- Children`s Cancer Research Institute
Transcription
- Children`s Cancer Research Institute
St. Anna Children’s Cancer Research Institute 25 Years. Science Report 2011–2012 St. Anna Kinderkrebsforschung 25 Jahre. Forschungsbericht 2011–2012 St. Anna Children’s Cancer Research Institute 25 Years. Science Report 2011–2012 St. Anna Kinderkrebsforschung 25 Jahre. Forschungsbericht 2011–2012 Quotes by Helmut Gadner Extracts from past Science Reports 25 Years / 25 Jahre A Brief Review Ein kurzer Rückblick Extracts from past CCRI Science Reports Auszüge aus vergangenen Jahresberichten der St. Anna Kinderkrebsforschung Helmut Gadner, Director of the Institute: Quotes from an Interview in October 2012 Helmut Gadner, Institutsleiter: Zitate aus einem Interview im Oktober 2012 Time Travel through Iconic Images Eine Zeitreise in Bildern Quotes by Helmut Gadner Extracts from past Science Reports Quotes by Helmut Gadner Extracts from past Science Reports 25 Years / 25 Jahre Among all types of malignant childhood cancer we had to deal with, leukaemia was certainly the biggest challenge. Leukämie war unter den bösartigen Erkrankungen im Kindes- und Jugendalter damals diejenige, wo es den größten Handlungsbedarf gab. After successful implementation of a novel treatment concept for children with cancer, I knew that we would need to see further progress. Being the Medical Director of the St. Anna Children’s Hospital, in 1985, I realised that all our therapeutic possibilities seemed to have hit the end of the road. Nach erfolgreicher Einführung eines neuen Behandlungskonzeptes für krebskranke Kinder habe ich als ärztlicher Direktor des St. Anna Kinderspitals im Jahr 1985 gewusst, dass wir noch mehr tun müssen, um weiter zu kommen. Die therapeutischen Möglichkeiten führten in eine Sackgasse. Thanks to the continuous financial support of our donors and various funds, it was possible to establish seven different research groups in the five labs that were available. Dank anhaltendem Spendenfluss und Unterstützung durch diverse Fonds war es möglich, in den fünf zur Verfügung stehenden Labors sieben Arbeitsgruppen zu beschäftigen. (Science Report 1990) After only one year of construction time, and financed exclusively by private donations, the Children’s Cancer Research Institute went operational in June 1988. Das Forschungsinstitut für krebskranke Kinder wurde im Juni 1988 nach einer einjährigen Bauzeit, deren Kosten ausschließlich aus Spenden der Öffentlichkeit getragen worden sind, seiner Bestimmung übergeben. (Science Report 1989) Quotes by Helmut Gadner Extracts from past Science Reports Quotes by Helmut Gadner Extracts from past Science Reports 25 Years / 25 Jahre In some areas, discoveries made at the CCRI have already been translated into new treatment strategies. In einzelnen Bereichen ist es gelungen, Erkenntnisse, die im Forschungsinstitut gewonnen wurden, in die Etablierung neuer Behandlungsstrategien umzusetzen. (Science Report 1994) The close cooperation of clinicians and researchers proves to be extremely beneficial to the patients. Die enge Zusammenarbeit von Klinikern und Forschern, wenn es um Belange des Patienten geht, hat sich hervorragend bewährt. (Science Report 1995/96) The primary goal of the newly founded institute was the improvement of diagnostics using novel technologies. In addition, we wanted to establish basic research and conduct clinical trials. Our expectations were exceeded in every respect. Die wichtigste Aufgabe des neu gegründeten Forschungsinstituts war die verbesserte Diagnostik mittels neuer Technologien. Zudem wollten wir auch die Grundlagenforschung etablieren und klinische Studien durchführen. Diese Ziele wurden bereits in wenigen Jahren mehr als übertroffen. In 1992 the CCRI’s company profile changed: applied research, sophisticated diagnostics, and basic research are now equally important. Im Jahr 1992 vertiefte sich das für das Forschungsinstitut charakteristische Profil: angewandte Forschung, verfeinerte Diagnostik und Grundlagenforschung stehen gleichberechtigt nebeneinander. (Science Report 1992) Quotes by Helmut Gadner Extracts from past Science Reports Quotes by Helmut Gadner Extracts from past Science Reports 25 Years / 25 Jahre ...the research activities of the CCRI are based partly on the work of diploma and PhD students from different disciplines…Thus we perform an important role in the training of future researchers... ... so baut die wissenschaftliche Arbeit zu einem guten Teil auf der Mitarbeit und dem Einsatz von Studenten … aus verschiedenen Disziplinen auf ... Damit übernimmt das Forschungsinstitut auch eine große Aufgabe in der Ausbildung dieser angehenden Wissenschafter ... (Science Report 2000/01) ...the fast development in haemato-oncology, which was observed in the last decade world-wide, led to a further diversification of the questions in this field also in our Institute...the transfer of examinations, which have in the meantime become routine examinations, to an especially equipped laboratory... ... weltweit beobachtete rasante Entwicklung auf dem Gebiet der Hämato-Onkologie hat auch in unserem Institut dazu geführt, dass eine weitere Diversifizierung der Fragestellungen aufgetreten ist … Auslagerung von inzwischen in Routine übergangenen Untersuchungen in ein speziell dafür eingerichtetes Labor ... (Science Report 1998/99) Quotes by Helmut Gadner Extracts from past Science Reports Enduring commitment of each individual research group builds the institute’s mission statement. The steadily increasing number of external grants makes the visions of CCRI scientists become reality. Das Engagement der einzelnen Forschungsgruppen ist heute das Leitbild des Instituts. Die zunehmenden Erfolge in der Einwerbung von Drittmitteln zeigen, dass man hier am CCRI sehr bemüht ist eigene Visionen zu realisieren. ...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...and international...cluster projects...as a result, it was possible to initiate in-house bioinformatic support ... konnte der Anteil extern finanzierter Projektmittel auf 38% gehoben werden was zu einem guten Teil auf die Teilnahme an nationalen … und internationalen …Kooperationsprojekten zurückzuführen ist ... in der Folge die Bioinformatik als Unterstützung im eigenen Haus zu etablieren ... (Science Report 2006/07/08) Quotes by Helmut Gadner Extracts from past Science Reports Good networking and international collaborations are still essential to make the best therapies available to young cancer patients. Gute Vernetzung sowie nationale und internationale Zusammenarbeit sind nach wie vor essentiell, um die Forschung weiter voran zu treiben und jungen Krebspatienten die beste Therapie zu ermöglichen. 25 Years / 25 Jahre Quotes by Helmut Gadner Extracts from past Science Reports Quotes by Helmut Gadner Extracts from past Science Reports Zukunftsweisend sind personalisierte Therapien sowie Genomanalysen, die uns Einblicke in die Ursachen einer bestimmten Krebserkrankung ermöglichen. In the future, genome analyses will enable us to further personalise therapies and reveal the mechanisms of cancer development and progression. Reflections on the past 25 years of children’s cancer research point to a promising future: There are many open questions that still need to be answered. Die Rückschau auf 25 Jahre Kinderkrebsforschung lässt uns hoffnungsvoll in die Zukunft blicken: Es sind noch so viele Fragen offen. ...In January 2009 we were able to relocate our research laboratories into the newly-constructed institutional building...it also opened up new paths as regards translational and applied research...more third-party funding than in previous years...networks enabled our researchers to gain access to very expensive genomic and post-genomic high-throughput technology... ... Im Jänner 2009 konnten die Forschungslabors in das neu errichtete Institutsgebäude … übersiedeln ... dies eröffnet neue Wege in der translatorischen und angewandten Forschung ... mehr Drittmittelgelder als in den Jahren zuvor ... Zugang zu sehr teuren genomischen und postgenomischen Hochdurchsatz-Technologien ... (Science Report 2009/10) My wish is that this institute can sustain its outstanding reputation by keeping up with novel developments and expanding its research efforts. Ich wünsche mir, dass das Institut auch in Zukunft mit dem starken Aufwärtstrend der letzte Jahre Schritt halten kann und die Forschung hier so weiter besteht wie bisher. 25 Years / 25 Jahre 3 25 Years Children’s Cancer Research Institute A Brief Review 16Preface Research Report Leukaemias 20 Immunological Diagnostics Michael N. Dworzak 24 Biology of Leukaemias Renate Panzer-Grümayer 30 Genetics of Leukaemia Sabine Strehl 36 42 Solid Tumours Tumour Biology Peter F. Ambros Molecular Biology of Solid Tumours Heinrich Kovar Immunology 48 Tumour Immunology Thomas Felzmann 54 Clinical Cell Biology and FACS Core Unit Gerhard Fritsch 60Transplantation-Immunology Andreas Heitger 66 Development of Cellular Therapeutics Wolfgang Holter 72 Molecular Microbiology and Labdia Labordiagnostik GmbH Thomas Lion 80 Clinical Research S2IRP, Studies & Statistics of Integrated Research and Projects Ruth Ladenstein 92 Services, Administration, PR and Donations Department Respective Departments Appendix 95 Scientific Advisory Board 96Awards 97 Completed MSc Diplomas and PhD Theses 98 External Grants and Research Funding Bodies 102Publications 110Imprint 111Acknowledgements 3 25 Jahre St. Anna Kinderkrebsforschung Ein kurzer Rückblick 17Vorwort Forschungsbericht Leukämien 20 Immunologische Diagnostik Michael N. Dworzak 24 Biologie der Leukämien Renate Panzer-Grümayer 30Leukämiegenetik Sabine Strehl Solide Tumoren 36Tumorbiologie Peter F. Ambros 42 Molekularbiologie Solider Tumoren Heinrich Kovar Immunologie 48Tumor-Immunologie Thomas Felzmann 54 Klinische Zellbiologie und FACS Core Unit Gerhard Fritsch 60Transplantations-Immunologie Andreas Heitger 66 Entwicklung zellulärer Therapeutika Wolfgang Holter 72 Molekulare Microbiologie und Labdia Labordiagnostik GmbH Thomas Lion 80 Klinische Forschung S2IRP, Studien und Statistik Ruth Ladenstein 92 Service-Einrichtungen, Administration, PR und Spendenabteilung Entsprechende Abteilungen Anhang 95 Wissenschaftlicher Beirat 96Preise 97 Abgeschlossene Diplomarbeiten und Dissertationen 98 Extern geförderte Projekte und Fördergeber 102Publikationen 110Impressum 111Danksagung While we are all very much looking forward to a new era in the history of this institute, we are also looking back with gratitude on those years in which Prof. Gadner nursed the CCRI from a mere idea in his head to an internationally renowned research institute that gives children with cancer in Austria access to research results, as well as state-of-the art diagnostics and treatments. In these past years, biomedical research has achieved significant progress and experienced several technological revolutions. The CCRI has always kept up with these important developments, and has contributed significantly to the translation of these achievements into paediatric cancer research. This also holds true for the period 2011/12, documented in this bi-annual report. The adaptation of modern genomic and biological concepts of cancerogenesis, immune surveillance, and targeted therapy to the specifics of childhood cancer is achieved by a highly motivated multidisciplinary team of CCRI researchers from different countries, of which ambitious graduate and PhD students form an indispensable part. Guided by our experienced group leaders and postdocs, several of these students successfully completed their theses in the reporting period. The strength of CCRI research is based on its close proximity to the clinics at St. Anna Kinderspital. To further strengthen the practical and intellectual relationship between these two institutions, we have installed an in-house program especially supporting clinical-translational research driven by treating physicians from the hospital. In addition, the area of clinical research has further expanded, and the CCRI has taken leadership of a European network (ENCCA) to improve multi-centric clinical and translational research through collaboration, coordination, and communication with European health authorities. ENCCA is only one example of several, in which CCRI researchers successfully applied to the European commission for funding of collaborative initiatives on an international level. This success has been greatly facilitated by our dedicated research support office which has been of invaluable help in the successful recruitment of a considerable number of competitive grants supporting high-end research at the CCRI. These grants and the continuous support of our donors and mentors allow the CCRI to continue growing and prospering. A new research group, working on the development of cellular therapeutics, was established with the arrival of the new director of the institute. The increasing needs for bioinformatics analyses of high throughput genomics data and for innovative in vivo models is met by the recruitment of an additional data analyst and a new junior research group, respectively, in 2013. In order to fulfill our responsibility towards our donors, we keep them informed on research outcomes and their application to the benefit of sick children and for this reason have also invested in a science communication team. This report is the most recent product of their important work. Heinrich Kovar Scientific Director Es ist erstaunlich: 2013 feiert das Children’s Cancer Research Institute (CCRI) bereits seinen 25. Geburtstag. Von Spenden und vielen kompetitiven Projektförderungen genährt, unterstützt von anhaltendem Wohlwollen wuchs das Institut heran. Ende 2012 ist nun der Gründungsvater und langjährige Leiter des CCRI, Prof. Helmut Gadner, in den wohlverdienten Ruhestand getreten, und ein neuer Direktor, Prof. Wolfgang Holter, hat das Institut übernommen. So markiert 2012 das Ende einer bemerkenswerten, fruchtbaren und auf regenden Periode in der Entwicklung des CCRI. Während wir uns schon sehr neugierig auf eine neue Ära in der Geschichte des Institutes freuen, blicken wir auch voll Dankbarkeit auf die Jahre zurück, in denen Prof. Gadner das CCRI von einer Idee in seinem Kopf zu einer international höchst anerkannten Forschungseinrichtung geführt hat, die krebskranken Kindern in Österreich Zugang zu den neuesten wissenschaftlichen Ergebnissen in Diagnostik und Therapie ermöglicht. In diesen vergangenen Jahren machte die biomedizinische Forschung signifikante Fortschritte und erlebte mehrere technologische Revolutionen. Das CCRI hat immer mit diesen Entwicklungen Schritt gehalten und trug maßgeblich zur Umsetzung dieser Errungenschaften für die Erforschung von und den Einsatz bei Krebserkrankungen des Kindesalters bei. Das gilt auch für die Periode 2011/12, die in diesem Jahresbericht dokumentiert wird. Die Anpassung moderner genomischer und biologischer Konzepte der Krebsentstehung, Immunüberwachung und gezielter Therapien an die speziellen Eigenheiten von Kinderkrebserkrankungen wird von einem hoch motivierten, multidisziplinären Team von CCRI Wissenschaftlern getragen, in dem ambitionierte Diplom-/Master und DissertationsstudentInnen eine wesentliche Rolle spielen. Angeleitet von erfahrenen GruppenleiterInnen und PostdoktorandInnen konnten mehrere StudentInnen ihre Dissertation im Berichtszeitraum abschließen. Die Stärke der Forschung am CCRI liegt in der großen Nähe zum St. Anna Kinderspital. Um die enge Zusammenarbeit von Klinikern und Forschern weiter zu stärken, haben wir ein Programm zur Förderung von klinisch-translationalen Projekten eingerichtet, welche durch behandelnde Ärzte des Kinderspitals vorangetrieben werden. Zudem wurde die klinische Forschung weiter ausgedehnt, und das CCRI übernahm die Führung eines europäischen Netzwerkes (ENCCA), dessen Ziel die Verbesserung multi-zentrischer klinischer und translationaler Studien durch Zusammenarbeit, Koordination und Kommunikation mit europäischen Gesundheitsbehörden ist. ENCCA ist nur ein Beispiel von mehreren, in denen CCRI Wissenschaftler erfolgreich Unterstützung für kollaborative Initiativen auf internationaler Ebene von der Europäischen Kommission einwerben konnten. Dieser Erfolg wurde durch die Einrichtung eines „Research Support Office“ erleichtert, welches von unschätzbarer Hilfe in der Akquisition einer beein druckenden Anzahl kompetitiver Drittmittelprojekte war. Diese Projekte und die fortgesetzte Unterstützung unserer SpenderInnen und MentorInnen ermöglichten ein weiteres Wachstum des CCRI. Mit der Ankunft des neuen Institutsdirektors wurde eine neue Arbeitsgruppe, die sich mit der Entwicklung zellulärer Therapeutika beschäftigt, etabliert. Der steigende Bedarf an bioinformatischen Analysen von Hoch durchsatz genomischen Daten sowie von innovativen in vivo Modellen erfordert 2013 sogar die Einstellung eines weiteren Datenanalysten und Anwerbung einer jungen neuen Arbeitsgruppe. Um der Verantwortung gegenüber unseren SpenderInnen gerecht zu werden, informieren wir sie über den Einsatz unserer Mittel für Forschung und deren Anwendung zum Wohl kranker Kinder und haben dafür auch in ein Wissenschafts kommunikationsteam investiert. Der vorliegende Jahresbericht ist das bislang letzte Produkt seiner wichtigen Arbeit. Heinrich Kovar Wissenschaftlicher Leiter 16–17 It is amazing: In 2013, the Children’s Cancer Research Institute celebrates its 25th anniversary. Bottle-fed by donations, supported by many benefactors, and raised on competitive grants and continuing philanthropy, the CCRI has come of age. At the end of 2012, the founding father and head of the CCRI, Prof. Helmut Gadner, retired from the directorship of the institute, and a new director, Prof. Wolfgang Holter, took over. Thus, 2012 marked the end of a remarkably fruitful and exciting era in the development of the CCRI. Vorwort St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Preface Research Report Forschungs bericht Leukaemias Immunological Diagnostics Leukämien Immunologische Diagnostik Group leader Michael N. Dworzak [email protected] Staff scientist Zvenyslava Husak Diploma/MSc student Romana Breitler1 Technician Angela Schumich Susanne Suhendra2 1 since Dec. 2012 2 since Aug. 2011 Our group’s research focus resides in developing and validating new diagnostic methods based on flow cytometry – mainly in the field of childhood leukaemia and lymphoma, which make up about 50% of all cancer cases in children and adolescents. Our working directions embrace investigations into disease-associated peculiarities of protein expression which can be exploited clinically for elaborate diagnostics [1], risk stratification and treatment tailoring to individual needs [2]: e.g. highly sensitive and specific detection of minimal residual disease in acute lymphoblastic as well as myeloid leukaemia1. In an additional but related focus we also aim at supporting novel therapeutic options against childhood leukaemias with new diagnostic approaches, e.g. by deciphering the activated signalling network of leukaemic cells potentially relevant for patient-tailored signal transduction inhibitor treatment2, or by unravelling signal regulation towards augmenting anti-leukaemic defence by the innate immune system [3]. 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 [1], Prognoseevaluation, Risikostratifizierung und indivi dualisierte Behandlungsplanung [2], basierend vor allem auf hochsensitiver und spezifischer Detektion minimaler Resterkrankung sowohl bei akuter lympho blastischer wie auch bei myeloischer Leukämie1. Andererseits beschäftigen wir uns auch mit leukämie biologischen Fragestellungen2. Wir analysieren die Zellsignalwegaktivierung in Hinblick auf den patienten gerechten Einsatz neuer Medikamente (Signal weginhibitoren) bzw. auf neue Möglichkeiten, die antileukämische Immunabwehr zu stimulieren [3]. CD99, an antigen implicated in cell survival and adhesion, is strongly expressed on Ewing tumours and on acute leukaemias. We previously found that modulation of this antigen induces cell death in TEL/AML1-positive ALL [1]. We also observed that CD99-modulation up-regulates the heat shock protein hsp70 in leukaemic cells. Based on these findings, we speculated that CD99-mediated hsp70 up-regulation would induce NK-cell cytotoxicity. In our subsequent investigations supported by the Austrian National Bank1 we demonstrated that CD99-mediated hsp70 up-regulation induces increased NK-cell cytotoxicity against ALL cells, both in primary samples and cell lines, but only weakly if at all against Ewing tumour cell lines [2]. This observation puts CD99-targeting forward as a potential tool to augment defence mechanisms of the immune system against ALL. 1 ENCCA grant 261474 (2011–2014) 2 WWTF grant Nr. LS07–037 (2008–2013) 1 ENCCA grant 261474 (2011–2014) 2 WWTF grant Nr. LS07–037 (2008–2013) In collaboration with Prof. Giusepe Basso, Laboratory of Pediatric Onco-Hematology, University of Padova, Italy. Prof. Andrea Biondi and Dr. Giuseppe Gaipa, Tettamanti Research Center, Monza – University Milano-Bicocca, Italy. Dr. Richard Ratei and Dr. Leonid Karawajew, Zellmarkerlabor, RRK, HELIOS Klinikum Berlin, Charite MS, Berlin, Germany. Prof. Dirk Reinhardt, Abteilung für Kinderheilkunde – Onko-Haematologie, Medizinische Hochschule Hannover, Hannover, Deutschland. Prof. Veronika Sexl, Vet. Med. Uni. Wien, Wien, Österreich In Zusammenarbeit mit Prof. Giusepe Basso, Laboratory of Pediatric Onco-Hematology, University of Padova, Italy. Prof. Andrea Biondi and Dr. Giuseppe Gaipa, Tettamanti Research Center, Monza – University Milano-Bicocca, Italy. Dr. Richard Ratei and Dr. Leonid Karawajew, Zellmarkerlabor, RRK, HELIOS Klinikum Berlin, Charite MS, Berlin, Germany. Prof. Dirk Reinhardt, Abteilung für Kinderheilkunde – Onko-Haematologie, Medizinische Hochschule Hannover, Hannover, Deutschland. Prof. Veronika Sexl, Vet. Med. Uni. Wien, Wien, Österreich For further reading [1] Fišer, Sieger et al. 2012 [2] Gaipa, Cazzaniga et al. 2012 [3] Husak and Dworzak 2012 Literaturangaben [1] Fišer, Sieger et al. 2012 [2] Gaipa, Cazzaniga et al. 2012 [3] Husak and Dworzak 2012 1 ÖNB grant 13081; 2008–2010 For further reading [1] Husak, Printz et al. 2010 [2] Husak and Dworzak 2012 s HSP70* s HSP70** cy HSP70** 22–23 CD99 and HSP70 crosstalk in ALL cells increases NK cytotoxicity 00 101 102 103 00 101 102 103 00 101 102 103 Fig. 1 – HSP up-regulation in primary ALL cells (red: CD99stimulation; blue and grey: (mock) controls. BCP-ALL 100 control DN16 80 60 40 20 0 0:1 2.5:1 10:1 E:T ratio Jurkat P<0.05 100 80 60 40 control DN16 20 0 0:1 1:1 2.5:1 10:1 E:T ratio Fig. 2 – Apoptosis induction by CD99 stimulation (grey line) versus control (black) with and without NK-cells in soaring ratios versus target ALL cells [2]. St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Innovative immunologische Diagnostik für Leukämie im Kindesalter % of apoptotic targeted cells Innovative diagnostic tools for paediatric leukaemia Leukaemias Specific project Cell counts Forschungsschwerpunkt % of apoptotic targeted cells Research focus Leukaemias Biology of Leukaemias Leukämien Biologie der Leukämien Group leader Renate Panzer-Grümayer [email protected] Postdoctoral fellows Reinhard Grausenburger Kamilla Malinowska-Ozdowy1 Maria Morak PhD students Stephan Daniel Bastelberger2 Gerhard Fuka Ulrike Kaindl Christine Nassimbeni3 Technicians Susanna Fischer Andrea Inthal4 Astrid Mecklenbräuker5 Marion Zeginigg6 1 since Aug. 2012 2 since May 2011 3 since Febr. 2011 4 maternity leave from Nov. 2011 5 since Aug. 2011 6 since May 2011 7 Vice Director Clinicians, St. Anna Children’s Hospital Andische Attarbaschi Georg Mann LabDia Labordiagnostik GmbH Oskar A. Haas7 Childhood acute lymphoblastic leukaemia (ALL), the most frequent cancer in childhood and adolescence, has reached an overall cure rate of approximately 80% when treated according to contemporary risk-directed protocols. Yet despite this remarkable progress, a substantial proportion of cases still suffers a relapse, making leukaemia the 4th most common cause of death in this age group. Consequently, current research focuses on poorly responding ALL subgroups and the identification of new genetic markers. Additionally, given the high cure rates of children with ALL, novel treatment strategies that are less toxic than the currently applied regimens are urgently needed. Therefore, the ultimate goal of our research is to identify and characterise new prognostic and predictive molecular lesions but also to understand how such changes collude to produce and sustain overt leukaemia, progress to relapse and cause drug resistance. To reach this ambitious goal, we i) try to understand why and how leukaemia develops and progresses, and ii) use molecular methods to assess the in vivo treatment response of children with ALL. What mutations drive leukaemia? The main focus of our basic and translational research lies on two large, genomically homogenous ALL subgroups, comprising approximately 20% of all ALL cases each. The first subgroup carries the ETV6/RUNX1, the second a high hyperdiploid karyotype (HD). They share similar clinical features, as both generally lack high risk criteria and respond rapidly to initial chemotherapy. They relapse, however, in up to 20% of cases from which a substantial proportion is resistant to treatment [1]. Both genetic entities have been shown to develop during fetal life, and need – in addition to the ETV6/RUNX1 gene fusion and the non-disjunction event that leads to the gain of additional chromosomes – cooperating aberrations for progression to overt leukaemia [3][4]. During the last two years, we specifically addressed the function of ETV6/RUNX1 in the fully malignant cell, as well as the timing and nature of presumably pivotal sec ondary aberrations in both ALL subgroups [1][2][5][6]. Entschlüsselung der Leukämieentstehung – Prognose verbessern Why do some leukaemias re-occur? To gain new insights into the relapse mechanisms, we have employed high resolution SNP arrays and, more recently, also next generation sequencing, for the unbiased genome-wide identification of copy number aberrations and somatic sequence mutations in various leukaemia subgroups. These studies are complemented by targeted re-sequencing and functional analysis of candidate genes. Thereby we detected CREBBP deletion and sequence mutations in more than half of all HD ALL cases at relapse. Moreover, these mutations seem to prevail at initial presentation of relapse cases – albeit to a lesser extent than at relapse. Thus, CREBBP mutations might eventually be explored as biomarkers for refined risk stratification and cus tomized treatment [2]. Similar strategies were applied to explore relapse mechanisms of ETV6/RUNX1-positive leukaemias and late relapsing T cell precursor ALL. The majority of relapses were either derived from a common ancestral clone or differed completely from the initial leukaemia [1][7]. All these studies required large patient cohorts and sample collections and were only possible through international