ST. ANNA KINDERKREBSFORSCHUNG

Transkript

1 FORSCHUNGSBERICHT 216 ST. ANNA KINDERKREBSFORSCHUNG kinderkrebsforschung.at science.ccri.at ST. ANNA KINDERKREBSFORSCHUNG FORSCHUNGSBERICHT 216

2 EINLEITUNG 7 SCIENCE REPORTS 33 CAREER 79 Vorwort des Institutsleiters 8 Introduction 36 Working at CCRI 82 Einleitung des wissenschaftlichen Direktors 1 Unseren Spendern sei Dank! 14 DATEN & FAKTEN 19 Kompetitive Drittmittel 22 Zuweisung der Geldmittel 22 Finanzierung 22 Personelle Zusammensetzung 23 Nationen 23 Forschungsnetzwerke 24 Klinische Forschung 26 Anstieg der 2-Jahres-Überlebensraten 27 5-Jahres-Überleben von krebskranken Kindern 28 Non-genetic plasticity and variability as therapeutic and prognostic targets in Ewing sarcoma 38 Improving the routine enumeration of hematopoietic stem cells: Multi-color analysis of CD34 subtypes reveals unexpected differences between various stem cell sources 44 Characterization of a novel fusion gene in juvenile myelomonocytic leukemia associated with resistance to tyrosine kinase inhibitors High resolution genomic and transcriptomic profiling of pediatric B-cell precursor acute lymphoblastic leukemia: implications for emergence of resistance and relapse New insights in neuroblastoma b iology from spontaneous maturation to the relapse seeding clone Chimeric antigen receptor (CAR)-based immunotherapy for treatment of reactivation of cytomegalovirus infection after stem cell transplantation Large scale European trial demonstrates survival advantage for high risk neuroblastoma patients receiving high dose busulphan and melphalan treatment 72 If you want to apply for a position 83 Scientific Staff 84 FINANZBERICHT 89 Richtlinien zur Spendenverwendung 9 Mittelherkunft 94 Mittelverwendung 95 ANHANG 97 Wissenschaftlicher Beirat 98 International fremdgeförderte Projekte 12 National fremdgeförderte Projekte 14 Danksagung 18 Diplom(Master)arbeiten /Dissertationen Publikationen Impressum 124

3 UNIV.-PROF. DR. WOLFGANG HOLTER Entwicklung zellulärer Therapie

4 EINLEITUNG

5 Einleitung VORWORT DES INSTITUTSLEITERS D ie Erfolge meiner Kolleginnen und Kollegen machen es mir leicht, diesen Jahresbericht einzuleiten. Doch trotz aller persönlichen Genugtuung über den wissenschaftlichen Fortschritt, trotz ermutigender Aner kennung unserer Arbeit auf internationaler Bühne, ist es doch immer der Zweck unserer Forschung, der uns antreibt. Es geht um das Überleben und Leben krebskranker Kinder und Jugendlicher und die Umsetzung unserer Erkenntnisse in die klinische Diagnostik und Therapie. Nichts keine Auszeichnungen oder wissenschaftlichen Ehren freut uns mehr als ein Beitrag zur Rettung der Betroffenen und zur Linderung von Leid und Schmerz bei deren Angehörigen. Dass die Heilungsraten bei manchen Kinderkrebsarten in lichte Höhen getrieben werden konnten, sodass wir nun die 1 % anpeilen können, hat die St. Anna Kinderkrebsforschung zu einem g e fragten Partner für staatenübergreifende Zusammen arbeit gemacht. So wurden wir für die Leitung eines dreijährigen Pilot-Referenzprojektes ausgewählt, das die Aufgabe hat, die unter schiedlichen Überlebensraten von Kindern mit Krebserkrankungen aufgrund ungleicher L eistungsfähigkeit der Gesundheitssysteme an zugleichen und zu verbessern. Neue, revolutionäre Möglichkeiten für die Diagnose des Neuroblastoms, einer besonders aggressiven Krebsart bei Kindern, hat unser Institut zur Leitung eines EU-weiten Forschungskonsortiums e mpfohlen. Die Flüssige Biopsie wird in weiterführende klinische Studien eingebunden, um diese exakte molekulare Bestimmung des individuellen Tumor genoms voranzutreiben. Eine weitere, vielversprechende Entdeckung der St. Anna Kinderkrebsforschung auf dem Gebiet der Immuntherapie gab grünes Licht zur Erforschung einer neuen Methode für die Behandlung lebens bedrohlicher Virusinfektionen nach einer Stamm zellentherapie, um nur einige Highlights aus der Forschung zu nennen. Zu guter Letzt sollte nicht unerwähnt b leiben, dass wir im wissenschaftlichen Wett bewerb ebenfalls gepunktet haben. Ein Elise-Richter-Forschungs stipendium, das hochkarätige Wissenschaftlerinnen auszeichnet, die eine Universitäts laufbahn an streben, ging 216 an eine Forscherin der St. Anna Kinderkrebsforschung. Ich möchte mich herzlich bei den vielen Unter stützerinnen und Unterstützern, den Mitgliedern des Ehrenkomitees, den Vorstandsmitgliedern, dem wissenschaftlichen Beirat und unseren vielen Förderern bedanken, die uns schon viele Jahre treu begleiten. Ich hoffe, dass dieser Leistungsbericht zur weiteren und so dringend notwendigen Spendenbereitschaft motiviert, für die ich mich im Namen aller Mitarbeiterinnen und Mitarbeiter herzlich bedanken möchte. Univ.-Prof. Dr. Wolfgang Holter Institutsleiter 8 9

6 Einleitung EINLEITUNG DES WISSENSCHAFTLICHEN DIREKTORS M it dem rasanten technologischen Fortschritt in der biomedizinischen Forschung eröffnen sich ungeahnte Dimensionen des Forschungs universums und ergeben sich immer neue, überraschende Erkenntnisse. Im Bereich der Krebsforschung ist dies zweifellos die Einsicht, dass Tumore keine homogene Zellmasse darstellen, sondern sich aus einer Vielzahl verschiedener Zelltypen zusammensetzen, die, jeder für sich, ein hohes Maß an Heterogenität und Plastizität auf weisen. Diese Erkenntnis trifft besonders auf Krebserkrankungen bei Kindern zu. Galt noch vor wenigen Jahren das Dogma, dass Krebs aus schließlich eine Erkrankung der Gene ist, welche sich in irreversiblen, starren Mutationsmustern manifestiert, so zeigt sich heute mehr und mehr, dass Krebs bei Kindern in der Regel mit verhältnis- mäßig wenigen genetischen Veränderungen einhergeht. Denn pädiatrische Krebserkrankungen müssen als Defekt in der normalen Entwicklungsbiologie unterschiedlicher Zelltypen gesehen werden. Um hier den Mechanismen als potentielle Therapie ansatzpunkte auf den Leib zu rücken, müssen wir die Regulationsmechanismen der Entwicklung verstehen. Die zunehmende Anzahl an Weiter entwicklungen neuer Technologien, wie etwa des Next Generation Sequencing (NGS), ermöglicht Einsichten in die normale und die krankhaft veränderte dreidimensionale Struktur des Chromatins, der Steuerzentrale der Genexpression, welches das Verhalten jeder Zelle bestimmt. Die hohe Sensitivität dieser Technologie erlaubt uns nicht nur genetische Zusammensetzung und Genexpression auf Einzelzellebene zu analysieren, sondern auch Tumorzellzerfallsprodukte in der Blutzirkulation nachzuweisen und zu bestimmen. So erhalten wir Auskunft über die genetische und nicht-genetische Variation von Tumoren und deren Komponenten im Wechselspiel der verschiedenen Zelltypen und ihres Stoffwechsels. Diese Entwicklungen spiegeln sich auch in den Forschungsaktivitäten der St. Anna Kinderkrebsforschung wider. Wie in diesem Jahresbericht dargestellt, gelang es uns auch 216 beachtliche Erfolge auf verschiedenen Gebieten der pädiatrisch-onkologischen Forschung zu erzielen, welche uns unserem Ziel einer für jeden Patienten maßgeschneiderten Therapie weiter näherbringen. Dies sei an einigen ausgewählten Beispielen aus den im Vorjahr veröffentlichten Forschungsergebnissen demonstriert. Univ.-Prof. Dr. Heinrich Kovar Wissenschaftlicher Direktor 1 11

7 DR. MARTIN DISTEL Innovative Krebsmodelle

8 Einleitung UNSEREN SPENDERN SEI DANK! Unsere Spenderfamilie ist groß und großartig! Wir sind dankbar für die langjährige, treue Unterstützung und wir freuen uns jedes Mal, ein neues Familienmitglied herzlich willkommen zu heißen! Das Faszinierende an der Arbeit im Spendenbüro der St. Anna Kinderkrebsforschung sind die Menschen, ihre Hilfsbereitschaft, ihre wunder baren Ideen und ihr großartiges Spendenengagement. Dank der konsequenten Forschung können heute bereits vier von fünf Kindern und Jugendlichen, die vor 4 Jahren noch als unheilbar galten, gerettet werden. Finanziert wird die St. Anna Kinderkrebsforschung, die seit 22 das Österreichische Spendengütesiegel führt und zum steuerlich begünstigten Empfängerkreis gehört, von Anfang an hauptsächlich durch Spenden. Dank gebührt daher unseren Spenderinnen und Spendern denn sie alle schenken krebskranken Kindern eine Chance auf eine gesunde Zukunft. BUNTE EINFÄLLE SPANNENDE AKTIONEN GROSSZÜGIGE SPENDEN Das Spektrum der Spendenmöglichkeiten ist viel fältig und die Unterstützung damit grenzenlos wie einige Beispiele aus 216 zeigen: Mit einer exklusiven Benefizgala im Goldenen Saal des Wiener Musikvereins feierte Aki Nuredini, prominenter und beliebter Patron des Wiener Nobel-Restaurants Sole, seinen 6. Geburtstag. Ein Teil des Reinerlöses des hochkarätig besetzten Konzerts kam der St. Anna Kinderkrebsforschung zugute. Die Besucher erlebten eine Musikgala der Extraklasse mit wunderbaren Interpreten, wie Ildikó Raimondi, Clemens Unterreiner, Ramón Vargas uvm. Sogar Starpianist Rudolf Buchbinder spielte ein Geburtstagsständchen. Das ganze Jahr über sammelten die Mitglieder der Kinderkrebshilfe Haid / Ansfelden durch den Verkauf von Flohmarktware Spenden. Der Einsatz war enorm und die Einnahmen rekordverdächtig. Bereits zum 11. Mal wurden nicht nur Familien mit kleinen Krebspatienten unterstützt, sondern auch der St. Anna Kinderkrebsforschung wieder eine namhafte Spende überreicht. Die Aschermittwochfeier der Künstlerinnen und Künstler in der Wiener Hofburgkapelle gilt als besondere Einstimmung auf die Fastenzeit. Die Wiener Hofmusikkapelle und alle Mitwirkenden agierten im Dienst der guten Sache und unter stützten auch im Jahr 216 mit den Einnahmen unsere Forschungs arbeit. Für einen wunderschönen Nachmittag mit vielen lachenden Gesichtern sorgte der Verein Öster reichische Journalistenwerkstätte. Dieter Wally gelang es gemeinsam mit seinen engagierten Kolleginnen und Kollegen, die Österreichpremiere des Kinderfilms Rettet Raffi zu einem speziellen Benefizerlebnis zu machen. Die Abenteuer des mutigen Hamsters und seines jungen Besitzers konnten auch Patienten aus dem St. Anna Kinderspital und deren Eltern genießen. Das Forscherteam freute sich, dank großzügiger Sponsoren, über einen hervorragenden Spendenbetrag. Die Unterstützung der Bastelrunde Hirtenberg, unter der Leitung von Marianne Brandtner, hat bereits eine langjährige Tradition. Das ganze Jahr über wird dort für die St. Anna Kinderkrebs forschung gebastelt, gemalt, gestrickt, genäht usw. Die Erlöse des jährlichen Oster- und Adventmarkts sind jedes Mal beeindruckend. Auch das spannende Wettpaddeln in Scharndorf hat bereits Tradition! Viel Spaß für Jung und Alt ist beim Benefiz-Sautrogrennen mit Kultcharakter garantiert. Der gesamte Erlös wird von Jahr zu Jahr unglaublich gesteigert und wurde auch 216 gespendet. Sogar das derzeit wahrscheinlich hippste Duo der österreichischen Musik- und Unterhaltungsbranche schaute schon vor einem Tourstart im Spendenbüro der St. Anna Kinderkrebsforschung vorbei. Die Kunst-Kampagne Horvathslos für eine gute Sache war die schöne Idee von Mama Speer. Der so gesammelte Betrag wurde von Seiler und Speer verdoppelt und eine tolle Summe persönlich übergeben. Jedes Jahr gestalten die Schülerinnen und S chüler der Schreibgruppe Kreatives Schreiben am Gymnasium Schärding, gemeinsam mit der r ührigen Initiatorin, Frau Prof. Mag. Thallinger, einen Lesezeichenkalender. Mit dem Reinerlös unter stützen die jungen Fabulanten auch 216 das Wiener Forschungsinstitut. L ANGE NACHT DER (KINDERKREBS)FORSCHUNG Forschung auf reizvolle Weise entdecken und Zukunft erleben! Dazu lädt die St. Anna Kinderkrebsforschung alljährlich ein. Im Rahmen der Langen Nacht der Kinderkrebsforschung wird ein spielerisches Kennenlernen des Laborall tages möglich und Wissenschaft zum A nfassen greifbar. Interaktiv, spannend und leicht ver ständlich wird Wissen über die Bausteine des Lebens und die molekularen Grundlagen der K rebsentstehung vermittelt. Vorführungen in den Labors und spannende wissenschaftliche Vorträge ver deutlichen, was unsere Forscherinnen und Forscher zur Verbesserung der Behandlungs qualität und E r höhung der Heilungschancen an Krebs erkrankter Kinder und Jugendlicher leisten. Mag. Andrea Prantl Leiterin Spendenbüro 14 15

9 Einleitung 216 öffneten wir bereits zum 12. Mal unsere Pforten, aber erstmalig im Rahmen von Österreichs größtem Forschungsfest: der Langen Nacht der Forschung. Rund 1 Besucherinnen und Besucher interessierten sich für unsere Arbeit, staunten über unsere Erfolge und nützten die Gelegenheit, sich zu informieren und einen Blick hinter die Forschungskulissen zu werfen. ST. ANNA KINDERKREBSFORSCHUNGUNTERSTÜTZUNGSKOMITEE Schon seit einiger Zeit begleitet ein prominent besetztes Ehrenkomitee die St. Anna Krebsforschung. Die Mentoren, Personen aus Politik, Wirtschaft und Kultur, zeichnen sich durch eine besondere Position aus und sind ehrenamtlich tätig. Durch ihre Präsenz in der Öffentlichkeit können sie viel zugunsten der St. Anna Kinderkrebsforschung bewegen. Seit einigen Jahren unterstützt Eva Angyan, Gattin des Intendanten der Gesellschaft der Musikfreunde in Wien, Dr. Thomas Angyan, als Komiteepräsidentin das Forschungsinstitut. Die Unterstützung der Kinderkrebsforschung ist ihr ein Herzensanliegen und deshalb bittet Frau Angyan regelmäßig eine ausgesuchte Gästeschar zu einem Treffen, um gemeinsame Projekte zugunsten der St. Anna Kinderkrebsforschung zu planen. Dr. Charlotte Rothensteiner, Mag. Maria PolstererKattus, Erste Bank Vorstand Willibald Cernko, KR Karl Javurek, Isabella Kapsch, General direktorin Dr. Elisabeth Gürtler, Parlamentsabgeordnete KR Brigitte Jank, Prof. Erwin Ortner, Bäcker meister Senator Kurt Mann, sowie BM Dr. Michael Häupl, Meinungsforscher Prof. Rudolf Bretschneider, Interspot-Chefin Inge Klingohr, Psychologin Mag. Ulla Konrad, Maestro Franz Welser-Möst und Kapsch-Vorstand Ing. Mag. Thomas Schöpf sind Teil des Unterstützungskomitees und freuen sich, die St. Anna Kinderkrebsforschung als Mentorinnen und Mentoren zu begleiten und tat kräftig zu unterstützen. Unsere Kuscheltiere: Kleine Lebensretter, die Freude schenken Bereits seit 24 Jahren sind die Kuscheltiere der St. Anna Kinderkrebsforschung bei Jung und Alt sehr beliebt und als Sammelobjekte auch heiß begehrt! Jedes Jahr im Oktober begrüßt die Maskottchen familie einen Neuzugang. 216 war es Gigi, der gutgelaunte Glückskäfer. Die kleinen Lebensretter freuen sich auf ein nettes Zuhause und jede Menge Spielgefährten. Für 12, schenkt man nicht nur krebskranken Kindern eine Chance, sondern auch sich selber oder seinen Lieben Freude. Bank Austria, Erste Bank und einige Sparkassen unterstützen die St. Anna Kinderkrebsforschung ebenso beim Vertrieb, wie die Raiffeisen Zentralbank. Sogar an der Rezeption des renommierten Wiener Innenstadt-Hotels Imperial ist das jeweils aktuelle Maskottchen erhältlich. Welche Stofftiere angeboten und wie sie zu bestellen sind, steht auf unserer Internetseite. Jede Spende hilft! Ob mit einer persönlichen Spende oder mit einer gemeinsamen Sammelaktion, ob per Zahlschein, Kreditkarte oder Onlinespende jede finanzielle Zuwendung ermöglicht die Fortsetzung unserer Forschungsarbeit im Kampf gegen K inderkrebs. Firmen spenden immer häufiger den für KundenWeihnachtsgeschenke vorgesehenen Betrag. Statt um Geschenke wird bei Geburtstagen, Jubiläen oder anderen Feiern gerne um Spenden gebeten. Auch der Verzicht auf Blumen und Kränze bei Begräbnissen, um stattdessen zu spenden, hilft krebskranken Kindern. Ein Testament oder ein Legat zugunsten der St. Anna Kinderkrebsforschung schenkt ebenfalls langfristig eine gesunde Zukunft. WOLLEN SIE INFORMATIONEN, UNTERL AGEN ODER HABEN SIE FRAGEN? Das Spendenteam und ich freuen uns auf Ihre Kontaktaufnahme: +43 () spende@kinderkrebsforschung.at Bank Austria IBAN: AT BIC: BKAUATWW Erste Bank IBAN: AT BIC: GIBAATWW Vielfältige Spendenmöglichkeiten: Onlinespende, Barspende, Spende mit Zahlschein, Spende mit Einziehungs- oder Dauerauftrag, Benefiz veranstaltungen, Sammelaktionen, Spenden statt Geburtstags- oder Weihnachtsgeschenken, Erwerb unserer Kuscheltiere, Kranzablösespenden, Legat / Testament etc. Das Team der St. Anna Kinderkrebsforschung ist dankbar für die langjährige spendenfreudige Unterstützung und ich persönlich für die Begegnung mit so vielen großzügigen, warmherzigen und hilfs bereiten Menschen. Herzlichen Dank! Mag. Andrea Prantl Leiterin Spendenbüro 16 17

