular Biology - unit is, just like the Cytogenetics-unit, of fundamental importance to the Children s Cancer Research Institute. After having developed and implemented modern molecular-genetic and biochemical methods, we have devoted ourselves to the study of bone-tumors of early childhood and adolescence, especially to the study of Ewing-Sarkoma. Twenty years ago, this malignant cancer was considered very difficult to diagnose and little was known about its origin and the mechanisms of this disease. Our group achieved international recognition by detecting and characterizing the first feature which was defined on a molecular level (MIC2, nowadays known as CD99) and by contributing to the discovery of the mutation which causes Ewing Sarkoma: a chromosomal translocation which leads to a novel genefusion (EWS-FLI1). Today, both features are accepted world-wide as standard diagnostic parameters for this disease. Under the leadership of our group the relevance of the molecular detection of the EWS-FLI1 genefusion for the follow-up of the disease (minimal residual disease) has been thoroughly investigated by international committees and first results are now available. Yet apart from the descriptive aspect it has always been our aim to find out more about the functional aspect of the specific tumor-development and the clinical course of the disease and we have in fact been quite successful in evaluating some of the most important growth-inhibiting factors. Our attention has been directed to the central regulators p53 and Rb which are connected by the loss of the INK4A gene which, we realized, is the second most common molecular-genetic mutation in Ewing Sarkoma. Recent results even define a completely new mechanism of p35 inactivation which might in fact not only be extremely important for Ewing Sarkoma but for other types of cancer as well. In this case we have finally succeeded in describing the complex functional interrelation within the Ewing tumor cell, thus creating new potential therapeutic approaches. The Molecular-Biology - unit has always emphasized the importance of imparting knowledge and experience to young researchers, to raise scientific awareness in clinicians and to facilitate access to up-todate molecular technologies. And once we have moved into our new building where we will share one big lab with other research groups this intellectual interaction will even further increase

18 Gruppenbeschreibung Das Molekularbiologie Labor beinhaltet eine der größten Gruppen des Forschungsinstitutes, bestehend aus einem ständigen wissenschaftlichen Mitarbeiter, vier Postdoktoranden, zwei Dissertantinnen, einem Diplomanden, und zwei Technikerinnen. Zusätzlich beherbergen und unterstützen wir in unserem Labor eine junge klinische Mitarbeiterin, welche weitgehend unabhängig an einem interessanten Forschungsprojekt über Langerhans Zell Histiozytose arbeitet. Während der vergangenen drei Jahre konnte diese Teamgröße nur durch zwei EU-geförderte (PROTHETS und E.E.T.-Pipeline) und ein vom Wissenschaftsministerium unterstütztes nationales Kollaborationsprojekt (GEN-AU Ch.I.L.D.), sowie zwei Projekte des österreichischen Wissenschaftsfonds FWF und zwei Projekte der Österreichischen Nationalbank aufrecht erhalten werden. Die kollaborativen Projekte stärkten nicht nur die interne Zusammenarbeit an unserem Institut (R. Panzer, S. Strehl), sondern auch mit anderen nationalen (Tiroler Krebsforschungsinstitut) und mehreren internationalen Forschungseinrichtungen. Sie ermöglichten die Anwendung sehr teurer Hochdurchsatz Screening-Technologien auf die Modellsysteme, mit welchen wir Ewing Sarkome (ESFT) im Labor studieren. Das GEN-AU Mobilitätsprogramm eröffnete uns die Möglichkeit, einen Mitarbeiter (M. Kauer) für acht Monate zu einem vertiefenden Bioinformatiktraining an das National Cancer Institute (NCI, Genetics Branch, NIH, Bethesda) zu entsenden. Dies war auch der Anfang einer engen und fruchtbaren Zusammenarbeit mit dem Labor von Paul Meltzer am NCI. Ein erstes