collaborations. Individualisation of leukaemia treatment Molecular detection of in vivo response to chemo therapy is currently one of the best predictive parameters and therefore an integral part of most treatment protocols [8][9]. Methods and applications are subjected to continuous improvement and adaptation within the EURO-MRD Group. For further reading [1] Kuster, Grausenburger et al. 2011 [2] Inthal, Zeitlhofer et al. 2012 [3] Panzer-Grümayer, Fasching et al. 2002 [4] Ford, Bennett et al. 1998 [5] Fuka, Kauer et al. 2011 [6] Fuka, Kantner et al. 2012 [7] Szczepanski, van der Velden et al. 2011 [8] Schrappe, Valsecchi et al. 2011 [9] Conter, Bartram et al. 2010 Die akute lymphoblastische Leukämie (ALL) ist als häufigste Krebsart des Kindes- und Jugendalters mit derzeitigen risiko-angepassten Therapieprotokollen in ungefähr 80% heilbar. Dieser Erfolg hat dazu geführt, dass nun auch die Lebensqualität bei Kindern mit ALL ein wichtiges Kriterium für die Beurteilung der Therapie darstellt und daher weniger toxische therapeutische Maßnahmen, als die bisher angewandten, notwendig geworden sind. Aus diesem Grund haben wir uns zum übergeordneten Forschungsziel gesetzt, einerseits neue prognostisch relevante molekulare Läsionen zu entdecken und zu charakterisieren, andererseits aber auch generell zum besseren Verständnis beizutragen, wie Mutationen zusammenwirken und zu einer klinisch manifesten Leukämie führen. Darüber hinaus möchten wir untersuchen, ob bestimmte genomische Ver änderungen für den Bestand der Leukämieerkrankung notwendig sind, welche Rolle sie in der Rezidiventwicklung spielen und wie sie zu einer Resistenz gegen Chemotherapeutika beitragen. Welche Mutationen sind für ALL wichtig? Der Hauptfokus unserer Grundlagen- und transla tionalen Forschung liegt auf zwei großen, genomisch einheitlich definierten Untergruppen der ALL, die jeweils ca. 20% aller Leukämien ausmachen. Die eine Gruppe ist durch das Vorhandensein des ETV6/RUNX1 Fusionsgens, die zweite durch einen hyperdiploiden Chromosomensatz charakterisiert; beide Leukämie formen sind generell nicht mit Hochrisiko-Kriterien assoziiert und sprechen sehr gut auf die Ersttherapie an. Nichtsdestoweniger treten bei bis zu 20% der Kinder Rezidive auf. Im Gegensatz zur früheren Annahme, dass diese Rezidive abermals gut auf die Therapie ansprechen, ist bei einer beträchtlichen Anzahl der Patienten nun eine Resistenz gegen die verabreichte Therapie beobachtet worden [1][2]. Beide Leukämieformen entstehen bereits während der fötalen Entwicklung im Mutterleib und benötigen weitere Mutationen, um eine klinisch manifeste Leukämie zu erzeugen [3][4]. Während der letzten zwei Jahre haben wir uns insbesondere auf die Funktion von ETV6/RUNX1 in der leukämischen Zelle, sowie die zeitliche Abfolge und Beschaffenheit von kritischen sekundären Veränderungen in beiden Leukämiegruppen konzentriert [1][2][5][6]. Warum treten Leukämien erneut auf? Um neue Erkenntnisse auf dem Gebiet der Rezidiventwicklung zu gewinnen, haben wir hochauflösende SNP Arrays und genomweite Sequenzierungen für den uneingeschränkten Nachweis von genetischen Veränderungen verwendet. Diese Studien wurden durch funktionelle Analysen ausgewählter Gene ergänzt. Wir konnten so einerseits bei hyperdiploiden Leukämien und ETV6/RUNX1-positiven, andererseits bei T ZellLeukämien feststellen, dass die meisten Rezidive von einer gemeinsamen Vorläuferzelle abstammen oder sogar komplett unterschiedlich sind [1][2][7]. Da alle diese Studien große Patientenzahlen benötigen, wurden sie im Rahmen von internationalen Kollabora tionen, insbesondere der BFM Studiengruppen, durchgeführt. Erfassung des Therapieansprechens Mittels hochauflösender molekularer Methoden bestimmen wir das individuelle Therapieansprechen aller Kinder mit ALL in Österreich. Diese Form der Diagnostik stellt derzeit einen der besten prädiktiven Parameter da und ist in die meisten Therapiestudien integriert [8][9]. Die Methoden und Anwendungen werden innerhalb der Europäischen MRD Gruppe ständig verbessert und erweitert. Literaturangaben [1] Kuster, Grausenburger et al. 2011 [2] Inthal, Zeitlhofer et al. 2012 [3] Panzer-Grümayer, Fasching et al. 2002 [4] Ford, Bennett et al. 1998 [5] Fuka, Kauer et al. 2011 [6] Fuka, Kantner et al. 2012 [7] Szczepanski, van der Velden et al. 2011 [8] Schrappe, Valsecchi et al. 2011 [9] Conter, Bartram et al. 2010 26–27 Unraveling leukaemia – pathogenesis to improve prognosis of affected children Leukaemias Forschungsschwerpunkt St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Research focus We also found that PI3K/mTOR inhibitors sensitised resistant cell lines to drugs. Our findings thus demonstrate that – together with a severely impaired repopulation capacity of leukaemia cell lines upon suppression of ETV6/RUNX1 in NOD/SCID mice – continuous expression of the ETV6/RUNX1 fusion gene i) interferes with key regulatory functions that shape the biology of this leukaemia subtype and ii) is essential for disease maintenance. Importantly, these results provide the first rationale and justification for targeting the fusion gene and the PI3K/AKT/mTOR pathway therapeutically [2]. This is of particular relevance given the enormous clonal heterogeneity of secondary aberrations that precludes their therapeutic targeting and leaves ETV6/RUNX1 as the only common marker [1][2][3]. A B Fig. 1 – Meta-groups of functional annotations for up-regulated genes upon E/R knock-down. Meta-groups were curated based on gene clustering of annotation terms. A: Top 100 annotation terms from knock-down-UP genes, their P-values and their affiliation to meta-groups. Similarity of the metagroups was based on the number of shared genes. For distance calculations between the meta-groups genes from all contributing terms were taken together. B: Change in expression of individual genes in meta-groups that contain significant annotation terms. The color code at the bottom of the figure indicates the extent of log2-fold changes in gene expression. The P2RY8-CRLF2 fusion defines a particular relapseprone subset of ALL in non-Down Syndrome (DS) children. It derives from an interstitial deletion in the PAR1 region, results in the overexpression of the cytokine receptor-like factor 2 (CRLF2) and practically never concurs with ETV6-RUNX1, MLL, TCF3 or BCR-ABL fusions. Previous reports suggested that P2RY8-CRLF2 represents a driver mutation and that the abrogation of its signaling may by therapeutically exploited. Prompted by the observation of subclonal distribution of the fusion, we investigate whether and to what extent different clone sizes influence disease and relapse development. For this purpose, we quantified the genomic P2RY8-CRLF2 fusion product and correlated it with the corresponding CRLF2 expression levels in patients enrolled in the BFM-ALL 2000 protocol in Austria. Of 268 cases without recurrent chromosomal translocations and high-hyperdiploidy (representing approximately 50% of all cases), 67 (25%) were P2RY8-CRLF2-positive [4]. The respective clone sizes were ≥20% in 27% and <20% in 73% of the cases. The cumulative incidence of relapse of the entire fusionpositive group was clone size independent and significantly higher than that of the fusion-negative group (35±8% vs. 13±3%, P=0.008); also, these cases were primarily confined to the non-high-risk groups. Of 22 P2RY8-CRLF2-positive diagnosis/relapse pairs, only 4/8 had the fusion-positive dominant clone conserved at relapse, whereas none of the original 14 fusion-positive small clones reappeared as the dominant relapse clone. We conclude that the majority of P2RY8-CRLF2positive clones are small at diagnosis and virtually never generate a dominant relapse clone. Our findings therefore suggest that P2RY8-CRLF2-positive clones do not have the necessary proliferative or selective advantage to evolve into a disease-relevant relapse clone [4][5]. In collaboration with Kofler R. and Rainer J., Tyrolian Cancer Research Institute and Biocenter – Division Molecular Pathophysiology, Innsbruck Medical University, Austria Bauer E., Kanter H.P. and Stoiber D., Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria Hall A. and Russel L., Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne UK Metzler M., Department of Paediatrics, University of Erlangen-Nuernberg, Erlangen, Germany Meyer L.H., Universitätsklinik für Kinder- und Jugendmedizin, Ulm, Germany Meyer C. and Marschalek R., Institute of Pharmacological Biology/DCAL, Goethe-University, Frankfurt, Germany Harbott J., Oncogenetic laboratory, Dept. Ped. Haematol/Oncol., Justus-Liebig University, Gießen, Germany Associazione Italiana di Ematologia ed Oncologia Pediatrica (AIEOP)-Berlin-Frankfurt-Münster (BFM) Study Group, European Study Group for the Detection of Minimal Residual Disease (Euro-MRD-Group). For further reading [1] Fuka, Kauer et al. 2011 [2] Fuka, Kantner et al. 2012 [3] Kuster, Grausenburger et al. 2011 [4] Attarbaschi, Morak et al. 2012 [5] Morak, Attarbaschi et al. 2012 28–29 To evaluate the impact of ETV6/RUNX1 on the regulation of genes and pathways in leukaemia, we performed shRNA-mediated knock-down of the endogeneous fusion gene in two leukaemic cell lines and investi gated the ensuing consequences on genome-wide gene expression patterns and deducible regulatory functions [1]. We thereby identified 777 genes whose expression was substantially altered with approximately equal numbers up- or down-regulated. The up- regulated set was significantly enriched with genes included in cell activation, immune response, apoptosis, signal transduction and development, and differentiation categories (Fig. 1). In the ETV6/RUNX1 knock-down set, only PI3K/AKT/mTOR signaling and haematopoietic stem cell category were observed. Comparable gene expression signatures obtained from primary ETV6/RUNX1-positive ALL samples also underlined the relevance of these pathways and molecular functions. As a consequence, we analysed the interaction between the fusion gene and PI3K/AKT/mTOR pathway activation in more detail and showed that ETV6/RUNX1 indeed activates this pathway and that pharmacological inhibition significantly increases the apoptosis rate in these leukaemias. Challenging P2RY8-CRLF2’s role as main relapse-driving force in leukaemia St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 ETV6/RUNX1 perturbs key processes and is essential for leukaemia Leukaemias Specific project Specific project Leukaemias Genetics of Leukaemia Leukämien Leukämiegenetik Group leader Sabine Strehl [email protected] Postdocs Dagma Denk1 Klaus Fortschegger Karin Nebral2 PhD students Stefanie Anderl3 Sara Colomer Lahiguera4 Dagmar Denk5 Diploma/MSc student Teresa Preglej6 Technician Margit König Clinicians, St. Anna Children’s Hospital Andishe Attarbaschi Michael N. Dworzak Georg Mann 1 since Aug. 2012 2 until Febr. 2012 3 since Apr. 2011 4 since Dec. 2011 5 until July 2012 6 since Sept. 2012 Although in the majority of leukaemia the primary genetic alterations responsible for the pathogenesis of the disease have already been unraveled [1][2], in many instances the underlying molecular genetic lesions are still unknown. Furthermore, the biological consequences as well the clinical impact of numerous specific genetic alterations remain to be determined. Therefore, the research of the Genetics of Leukaemia group is centred around two main aspects: On the one hand, we focus on the identification of novel genetic alterations that are involved in the pathogenesis and progression of childhood acute leukaemia and the evaluation of genetic lesions as potential predictive biomarkers for the refinement of risk stratification and risk-adapted therapy. On the other hand, our aim is to gain further insights into the functional consequences of specific genetic alterations, in particular those potentially oncogenic fusion genes that have been identified by our group [3][4]. Genetic alterations and prognosis To accomplish these tasks, we conduct comprehensive population-based screening studies for genetic alterations in leukaemia samples of patients enrolled in the Austrian BFM clinical trials employing a range of molecular genetic approaches. Genetic and clinical datasets are then used to determine whether some recurring genetic alteration may serve as biomarkers for treatment failure and whether its occurrence may, therefore, justify therapy intensification. The assessment of the prognostic relevance of specific genetic aberrations is performed in close collaboration with the clinicians of the St. Anna Children’s Hospital and within the framework of the International BFM Study Group. Genetische und biologische Charakterisierung akuter Leukämien PAX5 aberrations in B-cell leukaemia To gain further insights into the pathogenesis of B-cell precursor acute lymphoblastic leukaemia (BCP-ALL), we are focusing on the functional consequences of PAX5 alterations. The rationale for the selection of this particular gene is derived from the fact that PAX5 plays a pivotal role in B-cell commitment and maintenance and is affected by mutations – including deletions, amplifications, point mutations, and gene rearrangements – in about 30% of paediatric BCP-ALL, suggesting a crucial role in the pathogenesis of the disease. Based on our identification of several PAX5 fusion genes, which encode potentially oncogenic aberrant transcription factors [3–5], we now aim to elucidate their role in leukaemogenesis. References [1] Pui, Carroll et al. 2011 [2] Pui, Mullighan et al. 2012 [3] Nebral, König et al. 2007 [4] Nebral, Denk et al. 2009 [5] Denk, Nebral et al. 2012 In der Mehrheit der akuten Leukämien im Kindes- und Jugendalter wurden die für die Krankheitsentstehung verantwortlichen primären genetischen Veränderungen bereits aufgeklärt [1][2]. Dennoch sind in vielen Fällen die genetischen Aberrationen, die zur Leukämie entwicklung führen, nach wie vor unerforscht, und sowohl deren biologische Auswirkungen als auch deren Einfluss auf die Prognose der PatientInnen unbekannt. Deshalb fokussiert die Arbeitsgruppe für Leukämiegenetik ihre Arbeit auf zwei Schwerpunktthemen: Einerseits auf die Identifizierung genetischer Veränderungen, die in die Krankheitsentstehung und das Fortschreiten einer akuten Leukämie involviert sind, sowie die Bewertung der prognostischen Relevanz bestimmter genetischer Veränderungen, die in Folge als sogenannte Biomarker zur Risikobewertung und Therapieab stimmung herangezogen werden könnten. Andererseits ist es Ziel unserer Forschungsarbeit, die biologischen Auswirkungen bestimmter genetischer Veränderungen zu erforschen, insbesondere jener Fusionsgene, die unsere Arbeitsgruppe identifiziert hat [3][4]. Die Bedeutung genetischer Veränderungen Um diese Ziele zu erreichen, führen wir umfassende genetische Analysen der leukämischen Zellen aller PatientInnen durch, die in den österreichischen Leukämiestudien registriert sind. Dafür setzen wir eine Reihe molekularer Techniken ein. Um festzustellen, ob eine spezifische genetische Veränderung mit einem hohen Risiko eines Therapieversagens einhergeht und als prognostisch aufschlussreicher genetischer Biomarker eingesetzt werden kann, werden genetische und klinische Daten miteinander korreliert. Die Bewertung der prognostischen Relevanz der genetischen Marker, die zu einer verfeinerten Einteilung in Risikogruppen führen könnte, wird in enger Zusammenarbeit mit den ÄrztInnen des St. Anna Kinderspitals und der Internationalen BFM Studiengruppe durchgeführt. Leukämie und PAX5 Um neue Erkenntnisse in Bezug auf die Krankheitsentstehung der B-Vorläuferzell akuten lymphatischen Leukämie (B-ALL) zu gewinnen, erforschen wir schwer punktmäßig die Auswirkungen von Veränderungen des PAX5 Gens. Die Motivation, gerade dieses Gen zu untersuchen, begründet sich darauf, dass PAX5 eine zentrale Funktion in der Entwicklung normaler B-Zellen und deren Erhaltung spielt und in ungefähr 30% aller BALL von genetischen Veränderungen – Deletionen, Amplifikationen, Mutationen und Genrearrangements, die zu Genfusionen führen – betroffen ist. Nahe liegenderweise könnte PAX5 deshalb auch eine Rolle in der Leukämieentstehung spielen. Ausgehend von unserer Identifikation einiger PAX5 Partnergene [3–5] erforschen wir nun, welche Rolle diese potentiell onkogenen Fusionsgene in der Pathogenese der akuten Leukämie spielen. Literaturangaben [1] Pui, Carroll et al. 2011 [2] Pui, Mullighan et al. 2012 [3] Nebral, König et al. 2007 [4] Nebral, Denk et al. 2009 [5] Denk, Nebral et al. 2012 32–33 Genetic and biological characterisation of paediatric acute leukaemia Leukaemias Forschungsschwerpunkt St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Research focus Properties of PAX5 fusion proteins The consistent retention of the N-terminal PAX5 DNAbinding paired domain, which is fused to the C-terminal domains of one of the highly diverse partner proteins, indicates that PAX5 chimeric proteins may have properties both common to and distinct from those of wild-type PAX5. Indeed, independent of the genuine subcellular localisation of the individual partner protein, all PAX5 fusion proteins display a predominantly nuclear localisation (Fig. 1), which appears to be mainly dictated by the paired domain. Intriguingly, in contrast to the cytoplasmic localisation of all other JAK2 fusion proteins analysed to date, PAX5-JAK2 is also a nuclear protein. As a second common key characteristic of all PAX5 fusion proteins, the PAX5 paired domain mediates their DNA interactions at specific PAX5 target sequences, albeit with obviously variable affinities, pointing at potential differences in their effects on gene transcription. In contrast to these common biochemical properties, our experiments showed that only some PAX5 fusion proteins are capable of homooligomerisation while others are not. Since PAX5 wildtype protein is known to bind to DNA as monomer, the interfaces for self-interaction are provided by distinct moieties such as coiled-coil domains present in the respective partner proteins. In terms of their impact on PAX5 target gene expression, at least in reporter gene assays, some of the PAX5 fusion proteins appeared to have activation potential rather than mere antagonizing or repressive capacity. However, in a murine cell line model, the effect of PAX5 chimeric proteins on the transcriptional activation of the PAX5 target Cd79a could not be confirmed and thus, additional studies are required to determine the impact of PAX5 chimeric proteins on PAX5 target gene transcription. Constitutive kinase activity of PAX5-JAK2 Intriguingly, amongst all PAX5 fusion proteins, PAX5JAK2 appears to have a unique function, because it has constitutive kinase activity and activates the JAK-STAT signaling pathway. More specifically, PAX5JAK2 itself is phosphorylated and, in turn, phosphorylates and activates downstream STATs. Furthermore, gene expression profiling of PAX5-JAK2 positive patient samples revealed high similarities with BCR-ABL1 and JAK2-mutated BCP-ALL, further substantiating that in the primary leukaemia, the JAK-STAT pathway is indeed activated. Importantly, at least in vitro, the kinase activity of PAX5-JAK2 can be efficiently blocked by JAK2 inhibitors, rendering it a potential target for therapeutic intervention. References [1] Mullighan, Goorha et al. 2007 [2] Nebral, König et al. 2007 [3] Nebral, Denk et al. 2009 [4] Coyaud, Struski et al. 2010 [5] Denk, Nebral et al. 2012 In international collaborations, we continually contribute to studies regarding the prognostic relevance of specific genetic alterations in the context of the I-BFM and in comparison to other treatment protocols. These multicentre studies are a necessity to increase the size of patient cohorts to gain more statistical power, and to analyse rare disease entities. of leukaemia, we have analysed 350 cases lacking the major sentinel fusion genes by an IGH@ break-apart FISH assay and have identified IGH@ translocations in ~5% of patients. This study confirmed CRLF2 to be the most frequent IGH@ partner gene, however, in 50% of cases the IGH@ translocation partners remain unknown and their identification is still ongoing. MLL and CRLF2 rearrangements STAT5B-RARA-positive acute promyelocytic leukaemia The I-BFM study group has extended its meta-analysis assessing the clinical impact of specific MLL rearrangements in paediatric acute myeloid leukaemia (AML) and showed that not only the MLL translocation partner itself [1] but also secondary cytogenetic aberrations independently predict clinical outcome [2]. Moreover, in a comparative study it has been determined that the cumulative relapse incidence of P2RY8-CRLF2+ patients treated on the AIEOP-BFM or UK trials did not differ statistically. However, the relapse rate of IGH@-CRLF2+ patients was much higher in patients enrolled in UK trials suggesting treatment-related differences [3]. IGH@ translocations In another international project with the aim to ascertain the true incidence of IGH@ translocations in BCP-ALL and to further characterise this subtype Furthermore, we have determined that, in contrast to PML-RARA+ acute promyelocytic leukaemia (APL), which is responsive to treatment with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), the STAT5B-RARA fusion variant defines a highly relapse-prone APL subgroup, which is insensitive to both therapeutic agents. Hence, the accurate identification of this rare subtype of APL is therefore essential for therapeutic decision-making [4]. In collaboration with The Austrian (represented by Andishe Attarbaschi, Michael N. Dworzak, Georg Mann, all St. Anna Children’s Hospital) and the International Berlin-Frankfurt-Münster Study Group (I-BFM-SG). References [1] Balgobind, Raimondi et al. 2009 [2] Coenen, Raimondi et al. 2011 [3] Attarbaschi, Morak et al. 2012 [4] Strehl, König, et al. 2012 Fig. 1 – The indicated PAX5 fusion proteins predominantly localise to the nucleus as determined by immunofluorescence and confocal microscopy; white arrow points out punctate cytoplasmic staining and white bars indicate 20 μm. 34–35 In about 30% of B-cell precursor acute lymphoblastic leukaemia (BCP-ALL), PAX5, a master regulator of B-cell development, is affected by genetic alterations including deletions, amplifications, point mutations, and the formation of fusion genes [1]. While deletions and point mutations frequently coincide with known sentinel alterations such as the ETV6-RUNX1 and BCR-ABL1 fusion genes, and are therefore considered as secondary events, PAX5 rearrangements, which result in the expression of potentially oncogenic fusion genes, most likely represent primary genetic events. The chimeric proteins encoded by the fusion transcripts are hypothesised to act as trans-dominant aberrant transcription factors, which antagonize wild-type PAX5 function. Based on our own studies and those of others, in which it has been determined that the N-terminus of PAX5 is fused to a markedly heterogeneous group of C-terminal fusion partners including transcription factors, structural proteins or the tyrosine kinase JAK2 [1–5], we currently aim to investigate the biochemical and functional properties of these distinct chimeric proteins. Treatment outcome of childhood leukaemia with specific genetic alterations St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Functional consequences of PAX5 fusion proteins Leukaemias Specific project Specific project Solid Tumours Tumour Biology Solide Tumoren Tumorbiologie Group leader Peter F. Ambros [email protected] Staff scientist Ingeborg M. Ambros Sabine Taschner-Mandl PhD students M. Reza Abbasi1 Dominik Bogen2 Fikret Rifatbegovic3 Tamara Weiss4 Diploma/MSc student Magdalena Schwarz5 Technicians Bettina Brunner-Herglotz Andrea Ziegler Clinicians St. Anna Children’s Hospital Leo Kager Ruth Ladenstein 1 since Mar. 2012 2 currently at NIH 3 since May 2012 4 since Oct. 2011 5 since Sep. 2012 Our research is primarily devoted to understanding the genomic and cell biological aspects of neuro blastomas and other paediatric solid tumours to establish a sound basis for patient specific treatment options and to provide information on therapeutically useful compounds that induce tumour cell senescence and tumour cell death. Genomic data helps to direct therapy Neuroblastomas are the most frequent solid tumours of infancy and early childhood. Their biological behaviour is unique and does not necessarily correspond to tumour dissemination but is largely dependent on tumour genomics. A favourable course, which occurs in more than a third of patients, is based on spontaneous regression or maturation of tumour cells. In the majority of patients, however, neuro blastomas behave aggressively [1]. Over the last two decades a number of research teams, including ours, have provided evidence that genomic features help to differentiate between prognostically different subgroups. Austria was in fact one of the first countries to apply a genome based treatment stratification, resulting in a dramatic increase of surviving patients [2]. This worldwide accepted concept is now being applied and developed further in European clinical studies aiming at reducing or intensifying cytotoxic treatment in genetically defined patient groups [3]. Since these current clinical studies range from a “wait and see strategy” after complete/incomplete resection or even no resection at all to the application of high dose chemotherapy and stem cell transplantation, the tumour-genome based therapy stratification has to be as secure, reliable, sensitive and specific as possible. Hence, we apply the most advanced moleculargenomic diagnostic tools currently available to us. The detection of tumour genomic changes is performed by a high resolution array technique (besides the application of interphase FISH). The simultaneous quantification of more than 2.6 million probes on one array represents the highest genomic coverage Einblicke in die Tumorbiologie erlaubt individuelle Behandlung presently available, allowing detection of copy number changes that affect even single exons, exact breakpoint localisations, gene amplifications, uniparental disomies, low-level-mosaicisms and chromothripsis, a recently described state of genomic catastrophe indicating unfavourable prognosis [4]. This information, provided to the clinician in charge, enables a patient-tailored treatment strategy. Another crucial diagnostic pillar is the proof of tumour cells in the bone marrow, performed by a highly sophisticated combination of genetic and immunologic techniques and an automatic microscope [5]. International studies Currently, two European neuroblastoma studies cover the vast majority of patients throughout Europe by implementing genomic features to direct therapy: The Low/Intermediate Risk Neuroblastoma Study (LINES) and the High Risk Neuroblastoma (HR-NBL) Study. In both studies, our group has the role of chairman and co-chairman of the European Biology Group to guarantee the best possible quality of the genomic data [6]. The members of the Biology Group are from 12 European and 4 non-European countries (Australia, Hong