10 DATEN & FAKTEN

11 UNIV.-PROF. DR. RENATE PANZER-GRÜMAYER Leukämiebiologie

12 Daten & Fakten QUELLE DER KOMPETITIVEN DRITTMITTEL USA ISL AND FINNL AND RUSSL AND im Jahr 216 POLEN 9,42 % Europäische Union 26,12 % S onstige Drittmittelgeber DEUTSCHL AND SLOWAKEI 15,64 % F onds zur Förderung der wissenschaftlichen Forschung (FWF) ÖSTERREICH 43,41 % Österreichische Nationalbank 5,42 % Ö sterreichische Forschungsförderungs- gesellschaft (FFG) UNGARN Mitarbeiterinnen und Mitarbeiter der St. Anna Kinderkrebsforschung im Jahr 216 FRANKREICH SPANIEN ZUWEISUNG DER GELDMITTEL NATIONEN IRAN FINANZIERUNG im Jahr 216 TÜRKEI KROATIEN PORTUGAL im Jahr 216 GRIECHENL AND ITALIEN BOSNIENHERZEGOWINA SERBIEN & MONTENEGRO PERSONELLE ZUSAMMENSETZUNG der Mitarbeiterinnen und Mitarbeiter im Jahr % Frauen 9,51 % Forschung 5,92 % Spendenwerbung 79,66 % Spenden und Verlassenschaften 2,34 % Kompetitive Drittmittel 33 % Männer 3,6 % Verwaltungsaufwand,52 % Sonstiger Aufwand 22 23

13 FORSCHUNGSNETZWERKE National und international IE Daten & Fakten N N TI EN N I E G AL AR STR N E N AU L G I IE BE ASIL IEN R BR GA L BU LE I CH NA K I AR ND CH EM LA N DÄ T S C H U DE ND TL A D ES AN NL ICH N I F RE D NK A L AN N FR EN H NIE C IE TA N I GR R SSB GRO ONG GK N HO ND IRL A ND ISL A EL A ISR N L A IT IE J A PA N A K ANAD EN TI A KRO D AN TL T LE N BA AL N IE LIT AU EN LUXEM BURG M AL AYSIA NEUS EEL AND NIE DE RL AN DE NO RW EG EN Ö ST E R R E IC H POLEN PORTU GAL RUM Ä NIEN RUSS L AND SCH WED EN SCH WEI Z SER BIE N SIN GAP UR SLO WA KEI SLO WE NIE S PA N NIE SÜ N DK O TS RE CH A EC TÜ HI RK EN EI UK R UN AIN E UR GAR N US UGU AY A W EI SS RU SS LA ND 24 25

14 Daten & Fakten KLINISCHE FORSCHUNG ANSTIEG DER 2-JAHRES-ÜBERLEBENSRATEN Die St. Anna Kinderkrebsforschung fungiert als nationales Koodinierungszentrum und im Bereich der Langerhans-Zell-Histiozytose, des Neuro blastoms und der Stammzellentransplantation, als internationales Koordinierungszentrum für die hier abgebildeten Studien. Das Studienmanagement wurde von unserem Koordinierungszentrum, der Abteilung S2IRP, durchgeführt. PATIENTENAUFKOMMEN IN S 2 IRP ALS KOORDINIERUNGSZENTRUM FÜR KLINISCHE STUDIEN 14. ANSTIEG DER 2-JAHRES-ÜBERLEBENSRATEN VON KREBSKRANKEN KINDERN UND JUGENDLICHEN Morbus Hodgkin 1 % Maligne Keimzelltumoren GesamtpatientInnenanzahl % 1. 6 % Osteosarkom Rhabdomyosarkom 2 % Hirntumoren Ewing-Sarkom 195 Akute myeloische Leukämie 2. 2 PatientInnen in Österreich 4. 4 % Neuroblastom und Ganglioneuroblastom Non-Hodgkin-Lymphom 197 Internationale PatientInnen 8. Akute lymphoblastische Leukämie Wilms-Tumor Quelle: GPOH Kinderkrebsregister Mainz

15 Daten & Fakten 5-JAHRES-ÜBERLEBEN VON K REBSKRANKEN KINDERN 5-JAHRES-ÜBERLEBEN VON KREBSKRANKEN KINDERN IN EUROPA ZWISCHEN 2 UND 27 Auch in einer kürzlich erschienen Publikation zum 5-Jahres-Überleben von Kindern mit Leukämie (Bonaventure et al. Lancet Oncology 217, 4, e22-e216) liegt Österreich gemeinsam mit D eutschland über dem europäischen Durchschnitt. Dieses erfreuliche Ergebnis kann auf die stringente Zusammen arbeit im Studienumfeld zurückgeführt werden. NORTHERN EUROPE Denmark Finland Iceland Norway Sweden UK AND IRELAND Ireland UK (England and Wales) UK (Northern Ireland) UK (Scotland) CENTRAL EUROPE Austria Belgium France Germany Switzerland Netherlands SOUTHERN EUROPE Croatia Italy Malta Portugal Slovenia Spain EASTERN EUROPE Bulgaria Estonia Hungary Latvia Lithuania Poland Slovakia ALL EUROPE Europe* year survival (%) Quelle: Gatta et al. Lancet Oncology 214, 15,

16 DR. ALEX ANDER DOHNAL Tumorimmunologie

17 SCIENCE REPORTS

18 UNIV.-PROF. DR. HEINRICH KOVAR Molekularbiologie

19 Science Reports INTRODUCTION R apid technological progress in biomedical research explores unimagined dimensions of the scientific universe and constantly produces new, unexpected findings. In the field of cancer research thishas led to the insight that tumors are not simply a homogenous mass of cells, but comprise a multitude of different cell types, each of which demonstrating a high degree of heterogeneity and plasticity. This finding especially applies to pediatric cancers. Until recently, the dogma has been that cancer is a purely genetic disease, which manifests itself in irreversible, rigid mutation patterns. Now, however, it is becoming increasingly clear that childhood cancer is in fact associated with a relatively low number of genetic alterations. As a result, pediatric cancers are to be considered a consequence of a perturbed development of different cell types. In order to exploit these aberrant mechanisms for therapeutic purposes, we need to understand the regulatory mechanisms of normal and malignant development that lie behind these aberrations. The increasing number of refined applications for novel technologies such as next generation sequencing (NGS) enables insights into the three-dimensional organization and aberrations of chromatin, the control center of gene expression, which determine the fate of each cell. The high sensitivity and specificity of this technology allows us not only to address the genetic composition and gene expression on the single cell level, but also to analyse tumor cell break-down products in the blood circulation. This way, we are able to obtain information on the genetic and non-genetic v ariation of tumors and their components in their interplay with other cell types and metabolites. These develop ments are also reflected in the activities of the Children s Cancer Research Institute. As documented by this annual report, we continued being successful in several areas of pediatric oncology research in 216. These successes bring us a step closer to our goal of improved therapy tailored to the specific needs of individual patients. In support of this notion, this report includes several examples of research results published by the Children s Cancer Research Institute in the past year. Prof. Heinrich Kovar, PhD 36 37

20 Science Reports Figure 1 Figure 2 Levels of heterogeneity LOLA Core database DNA methylation NON-GENETIC PL ASTICIT Y AND VARIABILIT Y AS THERAPEUTIC AND PROGNOSTIC TARGETS IN EWING SARCOMA ENCODE DNase CODEX UCSC 1% % EwS-hypomethylated regions In a collaborative study between the CCRI, the CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences in Vienna, and the Institut Curie in Paris, led by CCRI scientist Eleni Tomazou with contributions from several hospitals in Austria, Germany, France and Spain, we assessed genome-wide epigenomic patterns of more than 14 Ewing sarcomas. Using novel bio informatic methods developed by Nathan Sheffield at CeMM, our team studied the tumors DNA methylation patterns one of the most important facets of the human epigenome. We applied a next-generation-sequencing (NGS) based high-throughput method, RRBS (Reduced Representation Bisulfite Sequencing) to all samples, thus analyzing DNA methylation patterns of CpG-rich genome regions at single-nucleotide resolution. In addition, a subset of samples was analysed by WGBS (Whole Genome Inter-individual EwS-specific Region sets Despite the low number of mutations, Ewing sarcoma shows significant biological, clinical, and immunophenotypic variation and plasticity, which have moved into our focus in the reporting period. To understand inter-tumor, inter-patient and intra-tumor variability in absence of significant genetic variation, we interrogated the epigenomes of more than 14 Ewing sarcomas in comparison to each other and to those of other tumors and normal tissues. As described below, this paradigmatic study revealed two dimensions of heterogeneity, providing unprecedented biological insights into the etiology of the disease. Inter-cancer LOLA Region set enrichment EwS samples Tumor cell plasticity is the basis for metastasis, the major threat to patients. Tumor progression is associated with different types of cellular stress imposed on the tumor cell by the microenvironment. We and others have previously demonstrated upregulation of gene products involved in handling of cellular stress, among them the metabolic sensor Sirtuin 1 (SIRT1) and the DNA repair enzyme poly(adp-ribose) polymerase 1 (PARP1). We hypothesize that interfering with the activity of these proteins may reduce the metastatic potential of Ewing sarcoma and potentially sensitize resistant tumor cells to a variety of chemotherapeutic drugs. As PARP1 and SIRT1 are responsive to changes in tumor and microenvironmental metabolic states, we interrogated the role of the Ewing sarcoma metabolome as a potential vulnerability of the disease. Other tissues Our group has been studying the molecular underpinnings of Ewing sarcoma for many years. We try to understand how a single genetic aberration, the EWS-FLI1 gene fusion, can drive the pathogenesis of this disease. Since recent genome sequencing studies have confirmed an extremely low number of mutations and the absence of recurrent aberrations other than the EWS-ETS gene rearrangement and facultative whole chromosome copy number changes, we hypothesize that, if activated in the right developmental, epigenomic and genomic context, EWS-FLI1 is sufficient to induce and maintain malignancy. We believe that a deep mechanistic understanding of the interaction between developmental cellular background, microenvironment, tumor epigenome and EWS-ETS activity on a systems level will allow us to better understand the biological and clinical variability of the disease, and ultimately lead to the identification of vulnerabilities with therapeutic potential. Prostate Intra-tumor EPIGENETIC DIVERSIT Y IN EWING SARCOMA Figure 1. DNA methylation profiling by RRBS i dentifies epigenetic diversity on different levels CpGs Odds ratio Figure 2. RRBS profiling identifies a unique Ewing sarcoma specific DNA methylation pattern Bisulfite Sequencing), and interrogated for open chromatin regions by ATAC-seq (Assay for Transposase-Accessible Chromatin with high throughput sequencing), and for histone modifications indicative of transcription-regulatory activity by ChIPseq (Chromatin Immunoprecipitation sequencing). Comparing our data to the Ewing sarcoma reference epigenome that we had established previously and to publicly accessible data banks for similarly obtained DNA methylation profiles of human cancers and normal tissues, we identified a diagnostic Ewing sarcoma specific DNA-methylation profile distinct from any other tumor and normal tissue, and predictive of activity of Ewing sarcoma specific transcriptional enhancers (Figure 2). In addition, we observed unexpected inter-individual diversity

21 Science Reports Figure 3 Figure 4 A A EwS specific chromatin accessible elements Activity score Ewing-like Viability(%) Other cells 5.1 EwS cell line 1 1 nmol/l Viability(%) MSC (BM) muscle Viability(%) Mesenchymal Activity score 1 1 TC IC nmol/l IC nmol/l 1 1 RM IC nmol/l 1 B B EwS cell line EwS tumor Figure 3. Two dimensions of inter-individual epigenetic heterogeneity in Ewing sarcoma 15 Viability(%).4. IC nmol/l pluripotent stem cell IC Viability(%) in regulatory regions of EWS-FLI1 anti-correlated genes (Figure 3). Additionally, RRBS identified significant intra-tumor heterogeneity, which was higher in metastatic tumors than in localized Ewing sarcoma. In summary, for the first time, our study published in Nature Medicine identified different levels of epigenetic heterogeneity in a childhood cancer that may explain the diverse clinical courses observed in patients. As DNA methy lation influences 15 PC However, no distinct subgroups of tumors could be defined that might have been used for biological or clinical stratification. Rather, inter-tumor hetero geneity defined a disease spectrum along two dimensions: The degree of similarity to the diagnostic Ewing sarcoma signature defined by methylation heterogeneity at Ewing sarcoma specific open c hromatin sites, and the degree of stemness defined by methylation heterogeneity SK-N-MC 5 STA-ET-11 muscle IC nmol/l 1 1 EwS-FLI1 anti-correlated enhancers MSC (BM) IC nmol/l Stem-like 1 IC nmol/l 1 STA-ET EwS tumor TC32 EWS-FLI1-high 1 EWS-FLI1-low IC IC pluripotent stem cell A673 A673sh 15 nmol/l IC nmol/l 1 IC U2OS 1 5 IC HeLa HEK293 nmol/l 1 1 CLB-MA IC nmol/l 1 1 MSC 5 nmol/l 1 1 IC nmol/l 1 Figure 4. Ewing sarcoma cell lines (A) are exquisitely sensitive to NAMPT inhibition by FK866 as compared to non-ewing sarcoma cell lines (B) 4 41

22 Science Reports gene activity, the combination of Ewing sarcoma specific and cell-of-origin specific patterns can lead to different outcomes. The epigenetic diversity also appears to correlate with the tumors aggressiveness and metastatic state. These new insights into the biology of Ewing sarcoma provide the basis for developing epigenetic biomarkers that can reliably predict disease course and therapy response. Our findings in Ewing sarcoma also provide an interesting concept for other cancers with low genetic complexity. Sheffield NC, Pierron G, Klughammer J, Datlinger P, Schönegger A, Schuster M, Hadler J, Surdez D, Guillemot D, L apouble E, Freneaux P, Champigneulle J, Bouvier R, Walder D, Ambros IM, Hutter C, Sorz E, Amaral AT, de Álava E, Schallmoser K, Strunk D, Rinner B, Liegl-Atzwanger B, Huppertz B, Leithner A, de Pinieux G, Terrier P, Laurence V, Michon J, Ladenstein R, Holter W, Windhager R, Dirksen U, Ambros PF, Delattre O, Kovar H, Bock C, Tomazou EM. (217). DNA methylation heterogeneity defines a disease spectrum in Ewing sarcoma. Nat Med; 23: THE NAD METABOLOME AS THERAPEUTIC TARGET IN EWING SARCOMA Several studies have previously indicated Ewing sarcoma specific overexpression of the DNA repair enzyme PARP1 and preclinical evidence for exquisite sensitivity of the disease to PARP1 inhibitors. However, clinical trials have, so far, failed to confirm therapeutic efficacy of PARP1 inhibitors in monotherapy, and the question, which drugs to use in combination therapy sensitizing to PARP inhibition remains to be answered. PARP1 is a protein-modifying enzyme that requires NAD (Nicotinamide Adenine Dinucleotide) as co-substrate for protein-adp-ribosylation. NAD is a key metabolite of energy metabolism involved in cellular redox reactions, DNA repair, and in the maintenance of genomic stability. We have previously demonstrated overexpression of the metabolic sensor SIRT1 associated particularly with Ewing sarcoma metastases. SIRT1 is also an enzyme competing for the co-substrate NAD to deacetylate acetylated proteins. In fact, PARP1 and SIRT1 are the major cellular consumers of NAD, and their high activity in Ewing sarcoma require continuous fuelling by the co-substrate. We therefore interrogated the major source of NAD in Ewing sarcoma and analysed the consequences of disrupting NAD supply. Analysing the transcriptional signature of EWS-FLI1, which drives Ewing sarcoma growth, we identified a number of genes in the NAD biosynthetic pathway to be deregulated in expression. NAD can be metabolized from the amino acid tryptophan in the microenvironment. We found that in the Ewing sarcoma model cell line A673 by activation of Tdo2 (tryptophan 2,3-dioxygenase) knockdown of EWS-FLI1 led to tryptophan break-down and synthesis of the primary metabolites kynurenine and kynurenic acid, which in an autocrine manner activated the aryl hydrocarbon receptor and its nuclear target genes. However, no further metabolization to NAD was observed and activation of TDO2 in response to low EWS-FLI1 levels was restricted to a subset of Ewing sarcoma cell lines only. Instead, we found the enzyme NAMPT (Nicotinamide Phosphoribosyltransferase) to be rate-limiting for NAD synthesis in Ewing sarcoma. We therefore investigated the consequences of NAMPT inhibition by the small molecule inhibitor FK866 on Ewing sarcoma growth. We observed that blocking NAMPT leads to exhaustive NAD depletion in Ewing sarcoma cells, followed by a metabolic collapse and cell death. Using conditional EWS-FLI1 knockdown by doxycycline-inducible shrna revealed that EWS-FLI1 depletion significantly reduces the sensitivity of Ewing sarcoma cells to NAMPT inhibition. Consistent with this finding, a comparison of 7 Ewing sarcoma cell lines of different genotypes with 5 Non-Ewing sarcoma cell lines and mesenchymal stem cells revealed significantly higher FK866 sensitivity of EWS-ETS positive Ewing sarcoma cells, with IC5 values mostly below 1nM (Figure 4). Taken together, our data reveal evidence of the important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of Ewing sarcoma cells and suggest NAMPT inhibition as a potential new treatment approach in this disease. (Mutz C, et al., 217, Oncotarget) Mutz CN, Schwentner R, Kauer MO, Katschnig AM, Kromp F, Aryee DN, Erhardt S, Goiny M, Alonso J, Fuchs D, Kovar H. (216). EWS-FLI1 impairs aryl hydrocarbon receptor activation by blocking tryptophan breakdown via the kynurenine pathway. FEBS Lett, 59:

