ST. ANNA KINDERKREBSFORSCHUNG FORSCHUNGSBERICHT 2016 FORSCHUNGSBERICHT 2016 FORSCHUNGSBERICHT

kinderkrebsforschung.at science.ccri.at ST. ANNA KINDERKREBSFORSCHUNG ANNA ST. EINLEITUNG 7 SCIENCE REPORTS 33 CAREER 79 Vorwort des Institutsleiters 8 Introduction 36 Working at CCRI 82 Einleitung des wissenschaftlichen Direktors 10 Non-genetic plasticity and variability If you want to apply for a position 83 as therapeutic and prognostic targets Unseren Spendern sei Dank! 14 Scientific Staff 84 in Ewing sarcoma 38 Improving the routine enumeration of DATEN & FAKTEN 19 ­hematopoietic stem cells: Multi-color analysis of FINANZBERICHT 89 Kompetitive Drittmittel 22 CD34 subtypes reveals unexpected differences Richtlinien zur Spendenverwendung 90 Zuweisung der Geldmittel 22 between various stem cell sources 44 Mittelherkunft 94 Finanzierung 22 Characterization of a novel fusion gene in ­ Mittelverwendung 95 juvenile myelomonocytic leukemia associated Personelle Zusammensetzung 23 with resistance to tyrosine kinase inhibitors 52 Nationen 23 High resolution genomic and transcriptomic ANHANG 97 Forschungsnetzwerke 24 ­profiling of pediatric B-cell precursor acute Wissenschaftlicher Beirat 98 ­lymphoblastic leukemia: implications for Klinische Forschung 26 International fremdgeförderte Projekte 102 ­emergence of resistance and relapse 56 Anstieg der 2-Jahres-Überlebensraten 27 National fremdgeförderte Projekte 104 New insights in neuroblastoma b­ iology 5-Jahres-Überleben von ­ – from spontaneous maturation to the ­ Danksagung 108 krebskranken Kindern 28 relapse seeding clone 64 Diplom(Master)arbeiten /Dissertationen 2016 109 Chimeric antigen receptor (CAR)-based Publikationen 2016 112 immunotherapy for treatment of reactivation Impressum 124 of cytomegalovirus infection after stem cell transplantation 68 Large scale European trial demonstrates survival advantage for high risk neuroblastoma patients receiving high dose busulphan and melphalan treatment 72 UNIV.-PROF. DR. WOLFGANG HOLTER Entwicklung zellulärer Therapie EINLEITUNG Einleitung

VORWORT DES INSTITUTSLEITERS

ie Erfolge meiner Kolleginnen und Eine weitere, vielversprechende Entdeckung der Kollegen machen es mir leicht, diesen St. Anna Kinderkrebsforschung auf dem Gebiet der Jahresbericht einzuleiten. Doch Immuntherapie gab grünes Licht zur Erforschung trotz aller persönlichen Genugtuung einer neuen Methode für die Behandlung lebens­ über den wissenschaftlichen Fort- bedrohlicher Virusinfektionen nach einer Stamm­ schritt, trotz ermutigender Aner­ zellentherapie, um nur einige Highlights aus der kennung unserer Arbeit auf internationaler Bühne, Forschung zu nennen. Dist es doch immer der Zweck unserer Forschung, der uns antreibt. Es geht um das Überleben und Zu guter Letzt sollte nicht unerwähnt b­ leiben, dass Leben krebskranker Kinder und Jugendlicher wir im wissenschaftlichen Wett­bewerb ebenfalls und die Umsetzung unserer Erkenntnisse in die gepunktet haben. Ein Elise-Richter-Forschungs­ klinische Diagnostik und Therapie. Nichts – keine stipendium, das hochkarätige Wissenschaftlerinnen Auszeichnungen oder wissenschaftlichen Ehren – auszeichnet, die eine Universitäts­laufbahn an­stre- freut uns mehr als ein Beitrag zur Rettung der ben, ging 2016 an eine Forscherin der St. Anna Betroffenen und zur Linderung von Leid und Kinderkrebsforschung. Schmerz bei deren Angehörigen. Ich möchte mich herzlich bei den vielen Unter­ Dass die Heilungsraten bei manchen Kinderkrebs- stützerinnen und Unterstützern, den Mitgliedern arten in lichte Höhen getrieben werden konnten, des Ehrenkomitees, den Vorstandsmitgliedern, sodass wir nun die 100 % anpeilen können, hat die dem wissenschaftlichen Beirat und unseren vielen St. Anna Kinderkrebsforschung zu einem g­ e­fragten Förderern bedanken, die uns schon viele Jahre Partner für staatenübergreifende Zusammen­arbeit treu begleiten. gemacht. So wurden wir für die Leitung eines drei- jährigen Pilot-Referenzprojektes ausgewählt, das Ich hoffe, dass dieser Leistungsbericht zur weiteren die Aufgabe hat, die unter­schiedlichen Überlebens- und so dringend notwendigen Spendenbereitschaft raten von Kindern mit Krebserkrankungen aufgrund motiviert, für die ich mich im Namen aller Mitarbei- ungleicher L­ eistungsfähigkeit der Gesundheitssys- terinnen und Mitarbeiter herzlich bedanken möchte. teme­ an­zugleichen und zu verbessern.

Neue, revolutionäre Möglichkeiten für die Diagnose Univ.-Prof. Dr. Wolfgang Holter des Neuroblastoms, einer besonders aggressiven Institutsleiter Krebsart bei Kindern, hat unser Institut zur Leitung eines EU-weiten Forschungskonsortiums e­ mpfohlen. Die „Flüssige Biopsie“ wird in weiterführende klini- sche Studien eingebunden, um diese exakte mole- kulare Bestimmung des individuellen Tumor­genoms voranzutreiben.

8 9 Einleitung

EINLEITUNG DES WISSENSCHAFTLICHEN DIREKTORS

it dem rasanten technologischen mäßig wenigen genetischen Veränderungen So erhalten wir Auskunft über die genetische und Fortschritt in der biomedizini- einhergeht. Denn pädiatrische Krebserkrankungen nicht-genetische Variation von Tumoren und deren schen Forschung eröffnen müssen als Defekt in der normalen Entwicklungs- Komponenten im Wechselspiel der verschiedenen sich ungeahnte Dimensionen biologie unterschiedlicher Zelltypen gesehen Zelltypen und ihres Stoffwechsels. Diese Entwick- des Forschungs­universums werden. Um hier den Mechanismen als potentielle lungen spiegeln sich auch in den Forschungsaktivi- und ergeben sich immer neue, Therapie ­ansatzpunkte auf den Leib zu rücken, täten der St. Anna Kinderkrebsforschung wider. überraschende Erkenntnisse. Im Bereich der müssen wir die Regulationsmechanismen der Wie in diesem Jahresbericht dargestellt, gelang es MKrebsforschung ist dies zweifellos die Einsicht, Entwicklung verstehen. Die zunehmende Anzahl uns auch 2016 beachtliche Erfolge auf verschiedenen dass Tumore keine homogene Zellmasse darstellen, an Weiter­entwicklungen neuer Technologien, wie Gebieten der pädiatrisch-onkologischen Forschung sondern sich aus einer Vielzahl verschiedener etwa des Next Generation Sequencing (NGS), zu erzielen, welche uns unserem Ziel einer für Zelltypen zusammensetzen, die, jeder für sich, ein ermöglicht Einsichten in die normale und die jeden Patienten maßgeschneiderten Therapie weiter hohes Maß an Heterogenität und Plastizität auf­- krankhaft veränderte dreidimensionale Struktur des näherbringen. Dies sei an einigen ausgewählten weisen. Diese Erkenntnis trifft besonders auf Chromatins, der Steuerzentrale der Genexpression, Beispielen aus den im Vorjahr veröffentlichten Krebserkrankungen bei Kindern zu. Galt noch vor welches das Verhalten jeder Zelle bestimmt. Die Forschungsergebnissen demonstriert. wenigen Jahren das Dogma, dass Krebs aus­- hohe Sensitivität dieser Technologie erlaubt uns schließlich eine Erkrankung der Gene ist, welche nicht nur genetische Zusammensetzung und sich in irreversiblen, starren Mutationsmustern Genexpression auf Einzelzellebene zu analysieren, Univ.-Prof. Dr. Heinrich Kovar ­manifestiert, so zeigt sich heute mehr und mehr, sondern auch Tumorzellzerfallsprodukte in der Wissenschaftlicher Direktor dass Krebs bei Kindern in der Regel mit verhältnis- Blutzirkulation nachzuweisen und zu bestimmen.

10 11 DR. MARTIN DISTEL Innovative Krebsmodelle Einleitung

UNSEREN SPENDERN SEI DANK!

Unsere Spenderfamilie ist groß und großartig! BUNTE EINFÄLLE – Für einen wunderschönen Nachmittag mit vielen Sogar das derzeit wahrscheinlich hippste Duo der Wir sind dankbar für die langjährige, treue Unter- SPANNENDE AKTIONEN – lachenden Gesichtern sorgte der Verein Öster­ österreichischen Musik- und Unterhaltungsbranche stützung und wir freuen uns jedes Mal, ein neues GROSSZÜGIGE SPENDEN reichische Journalistenwerkstätte. Dieter Wally schaute schon vor einem Tourstart im Spenden- Familienmitglied herzlich willkommen zu heißen! Das Spektrum der Spendenmöglichkeiten ist viel­ gelang es gemeinsam mit seinen engagierten büro der St. Anna Kinderkrebsforschung vorbei. Das Faszinierende an der Arbeit im Spendenbüro fältig und die Unterstützung damit grenzenlos – Kolleginnen und Kollegen, die Österreichpremiere Die Kunst-Kampagne „Horvathslos für eine gute der St. Anna Kinderkrebsforschung sind die wie einige Beispiele aus 2016 zeigen: des Kinderfilms „Rettet Raffi“ zu einem speziel- Sache“ war die schöne Idee von „Mama Speer“. Menschen, ihre Hilfsbereitschaft, ihre wunder­baren Mit einer exklusiven Benefizgala im Goldenen Saal len ­Benefizerlebnis zu machen. Die Abenteuer des Der so ­gesammelte Betrag wurde von „Seiler und Ideen und ihr großartiges Spendenengagement. des Wiener Musikvereins feierte Aki ­Nuredini, mutigen Hamsters und seines jungen Besitzers Speer“ verdoppelt und eine tolle Summe persönlich Dank der konsequenten Forschung können heute prominenter und beliebter Patron des Wiener konnten auch Patienten aus dem St. Anna Kinder- übergeben. bereits vier von fünf Kindern und Jugendlichen, Nobel-Restaurants „Sole“, seinen 60. Geburtstag. spital und deren Eltern genießen. Das Forscherteam die vor 40 Jahren noch als unheilbar galten, Ein Teil des Reinerlöses des hochkarätig besetzten freute sich, dank großzügiger Sponsoren, über einen Jedes Jahr gestalten die Schülerinnen und S­ chüler gerettet werden. Finanziert wird die St. Anna Konzerts kam der St. Anna Kinderkrebsforschung hervorragenden Spendenbetrag. der Schreibgruppe „Kreatives Schreiben“ am Kinderkrebsforschung, die seit 2002 das Österreichi- zugute. Die Besucher erlebten eine Musikgala der Gymnasium Schärding, gemeinsam mit der ­rührigen sche Spendengütesiegel führt und zum steuerlich Extraklasse mit wunderbaren Interpreten, wie Die Unterstützung der Bastelrunde Hirtenberg, Initiatorin, Frau Prof. Mag. Thallinger, einen begünstigten Empfängerkreis gehört, von Anfang Ildikó Raimondi, Clemens Unterreiner, Ramón unter der Leitung von Marianne Brandtner, hat ­Lesezeichenkalender. Mit dem Reinerlös unter­ an hauptsächlich durch Spenden. Dank gebührt Vargas uvm. Sogar Starpianist Rudolf Buchbinder bereits eine langjährige Tradition. Das ganze stützen die jungen Fabulanten auch 2016 das daher unseren Spenderinnen und Spendern – denn spielte ein Geburtstagsständchen. Jahr über wird dort für die St. Anna Kinderkrebs­ Wiener Forschungsinstitut. sie alle schenken krebskranken Kindern eine Chance forschung gebastelt, gemalt, gestrickt, genäht usw. auf eine gesunde Zukunft. Das ganze Jahr über sammelten die Mitglieder Die Erlöse des jährlichen Oster- und Adventmarkts der Kinderkrebshilfe Haid / Ansfelden durch den sind jedes Mal beeindruckend. LANGE NACHT DER Verkauf von Flohmarktware Spenden. Der Einsatz (KINDERKREBS)FORSCHUNG war enorm und die Einnahmen rekordverdächtig. Auch das spannende Wettpaddeln in Scharndorf Forschung auf reizvolle Weise entdecken und Bereits zum 11. Mal wurden nicht nur Familien mit hat bereits Tradition! Viel Spaß für Jung und Alt Zukunft erleben! Dazu lädt die St. Anna Kinder- kleinen Krebspatienten unterstützt, sondern auch ist beim Benefiz-Sautrogrennen mit Kultcharakter krebsforschung alljährlich ein. Im Rahmen der der St. Anna Kinderkrebsforschung wieder eine garantiert. Der gesamte Erlös wird von Jahr zu „Langen Nacht der Kinderkrebsforschung“ wird namhafte Spende überreicht. Jahr unglaublich gesteigert und wurde auch 2016 ein spielerisches Kennenlernen des Laborall­ gespendet. tages möglich und „Wissenschaft zum A­ nfassen“ Die Aschermittwochfeier der Künstlerinnen und greifbar. Interaktiv, spannend und leicht ver­ Künstler in der Wiener Hofburgkapelle gilt als ständlich wird Wissen über die Bausteine des besondere Einstimmung auf die Fastenzeit. Die Lebens und die ­molekularen Grundlagen der Wiener Hofmusikkapelle und alle Mitwirkenden ­Krebsentstehung vermittelt. Vorführungen agierten im Dienst der guten Sache und unter­ in den Labors und spannende wissenschaftliche stützten auch im Jahr 2016 mit den Einnahmen Vorträge ver­deutlichen, was unsere Forscherinnen unsere Forschungs­arbeit. und Forscher zur Verbesserung der Behandlungs­ qualität und E­ r­höhung der Heilungschancen an Krebs erkrankter Kinder und Jugendlicher leisten.

Mag. Andrea Prantl Leiterin Spendenbüro

14 15 Einleitung

2016 öffneten wir bereits zum 12. Mal unsere Dr. Charlotte Rothensteiner, Mag. Maria Polsterer- Jede Spende hilft! WOLLEN SIE INFORMATIONEN, Pforten, aber erstmalig im Rahmen von Österreichs Kattus, Erste Bank Vorstand Willibald Cernko, Ob mit einer persönlichen Spende oder mit einer UNTERLAGEN ODER HABEN SIE FRAGEN? größtem Forschungsfest: der „Langen Nacht der KR Karl Javurek, Isabella Kapsch, General­direktorin gemeinsamen Sammelaktion, ob per Zahlschein, Das Spendenteam und ich freuen uns auf Ihre Forschung“. Rund 1000 Besucherinnen und Besucher Dr. Elisabeth Gürtler, Parlamentsabgeordnete Kreditkarte oder Onlinespende – jede finanzielle Kontaktaufnahme: interessierten sich für unsere Arbeit, staunten über KR Brigitte Jank, Prof. Erwin Ortner, Bäcker­meister Zuwendung ermöglicht die Fortsetzung ­unserer unsere Erfolge und nützten die Gelegenheit, sich zu Senator Kurt Mann, sowie BM Dr. Michael Häupl, Forschungsarbeit im Kampf gegen ­Kinderkrebs. +43 (0)1 40 470 - 4000 informieren und einen Blick hinter die „Forschungs- Meinungsforscher Prof. Rudolf Bretschneider, Firmen spenden immer häufiger den für Kunden- [email protected] kulissen“ zu werfen. Interspot-Chefin Inge Klingohr, Psychologin Weihnachtsgeschenke vorgesehenen Betrag. Statt www.kinderkrebsforschung.at Mag. Ulla Konrad, Maestro Franz Welser-Möst um Geschenke wird bei Geburtstagen, Jubiläen und Kapsch-Vorstand Ing. Mag. Thomas Schöpf oder anderen Feiern gerne um Spenden gebeten. Bank Austria ST. ANNA KINDERKREBSFORSCHUNG- sind Teil des Unterstützungskomitees und freuen Auch der Verzicht auf Blumen und Kränze bei IBAN: AT79 1200 0006 5616 6600 UNTERSTÜTZUNGSKOMITEE sich, die St. Anna Kinderkrebsforschung als Begräbnissen, um stattdessen zu spenden, hilft BIC: BKAUATWW Schon seit einiger Zeit begleitet ein prominent ­Mentorinnen und Mentoren zu begleiten und krebskranken Kindern. Ein Testament oder ein besetztes Ehrenkomitee die St. Anna Krebs- ­tat­kräftig zu unterstützen. Legat zugunsten der St. Anna Kinderkrebsforschung Erste Bank forschung. Die Mentoren, Personen aus Politik, schenkt ebenfalls langfristig eine gesunde Zukunft. IBAN: AT66 2011 1000 0318 3777 Wirtschaft und Kultur, zeichnen sich durch eine Unsere Kuscheltiere: BIC: GIBAATWW besondere Position aus und sind ehrenamtlich tätig. Kleine Lebensretter, die Freude schenken Vielfältige Spendenmöglichkeiten: Durch ihre Präsenz in der Öffentlichkeit können sie Bereits seit 24 Jahren sind die Kuscheltiere der Onlinespende, Barspende, Spende mit Zahlschein, viel zugunsten der St. Anna Kinderkrebsforschung St. Anna Kinderkrebsforschung bei Jung und Spende mit Einziehungs- oder Dauerauftrag, Benefiz­ bewegen. Seit einigen Jahren unterstützt Eva Alt sehr beliebt und als Sammelobjekte auch veranstaltungen, Sammelaktionen, Spenden statt Angyan, Gattin des Intendanten der Gesellschaft heiß begehrt! Jedes Jahr im Oktober begrüßt die Geburtstags- oder Weihnachtsgeschenken, Erwerb der Musikfreunde in Wien, Dr. Thomas Angyan, Maskottchen­familie einen Neuzugang. 2016 war unserer Kuscheltiere, Kranzablösespenden, Legat / als Komiteepräsidentin das Forschungsinstitut. es „Gigi, der gutgelaunte Glückskäfer“. Die kleinen Testament etc. Die Unterstützung der Kinderkrebsforschung ist Lebensretter freuen sich auf ein nettes Zuhause ihr ein Herzensanliegen und deshalb bittet Frau und jede Menge Spielgefährten. Für 12,00 € schenkt Das Team der St. Anna Kinderkrebsforschung ist Angyan regelmäßig eine ausgesuchte Gästeschar zu man nicht nur krebskranken Kindern eine Chance, dankbar für die langjährige spendenfreudige Unter- einem Treffen, um gemeinsame Projekte zugunsten sondern auch sich selber oder seinen Lieben Freude. stützung und ich persönlich für die Begegnung der St. Anna Kinderkrebsforschung zu planen. Bank Austria, Erste Bank und einige Sparkassen mit so vielen großzügigen, warmherzigen und hilfs­ unterstützen die St. Anna Kinderkrebsforschung bereiten Menschen. Herzlichen Dank! ebenso beim Vertrieb, wie die Raiffeisen Zentral- bank. Sogar an der Rezeption des renommierten Wiener Innenstadt-Hotels Imperial ist das jeweils Mag. Andrea Prantl Leiterin Spendenbüro aktuelle Maskottchen erhältlich. Welche Stofftiere angeboten und wie sie zu bestellen sind, steht auf unserer Internetseite.