Ergebnis war die Erstellung zweier Genexpressionsdatenbanken für ESFT und für B-Zellvorläufer ALL (R. Panzers Gruppe), welche die im Haus generierten mitpublizierten Daten und einem Genexpressionsatlas von 79 Normalgeweben integrieren. Die ESFT Datenbank fokussiert auf die Funktion von EWS-FLI1, dem treibenden Onkogen in der ESFT Pathogenese. Sie basiert auf der experimentellen Ausschaltung von EWS-FLI1 in bisher 7 ESFT Zelllinien. Mit Hilfe dieser Modellsysteme konnten wir eine neue Funktion von EWS- FLI1 entdecken, welche zwei sehr wichtige Aktivitäten in der Krebsentwicklung miteinander in Verbindung bringt: den NOTCH Signalübertragungsweg und den Tumorsuppressor p53. Auch weisen preliminäre Ergebnisse auf die Beteiligung weiterer Mechanismen (TGFβ, IGF1, Wnt) hin, welche Signale von außen in das Zellinnere übertragen. Dies bewog uns Untersuchungen einzuleiten, welche den Einfluß der unmittelbaren Tumorumgebung und der Interaktion von Wirt und Tumor für die ESFT Pathogenese zum Inhalt haben. Unter anderem wurden zwei Projekte begonnen, die sich mit den Auswirkungen von Hypoxie und von Chemokinen auf ESFT befassen. Weiters erbrachte die bioinformatische Auswertung der ESFT Datenbank wertvolle Hinweise auf den Mechanismus der EWS-FLI1 Funktion, in dem sie Interaktionen mit kooperierenden Transkriptionsfaktoren auf EWS- FLI1 regulierten Genen offenlegte. In der Folge wurden Studien eingeleitet, die die spezifische Rolle von E2F und MYC für die ESFT Pathogenese untersuchen. Diese geben ein Beispiel für den hohen wissenschaftlichen Wert unserer ESFT Datenbank als eine wichtige Ressource, die von allen Labormitgliedern genutzt wird und ein unverzichtbares Instrument in all unseren Forschungsvorhaben geworden ist. Langfristig ist es unsere Absicht, die Bioinformatik als Service für alle Mitarbeiter des CCRI zu etablieren. Group description The molecular biology lab comprises one of the biggest groups of the CCRI including a staff scientist, four postdoctoral research fellows, two PhD students, one diploma student, and two technicians. In addition, we are mentoring and providing space to a young clinician who is independently working on an exciting Langerhans cell histiocytosis research project. During the last 3 years, maintenance of this group size was only possible through support by two collaborative EU funded research programs (PRO- THETS and E.E.T.-Pipeline), a national collaborative grant from the Austrian Ministery of Science (GEN-AU-Ch.I.L.D.), two individual grants by the Austrian Science Fund FWF, and two grants by the Austrian National Bank. The collaborative grants strengthened our in-house (i.e. R. Panzer and S. Strehl), national (Tyrolean Cancer Research Institute, R. Kofler), and several international co-operations. These funding sources were instrumental to the application of very expensive high-throughput genetic screening technologies to the model systems used in our studies on Ewing s sarcoma family tumors (ESFT). In addition, the GEN-AU mobility program enabled an 8-months training in bioinformatics of a lab member (M. Kauer) at the Genetics Branch of the National Cancer Institute (NCI, Bethesda, MD). This was also the starting point of a close and fruitful collaboration with the Meltzer lab at the NCI. As a first result, user friendly, easily searchable expression data bases were established for ESFT and for BCP-ALL (R. Panzer s group), combining in-house generated with published data and with a gene expression atlas of 78 normal tissues. The ESFT database focuses on the function of EWS-FLI1, the driving oncogene in ESFT pathogenesis. It is based on experimental silencing of EWS-FLI1 expression in, so far, 7 ESFT cell lines. Using these model systems we revealed a novel activity of EWS-FLI1 linking two prominent players in cancerogenesis, the NOTCH signalling pathway with the tumor suppressor p53. We also obtained preliminary evidence for the involvement of other pathways conferring outside signals to the inside of the cell (TGFβ, IGF1, Wnt), leading us to the investigation of the role of the tumor micro-environment and of tumor-host interactions for ESFT pathogenesis. Studies have been initiated evaluating the consequences of hypoxia and the role of chemokine signalling in ESFT. The bioinformatic mining of the ESFT database also yielded hints to the mechanism of EWS-FLI1 function by revealing transcription factor interactions on target gene promoters. Therefore, studies have been initiated to investigate the specific roles of E2F and MYC in ESFT pathogenesis. These give an example for the high scientific value of our ESFT database as a resource, which is used by all lab members and serves as an indispensible tool in all ongoing research activities of our lab. In the long run, it is our intention to establish bioinformatics as a service to all CCRI members in our institute

19 Mechanisms of EWS-FLI1 mediated gene regulation ESFT arise from a still undefined tissue. The unifying rate limiting mutation in ESFT is a fusion between the RNA-processing factor EWS and the ETS transcription factor FLI1. EWS-FLI1 is a DNA-binding protein with strong transcription stimulating activity. However, its in-vitro DNA binding specificity is not different from most other ETS factors, and transcriptionindependent functions have been proposed to contribute to the oncogenic activity of the fusion protein based on mutational analysis of the DNA binding domain [1]. To assess the mechanism of EWS-FLI1 mediated oncogenesis, we studied the genome-wide functional consequences of EWS-FLI1 silencing in 7 ESFT cell lines by stable and inducible RNA-interference. We hypothesized that most of the gene expression differences between the tissue of origin and ESFT should be related to EWS- FLI1 as the driving force in this disease, and thus gene expression in ESFT after EWS-FLI1 knockdown should be inversely correlated to gene expression in the candidate normal counterpart tissue. We found that mesenchymal stem cells (MSC) best fitted this assumption and corroborated the conclusions of a previous study [2]. Taking MSC as a reference tissue, we identified 344 and 237 genes to be consistently up- and down-regulated in the EWS-FLI1 knockdown and in primary tumors at a false discovery rate of < At a stringent cut-off (p=0.1), although significant, the overlap of this set of EWS-FLI1 activated and repressed genes with a recently published ESFT signature [3] was only 33% and 21%, respectively, and many more EWS-FLI1 regulated genes were identified. The functional annotation of these gene sets revealed that EWS-FLI1 activated genes are mainly involved in cell cycle, proliferation and repair, while repressed genes are related to differentiation and cell communication (M. Kauer, submitted). Studying the kinetics of the response to EWS-FLI1 modulation in an inducible ESFT cell line model, we further identified a difference in promoter structure of activated and repressed genes and obtained hints to the involvement of a small set of cooperating transcription factors in EWS-FLI1 mediated transcriptional activation, which are currently under study in a collaboration with P. Meltzer s lab (M. Kauer et al., manuscript in preparation). Cell cycle regulation in ESFT is also in the center of our contribution to the EU-funded project E.E.T.-Pipeline. So far, we found that part of the EWS-FLI1 transcriptional activity can be contributed to MYC acting downstream of and in a feed-back loop with EWS-FLI1. Importantly, we found that MYC overexpression can rescue ESFT cell viability in the absence of EWS-FLI1. Since EWS-FLI1 is considered the ideal therapeutic target which is however difficult to hit, our studies contribute to the identification of critical downstream pathways for future therapeutic intervention strategies. [1] Jaishankar S., Zhang J., Roussel M.F., Baker S.J. Transforming activity of EWS/FLI is not strictly dependent upon DNA- binding activity. Oncogene 1999;18: [2] Tirode F., Laud-Duval K., Prieur A., Delorme B., Charbord