Kong, Israel und Japan). Furthermore, our group is embedded in two international efforts to link genomic and MRD (minimal residual disease) information with clinical data to establish consensus criteria that will then be used worldwide (International Neuroblastoma Risk Group – INRG, and International Neuroblastoma Response Criteria – INRC) [7]. For further reading [1] Brodeur 2003; Maris, Hogarty et al. 2007 [2] Ladenstein, Ambros et al. 1996 [3] Cohn, Pearson et al. 2009 [4] Molenaar, Koster et al. 2012 [5] Ambros, Mehes et al. 2003 [6] Ambros, Benard et al. 2003; Ambros, Brunner et al. 2011 [7] Ambros, Ambros et al. 2009; Cohn, Pearson et al. 2009; Monclair, Brodeur et al. 2009; London, Castel et al. 2011; Moroz, Machin et al. 2011; Taggart, London et al. 2011; Angelini, London et al. 2012; Schleiermacher, Mosseri et al. 2012 Es ist unser Ziel, Einblicke in Genomveränderungen und die zellbiologischen Eigenschaften von Neuroblastomen und anderen soliden Tumoren bei Kindern und Jugendlichen zu gewinnen, um eine wissenschaftliche Grundlage für individuelle Therapien zu begründen. Zudem identifizieren wir Wirkstoffe, die Tumorzellen altern bzw. absterben lassen. Eine Basis für individuelle Therapien Neuroblastome sind die häufigsten Tumoren bei Säug lingen und Kleinkindern mit einem biologisch einzig artigen Verhalten, das oftmals von den Genomver änderungen des Tumors abhängig ist. In mehr als einem Drittel der Patienten verhalten sich die Neuroblastome durch spontane Rückbildung (Absterben der Tumor zellen) oder durch Ausreifung gutartig. Bei der Mehrzahl der Patienten werden allerdings aggressive Neuro blastome diagnostiziert [1]. Unsere Forschungsarbeiten haben dazu beigetragen, Patienten, die sich hinsichtlich ihrer Prognose erheblich unterscheiden, anhand tumorgenomischer Eigenschaften zu identifizieren. Österreich war weltweit eines der ersten Länder, in denen an Hand von Genomanalysen die Behandlungsstrategien festgesetzt wurden, was zu einem beachtlichen Anstieg der Überlebensraten führte [2]. Dieses Therapiekonzept ist heute weltweit anerkannt und wird in europäischen Studien stetig weiter entwickelt und verbessert [3]. Aktuelle klinische Studien verfolgen eine Strategie des Zuwartens ohne Chemotherapie nach kompletter, teilweiser oder gar keiner Resektion bis hin zu hochdosierter Chemotherapie und Stamm zelltransplantation. Aufgrund dieser Behandlungsunterschiede müssen die Untersuchungsmethoden, welche den Entscheidungsprozess mitbestimmen, sicher, zuverlässig, sensitiv und hochspezifisch sein. Wir verwenden höchstauflösende molekulargenetische Array Methoden, die nur an diesem Zentrum in der Neuroblastomdiagnostik eingesetzt werden. Derartige Analysen geben Aufschluss über die Organisation der Erbsubstanz und die Art und Anzahl von Abweichungen: Veränderungen einzelner Exons, Aufklärung exakter Bruchstellen, Nachweis von Genvervielfältigungen, und Chromothripsis, einer Form von sog. katastrophaler genomischer Umlagerung, die mit einer schlechten Prognose einhergeht [4]. Auch die klassische Methode der Interphase Fluoreszenz-in-situ-Hybridisierung wird in der Diagnostik angewandt. Mithilfe dieser Information können Therapien auf den Patienten maßgeschneidert werden. Ein weiterer Eckpfeiler der Diagnostik ist die B eurteilung des Therapieansprechens mittels Nachweis von Tumorzellen im Knochenmark bei Einsatz von genetischen und immunologischen Techniken und einem vollautomatischen Fluoreszenzmikroskop [5]. Internationale Studien Aktuell werden in zwei europäischen NeuroblastomStudien die Therapien auf Basis von Genominforma tionen angepasst: die Niedrig/Mittelhoch-Risiko Neuroblastom-Studie (LINES) und die Hoch-Risiko Neuroblastom-Studie (HR-NBL). Wir leiten die europäische Biologie Gruppe, die die Genomanalysen durchführt. Der Zusammenschluss internationaler Labors und permanente Qualitätskontrollen garantieren höchste Datenqualität. Zudem sind wir in zwei Initiativen aktiv, die genomische bzw. minimale Rest erkrankungsdaten mit klinischen Daten verbinden, um weltweit verbindliche, diagnostische Standards und Kriterien für den Therapieerfolg festzulegen (Inter national Neuroblastoma Risk Group – INRG, and Inter national Neuroblastoma Response Criteria – INRC) [6]. Literaturangaben [1] Brodeur 2003, Maris, Hogarty et al. 2007 [2] Ladenstein, Ambros et al. 1996 [3] Cohn, Pearson et al. 2009 [4] Molenaar, Koster et al. 2012 [5] Ambros, Mehes et al. 2003 [6] Ambros, Ambros et al. 2009; Cohn, Pearson et al. 2009; Monclair, Brodeur et al. 2009; London, Castel et al. 2011; Moroz, Machin et al. 2011; Taggart, London et al. 2011; Angelini, London et al. 2012; Schleiermacher, Mosseri et al. 2012 38–39 Understanding paediatric tumours facilitates patient-specific treatment Solid Tumours Forschungsschwerpunkt St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Research focus Disseminated tumour cells Induction of tumour cell senescence Disseminated tumour cells (DTCs) can be responsible for disease relapse in neuroblastoma patients. At diagnosis, virtually all stage 4 (M – metastatic) patients present with DTCs in the bone marrow (BM). After initial therapy, however, two patient subgroups can be identified: patients with a drastic reduction of the tumour cell load below the detection limit of the applied AIPF (automatic immune-fluorescence plus FISH) technique (<1 tumour cell/3 million BM cells) [1][2] and patients retaining tumour cells in the BM. In order to further elucidate the findings from the latter group, BM evaluation results of approximately three thousand BM samples from about seven hundred patients (from before, during and after chemotherapy or from the time of relapse) were generated and partly linked with clinical data. This correlation revealed significant better (p<0.0005) event-free and overall survival of patients that showed rapid elimination of DTCs from the BM as compared to the “non-clearing” group. Thus, monitoring and quantifying the tumour cell load during and after cytotoxic treatment is a reliable and sensitive way to monitor treatment success. It will no doubt vastly improve therapeutic strategies in the future. Another part of our research focuses on the potential therapeutic application of low dose cytotoxic drugs capable of inducing cellular senescence in a subset of genetically defined tumours. This permanent state of proliferative arrest can be induced in tumours with gene amplifications (MYCN amplification, in the case of neuroblastoma) by low dose hydroxyurea [3] and camptothecin, a topoisomerase I inhibitor, in vitro. Topotecan, a camptothecin derivate, is currently used only at high doses in neuroblastoma treatment regimens and, therefore, the effects evoked by lowdose topotecan and the involved pathways required further scrutiny. We found that senescence induction only works in cell lines with amplification of the MYCN oncogene and is linked to a drastic reduction of the MYCN amplification, frequently leading to a nonamplified state. In this manner, the tumour cells lose a crucial hallmark of aggressiveness and rapid growth. In two ongoing projects, we characterise DTCs on two levels: the genomic status and the global expression profile by applying SNParrays and expression arrays/RNA sequencing approaches. To facilitate these analyses, DTC fractions have to be enriched up to more than 1000-fold. The data from these studies are expected to identify genomic/expression markers that are able to further discriminate favourable from unfavourable tumours at diagnosis, to indicate a tumour’s sensitivity to therapy and to provide information for a targeted tumour therapy (e.g. ALK inhibitors, receptor tyrosine kinase inhibitors, CDK inhibitors). Moreover, consecutive array data obtained from DTCs of individual patients provide information on the genomic tumour evolution which helps to identify mechanisms leading to resistance against cytotoxic agents. In addition, besides a drastically reduced proliferation activity, marked changes in the expression profile were detected in low-dose hydroxyurea and topo tecan treated neuroblastoma cell lines. In co-cultures, senescent cells reduced cell growth and GD2 levels of non-senescent tumour cells. Moreover, senescent neuroblastoma-cells induced a strong immune reaction, probably mediated through cell-bound but not secreted factors. This is in line with an increased expression of MHCI and other immune-responserelated molecules. Senescent neuroblastoma-cells also showed elevated levels of MICA and MICB which are ligands of the activating receptor NKG2D, present on natural killer cells and other immune-reactive cells. In addition, CD44 a marker of favourable neuro blastoma, and several genes involved in the process of adhesion and negative regulation of cell proliferation as well as secreted factors were found to be up-regulated by senescent tumour cells. Our current work also addresses detailed evaluation of the expression profile with emphasis on the consequences for cell-cell interactions and analysis of the secretome of senescent neuroblastoma cells. Furthermore, we collaborate with the Institute of Pharmacology in Vienna to simulate the process of tumour cell senescence in a xenograft model. Preliminary data showed a reduced tumour growth and occurrence of tumour cell senescence in topotecan treated xenografts. On-going studies aim at verifying these first observations and will pave the way for long-term studies. However, further work is needed to clarify whether neuroblastoma patients may benefit from senescence-inducing, low-dose treatment. Schwann cell factors In contrast to aggressive neuroblastomas, spontane ously maturing and mature neuroblastomas (ganglio neuro-blastoma/ganglioneuroma) harbour a special characteristic, the Schwann cell (SC) stroma. Importantly, the presence and volume of Schwannian stroma closely correlate with neuroblastoma growth inhibition and a favourable prognosis (the exceptions are histologically unequivocally identifiable ganglioneuroblastoma subtypes, i.e. the nodular type of ganglioneuroblastoma) [4]. In previous studies, we showed that SCs in neuroblastomas are normal cells that supposedly have been recruited by neuro blastic cell signals [5]. Furthermore, we postulated a “cross-talk” between SCs and neuroblastoma cells causing SC attraction and proliferation, and, in turn, tumour cell apoptosis and/or differentiation into ganglionic cells. These assumptions were verified by us and others by in vitro and in vivo experiments where SCs have been shown to act also on aggressive neuroblastoma cells [6][7]. The ongoing project is dedicated to characterising the Schwann cell factors involved in tumour growth inhibition by applying a cytokine antibody array. Moreover, collaboration with the Institute of Analytical Chemistry shall result in a comprehensive view of the SC proteome by applying mass spectrometry. Using this approach, we aim at identifying the SC secretory protein profile responsible for inducing growth inhibition and/or apoptosis in aggressive neuroblastoma cells. For further reading [1] Mehes, Luegmayr et al. 2003 [2] Ambros, Mehes et al. 2003 [3] Narath, Ambros et al. 2007 [4] Shimada, Ambros et al. 1999 [5] Ambros, Zellner et al. 1996 [6] Ambros, Attarbaschi et al. 2001 [7] Chlenski, Liu et al. 2002 Fig. 1 – A fluorescence microscopic image of Schwann cells, which fulfill a broad range of tasks in the peripheral nervous system, in cell culture. Since this cell type plays a crucial role in the maturation process of neuroblastoma, Schwann cells were isolated, cleaned up for in vitro experiments and stained with S100 (green), cytokeratin (red) antibodies and nuclear staining (blue). St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Disease monitoring and novel therapeutic strategies 40–41 Solid Tumours Specific project Solid Tumours Molecular Biology of Solid Tumours Solide Tumoren Molekularbiologie Solider Tumore Group leader Heinrich Kovar [email protected] Staff scientist Dave Aryee Jozef Ban Postdoct. fellows Argyro Fourtouna1 David Herrero Martin1 Caroline Hutter2 Stefan Nieden3 Raphaela Schwentner3 Eleni Tomazou4 PhD students Anna Katschnig5 Cornelia Mutz5 Raphaela Schwentner Technicians Gunhild Jug Karin Mühlbacher 1 since Mar. 2011 2 since Oct. 2011 3 since Jan. 2013 4 since Mar. 2012 5 since Nov. 2012 Cancer is caused by mutations in key genes that withdraw body cells from regular growth control and immune surveillance. Typical cancers are characterised by hundreds of genetic alterations, which differ from patient to patient. In these instances it is frequently difficult to distinguish disease driving from passenger mutations. In contrast, Ewing sarcoma, a group of aggressive bone tumours in children and adolescents, is characterised by a specific recurrent gene fusion which is primarily responsible for the disease. Already since the nineties, our research group has been investigating the molecular pathogenesis of Ewing sarcoma. Based on the use of modern genomic high-throughput technologies, we are characterising the gene regulatory networks that are controlled by the specific gene fusion in this disease. Our goal is to develop basic concepts for novel biology-based therapeutic strategies. To achieve this goal, the Austrian Research Fund FWF1 and the collaborative European systems biology project ASSET2 enable us to generate and integrate large genomic data sets and to model and validate molecular disease mechanisms on a holistic network level. Our group’s basic research is complemented by our activities in clinical research networks3–5, which allow us to test and validate laboratory results in patients. Our aim is the prioritisation of novel biomarkers and novel therapeutic concepts for clinical use [1]. In the reporting period 2011–2012, significant progress has been achieved in the characterisation of the role of micro RNAs and of a developmental signaling pathway (NOTCH) involved in normal tissue differentiation for Ewing sarcoma pathogenesis [2][3]. Investigation of gene regulatory mechanisms in Ewing sarcoma led to the completion of three PhD theses6–8. Molekulare Mechanismen der Ewing-Sarkom-Pathogenese Langerhans cell histiocytosis’ mechanisms In addition to our focus on Ewing sarcoma, we have been investigating pathogenic mechanisms in Langerhans cell histiocytosis (LCH). This disease of enigmatic origin is characterised by the accumulation of Langerhans cell-like cells, and presents either localised or widely disseminated. It is still unknown if it has to be considered a reactive (inflammatory) or a malignant disease. We recently showed that in LCH, similar to many leukaemias, the NOTCH s ignaling pathway is activated [4]. Future investigations will explore the therapeutic potential of this finding. 1 P20665-B12; P223208-B09; P24708-B21 2 EU-FP7 259348 “Assessing and Striking the Sensitivities of Embryonal Tumours” 3 EU-FP7 “European Network for Cancer Research in Children and Adolescents”; 4 EuroEwing99; 5 ERA-NET TRANSCAN: Prospective Validation of Biomarkers in Ewing Sarcoma for personalized translational medicine “PROVABES” I1225-B19 6 Raphaela Schwentner: Mechanisms of target regulation by the chimeric oncogene EWS-FLI1 in Ewing’s sarcoma 7 Stephan Niedan: Mechanisms of transcriptional repression by EWS-FLI1 in Ewing sarcoma. 8 Lucia Riedmann: The effect of EWS and its oncogenic derivative EWS-FLI1 on transcriptional and post-transcriptional gene regulation in Ewing sarcoma. For further reading [1] Kovar, Alonso et al. 2012 [2] Ban, Jug et al. 2011 [3] Bennani-Baiti, Aryee et al. 2011 [4] Hutter, Kauer et al. 2012 Auslöser und Wirkung von Krebs sind Mutationen von Schlüsselgenen, welche Körperzellen der normalen Wachstumskontrolle und Immunüberwachung ent ziehen. Typische Krebserkrankungen sind durch mehrere hundert genetische Veränderungen gekennzeichnet, welche sich von Patient zu Patient unterscheiden. In diesen Fällen ist es vielfach schwierig, Schlüssel veränderungen von Trittbrettfahrermutationen zu unterscheiden. Ewing Sarkome, eine Gruppe äußerst bösartiger Knochentumore bei Kindern und Jugendlichen, sind hingegen durch eine spezifische, immer wiederkehrende Genfusion charakterisiert, die für die Erkrankung hauptverantwortlich ist. Seit den Neunzigerjahren befasst sich unsere Arbeitsgruppe mit der molekularen Pathogenese des Ewing Sarkoms. Basierend auf dem Einsatz moderner genomischer Hochdurchsatzmethoden charakteri sieren wir genregulatorische Netzwerke, welche von der für die Erkrankung typischen pathogenen Gen fusion gesteuert werden. Unser Ziel dabei ist, Grund lagen für neue therapeutische Ansätze zu entwickeln. Zur Erreichung unserer Ziele unterstützen uns der österreichische Forschungsförderungsfonds FWF1 und das Europäische Systembiologieprojekt ASSET2 und ermöglichen uns nicht nur, große Datensätze zu gewinnen und zu integrieren, sondern auch molekulare Krankheitsmechanismen auf einer ganzheitlichen Netzwerkebene zu modellieren und zu überprüfen. Die grundlagenwissenschaftliche Forschung unserer Arbeitsgruppe wird durch unsere aktive Rolle in Europäischen klinischen Forschungsnetzwerken3–5 ergänzt. Diese ermöglichen uns, die im Labor gewonnen Erkenntnisse auf ihre Gültigkeit in Patienten zu über prüfen. Ziel ist die Priorisierung von neuen Biomarkern und neuen therapeutischen Strategien für den Einsatz in der Klinik [1]. Im Berichtszeitraum 2011–2012 wurden vor allem Fortschritte in der Charakterisierung der Rolle von MikroRNAs [2] und eines in der normalen Embryonal entwicklung und Differenzierung vieler Gewebe wichtigen Signalweges (NOTCH) [3] für die Pathogenese des Ewing Sarkoms erzielt. Untersuchungen zu den Mechanismen der Genregulation in Ewing Sarkomen führten zum Abschluss dreier Dissertationen6–8. Langerhanszell-Histiozytose-Mechanismen Neben dem Forschungsschwerpunkt Ewing Sarkom beschäftigen wir uns mit den molekularen Mechanismen der Langerhanszell Histiozytose (LCH). Diese Erkrankung unbekannter Herkunft zeichnet sich durch die Anhäufung Langerhanszell-ähnlicher Zellen aus und kann sowohl lokalisiert als auch disseminiert auf treten. Bis heute ist unklar, ob es sich um eine reaktive (entzündliche) oder eine bösartige Tumorerkrankung handelt. Wir konnten zeigen, dass in dieser Erkrankung der NOTCH Signaltransduktionsweg, ähnlich wie bei vielen Leukämien, aktiviert vorliegt [4]. Zukünftige Untersuchungen werden das therapeutische Potential dieses Befundes erforschen. 1 P20665-B12; P223208-B09; P24708-B21 2 EU-FP7 259348 „Assessing and Striking the Sensitivities of Embryonal Tumours” 3 EU-FP7 „European Network for Cancer Research in Children and Adolescents”; 4 EuroEwing; 5 ERA-NET TRANSCAN: Prospective Validation of Biomarkers in Ewing Sarcoma for personalized translational medicine „PROVABES” I1225-B19 6 Raphaela Schwentner: Mechanismen der Zielgenregulation durch das chimäre Onkogen EWS-FLI1 im Ewing Sarkom 7 Stephan Niedan: Mechanismen der transkriptionellen Repression durch EWS-FLI1 im Ewing Sarkom. 8 Lucia Riedmann: Die Wirkung von EWS und seinem onkogenen Derivat EWS-FLI1 auf die transkriptionelle und post-transkriptionelle Genregulation im Ewing Sarkom Literaturangaben [1] Kovar, Alonso et al. 2012 [2] Ban, Jug et al. 2011 [3] Bennani-Baiti, Aryee et al. 2011 [4] Hutter, Kauer et al. 2012 44–45 Molecular mechanisms of Ewing sarcoma pathogenesis Solid Tumours Forschungsschwerpunkt St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Research focus From genomics to novel therapeutic concepts in Ewing sarcoma Re-activating the p53 tumour suppressor NOTCH in Langerhans cell histiocytosis The EWS-FLI1 chimeric protein is produced specifically in Ewing sarcoma as a result of a diagnostic chromosomal gene rearrangement. It functions as an aberrant sequence-specific ETS1 transcription factor with both gene activating and gene repressive consequences. Since this is specific to Ewing sarcoma and not existent in any normal tissue, EWS-FLI1 would constitute an ideal therapeutic target in this disease. However, efficient targeting of nuclear transcription factors still constitutes an unsolved pharmacological problem. We therefore used an integrated genomic approach to characterise mechanisms and downstream pathways of EWS-FLI1 activity. Two major patterns of gene regulation were observed: i) Gene activation was found predominantly associated with EWS-FLI1 binding proximal to the transcription start sites mainly of genes involved in cell cycle and growth regulation, RNA metabolism and translation. Here, we found functional cooperation with the family of E2F1 transcription factors [1]. ii) Gene repression was most frequently observed when EWS-FLI1 was binding to chromatin distal (>4kb) to transcriptional initiation sites and affected pre dominantly genes involved in mesenchymal differen tiation, neural development and bone remodeling. Activation of the p53 tumour suppressor pathway is central to different kinds of cellular stress responses. We have previously demonstrated modulation of p53 basal levels by EWS-FLI1 in Ewing sarcoma [3]. We found that EWS-FLI1 suppresses an important developmental signaling pathway, NOTCH2 [3][4]. Upon experimental silencing of EWS-FLI1, or ectopic stimulation of NOTCH activity, p53 was found to be reactivated leading to tumour cell growth arrest. Stability and activity of p53 is generally regulated by protein modifications. In the reporting period, we identified impaired acetylation as the cause of p53 in- activation in EWS-FLI1 expressing Ewing sarcoma cells. A deacetylase, SIRT1, which links cellular metabolism to p53 regulation, was identified as a NOTCH suppressed target. In the presence of EWS-FLI1, NOTCH signaling is repressed, and consequently SIRT1 expression is induced preventing p53 from activating acetylation. There is still relatively little known about the nature of the cells that are found in LCH, except that they seem to be immature dendritic cells (DC) that exhibit much of the surface markers of Langerhans cells – hence also the name Langerhans Cell Histiocytosis. In order to elucidate the relationship of the LCH-DCs with normally occurring DC subsets, we have performed comparative gene expression analysis of LCH cells and three major, functionally divergent human DC lineages, i.e. epidermal LC, myeloid dendritic cells (mDC1), and plasmacytoid dendritic cells (pDC) in close collabo ration with Ernst Kriehuber from the Department of Dermatology/Medical University of Vienna. We found that LCH cells are distinct from LC, mDC, and pDC and display a unique combination of hallmark lineage antigens, indicating that these cells form a separate DC entity. Interestingly, LCH cells seem to be equidistantly related to mDC1 and LC, challenging the idea that LCH cells are aberrant LCs. We found that the proximal regions of EWS-FLI1 repressed genes are enriched in recognition motifs of FOX1 transcription factors, suggesting an indirect mode of transcriptional repression of a subset of EWS-FLI1 controlled genes. We identified the responsible protein as the tumor suppressor FOXO1 and characterised a multilayered mechanism of FOXO1 repression by EWS-FLI1 through transcriptional and post-transcriptional mechanisms. Reactivation of FOXO1 in Ewing sarcoma cells resulted in cell death induction. We identified a small molecule capable of inducing active FOXO1 in Ewing sarcoma cells associated with significantly reduced tumour growth in an in-vivo disease model. These results provide a proof of principle for a successful pharmacological intervention downstream of EWS-FLI1 and potentially hold promise for innovative therapeutic strategies in Ewing sarcoma [2]. Using a pharmacological SIRT1 inhibitor, we were able to re-activate the tumour suppressor p53 and induce cell death in Ewing sarcoma cells in vitro. These findings suggest that SIRT1 inhibition may hold therapeutic promise, which now needs to be confirmed in an in vivo tumour model. About 10% of Ewing sarcomas carry p53 mutations that perturb its tumour suppressive function. This subgroup has a particularly bad prognosis [5]. P53 mutations frequently change the normal structure of the protein. We have been validating the potentially therapeutic efficacy of a p53 refolding drug. Preliminary in vitro experiments suggest that p53 refolding drugs can activate at least some normal p53 stress response functions and induce cell death. However, we also identified a so far unknown high variability in responsiveness to this drug which cannot merely be assigned to the specific type of p53 mutation but also to other, still uncharacterised host factors. Together, our studies identified several novel drug targets downstream of EWS-FLI1 in Ewing sarcoma. Future studies will focus on their therapeutic potential in combination chemotherapy. In addition, we have identified transcripts that are uniquely expressed by LCH cells. Among these transcripts is the NOTCH ligand JAG2, which is highly expressed in LCH. LCH cells also express NOTCH1 and because LCH cells within the lesions often form densely packed aggregates, it is conceivable that efficient stimulation of the NOTCH signaling pathway occurs in these cells in trans, leading to activation of NOTCH1 downstream targets. We demonstrated that JAG2 signaling can induce LCH-like features in vitro: JAG2 signaling induces the differentiation of monocytes into cells carrying the LCH hallmark markers CD1a and CD207 and can induce matrix metallo proteases which are highly expressed in LCH lesions and might be responsible for local tissue destruction. Since NOTCH inhibitors are currently evaluated in clinical trials and would therefore also be available for the treatment of LCH, the hypothesis that NOTCH signaling is a player in LCH pathogenesis is interesting also from a clinician’s perspective, since it could set off new approaches to the treatment of this perplexing disease [5]. Solid Tumours Specific project 1 ETS, E2F and FOX transcription factors are distinct families of gene regulatory proteins that bind to DNA at specific recognition motifs. 