16 17 DATEN & FAKTEN UNIV.-PROF. DR. RENATE PANZER-GRÜMAYER Leukämiebiologie Daten & Fakten

QUELLE DER USA ISLAND FINNLAND KOMPETITIVEN RUSSLAND DRITTMITTEL im Jahr 2016

9,42 % Europäische Union POLEN 26,12 % Sonstige Drittmittelgeber DEUTSCHLAND

15,64 % Fonds zur Förderung der SLOWAKEI wissenschaftlichen Forschung (FWF)

ÖSTERREICH UNGARN 43,41 % Österreichische 5,42 % Österreichische NATIONEN Nationalbank Forschungsförderungs- Mitarbeiterinnen gesellschaft (FFG) FRANKREICH und Mitarbeiter der St. Anna Kinderkrebsforschung IRAN im Jahr 2016 SPANIEN

ZUWEISUNG DER FINANZIERUNG TÜRKEI GELDMITTEL im Jahr 2016 KROATIEN PORTUGAL im Jahr 2016 GRIECHENLAND ITALIEN BOSNIEN- HERZEGOWINA SERBIEN & MONTENEGRO PERSONELLE ZUSAMMENSETZUNG der Mitarbeiterinnen und Mitarbeiter im Jahr 2016

67 % Frauen 90,51 % Forschung 79,66 % Spenden und Verlassenschaften 5,92 % Spendenwerbung 20,34 % Kompetitive Drittmittel 33 % Männer 3,06 % Verwaltungsaufwand 0,52 % Sonstiger Aufwand

22 23 Daten & Fakten

FORSCHUNGSNETZWERKE ALBANIEN ARGENTINIEN National und international AUSTRALIEN BELGIEN BRASILIEN BULGARIEN CHILE CHINA DÄNEMARK DEUTSCHLAND ESTLAND FINNLAND FRANKREICH GRIECHENLAND GROSSBRITANNIEN HONG KONG IRLAND ISLAND ISRAEL ITALIEN JAPAN KANADA KROATIEN LETTLAND LITAUEN LUXEMBURG MALAYSIA NEUSEELAND NIEDERLANDE NORWEGEN ÖSTERREICH POLEN PORTUGAL RUMÄNIEN RUSSLAND SCHWEDEN SCHWEIZ SERBIEN SINGAPUR SLOWAKEI SLOWENIEN SPANIEN SÜDKOREA TSCHECHIEN TÜRKEI UKRAINE UNGARN URUGUAY USA WEISSRUSSLAND

24 25 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 S2IRP ANSTIEG DER 2-JAHRES-ÜBERLEBENSRATEN ALS KOORDINIERUNGSZENTRUM FÜR KLINISCHE STUDIEN VON KREBSKRANKEN KINDERN UND JUGENDLICHEN Morbus Hodgkin 14.000 13.874 100 % GesamtpatientInnenanzahl Maligne Keimzelltumoren Wilms-Tumor 12.000 80 % Akute lymphoblastische Leukämie 10.000 60 % Non-Hodgkin-Lymphom Neuroblastom und 8.532 Ganglioneuroblastom 8.000 Internationale PatientInnen 40 % Osteosarkom Rhabdomyosarkom 6.000 20 % Hirntumoren 5.342 Ewing-Sarkom PatientInnen in Österreich 4.000 0 Akute myeloische Leukämie 1960 1970 1950 1990 1980 2000 Quelle: GPOH 2.000 Kinderkrebsregister Mainz

0 014 2011 2 1994 1996 2012 1991 1981 2015 2016 2010 2013 1995 1987 1997 1992 1982 1990 1989 1998 1999 1985 1986 1993 1988 1984 1983 2001 2007 2005 2002 2006 2009 2008 2000 2004 2003

26 27 Daten & Fakten

5-JAHRES-ÜBERLEBEN VON ­KREBSKRANKEN KINDERN

5-JAHRES-ÜBERLEBEN VON KREBSKRANKEN KINDERN NORTHERN EUROPE IN EUROPA ZWISCHEN 2000 UND 2007 Denmark Auch in einer kürzlich erschienen Publikation zum 5-Jahres-Überleben von Kindern mit Leukämie Finland (Bonaventure et al. Lancet Oncology 2017, 4, e202-e216) liegt Österreich gemeinsam mit D­ eutschland Iceland über dem europäischen Durchschnitt. Dieses erfreuliche Ergebnis kann auf die stringente Zusammen­arbeit im Studienumfeld zurückgeführt werden. 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* 0 10 20 30 40 50 60 70 80 90 100 5-year survival (%) Quelle: Gatta et al. Lancet Oncology 2014, 15, 35-47

28 29 DR. ALEXANDER DOHNAL Tumorimmunologie SCIENCE REPORTS UNIV.-PROF. DR. HEINRICH KOVAR Molekularbiologie Science Reports

INTRODUCTION

apid technological progress in biomed- alterations. As a result, pediatric cancers are to be circulation. This way, we are able to obtain infor- ical research explores unimagined considered a consequence of a perturbed develop- mation on the genetic and non-genetic v­ ariation of dimensions of the scientific universe ment of different cell types. In order to exploit these tumors and their components in their interplay with and constantly produces new, unex- aberrant mechanisms for therapeutic purposes, we other cell types and metabolites. These develop­ pected findings. In the field of cancer need to understand the regulatory mechanisms of ments are also reflected in the activities of the research thishas led to the insight normal and malignant development that lie behind ­Children´s Cancer Research Institute. As docu- that tumors are not simply a homogenous mass these aberrations. The increasing number of refined mented by this annual report, we continued being Rof cells, but comprise a multitude of different cell applications for novel technologies such as next successful in several areas of pediatric oncology types, each of which demonstrating a high degree generation sequencing (NGS) enables insights into research in 2016. These successes bring us a step of heterogeneity and plasticity. This finding espe- the three-dimensional organization and aberrations closer to our goal of improved therapy tailored to the cially applies to pediatric cancers. Until recently, of chromatin, the control center of gene expression, specific needs of individual patients. In support of the dogma has been that cancer is a purely genetic which determine the fate of each cell. The high this notion, this report includes several examples of disease, which manifests itself in irreversible, rigid sensitivity and specificity of this technology allows research results published by the Children´s Cancer mutation patterns. Now, however, it is becoming us not only to address the genetic composition and Research Institute in the past year. increasingly clear that childhood cancer is in fact gene expression on the single cell level, but also to associated with a relatively low number of genetic analyse tumor cell break-down products in the blood Prof. Heinrich Kovar, PhD

36 37 Science Reports

Figure 1 Figure 2 NON-GENETIC PLASTICITY AND VARIABILITY LOLA Core database Levels of heterogeneity ENCODE AS THERAPEUTIC AND PROGNOSTIC TARGETS 100% DNase CODEX

IN EWING SARCOMA DNA UCSC

methylation 0% EwS-hypomethylated regions Our group has been studying the molecular under- Tumor cell plasticity is the basis for metastasis, the LOLA Region set enrichment pinnings of Ewing sarcoma for many years. We try major threat to patients. Tumor progression is asso- to understand how a single genetic aberration, the ciated with different types of cellular stress imposed Inter-cancer EwS-specific EWS-FLI1 gene fusion, can drive the pathogenesis on the tumor cell by the microenvironment. We and of this disease. Since recent genome sequencing others have previously demonstrated upregulation of EwS samples studies have confirmed an extremely low number gene products involved in handling of cellular stress, of mutations and the absence of recurrent aberra- among them the metabolic sensor Sirtuin 1 (SIRT1) tions other than the EWS-ETS gene rearrangement and the DNA repair enzyme poly(ADP-ribose) poly- and facultative whole chromosome copy number merase 1 (PARP1). We hypothesize that interfering changes, we hypothesize that, if activated in the with the activity of these proteins may reduce the right developmental, epigenomic and genomic metastatic potential of Ewing sarcoma and poten- context, EWS-FLI1 is sufficient to induce and main- tially sensitize resistant tumor cells to a variety of Inter-individual tain malignancy. We believe that a deep mecha- chemotherapeutic drugs. As PARP1 and SIRT1 are nistic understanding of the interaction between responsive to changes in tumor and microenviron- Region sets developmental cellular background, microenviron- mental metabolic states, we interrogated the role ment, tumor epigenome and EWS-ETS activity on a of the Ewing sarcoma metabolome as a potential Other tissues Prostate systems level will allow us to better understand the vulnerability of the disease. biological and clinical variability of the disease, and Intra-tumor ultimately lead to the identification of vulnerabilities with therapeutic potential. EPIGENETIC DIVERSITY Figure 1. DNA methylation IN EWING SARCOMA profiling by RRBS i­dentifies epigenetic diversity on Despite the low number of mutations, Ewing In a collaborative study between the CCRI, the CpGs different­ levels sarcoma shows significant biological, clinical, CeMM Research Center for Molecular Medicine of Odds ratio and immunophenotypic variation and plasticity, the Austrian Academy of Sciences in Vienna, and Figure 2. RRBS profiling identifies which have moved into our focus in the reporting the Institut Curie in Paris, led by CCRI scientist a unique Ewing sarcoma specific period. To understand inter-tumor, inter-patient Eleni Tomazou with contributions from several DNA methylation pattern and intra-tumor variability in absence of significant hospitals in Austria, Germany, France and Spain, genetic variation, we interrogated the epigenomes we assessed genome-wide epigenomic patterns of of more than 140 Ewing sarcomas in comparison to more than 140 Ewing sarcomas. Using novel bio­ each other and to those of other tumors and normal informatic methods developed by Nathan Sheffield tissues. As described below, this paradigmatic study at CeMM, our team studied the tumors’ DNA meth- revealed two dimensions of heterogeneity, providing ylation patterns – one of the most important facets Bisulfite Sequencing), and interrogated for open and to publicly accessible data banks for similarly unprecedented biological insights into the etiology of the human epigenome. We applied a next-gen- chromatin regions by ATAC-seq (Assay for Trans- obtained DNA methylation profiles of human cancers of the disease. eration-sequencing (NGS) based high-throughput posase-Accessible Chromatin with high throughput and normal tissues, we identified a diagnostic method, RRBS (Reduced Representation Bisulfite sequencing), and for histone modifications indic- Ewing sarcoma specific DNA-methylation profile Sequencing) to all samples, thus analyzing DNA ative of transcription-regulatory activity by ChIP- distinct from any other tumor and normal tissue, methylation patterns of CpG-rich genome regions seq (Chromatin Immunoprecipitation sequencing). and predictive of activity of Ewing sarcoma specific at single-nucleotide resolution. In addition, a subset Comparing our data to the Ewing sarcoma reference transcriptional enhancers (Figure 2). In addition, we of samples was analysed by WGBS (Whole Genome epigenome that we had established previously observed unexpected inter-individual diversity.

38 39 Science Reports

Figure 3 EwS specific chromatin accessible elements Figure 4 A A 150 A673sh A673 TC32 EWS-FLI1-high 100 100 Other cells Activity score Ewing-like 100 EWS-FLI1-low IC50 2.65 -0.5 0.0 0.5 IC50 1.06 50 50 50 Viability(%)

IC50 0.46 IC50 0.15 0 0 0 pluripotent stem cell 0.1 1 10 100 0.01 0.1 1 10 0.01 0.1 1 10 nmol/L nmol/L nmol/L EwS cell line STA-ET-2.2 SK-N-MC TC32 100 100 100 EwS tumor

MSC (BM) 50 50 50 muscle Viability(%) IC50 0.25 IC50 0.58 IC50 0.22 0 0 0 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 nmol/L nmol/L nmol/L STA-ET-11 RM-82 EwS-FLI1 anti-correlated enhancers 100 100

50 50 Mesenchymal Stem-like Activity score Viability(%) IC50 0.60 IC50 0.77 0.4

0.8 0 0 0.0 1.2 0.01 0.1 1 10 0.01 0.1 1 10 nmol/L nmol/L

B pluripotent stem cell B 150 PC-3 150 HEK293 U2OS EwS cell line 100 100 100 EwS tumor Figure 3. Two dimensions MSC (BM) 50 50 50 of inter-individual epigenetic Viability(%) heterogeneity in Ewing muscle IC50 4.34 IC50 1.50 IC50 4.43 sarcoma 0 0 0 0.1 1 10 100 0.1 1 10 100 0.1 1 10 100 nmol/L nmol/L nmol/L 150 150 HeLa CLB-MA MSC 100 100 100

50 50 50

However, no distinct subgroups of tumors could in regulatory regions of EWS-FLI1 anti-correlated Viability(%) be defined that might have been used for biolog- genes (Figure 3). Additionally, RRBS identified IC50 1.62 IC50 0.87 IC 7.24 0 0 0 50 ical or clinical stratification. Rather, inter-tumor significant intra-tumor heterogeneity, which was 0.1 1 10 100 0.1 1 10 100 0.01 0.1 1 10 nmol/L nmol/L nmol/L hetero ­geneity defined a disease spectrum along higher in metastatic tumors than in localized Ewing two dimensions: The degree of similarity to the sarcoma. In summary, for the first time, our study Figure 4. Ewing sarcoma diagnostic Ewing sarcoma signature defined by published in Nature Medicine identified different cell lines (A) are exquisitely methylation heterogeneity at Ewing sarcoma levels of epigenetic heterogeneity in a childhood sensitive to NAMPT inhibition by FK866 as compared to specific open c­ hromatin sites, and the degree of cancer that may explain the diverse clinical courses non-Ewing sarcoma cell lines stemness defined by methylation heterogeneity observed in patients. As DNA methy­lation influences (B)

40 41 Science Reports

gene activity, the combination of Ewing sarcoma THE NAD METABOLOME AS THERAPEUTIC We found that in the Ewing sarcoma model cell line Mutz CN, Schwentner R, Kauer MO, Katschnig AM, Kromp F, specific and cell-of-origin specific patterns can lead TARGET IN EWING SARCOMA A673 by activation of Tdo2 (tryptophan 2,3-dioxy- Aryee DN, Erhardt S, Goiny M, Alonso J, Fuchs D, Kovar H. (2016). EWS-FLI1 impairs aryl hydrocarbon receptor activation by blocking to different outcomes. The epigenetic diversity also Several studies have previously indicated Ewing genase) knockdown of EWS-FLI1 led to tryptophan tryptophan breakdown via the kynurenine pathway. FEBS Lett, 590: appears to correlate with the tumors’ aggressive- sarcoma specific overexpression of the DNA repair break-down and synthesis of the primary metab- 2063-2075. ness and metastatic state. These new insights into enzyme PARP1 and preclinical evidence for exqui- olites kynurenine and kynurenic acid, which in an the biology of Ewing sarcoma provide the basis for site sensitivity of the disease to PARP1 inhibi- autocrine manner activated the aryl hydrocarbon developing epigenetic biomarkers that can reliably tors. However, clinical trials have, so far, failed to receptor and its nuclear target genes. However, no predict disease course and therapy response. Our confirm therapeutic efficacy of PARP1 inhibitors further metabolization to NAD was observed and findings in Ewing sarcoma also provide an inter- in monotherapy, and the question, which drugs to activation of TDO2 in response to low EWS-FLI1 esting concept for other cancers with low genetic use in combination therapy sensitizing to PARP levels was restricted to a subset of Ewing sarcoma complexity. inhibition remains to be answered. PARP1 is a cell lines only. Instead, we found the enzyme protein-modifying enzyme that requires NAD (Nico- NAMPT (Nicotinamide Phosphoribosyltransferase) Sheffield NC, Pierron G, Klughammer J, Datlinger P, tinamide Adenine Dinucleotide) as co-substrate for to be rate-limiting for NAD synthesis in Ewing Schönegger A, Schuster M, Hadler J, Surdez D, Guillemot D, protein-ADP-ribosylation. NAD is a key metabolite sarcoma. We therefore investigated the conse- ­Lapouble E, Freneaux P, Champigneulle J, Bouvier R, Walder D, of energy metabolism involved in cellular redox quences of NAMPT inhibition by the small molecule Ambros IM, Hutter C, Sorz E, Amaral AT, de Álava E, Schallmoser K, Strunk D, Rinner B, Liegl-Atzwanger B, reactions, DNA repair, and in the maintenance of inhibitor FK866 on Ewing sarcoma growth. We Huppertz B, Leithner A, de Pinieux G, Terrier P, Laurence V, genomic stability. We have previously demonstrated observed that blocking NAMPT leads to exhaustive Michon J, Ladenstein R, Holter W, Windhager R, Dirksen U, overexpression of the metabolic sensor SIRT1 associ- NAD depletion in Ewing sarcoma cells, followed by a Ambros PF, Delattre O, Kovar H, Bock C, Tomazou EM. (2017). DNA methylation heterogeneity defines a disease spectrum ated particularly with Ewing sarcoma metastases. metabolic collapse and cell death. Using conditional in Ewing sarcoma. Nat Med; 23:386-395. SIRT1 is also an enzyme competing for the co-sub- EWS-FLI1 knockdown by doxycycline-inducible strate NAD to deacetylate acetylated proteins. shRNA revealed that EWS-FLI1 depletion signifi- In fact, PARP1 and SIRT1 are the major cellular cantly reduces the sensitivity of Ewing sarcoma cells consumers of NAD, and their high activity in Ewing to NAMPT inhibition. Consistent with this finding, sarcoma require continuous fuelling by the co-sub- a comparison of 7 Ewing sarcoma cell lines of differ- strate. We therefore interrogated the major source ent genotypes with 5 Non-Ewing sarcoma cell lines of NAD in Ewing sarcoma and analysed the conse- and mesenchymal stem cells revealed significantly quences of disrupting NAD supply. Analysing the higher FK866 sensitivity of EWS-ETS positive Ewing transcriptional signature of EWS-FLI1, which drives sarcoma cells, with IC50 values mostly below 1nM Ewing sarcoma growth, we identified a number of (Figure 4). Taken together, our data reveal evidence genes in the NAD biosynthetic pathway to be dereg- of the important role of the NAMPT-mediated NAD ulated in expression. NAD can be metabolized from salvage pathway in the energy homeostasis of the amino acid tryptophan in the microenvironment. Ewing sarcoma cells and suggest NAMPT inhibi- tion as a potential new treatment approach in this disease. (Mutz C, et al., 2017, Oncotarget)

42 43 Science Reports

Figure 1 IMPROVING THE ROUTINE ENUMERATION CD45RA - MPP OF HEMATOPOIETIC STEM CELLS: CD133+ Multi- CD38 low potent MULTI-COLOR ANALYSIS OF CD34 SUBTYPES CD10- progenitors REVEALS UNEXPECTED DIFFERENCES BETWEEN VARIOUS STEM CELL SOURCES CD45RA + CD45RA - CD133 + CD133 -/low Lympho- LMPP EMP Erythro- myeloid myeloid + SUMMARY SPECIMEN COLLECTION progenitors CD38 low CD38 progenitors - Flow cytometric analysis of CD34+ hematopoie- All cell specimens used in this study were obtained CD10 - GMP CD10 tic stem- and progenitor cells commenced in the for routine CD34 enumeration. Donor BM samples CD38 + early nineteen nineties and became a standardized (BMd, n=31) were from healthy allogeneic BM CD10 - 3-color procedure in 1998. In the clinical setting, this donors aged from 2 to 48 (median 26) years. Patient Granulocytes method has since been used worldwide, without any BM specimens (BM1y, n=21) were from biopsies Multi- MLP and lymphoid macrophages changes or improvements, to enumerate CD34+ cells routinely drawn for clinical examination one year CD38 + EoBP MEP progenitors progenitors as such, irrespective of their developmental stage or after allografting. Six of these were paired samples, CD10 + their hematopoietic potential. Based on the results i.e. we analyzed both BMd and BM1y from these of different research groups and on recent adjust- patients. The BM recipients (11 males, 10 females) CD45RA + ments of the model of human hematopoiesis (Abb. 1, had been diagnosed with AML (4), ALL (6), MDS CD133- Görgens A. et al. 2013. Cell Rep.), we established (4), CML (2), RCC (2), SCID (1), SAA (1) and Hyper BLP Late GMP ++ a >10-color CD34 flow cytometric assay containing IG E syndrome (1). Their median age was 10 years B-lymphoid CD38 Eosinophils Megakaryocytes + + basophils erythrocytes antibodies like CD133, CD45RA, CD10, CD19, CD38 (range 0.8-18). Conditioning regimens were myelo- progenitors CD10 CD38 CD19 + CD10 - and CD33. We showed that it enables phenotyping ablative (n=11) and reduced intensity (n=10). Fifteen of at least 6 distinct CD34 subsets: The multipotent BM recipients had received antithymocyte glob- progenitors (MPP) are 133+/45RA-/10-/38low and ulin. PBSC samples (n=35) were from autologous represent the earliest of all CD34 subtypes. Differen- stem cell collections from adult patients (22 males, B-lymphocytes Neutrophils Figure 1. Model of the tiation of these MPP by asymmetrical division forms 13 females) with Multiple Myeloma (n=19) and monocytes human hematopoietic tree, adapted from the recent LMPP (lymphoid-primed multipotent progenitors) Non-Hodgkin´s lymphoma (n=16), with a median age version by Görgens et that acquired CD45RA (133+/45RA+/10-/38low), of 54 years (range 22-73). The mobilization regimen al. 2013. Only CD34+ cell and EMP (erythro myeloid progenitors) that lost comprised chemotherapy (CHT) and hematopoietic CELL PROCESSING, SINGLE PLATFORM stages are depicted. The + low - CD133 expression (133-/45RA-/10-/38+). LMPP growth factors (hGF; n=13), CHT+hGF and plerixafor PROTOCOL AND FLOW CYTOMETRY CD45RA-CD133 CD38 CD10 multipotent progenitors (MPP, represent progenitors of neutrophils and lymphoid (n=6), hGF alone (n=5) or hGF and plerixafor (n=11). Blood cell counts were obtained from a Sysmex upper) either remain CD45RA- blood cells while EMP can give rise to granulocytes Informed consent was obtained from all patients KX-21N (Sysmex Corporation, Kobe, Japan). If neces- and down-regulate CD133 other than neutrophils as well as to erythroid and and the extended staining experiments had been sary, cells were diluted with Dulbecco´s phosphate (middle right) to form cells of the erythro-myeloid lineage megacaryocytic lineages. Differentiation of LMPP approved by the local ethical committee. buffered saline (PBS, Carlsbad, CA, USA) and the (EMP), or they acquire CD45RA towards the more mature GMP (neutrophil gran- white blood cells (WBC) adjusted to 5-15 x106 cells/ and become CD133+ lympho- ulocyte and monocyte progenitors) goes along ml prior to immune staining. All monoclonal anti- myeloid progenitors (LMPP, with a loss of CD133 expression (133-/45RA+/10- bodies (mAB) were used at pretested concentrations middle left) that comprise all lymphoid but also neutrophil /38+), while gaining cell surface CD10 leads to the and after respective compensation. Isotype controls and monocyte precursors formation of MLP (myelo lymphoid progenitors; and fluorescence-minus-one analyses were used (GMP). Upon down-regulation 133+/45RA+/10+/38+). Further maturation of MLP to define gating and compensation. One hundred of CD133 (lower left), these towards the progenitors of B lymphocytes (BLP) is µl of the cell sample were reverse-pipetted into a form late GMP and, in case of CD10 acquisition, cells of the accompanied by a loss of surface CD133 resulting in Trucount tube (BD Biosciences, San Jose, CA, USA). B-lymphoid lineage (BLP). a 133-/45RA+/10+/38++/19+ phenotype. After adding the mAB cocktail, cells were mixed and incubated light-shielded at room temperature

44 45 Science Reports

Figure 2 105 4 PBSC BLP 10 MLP 3 5 10 10 late CD45RA+ 0 LMPP+GMP 4 GMP 10 -472 3 2 4 5 3 0 10 10 10 10 -807 for 20min. Red blood cells were lysed by adding (total number (events of (sample dilution of beads per tube) target population) factor) CD45RA- 5 2 ml of ammonium chloride working solution (BD × × 102 10 (number of (sample volume (µl) ) 1 4 Biosciences) for 10 min before samples were analyzed beads counted) 10 2 3 4 5 on the flow cytometer. The mAB cocktail contained CD 45 RA 10 10 10 10 103 the stem cell enumeration kit (BD Biosciences) To calculate absolute cell numbers in donor BM, an EMP 0 MPP comprising CD45 FITC, CD34 PE and 7AAD, as well additional dilution factor of 1.1 was considered to CD34 1 -472 as the following mAB: AC133-1 APC, CD7 PeCF594, compensate for the anticoagulant added. 3 4 5 0 10 10 10 CD10 BV421, CD19 APC-Cy7, CD38 PE-Cy7, and -807 CD45RA BV510, CD3 PerCPeFl710, and CD33 APC-R700. Acquisition of 150,000 CD45+events was STATISTICAL ANALYSES 105 done on a FACS Fortessa (BD Biosciences) equipped Differences in cell counts (both absolute and 4 5 BMd BLP 10 MLP with 4 solid state lasers with excitation wave lengths relative) between the three HSPC sources were 3 5 10 10 late (nm) of 488, 405, 561 and 640. The FACSDiVa 6 soft- assessed with the unpaired one-sided Wilcoxon CD45RA+ LMPP+GMP GMP 0 ware (BD Biosciences) was used for cell acquisition signed-rank test. A p-value <0.05 was considered 104 2 -472 and data evaluation. For quality control of the instru- statistically significant (*<0.05, **<0.01, ***<0.001). 3 4 5 3 0 10 10 10 ment’s performance, CS&T beads (BD Biosciences) Mean values (±SD) are provided in the text. In 10 -807 CD45RA- 5 were used at least weekly. depicted box-and-whisker plots, boxes range from 2 10 10 Figure 2. Multi-color­ CD34 first to third quartile (containing 50% of data points). 104 subtype analysis: Represen-