23 Science Reports IMPROVING THE ROUTINE ENUMERATION OF HEMATOPOIETIC STEM CELLS: MULTI-COLOR ANALYSIS OF CD34 SUBT YPES REVEALS UNEXPECTED DIFFERENCES BETWEEN VARIOUS STEM CELL SOURCES SUMMARY SPECIMEN COLLECTION Flow cytometric analysis of CD34+ hematopoietic stem- and progenitor cells commenced in the early nineteen nineties and became a standardized 3-color procedure in In the clinical setting, this method has since been used worldwide, without any changes or improvements, to enumerate CD34+ cells as such, irrespective of their developmental stage or their hematopoietic potential. Based on the results of different research groups and on recent adjustments of the model of human hematopoiesis (Abb. 1, Görgens A. et al Cell Rep.), we established a >1-color CD34 flow cytometric assay containing antibodies like CD133, CD45RA, CD1, CD19, CD38 and CD33. We showed that it enables phenotyping of at least 6 distinct CD34 subsets: The multipotent progenitors (MPP) are 133+/45RA-/1-/38low and represent the earliest of all CD34 subtypes. Differentiation of these MPP by asymmetrical division forms LMPP (lymphoid-primed multipotent progenitors) that acquired CD45RA (133+/45RA+/1-/38low), and EMP (erythro myeloid progenitors) that lost CD133 expression (133-/45RA-/1-/38+). LMPP represent progenitors of neutrophils and lymphoid blood cells while EMP can give rise to granulocytes other than neutrophils as well as to erythroid and megacaryocytic lineages. Differentiation of LMPP towards the more mature GMP (neutrophil granulocyte and monocyte progenitors) goes along with a loss of CD133 expression (133-/45RA+/1/38+), while gaining cell surface CD1 leads to the formation of MLP (myelo lymphoid progenitors; 133+/45RA+/1+/38+). Further maturation of MLP towards the progenitors of B lymphocytes (BLP) is accompanied by a loss of surface CD133 resulting in a 133-/45RA+/1+/38++/19+ phenotype. All cell specimens used in this study were obtained for routine CD34 enumeration. Donor BM samples (BMd, n=31) were from healthy allogeneic BM donors aged from 2 to 48 (median 26) years. Patient BM specimens (BM1y, n=21) were from biopsies routinely drawn for clinical examination one year after allografting. Six of these were paired samples, i.e. we analyzed both BMd and BM1y from these patients. The BM recipients (11 males, 1 females) had been diagnosed with AML (4), ALL (6), MDS (4), CML (2), RCC (2), SCID (1), SAA (1) and Hyper IG E syndrome (1). Their median age was 1 years (range.8-18). Conditioning regimens were myeloablative (n=11) and reduced intensity (n=1). Fifteen BM recipients had received antithymocyte globulin. PBSC samples (n=35) were from autologous stem cell collections from adult patients (22 males, 13 females) with Multiple Myeloma (n=19) and Non-Hodgkin s lymphoma (n=16), with a median age of 54 years (range 22-73). The mobilization regimen comprised chemotherapy (CHT) and hematopoietic growth factors (hgf; n=13), CHT+hGF and plerixafor (n=6), hgf alone (n=5) or hgf and plerixafor (n=11). Informed consent was obtained from all patients and the extended staining experiments had been approved by the local ethical committee. Figure 1 CD45RA CD133+ CD45RA + CD133 + Lymphomyeloid progenitors Multilymphoid progenitors MPP CD38 low CD1 - Multipotent progenitors LMPP EMP CD38 CD1 - CD38 + CD1 - MLP CD38 + CD1 + low GMP CD38 + CD1 Granulocytes and macrophages progenitors EoBP Erythromyeloid progenitors CD45RA CD133 -/low MEP CD45RA + CD133 B-lymphoid progenitors BLP CD38 ++ CD1 + CD19 + B-lymphocytes Late GMP CD38 + CD1 - Neutrophils monocytes CELL PROCESSING, SINGLE PL ATFORM PROTOCOL AND FLOW CY TOMETRY Blood cell counts were obtained from a Sysmex KX-21N (Sysmex Corporation, Kobe, Japan). If necessary, cells were diluted with Dulbecco s phosphate buffered saline (PBS, Carlsbad, CA, USA) and the white blood cells (WBC) adjusted to 5-15 x16 cells/ ml prior to immune staining. All monoclonal antibodies (mab) were used at pretested concentrations and after respective compensation. Isotype controls and fluorescence-minus-one analyses were used to define gating and compensation. One hundred µl of the cell sample were reverse-pipetted into a Trucount tube (BD Biosciences, San Jose, CA, USA). After adding the mab cocktail, cells were mixed and incubated light-shielded at room temperature Eosinophils basophils Megakaryocytes erythrocytes Figure 1. Model of the human hematopoietic tree, adapted from the recent version by Görgens et al Only CD34 + cell stages are depicted. The CD45RA-CD133 +CD38low CD1 multipotent progenitors (MPP, upper) either remain CD45RAand down-regulate CD133 (middle right) to form cells of the erythro-myeloid lineage (EMP), or they acquire CD45RA and become CD133 + lymphomyeloid progenitors (LMPP, middle left) that comprise all lymphoid but also neutrophil and monocyte precursors (GMP). Upon down-regulation of CD133 (lower left), these form late GMP and, in case of CD1 acquisition, cells of the B-lymphoid lineage (BLP)

24 Science Reports Figure CD 14 To calculate absolute cell numbers in donor BM, an additional dilution factor of 1.1 was considered to compensate for the anticoagulant added. CD4 5RA EMP CD CD45RA + 5RA late GMP 5RA BM1y + 5RA 4 CD 1 13 CD4 BLP late GMP 5RA LMPP+GMP MPP MLP LMPP+GMP MLP 14 EMP CD CD4 12 CD34 BLP MPP BMd CD34 STATISTICAL ANALYSES Differences in cell counts (both absolute and relative) between the three HSPC sources were assessed with the unpaired one-sided Wilcoxon signed-rank test. A p-value <.5 was considered statistically significant (*<.5, **<.1, ***<.1). Mean values (±SD) are provided in the text. In depicted box-and-whisker plots, boxes range from first to third quartile (containing 5% of data points). The median value is indicated by a thick horizontal line. Whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range (= box height) away from the box, and indicate the range that contains 95% of data points in a normally distributed sample. R version 3.2. ( ) was used for all statistical analyses. 3 LMPP+GMP (sample dilution factor) EMP 13 MPP -472 (events of target population) (sample volume (µl) ) CD45RA late GMP MLP CD1 GATING STRATEGY Viable WBC were defined by their CD45 expression, negativity for 7AAD, and typical position in the forward- and side scatter (FSC/SSC) dot plot. According to the ISHAGE guidelines, viable and true hematopoietic stem- and progenitor cells were determined by their positivity for CD34, their weak expression of CD45, their typical position in the lympho-monocytic area of the FSC/SSC dot plot and their negativity for 7AAD. To define subpopulations, CD34+ cells were first divided into an earlier CD45RA- and a more committed CD45RA+ cell fraction. These were then separately depicted in a CD133 vs. CD1 contour plot (Fig. 2). The resulting subpopulations were examined for their expression of CD38, CD33, CD1 and CD7. Beads were doublegated in two different dot plots (APC vs. SSC and APC-Cy7 vs. FITC) to exclude false-positive events, and the following formula was used to calculate the number of target cells per µl: (total number of beads per tube) (number of beads counted) 13 CD45RA for 2min. Red blood cells were lysed by adding 2 ml of ammonium chloride working solution (BD Biosciences) for 1 min before samples were analyzed on the flow cytometer. The mab cocktail contained the stem cell enumeration kit (BD Biosciences) comprising CD45 FITC, CD34 PE and 7AAD, as well as the following mab: AC133-1 APC, CD7 PeCF594, CD1 BV421, CD19 APC-Cy7, CD38 PE-Cy7, and CD45RA BV51, CD3 PerCPeFl71, and CD33 APC-R7. Acquisition of 15, CD45+events was done on a FACS Fortessa (BD Biosciences) equipped with 4 solid state lasers with excitation wave lengths (nm) of 488, 45, 561 and 64. The FACSDiVa 6 software (BD Biosciences) was used for cell acquisition and data evaluation. For quality control of the instrument s performance, CS&T beads (BD Biosciences) were used at least weekly. + 5RA BLP -472 PBSC CD Figure 2. M ulti-color CD34 subtype analysis: Representative examples of autologous PBSC (top), donor bone marrow (BMd, middle) and patient bone marrow one year after allogeneic transplantation (BM1y, bottom). Only viable and true CD34+ events are depicted (see supplementary Figure 1 for the gating strategy). Cells were first separated into fractions negative or positive for CD45RA (larger plots). The proportion of earlier CD45RA- progenitors was generally higher among PBSC than BMd and, particularly, BM1y cells. All CD34+CD45RA+ or CD34+CD45RA- events are shown in CD133 vs. CD1 contour plots, where they form the C D45RA-CD133+CD1- MPP, the CD45RA+CD133+CD1- LMPP, the CD45RA-CD133-CD1- EMP, the CD45RA+CD133-CD1late GMP, and the CD45RA+CD133+CD1+ MLP and CD45RA+CD133-CD1+ BLP

25 Science Reports afigure 3 a ** B b b 2 2 ** % of WBC % of WBC ** *** PBSC PBSC BMd BMd *** *** BM1y BM1y *** *** ** PBSC PBSC ns ns *** ns ns BMd BMd BM1y BM1y *** *** Cells /µi &HOOV ĂO CD34 subtyping was always started with CD45RA, as this marker allows defining 2 distinct subgroups in all materials, separating earlier CD45RA- from more committed CD45RA+ HSPC (Fig. 2). Both subpopulations were then further evaluated in CD133/CD1 contour plots where they formed at least 6 distinct CD34 subpopulations. Between the 3 cell sources analyzed, we observed considerable differences regarding the composition of CD34+ subsets, both for absolute cell numbers and relative values (Fig. 3B). The mean cell proportion (in % CD34+ cells ±SD) of the early MPP cells was significantly higher in PBSC (42% ±13.7) than in BMd (16% ±8; p<.1). In BM1y, their frequency was only 2.5% ±1.8 which was significantly lower than in BMd (p<.1) and roughly 17 fold less than in PBSC. Analogous results were obtained for absolute MPP numbers which were significantly higher in PBSC (68/µl, ±475) than in BMd (138/µl ±156; p<.1), and in BMd than in BM1y (5/µl ±6; p<.1) *** 1 1 *** *** ** ns ns %%CD34+ of CD34+ Enumeration of total HSPC irrespective of their CD34 subtype revealed significant differences between the 3 cell sources (Fig. 3A). Results are expressed as absolute numbers (viable CD34/µl) and as relative values (viable CD34+ cells as percentage of viable WBC). The mean percentage (±SD) of total CD34+ cells was highest in BMd (2.1% ±2), followed by BM1y (1.1% ±.6) and by autologous PBSC (.8% ±.9). In terms of absolute CD34 numbers, the highest values were obtained in PBSC (1,42/µl ±1,49), followed by BMd (794/µl ±753) and by BM1y (255/ µl ±253). The majority of PBSC samples described in the present work was from adult patients diagnosed with different lymphoid malignancies. Since most of the pediatric patients had been allografted with donor bone marrow, only few PBSC samples had been obtained and analyzed from healthy mobilized donors, but their comparison revealed a similar distribution of CD34 subtypes in patient and donor PBSC (data not shown). &' FHOOV ĂO CD34 cells /µi 4 4 RESULTS Total CD34 CD34 relative Total relative Total CD34 CD34 absolute Total absolute A MPP MPP *** *** *** EMP EMP * *** *** LMPP+GMP LMPP+GMP Late GMP Late GMP *** *** ns ns *** ns ns ** MLP+BLP MLP+BLP *** *** *** MPP MPP EMP EMP LMPP+GMP LMPP+GMP + Late GMP Late GMP MLP+BLP MLP+BLP Figure 3: Comparison of median absolute and relative values of (A) total CD34+ cells and (B) CD34 subpopulations in autologous PBSC (n=35), donor bone marrow (BMd, n=31) and patient bone marrow one year after allogeneic transplantation (BM1y, n=21). For total CD34+ cells (A), absolute numbers (left) were significantly higher in PBSC (open circles) than in BMd (grey), and higher in BMd than in BM1y (black), while relative values (right) were highest in BMd and lowest in PBSC. Regarding the CD34 subgroups (B), absolute numbers (upper graph) of MPP, EMP and LMPP were significantly higher in PBSC than in BMd, and higher in BMd than in BM1y. Relative values (lower graph) of MPP and EMP were also significantly more frequent in PBSC than in BMd, and more frequent in BMd than in BM1y, and LMPP fractions were higher in BMd than in BM1y. This was in contrast to late GMP which were clearly higher in BMd than PBSC, and particularly to the more mature MLP and BLP subsets which were much more frequent in BMd and BM1y than in PBSC, showing the highest proportions (mean 59%) in BM1y

26 Science Reports Figure 4 MPP differentiate to either LMPP (CD45RA+CD133+) or EMP (CD45RA-CD133-/low). Both subsets showed a higher CD38 expression than MPP supporting their higher differentiation (Fig. 4). As shown in Fig. 3B, the mean frequency of LMPP was similar between PBSC and BMd (25.6% ±11.1, and 23.7% ±1.1; ns), but significantly lower in BM1y (16.3% ±8.3) than in BMd (p<.1). Absolute LMPP cell numbers differed significantly between PBSC (383/µl ±412) and BMd (17/µl ±158; p<.1), and between BMd and BM1y (33/µl ±28; p<.1). This may suggest a higher proportion of neutrophil progenitors in favor of B-lymphoid progenitors in the LMPP subfraction of PBSC compared to BMd. EMP which give rise to erythrocytes, megakaryocytes and granulocytes other than neutrophils, showed results comparable to those obtained for LMPP. Their mean frequency was similar in PBSC (24.5% ±8.7) and BMd (19.5% ±6.5; p<.5) but differed significantly between BMd and BM1y (11.9% ±7.3; p<.1). Absolute EMP numbers differed clearly and were 331/µl ±261 for PBSC vs. 162/µl ±176 for BMd (p<.1), and 24/µl ±25 for BM1y vs. BMd (p<.1). In terms of LMPP and EMP, BM1y thus mediates an impression of exhaustion when compared with BMd. Late myeloid progenitors (late GMP) with a CD45RA+CD133-CD33+CD1phenotype differed mainly with regard to relative values, which were significantly lower in PBSC (4% ±3.1) than in BMd (8.5% ±4.1; p<.1), but similar between BMd and BM1y (11.1% ±6.5; ns). In terms of absolute values, they were 51/µl ±62 in PBSC vs. 72/µl ±91 in BMd (ns), and slightly lower in BM1y (23/µl ±2) than in BMd (p<.1). Due to the low frequency of the CD133+ MLP, this CD34 subset was evaluated together with the CD133- BLP. These cells were hardly detectable in PBSC (5% ±7.9) but clearly present in BMd (31.9% ±15.1; p<.1). In BM1y, they represented the largest CD34 subfraction (58.5% ±17.6) which was significantly higher than in BMd (p<.1). Despite the rather low proportions of the CD133dim MLP in the BM samples, it has to be noted that this CD34 subset, when expressed as percentage of the MLP/BLP fraction, represented a clearly lower median cell proportion (p=1.66e-9; one-sided unpaired Wilcoxon rank sum test) in BM1y (3.6%) than in BMd (11.9%). A representative example is depicted in Fig. 2. In terms of absolute CD1+ stem cell numbers, significant differences were observed between PBSC (43/µl ±83) and BMd (255/µl ±32; p<.1), whereas the values were similar between BMd and BM1y (171/µl ±191; ns). Out of the 31 BMd and 21 BM1y specimens analyzed, 6 were paired samples, i.e. we examined BMd and BM1y pairs from the same patients. The results were virtually identical to those obtained from the whole groups: The median proportions of the CD34 subsets for BMd/BM1y were 12.1%/1.8% (MPP), 22%/17.4% (LMPP), 2.1%/1% (EMP), 5.5%/9.1% (late GMP) and 34%/65.2% (MLP and BLP). The expression intensity of distinct markers often correlates with differentiation as shown for CD38 (see below). Such differences were also observed for CD33 and CD133. Expression of CD133 on MPP and LMPP was generally higher in PBSC than in BMd and BM1y (not depicted), suggesting that these cell stages are more differentiated in BM than in PBSC. Nevertheless, it was possible to distinguish the different CD133+/- subpopulations in all cell samples (Fig. 2). The myeloid marker CD33 was expressed in all CD34 subfractions, although it was weaker on MPP in PBSC with high CD133 expression than on MPP in BM with weaker CD133 expression (not depicted). Distinct CD1+ (and CD19+) HSPC subsets were detectable among both the CD45RA+ and the CD133- progenitors, and the CD33 expression was clearly higher in BM1y than PBSC (not shown). In all cell sources, a potential coexpression of CD7 as a marker of T- and NK-cell progenitors was only seen on % to 2.8% of CD34+ cells). As non-specific staining could not be excluded, this subtype was not pursued any further. We used the PE-Cy7-labelled H7 clone of CD38 in all experiments performed. Virtually all CD34+ cells were positive for this antibody, albeit at clearly different intensity (Fig. 4). MPP showed the lowest expression intensity, followed by LMPP and EMP. The intensity was higher among CD45RA+ late myeloid precursors, and highest among the CD45RA+CD1+CD19+ BLP. This differential CD38 expression was generally observed in all specimens examined although the differences were not always as clear as depicted in the BM1y sample shown in Fig. 4. Due to the considerable overlap between the different CD34 subgroups, CD38 was never used as first-line antibody for subgroup distinction. CONCLUSION We conclude that the presented analysis can identify and enumerate distinct CD34 subfractions in any conventional CD34 cell source. This approach may provide a solid basis for future studies to determine the impact of different CD34 subsets in the graft as well as in post-transplant bone marrow on engraftment kinetics and immune reconstitution. Whether or not the analysis will allow predicting engraftment kinetics on a routine basis remains to be examined. Dmytrus J, Matthes-Martin S, Pichler H, Worel N, Geyeregger R, Frank N, Frech C, Fritsch G. (216). Multi-color immune-pheno typing of CD34 subsets reveals unexpected differences between various stem cell sources. Bone Marrow Transplant, 51: CD38 CD34 subset MFI MPP LMPP EMP Late GMP BLP Figure 4. Rise of CD38 mean fluorescence intensity (MFI) values on HSPC with increasing differentiation of CD34+ subsets. Representative bone marrow sample from a patient one year after allogeneic transplantation (BM1y) depicting CD45RA-CD133+CD1multi-potent progenitors (MPP), CD45RA+CD133+CD1lymphoid-primed multi-potent progenitors (LMPP), CD45RA-CD133-CD1erythro-myeloid precursors (EMP), CD45RA+CD133-CD1late granulocyte monocyte progenitors (late GMP) and CD45RA+CD133-CD1+ B-lymphoid progenitors (BLP). 5 51