P., Delattre O. Mesenchy mal stem cell features of Ewing tumors. Cancer Cell 2007;11: [3] Hancock J.D., Lessnick S.L. A transcriptional profiling meta-analysis reveals a core EWS-FLI gene expression signature. Cell Cycle 2007;7. Linking EWS-FLI1 to the p53 tumor suppressor pathway Although the tumor suppressor p53 is widely mutated in human cancer and is therefore considered a central target generally impaired in cancerogenesis, about 50% of human malignancies express wildtype p53. In ESFT, p53 mutations occur at a frequency of less than 10% and are associated with bad prognosis [1]. However, p53 is mutated in more than 50% of ESFT cell lines. Therefore, EWS-FLI1 function has so far been studied exclusively in a p53 mutant background. In contrast, our ESFT cell line panel for EWS-FLI1 knockdown studies contained four wildtype p53 cell lines. In these, we identified consistent upregulation of p53 protein expression upon modulation of EWS-FLI1 by RNA interference. Studying the mechanism and consequences of p53 induction, we revealed the involvement of the NOTCH signaling pathway leading to p53 dependent upregulation of the cyclin dependent kinase inhibitor CDKN1Ap21/WAF1 and cell cycle arrest. Thus we identified a novel tumor suppressive function of NOTCH receptor signaling in ESFT, which is otherwise well known to be oncogenic in several other malignancies specifically in acute lymphoblastic leukemia (ALL). We identified the NOTCH ligand JAG1 and the NOTCH regulated transcription factor HEY1 as the key components suppressed by EWS-FLI1. Importantly, we found that providing a NOTCH ligand to the ESFT cell sufficed to reactivate the growth inhibitory function of NOTCH in the presence of EWS-FLI1, suggesting a growth regulatory role of the ESFT micro-environment (Ban et al, 2008). We also obtained preliminary evidence for the involvement of other signaling pathways (TGFβ, IGF1, Wnt) in p53 regulation. Therefore, current investigations aim at elucidating the complex interplay of these pathways and their targets within the p53 cell cycle regulatory network in ESFT. The relevance of our results is likely not restricted to ESFT, but also applicable to a plethora of other malignancies with wildtype p53 and NOTCH tumor suppressive function. Given the current involvement of many pharmaceutical companies in the development of p53 and NOTCH pathway targeting drugs, our studies may contribute to the development of novel treatment strategies for ESFT. [1] Huang H.Y., Illei P.B., Zhao Z., et al. Ewing sarcomas with p53 mutation or p16/p14arf homozygous deletion: a highly lethal subset associated with poor chemoresponse. JClinOncol 2005;23: Post-transcriptional and structural regulation of EWS-FLI1 in Ewing s sarcoma Transcription factor activity is frequently regulated by post-translational modifications (PTMs) integrating cellular and environmental signals and affecting protein structure. The EWS moiety of EWS-FLI1 is intrinsically unstructured and, therefore, protein crystallization efforts have been unsuccessful. So far, nothing has been known about PTMs of EWS-FLI1, while we know about various modifications of native EWS and little about FLI1. Previous studies by us and others [1] suggested distinct protein interactions of the EWS N-terminus in the context of native and rearranged EWS proteins. We generated single chain antibody fragments (scfv) against recombinant EWS-FLI1 from a combinatorial synthetic phage display library and found that all isolated specific scfvs recognized the same N-terminal epitope on the bacterially expressed recombinant EWS-FLI1 protein in the presence of mild detergents. This epitope was found to be easily accessible on native EWS from human cells, but remained inaccessible to the scfvs on EWS-FLI1 from ESFT cells, suggesting that PTMs might stabilize the N-terminal domain in a different structure in the context of the rearranged protein. While the scfvs proved to be a useful tool to inhibit the transcriptional co-activator function of