2 NOTCH is a transmembrane receptor involved in cellular communication playing a role during morphogenesis and differentiation of many tissues. In collaboration with Sven Bilke and Paul Meltzer, Genetics Branch, National Cancer Institute, NIH, Bethesda, USA Reinhard Kofler, Medical University, Innsbruck, Austria Udo Kontny, University Children’s Hospital Freiburg, Germany Ernst Kriehuber, Dept. Dermatology, Medical University, Vienna, Austria Elizabeth Lawlor, University of Michigan, Ann Arbor, USA Isidro Machado and Antonio Llombart-Bosch, Dept. Pathology, University of Valencia, Spain Katia Scotlandi, Rizzoli Institute, Bologna, Italy For further reading [1] Raphaela Schwentner PhD thesis: Mechanisms of target regulation by the chimeric oncogene EWS-FLI1 in Ewing’s sarcoma [2] Stephan Niedan PhD thesis: Mechanisms of transcriptional repression by EWS-FLI1 in Ewing sarcoma [3] Ban, Bennani-Baiti et al. 2008 [4] Bennani-Baiti, Aryee et al. 2011 [5] Hutter, Kauer et al. 2012 Fig. 1 – Immunohistochemical staining of a LCH lesion using an antibody against full-length NOTCH1. Our data indicate that the Notch signalling pathway could play an important role in the pathogenesis of LCH. 46–47 Specific project St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Specific project Immunology Tumour Immunology Immunologie Tumor-Immunologie Group leader Thomas Felzmann [email protected] Staff scientist Alexander Dohnal Postdoct. research fellow Caterina Vizzardelli1 Diploma/MSc student Melanie Floderer2 Natalie Knapp3 Klara Soukup4 Technician Angela Halfmann 1 from Jan. 2011 to Dec. 2012 2 Jul. 2011 to Aug. 2013 3 since Jun. 2011 4 since Jul. 2012 During the last decades, dendritic cells (DC) have been identified as the most important regulatory elements in orchestrating immune responses. Studies using primary mouse DC collected from lymphoid organs, skin and other tissues, suggest that immunity is directed by DC subsets, each separately executing a distinct function. Confirmation of such DC subset-mediated immune regulation in humans is complicated by the fact that primary human tissue DC are not directly available. The majority of information regarding human DC is derived from DC differentiated in vitro from monocytes. Such studies have revealed an interesting phenomenon: rather than a differentiation into DC subtypes, time-dependent changes of the DC function were observed. This pattern of DC differentiation might represent an additional layer of immune regulation. DC respond to the notion of danger that comes in different guises to initiate an activation or differentiation process, conventionally referred to as maturation. Maturation results from contact with pathogen- or damage-associated molecular patterns. Binding of microbial pattern molecules such as lipopolysaccha rides (LPS) to Toll-like receptors (TLR) on DC signal danger causing the DC to assume a potent immune stimulatory phenotype for approximately one day. LPS exposed DC trigger robust type 1 T-helper (Th1) cell and cytotoxic T-lymphocyte (CTL) dominated immune responses characterised by the release of interleukin (IL) -12 for approximately one day. The danger model is a helpful framework for understanding immune regulation, especially in the case of anti-tumour immunity. DC reside in the entire organism, where they continuously take up, process, and present material mainly from dead cells to T-lymphocytes. As long as the DC do not encounter a danger signal, they present the material they took up in a tolerance maintaining fashion. This holds true for DC situated in the tumour tissue as well. As opposed to microorganisms, however, tumour cells – although representing a serious threat – do not normally deliver danger signals to DC. Immunregulation durch die Krebsimmuntherapie Thus, tumour antigens taken up by DC are presented to T-cells in a tolerance inducing fashion, providing a useful loophole for tumour cells to evade immune surveillance. Adding a TLR derived danger signal to the equation has the capacity to tip the balance towards immune stimulation against tumour antigens. Early DC cancer vaccine designs advocated maturation of DC with a cocktail comprised of pro-inflammatory cytokines. More recent strategies for DC-based cancer immune therapy utilise TLR agonists in order to trigger IL-12 secretion from DC causing differentiation of Th1 and CTL priming human DC. In most of the early DC cancer vaccine studies, DC were inoculated after at least 24 hours of in vitro exposure to a danger molecule such as LPS. In contrast, in the design of our pre-clinical model systems and clinical DC cancer immune therapy trials, we elected to use the DC after only 6 hours of in vitro exposure to LPS in order to take advantage of the DC’s immune stimulatory time window characterised by IL-12 release. TLR engagement, however, induces not only proinflammatory activity but – with a delay of about one day – a switch into an anti-inflammatory mode of action. This switch plays a key role in priming regulatory T-cell (Treg) mediated anti-inflammatory functions. This feedback mechanism is a safeguard against immune responses running out of control and damaging healthy tissue. In cancer immune therapy, this feedback signalling limits the power of a DC-based cancer vaccine. Thus, our objective in designing a next generation DC cancer immune therapy is to circumvent DC-mediated negative feedback regulation in order to enhance the potency of a DC cancer vaccine. In collaboration with Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria Hannover Medical School, Germany Activartis Biotech GmbH, Vienna, Austria Während der letzten Jahrzehnte wurden Dentritische Zellen (DC) als wichtigstes regulatorisches Element in der Koordinierung von Abwehrreaktionen identifiziert. Studien in Mausmodellen mit DC aus lymphatischen Organen, Haut und anderen Geweben weisen darauf hin, dass spezielle Funktionen des Immunsystems von unterschiedlichen DC-Untergruppen gesteuert werden. Nachweise, dass auch im Menschen DC die Immunregulation leiten, sind komplizierter zu erbringen, da menschliches Gewebe für Experimente nur sehr eingeschränkt zu Verfügung steht. Für Studien beim Menschen müssen die DC in vitro aus Monozyten differenziert werden. Derartige Untersuchungen weisen auf ein interessantes Phänomen hin: Anstatt einer Differenzierung in DC-Untergruppen kommt es zu zeitabhängigen Funktionsveränderungen der DC. Dieses Muster der Differenzierung von DC könnte einen zusätzlichen Bereich der Immunregulation begründen. DC reagieren auf Gefahrensignale unterschiedlicher Erscheinung entweder mit Aktivierung oder Differen zierung, was üblicherweise als Reifungsprozess bezeichnet wird. Reifung erfolgt nach Kontakt mit Pathogenen oder molekularen Mustern, die mit Zellschädigung assoziiert sind. Die Bindung von mikro biellen Molekülen wie z. B. den Lipopolysacchariden (LPS) an Toll-like Rezeptoren (TLR) wird von den DC als Gefahrensignal interpretiert. Die so aktivierten DC lösen potente Immunreaktionen aus, die von Typ-1 T-Helfer Zellen (Th1) und von zytotoxischen T-Lymphozyten (CTL) ausgeführt werden. Diese DC sind durch die Freisetzung von Interleukin (IL)-12 für etwa einen Tag charakterisiert. Das Gefahrenmodell ist ein nützliches Bezugs system, um die Regulation der Antitumorimmunität zu verstehen. DC besiedeln den gesamten Organismus und präsentieren, solange sie kein Gefahrensignal empfangen, aufgenommenes Material auf eine Weise, dass die immunologische Toleranz aufrechterhalten wird. Ähnliches gilt für DC im Tumorgewebe. Im Gegensatz zu Mikroorganismen senden Tumorzellen, obwohl sie natürlich eine große Gefahr darstellen, keine für DC verwertbaren Gefahrensignale aus. Daher schaffen Tumorantigene, die von DC aufgenommen werden, für Tumorzellen ein Schlupfloch, durch das sie der Immunüberwachung entkommen können. Werden mikrobielle Gefahrenmoleküle zugeführt, kommt es zu einer Aktivierung der DC und zur Präsentation von im Tumorgewebe aufgenommenen Antigenen an T-Zellen. In frühen Konzepten von Krebsimpfungen auf Basis von DC wurden diese entweder gar nicht oder nur eingeschränkt mit pro-inflammatorischen Zytokinen aktiviert. Aktuelle Strategien verwenden TLR Agonisten zur Aktivierung von menschlichen DC, welche über IL-12 Sekretion Th1 Zellen und CTL stimulieren. In den meisten Studien mit DC-Tumorimpfstoffen werden die DC in vitro für mindestens 24 Stunden Gefahr signalisierenden Molekülen wie z. B. LPS ausgesetzt. Im Gegensatz dazu werden in unseren präklinischen Modellen und klinischen Studien die DC schon nach 6 stündiger Aktivierung mit LPS eingesetzt, um das Zeitfenster der IL-12 Sekretion optimal zu nutzen. TLR Agonisten lösen in DC nicht nur pro-inflammatorische Prozesse aus. Mit etwa eintägiger Verzögerung schalten die DC in einen anti-inflammatorischen Modus um. Dieser Wechsel spielt eine Schlüsselrolle in der Aktivierung von regulatorischen T-Zellen (Treg), welche anti-inflammatorische Funktionen ausüben und dient als Schutz vor Immunantworten, die sich unkontrolliert gegen gesundes Gewebe richten. In der Krebsimmuntherapie schwächt dieses Feedbacksignal aber die Wirksamkeit einer Krebsimpfung. Unser Ziel beim Design einer „Next Generation“ DC-Krebs immuntherapie ist es, eine DC-vermittelte negative Rückkopplung zu umgehen und somit die Wirksamkeit der DC-Krebsimpfung zu verbessern. In Kooperation mit Ludwig Boltzmann Institut für Krebsforschung, Wien, Österreich Medizinische Hochschule, Hannover, Deutschland Activartis Biotech GmbH, Wien, Österreich 50–51 Immune regulation in cancer immune therapy Immunology Forschungsschwerpunkt St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Research focus An anti-inflammatory DC master switch DC coordinate innate and adaptive immunity to fight infections and cancer (Fig. 1). Our observations reveal that DC exposed to LPS in the presence of interferon-γ (IFN-γ) acquire a continuously changing activation/ maturation phenotype [1]. The DC’s initial mode of action is pro-inflammatory via up-regulation among others the signalling molecule IL-12, which polarises IFN-γ secreting Th1 cells. Within 24 hours, the same DC switches from the pro- into an anti-inflammatory phenotype. This is mediated by autocrine IL-10 release and secretion of soluble IL-2 receptor alpha (sIL-2RA) molecules. T-cells, when contacted with DC during their anti-inflammatory phase lose their proliferative capacity and develop Treg-like anti-inflammatory functions indicated by IL-10 secretion and elevated FoxP3 levels. Studying the kinetics of IL-12 and IL-10 expression from LPS/IFN-γ activated myeloid DC on a single cell level confirmed these observations. When T-cells are separated from DC within 24 hours, they are spared from the anti-inflammatory DC activity. We conclude that, in addition to differentiation of DC into distinct subsets, the observed sequential functional phases of DC differentiation permit the fine-tuning of an immune response. A better understanding of time-kinetic DC features is required for optimally exploiting the therapeutic capacity of DC in cancer immune therapy. Having established that DC switch from a pro- into an anti-inflammatory mode of action, we were interested in the molecular mechanisms guiding that switch. DCmediated inflammation induced via LPS is promoted by mitogen-activated protein kinase (MAPK)-activated kinase (MK)-2, a substrate of p38 MAPK. We showed that MK2 consolidates LPS-driven anti-inflammatory DC functions by modulating p38- and Erk1/2-MAPK and STAT3 signalling. In the primary LPS/p38/MK2 axis, MK2-mediated feedback inhibits p38 and positively cross-regulates Erk1/2 activity. Impairment of secondary autocrine IL-1α signalling by MK2 further decreases the IL-1α/p38 but increases the anti-inflammatory IL-10/STAT3 signalling route. Thus, blocking of MK2 activity enables DC to strengthen pro-inflammatory effector mechanisms by promoting IL-1α-mediated Th1 and Th17 but blocking Treg responses. Further, MK2 deficient DC trigger enhanced cytotoxic activity. These data suggest that MK2 exerts a profound antiinflammatory effect that prevents DC from prolonging excessive effector T-cell functions. In collaboration with Activartis Biotech GmbH, Vienna, Austria For further reading [1] Luger, Valookaran et al. 2012 Our observations will need to be taken into consideration in designing future generations of DC-based cancer vaccines. In collaboration with Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria Hannover Medical School, Germany Activartis Biotech GmbH, Vienna, Austria Lymph node 52–53 Dodging anti-inflammatory DC activity Immunology Specific project adoptive Cell Therapy DC priming DC cytotoxicity DC Tumor Fig. 1 – Tumour-immune system interactions. Antiinflammatory factors released by tumour cells avoid priming and activation of tumourspecific effector T-cells. Adoptive transfer of ex vivo manipulated DC break the immune suppressive barrier in tumours. St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Specific project Immunology Clinical Cell Biology & FACS Core Unit Immunologie Klinische Zellbiologie & FACS Core Unit Group leader Gerhard Fritsch [email protected] Staff scientist René Geyeregger Technicians Nelli Frank1 Christine Freimüller Dieter Printz Daniela Scharner Julia Stemberger Dijana Trbojevic Elke Zipperer Clinicians, St. Anna Children’s Hospital Heidrun Boztug Susanne Matthes-Mann Herbert Pichler Volker Witt 1 since May 2012 Gruppe für „Klinische Zellbiologie & FACS Core Unit“ Forschung und FACS Core Unit The main task of our diagnostics department is to provide cytometric cell analyses and cell sorting services, primarily to the St. Anna Children’s Hospital, but also to the CCRI and external research institutes. The number of applications has increased and now also includes analyses that address spherocytosis and evaluate human sperm fertility. Every year, over 60,000 cytometric cell analyses and more than 2,000 cell sorts are carried out. The main purpose of the latter is to control the donor/recipient type of the different blood cells using FISH or PCR methods in post allo-transplantation patients. Internal quality control mechanisms are in place, which conform to internal standard operating procedures (SOPs) and to JACIE (Joint Accreditation Committee-ISCT & EBMT) guidelines. In addition, we participate in the regular Austrian and German quality control trials, organised by ÖQUASTA (Austrian Society for Quality Assurance and Standardisation of MedicalDiagnostics) and INSTAND (Society for Promotion of Quality Assurance in Medical Laboratories). Our task here is to measure subtypes of blood leukocytes, stem cells, and residual leukocytes in blood products. As a FACS (Fluorescence-activated cell sorting = flow cytometry) core unit, our laboratory presently has at its disposal 4 own flow cytometers for 6 to 16 parameter cell measurements, one of which is a multi-colour device (FACSAria) for sorting cells, the others being a 4 and 13 colour device (FACSCalibur and LSRII) for cell analyses. A fourth device (17-colour Fortessa) was acquired in 2011 to replace the 16-year old FACSCalibur. In close cooperation with clinicians, researchers of the CCRI and other scientists, our laboratory offers practical and theoretical support for all questions regarding flow cytometry. Our research is focused on the detection, quantification, enrichment and selection of donor virus-specific T-cells that can fight life-threatening complications after stem cell transplantation. In this field, progress was achieved by combining our T-cell expansion protocol with a magnetic selection step to highly purify adenovirusspecific T-cells (Fig. 1: A–C). This will further reduce patients’ risk for severe side effects. For those patients for whom a matched donor with Adenovirus-specific T cells is not available, we developed a method that converts non-specific donor lymphocytes into virus-specific T cells that can fight viral infections. These two strategies could have a major impact on future clinical treatment. Der größte Bereich unserer Abteilung stellt im Rahmen von Diagnostikaktivitäten durchflusszytometrische Analysen sowie ein Zell-Sort Service zur Verfügung, primär für das St. Anna Kinderspital, aber auch für die St. Anna Kinderkrebsforschung und externe Forschungsinstitute. Die Zahl der Anwendungen hat sich ausgeweitet und umfasst neuerdings auch Analysen zur Diagnose der Kugelzellanämie oder zur Fruchtbarkeit humaner Spermien. Jährlich werden über 60.000 durchflusszytometrische Analysen und mehr als 2.000 Zell-Sortierungen durchgeführt. Letztere dienen dazu, bei fremd-transplantierten Patienten den Spender/Empfängertyp der verschiedenen Blutzellen mittels FISH- oder PCR-Methode zu überprüfen. Unsere Arbeit unterliegt internen Qualitätskontrollen sowie jenen von JACIE1. Wir nehmen regelmäßig an Rundversuchen teil, die von ÖQUASTA2 und INSTAND3 organisiert werden. Dabei ist es unsere Aufgabe, Leukozytenuntergruppen, Stammzellen und Rest leukozyten in Blutprodukten zu messen. Als FACS (Fluorescence-activated cell sorting = Durchflusszytometrie)6 Core Unit verfügt unser Labor derzeit über vier Durchflusszytometriegeräte für 6 bis 16 Parameter Zellmessungen, von denen ein Vielfarbengerät (FACSAria) dem Sortieren von Zellen dient und ein 4- bzw. 13-Farbengerät (FACSCalibur und LSRII) der Zellanalyse. Ein neues 17-Farbengerät (Fortessa) wurde 2011 angeschafft, um den 16 Jahre alten FACSCalibur zu ersetzen. In enger Zusammenarbeit mit Klinik und Forschung bietet unser Labor Unterstützung für sämtliche Fragen der Durchfluss zytometrie. Unsere Forschungsaktivitäten betreffen die Messung, Quantifizierung, Anreicherung und Selektion humaner Abwehrzellen gegen klinisch relevante Viren, die im Rahmen der Stammzelltransplantation zu lebensbedrohlichen Komplikationen führen können. Hier wurden Erfolge erzielt, indem wir unsere Expansionsmethode mit einer zusätzlichen magnetischen Aufreinigung kombinierten. Dadurch gelang es uns, hochreine Adenovirus-spezifische Spenderzellen zu isolieren, bei deren Infusion in den Patienten potentielle Nebenwirkungen praktisch auszuschließen sind. Weil es für einige Patienten keinen passenden Spender gibt, der diese wichtigen Abwehrzellen im Blut hat, ent wickelten wir eine Methode, um unspezifische Abwehrzellen so umzuprogrammieren, dass sie in der Lage sind, Virusinfektionen zu erkennen und zu bekämpfen. Beide Strategien könnten einen bedeutenden Einfluss auf zukünftige klinische Fragestellungen haben. 103 104 Tetramer DSRed-A 105 100 AdV A01-CD3 50 100 AdV A01-CD3 50 102 Multimer 88C-A 150 Multimer 88C-A 150 88C-A 150 Streptamer 100 The second largest area encompasses clinical routine and concerns the manipulation of all cells or blood products that are administered to patients as part of treatment. This takes place under sterile conditions in our good manufacturing practice (GMP) facility. After successful validation, we introduced a new freezing medium for the storage of patient cells. The preparation of mononuclear cells for clinical use in extracorporeal photopheresis (“mini ECP”) proved successful for treatment of Graft-versus-Host Disease (GVHD) in very young patients. All these activities require a valid accreditation under JACIE and certifi cation of the laboratory by the AGES (Österreichische Agentur für Gesundheit und Ernährungssicherheit) PharmMed, which we were able to renew in June 2012. 50 Clinical routine (x 1.000) 200 250 Diagnostik (x 1.000) 200 250 Research, including FACS Core Unit (x 1.000) 200 250 Diagnostics 102 103 104 105 Tetramer DSRed-A Fig. 1: A–C – Magnetic selection to highly purify adenovirus-specific T-cells. 102 103 104 Tetramer DSRed-A 105 Klinische Routine Der zweitgrößte Bereich umfasst die klinische Routine. Manipulation von Zellen oder Blutprodukten, die Patienten zur Behandlung zugeführt werden, geschieht unter sterilen Bedingungen in unserem dafür speziell zertifizierten GMP 4-Labor. Zur Lagerung von Patienten zellen verwenden wir ein neu validiertes Einfrier medium. Die Reinigung und Verwendung von weißen Blutzellen in der Extracorporalen Photopherese („Mini ECP“) erwies sich als erfolgreich zur Bekämpfung der Graft-versus-Host-Erkrankung bei sehr jungen Patienten. Diese Tätigkeiten erfordern eine gültige Akkreditierung nach JACIE, die 2013 erneuert werden muss, sowie die Zertifizierung des Labors durch die Österreichische Agentur für Gesundheit und Ernährungssicherheit (AGES) Pharmed5, welche wir im Juni 2012 erneuern konnten. 1 JACIE: Joint Accreditation Committee-ISCT & EBMT. Akkreditierungsprogramm 2 ÖQUASTA: Österreichische Gesellschaft für Qualitätssicherung und Standardisierung medizinisch-diagnostischer Untersuchungen 3 INSTAND: Gesellschaft zur Förderung der Qualitätssicherung in medizinischen Laboratorien 4 GMP: Good Manufacturing Practice 5 AGES Pharmed: Nationale Zulassungsstelle für Arzneimittel 6 FACS, FACS Core Unit: Zentrum für Durchflusszytometrie 56–57 Group of “Clinical cell biology & FACS Core Unit” Immunology Forschungsschwerpunkt St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Research focus Improved magnetic selection of Adenovirus-specific T cells Based on our previously developed method to increase purity and number of virus-specific T cells, we further improved the purity of ADV-specific T cells using magnetic cell selection. As shown in Fig. 1, the purity significantly increased to almost 100%. The combination of both methods could therefore further reduce or even avoid potential side effects induced by residual non ADV-specific T cells. This is a further step towards safer T cell therapies for patients after stem cell transplantation. Impact of pre-transplant ATG on immune reconstitution Anti-thymocyte globulin (ATG) is widely used in the conditioning regimen of children undergoing haematopoietic stem cell transplantation (HSCT) to prevent graft-versus-host-disease and rejection of the graft. However, data on immune reconstitution following ATG are scarce. We retrospectively analysed the engraftment kinetics and clinical parameters of 113 paediatric/adolescent patients (median age 11.9 years) undergoing HSCT following myeloablative conditioning with or without in vivo T-cell depletion. Patients with matched unrelated donor received either ATG-Fresenius (n=45, group 1) or Thymoglobuline Genzyme (n=30, group 2), while patients with matched sibling donor (n=38, group 3) underwent SCT without in vivo T-cell depletion. Immune cell subsets (CD4, CD8, NK, CD19, naïve CD4) were determined on day +50, +100, +180 and +360 post HSCT. Immune reconstitution in the three groups was comparable in most cell subsets (Table 1). Important differences were lower average numbers of CD4+ cells following ATG-Fresenius compared to Thymoglobuline (p=0.049) and those not receiving ATG (p=0.002). By day +180, median CD4 cells were similar in all groups. Moreover, average naïve CD4+ cells were lower in groups 1 and 2 compared to group 3 (p<0.001). Concerning infectious complications, percentages of CMV or Adenovirus reactivation in the peripheral blood tended to be higher following ATG (group 1 and 2: 23/75, 31% vs. group 3: 3/38, 8%, p=0.29), while occurrence of graft versus host disease grade 3 and 4 was similar in the three groups (group 1 and 2: 10/75, 13% vs. group 3: 3/38, 8%, p=0.47).EBV lymphoproliferative disease occurred only in patients following in vivo T-cell depletion (group 1: 3/45, 7%, group 2: 3/30, 7%). Following myeloablative conditioning, immune reconstitution of most cell subsets after day +50 is similar with or without in vivo T-cell depletion. Occurrence of naïve CD4 cells as a sign for thymic recovery is faster without ATG. Apart from transiently lower CD4 cells following ATG-Fresenius, immune reconstitution is not majorly influenced by the ATG preparation used. Neutrophil engraftment after allogoeneic CD34 infusion Neutrophil engraftment (NE) following myeloablative conditioning was retrospectively analysed in 112 children transplanted 2000–2012 in a single centre. Graft source was bone marrow in 94 and peripheral blood in 18 patients. 38 patients received grafts from matched sibling donors (MSD), whereas 74 were transplanted from matched unrelated donors (MUD). The median donor age was 27.4 years and in 47 patients the donor was <25 years. A total body irradiation (TBI)-based conditioning was used in 72 patients, whereas 39 received chemotherapy. Virus prophylaxis consisted of acyclovir in 63 and gancyclovir in 32 patients. Engraftment was defined as the first of three consecutive days with neutrophil counts > 0.5 x 10^9/L peripheral blood. Median time to NE for all 112 patients was 20 days (range: 10 – 40) after receiving a median of 4.1 x 10^6/kg total CD34+ cells and 2.4 x 10^6/kg myeloid CD34+ cells respectively. Patients given a low dose of total CD34+ cells (< 2 x 10^6/kg) engrafted equally to those given 3 – 6 x 10^6/kg CD34+ cells or more. Likewise, the number of infused myeloid CD34+ cells did not correlate with NE (Fig. 2). Neutrophil Engraftment in 112 Patients 40 35 30 25 20 15 10 5 0 0 5 10 15 all CD34+ cells [n x 10ˆ6/kg] 20 Stem cell source, donor age, conditioning, and antiviral agents did not seem to have an impact on NE. Interestingly, patients transplanted from MSD engrafted earlier than those from MUD (median 17.5 days vs. 21 days; p=0.034). A subgroup analysis of MUD recipients moreover revealed a positive correlation between NE and total CD34+ cell numbers but not for myeloid CD34+ subsets. The median NE in our cohort was significantly faster for MSD recipients, whereas neither the graft content of total CD34+ nor of the myeloid CD34+ cells had an impact on engraftment kinetics. The evaluation of donor age, stem cell source, conditioning or antiviral agents revealed no significant influence on NE. In collaboration with The RNA-Group of Niels Schaft at the Universitätsklinikum Erlangen, Germany Fig. 2 – In contrast to the widely accepted opinion that 2 – 10 x 10^6 CD34+ cells per kg patient are necessary to guarantee timely engraftment, our data show no correlation between the number of CD34+ cells infused and the time span to neutrophil engraftment following allogeneic stem cell transplantation. 45 day ANC > 0.5 x 10ˆ9/L Several weeks or months after haematopoietic stem cell transplantation (HSCT) of children, an immune system (including T cells that protect against viral infections) does hardly exist. One option is to infuse specific T cells into patients to overcome viral infections. Unfortunately, in about 10% of all cases, Adenovirus-specific T cells are not present in the donor. Therefore, we developed a protocol to convert normal non-specific T cells into T cells that can fight against viral infections. To do so, we identified the T cell receptor expressed on the surface of ADV-specific T cells which they use to recognise and kill virusinfected cells. We transferred the genetic information of this receptor into non-specific T cells, making these T cells able to recognise and kill virus-infected cells. 