27 Science Reports CHARACTERIZATION OF A NOVEL FUSION GENE IN JUVENILE MYELOMONOCY TIC LEUKEMIA ASSOCIATED WITH RESISTANCE TO T YROSINE KINASE INHIBITORS Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative neoplasia occurring in young children, characterized by excessive proliferation of monocytic and granulocytic cells infiltrating different organs. About 9% of JMML patients have mutations in NF1, K-RAS, N-RAS, CBL, or PTPN11, all implicated in activation of the RAS-RAF-MAPK pathway. Despite recent efforts exploiting whole exome sequencing, the genetic alterations underlying the disease in the remaining 1% of patients remain elusive. Hematopoietic stem cell transplantation (HSCT) is currently the only curative therapy for most JMML patients, but advances in the understanding of the underlying molecular mechanisms in JMML have permitted the introduction of different therapeutic agents such as the DNA-hypomethylating substance azacitidine. We have identified a JMML case with a chromosomal translocation, t(5;17)(q33;p11.2), resulting in the fusion of the platelet-derived growth factor receptor β (PDGFRB) gene to a novel partner, the nuclear distribution protein nude-like 1 (NDEL1), which has not been implicated in any translocation event to date. In contrast to earlier data on fusion genes involving PDGRFB in myeloid malignancies, which were generally responsive to treatment with imatinib, the patient became refractory to both imatinib and nilotinib. This observation represented the first clinical finding of a PDGFRB gene fusion resistant to therapy with tyrosine kinase inhibitors (TKIs), and our work focused on elucidating the hitherto unknown mechanism of TKI resistance. IDENTIFICATION OF A NOVEL FUSION GENE INVOLVING PDGFRB AND MUTATIONAL ANALYSIS Sequencing of 5 -RACE-PCR products revealed an in-frame fusion between NDEL1 and PDGFRB. Screening of archived diagnostic specimens from 4 JMML patients provided no evidence for the occurrence of the NDEL1-PDGFRB fusion gene in other individuals. The observed development of resistance to two different TKIs including imatinib and nilotinib prompted screening of the entire tyrosine kinase domain (TKD) of PDGFRB for the presence of mutations. Sanger sequencing revealed the missense point mutation C255G in the activation loop of the TKD converting the aspartate residue at position 85 into glutamate (D85E). This mutation was identified at the time of both relapses but not in the diagnostic PB or BM samples. STRUCTURAL MODELLING OF THE PDGFRβ T YROSINE KINASE DOMAIN In order to elucidate the structural effects mediated by the D85E mutation in the PDGFRβ TKD, we have generated protein models of the kinase domain both in active and inactive conformations. Since the structure of PDGFRβ TKD is not available, we have modelled the kinase domain on the basis of established structures of highly homologous proteins from the PDGFR family including c-kit, CSF1R, and VEGFR2. All structural models indicated that the observed TKI type-ii resistance of cells expressing NDEL1-PDGFRβD85E was conceivably related to stabilization of the activation loop (A-loop) in the active conformation. In this conformation, three critical amino acids, D844, F845, and G846 (DFG) serving as a hypomochlion for the activation loop, adopt the so-called DFG-in position. The modelled structure of the inactive DFG-out conformation revealed BB + C C AA Figure 1 DD H857 H857 D85 D E85 E85 F F H H H657 PDGFRb A L M S E L K I M H657 S H LG PDGFRaA AL LMMSSEELLKKI IMMST HH LLGG PDGFRb FLT3A AL LMMS SE EL LKKI M MMT T HO LLGG PDGFRa M SOHLLGG CSF1R FLT3 AALLMMSSEELLKKMI MT G c-kita AL LMMSSEELLKKI VMLSSHY LL G CSF1R Consensus c-kit AALLMMSSEELLKKVI LM SS YHLLGG 1% Consensus A L M S E L K I M S H L G Conservation 1% Conservation % D85 D85 2.9A 2.9A G G H853 H853 R853 R853 E946 E946 K657 K657 3.A 3.A 4.1A 4.1A D85 D85 R853 R853 EE H657 H A 2.7A E946 E A 5.1A D D S N Y K I C D F G L A RD85 DIMR D FF G GL LA AR RD KK II CC D D II M MH RD D SS N N YY DF FG GL LA KK II CC D AR RD D II M M SH D D SS N N YY CD D F FG KK II C GL LA AR RD D II M M NS D D SS N N YY DF FG GL LA KK II CC D AR RD D II K M NNDDSSNNYY D FF G L A R D KK II CC D D II M KNDS SN N YY K I C D FGLARDIM N D SNY % αc-helix αc-helix A-loop A-loop the typical auto-inhibitory interaction between D85 and the amino acid at the +3 position, R853, which is commonly observed in inactive kinase domains of other receptor tyrosine kinases (RTKs) from the PDGFR family, and is believed to stabilize the A-loop in the inactive conformation (fig.1a, orange). However, modelling of the mutant PDGFRβ TKD in the inactive conformation could not explain the enhanced kinase activity and resistance to type-ii TKIs, because the negatively charged glutamate at position 85 is also able to form a salt bridge with the positively charged side chain of R853 (fig.1b). Moreover, the DFG-out model of the TKD did not display any indication of weakened electrostatic interaction between E85 and R853 which would destabilize the inactive conformation of the kinase domain. However, the DFG-in model suggested the occurrence of two intriguing amino acid interactions upon transition of the A-loop from inactive to the active state (fig.1a, green). One interaction implicated the negatively charged D85 and the positively charged, conserved H657 in the αc-helix Figure 1. Structural model of PDGFRβ TKD (fig. 1C), which is expected to stabilize the A-loop in the active conformation. This interaction can be further enhanced by the D85E mutation, because the longer side chain of glutamate in comparison to aspartate brings the negatively charged carboxylic group 1.1 Å closer to the positively charged histidine residue, thus increasing the stability of the activation loop in the active conformation (fig. 1D). The other interaction involved R853 and E946 in the C-lobe of the TKD (fig. 1F). The +3 position to D85 is one of the least conserved positions in the activation loop of receptor tyrosine kinases (RTKs) from the PDGFR family (fig. 1H), and the arginine at this position in PDGFRβ (R853) has the longest side chain among all members. The DFG-in model suggested that the positively charged side chain of R853 can reach a distance of approximately 2.5 Å to the negatively charged carboxyl group of E946, which may facilitate electrostatic bonds and provide additional stabilization of the DFG-in conformation of the PDGFRβ TKD (fig. 1G). The structural model therefore suggested resistance of 52 53

28 Science Reports Figure 2 A TYPE TYPE TYPE I I TYPE II II AA B BB TKI WT R853H TKI IMA WT 8 R853H 5 >5 D85E DR15 H657K >5 FIP1L1-PDGFRα HR FIP1L1-PDGFRα WT D842E HR 15 WT 2 D842E 4 IMA NIL > > NIL SOR SOR DAS N/A DAS MID N/A N/A PAC MID 1 PAC 25 3 R WT H R WT 1 HR H NDEL1-PDGFRβ DR H657K D85E NDEL1-PDGFRβ HR D D DR DR NIL,1 nm NIL,1 nm py751-pdgfrβ py751-pdgfrβ py857-pdgfrβ py857-pdgfrβ PDGFRβ PDGFRβ N/A N/A N/A N/A 12 N/A Figure 2. (A) Displayed are IC5 values of different TKIs against Ba/F3 cells expressing wildtype (wt) or mutant NDEL1-PDGFR? fusion proteins. The corresponding IC5 values for Ba/F3 expressing FIP1L1-PDGFR? WT and D842E are given for comparison. (B) Western blot analysis of Ba/F3 cells transduced with wildtype or mutant (R = R853H, H = H657K, HR = H657K/R853H, D = D85E, and DR = D85E/ R853H) NDEL1-PDGFRB genes. The phosphorylation levels of NDEL1-PDGFR? at Y751 and Y857, and Erk are displayed. Shown are also the total expression levels of NDEL1PDGFR?, Erk, and the control gene Gapdh upon mock treatment with DMSO (indicated by - ) or with 1 nm of nilotinib (indicated by + ) for 4 h. perk perk ErkErk Gapdh Gapdh NDEL1-PDGFRBD85E mutation to type-ii TKIs, which can only bind to the inactive conformation of the PDGFRβ TKD, but indicated sensitivity to type-i TKIs binding to the active conformation. To address the predictions provided by the protein model, we have introduced several mutations affecting the aforementioned interactions and tested the sensitivity of generated constructs against a panel of TKIs. TRANSFORMING ACTIVIT Y AND TKISENSITIVIT Y OF THE WILDT YPE AND MUTANT NDEL1-PDGFRB FUSION GENES To assess the oncogenic potential of the newly identified fusion gene, the murine cell line Ba/F3 was stably transduced with wildtype or mutant NDEL1-PDGFRB constructs by employing a transposon-based system (Byrgazov K et al. Oncotarget. 216). In addition to the clinically identified D85E mutant, we have made a construct carrying H657K mutation, which, according to our structural model, would strengthen the electrostatic interaction between D85 and αc-helix thus stabilizing the DFG-in conformation of the PDGFRβ TKD (fig.1e). In order to probe the influence of R853 on the kinase activity and TKI-sensitivity of PDGRFβ TKD, we have also generated the constructs carrying the R853H mutation. Ba/F3 cells expressing the mutants H657K and D85E versions of NDEL1-PDGFRB displayed a slightly higher proliferation rate, possibly reflecting an elevated kinase activity of the mutants, whereas the R853H mutation leveled the proliferation rate of these mutants to wildtype level. In line with this observation, the level of auto-phosphorylation of PDGFRβ and its target, ERK1/2, was higher in Ba/F3 cells carrying the H657K and D85E mutation (fig. 2B, lanes 5 and 9) in comparison to the wildtype and R853H-carrying versions of the fusion gene (fig. 2B, lanes 1, 3, 7, and 11). The in vitro responsiveness of Ba/F3 cells transduced with wildtype or mutant NDEL1-PDGFRB constructs to different TKIs of type-i (dasatinib (DAS), midostaurin (MID) and pacritinib (PAC)) and type-ii (imatinib (IMA), nilotinib (NIL) and sorafenib (SOR)) was determined by in vitro cell survival (MTT) assays. Oncogene-addicted proliferation of Ba/F3 cells carrying NDEL1-PDGFRBD85E could only be inhibited by the type-i TKIs dasatinib, midostaurin, and pacritinib at sub-micromolar concentrations, in line with predictions by the protein model. DIFFERENTIAL EFFECTS OF CORRESPONDING MUTATIONS IN PDGFRβ AND PDGFRα The intriguing observation of TKI resistance apparently induced by the D85E mutation in the kinase domain of PDGFRβ, which was in contrast to the same amino acid exchange at the corresponding site in PDGFRα (D842E), raised questions regarding important structural differences between the two highly homologous RTKs. While PDGFRβ displays an arginine in the +3 position to the mutation site (R853), PDGFRα has the much shorter and less basic histidine in the corresponding position (H845) (fig.1h). It appeared conceivable therefore that interaction between the side chains of R853 and E946 in the mutant PDGFRβ TKD could stabilize the active conformation in the mutants H657K and D85E, thus mediating resistance to type-ii TKIs. To address this notion, we introduced a mutation into NDEL1-PDGFRB constructs replacing arginine at position 853 by histidine (R853H), thus mimicking the sequence of the activation loop in the PDGFRα TKD (fig. 1G). This change reduced the NDEL1-PDGFRB-driven proliferation of Ba/F3 cells, and restored the sensitivity of cells carrying the H657K and D85E mutant of NDEL1-PDGFRβ to type-ii TKIs, in line with the properties of the D842E mutant of the FIP1L1-PDGFRα fusion (fig.2a). The restored sensitivity to nilotinib was also confirmed by western blot analysis (fig. 2B). Our data provide first evidence for the occurrence of a point mutation in the activation loop of PDGFRB mediating resistance to type-ii TKIs, and for a major alteration of responsiveness to TKI treatment mediated by an exchange between two negatively charged amino acids in a tyrosine kinase. The protein model indicated sensitivity of cells carrying the mutant NDEL1-PDGFRB to type-i TKIs, which was confirmed by sensitivity testing in vitro. Availability of the model at the time of relapse after failure of imatinib or nilotinib could have assisted in selecting potentially effective treatment options. The observations therefore provide new insights into specific amino acid interactions in mutant RTKs which are of clinical relevance for improved selection of appropriate TKI treatment (Preuner S et al. Int J Mol Sci 216). In addition, it provides a new molecular insight into activation of PDGFRβ which could serve for de novo design of specific inhibitors of this versatile RTK. Byrgazov K, Lucini CB, Berkowitsch B, Koenig M, Haas OA, Hoermann G, Valent P, Lion T. (216). Transposon-mediated generation of BCR-ABL1-expressing transgenic cell lines for unbiased sensitivity testing of tyrosine kinase inhibitors. Oncotarget, 7: Preuner S, Barna A, Frommlet F, Czurda S, Konstantin B, Alikian M, Machova Polakova K, Sacha T, Richter J, Lion T (corresp.author), Gabriel C. (216). Quantitative Analysis of Mutant Subclones in Chronic Myeloid Leukemia: Comparison of Different Methodo logical A pproaches. Int J Mol Sci, 17: 642 Byrgazov K, Kastner R, Gorna M, Hoermann G, Koenig M, Lucini CB, Ulreich R, Benesch M, Strenger V, Lackner H, Schwinger W, Sovinz P, Haas OA, van den Heuvel-Eibrink M, Niemeyer CM, Hantschel O, Valent P, Superti-Furga G, Urban C, Dworzak MN, Lion T. (217, Epub 216 Oct 7). NDEL1-PDGFRB fusion gene in a myeloid malignancy with eosinophilia associated with resistance to tyrosine kinase inhibitors. Leukemia, 31:

29 Science Reports HIGH RESOLUTION GENOMIC AND TRANSCRIPTOMIC PROFILING OF PEDIATRIC B-CELL PRECURSOR ACUTE LYMPHOBL ASTIC LEUKEMIA: IMPLICATIONS FOR EMERGENCE OF RESISTANCE AND REL APSE Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in childhood and adolescence. ALL comprises various disease entities characterized by chromosomal translocations, aneuploidy, structural variants, and sequence mutations that interfere with critical cellular pathways affecting lymphoid development, tumor suppression, cell cycle control, homing, as well as kinase and cytokine receptor signaling. With contemporary treatment protocols, up to 9% of the patients remain in longterm remission. Still, relapses are one of the leading causes of death in children and young people. Previous studies from others and our group have shown that deletions of genes involved in the glucocorticoid (GC) mediated signaling pathway prevail at relapse (Kuster et al). This finding was therefore taken as an indication that these alterations could render the affected cells resistant to GC an integral component of all major childhood ALL treatment protocols and would then constitute a significant precondition for disease recurrence. IMPLICATIONS OF GLUCOCORTICOID SIGNALING ALTERATIONS IN CHILDREN WITH REL APSED ET V6/RUNX1-POSITIVE LEUKEMIA Frequency of genetic deletions in E/R-positive relapses Similar to previous studies, we classified deletions into those that affect genes involved in the GC signaling pathway, B cell development and cell cycle. The overall incidence of deletions in the GC signaling gene components BTG1, NR3C1, NR3C2, BMF, MSH1 and MSH6 was, with 58% (18/31 cases), similar to the one in our previous report (Kuster et al.). The most common of all remaining recurrent deletions concerned the tumor-suppressor gene ETV6 (61%), followed by BCL2L14, a gene that encodes a mainly pro-apoptotic BH3-only family member, and CDKN1B, which generates the cyclin kinase inhibitor p27. Since both latter genes flank ETV6, they were co-deleted in 38% and 35% cases, respectively. Other common deletions affected genes that encode cell cycle regulators, such as CDKN2A, CDKN2B and RB1 in 35%, 29% and 1% cases, respectively. The ETV6/RUNX1 (E/R) gene fusion is the genetic hallmark of the largest subgroup of childhood B cell precursor acute lymphoblastic leukemia, which also has the overall most favorable prognostic outlook. Yet despite its low risk features and rapid response to current treatment regimens, up to 15% of cases still relapse. These disease recurrences are more difficult to treat and therefore also responsible for a dismal outcome in a considerable proportion of affected children. Association of genetic alterations with clinical characteristics and outcome Of all GC signaling pathway-associated gene deletions only those in NR3C1, which generates the glucocorticoid receptor (GR), were associated with a subsequent relapse (5 vs. 8%, p<.4) and tended to occur more frequently in cases with a poor MRD response to relapse treatment (25 vs. 7%; p<.4). ETV6 gene deletions prevailed among MRD poorly The goal of our basic and translational research is, therefore, to explore genomic and transcriptomic alterations in specific ALL subgroups to better understand the evolution of leukemia, the emergence of relapse, and the nature of the resistant clone. Thereby, not only new insight into the mechanisms of relapse development is gained, but also the biological impact of genomic alterations, their potential role in resistance mechanisms, as well as their applicability as biomarkers and drug targets can be inferred. Figure 1 Figure 1. Response of ETV6/ RUNX1-positive leukemic cell lines to glucocorticoids. (A) Western blot analysis of GR signaling components of AT-1, AT-2 (wt GR) and REH (mutant GR) cell lines after exposure to PRED. Protein abundance was determined using anti-gr, antibmf, anti-bim and anti-puma antibodies. GAPDH was used as loading control. (B) Viability of AT-1, AT-2 and REH cells upon exposure to PRED, measured by MTT assay. Values are means ± SD from four independent experiments. **p<.5 (paired t-test). (C) Quantification of GR, BMF, BIM and GILZ transcripts (RT-qPCR) in response to PRED exposure (expressed as fold-change of vehicle-treated cells) of all three cell lines. Specific mrna values were measured in triplicates and normalized to endogenous GUS. Bars represent mean values ± SD from four individual experiments. *p<.5; **p<.5 (paired t-test)

30 Science Reports responding cases (81 vs. 4%, p<.5). However, since ETV6 deletions are similarly frequent at diagnosis and at relapse and the gene is not expressed in the remaining, non-deleted cases, it is difficult to imagine that they can play a major role in the development of drug resistance. The concomitant deletions of the BCL2L14 and/or CDKN1B genes are in fact much better candidates, because even a partial loss of their function might affect apoptosis and drug response in a substantial way. The glucocorticoid receptor determines the response to GC in vitro We used GC-resistant (REH) and GC-sensitive (AT-1, AT-2) E/R-harboring leukemic cell lines to model and assess the consequences of a functional loss of the GR in E/R-positive leukemias. Consistent with the lack of a functional GR, REH cells were resistant to prednisolone (PRED) when exposed to clinically meaningful concentrations, as indicated by their unchanged viability as well as the inability to induce the GR downstream targets BCL2 modifying factor (BMF), the BCL2-like gene BIM, glucocorticoid-induced leucine zipper (GILZ) and BCL2 binding component 3 (PUMA) at the transcript and the protein levels [Fig. 1A C]. By contrast, both GC- sensitive cell lines showed a reduced viability upon exposure to the same PRED concentration and the concomitant up-regulation of GR, as well as of the downstream targets at both transcript and protein levels [Fig. 1. A C]. for instance, coordinating the cell cycle and cell survival. GC resistance at relapse can therefore not be viewed in isolation but must always be seen as part of a more global system of drug resistance. In such a context, a distinct form of GC resistance will lose its relevance as soon as effective drugs, such as obatoclax, become available for clinical use. Grausenburger R, Bastelberger S, Eckert C, Kauer M, Stanulla M, Frech C, Bauer E, Stoiber D, von Stackelberg A, Attarbaschi A, Haas OA, Panzer-Grümayer R. (216). Genetic alterations in glucocorticoid signaling pathway components are associated with adverse prognosis in children with relapsed ETV6/RUNX1-positive acute lymphoblastic leukemia. Leuk Lymphoma, 57: GENOMIC AND TRANSCRIPTIONAL L ANDSCAPE OF P2RY8-CRLF2-POSITIVE CHILDHOOD ACUTE LYMPHOBL ASTIC LEUKEMIA Recently, a novel subgroup of childhood ALL has been described whose defining characteristic is the deregulated expression of the cytokine receptorlike factor 2 (CRLF2) gene, which is located in the pseudoautosomal region 1 on the short arm of the X and Y chromosome. The two most common causative genetic defects are a small interstitial deletion that fuses the first non-coding exon P2RY8 to the entire coding region of CRLF2 and occurs in 5 8% of childhood B-cell precursor (BCP) ALL cases and a more rare translocation that places CRLF2 under the control of the IGH enhancer. P2RY8-CRLF2 fusion harboring leukemias often carry additional alterations in JAK/STAT pathway genes and they may cooperatively activate downstream pathways. They are associated with a significantly increased relapse risk in AIEOP/ BFM protocols, which is independent of the size of the P2RY8-CRLF2-positive clone (Morak et al.). Respective cases are primarily classified as non-high risk by clinical and molecular response criteria and relapses occur predominantly late. IKZF1 encodes the lymphoid transcription factor IKAROS, which is a key regulator in early lymphocyte development and prevails in poorly responding cases in major treatment protocols. IKZF1 deletions were also reported in small cohorts of P2RY8-CRLF2-positive leukemia cases recruited to various treatment protocols. It seems, therefore, likely that their presence contributes to relapse development in this particular subgroup, a notion that has, so far, not been systematically investigated in large and well-characterized cohorts. Therefore, we performed whole exome sequencing (WES) and transcriptional profiling (RNA-seg) in 41 relapsing and non-relapsing major clone P2RY8-CRLF2-positive cases that were treated primarily according to BFM protocols. Clonal heterogeneity and instability of kinase-activating pathway alterations At initial diagnosis, the overall frequency of JAK/ STAT pathway mutations was 51% and equally distributed between relapsing and non-relapsing cases. They were subclonal in 47% of cases. Irrespective of their original size, JAK/STAT pathway mutation carrying clones were lost at relapse in 6% of the cases (Figure 2A). Mutations in RTK/ Ras pathway genes were found in 29% of the cases at diagnosis, lost in almost half of the cases, and increased to 68% in relapses (P =.6). P2RY8CRLF2 was also lost in 32% of relapses. Overall, our findings, together with previous ones, corroborate the notion that the functional impairment of many GC signaling pathway elements is involved in the emergence of GC-resistant E/R-positive cell populations. Many of the affected genes are not only engaged in specific GC signaling alone but also in a variety of other pathways that are, 58 59

31 Science Reports Figure 2 A B INITIAL LEUKEMIA Normal cell Founder PAX5 PAR1 CDKN2A/B IKZF1 JAK/STAT RAS RELAPSE Figure 2. Clonal composition and stability of genomic alterations. (A) Clonal composition of JAK/STAT (blue) and RTK/ Ras pathway (red) signaling gene mutations according to individual genes (symbol code). Dots represent the adjusted allelic frequency (adj. AF) of mutations. Black dots mark conserved mutations at diagnosis and relapse. (B) Model for the evolution of leukemia and selection of relapse clones. Leukemia-initiating (founder) alterations occur in a hematopoietic stem/ progenitor cell, while the ensuing RAG-mediated microdeletions evolve later during early B cell differentiation. Microdeletions affect genes critical for B cell differentiation and tumor suppression (color code at the bottom of the graph). JAK/STAT or RTK/Ras pathway activating alterations continuously emerge but do not outcompete each other at initial presentation of leukemia. Chemotherapy then selects for resistant clones, which represent frequently only minor subclones at initial diagnosis, but they may vary regarding their proliferation driving mutation. IKZF1 alterations predict relapse in P2RY8-CRLF2-positive ALL and display distinctive transcriptional signatures Except for IKZF1, the frequency of alterations in genes implicated in lymphoid development and in tumor suppression/cell cycle regulation did not significantly differ between relapsing and non-relapsing cases. IKZF1 alterations prevailed in the relapsing cohort with a frequency of 41% versus 1% in non-relapsing cases (P =.1). Therefore, we profiled 22 leukemia samples by RNA-Seq according to their IKZF1 status. Transcriptional profiling revealed specific regulations in the IK6 deletions and biallelic alterations group and, albeit to a much lesser degree, in the group with larger deletions compared with the wtikzf1 one (Figure 3). Gene set enrichment analysis revealed a highly significant correlation of differentially expressed genes with various human hematopoietic and lymphoid stem cells sets, and concordantly for gene sets specifically expressed in immature B cells. This suggests that IKZF1 alterations lead to impaired B-cell differentiation and the acquisition of stem cell-like features. We also found enrichment in gene sets that are upregulated in the context of microenvironment, focal adhesion kinase and integrin pathways, as well as of genes that are higher expressed in response to hypoxia, downstream VEGF/VEGFR signaling and to EPO signaling. IKZF1 alterations are associated with dismal outcome Except for the genomic IKZF1 status and white blood cell count, which were not associated with each other (P =.66), we found no other biological or clinical parameters such as other genetic alterations, age at diagnosis, clinical risk group assignment or morphological and molecular response to treatment to be correlated with the occurrence of relapses. There was no difference between Down Syndrome (DS) and non-ds cases. Of note, only one of eight IKZF1-altered relapsing cases initially had a poor MRD response and was, therefore, assigned to high-risk treatment. Yet, IKZF1-mutated cases had a significantly poorer outcome than their IKZF1 wildtype counterparts as evidenced by an adverse pefs (P =.26) and pos (P =.51). The P2RY8-CRLF2 fusion was lost in one-third of the relapses. Together with previous observations that, for instance, these fusions frequently affect subclones that never evolve into major relapse clones this indicates that it is primarily a secondary change that may potentially supply the respective cells with a certain proliferative but certainly not with an evolutionary advantage ( Morak et al.). In line with other types of childhood BCP ALL, B-cell differentiation genes are also commonly deleted in cases with a P2RY8-CRLF2 fusion. These abnormalities are usually preserved in the corresponding relapses. Consistent with their role in drug resistance, IKZF1 alterations prevail already in relapse-prone cases at diagnosis but become even more abundant in relapses. IKZF1- deleted cases also seem to profit from other associated B-cell development and cell cycle gene defects, particularly those of PAX5 and/or CDKN2A/B, which are found in half of the cases, but also from mutations in specific proliferation-promoting pathway genes. 6 61

32 Science Reports Figure 3 Figure 3. Transcriptional signature of leukemias according to IKZF1 status. Cluster heatmap of the top 5 up- and downregulated genes in both IKZF1-altered groups ( IKN denotes IKZF1 alterations leading to a dominant-negative effect and biallelic alterations and IKD designates IKZF1 alterations resulting in haploinsufficiency) according to fold-change (P 1E 8 for IKN versus IKZF1 wt (IKC), P 2E 3 for IKD versus IKC); IKN cases are indicated in red, IKD ones in blue and IKC in gray at the top of the map. Thus, we consider the P2RY8-CRLF2 fusion as one of the many secondary proliferative driver alterations that - in line with those activating JAK/STAT and RTK/Ras pathways are highly instable at relapse, albeit to a lesser degree if initially present in the major clone, and not as a bona fide primary genetic alteration that is always stable at relapse and critical for the maintenance of the leukemia. Taken together and as schematically depicted in Figure 2B, we envision the following scenario of relapse evolution in P2RY8-CRLF2-positive leukemias. Predisposing constitutional or acquired genomic alterations as those affecting chromosome 21 facilitate the expansion of a pre/leukemic B cell precursor clone. Its regular development is then obstructed by defects in genes whose products are required for normal B cell differentiation. Cells that carry specific combinations of abnormalities may gain a competitive and selective advantage and eventually predominate in the pre-leukemic cell population. Parallel to these alterations and increasingly during later stages of leukemia evolution, mutations in specific genes activate signaling pathways that enable their unrestrained proliferation. Since chemotherapy primarily eliminates the bulk of rapidly proliferating cells, it spares those less active resistant stem-cell-like ones that eventually generate relapses. In this scenario, the P2RY8-CRLF2 fusion is only one of several proliferation activating alterations that merely serves as a common marker for an otherwise genetically heterogeneous group, whose other and probably more relevant features are IKZF1 alterations. These alterations are well-known disease drivers in many types of drug-resistant leukemias, such as BCR/ABL1-positive ones, which all share a similar gene expression signature. The transcriptional profile of IKZF1-altered cases reflects their strong homing preference to the bone marrow niche as well as their high repopulation capacity, attributes that also become apparent in mouse models in which IKZF1-/- pre-b cells acquire stem cell and adhesion properties including activation of the focal adhesion kinase pathway. Vesely C, Frech C, Eckert C, Cario G, Mecklenbrauker A, zur Stadt U, Nebral K, Kraler F, Fischer S, Attarbaschi A, Schuster M, Bock C, Cavé H, von Stackelberg A, Schrappe M, Horstmann MA, Mann G, Haas OA, Panzer-Grümayer R. (217). Genomic and transcriptional landscape of P2RY8-CRLF2-positive childhood acute lymphoblastic leukemia. Leukemia, Epub 217 Jan 6 Besides these biological insights, our findings also provide some clues that may become relevant in future treatment decisions. Apart from their prognostic implications, IKZF1 alterations may eventually serve as markers for specific therapeutic interventions. It appears reasonable to try to restore IKAROS signaling especially in those IKZF1-altered cases that still have retained a functional wild-type allele. For the other 2% of cases with biallelic IKZF1 alterations, inhibition of the activated focal adhesion kinase pathway may become a viable treatment option. Such approaches might perhaps be combined with a cocktail of other signaling inhibitors given the availability of various JAK/STAT, Ras/MEK/ERK and PI3K/mTOR pathway inhibitors

33 Science Reports MEDIATORS OF SPONTANEOUS MATURATION IN NEUROBL ASTOMA Neuroblastomas show a so far unique feature in oncology research, i.e., their ability to mature spontaneously into a benign tumor. Maturing neuroblastomas are composed of two cell populations 1) tumor cells differentiating into a mitotically quiescent, benign state and 2) Schwann cells forming a dense tumor stroma. Already in 1996, we have shown that these stromal Schwann cells are not of tumor origin and postulated a prominent role of Schwann cells in the maturation process of this favorable neuroblastoma subtype (Ambros et al NEJM). Schwann cells are known to closely interact with neurons regulating axon integrity in the adult as well as axon differentiation during development. Furthermore, it was demonstrated that Schwann cells are able to transform into dedicated repair cells with distinct functions essential to promote SCs in periphery blood vessel axonal re-growth after nerve injury. Based on these findings, we hypothesized that Schwann cells exert their physiological functions, i.e., the regulation of neuronal differentiation during development and nerve regeneration, on neuroblastoma cells facilitating spontaneous tumor maturation in vivo. Subsequent studies further demonstrated that Schwann cells are also able to impair the growth of aggressive neuroblastoma cell lines, obtained from tumors lacking a spontaneous maturation capacity in vitro, indicating a therapeutic potential of Schwann cells or the factors they express. To gain insights into the role of Schwann cells in the neuroblastoma maturation process, we tested our hypothesis on the similarities of physiological interactions between nerve regeneration and neuroblastoma maturation. In a first step, we analyzed the proteome and transcriptome and cellular processes active in repair Schwann cells after nerve injury using high-resolution mass spectrometry and RNA-sequencing of highly enriched human Schwann cells and injured nerve tissue (Figure 2). Our results revealed that cultured Schwann cells and injured nerves share a similar repair Schwann cell-associated expression signature including previously not described molecules involved in axonal differentiation and two novel repair Schwann cell functions, i.e., debris clearance via phagocytosis and a type II immune-regulation (Weiss et al. 216). These findings extended the functional spectrum of Schwann cells in regenerative processes after nerve injury and strengthened our hypothesis on their vital role during neuroblastoma maturation. Hence, two-follow up projects now explore the signaling events between Schwann cells and neuroblastoma and/or immune cells in detail. Our preliminary results using a FACS based read-out system and immunofluorescence, demonstrated growth-inhibitory, differenti- MSCs SC progenitors SC stroma Figure 1. Clinical and biological heterogeneity of neuroblastoma. favorable genetics aneuploidy no MNA ganglionic cells mostly no SCAs Schwann cell recruitment differentiating neurblastoma fully matures ganglioneuroma benignly behaving time unfavorable genetics neuroblastoma any ploidy ±MNA SCAs un- or poorly differentiated neuroblastoma mutations progressing neuroblastoma aggressive malignant progression Figure 2 hrms Proteomics Neurotrophic/Neuritogenic EGFL8 FLRT3 GFRA1 MDGA1 NRP2 SEM3B SHOT1 TEN3 3 (8%) 1 (52%) Nerve (control) Nerve (injured) Schwann cell Fibroblast Phagocytosis NB 2 (16%) MHCII upregulation HLA-ABC SCs FBs HLA-DRα1 SC NB cell/p1 SC co-culture Neuroblastoma is the most common extracranial solid tumor in childhood and accounts for around 15% of all pediatric oncology deaths. This embryonal tumor arises from sympathetic neuronal precursor cells and shows a unique biological and clinical spectrum, encompassing spontaneous regression, spontaneous maturation, or malignant progression. While most patients whose tumors undergo spontaneous regression or maturation (ganglioneuroblastomas, ganglioneuromas) have an excellent outcome, not all children with aggressive tumors can be cured (Figure 1). Thus, a better understanding of the biology of both tumor types, spontaneously regressing/ maturing and aggressive ones is of high interest to develop novel treatment approaches. Thus, our recent research has focused on two distinct aspects of neuroblastoma biology spontaneous maturation and relapse formation. spontaneous maturation Figure 1 counts NEW INSIGHTS IN NEUROBL ASTOMA B IOLOGY FROM SPONTANEOUS MATURATION TO THE R EL APSE SEEDING CLONE Figure 2. Schwann cells in spontaneous neuroblastoma maturation. Schwann cells adopt new functions upon nerve repair and as stromal cells in the tumor microenvironment. High-resolution mass spectrometry (proteomics) and functional tests revealed novel neuritogenic/neurotrophic factors, phagocytosis and MHCII up-regulation