native EWS in HNF4 and OCT4 mediated transcription, they failed to interfer with EWS-FLI1 transcriptional activity in ESFT (Aryee, 2006, Cancer Res). These results prompted us to investigate PTMs on EWS-FLI1. We identified O-linked N-Acetylglucosaminylation (GlcNAc) and phosphorylation of the fusion protein in the EWS N-terminus, and found that blocking phosphatase activity decreased the level of GlcNAc modification, suggesting a functional interplay of these two PTMs. Importantly, among EWS-FLI1 targets, blocking GlcNAcylation by the chemical inhibitor DON decreased EWS-FLI1 dependent transcriptional activation of Id2, while it did not affect transcriptional repression of TGFβR2 (Bachmaier et al., Oncogene, in revision). These results suggest that targeting of PTMs or of pathways involved in PTM might be considered as a therapeutic means to interfer with EWS-FLI1 function in ESFT. [1] Petermann R., Mossier B.M., Aryee D.N., Khazak V., Golemis E.A., Kovar H. Oncogenic EWS-Fli1 interacts with hsrpb7, a subunit of human RNA polymerase II [In Process Citation]. Oncogene 1998;17: Microenvironmental factors affecting Ewing s sarcoma progression Despite available effective treatments, most children suffering from metastatic ESFT die. The metastatic process is poorly understood in ESFT, and metastatic markers that would make early detection and treatment possible, are not available. It is likely that tumor-host interactions play a critical role in tumor spread and proliferation at metastatic sites. Using an unbiased, hypothesis-free approach, we surveyed the expression of more than genes in 3 non-metastatic and 3 metastatic ESFTderived cell lines. Only one gene showed a strict correlation with metastatic status, the chemokine receptor CXCR4. There is a perfect overlap between ESFT sites of metastasis, and tissues and organs that express the highest levels of CXCL12, CXCR4's only described ligand. Since CXCR4 is known to induce cell motility in-vitro and to promote metastasis in-vivo in all tumors and experimental models reported to date, and since CXCR4 expression correlates with metastasis and low survival rates in patients with tumors of various origins and etiologies, we are currently evaluating in a collaboration with C. Poremba (Düsseldorf) CXCR4 expression on tissue micro arrays (TMAs) of almost 60 clinically well documented primary tumors of different stages. Simultaneously, we also test for the expression of hypoxiainducible factor-1 (HIF-1). Hypoxia is an important phenomenon in the tumor cell microenvironment. While ESFT possess all the attributes of hypoxic tumors, studies on hypoxic factors in its histopathogenesis are lacking. The consequences of hypoxiainduced or hypoxia-repressed gene expression have important implications in several disease processes including tumor development. Tumor hypoxia is associated with a more aggressive phenotype. It is thought that this phenotype is the result of the hypoxic induction of a number of genes, the majority of which are regulated by HIF-1. While HIF-1 plays a major role in controlling the ubiquitous transcriptional response to hypoxia, it is clear that a number of other transcription factors are also activated either directly or indirectly. Here, we study the consequences of low oxygen tension on ESFT gene expression, proliferation, survival and migration. Preliminary results revealed that hypoxia induces EWS-FLI1 protein accumulation in a time- and dosedependent manner in ESFT cells. The accumulation of EWS-FLI1 under hypoxia was found to be HIF1α dependent and to influence the expression of downstream target genes as shown by gene expression profiling. A subset of genes modulated by hypoxia is dependent on EWS-FLI1 for hypoxic modulation. Thus, we postulate that EWS-FLI1 function is at least partially dependent on microenvironmental factors. Correlation of the in-vitro findings to primary tumors of different localizations and stages by screening for HIF1α expression on TMAs is currently done in an ongoing