The median value is indicated by a thick horizontal CD 45 RA 2 3 4 5 3 10 10 10 10 10 tative examples of autologous GATING STRATEGY line. Whiskers extend to the most extreme data EMP PBSC (top), donor bone marrow 0 MPP Viable WBC were defined by their CD45 expres- point which is no more than 1.5 times the interquar- CD34 1 (BMd, middle) and patient bone

-472 marrow one year after allo- sion, negativity for 7AAD, and typical position in tile range (= box height) away from the box, and 3 4 5 0 10 10 10 geneic transplantation (BM1y, the forward- and side scatter (FSC/SSC) dot plot. indicate the range that contains 95% of data points -807 bottom). Only viable and true According to the ISHAGE guidelines, viable and in a normally distributed sample. R version 3.2.0 CD34+ events are depicted (see 5 supplementary Figure 1 for the true hematopoietic stem- and progenitor cells were (2 015-04-16) was used for all statistical analyses. 10 gating strategy). Cells were first determined by their positivity for CD34, their weak 4 BM1y BLP 10 MLP separated into fractions negative expression of CD45, their typical position in the 3 5 10 or positive for CD45RA (larger lympho-monocytic area of the FSC/SSC dot plot and 10 late CD45RA+ LMPP+GMP plots). The proportion of earlier GMP 0 CD45RA- progenitors was gene- their negativity for 7AAD. To define subpopula- 104

-472 rally higher among PBSC than tions, CD34+ cells were first divided into an earlier 3 4 5 3 10 10 10 BMd and, particularly, -8070 CD45RA- and a more committed CD45RA+ cell 10 BM1y cells. All CD34+CD45RA+ CD45RA- 5 fraction. These were then separately depicted in a 2 10 or CD34+CD45RA- events 10 are shown in CD133 vs. CD10 CD133 vs. CD10 contour plot (Fig. 2). The resulting 4 10 contour plots, where they form

subpopulations were examined for their expression CD45RA 2 3 4 5 3 10 10 10 10 10 the C­ D45RA-CD133+CD10- MPP, of CD38, CD33, CD10 and CD7. Beads were double- EMP MPP the CD45RA+CD133+CD10- LMPP, gated in two different dot plots (APC vs. SSC and CD34 0 the CD45RA-CD133-CD10- EMP,

-472 the CD45RA+CD133-CD10- APC-Cy7 vs. FITC) to exclude false-positive events, 3 4 5 0 10 10 10 late GMP, and the CD1 0 -807 and the following formula was used to calculate the CD45RA+CD133+CD10+ MLP and number of target cells per µl: CD45RA+CD133-CD10+ BLP. CD133

46 47 PBSC (data shown). not PBSC donor and patient in subtypes ofCD34 distribution asimilar revealed comparison butdonors, their mobilized healthy from analyzed and obtained been had samples few PBSC only donor marrow, bone with allografted been had patients pediatric of the most Since malignancies. lymphoid different with diagnosed patients adult from present workthe was in described samples ofPBSC majority The ±253). µl BM1y by ±753) and (255/ (794/µl BMd by followed ±1,049), (1,402/µl PBSC in were obtained values est high the numbers, CD34 ofabsolute terms In ±0.9). BM1yby (1.1% (0.8% PBSC autologous by and ±0.6) (2.1% BMd in highest was ±2), followed cells CD34+ oftotal (±SD) percentage mean The WBC). of viable percentage as cells CD34+ (viable values relative as and CD34/µl) (viable numbers absolute as expressed are Results 3A). (Fig. sources 3cell the between differences significant revealed subtype CD34 oftheir irrespective HSPC oftotal Enumeration RESULTS - and in BMd than in BM1y (5/µl ±6; p<0.001). p<0.001). BM1y ±6; in (5/µl than BMd in and (138/µl ±156; BMd p<0.001), in than ±475) (608/µl, PBSC in higher were significantly which numbers MPP for absolute were obtained results Analogous PBSC. in 17 roughly and than foldless (p<0.001) BMd in than lower significantly was ±1.8 which 2.5% only was BM1y, In p<0.001). frequency ±8; their (16% (42% BMd ±13.7) in PBSC in than higher cantly signifi was cells MPP early ofthe ±SD) cells CD34+ % (in proportion cell 3B).mean The (Fig. values tive rela and numbers cell for absolute both subsets, ofCD34+ composition the regarding differences considerable we observed analyzed, sources 3 cell the Between subpopulations. CD34 6distinct least at where formed plots they contour CD133/CD10 in evaluated further were then subpopulations 2). Both (Fig. HSPC CD45RA+ more committed from CD45RA- earlier separating materials, all in subgroups distinct 2 defining allows marker this as CD45RA, with started always was subtyping CD34 - - b b a a Cells /µI B A 3 Figure &HOOVĂO &'FHOOVĂOCD34 cells /µI 1000 2000 3000 4000 1500 1000 2000 1000 1500 1000 2000 %CD34+ 2000 3000 4000 500

% of CD34+ 500 20 40 60 80 40 60 20 80 0 0 0 0 0 0 BCBdBM1y BMd PBSC BCBdBM1y BMd PBSC *** MPP MPP *** MPP MPP *** *** Total CD34absolute * Total CD34absolute *** ** ** * *** M MPGPLt M MLP+BLP LateGMP LMPP+GMP EMP EMP M MPGPLt M MLP+BLP LateGMP LMPP+GMP EMP EMP *** *** ** *** *** LMPP+GMP LMPP+GMP ns ** ns *** *** ***

** % of WBC 0 1 2 3 4 5 0 1 %2 of WBC3 4 5 + aeGPMLP+BLP Late GMP aeGPMLP+BLP Late GMP BCBdBM1y BMd PBSC BCBMd PBSC ns ns *** *** ns ns ns ** Total CD34relative ns Total CD34relative *** *** *** *** *** ** ns ns *** BM1y tions (mean 59%) in BM1y. in 59%) (mean tions showing the highest propor PBSC, in than BM1y and BMd in frequent more much were which subsets BLP and MLP mature more the to particularly and PBSC, than BMd in higher clearly were which GMP late to contrast in was This BM1y. in than BMd in higher were tions frac LMPP BM1y, and in than BMd in frequent more and BMd, in than PBSC in frequent more significantly also were EMP and MPP of graph) (lower values Relative BM1y. in than BMd in higher and BMd, in than PBSC in higher significantly were LMPP and MPP, of EMP graph) (B), subgroups CD34 the Regarding PBSC. in lowest and BMd in highest were (right) values tive rela while (black), BM1y in than BMd in higher and (grey), BMd in than circles) (open PBSC in higher significantly (left) were numbers absolute (A), cells CD34+ total For n=21). (BM1y, transplantation after allogeneic year one marrow bone patient and n=31) (BMd, marrow bone autologous donor (n=35), PBSC in subpopulations CD34 (B) and (A) of values relative and absolute median 3: Figure absolute numbers (upper absolute numbers mparison of ­Co total CD34+ cells cells CD34+ total Science Reports Science 48 - - 49 - Science Reports

Figure 4

CD34 subset MFI

MPP 18.191

MPP differentiate to either LMPP (CD45RA+CD133+) Despite the rather low proportions of the CD133dim of CD7 as a marker of T- and NK-cell progenitors was or EMP (CD45RA-CD133-/low). Both subsets showed MLP in the BM samples, it has to be noted that only seen on 0% to 2.8% of CD34+ cells). As non-spe- a higher CD38 expression than MPP supporting their this CD34 subset, when expressed as percentage cific staining could not be excluded, this subtype higher differentiation (Fig. 4). As shown in Fig. 3B, of the MLP/BLP fraction, represented a clearly was not pursued any further. LMPP 46.974 the mean frequency of LMPP was similar between lower median cell proportion (p=1.66e-09; one-sided PBSC and BMd (25.6% ±11.1, and 23.7% ±10.1; ns), unpaired Wilcoxon rank sum test) in BM1y (3.6%) We used the PE-Cy7-labelled H7 clone of CD38 in but significantly lower in BM1y (16.3% ±8.3) than in than in BMd (11.9%). A representative example is all experiments performed. Virtually all CD34+ BMd (p<0.01). Absolute LMPP cell numbers differed depicted in Fig. 2. In terms of absolute CD10+ stem cells were positive for this antibody, albeit at EMP 66.165 significantly between PBSC (383/µl ±412) and BMd cell numbers, significant differences were observed clearly different intensity (Fig. 4). MPP showed the (170/µl ±158; p<0.01), and between BMd and BM1y between PBSC (43/µl ±83) and BMd (255/µl ±302; lowest expression intensity, followed by LMPP and (33/µl ±28; p<0.001). This may suggest a higher p<0.001), whereas the values were similar between EMP. The intensity was higher among CD45RA+ proportion of neutrophil progenitors in favor of BMd and BM1y (171/µl ±191; ns). Out of the 31 BMd late myeloid precursors, and highest among the Late GMP 71.427 B-lymphoid progenitors in the LMPP subfraction and 21 BM1y specimens analyzed, 6 were paired CD45RA+CD10+CD19+ BLP. This differential CD38 of PBSC compared to BMd. EMP which give rise to samples, i.e. we examined BMd and BM1y pairs expression was generally observed in all specimens erythrocytes, megakaryocytes and granulocytes from the same patients. The results were virtually examined although the differences were not always other than neutrophils, showed results comparable to identical to those obtained from the whole groups: as clear as depicted in the BM1y sample shown in BLP 96.670 those obtained for LMPP. Their mean frequency was The median proportions of the CD34 subsets for Fig. 4. Due to the considerable overlap between the similar in PBSC (24.5% ±8.7) and BMd (19.5% ±6.5; BMd/BM1y were 12.1%/1.8% (MPP), 22%/17.4% different CD34 subgroups, CD38 was never used as p<0.05) but differed significantly between BMd and (LMPP), 20.1%/10% (EMP), 5.5%/9.1% (late GMP) and first-line antibody for subgroup distinction. CD38 BM1y (11.9% ±7.3; p<0.001). Absolute EMP numbers 34%/65.2% (MLP and BLP). Figure 4. ­Rise of CD38 mean fluorescence intensity (MFI) differed clearly and were 331/µl ±261 for PBSC vs. values on HSPC with increa- 162/µl ±176 for BMd (p<0.001), and 24/µl ±25 for The expression intensity of distinct markers often CONCLUSION sing differentiation of CD34+ BM1y vs. BMd (p<0.001). In terms of LMPP and EMP, correlates with differentiation as shown for CD38 We conclude that the presented analysis can identify subsets. Representative bone BM1y thus mediates an impression of exhaustion (see below). Such differences were also observed and enumerate distinct CD34 subfractions in any marrow sample from a patient one year after allogeneic when compared with BMd. Late myeloid progenitors for CD33 and CD133. Expression of CD133 on MPP conventional CD34 cell source. This approach may transplantation (BM1y) depic- (late GMP) with a CD45RA+CD133-CD33+CD10- and LMPP was generally higher in PBSC than in provide a solid basis for future studies to determine ting CD45RA-CD133+CD10- phenotype differed mainly with regard to relative BMd and BM1y (not depicted), suggesting that these the impact of different CD34 subsets in the graft as multi-potent progenitors (MPP), CD45RA+CD133+CD10- values, which were significantly lower in PBSC (4% cell stages are more differentiated in BM than in well as in post-transplant bone marrow on engraft- lymphoid-primed multi-po- ±3.1) than in BMd (8.5% ±4.1; p<0.001), but similar PBSC. Nevertheless, it was possible to distinguish ment kinetics and immune reconstitution. Whether tent progenitors (LMPP), between BMd and BM1y (11.1% ±6.5; ns). In terms the different CD133+/- subpopulations in all cell or not the analysis will allow predicting engraftment CD45RA-CD133-CD10- of absolute values, they were 51/µl ±62 in PBSC vs. samples (Fig. 2). The myeloid marker CD33 was kinetics on a routine basis remains to be examined. erythro-myeloid precursors (EMP), CD45RA+CD133-CD10- 72/µl ±91 in BMd (ns), and slightly lower in BM1y expressed in all CD34 subfractions, although it was late granulocyte monocyte (23/µl ±20) than in BMd (p<0.01). Due to the low weaker on MPP in PBSC with high CD133 expression Dmytrus J, Matthes-Martin S, Pichler H, Worel N, Geyeregger R, progenitors (late GMP) and frequency of the CD133+ MLP, this CD34 subset was than on MPP in BM with weaker CD133 expression Frank N, Frech C, Fritsch G. (2016). Multi-color immune-pheno­ CD45RA+CD133-CD10+ B-lym- typing of CD34 subsets reveals unexpected differences between evaluated together with the CD133- BLP. These cells (not depicted). Distinct CD10+ (and CD19+) HSPC phoid progenitors (BLP). various stem cell sources. Bone Marrow Transplant, 51: 1093-1100 were hardly detectable in PBSC (5% ±7.9) but clearly subsets were detectable among both the CD45RA+ present in BMd (31.9% ±15.1; p<0.001). In BM1y, they and the CD133- progenitors, and the CD33 expres- represented the largest CD34 subfraction (58.5% sion was clearly higher in BM1y than PBSC (not ±17.6) which was significantly higher than in BMd shown). In all cell sources, a potential coexpression (p<0.001).

50 51 Science Reports

Figure 1 AA C DD EE CHARACTERIZATION OF A NOVEL FUSION H857 H657 K657 H857 H657 K657

GENE IN JUVENILE MYELOMONOCYTIC LEUKEMIA 3.0A 4.1A 3.0A 4.1A 2.9A ASSOCIATED WITH RESISTANCE TO TYROSINE B D850 D850 2.9A B D850 E850 D850 D850 E850 + D850- KINASE INHIBITORS + -

R853 Juvenile myelomonocytic leukemia (JMML) is a rare R853 IDENTIFICATION OF A NOVEL FUSION F G myelodysplastic/myeloproliferative neoplasia occur- GENE INVOLVING PDGFRB AND F G ring in young children, characterized by excessive MUTATIONAL ANALYSIS proliferation of monocytic and granulocytic cells Sequencing of 5’-RACE-PCR products revealed R853 H853 R853 H853 5.1A 2.7A 5.1A infiltrating different organs. About 90% of JMML an in-frame fusion between NDEL1 and PDGFRB. E946 2.7A E946 patients have mutations in NF1, K-RAS, N-RAS, Screening of archived diagnostic specimens from E946 E946 CBL, or PTPN11, all implicated in activation of the 40 JMML patients provided no evidence for the RAS-RAF-MAPK pathway. Despite recent efforts occurrence of the NDEL1-PDGFRB fusion gene in H657 D850 +3 exploiting whole exome sequencing, the genetic other individuals. The observed development of H H PDGFRb ALMSELKI MSH657HLG KICDFGLA RDID850M RD+3 SNY alterations underlying the disease in the remain- resistance to two different TKIs including imati- PDGFRbPDGFRaALMSELKIAL MSELKIMSMTHLGHLG KICKICDFGLADFGLA RDIRDIMM HDRDSNYSNY ing 10% of patients remain elusive. Hematopoietic nib and nilotinib prompted screening of the entire PDGFRaFLT3ALALMSELKMMTOLGMSELKI MTHLG KICKICDFGLA RDIIMMSHDDSNYSNY CSF1RFLT3 ALMSELKMMTOLGALMSELKI MSHLG KICKICDFGLA RDIMNDSNYI MSDSNY stem cell transplantation (HSCT) is currently the ­tyrosine kinase domain (TKD) of PDGFRB for the CSF1Rc-KitALMSELKIALMSELKVLSYLGMSHLG KICKICDFGLA RDIKNDSNYIMNDSNY Consensusc-Kit ALMSELKVLSYLGALMSELKI MSHLG KICKICDFGLADFGLA RDIMNDSNYIKNDSNY only curative therapy for most JMML patients, but presence of mutations. Sanger sequencing revealed 100% Consensus ConservationALMSELKI MSHLG KICDFGLA RDIMNDSNY 100% advances in the understanding of the underlying the missense point mutation C2550G in the acti- 0% Conservation molecular mechanisms in JMML have permitted the vation loop of the TKD converting the aspartate 0% αC-helix A-loop Figure 1. Structural model of introduction of different therapeutic agents such as residue at position 850 into glutamate (D850E). This αC-helix A-loop PDGFRβ TKD the DNA-hypomethylating substance azacitidine. mutation was identified at the time of both relapses We have identified a JMML case with a chromo- but not in the diagnostic PB or BM samples. the typical auto-inhibitory interaction between D850 (fig. 1C), which is expected to stabilize the A-loop somal translocation, t(5;17)(q33;p11.2), resulting and the amino acid at the +3 position, R853, which in the active conformation. This interaction can be in the fusion of the platelet-derived growth factor is commonly observed in inactive kinase domains further enhanced by the D850E mutation, because receptor β (PDGFRB) gene to a novel partner, the STRUCTURAL MODELLING OF THE PDGFRβ of other receptor tyrosine kinases (RTKs) from the longer side chain of glutamate in comparison to nuclear distribution protein nudE-like 1 (NDEL1), TYROSINE KINASE DOMAIN the PDGFR family, and is believed to stabilize the aspartate brings the negatively charged carboxylic which has not been implicated in any translocation In order to elucidate the structural effects mediated A-loop in the inactive conformation (fig.1A, orange). group 1.1 Å closer to the positively charged histi- event to date. In contrast to earlier data on fusion by the D850E mutation in the PDGFRβ TKD, we However, modelling of the mutant PDGFRβ TKD dine residue, thus increasing the stability of the genes involving PDGRFB in myeloid malignancies, have generated protein models of the kinase domain in the inactive conformation could not explain the activation loop in the active conformation (fig. 1D). which were generally responsive to treatment with both in active and inactive conformations. Since the enhanced kinase activity and resistance to type-II The other interaction involved R853 and E946 in imatinib, the patient became refractory to both structure of PDGFRβ TKD is not available, we have TKIs, because the negatively charged glutamate at the C-lobe of the TKD (fig. 1F). The +3 position to imatinib and nilotinib. This observation represented modelled the kinase domain on the basis of estab- position 850 is also able to form a salt bridge with D850 is one of the least conserved positions in the the first clinical finding of a PDGFRB gene fusion lished structures of highly homologous proteins the positively charged side chain of R853 (fig.1B). activation loop of receptor tyrosine kinases (RTKs) resistant to therapy with tyrosine kinase inhibitors from the PDGFR family including c-KIT, CSF1R, and Moreover, the DFG-out model of the TKD did not from the PDGFR family (fig. 1H), and the arginine (TKIs), and our work focused on elucidating the hith- VEGFR2. All structural models indicated that the display any indication of weakened electrostatic at this position in PDGFRβ (R853) has the longest erto unknown mechanism of TKI resistance. observed TKI type-II resistance of cells expressing interaction between E850 and R853 which would side chain among all members. The DFG-in model NDEL1-PDGFRβD850E was conceivably related to destabilize the inactive conformation of the kinase suggested that the positively charged side chain stabilization of the activation loop (A-loop) in the domain. However, the DFG-in model suggested of R853 can reach a distance of approximately 2.5 Å active conformation. In this conformation, three crit- the occurrence of two intriguing amino acid inter- to the negatively charged carboxyl group of E946, ical amino acids, D844, F845, and G846 (DFG) serv- actions upon transition of the A-loop from inactive which may facilitate electrostatic bonds and ing as a hypomochlion for the activation loop, adopt to the active state (fig.1A, green). One interaction provide additional stabilization of the DFG-in the so-called DFG-in position. The modelled struc- implicated the negatively charged D850 and the conformation of the PDGFRβ TKD (fig. 1G). The ture of the inactive DFG-out conformation revealed positively charged, conserved H657 in the αC-helix structural model therefore suggested resistance of

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Figure 2 Figure 2. (A) Displayed are IC50 A values of different TKIs against A Ba/F3 cells expressing wildtype A NDEL1-PDGFRβ FIP1L1-PDGFRα (wt) or mutant NDEL1-PDGFR? TKI WT R853H D850E DR H657K HR WT D842E NDEL1-PDGFRβ FIP1L1-PDGFRα fusion proteins. The corres- IMATKI WT80 R853H50 D850E>5000 DR15 H657K>5000 HR15 WT2 D842E 4 ponding IC50 values for Ba/F3 NILIMA 9080 5040 >50002200 1512 >50002100 15 10 2 8 4 12 expressing FIP1L1-PDGFR? WT and D842E are given for compa- TYPE II NIL 90 40 2200 1215 2100 10 8 12 SOR 40 30 2100 1900 10 1 2 rison. (B) Western blot analysis

TYPE II 15 (DAS), midostaurin (MID) and pacritinib (PAC)) and Our data provide first evidence for the occurrence of DASSOR 4020 3010 210025 12 190025 10 10 1 7 2 N/A of Ba/F3 cells transduced with type-II (imatinib (IMA), nilotinib (NIL) and sorafenib a point mutation in the activation loop of PDGFRB DAS 20 10 25 12 25 10 7 N/A wildtype or mutant (R = R853H, MID 25 20 15 15 10 12 30 N/A