25 St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Conversion of non-specific T-cells into T-cells with anti ADV specificity 58–59 Immunology Specific project Immunology TransplantationImmunology Immunologie TransplantationsImmunologie Group leader Andreas Heitger [email protected] Postdoct. research fellow Ursula Hainz1 Markus Hölzl2 Birgit Jürgens Postdoct. medical fellow Sabine Heitzeneder3 PhD students Sarah Ahmadi4 Barbara Dillinger5 Edda Veith6 Diploma/MSc students Barbara Dillinger7 Mathias Hochgerner8 Margit Lanzinger9 Angelika Plach10 Stefanie Reinprecht11 Stefan Weinberger12 1 2 3 4 5 6 7 8 9 10 11 12 July 2010 – Dec. 2011 Oct. 2010 – Apr. 2012 Sept. 2008 – June 2012 since Sept. 2011 since Oct. 2012 Aug. 2007 – Apr. 2011 Jan. 2011 – Sept. 2012 since Jan. 2012 Oct. 2009 – Apr. 2011 Jan. 2012 – June 2012 July 2011 – Feb. 2012 since Oct. 2012 After hematopoietic stem cell transplantation (HSCT), the emergence of immunological tolerance is mandatory to ascertain the ultimate success of this therapeutic procedure. The particular challenge in HSCT is that donor-derived immune cells which are part of the hematopoietic system accommodate in the host and reconstitute immunocompetence against infectious pathogens, a state termed allo-specific tolerance. Human immune systems have evolved to strictly discriminate “self” from “non-self” and recognise microbiological invaders as “non-self”. After HSCT, the donor derived immune cells have to accept the host as a new self. Despite careful selection of HSCT donors and recipients for matching cellular components that are believed to define “self”, i.e. major histocompatibility (MHC) molecules, the transplanted immune cells often recognise the host organism as “non-self” and begin to mount immune reactions against HSCT recipients which can manifest as graft-versus-host disease (GvHD). GvHD occurs with an unpredictable grade of severity and an unpredictable outcome. Still, GvHD is the leading cause for transplant-related morbidity and mortality resulting in a 30 – 50% death rate and a debilitating course [1]. Additionally, the compromised immune system leaves recipients highly susceptible to otherwise harmless infectious pathogens and to a complicated course of infections. Thus, an improved understanding of the complex pathophysiology of post-transplant immunity and tolerance is of high demand to improve outcome and safety in HSCT. Regarding our focus on tolerogenesis by augmenting tryptophan metabolism, we recently showed that this capacity is limited [2]. Therefore, we expandend our efforts for a better understanding of allo-specific tolerance by initiation of new collaborations and projects with the aim to open new endeavours of targeting key processes in allo-specific tolerance. We continue to explore strategies of generating allo-specific tolerized T cells ex-vivo, i.e. to educate Umfassendes Verständnis der Stammzelltransplantation donor T cells ex-vivo to tolerate the recipient and thus make them suitable to be adoptively transferred to recipients in the post-transplant period to overcome the susceptibility to infections but not induce GvHD. In collaboration with the SME Effimune (Nantes, France) and the Havard University (Boston, USA), we have begun to investigate a unique combination of novel compounds modulating co-signals which are essential for T cell tolerance1. We also initiated an innovative project studying the autoantibody repertoire after HSCT. The auto-antibody repertoire (AAb repertoire) is an essential compartment defining the “immune self” [3]. Comparing the AAb-repertoire of HSCT donors and recipients will provide insight, whether the ability and the dynamics of restoring an appropriate AAb repertoire contribute to overall immune reconstitution and can serve as a biomarker predicting immune failure or GvHD, especially chronic GvHD. Finally, we concluded the study of the impact of mannan-binding lectin in HSCT [4] and provide solid evidence that low serum levels of MBL or MBL genotypes do not have any impact on the outcome of HSCT, thus precluding that patients with low MBL levels may profit from prophylactic MBL transfusion (manuscript in preparation). In addition, on the basic research level, we have started exploring the role of oxygen tension in governing immune reactions. Accumulating evidence suggests that low oxygen tension, as is found in most organ systems of the body, significantly impacts on the outcome of immune reactions. As a disease model, we include patients suffering from chronic granulomatous disease. 1 EU/FP-7 IAPP program “MODICELL” In collaboration with Effimune, Nantes, France Havard University, Boston, USA The Weizmann’ Institute, Rehovot, Israel Univ. Children’s Hospital Zürich, Switzerland For further reading [1] Blazar, Murphy et al. 2012 [2] Lanzinger, Jürgens et al. 2012 [3] Madi, Kenett et al. 2011 [4] Heitzeneder, Seidel et al. 2012 Die Ausbildung immunologischer Toleranz nach hämatopoietischer Stammzelltransplantation (HSZT) ist eine unabdingbare Voraussetzung für den Erfolg dieser Therapiemaßnahme. Das humane Immun system unterscheidet strikt zwischen „selbst“ und „fremd“. Nach HSZT müssen spenderabgeleitete Immunzellen lernen, den Empfänger-Organismus als „selbst“ zu akzeptieren. Trotz einer genauen Spender auswahl, bei der die molekularen Merkmale, die hauptsächlich das immunologische „Selbst“ definieren (major histocompatibility antigens, MHC Moleküle) mit dem Empfänger übereinstimmen, kann es zu Unverträglichkeitsreaktionen kommen. Diese manifestieren sich als Spender-gegen-Empfänger Krankheit (GvHD) in unterschiedlichen Schweregraden und mit unsicherem Ausgang. Die GvHD stellt immer noch die schwerste Komplikation nach HSZT dar und führt bei 30 – 50% der betroffenen Patienten zum Tod oder zu schweren Beeinträchtigungen [1]. Zudem leiden Patienten bis zur Wiederherstellung ihres Immunsystems an schwerer Immundefizienz, die zu einer erhöhten Inzidenz und Schwere von infektiösen Komplikationen nach HSZT führt. Ein verbessertes Verständnis der postHSZT Immunität ist notwendig, um die HSZT zu einem sicheren Therapieverfahren weiter zu entwickeln. Bezüglich unseres Schwerpunktes der Toleranzin duktion über den Tryptophanmetabolismus haben wir kürzlich gezeigt, dass dessen Wirkung limitiert ist [2]. Deshalb haben wir neue Forschungsprojekte initiiert. In Fortsetzung früherer Arbeiten forschen wir an Strategien zur Generierung allo-spezifisch toleranter T Zellen. T-Zellen sollen ex-vivo so ausgebildet werden, dass sie nach adoptivem Transfer HSZT Empfänger mit effizienter Immunität gegen Infektionserreger versorgen, ohne eine GvHD zu verursachen. In Zusammenarbeit mit Effimmune (Nantes, Frankreich) und der Harvard Universität (USA) testen wir eine innovative Kombination von Substanzen, die speziell in die Modulation von Ko-Signalen eingreifen. Ko-Signale sind für die endgültige Ausformung einer Immunantwort essentiell. Die getesteten Substanzen blocken Ko-stimulation über das T-Zell assoziierte CD28 Molekül einerseits und stimulieren die Ko-inhibition über den PD-1/PD-L1 Weg andererseits1. Des Weiteren erforschen wir die Rolle des Autoantikörper (AAk)-Repertoires nach HSZT. Das individuell gestaltete AAk-Repertoire stellt eine wesentliche Komponente der Definition des immunologischen „Selbst“ dar [3]. Der Vergleich des AAk-Repertoires von Spendern und Empfängern und der Verlauf seiner Rekonstitution nach HSZT kann Aufschluss darüber geben, ob und wie eine Veränderung des AAk-Repertoires mit der allgemeinen Immunrekon stitution assoziiert ist, und ob es bei der Entwicklung einer GvHD eine Rolle spielt. Im vergangenen Jahr haben wir die Studie über den Einfluss von Mannanbindenden Lektins (MBL) als Risikofaktor bei HSZT [4] abgeschlossen. Die Ergebnisse zeigen deutlich dass weder MBL Serum Spiegel noch MBL Genotypen den Transplantationsverlauf negativ beeinflussen und somit eine prophylaktische MBL Infusion keinen therapeutischen Wert bei pädiatrischer HSZT hat (Manuskript in Vorbereitung). In einem ergänzenden Projekt erforschen wir den Einfluss von Sauerstoffspannung auf Immunreaktionen. Jüngste Forschungsergebnisse zeigen, dass ein erniedrigter Sauerstoffgehalt, z. B. in entzündetem Gewebe, Immunreaktionen wesentlich beeinflussen kann. Als Krankheitsmodell inkludieren wir hier Patienten mit chronischer Granulomatose. 1 EU/FP7 Programm, „MODICELL“ In Kooperation mit Effimune, Nantes, Frankreich Havard University, Boston, USA Weizmann’ Institute, Rehovot, Israel Universitätskinderklinik Zürich, Schweiz Literaturangaben [1] Blazar, Murphy et al. 2012 [2] Lanzinger, Jürgens et al. 2012 [3] Madi, Kenett et al. 2011 [4] Heitzeneder, Seidel et al. 2012 62–63 Understanding transplantation immune tolerance across multiple levels Immunology Forschungsschwerpunkt St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Research focus The ongoing vision of the group “Transplantation Immunology” is the improvement of the feasibility and safety of paediatric hematopoietic stem cell transplantation (HSCT). We continue in our attempt to generate allo-specific tolerant T cells ex-vivo which can be safely transferred to HSCT recipients as a means to overcome post-HSCT immune insufficiency without the risk of inducing GvHD. In our in-vivo studies of co-stimulation blockade for inducing tolerance via amplified tryptophan metabolism, we discovered that a blockade of the costimulatory CD80/86 pathway by CTLA-4 fusion proteins (CTLA4-Ig) does not induce amplified tryptophan catabolism. Instead, CTLA4-Ig exerted a powerful tolerogenic effect by selectively decreasing allo-specific immune effector T cells while preserving T regulatory cells (manuscript in preparation). This prompted us to continue studying the tolerogenic potential of co-signal modulation also in human cells. The approach is based on the understanding that T cells recognising antigens receive two signals by antigenpresenting cells (APC). The first signal, generated by the stimulating antigen, activates T cells. The second signal, the co-signal, shapes the type of reaction. An ever-growing list of T cell surface molecules interacting with their ligands on APC have now been identified to serve as co-signals with either co-stimulatory or co-inhibitory function. The best studied co-stimulatory pathway is via the interaction of CD80/86 on APC and with CD 28 on T cells. CTLA4-Igs blocking this interaction have been pharmacologically designed to clinical grade and are already being used as immunosupressants in solid organ transplantation. A human CTLA4-Ig, belatacept [1], is currently probed in HSCT recipients in a phase 1 study in the USA. However, a drawback of CTLA4-Ig based treatments is a non-specific immunesuppressive effect, causing a generally immunosuppressed state in treated patients. In addition, CTLA4-Ig potentially also blocks the immune regulatory CTLA-4 pathways. In our laboratory, we found that belatacept is effective to induce a durable non-responsiveness in T cells to allogeneic stimulation. The ex-vivo tolerization approach chosen by us entails to exploit the effectiveness of belatacept but to spare patients from the general immune compromise, as belatacept will be removed from the cell product prior to adoptive transfer. However, our results so far also revealed some disadvantages of the CTLA4-Ig usage: In addition to blocking the immune regulatory pathway through CTLA-4 [2], we observed that CTLA4-Ig mainly affects the CD4+ T helper cell population. To overcome these disadvantages, we now focus on two new components. First, we currently included testing a newly developed specific CD28 antibody [2], which directly targets the CD28 molecule and leaves the counter-regulatory CTLA-4 pathway intact. Our preliminary findings show the efficacy of specific CD28 blockade across multiple donor-responder pairs in vitro. Like belatacept, however, direct CD28 blockade mainly targets CD4+ cells. Thus, to also target cytotoxic CD8+ cells, in future approaches we will include stimulation of co-inhibitory PD-1 agonist antibody in our ex-vivo cultures. This antibody is designed to stimulate the co-inhibitory PD-1/PD-L1 pathway [3], which is particularly effective in CD8 cells and promotes deletional tolerance of activated cytotoxic CD8 T cells [4]. Hence, within the next years, we will intensively study whether this innovative approach has the potential to stably and robustly generate T cell tolerance for the benefit of paediatric HSCT recipients. In collaboration with Effimmune, Nantes, France Harvard Medical University, Boston, USA. For further reading [1] Davies, Barbon et al. 2012 [2] Poirier, Mary et al. 2012 [3] Fife and Pauken 2011 [4] Keir, Butte et al. 2008 Chronic Granulomatous Disease1 Hypoxia Impacts Adaptive Immunity towards Fungal Pathogens1 Immunology Immune tolerance by co-signal modulation Specific project Chronic granulomatous disease (CGD) is an inherited immune disorder characterised by a disability to produce reactive oxygen intermediates (ROI). It is caused by a defect NADPH oxidase rendering patients highly susceptible to uncontrolled fungal or mycobacterial infections emerging on the background of hyperinflammation. Genetic defects may affect any subunit (e.g. gp91phox or p47phox) of the NADPH oxidase complex. In a murine p47phox-deficiency model, lethal pulmonary aspergillosis was reported to rely on a superoxide-dependent step in tryptophan metabolism, leading to dominant production of IL-17, defective regulatory T-cell activity and acute inflammatory lung injury [1]. In sharp contrast to these data, we showed that tryptophan metabolizing activity was fully preserved in human CGD [2]. We thus started examining whether an exaggerated Th17 response would underlie the hyperinflammatory phenotype in CGD patients. We confirmed published data showing that the induction of first-line pro-inflammatory cytokines IL-1 β, IL-6 and TNF- α was preserved in CGD patients. In a next step, we induced a Th17 response by using Th17-polarizing cytokine cocktails. Peripheral blood mononuclear cells (PBMC) from CGD patients showed decreased release of IL-17A and IFN-γ under polyclonal Th17-polarizing conditions. Furthermore, CGD patients showed genotype-dependent skewing of T helper cell response towards Th1 (gp91phox deficiency) or Th17 (p47phox deficiency) under polyclonal Th17-polarizing conditions. We are currently investigating the impact of CGD genotype on adaptive immune responses towards fungal pathogens. First evidence suggests that the genotype indeed influences the balance of T helper cell subsets (manuscript in preparation). Recent studies have shown that oxygen tensions in different tissues are often low (0.5 – 3% oxygen) and could be considered hypoxic. Furthermore, recent findings suggest that hypoxic conditions significantly affect immune responses [1]. Of note, most of our knowledge comes from experiments performed under normoxic conditions (21% oxygen). We designed a study to examine the impact of low oxygen tension on adaptive immune responses with emphasis on fungal infections. Our data show that low oxygen tension delays but does not inhibit immune response of human PBMC upon polyclonal stimulation. In contrast, antigen-specific stimulation is severely affected by hypoxia. Low oxygen culture conditions inhibit activation and proliferation of T cells upon challenge with C.albicans. Interestingly, hypoxia does not influence uptake and processing of C.albicans nor subsequent activation of APC but exerts its inhibitory potential only during following co-culture of such APC and T cells (Diploma thesis Mathias Hochgerner; manuscript in preparation). Moreover, hypoxia noticeably decreases the amount of CD4+IL-17A+ and CD4+IFN-γ+ T cells along with decreased release of IL-17A and IFN-γ upon stimulation with fungal pathogens in contrast to culturing under normoxic conditions. Current experiments focus on the direct impact of hypoxia-inducible factor 1 α on both, activation and cytokine release upon fungal stimulation. In a second approach, we investigate the influence of low oxygen tension on the balance of regulatory T cells and effector T-cell subsets (Diploma thesis Stefan Weinberger). 64–65 Specific project In collaboration with Janine Reichenbach, University Children’s Hospital Zürich, Switzerland Ronnie Chee, Royal Free Hospital & University College London, UK Gregor Dückers, HELIOS Klinikum Krefeld, Germany For further reading [1] Romani, Fallarino et al. 2008 [2] Jürgens, Fuchs et al. 2010 1 Coordinated by Birgit Jürgens For further reading [1] Nizet and Johnson, 2009 St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Specific project Immunology Development of Cellular Therapeutics Immunologie Entwicklung zellulärer Therapeutika Group leader Wolfgang Holter1 [email protected] Staff scientist Manfred Lehner1 PhD Student Julia Proff1 1 since Apr. 2012 The research of the group focuses on the development of adoptive therapies based on T cells, modified with chimeric antigen receptors (CARs). First CARs directed against antigens expressed on the surface of malignant and virally infected cells were developed and published [1][2] at the Children’s Hospital of the University Hospital Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, where the group worked until they moved to the CCRI in April 2012. The study of the biology of dendritic cells (DCs) with the aim of generating optimised vaccines was also part of the research in Erlangen and was recently published, too [3]. CAR modified T cells In clinical studies, it has been recently proven that adoptive immunotherapy based on the transfer of T cells modified with CARs mediates impressive regression and even cure of tumours. Thus, the approach is currently one of the most promising forms of cancer immunotherapy. Our prime goal is to establish new immuno-therapeutic options based on CAR modified T cells for several paediatric malignancies, including Ewing Sarcoma, glioma and certain types of leukaemia. Additionally, we hypothesise that the CAR-T cell approach could also be adapted for the treatment of infections with Cytomegalovirus (CMV), which is still an important complication in transplant patients. Entwicklung zellulärer Therapeutika Regulation of the survival of DCs The optimisation of the differentiation and maturation of DCs has been considered a key issue in the field of DC vaccination for a long time. Strong co-stimulatory potential of the DCs as well as the capacity for migration into the lymph nodes in conjunction with the ability to produce cytokines supporting the differentiation of Th1 T cells were defined as key criteria for this optimisation. In our recent research, we focused on the characterisation of conditions and factors regulating the lifespan of DCs, which might also be important for the efficacy of the vaccines. For further reading [1] Lehner, Götz et al. 2012 [2] Full, Lehner et al. 2010 [3] Lehner, Kellert et al. 2012 Der Forschungsschwerpunkt der Gruppe liegt auf der Entwicklung adoptiver Therapieansätze basierend auf T-Zellen, welche mit chimären Antigenrezeptoren (CARs) modifiziert werden. Bis zum Umzug an das CCRI im April 2012 arbeitete die Gruppe an der Kinderklinik des Universitätsklinikums Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg. Dort wurden erste CARs mit Spezifität für Antigene, welche auf malignen Zellen bzw. Virus-infizierten Zellen exprimiert werden, entwickelt und publiziert [1][2]. Darüber hinaus war in Erlangen auch die Untersuchung der Biologie Dendritischer Zellen (DCs) mit dem Ziel der Herstellung von optimierten DC-basierten Vakzinen im Fokus der Forschung [3]. CAR modifizierte T-Zellen scFv NKG2D lgG1 CM CD28 CD3ζ Fig. 1 – Domain structure of CARs with antigen binding domains derived from either antibodies (scFv, single chain variable region fragment directed against CMV-gB) or from the extracellular domain of the receptor NKG2D. The receptors contain IgG1 as stabilizing spacer and the signalling domains of CD3zeta and receptor CD28 (CM, cytoplasmic membrane). In kürzlich durchgeführten klinischen Studien bewies die adoptive Immuntherapie basierend auf dem Transfer von CAR-modifizierten T-Zellen eine beachtliche Wirksamkeit mit einer beeindruckenden Rückbildung bis hin zur Heilung von Tumoren. Dieser Ansatz zählt daher gegenwärtig zu einer der vielversprechendsten Formen der Tumor-Immuntherapie. Das Hauptziel unserer Forschung ist die Etablierung von neuen immuntherapeutischen Optionen für verschiedene kindliche maligne Erkrankungen, u.a. für Ewing Sarkome, Gliome und bestimmte Formen der Leukämie. Zusätzlich postulieren wir, dass sich CAR-modifizierte T-Zellen auch für die Behandlung von Infektionen mit dem Cytomegalovirus (CMV), welche immer noch eine bedeutende Komplikation in Transplant-Patienten darstellen, eignen könnten. Regulation der Lebensdauer von DCs Lange Zeit galt im Feld der DC Impfung die Optimierung der Differenzierung und Reifung von DCs als Schlüssel thema. Dabei betrachtete man die Fähigkeit zur starken Ko-stimulation sowie das Migrationspotential in die Lymphknoten und die Produktion von Th1-differenzierenden Zytokinen als entscheidende Parameter. Wir fokussierten uns zuletzt auf die Charakterisierung von Bedingungen und Faktoren, welche die Lebensdauer von DCs bestimmen, die wiederum für die Wirksamkeit von DC-Vakzinen essentiell sein dürfte. Literaturangaben [1] Lehner, Götz et al. 2012 [2] Full, Lehner et al. 2010 [3] Lehner, Kellert et al. 2012 68–69 Development of cellular therapeutics Immunology Forschungsschwerpunkt St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Research focus In order to redirect T cells to ES, we have generated and characterised a CAR based on the ectodomain of the activating NK cell receptor NKG2D. NKG2D recognises eight ligands, which are expressed on many transformed but not on most normal cells. NKG2D-ligands (NKG2D-Ls) are expressed by various tumours including ES and are important in tumour immunosurveillance. We analysed in detail the expression of MICA, MICB, ULBP-1, -2, and -3 in fourteen ES cell lines. All tested cell lines expressed at least one NKG2D-L. In order to retarget T cells to these proteins, we designed a NKG2D-based CAR of the second generation with an integrated CD3z/CD28-derived co-stimulatory signaling domain. Opposite transmembrane orientation of this signaling domain and of NKG2D required inverse orientation fusion of either of them. We hypothesised that the extracellular domain of NKG2D, due to its particularly located C-terminus, could be fused in inverse orientation and found indeed the resulting fusion protein to specifically recognise NKG2D-Ls. High level surface expression of the chimeric NKG2D (chNKG2D) in recipient T cells was achieved by RNA transfection but was followed by unexpected rapid down-modulation upon target-cell recognition. In contrast, lentivirally mediated chNKG2D expression was resistant to receptor down-modulation and allowed sustained ligand-dependent expansion of modified T cells. Target cell death was induced by activated but not by resting chNKG2D modified T cells. Importantly, ES cells with even low NKG2D-L expression were killed by both CD8pos and CD4pos cells (Fig. 2). Our data suggest that an adoptive T cell approach based on chNKG2D should be further pursued to target ES and possibly other malignomas. Thus, our next step will be the in vivo investigation of the approach in an ES xenotransplantation model. For further reading Lehner, Götz et al. 2012 % specific lysis Ewing sarcoma (ES) is the second most frequent type of primary bone cancer in children and young adults. Survival rates for localised ES have improved to about 70% with aggressive chemotherapy and local control. Long-term survival rates achieved by conventional cytotoxic chemotherapy in patients with metastatic or recurrent disease, however, have reached a plateau of around 20% over the last 20 years. effector/target ratio � chNKG2D � αCMV-gH-CAR control d1 control d4 Fig. 2 – The diagram shows the lytic activity of CD3 activated CD8 and CD4 positive T cells transfected with mRNA for either the chNKG2D or a similar CMV-specific control receptor. Shown is the specific lysis of the Ewing sarcoma cell line STA-ET-11, characterized by lowlevel expression of NKG2D-Ls, at the indicated effector:target ratio (mean ± S.D. of triplicates). gB-CAR CEA-CAR chNKG2D no CAR Fig. 3 – Supernatants from CAR expressing T cells inhibit CMV infection. Human fibroblasts were infected with CMV (encoding GFP) in presence or absence of supernatants. Progression of infection was monitored by counting green fluorescent fibroblasts one (d1) and four days (d4) later (white bars: no supernatant; gray bars: supernatant from T cell co-culture with non-infected fibroblasts; black bars: supernatants from T cell co-culture with infected fibroblasts). Retargeting T cells to viral glycoproteins for adoptive therapy of CMV TNF is critical for the survival of human dendritic cells Immunology Targeting Ewing sarcoma by T cells expressing a chimeric NKG2D receptor Specific project Cellular immunity is required for controlling human CMV infections in vivo. Despite close monitoring followed by preemptive therapy, reactivation of viral disease in immunocompromised patients can cause life-threatening complications. An attractive treatment strategy is the application of virus specific cytotoxic T cells isolated from blood of CMV positive donors and expanded in vitro for adoptive immunotherapy. However, this therapy is not applicable in the high risk subgroup of stem cell transplant patients characterised by donor CMV sero-negativity and patient CMV sero-positivity. Together with Prof. Dr. Armin Enssers group at the Institute for Clinical and Molecular Virology in Erlangen, we constructed a CAR targeting the CMV glycoprotein B (gB) which shall allow to develop an adoptive CMV directed immunotherapy. Activated T cells engrafted with the construct by electroporation of in vitro transcribed RNA showed specific effector functions like degranulation and cytotoxic activity after co-incubation with gB-expressing target cells. In addition, redirected T cells specifically released IFN-γ and TNF after stimulation with CMV infected human foreskin fibroblasts (HFF) and conferred inhibition of CMV replication by soluble factors as supernatants of co-cultures of T cells and infected HFFs inhibited CMV infection (Fig. 3). In ongoing experiments, we examine differentially activated T cells as well as NK cells engaged with our CAR for their potential to lyse infected HFF to identify the most suitable vehicles for CMV directed adoptive immunotherapy. The lifespan of DCs is determined by the balance of pro- and anti-apoptotic proteins. Using serum-free culture of human monocyte-derived DCs, we showed that DCs following TLR stimulation with poly(I:C) or LPS underwent apoptosis which was correlated with low TNF production. Apoptosis was prevented by the addition of exogenous TNF or by concomitant stimulation with R-848, which strongly amplified endogenous TNF production. In the contrary, neutralisation of TNF confirmed that DC survival was mediated by autocrine TNF induced either by stimulation with R-848 or also by ligation of CD40. 