34 Science Reports Figure 3 Tumor at diagnosis ation- and apoptosis-inducing effects of Schwann cells on co-cultivated aggressive neuroblastoma cells. Secretome analysis and RNA-sequencing of repair- and tumor-associated Schwann cells, neuroblastoma cell lines and primary tumors will aid to identify the signaling factors and receptors participating in this cross-talk. All in all, understanding the Schwann cell biology in neuronal differentiation is of utmost interest for new approaches in regenerative medicine as well as neuroblastoma therapy. TUMOR HETEROGENEIT Y AND IDENTIFICATION OF THE REL APSE SEEDING CLONE IN METASTATIC NEUROBL ASTOMA Although there has been substantial improvement in the outcome of patients with certain subsets of neuroblastoma, long-term survival of high-risk neuroblastoma patients is still less than 4% and effective relapse therapies are lacking. To improve the outcome of these patients, efforts are currently focusing on understanding fundamental genomic alterations driving neuroblastoma progression, therapy resistance and relapse development. Tumor relapse in patients with metastatic disease (stage M) is the main cause of mortality in these patients. Therefore, early and reliable detection and characterization of the relapse-seeding clone/s will help to monitor disease and to choose an appropriate treatment. However, due to intra-tumor heterogeneity, tumor biopsies may fail to identify the most aggressive tumor cell clone(s). In order to identify the most appropriate tissue for detecting the relapse-seeding clones, we recently studied the genomic evolution of neuroblastoma tumors and bone marrow-derived disseminated tumor cells by analyzing geographically and temporally separated samples of stage M neuroblastoma patients (Abbasi et al Clin. Cancer Research). In a single, well-characterized stage M patient, we found a unique aberration, deletion 1q, in the relapse samples besides a high number of concordant genomic aberrations present in all analyzed samples. Interestingly, this aberration was present in the disseminated tumor cells at diagnosis but not in the primary tumor, despite analyzing seven different tumor pieces (Figure 3). In a cohort of 154 patients, the highest incidence of this aberration was found in relapse samples and occurred in DTCs at diagnosis nearly twice as frequently as compared to primary tumors. Our results indicate that analysis of bone marrow-derived disseminated tumor cells at diagnosis besides the tumor biopsies may increase the probability for detecting the relapse-seeding clone and thus allow a more precise diagnosis of the most aggressive drivers of tumor progression. DTCs at diagnosis Tumor at relapse DTCs at relapse Figure 3. Tumor evolution in neuroblastoma. Graphical representation of clonal expan sion in a stage 4 neuroblastoma patient. Each quadrant represents the clonal architecture of a tissue/time point. Each color represents a group of chromosomal aberrations and the size of each colored area represents the pro portion of cells with these aberrations within the analyzed samples. A 1q terminal deletion (W) which was present in the diagnostic DTCs and also in both, DTCs and metastatic tumor, at relapse was not found in seven pieces of the primary tumor. Weiss T*, Taschner-Mandl S*, Bileck A, Slany A, Kromp F, Rifat begovic F, Frech C, Windhager R, Kitzinger H, Tzou CH and others. Proteomics and transcriptomics of peripheral nerve tissue and cells unravel new aspects of the human Schwann cell repair phenotype. Gila. 216 Dec; 64(12): doi: 1.12/ glia * Contributed equaly 66 67

35 Science Reports CHIMERIC ANTIGEN RECEPTOR (CAR)-BASED IMMUNOTHERAPY FOR TREATMENT OF REACTIVATION OF CY TOMEGALOVIRUS INFECTION AFTER STEM CELL TRANSPL ANTATION RESISTANCE OF HCMV-INFECTED CELLS TO T CELL CY TOTOXICIT Y DESPITE HL A-INDEPENDENT TARGETING Interestingly and unexpectedly, however, when we tested polyclonally activated T cells expressing the gb-specific CAR, we found that these T cells could not directly eliminate cells infected with HCMV (Figure 2). We have observed this with two well-characterized HCMV laboratory strains Ad169 and Towne and also after prolonged co-culture of infected and effector cells. In order to exclude that the observed lack of lysis of HCMV-infected fibroblasts was due to inhibition of T cell activation, we determined the level of T cell degranulation and cytokine production. However, we clearly found that the CAR triggers degranulation as well as release of cytokines IFN-g and TNF in the T cells. In order to further exclude that any possible defective function of our gb-specific CAR was responsible for the observed lack of lysis, we switched to a T cell Membrane Membrane gb gb Nucleocapsid Tegument Nucleocapsid gh Tegument gh CAR gb gb Figure 1. Schematics of C ytomegalovirus (A) and of the CAR-T cell approach (B). CAR gb gb Crough et al. 211 HCMV-infected cell Crough et al. 211 HCMV-infected cell Figure 2 HCMV-infected HFF T-gB 293T non-infected HFF A A counts In collaboration with our partner Armin Ensser (Institute of Virology, Universitätsklinikum Erlangen, Germany) we have thus previously constructed a CAR for targeting a conserved region in gb (Full F et al. 21), which is abundantly expressed on the surface of HCMV-infected cells and conserved among different viral strains. Targeting of HCMV-infected cells by CAR expressing T cells in an HLA-independent manner is attractive, because it obviates the need for enriching antigen-specific memory T cells and circumvents immune evasion by impaired antigen presentation. Originally, this approach has been proposed for the treatment of HIV and was tested in clinical phase II trials finally. Recent applications of a CAR-T cell approach also for fighting Hepatitis B and C have shown promising results both in vitro and in vivo in a preclinical model. B Cytomegalovirus HCMV is a complex virus with possibly 75 translational products encoded by its genome. Since many of these products are highly immunogenic, HCMV triggers immune responses from all arms of the immune system and leads to a high frequency of responding HCMV-specific CD8pos and CD4pos T cells (up to 4% of the whole T cell repertoire). Despite this strong T cell reaction HCMV persists and establishes lifelong latency, possibly explained by the fact that a large proportion of its genome encodes for RNAs and proteins interfering with the antiviral immune response. One of the most import and most intensively studied defense mechanism of HCMV is the prevention of recognition of infected cells by T and NK cells and by antibodies. Like many other viruses, HCMV escapes recognition by T cells through interfering with antigen processing and inhibiting antigen presentation by MHC class I and II molecules on the surface of infected cells. A gb isotype isotype B B specific lysis [%] Reactivation of cytomegalovirus (HCMV) infection after hematopoietic stem cell transplantation is still associated with substantial morbidity and mortality. Prophylactically and preemptively administered antiviral chemotherapy after transplantation frequently results in toxicity and selection of resistant virus variants. Adoptive transfer of HCMV-specific memory T cells has been successfully applied, however, generation of specific T cells from seronegative donors is difficult. We hypothesized that HCMV could be targeted by adoptive transfer of CAR-T cells in an HLA-independent manner, because HCMV-infected cells display intact viral proteins such as glycoprotein B (gb) on their surface. Figure 1 specific antibody Figure 2. CAR-T cells do not lyse HCMV-infected cells. (A) The histograms show the expression of gb in fibroblasts (HFF, infected or non-infected) and in 293T cells transduced or non-transduced with a gb-encoding vector. (B) Shown is the lytic activity of αcd3/αcd28-expanded T cells transfected with either the gb-specific CAR or an irrelevant CAR specific for the carcinoembroynic antigen (CEA) by electroporation of CAR-encoding mrna. Lytic activity of these CAR-T cells (effector:target ratio 25:1) was determined one day after electroporation using HFF (non-infected or 4 days after infection with AD169, MOI 5) and 293T cells transduced or non-transduced with gb (filled symbols: infected HFF or gb-transduced 293T cells; empty symbols: non-infected HFF or non-transduced 293T; three donors). gb-car CEA-CAR 293T cells cells 293T gb-transfection +/-+/gb-transfection -1 gb-car CEA-CAR Fibroblasts Fibroblasts HCMV-infection +/-+/HCMV-infection 68 69

36 Science Reports Figure 3 HCMV SHIELDS ITS HOST CELLS FROM T CELL CY TOTOXICIT Y BY VIRALLY ENCODED ANTI-APOPTOTIC PROTEINS UL36 AND UL37x1 In search for a possible explanation for the observed resistance to lysis we focused on previously described anti-apoptotic mechanisms, which so far have not yet been linked to evasion of T cell cytotoxicity. HCMV is a particular target of the most ancestral antiviral defense mechanism, namely premature elimination of infected cells by programmed cell death. This is a consequence of its slow replication cycle taking three days with sequentially ordered immediate-early, early and late phases of gene expression - a process which is paralleled by exponentially increasing levels of viral protein and the Figure 4 Annexin V + cells [%] normalized d d1 d d d2 d2 d1 d3 d3 d4 d4 p.i. p.i. d2 d3 d4 p.i UL UL37x1 + CMA HFF + chnkg2d T cells release of infectious HCMV particles increasing in vitro until day 5 after infection. In order to prevent termination of such slow replication by suicide of its host cells, HCMV has integrated in its genome a whole array of repressors blocking the host cell death machinery at several points HFF 8 6 * B IFN-y ng/ml B * 1 % A d IFN-y ng/ml CAR-independent experimental setup. For this purpose we employed EBV-peptide-specific cytotoxic lymphocytes, which were directed via their TCR to HLA-matched fibroblasts loaded with a saturating concentration of the same peptide used for the enrichment of the cells from PBMC. In addition, we used a recombinant variant of the HCMVstrain Ad169 (Ad169 US2-11), which contained extended deletions in the US-gene region in order B to prevent down-regulation of MHC class I molecules during infection. Also this system reproduced our previous observation of striking resistance of HCMV-infected cells to lysis despite strong activation of the T cells (Figure 3). Further we could show in this system that lysis inhibition of the peptideloaded fibroblasts gradually increased in the course of infection. Together, the data strongly suggested a so far unknown immune escape mechanism, which is independent from abrogating antigen-presentation and thereby inhibiting T cell activation but instead directly blocks the cytotoxic effector functions of the T cells. % specific lysis [%] A IFN-y [ng/ml] A d d1 d d2 d1 d d1 d2 d3d2 d3 peptide-pulsed Donor A non-pulsed Donor A d4 d3 p.i. d4 d4 p.i.,b,b peptide-pulsed Donor A,B,C non-pulsed Donor A,B,C p.i.,c,c Figure 3. HCMV infection results in gradually increasing resistance of host cells to cytotoxic effector functions. Peptide-specific cytotoxic lymphocytes were co-cultured with fibroblasts charged or non-charged with peptide at different time points after infection with a mutant variant of AD169 deficient in downregulation of HLA molecules (AD169 ΔUS2-11, MOI 5). Shown is the lysis of the fibroblasts (effector:target ratio 5:1) (A) and the secretion of IFN-γ (B). We hypothesized that some of these anti-suicide mechanisms could also inhibit T cell cytotoxicity and we thus investigated the role of UL37x1 and UL36, which were already known to block death receptor-mediated apoptosis and to inhibit cell death induction at different levels. Indeed, our data confirmed our hypothesis, because transfection of UL36 and UL37x1 in combination and even of UL37x1 alone into fibroblasts significantly inhibited the lysis of the cells by CAR-T cells (Figure 4). Notably, a major fraction of CAR-T cell cytotoxicity appeared to be mediated by the perforin/granzyme pathway, since addition of concanamycin A strongly inhibited cell death induction. These data finally proved that HCMV efficiently shields its infected host cells from T cell attack not only by preventing recognition and T cell activation, but additionally also by directly interfering with cytotoxic effector functions through factors of the viral anti-suicide machinery. Figure 4. Viral proteins inhibit CAR-T cell mediated apoptosis in infected cells. The diagram shows the induction of cell death in fibroblasts expressing UL36 and/or UL37x1 after co-incubation with αcd3/αcd28-activated T cells expressing a CAR directed against NKG2D-ligands (3 different T cell donors). The percentages of apoptotic fibroblasts obtained in the co-cultures of CAR-T cells plus fibroblasts without UL36/UL37x1 expression were set to 1%. Concanamycin A (CMA, 1 nm) was used to block perforin-induced apoptosis. Importantly, this newly discovered mechanism, however, does not totally preclude anti-viral efficacy of our HLA-independent approach. For example, there could exist non-cytotoxic effects of granzymes as, e.g., granzyme M, which cleaves cellular and viral proteins with essential function for HCMV replication. Moreover, only recently, we could show that the cytokines IFN-γ and TNF triggered by the gb-specific CAR in the T cells can efficiently inhibit the replication of HCMV. Meanwhile, we have reproduced this effect also with a bispecific antibody directed against gb, which we have developed as a further step towards clinical translation. Given the strong toxic side-effects and the occurrence of resistance in the current treatment of HCMV reactivation, our approach would be particularly attractive in the high-risk constellation of an HCMV seronegative donor and an HCMV seropositive transplant patient. Proff J, Walterskirchen C, Brey C, Geyeregger R, Full F, Ensser A, Lehner M, Holter W. (216). Cytomegalovirus-Infected Cells Resist T Cell Mediated Killing in an HLA-Recognition Independent Manner. Front Microbiol, 7:

37 Science Reports L ARGE SCALE EUROPEAN TRIAL DEMONSTRATES SURVIVAL ADVANTAGE FOR HIGH RISK NEUROBL ASTOMA PATIENTS RECEIVING HIGH DOSE BUSULPHAN AND MELPHAL AN TREATMENT Figure 1 (A RANDOMISED PHASE 3 TRIAL OF THE SIOP EUROPE NEUROBL ASTOMA GROUP (SIOPEN)). Neuroblastoma, the commonest paediatric extra-cranial solid tumour, is responsible for a large proportion of deaths from cancer in childhood. High-risk neuroblastoma, defined by metastatic disease over the age of 12 or 18 months and MYCN amplification (MNA) at any age remains associated with poor long-term survival rates. High-dose chemotherapy with haematopoietic stem cell rescue (HDT/SCR) improves event free survival (EFS) of patients with high-risk neuroblastoma (HR-NBL); however which regimen has the greatest patient benefit is under investigation. The high-risk neuroblastoma trial (HR-NBL1/ SIOPEN) opened in 22 and has tested, a number of hypo theses (Ladenstein et al., J Clin Oncol 21, 28(21): / Ladenstein et al., MAbs. 213; 5(5): 81 9). Here we report results of the hypothesis that HDT with busulphan and melphalan (BuMel) results in a superior EFS than HDT with carboplatin, etoposide and melphalan (CEM). METHODS Patients were enrolled and randomised by 128 SIOPEN member institutions/hospitals in 18 countries using the SIOPEN-R-NET web based, online remote data entry system ( siopen-r-net.org/). Randomisation was carried out just prior to HDT by minimisation balancing age at diagnosis, stage, MYCN amplification and national group between the arms. Tumour evaluations were carried out at the time of trial enrolment, after completion of induction, before HDT, following HDT and at the end of therapy. Eligibility and response was evaluated by 123ImIBG scintigraphy assessing the primary tumour and metastatic sites. In the case of mibg negative tumour, bone scintigraphy with 99mTc-hydroxy-methylenediphosphanate (MDP) scintigraphy was carried out; CTor MRI scan of primary tumour and radiological visualisation of any other evaluable disease; examination of bone marrow aspirates and trephines at two sites and measurement of urinary catecholamines. Patients were evaluated according to the National Cancer Institute Common Toxicity Criteria (CTC, Version 2) and by Bearman toxicity grades for pulmonary toxicity, veno-occclusive disease (VOD) and haemorrhagic cystitis. Monitoring for adverse event monitoring was continuous. The trial was approved as required by national regulatory authorities and by national as well as institutional ethical committees/review boards in participating countries. Three year EFS from randomisation was the primary endpoint. Analyses were done by intention to treat, but a per-protocol approach was used for the safety analysis. The trial was registered with ClinicalTrials.gov (number NCT174716) and EudraCT (number ). Trial efficacy results remained masked until release by the independent Data Monitoring and Safety Committee (DMSC) and only the DMSC and the study statistician were aware of interim efficacy monitoring results. At the pre-planned interim analysis of October 21 the DMSC recommended stopping randomisation as the Peto rule on EFS was met (p-value <.1 on the primary endpoint)