TYPE I H = H657K, HR = H657K/R853H, (SOR)) was determined by in vitro cell survival mediating resistance to type-II TKIs, and for a MID 25 20 15 15 10 12 30 N/A TYPE I PAC 30 25 10 12 15 13 N/A N/A D = D850E, and DR = D850E/ (MTT) assays. Oncogene-addicted proliferation of major alteration of responsiveness to TKI treatment PAC 30 25 10 12 15 13 N/A N/A R853H) NDEL1-PDGFRB genes. Ba/F3 cells carrying NDEL1-PDGFRBD850E could only mediated by an exchange between two negatively B WT R H HR D DR The phosphorylation levels of BB WT R H HR D DR NDEL1-PDGFR? at Y751 and be inhibited by the type-I TKIs dasatinib, midostau- charged amino acids in a tyrosine kinase. The NIL,100 nM NIL,100 nM Y857, and Erk are displayed. rin, and pacritinib at sub-micromolar concentrations, protein model indicated sensitivity of cells carry- Shown are also the total pY751-PDGFRβ in line with predictions by the protein model. ing the mutant NDEL1-PDGFRB to type-I TKIs, pY751-PDGFRβ expression levels of NDEL1- PDGFR?, Erk, and the control which was confirmed by sensitivity testing in vitro. pY857-PDGFR pY857-PDGFRβ β gene Gapdh upon mock treat- Availability of the model at the time of relapse after ment with DMSO (indicated by DIFFERENTIAL EFFECTS OF failure of imatinib or nilotinib could have assisted “-“) or with 100 nM of nilotinib PDGFRPDGFRβ β in selecting potentially effective treatment options. (indicated by “+”) for 4 h. CORRESPONDING MUTATIONS IN PDGFRβ AND PDGFRα The observations therefore provide new insights pErkpErk The intriguing observation of TKI resistance appar- into specific amino acid interactions in mutant RTKs 2016). In addition to the clinically identified D850E ently induced by the D850E mutation in the kinase which are of clinical relevance for improved selection ErkErk mutant, we have made a construct carrying H657K domain of PDGFRβ, which was in contrast to the of appropriate TKI treatment (Preuner S et al. Int J mutation, which, according to our structural model, same amino acid exchange at the corresponding Mol Sci 2016). In addition, it provides a new molec- GapdhGapdh would strengthen the electrostatic interaction site in PDGFRα (D842E), raised questions regarding ular insight into activation of PDGFRβ which could 9 11 2 2 3 4 5 5 6 6 7 7 8 8 9 1010 11 11 12 12 between D850 and αC-helix thus stabilizing the important structural differences between the two serve for de novo design of specific inhibitors of this DFG-in conformation of the PDGFRβ TKD (fig.1E). highly homologous RTKs. While PDGFRβ displays versatile RTK. In order to probe the influence of R853 on the an arginine in the +3 position to the mutation site NDEL1-PDGFRBD850E mutation to type-II TKIs, which kinase activity and TKI-sensitivity of PDGRFβ TKD, (R853), PDGFRα has the much shorter and less can only bind to the inactive conformation of the we have also generated the constructs carrying basic histidine in the corresponding position (H845) Byrgazov K, Lucini CB, Berkowitsch B, Koenig M, Haas OA, PDGFRβ TKD, but indicated sensitivity to type-I the R853H mutation. Ba/F3 cells expressing the (fig.1H). It appeared conceivable therefore that inter- ­Hoermann G, Valent P, Lion T. (2016). Transposon-mediated generation of BCR-ABL1-expressing transgenic cell lines for TKIs binding to the active conformation. To address mutants H657K and D850E versions of NDEL1-PDG- action between the side chains of R853 and E946 in unbiased sensitivity testing of tyrosine kinase inhibitors. the predictions provided by the protein model, we FRB displayed a slightly higher proliferation rate, the mutant PDGFRβ TKD could stabilize the active Oncotarget, 7: 78083-78094 have introduced several mutations affecting the possibly reflecting an elevated kinase activity of the conformation in the mutants H657K and D850E, thus Preuner S, Barna A, Frommlet F, Czurda S, Konstantin B, Alikian aforementioned interactions and tested the sensitiv- mutants, whereas the R853H mutation leveled the mediating resistance to type-II TKIs. To address this M, Machova Polakova K, Sacha T, Richter J, Lion T (corresp.author), ity of generated constructs against a panel of TKIs. proliferation rate of these mutants to wildtype level. notion, we introduced a mutation into NDEL1-PDG- Gabriel C. (2016). Quantitative Analysis of Mutant Subclones in Chronic Myeloid Leukemia: Comparison of Different Methodo­ In line with this observation, the level of auto-phos- FRB constructs replacing arginine at position 853 logical ­Approaches. Int J Mol Sci, 17: 642 phorylation of PDGFRβ and its target, ERK1/2, was by histidine (R853H), thus mimicking the sequence Byrgazov K, Kastner R, Gorna M, Hoermann G, Koenig M, Lucini CB, TRANSFORMING ACTIVITY AND TKI- higher in Ba/F3 cells carrying the H657K and D850E of the activation loop in the PDGFRα TKD (fig. 1G). Ulreich R, Benesch M, Strenger V, Lackner H, Schwinger W, SENSITIVITY OF THE WILDTYPE AND mutation (fig. 2B, lanes 5 and 9) in comparison to the This change reduced the NDEL1-PDGFRB-driven Sovinz P, Haas OA, van den Heuvel-Eibrink M, Niemeyer CM, MUTANT NDEL1-PDGFRB FUSION GENES wildtype and R853H-carrying versions of the fusion proliferation of Ba/F3 cells, and restored the sensitiv- Hantschel O, Valent P, Superti-Furga G, Urban C, Dworzak MN, Lion T. (2017, Epub 2016 Oct 7). NDEL1-PDGFRB fusion gene in a To assess the oncogenic potential of the newly gene (fig. 2B, lanes 1, 3, 7, and 11). ity of cells carrying the H657K and D850E mutant of myeloid malignancy with eosinophilia associated with resistance to identified fusion gene, the murine cell line Ba/F3 NDEL1-PDGFRβ to type-II TKIs, in line with the prop- tyrosine kinase inhibitors. Leukemia, 31: 237-240 was stably transduced with wildtype or mutant The in vitro responsiveness of Ba/F3 cells trans- erties of the D842E mutant of the FIP1L1-PDGFRα NDEL1-PDGFRB constructs by employing a trans- duced with wildtype or mutant NDEL1-PDGFRB fusion (fig.2A). The restored sensitivity to nilotinib poson-based system (Byrgazov K et al. Oncotarget. constructs to different TKIs of type-I (dasatinib was also confirmed by western blot analysis (fig. 2B).

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HIGH RESOLUTION GENOMIC AND TRANSCRIPTOMIC Figure 1 PROFILING OF PEDIATRIC B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA: IMPLICATIONS FOR EMERGENCE OF RESISTANCE AND RELAPSE

Acute lymphoblastic leukemia (ALL) is the most Previous studies from others and our group have Figure 1. Response of ETV6/ frequent malignancy in childhood and adolescence. shown that deletions of genes involved in the RUNX1-positive leukemic cell lines to glucocorticoids. ALL comprises various disease entities character- glucocorticoid (GC) mediated signaling pathway (A) Western blot analysis of GR ized by chromosomal translocations, aneuploidy, prevail at relapse (Kuster et al). This finding was signaling components of AT-1, structural variants, and sequence mutations that therefore taken as an indication that these altera- AT-2 (wt GR) and REH (mutant GR) cell lines after exposure to interfere with critical cellular pathways affecting tions could render the affected cells resistant to GC PRED. Protein abundance was lymphoid development, tumor suppression, cell cycle – an integral component of all major childhood ALL determined using anti-GR, anti- control, homing, as well as kinase and cytokine treatment protocols – and would then constitute a BMF, anti-BIM and anti-PUMA receptor signaling. With contemporary treatment significant precondition for disease recurrence. antibodies. GAPDH was used as loading control. protocols, up to 90% of the patients remain in long- (B) Viability of AT-1, AT-2 and term remission. Still, relapses are one of the leading Frequency of genetic deletions in REH cells upon exposure to causes of death in children and young people. E/R-positive relapses PRED, measured by MTT assay. Similar to previous studies, we classified deletions Values are means ± SD from four independent experiments. The goal of our basic and translational research is, into those that affect genes involved in the GC sign- **p<0.005 (paired t-test). therefore, to explore genomic and transcriptomic aling pathway, B cell development and cell cycle. (C) Quantification of GR, BMF, alterations in specific ALL subgroups to better The overall incidence of deletions in the GC signa- BIM and GILZ transcripts (RT-qPCR) in response to understand the evolution of leukemia, the emer- ling gene components BTG1, NR3C1, NR3C2, BMF, PRED exposure (expressed as gence of relapse, and the nature of the resistant MSH1 and MSH6 was, with 58% (18/31 cases), simi- fold-change of vehicle-treated clone. Thereby, not only new insight into the mech- lar to the one in our previous report (Kuster et al.). cells) of all three cell lines. anisms of relapse development is gained, but also The most common of all remaining recurrent dele- Specific mRNA values were measured in triplicates and the biological impact of genomic alterations, their tions concerned the tumor-suppressor gene ETV6 normalized to endogenous GUS. potential role in resistance mechanisms, as well as (61%), followed by BCL2L14, a gene that encodes a Bars represent mean values their applicability as biomarkers and drug targets mainly pro-apoptotic BH3-only family member, and ± SD from four individual ­experiments. can be inferred. CDKN1B, which generates the cyclin kinase inhib-

itor p27. Since both latter genes flank ETV6, they *p<0.05; were co-deleted in 38% and 35% cases, respectively. **p<0.005 (paired t-test). IMPLICATIONS OF GLUCOCORTICOID Other common deletions affected genes that encode SIGNALING ALTERATIONS IN CHILDREN WITH cell cycle regulators, such as CDKN2A, CDKN2B and RELAPSED ETV6/RUNX1-POSITIVE LEUKEMIA RB1 in 35%, 29% and 10% cases, respectively. The ETV6/RUNX1 (E/R) gene fusion is the genetic hallmark of the largest subgroup of childhood B cell Association of genetic alterations with clinical precursor acute lymphoblastic leukemia, which also characteristics and outcome has the overall most favorable prognostic outlook. Of all GC signaling pathway-associated gene Yet despite its low risk features and rapid response deletions only those in NR3C1, which generates the to current treatment regimens, up to 15% of cases glucocorticoid receptor (GR), were associated with a still relapse. These disease recurrences are more subsequent relapse (50 vs. 8%, p<0.04) and tended difficult to treat and therefore also responsible for to occur more frequently in cases with a poor MRD a dismal outcome in a considerable proportion of response to relapse treatment (25 vs. 7%; p<0.04). affected children. ETV6 gene deletions prevailed among MRD poorly

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responding cases (81 vs. 40%, p<0.05). However, for instance, coordinating the cell cycle and cell GENOMIC AND TRANSCRIPTIONAL IKZF1 encodes the lymphoid transcription factor since ETV6 deletions are similarly frequent at diag- survival. GC resistance at relapse can therefore not LANDSCAPE OF P2RY8-CRLF2-POSITIVE IKAROS, which is a key regulator in early lympho- nosis and at relapse and the gene is not expressed be viewed in isolation but must always be seen as CHILDHOOD ACUTE LYMPHOBLASTIC cyte development and prevails in poorly respond- in the remaining, non-deleted cases, it is difficult part of a more global system of drug resistance. In LEUKEMIA ing cases in major treatment protocols. IKZF1 to imagine that they can play a major role in the such a context, a distinct form of GC resistance will Recently, a novel subgroup of childhood ALL has deletions were also reported in small cohorts of development of drug resistance. The concomitant lose its relevance as soon as effective drugs, such as been described whose defining characteristic is the P2RY8-CRLF2-positive leukemia cases recruited deletions of the BCL2L14 and/or CDKN1B genes obatoclax, become available for clinical use. deregulated expression of the cytokine receptor- to various treatment protocols. It seems, therefore, are in fact much better candidates, because even a like factor 2 (CRLF2) gene, which is located in the likely that their presence contributes to relapse partial loss of their function might affect apoptosis pseudoautosomal region 1 on the short arm of the development in this particular subgroup, a notion and drug response in a substantial way. Grausenburger R, Bastelberger S, Eckert C, Kauer M, Stanulla M, X and Y chromosome. The two most common that has, so far, not been systematically investigated Frech C, Bauer E, Stoiber D, von Stackelberg A, Attarbaschi A, ­causative genetic defects are a small interstitial in large and well-characterized cohorts. Therefore, Haas OA, Panzer-Grümayer R. (2016). Genetic alterations in The glucocorticoid receptor determines the deletion that fuses the first non-coding exon P2RY8 we performed whole exome sequencing (WES) and glucocorticoid signaling pathway components are associated with response to GC in vitro adverse prognosis in children with relapsed ETV6/RUNX1-positive to the entire coding region of CRLF2 and occurs transcriptional profiling (RNA-seg) in 41 relapsing We used GC-resistant (REH) and GC-sensitive (AT-1, acute lymphoblastic leukemia. Leuk Lymphoma, 57: 1163-1173 in 5–8% of childhood B-cell precursor (BCP) ALL and non-relapsing major clone P2RY8-CRLF2-posi- AT-2) E/R-harboring leukemic cell lines to model cases and a more rare translocation that places tive cases that were treated primarily according to and assess the consequences of a functional loss CRLF2 under the control of the IGH enhancer. BFM protocols. of the GR in E/R-positive leukemias. Consistent P2RY8-CRLF2 fusion harboring leukemias often with the lack of a functional GR, REH cells were carry additional alterations in JAK/STAT path- Clonal heterogeneity and instability of resistant to prednisolone (PRED) when exposed to way genes and they may cooperatively activate kinase-activating pathway alterations clinically meaningful concentrations, as indicated by downstream pathways. They are associated with At initial diagnosis, the overall frequency of JAK/ their unchanged viability as well as the inability to a significantly increased relapse risk in AIEOP/ STAT pathway mutations was 51% and equally induce the GR downstream targets BCL2 modifying BFM protocols, which is independent of the size distributed between relapsing and non-relapsing factor (BMF), the BCL2-like gene BIM, glucocorti- of the P2RY8-CRLF2-positive clone (Morak et al.). cases. They were subclonal in 47% of cases. Irre- coid-induced leucine zipper (GILZ) and BCL2 binding ­Respective cases are primarily classified as non-high spective of their original size, JAK/STAT pathway component 3 (PUMA) at the transcript and the risk by clinical and molecular response criteria and mutation carrying clones were lost at relapse in protein levels [Fig. 1A–C]. By contrast, both GC-­sen- relapses occur predominantly late. 60% of the cases (Figure 2A). Mutations in RTK/ sitive cell lines showed a reduced viability upon Ras ­pathway genes were found in 29% of the cases exposure to the same PRED concentration and the at diagnosis, lost in almost half of the cases, and concomitant up-regulation of GR, as well as of the increased to 68% in relapses (P = 0.06). P2RY8- downstream targets at both transcript and protein CRLF2 was also lost in 32% of relapses. levels [Fig. 1. A–C].

Overall, our findings, together with previous ones, corroborate the notion that the functional impair- ment of many GC signaling pathway elements is involved in the emergence of GC-resistant E/R-pos- itive 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,

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Figure 2

A

IKZF1 alterations predict relapse in IKZF1 alterations are associated P2RY8-CRLF2-positive ALL and display with dismal outcome distinctive transcriptional signatures Except for the genomic IKZF1 status and white Except for IKZF1, the frequency of alterations in blood cell count, which were not associated with genes implicated in lymphoid development and each other (P = 0.66), we found no other biological in tumor suppression/cell cycle regulation did not or clinical parameters such as other genetic alter- significantly differ between relapsing and non-re- ations, age at diagnosis, clinical risk group assign- lapsing cases. IKZF1 alterations prevailed in the ment or morphological and molecular response to relapsing cohort with a frequency of 41% versus treatment to be correlated with the occurrence of Figure 2. Clonal composi- 10% in non-relapsing cases (P = 0.001). relapses. There was no difference between Down tion and stability of genomic Syndrome (DS) and non-DS cases. Of note, only one alterations. Therefore, we profiled 22 leukemia samples by of eight IKZF1-altered relapsing cases initially had a (A) Clonal composition of JAK/STAT (blue) and RTK/ RNA-Seq according to their IKZF1 status. Tran- poor MRD response and was, therefore, assigned to Ras pathway (red) signaling scriptional profiling revealed specific regulations in high-risk treatment. Yet, IKZF1-mutated cases had a gene mutations according the IK6 deletions and biallelic alterations group and, significantly poorer outcome than their IKZF1 wild- to individual genes (symbol code). Dots represent the albeit to a much lesser degree, in the group with type counterparts as evidenced by an adverse pEFS adjusted allelic frequency (adj. larger deletions compared with the wtIKZF1 one (P = 0.026) and pOS (P = 0.051). AF) of mutations. Black dots (Figure 3). Gene set enrichment analysis revealed mark conserved mutations at a highly significant correlation of differentially The P2RY8-CRLF2 fusion was lost in one-third of B INITIAL LEUKEMIA RELAPSE ­diagnosis and relapse. (B) Model for the evolution expressed genes with various human hematopoie- the relapses. Together with previous observations of leukemia and selection of tic and lymphoid stem cells sets, and concordantly – that, for instance, these fusions frequently affect relapse clones. Leukemia-in- for gene sets specifically expressed in immature B subclones that never evolve into major relapse itiating (founder) alterations occur in a hematopoietic stem/ cells. This suggests that IKZF1 alterations lead to clones – this indicates that it is primarily a second- progenitor cell, while the impaired B-cell differentiation and the acquisition ary change that may potentially supply the respec- ensuing RAG-mediated micro- of stem cell-like features. We also found enrichment tive cells with a certain proliferative but certainly deletions evolve later during in gene sets that are upregulated in the context of not with an evolutionary advantage ( Morak et al.). early B cell differentiation. Microdeletions affect genes microenvironment, focal adhesion kinase and inte- In line with other types of childhood BCP ALL, critical for B cell differentia- grin pathways, as well as of genes that are higher B-cell differentiation genes are also commonly tion and tumor suppression expressed in response to hypoxia, downstream deleted in cases with a P2RY8-CRLF2 fusion. These (color code at the bottom of the VEGF/VEGFR signaling and to EPO signaling. abnormalities are usually preserved in the corre- graph). JAK/STAT or RTK/Ras Normal cell pathway activating alterations sponding relapses. Consistent with their role in Founder continuously emerge but do drug resistance, IKZF1 alterations prevail already in PAX5 not outcompete each other at relapse-prone cases at diagnosis but become even initial presentation of leukemia. PAR1 Chemotherapy then selects more abundant in relapses. IKZF1- deleted cases CDKN2A/B for resistant clones, which also seem to profit from other associated B-cell represent frequently only minor IKZF1 development and cell cycle gene defects, particu- subclones at initial diagnosis, larly those of PAX5 and/or CDKN2A/B, which are JAK/STAT but they may vary regarding found in half of the cases, but also from mutations in RAS their proliferation driving mutation. specific proliferation-promoting pathway genes.

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Since chemotherapy primarily eliminates the bulk of Vesely C, Frech C, Eckert C, Cario G, Mecklenbrauker A, rapidly proliferating cells, it spares those less active zur Stadt U, Nebral K, Kraler F, Fischer S, Attarbaschi A, Schuster M, Bock C, Cavé H, von Stackelberg A, Schrappe M, resistant stem-cell-like ones that eventually gener- Horstmann MA, Mann G, Haas OA, Panzer-Grümayer R. (2017). Figure 3. Transcriptional ate relapses. In this scenario, the P2RY8-CRLF2 Genomic and transcriptional landscape of P2RY8-CRLF2-positive signature of leukemias accor- fusion is only one of several proliferation activating childhood acute lymphoblastic leukemia. Leukemia, Epub 2017 Jan 6 ding to IKZF1 status. alterations that merely serves as a common marker Cluster heatmap of the top 50 up- and downregulated genes for an otherwise genetically heterogeneous group, in both IKZF1-altered groups whose other and probably more relevant features are („IKN“ denotes IKZF1 altera- IKZF1 alterations. These alterations are well-known tions leading to a dominant-ne- gative effect and biallelic disease drivers in many types of drug-resistant alterations and „IKD“ designa- leukemias, such as BCR/ABL1-positive ones, which tes IKZF1 alterations resulting all share a similar gene expression signature. The in haploinsufficiency) according transcriptional profile of IKZF1-altered cases reflects to fold-change (P≤1E−8 for IKN versus IKZF1 wt (IKC), P≤2E−3 their strong homing preference to the bone marrow for IKD versus IKC); IKN cases niche as well as their high repopulation capacity, are indicated in red, IKD ones in attributes that also become apparent in mouse blue and IKC in gray at the top of the map. models in which IKZF1-/- pre-B cells acquire stem cell and adhesion properties including activation of the focal adhesion kinase pathway. Thus, we consider the P2RY8-CRLF2 fusion as one then obstructed by defects in genes whose products of the many secondary proliferative driver altera- are required for normal B cell differentiation. Cells Besides these biological insights, our findings also tions that - in line with those activating JAK/STAT that carry specific combinations of abnormalities provide some clues that may become relevant in and RTK/Ras pathways – are highly instable at may gain a competitive and selective advantage future treatment decisions. Apart from their prog- relapse, albeit to a lesser degree if initially present and eventually predominate in the pre-leukemic cell nostic implications, IKZF1 alterations may even- in the major clone, and not as a bona fide primary population. Parallel to these alterations and increas- tually serve as markers for specific therapeutic genetic alteration that is always stable at relapse ingly during later stages of leukemia evolution, interventions. It appears reasonable to try to restore and critical for the maintenance of the leukemia. mutations in specific genes activate signaling path- IKAROS signaling especially in those IKZF1-altered ways that enable their unrestrained proliferation. cases that still have retained a functional wild-type Taken together and as schematically depicted in allele. For the other 20% of cases with biallelic IKZF1 Figure 2B, we envision the following scenario of alterations, inhibition of the activated focal adhesion relapse evolution in P2RY8-CRLF2-positive leuke- kinase pathway may become a viable treatment mias. Predisposing constitutional or acquired option. Such approaches might perhaps be combined genomic alterations – as those affecting chromo- with a cocktail of other signaling inhibitors given some 21 – facilitate the expansion of a pre/leukemic the availability of various JAK/STAT, Ras/MEK/ERK B cell precursor clone. Its regular development is and PI3K/mTOR pathway inhibitors.