70–71 Specific project In collaboration with Prof. Armin Ensser, Institut für Klinische und Molekulare Virologie, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany Notably, DCs stimulated by poly(I:C) or IFN- α, another known inducer of DC apoptosis, were characterised by high levels and activation of the pro-apoptotic protein BAK. Further investigation showed that the ratio of anti-apoptotic BCL-2 to BAK correlated best with the survival of activated DCs. In line with this observation, the addition of TNF increased this ratio, but had little effect on BAX and XIAP. Knockdown experiments using siRNAs confirmed that the survival of activated and also of immature DCs was regulated by BAK and showed that TNF was protective only in the presence of FLIPL. Our study demonstrates that the survival of DCs during differentiation and activation strongly depends on autocrine TNF and that the inhibition of BAK plays an important role in this process. In collaboration with Prof. Eckhart Kämpgen, Department of Dermatology, Universitäts klinikum Erlangen; Prof. Martin Leverkus, Department of Dermatology, Venereology, and Allergology, Medical Faculty Mannheim, University of Heidelberg, Germany For further reading Full, Lehner et al. 2010 Lehner, Kellert et al. 2012 St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Specific project Molecular Microbiology and LabDia Labordiagnostik GmbH Molecular Microbiology Molekulare Mikrobiologie und LabDia Labordiagnostik GmbH Molekulare Mikrobiologie Group leader Thomas Lion [email protected] Staff scientist Reinhard Klein Postdoct. research fellow Renate Kastner1 PhD students Mirza Ibrisimovic2 Doris Kneidinger Diploma/MSc students Nikola Vidovic3 Florian Bellutti4 Technicians Helga Daxberger5 Elisabeth Geiger Dragana Jugovic5 Michaela Neßlböck5 Sandra Preuner Sophie Renner5 Clinicians, St. Anna Children’s Hospital Sabine Breuer Philipp Eickhoff Tamás Fazekas Andreas Vécsei 1 since Jan. 2012 2 until Dec. 2012 3 since Jan. 2012 4 until Sept. 2012 5 Dept. Molecular Genetic Analysis and Molecular Microbiology, LabDia LabDia Labordiagnostik GmbH The SME LabDia Labordiagnostik GmbH was established in the year 2006 (with Prof. Thomas Lion, MD, PhD, as Medical Director) as a non-profit subsidiary of the St. Anna Children’s Cancer Research Organisation, with the aim to promote development and performance of innovative diagnostics. The main areas of our activity include haematology/oncology and infection. Das Ambulatorium LabDia Labordiagnostik GmbH wurde im Jahr 2006 (mit Prof. DDr. Thomas Lion als ärztlichem Direktor) als gemeinnütziges Tochter unternehmen der St. Anna Kinderkrebsforschung mit dem Ziel gegründet, neue diagnostische Verfahren zu entwickeln und anzubieten. Die Schwerpunkte unserer Tätigkeit liegen in den Bereichen Hämatologie/ Onkologie und Infektiologie. In close collaboration with the St. Anna Children’s Cancer Research Institute (CCRI) and other national and international research centres, we continuously establish new assays and introduce them into clinical diagnostics. In enger Zusammenarbeit mit dem CCRI und anderen Forschungseinrichtungen werden laufend neue Methoden etabliert, validiert und in die klinische Diagnostik eingeführt. All diagnostic tests provided are based on our own developments, some of which have been patented. We actively participate in national and international boards focusing on the standardisation of different diagnostic methodologies, and have coordinated European activities in the area of diagnostic development in EU-funded projects. LabDia offers the newest technical know-how and high competence in the areas of diagnostics provided. In addition to our activities as a diagnostic reference centre for national and international therapeutic trials, LabDia also offers its services to individual patients treated at various centres in Austria and other countries. The most important diagnostic methodologies currently offered by LabDia include cytogenetic analyses, fluorescence in situ hybridization (FISH) assays, qualitative and quanti tative PCR tests, pharmacokinetic analyses, flow cytometry and cell sorting by FACS, and investigation of DNA mutations by various techniques. LabDia is a European-certified reference centre for comprehensive diagnostics of chronic myeloid leukaemia, which is an important focus within our spectrum of diagnostic activities. The current panel of diagnostic tests offered by individual divisions of LabDia Labordiagnostik is displayed on our website (www.labdia.at). Unsere diagnostischen Leistungen beruhen auf eigenen, zum Teil patentierten Entwicklungen. Wir arbeiten in nationalen und internationalen Gremien an der Standardisierung verschiedener diagnostischer Verfahren mit und haben auch europäische Aktivitäten in der Diagnostikentwicklung im Rahmen von EU-Projekten koordiniert. Das Ambulatorium LabDia verfügt über neuestes technisches Know-how und eine hohe fachliche Kompetenz in den Bereichen der angebotenen Diagnostik. Neben unserer Tätigkeit als diagnostisches Referenzzentrum in nationalen und internationalen Studien, bietet LabDia sein Diagnostikspektrum auch für individuelle Patienten aus dem In- und Ausland an. Unsere wichtigsten diagnostischen Methoden umfassen zytogene tische Analysen, Fluoreszenz in situ Hybridisierungs (FISH)-Assays, q ualitative und quantitative PCR Testmethoden, pharmakokinetische Bestimmungen, Durchfluss-Zytometrie und Zellsortierung mittels FACS sowie Analysen von DNA-Mutationen. LabDia ist unter anderem ein europäisch zertifiziertes Referenz zentrum für Diagnostik bei chronisch myeloischer Leukämie (CML). Aktuelle Leistungen der LabDia Labordiagnostik GmbH können der Website (www.labdia.at) entnommen werden. Medical Director Thomas Lion [email protected] Vice Director Oskar A. Haas Cytogenetics and Molecular Cytogenetics Gudrun Divoky1 Ulrike Engel Brigitte Grimm Susanna Koskela Margit König Bettina Nocker Gertrud Pass Eva Winkler Wohlmacher Sven Pharmacological Diagnostics Ulrike Kastner Clinical Cell Biology & FACS Core Unit 2 Gerhard Fritsch Christine Freimüller René Geyeregger Dieter Printz Julia Stemberger Dijana Trbojevic Elke Zipperer MRD Diagnostics2 Renate Panzer-Grümayer Susanne Fischer Andrea Inthal1 Ruth Joas Solid Tumour Diagnostics2 Peter Ambros Ingeborg Ambros Bettina Brunner Andrea Ziegler Immunological Diagnostics2 Michael Dworzak Angela Schumich Susanne Suhendra Molecular Genetic Analysis and Molecular Microbiology2 Helga Daxberger Elisabeth Geiger Dragana Jugovic Michaela Neßlböck Sandra Preuner Sophie Renner 1 maternity leave 2 CCRI groups that contribute to the LabDia diagnostic activities. 74–75 LabDia Labordiagnostik GmbH Molecular Microbiology Molekulare Mikrobiologie St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Molecular Microbiology Research, development (R&D) and diagnostics in the field of microbiology and chronic myeloid leukaemia (CML) are the major areas of activity in our division. The exploitation of diagnostic assays emanating from our R&D work and the performance of specific developmental tasks were transferred to LabDia Labordiagnostik, a non-profit institution established in 2006 as a subsidiary of the St. Anna Children’s Cancer Research Organisation. Our work is primarily focused on infectious problems in oncological patients undergoing allogeneic stem cell transplantation (allo-SCT) or chemotherapy. In addition to bacteria, viral and fungal pathogens frequently cause life-threatening infections in severely immunocompromised children. Early and reliable diagnosis is essential for successful therapy. We have therefore developed and, in part, patented quantitative molecular detection assays for many clinically relevant viruses and fungal species. We were able to demonstrate that the clinical implementation of various methods developed in our division permits early assess ment of impending infectious complications [1][2]. Our new diagnostic approaches facilitate improved treatment of life-threatening infections in immuno compromised patients. Moreover, we have developed the basis for novel therapeutic strategies against invasive infections with adenoviruses which are particularly dangerous for children undergoing allogeneic stem cell transplantation [3][4][5]. An additional focus of our activities is the diagnostic monitoring of patients after allo-SCT. In a European project coordinated by our group, we have established a standardised methodology for quantitative analysis of patient- and donor-derived cells (chimerism) [6]. The patented technique is already commercially available as a diagnostic kit. Moreover, another recently developed diagnostic approach to early prediction of graft rejection will facilitate timely therapeutic interventions [7]. Molekulare Mikrobiologie The recent development and clinical implementation of a molecular technique for the surveillance of mutant, therapy-resistant subclones in patients with CML [8] provided important information on clonal evolution of the disease, timely detection of resistance and clone-specific responses to treatment [9]. These new insights will facilitate the advancement of research to improve our understanding of the dynamics of CML. We will pursue this task in a recently granted long-term project (Special Research Area Program funded by the Austrian Science Fund). In addition to our R&D program, we provide services as a reference laboratory for molecular diagnostics within national and international therapy trials in the fields of infectious diseases and leukaemia (ALL-BFM, AML-BFM, CML-Ped, CML-11-CELSG, BFM-ALL-SCT, EWING, ENEST-1st, ENEST-Path, CARRAFLU), and we perform clinical studies addressing infectious disease-related issues. For further reading [1] Landlinger, Preuner et al. 2010 [2] Lion, Kosulin et al. 2010 [3] Kneidinger, Ibrišimović et al. 2012 [4] Ibrišimović, Kneidinger et al. 2012 [5] Ibrišimović, Kneidinger et al. 2012 [6] Lion, Watzinger et al. 2012 [7] Breuer, Preuner et al. 2012 [8] Preuner, Denk et al. 2008 [9] Preuner, Mitterbauer et al. 2012 Die Tätigkeitsschwerpunkte unserer Abteilung liegen im Bereich der mikrobiologischen Forschung und Entwicklung sowie der chronisch myeloischen Leukämie (CML). Die Entwicklung neuer diagnostischer Verfahren und deren klinische Implementierung wurden in das gemeinnützige Ambulatorium LabDia Labordiagnostik GmbH übertragen, das 2006 als Tochterunternehmen der St. Anna Kinderkrebsforschung gegründet wurde. Im Bereich der Mikrobiologie beschäftigen wir uns vor allem mit infektiologischen Problemen von allogen knochenmarktransplantierten und onkologischen Patienten unter Chemotherapie. Bei schwer immun geschwächten Kindern stellen neben bakteriellen Infektionen insbesondere Virus- und Pilzinfektionen eine sehr ernste Bedrohung dar. Eine frühzeitige und verlässliche Diagnostik ist eine wichtige Voraussetzung für erfolgreiche Therapien. Wir haben daher quanti tative molekulare Detektionsmethoden für viele pathogene Viren und klinisch relevante Pilzformen etabliert und zum Teil patentiert. Wir konnten bereits zeigen, dass der klinische Einsatz unserer Methoden eine frühzeitige Vorhersage drohender infektiologischer Komplikationen ermöglicht [1][2]. Diese neuen diagnos tischen Verfahren tragen maßgeblich zur Behandlungsverbesserung lebensbedrohlicher Infektionen von schwer immungeschwächten Patienten bei. Darüber hinaus wurden für invasive Infektionen mit Adenoviren, die insbesondere bei Kindern nach allogener Stammzelltransplantation zu schweren Erkrankungen führen können, Grundlagen für neue Therapiestrategien erarbeitet [3][4][5]. Ein weiterer wichtiger Bereich unserer Tätigkeiten ist die diagnostische Überwachung von Patienten nach allogener Stammzelltransplantation. Im Rahmen eines von uns koordinierten europäischen Projektes haben wir eine standardisierte Methodik für das quantitative Monitoring von Spender- und Empfängerzellen (Chimärismus) etabliert [6]. Das patentierte Verfahren steht bereits als kommerziell erhältlicher, diagnostischer Kit zur Verfügung. Darüber hinaus haben wir eine Methodik für die frühzeitige Vorhersage von Transplantatabstoßungen entwickelt, die künftig als Grundlage für zeitgerechte Therapiemaßnahmen dienen wird [7]. Die rezente Entwicklung und klinische Anwendung eines molekularen Verfahrens zur Überwachung mutierter Subklone bei Patienten mit CML, die mit Therapieresistenz einhergehen [8], hat es uns ermöglicht, wichtige Erkenntnisse über die klonale Evolution der Erkrankung, die frühzeitige Resistenzerkennung und das klon-spezifische Therapieansprechen zu gewinnen [9]. Auf der Grundlage dieser Erkenntnisse planen wir weiterführende Forschungsprojekte, die zu einem besseren Verständnis der Dynamik der CML beitragen sollen. Diese Untersuchungen werden im Rahmen eines kürzlich bewilligten Sonderforschungsbereichs (SFB-FWF) durchgeführt. Neben unserem Forschungs- und Entwicklungs programm stellen wir als Referenzlabor für molekulare Infektions- und Leukämiediagnostik Serviceleistungen für nationale und internationale Therapiestudien (ALL-BFM, AML-BFM, CML-Päd, BFM-ALL-SCT, EWING, ENEST-1st, ENEST-Path, CARRAFLU) zur Verfügung und führen eigene klinische Studien mit infektiologischen Fragestellungen durch. Literaturangaben [1] Landlinger, Preuner et al. 2010 [2] Lion, Kosulin et al. 2010 [3] Kneidinger, Ibrišimović et al. 2012 [4] Ibrišimović, Kneidinger et al. 2012 [5] Ibrišimović, Kneidinger et al. 2012 [6] Lion, Watzinger et al. 2012 [7] Breuer, Preuner et al. 2012 [8] Preuner, Denk et al. 2008 [9] Preuner, Mitterbauer et al. 2012 76–77 Molecular Microbiology Molecular Microbiology Forschungsschwerpunkt St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Research focus We hypothesised that sensitive detection of mutant subclones and monitoring of their proliferation kinetics could permit assessment of their biological behavior and resistance to ongoing treatment. To address this question, we have developed a quantitative PCR-based technique (LD-PCR), permitting the detection of individual mutations with a detection limit of ≥1%, and facilitating highly reproducible and accurate assessment of changes in the size of mutant subclones exceeding ± 5% [1]. We have subsequently investigated prospectively collected serial blood specimens derived from CML patients displaying a variety of mutations. The results provide new insights into the evolution of point-mutated subclones during the course of therapy. Our data demonstrate that the quantitative LD-PCR approach permits early detection of relevant mutant subclones, and enables accurate surveillance of subclone evolution. Mutant subclones may appear and expand rapidly after onset or change of TKI-therapy, and subclone-specific response to treatment can be readily documented [2]. The rapid appearance of mutant cells following changes of treatment supports the notion that CML is composed of various subclones. Many of them carry point mutations in the TKD, indi cating a non-linear, branching clonal architecture of the disease. Detection of expanding mutant subclones by LD-PCR mostly paralleled, but occasionally preceded the rise in BCR-ABL transcripts by several weeks. Quantitative monitoring of mutant subclones during treatment with TKIs provided information on their actual responsiveness to therapy, and facilitated early detection of imminent evolution of resistant disease. Implementation of quantitative approaches to the monitoring of mutant subclones in the surveillance of CML patients can therefore improve our current options for timely treatment decisions. This may help optimise disease management in patients displaying point mutations in the BCR-ABL TKD or other sites of potential relevance. The observations made in patients with CML may serve as a paradigm for similar studies in patients with other haematologic neoplasias to assess the role of mutant subclone formation and kinetics in the evolution of resistant disease. Further research into phenomena related to the subclonal architecture of specific leukaemias will be pursued with financial support of a recently awarded SFB grant from the Austrian Science Fund. Adenoviruses (AdVs) are a major threat to severely immunocompromised patients, including particularly children undergoing allogeneic stem cell trans plantation (allo-SCT). We have shown that AdVs can persist in several tissues in a latent state after primary infection. Reactivations in immunosuppressed patients can lead to life-threatening disseminated disease. We have demonstrated that invasive infections in the allo-SCT setting almost invariably originate from the gastrointestinal tract. Patients at risk of invasive infections need timely therapeutic intervention. We have therefore established diagnostic strategies facilitating early onset of treatment [1]. However, currently available therapy options do not always permit control of the disease, and novel approaches are therefore urgently needed. The current dilemma in treating AdV-mediated infections in severely immunocompromised patients prompted us to explore alternative treatment strategies. Small interfering RNAs (siRNAs) and artificial microRNAs (amiRNAs) are a class of synthetic RNAs resembling cellular miRNAs. Similar to their natural relatives, they can mediate the knockdown of endogenous gene expression. This process, termed RNA interference, can be harnessed to target and potentially silence both cellular and viral genes. We determined that silencing of the expression of several adenoviral genes that are essential for viral DNA synthesis can efficiently inhibit virus replication, and consequently, the generation of virus progeny in vitro [2][3]. Furthermore, a combination with conventional antiviral drugs such as cidofovir increased the cumulative antiviral effect. These insights suggest that a combination of two very distinct approaches may permit improved control of viral infections. In a second approach [4], we demonstrated that introduction of the herpes simplex virus thymidine kinase (HSV-TK) gene and its selective expression within AdV-infected cells can inhibit the replication of human AdV in vitro, when cells are concomitantly treated with the prodrug ganciclovir. This strategy of selectively sensitising AdV-infected cells to treatment with ganciclovir may constitute another way of controlling life-threatening AdV infections in patients with severely impaired immune response. For further reading [1] Lion, Kosulin et al. 2010 [2] Kneidinger, Ibrišimović et al. 2012 [3] Ibrišimović, Kneidinger et al. 2012 [4] Ibrišimović, Kneidinger et al. 2012 Fig. 1 – Surveillance of the evolution and expansion of leukaemic subclones. Proliferation kinetics of three different mutant subclones in a patient with chronic myeloid leukaemia (CML) in response to different treatment modalities. The differential kinetics indicates the presence of mutations within the BCR-ABL tyrosine kinase domain in separate subclones, and reveals the evolution of resistant disease. The monitoring of drug-resistant mutant subclones provides a potentially important diagnostic tool in the surveillance of patients with CML and other malignant disorders. Mut clone, BCR-ABL/ABL1 (%) Recent observations in different types of leukaemia revealed their composition of multiple subclones, carrying divergent genetic alterations which may mediate differential responses to treatment. Subclones resistant to the applied treatment can expand and give rise to disease recurrence or progression. Neoplasias in which activated tyrosine kinases (TKs) represent important driver mutations can be amenable to treatment with specific tyrosine kinase inhibitors (TKIs). However, the occurrence of subclones with mutations in the TK gene conferring TKI resistance can present a serious impediment to successful therapy. We use chronic myeloid leukaemia (CML) as a model disease to study this phenomenon. CML is characterised by the BCR-ABL fusion gene encoding a constitutively activated tyrosine kinase, and was the first disease for which treatment with a specific TKI has been developed. Despite the tremendous success of this therapeutic approach, the development of resistance remains an important problem. Point mutations within the BCR-ABL tyrosine kinase domain (TKD) are currently regarded as the most important mechanism of resistance to TKIs, and more than 100 different mutations have been described to date. The screening for resistant mutations is most commonly performed by direct sequencing of the entire BCR-ABL TKD upon amplification by PCR. This approach, however, does not reveal the presence of mutant subclones constituting less than 10 – 20% of the entire leukaemic cell pool. Despite the modest detection limit of this approach, the clinical relevance of mutations within individual subclones identified by this technique can be difficult to predict. Strategies to combat Adenovirus infections in the immunocompromised host T315l E255K G250E BCR-ABL1/ABL1 For further reading [1] Preuner, Denk et al. 2008 [2] Preuner, Mitterbauer et al. 2012 HU Dasa (100 mg) HU post SCT/no HU Nilo (800 mg) Nilo (? mg) Nilo (200 mg) 78–79 Surveillance of the evolution and expansion of leukaemic subclones Molecular Microbiology Specific Project St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Specific Project Clinical Research S2_IRP: Studies & Statistics for Integrated Research and Projects Klinische Forschung S2_IRP: Studien & Statistik Group leader Ruth Ladenstein [email protected] Scientific assistant Claudia Zeiner Statisticians Evgenia Glogova Ulrike Pötschger, senior International trials Alisa Alspach Inge Hirsch1 Ingrid Pribill Marlene Reisinger2 Elfriede Thiem Leukaemia & Lymphoma studies Saelde Baumgartner2,3 Corinne Grafl Susanne Karlhuber1,3,4 Nora Mühlegger3 Dasa Janousek5 Marek Nykiel6 Solid Tumour studies Eva Sorz3 Project manager, O.K.ids Andrea Mikolasek 1 Also: Stem Cell Transplantation studies 2 Quality Management 3 Phase I/II/III Pharmaceutical Industry studies for the St. Anna Children’s Hospital 4 Also Haematology studies 5 Data Management Support 6IT-support Senior Statistician U. Pötschger, MSc Statistician E. Glogova, MSc Langerhans Cell Histiocytosis Assoc. Prof. M. Minkov, MD RSA* E. Thiem, MSc EBMT/IBFM SCT Trials Assoc. Prof. S. Matthes-Leodolter, MD Prof. C. Peters, MD RSA* I. Hirsch, MSc SIOPEN Neuroblastoma Studies Assoc. Prof. R. Ladenstein, MD, MBA, cPM RSA* A. Alsbach, BSc I. Pribill, PhD M. Reisinger, BSc Scientific Guidance, Study Coordinators & Local Investigators Assoc. Prof. A. Attarbaschi, MD Assoc. Prof. M. Dworzak, MD Assoc. Prof. G. Mann, MD RSA* N. Mühlegger, MSc C. Grafl S. Karlhuber, PhD N. Mühlegger, MSc M. Nykiel Scientific Guidance, Study Coordinators & Local Investigators Assoc. Prof. L. Kager, MD Assoc. Prof. R. Ladenstein, MD, MBA, cPM RSA* E. Sorz Scientific Guidance, Study Coordinators & Local Investigators Prof. R. Ladenstein, MD, MBA, cPM A. Lawitschka, MD Assoc. Prof. S. Matthes-Leodolter, MD Prof. C. Peters, MD V. Witt, MD RSA* I. Hirsch, MSc S. Karlhuber, PhD Scientific Guidance, Study Coordinators & Local Investigators Assoc. Prof. M. Minkov, MD Assoc. Prof. L. Kager, MD RSA* S. Karlhuber, PhD International & National Paediatric Haemato-Oncology Groups International Groups International Trials Leukaemia & Lymphoma Task Team Solid Tumour Task Team Stem Cell Transplantation Task Team Haematology Task Team O.K.ids Project Manager A. Mikolasek Phase I/II/III Pharma Industry with St. Anna Children‘s Hospital RSA* S. Baumgartner, MSc S. Karlhuber, PhD N. Mühlegger, MSc E. Sorz Quality Management S. Baumgartner, MSc M. Reisinger, BSc Data Management Support IT Support RSA* D. Janousek, MSc M. Nykiel Fig. 1 – Complete organisation chart, including clinical study leaders and research and study assistants. *RSA: Research and Study Assistant AIEOP: Associazione Italiana Ematologia Oncologia Pediatrica; www.aieop.org ANZCHOG: Australia and New Zealand Children’s Haematology/Oncology Group; www.anzchog.org BSPHO: Belgian Society of Paediatric Haemato-Oncology; www.bspho.be CCLG: Children’s Cancer and Leukaemia Group, UK; www.cclg.org.uk COG: Children’s Oncology Group, USA DCOG-ECTC: Dutch Childhood Oncology Group – Early Clinical Trial Consortium DFCI: Dana-Farber Cancer Institute, USA; www.dana-farber.org EBMT: The European Group for Blood and MarrowTrans plantation; www.ebmt.org EnPREMA: European Medicines Agency; www.emea.europa.eu FRALLE: French Acute Lymphoblastic Leukaemia Group GPOH: German Group of Paediatric HaematoOncology; www.kinderkrebsinfo.de/gpoh HSPHO: Hellenic Society of Paediatric HaematologyOncology I BFM SG: The International BFM Study Group; www.bfm-international.org ICORG: The All Ireland Cooperative Oncology Research Group; www.icorg.ie ISPHO: Israeli Society of Paediatric Haematology Oncology ITCC: Innovative Therapies for Children with Cancer; www.itcc-consortium.org LCH: HS Histiocyte Society MRC: Medical Research Council, UK; www.mrc.ac.uk NOPHO: Nordic Society for Paediatric Haematology and Oncology, Denmark, Finland, Iceland, Norway, Sweden; www.nopho.org PINDA: National Chilean Pediatric Oncology Group PPLLSG: Polish Pediatric Leukemia Lymphoma Study Group SEOP: Spanish Society of Paediatric Oncology SFCE: Société Française des Cancers et Leucémies de l’Enfant et de l’Adolescent; http://sfce1.sfpediatrie.com SAKK: Swiss Group for Clinical Cancer Research; http://sakk.ch SIOP: International Society of Paediatric Oncology, (SIOP Office c/o Kenes Associations Worldwide, Switzerland); www.siop-online.org SIOP EUROPE: European Society for Paediatric Oncology (SIOPE Office Brussels, Belgium); www.siope.eu SIOPEN: International Society of Paediatric Oncology European Neuroblastoma Study Group; www.siopen-r-net.org National Groups AGPHO: Austrian Group of Paediatric HaematoOncology ÖGKJ: Austrian Society for Paediatrics (Österreichische Gesellschaft für Kinder- und Jugendheilkunde); www.docs4you.at Clinical Research Statistics Coordinating Operative And Research Assistant C. Zeiner, MSc 82–83 Operative & Strategic Management Assoc. Prof. R. Ladenstein, MD, MBA, cPM St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Networks and Groups S2IRP: Klinische Forschung bei internationalen Studien The Unit for Studies & Statistics for Integrated Research & Projects (S2IRP) has an important role at the interface of laboratory research activities at the CCRI and the clinical application of trials at the hospital site of the St. Anna Children’s hospital. As such the S2IRP has a practical agenda to facilitate international randomised trials and to foster clinical research in paediatric oncology. Prospective trials in paediatric-haemato oncology have enabled to advance the field and to improve the outcome for children and young people suffering of cancer over the last 50 years, now achieving survival chances of up to 80% and even more in some more favourable cancer types. These trials are also the backbone for associated translational research questions and may implement new stratifying diagnostics, which support optimised, risk-adapted treatments ultimately leading to improved overall survival at a lower treatment cost with regard to long term treatment side effects and quality of life. S2IRP as a centre of expertise for studies and statistics runs prospective trials pursuant to EU directives, ICH guidelines and the Austrian Pharmaceuticals Act (AMG). The team offers advice and expertise protocol development and case report form design, database design support and appropriate use of trial management software, gaining ethical and regulatory approvals and the necessary reporting follow-up procedures, trial co-ordination, randomisation procedures, support in patient recruitment and data management. Quality assurance procedures are in place. Austrian haemato-oncologists and the team of research and study assistants are trained annually according to GCP requirements. The statistical group supports clinical investigators of the St. Anna Children’s Hospital and researchers of the CCRI with all statistical issues. This includes the statistical