research project. Origin of Langerhans cell histiocytosis This project aims to elucidate pathogenetic mechanisms in Langerhans cell histiocytosis (LCH). The aetiology of LCH is unknown and it is even debated whether LCH is a neoplastic disease or merely represents an aberrant immune response. The disease is characterized by clonal proliferation and dissemination of cells that display much of the surface antigen profile of epidermal Langerhans cells (LC), which are considered to be the closest relatives if not progenitors of LCH histiocytes. LCH research was hampered particularly by the minute amounts of tissue specimens that were available for scientific purpo

20 ses. We have developed a high yield protocol for the isolation of highly purified LCH cells from small amounts of biopsy material. In collaboration with Dr. E. Kriehuber (Dept. Dermatology, Medical University, Vienna) we succeeded in hybridizing LCH and LC samples to Affymetrix human genome expression arrays in order to get a comprehensive view of the transcriptome of LCH cells. We found that the LCH samples were relatively homogeneous regarding their expression pattern and that in comparison to normal Langerhans cells, around 3000 genes are selectively up- or downregulated in LCH cells. Using bioinformatics tools, we identified a number of signalling pathways as being activated in LCH cells. One of these pathways is the NOTCH pathway, a highly conserved cell signalling system essential for development and implicated in a number of human diseases. We are therefore investigating the effect of NOTCH activation on LC differentiation in an in vitro model using NOTCH-ligand expressing stromal cells. Finally, we are trying to place LCH cells in context with other DC subsets, which we believe is essential to understanding the distinctive properties of the LCH cells. Our preliminary cluster analysis of microarray data show that the LCH cell transcriptome overlaps at least to a certain extent with other DC subsets. We are therefore currently analyzing different primary human DC subsets using Affymetrix arrays. S 2 IRP STUDIEN UND STATISTIK STUDIES AND STATISTICS for Integrated Research and Projects Gruppenleiterin (Group Leader): Statistikerin (Statistician) Wiss. Assistentin (Scientif. Assistant) Univ. Doz. Dr. Ruth LADENSTEIN (M.D.) Mag. Ulrike PÖTSCHGER (M.Sc.) Mag. Claudia ZEINER (M.Sc.) FSA/RSA: Forschungs- und StudienassistenInnen/Research and Study Assistents SA: Wissenschaftliche Assistentin/Scientific Assistant CC: Klinischer Studien-Leiter oder Mitarbeiter/Clinical Studies Leader and Collaborator CS: Freie Mitarbeiter/Collaborating Physicians and Students Univ. Doz. Dr. Ruth LADENSTEIN Leukämie und Lymphom Studien (leukaemia and lymphoma studies) FSA: Mag. Dasa JANOUSEK (M.Sc.) Mag. Nora MÜHLEGGER (M.Sc.) CC: Univ. Prof. Dr. Helmut GADNER (M.D., Professor) Dr. Georg MANN (M.D.) (ALL und Lymphom Studien) Univ. Doz. Dr. Andishe ATTARBASHI (M.D., Assoc. Professor) (ALL und Lymphom Studien) Univ. Doz. Dr. Michael DWORZAK (M.D., Assoc. Professor) (AML/MDS/CML Studien) Studien Solider Tumore (solid tumour studies) FSA: Cornelia SAX (M.Sc) CC: Univ. Doz. Dr. Ruth LADENSTEIN (M.D., Assoc. Professor) (Neuroblastome, Weichteilsarkome) Univ. Doz. Dr. Andreas ZOUBEK (M.D., Assoc. Professor) (Knochentumore, Nephroblastome) Univ. Doz. Dr. Leo KAGER (M.D., Assoc. Professor) (Osteosarkome) Langerhanszell Histiozytose Studien (LCH-studies) FSA: Elfriede THIEM Marek NYKIEL CS: Dr. Helmut PROSCH Dr. Bernhard FAHRNER Dr. Martha WNOROWSKI CC: Univ. Prof. Dr. Helmut GADNER (M.D.) Dr. Nicole GROIS (M.D.) Dr. Milen MINKOV (M.D.) Stammzelltransplantation Dokumentation und Studien (stem cell transplantation documentation and studies) FSA: Mag. Inge HIRSCH (M.Sc.) Dr. Susanne KARLHUBER (Ph.D.) CC: Univ. Doz. Dr. Christina PETERS (M.D., Assoc. Professor) Allogene SZT Univ. Doz. Dr. Susanne MATTHES-MARTIN (M.D., Assoc. Professor) Allogene SZT Univ. Doz. Dr. Ruth LADENSTEIN (M.D., Assoc. Professor) Autologe SZT 32 33