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NEW INSIGHTS IN NEUROBLASTOMA BIOL­ OGY Figure 1 spontaneous maturation – FROM SPONTANEOUS MATURATION TO THE SCs in periphery

­RELAPSE SEEDING CLONE blood vessel MSCs SC progenitors

Neuroblastoma is the most common extracranial axonal re-growth after nerve injury. Based on these Figure 1. Clinical and solid tumor in childhood and accounts for around findings, we hypothesized that Schwann cells exert biological­ heterogeneity of neuroblastoma. ­ 15% of all pediatric oncology deaths. This embryonal their physiological functions, i.e., the regulation of SC stroma tumor arises from sympathetic neuronal precursor neuronal differentiation during development and favorable genetics cells and shows a unique biological and clinical nerve regeneration, on neuroblastoma cells facilitat- aneuploidy spectrum, encompassing spontaneous regression, ing spontaneous tumor maturation in vivo. Subse- no MNA spontaneous maturation, or malignant progression. quent studies further demonstrated that Schwann mostly no SCAs ganglionic cells While most patients whose tumors undergo sponta- cells are also able to impair the growth of aggressive Schwann cell differentiating fully matures neous regression or maturation (ganglioneuroblasto- neuroblastoma cell lines, obtained from tumors recruitment neurblastoma ganglioneuroma mas, ganglioneuromas) have an excellent outcome, lacking a spontaneous maturation capacity in vitro, benignly behaving not all children with aggressive tumors can be cured indicating a therapeutic potential of Schwann cells time (Figure 1). Thus, a better understanding of the biol- or the factors they express. neuroblastoma unfavorable genetics ogy of both tumor types, spontaneously regressing/ any ploidy maturing and aggressive ones is of high interest To gain insights into the role of Schwann cells in to develop novel treatment approaches. Thus, our the neuroblastoma maturation process, we tested ±MNA recent research has focused on two distinct aspects our hypothesis on the similarities of physiological SCAs un- or poorly differentiated progressing of neuroblastoma biology – spontaneous maturation interactions between nerve regeneration and neuro- mutations neuroblastoma neuroblastoma and relapse formation. blastoma maturation. In a first step, we analyzed aggressive the proteome and transcriptome and cellular malignant progression processes active in repair Schwann cells after nerve Figure 2 MEDIATORS OF SPONTANEOUS MATURATION injury using high-resolution mass spectrometry and IN NEUROBLASTOMA RNA-sequencing of highly enriched human Schwann hrMS Proteomics Neurotrophic/Neuritogenic

Neuroblastomas show a so far unique feature in cells and injured nerve tissue (Figure 2). Our results EGFL8 oncology research, i.e., their ability to mature spon- revealed that cultured Schwann cells and injured FLRT3 3 (8%) GFRA1 taneously into a benign tumor. Maturing neuroblas- nerves share a similar repair Schwann cell-associ- MDGA1 1 (52%) NRP2 tomas are composed of two cell populations 1) tumor ated expression signature including previously not SEM3B SHOT1 cells differentiating into a mitotically quiescent, described molecules involved in axonal differenti- TEN3 benign state and 2) Schwann cells forming a dense ation and two novel repair Schwann cell functions, Nerve (control) Phagocytosis tumor stroma. Already in 1996, we have shown i.e., debris clearance via phagocytosis and a type II Nerve (injured) that these stromal Schwann cells are not of tumor immune-regulation (Weiss et al. 2016). These find- Schwann cell NB Figure 2. Schwann cells in Fibroblast origin and postulated a prominent role of Schwann ings extended the functional spectrum of Schwann 2 (16%) spontaneous neuroblastoma cells in the maturation process of this favorable cells in regenerative processes after nerve injury maturation. Schwann cells MHCII upregulation neuroblastoma subtype (Ambros et al. 1996. NEJM). and strengthened our hypothesis on their vital role adopt new functions upon nerve HLA-ABC HLA-DRα1 repair and as stromal cells Schwann cells are known to closely interact with during neuroblastoma maturation. Hence, two-fol- in the tumor microenviron- neurons regulating axon integrity in the adult as low up projects now explore the signaling events SCs ment. High-resolution mass well as axon differentiation during development. between Schwann cells and neuroblastoma and/or spectrometry (proteomics) Furthermore, it was demonstrated that Schwann immune cells in detail. Our preliminary results using and functional tests revealed FBs counts novel neuritogenic/neurotro- cells are able to transform into dedicated repair a FACS based read-out system and immunofluores- SC co-culture NB cell/p1 SC phic factors, phagocytosis and cells with distinct functions essential to promote cence, demonstrated growth-inhibitory, differenti- MHCII up-regulation.

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Figure 3

Tumor at diagnosis Tumor at relapse

ation- and apoptosis-inducing effects of Schwann stage M patient, we found a unique aberration, Figure 3. Tumor evolution in cells on co-cultivated aggressive neuroblastoma deletion 1q, in the relapse samples besides a high neuroblastoma. Graphical representation of clonal expan­ cells. Secretome analysis and RNA-sequencing of number of concordant genomic aberrations present sion in a stage 4 neuroblastoma repair- and tumor-associated Schwann cells, neuro- in all analyzed samples. Interestingly, this aberra- patient. Each quadrant repre- blastoma cell lines and primary tumors will aid to tion was present in the disseminated tumor cells sents the clonal architecture of identify the signaling factors and receptors partic- at diagnosis but not in the primary tumor, despite a tissue/time point. Each color represents a group of chromo- ipating in this cross-talk. All in all, understanding analyzing seven different tumor pieces (Figure 3). somal aberrations and the size the Schwann cell biology in neuronal differentiation In a cohort of 154 patients, the highest incidence of each colored area represents is of utmost interest for new approaches in regenera- of this aberration was found in relapse samples the pro­portion of cells with tive medicine as well as neuroblastoma therapy. and occurred in DTCs at diagnosis nearly twice these aberrations within the analyzed samples. A 1q terminal as frequently as compared to primary tumors. Our deletion (W) which was present results indicate that analysis of bone marrow-de- DTCs at diagnosis DTCs at relapse in the diagnostic DTCs and also rived disseminated tumor cells at diagnosis besides in both, DTCs and metastatic TUMOR HETEROGENEITY AND tumor, at relapse was not found IDENTIFICATION OF THE RELAPSE SEEDING the tumor biopsies may increase the probability for in seven pieces of the primary CLONE IN METASTATIC NEUROBLASTOMA detecting the relapse-seeding clone and thus allow tumor. Although there has been substantial improvement a more precise diagnosis of the most aggressive in the outcome of patients with certain subsets ‘drivers’ of tumor progression. of neuroblastoma, long-term survival of high-risk neuroblastoma patients is still less than 40% and effective relapse therapies are lacking. To improve Weiss T*, Taschner-Mandl S*, Bileck A, Slany A, Kromp F, Rifat ­begovic F, Frech C, Windhager R, Kitzinger H, Tzou CH and the outcome of these patients, efforts are currently others. Proteomics and transcriptomics of peripheral nerve focusing on understanding fundamental genomic tissue and cells unravel new aspects of the human Schwann cell alterations driving neuroblastoma progression, repair phenotype. Gila. 2016 Dec; 64(12):2133-2153. doi: 10.1002/ therapy resistance and relapse development. Tumor glia.23045. relapse in patients with metastatic disease (stage M) is the main cause of mortality in these patients. * Contributed equaly Therefore, early and reliable detection and charac- terization of the relapse-seeding clone/s will help to monitor disease and to choose an appropriate treat- ment. However, due to intra-tumor heterogeneity, tumor biopsies may fail to identify the most aggres- sive 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 geographi- cally and temporally separated samples of stage M neuroblastoma patients (Abbasi et al. 2017. Clin. Cancer Research). In a single, well-characterized

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CHIMERIC ANTIGEN RECEPTOR (CAR)-BASED Figure 1

A B

Cytomegalovirus T cell

IMMUNOTHERAPY FOR TREATMENT OF REACTIVATION

OF CYTOMEGALOVIRUS INFECTION AFTER

STEM CELL TRANSPLANTATION

Membrane Reactivation of cytomegalovirus (HCMV) infection Membrane Figure 1. Schematics of

RESISTANCE OF HCMV-INFECTED CELLS gB

gB ­Cytomegalovirus (A) and of after hematopoietic stem cell transplantation is still TO T CELL CYTOTOXICITY DESPITE ­ Nucleocapsid CAR NucleocapsidTegument CAR the CAR-T cell approach (B). associated with substantial morbidity and mortal- HLA-INDEPENDENT TARGETING TegumentgH gB ity. Prophylactically and preemptively adminis- gH gB Crough et al. 2011 tered antiviral chemotherapy after transplantation In collaboration with our partner Armin Ensser gBgB frequently results in toxicity and selection of resist- (Institute of Virology, Universitätsklinikum Erlan- Crough et al. 2011 HCMV-infected cell ant virus variants. Adoptive transfer of HCMV-spe- gen, Germany) we have thus previously constructed cific memory T cells has been successfully applied, a CAR for targeting a conserved region in gB (Full HCMV-infected cell however, generation of specific T cells from seron- F et al. 2010), which is abundantly expressed on egative donors is difficult. We hypothesized that the surface of HCMV-infected cells and conserved HCMV could be targeted by adoptive transfer among different viral strains. Targeting of HCMV-in- Figure 2 of CAR-T cells in an HLA-independent manner, fected cells by CAR expressing T cells in an HLA-in- Figure 2. CAR-T cells do not because HCMV-infected cells display intact viral dependent manner is attractive, because it obviates A lyse HCMV-infected cells. A non-infected HCMV-infected (A) The histograms show the proteins such as glycoprotein B (gB) on their surface. the need for enriching antigen-specific memory T HFF HFF 293T 293T-gB expression of gB in fibroblasts 350 450

cells and circumvents immune evasion by impaired 300 (HFF, infected or non-infected) 250 400 300

250 and in 293T cells transduced or

HCMV is a complex virus with possibly 750 trans- antigen presentation. Originally, this approach has 350 250 200

300 non-transduced with a gB-en- lational products encoded by its genome. Since been proposed for the treatment of HIV and was 200 200 150 250 coding vector. 150 150 many of these products are highly immunogenic, tested in clinical phase II trials finally. Recent appli- 200 (B) Shown is the lytic activity 100 100 150 HCMV triggers immune responses from all arms of cations of a CAR-T cell approach also for fighting 100 of αCD3/αCD28-expanded T 50 100 50 50 counts cells transfected with either the immune system and leads to a high frequency Hepatitis B and C have shown promising results 50 0 0 0 0 the gB-specific CAR or an of responding HCMV-specific CD8pos and CD4pos both in vitro and in vivo in a preclinical model. 102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105 irrelevant CAR specific for gB T cells (up to 40% of the whole T cell repertoire). the carcinoembroynic antigen Despite this strong T cell reaction HCMV persists Interestingly and unexpectedly, however, when (CEA) by electroporation of isotypeisotype specific antibody and establishes lifelong latency, possibly explained we tested polyclonally activated T cells expressing CAR-encoding mRNA. Lytic activity of these CAR-T cells by the fact that a large proportion of its genome the gB-specific CAR, we found that these T cells B B 80 80 (effector:target ratio 25:1) was encodes for RNAs and proteins interfering with the could not directly eliminate cells infected with determined one day after elec- antiviral immune response. One of the most import HCMV (Figure 2). We have observed this with two 70 70 troporation using HFF (non-in- and most intensively studied defense mechanism of well-characterized HCMV laboratory strains Ad169 60 60 fected or 4 days after infection with AD169, MOI 5) and 293T HCMV is the prevention of recognition of infected and Towne and also after prolonged co-culture of 50 50 cells transduced or non-trans- cells by T and NK cells and by antibodies. Like many infected and effector cells. In order to exclude that 40 40 duced with gB (filled symbols: other viruses, HCMV escapes recognition by T cells the observed lack of lysis of HCMV-infected fibro- infected HFF or gB-transduced specific lysis [ % ] specific lysis 30 30 293T cells; empty symbols: through interfering with antigen processing and blasts was due to inhibition of T cell activation, we 20 20 non-infected HFF or non-trans- inhibiting antigen presentation by MHC class I and determined the level of T cell degranulation and duced 293T; three donors). II molecules on the surface of infected cells. cytokine production. However, we clearly found that 10 10 the CAR triggers degranulation as well as release of 0 0 cytokines IFN-g and TNF in the T cells. In order to -10 gB-CAR CEA-CAR -10 gB-CAR CEA-CAR further exclude that any possible defective function 293T293T cells FibroblastsFibroblasts of our gB-specific CAR was responsible for +/-+/- gB-transfection gB-transfection +/-+/- HCMV-infection HCMV-infection the observed lack of lysis, we switched to a

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A Figure 3 Figure 4 * * A 100100 100 9090 A 8080 80 7070 100 %

90 cells [%] 60 6060 + 80 50 CAR-independent experimental setup. For this 50 70 Figure 4. Viral proteins inhibit

% 40 purpose we employed EBV-peptide-specific cyto- 4040 60 normalized CAR-T cell mediated apoptosis 3030 50 in infected cells. The diagram specific lysis [ % ] specific lysis toxic lymphocytes, which were directed via their Annexin V 20 2020 40 shows the induction of cell death TCR to HLA-matched fibroblasts loaded with a 30 in fibroblasts expressing UL36 1010 0 ­saturating concentration of the same peptide used 20 and/or UL37x1 after co-incu- 0 - UL36 for the enrichment of the cells from PBMC. In addi- 10 - + - + bation with αCD3/αCD28-acti- d0d0 0 d1 d2d2 d3 d4 p.i. vated T cells expressing a CAR tion, we used a recombinant variant of the HCMV- - - + + - UL37x1 d0 d1 d2 d3 d4 p.i. directed against NKG2D-ligands strain Ad169 (Ad169∆US2-11), which contained + CMA (3 different T cell donors). extended deletions in the US-gene region in order B HFF HFF + chNKG2D T cells The percentages of apoptotic B B fibroblasts obtained in the to prevent down-regulation of MHC class I mole- 80 co-cultures of CAR-T cells plus cules during infection. Also this system reproduced fibroblasts without UL36/UL37x1 our previous observation of striking resistance of 8060 80 expression were set to 100%. HCMV-infected cells to lysis despite strong activa- release of infectious HCMV particles increasing in Concanamycin A (CMA, 100 nM) 60 was used to block perforin-in- tion of the T cells (Figure 3). Further we could show 6040 vitro until day 5 after infection. In order to prevent duced apoptosis. in this system that lysis inhibition of the peptide- termination of such slow replication by suicide of

IFN-y [ ng/ml ] 40

loaded fibroblasts gradually increased in the course 40 IFN-y ng/ml its host cells, HCMV has integrated in its genome Importantly, this newly discovered mechanism, 20 of infection. Together, the data strongly suggested a IFN-y ng/ml 20 a whole array of repressors blocking the host cell however, does not totally preclude anti-viral efficacy so far unknown immune escape mechanism, which death machinery at several points. of our HLA-independent approach. For example, 200 is independent from abrogating antigen-presentation 0 there could exist non-cytotoxic effects of granzymes d0 d1 d0 d2 d1 d3 d2 d4d3 p.i. d4 p.i. and thereby inhibiting T cell activation but instead 0 We hypothesized that some of these anti-suicide as, e.g., granzyme M, which cleaves cellular and directly blocks the cytotoxic effector functions of the d0 d1 d2 d3 d4 p.i. mechanisms could also inhibit T cell cytotoxicity viral proteins with essential function for HCMV peptide-pulsed Donor A , B , C T cells. and we thus investigated the role of UL37x1 and replication. Moreover, only recently, we could show non-pulsed Donor A , B , C UL36, which were already known to block death that the cytokines IFN-γ and TNF triggered by the peptide-pulsed Donor A , B , C receptor-mediated apoptosis and to inhibit cell gB-specific CAR in the T cells can efficiently inhibit non-pulsed Donor A , B , C HCMV SHIELDS ITS HOST CELLS FROM death induction at different levels. Indeed, our data the replication of HCMV. Meanwhile, we have repro- T CELL CYTOTOXICITY BY VIRALLY ENCODED Figure 3. HCMV infection confirmed our hypothesis, because transfection duced this effect also with a bispecific antibody ANTI-APOPTOTIC PROTEINS UL36 AND UL37x1 results in gradually increasing of UL36 and UL37x1 in combination and even of directed against gB, which we have developed as a resistance of host cells to In search for a possible explanation for the observed UL37x1 alone into fibroblasts significantly inhib- further step towards clinical translation. Given the cytotoxic effector functions. resistance to lysis we focused on previously Peptide-specific cytotoxic ited the lysis of the cells by CAR-T cells (Figure 4). strong toxic side-effects and the occurrence described anti-apoptotic mechanisms, which so far lymphocytes were co-cultu- Notably, a major fraction of CAR-T cell cytotoxicity of resistance in the current treatment of HCMV have not yet been linked to evasion of T cell cytotox- red with fibroblasts charged appeared to be mediated by the perforin/granzyme reactivation, our approach would be particularly or non-charged with peptide icity. HCMV is a particular target of the most ances- at different time points after pathway, since addition of concanamycin A strongly attractive in the high-risk constellation of an HCMV tral antiviral defense mechanism, namely premature infection with a mutant variant inhibited cell death induction. These data finally seronegative donor and an HCMV seropositive elimination of infected cells by programmed cell of AD169 deficient in downregu- proved that HCMV efficiently shields its infected transplant patient. death. This is a consequence of its slow replication lation of HLA molecules (AD169 host cells from T cell attack not only by preventing ΔUS2-11, MOI 5). Shown is the cycle taking three days with sequentially ordered lysis of the fibroblasts (effec- recognition and T cell activation, but additionally Proff J, Walterskirchen C, Brey C, Geyeregger R, Full F, Ensser A, immediate-early, early and late phases of gene tor:target ratio 5:1) (A) and the also by directly interfering with cytotoxic effector Lehner M, Holter W. (2016). Cytomegalovirus-Infected Cells Resist secretion of IFN-γ (B). expression - a process which is paralleled by expo- functions through factors of the viral anti-suicide T Cell Mediated Killing in an HLA-Recognition Independent Manner. nentially increasing levels of viral protein and the machinery. Front Microbiol, 7: 844

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LARGE SCALE EUROPEAN TRIAL DEMONSTRATES Figure 1 SURVIVAL ADVANTAGE FOR HIGH RISK NEUROBLASTOMA PATIENTS RECEIVING HIGH DOSE BUSULPHAN AND MELPHALAN TREATMENT (A RANDOMISED PHASE 3 TRIAL OF THE SIOP EUROPE NEUROBLASTOMA GROUP (SIOPEN)).

Neuroblastoma, the commonest paediatric extra-cra- Eligibility and response was evaluated by 123I- nial solid tumour, is responsible for a large propor- mIBG scintigraphy assessing the primary tumour tion of deaths from cancer in childhood. High-risk and metastatic sites. In the case of mIBG nega- neuroblastoma, defined by metastatic disease over tive tumour, bone scintigraphy with 99mTc-hy- the age of 12 or 18 months and MYCN amplification droxy-methylenediphosphanate (MDP) scintigraphy (MNA) at any age remains associated with poor was carried out; CTor MRI scan of primary tumour long-term survival rates. High-dose chemotherapy and radiological visualisation of any other evaluable with haematopoietic stem cell rescue (HDT/SCR) disease; examination of bone marrow aspirates and improves event free survival (EFS) of patients with trephines at two sites and measurement of urinary high-risk neuroblastoma (HR-NBL); however which catecholamines. Patients were evaluated according regimen has the greatest patient benefit is under to the National Cancer Institute Common Toxicity investigation. Criteria (CTC, Version 2) and by Bearman toxic- ity grades for pulmonary toxicity, veno-occclusive The high-risk neuroblastoma trial (HR-NBL1/ disease (VOD) and haemorrhagic cystitis. Monitor- SIOPEN) opened in 2002 and has tested, a number ing for adverse event monitoring was continuous. of hypo­theses (Ladenstein et al., J Clin Oncol 2010, 28(21):3516–24 / Ladenstein et al., MAbs. 2013; 5(5): The trial was approved as required by national 801–9). Here we report results of the hypothesis regulatory authorities and by national as well as that HDT with busulphan and melphalan (BuMel) institutional ethical committees/review boards results in a superior EFS than HDT with carboplatin, in participating countries. Three year EFS from etoposide and melphalan (CEM). randomisation was the primary endpoint. Analyses were done by intention to treat, but a per-protocol approach was used for the safety analysis. The METHODS trial was registered with ClinicalTrials.gov Patients were enrolled and randomised by (number NCT01704716) and EudraCT (number 128 SIOPEN member institutions/hospitals in 2006-001489-17). 18 countries using the SIOPEN-R-NET web based, online remote data entry system (https://www. Trial efficacy results remained masked until release siopen-r-net.org/). Randomisation was carried out by the independent Data Monitoring and Safety just prior to HDT by minimisation balancing age at Committee (DMSC) and only the DMSC and the diagnosis, stage, MYCN amplification and national study statistician were aware of interim efficacy group between the arms. Tumour evaluations monitoring results. At the pre-planned interim were carried out at the time of trial enrolment, analysis of October 2010 the DMSC recommended after completion of induction, before HDT, following stopping randomisation as the Peto rule on EFS was HDT and at the end of therapy. met (p-value <0.001 on the primary endpoint).