consultation at the design stage (statistical section of the protocol), the data analysis and the statistical support of the final publication. Within the established national and international networks of investigators in paediatric hemato-oncology (e.g. I-BFM, SIOPEN, Histiocyte-Society and EBMT) statistical groups have been formed to allow a close collaboration between statisticians in all issues related to statistical methodology. In these statistical collaborations, the CCRIstatistics is an active member. According to the requirements of the EU directive 2001 and its implementation to the Austrian Pharmaceuticals Act (AMG) in 2004, the St. Anna Kinderkrebsforschung e.V. has undertaken major efforts to secure the continuation of academicinvestigator driven trials. The St. Anna Kinderkrebsforschung e.V. assumed the responsibility as sponsor on the international and national level for these trials. Since September 2005, the research institute holds a master policy for Austria to cover trial insurance for patients on trials and investigators involved. The S2IRP is a coordinating clinical trials unit, administering registered data of more than 10.000 patients from ongoing and past national and international trials. Current AGM-conform academic trials are run in the areas of Langerhans Cell Histiocytosis (2), Leukaemias & Lymphomas (6), Solid Tumours (7), and Stem Cell Transplantation (4). In addition a number of registries are supported: Leukaemias & Lymphomas (6), Solid Tumours (4), Haematology (5) and Stem Cell Transplantation (4). A limited number of industry sponsored trials – partly (3), completely (2) – are run with help of the unit. International trials with the St. Anna Kinderkrebsforschung e.V. in the sponsor role focus on the fields of Langerhans’ cell disease (international study centre of LCH trials I, II, III), Neuroblastoma (international study centre of the European High Risk Neuroblastoma trial HR-NBL-1/SIOPEN and the LTI /ch14.18/CHO SIOPEN trial) and Stem Cell Transplantation for Acute Lymphoblastic Leukaemia (international study centre of the international ALL-SCT Study). The cumulative patient numbers within these three international studies are now: 2.893 patients in LCH trials (I, II and III), 2144 patients in the HR-NBL-1/SIOPEN trial and 1.106 patients in the ALL-SCT trials. Die Abteilung für Studien und Statistik (S2IRP) ist ein wichtiges Bindeglied zwischen dem Forschungsinstitut und dem St. Anna Kinderspital. Kernaufgaben sind die Durchführung von internationalen, randomisierten Studien und die Förderung der klinischen Forschung in der pädiatrischen Hämato-Onkologie. Prospektive Studien ermöglichten es, die Überlebenschancen für Kinder und Jugendliche mit Krebserkrankungen in den letzten 50 Jahren entscheidend zu verbessern. Die Überlebenschancen liegen bei etwa 80%, bei einigen Krebsarten mit günstigerer Prognose sogar höher. Diese Studien sind auch die Basis für assoziierte translationale Forschungsfragen. Neue, stratifizierende Diagnostik kann so implementiert werden und ermöglicht optimierte, risiko-adaptierte Therapien, welche letztendlich zu einem verbessertem Gesamtüberleben mit einer reduzierten Nebenwirkungsrate und verbesserter Lebensqualität führen. S2IRP ist ein Koordinierungszentrum (KKS) für Studien und Statistik und führt prospektive Studien gemäß EU-Direktiven, ICH Richtlinien und dem österreichischen Arzneimittelgesetz (AMG) durch. Das Team stellt Beratung und Expertise bei Protokollentwicklung und Erhebungsbögen zur Verfügung und unterstützt bei Datenbankdesign sowie Anwendung von Studien managementsoftware. Es führt Einreichungen bei Ethikkommissionen und Behörden durch und betreut die Datenerhebung, das Datenmanagement, Randomi sierungen und die Nachverfolgung. Außerdem unterstützt das Team bei der Patientenrekrutierung. Weiters verfügt das S2IRP über ein Qualitätsmanagement system. Österreichische Hämato-Onkologen und die Forschungs- und StudienassistentInnen werden einmal jährlich gemäß GCP-Auflagen fortgebildet. Die Statistik gruppe unterstützt Kliniker am St. Anna Kinderspital und Wissenschaftler der St. Anna Kinderkrebsforschung in allen statistischen Belangen. Dazu zählen statistische Beratungen in der Designphase (Statistikteil des Pro tokolls), Datenanalyse und statistische Unterstützung bei der finalen Publikation. Innerhalb national und international etablierter Netzwerke von Wissenschaftlern in der pädiatrischen Hämato-Onkologie (z. B. I-BFM, SIOPEN, Histiocyte Society und EBMT) wurden Statistikgruppen gegründet, die eine enge Zusammenarbeit im Bereich der statistischen Methodologie pflegen. Die Statistikgruppe ist aktives Mitglied dieser Kooperationen im Statistik bereich. Gemäß den Auflagen der EU-Direktive 2001 und deren Umsetzung im AMG im Jahr 2004, setzte sich die St. Anna Kinderkrebsforschung dafür ein, die Durchführung von akademischen Studien sicherzu stellen. Die St. Anna Kinderkrebsforschung übernahm die Sponsorverantwortung für diese Studien auf nationaler und internationaler Ebene. Seit September 2005 verfügt das Forschungsinstitut über eine Rahmen versicherung für österreichische Studienpatienten und Prüfärzte. S2IRP ist Koordinierungszentrum für klinische Studien und betreut Daten von mehr als 10.000 Patienten aus laufenden und geschlossenen nationalen und internationalen Studien. Derzeit laufen akademische AMG Studien in folgenden Gebieten: Langerhanszell Histiocytose (2), Leukämien & Lymphome (6), Solide Tumoren und Stammzelltransplantation (4). Zusätzlich werden folgende Register betreut: Leukämien & Lymphome (6), Solide Tumoren (4), Hämatologie (5) und Stammzelltransplantation (4). Einige Industrie studien (5) werden durch das S2IRP unterstützt. Für drei internationale Studien hat die St. Anna Kinderkrebsforschung e.V. die Sponsorrolle übernommen, nämlich für die Bereiche der Langerhanszell Histiozytose (internationale Studienzentrale der LCH-Studien I, II,III), dem Neuroblastom (internationale Studien zentrale der Europäischen Hochrisiko Neuroblastomstudie HR-NBL-1/SIOPEN und der LTI/ch14.18/CHO SIOPEN Studie) sowie für die Stammzelltransplantation für akute lymphoblastische Leukämie (internationale Studienzentrale der internationalen ALL-SCT Studie). Die kumulative Anzahl der Patienten dieser drei Studien beträgt derzeit: 2.893 Patienten in den LCH-Studien (I, II, III), 2.144 Patienten in der HR-NBL-1/SIOPEN Studie und 1.106 Patienten in den ALL-SCT Studien. 84–85 S IRP Clinical Research in International Trials Clinical Research Forschungsschwerpunkt 2 St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Research Focus Langerhans Cell Histiocytosis Study Reference Centre EHN (EuroHistioNet) project 2008–2011 Stem Cell Transplantation Study Centre Main recent tasks of the Langerhans Cell Histiocytosis (LCH)-study reference centre were the planning, designing and organising of the study protocol for the next International LCH-IV trial which opened in Austria in December 2012. Other international participating subcentres are expected to follow in 2013. The results of the LCH-III data analysis showed no benefit for patients treated with Prednisone, Vinblastine and additional Methotrexate as compared to the standard treatment arm with Prednisone, Vinblastine and 6-Mercaptopurine when referring to time to non-active disease, overall response or numbers of reactivation. In low-risk patients treated for 12 months with low-risk treatment, the reactivation rate was significantly lower than in patients treated for only 6 months. A manuscript describing the results of the LCH-III study is in preparation. Euro-Histio-Net was brought up to join efforts in increasing the knowledge of the disease LCH, improving quantity and quality of knowledge exchange and producing guidelines. Any new information will be distributed by a web portal for LCH professionals, doctors and patients at www.eurohistio.net. Acute lymphoblastic leukaemia (ALL) is the most frequent haemato-oncological disease in children. In spite of excellent survival rates of more than 80% after risk adapted front line studies according to BFM strategies, some children will suffer a relapse. Allogeneic stem cell transplantation (SCT) is a therapeutic option opening a second chance for long-term survival. The LCH study reference centre is supported i.a. by the Histiocytosis Association of America (HAA), a grant of the Österreichische Nationalbank (ÖNB) and by the European Commission. The current LCH-IV study protocol committee (chair, Assoc. Prof. Milen Minkov, MD; team, B. Fahrner, MD, Prof. Gardner, MD, C. Hutter, MD, PhD, L. Ronceray, MD) agreed upon diagnostic guidelines for LCH-IV patients. Especially, criteria for the diagnostic and follow-up evaluations of LCH lung disease, bone or central nervous system involvement were revised. On the laboratory side, Dr. Caroline Hutter investigates pathogenetic mechanisms in LCH (for details, see report of the Molecular Biology group). The associated partners of the project are four European research institutes from Austria, France, Italy and Spain and one patient association from Great Britain. The project aimed to merge information in an international database which can serve as a common platform for many different groups collecting data on LCH and associated syndromes, on paediatric and adult patients. Several people and teams worldwide produced guidelines for diagnosis, treatment and follow-up of LCH. One of the ambitious goals of Euro-Histio-Net was to review these efforts, to generate a synthesis of this information and to provide guidelines after approval of many involved specialists. In several European countries, patient associations have developed websites dealing with histiocytic diseases. Euro-Histio-Net aimed to coordinate these existing resources and to extend this network. The European patient associations will decide jointly about the exact contents of this part of the Euro-Histio-Net web portal (Patients Web Site). The ALL SZT BFM 2003 became the biggest prospective multinational trial (Germany, Austria, and Switzerland) on the value of allogeneic haematopoietic stem cell transplantation for children and adolescents. This was made possible through the establishment, for the first time, of common strategies for conditioning regimen, donor recruitment, GVHD-prophylaxis and methods of T-cell depletion according to a given indication for an allogeneic transplantation. From 2003 until 2011, 624 patients with a median age of 9 years at diagnosis were entered on trial. It was demonstrated that with the given measures, unrelated matched donor-HSCT is equivalent to allogeneic HSCT from HLA identical siblings. Incidence of severe acute and chronic GVHD is low and the treatment-related mortality after one year is below 9% in both groups. To confirm these excellent results, the protocol is running since 2007 in 11 other European countries (ALL-SCT BFM international). So far 482 patients have been registered on this GCP study. Data collection runs on an internet-based remote data entry system developed in collaboration with the Austrian Research Centre Seibersdorf (AIT). Both studies (ALL-SZT BFM 2003 and ALL SCT BFM international) closed their patient registration in 2011. The results of these two studies are currently analysed and the publication is in preparation. Clinical Research Specific project The successor trial ALL SCTped 2012 focuses on a new approach to reduce toxicity and late effects after TBI. It has been approved by the ethics committee and the consent of the Austrian competent authority by Feb. 11th 2013 and will open soon. Since March 2008, Prof. C. Peters has been elected chair of the Paediatric Disease Working Party of the European Marrow and Bone Transplantation Group (EBMT). This is an important strategic position to further enhance international collaboration for patientoriented clinical research and studies in the field of stem cell transplantation to ultimately improve survival and quality of life in children subject to stem cell transplantation. Many activities have taken place in these last years, mainly focusing on education and consensus treatment for rare diseases. Experts from different fields were brought together and new projects and studies were launched. Since autumn 2012, Prof. Peters is also responsible for education within EBMT, and became part of the EBMT Executive Board. 86–87 Specific project St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Specific project Clinical Research The mission of the European International Society for Pediatric Oncology Neuroblastoma Group (SIOPEN) is to ultimately improve survival in children suffering from high-risk neuroblastoma (NBL) with improved treatment concepts including immunotherapy after intensive induction and stem cell transplanation. Moreover, futherfinetuning of risk-adapted therapy advances treatment deescalation strategies and thus, assurance of quality of life in children with inter mediate- and, in particular, low-risk disease. Supported by the European Commission1, the SIOPEN-R-NET research network (www.siopen-r-net.org) was built. An internet and web-based centralised remote data entry database and communication system was developed by AIT Austrian Institute of Technology GmbH, Austria (www.ait.ac.at) serving as common trial platform for SIOPEN. The SIOPEN Association was established in 2009 with a registered office in Vienna to foster Neuroblastoma research and enables the long-term sustainability of the group. The liaison office at the S2IRP acts as the members’ contact point. (http://membership.siopen-r-net.org). In 2012 the SIOPEN Association has active members in 27 countries including currently Australia, Austria, Belgium, Croatia, Czech Republic, Denmark, Finland, France, Germany, Greece, Hong Kong, Hungary, Ireland, Israel, Italy, Japan, New Zealand Norway, Poland, Portugal, Serbia, Singapour, Slovakia, Spain, Sweden, Switzerland and United Kingdom. This network application to the Austrian Ministry of Health and Pharmaceutical Industry was successfully approved and announced at the Forum Alpach/Tyrol in 2012 in a press conference by the minister of health (BM Alois Stöger) and, based on a privat-public funding partnership, will be supported over 3 years. The expertise of 5 coordinating clinical trial units of Austrian universities and institutes will network with paediatric experts and clinical sites to foster drug development and approval of medicines for children and adolescents in close collaboration with industry but will equally support and enhance academic research trials to establish treatment guidelines. These efforts were driven by the vision to support solutions to overcome off-label use in clinical trials for children and adolescents. O.K.ids aims to become part of EnprEMA, the European Network of paediatric networks. The web page is currently under construction and will be available soon for further, more detailed information on aims and progress: www.Okids-net.at 88–89 European SIOPEN Neuroblastoma Study Reference Centre Specific project O.K.ids – Medicines for Children Research Organisation – Austria In 2002, SIOPEN launched the HR-NBL-1 (1.5)/ SIOPEN trial for the treatment of high-risk neuro blastoma under the leadership of Prof. R. Ladenstein as international chair. In 2010, the use of G-CSF during induction was published as newly established treatment standard reducing significantly side effects as a first result of the trial [1]. At the ASCO Annual meeting in 2011, the randomisation result on 598 patients comparing two high-dose treatment (HDT) approaches followed by stem cell reinfusion was awarded by a presentation in the plenary session. A significant difference in favour of the European approach with Busulfan-Melphalan (Bu-Mel) high dose chemotherapy over the North American (Children’s Oncology group) regimen carboplatin, etoposide, melphalane was observed (3-years event free survival 49% vs 33%; 3-years overall survival 60% vs 48%, p=0.004). This relapse and progression incidence was significantly lower with BuMel (48% vs 60%, p<0.001) as well as the severe toxicity rate [2]. The BuMel regime is now the recommended new treatment standard. The data is now matured, results maintained and final publication in preparation. The trial also enables access to immunotherapy for children with High-Risk NBL in Europe based on a purely investigator-driven effort and a European wide SIOPEN funding campaign managed by Prof. R. Ladenstein to allow for the ch14.18/CHO anti-GD2 antibody development. Having been given Scientific Advice as Academia at the European Medical Agency (EMA) in 2010, a small biotech company APEIRON (http://www.apeiron-biologics.com) has shown interest and contracted in 2011 with the CCRI and the SIOPEN group to develop the antibody for the market. As a result of this cooperation with APEIRON, the ch14.18/CHO antibody has been granted Orphan Status in 2012, both in Europe by the EMA and in the US by the FDA. The part of the trial is expected to answer the randomised immunotherapy question towards the end of 2013 having accrued already 342 patients out of a planned sample of 400 patients. To allow antibody access also for patients with primary refractory or relapsing NBL, the “Long-term Infusion Study (LTI)” using the antibody in combination with subcutaneous interleukin 2 with Prof. H. Lode (Greifswald/Germany) and Prof. R. Ladenstein as main investigators was developed as a phase I/II trial with support of the S2IRP team and is run by the unit with the St. Anna Kinderkrebsforschung e.V. as a sponsor. 1 EC FP5 Grant No. QLRI-CT-2002–01768 (project coordination: Prof. Ladenstein, MD, MBA, cPM) For further reading [1] Ladenstein, Valteau-Couanet et al. 2010, J Clin Oncol. 28(21):3516–24 [2] Ladenstein, Poetschger, et al., J Clin Oncol 29: 2011 (suppl; abstr 2) St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 Specific project This European Commission-funded project run through the CCRI is coordinated by Prof. R. Ladenstein and was established during her presidency of SIOPE (2010–2012). Starting in 2011 with 33 European partner institutions in 16 countries, it will deliver 80 milestones and manifold associated tasks within 18 work packages over 4 years. ENCCA aims are to • Ensure children and adolescents in Europe receive the best treatment and caring support. • Establish a durable, integrated clinical and translational research infrastructure for Europe. • Improve access to new anti-cancer drugs for children/adolescents in Europe through a biology-driven drug development strategy. • Address inequalities by addressing the differences in terms of ethics, attitudes to experimental therapies and quality of care for children with cancer. • Build for the future through investment in the cancer control workforce, methodology and infrastructure and enhancement of relationships between industry and clinical research. • Improve substantially the quality of life of children and adolescents with cancer and cancer survivors through improved, risk-adapted treatment strategies and enhanced, personalised guidance through risk-adapted long term follow up and care. • Enhance communication and increase under standing among childhood cancer patients, their families and the research community. Major milestones have been achieved since the Kickoff in January 2011 and what follows is just to highlight a few. A European Clinical Research Council (ECRC) was founded as a stakeholder platform of presidents of national paediatric haemato-oncological Societies and chairs of European clinical paediatric haematooncological trials, dealing with common burning issues of the field (i.e. revision of the European Clinical Trial Directive and of suggested possible solutions for its revision, engagement with the European Parliament with specific events held there on invitation). The founded Parent-Patient Advocacy Committee (PPAC) will engage in close collaboration with the ECRC in the policy agenda of burning needs of children and young people with cancer. ENCCA key awareness actions include the establishment of strong relations with policy makers (DG Sanco, EMA, members of the European Parliament – 3 events in the EU Parliament). A strategy for the development of new drugs in four malignancies (Rhabdomyosarcoma, Acute Myeloid Leukaemia, Hepatoblastoma and Medulloblastoma) was established. A prototype of the Survivorship Passport originating from the idea of childhood cancer survivors was developed with the FP7 partner Project PanCareSurFup (www.pancaresurfup.eu) and support of the International Confederation of Childhood Cancer Parent Organizations ICCPO (http://icccpo.org), to provide a detailed and up-to-date description of the tumour type, treatment, complication during treatment and any other relevant data required for counseling or assessing health need. A European steering group to develop a Teenage Young Adult (TYA) Oncology framework with key leaders and stakeholders identified in 18 Countries, dealing with early diagnosis and better healthcare models to improve the currently poor outcome of the TYA population was established. For detailed information on activities and results please visit the ENCCA web page: www.encca.eu One ENCCA work package on “Standardised and innovative methodology for clinical trial design and analysis” addresses the needs for innovative approaches to meet the requirements and unsolved methodological challenges of trials in Paediatric Haemato-Oncology. Ulrike Pötschger, the senior S2IRP statistician, has her main statistical research focus in the evaluation of long-term outcome. Her tasks within this work package complement an ongoing collaboration on this topic with the Institute of Clinical Biometry (Medical University of Vienna) that started in 2008. Although Cox-regression and the Log-Rank test are commonly applied, neither method directly addresses longterm outcome. Cure models and the pseudo value regression technique are promising approaches and in many situations, these non-standard methods clearly outperform the commonly used approaches. Innovative study designs based on these models have been recently implemented in the new LCH-IV study. St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012 A European Network for Cancer Research in Children and Adolescents: ENCCA 90–91 Clinical Research Specific project Services, Administration, PR and Donations Department Service-Einrichtungen, Administration, PR und Spendenabteilung Bioinformatics Maximilian Kauer Science communication Sandra Brezina-Krivda Lisa Glenk1 Research Support Barbara Brunmair Nuno Andrade Antonella Chiucchiuini2 Ivona Brasnjevic3 Finance & Administration Director Karla Valdés Rodríguez4 Raoul Lavaulx-Vrécourt5 Secretaries Marion Zavadil (CCRI Research) Brigitte Glatz (LabDia) Claudia Zeiner (S 2–IRP)6 IT Department Group leader Johann Kalhs Team Navid Kamalejan Florian Kromp Mark Rossiwall Lukas Schneider PR and Donations Department Group leader Andrea Prantl Team Elisabeth Tax Katarina Krizanac11 Anja Fennes12 Bettina Nistler Beatrix Wanek13 Personnel office & accounting Caroline Schmid Accountancy, book-keeping, balance-sheet accounting & controlling Monika Beran7 Claudia Hochweis8 Alexandra Lidy Shideh Karvandi Quality management Michaela Artwohl Sandra Weinzettl9 Facility management and pursuing Corvin Fiala10 Klaus Kienzer 1 2 3 4 5 6 7 8 9 10 11 12 13 since Aug. 2012 since June 2012 until Sept. 2012 until Nov. 2011 since Apr. 2012 coordinating operative and research assistant S2IRP since Nov. 2011 until Dec. 2011 since Febr. 2012 since Nov. 2012 on maternity leave since Oct. 2012 Jan. – Dec. 2012 James R. Downing Scientific Director. Associate Director of Basic Research. Cancer Center. Co-Leader. Haematological Malignancies Laboratory. Departments: Administration, Pathology, St. Jude Children’s Research Hospital, Memphis, USA Lee J. Helman Scientific Director for Clinical Research. Head of the Molecular Oncology Section Department: Paediatric Oncology Branch. National Cancer Institute (NCI), Bethesda, USA Stephan Ladisch Vice Chair for External Affairs. Principal investigator. Department of Paediatrics; Children’s Research Institute; Center for Cancer and Immunology Research (CCIR); Children’s National Medical Center, Washington, USA. Professor Biochemistry and Molecular Biology, George Washington University, USA Crystal L. Mackall Chief Pediatric Oncology Branch/Head of Immunology Section. Paediatric Oncology Branch National Cancer Institute (NCI), Bethesda, USA Andrew Pearson Chairman, Paediatric Oncology Section. Head of Paediatric Research Team, Section of Paediatric Oncology, Institute of Cancer. The Royal Marsden Hospital, Sutton, UK Gregory H. Reaman Principal Investigator: Center for Cancer and Immunology Research (CCIR), Children’s National Medical Center, Washington, USA Maria Grazia Valsecchi Professore Ordinario, Biostatistics Centre Department of Clinical Medicine and Prevention University of Milano-Bicocca, Italy 94–95 Klaus-Michael Debatin Medical Director. University of Ulm, Clinic for Paediatrics and Adolescent Medicine, Germany St. Anna Kinderkrebsforschung /CCRI Scientific Report 2011–2012 Appendix Anhang Scientific Advisory Board Wissenschaftlicher Beirat Caroline Hutter was awarded the 2012 Dr. Jon Pritchard Award organised by The Histiocyte Society. Hutter, C. et al. Blood 2012. Volume 120, Issue 26, Pages 5199–5208. Notch is active in Langerhans cell histiocytosis and confers pathognomonic features on dendritic cells. Heinrich Kovar was awarded the 2011 Science Award of the Austrian Paediatric Society. Le Deley et al. 2010: Journal of Clinical Oncology: Volume 28, Issue 12, Pages 1982–1988. Impact of EWS-ETS fusion type on disease progression in Ewing’s sarcoma/peripheral primitive neuroecto dermal tumor: prospective results from the cooperative Euro-E.W.I.N.G. 99 trial. Lilian Kuster was awarded the 2011 first prize for the best work in paediatric oncology in Austria Kuster, L. et al. 2011. Blood: Volume 117, Issue 9, Pages 2658–2667. ETV6/RUNX1positive relapses evolve from an ancestral clone and frequently acquire deletions of genes implicated in glucocorticoid signaling. Ruth Ladenstein was awarded the 2011 Science Award of the Working Party Science and Research of the Austrian Society for Paediatrics, Section: Haematological – Oncological Research Ladenstein, R. et al, Journal of Clinical Oncology 2010. Volume 28, Issue 20, Pages 3284–3291. Primary Disseminated Multifocal Ewing Sarcoma: Results of the Euro-EWING 99 Trial Ladenstein, R. et al, Journal of Clinical Oncology 2010. Volume 28, Issue 21, Pages 3516–3524. Randomized Trial of Prophylactic Granulocyte ColonyStimulating Factor During Rapid COJEC Induction in Pediatric Patients With High-Risk Neuroblastoma: The European HR-NBL1/SIOPEN Study Stephan Niedan was awarded the 2012 AACR-Pezcoller Foundation Scholar in Training Award. Niedan, S. et al. 2012. Re-activation of EWS-FLI1 suppressed FOXO1 expression as a novel therapeutic strategy for Ewing’s sarcoma Raphaela Schwentner was awarded the 2012 AACR Woman in