38 598 patients (88% of all eligible) were randomised to receive either BuMel or CEM as HDT. Supportive care followed institutional guidelines. VOD management was similar to published. Defibrotide was not recommended, because the study was designed before defibrotide was approved. Baseline characteristics were the same in each group including length of induction treatments, additional TVD, time of surgery and radiotherapy. All randomised patients were included in the analysis and no patients discontinued BuMel or CEM once commenced. Of the 598 randomised patients, 79 (13.2%) were subsequently enrolled in immunotherapy randomisations, of these only 64 patients (11%) were randomised to ch14.18/cho immunotherapy. The ch14.18/cho immunotherapy randomisations have been reported elsewhere. The median age of patients at randomisation was 3 years (range, 1 to 17 years). The median follow-up time of patients since randomisation is 7.2 years (IQR ). The 5yr-EFS (event free survival) and 5yr-OS (overall survival) for the entire 1347 population were 33 [31-35%] and 43 [41-45%] respectively from enrolment. In all randomised patients, independently of randomised HDT, the 5yr-EFS and OS of the 72 patients with localised stage MNA neuroblastoma was significantly higher (71 [58-8%] and 75 [63-83%] respectively) compared to the 526 stage 4 patients (35 [31-39%] and 44 [39-48%]) (both p<.1). In the age group of months, 18 stage 4 patients without MNA had a superior prognosis (5yr-EFS 72 [46-87%]) compared to those with MNA (5yr-EFS 32 [18-47%]) (p=.114). The six infants <12 months of age with stage 4 MNA tumours had a 5yr-EFS of 33 [4-67%]. Severe toxicities (need for intensive care and toxic deaths) were lower with BuMel (4%) compared to CEM (1%). BuMel had fewer grade 3 and 4 non-haematological toxicities, but was associated with 22% veno-occlusive disease Bearman grades 1-3 (4% grade 3) versus 9% with CEM (1% grade 3). Patients receiving CEM had higher median numbers of days with fever (CEM 7 [IQR, 3-12], BuMel 4 [3-6], and intravenous antibiotics (CEM 13 [IQR, 7-14], BuMel 1 [IQR, 7-14]). BuMel was associated with fewer CTC grade 3 and 4 non-haematological toxicities (fever, stomatitis, nausea and vomiting, diarrhoea, reduced cardiac function, raised serum creatinine and reduced GFR). Bearman pulmonary toxicity CEM 6 1 BUMEL CEM 4 8 Patients Events BUMEL CEM BUMEL 8 5-yrs. EFS 41 [35-47] 29 [24-35] p-value Patients Events 5-yrs. EFS p-value Follow-up (years) BUMEL [35-47].6 CEM (1)29 [24-35] 184 () (2) 118 (4) 85 (23) 7 (37) 51 (55) (1) () 133 (2) (1) 97 (1) 2 Event Free Survival Event Free (%)Survival (%) Between 22 and 21, 1347 patients were enrolled and 676 were eligible for randomisation. Eligibility for this randomisation included the completion of a multi-drug induction regimen (COJEC with or without topotecan, vincristine and doxorubicin) and achievement of an adequate disease response after induction (complete bone marrow response and at least a partial response at skeletal sites with three or fewer abnormal sites on 123iodine-metaiodobenzylguanidine scintigraphy). The primary endpoint, EFS (95% CI) at 3 and 5 years was 5 [45-56%] and 45 [39-51%] in 296 patients randomised to BuMel versus 38 [32-43%] and 33 [28-39%] in 32 patients randomised to CEM (p=.5). The post-hoc analysis of cumulative incidence of relapses (CIR) was significantly lower with BuMel (52 [46-56%])) compared to CEM (63 [57-68%]) (p=.33), suggesting a better drug action on residual tumor cells with the BuMel regimen. localisedlocalised localised FINDINGS (1) 2 87 (1) 73 (9) 56 (22) 49 (29) 118 (4) 12 (1) 85 (23) 7 (37) 51 (55) 87 (1) 73 (9) 56 (22) 49 (29) 39 (37) Follow-up (years) 7 39 (37) (66) 1 Overall Survival Overall (%)Survival (%) Stage 4 Stage 4 Stage 4 Figure 2 25 (79) 3 (44) 9 38 (66) BUMEL CEM BUMEL 227 () CEM 264 Patients Events yrs. OS 51 [44-57] 37 [31-43] p-value Patients Events 5-yrs.(years) OS p-value Follow-up [44-57] (2) (4) 129(13) 17 (28) 83 (46) 61(65) [31-43] 47 (77) 25 (1) 186 (1) 128 (1) 34 (56) () 186 (2) (1) 186 (1) 2 23 (51) (92) 98(1) 75 (25) 61 (33) 151 (4) 129(13) 17 (28) 83 (46) 61(65) 47 (77) 31 (92) 128 (1) 98(1) 75 (25) 61 (33) 47(77) 34 (56) 25 (64) Follow-up (years) 47(77) (64) Patients Events BUMEL 34 7 CEM (79) 3 (44) (51) BUMEL CEM 29 () 3 1 () 1 5-yrs. EFS 79 [62-9] 62 [45-76] p-value Patients Events 5-yrs. EFS p-value Follow-up (years) [62-9] ()14 27 () 62 [45-76] 28 () 26 (1) 21 (6) 17(1) 27 () 2 25 (1) 3 23 (3) 2 (4) 18 (4) Follow-up (years) 15 (9) () 28 () 28 () 27 () 26 (1) 21 (6) 17(1) () 27 () 25 (1) 23 (3) 2 (4) 18 (4) 15 (9) (13) 9 (15) 1 1 (17) 9 7 (17) 1 14 (13) 1 (17) 9 (15) 7 (17) was significantly higher with CEM (CEM 1% vs. BuMel 5%) and central neurotoxicity tended to be higher with CEM (CEM 4% vs. 1% BuMel (seizures associated with fever and metabolic disturbances). Twenty-two percent of patients receiving BuMel developed VOD with Bearman toxicity grades 1-3 compared to 8% receiving CEM. Only 4% of patients who received BuMel and 1% with CEM developed grade 3 VOD. Severe VOD was the cause of death in one patient on BuMel. The 5-year non relapse related mortality 3 [1-6%] for BuMel and 4 [2-7%] for CEM. Overall Survival Overall (%)Survival (%) Event Free Survival Event Free (%)Survival (%) Science Reports BUMEL CEM BUMEL 31 () CEM 32 () 1 Patients Events yrs. OS 79 [61-9] 71 [53-83] p-value Patients Events 5-yrs. OS p-value Follow-up (years) [61-9] () () 27 ()71 [53-83] 26 () 26 (1) 21(6) 3 () 2 27 (1) 3 25 (3) 23 (4) 2 (6) Follow-up (years) 16 (1) () 29 () 28 () 27 () 26 () 26 (1) 21(6) () 3 () 27 (1) 25 (3) 23 (4) 2 (6) 16 (1) (1) 8 1 (16) 1 1 (17) 9 17 (1) 1 (16) 8 (18) 1 1 (17) 8 (18) In multivariable analysis of all randomised patients, CEM, stage 4, age >5 years, and failure to achieve CR prior to HDT/SCR were identified as independent unfavourable prognostic factors (Model 1), but not age years or whether surgery was performed before HDT. Stage 2 and 3 MNA patients strongly influence the response result in this multivariable analysis as patients in CR (38) prior to HDT/SCR had a very good prognosis (5yr-EFS 89 [74-95%]) whilst patients achieving a VGPR (2) or PR (13) had 5yr-EFS of 5 [27-69%] and 46[19-69%], respectively. A multivariable analysis model limited to randomised stage 4 patients showed CEM, age >5 years and involvement of more than one metastatic compartment (MC) at diagnosis as independent unfavourable factors but not failure to achieve CR prior to HDT/SCR, MNA, age years

39 Science Reports INTERPRETATION FUNDING SUPPORT This randomised trial has evaluated a critical component of high risk neuroblastoma therapy and compared BuMel with CEM. After COJEC induction, BuMel significantly improved EFS in children with HR-NBL with an adequate response to induction therapy and induced less severe toxicities. BuMel showed an advantage in patients with stage 4 metastatic disease and less than 5 years. The improved survival of patients receiving BuMel was supported by the post-hoc analysis demonstrating that the cumulative incidence of relapse (CIR) was significantly lower with BuMel. The outcome of patients with stage 2 and 3 MNA appeared to be improved (79% 5yr-EFS) compared to approaches without HDT/SCR. European Commission 5th Frame Work Grant (SIOPEN-R-NET EC grant No. QLRI-CT , Pierre Fabre Médicament providing Busilvex and the St. Anna Kinderkrebsforschung. Neither the European Commission nor Pierre Fabre had a role in study design, data collection, data analysis, data interpretation or writing of the report. All authors participated in the report and had full access to all the data in the study. The corresponding author had final responsibility for the decision to submit for publication. BuMel was found tolerable in spite of more frequent VOD and spared important other life-threatening toxicities observed with CEM. Both in COG and SIOPEN further randomised studies are evaluating additional intensification of consolidation including two HDT procedures in tandem. PUBLICATION ON THE HR-NBL1/SIOPEN TRIAL: Ladenstein R, Pötschger U, Pearson ADJ, et al. For the SIOP Europe Neuroblastoma Group (SIOPEN). (217). Busulfan and melphalan versus carboplatin, etoposide, and melphalan as high-dose chemo therapy for high-risk neuroblastoma (HR-NBL1/ SIOPEN): an international, randomised, multi-arm, open-label, phase 3 trial. Lanct Oncology, Volume 18, No. 4 Ladenstein R, Poetschger U, Gray J, et al. (216). Toxicity and outcome of anti-gd2 antibody ch14.18/ CHO in front-line, high-risk patients with neuro blastoma: Final results of the phase III immuno therapy randomisation (HR-NBL1/SIOPEN trial). ASCO Annual Meeting Abstracts. ASCO Annual Meeting, J Clin Oncol 34, (suppl; abstr 15.) BuMel is now considered standard HDT/SCR for SIOPEN and ongoing randomised SIOPEN studies will continue to optimise therapy for high-risk neuroblastoma

40 CAREER

41 UNIV.-PROF. DR. RUTH L ADENSTEIN S2IRP: Studien und Statistik

42 Career WORKING AT CCRI A SHARED MISSION MAKES A DIFFERENCE IN RESEARCH! The Children s Cancer Research Institute (CCRI) is an internationally renowned biomedical research institute, dedicated to the advancement of diagnosis, prognosis and treatment of childhood cancer. Science at the CCRI addresses a broad range of topics, covered by 12 research groups whose members work closely t ogether and in c ooperation with clinicians from St. Anna Children s Hospital. We are at the forefront of international pediatric cancer research and scientists at CCRI are striving to make the most innovative and effective therapies available to young patients. IF YOU WANT TO APPLY FOR A POSITION The CCRI offers an intellectually stimulating environ ment with lots of opportunities for s cientific exchange and discussions in regular seminars, retreats, and national and international conference attendances. The quality of our work is p eriodically reviewed by a panel of top international experts guaranteeing consistently high standards of CCRI research. PhD students are accompanied by PhD-committees to guide them through their thesis and postdocs have the option to exercise their ability to teach by mentoring and training younger colleagues. please proceed according to the details given on our web site. At CCRI, we are an international team with employees from all over the world, offering students and postdocs an ideal combination of freedom and guidance to foster their personal and i ntellectual development while at the same time introducing them to the world of science and academic competition

43 Career SCIENTIFIC STAFF GROUP LEADER Peter Ambros Martin Distel Alexander Dohnal Michael Dworzak Gerhard Fritsch Oskar Haas Wolfgang Holter Heinrich Kovar Ruth Ladenstein Thomas Lion Renate Panzer-Grümayer Sabine Strehl AFFILIATED CLINICIANS Andishe Attarbaschi Heidrun Boztug Kaan Boztug Christofer Diakos Bernhard Fahrner Caroline Hutter Leo Kager Ulrike Kastner Anita Lawitschka Georg Mann Susanne MatthesLeodolter Milen Minkov Christina Peters Herbert Pichler Christian Posch Stefan Riegler Andreas Vécsei Volker Witt POSTDOCTORAL FELLOWS & STAFF SCIENTISTS Reza Abbasi M. Sarah Ahmadi-Erber Ingeborg Ambros Dave Aryee Jozef Ban Dominik Bogen Konstantin Byrgazov Sara Colomer Lahiguera Stefan Czurda Filomena De Almeida Nogueira Markus Diem Klaus Fortschegger Christian Frech René Geyeregger Zvenyslava Husak Maximilian Otto Kauer Markus Abraham Kernbauer-Hölzl Stefanie Kirchberger Karin Kosulin Zsuzsanna Lehner Manfred Lehner Chantal Lucini Karin Nebral Michael Reiter Dagmar Schinnerl Raphaela Schwentner Caterina Sturtzel Sabine Taschner-Mandl Eleni Tomazou-Bock Murat Tugrul Cornelia Vesely CLINICAL RESEARCH PHD STUDENTS DIPLOMA STUDENTS Helga Björk Arnardóttir Saelde Baumgartner Tijana Frank Evgenia Glogova Corinne Grafl Ingeborg Hirsch Dasa Janousek Susanne Karlhuber Monica Kiesewetter Barbara Kristufek Nora Mühlegger Marek Nykiel Ulrike Pötschger Ingrid Pribill Marion Sebek Eva Sorz Elfriede Thiem Charlotte Brey Barbara Dillinger Friedrich Erhart Teresa Gerber Clara Hechenberger Anna-Maria Husa Anna Maria Katschnig Florian Kromp Cornelia Mutz Leonel Pereira Katarzyna Pietrzykowska Fikret Rifatbegovic Benjamin Salzer Klara Soukup Tamara Weiss Martin Zeppetzauer Jakob Berner Bernadette Blauensteiner Clemens Brunner Helena Dodig Kristin Fischer Katharina Martin Magdalena Reiter Maria Regina Strobl Jakob Winkler TECHNICIANS IN RESEARCH & DIAGNOSTICS Bettina Berkowitsch Maria Berneder Bettina BrunnerHerglotz Helga Daxberger Gudrun Divoky Ulrike Engel Susanna Fischer Michaela Fortschegger Nelli Frank Brigitte Grimm Angela Halfmann Sabrina Haslinger Christine HoffmannFreimüller Stefanie Hosiner Andrea Inthal Jovana Jovanovic Gunhild Jug Dragana Jugovic Margit König Susanna Koskela Fiona Anna-Maria Kraler Isabella Krickl Erika Marton Astrid Mecklenbräuker Gerda Modarres Karin Mühlbacher Nadine Nirtl Bettina Nocker Susana Pascoal Maya-Marisol Plank Michaela Pregesbauer Sandra Preuner-Stix Dieter Printz Christina Satke Daniela Scharner Angela Schumich Manuela Stadler Julia Stemberger Susanne Suhendra Dijana Trbojevic Eva Winkler Sven Wohlmacher Marion Zeginigg Andrea Ziegler Elke Zipperer RESEARCH SUPPORT OFFICE Nuno-Miguel Andrade Gomes Caterina Barresi Barbara Brunmair Zoltán Dobai Melanie Brunhofer 84 85

44 UNIV.-DOZ. DR. MICHAEL DWORZAK Immunologische Diagnostik

45 FINANZBERICHT

46 Finanzbericht RICHTLINIEN ZUR SPENDENVERWENDUNG D ie St. Anna Kinderkrebsforschung wird zum über wiegenden Teil durch private Spenden finanziert. Für den Betrieb des Forschungs institutes werden jährlich mehr als sieben M illionen Euro benötigt, der Verein verfügt jedoch über keine Basisfinanzierung durch die öffentliche Hand. Zusätzliche Mittel werden im Rahmen von kompetitiv ausgeschriebenen P rojektförderungen von anerkannten nationalen und internationalen Stellen akquiriert. Wir fühlen uns unseren Spenderinnen und Spendern gegenüber zu einer sparsamen und effizienten Verwendung der uns anvertrauten Gelder ver pflichtet. Aus diesem Grund verwenden wir weniger als 1 % für die Verwaltung und das Fundraising, das bedeutet: mehr als 9 % der Spenden fließen direkt in die Forschung. SPENDENGÜTESIEGEL UND STEUERLICHE ABSETZBARKEIT QUALITÄTSSICHERUNG DER WISSENSCHAFTLICHEN ARBEIT Seit dem Jahr 22 trägt die St. Anna Kinderkrebs forschung als eine der ersten Organisationen Öster reichs das Spendengütesiegel der Kammer der Wirtschaftstreuhänder. Für die jährliche Neu verleihung führt ein Wirtschaftsprüfer zusätzlich eine Prüfung der transparenten und ordnungs ge mäßen Verwendung der Mittel gemäß den strengen R icht linien des Spendengütesiegels durch. Das Forschungsinstitut verfügt über ein Scientific Advisory Board ein Gremium aus externen Experten mit der Aufgabe der laufenden Evalu ierung der wissenschaftlichen Arbeiten und B eratung der Institutsleitung. Darüber hinaus werden regelmäßig neue wissenschaftliche Projekte bei renommierten forschungsfördernden nationalen und internatio nalen Stellen eingereicht und Forschungsergebnisse in international anerkannten, wissenschaftlichen Journalen publiziert. In regelmäßigen Abständen fi ndet zusätzlich eine o bjektive Beurteilung der wissenschaftlichen Leistung durch ausgewiesene externe Fachleute auf dem Gebiet statt. Auf Grundlage eines von der F inanzlandes direk tion für Wien erlassenen Bescheides zählt die St. Anna Kinderkrebsforschung zum begünstigten Empfänger kreis, sodass Spenden sowohl von der Lohnsteuer als Sonderausgabe, als auch von der Einkommensteuer als Betriebsausgabe steuerlich absetzbar sind. Der Jahresabschluss wird gemäß den Bestimmungen des Vereinsgesetzes für große Vereine erstellt, wobei die gleichen Richtlinien wie für Kapitalgesellschaften gelten. Die Finanzgebarung und der Jahresabschluss des Vereins werden jährlich von einem beeideten Wirtschaftsprüfer kontrolliert und mit einem uneingeschränkten Bestätigungsvermerk versehen. Damit wird der sach- und zweckgemäße Umgang mit den erhaltenen Spenden sichergestellt und bestätigt. 9 91