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100 100 Figure 2 BUMEL CEM 80 80

10060 10060 BUMEL Stage 4 Stage CEM 8040 8040 Patients Events 5-yrs. EFS p-value Event Free Survival (%) Survival Free Event 20 (%) Survival Overall 20 Patients Events 5-yrs. OS p-value 60 BUMEL 262 158 41 [35-47] 0.0006 60 BUMEL 262 139 51 [44-57] 0.0010 CEM 264 190 29 [24-35]

Stage 4 Stage CEM 264 176 37 [31-43]

Stage 4 400 400 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 Patients Events 5-yrs. EFS p-value Event Free Survival (%) Survival Free Event 20 Follow-up (years) (%) Survival Overall 20 Patients Events Follow-up 5-yrs. (years) OS p-value FINDINGS The primary endpoint, EFS (95% CI) at 3 and 5 years BUMEL 262 158 41 [35-47] 0.0006 BUMEL 262 139 51 [44-57] 0.0010 Between 2002 and 2010, 1347 patients were enrolled was 50 [45-56%] and 45 [39-51%] in 296 patients 262 184CEM (0) 133 264(2) 118 (4) 190 102 (10) 29 [24-35] 85 (23) 70 (37) 51 (55) 38 (66) 25 (79) 262 CEM227 (0) 186 264 (2) 151 176(4) 129(13) 37 [31-43] 107 (28) 83 (46) 61(65) 47 (77) 31 (92) 0 264 150 (1) 97 (1) 87 (1) 73 (9) 56 (22) 49 (29) 39 (37) 30 (44) 23 (51) 0 264 205 (1) 186 (1) 128 (1) 98(10) 75 (25) 61 (33) 47(77) 34 (56) 25 (64) 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 and 676 were eligible for randomisation. Eligibil- randomised to BuMel versus 38 [32-43%] and Follow-up (years) Follow-up (years) ity for this randomisation included the completion 33 [28-39%] in 302 patients randomised to CEM 262 184 (0) 133 (2) 118 (4) 102 (10) 85 (23) 70 (37) 51 (55) 38 (66) 25 (79) 262 227 (0) 186 (2) 151 (4) 129(13) 107 (28) 83 (46) 61(65) 47 (77) 31 (92) of a multi-drug induction regimen (COJEC with or (p=0.0005). The post-hoc analysis of cumulative inci- 264 150 (1) 97 (1) 87 (1) 73 (9) 56 (22) 49 (29) 39 (37) 30 (44) 23 (51) 264 205 (1) 186 (1) 128 (1) 98(10) 75 (25) 61 (33) 47(77) 34 (56) 25 (64) without topotecan, vincristine and doxorubicin) and dence of relapses (CIR) was significantly lower with achievement of an adequate disease response after BuMel (52 [46-56%])) compared to CEM (63 [57-68%]) 100 100 induction (complete bone marrow response and at (p=0.0033), suggesting a better drug action on resid- 80 least a partial response at skeletal sites with three ual tumor cells with the BuMel regimen. 80 60 or fewer abnormal sites on 123iodine-metaiodoben- 10060 100 localised 8040 zylguanidine scintigraphy). In all randomised patients, independently of localised 8040

Patients Events 5-yrs. EFS p-value (%) Survival Overall Patients Events 5-yrs. OS p-value

randomised HDT, the 5yr-EFS and OS of the (%) Survival Free Event 6020 6020 BUMEL 34 7 79 [62-90] 0.1232 BUMEL 34 7 79 [61-90] 0.2938 CEM 38 14 62 [45-76] CEM 38 12 71 [53-83] 598 patients (88% of all eligible) were randomised 72 patients with localised stage MNA neuroblas- localised 0 400 40 to receive either BuMel or CEM as HDT. Supportive toma was significantly higher (71 [58-80%] and 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10

Patients Events 5-yrs. EFS p-value (%) Survival Overall Patients Events Follow-up 5-yrs. (years) OS p-value

Event Free Survival (%) Survival Free Event Follow-up (years) 20 care followed institutional guidelines. VOD manage- 75 [63-83%] respectively) compared to the 526 stage 20 BUMEL 34 7 79 [62-90] 0.1232 BUMEL 34 7 79 [61-90] 0.2938 34 29CEM (0) 28 (0) 38 28 (0) 14 27 (0) 62 [45-76] 26 (1) 21 (6) 17(10) 14 (13) 10 (17) 34 31CEM (0) 29 38 (0) 28 12(0) 27 (0) 71 [53-83] 26 (0) 26 (1) 21(6) 17 (10) 10 (17) 0 38 32 (0) 30 (0) 27 (1) 25 (3) 23 (4) 20 (6) 16 (10) 10 (16) 8 (18) ment was similar to published. Defibrotide was not 4 patients (35 [31-39%] and 44 [39-48%]) (both 0 38 3 1 (0) 27 (0) 25 (1) 23 (3) 20 (4) 18 (4) 15 (9) 9 (15) 7 (17) 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 recommended, because the study was designed p<0.0001). Follow-up (years) Follow-up (years) 34 31 (0) 29 (0) 28 (0) 27 (0) 26 (0) 26 (1) 21(6) 17 (10) 10 (17) before defibrotide was approved. Baseline char- 34 29 (0) 28 (0) 28 (0) 27 (0) 26 (1) 21 (6) 17(10) 14 (13) 10 (17) 38 3 1 (0) 27 (0) 25 (1) 23 (3) 20 (4) 18 (4) 15 (9) 9 (15) 7 (17) 38 32 (0) 30 (0) 27 (1) 25 (3) 23 (4) 20 (6) 16 (10) 10 (16) 8 (18) acteristics were the same in each group including In the age group of 12-18 months, 18 stage 4 patients length of induction treatments, additional TVD, time without MNA had a superior prognosis (5yr-EFS 72 of surgery and radiotherapy. All randomised patients [46-87%]) compared to those with MNA (5yr-EFS 32 were included in the analysis and no patients [18-47%]) (p=0.0114). The six infants <12 months of was significantly higher with CEM (CEM 10% vs. In multivariable analysis of all randomised patients, discontinued BuMel or CEM once commenced. Of age with stage 4 MNA tumours had a 5yr-EFS of 33 BuMel 5%) and central neurotoxicity tended to be CEM, stage 4, age >5 years, and failure to achieve the 598 randomised patients, 79 (13.2%) were subse- [4-67%]. higher with CEM (CEM 4% vs. 1% BuMel (seizures CR prior to HDT/SCR were identified as independent quently enrolled in immunotherapy randomisations, associated with fever and metabolic disturbances). unfavourable prognostic factors (Model 1), but not of these only 64 patients (11%) were randomised Severe toxicities (need for intensive care and toxic Twenty-two percent of patients receiving BuMel age 1.5-5 years or whether surgery was performed to ch14.18/CHO immunotherapy. The ch14.18/CHO deaths) were lower with BuMel (4%) compared to developed VOD with Bearman toxicity grades 1-3 before HDT. Stage 2 and 3 MNA patients strongly immunotherapy randomisations have been reported CEM (10%). BuMel had fewer grade 3 and 4 non-hae- compared to 8% receiving CEM. Only 4% of patients influence the response result in this multivariable elsewhere. The median age of patients at randomi- matological toxicities, but was associated with 22% who received BuMel and 1% with CEM developed analysis as patients in CR (38) prior to HDT/SCR sation was 3 years (range, 1 to 17 years). The median veno-occlusive disease Bearman grades 1-3 (4% grade 3 VOD. Severe VOD was the cause of death in had a very good prognosis (5yr-EFS 89 [74-95%]) follow-up time of patients since randomisation is grade 3) versus 9% with CEM (1% grade 3). Patients one patient on BuMel. The 5-year non relapse related whilst patients achieving a VGPR (20) or PR (13) had 7.2 years (IQR 5.3-9.2). receiving CEM had higher median numbers of days mortality 3 [1-6%] for BuMel and 4 [2-7%] for CEM. 5yr-EFS of 50 [27-69%] and 46[19-69%], respectively. with fever (CEM 7 [IQR, 3-102], BuMel 4 [3-6], and A multivariable analysis model limited to The 5yr-EFS (event free survival) and 5yr-OS (over- intravenous antibiotics (CEM 13 [IQR, 7-14], BuMel randomised stage 4 patients showed CEM, age >5 all survival) for the entire 1347 population were 10 [IQR, 7-14]). BuMel was associated with fewer years and involvement of more than one metastatic 33 [31-35%] and 43 [41-45%] respectively from enrol- CTC grade 3 and 4 non-haematological toxicities compartment (MC) at diagnosis as independent ment. (fever, stomatitis, nausea and vomiting, diarrhoea, unfavourable factors but not failure to achieve CR reduced cardiac function, raised serum creatinine prior to HDT/SCR, MNA , age 1.5- 5 years. and reduced GFR). Bearman pulmonary toxicity

74 75 Science Reports

INTERPRETATION FUNDING SUPPORT PUBLICATION ON THE This randomised trial has evaluated a critical European Commission 5th Frame Work Grant HR-NBL1/SIOPEN TRIAL: component of high risk neuroblastoma therapy and (SIOPEN-R-NET EC grant No. QLRI-CT-2002-01768, Ladenstein R, Pötschger U, Pearson ADJ, et al. For compared BuMel with CEM. After COJEC induction, www.siopen-r-net.org), Pierre Fabre Médicament the SIOP Europe Neuroblastoma Group (SIOPEN). BuMel significantly improved EFS in children with providing Busilvex® and the St. Anna Kinder- (2017). Busulfan and melphalan versus carboplatin, HR-NBL with an adequate response to induction krebsforschung. Neither the European Commission etoposide, and melphalan as high-dose chemo­ therapy and induced less severe toxicities. nor Pierre Fabre had a role in study design, data therapy for high-risk neuroblastoma (HR-NBL1/ BuMel showed an advantage in patients with stage collection, data analysis, data interpretation or SIOPEN): an international, randomised, multi-arm, 4 metastatic disease and less than 5 years. The writing of the report. All authors participated in open-label, phase 3 trial. Lanct Oncology, Volume 18, improved survival of patients receiving BuMel was the report and had full access to all the data in the No. 4 supported by the post-hoc analysis demonstrating study. The corresponding author had final responsi- that the cumulative incidence of relapse (CIR) was bility for the decision to submit for publication. Ladenstein R, Poetschger U, Gray J, et al. (2016). significantly lower with BuMel. The outcome of Toxicity and outcome of anti-GD2 antibody ch14.18/ patients with stage 2 and 3 MNA appeared to be CHO in front-line, high-risk patients with neuro­ improved (79% 5yr-EFS) compared to approaches blastoma: Final results of the phase III immuno­ without HDT/SCR. therapy randomisation (HR-NBL1/SIOPEN trial). ASCO Annual Meeting Abstracts. ASCO Annual BuMel was found tolerable in spite of more frequent Meeting, J Clin Oncol 34, (suppl; abstr 10500.) 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.

BuMel is now considered standard HDT/SCR for SIOPEN and ongoing randomised SIOPEN studies will continue to optimise therapy for high-risk neuro- blastoma.

76 77 CAREER UNIV.-PROF. DR. RUTH LADENSTEIN S2IRP: Studien und Statistik Career

WORKING AT CCRI IF YOU WANT TO APPLY FOR A POSITION

A SHARED MISSION MAKES The CCRI offers an intellectually stimulating please proceed according to the details given A DIFFERENCE IN RESEARCH! environ ­ment with lots of opportunities for s­ cientific on our web site. The Children’s Cancer Research Institute (CCRI) exchange and discussions in regular seminars, is an internationally renowned biomedical research retreats, and national and international conference http://science.ccri.at institute, dedicated to the advancement of diagno- attendances. The quality of our work is p­ eriodically sis, prognosis and treatment of childhood cancer. reviewed by a panel of top international experts Science at the CCRI addresses a broad range guaranteeing consistently high standards of of topics, covered by 12 research groups whose CCRI research. PhD students are accompanied members work closely t­ogether and in ­cooperation by PhD-committees to guide them through their with clinicians from St. Anna Children’s Hospital. thesis and postdocs have the option to exercise We are at the forefront of international pediatric their ability to teach by mentoring and training cancer research and scientists at CCRI are striving younger colleagues. to make the most innovative and effective therapies available to young patients.

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 i­ntellectual development while at the same time introducing them to the world of science and academic competition.

82 83 Career

SCIENTIFIC STAFF

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

84 85 UNIV.-DOZ. DR. MICHAEL DWORZAK Immunologische Diagnostik FINANZBERICHT Finanzbericht

RICHTLINIEN ZUR SPENDENVERWENDUNG

ie St. Anna Kinderkrebsforschung SPENDENGÜTESIEGEL UND QUALITÄTSSICHERUNG DER wird zum über­wiegenden Teil STEUERLICHE ABSETZBARKEIT WISSENSCHAFTLICHEN ARBEIT durch private Spenden finanziert. Seit dem Jahr 2002 trägt die St. Anna Kinderkrebs­ Das Forschungsinstitut verfügt über ein ­Scientific Für den Betrieb des Forschungs­ forschung als eine der ersten Organisationen Advisory Board – ein Gremium aus externen Exper- institutes werden jährlich mehr als ­Öster ­reichs das Spendengütesiegel der Kammer ten – mit der Aufgabe der laufenden Evalu­ierung sieben M­ illionen Euro benötigt, der der ­Wirtschaftstreuhänder. Für die jährliche Neu­ der wissenschaftlichen Arbeiten und B­ eratung der Verein verfügt jedoch über keine Basisfinanzie- verleihung führt ein Wirtschaftsprüfer zusätzlich Institutsleitung. Darüber hinaus werden regelmäßig Drung durch die öffentliche Hand. Zusätzliche Mittel eine Prüfung der transparenten und ordnungs­­­ge­ neue wissenschaftliche Projekte bei renommierten werden im Rahmen von kompetitiv ausgeschriebe- mäßen Verwendung der Mittel gemäß den strengen forschungsfördernden nationalen und internatio­ nen P­ rojektförderungen von anerkannten nationalen ­Richt ­linien des Spendengütesiegels durch. nalen Stellen eingereicht und Forschungsergebnisse und internationalen Stellen akquiriert. in international anerkannten, wissenschaftlichen Auf Grundlage eines von der ­Finanzlandes- ­Journalen publiziert. In regelmäßigen Abständen Wir fühlen uns unseren Spenderinnen und Spen- ­direk ­tion für Wien erlassenen Bescheides zählt ­findet zusätzlich eine o­ bjektive Beurteilung der dern gegenüber zu einer sparsamen und effizienten die St. Anna Kinderkrebsforschung zum begünstig- wissenschaftlichen Leistung durch ausgewiesene Verwendung der uns anvertrauten Gelder ver­- ten Empfänger­kreis, sodass Spenden sowohl externe Fachleute auf dem Gebiet statt. pflichtet. Aus diesem Grund verwenden wir weniger von der Lohnsteuer als Sonderausgabe, als auch als 10 % für die Verwaltung und das Fundraising, von der Einkommensteuer als Betriebsausgabe das bedeutet: mehr als 90 % der Spenden fließen ­steuerlich absetzbar sind. direkt in die Forschung.

Der Jahresabschluss wird gemäß den Bestimmun- gen des Vereinsgesetzes für große Vereine erstellt, wobei die gleichen Richtlinien wie für Kapitalge- sellschaften 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.

90 91 UNIV.-DOZ. DR. GERHARD FRITSCH Klinische Zellbiologie Finanzbericht

MITTELHERKUNFT MITTELVERWENDUNG

2015 2016 2015 2016

I. Spenden I. Leistungen für die statutarisch festgelegten Zwecke € 10.950.509,20 € 10.645.229,50 a) ungewidmete € 0,00 € 0,00 II. Spendenwerbung € 598.537,96 € 695.899,94 b) gewidmete € 13.202.063,51 € 7.831.440,93 III. Verwaltungsaufwand € 346.652,69 € 359.480,99 II. Mitgliedsbeiträge € 0,00 € 0,00 IV. Sonstiger Aufwand sofern nicht unter € 2.639,13 € 60.763,57 III. Betriebliche Einnahmen Punkt I bis III festgehalten a) betriebliche Einnahmen aus öffentlichen Mitteln € 1.606.752,78 € 1.480.409,97 b) sonstige betriebliche Einnahmen € 2.740.734,54 € 2.567.045,14 V. Zuführung zu Passivposten für noch nicht € 6.092.757,29 € 674.587,50 widmungsgemäß verwendete Spenden bzw. Subventionen IV. Subventionen und Zuschüsse der öffentlichen Hand € 0,00 € 0,00 VI. Zuführung zu Rücklagen € 0,00 € 0,00 V. Sonstige Einnahmen a) Vermögensverwaltung € 0,00 € 0,00 VII. Jahresüberschuss € 0,00 € 0,00 b) sonstige andere Einnahmen sofern nicht € 441.545,44 € 557.065,46 in Punkt I bis IV festgehalten TOTAL € 17.991.096,27 € 12.435.961,50

VI. Auflösung von Passivposten für noch nicht € 0,00 € 0,00 widmungsgemäß verwendete Spenden bzw. JAHRESERGEBNIS € 0,00 € 0,00 Subventionen

VII. Auflösung von Rücklagen € 0,00 € 0,00 VIII. Jahresverlust € 0,00 € 0,00

TOTAL € 17.991.096,27 € 12.435.961,50

94 95 ANHANG Anhang

WISSENSCHAFTLICHER BEIRAT

PROF. DR. KLAUS-MICHAEL DEBATIN PROF. MEGAN SYKES, MD Universitätsklinik für Columbia University Medical Center Kinder- und Jugendmedizin New York, NY 10032, USA 89075 Ulm, Germany PROF. JOSEF VORMOOR, MD PROF. JAMES R. DOWNING, MD Newcastle Cancer Centre at the Scientific Director Northern Institute for Cancer Research St. Jude Children’s Research Hospital Newcastle University, Paul O’Gorman Building, Memphis, TN 38105-3678, USA Medical School, Framlington Place Newcastle upon Tyne, NE2 4HH PROF. LEE J. HELMAN, MD Scientific Director for Clinical Research Center for Cancer Research National Cancer Institute, National Institutes of Health Bethesda, MD 20892-2440, USA

PROF. STEPHAN LADISCH, MD Bosworth Chair for Cancer Biology Center for Cancer and Immunology Research Children’s National Medical Center Washington, DC 20010, USA

98 99 UNIV.-PROF. DDR. THOMAS LION Molekulare Mikrobiologie Anhang

INTERNATIONAL FREMDGEFÖRDERTE PROJEKTE

The interplay of NOTCH and MAPK pathway in LCH Molecular mechanisms of human fungal pathogen Analysing and Striking the Sensitivities of Principal investigator: Raphaela Schwentner host interaction (ImResFun) Embryonal Tumours (ASSET) Grant from the Histiocytosis Association CCRI partner: Thomas Lion CCRI partner: Heinrich Kovar Duration: 01/01/2016 to 31/12/2016 Coordinator: Karl Kuchler (Medical University of Coordinator: Walter Koch (University College Dublin, Vienna, Austria) Ireland) Optimized diagnostics for improved treatment European Commission Grant – FP7 Initial Training European Commission Grant – FP7 Cooperation stratification in invasive fungal diseases Network (Marie Curie Actions) Project (FUNGITECT) N°: 606786 | Duration: 01/10/2013 to 30/09/2017 N°: 259348 | Duration: 01/11/2010 to 30/04/2016 Coordinator: Thomas Lion (CCRI/Labdia) European Commission Grant – FP7 Cooperation Expert paediatric oncology reference network for PanCare childhood and adolescent cancer survivor Project diagnostics and treatment (ExPO-r-Net) care and follow-up studies (PanCareSurFup) N°: 602125 | Duration: 01/02/2014 to 31/01/2019 Coordinator: Ruth Ladenstein (CCRI) CCRI/St Anna Spital partners: Eva Frey Grant from the European Consumers, Health, Coordinator: Lars Hjorth (Lund University, Sweden) Automation of flow cytometric analysis for Agriculture and Food Executive Agency (CHAFEA) European Commission Grant – FP7 Cooperation quality-assured follow-up assessment to guide N°: 20131207 | Duration: 01/03/2014 to 31/08/2017 Project curative therapy for acute lymphoblastic leukaemia N°: 257505 | Duration: 01/02/2011 to 31/01/2017 in children (AutoFLOW) Targeted modulation of immune-system responses Labdia/CCRI partner: Michael Dworzak in cell therapies (MODICELL) Coordinator: Martin Kampel (Technical University of Coordinator: Originally Andreas Heitger (5 May Vienna, Austria) 2014), presently Wolfgang Holter European Commission Grant – FP7 Industry- European Commission Grant – FP7 Industry- Academia Partnerships and Pathways Academia Partnerships and Pathways (Marie Curie Actions) (Marie Curie Actions) N°: 610872 | Duration: 01/02/2014 to 31/01/2018 N°: 285875 | Duration: 01/01/2013 to 31/12/2016

EURO EWING Consortium – International clinical International study for treatment of childhood trials to improve survival from Ewing sarcoma (EEC) relapsed ALL 2010 (IntReALL) CCRI partner: Heinrich Kovar CCRI partners: Georg Mann, Andishe Attarbaschi Coordinator: Jeremy Whelan (University College and Ruth Ladenstein London, UK) Coordinator: Jeremy Whelan (University College European Commission Grant – FP7 Cooperation London, UK) Project European Commission Grant – FP7 Cooperation N°: 602586 | Duration: 01/10/2013 to 31/09/2018 Project N°: 278514 | Duration: 01/10/2011 to 30/09/2017