Cancer Research (WICR) Scholar in Cancer Research Award. Schwentner, R. et al. 2012. A functional ETS/E2F module in cancers expressing ETS fusion genes Florian Bellutti Molecular interactions between adenoviral RNAs and the cellular RNA interference machinery. Supervised by Reinhard Klein, PhD. Diploma thesis Ulrike Kaindl Modulation of the p53 pathway by ETV6/RUNX1 in childhood acute lymphoblastic leukemia. Supervised by Prof. Renate Panzer-Grümayr, MD. PhD Thesis Dagmar Denk The role of PAX5 fusion genes in childhood acute lymphoblastic leukemia. Supervised by Sabine Strehl, PhD. PhD Thesis Stephan Niedan Mechanisms of transcriptional repression by EWS-FLI1 in Ewing sarcoma. Supervised by Prof. Heinrich Kovar, PhD. PhD Thesis Barbara Dillinger The of IDO induced tryptophan depletion and kynurenine accumulation in inhibition of translation initiation and apoptotic decline in activated human T-cells. Supervised by Assoc. Prof. Andreas Heitger, MD. MSc Diploma Thesis Angelika Plach Donor T cells – Prevention of rejection through induction of immunological tolerance. Supervised by Assoc. Prof. Andreas Heitger, MD. BSc Bachelor Thesis Friedrich Erhart The molecular biology of dendritic cells and their application in cell therapy. Supervised by Alexander M. Dohnal, PhD. MD Thesis Nelli Frank Analysis of senescence markers and impact of drug-induced senescent neuroblastoma cells on tumour cell proliferation and immunomodulation. Supervised by Sabine Taschner-Mandl, PhD, and Assoc. Prof. Peter Ambros, PhD. MSc Thesis Gerhard Fuka Exploring ETV6/RUNX1 function in childhood acute lymphoblastic leukemia. Supervised by Prof. Renate Panzer-Grümayr, MD. PhD Thesis Mirza Ibrisimovic Alternative approaches for the treatment of adenovirus infections. Supervised by Reinhard Klein, PhD. PhD Thesis Stefanie Reinprecht The influence of reactive oxygen intermediates on the polarization and behaviour of human T-cells. Supervised by Birgit Jürgens, PhD. BSc Bachelor Thesis Lucia Riedmann The effect of EWS and its oncogenic derivative EWS-FLI1 on transcriptional and post-transcriptional gene regulation in Ewing sarcoma. Supervised by Prof. Heinrich Kovar, PhD. PhD Thesis Raphaela Schwentner Mechanisms of target regulation by the chimeric oncogene EWS-FLI1 in Ewing’s sarcoma. Supervised by Prof. Heinrich Kovar, PhD. PhD Thesis Sneha Valookaran Importance of timing in immune modulation: A study on toll like receptor-4 activated dendritic cells. Supervised by Alexander M. Dohnal, PhD. MSc Thesis Edda Veith Immune-regulation of allogeneic stimulation by CTLA-4Ig: Propagating CD4+CD25+FOXP3 + T cells in an IDO independent fashion. Supervised by Assoc. Prof. Andreas Heitger, MD. PhD Thesis Sarah Vittori Characterization of a putative immunosuppressive MAPKAP kinase family member in human Dendritic Cells. Supervised by Alexander M. Dohnal, PhD. MSc Thesis 96–97 Jozef Ban was awarded the 2012 Best Basic Science Paper of the European Muscoskeletal Oncology Society Award. Ban, J. et al. 2012. Deacetylase SIRT1 links tumor suppressive NOTCH signaling to p53 in Ewing sarcoma. Completed MSc Diplomas and PhD Theses Abgeschlossene Diplomarbeiten und Dissertationen St. Anna Kinderkrebsforschung /CCRI Scientific Report 2011–2012 Awards Preise High-resolution genomic profiling of disseminated tumour cells for a refined risk assessment. Coordinated by Peter Ambros. Project no. 151141. Clinical and biological relevance of novel genomic aberrations affecting the glucocorticoid response in childhood leukemia. Coordinated by Renate Panzer-Grümayer. Project no. 14500 Period covered: 01.01.2012 – 31.12.2012 Interference of immunoregulatory tryptophan metabolism with translation initiation. Coordinated by Birgit Jürgens, group leader Andreas Heitger. Project no. 14225 Period covered: 01.01.2011 – 30.06.2012 Clinical and biological relevance of the P2RY8-CRLF2 fusion gene in childhood acute lymphoblastic leukemia. Coordinated by Maria Morak, group leader Renate Panzer-Grümayer. Project no. 14129 Period covered: 01.01.2011 – 30.06.2012 Mannan-binding lectin deficiency in pediatric stem cell transplantation. Coordinated by Andreas Heitger. Project no. 13075 Period covered: 01.07.2008 – 30.04.2012 Control of Ewing sarkoma stem cell growth. Coordinated by Heinrich Kovar. Project no. 13349 Period covered: 01.01.2009 – 30.06.2011 Origin of relapses from childhood hyperdiploid B-lineage leukemias. Coordinated by Georg Mann, principle investigator Renate Panzer-Grümayer. Project no. 13881 Period covered: 01.08.2010 – 31.12.2011 Differential expression of chromatin fractions in aggressive/benign neuro blastomas. Coordinated by Eva Bozsaky, group leader Peter Ambros. Project no. 13835 Period covered: 01.08.2010 – 30.09.2012 Therapeutic targeting of the tumor suppressor p53 in Ewing’s sarcoma family tumors. Coordinated by Dave Aryee, group leader Heinrich Kovar. Project no 14205 Period covered: 01.01.2011 – 30.06. 2012 Evaluation of novel potential predictive biomarkers in pediatric acute lymphoblastic leukemia. Coordinated by Karin Nebral, group leader Sabine Strehl. Project no. 14133 Period covered: 01.01.2011 – 30.07.2012 The role of CDKN1B-p27KIP1 deregulation in the pathogenesis of pediatric T-cell acute lymphoblastic leukemia. Coordinated by Sabine Strehl. Project no. 14444 Period covered: 01.12.2011 – 30.11.2013 Exploring the genetic base of relapsing high-hyperdiploid acute lymphoblastic leukemia in children with a genome-wide next generation sequencing approach. Coordinated by Georg Mann, principal investigator Renate Panzer-Grümayer. Project no. 14850. Period covered: 01.07.2012 – 31.12.2013 The role of hypoxia in ETV6/RUNX1 positive childhood leukemia: implications for biology and clinic. Coordinated by Gerd Krapf, group leader Renate Panzer-Grümayer. Project no. 13665. Period covered: 01.01.2010 – 31.12.2011 Uncovering Clinical Markers for Langerhans Cell Histiocytosis. Coordinated by Caroline Hutter, group leader Heinrich Kovar. Project no. 13434 Period covered: 01.07.2009 – 31.12. 2011 Austrian Academy of Sciences Österreichische Akademie der Wissenschaften (ÖAW) Austrian Science Fund Fonds zur Förderung der wissen schaftlichen Forschung (FWF) Mechanisms of target regulation by the chimeric oncogene EWS-FLI1 in Ewing’s sarcoma. Applicant Raphaela Schwendtner, group leader Heinrich Kovar. DOC-fFORTE fellowship no. 22882 Period covered: 01.01.2010 – 31.12.2012 Allo-antigen-specific-T-cell-tolerance induced by human dendritic cells expressing the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase. Coordinated by Andreas Heitger. Stand alone project no. P20865-B13. Period covered: 01.08.2008 – 31.12.2011. Austrian Research Promotion Agency Österreichische Forschungsförderungsgesellschaft (FFG) ADENOTAG – Validation of a novel powerful technology for clinical-scale isolation of T-cells for adenovirus therapy. Coordinated by René Geyeregger, group leader Gerhard Fritsch. Eurostars/FFG project no. E! 5744/829495. Period covered: 01.06.2011 – 31.03.2013 INTERACCT – Integrating Entertainment and reaction assessment into child therapy. Coordinated by Helmut Hlavacs (Research Group Entertainment Computing, University of Vienna, Austria), partner Anita Lawitschka Bridge project no. 8385941 In SITU DC-CIT – Pre-clinical development of an Off-the-Shelf individualised cancer immune therapy. Coordinated by Wolfgang Schöfberger (Institute for Anorganic Chemistry, Johannes Kepler University Linz, Austria), partner Alexander Dohnal, group leader Thomas Felzmann. Bridge project no. 836532 Period covered: 01.10.2012 – 30.09.2015 1 Operational start in 2013 A phase I/IIdose schedule finding study of ch14.18/CHO continuous infusion combined with subcutaneous IL-2 in patients with primary refractory or relapsed neuro blastoma. A SIOPEN study. Coordinated by Ruth Ladenstein.KLI project no. 175-B00. Period covered: 01.08.2011 – 31.07.2013 Elucidating the role of PAX5 chimeric proteins in the pathogenesis of childhood B-cell precursor acute lymphoblastic leukemia. Coordinated by Sabine Strehl. Stand alone project no. P 21554-B19 Period covered: 01.11.2009 – 31.07.2013 Mechanisms of transcriptional control by EWS-FLI.1 Coordinated by Heinrich Kovar. Stand alone project no. 22328-B09 Period covered: 04.01.2010 – 30.06.2013 MK2/3 in immune regulation by dendritic cells – DC feedback loops. Coordinated by Alexander Dohnal, group leader Thomas Felzmann. Stand alone project no. P 23271-B11 Period covered: 01.04.2011 – 31.03.2014 Oncogenic fusion proteins & epigenome: partners in crime? Applicant Eleni Tomazou, group leader Heinrich Kovar. Lise Meitner fellowship no. M 1448-B13. Period covered: 01.10.2012 – 30.09.2014 PROVABES – Prospective validation of Biomarkers in Ewing Sarcoma for personalised translational medicine. Coordinated by Uta Dirksen (University Hospital Münster, Pediatric Haematology and Oncology, Germany), partner Heinrich Kovar. EraNET-TRANSCAN 2012 project no. I225-B191. RNA interference-mediated inhibition of adenoviruses. Coordinated by Reinhard Klein, group leader Thomas Lion. Translational research project no. L665-B13. Period covered: 01.09.2009 – 31.12.2012. Subclone expansion and evolution in CML and other MPNs. Coordinated by Thomas Lion, Subproject of the SFB-F47 research cluster Myeloproliferative neoplasmspathogenesis and development of new therapeutic strategies (Coordinator Peter Valent, Medical University of Vienna). SFB-project no. F4705-B201. Role of EWS-FLI1 in post-transcriptional gene regulation Coordinated by Heinrich Kovar. Stand alone project no. 20665-B12. Period covered: 01.03.2008 – 30.09.2011 The role of microRNAs in the gene regulatory network of Ewing’s sarcoma Coordinated by Heinrich Kovar. Stand alone project no. 24708-B21 Period covered: 01.08.2012 – 31.07.2015 Timing of genetic “second hit” alterations in childhood acute lymphoblastic leukemia. Coordinated by Renate Panzer-Grümayer. Stand alone project no. 22073-B19. Period covered: 01.01.2010 – 31.06.2013 TRANSCALL – Translational Research in Childhood Acute Lymphoblastic Leukemia. Coordinated by Martin Stanulla (University Hospital Schleswig Holstein, Dept. Pediatrics, Kiel, Germany), partner Renate Panzer-Grümayer. EraNET-TRANSCAN 2012 project no. I226-B191. Herzfelder’sche Familienstiftung Herzfelder’sche Familienstiftung Analysis of the secretory profile and immunogenicity of drug-induced senescent tumor cells in aggressive neuroblastoma. Coordinated by Sabine Taschner-Mandl, group leader Peter Ambros. Period covered: 01.07.2012 – 30.06.2014. Liddy Shriver Sarcoma Initiative Liddy Shriver Sarkom Initiative Molecular mechanisms and pharmacologic inhibition of bone sarcoma metastasis Coordinated by David Loeb (Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore) partner Heinrich Kovar. Period covered: 01.11.2012 – 31.10.2014 1 Operational start in 2013 St. Anna Kinderkrebsforschung /CCRI Scientific Report 2011–2012 Anniversary Fund of the Österreichische Nationalbank (Austrian National Bank) Jubiläumsfonds der Österreichischen Nationalbank (OeNB) 98–99 External Grants and Research Funding Bodies Fremdgeförderte Projekte und Fördergeber ASSET – Analysing and stryking the sensitivities of embryonal tumors Coordinated by Walter Kölch (Systems Biology, Univ. College Dublin, Ireland). Partner Heinrich Kovar. FP7-Health-2010 project no. 259348 Period covered: 01.11.2010 – 31.10.2015 ENCCA – European network for cancer research in children and adolescents. Coordinated by Ruth Ladenstein in collab oration with Peter Ambros, Heinrich Kovar, Michael Dworzak and external partners. FP7-Health-2010 project no. 261474 Period covered: 01.01.2011 – 31.12.2014 EURO-HISTIO-NET – A reference network for Langerhans Cell Histiocytosis and associated syndromes. Coordinated by Milen Minkov. Public Health Executive Agency (PHEA). Project no. 2007120 Period covered: 01.01.2008 – 31.08.2011 IntReALL – International study for treatment of childhood relapsed ALL2010 with standard therapy, systematic integration of new agents and establishment of standardized diagnostic and research. FP7-Health-2011 project no. 278514 Coordinated by Arend von Stackelberg (Charité-Universitätsmedizin Berlin, Germany), partner Andishe Attarbaschi. Period covered 01.10.2011 – 30.09.2016 MODICELL – Targeted modulation of immune-system responses in cell therapies. Coordinated by Andreas Heitger in collaboration with Thomas Felzmann and external partners. FP7-People-2011 project no. 2858751 Vienna Science and Technology Fund Wiener Wissenschafts-, Forschungs-, und Technologiefonds (WWTF) Flow cytometric signal typing for therapy response prediction in pediatric myeloid leukaemia. Coordinated by Michael N. Dworzak. Life Sciences 2007 project no. LS07–037. Period covered: 01.03.2008 – 31.09.2013 Wilhelm Sander foundation, Munich Wilhelm Sander-Stiftung, München Chimäre T-Zellrezeptoren für die CMV-Therapie nach Stammzell transplantation in Hochrisikopatienten. Coordinated by Wolfgang Holter, Armin Ensser, Manfred Lehner. Project 2009.002.1 Period covered: 08.2009 – 07.2011 Histiocytosis association, New Jersey, USA Histiozytose-Gesellschaft, New Jersey, USA Analysis of Jagged2 Signaling in Langerhans Cell Histiocytosis. Coordinated by Caroline Hutter, group leader Heinrich Kovar. Histiocytosis association grant 20121. 1 Operational start in 2013 AIEOP-BFM ALL 2009, PEG-Asparaginase (Oncaspar®).Coordinated by Georg Mann. Medac Gesellschaft für klinische Spezial präparate GmbH, Germany Period covered 2010–2012 ALL-SZT-Studies. Coordinated by Christina Peters. DKMS Germany Period covered 2008–2011 Annual contribution. Umbrella organisation of the Austrian Childhood Cancer Aid (Dachverband der österreichischen Kinderkrebshilfe) Period covered: 2011–2012 EBMT-PDWP. Coordinated by Christina Peters. Period covered 2008–2013 OKIDS GmbH, O.K.ids – Medicines for Children Research Organisation. Coordinated by Ruth Ladenstein. “Federal Ministery of Health” grant approval: summer 2012, “Health aims” grant approval: November/December 2012, Pharmig’ grant approval December 2012. Period covered 2013–2016 SIOPEN Association to foster Neuroblastoma Research (SIOP Europe Neuroblastoma Group), Vienna. Coordinated by Ruth Ladenstein. Fundraising SIOPEN Association. Period covered 2011–2012 2 Group leader of the S2IRP Coordinating Centre for Paediatric-Oncological Traials at the CCRI, Ruth Ladenstein St. Anna Kinderkrebsforschung /CCRI Scientific Report 2011–2012 Seventh Framework Programme (FP7) of the European Commission 7. Rahmenprogramm der Europäischen Union 100–101 National and International Trusts for Funding Clinical Trials2 Nationale und internationale Fördergeber für klinische Studien2 Andreou, D., Bielack, S.S., Carrle, D., Kevric, M., Kotz, R., Winkelmann, W., Jundt, G., Werner, M., Fehlberg, S., Kager, L., Kuhne, T., Lang, S., Dominkus, M., Exner, G.U., Hardes, J., Hillmann, A., Ewerbeck, V., Heise, U., Reichardt, P. & Tunn, P.U. (2011). The influence of tumor- and treatment-related factors on the development of local recurrence in osteosarcoma after adequate surgery. An analysis of 1355 patients treated on neoadjuvant Cooperative Osteosarcoma Study Group protocols. Ann Oncol. 22(5), 1228–35. Angelini, P., London, W.B., Cohn, S.L., Pearson, A.D., Matthay, K.K., Monclair, T., Ambros, P.F., Shimada, H., Leuschner, I., Peuchmaur, M., Irwin, M.S. & Baruchel, S. (2012). Characteristics and outcome of patients with ganglioneuroblastoma, nodular subtype: a report from the INRG project. Eur J Cancer. 48(8), 1185–91. Attarbaschi, A., Mann, G., Rosolen, A., Williams, D., Uyttebroeck, A., Marky, I., Lamant, L., Horibe, K., Wrobel, G., Beishuizen, A., Wossmann, W., Reiter, A., Mauguen, A., Le Deley, M.C., Brugieres, L., European Intergroup for Childhood Non-Hodgkin Lymphoma AT. (2011). Limited stage I disease is not necessarily indicative of an excellent prognosis in childhood anaplastic large cell lymphoma. Blood. (Multicenter Study Research Support, Non-U.S. Gov’t). 117(21), 5616–9. Attarbaschi, A., Morak, M., Cario, G., Cazzaniga, G., Ensor, H. M., Te Kronnie, T., Bradtke, J., Mann, G., Vendramini, E., Palmi, C., Schwab, C., Russell, L. J., Schrappe, M., Conter, V., Mitchell, C. D., Strehl, S., Zimmermann, M., Pötschger, U., Harrison, C. J., Stanulla, M., PanzerGrümayer, R., Haas, O. A., & Moorman, A. V.; on behalf of the Associazione Italiana di Ematologia ed Oncologia Pediatrica (AIEOP)-Berlin-Frankfurt-Münster (BFM) Study Group and National Cancer Research Institute (NCRI)-Children’s Cancer and Leukaemia (CCLG) Study Group. (2012). Treatment outcome of CRLF2 -rearranged childhood acute lymphoblastic leukaemia: a comparative analysis of the AIEOP-BFM and UK NCRI-CCLG study groups. Br J Haematol, 158(6), 772–777. Ban, J., Jug, G., Mestdagh, P., Schwentner, R., Kauer, M., Aryee, D.N., Schaefer, K.L., Nakatani, F., Scotlandi, K., Reiter, M., Strunk, D., Speleman, F., Vandesompele, J. & Kovar H. (2011). Hsa-mir-145 is the top EWS-FLI1-repressed microRNA involved in a positive feedback loop in Ewing’s sarcoma. Oncogene. 30(18), 2173–80. Beier, R., Albert, M.H., Bader, P., Borkhardt, A., Creutzig, U., Eyrich, M., Ehlert, K., Gruhn, B., Greil, J., Handgretinger, R., Holter, W., Klingebiel, T., Kremens, B., Lang, P., MauzKorholz, C., Meisel, R., Muller, I., Peters, C., Reinhardt, D., Sedlacek, P., Schulz, A., Schuster, F.R., Schrauder, A., Strahm, B., Sykora, K.W., Wossmann, W., Zimmermann, M. & Sauer, M.G. (2012). Allo-SCT using BU, CY and melphalan for children with AML in second CR. Bone Marrow Transplant. (Epub ahead of print) Bennani-Baiti, I.M., Aryee, D.N., Ban, J., Machado, I., Kauer, M., Mühlbacher, K., Amann, G., Llombart-Bosch, A. & Kovar, H. (2011). Notch signaling is off and is uncoupled from HES1 expression in Ewing’s sarcoma. J. Pathol., 225(2), 353–63. Bennani-Baiti, I.M., Machado, I., LlombartBosch, A. & Kovar H. (2012). Lysine-specific demethylase 1 (LSD1/KDM1A/AOF2/BHC110) is expressed and is an epigenetic drug target in chondrosarcoma, Ewing’s sarcoma, osteosarcoma, and rhabdomyosarcoma. Human. Pathol. 43, 1300–7. Booth, C., Gilmour, K.C., Veys, P., Gennery, A.R., Slatter, M.A., Chapel, H., Heath, P.T., Steward, C.G., Smith, O., O’Meara, A., Kerrigan, H., Mahlaoui, N., Cavazzana-Calvo, M., Fischer, A., Moshous, D., Blanche, S., Pachlopnik Schmid, J., Latour, S., de Saint-Basile, G., Albert, M., Notheis, G., Rieber, N., Strahm, B., Ritterbusch, H., Lankester, A., Hartwig, N.G., Meyts, I., Plebani, A., Soresina, A., Finocchi, A., Pignata, C., Cirillo, E., Bonanomi, S., Peters, C., Kalwak, K., Pasic, S., Sedlacek, P., Jazbec, J., Kanegane, H., Nichols, K.E., Hanson, I.C., Kapoor, N., Haddad, E., Cowan, M., Choo, S., Smart, J., Arkwright, P.D. & Gaspar, H.B. (2011). X-linked lymphoproliferative disease due to SAP/SH2D1A deficiency: a multicenter study on the manifestations, management and outcome of the disease. Blood, 117(1), 53–62. Boztug, H., Karitnig-Weiss, C., Ausserer, B., Renner, E.D., Albert, M.H., SawalleBelohradsky, J., Belohradsky, B.H., Mann, G., Horcher, E., Rummele-Waibel, A., Geyeregger, R., Lakatos, K., Peters, C., Lawitschka, A. & Matthes-Martin, S. (2012). Clinical and immunological correction of DOCK8 deficiency by allogeneic hematopoietic stem cell transplantation following a reduced toxicity conditioning regimen. Pediatric hematology and oncology. 29(7), 585–94. Breuer, S., Preuner, S., Fritsch, G., Daxberger, H., Koenig, M., Poetschger, U., Lawitschka, A., Peters, C., Mann, G., Lion, T. & Matthes-Martin. S. (2012). Early recipient chimerism testing in the T- and NK-cell lineages for risk assessment of graft rejection in pediatric patients undergoing allogeneic stem cell transplantation. Leukemia. 26(3), 509–19. Breuer, S., Rauch, M., Matthes-Martin, S. & Lion, T. (2012). Molecular diagnosis and management of viral infections in hema topoietic stem cell transplant recipients. Mol Diagn Ther. 16(2), 63–77. Burkhardt, B., Oschlies, I., Klapper, W., Zimmermann, M., Woessmann, W., Meinhardt, A., Landmann, E., Attarbaschi, A., Niggli, F., Schrappe, M. & Reiter, A. (2011). Non-Hodgkin’s lymphoma in adolescents: experiences in 378 adolescent NHL patients treated according to pediatric NHL-BFM protocols. Leukemia. 25(1), 153–60. Coenen, E. A., Raimondi, S. C., Harbott, J., Zimmermann, M., Alonzo, T. A., Auvrignon, A., Beverloo, H. B., Chang, M., Creutzig, U., Dworzak, M. N., Forestier, E., Gibson, B., Hasle, H., Harrison, C. J., Heerema, N. A., Kaspers, G. J., Leszl, A., Litvinko, N., Lo Nigro, L., Morimoto, A., Perot, C., Reinhardt, D., Rubnitz, J. E., Smith, F. O., Stary, J., Stasevich, I., Strehl, S., Taga, T., Tomizawa, D., Webb, D., Zemanova, Z., Pieters, R., Zwaan, C. M., & van den HeuvelEibrink, M. M. (2011). Prognostic significance of additional cytogenetic aberrations in 733 de novo pediatric 11q23/MLL-rearranged AML patients: results of an international study. Blood, 117(26), 7102–7111. Collins, M., Wilhelm, M., Conyers, R., Herschtal, A., Whelan, J., Bielack, S. & Kager, L. (2012). Benefits and adverse events in younger versus older patients receiving (neo)-adjuvant chemotherapy for osteosarcoma. Clin-Oncol. Corbacioglu, S., Cesaro, S., Faraci, M., Valteau-Couanet, D., Gruhn, B., Rovelli, A., Boelens, J.J., Hewitt, A., Schrum, J., Schulz, A.S., Muller, I., Stein, J., Wynn, R., Greil, J., Sykora, K.W., Matthes-Martin, S., Fuhrer, M., O’Meara, A., Toporski, J., Sedlacek, P., Schlegel, P.G., Ehlert, K., Fasth, A., Winiarski, J., Arvidson, J., Mauz-Korholz, C., Ozsahin, H., Schrauder, A., Bader, P., Massaro, J., D’Agostino, R., Hoyle, M., Iacobelli, M., Debatin, K.M., Peters, C. & Dini, G. (2012). Defibrotide for prophylaxis of hepatic veno-occlusive disease in paediatric haemopoietic stem-cell transplantation: an open-label, phase 3, randomised controlled trial. Lancet. 379(9823), 1301–9. Creutzig, U., van den Heuvel-Eibrink, M.M., Gibson, B., Dworzak, M.N., Adachi, S., de Bont, E., Harbott, J., Hasle, H., Johnston, D., Kinoshita, A., Lehrnbecher, T., Leverger, G., Mejstrikova, E., Meshinchi, S., Pession, A., Raimondi, S.C., Sung, L., Stary, J., Zwaan, C.M., Kaspers, G.J. & Reinhardt, D.; on behalf of the AML Committee of the International BFM Study Group (2012). Diagnosis and management of acute myeloid leukemia in children and adolescents: recommendations from an international expert panel. Blood. 120(16), 3187–3205. Creutzig, U., Zimmermann, M., Bourquin, J.P., Dworzak, M.N., Fleischhack, G., von Neuhoff, C., Sander, A., Schrauder, A., von Stackelberg, A., Ritter, J., Starý, J. & Reinhardt, D. (2011). 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No improvement of survival with reduced-versus highintensity conditioning for allogeneic stem cell transplants in Ewing tumor patients. Ann Oncol. 22(7), 1614–21. Publisher St. Anna Kinderkrebsforschung e.V. Zimmermannplatz 10, A-1090 Vienna www.ccri.at Herausgeber St. Anna Kinderkrebsforschung e.V. Zimmermannplatz 10, A-1090 Vienna www.ccri.at Contact Marion Zavadil CCRI Secretariat [email protected] Kontakt Marion Zavadil CCRI Sekretariat [email protected] We would like to express our gratitude to the numerous private donors who have been loyal in their support for many years, as well as the following national and inter national funding bodies and organisations: EU 7th Research Programme (FP7) Austrian Science Fund (FWF) Austrian Research Promotion Agency (FFG) Responsibility for scientific content Heinrich Kovar Scientific Director [email protected] Verantwortlich für den wissenschaftlichen Inhalt Heinrich Kovar Scientific Director [email protected] Text Editor & Project Management Sandra Brezina-Krivda Lisa Glenk Science Communication [email protected] [email protected] Redaktion & Projektmanagement Sandra Brezina-Krivda Lisa Glenk Science Communication [email protected] [email protected] Vienna Science and Technology Fund (WWTF) Federal Ministry of Health Austrian National Bank (OeNB) Austrian Academy of Sciences Art Direction & Design Lichtwitz Leinfellner visuelle Kultur KG Konzeption & Gestaltung Lichtwitz Leinfellner visuelle Kultur KG Photography Gerhard Wasserbauer Fotografie Gerhard Wasserbauer Print Ueberreuter Print GmbH Paper Maxioffset 120/130 g Font Omnes Pro Druck Ueberreuter Print GmbH Papier Maxioffset 120/130 g Schrift Omnes Pro SIOPEN Association (Society of Paediatric Oncology European Neuroblastoma Network) Liddy Shirver Sarcoma Initiative EMBT (European Group for Blood and Marrow Transplantation) Wilhelm Sander Foundation, Munich Umbrella organisation of the Austrian Childhood Cancer Aid Working Party Science and Research of the Austrian Society for Paediatrics, Section: Haematological-Oncological Research Medac Gesellschaft für klinische Spezialpräparate GmbH, Germany Herzfelder’sche Familienstiftung Download of the Scientific Report as pdf file: http://science.ccri.at Download des Forschungsberichtes als PDF-Datei: http://science.ccri.at DKMS, Germany Support Unterstützung Wir möchten uns gerne an dieser Stelle bei unseren zahlreichen privaten Spendern und Spenderinnen bedanken, die uns seit vielen Jahren treu unterstützen. Des Weiteren bedanken wir uns bei den folgenden nationalen und internationalen Fördergebern und Organisationen: 7. Forschungsrahmenprogramm der Europäischen Kommission (FP7) Fonds zur Förderung der wissenschaftlichen Forschung (FWF) Bank Account St. Anna Kinderkrebsforschung e.V. Bank Austria, BLZ 12000 Konto: 00 656 166 600 IBAN: AT 79 12000 00656166600 BIC: BKAUATWW Further details: Page 92–93 www.ccri.at http://science.ccri.at Österreichische Forschungsförderungsgesellschaft (FFG) Wiener Wissenschafts-, Forschungsund Technologiefonds (WWTF) Bundesministerium für Wissenschaft und Forschung (BMWF) Österreichische Nationalbank (OeNB) Bankverbindung St. Anna Kinderkrebsforschung e.V. Bank Austria, BLZ 12000 Konto: 00 656 166 600 IBAN: AT 79 12000 00656166600 BIC: BKAUATWW Akademie der Wissenschaften SIOPEN Association (Internationale Gesell. des Europäischen pädiatrisch-onkolog. Neuroblastom-Forschungsnetzwerks) Liddy Shirver Sarcoma Initiative EMBT (Europäische Gruppe für Blut- und Knohenmarktransplantation) Wilhelm Sander Foundation, München Dachverband der österr. Kinderkrebshilfe AG Wissenschaft und Forschung der Österr. Gesell. für Kinder- und Jugend heilkunde ÖGKJ im Bereich der hämatoonkolog. Forschung – Arbeitsgruppe für Wissenschaft und Forschung der Österr. Gesell. für Kinder- und Jugendheilkunde, Sektion: Hämatolog.-onkolog. Forschung Medac Gesellschaft für klinische Spezialpräparate GmbH, Deutschland Herzfelder’sche Familienstiftung DKMS, Deutschland Weitere Details: Seite 92–93 www.ccri.at http://science.ccri.at 110–111 Acknowledgements Danksagung St. Anna Kinderkrebsforschung /CCRI Scientific Report 2011–2012 Imprint Impressum Reflections on the past 25 years of children’s cancer research point to a promising future: However, there are still many open questions that still need to be answered. Die Rückschau auf 25 Jahre Kinderkrebsforschung lässt uns hoffnungsvoll in die Zukunft blicken: Aber es sind auch noch sehr viele Fragen offen. http://science.ccri.at www.ccri.at