47 UNIV.-DOZ. DR. GERHARD FRITSCH Klinische Zellbiologie

48 Finanzbericht MITTELHERKUNFT I. Spenden a) ungewidmete b) gewidmete II. Mitgliedsbeiträge III. Betriebliche Einnahmen a) betriebliche Einnahmen aus öffentlichen Mitteln b) sonstige betriebliche Einnahmen IV. Subventionen und Zuschüsse der öffentlichen Hand V. Sonstige Einnahmen a) Vermögensverwaltung b) sonstige andere Einnahmen sofern nicht in Punkt I bis IV festgehalten VI. Auflösung von Passivposten für noch nicht widmungsgemäß verwendete Spenden bzw. Subventionen MITTELVERWENDUNG , ,51, ,93,, , , , ,14,,, ,44, ,46,,,, VIII. J ahresverlust,, , ,5 TOTAL , ,5 I. Leistungen für die statutarisch festgelegten Zwecke II. Spendenwerbung , ,94 III. Verwaltungsaufwand , , , , , ,5 VI. Zuführung zu Rücklagen,, VII. Jahresüberschuss,, , ,5,, IV. Sonstiger Aufwand sofern nicht unter Punkt I bis III festgehalten V. Zuführung zu Passivposten für noch nicht widmungsgemäß verwendete Spenden bzw. Subventionen TOTAL JAHRESERGEBNIS VII. Auflösung von Rücklagen

49 ANHANG

50 Anhang WISSENSCHAFTLICHER BEIRAT PROF. DR. KL AUS-MICHAEL DEBATIN PROF. MEGAN SYKES, MD Universitätsklinik für Kinder- und Jugendmedizin 8975 Ulm, Germany Columbia University Medical Center New York, NY 132, USA PROF. JAMES R. DOWNING, MD Newcastle Cancer Centre at the Northern Institute for Cancer Research Newcastle University, Paul O Gorman Building, Medical School, Framlington Place Newcastle upon Tyne, NE2 4HH Scientific Director St. Jude Children s Research Hospital Memphis, TN , USA PROF. LEE J. HELMAN, MD PROF. JOSEF VORMOOR, MD Scientific Director for Clinical Research Center for Cancer Research National Cancer Institute, National Institutes of Health Bethesda, MD , USA PROF. STEPHAN L ADISCH, MD Bosworth Chair for Cancer Biology Center for Cancer and Immunology Research Children s National Medical Center Washington, DC 21, USA 98 99

51 UNIV.-PROF. DDR. THOMAS LION Molekulare Mikrobiologie

52 Anhang INTERNATIONAL FREMDGEFÖRDERTE PROJEKTE The interplay of NOTCH and MAPK pathway in LCH Principal investigator: Raphaela Schwentner Grant from the Histiocytosis Association Duration: 1/1/216 to 31/12/216 Optimized diagnostics for improved treatment stratification in invasive fungal diseases (FUNGITECT) Coordinator: Thomas Lion (CCRI/Labdia) European Commission Grant FP7 Cooperation Project N : Duration: 1/2/214 to 31/1/219 Automation of flow cytometric analysis for quality-assured follow-up assessment to guide curative therapy for acute lymphoblastic leukaemia in children (AutoFLOW) Labdia/CCRI partner: Michael Dworzak Coordinator: Martin Kampel (Technical University of Vienna, Austria) European Commission Grant FP7 IndustryAcademia Partnerships and Pathways (Marie Curie Actions) N : Duration: 1/2/214 to 31/1/218 EURO EWING Consortium International clinical trials to improve survival from Ewing sarcoma (EEC) CCRI partner: Heinrich Kovar Coordinator: Jeremy Whelan (University College London, UK) European Commission Grant FP7 Cooperation Project N : Duration: 1/1/213 to 31/9/218 Molecular mechanisms of human fungal pathogen host interaction (ImResFun) CCRI partner: Thomas Lion Coordinator: Karl Kuchler (Medical University of Vienna, Austria) European Commission Grant FP7 Initial Training Network (Marie Curie Actions) N : Duration: 1/1/213 to 3/9/217 Analysing and Striking the Sensitivities of Embryonal Tumours (ASSET) CCRI partner: Heinrich Kovar Coordinator: Walter Koch (University College Dublin, Ireland) European Commission Grant FP7 Cooperation Project N : Duration: 1/11/21 to 3/4/216 Expert paediatric oncology reference network for diagnostics and treatment (ExPO-r-Net) Coordinator: Ruth Ladenstein (CCRI) Grant from the European Consumers, Health, Agriculture and Food Executive Agency (CHAFEA) N : Duration: 1/3/214 to 31/8/217 PanCare childhood and adolescent cancer survivor care and follow-up studies (PanCareSurFup) CCRI/St Anna Spital partners: Eva Frey Coordinator: Lars Hjorth (Lund University, Sweden) European Commission Grant FP7 Cooperation Project N : Duration: 1/2/211 to 31/1/217 Targeted modulation of immune-system responses in cell therapies (MODICELL) Coordinator: Originally Andreas Heitger (5 May 214), presently Wolfgang Holter European Commission Grant FP7 IndustryAcademia Partnerships and Pathways (Marie Curie Actions) N : Duration: 1/1/213 to 31/12/216 International study for treatment of childhood relapsed ALL 21 (IntReALL) CCRI partners: Georg Mann, Andishe Attarbaschi and Ruth Ladenstein Coordinator: Jeremy Whelan (University College London, UK) European Commission Grant FP7 Cooperation Project N : Duration: 1/1/211 to 3/9/

53 Einleitung NATIONAL FREMDGEFÖRDERTE PROJEKTE Overcoming Neuroblastoma Tumour HETerogeneity, Resistance and RecurrAnCe_ (ONTHETRRAC) CCRI responsible principal investigator: Peter Ambros Grant from the Austrian Science Fund (FWF), ERA-Net Transcan N : 2799 B28 Duration: 1/1/216 to 3/6/218 Myeloproliferative neoplasms CCRI partner: Thomas Lion Coordinator: Peter Valent (Medical University of Vienna, Austria) Grant from the Austrian Science Fund (FWF), Special Research Programme (SFB) N : F475-B2 Duration: 1/3/213 to 28/2/217 Ewing sarcoma an enhancer disease? CCRI responsible principal investigator: Eleni Tomazou Grant from the Austrian Science Fund (FWF), Elise Richter Programme N : V 56 B28 Duration: 1/4/216 to 31/1/221 Single molecule array platform for sensitive diagnostics (SmardScout) CCRI partner: Thomas Lion Coordinator: Jan Hesse (Center for Advanced Bioanalysis GmbH, Linz, Austria) Grant from the Austrian Research Promotion Agency (FFG), Research Studios Austria, 4th Call N : Duration: 1/9/214 to 22/11/216 TRANSCALL (Translational research in childhood acute lymphoblastic leukemia) CCRI responsible principal investigator: Renate Panzer-Grümayer Grant from the Austrian Science Fund (FWF), ERA-Net Transcan N : I1226-B19 Duration: 1/7/213 to 3/6/217 PROVABES (prospective validation of biomarkers in Ewing sarcoma for personalized treatment) CCRI responsible principal investigator: Heinrich Kovar Grant from the Austrian Science Fund (FWF), ERA-Net Transcan N : I1225-B19 Duration: 1/4/213 to 31/3/217 Verfahren zur hochautomatisierten Bewertung und Klassifikation von Zellen in Gewebeschnitten anhand räumlicher Markerprofile (TisQuant) LABDIA responsible principal investigator: Peter Ambros Grant from the Austrian Research Promotion Agency (FFG), ERA-SME N : Duration: 1/6/214 to 31/5/217 Directed in vitro differentiation of induced pluripotent stem cells towards the B lymphoid lineage (B-different) CCRI coordinator: Klaus Fortschegger Grant from the Austrian Research Promotion Agency (FFG), Call Bridge (Brückenschlagprogramm) N : Duration: 1/6/214 to 22/12/217 Integrating entertainment and reaction assessment into child cancer therapy (INTERACCT) CCRI/St Anna Spital partner: Anita Lawitschka Coordinator: Helmut Hlavacs (University of Vienna, Austria) Grant from the Austrian Research Promotion Agency (FFG), Call Bridge (Brückenschlagprogramm) N : Duration: 1/5/213 to 3/4/216 Prä-klinische Entwicklung einer Off-the-Shelf individualisierten Krebsimmuntherapie (IN SITU DC-CIT) CCRI partner: Alexander Dohnal Coordinator: Wolfgang Schöfberger (University of Linz, Austria) Grant from the Austrian Research Promotion Agency (FFG), Call Bridge (Brückenschlagprogramm) N : Duration: 1/1/212 to 3/9/216 Virus-specific immunotherapy (VISIT) Labdia/CCRI: coordinator René Geyeregger (Labdia), partner: Matthes-Leodolter (CCRI) Grant from the Wirtschaftsagentur Wien, Call Life Sciences 214 N : Duration: 1/4/215 to 31/3/218 Automated MRD-assessment in AML (flowcluster) Labdia coordinator: Michael Dworzak Grant from the Wirtschaftsagentur Wien, Call Life Sciences 214 N : Duration: 1/3/215 to 28/2/218 Liquid biopsy in neuroblastoma: chance for diagnosis, prognosis and disease monitoring CCRI responsible principal investigator: Peter Ambros Grant from the Austrian National Bank (OeNB), Jubiläumsfonds N : OeNB Duration: 1/1/216 to 31/12/217 Permanent consequences in childhood Langerhans cell histiocytosis CCRI responsible principal investigator: Milen Minkov Grant from the Austrian National Bank (OeNB), Jubiläumsfonds N : Duration: 1/9/215 to 31/8/217 Regulation of the MYCN oncogene by Nuclear Lamina Proteins upon therapy-induced senescence in aggressive neuroblastoma CCRI responsible principal investigator: Sabine Taschner-Mandl Grant from the Herzfelder sche Familienstiftung Duration: 1/5/215 to 31/1/216 Verbesserte Patientenkommunikation in der Onkologie mittels INTERACCT App(OCCURSUS) CCRI responsible principal investigator: Anita Lawitschka Grant from the Österreichische Gesellschaft für Hämatologie und Medizinische Onkologie Duration: 1/5/216 to 31/7/

54 DR. SABINE STREHL Leukämiegenetik

55 Anhang DANKSAGUNG Wir möchten uns an dieser Stelle gerne bei unseren zahlreichen privaten Spenderinnen und Spendern bedanken, die uns seit vielen Jahren treu unter stützen. Des Weiteren bedanken wir uns bei den folgenden nationalen und internationalen Förder gebern und Organisationen: DIPLOM(MASTER)ARBEITEN / DISSERTATIONEN Forschungsrahmenprogramm der Europäischen Kommission (FP7) 3rd Health Programme of the European Commission Bundesministerium für Wissenschaft, Forschung und Wirtschaft (BMWFW) Fonds zur Förderung der wissenschaftlichen Forschung (FWF) Österreichische Forschungsförderungsgesellschaft (FFG) Österreichische Nationalbank (OeNB) Wirtschaftsagentur Wien Histiocytosis Association Herzfelder sche Familienstiftung Gigax Privatstiftung FL Kapsch AG Österreichische Gesellschaft für Hämatologie & Medizinische Onkologie (ÖGHO) Österreichischen Gesellschaft für Kinder- und Jugendheilkunde (ÖGKJ) Dachverband der Österreichischen Kinder-Krebs-Hilfe Medac Gesellschaft für klinische Spezialpräparate mbh, Deutschland Verein für Dermatologie, Wien REZA ABBASI Genomic Analysis of Bone Marrow-Derived Disseminated Neuroblastoma Cells Supervised by Assoc.-Prof. Peter F. Ambros, PhD PhD Thesis STEFANIE ANDERL The role of PAX5 fusion genes in the pathogenesis of childhood B-cell precursor acute lymphoblastic leukemia. Supervised by Sabine Strehl, PhD and Klaus Fortschegger, PhD. PhD Thesis L ARISSA KOLLER Generierung von BCR-ABL1-exprimierenden Zelllinien zur Untersuchung der Ph+ Leukämie mutierten Subklon-Evolution Supervised by Dr. Konstantin Byrgazov, PhD and Univ.-Prof. Thomas Lion, MD, PhD Bachelor Thesis VANESSA MAYR Dynamic in vivo MAP kinase reporters to study development and disease in zebrafish Supervised by Martin Distel, PhD CORNELIA MUTZ Investigating the NAD metabolome in Ewing sarcoma Supervised by Univ.-Prof. Heinrich Kovar, PhD PhD thesis MAGDALENA REITER Function of ETV6/RUNX1 in leukaemia Supervised by Univ.-Prof. Dr. Renate Panzer-Grümayer, MD Master Thesis 18 19

56 UNIV.-DOZ. DR. PETER F. AMBROS Tumorbiologie

57 Anhang PUBLIKATIONEN 216 Araki A, Chocholous M, Gojo J, Dorfer C, Czech T, Heinzl H, Dieckmann K, Ambros IM, Ambros PF, Slavc I, Haberler C. (216). Chromosome 1q gain and tenascin-c expression are candidate markers to define different risk groups in pediatric posterior fossa ependymoma. Acta Neuropathol Commun, 4: 88 Bogen D, Brunner C, Walder D, Ziegler A, Abbasi R, Ladenstein RL, Noguera R, Martinsson T, Amann G, Schilling FH, Ussowicz M, Benesch M, Ambros PF, Ambros IM. (216). The genetic tumor background is an important determinant for heterogeneous MYCN-amplified neuroblastoma. Int J Cancer, 139: Berbegall AP, Villamon E, Piqueras M, Tadeo I, Djos A, Ambros PF, Martinsson T, Ambros IM, Canete A, Castel V, Navarro S, Noguera R. (216). Comparative genetic study of intratumoral hetero genous MYCN amplified neuroblastoma versus aggressive genetic profile neuroblastic tumors. Oncogene, 35: Boztug H, Hirschmugl T, Holter W, Lakatos K, Kager L, Trapin D, Pickl W, Förster-Waldl E, Boztug K. (216). NF-kappaB1 Haploinsufficiency Causing Immunodeficiency and EBV-Driven Lympho proliferation. J Clin Immunol, 36: Bileck A, Mayer RL, Kreutz D, Weiss T, TaschnerMandl S, Meier SM, Slany A, Gerner C. (217, Epub 216 Nov 13). Evaluation of inflammation-related signaling events covering phosphorylation and nuclear translocation of proteins based on mass spectrometry data. J Proteomics, 152: Boer JM, van der Veer A, Rizopoulos D, Fiocco M, Sonneveld E, de Groot-Kruseman HA, Kuiper RP, Hoogerbrugge P, Horstmann M, Zaliova M, Palmi C, Trka J, Fronkova E, Emerenciano M, do Socorro Pombo-de-Oliveira M, Mlynarski W, Szczepanski T, Nebral K, Attarbaschi A, Venn N, Sutton R, Schwab CJ, Enshaei A, Vora A, Stanulla M, Schrappe M, Cazzaniga G, Conter V, Zimmermann M, Moorman AV, Pieters R, den Boer ML. (216). Prognostic value of rare IKZF1 deletion in childhood B-cell precursor acute lymphoblastic leukemia: an international collaborative study. Leukemia, 3: Boztug H, Mühlegger N, Pötschger U, Attarbaschi A, Peters C, Mann G, Dworzak M. (217, Epub 216 Oct 4). Antibiotic prophylaxis with teicoplanin on alternate days reduces rate of viridans sepsis and febrile neutropenia in pediatric patients with acute myeloid leukemia. Ann Hematol, 96: Brunner C, Brunner-Herglotz B, Ziegler A, Frech C, Amann G, Ladenstein R, Ambros IM, Ambros PF. (216). Tumor Touch Imprints as Source for Whole Genome Analysis of Neuroblastoma Tumors. PLoS One, 11:8 Burchill SA, Beiske K, Shimada H, Ambros PF, Seeger R, Tytgat GA, Brock PR, Haber M, Park JR, Berthold F. (217, Epub 216 Dec 16). Recommendations for the standardization of bone marrow disease assessment and reporting in children with neuroblastoma; on behalf of the International Neuroblastoma Response Criteria Bone Marrow Working Group. Cancer, 123: Byrgazov K, Kastner R, Gorna M, Hoermann G, Koenig M, Lucini CB, Ulreich R, Benesch M, Strenger V, Lackner H, Schwinger W, Sovinz P, Haas OA, van den Heuvel-Eibrink M, Niemeyer CM, Hantschel O, Valent P, Superti-Furga G, Urban C, Dworzak MN, Lion T. (217, Epub 216 Oct 7). NDEL1-PDGFRB fusion gene in a myeloid malignancy with eosinophilia associated with resistance to tyrosine kinase inhibitors. Leukemia, 31: Byrgazov K, Lucini CB, Berkowitsch B, Koenig M, Haas OA, Hoermann G, Valent P, Lion T. (216). Transposon-mediated generation of BCR-ABL1- expressing transgenic cell lines for unbiased sensitivity testing of tyrosine kinase inhibitors. Oncotarget, 7: Chagtai T, Zill C, Dainese L, Wegert J, Savola S, Popov S, Mifsud W, Vujanic G, Sebire N, Le Bouc Y, Ambros PF, Kager L, O'Sullivan MJ, Blaise A, Bergeron C, Mengelbier LH, Gisselsson D, Kool M, Tytgat GA, van den Heuvel-Eibrink MM, Graf N, van Tinteren H, Coulomb A, Gessler M, Williams RD, rognostic Pritchard-Jones K. 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59 UNIV.-PROF. DR. OSK AR HA AS Zytogenetik und molekulare Genetik