102 103 Einleitung

NATIONAL FREMDGEFÖRDERTE PROJEKTE

Overcoming Neuroblastoma Tumour HETerogeneity, Myeloproliferative neoplasms Integrating entertainment and reaction assessment Liquid biopsy in neuroblastoma: chance for Resistance and RecurrAnCe_ (ONTHETRRAC) CCRI partner: Thomas Lion into child cancer therapy (INTERACCT) diagnosis, prognosis and disease monitoring CCRI responsible principal investigator: Coordinator: Peter Valent (Medical University CCRI/St Anna Spital partner: Anita Lawitschka CCRI responsible principal investigator: Peter Ambros of Vienna, Austria) Coordinator: Helmut Hlavacs (University of Peter Ambros Grant from the Austrian Science Fund (FWF), Grant from the Austrian Science Fund (FWF), Vienna, Austria) Grant from the Austrian National Bank (OeNB), ERA-Net Transcan Special Research Programme (SFB) Grant from the Austrian Research Promotion Agency Jubiläumsfonds N°: 2799 B28 | Duration: 01/01/2016 to 30/06/2018 N°: F4705-B20 | Duration: 01/03/2013 to 28/02/2017 (FFG), Call Bridge (Brückenschlagprogramm) N°: OeNB 16611 | Duration: 01/01/2016 to 31/12/2017 N°: 838594 | Duration: 01/05/2013 to 30/04/2016 Ewing sarcoma – an enhancer disease? Single molecule array platform for sensitive Permanent consequences in childhood Langerhans CCRI responsible principal investigator: diagnostics (SmardScout) Prä-klinische Entwicklung einer Off-the-Shelf cell histiocytosis Eleni Tomazou CCRI partner: Thomas Lion individualisierten Krebsimmuntherapie CCRI responsible principal investigator: Grant from the Austrian Science Fund (FWF), Coordinator: Jan Hesse (Center for Advanced (IN SITU DC-CIT) Milen Minkov Elise Richter Programme Bioanalysis GmbH, Linz, Austria) CCRI partner: Alexander Dohnal Grant from the Austrian National Bank (OeNB), N°: V 506 B28 | Duration: 01/04/2016 to 31/01/2021 Grant from the Austrian Research Promotion Coordinator: Wolfgang Schöfberger (University Jubiläumsfonds Agency (FFG), of Linz, Austria) Grant from the Austrian N°: 16431 | Duration: 01/09/2015 to 31/08/2017 TRANSCALL (Translational research in childhood Research Studios Austria, 4th Call Research Promotion Agency (FFG), Call Bridge acute lymphoblastic leukemia) N°: 844738 | Duration: 01/09/2014 to 22/11/2016 (Brückenschlagprogramm) Regulation of the MYCN oncogene by Nuclear CCRI responsible principal investigator: N°: 836532 | Duration: 01/10/2012 to 30/09/2016 Lamina Proteins upon therapy-induced senescence Renate Panzer-Grümayer Verfahren zur hochautomatisierten Bewertung in aggressive neuroblastoma Grant from the Austrian Science Fund (FWF), und Klassifikation von Zellen in Gewebeschnitten Virus-specific immunotherapy (VISIT) CCRI responsible principal investigator: ERA-Net Transcan anhand räumlicher Markerprofile (TisQuant) Labdia/CCRI: coordinator René Geyeregger Sabine Taschner-Mandl N°: I1226-B19 | Duration: 01/07/2013 to 30/06/2017 LABDIA responsible principal investigator: (Labdia), partner: Matthes-Leodolter (CCRI) Grant from the Herzfelder‘sche Familienstiftung Peter Ambros Grant from the Wirtschaftsagentur Wien, Duration: 01/05/2015 to 31/10/2016 PROVABES (prospective validation of biomarkers Grant from the Austrian Research Promotion Call Life Sciences 2014 in Ewing sarcoma for personalized treatment) Agency (FFG), ERA-SME N°: 1207846 | Duration: 01/04/2015 to 31/03/2018 Verbesserte Patientenkommunikation in der CCRI responsible principal investigator: N°: 844198 | Duration: 01/06/2014 to 31/05/2017 Onkologie mittels INTERACCT App(OCCURSUS) Heinrich Kovar Automated MRD-assessment in AML CCRI responsible principal investigator: Grant from the Austrian Science Fund (FWF), Directed in vitro differentiation of induced (flowCLUSTER) Anita Lawitschka ERA-Net Transcan pluripotent stem cells towards the B lymphoid Labdia coordinator: Michael Dworzak Grant from the „Österreichische Gesellschaft für N°: I1225-B19 | Duration: 01/04/2013 to 31/03/2017 lineage (B-different) Grant from the Wirtschaftsagentur Wien, Hämatologie und Medizinische Onkologie“ CCRI coordinator: Klaus Fortschegger Call Life Sciences 2014 Duration: 01/05/2016 to 31/07/2017 Grant from the Austrian Research Promotion N°: 1207843 | Duration: 01/03/2015 to 28/02/2018 Agency (FFG), Call Bridge (Brückenschlagprogramm) N°: 843456 | Duration: 01/06/2014 to 22/12/2017

104 105 DR. SABINE STREHL Leukämiegenetik Anhang

DANKSAGUNG DIPLOM(MASTER)ARBEITEN / DISSERTATIONEN 2016

Wir möchten uns an dieser Stelle gerne bei unseren • 7. Forschungsrahmenprogramm der REZA ABBASI zahlreichen privaten Spenderinnen und Spendern Europäischen Kommission (FP7) Genomic Analysis of Bone Marrow-Derived bedanken, die uns seit vielen Jahren treu unter­ • 3rd Health Programme of the European Disseminated Neuroblastoma Cells stützen. Des Weiteren bedanken wir uns bei den Commission Supervised by Assoc.-Prof. Peter F. Ambros, PhD folgenden nationalen und internationalen Förder­ • Bundesministerium für Wissenschaft, PhD Thesis gebern und Organisationen: Forschung und Wirtschaft (BMWFW) • Fonds zur Förderung der wissenschaftlichen STEFANIE ANDERL Forschung (FWF) The role of PAX5 fusion genes in the • Österreichische pathogenesis of childhood B-cell precursor Forschungsförderungsgesellschaft (FFG) acute lymphoblastic leukemia. • Österreichische Nationalbank (OeNB) Supervised by Sabine Strehl, PhD and • Wirtschaftsagentur Wien Klaus Fortschegger, PhD. • Histiocytosis Association PhD Thesis • Herzfelder’sche Familienstiftung • Gigax Privatstiftung FL LARISSA KOLLER • Kapsch AG Generierung von BCR-ABL1-exprimierenden • Österreichische Gesellschaft für Hämatologie & Zelllinien zur Untersuchung der Ph+ Leukämie Medizinische Onkologie (ÖGHO) mutierten Subklon-Evolution • Österreichischen Gesellschaft für Supervised by Dr. Konstantin Byrgazov, PhD and Kinder- und Jugendheilkunde (ÖGKJ) Univ.-Prof. Thomas Lion, MD, PhD • Dachverband der Österreichischen Bachelor Thesis Kinder-Krebs-Hilfe • Medac Gesellschaft für klinische VANESSA MAYR Spezialpräparate mbH, Deutschland Dynamic in vivo MAP kinase reporters to study • Verein für Dermatologie, Wien 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

108 109 UNIV.-DOZ. DR. PETER F. AMBROS Tumorbiologie Anhang

PUBLIKATIONEN 2016

Araki A, Chocholous M, Gojo J, Dorfer C, Czech T, Bogen D, Brunner C, Walder D, Ziegler A, Abbasi R, Byrgazov K, Kastner R, Gorna M, Hoermann G, Crazzolara R, Kropshofer G, Haas OA, Matthes-­ Heinzl H, Dieckmann K, Ambros IM, Ambros PF, Ladenstein RL, Noguera R, Martinsson T, Amann G, Koenig M, Lucini CB, Ulreich R, Benesch M, Strenger Martin S, Kager L. (2017, Epub 2016 Dec 7). Slavc I, Haberler C. (2016). Chromosome 1q gain Schilling FH, Ussowicz M, Benesch M, Ambros PF, V, Lackner H, Schwinger W, Sovinz P, Haas OA, van Reduced-intensity conditioning and stem cell trans- and tenascin-C expression are candidate markers Ambros IM. (2016). The genetic tumor background den Heuvel-Eibrink M, Niemeyer CM, Hantschel O, plantation in infants with Diamond Blackfan anemia. to define different risk groups in pediatric posterior is an important determinant for heterogeneous Valent P, Superti-Furga G, Urban C, Dworzak MN, Haematologica, 102: e73-e75 fossa ependymoma. Acta Neuropathol Commun, MYCN-amplified neuroblastoma. Int J Cancer, Lion T. (2017, Epub 2016 Oct 7). NDEL1-PDGFRB 4: 88 139: 153-163 fusion gene in a myeloid malignancy with eosino- Creutzig U, Rössig C, Dworzak M, Stary J, von Stack- philia associated with resistance to tyrosine kinase elberg A, Wössmann W, Zimmermann M, Reinhardt Berbegall AP, Villamon E, Piqueras M, Tadeo I, Boztug H, Hirschmugl T, Holter W, Lakatos K, inhibitors. Leukemia, 31: 237-240 D. (2016). Exchange Transfusion and Leukapheresis Djos A, Ambros PF, Martinsson T, Ambros IM, Kager L, Trapin D, Pickl W, Förster-Waldl E, in Pediatric Patients with AML With High Risk of Canete A, Castel V, Navarro S, Noguera R. (2016). Boztug K. (2016). NF-kappaB1 Haploinsufficiency Byrgazov K, Lucini CB, Berkowitsch B, Koenig M, Early Death by Bleeding and Leukostasis. Pediatr Comparative genetic study of intratumoral hetero­ Causing Immunodeficiency and EBV-Driven Lympho­ Haas OA, Hoermann G, Valent P, Lion T. (2016). Blood Cancer, 63: 640-645 genous MYCN amplified neuroblastoma versus proliferation. J Clin Immunol, 36: 533-540 Transposon-mediated generation of BCR-ABL1-­ aggressive genetic profile neuroblastic tumors. expressing transgenic cell lines for unbiased Creutzig U, Zimmermann M, Reinhardt D, Rasche M, Oncogene, 35: 1423-1432 Boztug H, Mühlegger N, Pötschger U, Attarbaschi sensitivity testing of tyrosine kinase inhibitors. von Neuhoff C, Alpermann T, Dworzak M, ­Perglerova A, Peters C, Mann G, Dworzak M. (2017, Epub 2016 Oncotarget, 7: 78083-78094 K, Zemanova Z, Tchinda J, Bradtke J, Thiede C, Bileck A, Mayer RL, Kreutz D, Weiss T, Taschner- Oct 4). Antibiotic prophylaxis with teicoplanin on Haferlach C. (2016). Changes in cytogenetics and Mandl S, Meier SM, Slany A, Gerner C. (2017, Epub alternate days reduces rate of viridans sepsis and Chagtai T, Zill C, Dainese L, Wegert J, Savola S, molecular genetics in acute myeloid leukemia from 2016 Nov 13). Evaluation of inflammation-related febrile neutropenia in pediatric patients with acute Popov S, Mifsud W, Vujanic G, Sebire N, Le Bouc Y, childhood to adult age groups. Cancer, 122: 3821- signaling events covering phosphorylation and myeloid leukemia. Ann Hematol, 96: 99-106 Ambros PF, Kager L, O'Sullivan MJ, Blaise A, 3830 nuclear translocation of proteins based on mass Bergeron C, Mengelbier LH, Gisselsson D, Kool M, spectrometry data. J Proteomics, 152: 161-171 Brunner C, Brunner-Herglotz B, Ziegler A, Frech C, Tytgat GA, van den Heuvel-Eibrink MM, Graf N, Czurda S, Lion T. (2017, Epub 2016 Nov 12). Amann G, Ladenstein R, Ambros IM, Ambros PF. van Tinteren H, Coulomb A, Gessler M, Williams RD, Broad-Spectrum Molecular Detection of Fungal Boer JM, van der Veer A, Rizopoulos D, Fiocco M, (2016). Tumor Touch Imprints as Source for Whole Pritchard-Jones K. (2016). Gain of 1q As a P­ rognostic Nucleic Acids by PCR-Based Amplification Tech- Sonneveld E, de Groot-Kruseman HA, Kuiper RP, Genome Analysis of Neuroblastoma Tumors. PLoS Biomarker in Wilms Tumors (WTs) Treated With niques. Methods Mol Biol, 1508: 257-266 Hoogerbrugge P, Horstmann M, Zaliova M, Palmi C, One, 11:8 Preoperative Chemotherapy in the International Trka J, Fronkova E, Emerenciano M, do Socorro Society of Paediatric Oncology (SIOP) WT 2001 Trial: Czurda S, Lion T. (2017, Epub 2016 Nov 12). Pombo-de-Oliveira M, Mlynarski W, Szczepanski T, Burchill SA, Beiske K, Shimada H, Ambros PF, A SIOP Renal Tumours Biology Consortium Study. Prerequisites for Control of Contamination in Fungal Nebral K, Attarbaschi A, Venn N, Sutton R, Schwab Seeger R, Tytgat GA, Brock PR, Haber M, Park JR, J Clin Oncol, 34: 3195-3203 Diagnosis. Methods Mol Biol, 1508: 249-255 CJ, Enshaei A, Vora A, Stanulla M, Schrappe M, Berthold F. (2017, Epub 2016 Dec 16). Recommenda- Cazzaniga G, Conter V, Zimmermann M, Moorman tions for the standardization of bone marrow Colomer-Lahiguera S, Strehl S. (2016). Complexity­ Czurda S, Smelik S, Preuner-Stix S, Nogueira F, AV, Pieters R, den Boer ML. (2016). Prognostic value disease assessment and reporting in children with of NOTCH1 juxtamembrane insertion mutations Lion T. (2016). Occurrence of Fungal DNA Contami- of rare IKZF1 deletion in childhood B-cell precursor neuroblastoma; on behalf of the International Neuro- in T-cell acute lymphoblastic leukemia. Leuk nation in PCR Reagents: Approaches to Control and acute lymphoblastic leukemia: an international blastoma Response Criteria Bone Marrow Working Lymphoma, 57: 1219-1222 Decontamination. J Clin Microbiol, 54: 148-152 collaborative study. Leukemia, 30: 32-38 Group. Cancer, 123: 1095-1105

112 113 Anhang

Dittrich C, Kosty M, Jezdic S, Pyle D, Berardi R, Dittrich C, Kosty M, Jezdic S, Pyle D, Berardi R, Bergh J, El Saghir N, Lotz JP, Österlund P, Pavlidis N, Bergh J, El-Saghir N, Lotz JP, Österlund P, Pavlidis Ebinger S, Özdemir EZ, Ziegenhain C, Tiedt S, Grausenburger R, Bastelberger S, Eckert C, Kauer M, Purkalne G, Awada A, Banerjee S, Bhatia S, N, Purkalne G, Awada A, Banerjee S, Bhatia S, Castro Alves C, Grunert M, Dworzak M, Lutz C, Stanulla M, Frech C, Bauer E, Stoiber D, von Stackel- Bogaerts J, Buckner J, Cardoso F, Casali P, Chu E, Bogaerts J, Buckner J, Cardoso F, Casali P, Chu E, Turati VA, Enver T, Horny HP, Sotlar K, Parekh S, berg A, Attarbaschi A, Haas OA, Panzer-Grümayer Close JL, Coiffier B, Connolly R, Coupland S, De Close JL, Coiffier B, Connolly R, Coupland S, De Spiekermann K, Hiddemann W, Schepers A, Polzer B, R. (2016). Genetic alterations in glucocorticoid Petris L, De Santis M, de Vries EG, Dizon DS, Duff J, Petris L, De Santis M, de Vries EG, Dizon DS, Duff Kirsch S, Hoffmann M, Knapp B, Hasenauer J, Pfeifer signaling pathway components are associated with Duska LR, Eniu A, Ernstoff M, Felip E, Fey MF, J, Duska LR, Eniu A, Ernstoff M, Felip E, Fey MF, H, Panzer-Grümayer R, Enard W, Gires O, Jeremias adverse prognosis in children with relapsed ETV6/ Gilbert J, Girard N, Glaudemans AW, Gopalan PK, Gilbert J, Girard N, Glaudemans AW, Gopalan PK, I. (2016). Characterization of Rare, Dormant, and RUNX1-positive acute lymphoblastic leukemia. Leuk Grothey A, Hahn SM, Hanna D, Herold C, Grothey A, Hahn SM, Hanna D, Herold C, Herrstedt Therapy-Resistant Cells in Acute Lymphoblastic Lymphoma, 57: 1163-1173 Herrstedt J, Homicsko K, Jones DV Jr, Jost L, J, Homicsko K, Jones DV Jr, Jost L, Keilholz U, Khan Leukemia. Cancer Cell, 30: 849-862 Keilholz U, Khan S, Kiss A, Köhne CH, Kunstfeld R, S, Kiss A, Köhne CH, Kunstfeld R, Lenz HJ, Licht- Grelpois G, Sabbagh C, Cosse C, Robert B, Chapu- Lenz HJ, Lichtman S, Licitra L, Lion T, Litière S, man S, Licitra L, Lion T, Litière S, Liu L, Loehrer PJ, Ebner H, Hayn D, Falgenhauer M, Nitzlnader M, is-Roux E, Ntouba A, Lion T, Regimbeau JM. (2016). Liu L, Loehrer PJ, Markham MJ, Markman B, Markham MJ, Markman B, Mayerhoefer M, Meran Schleiermacher G, Haupt R, Erminio G, Defferrari Management of Uncomplicated Acute Appendicitis Mayerhoefer M, Meran JG, Michielin O, Moser JG, Michielin O, Moser EC, Mountzios G, Moynihan R, Mazzocco K, Kohler J, Tonini GP, Ladenstein R, as Day Case Surgery: Feasibility and a Critical EC, Mountzios G, Moynihan T, Nielsen T, Ohe Y, T, Nielsen T, Ohe Y, Öberg K, Palumbo A, Peccatori Schreier G. (2016). Piloting the European Unified Analysis of Exclusion Criteria and Treatment Öberg K, Palumbo A, Peccatori FA, Pfeilstöcker M, FA, Pfeilstöcker M, Raut C, Remick SC, Robson M, Patient Identity Management (EUPID) Concept to Failure. J Am Coll Surg, 223:694-703 Raut C, Remick SC, Robson M, Rutkowski P, Rutkowski P, Salgado R, Schapira L, Schernham- Facilitate Secondary Use of Neuroblastoma Data Salgado R, Schapira L, S­ chernhammer E, Schlum- mer E, Schlumberger M, Schmoll HJ, Schnipper from Clinical Trials and Biobanking. Stud Health Grevener K, Haveman LM, Ranft A, van den Berg berger M, Schmoll HJ, Schnipper L, Sessa C, L, Sessa C, Shapiro CL, Steele J, Sternberg CN, Technol Inform, 223: 31-38 H, Jung S, Ladenstein R, Klco-Brosius S, Juergens ­Shapiro CL, Steele J, Sternberg CN, Stiefel F, Stiefel F, Strasser F, Stupp R, Sullivan R, Tabernero H, Merks JH, Dirksen U. (2016). Management and Strasser F, Stupp R, Sullivan R, Tabernero J, J, Travado L, Verheij M, Voest E, Vokes E, Von Eder J, Kammerstatter M, Erhart F, Mairhofer-Muri Outcome of Ewing Sarcoma of the Head and Neck. Travado L, Verheij M, Voest E, Vokes E, Von Roenn J, Roenn J, Weber JS, Wildiers H, Yarden Y. (2016). D, Trautinger F. (2016). Illness Perception in Primary Pediatr Blood Cancer, 63: 604-610 Weber JS, Wildiers H, Yarden Y. (2016). ESMO/ASCO ESMO / ASCO Recommendations for a Global Cutaneous T-cell Lymphomas: What Patients Believe recommendations for a Global Curriculum (GC) in Curriculum in Medical Oncology Edition 2016. About Their Disease. Acta Derm Venereol, 96: Hartz B, Marsh R, Rao K, Henter JI, Jordan M, medical oncology-edition 2016. Ann Oncol, 27: 1378- ESMO Open, 1: e000097 381-385 Filipovich L, Bader P, Beier R, Burkhardt B, Meisel 1381 R, Schulz A, Winkler B, Albert MH, Greil J, Karasu Dmytrus J, Matthes-Martin S, Pichler H, Worel N, Eder J, Rogojanu R, Jerney W, Erhart F, Dohnal A, G, Woessmann W, Corbacioglu S, Gruhn B, Holter Geyeregger R, Frank N, Frech C, Fritsch G. (2016). Kitzwögerer M, Steiner G, Moser J, Trautinger F. W, Kühl JS, Lang P, Seidel MG, Veys P, Löfstedt A, Multi-color immune-phenotyping of CD34 subsets (2016). Mast Cells Are Abundant in Primary Ammann S, Ehl S, Janka G, Müller I, Lehmberg K. reveals unexpected differences between various Cutaneous T-Cell Lymphomas: Results from a (2016). The minimum required level of donor stem cell sources. Bone Marrow Transplant, 51: Computer-Aided Quantitative Immunohistological chimerism in hereditary hemophagocytic 1093-1100 Study. PLoS One, 11: e0163661 lymphohistiocytosis. Blood, 127: 3281-3290

Duregger K, Hayn D, Nitzlnader M, Kropf M, Falgen- Fritsch G, Frank N, Dmytrus J, Frech C, Pichler Hayn D, Falgenhauer M, Kropf M, Nitzlnader M, hauer M, Ladenstein R, Schreier G. (2016). Electronic H, Witt V, Geyeregger R, Scharner D, Trbojevic D, Welte S, Ebner H, Ladenstein R, Schleiermacher Patient Reported Outcomes in Paediatric Oncology - Zipperer E, Printz D, Worel N. (2016). Relevance of G, Hero B, Schreier G. (2016). IT Infrastructure for Applying Mobile and Near Field Communication flow cytometric enumeration of post-thaw leuco- Merging Data from Different Clinical Trials and Technology. Stud Health Technol Inform, 223: cytes: influence of temperature during cell staining Across Independent Research Networks. Stud 281-288 on viable cell recovery. Vox Sang, 111: 187-196 Health Technol Inform, 228: 287-291

114 115 UNIV.-PROF. DR. OSKAR HAAS Zytogenetik und molekulare Genetik Anhang

Kosulin K, Dworzak S, Lawitschka A, Matthes-Le- Kromp F, Ambros IM, Weiss T, Bogen D, Dodig H, He T, Surdez D, Rantala JK, Haapa-Paananen S, Kager L, Minkov M, Zeitlhofer P, Fahrner B, odolter S, Lion T. (2016). Comparison of different Berneder M, Gerber T, Taschner-Mandl S, Ambros Ban J, Kauer M, Tomazou E, Fey V, Alonso J, Kovar Ratzinger F, Boztug K, Dossenbach-Glaninger A, approaches to quantitative adenovirus detection in PF, Hanbury A. (2016). Machine Learning Frame- H, H, Delattre O, Iljin K. (2017, Epub 2016 Oct 17). Haas OA. (2016). Two Novel Missense Mutations stool specimens of hematopoietic stem cell trans- work incorporating Expert Knowledge in Tissue High-throughput RNAi screen in Ewing sarcoma and a 5bp Deletion in the Erythroid-Specific plant recipients. J Clin Virol, 85: 31-36 Image Annotation. Proceedings of the International cells identifies leucine rich repeats and WD repeat Promoter of the PKLR Gene in Two Unrelated Conference on Pattern Recognition, Cancun. domain containing 1 (LRWD1) as a regulator of Patients With Pyruvate Kinase Deficient Transfusion- Kosulin K, Geiger E, Vecsei A, Huber WD, Rauch EWS-FLI1 driven cell viability. Gene, 596: 137-146 Dependent Chronic Nonspherocytic Hemolytic M, Brenner E, Wrba F, Hammer K, Innerhofer A, Kuttke M, Sahin E, Pisoni J, Percig S, Vogel A, Anemia. Pediatr Blood Cancer, 63: 914-916 Pötschger U, Lawitschka A, Matthes-Leodolter S, Kraemmer D, Hanzl L, Brunner JS, Paar H, Soukup Heller G, Topakian T, Altenberger C, Cerny-Reiterer Fritsch G, Lion T. (2016). Persistence and reactiva- K, Halfmann A, Dohnal AM, Steiner CW, Blüml S, Herndlhofer S, Ziegler B, Datlinger P, Byrgazov K, Kager L, Whelan J, Dirksen U, Hassan B, Anninga J, tion of human adenoviruses in the gastrointestinal S, Basilio J, Hochreiter B, Salzmann M, Hoesel B, Bock C, Mannhalter C, Hörmann G, Sperr WR, Lion Bennister L, Bovee JV, Brennan B, Broto JM, tract. Clin Microbiol Infect, 22: 381.e1-8 Lametschwandtner G, Eferl R, Schmid JA, Schab- T, Zielinski CC, Valent P, Zöchbauer-Müller S. (2016). Brugieres L, Cleton-Jansen AM, Copland C, Dutour bauer G. (2016). Myeloid PTEN deficiency impairs Next-generation sequencing identifies major A, Fagioli F, Ferrari S, Fiocco M, Fleuren E, Gaspar Kovar H, Amatruda J, Brunet E, Burdach S, Cidre- tumor-immune surveillance via immune-checkpoint DNA methylation changes during progression of Ph+ N, Gelderblom H, Gerrand C, Gerß J, Gonzato O, Aranaz F, de Alava E, Dirksen U, van der Ent W, inhibition. Oncoimmunology, 5: e1164918 chronic myeloid leukemia. Leukemia, 30: 1861-1868 van der Graaf W, Hecker-Nolting S, Herrero-Martín Grohar P, Grünewald TG, Helman L, Houghton P, D, Klco-Brosius S, Kovar H, Ladenstein R, Lancia C, Iljin K, Korsching E, Ladanyi M, Lawlor E, Lessnick Lewington V, Lambert B, Poetschger U, Sever ZB, Hutter C, Minkov M. (2016). Insights into the LeDeley MC, McCabe MG, Metzler M, Myklebost S, Ludwig J, Meltzer P, Metzler M, Mora J, Moriggl R, Giammarile F, McEwan AJ, Castellani R, Lynch T, pathogenesis of Langerhans cell histiocytosis: the O, Nathrath M, Picci P, Potratz J, Redini F, Richter Nakamura T, Papamarkou T, Radic Sarikas B, Rédini Shulkin B, Drobics M, Staudenherz A, Ladenstein R. development of targeted therapies. Immunotargets GH, Reinke D, Rutkowski P, Scotlandi K, Strauss S, F, Richter GH, Rossig C, Schadler K, Schäfer BW, (2017, Epub 2016 Sep 24). 123I-mIBG ­scintigraphy Ther, 5: 81-91 Thomas D, Tirado OM, Tirode F, Vassal G, Bielack Scotlandi K, Sheffield NC, Shelat A, Snaar-Jagalska in neuroblastoma: development of a SIOPEN SS. (2016). The ENCCA-WP7/EuroSarc/EEC/PROVA- E, Sorensen P, Stegmaier K, Stewart E, Sweet-Cor- semi-quantitative reporting method by an inter- Javaheri T, Kazemi Z, Pencik J, Pham HT, Kauer BES/EURAMOS 3rd European Bone Sarcoma dero A, Szuhai K, Tirado OM, Tirode F, Toretsky J, national panel. Eur J Nucl Med Mol Imaging, 44: M, Noorizadeh R, Sax B, Nivarthi H, Schlederer M, Networking Meeting/Joint Workshop of EU Bone Tsafou K, Üren A, Zinovyev A, Delattre O. (2016). 234-241 Maurer B, Hofbauer M, Aryee DN, Wiedner M, Toma- Sarcoma Translational Research Networks; Vienna, The second European interdisciplinary Ewing zou EM, Logan M, Hartmann C, Tuckermann JP, Austria, September 24-25, 2015. Workshop Report. sarcoma research summit--A joint effort to decon- Li J, Casteels T, Frogne T, Ingvorsen C, Honore C, Kenner L, Mikula M, Dolznig H, Üren A, Richter GH, Clin Sarcoma Res, 6: 3 structing the multiple layers of a complex disease. Courtney M, Huber KV, Schmitner N, Kimmel RA, Grebien F, Kovar H, Moriggl R. (2016). Increased Oncotarget, 7: 8613-8624 Romanov RA, Sturtzel C, Lardeau CH, Klugham- survival and cell cycle progression pathways are Karim-Kos HE, Hackl M, Mann G, Urban C, Woehrer mer J, Farlik M, Sdelci S, Vieira A, Avolio F, Briand required for EWS/FLI1-induced malignant trans­ A, Slavc I, Ladenstein R. (2016). Trends in incidence, Kowalczyk JR, Samardakiewicz M, Pritchard-Jones F, Baburin I, Májek P, Pauler FM, Penz T, Stukalov formation. Cell Death Dis, 7: e2419 survival and mortality of childhood and adolescent K, Ladenstein R, Essiaf S, Fitzgerald E, Petrarulo G, A, Gridling M, Parapatics K, Barbieux C, Berish- cancer in Austria, 1994-2011. Cancer Epidemiol, 42: Vassal G. (2016). European Survey on Standards of vili E, Spittler A, Colinge J, Bennett KL, Hering S, Kager L, Bruce LJ, Zeitlhofer P, Flatt JF, Maia TM, 72-81 Care in paediatric oncology centres. Eur J Cancer, Sulpice T, Bock C, Distel M, Harkany T, Meyer D, Ribeiro ML, Fahrner B, Fritsch G, Boztug K, Haas 61: 11-19 Superti-Furga G, Collombat P, Hecksher-Sørensen J, OA. (2017, Epub 2016 Oct 8). Band 3 nullVIENNA, a Kosulin K, Berkowitsch B, Lion T. (2016). Modified­ Kubicek S. (2017, Epub 2016 Dec 1). Artemisinins novel homozygous SLC4A1 p.Ser477X variant caus- pan-adenovirus real-time PCR assay based on Target GABAA Receptor Signaling and Impair alpha ing severe hemolytic anemia, dyserythropoiesis and genome analysis of seventy HAdV types. J Clin Cell Identity. Cell, 168: 86-100 complete distal renal tubular acidosis. Pediatr Blood Virol, 80: 60-61 Cancer, 64: e26227

118 119 Anhang

Lion T. (2017, Epub 2016 Nov 12). Human Fungal Perwein T, Strehl S, König M, Lackner H, Panzer-­ Preuner S, Peters C, Pötschger U, Daxberger H, Rieder FJ, Gröschel C, Kastner MT, Kosulin K, Pathogen Identification: Methods and Protocols: Grümayer R, Mann G, Attarbaschi A, Urban EC, Fritsch G, Geyeregger R, Schrauder A, von Stack- Laengle J, Zadnikar R, Marculescu R, Schneider M, Springer New York. Haas OA. (2016). Imatinib-induced long-term remis- elberg A, Schrappe M, Bader P, Ebell W, Eckert C, Lion T, Bergmann M, Kallay E, Steininger C. (2017, sion in a relapsed RCSD1-ABL1-positive acute lymph- Lang P, Sykora KW, Schrum J, Kremens B, Ehlert Epub 2016 Aug 9). Human cytomegalovirus infection Lopes SS, Distel M, Linker C, Fior R, Monteiro R, oblastic leukemia. Haematologica, 101: e332-335 K, Albert MH, Meisel R, Lawitschka A, Mann G, downregulates vitamin-D receptor in mammalian Bianco IH, Portugues R, Strahle U, Saude L. (2016). Panzer-Grümayer R, Güngör T, Holter W, Strahm B, cells. J Steroid Biochem Mol Biol, 165: 356-362 Report of the 4th European Zebrafish Principal Pichler H, Fritsch G, König M, Daxberger H, Gruhn B, Schulz A, Woessmann W, Lion T. (2016). Investigator Meeting. Zebrafish, 13: 590-595 Glogova E, Pötschger U, Breuer S, Lawitschka A, Risk assessment of relapse by lineage-specific Ruiz-Pinto S, Pita G, Patino-Garcia A, Garcia- Güclü ED, Karlhuber S, Holter W, Haas OA, Lion T, monitoring of chimerism in children undergoing Miguel P, Alonso J, Perez-Martinez A, Sastre A, Mann G, Holter W. (2016). [Treatment of low-risk Matthes-Martin S. (2016). Peripheral blood late allogeneic stem cell transplantation for acute Gomez-Mariano G, Lissat A, Scotlandi K, Serra M, pediatric lymphocyte-predominant Hodgkin mixed chimerism in leucocyte subpopulations lympho­blastic leukemia. Haematologica, 101: 741-746 Ladenstein R, Lapouble E, Pierron G, Kontny U, Picci lymphoma]. Strahlenther Onkol, 192: 827-829 following allogeneic stem cell transplantation for P, Kovar H, Delattre O, González-Neira A. (2016). childhood malignancies: does it matter? Br J Haema- Proff J, Walterskirchen C, Brey C, Geyeregger R, Identification of genetic variants in pharmacokinetic Minas TZ, Surdez D, Javaheri T, Tanaka M, Howarth tol, 173: 905-917 Full F, Ensser A, Lehner M, Holter W. (2016). Cyto- genes associated with Ewing Sarcoma treatment M, Kang HJ, Han J, Han ZY, Sax B, Kream BE Hong megalovirus-Infected Cells Resist T Cell Mediated outcome. Ann Oncol, 27: 1788-1793 SH, Çelik H, Tirode F, Tuckermann J, Toretsky JA, Posch C, Moslehi H, Sanlorenzo M, Green G, Vujic I, Killing in an HLA-Recognition Independent Manner. Kenner L, Kovar H, Lee S, Sweet-Cordero EA, Naka- Panzer-Grümayer R, Rappersberger K, Ortiz-Urda S. Front Microbiol, 7: 844 Scheer M, Dantonello T, Hallmen E, Vokuhl C, mura T, Moriggl R, Delattre O, Üren A. (2017, Epub (2016). Pharmacological inhibitors of c-KIT block Leuschner I, Sparber-Sauer M, Kazanowska B, Niggli 2016 May 15). Combined experience of six independ- mutant c-KIT mediated migration of melanocytes Radic-Sarikas B, Tsafou KP, Emdal KB, Papamarkou F, Ladenstein R, Bielack SS, Klingebiel T, Koscielniak ent laboratories attempting to create an Ewing and melanoma cells in vitro and in vivo. Oncotarget, T, Huber KVM, Mutz C, Toretsky JA, Bennett KL, E. (2016). Primary Metastatic Synovial Sarcoma: sarcoma mouse model. Oncotarget, 8: 34141-34163 7: 45916-45925 Olsen JV, Brunak S, Kovar H, Superti-Furga G. (2017, Experience of the CWS Study Group. Pediatr Blood Epub 2016 Nov 15). Combinatorial drug screening Cancer, 63: 1198-1206 Mutz CN, Schwentner R, Kauer MO, Katschnig AM, Posch C, Sanlorenzo M, Vujic I, Oses-Prieto JA, identifies Ewing sarcoma-specific sensitivities. Kromp F, Aryee DN, Erhardt S, Goiny M, Alonso J, Cholewa BD, Kim ST, Ma J, Lai K, Zekhtser M, Mol Cancer Ther, 16: 88-101 Schüller S, Attarbaschi A, Berger A, Hutter C, Fuchs D, Kovar H. (2016). EWS-FLI1 impairs aryl Esteve-Puig R, Green G, Chand S, Burlingame AL, Klebermass-Schrehof K, Steiner M. (2016). hydrocarbon receptor activation by blocking trypto- Panzer-Grümayer R, Rappersberger K, Ortiz-Urda S. Reismüller B, Steiner M, Pichler H, Dworzak M, Hemophagocytic lymphohistiocytosis triggered by phan breakdown via the kynurenine pathway. FEBS (2016). Phosphoproteomic Analyses of NRAS(G12) Urban C, Meister B, Schmitt K, Pötschger U, Gaucher disease in a preterm neonate. Pediatric Lett, 590: 2063-2075. and NRAS(Q61) Mutant Melanocytes Reveal König M, Mann G, Haas OA, Attarbaschi A; Austrian Hematology and Oncology, 33: 462-467 Increased CK2alpha Kinase Levels in NRAS(Q61) ALL-BFM (Berlin-Frankfurt-Münster) Study Group. Nitzlnader M, Canete Nieto A, Ribelles AJ, Brunmair Mutant Cells. J Invest Dermatol, 136: 2041-2048 (2017, Epub 2016 Nov 2). High hyperdiploid acute Schwentner R, Herrero-Martin D, Kauer MO, Mutz B, Ladenstein R, Schreier G. (2016). Interoperability lymphoblastic leukemia (ALL)-A 25-year population- CN, Katschnig AM, Sienski G, Alonso J, Aryee DN, Architecture for a Paediatric Oncology European Preuner S, Barna A, Frommlet F, Czurda S, Konstan- based survey of the Austrian ALL-BFM Kovar H. (2017, Epub 2016 Dec 22). The role of Reference Network. Stud Health Technol Inform, tin B, Alikian M, Machova Polakova K, Sacha T, Rich- (Berlin-Frankfurt-Munster) Study Group. miR-17-92 in the miRegulatory landscape of Ewing 223: 39-45 ter J, Lion T, Gabriel C. (2016). Quantitative Analysis Pediatr Blood Cancer, 64: e26327 sarcoma. Oncotarget, 8: 10980-10993 of Mutant Subclones in Chronic Myeloid Leukemia: Comparison of Different Methodological Approaches. Reiter M, Rota P, Kleber F, Diem M, Groeneveld-­ Seidel MG, Boztug K, Haas OA. (2016). Immune Int J Mol Sci, 17: 642 Krentz S, Dworzak M. (2016). Clustering of cell Dysregulation Syndromes (IPEX, CD27 Deficiency, populations in flow cytometry data using a combina- and Others): Always Doomed from the Start? tion of Gaussian mixtures. Pattern Recognition, 60: J Clin Immunol, 36: 6-7 1029-1040

120 121 Einleitung

Siebert N, Eger C, Seidel D, Juttner M, Zumpe M, Thompson D, Vo KT, London WB, Fischer M, Weilner S, Schraml E, Wieser M, Messner P, Schnei- Worel N, Fritsch G, Agis H, Bohm A, Engelich Wegner D, Kietz S, Ehlert K, Veal GJ, Siegmund Ambros PF, Nakagawara A, Brodeur GM, Matthay der K, Wassermann K, Micutkova L, Fortschegger K, G, Leitner GC, Geissler K, Gleixner K, Kalhs P, W, Weiss M, Loibner H, Ladenstein R, Lode HN. KK, DuBois SG. (2016). Identification of patient Maier AB, Westendorp R, Resch H, Wolbank S, Redl Buxhofer-Ausch V, Keil F, Kopetzky G, Mayr V, (2016). Pharmacokinetics and pharmacodynamics of subgroups with markedly disparate rates of MYCN H, Jansen-Dürr P, Pietschmann P, Grillari-Voglauer Rabitsch W, Reisner R, Rosskopf K, Ruckser R, ch14.18/CHO in relapsed/refractory high-risk neuro- amplification in neuroblastoma: A report from the R, Grillari J. (2016). Secreted microvesicular miR-31 ­Zoghlami C, Zojer N, Greinix HT. (2016). Plerixafor blastoma patients treated by long-term infusion in International Neuroblastoma Risk Group project. inhibits osteogenic differentiation of mesenchymal as preemptive strategy results in high success rates combination with IL-2. MAbs, 8: 604-616 Cancer, 122: 935-945 stem cells. Aging Cell, 15: 744-754 in autologous stem cell mobilization failure. J Clin Apher, Epub 2016 Aug 31 Sipurzynski J, Fahrner B, Kerbl R, Crazzolara R, Tischer S, Geyeregger R, Kwoczek J, Heim A, Figue- Weiss T, Taschner-Mandl S, Bileck A, Slany A, Jones N, Ebetsberger G, Jauk B, Strenger V, Wohl- iredo C, Blasczyk R, Maecker-Kolhoff B, Eiz-Vesper B. Kromp F, Rifatbegovic F, Frech C, Windhager R, Zaliova M, Moorman AV, Cazzaniga G, Stanulla muther B, Schwinger W, Lackner H, Urban C, Holter (2016). Discovery of immunodominant T-cell epitopes Kitzinger H, Tzou CH, Ambros PF, Gerner C, Ambros M, Harvey RC, Roberts KG, Heatley SL, Loh ML, W, Minkov M, Kager L, Benesch M, Seidel MG. reveals penton protein as a second immunodominant IM. (2016). Proteomics and transcriptomics of periph- Konopleva M, Chen IM, Zimmermannova O, Schwab (2016). Management of chronic immune thrombocy- target in human adenovirus infection. J Transl Med, eral nerve tissue and cells unravel new aspects of C, Smith O, Mozziconacci MJ, Chabannon C, Kim topenia in children and adolescents: lessons from 14: 286 the human Schwann cell repair phenotype. Glia, 64: M, Frederik Falkenburg JH, Norton A, Marshall K, an Austrian national cross-sectional study of 81 2133-2153 Haas OA, Starkova J, Stuchly J, Hunger SP, White D, patients. Semin Hematol, 53: Suppl 1, S43-47 Valent P, Groner B, Schumacher U, Superti-Furga G, Mullighan CG, Willman CL, Stary J, Trka J, Zuna J. Busslinger M, Kralovics R, Zielinski C, Penninger Witt V, Pichler H, Fritsch G, Peters C. (2016). (2016). Characterization of leukemias with ETV6- Smidak R, Aradska J, Kirchberger S, Distel M, JM, Kerjaschki D, Stingl G, Smolen JS, Valenta R, ­Multiple small versus few large amount aspirations ABL1 fusion. Haematologica, 101: 1082-1093 Sialana FJ, Wackerlig J, Mechtcheriakova D, Lubec Lassmann H, Kovar H, Jäger U, Kornek G, Müller M, for bone marrow harvesting in autologous and G. (2016). A detailed proteomic profiling of plasma Sörgel F. (2016). Paul Ehrlich (1854-1915) and His allogeneic bone marrow transplantation. Transfus Zuna J, Moericke A, Arens M, Koehler R, membrane from zebrafish brain. Proteomics Clin Contributions to the Foundation and Birth of Trans- Apher Sci, 55: 221-224 Panzer-Grümayer R, Bartram CR, Fischer S, Fronk- Appl, 10: 1264-1268 lational Medicine. J Innate Immun, 8: 111-120 ova E, Zaliova M, Schrauder A, Stanulla M, Zimmer- Wlodarski MW, Hirabayashi S, Pastor V, Stary J, mann M, Trka J, Stary J, Attarbaschi A, Mann G, Stegmaier S, Leuschner I, Poremba C, Ladenstein Ventura S, Aryee DN, Felicetti F, De Feo A, Mancar- Hasle H, Masetti R, Dworzak M, Schmugge M, van Schrappe M, Cario G. Implications of delayed bone R, Kazanowska B, Ljungman G, Scheer M, Blank B, ella C, Manara MC, Picci P, Colombo MP, Kovar H, den Heuvel-Eibrink M, Ussowicz M, De Moerloose marrow aspirations at the end of treatment induc- Bielack S, Klingebiel T, Koscielniak E. (2017, Epub Care A, Scotlandi K. (2016). CD99 regulates neural B, Catala A, Smith OP, Sedlacek P, Lankester AC, tion for risk stratification and outcome in children 2016 Sep 13). The prognostic impact of SYT-SSX differentiation of Ewing sarcoma cells through Zecca M, Bordon V, Matthes-Martin S, Abrahamsson with acute lymphoblastic leukaemia. Br J Haematol, fusion type and histological grade in pediatric miR-34a-Notch-mediated control of NF-kappaB J, Kühl JS, Sykora KW, Albert MH, Przychodzien B, 173: 742-748 patients with synovial sarcoma treated according signaling. Oncogene, 35: 3944-3954 Maciejewski JP, Schwarz S, Göhring G, Schlegel- to the CWS (Cooperative Weichteilsarkom Studie) berger B, Cseh A, Noellke P, Yoshimi A, Locatelli F, trials. Pediatr Blood Cancer, 64: 89-95 Vesely C, Frech C, Eckert C, Cario G, Mecklen- Baumann I, Strahm B, Niemeyer CM; EWOG-MDS. brauker A, zur Stadt U, Nebral K, Kraler F, Fischer (2016). Prevalence, clinical characteristics, and prog- Taschner-Mandl S, Schwarz M, Blaha J, Kauer M, S, Attarbaschi A, Schuster M, Bock C, Cavé H, von nosis of GATA2-related myelodysplastic syndromes Kromp F, Frank N, Rifatbegovic F, Weiss T, Laden- Stackelberg A, Schrappe M, Horstmann MA, Mann in children and adolescents. Blood, 127: 1387-1397 stein R, Hohenegger M, Ambros IM, Ambros PF. G, Haas OA, Panzer-Grümayer R. (2017). Genomic (2016). Metronomic topotecan impedes tumor and transcriptional landscape of P2RY8-CRLF2- growth of MYCN-amplified neuroblastoma cells in positive childhood acute lymphoblastic leukemia. vitro and in vivo by therapy induced senescence. Leukemia, Epub 2017 Jan 6 Oncotarget, 7: 3571-3586

122 123 IMPRESSUM UNTERSTÜTZUNG

HERAUSGEBER SPENDENKONTO St. Anna Kinderkrebsforschung e.V. St. Anna Kinderkrebsforschung e.V. Zimmermannplatz 10, A-1090 Wien Bank Austria www.ccri.at, www.kinderkrebsforschung.at IBAN: AT79 1200 0006 5616 6600 KONTAKT BIC: BKAUATWW Mag. Marion Zavadil, CCRI Sekretariat [email protected]

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